Insulin and contraction directly stimulate UCP2 and UCP3 mRNA expression in rat skeletal muscle in vitro

To study the regulation of the mitochondrial uncoupling protein 2 and 3 (UCP2 and UCP3), we studied the effect of insulin and muscle contraction on UCP mRNA expression in rat skeletal muscle in vitro. Insulin dose-dependently increased skeletal muscle UCP2 and UCP3 mRNA expression in m. extensor digitorum longus (EDL) with maximal stimulation obtained at around 0.6-6 nM. The concentration of insulin giving half-maximal stimulation was 60 pM for the UCP2 and 48 pM for the UCP3 mRNA expression. The effect of insulin was maximal after 2 h and the effect was sustained during the whole study period (6 h). The insulin-induced increase in UCP mRNA was independent of the glucose uptake (as UCP mRNA was stimulated even in incubations without glucose). In addition, electrically induced contractions (in vitro) increased UCP2 and UCP3 mRNA expression 60-120 min after a single bout of contraction (for 10 min). Both the increment of UCP2 and UCP3 mRNA were sustained throughout the study period (4 h) (153 +/- 62 and 216 +/- 71% above basal, P < 0.05 respectively). Finally, 5-aminoimidazole-4-carboxamid-ribosid (AICAR), an activator of the AMP-activated protein kinase (AMPK), that is activated during exercise, was able to mimic the increase in UCP2 and UCP3 mRNA expression. In conclusion, UCP2 and UCP3 mRNA expression in skeletal muscle are stimulated rapidly by insulin and contraction in vitro, thus the stimulation is direct and not caused by changes in other hormones or metabolites. Even a brief bout of contraction induces an increase in UCP2 and UCP3 expression, an effect that could be mimicked by activation of the AMP-activated protein kinase by AICAR.     

Respiratory uncoupling by UCP1 and UCP2 and superoxide generation in endothelial cell mitochondria

Mitochondria represent a major source of reactive oxygen species (ROS), particularly during resting or state 4 respiration wherein ATP is not generated. One proposed role for respiratory mitochondrial uncoupling proteins (UCPs) is to decrease mitochondrial membrane potential and thereby protect cells from damage due to ROS. This work was designed to examine superoxide production during state 4 (no ATP production) and state 3 (active ATP synthesis) respiration and to determine whether uncoupling reduced the specific production of this radical species, whether this occurred in endothelial mitochondria per se, and whether this could be modulated by UCPs. Superoxide formation by isolated bovine aortic endothelial cell (BAE) mitochondria, determined using electron paramagnetic resonance spectroscopy, was approximately fourfold greater during state 4 compared with state 3 respiration. UCP1 and UCP2 overexpression both increased the proton conductance of endothelial cell mitochondria, as rigorously determined by the kinetic relationship of respiration to inner membrane potential. However, despite uncoupling, neither UCP1 nor UCP2 altered superoxide formation. Antimycin, known to increase mitochondrial superoxide, was studied as a positive control and markedly enhanced the superoxide spin adduct in our mitochondrial preparations, whereas the signal was markedly impaired by the powerful chemical uncoupler p-(trifluoromethoxyl)-phenyl-hydrazone. In summary, we show that UCPs do have uncoupling properties when expressed in BAE mitochondria but that uncoupling by UCP1 or UCP2 does not prevent acute substrate-driven endothelial cell superoxide as effluxed from mitochondria respiring in vitro.     

鸡解偶联蛋白(UCP)基因内含子的克隆与系统发生树的构建

解偶联蛋白基因是新近发现的能够增加能量的消耗,与脂肪代谢和能量调控密切相关的一组基因。本研究根据小鼠UCP2基因的剪切方式,设计4对引物成功克隆测序了鸡UCP基因的全部5个内含子,发现都是GT-AG类型的内含子,鸡UCP基因的结构和小鼠的UCP2基因结构一致。以不同物种UCP基因的cds 区域序列和内含子2、内含子3序列进行系统发生树的构建,结果表明：以UCP基因cds区域序列构建的系统发生树与物种树是一致的,UCP基因可以作为研究动物群体系统演化研究的有效基因;但以内含子2与内含子3序列构建的系统发生树的结构则完全不是这样,与物种树的差别比较大。 Abstract:The UCP genes were the newly discovered genes that can increase the energy expenditure and involve in the metabolism of fat and regulation of energy.Four pairs of primers in chicken UCP exon region were designed to amplify the introns of chicken UCP gene according to the splice ways of the mouse UCP2 gene （Accession No.AF096288）.The sequence results showed that the chicken UCP gene also had five GT-AG type introns.The molecular phylogenetic tree was constructed based on the sequence of cds,intron 2 and intron 3 region,respectively.The phylogenetic tree based on the UCP cds region was consistent with the species phylogenetic tree.This result implicated that UCP gene can be regarded as the useful gene for the study of animal phylogenesis.On the contrast,the phylogenetic tree based on the intron 2 and intron 3 region was different from the species phylogenetic tree,which showed that the evolution of intron and cds region is different.     

Molecular identification of uncoupling proteins (UCP2 and UCP3) and absence of UCP1 in the marsupial Tasmanian bettong,Bettongia gaimardi

This study has identified the expression of uncoupling proteins in a marsupial using molecular techniques. The Tasmanian bettong, Bettongia gaimardi, increases non-shivering thermogenesis (NST) in response to cold exposure and norepinephrine, although previous studies have been unable to demonstrate the presence of brown adipose tissue or uncoupling protein 1 (UCP1). This study used molecular techniques to confirm the absence of UCP1 as well as ascertain if this species expresses UCP2 and/or UCP3. Tissue samples from four B. gaimardi were taken prior to and post-cold exposure at 4-5 degrees C for 2 weeks. The tissues were then examined for UCP1, UCP2 and UCP3 expression using Western blotting. UCP2 and UCP3 were amplified through RT-PCR and subsequently sequenced to confirm molecular identity. Our work confirms that B. gaimardi does not express UCP1 and that this species expresses both uncoupling proteins 2 and 3. The sequencing of the amplified B. gaimardi UCP2 and UCP3 cDNAs have revealed a 74% homology with rat UCP2 cDNA, and 65% homology with rat UCP3 cDNA. Although this work has not yet characterised the functional properties of these proteins in the marsupial, it does suggest a possible mechanism to explain the existence of NST in B. gaimardi.     

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.     

Toward understanding the mechanism of ion transport activity of neuronal uncoupling proteins UCP2, UCP4, and UCP5

Neuronal uncoupling proteins (UCP2, UCP4, and UCP5) have crucial roles in the function and protection of the central nervous system (CNS). Extensive biochemical studies of UCP2 have provided ample evidence of its participation in proton and anion transport. To date, functional studies of UCP4 and UCP5 are scarce. In this study, we show for the first time that, despite a low level of amino acid sequence identity with the previously characterized UCPs (UCP1-UCP3), UCP4 and UCP5 share their functional properties. Recombinantly expressed in Escherichia coli, UCP2, UCP4, and UCP5 were isolated and reconstituted into liposome systems, where their conformations and ion (proton and chloride) transport properties were examined. All three neuronal UCPs are able to transport protons across lipid membranes with characteristics similar to those of the archetypal protein UCP1, which is activated by fatty acids and inhibited by purine nucleotides. Neuronal UCPs also exhibit transmembrane chloride transport activity. Circular dichroism spectroscopy shows that these three transporters exist in different conformations. In addition, their structures and functions are differentially modulated by the mitochondrial lipid cardiolipin. In total, this study supports the existence of general conformational and ion transport features in neuronal UCPs. On the other hand, it also emphasizes the subtle structural and functional differences between UCPs that could distinguish their physiological roles. Differentiation between structure-function relationships of neuronal UCPs is essential for understanding their physiological functions in the CNS.     

Reconstitution of novel mitochondrial uncoupling proteins UCP2 and UCP3

Reconstitution of novel mitochondrial uncoupling proteins, human UCP2 and UCP3, expressed in yeast, was performed to characterize fatty acid (FA)-induced H⁺ efflux in the resulted proteoliposomes. We now demonstrate for the first time that representatives of physiologically abundant long chain FAs, saturated or unsaturated, activate H⁺ translocation in UCP2- and UCP3-proteoliposomes. Efficiency of lauric, palmitic or linoleic acid was roughly the same, but oleic acid induced faster H⁺ uniport. We have confirmed that ATP and GTP inhibit such FA-induced H⁺ uniport mediated by UCP2 and UCP3. Coenzyme Q₁₀ did not further significantly activate the observed H⁺ efflux. In conclusion, careful instant reconstitution yields intact functional recombinant proteins, UCP2 and UCP3, the activity of which is comparable with UCP1.     

Up-regulation of uterine UCP2 and UCP3 in pregnant rats.

Pregnancy produces profound changes in hormone dynamics, thermoregulation and energy metabolism. Uncoupling proteins (UCPs) have been identified in a variety of tissues and UCP1 is known to play important roles in energy homeostasis, while the regulation of UCP2 and UCP3 is still unclear. The present study aimed to investigate the effects of the changes during pregnancy on UCP gene expression in the uterus, as well as in brown adipose tissue (BAT), white adipose tissue (WAT), soleus muscle (Muscle), and liver, throughout the estrus and metestrus periods, at early, middle and late stages in pregnancy, and during post-gestational stages. The expression of uterine UCP2 and UCP3 were up-regulated by 3.2- and 1. 5-fold, respectively, during the late stage of pregnancy with an increase of WAT leptin mRNA expression and exogenous administration of leptin resulted in induction of the uterine UCP2 and UCP3 levels. Contrary to uterine UCPs, UCP1 mRNA expression in BAT was down-regulated by 0.5-fold and there were no remarkable changes in WAT or liver UCP2, or Muscle UCP3 expression throughout the periods. These results indicate that UCP gene expressions during pregnancy are regulated tissue-dependently, and up-regulation of uterine UCP2 and UCP3 mRNA may be due to increased leptin levels.     

Seasonal effects of the UCP3 and the RPTOR gene polymorphisms on obesity traits in Japanese adults

Background

Non-shivering thermogenesis (NST) involves a substantial amount of energy expenditure in humans and, thus, contributes to reducing the risk for obesity. Molecular evolutionary studies have reported that SNPs in/near the uncoupling protein 3 gene (UCP3) and the regulatory associated protein of mTOR complex 1 gene (RPTOR) might influence NST and confer adaptive advantages for modern human dispersal into cold environments. In the present study, the impact of these SNPs on obesity-related traits was investigated.

Methods

Study subjects consisted of 2,834 Japanese adults (percentage of female: 46%, mean age: 51.5). Associations of the UCP3-55C/T and the RPTOR-26934C/T - the 2 potential genetic variations involved in cold adaptation and thermogenic mechanisms in mammals, with quantitative obesity-related traits including body mass index (BMI), waist circumference, visceral fat area (VFA), VFA adjusted for BMI, and selected blood parameters - were tested using multiple linear regression models. Sliding windowsampling analysis was applied to depict seasonal effects of the SNPs on the obesity-related phenotypes.

Results

UCP3-55C/T and the RPTOR-26934C/T did not show any association with obesity traits and blood chemical parameters in multiple linear regression models consisting of the whole subjects. Moreover, sliding window sampling-based association analyses involving seasonality also failed to find associations between these two SNPs and obesity-related traits.

Conclusions

UCP3-55C/T and the RPTOR-26934C/T may only have subtle effects on the development of obesity-related traits in the present humans. These two SNPs might be irrelevant to inter-individual variations in energy metabolism and efficiency of NST.     

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 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 Hanak 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.     

Structure-function relationships in UCP1, UCP2 and chimeras: EPR analysis and retinoic acid activation of UCP2.

Uncoupling proteins (UCPs) are composed of three repeated domains of approximately 100 amino acids each. We have used chimeras of UCP1 and UCP2, and electron paramagnetic resonance (EPR), to investigate domain specific properties of these UCPs. Questions include: are the effects of nucleotide binding on proton transport solely mediated by amino acids in the third C-terminal domain, and are the amino acids in the first two domains involved in retinoic or fatty acid activation? We first confirmed that our reconstitution system produced UCP1 that exhibited known properties, such as activation by fatty acids and inhibition of proton transport by purine nucleotides. Our results confirm the observations reported for recombinant yeast that retinoic acid, but not fatty acids known to activate UCP1, activates proton transport by UCP2 and that this activation is insensitive to nucleotide inhibition. We constructed chimeras in which the last domains of UCP1 or UCP2 were switched and tested for activation by fatty acids or retinoic acid and inhibition by nucleotides. U1U2 is composed of mUCP1 (amino acids 1-198) and hUCP2 (amino acids 211-309). Fatty acids activated proton transport of U1U2 and GTP mediated inhibition. In the other chimeric construct U2U1, hUCP2 (amino acids 1-210) and mUCP1 (amino acids 199-307), retinoic acid still acted as an activator, but no inhibition was observed with GTP. Using EPR, a method well suited to the analysis of the structure of membrane proteins such as UCPs, we confirmed that UCP2 binds nucleotides. The EPR data show large structural changes in UCP1 and UCP2 on exposure to ATP, implying that a putative nucleotide-binding site is present on UCP2. EPR analysis also demonstrated changes in conformation of UCP1/UCP2 chimeras following exposure to purine nucleotides. These data demonstrate that a nucleotide-binding site is present in the C-terminal domain of UCP2. This domain was able to inhibit proton transport only when fused to the N-terminal part of UCP1 (chimera U1U2). Thus, residues involved in nucleotide inhibition of proton transport are located in the two first carrier motifs of UCP1. While these results are consistent with previously reported effects of the C-terminal domain on nucleotide binding, they also demonstrate that interactions with the N-terminal domains are necessary to inhibit proton transport. Finally, the results suggest that proteins such as UCP2 may transport protons even though they are not responsible for basal or cold-induced thermogenesis.     

The uncoupling protein 1 gene, UCP1, is expressed in mammalian islet cells and associated with acute insulin response to glucose in African American families from the IRAS Family Study

Background

Variants of uncoupling protein genes UCP1 and UCP2 have been associated with a range of traits. We wished to evaluate contributions of known UCP1 and UCP2 variants to metabolic traits in the Insulin Resistance and Atherosclerosis (IRAS) Family Study.

Methods

We genotyped five promoter or coding single nucleotide polymorphisms (SNPs) in 239 African American (AA) participants and 583 Hispanic participants from San Antonio (SA) and San Luis Valley. Generalized estimating equations using a sandwich estimator of the variance and exchangeable correlation to account for familial correlation were computed for the test of genotypic association, and dominant, additive and recessive models. Tests were adjusted for age, gender and BMI (glucose homeostasis and lipid traits), or age and gender (obesity traits), and empirical P-values estimated using a gene dropping approach.

Results

UCP1 A-3826G was associated with AIR_g in AA (P = 0.006) and approached significance in Hispanic families (P = 0.054); and with HDL-C levels in SA families (P = 0.0004). Although UCP1 expression is reported to be restricted to adipose tissue, RT-PCR indicated that UCP1 is expressed in human pancreas and MIN-6 cells, and immunohistochemistry demonstrated co-localization of UCP1 protein with insulin in human islets. UCP2 A55V was associated with waist circumference (P = 0.045) in AA, and BMI in SA (P = 0.018); and UCP2 G-866A with waist-to-hip ratio in AA (P = 0.016).

Conclusion

This study suggests a functional variant of UCP1 contributes to the variance of AIR_g in an AA population; the plausibility of this unexpected association is supported by the novel finding that UCP1 is expressed in islets.     

Effects of Obesity and Stable Weight Reduction on UCP2 and UCP3 Gene Expression in Humans

VIDAL-PUIG, ANTONIO, MICHAEL ROSENBAUM, ROBERT C. CONSIDINE, RUDOLPH L. LEIBEL, G. LYNIS DOHM, AND BRADFORD B. LOWELL. Effects of obesity and stable weight reduction on UCP2 and UCP3 gene expression in humans. Obes Res. Objectives: The molecular determinants of energy expenditure are presently unknown. Recently, two uncoupling protein homologues, UCP2 and UCP3, have been identified. UCP2 is expressed widely, and UCP3 is expressed abundantly in skeletal muscle. Both could be important regulators of energy balance. In this paper, we investigated whether altered UCP2 and UCP3 mRNA levels are associated with obesity or weight reduction. Research Methods and Procedures: UCP2, UCP3 long and short mRNA levels were examined in skeletal muscle and in white adipose tissue of lean, obese, and weight-reduced individuals by RNase protection assay. Results: Expression of UCP2, UCP3S, and UCP3L mRNA in skeletal muscle was similar in lean individuals and in individuals with obesity at stable weight. In contrast, UCP3L and UCP3S mRNAs were decreased by 38% (p < 0.0059) and 48% (p<0.0047), respectively, in 20% weight-reduced patients with obesity at stable weight. In contrast, UCP2 mRNA levels were increased by 30% in skeletal muscle of 20% weight-reduced subjects with obesity. In a different set of patients, mostly lean, UCP3L mRNA in skeletal muscle was decreased by 28% (p = 0.0425) after 10% weight reduction at stable weight. Expression of UCP2 mRNA in subcutaneous adipose tissue was similar in lean individuals and in individuals with obesity, and was increased by 58% during active weight loss. Discussion: Stabilization at reduced body weight in humans is associated with a decrease in UCP3 mRNA in muscle. It is possible that reduced UCP3 expression could contribute to decreased energy expenditure in weight-stable, weight-reduced individuals.     

Physiological regulation of the transport activity in the uncoupling proteins UCP1 and UCP2

Brown fat is a thermogenic organ that allows newborns and small mammals to maintain a stable body temperature when exposed to cold. The heat generation capacity is based on the uncoupling of respiration from ATP synthesis mediated by the uncoupling protein UCP1. The first studies on the properties of these mitochondria revealed that fatty acid removal was an absolute prerequisite for respiratory control. Thus fatty acids, that are substrate for oxidation, were proposed as regulators of respiration. However, their ability to uncouple all types of mitochondria and the demonstration that several mitochondrial carriers catalyze the translocation of the fatty acid anion have made them unlikely candidates for a specific role in brown fat. Nevertheless, data strongly argue for a physiological function. First, fatty acids mimic the noradrenaline effects on adipocytes. Second, there exists a precise correlation between fatty acid sensitivity and the levels of UCP1. Finally, fatty acids increase the conductance by facilitating proton translocation, a mechanism that is distinct from the fatty acid uncoupling mediated by other mitochondrial carriers. The regulation of UCP1 and UCP2 by retinoids and the lack of effects of fatty acids on UCP2 or UCP3 are starting to set differences among the new uncoupling proteins.     

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.     

Uncoupling proteins UCP2 and UCP3 from mitochondria of liver and skeletal muscle of ground squirrel Spermophilus undulatus do not transport pyruvate in contrast to UCP1 from brown adipose tissue

Physiological role of mitochondrial uncoupling proteins UCP2 and UCP3, homologous to UCP1 from brown adipose tissue, is unclear. It was proposed recently that UCP2 and UCP3 are metabolic triggers that switch oxidation of glucose to oxidation of fatty acids, exporting pyruvate from mitochondria. In the present study we tried to verify this hypothesis using ground squirrels (Spermophilus undulatus), since expression of all UCPs in different tissues increases during winter season, and UCP1 is abundant in brown fat. We confirmed the possibility of nonspecific transport of pyruvate through UCP1 in brown fat mitochondria and tried to identify similar transport in liver and skeletal muscle mitochondria where UCP2 and UCP3 are expressed. Transport of pyruvate mediated by UCP1 in mitochondria of brown fat was observed using valinomycin-induced swelling of non-respiring mitochondria in 55 mM potassium pyruvate and was inhibited by GDP. In contrast, mitochondria of liver and skeletal muscles in similar conditions did not exhibit electrogenic transport of pyruvate anions that could be related to functioning of UCP2 and UCP3. At the same time, functioning of pyruvate carrier was detected in these mitochondria by nigericin-induced passive swelling or valinomycin-induced active swelling in potassium pyruvate that was inhibited by α-CHC, a specific inhibitor of the pyruvate carrier. Thus, our results suggest that in contrast to UCP1 of brown fat, UCP2 and UCP3 from intact liver and skeletal muscle mitochondria of winter active ground squirrels are unable to carry out pyruvate transport.     

Differential regulation of the promoter activity of the mouse UCP2 and UCP3 genes by MyoD and myogenin

UCP2 and UCP3 are members of the uncoupling protein family, which may play roles in energy homeostasis. In order to determine the regulation of the predominant expression of UCP3 in skeletal muscle, the effects of differentiation and myogenic regulatory factors on the promoter activities of the mouse UCP2 and UCP3 genes were studied. Reporter plasmids, containing approximately 3 kb of the 5'-upstream region of the mouse UCP2 and UCP3 genes, were transfected into C2C12 myoblasts, which were then induced to differentiate. Differentiation positively induced the reporter expression about 20-fold via the UCP3 promoter, but by only 2-fold via the UCP2 promoter. C2C12 myoblasts were cotransfected with expression vectors for myogenin and/or MyoD as well as reporter constructs. The simultaneous expression of myogenin and MyoD caused an additional 20-fold increase in the reporter expression via the UCP3 promoter, but only a weak effect via the UCP2 promoter. In L6 myoblasts, only MyoD activated the UCP3 promoter, but in 3T3-L1 cells neither factor activated the UCP3 promoter, indicating that additional cofactors are required, which are present only in C2C12 myoblasts. The expression of UCP2 and UCP3 is differentially regulated during muscle differentiation due to the different responsiveness of their promoter regions to myogenin and MyoD.     

Inhibition of mitochondrial UCP1 and UCP3 by purine nucleotides and phosphate

Mitochondrial membrane uncoupling protein 3 (UCP3) is not only expressed in skeletal muscle and heart, but also in brown adipose tissue (BAT) alongside UCP1, which facilitates a proton leak to support non-shivering thermogenesis. In contrast to UCP1, the transport function and molecular mechanism of UCP3 regulation are poorly investigated, although it is generally agreed upon that UCP3, analogous to UCP1, transports protons, is activated by free fatty acids (FFAs) and is inhibited by purine nucleotides (PNs). Because the presence of two similar uncoupling proteins in BAT is surprising, we hypothesized that UCP1 and UCP3 are differently regulated, which may lead to differences in their functions. By combining atomic force microscopy and electrophysiological measurements of recombinant proteins reconstituted in planar bilayer membranes, we compared the level of protein activity with the bond lifetimes between UCPs and PNs. Our data revealed that, in contrast to UCP1, UCP3 can be fully inhibited by all PNs and IC50 increases with a decrease in PN-phosphorylation. Experiments with mutant proteins demonstrated that the conserved arginines in the PN-binding pocket are involved in the inhibition of UCP1 and UCP3 to different extents. Fatty acids compete with all PNs bound to UCP1, but only with ATP bound to UCP3. We identified phosphate as a novel inhibitor of UCP3 and UCP1, which acts independently of PNs. The differences in molecular mechanisms of the inhibition between the highly homologous transporters UCP1 and UCP3 indicate that UCP3 has adapted to fulfill a different role and possibly another transport function in BAT.