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The successful marriage policy of margrave Leopold III increased the importance of the House of Babenberg in late medieval Austria (12th century). Historical documentation is inconclusive in providing evidence whether or not his eldest son Adalbert derived from an earlier relationship or from the marriage with King Henry IV's daughter Agnes of Waiblingen, with whom Leopold is considered to have had 17 children. As a matter of fact Adalbert was ignored in the line of succession in favor of a younger brother, Leopold IV, which has led to long term historical discussions. Human remains attributed to these individuals were subjected to DNA analysis. Autosomal, Y-chromosomal and mitochondrial DNA analyses brought successful results, which suggested that Leopold III, Agnes and Adalbert were related in parent-son constellation, in contrast to historical considerations. A possible mix-up of Adalbert's remains with those of his younger brother Ernst could not be confirmed by DNA analysis. +
Archaeological excavations conducted at an early mediaeval cemetery in Volders (Tyrol, Austria) produced 141 complete skeletal remains dated between the 5th/6th and 12th/13th centuries. These skeletons represent one of the largest historical series of human remains ever discovered in the East Alpine region. Little historical information is available for this region and time period. The good state of preservation of these bioarchaeological finds offered the opportunity of performing molecular genetic investigations. Adequate DNA extraction methods were tested in the attempt to obtain as high DNA yields as possible for further analyses. Molecular genetic sex-typing using a dedicated PCR multiplex ("Genderplex") gave interpretable results in 88 remains, 78 of which had previously been sexed based on morphological features. We observed a discrepancy in sex determination between the two methods in 21 cases. An unbiased follow-up morphological examination of these finds showed congruence with the DNA results in all but five samples. +
Mitochondria from diverse organisms are capable of transporting large amounts of Ca<sup>2+</sup> via a ruthenium-red-sensitive, membrane-potential-dependent mechanism called the uniporter. Although the uniporter's biophysical properties have been studied extensively, its molecular composition remains elusive. We recently used comparative proteomics to identify MICU1 (also known as CBARA1), an EF-hand-containing protein that serves as a putative regulator of the uniporter. Here, we use whole-genome phylogenetic profiling, genome-wide RNA co-expression analysis and organelle-wide protein coexpression analysis to predict proteins functionally related to MICU1. All three methods converge on a novel predicted transmembrane protein, CCDC109A, that we now call 'mitochondrial calcium uniporter' (MCU). MCU forms oligomers in the mitochondrial inner membrane, physically interacts with MICU1, and resides within a large molecular weight complex. Silencing MCU in cultured cells or in vivo in mouse liver severely abrogates mitochondrial Ca<sup>2+</sup> uptake, whereas mitochondrial respiration and membrane potential remain fully intact. MCU has two predicted transmembrane helices, which are separated by a highly conserved linker facing the intermembrane space. Acidic residues in this linker are required for its full activity. However, an S259A point mutation retains function but confers resistance to Ru360, the most potent inhibitor of the uniporter. Our genomic, physiological, biochemical and pharmacological data firmly establish MCU as an essential component of the mitochondrial Ca<sup>2+</sup> uniporter. +
The recent correspondence about intramitochondrial [1–3] pH and intra-extramitochondrial pH-gradients [4] does not touch on a related (and possibly naive) problem arising out of the extreme smallness of the space within a single mitochondrion. +
Efficient mitochondrial function is required in tissues with high energy demand such as the heart, and mitochondrial dysfunction is associated with cardiovascular disease. Expression of mitochondrial proteins is tightly regulated in response to internal and external stimuli. Here we identify a novel mechanism regulating mitochondrial content and function, through BUD23-dependent ribosome generation. BUD23 was required for ribosome maturation, normal 18S/28S stoichiometry and modulated the translation of mitochondrial transcripts in human A549 cells. Deletion of Bud23 in murine cardiomyocytes reduced mitochondrial content and function, leading to severe cardiomyopathy and death. We discovered that BUD23 selectively promotes ribosomal interaction with low GC-content 5'UTRs. Taken together we identify a critical role for BUD23 in bioenergetics gene expression, by promoting efficient translation of mRNA transcripts with low 5'UTR GC content. BUD23 emerges as essential to mouse development, and to postnatal cardiac function.
<small>© 2019, Baxter et al.</small> +
Down syndrome is the most common genomic disorder of intellectual disability and is caused by trisomy of chromosome 21. Several genes in this chromosome repress mitochondrial biogenesis. The goal of this study was to evaluate whether early overexpression of these genes may cause a prenatal impairment of oxidative phosphorylation negatively affecting neurogenesis. Reduction in the mitochondrial energy production and a lower mitochondrial function have been reported in diverse tissues or cell types, and also at any age, including early fetuses, suggesting that a defect in oxidative phosphorylation is an early and general event in Down syndrome individuals. Moreover, many of the medical conditions associated with Down syndrome are also frequently found in patients with oxidative phosphorylation disease. Several drugs that enhance mitochondrial biogenesis are nowadays available and some of them have been already tested in mouse models of Down syndrome restoring neurogenesis and cognitive defects. Because neurogenesis relies on a correct mitochondrial function and critical periods of brain development occur mainly in the prenatal and early neonatal stages, therapeutic approaches intended to improve oxidative phosphorylation should be provided in these periods. +
ATP-dependent proteases are currently emerging as key regulators of mitochondrial functions. Among these proteolytic systems, Lon protease is involved in the control of selective protein turnover in the mitochondrial matrix. In the absence of Lon, yeast cells have been shown to accumulate electron-dense inclusion bodies in the matrix space, to loose integrity of mitochondrial genome and to be respiratory deficient. In order to address the role of Lon in mitochondrial functionality in human cells, we have set up a HeLa cell line stably transfected with a vector expressing a shRNA under the control of a promoter which is inducible with doxycycline. We have demonstrated that reduction of Lon protease results in a mild phenotype in this cell line in contrast with what have been observed in other cell types such as WI-38 fibroblasts. Nevertheless, deficiency in Lon protease led to an increase in ROS production and to an accumulation of carbonylated protein in the mitochondria. Our study suggests that Lon protease has a wide variety of targets and is likely to play different roles depending of the cell type. +
We use the yeast ''Saccharomyces cerevisiae'' as a model to study the molecular mechanisms by which age-related changes in mitochondrial membrane lipids regulate longevity [1-8].
We found that an exogenously added bile acid called lithocholic acid (LCA) extends yeast chronological lifespan and accumulates in the inner mitochondrial membrane (IMM). To define the mechanism by which LCA extends yeast longevity, we 1) examined how LCA influences mitochondrial proteome and lipidome; 2) investigated the effect of LCA on the composition and stoichiometry of respiratory complexes and supercomplexes in the IMM; 3) assessed how LCA affects mitochondrial oxygen consumption, membrane potential and reactive oxygen species (ROS); 4) examined how LCA influences mitochondrial morphology and how it affects the chronology of events characteristic of age-related, mitochondria-controlled apoptosis; and 5) investigated the effect of LCA on the lifespans of long- and short-lived mutants lacking individual components of the mitochondrial fission and fusion machines.
Our findings imply that LCA delays yeast aging by increasing the level of phosphatidylserine (PS) and decreasing the levels of phosphatidylethanolamine (PE) and cardiolipin in the IMM. By altering the abundance of these lipid species, LCA greatly expands mitochondrial membrane cristae. In addition, LCA enhances the positive effect of PS and weakens the negative effect of PE on membrane protein machines whose activity they modulate – thereby 1) stimulating protein machines driving mitochondrial respiration, the maintenance of mitochondrial membrane potential and ROS homeostasis, and mitochondrial fusion; and 2) inhibiting protein machines promoting mitochondrial fission and mitochondria-controlled apoptosis.
We found that LCA also functions as a potent and selective anti-tumor compound in cultured human neuroblastoma, glioma and breast cancer cells by modulating mitochondrial processes playing essential roles in both cancer and aging. The effects of LCA on these processes seen in cancer cell cultures are opposite of those observed in non-cancerous human cells and in chronologically aged, quiescent yeast.
Mitochondria-shaping proteins modulate bioenergetics, apoptosis, Ca<sup>2+</sup> signalling and autophagy. The inner membrane pro-fusion and cristae shaping protein Optic atrophy 1 (Opa1) protects multiple tissues from damage by regulating cytochrome c release and mitochondrial respiratory efficiency, but whether this is mirrored by systemic changes in intermediary metabolism is unknown. Here we identify Opa1 as a key regulator of insulin sensitivity and adipose tissue function. Controlled Opa1 overexpression in the mouse reduces weight, improves glucose metabolism and insulin sensitivity, by reducing fat depots and favoring brownization of white adipose cells ''in vivo'' and ''in vitro''. Adipocyte-specific Opa1 deletion triggers a lipodystrophic phenotype with hyperglycemia, insulin resistance, brown adipose tissue whitening and hepatosteatosis. Our findings identify the genetic and metabolic basis for Opa1 role in a lean and insulin-sensitive phenotype, paving the way for novel therapeutic strategies to treat obesity and diabetes. +
A computational model for the mitochondrial respiratory chain that appropriately balances mass, charge, and free energy transduction is introduced and analyzed based on a previously published set of data measured on isolated cardiac mitochondria. The basic components included in the model are the reactions at Complexes I, III, and IV of the electron transport system, ATP synthesis at F1FO ATPase, substrate transporters including adenine nucleotide translocase and the phosphate-hydrogen co-transporter, and cation fluxes across the inner membrane including fluxes through the K<sup>+</sup>/H<sup>+</sup> antiporter and passive H<sup>+</sup> and K<sup>+</sup> permeation. Estimation of 16 adjustable parameter values is based on fitting model simulations to nine independent data curves. The identified model is further validated by comparison to additional datasets measured from mitochondria isolated from rat heart and liver and observed at low oxygen concentration. To obtain reasonable fits to the available data, it is necessary to incorporate inorganic-phosphate-dependent activation of the dehydrogenase activity and the electron transport system. Specifically, it is shown that a model incorporating phosphate-dependent activation of Complex III is able to reasonably reproduce the observed data. The resulting validated and verified model provides a foundation for building larger and more complex systems models and investigating complex physiological and pathophysiological interactions in cardiac energetics. +
Chemical reaction systems operating in nonequilibrium open-system states arise in a great number of contexts, including the study of living organisms, in which chemical reactions, in general, are far from equilibrium. Here we introduce a theorem that relates forward and reverse fluxes and free energy for any chemical process operating in a steady state. This relationship, which is a generalization of equilibrium conditions to the case of a chemical process occurring in a nonequilibrium steady state in dilute solution, provides a novel equivalent definition for chemical reaction free energy. In addition, it is shown that previously unrelated theories introduced by Ussing and Hodgkin and Huxley for transport of ions across membranes, Hill for catalytic cycle fluxes, and Crooks for entropy production in microscopically reversible systems, are united in a common framework based on this relationship. +
Inorganic phosphorus (P<sub>i</sub>) is often the primary limiting nutrient in freshwater ecosystems. Since P<sub>i</sub>-limitation affects energy transduction, and inorganic carbon (C<sub>i</sub>) acquisition can be energy demanding, C<sub>i</sub>-acquisition strategies were compared in four species of green algae grown under P<sub>i</sub>-replete and P<sub>i</sub>-limited conditions predominantly at low and partly at high CO<sub>2</sub>. Although P<sub>i</sub>-limitation was evident by the 10-fold higher cellular C:P ratio and enhanced phosphatase activity, it only decreased C<sub>i</sub>-acquisition to a small extent. Nonetheless, the effects of P<sub>i</sub>-limitation on both CO<sub>2</sub> and acquisition were demonstrated. Decreased CO<sub>2</sub> acquisition under conditions of P<sub>i</sub> limitation was mainly visible in the maximum uptake rate (''V''<sub>max</sub>) and, for the neutrophile ''Scenedesmus vacuolatus'', in the affinity for CO<sub>2</sub> acquisition. Discrimination against <sup>13</sup>C was higher under P<sub>i</sub>-limited, high CO<sub>2</sub> conditions, compared with P<sub>i</sub>-replete, high CO<sub>2</sub> conditions, in ''Chlamydomonas acidophila'' and ''S. vacuolatus''. In the pH-drift experiments, acquisition was reduced in P<sub>i</sub>-limited ''C. reinhardtii''. In general, energy demanding bicarbonate uptake was indicated by the less strong discrimination against <sup>13</sup>C under low CO<sub>2</sub> conditions in the neutrophiles (HCO<sub>3</sub><sup>-</sup> users), separating them from the acidophilic or acidotolerant species (CO<sub>2</sub> users). The high variability of the influence of P<sub>i</sub> supply among different green algal species is linked to their species-specific C<sub>i</sub>-acquisition strategies. +
Beatson International Cancer Conference, Glasgow, United Kingdom, 2018 +
Intrauterine growth restriction is associated with an increased risk of developing obesity, insulin resistance, and cardiovascular disease. However its effect on energetics in heart remains unknown. In this study, we examined respiration in cardiac muscle and liver from adult mice that were undernourished ''in utero''. We report that ''in utero'' undernutrition is associated with impaired cardiac muscle energetics, including decreased fatty acid oxidative capacity, decreased maximum oxidative phosphorylation rate, and decreased proton leak respiration. No differences in oxidative characteristics were detected in liver. We also measured plasma acylcarnitine levels and found that short-chain acylcarnitines are increased with ''in utero'' undernutrition. Results reveal the negative impact of suboptimal maternal nutrition on adult offspring cardiac energy metabolism, which may have lifelong implications for cardiovascular function and disease risk. +
Resveratrol (RSV) is a polyphenolic compound suggested to have anti-diabetic properties. Surprisingly, little is known regarding the effects of RSV supplementation on adipose tissue (AT) metabolism ''in vivo''. The purpose of this study was to assess the effects of RSV on mitochondrial content and respiration, glyceroneogenesis (GNG), and adiponectin secretion in adipose tissue from Zucker diabetic fatty (ZDF) rats. Five-week-old ZDF rats were fed a chow diet with (ZDF RSV) or without (ZDF chow) RSV (200 mg/kg body wt) for 6 wk. Changes in adipose tissue metabolism were assessed in subcutaneous (scAT) and intra-abdominal [retroperitoneal (rpWAT), epididymal (eWAT)] adipose tissue depots. ZDF RSV rats showed lower fasting glucose and higher circulating adiponectin, as well as lower glucose area under the curve during intraperitoneal glucose and insulin tolerance tests than ZDF chow. [¹⁴C]pyruvate incorporation into triglycerides and adiponectin secretion were higher in scAT from ZDF RSV rats, concurrent with increases in adipose tissue triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and the phosphorylation of pyruvate dehydrogenase-E1α (PDH) (Ser293) protein content in this depot. Moreover, uncoupled mitochondrial respiration and complex I and II-supported respiration were increased in both scAT and rpWAT, which correlated with increases in cytochrome c oxidase subunit IV (COX4) protein content. ''In vitro'' treatment of scAT with RSV (50 μmol/l; 24 h) induced pyruvate dehydrogenase kinase 4 (PDK4) and peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC-1α) mRNA expression. Collectively, these data demonstrate that RSV can induce adipose tissue mitochondrial biogenesis in parallel with increases in GNG and adiponectin secretion. +
Alterations in lipid metabolism within the heart may have a causal role in the establishment of diabetic cardiomyopathy, however this remains equivocal. Therefore, in the current study we determined cardiac mitochondrial bioenergetics in ZDF rats before overt type 2 diabetes and diabetic cardiomyopathy developed. In addition, we utilized resveratrol, a compound previously shown to improve prevent or reverse cardiac dysfunction in high fat-fed rodents, as a tool to potential recover dysfunctions within mitochondria. Fasting blood glucose and invasive left ventricular hemodynamic analysis confirmed the absence of type 2 diabetes and diabetic cardiomyopathy. However, fibrosis was already increased (P<0.05) ~70% in ZDF rats at this early stage in disease progression. Assessments of mitochondrial ADP and pyruvate respiratory kinetics in permeabilized fibres from the left ventricle revealed normal electron transport chain function and content. In contrast, the apparent Km to palmitoyl-CoA (P-CoA) was increased (P<0.05) ~60%, which was associated with an accumulation of intracellular triacylgycerol, diacylglycerol and ceramide species. In addition, the capacity for mitochondrial ROS emission was increased (P<0.05) ~3-fold in ZDF rats. The provision of resveratrol recovered fibrosis, P-CoA respiratory sensitivity, reactive lipid accumulation and mitochondrial reactive oxygen species emission rates. Altogether the current data supports the supposition that a chronic dysfunction within mitochondrial lipid-supported bioenergetics contributes to the development of diabetic cardiomyopathy, as this was present before overt diabetes or cardiac dysfunction. In addition, we show resveratrol supplementation prevents these changes, supporting the belief that resveratrol is a potent therapeutic approach for preventing diabetic cardiomyopathy. +
Bronchial remodeling is a key feature of asthma that is already present in preschoolers with wheezing. Moreover, bronchial smooth muscle (BSM) remodeling at preschool age is predictive of asthma at school age. However, the mechanism responsible for BSM remodeling in preschoolers with wheezing remains totally unknown. In contrast, in adult asthma, BSM remodeling has been associated with an increase in BSM cell proliferation related to increased mitochondrial mass and biogenesis triggered by an altered calcium homeostasis. Indeed, BSM cell proliferation was decreased ''in vitro'' by the calcium channel blocker gallopamil.
Our aim was to investigate the mechanisms involved in BSM cell proliferation in preschoolers with severe wheezing, with special attention to the role of mitochondria and calcium signaling.
Bronchial tissue samples obtained from 12 preschool controls without wheezing and 10 preschoolers with severe wheezing were used to measure BSM mass and establish primary BSM cell cultures. BSM cell proliferation was assessed by manual counting and flow cytometry, ATP content was assessed by bioluminescence, mitochondrial respiration was assessed by using either the Seahorse or Oroboros technique, mitochondrial mass and biogenesis were assessed by immunoblotting, and calcium response to carbachol was assessed by confocal microscopy. The effect of gallopamil was also evaluated.
BSM mass, cell proliferation, ATP content, mitochondrial respiration, mass and biogenesis, and calcium response were all increased in preschoolers with severe wheezing compared with in the controls. Gallopamil significantly decreased BSM mitochondrial biogenesis and mass, as well as cell proliferation.
Mitochondria are key players in BSM cell proliferation in preschoolers with severe wheezing and could represent a potential target to treat BSM remodeling at an early stage of the disease. +
Hepatic insulin resistance in obesity and type 2 diabetes was recently associated with endoplasmic reticulum (ER)-mitochondria miscommunication. These contact sites (mitochondria-associated membranes: MAMs) are highly dynamic and involved in many functions; however, whether MAM dysfunction plays a causal role in hepatic insulin resistance and steatosis is not clear. Thus, we aimed to determine whether and how organelle miscommunication plays a role in the onset and progression of hepatic metabolic impairment.
We analyzed hepatic ER-mitochondria interactions and calcium exchange in a time-dependent and reversible manner in mice with diet-induced obesity. Additionally, we used recombinant adenovirus to express a specific organelle spacer or linker in mouse livers, to determine the causal impact of MAM dysfunction on hepatic metabolic alterations.
Disruption of ER-mitochondria interactions and calcium exchange is an early event preceding hepatic insulin resistance and steatosis in mice with diet-induced obesity. Interestingly, an 8-week reversal diet concomitantly reversed hepatic organelle miscommunication and insulin resistance in obese mice. Mechanistically, disrupting structural and functional ER-mitochondria interactions through the hepatic overexpression of the organelle spacer FATE1 was sufficient to impair hepatic insulin action and glucose homeostasis. In addition, FATE1-mediated organelle miscommunication disrupted lipid-related mitochondrial oxidative metabolism and induced hepatic steatosis. Conversely, reinforcement of ER-mitochondria interactions through hepatic expression of a synthetic linker prevented diet-induced glucose intolerance after 4 weeks' overnutrition. Importantly, ER-mitochondria miscommunication was confirmed in the liver of obese patients with type 2 diabetes, and correlated with glycemia, HbA1c and HOMA-IR index.
ER-mitochondria miscommunication is an early causal trigger of hepatic insulin resistance and steatosis, and can be reversed by switching to a healthy diet. Thus, targeting MAMs could help to restore metabolic homeostasis.
The role of the nuclear-encoded subunit VIa in the regulation of cytochrome oxidase by ATP was investigated in isolated yeast mitochondria. As the subunit VIa-null strain possesses a fully active and assembled cytochrome oxidase, multiple ATP-regulating sites were characterized with respect to their location and their kinetic effect:
(a) intra-mitochondrial ATP inhibited the complex IV activity of the null strain, whereas the prevailing effect of
ATP on the wild-type strain, at low ionic strength, was activation on the cytosolic side of complex IV, mediated
by subunit VIa. However, at physiological ionic strength (i.e. <200 mm), activation by ATP was absent but
inhibition was not impaired; (b) in ethanol-respiring mitochondria, when the electron flux was modulated using a
protonophoric uncoupler, the redox state of aa3 cytochromes varied with respect to activation (wild-type) or
inhibition (null-mutant) of the cytochrome oxidase by ATP; (c) consequently, the control coefficient of
cytochrome oxidase on respiratory flux, decreased (wild-type) or increased (null-mutant) in the presence of ATP;
(d) considering electron transport from cytochrome c to oxygen, the response of cytochrome oxidase to its thermodynamic driving force was increased by ATP for the wild-type but not for the mutant subunit. Taken together, these findings indicate that at physiological concentration, ATP regulates yeast cytochrome oxidase via subunit-mediated interactions on both sides of the inner membrane, thus subtly tuning the thermodynamic and kinetic control of respiration. This study opens up new prospects for understanding the feedback regulation of the respiratory chain by ATP. +
The amount of light scattered by a mitochondrial suspension depends on matrix volume (Tedeschi, H., and Harris, D.L. (1955) Arch. Biochem. Biophys. 58, 52-67), a correlation which has been extensively exploited for qualitative studies of solute transport across the inner membrane. To obtain reliable, quantitative estimates of solute transport, it is first necessary to characterize the factors determining mitochondrial light scattering. We show that the dependence of absorbance on mitochondrial concentration can be linearized, resulting in an intrinsic light scattering parameter which is independent of the concentration and source of mitochondria. We show that the absorbance osmotic curve is segmentally linear, exhibiting discontinuities which disappear irreversibly following preswelling. In contrast, direct measurements reveal matrix volume to be reversibly and linearly dependent on inverse osmolality. This divergence is a consequence of the fact that the optical technique samples total particle volume, including contributions from folded membranes and trapped medium. These contributions are minimized by structural components, such as intermembrane connections and the outer membrane, which contribute to efficient packaging of the mitochondrion. When these structures are broken, the mitochondrion cannot return to its native state. We observe that the swelling-induced, irreversible transition from efficient packaging to a random packing state begins at a matrix volume of 1.9 microliter/mg and is complete at 3.1 microliter/mg. These findings complicate the interpretation of light scattering results but do not appear to present an insurmountable obstacle to the quantitative application of this technique to transport kinetics. +
Although a burst of oxidants has been well described with reperfusion, less is known about the oxidants generated by the highly reduced redox state and low O(2) of ischemia. This study aimed to further identify the species and source of these oxidants. Cardiomyocytes were exposed to 1 h of simulated ischemia while oxidant generation was assessed by intracellular dihydroethidine (DHE) oxidation. Ischemia increased DHE oxidation significantly (0.7 +/- 0.1 to 2.3 +/- 0.3) after 1 h. Myxothiazol (mitochondrial site III inhibitor) attenuated oxidation to 1.3 +/- 0.1, as did the site I inhibitors rotenone (1.0 +/- 0.1), amytal (1.1 +/- 0.1), and the flavoprotein oxidase inhibitor diphenyleneiodonium (0.9 +/- 0.1). By contrast, the site IV inhibitor cyanide, as well as inhibitors of xanthine oxidase (allopurinol), nitric oxide synthase (nitro-L-arginine methyl ester), and NADPH oxidase (apocynin), had no effect. Finally, DHE oxidation increased with Cu- and Zn-containing superoxide dismutase (SOD) inhibition using diethyldithiocarbamate (2.7 +/- 0.1) and decreased with exogenous SOD (1.1 +/- 0.1). We conclude that significant superoxide generation occurs during ischemia before reperfusion from the ubisemiquinone site of the mitochondrial electron transport chain. +
Mitochondria are fundamental for cellular metabolism as they are both a source and a target of nutrient intermediates originating from converging metabolic pathways, and their role in the regulation of systemic metabolism is increasingly recognized. Thus, maintenance of mitochondrial homeostasis is indispensable for a functional energy metabolism of the whole organism. Here, we report that loss of the mitochondrial matrix protease CLPP results in a lean phenotype with improved glucose homeostasis. Whole-body CLPP-deficient mice are protected from diet-induced obesity and insulin resistance, which was not present in mouse models with either liver- or muscle-specific depletion of CLPP. However, CLPP ablation also leads to a decline in brown adipocytes function leaving mice unable to cope with a cold-induced stress due to non-functional adaptive thermogenesis. These results demonstrate a critical role for CLPP in different metabolic stress conditions such as high-fat diet feeding and cold exposure providing tools to understand pathologies with deregulated ''Clpp'' expression and novel insights into therapeutic approaches against metabolic dysfunctions linked to mitochondrial diseases. +
In this work, ion-selective electrodes for calcium ion were investigated. Two ionophores were used in the membranes: ETH 1001 and ETH 129. An internal filling solution buffered for primary ion was used that allowed the lower detection limit to be decreased down to 10(-8.8) M. Theoretical and experimental electrode characteristics pertaining to both primary and interfering ions are discussed. Better behavior was obtained with the electrode prepared with ETH 129 in the membrane. This electrode would be the most likely candidate for obtaining a low Ca(2+) detection limit in measurements performed with high K(+), Na(+), Mg(2+) background, which is found inside the cells of living organisms, for example. The potentiometric response of the electrode in solutions containing main and interfering ions is in good agreement with simulated curves obtained using the Nernst-Planck-Poisson (NPP) model. +
Potassium channels as present in the plasma membrane of various cells have also been found in the inner mitochondrial membrane. Potassium channels have been proposed to regulate the mitochondrial membrane potential, respiration, matrix volume and Ca2+ ion homeostasis. It has been suggested that mitochondrial potassium channels participate in ischemic preconditioning and neurodegenerative disorders.
In our study single channel activity of a large conductance Ca2+-regulated potassium channel was measured by patch-clamp of mitoplasts isolated from an astrocytoma cell line. A potassium selective current was recorded with a mean conductance of 290 pS in symmetrical 150 mM KCl solution. The channel was activated by Ca2+ at micromolar concentrations and inhibited irreversibly by iberiotoxin, an selective inhibitor of the BKCa channel. Additionally, we showed that substrates of the respiratory chain like NADH, succinate, and glutamate/malate, decrease the activity of the channel at positive voltages. The effect was abolished by rotenone, antimycin and cyanide, being inhibitors of respiratory chain.
Our findings indicate that mitochondrial large conductance Ca2+-regulated potassium channels with properties similar to the surface membrane BKCa channel are present in human astrocytoma mitochondria and can be stimulated by redox status of the respiratory chain.
Details: Bednarczyk P, Wieckowski MR, Broszkiewicz M, Skowronek K, Siemen D, Szewczyk A. Putative Structural and Functional Coupling of the Mitochondrial BKCa Channel to the Respiratory Chain. PLoS One. 2013 Jun 27;8(6):e68125. +
Direct comparisons of synaptic functional parameters in brain tissues from different groups of experimental animals and different samples from post mortem human brain are often hindered by the inability to perform assays at the same time. To circumvent these difficulties we developed methods for cryopreservation and long-term storage of neocortical synaptosomes. The synaptosomes are suspended in a cryopreservation medium containing 10% dimethylsulfoxide and 10% fetal bovine serum, and are slowly cooled to -80 degreesC and then stored in liquid nitrogen. The function of plasma membrane glucose and glutamate transporters, and mitochondrial electron transport activity and membrane potential were measured in fresh, cryopreserved (CP), and non-cryopreserved freeze-thawed (NC) synaptosomes. Glucose and glutamate transporter activities, and mitochondrial functional parameters in CP synaptosomes were essentially identical to those in fresh unfrozen synaptosomes. Glucose and glutamate transport were severely compromised in NC synaptosomes, whereas mitochondrial function and cellular esterase activity were largely maintained. Electron paramagnetic resonance studies in conjunction with a protein-specific spin label indicated that cryopreservation did not alter the physical state of synaptosomal membrane proteins. These methods provide the opportunity to generate stocks of functional synaptosomes from different experiments or post mortem samples collected over large time intervals. +
Medical and scientific advances are predicated on new knowledge that is robust and reliable and that serves as a solid foundation on which further advances can be built. In biomedical research, we are in the midst of a revolution with the generation of new data and scientific publications at a previously unprecedented rate. However, unfortunately, there is compelling evidence that the majority of these discoveries will not stand the test of time. To a large extent, this reproducibility crisis in basic and preclinical research may be as a result of failure to adhere to good scientific practice and the desperation to publish or perish. This is a multifaceted, multistakeholder problem. No single party is solely responsible, and no single solution will suffice. Here we review the reproducibility problems in basic and preclinical biomedical research, highlight some of the complexities, and discuss potential solutions that may help improve research quality and reproducibility. +
Numerous investigations have shown that mitochondrial dysfunction is a major mechanism of drug-induced liver injury, which involves the parent drug or a reactive metabolite generated through cytochromes P450. Depending of their nature and their severity, the mitochondrial alterations are able to induce mild to fulminant hepatic cytolysis and steatosis (lipid accumulation), which can have different clinical and pathological features. Microvesicular steatosis, a potentially severe liver lesion usually associated with liver failure and profound hypoglycemia, is due to a major inhibition of mitochondrial fatty acid oxidation (FAO). Macrovacuolar steatosis, a relatively benign liver lesion in the short term, can be induced not only by a moderate reduction of mitochondrial FAO but also by an increased hepatic de novo lipid synthesis and a decreased secretion of VLDL-associated triglycerides. Moreover, recent investigations suggest that some drugs could favor lipid deposition in the liver through primary alterations of white adipose tissue (WAT) homeostasis. If the treatment is not interrupted, steatosis can evolve toward steatohepatitis, which is characterized not only by lipid accumulation but also by necroinflammation and fibrosis. Although the mechanisms involved in this aggravation are not fully characterized, it appears that overproduction of reactive oxygen species by the damaged mitochondria could play a salient role. Numerous factors could favor drug-induced mitochondrial and metabolic toxicity, such as the structure of the parent molecule, genetic predispositions (in particular those involving mitochondrial enzymes), alcohol intoxication, hepatitis virus C infection, and obesity. In obese and diabetic patients, some drugs may induce acute liver injury more frequently while others may worsen the pre-existent steatosis (or steatohepatitis). +
Cancer cells have an abnormally high mitochondrial membrane potential (ΔΨm ), which is associated with enhanced invasive properties in vitro and increased metastases in vivo. The mechanisms underlying the abnormal ΔΨm in cancer cells remain unclear. Research on different cell types has shown that ΔΨm is regulated by various intracellular mechanisms such as by mitochondrial inner and outer membrane ion transporters, cytoskeletal elements, and biochemical signaling pathways. On the other hand, the role of extrinsic, tumor microenvironment (TME) derived cues in regulating ΔΨm is not well defined. In this review, we first summarize the existing literature on intercellular mechanisms of ΔΨm regulation, with a focus on cancer cells. We then offer our perspective on the different ways through which the microenvironmental cues such as hypoxia and mechanical stresses may regulate cancer cell ΔΨm . +
Conventional T (Tcon) cells and Foxp3(+) T-regulatory (Treg) cells are thought to have differing metabolic requirements, but little is known of mitochondrial functions within these cell populations in vivo. In murine studies, we found that activation of both Tcon and Treg cells led to myocyte enhancer factor 2 (Mef2)-induced expression of genes important to oxidative phosphorylation (OXPHOS). Inhibition of OXPHOS impaired both Tcon and Treg cell function compared to wild-type cells but disproportionally affected Treg cells. Deletion of Pgc1α or Sirt3, which are key regulators of OXPHOS, abrogated Treg-dependent suppressive function and impaired allograft survival. Mef2 is inhibited by histone/protein deacetylase-9 (Hdac9), and Hdac9 deletion increased Treg suppressive function. Hdac9(-/-) Treg showed increased expression of Pgc1α and Sirt3, and improved mitochondrial respiration, compared to wild-type Treg cells. Our data show that key OXPHOS regulators are required for optimal Treg function and Treg-dependent allograft acceptance. These findings provide a novel approach to increase Treg function and give insights into the fundamental mechanisms by which mitochondrial energy metabolism regulates immune cell functions in vivo. +
No Abstract +
Tissue damage by ischemia/reperfusion (I/R) results from a temporary cessation of blood flow followed by restoration of circulation. The injury depresses mitochondrial respiration, increases the production of reactive oxygen species (ROS), decreases the mitochondrial transmembrane potential and stimulates invasion by inflammatory cells. The primary objective of this work was to address the potential use of bone marrow stem cells (BMSCs) to preserve/restore mitochondrial function in the kidney after I/R. Mitochondria from renal proximal tubule cells were isolated by differential centrifugation from rat kidneys subjected to I/R (clamping of renal arteries followed by release of circulation after 30 min), without or with subcapsular administration of BMSCs. Respiration starting from mitochondrial complex II was strongly affected following I/R. However, when BMSCs were injected before ischemia or together with reperfusion, normal electron fluxes, electrochemical gradient for protons and ATP synthesis were almost completely preserved, and mitochondrial ROS formation occurred at low rate. In homogenates from cultured renal cells transiently treated with antimycin A, the co-culture with BMSCs induced a remarkable increase in protein S-nitrosylation that was similar to that found in mitochondria isolated from I/R rats, evidence that BMSCs protected against both superoxide anion and peroxynitrite. Labeled BMSCs migrated to damaged tubules, suggesting that the injury functions as a signal to attract and host the injected BMSCs. Structural correlates of BMSC injection in kidney tissue included stimulus of tubule cell proliferation, inhibition of apoptosis and decreased inflammatory response. Histopathological analysis demonstrated a score of complete preservation of tubular structures by BMSCs, associated with normal plasma creatinine and urinary osmolality. The key findings shed light on the mechanisms that explain, at the mitochondrial level, how stem cells prevent damage by I/R. The action of BMSCs on mitochondrial functions raises the possibility that autologous BMSCs may help prevent I/R injuries associated with transplantation and acute renal diseases.
To gain understanding on the mechanisms that drive immunosenescence in humans, we examined CD4+ T cells obtained from younger (20-39 years-old) and older (70+ years-old) healthy participants of the Baltimore Longitudinal Study on Aging (BLSA). We found that mitochondrial proteins involved in the electron transport chain were overrepresented in cells from older participants, with prevalent dysregulation of oxidative phosphorylation and energy metabolism molecular pathways. Surprisingly, gene transcripts coding for mitochondrial proteins pertaining to oxidative phosphorylation and electron transport chain pathways were underrepresented in older individuals. Paralleling the observed decrease in gene expression, mitochondrial respiration was impaired in CD4+ T cells from older subjects. Though mitochondrial number in both naïve and memory cells visualized with electron microcopy was similar in older versus younger participants, there were a significantly higher number of autophagosomes, many of them containing undegraded mitochondria, in older individuals. The presence of mitochondria inside the accumulated autophagic compartments in CD4+ T cells from older individuals was confirmed by immunofluorescence. These findings suggest that older age is associated with persistence of dysfunctional mitochondria in CD4+ T lymphocytes caused by defective mitochondrial turnover by autophagy, which may trigger chronic inflammation and contribute to the impairment of immune defense in older persons. +
The development of mitochondrial medicine has been severely impeded by a lack of effective therapies.
To better understand Mitochondrial Encephalopathy Lactic Acidosis Syndrome Stroke-like episodes (MELAS) syndrome, neuronal cybrid cells carrying different mutation loads of the m.3243A > G mitochondrial DNA variant were analysed using a multi-omic approach.
Specific metabolomic signatures revealed that the glutamate pathway was significantly increased in MELAS cells with a direct correlation between glutamate concentration and the m.3243A > G heteroplasmy level. Transcriptomic analysis in mutant cells further revealed alterations in specific gene clusters, including those of the glutamate, gamma-aminobutyric acid pathways, and tricarboxylic acid (TCA) cycle. These results were supported by post-mortem brain tissue analysis from a MELAS patient, confirming the glutamate dysregulation. Exposure of MELAS cells to ketone bodies significantly reduced the glutamate level and improved mitochondrial functions, reducing the accumulation of several intermediate metabolites of the TCA cycle and alleviating the NADH-redox imbalance.
Thus, a multi-omic integrated approach to MELAS cells revealed glutamate as a promising disease biomarker, while also indicating that a ketogenic diet should be tested in MELAS patients. +
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The development of gestational diabetes mellitus (GDM) during pregnancy is associated with impaired glucose tolerance and insulin resistance, which may be transgenerationally inherited by the offspring (F1). The aim of our study was to assess the effects of maternal physical exercise (PE) during pregnancy on F1 (16 wks-old) liver mitochondrial function in a model of GDM.
Female Sprague-Dawley fed with control (C) or high-fat-high-sugar (HFHS) diets were submitted to PE during the 3 weeks of pregnancy. Oral glucose tolerance tests (OGTT) were performed before and during pregnancy to assess the GMD condition. F1 body weight (BW) was weekly monitored and liver mitochondrial function was determined at 16th wks of age using complex I and II-related substrates.
No pre-mating OGTT differences were observed between C and HFHS groups. In contrast, increased impaired glucose tolerance was detected during pregnancy in HFHS groups, regardless of PE. Although F1 of HFHS mothers had significantly higher BW compared to C, PE during pregnancy decreased this adverse effect of HFHS. Regarding F1 liver mitochondrial function, no alterations were found in state 3 and 4 respiration between groups, using complex I and II-related substrates, while a decrease in the respiratory control ratio (RCR) of the HFHS animals compared to C was observed using substrates for complex I. The PE program was able to improve the F1-related RCR in the HFHS animals back to C values.
In conclusion, HFHS during pregnancy induced GDM; however, the negative impact of this fed condition during pregnancy on liver mitochondrial function was significantly attenuated by the PE program. +
In this review, we discuss the concept of metabolic remodeling and signaling in tumors, specifically the various metabolites that participate in the regulation of gene expression in cancer cells. In particular, pyruvate, oxaloacetate, succinate and fumarate, four mitochondrial metabolites, activate genes relevant for tumor progression. When the balance between glycolysis and oxidative phosphorylation is altered, these metabolites accumulate in the cytoplasm and regulate the activity of the Hypoxia Inducible Factor 1alpha (HIF-1alpha). HIF is one of the main factors that orchestrate the metabolic switch observed during oncogenesis. There is also an important role for lactate, fructose 1-6 bisphosphate or citrate that leads to the diversion of glucose metabolites to anabolism. In addition reactive oxygen species, which are produced by the respiratory chain, could serve as an endogenous source of DNA-damaging agents to promote genetic instability. Accordingly, several mitochondrial DNA mutations were reported in tumors, and the construction of cybrids recently demonstrated their role in the control of tumor progression. +
Little is known on the metabolic profile of lung tumors and the reminiscence of embryonic features. Herein, we determined the bioenergetic profiles of human fibroblasts taken from lung epidermoid carcinoma (HLF-a) and fetal lung (MRC5). We also analysed human lung tumors and their surrounding healthy tissue from four patients with adenocarcinoma. On these different models, we measured functional parameters (cell growth rates in oxidative and glycolytic media, respiration, ATP synthesis and PDH activity) as well as compositional features (expression level of various energy proteins and upstream transcription factors). The results demonstrate that both the lung fetal and cancer cell lines produced their ATP predominantly by glycolysis, while oxidative phosphorylation was only capable of poor ATP delivery. This was explained by a decreased mitochondrial biogenesis caused by a lowered expression of [[PGC1α]] (as shown by RT-PCR and Western blot) and mtTFA. Consequently, the relative expression of glycolytic versus OXPHOS markers was high in these cells. Moreover, the re-activation of mitochondrial biogenesis with resveratrol induced cell death specifically in cancer cells. A consistent reduction of mitochondrial biogenesis and the subsequent alteration of respiratory capacity was also observed in lung tumors, associated with a lower expression level of bcl2. Our data give a better characterization of lung cancer cells' metabolic alterations which are essential for growth and survival. They designate mitochondrial biogenesis as a possible target for anti-cancer therapy. +
Patients with acute alcohol-associated hepatitis (AH) have immune dysfunction. Mitochondrial function is critical for immune cell responses and regulates senescence. Clinical translational studies using complementary bioinformatics-experimental validation of mitochondrial responses were performed in peripheral blood mononuclear cells (PBMC) from patients with AH, healthy controls (HC), and heavy drinkers without evidence of liver disease (HD).
Feature extraction for differentially expressed genes (DEG) in mitochondrial components and telomere regulatory pathways from single-cell RNAseq (scRNAseq) and integrated 'pseudobulk' transcriptomics from PBMC from AH and HC (n = 4 each) were performed. After optimising isolation and processing protocols for functional studies in PBMC, mitochondrial oxidative responses to substrates, uncoupler, and inhibitors were quantified in independent discovery (AH n = 12; HD n = 6; HC n = 12) and validation cohorts (AH n = 10; HC n = 7). Intermediary metabolites (gas-chromatography/mass-spectrometry) and telomere length (real-time PCR) were quantified in subsets of subjects (PBMC/plasma AH n = 69/59; HD n = 8/8; HC n = 14/27 for metabolites; HC n = 13; HD n = 8; AH n = 72 for telomere length).
Mitochondrial, intermediary metabolite, and senescence-regulatory genes were differentially expressed in PBMC from AH and HC in a cell type-specific manner at baseline and with lipopolysaccharide (LPS). Fresh PBMC isolated using the cell preparation tube generated optimum mitochondrial responses. Intact cell and maximal respiration were lower (p ≤ .05) in AH than HC/HD in the discovery and validation cohorts. In permeabilised PBMC, maximum respiration, complex I and II function were lower in AH than HC. Most tricarboxylic acid (TCA) cycle intermediates in plasma were higher while those in PBMC were lower in patients with AH than those from HC. Lower telomere length, a measure of cellular senescence, was associated with higher mortality in AH.
Patients with AH have lower mitochondrial oxidative function, higher plasma TCA cycle intermediates, with telomere shortening in nonsurvivors.
[[File:Bellissimo 2022 MitoFit graphic.jpg|right|350px|Graphical abstract]]In isolated mitochondria, calcium-induced mitochondrial permeability transition pore (mPTP) opening is thought to be regulated by adenylates but the relative effects of adenylate cycling in permeabilized tissues is less explored. To determine the effect of adenylates on calcium retention capacity (CRC) as an index of mPTP in permeabilized muscle fibres, separate in vitro assessments of CRC were compared in media that did not contain ADP or received exogenous ADP that naturally equilibrated with ATP through endogenous ATP-dependent pathways (presence of both ADP and ATP). Comparisons were made to a hexokinase 2-deoxyglucose system that recycles ATP to ADP (depletion of ATP). In permeabilized mouse quadriceps fibres, we found that CRC was increased by ADP suggesting endogenous ADP-ATP equilibria attenuate mPTP. Supplementing ADP with the hexokinase ATP recycling clamp lowered CRC relative to ADP alone but had no effect relative to the absence of adenylates. This finding suggests ADP does not alter calcium-induced mPTP and that its regulation by adenylates is specific to ATP in permeabilized mouse quadriceps fibres. Accelerating matrix ADP/ATP cycling with creatine had no effect on CRC when combined with 5mM ADP but more than doubled CRC (desensitized mPTP) when the hexokinase clamp was included. These results demonstrate the importance of considering adenylate equilibria during the design of in vitro assessments of calcium-induced mPTP in permeabilized muscle fibres.<br> +
[[File:BEC.png|25px|link=https://doi.org/10.26124/bec:2023-0001]] https://doi.org/10.26124/bec:2023-0001
[[File:Bellissimo 2022 MitoFit graphic.jpg|right|350px|Graphical abstract]] In isolated mitochondria, calcium-induced mitochondrial permeability transition pore (mtPTP) opening is thought to be regulated by adenylates but the relative effects of inherent adenylate cycling unique to permeabilized tissues is less explored. To determine the effect of adenylate cycling on calcium retention capacity (CRC) as an index of mt-permeability transition (mtPT) in permeabilized muscle fibers, separate ''in vitro'' assessments of CRC were compared in media that did not contain ADP or received exogenous ADP that naturally equilibrated with ATP through endogenous ATP-dependent pathways (ADP/ATP cycling). Comparisons were made to ADP combined with a hexokinase 2-deoxyglucose system recycling ATP back to ADP (ATP depletion). In mouse quadriceps, ADP increased CRC suggesting endogenous ADP-ATP cycling attenuates mtPT. ADP with hexokinase lowered CRC relative to ADP alone but had no effect compared to the absence of ADP (no adenylates). This finding suggests ADP does not alter calcium-induced mtPT and that its regulation by adenylates is specific to ATP in permeabilized mouse quadriceps fibers. Accelerating matrix ADP/ATP cycling with creatine and 5 mM ADP did not affect CRC but more than doubled CRC (desensitized mtPT) when the hexokinase clamp was included. CRC was similar in wildtype and D2.''mdx'' mouse cardiac left ventricle fibers whether assessed with ADP and creatine (adenylate cycling) or without ADP, confirming adenylate-dependent and -independent control of mtPT is not altered in this model of myopathy. These results demonstrate the natural cycling of ADP with ATP in permeabilized muscle fibers attenuates mtPT, but not due to ADP itself. Repeating assessments with and without adenylate cycling can add further insight to experimental results. +
What is the central question of this study? Can adiponectin receptor agonism improve recognition memory in a mouse model of Duchenne muscular dystrophy? What is the main finding and its importance? Short-term treatment with the new adiponectin receptor agonist ALY688 improves recognition memory in D2.mdx mice. This finding suggests that further investigation into adiponectin receptor agonism is warranted, given that there remains an unmet need for clinical approaches to treat this cognitive dysfunction in people with Duchenne muscular dystrophy.
Memory impairments have been well documented in people with Duchenne muscular dystrophy (DMD). However, the underlying mechanisms are poorly understood, and there is an unmet need to develop new therapies to treat this condition. Using a novel object recognition test, we show that recognition memory impairments in D2.mdx mice are completely prevented by daily treatment with the new adiponectin receptor agonist ALY688 from day 7 to 28 of age. In comparison to age-matched wild-type mice, untreated D2.mdx mice demonstrated lower hippocampal mitochondrial respiration (carbohydrate substrate), greater serum interleukin-6 cytokine content and greater hippocampal total tau and Raptor protein contents. Each of these measures was partly or fully preserved after treatment with ALY688. Collectively, these results indicate that adiponectin receptor agonism improves recognition memory in young D2.mdx mice. +
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The effect of the lipophilic penetrating cation dequalinium on rat liver mitochondria was studied. It was found that dequalinium dose-dependently inhibits the respiration rate of rat liver mitochondria in ADP-stimulated (V<sub>3</sub>) and DNP-stimulated (uncoupled) states. This can be due to the fact that dequalinium is a potent inhibitor of complex III of the mitochondrial respiratory chain. It was shown that dequalinium induces a high-amplitude swelling of rat liver mitochondria. The dequalinium-induced swelling of the organelles depends on the presence of inorganic phosphate in the incubation medium: in the absence of phosphate or in the presence of the phosphate carrier inhibitor mersalyl in the phosphate-containing medium, no swelling of the mitochondria was observed. At low concentrations of dequalinium (≤10 μM), this swelling is inhibited by cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. At the same time, at high concentrations of dequalinium (>10 μM), cyclosporin A becomes ineffective. It was found that in the presence of dequalinium the rate of the H<sub>2</sub>O<sub>2</sub> production increased in rat liver mitochondria. Possible mechanisms of toxic effect of dequalinium chloride are discussed. +
The paper considers the effects of bedaquiline (BDQ), an antituberculous preparation of the new generation, on rat liver mitochondria. It was shown that 50 μM BDQ inhibited mitochondrial respiration measured with substrates of complexes I and II (glutamate/malate and succinate/rotenone systems respectively) in the states V<sub>3</sub> and V<sub>DNP</sub>. At the same time, at concentrations below 50 μM, BDQ slightly stimulated respiration with substrates of complex I in the state V<sub>2</sub>. BDQ was also found to suppress, in a dose-dependent manner, the activity of complex II and the total activity of complexes II + III of the mitochondrial transport chain. It was discovered that at concentrations up to 10 μM, BDQ inhibited H<sub>2</sub>O<sub>2</sub> production in mitochondria. BDQ (10-50 μM) suppressed the opening of Ca<sup>2+</sup>-dependent CsA-sensitive mitochondrial permeability transition pore. The latter was revealed experimentally as the inhibition of Ca<sup>2+</sup>/P<sub>i</sub>-dependent swelling of mitochondria, suppression of cytochrome c release, and an increase in the Ca<sup>2+</sup> capacity of the organelles. BDQ also decreased the rate of mitochondrial energy-dependent K<sup>+</sup> transport, which was evaluated by the energy-dependent swelling of mitochondria in a K<sup>+</sup> buffer and DNP-induced K<sup>+</sup> efflux from the organelles. The possible mechanisms of BDQ effect of rat liver mitochondria are discussed. +
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Bedaquiline (BDQ) and delamanid have recently been approved for the treatment of multidrug- and extensively drug-resistant tuberculosis. The antibacterial effect of these compounds has been well studied. The mechanism of BDQ antibacterial effect is based on the inhibition of bacterial F<sub>1</sub>/F<sub>O</sub> ATP-synthase, which leads to suppression of the process of oxidative phosphorylation, poor ATP production and, eventually, cell death. Delamanid inhibits the biosynthesis of mycolic acids of cell wall of ''Mycobacterium tuberculosis'' [1].
An important step in the introduction of a new antibacterial preparation into medical practice is the study of its influence on the eukaryotic cell, in order to reveal its possible side effects in the human and animal organisms. At the same time, data on the interaction of BDQ and delamanid with cells of eukaryotic organisms are scarce: these drugs were introduced into practice not a long time ago and in quite a hasty manner [2]. The available literature data indicate that the influence of BDQ on eukaryotic cells is based on, at least, two mechanisms: (1) its effect on cell energetics and (2) its effect on the operation of membrane enzymes and ion channels. In this connection, of special interest is the question of how BDQ and delamanid interact with mitochondria.
Mitochondria were isolated from the liver of Wistar rats by differential centrifugation. The rate of oxygen consumption of rat liver mitochondria was measured polarographically with a Clark-type gold electrode (Oroboros Instruments, Oxygraph-2k, Austria) at 25 °C under continuous stirring. The swelling of mitochondria (0.4 mg/mL) was measured as a decrease in A<sub>540</sub> in a stirred cuvette at room temperature (~22 °C) using a USB-2000 spectroscopy fiber-optic system (Ocean Optics, USA). The release of cytochrome ''c'' from mitochondria was determined using western-blot analysis.
The effect of BDQ on the functional state of rat liver mitochondria was evaluated by the rate of mitochondrial respiration with glutamate/malate (substrates of Complex I of the respiratory chain) or succinate (a substrate of Complex II) in the presence of rotenone [3]. 10-20 µM BDQ had little effect on the succinate-fueled respiration (less than a 10% decrease) in all the functional states (''J''<sub>ROX</sub>, ''V''<sub>''P''</sub>, ''J''<sub>''L''</sub> and ''J''<sub>''E''</sub>). However, raising BDQ concentration to 50 µM resulted in a marked inhibition of mitochondrial respiration: the rates of respiration in OXPHOS- and ET(DNP)-states were lowered by 40 and 44% of control, respectively. The parameter of respiratory control decreased by 20%. At the same time, the efficiency of ATP synthesis estimated as ADP/O ratio remained practically the same (1.90 ± 0.09 in control measurements and 1.82 ± 0.11 in the presence of BDQ). The time of phosphorylation, however, increased almost 2-fold (from 109 ± 5 s to 208 ± 15 s) when BDQ was added. With NAD-dependent substrates (2.5 mM glutamate and 2.5 mM malate), the effect of BDQ on mitochondrial respiration was different from the effect observed with succinate. 50 µM BDQ caused a slight inhibition of ''J''<sub>''P''</sub> and ''J''<sub>''E''</sub> respiration (approximately, by 10-15% of control). This was accompanied by a decrease of the respiratory control level by less than 1.
The inhibitory effect of BDQ on ATP synthesis seems to be associated with the suppression of the functioning of the mitochondrial respiratory chain BDQ practically did not affect the activity of Complex III and Complex IV. At the same time, it suppressed the activity of Complex II, which was especially evident when the combined activity of Complex II + III was assessed (a 60% inhibition by 50 µM BDQ). These results indicate that BDQ hinders the access of coenzyme Q to the respiratory complexes.
In contrast to BDQ, delamanid caused a marked inhibition of mitochondrial respiration using substrates of Complex I and II of the respiratory system. The rates of respiration in OXPHOS- and ET-states in the presence of 20-100 µM delamanid were lowered by almost 2-fold compared with the control ones.
BDQ and delamanid can result in the inhibition of H<sub>2</sub>O<sub>2</sub> production by rat liver mitochondria. The effect of different concentrations of these compounds on ROS production fits well in the scheme of action of antioxidants, which exhibit antioxidant properties at low concentrations but exert a prooxidant effect at higher concentrations.
The development of tuberculosis is known to be accompanied by the opening of MPT pore in mitochondria of infected cells. BDQ inhibited swelling of rat liver mitochondria induced by 50 µM Ca<sup>2+</sup> in the presence of 1 mM P<sub>i</sub>. The inhibition was dose-dependent, with 50 µM BDQ almost completely suppressing the swelling. When mitochondria are preincubated with BDQ, a significant part of the organelles does not undergo structural changes upon the Ca<sup>2+</sup>/P<sub>i</sub>-induced opening of the pore. In addition, As it is known, the opening of MPT pore is one of the major pathways for the induction of cell death, with the mechanism being related to the release of proapoptotic proteins from the organelles (cytochrome ''c'', AIF etc.). BDQ prevents the release of cytochrome ''c'' from mitochondria.
At the same time, delamanid stimulated swelling of rat liver mitochondria induced by 50 µM Ca<sup>2+</sup> in the presence of 1 mM P<sub>i</sub>. Thus, it can be concluded that delamanid in the used concentrations is able to exert a toxic effect on mitochondria of rat liver.
The results obtained in the present work indicate a complex effect of BDQ on mitochondria. BDQ causes inhibition of both physiologically relevant and pathology-related mitochondrial processes. It is important to note that the data of most of the BDQ tests conducted by now indicate that BDQ has no toxic effects on normal eukaryotic cells, only affecting bacterial and abnormal (both tuberculosis-infected and cancer) eukaryotic cells [4,5]. In this respect, the results of our work obtained on the suspensions of mitochondria of healthy animals confirm those data and allow us to conclude that BDQ can protect normal cells from the development of oxidative stress and cell death.
The paper considers the effects of bedaquiline (BDQ), an antituberculous preparation of the new generation, on rat liver mitochondria. It was shown that 50 μM BDQ inhibited mitochondrial respiration measured with substrates of complexes I and II (glutamate/malate and succinate/rotenone systems respectively) in the states V<sub>3</sub> and V<sub>DNP</sub>. At the same time, at concentrations below 50 μM, BDQ slightly stimulated respiration with substrates of complex I in the state V<sub>2</sub>. BDQ was also found to suppress, in a dose-dependent manner, the activity of complex II and the total activity of complexes II + III of the mitochondrial transport chain. It was discovered that at concentrations up to 10 μM, BDQ inhibited H<sub>2</sub>O<sub>2</sub> production in mitochondria. BDQ (10-50 μM) suppressed the opening of Ca<sup>2+</sup>-dependent CsA-sensitive mitochondrial permeability transition pore. The latter was revealed experimentally as the inhibition of Ca<sup>2+</sup>/P<sub>i</sub>-dependent swelling of mitochondria, suppression of cytochrome c release, and an increase in the Ca<sup>2+</sup> capacity of the organelles. BDQ also decreased the rate of mitochondrial energy-dependent K<sup>+</sup> transport, which was evaluated by the energy-dependent swelling of mitochondria in a K<sup>+</sup> buffer and DNP-induced K<sup>+</sup> efflux from the organelles. The possible mechanisms of BDQ effect of rat liver mitochondria are discussed.
<small>Copyright © 2018 Elsevier B.V. All rights reserved.</small> +
Although diabetes mellitus is known to be a disease associated with mitochondrial dysfunction, not everything is clear about mitochondrial Ca<sup>2+</sup> transport and Ca<sup>2+</sup>-induced permeability transition in diabetic cells. The objective of this work was to study the operation of MCU and Ca<sup>2+</sup>-dependent mitochondrial permeabilization in the liver cells of Sprague-Dawley rats under the streptozotocin-induced type I diabetes. It was shown that two weeks after the induction of diabetes, the rate of Ca<sup>2+</sup> uptake by the mitochondria of diabetic animals increased ~1.4-fold. The expression of MCU and MICU1 subunits did not change, yet the quantity of dominant-negative MCUb channel subunits was almost twice as lower. The organelles also became more resistant to the induction of CsA-sensitive MPT pore and less resistant to the induction of CsA-insensitive palmitate/Ca<sup>2+</sup>-induced pore. The mitochondria of diabetic liver cells also showed changes in the lipid matrix of their membranes. The content of fatty acids in the membranes grew, and microviscosity of the lipid bilayer (assessed with laurdan) increased. At the same time, lipid peroxidation (assessed by the production of malonic dialdehyde) was stimulated. The paper discusses the consequences of the diabetes-related changes in mitochondria in the context of cell physiology. +
Itaconic acid (methylene-succinic acid, ItA) is an unsaturated dicarboxylic acid that is secreted by mammalian macrophages in response to a pro-inflammatory stimulus and shows an anti-inflammatory/antibacterial effect. Being a mitochondrial metabolite, it exhibits an inhibitory activity on succinate dehydrogenase and subsequently induces mitochondrial dysfunction. The present study has shown that ItA dose-dependently inhibited ADP- and DNP-stimulated (uncoupled) respiration of rat liver mitochondria energized with succinate. This effect of ItA could be related to the suppression of the activity of complex II and the combined activity of complexes II + III of the respiratory chain. At the same time, ItA had no effect on the activity of the dicarboxylate carrier, which catalyzes the transport of succinate across the inner mitochondrial membrane. It was found that 4 mM ItA diminished the rates of ADP- and DNP-stimulated mitochondrial respiration supported by the substrates of complex I glutamate and malate. A study of the effect of ItA on the activity of complexes of the respiratory chain showed that it significantly decreases the activity of complex IV. It was observed that 4 mM ItA inhibited the rate of H<sub>2</sub>O<sub>2</sub> production by mitochondria. At the same time, ItA promoted the opening of the cyclosporin A-sensitive Ca<sup>2+</sup>-dependent permeability transition pore. The latter was revealed as the decrease in the calcium retention capacity of mitochondria and the stimulation of release of cytochrome c from the organelles. ItA by itself promotes the cytochrome c release from mitochondria. Possible mechanisms of the effect of ItA on mitochondrial function are discussed. +
We analyzed structural and functional features of the main mitochondrial Ca2+-transporting systems, mitochondrial Ca2+ uniporter complex (MCUC) and Ca<sup>2+</sup>-dependent cyclosporin A-sensitive mitochondrial permeability transition pore (MPT pore), in rats with hyperthyroid state. It was found that, the rate of Ca<sup>2+</sup> accumulation by rat liver mitochondria in this pathology increases by 1.3 times, which can be associated with higher level of the channel-forming subunit of the uniporter MCU and lower content of dominant-negative subunit of this complex MCUb. At the same time, the level of the regulatory subunit MICU1 remained unchanged. It was shown that calcium retention capacity of liver mitochondria in rats with experimental hyperthyroidism decreased by 2 times in comparison with the control, which attested to reduced resistance of liver mitochondria of hyperthyroid rats to induction of the MPT pore. The observed changes are consistent with the data on increased amount of cyclophilin D, a mitochondrial matrix peptidyl-prolyl isomerase that is known to modulate the MPT pore opening and expression of the Ppif gene that encodes mitochondrial cyclophilin D in rats with experimental hyperthyroidism. +
We studied the effect of a new hypoglycemic compound dapagliflozin on the functioning of rat liver mitochondria. Dapagliflozin in concentrations of 10-20 μM had no effect on the parameters of respiration and oxidative phosphorylation of rat liver mitochondria. Increasing dapagliflozin concentration to 50 μM led to a significant inhibition of mitochondrial respiration in states 3 and 3UDNP. Dapagliflozin in this concentration significantly reduced calcium retention capacity of rat liver mitochondria. These findings indicate a decline in the resistance of rat liver mitochondria to induction of Ca2+-dependent mitochondrial permeability transition pore. In a concentration of 10 μM, dapagliflozin significantly decreases the rate of H2O2 formation in rat liver mitochondria, which attested to an antioxidant effect of this compound. Possible mitochondrion-related mechanisms of the protective action of dapagliflozin on liver cells are discussed. +
Mitochondria are capable of synchronized oscillations in many variables, but the underlying mechanisms are still unclear. In this study, we demonstrated that rat liver mitochondria, when exposed to a pulse of Sr<sup>2+</sup> ions in the presence of valinomycin (a potassium ionophore) and cyclosporin A (a specific inhibitor of the permeability transition pore complex) under hypotonia, showed prolonged oscillations in K<sup>+</sup> and Sr<sup>2+</sup> fluxes, membrane potential, pH, matrix volume, rates of oxygen consumption and H<sub>2</sub>O<sub>2</sub> formation. The dynamic changes in the rate of H<sub>2</sub>O<sub>2</sub> production were in a reciprocal relationship with the respiration rate and in a direct relationship with the mitochondrial membrane potential and other indicators studied. The pre-incubation of mitochondria with Ca<sup>2+</sup>(Sr<sup>2+</sup>)-dependent phospholipase A<sub>2</sub> inhibitors considerably suppressed the accumulation of free fatty acids, including palmitic and stearic acids, and all spontaneous Sr<sup>2+</sup>-induced cyclic changes. These data suggest that the mechanism of ion efflux from mitochondria is related to the opening of short-living pores, which can be caused by the formation of complexes between Sr<sup>2+</sup>(Ca<sup>2+</sup>) and endogenous long-chain saturated fatty acids (mainly, palmitic acid) that accumulate due to the activation of phospholipase A<sub>2</sub> by the ions. A possible role for transient palmitate/Ca<sup>2+</sup>(Sr<sup>2+</sup>)-induced pores in the maintenance of ion homeostasis and the prevention of calcium overload in mitochondria under pathophysiological conditions is discussed. +
Schizophrenia is currently believed to result from variations in multiple genes, each contributing a subtle effect, which combines with each other and with environmental stimuli to impact both early and late brain development. At present, schizophrenia clinical heterogeneity as well as the difficulties in relating cognitive, emotional and behavioral functions to brain substrates hinders the identification of a disease-specific anatomical, physiological, molecular or genetic abnormality. Mitochondria play a pivotal role in many essential processes, such as energy production, intracellular calcium buffering, transmission of neurotransmitters, apoptosis and ROS production, all either leading to cell death or playing a role in synaptic plasticity. These processes have been well established as underlying altered neuronal activity and thereby abnormal neuronal circuitry and plasticity, ultimately affecting behavioral outcomes. The present article reviews evidence supporting a dysfunction of mitochondria in schizophrenia, including mitochondrial hypoplasia, impairments in the oxidative phosphorylation system (OXPHOS) as well as altered mitochondrial-related gene expression. Abnormalities in mitochondrial complex I, which plays a major role in controlling OXPHOS activity, are discussed. Among them are schizophrenia specific as well as disease-state-specific alterations in complex I activity in the peripheral tissue, which can be modulated by DA. In addition, CNS and peripheral abnormalities in the expression of three of complex I subunits, associated with parallel alterations in their transcription factor, specificity protein 1 (Sp1) are reviewed. Finally, this review discusses the question of disease specificity of mitochondrial pathologies and suggests that mitochondria dysfunction could cause or arise from anomalities in processes involved in brain connectivity. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MITOEAGLE]] Mitochondrial complex I (CoI) deficit, associated with mitochondrial dysfunction, has been observed in schizophrenia (SZ), a devastating psychotic disorder characterized by cognitive, emotional and behavioral abnormalities with a world-wide prevalence of 1%. Altered expression of CoI nuclear DNA encoded subunits NDUFV1, NDUFV2 and NDUFS1, has been consistently reported in SZ patients. These three interchangeable (labile) subunits comprise the N module of CoI, involved in binding and oxidation of NADH. The aim of the present study was to unravel the defects in holo-CoI homeostasis that are involved in mitochondrial dysfunction in SZ. Epstein-Barr virus transformed lymphocyte cell lines from SZ patients and healthy subjects were used. Deficits, specifically in CoI-driven respiration in SZ-permeabilized cells were associated with reduced in-gel activity of isolated holo-CoI, indicating an impairment of CoI ''per-se''. CoI and CoI-bound labile subunits level were similar in both cohorts, yet CoI synthesis rate was decreased in patients. This may be due to a decrease in subunits degradation rates as observed for NDUFV1 and NDUFV2 in patient cells. The latter is in line with a higher level of mitochondrial cAMP, which inhibits the degradation of these subunits, and the reduced variability in CoI-bound labile subunits between patients' cell lines compared to controls. To study the mechanism involved in the hitherto described deficits, we focused on NDUFV2, the most affected subunit in SZ. Although CoI-bound NDUFV2 level did not alter in patients, its mitochondrial level was lower. We therefore studied NDUFV2 import into the mitochondria following ''in-vitro'' translation of its RT-PCR product. We observed an impaired import in the patients, which was due to both protein and mitochondria source (patients or controls). As translation was performed ''in-vitro'', we assumed that NDUFV2 transcript is impaired in patients. While Sanger sequencing showed no difference in transcript sequence between SZ and controls, it showed a mix of products in SZ cells. Different factors can interfere with the reverse transcriptase (RT) and hamper cDNA synthesis. In patients' samples, RT reaction at high and low dNTPs concentration followed by PCR resulted in a significantly greater difficulty in synthesizing cDNA than in controls. Notably, in the control but not in SZ samples, the extent of difficulty showed a strong and significant correlation with NDUFV2 cytosolic pre-protein level, suggesting that the interference with mRNA transcription has a functional effect on translation. NDUFV2 has a pseudogene (NDUFV2P1), which is an attractive candidate for the regulation of RNA transcription as are other non-coding RNAs. We therefore analyzed NDUFV2P1 expression in lymphoblast and frontal cortex postmortem specimens from patients and controls. We observed a significantly higher NDUFV2P1 transcript levels in patients' samples. Increased NDUFV2P1 transcripts can interfere with NDUFV2 reverse transcription, translation and import, lead to changes in synthesis and degradation rates of CoI, its abnormal activity and mitochondrial dysfunction in SZ.
The ability for the brain to sense peripheral fuel availability is mainly accomplished within the hypothalamus, which detects ongoing systemic nutrients and adjusts food intake and peripheral metabolism as needed. Here, we hypothesized that mitochondrial reactive oxygen species (ROS) could trigger sensing of nutrients within the hypothalamus. For this purpose, we induced acute hypertriglyceridemia in rats and examined the function of mitochondria in the hypothalamus. Hypertriglyceridemia led to a rapid increase in the mitochondrial respiration in the ventral hypothalamus together with a transient production of ROS. Cerebral inhibition of fatty acids-CoA mitochondrial uptake prevented the hypertriglyceridemia-stimulated ROS production, indicating that ROS derived from mitochondrial metabolism. The hypertriglyceridemia-stimulated ROS production was associated with change in the intracellular redox state without any noxious cytotoxic effects, suggesting that ROS function acutely as signaling molecules. Moreover, cerebral inhibition of hypertriglyceridemia-stimulated ROS production fully abolished the satiety related to the hypertriglyceridemia, suggesting that hypothalamic ROS production was required to restrain food intake during hypertriglyceridemia. Finally, we found that fasting disrupted the hypertriglyceridemia-stimulated ROS production, indicating that the redox mechanism of brain nutrient sensing could be modulated under physiological conditions. Altogether, these findings support the role of mitochondrial ROS as molecular actors implied in brain nutrient sensing. +
Different roles of mitochondria in brain function according to brain area are now clearly emerging. Unfortunately, no technique is yet described to investigate mitochondria function in specific brain area. In this article, we provide a complete description of a procedure to analyze the mitochondrial function in rat brain biopsies. Our two-step method consists in a saponin permeabilization of fresh brain tissues in combination with high-resolution respirometry to acquire the integrated respiratory rate of the biopsy.
In the first part, we carefully checked the mitochondria integrity after permeabilization, defined experimental conditions to determine the respiratory control ratio (RCR), and tested the reproducibility of this technique. In the second part, we applied our method to test its sensitivity. As a result, this method was sensitive enough to reveal region specificity of mitochondrial respiration within the brain. Moreover, we detected physiopathological modulation of the mitochondrial function in the hypothalamus. Thus this
new technique that takes all cell types into account, and does not discard or select any mitochondria sub-population is very suitable to analyze the integrated mitochondrial respiration of brain biopsies. +
Activity defects in respiratory chain complexes are responsible for a large variety of pathological situations, including neuromuscular diseases and multisystemic disorders. Their impact on energy production is highly variable and disproportional. The same biochemical or genetic defect can lead to large differences in clinical symptoms and severity between tissues and patients, making the pathophysiological analysis of mitochondrial diseases difficult. The existence of compensatory mechanisms operating at the level of the respiratory chain might be an explanation for the biochemical complexity observed for respiratory defects. Here, we analyzed the role of cytochrome c and coenzyme Q in the attenuation of complex III and complex IV pharmacological inhibition on the respiratory flux. Spectrophotometry, HPLC-EC, polarography and enzymology permitted the calculation of molar ratios between respiratory chain components, giving values of 0.8:61:3:12:6.8 in muscle and 1:131:3:9:6.5 in liver, for CII:CoQ:CIII:Cyt c:CIV. The results demonstrate the dynamic functional compartmentalization of respiratory chain substrates, with the existence of a substrate pool that can be recruited to maintain energy production at normal levels when respiratory chain complexes are inhibited. The size of this reserve was different between muscle and liver, and in proportion to the magnitude of attenuation of each respiratory defect. Such functional compartmentalization could result from the recently observed physical compartmentalization of respiratory chain substrates. The dynamic nature of the mitochondrial network may modulate this compartmentalization and could play a new role in the control of mitochondrial respiration as well as apoptosis. +
In this chapter we describe the fundamental mechanisms by which mammalian cells regulate energy production, and we put emphasis on the importance of mitochondrial dynamics for the regulation of bioenergetics. We discuss both the impact of shape changes of the mitochondrion on organellar energy production, and the existence of reverse mechanisms of regulation of mitochondrial fusion and fission by the cellular energy state. Hence, in complement to pioneering concepts of metabolic control which only considered the key controlling steps of energy fluxes at the level of the respiratory chain, the recent study of mitochondrial dynamics highlights new possibilities for OXPHOS control. The implications of such a regulatory loop between mitochondrial dynamics and bioenergetics impacts several fields of human biology, as diverse as embryonic development, energy storage, cell motility, lipid and membrane biogenesis, intracellular trafficking and cell death. In addition, most neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and Hereditary Spastic Paraplegia are associated with defects in mitochondrial dynamics and bioenergetics. Therefore, to unravel the fundamental mechanisms by which mitochondrial form interacts with mitochondrial function could permit to increase our basic knowledge on the regulation of energy metabolism and to decipher the pathophysiology of a group of rare neuronal diseases. +
The consumption of garlic is inversely correlated with the
progression of cardiovascular disease, although the responsible
mechanisms remain unclear. Here we show that human RBCs
convert garlic-derived organic polysulfides into hydrogen sulfide
(H2S), an endogenous cardioprotective vascular cell signaling
molecule. This H2S production, measured in real time by
a novel polarographic H2S sensor, is supported by glucosemaintained
cytosolic glutathione levels and is to a large extent
reliant on reduced thiols in or on the RBC membrane. H2S
production from organic polysulfides is facilitated by allyl substituents
and by increasing numbers of tethering sulfur atoms.
Allyl-substituted polysulfides undergo nucleophilic substitution
at the alfa-carbon of the allyl substituent, thereby forming a
hydropolysulfide (RSnH), a key intermediate during the formation
of H2S. Organic polysulfides (R-Sn-R'; n > 2) also undergo
nucleophilic substitution at a sulfur atom, yielding RSnH and H2S.
Intact aorta rings, under physiologically relevant oxygen levels,
also metabolize garlic-derived organic polysulfides to liberate
H2S. The vasoactivity of garlic compounds is synchronous with
H2S production, and their potency to mediate relaxation increases
with H2S yield, strongly supporting our hypothesis that
H2S mediates the vasoactivity of garlic. Our results also suggest
that the capacity to produce H2S can be used to standardize
garlic dietary supplements. +
Advanced HF (heart failure) is associated with altered substrate metabolism. Whether modification of substrate use improves the course of HF remains unknown. The antihyperglycaemic drug MET (metformin) affects substrate metabolism, and its use might be associated with improved outcome in diabetic HF. The aim of the present study was to examine whether MET would improve cardiac function and survival also in non-diabetic HF. Volume-overload HF was induced in male Wistar rats by creating ACF (aortocaval fistula). Animals were randomized to placebo/MET (300 mg·kg(-1) of body weight·day(-1), 0.5% in food) groups and underwent assessment of metabolism, cardiovascular and mitochondrial functions (n=6-12/group) in advanced HF stage (week 21). A separate cohort served for survival analysis (n=10-90/group). The ACF group had marked cardiac hypertrophy, increased LVEDP (left ventricular end-diastolic pressure) and lung weight confirming decompensated HF, increased circulating NEFAs (non-esterified 'free' fatty acids), intra-abdominal fat depletion, lower glycogen synthesis in the skeletal muscle (diaphragm), lower myocardial triacylglycerol (triglyceride) content and attenuated myocardial (14)C-glucose and (14)C-palmitate oxidation, but preserved mitochondrial respiratory function, glucose tolerance and insulin sensitivity. MET therapy normalized serum NEFAs, decreased myocardial glucose oxidation, increased myocardial palmitate oxidation, but it had no effect on myocardial gene expression, AMPK (AMP-activated protein kinase) signalling, ATP level, mitochondrial respiration, cardiac morphology, function and long-term survival, despite reaching therapeutic serum levels (2.2±0.7 μg/ml). In conclusion, MET-induced enhancement of myocardial fatty acid oxidation had a neutral effect on cardiac function and survival. Recently reported cardioprotective effects of MET may not be universal to all forms of HF and may require AMPK activation or ATP depletion. No increase in mortality on MET supports its safe use in diabetic HF.
Mitochondria provide ATP through the process of oxidative phosphorylation, physically located in the inner mitochondrial membrane (IMM). The mitochondrial contact site and organising system (MICOS) complex is known as the 'mitoskeleton' due to its role in maintaining IMM architecture. APOO encodes MIC26, a component of MICOS, whose exact function in its maintenance or assembly has still not been completely elucidated.
We have studied a family in which the most affected subject presented progressive developmental delay, lactic acidosis, muscle weakness, hypotonia, weight loss, gastrointestinal and body temperature dysautonomia, repetitive infections, cognitive impairment and autistic behaviour. Other family members showed variable phenotype presentation. Whole exome sequencing was used to screen for pathological variants. Patient-derived skin fibroblasts were used to confirm the pathogenicity of the variant found in APOO. Knockout models in Drosophila melanogaster and Saccharomyces cerevisiae were employed to validate MIC26 involvement in MICOS assembly and mitochondrial function.
A likely pathogenic c.350T>C transition was found in APOO predicting an I117T substitution in MIC26. The mutation caused impaired processing of the protein during import and faulty insertion into the IMM. This was associated with altered MICOS assembly and cristae junction disruption. The corresponding mutation in MIC26 or complete loss was associated with mitochondrial structural and functional deficiencies in yeast and D. melanogaster models.
This is the first case of pathogenic mutation in APOO, causing altered MICOS assembly and neuromuscular impairment. MIC26 is involved in the assembly or stability of MICOS in humans, yeast and flies. +
Seventy years from the formalization of the Krebs cycle as the central metabolic turntable sustaining the cell respiratory process, key functions of several of its intermediates, especially succinate and fumarate, have been recently uncovered. The presumably immutable organization of the cycle has been challenged by a number of observations, and the variable subcellular location of a number of its constitutive protein components is now well recognized, although yet unexplained. Nonetheless, the most striking observations have been made in the recent period while investigating human diseases, especially a set of specific cancers, revealing the crucial role of Krebs cycle intermediates as factors affecting genes methylation and thus cell remodeling. We review here the recent advances and persisting incognita about the role of Krebs cycle acids in diverse aspects of cellular life and human pathology. +
Research focused on succinate dehydrogenase (SDH) and its substrate, succinate, culminated in the 1950s accompanying the rapid development of research dedicated to bioenergetics and intermediary metabolism. This allowed researchers to uncover the implication of SDH in both the mitochondrial respiratory chain and the Krebs cycle. Nowadays, this theme is experiencing a real revival following the discovery of the role of SDH and succinate in a subset of tumors and cancers in humans. The aim of this review is to enlighten the many questions yet unanswered, ranging from fundamental to clinically oriented aspects, up to the danger of the current use of SDH as a target for a subclass of pesticides. +
The aim of the present study was to compare the effects of different physical activity programs, in combination with a hypocaloric diet, on anthropometric variables and body composition in obese subjects. Ninety-six obese (men: ''N'' = 48; women: ''N'' = 48; age range: 18-50 yr) participated in a supervised 22-wk program. They were randomized into four groups: strength training (S; ''N'' = 24), endurance training (E; ''N'' = 26), combined strength + endurance training (SE; ''N'' = 24), and physical activity recommendations (C; ''N'' = 22). In addition, all groups followed the same hypocaloric diet. At baseline and at the end of the intervention, dietetic and physical activity variables were assessed using validated questionnaires. Anthropometric variables were recorded along with body composition variables measured using dual-energy X-ray absorptiometry techniques. At the end of the intervention, significant improvements were seen within groups in terms of body weight (S: -9.21 ± 0.83 kg; E: -10.55 ± 0.80 kg; SE: -9.88 ± 0.85 kg; C: -8.69 ± 0.89 kg), and total fat mass (S: -5.24 ± 0.55%; E: -5.35 ± 0.55%; SE: -4.85 ± 0.56%; C: -4.89 ± 0.59%). No differences were seen between groups at this time in terms of any other anthropometric or body composition variables examined. All groups increased their total physical activity in metabolic equivalents (MET) per week during the intervention, but with no difference between groups (S: 976 ± 367 MET-min/wk; E: 954 ± 355 MET-min/wk; SE: 1 329 ± 345 MET-min/wk; C: 763 ± 410 MET-min/wk). This study shows that, when combined with a hypocaloric diet, exercise training and adherence to physical activity recommendations are equally effective at reducing body weight and modifying body composition in the treatment of obesity (Clinical Trials Gov. number: NCT01116856). +
The synthetic strobilurin fungicides are derived from the naturally occurring strobilurin A and B. The strobilurins bind to the quinol oxidation site of cytochrome ''b'' of complex III (CIII) of the mitochondria which is also their fungicidal mode of action. There are some signals of potential neurotoxicity from in vitro studies by a CIII-mediated mechanism. +
This chapter focuses on the methods to measure unique metabolites, specific enzymes, and metabolic flux in fatty acid β-oxidation, and on biochemical assays of tricarboxylic acid (TCA) cycle enzymes and the pyruvate dehydrogenase complex. These assays play an important role in the diagnosis of genetic diseases, newborn screening, and in cancer and metabolism research. The rationale, protocol, pros and cons, and alternative methods are discussed. Nevertheless, each laboratory should adapt the preferred method optimizing sample preparation and assay conditions for linearity and a low signal-to-noise ratio. The reader is also referred to the additional literature citing methods and clinical descriptions of the various diseases. +
Adult human brains consume a disproportionate amount of energy substrates (2-3 % of body weight; 20-25 % of total glucose and oxygen). Adenosine triphosphate (ATP) is a universal energy currency in brains and is produced by oxidative phosphorylation (OXPHOS) using ATP synthase, a nano-rotor powered by the proton gradient generated from proton-coupled electron transfer (PCET) in the multi-complex electron transport chain (ETC). ETC catalysis rates are reduced in brains from humans with neurodegenerative diseases (NDDs). Declines of ETC function in NDDs may result from combinations of nitrative stress (NS)-oxidative stress (OS) damage; mitochondrial and/or nuclear genomic mutations of ETC/OXPHOS genes; epigenetic modifications of ETC/OXPHOS genes; or defects in importation or assembly of ETC/OXPHOS proteins or complexes, respectively; or alterations in mitochondrial dynamics (fusion, fission, mitophagy). Substantial free energy is gained by direct O2-mediated oxidation of NADH. Traditional ETC mechanisms require separation between O2 and electrons flowing from NADH/FADH2 through the ETC. Quantum tunneling of electrons and much larger protons may facilitate this separation. Neuronal death may be viewed as a local increase in entropy requiring constant energy input to avoid. The ATP requirement of the brain may partially be used for avoidance of local entropy increase. Mitochondrial therapeutics seeks to correct deficiencies in ETC and OXPHOS. +
Mitochondrial energetic adaptations encompass a plethora of conserved processes that maintain cell and organismal fitness and survival in the changing environment by adjusting the respiratory capacity of mitochondria. These mitochondrial responses are governed by general principles of regulatory biology exemplified by changes in gene expression, protein translation, protein complex formation, transmembrane transport, enzymatic activities and metabolite levels. These changes can promote mitochondrial biogenesis and membrane dynamics that in turn support mitochondrial respiration. The main regulatory components of mitochondrial energetic adaptation include: the transcription coactivator peroxisome proliferator-activated receptor-γ (PPARγ) coactivator 1α (PGC1α) and associated transcription factors; mTOR and endoplasmic reticulum stress signalling; TOM70-dependent mitochondrial protein import; the cristae remodelling factors, including mitochondrial contact site and cristae organizing system (MICOS) and OPA1; lipid remodelling; and the assembly and metabolite-dependent regulation of respiratory complexes. These adaptive molecular and structural mechanisms increase respiration to maintain basic processes specific to cell types and tissues. Failure to execute these regulatory responses causes cell damage and inflammation or senescence, compromising cell survival and the ability to adapt to energetically demanding conditions. Thus, mitochondrial adaptive cellular processes are important for physiological responses, including to nutrient availability, temperature and physical activity, and their failure leads to diseases associated with mitochondrial dysfunction such as metabolic and age-associated diseases and cancer. +
Sterile inflammation is a key determinant of myocardial reperfusion injuries. It participates in infarct size determination in acute myocardial infarction and graft rejection following heart transplantation. We previously showed that P2Y11 exerted an immunosuppressive role in human dendritic cells, modulated cardiofibroblasts' response to ischemia/reperfusion ''in vitro'' and delayed graft rejection in an allogeneic heterotopic heart transplantation model. We sought to investigate a possible role of P2Y11 in the cellular response of cardiomyocytes to ischemia/reperfusion. We subjected human AC16 cardiomyocytes to 5 h hypoxia/1 h reoxygenation (H/R). P2Y11R (P2Y11 receptor) selective agonist NF546 and/or antagonist NF340 were added at the onset of reoxygenation. Cellular damages were assessed by LDH release, MTT assay and intracellular ATP level; intracellular signaling pathways were explored. The role of P2Y11R in mitochondria-derived ROS production and mitochondrial respiration was investigated. ''In vitro'' H/R injuries were significantly reduced by P2Y11R stimulation at reoxygenation. This protection was suppressed with P2Y11R antagonism. P2Y11R stimulation following H<sub>2</sub>O<sub>2</sub>-induced oxidative stress reduced mitochondria-derived ROS production and damages through PKCε signaling pathway activation. Our results suggest a novel protective role of P2Y11 in cardiomyocytes against reperfusion injuries. Pharmacological post-conditioning targeting P2Y11R could therefore contribute to improve myocardial ischemia/reperfusion outcomes in acute myocardial infarction and cardiac transplantation. +
C75 is a potential drug for the treatment of obesity. It was first identified as a competitive, irreversible inhibitor of fatty acid synthase (FAS). It has also been described as a malonyl-CoA analogue that antagonizes the allosteric inhibitory effect of malonyl-CoA on carnitine palmitoyltransferase I (CPT I), the main regulatory enzyme involved in fatty acid oxidation. On the basis of MALDI-TOF analysis, we now provide evidence that C75 can be transformed to its C75-CoA derivative. Unlike the activation produced by C75, the CoA derivative is a potent competitive inhibitor that binds tightly but reversibly to CPT I. IC50 values for yeast-overexpressed L- or M-CPT I isoforms, as well as for purified mitochondria from rat liver and muscle, were within the same range as those observed for etomoxiryl-CoA, a potent inhibitor of CPT I. When a pancreatic INS(823/13), muscle L6E9, or kidney HEK293 cell line was incubated directly with C75, fatty acid oxidation was inhibited. This suggests that C75 could be transformed in the cell to its C75-CoA derivative, inhibiting CPT I activity and consequently fatty acid oxidation. In vivo, a single intraperitoneal injection of C75 in mice produced short-term inhibition of CPT I activity in mitochondria from the liver, soleus, and pancreas, indicating that C75 could be transformed to its C75-CoA derivative in these tissues. Finally, in silico molecular docking studies showed that C75-CoA occupies the same pocket in CPT I as palmitoyl-CoA, suggesting an inhibiting mechanism based on mutual exclusion. Overall, our results describe a novel role for C75 in CPT I activity, highlighting the inhibitory effect of its C75-CoA derivative. +
Cellular transformation and proliferation are fostered by a major rearrangement of cellular metabolism, resulting in unique expression patterns along a series of metabolic pathways in cancer. Mitochondria are central to many of these pathways, suggesting that the transcriptome underlying mitochondrial biogenesis will also follow a distinctive pattern in tumour tissues. Nonetheless, few studies have addressed this question so far.
In order to explore the expression pattern of nuclear encoded mitochondrial genes in cancer, we applied a novel bi-clustering algorithm together with supervised machine learning to predict gene correlations between mitochondrial genes and oncogenic pathways. ''In silico'' analysis of a large set of exome and mRNA expression data of BRCA in The Cancer Genome Atlas revealed a striking mosaic pattern of mitochondrial gene expression, which was specific to breast cancer subtypes. Functional validation of the bioinformatic analysis in subtype specific cancer cell line models showed unique mitochondrial metabolism correlating with gene expression profiles. Moreover, we have identified tumour subtypes of which the proliferation and survival relies on the expression of specific mitochondrial genesets, presumably driven by oncogene driven activation of transcriptional factors and co-regulators. The results provide a novel framework to understand the role of mitochondria in cancer and help to identify cancer subtypes that could be targeted by mitochondrion specific therapeutic approaches. +
A number of functions for coenzyme Q (CoQ) have been established during the years but its role as an effective antioxidant of the cellular membranes remains of dominating interest. This compound is our only endogenously synthesized lipid soluble antioxidant, present in all membranes and exceeding both in amount and efficiency that of other antioxidants. The protective effect is extended to lipids, proteins and DNA mainly because of its close localization to the oxidative events and the effective regeneration by continuous reduction at all locations. Its biosynthesis is influenced by nuclear receptors which may give the possibility, in the future, by using agonists or antagonists, of reestablishing the normal level in deficiencies caused by genetic mutations, aging or cardiomyopathy. An increase in CoQ concentration in specific cellular compartments in the presence of various types of oxidative stress appears to be of considerable interest. +
In addition to its role as a component of the mitochondrial respiratory chain and our only lipid-soluble antioxidant synthesized endogenously, in recent years coenzyme Q (CoQ) has been found to have an increasing number of other important functions required for normal metabolic processes. A number of genetic mutations that reduce CoQ biosynthesis are associated with serious functional disturbances that can be eliminated by dietary administration of this lipid, making CoQ deficiencies the only mitochondrial diseases which can be successfully treated at present. In connection with certain other diseases associated with excessive oxidative stress, the level of CoQ is elevated as a protective response. Aging, certain experimental conditions and several human diseases reduce this level, resulting in serious metabolic disturbances. Since dietary uptake of this lipid is limited, up-regulation of its biosynthetic pathway is of considerable clinical interest. One approach for this purpose is administration of epoxidated all-trans polyisoprenoids, which enhance both CoQ biosynthesis and levels in experimental systems. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]]
Human urine contains a small population of cells with stemness properties, urine-derived stem cells (USCs). USCs can be non-invasively collected and used for personalized therapeutics. However, only a small number of studies about USCs is available. Thus, a deeper characterization of these cells is essential, including in what regards to their mitochondrial function profile. Our aim is to establish a protocol to isolate and expand USCs, enabling for a mitochondrial metabolic characterization.
Urine from female volunteers was used to isolate and expand USCs using the commonly used mixture of Keratinocyte Serum Free Medium and Embryonic Fibroblast Medium (KSFM+EFM) and a mixture of KSFM and DMEM, 10%FBS, 1% MEM-NEAA (KSFM+DMEM). Isolated cells were characterized by flow cytometry for mesenchymal stem cells markers (CD44, CD73, CD90 and CD105) and hematopoietic stem cells markers (CD14, CD20, CD34 and CD45). Moreover, the renal progenitor/multipotent marker CD24 was also assessed. For mitochondrial function profiling, different cell densities were used to determine the oxygen consumption rate (OCR) using the Seahorse XFe96 Extracellular Flux Analyzer. The optimized conditions used to measure OCR involved 20,000 cells/well, with oligomycin 2 μM, FCCP 0.5 μM and rotenone/antimycin A 1 μM used to titrate respiration.
The phenotyping results for USCs isolated from one volunteer using the two media described did not show statistically significant differences. Using the KSFM+DMEM medium, the USCs from six volunteers were ˃96% positive for CD44, CD73 and CD24. The expression of CD90 and CD105 was unstable with average values for positive cells of 60 and 68%, respectively, and the expression of hematopoietic stem cells markers is ˂ 1%. We observed that 77.5% of the OCR was used by USC to sustain ADP phosphorylation and the basal respiration corresponds to 57.2% of their maximal capacity.
In our study, the KSFM+EFM could be replaced by KSFM+DMEM for USCs isolation and expansion from human urine, with similar results regarding stem cells markers. Isolated USCs showed a respiratory reserve capacity which may be used for boosting mitochondrial metabolism after cell differentiation. Future studies will be performed in order to characterize the glycolytic and mitochondrial function profile of USCs.
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]]
[[Bento 2017 MITOEAGLE Obergurgl]] +
Charcot-Marie-Tooth disease type 2A (CMT2A) is an autosomal dominant axonal peripheral neuropathy caused by mutations in the mitofusin 2 gene (''MFN2''). Mitofusin 2 is a GTPase protein present in the outer mitochondrial membrane and responsible for regulation of mitochondrial network architecture via the fusion of mitochondria. As that fusion process is known to be strongly dependent on the GTPase activity of mitofusin 2, it is postulated that the MFN2 mutation within the GTPase domain may lead to impaired GTPase activity, and in turn to mitochondrial dysfunction. The work described here has therefore sought to verify the effects of MFN2 mutation within its GTPase domain on mitochondrial and endoplasmic reticulum morphology, as well as the mtDNA content in a cultured primary fibroblast obtained from a CMT2A patient harboring a ''de novo'' Arg274Trp mutation. In fact, all the parameters studied were affected significantly by the presence of the mutant MFN2 protein. However, using the stable model for mitofusin 2 obtained by us, we were next able to determine that the Arg274Trp mutation does not impact directly upon GTP binding. Such results were also confirmed for GTP-hydrolysis activity of MFN2 protein in patient fibroblast. We therefore suggest that the biological malfunctions observable with the disease are not consequences of impaired GTPase activity, but rather reflect an impaired contribution of the GTPase domain to other MFN2 activities involving that region, for example protein-protein interactions. +
Charcot-Marie-Tooth disease type 2A (CMT2A) is an autosomal dominant axonal peripheral neuropathy caused by mutations in the mitofusin 2 gene (MFN2). Mitofusin 2 is a GTPase protein present in the outer mitochondrial membrane and responsible for regulation of mitochondrial network architecture via the fusion of mitochondria. As that fusion process is known to be strongly dependent on the GTPase activity of mitofusin 2, it is postulated that the MFN2 mutation within the GTPase domain may lead to impaired GTPase activity, and in turn to mitochondrial dysfunction. The work described here has therefore sought to verify the effects of MFN2 mutation within its GTPase domain on mitochondrial and endoplasmic reticulum morphology, as well as the mtDNA content in a cultured primary fibroblast obtained from a CMT2A patient harboring a ''de novo'' Arg274Trp mutation. In fact, all the parameters studied were affected significantly by the presence of the mutant MFN2 protein. However, using the stable model for mitofusin 2 obtained by us, we were next able to determine that the Arg274Trp mutation does not impact directly upon GTP binding. Such results were also confirmed for GTP-hydrolysis activity of MFN2 protein in patient fibroblast. We therefore suggest that the biological malfunctions observable with the disease are not consequences of impaired GTPase activity, but rather reflect an impaired contribution of the GTPase domain to other MFN2 activities involving that region, for example protein-protein interactions. +
A preprint is a complete scientific manuscript (often one also being submitted to a peer-reviewed journal) that is uploaded by the authors to a public server without formal review. After a brief inspection to ensure that the work is scientific in nature, the posted scientific manuscript can be viewed without charge on the Web.
Conclusions: Preprints could play important roles in accelerating scientific progress; they could serve the needs and foster the careers of scientists; and, in cooperation with existing journals, they could enhance the current system for communicating results and ideas in the life sciences. However, preprints are relatively new to biology, and many questions remain unanswered. Will funding agencies encourage the use of preprint servers? Will all journals accept manuscripts for publication after they have been disseminated as preprints? Will the life sciences community find ways to make biology preprints easily discoverable? And will researchers themselves decide to submit, cite, and evaluate work presented in preprint form? The cooperative spirit displayed by the attendees at ASAPbio gives hope that these complex issues, as well as others that limit the communication of scientific ideas and results, can be addressed in a productive and thoughtful manner. +
In 1991, an electronic system through which interested parties could access non-peer-reviewed physics papers was launched by Paul Ginsparg at Los Alamos National Laboratory. This evolved into the “preprint” server arXiv, which has played a central role in information sharing in physics, mathematics, and related fields over many years. Recently, other preprint servers have been or are being developed to serve other fields including biological sciences, chemistry, and medicine (see the News story on p. [[Kaiser 2017 Science 357 |1344]]). Given the different cultures associated with these disciplines, and other external factors, policies and practices must evolve as these servers become integrated into their communities. +
The main purposes of this study were to compare mitochondrial respiration in ''M. triceps brachii'' and ''M. vastus lateralis'' between elite cross-country (XC) skiers and physically active controls (CON), and to explore the associations between mitochondrial respiration in these muscles and peak oxygen uptake (VO<sub>2peak</sub>) in arm- and leg-dominant exercise modes.
Thirteen male elite XC skiers (age: 25 ± 4; peak oxygen uptake (VO<sub>2peak</sub>): 75.5 ± 4.2 mL⋅kg<sup>-1</sup>⋅min<sup>-1</sup>) and twelve CON (age: 26 ± 3; VO<sub>2peak</sub>: 57.2 ± 6.4 mL⋅kg<sup>-1</sup>⋅min<sup>-1</sup>) had microbiopsies taken from ''M. vastus lateralis'' and ''M. triceps brachii'', which were analyzed for various measures of mitochondrial respiration using high-resolution respirometry. Thereafter, all participants tested VO<sub>2peak</sub> in both running (RUN) and upper body poling (UBP).
XC skiers had generally higher mitochondrial respiration in ''M. triceps brachii'' compared to CON (P < 0.001), whereas no significant group-differences in mitochondrial respiration in ''M. vastus lateralis'' were revealed. XC skiers had higher mitochondrial respiration in ''M. triceps brachii'' compared to ''M. vastus lateralis'' (P = 0.005-0.058), whereas in CON, most mitochondrial respiration measures were higher in ''M. vastus lateralis'' than in ''M. triceps brachii'' (P < 0.01). When all athletes were pooled, there was a strong positive correlation between VO<sub>2peak</sub> in UBP and mitochondrial respiration in ''M. triceps brachii'' on several measures (P < 0.01), whereas no correlation was found for RUN.
The higher mitochondrial respiration found in ''M. triceps brachii'' compared to ''M. vastus lateralis'' among our elite XC skiers demonstrates the potential for the arm muscles to adapt to aerobic endurance training. The opposite pattern found in CON, clearly showed lower mitochondrial respiration in ''M. triceps brachii'' compared to XC skiers, whereas respiration in ''M. vastus lateralis'' did not differ between groups. The strong positive correlation between mitochondrial respiration in ''M. triceps brachii'' and VO<sub>2peak</sub> in UBP indicate that arm muscles' respiratory function may be a limiting factor for VO<sub>2peak</sub> in arm-dominant exercise modes.
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Control of intestinal epithelial stemness is crucial for tissue homeostasis. Disturbances in epithelial function are implicated in inflammatory and neoplastic diseases of the gastrointestinal tract. Here we report that mitochondrial function plays a critical role in maintaining intestinal stemness and homeostasis. Using intestinal epithelial cell (IEC)-specific mouse models, we show that loss of HSP60, a mitochondrial chaperone, activates the mitochondrial unfolded protein response (MT-UPR) and results in mitochondrial dysfunction. HSP60-deficient crypts display loss of stemness and cell proliferation, accompanied by epithelial release of WNT10A and RSPO1. Sporadic failure of Cre-mediated Hsp60 deletion gives rise to hyperproliferative crypt foci originating from OLFM4<sup>+</sup> stem cells. These effects are independent of the MT-UPR-associated transcription factor CHOP. In conclusion, compensatory hyperproliferation of HSP60<sup>+</sup> escaper stem cells suggests paracrine release of WNT-related factors from HSP60-deficient, functionally impaired IEC to be pivotal in the control of the proliferative capacity of the stem cell niche. +
A major international movement is in progress to extend metrication using the International System of Units. Significantly involved is the field of medicine. Extensive changes adopted abroad now appear in foreign medical literature, and physicians in the United States commonly are unprepared to interpret medical information from abroad because the data are reported in unfamiliar terms. The system has broad immediate and future implications to American physicians. +
Obesity is a growing problem in the United States and throughout the world. It is a risk factor for many chronic diseases. The BMI has been used to assess body fat for almost 200 years. BMI is known to be of limited accuracy, and is different for males and females with similar %body adiposity. Here, we define an alternative parameter, the body adiposity index (BAI = ((hip circumference)/((height)(1.5))-18)). The BAI can be used to reflect %body fat for adult men and women of differing ethnicities without numerical correction. We used a population study, the "BetaGene" study, to develop the new index of body adiposity. %Body fat, as measured by the dual-energy X-ray absorptiometry (DXA), was used as a "gold standard" for validation. Hip circumference (R = 0.602) and height (R = -0.524) are strongly correlated with %body fat and therefore chosen as principal anthropometric measures on which we base BAI. The BAI measure was validated in the "Triglyceride and Cardiovascular Risk in African-Americans (TARA)" study of African Americans. Correlation between DXA-derived %adiposity and the BAI was R = 0.85 for TARA with a concordance of C_b = 0.95. BAI can be measured without weighing, which may render it useful in settings where measuring accurate body weight is problematic. In summary, we have defined a new parameter, the BAI, which can be calculated from hip circumference and height only. It can be used in the clinical setting even in remote locations with very limited access to reliable scales. The BAI estimates %adiposity directly. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]] +
Hypoxic preconditioning (HPC) is the application of mild transient hypoxia which protects the brain against a following more severe hypoxic insult as it occurs during epileptic seizures. HPC decreases seizure susceptibility and severity as well as neuronal damage in the hippocampus. The delta opioid receptor (DOR) and its primary endogenous ligands, the neuropeptides met- and leu-enkephalin (Enk), are thought to be involved in the neuroprotective actions of HPC. Recently, we showed that Enk influences mitochondrial respiration that may contribute to the neuroprotective effects of the Enk/DOR system. The present study aims at investigating the effects of the Enk/DOR system on structural and functional alterations of mitochondria in HPC.
Wild type (WT) and met-Enk-knockout (met-Enk-KO) mice were exposed to hypoxia. Subsequently, we determined the seizure threshold, analyzed mitochondrial function and dynamics.
In WT mice after HPC we observed an elevated seizure threshold, improved mitochondrial reserve capacity and increased mitochondrial fusion. In addition, our results suggest mitochondrial biogenesis after HPC in WT mice. Naïve met-Enk-KO mice had an increased seizure threshold and increased mitochondrial fusion but no changes upon HPC.
The observed mitochondrial alterations after HPC in WT mice could explain improved neuronal survival and increased seizure threshold. Enhanced mitochondrial reserve capacity improves energy supply in stress situations and increased mitochondrial fusion is associated with neuronal survival and elevated Ca<sup>2+</sup> storage capacities. However, the precise role of met-Enk in HPC is unclear but we observed adaptive mechanisms in WT mice upon hypoxia which are absent in met-Enk-KO mice. +
Pulmonary arterial hypertension (PAH) is characterized by pulmonary vessel remodeling; however, its severity and impact on survival depend on right ventricular (RV) failure. Resveratrol (RES), a polyphenol found in red wine, exhibits cardioprotective effects on RV dysfunction in PAH. However, most literature has focused on RES protective effect on lung vasculature; recent finding indicates that RES has a cardioprotective effect independent of pulmonary arterial pressure on RV dysfunction, although the underlying mechanism in RV has not been determined. Therefore, this study is aimed at evaluating sirtuin-3 (SIRT3) modulation by RES in RV using a monocrotaline- (MC-) induced PAH rat model. Myocyte function was evaluated by confocal microscopy as cell contractility, calcium signaling, and mitochondrial membrane potential (ΔΨ''m''); cell energetics was assessed by high-resolution respirometry, and western blot and immunoprecipitation evaluated posttranslational modifications. PAH significantly affects mitochondrial function in RV; PAH is prone to mitochondrial permeability transition pore (mPTP) opening, thus decreasing the mitochondrial membrane potential. The compromised cellular energetics affects cardiomyocyte function by decreasing sarco-endoplasmic reticulum Ca<sup>2+</sup>-ATPase (SERCA) activity and delaying myofilament unbinding, disrupting cell relaxation. RES partially protects mitochondrial integrity by deacetylating cyclophilin-D, a critical component of the mPTP, increasing SIRT3 expression and activity and preventing mPTP opening. The preserved energetic capability rescues cell relaxation by maintaining SERCA activity. Avoiding Ca<sup>2+</sup> transient and cell contractility mismatch by preserving mitochondrial function describes, for the first time, impairment in excitation-contraction-energetics coupling in RV failure. These results highlight the importance of mitochondrial energetics and mPTP in PAH. +
This review provides a selective history of how studies of mitochondrial cation transport (K+, Na+, Ca2+) developed in relation to the major themes of research in bioenergetics. It then covers in some detail specific transport pathways for these cations, and it introduces and discusses open problems about their nature and physiological function, particularly in relation to volume regulation and Ca2+ homeostasis. The review should provide the basic elements needed to understand both earlier mitochondrial literature and current problems associated with mitochondrial transport of cations and hopefully will foster new interest in the molecular definition of mitochondrial cation channels and exchangers as well as their roles in cell physiology. +
Lipoyl(Octanoyl) Transferase 2 (LIPT2) is a protein involved in the post-translational modification of key energy metabolism enzymes in humans. Defects of lipoic acid synthesis and transfer start to emerge as causes of fatal or severe early-onset disease. We show that the first 31 amino acids of the N-terminus of LIPT2 represent a mitochondrial targeting sequence and inhibition of the transit of LIPT2 to the mitochondrion results in apoptotic cell death associated with activation of the apoptotic volume decrease (AVD) current in normotonic conditions, as well as over-activation of the swelling-activated chloride current (IClswell), mitochondrial membrane potential collapse, caspase-3 cleavage and nuclear DNA fragmentation. The findings presented here may help elucidate the molecular mechanisms underlying derangements of lipoic acid biosynthesis. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]]
Carbonic anhydrases (CAs) are metalloenzymes present in all prokaryotes and eukaryotes. These enzymes catalyse the conversion of CO<sub>2</sub> to HCO<sub>3-</sub> and H<sup>+</sup>. This reaction is involved in multiple physiological processes [1]. CAs have been identified in the reproductive tract of various species [2,3], but their precise physiological roles in the male reproductive tract is still unclear.
Herein, we determined the impact of CAs inhibition with acetazolamide, a general inhibitor of CAs, on the metabolism (by means of nuclear magnetic resonance spectroscopy), and on the mitochondrial morphology (by means of electron microscopy), biogenesis (using quantitative reverse transcriptase polymerase chain reaction) and bioenergetics (using the fluorescent probe JC1) of primary cultures of human Sertoli cells (hSCs), obtained from biopsies from men with conserved spermatogenesis.
When CAs were inhibited in hSCs (by exposure to acetazolamide during 24 hours), we observed a decrease in the number of mitochondria and an alteration on their morphology (Figure 1) though the average mitochondrial membrane potential was not affected. Still, cells treated with acetazolamide exhibited an accumulation of cytoplasmic lipid droplets, suggesting a diminished mitochondrial function. In fact, when assessing the expression of mitochondrial biogenesis markers, we observed a striking decrease in the abundance of HIF-1α, Sirt1, PGC-1α and NRF-1 mRNAs, when CAs were inhibited (Figure 2). All these mitochondria-associated alterations were accompanied by an increased production of lactate and alanine (although the consumption of glucose was maintained) indicating a reduced conversion of pyruvate to acetyl-coA, possibly due to the diminished mitochondrial function.
Our results suggest that, in hSc, CAs are essential for mitochondrial biogenesis, morphology and physiology. Since Sertoli cells are essential for the occurrence of spermatogenesis, CAs should play an important role in the establishment of male reproductive potential.
Cardiovascular diseases are coupled to decreased nitric oxide (NO) bioavailability, and there is a constant search for novel and better NO-donors. Here we synthesized and characterized the cardiovascular effects of the new organic nitrate 2-nitrate-1,3-dioctanoxypropan (NDOP).
A combination of ''in vitro'' and ''in vivo'' experiments was performed in C57BL/6 mice and Wistar rats. Thus, the ability of NDOP in donating NO in a cell-free system and in vascular smooth muscles cells (VSMC) and its ability to induce vasorelaxation in aortic rings from mice were evaluated. In addition, changes in blood pressure and heart rate to different doses of NDOP were evaluated in conscious rats. Finally, acute pre-clinical toxicity to oral administration of NDOP was assessed in mice.
In cell-free system, NDOP increased NO levels, which was dependent on xanthine oxidoreductase (XOR). NDOP also increased NO levels in VSMC, which was not influenced by endothelial NO synthase. Furthermore, incubation with the XOR inhibitor febuxostat blunted the vasorelaxation in aortic ring preparations. In conscious rats, NDOP elicited dose-dependent reduction in blood pressure accompanied with increased heart rate. In vessel preparations, NDOP (10<sup>-8</sup>-10<sup>-3</sup> mol/L) induced endothelium-independent vasorelaxation, which was inhibited by the NO scavengers 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and hydroxocobalamin or by inhibition of soluble guanylyl cyclase using H- [1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one. To investigate if NDOP acts through potassium channels, selective blockers were used. Inhibition of BK<sub>Ca</sub>, K<sub>v</sub> or K<sub>ATP</sub> subtypes of potassium channels had no effect, but inhibition of inward-rectifier potassium channels (KIR) significantly reduced NDOP-mediated vasorelaxation. Lastly, NDOP showed low toxicity (LD<sub>50</sub> ~5000 mg/kg).
Conclusion: Bioactivation of NDOP involves functional XOR, and this new organic nitrate elicits vasorelaxation via NO-cGMP-PKG signaling and activation of K<sub>IR</sub> channels. Future studies should further characterize the underlying mechanism and evaluate the therapeutic benefits of chronic NDOP treatment in relevant cardiovascular disease models.
Peroxisome proliferator-activated receptor-γ (PPAR-γ) is one of the most studied nuclear receptor since its identification as a target to treat metabolic and neurological diseases. In addition to exerting anti-inflammatory and neuroprotective effects, PPAR-γ agonists, such as the insulin-sensitizing drug pioglitazone, promote the differentiation of oligodendrocytes (OLs), the myelin-forming cells of the central nervous system (CNS). In addition, PPAR-γ agonists increase OL mitochondrial respiratory chain activity and OL's ability to respond to environmental signals with oscillatory Ca2+ waves. Both OL maturation and oscillatory Ca2+ waves are prevented by the mitochondrial inhibitor rotenone and restored by PPAR-γ agonists, suggesting that PPAR-γ promotes myelination through mechanisms involving mitochondria. +
Several studies have demonstrated that overnutrition during early postnatal period can increase the long term risk of developing obesity and cardiac disorders, yet the short-term effects of postnatal overfeeding in cardiac metabolism remains unknown. The aim of our study was to investigate the cardiac metabolism of weaned mice submitted to overnutrition during lactation, particularly as to mitochondrial function, substrate preference and insulin signaling. Postnatal overfeeding was induced by litter size reduction in mice at postnatal day 3. At 21 days of age (weaning), mice in the Overfed group (OG) presented biometric and biochemical parameters of obesity, including increased body weight, visceral fat, liver weight and increased left ventricle weight/tibia length ratio; indicating cardiac hypertrophy; hyperglycemia, hyperinsulinemia and increased liver glycogen content compared to control group (CG). In the heart, we detected impaired insulin signaling, mainly due to decreased IRβ, pTyr-IRS1, PI3K, GLUT4 and pAkt/Akt, and increased PTP1B, GLUT1 and pAMPKα/AMPKα content. Activities of lactate dehydrogenase and citrate synthase were increased, accompanied by enhanced carbohydrate oxidation, as observed by high-resolution respirometry. Moreover, OG hearts had lower CPT1, PPARα and increased UCP2 mRNA expression, associated with increased oxidative stress (4-HNE content), BAX/BCL2 ratio and cardiac fibrosis. Ultrastructural analysis of OG hearts demonstrated mild mitochondrial damage without alterations in OxPhos complexes. In conclusion, overnutrition during early life induces short term metabolic disturbances, impairment in heart insulin signaling, upregulates GLUT-1 and switch cardiac fuel preference in juvenile mice. +
Activation of mammalian embryonic development relies on a series of fertilization-induced increases in intracellular Ca<sup>2+</sup>. Full egg activation also requires influx of extracellular Ca<sup>2+</sup>, but the channel or channels mediating this influx remain unknown. In these studies we examined whether T-type Ca<sup>2+</sup> channels, including CACNA1H subunit-containing CaV3.2 channels, mediate Ca<sup>2+</sup> entry after fertilization. We found that female mice lacking CACNA1H have reduced litter size. Careful analysis of Ca<sup>2+</sup> oscillation patterns following ''in vitro'' fertilization (IVF) of ''Cacna1h<sup>-/-</sup>'' eggs revealed shortening of the first Ca<sup>2+</sup> transient length and reduction in Ca<sup>2+</sup> oscillation persistence. Both total and endoplasmic reticulum (ER) Ca<sup>2+</sup> stores in ''Cacna1h<sup>-/-</sup>'' eggs were reduced, showing an impairment of Ca<sup>2+</sup> accumulation during oocyte maturation in ''Cacna1h<sup>-/-</sup>'' eggs. Pharmacological inhibition of T-type channels during ''in vitro'' maturation also reduced Ca<sup>2+</sup> store accumulation, indicating that T-type channels are responsible for mediating Ca<sup>2+</sup> entry and ER store accumulation during meiotic maturation. T-type channel inhibition also reduced oscillation persistence, frequency, and duration following IVF in wild-type eggs. Together, these data support previously unrecognized roles for T-type Ca<sup>2+</sup> channels in mediating the maturation-associated increase in ER Ca<sup>2+</sup> stores and allowing Ca<sup>2+</sup> influx required for the activation of embryo development. In future studies, we plan to investigate how fluxes in oocyte Ca<sup>2+</sup> and Zn<sup>2+</sup> influence mitochondrial function, which is a critical determinant of oocyte and embryo quality. Developing better understanding of the interplay between these pathways may translate into clinical application to improve assisted reproductive technologies.
Aging of biological systems is accompanied by degeneration of mitochondrial functions. Different pathways are active to counteract the processes which lead to mitochondrial dysfunction. Mitochondrial dynamics, the fission and fusion of mitochondria, is one of these quality control pathways. Mitophagy, the controlled degradation of mitochondria, is another one. Here we show that these pathways are linked. A double deletion mutant of Saccharomyces cerevisiae in which two essential components of the fission and fusion machinery, Dnm1 and Mgm1, are simultaneously ablated, contain wild-type like filamentous mitochondria, but are characterized by impaired respiration, an increased sensitivity to different stressors, increased mitochondrial protein carbonylation, and a decrease in mitophagy and replicative lifespan. These data show that a balanced mitochondrial dynamics and not a filamentous mitochondrial morphotype per se is the key for a long lifespan and demonstrate a cross-talk between two different mitochondrial quality control pathways. +
Single long-chain omega-3 fatty acids (e.g. docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA)) are known for their neuroprotective properties associated with ischemic stroke. This pilot study aimed to test the effectiveness of an acute treatment with a long-chain omega-3 lipid emulsion (Omegaven 10%®, OGV) that contains fish oil (DHA 18 mg/ml; EPA 21 mg/ml) and α-tocopherol (0.2 mg/ml) in a transient middle cerebral artery occlusion (MCAO) model of ischemic stroke in mice. For this purpose, female CD-1 mice were anesthetized and subjected to 90 minutes of MCAO. To reflect a clinically relevant situation for an acute treatment, either after induction of stroke or after reperfusion, a single dose of OGV was injected intravenously into the tail vein (5 ml/kg b.w.). A neurological severity score was used to assess motor function and neurological outcome. Stroke-related parameters were determined 24 hours after MCAO. Microdialysis was used to collect samples from extracellular space of the striatum. Mitochondrial function was determined in isolated mitochondria or dissociated brain cells. Inflammation markers were measured in brain homogenate. According to control experiments, neuroprotective effects could be attributed to the long-chain omega-3 content of the emulsion. Intravenous injection of OGV reduced size and severity of stroke, restored mitochondrial function, and prevented excitotoxic glutamate release. Increases of pro-inflammatory markers (COX-2 and IL-6) were attenuated. Neurological severity scoring and neurochemical data demonstrated that acute OGV treatment shortly after induction of stroke was most efficient and able to improve short-term neurological outcome, reflecting the importance of an acute treatment to improve the outcome. Summarising, acute treatment of stroke with a single intravenous dose of OGV provided strong neuroprotective effects and was most effective when given immediately after onset of ischemia. As OGV is an approved fishoil emulsion for parenteral nutrition in humans, our results may provide first translational data for a possible early management of ischemic stroke with administration of OGV to prevent further brain damage.
Chronic kidney disease (CKD) substantially increases the severity of peripheral arterial disease (PAD) symptomology, however, the biological mechanisms remain unclear. The objective herein was to determine the impact of CKD on PAD pathology in mice. C57BL6/J mice were subjected to a diet-induced model of CKD by delivery of adenine for six weeks. CKD was confirmed by measurements of glomerular filtration rate, blood urea nitrogen, and kidney histopathology. Mice with CKD displayed lower muscle force production and greater ischemic lesions in the ''tibialis anterior'' muscle (78.1 ± 14.5% vs. 2.5 ± 0.5% in control mice, P < 0.0001, N = 5-10/group) and decreased myofiber size (1661 ± 134 μm<sup>2</sup> vs. 2221 ± 100 μm<sup>2</sup> in control mice, P < 0.01, N = 5-10/group). This skeletal myopathy occurred despite normal capillary density (516 ± 59 vs. 466 ± 45 capillaries/20x field of view) and limb perfusion. CKD mice displayed a ~50-65% reduction in muscle mitochondrial respiratory capacity in ischemic muscle, whereas control mice had normal mitochondrial function. Hydrogen peroxide emission was modestly higher in the ischemic muscle of CKD mice, which coincided with decreased oxidant buffering. Exposure of cultured myotubes to CKD serum resulted in myotube atrophy and elevated oxidative stress, which were attenuated by mitochondrial-targeted therapies. Taken together, these findings suggest that mitochondrial impairments caused by CKD contribute to the exacerbation of ischemic pathology. +
When isolated liver cells from starved rats were incubated with fatty acids, the rates of O2 uptake and ketone body production in the presence of hexanoate were somewhat greater than with added palmitate. However, the 3-hydroxybutyrate/acetoacetate ratio was consistently higher in the presence of palmitate. Moreover, palmitate was much more effective than hexanoate in slowing the restoration to normal of an elevated lactate/pyruvate ratio. This action of palmitate was counteracted by inhibitors of long-chain fatty acid oxidation to ketone bodies. Palmitate, but not hexanoate, partially inhibited lactate-stimulated ethanol oxidation. These effects of palmitate were in most instances exaggerated in cells from clofibrate-treated rats, which had somewhat higher rates of ketogenesis.
When cells were incubated with rotenone, ketone bodies were produced at about 30% of control rates and accumulated almost entirely as 3-hydroxybutyrate. In the presence of antimycin, ketone body accumulation from added palmitate again occurred at about 30 % of control rates, but ketogenesis from endogenous substrates or hexanoate was inhibited to a greater extent. Cells from clofibrate-treated rats were equally sensitive to antimycin but considerably less inhibited by rotenone than control cells, although the 3-hydroxybutyrate/acetoacetate ratio remained very high.
The depression of fatty acid oxidation in cells incubated with rotenone could be relieved to a considerable degree by coupling the dehydrogenation of hydroxyacyl-CoA to the reduction of acetoacetate. This observation was found to be valid only for longer chain fatty acids (C10–C18). The coupling of octanoate oxidation to acetoacetate reduction was promoted by the addition of carnitine. Carnitine did not increase the coupling of the oxidation of other fatty acids tested to acetoacetate reduction.
The formation of labelled ketone bodies from [1-14C]palmitate and [1-14C]hexanoate was also examined. When the two fatty acids were presented together to rotenone-poisoned cells in the presence of acetoacetate, hexanoate caused some inhibition of 14CO2 formation and ketone body production from [1-14C]palmitate, but the converse did not apply. In fact palmitate stimulated the oxidation of [1-14C]hexanoate to 14CO2 and labelled ketone bodies. This stimulation was abolished by low levels of 2,4-dinitrophenol.
The results of these experiments are explained on the basis of compartmentation of fatty acid oxidation between mitochondrial matrix and peroxisomes. Whereas both long-chain and short-chain fatty acids can be oxidized within the mitochondrial matrix, an additional site of long-chain acyl-CoA oxidation exists in the peroxisomes. Thus, peroxisomes and mitochondria co-operate in the oxidation of long-chain fatty acids. The oxidation of long-chain acyl groups within the peroxisomes and the subsequent transfer of reducing equivalents to the mitochondria, probably via a glycerol 1-phosphate shuttle, explains why long-chain fatty acids induce a more reduced ‘redox state’ than short-chain species in isolated liver cells.
A simple method for the accurate determination of free [Ca] in ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA)-buffered Ca solutions is described. This method is useful for calibration of Ca macro- and microelectrodes to low free [Ca] and should improve the reliability of calculated free [Ca] in more complex solutions. Briefly, free [Ca] in Ca-EGTA solutions is measured with a Ca electrode, bound Ca is calculated, and Scatchard and double-reciprocal plots are resolved for the total [EGTA] and the apparent Ca-EGTA association constant (K'Ca) in the solutions used. The free [Ca] is then recalculated using the determined parameters, giving a more accurate knowledge of the free [Ca] in these solutions and providing an accurate calibration curve for the Ca electrode. These solutions can then be used to calibrate other Ca electrodes (e.g., Ca microelectrodes) or the calibrated Ca electrode can be used to measure free [Ca] in solutions containing multiple metal ligands. This method allows determination of free [Ca], K'Ca, and total [EGTA] in the actual solutions used regardless of pH, temperature, or ionic strength. It does not require accurate knowledge of K'Ca or EGTA purity and circumvents many potential errors due to assumption of binding parameters. K'Ca was found to be 2.45 +/- 0.04 X 10(6) M-1 in 100 mM KCl, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and 1 mM EGTA at pH 7.00 and 23 degrees C. Total [EGTA] varied with supplier but was always less than quoted. +
In living cells the cellular proteome is under constant remodeling as cells adapt to altered physiological states or as cellular proteins become misfolded and need to be degraded. The cellular processes which grant maintenance of a functional proteome are collectively called proteostasis. The proteasome constitutes one of the major intracellular protein degradation systems and is responsible for the turnover of damaged or unwanted proteins with almost 90% of the intracellular proteins being potential proteasome substrates. Especially for misfolded proteins, a timely degradation is necessary to avoid proteotoxic stress, e.g. by formation of protein aggregates. Degradation of proteins by the proteasome system is a very energy demanding process. Hence it was speculated that proteasomal function is interconnected with the function of mitochondria. Additionally, a correlation between mitochondrial and proteasomal dysfunction exists since a decline in the function of both systems is well recognized as hallmark of aging. However, it is unclear how proteasomal and mitochondrial function are directly linked together, especially in settings of chronic mitochondrial respiratory chain dysfunction. Moreover, limited knowledge is available to which extent proteasomal and mitochondrial functions contribute to aging processes specifically in lung tissue during healthy aging or in the development of age-related lung diseases such as chronic obstructive pulmonary disease. +
The heart consumes large amounts of energy in the form of ATP that is continuously replenished by oxidative phosphorylation in mitochondria and, to a lesser extent, by glycolysis. To adapt the ATP supply efficiently to the constantly varying demand of cardiac myocytes, a complex network of enzymatic and signalling pathways controls the metabolic flux of substrates towards their oxidation in mitochondria. In patients with heart failure, derangements of substrate utilization and intermediate metabolism, an energetic deficit, and oxidative stress are thought to underlie contractile dysfunction and the progression of the disease. In this Review, we give an overview of the physiological processes of cardiac energy metabolism and their pathological alterations in heart failure and diabetes mellitus. Although the energetic deficit in failing hearts - discovered >2 decades ago - might account for contractile dysfunction during maximal exertion, we suggest that the alterations of intermediate substrate metabolism and oxidative stress rather than an ATP deficit per se account for maladaptive cardiac remodelling and dysfunction under resting conditions. Treatments targeting substrate utilization and/or oxidative stress in mitochondria are currently being tested in patients with heart failure and might be promising tools to improve cardiac function beyond that achieved with neuroendocrine inhibition. +
In the current issue of Function, Juhaszova and colleagues3 substantially challenge—or rather expand, but do not tumble—this concept in revealing that in addition to H+, potassium ion (K+) flux through the F1Fo-ATP synthase (working the same way as H+) provides the majority of energy to produce ATP (Figure 1). Why was this was overlooked for more than six decades? Presumably because the F1Fo-ATP synthase has a > 107-fold selectivity for H+ over other cations.4 But what was not sufficiently considered is that due to the 106-fold higher cytosolic concentration for K+ (∼100 mM) than for H+(∼100 nM), such that K+ flux—driven mostly by the same high electrical driving force (∆Ψm) - could be comparable to H+ flux via the ATP synthase. +
The mitochondrial Ca<sup>2+</sup> uptake in trypanosomatids, which belong to the eukaryotic supergroup Excavata, shares biochemical characteristics with that of animals, which, together with fungi, belong to the supergroup Opisthokonta. However, the composition of the mitochondrial calcium uniporter (MCU) complex in trypanosomatids is quite peculiar, suggesting lineage-specific adaptations. In this work, we used ''Trypanosoma cruzi'' to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca<sup>2+</sup> uptake. ''T. cruzi'' MICU1 (TcMICU1) and TcMICU2 have mitochondrial targeting signals, two canonical EF-hand calcium-binding domains, and localize to the mitochondria. Using the CRISPR/Cas9 system (i.e., clustered regularly interspaced short palindromic repeats with Cas9), we generated TcMICU1 and TcMICU2 knockout (-KO) cell lines. Ablation of either TcMICU1 or TcMICU2 showed a significantly reduced mitochondrial Ca<sup>2+</sup> uptake in permeabilized epimastigotes without dissipation of the mitochondrial membrane potential or effects on the AMP/ATP ratio or citrate synthase activity. However, none of these proteins had a gatekeeper function at low cytosolic Ca<sup>2+</sup> concentrations ([Ca<sup>2+</sup>]<sub>cyt</sub>), as occurs with their mammalian orthologs. TcMICU1-KO and TcMICU2-KO epimastigotes had a lower growth rate and impaired oxidative metabolism, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes. The findings of this work, which is the first to study the role of MICU1 and MICU2 in organisms evolutionarily distant from animals, suggest that, although these components were probably present in the last eukaryotic common ancestor (LECA), they developed different roles during evolution of different eukaryotic supergroups. The work also provides new insights into the adaptations of trypanosomatids to their particular life styles.
''Trypanosoma cruzi'' is the etiologic agent of Chagas disease and belongs to the early-branching eukaryotic supergroup Excavata. Its mitochondrial calcium uniporter (MCU) subunit shares similarity with the animal ortholog that was important to discover its encoding gene. In animal cells, the MICU1 and MICU2 proteins act as Ca<sup>2+</sup> sensors and gatekeepers of the MCU, preventing Ca<sup>2+</sup> uptake under resting conditions and favoring it at high cytosolic Ca<sup>2+</sup> concentrations ([Ca<sup>2+</sup>]<sub>cyt</sub>). Using the CRISPR/Cas9 technique, we generated TcMICU1 and TcMICU2 knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low [Ca<sup>2+</sup>]<sub>cyt</sub> but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations.
<small>Copyright © 2019 Bertolini et al.</small>
Background: Our aim is to highlight the benefits and limitations of open and non-anonymized peer review. Our argument is based on the literature and on responses to a survey on the reviewing process of alt.chi, a more or less open review track within the so-called Computer Human Interaction (CHI) conference, the predominant conference in the field of human-computer interaction. This track currently is the only implementation of an open peer review process in the field of human-computer interaction while, with the recent increase in interest in open scientific practices, open review is now being considered and used in other fields.
Methods: We ran an online survey with 30 responses from alt.chi authors and reviewers, collecting quantitative data using multiple-choice questions and Likert scales. Qualitative data were collected using open questions.
Results: Our main quantitative result is that respondents are more positive to open and non-anonymous reviewing for alt.chi than for other parts of the CHI conference. The qualitative data specifically highlight the benefits of open and transparent academic discussions. The data and scripts are available on https://osf.io/vuw7h/, and the figures and follow-up work on http://tiny.cc/OpenReviews.
Conclusion: While the benefits are quite clear and the system is generally well-liked by alt.chi participants, they remain reluctant to see it used in other venues. This concurs with a number of recent studies that suggest a divergence between support for a more open review process and its practical implementation. +
In the last decade Open Science principles have been successfully advocated for and are being slowly adopted in different research communities. In response to the COVID-19 pandemic many publishers and researchers have sped up their adoption of Open Science practices, sometimes embracing them fully and sometimes partially or in a sub-optimal manner. In this article, we express concerns about the violation of some of the Open Science principles and its potential impact on the quality of research output. We provide evidence of the misuses of these principles at different stages of the scientific process. We call for a wider adoption of Open Science practices in the hope that this work will encourage a broader endorsement of Open Science principles and serve as a reminder that science should always be a rigorous process, reliable and transparent, especially in the context of a pandemic where research findings are being translated into practice even more rapidly. We provide all data and scripts at https://osf.io/renxy/ . +
Both positive and negative (null or neutral) results are essential for the progress of science and its self-correcting nature. However, there is general reluctance to publish negative results, and this may be due a range of factors (e.g., the widely held perception that negative results are more difficult to publish, the preference to publish positive findings that are more likely to generate citations and funding for additional research). It is particularly challenging to disclose negative results that are not consistent with previously published positive data, especially if the initial publication appeared in a high impact journal. Ideally, there should be both incentives and support to reduce the costs associated with investing efforts into preparing publications with negative results. We describe here a set of criteria that can help scientists, reviewers and editors to publish technically sound, scientifically high-impact negative (or null) results originating from rigorously designed and executed studies. Proposed criteria emphasize the importance of collaborative efforts and communication among scientists (also including the authors of original publications with positive results). +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]]
Amiodarone is one of the most commonly prescribed anti-arrhythmic drugs in the management of both supraventricular and ventricular arrhythmias [1]. Despite its beneficial properties, several adverse effects have been classically reported. Mitochondrial dysfunction has recently emerged as a major pathomechanism underlying amiodarone toxicity [2]. Studies conducted in mouse liver, hamster lung and human hepatocytes cells reported that amiodarone mitochondrial toxicity was caused by an impairment of Complexes I and II of respiratory chain [2, 3]. Succinate is a Complex II substrate that potentially can alleviate such metabolic dysfunction; however, it does not readily permeate the cell membrane. Recently, cell-permeable succinate prodrugs have been developed in order to bypass mitochondrial Complex I deficiency of different etiology [4].
The present study was purported to assess the effects of amiodarone on mitochondrial respiration in intact and permeabilized human platelets in the presence vs. the absence of NV118, a cell-permeable succinate prodrug. To this aim peripheral blood platelets were isolated from healthy volunteers by differential centrifugations (using K<sub>2</sub>-EDTA as anticoagulant) and exposed to increasing concentrations of amiodarone in the presence vs. absence of NV118; the compound was generously provided by NeuroVive Pharmaceutical AB (Lund, Sweden), also available as the MitoKit-CII from Oroboros Instruments GmbH, Innsbruck, Austria. Respiratory capacities of intact and digitonin-permeabilized platelets (200 x 10<sup>6</sup> cells/mL) were analyzed using the substrate-uncoupler-inhibitor titration protocol.
In both intact and permeabilized cells, amiodarone elicited a dose-dependent (15-240 μM) inhibition of both NADH- and succinate-linked respiration. The addition of the cell-permeable succinate prodrug normalized the respiratory capacity at the electron transport system.
Amiodarone induced an acute inhibition of respiration in human non-cultured cells that was alleviated by cell-permeable succinate. Whether these results (drug effect and treatment response) can be seen in platelets isolated from patients chronically treated with amiodarone warrants further investigation.
Mitochondria are central organelles in the homeostasis of the cardiovascular system via the integration of several physiological processes, such as ATP generation via oxidative phosphorylation, synthesis/exchange of metabolites, calcium sequestration, reactive oxygen species (ROS) production/buffering and control of cellular survival/death. Mitochondrial impairment has been widely recognized as a central pathomechanism of almost all cardiovascular diseases, rendering these organelles important therapeutic targets. Mitochondrial dysfunction has been reported to occur in the setting of drug-induced toxicity in several tissues and organs, including the heart. Members of the drug classes currently used in the therapeutics of cardiovascular pathologies have been reported to both support and undermine mitochondrial function. For the latter case, mitochondrial toxicity is the consequence of drug interference (direct or off-target effects) with mitochondrial respiration/energy conversion, DNA replication, ROS production and detoxification, cell death signaling and mitochondrial dynamics. The present narrative review aims to summarize the beneficial and deleterious mitochondrial effects of common cardiovascular medications as described in various experimental models and identify those for which evidence for both types of effects is available in the literature. +
In non-thermoregulating and sessile organisms, such as the imperiled freshwater mussels (Bivalvia: Unionida), thermal sensitivity of mitochondria is a key factor for survival to global warming. Given the protected status of many unionids, non-destructive biopsies and subsequent cryopreservation are advisable procedures for further investigation of their mitochondrial function. To address whether long-term cryopreservation affects mitochondria in freshwater mussels, the mitochondrial respiration in permeabilized somatic cells of ''Elliptio complanata'' has been fully characterized through high-resolution respirometry. Our results indicate that cryopreservation does affect the absolute rate of respiration, which significantly decrease compared to fresh tissues, independently of substrates combination, respiratory state and normalizing factor. However, the negative impact is not reflected at the level of flux control ratios, suggesting that, even in front of a sharp decline in the aerobic capacity, cryopreserved tissues preserve the mitochondrial organization and could be thus employed for the qualitative analysis of mitochondrial function.
<small>Copyright © 2019. Published by Elsevier Inc.</small> +
Mitochondria produce energy through oxidative phosphorylation (OXPHOS), which depends on the expression of both nuclear and mitochondrial DNA (mtDNA). In metazoans, a striking exception from strictly maternal inheritance of mitochondria is doubly uniparental inheritance (DUI). This unique system involves the maintenance of two highly divergent mtDNAs (F- and M-type, 8–40% of nucleotide divergence) associated with gametes, and occasionally coexisting in somatic tissues. To address whether metabolic differences underlie this condition, we characterized the OXPHOS activity of oocytes, spermatozoa, and gills of different species through respirometry. DUI species express different gender-linked mitochondrial phenotypes in gametes and partly in somatic tissues. The M-phenotype is specific to sperm and entails (i) low coupled/uncoupled respiration rates, (ii) a limitation by the phosphorylation system, and (iii) a null excess capacity of the final oxidases, supporting a strong control over the upstream complexes. To our knowledge, this is the first example of a phenotype resulting from direct selection on sperm mitochondria. This metabolic remodelling suggests an adaptive value of mtDNA variations and we propose that bearing sex-linked mitochondria could assure the energetic requirements of different gametes, potentially linking male-energetic adaptation, mitotype preservation and inheritance, as well as resistance to both heteroplasmy and ageing. +
Despite playing a key role in energy metabolism, mitochondria are uniquely exposed to perturbation because their functions depend on the correct interaction between two distinct genomes, the mitochondrial and the nuclear DNA. Even mild incompatibilities between the two genomes could impact mitochondrial functions with downstream repercussions on individual fitness. Climate change predictions estimate an increase in temperature and its variability, changes in food web structures, but also in the distribution of populations. Events that may generate mitonuclear mismatches (e.g. hybridization between separate populations) are therefore expected to increase in frequency following the shifts in thermal niches. In addition, temperature and dietary regimes are well-known metabolic stressors whose variation can potentially exacerbate mitonuclear incompatibilities. The aim of this research was to test how far mild mitonuclear variations, of the kind that could be found in natural populations following introgression in shifting niches, might affect organismal fitness.
I employed four experimental lines of the fruitfly ''Drosophila melanogaster'', characterized by mitonuclear match or mismatch, to investigate the impact of mitonuclear genotype over a wide array of phenotypic traits, including mitochondrial functions, reactive oxygen species metabolism and life-history traits.
Results indicate a general trade-off between bioenergetic capacity and fecundity in mitonuclear mismatched lines, most prominently in females than males. Cybrids tend to have a higher OXPHOS activity compared to the matched parental populations, as well as generally lower H<sub>2</sub>O<sub>2</sub> efflux. The high mitochondrial respiration rate also links with a trend of higher locomotor activity, but with lower fecundity parameters. Finally, differences in thermal tolerance also exist, but link solely to the nuclear component, and not to the mitonuclear combination. Overall, results suggest that mitonuclear interactions might impact organismal fitness in an unpredictable way, potentially influencing local adaptation in a mutating world.
Mitochondrial function has been predominantly measured ''ex vivo''. Due to isolation and preservation procedures ''ex vivo'' measurements might misrepresent ''in vivo'' mitochondrial conditions. Direct measurement of ''in vivo'' mitochondrial oxygen tension (mitoPO2) and oxygen disappearance rate (ODR) with the protoporphyrin IX-triplet state lifetime technique (PpIX-TSLT) might increase our understanding of mitochondrial dysfunction in the pathophysiology of acute disease. LPS administration decreased mitochondrial respiration (ODR) ''in vivo'' but did not alter mitochondrial function as assessed with ''ex vivo'' techniques (high resolution respirometry and specific complex determinations). PpIX-TSLT measures ''in vivo'' mitoPO2 and ODR and can be applied non-invasively at the skin.
<small>Copyright © 2019. Published by Elsevier B.V.</small> +
Mitochondria provide energy in form of ATP in eukaryotic cells. However, it is not known when, during embryonic cardiac development, mitochondria become able to fulfill this function. To assess this, we measured mitochondrial oxygen consumption and the activity of the complexes (Cx) 1 and 2 of the electron transport chain (ETC) and used immunoprecipitation to follow the generation of mitochondrial supercomplexes. We show that in the heart of mouse embryos at embryonic day (E) 9.5, mitochondrial ETC activity and oxidative phosphorylation (OXPHOS) are not coupled, even though the complexes are present. We show that Cx-1 of the ETC is able to accept electrons from the Krebs cycle, but enzyme assays that specifically measure electron flow to ubiquinone or Cx-3 show no activity at this early embryonic stage. At E11.5, mitochondria appear functionally more mature; ETC activity and OXPHOS are coupled and respond to ETC inhibitors. In addition, the assembly of highly efficient respiratory supercomplexes containing Cx-1, -3, and -4, ubiquinone, and cytochrome c begins at E11.5, the exact time when Cx-1 becomes functional activated. At E13.5, ETC activity and OXPHOS of embryonic heart mitochondria are indistinguishable from adult mitochondria. In summary, our data suggest that between E9.5 and E11.5 dramatic changes occur in the mitochondria of the embryonic heart, which result in an increase in OXPHOS due to the activation of complex 1 and the formation of supercomplexes. +
In the past three decades the consumption of fructose, in the form of high fructose corn syrup (HFCS), has increased considerably with a concurrent rise in obesity and insulin resistance (IR) [1], however the exact mechanisms remain unclear. Evidence suggests elevated reactive oxygen species (ROS) production
and down regulation of Sirt3 to be a likely cause [2]. Sirt3 a key regulator
of the enzymes involved in energy homeostasis, has been shown to be
associated with the development of these diseases [3]. Sirt3 expression
is known to be regulated by the binding of the estrogen related receptor
alpha (ERR) on the Sirt3 estrogen related receptor binding element (ERRE) and up-regulated by exercise, which has been shown to protect
against IR [4]. We therefore hypothesize that Sirt3 down regulation
in high fructose feeding is due to increased ROS production, and is
associated with hypoacetylation of the histones at the ERRE of the Sirt3
gene. Furthermore we propose that exercise will increase the expression
of Sirt3 through hyperacetylation of the histones at the same promoter
region and enhance antioxidant capacity to restore Sirt3 expression
and mitochondrial function. . Adult male Wistar rats (200-300g; 90
days old), will be divided into four groups, control (10% glucose), HFCS
(8%), exercise (10% glucose) and exercise (8% HFCS). All groups will
have ad libitum access to standard rat chow and drinking water and
the sugars administered in the drinking water for a period of 8 weeks.
Hereafter the exercise groups will undergo high-intensity intermittent
swimming exercises. Western blot analysis and RT-PCR will be used
to determine the levels of expression of Sirt3 and various antioxidant
enzymes. Mitochondrial respiration, with pyruvate/malate, glutamate/
malate, malate/octanoyl carnitine as substrates, will be assessed to
determine mitochondrial dysfunction with regard to glucose and fatty
acid metabolism, using the Oroboros Oxygraph-2k. Additionally mitochondrial
ROS levels will be determined with the use of immunohistochemistry
and fluorometry. Binding of the estrogen related receptor to ERRE
will be measured using chromatin immunoprecipitation and nuclease
accessibility assay techniques.
It has been reported that the oxygen (O<sub>2</sub>) dependence of mitochondrial respiration in permeabilized fibers (Pfi) is ~100-fold higher compared to isolated mitochondria [1]. Pfi are sensitive to low oxygen supply due to diffusion restrictions that limit the supply of oxygen to the core of the fiber bundle. Therefore, hyperoxic conditions (oxygen range 400-250 μM) have been employed to counteract this limitation. Further studies have shown that the addition of a myosin II-specific inhibitor, blebbistatin (BLEB) in the respiration medium reduces this sensitivity and allows the study of ADP kinetics in Pfi at normoxic oxygen levels (250-200 μM). Moreover, it has been described that the use of BLEB prevents fiber contraction and yields high Km values for ADP [2].
In the present study, high-resolution respirometry (HRR) was used to test ADP respiratory kinetics in skeletal muscle Pfi from wild-type C57BL/6 mice under three different oxygen regimens: hypoxia (100-80 μM), normoxia (250-200 μM) and hyperoxia (400 μM) comparing two respiration media: Buffer Z with catalase (Ctl), creatine (Cr) and BLEB (BufferZCtlCr+BLEB) and MiR06+Cr (MiR06Cr) at a physiological temperature of 37 °C. Data were normalized to wet weight (''W''w) (mg of tissue), complex IV (COX) and citrate synthase (CS) activities.
In our experiments, we found no differences in O<sub>2</sub> flux when comparing rates determined in Buffer ZCtlCr + BLEB and MiR06Cr. However, our results do show that different O<sub>2</sub> regimens such as hypoxic and normoxic conditions in the O2k-chamber may lead to a relevant underestimation of OXPHOS capacity due to the oxygen limitation in both media (BufferZCtlCr+BLEB and MiR06Cr). It would therefore appear that, in our hands, the use of BufferZCtlCr with the addition of BLEB does not alleviate O<sub>2</sub> dependency in permeabilized skeletal muscle fibers. +
Permeabilized muscle fibers is the most widely used sample preparation technique when assessing mitochondrial function in skeletal and cardiac muscle tissue. A cooling plate was developed in an attempt to standardize mechanical separation of fibers, through close regulation of temperature and optimization of visualization. Here, we wanted to investigate if using the cooling plate would influence mitochondrial function.
Mitochondrial oxygen consumption was measured using high resolution respirometry (HRR, Oroboros Oxygraph-2K) in skeletal muscle permeabilized fibers from adult male mice (1.5-2.5 mg tissue/2 ml chamber). MiR05 + catalase + creatine (MiR06Cr) respiration medium was used, the oxygen (O<sub>2</sub>) range was maintained between 400-250 µM, and mitochondrial respiratory capacities were determined using a SUIT (substrate-uncoupler-inhibitor-titrations) protocol. Data were normalized to wet weight (''W''w) (mg tissue), complex IV (COX) and citrate synthase (CS) activities.
No significant differences were found in any of the respiratory states determined with fibers prepared on ice versus the cooling plate when data were normalized using ''W''w and COX activity, whereas data normalized using CS activity were significantly different across all respiratory states between the groups when compared to data normalized to ''W''w and COX activity. The use of the cooling plate for preparing permeabilized fibers maintains mitochondrial function and it improves various practical aspects of mechanical separation. These improvements contributed to faster, more accurate sample preparation and increased picture quality. Further studies have been carried out using a combination of ice and the cooling plate, and suggestions have been made for improvements of the cooling plate to allow its sole use when preparing fibers. +
Mitochondria are dynamic organelles that play central roles in eukaryotic cellular energy metabolism. They harbor an electron transport chain (ETC) that couples electron transfer to the movement of protons across the mitochondrial inner membrane, forming an electrochemical gradient that captures chemical energy in the form of adenosine triphosphate (ATP). The biochemical and biophysical properties of the ETC have been studied in detail (1). On page 1306 of this issue, Vos et al. (2) report a new constituent of this chain. The authors show that vitamin K<sub>2</sub> is an electron carrier, suggesting this small organic molecule as a possible treatment for mitochondrial pathologies such as Parkinson’s disease and amyotrophic lateral sclerosis. +
Preprints, non-peer-reviewed drafts of manuscripts available on the Internet, have been used in conjunction with peer review and publication in journals in the physical sciences for almost 25 years. Recently, more scientists have been discussing whether preprints can play a similar role in biological and biomedical research. Here, I discuss my excitement and concerns about the role that preprints can play in disseminating research findings in the life sciences. +
The kidney requires a large number of mitochondria to remove waste from the blood and regulate fluid and electrolyte balance. Mitochondria provide the energy to drive these important functions and can adapt to different metabolic conditions through a number of signalling pathways (for example, mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) pathways) that activate the transcriptional co-activator peroxisome proliferator-activated receptor-γ co-activator 1α (PGC1α), and by balancing mitochondrial dynamics and energetics to maintain mitochondrial homeostasis. Mitochondrial dysfunction leads to a decrease in ATP production, alterations in cellular functions and structure, and the loss of renal function. Persistent mitochondrial dysfunction has a role in the early stages and progression of renal diseases, such as acute kidney injury (AKI) and diabetic nephropathy, as it disrupts mitochondrial homeostasis and thus normal kidney function. Improving mitochondrial homeostasis and function has the potential to restore renal function, and administering compounds that stimulate mitochondrial biogenesis can restore mitochondrial and renal function in mouse models of AKI and diabetes mellitus. Furthermore, inhibiting the fission protein dynamin 1-like protein (DRP1) might ameliorate ischaemic renal injury by blocking mitochondrial fission. +
Caseinolytic peptidase P (ClpP) is a mammalian quality control protease that is proposed to play an important role in the initiation of the mitochondrial unfolded protein response (UPR<sup>mt</sup>), a retrograde signaling response that helps to maintain mitochondrial protein homeostasis. Mitochondrial dysfunction is associated with the development of metabolic disorders, and to understand the effect of a defective UPR<sup>mt</sup> on metabolism, ClpP knockout (''ClpP<sup>-/-</sup>'') mice were analyzed. ''ClpP<sup>-/-</sup>'' mice fed ''ad libitum'' have reduced adiposity and paradoxically improved insulin sensitivity. Absence of ClpP increased whole-body energy expenditure and markers of mitochondrial biogenesis are selectively up-regulated in the white adipose tissue (WAT) of ''ClpP<sup>-/-</sup>'' mice. When challenged with a metabolic stress such as high-fat diet, despite similar caloric intake, ''ClpP<sup>-/-</sup>'' mice are protected from diet-induced obesity, glucose intolerance, insulin resistance, and hepatic steatosis. Our results show that absence of ClpP triggers compensatory responses in mice and suggest that ClpP might be dispensable for mammalian UPR<sup>mt</sup> initiation. Thus, we made an unexpected finding that deficiency of ClpP in mice is metabolically beneficial. +
Age-associated loss of muscle mass and function (sarcopenia) has a profound effect on the quality of life in the elderly. Our previous studies show that CuZnSOD deletion in mice (Sod1<sup>-/-</sup> mice) recapitulates sarcopenia phenotypes, including elevated oxidative stress and accelerated muscle atrophy, weakness, and disruption of neuromuscular junctions (NMJs). To determine whether deletion of Sod1 initiated in neurons in adult mice is sufficient to induce muscle atrophy, we treated young (2- to 4-month-old) Sod1flox/SlickHCre mice with tamoxifen to generate i-mn-Sod1KO mice. CuZnSOD protein was 40-50% lower in neuronal tissue in i-mn-Sod1KO mice. Motor neuron number in ventral spinal cord was reduced 28% at 10 months and more than 50% in 18- to 22-month-old i-mn-Sod1KO mice. By 24 months, 22% of NMJs in i-mn-Sod1KO mice displayed a complete lack of innervation and deficits in specific force that are partially reversed by direct muscle stimulation, supporting the loss of NMJ structure and function. Muscle mass was significantly reduced by 16 months of age and further decreased at 24 months of age. Overall, our findings show that neuronal-specific deletion of CuZnSOD is sufficient to cause motor neuron loss in young mice, but that NMJ disruption, muscle atrophy, and weakness are not evident until past middle age. These results suggest that loss of innervation is critical but may not be sufficient until the muscle reaches a threshold beyond which it cannot compensate for neuronal loss or rescue additional fibers past the maximum size of the motor unit. +
Background: Community transmission of coronavirus 2019 (Covid-19) was detected in the state of Washington in February 2020.
Methods: We identified patients from nine Seattle-area hospitals who were admitted to the intensive care unit (ICU) with confirmed infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Clinical data were obtained through review of medical records. The data reported here are those available through March 23, 2020. Each patient had at least 14 days of follow-up.
Results: We identified 24 patients with confirmed Covid-19. The mean (±SD) age of the patients was 64±18 years, 63 % were men, and symptoms began 7±4 days before admission. The most common symptoms were cough and shortness of breath; 50 % of patients had fever on admission, and 58 % had diabetes mellitus. All the patients were admitted for hypoxemic respiratory failure; 75 % (18 patients) needed mechanical ventilation. Most of the patients (17) also had hypotension and needed vasopressors. No patient tested positive for influenza A, influenza B, or other respiratory viruses. Half the patients (12) died between ICU day 1 and day 18, including 4 patients who had a do-not-resuscitate order on admission. Of the 12 surviving patients, 5 were discharged home, 4 were discharged from the ICU but remained in the hospital, and 3 continued to receive mechanical ventilation in the ICU.
Conclusions: During the first 3 weeks of the Covid-19 outbreak in the Seattle area, the most common reasons for admission to the ICU were hypoxemic respiratory failure leading to mechanical ventilation, hypotension requiring vasopressor treatment, or both. Mortality among these critically ill patients was high. (Funded by the National Institutes of Health.). +
Epithelial ovarian cancer is the leading cause of gynecologic cancer deaths. Most patients respond initially to platinum-based chemotherapy after surgical debulking, however relapse is very common and ultimately platinum resistance emerges. Understanding the mechanism of tumor growth, metastasis and drug resistant relapse will profoundly impact the therapeutic management of ovarian cancer.
Using patient tissue microarray (TMA), ''in vitro'' and ''in vivo'' studies we report a role of of cystathionine-beta-synthase (CBS), a sulfur metabolism enzyme in ovarian carcinoma. We report here that the expression of cystathionine-beta-synthase (CBS), a sulfur metabolism enzyme, is common in primary serous ovarian carcinoma. The ''in vitro'' effects of CBS silencing can be reversed by exogenous supplementation with the GSH and H2S producing chemical Na2S. Silencing CBS in a cisplatin resistant orthotopic model ''in vivo'' by nanoliposomal delivery of CBS siRNA inhibits tumor growth, reduces nodule formation and sensitizes ovarian cancer cells to cisplatin. The effects were further corroborated by immunohistochemistry that demonstrates a reduction of H&E, Ki-67 and CD31 positive cells in si-RNA treated as compared to scrambled-RNA treated animals. Furthermore, CBS also regulates bioenergetics of ovarian cancer cells by regulating mitochondrial ROS production, oxygen consumption and ATP generation. This study reports an important role of CBS in promoting ovarian tumor growth and maintaining drug resistant phenotype by controlling cellular redox behavior and regulating mitochondrial bioenergetics.
The present investigation highlights CBS as a potential therapeutic target in relapsed and platinum resistant ovarian cancer. +
Burn victims undergo prolonged immobilization and become profoundly cachectic after injury [1,2]. Loss of muscle mass and function leads to reduced quality of life in burn survivors. Rehabilitative exercise training (RET) has been shown to increase muscle mass and strength while also increasing peak oxygen uptake (peak ''V''<sub>O2</sub>) in burn survivors [2]. However, the mechanisms responsible for these improvements in physiological function are not fully understood. Here, we studied the impact of chronic RET on whole body (peak ''V''<sub>O2</sub>) and skeletal muscle ([[OXPHOS]]) oxidative capacity.
Fourteen children (7-17 years) with burns covering ≥30% of their total body surface area were studied. After discharge from hospital, patients performed a 6-week supervised RET program. RET consisted of both resistive and aerobic exercise performed at least 3-times weekly on non-consecutive days. Peak ''V''<sub>O2</sub> and muscle OXPHOS capacity were determined before and after the RET program. Peak ''V''<sub>O2</sub> was determined using a modified Bruce protocol treadmill test. Muscle OXPHOS capacity was determined in permeabilized muscle fibers by high-resolution respirometry.
Peak ''V''<sub>O2</sub> increased significantly after RET (33.0±2.2 vs. 28.1± 1.7 ml.min<sup>-1</sup>.kg<sup>-1</sup>; ''P''<0.001). Muscle OXPHOS capacity increased by 43% after RET (46.7±4.5 vs. 32.5±4.9 pmol.s<sup>-1</sup>.mg<sup>-1</sup>, ''P''<0.05).
In agreement with the published literature, RET increases aerobic exercise capacity in burn survivors. Here, we show that this is accompanied by an increase in skeletal muscle OXPHOS capacity. We suggest that RET improves whole body physiological function in burn survivors at least part, by improving skeletal muscle OXPHOS capacity. +
COPD represents a major global health issue, which is often accompanied by cardiovascular diseases. A considerable body of evidence suggests that cardiovascular risk is elevated by the activation of blood platelets, which in turn is exacerbated by inflammation. As reactive oxygen species are believed to be an important factor in platelet metabolism and functioning, the aim of our study was to perform a complex assessment of mitochondrial function in platelets in chronic smoke exposed animals with COPD-like lung lesions.
Eight-week-old, male Dunkin Hartley guinea pigs (the study group) were exposed to the cigarette smoke from commercial unfiltered cigarettes (0.9 mg/cig of nicotine content) or to the air without cigarette smoke (control group), using the Candela Constructions® exposure system. The animals were exposed for 4 hours daily, 5 days a week, with 2×70 mL puff/minute, until signs of dyspnea were observed. The animals were bled, and isolated platelets were used to monitor blood platelet respiration. The mitochondrial respiratory parameters of the platelets were monitored ''in vitro'' based on continuous recording of oxygen consumption by high-resolution respirometry.
An elevated respiration trend was observed in the LEAK-state (adjusted for number of platelets) in the smoke-exposed animals: 6.75 (5.09) vs 2.53 (1.28) (pmol O<sub>2</sub>/[s · 1×10<sup>8</sup> platelets]); bootstrap-boosted P<sub>1α</sub>=0.04. The study group also demonstrated lowered respiration in the ET-state (normalized for protein content): 12.31 (4.84) vs 16.48 (1.72) (pmol O<sub>2</sub>/[s · mg of protein]); bootstrap-boosted P<sub>1α</sub>=0.049.
Our results suggest increased proton and electron leak and decreased electron transfer system capacity in platelets from chronic smoke-exposed animals. These observations may also indicate that platelets play an important role in the pathobiology of COPD and its comorbidities and may serve as a background for possible therapeutic targeting. However, these preliminary outcomes should be further validated in studies based on larger samples.
The invasion and metastasis of malignant tumors are major causes of death. The most common metastases of cancer are lymphatic metastasis and hematogenous metastasis. Hematogenous metastasis often leads to rapid tumor dissemination. The mechanism of hematogenous metastasis of malignant tumors is very complex. Some experts have found that platelets play an important role in promoting tumor hematogenous metastasis. Platelets may be involved in many processes, such as promoting tumor cell survival, helping tumor cells escape immune surveillance, helping tumors attach to endothelial cells and penetrating capillaries for distant metastasis. However, recent studies have shown that platelets can also inhibit tumor metastasis. At present, the function of platelets in tumor progression has been widely studied, and they not only promote tumor cell metastasis, but also have an inhibitory effect. Therefore, in-depth and summary research of the molecular mechanism of platelets in tumor cell metastasis is of great significance for the screening and treatment of cancer patients. The following is a brief review of the role of platelets in the process of malignant tumor metastasis. +
Metabolic flux control analysis of NADH oxidation in bovine heart submitochondrial particles revealed high flux control coefficients for both Complex I and Complex III, suggesting that the two enzymes are functionally associated as a single enzyme, with channelling of the common substrate, Coenzyme Q. This is in contrast with the more accepted view of a mobile diffusable Coenzyme Q pool between these enzymes. Dilution with phospholipids of a mitochondrial fraction enriched in Complexes I and III, with consequent increased theoretical distance between complexes, determines adherence to pool behavior for Coenzyme Q, but only at dilution higher than 1:5 (protein:phospholipids), whereas, at lower phospholipid content, the turnover of NADH cytochrome c reductase is higher than expected by the pool equation. +
The model of the respiratory chain in which the enzyme complexes are independently embedded in the lipid bilayer of the inner mitochondrial membrane and connected by randomly diffusing coenzyme Q and cytochrome c is mostly favored. However, multicomplex units can be isolated from mammalian mitochondria, suggesting a model based on direct electron channeling between complexes. Kinetic testing using metabolic flux control analysis can discriminate between the two models: the former model implies that each enzyme may be rate-controlling to a different extent, whereas in the latter, the whole metabolic pathway would behave as a single supercomplex and inhibition of any one of its components would elicit the same flux control. In particular, in the absence of other components of the oxidative phosphorylation apparatus (i.e. ATP synthase, membrane potential, carriers), the existence of a supercomplex would elicit a flux control coefficient near unity for each respiratory complex, and the sum of all coefficients would be well above unity. Using bovine heart mitochondria and submitochondrial particles devoid of substrate permeability barriers, we investigated the flux control coefficients of the complexes involved in aerobic NADH oxidation (I, III, IV) and in succinate oxidation (II, III, IV). Both Complexes I and III were found to be highly rate-controlling over NADH oxidation, a strong kinetic evidence suggesting the existence of functionally relevant association between the two complexes, whereas Complex IV appears randomly distributed. Moreover, we show that Complex II is fully rate-limiting for succinate oxidation, clearly indicating the absence of substrate channeling toward Complexes III and IV. +
Natural products are a valuable source of new molecules and are important for drug discovery. Many chemotherapeutics currently in clinical use were originated from natural sources and are effective cytotoxic agents. In this study, we investigated the cytotoxic and pro-apoptotic effects of achyrobichalcone (ACB) and 3-O-methylquercetin (3OMQ), two novel compounds isolated from the ''Achyrocline satureioides'' plant. Because extracts from this plant have been shown to have anticancer activity ''in vitro'', we evaluated ACB and 3OMQ using a human breast cancer cell line, MDA-MB-231, and a nontumorigenic human breast epithelial cell line, MCF-12A. We found that ACB demonstrates cytotoxic effects on MDA-MB-231 cells, but not MCF-12A cells. 3OMQ also demonstrated cytotoxic effects on MDA-MB-231 cells, but with lower selectivity compared to treated MCF-12A cells. Cell death by both compounds was associated with caspase-9 and caspase-3/7 activation. Using high-resolution respirometry, we found that ACB and 3OMQ were able to cause acute mitochondrial dysfunction in MDA-MB-231-treated cells. These results suggest that apoptosis in MDA-MB-231 cells is induced through the activation of the mitochondrial-dependent pathway. Collectively, these findings suggest that ACB is a strong candidate for further anticancer ''in vivo'' tests. +
In unilateral ureteral obstruction (UUO), both oxidative stress and mitochondrial dysfunction are related to cell death. The aim of this study has been to characterize profiles of enzyme antioxidant activities and mitochondrial functioning of the contralateral (CL) compared to UUO and Sham (false-operated) kidneys of Balb/c mice. Kidneys were resected 14 days after obstruction for immunohistochemical and cortical mitochondrial functioning assays. Antioxidant enzymes activities were investigated in mitochondria and cytosol. Oxygen consumption (QO<sub>2</sub>) and formation of O<sub>2</sub> reactive species (ROS) were assessed with pyruvate plus malate or succinate as the respiratory substrates. QO<sub>2</sub> decreased in CL and UUO in all states using substrates for complex II, whereas it was affected only in UUO when substrates for complex I were used. Progressive decrease in mitochondrial ROS formation-in the forward and reverse pathway at complex I-correlates well with the inhibition of QO<sub>2</sub> and, therefore, with decreased electron transfer at the level of complexes upstream of cytochrome c oxidase. CL and UUO transmembrane potential responses to ADP were impaired with succinate. Intense Ca<sup>2+</sup>-induced swelling was elicited in CL and UUO mitochondria. Important and selective differences exist in CL antioxidant enzymes with respect to either Sham or UUO kidneys: CL kidneys had increased mitochondrial glutathione peroxidase and cytosolic catalase activities, indicative of compensatory responses in the face of an early altered ROS homeostasis (as detected by 4-hydroxynonenal), and of a significant tendency to apoptosis. In CL and UUO, upregulation of nuclear (erythroid-derived 2)-like 2 transcription factor (Nrf2), as well as of cytoplasmic and nuclear Kelch-like ECH-associated protein 1 (Keap1) in opposition to decreased heme oxygenase-1 (HO-1), suggest impairment of the Nrf2/Keap1/HO-1 system. It is concluded that chronic obstruction impairs mitochondrial function in CL and UUO, preferentially affecting complex II.
Hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) belongs to the reactive oxygen species (ROS), known as oxidants that can react with various cellular targets thereby causing cell damage or even cell death. On the other hand, recent work has demonstrated that H2O2 also functions as a signalling molecule controlling different essential processes in plants and mammals. Because of these opposing functions the cellular level of H<sub>2</sub>O<sub>2</sub> is likely to be subjected to tight regulation via processes involved in production, distribution and removal. Substantial progress has been made exploring the formation and scavenging of H2O2, whereas little is known about how this signal molecule is transported from its site of origin to the place of action or detoxification. From work in yeast and bacteria it is clear that the diffusion of H<sub>2</sub>O<sub>2</sub> across membranes is limited. We have now obtained direct evidence that selected aquaporin homologues from plants and mammals have the capacity to channel H<sub>2</sub>O<sub>2</sub> across membranes. The main focus of this review is (i) to summarize the most recent evidence for a signalling role of H<sub>2</sub>O<sub>2</sub> in various pathways in plants and mammals and (ii) to discuss the relevance of specific transport of H<sub>2</sub>O<sub>2</sub>. +
BACKGROUND: Hypoxia/reoxygenation (H/R) of proximal tubules leads to persistent ATP depletion due to decreased mitochondrial membrane potential (MMP) resulting from nonesterified fatty acid (NEFA)-mediated uncoupling that is paradoxically accompanied by respiratory inhibition rather than the stimulation expected for uncoupled states.
METHODS: Since NEFA have been reported to directly inhibit electron transport in some settings we assessed respiratory function in isolated, permeabilized rabbit tubules after H/R as a function of NEFA availability.
RESULTS: Compared to respiration supported by the complex II-dependent substrate, succinate, which was highly uncoupled after H/R but relatively well preserved (ADP-stimulated respiration (S3) of permeabilized tubules 71.0±8.5% of normoxic control (NC)), respiration supported by complex I-dependent substrates that normally predominate in cells was also uncoupled, but S3 was reduced to 26.9±3.3% of NC, P < 0.001 vs. succinate, N=5. With complex I substrates, acutely lowering NEFA after permeabilization improved coupling but only minimally increased S3. In contrast, lowering NEFA during 60 min. of reoxygenation prior to permeabilization increased S3 supported by complex I substrates, but it remained lower (55.7±7.5% of NC) than with succinate after the same treatment, 80.0±4.8%, p < 0.02. MMP at the end of H/R was much lower with complex I substrates (30.7±9.2% NC) than with succinate (67.4±4.5%), P < 0.004. Lowering NEFA during 60 min. of reoxygenation strongly improved recovery and decreased the MMP difference between complex I substrates (73.3±5.1% of NC) and succinate (83.4±6.6%).
CONCLUSION: The studies indicate that selectively impaired utilization of complex I substrates to support respiration after H/R promotes NEFA-induced deenergization and is only minimally improved by acutely removing NEFA. In the presence of NEFA, the higher efficiency of complex I substrates to support electron transport does not mitigate the impact of the impaired respiration on MMP. However, lowering NEFA within cells for 60 min. allows strong recovery of MMP despite persistence of some respiratory impairment.
Inaccurate data in scientific papers can result from honest error or intentional falsification. This study attempted to determine the percentage of published papers that contain inappropriate image duplication, a specific type of inaccurate data. The images from a total of 20,621 papers published in 40 scientific journals from 1995 to 2014 were visually screened. Overall, 3.8% of published papers contained problematic figures, with at least half exhibiting features suggestive of deliberate manipulation. The prevalence of papers with problematic images has risen markedly during the past decade. Additional papers written by authors of papers with problematic images had an increased likelihood of containing problematic images as well. As this analysis focused only on one type of data, it is likely that the actual prevalence of inaccurate data in the published literature is higher. The marked variation in the frequency of problematic images among journals suggests that journal practices, such as prepublication image screening, influence the quality of the scientific literature. +
Adipocyte mitochondrial respiration may influence metabolic fuel partitioning into oxidation versus storage, with implications for whole-body energy expenditure. Although insulin has been shown to influence mitochondrial respiration, the effects of dietary macronutrient composition have not been well characterized. The aim of this exploratory study was to test the hypothesis that a high-carbohydrate diet lowers the oxygen flux of adipocyte mitochondria ''ex vivo''. Among participants in a randomized-controlled weight-loss maintenance feeding trial, those consuming a high-carbohydrate diet (60% carbohydrate as a proportion of total energy, n = 10) had lower rates of maximal adipose tissue mitochondrial respiration than those consuming a moderate-carbohydrate diet (40%, n = 8, p = 0.039) or a low-carbohydrate diet (20%, n = 9, p = 0.005) after 10 to 15 weeks. This preliminary finding may provide a mechanism for postulated calorie-independent effects of dietary composition on energy expenditure and fat deposition, potentially through the actions of insulin on fuel partitioning. +
Background: The ratio of NAD(+)/NADH is a key indicator that reflects the overall redox state of the cells. Until recently, there were no methods for real time NAD(+)/NADH monitoring in living cells. Genetically encoded fluorescent probes for NAD(+)/NADH are fundamentally new approach for studying the NAD(+)/NADH dynamics.
Methods: We developed a genetically encoded probe for the nicotinamide adenine dinucleotide, NAD(H), redox state changes by inserting circularly permuted YFP into redox sensor T-REX from Thermus aquaticus. We characterized the sensor in vitro using spectrofluorometry and in cultured mammalian cells using confocal fluorescent microscopy.
Results: The sensor, named RexYFP, reports changes in the NAD(+)/NADH ratio in different compartments of living cells. Using RexYFP, we were able to track changes in NAD(+)/NADH in cytoplasm and mitochondrial matrix of cells under a variety of conditions. The affinity of the probe enables comparison of NAD(+)/NADH in compartments with low (cytoplasm) and high (mitochondria) NADH concentration. We developed a method of eliminating pH-driven artifacts by normalizing the signal to the signal of the pH sensor with the same chromophore.
Conclusion: RexYFP is suitable for detecting the NAD(H) redox state in different cellular compartments.
General significance: RexYFP has several advantages over existing NAD(+)/NADH sensors such as smallest size and optimal affinity for different compartments. Our results show that normalizing the signal of the sensor to the pH changes is a good strategy for overcoming pH-induced artifacts in imaging. +
Everything we have gained by opening content and data will be under threat if we allow the enclosure of scholarly infrastructures. We propose a set of principles by which Open Infrastructures to support the research community could be run and sustained.
What should a shared infrastructure look like? Infrastructure at its best is invisible. We tend to only notice it when it fails. If successful, it is stable and sustainable. Above all, it is trusted and relied on by the broad community it serves. Trust must run strongly across each of the following areas: running the infrastructure (governance), funding it (sustainability), and preserving community ownership of it (insurance). In this spirit, we have drafted a set of design principles we think could support the creation of successful shared infrastructures. +
The NLRP3 inflammasome is linked to sterile and pathogen-dependent inflammation, and its dysregulation underlies many chronic diseases. Mitochondria have been implicated as regulators of the NLRP3 inflammasome through several mechanisms including generation of mitochondrial reactive oxygen species (ROS). Here, we report that mitochondrial electron transport chain (ETC) complex I, II, III and V inhibitors all prevent NLRP3 inflammasome activation. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI1) or Ciona intestinalis alternative oxidase, which can complement the functional loss of mitochondrial complex I or III, respectively, without generation of ROS, rescued NLRP3 inflammasome activation in the absence of endogenous mitochondrial complex I or complex III function. Metabolomics revealed phosphocreatine (PCr), which can sustain ATP levels, as a common metabolite that is diminished by mitochondrial ETC inhibitors. PCr depletion decreased ATP levels and NLRP3 inflammasome activation. Thus, the mitochondrial ETC sustains NLRP3 inflammasome activation through PCr-dependent generation of ATP, but via a ROS-independent mechanism. +
The Zn<sup>2+</sup> in cardiomyocytes is buffered by structures near T-tubulus and/or sarcoplasmic/endoplasmic reticulum (S(E)R) while playing roles as either an antioxidant or a toxic agent, depending on the concentration. Therefore, we aimed first to examine a direct effect of ZnPO<sub>4</sub> (extracellular exposure) or Zn<sup>2+</sup> pyrithione (ZnPT) (intracellular exposure) application on the structure of the mitochondrion in ventricular cardiomyocytes by using histological investigations. The light microscopy data demonstrated that Zn<sup>2+</sup> exposure induced marked increases on cellular surface area, an indication of hypertrophy, in a concentration-dependent manner. Furthermore, a whole-cell patch-clamp measurement of cell capacitance also supported the hypertrophy in the cells. We observed marked increases in mitochondrial matrix/cristae area and matrix volume together with increased lysosome numbers in ZnPO<sub>4-</sub> or ZnPT-incubated cells by using transmission electron microscopy, again in a concentration-dependent manner. Furthermore, we observed notable clustering and vacuolated mitochondrion, markedly disrupted and damaged myofibrils, and electron-dense small granules in Zn<sup>2+</sup>-exposed cells together with some implications of fission-fusion defects in the mitochondria. Moreover, we observed marked depolarization in mitochondrial membrane potential during 1-μM ZnPT minute applications by using confocal microscopy. We also showed that 1-μM ZnPT incubation induced significant increases in the phosphorylation levels of GSK3β (Ser21 and Ser9), Akt (Ser473), and NFκB (Ser276 and Thr254) together with increased expression levels in ER stress proteins such as GRP78 and calregulin. Furthermore, a new key player at ER-mitochondria sites, promyelocytic leukemia protein (PML) level, was markedly increased in ZnPT-incubated cells. As a summary, our present data suggest that increased cytosolic free Zn<sup>2+</sup> can induce marked alterations in mitochondrion morphology as well as depolarization in mitochondrion membrane potential and changes in some cytosolic signaling proteins as well as a defect in ER-mitochondria cross talk.
Preeclampsia (PE) is a major complication of pregnancy with partially elucidated pathophysiology. Placental mitochondrial dysfunction has been increasingly studied as major pathomechanism in both early- and late-onset PE. Impairment of mitochondrial respiration in platelets has recently emerged as a peripheral biomarker that may mirror organ mitochondrial dysfunction in several acute and chronic pathologies. The present study was purported to assess mitochondrial respiratory dys/function in both platelets and placental mitochondria in PE pregnancies. To this aim, a high-resolution respirometry SUIT (Substrate-Uncoupler-Inhibitor-Titration) protocol was adapted to assess complex I (glutamate + malate)- and complex II (succinate)-supported respiration. A decrease in all respiratory parameters (basal, coupled, and maximal uncoupled respiration) in peripheral platelets was found in preeclamptic as compared to healthy pregnancies. At variance, placental mitochondria showed a dichotomous behavior in preeclampsia in relation to the fetal birth weight. PE pregnancies with fetal growth restriction were associated with decreased in coupled respiration (oxidative phosphorylation/OXPHOS capacity) and maximal uncoupled respiration (electron transfer/ET capacity). At variance, these respiratory parameters were increased for both complex I- and II-supported respiration in PE pregnancies with normal weight fetuses. Large randomized controlled clinical studies are needed in order to advance our understanding of mitochondrial adaptive vs. pathological changes in preeclampsia. +
Malignant tumor cells exhibit mitochondrial alterations and are also influenced by biobehavioral processes, but the intersection of biobehavioral factors and mitochondria in malignant tumors remains unexplored. Here we examined multiple biochemical and molecular markers of mitochondrial content and function in benign tissue and in high-grade epithelial ovarian carcinoma (EOC) in parallel with exploratory analyses of biobehavioral factors. First, analysis of a publicly-available database (n = 1435) showed that gene expression of specific mitochondrial proteins in EOC is associated with survival. Quantifying multiple biochemical and molecular markers of mitochondrial content and function in tissue from 51 patients with benign ovarian masses and 128 patients with high-grade EOC revealed that compared to benign tissue, EOCs exhibit 3.3-8.4-fold higher mitochondrial content and respiratory chain enzymatic activities (P < 0.001) but similar mitochondrial DNA (mtDNA) levels (- 3.1%), documenting abnormal mitochondrial phenotypes in EOC. Mitochondrial respiratory chain activity was also associated with interleukin-6 (IL-6) levels in ascites. In benign tissue, negative biobehavioral factors were inversely correlated with mitochondrial content and respiratory chain activities, whereas positive biobehavioral factors tended to be positively correlated with mitochondrial measures, although effect sizes were small to medium (r = - 0.43 to 0.47). In contrast, serous EOCs showed less pronounced biobehavioral-mitochondrial correlations. These results document abnormal mitochondrial functional phenotypes in EOC and warrant further research on the link between biobehavioral factors and mitochondria in cancer. +
Our group at the University of Bern uses biochemical and biophysical techniques to unravel details of the molecular mechanism of membrane proteins. Of special interest are the large multi-subunit complexes of the universally conserved respiratory chain and the ATP synthase that are found in mitochondria and aerobic bacteria. In a bottom-up approach using purified membrane proteins and synthetic lipids, we aim to mimic the basic processes of oxidative phosphorylation. We further develop methodologies to increase the complexity of such artificial systems, paving the way for a synthetic mitochondrion. In this minireview, we summarize recent efforts of our groups and others towards a synthetic respiratory chain. +
Mitochondria, besides the key role in bioenergetics, carry out a lot of functions essential for cell viability, thus impairment of any of them can result in a wide spectrum of severe abnormalities in humans known as mitochondrial diseases. The diagnosis is difficult due to multiplicity of clinical manifestation depending on involved function and affected tissues. Additionally it is complicated by heteroplasmy of mitochondrial DNA (mtDNA) in human cells. The yeast ''Saccharomyces cerevisiae'' is the organism of choice to uncover cellular and molecular mechanisms underlying the mitochondrial diseases. The most important is the capability to use fermentable carbon substrates as energy source, resulting in ability to survive even when mtDNA has been completely depleted. What more, site-direct mutagenesis of yeast mtDNA is possible by biolistic transformation and the population of mutated mtDNA will be 100% homoplasmic.
ATP synthase is multi-subunit enzyme located in inner mitochondrial membrane. The enzyme uses the energy provided by the proton electrochemical gradient as a force to drive ATP synthesis. Point mutations in ATP6 gene were identified in patients suffering the neurological defects.
The mitochondrially encoded Atp6 subunit of ATP synthase is evolutionary conserved, therefore it is possible to create yeast models of human diseases bearing the particular pathogenic mutations for analysis of their consequences. Here we present the results of systematic investigation on cellular effects of 9 pathogenic mutations introduced to ATP6 gene of ''S. cerevisiae'' leading in human to Neurogenic Ataxia and Retinitis Pigmentosa (NARP), Leigh syndrome (LS), Charcot-Marie-Tooth (CMT), NARP or Familial Bilateral Striatal Necrosis (FBSN) syndromes.
Importantly, chemical screens of drugs using yeast have pointed to potential therapeutic targets. Through selection of intragenic revertants in respiratory deficient mutants of ATP6 gene, the identification of amino acids important for the mechanism of proton transport was possible. Thus from study of the pathogenic mutations yeast has brought us to the fundamental mechanism of the enzyme function.
'''Bioblast 2022: Inaugural Conference of ''Bioenergetics Communications''''' +
Bioenergetics - Art meets Gentle Science in Sickness and in Health. A [[Gentle Science]] project initiated by [[Iyer S |Shilpa Iyer]]. Science Museum of Virginia, Richmond VA, US. +
'''Bioenergetics Vienna''', 1st Bioenergetics DE-CH-AT Meeting +
MitoGlobal
Bioenergetika conference, Kacov, Czech Republic, 2020 +
Biophysical Society – Bioenergetics Subgroup Mini-Symposium, Baltimore, MD, USA +
α-Synucleinopathy and mitochondrial dysfunction are important elements of sporadic Parkinson’s disease (PD) pathogenesis [1]. It is, however, not clear whether the accumulated α-synuclein in degenerating dopaminergic neurons in PD causes mitochondrial injury and subsequent cell death. Our earlier study has shown that α-synuclein causes functional impairment of rat brain mitochondria incubated in vitro [2].
Mitochondrial membrane potential was measured using the carbocyanine dye JC1, and the phosphorylation capacity determined spectrophotometrically from inorganic phosphate utilization [2,3]. The respiratory functions of mitochondria in isolated preparations and within intact cells were analyzed by high-resolution respirometry. α-Synuclein accumulation within SHSY5Y cells was induced by lactacystin treatment and detected by immunoblotting. The transfection of SHSY5Y cells with α synuclein specific SiRNA was carried out using the lipofectamine kit (Invitrogen).
Our results show that α-synuclein causes a loss of membrane potential and phosphorylation capacity with alterations in respiratory parameters in isolated rat brain mitochondria. Some of these effects were inhibited very significantly by cyclosporine (1 μM). When SHSY5Y cells were exposed to 5 μM lactacystin for 24 h, α-synuclein accumulation occured intracellularly as detected by immunoblotting experiments. Further, lactacystin treatment of SHSY5Y cells also leads to mitochondrial dysfunction and cell death concomitant with α synuclein accumulation. To confirm the involvement of α synuclein in lactacystin induced mitochondrial dysfunction, the effects of cyclosporine and the gene silencing of α-synuclein with specific SiRNA on these phenomena are being investigated. +
Using fresh (and frozen?) patient: muscle, liver and fibroblasts, we aim to.
#Establish reference ranges
#Validate the technique in biopsies and cultured fibroblasts from patients with a confirmed inherited mitochondrial disorder and patients with non alcoholic liver steatosis AND
#Prospectively validate the assay in 150 patients suspected of a mitochondrial disorder
In parallel, we will test these samples for mitochondrial function using established protocols to measure ATP production, and respiratory chain enzyme activities, such that we can compare the diagnostic capabilities of these parallel diagnostic tools. +
The metabolic properties of lymphomas derived from germinal center (GC) B cells have important implications for therapeutic strategies. In this study, we have compared metabolic features of Hodgkin-Reed-Sternberg (HRS) cells, the tumor cells of classical Hodgkin's lymphoma (cHL), one of the most frequent (post-)GC-derived B-cell lymphomas, with their normal GC B cell counterparts. We found that the ratio of oxidative to nonoxidative energy conversion was clearly shifted toward oxidative phosphorylation (OXPHOS)-linked ATP synthesis in HRS cells as compared to GC B cells. Mitochondrial mass, the expression of numerous key proteins of oxidative metabolism and markers of mitochondrial biogenesis were markedly upregulated in cHL cell lines and in primary cHL cases. NFkappaB promoted this shift to OXPHOS. Functional analysis indicated that both cell growth and viability of HRS cells depended on OXPHOS. The high rates of OXPHOS correlated with an almost complete lack of lactate production in HRS cells not observed in other GC B-cell lymphoma cell lines. Overall, we conclude that OXPHOS dominates energy conversion in HRS cells, while nonoxidative ATP production plays a subordinate role. Our results suggest that OXPHOS could be a new therapeutic target and may provide an avenue toward new treatment strategies in cHL. +
Endoreplication, duplication of the nuclear genome without cell division, occurs in disease to drive morphologic growth, cell fate, and function. Despite its criticality, the metabolic underpinnings of disease-induced endoreplication and its link to morphologic growth are unknown. Heart disease is characterized by endoreplication preceding cardiac hypertrophy. We identify ATP synthase as a central control node and determinant of cardiac endoreplication and hypertrophy by rechanneling free mitochondrial ADP to methylenetetrahydrofolate dehydrogenase 1 L (MTHFD1L), a mitochondrial localized rate-limiting enzyme of formate and ''de novo'' nucleotide biosynthesis. Concomitant activation of the adenosine monophosphate–activated protein kinase (AMPK)–retinoblastoma protein (Rb)-E2F axis co-opts metabolic products of MTHFD1L function to support DNA endoreplication and pathologic growth. Gain- and loss-of-function studies in genetic and surgical mouse heart disease models and correlation in individuals confirm direct coupling of deregulated energetics with endoreplication and pathologic overgrowth. Together, we identify cardiometabolic endoreplication as a hitherto unknown mechanism dictating pathologic growth pro-gression in the failing myocardium. +
In the phylum Chordata, only members of the subphylum Vertebrata were thought to express hemoglobin (Hb). Here we document the existence of intracellular Hb expressed in members of the subphylum Cephalochordata. Hemoglobin is expressed in myotome tissue and in notochord cells within the body of amphioxus. Both notochord and myotome tissue Hbs have a molecular size consistent with a dimeric molecule made up of two non-covalently linked monomers each of approximately 19 kD. The notochord Hb has a relatively high oxygen-binding affinity, with an apparent P5O of 0.036 kPa (0.27mm Hg), and it does not bind oxygen cooperatively. The notochord Hb may be involved in facilitating oxygen delivery and providing a short-term oxygen store within the notochord cells in order to maintain a high level of aerobic metabolism in support of the sustained contraction necessary for notochord function. +
Prediabetes is associated with postprandial hypertriacylglycerolaemia. Resistance exercise acutely lowers postprandial plasma triacylglycerol (TG); however, the changes in lipid metabolism that mediate this reduction are poorly understood. The aim of this study was to identify the constitutive metabolic mechanisms underlying the changes in postprandial lipid metabolism after resistance exercise in obese men with prediabetes.
We evaluated the effect of a single bout of whole-body resistance exercise (seven exercises, three sets, 10-12 repetitions at 80% of one-repetition maximum) on postprandial lipid metabolism in ten middle-aged (50 ± 9 years), overweight/obese (BMI: 33 ± 3 kg/m<sup>2</sup>), sedentary men with prediabetes (HbA<sub>1c</sub> >38 but <48 mmol/mol [>5.7% but <6.5%]), or fasting plasma glucose >5.6 mmol/l but <7.0 mmol/l or 2 h OGTT glucose >7.8 mmol/l but <11.1 mmol/l). We used a randomised, crossover design with a triple-tracer mixed meal test (ingested [(<sup>13</sup>C<sub>4</sub>)<sub>3</sub>]tripalmitin, i.v. [U-<sup>13</sup>C<sub>16</sub>]palmitate and [<sup>2</sup>H<sub>5</sub>]glycerol) to evaluate chylomicron-TG and total triacylglycerol-rich lipoprotein (TRL)-TG kinetics. We used adipose tissue and skeletal muscle biopsies to evaluate the expression of genes regulating lipolysis and lipid oxidation, skeletal muscle respirometry to evaluate oxidative capacity, and indirect calorimetry to assess whole-body lipid oxidation.
The single bout of resistance exercise reduced the lipaemic response to a mixed meal in obese men with prediabetes without changing chylomicron-TG or TRL-TG fractional clearance rates. However, resistance exercise reduced endogenous and meal-derived fatty acid incorporation into chylomicron-TG and TRL-TG. Resistance exercise also increased whole-body lipid oxidation, skeletal muscle mitochondrial respiration, oxidative gene expression in skeletal muscle, and the expression of key lipolysis genes in adipose tissue.
A single bout of resistance exercise improves postprandial lipid metabolism in obese men with prediabetes, which may mitigate the risk for cardiovascular disease and type 2 diabetes.
Two-thirds of people with type 2 diabetes mellitus (T2DM) have or will develop chronic kidney disease (CKD), which is characterized by rapid renal decline that, together with superimposed T2DM-related metabolic sequelae, synergistically promotes early frailty and mobility deficits that increase the risk of mortality. Distinguishing the mechanisms linking renal decline to mobility deficits in CKD progression and/or increasing severity in T2DM is instrumental both in identifying those at high risk for functional decline and in formulating effective treatment strategies to prevent renal failure. While evidence suggests that skeletal muscle energetics may relate to the development of these comorbidities in advanced CKD, this has never been assessed across the spectrum of CKD progression, especially in T2DM-induced CKD. Here, using next-generation sequencing, we first report significant downregulation in transcriptional networks governing oxidative phosphorylation, coupled electron transport, electron transport chain (ETC) complex assembly, and mitochondrial organization in both middle- and late-stage CKD in T2DM. Furthermore, muscle mitochondrial coupling is impaired as early as stage 3 CKD, with additional deficits in ETC respiration, enzymatic activity, and increased redox leak. Moreover, mitochondrial ETC function and coupling strongly relate to muscle performance and physical function. Our results indicate that T2DM-induced CKD progression impairs physical function, with implications for altered metabolic transcriptional networks and mitochondrial functional deficits as primary mechanistic factors early in CKD progression in T2DM. +
Small zooplankton, rotatoria and nauplii decomposed rapidly during in-vitro incubation in the gut fluid of silvercarp. Algae remained unchanged. Gut content analysis, therefore, erroneously suggests a phytoplanktivorous strategy, whereas omnivorous feeding may actually be required for maintaining a positive energy balance in these stomachless fish. +
The 4-thia fatty acid tetradecylthiopropionic acid (TTP) is known to inhibit mitochondrial β-oxidation, and can be used as chemically induced hepatic steatosis-model in rodents, while 3-thia fatty acid tetradecylthioacetic acid (TTA) stimulates fatty acid oxidation through activation of peroxisome proliferator activated receptor alpha (PPARα). We wished to determine how these two compounds affected ''in vivo'' respiration and mitochondrial efficiency, with an additional goal to elucidate whether mitochondrial function is reflected in plasma acylcarnitine levels.
C57BL/6 mice were divided in 4 groups of 10 mice and fed a control low-fat diet, low-fat diets with 0.4% (w/w) TTP, 0.4% TTA or a combination of these two fatty acids for three weeks (n = 10). At sacrifice, β-oxidation and oxidative phosphorylation (OXPHOS) capacity was analysed in fresh liver samples. Hepatic mitochondria were studied using transmission electron microscopy. Lipid classes were measured in plasma, heart and liver, acylcarnitines were measured in plasma, and gene expression was measured in liver.
The TTP diet resulted in hepatic lipid accumulation, plasma L-carnitine and acetylcarnitine depletion and elevated palmitoylcarnitine and non-esterified fatty acid levels. No significant lipid accumulation was observed in heart. The TTA supplement resulted in enhanced hepatic β-oxidation, accompanied by an increased level of acetylcarnitine and palmitoylcarnitine in plasma. Analysis of mitochondrial respiration showed that TTP reduced oxidative phosphorylation, while TTA increased the maximum respiratory capacity of the electron transport system. Combined treatment with TTP and TTA resulted in a profound stimulation of genes involved in the PPAR-response and L-carnitine metabolism, and partly prevented triacylglycerol accumulation in the liver concomitant with increased peroxisomal β-oxidation and depletion of plasma acetylcarnitines. Despite an increased number of mitochondria in the liver of TTA + TTP fed mice, the OXPHOS capacity was significantly reduced.
This study indicates that fatty acid β-oxidation directly affects mitochondrial respiratory capacity in liver. As plasma acylcarnitines reflected the reduced mitochondrial β-oxidation in TTP-fed mice, they could be useful tools to monitor mitochondrial function. As mitochondrial dysfunction is a major determinant of metabolic disease, this supports their use as plasma markers of cardiovascular risk in humans. Results however indicate that high PPAR activation obscures the interpretation of plasma acylcarnitine levels.
The inherent drug susceptibility of microorganisms is determined by multiple factors, including growth state, the rate of drug diffusion into and out of the cell, and the intrinsic vulnerability of drug targets with regard to the corresponding antimicrobial agent. Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a significant source of global morbidity and mortality, further exacerbated by its ability to readily evolve drug resistance. It is well accepted that drug resistance in M. tuberculosis is driven by the acquisition of chromosomal mutations in genes encoding drug targets/promoter regions; however, a comprehensive description of the molecular mechanisms that fuel drug resistance in the clinical setting is currently lacking. In this context, there is a growing body of evidence suggesting that active extrusion of drugs from the cell is critical for drug tolerance. M. tuberculosis encodes representatives of a diverse range of multidrug transporters, many of which are dependent on the proton motive force (PMF) or the availability of ATP. This suggests that energy metabolism and ATP production through the PMF, which is established by the electron transport chain (ETC), are critical in determining the drug susceptibility of M. tuberculosis. In this review, we detail advances in the study of the mycobacterial ETC and highlight drugs that target various components of the ETC. We provide an overview of some of the efflux pumps present in M. tuberculosis and their association, if any, with drug transport and concomitant effects on drug resistance. The implications of inhibiting drug extrusion, through the use of efflux pump inhibitors, are also discussed. +
The redox states of the NAD and NADP pyridine nucleotide pools play critical roles in defining the activity of energy producing pathways, in driving oxidative stress and in maintaining antioxidant defences. Broadly speaking, NAD is primarily engaged in regulating energy-producing catabolic processes, whilst NADP may be involved in both antioxidant defence and free radical generation. Defects in the balance of these pathways are associated with numerous diseases, from diabetes and neurodegenerative disease to heart disease and cancer. As such, a method to assess the abundance and redox state of these separate pools in living tissues would provide invaluable insight into the underlying pathophysiology. Experimentally, the intrinsic fluorescence of the reduced forms of both redox cofactors, NADH and NADPH, has been used for this purpose since the mid-twentieth century. In this review, we outline the modern implementation of these techniques for studying mitochondrial redox state in complex tissue preparations. As the fluorescence spectra of NADH and NADPH are indistinguishable, interpreting the signals resulting from their combined fluorescence, often labelled NAD(P)H, can be complex. We therefore discuss recent studies using fluorescence lifetime imaging microscopy (FLIM) which offer the potential to discriminate between the two separate pools. This technique provides increased metabolic information from cellular autofluorescence in biomedical investigations, offering biochemical insights into the changes in time-resolved NAD(P)H fluorescence signals observed in diseased tissues. +
Surgery for urological cancers is associated with high complication rates and survivors commonly experience fatigue, reduced physical ability and quality of life. High-intensity interval training (HIIT) as surgical prehabilitation has been proven effective for improving the cardiorespiratory fitness (CRF) of urological cancer patients, however the mechanistic basis of this favourable adaptation is undefined. Thus, we aimed to assess the mechanisms of physiological responses to HIIT as surgical prehabilitation for urological cancer.
Nineteen male patients scheduled for major urological surgery were randomised to complete 4-weeks HIIT prehabilitation (71.6 ± 0.75 years, BMI: 27.7 ± 0.9 kg·m<sup>2</sup>) or a no-intervention control (71.8 ± 1.1 years, BMI: 26.9 ± 1.3 kg·m<sup>2</sup>). Before and after the intervention period, patients underwent m. vastus lateralis biopsies to quantify the impact of HIIT on mitochondrial oxidative phosphorylation (OXPHOS) capacity, cumulative myofibrillar muscle protein synthesis (MPS) and anabolic, catabolic and insulin-related signalling.
OXPHOS capacity increased with HIIT, with increased expression of electron transport chain protein complexes (C)-II (p = 0.010) and III (p = 0.045); and a significant correlation between changes in C-I (r = 0.80, p = 0.003), C-IV (r = 0.75, p = 0.008) and C-V (r = 0.61, p = 0.046) and changes in CRF. Neither MPS (1.81 ± 0.12 to 2.04 ± 0.14%·day<sup>-1</sup>, p = 0.39) nor anabolic or catabolic proteins were upregulated by HIIT (p > 0.05). There was, however, an increase in phosphorylation of AS160<sup>Thr642</sup> (p = 0.046) post-HIIT.
A HIIT surgical prehabilitation regime, which improved the CRF of urological cancer patients, enhanced capacity for skeletal muscle OXPHOS; offering potential mechanistic explanation for this favourable adaptation. HIIT did not stimulate MPS, synonymous with the observed lack of hypertrophy. Larger trials pairing patient-centred and clinical endpoints with mechanistic investigations are required to determine the broader impacts of HIIT prehabilitation in this cohort, and to inform on future optimisation (i.e., to increase muscle mass).
Thickening of the airway smooth muscle is central to bronchial hyperreactivity. We have shown that the sphingosine analog AAL-R can reverse pre-established airway hyperreactivity in a chronic asthma model. Since sphingosine analogs can be metabolized by sphingosine kinase 2, we investigated whether this enzyme was required for AAL-R to perturb mechanisms sustaining airway smooth muscle cell proliferation. We found that AAL-R pre-treatment reduced the capacity of live airway smooth muscle cells to use oxygen for oxidative phosphorylation and increased lactate dehydrogenase activity. We also determined that sphingosine kinase 2 was upregulated in airway smooth muscle cells bearing the proliferation marker Ki67, relative to their Ki67-negative counterpart. Comparing different stromal cell subsets of the lung, we found that high sphingosine kinase 2 levels were associated with the ability of AAL-R to inhibit metabolic activity assessed by conversion of the tetrazolium dye MTT. Knock down or pharmacologic inhibition of sphingosine kinase 2 reversed the effect of AAL-R on MTT conversion, indicating the essential role for this kinase in the metabolic perturbations induced by sphingosine analogs. Our results support the hypothesis that increased sphingosine kinase 2 levels in proliferating airway smooth muscle cells could be exploited to counteract airway smooth muscle thickening with synthetic substrates. +
Mutations of mitochondrial DNA (mtDNA) are an important cause of genetic disease, yet rarely present in the neonatal period. Here we report the clinical, biochemical, and molecular genetic findings of an infant who died at the age of 1 mo with marked biventricular hypertrophy, aortic coarctation, and severe lactic acidosis due to a previously described but unusual mtDNA mutation, a 7-bp intragenic inversion within the mitochondrial gene encoding ND1 protein of complex I (MTND1). In direct contrast to the previous case, an adult with exercise intolerance who only harbored the mutation in muscle, the MTND1 inversion in our patient was present at high levels in several tissues including the heart, muscle, liver, and cultured skin fibroblasts. There was no evidence of the mutation or respiratory complex I defect in a muscle biopsy from the patient's mother. Transmitochondrial cytoplasmic hybrids (cybrids) containing high mutant loads of the inversion expressed the biochemical defect but apparently normal levels of the assembled complex. Our report highlights the enormous phenotypic diversity that exists among pathogenic mtDNA mutations and reemphasizes the need for appropriate genetic counseling for families affected by mtDNA disease. +
In primary fibroblasts from Leigh Syndrome (LS) patients, isolated mitochondrial complex I deficiency is associated with increased reactive oxygen species levels and mitochondrial morpho-functional changes. Empirical evidence suggests these aberrations constitute linked therapeutic targets for small chemical molecules. However, the latter generally induce multiple subtle effects, meaning that ''in vitro'' potency analysis or single-parameter high-throughput cell screening are of limited use to identify these molecules. We combine automated image quantification and artificial intelligence to discriminate between primary fibroblasts of a healthy individual and a LS patient based upon their mitochondrial morpho-functional phenotype. We then evaluate the effects of newly developed Trolox variants in LS patient cells. This revealed that Trolox ornithylamide hydrochloride best counterbalanced mitochondrial morpho-functional aberrations, effectively scavenged ROS and increased the maximal activity of mitochondrial complexes I, IV and citrate synthase. Our results suggest that Trolox-derived antioxidants are promising candidates in therapy development for human mitochondrial disorders. +
Biological oxidations do not take place by direct transfer of electrons (e−) from substrate to oxygen. They are carried out in successive stages by different e− acceptors with increasing reduction potential. This allows for a stepwise release of energy and its best utilization by the cell. The respiratory chain, located in the mitochondrial inner membrane, comprises a series of H or electrons (e−) acceptors arranged according to increasing reduction potential, associated with enzymes that catalyze e− transfer. It is composed of complex I or NADH–ubiquinone reductase, complex II or succinate–ubiquinone reductase, ubiquinone or coenzyme Q, complex III or ubiquinone–cytochrome c reductase, cytochrome c, located on the outer face of inner membrane, complex IV or cytochrome oxidase. Finally 4 e− are transferred to 2 O atoms, which with 4 H+ form 2 H2O. The energy produced by the flow of e− is coupled to phosphoryl transfer, synthesizing adenosine triphosphate (ATP) from ADP in a process known as oxidative phosphorylation. Each e− pair from substrates of NAD-linked dehydrogenases generates three molecules of ATP, while substrates oxidized by FAD-dependent enzymes produce two ATP. The chemio-osmotic hypothesis explains the mechanism underlying oxidative phosphorylation. The energy generated by the flow of reducing equivalents is used to pump protons from the mitochondrial matrix outward into the inner membrane, at the site of complexes I, III, and IV. The proton gradient created across the mitochondrial inner membrane drives proton flux through the F1F0 or ATP synthase complex, which couples proton transport to phosphate addition to ADP. Compounds that reduce or eliminate the proton gradient inhibit phosphorylation. Inhibitors can block e− transfer at different levels of the respiratory chain. Rotenone, amytal, and other barbiturates act at the level of complex I; antimycin A, at complex III; and cyanide, carbon monoxide, and azide, on complex IV. Inhibitors of oxidative phosphorylation include proton and K+ ionophores, which suppress the mitochondrial electrical potential gradient, acting as uncoupling agents, and compounds, such as oligomycin, which interfere with the function of the F1F0 ATPase. Brown fat present in infants and hibernating animals has thermogenin, a protein that inhibits ATP synthesis, uncoupling mitocondrial function and contributing to maintain body temperature. Oxidative phosphorylation is mainly controlled by ADP levels. Phosphorylation at substrate level is another way to generate ATP by phosphoryl transfer from high energy metabolites.
Invasive fungal infections have significantly increased over the past decades in immune-compromised individuals and high-risk patients. Amphotericin B (AmB) exerts a powerful and broad activity against a vast array of fungi and has a remarkably low rate of microbial resistance. However, most isolates of ''A. terreus'' developed an intrinsic resistance against AmB and during this study we characterized the mode of action of this polyene antifungal drug in more detail in resistant (ATR) and rare susceptible (ATS) clinical isolates of A. terreus.
We illustrate that AmB treatment changes cellular redox status and promotes the generation of high levels of reactive oxygen species (ROS) in ATS. In contrast, ATR were able to cope better with AmB-induced oxidative stress.
Most importantly, we demonstrate in this study that co-application of anti- and pro-oxidants significantly affects AmB efficacy in an antithetic manner - antioxidants and ROS-scavenging agents increase AmB tolerance in susceptible strains, while pro-oxidants render formerly resistant isolates considerably susceptible to the antifungal drug also ''in vivo'' in a Galleria animal model.
Thereby, our study provides novel therapeutic options to treat formerly resistant fungal strains by a combination of AmB and pro-oxidant compounds.
<br><br> +
Mitochondrial electron transport chain (ETC) drives ATP production and is the major source of reactive oxygen species (ROS). We have previously shown that mitochondrially targeted vitamin E succinate (MitoVES) induces cell death by inhibiting complex II of ETC, leading to considerable ROS production. In addition, MitoVES selectively eliminates proliferating but not quiescent (confluent) endothelial cells (ECs) and suppresses tumorigenic angiogenesis ''in vivo''. This suggests that modulation of ETC activity in proliferating and quiescent cells might have different outcomes with respect to cell death induction. To investigate the role of ROS generation and inhibition of ATP production (ETC inhibition may result in both), we cultured ECs in low (1 g/L) and high glucose (4.5 g/L) that promotes and suppresses mitochondrial respiration/ATP production, respectively. We exposed these cells to agents that induce ROS without ETC inhibition (phenethylisothiocyanate - PEITC, and hydrogen peroxide), inhibit ETC (rotenone, antimycin A) or directly interfere with mitochondrial ATP production (FCCP, oligomycin). Interestingly, PEITC and hydrogen peroxide induced cell death and ROS preferentially in proliferating cells irrespective of cell culture conditions. In contrast, treatment with the other compounds resulted in more cell death in proliferating than in quiescent cells when glucose was high, but this pattern was reversed when glucose was low. In addition, ROS generation only correlated with cell death induction in high glucose. Respiration measurements showed that cells grown in high glucose slightly reduced, and cells grown in low glucose significantly increased respiration on entering the quiescence state. This occurred despite the fact that ETC was primed for high activity in quiescent cells irrespective of glucose concentration, as evidenced by elevated expression of protein subunits and increased enzymatic activity of ETC complexes as well as enhanced supercomplex assembly. These data suggest that in low glucose interference with ETC activity is a major factor for cell death induction, whereas in high glucose the level of ROS generation becomes dominant. We therefore propose that ETC inhibition differentially affects proliferating and quiescent cells and might be the key determinant of proliferation-responsive cell death sensitivity. Experiments to confirm this scenario are ongoing.
Mitochondrial electron transport chain (ETC) targeting shows a great promise in cancer therapy. It is particularly effective in tumors with high ETC activity where ETC-derived reactive oxygen species (ROS) are efficiently induced. Why modern ETC-targeted compounds are tolerated on the organismal level remains unclear. As most somatic cells are in non-proliferative state, the features associated with the ETC in quiescence could account for some of the specificity observed. Here we report that quiescent cells, despite increased utilization of the ETC and enhanced supercomplex assembly, are less susceptible to cell death induced by ETC disruption when glucose is not limiting. Mechanistically, this is mediated by the increased detoxification of ETC-derived ROS by mitochondrial antioxidant defense, principally by the superoxide dismutase 2 - thioredoxin axis. In contrast, under conditions of glucose limitation, cell death is induced preferentially in quiescent cells and is correlated with intracellular ATP depletion but not with ROS. This is related to the inability of quiescent cells to compensate for the lost mitochondrial ATP production by the upregulation of glucose uptake. Hence, elevated ROS, not the loss of mitochondrially-generated ATP, are responsible for cell death induction by ETC disruption in ample nutrients condition, e.g. in well perfused healthy tissues, where antioxidant defense imparts specificity. However, in conditions of limited glucose, e.g. in poorly perfused tumors, ETC disruption causes rapid depletion of cellular ATP, optimizing impact towards tumor-associated dormant cells. In summary, we propose that antioxidant defense in quiescent cells is aided by local glucose limitations to ensure selectivity of ETC inhibition-induced cell death.
Copyright © 2017 Elsevier Inc. All rights reserved. +
To understand the role of reactive oxygen species (ROS) in oxidative stress and redox signaling it is necessary to link their site of generation to the oxidative modification of specific targets. Here we have studied the selective modification of protein thiols by mitochondrial ROS that have been implicated as deleterious agents in a number of degenerative diseases and in the process of biological aging, but also as important players in cellular signal transduction. We hypothesized that this bipartite role might be based on different generator sites for "signaling" and "damaging" ROS and a directed release into different mitochondrial compartments. Because two main mitochondrial ROS generators, complex I (NADH:ubiquinone oxidoreductase) and complex III (ubiquinol:cytochrome ''c'' oxidoreductase; cytochrome bc1 complex), are known to predominantly release superoxide and the derived hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) into the mitochondrial matrix and the intermembrane space, respectively, we investigated whether these ROS generators selectively oxidize specific protein thiols. We used redox fluorescence difference gel electrophoresis analysis to identify redox-sensitive targets in the mitochondrial proteome of intact rat heart mitochondria. We observed that the modified target proteins were distinctly different when complex I or complex III was employed as the source of ROS. These proteins are potential targets involved in mitochondrial redox signaling and may serve as biomarkers to study the generator-dependent dual role of mitochondrial ROS in redox signaling and oxidative stress. +
Changes in environmental temperature can pose significant challenges to animals. Shifts in thermal habitat have been shown to be a major force driving species adaptation. These adaptations have been the focus of major research efforts to delineate the physiological or metabolic constraints related to temperature and to reveal the phenotypic characters that can or should adjust. Considering the current consensus on climate change, the focus of research will likely move on questioning if they will survive to future modifications of their thermal niches. Adjustments to temperature can either be through physiological plasticity (e.g. acclimation) or via genetic adaptation. Therefore we will have to specify what are the genetic and phenotypic attributes (at the level of individual, population and species) that could grant survival success. These questions are particularly important for ectotherms, which are in thermal equilibrium with the surrounding environment. To start answering these queries, we should wonder if any physiological or metabolic function set the temperature impact on organisms. Some recent developments point to mitochondria as a key metabolic structure that partly delineates the thermal range that organism can tolerate [1]. The catalytic capacity of mitochondria is highly sensitive to thermal variation and therefore should partly dictate the temperature dependence of biological functions. Mitochondria are a complex network of pathways of different enzymatic reactions that synergistically interact. The fine regulation of both ATP and ROS production depends on this integration of different enzymes and pathways. Here, we will scrutinize the temperature dependence of different parts of the mitochondrial pathways and evaluate the evolutionary challenges that should be overcome to insure mitochondrial adaptations to new thermal environments. +
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Delineating the physiological and biochemical causes of aging process in the animal kingdom is a highly active area of research not only because of potential benefits for human health but also because aging process is related to life history strategies (growth and reproduction) and to responses of organisms to environmental conditions and stress. In this presentation, I advocate studying bivalve species as models for revealing the determinants of species divergences in maximal longevity. This taxonomic group includes the longest living metazoan on earth (''Arctica islandica''), which insures the widest range of maximum life span when shorter living species are also included in the comparative model. This model can also be useful for uncovering factors modulating the pace of aging in given species by taking advantages of the wide disparity of lifespan among different populations of the same species. For example, maximal lifespan in different populations of ''A islandica'' range from approximately 36 years to over 500 years.
We compared membrane fatty acid as well as metabolic capacities (respiration rates) and ROS production from mitochondria of five species of bivalves and different populations of ''A islandica''. Fatty acid profiles were determined by Gas Chromatography with FID detection, while oxygen consumption and peroxide efflux were assayed by high resolution respirometry (O2K) and fluorescence detection with Amplex Red<sup>®</sup>.
Our analysis revealed that either regulation or tolerance to oxidative stress is tightly correlated to longevity of species [1,2,3] but when comparing different populations of ''A islandica'', the relationship of membrane fatty acid composition or Peroxidation Index with maximum lifespan vanishes [4]. This led us to conclude that mitochondrial membrane robustness to ROS attack is required for a species to be able to reach long lifespan but it is not sufficient at the population level to express this life history trait.
Neurodegenerative diseases, including Alzheimer´s Disease (AD) and Parkinson´s Disease (PD), lack efficient medications to modify pathogenetic mechanisms. Affecting millions of people worldwide every year, the need for disease-modifying therapies is pressing.
There is strong evidence for mitochondrial dysfunction playing a critical role in the development of AD and PD, implicated by the accumulation of amyloid-β and α-synuclein respectively. Synaptic failure and neuronal death are also consequences of impaired mitochondrial biogenesis, bioenergetics and transport [1,2].
Studies have shown that synthetic heteroatom-substituted fatty acids in β-position such as tetradecylthioacetic acid (TTA) have favorable effects on mitochondrial function. This includes stimulation of mitochondrial and peroxisomal fatty acid oxidation [3], antioxidant capacity and mild uncoupling by UCP2 and UCP3. Induction of mitochondrial biogenesis and respiration by TTA have the potential to repopulate neurites with mitochondria, possibly preventing neurodegeneration, synaptic failure and neuronal death.
During the work with my master´s thesis, I wish to investigate the effects of TTA along with other novel modified fatty acids on neuronal cells. These include triple-TTA with a triple bond at the methyl end, possibly slowing the catabolism, and N-TTA which has a nitrogen atom in β-position instead of sulphur.
Starting procedures have included viability tests on the cell lines used in the project, and determination of cell toxicity of the fatty acids using WST-1-assay and spectrophotometric detection. When appropriate concentrations of the fatty acids are known, the plan is to perform ''in vitro'' respiration assays to determine mitochondrial activity in the cell lines after treatment with the selected compounds. Oxygen utilization in response to the treatment will be quantified by polarographic respirometry (OROBOROS® Oxygraph), after permeabilization of the cells. By employing various metabolic substrates and molecular manipulators we can differentiate functional and regulatory aspects of single components of the respiratory chain. Specifically, we will examine if the fatty acids alter the capacity or coupling state of the mitochondria.
Sepsis can cause the nonthyroidal illness syndrome (NTIS), resulting in perturbed thyroid hormone (TH) signaling and reduced thyroxine (T4) levels. TH is a major regulator of muscle function, via its influence on mitochondria. The present study aimed to evaluate the relationship among TH signaling, mitochondrial function and the antioxidant defense system in the diaphragms of septic mice.
Male C57Bl/6 mice were divided into two groups: cecal ligation and puncture (CLP) and sham. Twenty-four hours after surgery, plasma, diaphragms and livers were collected. TH metabolism and responses were analyzed by measuring mRNA expression of Dio1 in the liver, and Thra, Thrb, Dio2, Slc16a10 and Slc16a2 (encodes MCT 10 and 8), in the diaphragm. T4 plasma levels were measured by radioimmunoassay. Damage to diaphragm mitochondria was assessed by electron microscopy and qPCR, and function with oxygraphy. The diaphragm anti-oxidative defense system was examined by qPCR, analyzing Sod1, Sod2, Sod3, Gpx1 and Cat expression. The effect of TH replacement was tested by treating the mice with T4 and tri-iodothyronine (T3) (CLP+TH) after surgery.
CLP mice presented reduced total plasma T4 concentrations, as well as downregulated Dio1 and upregulated Il1b mRNA expression in the liver. CLP mice also displayed downregulated Thra, Thrb, Slc16a10 and Slc16a2 expression in the diaphragm, suggesting that TH signaling was compromised. The expression of Ppargc1a (encodes PGC1a) was downregulated, which correlated with the decrease in the number of total mitochondria, increase in the percentage of injured mitochondria, downregulation of respiratory chain complex 2 and 3 mRNA expression and reduced maximal respiration. Additionally, septic animals presented a 3-fold increase in Ucp3 and G6pdh expression, downregulated Sod3, Gpx1 and Cat expression; and upregulated Sod2 expression, potentially due to elevated ROS levels. The mitochondrial number and the percentage of injured mitochondrial were similar between sham and CLP+TH mice.
Sepsis induced responses consistent with NTIS, resulted in mitochondrial damage and functional impairment, and modulated the expression of key antioxidant enzymes in the diaphragm. Thus, impaired diaphragm function during sepsis seems to involve altered local TH signaling, mitochondrial dysfunction and oxidative stress defense.
CDP diacylglycerol synthase (CDS) catalyses the conversion of phosphatidic acid (PA) to CDP-diacylglycerol, an essential intermediate in the synthesis of phosphatidylglycerol, cardiolipin and phosphatidylinositol (PI). CDS activity has been identified in mitochondria and endoplasmic reticulum of mammalian cells apparently encoded by two highly-related genes, CDS1 and CDS2. Cardiolipin is exclusively synthesised in mitochondria and recent studies in cardiomyocytes suggest that the peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1α and β) serve as transcriptional regulators of mitochondrial biogenesis and up-regulate the transcription of the CDS1 gene. Here we have examined whether CDS1 is responsible for the mitochondrial CDS activity. We report that differentiation of H9c2 cells with retinoic acid towards cardiomyocytes is accompanied by increased expression of mitochondrial proteins, oxygen consumption, and expression of the PA/PI binding protein, PITPNC1, and CDS1 immunoreactivity. Both CDS1 immunoreactivity and CDS activity were found in mitochondria of H9c2 cells as well as in rat heart, liver and brain mitochondria. However, the CDS1 immunoreactivity was traced to a peripheral p55 cross-reactive mitochondrial protein and the mitochondrial CDS activity was due to a peripheral mitochondrial protein, TAMM41, not an integral membrane protein as expected for CDS1. TAMM41 is the mammalian equivalent of the recently identified yeast protein, Tam41. Knockdown of TAMM41 resulted in decreased mitochondrial CDS activity, decreased cardiolipin levels and a decrease in oxygen consumption. We conclude that the CDS activity present in mitochondria is mainly due to TAMM41, which is required for normal mitochondrial function. +
Transfection of genes that code for enzymes of energy metabolism provides alternative models to study the adaptive response to energy restriction induced by endogenous changes instead of by unfavorable environmental conditions. Overexpression of the glycolytic enzyme fructose-2,6-bisphosphatase reduced the content of fructose 2,6-bisphosphate, inducing energy limitation in the mink lung epithelial cell line Mv1Lu. This metabolic stress reduced the ATP available in transfected cells by 20%, which downregulated active ion transport and protein turnover. Ion homeostasis and cell function require concomitant reductions in cell membrane ion permeability and protein damage. Our results indicate that glutathione content linked these features of the adaptive response to the endogenously induced metabolic downregulation. +
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An ischemic insult is associated with increased circulating fatty acids (FA) due to an adrenergic activation of adipose tissue lipolysis. Therefore, hearts will not only be challenged by hypoxia, but also by an acute FA-load, which has been shown to induce adverse cardiac effects such as mitochondrial dysfunction, oxidative stress, oxygen wasting and inefficiency. Although obesity is a contributing factor to the development of type 2 diabetes and heart failure it remains unclear if and how obesity-associated chronic hyperlipidemia affects the cardiac response to an acute FA-load. Thus, we have examined the effect of high FA on hearts from a murine model of obesity.
Diet-induced obesity (DIO) was obtained by feeding 5-wk old male C57BL/6J mice obesogenic diet for 20 wks. Age-matched chow-fed mice were included as controls (CON). ''Ex vivo'' left ventricular (LV) function (working heart perfusions, n=8-11) and ischemic susceptibility (LV post-ischemic functional recovery and infarct size, Langendorff perfusions, n=12-15) were examined in hearts exposed to normal (0.35 mM) or high (1.8 mM) palmitate levels. We also assessed myocardial O<sub>2</sub> consumption (MVO<sub>2</sub>), FA oxidation and mechanical efficiency (n=12-15), as well as myocardial ROS content (DHE tissue staining) and mitochondrial respiration (high-resolution respirometry, ''n''=6-8).
DIO mice demonstrated elevated plasma FA levels (0.37±0.03 vs 0.58±0.04 mM, p<0.01) and insulin resistance (4.4 fold higher HOMA-IR). They also developed diastolic dysfunction with only a mild systolic dysfunction. High FA perfusion did not alter LV function in neither CON nor DIO hearts. However, elevated FA decreased mechanical efficiency (due to increased MVO<sub>2</sub>, 28±2 vs 37±2 μmol/min/g, p<0.01), induced oxidative stress and reduced mitochondrial OXPHOS rate and coupling (RCR) in CON hearts. These FA-mediated changes were not found in DIO hearts. Furthermore, in contrast to CON hearts (where ischemic tolerance was not altered by the FA-load), DIO hearts exposed to high FA levels showed increased functional recovery (53±5 vs 36±5 % Rate-Pressure-Product, p<0.01) and decreased infarct size (47±2 vs 62±5%, p<0.02). This cardioprotective effect was corroborated in hearts from obese, type 2 diabetic (db/db) mice (54±6 vs 36±5 % recovery of RPP, p<0.05, and 55±5 vs72±2 % infarction, p<0.01, respectively).
This study shows that hearts from obese/diabetic mice are resistant to the adverse effects an acute FA-load. Although dyslipidemia plays a role in the development of obesity/diabetes-mediated heart failure, we suggest that these hearts undergo adaptive changes where elevated FA levels exert cardioprotection.
Uric acid is a purine degradation product but also an important antioxidant and ROS scavenger. Experimental settings that mimic myocardial ischemia-reperfusion have not included uric acid despite that it is always present in human extracellular fluid and plasma. We hypothesized that uric acid has an important role in myocardial ROS scavenging. Here, we tested the cardiac response to uric acid on infarct size following ischemia-reperfusion with and without exacerbated oxidative stress due to acute pressure overload and during preconditioning. We also examined mitochondrial respiration and ROS-induced mitochondrial permeability transition pore opening. Under exacerbated ROS stress induced by high pressure perfusion, uric acid lowered oxidative stress and reduced infarct size. In contrast, uric acid blocked cardioprotection induced by ischemic preconditioning. However, this effect was reversed by probenecid, an inhibitor of cellular uptake of uric acid. In accordance, in intact cardiomyocytes, extracellular uric acid reduced the susceptibility of mitochondria towards opening of the permeability transition pore, suggesting that uric acid may prevent ischemia-reperfusion injury due to scavenging of maladaptive ROS. Moreover, as uric acid also scavenges also adaptive ROS, this may interfere with preconditioning. Altogether, uric acid might be a confounder when translating preclinical experimental results into clinical treatment. +
An ischemic insult is accompanied by an acute increase in circulating fatty acid (FA), which can induce adverse changes related to cardiac metabolism/energetics. Although chronic hyperlipidemia contributes to the pathogenesis of obesity/diabetes-related cardiomyopathy, it unclear how these hearts are affected by an acute high FA-load. We hypothesize that adaptation to chronic FA exposure enhances the obese hearts' ability to handle an acute high FA-load. Diet-induced obese (DIO) and age-matched control (CON) mouse hearts were perfused in the presence of low or high FA-load (0.4 and 1.8 mM). Left ventricular (LV) function, FA oxidation rate, myocardial oxygen consumption and mechanical efficiency were assessed, followed by analysis of myocardial oxidative stress, mitochondrial respiration, protein acetylation as well as gene expression. Finally, ischemic tolerance was determined by examining LV functional recovery and infarct size. Under low FA conditions, DIO hearts showed mild LV dysfunction, oxygen wasting, mechanical inefficiency, and reduced mitochondrial OxPhos. High FA-load increased FA oxidation rates in both groups, but this did not alter any of the above parameters in DIO hearts. In contrast, CON hearts showed FA-induced mechanical inefficiency, oxidative stress and reduced OxPhos, as well as enhanced acetylation and activation of PPARα-dependent gene expression. While high FA-load did not alter functional recovery and infarct size in CON hearts, it increased ischemic tolerance in DIO hearts. Thus, this study demonstrates that acute FA-load affects normal and obese hearts differently, and that chronically elevated circulating FA levels render the DIO heart less vulnerable to the disadvantageous effects of an acute FA-load. +
Alzheimer’s disease (AD) and cancer proceed
via one or more common molecular mechanisms: a metabolic
shift from oxidative phosphorylation to glycolysis—
corresponding to the activation of the Warburg effect—
occurs in both diseases. The findings reported in this paper
demonstrate that, in the early phase of apoptosis, glucose
metabolism is enhanced, i.e. key proteins which internalize
and metabolize glucose—glucose transporter, hexokinase
and phosphofructokinase—are up-regulated, in concomitance
with a parallel decrease in oxygen consumption by
mitochondria and increase of L-lactate accumulation.
Reversal of the glycolytic phenotype occurs in the presence
of dichloroacetate, inhibitor of the pyruvate dehydrogenase
kinase enzyme, which speeds up apoptosis of cerebellar
granule cells, reawakening mitochondria and then modulating
glycolytic enzymes. Loss of the adaptive advantage
afforded by aerobic glycolysis, which occurs in the late
phase of apoptosis, exacerbates the pathological processes
underlying neurodegeneration, leading inevitably the cell
to death. In conclusion, the data propose that both aerobic,
i.e. Warburg effect, essentially due to the protective
numbness of mitochondria, and anaerobic glycolysis, rather
due to the mitochondrial impairment, characterize the
entire time frame of apoptosis, from the early to the late
phase, which mimics the development of AD. +
Background: The iatrogenic, HIV-related lipodystrophy is associated with development of the significant metabolic and cardiovascular complications. The underlying mechanisms of antiretroviral (ARV) drugs are not completely explored.
Methods: The aim of the study was to characterize effects of the protease inhibitor (PI) - saquinavir (SQV) on metabolic functions, and gene expression during differentiation in cells (Chub-S7) culture. Results: SQV in concentrations observed during antiretroviral therapy (ART) significantly decreased mitochondrial membrane potential (MMP), oxygen consumption and ATP generation. The effects were greater in already differentiated cells. This was accompanied by characteristic changes in the expression of the genes involved in endoplasmic reticulum (ER) stress, and differentiation (lipid droplet formation) process such as: WNT10a, C/EBPa, AFT4, CIDEC, ADIPOQ, LPIN1.
Conclusions: The results indicate that SQV affects not only metabolic (mitochondrial) activity of adipocytes, but affects the expression of genes related to differentiation and to a lesser extent to cell apoptosis. +
Sauerstoff wurde erstmals im 18. Jahrhundert mit therapeutischer Zielsetzung
eingesetzt. [72] Zunächst bedurfte es vieler Erfahrungen, bis dann zu Beginn des 20. Jahrhunderts der Durchbruch gelang und Sauerstoff als Basistherapeutikum
eingesetzt wurde. [63, 88]
Sauerstoff hat auch in der heutigen Medizin weiterhin einen festen Stellenwert.
... +
A close link between Ca<sup>2+</sup>, ATP level, and neurogenesis is apparent; however, the molecular mechanisms of this relationship have not been completely elucidated. Transient elevations of cytosolic Ca<sup>2+</sup> may boost ATP synthesis, but ATP is also consumed by ion pumps to maintain a low Ca<sup>2+</sup> in cytosol. In differentiation process plasma membrane Ca<sup>2+</sup> ATPase (PMCA) is considered as one of the major players for Ca<sup>2+</sup> homeostasis. From four PMCA isoforms, the fastest PMCA2 and PMCA3 are expressed predominantly in excitable cells.
In the present study we assessed whether PMCA isoform composition may affect energy balance in differentiating PC12 cells. We found that PMCA2-downregulated cells showed higher basal O<sup>2</sup> consumption, lower NAD(P)H level, and increased activity of ETC. These changes associated with higher [Ca<sup>2+</sup>]<sub>c</sub> resulted in elevated ATP level. Since PMCA2-reduced cells demonstrated greatest sensitivity to ETC inhibition, we suppose that the main source of energy for PMCA isoforms 1, 3, and 4 was oxidative phosphorylation. Contrary, cells with unchanged PMCA2 expression exhibited prevalence of glycolysis in ATP generation. Our results with PMCA2- or PMCA3-downregulated lines provide an evidence of a novel role of PMCA isoforms in regulation of bioenergetic pathways, and mitochondrial activity and maintenance of ATP level during PC12 cells differentiation. +
Mesenchymal stem cells (MSCs) have been explored as promising tools for treatment of several neurological and neurodegenerative diseases. MSCs release abundant extracellular vesicles (EVs) containing a variety of biomolecules, including mRNAs, miRNAs, and proteins. We hypothesized that EVs derived from human Wharton's jelly would act as mediators of the communication between hMSCs and neurons and could protect hippocampal neurons from damage induced by Alzheimer's disease-linked amyloid beta oligomers (AβOs).
We isolated and characterized EVs released by human Wharton's jelly mesenchymal stem cells (hMSC-EVs). The neuroprotective action of hMSC-EVs was investigated in primary hippocampal cultures exposed to AβOs.
hMSC-EVs were internalized by hippocampal cells in culture, and this was enhanced in the presence of AβOs in the medium. hMSC-EVs protected hippocampal neurons from oxidative stress and synapse damage induced by AβOs. Neuroprotection by hMSC-EVs was mediated by catalase and was abolished in the presence of the catalase inhibitor, aminotriazole.
hMSC-EVs protected hippocampal neurons from damage induced by AβOs, and this was related to the transfer of enzymatically active catalase contained in EVs. Results suggest that hMSC-EVs should be further explored as a cell-free therapeutic approach to prevent neuronal damage in Alzheimer's disease. +
Impaired adipose tissue (AT) function might induce recent-onset type 2 diabetes (T2D). Understanding AT energy metabolism could yield novel targets for the treatment of T2D.
Recently diagnosed male T2D patients and healthy humans (controls, CON) of similar abdominal subcutaneous AT (SAT)-thickness, fat mass and age (n=14 each), underwent hyperinsulinemic-euglycemic clamps with [6,6-<sup>2</sup>H<sub>2</sub>]glucose and indirect calorimetry. We assessed mitochondrial efficiency (coupling: state 3/4o; proton leak: state 4o/u) via high-resolution respirometry in superficial (SSAT) and deep (DSAT) SAT-biopsies, hepatocellular lipids (HCL) and fat mass by proton-magnetic-resonance-spectroscopy and -imaging.
T2D patients (known diabetes duration: 2.5 [0.1; 5.0] years) had 43%, 44% and 63% lower muscle insulin sensitivity (IS), metabolic flexibility (p<0.01) and AT IS (p<0.05), 73% and 31% higher HCL (p<0.05) and DSAT-thickness (p<0.001), but similar hepatic IS compared to CON. Mitochondrial efficiency was ~22% lower in SSAT and DSAT of T2D (p<0.001) and ~8% lower in SSAT vs DSAT (p<0.05). In both fat depots, mitochondrial coupling correlated positively with muscle IS and metabolic flexibility (r≥0.40, p<0.05), proton leak correlated positively (r≥0.51, p<0.01) and oxidative capacity negatively (r≤-0.47, p<0.05) with fasting FFA. Metabolic flexibility correlated positively with SAT-oxidative capacity (r≥0.48, p<0.05) and negatively with DSAT-thickness (r=-0.48, p<0.05). DSAT-thickness correlated negatively with mitochondrial coupling in both depots (r≤-0.50, p<0.01) and muscle IS (r=-0.59, p<0.01), positively with FFA during clamp (r=0.63, p<0.001) and HCL (r=0.49, p<0.01).
Impaired mitochondrial function, insulin resistance and DSAT expansion are AT abnormalities in recent-onset T2D that might promote whole-body insulin resistance and increased substrate flux to the liver.
<small>© Endocrine Society 2019.</small> +
Exercise training induces white adipose tissue (WAT) beiging and improves glucose homeostasis and mitochondrial function in rodents. This could be relevant for type 2 diabetes in humans, but the effect of physical fitness on beiging of subcutaneous WAT (scWAT) remains unclear. This translational study investigates if beiging of scWAT associates with physical fitness in healthy humans and recent-onset type 2 diabetes and if a voluntary running wheel intervention is sufficient to induce beiging in mice.
Gene expression levels of established beiging markers were measured in scWAT biopsies of humans with (n = 28) or without type 2 diabetes (n = 28), stratified by spiroergometry into low (L-FIT; n = 14 each) and high physical fitness (H-FIT; n = 14 each). High-fat diet-fed FVB/N mice underwent voluntary wheel running, treadmill training or no training (n = 8 each group). Following the training intervention, mitochondrial respiration and content of scWAT were assessed by high-resolution respirometry and citrate synthase activity, respectively.
Secreted CD137 antigen (Tnfrsf9/Cd137) expression was three-fold higher in glucose-tolerant H-FIT than in L-FIT, but not different between H-FIT and L-FIT with type 2 diabetes. In mice, both training modalities increased Cd137 expression and enhanced mitochondrial content without changing respiration in scWAT. Treadmill but not voluntary wheel running led to improved whole-body insulin sensitivity.
Higher physical fitness and different exercise interventions associated with higher gene expression levels of the beiging marker CD137 in healthy humans and mice on a high-fat diet. Humans with recent-onset type 2 diabetes show an impaired adipose tissue-specific response to physical activity. +
The experience of maltreatment during childhood is associated with chronic low-grade inflammation in adulthood. However, the molecular mechanisms underlying this pro-inflammatory phenotype remain unclear. Mitochondria were recently found to principally coordinate inflammatory processes via both inflammasome activation and inflammasome-independent pathways. To this end, we hypothesized that alterations in immune cell mitochondrial functioning and oxidative stress might be at the interface between the association of maltreatment experiences during childhood and inflammation. We analyzed pro-inflammatory biomarkers (levels of C-reactive protein, cytokine secretion by peripheral blood mononuclear cells (PBMC) ''in vitro'', PBMC composition, lysophosphatidylcholine levels), serum oxidative stress levels (arginine:citrulline ratio, l-carnitine and acetylcarnitine levels) and mitochondrial functioning (respiratory activity and density of mitochondria in PBMC) in peripheral blood samples collected from 30 women (aged 22-44 years) with varying degrees of maltreatment experiences in form of abuse and neglect during childhood. Exposure to maltreatment during childhood was associated with an increased ROS production, higher levels of oxidative stress and an increased mitochondrial activity in a dose-response relationship. Moreover, the increase in mitochondrial activity and ROS production were positively associated with the release of pro-inflammatory cytokines by PBMC. Decreased serum levels of lysophosphatidylcholines suggested higher inflammasome activation with increasing severity of child maltreatment experiences. Together these findings offer preliminary evidence for the association of alterations in immune cell mitochondrial functioning, oxidative stress and the pro-inflammatory phenotype observed in individuals with a history of maltreatment during childhood. The results emphasize that the early prevention of child abuse and neglect warrants more attention, as the experience of maltreatment during childhood might have life-long consequences for physical health.
Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
Childhood maltreatment (CM) is associated with an increased risk for the development of psychiatric and somatic diseases in later life. Individual risk and resilience factors may, however, influence how deep psychological stress gets under the skin. We hypothesized that the stress-related hormone cortisol and the attachment-related hormone oxytocin constitute biological factors that might moderate the biological sequelae and long-term health outcomes associated with CM. As biological outcome, we thereby focused on immunocellular oxygen consumption, which we previously found to be increased with a higher severity of CM experiences. In a study cohort of N = 49 postpartum women, we investigated the interaction between CM experiences, serum cortisol and plasma oxytocin levels, and the cellular oxygen consumption of intact peripheral blood mononuclear cells (PBMC) by high-resolution respirometry. Regression analyses revealed a significant interaction between the severity of CM experiences and cortisol as well as oxytocin on cellular oxygen consumption of PBMC three months postpartum: higher cortisol levels were thereby associated with an increase in oxygen consumption related to basal mitochondrial respiration and ATP turnover, while oxygen consumption related to basal mitochondrial respiration and ATP turnover were reduced with higher oxytocin levels in individuals with higher CM severity. These associations were not seen among women with no or low CM experiences. Together, the results suggest that cortisol and oxytocin might be associated with opposite effects on CM-related alterations in the bioenergetic profile of peripheral immune cells. +
Major Depressive Disorder (MDD) has been associated with telomere dysfunction and alterations in mitochondrial activity, which seem to be co-regulated in human cells. To investigate this co-regulation in MDD, we assessed telomere length (TL) in peripheral blood mononuclear cells (PBMC) and selected immune cell subsets by quantitative fluorescence ''in situ'' hybridization and mitochondrial respiratory activity in PBMC by high-resolution respirometry in a study cohort of 18 MDD patients and 21 non-depressed controls. We provide initial evidence for a differential vulnerability to telomere attrition in selected adaptive immune cell populations. Here we found the highest difference in TL between depressed and control subjects for memory cytotoxic T cells. Depression was associated with reduced mitochondrial activity (mitochondrial bioenergetics), but increased mitochondrial density (mitochondrial biogenesis) in PBMC. Exploratory ''post-hoc'' analyses indicated that the changes in TL and immune cell bioenergetics were most pronounced in MDD patients who reported experiences of childhood sexual abuse. Among MDD patients, PBMC TL was as a trend positively associated with mitochondrial density and negatively associated with mitochondrial leak respiration, but not with mitochondrial activity related to biological energy production. These initial findings support the hypothesis of a co-regulation between telomeres and mitochondrial biogenesis but not mitochondrial bioenergetics among MDD patients. +
The chaperone-related AAA ATPase Cdc48 (p97/VCP in higher eukaryotes) segregates ubiquitylated proteins for subsequent degradation by the 26S proteasome or for nonproteolytic fates. The specific outcome of Cdc48 activity is controlled by the evolutionary conserved cofactors Ufd2 and Ufd3, which antagonistically regulate the substrates' ubiquitylation states. In contrast to the interaction of Ufd3 and Cdc48, the interaction between the ubiquitin chain elongating enzyme Ufd2 and Cdc48 has not been precisely mapped. Consequently, it is still unknown whether physiological functions of Ufd2 in fact require Cdc48 binding. Here, we show that Ufd2 binds to the C-terminal tail of Cdc48, unlike the human Ufd2 homologue E4B, which interacts with the N domain of p97. The binding sites for Ufd2 and Ufd3 on Cdc48 overlap and depend critically on the conserved residue Y834 but are not identical. ''Saccharomyces cerevisiae'' cdc48 mutants altered in residue Y834 or lacking the C-terminal tail are viable and exhibit normal growth. Importantly, however, loss of Ufd2 and Ufd3 binding in these mutants phenocopies defects of Δufd2 and Δufd3 mutants in the ubiquitin fusion degradation (UFD) and Ole1 fatty acid desaturase activation (OLE) pathways. These results indicate that key cellular functions of Ufd2 and Ufd3 in proteasomal protein degradation require their interaction with Cdc48. +
Conditions during blood product storage and transportation should maintain quality. The aim of this ''in vitro'' study was to investigate the effect of interruption of agitation, temporary cooling (TC), and pneumatic tube system transportation (PTST) on the aggregation ability (AA) and mitochondrial function (MF) of platelet concentrates (PC).
A PC was divided equally into four subunits and then allocated to four test groups. The control group (I) was stored as recommended (continuous agitation, 22 ± 2°C) for 4 days. The test groups were stored without agitation (II), stored as recommended, albeit 4°C for 60 minutes on day (d)2 (III) and PTST (IV). Aggregometry was measured using Multiplate (RocheAG; ADPtest, ASPItest, TRAPtest, COLtest) and MF using Oxygraph-2k (Oroboros Instruments). The basal and maximum mitochondrial respiratory rate (MMRR) were determined. AA and MF were measured daily in I and II and AA in III and IV on d2 after TC/PTST. Statistical analysis was performed using tests for matched observations.
Eleven PCs were used. TRAP-6 induced AA was significantly lower in II when compared to I on d4 (P = 0.015*). In III the ASPItest was significantly lower (P = 0.032*). IV showed no significant differences. The basal and MMRR were significantly reduced over 4 days in I and II (for both rates in both groups: P = <0.0001*). No significant differences occurred on d4 (P = 0.495).
Our results indicate that ex vivo AA and MF of PCs are unaffected, even in no-ideal storage and transport circumstances with respect to agitation, temperature, and force.
<small>© 2020 The Authors. Transfusion published by Wiley Periodicals LLC. on behalf of AABB.</small> +
Mass-specific metabolic rate negatively co-varies with body mass from the whole-animal to the mitochondrial levels. Mitochondria are the mainly consumers of oxygen inspired by mammals to generate ATP or compensate for energetic losses dissipated as the form of heat (proton leak) during oxidative phosphorylation. Consequently, ATP synthesis and proton leak compete for the same electrochemical gradient. Because proton leak co-varies negatively with body mass, it is unknown whether extremely small mammals further decouple their mitochondria to maintain their body temperature or whether they implement metabolic innovations to ensure cellular homeostasis. The present study investigated the impact of body mass variation on cellular and mitochondrial functioning in small mammals, comparing two extremely small African pygmy mice (''Mus mattheyi'', ∼5 g, and ''Mus minutoides'', ∼7 g) with the larger house mouse (''Mus musculus'', ∼22 g). Oxygen consumption rates were measured from the animal to the mitochondrial levels. We also measured mitochondrial ATP synthesis in order to appreciate the mitochondrial efficiency (ATP/O). At the whole-animal scale, mass- and surface-specific metabolic rates co-varied negatively with body mass, whereas this was not necessarily the case at the cellular and mitochondrial levels. ''Mus mattheyi'' had generally the lowest cellular and mitochondrial fluxes, depending on the tissue considered (liver or skeletal muscle), as well as having more-efficient muscle mitochondria than the other two species. ''Mus mattheyi'' presents metabolic innovations to ensure its homeostasis, by generating more ATP per oxygen consumed.
<small>© 2020. Published by The Company of Biologists Ltd.</small> +
Cardiac mitochondrial matrix (m) free Ca<sup>2+</sup> ([Ca<sup>2+</sup>]<sub>m</sub>) increases primarily by Ca<sup>2+</sup> uptake through the Ca<sup>2+</sup> uniporter (CU). Ca<sup>2+</sup> uptake via the CU is attenuated by extra-matrix (e) Mg<sup>2+</sup> ([Mg<sup>2+</sup>]<sub>e</sub>). How [Ca<sup>2+</sup>]<sub>m</sub> is dynamically modulated by interacting physiological levels of [Ca<sup>2+</sup>]<sub>e</sub> and [Mg<sup>2+</sup>]<sub>e</sub> and how this interaction alters bioenergetics are not well understood. We postulated that as [Mg<sup>2+</sup>]<sub>e</sub> modulates Ca<sup>2+</sup> uptake via the CU, it also alters bioenergetics in a matrix Ca<sup>2+</sup>-induced and matrix Ca<sup>2+</sup>-independent manner. To test this, we measured changes in [Ca<sup>2+</sup>]<sub>e</sub>, [Ca<sup>2+</sup>]<sub>m</sub>, [Mg<sup>2+</sup>]<sub>e</sub> and [Mg<sup>2+</sup>]<sub>m</sub> spectrofluorometrically in guinea pig cardiac mitochondria in response to added CaCl<sub>2</sub> (0-0.6 mM; 1 mM EGTA buffer) with/without added MgCl<sub>2</sub> (0-2 mM). In parallel, we assessed effects of added CaCl<sub>2</sub> and MgCl<sub>2</sub> on NADH, membrane potential (ΔΨm), and respiration. We found that ≥0.125 mM MgCl<sub>2</sub> significantly attenuated CU-mediated Ca<sup>2+</sup> uptake and [Ca<sup>2+</sup>]<sub>m</sub>. Incremental [Mg<sup>2+</sup>]<sub>e</sub> did not reduce initial Ca<sup>2+</sup> uptake but attenuated the subsequent slower Ca<sup>2+</sup> uptake, so that [Ca<sup>2+</sup>]<sub>m</sub> remained unaltered over time. Adding CaCl<sub>2</sub> without MgCl<sub>2</sub> to attain a [Ca<sup>2+</sup>]<sub>m</sub> from 46 to 221 nM enhanced state 3 NADH oxidation and increased respiration by 15 %; up to 868 nM [Ca<sup>2+</sup>]<sub>m</sub> did not additionally enhance NADH oxidation or respiration. Adding MgCl<sub>2</sub> did not increase [Mg<sup>2+</sup>]<sub>m</sub> but it altered bioenergetics by its direct effect to decrease Ca<sup>2+</sup> uptake. However, at a given [Ca<sup>2+</sup>]<sub>m</sub>, state 3 respiration was incrementally attenuated, and state 4 respiration enhanced, by higher [Mg<sup>2+</sup>]<sub>e</sub>. Thus, [Mg<sup>2+</sup>]<sub>e</sub> without a change in [Mg<sup>2+</sup>]<sub>m</sub> can modulate bioenergetics independently of CU-mediated Ca<sup>2+</sup> transport.
Glutathione S-transferase alpha 4 (GSTA4-4) is one of the enzymes responsible for the removal of 4-hydroxynonenal (4-HNE), an electrophilic product of lipid peroxidation in cellular membranes during oxidative stress. 4-HNE is a direct activator of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a transcription factor with many target genes encoding antioxidant and anti-electrophile enzymes. We have previously shown that ''Gsta4''-null mice on a 129/Sv background exhibited increased activity of Nrf2 in the heart. Here we examined the sensitivity of this ''Gsta4''-null mouse model towards cardiac function and structure loss due to local heart irradiation. Male ''Gsta4''-null and wild-type mice were exposed to a single X-ray dose of 18 Gy to the heart. Six months after irradiation, immunohistochemical staining for respiratory complexes 2 and 5 indicated that radiation exposure had caused most pronounced alterations in mitochondrial morphology in ''Gsta4''-null mice. On the other hand, wild-type mice showed a decline in cardiac function and an increase in plasma levels of troponin-I, while no such changes were observed in ''Gsta4''-null mice. Radiation-induced Nrf2-target gene expression only in ''Gsta4''-null mice. In conclusion, although loss of GSTA4-4 led to enhanced susceptibility of cardiac mitochondria to radiation-induced loss of morphology, cardiac function was preserved in ''Gsta4''-null mice. We propose that this protection against cardiac function loss may occur, at least in part, by upregulation of the Nrf2 pathway. +
A complete understanding of the role of the liver in burn-induced hypermetabolism is lacking. We investigated the acute effect of severe burn trauma on liver mitochondrial respiratory capacity and coupling control as well as the signaling events underlying these alterations.
Male BALB/c mice (8-12 weeks) received full-thickness scald burns on ∼30% of the body surface. Liver tissue was harvested 24 hours post injury. Mitochondrial respiration was determined by high-resolution respirometry. Citrate synthase activity was determined as a proxy of mitochondrial density. Male Sprague-Dawley rats received full-thickness scald burns to ∼60% of the body surface. Serum was collected 24 hours post injury. HepG2 cells were cultured with serum-enriched media from either sham or burn treated rats. Protein levels were analyzed via western blot.
Mass-specific (p = 0.01) and mitochondrial-specific (p = 0.01) respiration coupled to ATP production significantly increased in the liver after burn. The respiratory control ratio for ADP (p = 0.04) and the mitochondrial flux control ratio (p = 0.03) were elevated in the liver of burned animals. Complex III and Complex IV protein abundance in the liver increased after burn by 17% and 14%, respectively. Exposure of HepG2 cells to serum from burned rats increased the pAMPKα:AMPKα ratio (p < 0.001) and levels of SIRT1 (p = 0.01), Nrf2 (p < 0.001), and PGC1α (p = 0.02).
Severe burn trauma augments respiratory capacity and function of liver mitochondria, adaptations that augment ATP production. This response may be mediated by systemic factors that activate signaling proteins responsible for regulating cellular energy metabolism and mitochondrial biogenesis. +
The activation of thermogenesis in adipose tissue has emerged as an important target for the development of novel anti-obesity therapies. Using multi-well isothermal microcalorimetry, we have demonstrated that mature murine brown and brite adipocytes produce quantifiable heat upon β3-AR stimulation, independently of any anaerobic mechanisms. Additionally, in brite adipocytes lacking UCP1 protein, β3-AR stimulation still induces heat production, albeit to a much lower extent than in their wildtype counterparts, suggesting that UCP1 is an essential component of adrenergic induced thermogenesis in murine brite adipocytes exvivo. Similarly, we could observe an increase in heat production in human-derived adipocytes (hMADS) upon β-AR stimulation. Collectively, these results establish the use of isothermal microcalorimetry as a sensitive and accurate technique for measuring thermogenic responses in intact mature brite adipocytes from murine and human origin. +
Schon oft wurde mir nahegelegt, ein Lehrbuch über Gastheorie zu schreiben. .. Man wird mir wohl nicht übelnehmen, dass ich auch meinen eigenen Arbeiten einigen Platz gegönnt habe. .. Etwas weitschweifige Formeln waren zum Ausdrucke complicirter Gedankenreihen leider manches Mal nicht zu vermeiden .. +
Reactive oxygen species (ROS) are toxic molecules involved in several biological processes such as cellular signaling, proliferation, differentiation and cell death. Adaptations to oxidative environments are crucial for the success of the colonization of insects by protozoa. ''Strigomonas culicis'' is a monoxenic trypanosomatid found in the midgut of mosquitoes and presenting a life cycle restricted to the epimastigote form. Among ''S. culicis peculiarities'', there is an endosymbiotic bacterium in the cytoplasm, which completes essential biosynthetic routes of the host cell and may represent an intermediary evolutive step in organelle origin, thus constituting an interesting model for evolutive researches. In this work, we induced ROS resistance in wild type ''S. culicis'' epimastigotes by the incubation with increasing concentrations of hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>), and compared the oxidative and energetic metabolisms among wild type, wild type-H<sub>2</sub>O<sub>2</sub> resistant and aposymbiotic strains. Resistant protozoa were less sensitive to the oxidative challenge and more dependent on oxidative phosphorylation, which was demonstrated by higher oxygen consumption and mitochondrial membrane potential, increased activity of complexes II-III and IV, increased complex II gene expression and higher ATP production. Furthermore, the wild type-H<sub>2</sub>O<sub>2</sub> resistant strain produced reduced ROS levels and showed lower lipid peroxidation, as well as an increase in gene expression of antioxidant enzymes and thiol-dependent peroxidase activity. On the other hand, the aposymbiotic strain showed impaired mitochondrial function, higher H<sub>2</sub>O<sub>2</sub> production and deficient antioxidant response. The induction of H<sub>2</sub>O<sub>2</sub> resistance also led to a remarkable increase in ''Aedes aegypti'' midgut binding ''in vitro'' and colonization ''in vivo'', indicating that both the pro-oxidant environment in the mosquito gut and the oxidative stress susceptibility regulate ''S. culicis'' population in invertebrates.
Chagas disease is caused by the hemoflagellate protozoa ''Trypanosoma cruzi'' and is one of the most important neglected tropical diseases, especially in Latin American countries, where there is an association between low-income populations and mortality. The nitroderivatives used in current chemotherapy are far from ideal and present severe limitations, justifying the continuous search for alternative drugs. Since the1990s, our group has been investigating the trypanocidal activity of natural naphthoquinones and their derivatives, and three naphthoimidazoles (N1, N2 and N3) derived from β-lapachone were found to be most effective ''in vitro''. Analysis of their mechanism of action via cellular, molecular and proteomic approaches indicates that the parasite mitochondrion contains one of the primary targets of these compounds, trypanothione synthetase (involved in trypanothione production), which is overexpressed after treatment with these compounds. Here, we further evaluated the participation of the mitochondria and reactive oxygen species (ROS) in the anti-''T. cruzi'' action of naphthoimidazoles. Preincubation of epimastigotes and trypomastigotes with antioxidants (α-tocopherol and urate) strongly protected the parasites from the trypanocidal effect of naphthoimidazoles, decreasing the ROS levels produced and reverting the mitochondrial swelling phenotype. The addition of pro-oxidants (menadione and H<sub>2</sub>O<sub>2</sub>) before the treatment induced an increase in parasite lysis. Despite the O<sub>2</sub> uptake and mitochondrial complex activity being strongly reduced by N1, N2 and N3, urate partially restored the mitochondrial metabolism only in N1-treated parasites. In parallel, MitoTEMPO, a mitochondrial-targeted antioxidant, protected the functionality of the mitochondria in N2- and N3-treated parasites. In addition, the trypanothione reductase activity was remarkably increased after treatment with N1 and N3, and molecular docking demonstrated that these two compounds were positioned in pockets of this enzyme. Based on our findings, the direct impairment of the mitochondrial electron transport chain by N2 and N3 led to an oxidative misbalance, which exacerbated ROS generation and led to parasite death. Although other mechanisms cannot be discounted, mainly in N1-treated parasites, further investigations are required.
During their life cycle, trypanosomatids are exposed to stress conditions and adapt their energy and antioxidant metabolism to colonize their hosts. ''Strigomonas culicis'' is a monoxenous protist found in invertebrates with an endosymbiotic bacterium that completes essential biosynthetic pathways for the trypanosomatid. Our research group previously generated a wild-type H<sub>2</sub>O<sub>2</sub>-resistant (WTR) strain that showed improved mitochondrial metabolism and antioxidant defenses, which led to higher rates of ''Aedes aegypti'' infection. Here, we assess the biological contribution of the ''S. culicis'' endosymbiont and reactive oxygen species (ROS) resistance to oxidative and energy metabolism processes. Using high-throughput proteomics, several proteins involved in glycolysis and gluconeogenesis, the pentose phosphate pathway and glutathione metabolism were identified. The results suggest that ROS resistance decreases glucose consumption and indicate that the metabolic products from gluconeogenesis are key to supplying the protist with high-energy and reducing intermediates. Our hypothesis was confirmed by biochemical assays showing opposite profiles for glucose uptake and hexokinase and pyruvate kinase activity levels in the WTR and aposymbiotic strains, while the enzyme glucose-6P 1-dehydrogenase was more active in both strains. Regarding the antioxidant system, ascorbate peroxidase has an important role in H<sub>2</sub>O<sub>2</sub> resistance and may be responsible for the high infection rates previously described for ''A. aegypti''. In conclusion, our data indicate that the energy-related and antioxidant metabolic processes of ''S. culicis'' are modulated in response to oxidative stress conditions, providing new perspectives on the biology of the trypanosomatid-insect interaction as well as on the possible impact of resistant parasites in accidental human infection.
<small>Copyright © 2019 Elsevier Inc. All rights reserved.</small> +
[[File:BEC.png|25px|link=https://doi.org/10.26124/bec:2022-0020]] https://doi.org/10.26124/bec:2022-0020
[[File:Bombaca 2022 MitoFit graphical abstract.png|right|300px|Graphical abstract]] Trypanosomatids colonize different environments and are submitted to several stress situations in their hosts, which trigger intense metabolic remodeling to ensure the parasites survival in hostile environments. Some trypanosomatids can avoid the host microbicidal mechanisms, exhibiting natural resistance to oxidative and nitrosative stresses, in addition to resistance to current drug treatment. Beyond the classical role in bioenergetics, mitochondria contribute decisively to oxidative stress due to electron leakage from the electron transfer system. Several functional peculiarities made trypanosomatids’ organelle an excellent target for drug intervention. Here, we discuss data on mitochondrial susceptibility and adaptative processes obtained by our group in the last 17 years. Different pathways are evaluated associated with metabolic and mitochondrial remodeling during the life cycle of trypanosomatids, and its impact on the interaction with vertebrate and invertebrate hosts. In addition, mechanistic proposals of preclinical drugs are reviewed. +
[[File:Bombaca 2022 MitoFit graphical abstract.png|right|300px|Graphical abstract]] [[Menna-Barreto 2022 Abstract Bioblast]] Neglected tropical diseases impact more than a billion people globally, with millions of them at risk of infection by parasites of the Trypanosomatidae family. The need to colonize different environments in their hosts means that trypanosomatids are constantly subjected to stress situations, among which the presence of reactive oxygen (ROS) and nitrogen (RNS) species, requiring intense metabolic remodeling to ensure the parasites survival in hostile environments. Additionaly to the classical role in bioenergetics, mitochondrion has a decisive contribution to the oxidative stress, due to the electron leakage from the electron transfer system (ETS). The presence of several functional peculiarities made the mitochondrion of trypanosomatids an unique organelle, considered an excellent target for drug intervention. Some trypanosomatids such as ''Leishmania'' spp. can avoid the microbicidal mechanisms of the host cells, exhibiting a profile of natural resistance to oxidative and nitrosative stresses. Here, we discussed data about mitochondrial susceptibility and adaptative processes obtained by our group in the last 17 years. Mechanistic proposals of preclinical drugs was reviewed, as well as different pathways associated with metabolic and mitochondrial remodeling during the life cycle of trypanosomatids, including the possible biological role of ROS and RNS resistance and its impact on the interaction with vertebrate and invertebrate hosts. +
Heart phosphorylating electron transfer particles (ETPH) produced NO at 1.2 ± 0.1 nmol NO. min<sup>-1</sup> mg protein<sup>-1</sup> by the mtNOS catalyzed reaction. These particles showed a NAD<sup>+</sup> reductase activity of 64 ± 3 nmol min<sup>-1</sup> mg protein<sup>-1</sup> sustained by reverse electron transfer (RET) at expenses of ATP and succinate. The same particles, without NADPH and in conditions of RET produced 0.97 ± 0.07 nmol NO. min<sup>-1</sup> mg protein<sup>-1</sup>. Rotenone inhibited NO production supported by RET measured in ETPH and in coupled mitochondria, but did not reduce the activity of recombinant nNOS, indicating that the inhibitory effect of rotenone on NO production is due to an electron flow inhibition and not to a direct action on mtNOS structure. NO production sustained by RET corresponds to 20% of the total amount of NO released from heart coupled mitochondria. A mitochondrial fraction enriched in complex I produced 1.7 ± 0.2 nmol NO. min<sup>-1</sup> mg protein<sup>-1</sup> and reacted with anti-75 kDa complex I subunit and anti-nNOS antibodies, suggesting that complex I and mtNOS are located contiguously. These data show that mitochondrial NO production can be supported by RET, and suggest that mtNOS is next to complex I, reaffirming the idea of a functional association between these proteins.
Copyright © 2016 Elsevier Inc. All rights reserved. +
Energy exchange in the cell is associated with mitochondria, which play an important role in vital processes. The Oxidative Phosphorylation System (OxРhoS), localized in the inner mitochondrial membrane, consists of five membrane enzymes. Four of the five protein complexes make up the "respiratory chain" and are involved in the transfer of electrons, which at three points is coupled with the translocation of protons across the inner mitochondrial membrane. The resulting proton gradient is used by the ATP synthase complex (the fifth enzyme complex) to phosphorylate ADP. The methods for studying the activity of the electron transport chain of mitochondria described in the article, especially their use in a complex, makes it possible to significantly detail the understanding of the pathogenesis of disorders of cell energy exchange that occurs in various diseases, which will improve the prevention and correction of mitochondrial dysfunction. +
In photosynthetic organisms, feedback dissipation of excess absorbed light energy balances harvesting of light with metabolic energy consumption. This mechanism prevents photodamage caused by reactive oxygen species produced by the reaction of chlorophyll (Chl) triplet states with O₂. Plants have been found to perform the heat dissipation in specific proteins, binding Chls and carotenoids (Cars), that belong to the Lhc family, while triggering of the process is performed by the PsbS subunit, needed for lumenal pH detection. PsbS is not found in algae, suggesting important differences in energy-dependent quenching (qE) machinery. Consistent with this suggestion, a different Lhc-like gene product, called LhcSR3 (formerly known as LI818) has been found to be essential for qE in ''Chlamydomonas reinhardtii''. In this work, we report the production of two recombinant LhcSR isoforms from ''C. reinhardtii'' and their biochemical and spectroscopic characterization. We found the following: (i) LhcSR isoforms are Chl a/b- and xanthophyll-binding proteins, contrary to higher plant PsbS; (ii) the LhcSR3 isoform, accumulating in high light, is a strong quencher of Chl excited states, exhibiting a very fast fluorescence decay, with lifetimes below 100 ps, capable of dissipating excitation energy from neighbor antenna proteins; (iii) the LhcSR3 isoform is highly active in the transient formation of Car radical cation, a species proposed to act as a quencher in the heat dissipation process. Remarkably, the radical cation signal is detected at wavelengths corresponding to the Car lutein, rather than to zeaxanthin, implying that the latter, predominant in plants, is not essential; (iv) LhcSR3 is responsive to low pH, the trigger of non-photochemical quenching, since it binds the non-photochemical quenching inhibitor dicyclohexylcarbodiimide, and increases its energy dissipation properties upon acidification. This is the first report of an isolated Lhc protein constitutively active in energy dissipation in its purified form, opening the way to detailed molecular analysis. Owing to its protonatable residues and constitutive excitation energy dissipation, this protein appears to merge both pH-sensing and energy-quenching functions, accomplished respectively by PsbS and monomeric Lhcb proteins in plants.
MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1(-/-) mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1(-/-) mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD(+), and is associated with obesity resistance. Consistent with this, NAD(+) levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences. +
Simvastatin is effective and well tolerated, with adverse reactions mainly affecting skeletal muscle. Important mechanisms for skeletal muscle toxicity include mitochondrial impairment and increased expression of atrogin-1. The aim was to study the mechanisms of toxicity of simvastatin on H9c2 cells (a rodent cardiomyocyte cell line) and on the heart of male C57BL/6 mice. After, exposure to 10 μmol/L simvastatin for 24 h, H9c2 cells showed impaired oxygen consumption, a reduction in the mitochondrial membrane potential and a decreased activity of several enzyme complexes of the mitochondrial electron transfer system (ETS). The cellular ATP level was also decreased, which was associated with phosphorylation of AMPK, dephosphorylation and nuclear translocation of FoxO3a as well as increased mRNA expression of atrogin-1. Markers of apoptosis were increased in simvastatin-treated H9c2 cells. Treatment of mice with 5 mg/kg/day simvastatin for 21 days was associated with a 5 % drop in heart weight as well as impaired activity of several enzyme complexes of the ETS and increased mRNA expression of atrogin-1 and of markers of apoptosis in cardiac tissue. Cardiomyocytes exposed to simvastatin in vitro or in vivo sustain mitochondrial damage, which causes AMPK activation, dephosphorylation and nuclear transformation of FoxO3a as well as increased expression of atrogin-1. Mitochondrial damage and increased atrogin-1 expression are associated with apoptosis and increased protein breakdown, which may cause myocardial atrophy. +
Though defective genome maintenance and DNA repair have long been known to promote phenotypes of premature aging, the role protein methylation plays in these processes is only now emerging. We have recently identified the first N-terminal methyltransferase, NRMT1, which regulates protein-DNA interactions and is necessary for both accurate mitotic division and nucleotide excision repair. To demonstrate if complete loss of NRMT1 subsequently resulted in developmental or aging phenotypes, we constructed the first NRMT1 knockout (Nrmt1(-/-)) mouse. The majority of these mice die shortly after birth. However, the ones that survive, exhibit decreased body size, female-specific infertility, kyphosis, decreased mitochondrial function, and early-onset liver degeneration; phenotypes characteristic of other mouse models deficient in DNA repair. The livers from Nrmt1(-/-) mice produce less reactive oxygen species (ROS) than wild type controls, and Nrmt1(-/-) mouse embryonic fibroblasts show a decreased capacity for handling oxidative damage. This indicates that decreased mitochondrial function may benefit Nrmt1(-/-) mice and protect them from excess internal ROS and subsequent DNA damage. These studies position the NRMT1 knockout mouse as a useful new system for studying the effects of genomic instability and defective DNA damage repair on organismal and tissue-specific aging. +
BACKGROUND: In children with either delayed or accelerated growth, expressing the body mass index (BMI) to chronological age might lead to invalid body composition estimates. Reference to height-age has been suggested for such populations; however its validity has not been demonstrated.
METHODS: Anthropometric data of healthy children were obtained from the German KiGGS survey. We selected three samples with different height distributions representing short stature (mean height SDS: -1.6), normal stature (height SDS: 0), and tall stature (height SDS: +1.6), and compared BMI-for-age and BMI-for-height-age between these samples across the paediatric age range. Differences between samples were tested using Kruskal-Wallis one-way analysis of variance and permutation tests.
RESULTS: At a given age, BMI was distributed towards lower values in short, and towards higher values in tall subjects as compared to a population with average height distribution. Expressing BMI to height-age eliminated these differences in boys with a short stature from 4 years to 14 years of age, in tall boys from 4 to 16 years, in short girls aged 2-10 years or tall girls aged 2-17 years.
CONCLUSION: From late infancy to adolescent age, BMI distribution co-varies with height distribution and referencing to height-age appears appropriate within this age period. However, caution is needed when data about pubertal status are absent. +
Gas exchange characteristics of the halotolerant unicellular alga ''Dunaliella salina'' (Dunal) Teodoresco (Chlorophyta) were examined over a range of 0.21 m to 2.22 m NaCl. Cells of D. salina demonstrated a high affinity in photosynthesis for CO2 and HCO3−, suggesting that ribulose-1,5-P2 oxygenase activity is inhibited and photorespiration is consequently suppressed as a result of the activity of a CO2-concentrating mechanism (CCM). The affinity in photosynthesis for CO2 was more pronounced in cells grown at higher NaCl concentrations, with a K½(Co2) for photosynthesis of 0.15 μm at the highest salinity employed (2.22 m NaCl). The activity (and percentage total activity) of carbonic anhydrase accessible at the cell surface was observed to increase substantially with increasing NaCl concentration. Increases in external carbonic anhydrase activity correlated closely with increased affinity for CO2 and HCO3− in photosynthesis, reflecting the importance of external carbonic anhydrase for the effective functioning of the CCM in D. salina. It is suggested that CO2 is the species of inorganic carbon that crosses the plasmalemma in a process facilitated by the action of extracellular carbonic anhydrase. +
The objectives of this study are to test the hypothesis that the fatigue and accompanying symptoms of Chronic Myalgic Encephalomyelitis/Fatigue Syndrome are in part due to defects in energy provision at the cellular level, and to understand the pathophysiology of the defects so that effective medical intervention can be implemented. We performed an audit of 138 patients (ages 18-65) diagnosed with ME/CFS and attending a private practice. The patients and 53 normal, healthy controls had the ATP Profile test carried out on neutrophils from a 3-ml venous blood sample. This test yields 6 numerical factors that describe the availability of ATP and the efficiency of oxidative phosphorylation in mitochondria. Other biomedical measurements, including the concentration of cell-free DNA in plasma, were made. The results of the audit are compared with the controls and a previous cohort of 61 patients. We find that all patients tested have measureable mitochondrial dysfunction which correlates with the severity of the illness. The patients divide into two main groups differentiated by how cellular metabolism attempts to compensate for the dysfunction. Comparisons with exercise studies suggest that the dysfunction in neutrophils also occurs in other cells. This is confirmed by the cell-free DNA measurements which indicate levels of tissue damage up to 3.5 times the normal reference range. The major immediate causes of the dysfunction are lack of essential substrates and partial blocking of the translocator protein sites in mitochondria. The ATP Profile is a valuable diagnostic tool for the clinical management of ME/CFS. +
Reactive oxygen species (ROS) comprise the superoxide anion (O2<sup>•-</sup>), hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>), hydroxyl radical (<sup>•</sup>OH), and singlet oxygen (<sup>1</sup>O2). ROS can damage a variety of macromolecules, including DNA, RNA, proteins, and lipids, and compromise cell viability. To prevent or reduce ROS-induced oxidative stress, bacteria utilize different ROS defense mechanisms, of which ROS scavenging enzymes, such as superoxide dismutases, catalases, and peroxidases, are the best characterized. Recently, evidence has been accumulating that some of the terminal oxidases in bacterial respiratory chains may also play a protective role against ROS. The present review covers this role of terminal oxidases in light of recent findings. +
We have studied sporadic Parkinson's disease (sPD) from expression of patient mitochondrial DNA (mtDNA) in neural cells devoid of their own mtDNA, the "cybrid" model. In spite of reproducing several properties of sPD brain, it remains unclear whether sPD cybrid cells reflect more complex sPD brain bioenergetic pathophysiology. We characterized and correlated respiration of intact sPD cybrid cells with electron transport chain (ETC) protein assembly, complex I ETC gene expression and ETC protein levels in sPD brain. We also assayed expression for multiple ETC genes coded by mtDNA and nuclear DNA (nDNA) in sPD cybrid cells and brain. sPD cybrid cells have reduced levels of mtDNA genes, variable compensatory normalization of mitochondrial gene expression and show robust correlations with mitochondrial ETC gene expression in sPD brains. Relationships among ETC protein levels predict impaired complex I-mediated respiration in sPD brain. That sPD cybrid cells and sPD brain samples show very correlated regulation of nDNA and mtDNA ETC transcriptomes suggests similar bioenergetic physiologies. We propose that further insights into sPD pathogenesis will follow elucidation of mechanisms leading to reduced mtDNA gene levels in sPD cybrids. This will require characterization of the abnormalities and dynamics of mtDNA changes propagated through sPD cybrids over time. +
We have investigated the differential mitochondrial oxidative stress between males and females to understand the molecular mechanisms enabling females to live longer than males. Mitochondria are a major source of free radicals in cells. Those from female rats generate half the amount of peroxides than those of males. This does not occur in ovariectomized animals. Estrogen replacement therapy prevents the effect of ovariectomy. Mitochondria from females have higher levels of reduced glutathione than those from males. Those from ovariectomized rats have similar levels to males, and estrogen therapy prevents the fall in glutathione levels that occurs in ovariectomized animals. Oxidative damage to mitochondrial DNA in males is 4-fold higher than that in females. This is due to higher expression and activities of Mn-superoxide dismutase and of glutathione peroxidase in females, which behave as double transgenics overexpressing superoxide dismutase and glutathione peroxidase, conferring protection against free-radical-mediated damage in aging. Moreover, 16S rRNA expression, which decreases significantly with aging, is four times higher in mitochondria from females than in those from males of the same chronological age. The facts reported here provide molecular evidence to explain the different life span in males and females. +
Females live longer than males. Oestrogens protect females against aging by up-regulating the expression of antioxidant, longevity-related genes such as glutathione peroxidase (GPx) and Mn-superoxide dismutase (Mn-SOD). The mechanism through which oestrogens up-regulate those enzymes remains unidentified, but may have implications for gender differences in lifespan. We show that physiological concentrations of oestradiol act through oestrogen receptors to reduce peroxide levels in MCF-7 cells (a mammary gland tumour cell line). Oestradiol increases MAP kinase (MAPK) activation as indicated by ERK1 and ERK2 phosphorylation in MCF-7 cells, which in turn activates the nuclear factor kappa B (NFkappaB) signalling pathways as indicated by an increase in the p50 subunit of NFkappaB in nuclear extracts. Blockade of MAPK and NFkappaB signalling reduces the antioxidant effect of oestradiol. Finally, we show that activation of MAPK and NFkappaB by oestrogens drives the expression of the antioxidant enzymes Mn-SOD and GPx. We conclude that oestradiol sequentially activates MAPK and NFkappaB following receptor activation to up-regulate the expression of antioxidant enzymes, providing a cogent explanation for the antioxidant properties of oestrogen and its effects on longevity-related genes. +
The validity of the free radical theory of aging has been recently questioned. Our aim was to test whether there is oxidative stress in tissues critically involved in accelerated aging (senescence-accelerated mice, SAM) and whether this correlates with lower glucose consumption ''in vivo'' and behavioural tests. Positron emission tomography shows that brains of old SAM-prone animals consume less glucose than young ones. Behavioural characteristics, mitochondrial peroxide production, and damage in both the central nervous system and bone marrow stem cells also indicate that SAM-prone animals age faster than SAM-resistant ones. Our results support the role of the free radical theory of aging in critical tissues involved in aging and that this correlates with glucose consumption. +
Estrogens have antioxidant properties which are due to their ability to bind to estrogen receptors and to up-regulate the expression of antioxidant enzymes via intracellular signalling pathways. Mitochondria are key organelles in the development of age-associated cellular damage. Recently, estrogen receptors were identified in mitochondria. The aim of this paper was to test whether estradiol directly affects mitochondria by preventing oxidative stress and protecting frail mitochondria. Incubation with estradiol at normal intracellular concentrations prevents the formation of reactive oxygen species by mitochondria in a saturable manner. Moreover, estradiol protects mitochondrial integrity as indicated by an increase in mitochondrial membrane potential. It also prevents the apoptogenic leakage of cytochrome c from mitochondria and as a result the mitochondrial content of this cytochrome c is maintained high. Thus, estradiol prevents the onset of the mitochondrial pathway of apoptosis by a direct effect on the organelle. Genistein, a phytoestrogen present at high concentration in soy, mimics the protective effect of estradiol by both decreasing the rate of formation of reactive oxygen species and preventing the release of cytochrome c from mitochondria. +
We compared three models of cerebral ischemia in piglets – unilateral and bilateral carotid artery occlusion as well as bilateral occlusion with hypotension for 3 h. These surgical procedures produced different effects on microcirculation which were accompanied by the gradual decline in the activity of mitochondrial oxidative phosphorylation. We found that LEAK respiration (measured in the presence of pyruvate plus malate but without ADP) was not affected by ischemia in any experimental model. The OXPHOS capacity with pyruvate+malate as substrates decreased by 20% and 80% compared to the control level after bilateral carotid artery occlusion and bilateral carotid artery occlusion plus hypotension, respectively, resulting in the decrease of respiratory control index. OXPHOS capacity with succinate as substrate remained constant after unilateral carotid artery occlusion or bilateral carotid artery occlusion but decreased by 50% after bilateral carotid artery occlusion and hypotension. No loss of cytochrome ''c'' from mitochondria was observed in any model of cerebral ischemia. This suggests that damage to Complex I of the mitochondrial respiratory system is the primary target of ischemic insult and may lead to subsequent delayed neuronal death in piglet model of global cerebral ischemia.
In the in vitro model of rat global brain ischemia, mitochondrial OXPHOS activity with pyruvate plus malate and to a lesser extent with succinate was decreased after 30 min ischemia. OXPHOS activity with both substrates decreased further during 60-120 min period. This inhibition was not reversed in the presence of added cytochrome ''c'' suggesting that inhibition of OXPHOS was not caused by the loss of cytochrome ''c''. Measurements of mitochondrial content of cytochrome ''c'' confirmed this as there was no change in cytochrome ''c'' levels during 30-120 min ischemic period. These findings are in contrast to reported previously for heart ischemia where it was found that the loss of cytochrome ''c'' from mitochondria is the earliest event in ischemic mitochondrial damage leading to caspase activation and cell death [1].
In conclusion, our data suggest that brain and heart ischemia causes mitochondrial damage, however, the mechanisms involved are different: Ischemic damage to heart mitochondria is primarily related to opening of mitochondrial permeability transition pore and the release of cytochrome ''c'', whereas during brain ischemia the earliest event is inhibition of Complex I.
Cytochrome ''c'' is a small soluble protein which has two main functions: transfer of electrons within the mitochondrial respiratory system and formation of apoptosomes when released into cytosol. Under normal conditions, the outer mitochondrial (mt) membrane is not permeable to cytochrome ''c''. Therefore, stimulation of mitochondrial respiration by exogenous cytochrome ''c'' is widely used as a test for outer mt-membrane damage. Using this test, we and others have shown that heart ischemia causes rapid permeabilization of the outer mt-membrane, resulting in loss of cytochrome ''c'' from mitochondria and subsequent inhibition of mitochondrial respiration. In contrast, addition of exogenous cytochrome ''c'' to mitochondria isolated from ischemia-damaged brains does not stimulate mitochondrial respiration, suggesting that during brain ischemia the outer mt-membrane remains intact. We also found that in the presence of certain polyphenolic plant compounds (anthocyanins), addition of cytochrome ''c'' to isolated mitochondria results in acute stimulation of mitochondrial respiration. This effect is not linked to the permeabilization of the outer mt-membrane, as these anthocyanins can directly reduce cytochrome ''c'', thus facilitating electron transfer to cytochrome ''c'' oxidase.
Certain anthocyanins, such as delphinidin-3-O-glucoside or cyaniding-3-O-glucoside, can serve as electron acceptors at Complex I of the mitochondrial respiratory system and, therefore, in pathological conditions related to inhibition of Complex I, facilitate an alternative electron transfer from Complex I to cytochrome ''c'' and cytochrome ''c'' oxidase.
Supported by European Social Fund under the Global grant measure; project No VP1-3.1-SMM-07-K-01-130. +
Opening of mitochondrial permeability transition pore (MPTP) is
considered as one of the main determinants in ischemia-reperfusion induced
myocardial injury. The involvement of MPTP in brain ischemia induced
damage is less clear. MPTP causes permeabilization of
mitochondrial membranes leading to the release of cytochrome c
from mitochondria, mitochondrial dysfunction, caspase activation and
apoptotic or necrotic death of cardiac or neuronal cells depending on
cellular resources of ATP. In recent years various strategies aiming at
pharmacological inhibition of MPTP during ischemia or reperfusion
have been proposed, though the molecular mechanisms of regulation
of MPTP are still unclear. The molecular composition of MPTP is also
unresolved question. We have recently shown that low levels of NO may
activate signalling pathways in cardiac and neuronal cells that involve
protein kinases C and G which, in turn, may increase resistance of
mitochondria to Ca<sup>2+</sup>- and ischemia-induced opening of MPTP. Analysis
of phosphoproteome of mitochondria isolated from hearts treated with
NO revealed several mitochondrial proteins, phosphorylation of which is
affected by this treatment and which may be involved in MPTP formation.
In this lecture, we will discuss current knowledge on structure of MPTP,
experimental approaches to investigate MPTP functions and possible
role of MPTP in ischemic heart and brain damage. +
A growing body of evidence suggests that neurodegeneration in Alzheimer‘s disease (AD) is related to extracellular and intracellular accumulation of amyloid beta peptide (Aβ), mitochondrial dysfunction, increased neuronal loss, however the molecular pathways from Aβ to the main pathological hallmarks of AD are still elusive. Aβ molecules tend to aggregate and form complexes of varying size - from small soluble oligomers, bigger protofibrils and large insoluble fibrils. It is commonly assumed that formation of Aβ fibrils is the crucial event in the pathogenesis of AD. However, there is accumulating evidence that soluble oligomers are the most cytotoxic forms of Aβ though it is still unclear particles of which size and morphology exert most neurotoxicity. In our study we aimed to investigate a link between the size of soluble Aβ oligomers and their toxicity to rat cerebellar granule cells (CGC), cortical neurons and other non-neuronal cells. Variation in conditions during ''in vitro'' oligomerization of Aβ1-42 resulted in peptide assemblies with different particle size. Small oligomeric forms of Aβ1-42 with a particle z-height of 1-2 nm (as measured by atomic force microscopy) were found to be the most toxic species, inducing rapid neuronal necrosis at submicromolar concentrations, whereas the bigger aggregates (above 4-5 nm) did not cause detectable neuronal death. Aβ1-42 oligomers, monomers and fibrils were non-toxic to glial cells in CGC cultures or macrophage J774 cells. Small oligomers of Aβ exhibited tendency to bind to the phospholipid vesicles which composition was similar to reported neuronal plasma membrane composition. In contrast, bigger, non-toxic oligomers did not bind to phospholipid vesicles.
We also found that mitochondrial respiratory functions were not affected by Aβ1-42 irrespective of the aggregate state: monomers, oligomers or fibrils of Aβ at concentrations up to 2 µM did not inhibit state 3 and state 4 respiration of isolated brain mitochondria and did not cause permeabilization of mitochondrial outer membrane as measured by the exogenous cytochrome c test on mitochondrial respiration. This suggests that Aβ1-42 at pathophysiologically relevant concentrations has no acute effect on mitochondria.
In conclusion, our data demonstrate that small oligomers of Aβ at submicromolar concentrations induce rapid neuronal necrosis most likely due to the effect on neuronal plasma membranes, whereas bigger aggregates are not directly toxic to neurons.
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The lactate / pyruvate ratio is an important criterion for determining possible disturbances in mitochondrial metabolism, especially in patients with neurological dysfunctions [3].
Lactate elevation occurs because the flux through glycolysis overwhelms the utilization of pyruvate in the mitochondria. Several studies have shown that in patients with primary mitochondrial disease, truly elevated lactate levels have sensitivity between 34 and 62% and specificity between 83 and 100%. The blood lactate/pyruvate ratio is most reliable in differentiating electron transport chain (ETC) disease from disorders of pyruvate metabolism, but it’s a question whether it’s important only when lactate levels are high [1]. The sensitivity of this ratio is 31%, with a specificity of 100 [4].
A consensus statement from the Mitochondrial Medicine Society from 2014 states lactate:pyruvate ratio as one of the important diagnostic tools for the mitochondrial diseases [2].
We determined the level of lactate, pyruvate and the lactate / pyruvate ratio in 30 children with neurological disorders and suspected mitochondrial dysfunction. Blood sampling was performed with a test tube with NaF system Venosafe. The analysis was performed by spectrophotometry.
According to the obtained data, 83 % of patients have elevated lactate-pyruvate ratio, but only 20% from them have also elevated lactate level. Patients with elevated lactate level had lactate/pyruvate ratio elevation about 3 times higher than upper reference, estimated by the laboratory.
So lactate/pyruvate ratio is a more sensitive marker, but it needs further investigation for approving its’ clinical significance. It is also important to re-estimate reference for this indicator. +
Age-associated mitochondrial dysfunction and oxidative damage are primary causes for multiple health problems including sarcopenia and cardiovascular disease (CVD). Though the role of Nrf2, a transcription factor that regulates cytoprotective gene expression, in myopathy remains poorly defined, it has shown beneficial properties in both sarcopenia and CVD. Sulforaphane (SFN), a natural compound Nrf2-related activator of cytoprotective genes, provides protection in several disease states including CVD and is in various stages of clinical trials, from cancer prevention to reducing insulin resistance. This study aimed to determine whether SFN may prevent age-related loss of function in the heart and skeletal muscle. Cohorts of 2-month-old and 21- to 22-month-old mice were administered regular rodent diet or diet supplemented with SFN for 12 weeks. At the completion of the study, skeletal muscle and heart function, mitochondrial function, and Nrf2 activity were measured. Our studies revealed a significant drop in Nrf2 activity and mitochondrial functions, together with a loss of skeletal muscle and cardiac function in the old control mice compared to the younger age group. In the old mice, SFN restored Nrf2 activity, mitochondrial function, cardiac function, exercise capacity, glucose tolerance, and activation/differentiation of skeletal muscle satellite cells. Our results suggest that the age-associated decline in Nrf2 signaling activity and the associated mitochondrial dysfunction might be implicated in the development of age-related disease processes. Therefore, the restoration of Nrf2 activity and endogenous cytoprotective mechanisms by SFN may be a safe and effective strategy to protect against muscle and heart dysfunction due to aging. +
Perilipin 5 (PLIN5/OXPAT) is a lipid droplet (LD) coat protein mainly present in tissues with a high fat-oxidative capacity, suggesting a role for PLIN5 in facilitating fatty acid oxidation. Here, we investigated the role of PLIN5 in fat oxidation in skeletal muscle. In human skeletal muscle, we observed that PLIN5 (but not PLIN2) protein content correlated tightly with OXPHOS content and in rat muscle PLIN5 content correlated with mitochondrial respiration rates on a lipid-derived substrate. This prompted us to examine PLIN5 protein expression in skeletal muscle mitochondria by means of immunogold electron microscopy and Western blots in isolated mitochondria. These data show that PLIN5, in contrast to PLIN2, not only localizes to LD but also to mitochondria, possibly facilitating fatty acid oxidation. Unilateral overexpression of PLIN5 in rat anterior tibialis muscle augmented myocellular fat storage without increasing mitochondrial density as indicated by the lack of change in protein content of five components of the OXPHOS system. Mitochondria isolated from PLIN5 overexpressing muscles did not possess increased fatty acid respiration. Interestingly though, (14)C-palmitate oxidation assays in muscle homogenates from PLIN5 overexpressing muscles revealed a 44.8% (''P'' = 0.05) increase in complete fatty acid oxidation. Thus, in mitochondrial isolations devoid of LD, PLIN5 does not augment fat oxidation, while in homogenates containing PLIN5-coated LD, fat oxidation is higher upon PLIN5 overexpression. The presence of PLIN5 in mitochondria helps to understand why PLIN5, in contrast to PLIN2, is of specific importance in fat oxidative tissues. Our data suggests involvement of PLIN5 in directing fatty acids from the LD to mitochondrial fatty acid oxidation. +
This invention relates to brown adipose tissue (BAT) progenitor cells and methods for isolating BAT progenitor cells from skeletal muscle. BAT progenitor cell surface markers and medium and agents for inducing cell differentiation into brown adipocytes are also provided. In some embodiments, the BAT progenitor cell expresses a first cell surface marker associated with endothelial cells, the first cell surface marker being detectable in an antibody based assay using a first antibody. In addition, the BAT progenitor cell can be substantially free of a second cell surface marker associated with endothelial cells, the second cell surface marker being substantially undetectable in said antibody based assay using a second antibody. The BAT progenitor cell can also be substantially free of additional cell surface markers. +
Nitrosothiols (RSNO), formed from thiols and metabolites of nitric oxide (•NO), have been implicated in a diverse set of physiological and pathophysiological processes, although the exact mechanisms by which they are formed biologically are unknown. Several candidate nitrosative pathways involve the reaction of •NO with O<sub>2</sub>, reactive oxygen species (ROS), and transition metals. We developed a strategy using extracellular ferrocyanide to determine that under our conditions intracellular protein RSNO formation occurs from reaction of •NO inside the cell, as opposed to cellular entry of nitrosative reactants from the extracellular compartment. Using this method we found that in RAW 264.7 cells RSNO formation occurs only at very low (<8 μM) O<sub>2</sub> concentrations and exhibits zero-order dependence on •NO concentration. Indeed, RSNO formation is not inhibited even at O<sub>2</sub> levels <1 μM. Additionally, chelation of intracellular chelatable iron pool (CIP) reduces RSNO formation by >50%. One possible metal-dependent, O<sub>2</sub>-independent nitrosative pathway is the reaction of thiols with dinitrosyliron complexes (DNIC), which are formed in cells from the reaction of •NO with the CIP. Under our conditions, DNIC formation, like RSNO formation, is inhibited by ≈50% after chelation of labile iron. Both DNIC and RSNO are also increased during overproduction of ROS by the redox cycler 5,8-dimethoxy-1,4-naphthoquinone. Taken together, these data strongly suggest that cellular RSNO are formed from free •NO via transnitrosation from DNIC derived from the CIP. We have examined in detail the kinetics and mechanism of RSNO formation inside cells. +
BACKGROUND:
In normal-weight subjects, resting energy expenditure (REE) can be accurately calculated from organ and tissue masses applying constant organ-specific metabolic rates. This approach allows a precise correction for between-subjects variation in REE, explained by body composition. Since a decrease in organ metabolic rate with increasing organ mass has been deduced from interspecies comparison including human studies, the validity of the organ- and tissue-specific REE calculation remains to be proved over a wider range of fat-free mass (FFM).
DESIGN:
In a cross-sectional study on 57 healthy adults (35 females and 22 males, 19-43 y; 14 underweight, 25 intermediate weight and 18 obese), magnetic resonance imaging (MRI) and dual-energy X-ray absorptiometry (DXA) were used to assess the masses of brain, internal organs, skeletal muscle (MM), bone and adipose tissue. REE was measured by indirect calorimetry (REEm) and calculated from detailed organ size determination by MRI and DXA (REEc1), or in a simplified approach exclusively from DXA (REEc2).
RESULTS:
We found a high agreement between REEm and REEc1 over the whole range of FFM (28-86 kg). REE prediction errors were -17 +/- 505, -145 +/- 514 and -141 +/- 1058 kJ/day in intermediate weight, underweight and obese subjects, respectively (n.s.). Regressing REEm on FFM resulted in a significant positive intercept of 1.6 MJ/day that could be reduced to 0.5 MJ/day by adjusting FFM for the proportion of MM/organ mass. In a multiple regression analysis, MM and liver mass explained 81% of the variance in REEm. DXA-derived REE prediction showed a good agreement with measured values (mean values for REEm and REEc2 were 5.72 +/- 1.87 and 5.82 +/- 1.51 MJ/day; difference n.s.).
CONCLUSION:
Detailed analysis of metabolically active components of FFM allows REE prediction over a wide range of FFM. The data provide indirect evidence for a view that, for practical purposes within humans, the specific metabolic rate is constant with increasing organ mass. Nonlinearity of REE on FFM was partly explained by FFM composition. A simplified REE prediction algorithm from regional DXA measurements has to be validated in future studies.
Shorter than average adults are at a higher risk for obesity and are also more susceptible to diabetes and CVD, independent of BMI. In contrast, taller children have a higher risk of obesity. We hypothesised that short stature is related to adverse body composition and that the association between stature and obesity differs between generations. In a cross-sectional German database of 213 804 adults and 12 411 children and adolescents, the prevalence of overweight and obesity was compared between percentiles of height. The association between stature and percentage of fat mass (%FM), lean BMI (LBMI; kg/m2) or waist:hip ratio (in children only) was analysed within BMI groups. In adults, the prevalence of BMI >30 kg/m2 gradually increased with decreasing percentile of height whereas in children and adolescents, a positive association between height and weight status was observed. Short-stature women and girls had a 0.8-3.2 % lower %FM than tall subjects (''P'' < 0.05), whereas no trend for %FM was observed in males. When compared with tall subjects, LBMI was 0.2-0.6 kg/m2 lower in short-stature men, as well as obese women (''P'' < 0.05). There was a non-significant trend for a lower LBMI and a higher waist:hip ratio in shorter children. In conclusion, short stature is associated with an increased risk of obesity in adults. Cardiometabolic risk in short stature is not explained by an adverse body composition. +
Mitochondria are responsible for roughly 90% of the ATP produced in a cell. A consequence of aerobic metabolism is oxidative stress that results from production of mitochondrial reactive oxygen species (ROS) due to inefficiency of electron transport. Several antioxidant-redox coupled reactions in the mitochondria help minimize oxidative damage in the mitochondria. These redox reactions not only protect mitochondria from oxidative damage but also are important in regulating cellular redox status. Oxidative stress from mitochondrial ROS occurs in broilers in pulmonary hypertension syndrome, heat stress, and in the phenotypic expression of feed efficiency. Low levels of mitochondrial ROS are now recognized to play important roles in signal transduction mechanisms. A topology of ROS production has been reported that indicates that ROS derived from Complex I primarily cause oxidative damage, whereas ROS generated from Complex III are primarily involved in cell signaling. Reverse electron transport, once considered an artifact of in vitro conditions, now plays significant roles in physiological conditions including inflammation, ischemia-reperfusion, muscle differentiation, and energy utilization. Understanding the balancing act that mitochondria play in health and disease will continue to be vital biological component of improving efficiency in animal production. +
Given the role of mitochondria in modulating many cellular functions, it is not surprising that they can play a crucial role also in molecular pathophysiology of cancer. In particular, the discovery in recent decades of a link between cancer metabolic processes, alterations of mitochondrial DNA, oncogenes and tumor suppressors has led not only to a renaissance of interest in Warburg's pioneering work, but also to a reexamination of his original observations above all in relation to the current knowledge in cancer cell metabolism. It follows that, although mitochondrial contribution to the pathogenesis of cancer has historically tended to be neglected, it is now evident that reprogrammed mitochondria can contribute to a complex bioenergetic adjustment that sustains not only tumor formation but also its progression. Most importantly, cancer cell metabolism seems to have a role in diversified aspects related to cancer pathophysiology (i.e., aggressiveness, recurrence, metastatic dissemination). Hence, it is imperative to always consider cancer cell metabolism, its adaptability, its influences but, above all, its functional heterogeneity in a single tumor, for a really rational and valid approach towards molecular biology of cancer. +
Mitochondrial dynamics is a possible modulator of myocardial ischemia/reperfusion injuries (IRI). We previously reported that mice partially deficient in the fusion protein OPA1
exhibited higher IRI. Therefore, we investigated whether deficiency in the fission protein
DRP1 encoded by Dnm1l gene would affect IRI in Dnm1l<sup>+/-</sup> mouse. After baseline characterization of the Dnm1l<sup>+/-</sup> mice heart, using echocardiography, electron microscopy, and
oxygraphy, 3-month-old Dnm1l<sup>+/-</sup> and wild type (WT) mice were exposed to myocardial
ischemia/reperfusion (I/R). The ischemic area-at-risk (AAR) and area of necrosis (AN)
were delimited, and the infarct size was expressed by AN/AAR. Proteins involved in mitochondrial dynamics and autophagy were analyzed before and after I/R. Mitochondrial permeability transition pore (mPTP) opening sensitivity was assessed after I/R. Heart weight
and left ventricular function were not significantly different in 3-, 6- and 12-month-old
Dnm1l<sup>+/-</sup> mice than in WT. The cardiac DRP1 protein expression levels were 60% lower,
whereas mitochondrial area and lipid degradation were significantly higher in Dnm1l<sup>+/-</sup>
mice than in WT, though mitochondrial respiratory parameters and mPTP opening did not
significantly differ. Following I/R, the infarct size was significantly smaller in Dnm1l<sup>+/-</sup> mice
than in WT (34.6±3.1% vs. 44.5±3.3%, respectively; p<0.05) and the autophagic markers,
LC3 II and P62 were significantly increased compared to baseline condition in Dnm1l<sup>+/-</sup>
mice only. Altogether, data indicates that increasing fusion by means of Dnm1l deficiency
was associated with protection against IRI, without alteration in cardiac or mitochondrial
functions at basal conditions. This protection mechanism due to DRP1 haploinsufficiency
increases the expression of autophagic markers. +
The developmental origins of health and disease (DoHAD) hypothesis suggests that negative maternal lifestyle choices, such as obesity, affect the health of her offspring. Clinical and laboratory studies support this hypothesis – offspring born to obese mothers are at increased risk for health conditions including cardiometabolic syndrome and congenital abnormalities. Maternal obesity damages the oocytes, contributing to the increased disease risk by transmitting damaged organelles and epigenetic modifications to the offspring. Mitochondria, the most abundant organelle in the oocyte, are damaged in oocytes from obese females. However, we do not understand if mitochondrial damage in oocytes is reversible nor why offspring are at increased risk for cardiometabolic syndrome like cardiomyopathy. Here we show that in mice fed a high fat/high sugar (HF/HS diet), improving female health with moderate, voluntary exercise does not reverse oocyte damage. We also tested if oocytes could activate mitophagy to repair obesity induced mitochondrial damage. Finally, we show that female offspring from obese mothers have mitochondrial damage in the heart that persists into adulthood. This damage causes dilated cardiomyopathy that worsens with age. These results provide an explanation for the persistence of damaged mitochondria in the oocytes of obese females. Additionally, they suggest that maternal obesity promotes the development of heart failure in offspring by inducing mitochondrial damage in the heart. Together, this data suggests mitochondrial damage caused by maternal obesity is non-reversible and contributes to cardiometabolic syndrome. The research provides potential mechanisms that support the DoHAD hypothesis and open new questions about how the changes to offspring health occur. +
[[File:Bouillaud-photo.JPG|right|200px|Frédéric Bouillaud]]Sulfide (H<sub>2</sub>S gas, HS<sup>-</sup> anion) shows the same toxicity as cyanide, NO or CO for Complex IV (cytochrome oxidase; CIV). NO, CO and H<sub>2</sub>S are considered as gasotransmitters of physiological relevance. While signaling is expected to occur by different pathways, the question of involvement of mitochondria remains sometimes unresolved and particularly because some experiments used concentrations relevant to bioenergetics. Cellular metabolism generates low amount of sulfide and the activity of bacteria in the colonic lumen exposes the colonic wall to extracellular concentrations of sulfide [#60 µM], large enough to inhibit cellular respiration. Therefore, the question of sulfide disposal needs to be adressed. Mitochondria themselves appear as best candidate to explain sulfide disposal: a ''Sulfide Oxidation Unit'' (SOU) oxidizes sulfide into thiosulfate in many cell types in culture and in mitochondria from liver, heart or kidney. When sulfide is infused to mitochondria or cells at rates that stay within their sulfide oxidation capacities they oxidize it and maintain a low (<500 nM with cells) external concentration of sulfide well behind the toxic level (IC50 10-20 µM with cells). Notably, SOU activity could not be detected in brain mitochondria or neuroblastoma cells making them intolerant to sulfide. SOU is constituted by a sulfide quinone reductase (SQR) associated with a sulfur transferase and a dioxygenase. Present knowledge indicates that two molecules of H<sub>2</sub>S and one of oxygen (O<sub>2</sub>) are consumed by SOU to deliver two electrons to quinone. Then the stoichiometry for mitochondrial respiration based on sulfide oxidation is (1+0.5) O<sub>2</sub> / 2 H<sub>2</sub>S = 0.75 and for the same rate of electron transfer three times more oxygen are needed than with NADH/FADH<sub>2</sub> coenzymes. In consequence, infusing sulfide immediately and significantly increases oxygen consumption of respiring cells/mitochondria.
Mitochondria show a high affinity for sulfide and its oxidation usually takes priority over ongoing mitochondrial oxidation processes. Something that is mandatory to ensure efficient sulfide disposal in presence of largely greater intracellular concentrations of other substrates. Extracellular sulfide is therefore a remarkable substrate: it could be used at nanomolar concentrations, its gaseous nature allows fast transfer to mitochondria without transporters, it is directly usable to reduce quinone without metabolic processing (including for example initial ATP consuming steps). This metabolic role of sulfide and its reductant properties contrast sharply with the other two gazotransmitters and particularly with NO which is pro-oxidant. There are still uncertainties about the lowest concentration of sulfide that SQR would ''detect'' but, in physiological conditions, the overlap between bioenergetically relevant and signaling concentrations appears even more likely with sulfide than with NO or CO.
Colonocytes are adapted to high sulfide exposure. In these cells ''reverse bioenergetic reactions'' including reverse electron flux in Complex I are taking place to ensure continuation of a fast sulfide oxidation even if CIV is inhibited.
Sulfide (H2S, HS-, S2-) is highly toxic because it is an inhibitor of mitochondrial Complex IV similar to cyanide, NO or CO [1] and high nanomolar concentration of sulfide are sufficient to inhibit 50% of cytochrome oxidase activity [1,2]. However, when respiration of intact cells is measured, concentrations above 10 µM are needed to reach a significant inhibition. In mammals the metabolism of sulfur containing amino acids releases sulfide that if unchecked would be rising to micromolar values within minutes. Moreover, the anaerobic metabolism of the bacteria in the colonic lumen raises concentrations of free sulfide to 60 µM or even to mM concentrations if one considers the bound sulfide. Protection of cells and particularly of colonocytes against sulfide toxicity is thus required [3,4].
Sulfide at least in the H2S form (20-30% of free sulfide at physiological pH) is a hydrogen donor and is used by the mitochondrial respiratory system as a substrate [4,5]. The enzyme involved is the [[sulfide quinone reductase]] (SQR), associated with two other enzymes (a dioxygenase and a sulfur transferase) to release thiosulfate (H2S2O3). Initiation of SQR activity is detectable with nM concentration of sulfide. Two sulfide molecules are engaged, one molecule of dioxygen is used by the dioxygenase and one atom of oxygen by the cytochrome oxidase and thus for the same electron transfer through Complexes III and IV three times more oxygen are needed. The SQR activity was first demonstrated in colonocytes [4] but later on it was recognized that it is present in a large variety of cells/organs [5]. It is thought to reflect the need of a continuous sulfide disposal to eliminate endogenous release. Moreover, sulfide like NO or CO is though to be a gasotransmitter and SQR activity appears relevant to sulfide signaling/pharmacology [6].
As external concentration requires to be lower than 10 µM the establishment of a steady state for sulfide oxidation requires a continuous infusion of sulfide [4]. Because of the stoichiometry of sulfide oxidation this infusion increases the oxygen consumption of the cells/mitochondria even if a “normal” carbon based oxidation was taking place [5]. If the flux of sulfide (JH2S) is too high inhibition of respiration and of oxygen consumption develops quickly [5]. Therefore according to the intensity of sulfide exposure (concentration and flux) sulfide could either stimulate or inhibit cellular oxygen consumption [5]. A convenient way to express the sulfide challenge is the ratio between the flux of sulfide delivered and the respiratory rate before sulfide infusion (''J''H2S/''J''O2) indicating a relative sulfide exposure [5]. A few cell types including colonocytes tolerate values above 2 [5].
Statins protect against cardiovascular-related mortality but induce skeletal muscle toxicity [1]. To investigate mechanisms of statins, we tested the hypothesis that statins optimized cardiac mitochondrial function but impaired vulnerable skeletal muscle by inducing different level of reactive oxygen species (ROS). In atrium of patients treated with statins, ROS production was decreased and oxidative capacities were enhanced together with an extensive augmentation of mRNAs expression of PGC-1 family. However, in deltoid biopsies from patients with statin-induced muscular myopathy, oxidative capacities were decreased together with ROS increase and a collapse of PGC-1 mRNA expression. Several animal and cell culture experiments were conducted and showed by using ROS scavengers that ROS production was the triggering factor responsible of atorvastatin-induced activation of mitochondrial biogenesis pathway and improvement of antioxidant capacities. Conversely, in skeletal muscle, the large augmentation of ROS production following treatment induced mitochondrial impairments, and reduced mitochondrial biogenesis mechanisms. Quercetin, an antioxidant molecule, was able to counteract skeletal muscle deleterious effects of atorvastatin in rat [2].
Our findings identify statins as a new activating factor of cardiac mitochondrial biogenesis and antioxidant capacities, and suggest the importance of ROS/PGC-1 signalling pathway as a key element in regulation of mitochondrial function in cardiac as well as skeletal muscles.
References:
1. Bouitbir J, Charles AL, Rasseneur L, Dufour S, Piquard F, Geny B, Zoll J (2011) Atorvastatin treatment reduces exercise capacities in rats: involvement of mitochondrial impairments and oxidative stress. J Appl Physiol 111: 1477-1483.
2. Bouitbir J, Charles AL, Echaniz-Laguna A, Kindo M, Daussin F, Auwerx J, Piquard F, Geny B, Zoll J (2011) Opposite effects of statins on mitochondria of cardiac and skeletal muscles: a “mitohormesis” mechanism involving reactive oxygen species and PGC-1. Eur Heart J (in press).
AIMS: Although statins are the most widely used cholesterol-lowering agents, they are associated with a variety of muscle complaints. The goal of this study was to characterize the effects of statins on the mitochondrial apoptosis pathway induced by mitochondrial oxidative stress in skeletal muscle using human muscle biopsies as well as ''in vivo'' and ''in vitro'' models.
RESULTS:
Statins increased mitochondrial H<sub>2</sub>O<sub>2</sub> production, the Bax/Bcl-2 ratio and TUNEL staining in deltoid biopsies of patients with statin-associated myopathy. Furthermore, atorvastatin treatment for two weeks at 10 mg/kg/day in rats increased H<sub>2</sub>O<sub>2</sub> accumulation, and mRNA levels and immunostaining of the Bax/Bcl-2 ratio, as well as TUNEL staining and caspase 3 cleavage in glycolytic (plantaris) skeletal muscle but not in oxidative (soleus) skeletal muscle, which has a high antioxidative capacity. Atorvastatin also decreased the GSH/GSSG ratio, but only in glycolytic skeletal muscle. Co-treatment with the antioxidant quercetin at 25 mg/kg/d abolished these effects in plantaris. An ''in vitro'' study with L6 myoblasts directly demonstrated the link between mitochondrial oxidative stress following atorvastatin exposure and activation of the mitochondrial apoptosis signaling pathway.
INNOVATION:
Treatment with atorvastatin is associated with mitochondrial oxidative stress, which activates apoptosis and contributes to myopathy. Glycolytic muscles are more sensitive to atorvastatin than oxidative muscles, which may be due to the higher antioxidative capacity in oxidative muscles.
CONCLUSION:
There is a link between statin-induced mitochondrial oxidative stress and activation of the mitochondrial apoptosis signaling pathway in glycolytic skeletal muscle, which may be associated with statin-associated myopathy. +
Sunitinib is cardiotoxic, but the mechanisms are not entirely clear. We aimed to enlarge our knowledge about the role of mitochondria in cardiac toxicity of sunitinib ''in vitro'' and ''in vivo''. For this reason, we studied the toxicity of sunitinib on cardiac H9c2 cells exposed for 24 h, permeabilized rat cardiac fibers exposed for 15 minutes and in mice treated orally with sunitinib for 2 weeks (7.5 mg/kg/day). In H9c2 cells exposed for 24 h, sunitinib was more cytotoxic under galactose (favoring mitochondrial metabolism) compared to glucose conditions (favoring glycolysis). Sunitinib dissipated the mitochondrial membrane potential starting at 10 μM under glucose and at 5 μM under galactose conditions. Sunitinib reduced activities of mitochondrial enzyme complexes of the electron transport chain (ETC), increased mitochondrial ROS accumulation and decreased the cellular GSH pool. Electron microscopy revealed swollen mitochondria with loss of cristae. Accordingly, sunitinib caused caspase 3 activation and DNA fragmentation in H9c2 cells. Co-exposure with mito-TEMPO (mitochondrial-specific ROS scavenger) for 24 h prevented ATP and GSH depletion, as well as the increases in H<sub>2</sub>O<sub>2</sub> and caspase 3/7 activity observed with sunitinib. In mice, treatment with sunitinib for two weeks increased plasma concentrations of troponin I and creatine kinase MB, indicating cardiomyocyte damage. The activity of enzyme complexes of the ETC was decreased, mitochondrial ROS were increased and cleavage of caspase 3 was increased, suggesting cardiomyocyte apoptosis. In conclusion, mitochondrial damage with ROS accumulation appears to be an important mechanism of cardiotoxicity associated with sunitinib, eventually leading to apoptotic cell death.
<small>Copyright © 2019. Published by Elsevier B.V.</small> +
Tyrosine kinase inhibitors (TKIs) are associated with cardiac toxicity, which may be caused by mitochondrial toxicity. The underlying mechanisms are currently unclear and require further investigation. In the present study, we aimed to investigate in more detail the role of the enzyme Complexes of the electron transfer system (ETS), mitochondrial oxidative stress, and mechanisms of cell death in cardiac toxicity associated with imatinib and sorafenib. Cardiac myoblast H9c2 cells were exposed to imatinib and sorafenib (1 to 100 µM) for 24 h. Permeabilized rat cardiac fibers were treated with both drugs for 15 min. H9c2 cells exposed to sorafenib for 24 h showed a higher membrane toxicity and ATP depletion in the presence of galactose (favoring mitochondrial metabolism) compared to glucose (favoring glycolysis) but not when exposed to imatinib. Both TKIs resulted in a higher dissipation of the mitochondrial membrane potential in galactose compared to glucose media. Imatinib inhibited Complex I (CI)- and CIII-linked respiration under both conditions. Sorafenib impaired CI-, CII-, and CIII-linked respiration in H9c2 cells cultured with glucose, whereas it inhibited all ETS complexes with galactose. In permeabilized rat cardiac myofibers, acute exposure to imatinib and sorafenib decreased CI- and CIV-linked respiration in the presence of the drugs. Electron microscopy showed enlarged mitochondria with disorganized cristae. In addition, both TKIs caused mitochondrial superoxide accumulation and decreased the cellular GSH pool. Both TKIs induced caspase 3/7 activation, suggesting apoptosis as a mechanism of cell death. Imatinib and sorafenib impaired the function of cardiac mitochondria in isolated rat cardiac fibers and in H9c2 cells at plasma concentrations reached in humans. Both imatinib and sorafenib impaired the function of enzyme complexes of the ETS, which was associated with mitochondrial ROS accumulation and cell death by apoptosis. +
Dihydroorotate dehydrogenase (DHODH) is an enzyme of the de novo pyrimidine synthesis pathway that provides nucleotides for RNA/DNA synthesis essential for proliferation. In mammalian cells, DHODH is localized in mitochondria, linked to the respiratory chain via the coenzyme Q pool. Here we discuss the role of DHODH in the oxidative phosphorylation system and in the initiation and progression of cancer. We summarize recent findings on DHODH biology, the progress made in the development of new, specific inhibitors of DHODH intended for cancer therapy, and the mechanistic insights into the consequences of DHODH inhibition. +
For the purposes of these rules, a preprint is defined as a complete written description of a body of scientific work that has yet to be published in a journal. Typically, a preprint is a research article, editorial, review, etc. that is ready to be submitted to a journal for peer review or is under review. It could also be a commentary, a report of negative results, a large data set and its description, and more. Finally, it could also be a paper that has been peer reviewed and either is awaiting formal publication by a journal or was rejected, but the authors are willing to make the content public. In short, a preprint is a research output that has not completed a typical publication pipeline but is of value to the community and deserving of being easily discovered and accessed. We also note that the term preprint is an anomaly, since there may not be a print version at all. The rules that follow relate to all these preprint types unless otherwise noted. +
Aims/hypothesis: Insulin resistance and type 2 diabetes are associated with mitochondrial dysfunction. The aim of the present study was to test the hypothesis that oxidative phosphorylation and electron transport capacity are diminished in the skeletal muscle of type 2 diabetic subjects, as a result of a reduction in the mitochondrial content.
Materials and methods: The O<sub>2</sub> flux capacity of permeabilised muscle fibres from biopsies of the quadriceps in healthy subjects (''n''=8; age 58±2 years [mean±SEM]; BMI 28±1 kg/m<sup>2</sup>; fasting plasma glucose 5.4±0.2 mmol/l) and patients with type 2 diabetes (''n''=11; age 62±2 years; BMI 32±2 kg/m<sup>2</sup>; fasting plasma glucose 9.0±0.8 mmol/l) was measured by high-resolution respirometry.
Results: O<sub>2</sub> flux expressed per mg of muscle (fresh weight) during ADP-stimulated state 3 respiration was lower ( ''p''<0.05) in patients with type 2 diabetes in the presence of complex I substrate (glutamate) (31±2 vs 43±3 pmol O<sub>2</sub> s<sup>-1</sup> mg<sup>-1</sup>) and in response to glutamate + succinate (parallel electron input from complexes I and II) (63±3 vs 85±6 pmol s<sup>-1</sup> mg<sup>-1</sup>). Further increases in O<sub>2</sub> flux capacity were observed in response to uncoupling by FCCP, but were again lower ( ''p''<0.05) in type 2 diabetic patients than in healthy control subjects (86±4 vs 109±8 pmol s<sup>-1</sup> mg<sup>-1</sup>). However, when O<sub>2</sub> flux was normalised for mitochondrial DNA content or citrate synthase activity,there were no differences in oxidative phosphorylation or electron transport capacity between patients with type 2 diabetes and healthy control subjects.
Conclusions/interpretation: Mitochondrial function is normal in type 2 diabetes. Blunting of coupled and uncoupled respiration in type 2 diabetic patients can be attributed to lower mitochondrial content. +
Across a wide range of species and body mass a close matching exists between maximal conductive oxygen delivery and mitochondrial respiratory rate. In this study we investigated in humans how closely ''in vivo'' maximal oxygen consumption (''V''O(2)max) is matched to muscle tissue-specific OXPHOS capacity ([[State 3]]) respiration. High-resolution respirometry was used to quantify mitochondrial respiration from the biopsies of arm and leg muscles while ''in vivo'' arm and leg ''V''O(2) were determined by the Fick method during leg cycling and arm cranking. We hypothesized that muscle mitochondrial respiratory rate exceeds that of systemic oxygen delivery. OXPHOS capacity of the deltoid muscle (4.3±0.4 mmol O(2)kg(-1)min(-1)) was similar to the ''in vivo'' ''V''O(2) during maximal arm cranking (4.7±0.5 mmol O(2)kg(-1)min(-1)) with 6 kg muscle. In contrast, the mitochondrial OXPHOS capacity of the quadriceps was 6.9±0.5 mmol O(2)kg(-1)min(-1), exceeding the ''in vivo'' leg ''V''O(2)max (5.0±0.2mmolO(2)kg(-1)min(-1)) during leg cycling with 20 kg muscle (''P''<0.05). Thus, when half or more of the body muscle mass is engaged during exercise, muscle mitochondrial respiratory capacity surpasses ''in vivo'' ''V''O(2)max. The findings reveal an excess capacity of muscle mitochondrial respiratory rate over O(2) delivery by the circulation in the cascade defining maximal oxidative rate in humans. +
Nitric oxide (NO) and prostaglandins (PG) together play a role in regulation blood flow during exercise. NO also regulates mitochondrial oxygen consumption through competitive binding to cytochrome ''c'' oxidase. Indomethacin both uncouples and inhibits the electron transport chain in a concentration-dependent manner, and thus inhibition of NO and PG may regulate both muscle oxygen delivery and utilization. The purpose of this study was to examine the independent and combined effects of NO and PG blockade (L-NMMA and indomethacin respectively) on mitochondrial respiration in human muscle following knee extension (KE) exercise. Mitochondrial respiration was measured ''ex-vivo'' by high-resolution respirometry in saponin-permeabilized fibers following 6 min KE in control (CON, ''n''=8), arterial infusion of LNMMA (''n''=4) and Indo (''n''=4) followed by combined inhibition of NO and PG (L-NMMA + Indo, n=8). ADP-stimulated [[State 3]] respiration with substrates for Complex I (glutamate, malate) was reduced 50% by Indo. State 3 O(2) flux with Complex I and II substrates was reduced less with both Indo (20%) and L-NMMA + Indo (15%) compared to CON. The results indicate that indomethacin reduces State 3 mitochondrial respiration primarily at Complex I of the respiratory chain while blockade of NO by addition of L-NMMA counteracts the inhibition of Indo. This metabolic effect in concert with a reduction of blood flow likely accounts for ''in-vivo'' changes in muscle O<sub>2</sub> consumption during combined blockade of NO and PG. +
Maximal oxygen consumption (''V''<sub>O2</sub>) in the organism is defined by the integrated functional capacities of multiple organ systems and characterized by proportionate design of the structural components of the O2 cascade from lung to mitochondria across a wide range of species. Yet, humans exhibit an excess capacity of muscle mitochondrial OXPHOS capacity relative to convective O2 delivery [1,2]. This pattern holds with aging and in chronic diseases such as chronic obstructive lung disease and type 2 diabetes despite a lower expression and/or dysfunction of mitochondria. Muscle diffusional O<sub>2</sub> conductance (''D''<sub>O2</sub>) is largely dependent on the capillary volume:muscle interface which influences the mean transit time of erythrocytes and off-loading of O2 from haemoglobin at a given ''p''<sub>50</sub> and blood flow. Convective O<sub>2</sub> delivery remains a dominant factor defining maximal ''V''<sub>O2</sub> as revealed by comparison of muscle ''D''<sub>O2</sub>, mean capillary ''p''<sub>O2</sub>, and ''V''<sub>O2</sub> during exercise engaging small to large muscle groups. The increase in muscle ''V''<sub>O2</sub> with endurance training in young, healthy humans is characterized by proportional increases in O<sub>2</sub> convection and diffusion, with variable enhancement of mitochondrial OXPHOS capacity, which remains in excess to maximize the ''p''<sub>O2</sub> gradient from the red blood cell to cytochrome ''c'' oxidase. +
Muscle mitochondrial respiratory capacity measured ''ex vivo'' provides a physiological reference to assess cellular oxidative capacity as a component in the oxygen cascade ''in vivo''. In this article, the magnitude of muscle blood flow and oxygen uptake during exercise involving a small-to-large fraction of the body mass will be discussed in relation to mitochondrial capacity measured ''ex vivo''. These analyses reveal that as the mass of muscle engaged in exercise increases from one-leg knee extension, to 2-arm cranking, to 2-leg cycling and x-country skiing, the magnitude of blood flow and oxygen delivery decrease. Accordingly, a 2-fold higher oxygen delivery and oxygen uptake per unit muscle mass are seen ''in vivo'' during 1-leg exercise compared to 2-leg cycling indicating a significant limitation of the circulation during exercise with a large muscle mass. This analysis also reveals that mitochondrial capacity measured ''ex vivo'' underestimates the maximal ''in vivo'' oxygen uptake of muscle by up to ∼2-fold. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy. +
It is an ongoing discussion the extent to which oxygen delivery and oxygen extraction contribute to an elevated muscle oxygen uptake during dynamic exercise. It has been proposed that local muscle factors including the capillary bed and mitochondrial oxidative capacity play a large role in prolonged low intensity training of a small muscle group when the cardiac output capacity is not directly limiting. The purpose of this study was to investigate the relative roles of circulatory and muscle metabolic mechanisms by which prolonged low-intensity exercise training alters regional muscle ''V''<sub>O<sub>2</sub></sub>.
In 9 healthy volunteers (7 male, 2 female), hemodynamic and metabolic responses to incremental arm cycling were measured by the Fick method and biopsy of the deltoid and triceps muscles before and after 42 days of skiing for 6 h.day<sup>-1</sup> at 60 % max heart rate.
Peak pulmonary ''V''<sub>O<sub>2</sub></sub> during arm crank was unchanged after training (2.38±0.19 vs. 2.18±0.2 L.min<sup>-1</sup> pre-training) yet arm ''V''<sub>O<sub>2</sub></sub> (1.04±0.08 vs. 0.83±0.1 L.min<sup>-1</sup>, ''P''<0.05) and power output (137±9 vs. 114±10 W) were increased along with a higher arm blood flow (7.9±0.5 vs. 6.8±0.6 L.min<sup>-1</sup>, ''P''<0.05) and expanded muscle capillary volume (76±7 vs. 62±4 mL, ''P''<0.05). Muscle O<sub>2</sub> diffusion capacity (16.2±1 vs. 12.5 ±0.9 mL.min<sup>-1</sup>.mmHg<sup>-1</sup>, ''P''<0.05) and O<sub>2</sub> extraction (68±1 vs. 62±1 %, ''P''<0.05) were enhanced at a similar mean capillary transit time (569±43 vs. 564±31 ms) and ''p''<sub>50</sub> (35.8±0.7 vs. 35±0.8), whereas mitochondrial O<sub>2</sub> flux capacity was unchanged (147±6 mL.min<sup>-1</sup>.kg<sup>-1</sup> vs. 146±8 mL.min<sup>-1</sup>.kg<sup>-1</sup>).
The mechanisms underlying the increase in peak arm ''V''<sub>O<sub>2</sub></sub> with prolonged low intensity training in previously untrained subjects are an elevated convective O<sub>2</sub> delivery specifically to the muscles of the arm combined with a larger capillary-muscle surface area that enhance diffusional O<sub>2</sub> conductance, with no apparent role of mitochondrial respiratory capacity. This article is protected by copyright. All rights reserved.
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Aging is associated with diminished cardiovascular function and sarcopenia, and loss of muscle oxidative capacity is considered a salient feature of aging. While moderate-to-high intensity training evokes mitochondrial biogenesis in skeletal muscle, it remains unclear to what extent aging in itself or rather a lower training stimulus that accompanies aging contributes to loss of skeletal muscle mitochondrial function. To address this question leg muscle mitochondrial respiratory capacity in 8 older men (65±2 yrs) who had maintained road cycling training 200 km/week for 50 years was compared to that of 8 age-matched sedentary (UT) controls (67±1 yrs).V˙ O2 max was measured on a bicycle ergometer and a biopsy obtained from vastus lateralis muscle was permeabilized and prepared for high resolution respirometry (Oxygraph, Oroboros, AT). V˙ O2 max was substantially higher (p<0.05) in lifelong trained (45±2 ml/kg/min) compared to UT (27±2 ml/kg/min). Mitochondrial LEAK respiration was higher in ET, and Vmax of mitochondrial respiration (OXPHOS) with mixed substrates was 2-fold higher in the ET (132±6 pmol/sec/mg) compared to UT (72±4 pmol/sec/mg, p<0.01). Higher fatty acid oxidation and substrate control ratios in ET indicate regulatory changes in mitochondria in addition to a larger mitochondrial volume. The findings indicate that skeletal muscle mitochondrial respiratory capacity of ‘lifelong trained’ older males is retained at a level comparable to young athletic individuals, and suggest that decrements in aerobic performance with age are primarily attributed to diminished cardiovascular function. +
We recently reported the circulatory and muscle oxidative capacities of the arm after prolonged low-intensity skiing in the arctic (Boushel et al., 2014). In the present study, leg ''V''<sub>O2</sub> was measured by the Fick method during leg cycling while muscle mitochondrial capacity was examined on a biopsy of the vastus lateralis in healthy volunteers (7 male, 2 female) before and after 42 days of skiing at 60% HR max. Peak pulmonary ''V''<sub>O2</sub> (3.52 ± 0.18 L.min<sup>-1</sup> pre vs 3.52 ± 0.19 post) and ''V''<sub>O2</sub> across the leg (2.8 ± 0.4 L.min<sup>-1</sup> pre vs 3.0 ± 0.2 post) were unchanged after the ski journey. Peak leg O<sub>2</sub> delivery (3.6 ± 0.2 L.min<sup>-1</sup> pre vs 3.8 ± 0.4 post), O<sub>2</sub> extraction (82 ± 1% pre vs 83 ± 1 post), and muscle capillaries per mm<sup>2</sup> (576 ± 17 pre vs 612 ± 28 post) were also unchanged; however, leg muscle mitochondrial OXPHOS capacity was reduced (90 ± 3 pmol.s<sup>-1</sup>.mg<sup>-1</sup> pre vs 70 ± 2 post, ''P'' < 0.05) as was citrate synthase activity (40 ± 3 µmol.min<sup>-1</sup>.g<sup>-1</sup> pre vs 34 ± 3; ''P'' < 0.05). These findings indicate that peak muscle ''V''<sub>O2</sub> can be sustained with a substantial reduction in mitochondrial OXPHOS capacity. This is achieved at a similar O<sub>2</sub> delivery and a higher relative ADP-stimulated mitochondrial respiration at a higher mitochondrial ''p''<sub>50</sub>. These findings support the concept that muscle mitochondrial respiration is submaximal at ''V''<sub>O2max</sub>, and that mitochondrial volume can be downregulated by chronic energy demand. +
Angiogenesis is a complex process leading to the growth of new blood vessels from existing vasculature, triggered by local proangiogenic factors such as VEGF. An excess of angiogenesis is a recurrent feature of various pathologic conditions such as tumor growth. Phostines are a family of synthetic glycomimetic compounds that exhibit anticancer properties, and the lead compound 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl2-oxo-2λ5-[1,2]oxaphosphinane (PST 3.1a) shows antiglioblastoma properties both ''in vitro'' and ''in vivo''. In the present study, we assessed the effect of PST 3.1a on angiogenesis and endothelial metabolism. ''In vitro'', PST 3.1a (10 µM) inhibited all steps that regulate angiogenesis, including migration, proliferation, adhesion, and tube formation. ''In vivo'', PST 3.1a reduced intersegmental vessel formation and vascularization of the subintestinal plexus in zebrafish embryos and also altered pathologic angiogenesis and glioblastoma progression ''in vivo''. Mechanistically, PST 3.1a altered interaction of VEGF receptor 2 and glycosylation-regulating protein galectin-1, a key component regulating angiogenesis associated with tumor resistance. Thus, these data show that use of PST 3.1a is an innovative approach to target angiogenesis. +
OBJECTIVE:
SIRT1 has been proposed to be a key signaling node linking changes in energy metabolism to transcriptional adaptations. Although SIRT1 overexpression is protective against diverse metabolic complications, especially in response to high-fat diets, studies aiming to understand the etiology of such benefits are scarce. Here, we aimed to identify the key tissues and mechanisms implicated in the beneficial effects of SIRT1 on glucose homeostasis.
METHODS:
We have used a mouse model of moderate SIRT1 overexpression, under the control of its natural promoter, to evaluate glucose homeostasis and thoroughly characterize how different tissues could influence insulin sensitivity.
RESULTS:
Mice with moderate overexpression of SIRT1 exhibit better glucose tolerance and insulin sensitivity even on a low fat diet. Euglycemic-hyperinsulinemic clamps and in-depth tissue analyses revealed that enhanced insulin sensitivity was achieved through a higher brown adipose tissue activity and was fully reversed by housing the mice at thermoneutrality. SIRT1 did not influence brown adipocyte differentiation, but dramatically enhanced the metabolic transcriptional responses to β3-adrenergic stimuli in differentiated adipocytes.
CONCLUSIONS:
Our work demonstrates that SIRT1 improves glucose homeostasis by enhancing BAT function. This is not consequent to an alteration in the brown adipocyte differentiation process, but as a result of potentiating the response to β3-adrenergic stimuli. +
Caloric restriction (CR) has been shown to prevent the onset of insulin resistance and to delay age-related physiological decline in mammalian organisms. SIRT1, a NAD(+)-dependent deacetylase enzyme, has been suggested to mediate the adaptive responses to CR, leading to the speculation that SIRT1 activation could be therapeutically used as a CR-mimetic strategy. Here, we used a mouse model of moderate SIRT1 overexpression to test whether SIRT1 gain of function could mimic or boost the metabolic benefits induced by every-other-day feeding (EODF). Our results indicate that SIRT1 transgenesis does not affect the ability of EODF to decrease adiposity and improve insulin sensitivity. Transcriptomic analyses revealed that SIRT1 transgenesis and EODF promote very distinct adaptations in individual tissues, some of which can even be metabolically opposite, as in brown adipose tissue. Therefore, whereas SIRT1 overexpression and CR both improve glucose metabolism and insulin sensitivity, the etiologies of these benefits are largely different.
Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved. +
Mitochondrial fusion and fission events, collectively known as mitochondrial dynamics, act as quality control mechanisms to ensure mitochondrial function and fine-tune cellular bioenergetics. Defective mitofusin 2 (Mfn2) expression and enhanced mitochondrial fission in skeletal muscle are hallmarks of insulin-resistant states. Interestingly, Mfn2 is highly expressed in brown adipose tissue (BAT), yet its role remains unexplored. Using adipose-specific Mfn2 knockout (Mfn2-adKO) mice, we demonstrate that Mfn2, but not Mfn1, deficiency in BAT leads to a profound BAT dysfunction, associated with impaired respiratory capacity and a blunted response to adrenergic stimuli. Importantly, Mfn2 directly interacts with perilipin 1, facilitating the interaction between the mitochondria and the lipid droplet in response to adrenergic stimulation. Surprisingly, Mfn2-adKO mice were protected from high-fat diet-induced insulin resistance and hepatic steatosis. Altogether, these results demonstrate that Mfn2 is a mediator of mitochondria to lipid droplet interactions, influencing lipolytic processes and whole-body energy homeostasis.
© 2017 The Authors. Published under the terms of the CC BY NC ND 4.0 license. +
The cold submerged frog (''Rana temporaria'') serves as a useful model for many hibernating ectotherms that take refuge in hypoxic ponds and lakes until more favourable conditions of climate and food availability return. In all such animals, entry into a hypometabolic state effectively extends their survival time by lessening the impact of ATP demands on endogenous substrates. At the cellular level, metabolic depression may be brought about by decreasing energy-consuming processes and/or by increasing the efficiency of energy-producing pathways. Since the mitochondrion is the major contributor to the total energy production during aerobic metabolism and frog survival during winter depends on entry into a hypometabolic state, this review focuses on the respiratory properties of mitochondria that serve to increase the efficiency of energy production in hibernation. Energy conservation during overwintering also occurs through decreases in the ATP demand of the energy-consuming processes. For example, hibernating frogs decrease their ATP demands for Na<sup>+</sup>/K<sup>+</sup>-ATPase activity as part of a coordinated process of energy conservation wherein O<sub>2</sub>-limitation initiates a generalised suppression of ion channel densities and/or channel leak activities. The net result is that cell membrane permeabilities are reduced, thereby lowering the energetic costs of maintaining transmembrane ion gradients. +
Human proteins MTO1 and GTPBP3 are thought to jointly catalyze the modification of the wobble uridine in mitochondrial tRNAs. Defects in each protein cause infantile hypertrophic cardiomyopathy with lactic acidosis. However, the underlying mechanisms are mostly unknown. Using fibroblasts from an MTO1 patient and MTO1 silenced cells, we found that the MTO1 deficiency is associated with a metabolic reprogramming mediated by inactivation of AMPK, down regulation of the uncoupling protein 2 (UCP2) and transcription factor PPARγ, and activation of the hypoxia inducible factor 1 (HIF-1). As a result, glycolysis and oxidative phosphorylation are uncoupled, while fatty acid metabolism is altered, leading to accumulation of lipid droplets in MTO1 fibroblasts. Unexpectedly, this response is different from that triggered by the GTPBP3 defect, as GTPBP3-depleted cells exhibit AMPK activation, increased levels of UCP2 and PPARγ, and inactivation of HIF-1. In addition, fatty acid oxidation and respiration are stimulated in these cells. Therefore, the HIF-PPARγ-UCP2-AMPK axis is operating differently in MTO1- and GTPBP3-defective cells, which strongly suggests that one of these proteins has an additional role, besides mitochondrial-tRNA modification. This work provides new and useful information on the molecular basis of the MTO1 and GTPBP3 defects and on putative targets for therapeutic intervention. +
Human proteins MTO1 and GTPBP3 are thought to jointly catalyze the modification of the wobble uridine in mitochondrial tRNAs. Defects in each protein cause infantile hypertrophic cardiomyopathy with lactic acidosis. However, the underlying mechanisms are mostly unknown. Using fibroblasts from an MTO1 patient and MTO1 silenced cells, we found that the MTO1 deficiency is associated with a metabolic reprogramming mediated by inactivation of AMPK, down regulation of the uncoupling protein 2 (UCP2) and transcription factor PPARγ, and activation of the hypoxia inducible factor 1 (HIF-1). As a result, glycolysis and oxidative phosphorylation are uncoupled, while fatty acid metabolism is altered, leading to accumulation of lipid droplets in MTO1 fibroblasts. Unexpectedly, this response is different from that triggered by the GTPBP3 defect, as GTPBP3-depleted cells exhibit AMPK activation, increased levels of UCP2 and PPARγ, and inactivation of HIF-1. In addition, fatty acid oxidation and respiration are stimulated in these cells. Therefore, the HIF-PPARγ-UCP2-AMPK axis is operating differently in MTO1- and GTPBP3-defective cells, which strongly suggests that one of these proteins has an additional role, besides mitochondrial-tRNA modification. This work provides new and useful information on the molecular basis of the MTO1 and GTPBP3 defects and on putative targets for therapeutic intervention. +
To identify the clinical characteristics and genetic etiology of a family affected with hereditary spastic paraplegia (HSP).
Clinical, genetic, and functional analyses involving genome-wide linkage coupled to whole-exome sequencing in a consanguineous family with complicated HSP.
A homozygous missense mutation was identified in the ''ACO2'' gene (c.1240T>G p.Phe414Val) that segregated with HSP complicated by intellectual disability and microcephaly. Lymphoblastoid cell lines of homozygous carrier patients revealed significantly decreased activity of the mitochondrial aconitase enzyme and defective mitochondrial respiration. ''ACO2'' encodes mitochondrial aconitase, an essential enzyme in the Krebs cycle. Recessive mutations in this gene have been previously associated with cerebellar ataxia.
Our findings nominate ''ACO2'' as a disease-causing gene for autosomal recessive complicated HSP and provide further support for the central role of mitochondrial defects in the pathogenesis of HSP. +
Physical activity is a necessity for healthy living. Essential to this is the assessment of cardiorespiratory fitness by measuring maximal oxygen uptake (VO<sub>2</sub>max), which is the one of the strongest predictors of morbidity and mortality. While classically thought to be determined by oxygen delivery to working muscle, the adaptive responses of muscle oxidative capacity and therefore mitochondrial contributions are not fully understood. Moreover, changes in VO<sub>2</sub>max with standardized training programs vary substantially. A greater understanding of this variation may be achieved by a systems biology approach characterizing the biomolecular response to exercise (“the exercise responsome”), including differences in arterial and venous concentrations of proteins and metabolites (i.e., fluxomics). Given the “drug-like” effects of molecules secreted by muscle during exercise, characterizing the exercise responsome can highlight exercise dosages that optimize circulating biomolecule levels, adaptations to training, and therefore health benefits of exercise. Thus, the purposes of this study are three-fold: (1) To understand the relative and integrated contributions of the circulatory and muscle oxidative components to oxygen uptake with exercise training; (2) to assess the “exercise responsome”; and (3) to associate determinants of oxygen uptake with biomolecular markers of health.
Trained and untrained individuals will be recruited. At Visit 1, maximal oxygen uptake and critical power will be assessed. At Visit 2, blood samples will be drawn in the morning (fasted), prior to an exhaustive bout of exercise, and at multiple post-exercise time points to assess the proteomic and metabolomic responses to exercise. Body composition will be assessed, and muscle biopsies will be taken prior to and after exercise to assess mitochondrial function and oxidative stress. Specifically, a substrate and inhibitor protocol will be applied to assess OXPHOS, substrate and coupling control, LEAK respiration, mitochondrial p50, and COX excess capacity. At Visit 3, subjects will be instrumented with femoral arterial and venous catheters, as well as antecubital venous catheterization, and complete multiple incremental exercise tests on 2-legged cycling and 1-leg knee extension ergometers. During each exercise stage, blood samples will be drawn to measure fluxomics and circulatory responses to exercise will be determined. Integrative determinants of oxygen uptake will be modeled to include muscle mass-normalized O<sub>2</sub> delivery, mitochondrial excess capacity, relative activation of mitochondria, and the role of p50 in O<sub>2</sub> extraction. Bioinformatic analysis of omic responses alongside integrative determinants will investigate molecular-to-organ signaling networks. Trained vs. untrained groups and males vs. females will be compared. Untrained subjects will then complete a 12-16-week exercise training program, including aerobic intervals and resistance exercise, before repeating the 3 visits. Pre- and post-training will be compared.
The ASBMR Publication Committee, together with the Editors‐in‐Chief of JBMR, JBMR Plus, and members of the editorial boards, have implemented a new policy that allows manuscripts that have been posted on noncommercial preprint servers to be submitted to the journals for consideration. The use of preprint servers is becoming more common, but, because it is still rare in the field of bone and mineral research, the concept is most likely unfamiliar to many readers of JBMR and JBMR Plus. To clarify the new policy decision, this editorial provides a brief overview of preprint servers and describes their role in biomedical research.
Picture this common scenario: a young biomedical researcher has spent years collecting data for a very exciting project and is finally ready to submit a manuscript. This researcher is also applying for an NIH grant. Do they (i) hold off on submitting the grant application, knowing that they will have a greater chance of receiving funding once their paper is accepted; (ii) submit the application right away, even though they are missing the crucial citation; or (iii) upload a draft of their manuscript to a preprint server, using the citation for their application while they wait for a response from the journal? Many researchers are unaware of option iii, but it demonstrates one of the primary utilities of preprint servers: rapid dissemination of results within the scientific community. +
1. Pigeon heart mitochondria produce H(2)O(2) at a maximal rate of about 20 nmol/min per mg of protein.
2. Succinate-glutamate and malate-glutamate are substrates which are able to support maximal H(2)O(2) production rates. With malate-glutamate, H(2)O(2) formation is sensitive to rotenone. Endogenous substrate, octanoate, stearoyl-CoA and palmitoyl-carnitine are by far less efficient substrates.
3. Antimycin A exerts a very pronounced effect in enhancing H(2)O(2) production in pigeon heart mitochondria; 0.26 nmol of antimycin A/mg of protein and the addition of an uncoupler are required for maximal H(2)O(2) formation.
4. In the presence of endogenous substrate and of antimycin A, ATP decreases and uncoupler restores the rates of H(2)O(2) formation.
5. Reincorporation of ubiquinone-10 and ubiquinone-3 to ubiquinone-depleted pigeon heart mitochondria gives a system in which H(2)O(2) production is linearly related to the incorporated ubiquinone.
6. The generation of H(2)O(2) by pigeon heart mitochondria in the presence of succinate-glutamate and in metabolic [[State 4]] has an optimum pH value of 7.5. In States 1 and 3u, and in the presence of antimycin A and uncoupler, the optimum pH value is shifted towards more alkaline values.
7. With increase of the partial pressure of O(2) to the hyperbaric region the formation of H(2)O(2) is markedly increased in pigeon heart mitochondria and in rat liver mitochondria. With rat liver mitochondria and succinate as substrate in State 4, an increase in the ''p''O(2) up to 1.97 MPa (19.5 atm) increases H(2)O(2) formation 10-15-fold. Similar ''p''O(2) profiles were observed when rat liver mitochondria were supplemented either with antimycin A or with antimycin A and uncoupler. No saturation of the system with O(2) was observed up to 1.97 MPa (19.5 atm). By increasing the ''p''O(2) to 1.97 MPa (19.5atm), H(2)O(2) formation in pigeon heart mitochondria with succinate as substrate increased fourfold in metabolic State 4, with antimycin A added the increase was threefold and with antimycin A and uncoupler it was 2.5-fold. In the last two saturation of the system with oxygen was observed, with an apparent ''K''(m) of about 71 kPa (0.7-0.8 atm) and a ''V''(max) of 12 and 20 nmol of H(2)O(2)/min per mg of protein.
8. It is postulated that in addition to the well-known flavin reaction, formation of H(2)O(2) may be due to interaction with an energy-dependent component of the respiratory chain at the cytochrome ''b'' level.
Although the regulation of mitochondrial respiration and energy production in mammalian tissues has been exhaustively studied and extensively reviewed, a clear understanding of the regulation of cellular respiration has not yet been achieved. In particular, the role of tissue pO2 as a factor regulating
cellular respiration remains controversial. The concept of a complex and multisite regulation of cellular respiration and energy production signaled by
cellular and intercellular messengers has evolved in the last few years and is
still being researched. A recent concept that regulation of cellular respiration
is regulated by ADP, O2 and NO preserves the notion that energy demands
drive respiration but places the kinetic control of both respiration and energy
supply in the availability of ADP to F1-ATPase and of O2 and NO to cytochrome
oxidase. In addition, recent research indicates that NO participates in
redox reactions in the mitochondrial matrix that regulate the intramitochondrial
steady state concentration of NO itself and other reactive species such as
superoxide radical (O2−) and peroxynitrite (ONOO−). In this way, NO acquires
an essential role as a mitochondrial regulatory metabolite. NO exhibits a rich
biochemistry and a high reactivity and plays an important role as intercellular
messenger in diverse physiological processes, such as regulation of blood flow,
neurotransmission, platelet aggregation and immune cytotoxic response. +
Peripheral muscle dysfunction is a key mechanism contributing to exercise intolerance (i.e. breathlessness and fatigue) in heart failure patients with preserved ejection fraction (HFpEF); however, the underlying molecular and cellular mechanisms remain unknown. We therefore used an animal model to elucidate potential molecular, mitochondrial, histological, and functional alterations induced by HFpEF in the diaphragm and soleus, while also determining the possible benefits associated with exercise training.
Female Dahl salt-sensitive rats were fed a low (CON; n = 10) or high salt (HFpEF; n = 11) diet of 0.3% or 8% NaCl, respectively, or a high salt diet in combination with treadmill exercise training (n = 11). Compared with low-salt rats, high-salt rats developed (''P'' < 0.05) HFpEF. Compared with CON, the diaphragm of HFpEF rats demonstrated (''P'' < 0.05): a fibre type shift from fast-to-slow twitch; fibre atrophy; a decreased pro-oxidative but increased anti-oxidant capacity; reduced proteasome activation; impaired ''in situ'' mitochondrial respiration; and ''in vitro'' muscle weakness and increased fatigability. The soleus also demonstrated numerous alterations (''P'' < 0.05), including fibre atrophy, decreased anti-oxidant capacity, reduced mitochondrial density, and increased fatigability. Exercise training, however, prevented mitochondrial and functional impairments in both the diaphragm and soleus (''P'' < 0.05).
Our findings are the first to demonstrate that HFpEF induces significant molecular, mitochondrial, histological, and functional alterations in the diaphragm and soleus, which were attenuated by exercise training. These data therefore reveal novel mechanisms and potential therapeutic treatments of exercise intolerance in HFpEF. +
Respiratory muscle weakness contributes to exercise intolerance in patients with heart failure with a preserved ejection fraction (HFpEF)-a condition characterized by multiple comorbidities with few proven treatments. We aimed, therefore, to provide novel insight into the underlying diaphragmatic alterations that occur in HFpEF by using an obese cardiometabolic rat model and further assessed whether exercise training performed only after the development of overt HFpEF could reverse impairments.
Obese ZSF1 rats (''N''=12) were compared with their lean controls (''N''=8) at 20 weeks, with 3 additional groups of obese ZSF1 rats compared at 28 weeks following 8 weeks of either sedentary behavior (''N''=13), high-intensity interval training (''N''=11), or moderate-continuous training (''N''=11). Obese rats developed an obvious HFpEF phenotype at 20 and 28 weeks. In the diaphragm at 20 weeks, HFpEF induced a shift towards an oxidative phenotype and a fiber hypertrophy paralleled by a lower protein expression in MuRF1 and MuRF2, yet mitochondrial and contractile functional impairments were observed. At 28 weeks, neither the exercise training regimen of high-intensity interval training or moderate-continuous training reversed any of the diaphragm alterations induced by HFpEF.
This study, using a well-characterized rat model of HFpEF underpinned by multiple comorbidities and exercise intolerance (ie, one that closely resembles the patient phenotype), provides evidence that diaphragm alterations and dysfunction induced in overt HFpEF are not reversed following 8 weeks of aerobic exercise training. As such, whether alternative therapeutic interventions are required to treat respiratory muscle weakness in HFpEF warrants further investigation.
© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley. +
Marine organisms are under threat from a simultaneous combination of climate change stressors, including warming sea surface temperatures (SST), marine heatwave (MHW) episodes, and hypoxic events. This study sought to investigate the impacts of these stressors on the Australasian snapper (''C. auratus'') - a finfish species of high commercial and recreational importance, from the largest snapper fishery in Aotearoa New Zealand (SNA1). A MHW scenario was simulated from 21°C (current February SST average for north-eastern New Zealand) to a future predicted level of 25°C, with the whole-animal and mitochondrial metabolic performance of snapper in response to hypoxia and elevated temperature tested after 1-, 10-, and 30-days of thermal challenge. It was hypothesised that key indicators of snapper metabolic performance would decline after 1-day of MHW stress, but that partial recovery might arise as result of thermal plasticity after chronic (e.g., 30-day) exposures. In contrast to this hypothesis, snapper performance remained high throughout the MHW: 1) Aerobic metabolic scope increased after 1-day of 25°C exposure and remained high. 2) Hypoxia tolerance, measured as the critical O<sub>2</sub> pressure and O<sub>2</sub> pressure where loss of equilibrium occurred, declined after 1-day of warm-acclimation, but recovered quickly with no observable difference from the 21°C control following 30-days at 25°C. 3) The performance of snapper mitochondria was also maintained, with oxidative phosphorylation respiration and proton leak flux across the inner mitochondrial membrane of the heart remaining mostly unaffected. Collectively, the results suggest that heart mitochondria displayed resilience, or plasticity, in snapper chronically exposed to 25°C. Therefore, contrary to the notion of climate change having adverse metabolic effects, future temperatures approaching 25°C may be tolerated by ''C. auratus'' in Northern New Zealand. Even in conjunction with supplementary hypoxia, 25°C appears to represent a metabolically optimal temperature for this species.
Hydrogenosomes are organelles that produce ATP and hydrogen, and are found in various unrelated eukaryotes, such as anaerobic flagellates, chytridiomycete fungi and ciliates. Although all of these organelles generate hydrogen, the hydrogenosomes from these organisms are structurally and metabolically quite different, just like mitochondria where large differences also exist. These differences have led to a continuing debate about the evolutionary origin of hydrogenosomes. Here we show that the hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis, which thrives in the hindgut of cockroaches, have retained a rudimentary genome encoding components of a mitochondrial electron transport chain. Phylogenetic analyses reveal that those proteins cluster with their homologues from aerobic ciliates. In addition, several nucleus-encoded components of the mitochondrial proteome, such as pyruvate dehydrogenase and complex II, were identified. The N. ovalis hydrogenosome is sensitive to inhibitors of mitochondrial complex I and produces succinate as a major metabolic end product--biochemical traits typical of anaerobic mitochondria. The production of hydrogen, together with the presence of a genome encoding respiratory chain components, and biochemical features characteristic of anaerobic mitochondria, identify the ''N. ovalis'' organelle as a missing link between mitochondria and hydrogenosomes. +
THE importance of reactions in which electron transport from reduced pyridine nucleotides to oxygen is coupled with the formation of adenosine triphosphate from adenosine diphosphate and inorganic phosphate is well recognized by biochemists. Although much pertinent information has been accumulated, no clear picture of the sites and mechanism of uptake of inorganic phosphate coupled with electron transport has yet been obtained. This communication gives some results of exchange reactions of inorganic phosphate labelled with phosphorus-32 and oxygen-18 which may give some additional insight concerning oxidative phosphorylation, together with a brief presentation of a hypothesis for the mechanism of coupling of electron transport with inorganic phosphate uptake. +
Targeting mitochondrial oxidative phosphorylation (OXPHOS) to treat cancer has been hampered due to serious side-effects potentially arising from the inability to discriminate between non-cancerous and cancerous mitochondria. Herein, comprehensive mitochondrial phenotyping was leveraged to define both the composition and function of OXPHOS across various murine cancers and compared to both matched normal tissues and other organs. When compared to both matched normal tissues, as well as high OXPHOS reliant organs like heart, intrinsic expression of the OXPHOS complexes, as well as OXPHOS flux were discovered to be consistently lower across distinct cancer types. Assuming intrinsic OXPHOS expression/function predicts OXPHOS reliance ''in vivo'', these data suggest that pharmacologic blockade of mitochondrial OXPHOS likely compromises bioenergetic homeostasis in healthy oxidative organs prior to impacting tumor mitochondrial flux in a clinically meaningful way. Although these data caution against the use of indiscriminate mitochondrial inhibitors for cancer treatment, considerable heterogeneity was observed across cancer types with respect to both mitochondrial proteome composition and substrate-specific flux, highlighting the possibility for targeting discrete mitochondrial proteins or pathways unique to a given cancer type. +
The skeletal muscle of obese humans is characterized by an inability to appropriately respond to alterations in substrate availability. The purpose of this study was to determine if this metabolic inflexibility with obesity is retained in mitochondria of human skeletal muscle cells raised in culture (HSkMC) and to identify potential mechanisms involved.
Mitochondrial respiration was measured in permeabilized myotubes cultured from lean and obese individuals before and after a 24-h lipid incubation. Mitochondrial respiration ([[State 3]]) in the presence of lipid substrate (palmitoyl carnitine) increased by almost twofold after lipid incubation in HSkMC from lean, but not obese subjects, indicative of metabolic inflexibility with obesity. The 24-h lipid incubation increased mitochondrial DNA (mtDNA) copy number in HSkMC from lean subjects by +16% (''P''<0.05); conversely, mtDNA copy number decreased in myotubes cultured from obese individuals (-13%, ''P''=0.06). When respiration data were normalized to mtDNA copy number and other indices of mitochondrial content (COX-IV protein content and CS activity), the significant treatment effects of lipid incubation persisted in the lean subjects, suggesting concomitant alterations in mitochondrial function; no similar adjustment was evident in HSkMC from obese individuals.
These data indicate that the skeletal muscle of obese individuals inherently lacks metabolic flexibility in response to lipid exposure, which consists of an inability to increase mitochondrial respiration in the presence of lipid substrate and perhaps by an inability to induce mitochondrial proliferation. +
Mutations in the presenilin 1 (PS1) gene lead to early-onset Alzheimer's disease with the S170F mutation causing the earliest reported age of onset. Expression of this, and other PS1 mutations, in SH-SY5Y cells resulted in significant loss of cellular viability compared to control cells. Basal Ca<sup>2+</sup> concentrations in PS1 mutants were never lower than controls and prolonged incubation in Ca<sup>2+</sup>-free solutions did not deplete Ca<sup>2+</sup> stores, demonstrating there was no difference in Ca<sup>2+</sup> leak from endoplasmic reticulum (ER) stores in PS1 mutants. Peak muscarine-evoked rises of [Ca<sup>2+</sup>](i) were variable, but the integrals were not significantly different, suggesting, while kinetics of Ca<sup>2+</sup> store release might be affected in PS1 mutants, store size was similar. However, when Ca<sup>2+</sup>-ATPase activity was irreversibly inhibited with thapsigargin, the S170F and ΔE9 cells showed larger capacitative calcium entry indicating a direct effect on Ca<sup>2+</sup> influx pathways. There was no significant effect of any of the mutations on mitochondrial respiration. Amyloid β(Aβ(1-40)) secretion was reduced, and Aβ(1-42) secretion increased in the S170F cells resulting in a very large increase in the Aβ42/40 ratio. This, rather than any potential disruption of ER Ca<sup>2+</sup> stores, is likely to explain the extreme pathology of this mutant. +
Kv1.3 is a member of the delayed rectifier family of voltage-activated potassium channels and has become a major therapeutic target because of its role in autoimmune diseases, in leukaemia, atherosclerosis and obesity and type 2 diabetes. Kv1.3 is not only expressed on the plasma membrane but also on the inner mitochondrial membrane [1] suggesting that some of its actions might be via modulation of mitochondrial function.
This was investigated in HEK293/Kv1.3 cells and human saphenous vein smooth muscle cells (HSVSMCs), using proliferation assays, immunocytochemistry and high resolution respirometry.
HEK293/Kv1.3 cells had significantly increased rates of proliferation compared to WT HEK293 cells. PAP-1, a selective, cell permeant Kv1.3 inhibitor, reduced proliferation in both HEK293/Kv1.3 and HSVSMCs. Channel expression in both the plasma membrane and mitochondria was confirmed using mitotracker in conjunction with immunocytochemical detection of Kv1.3. Mitochondrial expression of the channel was confirmed in both cell types. In addition, the functional expression of the Kv1.3 channel in the plasma membrane was confirmed using patch clamp electrophysiology. High resolution respirometry demonstrated that HEK293/Kv1.3 cells were significantly more metabolically active than WT HEK cells with both increased OXPHOS and glycolytic activity.
Thus mitochondrial Kv1.3 may contribute to increased mitochondrial respiration. This will be further investigated using additional permeant and impermeant inhibitors of the Kv1.3 channel. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MITOEAGLE]] Chronic heart failure (CHF) is characterised by exercise intolerance, which is exacerbated in patients with concomitant type 2 diabetes (CHF+DM) independent of left ventricular ejection fraction. This may be attributed to non-cardiac alterations, with greater skeletal muscle mitochondrial dysfunction a potential underlying mechanism. However, previous studies in CHF have used single isolated biopsies from peripheral locomotor muscles, which are susceptible to detraining, thereby confounding the results.
We assessed the hypothesis that CHF+DM patients are characterised by systemic skeletal muscle mitochondrial dysfunction by taking biopsies from two muscles.
Skeletal muscle biopsies were sampled consecutively from ''vastus lateralis'' and ''pectoralis major'' from age-matched healthy controls (n=6), CHF (n=12), and CHF+DM patients (n=7). Mitochondrial oxygen flux of permeabilized fibres was subsequently assessed using a standard substrate, uncoupler and inhibitor titration protocol to selectively measure isolated components of the electron transfer chain.
After correcting for mitochondrial content, we found a significant main effect for group for complex I oxygen flux, F (2, 22) = 4.16, p = 0.029, with a significant difference between CHF+DM and healthy controls in both muscles (Figure 1). In contrast, CHF patients alone were not different to controls.
In conclusion, CHF+DM patients present with a systemic skeletal muscle mitochondrial dysfunction, which is not apparent in CHF patients. These findings provide an initial rationale for why exercise intolerance is exacerbated in CHF+DM. +
Metabolite concentrations reflect the physiological states of tissues and cells. However, the role of metabolic changes in species evolution is currently unknown. Here, we present a study of metabolome evolution conducted in three brain regions and two non-neural tissues from humans, chimpanzees, macaque monkeys, and mice based on over 10,000 hydrophilic compounds. While chimpanzee, macaque, and mouse metabolomes diverge following the genetic distances among species, we detect remarkable acceleration of metabolome evolution in human prefrontal cortex and skeletal muscle affecting neural and energy metabolism pathways. These metabolic changes could not be attributed to environmental conditions and were confirmed against the expression of their corresponding enzymes. We further conducted muscle strength tests in humans, chimpanzees, and macaques. The results suggest that, while humans are characterized by superior cognition, their muscular performance might be markedly inferior to that of chimpanzees and macaque monkeys. +
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls. +
Lysophosphatidic acid acyltransferase (LPAAT) δ/acylglycerophosphate acyltransferase 4 is a mitochondrial enzyme and one of five homologues that catalyze the acyl-CoA-dependent synthesis of phosphatidic acid (PA) from lysophosphatidic acid. We studied skeletal muscle LPAATδ and found highest levels in soleus, a red oxidative fibre-type that is rich in mitochondria, and lower levels in extensor digitorum longus (EDL) (white glycolytic) and gastrocnemius (mixed fibre-type). Using Lpaatδ-deficient mice, we found no change in soleus or EDL mass, or in treadmill time-to-exhaustion compared to wildtype littermates. There was, however, a significant reduction in the proportion of type I and type IIA fibres in EDL but, surprisingly, not soleus, where these fibre-types predominate. Also unexpectedly, there was no impairment in force generation by EDL, but a significant reduction by soleus. Oxidative phosphorylation and activity of complexes I, I + II, III, and IV in soleus mitochondria was unchanged and therefore could not explain this effect. However, pyruvate dehydrogenase activity was significantly reduced in Lpaatδ-/- soleus and EDL. Analysis of cellular lipids indicated no difference in soleus triacylglycerol, but specific elevations in soleus PA and phosphatidylethanolamine levels, likely due to a compensatory upregulation of Lpaatβ and Lpaatε in Lpaatδ-/- mice. An anabolic effect for PA as an activator of skeletal muscle mTOR has been reported, but we found no change in serine 2448 phosphorylation, indicating reduced soleus force generation is unlikely due to the loss of mTOR activation by a specific pool of LPAATδ-derived PA. Our results identify an important role for LPAATδ in soleus and EDL. +
The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) protects against redox stress by providing reducing equivalents to antioxidants such as glutathione and thioredoxin. NADPH levels decline with aging in several tissues, but whether this is a major driving force for the aging process has not been well established. Global or neural overexpression of several cytoplasmic enzymes that synthesize NADPH have been shown to extend lifespan in model organisms such as Drosophila suggesting a positive relationship between cytoplasmic NADPH levels and longevity. Mitochondrial NADPH plays an important role in the protection against redox stress and cell death and mitochondrial NADPH-utilizing thioredoxin reductase 2 levels correlate with species longevity in cells from rodents and primates. Mitochondrial NADPH shuttles allow for some NADPH flux between the cytoplasm and mitochondria. Since a decline of nicotinamide adenine dinucleotide (NAD⁺) is linked with aging and because NADP⁺ is exclusively synthesized from NAD⁺ by cytoplasmic and mitochondrial NAD⁺ kinases, a decline in the cytoplasmic or mitochondrial NADPH pool may also contribute to the aging process. Therefore pro-longevity therapies should aim to maintain the levels of both NAD⁺ and NADPH in aging tissues. +
The maintenance of mitochondrial activity in hypothalamic neurons is determinant to the control of energy homeostasis in mammals. Disturbs in the mitochondrial proteostasis can trigger the mitonuclear imbalance and mitochondrial unfolded protein response (UPR<sup>mt</sup>) to guarantee the mitochondrial integrity and function. However, the role of mitonuclear imbalance and UPR<sup>mt</sup> in hypothalamic cells are unclear. Combining the transcriptomic analyses from BXD mice database and ''in vivo'' experiments, we demonstrated that physical training alters the mitochondrial proteostasis in the hypothalamus of C57BL/6J mice. This physical training elicited the mitonuclear protein imbalance, increasing the mtCO-1/Atp5a ratio, which was accompanied by high levels of UPR<sup>mt</sup> markers in the hypothalamus. Also, physical training increased the maximum mitochondrial respiratory capacity in the brain. Interestingly, the transcriptomic analysis across several strains of the isogenic BXD mice revealed that hypothalamic mitochondrial DNA-encoded genes were negatively correlated with body weight and several genes related to the orexigenic response. As expected, physical training reduced body weight and food intake. Interestingly, we found an abundance of mt-CO1, a mitochondrial DNA-encoded protein, in NPY-producing neurons in the lateral hypothalamus nucleus of exercised mice. Collectively, our data demonstrated that physical training altered the mitochondrial proteostasis and induced the mitonuclear protein imbalance and UPR<sup>mt</sup> in hypothalamic cells. +
Physical function decreases with age, and though bioenergetic alterations contribute to this decline, the mechanisms by which mitochondrial function changes with age remains unclear. This is partially because human mitochondrial studies require highly invasive procedures, such as muscle biopsies, to obtain live tissue with functional mitochondria. However, recent studies demonstrate that circulating blood cells are potentially informative in identifying systemic bioenergetic changes. Here, we hypothesize that human platelet bioenergetics reflect bioenergetics measured in muscle biopsies.
We demonstrate that maximal and ATP-linked respiratory rate measured in isolated platelets from older adults (86-93 years) correlates significantly with maximal respiration (''r'' = 0.595; ''P'' = 0.003) measured by muscle biopsy respirometry and maximal ATP production (''r'' = 0.643; ''P'' = 0.004) measured by <sup>31</sup>P-MRS respectively, in the same individuals. Comparison of platelet bioenergetics in this aged cohort to platelets from younger adults (18-35 years) shows aged adults demonstrate lower basal and ATP-linked respiration. Platelets from older adults also show enhanced proton leak, which is likely due to increased protein levels of uncoupling protein 2, and correlates with increased gate speed in this cohort (''r'' = 0.58; ''P'' = 0.0019). While no significant difference in glycolysis was observed in older adults compared to younger adults, platelet glycolytic rate correlated with fatigability (''r'' = 0.44; ''P'' = 0.016).
These data advance the mechanistic understanding of age-related changes in mitochondrial function. Further, they suggest that measuring platelet bioenergetics provides a potential supplement or surrogate for muscle biopsy measurement and may be a valuable tool to study mitochondrial involvement in age-related decline of physical function. +
Accumulating studies demonstrate that mitochondrial genetics and function are central to determining the susceptibility to, and prognosis of numerous diseases across all organ systems. Despite this recognition, mitochondrial function remains poorly characterized in humans primarily due to the invasiveness of obtaining viable tissue for mitochondrial studies. Recent studies have begun to test the hypothesis that circulating blood cells, which can be obtained by minimally invasive methodology, can be utilized as a biomarker of systemic bioenergetic function in human populations. Here we present the available methodologies for assessing blood cell bioenergetics and review studies that have applied these techniques to healthy and disease populations. We focus on the validation of this methodology in healthy subjects, as well as studies testing whether blood cell bioenergetics are altered in disease, correlate with clinical parameters, and compare with other methodology for assessing human mitochondrial function. Finally, we present the challenges and goals for the development of this emerging approach into a tool for translational research and personalized medicine.
<small>Copyright © 2019 Elsevier Ltd. All rights reserved.</small> +
Fibroblast growth factor 21 (FGF21) is a PPARα-regulated gene elucidated in the liver of PPARα-deficient mice or PPARα agonist-treated mice. Mice globally lacking adipose triglyceride lipase (ATGL) exhibit a marked defect in TG catabolism associated with impaired PPARα-activated gene expression in the heart and liver, including a drastic reduction in hepatic FGF21 mRNA expression. Here we show that FGF21 mRNA expression is markedly increased in the heart of ATGL-deficient mice accompanied by elevated expression of endoplasmic reticulum (ER) stress markers, which can be reversed by reconstitution of ATGL expression in cardiac muscle. In line with this assumption, the induction of ER stress increases FGF21 mRNA expression in H9C2 cardiomyotubes. Cardiac FGF21 expression was also induced upon fasting of healthy mice, implicating a role of FGF21 in cardiac energy metabolism. To address this question, we generated and characterized mice with cardiac-specific overexpression of FGF21 (CM-Fgf21). FGF21 was efficiently secreted from cardiomyocytes of CM-Fgf21 mice, which moderately affected cardiac TG homeostasis, indicating a role for FGF21 in cardiac energy metabolism. Together, our results show that FGF21 expression is activated upon cardiac ER stress linked to defective lipolysis and that a persistent increase in circulating FGF21 levels interferes with cardiac and whole body energy homeostasis. +
Cadmium (Cd) is a well-known heavy metal and environmental toxicant and pollutant worldwide, being largely present in every kind of item such as plastic (toys), battery, paints, ceramics, contaminated water, air, soil, food, fertilizers, and cigarette smoke. Nowadays, it represents an important research area for the scientific community mainly for its effects on public health. Due to a half-life ranging between 15 and 30 years, Cd owns the ability to accumulate in organs and tissues, exerting deleterious effects. Thus, even at low doses, a Cd prolonged exposure may cause a multiorgan toxicity. Mitochondria are key intracellular targets for Cd-induced cytotoxicity, but the underlying mechanisms are not fully elucidated. The present review is aimed to clarify the effects of Cd on mitochondria and, particularly, on the mitochondrial electron transport chain. +
Since the discovery of insulin as treatment for diabetes, observations showed that diabetic patients have higher chances to develop neurodegenerative disorders as Alzheimer’s disease. The role of insulin in the central nervous system is an important topic of interest in the molecular and cellular neuroscience research field. Microglia are the macrophages of the central nervous system with immunological functions and play an important role in neuroinflammation. Insulin signaling resistance in the aging brain and insulin signaling in astrocytes are more recent findings that broaden the perspective of the role of insulin in the brain. Insulin is an important factor that in addition to astrocytes also affects neurons, and its function can be altered or (partially) lost during life. In the studies presented in this thesis, new molecular and cellular mechanisms of insulin signaling in the hippocampus and its effects on microglia and neuroinflammation in both young and aged brains were identified. New insights into insulin effects on microglia metabolism followed by a new strategy to access microglia oxygen metabolism and mitochondrial function were also objects of study. Present data show that microglia are sensitive to insulin in both cell culture and in vivo with a protective pro-inflammatory effect. Complementarily, it was found that insulin modulates microglia oxygen metabolism and the production of reactive oxygen species in both a mitochondrial-dependent and -independent manner. In this thesis, the inflammatory signaling of insulin and the metabolism of microglia was investigated in the brain. +
The number of protons ejected during electron transport per pair of electrons per energy-conserving site (the H<sup>+</sup>/site ratio) was measured in rat liver mitochondria by three different methods under conditions in which transmembrane movements of endogenous phosphate were minized or eliminated.
# In the Ca<sup>2+</sup> pulse method, between 3.5 and 4.0 molecules of 3-hydroxybutyrate and 1.75 to 2.0 Ca<sup>2+</sup> ions were accumulated per 2 e<sup>-</sup> per site during Ca<sup>2+</sup> induced electron transport in the presence of rotenone, when measured under conditions in which movements of endogenous phosphate were negligible. Since entry of 3-hydroxybutyrate requires its protonation to the free acid these data correspond to an H<sup>+</sup>/site ratio of 3.5-4.0
# In the oxygen pulse method addition of known amounts of oxygen to anaerobic mitochondria in the presence of substrate yielded H<sup>+</sup>/site ratios of 3.0 when phosphate transport was eliminated by addition of N-ethylmaleimide or by anaerobic washing to remove endogenous phosphate. In the absence of such measures the observed H<sup>+</sup>/site ratio was 2.0.
# In the reductant pulse method measurement of the initial steady rates of H<sup>+</sup> ejection and oxygen consumption by mitochondria in an aerobic medium after addition of substrate gave H<sup>+</sup>/site near 4.0 in the presence of N-ethylmaleimide; in the absence of the inhibitor the observed ratio was only 2.0.
Abstract Continued in Free Text +
No Abstract +
Assessing mitochondrial dysfunction requires definition of the dysfunction to be investigated. Usually, it is the ability of the mitochondria to make ATP appropriately in response to energy demands. Where other functions are of interest, tailored solutions are required. Dysfunction can be assessed in isolated mitochondria, in cells or in vivo, with different balances between precise experimental control and physiological relevance. There are many methods to measure mitochondrial function and dysfunction in these systems. Generally, measurements of fluxes give more information about the ability to make ATP than do measurements of intermediates and potentials. For isolated mitochondria, the best assay is mitochondrial respiratory control: the increase in respiration rate in response to ADP. For intact cells, the best assay is the equivalent measurement of cell respiratory control, which reports the rate of ATP production, the proton leak rate, the coupling efficiency, the maximum respiratory rate, the respiratory control ratio and the spare respiratory capacity. Measurements of membrane potential provide useful additional information. Measurement of both respiration and potential during appropriate titrations enables the identification of the primary sites of effectors and the distribution of control, allowing deeper quantitative analyses. Many other measurements in current use can be more problematic, as discussed in the present review. +
Occupational burnout is both a serious public and individual health concern. Psychopharmacological and psychological interventions are often employed, while interventions involving physical activity have been less frequently studied. The aims of the present study were (1) to investigate the effects of physical activity on mitochondrial activity levels and symptoms of burnout, (2) to compare the mitochondrial activity levels and symptoms of burnout of individuals suffering burnout with those of healthy controls (HCs), and (3) to explore the associations between mitochondrial activity and burnout symptoms.
Twelve males with burnout (mean age: M = 45.8 years) took part in the study. At baseline and after 12 weeks of an intervention involving physical activity, participants completed questionnaires covering symptoms of burnout and depression. In parallel, blood samples were taken to measure changes in mitochondrial functional outcomes, such as ATP levels, oxygen consumption and complex I. For comparison, baseline values of healthy controls (HCs; depression and burnout questionnaires; blood samples) were assessed.
Over time, symptoms of burnout (emotional exhaustion and depersonalization) and depression significantly decreased in participants with burnout (large effect sizes) but remained significantly higher than those of HCs (medium to large effect sizes). Personal accomplishment increased over time (medium effect size) but was still lower than for HCs (large effect size). At baseline and compared to HCs, individuals with burnout had significantly lower ATP levels of mitochondrial functional outcomes. Over time, mitochondrial activity levels increased among individuals with burnout. High baseline mitochondrial activity was significantly correlated with lower depression and burnout scores both at baseline and at the end of the study.
In individuals with burnout, regular physical activity had positive effects on mitochondrial activity and on symptoms of burnout and depression. However, when compared to healthy controls, full remission was not achieved.
Free fatty acid (FFA)- and obesity-induced insulin resistance has been associated with disturbed mitochondrial function. Elevated plasma FFA can impair insulin-induced increase of adenosine triphosphate synthesis and downregulate the expression of genes important in the biogenesis of mitochondria in human skeletal muscle. Whether FFAs have a direct effect on intrinsic mitochondrial capacity remains to be established. Therefore, we measured ex vivo mitochondrial respiratory capacity in human skeletal muscle after exposure to hyperinsulinemia and high levels of plasma FFA. Nine healthy lean men were studied during a 6-hour hyperinsulinemic (600 pmol/L) euglycemic clamp with concomitant infusion of Intralipid (Fresensius Kabi Nederland, Den Bosch, the Netherlands) (FFA clamped at 0.5 mmol/L) or saline. Mitochondrial respiratory capacity was measured by high-resolution respirometry in permeabilized muscle fibers using an Oxygraph (Oroboros Instruments, Innsbruck, Austria). Each participant served as his own control. Peripheral glucose uptake (rate of disappearance) was significantly lower during infusion of the lipid emulsion compared with the control saline infusion (68 μmol/kg·min [saline] vs 40 μmol/kg·min [lipid], ''P'' = .008). However, adenosine diphosphate-stimulated and maximal carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone-stimulated uncoupled respiration rates were not different in permeabilized skeletal muscle fibers after exposure to high levels of FFA compared with the control condition. We conclude that short-term elevation of FFA within the physiological range induces insulin resistance but does not affect intrinsic mitochondrial capacity in skeletal muscle in humans. +
Mitochondria are the energy-converting cell organelles. Their dysfunction has been described as a cause or symptom of ageing. Mutations of mitochondrial DNA lead to respiratory system defects, possibly causing an increased release of reactive oxygen species. Affected cells accumulate, resulting in tissues with mosaic respiratory system deficiency that impairs the efficiency of affected organs [1].
We have previously found dramatic morphological changes in mitochondria of the fast-ageing model organism ''Podospora anserina'' [2]. Here, we investigated the morphology and function of aged mammalian mitochondria. We used mouse models that would reveal changes in mitochondrial structure and function in dependence of organismal age. Mitochondrial morphology was examined by electron cryo-tomography, and function was assessed by respirometry in parallel experiments. Thus, in contrast to electron microscopy of resin-embedded samples, we were able to obtain three-dimensional volumes of mitochondria at higher resolution in a close-to-native state, and to correlate our findings to respiratory activity.
We analysed isolated mitochondria from heart, kidney and liver tissue of young and aged mice. Interestingly, we observed clear tissue-specific differences. Whereas heart mitochondria were functionally and structurally unchanged in aged animals, mitochondria from old mouse kidney and liver showed increased populations with altered cristae morphology. Furthermore, we analysed samples from the mtDNA mutator mouse, an established ageing model. In these animals, the highly mutated mitochondrial DNA caused a wide range of structural defects that were accompanied by loss of function.
In summary, mouse mitochondria show subtle, tissue-dependent age-related changes, the molecular causes of which remain to be elaborated. In contrast to a fungal ageing model, a drastic rearrangement of the mitochondrial inner membrane was not observed. +
Inner workings: The X-ray crystal structure of the entire bacterial complex I at 3.3 Å resolution offers fascinating insights into a giant 536 kDa molecular machine. The respiratory chain complex seems to employ unique mechanisms of energetic coupling that are entirely different from those found in all other enzymes using redox energy to drive vectorial proton transport across a bioenergetic membrane. +
Previously, we demonstrated that sydnone SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) impairs the mitochondrial functions linked to energy provision and suggested that this effect could be associated with its antitumor activity. Herein, we evaluated the effects of SYD-1 (25 and 50μM) on rat hepatocytes to determine its cytotoxicity on non-tumor cells. SYD-1 (25 and 50 μM) did not affect the viability of hepatocytes in suspension after 1-40 min of incubation. However, the viability of the cultured hepatocytes was decreased by ∼66% as a consequence of treatment with SYD-1 (50 μM) for 18 h. Under the same conditions, SYD-1 promoted an increase in the release of LDH by ∼19%. The morphological changes in the cultured cells treated with SYD-1 (50 μM) were suggestive of cell distress, which was demonstrated by the presence of rounded hepatocytes, cell fragments and monolayer impairment. Furthermore, fluorescence microscopy showed an increase in the annexin label after treatment with SYD-1 (50 μM), suggesting that apoptosis had been induced in these cells. SYD-1 did not affect the states of respiration in the suspended hepatocytes, but the pyruvate levels were decreased by ∼36%, whereas the lactate levels were increased by ∼22% (for the 50 μM treatment). The basal and uncoupled states of respiration of the cultured hepatocytes were inhibited by ∼79% and ∼51%, respectively, by SYD-1 (50 μM). In these cells, SYD-1 (50 μM) increased the pyruvate and lactate levels by ∼84% and ∼16%, respectively. These results show that SYD-1 affects important metabolic functions related to energy provision in hepatocytes and that this effect was more pronounced on cells in culture than those in suspension. +
Toxicity of the SYD-1 mesoionic compound (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) was evaluated on human liver cancer cells (HepG2) grown in either high glucose (HG) or galactose (GAL) medium, and also on suspended cells kept in HG medium. SYD-1 was able to decrease the viability of cultured HepG2 cells in a dose-dependent manner, as assessed by MTT, LDH release and dye with crystal violet assays, but no effect was observed on suspended cells after 1-40 min of treatment. Respiration analysis was performed after 2 min (suspended cells) or 24 h (cultured cells) of treatment: no change was observed in suspended cells, whereas SYD-1 inhibited as well basal, leak and uncoupled states of the respiration in cultured cells with HG medium. These inhibitions were consistent with the decrease in pyruvate level and increase in lactate level. Even more extended results were obtained with HepG2 cells grown in GAL medium where, additionally, the ATP amount was reduced. Furthermore, SYD-1 appears not to be transported by the main ABC multidrug transporters. These results show that SYD-1 is able to change the metabolism of HepG2 cells, and suggest that its cytotoxicity is related to impairment of mitochondrial metabolism. Therefore, we may propose that SYD-1 is a potential candidate for hepatocarcinoma treatment.
Copyright © 2016 Elsevier Ireland Ltd. All rights reserved. +
We show that elevation of mitochondrial superoxide generation increases ''Caenorhabditis elegans'' life span by enhancing a RAS-dependent ROS (reactive oxygen species) signaling pathway (RDRS) that controls the expression of half of the genome as well as animal composition and physiology. RDRS stimulation mimics a program of change in gene expression that is normally observed at the end of postembryonic development. We further show that RDRS is regulated by negative feedback from the superoxide dismutase 1 (SOD-1)-dependent conversion of superoxide into cytoplasmic hydrogen peroxide, which, in turn, acts on a redox-sensitive cysteine (C118) of RAS. Preventing C118 oxidation by replacement with serine, or mimicking oxidation by replacement with aspartic acid, leads to opposite changes in the expression of the same large set of genes that is affected when RDRS is stimulated by mitochondrial superoxide. The identities of these genes suggest that stimulation of the pathway extends life span by boosting turnover and repair while moderating damage from metabolic activity. +
The bicoid stability factor (BSF) of ''Drosophila melanogaster'' has been reported to be present in the cytoplasm, where it stabilizes the maternally contributed bicoid mRNA and binds mRNAs expressed from early zygotic genes. BSF may also have other roles, as it is ubiquitously expressed and essential for survival of adult flies. We have performed immunofluorescence and cell fractionation analyses and show here that BSF is mainly a mitochondrial protein. We studied two independent RNAi knockdown fly lines and report that reduced BSF protein levels lead to a severe respiratory deficiency and delayed development at the late larvae stage. Ubiquitous knockdown of BSF results in a severe reduction of the polyadenylation tail lengths of specific mitochondrial mRNAs, accompanied by an enrichment of unprocessed polycistronic RNA intermediates. Furthermore, we observed a significant reduction in mRNA steady state levels, despite increased ''de novo'' transcription. Surprisingly, mitochondrial ''de novo'' translation is increased and abnormal mitochondrial translation products are present in knockdown flies, suggesting that BSF also has a role in coordinating the mitochondrial translation in addition to its role in mRNA maturation and stability. We thus report a novel function of BSF in flies and demonstrate that it has an important intra-mitochondrial role, which is essential for maintaining mtDNA gene expression and oxidative phosphorylation. +
Over the last decade, accumulating evidence has suggested a causative link between mitochondrial dysfunction and major phenotypes associated with aging. Somatic mitochondrial DNA (mtDNA) mutations and respiratory chain dysfunction accompany normal aging, but the first direct experimental evidence that increased mtDNA mutation levels contribute to progeroid phenotypes came from the mtDNA mutator mouse. Recent evidence suggests that increases in aging-associated mtDNA mutations are not caused by damage accumulation, but rather are due to clonal expansion of mtDNA replication errors that occur during development. Here we discuss the caveats of the traditional mitochondrial free radical theory of aging and highlight other possible mechanisms, including insulin/IGF-1 signaling (IIS) and the target of rapamycin pathways, that underlie the central role of mitochondria in the aging process. +
Replication errors are the main cause of mitochondrial DNA (mtDNA) mutations and a compelling approach to decrease mutation levels would therefore be to increase the fidelity of the catalytic subunit (POLγA) of the mtDNA polymerase. Here we genomically engineer the ''tamas'' locus, encoding fly POLγA, and introduce alleles expressing exonuclease- (exo(-)) and polymerase-deficient (pol(-)) POLγA versions. The exo(-) mutant leads to accumulation of point mutations and linear deletions of mtDNA, whereas pol(-) mutants cause mtDNA depletion. The mutant ''tamas'' alleles are developmentally lethal but can complement each other in trans resulting in viable flies with clonally expanded mtDNA mutations. Reconstitution of human mtDNA replication ''in vitro'' confirms that replication is a highly dynamic process where POLγA goes on and off the template to allow complementation during proofreading and elongation. The created fly models are valuable tools to study germ line transmission of mtDNA and the pathophysiology of POLγA mutation disease. +
Polyadenylation has well characterised roles in RNA turnover and translation in a variety of biological systems. While polyadenylation on mitochondrial transcripts has been suggested to be a two-step process required to complete translational stop codons, its involvement in mitochondrial RNA turnover is less well understood. We studied knockdown and knockout models of the mitochondrial poly(A) polymerase (MTPAP) in ''Drosophila melanogaster'' and demonstrate that polyadenylation of mitochondrial mRNAs is exclusively performed by MTPAP. Further, our results show that mitochondrial polyadenylation does not regulate mRNA stability but protects the 3' terminal integrity, and that despite a lack of functioning 3' ends, these trimmed transcripts are translated, suggesting that polyadenylation is not required for mitochondrial translation. Additionally, loss of MTPAP leads to reduced steady-state levels and disturbed maturation of tRNACys, indicating that polyadenylation in mitochondria might be important for the stability and maturation of specific tRNAs. +
The potential role of dystrophin-mediated control of systems integrating mitochondria with ATPases was assessed in muscle cells. Mitochondrial distribution and function in skinned cardiac and skeletal muscle fibers from dystrophin-deficient (MDX) and wild-type mice were compared. Laser confocal microscopy revealed disorganized mitochondrial arrays in m. gastrocnemius in MDX mice, whereas the other muscles appeared normal in this group. Irrespective of muscle type, the absence of dystrophin had no effect on the maximal capacity of oxidative phosphorylation, nor on coupling between oxidation and phosphorylation. However, in the myocardium and ''m. soleus'', the coupling of mitochondrial creatine kinase to adenine nucleotide translocase was attenuated as evidenced by the decreased effect of creatine on the Km for ADP in the reactions of oxidative phosphorylation. In ''m. soleus'', a low Km for ADP compared to the wild-type counterpart was found, which implies increased permeability for that nucleotide across the mitochondrial outer membrane. In normal cardiac fibers 35% of the ADP flux generated by ATPases was not accessible to the external pyruvate kinase-phosphoenolpyruvate system, which suggests the compartmentalized (direct) channeling of that fraction of ADP to mitochondria. Compared to control, the direct ADP transfer was increased in MDX ventricles. In conclusion, our data indicate that in slow-twitch muscle cells, the absence of dystrophin is associated with the rearrangement of the intracellular energy and feedback signal transfer systems between mitochondria and ATPases. As the mechanisms mediated by creatine kinases become ineffective, the role of diffusion of adenine nucleotides increases due to the higher permeability of the mitochondrial outer membrane for ADP and enhanced compartmentalization of ADP flux. +
This book uses an array of different approaches to describe photosynthesis, ranging from the subjectivity of human perception to the mathematical rigour of quantum electrodynamics. This interdisciplinary work draws from fields as diverse as astronomy, agriculture, classical and quantum optics, and biology in order to explain the working principles of photosynthesis in plants and cyanobacteria. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoeagle.org/index.php/MitoEAGLE|COST Action MitoEAGLE]]
Contact points between the endoplasmic reticulum (ER) and mitochondria enable Ca<sup>2+</sup> transfer between both organelles, which is a key mechanism in the modulation of mitochondrial metabolism. During early ER stress, this communication increases as an adaptive mechanism [1]. The signalling pathways controlling this response, however, are yet to be fully characterized. As candidates, we hypothesised that Caveolin-1 (CAV1) may be implicated, as it is enriched at ER–mitochondria contact sites [2]. PKA was also a candidate, as its activity has been shown to regulate organelle dynamics [3].
We used wild type HeLa cells for overexpressing CAV1. Early ER stress was induced with tunicamycin 0,5 µg/mL for 4 h. We measured ER–mitochondria contacts via electron microscopy and confocal microscopy. For Ca<sup>2+</sup> transfer, we used the fluorescent probe Rhod-FF. To evaluate mitochondrial respiration, we measured a Clark’s electrode. DRP1 phosphorylation was analysed through western blot. Cell viability was determined though annexin V staining using flow cytometry.
Early ER stress augmented ER–mitochondria contacts, which was prevented by CAV1 overexpression. This rendered ER-to-mitochondria Ca<sup>2+</sup> transfer and mitochondrial bioenergetics unresponsive to ER stress. PKA inhibition with H89 or siRNA also impaired the increase in organelle contacts, Ca<sup>2+</sup> transfer and mitochondrial respiration. CAV1 overexpression reduced PKA-mediated DRP1 phosphorylation, thereby enhancing ER stress-induced cell death. Increasing ER-mitochondria contacts with a synthetic linker restored cell survival.
Thus, PKA promotes the increase of ER–mitochondria contacts that occurs during ER stress. CAV1, in turn, prevents PKA-mediated phosphorylation, also impairing said remodelling. Increasing ER–mitochondria contacts is necessary for the increase in mitochondrial bioenergetics and cell adaptation to ER stress [4].
Zika virus (ZIKV) has a strong tropism for the nervous system and has been related to post-infection neurological syndromes. Once neuronal cells are infected, the virus is capable of modulating cell metabolism, leading to neurotoxicity and cellular death. The negative effect of ZIKV in neuron cells has been characterized. However, the description of molecules capable of reversing these cytotoxic effects is still under investigation. In this context, it has been largely demonstrated that docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid, is highly neuroprotective. Here, we hypothesized that DHA's neuroprotective proprieties could have an influence on ZIKV-induced neurotoxicity in SH-SY5Y cells. Our data showed that pre-treatment of SH-SY5Y cells with DHA increased the cell viability and proliferation in ZIKV-infected cells. Moreover, DHA triggered an anti-inflammatory response in those infected cells. Besides, DHA was capable of restoring mitochondria function and number in ZIKV-infected SH-SY5Y cells. In addition, cells pre-treated with DHA prior to ZIKV infection presented a lower viral load at different times of infection. Taking together, these results demonstrated that DHA has a potential anti-inflammatory and neuroprotective effect against ZIKV infection in these neuron-like cells and could be a useful tool in the treatment against this virus. +
Copper oxide nanoparticles (nCuO) are widely used in boat antifouling paints, and are released into the environment, inducing toxicity to aquatic organisms. The present study aimed to understand the effects of nCuO and dissolved copper on two ornamental Amazon fish species: ''Apistogramma agassizii'' and ''Paracheirodon axelrodi''. Fish were exposed to 50% nCuO LC<sub>50</sub> (''A. agassizii'' 375 μg L<sup>-1</sup> and ''P. axelrodi'' 460 μg L-1) and 50% Cu LC<sub>50</sub> (''A. agassizii'' 20 mg L<sup>-1</sup> and ''P. axelrodi'' 22.9 μg L-1) for 24, 48, 72 and 96 hours. Metabolic rate (MO<sub>2</sub>), gill osmorregulatory processes, gill mitochondria oxidative phosphorylation capacity and ROS generation, oxidative stress defense and morfological damages were evaluated. Our results showed a strong increase in MO<sub>2</sub> and a higher impairment in its gill’s morphology in ''P. axelrodi'' after the copper exposures. Differently, ''A. agassizii'' presented an increased proton leak (i.e. uncoupling between respiration and ATP production) in response to nCuO and Cu exposure, thus decreasing their Respiratory Control Rate (RCR). Interestingly, this uncoupling was directly related to an increase in ROS levels. Our findings reveal that the metabolic responses of these two species in response to nCuO and Cu, which are probably caused by the differences between species natural histories, indicating that different mechanisms of toxic action of the contaminants are associated to differences in the sensibility of these two species. +
Copper oxide nanoparticles (nCuO) are widely used in boat antifouling paints and are released into the environment, potentially inducing toxicity to aquatic organisms. The present study aimed to understand the effects of nCuO and dissolved copper (Cu) on two ornamental Amazon fish species: dwarf cichlid (''Apistogramma agassizii'') and cardinal tetra (''Paracheirodon axelrodi''). Fish were exposed to 50% of the LC<sub>50</sub> for nCuO (dwarf cichlid 58.31μgL<sup>-1</sup> and cardinal tetra 69.6μgL<sup>-1</sup>) and Cu (dwarf cichlid 20μgL<sup>-1</sup> and cardinal tetra 22.9μgL<sup>-1</sup>) for 24, 48, 72 and 96h. Following exposure, aerobic metabolic rate (''ṀO<sub>2</sub>''), gill osmoregulatory physiology and mitochondrial function, oxidative stress markers, and morphological damage were evaluated. Our results revealed species specificity in metabolic stress responses. An increase of ''ṀO<sub>2</sub>'' was noted in cardinal tetra exposed to Cu, but not nCuO, whereas ''ṀO<sub>2</sub>'' in dwarf cichlid showed little change with either treatment. In contrast, mitochondria from dwarf cichlid exhibited increased proton leak and a resulting decrease in respiratory control ratios in response to nCuO and Cu exposure. This uncoupling was directly related to an increase in reactive oxygen species (ROS) levels. Our findings reveal different metabolic responses between these two species in response to nCuO and Cu, which are probably caused by the differences between species natural histories, indicating that different mechanisms of toxic action of the contaminants are associated to differential osmoregulatory strategies among species. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MITOEAGLE]] Metformin is widely prescribed as a first-choice anti-hyperglycemic drug for treatment of type 2 diabetes mellitus and recent epidemiological studies demonstrated its utility also in cancer therapy. Despite its beneficial therapeutic effects and long term use in clinical practice, its molecular target, either for anti-hyperglycemic or anti-neoplastic action, remains elusive. However, the body of the research on metformin effect oscillates around mitochondrial metabolism, including the function of oxidative phosphorylation (OXPHOS) apparatus.
We focused on direct inhibitory mechanism of biguanides (metformin and phenformin) on OXPHOS complexes and its functional impact using the model of isolated brown adipose tissue mitochondria. We demonstrated that biguanides non-specifically target the activities of all respiratory chain dehydrogenases (mitochondrial NADH, succinate and glycerophosphate dehydrogenases), but only at very high concentrations (10<sup>-2</sup> – 10<sup>-1</sup> M) that highly exceeded cellular concentrations observed during the treatment. In addition, these concentrations of biguanides also triggered burst of reactive oxygen species production which, in combination with pleiotropic OXPHOS inhibition, could be toxic for the organism.
We conclude that the favorable effect of biguanides should probably be associated with subtler mechanism, different from the generalized inhibition of the respiratory chain. +
Arctigenin has previously been identified as a potential anti-tumor treatment for advanced pancreatic cancer. However, the mechanism of how arctigenin kills cancer cells is not fully understood. In the present work we studied the mechanism of toxicity by arctigenin in the human pancreatic cell line, Panc-1, with special emphasis on the mitochondria. A comparison of Panc-1 cells cultured in glucose versus galactose medium was applied, allowing assessments of effects in glycolytic versus oxidative phosphorylation (OXPHOS)-dependent Panc-1 cells. For control purposes, the mitochondrial toxic response to treatment with arctigenin was compared to the anti-cancer drug, sorafenib, which is a tyrosine kinase inhibitor known for mitochondrial toxic off-target effects (Will et al., 2008). In both Panc-1 OXPHOS-dependent and glycolytic cells, arctigenin dissipated the mitochondrial membrane potential, which was demonstrated to be due to inhibition of the mitochondrial complexes II and IV. However, arctigenin selectively killed only the OXPHOS-dependent Panc-1 cells. This selective killing of OXPHOS-dependent Panc-1 cells was accompanied by generation of ER stress, mitochondrial membrane permeabilization and caspase activation leading to apoptosis and aponecrosis.
Copyright © 2016 Elsevier Ltd. All rights reserved. +
BACKGROUND: An organic extract of the recreational herb khat (''Catha edulis'' Forsk.) triggers cell death in various leukemia cell lines ''in vitro''. The chemotherapeutics camptothecin, a plant alkaloid topoisomerase I inhibitor, was tested side-by-side with khat in a panel of acute myeloid leukemia cell lines to elucidate mechanisms of toxicity.
RESULTS: Khat had a profound effect on MOLM-13 cells inducing mitochondrial damage, chromatin margination and morphological features of autophagy. The effects of khat on mitochondrial ultrastructure in MOLM-13 correlated with strongly impaired routine respiration, an effect neither found in the khat-resistant MV-4-11 cells nor in camptothecin treated cells. Enforced expression of anti-apoptotic Bcl-2 protein provided protection against camptothecin-induced cell death and partly against khat toxicity. Khat-induced cell death in MOLM-13 cells included reduced levels of anti-apoptotic Mcl-1 protein, while both khat and camptothecin induced c-FLIPL cleavage and procaspase-8 activation.
CONCLUSION: Khat activated a distinct cell death pathway in sensitive leukemic cells as compared to camptothecin, involving mitochondrial damage and morphological features of autophagy. This suggests that khat should be further explored in the search for novel experimental therapeutics. +
Native uncoupling protein 1 (UCP 1) was purified from rat mitochondria by hydroxyapatite chromatography and identified by peptide mass mapping and tandem mass spectrometry. Native and expressed UCP 1 were reconstituted into liposomes, and proton flux through UCP 1 was shown to be fatty acid-dependent and GDP-sensitive. To investigate the mechanism of action of UCP 1, we determined whether hydrophilic modification of the omega-carbon of palmitate effected its transport function. We show that proton flux was greater through native UCP 1-containing proteoliposomes when facilitated by less hydrophilically modified palmitate (palmitate > omega-methoxypalmitate > omega-hydroxypalmitate with little or no proton flux due to glucose-O-omega-palmitate or undecanesulfonate). We show that non-proton-dependent charge transfer was greater when facilitated by less hydrophilically modified palmitate (palmitate/undecanesulfonate > omega-methoxypalmitate > omega-hydroxypalmitate, with no non-proton-dependent charge transfer flux due to glucose-O-omega-palmitate). We show that the GDP-inhibitable oxygen consumption rate in brown adipose tissue mitochondria was reversed by palmitate (as expected) but not by glucose-O-omega-palmitate. Our data are consistent with the model that UCP 1 flips long-chain fatty acid anions and contradict the "cofactor" model of UCP 1 function. +
[[File:Bild2010.jpg|right|150px|Michael Breitenbach]]
A yeast deletion mutant in the gene AFO1, coding for a mitochondrial ribosomal protein, confers respiratory deficiency, resistance to several oxidants, a 60% increased replicative lifespan, and very surprisingly, rapid growth on glucose and a highly efficient energy metabolism. The gene defect induces loss of the mitochondrial genome. We showed that the effect of the mutation on lifespan is independent of the retrograde response, and defines a longevity signaling mechanism from the mitochondria to the nuclear/cytoplasmic gene expression system which depends on the presence of an intact TOR1 gene and glucose as a carbon source. The mutant displays an extraordinarily low level of oxygen radicals. We show that this mutation grows rapidly and produces ethanol and biomass on glucose with a kinetics comparable to wild type, in stark contrast to a ''bona fide'' ethidium bromide induced rho-zero strain, which grows slowly. The growth phenotypes were shown to be the same in two quite different genetic backgrounds, one of them completely prototrophic. Transcriptome and metabolic analysis of wild type and mutant confirms metabolic similarity of the two strains and points to futile metabolic cycles in the ''bona fide'' rho-zero strain, which could be reponsible for slow growth of the rho-zero strain. Taken together, the phenotype of the mutant points to the fact that slow growth of rho-zero strains is not caused by slow and inefficient production of ATP, as is often maintained in the textbooks, but is rather a metabolic regulatory phenomenon. It is the intention of this contribution to aid understanding of the role of oxidative stress response and the mitochondria in the mother cell-specific aging process.
# [http://www.ncbi.nlm.nih.gov/pubmed/11251834 Laun P, Pichova A, Madeo F, Fuchs J, Ellinger A, Kohlwein S, Dawes I, Fröhlich KU, Breitenbach M (2001) Mol Microbiol 39: 1166-1173.]
# [http://www.ncbi.nlm.nih.gov/pubmed/20157544 Heeren G, Rinnerthaler M, Laun P, von Seyerl P, Kössler S, Klinger H, Hager M, Bogengruber E, Jarolim S, Simon-Nobbe B, Schüller C, Carmona-Gutierrez D, Breitenbach-Koller L, Mück C, Jansen-Dürr P, Criollo A, Kroemer G, Madeo F, Breitenbach M (2009) The mitochondrial ribosomal protein of the large subunit, Afo1p, determines cellular longevity through mitochondrial back-signaling via TOR1. Aging (Albany NY) 1: 622-636 Open Access]
# [http://www.ncbi.nlm.nih.gov/pubmed/22586098 Rinnerthaler M, Büttner S, Laun P, Heeren G, Felder TK, Klinger H, Weinberger M, Stolze K, Grousl T, Hasek J, Benada O, Frydlova I, Klocker A, Simon-Nobbe B, Jansko B, Breitenbach-Koller H, Eisenberg T, Gourlay CW, Madeo F, Burhans WC, Breitenbach M (2012) Yno1p/Aim14p, a NADPH-oxidase ortholog, controls extramitochondrial reactive oxygen species generation, apoptosis, and actin cable formation in yeast. Proc Natl Acad Sci U S A 109: 8658-8663. Open Access]
Oxidative stress is now a well-researched area with thousands of new articles
appearing every year. We want to give the reader here an overview of the topics in biomedical
and basic oxidative stress research which are covered by the authors of this thematic issue.
We also want to give the newcomer a short introduction into some of the basic concepts,
definitions and analytical procedures used in this field.
... +
Mitochondrial uncoupling protein 1 is usually associated with brown adipose tissue but has recently been discovered in rat and mouse thymus. We wished to establish whether there was a thermogenic role for UCP 1 in thymus and thus examined the effect of 5 weeks cold-acclimation on rat thymus tissue abundance, thymocyte oxygen consumption, thymus mitochondrial abundance, uncoupling protein 1 expression and function. We found that thymocytes from cold-acclimated rats had oxygen consumption rates 8 times less than those from rats held at room temperature and that thymocytes from cold-acclimated rats or rats kept at room temperature were noradrenaline insensitive. In addition, we found that thymus tissue or mitochondrial abundance was not increased after cold-acclimation. However uncoupling protein 1 expression per unit mass of mitochondria was increased after cold-acclimation, as determined by immunoblotting (not, vert, similar 1.7-fold) and GDP binding (not, vert, similar 1.5-fold). Consistent with our protein expression data, we also observed an increased, state 4 (not, vert, similar 1.5-fold), GDP-inhibitable (not, vert, similar 1.3-fold) and palmitate activatable (not, vert, similar 1.6-fold) oxygen consumption rates in isolated thymus mitochondria. However, extrapolation of our data showed that cold-acclimation only increased the amount of UCP 1 per gram of thymus tissue not, vert, similar 1.2-fold. Taken together, we conclude that UCP 1 does not have a thermogenic role in thymus. +
Transporters of the SLC25 mitochondrial carrier superfamily bridge cytoplasmic and mitochondrial metabolism by channeling metabolites across mitochondrial membranes and are pivotal for metabolic homeostasis. Despite their physiological relevance as gatekeepers of cellular metabolism, most of the SLC25 family members remain uncharacterized. We undertook a comprehensive tissue distribution analysis of all Slc25 family members across metabolic organs and identified SLC25A47 as a liver-specific mitochondrial carrier.
We used a murine loss-of-function model to unravel the role of this transporter in mitochondrial and hepatic homeostasis. We performed extensive metabolic phenotyping and molecular characterization of newly generated Slc25a47<sup>hep-/-</sup> and Slc25a47-Fgf21<sup>hep-/-</sup> mice.
Slc25a47<sup>hep-/-</sup> mice displayed a wide variety of metabolic abnormalities, as a result of sustained energy deficiency in the liver originating from impaired mitochondrial respiration. This mitochondrial phenotype was associated with an activation of the mitochondrial stress response (MSR) in the liver, and the development of fibrosis, which was exacerbated upon feeding a high-fat high-sucrose diet. The MSR induced the secretion of several mitokines, amongst which FGF21 played a preponderant role on systemic physiology. To dissect the FGF21-dependent and -independent physiological changes induced in Slc25a47<sup>hep-/-</sup> mice, we generated a double Slc25a47-Fgf21<sup>hep-/-</sup> mouse model and demonstrated that several aspects of the hypermetabolic state were driven by hepatic secretion of FGF21. On the other hand, the metabolic fuel inflexibility observed in Slc25a47<sup>hep-/-</sup> mice could not be rescued with the genetic removal of Fgf21.
Collectively, our data place the Slc25a47 locus at the center of mitochondrial homeostasis, which upon dysfunction triggers robust liver-specific and systemic adaptive stress responses. The prominent role of the Slc25a47 locus in hepatic fibrosis identifies this carrier, or its transported metabolite, as a potential target for therapeutic intervention.
White adipose tissue (WAT) has a crucial role in the development of obesity and related diseases, and the relevance of WAT mitochondrial function has been highlighted in the literature during the last decade [1,2,4]. Mitochondrial parameters, such as reactive oxygen species, biogenesis, fatty acid oxidation, respiration and uncoupling have been implicated in white adipocyte proliferation, adipogenesis, transdifferentiation, lipolysis and lipogenesis [1,2,4]. Therefore, WAT mitochondria function regulation is a promising target for the development of therapies tackling insulin resistance, obesity and related diseases.
Palmitoleic acid is a monounsaturated n-7 fatty acid (16:1n7), produced and released by adipocytes, that has been shown to enhance whole body glucose disposal, to attenuate high-fat-fed mice hepatic steatosis, to protect pancreatic beta-cells from palmitic acid-induced death and to improve circulating lipid profile in both rodents and humans [3]. Our group has recently found strong evidence that palmitoleic acid is an important positive modulator of white adipocyte lipolysis and the content of the major lipases ATGL and HSL through a PPAR alpha-dependent mechanism in vitro and in vivo. Acute and chronic palmitoleic treatment led to enhanced lipolysis and inhibited lipogenesis [3].
To study the correlation of the previously described effects of palmitoleic acid in WAT with mitochondrial function, we performed oxygen consumption experiments using the Oroboros Oxygraph-2k. Our results show that both acute and chronic treatments with palmitoleic acid enhanced basal oxygen consumption in 3T3-L1 adipocytes by 7.6% and 12.8%, respectively. Experiments were carried out to test whether lipolysis and respiration enhancement by palmitoleic acid are linked to improved mitochondrial fatty acid oxidation and/or uncoupling. +
Interactions of chemicals with cerebral cellular systems are often accompanied by similar changes involving components in non-neural tissues. On this basis, indirect strategies have been developed to investigate neural cell function parameters by methods using accessible cells, including platelets and/or peripheral blood lymphocytes. Therefore, here it was investigated whether peripheral blood markers may be useful for assessing the central toxic effects of methylmercury (MeHg). For this purpose, we investigated platelet mitochondrial physiology in a well-established mouse model of MeHg-induced neurotoxicity, and correlated this peripheral activity with behavioural and central biochemical parameters. In order to characterize the cortical toxicity induced by MeHg (20 and 40 mg/L in drinking water, 21 days), the behavioral parameter namely, short-term object recognition, and the central mitochondrial impairment assessed by measuring respiratory complexes I-IV enzyme activities were determined in MeHg-poisoned animals. Neurotoxicity induced by MeHg exposure provoked compromised cortical activity (memory impairment) and reduced NADH dehydrogenase, complex II and II-III activities in the cerebral cortex. These alterations correlated with impaired systemic platelet oxygen consumption of intoxicated mice, which was characterized by reduced electron transfer activity and uncoupled mitochondria. The data brought here demonstrated that impaired systemic platelet oxygen consumption is a sensitive and non-invasive marker of the brain energy deficits induced by MeHg poisoning. Finally, brain and platelets biochemical alterations significantly correlated with cognitive behavior in poisoned mice. Therefore, it could be proposed the use of platelet oxygen consumption as a peripheral blood marker of brain function in a mouse model MeHg-induced neurotoxicity. +
Endothelial cells in the vascular system are constantly subjected to the frictional force of shear stress due to the pulsatile nature of blood flow. Although several proteins form part of the shear stress mechano-sensing pathway, the identification of mechano-transducing pathways is largely unknown. Given the increasing evidence for a signaling function of mitochondria in endothelial cells, the aim of this study was to investigate their role as mechano-sensor organelles during laminar shear stress (LSS). We demonstrated that LSS activates intracellular signaling pathways that modulate not only mitochondrial dynamics but also mitochondrial function. At early time points of LSS, the fission-related protein Drp1 was recruited from the cytosol to mitochondria and activated mitochondrial fission. LSS-dependent increase in intracellular Ca2+ concentration was indispensable for mitochondrial fission. As alterations in mitochondrial dynamics have been related to changes in bioenergetics profiles, we studied mitochondrial function after LSS. We found that LSS decreased respiration rate, increased mitochondrial membrane potential and promoted the mitochondrial generation of ROS with the subsequent oxidation and activation of the antioxidant enzyme PRX3. Our data support a novel and active role for mitochondria in endothelial cells as active players, able to transduce the mechanical force of shear stress in the vascular endothelium into a biological response. +
The development of neurologic disease is a complex and multi-faceted process. Several factors, such as physiology, environment and genetics may play key roles in the manifestation of the associated illnesses. During the past decades, it has become clear that, at the cellular level, mitochondria function as more than "just" an energy source for our cells and plays a significant role in such aspects as neuronal development, maintenance and degeneration. Malfunctions in mitochondrial respiration and ATP production may prove disastrous for our cells and neurons, ultimately resulting in apoptosis, neurodegeneration and consequently, neurodegenerative diseases. +
We tested the effect of an anti-oxidant mixture on respiration in isolated rat brain mitochondria. Mitochondria were isolated in mannitol/sucrose/EGTA/BSA±SCAVEGR™ anti-oxidants (SOD, catalase, vitamin E, vitamin E acetate, and glutathione reduced). TBARS were reduced by greater than 40% with SCAVEGR. Respiration driven by ADP showed a two-fold higher Vmax and a 15% higher respiratory control ratio when mitochondria were prepared with SCAVEGR. SCAVEGR also stabilized the octameric state of mitochondrial creatine kinase and thus improved creatine-stimulated respiration. These results suggest that significant improvements in brain mitochondrial function are obtained by isolation in the presence of an anti-oxidants mixture. +
The most interesting property of neurons is their long-distance propagation of signals as spiking action potentials. Since 1993, Neurobasal/B27 has been used as a serum-free medium optimized for hippocampal neuron survival. Neurons on microelectrode arrays (MEA) were used as an assay system to increase spontaneous spike rates in media of different compositions. We find spike rates of 0.5 s(-1) (Hz) for rat embryonic hippocampal neurons cultured in Neurobasal/B27, lower than cultures in serum-based media and offering an opportunity for improvement. NbActiv4 was formulated by addition of creatine, cholesterol and estrogen to Neurobasal/B27 that synergistically produced an eightfold increase in spontaneous spike activity. The increased activity with NbActiv4 correlated with a twofold increase in immunoreactive synaptophysin bright puncta and GluR1 total puncta. Characteristic of synaptic scaling, immunoreactive GABAAbeta puncta also increased 1.5-fold and NMDA-R1 puncta increased 1.8-fold. Neuron survival in NbActiv4 equaled that in Neurobasal/B27, but with slightly higher astroglia. Resting respiratory demand was decreased and demand capacity was increased in NbActiv4, indicating less stress and higher efficiency. These results show that NbActiv4 is an improvement to Neurobasal/B27 for cultured networks with an increased density of synapses and transmitter receptors which produces higher spontaneous spike rates in neuron networks. +
Aberrant histone methylation profile is reported to correlate with the development and progression of NAFLD during obesity. However, the identification of specific epigenetic modifiers involved in this process remains poorly understood. Here, we identify the histone demethylase Plant Homeodomain Finger 2 (Phf2) as a new transcriptional co-activator of the transcription factor Carbohydrate Responsive Element Binding Protein (ChREBP). By specifically erasing H3K9me2 methyl-marks on the promoter of ChREBP-regulated genes, Phf2 facilitates incorporation of metabolic precursors into mono-unsaturated fatty acids, leading to hepatosteatosis development in the absence of inflammation and insulin resistance. Moreover, the Phf2-mediated activation of the transcription factor NF-E2-related factor 2 (Nrf2) further reroutes glucose fluxes toward the pentose phosphate pathway and glutathione biosynthesis, protecting the liver from oxidative stress and fibrogenesis in response to diet-induced obesity. Overall, our findings establish a downstream epigenetic checkpoint, whereby Phf2, through facilitating H3K9me2 demethylation at specific gene promoters, protects liver from the pathogenesis progression of NAFLD. +
[[File:Gnaiger&Forstner POS 1983.jpg|right|140px|link=Gnaiger 1983 Springer POS|Gnaiger 1983 Springer POS]] A major function of blood in most organisms is to supply the tissues with oxygen. The measurement of oxygen partial pressure (''p''<sub>O<sub>2</sub></sub>) and oxygen content (''c''<sub>O<sub>2</sub></sub>) in blood are therefore important criteria through which the state of the gas exchange system of the organism can be assessed.
In: [[Gnaiger 1983 Springer POS |Gnaiger E, Forstner H, eds (1983) Polarographic Oxygen Sensors. Aquatic and Physiological Applications. Springer, Berlin, Heidelberg, New York:370 pp.]] +
Obesity is a growing health issue in Canada and the identification of the determinants of obesity is important for the development of prevention strategies. The purpose of this investigation was to determine the relationships between physical activity, cardiorespiratory fitness, body mass index (BMI), and the development of future obesity.
The sample included 459 adults (18+ y; 223 men, 236 women) from the Canadian Physical Activity Longitudinal Study (PALS; 2002-04). Data on physical activity, smoking, alcohol consumption, BMI, and cardiorespiratory fitness (VO2max) were collected in 1981 and 1988. The mean BMI, physical activity, and VO2max were calculated across the 1981 and 1988 measures. Self-reported height and weight were collected in the 2002-04 survey, and participants were classified as overweight (BMI 25 to 29.9 kg/m2) or obese (BMI 230 kg/m2). Logistic regression was used to predict overweight, obesity or substantial weight gain (10 kg or more) in 2002-04, controlling for age, sex, smoking and alcohol use.
Higher VO2max in 1981-88 was associated with lower odds of obesity in 2002-04 (OR = 0.87; 95% Cl: 0.76-0.99, p < 0.05), and higher BMI in 1981-88 was associated with higher odds of obesity in 2002-04 (1.84; 1.52-2.20, p < 0.0001). In women, higher VO2max (0.82; 0.72-0.93) resulted in lower odds of a 10 kg weight gain.
The results indicate that cardiorespiratory fitness and previous BMI are important predictors of future weight gain and obesity, and should be incorporated in strategies to identify individuals at increased risk of obesity. +
No abstract supplied. +
It is now well established that mitochondria contain three antiporters that transport monovalent cations. A latent, allosterically regulated K+/H+ antiport appears to serve as a cation-extruding device that helps maintain mitochondrial volume homeostasis. An apparently unregulated Na+/H+ antiport keeps matrix [Na+] low and the Na(+)-gradient equal to the H(+)-gradient. A Na+/Ca2+ antiport provides a Ca(2+)-extruding mechanism that permits the mitochondrion to regulate matrix [Ca2+] by balancing Ca2+ efflux against influx on the Ca(2+)-uniport. All three antiports have well-defined physiological roles and their molecular properties and regulatory features are now being determined. Mitochondria also contain monovalent cation uniports, such as the recently described ATP- and glibenclamide-sensitive K+ channel and ruthenium red-sensitive uniports for Na+ and K+. A physiological role of such uniports has not been established and their properties are just beginning to be defined. +
Maleic acid (MA)-induced nephropathy that is characterized by proteinuria, glycosuria, phosphaturia and a deficient urinary acidification and concentration. Sulforaphane (SF) is an indirect antioxidant that shows nephroprotective effects. The aim of the present work was to test the pre-treatment with SF against the MA-induced nephropathy.
Wistar rats (230-260 g) were separated in the following groups: control, MA (which received 400 mg/kg of MA), SF + MA (which received MA and 1 mg/kg of SF each day for four days) and SF (which only received SF). MA induced proteinuria, an increase in urinary excretion of N-acetyl-β-d-glucosaminidase, and a decrease in plasma glutathione peroxidase activity, renal blood flow, and oxygenation and perfusion of renal cortex. All these impairments correlated with higher levels of oxidative damage markers and exacerbated superoxide anion production on renal cortex. Moreover, MA impaired mitochondrial bioenergetics associated to complex I, mitochondrial membrane potential and respiratory control index and increased the mitochondrial production of hydrogen peroxide. Further it disrupted mitochondrial morphology. SF prevented all the above-described alterations.
In conclusion, the protective effect of SF against MA-induced nephropathy is associated with preservation of mitochondrial bioenergetics, amelioration of oxidative stress and improvement of renal hemodynamics and renal cortex oxygenation. +
The kidney proximal tubule function relies on oxidative phosphorylation (OXPHOS), thus mitochondrial dysfunction is characteristic of acute kidney injury (AKI). Maleic acid (MA) can induce an experimental model of Fanconi syndrome that is associated to oxidative stress and decreased oxygen consumption. Sulforaphane (SF) is an antioxidant known to protect against MA-induced AKI. The molecular basis by which SF maintains the bioenergetics in MA-induced AKI is not fully understood. To achieve it, rats were submitted to a protective scheme: SF (1 mg/kg/day i.p.) for four days and, at the fourth day, they received a single dose of MA (400 mg/kg i.p.), getting four main experimental groups: (1) control (CT), (2) MA-nephropathy (MA), (3) SF-protected and (4) SF-control (SF). Additionally, a similar protective schema was tested in cultured NRK-52E cells with different concentrations of SF and MA. In the animal model, SF prevented the MA-induced alterations: decrease in fatty acid-related oxygen consumption rate, OXPHOS capacity, mitochondrial membrane potential (Ψ<sub>mt</sub>), and the activity of complex I (CI) as its monomeric and supercomplexes forms; the antioxidant also increased the activity of cytochrome c oxidase as well as mitochondrial biogenesis markers. Thus, SF prevented the MA-induced increase in fission, mitophagy and autophagy markers. In NRK-52E cells, we found that SF prevented the MA-induced cell death, increased mitochondrial mass and ameliorated the loss of Ψ<sub>mt</sub>. We concluded that SF-induced biogenesis protects against mitochondrial dysfunction maintaining Ψmt, activities of mitochondrial complexes and supercomplexes, and prevents the extensive fission and mitophagy.
<small>Copyright © 2020 Elsevier Inc. All rights reserved.</small> +
Cytochrome c oxidase (COX) deficiency is characterized by a high degree of genetic and phenotypic heterogeneity, partly reflecting the extreme structural complexity, multiple post-translational modification, variable, tissue-specific composition, and the high number of and intricate connections among the assembly factors of this enzyme. In fact, decreased COX specific activity can manifest with different degrees of severity, affect the whole organism or specific tissues, and develop a wide spectrum of disease natural history, including disease onsets ranging from birth to late adulthood. More than 30 genes have been linked to COX deficiency, but the list is still incomplete and in fact constantly updated. We here discuss the current knowledge about COX in health and disease, focusing on genetic aetiology and link to clinical manifestations. In addition, information concerning either fundamental biological features of the enzymes or biochemical signatures of its defects have been provided by experimental in vivo models, including yeast, fly, mouse and fish, which expanded our knowledge on the functional features and the phenotypical consequences of different forms of COX deficiency. +
Growing evidence suggests persistent mitochondrial permeability transition pore (mPTP) opening is a key pathophysiological event in cell death underlying a variety of diseases. While it has long been clear the mPTP is a druggable target, current agents are limited by off-target effects and low therapeutic efficacy. Therefore identification and development of novel inhibitors is necessary. To rapidly screen large compound libraries for novel mPTP modulators, a method was exploited to cryopreserve large batches of functionally active mitochondria from cells and tissues. The cryopreserved mitochondria maintained respiratory coupling and ATP synthesis, Ca<sup>2+</sup> uptake and transmembrane potential. A high-throughput screen (HTS), using an assay of Ca<sup>2+</sup>-induced mitochondrial swelling in the cryopreserved mitochondria identified ER-000444793, a potent inhibitor of mPTP opening. Further evaluation using assays of Ca<sup>2+</sup> induced membrane depolarisation and Ca<sup>2+</sup> retention capacity also indicated that ER-000444793 acted as an inhibitor of the mPTP. ER-000444793 neither affected cyclophilin D (CypD) enzymatic activity, nor displaced of CsA from CypD protein, suggesting a mechanism independent of CypD inhibition. Here we identified a novel, CypD-independent inhibitor of the mPTP. The screening approach and compound described provides a workflow and additional tool to aid the search for novel mPTP modulators and to help understand its molecular nature. +
Mitochondrial Ca<sup>2+</sup> uptake has a key role in cellular Ca<sup>2+</sup> homeostasis. Excessive matrix Ca<sup>2+</sup> concentrations, especially when coincident with oxidative stress, precipitate opening of an inner mitochondrial membrane, high-conductance channel: the mitochondrial permeability transition pore (mPTP). mPTP opening has been implicated as a final cell death pathway in numerous diseases and therefore understanding conditions dictating mPTP opening is crucial for developing targeted therapies. Here, we have investigated the impact of mitochondrial metabolic state on the probability and consequences of mPTP opening. Isolated mitochondria were energised using NADH- or FADH<sub>2</sub>-linked substrates. The functional consequences of Ca<sup>2+</sup>-induced mPTP opening were assessed by Ca<sup>2+</sup> retention capacity, using fluorescence-based analysis, and simultaneous measurements of mitochondrial Ca<sup>2+</sup> handling, membrane potential, respiratory rate and production of reactive oxygen species (ROS). Succinate-induced, membrane potential-dependent reverse electron transfer sensitised mitochondria to mPTP opening. mPTP-induced depolarisation under succinate subsequently inhibited reverse electron transfer. Complex I-driven respiration was reduced after mPTP opening but sustained in the presence of complex II-linked substrates, consistent with inhibition of complex I-supported respiration by leakage of matrix NADH. Additionally, ROS generated at complex III did not sensitise mitochondria to mPTP opening. Thus, cellular metabolic fluxes and metabolic environment dictate mitochondrial functional response to Ca<sup>2+</sup> overload. +
Li-Fraumeni Syndrome (LFS) and Li-Fraumeni-like Syndrome (LFL) are inherited disorders, associated to TP53 germline mutations and characterized by increased predisposition to multiple early-onset cancers [1]. Studies in families from Southern and Southeastern Brazil with LFS/LFL phenotype have identified a germline founder mutation in the TP53 gene, the p.R337H mutation (c.1010G>A), in a high population prevalence (~0.3%) [2]. Unlike the majority of the mutations in TP53, which are missense mutations located in the DNA-binding domain (DBD) of the protein (exons 5-8), the TP53 p.R337H (c.1010G>A) is located in exon 10, corresponding to the oligomerization domain (OD). The p53 nuclear phosphoprotein is known for its functions in the DNA damage response and apoptosis. Recently, this protein has been shown to regulate many aspects of energy metabolism as well as enzymes that are involved in cell responses to oxidative stress, manly through TIGAR activation [3].
In a previous work, we analyzed the levels of several markers of oxidative stress responses in blood samples of p.R337H mutation carries and non-carries. We observed oxidative damage in lipids and proteins. Moreover, there was increased erythrocyte GPx activity, as well as increased total antioxidant status in the p.R337H mutation carries [4]. Therefore, our study was able to establish the relationship of oxidative stress with the loss of function of p53.
The aim of this work was to evaluate the association between TP53 germline mutations with deregulation of cell bioenergetics. For this purpose, we performed high-resolution respirometry (HHR) of intact human fibroblast cells, derived from patients. Preliminary results showed a distinct pattern of HHR in different TP53 germline mutations genotypes. Fibroblasts from carriers of DBD mutations and wt/p.R337H showed higher ROUTINE, total and extramitochodrial respiration, as well as LEAK respiration, compared to p.R377H/p.R337H mutants and WT/WT cells. In agreement with HHR results, cells with DBD mutation showed increased ROS (reactive oxygen species) by DCF assay. On the other hand an unexpectedly high production was found of ROS by p.R377H/p.R337H. These data were correlated with antimetabolic drug sensitivity, mitochondrial membrane potential and cellular doubling time to better evaluate the potential role of these findings for the increased predisposition to multiple early-onset cancers presented by Li-Fraumeni patients.
Cell-to-cell fusion is emerging as a key element of the metastatic process in various cancer types. We recently showed that hybrids made from the spontaneous merging of pre-malignant (IMR90 E6E7, i.e. E6E7) and malignant (IMR90 E6E7 RST, i.e. RST) mesenchymal cells recapitulate the main features of human undifferentiated pleomorphic sarcoma (UPS), with a highly rearranged genome and increased spreading capacities. To better characterize the intrinsic properties of these hybrids, we investigated here their metabolic energy profile compared to their parents.
Our results unveiled that hybrids harbored a Warburg-like metabolism, like their RST counterparts. However, hybrids displayed a much greater metabolic activity, enhancing glycolysis to proliferate. Interestingly, modifying the metabolic environmental conditions through the use of 5-aminoimidazole-4-carbox-amide-1-β-D-ribofuranoside (AICAR), an activator of the 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK), specifically reduced the growth of hybrids, and also abrogated the invasive capacity of hybrids displaying enhanced glycolysis. Furthermore, AICAR efficiently blocked the tumoral features related to the aggressiveness of human UPS cell lines.
Altogether, our findings strongly suggest that hybrids rely on higher energy flux to proliferate and that a drug altering this metabolic equilibrium could impair their survival and be potentially considered as a novel therapeutic strategy. +
Skeletal muscle atrophy is a common feature of numerous chronic pathologies and is correlated with patient mortality. The REDD1 protein is currently recognized as a negative regulator of muscle mass through inhibition of the Akt/mTORC1 signaling pathway. REDD1 expression is notably induced following glucocorticoid secretion, which is a component of energy stress responses.
Unexpectedly, we show here that REDD1 instead limits muscle loss during energetic stresses such as hypoxia and fasting by reducing glycogen depletion and AMPK activation. Indeed, we demonstrate that REDD1 is required to decrease O<sub>2</sub> and ATP consumption in skeletal muscle via reduction of the extent of mitochondrial-associated endoplasmic reticulum membranes (MAMs), a central hub connecting energy production by mitochondria and anabolic processes. In fact, REDD1 inhibits ATP-demanding processes such as glycogen storage and protein synthesis through disruption of the Akt/Hexokinase II and PRAS40/mTORC1 signaling pathways in MAMs. Our results uncover a new REDD1-dependent mechanism coupling mitochondrial respiration and anabolic processes during hypoxia, fasting, and exercise.
Therefore, REDD1 is a crucial negative regulator of energy expenditure that is necessary for muscle adaptation during energetic stresses. This present study could shed new light on the role of REDD1 in several pathologies associated with energetic metabolism alteration, such as cancer, diabetes, and Parkinson's disease. +
We investigated the underlying molecular mechanisms by which postexercise cold-water immersion (CWI) may alter key markers of mitochondrial biogenesis following both a single session and 6 wk of sprint interval training (SIT). Nineteen men performed a single SIT session, followed by one of two 15-min recovery conditions: cold-water immersion (10°C) or a passive room temperature control (23°C). Sixteen of these participants also completed 6 wk of SIT, each session followed immediately by their designated recovery condition. Four muscle biopsies were obtained in total, three during the single SIT session (preexercise, postrecovery, and 3 h postrecovery) and one 48 h after the last SIT session. After a single SIT session, phosphorylated (p-)AMPK, p-p38 MAPK, p-p53, and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) mRNA were all increased (P < 0.05). Postexercise CWI had no effect on these responses. Consistent with the lack of a response after a single session, regular postexercise CWI had no effect on PGC-1α or p53 protein content. Six weeks of SIT increased peak aerobic power, ''V''<sub>O2peak</sub>, maximal oxygen consumption, maximal uncoupled respiration (complexes I and II), and 2-km time trial performance (P < 0.05). However, regular CWI had no effect on changes in these markers, consistent with the lack of response in the markers of mitochondrial biogenesis. Although these observations suggest that CWI is not detrimental to endurance adaptations following 6 wk of SIT, they question whether postexercise CWI is an effective strategy to promote mitochondrial biogenesis and improvements in endurance performance.
Copyright © 2017 the American Physiological Society. +
The aim of our work was to study effect of antidepressant imipramine on both thapsigargin- and tunicamycin-induced ER stress and mitochondrial dysfunction in neuroblastoma SH-SY5Y cells. ER stress in SH-SY5Y cells was induced by either tunicamycin or thapsigargin in the presence or absence of imipramine. Cell viability was tested by the MTT assay. Splicing of XBP1 mRNA was studied by RT-PCR. Finally, expression of Hrd1 and Hsp60 was determined by Western blot analysis.
Our findings provide evidence that at high concentrations imipramine potentiates ER stress-induced death of SH-SY5Y cells. The effect of imipramine on ER stress-induced death of SH-SY5Y cells was stronger in combination of imipramine with thapsigargin. In addition, we have found that treatment of SH-SY5Y cells with imipramine in combination of either thapsigargin or tunicamycin is associated with the alteration of ER stress-induced IRE1α-XBP1 signalling. Despite potentiation of ER stress-induced XBP1 splicing, imipramine suppresses both thapsigargin- and tunicamycin-induced expression of Hrd1. Finally, imipramine in combination with thapsigargin, but not tunicamycin, aggravates ER stress-induced mitochondrial dysfunction without significant impact on intracellular mitochondrial content as indicated by the unaltered expression of Hsp60.
Our results indicate the possibility that chronic treatment with imipramine might be associated with a higher risk of development and progression of neurodegenerative disorders, in particular those allied with ER stress and mitochondrial dysfunction like Parkinson's and Alzheimer's disease. +
Renal Fanconi syndrome is defined as an impaired transport capacity of the proximal tubule and can be caused by a variety of factors. Besides secondary Fanconi syndromes caused by another disease or as side effects of medication primary forms exist as a consequence of a mutation. Here, two distinct forms of renal Fanconi syndrome are described which are both inherited in an autosomal dominant fashion. Firstly, EHHADH, a protein involved in peroxisomal β-oxidation, and secondly FAP2, a component of creatine synthesis.
EHHADH was already known to be expressed in the late proximal tubule. This was confirmed by staining with two commercial antibodies, one staining for EHHADH and another staining for the peroxisomal protein D-amino acid oxidase. In both cases, a positive staining was visible in the kidney cortex in the late proximal tubule (S2/S3 segment). The observed granular staining was consistent with peroxisomal localization. I therefore concluded that EHHADH is expressed in peroxisomes of the late proximal tubule of the kidney. Previous experiments indicated that mutated EHHADH is also imported into the mitochondria. Intramitochondrial localization was evaluated electron microscopically using the ReAsH method. In EHHADHWT-transfected cells only the peroxisomes were positive. No electron-dense precipitates were visible in mitochondria. In contrast, in EHHADHMUT-transfected cells both peroxisomes and mitochondria were positive. In my diploma thesis I could already determine that mitochondrial import of EHHADHMUT leads to a decreased activity of oxidative phosphorylation. To evaluate if this affects transcellular transport α-D-methyl glucoside was added to the medium of cells which were grown on filters. This glucose derivative is transported but not metabolized by cells. Transport in EHHADHMUT-expressing cells was reduced to 25% of the capacity of EHHADHWT-expressing cells.
These data reveal new insights into the pathogenesis of this form of Fanconi syndrome. Mutated EHHADH is mistargeted to the mitochondria where it interferes with mitochondrial β-oxidation. This in turn leads to a decreased ATP production and subsequently to an impaired transepithelial transport capacity in the proximal tubule of affected patients.
When organic compounds are irradiated with UV light at 254 nm, part of their covalent bonds can dissociate if the compound absorbs light at that wavelength. Therefore, photo-degradation depends strongly on the wavelength used. The energy of a light quanta at 254 nm amounts to approximately 110 kcal/mol quanta, which is in many cases higher than the binding energy of a variety of covalent bonds. As a consequence, the absorbing molecule is degraded. As melatonin absorbs light at 254 nm, this compound is vulnerable to UV light. In order to minimize undesired effects of other absorbing substances, we used as solvent mostly pure water and analyzed the influence of λirr = 254 nm on the disappearance of the educt (melatonin) as well as on the appearance of products derived from melatonin in the presence of oxygen, argon, hydrogen peroxide, and ethanol by UV–VIS spectroscopy and high-performance liquid chromatography (HPLC) technique. N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) appears to be the main, but obviously not the only product of photo-degradation of melatonin, independently of whether the system contains oxygen or not. If the system contains additionally hydrogen peroxide, a very strong oxidant, the hydroxyl radical (•OH), is formed. Under such conditions, melatonin is not solely photo-degraded but also attacked by the formed •OH which interact similarly with the formed main photo-product AFMK. Ethanol, as a potent scavenger of •OH, efficiently blocks the effect of this aggressive radical even at low concentrations of that scavenger (0.1% v/v) but is less effective in preventing photo-degradation of melatonin. +
This ''in vitro'' study compares the frequency of redox cycling between alloxan and dialuric acid at different initial ratios of glutathione and alloxan. Alloxan oxidizes GSH to GSSG. The rate of GSH oxidation at a given initial GSH concentration of 2.0 mmol/L depends on the initial concentration of alloxan added. The higher the concentration of alloxan in relation to the initial concentration of GSH, the faster GSH oxidation proceeds, as well as oxygen consumption, and therefore, formation of reactive oxygen species. The highest rates of GSH oxidation, i. e. GSSG formation, were found at concentration ratios of between 2.0 mmol/L GSH and 0.2 and 0.04 mmol/L alloxan, respectively. Because 0.04 mmol/L alloxan oxidizes 2.0 mmol/L GSH completely, a frequency of at least 25 cycles between alloxan and dialuric acid within 3 hours can be assumed. During each redox cycle, two molecules of GSH are oxidized to one molecule of GSSG, and during each cycle one molecule of oxygen is reduced simultaneously to one molecule of hydrogen peroxide. In total, therefore, one molecule of alloxan oxidizes at least 50 molecules of GSH and forms about 25 molecules of hydrogen peroxide. +
When organic compounds are irradiated with UV light at 254 nm, part of their covalent bonds can dissociate if the compound absorbs light at that wavelength. Therefore, photo-degradation depends strongly on the wavelength used. The energy of a light quanta at 254 nm amounts to approximately 110 kcal/mol quanta, which is in many cases higher than the binding energy of a variety of covalent bonds. As a consequence, the absorbing molecule is degraded. As melatonin absorbs light at 254 nm, this compound is vulnerable to UV light. In order to minimize undesired effects of other absorbing substances, we used as solvent mostly pure water and analyzed the influence of lambda irr = 254 nm on the disappearance of the educt (melatonin) as well as on the appearance of products derived from melatonin in the presence of oxygen, argon, hydrogen peroxide, and ethanol by UV-VIS spectroscopy and high-performance liquid chromatography (HPLC) technique. N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) appears to be the main, but obviously not the only product of photo-degradation of melatonin, independently of whether the system contains oxygen or not. If the system contains additionally hydrogen peroxide, a very strong oxidant, the hydroxyl radical (*OH), is formed. Under such conditions, melatonin is not solely photo-degraded but also attacked by the formed *OH which interact similarly with the formed main photo-product AFMK. Ethanol, as a potent scavenger of *OH, efficiently blocks the effect of this aggressive radical even at low concentrations of that scavenger (0.1% v/v) but is less effective in preventing photo-degradation of melatonin. +
After a century, it's time to turn the page on understanding of lactate metabolism and appreciate that lactate shuttling is an important component of intermediary metabolism in vivo. Cell-cell and intracellular lactate shuttles fulfil purposes of energy substrate production and distribution, as well as cell signalling under fully aerobic conditions. Recognition of lactate shuttling came first in studies of physical exercise where the roles of driver (producer) and recipient (consumer) cells and tissues were obvious. Moreover, the presence of lactate shuttling as part of postprandial glucose disposal and satiety signalling has been recognized. Mitochondrial respiration creates the physiological sink for lactate disposal in vivo. Repeated lactate exposure from regular exercise results in adaptive processes such as mitochondrial biogenesis and other healthful circulatory and neurological characteristics such as improved physical work capacity, metabolic flexibility, learning, and memory. The importance of lactate and lactate shuttling in healthful living is further emphasized when lactate signalling and shuttling are dysregulated as occurs in particular illnesses and injuries. Like a phoenix, lactate has risen to major importance in 21st century biology. +
The role of mitochondrial ROS in signalling muscle adaptations to exercise training has not been explored in detail. We investigated the effect of supplementation with the mitochondria-targeted antioxidant MitoQ on a) the skeletal muscle mitochondrial and antioxidant gene transcriptional response to acute high-intensity exercise and b) skeletal muscle mitochondrial content and function following exercise training. In a randomised, double-blind, placebo-controlled, parallel design study, 23 untrained men (age: 44 ± 7 years, VO<sub>2peak</sub>: 39.6 ± 7.9 ml/kg/min) were randomised to receive either MitoQ (20 mg/d) or a placebo for 10 days before completing a bout of high-intensity interval exercise (cycle ergometer, 10 × 60 s at VO<sub>2peak</sub> workload with 75 s rest). Blood samples and ''vastus lateralis'' muscle biopsies were collected before exercise and immediately and 3 h after exercise. Participants then completed high-intensity interval training (HIIT; 3 sessions per week for 3 weeks) and another blood sample and muscle biopsy were collected. There was no effect of acute exercise or MitoQ on systemic (plasma protein carbonyls and reduced glutathione) or skeletal muscle (mtDNA damage and 4-HNE) oxidative stress biomarkers. Acute exercise-induced increases in skeletal muscle peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α) mRNA expression were augmented in the MitoQ group. Despite this, training-induced increases in skeletal muscle mitochondrial content were similar between groups. HIIT-induced increases in VO<sub>2peak</sub> and 20 km time trial performance were also similar between groups while training-induced increases in peak power achieved during the VO<sub>2peak</sub> test were augmented in the MitoQ group. These data suggest that training-induced increases in peak power are enhanced following MitoQ supplementation, which may be related to the augmentation of skeletal muscle PGC1α expression following acute exercise. However, these effects do not appear to be related to an effect of MitoQ supplementation on exercise-induced oxidative stress or training-induced mitochondrial biogenesis in skeletal muscle.
Sarcopenia is thought to be associated with mitochondrial (Mito) loss. It is unclear whether the decrease in Mito content is consequent to aging per se or to decreased physical activity.
The objective of the study was to examine the influence of fitness on Mito content and function and to assess whether exercise could improve Mito function in older adults.
Three distinct studies were conducted: 1) a cross-sectional observation comparing Mito content and fitness in a large heterogeneous cohort of older adults; 2) a case-control study comparing chronically endurance-trained older adults and sedentary (S) subjects matched for age and gender; and 3) a 4-month exercise intervention in S.
The study was conducted at a university-based clinical research center.
Mito volume density (MitoVd) was assessed by electron microscopy from vastus lateralis biopsies, electron transport chain proteins by Western blotting, mRNAs for transcription factors involved in M biogenesis by quantitative RT-PCR, and in vivo oxidative capacity (ATPmax) by (31)P-magnetice resonance spectroscopy. Peak oxygen uptake was measured by graded exercise test.
Peak oxygen uptake was strongly correlated with MitoVd in 80 60- to 80-year-old adults. Comparison of chronically endurance-trained older adults vs S revealed differences in MitoVd, ATPmax, and some electron transport chain protein complexes. Finally, exercise intervention confirmed that S subjects are able to recover MitoVd, ATPmax, and specific transcription factors.
These data suggest the following: 1) aging per se is not the primary culprit leading to Mito dysfunction; 2) an aerobic exercise program, even at an older age, can ameliorate the loss in skeletal muscle Mito content and may prevent aging muscle comorbidities; and 3) the improvement of Mito function is all about content. +
Oxidative phosphorylation (OXPHOS) is the primary source of ATP in eukaryotes and serves as a mechanistic link between variation in genotypes and energetic phenotypes. Increased aerobic capacity may be facilitated by numerous factors, including increased expression of OXPHOS genes, increase in number and morphology of mitochondria, enhanced O<sup>2</sup> transport within the body, and structural variation in OXPHOS proteins. Directional (positive) natural selection on particular amino acid sites may reveal important functional variation in OXPHOS proteins. Although evidence of natural selection on mitochondrial OXPHOS genes has been documented in several animal groups, the relationship between selection and OXPHOS function has not been well characterized. Because organismal performance should be a function of OXPHOS functional efficiency, we investigated this relationship by examining patterns of natural selection on OXPHOS genes in several vertebrate lineages with extreme differences in aerobic performance.
We assembled OXPHOS genes for several groups of taxa, including 40 mammal species from diverse taxonomic orders as well as trees for several fish groups each including one or more orders. For the mammals, all OXPHOS genes (both mitochondrial and nuclear encoded) were examined and species represented a range of energetic habits. In fishes, mitochondrial and some nuclear genes were examined from largely sedentary groups as well as highly active, high-performance swimmers. We used estimates of the synonymous (dS) to non-synonymous (dN) amino acid substitution rate ratios to provide evidence of natural selection. Directional (positive) selection was inferred where the dN/dS ratio was greater than 1.0, while purifying (negative) selection was indicated by dN/dS ratios much less than 1.0. Specific amino acid sites under positive selection were identified using a Bayesian procedure and for several groups these were localized on the 3-dimensional structure of their respective OXPHOS complexes.
We found that patterns of natural selection on OXPHOS genes are complex among fishes and mammals with different locomotive performance or other energetic demands. Positive selection is associated with several high performance groups but is also associated with some low performance taxa. For example among mammals positive selection was detected for many genes on the horse and primate lineages but was also detected on the lineage leading to sloths and armadillos. Some high performance taxa such as dolphins did not exhibit evidence of positive selection. In fishes positive selection was strongest on low-performance seahorses and flounders, while the high-performance tunas and marlins exhibited strong purifying (negative) selection. Thus, positive natural selection was not consistently associated with high performance taxa and in several cases selection was quite strong on low-performance taxa. Within some high-performance lineages, negative selection in the form of functional constraint appears to predominate.
Mice overexpressing NAMPT in skeletal muscle (NamptTg mice) develop higher exercise endurance and maximal aerobic capacity (VO<sub>2</sub>max) following voluntary exercise training compared to wild-type (WT) mice. Here, we aimed to investigate the mechanisms underlying by determining skeletal muscle mitochondrial respiratory capacity in NamptTg and WT mice. Body weight and body composition, tissue weight (gastrocnemius, quadriceps, soleus, heart, liver, and epididymal white adipose tissue), skeletal muscle and liver glycogen content, VO<sub>2</sub>max, skeletal muscle mitochondrial respiratory capacity (measured by high-resolution respirometry), skeletal muscle gene expression (measured by microarray and qPCR), and skeletal muscle protein content (measured by Western blot) were determined following 6 weeks of voluntary exercise training (access to running wheel) in 13-week-old male NamptTg (exercised NamptTg) mice and WT (exercised WT) mice. Daily running distance and running time during the voluntary exercise training protocol were recorded. Daily running distance (p = 0.51) and running time (p = 0.85) were not significantly different between exercised NamptTg mice and exercised WT mice. VO<sub>2</sub>max was higher in exercised NamptTg mice compared to exercised WT mice (p = 0.02). Body weight (p = 0.92), fat mass (p = 0.49), lean mass (p = 0.91), tissue weight (all p > 0.05), and skeletal muscle (p = 0.72) and liver (p = 0.94) glycogen content were not significantly different between exercised NamptTg mice and exercised WT mice. Complex I oxidative phosphorylation (OXPHOS) respiratory capacity supported by fatty acid substrates (p < 0.01), maximal (complex I+II) OXPHOS respiratory capacity supported by glycolytic (p = 0.02) and fatty acid (p < 0.01) substrates, and maximal uncoupled respiratory capacity supported by fatty acid substrates (p < 0.01) was higher in exercised NamptTg mice compared to exercised WT mice. Transcriptomic analyses revealed differential expression for genes involved in oxidative metabolism in exercised NamptTg mice compared to exercised WT mice, specifically, enrichment for the gene set related to the SIRT3-mediated signaling pathway. SIRT3 protein content correlated with NAMPT protein content (r = 0.61, p = 0.04). In conclusion, NamptTg mice develop higher exercise capacity following voluntary exercise training compared to WT mice, which is paralleled by higher mitochondrial respiratory capacity in skeletal muscle. The changes in SIRT3 targets suggest that these effects are due to remodeling of mitochondrial function.
We have seen that there is no simple answer to the question 'what controls respiration?' The answer varies with (a) the size of the system examined (mitochondria, cell or organ), (b) the conditions (rate of ATP use, level of hormonal stimulation), and (c) the particular organ examined. Of the various theories of control of respiration outlined in the introduction the ideas of Chance & Williams (1955, 1956) give the basic mechanism of how respiration is regulated. Increased ATP usage can cause increased respiration and ATP synthesis by mass action in all the main tissues. Superimposed on this basic mechanism is calcium control of matrix dehydrogenases (at least in heart and liver), and possibly also of the respiratory chain (at least in liver) and ATP synthase (at least in heart). In many tissues calcium also stimulates ATP usage directly; thus calcium may stimulate energy metabolism at (at least) four possible sites, the importance of each regulation varying with tissue. Regulation of multiple sites may occur (from a teleological point of view) because: (a) energy metabolism is branched and thus proportionate regulation of branches is required in order to maintain constant fluxes to branches (e.g. to proton leak or different ATP uses); and/or (b) control over fluxes is shared by a number of reactions, so that large increases in flux requires stimulation at multiple sites because each site has relatively little control. Control may be distributed throughout energy metabolism, possibly due to the necessity of minimizing cell protein levels (see Brown, 1991). The idea that energy metabolism is regulated by energy charge (as proposed by Atkinson, 1968, 1977) is misleading in mammals. Neither mitochondrial ATP synthesis nor cellular ATP usage is a unique function of energy charge as AMP is not a significant regulator (see for example Erecinska et al., 1977). The near-equilibrium hypothesis of Klingenberg (1961) and Erecinska & Wilson (1982) is partially correct in that oxidative phosphorylation is often close to equilibrium (apart from cytochrome oxidase) and as a consequence respiration and ATP synthesis are mainly regulated by (a) the phosphorylation potential, and (b) the NADH/NAD+ ratio. However, oxidative phosphorylation is not always close to equilibrium, at least in isolated mitochondria, and relative proximity to equilibrium does not prevent the respiratory chain, the proton leak, the ATP synthase and ANC having significant control over the fluxes. Thus in some conditions respiration rate correlates better with [ADP] than with phosphorylation potential, and may be relatively insensitive to mitochondrial NADH/NAD+ ratio.
Most textbooks still show the oxidation of succinate in the tricarboxylic acid (TCA) cycle resulting in the reduction of FADH<sub>2</sub>. Such a presentation does not reflect the reaction catalysed by the enzyme in vivo or in vitro, does not simplify the treatment of the reaction, and is unnecessarily misleading and confusing. +
Insulin-resistant type 2 diabetic patients have been reported to have impaired skeletal muscle mitochondrial respiratory function. A key question is whether decreased mitochondrial respiration contributes directly to the decreased insulin action. To address this, a model of impaired cellular respiratory function was established by incubating human skeletal muscle cell cultures with the mitochondrial inhibitor sodium azide and examining the effects on insulin action. Incubation of human skeletal muscle cells with 50 and 75 microM azide resulted in 48 +/- 3% and 56 +/- 1% decreases, respectively, in respiration compared with untreated cells mimicking the level of impairment seen in type 2 diabetes. Under conditions of decreased respiratory chain function, insulin-independent (basal) glucose uptake was significantly increased. Basal glucose uptake was 325 +/- 39 pmol/min/mg (mean +/- SE) in untreated cells. This increased to 669 +/- 69 and 823 +/- 83 pmol/min/mg in cells treated with 50 and 75 microM azide, respectively (vs. untreated, both P < 0.0001). Azide treatment was also accompanied by an increase in basal glycogen synthesis and phosphorylation of AMP-activated protein kinase. However, there was no decrease in glucose uptake following insulin exposure, and insulin-stimulated phosphorylation of Akt was normal under these conditions. GLUT1 mRNA expression remained unchanged, whereas GLUT4 mRNA expression increased following azide treatment. In conclusion, under conditions of impaired mitochondrial respiration there was no evidence of impaired insulin signaling or glucose uptake following insulin exposure in this model system. +
Inflammatory neurodegeneration is neuronal degeneration due to inflammation, and is thought to contribute to neuronal loss in infectious, ischemic, traumatic and neurodegenerative brain pathologies. We have identified three mechanisms by which inflamed glia kill neurons: iNOS, PHOX and phagocytosis.
A variety of inflammatory mediators induce the expression in microglia and astrocytes of inducible nitric oxide synthase (iNOS), which produces high levels of NO. NO acutely and potently inhibits mitochondrial respiration at cytochrome oxidase in competition with oxygen, while NO derivatives peroxynitrite and S-nitrosothiols inactivate mitochondrial Complex I, resulting in a stimulation of oxidant production by mitochondria. We find that a high level of glial iNOS expression induces neuronal death in synergy with hypoxia, basically by NO inhibition of neuronal respiration resulting in glutamate release and excitotoxicity. This suggests that the inflamed brain may be more sensitive to hypoxic damage. NO from nNOS can also synergise with hypoxia to induce neuronal death via inhibition of mitochondrial respiration (if glycolysis is blocked).
The phagocyte NADPH oxidase (PHOX) is constitutively expressed primarily on the plasma membrane of microglia. Acute activation of PHOX (by e.g. PMA, ATP or Aβ) produces superoxide and hydrogen peroxide. This stimulates microglial production of TNF-α and IL-1β and microglial proliferation, which is blocked by inhibiting PHOX or by removing H2O2 with catalase, and is replicated by adding H2O2. Thus PHOX appears to regulate microglial proliferation and activation through H2O2 production, but has no apparent direct effect on neuronal death. However, if we activated PHOX in glia where iNOS had previously been induced there was considerable synergy in inducing apoptosis of co-cultured neurons. And this apoptosis was prevented either by inhibiting iNOS or PHOX, or scavenging peroxynitrite (the neurotoxic product of NO reacting with superoxide).
However, we find that apoptosis, as measured by phosphatidyserine (PS) exposure, can be reversible in neurons. Temporary exposure of neurons to low levels of H2O2, glutamate or peroxynitrite results in reversible PS exposure on neurons. And such neurons go on to survive, if their phagocytosis by inflamed microglia is prevented at the time of PS exposure. Inflammatory activation of neuronal-glial co-cultures with LPS, LTA or β-amyloid (in the absence of pro-inflammatory cytokines) results in progressive loss of neurons (without any apparent cell death) which is accompanied by microglial phagocytosis of neurons, and is prevented by blocking phagocytosis, in culture and in vivo.
1. Borutaite V, Brown GC (2006) S-nitrosothiols induce inhibition of Complex I and ROS production by mitochondria. Biochim. Biophys. Acta 1757: 405.
2. Jekabsone A, Nehrer J, Borutaite V, Brown GC (2007) NO from neuronal nitric oxide synthase sensitises neurons to hypoxia-induced death via competitive inhibition of cytochrome oxidase. J. Neurochem. 103: 346-356.
3. Brown GC, Neher JJ (2010) Inflammatory neurodegeneration and mechanisms of microglial killing of neurons. Mol. Neurobiol. Mar 2.
During hibernation, animals cycle between periods of torpor, where body temperature (T(b)) and metabolic rate (MR) are suppressed for days, and interbout euthermia (IBE), where T(b) and MR return to resting levels for several hours. In this study, we measured respiration rates, membrane potentials, and reactive oxygen species (ROS) production of liver and skeletal muscle mitochondria isolated from ground squirrels (''Ictidomys tridecemlineatus'') during torpor and IBE to determine how mitochondrial metabolism is suppressed during torpor and how this suppression affects oxidative stress. In liver and skeletal muscle, [[State 3]] respiration measured at 37°C with succinate was 70% and 30% lower, respectively, during torpor. In liver, this suppression was achieved largely via inhibition of substrate oxidation, likely at succinate dehydrogenase. In both tissues, respiration by torpid mitochondria further declined up to 88% when mitochondria were cooled to 10°C, close to torpid T(b). In liver, this passive thermal effect on respiration rate reflected reduced activity of all components of oxidative phosphorylation (substrate oxidation, phosphorylation and proton leak). With glutamate+malate and succinate, mitochondrial free radical leak (FRL; proportion of electrons leading to ROS production) was higher in torpor than IBE, but only in liver. With succinate, higher FRL likely resulted from increased reduction state of Complex III during torpor. With glutamate+malate, higher FRL resulted from active suppression of Complex I ROS production during IBE, which may limit ROS production during arousal. In both tissues, ROS production and FRL declined with temperature, suggesting ROS production is also reduced during torpor by passive thermal effects. +
[[File:GuyBrown.JPG|right|150px|Guy Brown]]
There is a much lower effective selective pressure on our mitochondrial genome relative to nuclear-encoded mitochondrial genes because the mitochondrial genome: 1) is present at high copy numbers per cells, 2) does not undergo recombination; 3) is selected in females only, and 4) is not selected by the sperm race. In addition, the mitochondrial genome may mutate at a higher rate. The large mismatch between mitochondrial and nuclear genes in the ability of evolution to select beneficial and eliminate detrimental variants might be a cause of the rapid evolution and poor adaption of mitochondrial genes. As mammals and humans became larger, brainier, with more skills to pass on and delayed sexual-maturity, and decreased extrinsic causes of death, there would have been selection pressure to delay ageing and age-related disease. But that selection pressure would have had relatively little effect on the mitochondrial relative to nuclear genome, causing mitochondria to be a major contributor to aging. +
During hibernation, animals cycle between periods of torpor, during which body temperature (T(b)) and metabolic rate (MR) are suppressed for days, and interbout euthermia (IBE), during which T(b) and MR return to resting levels for several hours. In this study, we measured respiration rates, membrane potentials, and reactive oxygen species (ROS) production of liver and skeletal muscle mitochondria isolated from ground squirrels (''Ictidomys tridecemlineatus'') during torpor and IBE to determine how mitochondrial metabolism is suppressed during torpor and how this suppression affects oxidative stress. In liver and skeletal muscle, state 3 respiration measured at 37 °C with succinate was 70% and 30% lower, respectively, during torpor. In liver, this suppression was achieved largely via inhibition of substrate oxidation, likely at succinate dehydrogenase. In both tissues, respiration by torpid mitochondria further declined up to 88% when mitochondria were cooled to 10 °C, close to torpid T(b). In liver, this passive thermal effect on respiration rate reflected reduced activity of all components of oxidative phosphorylation (substrate oxidation, phosphorylation, and proton leak). With glutamate & malate & succinate, mitochondrial free radical leak (FRL; proportion of electrons leading to ROS production) was higher in torpor than IBE, but only in liver. With succinate, higher FRL likely resulted from increased reduction state of Complex III during torpor. With glutamate & malate, higher FRL resulted from active suppression of Complex I ROS production during IBE, which may limit ROS production during arousal. In both tissues, ROS production and FRL declined with temperature, suggesting ROS production is also reduced during torpor by passive thermal +
It is often assumed that mitochondria are the main source of reactive oxygen species (ROS) in mammalian cells, but there is no convincing experimental evidence for this in the literature. What evidence there is suggests mitochondria are a significant source for ROS, which may have physiological and pathological effects. But quantitatively, endoplasmic reticulum and peroxisomes have a greater capacity to produce ROS than mitochondria, at least in liver. In most cells and physiological or pathological conditions there is a lack of evidence for or against mitochondria being the main source of cellular ROS. Mitochondria can rapidly degrade ROS and thus are potential sinks for ROS, but whether mitochondria act as net sources or sinks within cells in particular conditions is unknown. +
Hibernating ground squirrels (''Ictidomys tridecemlineatus'') alternate between two distinct metabolic states throughout winter: torpor, during which metabolic rate (MR) and body temperature (T(b)) are considerably suppressed, and interbout euthermia (IBE), during which MR and T(b) briefly return to euthermic levels. Previous studies showed suppression of succinate-fueled respiration during torpor in liver and skeletal muscle mitochondria; however, these studies used only a single, saturating succinate concentration. Therefore, they could not address whether mitochondrial metabolic suppression occurs under physiological substrate concentrations or whether differences in the kinetics of mitochondrial responses to changing substrate concentration might also contribute to mitochondrial metabolic regulation during torpor. The present study confirmed that succinate oxidation is reduced during torpor in liver and skeletal muscle at 37°C and 10°C over a 100-fold range of succinate concentrations. At 37°C, this suppression resulted from inhibition of succinate dehydrogenase (SDH), which had a greater affinity for oxaloacetate (an SDH inhibitor) during torpor. At 10°C, SDH was not inhibited, suggesting that SDH inhibition initiates but does not maintain mitochondrial suppression during torpor. Moreover, in both liver and skeletal muscle, mitochondria from torpid animals maintained relatively higher respiration rates at low succinate concentrations, which reduces the extent of energy savings that can be achieved during torpor but may also maintain mitochondrial oxidative capacity above some lower critical threshold, thereby preventing cellular and/or mitochondrial injury during torpor and facilitating rapid recruitment of oxidative capacity during arousal. +
We recently showed that [[Bendavia]], a novel mitochondria-targeting peptide, reduced infarction and no-reflow across several experimental models. The purpose of this study was to determine the therapeutic timing and mechanism of action that underlie Bendavia's cytoprotective property. In rabbits exposed to ''in vivo'' ischemia/reperfusion (30/180 min), Bendavia administered 20 minutes prior to reperfusion (0.05 mg/kg/h, intravenously) reduced myocardial infarct size by ∼50% when administered for either 1 or 3 hours of reperfusion. However, when Bendavia perfusion began just 10 minutes after the onset of reperfusion, the protection against infarction and no-reflow was completely lost, indicating that the mechanism of protection is occurring early in reperfusion. Experiments in isolated mouse liver mitochondria found no discernible effect of Bendavia on blocking the permeability transition pore, and studies in isolated heart mitochondria showed no effect of Bendavia on respiratory rates. As Bendavia significantly lowered reactive oxygen species (ROS) levels in isolated heart mitochondria, the ROS-scavenging capacity of Bendavia was compared to well-known ROS scavengers using ''in vitro'' (cell-free) systems that enzymatically generate ROS. Across doses ranging from 1 nmol/L to 1 mmol/L, Bendavia showed no discernible ROS-scavenging properties, clearly differentiating itself from prototypical scavengers. In conclusion, Bendavia is a promising candidate to reduce cardiac injury when present at the onset of reperfusion but not after reperfusion has already commenced. Given that both infarction and no-reflow are related to increased cellular ROS, Bendavia's protective mechanism of action likely involves reduced ROS generation (as opposed to augmented scavenging) by endothelial and myocyte mitochondria. +
In this study we determined the efficacy of the cardiolipin-targeting peptide MTP-131 (Bendavia) on mitochondrial function in isolated hearts, permeabilized fibers, and isolated mitochondria. We hypothesized that this peptide would improve mitochondrial membrane fluidity during ischemia-reperfusion (I/R). Fluidity is a biophysical parameter of the membrane that influences the assembly of mitochondrial respiratory supercomplexes. MTP-131 is a cell-permeable peptide known to confer cardioprotection in pre-clinical models and is currently being investigated in Phase 2 clinical trials for cardiovascular disease1-4.
Mitochondria from rat ventricle were isolated and placed in the chamber of a modified Oroboros Instruments O2k high-resolution respirometer. Seeking to mimic physiological state 3 respiration in heart, we clamped ADP levels at 75uM using a hexokinase/2-deoxyglucose clamp. Mitochondria were treated with either saline or 1uM MTP-131 prior to hypoxia. In this protocol, mitochondria make themselves hypoxic by consuming the oxygen in the chamber in about 10 minutes. Hypoxia lasted for 25 minutes, followed by reoxygenation. Mitochondrial membrane fluidity was monitored continuously throughout the protocol using the fluidity-dependent fluorophore MC540. Membrane fluidity decreased sharply at the onset of reoxygenation (32,335 ± 3948 AU). Mitochondria treated with MTP-131 displayed higher membrane fluidity in early reperfusion (43,322 ± 6862 AU). These data were corroborated in intact heart studies, where both lipid head-group and acyl side-chain fluidity were restored with MTP-131. The improvement in fluidity was associated with greater density of mitochondrial respiratory supercomplexes and decreased degradation of respiratory protein complexes assessed with blue-native gel electrophoresis.
Mitochondrial respiration in permeabilized ventricular fibers was significantly impaired after I/R. Complex I-dependent respiration after reperfusion was 208±19 v 42±9 pmol O2 mg<sup>-1</sup>∙s<sup>-1</sup> in control v I/R, respectively; ''p''<0.05. Complex II-dependent respiration was also lower (753±41 v 168±13 pmol∙mg<sup>-1</sup>∙s<sup>-1</sup> in control versus I/R; ''p''<0.05). Perfusion with MTP-131 during reperfusion significantly increased Complexes I- (100±13 pmol O2 mg<sup>-1</sup>∙s<sup>-1</sup>) and II-dependent (334±63 pmol O2 mg-1∙s-1) respiration (''p''<0.05 versus untreated for both). Finally, this recovery of bioenergetics was associated with a significant reduction in infarct size in MTP-131 treated animals.
In summary, our results provide evidence that post-ischemic mitochondrial function can be rescued by targeting the cardiolipin-dependent assembly of mitochondrial supercomplexes.
Heart failure is a pressing worldwide public-health problem with millions of patients having worsening heart failure. Despite all the available therapies, the condition carries a very poor prognosis. Existing therapies provide symptomatic and clinical benefit, but do not fully address molecular abnormalities that occur in cardiomyocytes. This shortcoming is particularly important given that most patients with heart failure have viable dysfunctional myocardium, in which an improvement or normalization of function might be possible. Although the pathophysiology of heart failure is complex, mitochondrial dysfunction seems to be an important target for therapy to improve cardiac function directly. Mitochondrial abnormalities include impaired mitochondrial electron transport chain activity, increased formation of reactive oxygen species, shifted metabolic substrate utilization, aberrant mitochondrial dynamics, and altered ion homeostasis. In this Consensus Statement, insights into the mechanisms of mitochondrial dysfunction in heart failure are presented, along with an overview of emerging treatments with the potential to improve the function of the failing heart by targeting mitochondria. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]]
Mitochondrial and peroxisomal function are inextricably interlinked[1]. Peroxisomes have multiple functions including, as arguable key partners in mitochondrial metabolic and redox function. Consideration here is limited to their role in providing substrate to mitochondria, their impact on redox regulation pathways, and consequences of imbalances in dietary polyunsaturated fat intakes[2].
Human peroxisomes can beta-oxidise a number of substrates including branched chain fats, ‘oxidised’ fats such as LOX COX P450 products, and all fats longer than 8 carbons. The products are peroxide, heat, acyl-CoA, and medium chain fats, which can be fed to mitochondria as fuel, and / or diverted for creation of substrate, including lipids and sterols, to support tissue function and repair. In contrast mitochondria beta-oxidise substrate primarily to energy and related pathways.
Crucially, the preferred peroxisomal substrate appears to be 18 carbon polyunsaturated fats, ranked by number of double bonds, thus; Linolenic acid (ALA)>Gamma linoleic>Linoleic (LA)>Oleic,[3] which is consistent with the plant; ‘origins’ of peroxisomes, and metabolic prominence of 18 carbon polyunsaturated fats, including in photosynthesis and germination. LA and ALA together, due to their dominance in green and plant reproductive material, are the predominant terrestrial fats.
Mitochondria oxidise medium chain fats avidly, saturated fats well, and polyunsaturates less well, but synergistically struggle or cannot beta-oxidise longer fats e.g. docosahexaenoic acid that peroxisomes beta-oxidise well. Again synergistically, peroxisomes unlike mitochondria oxidise medium-fats poorly.
They are working in concert; peroxisomes provide medium fats and ACoA to the mitochondria. Both likely bypass the need for carnitine to enter the mitochondria (but not MCAD see below), bypassing the insulin / malonyl-CoA related regulation of entry of fats into the mitochondria (Randle Cycle), providing alternative energy pathways, during glucose shortage and or blockage of entry of long fats, and in situations of glucose pathways dysbiosis e.g. diabetes and Alzheimer’s.
Further peroxisomes impact redox by producing peroxide and catalase. Catalase is the most efficient reducer of peroxide, and catalase production is believed limited or absent from mitochondria. Peroxisomal catalase and peroxide production both likely help regulate mitochondrial redox status.
The crucial importance of interaction between peroxisomes and mitochondria is deduced from the metabolic effects of MCAD (medium-chain acyl-coenzyme deficiency disorder): failure to access food containing glucose substrate results in brain damage, even death, in hours, because ‘ketosis’ does not develop in short time frames. However new babies with limited access to glucose in new breast milk, and Inuit with a CPT1A polymorphism inhibiting import of long chain fats into the mitochondria on a native marine fat rich diet, with functioning MCAD, both not in significant ketosis, function perfectly normally, suggesting alternative fuels to glucose or ketones must exist; logically medium fats/ACoA. The peroxisomes are the only available logical endogenous source of these mitochondrial substrates, through peroxisomal beta-oxidation of stored lipid adipose sources including polyunsaturated fats.
Further and synergistically PPAR (peroxisome proliferator alpha) pathways are activated in fasting and exercise, and for both, peroxisomal beta-oxidation is arguably an important energy resource. Peroxisomes proliferation, whilst not as marked as in rodents, is seen in humans and pigs. Interestingly migrating birds, possibly migrating monogastrics, and diving mammals, may utilise stored lipids, particularly polyunsaturated fats, via peroxisomal beta-oxidation for energy. Further, opportunity exists to recycle peroxide through catalase back to oxygen, so improving oxygen efficiency, which would be a useful adaptation at altitude, during flight, or whilst diving.
LA has wider metabolic roles; the oxidation of LA within cardiolipin modulates mitochondrial function, including energy production, ultimately inducing damage[4] and apoptosis. In contrast, but to a lesser degree, Omega 3 ALA uprates PPAR alpha activity, and is preferentially metabolised by peroxisomes compared to Omega 6 LA. Further ALA derivative DHA presence in cardiolipin likely alters energetics, and modulates mitochondrial oxidative pathways including apoptosis.
Interestingly, and arguably with crucial metabolic and wider health consequences, including in western inflammatory, lipid deposition related comorbid non-infective western conditions, the primary activators of PPAR gamma pathways are oxidised products of LA. Thus, excess dietary intake of LA and preoxidised LA, imbalance of LA / ALA, combined with calorific excess, significantly impact, metabolic regulation through manipulation of the peroxisomal metabolic pathways, as well as redox regulation. Peroxisomal product of PPAR gamma activation is likely directed to tissue creation repair and oxidative stress related pathways, directing substrate to repair or energy pathways, promoting inflammation, and intracellular lipid deposition. Significant LA is stored in human adipose tissue.
Alternatively in energy shortage such as during exercise or fasting, PPAR alpha activation enables mitochondrial function via significant peroxisomal supply of substrates ACoA and medium chain fats.
Peroxisomes through ACoA, and short chain fat provision, are arguably; central to human energy provision including pre-ketosis, and or repair substrate supply, so ultimately survival, and via astrocytic pathways crucial in fuelling, and for supply of substrate to the brain, the seat of humanity.
'''Call for research - We all need oxygen – “The oceans are gasping for air”''' 1, 2, 3
“Human dominion over planet Earth is driving profound changes that may culminate in extinction.” 4 Whilst there is wider research into the oceanic impact of climate change including warming and acidification,5, 6 and on oxygen content of oceans, there is very little research into the specific impact of acidification and related carbon dioxide changes on marine photosynthetic oxygen production.
This is an important field of research as it also involves consideration of the consequent effects of excess atmospheric carbon dioxide, including warming, on oceanic and atmospheric oxygen, oxygen exchange between them and possibilities of tipping points whereby photosynthetic marine organisms may rapidly die off, potentially leading to severe existential consequences for aerobic life forms.
We bemoan the loss of polar bears and rare alpine plants along with changes to weather and food, but as societies and individuals we are reluctant to severely moderate the day-to-day fossil fuel energy consumption that underlies 21st century life. When faced by the choice of polar bears vs cars, heating, laptops and phones, the polar bears lose!
Ocean acidification is a more empirically evidenced phenomenon than climate change, however, it is also less prominent in the public psyche even though it springs from the same increased atmospheric carbon dioxide levels. Were research to be commissioned, though, that provides clear evidence of risk to oceanic oxygen production and therefore atmospheric oxygen levels, the conclusions could be far-reaching, including identifying a potential tipping point that may result in human extinction.
This stark prospect would, arguably, be easier to convey to, and fix in the wider public consciousness than the more diffuse issues around climate change. Humans are reminded with every breath they take that oxygen is essential to their health function and, ultimately, their survival and existence as a species.7 The importance of the prospect of oxygen depletion for future generations would be easily understood by all, and so promote greater public engagement and cohesive demand for a global response to try and find viable energy alternatives to fossil fuels.
This discussion article begins by highlighting the benefits of the mole's incorporation within the international system of units (SI), in particular by bringing chemical measurement within formal metrology structures. The origins of the confusion that has consistently existed between amount of substance (the base quantity of which the mole is the SI base unit) and counting quantities are examined in detail and their differentiating characteristics fully elaborated on. The importance and benefits of distinguishing between these different quantities and the role that the Avogadro constant plays in doing this are highlighted. It is proposed that these issues are becoming increasingly important for two reasons. First, as chemistry and biology consider increasingly small size domains, measurements are being made of significantly reduced collections of entities. Second, the proposed re-definition of the mole makes the link between amount of substance and the number of elementary entities more transparent. Finally, proposals for new ways of expressing very low amounts of substance in terms of new prefixes based on the numerical value of the Avogadro constant are presented as a way to encourage the use of the mole, when appropriate, even for ultra-low level chemical measurement. +
COVID-19 (Coronavirus) mortality disproportionately impacts BAME (Black, Asian and Minority Ethnic) UK individuals, African Americans, Swedish Somalis,[1] and the institutionalised; particularly care-home residents. COVID-19 severity and mortality, appear related to vitamin D deficiency, [2-12] helping explain higher COVID-19 mortality rates in BAME and the obese.[13] Obesity is a strong COVID-19 risk factor, as are co-morbidities, including diabetes, cardio-vascular disease; and sedentary lifestyle; all are dependent on mitochondrial functionality.[14] Fat cells accrete vitamin D.[15] The obese consistently have proportionately lower vitamin D status (serum 25-hydroxyvitamin D [25(OH)D]).[16] +
Version 1 ('''v1''') '''2020-03-24''' [https://www.mitofit.org/images/e/ec/Brown_et_al_2020_MitoFit_Preprint_Arch_doi_10.26214_mitofit_200001.pdf doi:10.26124/mitofit:200001]
The COVID-19 virus emerged in 2019. Mortality rates as at 20th March 2020 are much higher in southern than northern Europe. The elderly, and those with pre-existing conditions, are at greatest risk. It is hypothesised, vitamin D deficiency may significantly compromise, respiratory immune response function, thus greatly increasing risk of COVID-19 hospitalisation, severity and mortality. Winter vitamin D levels: based on; limited data, including; historical measured regional vitamin D deficiency rates (<25 nmol/L), intakes, and plasma vitamin D levels; fortification and supplementation policies; and public vitamin D awareness: appear to be significantly lower in southern, than northern Europe. In respiratory system conditions, such as influenza, vitamin D has wide-ranging and fundamental roles, including through: gene transcription via COVID-19 relevant VDR (Vitamin D Receptor) pathways; ACE1 and ACE2 pathways; wider immune function; airway epithelial cell tight-junction function and integrity; and mitochondrial related, energetics, apoptosis and inflammation, management. Studies suggest vitamin D supplementation may be protective against respiratory conditions, in ‘D’ deficient persons. Would vitamin D supplementation of the deficient, mitigate the severity of the current COVID-19 outbreak; and reduce future, likely upcoming, seasonal amplification effects? +
There has long been debate over how to treat dimensionless quantities, or quantities with the unit one, within the International System of Units (SI). These arguments have been brought into sharper focus because of the increasing application of metrological principles in areas such as chemistry, biology and nanoscience where counting measurements are common. This has caused debates about how the SI should address counting quantities and the unit one (symbol 1). This article reviews the types of quantities with the unit one, how these quantities may be expressed together with their uncertainty and how this relates to counting. The qualities of counting quantities are explored in more detail and the range of possibilities for dealing with the unit one for counting are discussed. It is proposed that the SI should allow only the unit one for counting, but that downstream of the SI there may well be benefits from standardising the use of more descriptive, technical area specific 'units' for expressing the results of counting. As with all measurement it is essential that a full description, in words, of the counting quantity being expressed accompanies the measurement result. +
Loss of innervation is a key driver of age associated muscle atrophy and weakness (sarcopenia). Our laboratory has previously shown that denervation induced atrophy is associated with the generation of mitochondrial hydroperoxides and lipid mediators produced downstream of cPLA<sub>2</sub> and 12/15 lipoxygenase (12/15-LOX). To define the pathological impact of lipid hydroperoxides generated in denervation-induced atrophy ''in vivo'', we treated mice with liproxstatin-1, a lipid hydroperoxide scavenger. We treated adult male mice with 5 mg/kg liproxstain-1 or vehicle one day prior to sciatic nerve transection and daily for 7 days post-denervation before tissue analysis. Liproxstatin-1 treatment protected gastrocnemius mass and fiber cross sectional area (∼40% less atrophy post-denervation in treated versus untreated mice). Mitochondrial hydroperoxide generation was reduced 80% ''in vitro'' and by over 65% ''in vivo'' by liproxstatin-1 treatment in denervated permeabilized muscle fibers and decreased the content of 4-HNE by ∼25% post-denervation. Lipidomic analysis revealed detectable levels of 25 oxylipins in denervated gastrocnemius muscle and significantly increased levels for eight oxylipins that are generated by metabolism of fatty acids through 12/15-LOX. Liproxstatin-1 treatment reduced the level of three of the eight denervation-induced oxylipins, specifically 15-HEPE, 13-HOTrE and 17-HDOHE. Denervation elevated protein degradation rates in muscle and treatment with liproxstatin-1 reduced rates of protein breakdown in denervated muscle. In contrast, protein synthesis rates were unchanged by denervation. Targeted proteomics revealed a number of proteins with altered expression after denervation but no effect of liproxstain-1. Transcriptomic analysis revealed 203 differentially expressed genes in denervated muscle from vehicle or liproxstatin-1 treated mice, including ER stress, nitric oxide signaling, Gαi signaling, glucocorticoid receptor signaling, and other pathways. Overall, these data suggest lipid hydroperoxides and oxylipins are key drivers of increased protein breakdown and muscle loss associated with denervation induced atrophy and a potential target for sarcopenia intervention.
Bendavia is a cytoprotective mitochondria-targeting peptide [1-4], currently being tested in the EMBRACE-STEMI trial for reducing injury during acute coronary syndromes. We previously showed that the cardioprotective effects of Bendavia involved improving cardiolipin-dependent mitochondrial membrane fluidity. As membrane fluidity influences the ability of proteins to assemble, we hypothesized that a consequence of augmented membrane fluidity would be higher expression of mitochondrial respiratory supercomplexes.
Rat hearts (''N''=42) were subjected to ischemia-reperfusion (I/R) with our without 1 nM Bendavia perfusion, beginning at the onset of reperfusion. Left ventricular tissue was split into one of two study arms: 1. Supercomplex expression using blue-native gel electrophoresis (BN-PAGE), or 2. High-resolution respirometry using permeabilized ventricular fibers. For BN-PAGE studies, respiratory supercomplex bands were decreased with I/R, and restored with Bendavia (Fig. 1). High-resolution respirometry studies indicated suppressed Complex I-dependent respiration after I/R (208±19 v 42±9 pmol O<sub>2</sub>∙s<sup>-1</sup>.mg<sup>-1</sup>) in control v I/R, respectively; ''P''<0.05. Complex II-dependent respiration was also lower (753±41 v 168±13 pmol∙s<sup>-1</sup>∙mg<sup>-1</sup> in control versus I/R; ''P''<0.05). Perfusion with Bendavia during reperfusion significantly increased Complex I- (100±13 pmol O<sub>2</sub>∙s<sup>-1</sup>.mg<sup>-1</sup>) and II-dependent (334±63 pmol O<sub>2</sub>∙s<sup>-1</sup>.mg<sup>-1</sup>) respiration (''P''<0.05 versus untreated IR for both).
Taken together, these data suggest that Bendavia’s protective mechanism of action involves preserving supercomplex-dependent mitochondrial function during cardiac reperfusion. +
Brain inflammation may contribute to neuronal loss in infectious, ischemic, traumatic and neurodegenerative brain pathologies. We and others have shown that: a) brain inflammation induces the expression in microglia and astrocytes of inducible nitric oxide synthase (iNOS), which produces high levels of NO, b) NO derivatives peroxynitrite and S-nitrosothiols inactivate mitochondrial Complex I, resulting in a stimulation of oxidant production by mitochondria, c) oxidant production by microglia contributes to their inflammatory activation, and d) activated microglia can cause neuronal loss by eating them alive [1-4]. Thus, we were interested in whether activated microglia may inhibit their mitochondrial Complex I, resulting in sustained activation and phagocytosis of live neurons.
There is evidence that in Parkinson’s disease and general brain aging mitochondrial Complex I is inhibited in affected parts of the brain. Rotenone is an environmental toxin and Complex I inhibitor that can cause activation of microglia and a Parkinson’s like pathology in rodents. We, therefore, tested whether it could cause microglia to phagocytose live neurons.
We found that low nanomolar levels of rotenone could indeed activate microglial phagocytosis and cause neurons to phagocytose co-cultured neurons [5]. Removal of microglia or inhibition of phagocytic signalling prevented rotenone-induced neuronal loss, leaving viable neurons [5].
Low levels of brain inflammation during ageing may cause partial inhibition of Complex I, resulting in oxidant production which sustains inflammation, and induces microglia to phagocytose synapses and cell bodies of live neurons. This process may be exacerbated in Parkinson’s disease and prevented by blocking inflammation or phagocytic signalling. +
Diabetes-specific microvascular disease is a leading cause of blindness, renal failure and nerve damage, and diabetes-accelerated atherosclerosis leads to increased risk of myocardial infarction, stroke and limb amputation. Four main molecular mechanisms have been implicated in glucose-mediated vascular damage. All seem to reflect a single hyperglycaemia-induced process of overproduction of superoxide by the mitochondrial electron-transport chain. This integrating paradigm provides a new conceptual framework for future research and drug discovery. +
The development of type 2 diabetes requires impaired beta cell function. Hyperglycemia itself causes further decreases in glucose-stimulated insulin secretion. A new study demonstrates that hyperglycemia-induced mitochondrial superoxide production activates uncoupling protein 2, which decreases the ATP/ADP ratio and thus reduces the insulin-secretory response. These data suggest that pharmacologic inhibition of mitochondrial superoxide overproduction in beta cells exposed to hyperglycemia could prevent a positive feed-forward loop of glucotoxicity that drives impaired glucose tolerance toward frank type 2 diabetes. +
Lung capillary endothelial cells (ECs) are a critical target of oxygen toxicity and play a central role in the pathogenesis of hyperoxic lung injury. To determine mechanisms and time course of EC activation in normobaric hyperoxia, we measured endothelial concentration of reactive oxygen species (ROS) and cytosolic calcium ([Ca(2+)](i)) by in situ imaging of 2',7'-dichlorofluorescein (DCF) and fura 2 fluorescence, respectively, and translocation of the small GTPase Rac1 by immunofluorescence in isolated perfused rat lungs. Endothelial DCF fluorescence and [Ca(2+)](i) increased continuously yet reversibly during a 90-min interval of hyperoxic ventilation with 70% O(2), demonstrating progressive ROS generation and second messenger signaling. ROS formation increased exponentially with higher O(2) concentrations. ROS and [Ca(2+)](i) responses were blocked by the mitochondrial complex I inhibitor rotenone, whereas inhibitors of NAD(P)H oxidase and the intracellular Ca(2+) chelator BAPTA predominantly attenuated the late phase of the hyperoxia-induced DCF fluorescence increase after > 30 min. Rac1 translocation in lung capillary ECs was barely detectable at normoxia but was prominent after 60 min of hyperoxia and could be blocked by rotenone and BAPTA. We conclude that hyperoxia induces ROS formation in lung capillary ECs, which initially originates from the mitochondrial electron transport chain but subsequently involves activation of NAD(P)H oxidase by endothelial [Ca(2+)](i) signaling and Rac1 activation. Our findings demonstrate rapid activation of ECs by hyperoxia in situ and identify mechanisms that may be relevant in the initiation of hyperoxic lung injury. +
Fibromyalgia (FM) is one of the most common musculoskeletal pain conditions. Although the aetiology of FM is still unknown, mitochondrial dysfunction and the overproduction of reactive oxygen intermediates (ROI) are common characteristics in its pathogenesis. The reserpine experimental model can induce FM-related symptoms in rodents by depleting biogenic amines. However, it is unclear whether reserpine causes other pathophysiologic characteristics of FM. So far, no one has investigated the relevance of mitochondrial dysfunction in the reserpine-induced experimental FM model using protection- and insult-based mitochondrial modulators. Reserpine (1 mg/kg) was subcutaneously injected once daily for three consecutive days in male Swiss mice. We carried out analyses of reserpine-induced FM-related symptoms, and their modulation by using mitochondrial insult on ATP synthesis (oligomycin; 1 mg/kg, intraperitoneally) or mitochondrial protection (coenzyme Q10; 150 mg/kg/5 days, orally). We also evaluated the effect of reserpine on mitochondrial function using high-resolution respirometry and oxidative status. Reserpine caused nociception, loss in muscle strength, and anxiety- and depressive-like behaviours in mice that were consistent with clinical symptoms of FM, without inducing body weight and temperature alterations or motor impairment. Reserpine-induced FM-related symptoms were increased by oligomycin and reduced by coenzyme Q10 treatment. Reserpine caused mitochondrial dysfunction by negatively modulating the electron transport system and mitochondrial respiration (ATP synthesis) mainly in oxidative muscles and the spinal cord. These results support the role of mitochondria in mediating oxidative stress and FM symptoms in this model. In this way, reserpine-inducing mitochondrial dysfunction and increased production of ROI contribute to the development and maintenance of nociceptive, fatigue, and depressive-like behaviours. +
Iron (Fe) is an essential metal ion that plays a major role as a cofactor in many biological processes. The balance between the Fe<sup>2+</sup> and Fe<sup>3+</sup> forms is central for cellular Fe homeostasis because it regulates its transport, utilization, and storage. Contrary to Fe<sup>3+</sup> reduction that is crucial for Fe uptake by roots in deficiency conditions, ferroxidation has been much less studied. In this work, we have focused on the molecular characterization of two members of the MultiCopper Oxidase family (MCO1 and MCO3) that share high identity with the ''Saccharomyces cerevisiae'' ferroxidase Fet3. The heterologous expression of MCO1 and MCO3 restored the growth of the yeast fet3fet4 mutant, impaired in high and low affinity Fe uptake and otherwise unable to grow in Fe deficient media, suggesting that MCO1 and MCO3 were functional ferroxidases. The ferroxidase enzymatic activity of MCO3 was further confirmed by the measurement of Fe<sup>2+</sup>-dependent oxygen consumption, because ferroxidases use oxygen as electron acceptor to generate water molecules. In planta, the expression of MCO1 and MCO3 was induced by increasing Fe concentrations in the medium. Promoter-GUS reporter lines showed that MCO1 and MCO3 were mostly expressed in shoots and histochemical analyses further showed that both promoters were highly active in mesophyll cells. Transient expression of MCO1-RFP and MCO3-RFP in tobacco leaves revealed that both proteins were localized in the apoplast. Moreover, cell plasmolysis experiments showed that MCO1 remained closely associated to the plasma membrane whereas MCO3 filled the entire apoplast compartment. Although the four knock out mutant lines isolated (mco1-1, mco1-2, mco3-1, and mco3-2) did not display any macroscopic phenotype, histochemical staining of Fe with the Perls/DAB procedure revealed that mesophyll cells of all four mutants overaccumulated Fe inside the cells in Fe-rich structures in the chloroplasts, compared with wild-type. These results suggested that the regulation of Fe transport in mesophyll cells had been disturbed in the mutants, in both standard condition and Fe excess. Taken together, our findings strongly suggest that MCO1 and MCO3 participate in the control of Fe transport in the mesophyll cells, most likely by displacing the Fe<sup>2+</sup>/Fe<sup>3+</sup> balance toward Fe<sup>3+</sup> in the apoplast and therefore limiting the accumulation of Fe<sup>2+</sup>, which is more mobile and prone to be transported across the plasma membrane.
Mammalian cells detect decreases in oxygen concentrations to activate a variety of responses that help cells adapt to low oxygen levels (hypoxia). One such response is stabilization of the protein HIF-1 alpha, a component of the transcription factor HIF-1. Here we show that a small interfering RNA (siRNA) against the Rieske iron-sulfur protein of mitochondrial complex III prevents the hypoxic stabilization of HIF-1 alpha protein. Fibroblasts from a patient with Leigh's syndrome, which display residual levels of electron transport activity and are incompetent in oxidative phosphorylation, stabilize HIF-1 alpha during hypoxia. The expression of glutathione peroxidase or catalase, but not superoxide dismutase 1 or 2, prevents the hypoxic stabilization of HIF-1 alpha. These findings provide genetic evidence that oxygen sensing is dependent on mitochondrial-generated reactive oxygen species (ROS) but independent of oxidative phosphorylation. +
Recently, significant attention has been given to the role of muscle mitochondrial function in the development of insulin resistance associated with obesity. Our aim was to investigate temporal alterations in mitochondrial respiration, H<sub>2</sub>O<sub>2</sub> emission, and mitochondrial responsivity to insulin in permeabilized skeletal muscle fibers during the development of obesity in mice. Swiss male mice (5-6 weeks old) were fed with a high-fat diet (60 % calories from fat) or standard diet for 7, 14 and 28 days to induce obesity and insulin resistance. Diet-induced obese (DIO) mice presented reduced glucose tolerance and hyperinsulinemia after 7 days of HFD. After 14 days, the expected increase in muscle glycogen content after systemic injection of glucose and insulin was not observed in DIO mice. At 28 days, blood glucose decay after insulin injection was significantly impaired. Complex I (pyruvate + malate) and CII (succinate) linked respiration, and oxidative phosphorylation (ADP) were decreased after 7 days of HFD and remained low in DIO mice after 14 and 28 days of treatment. Moreover, mitochondria from DIO mice were incapable of increasing respiratory coupling and ADP responsivity after insulin-stimulation in all observed periods. Mitochondrial content markers were reduced only after 28 days of treatment. Mitochondrial H<sub>2</sub>O<sub>2</sub> emission profile varied during the time course of DIO, with a reduction of H<sub>2</sub>O<sub>2</sub> emission in the early stages of DIO and an increased emission after 28 days of treatment. Our data demonstrate that DIO promotes transitory alterations in mitochondrial physiology during the early and late stages of insulin-resistance related to obesity.
<small>This article is protected by copyright. All rights reserved.</small> +
Leucine is an essential amino acid that has been investigated by its participation in the regulation of whole-body metabolism and mitochondrial function. Here, we evaluated acute leucine effects on mitochondrial respiration of skeletal muscle from male Swiss mice ''in vitro''. Additionally, we further investigated the effects of 4-wk leucine ingestion (2.5% on drinking water) on skeletal muscle mitochondrial respiration and morphology of diet-induced obesity (DIO) mice. ''In vitro'', acute leucine increased mitochondrial respiration, and these effects were abolished in the presence of rapamycin. In DIO mice, ingestion of leucine for 4-wk improved glucose tolerance and insulin responsivity. Leucine supplementation also prevented the reduction in mitochondrial respiration, size, and complexity in the soleus skeletal muscle. We conclude that the positive effects of leucine on whole-body metabolism in DIO mice are associated with improvements in skeletal muscle mitochondrial function and morphology. Furthermore, leucine acute effects on mitochondrial respiration are mTORC1 dependent. +
Evidence has accumulated to indicate that dietary nitrate alters energy expenditure and the metabolic derangements associated with a high-fat diet, however, the mechanism(s) of action remain incompletely elucidated. Therefore, we aimed to determine if dietary nitrate (4 mm sodium nitrate via drinking water) could prevent high-fat diet (HFD) mediated glucose intolerance in association with improved mitochondrial bioenergetics within both white (WAT) and brown (BAT) adipose tissue. HFD-feeding caused glucose intolerance (P < 0.05) and increased body weight. As a result of higher body weight, energy expenditure increased proportionally. HFD-fed mice displayed greater mitochondrial uncoupling and a 2-fold increase in UCP-1 content within BAT. Within epididymal adipose tissue (eWAT), HFD increased cell size (i.e. hypertrophy), mitochondrial H<sub>2</sub>O<sub>2</sub> emission, oxidative stress, JNK phosphorylation, leucocyte infiltration, and induced insulin resistance. Remarkably, dietary nitrate consumption attenuated and/or mitigated all these responses, including rendering mitochondria more coupled within BAT, and normalizing mitochondrial H<sub>2</sub>O<sub>2</sub> emission and insulin-mediated Akt-Thr308 phosphorylation within eWAT. Intriguingly, the positive effects of dietary nitrate appear to be independent of eWAT mitochondrial respiratory capacity and content. Altogether, these data suggest that dietary nitrate attenuates the development of HFD-induced insulin resistance in association with attenuating WAT inflammation and redox balance, independent of changes within either WAT or BAT mitochondrial respiratory capacity/content. This article is protected by copyright. All rights reserved. +
Dietary nitrate supplementation, and the subsequent serial reduction to nitric oxide, has been shown to improve glucose homeostasis in several pre-clinical models of obesity and insulin resistance. While the mechanisms remain poorly defined, the beneficial effects of nitrate appear to be partially dependent on AMPK-mediated signaling events, a central regulator of metabolism and mitochondrial bioenergetics. Since AMPK can activate SIRT1, we aimed to determine if nitrate supplementation (4 mM sodium nitrate via drinking water) improved skeletal muscle mitochondrial bioenergetics and acetylation status in mice fed a high-fat diet (HFD: 60% fat). Consumption of HFD induced whole-body glucose intolerance, and within muscle attenuated insulin-induced Akt phosphorylation, mitochondrial ADP sensitivity (higher apparent Km), submaximal ADP-supported respiration, mitochondrial hydrogen peroxide (mtH<sub>2</sub>O<sub>2</sub>) production in the presence of ADP and increased cellular protein carbonylation alongside mitochondrial-specific acetylation. Consumption of nitrate partially preserved glucose tolerance and, within skeletal muscle, normalized insulin-induced Akt phosphorylation, mitochondrial ADP sensitivity, mtH<sub>2</sub>O<sub>2</sub>, protein carbonylation and global mitochondrial acetylation status. Nitrate also prevented the HFD-mediated reduction in SIRT1 protein, and interestingly, the positive effects of nitrate ingestion on glucose homeostasis and mitochondrial acetylation levels were abolished in SIRT1 inducible knock-out mice, suggesting SIRT1 is required for the beneficial effects of dietary nitrate. Altogether, dietary nitrate preserves mitochondrial ADP sensitivity and global lysine acetylation in HFD-fed mice, while in the absence of SIRT1, the effects of nitrate on glucose tolerance and mitochondrial acetylation were abrogated. +
'''Authors:''' [[Brunetta Henver Simionato]], [[Palermo Ruiz Gabriel]], [[Ludwig Raissa]], [[Ruberti Olivia]], [[Bechara Luiz]], [[Consonni Silvio]], [[Rodrigues Bruno]], [[Ferreira Julio Cesar B]], [[Mori Marcelo AS]] <br><br>
The negative effects of high-fat high-sucrose (HFHS) diet consumption on heart function are exacerbated in mice lacking DICER in adipocytes (AdicerKO). These findings suggest a protective role of adipocyte-derived microRNAs on heart physiology. Exercise training is known to have a protective role in cardiometabolic diseases. However, it is not known whether chronic aerobic training is able to rescue heart dysfunction in HFHS-fed AdicerKO mice. Here, we fed AdicerKO mice with a HFHS diet for 12 weeks, after confirming the deleterious effects of the diet on these mice, we submitted them to moderate aerobic training for 8 weeks, 5 days/week for 60 minutes each section while keeping them on HFHS-diet. Chronic aerobic training restored end-systolic volume and stroke volume in the hearts of HFHS-fed AdicerKO mice without changing ejection fraction. In addition, aerobic exercise increased left ventricle diameter in both, systolic and diastolic, phases. Notably, HFHS-fed AdicerKO-trained mice presented lower heart rate with no differences in systolic blood pressure compared to HFHS-fed AdicerKO sedentary mice. Mechanistically, chronic exercise training lowered mitochondrial H<sub>2</sub>O<sub>2</sub> emission and oxidative stress alongside greater lipid- and succinate-supported mitochondrial respiration. Importantly, these effects were not followed by changes in triacylglycerol content within the left ventricle or fibrosis. In summary, chronic aerobic training is capable to rescue heart function of HFHS-fed AdicerKO mice in association with improvements in mitochondrial bioenergetics and redox balance. +
In skeletal muscle, mitochondria adapt to physiological (i.e. exercise, aging) and pathological scenarios (i.e. insulin resistance, muscle atrophy). Due to the kinetic regulation by adenylates within the oxidative phosphorylation system, a small increase in free ADP (f[ADP]) within the cell results in a rapid compensatory increment in ATP production and oxygen consumption, conferring to mitochondria the unique ability to detect, and respond, to small changes in the energetic status of the cells. The advent of high-resolution oxygraphy potentiated the studies on mitochondrial bioenergetics where it is now possible to record mitochondrial O<sub>2</sub> consumption in real-time with high sensitivity in various tissues and substrate protocols. While most of the studies rely on saturating concentrations of substrates and ADP to test the maximal respiratory capacity of mitochondria, such approaches may not fully recapitulate physiological conditions by which these organelles are exposed within skeletal muscle cells. Over the years, we and others have employed a mitochondrial ADP sensitivity assay, where we determine mitochondrial bioenergetic responses to a wide range of ADP concentrations, scaling from the physiological levels found in resting skeletal muscle cells (μM) to saturating values (mM). Here, we reviewed this methodology by offering practical guidance and insights from experiments performed in our laboratory, as well as examples of the applicability of such a protocol. <br> +
[[Metformin]] and thiazolidinediones (TZDs) are believed to exert their antidiabetic effects via different mechanisms. As evidence suggests that both impair cell respiration ''in vitro'', this study compared their effects on mitochondrial functions. The activity of [[Complex I]] of the respiratory chain, which is known to be affected by metformin, was measured in tissue homogenates that contained disrupted mitochondria. In homogenates of skeletal muscle, metformin and TZDs reduced the activity of Complex I (30 mmol/L metformin, -15 +/- 2 %; 100 µmol/L rosiglitazone, -54 +/- 7; and 100 µmol/L pioglitazone, -12 +/- 4; ''P'' < 0.05 each). Inhibition of Complex I was confirmed by reduced State 3 respiration of isolated mitochondria consuming glutamate + malate as substrates for Complex I (30 mmol/L metformin, -77 +/- 1 %; 100 µmol/l rosiglitazone, -24 +/- 4; and 100 µmol/l pioglitazone, -18 +/- 5; ''P'' < 0.05 each), whereas respiration with succinate feeding into Complex II was unaffected. In line with inhibition of Complex I, 24-h exposure of isolated rat soleus muscle to metformin or TZDs reduced cell respiration and increased anaerobic glycolysis (glucose oxidation: 270 µmol/L metformin, -30 +/- 9 %; 9 µmol/L rosiglitazone, -25 +/- 8; and 9 µmol/L pioglitazone, -45 +/- 3; lactate release: 270 µmol/L metformin, +84 +/- 12; 9 µmol/L rosiglitazone, +38 +/- 6; and 9 µmol/L pioglitazone, +64 +/- 11; ''P'' < 0.05 each). As both metformin and TZDs inhibit Complex I activity and cell respiration ''in vitro'', similar mitochondrial actions could contribute to their antidiabetic effects. +
Environmental temperature can greatly impact the functioning of ectothermic organisms through effects on mitochondria, which are crucial to aerobic metabolism. Changes in temperature have the potential to influence mitochondrial ATP production and production of reactive oxygen species (ROS), both of which are influenced by the activity of the mitochondrial electron transport system, which generates the proton gradient necessary for mitochondrial ATP production. Thus, I hypothesized that ectothermic organisms have a mechanism for modulating the proton gradient in the face of changes in environmental temperature to maintain ATP production, and that this mechanism may act through uncoupling proteins (UCPs) which can cause a decrease in the proton gradient independent of the production of ATP. Here, I investigate changes in UCPs and mitochondrial function following thermal acclimation in two populations of the eurythermal Atlantic killifish, ''Fundulus heteroclitus''. I show that UCP mRNA expression is tissue-specific, changes with thermal acclimation, and differs between two populations of killifish. However, these changes vary depending on the isoform, tissue, and population (Chapter 2). I also demonstrate that changes in UCP function are not necessarily consistent with changes in mRNA expression in isolated liver and brain mitochondria, but that UCP function may differ in liver between the two populations (Chapter 3). Cold-acclimated northern killifish increase liver mitochondrial capacity and coupling as indicated by increases in state III, respiratory control and ADP/O ratios (Chapter 3). Interestingly, I also observed increases in proton conductance in isolated liver mitochondria from cold-acclimated northern killifish as indicated by increased O<sub>2</sub> consumption rate at a common membrane potential (Chapter 3). Mitochondrial properties in southern killifish did not differ with thermal acclimation. Taken together, my data suggest that UCPs may play a role in thermal acclimation, although there is not a clear connection between UCP mRNA expression and function. Furthermore, my data indicate that northern killifish may have a greater capacity to respond to low temperature acclimation than southern killifish, suggesting a potential role for adaptive variation in mitochondrial responses to temperature.
In the final steps of energy conservation in aerobic organisms, free energy from electron transfer through the respiratory chain is transduced into a proton electrochemical gradient across a membrane. In mitochondria and many bacteria, reduction of the dioxygen electron acceptor is catalyzed by cytochrome ''c'' oxidase (Complex IV), which receives electrons from cytochrome ''bc''1 (Complex III), via membrane-bound or water-soluble cytochrome ''c''. These complexes function independently, but in many organisms they associate to form supercomplexes. Here, we review the structural features and the functional significance of the nonobligate III2IV1/2 ''Saccharomyces cerevisiae'' mitochondrial supercomplex as well as the obligate III2IV2 supercomplex from actinobacteria. The analysis is centered around the Q-cycle of Complex III, proton uptake by CytcO, as well as mechanistic and structural solutions to the electronic link between Complexes III and IV. +
The aim of the study was to investigate the impact of autophagy inhibitioin on skeletal muscle mitochondrial function and glucose homeostasis in young and aged mice. The transcriptional co-activator PGC-1α regulates muscle oxidative phenotype which has been shown to be linked with basal autophagic capacity. Therefore, young and aged inducible muscle-specific PGC-1α knockout (iMKO) mice and littermate lox/lox controls were used in three separate experiments performed after either saline or colchicine injections on two consecutive days: (1) Euthanization in the basal state obtaining skeletal muscle for mitochondrial respirometry, (2) whole body glucose tolerance test, and (3) ''in vivo'' insulin-stimulated 2-deoxyglucose (2-DG) uptake into skeletal muscle. Muscle PGC-1α was not required for maintaining basal autophagy flux, regardless of age. Colchicine-induced inhibition of autophagy was associated with impairments of skeletal muscle mitochondrial function, including reduced ADP sensitivity and altered mitochondrial redox balance in both young and aged mice. Colchicine treatment reduced the glucose tolerance in aged, but not young mice, and similarly in iMKO and lox/lox mice. Colchicine reduced insulin-stimulated 2-DG uptake in soleus muscle in aged mice, independently of PGC-1α, and without affecting insulin-regulated phosphorylation of proximal or distal mediators of insulin signaling. In conclusion, the results indicate that autophagy regulates the mitochondrial ADP sensitivity and redox balance as well as whole body glucose tolerance and skeletal muscle insulin sensitivity in aged mice, with no additional effects of inducible PGC-1α deletion.
<small>© 2020 Federation of American Societies for Experimental Biology.</small> +
The metabolic suppression due to anoxia in hepatocytes from the anoxia-tolerant turtle ''Chrysemys picta bellii'' was measured directly using microcalorimetric techniques. The normoxic heat flux from hepatocytes in suspension (25 °C) was 1.08 +/- 0.08 mW/g cells and decreased by 76% to 0.26 +/- 0.03 mW/g cells in response to anoxic incubation. After an acute decrease in temperature (to 10 °C) anoxic heat flux dropped by 96% relative to the normoxic control at 25 degrees C. The relative decrease in heat flux at both temperatures was similar, 76% at 25 °C and 68% at 10 °C. From the caloric equivalent of glycogen fermentation to lactate the heat flux from lactate production was calculated to be -93 µW/g cells (25 °C), and this accounted for 36% of the anoxic heat flux. When the enthalpy change associated with the release of free glucose (from glycogen breakdown) is considered, an additional 6% of the anoxic heat flux can be accounted for. Therefore, a portion of the anoxic heat flux is unaccounted for (58%), resulting in an "exothermic gap." This differs from the normoxically incubated hepatocytes where the indirect calorimetric measurement of heat flux (hepatocyte O<sub>2</sub> consumption) could fully account for the calorimetrically measured heat flux. When normoxic hepatocytes were inhibited with cyanide, a rapid suppression in heat flux was observed. Because rapid reequilibration to a lower, cyanide-induced steady state occurred in < 15 min, it is also assumed that there is no short-term Pasteur effect in this tissue. (ABSTRACT TRUNCATED AT 250 WORDS) +
Anoxia rapidly elicits hyper-excitability and cell death in mammal brain but this is not so in anoxia-tolerant turtle brain where spontaneous electrical activity is suppressed by anoxia (i.e. spike arrest; SA). In anoxic turtle brain extracellular GABA concentrations increase dramatically and impact GABAergic synaptic transmission in a way that results in SA. Here we briefly review what is known about the regulation of glutamatergic signalling during anoxia and investigate the possibility that in anoxic turtle cortical neurons GABA(A/B) receptors play an important role in neuroprotection. Both AMPA and NMDA receptor currents decrease by about 50% in anoxic turtle cerebrocortex and therefore exhibit channel arrest, whereas GABA-A receptor currents increase twofold and increase whole-cell conductance. The increased post synaptic GABA-A receptor current is contrary to the channel arrest hypothesis but it does serve an important function. The reversal potential of the GABA-A receptor (E(GABA)) is only slightly depolarized relative to the resting membrane potential of the neuron and not sufficient to elicit an action potential. Therefore, when GABA-A receptors are activated, membrane potential moves to E(GABA) and prevents further depolarization by glutamatergic inputs during anoxia by a process termed shunting inhibition. Furthermore we discuss the presynaptic role of GABA-B receptors and show that increased endogenous GABA release during anoxia mediates SA by activating both GABA-A and B receptors and that this represents a natural oxygen-sensitive adaptive mechanism to protect brain from anoxic injury. +
<big>'''Peter Hochachka lecture'''</big>
Earth’s changing environment has been a major evolutionary force shaping the diversity of species both in the past and present. In particular, seasonal ice cover in northern latitudes has selected for hypoxia and anoxia tolerance in some species, such as freshwater turtles. At the northern reaches of their range North American western painted turtles spend 4 months or more buried in the mud bottom of ice covered lakes and ponds [1]. This offers a unique opportunity to understand how a vertebrate brain, an organ extremely sensitive to reduced oxygen availability in mammals, can function without oxygen [2]. Through oxidative phosphorylation mitochondria fuel the inherently high energetic demands of brain and in mammals mitochondria also play a key role in injury from hypoxic stress – including loss of calcium homeostasis and production of reactive oxygen species (ROS) leading to apoptosis and necrosis. Hypoxic or anoxic stress does not signal stress in turtle brain but rather protective mechanisms with the onset of anoxia. Indeed our data show that mitochondria play a key role in low oxygen signaling in turtle brain by a reduction in mitochondrial membrane potential and release of a relatively small but significant amount of calcium. The increase in cytosolic calcium signals a phosphatase based mechanism to decrease whole-cell glutamatergic (NMDA and AMPA) excitatory currents in pyramidal neurons. While in stellate neurons anoxia results in a large reduction in mitochondrial ROS production that increases the magnitude of GABAergic inhibitory neurotransmission. The increased GABA activity produces a chloride based shunting current that “arrests” action potentials in pyramidal cells resulting in metabolic depression and neuroprotection. +
The neurodegenerative genetic disorder of Huntington’s disease (HD) is characterised by mitochondrial impairments of the respiratory chain. The ubiquitous expression of the disease causing mutant huntingtin gene raises the question to which extent changes in mitochondrial respiration are evident in the human skeletal muscle. In addition characterisation of mitochondrial respiration in the muscle might allow conclusions about the respiratory status in the brain.
The integrated respiratory chain function of the human quadriceps ''vastus lateralis'' was measured by high-resolution respirometry in fine-needle biopsies of four pre-symptomatic HD mutation carriers and seven controls. The respiratory parameters indicated a trend towards a reduction in the respiratory control ratio (RCR) of the HD carriers. In parallel, murine cortex, liver, soleus muscle and heart of male HD knock-in mice (HdhQ111), were examined by the same method. Significant changes of the respiration were restricted to the liver and the cortex. In addition mitochondrial DNA copy number and citrate synthase activity were determined to quantify the mitochondrial mass, showing no differences. From the murine tissues mRNA levels of key enzymes characterised the mitochondrial metabolic pathways.
We demonstrated the feasibility to perform high-resolution respirometry measurements from small human HD muscle biopsies. Furthermore, we conclude that differences in respiratory parameters of pre-symptomatic human muscle biopsies are rather limited, which is confirmed by the analysis of murine skeletal muscle tissue. The murine cortex and liver turned out to show respiratory changes in the HdhQ111 mouse model, which indicates that respiratory capacities are different between tissues. +
Alterations in mitochondrial respiration are an important hallmark of Huntington's disease (HD), one of the most common monogenetic causes of neurodegeneration. The ubiquitous expression of the disease causing mutant huntingtin gene raises the prospect that mitochondrial respiratory deficits can be detected in skeletal muscle. While this tissue is readily accessible in humans, transgenic animal models offer the opportunity to cross-validate findings and allow for comparisons across organs, including the brain. The integrated respiratory chain function of the human ''vastus lateralis'' muscle was measured by high-resolution respirometry (HRR) in freshly taken fine-needle biopsies from seven pre-manifest HD expansion mutation carriers and nine controls. The respiratory parameters were unaffected. For comparison skeletal muscle isolated from HD knock-in mice (HdhQ111) as well as a broader spectrum of tissues including cortex, liver and heart muscle were examined by HRR. Significant changes of mitochondrial respiration in the HdhQ knock-in mouse model were restricted to the liver and the cortex. Mitochondrial mass as quantified by mitochondrial DNA copy number and citrate synthase activity was stable in murine HD-model tissue compared to control. mRNA levels of key enzymes were determined to characterize mitochondrial metabolic pathways in HdhQ mice. We demonstrated the feasibility to perform high-resolution respirometry measurements from small human HD muscle biopsies. Furthermore, we conclude that alterations in respiratory parameters of pre-manifest human muscle biopsies are rather limited and mirrored by a similar absence of marked alterations in HdhQ skeletal muscle. In contrast, the HdhQ111 murine cortex and liver did show respiratory alterations highlighting the tissue specific nature of mutant huntingtin effects on respiration. +
Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) is a two-stage strategy to induce rapid regeneration of the remnant liver. The technique has been associated with high mortality and morbidity rates. This study aimed to evaluate mitochondrial function, biogenesis and morphology during ALPPS-induced liver regeneration.
Male Wistar rats (n = 100) underwent portal vein ligation (PVL) or ALPPS. The animals were killed at 0 h (without operation), and 24, 48, 72 or 168 h after intervention. Regeneration rate and proliferation index were assessed. Mitochondrial oxygen consumption and adenosine 5'-triphosphate (ATP) production were measured. Mitochondrial biogenesis was evaluated by protein level measurements of peroxisome proliferator-activated receptor γ co-activator (PGC) 1-α, nuclear respiratory factor (NRF) 1 and 2, and mitochondrial transcription factor α. Mitochondrial morphology was evaluated by electron microscopy.
Regeneration rate and Ki-67 index were significantly raised in the ALPPS group compared with the PVL group (regeneration rate at 168 h: mean(s.d.) 291·2(21·4) versus 245·1(13·8) per cent, P < 0·001; Ki-67 index at 24 h: 86·9(4·6) versus 66·2(4·9) per cent, P < 0·001). In the ALPPS group, mitochondrial function was impaired 48 h after the intervention compared with that in the PVL group (induced ATP production); (complex I: 361·9(72·3) versus 629·7(165·8) nmol per min per mg, P = 0·038; complex II: 517·5(48·8) versus 794·8(170·4) nmol per min per mg, P = 0·044). Markers of mitochondrial biogenesis were significantly lower 48 and 72 h after ALPPS compared with PVL (PGC1-α at 48 h: 0·61-fold decrease, P = 0·045; NRF1 at 48 h: 0·48-fold decrease, P = 0·028). Mitochondrial size decreased significantly after ALPPS (0·26(0·05) versus 0·40(0·07) μm<sup>2</sup> ; P = 0·034).
Impaired mitochondrial function and biogenesis, along with the rapid energy-demanding cell proliferation, may cause hepatocyte dysfunction after ALPPS. Surgical relevance Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) is a well known surgical strategy that combines liver partition and portal vein ligation. This method induces immense regeneration in the future liver remnant. The rapid volume increase is of benefit for resectability, but the mortality and morbidity rates of ALPPS are strikingly high. Moreover, lagging functional recovery of the remnant liver has been reported recently. In this translational study, ALPPS caused an overwhelming inflammatory response that interfered with the peroxisome proliferator-activated receptor γ co-activator 1-α-coordinated, stress-induced, mitochondrial biogenesis pathway. This resulted in the accumulation of immature and malfunctioning mitochondria in hepatocytes during the early phase of liver regeneration (bioenergetic destabilization). These findings might explain some of the high morbidity if confirmed in patients.
<small>© 2018 BJS Society Ltd Published by John Wiley & Sons Ltd.</small>
During myocardial hibernation, decreases in coronary perfusion elicit inhibition of contraction, suggesting that energy demand is attenuated. We previously found an inhibition of contraction and O2 consumption during hypoxia (3% O2; PO2 = 20 torr for >2 h) in cardiomyocytes, which was reversible after reoxygenation. This study sought to determine whether mitochondria function as cellular O2 sensors mediating this response. Embryonic cardiomyocytes were studied under controlled O2 conditions. Hypoxia produced no acute decrease in mitochondrial potential as assessed using tetramethylrhodamine ethylester (TMRE). Cellular [ATP] was preserved throughout hypoxia, as assessed using the probe Magnesium Green. Thus, ATP synthesis and utilization remained closely coupled. Cells adapted to hypoxia for >2 h exhibited a 4% increase in mitochondrial potential upon reoxygenation, suggesting that a partial inhibition of cytochrome c oxidase had existed. To test whether the oxidase serves as an O2 sensor, azide was administered (1 mM) to simulate the effects of hypoxia by lowering the Vmax of the oxidase. The effects of azide on contraction and mitochondrial potential mimicked the response to hypoxia. We conclude that partial inhibition of cytochrome oxidase during hypoxia allows mitochondria to function as the O2 sensor mediating the decreases in ATP utilization and O2 consumption during hypoxia. +
Die Wechselbeziehungen innerhalb des Netzwerks von Redox-Reaktionen, das sich ueber einen großen Teil der metabolisch wesentlichen Funktionen lebender Zellen erstreckt, werden eroertert. Anschließend an die Darlegung grundsaetzlicher Gegebenheiten bei der Zerlegung der Brennstoffe werden einige Gruppen von Redox-Systemen in verschiedenen Raeumen der Zellen und Gewebe in ihren Beziehungen zur Biosynthese, zur Bioenergetik und zur Zellatmung behandelt. Neuere Ergebnisse aus dem Arbeitskreis der Verfasser stehen dabei im Vordergrund. Die Beispiele zeigen, wie weitgehend die Entwicklung der Problematik der dynamischen Biochemie an den Fortschritt der Cytologie gebunden ist. +
Mitochondria play vital cellular functions. These organelles functions and
roles in cellular metabolism not surprisingly have been implicated in a broad
spectrum of diseases, including neurodegeneration and cancer. Lately, it has been
given an important role for mitochondria and cellular metabolism in response and
resistance of tumour cells to therapy. Given the central role of mitochondria in
cellular homeostasis, we evaluated mitochondria network alterations in
glioblastoma cells in response to treatment with the chemotherapeutic
Temozolomide (TMZ). The acute treatment with TMZ induced an increase in both
mitochondrial mass and mitochondrial membrane potential (MMP) 5 days after
treatment, followed by a decrease in the same parameters on day 7. The increase
in these parameters is accompanied by a decrease in PGC1a expression on day 3
and a progressive increase in autophagy levels, suggesting that the increase in
mitochondrial mass is independent of PGC1a and may be due to an accumulation of
mitochondria. Also, the increase in mitochondrial mass and MMP correlates with
higher levels of oxidative stress and senescence on day 5. We also evaluated
mitochondrial oxygen consumption in both control and treated with TMZ cells,
observing that 5 days after treatment with TMZ cells have higher oxygen
consumption rates and a higher reserve capacity. In order to understand how
mitochondrial alterations influence the response to chemotherapy, we treated the
cells previously treated with TMZ on D5 with glycolysis and OXPHOS inhibitors
and followed cell number. Results suggest that these inhibitors abrogate TMZ treated
cells proliferation. Also, TMZ-treated cells are more sensitive to OXPHOS
inhibition than control cells, suggesting that TMZ reprograms the cells metabolism
to a more oxidative state instead of a glycolytic one. +
Systems biology is now recognized as a needed approach to understand the dynamics of inter- and intra-cellular processes. Redox processes are at the foundation of nearly all aspects of biology. Free radicals, related oxidants, and antioxidants are central to the basic functioning of cells and tissues. They set the cellular redox environment and therefore are key to regulation of biochemical pathways and networks, thereby influencing organism health. To understand how short-lived, quasi-stable species, such as superoxide, hydrogen peroxide, and nitric oxide, connect to the metabolome, proteome, lipidome, and genome we need absolute quantitative information on all redox active compounds as well as thermodynamic and kinetic information on their reactions, i.e. knowledge of the complete redoxome. Central to the state of the redoxome are the interactive details of the superoxide/peroxide formation and removal systems. Quantitative information is essential to establish the dynamic mathematical models needed to reveal the temporal evolution of biochemical pathways and networks. This new field of Quantitative Redox Biology will allow researchers to identify new targets for intervention to advance our efforts to achieve optimal human health. +
To this day, cancer is still among the leading causes of death worldwide and predicted to catch up with heart diseases, which holds the first position for now, very soon <sup>1</sup>. The treatment is complex and in many cases ineffective. Especially for patients diagnosed with oesophageal cancer (OC) the odds are bad, which could be told by a 5-year survival rate of only about 18%. Furthermore, the number of new cases and deaths did not markedly decrease over the last years for this type of cancer <sup>2</sup>. One reason for this is that up to this point no clinically useful molecular prognostic biomarkers exist and treatment options are very poor <sup>3</sup>. Therefore, it is strongly required to explore new ways and tools to carry on the fight against OC and cancerous diseases in general.
As it was found out during the last years that cells undergo a distinct metabolic remodeling when turning into tumour cells, the investigation of this process might be highly relevant for future cancer research <sup>4</sup>. Pilot studies performed in the Queen’s University of Belfast (QUB) using a pathway based approach to identify determinants of drug resistance in oral and oesophageal cancer (OOC) have already identified the glycolytic pathway as a potential targetable pathway. With this, particularly mitochondria, which are the main energy producers of the cell and are involved in multiple regulatory pathways, may play key factors in the carcinogenesis and therefore should be focused over the course of this work.
The stated PhD project will be conducted in the frame of the European Union's program “TRACT”, funded by a Marie Skłodowska-Curie fellowship. With the company Oroboros Instruments as a partner, it will aim at the examination of metabolic transformation mechanisms in OC with the attempt of identifying new drug targets for future therapeutic development and new diagnostic strategies.
The work will further characterize the bioenergetic and metabolic properties of normal, metaplastic, dysplastic and cancerous oesophageal cells and identify differences between these stages of carcinogenesis. The study will include comparison of oxygen consumption, extracellular acidity and metabolic flux under normoxic and hypoxic conditions. The measurements will be conducted using an Oroboros Oxygraph 2k (O2k)-Fluorometer enabling the screening of real-time bioenergetics and metabolism of cells by the combination of high resolution respirometry (HRR) with fluorometry. By this, not only the cellular respiration (O<sub>2</sub> consumption) of cells or mitochondria can be analyzed, but also multiple other metabolic parameters including the mitochondrial membrane potential, ATP production (indirectly via the concentration of free Mg<sup>2+</sup>), Ca<sup>2+</sup> production and reactive oxygen (ROS) production (via H<sub>2</sub>O<sub>2</sub> concentration) <sup>5</sup>. These parameters are often altered in cancerous cells and might therefore be interesting for diagnostic and therapeutic interventions. To name an example, the production of ROS in mitochondria is known to be an essential component of multiple cellular pathways; however, an overload of oxidative stress can also cause genetic and functional damage, probably leading to degenerative diseases or cancer <sup>6</sup>. By measuring the oxidative stress as a signal of hydrogen peroxide production and decreasing or increasing it by adding specific chemicals, it could be further evaluated how significant the effects of ROS production might be in the development of OC.
Over the course of a 9-month secondment at the Trinity College of Dublin (TCD) under the supervision of Prof. Richard Porter, metabolic flux will be measured additionally through glycolysis, pentose phosphate pathway and glutaminolysis using <sup>2</sup>H/<sup>13</sup>C NMR to complement the respiratory and fluorometric measurements conducted with the O2k at Oroboros Instruments. The stated experiments and investigations will be performed with cell culture models of oesophageal adenocarcinoma as well as human tissue biopsies of different stages of cancer -namely intestinal metaplasia, dysplasia, adenocarcinoma and non-cancerous tissue as control. For the conduction of the experiments, an ethics committee vote is obtained. To enable the repeated measurement of the samples, parts of all received samples are intended to be cryopreserved. Anyway, the feasibility of the cryopreservation of these tissues remains to be tested and will be proven by simultaneous comparative measurements of the cellular functions and metabolism of cryopreserved as well as fresh tissues with the O2k.
By comparing the different types of cancerous tissue with the cell culture models of adenocarcinoma, the representativeness and reliability of the utilized cell models regarding their metabolic properties will be tested. With the inclusion of human tissues, the relevance and applicability of this study for the clinic is particularly ensured.
On the whole, the main aim of this thesis is the identification of differences in the metabolic profiles of the examined states of cancer and, in hand with this, the metabolic transformation mechanisms taking place in OC. In so doing a basis of differential novel drug targets will be established and the progression of means to enhance the chemotherapeutic sensitivity of cancer cells identified.
== References ==
# Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C et al. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide, IARC CancerBase, No. 11 (2013).
# Howlader N, Noone AM, Krapcho M, Miller D, Bishop K, Altekruse SF, Kosary CL, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA. SEER Cancer Statistics Review, 1975-2013, National Cancer Institute (2016).
# McCormick Matthews LH, Noble F, Tod J, et al. Systematic review and meta-analysis of immunohistochemical prognostic biomarkers in resected oesophageal adenocarcinoma, British Journal of Cancer, 113(1):107-118 (2015), doi:10.1038/bjc.2015.179.
# Smolková K, Plecitá-Hlavatá L, Bellance N, Benard G, Rossignol R, Ježek P. Waves of gene regulation suppress and then restore oxidative phosphorylation in cancer cells, The International Journal of Biochemistry & Cell Biology, 43(7):950-968 (2011), ISSN 1357-2725, doi: 10.1016/j.biocel.2010.05.003.
# Fasching M, Gradl P and Gnaiger E. The O2k-Fluo LED2-Module, Mitochondrial Physiology Network, 17.05(08):1-6 (2015).
# Camara AKS, Lesnefsky EJ, Stowe DF. Potential Therapeutic Benefits of Strategies Directed to Mitochondria, Antioxidants & Redox Signaling, 13(3):279-347 (2010), doi:10.1089/ars.2009.2788.
Kidney proximal tubules (PTs) are densely packed with mitochondria, and defects in mitochondrial function are implicated in many kidney diseases. However, little is known about intrinsic mitochondrial function within PT cells. Here, using intravital multiphoton microscopy and live slices of mouse kidney cortex, we show that autofluorescence signals provide important functional readouts of redox state and substrate metabolism and that there are striking axial differences in signals along the PT. Mitochondrial NAD(P)H intensity was similar in both PT segment (S)1 and S2 and was sensitive to changes in respiratory chain (RC) redox state, whereas cytosolic NAD(P)H intensity was significantly higher in S2. Mitochondrial NAD(P)H increased in response to lactate and butyrate but decreased in response to glutamine and glutamate. Cytosolic NAD(P)H was sensitive to lactate and pyruvate and decreased dramatically in S2 in response to inhibition of glucose metabolism. Mitochondrial flavoprotein (FP) intensity was markedly higher in S2 than in S1 but was insensitive to changes in RC redox state. Mitochondrial FP signal increased in response to palmitate but decreased in response to glutamine and glutamate. Fluorescence lifetime decays were similar in both S1 and S2, suggesting that intensity differences are explained by differences in abundance of the same molecular species. Expression levels of known fluorescent mitochondrial FPs were higher in S2 than S1. In summary, substantial metabolic information can be obtained in kidney tissue using a label-free live imaging approach, and our findings suggest that metabolism is tailored to the specialized functions of S1 and S2 PT segments. +
There is growing evidence for the contribution of the activated coagulation factor X (FXa) in the development of chronic inflammatory lung diseases. Therefore, we aimed to investigate effects of exogenous FXa on mitochondrial and metabolic function as well as the induction of inflammatory molecules in type II alveolar epithelial cells. Effects of FXa on epithelial cells were investigated in A549 cell line. Activation of extracellular signal-regulated kinase (ERK) and induction of inflammatory molecules were examined by immunoblot and gene expression analysis. Mitochondrial function was assessed by the measurement of oxygen consumption during maximal oxidative phosphorylation and quantitative determination of cardiolipin oxidation. Apoptosis was tested using a caspase 3 antibody. Metabolic activity and lactate dehydrogenase assay were applied for the detection of cellular viability.
FXa activated ERK1/2 and induced an increase in the expression of pro-inflammatory cytokines, which was prevented by an inhibitor of FXa, edoxaban, or an inhibitor of protease-activated receptor 1, vorapaxar. Exposure to FXa caused mitochondrial alteration with restricted capacity for ATP generation, which was effectively prevented by edoxaban, vorapaxar and GB83 (inhibitor of protease-activated receptor 2). Of note, exposure to FXa did not initiate apoptosis in epithelial cells.
FXa-dependent pro-inflammatory state and impairment of mitochondria did not reach the level of significance in lung epithelial cells. However, these effects might limit regenerative potency of lung epithelial cells, particular under clinical circumstances where lung injury causes exposure to clotting factors.
<small>Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.</small> +
Oxidative stress is one of the factors that could explain the pathophysiological mechanism of inflammatory conditions that occur in cardiovascular disease (CVD) and periodontitis. Such inflammatory response is often evoked by specific bacteria, as the lipopolysaccharide (LPS) of ''Porphyromonas gingivalis'' is a key factor in this process. The aim of this research was to study the role of mitochondrial dysfunction in peripheral blood mononuclear cells (PBMCs) from periodontitis patients and to evaluate the influence of LPS on fibroblasts to better understand the pathophysiology of periodontitis and its relationship with CVD. PBMCs from patients showed lower CoQ10 levels and citrate synthase activity, together with high levels of ROS production. LPS-treated fibroblasts provoked increased oxidative stress and mitochondrial dysfunction by a decrease in mitochondrial protein expression, mitochondrial mass, and mitochondrial membrane potential. Our study supports the hypothesis that LPS-mediated mitochondrial dysfunction could be at the origin of oxidative stress in periodontal patients. Abnormal PBMC performance may promote oxidative stress and alter cytokine homeostasis. In conclusion, mitochondrial dysfunction could represent a possible link to understanding the interrelationships between two prominent inflammatory diseases: periodontitis and CVD.
Copyright © 2011 Elsevier Inc. All rights reserved. +
Oxidative stress is implicated in several infectious diseases. In this regard, lipopolysaccharide (LPS), an endotoxic component, induces mitochondrial dysfunction and oxidative stress in several pathological events such as periodontal disease or sepsis. In our experiments, LPS-treated fibroblasts provoked increased oxidative stress, mitochondrial dysfunction, reduced oxygen consumption and mitochondrial biogenesis. After comparing coenzyme Q10 (CoQ10) and N-acetylcysteine (NAC), we observed a more significant protection of CoQ10 than of NAC, which was comparable with other lipophilic and hydrophilic antioxidants such as vitamin E or BHA respectively. CoQ10 improved mitochondrial biogenesis by activating PGC-1α and TFAM. This lipophilic antioxidant protection was observed in mice after LPS injection. These results show that mitochondria-targeted lipophilic antioxidants could be a possible specific therapeutic strategy in pharmacology in the treatment of infectious diseases and their complications.
Copyright © 2014 Elsevier Ltd. All rights reserved. +
As many diseases have been shown to have several or indirect causes (i.e. are multifactorial) the question is what is the relative importance of each factor in a given disease? Also, what happens when some diseases, although apparently disparate, share causative factors and/or tissue pathologies? Host inflammation response mechanisms are largely shared by the body's different tissues and systems and only recently has special attention been paid to the possible linkages among chronic periodontitis and other chronic systemic diseases. The aim of this review was to consider and discuss the mounting evidence that the basis for the inter-relationships between chronic periodontitis and atheromatous disease and diabetes lie at a fundamental intracellular level, namely oxidative stress and mitochondrial dysfunction, as a meeting background among such chronic diseases and periodontitis.
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. +
Nitrite protects the heart from toxic oxygen radicals when oxygen returns after a period of oxygen deprivation, such as after heart attack or infarct. During anoxia, nitrite can inhibit inactive proteins, such as complex I in the electron transport chain, by a post-translational modification termed S-nitrosation, where an NO-moiety binds protein cysteines. Inhibition of complex I have been shown to limit the production of oxygen radicals, thereby protecting the heart from oxidative damage [1].
Some extreme animals, such as the red-eared slider turtle, survive the winter at the bottom of frozen ponds, and remain completely deprived of oxygen for several months. Unlike mammals, these turtles are not maimed by reoxygenation, but wake up in the spring with healthy hearts. Nitrite is naturally accumulated in the hearts of these animals during anoxia, which might protect them from reperfusion damage [2].
In this study, we investigate the protective effects of nitrite on the turtle heart. ''In vitro'' studies on isolated mitochondria have shown that the artificial S-nitrosating agent MitoSNO S-nitrosates turtle complex I which decreases activity of the enzyme and reduces ROS production upon reoxygenation, but does not affect respiration rate. Further, we have shown that succinate is accumulated in the anoxic turtle heart, which has been shown in mice to fuel the ROS production that occurs upon reoxygenation. This would corroborate the need for inhibition of complex I in the turtle.
We further wish to investigate whether the accumulated nitrite in the anoxic turtle mimics the protective effect of S-nitrosation ''in vitro'' and whether this is involved in keeping the turtle heart healthy upon awakening in the spring after a long, oxygen-deprived winter. Using extreme animals such as the turtle as models for coping with extreme situations like oxygen deprivation might teach us how to protect the more sensitive human heart. +
ATP depletion and succinate accumulation during ischemia lead to oxidative damage to mammalian organs upon reperfusion. In contrast, freshwater turtles survive weeks of anoxia at low temperatures without suffering from oxidative damage upon reoxygenation, but the mechanisms are unclear. To determine how turtles survive prolonged anoxia, we measured ~80 metabolites in hearts from cold-acclimated (5 °C) turtles exposed to 9 days anoxia and compared the results with those for normoxic turtles (25 °C) and mouse hearts exposed to 30 min of ischemia. In turtles, ATP and ADP decreased to new steady-state levels during fasting and cold-acclimation and further with anoxia, but disappeared within 30 min of ischemia in mouse hearts. High NADH/NAD<sup>+</sup> ratios were associated with succinate accumulation in both anoxic turtles and ischemic mouse hearts. However, succinate concentrations and succinate/fumarate ratios were lower in turtle than in mouse heart, limiting the driving force for production of reactive oxygen species (ROS) upon reoxygenation in turtles. Furthermore, we show production of ROS from succinate is prevented by re-synthesis of ATP from ADP. Thus, maintenance of an ATP/ADP pool and low succinate accumulation likely protects turtle hearts from anoxia/reoxygenation injury and suggests metabolic interventions as a therapeutic approach to limit ischemia/reperfusion injury in mammals. +
Mitochondria are important to cellular homeostasis, but can become a dangerous liability when cells recover from hypoxia. Anoxia-tolerant freshwater turtles show reduced mitochondrial respiratory capacity and production of reactive oxygen species (ROS) after prolonged anoxia, but the mechanisms are unclear. Here, we investigated whether this mitochondrial suppression originates from downregulation of mitochondrial content or intrinsic activity by comparing heart mitochondria from (1) warm (25°C) normoxic, (2) cold-acclimated (4°C) normoxic and (3) cold-acclimated anoxic turtles. Transmission electron microscopy of heart ventricle revealed that these treatments did not affect mitochondrial volume density and morphology. Furthermore, neither enzyme activity, protein content nor supercomplex distribution of electron transport chain (ETC) enzymes changed significantly. Instead, our data imply that turtles inhibit mitochondrial respiration rate and ROS production by a cumulative effect of slight inhibition of ETC complexes. Together, these results show that maintaining mitochondrial integrity while inhibiting overall enzyme activities are important aspects of anoxia tolerance. +
Mitochondrial 2-oxoacid dehydrogenase complexes oxidize 2-oxoglutarate, pyruvate, branched-chain 2-oxoacids and 2-oxoadipate to the corresponding acyl-CoAs and reduce NAD+ to NADH. The isolated enzyme complexes generate superoxide anion radical or hydrogen peroxide in defined reactions by leaking electrons to oxygen. Studies using isolated mitochondria in media mimicking cytosol suggest that the 2-oxoacid dehydrogenase complexes contribute little to the production of superoxide or hydrogen peroxide relative to other mitochondrial sites at physiological steady states. However, the contributions may increase under pathological conditions, in accordance with the high maximum capacities of superoxide or hydrogen peroxide-generating reactions of the complexes, established in isolated mitochondria. We assess available data on the use of modulations of enzyme activity to infer superoxide or hydrogen peroxide production from particular 2-oxoacid dehydrogenase complexes in cells, and limitations of such methods to discriminate specific superoxide or hydrogen peroxide sources in vivo. +
Ion-stabilized gas nanobubbles (the so-termed "bubstons") and their clusters are investigated in bulk aqueous solutions of NaCl at different ion concentrations by four independent laser diagnostic methods. It turned out that in the range of NaCl concentration 10(-6) < C < 1 M the radius of bubston remains virtually unchanged at a value of 100 nm. Bubstons and their clusters are a thermodynamically nonequilibrium phase, which has been demonstrated in experiments with magnetic stirrer at different stirring rates. Different regimes of the bubston generation, resulting from various techniques of processing the liquid samples, were explored. +
The names at present available for hydrogen ions or groups, and for reactions involving them, are not always adequate for describing isotopic differences. For example, the word proton is used not only for the 'H<sup>+</sup> ion but commonly, and incorrectly, for H<sup>+</sup> in natural abundance. In many contexts this creates no ambiguity and it is likely that this usage will continue. However, in discussions of isotope effects and in several areas of nomenclature the ability to make a distinction is essential, and the Commission on Physical Organic Chemistry recommends the terms set out below to avoid such ambiguities. +
Mitochondria strongly accumulate amphiphilic cations. We report here a study of the association of respiring rat liver mitochondria with several fluorescent cationic dyes from differing structural classes. Using gravimetric and fluorometric analysis of dye partition, we find that dyes and mitochondria interact in three ways: (a) uptake with fluorescence quenching, (b) uptake without change in fluorescence intensity, and (c) lack of uptake. For dyes that quench upon uptake, the extent of quenching correlates with the degree of aggregation of the dye to dimers, as predicted by theory (Tomov, T.C. 1986. J. Biochem. Biophys. Methods. 13:29-38). Also predicted is the relationship observed between quenching and the mitochondria concentration when constant dye is titrated with mitochondria. Not predicted is the relationship observed between quenching and dye concentration when constant mitochondria are titrated with dye. Because a limit to dye uptake exists, in this case, the degree of quenching decreases as dye is added. A Langmuir isotherm analysis gives phenomenological parameters that predict quenching when it is observed as a function of dye concentration. By allowing for a decrease in membrane potential, caused by incorporation of cationic dye into the lipid bilayer, a modification of the Tomov theory predicts the dye titration data. We present a model of cationic dye-mitochondria interaction and discuss the use of these as probes of mitochondrial membrane potential. +
Scientists create work under their own direction – funded largely by governments – and give it to publishers for free; the publisher pays scientific editors who judge whether the work is worth publishing and check its grammar, but the bulk of the editorial burden – checking the scientific validity and evaluating the experiments, a process known as peer review – is done by working scientists on a volunteer basis. The publishers then sell the product back to government-funded institutional and university libraries, to be read by scientists – who, in a collective sense, created the product in the first place. +
The [[International System of Units]], the SI, has been used around the world as the preferred system of units, the basic language for science, technology, industry and trade since it was established in 1960 by a resolution at the 11th meeting of the Conférence Générale des Poids et Mesures, the CGPM (known in English as the General Conference on Weights and Measures). +
Mitochondria provide the main source of energy for eukaryotic cells, oxidizing fatty acids and sugars to generate ATP. Mitochondrial fatty acid β-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are two key pathways involved in this process. Disruption of FAO can cause human disease, with patients commonly presenting with liver failure, hypoketotic glycaemia and rhabdomyolysis. However, patients with deficiencies in the FAO enzyme short-chain enoyl-CoA hydratase 1 (ECHS1) are typically diagnosed with Leigh syndrome, a lethal form of subacute necrotizing encephalomyelopathy that is normally associated with OXPHOS dysfunction. Furthermore, some ECHS1-deficient patients also exhibit secondary OXPHOS defects. This sequela of FAO disorders has long been thought to be caused by the accumulation of inhibitory fatty acid intermediates. However, new evidence suggests that the mechanisms involved are more complex, and that disruption of OXPHOS protein complex biogenesis and/or stability is also involved. In this review, we examine the clinical, biochemical and genetic features of all ECHS1-deficient patients described to date. In particular, we consider the secondary OXPHOS defects associated with ECHS1 deficiency and discuss their possible contribution to disease pathogenesis. +
The lack of effective treatments for mitochondrial disease has seen the development of new approaches, including those that aim to stimulate mitochondrial biogenesis to boost ATP generation above a critical disease threshold. Here, we examine the effects of the peroxisome proliferator-activated receptor γ (PPARγ) activator pioglitazone (PioG), in combination with deoxyribonucleosides (dNs), on mitochondrial biogenesis in cybrid cells containing >90% of the m.3243A>G mutation associated with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). PioG + dNs combination treatment increased mtDNA copy number and mitochondrial mass in both control (CON) and m.3243A>G (MUT) cybrids, with no adverse effects on cell proliferation. PioG + dNs also increased mtDNA-encoded transcripts in CON cybrids, but had the opposite effect in MUT cybrids, reducing the already elevated transcript levels. Steady-state levels of mature oxidative phosphorylation (OXPHOS) protein complexes were increased by PioG + dNs treatment in CON cybrids, but were unchanged in MUT cybrids. However, treatment was able to significantly increase maximal mitochondrial oxygen consumption rates and cell respiratory control ratios in both CON and MUT cybrids. Overall, these findings highlight the ability of PioG + dNs to improve mitochondrial respiratory function in cybrid cells containing the m.3243A>G MELAS mutation, as well as their potential for development into novel therapies to treat mitochondrial disease. +
The use of acoustic forces to manipulate particles or cells at the microfluidic scale (i.e. acoustophoresis), enables non-contact, label-free separation based on intrinsic cell properties such as size, density and compressibility. Acoustophoresis holds great promise as a cell separation technique in several research and clinical areas. However, it has been suggested that the force acting upon cells undergoing acoustophoresis may impact cell viability, proliferation or cell function via subtle phenotypic changes. If this were the case, it would suggest that the acoustophoresis method would be a less useful tool for many cell analysis applications as well as for cell therapy.
We investigate, for the first time, several key aspects of cellular changes following acoustophoretic processing. We used two settings of ultrasonic actuation, one that is used for cell sorting (10 Vpp operating voltage) and one that is close to the maximum of what the system can generate (20 Vpp). We used microglial cells and assessed cell viability and proliferation, as well as the inflammatory response that is indicative of more subtle changes in cellular phenotype. Furthermore, we adapted a similar methodology to monitor the response of human prostate cancer cells to acoustophoretic processing. Lastly, we analyzed the respiratory properties of human leukocytes and thrombocytes to explore if acoustophoretic processing has adverse effects.
BV2 microglia were unaltered after acoustophoretic processing as measured by apoptosis and cell turnover assays as well as inflammatory cytokine response up to 48 h following acoustophoresis. Similarly, we found that acoustophoretic processing neither affected the cell viability of prostate cancer cells nor altered their prostate-specific antigen secretion following androgen receptor activation. Finally, human thrombocytes and leukocytes displayed unaltered mitochondrial respiratory function and integrity after acoustophoretic processing.
We conclude that microchannel acoustophoresis can be used for effective continuous flow-based cell separation without affecting cell viability, proliferation, mitochondrial respiration or inflammatory status.
The prevalence of obesity is increasing worldwide, causing a subsequent increase in insulin resistance (IR), metabolic disorders, and cardiovascular diseases. Although these conditions significantly impact both the individual and society, their etiologies have not been discovered, inhibiting potential prevention and treatment. Alterations to mitochondrial metabolism are implicated in the development of IR and progression to Type 2 Diabetes (T2D), raising the need for an investigation of mitochondrial metabolism in skeletal muscle. Understanding metabolism on an individual substrate basis within a model system enables identification of each nutrient’s contribution to respiration and potential mitochondrial dysfunction. As such, the purpose of this investigation was to characterize mitochondrial metabolism and determine mitochondrial respiratory control in L6 rat myoblasts. High-resolution respirometry and three Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocols were used to examine respiration. Mitochondrial capacity was not limited by the electron transfer system, although the presence of multiple substrates potentially flooded the Q-junction, resulting in lower respiratory control and indicating the presence of a potential limiting factor. In sum, L6 myoblasts exhibit no overt defects in mitochondrial metabolism, but may have a propensity for substrate overload, thereby limiting respiration. +
The mitochondrial respiratory chain (MRC) complex III (CIII) associates with complexes I and IV (CI and CIV) into supercomplexes. We identified a novel homozygous missense mutation (c.665G>C; p.Gly222Ala) in UQCRC2 coding for structural subunit Core 2 in a patient with severe encephalomyopathy. The structural data suggest that the Gly222Ala exchange might result in an altered spatial arrangement in part of the UQCRC2 subunit, which could impact specific protein-protein interactions. Accordingly, we have found decreased levels of CIII and accumulation of CIII-specific subassemblies comprising MT-CYB, UQCRB, UQCRQ, UQCR10 and CYC1 subunits, but devoid of UQCRC1, UQCRC2, and UQCRFS1 in the patient's fibroblasts. The lack of UQCRC1 subunit-containing subassemblies could result from an impaired interaction with mutant UQCRC2<sup>Gly222Ala</sup> and subsequent degradation of both subunits by mitochondrial proteases. Indeed, we show an elevated amount of matrix CLPP protease, suggesting the activation of the mitochondrial protein quality control machinery in UQCRC2<sup>Gly222Ala</sup> fibroblasts. In line with growing evidence, we observed a rate-limiting character of CIII availability for the supercomplex formation, accompanied by a diminished amount of CI. Furthermore, we found impaired electron flux between CI and CIII in skeletal muscle and fibroblasts of the UQCRC2<sup>Gly222Ala</sup> patient. The ectopic expression of wild-type UQCRC2 in patient cells rescued maximal respiration rate, demonstrating the deleterious effect of the mutation on MRC. Our study expands the phenotypic spectrum of human disease caused by CIII Core protein deficiency, provides insight into the assembly pathway of human CIII, and supports the requirement of assembled CIII for a proper accumulation of CI. +
Mutations in coiled-coil-helix-coiled-coil-helix-domain containing 10 (CHCHD10), a mitochondrial twin CX9C protein whose function is still unknown, cause myopathy, motor neuron disease, frontotemporal dementia, and Parkinson's disease. Here, we investigate CHCHD10 topology and its protein interactome, as well as the effects of CHCHD10 depletion or expression of disease-associated mutations in wild-type cells. We find that CHCHD10 associates with membranes in the mitochondrial intermembrane space, where it interacts with a closely related protein, CHCHD2. Furthermore, both CHCHD10 and CHCHD2 interact with p32/GC1QR, a protein with various intra and extra-mitochondrial functions. CHCHD10 and CHCHD2 have short half-lives, suggesting regulatory rather than structural functions. Cell lines with CHCHD10 knockdown do not display bioenergetic defects, but, unexpectedly, accumulate excessive intramitochondrial iron. In mice, CHCHD10 is expressed in many tissues, most abundantly in heart, skeletal muscle, liver, and in specific CNS regions, notably the dopaminergic neurons of the substantia nigra and spinal cord neurons, which is consistent with the pathology associated with CHCHD10 mutations. Homozygote CHCHD10 knockout mice are viable, have no gross phenotypes, no bioenergetic defects or ultrastructural mitochondrial abnormalities in brain, heart or skeletal muscle, indicating that functional redundancy or compensatory mechanisms for CHCHD10 loss occur ''in vivo''. Instead, cells expressing S59L or R15L mutant versions of CHCHD10, but not WT, have impaired mitochondrial energy metabolism. Taken together, the evidence obtained from our ''in vitro'' and ''in vivo'' studies suggest that CHCHD10 mutants cause disease through a gain of toxic function mechanism, rather than a loss of function.
<small>© The Author 2017. Published by Oxford University Press. All rights reserved.</small> +
Investigations of oxidative phosphorylation (OXPHOS) play an increasingly important role in relating mitochondrial health to life style (exercise and nutrition), mitochondrial haplogroups and heteroplasmy, and individual training programs of competitive athletes. +
[[File:Martin.jpg|right|150px|Martin Burtscher]]
The individual level of exercise tolerance is closely associated with mortality and quality of life. Thus, to maintain or improve exercise tolerance is one of the most important goals in the elderly or those suffering from various diseases. Exercise training has been evidenced as the most effective way to achieve this goal. Exercise tolerance is related to the ability to use a high percentage of the individual maximum oxygen uptake which is thought predominantly to result from chronic adaptations in skeletal muscle. These adaptations include the increase of key enzyme activities of the mitochondrial electron transport chain and an associated increase in mitochondrial protein accumulation and increased capillary supply. The resulting improvements in performance seem mainly due to a higher rate of fat oxidation and a concomitant reduction in glycolytic flux, and a tighter control of the acid-base status. However, in certain cases, the rapid development of fatigue which cannot simply be explained by metabolic aspects of working muscles affects exercise performance. Recent studies suggest that exercise increases not only muscle but also brain mitochondrial biogenesis thereby likely contributing to reduced fatigue and improved exercise performance [1]. Beside exercise training, repeated passive exposures to short-term hypoxia (interval hypoxia) have also been demonstrated to increase exercise tolerance, e.g. in patients suffering from respiratory or heart diseases [2]. Similar to exercise, transient hypoxia with and without exercise may be capable to stimulate mitochondrial biogenesis in the skeletal muscle as well as the brain [3], thereby contributing to the observed changes in muscle metabolism and the rating of perceived exertion after interval hypoxia [2]. Our observations, derived from measurements of cerebral and muscle oxygenation by NIRS, indicate that beneficial effects on exercise tolerance following exercise training might rather be due to reduced peripheral fatigue and following interval hypoxia due to diminished central fatigue.
# Steiner JL, Murphy EA, McClellan JL, Carmichael MD, Davis JM (2011) Exercise training increases mitochondrial biogenesis in the brain. J Appl Physiol 111: 1066-1071.
# Burtscher M, Gatterer H, Szubski C, Pierantozzi E, Faulhaber M (2010) Effects of interval hypoxia on exercise tolerance: special focus on patients with CAD or COPD. Sleep Breath 14: 209-220.
# Gutsaeva DR, Carraway MS, Suliman HB, Demchenko IT, Shitara H, Yonekawa H, Piantadosi CA (2008) Transient hypoxia stimulates mitochondrial biogenesis in brain subcortex by a neuronal nitric oxide synthase-dependent mechanism. J Neurosci 28: 2015-2204.
Arnold Durig (1872-1961) grew up in the Austrian mountains in the period when intense exploration of the Alps started. As an enthusiastic mountaineer, scientist, and physician, he became one of the pioneers exploring physiological and pathophysiological aspects of humans sojourning to high altitudes. At the beginning of the 20<sup>th</sup> century, Durig was one of the great physiologists whose knowledge covered the whole field of physiology. Durig founded a renowned School and his students spread all over the world. He stayed in close contact with many colleagues and famous scientists, such as Albert Einstein and Sigmund Freud. Although he was an extremely productive and acknowledged physiologist and teacher at that time, his work and life are not very well known at the beginning of the 3<sup>rd</sup> millennium, even by high altitude physiologists. Thus, this article provides an overview on Durig's life and work, highlighting the most important scientific studies he performed at moderate and high altitudes, in an attempt to provide a few links to the development of high altitude research in the late 19<sup>th</sup> and early 20<sup>th</sup> centuries, complemented by some comments from a current Point of view. +
Mitochondrial dysfunction appears to be a common factor in neurodegenerative diseases. However, such diseases differ markedly in the nervous tissue affected. To test potential differences in mitochondrial respiratory capacity of different brain tissues under physiological or pathological conditions, we established a SUIT protocol for the analysis of oxidative phosphorylation (OXPHOS) and electron transfer-pathway capacity (ET-pathway) of small amounts of defined brain-tissues of mice. This protocol enables us to measure, independently, Complex I-, II- and IV-linked (CI, CII, and CIV, respectively) respiration, as well as the combined CI&II-linked OXPHOS- and electron transfer-pathway (ET-pathway) capacity in a single run from as little as 2 mg tissue applying the Oroboros high-resolution respirometry system [1].
The reproducibility within one experiment (two replica from the same tissue sample) and between experiments was very high. We observed significantly higher CI-linked oxygen fluxes in the motorcortex and CII-linked respiration in the striatum, when comparing motorcortex, striatum, hippocampus and brainstem obtained from young, healthy, adult, male C57BL6/J mice. No differences were found for CI&II-linked ET capacity and CIV activity expressed as oxygen consumption per tissue mass or as CIV/CI&II<sub>''E''</sub> flux control ratios. The ''P/E'' coupling control ratio (CI&II), an index of the limitation of OXPHOS capacity by the phorphorylation system, was significantly different between motorcortex and hippocampus.
The established protocol allows detailed analysis of mitochondrial function from small amounts of specific tissues. It thus enables comparison of different brain regions implicated in neurodegenerative diseases of the healthy mouse and disease models while leaving sufficient amounts of sample for additional analysis of the tissues. +
Mitochondrial dysfunction and oxidative stress are strongly implicated in neurodegenerative diseases and epilepsy. Strikingly, neurodegenerative diseases show regional specificity in vulnerability and follow distinct patterns of neuronal loss. A challenge is to understand, why mitochondria fail in particular brain regions under specific pathological conditions. A potential explanation could be provided by regional or cellular specificity of mitochondrial function.
We applied high-resolution respirometry to analyze the integrated Complex I- and II (CI and CII)-linked respiration, the activity of Complex IV, and the combined CI&II-linked oxidative phosphorylation (OXPHOS)- and electron-transfer system (ET-pathway)-capacity in microsamples obtained from distinct regions of the mouse brain. We compared different approaches to assess mitochondrial density and suggest flux control ratios as a valid method to normalize respiration to mitochondrial density.
This approach revealed significant differences of CI- and CII-linked OXPHOS capacity and coupling control between motor cortex, striatum, hippocampus and pons of naïve mice. CI-linked respiration was highest in motor cortex, while CII-linked respiration predominated in the striatum. To investigate if this method could also determine differences in normal and disease states within the same brain region, we compared hippocampal homogenates in a chronic epilepsy model. Three weeks after stereotaxic injection of kainate, there was a down-regulation of CI- and upregulation of CII-linked respiration in the resulting epileptic ipsilateral hippocampus compared to the contralateral one.
In summary, respirometric OXPHOS analysis provides a very sensitive diagnostic approach using small amounts of distinct brain tissues. In a single assay, information is obtained on numerous OXPHOS parameters as indicators of tissue-specific mitochondrial performance.
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Epilepsies are a group of common neurological diseases exerting a strong burden on patients and society, often lacking clear etiology and effective therapeutical strategies. Early intervention during the development of epilepsy (epileptogenesis) is of great medical interest, though hampered by poorly characterized epileptogenetic processes.
Using the intrahippocampal kainic acid mouse model of temporal lobe epilepsy, we investigated the functional role of the endogenous opioid enkephalin during epileptogenesis. We addressed 3 sequential questions: (1) How does enkephalin affect seizure threshold and how is it regulated during epileptogenesis? (2) Does enkephalin influence detrimental effects during epileptogenesis? (3) How is enkephalin linked to mitochondrial function during epileptogenesis?
In contrast to other neuropeptides, the expression of enkephalin is not regulated in a seizure dependent manner. The pattern of regulation, and enkephalin’s proconvulsive effects suggested it as a potential driving force in epileptogenesis. Surprisingly, enkephalin deficiency aggravated progressive granule cell dispersion in kainic acid induced epileptogenesis. Based on reported beneficial effects of enkephalin on mitochondrial function in hypoxic/ischemic states, we hypothesized that enkephalin may be involved in the adaptation of mitochondrial respiration during epileptogenesis. Using high-resolution respirometry, we observed dynamic improvement of hippocampal mitochondrial respiration after kainic acid-injections in wild-type, but not in enkephalin-deficient mice. Thus, wild-type mice displayed higher efficiency in the use of mitochondrial capacity as compared to enkephalin-deficient mice.
Our data demonstrate a Janus-headed role of enkephalin in epileptogenesis. In naive mice, enkephalin facilitates seizures, but in subsequent stages it contributes to neuronal survival through improved mitochondrial respiration.
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Mitochondrial dysfunction is crucially involved in aging and neurodegenerative diseases, such as Huntington's Disease (HD). How mitochondria become compromised in HD is poorly understood but instrumental for the development of treatments to prevent or reverse resulting deficits. In this paper, we investigate whether oxidative phosphorylation (OXPHOS) differs across brain regions in juvenile as compared to adult mice and whether such developmental changes might be compromised in the R6/2 mouse model of HD. We study OXPHOS in the striatum, hippocampus, and motor cortex by high resolution respirometry in female wild-type and R6/2 mice of ages corresponding to pre-symptomatic and symptomatic R6/2 mice. We observe a developmental shift in OXPHOS-control parameters that was similar in R6/2 mice, except for cortical succinate-driven respiration. While the LEAK state relative to maximal respiratory capacity was reduced in adult mice in all analyzed brain regions, succinate-driven respiration was reduced only in the striatum and cortex, and NADH-driven respiration was higher as compared to juvenile mice only in the striatum. We demonstrate age-related changes in respirational capacities of different brain regions with subtle deviations in R6/2 mice. Uncovering in situ oxygen conditions and potential substrate limitations during aging and HD disease progression are interesting avenues for future research to understand brain-regional vulnerability in HD. +
Increasing evidence suggests that cross talk between α-synuclein pathology formation and mitochondrial dysfunction plays a central role in the pathogenesis of Parkinson's disease (PD) and related synucleinopathies. While mitochondrial dysfunction is a well-studied phenomenon in the substantia nigra, which is selectively vulnerable in PD and some models thereof, less information is available in other brain regions that are also affected by synuclein pathology. Therefore, we sought to test the hypothesis that early α-synuclein pathology causes mitochondrial dysfunction and that this effect might be exacerbated in conditions of increased vulnerability in affected brain regions, such as the amygdala. We combined a model of intracerebral α-synuclein pathology seeding with chronic glucocorticoid treatment, which models non-motor symptoms of PD and affects amygdala physiology. We measured mitochondrial respiration, reactive oxygen species (ROS) generation and protein abundance as well as α-synuclein pathology in male mice. Chronic corticosterone administration induced mitochondrial hyperactivity in the amygdala. Although injection of α-synuclein preformed fibrils (PFFs) into the striatum resulted in pronounced α-synuclein pathology in both striatum and amygdala, mitochondrial respiration in these brain regions was not compromised, regardless of corticosterone treatment. Our results suggest that early stage α-synuclein pathology does not influence mitochondrial respiration in the striatum and amygdala, even in corticosterone-induced respirational hyperactivity. We discuss our findings in light of relevant literature, warn of a potential publication bias and encourage scientists to report their negative results within the framework of this model. +
The pathophysiology, immune reaction, differential vulnerability of different population groups and viral host immune system evasion strategies of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection are not yet well understood. Here, we reviewed the multitude of known strategies of coronaviruses and other viruses to usurp mitochondria-associated mechanisms involved in the host innate immune response and put them in context with the current knowledge on SARS-CoV-2. We argue that maintenance of mitochondrial integrity is essential for adequate innate immune system responses and to blunt mitochondrial modulation by SARS-CoV-2. Mitochondrial health thus may determine differential vulnerabilities to SARS-CoV-2 infection rendering markers of mitochondrial functions promising potential biomarkers for SARS-CoV-2 infection risk and severity of outcome. Current knowledge gaps on our understanding of mitochondrial involvement in SARS-CoV-2 infection, life-style and pharmacological strategies to improve mitochondrial integrity and potential reciprocal interactions with chronic and age-related diseases, e.g. Parkinson's Disease, are pointed out.
<small>© 2020 The Author(s). </small> +
Mitochondria are central regulators of cellular metabolism, most known for their role in energy production. They can be "enhanced" by physical activity (including exercise), which increases their integrity, efficiency and dynamic adaptation to stressors, in short "mitochondrial fitness". Mitochondrial fitness is closely associated with cardiorespiratory fitness and physical activity. Given the importance of mitochondria in immune functions, it is thus not surprising that cardiorespiratory fitness is also an integral determinant of the antiviral host defense and vulnerability to infection. Here, we first briefly review the role of physical activity in viral infections. We then summarize mitochondrial functions that are relevant for the antiviral immune response with a particular focus on the current Coronavirus Disease (COVID-19) pandemic and on innate immune function. Finally, the modulation of mitochondrial and cardiorespiratory fitness by physical activity, aging and the chronic diseases that represent the most common comorbidities of COVID-19 is discussed. We conclude that a high mitochondrial - and related cardiorespiratory - fitness should be considered as protective factors for viral infections, including COVID-19. This assumption is corroborated by reduced mitochondrial fitness in many established risk factors of COVID-19, like age, various chronic diseases or obesity. We argue for regular analysis of the cardiorespiratory fitness of COVID-19 patients and the promotion of physical activity - with all its associated health benefits - as preventive measures against viral infection. +
'''''Significance:''''' Oxygen is indispensable for aerobic life, but its utilization exposes cells and tissues to oxidative stress; thus, tight regulation of cellular, tissue, and systemic oxygen concentrations is crucial. Here, we review the current understanding of how the human organism (mal-)adapts to low (hypoxia) and high (hyperoxia) oxygen levels and how these adaptations may be harnessed as therapeutic or performance enhancing strategies at the systemic level.
'''''Recent Advances:''''' Hyperbaric oxygen therapy is already a cornerstone of modern medicine, and the application of mild hypoxia, that is, hypoxia conditioning (HC), to strengthen the resilience of organs or the whole body to severe hypoxic insults is an important preparation for high-altitude sojourns or to protect the cardiovascular system from hypoxic/ischemic damage. Many other applications of adaptations to hypo- and/or hyperoxia are only just emerging. HC-sometimes in combination with hyperoxic interventions-is gaining traction for the treatment of chronic diseases, including numerous neurological disorders, and for performance enhancement.
'''''Critical Issues:''''' The dose- and intensity-dependent effects of varying oxygen concentrations render hypoxia- and/or hyperoxia-based interventions potentially highly beneficial, yet hazardous, although the risks versus benefits are as yet ill-defined.
'''''Future Directions:''''' The field of low and high oxygen conditioning is expanding rapidly, and novel applications are increasingly recognized, for example, the modulation of aging processes, mood disorders, or metabolic diseases. To advance hypoxia/hyperoxia conditioning to clinical applications, more research on the effects of the intensity, duration, and frequency of altered oxygen concentrations, as well as on individual vulnerabilities to such interventions, is paramount. +
[[File:Johannes.jpg|right|150px|Johannes Burtscher]]
Epilepsy is one of the most common neurological diseases featuring a prevalence of 1-2%. A high percentage of patients is refractory to antiepileptic medication, especially in mesial temporal lobe epilepsy (mTLE). Epilepsy is characterized by seizures, in which a lot of glutamate is released leading to excitotoxicity and neuronal loss. Seizure related alterations in neurons are often associated with damaged mitochondria and with impaired functions of distinct complexes of the electron transport chain in human patients and animal models. However, mitochondrial alterations during the development of epilepsy (epileptogenesis) are not well characterized and it is not yet known, whether mitochondrial alterations are cause or consequence of epileptogenesis. Answers to these questions are important to learn more about the neurochemical processes underlying epileptogenesis and to assess implications on the development of antiepilept(ogen)ic medication. Therefore, we are in the process of developing protocols to analyze different mitochondrial parameters using the Oxygraph-2K (Oroboros Instruments, Innsbruck) in hippocampal tissue - which is strongly affected in mTLE - of mice. We apply the kainic acid model of TLE in mice. Injection of kainic acid into the hippocampal CA1 region results in ''Status epilepticus'', a subsequent silent phase and ultimately recurrent seizures. We want to study the activities of electron transport chain (ETC) complexes I, II and IV across different time points of these phases of epileptogenesis.
# [[Kudin 2002 Eur J Neurosci|Kudin AP, Kudina TA, Seyfried J, Vielhaber S, Beck H, Elger CE, Kunz WS (2002) Seizure-dependent modulation of mitochondrial oxidative phosphorylation in rat hippocampus. Eur J Neurosci 15: 1105-1114.]]
# [http://www.ncbi.nlm.nih.gov/pubmed?term=Emerging%20insights%20into%20the%20genesis%20of%20epilepsy.%20Nature McNamara JO (1999) Emerging insights into the genesis of epilepsy. Nature 399: A15-22.]
# [http://www.ncbi.nlm.nih.gov/pubmed?term=Alterations%20in%20cytochrome%20c%20oxidase%20activity%20and%20energy%20metabolites%20in%20response%20to%20kainic%20acid-induced%20status%20epilepticus Milatovic D, Zivin M, Gupta RC, Dettbarn WD (2001) Alterations in cytochrome ''c'' oxidase activity and energy metabolites in response to kainic acid-induced status epilepticus. Brain Res 912: 67-78.]
Mitochondrial dysfunction appears to be a common factor in neurodegenerative diseases. Strikingly, neurodegenerative diseases show regional specificity in vulnerability and follow distinct patterns of neuronal loss. A challenge is to understand, how mitochondrial failure in particular brain regions contributes to specific pathological conditions [1].
High-resolution respirometry revealed significant differences of Complex I- and II- (CI and CII) linked oxidative phosphorylation (OXPHOS) capacity and coupling control between motor cortex, striatum, hippocampus and pons of naïve mice. CI-linked respiration was highest in motor cortex. In contrast, CII-linked capacity was especially important in the striatum. Apparent excess capacities of the electron transfer-pathway (ET-pathway) over OXPHOS also differed between regions. These differences may indicate risk factors for region-specific neuronal vulnerabilities.
In the kainic acid (KA) model of temporal lobe epilepsy in mice, we observed markedly decreased absolute CI- and CII- linked oxygen consumption and also decreased ET-capacity in the injected dorsal hippocampus 2 days after KA. When normalized to ET-capacity, CII-linked respiration was significantly increased compared to controls. 3 weeks after KA-injection, tissue-mass specific CII-linked oxygen consumption reached control levels, but was elevated when normalized to ET-capacity. Tissue-mass specific CI-linked oxygen consumption and ET-capacity remained decreased.
In summary, respirometric OXPHOS analysis allows detailed analysis of mitochondrial function from small amounts of specific brain regions (about 2 mg). It thus enables comparison of different brain tissues implicated in neurodegenerative diseases of the healthy mouse and disease models, while leaving enough material for further studies on the tissues.
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Worldwide, approximately 400 million people are permanently residing at elevations above 1,500 m and more than 100 million tourists visit altitudes up to and higher than 2,500 m annually. Environmental factors, i.e. changing climate conditions with increasing altitude, and life-style factors like diet and physical activity, may have an inherent potential to modify mortality and life expectancy. Available data suggest lower mortality from cardiovascular diseases, stroke, dementia, and certain types of cancer in residents of moderate altitudes [1]. From these data it may also be argued that moderate altitudes, e.g. up to 2,500 m, are more protective than high or even very high altitudes. With regard to mountain tourists, acute exercise at altitude may on the one hand trigger cardiorespiratory adverse events but may on the other hand evoke beneficial effects from hypoxia preconditioning. Such effects result from a single or several short exposures (2 – 10 min) to hypoxia occurring immediately, persisting for a few hours, or delayed after a vulnerable phase of about 24 h, and then persisting for several days. Actually, our data on sudden cardiac deaths in male downhill skiers revealed an episode of early protection for about three hours and a subsequent vulnerable episode during the first skiing day at moderate altitude [2]. Expanding these findings and better understanding of underlying mechanisms would help to optimize the ratio between the health risks and benefits of living and exercising at moderate altitudes. +
Statins are efficient cholesterol-lowering medicines utilized worldwide. However, 10% of patients suffer from adverse effects specially related to skeletal muscle function. Pro- or anti-oxidant effects of statins have been reported. Here we hypothesized that statins induce muscle mitochondrial oxidative stress leading to mitochondrial permeability transition (MPT) which may explain statin muscle toxicity. Thus, our aims were to investigate the effects of statin chronic treatment on muscle mitochondrial respiration rates, MPT and redox state indicators in the context of hypercholesterolemia. For this purpose, we studied muscle biopsies of the hypercholesterolemic LDL receptor knockout mice (LDLr-/-) treated with pravastatin during 3 months. Plantaris, but not soleus muscle of treated mice showed significant inhibition of respiration rates induced by ADP (-14%), oligomycin (-20%) or FCCP (-40%). Inhibitions of respiratory rates were sensitive to EGTA (Ca<sup>2+</sup> chelator), cyclosporin A (MPT inhibitor), ruthenium red (inhibitor of mitochondria Ca<sup>2+</sup> uptake) and coenzyme Q10 (antioxidant), indicating that pravastatin treatment favors Ca<sup>2+</sup> induced MPT. Diet supplementation with creatine (antioxidant) also protected treated mice against pravastatin sensitization to Ca<sup>2+</sup> induced MPT. Among several antioxidant enzymes analyzed, only catalase activity was increased by 30% in plantaris muscle of pravastatin treated mice. Oxidized lipids, but not proteins biomarkers were identified in treated LDLr-/- plantaris muscle. Taken together, the present results suggest that chronic pravastatin administration to a model of familial hypercholesterolemia promotes mitochondrial dysfunctions in plantaris muscle that can be counteracted by antioxidants administered either ''in vitro'' (CoQ10) or ''in vivo'' (creatine). Therefore, we propose that inhibition of muscle mitochondrial respiration by pravastatin leads to an oxidative stress that, in the presence of calcium, opens the permeability transition pore. This mitochondrial oxidative stress caused by statin treatment also signals for cellular antioxidant system responses such as catalase upregulation. These results suggest that the detrimental effects of statins on muscle mitochondria could be prevented by co-administration of a safe antioxidant such as creatine or CoQ10.
Mitochondrion is an important organelle for cells survival. In fact, it is responsible for
many processes such as cellular metabolism, i.e. oxidative phosphorylation for ATP
production, energy homeostasis and regulation of apoptosis and autophagy. Mitochondrion,
due to this role, needs to be “plastic” in order to respond and adapt quickly to any perturbation
and change of conditions in the different tissues of the human body. The induction of
mitochondria biogenesis is required to meet different energetic demands under stress
conditions. Thus, mitochondrial plasticity is the mechanism that controls modification in
conditions of cellular stress or in response to environmental stimuli like exercise, caloric
restriction, cold exposure, oxidative stress, cell division and renewal, and differentiation.
Recently, mitochondrial modulation has become also a topic of interest as a therapeutic target.
The master regulator gene of mitochondrial biogenesis is PGC1α that, through nuclear
transcription factors and subsequent metabolic sensors and other signalling proteins, is capable
to modulate mitochondrial abundance, activity and oxidative phosphorylation as a
consequence of energy homeostasis unbalance. Mitochondrial plasticity during the last few
years was extensively studied in skeletal muscle models, due to its fast adaptation in exercise
and rest condition, but also in cancer cachexia, ageing and heart disease. Also in cancer,
mitochondrial adaptations have become a fundamental topic, in particular to understand the
underling pathogenic mechanism of disease progression, to identify prognostic factors and to
design adjuvant therapies targeting mitochondria.
In this frame, this PhD Thesis investigates the role and adaptations of mitochondria in
different pathophysiological models of skeletal muscle and brain tumors.
The expression of some key proteins of the signalling pathways involved in
mitochondrial biogenesis regulation, such as PGC1α, LKB1-AMPK an energy sensing axis,
Sirt3 a regulator of mitochondrial enzymes functionality, are investigated together with the
OXPHOS complexes, HSP60, CS and TOM20 as mitochondrial mass markers.
The first model is aimed at testing the expression levels of the protein panel in skeletal
muscle biopsies from a cohort of 16 elderly and 7 young people subjected to immobility (bedrest)
causing hypotrophy and subsequent rehabilitation via exercise training. Based on
quantitative immunoblot data, there is a down-regulation of PGC1α, Sirt3 and OXPHOS
complexes II, III and IV occurring during bed-rest with a subsequent up-regulation after
rehabilitation in both groups. AMPK and LKB1 do not change during bed-rest and
rehabilitation in elderly and young subjects suggesting that there is not energetic impairment.
Age related changes in brain cortex NO metabolism were investigated in mitochondria and cytosolic extracts from youth to adulthood. Decreases of 19%, 40% and 71% in NO production were observed in mitochondrial fractions from 3, 7, and 14 months old rats, respectively, as compared with 1-month-old rats. Decreased nNOS protein expression in 14 months old rats was also observed in mitochondria as compared with the nNOS protein expression in 1-month-old rats. Low levels of eNOS protein expression close to the detection limits and no iNOS protein expression were significantly detected in mitochondrial fraction for both groups of age. NO production in the cytosolic extracts also showed a marked decreasing tendency, showing higher levels than those observed in mitochondrial fractions for all groups of age. In the cytosolic extracts, however, the levels were stabilized in adult animals from 7 to 14 months. nNOS protein expression showed a similar age-pattern in cytosolic extracts for both groups of age, while the protein expression pattern for eNOS was higher expressed in adult rats (14 months) than in young animals. As well as in mitochondrial extracts iNOS protein expression was not significantly detected in cytosolic extracts at any age. RT-PCR assays indicated increased levels of nNOS mRNA in 1-month-old rats as compared with 14 months old rats, showing a similar pattern to that one observed for protein nNOS expression. A different aged pattern was observed for eNOS mRNA expression, being lower in 1-month-old rats as compared with 14 months old animals. iNOS mRNA was very low expressed in both groups of age, showing a residual iNOS mRNA that was not significantly detected. State 3 respiration rates were 78% and 85% higher when succinate and malate-glutamate were used as substrates, respectively, in 14 months rats as compared with 1-month-old rats. No changes were observed in state 4 respiration rates. These results could indicate 1 that nNOS and eNOS mRNA and protein expression can be age-dependent, and confirmed the nNOS origin for the mitochondrial NOS. During rat growth, the respiratory function seems to be modulated by NO produced by the different NOS enzymes: nNOS, eNOS and mtNOS present in the cytosol and in the mitochondria.
Ethanol has been known to affect various behavioral parameters in experimental animals, even several hours after ethanol (EtOH) is absent from blood circulation, in the period known as hangover. The aim of this study was to assess the effects of acute ethanol hangover on motor performance in association with the brain cortex energetic metabolism. Evaluation of motor performance and brain cortex mitochondrial function during alcohol hangover was performed in mice 6 hours after a high ethanol dose (hangover onset). Animals were injected i.p. either with saline (control group) or with ethanol (3.8 g/kg BW) (hangover group). Ethanol hangover group showed a bad motor performance compared with control animals (''P'' < .05). Oxygen uptake in brain cortex mitochondria from hangover animals showed a 34% decrease in the respiratory control rate as compared with the control group. Mitochondrial complex activities were decreased being the Complex I-III the less affected by the hangover condition; Complex II-III was markedly decreased by ethanol hangover showing 50% less activity than controls. Complex IV was 42% decreased as compared with control animals. Hydrogen peroxide production was 51% increased in brain cortex mitochondria from the hangover group, as compared with the control animals. Quantification of the mitochondrial transmembrane potential indicated that ethanol injected animals presented 17% less ability to maintain the polarized condition as compared with controls. These results indicate that a clear decrease in proton motive force occurs in brain cortex mitochondria during hangover conditions. We can conclude that a decreased motor performance observed in the hangover group of animals could be associated with brain cortex mitochondrial dysfunction and the resulting impairment of its energetic metabolism. +
Platelets (PLTs) are stored at room temperature (RT) to preserve ''in vivo'' circulation time, but PLT quality is degraded. The PLT storage lesion is mitigated by refrigeration, but questions remain regarding effects of cold storage (4°C) on mitochondrial function. Mitochondrial reactive oxygen species (ROS) generation may adversely affect PLT function and viability during storage, and refrigeration may mitigate these effects.
PLTs were stored under two temperature conditions (RT, 20-24°C; or 4°C, 1-6°C) and four storage durations (baseline [BL] and Days 3, 5, and 7). Mitochondrial respiration and maximal oxygen utilization were assessed with high-resolution respirometry. Mitochondrial ROS generation was assessed using a superoxide stain. Rotational thromboelastometry (ROTEM) was performed at BL and on Day 5 to assess PLT function. Collagen-induced PLT aggregation was measured by impedance aggregometry.
Mitochondrial ROS in 4°C-stored samples were lower compared to RT and retained a greater capacity to generate ROS after activation. Mitochondrial respiration and maximal mitochondrial utilization was conserved in PLTs stored at 4°C. ROTEM data demonstrated that net maximum clot firmness was higher in 4°C samples compared to RT and prevented fibrinolysis. The aggregation response to collagen was preserved in the 4°C samples versus RT-stored PLTs. Aggregation impairment correlated well with attenuated mitochondrial respiration and elevated production of intracellular mitochondrial ROS in the RT PLTs.
Mitochondrial damage and ROS production may contribute to loss of PLT viability during storage, whereas cold storage is known to preserve PLT function. Here we demonstrate that 4°C storage results in less oxidant stress and preserves mitochondrial function and potential compared to RT. +
When exposed to air, the freshwater bivalve, ''Corbicula jluminea'', displayed valve movement behaviors, such as mantle edge exposure, wider gaping “ventilatory” response, and an escape or “burrowing” response. The proportion of the emersion period spent in these behaviors, relative to valve closure, increased with decreasing temperature. Emersion at 35 °C inhibited valve movement behaviors, whereas emersion in a nitrogen atmosphere stimulated ventilatory activity. High rates of aerial oxygen uptake (MO,) were associated with initial valve opening and ventilatory behaviors, and lower MO, occurred during bouts of mantle edge exposure. Heart rate was affected by temperature, but not by mantle edge exposure. Heart rate increased during burrowing and ventilatory behaviors suggesting a hydraulic function for hemolymph. Emersed ''C. jluminea'' had short bursts of heat production followed by longer periods of lower heat flux when measured by direct calorimetry. The mean heat production rate was 1.11 mW/(g dry tissue), significantly higher than the mean value for clams exposed in a nitrogen atmosphere, 0.50 mW/(g dry tissue). On reimmersion, ''C. jluminea'' showed no significant “oxygen debt” until after three days aerial exposure. The bursts of activity, while emersed, may be the result of periodic renewal of oxygen stores followed by immediate oxygen use. +
With brown adipose tissue (BAT) becoming a possible therapeutic target to counteract obesity, the prenatal environment could represent a critical window to modify BAT function and browning of white AT. We investigated if levels of uncoupling protein 1 (UCP1) and UCP1-mediated thermogenesis are altered in offspring exposed to prenatal obesity. Female CD-1 mice were fed a high-fat (HF) or standard-fat (SF) diet for 3 months before breeding. After weaning, all pups were placed on SF. UCP1 mRNA and protein levels were quantified using quantitative real-time PCR and Western blot analysis, respectively, in brown (BAT), subcutaneous (SAT) and visceral (VAT) adipose tissues at 6 months of age. Total and UCP1-dependent mitochondrial respiration were determined by high-resolution respirometry. A Student's t-test and Mann-Whitney test were used (significance: P<0.05). UCP1 mRNA levels were not different between the HF and SF offspring. UCP1 protein levels, total mitochondrial respiration and UCP1-dependent respiration were significantly higher in BAT from HF males (P=0.02, P=0.04, P=0.005, respectively) and females (P=0.01, P=0.04, P=0.02, respectively). In SAT, the UCP1 protein was significantly lower in HF females (P=0.03), and the UCP1-dependent thermogenesis was significantly lower from HF males (P=0.04). In VAT, UCP1 protein levels and UCP1-dependent respiration were significantly lower only in HF females (P=0.03, P=0.04, respectively). There were no differences in total respiration in SAT and VAT. Prenatal exposure to maternal obesity leads to significant increases in UCP1 levels and function in BAT in offspring with little impact on UCP1 levels and function in SAT and VAT. +
C
Chinese Academic Congress on Tumor Biomarkers, Nanjing, China, 2024 +
International Conference on Cell Death in Cancer and Toxicology (CDCT), Lucknow, India, 2018 +
CFAS 2023 Symposium on “Exercise as Medicine”, Copenhagen, Denmark, 2023 +
CRUK Beatson Institute Workshop, Glasgow, United Kingdom, 2022 +
National Academic Conference of the Chinese Society of Biochemistry and Molecular Biology - Biochemistry in Transformation, Changsha, China, 2024 +
'''The Cold Spring Harbor Asia conference on Mitochondria'''. Suzhou, China; 2017 October. +
The second annual conference of theChinese Society for Lipid Metabolism and Bioenergetics, Shanghai, China, 2018 +
The 11<sup>th</sup> National Toxicology Congress of Chinese Society of Toxicology, Suzhou, China, 2024 +
An international symposium on cellular therapy in cardiovascular medicine (CTCM): stem cell opportunity, Ankara, Turkey, 2019 +
Astrocytes are a heterogenous population of macroglial cells spread throughout the central nervous system with diverse functions, expression signatures, and intricate morphologies. Their subcellular compartments contain a distinct range of mitochondria, with functional microdomains exhibiting widespread activities, such as controlling local metabolism and Ca<sup>2+</sup> signaling. Ca<sup>2+</sup> is an ion of utmost importance, both physiologically and pathologically, and participates in critical central nervous system processes, including synaptic plasticity, neuron-astrocyte integration, excitotoxicity, and mitochondrial physiology and metabolism. The mitochondrial Ca<sup>2+</sup> handling system is formed by the mitochondrial Ca<sup>2+</sup> uniporter complex (MCUc), which mediates Ca<sup>2+</sup> influx, and the mitochondrial Na<sup>+</sup>/Ca<sup>2+</sup> exchanger (NCLX), responsible for most mitochondrial Ca<sup>2+</sup> efflux, as well as additional components, including the mitochondrial permeability transition pore (mtPTP). Over the last decades, mitochondrial Ca<sup>2+</sup> handling has been shown to be key for brain homeostasis, acting centrally in physiopathological processes such as astrogliosis, astrocyte-neuron activity integration, energy metabolism control, and neurodegeneration. In this review we discuss the current state of knowledge of the mitochondrial Ca<sup>2+</sup> handling system molecular composition, highlighting its impact on astrocytic homeostasis.<br> +
In this meeting report, particularly addressing the topic of protection of the cardiovascular system from ischemia/reperfusion injury, highlights are presented that relate to conditioning strategies of the heart with respect to molecular mechanisms and outcome in patients' cohorts, the influence of co-morbidities and medications, as well as the contribution of innate immune reactions in cardioprotection. Moreover, developmental or systems biology approaches bear great potential in systematically uncovering unexpected components involved in ischemia-reperfusion injury or heart regeneration. Based on the characterization of particular platelet integrins, mitochondrial redox-linked proteins, or lipid-diol compounds in cardiovascular diseases, their targeting by newly developed theranostics and technologies opens new avenues for diagnosis and therapy of myocardial infarction to improve the patients' outcome. +
Uncoupling proteins (UCPs) are mitochondrial membrane proton transporters that uncouple respiration from oxidative phosphorylation by dissipating the proton gradient across the membrane. Treatment of primary culture of rat preadipocytes for 24 h with 40 microM etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase I (CPT-I), up-regulated UCP-3 mRNA levels (3. 6-fold induction), whereas changes in UCP-2 mRNA levels were not significant. As a consequence of increased UCP-3 expression, a fall in the mitochondrial membrane potential was detected by flow cytometry. Etomoxir treatment modified neither L-CPT-I (liver-type) nor PPARalpha mRNA levels in preadipocytes. In contrast, mRNA expression of acyl-CoA oxidase (ACO), the rate-limiting enzyme of peroxisomal fatty acid beta-oxidation, whose transcription is controlled by PPARalpha, was significantly induced (1.3-fold induction, P = 0.015). These findings suggest that the effects of etomoxir were mediated by PPARalpha. Since it has been reported that the intracellular accumulation of lipids following the inhibition of CPT-I by etomoxir leads to a PPARalpha-mediated metabolic response that increases the expression of genes involved in alternate fatty acid oxidation pathways, these results seem to implicate UCP-3 in this protective metabolic response. It remains to be studied whether reductions in the expression of UCP-3 could compromise this response, giving rise to lipotoxic effects on cells. +
Amyotrophic lateral sclerosis (ALS) is a motor neuron disease with a gender bias towards major prevalence in male individuals. Several data suggest the involvement of oxidative stress and mitochondrial dysfunction in its pathogenesis, though differences between genders have not been evaluated. For this reason, we analysed features of mitochondrial oxidative metabolism, as well as mitochondrial chain complex enzyme activities and protein expression, lipid profile, and protein oxidative stress markers, in the Cu,Zn superoxide dismutase with the G93A mutation (hSOD1-G93A)- transgenic mice and Neuro2A(N2A) cells overexpressing hSOD1-G93A.
Our results show that overexpression of hSOD1-G93A in transgenic mice decreased efficiency of mitochondrial oxidative phosphorylation, located at complex I, revealing a temporal delay in females with respect to males associated with a parallel increase in selected markers of protein oxidative damage. Further, females exhibit a fatty acid profile with higher levels of docosahexaenoic acid at 30 days. Mechanistic studies showed that hSOD1-G93A overexpression in N2A cells reduced complex I function, a defect prevented by 17β-estradiol pretreatment. In conclusion, ALS-associated SOD1 mutation leads to delayed mitochondrial dysfunction in female mice in comparison with males, in part attributable to the higher oestrogen levels of the former. This study is important in the effort to further understanding of whether different degrees of spinal cord mitochondrial dysfunction could be disease modifiers in ALS. +
Docosahexaenoic acid (DHA), a key lipid in nervous system homeostasis, is depleted in the spinal cord of sporadic amyotrophic lateral sclerosis (sALS) patients. However, the basis for such loss was unknown.
DHA synthetic machinery was evaluated in spinal cord samples from ALS patients and controls by immunohistochemistry and western blot. Further, lipid composition was measured in organotypic spinal cord cultures by gas chromatography and liquid chromatography coupled to mass spectrometry. In these samples, mitochondrial respiratory functions were measured by high resolution respirometry. Finally, Neuro2-A and stem cell-derived human neurons were used for evaluating mechanistic relationships between TDP-43 aggregation, oxidative stress and cellular changes in DHA-related proteins.
ALS is associated to changes in the spinal cord distribution of DHA synthesis enzymatic machinery comparing ten ALS cases and eight controls. We found increased levels of desaturases (ca 95% increase, ''p''<0.001), but decreased amounts of DHA-related β-oxidation enzymes in ALS samples (40% decrease, ''p''<0.05). Further, drebrin, a DHA-dependent synaptic protein, is depleted in spinal cord samples from ALS patients (around 40% loss, ''p''<0.05). In contrast, chronic excitotoxicity in spinal cord increases DHA acid amount, with both enhanced concentrations of neuroprotective docosahexaenoic acid-derived resolvin D, and higher lipid peroxidation-derived molecules such as 8-iso-prostaglandin-F2-α (8-iso-PGF2α) levels. Since α-tocopherol improved mitochondrial respiratory function and motor neuron survival in these conditions, it is suggested that oxidative stress could boost motor neuron loss. Cell culture and metabolic flux experiments, showing enhanced expression of desaturases (FADS2) and β-oxidation enzymes after H<sub>2</sub>O<sub>2</sub> challenge suggest that DHA production can be an initial response to oxidative stress, driven by TDP-43 aggregation and drebrin loss. Interestingly, these changes were dependent on cell type used, since human neurons exhibited losses of FADS2 and drebrin after oxidative stress. These features (drebrin loss and FADS2 alterations) were also produced by transfection by aggregation prone C-terminal fragments of TDP-43.
sALS is associated with tissue-specific DHA-dependent synthetic machinery alteration. Furthermore, excitotoxicity synergizes with oxidative stress to increase DHA levels, which could act as a response over stress, involving the expression of DHA synthetic enzymes. Later on, this allostatic overload could exacerbate cell stress by contributing to TDP-43 aggregation. This, at its turn, could blunt this protective response, overall leading to DHA depletion and neuronal dysfunction.
At low oxygen levels, mitochondrial respiration is controlled by the nitric oxide (NO)-cytochrome c oxidase (COX) signaling pathway, since NO is a membrane-permeant second messenger and competitive inhibitor of COX (1). It is now well established that oxygraphs, with Teflon-coated stirrer bars and other plastic materials of high oxygen solubility, yield high rates of oxygen back-diffusion into the chamber when oxygen levels decline, causing artefacts of respiratory measurements. High-resolution respirometry with the Oroboros O2k reduces such back-diffusion by at least an order of magnitude, and incorporates automatic instrumental background corrections, treating the ‘closed’ chamber essentially as an open system with oxygen transport between the aqueous phase and the system boundary (2). For measurement of NO in experimental chambers, however, the same instrumental problem of gas exchange between hydrophobic plastic materials and the aqueous medium has not been addressed, despite the high partition coefficient of NO between aqueous and organic phases (3). To address these problems, we incorporated an NO sensor (ISO-NOP, WPI) into a Hansatech oxygraph chamber and a high-resolution respirometer (O2k), for simultaneous recording of respiration and NO. The NO sensor was calibrated by addition of known concentrations of KNO2 under reducing conditions (KI/H<sub>2</sub>SO<sub>4</sub>) at 37 ºC and the response of the NO sensor in terms of accuracy, stability and reproducibility of the signal was compared between the two chambers. Measurements were taken in 1 ml (Hansatech) or 2 ml (O2k) closed chambers at 37 ºC, using their standard Teflon- or PEEK-coated stirrer bars, respectively. The titanium stopper of the O2k chamber was replaced by a polyvinylidenfluorid (PVDF) stopper, including a second inlet (2 mm diameter) for the NO sensor in addition to the capillary used for extrusion of gas bubbles and titration of chemicals. The PVDF stopper showed identical characteristics to titanium in terms of minimum back-diffusion of oxygen in aerobic-anaerobic transitions, can be cleaned with 70 % and pure ethanol, and offers increased flexibility for accommodation of various additional electrodes for multi-sensor applications. We compared the response of the NO sensor in the determination of the release of NO from a chemical source (DETA-NO) and the endogenous release from controlled intracellular NO production. We determined the inhibition of respiration caused by NO under physiological oxygen concentrations using conventional and high-resolution respirometry (2).
Zebrafish (''Danio rerio'') are widely used animal models. Nevertheless, the mechanisms underlying hypoxia tolerance in this species have remained poorly understood. In the present study, we have determined the effects of hypoxia on blood-O<sub>2</sub> transport properties and mitochondrial respiration rate in permeabilized muscle fibres of adult zebrafish exposed to either 1) a gradual decrease in O<sub>2</sub> levels until fish lost equilibrium (~1 h, acute hypoxia), or 2) severe hypoxia (PO<sub>2</sub> ∼ 15 Torr) for 48 h (prolonged hypoxia). Acute, short-term hypoxia caused an increase in hemoglobin (Hb) O<sub>2</sub> affinity (decrease in P<sub>50</sub>), due to a decrease in erythrocyte ATP after erythrocyte swelling. No changes in iso-Hb expression patterns were observed between hypoxic and normoxic treatments. Prolonged hypoxia elicited additional reponses on O<sub>2</sub> consumption: lactate accumulated in the blood, indicating that zebrafish relied on glycolysis for ATP production, and mitochondrial respiration of skeletal muscle was overall significantly inhibited. In addition, male zebrafish had higher hypoxia tolerance (measured as time to loss of equilibrium) than females. The present study contributes to our understanding of the adaptive mechanisms that allow zebrafish, and by inference other fish species, to cope with low O<sub>2</sub> levels.
<small>Copyright © 2019 Elsevier Inc. All rights reserved.</small> +
Mitochondria are the primary source for energy generation in the cell, which manifests itself in the form of the adenosine triphosphate (ATP). Nicotinamide dinucleotide (NADH) molecules are the first to enter the so-called electron transport chain or ETC of the mitochondria. The ETC represents a chain of reducing agents organized into four major protein-metal complexes (I-IV) that utilize the flow of electrons to drive the production of ATP. An additional integral protein that is related to oxidative phosphorylation is ATP synthase, referred to as complex V. Complex V carries out ATP synthesis as a result of the electron flow through the ETC. The coupling of electron flow from NADH to molecular oxygen to the production of ATP represents a process known as oxidative phosphorylation. In this review, we describe mainly the bioenergetic properties of mitochondria, such as those found in the ETC that may be altered in Alzheimer's disease (AD). Increasing evidence points to several mitochondrial functions that are affected in AD. Furthermore, it is becoming apparent that mitochondria are a potential target for treatment in early-stage AD. With growing interest in the mitochondria as a target for AD, it has been hypothesized that deficit in this organelle may be at the heart of the progression of AD itself. The role of mitochondria in AD may be significant and is emerging as a main area of AD research. +
Mitochondrial DNA is a 16.6 kb double-stranded circular DNA molecule which can be found in various copy numbers in a tissue specific manner. mtDNA encodes for 37 genes whith only 13 of them being polypeptides, all having functions in oxidative phosphorylation. The rest of the proteins which have roles in mitochondria are encoded by nuclear genome. Therefore, controlled expression of both genomes (nuclear and mitochondrial) is a very important process for the well-being of the cell. Furthermore, mitochondria to nucleus cross-talk, also known as retrograde response, is conserved between species and can interact with most of the intracellular signaling pathways and processes [1].
Interaction between the two genomes of the cell is currently studied by depletion of mtDNA and one of the widely used models for studying effects of mtDNA depletion is rho-zero cells. Mammalian derived Rhoº cells were firstly characterized in 1989 by G. Attardi [2]. We have decided to use mtDNA depleted cells in order to study details of nuclear involvement in mitochondrial dysfunction. Here, we use RNA sequencing strategy to compare nuclear gene expression induced by the complete depletion of mtDNA in cell lines of different origin.
Rhoº cells completely lack mtDNA, therefore they can not form respiratory chain complexes. As a result, these cells cannot synthesize ATP by OXPHOS and they are dependent on glycolysis. However, there are more consequences of complete depletion of mtDNA. For example, mitochondria of Rhoº cells are mostly fragmented compared to tubular/filamentous mitochondria observed in wild-type cells. Several microarray studies also showed that cellular transcriptome is changed upon mtDNA depletion. However, there is not a complete consensus on the current published results of other groups. This suggests that the details of retrograde response are complex and still waiting to be uncovered [3]. We find common Rhoº transcriptomic signatures as well as particular modifications associated with the cellular origin.
We are currently performing detailed functional and bioinformatic analysis dissecting basis of common and cell specific responses. We strongly believe the outcome of this study can propose novel ways to treat mitochondrial diseases.
The mitochondrial genome resides in the mitochondrion of nearly all mammalian cells. It is important for energy production as it encodes 13 of the key subunits of the electron transfer chain, which generates the vast majority of cellular ATP through the process of oxidative phosphorylation. As cells establish pluripotency, they regulate their mtDNA copy number so that they possess few copies but sufficient that they can be replicated to match the differentiated cell-specific requirements for ATP derived through oxidative phosphorylation. However, the failure to strictly regulate this process prevents pluripotent cells from differentiating. We describe a series of protocols that analyze mtDNA copy number, DNA methylation within the nuclear-encoded mtDNA-specific polymerase, and gene expression of the other factors that drive replication of the mitochondrial genome. We demonstrate how to measure ATP-generating capacity through oxygen respiratory capacity and total cellular ATP and lactate levels. Finally, we also describe how to detect mtDNA variants in pluripotent and differentiating cells using next-generation sequencing protocols and how the variants can be confirmed by high-resolution melt analysis. +
An increasing number of women fail to achieve pregnancy due to either failed fertilization or embryo arrest during preimplantation development. This often results from decreased oocyte quality. Indeed, reduced mitochondrial DNA copy number (mitochondrial DNA deficiency) may disrupt oocyte quality in some women. To overcome mitochondrial DNA deficiency, whilst maintaining genetic identity, we supplemented pig oocytes selected for mitochondrial DNA deficiency, reduced cytoplasmic maturation and lower developmental competence, with autologous populations of mitochondrial isolate at fertilization. Supplementation increased development to blastocyst, the final stage of preimplantation development, and promoted mitochondrial DNA replication prior to embryonic genome activation in mitochondrial DNA deficient oocytes but not in oocytes with normal levels of mitochondrial DNA. Blastocysts exhibited transcriptome profiles more closely resembling those of blastocysts from developmentally competent oocytes. Furthermore, mitochondrial supplementation reduced gene expression patterns associated with metabolic disorders that were identified in blastocysts from mitochondrial DNA deficient oocytes. These results demonstrate the importance of the oocyte's mitochondrial DNA investment in fertilization outcome and subsequent embryo development to mitochondrial DNA deficient oocytes. +
The natural chemical compounds radicicol, polygodial and ubiquinone-10 (Q10) have previously been identified as inhibitors of metamorphosis in ascidian larvae. Accordingly, they have potential as a specific remedy for the costly problem of fouling ascidians in bivalve aquaculture. In this study, these compounds were screened for their effects on the physiological health of an aquaculture species, the green-lipped mussel, ''Perna canaliculus'' Gmelin, at or above the 99% effective dose (IC(99)) in ascidians. Three physiological biomarkers of mussel health were screened: growth (increases in shell height and wet weight), condition (condition index) and mitochondrial respirational function (Complex I-mediated respiration, Complex II-mediated respiration, maximum uncoupled respiration, leak respiration, respiratory control ratios and phosphorylation system control ratios). While polygodial and Q10 had no effect on mussel growth or the condition index, radicicol retarded growth and decreased the condition index. Mitochondrial respirational function was unaffected by radicicol and polygodial. Conversely, Q10 enhanced Complex I-mediated respiration, highlighting the fundamental role of this compound in the electron transport system. The present study suggests that polygodial and Q10 do not negatively affect the physiological health of ''P. canaliculus'' at the IC(99) in ascidians, while radicicol is toxic. Moreover, Q10 is of benefit in biomedical settings as a cellular antioxidant and therefore may also benefit ''P. canaliculus''. Accordingly, polygodial and Q10 should be progressed to the next stage of testing where possible negative effects on bivalves will be further explored, followed by development of application techniques and testing in a laboratory and aquaculture setting. +
We previously reported that a missense mutation in the mitochondrial fission gene Dynamin-related protein 1 (Drp1) underlies the ''Python'' mouse model of monogenic dilated cardiomyopathy (DCM). The aim of this study was to investigate the consequences of the C452F mutation on Drp1 protein function and to define the cellular sequelae leading to heart failure in the ''Python'' DCM model. We found that the C452F mutation increased Drp1 GTPase activity. The mutation also conferred resistance to oligomer disassembly by guanine nucleotides and high ionic strength solutions. In a mouse embryonic fibroblast (MEF) model, Drp1 C452F cells exhibited abnormal mitochondrial morphology and defective mitophagy. Mitochondria in C452F MEFs were depolarized and had reduced calcium uptake, with impaired ATP production by oxidative phosphorylation. In the Python heart, we found a corresponding progressive decline in oxidative phosphorylation with age, and activation of sterile inflammation. As a corollary, enhancing autophagy by exposure to a prolonged low protein diet improved cardiac function in ''Python'' mice. In conclusion, failure of Drp1 disassembly impairs mitophagy, leading to a downstream cascade of mitochondrial depolarization, aberrant calcium handling, impaired ATP synthesis and activation of sterile myocardial inflammation resulting in heart failure. +
The aim of study was to estimate the effect of carnitine supplementation on lipid disorders and peripheral tissue insulin sensitivity in a non-obese animal model of insulin resistance, hereditary hypertriglyceridemic rats (HHTg). Male HHTg rats were fed a standard diet, and half of them were administered carnitine 500mg/kg b.wt. daily for 8 weeks. Rats of the original Wistar strain were used for comparison. HHTg rats exhibited an increased urinary excretion of free carnitine together with reduced carnitine content in the liver and blood. Carnitine supplementation compensated for this shortage and promoted urinary excretion of acetylcarnitine without any signs of (acyl)carnitine accumulation in skeletal muscle. Carnitine-treated HHTg rats exhibited lower weight gain, reduction of liver steatosis, lower fasting triglyceridemia and greater reduction of free fatty acid serum content after glucose load, compared with their untreated littermates. Carnitine treatment was associated with increased mitochondrial biogenesis and oxidative capacity for fatty acids, amelioration of oxidative stress, and restored substrate switching in the liver. In skeletal muscle (diaphragm) carnitine supplementation was associated with significantly higher palmitate oxidation and more favorable complete to incomplete oxidation products ratio. It further enhanced insulin sensitivity ex vivo. No effects on whole body glucose tolerance were observed. Our data suggest that some metabolic syndrome related disorders, particularly fatty acid oxidation, steatosis, and oxidative stress in the liver, could be attenuated by carnitine supplementation. The effect of carnitine could be explained, at least partly, by enhanced substrate oxidation and increased fatty acid transport from tissues in the form of short-chain acylcarnitines. +
Nonalcoholic fatty liver disease is associated with chronic oxidative stress. In our study, we explored the antioxidant effect of antidiabetic metformin on chronic [high-fat diet (HFD)-induced] and acute oxidative stress induced by short-term warm partial ischemia-reperfusion (I/R) or on a combination of both in the liver. Wistar rats were fed a standard diet (SD) or HFD for 10 wk, half of them being administered metformin (150 mg·kg body wt(-1)·day(-1)). Metformin treatment prevented acute stress-induced necroinflammatory reaction, reduced alanine aminotransferase and aspartate aminotransferase serum activity, and diminished lipoperoxidation. The effect was more pronounced in the HFD than in the SD group. The metformin-treated groups exhibited less severe mitochondrial damage (markers: cytochrome c release, citrate synthase activity, mtDNA copy number, mitochondrial respiration) and apoptosis (caspase 9 and caspase 3 activation). Metformin-treated HFD-fed rats subjected to I/R exhibited increased antioxidant enzyme activity as well as attenuated mitochondrial respiratory capacity and ATP resynthesis. The exposure to I/R significantly increased NADH- and succinate-related reactive oxygen species (ROS) mitochondrial production in vitro. The effect of I/R was significantly alleviated by previous metformin treatment. Metformin downregulated the I/R-induced expression of proinflammatory (TNF-α, TLR4, IL-1β, Ccr2) and infiltrating monocyte (Ly6c) and macrophage (CD11b) markers. Our data indicate that metformin reduces mitochondrial performance but concomitantly protects the liver from I/R-induced injury. We propose that the beneficial effect of metformin action is based on a combination of three contributory mechanisms: increased antioxidant enzyme activity, lower mitochondrial ROS production, and reduction of postischemic inflammation. +
Although it is well known that selective intra-arterial cooling (SI-AC) elicits cerebral protection against ischemia/reperfusion (I/R) injury, the underlying mechanism remains unclear. This study aimed to determine whether SI-AC can protect against cerebral I/R injury by inhibiting oxidative stress and mitochondrial dysfunction through regulation of Sirt3 deSUMOylation via SENP1.
All mice were subjected to 2 h of cerebral ischemia followed by 24 h of reperfusion. SI-AC treatment was performed by infusion with cold saline (10 °C, 20 mL/kg) for 15 min through a microcatheter placed in the internal carotid artery immediately before reperfusion. The infarct volume, survival rate, neurological deficit scores, behavioral parameters, histopathology findings, and apoptosis were assessed. HT22 cells were subjected to 2 h of oxygen and sugar deprivation (OGD) and 22 h of reoxygenation. HA-SUMO1, Flag-Sirt3, a Sirt3 mutation plasmid (Flag-Sirt3 K288R), His-SENP1, and SENP1 small interfering RNA were transfected into HT22 cells 48 h before OGD. Apoptosis-related proteins were analyzed by western blotting. SUMOylation of Sirt3, acetylation of cyclooxygenase 1 (COX1), superoxide dismutase 2 (SOD2), and isocitrate dehydrogenase 2 (IDH2), the activities of COX1, SOD2, and IDH2, oxidative stress, and mitochondrial dysfunction were evaluated.
Compared with the I/R group, SI-AC decreased cerebral infarct volume and neurological deficit scores and increased motor coordination, exploratory behavior, and memory. Hematoxylin and eosin and Nissl staining showed that SI-CA decreased karyopyknosis, nuclear fragmentation, and nucleolysis, increased neuron density, and decreased the cell apoptosis rate. In addition, Sirt3 was revealed as a target protein of SUMO1. SI-AC attenuated cerebral I/R injury through Sirt3 deSUMOylation via SENP1.
SENP1-mediated deSUMOylation of Sirt3 plays an essential role in SI-AC-induced cerebral protection against I/R injury. Our findings provide a promising therapeutic approach for treatment of acute cerebral I/R injury.
Mitochondria are structurally and biochemically diverse, even within a single type of cell. Protein complexes localized to the inner mitochondrial membrane synthesize ATP by coupling electron transport and oxidative phosphorylation. The organelles produce reactive oxygen species (ROS) from mitochondrial oxygen and ROS can, in turn, alter the function and expression of proteins used for aerobic respiration by post-translational and transcriptional regulation. Recent Advances: New interest is emerging not only into the roles of mitochondria in disease development and progression but also as a target for environmental toxicants.
Dysregulation of respiration has been linked to cell death and is a major contributor to acute neuronal trauma, peripheral diseases, as well as chronic neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease.
Here, we discuss the mechanisms underlying the sensitivity of the mitochondrial respiratory complexes to redox modulation, as well as examine the effects of environmental contaminants that have well-characterized mitochondrial toxicity. The contaminants discussed in this review are some of the most prevalent and potent environmental contaminants that have been linked to neurological dysfunction, altered cellular respiration, and oxidation. +
High Intensity Interval Training (HIT) has been shown to improve cardiovascular fitness and seems to induce beneficial modifications of cardiometabolic risk factors in healthy subjects and patients. Less is known about the efficacy of HIT applied to healthy older adults, and the adaptations induced at the central and periprheral level.
This study tested the hypothesis that 8 weeks of HIT can induce significant improvements of cardiovascular fitness, exercise capacity and of selected cardiometabolic risk factors in healthy older adults. In 12 healthy elderly male volunteers, we measured V’O2max, gas exchange threshold (GET ), respiratory compensation point (RCP), resting mean, systolic and diastolic blood pressures (MBP, SDP, DBP), fasting blood glucose concentration (GLU), total cholesterol/HDL ratio (CHOLtot/HDL), % body fat (BF) and waist circumference (WC) before (PRE) and after (POST) an 8-week of HIT. The training program consisted of 7 bouts of 2-min near-maximal cycling (i.e. 85-90%V’O2max) interspersed with 2 minutes of recovery performed 3 times a week.
Absolute and relative V’O significantly increased by 5.4 % and 11.7 % respectively. V’O at GET and RCP increased by 7.2 % respectively. MBP and SDP significantly decreased by 7 % and 9 % respectively. GLU was diminished by 7 % and TC/HDL decreased by 5 %. BF and WC decreased by 4 % and 1.4 % respectively. Surprisingly, analysis of the quadriceps muscle showed that both CSA and muscle volume significantly increased (5.0 and 5.4% respectively).
Thus we can conclude that 8 weeks of HIT promote significant changes of maximal aerobic power and exercise resistance in healthy, male, elderly subjects. In addition, they induce significant improvements of some selected cardiometabolic risk factors.
Further studies are now needed to investigate how peripheral modifications in the skeletal muscle tissue may contribute to the described adaptations. Changes in the mass and/or in the function of the mitochondrial network will be evaluated on skeletal muscle biopsies in association to the assessment of muscle fiber type expression and capillary density.
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Measures of physical function are good predictors of morbidity and mortality. Aging is associated with a progressive decline of physical function often with health consequences. In healthy aging the main limiting factors are the progressive loss of muscle mass (sarcopenia), immunosenescence and cognitive impairment. The biological mechanisms underlying this decline are not yet understood. It has been proposed that mitochondrial dys-function is at the basis of the aging process. Frailty is a condition that is also associated with aging, however the information linking aging, frailty and mitochondrial bioenergetics are still lacking.
Recent studies showed that it is possible to measure of mitochondrial function in human blood cells and that this can be associates to the level of fitness[1]. We have recently shown that aging results also in the down-regulation of a set of genes associated with mitochondrial electron transport system in blood cells [2].
The aim of our study is to investigate the reciprocal connections existing between human aging, frailty, progressive inactivity and mitochondrial function. We are using an interdisciplinary approach evaluating both whole body physiological parameters and mitochondrial respiratory capacity in PBMCs in two groups of men and women of different ages (adult - 40 years old ; elderly 69 years old). Evaluations consist in a battery of test to assess physical performance (SPPB test), cardio-respiratory capacity (''V''<sub>O2peak</sub>) measured with a ramp test on the cycle-ergometer and mitochondrial function with ''in vivo'' high-resolution respiration (HRR) on PBMCs. Preliminary data collected show that there is a progressive decay of mitochondrial coupling efficiency (1-L/E) is negatively correlated to age for men (p 0.030; r -0.561) and it is particularly evident in elderly men when compared to the group of adults or elderly women (p 0.044; p 0.026 respectively). The concentration of lactate accumulated at the peak is negatively correlated to mitochondrial ROUTINE metabolism (p 0.024; r -0.786). Furthermore in these groups of men and women, as expected, “age” negatively correlates also with functional parameters associated to the cardio-respiratory function and to mechanical power (''V''<sub>O2peak</sub> and Watt<sub>peak</sub>).
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MITOEAGLE]] Aging is associated to the progressive decline of physiological functions related to physical activity, cardio-respiratory function, cognitive capacities and immunity. All this is accompanied by reduced mobility, or ''viceversa'', and often leads to changes in the quality of life and health. Frailty is a further condition that is also associated with aging, however the information linking aging, frailty and mitochondrial bioenergetics are still unclear. It has been proposed that progressive mitochondrial dys-function with increased accumulation of oxidative stress could be at the basis of the aging process [1]. Comparing two groups of healthy men of different age, we have shown that healthy aging is associated with the down-regulation of a set of genes associated with the mitochondrial electron transport system in blood cells [2]. Recent studies indicate that the measurement of mitochondrial function in human blood cells is associated with the level of fitness [3].
To untangle the links existing between human aging, frailty, physical activity and mitochondrial function we are evaluating whole body physiological parameters and mitochondrial respiratory capacity in blood cells (PBMCs) in three groups of 10 volunteers (men and women) of different ages (40, 70 and 80 years old). We collected data of physical performance (SPPB test), cardio-respiratory capacity (''V''<sub>O2peak</sub>; HR), strength of upper and lower limbs, and mitochondrial function with ''in vivo'' high-resolution respirometry (HRR) on PBMCs. Data collected show the well known age-relate decline of ''V''<sub>O2peak</sub> (p=0.001; r=-0.632), but also strength and physical performance parameters are negatively associated with age.
The values of mitochondrial respiration (FCR) related to ETS supported by complex II (ETS_cII) are significantly reduced in the most aged group (80s). ETS coupling efficiency (1-L/E) is significantly lower (-13%) in men versus women in the 70s groups (p 0.03). ETS_cII resulted also to be negatively associated with the % of body fat (%BFAT) (p 0.038; r -0.417). The values of leak respiration following cell permeabilization resulted positively associated with ''V''<sub>O2peak</sub>.
In conclusion the results reported are still preliminary, but it is interesting to observe that PBMCs mitochondrial function and efficiency is modulated by aging and gender. Although mitochondrial oxygen consumption is evaluated in circulating PBMCs, our data support the idea that metabolic adaptations occurring in blood cells are reflecting systemic metabolic properties, and the association between the ETS_cII and LEAK dissipative respiration with measures of body composition and whole body oxygen consumption point in this direction.
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In our laboratory we are mainly focused on PBMCs blood cells. One of the main issues of working
with PBMCs is the quality of the final batch of cells. In particular the contamination of platelets in the final sample could affect the respiratory profile of the sample when evaluating mitochondrial
function. We have monitored all the steps of the PBMCs purification procedure with a Sysmex
XN-1000 hematology analyzer. This instruments allows a high degree of accuracy of platelets
counts, also using a specific fluorescent reagent [1]. The data collected show
that we achieve 97% depletion of platelets after two washing steps in RPMI, and we collect the
30% of the initial amount of PBMCs.
One of the main aim of [[WG4]] is also protocols in the harmonization to SUIT reference protocols
(RP1 and RP2). Since we are not currently running exactly the suggested RP protocols, we tried to verify if at least some common functional steps of the protocol are comparable to RP1 and RP2 protocols. Preliminary data show that at least in some common steps there is no significant
difference between the old protocol and the suggested RP1 and protocol in PBMCs human cells,
further experiments are needed to evaluate RP2. +
The amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motoneurons death. Mutations in the superoxide dismutase 1 (SOD1) protein have been identified to be related to the disease. Beyond the different altered pathways, the mitochondrial dysfunction is one of the major features that leads to the selective death of motoneurons in ALS. The NSC-34 cell line, overexpressing human SOD1(G93A) mutant protein [NSC-34(G93A)], is considered an optimal ''in vitro'' model to study ALS. Here we investigated the energy metabolism in NSC-34(G93A) cells and in particular the effect of the mutated SOD1(G93A) protein on the mitochondrial respiratory capacity (complexes I-IV) by high resolution respirometry (HRR) and cytofluorimetry. We demonstrated that NSC-34(G93A) cells show a reduced mitochondrial oxidative capacity. In particular, we found significant impairment of the complex I-linked oxidative phosphorylation, reduced efficiency of the electron transfer system (ETS) associated with a higher rate of dissipative respiration, and a lower membrane potential. In order to rescue the effect of the mutated SOD1 gene on mitochondria impairment, we evaluated the efficacy of the exosomes, isolated from adipose-derived stem cells, administrated on the NSC-34(G93A) cells. These data show that ASCs-exosomes are able to restore complex I activity, coupling efficiency and mitochondrial membrane potential. Our results improve the knowledge about mitochondrial bioenergetic defects directly associated with the SOD1(G93A) mutation, and prove the efficacy of adipose-derived stem cells exosomes to rescue the function of mitochondria, indicating that these vesicles could represent a valuable approach to target mitochondrial dysfunction in ALS.
<small>Copyright © 2019 Calabria, Scambi, Bonafede, Schiaffino, Peroni, Potrich, Capelli, Schena and Mariotti.</small> +
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The sensitivity of high-resolution respirometry (HRR) has made it possible to carry out reliable measurements of oxygen consumption in blood cells, opening up new scenarios for monitoring mitochondrial function / dysfunction in health conditions, but also to study the pathophysiology of many diseases or the effects of interventions. Essential elements of this approach are (''1'') the validation and harmonization of preparatory procedures, from blood collection to cell purification, (''2'') and standardization of SUIT protocols applied in the respirometric analysis. We present the procedures adopted for peripheral blood mononuclear cells (PBMC) [1,2] and platelets (PLT) [3, 4], and discuss them in relation to procedures used by other laboratories participating in WG4. We found that comparable results can be obtained despite the use of different approaches in the isolation of cells. +
Type 2 diabetes (T2D) is a multisystem disease that is the subject of many studies, but the earliest cause of the disease has yet to be elucidated. Mitochondrial impairment has been associated with diabetes in several tissues. To extend the association between T2D and mitochondrial impairment to blood cells, we investigated T2D-related changes in peripheral mononucleated blood cells’ (PBMCs) mitochondrial function in two groups of women (CTRL vs. T2D; mean age: 54.1 ± 3.8 vs. 60.9 ± 4.8; mean BMI 25.6 ± 5.2 vs. 30.0 ± 5), together with a panel of blood biomarkers, anthropometric measurements and physiological parameters (VO<sub>2max</sub> and strength tests). Dual-energy X-ray absorptiometry (DXA) scan analysis, cardio-pulmonary exercise test and blood biomarkers confirmed hallmarks of diabetes in the T2D group. Mitochondrial function assays performed with high resolution respirometry highlighted a significant reduction of mitochondrial respiration in the ADP-stimulated state (OXPHOS; −30%, p = 0.006) and maximal non-coupled respiration (ET; −30%, p = 0.004) in PBMCs samples from the T2D group. The total glutathione antioxidant pool (GSHt) was significantly reduced (−38%: p = 0.04) in plasma samples from the T2D group. The fraction of glycated hemoglobin (Hb1Ac) was positively associated with markers of inflammation (C-reactive protein-CRP r = 0.618; p = 0.006) and of dyslipidemia (triglycerides-TG r = 0.815; p < 0.0001). The same marker (Hb1Ac) was negatively associated with mitochondrial activity levels (OXPHOS r = −0.502; p = 0.034; ET r = −0.529; p = 0.024). The results obtained in overweight postmenopausal women from analysis of PBMCs mitochondrial respiration and their association with anthropometric and physiological parameters indicate that PBMC could represent a reliable model for studying T2D-related metabolic impairment and could be useful for testing the effectiveness of interventions targeting mitochondria. +
Acute hypoxia (AH) reduces maximal O<sub>2</sub> consumption (''V''<sub>O<sub>2</sub>max</sub>), but after acclimatization, and despite increases in both hemoglobin concentration and arterial O<sub>2</sub> saturation that can normalize arterial O<sub>2</sub> concentration ([O<sub>2</sub>]), ''V''<sub>O<sub>2</sub>max</sub> remains low. To determine why, seven lowlanders were studied at ''V''<sub>O<sub>2</sub>max</sub> (cycle ergometry) at sea level (SL), after 9-10 wk at 5260 m [chronic hypoxia (CH)], and 6 mo later at SL in AH (''F''<sub>i</sub>O<sub>2</sub> = 0.105) equivalent to 5260 m. Pulmonary and leg indexes of O<sub>2</sub> transport were measured in each condition. Both cardiac output and leg blood flow were reduced by approximately 15 % in both AH and CH (''P'' < 0.05). At maximal exercise, arterial [O<sub>2</sub>] in AH was 31 % lower than at SL (''P'' < 0.05), whereas in CH it was the same as at SL due to both polycythemia and hyperventilation. O<sub>2</sub> extraction by the legs, however, remained at SL values in both AH and CH. Although at both SL and in AH, 76 % of the cardiac output perfused the legs, in CH the legs received only 67 %. Pulmonary ''V''<sub>O<sub>2</sub>max</sub> (4.1 +/- 0.3 L/min at SL) fell to 2.2 +/- 0.1 L/min in AH (''P'' < 0.05) and was only 2.4 +/- 0.2 L/min in CH (''P'' < 0.05). These data suggest that the failure to recover ''V''<sub>O<sub>2</sub>max</sub> after acclimatization despite normalization of arterial [O<sub>2</sub>] is explained by two circulatory effects of altitude: 1) failure of cardiac output to normalize and 2) preferential redistribution of cardiac output to nonexercising tissues. Oxygen transport from blood to muscle mitochondria, on the other hand, appears unaffected by CH. +
Sensory neurons have the capacity to produce, release, and respond to acetylcholine (ACh), but the functional role of cholinergic systems in adult mammalian peripheral sensory nerves has not been established. Here, we have reported that neurite outgrowth from adult sensory neurons that were maintained under subsaturating neurotrophic factor conditions operates under cholinergic constraint that is mediated by muscarinic receptor-dependent regulation of mitochondrial function via AMPK. Sensory neurons from mice lacking the muscarinic ACh type 1 receptor (M1R) exhibited enhanced neurite outgrowth, confirming the role of M1R in tonic suppression of axonal plasticity. M1R-deficient mice made diabetic with streptozotocin were protected from physiological and structural indices of sensory neuropathy. Pharmacological blockade of M1R using specific or selective antagonists, pirenzepine, VU0255035, or muscarinic toxin 7 (MT7) activated AMPK and overcame diabetes-induced mitochondrial dysfunction ''in vitro'' and ''in vivo''. These antimuscarinic drugs prevented or reversed indices of peripheral neuropathy, such as depletion of sensory nerve terminals, thermal hypoalgesia, and nerve conduction slowing in diverse rodent models of diabetes. Pirenzepine and MT7 also prevented peripheral neuropathy induced by the chemotherapeutic agents dichloroacetate and paclitaxel or HIV envelope protein gp120. As a variety of antimuscarinic drugs are approved for clinical use against other conditions, prompt translation of this therapeutic approach to clinical trials is feasible. +
The rediscovery of brown adipose tissue (BAT) in humans and its capacity to oxidize fat and dissipate energy as heat has put the spotlight on its potential as a therapeutic target in the treatment of several metabolic conditions including obesity and diabetes. To date the measurement of bioenergetics parameters has required the use of cultured cells or extracted mitochondria with the corresponding loss of information in the tissue context. Herein, we present a method to quantify mitochondrial bioenergetics directly in BAT. Based on XF Seahorse Technology, we assessed the appropriate weight of the explants, the exact concentration of each inhibitor in the reaction, and the specific incubation time to optimize bioenergetics measurements. Our results show that BAT basal oxygen consumption is mostly due to proton leak. In addition, BAT presents higher basal oxygen consumption than white adipose tissue and a positive response to b-adrenergic stimulation. Considering the whole tissue and not just subcellular populations is a direct approach that provides a realistic view of physiological respiration. In addition, it can be adapted to analyze the effect of potential activators of thermogenesis, or to assess the use of fatty acids or glucose as a source of energy. +
Due to the dynamic nature of organelle contact sites, it is difficult to monitor their behavior in living cells in a simple, one-step manner. Organelle proximity represents an important mechanism employed by the cells to ensure the tight coordination of several cellular activities. Indeed, a network of contact sites between the membranes of different organelles guarantee their mutual communication by creating microdomains that favor both signaling and the passage of lipids and ions. Because of their central role in the most essential cellular activities, the contact sites between mitochondria and the endoplasmic reticulum (ER) are, so far, the best characterized. However, the lack of a general and reliable method to properly monitor their proximity often prevented the possibility to draw clear conclusions. Here we describe a split-GFP based method for quick and reliable monitoring of organelle proximity adapted for ER-mitochondria contact sites evaluation. It is based on the ability of two non-fluorescent portions (the GFP 1-10 moiety and the GFP β-strand 11) of the extremely well folded superfolder GFP variant to restore a fully fluorescent GFP upon self-assembly.
To this aim we targeted the 16-amino-acid GFP β-strand 11 spaced by a 13 amino-acid (Sac Short β-11) and a 130 amino-acid long (Sac Long β-11) linker, to the cytosolic face of the ER and the GFP 1-10 detector fragment the outer mitochondrial membrane (OMM) in order to follow the appearance of tight (<15 nm) as well as loose contacts (<100 nm). We reasoned that upon co-expression in mammalian cells this one-step imaging technique would result in fluorescence emission specifically in the sites of apposition of the two organelles lying in a range between 15 and 100 nm. +
A dedicated website for sharing biology papers before peer review leaves journals divided. What are biologists so afraid of? Physicists, mathematicians and social scientists routinely post their research to preprint servers such as arXiv.org before publication, yet few life scientists follow suit. A website that goes live this week is hoping to change that. The site, bioRχiv.org, launched by Cold Spring Harbor Laboratory Press in New York, bills itself as “the preprint server for biology”. It will operate similarly to arXiv, with scientists depositing papers as soon as they are ready to share them, weeks or months before formal publication. +
Mitochondrial membrane proteins with internal targeting signals are inserted into the inner membrane by the carrier translocase (TIM22 complex). For this, precursors have to be initially directed from the TOM complex in the outer mitochondrial membrane across the intermembrane space towards the TIM22 complex. How these two translocation processes are topologically coordinated is still unresolved. Using proteomic approaches, we find that the human TIM22 complex associates with the Mitochondrial Contact site and Cristae Organizing System (MICOS) complex. This association does not appear to be conserved in yeast, whereby the yeast MICOS complex instead interacts with the presequence translocase. Using a yeast ''mic10''Δ strain and a HEK293T MIC10 knockout cell line, we characterize the role of MICOS for protein import into the mitochondrial inner membrane and matrix. We find that a physiological cristae organization promotes efficient import via the presequence pathway in yeast, while in human mitochondria, the MICOS complex is dispensable for protein import along the presequence pathway. However, in human mitochondria the MICOS complex is required for the efficient import of carrier proteins into the mitochondrial inner membrane. Our analyses suggest that in human mitochondria, positioning of the carrier translocase at the crista junction, and potentially in vicinity to the TOM complex, is required for efficient transport into the inner membrane.
<small>Copyright © 2019. Published by Elsevier Ltd.</small> +
In humans, liver phenylalanine hydroxylase (PAH) has an established catabolic function, and mutations in PAH cause phenylketonuria, a genetic disease characterized by neurological damage, if not treated. To obtain novel evolutionary insights and information on molecular mechanisms operating in phenylketonuria, we investigated PAH in the nematode ''Caenorhabditis elegans'' (cePAH), where the enzyme is coded by the ''pah-1'' gene, expressed in the hypodermis. CePAH presents similar molecular and kinetic properties to human PAH [S(0.5)(L-Phe) approximately 150 microM; ''K''(m) for tetrahydrobiopterin (BH(4)) approximately 35 microM and comparable ''V''<sub>max</sub>], but cePAH is devoid of positive cooperativity for L-Phe, an important regulatory mechanism of mammalian PAH that protects the nervous system from excess L-Phe. Pah-1 knockout worms show no obvious neurological defects, but in combination with a second cuticle synthesis mutation, they display serious cuticle abnormalities. We found that pah-1 knockouts lack a yellow-orange pigment in the cuticle, identified as melanin by spectroscopic techniques, and which is detected in ''C. elegans'' for the first time. Pah-1 mutants show stimulation of superoxide dismutase activity, suggesting that cuticle melanin functions as oxygen radical scavenger. Our results uncover both an important anabolic function of PAH and the change in regulation of the enzyme along evolution. +
[[File:Enrico Calzia.JPG|right|300px|Enrico Calzia]]
Even in higher species sulfide is quickly metabolised and thus degraded by an ancient metabolic pathway linked to the mitochondrial respiratory chain [1]. Since this metabolic process is strictly aerobic, the efficiency of sulfide metabolism is expected to decrease at low oxygen. This mechanism may explain how sulfide acts as an oxygen sensor within cells [2]. Therefore, in a series of preliminary experiments we quantified the sulfide metabolism in a stable cell line derived from alveolar macrophages (AMJ2-C11), which previously proved to efficiently metabolise sulfide under aerobic conditions [3], at O<sub>2</sub> partial pressures approaching hypoxia from 0.8 kPa down to 0.4 and 0.1 kPa.
'''Methods''': All measurements were conducted using an Oxygraph-2k together with a TIP2k® titration pump (Oroboros Instruments, Austria). For each O<sub>2</sub>-partial pressure we performed 6 – 8 separate experiments. Before performing the experiments we recorded several transitions to anoxia in order to quantify the un-inhibited respiratory capacity close to anoxia [4] under the O<sub>2</sub>-partial pressures of 0.1, 0.4, and 0.8 KPa respectively. The inhibition of mitochondrial respiration was quantified in terms of the total amount of sulfide required to reduce the routine oxygen flux (J<sub>O2</sub>) to 50% by means of a continuous sulfide injection at a rate of 10 nM/s. A second titration pump was used to simultaneously maintain the oxygen concentration rate at the predefined level adopting a previously described technique [5].
'''Results''': Figure 1 shows representative records of 2 anoxic transition experiments in AMJ2-C11 cells; the JO<sub>2</sub> was 74%, 92%, and 96% of maximum J<sub>O2</sub> [JO<sub>2</sub><sub>max</sub>] at 0.1 kPa, 0.4 kPa, and 0.8 kPa. The total amount of sulfide required for achieving a 50%-inhibition of mitochondrilal respiration was about 10% at 0.1 kPa when compared to that needed at 0.8 kPa. (0.2 ± 0.03 µmol at 0.1 kPa vs. 1.30 ± 0.4 µmol at 0.4 kPa, and 2.2 ± 1.1 µmol at 0.8 kPa; see Figure 2).
'''Discussion''': These results confirm a much less efficient degradation of sulfide at the lower oxygen level suggesting potential interactions between sulfide and cell metabolism when approaching the anoxic condition. In theory, sulfide concentration should increase and thus potentially inhibit mitochondrial respiration at low oxygen, provided that the production rate is sufficiently high and remains constant under these conditions, and that the excess sulfide is not chemically bound by proteins (''sulfhydration''). On the other hand, the inhibition of mitochondrial respiration should in turn decrease the oxygen consumption of the cell, thus potentially decelerating or even impeding a further decrease of the oxygen concentration. So far, however, whether and how sulfide interacts with metabolism at low oxygen remains the subject of future investigations.
# [http://www.ncbi.nlm.nih.gov/pubmed?term=Bouillaud%20F%20Antioxid%20Redox%20Signal%202011 Bouillaud F, Blachier F (2011) Mitochondria and sulfide: a very old story of poisoning, feeding, and signaling? Antioxid Redox Signal 15: 379-391.]
# [http://www.ncbi.nlm.nih.gov/pubmed?term=Olson%20KR.%20J%20Comp%20Physiol%20B%202012 Olson KR (2012) Mitochondrial adaptations to utilize hydrogen sulfide for energy and signaling. J Comp Physiol B 182: 881-897.]
# [http://www.ncbi.nlm.nih.gov/pubmed?term=Groeger%20M%20et%20al.%3A%20Shock.%202012%3B%2038%3A%20367%20-%2074 Groeger M, Matallo J, McCook O, Wagner F, Wachter U, Bastian O, Gierer S, Reich V, Stahl B, Huber-Lang M, Szabó C, Georgieff M, Radermacher P, Calzia E, Wagner K (2012) Temperature and cell-type dependency of sulfide effects on mitochondrial respiration. Shock 38: 367-374.]
Several investigations demonstrated that a hibernation-like metabolic state characterized by reduced energy expenditure and hypothermia can be induced in mice that have inhaled 80-100 ppm of H2S [1,2,3]. These observations suggest the exciting prospect of pharmacologically controlling energy expenditure in severe trauma and disease states by protecting against transitory hypoxia. However, equivocal data available from studies on large animals still raise serious doubts on the genuine effects of H2S on cell metabolism and mitochondrial function. In fact, data from sheep or swine failed to show any metabolic effect [4,5], and the effects observed injecting the intravenous sulfide donor Na2S in pigs are still controversial [6,7]. Obviously, body temperature is a major confounder in most animal studies on sulfide, as it can be easily modified, deliberately or accidentally, in murine experiments, but is hard to control in larger species. Therefore, it is still not clear which impact non-toxic doses of sulfide may have in-vivo on metabolic or mitochondrial functions beyond eventual temperature related effects.
In the last few years we performed experiments on the effects of sulfide in anesthetized and mechanically ventilated mice using a setting closely resembling an intensive care unit [8]. A closed loop control of body temperature allowed to efficiently separate genuine effects of sulfide and body temperature. Our data obtained ex-vivo by measuring mitochondrial respiration in small liver biopsies by means of high-resolution respirometry performed with the O2k-Oxygraph (Oroboros Instruments, Innsbruck, AT) suggest that low (non-toxic) sulfide concentrations per se do not suppress mitochondrial respiratory activity (Figure 1, A and B). Nevertheless, sulfide normalised the amount of LEAK-respiration, which was increased in septic animals under normothermia, an effect which was not related to any change in body temperature (Figure 1, C and D).
In conclusion, the main premise for studying mitochondrial effects of sulfide in animal models should be an efficient control of body temperature in order to clearly separate the effects of both factors. Technically, this can be done most easily in small animals like mice. Of course, the question remains to which extent data obtained in mice may be translated to larger mammalian species. Further preliminary data obtained on tissue biopsies from mice and pigs, however, indicate that sulfide is processed in a very similar way at the cellular level in different mammalian species, thus suggesting that the effects of sulfide at the mitochondrial level may be comparable between the species.
Mitochondrial respiration is assumed to be severely affected by the presence of mutant huntingtin in HD patients. However, mitochondrial function remains difficult to be quantified ''in-vivo''. Therefore, we used minimal volume tissue biopsies (4-8 mg) obtained from the ''m. vastus lateralis'' of HD subjects (mutation carriers) who volunteered to participate to our study, for quantifying mitochondrial respiration by means of the high-resolution respirometry before and after a standardized cycling exercise.
All patients gave written consent to participate to our study; the study protocol has been approved by the ethical committee of our institution. The tissue samples homogenates were put into the chambers of the O2K®-Oxygraph (Oroboros Instruments, Austria) and continuously stirred at 37°C. Mitochondrial respiration was quantified by adding complex I (10 mM Pyruvate, 5 mM Malate, and 10 mM Glutamate) and complex II (10 mM Succinate) substrates in the medium containing the homogenate and 5 mM ADP. Then 5 µM oligomycine was added to inhibit the ATP-synthase in order to obtain the LEAK-respiration state as an indicator of mitochondrial coupling. This step was followed by the addition of 1 µM of the uncoupler FCCP in order to achive the maximum respiration in the uncoupled state and the coupling (LEAK/ET-pathway)-ratio. After blocking mitochondrial respiration by 0.5 µM rotenone and 5 µM Antimycine A, the complex IV activity was selectively quantified by adding 2 mM Ascorbate and 0,5 mM TMPD. Here we present preliminary data from the first 3 patients included into the study.
A typical experiment as well as the preliminary data obtained yet in healthy controls and HD mutation carriers are presented in the figures below.
Our preliminary data do not allow definitive conclusions yet but they suggest that mitochondrial respiration can be reliably quantified in minimal volume needle biopsies from the ''m. vastus lateralis'' of human subjects. This test may therefore be used to quantify mitochondrial dysfunction as well as the effects of physical training during the course of the disease.
The turquoise killifish ''Nothobranchius furzeri'' is a short-lived vertebrate who inhabits transient freshwater ponds in the southeast of Africa. Due to its short lifespan this species has advanced to a vertebrate model for the biology and genetics of aging [1]. In our present study we quantified mitochondrial respiration in brain, liver, heart, and skeletal muscle of a short- and a long-lived strain of the fish at sequential points in time along to their specific lifespan duration.
Tissue samples were obtained at day 35, 68, and 100 in fishes of the short-lived strain and at day 35, 68, 100, 147, and 287 in the long-lived animals. Mitochondrial respiration was measured in terms of LEAK-, OXPHOS-, and ET-capacities by means of the high-resolution respirometry using an Oxygraph-2k (Oroboros Instruments, Austria). Respiratory activity of the homogenized tissue samples was simultaneously supported by complex I and II substrates (malate, glutamate, pyruvate, and succinate), as well as by ADP and quantified in terms of oxygen flux [''J''<sub>O2</sub>] per wet weight with the unit of pmol O<sub>2</sub>/(s·mg). OXPHOS capacity was obtained as the maximum activity under all substrates and ADP, LEAK respiration by inhibition of the ATP-synthase obtained by further injection of oligomycin, and, finally, ET-capacity by titration of the uncoupler FCCP.
We found a particularly pronounced decrease in mitochondrial respiration with age in the skeletal muscle of both strains; a more moderate decrease was also observed in the brain. In contrast, mitochondrial respiration in liver and heart was almost constant over the whole lifespan duration.
Our results suggest that aging-related changes in mitochondrial respiration of the ''Nothobranchius furzeri'' fish species are organ specific. These results will be discussed in relation to the biology of aging in general, and, in particular, to the most recently identified relevance of Complex I for determining the lifespan of this species [2].
Nicotinamide adenine dinucleotide (NAD) is a key metabolite involved in many cellular signaling in different metabolic conditions. NAD, as a cofactor of key enzymes in glycolisis, tricarboxilic acid cycle and oxidative phosphorylation, is important to cell energy generation, but also NAD is a substrate for generation of second messengers such as cyclic-ADP-ribose (cADPR) and has a role as a substrate and regulator of the NAD dependent deacetylases sirtuins (SIRTs). Lately, many studies have been demonstrating that regulating NAD levels can be used as therapy for many conditions as aging, high caloric diet, diabetes and other metabolic diseases, but further investigations are required to elucidate how the steady state of NAD regulates various aspects of mitochondrial function [1]. CD38 is a surface enzyme involved in different mechanism as calcium signaling, cell adhesion and signal transduction. In our scenario, CD 38 emerges as the main NADase in mammalian cells, and a regulator of intracellular NAD levels [2]. Our group demonstrated that CD38-deficient mice (CD38-/-) are protected against high-fat diet-induced obesity indicating that CD38-/- animals have a higher metabolic rate compared to wild-type mice (WT) [3]. As a regulator of NAD levels, CD38 can be a potential target for the regulation of mitochondrial function derived by NAD metabolism. We were able to demonstrate that the oxygen consumption in mitochondria isolated from CD38-/- mice is 2.5 times higher compared to the WT. To further confirm the regulation of mitochondria function promoted by CD38, we over expressed the CD38 vector in HEK 293T cells, which constitutively does not express this protein. Intact cells over expressing CD38 have 25% less oxygen consumption than the control vector. The decrease in respiration levels is mostly observed in maximum electron transport capacity (ETC). With the increase in CD38 expression, NAD levels in HEK293T decreases by 80%. The mitochondrial number is apparently increased in CD38 transfected cells but an abnormally mitochondria shape is observed by electron microscope. Other process that involves decrease in mitochondria respiration is immune response. Macrophages isolated from WT mice treated with 50ng/ml of LPS increased CD38 expression which was followed by a decreased in oxygen consumption, mostly in basal and ETC respiration. When this experiment was performed in macrophages isolated from CD38-/- animals, the decrease in oxygen consumption was abolished, confirming that CD38 could be an important key in the regulation of mitochondria respiration by LPS.
# Stein LR, Imai S (2012) The dynamic regulation of NAD metabolism in mitochondria. Trends Endocrinol Metab 23: 420-428.
# Chini EN (2009) CD38 as a regulator of cellular NAD: a novel potential pharmacological target for metabolic conditions. Curr Pharm Des 15: 57-63.
# Barbosa MT, Soares SM, Novak CM, Sinclair D, Levine JA, Aksoy P, Chini EN (2007) The enzyme CD38 (a NAD glycohydrolase, EC 3.2.2.5) is necessary for the development of diet-induced obesity. FASEB J 21: 3629-3639.
Nicotinamide adenine dinucleotide (NAD) levels decrease during aging and are involved in age-related metabolic decline. To date, the mechanism responsible for the age-related reduction in NAD has not been elucidated. Here we demonstrate that expression and activity of the NADase CD38 increase with aging and that CD38 is required for the age-related NAD decline and mitochondrial dysfunction via a pathway mediated at least in part by regulation of SIRT3 activity. We also identified CD38 as the main enzyme involved in the degradation of the NAD precursor nicotinamide mononucleotide (NMN) ''in vivo'', indicating that CD38 has a key role in the modulation of NAD-replacement therapy for aging and metabolic diseases.
Copyright © 2016 Elsevier Inc. All rights reserved. +
The mitochondrion is a vital component in cellular energy metabolism and intracellular signaling processes. Mitochondria are involved in a myriad of complex signaling cascades regulating cell death vs. survival. Importantly, mitochondrial dysfunction and the resulting oxidative and nitrosative stress are central in the pathogenesis of numerous human maladies including cardiovascular diseases, neurodegenerative diseases, diabetes, and retinal diseases, many of which are related. This review will examine the emerging understanding of the role of mitochondria in the etiology and progression of cardiovascular diseases and will explore potential therapeutic benefits of targeting the organelle in attenuating the disease process. Indeed, recent advances in mitochondrial biology have led to selective targeting of drugs designed to modulate or manipulate mitochondrial function, to the use of light therapy directed to the mitochondrial function, and to modification of the mitochondrial genome for potential therapeutic benefit. The approach to rationally treat mitochondrial dysfunction could lead to more effective interventions in cardiovascular diseases that to date have remained elusive. The central premise of this review is that if mitochondrial abnormalities contribute to the etiology of cardiovascular diseases (e.g., ischemic heart disease), alleviating the mitochondrial dysfunction will contribute to mitigating the severity or progression of the disease. To this end, this review will provide an overview of our current understanding of mitochondria function in cardiovascular diseases as well as the potential role for targeting mitochondria with potential drugs or other interventions that lead to protection against cell injury. +
When mitochondrial function is compromised and the mitochondrial membrane potential (Deltapsi(m)) falls below a threshold, the F(1)F(o)-ATP synthase can reverse, hydrolysing ATP to pump protons out of the mitochondrial matrix. Although this activity can deplete ATP and precipitate cell death, it is limited by the mitochondrial protein IF(1), an endogenous F(1)F(o)-ATPase inhibitor. IF(1), therefore, preserves ATP at the expense of Deltapsi(m). Despite a wealth of detailed knowledge on the biochemistry of the interaction of IF(1) and the F(1)F(o)-ATPase, little is known about its physiological activity. Emerging research suggests that IF(1) has a wider ranging impact on mitochondrial structure and function than previously thought. +
The decline in skeletal muscle performance with age (sarcopenia) is a significant public health concern due to the effect on quality of life and loss of independence. Mitochondrial oxidative stress has been thought to be a key mediator of age-related degeneration in skeletal muscle, although recent reports have challenged this view. Previously our lab has demonstrated that directly targeting mitochondrial oxidative stress and energetics using a single acute treatment with the mitochondrial-targeted peptide SS-31 reduces oxidative stress and improves muscle function in aged mice. However, whether long-term treatment with SS-31 peptide prevents sarcopenia and improves exercise tolerance is still unknown. We used osmotically controlled pumps to deliver a dose of SS-31 peptide equivalent to 3 mg/kg of body mass per day for 4 or 8 weeks to 7 and 26-month old mice. After 4 and 8 weeks SS-31 treatment led to an increase in both running distance and time in 26-month old mice using a ramped treadmill protocol compared to saline treated controls. The increase in exercise tolerance also correlated with an improvement of in-situ fatigue resistance among aged mice treated with SS-31 peptide for 8 weeks. To test whether changes in redox signaling could underlie mitochondrial and muscle deficits with age we used a thiol redox proteomics approach. Aging results in a significant increase in protein S-glutathionylation (PSSG) that was partially reversed with SS-31 treatment. Cysteine residues sensitive to PSSG with age and reversal by SS-31 treatment exist in many different cellular systems including but not limited to cellular contractility, glycolysis, oxidative phosphorylation, membrane repair, and control of redox signaling. Our preliminary data supports the conclusion that SS-31 represents a novel intervention with excellent translational potential to improve skeletal muscle function in the elderly. +
Sarcopenia and exercise intolerance are major contributors to reduced quality of life in the elderly for which there are few effective treatments. We tested whether enhancing mitochondrial function and reducing mitochondrial oxidant production with SS-31 (elamipretide) could restore redox balance and improve skeletal muscle function in aged mice. Young (5 mo) and aged (26 mo) female C57BL/6Nia mice were treated for 8-weeks with 3mg/kg/day SS-31. Mitochondrial function was assessed ''in vivo'' using <sup>31</sup>P and optical spectroscopy. SS-31 reversed age-related decline in maximum mitochondrial ATP production (ATPmax) and coupling of oxidative phosphorylation (P/O). Despite the increased ''in vivo'' mitochondrial capacity, mitochondrial protein expression was either unchanged or reduced in the treated aged mice and respiration in permeabilized gastrocnemius (GAS) fibers was not different between the aged and aged+SS-31 mice. Treatment with SS-31 also restored redox homeostasis in the aged skeletal muscle. The glutathione redox status was more reduced and thiol redox proteomics indicated a robust reversal of cysteine S-glutathionylation post-translational modifications across the skeletal muscle proteome. The gastrocnemius in the age+SS-31 mice was more fatigue resistant with significantly greater mass compared to aged controls. This contributed to a significant increase in treadmill endurance compared to both pretreatment and untreated control values. These results demonstrate that the shift of redox homeostasis due to mitochondrial oxidant production in aged muscle is a key factor in energetic defects and exercise intolerance. Treatment with SS-31 restores redox homeostasis, improves mitochondrial quality, and increases exercise tolerance without an increase in mitochondrial content. Since elamipretide is currently in clinical trials these results indicate it may have direct translational value for improving exercise tolerance and quality of life in the elderly.
Huntington's disease (HD) is a fatal neurodegenerative disease with limited treatment options. Human and animal studies have suggested that metabolic and mitochondrial dysfunctions contribute to HD pathogenesis. Here, we use high-resolution respirometry to uncover defective mitochondrial oxidative phosphorylation and electron transfer capacity when a mutant huntingtin fragment is targeted to neurons or muscles in ''Drosophila'' and find that enhancing mitochondrial function can ameliorate these defects. In particular, we find that co-expression of parkin, an E3 ubiquitin ligase critical for mitochondrial dynamics and homeostasis, produces significant enhancement of mitochondrial respiration when expressed either in neurons or muscles, resulting in significant rescue of neurodegeneration, viability and longevity in HD model flies. Targeting mutant HTT to muscles results in larger mitochondria and higher mitochondrial mass, while co-expression of parkin increases mitochondrial fission and decreases mass. Furthermore, directly addressing HD-mediated defects in the fly's mitochondrial electron transport system, by rerouting electrons to either bypass mitochondrial complex I or complexes III-IV, significantly increases mitochondrial respiration and results in a striking rescue of all phenotypes arising from neuronal mutant huntingtin expression. These observations suggest that bypassing impaired mitochondrial respiratory complexes in HD may have therapeutic potential for the treatment of this devastating disorder. +
Fibroblast growth factor 21 (FGF21) is a potent regulator of glucose and lipid metabolism and is currently being pursued as a therapeutic agent for insulin resistance and type 2 diabetes. However, the cellular mechanisms by which FGF21 modifies insulin action ''in vivo'' are unclear. To address this question, we assessed insulin action in regular chow (RC) and high-fat (HFD) fed wild-type mice chronically infused with FGF21 or vehicle. Here, we show that FGF21 administration results in improvements in both hepatic and peripheral insulin sensitivity in both RC and HFD mice. This improvement in insulin responsiveness in FGF21 treated HFD fed mice was associated with decreased hepatocellular and myocellular diacylglycerol (DAG) content and reduced protein kinase C (PKC)∈ activation in liver and PKCθ in skeletal muscle. In contrast there were no effects of FGF21 on liver or muscle ceramide content. These effects may be attributed, in part, to increased energy expenditure in the liver and white adipose tissue. Taken together, these data provide a mechanism by which FGF21 protects mice from lipid-induced liver and muscle insulin resistance and support its development as a novel therapy for the treatment of nonalcoholic fatty liver disease, insulin resistance, and type 2 diabetes. +
We previously reported that facilitating the clearance of damaged mitochondria through macroautophagy/autophagy protects against acute myocardial infarction. Here we characterize the impact of exercise, a safe strategy against cardiovascular disease, on cardiac autophagy and its contribution to mitochondrial quality control, bioenergetics and oxidative damage in a post-myocardial infarction-induced heart failure animal model. We found that failing hearts displayed reduced autophagic flux depicted by accumulation of autophagy-related markers and loss of responsiveness to chloroquine treatment at 4 and 12 wk after myocardial infarction. These changes were accompanied by accumulation of fragmented mitochondria with reduced O<sub>2</sub> consumption, elevated H<sub>2</sub>O<sub>2</sub> release and increased Ca<sup>2+</sup>-induced mitochondrial permeability transition pore opening. Of interest, disruption of autophagic flux was sufficient to decrease cardiac mitochondrial function in sham-treated animals and increase cardiomyocyte toxicity upon mitochondrial stress. Importantly, 8 wk of exercise training, starting 4 wk after myocardial infarction at a time when autophagy and mitochondrial oxidative capacity were already impaired, improved cardiac autophagic flux. These changes were followed by reduced mitochondrial number:size ratio, increased mitochondrial bioenergetics and better cardiac function. Moreover, exercise training increased cardiac mitochondrial number, size and oxidative capacity without affecting autophagic flux in sham-treated animals. Further supporting an autophagy mechanism for exercise-induced improvements of mitochondrial bioenergetics in heart failure, acute ''in vivo'' inhibition of autophagic flux was sufficient to mitigate the increased mitochondrial oxidative capacity triggered by exercise in failing hearts. Collectively, our findings uncover the potential contribution of exercise in restoring cardiac autophagy flux in heart failure, which is associated with better mitochondrial quality control, bioenergetics and cardiac function.
Since the industrial revolution, the earth has been facing increases in CO<sub>2</sub> concentration which, consequently, have led to an increase in global average temperatures. Of the last 23 years, 22 have had temperatures above the global average. Temperature is the main factor affecting the life of ectothermic species. In addition, tropical species are expected to be especially vulnerable to temperature increases, since many of them appear to have a narrower thermal tolerance range and live closer to their thermal limits. However, to date, studies that assess the vulnerability of Amazonian species are scarcest. Among these species, fish from igarapé appear to be especially vulnerable, since they occur in an extremely stable thermal environment. Therefore, the present thesis aims to evaluate the adaptability to the increase of temperature and CO<sub>2</sub> on the physiological parameters in Igarapé fish species. In chapter I our results showed that active species present high energy demand in order to supply maintenance costs for a high activity and, therefore, have a low thermal tolerance. In Chapter II, ''Hyphessobrychon melazonatus'', an active species, acclimated to the extreme climate change scenario, presented great osmoregulatory disturbances, which may have important impacts on the long-term survival capacity. In fact, in chapter III we observed that all three species present metabolic alterations and acclimate cellular damages the future climatic conditions, however, ''H. melazonatus'' was the species that presented the highest mortality rates, lipid damages and reduction in the thermal safety margin. In chapter IV we showed that ''Pyrrhullina brevis'' acclimated for 180 days in the extreme scenario presents a reduction in body size, according to the third universal response to temperature. However, contrary to the OCLTT hypothesis, reduction in size is not linked to an inability to supply oxygen to tissues, but rather appears to be related to an increase in levels of cellular damage that impede growth according to life-history trade-off theory. Our results highlight the importance of public policies aimed at reducing the agents that cause climate change and the preservation of forest areas that play a fundamental role in maintaining the temperature of the Igarapés.
At the request of the author, this abstract is not made available online. +
Acute lymphoblastic leukemia (ALL) is the most common type of childhood cancer, accounting for 25% of all cancers in this age group. One of the chemotherapeutics used in the therapy of ALL (and autoimmune diseases such as rheumatoid arthritis) is methotrexate (MTX), a folic acid antagonist (antifolate). As a chemotherapeutic agent, MTX´s mechanism of action is primarily attributed to the inhibition of the dihydrophate reductase enzyme, which synthesizes tetrahydrofolate from dihydrofolate – a key step in the ''de novo'' synthesis of purine nucleotides used in cell division. In rheumatoid arthritis, lower doses of MTX inhibit the 5-aminoimidazole-4-ribonucleotide-carboxamide formyltransferase (ATIC) enzyme, which culminates in the production of high levels of adenosine, a potent anti-inflammatory. However, recent works continue to present previously unknown mechanisms and effects through which MTX acts within the cell, attesting that MTX´s mechanisms of action appear to be as multiple as complex. Using several techniques of molecular biology, this work sought to expand the existing knowledge of the action of MTX in ALL. For this purpose, several
biological parameters were measured under or without MTX treatment in a panel of 13 ALL
cell lines. Proliferation tests, metabolic studies, drug synergism, quantification of cellular
respiration and the production of reactive oxygen species (ROS) were performed, as well as
the measurement of the activation of the NF-κB signaling pathway. Resistance of the MTX
strains within 48 h of treatment (but not 96 h) was related to the proliferation rate of the cells.
Treatment with MTX altered the concentration of 28 intracellular metabolites, highlights for a
consistent increase in glycine concentration. Intracellular concentrations of asparagine,
guanosine and glutathione – including the expression of genes from glutathione pathway –
were associated with MTX resistance. Supplementation of the culture medium with Nacetylcysteine,
a precursor metabolite of glutathione, promoted proliferation and resistance to
MTX; however, cell treatment with piperlongumine or hydrogen peroxide, two glutathione
scavengers and ROS promoters, did not potentiate the effect of MTX. MTX induced ROS in
ALL after 6 h of treatment with low fold change, though. Paradoxically, higher ROS
production was found in cell lines with high MTX resistance and intracellular glutathione.
The oxygen uptake of the cell lines was not associated with MTX resistance and a preliminary
test showed that MTX did not alter cellular respiration. MTX activated the transcription factor
NF-κB in some ALL cell lines and, interestingly, the activation of this transcription factor by
tumor necrosis factor alpha (TNF-α) was positively correlated with the resistance of leukemic
lines to MTX. A wide bibliographic review allowed both the integration of the obtained
results to the most current knowledge on the subject, and the identification of new paths to be
explored in future stages.
Skeletal muscle atrophy occurs as a result of disuse. Although several studies have established that a decrease in protein synthesis and increase in protein degradation lead to muscle atrophy, little is known about the triggers underlying such processes. A growing body of evidence challenges oxidative stress as a trigger of disuse atrophy; furthermore, it is also becoming evident that mitochondrial dysfunction may play a causative role in determining muscle atrophy. Mitochondrial fusion and fission have emerged as important processes that govern mitochondrial function and PGC-1α may regulate fusion/fission events. Although most studies on mice have focused on the anti-gravitary slow soleus muscle as it is preferentially affected by disuse atrophy, several fast muscles (including gastrocnemius) go through a significant loss of mass following unloading. Here we found that in fast muscles an early down-regulation of pro-fusion proteins, through concomitant AMP-activated protein kinase (AMPK) activation, can activate catabolic systems, and ultimately cause muscle mass loss in disuse. Elevated muscle PGC-1α completely preserves muscle mass by preventing the fall in pro-fusion protein expression, AMPK and catabolic system activation, suggesting that compounds inducing PGC-1α expression could be useful to treat and prevent muscle atrophy.
The mechanisms triggering disuse muscle atrophy remain of debate. It is becoming evident that mitochondrial dysfunction may regulate pathways controlling muscle mass. We have recently shown that mitochondrial dysfunction plays a major role in disuse atrophy of soleus, a slow, oxidative muscle. Here we tested the hypothesis that hindlimb unloading-induced atrophy could be due to mitochondrial dysfunction in fast muscles too, notwithstanding their much lower mitochondrial content. Gastrocnemius displayed atrophy following both 3 and 7 days of unloading. SOD1 and catalase up-regulation, no H<sub>2</sub>O<sub>2</sub> accumulation and no increase of protein carbonylation suggest the antioxidant defence system efficiently reacted to redox imbalance in the early phases of disuse. A defective mitochondrial fusion (Mfn1, Mfn2 and OPA1 down-regulation) occurred together with an impairment of OXPHOS capacity. Furthermore, at 3 days of unloading higher acetyl-CoA carboxylase (ACC) phosphorylation was found, suggesting AMP-activated protein kinase (AMPK) pathway activation. To test the role of mitochondrial alterations we used Tg-mice overexpressing PGC-1α because of the known effect of PGC-1α on stimulation of Mfn2 expression. PGC-α overexpression was sufficient to prevent (i) the decrease of pro-fusion proteins (Mfn1, Mfn2 and OPA1), (ii) activation of the AMPK pathway, (iii) the inducible expression of MuRF1 and atrogin1 and of authopagic factors, and (iv) any muscle mass loss in response to disuse. As the effects of increased PGC-1α activity were sustained throughout disuse, compounds inducing PGC-1α expression could be useful to treat and prevent muscle atrophy also in fast muscles.
Brown and white adipose tissues in mammals have a number of similar properties, such as lipid storage and adipokine production, but also distinctive properties. The energy-storing white adipose tissue has few mitochondria and low oxidative capacity. The heat-producing brown adipose tissue has a high density of mitochondria and high oxidative capacity. Mitochondrial function can be investigated in cells and organelles isolated from both brown and white adipose tissues. This chapter describes methods for successful isolation of suitable preparations of adipose tissues and their subsequent use. Questions concerning thermogenic capacity of the tissues, their potential influence on whole body metabolism, and specific properties of the mitochondria and their mode of function may be addressed using these methods. +
Primary pulmonary arterial hypertension (PAH) is a progressive disease of the pulmonary circulation resulting in increased pulmonary vascular resistance, right ventricular hypertrophy and right ventricular failure. PAH results in poor quality of life due to dyspnoea and exercise intolerance, and unsurprisingly, an increased mortality risk. Therefore, therapies to improve muscle metabolic function and exercise tolerance are crucial to preserve quality of life and patient prognosis. Numerous studies support the safety and efficacy of exercise training in patients with COPD, and early concerns about accelerating the rate of decline in patient health have mostly not been substantiated. Evidence for benefits with rehabilitative exercise training in PAH have recently been reported (Mereles et al 2006). While limited data are available for exercise training in PAH, none have examined mitochondrial function after exercise training. Recently, our group have demonstrated reduced maximum respiratory rate and Complex I dysfunction using high-resolution respirometry in an animal model of PAH (Wuest et al 2012). However, whether this mitochondrial dysfunction is reversible through exercise training or pharmacologic intervention (Piao et al 2010) remains to be determined.
Therefore, our study aims to exploit a multi-disciplinary exercise and education intervention study of PAH. This investigation will explore the efficacy of exercise training in mild and severe PAH. Control groups of mild and severe patients will receive health education and exercise training in a cross-over design. In conjunction with traditional measures of cardiorespiratory fitness, pulmonary function, muscle oxygenation during exercise, cardiac MR imaging, and quality-of-life assessment, knee-extensor muscle biopsies will be taken for measurement of mitochondrial function. This is to be done using a substrate-uncoupler-inhibitor-titration protocol of high-resolution respirometry. Additionally, biochemical analyses for respiratory enzymes and mitochondrial protein expression will be made.
References:
Mereles D, Ehlken N, Kreuscher S, Ghofrani S, Hoeper MM, Halank M, Meyer FJ, Karger G, Buss J, Juenger J, Holzapfel N, Opitz C, Winkler J, Herth FF, Wilkens H, Katus HA, Olschewski H, Grunig E (2006) Exercise and respiratory training improve exercise capacity and quality of life in patients with severe chronic pulmonary hypertension. Circulation 114: 1482-1489.
Piao L, Marsboom G, Archer SL (2010) Mitochondrial metabolic adaptation in right ventricular hypertrophy and failure. J Mol Med 88: 1011-1020.
[[Wuest_2012_Am J Physiol Heart Circ Physiol|Wuest RC, Myers DS, Stones R, Benoist D, Robinson PA, Boyle JP, Peers C, White E, Rossiter HB (2012) Regional skeletal muscle remodeling and mitochondrial dysfunction in right ventricular heart failure. Am J Physiol Heart Circ Physiol 302: H402-H411.]]
*Saponin permeabilized skeletal muscle fiber bundles
*'''Protocol''':
#0.5mM Malate
#50 µM palmitoyl-CoA + 2mM carnitine
#5mM ADP
#From here on out, various combinations for titration protocol
*Coupling states:
#LEAK_M+Palmitoylcarnitine
#P_M+Palmitoylcarnitine
#P_M+Palmitoylcarnitine+S
#E_O+CCCP titrations
#E_S+Rot
#ROX_AntimycinA
+
Perinatal smoke/nicotine exposure predisposes to chronic lung disease and morbidity. Mitochondrial abnormalities may contribute as the PPARγ pathway is involved in structural and functional airway deficits after perinatal nicotine exposure. We hypothesized perinatal nicotine exposure results in lung mitochondrial dysfunction that can be rescued by rosiglitazone (RGZ; PPARγ receptor agonist). Sprague-Dawley dams received placebo (CON), nicotine (NIC, 1 mg kg(-1)), or NIC + RGZ (3 mg kg(-1)) daily from embryonic day 6 to postnatal day 21. Parenchymal lung (~10 mg) was taken from adult male offspring for mitochondrial assessment ''in situ''. ADP-stimulated O<sub>2</sub> consumption was less in NIC and NIC + RGZ compared to CON (F[2,14] = 17.8; 4.5 ± 0.8 and 4.1 ± 1.4 vs. 8.8 ± 2.5 pmol s mg(-1); ''p'' < 0.05). The respiratory control ratio for ADP, an index of mitochondrial coupling, was reduced in NIC and remediated in NIC + RGZ (F[2,14] = 3.8; ''p'' < 0.05). Reduced mitochondrial oxidative capacity and abnormal coupling were evident after perinatal nicotine exposure. RGZ improved mitochondrial function through tighter coupling of oxidative phosphorylation. +
Diaphragm dysfunction accompanies cardiopulmonary disease and impaired oxygen delivery. Vascular endothelial growth factor (VEGF) regulates oxygen delivery through angiogenesis, capillary maintenance, and contraction-induced perfusion. We hypothesized that myofiber-specific VEGF deficiency contributes to diaphragm weakness and fatigability. Diaphragm protein expression, capillarity and fiber morphology, mitochondrial respiration and hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) generation, and contractile function were compared between adult mice with conditional gene ablation of skeletal myofiber VEGF (SkmVEGF<sup>-/-</sup>; n=12) and littermate controls (n=13). Diaphragm VEGF protein was ~50 % lower in SkmVEGF<sup>-/-</sup> than littermate controls (1.45±0.65 vs. 3.04±1.41 pg/total protein; P=0.001). This was accompanied by an ~15% impairment in maximal isometric specific force (F[1,23] = 15.01, P=0.001) and a trend for improved fatigue resistance (P=0.053). Mean fiber cross-sectional area and type I fiber cross-sectional area were lower in SkmVEGF<sup>-/-</sup> by ~40 % and ~25% (P<0.05). Capillary-to-fiber ratio was also lower in SkmVEGF<sup>-/-</sup> by ~40% (P<0.05), thus capillary density was not different. Sarcomeric actin expression was ~30% lower in SkmVEGF<sup>-/-</sup> (P<0.05), while myosin heavy chain and MAFbx were similar (measured via immunoblot). Mitochondrial respiration, citrate synthase activity, PGC-1α, and HIF-1α were not different in SkmVEGF<sup>-/-</sup> (P>0.05). However mitochondrial-derived reactive oxygen species (ROS) flux was lower in SkmVEGF<sup>-/-</sup> (P=0.0003). In conclusion, myofiber-specific VEGF gene deletion resulted in a lower capillary-to-fiber ratio, type I fiber atrophy, actin loss, and contractile dysfunction in the diaphragm. In contrast, mitochondrial respiratory function was preserved alongside lower ROS generation, which may play a compensatory role to preserve fatigue resistance in the diaphragm. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]] +
Pulmonary arterial hypertension (PAH) is a progressive disease of the pulmonary vasculature that leads to right ventricular failure. Skeletal muscle maladaptations limit physical activity and may contribute to disease progression. The role of alarmin/inflammatory signaling in PAH respiratory muscle dysfunction is unknown. We hypothesized that diaphragm mitochondrial and contractile functions are impaired in SU5416/hypoxia-induced pulmonary hypertension due to increased systemic IL-33 signaling. We induced pulmonary hypertension in adult C57Bl/6 J (WT) and ST2 (IL1RL1) gene ablated mice by SU5416/hypoxia (SuHx). We measured diaphragm fiber mitochondrial respiration, inflammatory markers, and contractile function ex vivo. SuHx reduced coupled and uncoupled permeabilized myofiber respiration by ∼40 %. During coupled respiration with complex I substrates, ST2<sup>-/-</sup> attenuated SuHx inhibition of mitochondrial respiration (genotype × treatment interaction F[1,67] = 3.3, p = 0.07, η<sup>2</sup> = 0.04). Flux control ratio and coupling efficiency were not affected by SuHx or genotype. A higher substrate control ratio for succinate was observed in SuHx fibers and attenuated in ST2<sup>-/-</sup> fibers (F[1,67] = 5.3, p < 0.05, η<sup>2</sup> = 0.07). Diaphragm TNFα, but not IL-33 or NFkB, was increased in SuHx vs. DMSO in both genotypes (F[1,43] = 4.7, p < 0.05, η<sup>2</sup> = 0.1). Diaphragm force-frequency relationships were right-shifted in SuHx vs. WT (F[3,440] = 8.4, p < 0.05, η<sup>2</sup> = 0.0025). There was no effect of ST2<sup>-/-</sup> on the force-frequency relationship. Force decay during a fatigue protocol at 100 Hz, but not at 40 Hz, was attenuated by SuHx vs. DMSO in both genotypes (F[1,41] = 5.6, p < 0.05, η<sup>2</sup> = 0.11). SuHx mice exhibit a modest compensation in diaphragm contractility and mitochondrial dysfunction during coupled respiration; the latter partially regulated through ST2 signaling. +
Primary brain cell cultures are a useful tool for understanding the physiopathology of epilepsy and for searching new potential antiepileptic drugs. These cell types are usually prepared from murine species and few human models have been described. The main goal of this study is the establishment of experimental conditions to isolate and culture neurons and astrocytes from human brain and to test its functionality. The tissues came from antiepileptic drug-resistant epileptic patients undergoing surgery. Human neurons and astrocytes were isolated following an enzymatic and mechanical dissociation protocol. Cultures were viable for 3-6 weeks. Cytological characterization was performed by immunocytochemistry using specific antibodies against both neuron (anti-NeuN) and astrocyte (anti-GFAP) protein markers. In order to test their viability and functionality, cells were loaded with the fluorescent calcium probe fura-2 and variations in cytosolic calcium concentrations ([Ca<sup>2+</sup>]c) were measured by cell imaging. [Ca<sup>2+</sup>]c increases were evoked upon cell stimulation with high Ka<sup>+</sup> (KCl 75 mM), glutamate (500 μM) or bicuculline (100 μM). Interestingly, spontaneous [Ca<sup>2+</sup>]c transients were also observed in some neuron-like cells. A novel unreported finding in this study has been the incorporation of human serum that was critical for cell functionality. The setting of these human cultures open the opportunity to new insights on culture and calcium signalling studies on the mechanism(s) of cell resistance to antiepileptic drugs, as well as to studies on plasticity, maturation and possible neurite emission for graft studies.
Copyright © 2011 ISDN. Published by Elsevier Ltd. All rights reserved. +
Envenomations by ''Bothrops'' snakebites can induce overwhelming systemic inflammation ultimately leading to multiple organ system failure and death. Release of damage-associated molecular pattern molecules (DAMPs), in particular of mitochondrial origin, has been implicated in the pathophysiology of the deregulated innate immune response.
The objective was to test whether whole ''Bothrops lanceolatus'' venom would induce mitochondrial dysfunction and DAMPs release in human heart preparations.
Human atrial trabeculae were obtained during cannulation for cardiopulmonary bypass from patients who were undergoing routine coronary artery bypass surgery. Cardiac fibers were incubated with vehicle and whole ''Bothrops lanceolatus'' venom for 24hr before high-resolution respirometry, mitochondrial membrane permeability evaluation and quantification of mitochondrial DNA.
Compared with vehicle, incubation of human cardiac muscle with whole ''Bothrops lanceolatus'' venom for 24hr impaired respiratory control ratio and mitochondrial membrane permeability. Levels of mitochondrial DNA increased in the medium of cardiac cell preparation incubated with venom of ''Bothrops lanceolatus''.
Our study suggests that whole venom of ''Bothrops lanceolatus'' impairs mitochondrial oxidative phosphorylation capacity and increases mitochondrial membrane permeability. Cardiac mitochondrial dysfunction associated with mitochondrial DAMPs release may alter myocardium function and engage the innate immune response, which may both participate to the cardiotoxicity occurring in patients with severe envenomation. +
AMP-activated protein kinase (AMPK) is a metabolic fuel gauge conserved along the evolutionary scale in eukaryotes that senses changes in the intracellular AMP/ATP ratio. Recent evidence indicated an important role for AMPK in the therapeutic benefits of metformin, thiazolidinediones and exercise, which form the cornerstones of the clinical management of type 2 diabetes and associated metabolic disorders. In general, activation of AMPK acts to maintain cellular energy stores, switching on catabolic pathways that produce ATP, mostly by enhancing oxidative metabolism and mitochondrial biogenesis, while switching off anabolic pathways that consume ATP. This regulation can take place acutely, through the regulation of fast post-translational events, but also by transcriptionally reprogramming the cell to meet energetic needs. Here we demonstrate that AMPK controls the expression of genes involved in energy metabolism in mouse skeletal muscle by acting in coordination with another metabolic sensor, the NAD+-dependent type III deacetylase SIRT1. AMPK enhances SIRT1 activity by increasing cellular NAD+ levels, resulting in the deacetylation and modulation of the activity of downstream SIRT1 targets that include the [[PGC-1alpha|peroxisome proliferator-activated receptor-gamma coactivator 1alpha]] and the forkhead box O1 (FOXO1) and O3 (FOXO3a) transcription factors. The AMPK-induced SIRT1-mediated deacetylation of these targets explains many of the convergent biological effects of AMPK and SIRT1 on energy metabolism. +
This article describes methodologies to examine mitochondrial respiration in fresh preparations of mouse tissues, including skeletal muscle, heart, liver, white and brown adipose tissue, and brain. Reference values and tips to maximize experimental efficiencies are also provided. Finally, correction methods and complementary techniques to properly interpret the results are presented and contrasted. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]]
Mitochondrial dysfunction is a hallmark for multiple metabolic and age-related diseases [1]. Mitochondrial quality control mechanisms are largely influenced by mitochondrial fusion and fission events, referred to as mitochondrial dynamics, which also influence mitochondrial bioenergetics properties [2]. Nevertheless most of this knowledge derives from the use of cultured cell lines, and the role of different mitochondrial fusion and fission regulators in differentiated tissues remains unclear.
In order to explore the role of Mitofusins (Mfn1 and Mfn2) and Drp1 in tissue metabolism, we have generated a collection of conditional, tissue-specific, mouse models for defective fusion and fission mechanisms. Further, we have examined how these mice respond to dietary (e.g.: high-fat feeding) and physiological challenges. We will have a special focus on their impact on brown adipose tissue metabolism and thermogenesis.
Our results illustrate the Mfn1 and Mfn2, despite being both involved in mitochondrial fusion, have critically different roles in brown adipose tissue physiology. In particular, Mfn2 plays a unique role in linking mitochondria to lipid droplets. We will also discuss how Drp1 phosphorylation impacts mitochondrial fusion/fission in brown adipose tissue by using phospho-null knockin mice.
As a whole, our results illustrate that mitochondrial dynamics-related proteins critically influence thermogenic capacity and energy substrate utilization in brown adipose tissue. In addition, we show that this occurs not just through the modulation of mitochondrial architecture, but also by enabling the communication of mitochondria with other cellular organelles. +
A new criterion is utilized for the interpretation of flow-force relationships in rat liver mitochondria. The criterion is based on the view that the nature of the relationship between the H+/O ratio and the membrane potential can be inferred from the relationship between ohmic-uncoupler-induced extra respiration and the membrane potential. Thus a linear relationship between extra respiration and membrane potential indicates unequivocally the independence of the H+/O ratio from the membrane potential and the leak nature of the resting respiration [Brand, Chien, and Diolez (1994) Biochem. J. 297, 27-29]. On the other hand, a non-linear relationship indicates that the H+/O ratio is dependent on the membrane potential. The experimental assessment of this relationship in the presence of an additional ohmic leak, however, is rendered difficult by both the uncoupler-induced depression of membrane potential and the limited range of dependence of the H+/O ratio on the membrane potential. We have selected conditions, i.e. incubation of mitochondria at low temperatures, where the extent of non-linearity is markedly increased. It appears that the nature of the resting respiration of mitochondria in vitro is markedly dependent on the temperature: at low temperatures the percentage of resting respiration due to membrane leak decreases and that due to intrinsic uncoupling of the proton pumps increases. +
The LHCSR protein belongs to the light harvesting complex family of pigment-binding proteins found in oxygenic photoautotrophs. Previous studies have shown that this complex is required for the rapid induction and relaxation of excess light energy dissipation in a wide range of eukaryotic algae and moss. The ability of cells to rapidly regulate light harvesting between this dissipation state and one favoring photochemistry is believed to be important for reducing oxidative stress and maintaining high photosynthetic efficiency in a rapidly changing light environment. We found that a mutant of Chlamydomonas reinhardtii lacking LHCSR, npq4lhcsr1, displays minimal photoinhibition of photosystem II and minimal inhibition of short term oxygen evolution when grown in constant excess light compared to a wild type strain. We also investigated the impact of no LHCSR during growth in a sinusoidal light regime, which mimics daily changes in photosynthetically active radiation. The absence of LHCSR correlated with a slight reduction in the quantum efficiency of photosystem II and a stimulation of the maximal rates of photosynthesis compared to wild type. However, there was no reduction in carbon accumulation during the day. Another novel finding was that npq4lhcsr1 cultures underwent fewer divisions at night, reducing the overall growth rate compared to the wild type. Our results show that the rapid regulation of light harvesting mediated by LHCSR is required for high growth rates, but it is not required for efficient carbon accumulation during the day in a sinusoidal light environment. This finding has direct implications for engineering strategies directed at increasing photosynthetic productivity in mass cultures. +
The subunit structure of oligomycin sensitive ATPase has been determined. In addition to the components of F1, and the so-called oligomycin sensitivity conferring protein, there are four other polypeptides of molecular weights 55,000, 29,000, 20,000 and 10,000 which together form the intrinsic membrane portion of the enzymic complex. +
We report a resonance Raman and UV-vis characterization of the active site structure of oxidatively modified forms of cytochrome c (Cyt-c) free in solution and in complexes with cardiolipin (CL). The studied post-translational modifications of Cyt-c include methionine sulfoxidation and tyrosine nitration, which lead to altered heme axial ligation and increased peroxidase activity with respect to those of the wild-type protein. In spite of the structural and activity differences between the protein variants free in solution, binding to CL liposomes induces in all cases the formation of a spectroscopically identical bis-His axial coordination conformer that more efficiently promotes lipid peroxidation. The spectroscopic results indicate that the bis-His form is in equilibrium with small amounts of high-spin species, thus suggesting a labile distal His ligand as the basis for the CL-induced increase in enzymatic activity observed for all protein variants. For Cyt-c nitrated at Tyr74 and sulfoxidized at Met80, the measured apparent binding affinities for CL are ∼4 times larger than for wild-type Cyt-c. On the basis of these results, we propose that these post-translational modifications may amplify the pro-apoptotic signal of Cyt-c under oxidative stress conditions at CL concentrations lower than for the unmodified protein. +
Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals owing to the expression of uncoupling protein-1 (UCP-1), which uncouples mitochondrial respiration from ATP production. It has recently been observed that adult humans have functional BAT. While it has been theorized that uncoupled mitochondrial respiration contributes to the hypermetabolic response to burns, whether patients with severe burns have functional BAT remains unknown.
We collected sub-platysmal adipose tissue (sPAT) (n=5 samples) from patients undergoing reconstructive surgeries and peri-renal adipose tissue (pRAT) (n=2 samples) from patients at autopsy. Sub-cutaneous white adipose tissue (scWAT) samples were also collected. High-resolution respirometry was performed on permeabilized tissue samples to determine respiration. Titration of the UCP-1 inhibitor guanosine diphosphate (GDP) was used to determine the presence or absence of BAT in sPAT and scWAT. Histology was also performed on pRAT and scWAT samples.
The average of patients providing sPAT was 9 ± 1 years. sPAT adipose tissue had a respiratory capacity 26-fold higher than scWAT (68.5 ± 39.7 vs. 2.6 ± 1.5 pmol/s/mg, p<0.001). GDP titration reduced respiration in sPAT (-38.9 ± 17.7 pmol/s/mg) but not scWAT mitochondria (-0.08 ± 0.07 pmol/s/mg), providing direct evidence of functional BAT within sPAT. Histological analysis showed that pRAT had distinct areas with an abundance of small multi-locular cells (adipocytes containing numerous small lipid droplets), whereas scWAT exhibited larger mainly uni-locular cells (adipocytes containing one large lipid droplet).
We provide novel functional and histological evidence of BAT in patients with severe burns. The functional signature of UCP-1 in sPAT of burned patients indicates that this BAT is thermogenic, and therefore may contribute to the hypermetabolic response to burn injury.
We have identified BAT in burned patients as a component of the metabolic response to burn injury. The regulatory and homeostatic qualities of this tissue render it a potential target to modulate the hypermetabolic response to burn injury.
This book offers a state-of-the-art description of the discipline of nonequilibrium thermodynamics as applied to biophysics and bioenergetics in the linear domain. It is replete with experimental examples not only from the authors’ laboratories but from those of other workers in the field, providing a logical and compact framework for organizing data, from the level of whole tissues to that of sub-cellular organelles. The authors provide the necessary grounding in classical as well as nonequilibrium thermodynamics before moving on to proper pathways, degrees of coupling and energy conversion, isotope interactions and kinetic interpretations, and practical applications. Throughout, experimental and theoretical aspects are fully integrated, and kinetic methods are used to complement the thermodynamic approach.
The systematic correlation of diverse data underlines the strong analogy that often exists between seemingly different systems. Biophysicists, biochemists, bioenergeticists, and physiologists will find that Caplan and Essig offer a remarkably useful tool for application to a wide range of biological problems. +
Pharmacological approaches to reduce obesity have not resulted in dramatic reductions in the risk of coronary heart disease (CHD). Exercise, in contrast, reduces CHD risk even in the setting of obesity. Cholesteryl Ester Transfer Protein (CETP) is a lipid transfer protein that shuttles lipids between serum lipoproteins and tissues. There are sexual-dimorphisms in the effects of CETP in humans. Mice naturally lack CETP, but we previously reported that transgenic expression of CETP increases muscle glycolysis in fasting and protects against insulin resistance with high-fat diet (HFD) feeding in female but not male mice. Since glycolysis provides an important energy source for working muscle, we aimed to define if CETP expression protects against the decline in exercise capacity associated with obesity. We measured exercise capacity in female mice that were fed a chow diet and then switched to a HFD. There was no difference in exercise capacity between lean, chow-fed CETP female mice and their non-transgenic littermates. Female CETP transgenic mice were relatively protected against the decline in exercise capacity caused by obesity compared to WT. Despite gaining similar fat mass after 6 weeks of HFD-feeding, female CETP mice showed a nearly two-fold increase in run distance compared to WT. After an additional 6 weeks of HFD-feeding, mice were subjected to a final exercise bout and muscle mitochondria were isolated. We found that improved exercise capacity in CETP mice corresponded with increased muscle mitochondrial oxidative capacity, and increased expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). These results suggest that CETP can protect against the obesity-induced impairment in exercise capacity and may be a target to improve exercise capacity in the context of obesity. +
Mammalian cells synthesize H<sub>2</sub>S from sulfur containing amino acids and are also exposed to exogenous sources of this signaling molecule, notably from gut microbes. As an inhibitor of complex IV in the electron transport chain, H<sub>2</sub>S can have a profound impact on metabolism, suggesting the hypothesis that metabolic reprogramming is a primary mechanism by which H<sub>2</sub>S signals. In this study, we report that H<sub>2</sub>S increases lipogenesis in many cell types, using carbon derived from glutamine rather than from glucose. H<sub>2</sub>S-stimulated lipid synthesis is sensitive to the mitochondrial NAD(P)H pools and is enabled by reductive carboxylation of α-ketoglutarate. Lipidomics analysis revealed that H<sub>2</sub>S elicits time-dependent changes across several lipid classes, e.g., upregulating triglycerides while down regulating phosphatidylcholine. Direct analysis of triglyceride concentration revealed that H<sub>2</sub>S induces a net increase in the size of this lipid pool. These results provide a mechanistic framework for understanding the effects of H<sub>2</sub>S on increasing lipid droplets in adipocytes and population studies that have pointed to a positive correlation between cysteine (a substrate for H<sub>2</sub>S synthesis) and fat mass. +
The current study aimed to examine the effects of resistance exercise with concomitant consumption of high vs. low daily doses of non-steroidal anti-inflammatory drugs (NSAIDs) on mitochondrial oxidative phosphorylation in skeletal muscle. As a secondary aim, we compared the effects of eccentric overload with conventional training.
Twenty participants were randomized to either a group taking high doses (3 × 400 mg/day) of ibuprofen (IBU; 27 ± 5 year; n = 11) or a group ingesting a low dose (1 × 75 mg/day) of acetylsalicylic acid (ASA; 26 ± 4 year; n = 9) during 8 weeks of supervised knee extensor resistance training. Each of the subject's legs were randomized to complete the training program using either a flywheel (FW) device emphasizing eccentric overload, or a traditional weight stack machine (WS). Maximal mitochondrial oxidative phosphorylation (CI+IIP) from permeabilized skeletal muscle bundles was assessed using high-resolution respirometry. Citrate synthase (CS) activity was assessed using spectrophotometric techniques and mitochondrial protein content using western blotting.
After training, CI+IIP decreased (P < 0.05) in both IBU (23%) and ASA (29%) with no difference across medical treatments. Although CI+IIP decreased in both legs, the decrease was greater (interaction p = 0.015) in WS (33%, p = 0.001) compared with FW (19%, p = 0.078). CS activity increased (p = 0.027) with resistance training, with no interactions with medical treatment or training modality. Protein expression of ULK1 increased with training in both groups (p < 0.001). The increase in quadriceps muscle volume was not correlated with changes in CI+IIP (R = 0.16).
These results suggest that 8 weeks of resistance training with co-ingestion of anti-inflammatory drugs reduces mitochondrial function but increases mitochondrial content. The observed changes were not affected by higher doses of NSAIDs consumption, suggesting that the resistance training intervention was the prime mediator of the decreased mitochondrial phosphorylation. Finally, we noted that flywheel resistance training, emphasizing eccentric overload, rescued some of the reduction in mitochondrial function seen with conventional resistance training.
