Effects of short- and medium-term calorie restriction on muscle mitochondrial proton leak and reactive oxygen species production

Reductions in cellular oxygen consumption (Vo2) and reactive oxygen species (ROS) production have been proposed as mechanisms underlying the anti-aging effects of calorie restriction (CR). Mitochondria are a cell's greatest "sink" for oxygen and also its primary source of ROS. The mitochondrial proton leak pathway is responsible for 20-30% of Vo2 in resting cells. We hypothesized that CR leads to decreased proton leak with consequential decreases in Vo2, ROS production, and cellular damage. Here, we report the effects of short-term (2-wk, 2-mo) and medium-term (6-mo) CR (40%) on rat muscle mitochondrial proton leak, ROS production, and whole animal Vo2. Whole body Vo2 decreased with CR at all time points, whereas mass-adjusted Vo2 was normal until the 6-mo time point, when it was 40% lower in CR compared with control rats. At all time points, maximal leak-dependent Vo2 was lower in CR rats compared with controls. Proton leak kinetics indicated that mechanisms of adaptation to CR were different between short- and medium-term treatments, with the former leading to decreases in protonmotive force (Deltap) and state 4 Vo2 and the latter to increases in Deltap and decreases in state 4 Vo2. Results from metabolic control analyses of oxidative phosphorylation are consistent with the idea that short- and medium-term responses are distinct. Mitochondrial H2O2 production was lower in all three CR groups compared with controls. Overall, this study details the rapid effects of short- and medium-term CR on proton leak, ROS production, and metabolic control of oxidative phosphorylation. Results indicate that a reduction in mitochondrial Vo2 and ROS production may be a mechanism for the actions of CR.     

Long-term calorie restriction reduces proton leak and hydrogen peroxide production in liver mitochondria

Calorie restriction (CR) without malnutrition increases maximal life span in diverse species. It has been proposed that reduction in energy expenditure and reactive oxygen species (ROS) production could be a mechanism for life span extension with CR. As a step toward testing this theory, mitochondrial proton leak, H2O2 production, and markers of oxidative stress were measured in liver from FBNF1 rats fed control or 40% CR diets for 12 or 18 mo. CR was initiated at 6 mo of age. Proton leak kinetics curves, generated from simultaneous measures of oxygen consumption and membrane potential, indicated a decrease in proton leak after 18 mo of CR, while only a trend toward a proton leak decrease was observed after 12 mo. Significant shifts in phosphorylation and substrate oxidation curves also occurred with CR; however, these changes occurred in concert with the proton leak changes. Metabolic control analysis indicated no difference in the overall pattern of control of the oxidative phosphorylation system between control and CR animals. At 12 mo, no significant differences were observed between groups for H2O2 production or markers of oxidative stress. However, at 18 mo, protein carbonyl content was lower in CR animals, as was H2O2 production when mitochondria were respiring on either succinate alone or pyruvate plus malate in the presence of rotenone. These results indicate that long-term CR lowers mitochondrial proton leak and H2O2 production, and this is consistent with the idea that CR may act by decreasing energy expenditure and ROS production.     

Absence of uncoupling protein-3 leads to greater activation of an adenine nucleotide translocase-mediated proton conductance in skeletal muscle mitochondria from calorie restricted mice

Calorie restriction (CR), without malnutrition, consistently increases lifespan in all species tested, and reduces age-associated pathologies in mammals. Alterations in mitochondrial content and function are thought to underlie some of the effects of CR. Previously, we reported that rats subjected to variable durations of 40% CR demonstrated a rapid and sustained decrease in maximal leak-dependent respiration in skeletal muscle mitochondria. This was accompanied by decreased mitochondrial reactive oxygen species generation and increased uncoupling protein-3 protein (UCP3) expression. The aim of the present study was to determine the contribution of UCP3, as well as the adenine nucleotide translocase to these functional changes in skeletal muscle mitochondria. Consistent with previous findings in rats, short-term CR (2 weeks) in wild-type (Wt) mice resulted in a lowering of the maximal leak-dependent respiration in skeletal muscle mitochondria, without any change in proton conductance. In contrast, skeletal muscle mitochondria from Ucp3-knockout (KO) mice similarly subjected to short-term CR showed no change in maximal leak-dependent respiration, but displayed an increased proton conductance. Determination of ANT activity (by measurement of inhibitor-sensitive leak) and protein expression revealed that the increased proton conductance in mitochondria from CR Ucp3-KO mice could be entirely attributed to a greater acute activation of ANT. These observations implicate UCP3 in CR-induced mitochondrial remodeling. Specifically, they imply the potential for an interaction, or some degree of functional redundancy, between UCP3 and ANT, and also suggest that UCP3 can minimize the induction of the ANT-mediated ‘energy-wasting’ process during CR.     

The influence of dietary lipid composition on liver mitochondria from mice following 1 month of calorie restriction

To investigate the role mitochondrial membrane lipids play in the actions of CR (calorie restriction), C57BL/6 mice were assigned to four groups (control and three 40% CR groups) and the CR groups were fed diets containing soya bean oil (also in the control diet), fish oil or lard. The fatty acid composition of the major mitochondrial phospholipid classes, proton leak and H₂O₂ production were measured in liver mitochondria following 1 month of CR. The results indicate that mitochondrial phospholipid fatty acids reflect the PUFA (polyunsaturated fatty acid) profile of the dietary lipid sources. CR significantly decreased the capacity of ROS (reactive oxygen species) production by Complex III but did not markedly alter proton leak and ETC (electron transport chain) enzyme activities. Within the CR regimens, the CR-fish group had decreased ROS production by both Complexes I and III, and increased proton leak when compared with the other CR groups. The CR-lard group showed the lowest proton leak compared with the other CR groups. The ETC enzyme activity measurements in the CR regimens showed that Complex I activity was decreased in both the CR-fish and CR-lard groups. Moreover, the CR-fish group also had lower Complex II activity compared with the other CR groups. These results indicate that dietary lipid composition does influence liver mitochondrial phospholipid composition, ROS production, proton leak and ETC enzyme activities in CR animals.     

Proton leak and hydrogen peroxide production in liver mitochondria from energy-restricted rats

Energy restriction (ER), without malnutrition, is the only environmental intervention that consistently increases maximum life span in laboratory rodents. One theory proposes that a reduction in energy expenditure and reactive oxygen species production is the mechanism responsible for this action of ER. To further test this theory, proton leak, H2O2 production, lipid peroxidation, and protein carbonyls were measured in mitochondria from FBNF1 rats fed either a control or 40% ER diet (onset at 6 mo of age). Liver mitochondria were isolated at 7 and 12 mo of age. Liver weight decreased 25 and 36% at 1 and 6 mo of ER, respectively (P < 0.05). ER resulted in an increase (P < 0.05) in percent total polyunsaturates, n-6 polyunsaturates, and total unsaturates (6 mo only) in mitochondrial lipids. These changes, however, were not associated with significant alterations in mitochondrial function. State 4 respiration and membrane potential were not different (P > 0.05) between groups at either assessment period. Similarly, proton leak kinetics were not different between control and ER animals. Top-down metabolic control analysis and its extension, elasticity analysis, were used at the 6-mo assessment and revealed no difference in control of the oxidative phosphorylation system between control and ER rats. H2O2 production with either succinate or pyruvate/malate substrates was also not different (P > 0.05) between groups at either time point. In conclusion, ER did not alter proton leak or H2O2 production at this age or stage of restriction in liver.     

Increased mitochondrial proton leak in skeletal muscle mitochondria of UCP1-deficient mice

Mice having targeted inactivation of uncoupling protein 1 (UCP1) are cold sensitive but not obese (Enerb?ck S, Jacobsson A, Simpson EM, Guerra C, Yamashita H, Harper M-E, and Kozak LP. Nature 387: 90-94, 1997). Recently, we have shown that proton leak in brown adipose tissue (BAT) mitochondria from UCP1-deficient mice is insensitive to guanosine diphosphate (GDP), a well known inhibitor of UCP1 activity (Monemdjou S, Kozak LP, and Harper M-E. Am J Physiol Endocrinol Metab 276: E1073-E1082, 1999). Moreover, despite a fivefold increase of UCP2 mRNA in BAT of UCP1-deficient mice, we found no differences in the overall kinetics of this GDP-insensitive proton leak between UCP1-deficient mice and controls. Based on these findings, which show no adaptive increase in UCP1-independent leak in BAT, we hypothesized that adaptive thermogenesis may be occurring in other tissues of the UCP1-deficient mouse (e.g., skeletal muscle), thus allowing them to maintain their normal resting metabolic rate, feed efficiency, and adiposity. Here, we report on the overall kinetics of the mitochondrial proton leak, respiratory chain, and ATP turnover in skeletal muscle mitochondria from UCP1-deficient and heterozygous control mice. Over a range of mitochondrial protonmotive force (Deltap) values, leak-dependent oxygen consumption is higher in UCP1-deficient mice compared with controls. State 4 (maximal leak-dependent) respiration rates are also significantly higher in the mitochondria of mice deficient in UCP1, whereas state 4 Deltap is significantly lower. No significant differences in state 3 respiration rates or Deltap values were detected between the two groups. Thus the altered kinetics of the mitochondrial proton leak in skeletal muscle of UCP1-deficient mice indicate a thermogenic mechanism favoring the lean phenotype of the UCP1-deficient mouse.     

Targeted expression of the human uncoupling protein 2 (hUCP2) to adult neurons extends life span in the fly

The oxidative stress hypothesis of aging predicts that a reduction in the generation of mitochondrial reactive oxygen species (ROS) will decrease oxidative damage and extend life span. Increasing mitochondrial proton leak-dependent state 4 respiration by increasing mitochondrial uncoupling is an intervention postulated to decrease mitochondrial ROS production. When human UCP2 (hUCP2) is targeted to the mitochondria of adult fly neurons, we find an increase in state 4 respiration, a decrease in ROS production, a decrease in oxidative damage, heightened resistance to the free radical generator paraquat, and an extension in life span without compromising fertility or physical activity. Our results demonstrate that neuronal-specific expression of hUCP2 in adult flies decreases cellular oxidative damage and is sufficient to extend life span.     

The proton leak across the mitochondrial inner membrane

The proton conductance of the mitochondrial inner membrane increases at high protonmotive force in isolated mitochondria and in mitochondria in situ in rat hepatocytes. Quantitative analysis of its importance shows that about 20-30% of the oxygen consumption by resting hepatocytes is used to drive a heat-producing cycle of proton pumping by the respiratory chain and proton leak back to the matrix. The flux control coefficient of the proton leak pathway over respiration rate varies between 0.9 and zero in mitochondria depending on the rate of respiration, and has a value of about 0.2 in hepatocytes. Changes in the proton leak pathway in situ will therefore change respiration rate. Mitochondria isolated from hypothyroid animals have decreased proton leak pathway, causing slower state 4 respiration rates. Hepatocytes from hypothyroid rats also have decreased proton leak pathway, and this accounts for about 30% of the decrease in hepatocyte respiration rate. Mitochondrial proton leak may be a significant contributor to standard metabolic rate in vivo.     

疲劳性运动中线粒体电子漏引起质子漏增加

以大鼠递增强度力竭性竭性跑台运动为疲劳运动模型,观察了运动后大鼠骨骼肌线粒体电子漏和质子漏的变化。结果表明,运动性疲劳状态下大鼠骨骼肌线粒体超氧阴离子生成增加,脂质过氧线粒体质子漏增多是氧化磷酸化偶联程度下降的重要因素。实验结果支持电子漏引起质子漏的假说。     

Glutaredoxin-2 Is Required to Control Proton Leak through Uncoupling Protein-3

Glutathionylation has emerged as a key modification required for controlling protein function in response to changes in cell redox status. Recently, we showed that the glutathionylation state of uncoupling protein-3 (UCP3) modulates the leak of protons back into the mitochondrial matrix, thus controlling reactive oxygen species production. However, whether or not UCP3 glutathionylation is mediated enzymatically has remained unknown because previous work relied on the use of pharmacological agents, such as diamide, to alter the UCP3 glutathionylation state. Here, we demonstrate that glutaredoxin-2 (Grx2), a matrix oxidoreductase, is required to glutathionylate and inhibit UCP3. Analysis of bioenergetics in skeletal muscle mitochondria revealed that knock-out of Grx2 (Grx2^−/−) increased proton leak in a UCP3-dependent manner. These effects were reversed using diamide, a glutathionylation catalyst. Importantly, the increased leak did not compromise coupled respiration. Knockdown of Grx2 augmented proton leak-dependent respiration in primary myotubes from wild type mice, an effect that was absent in UCP3^−/− cells. These results confirm that Grx2 deactivates UCP3 by glutathionylation. To our knowledge, this is the first enzyme identified to regulate UCP3 by glutathionylation and is the first study on the role of Grx2 in the regulation of energy metabolism.     

Lower extremity muscle size and strength and aerobic capacity decrease with caloric restriction but not with exercise-induced weight loss.

Caloric restriction (CR) results in fat loss; however, it may also result in loss of muscle and thereby reduce strength and aerobic capacity (VO2 max). These effects may not occur with exercise-induced weight loss (EX) because of the anabolic effects of exercise on heart and skeletal muscle. We tested the hypothesis that CR reduces muscle size and strength and VO2 max, whereas EX preserves or improves these parameters. Healthy 50- to 60-yr-old men and women (body mass index of 23.5-29.9 kg/m2) were studied before and after 12 mo of weight loss by CR (n = 18) or EX (n = 16). Lean mass was assessed by dual-energy X-ray absorptiometry, thigh muscle volume by MRI, isometric and isokinetic knee flexor strength by dynamometry, and treadmill VO2 max by indirect calorimetry. Both interventions caused significant decreases in body weight (CR: -10.7 +/- 1.4%, EX: -9.5 +/- 1.5%) and lean mass (CR: -3.5 +/- 0.7%, EX: -2.2 +/- 0.8%), with no significant differences between groups. Significant decreases in thigh muscle volume (-6.9 +/- 0.8%) and composite knee flexion strength (-7.2 +/- 3%) occurred in the CR group only. Absolute VO2 max decreased significantly in the CR group (-6.8 +/- 2.3%), whereas the EX group had significant increases in both absolute (+15.5 +/- 2.4%) and relative (+28.3 +/- 3.0%) VO2 max. These data provide evidence that muscle mass and absolute physical work capacity decrease in response to 12 mo of CR but not in response to a similar weight loss induced by exercise. These findings suggest that, during EX, the body adapts to maintain or even enhance physical performance capacity.     

Caloric restriction influences hydrogen peroxide generation in mitochondrial sub-populations from mouse liver

Calorie restriction (CR) has been shown to decrease H₂O₂ production in liver mitochondria, although it is not known if this is due to uniform changes in all mitochondria or changes in particular mitochondrial sub-populations. To address this issue, liver mitochondria from control and CR mice were fractionated using differential centrifugation at 1,000 g, 3,000 g and 10,000 g into distinct populations labeled as M1, M3 and M10, respectively. Mitochondrial protein levels, respiration and H₂O₂ production were measured in each fraction. CR resulted in a decrease in total protein (mg) in each fraction, although this difference disappeared when adjusted for liver weight (mg protein/g liver weight). No differences in respiration (State 3 or 4) were observed between control and CR mice in any of the mitochondrial fractions. CR decreased H₂O₂ production in all fractions when mitochondria respired on succinate (Succ), succ+antimycin A (Succ+AA) or pyruvate/malate+rotenone (P/M+ROT). Thus, CR decreased reactive oxygen species (ROS) production under conditions which stimulate mitochondrial complex I ROS production under both forward (P/M+ROT) and backward (Succ & Succ+AA) electron flow. The results indicate that CR decreases H₂O₂ production in all liver mitochondrial fractions due to a decrease in capacity for ROS production by complex I of the electron transport chain.     

Ischemic preconditioning preserves proton leakage from mitochondrial membranes but not oxidative phosphorylation during heart reperfusion

The aim of this study was to evaluate the role of mitochondria in the recovery of cardiac energetics induced by ischaemic preconditioning at reperfusion. Isolated rat hearts were aerobically perfused (control), subjected to global ischaemia and reperfusion (reperfusion), or subjected to 3 brief cycles of ischaemia/reperfusion and then to the protocol of reperfusion (preconditioning). At the end of the perfusion, antimycin A was delivered to the heart for 25 min, to inhibit mitochondrial respiration and stimulate glycolysis. The increased amount of lactate released in the coronary effluent was correlated with the number of viable cells producing this end-product of glycolysis. Preconditioned hearts released 18% more lactate than reperfused hearts (p < 0.05). This result indicates that preconditioning partially preserved cell viability, as was also evidenced by the MTT assay performed on cardiac biopsies. The difference between antimycin A-stimulated and basal lactate concentration, representing the contribution of mitochondria to the overall energetics of cardiac tissue, was also 18% more elevated in the preconditioned hearts than in the reperfused hearts (p < 0.01). The study of the respiratory function of mitochondria isolated at the end of perfusion, showed that preconditioning did not improve the oxygen-dependent production of ATP (state 3 respiration, ADP/O). On the contrary, state 4 respiration, which is related to proton leakage, was 35.0% lower in the preconditioned group than reperfusion group (p < 0.05). Thus, preconditioning ameliorates cardiac energetics by preserving cell death, but without affecting mitochondrial oxidative phosphorylation. Mitochondria can contribute to cell survival by the attenuation of proton leak from inner membrane.     

Undernutrition during pregnancy in mice leads to dysfunctional cardiac muscle respiration in adult offspring

Intrauterine growth restriction (IUGR) is associated with an increased risk of developing obesity, insulin resistance and cardiovascular disease. However, its effect on energetics in heart remains unknown. In the present 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 life-long implications for cardiovascular function and disease risk.     

Hypothyroidism in rats decreases mitochondrial inner membrane cation permeability

We investigated the cation permeability of liver mitochondria isolated from hypothyroid or euthyroid rats by measuring the rate of swelling of respiring mitochondria in acetate salts as a function of membrane potential. Mitochondria from hypothyroid rats have a decreased permeability of roughly 3-fold in the presence of monovalent cations K and tetramethylammonium at any (measured) membrane potential. Since the monovalent cation leak and the proton leak are known to respond similarly to membrane potential our results support the theory that the difference in non-phosphorylating respiration rate between mitochondria from hypothyroid and euthyroid rats is due to a difference in proton leak.     

Dietary Restriction at Old Age Lowers Mitochondrial Oxygen Radical Production and Leak at Complex I and Oxidative DNA Damage in Rat Brain

Previous studies in mammalian models indicate that the rate of mitochondrial reactive oxygen species ROS production and the ensuing modification of mitochondrial DNA (mtDNA) link oxidative stress to aging rate. However, there is scarce information concerning this in relation to caloric restriction (CR) in the brain, an organ of maximum relevance for ageing. Furthermore, it has never been studied if CR started late in life can improve those oxidative stress-related parameters. In this investigation, rats were subjected during 1 year to 40% CR starting at 24 months of age. This protocol of CR significantly decreased the rate of mitochondrial H₂O₂ production (by 24%) and oxidative damage to mtDNA (by 23%) in the brain below the level of both old and young ad libitum-fed animals. In agreement with the progressive character of aging, the rate of H₂O₂ production of brain mitochondria stayed constant with age. Oxidative damage to nuclear DNA increased with age and this increase was fully reversed by CR to the level of the young controls. The decrease in ROS production induced by CR was localized at Complex I and occurred without changes in oxygen consumption. Instead, the efficiency of brain mitochondria to avoid electron leak to oxygen at Complex I was increased by CR. The mechanism involved in that increase in efficiency was related to the degree of electronic reduction of the Complex I generator. The results agree with the idea that CR decreases aging rate in part by lowering the rate of free radical generation of mitochondria in the brain.     

Mitochondrial metabolic suppression and reactive oxygen species production in liver and skeletal muscle of hibernating thirteen-lined ground squirrels

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 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.     

Mitochondrial uncoupling,ROS generation and cardioprotection

Mitochondrial oxidative phosphorylation is incompletely coupled, since protons translocated to the intermembrane space by specific respiratory complexes of the electron transport chain can return to the mitochondrial matrix independently of the ATP synthase —a process known as proton leak— generating heat instead of ATP. Proton leak across the inner mitochondrial membrane increases the respiration rate and decreases the electrochemical proton gradient (Δp), and is an important mechanism for energy dissipation that accounts for up to 25% of the basal metabolic rate. It is well established that mitochondrial superoxide production is steeply dependent on Δp in isolated mitochondria and, correspondingly, mitochondrial uncoupling has been identified as a cytoprotective strategy under conditions of oxidative stress, including diabetes, drug-resistance in tumor cells, ischemia-reperfusion (IR) injury or aging. Mitochondrial uncoupling proteins (UCPs) are able to lower the efficiency of oxidative phosphorylation and are involved in the control of mitochondrial reactive oxygen species (ROS) production. There is strong evidence that UCP2 and UCP3, the UCP1 homologues expressed in the heart, protect against mitochondrial oxidative damage by reducing the production of ROS. This review first analyzes the relationship between mitochondrial proton leak and ROS generation, and then focuses on the cardioprotective role of chemical uncoupling and uncoupling mediated by UCPs. This includes their protective effects against cardiac IR, a condition known to increase ROS production, and their roles in modulating cardiovascular risk factors such as obesity, diabetes and atherosclerosis.     

Influence of intensity of food restriction on skeletal muscle mitochondrial energy metabolism in rats

Variable durations of food restriction (FR; lasting weeks to years) and variable FR intensities are applied to animals in life span-prolonging studies. A reduction in mitochondrial proton leak is suggested as a putative mechanism linking such diet interventions and aging retardation. Early mechanisms of mitochondrial metabolic adaptation induced by FR remain unclear. We investigated the influence of different degrees of FR over 3 days on mitochondrial proton leak and mitochondrial energy metabolism in rat hindlimb skeletal muscle. Animals underwent 25, 50, and 75% and total FR compared with control rats. Proton leak kinetics and mitochondrial functions were investigated in two mitochondrial subpopulations, intermyofibrillar (IMF) and subsarcolemmal (SSM) mitochondria. Regardless of the degree of restriction, skeletal muscle mass was not affected by 3 days of FR. Mitochondrial basal proton conductance was significantly decreased in 50% restricted rats in both mitochondrial subpopulations (46 and 40% for IMF and SSM, respectively) but was unaffected in other groups compared with controls. State 3 and uncoupled state 3 respiration rates were decreased in SSM mitochondria only for 50% restricted rats when pyruvate + malate was used as substrate (-34.5 and -38.9% compared with controls, P < 0.05). IMF mitochondria respiratory rates remained unchanged. Three days of FR, particularly at 50% FR, were sufficient to lower mitochondria energetic metabolism in both mitochondrial populations. Our study highlights an early step in mitochondrial adaptation to FR and the influence of the severity of restriction on this adaptation. This step may be involved in an aging-retardation process.     

Upregulation of uncoupling protein-3 in skeletal muscle during exercise: a potential antioxidant function

Uncoupling protein-3 (UCP3) expression has been shown to increase dramatically in response to muscular contraction, but the physiological significance of UCP3 upregulation is still elusive. In this study, UCP3 mRNA and protein expression were investigated along with mitochondrial respiratory function, reactive oxygen species (ROS) generation, and antioxidant defense in rat skeletal muscle during and after an acute bout of prolonged exercise. UCP3 mRNA expression was elevated sharply at 45 min of exercise, reaching 7- to 8-fold above resting level at 150 min. The increase in UCP3 protein content showed a latent response but was elevated approximately 1.9-fold at 120 min of exercise. Both UCP3 mRNA and UCP3 protein gradually returned to resting levels 24 h postexercise. Mitochondrial ROS production was progressively increased during exercise. However, ROS showed a dramatic drop at 150 min although their levels remained severalfold higher during the recovery. Mitochondrial State 4 respiration rate was increased by 46 and 58% (p < 0.05) at 90 and 120 min, respectively, but returned to resting rate at 150 min, when State 3 respiration and respiratory control index (RCI) were suppressed. ADP-to-oxygen consumption (P/O) ratio and ATP synthase activity were lowered at 3 h postexercise, whereas proton motive force and mitochondrial malondialdehyde content were unchanged. Manganese superoxide dismutase gene expression was not affected by exercise except for an increase in mRNA abundance at 3 h postexercise. These data demonstrate that UCP3 expression in rat skeletal muscle can be rapidly upregulated during prolonged exercise, possibly owing to increased ROS generation. Increased UCP3 may partially alleviate the proton gradient across the inner membrane, thereby reducing further ROS production by the electron transport chain. However, prolonged exercise caused a decrease in energy coupling efficiency in muscle mitochondria revealed by an increased respiration rate due to proton leak (State 4/State 3 ratio) and decreased RCI. We thus propose that the compromise of the oxidative phosphorylation efficiency due to UCP3 upregulation may serve an antioxidant function to protect the muscle mitochondria from exercise-induced oxidative stress