International Journal of Obesity (1999) 23, Suppl 6, S46±S48 ß 1999 Stockton Press All rights reserved 0307±0565/99 $12.00 http://www.stockton-press.co.uk/ijo
Genetics of uncoupling proteins in humans C Warden1,* 1
Rowe Program in Human Genetics, University of California at Davis, Davis, California 95616, USA
Genetic studies in humans provide a method to test hypotheses about the biological roles of speci®c genes. So far, ten published papers have chosen to examine the hypothesis that uncoupling protein-2 (UCP2) and=or UCP3 in¯uence energy expenditure and=or body fat accumulation. These genes were chosen because they are candidate energy expenditure genes, based on their homology to UCP1. Studies of UCP2 and UCP3 are intrinsically intertwined because the two genes are separated by only 6000 base pairs on human chromosome 11. Linkage studies in families have suggested that UCP2 and=or UCP3, or a closely linked gene, may in¯uence resting metabolic rate (RMR) Some association studies using a 30 untranslated region insertion=deletion variant of UCP2 have produced statistically positive evidence for association with body mass index (BMI) and RMR. In contrast, association studies of UCP2 using an Ala to Val variant at amino acid 55 have produced negative results. Positive results have also been reported for association of a UCP3 splice variant with respiratory quotient in African Americans. In addition, no studies have reported linkage or association of UCP2 or UCP3 with diabetes. Overall, the results suggest that some variants of UCP2 and UCP3 may be associated with obesity traits in some populations. The UCPs, to date, show positive results in associations with obesity traits but not with diabetes traits. Further work will be needed to settle the role of UCP2 and UCP3 alleles in human body weight regulation. Keywords: linkage; human; obesity; diabetes; metabolic rate
Introduction As other reviews in this supplement discuss, proton leaks exist, are regulated, and vary between tissues and species.1 However, the molecular bases of these proton leaks remain mostly unknown. Genetic approaches examine the importance of speci®c genes on traits, regardless of their function by searching for co-incidence of traits and genes. Furthermore, genetic assays test for lifetime effects of speci®c alleles on traits, therefore the effects of any one gene may be small at any one time, but may be large when summed over a period of time. Genetic studies can detect effects of genes causing small effects over years. This is ideal for obesity genes that may promote obesity by decreasing resting metabolic rate (RMR) by as little as one percent over a lifetime.
Energy balance and UCPs The most basic principle of an organism's weight regulation is that changes in body weight result from an imbalance between energy intake and energy expenditure. Energy expenditure is a complex trait, which includes the RMR, the thermic effect of food and the energy expenditure of activity.2 Several human studies suggest that RMR has a genetic
*Correspondence: Dr C Warden, Rowe, Program in Human Genetics, University of California at Davis, Davis, California 95616, USA. E-mail:
[email protected]
component.2 Longitudinal studies of Pima Indians show that people with low RMRs are likely to gain more weight than people with high RMRs.3 However, the molecular bases for RMR and for individual differences in RMR are uncertain. Many hypotheses exist for the molecular basis for calorie partitioning. The recent discovery of widely distributed and regulated mitochondrial uncoupling proteins (UCPs) opens up new possibilities. The UCPs allow partial uncoupling of the mitochondrial proton gradient from ATP production. Therefore, increased UCP activity may increase energy expenditure through increased heat loss and thus decrease body weight. The three mammalian UCPs include UCP1, UCP2, and UCP3. The UCPs share some structural elements and all partially uncouple mitochondrial respiration, but they differ greatly in tissue distribution and regulation. The UCPs probably have different physiological roles and vary in potential value for drug targeting of obesity. To date, ten published studies examine the UCP2 and UCP3 genes for linkage or association with diabetes or obesity.4 ± 13 These studies study a diverse collection of ethnic groups, examine a variety of alleles, and apply widely divergent ascertainment schemes. Therefore, it is dif®cult to combine data from the whole set, although some patterns are apparent.
Linkage of the UCP2- UCP3 locus to obesity traits Linkage of the UCP2- UCP3 locus to RMR has been examined in more than 600 French Canadians
UCPs in humans C Warden Table 1
S47
Summary of linkage and association studies for UCP2 and UCP3
Study type
Ascertainment
Alleles
Results
Linkage
French Canadian
D11S916, D11S1321
American Caucasian NIDDM
7 markers on chromosome 11 UCP2 (A55V, ID) UCP3 (C ± T) UCP2 (A55V, A232T) A55V
RMR P < 0.000002 no linkage to BMI No linkage to BMI or WHR
Association
Pima Indians Japanese NIDDM or obese Random Danish young adults American Caucasian and African American Obese Swedish with low BMR and healthy controls French NIDDM, morbidly obese or healthy controls Caucasian and African American Random Danish young adults
Reference 11 12
A55V
Sleeping Metabolic Rate with A55V, BMI in Pimas > 45 y old with ID No association with NIDDM or obesity No association to BMI, WHR, fat mass, weight gain, or insulin. No signi®cant associations
5
A55V
No association with BMR
6
6 UCP2 alleles, including A55V, and exon 8 ID UCP3 splice
No signi®cant associations
4
Respiratory quotient
8
UCP3 (G84S)
No signi®cant association with BMI
9
10 13 7
NIDDM non-insulin dependent diabetes mellitus; RMR resting metabolic rate; BMI body mass index; WHR waist to hip ratio; ID BMR Basol metabolic rate
participating in the Quebec Family Study.11 Three microsatellite markers surrounding the UCP2-UCP3 locus were genotyped. Relative pair analysis determined linkage to RMR (Table 1). The human UCP2UCP3 locus linked to resting metabolic rate with a Pvalue of 0.000002.11 This linkage suggests that UCP2, UCP3 or another closely linked gene in¯uences RMR in humans.
Association studies of UCP2-UCP3 Sequencing of human UCP2 reveals several polymorphic sites. Two of these sites are very polymorphic: 1) an arginine for valine substitution at amino acid 55 in exon 4 (A55V) and 2) a 45 base pair insertion=deletion in the untranslated region of exon 8. Association studies using these alleles, as well as others, are summarized in Table 1. A study of 82 unrelated Pima Indians found an association between UCP2 A55V and metabolic rate during sleep (P 0.007). A silent UCP3 variant (C - T in exon 3) exhibited no associations with BMI or metabolic rate. Study of a further 790 full-blooded Pima Indians revealed that individuals > 45 years of age who were UCP2 exon 8 insertion=deletion heterozygotes had the lowest body mass indices (BMIs) (P 0.04).5 In contrast, a study of morbidly obese French identi®ed six UCP2 variants (2 alleles in exons 1 and 4, and 1 allele each in exons 2 and 8).4 Comparison of allele frequencies in morbidly obese and normal controls failed to reveal association with any of the six alleles. Examination of 966 people, including many morbidly obese, for the exon 8 insertion=deletion variant did not reveal any association with weight gain, BMI, RMR or body composition characteristics.
Two studies using Danish Caucasians have examined UCP2 and UCP3 association.1314 These studies searched for alleles in 35 non-insulin dependent diabetes mellitus (NIDDM) patients and then screened for genotype-phenotype association in 380 randomly recruited healthy young 18 ± 32 y old individuals. These studies did not ®nd any association with BMI and A55V or the UCP3 Glycine to Serine at amino acid 84 variant. In a study from Finland, 55 patients with MSDR (metabolic syndrome of diabetes resistance) and 46 healthy controls were examined for the frequency of the UCP2 A55V variant.6 No association was found with BMR. A study from Japan examined 210 people with NIDDM, 42 obese individuals and 218 normal controls for association with UCP2 A55V.10 No differences in allele frequency were found in the three groups. This study also reported an Ala232 to Thr allele, but found no association with BMR, nor did this allele exhibit functional activity different from the wild type allele when expressed in yeast. A study of Caucasians and African Americans identi®ed 3 variants in UCP3: a missense polymorphism in exon 3 (V102I), a stop codon in exon 4 (R143X), and a polymorphism of a splice donor junction of exon 6 that results in exclusive production of UCP3S.8 The V120I and R143X alleles were rare, but were identi®ed in individuals with early-onset severe obesity. The exon 6 splice donor variant was found only in African Americans and was associated with reduced fat oxidation (P 0.018) and increased respiratory quotient (P 0.008).
Conclusion Some patterns are apparent from published results for UCP2 and UCP3 association and linkage studies. The
UCPs in humans C Warden
S48
published data suggest that alleles of UCP2 do not in¯uence diabetes. Further, the common UCP2 exon 4 variant, Ala to Val variant at amino acid 55 (A55V) does not exhibit association to obesity in any published work. In contrast, the UCP2 exon 8 insertion=deletion variant sometimes shows associations to obesity, especially in populations ascertained for obesity. In populations ascertained for diabetes, the results are mixed. Therefore, UCP2 remains a candidate obesity gene. Fewer studies have been published with UCP3 association, but current data suggest that a splice variant eliminating the long form may in¯uence fat oxidation. Overall, the results suggest that association studies of UCP2 are most productive when conducted in populations ascertained for obesity, and that one should look particularly for effects of the UCP2 45 base pair insertion=deletion in the untranslated region of exon 8 variant. These results need to be followed by studies of the insertion=deletion variant effect on UCP2 protein levels. Several rare alleles of UCP2 and UCP3 have been identi®ed in individuals with obesity or NIDDM. These may warrant further functional studies or examination for association with obesity in additional populations. Acknowledgements
The author thanks Juliet Easlick and Janis Fisler for careful reading of the manuscript. References
1 Rolfe DF, Hulbert AJ, Brand MD. Characteristics of mitochondrial proton leak and control of oxidative phosphorylation in the major oxygen-consuming tissues of the rat. Biochim Biophys Acta 1994; 1188: 405 ± 416. 2 Bouchard C, DespreÂs J-P, Tremblay A. Genetics of obesity and human energy metabolism Proc Nutr Soc 1991; 50: 139 ± 147. 3 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. 4 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 UCP2 gene in normoglycemic and NIDDM morbidly obese patients: lack of association between new UCP 2 polymorphisms and obesity in French Caucasians. Diabetes 1998; 47: 840 ± 842.
5 Walder K, Norman RA, Hanson RL, Schrauwen P, Neverova M, Jenkinson CP, Easlick J, Warden CH, Pecqueur C, Raimbault S, Ricquier D, Silver MHK, Shuldiner AR, Solanes G, Lowell BB, Chung WK, Leibel RL, Pratley R, Ravussin E. Association between uncoupling protein polymorphisms (UCP2 ± UCP3) and energy metabolism=obesity in Pima indians. Hum Mol Genet 1998; 7: 1431 ± 1435. 6 Klannemark M, Orho M, Groop L. No relationship between identi®ed variants in the uncoupling protein 2 gene and energy expenditure. Eur J Endocrinol 1998; 139: 217 ± 223. 7 Argyropulos G, Brown AM, Peterson R, Likes CE, Watson DK, Garvey WT. Structure and organization of the human uncoupling protein 2 gene and identi®cation of a common biallelic variant in Caucasian and African- American subjects. Diabetes 1998; 47: 685 ± 687. 8 Argyropoulos G, Brown AM, Willi SM, Zhu J, He Y, Reitman M, Gevao SM, Spruill I, Timothy 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. 9 Urhammer SA, Dalgaard LT, Sorensen TI, TybjaergHansen A, Echwald SM, Andersen T, Clausen JO, Pedersen O. Organisation of the coding exons and mutational screening of the uncoupling protein 3 gene in subjects with juvenile-onset obesity. Diabetologia 1998; 41: 241 ± 244. 10 Kubota T, Mori H, Tamori Y, Okazawa H, Fukuda T, Miki M, Ito C, Fleury C, Bouillaud F, Kasuga M. Molecular screening of uncoupling protein 2 gene in patients with non insulindependent diabetes mellitus or obesity. J Clin Endocrinol Metab 1998; 83: 2800 ± 2804. 11 Bouchard C, Perusse L, Chagnon YC, Warden C, Ricquier D. Linkage between markers in the vicinity of the uncoupling protein 2 gene and resting metabolic rate in humans. Hum Mol Genet 1997; 6: 1887 ± 1889. 12 Elbein SC, Leppert M, Hasstedt S. Uncoupling protein 2 region on chromosome 11q13 is not linked to markers of obesity in familial type 2 diabetes. Diabetes 1997; 46: 2105 ± 2107. 13 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. Diabetologia 1997; 40: 1227 ± 1230. 14 Urhammer SA, Dalgaard LT, Sorensen TI, TybjaergHansen A, Echwald SM, Andersen T, Clausen JO, Pedersen O. Organization of the coding exons and mutational screening of the uncoupling protein 3 gene in subjects with juvenile-onset obesity. Diabetologia 1998; 41: 241 ± 244.
