Lack of obesity and normal response to fasting and thyroid hormone in mice lacking uncoupling protein-3

Uncoupling protein-3 (UCP3) is a mitochondrial protein that can diminish the mitochondrial membrane potential. Levels of muscle Ucp3 mRNA are increased by thyroid hormone and fasting. Ucp3 has been proposed to influence metabolic efficiency and is a candidate obesity gene. We have produced a Ucp3 knockout mouse to test these hypotheses. The Ucp3 (-/-) mice had no detectable immunoreactive UCP3 by Western blotting. In mitochondria from the knockout mice, proton leak was greatly reduced in muscle, minimally reduced in brown fat, and not reduced at all in liver. These data suggest that UCP3 accounts for much of the proton leak in skeletal muscle. Despite the lack of UCP3, no consistent phenotypic abnormality was observed. The knockout mice were not obese and had normal serum insulin, triglyceride, and leptin levels, with a tendency toward reduced free fatty acids and glucose. Knockout mice showed a normal circadian rhythm in body temperature and motor activity and had normal body temperature responses to fasting, stress, thyroid hormone, and cold exposure. The base-line metabolic rate and respiratory exchange ratio were the same in knockout and control mice, as were the effects of fasting, a beta3-adrenergic agonist (CL316243), and thyroid hormone on these parameters. The phenotype of Ucp1/Ucp3 double knockout mice was indistinguishable from Ucp1 single knockout mice. These data suggest that Ucp3 is not a major determinant of metabolic rate but, rather, has other functions.     

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.     

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.     

Thermogenic responses in brown fat cells are fully UCP1-dependent. UCP2 or UCP3 do not substitute for UCP1 in adrenergically or fatty scid-induced thermogenesis

To examine the thermogenic significance of the classical uncoupling protein-1 (UCP1), the thermogenic potential of brown adipocytes isolated from UCP1-ablated mice was investigated. Ucp1(-/-) cells had a basal metabolic rate identical to wild-type; the mitochondria within them were coupled to the same degree. The response to norepinephrine in wild-type cells was robust ( approximately 10-fold increase in thermogenesis); Ucp1(-/-) cells only responded approximately 3% of this. Ucp1(-/-) cells were as potent as wild-type in norepinephrine-induced cAMP accumulation and lipolysis and had a similar mitochondrial respiratory complement. In wild-type cells, fatty acids induced a thermogenic response similar to norepinephrine, but fatty acids (and retinoate) were practically without effect in Ucp1(-/-) cells. It is concluded that no other adrenergically induced thermogenic mechanism exists in brown adipocytes except that mediated by UCP1 and that entopic expression of UCP1 does not lead to overt innate uncoupling, and it is suggested that fatty acids are transformed to an intracellular physiological activator of UCP1. High expression of UCP2 and UCP3 in the tissue was not associated with an overt innate highly uncoupled state of mitochondria within the cells, nor with an ability of norepinephrine or endo- or exogenous fatty acids to induce uncoupled respiration in the cells. Thus, UCP1 remains the only physiologically potent thermogenic uncoupling protein in these cells.     

Long-term high-fat feeding induces greater fat storage in mice lacking UCP3

Uncoupling protein-3 (UCP3) is a mitochondrial inner-membrane protein highly expressed in skeletal muscle. While UCP3's function is still unknown, it has been hypothesized to act as a fatty acid (FA) anion exporter, protecting mitochondria against lipid peroxidation and/or facilitating FA oxidation. The aim of this study was to determine the effects of long-term feeding of a 45% fat diet on whole body indicators of muscle metabolism in congenic C57BL/6 mice that were either lacking UCP3 (Ucp3(-/-)) or had a transgenically induced approximately twofold increase in UCP3 levels (UCP3tg). Mice were fed the high-fat (HF) diet for a period of either 4 or 8 mo immediately following weaning. After long-term HF feeding, UCP3tg mice weighed an average of 15% less than wild-type mice (P < 0.05) and were 20% less metabolically efficient than both wild-type and Ucp3(-/-) mice (P < 0.01). Additionally, wild-type mice had 21% lower, whereas UCP3tg mice had 36% lower, levels of adiposity compared with Ucp3(-/-) mice (P < 0.05 and P < 0.001, respectively), indicating a protective effect of UCP3 against fat gain. No differences in whole body oxygen consumption were detected following long-term HF feeding. Glucose and insulin tolerance tests revealed that both the UCP3tg and Ucp3(-/-) mice were more glucose tolerant and insulin sensitive compared with wild-type mice after short-term HF feeding, but this protection was not maintained in the long term. Findings indicate that UCP3 is involved in protection from fat gain induced by long-term HF feeding, but not in protection from insulin resistance.     

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.     

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.     

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.     

The bioenergetics of brown fat mitochondria from UCP1-ablated mice. Ucp1 is not involved in fatty acid-induced de-energization ("uncoupling").

The bioenergetics of brown fat mitochondria isolated from UCP1-ablated mice were investigated. The mitochondria had lost the high GDP-binding capacity normally found in brown fat mitochondria, and they were innately in an energized state, in contrast to wild-type mitochondria. GDP, which led to energization of wild-type mitochondria, was without effect on the brown fat mitochondria from UCP1-ablated mice. The absence of thermogenic function did not result in reintroduction of high ATP synthase activity. Remarkably and unexpectedly, the mitochondria from UCP1-ablated mice were as sensitive to the de-energizing ("uncoupling") effect of free fatty acids as were UCP1-containing mitochondria. Therefore, the de-energizing effect of free fatty acids does not appear to be mediated via UCP1, and free fatty acids would not seem to be the intracellular physiological activator involved in mediation of the thermogenic signal from the adrenergic receptor to UCP1. In the UCP1-ablated mice, Ucp2 mRNA levels in brown adipose tissue were 14-fold higher and Ucp3 mRNA levels were marginally lower than in wild-type. The Ucp2 and Ucp3 mRNA levels were therefore among the highest found in any tissue. These high mRNA levels did not confer on the isolated mitochondria any properties associated with de-energization. Thus, the mere observation of a high level of Ucp2 or Ucp3 mRNA in a tissue cannot be taken as an indication that mitochondria isolated from that tissue will display innate de-energization or thermogenesis.     

Effects of the presence, absence, and overexpression of uncoupling protein-3 on adiposity and fuel metabolism in congenic mice

Uncoupling protein-3 (UCP3) is a poorly understood mitochondrial inner membrane protein expressed predominantly in skeletal muscle. The aim of this study was to examine the effects of the absence or constitutive physiological overexpression of UCP3 on whole body energy metabolism, glucose tolerance, and muscle triglyceride content. Congenic male UCP3 knockout mice (Ucp3-/-), wild-type, and transgenic UCP3 overexpressing (UCP3Tg) mice were fed a 10% fat diet for 4 or 8 mo after they were weaned. UCP3Tg mice had lower body weights and were less metabolically efficient than wild-type or Ucp3-/- mice, but they were not hyperphagic. UCP3Tg mice had smaller epididymal white adipose tissue and brown adipose tissue (BAT) depots; however, there were no differences in muscle weights. Glucose and insulin tolerance tests revealed that both UCP3Tg and Ucp3-/- mice were protected from development of impaired glucose tolerance and were more sensitive to insulin. 2-Deoxy-D-[1-3H]glucose tracer studies showed increased uptake of glucose into BAT and increased storage of liver glycogen in Ucp3-/- mice. Assessments of intramuscular triglyceride (IMTG) revealed decreases in quadriceps of UCP3Tg mice compared with wild-type and Ucp3-/- mice. When challenged with a 45% fat diet, Ucp3-/- mice showed increased accumulation of IMTG compared with wild-type mice, which in turn had greater IMTG than UCP3Tg mice. Results are consistent with a role for UCP3 in preventing accumulation of triglyceride in both adipose tissue and muscle.     

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.     

UCP1 deficiency increases susceptibility to diet-induced obesity with age

Loss of nonshivering thermogenesis in mice by inactivation of the mitochondrial uncoupling protein gene (Ucp1-/- mice) causes increased sensitivity to cold and unexpected resistance to diet-induced obesity at a young age. To clarify the role of UCP1 in body weight regulation throughout life and influence of UCP1 deficiency on longevity, we longitudinally analyzed the phenotypes of Ucp1-/- mice maintained in a room at 23 degrees C. There was no difference in body weight and lifespan between genotypes under the standard chow diet condition, whereas the mutant mice developed obesity with age under the high-fat (HF) diet condition. Compared with Ucp1+/+ mice, Ucp1-/- mice showed increased expression of genes related to thermogenesis and fatty acid metabolism, such as beta3-adrenergic receptor, in adipose tissues of the 3-month-old mutants; however, the augmented expression was reduced in Ucp1+/+ mice in 11-month-old Ucp1-/- mice fed the HF diet. Likewise, the increased levels of UCP3 and cAMP-dependent protein kinase in the brown adipose tissue of Ucp1-/- mice given the standard diet were decreased significantly in that of Ucp1-/- mice fed the HF diet, which animals showed impaired norepinephrine-induced lipolysis in their adipose tissues. These results suggest profound attenuation of beta-adrenergic responsiveness and fatty acid utilization in Ucp1-/- mice fed the HF diet, bringing them to late-onset obesity. Our findings provide evidence that UCP1 is neither essential for body weight regulation nor for longevity under conditions of standard diet and normal housing temperature, but deficiency increases susceptibility to obesity with age in combination with HF diet.     

Essential role for uncoupling protein-3 in mitochondrial adaptation to fasting but not in fatty acid oxidation or fatty acid anion export

Uncoupling protein-3 (UCP3) is a mitochondrial inner membrane protein expressed most abundantly in skeletal muscle and to a lesser extent in heart and brown adipose tissue. Evidence supports a role for UCP3 in fatty acid oxidation (FAO); however, the underlying mechanism has not been explored. In 2001 we proposed a role for UCP3 in fatty acid export, leading to higher FAO rates (Himms-Hagen, J., and Harper, M. E. (2001) Exp. Biol. Med. (Maywood) 226, 78-84). Specifically, this widely held hypothesis states that during elevated FAO rates, UCP3 exports fatty acid anions, thereby maintaining mitochondrial co-enzyme A availability; reactivation of exported fatty acid anions would ultimately enable increased FAO. Here we tested mechanistic aspects of this hypothesis as well as its functional implications, namely increased FAO rates. Using complementary mechanistic approaches in mitochondria from wild-type and Ucp3(-)(/)(-) mice, we find that UCP3 is not required for FAO regardless of substrate type or supply rate covering a 20-fold range. Fatty acid anion export and reoxidation during elevated FAO, although present in skeletal muscle mitochondria, are independent of UCP3 abundance. Interestingly, UCP3 was found to be necessary for the fasting-induced enhancement of FAO rate and capacity, possibly via mitigated mitochondrial oxidative stress. Thus, although our observations indicate that UCP3 can impact FAO rates, the mechanistic basis is not via an integral function for UCP3 in the FAO machinery. Overall our data indicate a function for UCP3 in mitochondrial adaptation to perturbed cellular energy balance and integrate previous observations that have linked UCP3 to reduced oxidative stress and FAO.     

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.     

The role of uncoupling protein 1 in the metabolism and adiposity of RII beta-protein kinase A-deficient mice

Mice lacking the RII beta regulatory subunit of protein kinase A exhibit a 50% reduction in white adipose tissue stores compared with wild-type littermates and are resistant to diet-induced obesity. RII beta(-/-) mice also have an increase in resting oxygen consumption along with a 4-fold increase in the brown adipose-specific mitochondrial uncoupling protein 1 (UCP1). In this study, we examined the basis for UCP1 induction and tested the hypothesis that the induced levels of UCP1 in RII beta null mice are essential for the lean phenotype. The induction of UCP1 occurred at the protein but not the mRNA level and correlated with an increase in mitochondria in brown adipose tissue. Mice lacking both RII beta and UCP1 (RII beta(-/-)/Ucp1(-/-)) were created, and the key parameters of metabolism and body composition were studied. We discovered that RII beta(-/-) mice exhibit nocturnal hyperactivity in addition to the increased oxygen consumption at rest. Disruption of UCP1 in RII beta(-/-) mice reduced basal oxygen consumption but did not prevent the nocturnal hyperactivity. The double knockout animals also retained the lean phenotype of the RII beta null mice, demonstrating that induction of UCP1 and increased resting oxygen consumption is not the cause of leanness in the RII beta mutant mice.     

Uncoupling protein 3 protects aconitase against inactivation in isolated skeletal muscle mitochondria

Mitochondrial uncoupling proteins only catalyse proton transport when they are activated. Activators include superoxide and reactive alkenals, suggesting new physiological functions for UCP2 and UCP3: their activation by superoxide when protonmotive force is high causes mild uncoupling, which lowers protonmotive force and attenuates superoxide generation by the electron transport chain. This feedback loop acts to prevent excessive mitochondrial superoxide production. Superoxide inactivates aconitase in the mitochondrial matrix, so aconitase activity provides a sensitive measure of the effects of UCPs on matrix superoxide. We find that inhibition of UCP3 in isolated skeletal muscle mitochondria by GDP decreases aconitase activity by 25% after 20 min incubation. The GDP effect is absent in skeletal muscle mitochondria from UCP3 knockout mice, showing that it is mediated by UCP3. Protection of aconitase by UCP3 in the absence of nucleotides does not require added fatty acids. The purine nucleoside diphosphates and triphosphates cause aconitase inactivation, but the monophosphates and CDP do not, consistent with the known nucleotide specificity of UCP3. The IC(50) for GDP is about 100 microM. These findings support the proposal that UCP3 attenuates endogenous radical production by the mitochondrial electron transport chain at high protonmotive force.     

Absence of mitochondrial uncoupling protein 3: effect on thymus and spleen in the fed and fasted mice

Mitochondrial uncoupling protein 3 (UCP3) is constitutively expressed in mitochondria from thymus and spleen of mice, and confocal microscopy has been used to visualize UCP3 in situ in mouse thymocytes. UCP3 is present in mitochondria of thymus and spleen up to at least 16 weeks after birth, but levels decrease by a half in thymus and a fifth in spleen after three weeks, probably reflecting the suckling to weaning transition. UCP3 protein levels increase approximately 3-fold in thymus on starvation, but expression levels in spleen were unaffected by starvation. Lack of UCP3 had little effect on thymus mass or thymocyte number. However, lack of UCP3 affected spleen mass and splenocyte number (in the fasted state) and results in reduced CD4+ single positive cell numbers and reduced double negative cells in the thymus, but as a 2-fold increase in the proportion of CD4(+), CD8(+) and DP cells in spleen. Starvation attenuates these proportionate differences in the spleen. A lack of UCP3 had no apparent effect on basal oxygen consumption of thymocytes or splenocytes or on oxygen consumption due to mitochondrial proton leak. Splenocytes from UCP3 knock-out mice are also more resistant to apoptosis than those from wild-type mice. Overall we can conclude that UCP3 affects thymocyte and spleen cell profiles in the fed and fasted states.     

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     

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.