The Common -866 G/A Polymorphism in the Promoter of Uncoupling Protein 2 Is Associated With Increased Carbohydrate and Decreased Lipid Oxidation in Juvenile Obesity
Sophie Le Fur,
Catherine Le Stunff,
Christine Dos Santos, and
Pierre Bougnères
From the Department of Pediatric Endocrinology, Hôpital St-Vincent de Paul, Paris, France
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ABSTRACT
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Uncoupling protein (UCP) 2 is a member of the mitochondrial transporter superfamily that uncouples proton entry in the mitochondrial matrix from ATP synthesis. Although its physiological role remains to be established, UCP2 is considered a candidate gene for association with energy metabolism and obesity. A common promoter polymorphism, -866 G/A, has been associated with increased UCP2 gene expression and middle-aged adult obesity. In fact, our analysis of 296 juvenile obese and 568 nonobese control subjects revealed no difference in the prevalence of this polymorphism. Insulin and glucose response to oral glucose was comparable across the -866 genotypes. Metabolic studies in 147 of these juvenile obese subjects showed that homozygosity for the UCP2 promoter variant A was associated with important changes in energy metabolism compared with other genotypes, i.e., a 34% increase of carbohydrate oxidation (94 ± 10 vs. 70 ± 3 mg · min-1 · m-2, P = 0.004) and a 23% decrease of lipid oxidation (26 ± 3 vs. 34 ± 1 mg · min-1 · m-2, P = 0.03). Therefore, the juvenile obese subjects who are homozygous for the A variant have an increased ratio (3.6 ± 1.2) of calories derived from carbohydrates to those from lipids compared with G/A or G/G obese children (1.4 ± 0.2, P = 0.003), suggesting a role for UCP2 in the partitioning of metabolic fuels.
The epidemic of childhood obesity that is currently taking over many parts of the world will contribute to a large increase of adult obesity and metabolic complications. It is thus important to study the early metabolic alterations characterizing juvenile obesity and the associated genetic factors. Because of its suspected function, the uncoupling protein (UCP) 2 is one of the many genes that might be associated with early obesity or with obesity effects on metabolic tissues. The human UCP2 gene encodes a mitochondrial transmembrane carrier protein that shows high amino acid similarities with the other two human UCPs, UCP1 and -3. UCP3 is predominantly expressed in skeletal muscle (1), but UCP2 appears to be widely expressed (24). UCP2 and UCP3 are located within 150 kb of each other on chromosome 11q13 (5,6). As a potential uncoupler of oxidative phosphorylation, UCP2 could be expected to play a role in energy balance and the regulation of body weight. However, it should be kept in mind that the physiological function of UCP2 is still a matter of debate and research (3). A number of genetic studies have investigated the relationship between the UCP2 locus and obesity propensity, with a specific focus on coding variants of this protein (7). Esterbauer et al. (8) recently reported that a functional G/A polymorphism in the promoter of UCP2 is associated with obesity in middle-aged European adults. The variant A allele of the -866 polymorphism was associated with enhanced transcription of the UCP2 gene and a decreased risk of obesity (8).
Since a large proportion of adult obesity starts during childhood (9,10), we examined whether the decreased prevalence of allele A seen in obese adults is already observable in juvenile obesity. The distribution of -866 G/G (0.385), G/A (0.490), and A/A (0.125) genotypes was in fact comparable in the 296 juvenile obese subjects (Table 1) and in the 568 never obese control subjects (not shown). The -866 polymorphism fulfilled Hardy-Weinberg expectations in both cohorts. Genotype prevalence was exactly similar to that found in the nonobese Caucasians by Esterbauer et al. (8). Age, sex, and BMI did not differ significantly between -866 genotypic groups of obese children (Table 1). These observations suggest that genomic variations nearby the UCP2 gene do not play a major role in the predisposition to juvenile obesity, in contrast with middle-aged obese adults, who show an increased proportion of G/G genotypes (8). Our interpretation is that juvenile forms of obesity studied here may not share all of their genetic factors with the late adulthood forms studied by Esterbauer et al. (8) ("early-onset obesity" versus "late-onset obesity"). According to this view, the -866 G allele would predispose an increased proportion of obese juveniles to maintain their obesity status during adulthood.
UCP2 is a negative regulator of insulin secretion (11) and could thus be a link between obesity and ß-cell dysfunction in obesity-induced type 2 diabetes (11). Alterations in fatty acid metabolism are likely involved in the mechanisms of this ß-cell dysfunction (11). Because of the clinical (12,13) and experimental (14) association of ß-cell UCP2 expression with insulin secretion (15), we searched for effects of -866 G/A polymorphism on the insulin response to glucose by performing oral glucose tolerance tests (OGTTs) in 129 obese children. We found that fasting glucose, fasting insulin, and insulin and glucose areas did not differ between the -866 genotypic groups (Fig. 1 and Table 2). These findings support the theory that glucose-stimulated insulin secretion is independent of the -866 UCP2 genotype in young obese patients (16,17). In addition, the close similarity of insulin and glucose curves across genotypes during OGTT (Fig. 1) indicates that the -866 variants of the UCP2 promoter are not associated with variations in insulin sensitivity at the whole-body level. In middle-aged adults, the -866 A/A genotype was found to be associated with slightly higher fasting insulin levels (8) but with a decreased acute insulin response to intravenous glucose (18). None of these genotypic differences, however, were of sufficient magnitude to reach significance (8,18). It is possible that, during evolution from adolescence to late adulthood, the -866 A allele of the UCP2 promoter is associated with a diminution of insulin secretion, which could have two effects. One would be to reduce the insulin-dependent deposition of fat and thus to account for the decreased prevalence of middle-age obesity observed in A/A homozygous subjects (18). The other would be to increase the risk of type 2 diabetes in these individuals (18).

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FIG. 1. Plasma insulin and glucose during OGTT in the 129 obese children according to their -866 UCP2 genotype.
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TABLE 2 Glucose and insulin parameters during OGTT in a subgroup of obese children; a group of age-matched nonobese children is presented for comparison
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We previously reported that postabsorptive energy metabolism in juvenile obese subjects is characterized by an early increase in lipid oxidation followed by a progressive decrease in glucose oxidation (19). Obese children derive
61% of their energy production from lipids compared with 34% in lean children. The present results show that the -866 G/A polymorphism is associated with changes in the rates of oxidation of carbohydrate and fatty acid fuels in the current cohort of young obese subjects. Obese children with -866 A/A genotypes have a modest but significant increase of resting energy expenditure, possibly consistent with the decreased risk of middle-age obesity observed in these subjects by Esterbauer et al. (8). Astrup et al. (20) and Ukkola et al. (21) both reported that variations in lipid fuel oxidation, reflected by respiratory quotient measurement, is associated with the A/V polymorphism of UCP2 protein in healthy adults. We also observed that despite comparable glucose, free fatty acid, and insulin levels, the obese children with -866 A/A genotypes oxidize more glucose and less lipids than the -866 G/G or G/A groups (Table 3). We have no direct explanation for this phenomenon. According to a recent hypothesis (22), UCP2 and -3 may play a role in the export of fatty acids from mitochondria (23) when fatty acid oxidation predominates. An increase of UCP2 expression would thus be associated with an increase of fatty acid oxidation. However, if UCP2 is truly an uncoupling molecule, it should instead favor an increase of intracellular ATP, an increase of glucose oxidation, and a decrease of lipid oxidation ("Pasteur" effect). In obese subjects, most of glucose and lipid oxidation takes place in the skeletal muscle, a tissue in which ethics do not allow us to measure UCP2 gene expression. It is possible that -866 G/A genotypes are associated with changes in UCP2 expression in muscle comparable with those observed in the adipose tissue of obese adults with this genotype (8). In obese adults, however, the expression of the UCP2 gene shows discordant changes in muscle and adipose tissue (2428). It is also notable that UCP2 expression seems to be much less widespread as a protein than as mRNA species (3,4) and that most experimental and clinical reports on UCP2 expression levels are based only on mRNA measurements.
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TABLE 3 Main clinical and metabolic characteristics in the UCP2 genotypic subgroups of 147 obese children and in 17 age-matched nonobese children.
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In summary, the -866 G/A polymorphism in the UCP2 promoter seems to underlie modifications in the fluxes of substrates that are oxidized in the mitochondria of young obese patients. Randle et al. (29) proposed the possibility that the impairment in glucose metabolism that characterizes obese subjects is caused by an excessive utilization of fatty substrates. The conditions on which Randle et al.s hypothesis can hold in clinical situations are 1) lipid oxidation is increased, 2) glucose oxidation is decreased, and 3) the two changes are commensurate to one another. Our observations with UCP2 genotypes suggest that UCP2 could play a significant role in the regulation of the phenomenon of Randle et al. in young obese subjects. Commonly, genetic association studies try to interpret positive results in the light of the physiology of the considered molecule. In the case of UCP2, such an attempt should remain very cautious since the precise role of this carrier protein in mitochondrial lipid metabolism remains to be defined. On the other side, further epidemiogenetic studies will be necessary to determine whether the -866 UCP2 promoter polymorphism influences insulin secretion and substrate oxidation during evolution from juvenile to late-adulthood obesity and if it is truly implicated in the natural history of obesity-dependent diabetes.
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RESEARCH DESIGN AND METHODS
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The 296 obese patients came from a previously described cohort (30) originating from Mediterranean and Central European countries. The geographic origin of the patients was assessed through family history, analysis of patronymic names, and grandparents birthplace. Inclusion criteria were a BMI exceeding the 85th centile before the age of 6 years, a monotonic weight curve since birth, and the lack of any weight reduction during obesity course. There were 147 obese children from this cohort who underwent indirect calorimetry, and another group of 129 obese patients had OGTTs.
A total of 568 young adults aged 2240 years, of comparable Caucasian origin (30), and who had never been obese, were studied as control subjects.
The obese and lean children were genotyped at position -866 of the UCP2 locus as reported (31). Single nucleotide polymorphism genotypes were determined by the analysis of PCR products.
To minimize experimental variability in measuring insulin levels, we used codified conditions. After 12 h of overnight fasting following 3 days of standardized diet (caloric and carbohydrate content) in hospital, plasma insulin was measured in unstressed conditions (an intravenous microcatheter was placed in a peripheral vein 24 h before sampling) (30). The OGTT consisted of the ingestion of 1.75 g/kg glucose (75 g maximum) in 200 ml lemon-flavored water at 10°C and venous blood sampling at 0, 30, 60, 90, and 120 min after the end of the ingestion. Plasma insulin was measured in each sample in duplicate with standard radioassay (30). As a reproductibility test, duplicate OGTT measurements were performed on following days in 20 children. The intraindividual coefficient of variation of insulin values at the various time points was 1318%.
We used insulin (µU/ml) and glucose (mg/dl) values during the OGTT to calculate the composite whole-body index insulin sensitivity index (ISIcomp) described by Matsuda and DeFronzo (32), according to the formula ISIcomp = 10,000/square root of (Ins 0 x Glu 0 x mean OGTT Ins x mean OGTT Glu).
Respiratory calorimetry, energy expenditure, resting metabolic rate, percent body fat, glucose and fat oxidation, and lean body mass were measured as previously described (33).
All data are presented as means ± SE. The statistical comparisons used ANOVA among the various subgroups.
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ACKNOWLEDGMENTS
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S.L.F. is a recipient of the Research Award from the Danone Institute.
We thank A. Dermane for the clinical care of the patients and D. Riquier and J. Girard for helpful discussions.
Address correspondence and reprint requests to Pierre Bougnères, Département dEndocrinologie, Hôpital Saint-Vincent de Paul, 82, avenue Denfert Rochereau 75014 Paris, France. E-mail: bougneres{at}cochin.inserm.fr
Received for publication June 27, 2003
and accepted in revised form September 22, 2003
OGTT, oral glucose tolerance test; UCP, uncoupling protein
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REFERENCES
|
---|
- Boss O, Samec S, Paoloni-Giacobino A, Rossier C, Dulloo A, Seydoux J, Muzzin P, Giacobino JP: Uncoupling protein-3: a new member of the mitochondrial carrier family with tissue-specific expression.
FEBS Lett408
:39
42,1997[Medline]
- 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 Genet15
:269
272,1997[Medline]
- Nedergaard J, Cannon B: Pros and cons for suggested functions.
Exp Physiol88
:65
84,2003[Abstract/Free Full Text]
- Pecqueur C, Alves-Guerra MC, Gelly C, Levi-Meyrueis C, Couplan E, Collins S, Ricquier D, Bouillaud F, Miroux B: Uncoupling protein 2, in vivo distribution, induction upon oxidative stress, and evidence for translational regulation.
J Biol Chem276
:8705
8712,2001[Abstract/Free Full Text]
- Argyropoulos G, Brown AM, Peterson R, Likes CE, Watson DK, Garvey WT: Structure and organization of the human uncoupling protein 2 gene and identification of a common biallelic variant in Caucasian and African-American subjects.
Diabetes47
:685
687,1998[Free Full Text]
- Pecqueur C, Cassard-Doulcier AM, Raimbault S, Miroux B, Fleury C, Gelly C, Bouillaud F, Ricquier D: Functional organization of the human uncoupling protein-2 gene, and juxtaposition to the uncoupling protein-3 gene.
Biochem Biophys Res Commun255
:40
46,1999[Medline]
- Schonfeld-Warden NA, Warden CH: Physiological effects of variants in human uncoupling proteins: UCP2 influences body-mass index.
Biochem Soc Trans29
:777
784,2001[Medline]
- Esterbauer H, Schneitler C, Oberkofler H, Ebenbichler C, Paulweber B, Sandhofer F, Ladurner G, Hell E, Strosberg AD, Patsch JR, Krempler F, Patsch W: A common polymorphism in the promoter of UCP2 is associated with decreased risk of obesity in middle-aged humans.
Nat Genet28
:178
183,2001[Medline]
- Sorensen TI, Sonne-Holm S: Risk in childhood of development of severe adult obesity: retrospective, population-based case-cohort study.
Am J Epidemiol127
:104
113,1988[Abstract]
- Mijailovic M, Mijailovic V, Micic D: Childhood onset of obesity: does an obese child become an obese adult?
J Pediatr Endocrinol Metab14 (Suppl. 5)
:S1335
S1338; 2001
- Zhang CY, Baffy G, Perret P, Krauss S, Peroni O, Grujic D, Hagen T, Vidal-Puig AJ, Boss O, Kim YB, Zheng XX, Wheeler MB, Shulman GI, Chan CB, Lowell BB: Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes.
Cell105
:745
755,2001[Medline]
- Chan CB: Endogenous regulation of insulin secretion by UCP2.
Clin Lab48
:599
604,2002[Medline]
- Chan CB, De Leo D, Joseph JW, McQuaid TS, Ha XF, Xu F, Tsushima RG, Pennefather PS, Salapatek AM, Wheeler MB: Increased uncoupling protein-2 levels in ß-cells are associated with impaired glucose-stimulated insulin secretion: mechanism of action.
Diabetes50
:1302
1310,2001[Abstract/Free Full Text]
- Chan CB, MacDonald PE, Saleh MC, Johns DC, Marban E, Wheeler MB: Overexpression of uncoupling protein 2 inhibits glucose-stimulated insulin secretion from rat islets.
Diabetes48
:1482
1486,1999[Abstract]
- Polonsky KS, Semenkovich CF: The pancreatic beta cell heats up: UCP2 and insulin secretion in diabetes.
Cell105
:705
707,2001[Medline]
- Otabe S, Clement K, Rich N, Warden C, Pecqueur C, Neverova M, Raimbault S, Guy-Grand B, Basdevant A, Ricquier D, Froguel P, Vasseur F: Mutation screening of the human UCP 2 gene in normoglycemic and NIDDM morbidly obese patients: lack of association between new UCP 2 polymorphisms and obesity in French Caucasians.
Diabetes47
:840
842,1998[Free Full Text]
- Urhammer SA, Dalgaard LT, Sorensen TI, Moller AM, Andersen T, Tybjaerg-Hansen A, Hansen T, Clausen JO, Vestergaard H, Pedersen O: Mutational analysis of the coding region of the uncoupling protein 2 gene in obese NIDDM patients: impact of a common amino acid polymorphism on juvenile and maturity onset forms of obesity and insulin resistance.
Diabetologia40
:1227
1230,1997[Medline]
- Krempler F, Esterbauer H, Weitgasser R, Ebenbichler C, Patsch JR, Miller K, Xie M, Linnemayr V, Oberkofler H, Patsch W: A functional polymorphism in the promoter of UCP2 enhances obesity risk but reduces type 2 diabetes risk in obese middle-aged humans.
Diabetes51
:3331
3335,2002[Abstract/Free Full Text]
- Felber JP, Meyer HU, Curchod B, Iselin HU, Rousselle J, Maeder E, Pahud P, Jequier E: Glucose storage and oxidation in different degrees of human obesity measured by continuous indirect calorimetry.
Diabetologia20
:39
44,1981[Medline]
- Astrup A, Toubro S, Dalgaard LT, Urhammer SA, Sorensen TI, Pedersen O: Impact of the v/v 55 polymorphism of the uncoupling protein 2 gene on 24-h energy expenditure and substrate oxidation.
Int J Obes Relat Metab Disord23
:1030
1034,1999[Medline]
- Ukkola O, Tremblay A, Sun G, Chagnon YC, Bouchard C: Genetic variation at the uncoupling protein 1, 2 and 3 loci and the response to long-term overfeeding.
Eur J Clin Nutr55
:1008
1015,2001[Medline]
- Himms-Hagen J, Harper ME: Physiological role of UCP3 may be export of fatty acids from mitochondria when fatty acid oxidation predominates: an hypothesis.
Exp Biol Med (Maywood)226
:78
84,2001[Abstract/Free Full Text]
- Jaburek M, Varecha M, Gimeno RE, Dembski M, Jezek P, Zhang M, Burn P, Tartaglia LA, Garlid KD: Transport function and regulation of mitochondrial uncoupling proteins 2 and 3.
J Biol Chem274
:26003
26007,1999[Abstract/Free Full Text]
- Oberkofler H, Liu YM, Esterbauer H, Hell E, Krempler F, Patsch W: Uncoupling protein-2 gene: reduced mRNA expression in intraperitoneal adipose tissue of obese humans.
Diabetologia41
:940
946,1998[Medline]
- Bao S, Kennedy A, Wojciechowski B, Wallace P, Ganaway E, Garvey WT: Expression of mRNAs encoding uncoupling proteins in human skeletal muscle: effects of obesity and diabetes.
Diabetes47
:1935
1940,1998[Abstract]
- 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 Invest100
:2665
2670,1997[Abstract/Free Full Text]
- Pinkney JH, Boss O, Bray GA, Bulmer K, Coppack SW, Mohamed-Ali V: Physiological relationships of uncoupling protein-2 gene expression in human adipose tissue in vivo.
J Clin Endocrinol Metab85
:2312
2317,2000[Abstract/Free Full Text]
- Nordfors L, Hoffstedt J, Nyberg B, Thorne A, Arner P, Schalling M, Lonnqvist F: Reduced gene expression of UCP2 but not UCP3 in skeletal muscle of human obese subjects.
Diabetologia41
:935
939,1998[Medline]
- Randle PJ GP, Hales CN, Newsholme EA: The glucose fatty-acid cycle: its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus.
Lancet1
:785
789,1963
- Le Stunff C, Fallin D, Schork NJ, Bougneres P: The insulin gene VNTR is associated with fasting insulin levels and development of juvenile obesity.
Nat Genet26
:444
446,2000[Medline]
- Sesti G, Cardellini M, Marini MA, Frontoni S, DAdamo M, Del Guerra S, Lauro D, De Nicolais P, Sbraccia P, Del Prato S, Gambardella S, Federici M, Marchetti P, Lauro R: A common polymorphism in the promoter of UCP2 contributes to the variation in insulin secretion in glucose-tolerant subjects.
Diabetes52
:1280
1283,2003[Abstract/Free Full Text]
- Matsuda M, DeFronzo RA: Insulin sensitivity indices obtained from oral glucose tolerance testing: comparison with the euglycemic insulin clamp.
Diabetes Care22
:1462
1470,1999[Abstract]
- Le Stunff C, Bougneres PF: Time course of increased lipid and decreased glucose oxidation during early phase of childhood obesity.
Diabetes42
:1010
1016,1993[Abstract]