(UCP2) and uncoupling protein-3 (UCP3) - Nature

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Uncoupling protein-2 (UCP2) and uncoupling protein-3 (UCP3) are mitochondrial proteins that may play a role in the control of energy expenditure by ...

International Journal of Obesity (1999) 23, Suppl 6, S64±S67 ß 1999 Stockton Press All rights reserved 0307±0565/99 $12.00 http://www.stockton-press.co.uk/ijo

Uncoupling protein-2 (UCP2) and uncoupling protein-3 (UCP3) expression in adipose tissue and skeletal muscle in humans D Langin1*, D Larrouy1, P Barbe1, L Millet1, N Viguerie-Bascands1, F Andreelli2, M Laville2 and H Vidal2 1

Unite INSERM 317, Institut Louis Bugnard, Universite Paul Sabatier, HoÃpital Rangueil, Toulouse, France; and 2Unite INSERM 449 et Centre de Recherche en Nutrition Humaine de Lyon, Faculte de MeÂdecine LaeÈnnec, Lyon, France

Uncoupling protein-2 (UCP2) and uncoupling protein-3 (UCP3) are mitochondrial proteins that may play a role in the control of energy expenditure by uncoupling respiration from ATP synthesis. The present review focuses on data obtained in humans. UCP2 is widely expressed in the body, whereas UCP3 expression is restricted to skeletal muscle. Positive correlations have been reported between UCP2 mRNA concentrations in adipose tissue, UCP3 mRNA concentrations in skeletal muscle, and components of the metabolic rate. Fasting induces an up-regulation of UCP2 and UCP3 mRNA expression. In vivo and in vitro studies suggest that fatty acids could modulate uncoupling protein gene expression. The putative relationship between obesity, energy expenditure and uncoupling protein expression, and the unexpected rise in UCP2 and UCP3 mRNA concentrations during short-term fasting, are discussed in view of the recent data obtained in rodents and cell lines. Keywords: energy expenditure; fatty acid; adipose tissue; skeletal muscle; obesity;

Uncoupling protein-2 (UCP2) and uncoupling protein-3 (UCP3) expression and isoforms in humans The two novel uncoupling proteins known as UCP2 and UCP3 may be the long-awaited `missing link' in the understanding of the molecular basis of energy expenditure in humans. The resting metabolic rate (RMR), that is, the obligatory energy expenditure required to maintain physiological tissue function in the resting state, is the largest component of daily energy expenditure. A substantial part of the RMR results from a leaking of protons across the mitochondrial inner membrane, which results in energy dissipation because of uncoupling of oxygen consumption to ATP synthesis.1,2 UCP2 and UCP3 are candidates to explain the proton leak in many tissues.3 ± 6 UCP2 and UCP3 expression in yeast indeed causes a decrease in mitochondrial membrane potential. The two proteins show sequence identity with UCP1, an uncoupling protein expressed in brown adipose tissue (BAT). In rodents, but probably not in adult humans, BAT is an important site of adaptive thermogenesis.7 The tissue distribution of UCP2 and

Correspondence: Dr Dominique Langin, INSERM U317, Institut Louis Bugnard, BaÃtiment L3, CHU Rangueil, 31403 Toulouse Cedex 4, France.

UCP3 mRNAs are markedly different. UCP2 mRNA is widely expressed in the body whereas the main site of UCP3 mRNA expression is skeletal muscle in adult humans. UCP3 mRNA exists as long and short form transcripts.3 The two transcripts are generated from a single gene, through alternative splicing and use of polyadenylation signals.8 The short form transcript encodes a putative protein, designated UCP3S, that does not contain the last 37 amino acids present in the long form UCP3 (UCP3L), UCP1 and UCP2. This region contains the putative sixth transmembrane domain and motifs likely to be critical for uncoupling activity. UCP3S could show an increased uncoupling activity because of the lack of a conserved motif that mediates inhibition of UCP1 uncoupling activity by purine nucleotides. It is also possible that UCP3S may not be stable or may not be functional in the mitochondrial inner membrane. The effect of mutations recently found in the human UCP3 gene9 is in accordance with a defective function of UCP3S. A mutation at the exon 6-splice donor site, detected in African-Americans, results in the premature termination of the protein product which is identical to UCP3S. Heterozygotes for the mutation showed a 50% reduction in fat oxidation and an elevation of the nonprotein respiratory quotient (RQ), compared with wild-type subjects. The frequency of the mutation was twice as high in obese, compared with lean, individuals. Variation in the ratio between UCP3L and UCP3S could therefore modulate skeletal muscle uncoupling activity.

Tissue UCP2 and UCP3 D Langin et al

Uncoupling protein mRNA concentrations, energy expenditure and obesity Because a low rate of energy expenditure is a predisposing factor for weight gain and the potential link between uncoupling protein expression and RMR, several laboratories, including ours, have sought to determine the factors modulating UCP2 and UCP3 gene expression in human skeletal muscle and adipose tissue. We have developed a reverse transcriptioncompetitive polymerase chain reaction (RT-cPCR) assay to determine UCP2 and UCP3 mRNA concentrations.11 Because of the important differences between UCP3L and UCP3S, the two mRNAs were quanti®ed separately.12 In the vastus lateralis muscle, UCP3 mRNA is more abundant than UCP2 mRNA, as also found in rodents.3,6 UCP3L and UCP3S are expressed in equal amounts. No large difference in the level of the three transcripts was observed between Caucasian obese and lean individuals.11,12 Using in situ hybridisation, a 30% lower UCP2 mRNA expression was reported by Norfors et al13 in skeletal muscle of obese subjects. In agreement with our data,11 UCP3 mRNA concentrations were similar in the two groups. In contrast, Schrauwen et al14 showed a negative correlation between UCP3L mRNA concentrations and body mass index (BMI) in Pima Indians that was not found with UCP2 mRNA levels. The lower susceptibility to obesity of Caucasians compared with Pima Indians might explain such a discrepancy. Studies on a large number of individuals with a wide range of BMI are clearly needed to clarify this point. Nevertheless, in line with a putative role of skeletal muscle UCP3 in the control of energy expenditure, a positive correlation was found in Pima Indians between sleeping metabolic rate (SMR) adjusted for fat-free mass (FFM) and fat mass (FM) and UCP3L mRNA concentrations.14 In subcutaneous adipose tissue, we found a positive correlation between BMI and UCP2 mRNA concentrations.11 This data is consistent with the higher UCP2 mRNA concentration found in white adipose tissue of ob=ob and db=db obese mice, compared with lean littermates,5 suggesting a possible relationship between the concentration of UCP2 mRNA and fat cell hypertrophy. A potential link between UCP2 and energy expenditure was studied in obese premenopausal women.15 After standardisation of food intake by a four week very-low-calorie diet (VLCD), a positive correlation was found between subcutaneous adipose tissue UCP2 mRNA concentration and RMR adjusted for FFM, but the correlation failed to reach signi®cance before the diet. Factors other than RMR (for example, the diet composition) are therefore likely to be associated with UCP2 mRNA concentrations in adipose tissue. A strong correlation was found between subcutaneous and visceral adipose tissue UCP2 mRNA concentrations. The two fat depots

represent most of the adipose tissue mass in humans. Therefore, the data suggest that the concentration of UCP2 mRNA in subcutaneous adipose tissue re¯ects the overall level of UCP2 gene expression in total body fat. Hence, the positive correlation found between RMR and subcutaneous adipose tissue UCP2 mRNA concentration might be extended to UCP2 mRNA concentration in whole body fat. Oberko¯er et al16 have reported a reduced UCP2 mRNA expression in visceral adipose tissue of a large number of morbidly obese subjects. As the authors indicate, the possible role of the lower UCP2 expression in the pathophysiology of obesity remains to be proven. In rodents, a potential role for white adipose tissue (WAT) UCP2 in the regulation of body weight and energy expenditure is suggested by several lines of evidence. A high fat diet increases WAT UCP2 gene expression in the obesity-resistant A=J and C57BL= KsJ strains, but not in the obesity-prone C57BL=6J mice. Interestingly, the diet does not affect UCP2 and UCP3 mRNA expression in skeletal muscle.4,17 UCP1-de®cient mice do not become obese and it was proposed that the lack of UCP1 may be compensated by UCP2.18 Moreover, the ectopic expression of UCP1 in WAT results, in transgenic mice, in a decrease of adiposity attributed to an increase of energy dissipation in this tissue.19, 20 A similar role for adipose tissue UCP2 in energy dissipation remains to be demonstrated.

Variations in uncoupling protein gene expression during fasting Possible modulation of uncoupling protein gene expression was studied during a ®ve-day severe calorie restriction, a situation associated with a decrease in energy expenditure.11,12 Unexpectedly, the fasting condition induced a 2 ± 3 fold increase of UCP2, UCP3L and UCP3S mRNA concentrations. Similar ®ndings have been reported in rodents.21,22 The induction was similar in lean and obese subjects suggesting that, during fasting, there is no major alteration of uncoupling protein gene regulation in obesity. The signi®cance of this observation is not clear at the present time. Study of UCP2 and UCP3 gene expression during fasting and refeeding in rodents has led to the proposal that UCP2 and UCP3 would act as regulators of lipids as a fuel substrate rather than as a mediator of regulatory thermogenesis.22 In agreement with this hypothesis, mutations in the human UCP3 gene have been associated with a decreased capacity of fat oxidation.9 The hypothesis needs, however, to be substantiated by studying the relationship between mitochondrial uncoupling proteins and intracellular lipid metabolism. It is important to stress that the increase in uncoupling protein mRNA concentrations might be transient and modulated by


Tissue UCP2 and UCP3 D Langin et al


calorie intake. During a four week VLCD providing more energy per day than the ®ve day severe calorie restricted diet, no changes in subcutaneous adipose tissue UCP2 mRNA concentrations were observed.15 This situation is reminiscent of the effect of food intake on the UCP3 mRNA concentration in rodent skeletal muscle.23 A 48 h fast markedly increased the UCP3 mRNA concentration, whereas one week of 50% food restriction resulted in a decrease of UCP3 mRNA expression. A biphasic regulation is also found during exercise and endurance training in rodents. A single bout of exercise results in a transient increase of UCP3 mRNA concentrations.24 On the other hand, skeletal muscle UCP2 and UCP3 mRNA concentrations were reduced after an eight week endurance training.25 In both calorie restriction and exercise, the short-term up-regulation of uncoupling protein mRNA concentrations seems to be transient, maybe as discussed below, in response to acute metabolic and hormonal changes. The long-term down-regulation is consistent with the increased feeding ef®ciency which contributes to the increased weight gain observed after cessation of endurance training or calorie restriction. Fasting provokes a complex physiological adaptation with numerous hormonal and metabolic changes that could explain the increase in UCP2 and UCP3 gene expression. Among the hormones that have been shown to upregulate UCP2 and UCP3 gene expression, leptin and triiodothyronine are not likely to play a role because their plasma concentrations decrease during fasting.26 ± 30 Moreover, we have shown that insulin does not acutely regulate UCP2 and UCP3 mRNA concentrations in humans.11 Calorie restriction induces an increase in adipose tissue lipolysis,31 resulting in an important fatty acid release from body fat stores. Fatty acids and other ligands of the peroxisome proliferator-activated receptors stimulate UCP2 gene expression in adipocyte and skeletal muscle cell lines,32,33 and UCP2 and UCP3 mRNA concentrations are increased by high-fat feeding in mice. 17,34 In rats, elevation of plasma free fatty acids (FFA) concentrations by Intralipid plus heparin infusion induces UCP3 mRNA expression in skeletal muscle.35 A positive correlation was found between UCP3 mRNA concentration and plasma FFA concentration in 10 h fasted obese subjects.36 Further investigation is required to determine whether the increase in plasma FFA concentrations occurring during fasting contributes to the up-regulation of uncoupling protein mRNAs in humans. Acknowledgements

The laboratories are parts of the FATLINK Concerted Action supported by the European Commission (FAIR programme). References

1 Porter RK, Brand MD. Body mass dependence of H‡ leak in mitochondria and its relevance to metabolic rate. Nature 1993; 362: 628 ± 630.

2 Rolfe DF, Brand MD. Contribution of mitochondrial proton leak to skeletal muscle respiration and to standard metabolic rate. Am J Physiol 1996; 271: C1380 ± 1389. 3 Boss O, Samec S, Paolini-Giacobino A, Rossier C, Dulloo A, Seydoux J, Muzzin P, Giacobino J-P. Uncoupling protein-3: a new member of the mitochondrial carrier family with tissue-speci®c expression. FEBS Lett 1997; 408: 39 ± 42. 4 Fleury C, Neverova M, Collins S, Raimbault S, Champigny O, Levi-Meyrueis C, Bouillaud F, Seldin MF, Surwit RS, Ricquier D, Warden CH. Uncoupling protein-2; a novel gene linked to obesity and hyperinsulinemia. Nat Genet 1997; 15: 269 ± 272. 5 Gimeno RE, Dembski M, Weng X, Deng N, Shyjan AW, Gimeno CJ, Iris F, Ellis SJ, Woolf EA, Tartaglia LA. Cloning and characterization of an uncoupling protein homolog. A potential molecular mediator of human thermogenesis. Diabetes 1997; 46: 900 ± 906. 6 Vidal-Puig A, Solanes G, Grujic D, Flier JS, Lowell BB. UCP3: an uncoupling protein homologue expressed preferentially and abundantly in skeletal muscle and brown adipose tissue. Biochem Biophys Res Commun 1997; 235: 79 ± 82. 7 Garruti G, Ricquier D. Analysis of uncoupling protein and its mRNA in adipose tissue deposits of adult humans. Int J Obes 1992; 16: 383 ± 390. 8 Solanes G, Vidal-Puig A, Grujic D, Flier JS, Lowell BB. The human uncoupling protein-3 gene. Genomic structure, chromosomal localization, and genetic basis for short and long form transcripts. J Biol Chem 1997; 272: 25433 ± 25436. 9 Argyropoulos G, Brown A, Willi S, Zhu J, He Y, Reitman M, Gevao S, Spruill I, Garvey W. Effects of mutations in the human uncoupling protein 3 gene on the respiratory quotient and fat oxidation in severe obesity and type 2 diabetes. J Clin Invest 1998; 102: 1345 ± 1351. 10 Ravussin E, Lillioja S, Knowler WC, Christin L, Freymond D, Abbott WG, Boyce V, Howard BV, Bogardus C. Reduced rate of energy expenditure as a risk factor for body-weight gain. N Engl J Med 1988; 318: 467 ± 472. 11 Millet L, Vidal H, Andreelli F, Larrouy D, Riou JP, Ricquier D, Laville M, Langin D. Increased uncoupling protein-2 and -3 mRNA expression during fasting in obese and lean humans. J Clin Invest 1997; 100: 2665 ± 2670. 12 Millet L, Vidal H, Larrouy D, Andreelli F, Laville M, Langin D. mRNA expression of the long and short forms of uncoupling protein-3 in obese and lean humans. Diabetologia 1998; 41: 829 ± 832. 13 Nordfors L, Hoffstedt J, Nyberg B, ThoÈrne A, Arner P, Schalling M, LoÈnnqvist F. Reduced gene expression of UCP2 but not UCP3 in skeletal muscle of human obese subjects. Diabetologia 1998; 41: 935 ± 939. 14 Schrauwen P, Xia J, Bogardus C, Pratley R, Ravussin E. Skeletal muscle UCP3 expression is a determinant of energy expenditure in Pima Indians. Diabetes 1999; 48: 146 ± 149. 15 Barbe P, Millet L, Larrouy D, Galitzky J, Berlan M, Louvet JP, Langin D. Uncoupling protein-2 messenger ribonucleic acid expression during very-low-calorie diet in obese premenopausal women. J Clin Endocrinol Metab 1998; 83: 2450 ± 2453. 16 Oberko¯er H, Liu Y, Esterbauer H, Hell E, Krempler F, Patsch W. Uncoupling protein-2 gene: reduced mRNA expression in intraperitoneal adipose tissue of obese humans. Diabetologia 1998; 41: 940 ± 946. 17 Surwit RS, Wang S, Petro AE, Sanchis D, Raimbault S, Ricquier D, Collins S. Diet-induced changes in uncoupling proteins in obesity-prone and obesity-resistant strains of mice. Proc Natl Acad Sci USA 1998; 95: 4061 ± 4065. 18 EnerbaÈck S, Jacobsson A, Simpson EM, Guerra C, Yamashita H, Harper M-E, Kozak LP. Mice lacking mitochondrial uncoupling protein are cold-sensitive but not obese. Nature 1997; 387: 90 ± 94.

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19 Kopecky J, Clarke G, EnerbaÈck S, Spiegelman B, Kozak LP. Expression of the mitochondrial uncoupling protein gene from the aP2 gene promoter prevents genetic obesity. J Clin Invest 1995; 96: 2914 ± 2923. 20 Kopecky J, Rossmeisl M, Hodny Z, Syrovy I, Horakova M, Kolarova P. Reduction of dietary obesity in aP2-Ucp transgenic mice: mechanism and adipose tissue morphology. Am J Physiol 1996; 270: E776 ± E786. 21 Boss O, Samec S, Dulloo A, Seydoux J, Muzzin P, Giacobino J. Tissue-dependent up-regulation of rat uncoupling protein-2 expression in response to fasting or cold. FEBS Lett 1997; 412: 111 ± 114. 22 Samec S, Seydoux J, Dulloo A. Role of UCP homologues in skeletal muscles and brown adipose tissue: mediators of thermogenesis or regulators of lipids as fuel substrate? FASEB J 1998; 12: 715 ± 724. 23 Boss O, Samec S, KuÈhne F, Bijlenga P, AssimacopoulosJeannet F, Seydoux J, Giacobino JP, Muzzin P. Uncoupling protein-3 expression in rodent skeletal muscle is modulated by food intake but not by changes in environmental temperature. J Biol Chem 1998; 273: 5 ± 8. 24 Tsuboyama-Kasaoka N, Tsunoda N, Maruyama K, Takahashi M, Kim H, Ikemoto S, Ezaki O. Up-regulation of uncoupling protein 3 (UCP3) mRNA by exercise training and downregulation of UCP3 by denervation in skeletal muscles. Biochem Biophys Res Commun 1998; 247: 498 ± 503. 25 Boss O, Samec S, Desplanches D, Mayet MH, Seydoux J, Muzzin P, Giacobino JP. Effect of endurance training on mRNA expression of uncoupling proteins 1, 2 and 3 in the rat. FASEB 1998; 12: 335 ± 339. 26 Cusin I, Zakrzewska K, Boss O, Muzzin P, Giacobino JP, Ricquier D, Jeanrenaud B, Rohner-Jeanrenaud F. Chronic central leptin infusion enhances insulin-stimulated glucose metabolism and favors the expression of uncoupling proteins. Diabetes 1998; 47: 1014 ± 1019. 27 Gong DW, He Y, Karas M, Reitman M. Uncoupling protein-3 is a mediator of thermogenesis regulated by thyroid hormone, beta3-adrenergic agonists and leptin. J Biol Chem 1997; 272: 24129 ± 24132. 28 Liu Q, Bai C, Chen F, Wang R, MacDonald T, Gu M, Zhang Q, Morsy M, Caskey C. Uncoupling protein-3 : a musclespeci®c gene upregulated by leptin in ob=ob mice. Gene 1998; 207: 1 ± 7.

29 Masaki T, Yoshimatsu H, Kakuma T, Hidaka S, Kurokawa M, Sakata T. Enhanced expression of uncoupling protein 2 in rat white adipose tissue and skeletal muscle following chronic treatment with thyroid hormone. FEBS Lett 1997; 418: 323 ± 326. 30 Zhou YT, Shimabukuro M, Koyama K, Lee Y, Wang MY, Trieu F, Newgard CB, Unger RH. Induction by leptin of uncoupling protein-2 and enzymes of fatty acid oxidation. Proc Natl Acad Sci USA 1997; 94: 6386 ± 6390. 31 Stich V, Harant I, de Glizesinski I, Crampes F, Berlan M, Kunesova M, Hainer V, Dauzats M, RivieÁre D, Garrigues M, Holm C, Lafontan M, Langin D. Adipose tissue lipolysis and hormone-sensitive lipase expression during very-low-calorie diet in obese female identical twins. J Clin Endocrinol Metab 1997; 82: 739 ± 744. 32 Aubert J, Champigny O, Saint-Marc P, NeÂgrel R, Collins S, Ricquier D, Ailhaud G. Up-regulation of UCP-2 gene expression by PPAR agonists in preadipose and adipose cells. Biochem Biophys Res Commun 1997; 238: 606 ± 611. 33 Camirand A, Marie V, Rabelo R, Silva J. Thiazolidinediones stimulate uncoupling protein-2 expression in cell lines representing white and brown adipose tissues and skeletal muscle. Endocrinology 1998; 139: 428 ± 431. 34 Matsuda J, Hosoda K, Itoh H, Son C, Doi K, Tanaka T, Fukunaga Y, Inoue G, Nishimura H, Yoshimasa Y, Yamori Y, 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. 35 Weigle DS, Selfridge LE, Schwartz MW, Seeley RJ, Cummings DE, Havel PJ, Kuijper JL, BeltrandeRio H. Elevated fatty acids induce uncoupling protein-3 expression in muscle. A potential explanation for the effect of fasting. Diabetes 1998; 47: 298 ± 302. 36 Boss O, Bobbioni-Harsch E, Assimacopoulos-Jeannet F, Muzzin P, Munger R, Giacobino J, Golay A. Uncoupling protein-3 expression in skeletal muscle and free fatty acids in obesity. Lancet 1998; 351: 1933.


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