Mitochondrial H2O2 Emission Research Articles

Targeted overexpression of mitochondrial catalase protects against cancer chemotherapy-induced skeletal muscle dysfunction.

The loss of strength in combination with constant fatigue is a burden on cancer patients undergoing chemotherapy. Doxorubicin, a standard chemotherapy drug used in the clinic, causes skeletal muscle dysfunction and increases mitochondrial H2O2 We hypothesized that the combined effect of cancer and chemotherapy in an immunocompetent breast cancer mouse model (E0771) would compromise skeletal muscle mitochondrial respiratory function, leading to an increase in H2O2-emitting potential and impaired muscle function. Here, we demonstrate that cancer chemotherapy decreases mitochondrial respiratory capacity supported with complex I (pyruvate/glutamate/malate) and complex II (succinate) substrates. Mitochondrial H2O2-emitting potential was altered in skeletal muscle, and global protein oxidation was elevated with cancer chemotherapy. Muscle contractile function was impaired following exposure to cancer chemotherapy. Genetically engineering the overexpression of catalase in mitochondria of muscle attenuated mitochondrial H2O2 emission and protein oxidation, preserving mitochondrial and whole muscle function despite cancer chemotherapy. These findings suggest mitochondrial oxidants as a mediator of cancer chemotherapy-induced skeletal muscle dysfunction.

Pharmacological targeting of mitochondrial reactive oxygen species counteracts diaphragm weakness in chronic heart failure.

Diaphragm muscle weakness in chronic heart failure (CHF) is caused by elevated oxidants and exacerbates breathing abnormalities, exercise intolerance, and dyspnea. However, the specific source of oxidants that cause diaphragm weakness is unknown. We examined whether mitochondrial reactive oxygen species (ROS) cause diaphragm weakness in CHF by testing the hypothesis that CHF animals treated with a mitochondria-targeted antioxidant have normal diaphragm function. Rats underwent CHF or sham surgery. Eight weeks after surgeries, we administered a mitochondrial-targeted antioxidant (MitoTEMPO; 1 mg·kg(-1)·day(-1)) or sterile saline (Vehicle). Left ventricular dysfunction (echocardiography) pre- and posttreatment and morphological abnormalities were consistent with the presence of CHF. CHF elicited a threefold (P < 0.05) increase in diaphragm mitochondrial H2O2 emission, decreased diaphragm glutathione content by 23%, and also depressed twitch and maximal tetanic force by ∼20% in Vehicle-treated animals compared with Sham (P < 0.05 for all comparisons). Diaphragm mitochondrial H2O2 emission, glutathione content, and twitch and maximal tetanic force were normal in CHF animals receiving MitoTEMPO. Neither CHF nor MitoTEMPO altered the diaphragm protein levels of antioxidant enzymes: superoxide dismutases (CuZn-SOD or MnSOD), glutathione peroxidase, and catalase. In both Vehicle and MitoTEMPO groups, CHF elicited a ∼30% increase in cytochrome c oxidase activity, whereas there were no changes in citrate synthase activity. Our data suggest that elevated mitochondrial H2O2 emission causes diaphragm weakness in CHF. Moreover, changes in protein levels of antioxidant enzymes or mitochondrial content do not seem to mediate the increase in mitochondria H2O2 emission in CHF and protective effects of MitoTEMPO.

High-intensity sprint training inhibits mitochondrial respiration through aconitase inactivation.

Intense exercise training is a powerful stimulus that activates mitochondrial biogenesis pathways and thus increases mitochondrial density and oxidative capacity. Moderate levels of reactive oxygen species (ROS) during exercise are considered vital in the adaptive response, but high ROS production is a serious threat to cellular homeostasis. Although biochemical markers of the transition from adaptive to maladaptive ROS stress are lacking, it is likely mediated by redox sensitive enzymes involved in oxidative metabolism. One potential enzyme mediating such redox sensitivity is the citric acid cycle enzyme aconitase. In this study, we examined biopsy specimens of vastus lateralis and triceps brachii in healthy volunteers, together with primary human myotubes. An intense exercise regimen inactivated aconitase by 55-72%, resulting in inhibition of mitochondrial respiration by 50-65%. In the vastus, the mitochondrial dysfunction was compensated for by a 15-72% increase in mitochondrial proteins, whereas H2O2 emission was unchanged. In parallel with the inactivation of aconitase, the intermediary metabolite citrate accumulated and played an integral part in cellular protection against oxidative stress. In contrast, the triceps failed to increase mitochondrial density, and citrate did not accumulate. Instead, mitochondrial H2O2 emission was decreased to 40% of the pretraining levels, together with a 6-fold increase in protein abundance of catalase. In this study, a novel mitochondrial stress response was highlighted where accumulation of citrate acted to preserve the redox status of the cell during periods of intense exercise.

Skeletal muscle mitochondrial H2 O2 emission increases with immobilization and decreases after aerobic training in young and older men.

Currently, it is not known whether impaired mitochondrial function contributes to human ageing or whether potential impairments in mitochondrial function with age are secondary to physical inactivity. The present study investigated mitochondrial respiratory function and reactive oxygen species emission at a predefined membrane potential in young and older men subjected to 2 weeks of one-leg immobilization followed by 6 weeks of aerobic cycle training. Immobilization increased reactive oxygen species emission and decreased ATP generating respiration. Subsequent aerobic training reversed these effects. By contrast, age had no effect on the measured variables. The results of the present study support the notion that increased mitochondrial reactive oxygen species production mediates the detrimental effects seen after physical inactivity and that ageing per se does not cause mitochondrial dysfunction. Mitochondrial dysfunction, defined as increased oxidative stress and lower capacity for energy production, may be seen with ageing and may cause frailty, or it could be that it is secondary to physical inactivity. We studied the effect of 2 weeks of one-leg immobilization followed by 6 weeks of supervised cycle training on mitochondrial function in 17 young (mean ± SEM: 23 ± 1 years) and 15 older (68 ± 1 years) healthy men. Submaximal H2 O2 emission and respiration were measured simultaneously at a predefined membrane potential in isolated mitochondria from skeletal muscle using two protocols: pyruvate + malate (PM) and succinate + rotenone (SR). This allowed measurement of leak and ATP generating respiration from which the coupling efficiency can be calculated. The protein content of the anti-oxidants manganese superoxide dismuthase (MnSOD), CuZn superoxide dismuthase, catalase and gluthathione peroxidase 1 was measured by western blotting. Immobilization decreased ATP generating respiration using PM and increased H2 O2 emission using both PM and SR similarly in young and older men. Both were restored to baseline after the training period. Furthermore, MnSOD and catalase content increased with endurance training. The young men had a higher leak respiration at inclusion using PM and a higher membrane potential in State 3 using both substrate combinations. Collectively, the findings of the present study support the notion that increased mitochondrial reactive oxygen species mediates the detrimental effects seen after physical inactivity. Age, on the other hand, was not associated with impairments in anti-oxidant protein levels, mitochondrial respiration or H2 O2 emission using either protocol.

Rapid Repression of ADP Transport by Palmitoyl-CoA Is Attenuated by Exercise Training in Humans: A Potential Mechanism to Decrease Oxidative Stress and Improve Skeletal Muscle Insulin Signaling.

Mitochondrial ADP transport may represent a convergence point unifying two prominent working models for the development of insulin resistance, as reactive lipids (specifically palmitoyl-CoA [P-CoA]) can inhibit ADP transport and subsequently increase mitochondrial reactive oxygen species emissions. In the current study, we aimed to determine if exercise training in humans diminished P-CoA attenuation of mitochondrial ADP respiratory sensitivity. Six weeks of exercise training increased whole-body glucose homeostasis and skeletal muscle Akt signaling and reduced markers of oxidative stress without reducing maximal mitochondrial H2O2 emissions. To ascertain if enhanced mitochondrial ADP transport contributed to the improvement in the in vivo oxidative state, we determined mitochondrial ADP sensitivity in the presence and absence of P-CoA. In the absence of P-CoA, exercise training reduced mitochondrial ADP sensitivity. In contrast, exercise training increased mitochondrial ADP sensitivity with P-CoA present. We further show that P-CoA noncompetitively inhibits mitochondrial ADP transport and the ability of ADP to attenuate mitochondrial H2O2 emission. Altogether, the current data provide a potential mechanism for how P-CoA contributes to insulin resistance and highlight the ability of exercise training to diminish P-CoA attenuation in mitochondrial ADP transport.

Linoleic and α‐Linolenic Acid Prevent Insulin Resistance in Obese Zucker Rats but have Different Impacts on Skeletal Muscle Metabolism

The use of long‐chain polyunsaturated fatty acids (LC‐PUFAs) to prevent the development of insulin resistance (IR) has recently gained considerable interest. However, the roles of essential PUFAs (i.e., linoleic acid, LA; α‐linolenic acid, ALA) remain poorly understood. We investigated the efficacy of diets enriched with either LA or ALA on preventing IR in obese Zucker rats. After the 12‐wk intervention, both LA‐ and ALA‐enriched diets protected against IR compared to obese control animals. The maintenance of whole‐body glucose homeostasis was linked to the preservation of muscle‐specific insulin sensitivity. LA and ALA differentially regulated markers of skeletal muscle mitochondrial content, respiratory function and H2O2 emission. However, both LA and ALA prevented increases in 4‐HNE content (i.e., a marker of oxidative stress) compared to obese control animals. Collectively, the protective effects of LA and ALA appear independent of mitochondrial bioenergetics, but may involve improvements in oxidative stress. Subsequent lipid profiling in skeletal muscle by gas chromatography revealed that LA and ALA increased omega‐6 and omega‐3 PUFA content, as expected. However, diacylglycerol (DAG) and ceramide accumulation was similar between obese control, LA and ALA groups; suggesting that preservation of skeletal muscle insulin sensitivity occurred independent of changes in reactive lipid content. Overall, LA and ALA are efficacious in preventing obesity‐related IR, but further work is necessary to elucidate the mechanism(s) of action.Grant Funding Source: Supported by OMAFRA

Isoflurane anesthesia initiated at the onset of reperfusion attenuates oxidative and hypoxic-ischemic brain injury.

This study demonstrates that in mice subjected to hypoxia-ischemia (HI) brain injury isoflurane anesthesia initiated upon reperfusion limits a release of mitochondrial oxidative radicals by inhibiting a recovery of complex-I dependent mitochondrial respiration. This significantly attenuates an oxidative stress and reduces the extent of HI brain injury. Neonatal mice were subjected to HI, and at the initiation of reperfusion were exposed to isoflurane with or without mechanical ventilation. At the end of HI and isoflurane exposure cerebral mitochondrial respiration, H2O2 emission rates were measured followed by an assessment of cerebral oxidative damage and infarct volumes. At 8 weeks after HI navigational memory and brain atrophy were assessed. In vitro, direct effect of isoflurane on mitochondrial H2O2 emission was compared to that of complex-I inhibitor, rotenone. Compared to controls, 15 minutes of isoflurane anesthesia inhibited recovery of the compex I-dependent mitochondrial respiration and decreased H2O2 production in mitochondria supported with succinate. This was associated with reduced oxidative brain injury, superior navigational memory and decreased cerebral atrophy compared to the vehicle-treated HI-mice. Extended isoflurane anesthesia was associated with sluggish recovery of cerebral blood flow (CBF) and the neuroprotection was lost. However, when isoflurane anesthesia was supported with mechanical ventilation the CBF recovery improved, the event associated with further reduction of infarct volume compared to HI-mice exposed to isoflurane without respiratory support. Thus, in neonatal mice brief isoflurane anesthesia initiated at the onset of reperfusion limits mitochondrial release of oxidative radicals and attenuates an oxidative stress. This novel mechanism contributes to neuroprotective action of isoflurane. The use of mechanical ventilation during isoflurane anesthesia counterbalances negative effect of isoflurane anesthesia on recovery of cerebral circulation which potentiates protection against reperfusion injury.

Both linoleic and α-linolenic acid prevent insulin resistance but have divergent impacts on skeletal muscle mitochondrial bioenergetics in obese Zucker rats.

The therapeutic use of polyunsaturated fatty acids (PUFA) in preserving insulin sensitivity has gained interest in recent decades; however, the roles of linoleic acid (LA) and α-linolenic acid (ALA) remain poorly understood. We investigated the efficacy of diets enriched with either LA or ALA on attenuating the development of insulin resistance (IR) in obesity. Following a 12-wk intervention, LA and ALA both prevented the shift toward an IR phenotype and maintained muscle-specific insulin sensitivity otherwise lost in obese control animals. The beneficial effects of ALA were independent of changes in skeletal muscle mitochondrial content and oxidative capacity, as obese control and ALA-treated rats showed similar increases in these parameters. However, ALA increased the propensity for mitochondrial H2O2 emission and catalase content within whole muscle and reduced markers of oxidative stress (4-HNE and protein carbonylation). In contrast, LA prevented changes in markers of mitochondrial content, respiratory function, H2O2 emission, and oxidative stress in obese animals, thereby resembling levels seen in lean animals. Together, our data suggest that LA and ALA are efficacious in preventing IR but have divergent impacts on skeletal muscle mitochondrial content and function. Moreover, we propose that LA has value in preserving insulin sensitivity in the development of obesity, thereby challenging the classical view that n-6 PUFAs are detrimental.

Mitochondrial-targeted peptide rapidly improves mitochondrial energetics and skeletal muscle performance in aged mice.

Mitochondrial dysfunction plays a key pathogenic role in aging skeletal muscle resulting in significant healthcare costs in the developed world. However, there is no pharmacologic treatment to rapidly reverse mitochondrial deficits in the elderly. Here, we demonstrate that a single treatment with the mitochondrial-targeted peptide SS-31 restores in vivo mitochondrial energetics to young levels in aged mice after only one hour. Young (5month old) and old (27month old) mice were injected intraperitoneally with either saline or 3mg kg(-1) of SS-31. Skeletal muscle mitochondrial energetics were measured in vivo one hour after injection using a unique combination of optical and (31) P magnetic resonance spectroscopy. Age-related declines in resting and maximal mitochondrial ATP production, coupling of oxidative phosphorylation (P/O), and cell energy state (PCr/ATP) were rapidly reversed after SS-31 treatment, while SS-31 had no observable effect on young muscle. These effects of SS-31 on mitochondrial energetics in aged muscle were also associated with a more reduced glutathione redox status and lower mitochondrial H2 O2 emission. Skeletal muscle of aged mice was more fatigue resistant in situ one hour after SS-31 treatment, and eight days of SS-31 treatment led to increased whole-animal endurance capacity. These data demonstrate that SS-31 represents a new strategy for reversing age-related deficits in skeletal muscle with potential for translation into human use.

High-fat load: mechanism(s) of insulin resistance in skeletal muscle.

Skeletal muscle from sedentary obese patients is characterized by depressed electron transport activity, reduced expression of genes required for oxidative metabolism, altered mitochondrial morphology and lower overall mitochondrial content. These findings imply that obesity, or more likely the metabolic imbalance that causes obesity, leads to a progressive decline in mitochondrial function, eventually culminating in mitochondrial dissolution or mitoptosis. A decrease in the sensitivity of skeletal muscle to insulin represents one of the earliest maladies associated with high dietary fat intake and weight gain. Considerable evidence has accumulated to suggest that the cytosolic ectopic accumulation of fatty acid metabolites, including diacylglycerol and ceramides, underlies the development of insulin resistance in skeletal muscle. However, an alternative mechanism has recently been evolving, which places the etiology of insulin resistance in the context of cellular/mitochondrial bioenergetics and redox systems biology. Overnutrition, particularly from high-fat diets, generates fuel overload within the mitochondria, resulting in the accumulation of partially oxidized acylcarnitines, increased mitochondrial hydrogen peroxide (H2O2) emission and a shift to a more oxidized intracellular redox environment. Blocking H2O2 emission prevents the shift in redox environment and preserves insulin sensitivity, providing evidence that the mitochondrial respiratory system is able to sense and respond to cellular metabolic imbalance. Mitochondrial H2O2 emission is a major regulator of protein redox state, as well as the overall cellular redox environment, raising the intriguing possibility that elevated H2O2 emission from nutrient overload may represent the underlying basis for the development of insulin resistance due to disruption of normal redox control mechanisms regulating protein function, including the insulin signaling and glucose transport processes.

Intrinsic aerobic capacity correlates with greater inherent mitochondrial oxidative and H2O2 emission capacities without major shifts in myosin heavy chain isoform.

Exercise capacity and performance strongly associate with metabolic and biophysical characteristics of skeletal muscle, factors that also relate to overall disease risk. Despite its importance, the exact mechanistic features that connect aerobic metabolism with health status are unknown. To explore this, we applied artificial selection of rats for intrinsic (i.e., untrained) aerobic treadmill running to generate strains of low- and high-capacity runners (LCR and HCR, respectively), subsequently shown to diverge for disease risk. Concurrent breeding of LCR and HCR per generation allows the lines to serve as reciprocal controls for unknown environmental changes. Here we provide the first direct evidence in mitochondria isolated from skeletal muscle that intrinsic mitochondrial capacity is higher in HCR rats. Maximal phosphorylating respiration was ~40% greater in HCR mitochondria, independent of substrate and without altered proton leak or major changes in protein levels or muscle fiber type, consistent with altered control of phosphorylating respiration. Unexpectedly, H(2)O(2) emission was ~20% higher in HCR mitochondria, due to greater reduction of more harmful reactive oxygen species to H(2)O(2); indeed, oxidative modification of mitochondrial proteins was lower. When the higher mitochondrial yield was considered, phosphorylating respiration and H(2)O(2) emission were 70-80% greater in HCR muscle. Greater capacity of HCR muscle for work and H(2)O(2) signaling may result in enhanced and more immediate cellular repair, possibly explaining lowered disease risks.

Differential Adaptations to High‐calorie, Western‐pattern Diet and Exercise Training in Redox Status and Mitochondria of Heart and Skeletal Muscle

Two interventions known to cause profound changes in cellular metabolism and redox enzyme expression/activity in heart and skeletal muscle are exercise (EX), and high‐fat, high sugar “Western pattern” diet (HFHS). To determine whether heart and skeletal muscle are differentially impacted by HFHS diet and EX, Sprague‐Dawley rats were fed either standard chow (CON) or a HFHS diet for 12 weeks (HFHS). A sub‐group of HFHS rats exercise‐trained 5 days/week for 2 hours/day (HFHS+EX). Following these interventions, mitochondrial H2O2 emission (mH2O2) and Ca2+ tolerance (mCa2+) supported by pyruvate + malate + succinate, and O2 consumption (mO2) supported by glutamate+malate (GM) and palmitoyl‐carnitine (PC) was determined in permeabilized red gastrocnemius (RG) and cardiac fibers. HFHS diet increased mH2O2 ~2‐fold, and HFHS+EX decreased in RG (P&lt;0.05) compared to CON. Surprisingly, the HFHS diet decreased mH2O2 ~2‐fold in heart compared to CON (P&lt;0.05), and was even further decreased in HFHS+EX. Maximal PC‐supported mO2 increased in RG in both HFHS and HFHS+Ex compared to CON (P&lt;0.05), but in heart this was only increased in HFHS+EX. Maximal GM‐supported mO2 decreased in both RG and heart with HFHS, and this decrease was prevented in HFHS+EX. Total mCa2+ increased only with HFHS+EX in both RG and heart compared to CON. These findings suggest that heart and skeletal muscle mitochondria are differentially affected by diet and exercise, which point to adaptations in cellular redox state. (Supported by NIH grant HL098780 to EJA)

Doxorubicin impairs skeletal muscle mitochondrial respiratory capacity in skeletal muscle

Debilitating muscle weakness and fatigue are common side effects of chemotherapy in cancer patients, limiting recovery and increasing morbidity. Our previous work shows that doxorubicin, a common chemotherapy drug, elevates reactive oxygen species (ROS) and decreases contractile force in skeletal muscle. As mitochondria represent a primary source of oxidant generation in muscle, we hypothesized that doxorubicin may compromise mitochondrial respiratory control and increase ROS production. 72 hrs following a single doxorubicin injection (20 mg/kg), maximal ADP‐stimulated O2 consumption was decreased during respiration supported by palmitoylcarnitine (PC) (−46 ± 26 %, p&lt;0.05) and pyruvate/malate (−43 ± 21 %, p&lt;0.05, n=6/group) in permeabilized fiber bundles (PmFB) from red gastrocnemius muscle. PmFB from doxorubicin treated animals also display increased rates of mitochondrial H2O2 emission with PC (23 ± 2 %) and 3‐phosphoglycerate (49 ± 2 %, p=0.1). Our data indicate that doxorubicin impairs both maximal State 3, and fatty‐acid‐supported respiration. In addition, doxorubicin promotes mitochondrial ROS generation, a potential underlying cause of doxorubicin‐induced muscle dysfunction.Supported by DK073488

Progesterone increases skeletal muscle mitochondrial H2O2emission in nonmenopausal women

The luteal phase of the female menstrual cycle is associated with both 1) elevated serum progesterone (P4) and estradiol (E2), and 2) reduced insulin sensitivity. Recently, we demonstrated a link between skeletal muscle mitochondrial H(2)O(2) emission (mE(H2O2)) and insulin resistance. To determine whether serum levels of P4 and/or E(2) are related to mitochondrial function, mE(H2O2) and respiratory O(2) flux (Jo(2)) were measured in permeabilized myofibers from insulin-sensitive (IS, n = 24) and -resistant (IR, n = 8) nonmenopausal women (IR = HOMA-IR > 3.6). Succinate-supported mE(H2O2) was more than 50% greater in the IR vs. IS women (P < 0.05). Interestingly, serum P4 correlated positively with succinate-supported mE(H2O2) (r = 0. 53, P < 0.01). To determine whether P4 or E2 directly affect mitochondrial function, saponin-permeabilized vastus lateralis myofibers biopsied from five nonmenopausal women in the early follicular phase were incubated in P4 (60 nM), E2 (1.4 nM), or both. P4 alone inhibited state 3 Jo(2), supported by multisubstrate combination (P < 0.01). However, E2 alone or in combination with P4 had no effect on Jo(2). In contrast, during state 4 respiration, supported by succinate and glycerophosphate, mE(H2O2) was increased with P4 alone or in combination with E2 (P < 0.01). The results suggest that 1) P4 increases mE(H2O2) with or without E2; 2) P4 alone inhibits Jo(2) but not when E2 is present; and 3) P4 is related to the mE(H2O2) previously linked to skeletal muscle insulin resistance.

Increased propensity for cell death in diabetic human heart is mediated by mitochondrial-dependent pathways

Progressive energy deficiency and loss of cardiomyocyte numbers are two prominent factors that lead to heart failure in experimental models. Signals that mediate cardiomyocyte cell death have been suggested to come from both extrinsic (e.g., cytokines) and intrinsic (e.g., mitochondria) sources, but the evidence supporting these mechanisms remains unclear, and virtually nonexistent in humans. In this study, we investigated the sensitivity of the mitochondrial permeability transition pore (mPTP) to calcium (Ca(2+)) using permeabilized myofibers of right atrium obtained from diabetic (n = 9) and nondiabetic (n = 12) patients with coronary artery disease undergoing nonemergent coronary revascularization surgery. Under conditions that mimic the energetic state of the heart in vivo (pyruvate, glutamate, malate, and 100 μM ADP), cardiac mitochondria from diabetic patients show an increased sensitivity to Ca(2+)-induced mPTP opening compared with nondiabetic patients. This increased mPTP Ca(2+) sensitivity in diabetic heart mitochondria is accompanied by a substantially greater rate of mitochondrial H(2)O(2) emission under identical conditions, despite no differences in respiratory capacity under these conditions or mitochondrial enzyme content. Activity of the intrinsic apoptosis pathway mediator caspase-9 was greater in diabetic atrial tissue, whereas activity of the extrinsic pathway mediator caspase-8 was unchanged between groups. Furthermore, caspase-3 activity was not significantly increased in diabetic atrial tissue. These data collectively suggest that the myocardium in diabetic patients has a greater overall propensity for mitochondrial-dependent cell death, possibly as a result of metabolic stress-imposed changes that have occurred within the mitochondria, rendering them more susceptible to insults such as Ca(2+) overload. In addition, they lend further support to the notion that mitochondria represent a viable target for future therapies directed at ameliorating heart failure and other comorbidities that come with diabetes.

β-GPA Prevents High-fat Induced Mitochondrial H2O2 Emission And Insulin Resistance Independent of AMPK In Mice

We recently found that treatment with the creatine analogue β-guanidine propionic acid (βGPA) is sufficient to prevent increased mitochondrial H2O2 emission (mEH2O2) potential and to maintain insulin sensitivity in high fat (HF) fed SD rats. βGPA treatment lowers cellular energy charge raising the question as to whether AMPK or mEH2O2 is the primary factor linking βGPA to the prevention of insulin resistance. PURPOSE: To determine if βGPA induced improvements in mitochondrial function and insulin sensitivity during a HF diet are mediated by AMPK. METHODS: AMPK·2 subunit dominant negative mice and their wild-type littermates were fed (10 wks) standard chow (SC), 60% HF or HF plus βGPA (HF βGPA, 0.4 mg/g/d by gavage). RESULTS: Regardless of genotype, βGPA prevented the HF-induced body weight gain while both food intake and whole body oxygen consumption were significantly (P<0.05) increased. Respiratory quotient was lower with HF and further lowered by βGPA but unaffected by genotype. Locomotor activity was similar in all groups. HF decreased both whole body and muscle specific glucose uptake whereas both were preserved in HF- βGPA regardless of genotype. In permeabilized red gastrocnemius myofibers, even though mitochondrial maximal respiration was similar between all groups, mEH2O2 potential was ∼60% higher in HF vs SC. βGPA treatment prevented the HF effect and, remarkably, reduced mEH2O2 potential to levels ∼50% lower than SC, regardless of genotype. CONCLUSION: These data indicate that increased energy expenditure induced by βGPA, independent of AMPK, prevents the HF-induced increase in mEH2O2 potential and the development of insulin resistance, providing further evidence that the governance of mEH2O2 is a primary factor regulating insulin sensitivity in skeletal muscle. Supported by NIH DK073488TABLE

Electron Transport Chain-dependent and -independent Mechanisms of Mitochondrial H2O2 Emission during Long-chain Fatty Acid Oxidation

Oxidative stress in skeletal muscle is a hallmark of various pathophysiologic states that also feature increased reliance on long-chain fatty acid (LCFA) substrate, such as insulin resistance and exercise. However, little is known about the mechanistic basis of the LCFA-induced reactive oxygen species (ROS) burden in intact mitochondria, and elucidation of this mechanistic basis was the goal of this study. Specific aims were to determine the extent to which LCFA catabolism is associated with ROS production and to gain mechanistic insights into the associated ROS production. Because intermediates and by-products of LCFA catabolism may interfere with antioxidant mechanisms, we predicted that ROS formation during LCFA catabolism reflects a complex process involving multiple sites of ROS production as well as modified mitochondrial function. Thus, we utilized several complementary approaches to probe the underlying mechanism(s). Using skeletal muscle mitochondria, our findings indicate that even a low supply of LCFA is associated with ROS formation in excess of that generated by NADH-linked substrates. Moreover, ROS production was evident across the physiologic range of membrane potential and was relatively insensitive to membrane potential changes. Determinations of topology and membrane potential as well as use of inhibitors revealed complex III and the electron transfer flavoprotein (ETF) and ETF-oxidoreductase, as likely sites of ROS production. Finally, ROS production was sensitive to matrix levels of LCFA catabolic intermediates, indicating that mitochondrial export of LCFA catabolic intermediates can play a role in determining ROS levels.

Mitochondrial H2O2 emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans

High dietary fat intake leads to insulin resistance in skeletal muscle, and this represents a major risk factor for type 2 diabetes and cardiovascular disease. Mitochondrial dysfunction and oxidative stress have been implicated in the disease process, but the underlying mechanisms are still unknown. Here we show that in skeletal muscle of both rodents and humans, a diet high in fat increases the H(2)O(2)-emitting potential of mitochondria, shifts the cellular redox environment to a more oxidized state, and decreases the redox-buffering capacity in the absence of any change in mitochondrial respiratory function. Furthermore, we show that attenuating mitochondrial H(2)O(2) emission, either by treating rats with a mitochondrial-targeted antioxidant or by genetically engineering the overexpression of catalase in mitochondria of muscle in mice, completely preserves insulin sensitivity despite a high-fat diet. These findings place the etiology of insulin resistance in the context of mitochondrial bioenergetics by demonstrating that mitochondrial H(2)O(2) emission serves as both a gauge of energy balance and a regulator of cellular redox environment, linking intracellular metabolic balance to the control of insulin sensitivity.

QS8. Reduced Fatty Acid Metabolism and Elevated Mitochondrial H2O2 Emission Are Associated With Decreased PPARα in Diabetic Human Myocardium

QS8. Reduced Fatty Acid Metabolism and Elevated Mitochondrial H2O2 Emission Are Associated With Decreased PPARα in Diabetic Human Myocardium

Metformin Attenuates a Complex I Mediated Increase in Mitochondrial Oxidant Emitting Potential in Skeletal Muscle of Obese Zucker Rats

Metformin (MET) is currently the most popular antidiabetic drug, although its insulin sensitizing mechanism of action is unknown. To test the effects of metformin (MET) on skeletal muscle mitochondrial function, we measured both mitochondrial respiration and H2O2 emission in saponin‐permeabilized red gastrocnemius muscle fiber bundles from lean and obese Zucker (fa/fa) rats that had been treated with MET for 4 wks. MET treatment significantly (p=.03) improved insulin sensitivity (HOMA) in obese, but not lean rats. In permeabilized fibers, ADP‐stimulated state 3, but not basal state 4, respiration rates were greater in obese vs. lean rats. However, MET treatment did not significantly affect permeabilized fibers respiring on palmitoyl‐carnitine (PC), complex I‐ or complex II‐linked substrates. Surprisingly, H2O2 emission induced by reverse electron flow (titration of complex II substrate succinate) was more than 2‐fold higher in obese vs lean rats. MET treatment significantly (p&lt;.05) attenuated succinate‐ and PC‐induced H2O2 emission in both lean and obese rats. These results suggest that 1) obesity is associated with a marked increase in mitochondrial ROS emitting potential in skeletal muscle, and 2) metformin may exert its antidiabetic effects by inhibiting complex I mediated ROS production, thereby decreasing the susceptibility to conditions favoring ROS emission. Support: NIH DK061314 and DK073488