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Home , Mitochondrial carrier, Thermogenesis, UCP2, Uncoupling protein

International Journal of Obesity (1999) 23, Suppl 6, S38±S42 ß 1999 Stockton Press All rights reserved 0307±0565/99 $12.00 http://www.stockton-press.co.uk/ijo Uncoupling -2 (UCP2): Molecular and genetic studies

D Ricquier1*

1CEREMOD, CRNS, Meudon, France.

Thermogenesis is associated to oxygen consumption and cellular respiration. This process is coupled to adenosine- diphosphate (ADP) phosphorylation through the existence of a proton gradient across the inner mitochondrial membrane. It was postulated that proton leaks through this membrane would uncouple respiration from adeno- sine-triphosphate (ATP) synthesis and induce energy dissipation as heat. Such a mechanism was identi®ed in thermogenic brown mitochondria which contain a unique proton carrier referred to as (UCP). This UCP is activated by fatty acids and its synthesis is positively controlled by retinoids, hormones, catecholamines and rexinoids. In fact, in most types of cells, respiring mitochondria release heat and the coupling of substrate oxidation to ADP phosphorylation is under 100%. It suggested that the partial coupling of respiration to ADP phosphorylation was due to proton leaks possibly related to the brown fat UCP. This approach led to the identi®cation of UCP2 and UCP3, two homologues of the brown fat UCP (renamed UCP1). UCP2 is widely expressed in tissues and cell types, whereas the UCP3 gene is dominantly expressed in skeletal muscles (and brown fat in mice). Recent genetic, biochemical and physiological studies suggest that these novel UCP2 contribute to resting metabolic rate, fat oxidation and may represent new targets for anti-obesity compounds.

Keywords: ; ; fatty acids; thyroid hormone; proton leaks;

Introduction: contribution of exergonic processes release heat. Apart from the `futile' cycles, uncoupling of mitochondrial respiration mitochondrial proton leaks to from adenosine-diphosphate (ADP) phosphorylation is a highly thermogenic mechanism observed in BAT.

Most mammals maintain their body temperature around 37C even in a cold environment. This is The mitochondrial uncoupling achieved by the ability of mammals to decrease heat loss and activate thermogenic mechanisms. Obliga- protein (UCP1) of brown adipose tory thermogenesis corresponds to resting energy tissue: the ®rst example of expenditure, in the absence of food intake, and at thermogenic mitochondrial proton thermoneutrality. Regulatory or adaptive thermogen- esis is the extra-heat induced by exposure to the cold leak or food intake. Apart obligatory or adaptive thermo- genesis, and are two major situations The brown adipocytes are characterized by the pre- generating heat. sence of UCP1. This protein is located in the inner In large mammals, skeletal muscle is the dominant mitochondrial membrane where it uncouples respira- thermogenic organ although other organs such as liver tion from phosphorylation and leads to the dissipation have a high heat production. In rodents, the major site of energy as heat.1±4 In large mammals, BAT is easily of thermogenesis induced by the cold (and mediated observed only in neonates,1,3 and is poorly developed by noradrenaline) is the brown adipose tissue (BAT). in adults, excepted in adult human patients with It is known that thermogenesis is proportional to phaechromocytoma.2 However, studies on Finnish oxygen consumption and is directly related to the outdoor workers, and recent studies made on adult level of mitochondrial oxidation of substrates in patients with various pathologies or alcohol consu- tissues. A number of biochemical reactions involving mers, suggested that a low, but signi®cant amount of adenosine-triphosphate (ATP) hydrolysis and ion BAT can exist in human adults.2 In situations not transport and partial coupling of endergonic and requiring extra-heat production, the UCP1 is inactive and is inhibited by purine . When a rodent is exposed to the cold, the sympathetic ®bres innervat- ing the brown adipocytes are activated. Noradrenaline *Correspondence: Dr Daniel Ricquier, CEREMOD, CRNS, 9 rue Jules Hetzel, 92190 Meudon, France. is released at the surface of brown adipocytes and bind Email: [email protected] adrenergic receptors provoking in brown Molecular and genetic aspects of UCP2 D Ricquier S39 adipocytes and increased of free fatty acids which, in lymphocytes.8 In rat liver, only KuÈpffer cells were turn, activate UCP1. UCP1 activation promotes proton shown to signi®cantly express UCP2 mRNA.10 The re-entry in the matrix, decrease in mitochondrial level of homology between UCP2 and UCP1 sug- membrane potential and increase in respiratory rate gested a possible uncoupling activity of UCP2. In in such a way that respiration is not coupled to ADP agreement with this hypothesis, functional studies of phosphorylation. In such conditions, most of oxidation UCP2 expressed in yeasts revealed that UCP2 lowered energy is dissipated as heat.1±4 When animals are the mitochondrial membrane potential, suggesting it exposed to the cold for several hours or days, the decreased the level of coupling of respiration.8,9 catecholamines also activate UCP1 gene transcrip- Human and mouse cDNAs corresponding to another tion.1±4 In addition to catecholamines, thyroid homolog of UCP1 were recently cloned.11 ± 13 This new hormones, retinoids and ligands of -pro- protein was referred to as UCP3. The predicted amino liferator-activated-receptors also activate UCP1 gene acid sequence of human UCP3 is 57% identical to transcription.2,5 human UCP1 and 73% identical to human UCP2. It was proposed that UCP3 was another mitochondrial protein able to modulate the respiratory control of The novel uncoupling protein UCP2 mitochondria. In comparison with UCP1 which is unique to brown adipocytes and UCP2 which is widely expressed in organs, UCP3 mRNA is domi- Actually, the coupling of respiration to ADP phos- nantly expressed in skeletal muscles of humans or phorylation in any type of mitochondria is not perfect. rodents, although it is also present in heart and brown It is known that only 50% of the energy released from adipose tissue, at least in rodents.11 ± 13 respiratory substrate oxidation is recovered as ATP and also that any respiring releases heat. To explain the partial coupling of respiration to ATP synthesis in mitochondria, bioenergeticists are Physiological situations, factors or debating between the importance of slippage of hormones controlling UCP2 respiratory chains, and the existence of proton leaks. expression Slippage of respiratory chain decreases the value of the proton and lowers the proton motive force available for ADP phosphoryla- In the original paper describing UCP2, it was reported tion. Proton leaks through the inner mitochondrial that a hyperlipidic diet increased UCP2 mRNA membrane decrease the membrane potential, which expression in adipose tissues of mice and in particular in turn stimulates respiration, and also decreased the in mice resistant to diet-induced obesity, and also that proton motive force and ADP phosphorylation. Mea- exposure to the cold of mice did not induce UCP2 surement of proton leaks in liver and muscle mito- mRNA.8,11 A similar effect of hyperlipidic diet in rats chondrial suggests a role for proton leak in the partial was also reported by others.15 These data suggested 6,7 coupling of O2 consumption to ATP synthesis. that UCP2 gene was rather involved in diet-induced Brand et al calculated that proton leak contributed thermogenesis than in cold-induced thermogenesis. to 27% of the resting O2 consumption of hepatocytes However, Boss et al measured an increased expres- and up to 52% of the resting O2 consumption of rat sion of UCP2 mRNA in muscles of rats exposed to the perfused muscle.7 The existence of proton leaks cold for 2 d.16 There is now a number of data linking suggested that mechanisms similar to the UCP1 UCP2 mRNA level to metabolism and fatty mechanism detected in BAT could exist in many acid ¯ux into skeletal muscles. Starvation, which tissues. It also suggested that related to increases the level of circulating free fatty acids, UCP1 might exist in most tissues. This proposal led markedly increases UCP2 mRNA level in mus- us to screen a skeletal muscle library using UCP1 cles.13,17,18 In man, hypocaloric diet increases UCP2 cDNA as a probe in low stringency conditions, and let mRNA level in white adipose tissue or skeletal to the cloning of a UCP homologue referred to as muscle.19 Taken together, these data strongly support uncoupling protein-2 or UCP2.8 An UCP2 cDNA was a regulation of UCP2 gene expression by fatty acids also isolated by another group using differential dis- and also indicate that UCP2 activity could be related play analysis of tissues from lean or obese animals.9 to fatty acid metabolism. The up-regulation of UCP2 The predicted sequence of UCP2 indi- mRNA in muscles during starvation does not ®t with a cated that UCP2 was 60% homologous to UCP1.8,9 primary role for UCP2 in energy dissipation but Northern analysis of UCP2 in mouse and human suggests that the primary role of UCP2 could be to tissues showed a wide expression in a number of regulate fatty acid oxidation.18 tissues such as white adipose tissue, brown adipose Based on the assumption that UCP2 is an effector tissue, skeletal muscles, heart, placenta, brain, sto- of basal or regulated thermogenesis, several groups mach, kidney, lung and liver.8,9 We also noticed that examined the effect of the thyroid status on UCP2 UCP2 mRNA was present at high level in spleen, mRNA level. These studies were in agreement with thymus and all organs containing macrophages and this hypothesis since the UCP2 mRNA level was Molecular and genetic aspects of UCP2 D Ricquier S40 shown to be up-regulated in heart20 or skeletal or 50 side; the 50 region of the gene behaves as a white fat of hyperthyroid rodents.21 A stronger effect promoter when it is associated to a reported gene of on UCP3 mRNA level was also and introduced in cells (manuscript under revision). noticed.13,22 Several studies also demonstrated that The identi®cation of cis-regulatory elements is under UCP2 expression was not markedly regulated by the progress. In particular we demonstrated that both in sympathetic nervous system as is UCP1. Analysis of mouse14 and man (manuscript under revision), the the various roles of showed that this hormone UCP2 gene is 7-8 kb downstream of the UCP3 gene elicited a thermogenic response mediated by the which is in agreement with a duplication event. In central nervous system. In agreement with this ®nd- order to appreciate the contribution of the UCP2 ing, several groups reported a positive effect of leptin gene to metabolism or energy expenditure, several on UCP2 expression in white fat BAT and pancreatic approaches were used: measurement of correlations islet associated to increased oxidation of fat.23,24 between UCP2 mRNA level in human tissues and The positive effect of fatty acids on UCP2 mRNA starvation or resting metabolic rate, linkage studies of expression in animals suggested that lipidic com- markers encompassing the UCP2 genomic region, pounds such as rexinoids could regulate this gene. association studiesbetweenhuman UCP2 polymorphism In agreement with this proposal, it was shown that and various traits. thiazolidinediones were able to activate UCP2 gene transcription in adipose cells,25 muscle cells,26 or Correlations between UCP2 mRNA level in human pancreatic islets.27 In other respects, it was reported that administration of pyretic compounds such as tissues and physiological parameters Millet et al have described an increased UCP2 mRNA lipopolysaccharides (endotoxin) to mice induced expression during fasting in white fat and skeletal UCP2 mRNA 28-fold in liver.28 These data illustrate muscle of lean or obese humans under calorie restric- a potential role for UCP2 in in¯ammation or fever as tion (104 kJ=kg=d) for 5 d.32 In the same study, a originally postulated.8 positive correlation between UCP2 mRNA level in adipose tissue and body mass index was calculated. In another study, the same group of researchers mea- sured a positive correlation between UCP2 mRNA UCP2: molecular and genetic level in human adipose tissue and resting metabolic aspects rate.33 Oberko¯er et al measured a smaller level of UCP2 mRNA in intraperitoneal adipose tissue of obese patients compared to non-obese individuals.34 The ®rst question concerning UCP2 is the question of In agreement with this ®nding, Nordfors et al mea- its uncoupling activity. The high level of homology sured a reduced expression of UCP2 gene in skeletal between UCP2 and UCP1 as well as its ability to muscle of human obese subjects.35 At the opposite, strongly decrease the mitochondrial membrane poten- using antibodies, Simoneau et al measured a higher tial when it is expressed in yeasts, are in favour of an level of UCP2 in skeletal muscle of obese indivi- uncoupling activity of UCP2.8 In similar conditions duals.36 UCP2 increased the thermogenesis of recombinant yeasts.29 In other experiments, we were able to demonstrate that UCP2 markedly decrease the respira- Linkage and association studies tory control ratio and the effect of chemical Using markers encompassing the UCP2 gene (and in association with a decrease if the mitochondrial therefore in the vicinity of UCP3 gene), Bouchard et membrane potential and an increase rate of respiration al calculated a very signi®cant linkage of the UCP2 (Bouillaud, Goubern and Rial, unpublished data). to resting metabolic rate in humans.37 These These data support a true uncoupling activity of data strongly support a role for the UCP2=UCP3 locus UCP2. These data are reinforced by the recent dis- in in man. Elbein et al concluded covery of natural ligands which activate the uncou- that the UCP2 region was not linked to markers of pling activity of UCP2 expression is related to fatty obesity in familial type 2 diabetes,38 whereas Kaisaki acid metabolism. It is therefore tempting to propose et al localized UCP2 gene to a region linked to that the primary role for UCP2 is to uncouple respira- glucose intolerance and adiposity in the Goto-Kaki- tion in order to adapt the activity of the respiratory zaki type-2 diabetic rat.39 We, Craig Warden and chains to an excess of substrates. Such an uncoupling others identi®ed several polymorphic sites in the would decrease the amount of superoxide ions as human UCP2 gene (unpublished data). These poly- proposed by Skulachev.30 Others have recently pro- morphisms were analyzed by several groups of posed such a role for UCP2 in liver parenchymal researchers working on different cohorts of lean, cells.31 UCP2 could also be involved in the regulation obese or diabetic individuals. The data differ accord- of ATP level or of the NADH=NAD‡ ratio. ing to the type of cohort and the trait which were We cloned and entirely sequenced the human and analyzed. No relationship between UCP2 variants and mouse UCP2 gene. In comparison to the UCP1 gene, energy expenditure was noticed in a Swedish popula- this gene has 2 additional non-translated exons on its tion.40 A negative conclusion was also reached for Molecular and genetic aspects of UCP2 D Ricquier S41 type-2 diabetes in a Danish cohort41 and by Otabe et 13 Gong DW, He Y, Karas M, Reitman M. Uncoupling protein-3 al analyzing morbidly obese French subjects.42 How- is a mediator of thermogenesis regulated by thyroid hormone, beta3-adrenergic agonists, and leptin. J Biol Chem 1997, 272: ever, an association study of UCP2 polymorphism to 24129 ± 24132. energy metabolism in Pima Indians concluded that 14 Surwit RS, Wang S, Petro AE, Sanchis D, Raimbault S, two UCP2 polymorphisms were associated with sleep- Ricquier D, Collins S. Diet-induced changes in the Uncoupling ing or 24-h metabolic rate.43 Proteins in obesity-prone and obesity-resistant strains of mice. PNAS-USA 1998, 95: 4061 ± 4065. 15 Matsuda J, Hosoda K, Itoh H, Son C, Doi K, Tanaka T, Fukunaga Y, Inoue G, Nishimura H, Yoshimasa Y, Yamori Y, Conclusion Nakao K. Cloning of rat uncoupling protein-3 and uncoupling protein-2 cDNAs: their gene expression in rats fed high-fat diet. FEBS Lett 1997, 418: 200 ± 204. The exact function of UCP2 in cells or in the whole 16 Boss O, Samec S, Dulloo S, Seydoux J,Muzzin P,Giacobino organism is not well understood yet. As an example, JPTissue-dependent regulation of rat uncoupling protein-2 UCP2 mRNA is highly expressed in certain brain expression in response to fasting or cold. FEBS Lett 1997, 412: 111 ± 114. nuclei and the exact activity of UCP2 in these 17 Weigle DS, Selfridge LE, Schwartz MW, Seeley RJ, Cum- 44 nuclei is presently ignored. A second example is mings DE, Havel PJ, Kuijper JL, Beltrandel Rio H. Elevated the high expression of UCP2 in fetal liver in mono- free fatty acids induce uncoupling protein 3 expression in cyte=macrophage cells suggesting a role for UCP2 muscle. Diabetes 1998, 47: 298 ± 302. associated to haematopoietic development.45 Further 18 Samec S, Seydoux J, Dulloo A. Role of UCP homologues in skeletal muscles and brown adipose tissue: mediators of progress in the knowledge of UCP2 biology will come thermogenesis or regulators of lipids as fuel substrate? from different approaches which have been under- FASEB J 1998, 12: 715 ± 724. taken: immuno-assay of UCP2, puri®cation of UCP2, 19 Millet L, Vidal H, Andrealli F, Larrouy D, Riou JP, Ricquier reconstitution of its transport activity in liposomes and D, Laville M, Langin D. Increased uncoupling protein-2 and mutagenesis of the UCP2 gene in mouse using homo- uncoupling protein-3 mRNA expression during fasting in obese and lean humans. J Clin Invest 1997, 100: 26654 ± 2670. logous recombination. 20 Lanni A, De Felice M, Lombardi A, Moreno A, Fleury C, Ricquier D, Goglia F. Induction of UCP2 mRNA in rat heart References by thyroid hormones. FEBS Lett 1997, 418: 171 ± 174. 1 Himms-Hagen J. 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