Mice Deficient in the Respiratory Chain Gene Cox6a2 Are Protected against High-Fat Diet-Induced Obesity and Insulin Resistance

Oxidative phosphorylation in mitochondria is responsible for 90% of ATP synthesis in most cells. This essential housekeeping function is mediated by nuclear and mitochondrial genes encoding subunits of complex I to V of the respiratory chain. Although complex IV is the best studied of these complexes, the exact function of the striated muscle-specific subunit COX6A2 is still poorly understood. In this study, we show that Cox6a2-deficient mice are protected against high-fat diet-induced obesity, insulin resistance and glucose intolerance. This phenotype results from elevated energy expenditure and a skeletal muscle fiber type switch towards more oxidative fibers. At the molecular level we observe increased formation of reactive oxygen species, constitutive activation of AMP-activated protein kinase, and enhanced expression of uncoupling proteins. Our data indicate that COX6A2 is a regulator of respiratory uncoupling in muscle and we demonstrate that a novel and direct link exists between muscle respiratory chain activity and diet-induced obesity/insulin resistance.     

Yeast cells depleted in Atp14p fail to assemble Atp6p within the ATP synthase and exhibit altered mitochondrial cristae morphology

Within the yeast mitochondrial ATP synthase, subunit h is a small nuclear encoded protein belonging to the so-called "peripheral stalk" that connects the enzyme catalytic F(1) component to the mitochondrial inner membrane. This study examines the role of subunit h in ATP synthase function and assembly using a regulatable, doxycycline-repressible subunit h gene to overcome the strong instability of the mtDNA previously observed in strains lacking the native subunit h gene. Yeast cells expressing less than 3% of subunit h, but still containing intact mitochondrial genomes, grew poorly on respiratory substrates because of a major impairment of ATP synthesis originating from the ATP synthase, whereas the respiratory chain complexes were not affected. The lack of ATP synthesis in the subunit h-depleted (deltah) mitochondria was attributed to defects in the assembly/stability of the ATP synthase. A main feature of deltah-mitochondria was a very low content (<6%) in the mitochondrially encoded Atp6p subunit, an essential component of the enzyme proton channel, which was in large part because of a slowing down in translation. Interestingly, depletion of subunit h resulted in dramatic changes in mitochondrial cristae morphology, which further supports the existence of a link between the ATP synthase and the folding/biogenesis of the inner mitochondrial membrane.     

Mitochondrial respiratory rates and activities of respiratory chain complexes correlate linearly with heteroplasmy of deleted mtDNA without threshold and independently of deletion size

To clarify the importance of deleted protein and tRNA genes on the impairment of mitochondrial function, we performed a quantitative analysis of biochemical, genetic and morphological findings in skeletal muscles of 16 patients with single deletions and 5 patients with multiple deletions of mtDNA. Clinically, all patients showed chronic progressive external ophthalmoplegia (CPEO). The size of deletions varied between 2.5 and 9 kb, and heteroplasmy between 31% and 94%. In patients with single deletions, the citrate synthase (CS) activity was nearly doubled. Decreased ratios of pyruvate- and succinate-dependent respiration were detected in fibers of all patients in comparison to controls. Inverse and linear correlations without thresholds were established between heteroplasmy and (i) CS referenced activities of the complexes of respiratory chain, (ii) CS referenced maximal respiratory rates, (iii) and cytochrome-c-oxidase (COX) negative fibers. In patients with single and multiple deletions, all respiratory chain complexes as well as the respiratory rates were decreased to a similar extent. All changes detected in patients with single deletions were independent of deletion size. In one patient, only genes of ND5, ND4L as well as tRNA(Leu(CUN)), tRNA(Ser(AGY)), and tRNA(His) were deleted. The pronounced decrease in COX activity in this patient points to the high pathological impact of these missing tRNA genes. The activity of nuclear encoded SDH was also significantly decreased in patients, but to a lesser extent. This is an indication of secondary disturbances of mitochondria at CPEO.In conclusion, we have shown that different deletions cause mitochondrial impairments of the same phenotype correlating with heteroplasmy. The missing threshold at the level of mitochondrial function seems to be characteristic for large-scale deletions were tRNA and protein genes are deleted.     

Effects of dietary methylmercury on the zebrafish brain: histological, mitochondrial, and gene transcription analyses

The neurotoxic compound methylmercury (MeHg) is a commonly encountered pollutant in the environment, and constitutes a hazard for wildlife and human health through fish consumption. To study the neurotoxic impact of MeHg on piscivorous fish, we contaminated the model fish species Danio rerio for 25 and 50 days with food containing 13.5 μg/g dry weight (dw) of MeHg (0.6 μg MeHg/fish/day), an environmentally relevant dose leading to brain mercury concentrations of 30 ± 4 μg of Hg g⁻¹ (dw) after 25 days of exposure and 46 ± 7 μg of Hg g⁻¹ (dw) after 50 days. Brain mitochondrial respiration was not modified by exposure to MeHg, contrary to what happens in skeletal muscles. A 6-fold increase in the expression of the sdh gene encoding the succinate dehydrogenase Fe/S protein subunit was detected in the contaminated brain after 50 days of exposure. An up regulation of 3 genes, atp2b3a, atp2b3b, and slc8a2b, encoding for calcium transporters was noticed after 25 days of exposure but the atp2b3a and atp2b3b were repressed and the slc8a2b gene expression returned to its basal level after 50 days, suggesting a perturbation of calcium homeostasis. After 50 days, we detected the up regulation of glial fibrillary acidic protein and glutathione S-transferase genes (gfap and gst), along with a repression of the glutathione peroxidase gene gpx1. These results match well with a MeHg-induced onset of oxidative stress and inflammation. A transmission electron microscopic observation confirmed an impairment of the optical tectum integrity, with a decrease of the nucleal area in contaminated granular cells compared to control cells, and a lower density of cells in the contaminated tissue. A potential functional significance of such changes observed in optical tectum when considering wild fish contaminated in their natural habitat might be an impaired vision and therefore a lowered adaptability to their environment.     

Oxidative phosphorylation in mitochondria from different fiber types of chicken muscles

Isolated mitochondria from different types of muscle fibers from chickens 3 to 5 weeks were studied to evaluate the comparative oxidation of various substrates. Pectoralis (alphaW fibers), lateral adductor (betaR fibers), and medial adductor (alphaR fibers) were the muscles used. Oxygen consumption rates, RCR, and ADP/O ratios were measured to study mitochondrial function. Mitochondria from pectoralis muscle utilized pyruvate, succinate, L-glutamate, alpha-glycerophosphate, and beta-hydroxybutyrate. Mitochondria from the other two muscle types utilized all of those substrates except alpha-glycerophosphate. In each muscle type utilization of NADH was minimum and was not coupled with phosphorylation of ADP. Thus, in alphaW muscles oxidation of alpha-glycerophosphate may play an important role in transport of cytoplasmic NADH to the mitochondrial respiratory chain. In alphaR and betaR muscles "shuttle" systems other than alpha-glycerophosphate oxidation, e.g., beta-hydroxybutyrate, may perform that important role.     

On the stability of key enzymes of energy metabolism in muscle biopsies

Enzymes of energy metabolism were tested for stability depending on different storage conditions (-20, -80 degrees C). To avoid problems due to the different fiber type composition of human muscle, we selected two muscles from rabbit. The m. psoas consists almost exclusively of type 2B fibers, and the m. soleus consists almost exclusively of type 1 fibers. Enzyme activities were measured from small aliquots of these muscles at various time points up to 1 year after sacrificing the animal. Enzymes from anaerobic metabolism were stable for more than 1 year, independent of whether the muscle was stored at -20 or -80 degrees C. Oxidative enzymes, such as succinate dehydrogenase, citrate synthetase, or cytochrome c oxidase (COX) decrease in activity at -20 degrees C and, to a lesser degree, at -80 degrees C. In addition, mitochondria were isolated from freshly taken muscle and stored at -80 degrees C. Oxidative enzymes were surprisingly stable for more than 1 year, with the exception of COX which decreased by 60% of its original activity in mitochondria from m. soleus.     

An mtDNA mutation in the initiation codon of the cytochrome C oxidase subunit II gene results in lower levels of the protein and a mitochondrial encephalomyopathy

A novel heteroplasmic 7587T-->C mutation in the mitochondrial genome which changes the initiation codon of the gene encoding cytochrome c oxidase subunit II (COX II), was found in a family with mitochondrial disease. This T-->C transition is predicted to change the initiating methionine to threonine. The mutation load was present at 67% in muscle from the index case and at 91% in muscle from the patient's clinically affected son. Muscle biopsy samples revealed isolated COX deficiency and mitochondrial proliferation. Single-muscle-fiber analysis revealed that the 7587C copy was at much higher load in COX-negative fibers than in COX-positive fibers. After microphotometric enzyme analysis, the mutation was shown to cause a decrease in COX activity when the mutant load was >55%-65%. In fibroblasts from one family member, which contained >95% mutated mtDNA, there was no detectable synthesis or any steady-state level of COX II. This new mutation constitutes a new mechanism by which mtDNA mutations can cause disease-defective initiation of translation.     

Flux control of cytochrome c oxidase in human skeletal muscle

In the present work, by titrating cytochrome c oxidase (COX) with the specific inhibitor KCN, the flux control coefficient and the metabolic reserve capacity of COX have been determined in human saponin-permeabilized muscle fibers. In the presence of the substrates glutamate and malate, a 2.3 +/- 0.2-fold excess capacity of COX was observed in ADP-stimulated human skeletal muscle fibers. This value was found to be dependent on the mitochondrial substrate supply. In the combined presence of glutamate, malate, and succinate, which supported an approximately 1.4-fold higher rate of respiration, only a 1.4 +/- 0.2-fold excess capacity of COX was determined. In agreement with these findings, the flux control of COX increased, in the presence of the three substrates, from 0.27 +/- 0.03 to 0.36 +/- 0.08. These results indicate a tight in vivo control of respiration by COX in human skeletal muscle. This tight control may have significant implications for mitochondrial myopathies. In support of this conclusion, the analysis of skeletal muscle fibers from two patients with chronic progressive external ophthalmoplegia, which carried deletions in 11 and 49% of their mitochondrial DNA, revealed a substantially lowered reserve capacity and increased flux control coefficient of COX, indicating severe rate limitations of oxidative phosphorylation by this enzyme.     

The contribution of uncoupling protein and ATP synthase to state 3 respiration in Acanthamoeba castellanii mitochondria

Mitochondria of the amoeba Acanthamoeba castellanii possess a free fatty acid-activated uncoupling protein (AcUCP) that mediates proton re-uptake driven by the mitochondrial proton electrochemical gradient. We show that AcUCP activity diverts energy from ATP synthesis during state 3 mitochondrial respiration in a fatty acid-dependent way. The efficiency of AcUCP in mitochondrial uncoupling increases when the state 3 respiratory rate decreases as the AcUCP contribution is constant at a given linoleic acid concentration while the ATP synthase contribution decreases with respiratory rate. Respiration sustained by this energy-dissipating process remains constant at a given linoleic acid concentration until more than 60% inhibition of state 3 respiration by n-butyl malonate is achieved. The present study supports the validity of the ADP/O method to determine the actual contributions of AcUCP (activated with various linoleic acid concentrations) and ATP synthase in state 3 respiration of A.castellanii mitochondria fully depleted of free fatty acid-activated and describes how the two contributions vary when the rate of succinate dehydrogenase is decreased by succinate uptake limitation.     

Bioenergetic remodeling during cellular differentiation: changes in cytochrome c oxidase regulation do not affect the metabolic phenotype.

Myogenesis induces mitochondrial proliferation, a decrease in reactive oxygen species (ROS) production, and an increased reliance upon oxidative phosphorylation. While muscles typically possess 20%-40% excess capacity of cytochrome c oxidase (COX), undifferentiated myoblasts have only 5%-20% of the mitochondrial content of myotubes and muscles. We used two muscle lines (C2C12, Sol8) and 3T3-L1 pre-adipocytes to examine if changes in COX regulation or activity with differentiation cause a shift in metabolic phenotype (i.e., more oxidative, less glycolytic, less ROS). COX activity in vivo can be suppressed by its inhibitor, nitric oxide, or sub-optimal substrate (cytochrome c) concentrations. Inhibition of nitric oxide synthase via L-NAME had no effect on the respiration of adherent undifferentiated cells, although it did stimulate respiration of myoblasts in suspension. While cytochrome c content increased during differentiation, there was no correlation with respiratory rate or reliance on oxidative metabolism. There was no correlation between COX specific activity and oxidative metabolism between cell type or in relation to differentiation. These studies show that, despite the very low activities of COX, undifferentiated myoblasts and pre-adipocytes possess a reserve of COX capacity and changes in COX with differentiation do not trigger the shift in metabolic phenotype.     

Antagonistic Regulation of Parvalbumin Expression and Mitochondrial Calcium Handling Capacity in Renal Epithelial Cells

Parvalbumin (PV) is a cytosolic Ca²⁺-binding protein acting as a slow-onset Ca²⁺ buffer modulating the shape of Ca²⁺ transients in fast-twitch muscles and a subpopulation of neurons. PV is also expressed in non-excitable cells including distal convoluted tubule (DCT) cells of the kidney, where it might act as an intracellular Ca²⁺ shuttle facilitating transcellular Ca²⁺ resorption. In excitable cells, upregulation of mitochondria in “PV-ergic” cells in PV-/- mice appears to be a general hallmark, evidenced in fast-twitch muscles and cerebellar Purkinje cells. Using Gene Chip Arrays and qRT-PCR, we identified differentially expressed genes in the DCT of PV-/- mice. With a focus on genes implicated in mitochondrial Ca²⁺ transport and membrane potential, uncoupling protein 2 (Ucp2), mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu), mitochondrial calcium uniporter regulator 1 (Mcur1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase subunit β (Atp5b) were found to be up-upregulated. At the protein level, COX1 was increased by 31 ± 7%, while ATP-synthase subunit β was unchanged. This suggested that these mitochondria were better suited to uphold the electrochemical potential across the mitochondrial membrane, necessary for mitochondrial Ca²⁺ uptake. Ectopic expression of PV in PV-negative Madin-Darby canine kidney (MDCK) cells decreased COX1 and concomitantly mitochondrial volume, while ATP synthase subunit β levels remained unaffected. Suppression of PV by shRNA in PV-expressing MDCK cells led subsequently to an increase in COX1 expression. The collapsing of the mitochondrial membrane potential by the uncoupler CCCP occurred at lower concentrations in PV-expressing MDCK cells than in control cells. In support, a reduction of the relative mitochondrial mass was observed in PV-expressing MDCK cells. Deregulation of the cytoplasmic Ca²⁺ buffer PV in kidney cells was counterbalanced in vivo and in vitro by adjusting the relative mitochondrial volume and modifying the mitochondrial protein composition conceivably to increase their Ca²⁺-buffering/sequestration capacity.     

The role of nitric oxide in muscle fibers with oxidative phosphorylation defects

NO has been pointed as an important player in the control of mitochondrial respiration, especially because of its inhibitory effect on cytochrome c oxidase (COX). However, all the events involved in this control are still not completely elucidated. We demonstrate compartmentalized abnormalities on nitric oxide synthase (NOS) activity on muscle biopsies of patients with mitochondrial diseases. NOS activity was reduced in the sarcoplasmic compartment in COX deficient fibers, whereas increased activity was found in the sarcolemma of fibers with mitochondrial proliferation. We observed increased expression of neuronal NOS (nNOS) in patients and a correlation between nNOS expression and mitochondrial content. Treatment of skeletal muscle culture with an NO donor induced an increase in mitochondrial content. Our results indicate specific roles of NO in compensatory mechanisms of muscle fibers with mitochondrial deficiency and suggest the participation of nNOS in the signaling process of mitochondrial proliferation in human skeletal muscle.     

Cellular phenotypes of age-associated skeletal muscle mitochondrial abnormalities in rhesus monkeys

Rhesus monkey vastus lateralis muscle was examined histologically for age-associated electron transport system (ETS) abnormalities: fibers lacking cytochrome c oxidase activity (COX(-)) and/or exhibiting succinate dehydrogenase hyperreactivity (SDH(++)). Two hundred serial cross-sections (spanning 1600 microm) were obtained and analyzed for ETS abnormalities at regular intervals. The abundance and length of ETS abnormal regions increased with age. Extrapolating the data to the entire length of the fiber, up to 60% of the fibers were estimated to display ETS abnormalities in the oldest animal studied (34 years) compared to 4% in a young adult animal (11 years). ETS abnormal phenotypes varied with age and fiber type. Middle-aged animals primarily exhibited the COX(-) phenotype, while COX(-)/SDH(++) abnormalities were more common in old animals. Transition region phenotype was affected by fiber type with type 2 fibers first displaying COX(-) and then COX(-)/SDH(++) while type 1 fibers progressed from normal to SDH(++) and then to COX(-)/SDH(++). In situ hybridizations studies revealed an association of ETS abnormalities with deletions of the mitochondrial genome. By measuring cross-sectional area along the length of ETS abnormal fibers, we demonstrated that some of these fibers exhibit atrophy. Our data suggest mitochondrial (mtDNA) deletions and associated ETS abnormalities are contributors to age-associated fiber atrophy.     

Control of skeletal muscle mitochondria respiration by adenine nucleotides: differential effect of ADP and ATP according to muscle contractile type in pigs

Skeletal muscle exhibits considerable variation in mitochondrial content among fiber types, but it is less clear whether mitochondria from different fiber types also present specific functional and regulatory properties. The present experiment was undertaken on ten 170-day-old pigs to compare functional properties and control of respiration by adenine nucleotides in mitochondria isolated from predominantly slow-twitch (Rhomboideus (RM)) and fast-twitch (Longissimus (LM)) muscles. Mitochondrial ATP synthesis, respiratory control ratio (RCR) and ADP-stimulated respiration with either complex I or II substrates were significantly higher (25-30%, P<0.05) in RM than in LM mitochondria, whereas no difference was observed for basal respiration. Based on mitochondrial enzyme activities (cytochrome c oxidase [COX], F0F1-ATPase, mitochondrial creatine kinase [mi-CK]), the higher ADP-stimulated respiration rate of RM mitochondria appeared mainly related to a higher maximal oxidative capacity, without any difference in the maximal phosphorylation potential. Mitochondrial K(m) for ADP was similar in RM (4.4+/-0.9 microM) and LM (5.9+/-1.2 microM) muscles (P>0.05) but the inhibitory effect of ATP was more marked in LM (P<0.01). These findings demonstrate that the regulation of mitochondrial respiration by ATP differs according to muscle contractile type and that absolute muscle oxidative capacity not only relies on mitochondrial density but also on mitochondrial functioning per se.     

Protein phosphorylation and prevention of cytochrome oxidase inhibition by ATP: coupled mechanisms of energy metabolism regulation

Rapid regulation of oxidative phosphorylation is crucial for mitochondrial adaptation to swift changes in fuels availability and energy demands. An intramitochondrial signaling pathway regulates cytochrome oxidase (COX), the terminal enzyme of the respiratory chain, through reversible phosphorylation. We find that PKA-mediated phosphorylation of a COX subunit dictates mammalian mitochondrial energy fluxes and identify the specific residue (S58) of COX subunit IV-1 (COXIV-1) that is involved in this mechanism of metabolic regulation. Using protein mutagenesis, molecular dynamics simulations, and induced fit docking, we show that mitochondrial energy metabolism regulation by phosphorylation of COXIV-1 is coupled with prevention of COX allosteric inhibition by ATP. This regulatory mechanism is essential for efficient oxidative metabolism and cell survival. We propose that S58 COXIV-1 phosphorylation has evolved as a metabolic switch that allows mammalian mitochondria to rapidly toggle between energy utilization and energy storage.     

Mitochondrial respiratory rates and activities of respiratory chain complexes correlate linearly with heteroplasmy of deleted mtDNA without threshold and independently of deletion size

To clarify the importance of deleted protein and tRNA genes on the impairment of mitochondrial function, we performed a quantitative analysis of biochemical, genetic and morphological findings in skeletal muscles of 16 patients with single deletions and 5 patients with multiple deletions of mtDNA. Clinically, all patients showed chronic progressive external ophthalmoplegia (CPEO). The size of deletions varied between 2.5 and 9 kb, and heteroplasmy between 31% and 94%. In patients with single deletions, the citrate synthase (CS) activity was nearly doubled. Decreased ratios of pyruvate- and succinate-dependent respiration were detected in fibers of all patients in comparison to controls. Inverse and linear correlations without thresholds were established between heteroplasmy and (i) CS referenced activities of the complexes of respiratory chain, (ii) CS referenced maximal respiratory rates, (iii) and cytochrome-c-oxidase (COX) negative fibers. In patients with single and multiple deletions, all respiratory chain complexes as well as the respiratory rates were decreased to a similar extent. All changes detected in patients with single deletions were independent of deletion size. In one patient, only genes of ND5, ND4L as well as tRNA_Leu(CUN), tRNA_Ser(AGY), and tRNA_His were deleted. The pronounced decrease in COX activity in this patient points to the high pathological impact of these missing tRNA genes. The activity of nuclear encoded SDH was also significantly decreased in patients, but to a lesser extent. This is an indication of secondary disturbances of mitochondria at CPEO.In conclusion, we have shown that different deletions cause mitochondrial impairments of the same phenotype correlating with heteroplasmy. The missing threshold at the level of mitochondrial function seems to be characteristic for large-scale deletions were tRNA and protein genes are deleted.     

Regulation of mitochondrial respiration and apoptosis through cell signaling: cytochrome c oxidase and cytochrome c in ischemia/reperfusion injury and inflammation

Cytochrome c (Cytc) and cytochrome c oxidase (COX) catalyze the terminal reaction of the mitochondrial electron transport chain (ETC), the reduction of oxygen to water. This irreversible step is highly regulated, as indicated by the presence of tissue-specific and developmentally expressed isoforms, allosteric regulation, and reversible phosphorylations, which are found in both Cytc and COX. The crucial role of the ETC in health and disease is obvious since it, together with ATP synthase, provides the vast majority of cellular energy, which drives all cellular processes. However, under conditions of stress, the ETC generates reactive oxygen species (ROS), which cause cell damage and trigger death processes. We here discuss current knowledge of the regulation of Cytc and COX with a focus on cell signaling pathways, including cAMP/protein kinase A and tyrosine kinase signaling. Based on the crystal structures we highlight all identified phosphorylation sites on Cytc and COX, and we present a new phosphorylation site, Ser126 on COX subunit II. We conclude with a model that links cell signaling with the phosphorylation state of Cytc and COX. This in turn regulates their enzymatic activities, the mitochondrial membrane potential, and the production of ATP and ROS. Our model is discussed through two distinct human pathologies, acute inflammation as seen in sepsis, where phosphorylation leads to strong COX inhibition followed by energy depletion, and ischemia/reperfusion injury, where hyperactive ETC complexes generate pathologically high mitochondrial membrane potentials, leading to excessive ROS production. Although operating at opposite poles of the ETC activity spectrum, both conditions can lead to cell death through energy deprivation or ROS-triggered apoptosis.