Impact of polymorphisms of the human β2-adrenoceptor gene on obesity in a French population

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Abstract

OBJECTIVE: To investigate the impact of two common polymorphisms in the human β2-adrenoceptor gene (Gly16Arg and Gln27Glu substitutions) on obesity and anthropometric measurements as well as blood variables in a large sample of a French population.

DESIGN AND SUBJECTS: Within the framework of the WHO-MONICA project, a population study composed of 1195 subjects aged 35–64 y was randomly sampled from the electoral rolls of the Urban Community of Lille, in northern France. Subjects without any medical treatment (for hypercholesterolaemia, hypertension or diabetes mellitus) susceptible to interfere with body weight and biological variables were selected (n=836, 419 men/417 women, age=49.5±8.1 y, body mass index (BMI)=25.7±4.4 kg/m2). Subjects with a body mass index ≥30 kg/m2 were considered as obese (n=119, age=49.5±8.2 y, BMI=33.9±3.3 kg/m2 range 30–44).

MEASUREMENTS: Genotyping was carried out with allele-specific oligonucleotides hybridization. Association between genotypes and various obesity markers (body weight, body mass index, waist and waist-to-hip ratio), lipid, glucose and insulin variables were studied.

RESULTS: The Gly16Arg and Gln27Glu polymorphisms were in complete linkage disequilibrium. Gln27Gln subjects had an increased risk of obesity (odds ratio (OR)=1.77, 95% CI 1.19–2.62, P=0.005). This effect was mainly detected in men (OR=2.40, 95% CI 1.34–4.27, P=0.003). Men bearing the Gln27Gln genotype had higher body weight, BMI, waist and hip circumferences and waist-to-hip ratio than others. Moreover, if Gln27Gln men carried in addition the Arg16 allele, the increase in body weight, BMI and waist-to-hip ratio was more important.

CONCLUSION: Our results suggest that genetic variability of the β2-adrenoceptor gene is implicated in body weight regulation and in the onset of obesity in French men.

Introduction

Four adrenoceptor subtypes (β1, β2, β3, α2), activated by catecholamines, modulate a large number of biological responses, including adipose tissue lipolysis.1,2 β1-, β2- and β3-adrenoceptors stimulate while α2-adrenoceptors inhibit lipolysis in human fat. Genes encoding these receptors may constitute interesting candidates to explain part of the genetic predisposition to human obesity. Reihsaus and collaborators detected nine polymorphisms in the human β2-adrenoceptor (BAR-2) gene in normal and asthmatic subjects.3 The impacts of two common polymorphisms, the Gly16Arg and Gln27Glu substitutions, have been explored in several diseases, such as asthma,4,5,6 parental hypertension7 and obesity.8,9,10,11,12 Large and colleagues described the impacts of these two polymorphisms in a group of 140 Swedish women with body mass index between 17.8 and 60.0 kg/m2. In this study, the Glu27 allele was associated with obesity, increased body mass index and body fat, while the Gly16Arg polymorphism was not. However, this last polymorphism was associated with altered BAR-2 function, Arg16 carriers showing a five-fold decreased agonist sensitivity.8

To approach the impact of genetic variability at the BAR-2 locus in the general population, we investigated whether the Gly16Arg and Gln27Glu polymorphisms were associated with obesity and various anthropometric and biological variables in a large representative sample of the northern French population.

Research design and methods

Population study

Within the framework of the WHO-MONICA (Multinational MONItoring of trends and determinants in CArdiovascular diseases) project,13 we constituted in 1995–1997 a representative sample of 1195 men (n=601) and women (n=594) living in the urban community of Lille (northern France), aged 35–64 y. This study was randomly sampled from the electoral rolls, and equally distributed in 10 y age groups and gender14 (overall response rate was 77%). There were no related subjects in our sample. This study was approved by the Ethics Committee of the Centre Hospitalier et Universitaire de Lille. Each individual signed an informed consent form. A detailed questionnaire was filled out including alcohol and smoking consumption estimations, and personal medical history. Body mass index (BMI kg/m2), waist-to-hip ratio and blood pressure were measured and a fasting blood sample was drawn. DNA sample was available for 1155 subjects.

Biological measurements

Glucose was measured by the glucose oxidase method (DuPont Dimension). Plasma total cholesterol and triglycerides levels were measured by enzymatic methods (DuPont Dimension). Plasma insulin was measured by radio-immunoassay (BiInsuline, ERIA Pasteur).

Genetic analysis

Genomic DNA was extracted from white blood cells as previously described.15 DNA amplification was performed using Polymerase Chain Reaction (PCR) for 1152 subjects (three could not be genotyped for technical reasons). The primers that were used to amplify simultaneously the codons 16 and 27 were derived from the genomic sequence of the BAR-2 gene.16 The upstream primer was 5′-CTTCTTGCTGGCACGCAAT-3′ and the downstream primer was 5′-CCAGTGAAGTGATGAAGTAGTTGG-3′. The PCR conditions were 94°C 1 min, 56°C 1 min, 72°C 1 min for 30 cycles, and final extension at 72°C for 10 min. These primers gave a PCR product of 199 bp. The Gly16Arg polymorphism was detected using allele specific oligonucleotides hybridization with the following primers: 5′-CATGGCTTCCATTGCGTGCC-3′ for the Gly16 allele and 5′-CATGGCTTC TATTGCGTGCC-3′ for the Arg16 allele (polymorphism underlined). Membranes were hybridized at 58°C for 1 h, washed twice in 1 XSSC, 10% SDS buffer for 5 min, washed in 0.5 XSSC, 10% SDS buffer for 5 min, and washed in 0.5 XSSC, 10% SDS buffer at 62°C for 3 min. A random sample of the Gly16Arg genotypes (n=250) was verified using procedure as previously described.8 The Gln27Glu polymorphism was detected using allele specific oligonucleotides hybridization with the following primers: 5′-CGTCCCTTTGCTGC-GTGACG-3′ for the Gln27 allele and 5′-CGTCCCTTTCCTGCGTGACG-3′ for the Glu27 allele (polymorphism underlined). Membranes were hybridized at 61°C for 1 h, washed twice in 1 XSSC, 10% SDS buffer for 5 min, washed in 0.5 XSSC, 10% SDS buffer for 5 min, and washed in 0.5 XSSC, 10% SDS buffer at 65°C for 3 min for the Gln27 membrane or 63°C for the Glu27 membrane. A random sample of 200 genotypes were verified using previously described procedure.8

Statistical analysis

Statistical analyses were performed with the SAS statistical software, version 6.12 (SAS Institute, Inc., Cary, NC). Genotypic and allelic distributions were compared with Pearson χ2 statistical tests. The effect of the polymorphisms on quantitative variables was tested with a multivariate analysis of covariance using a general linear model (proc GLM, Type III SS). Linkage disequilibrium was computed according to Thomson et al17 and tested.18 Multivariate logistic regression was used to calculate adjusted odds ratio (OR). Because of the potential influence of medication on blood variables and body weight, individuals treated for hyperlipidaemia, hypertension or diabetes mellitus were excluded from these analyses (n=316). Insulin, triglyceride and leptin values were log transformed to normalize their distributions.

Results

Gly16Arg polymorphism

The genotype distribution of the BAR-2 Gly16Arg polymorphism in the whole population (n=836) was 38.0% (n=320), 48.3% (n=406) and 13.7% (n=110) for Gly16Gly, Gly16Arg and Arg16Arg, respectively (Arg16 allele frequency: 0.37). These distributions did not differ from the Hardy–Weinberg equilibrium. We divided the population according to obesity using a BMI cut-off of 30 kg/m2. There was no significant difference in the Arg16 allele frequency between obese (n=119, frequency=0.40) and non-obese (n=717, frequency=0.37) subjects, although a trend could be detected in men: Arg16 allele frequency was slightly more frequent in obese men (OR=1.48, 95% CI 0.96–2.26, P=0.059; data not shown).

The phenotypic characteristics of the polymorphism at codon 16 according to gender are shown in Table 1. Slight effects were detected in men: Arg16Arg men had higher body weight (+7%) and waist circumference (+6%) with preferential upper body fat distribution characterized by increased waist-to-hip ratio (+3%) than Gly16 homozygous men, in an allele dose-dependent manner. Plasma insulin levels were also higher in Arg16Arg men (+20%) than in Gly16Gly subjects. No association could be detected for plasma glucose or lipid variables. No association was found in women, both in the subgroup of pre- or post-menopausal women.

Table 1 Effects of the Gly16Arg polymorphism according to gender

Gln27Glu polymorphism

The genotype distribution of the BAR-2 Gln27Glu polymorphism in the whole population was 33.1%, 51.0% and 15.9% for Gln27Gln, Gln27Glu and Glu27Glu genotypes, respectively (Glu27 allele frequency: 0.41; Table 2). The distributions were in Hardy–Weinberg equilibrium. Gln27Gln subjects had an increased risk of obesity (OR=1.77, 95% CI 1.19–2.62, P=0.005; Table 2). When stratified on gender, only Gln27Gln men had an increased risk of obesity (OR=2.40, 95% CI 1.34–4.27, P=0.003).

Table 2 Genotype and allele frequencies of the Gln27Glu polymorphism according to obesity

The phenotypic characteristics associated with polymorphism at codon 27 are shown in Table 3. Gln27Gln homozygous men had higher body weight (+5%), BMI (+2%), waist circumference (+3%), and waist-to-hip ratio (+2%) compared to Glu27Glu subjects. Plasma triglyceride levels were increased in Gln27Gln men (+30%) compared to Glu27Glu men as well as plasma insulin levels (+18%), but this last association was no longer statistically significant after adjustment on body mass index. No association could be detected for glucose or lipid variables. No association was found in women, both in the subgroup of pre- or post-menopausal women.

Table 3 Effects of the Gln27Glu polymorphism according to gender

Combined analyses

The two polymorphisms were in complete linkage disequilibrium in our sample, the Arg16 allele being associated with the Gln27 allele. When the impact of these two polymorphisms were studied simultaneously in a regression model, only the Gln27Glu polymorphism modified the risk of developing obesity both in the whole sample (OR=1.70, 95% CI 1.07–2.72, P=0.026) and in men (OR=2.40, 95% CI 1.25–4.58, P=0.008). Moreover, the increases in body weight, BMI, waist-to-hip ratio and plasma triglyceride levels observed in Gln27Gln men were still statistically significant after adjustment on the Gly16Arg polymorphism. Conversely, the impact of the Gly16Arg polymorphism on anthropometric variables disappeared after adjustment on the Gln27Glu polymorphism.

Table 4 gives mean values of body weight, BMI, waist-to-hip ratio and plasma insulin levels in men for combined genotypes. Men bearing both the Arg16 allele and the Gln27Gln genotype (n=51) had higher body weight (+10%), BMI (+8%), and waist-to-hip ratio (+3%) than men bearing other genotypes.

Table 4 Combined analyses of both polymorphisms in men

Discussion

In this study, we analysed the impacts of two common polymorphisms of the human β2-adrenoceptor gene (Gly16Arg and Gln27Glu substitutions) on obesity in a large representative sample of the northern French population. As previously described,7,8,10,28 we showed that the two polymorphisms were in complete linkage disequilibrium, the rare Arg16 allele being associated with the frequent Gln27 allele. When the impacts of these two polymorphisms were studied simultaneously, only the Gln27Glu polymorphism modified the risk of developing obesity. Men bearing the Gln27Gln genotype had higher body weight, BMI, waist and hip circumferences, and waist-to-hip ratio than others. If Gln27Gln men carried in addition the Arg16 allele, the increase in body weight, BMI and waist-to-hip ratio was more important. This suggests that the Arg16 allele increases the deleterious effect of the Gln27Glu polymorphism on body fat mass.

Published searches for associations between β2-adrenoceptor 16 and/or 27 polymorphisms and obesity were conducted in five other independent populations.8,9,10,11,12 Large et al reported that the Gln27 allele frequency was higher in obese Swedish males compared with non-obese males but was lower in obese Swedish females than in non-obese females.8,11 In our study, we confirmed that the Gln27 allele was a risk factor of obesity in men and that Gln27Gln homozygous men had increased BMI with fat accumulation in upper body region but we were not able to detect an effect of polymorphisms of the BAR-2 gene on obesity in pre- or post-menopausal women. These findings suggest that genetic factors contributing to obesity are different between men and women. Moreover, it has been shown that in Japanese subjects the frequency of the Glu27 allele was higher in obese subjects compared to non-obese subjects.10,12 However, the frequency of Glu27 allele in non-obese Japanese for both genders was much lower ( f≈0.07) compared with that found in French or Swedish subjects ( f≈0.40), and this difference may explain the discrepancy observed between Japanese and European subjects.

In our study, no effect of the BAR-2 polymorphisms could be detected among women. In fact, many studies reported sex and fat regional-related differences in lipolysis.19 In human omental fat cells, the lipolytic activity is higher than in human subcutaneous fat cells, owing to the lower α2-adrenergic receptor efficiency and to site variations in the β-adrenoceptor subtypes.20,21 In obese22 or non-obese23 subjects, the β-adrenergic lipolytic response is greater in subcutaneous abdominal than in gluteal adipocytes from both sexes. However, abdominal adipocytes of women showed lower α2-adrenergic antilipolytic sensitivity than did gluteal adipocytes, leading to a more pronounced regional difference in lipolysis in women than in men. In addition, men seem to have higher abdominal α2-adrenoceptor efficiency (antilipolysis) than women, implicating for men an accumulation of adipose tissue in the abdominal region. So, if a mutation (Arg16 or Glu27 or others) alters the β2-adrenergic pathway, the induced loss of lipolysis efficiency may be more efficiently compensated by a decrease in the α2-adrenergic pathway in women than in men. Recently, it has been shown that physical activity, another compensation mechanism, was able to counterbalance the effect of the Gln27Glu polymorphism to increase body weight, body fat and obesity in men.24 Moreover, in Japanese obese women, the weight loss occurring during a 3 month combined low-energy diet and exercise regimen was greater among women bearing the Gly16 allele.25

In our study, Gln27Gln men had a significant increase in plasma triglyceride concentrations compared to others. We know that the Glu27 isoform does not reach the mature wild-type conformation and this may result in an altered ability of this receptor to be degraded.26 Therefore, the lipolysis may be more efficient in Glu27 carriers than in Gln27 carriers, leading to an accumulation of triglycerides in these last subjects.

Several studies on the activity of the different BAR-2 isoforms have already been performed in vitro. Large et al reported a five-fold decreased agonist sensitivity (terbutaline) in adipocytes from Arg16Arg subjects compared to adipocytes from Gly16Gly subjects.8 This observation may be explained because Asn15 is one of the four sites of N-glycosylation for BAR-2, a post-translational event that occurs in β-turns,27 and the change of conformation due to the Gly16Arg mutation may explain the association with obesity observed in the present work. McGraw and colleagues recently studied a Cys19Arg polymorphism located in the BAR-2 5′ leader cistron,28 a 19-amino-acid peptide which regulates cellular expression of the receptor at a translational level.29 The Cys19, Arg16 and Gln27 alleles are in linkage disequilibrium and in vitro the BAR-2 receptor expression was almost two-fold higher when the receptor bore the Cys19 allele instead of the Arg19 allele. However, due to the presence of complete linkage disequilibrium between these polymorphisms and to the different functional properties of each polymorphism in vitro, it is highly speculative to bring these data together.

In conclusion, our results suggest that the β2-adrenoceptor gene plays a central role in adipose tissue metabolism and body weight regulation, and that its genetic variability may contribute to the development of obesity in French men.

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Acknowledgements

We would like to thank Selaiman Rahimi, Valérie Codron and Xavier Hermant for technical assistance. AM was supported by a grant from the Fondation pour la Recherche Médicale. The WHO-MONICA population study developed in the North of France was supported by grants from the Conseil Régional du Nord-Pas de Calais, the Fondation pour la Recherche Médicale, ONIVINS, the Parke–Davis Laboratory, the Mutuelle Générale de l'Education Nationale (MGEN), the Réseau National de Santé Publique, the Direction Générale de La Santé, the Institut National de la Santé Et de la Recherche Médicale (INSERM), the Institut Pasteur de Lille and the Unité d'Evaluation du Centre Hospitalier et Universitaire de Lille.

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Correspondence to P Amouyel.

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Keywords

  • β-adrenergic receptors
  • obesity
  • fat mass
  • polymorphism

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