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The Journal of Clinical Endocrinology & Metabolism 88(10):4862– 4866 Copyright © 2003 by The Endocrine Society doi: 10.1210/jc.2003-030173
The Gly163 Arg16 and Gln273 Glu27 Polymorphisms of ␤2-Adrenergic Receptor Are Associated with Metabolic Syndrome in Men JEAN DALLONGEVILLE, NICOLE HELBECQUE, DOMINIQUE COTTEL, PHILIPPE AMOUYEL, ALINE MEIRHAEGHE
Institut National de la Sante´ et de la Recherche Me´dicale, U-508, Institut Pasteur de Lille (J.D., N.H., D.C., P.A., A.M.), and Faculte´ de Me´decine, Universite´ Lille II (P.A.), 59019 Lille Cedex, France Endogenous catecholamines contribute to regulation of adipose tissue lipolysis, glucose homeostasis, and vascular tone. The goal of the present study was to assess the association between the Gly163 Arg16 and Gln273 Glu27 polymorphisms of the ␤2-adrenergic receptor and metabolic syndrome. Participants were recruited in a population survey and included 1195 men and women. Metabolic syndrome was defined according to National Cholesterol Education Program Adult Treatment Panel III guidelines. There were 276 patients with metabolic syndrome and 872 controls. The Gly163 Arg16 (P < 0.005) and Gln273 Glu27 (P < 0.04) polymorphisms were associated with metabolic syndrome in men, but not in women. In multivariate analyses adjusting for age, physical activity, smoking habits, alcohol consumption, and body mass index, the odds ratio of
metabolic syndrome was 1.83 (95% confidence interval, 1.10 – 3.05) and 2.43 (95% confidence interval, 1.19 – 4.95) in men bearing the Gly16/Arg16 and Arg16/Arg16 genotypes, respectively. Similarly, the odds ratios of metabolic syndrome were 0.99 (95% confidence interval, 0.50 –1.93) and 1.67 (95% confidence interval, 0.84 –3.33) in men bearing the Gln27/Glu27 and Gln27/ Gln27 genotypes, respectively. Because both variants were in linkage disequilibrium, a haplotype analysis was performed. There was no evidence of any statistically significant association between ␤2-adrenergic receptor haplotypes and metabolic syndrome. In conclusion, these data suggest that the Arg16 and Gln27 variants of the ␤2-adrenergic receptor gene contribute to metabolic syndrome susceptibility in men. (J Clin Endocrinol Metab 88: 4862– 4866, 2003)
domain of the receptor: Gly163 Arg16 and Gln273 Glu27 (8). Earlier studies have reported associations between the Gly163 Arg16 and Gln273 Glu27 polymorphisms and obesity, insulin resistance, dyslipidemia, and hypertension (9 –24). Because these pathophysiological disorders are major components of the metabolic syndrome, the Gly163 Arg16 and Gln273 Glu27 polymorphisms appear as possible genetic susceptibility markers for the metabolic syndrome. In the present study we used the National Cholesterol Education Program Adult Treatment Panel III (NCEP III) definition of metabolic syndrome (25) to analyze the association between the Gly163 Arg16 and Gln273 Glu27 polymorphisms and metabolic syndrome in a population-based study.
ETABOLIC SYNDROME IS a complex disease characterized by the clustering of several metabolic disorders (1, 2). Increased body weight, insulin resistance, alteration of plasma lipid levels and glucose homeostasis, and increased blood pressure are the principal components of the cluster. Environmental influences, such as low physical activity and inappropriate diet, play a major role in the development of metabolic syndrome. In addition, familial aggregation of metabolic disorders has been reported, suggesting a genetic component to the syndrome (3–5). Endogenous catecholamines are involved in the regulation of adipose tissue lipolysis, nonesterified fatty acid distribution, lipoprotein metabolism, glucose homeostasis, vascular tone, and blood pressure (6). In this respect, catecholamines control, directly or indirectly, several metabolic pathways of the metabolic syndrome. Catecholamines exert their effects through different ␤-adrenergic receptors (BAR). The BARs are members of the large family of G protein-coupled receptors. There are three BAR subtypes (BAR1, BAR2, and BAR2). The BAR2 is broadly expressed throughout tissues of the body where catecholamines exert their effects on metabolic systems. Therefore, BAR2 genes appear as potential candidate genes for metabolic syndrome. Several polymorphisms of the human BAR2 gene have been described (7). Among these, two common mutations result in the substitution of an amino acid in the extracellular Abbreviations: BAR, ␤-Adrenergic receptor; BMI, body mass index; HDL, high-density lipoprotein; NCEP, National Cholesterol Education Program; OR, odds ratio; SNP, single nucleotide polymorphism.
Subjects and Methods Subjects Participants were recruited in the framework of the WHO-MONICA Population Survey conducted from 1995–1997 in the urban community of Lille in the north of France. The sample included representative subjects, aged 35– 64 yr, stratified by town size, randomly selected from the electoral rolls to obtain 200 participants for each gender and each 10-yr age group [WHO-MONICA Project protocol (26)]. A total of 573 men and 575 women completed the recruitment procedure. The ethical committee of Lille University Hospital approved the protocol. After signing an informed consent, participants were administered a standard questionnaire, and physical measurements were made by a specially trained nurse. The questionnaire covered questions on socioeconomic factors, physical activity, alcohol consumption, smoking status, personal medical history, family history, attitudes, and knowledge concerning several diseases and current drug therapy. The level of physical activity was defined as walking or riding 15 min or more/d, lifting or carrying heavy objects at work daily, and/or participating in
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a sport or physical exercise more than 2 h/wk. Current cigarette smokers were defined as subjects reporting at least one cigarette per day. Total alcohol intake was expressed as the sum of ml alcohol per week from wine, beer, cider, and spirits. The anthropometric measurements included body weight (rounded to the nearest even decimal) and waist (at a level midway between lower rib margin and iliac crest; to the nearest 0.5 cm) and were made on subjects in light clothing without shoes. The body mass index (BMI) was calculated according to the Quetelet equation. Blood pressure was measured on the right arm with the subject in a sitting position and after a minimum 5-min rest, using a standard mercury sphygmomanometer. Two consecutive measures of systolic and diastolic blood pressures were recorded to the nearest 2 mm Hg. The second blood pressure recording was taken at least 1 min after the first one. The mean of the two blood pressure readings was used. Metabolic syndrome was defined, according to the NCEP III recommendations (25), by the presence of at least three or more of the following abnormalities: waist girth greater than 102 cm in men and greater than 88 cm in women, triglycerides 150 mg/dl or more or treatment with fibrates or fish oil, high density lipoprotein (HDL) cholesterol less than 40 mg/dl in men and less than 50 mg/dl in women, blood pressure of 130/85 mm Hg or more or treatment with blood pressure-lowering medications, and fasting glucose of 110 mg/dl or more or type II diabetes.
Methods A blood sample of 20 ml was drawn on disodium EDTA after the subjects had fasted for at least 10 h; samples were kept at room temperature and centrifuged within 4 h. Lipid and lipoprotein levels were measured in a central laboratory (Purpan Hospital Biochemical Laboratory, Toulouse, France). The quality of biological measurements was assessed within the framework of the MONICA Project. Glucose was measured by the glucose oxidase method (Dimension, DuPont, Wilmington, DE). Plasma insulin was measured by RIA (Bi-Insuline, RIA, Pasteur, Paris, France). Serum triglyceride and HDL cholesterol levels were measured enzymatically (DuPont Dimension). Leptin levels were measured in duplicate by RIA (human leptin RIA kit, Wak-Chemie Medical, Steinbach, Germany). Polymorphisms were detected as previously described (19).
Statistical analyses Linkage disequilibrium was assessed with SYSTAT version 3, 1986 (Systat, Inc., Evanston, IL). 2 analysis was used to compare the distribution of gene variants between the control and metabolic syndrome groups. Logistic regression analysis was used to calculate the odds ratio (OR) of metabolic syndrome in carriers of gene variants. Linkage disequilibrium was assessed using a standardized r coefficient (27). The haplotype frequencies were estimated using the myriad haplotype al-
gorithm described by MacLean and Morton (28) and implemented in a computer program by Cox et al. (29).
Table 1 shows the clinical characteristics of the subjects according to gender and metabolic syndrome status. According to the NCEP III definition, 26.2% of men and 21.9% of women had the metabolic syndrome. BMI, leptin, waist circumference, insulin, glucose, triglycerides, and blood pressure were higher, and HDL cholesterol was lower in both men and women with the metabolic syndrome than in those without the metabolic syndrome (controls). Table 2 shows the allele distributions of the Gly163 Arg16 and Gln273 Glu27 polymorphisms in men and women. Both polymorphisms were in Hardy-Weinberg equilibrium. The Arg16 and Gln27 variants were in allelic association, indicating a linkage disequilibrium (r2 ⫽ 0.42 and 0.45 in men and women, respectively). There was no difference in allelic distribution between men and women. Table 3 presents the Gly163 Arg16 and Gln273 Glu27 polymorphism distributions according to metabolic syndrome status and gender. In men, the Gly163 Arg16 (P ⬍ 0.005) and Gln273 Glu27 (P ⬍ 0.04) polymorphisms were associated with the metabolic syndrome. The prevalence of the metabolic syndrome increased progressively in men with the Gly16/Arg16 (29.0%) and Arg16/Arg16 (36.1%) genotypes compared with that in men who were Gly16/Gly16 (18.9%) TABLE 2. Distribution of BAR2 genotypes in men and women Gln27/Gln27
Men Gly16/Gly16 Gly16/Arg16 Arg16/Arg16 n (%)
28 95 72 195 (34.4)
92 188 0 280 (49.4)
92 0 0 92 (16.2)
212 (37.4) 283 (49.9) 72 (12.7)
Women Gly16/Gly16 Gly16/Arg16 Arg16/Arg16 n (%)
30 63 85 178 (31.7)
98 197 0 295 (52.5)
89 0 0 89 (15.8)
217 (38.6) 260 (46.3) 85 (15.1)
TABLE 1. Characteristics of the subjects with the metabolic syndrome and control Men
n (%) Age (yr) Weight (kg) BMI (kg/m2) Waist girth (cm) Leptin (ng/ml) Glucose (mg/dl) Insulin (U/ml) Triglycerides (mg/dl) HDL cholesterol (mg/dl) Systolic BP (mm Hg) Diastolic BP (mm Hg) Data are the mean ⫾ Blood pressure. a P ⬍ 0.01. b P ⬍ 0.0001.
423 (73.8) 50.5 ⫾ 8.6 76.3 ⫾ 11.2 25.4 ⫾ 3.3 92.7 ⫾ 8.9 7.7 ⫾ 6.2 97.6 ⫾ 15.4 10.1 ⫾ 5.1 111.7 ⫾ 71.3 55.5 ⫾ 15.6 133.4 ⫾ 18.6 83.4 ⫾ 11.2
150 (26.2) 52.9 ⫾ 8.7a 90.8 ⫾ 14.4b 30.2 ⫾ 4.2b 106.6 ⫾ 9.7b 14.0 ⫾ 9.1b 120.9 ⫾ 40.8b 18.2 ⫾ 14.2b 271.0 ⫾ 338.2b 40.4 ⫾ 10.5b 145.8 ⫾ 18.2b 90.9 ⫾ 12.1b
449 (78.1) 50.4 ⫾ 8.2 64.7 ⫾ 12.3 25.0 ⫾ 4.5 81.1 ⫾ 11.3 21.1 ⫾ 12.8 91.8 ⫾ 14.3 10.4 ⫾ 4.5 82.9 ⫾ 36.6 68.2 ⫾ 17.5 126.2 ⫾ 16.7 78.6 ⫾ 10.1
126 (21.9) 54.6 ⫾ 7.7b 82.6 ⫾ 15.7b 32.1 ⫾ 6.0b 82.6 ⫾ 15.7b 33.1 ⫾ 14.3b 124.7 ⫾ 50.0b 15.7 ⫾ 8.2b 204.7 ⫾ 287.4b 50.2 ⫾ 15.6b 146.2 ⫾ 18.7b 87.2 ⫾ 10.0b
percentages are in parentheses. A t test was used for comparison between controls and metabolic syndrome. BP,
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TABLE 3. Distribution of BAR2 Gly16⬎Arg16 and Gln27⬎Glu27 genotypes in controls and metabolic syndrome Men
Gly16/Gly16 Gly16/Arg16 Arg16/Arg16 Gln27/Gln27 Gln27/Glu27 Glu27/Glu27
172 (41.0) 201 (47.9) 46 (10.1) 131 (31.3) 217 (51.8) 71 (16.9)
40 (27.0) 82 (55.4) 26 (17.6) 64 (43.2) 63 (42.6) 21 (14.2)
168 (38.4) 203 (46.3) 67 (15.3) 141 (32.2) 224 (51.1) 73 (16.7)
49 (39.5) 57 (46.0) 18 (14.5) 37 (29.8) 71 (57.3) 16 (12.9)
Values are the number of subjects, with percentages in parentheses.
homozygotes. Compared with the Glu27/Glu27 (22.8%) homozygotic men, the prevalence of the metabolic syndrome was similar in men with the Gln27/Glu27 (22.5%) genotype and was higher in those with the Gln27/Gln27 (32.8%) genotype. In multivariate logistic regression analyses using the Gly16/Gly16 carriers as a reference group and adjusting for age, physical activity, smoking habits, alcohol consumption, and BMI, the OR of the metabolic syndrome was 1.83 (95% confidence interval, 1.10 –3.05) and 2.43 (95% confidence interval, 1.19 – 4.95) in men bearing the Gly16/Arg16 and Arg16/ Arg16 genotypes, respectively. Similarly, compared with Glu27/Glu27 carriers, the ORs of the metabolic syndrome were 0.99 (95% confidence interval, 0.50 –1.93) and 1.67 (95% confidence interval, 0.84 –3.33) in carriers of the Gln27/Glu27 and Gln27/Gln27 genotypes, respectively. In contrast, there was no evidence of any association between the Gly163 Arg16 and Gln273 Glu27 polymorphisms and the metabolic syndrome in women. Because the metabolic syndrome is defined by the clustering of at least three metabolic disorders among five, we looked at the relationship between the BAR2 polymorphisms and each of these metabolic disorders separately. Abdominal obesity, hypertriglyceridemia, glucose intolerance, hypertension, and decreased HDL cholesterol levels were defined according to NCEP III guidelines for the metabolic syndrome. In men, but not in women, there was a tendency to a higher prevalence of low HDL cholesterol levels (16.5%, 29.3%, and 38.9%; P ⬍ 0.05) and glucose intolerance (4.7%, 11.0%, and 15.3%; P ⬍ 0.05) across BAR2 Gly16/Gly16, Gly16/ Arg16, and Arg16/Arg16 genotypes, respectively (Fig. 1; data not shown in women). Similarly, there was a tendency to a higher prevalence of low HDL cholesterol levels (17.4%, 22.9%, and 33.9%; P ⬍ 0.05) and high blood pressure (60.9%, 71.1%, and 73.9%; P ⬍ 0.05) across BAR2 Glu27/Glu27, Gln27/ Glu27, and Gln27/Gln27 genotypes. In contrast, other metabolic markers were not associated with BAR2 polymorphisms in either men or women. Because the Gly163 Arg16 and Gln273 Glu27 polymorphisms are in linkage disequilibrium, the association with the metabolic syndrome was examined further using haplotype analysis (Table 4). Of the four theoretical haplotypes derived from the two polymorphic sites, only three were represented due to the linkage disequilibrium. In men, the prevalence of metabolic syndrome was 23, 23, and 32% in the Gly16Glu27, Gly16Gln27, and Arg16Gln27 haplotypes, respectively (P ⫽ 0.062). In women, these numbers were 27.6, 30.1, and 27.9% in the Gly16Glu27, Gly16Gln27, and Arg16Gln27 haplotypes, respectively (P ⫽ 0.91).
FIG. 1. Prevalence of metabolic disorders according to Gly163 Arg16 and Gln273 Glu27 BAR2 polymorphisms in men. Ab., Abdominal; TG, triglycerides; Gluc., glucose; BP, blood pressure.
Endogenous catecholamines are important modulators of adipose tissue lipolysis, glucose homeostasis, and vascular tone. In human tissues catecholamines exert their effects through BAR receptors. Two single nucleotide polymorphisms (SNPs) at nucleotides 46 and 79 of the BAR2 gene result in changes in amino acid residues 16 and 27. In cultured cell experiments, the Gly163 Arg16 and Gln273 Glu27 polymorphisms alter cellular trafficking of the receptor, such that the agonist-dependent down-regulation varies with certain alleles (8, 30). Given the pleiotropic properties of catecholamines and the functional consequences of Gly163 Arg16 and Gln273 Glu27 polymorphisms, we have speculated that these SNPs could contribute to metabolic syndrome susceptibility. The findings of the present study showed that Arg16 and, to a lesser extent, Gln27 BAR2 variants are associated with increased risk of metabolic syndrome in men, but not in women. Together, these results suggest that BAR2 polymorphisms affect susceptibility to the metabolic syndrome in a gender-dependent manner. The association between metabolic syndrome and BAR2 polymorphisms was found in men, but not in women, suggesting that BAR2 genetic susceptibility to the metabolic
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TABLE 4. Distribution of BAR2 haplotypes in controls and patients with the metabolic syndrome Men
Gly16Glu27 Gly16Gln27 Arg16Gln27
179 (42.9) 93 (22.3) 145 (34.8)
52 (35.1) 28 (18.9) 68 (45.9)
185 (42.3) 84 (19.2) 168 (38.4)
51 (41.1) 26 (21.0) 47 (37.9)
Values are the number of subjects, with percentages in parentheses. NS, Not significant.
syndrome is dependent on gender. This observation is consistent with previous studies that have reported specific associations between BAR2 polymorphisms and obesity in men and women (17–19). The reasons for these differences are not known. One possible explanation is that free fatty acid metabolism regulation differs substantially between genders (31). For instance, we have previously shown that the BAR2 Gly163 Arg16 polymorphism has a different effect on postprandial free fatty acid metabolism in men and women (32). Another possibility could be that abdominal adipose tissue, which is a major source of free fatty acids and an important target for catecholamine-mediated lipolysis (31), is more abundant in men than in women (33), resulting in higher fluxes of plasma fatty acids to the liver and peripheral tissues. Furthermore, higher lipolytic rates have also been reported in visceral adipose tissue from obese men than women (34). Therefore, BAR2 polymorphisms might have more metabolic consequences in men than in women due to the increased volume of more active adipose tissue. Earlier studies found that unique interactions of multiple BAR2 SNPs within a haplotype affect biological and therapeutic phenotypes. Therefore, we performed analysis aimed at comparing Gly163 Arg16 and Gln273 Glu27 haplotype distribution in control and metabolic syndrome groups. Due to linkage disequilibrium between the Gly163 Arg16 and Gln273 Glu27 polymorphisms, only three of the four possible haplotypes were found in this population sample. In men and women, there was no statistically significant difference in haplotype distribution between metabolic syndrome and controls. Therefore, further structure-function studies are necessary to understand the possible implications of BAR2 haplotypes in the susceptibility to the metabolic syndrome. The metabolic syndrome is characterized by several closely correlated metabolic disorders. Therefore, we looked at the relationship between BAR2 polymorphisms and each component of the metabolic syndrome individually. In men, the results showed a gene-dose association between BAR2 SNPs and low HDL cholesterol, glucose intolerance, and high blood pressure. These findings support the concept that BAR2 polymorphisms have ubiquitous effects. A small impact on each component of the metabolic syndrome results in a significant clustering of metabolic disorders. This hypothesis is consistent with the concept that polymorphisms on a unique gene with ubiquitous effects on metabolic pathways may increase the susceptibility to the metabolic syndrome in men. However, these findings do not preclude the possibility that these multiple associations reflect a relationship between BAR2 SNPs and a single risk factor, which is itself related to other risk factors. For instance, we have previously shown that Gln273 Gln27 homozygous subjects have an increased risk of obesity, of increased waist girth and
waist to hip ratio (19). Given the close relationship between abdominal obesity and metabolic disorders, it could be possible that the association between BAR2 polymorphisms and the metabolic syndrome results from a primary relationship with abdominal fat. The design of the present study does not allow clarification of this issue. Obesity is a major determinant of the metabolic syndrome (2). The adipose cell secretes free fatty acids, which stimulate hepatic triglyceride and glucose production. Obesity is associated with decreased peripheral insulin sensitivity and peripheral glucose uptake. It also induces the sympathetic nervous system and changes in kidney structure and increases sodium reabsorption, resulting in increased blood pressure. The BAR2 gene is a strong candidate for obesity. The Gly163 Arg16 and Glu273 Gln27 SNPs have been associated with an increased risk of obesity in most studies (16 – 24). In this population we have shown that Gln27/Gln27 homozygous subjects have nearly a doubling of obesity risk (19). Therefore, it appears that obesity could be an intermediary factor between BAR2 SNPs and the metabolic syndrome. The results of the logistic regression analysis are not in favor of this hypothesis, because BAR2 SNPs remained significantly associated with the metabolic syndrome after inclusion of BMI in the model. However, because BMI is an incomplete surrogate of body fat mass, this result does not preclude a possible role for fat tissue in the association between BAR2 SNPs and the metabolic syndrome. Finally, in this population the association between BAR2 SNPs and obesity is stronger for Glu273 Gln27 SNP (19), whereas the association with the metabolic syndrome is more marked for Gly163 Arg16 SNP, suggesting possible different influences of these SNPs on both metabolic disorders. Earlier studies have reported significant associations between the Gly163 Arg16 and Gln273 Glu27 polymorphisms of the BAR2 gene and several components of the metabolic syndrome, such as obesity (16 –24), hypertension (10, 11), diabetes (10, 12), and dyslipidemia (13, 14). However, the associations were not always consistent among studies. One possible explanation for these discrepancies might be related to differences in gender distribution among studies, recruitment bias of patients and controls, or confounding effects of variables, such as physical activity or smoking habits. For example, in the study by Large et al. (16), the nonobese group was defined by a BMI less than 27 kg/m2 and was recruited among women referred to the hospital for surgery. Additional studies by the same group found different results in men than in women (17). The present study, in contrast, was performed in a population-based sample stratified for gender and age. This allowed us to assess the impact of BAR2 polymorphisms, adjusting for possible confounding vari-
J Clin Endocrinol Metab, October 2003, 88(10):4862– 4866
ables, such as physical activity, alcohol consumption, and smoking habits. In conclusion, complex interactions among environmental, familial, and genetic factors play a major role in the development of the metabolic syndrome. In the present population-based study we found that Gly163 Arg16 and Glu273 Gln27 polymorphisms are associated with the metabolic syndrome in men, but not in women, illustrating such complex interactions. These data suggest that BAR2 genetic variability contributes to metabolic syndrome susceptibility in men. Acknowledgments We thank Mrs. V. Codron and Mr. X. Hermant for technical assistance. Received February 3, 2003. Accepted July 7, 2003. Address all correspondence and requests for reprints to: Dr. Jean Dallongeville, Institut National de la Sante´ et de la Recherche Me´ dicale, U-508, 1 rue du Prof. Calmette, Institut Pasteur de Lille, 59019 Lille Cedex, France. E-mail: [email protected]
The WHO-MONICA population study developed in the North of France was supported by unrestricted grants from the Conseil Re´ gional du Nord-Pas de Calais, ONIVINS, Parke-Davies Laboratory, the Mutuelle Ge´ ne´ rale de l’Education Nationale, Groupe Fournier, the Re´ seau National de Sante´ Publique, the Direction Ge´ ne´ rale de la Sante´ , Institut National de la Sante´ et de la Recherche Me´ dicale, the Institut Pasteur de Lille and the Unite´ d’Evaluation du Centre Hospitalier et Universitaire de Lille.
Dallongeville et al. • Metabolic Syndrome and BAR2 Polymorphism
17. 18. 19. 20. 21. 22.
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