Mitochondrial Proton Leak and the Uncoupling Proteins

An energetically significant leak of protons occurs across the mitochondrial inner membranesof eukaryotic cells. This seemingly wasteful proton leak accounts for at least 20% of thestandard metabolic rate of a rat. There is evidence that it makes a similar contribution tostandard metabolic rate in a lizard. Proton conductance of the mitochondrial inner membranecan be considered as having two components: a basal component present in all mitochondria,and an augmentative component, which may occur in tissues of mammals and perhaps ofsome other animals. The uncoupling protein of brown adipose tissue, UCP1, is a clear exampleof such an augmentative component. The newly discovered UCP1 homologs, UCP2, UCP3,and brain mitochondrial carrier protein 1 (BMCP1) may participate in the augmentativecomponent of proton leak. However, they do not appear to catalyze the basal leak, as this isobserved in mitochondria from cells which apparently lack these proteins. Whereas UCP1plays an important role in thermogenesis, the evidence that UCP2 and UCP3 do likewiseremains equivocal.     

GDP and carboxyatractylate inhibit 4-hydroxynonenal-activated proton conductance to differing degrees in mitochondria from skeletal muscle and heart

The lipid peroxidation product 4-hydroxynonenal (HNE) increases the proton conductance of the inner mitochondrial membrane through effects on uncoupling proteins (UCPs) and the adenine nucleotide translocase (ANT); however, the relative contribution of the two carriers to these effects is unclear. To clarify this we isolated mitochondria from skeletal muscle and heart of wild-type and Ucp3 knockout (Ucp3KO) mice. To increase UCP3 expression, some mice were i.p. injected with LPS (12 mg/kg body weight). In spite of the increased UCP3 expression levels, basal proton conductance did not change. HNE increased the proton conductance of skeletal muscle and heart mitochondria. In skeletal muscle, this increase was lower in Ucp3KO mice and higher in LPS-treated wild-type mice, and was partially abolished by GDP (UCPs inhibitor) and completely abolished by carboxyatractylate (ANT inhibitor) or addition of both inhibitors. GDP had no effect on HNE-induced conductance in heart mitochondria, but carboxyatractylate or administration of both inhibitors had a partial effect. GDP-mediated inhibition of HNE-activated proton conductance in skeletal muscle mitochondria was not observed in Ucp3KO mice, indicating that GDP is specific for UCP3, at least in muscle. Carboxyatractylate was able to inhibit UCP3, probably through an indirect mechanism. Our results are consistent with the conclusion that, in skeletal muscle, HNE-induced increase in proton conductance is mediated by UCP3 (30%) and ANT, whereas in the heart the increase is mediated by ANT and other carriers, possibly including UCP3.     

UCP2-dependent proton leak in isolated mammalian mitochondria.

A role for uncoupling protein (UCP) homologues in mediating the proton leak in mammalian mitochondria is controversial. We subjected insulinoma (INS-1) cells to adenoviral expression of UCP2 or UCP1 and assessed the proton leak as the kinetic relationship between oxygen use and the inner mitochondrial membrane potential. Cells were infected with different amounts of rat UCP2, and, in other experiments, with either UCP2 or UCP1. The relative molar expression of these subtypes was quantified through comparison with histidine-tagged UCP1 or UCP2 proteins engineered by expression in Escherichia coli. Adenoviral infection with UCP2, compared with beta-galactosidase, resulted in a dose-dependent shift in kinetics indicating increased H(+) flux at any given membrane potential. UCP1 also enhanced H(+) flux, but, on a relative molar basis, the overexpression of the endogenous protein, UCP2, was more potent than UCP1. These results were not due to nonspecific overexpression of mitochondrial protein since UCP1 activity was inhibited by GDP and because overexpression of another membrane carrier protein, the oxoglutarate malate carrier had no effect. UCP2-mediated H(+) conduction was not GDP sensitive. These data suggest that the UCP homologue, UCP2, mediates the proton leak in mitochondria of a mammalian cell wherein UCP2 is the native subtype.     

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.     

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.     

A sequence related to a DNA recognition element is essential for the inhibition by nucleotides of proton transport through the mitochondrial uncoupling protein.

The uncoupling protein (UCP) is uniquely expressed in brown adipose tissue, which is a thermogenic organ of mammals. The UCP uncouples mitochondrial respiration from ATP production by introducing a proton conducting pathway through the mitochondrial inner membrane. The activity of the UCP is regulated: nucleotide binding to the UCP inhibits proton conductance whereas free fatty acids increase it. The similarities between the UCP, the ADP/ATP carrier and the DNA recognition element found in the DNA binding domain of the estrogen receptor suggested that these proteins could share common features in their respective interactions with free nucleotides or DNA, and thus defined a putative 'nucleotide recognition element' in the UCP. This article provides demonstration of the validity of this hypothesis. The putative nucleotide recognition element corresponding to the amino acids 261-269 of the UCP was gradually destroyed, and these mutant proteins were expressed in yeast. Flow cytometry, measuring the mitochondrial membrane potential in vivo, showed increased uncoupling activities of these mutant proteins, and was corroborated with studies with isolated mitochondria. The deletion of the three amino acids Phe267, Lys268 and Gly269, resulted in a mutant where proton leak could be activated by fatty acids but not inhibited by nucleotides.     

A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling

Oxidative stress and mitochondrial dysfunction are associated with disease and aging. Oxidative stress results from overproduction of reactive oxygen species (ROS), often leading to peroxidation of membrane phospholipids and production of reactive aldehydes, particularly 4-hydroxy-2-nonenal. Mild uncoupling of oxidative phosphorylation protects by decreasing mitochondrial ROS production. We find that hydroxynonenal and structurally related compounds (such as trans-retinoic acid, trans-retinal and other 2-alkenals) specifically induce uncoupling of mitochondria through the uncoupling proteins UCP1, UCP2 and UCP3 and the adenine nucleotide translocase (ANT). Hydroxynonenal-induced uncoupling was inhibited by potent inhibitors of ANT (carboxyatractylate and bongkrekate) and UCP (GDP). The GDP-sensitive proton conductance induced by hydroxynonenal correlated with tissue expression of UCPs, appeared in yeast mitochondria expressing UCP1 and was absent in skeletal muscle mitochondria from UCP3 knockout mice. The carboxyatractylate-sensitive hydroxynonenal stimulation correlated with ANT content in mitochondria from Drosophila melanogaster expressing different amounts of ANT. Our findings indicate that hydroxynonenal is not merely toxic, but may be a biological signal to induce uncoupling through UCPs and ANT and thus decrease mitochondrial 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.     

Energization-dependent endogenous activation of proton conductance in skeletal muscle mitochondria

Leak of protons into the mitochondrial matrix during substrate oxidation partially uncouples electron transport from phosphorylation of ADP, but the functions and source of basal and inducible proton leak in vivo remain controversial. In the present study we describe an endogenous activation of proton conductance in mitochondria isolated from rat and mouse skeletal muscle following addition of respiratory substrate. This endogenous activation increased with time, required a high membrane potential and was diminished by high concentrations of serum albumin. Inhibition of this endogenous activation by GDP [classically considered specific for UCPs (uncoupling proteins)], carboxyatractylate and bongkrekate (considered specific for the adenine nucleotide translocase) was examined in skeletal muscle mitochondria from wild-type and Ucp3-knockout mice. Proton conductance through endogenously activated UCP3 was calculated as the difference in leak between mitochondria from wild-type and Ucp3-knockout mice, and was found to be inhibited by carboxyatractylate and bongkrekate, but not GDP. Proton conductance in mitochondria from Ucp3-knockout mice was strongly inhibited by carboxyatractylate, bongkrekate and partially by GDP. We conclude the following: (i) at high protonmotive force, an endogenously generated activator stimulates proton conductance catalysed partly by UCP3 and partly by the adenine nucleotide translocase; (ii) GDP is not a specific inhibitor of UCP3, but also inhibits proton translocation by the adenine nucleotide translocase; and (iii) the inhibition of UCP3 by carboxyatractylate and bongkrekate is likely to be indirect, acting through the adenine nucleotide translocase.     

Regulation of UCP1 and UCP3 in arctic ground squirrels and relation with mitochondrial proton leak.

Uncoupling protein (UCP) 1 (UCP1) catalyzes a proton leak in brown adipose tissue (BAT) mitochondria that results in nonshivering thermogenesis (NST), but the extent to which UCP homologs mediate NST in other tissues is controversial. To clarify the role of UCP3 in mediating NST in a hibernating species, we measured Ucp3 expression in skeletal muscle of arctic ground squirrels in one of three activity states (not hibernating, not hibernating and fasted for 48 h, or hibernating) and housed at 5 degrees C or -10 degrees C. We then compared Ucp3 mRNA levels in skeletal muscle with Ucp1 mRNA and UCP1 protein levels in BAT in the same animals. Ucp1 mRNA and UCP1 protein levels were increased on cold exposure and decreased with fasting, with the highest UCP1 levels in thermogenic hibernators. In contrast, Ucp3 mRNA levels were not affected by temperature but were increased 10-fold during fasting and >3-fold during hibernation. UCP3 protein levels were increased nearly fivefold in skeletal muscle mitochondria isolated from fasted squirrels compared with nonhibernators, but proton leak kinetics in the presence of BSA were unchanged. Proton leak in BAT mitochondria also did not differ between fed and fasted animals but did show classical inhibition by the purine nucleotide GDP. Levels of nonesterified fatty acids were highest during hibernation, and tissue temperatures during hibernation were related to Ucp1, but not Ucp3, expression. Taken together, these results do not support a role for UCP3 as a physiologically relevant mediator of NST in muscle.     

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.     

UCP1 muscle gene transfer and mitochondrial proton leak mediated thermogenesis

Mitochondrial uncoupling protein 1 (UCP1) mediates the thermogenic transport of protons through the inner mitochondrial membrane. This proton leak uncouples respiration from ATP synthesis. The current study assessed the possible contribution of UCP1 muscle gene transfer to impair mitochondrial respiration in a tissue lacking UCP1 gene expression. Rats received an intramuscular injection of plasmid pXC1 containing UCP1 cDNA in the right tibialis muscles, while left tibialis muscles were injected with empty plasmid as control. Ten days after DNA injection, mitochondria from tibialis anterior muscles were isolated and analyzed. UCP1 gene transfer resulted in protein expression as analyzed by inmunoblotting. Mitochondria isolated from UCP1-injected muscles showed a significant increase in state 2 and state 4 oxygen consumption rates and a decreased respiration control ratio in comparison to mitochondria from control muscles. Furthermore, UCP1-containing mitochondria had a lower membrane potential in those states (2 and 4) when compared with control mitochondria. Our results revealed that UCP1 muscle gene transfer is associated with an induced mitochondrial proton leak, which could contribute to increase energy expenditure.     

Mitochondrial proton leak in obesity-resistant and obesity-prone mice

We quantified uncoupling proteins (UCPs) in molar amounts and assessed proton conductance in mitochondria isolated from interscapular brown adipose tissue (IBAT) and hindlimb muscle [known from prior work to contain ectopic brown adipose tissue (BAT) interspersed between muscle fibers] of obesity-resistant 129S6/SvEvTac (129) and obesity-prone C57BL/6 (B6) mice under conditions of low (LF) and high-fat (HF) feeding. With usual feeding, IBAT mitochondrial UCP1 content and proton conductance were greater in 129 mice than B6. However, with HF feeding, UCP1 and proton conductance increased more in B6 mice. Moreover, with HF feeding GDP-inhibitable proton conductance, specific for UCP1, equaled that seen in the 129 strain. UCP1 expression was substantial in mitochondria from hindlimb muscle tissue (ectopic BAT) of 129 mice as opposed to B6 but did not increase with HF feeding in either strain. As expected, muscle UCP3 expression increased with HF feeding in both strains but did not differ by strain. Moreover, the proton conductance of mitochondria isolated from hindlimb muscle tissue did not differ by strain or diet. Our data uncover a response to weight gain in obesity-prone (compared to resistant) mice unrecognized in prior studies that examined only UCP1 mRNA. Obesity-prone mice have the capacity to increase both IBAT UCP1 protein and mitochondrial proton conductance as much or more than obesity-resistant mice. But, this is only achieved only at a higher body mass and, therefore, may be adaptive rather than preventative. Neither obesity-prone nor resistant mice respond to HF feeding by expressing more UCP1 in ectopic BAT within muscle tissue.     

Mitochondrial proton leak and the uncoupling protein 1 homologues

Mitochondrial proton leak is the largest single contributor to the standard metabolic rate (SMR) of a rat, accounting for about 20% of SMR. Yet the mechanisms by which proton leak occurs are incompletely understood. The available evidence suggests that both phospholipids and proteins in the mitochondrial inner membrane are important determinants of proton conductance. The uncoupling protein 1 homologues (e.g. UCP2, UCP3) may play a role in mediating proton leak, but it is unlikely they account for all of the observed proton conductance. Experimental data regarding the functions of these proteins include important ambiguities and contradictions which must be addressed before their function can be confirmed. The physiological role of the proton leak, and of the uncoupling protein 1 homologues, remains similarly unclear.     

Carboxyatractyloside effects on brown-fat mitochondria imply that the adenine nucleotide translocator isoforms ANT1 and ANT2 may be responsible for basal and fatty-acid-induced uncoupling respectively

In brown-fat mitochondria, fatty acids induce thermogenic uncoupling through activation of UCP1 (uncoupling protein 1). However, even in brown-fat mitochondria from UCP1-/- mice, fatty-acid-induced uncoupling exists. In the present investigation, we used the inhibitor CAtr (carboxyatractyloside) to examine the involvement of the ANT (adenine nucleotide translocator) in the mediation of this UCP1-independent fatty-acid-induced uncoupling in brown-fat mitochondria. We found that the contribution of ANT to fatty-acid-induced uncoupling in UCP1-/- brown-fat mitochondria was minimal (whereas it was responsible for nearly half the fatty-acid-induced uncoupling in liver mitochondria). As compared with liver mitochondria, brown-fat mitochondria exhibit a relatively high (UCP1-independent) basal respiration ('proton leak'). Unexpectedly, a large fraction of this high basal respiration was sensitive to CAtr, whereas in liver mitochondria, basal respiration was CAtr-insensitive. Total ANT protein levels were similar in brown-fat mitochondria from wild-type mice and in liver mitochondria, but the level was increased in brown-fat mitochondria from UCP1-/- mice. However, in liver, only Ant2 mRNA was found, whereas in brown adipose tissue, Ant1 and Ant2 mRNA levels were equal. The data are therefore compatible with a tentative model in which the ANT2 isoform mediates fatty-acid-induced uncoupling, whereas the ANT1 isoform may mediate a significant part of the high basal proton leak in brown-fat mitochondria.     

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.     

No evidence for a basal,retinoic, or superoxide-induced uncoupling activity of the uncoupling protein 2 present in spleen or lung mitochondria

The phenotypes observed in mice whose uncoupling protein (Ucp2) gene had been invalidated by homologous recombination (Ucp2(-/-) mice) are consistent with an increase in mitochondrial membrane potential in macrophages and pancreatic beta cells. This could support an uncoupling (proton transport) activity of UCP2 in the inner mitochondrial membrane in vivo. We used mitochondria from lung or spleen, the two organs expressing the highest level of UCP2, to compare the proton leak of the mitochondrial inner membrane of wild-type and Ucp2(-/-) mice. No difference was observed under basal conditions. Previous reports have concluded that retinoic acid and superoxide activate proton transport by UCP2. Spleen mitochondria showed a higher sensitivity to retinoic acid than liver mitochondria, but this was not caused by UCP2. In contrast with a previous report, superoxide failed to increase the proton leak rate in kidney mitochondria, where no UCP2 expression was detected, and also in spleen mitochondria, which does not support stimulation of UCP2 uncoupling activity by superoxide. Finally, no increase in the ATP/ADP ratio was observed in spleen or lung of Ucp2(-/-) mice. Therefore, no evidence could be gathered for the uncoupling activity of the UCP2 present in spleen or lung mitochondria. Although this may be explained by difficulties with isolated mitochondria, it may also indicate that UCP2 has another physiological significance in spleen and lung.     

UCP2 and UCP3 rise in starved rat skeletal muscle but mitochondrial proton conductance is unchanged

The relationship between UCP2 and UCP3 expression and mitochondrial proton conductance of rat skeletal muscle was examined. Rats were starved for 24 h and the levels of UCP2 and UCP3 mRNA and UCP3 protein were determined by Northern and Western blots. Proton conductance was measured by titrating mitochondrial respiration rate and membrane potential with malonate. Starvation increased UCP2 and UCP3 mRNA levels more than 5-fold and 4-fold, respectively, and UCP3 protein levels by 2-fold. However, proton conductance remained unchanged. These results suggest either that Northern and Western blots do not reflect the levels of active protein or that these UCPs do not catalyse the basal proton conductance in skeletal muscle mitochondria.     

Mitochondrial UCPs: new insights into regulation and impact

Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins sustaining an inducible proton conductance. They weaken the proton electrochemical gradient built up by the mitochondrial respiratory chain. Brown fat UCP1 sustains a free fatty acid (FA)-induced purine nucleotide (PN)-inhibited proton conductance. Inhibition of the proton conductance by PN has been considered as a diagnostic of UCP activity. However, conflicting results have been obtained in isolated mitochondria for UCP homologues (i.e., UCP2, UCP3, plant UCP, and protist UCP) where the FFA-activated proton conductance is poorly sensitive to PN under resting respiration conditions. Our recent work clearly indicates that the membranous coenzyme Q, through its redox state, represents a regulator of the inhibition by PN of FFA-activated UCP1 homologues under phosphorylating respiration conditions. Several physiological roles of UCPs have been suggested, including a control of the cellular energy balance as well as the preventive action against oxidative stress. In this paper, we discuss new information emerging from comparative proteomics about the impact of UCPs on mitochondrial physiology, when recombinant UCP1 is expressed in yeast and when UCP2 is over-expressed in hepatic mitochondria during steatosis.     

Proton leak modulation in testicular mitochondria affects reactive oxygen species production and lipid peroxidation

Mitochondrial proton leak can account for almost 20% of oxygen consumption and it is generally accepted that this process contributes to basal metabolism. In order to clarify the role of basal proton leak in testicular mitochondria, we performed a comparative study with kidney and liver mitochondrial fractions. Proton leak stimulated by linoleic acid and inhibited by guanosine diphosphate (GDP) was detected, in a manner that was correlated with protein levels for uncoupling protein 2 (UCP2) in the three fractions. Modulation of proton leak had an effect on reactive oxygen species production as well as on lipid peroxidation, and this effect was also tissue‐dependent. However, a possible role for the adenine nucleotide transporter (ANT) in testicular mitochondria proton leak could not be excluded. The modulation of proton leak appears as a possible and attractive target to control oxidative stress with implications for male gametogenesis. Copyright © 2010 John Wiley & Sons, Ltd.