Uncoupling protein-1 (UCP1) contributes to the basal proton conductance of brown adipose tissue mitochondria

Proton leak pathways uncouple substrate oxidation from ATP synthesis in mitochondria. These pathways are classified as basal (not regulated) or inducible (activated and inhibited). Previously it was found that over half of the basal proton conductance of muscle mitochondria was catalyzed by the adenine nucleotide translocase (ANT), an abundant mitochondrial anion carrier protein. To determine whether ANT is the unique protein catalyst, or one of many proteins that catalyze basal proton conductance, we measured proton leak kinetics in mitochondria isolated from brown adipose tissue (BAT). BAT can express another mitochondrial anion carrier, UCP1, at concentrations similar to ANT. Basal proton conductance was measured under conditions where UCP1 and ANT were catalytically inactive and was found to be lower in mitochondria from UCP1 knockout mice compared to wild-type. Ablation of another abundant inner membrane protein, nicotinamide nucleotide transhydrogenase, had no effect on proton leak kinetics in mitochondria from liver, kidney or muscle, showing that basal proton conductance is not catalyzed by all membrane proteins. We identify UCP1 as a second protein propagating basal proton leak, lending support to the hypothesis that basal leak pathways are perpetrated by members of the mitochondrial anion carrier family but not by other mitochondrial inner membrane proteins.     

Long-term fasting decreases mitochondrial avian UCP-mediated oxygen consumption in hypometabolic king penguins

In endotherms, regulation of the degree of mitochondrial coupling affects cell metabolic efficiency. Thus it may be a key contributor to minimizing metabolic rate during long periods of fasting. The aim of the present study was to investigate whether variation in mitochondrial avian uncoupling proteins (avUCP), as putative regulators of mitochondrial oxidative phosphorylation, may contribute to the ability of king penguins (Aptenodytes patagonicus) to withstand fasting for several weeks. After 20 days of fasting, king penguins showed a reduced rate of whole animal oxygen consumption (Vo2; -33%) at rest, together with a reduced abundance of avUCP and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC1-alpha) mRNA in pectoralis muscle (-54%, -36%, respectively). These parameters were restored after the birds had been refed for 3 days. Furthermore, in recently fed, but not in fasted penguins, isolated muscle mitochondria showed a guanosine diphosphate-inhibited, fatty acid plus superoxide-activated respiration, indicating the presence of a functional UCP. It was calculated that variation in mitochondrial UCP-dependent respiration in vitro may contribute to nearly 20% of the difference in resting Vo2 between fed or refed penguins and fasted penguins measured in vivo. These results suggest that the lowering of avUCP activity during periods of long-term energetic restriction may contribute to the reduction in metabolic rate and hence the ability of king penguins to face prolonged periods of fasting.     

Meat-type chickens have a higher efficiency of mitochondrial oxidative phosphorylation than laying-type chickens

Meat-type chickens show high feed efficiency and have a very rapid growth rate compared with laying-type chickens. To clarify whether the type-specific difference in feed conversion efficiency is involved in mitochondrial bioenergetics, modular kinetic analysis was applied to oxidative phosphorylation in skeletal muscle mitochondria of both type chickens. Mitochondria from skeletal muscle of meat-type chickens showed greater substrate oxidation and phosphorylating activities, and less proton leak than those of the laying-type, resulting in a higher efficiency of oxidative phosphorylation. Gene expression and protein content of uncoupling protein (avUCP) but not adenine nucleotide translocase (avANT) gene expression were lower in skeletal muscle mitochondria of meat-type chickens than the laying-type. The current results regarding a higher efficiency of oxidative phosphorylation and UCP content may partially support the high feed efficiency of meat-type chickens.     

The basal proton conductance of skeletal muscle mitochondria from transgenic mice overexpressing or lacking uncoupling protein-3.

The ability of native uncoupling protein-3 (UCP3) to uncouple mitochondrial oxidative phosphorylation is controversial. We measured the expression level of UCP3 and the proton conductance of skeletal muscle mitochondria isolated from transgenic mice overexpressing human UCP3 (UCP3-tg) and from UCP3 knockout (UCP3-KO) mice. The concentration of UCP3 in UCP3-tg mitochondria was approximately 3 microg/mg protein, approximately 20-fold higher than the wild type value. UCP3-tg mitochondria had increased nonphosphorylating respiration rates, decreased respiratory control, and approximately 4-fold increased proton conductance compared with the wild type. However, this increased uncoupling in UCP3-tg mitochondria was not caused by native function of UCP3 because it was not proportional to the increase in UCP3 concentration and was neither activated by superoxide nor inhibited by GDP. UCP3 was undetectable in mitochondria from UCP3-KO mice. Nevertheless, UCP3-KO mitochondria had unchanged respiration rates, respiratory control ratios, and proton conductance compared with the wild type under a variety of assay conditions. We conclude that uncoupling in UCP3-tg mice is an artifact of transgenic expression, and that UCP3 does not catalyze the basal proton conductance of skeletal muscle mitochondria in the absence of activators such as superoxide.     

Cold-induced mitochondrial uncoupling and expression of chicken UCP and ANT mRNA in chicken skeletal muscle

Although bird species studied thus far have no distinct brown adipose tissue (BAT) or a related thermogenic tissue, there is now strong evidence that non-shivering mechanisms in birds may play an important role during cold exposure. Recently, increased expression of the duckling homolog of the avian uncoupling protein (avUCP) was demonstrated in cold-acclimated ducklings [Raimbault et al., Biochem. J. 353 (2001) 441-444]. Among the mitochondrial anion carriers, roles for the ATP/ADP antiporter (ANT) as well as UCP variants in thermogenesis are proposed. The present experiments were conducted (i) to examine the effects of cold acclimation on the fatty acid-induced uncoupling of oxidative phosphorylation in skeletal muscle mitochondria and (ii) to clone the cDNA of UCP and ANT homologs from chicken skeletal muscle and study differences compared to controls in expression levels of their mRNAs in the skeletal muscle of cold-acclimated chickens. The results obtained here show that suppression of palmitate-induced uncoupling by carboxyatractylate was greater in the subsarcolemmal skeletal muscle mitochondria from cold-acclimated chickens than that for control birds. An increase in mRNA levels of avANT and, to lesser degree, of avUCP in the skeletal muscle of cold-acclimated chickens was also found. Taken together, the present studies on cold-acclimated chickens suggest that the simultaneous increments in levels of avANT and avUCP mRNA expression may be involved in the regulation of thermogenesis in skeletal muscle.     

Tissue variation of mitochondrial oxidative phosphorylation efficiency in cold-acclimated ducklings

We investigated the oxidative phosphorylation efficiency of liver and gastrocnemius muscle mitochondria in thermoneutral and cold-acclimated ducklings. The yield of oxidative phosphorylation was lower in muscle than in liver mitochondria, a difference that was associated with a higher proton conductance in muscle mitochondria. Cold exposure did not affect oxidative phosphorylation efficiency or basal proton leak in mitochondria. We conclude that the basal proton conductance of mitochondria may regulate mitochondrial oxidative phosphorylation efficiency, but is not an important contributor to thermogenic processes in cold-acclimated ducklings.     

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.     

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.     

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.     

Production of endogenous matrix superoxide from mitochondrial complex I leads to activation of uncoupling protein 3

Superoxide generated using exogenous xanthine oxidase indirectly activates an uncoupling protein (UCP)-mediated proton conductance of the mitochondrial inner membrane. We investigated whether endogenous mitochondrial superoxide production could also activate proton conductance. When respiring on succinate, rat skeletal muscle mitochondria produced large amounts of matrix superoxide. Addition of GDP to inhibit UCP3 markedly inhibited proton conductance and increased superoxide production. Both superoxide production and the GDP-sensitive proton conductance were suppressed by rotenone plus an antioxidant. Thus, endogenous superoxide can activate the proton conductance of UCP3, which in turn limits mitochondrial superoxide production. These observations provide a departure point for studies under more physiological conditions.     

Ontogeny of thermoregulatory mechanisms in king penguin chicks (Aptenodytes patagonicus)

The rapid maturation of thermoregulatory mechanisms may be of critical importance for optimising chick growth and survival and parental energy investment under harsh climatic conditions. The ontogeny of thermoregulatory mechanisms was studied in growing king penguin chicks from hatching to the full emancipation observed at 1 month of age in the sub-Antarctic area (Crozet Archipelago). Newly hatched chicks showed small, but significant regulatory thermogenesis (21% rise in heat production assessed by indirect calorimetry), but rapidly became hypothermic. Within a few days, both resting (+32%) and peak (+52%) metabolic rates increased. The first week of life was characterised by a two-fold rise in thermogenic capacity in the cold, while thermal insulation was not improved. During the second and third weeks of age, thermal insulation markedly rose (two-fold drop in thermal conductance) in relation to down growth, while resting heat production was slightly reduced (-13%). Shivering (assessed by electromyography) was visible right after hatching, although its efficiency was limited. Thermogenic efficiency of shivering increased five-fold with age during the first weeks of life, but there was no sign of non-shivering thermogenesis. We conclude that thermal emancipation of king penguin chicks may be primarily determined by improvement of thermal insulation after thermogenic processes have become sufficiently matured. Both insulative and metabolic adaptations are required for the rapid ontogeny of thermoregulation and thermal emancipation in growing king penguin chicks.     

Superoxide stimulates a proton leak in potato mitochondria that is related to the activity of uncoupling protein

The ability of plant mitochondrial uncoupling proteins to catalyze a significant proton conductance in situ is controversial. We have re-examined conditions that lead to uncoupling of mitochondria isolated from the tubers of potato (Solanum tuberosum). Specifically, we have investigated the effect of superoxide. In the absence of superoxide, linoleic acid stimulated a proton leak in mitochondria respiring NADH that was insensitive to GTP. However, when exogenous superoxide was generated by the addition of xanthine and xanthine oxidase, there was an additional linoleic acid-stimulated proton leak that was specifically inhibited by GTP. Under these conditions of assay (NADH as a respiratory substrate, in the presence of linoleic acid and xanthine/xanthine oxidase) there was a higher rate of proton conductance in mitochondria from transgenic potato tubers overexpressing the StUCP gene than those from wild type. The increased proton leak in the transgenic mitochondria was completely abolished by the addition of GTP. This suggests that superoxide and linoleic acid stimulate a proton leak in potato mitochondria that is related to the activity of uncoupling protein. Furthermore, it demonstrates that changes in the amount of StUCP can alter the rate of proton conductance of potato mitochondria.     

Physiological levels of mammalian uncoupling protein 2 do not uncouple yeast mitochondria

We assessed the ability of human uncoupling protein 2 (UCP2) to uncouple mitochondrial oxidative phosphorylation when expressed in yeast at physiological and supraphysiological levels. We used three different inducible UCP2 expression constructs to achieve mitochondrial UCP2 expression levels in yeast of 33, 283, and 4100 ng of UCP2/mg of mitochondrial protein. Yeast mitochondria expressing UCP2 at 33 or 283 ng/mg showed no increase in proton conductance, even in the presence of various putative effectors, including palmitate and all-trans-retinoic acid. Only when UCP2 expression in yeast mitochondria was increased to 4 microg/mg, more than an order of magnitude greater than the highest known physiological concentration, was proton conductance increased. This increased proton conductance was not abolished by GDP. At this high level of UCP2 expression, an inhibition of substrate oxidation was observed, which cannot be readily explained by an uncoupling activity of UCP2. Quantitatively, even the uncoupling seen at 4 microgram/mg was insufficient to account for the basal proton conductance of mammalian mitochondria. These observations suggest that uncoupling of yeast mitochondria by UCP2 is an overexpression artifact leading to compromised mitochondrial integrity.     

Fatty acid activation of the reconstituted brown adipose tissue mitochondria uncoupling protein

The effect of fatty acids, palmitoyl-CoA, and N',N-dicyclohexylcarbodiimide on the ion conductance of the reconstituted brown adipose tissue mitochondria uncoupling protein was investigated. 1, 5, and 10 microM palmitic acid induced a specific, GDP inhibited, increase in proton conductance in proteoliposomes containing the uncoupling protein but not in proteoliposomes prepared with purified protein extracts of liver mitochondria. 10 microM oleic acid, like palmitic acid, increased proton conductance in proteoliposomes prepared with the uncoupling protein. Palmitoyl-CoA and caprylic acid had no effect on increasing proton conductance. Similar to the observation in mitochondria, there was no effect of palmitic acid on Cl-conductance, but unlike mitochondria its activation by palmitoyl-CoA or inhibition by N',N-dicyclohexylcarbodiimide was lost. The results, obtained in an isolated system, provide support for the contention that long chain fatty acids act as an acute physiological activator of the uncoupling protein.     

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.     

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.     

Up-regulation of avian uncoupling protein in cold-acclimated and hyperthyroid ducklings prevents reactive oxygen species production by skeletal muscle mitochondria

Background  

Although identified in several bird species, the biological role of the avian homolog of mammalian uncoupling proteins (avUCP) remains extensively debated. In the present study, the functional properties of isolated mitochondria were examined in physiological or pharmacological situations that induce large changes in avUCP expression in duckling skeletal muscle.     

Uncoupling protein-2 contributes significantly to high mitochondrial proton leak in INS-1E insulinoma cells and attenuates glucose-stimulated insulin secretion

Proton leak exerts stronger control over ATP/ADP in mitochondria from clonal pancreatic beta-cells (INS-1E) than in those from rat skeletal muscle, due to the higher proton conductance of INS-1E mitochondria [Affourtit and Brand (2006) Biochem. J. 393, 151-159]. In the present study, we demonstrate that high proton leak manifests itself at the cellular level too: the leak rate (measured as myxothiazol-sensitive, oligomycin-resistant respiration) was nearly four times higher in INS-1E cells than in myoblasts. This relatively high leak activity was decreased more than 30% upon knock-down of UCP2 (uncoupling protein-2) by RNAi (RNA interference). The high contribution of UCP2 to leak suggests that proton conductance through UCP2 accounts for approx. 20% of INS-1E respiration. UCP2 knock-down enhanced GSIS (glucose-stimulated insulin secretion), consistent with a role for UCP2 in beta-cell physiology. We propose that the high mitochondrial proton leak in beta-cells is a mechanism which amplifies the effect of physiological UCP2 regulators on cytoplasmic ATP/ADP and hence on insulin secretion.