Subsarcolemmal and intermyofibrillar mitochondria proteome differences disclose functional specializations in skeletal muscle

Skeletal muscle is a highly specialized tissue that contains two distinct mitochondria subpopulations, the subsarcolemmal (SS) and the intermyofibrillar (IMF) mitochondria. Although it is established that these mitochondrial subpopulations differ functionally in several ways, limited information exists about the proteomic differences underlying these functional differences. Therefore, the objective of this study was to biochemically characterize the SS and IMF mitochondria isolated from rat red gastrocnemius skeletal muscle. We separated the two mitochondrial subpopulations from skeletal muscle using a refined method that provides an excellent division of these unique mitochondrial subpopulations. Using proteomics of mitochondria and its subfractions (intermembrane space, matrix and inner membrane), a total of 325 distinct proteins were identified, most of which belong to the functional clusters of oxidative phosphorylation, metabolism and signal transduction. Although more gel spots were observed in SS mitochondria, 38 of the identified proteins were differentially expressed between the SS and IMF subpopulations. Compared to the SS mitochondrial, IMF mitochondria expressed a higher level of proteins associated with oxidative phosphorylation. This observation, coupled with the finding of a higher respiratory chain complex activity in IMF mitochondria, suggests a specialization of IMF mitochondria toward energy production for contractile activity.     

Differences in proton leak kinetics, but not in UCP3 protein content, in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria from fed and fasted rats

We have investigated the effect of 24-h fasting on basal proton leak and uncoupling protein (UCP) 3 expression at the protein level in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria. In fed rats, the two mitochondrial populations displayed different proton leak, but the same protein content of UCP3. In addition, 24-h fasting, both at 24 and 29 degrees C, induced an increase in proton leak only in subsarcolemmal mitochondria, while UCP3 content increased in both the populations. From the present data, it appears that UCP3 does not control the basal proton leak of skeletal muscle mitochondria.     

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。     

Tissue-specific depression of mitochondrial proton leak and substrate oxidation in hibernating arctic ground squirrels

A significant proportion of standard metabolic rate is devoted to driving mitochondrial proton leak, and this futile cycle may be a site of metabolic control during hibernation. To determine if the proton leak pathway is decreased during metabolic depression related to hibernation, mitochondria were isolated from liver and skeletal muscle of nonhibernating (active) and hibernating arctic ground squirrels (Spermophilus parryii). At an assay temperature of 37 degrees C, state 3 and state 4 respiration rates and state 4 membrane potential were significantly depressed in liver mitochondria isolated from hibernators. In contrast, state 3 and state 4 respiration rates and membrane potentials were unchanged during hibernation in skeletal muscle mitochondria. The decrease in oxygen consumption of liver mitochondria was achieved by reduced activity of the set of reactions generating the proton gradient but not by a lowered proton permeability. These results suggest that mitochondrial proton conductance is unchanged during hibernation and that the reduced metabolism in hibernators is a partial consequence of tissue-specific depression of substrate oxidation.     

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.     

Role of UCP3 in state 4 respiration during contractile activity-induced mitochondrial biogenesis.

In an effort to better characterize uncoupling protein-3 (UCP3) function in skeletal muscle, we assessed basal UCP3 protein content in rat intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondrial subfractions in conjunction with measurements of state 4 respiration. UCP3 content was 1.3-fold (P < 0.05) greater in IMF compared with SS mitochondria. State 4 respiration was 2.6-fold greater (P < 0.05) in the IMF subfraction than in SS mitochondria. GDP attenuated state 4 respiration by approximately 40% (P < 0.05) in both subfractions. The UCP3 activator oleic acid (OA) significantly increased state 4 respiration in IMF mitochondria only. We used chronic electrical stimulation (3 h/day for 7 days) to investigate the relationship between changes in UCP3 protein expression and alterations in state 4 respiration during contractile activity-induced mitochondrial biogenesis. UCP3 content was increased by 1.9- and 2.3-fold in IMF and SS mitochondria, respectively, which exceeded the concurrent 40% (P < 0.05) increase in cytochrome-c oxidase activity. Chronic contractile activity increased state 4 respiration by 1.4-fold (P < 0.05) in IMF mitochondria, but no effect was observed in the SS subfraction. The uncoupling function of UCP3 accounted for 50-57% of the OA-induced increase in state 4 respiration in IMF mitochondria, which was independent of the induced twofold difference in UCP3 content due to chronic contractile activity. Thus modifications in UCP3 function are more important than changes in UCP3 expression in modifying state 4 respiration. This effect is evident in IMF but not SS mitochondria. We conclude that UCP3 at physiological concentrations accounts for a significant portion of state 4 respiration in both IMF and SS mitochondria, with the contribution being greater in the IMF subfraction. In addition, the contradiction between human and rat training studies with respect to UCP3 protein expression may partly be explained by the greater than twofold difference in mitochondrial UCP3 content between rat and human skeletal muscle.     

Food restriction affects energy metabolism in rat liver mitochondria

To examine the effect of 50% food restriction over a period of 3 days on mitochondrial energy metabolism, liver mitochondria were isolated from ad libitum and food-restricted rats. Mitochondrial enzyme activities and oxygen consumption were assessed spectrophotometrically and polarographically. With regard to body weight loss (-5%), food restriction decreased the liver to body mass ratio by 7%. Moreover, in food-restricted rats, liver mitochondria displayed diminished state 3 (-30%), state 4-oligomycin (-26%) and uncoupled state (-24%) respiration rates in the presence of succinate. Furthermore, "top-down" elasticity showed that these decreases were due to an inactivation of reactions involved in substrate oxidation. Therefore, it appears that rats not only adapt to food restriction through simple passive mechanisms, such as liver mass loss, but also through decreased mitochondrial energetic metabolism.     

High‐fat diet affects skeletal muscle mitochondria comparable to pressure overload‐induced heart failure

In heart failure, high‐fat diet (HFD) may exert beneficial effects on cardiac mitochondria and contractility. Skeletal muscle mitochondrial dysfunction in heart failure is associated with myopathy. However, it is not clear if HFD affects skeletal muscle mitochondria in heart failure as well. To induce heart failure, we used pressure overload (PO) in rats fed normal chow or HFD. Interfibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM) from gastrocnemius were isolated and functionally characterized. With PO heart failure, maximal respiratory capacity was impaired in IFM but increased in SSM of gastrocnemius. Unexpectedly, HFD affected mitochondria comparably to PO. In combination, PO and HFD showed additive effects on mitochondrial subpopulations which were reflected by isolated complex activities. While PO impaired diastolic as well as systolic cardiac function and increased glucose tolerance, HFD did not affect cardiac function but decreased glucose tolerance. We conclude that HFD and PO heart failure have comparable effects leading to more severe impairment of IFM. Glucose tolerance seems not causally related to skeletal muscle mitochondrial dysfunction. The additive effects of HFD and PO may suggest accelerated skeletal muscle mitochondrial dysfunction when heart failure is accompanied with a diet containing high fat.     

Effects of fasting on muscle mitochondrial energetics and fatty acid metabolism in Ucp3(-/-) and wild-type mice

Uncoupling protein-3 (UCP3) is a mitochondrial carrier protein of as yet undefined physiological function. To elucidate characteristics of its function, we studied the effects of fasting on resting metabolic rate, respiratory quotient, muscle Ucp3 expression, and mitochondrial proton leak in wild-type and Ucp3(-/-) mice. Also analyzed were the fatty acid compositions of skeletal muscle mitochondria in fed and fasted Ucp3(-/-) and wild-type mice. In wild-type mice, fasting caused significant increases in Ucp3 (4-fold) and Ucp2 (2-fold) mRNA but did not significantly affect mitochondrial proton leak. State 4 oxygen consumption was not affected by fasting in either of the two groups. However, protonmotive force was consistently higher in mitochondria of Ucp3(-/-) animals (P = 0.03), and fasting further augmented protonmotive force in Ucp3(-/-) mice; there was no effect in wild-type mitochondria. Resting metabolic rates decreased with fasting in both groups. Ucp3(-/-) mice had higher respiratory quotients than wild-type mice in fed resting states, indicating impaired fatty acid oxidation. Altogether, results show that the fasting-induced increases in Ucp2 and Ucp3 do not correlate with increased mitochondrial proton leak but support a role for UCP3 in fatty acid metabolism.     

棕榈酸对模拟高原低氧大鼠离体脑线粒体解耦联蛋白活性及质子漏的影响

本文旨在通过观察棕榈酸对模拟高原低氧大鼠离体脑线粒体解耦联蛋白(uncoupling proteins,UCPs)活性的影响及脑线粒体质子漏与膜电位的改变,探讨UCPs在介导游离脂肪酸对低氧时线粒体氧化磷酸化功能改变中的作用.将SpragueDawley大鼠随机分为对照组、急性低氧组和慢性低氧组.低氧大鼠于低压舱内模拟海拔5 000 m高原23 h/d作低氧暴露,分别连续低氧3 d和30 d.用差速密度梯度离心法提取脑线粒体,[3H-GTP法测定UCPs含量与活性,TPMP 电极与Clark氧电极结合法测量线粒体质子漏,罗丹明123荧光法测定线粒体膜电位.结果显示,低氧使脑线粒体内UCPs含量与活性升高、质子漏增加、线粒体膜电位降低;同时,低氧暴露降低脑线粒体对棕榈酸的反应性,UCPs活性的改变率低于对照组,且线粒体UCPs含量、质子漏、膜电位变化率亦出现相同趋势.线粒体质子漏与反映UCPs活性的Kd值呈线性负相关(P<0.01 r=-0.906),与反映UCPs含量的Bmax呈线性正相关(P<0.01,r=0.856),与膜电位呈线性负相关(P<0.01,r=-0.880).以上结果提示,低氧导致的脑线粒体质子漏增加及膜电位降低与线粒体内UCPs活性升高有关,同时低氧暴露能降低脑线粒体对棕榈酸的反应性,提示在高原低氧环境下,游离脂肪酸升高在维持线粒体能量代谢中起着自身保护和调节机制.     

Nucleotide effects on liver and muscle mitochondrial non-phosphorylating respiration and membrane potential

Uncoupling protein-1 homologs are hypothesized to mediate mitochondrial proton leak. To test this hypothesis, we determined the effects of ATP and other nucleotides on liver and skeletal muscle mitochondrial non-phosphorylating respiration (VO(2)), membrane potential, FCCP-stimulated respiratory control ratios, and swelling. Neither ATP nor CTP affected liver or muscle proton leak, but both inhibited the respiratory chain. Unexpectedly, CMP stimulated liver proton leak (EC(50) approximately 4.4+/-0.5 mM). Using CMP chromatography, we identified two proteins (M(r)=31.2 and 32.6 kDa) from liver mitochondria that are similar in size to members of the mitochondrial carrier protein family. We conclude (a) liver and muscle mitochondrial proton leak is insensitive to ATP and CTP, and (b) CMP activates a leak in liver mitochondria. The CMP-inducible leak may be mediated by a 30-32 kDa protein. Based on the high concentrations required, CMP is unlikely to be a physiologically important leak regulator. Nonetheless, our results show that tissues other than brown fat have inducible leaks that may be protein-mediated.     

Two subpopulations of mitochondria in the aging rat heart display heterogenous levels of oxidative stress

Cardiac mitochondria are composed of two distinct subpopulations: one beneath the sarcolemma (subsarcolemmal mitochondria: SSM), and another along the myofilaments (interfibrillary mitochondria: IFM). Previous studies suggest a preferential loss of IFM function with age; however, the age-related changes in oxidative stress in these mitochondrial subpopulations have not been examined. To this end, the changes in mitochondrial antioxidant capacity, oxidant output, and oxidative damage to Complex IV in IFM and SSM from young and old rats were studied. Results show no apparent differences in any parameters examined between IFM and SSM from young rats. However, relative to young, only IFM from old rats had a significantly higher rate of oxidant production and a decline in mitochondrial ascorbate levels and GSH redox status. The age-related decline in mitochondrial antioxidant capacity in IFM was accompanied by a marked loss in glutaredoxin and GSSG reductase activities, suggesting a diminished reductive capacity in IFM with age. Moreover, the loss in Complex IV activity was limited to the IFM of old rats, which was accompanied by a 4-fold increase in 4-hydroxynonenal-modified Complex IV. Thus, mitochondrial decay is not uniform and further indicates that myofibrils may be uniquely under oxidative stress in the aging heart.     

Mitochondrial proton leak: a role for uncoupling proteins 2 and 3?

In mitochondria ATP synthesis is not perfectly coupled to oxygen consumption due to proton leak across the mitochondrial inner membrane. Quantitative studies have shown that proton leak contributes to approximately 25% of the resting oxygen consumption of mammals. Proton leak plays a role in accounting for differences in basal metabolic rate. Thyroid studies, body mass studies, phylogenic studies and obesity studies have all shown that increased mass-specific metabolic rate is linked to increased mitochondrial proton leak. The mechanism of the proton leak is unclear. Evidence suggests that proton leak occurs by a non-specific diffusion process across the mitochondrial inner membrane. However, the high degree of sequence homology of the recently cloned uncoupling proteins UCP 2 and UCP 3 to brown adipose tissue UCP 1, and their extensive tissue distribution, suggest that these novel uncoupling proteins play a role in proton leak. Early indications from reconstitution experiments and several in vitro expression studies suggest that the novel uncoupling proteins uncouple mitochondria. Furthermore, mice overexpressing UCP 3 certainly show a phenotype consistent with increased metabolism. The evidence for a role for these novel UCPs in mitochondrial proton leak is reviewed.     

Dexamethasone treatment specifically increases the basal proton conductance of rat liver mitochondria

We investigated the role that mitochondrial proton leak may play in the glucocorticoid-induced hypermetabolic state. Sprague-Dawley rats were injected with dexamethasone over a period of 5 days. Liver mitochondria and gastrocnemius subsarcolemmal and intermyofibrillar mitochondria were isolated from dexamethasone-treated, pair-fed and control rats. Respiration and membrane potential were measured simultaneously using electrodes sensitive to oxygen and to the potential-dependent probe triphenylmethylphosphonium, respectively. Five days of dexamethasone injection resulted in a marked increase in the basal proton conductance of liver mitochondria, but not in the muscle mitochondrial populations. This effect would have a modest impact on energy expenditure in rats.     

Effects of chronic caloric restriction on mitochondrial respiration in the ischemic reperfused rat heart

Dietary restriction increases life span and delays the development of age-related diseases in rodents. We have recently demonstrated that chronic dietary restriction is beneficial on recovery of heart function following ischemia. We studied whether the metabolic basis of this benefit is associated with alterations in mitochondrial respiration. Male Wistar rats were assigned to an ad libitum-fed (AL) group and a food restricted (FR) group, in which food intake was reduced to 55% of the amount consumed by the AL group. Following an 8-month period of restricted caloric intake, isolated working hearts perfused with glucose and high levels of fatty acids were subjected to global ischemia followed by reperfusion. At the end of reperfusion, total heart mitochondria was respiration was assessed in the presence of pyruvate, tricarboxylic acid intermediates, and palmitoylcarnitine. Recovery of heart function following ischemia was greater in FR hearts compared to AL hearts. Paralleling these changes in heart function was in increase in state 3 respiration with pyruvate. The respiratory control ratios in the presence of pyruvate and tricarboxylic acid intermediates were higher in FR hearts compared to AL hearts, indicating well-coupled mitochondria. Overall energy production, expressed as the ADP:O ratio and the oxidative phosphorylation rate, was also improved in FR hearts. Our results indicate that the beneficial effect of FR on recovery of heart function following ischemia is associated with changes in mitochondrial respiration.     

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.     

The preservation of in vivo phosphorylated and activated uncoupling protein 3 (UCP3) in isolated skeletal muscle mitochondria following administration of 3,4-methylenedioxymethamphetamine (MDMA aka ecstasy) to rats/mice

Previous researchers have demonstrated that 3,4-methylenedioxymethamphetamine (MDMA) induced hyperthermia, in skeletal muscle of animals, is uncoupling protein 3 (UCP3) dependent. In light of our investigations that in vivo phosphorylation of UCP1 is augmented under conditions of cold-acclimation, we set out to investigate whether (a) UCP3 was phosphorylated in vivo and (b) whether in vivo phosphorylation of UCP3 resulted in increased proton leak following MDMA administration to animals. Our data demonstrate that MDMA treatment (but not PBS treatment) of animals results in both in vivo serine and tyrosine phosphorylation of UCP3 in skeletal muscle mitochondria, isolated in the presence of phosphatase inhibitors to preserve in vivo phosphorylation. In addition, proton leak is only increased in skeletal muscle mitochondria isolated from MDMA treated animals (in the presence of phosphatase inhibitors) and the increased proton leak is due to phosphorylated UCP3. UCP3 abundance in skeletal muscle mitochondria is unaffected by MDMA administration. Preservation of UCP3 phosphorylation and increased proton leak is lost when skeletal muscle mitochondria are isolated in the absence of phosphatase inhibitors. We conclude that MDMA treatment of animals increases proton leak in skeletal muscle mitochondria by activating UCP3 through in vivo covalent modification of UCP3 by phosphorylation. Furthermore, we deduce that the MDMA induced hyperthermia in skeletal muscle is due to increased proton leak in vivo as a result of activation of UCP3 through phosphorylation.     

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.     

Bioenergetics in aging: mitochondrial proton leak in aging rat liver, kidney and heart

Aging is associated with a decline in performance in many organs and loss of physiological performance can be due to free radicals. Mitochondria are incompletely coupled: during oxidative phosphorylation some of the redox energy is dissipated as natural proton leak across the inner membrane. To verify whether proton leak occurs in mitochondria during aging, we measured the mitochondrial respiratory chain activity, membrane potential and proton leak in liver, kidneys and heart of young and old rats. Mitochondria from old rats showed normal rates of Complex I and Complex II respiration. However, they had a lower membrane potential compared to mitochondria from younger rats. In addition, they exhibited an increased rate of proton conductance which partially dissipated the mitochondrial membrane potential when the rate of electron transport was suppressed. This could compromise energy homeostasis in aging cells in conditions that require additional energy supply and could minimize oxidative damage to DNA.     

Stimulation of mitochondrial proton conductance by hydroxynonenal requires a high membrane potential

Mild uncoupling of oxidative phosphorylation, caused by a leak of protons back into the matrix, limits mitochondrial production of ROS (reactive oxygen species). This proton leak can be induced by the lipid peroxidation products of ROS, such as HNE (4-hydroxynonenal). HNE activates uncoupling proteins (UCP1, UCP2 and UCP3) and ANT (adenine nucleotide translocase), thereby providing a negative feedback loop. The mechanism of activation and the conditions necessary to induce uncoupling by HNE are unclear. We have found that activation of proton leak by HNE in rat and mouse skeletal muscle mitochondria is dependent on incubation with respiratory substrate. In the presence of HNE, mitochondria energized with succinate became progressively more leaky to protons over time compared with mitochondria in the absence of either HNE or succinate. Energized mitochondria must attain a high membrane potential to allow HNE to activate uncoupling: a drop of 10-20 mV from the resting value is sufficient to blunt induction of proton leak by HNE. Uncoupling occurs through UCP3 (11%), ANT (64%) and other pathways (25%). Our findings have shown that exogenous HNE only activates uncoupling at high membrane potential. These results suggest that both endogenous HNE production and high membrane potential are required before mild uncoupling will be triggered to attenuate mitochondrial ROS production.