Uncoupling Protein Homologues Research Articles

A biophysical study on molecular physiology of the uncoupling proteins of the central nervous system.

Mitochondrial inner membrane uncoupling proteins (UCPs) facilitate transmembrane (TM) proton flux and consequently reduce the membrane potential and ATP production. It has been proposed that the three neuronal human UCPs (UCP2, UCP4 and UCP5) in the central nervous system (CNS) play significant roles in reducing cellular oxidative stress. However, the structure and ion transport mechanism of these proteins remain relatively unexplored. Recently, we reported a novel expression system for obtaining functionally folded UCP1in bacterial membranes and applied this system to obtain highly pure neuronal UCPs in high yields. In the present study, we report on the structure and function of the three neuronal UCP homologues. Reconstituted neuronal UCPs were dominantly helical in lipid membranes and transported protons in the presence of physiologically-relevant fatty acid (FA) activators. Under similar conditions, all neuronal UCPs also exhibited chloride transport activities that were partially inhibited by FAs. CD, fluorescence and MS measurements and semi-native gel electrophoresis collectively suggest that the reconstituted proteins self-associate in the lipid membranes. Based on SDS titration experiments and other evidence, a general molecular model for the monomeric, dimeric and tetrameric functional forms of UCPs in lipid membranes is proposed. In addition to their shared structural and ion transport features, neuronal UCPs differ in their conformations and proton transport activities (and possibly mechanism) in the presence of different FA activators. The differences in FA-activated UCP-mediated proton transport could serve as an essential factor in understanding and differentiating the physiological roles of UCP homologues in the CNS.

On the Role of Positively Charged Residues of TM2 Domain in the Chloride Transport of Human UCP2

Located in the inner mitochondrial membrane, uncoupling proteins (UCPs) dissipate the proton electrochemical gradient causing reduction in the rate of ATP synthesis. Among five human UCP homologues, UCP2 is unique with its ubiquitous expression in various tissues. This important feature has been attributed to UCP2's multiple physiological roles in tissues, including its involvement in protective mechanisms against oxidative stress, glucose and lipid metabolisms. Despite numerous physiological studies, UCP2 function in cell remains poorly understood. UCP2 proton transport is regulated by purine nucleotides such as ATP, ADP, GTP and GDP. In addition, UCP2 has also been observed to transport chlorides and other small anions. Identification of the key amino acid residues in UCP2 in proton, anion transport and regulation will help determine the protein's mechanism of action in cells. It has been established that positively charged residues on transmembrane helix 2 (TM2) of UCP1 and UCP2 are crucial for chloride transport. However, a full understanding of the transport mechanism is yet to be achieved. More importantly, some of these residues are also involved in the UCP2 proton transport regulation. To further understand the ion transport of UCP2, four TM2 mutants have been made (R76Q, R88Q, R96Q, and K104Q). Over-expressed proteins were purified and reconstituted into liposomes for structural and functional studies. All mutants share an overall helical conformation with wtUCP2. using anion-sensitive fluorescent probes, proton and chloride transport of UCP2 mutants are examined to determine the effect of each mutation on the ion transport of UCP2. In addition, Mant-modified purine nucleotide will be used to study the binding of UCP2 and its mutants to purine nucleotides. Overall, the outcome of this study will provide a more detailed molecular image of UCP2 ion transport mechanism.

Conformation and Ion Transport of Neuronal Uncoupling Proteins

Located in the inner mitochondrial membrane, uncoupling proteins (UCPs) dissipate the proton electrochemical gradient causing reduction in the rate of ATP synthesis. Three UCP homologues (UCP2, UCP4 and UCP5), expressed in neurons, are suggested to have potential roles in the function and protection of the central nervous system (CNS). Extensive biochemical studies on UCP2 have provided adequate evidence for its participation in proton and anion transport. So far, no functional studies in proteoliposome systems have been performed on UCP4 and UCP5. Due to the lack of high resolution structures, structural information for UCPs remains limited. The goals of this study are to gain further information on the conformations and functional properties of neuronal UCPs reconstituted in liposomes. The emphasis is on UCP4 and UCP5 and their comparison to UCP2. Recombinant versions of all five UCPs have been successfully expressed, purified and reconstituted in liposomes. All UCPs formed dominantly helical conformations in negatively charged phospholipid vesicles as detected by CD spectroscopy [Ivanova et al (2010) Biochemistry 49(3): 512-21]. Neuronal UCPs also share common structural and binding properties with UCPs 1 and 3, implying a common physiological role in addition to their specific roles in the CNS. Ion transport assays (proton and chloride) for reconstituted neuronal UCPs have been developed using anion-sensitive fluorescent probes. Purine nucleotide inhibitions of these proteins resulted in decrease in the initial transport rate. Preliminary results show that reconstituted UCP5 has proton transport activity that is inhibited by ATP. Similar experiments are planned for UCP4. Transport activities of the all neuronal UCPs will be compared to those of the prototypical UCP1. The outcome of this study will clarify several aspects of the structure-function relationships of the least studied UCPs 4 and 5 and their possible physiological roles in the CNS.

Mitochondrial Uncoupler Proteins

Mitochondrial uncoupling proteins (UCPs), members of a family of mitochondrial anion carrier proteins (MACP), are nuclear-encoded transmembrane transporter proteins located in the mitochondrial inner membrane. UCP1, mainly expressed in brown adipose tissue (BAT), was the first to be discovered and is responsible for animal thermogenesis; UCP2, originally thought to play a role in nonshivering thermogenesis, obesity and diabetes, its main function appears to be in the control of mitochondria-derived reactive oxygen species (ROS). Another uncoupling protein homologue, the UCP3 is mainly expressed in skeletal muscle and brown adipose tissue and its gene is transcribed from tissue-specific promoters in humans but not in rodents. All the members of this protein family possess a common feature of shunting protons across the mitochondrial inner membrane and reduce ATP synthesis; however, this common mechanism of action is used to carry out different functions by the different UCPs. The distribution and abundance of UCPs are tissue specific, which is also reflected into the processes that these proteins are thought to be participating. UCPs other than UCP1 are involved in several biological processes such as fatty acid (FA) metabolism, insulin secretion, oxidative stress (OS), heart pathophysiology and macrophage activation. New discoveries are advancing our understanding of UCPs roles in cardiovascular physiology.

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.

Tough love: left out in the cold, but not abandoned, by UCP3

In rodents, the challenge of maintaining core body temperature in a cold environment is partially met by increased activity of mitochondrial uncoupling protein (UCP) 1 in brown adipose tissue (BAT) ([1][1]). The energy that would normally be preserved by coupling ATP synthesis with the translocation

Uncoupling protein homologs may provide a link between mitochondria, metabolism and lifespan

Uncoupling proteins (UCPs), which dissipate the mitochondrial proton gradient, have the ability to decouple mitochodrial respiration from ATP production. Since mitochondrial electron transport is a major source of free radical production, it is possible that UCP activity might impact free radical production. Free radicals can react with and damage cellular proteins, DNA and lipids. Accumulated damage from oxidative stress is believed to be a major contributor to cellular decline during aging. If UCP function were to impact mitochondrial free radical production, then one would expect to find a link between UCP activity and aging. This theory has recently been tested in a handful of organisms whose genomes contain UCP1 homologs. Interestingly, these experiments indicate that UCP homologs can affect lifespan, although they do not support a simple relationship between UCP activity and aging. Instead, UCP-like proteins appear to have a variety of effects on lifespan, and on pathways implicated in lifespan regulation. One possible explanation for this complex picture is that UCP homologs may have tissue-specific effects that complicate their effects on aging. Furthermore, the functional analysis of UCP1 homologs is incomplete. Thus, these proteins may perform functions in addition to, or instead of, mitochondrial uncoupling. Although these studies have not revealed a clear picture of UCP effects on aging, they have contributed to the growing knowledge base for these interesting proteins. Future biochemical and genetic investigation of UCP-like proteins will do much to clarify their functions and to identify the regulatory networks in which they are involved.

Mitochondrial metabolism in mammalian cold-acclimation: Magnitude and mechanisms of fatty-acid uncoupling

Cold-acclimation did not alter uncoupling (state 4 respiration) in rat liver or skeletal muscle mitochondria. Palmitate significantly uncoupled mitochondria, but neither the magnitude of this uncoupling nor the contribution of different inner mitochondrial membrane transporters to uncoupling was altered by cold-acclimation. Guanosine diphosphate did not reduce uncoupling, suggesting no role for uncoupling protein homologues. The adenine nucleotide transporter and the permeability transition pore, either alone or in combination, appear to contribute significantly to free fatty-acid (FFA)-induced uncoupling. Evidence suggests that these two elements may be working together, as components of the same mechanism, to mediate FFA uncoupling.

Uncoupling Proteins 2 and 3 Function in Concert to Augment Tolerance to Cardiac Ischemia

Transient cardiac ischemia activates cell survival signaling, conferring subsequent ischemia tolerance to the heart. This biological phenomenon, termed ischemic preconditioning, results in improved clinical outcome and attenuated infarct size following myocardial infarction. To explore genomic modifications underpinning this ischemia tolerance, we delineated the regulation and function of the cardiac enriched mitochondrial uncoupling proteins 2 and 3 during delayed ischemic preconditioning in the rat. Cardiac transcripts of genes encoding uncoupling proteins 2 and 3 are up-regulated in parallel with infarct size reduction in preconditioned hearts. Mitochondria isolated from preconditioned hearts exhibit an augmented inducible proton leak. In parallel, following anoxia-reoxygenation these mitochondria generate less hydrogen peroxide compared with non-preconditioned mitochondria. Preconditioning in rat cardiac derived myoblasts is abolished following uncoupling protein-2 depletion by RNA-interference. RNAi of uncoupling protein-3 partially attenuates the capacity to precondition these cells. Functional characterization of anoxia and reoxygenation tolerance following uncoupling protein 2 or 3 and combined 2 and 3 RNAi shows the largest reduction in viability follows depletion of both homologues. Uncoupling protein-2 depletion alone significantly attenuates anoxia-reoxygenation tolerance but uncoupling protein-3 depletion does not reduce anoxia tolerance. In parallel combined uncoupling protein depletion and isolated uncoupling protein-2 depletion augments ROS production in viable cardiomyocytes following anoxia-reoxygenation. Concurrent anti-oxidant administration ameliorates the uncoupling protein-depleted anoxia-susceptible phenotype. In conclusion, mitochondrial uncoupling proteins are necessary components of ischemia tolerance and function as components of the cellular antioxidant defense program. In the cytoprotective hierarchy, uncoupling protein-2 appears to play a greater role than uncoupling protein-3 in modulating ischemia/anoxia tolerance in heart-derived cells.

The mitochondrial uncoupling-protein homologues

Uncoupling protein(UCP)1 is an integral membrane protein that is located in the mitochondrial inner membrane of brown adipocytes. Its physiological role is to mediate a regulated, thermogenic proton leak. UCP2 and UCP3 are recently identified UCP1 homologues. They also mediate regulated proton leak, and might function to control the production of superoxide and other downstream reactive oxygen species. However, their role in normal physiology remains unknown. Recent studies have shown that UCP2 has an important part in the pathogenesis of type-2 diabetes. The obscure roles of the UCP homologues in normal physiology, together with their emerging role in pathophysiology, provide exciting potential for further investigation.

Long–term dietary high protein intake up–regulates tissue specific gene expression of uncoupling proteins 1 and 2 in rats

The consequences of chronic high protein (HP) diets are discussed controversially and are not well understood. Rats adapted to HP exposure show an increased amino acid and fat oxidation and lower feed energy efficiency. We hypothesized that the dietary protein level can affect gene expression of uncoupling protein (UCP) homologues which is suggested to be involved in thermogenesis, substrate oxidation, and energy expenditure. To assess the mRNA expression of UCP homologues in various tissues of rats fed HP diets and to relate UCP gene expression to various parameters of substrate and energy metabolism. To obtain further indications for the possible involvement of UCP in reducing feed energy efficiency under conditions of HP exposure. Adult rats were adapted to casein based diets containing either 13.8% (adequate, AP), 25.7% (medium, MP), or 51.3 % (high, HP) crude protein. Rats were fed for 8 wk and killed in the postabsorptive state. Energy expenditure and mRNA expression were measured using indirect calorimetry and Northern blot analysis, respectively. Pearson correlation coefficients were calculated to determine relationships between UCP mRNA expression and metabolic parameters. Hepatic UCP2 mRNA expression was increased by MP and HP diets compared to AP diet. In skeletal muscle UCP2 mRNA expression was lowest under MP conditions. UCP1 mRNA expression in brown adipose tissue (BAT) was significantly increased by HP exposure. The values were inversely associated with feed energy efficiency and positively with energy expenditure and oxygen consumption in the dark period. Skeletal muscle UCP2 and -3 mRNA expression strongly correlated with the plasma free fatty acid concentration, whereas BAT UCP1 and hepatic UCP2 gene expression did not. Our results indicate that hepatic UCP2 and BAT UCP1 mRNA expression is related to the level of dietary protein intake. This suggests a role of UCPs in substrate oxidation and in thermogenesis under conditions of HP exposure.

Evolution of mitochondrial uncoupling proteins: novel invertebrate UCP homologues suggest early evolutionary divergence of the UCP family

Current hypothesis about the evolution of uncoupling proteins (UCPs) proposed by Hának and Jezek (2001) [FEBS Lett. 495, 137–141] suggests that UCP4 is the earliest form of UCP ancestral to all other UCP orthologues. However, this hypothesis is difficult to reconcile with a narrow tissue distribution of UCP4 (which is a brain-specific isoform), suggesting highly specialized rather than anfcestral function for this protein. We searched for UCP2, UCP3, and UCP5 homologues in invertebrate genomes using amplification with degenerate primers designed against UCP2-specific conserved sequences and/or BLASTP search with stringent ad hoc criteria to distinguish between homologues and orthologues of different UCPs. Our study identified invertebrate UCP homologues similar to UCP2 and 3 (which we termed UCP6) and an invertebrate homologue of UCP5. Phylogenetic analysis indicates that there are at least three clades of UCPs in invertebrates, which are closely related to vertebrate UCP1-3, UCP4, and UCP5, respectively, and shows early evolutionary divergence of UCPs, which pre-dates the divergence of protostomes and deuterostomes. It also suggests that the newly identified UCP6 proteins from invertebrates are ancestral to the vertebrate UCP1, UCP2, and UCP3, and that divergence of these three vertebrate orthologues occurred late in evolution of the vertebrates. This study refutes the hypothesis of Hának and Jezek (2001) that UCP4 is an ancestral form for all UCPs, and shows early evolutionary diversification of this protein family, which corresponds to their proposed functional diversity in regulation of proton leak, antioxidant defense and apoptosis.

The plant uncoupling protein homologues: a new family of energy-dissipating proteins in plant mitochondria

Uncoupling proteins (UCPs) form a subfamily within the mitochondrial carrier protein family, which catalyze a free fatty acid-mediated proton recycling and can modulate the tightness of coupling between mitochondrial respiration and ATP synthesis. As in mammalian tissues, UCPs are rather ubiquitous in the plant kingdom and widespread in plant tissues in which they could have various physiological roles, such as heat production or protection against free oxygen radicals. The simultaneous occurrence in plant mitochondria of two putative energy-dissipating systems, namely UCP which dissipates the proton motive force, and alternative oxidase (AOX) which dissipates the redox potential, raises the question of their functional interactions.

Effects of dietary macronutrient content on energy metabolism and uncoupling protein mRNA expression in broiler chickens

The objective of the present study was to investigate the effects of dietary macronutrient ratio on energy metabolism and on skeletal muscle mRNA expression of avian uncoupling protein (UCP), thought to be implicated in thermogenesis in birds. Broiler chickens from 2 to 6 weeks of age received one of three isoenergetic diets containing different macronutrient ratios (low-lipid (LL) 30 v. 77 g lipid/kg; low-protein (LP) 125 v. 197 g crude protein (Nx6.25)/kg; low-carbohydrate (LC) 440 v. 520 g carbohydrate/kg). LP chickens were characterised by significantly lower body weights and food intakes compared with LL and LC chickens (-47 and -38 % respectively) but similar heat production/kg metabolic body weight, as measured by indirect calorimetry, in the three groups. However, heat production/g food ingested was higher in animals receiving the LP diet (+41 %, P<0.05). These chickens also deposited 57 % less energy as protein (P<0.05) and 33 % more as fat. No significant differences in energy and N balances were detected between LL and LC chickens. The diets with the higher fat contents (i.e. the LP and LC diets) induced slightly but significantly higher relative expressions of avian UCP mRNA in gastrocnemius muscle, measured by reverse transcription-polymerase chain reaction, than the LL diet (88 and 90 v. 78 % glyceraldehyde-3-phosphate dehydrogenase respectively, P<0.05). Our present results are consistent with the recent view that UCP homologues could be involved in the regulation of lipid utilisation as fuel substrate and provide evidence that the macronutrient content of the diet regulates energy metabolism and especially protein and fat deposition.

A significant portion of mitochondrial proton leak in intact thymocytes depends on expression of UCP2.

The uncoupling protein homologue UCP2 is expressed in a variety of mammalian cells. It is thought to be an uncoupler of oxidative phosphorylation. Uncoupling proteins previously have been shown to be capable of translocating protons across phospholipid bilayers in proteoliposome systems. Furthermore, studies in mitochondria from yeast overexpressing the proteins have led to suggestions that they may act as uncouplers in cells. However, this issue is controversial, and to date, definitive experimental evidence is lacking as to whether UCP2 mediates part or all of the basal mitochondrial proton leak in mammalian cells in situ. In the present study, by using thymocytes isolated from UCP2-deficient and wild-type (WT) mice, we addressed the question whether UCP2 is directly involved in catalyzing proton leak in intact cells. Over a range of mitochondrial membrane potentials (DeltaPsi(m)), proton leak activity was lower in thymocytes from UCP2-deficient mice compared with WT mice. At physiological levels of DeltaPsi(m), a significant portion (50%) of basal proton leak in resting cells depended on UCP2. Of note, proton leak in whole cells from WT mice, but not UCP2-deficient mice, responded to stimulation by 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-1-propenyl]benzoic acid (TTNPB), a known activator of UCP2 activity. Consistent with the observed changes in proton leak, DeltaPsi(m) and ATP levels were increased in untreated thymocytes from UCP2-deficient mice. Interestingly, resting respiration was unaltered, suggesting that UCP2 function in resting cells may be concerned with the control of ATP production rather than substrate oxidation. This study establishes that UCP2, expressed at endogenous levels, mediates proton leak in intact cells.

Perspectives on the biology of uncoupling protein (UCP) homologues.

In mammals, it is believed that a portion of tissue metabolic rate is driven by counteraction of uncoupling, in which the energetically inefficient process of proton leak acts to diminish the mitochondrial electrochemical membrane potential. It is proposed that specific proteins associated with the mitochondrion catalyse uncoupling, and the biology of such putative uncoupling proteins (UCPs) is the subject of active research efforts. UCP4 and UCP5 are interesting in light of their abundant expression in the brain, which may signal an important metabolic function in thermogenesis or regulation of reactive oxygen species in that tissue. While each is expressed to various degrees outside of the brain, their impact on whole-animal metabolism remains to be clarified further. Transgenic mice expressing murine UCP5(L), the long isoform of UCP5, using an inducible metallothionine promoter (to drive expression of the transgene in liver, testis, heart, lung, spleen, intestine, kidney and brain) did not display any overt metabolic phenotype, despite liver UCP5(L) mRNA expression equivalent to that of normal mouse brain. This highlights the need for further studies to examine the nature of UCP5 physiology. Evidence for uncoupling behaviour has recently emerged from studies of the human 2-oxoglutarate carrier (OGC), indicating that the possibility of physiological proton leak elicited by the OGC and other mitochondrial carriers warrants further experimental evaluation.

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.

Perspectives on the biology of uncoupling protein (UCP) homologues

In mammals, it is believed that a portion of tissue metabolic rate is driven by counteraction of uncoupling, in which the energetically inefficient process of proton leak acts to diminish the mitochondrial electrochemical membrane potential. It is proposed that specific proteins associated with the mitochondrion catalyse uncoupling, and the biology of such putative uncoupling proteins (UCPs) is the subject of active research efforts. UCP4 and UCP5 are interesting in light of their abundant expression in the brain, which may signal an important metabolic function in thermogenesis or regulation of reactive oxygen species in that tissue. While each is expressed to various degrees outside of the brain, their impact on whole-animal metabolism remains to be clarified further. Transgenic mice expressing murine UCP5L, the long isoform of UCP5, using an inducible metallothionine promoter (to drive expression of the transgene in liver, testis, heart, lung, spleen, intestine, kidney and brain) did not display any overt metabolic phenotype, despite liver UCP5L mRNA expression equivalent to that of normal mouse brain. This highlights the need for further studies to examine the nature of UCP5 physiology. Evidence for uncoupling behaviour has recently emerged from studies of the human 2-oxoglutarate carrier (OGC), indicating that the possibility of physiological proton leak elicited by the OGC and other mitochondrial carriers warrants further experimental evaluation.

Uncoupling proteins: functional characteristics and role in the pathogenesis of obesity and Type II diabetes

Uncoupling proteins are mitochondrial carrier proteins which are able to dissipate the proton gradient of the inner mitochondrial membrane. This uncoupling process reduces the amount of ATP generated through an oxidation of fuels. The hypothesis that uncoupling proteins (UCPs) are candidate genes for human obesity or Type II (non-insulin-dependent) diabetes mellitus is based on the finding that a chemical uncoupling of the mitochondrial membrane reduces body adiposity, and that lower metabolic rates predict weight gain. It is straightforward to hypothesize that common polymorphisms of UCP1, UCP2 and UCP3 genes lower metabolic rate by a more efficient energy coupling in the mitochondria. Furthermore, genetically engineered mice over expressing different UCP homologues are lean and resistant to diet-induced obesity. The three uncoupling protein homologue genes UCP1, UCP2, and UCP3 have been investigated for polymorphisms and mutations and their impact on Type II diabetes mellitus, obesity, and body weight gain or BMI. The main conclusion is that variation in the UCP1, UCP2 or UCP3 genes is not associated with major alterations of body weight gain. The contribution of UCP genes towards polygenic obesity and Type II diabetes is evaluated and discussed.

Cloning and functional characterization of an uncoupling protein homolog in hummingbirds

The cDNA of an uncoupling protein (UCP) homolog has been cloned from the swallow-tailed hummingbird, Eupetomena macroura. The hummingbird uncoupling protein (HmUCP) cDNA was amplified from pectoral muscle (flight muscle) using RT-PCR and primers for conserved domains of various known UCP homologs. The rapid amplification of cDNA ends (RACE) method was used to complete the cloning of the 5' and 3' ends of the open reading frame. The HmUCP coding region contains 915 nucleotides, and the deduced protein sequence consists of 304 amino acids, being approximately 72, 70, and 55% identical to human UCP3, UCP2, and UCP1, respectively. The uncoupling activity of this novel protein was characterized in yeast. In this expression system, the 12CA5-tagged HmUCP fusion protein was detected by Western blot in the enriched mitochondrial fraction. Similarly to rat UCP1, HmUCP decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3',3-dihexyloxacarbocyanine iodide. The HmUCP mRNA is primarily expressed in skeletal muscle, but high levels can also be detected in heart and liver, as assessed by Northern blot analysis. Lowering the room's temperature to 12-14 degrees C triggered the cycle torpor/rewarming, typical of hummingbirds. Both in the pectoral muscle and heart, HmUCP mRNA levels were 1.5- to 3.4-fold higher during torpor. In conclusion, this is the first report of an UCP homolog in birds. The data indicate that HmUCP has the potential to function as an UCP and could play a thermogenic role during rewarming.