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Lack of association between the uncoupling protein-2 Ala55Val gene polymorphism and phenotypic features of the Metabolic Syndrome
Biochimica et Biophysica Acta 1588 (2002) 103 – 105 www.bba-direct.com
Lack of association between the uncoupling protein-2 Ala55Val gene polymorphism and phenotypic features of the Metabolic Syndrome Roland Rosmond a,b,*, Claude Bouchard b, Per Bjo¨rntorp c a
Department of Clinical Chemistry, Sahlgrenska University Hospital, S-413 45 Go¨teborg, Sweden b Pennington Biomedical Research Center, Baton Rouge, LA, USA c Department of Cardiovascular Diseases, Sahlgrenska University Hospital, Go¨teborg, Sweden Received 13 February 2002; received in revised form 29 May 2002; accepted 3 June 2002
Abstract The uncoupling protein (UCP) 2 gene is expressed in adipose tissues and skeletal muscles, which are important sites for variations in energy expenditure. The objective of the current study was to examine the potential impact of a C!T substitution in exon 4, resulting in an alanine to valine substitution at codon 55, on the Metabolic Syndrome in 284 unrelated Swedish men born in 1944. The subjects were genotyped using PCR amplification of the exon 4 region of the UCP2 gene followed by digestion with the restriction enzyme EclHK1. The allelic frequencies were 0.56 for allele Ala and 0.44 for allele Val. No association was found between the Ala55Val SNP and obesity and blood levels of insulin, glucose, and lipids as well as blood pressure and circulating hormones. From these data, we conclude that the C!T substitution in exon 4 of the UCP2 gene does not contribute to the predisposition to be affected by the Metabolic Syndrome. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Ala55Val; Obesity; UCP2; SNP; Hormone; Lipid; Blood pressure
1. Introduction The mitochondrial uncoupling proteins (UCP) are thought to play an important role in the molecular basis for energy expenditure. However, the precise function of these proteins in human metabolism is still unclear [1]. In newborn infants, UCP1 is potentially a major contributor to the energy expenditure [1], but is unlikely to be involved in weight regulation in adult humans. In 1997, two new mitochondrial UCPs, UCP2 and UCP3, were discovered. It has been proposed that UCP2 and UCP3 have thermogenic properties and that they may thus be involved in energy metabolism [2,3]. The UCP2 –UCP3 gene cluster maps to 11q13 in humans [4]. A number of single nucleotide polymorphisms (SNPs) in the UCP1, UCP2 and UCP3 genes have been identified, and they have been studied for linkages and associations with obesity traits [1]. In Pima Indians, a C!T substitution in exon 4 of the UCP2 gene,
*
Corresponding author. Department of Clinical Chemistry, Sahlgrenska University Hospital, S-413 45 Go¨teborg, Sweden. Tel.: +46-31-3426272; fax: +46-31-82-8458. E-mail address: [email protected] (R. Rosmond).
resulting in an alanine to valine substitution at codon 55, has been found to be associated with metabolic rate during sleep and over 24 h [4]. In addition, the valine substitution in the homozygous state exhibits an enhanced metabolic efficiency and a lower fat oxidation rate [5]. However, several studies do not support an impact of the UCP2 Ala55Val SNP on either obesity or Type 2 diabetes [1]. There is a clustering of risk factors, including obesity, hypertension, glucose intolerance, diabetes mellitus, and hyperlipidemia, which is observed more frequently in some individuals than by chance alone. This has led to the suggestion that this cluster represents a single syndrome, which has been referred to as the Metabolic Syndrome [6]. Hence, the objective of the present study was to evaluate the potential impact of the Ala55Val variant of the UCP2 gene on obesity and blood levels of insulin, glucose, and lipids as well as blood pressure and circulating hormones, including salivary cortisol.
2. Subjects and methods The subjects (n=284) were randomly selected from a larger geographically defined total population cohort of men
0925-4439/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 4 3 9 ( 0 2 ) 0 0 1 5 2 - 7
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R. Rosmond et al. / Biochimica et Biophysica Acta 1588 (2002) 103–105
born in Gothenburg, Sweden in 1944. The design has been described elsewhere [7]. All subjects gave written informed consent before participating in the study, which was approved by the Ethics Committee of Go¨teborg University. Body mass index (BMI, kg/m2), waist-to-hip ratio (WHR), and abdominal sagittal diameter were measured as described previously [7]. Salivary cortisol was measured repeatedly over a random working day. Endocrine measurements, beside cortisol, included testosterone, insulin-like growth factor I, and leptin as described previously [7]. Blood levels of insulin, glucose, triglycerides, total, high (HDL) and low (LDL) density lipoprotein cholesterol were measured in the overnight fasting state as described previously [7]. The serum lipids were determined by an enzymatic procedure in a Cobas Fara II (Roche Molecular Biochemicals, Mannheim, Germany). Two blood pressure readings were recorded on the right arm with the participants sitting, using a random-zero mercury sphygmomanometer, after 5 min resting, with the auscultation site at heart level, a peak inflation level of 30 mm Hg above radial pulse disappearance, and a cuff-deflation rate of 2– 3 mm Hg/s. Values were recorded to the nearest even digit. Heart rate was recorded simultaneously, and systolic and diastolic blood pressure was calculated as the mean of the two measurements. Genotyping of the DNA sequence variant in exon 4 was carried out by PCR and restriction enzyme digestion. Genomic leukocyte DNA (150 ng in a final volume of 10 Al) was amplified by PCR (annealing temperature: 56 jC) using primers described previously [4]. The PCR reaction product was digested at 37 jC for 3 – 4 h with 2 U of the restriction enzyme EclHK1, and the fragments were separated on a 3% agarose gel. All statistical analyses were performed using SPSS for Windows, release 10.0 (SPSS Inc., Chicago, IL). P values are two-sided throughout, and a P<0.05 was considered significant. Analyses were carried out with the General Linear Model, with genotype as independent factor and BMI and WHR as covariates whenever appropriate. All P values were adjusted for multiple tests by using the Spjotvoll – Stoline post hoc correction [8].
3. Results and discussion The frequency of the Ala allele was 0.56 and that of the Val allele 0.44. The observed genotype frequencies were 29.3%, 54.2% and 16.5% for Ala/Ala, Ala/Val and Val/Val, respectively. Genotype frequencies were in a Hardy– Weinberg equilibrium (v2=1.6, df=2). The allelic frequencies are in accordance with those observed in other studies on Caucasian populations [9,10]. In the present study, there were no significant differences between the genotype groups with respect to phenotypes of obesity (BMI) and body fat distribution (WHR and abdominal sagittal diameter). Interestingly, the UCP2 knock-out
mice is neither obese nor resistant to diet-induced obesity [11]. In fact, these mice have a normal body temperature regulation, suggesting that UCP2 is not vital in the maintenance of normal body weight [11]. However, it has been recently shown that UCP2-deficient ob/ob mice have increased serum insulin levels, and greatly decreased levels of glycemia [12]. In contrast, the results of our study indicate no significant difference in serum insulin levels and fasting glucose, either before or after adjusting for BMI and WHR, among the genotypes of UCP2 Ala55Val. Moreover, our data indicate no significant differences between the genotype groups regarding salivary cortisol, testosterone, insulin-like growth factor I, leptin, serum lipids and blood pressure (Table 1). Similar findings have previously been reported in another Swedish sample [10]. Although one study related the Ala55Val polymorphism to measures of energy expenditure [5], the majority of studies do not support a functional impact of the Ala55Val polymorphism on polygenic obesity or Type 2 diabetes [1]. In conclusion, the Ala55Val UCP2 SNP is not associated with any of the phenotypic features of the Metabolic Syndrome in a general Swedish population of middle-aged men. The Ala55Val variant might, however, influence different components of the Metabolic Syndrome in other ethnic groups.
Table 1 Differences in anthropometric, endocrine, metabolic and circulatory measurements between genotypes of the Ala55Val SNP in exon 4 of the UCP2 gene Genotypes Ala55Ala (n=78) BMI (kg/m2) WHR Abdominal sagittal diameter (cm) Dexamethasone suppression test (nmol/l) Testosterone (nmol/l) Insulin-like growth factor I (Ag/l) Leptin (Ag/l) Fasting insulin (mU/l) Fasting glucose (mmol/l) Triglycerides (mmol/l) Total cholesterol (mmol/l) High-density lipoprotein cholesterol (mmol/l) Low-density lipoprotein cholesterol (mmol/l) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Heart rate (beats/min)
Ala55Val (n=144)
Val55Val (n=44)
26.5F4.2 0.94F0.09 22.8F4.0
26.1F3.8 0.94F0.06 22.7F3.5
25.7F3.8 0.93F0.06 22.3F3.5
11.3F4.4
12.1F5.7
13.2F5.5
19.5F5.2 214.6F67.9
19.8F5.6 199.5F64.7
19.6F5.7 209.6F61.1
6.3F4.7 14.2F14.0 4.5F1.0 1.8F1.1 6.1F1.1 1.3F0.3
6.2F4.2 11.4F7.0 4.6F0.9 1.8F1.1 6.2F1.0 1.2F0.3
5.7F3.8 14.2F14.5 4.6F1.2 1.7F0.9 6.2F1.3 1.3F0.4
4.0F1.0
4.1F1.0
4.2F1.2
130.3F16.8
129.4F18.6
128.2F15.2
83.7F9.4
83.8F11.5
82.5F8.9
68.8F10.8
68.0F10.0
72.2F11.2
Data (meanFSD) were adjusted for BMI and WHR when appropriate. All P values are > 0.20.
R. Rosmond et al. / Biochimica et Biophysica Acta 1588 (2002) 103–105
Acknowledgements This study was supported by grants from the Swedish Medical Research Council (K97-19X-00251-35A) and the Pennington Biomedical Research Center. R. Rosmond would also like to acknowledge the Henning and Johan Throne-Holst Foundation for the support of a postdoctoral fellowship at the Pennington Biomedical Research Center. C. Bouchard is partially supported by the George A. Bray Chair in Nutrition.
References [1] L.T. Dalgaard, O. Pedersen, Uncoupling proteins: functional characteristics and role in the pathogenesis of obesity and Type II diabetes, Diabetologia 44 (2001) 946 – 965. [2] C. Fleury, M. Neverova, S. Collins, S. Raimbault, O. Champigny, C. Levi-Meyrueis, F. Bouillaud, M.F. Seldin, R.S. Surwit, D. Ricquier, C.H. Warden, Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia, Nat. Genet. 15 (1997) 269 – 272. [3] O. Boss, S. Samec, A. Paoloni-Giacobino, C. Rossier, A. Dulloo, J. Seydoux, P. Muzzin, J.P. Giacobino, Uncoupling protein-3: a new member of the mitochondrial carrier family with tissue-specific expression, FEBS Lett. 408 (1997) 39 – 42. [4] K. Walder, R.A. Norman, R.L. Hanson, P. Schrauwen, M. Neverova, C.P. Jenkinson, J. Easlick, C.H. Warden, C. Pecqueur, S. Raimbault, D. Ricquier, M.H. Silver, A.R. Shuldiner, G. Solanes, B.B. Lowell, W.K. Chung, R.L. Leibel, R. Pratley, E. Ravussin, Association between uncoupling protein polymorphisms (UCP2 – UCP3) and energy metabolism/obesity in Pima indians, Hum. Mol. Genet. 7 (1998) 1431 – 1435.
105
[5] A. Astrup, S. Toubro, L.T. Dalgaard, S.A. Urhammer, T.I. Sorensen, O. Pedersen, 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. Disord. 23 (1999) 1030 – 1034. [6] P. Bjo¨rntorp, The associations between obesity, adipose tissue distribution and disease, Acta Med. Scand., Suppl. 723 (1988) 121 – 134. [7] R. Rosmond, Y.C. Chagnon, G. Holm, M. Chagnon, L. Perusse, K. Lindell, B. Carlsson, C. Bouchard, P. Bjo¨rntorp, Hypertension in obesity and the leptin receptor gene locus, J. Clin. Endocrinol. Metab. 85 (2000) 3126 – 3131. [8] E. Spjotvoll, M. Stoline, An extension of the T-method of multiple comparison to include the cases with unequal sample sizes, J. Am. Stat. Assoc. 68 (1973) 976 – 978. [9] S.A. Urhammer, L.T. Dalgaard, T.I. Sorensen, A.M. Moller, T. Andersen, A. Tybjaerg-Hansen, T. Hansen, J.O. Clausen, H. Vestergaard, O. Pedersen, 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 40 (1997) 1227 – 1230. [10] M. Klannemark, M. Orho, L. Groop, No relationship between identified variants in the uncoupling protein 2 gene and energy expenditure, Eur. J. Endocrinol. 139 (1998) 217 – 223. [11] D. Arsenijevic, H. Onuma, C. Pecqueur, S. Raimbault, B.S. Manning, B. Miroux, E. Couplan, M.C. Alves-Guerra, M. Goubern, R. Surwit, F. Bouillaud, D. Richard, S. Collins, D. Ricquier, Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production, Nat. Genet. 26 (2000) 435 – 439. [12] C.Y. Zhang, G. Baffy, P. Perret, S. Krauss, O. Peroni, D. Grujic, T. Hagen, A.J. Vidal-Puig, O. Boss, Y.B. Kim, X.X. Zheng, M.B. Wheeler, G.I. Shulman, C.B. Chan, B.B. Lowell, Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes, Cell 105 (2001) 745 – 755.
Lack of association between the uncoupling protein-2 Ala55Val gene polymorphism and phenotypic features of the Metabolic Syndrome Roland Rosmond a,b,*, Claude Bouchard b, Per Bjo¨rntorp c a
Department of Clinical Chemistry, Sahlgrenska University Hospital, S-413 45 Go¨teborg, Sweden b Pennington Biomedical Research Center, Baton Rouge, LA, USA c Department of Cardiovascular Diseases, Sahlgrenska University Hospital, Go¨teborg, Sweden Received 13 February 2002; received in revised form 29 May 2002; accepted 3 June 2002
Abstract The uncoupling protein (UCP) 2 gene is expressed in adipose tissues and skeletal muscles, which are important sites for variations in energy expenditure. The objective of the current study was to examine the potential impact of a C!T substitution in exon 4, resulting in an alanine to valine substitution at codon 55, on the Metabolic Syndrome in 284 unrelated Swedish men born in 1944. The subjects were genotyped using PCR amplification of the exon 4 region of the UCP2 gene followed by digestion with the restriction enzyme EclHK1. The allelic frequencies were 0.56 for allele Ala and 0.44 for allele Val. No association was found between the Ala55Val SNP and obesity and blood levels of insulin, glucose, and lipids as well as blood pressure and circulating hormones. From these data, we conclude that the C!T substitution in exon 4 of the UCP2 gene does not contribute to the predisposition to be affected by the Metabolic Syndrome. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Ala55Val; Obesity; UCP2; SNP; Hormone; Lipid; Blood pressure
1. Introduction The mitochondrial uncoupling proteins (UCP) are thought to play an important role in the molecular basis for energy expenditure. However, the precise function of these proteins in human metabolism is still unclear [1]. In newborn infants, UCP1 is potentially a major contributor to the energy expenditure [1], but is unlikely to be involved in weight regulation in adult humans. In 1997, two new mitochondrial UCPs, UCP2 and UCP3, were discovered. It has been proposed that UCP2 and UCP3 have thermogenic properties and that they may thus be involved in energy metabolism [2,3]. The UCP2 –UCP3 gene cluster maps to 11q13 in humans [4]. A number of single nucleotide polymorphisms (SNPs) in the UCP1, UCP2 and UCP3 genes have been identified, and they have been studied for linkages and associations with obesity traits [1]. In Pima Indians, a C!T substitution in exon 4 of the UCP2 gene,
*
Corresponding author. Department of Clinical Chemistry, Sahlgrenska University Hospital, S-413 45 Go¨teborg, Sweden. Tel.: +46-31-3426272; fax: +46-31-82-8458. E-mail address: [email protected] (R. Rosmond).
resulting in an alanine to valine substitution at codon 55, has been found to be associated with metabolic rate during sleep and over 24 h [4]. In addition, the valine substitution in the homozygous state exhibits an enhanced metabolic efficiency and a lower fat oxidation rate [5]. However, several studies do not support an impact of the UCP2 Ala55Val SNP on either obesity or Type 2 diabetes [1]. There is a clustering of risk factors, including obesity, hypertension, glucose intolerance, diabetes mellitus, and hyperlipidemia, which is observed more frequently in some individuals than by chance alone. This has led to the suggestion that this cluster represents a single syndrome, which has been referred to as the Metabolic Syndrome [6]. Hence, the objective of the present study was to evaluate the potential impact of the Ala55Val variant of the UCP2 gene on obesity and blood levels of insulin, glucose, and lipids as well as blood pressure and circulating hormones, including salivary cortisol.
2. Subjects and methods The subjects (n=284) were randomly selected from a larger geographically defined total population cohort of men
0925-4439/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 4 3 9 ( 0 2 ) 0 0 1 5 2 - 7
104
R. Rosmond et al. / Biochimica et Biophysica Acta 1588 (2002) 103–105
born in Gothenburg, Sweden in 1944. The design has been described elsewhere [7]. All subjects gave written informed consent before participating in the study, which was approved by the Ethics Committee of Go¨teborg University. Body mass index (BMI, kg/m2), waist-to-hip ratio (WHR), and abdominal sagittal diameter were measured as described previously [7]. Salivary cortisol was measured repeatedly over a random working day. Endocrine measurements, beside cortisol, included testosterone, insulin-like growth factor I, and leptin as described previously [7]. Blood levels of insulin, glucose, triglycerides, total, high (HDL) and low (LDL) density lipoprotein cholesterol were measured in the overnight fasting state as described previously [7]. The serum lipids were determined by an enzymatic procedure in a Cobas Fara II (Roche Molecular Biochemicals, Mannheim, Germany). Two blood pressure readings were recorded on the right arm with the participants sitting, using a random-zero mercury sphygmomanometer, after 5 min resting, with the auscultation site at heart level, a peak inflation level of 30 mm Hg above radial pulse disappearance, and a cuff-deflation rate of 2– 3 mm Hg/s. Values were recorded to the nearest even digit. Heart rate was recorded simultaneously, and systolic and diastolic blood pressure was calculated as the mean of the two measurements. Genotyping of the DNA sequence variant in exon 4 was carried out by PCR and restriction enzyme digestion. Genomic leukocyte DNA (150 ng in a final volume of 10 Al) was amplified by PCR (annealing temperature: 56 jC) using primers described previously [4]. The PCR reaction product was digested at 37 jC for 3 – 4 h with 2 U of the restriction enzyme EclHK1, and the fragments were separated on a 3% agarose gel. All statistical analyses were performed using SPSS for Windows, release 10.0 (SPSS Inc., Chicago, IL). P values are two-sided throughout, and a P<0.05 was considered significant. Analyses were carried out with the General Linear Model, with genotype as independent factor and BMI and WHR as covariates whenever appropriate. All P values were adjusted for multiple tests by using the Spjotvoll – Stoline post hoc correction [8].
3. Results and discussion The frequency of the Ala allele was 0.56 and that of the Val allele 0.44. The observed genotype frequencies were 29.3%, 54.2% and 16.5% for Ala/Ala, Ala/Val and Val/Val, respectively. Genotype frequencies were in a Hardy– Weinberg equilibrium (v2=1.6, df=2). The allelic frequencies are in accordance with those observed in other studies on Caucasian populations [9,10]. In the present study, there were no significant differences between the genotype groups with respect to phenotypes of obesity (BMI) and body fat distribution (WHR and abdominal sagittal diameter). Interestingly, the UCP2 knock-out
mice is neither obese nor resistant to diet-induced obesity [11]. In fact, these mice have a normal body temperature regulation, suggesting that UCP2 is not vital in the maintenance of normal body weight [11]. However, it has been recently shown that UCP2-deficient ob/ob mice have increased serum insulin levels, and greatly decreased levels of glycemia [12]. In contrast, the results of our study indicate no significant difference in serum insulin levels and fasting glucose, either before or after adjusting for BMI and WHR, among the genotypes of UCP2 Ala55Val. Moreover, our data indicate no significant differences between the genotype groups regarding salivary cortisol, testosterone, insulin-like growth factor I, leptin, serum lipids and blood pressure (Table 1). Similar findings have previously been reported in another Swedish sample [10]. Although one study related the Ala55Val polymorphism to measures of energy expenditure [5], the majority of studies do not support a functional impact of the Ala55Val polymorphism on polygenic obesity or Type 2 diabetes [1]. In conclusion, the Ala55Val UCP2 SNP is not associated with any of the phenotypic features of the Metabolic Syndrome in a general Swedish population of middle-aged men. The Ala55Val variant might, however, influence different components of the Metabolic Syndrome in other ethnic groups.
Table 1 Differences in anthropometric, endocrine, metabolic and circulatory measurements between genotypes of the Ala55Val SNP in exon 4 of the UCP2 gene Genotypes Ala55Ala (n=78) BMI (kg/m2) WHR Abdominal sagittal diameter (cm) Dexamethasone suppression test (nmol/l) Testosterone (nmol/l) Insulin-like growth factor I (Ag/l) Leptin (Ag/l) Fasting insulin (mU/l) Fasting glucose (mmol/l) Triglycerides (mmol/l) Total cholesterol (mmol/l) High-density lipoprotein cholesterol (mmol/l) Low-density lipoprotein cholesterol (mmol/l) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Heart rate (beats/min)
Ala55Val (n=144)
Val55Val (n=44)
26.5F4.2 0.94F0.09 22.8F4.0
26.1F3.8 0.94F0.06 22.7F3.5
25.7F3.8 0.93F0.06 22.3F3.5
11.3F4.4
12.1F5.7
13.2F5.5
19.5F5.2 214.6F67.9
19.8F5.6 199.5F64.7
19.6F5.7 209.6F61.1
6.3F4.7 14.2F14.0 4.5F1.0 1.8F1.1 6.1F1.1 1.3F0.3
6.2F4.2 11.4F7.0 4.6F0.9 1.8F1.1 6.2F1.0 1.2F0.3
5.7F3.8 14.2F14.5 4.6F1.2 1.7F0.9 6.2F1.3 1.3F0.4
4.0F1.0
4.1F1.0
4.2F1.2
130.3F16.8
129.4F18.6
128.2F15.2
83.7F9.4
83.8F11.5
82.5F8.9
68.8F10.8
68.0F10.0
72.2F11.2
Data (meanFSD) were adjusted for BMI and WHR when appropriate. All P values are > 0.20.
R. Rosmond et al. / Biochimica et Biophysica Acta 1588 (2002) 103–105
Acknowledgements This study was supported by grants from the Swedish Medical Research Council (K97-19X-00251-35A) and the Pennington Biomedical Research Center. R. Rosmond would also like to acknowledge the Henning and Johan Throne-Holst Foundation for the support of a postdoctoral fellowship at the Pennington Biomedical Research Center. C. Bouchard is partially supported by the George A. Bray Chair in Nutrition.
References [1] L.T. Dalgaard, O. Pedersen, Uncoupling proteins: functional characteristics and role in the pathogenesis of obesity and Type II diabetes, Diabetologia 44 (2001) 946 – 965. [2] C. Fleury, M. Neverova, S. Collins, S. Raimbault, O. Champigny, C. Levi-Meyrueis, F. Bouillaud, M.F. Seldin, R.S. Surwit, D. Ricquier, C.H. Warden, Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia, Nat. Genet. 15 (1997) 269 – 272. [3] O. Boss, S. Samec, A. Paoloni-Giacobino, C. Rossier, A. Dulloo, J. Seydoux, P. Muzzin, J.P. Giacobino, Uncoupling protein-3: a new member of the mitochondrial carrier family with tissue-specific expression, FEBS Lett. 408 (1997) 39 – 42. [4] K. Walder, R.A. Norman, R.L. Hanson, P. Schrauwen, M. Neverova, C.P. Jenkinson, J. Easlick, C.H. Warden, C. Pecqueur, S. Raimbault, D. Ricquier, M.H. Silver, A.R. Shuldiner, G. Solanes, B.B. Lowell, W.K. Chung, R.L. Leibel, R. Pratley, E. Ravussin, Association between uncoupling protein polymorphisms (UCP2 – UCP3) and energy metabolism/obesity in Pima indians, Hum. Mol. Genet. 7 (1998) 1431 – 1435.
105
[5] A. Astrup, S. Toubro, L.T. Dalgaard, S.A. Urhammer, T.I. Sorensen, O. Pedersen, 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. Disord. 23 (1999) 1030 – 1034. [6] P. Bjo¨rntorp, The associations between obesity, adipose tissue distribution and disease, Acta Med. Scand., Suppl. 723 (1988) 121 – 134. [7] R. Rosmond, Y.C. Chagnon, G. Holm, M. Chagnon, L. Perusse, K. Lindell, B. Carlsson, C. Bouchard, P. Bjo¨rntorp, Hypertension in obesity and the leptin receptor gene locus, J. Clin. Endocrinol. Metab. 85 (2000) 3126 – 3131. [8] E. Spjotvoll, M. Stoline, An extension of the T-method of multiple comparison to include the cases with unequal sample sizes, J. Am. Stat. Assoc. 68 (1973) 976 – 978. [9] S.A. Urhammer, L.T. Dalgaard, T.I. Sorensen, A.M. Moller, T. Andersen, A. Tybjaerg-Hansen, T. Hansen, J.O. Clausen, H. Vestergaard, O. Pedersen, 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 40 (1997) 1227 – 1230. [10] M. Klannemark, M. Orho, L. Groop, No relationship between identified variants in the uncoupling protein 2 gene and energy expenditure, Eur. J. Endocrinol. 139 (1998) 217 – 223. [11] D. Arsenijevic, H. Onuma, C. Pecqueur, S. Raimbault, B.S. Manning, B. Miroux, E. Couplan, M.C. Alves-Guerra, M. Goubern, R. Surwit, F. Bouillaud, D. Richard, S. Collins, D. Ricquier, Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production, Nat. Genet. 26 (2000) 435 – 439. [12] C.Y. Zhang, G. Baffy, P. Perret, S. Krauss, O. Peroni, D. Grujic, T. Hagen, A.J. Vidal-Puig, O. Boss, Y.B. Kim, X.X. Zheng, M.B. Wheeler, G.I. Shulman, C.B. Chan, B.B. Lowell, Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes, Cell 105 (2001) 745 – 755.