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Keywords:

  • genetics;
  • epidemiology;
  • central adiposity;
  • waist circumference

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Objective: To study the association between the Gly40Ser polymorphism of the glucagon receptor gene (GCG-R) and central adiposity.

Research Methods and Procedures: Data from 985 working men (The Olivetti Heart Study) examined in 1994 were used in a cross-sectional design. A complete anthropometry was performed; body mass index and waist circumference were taken as measures of total and central adiposity, respectively. The GCG-R Gly40Ser polymorphism was characterized. Biochemical variables linked to energy metabolism were measured.

Results: The GCG-R Gly40Ser variant was present in 37 individuals only in heterozygous form and was significantly associated with anthropometric indices of central adiposity, accounting for age and body mass (odds ratio for waist circumference > 94 cm; 95% confidence interval: 3.14, 1.26 to 7.81), whereas no difference between the two groups was found with regard to biochemical indices of insulin resistance or plasma leptin levels.

Discussion: The Gly40Ser polymorphism of the GCG-R gene is associated with central adiposity independently from total body mass in men.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

A large proportion of the variation in obesity-related phenotypes is under the genetic control of anonymous major genes (1). The number of putative loci affecting human obesity is increasing rapidly (2). Although some mutations in candidate genes explain a few rare cases of monogenic forms of human obesity, major genes conferring susceptibility to obesity remain mostly unidentified. However, the identification of common alleles with moderate but measurable effect on obesity might have important public health implications. In particular, relatively few studies have addressed the hypothesis that regional fat deposition and, in particular, central obesity—an independent cardiovascular risk factor (3) —might be under the control of genes different from those responsible for total adiposity (4) (5). Among genes contributing to fat accumulation, genes regulating lipolysis, thermogenesis, and satiety signaling remain prime candidates (1).

A single heterozygous Gly40Ser missense polymorphism in the glucagon receptor gene (GCG-R) has been described in a few population samples of white descent (6) (7) (8) (9). The human GCG-R is located on chromosome 17q25 (6), and the variant described is the result of a missense mutation at codon 40 in the exon 2 of the GCG-R, leading to the substitution of a serine for a glycine aminoacidic residue in the encoded protein (6). This variant is relatively rare, ranging from 2% to 8% in white populations (6) (7) (8) (9), whereas other studies failed to report the polymorphism in other populations samples (10). Genetic variants resulting in abnormalities in the structure and function of the glucagon receptor might be reasonably expected to result in alterations of body fat accumulation and/or distribution because of the central role of glucagon in several metabolic pathways related to energy use and storage (11) and because of its putative activity in the central regulation of the satiety signaling (12).

Thus, we investigated the association of the GCG-R Gly40Ser with body mass and different measures of central adiposity in a large population of working men (The Olivetti Heart Study) examined from 1994 to 1995.

Research Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The study protocol has been described in detail previously (13) (14). Briefly, anthropometric and metabolic variables (including fasting and post-load serum insulin and plasma leptin levels) were measured on a sample of 1075 working men (25 to 75 years old). The study protocol was approved by the local ethics committee, and participants gave their informed consent to participate. Body weight and height were measured on a standard beam-balance scale with an attached ruler. Body weight was measured at the nearest 0.1 kg, and height was measured at the nearest centimeter, with subjects wearing only light indoor clothing without shoes. The body mass index (BMI) was calculated as weight in kilograms divided by the square of the height in meters. Abdominal circumferences were measured according to standardized methods (15). The measurements were performed at the nearest 0.1 cm with a flexible inextensible plastic tape. The ratio of waist-to-hip circumference was calculated. The sagittal (i.e., antero–posterior) abdominal diameter was measured with the Holtain-Kahn abdominal caliper (Holtain Ltd., Crosswell, Wales, UK) (16). Waist circumference was taken as a reference measure of abdominal obesity, according to the National Institutes of Health Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity (15).

The GCG-R Gly40Ser polymorphism was characterized in 985 participants. Genomic DNA was isolated from leukocytes with a rapid, nonenzymatic, salting out procedure (17). Amplification of DNA by the polymerase chain reaction (PCR) was carried out according to Hager et al. (6) as described, with few modifications. Briefly, PCR conditions were: initial denaturation at 94 °C for 5 minutes; then, 45 cycles at 94 °C for 30 seconds, at 62 °C for 30 seconds and at 72 °C for 30 seconds; final extension step at 72 °C for 5 minutes using a GeneAmp PCR System 9600 (Perkin-Elmer, Milan, Italy). The PCR product, a 196-base pair (bp) fragment, was checked on a 1.5% agarose gel with a Gel Electrophoresis Apparatus GNA-200 (Pharmacia Biotech, Milan, Italy). Restriction fragment length polymorphism analysis was effected by adding 10 units of BstEII (Life Technologies Italia, Milan, Italy), in the appropriate buffer, to PCR product and by incubating at 60 °C for 3 hours: this restriction enzyme is specific for the sequence G/GT(A or C or G or T)ACC. The digested samples were separated by electrophoresis on 4% metaphor gel, ethidium bromide stained, and analyzed under ultraviolet light. Digestion of PCR product with BstEII produced two fragments of different size: 108 and 88 bp, but, in the presence of G∏ A mutation, changing the codon 40 GGTGly to AGTSer, the restriction site is lost and, therefore, these individuals show an additional band (196 bp) corresponding to the undigested mutant allele.

Statistical analysis was performed using the Statistical Package for Social Sciences (version 8.0; SPSS, Chicago, IL). The distributions of serum glucose and triglycerides, serum insulin, and the homeostatic model of assessment were normalized by log transformation, and log-transformed values were used in the analysis. All phenotype data are expressed as means and SE. Age- and BMI-adjusted analysis of covariance was used to assess differences between group means by genotype. Logistic regression was used to determine the independent risk of central adiposity (expressed as odds ratios [ORs] and 95% confidence intervals [CIs]) attributable to the GCG-R variant, accounting for confounders (age and BMI). Two-sided p values <0.05 were considered statistically significant.

Because the Hardy–Weinberg equilibrium cannot be tested in this sample by the use of χ2 statistics, the goodness of fit of the genotype distribution with the Hardy–Weinberg equilibrium was tested by the exact significance statistics using STATA, version 6.0 (STATA Corp., College Station, TX), which provided the direct calculation of the Hardy–Weinberg equilibrium both by the χ2 and the exact significance statistics (STATA, version 6.0; command, “genhwi”). The latter obtains an accurate significance in the presence of sparse or unbalanced tables, as occurs in the case of very rare variants when one of the cells may actually be empty.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Among the 985 participants genotyped, the Gly40Ser variant was present in 37 individuals (3.75%), a proportion similar to that found in other white populations (6) (7) (8) (9). The allele distribution followed the Hardy–Weinberg equilibrium. The genotype observed and expected frequencies are reported in Table 1. A complete dataset was available for 950 individuals, and they are included in the present analysis.

Table 1.  Observed and expected frequencies of GCG-R polymorphism
GenotypeObservedExpected
  1. Hardy–Weinberg Equilibrium test: exact significance probability = 1.000 (not significant).

Gly40Gly948948.35
Gly40Ser3736.31
Ser40Ser00.35

Participants with the Gly40Ser genotype tended to be younger (49.5 ± 1.3 vs. 51.9 ± 0.2 years old; p = 0.06) and more overweight than did controls (BMI: 27.5 ± 0.4 vs. 26.9 ± 0.1 kg/m2; p = 0.1). Thus, age- and BMI-adjusted analysis of covariance was used to assess differences in the anthropometric variables relevant to body fat distribution (waist circumference, waist to hip circumference ratio, and sagittal abdominal diameter) between Gly40Ser carriers and homozygotes for wild-type alleles accounting for these confounders (Table 2). The participants with the Gly40Ser genotype showed significantly higher values of all the measures of central adiposity under consideration. Logistic regression analysis was used to determine the association of the Gly40Ser variant with central adiposity accounting for age and BMI. This variant was independently and significantly associated with an increased risk of having both a waist circumference > 94 cm (median of our population; OR: 3.14; 95% CI: 1.26 to 7.81,) and a waist-to-hip ratio > 1.0 (upper tertile for our population; OR: 2.72; 95% CI: 1.30 to 5.68). The values adopted to define central adiposity in our population match the gender-specific cutoff levels for central adiposity (18) (19). No difference between the two groups was found with regard to biochemical indices of insulin resistance (fasting and post-load serum glucose and insulin concentration; insulin resistance as calculated from the homeostatic model of assessment index) as well as to plasma leptin and serum cholesterol and triglyceride levels (Table 2).

Table 2.  Anthropometric and metabolic variables by GCG-R polymorphism
 Gly40Gly (n = 913)Gly40Ser (n = 37)p Value
  • *

    Homeostatic model of assessment index: fasting serum insulin (μIU/mL) × fasting serum glucose (mM)/22.5.

  • One-hour oral glucose tolerance test was performed on a randomly selected sample of 446 participants (Gly40Gly: n = 431; Gly40Ser: n = 15).

  • Plasma leptin was measured on 413 participants (Gly40Gly: n = 398; Gly40Ser: n = 15).

Age (years)51.9 ± 0.249.5 ± 1.30.06
 (51.4 to 52.4)(46.8 to 52.3) 
BMI (kg/m2)26.9 ± 0.127.5 ± 0.40.16
 (26.7 to 27.1)(26.7 to 28.3) 
Waist circumference (cm)94.4 ± 0.196.5 ± 0.70.007
 (94.1 to 94.7)(95.0 to 97.9) 
Waist-to-hip ratio0.976 ± 0.0011.002 ± 0.007<0.001
 (0.973 to 0.979)(0.988 to 1.015) 
Sagittal abdominal diameter (cm)21.3 ± 0.0521.8 ± 0.20.04
 (21.0 to 21.4)(21.4 to 22.3) 
Serum cholesterol (mM)5.73 ± 0.035.58 ± 0.180.38
 (5.66 to 5.80)(5.21 to 5.95) 
Serum triglyceride (mM)1.73 ± 0.031.70 ± 0.130.83
 (1.66 to 1.80)(1.44 to 1.96) 
Fasting serum glucose (mM)5.64 ± 0.045.62 ± 0.040.91
 (5.56 to 5.73)(5.29 to 5.74) 
Fasting serum insulin (μIU/mL)9.4 ± 0.29.8 ± 0.80.79
 (9.0 to 9.8)(8.2 to 11.5) 
Homeostatic model of assessment index*2.42 ± 0.062.52 ± 0.260.77
 (2.30 to 2.54)(1.99 to 3.05) 
Postload serum insulin (μIU/mL)50.6 ± 1.663.4 ± 9.80.10
 (47.4 to 53.7)(42.4 to 84.6) 
Plasma leptin (ng/mL)4.2 ± 0.24.4 ± 0.70.68
 (3.9 to 4.5)(2.8 to 6.0) 

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Although the structural–functional analysis of the glucagon receptor family is still at a relatively early stage (20), the effect of Gly40Ser mutation on biological functions is already apparent. The mutation is located within an extracellular domain of the receptor (6) and might play a role in the functional activity of the receptor. In vitro studies suggest that this variant results in a reduced affinity of the receptor for the agonist and in a concomitant reduced synthesis of cyclic-adenosine monophosphate by target cells (21). Furthermore, in vivo studies show a lower hyperglycemic effect of systemic glucagon infusion in volunteers carrying the mutation (9). The glucagon receptor is expressed in liver, pancreatic β-cells, kidney, adipose tissue, heart, and in some regions of the central nervous system (20). A decrease in receptor activity in vivo may affect fat deposition by diverse mechanisms. A reduced cyclic-adenosine monophosphate-mediated lypolitic response (22) (23) or, alternatively, a deficiency of glucagon counter-regulatory activity could potentially lead to an increase of the anabolic action of insulin, even in the absence of differences in insulin level, as was the case in our population. Because visceral fat is metabolically very active with a high rate of free fatty acid turnover, a possible explanation for the preferential abdominal fat deposition associated with the Gly40Ser variant might be an impaired glucagon-dependent regulation of free fatty acid metabolism (24).

Glucagon may also combine with other signals to influence meal size, acting as a satiety factor, as shown by infusion studies in rat models (12).

An association between the Gly40Ser variant of the GCG-R and both noninsulin-dependent diabetes mellitus (6) (9) and hypertension (7) (8) has been described in white population samples. Furthermore, we showed that in Gly40Ser heterozygotes, hypertension is associated with an altered renal sodium handling, a factor possibly contributing to hypertension in this group (25). Thus, the combined results of these studies suggest that this polymorphism might have a relevant role in the genetics of the metabolic syndrome.

In conclusion, this is the first report showing that GCG-R Gly40Ser is significantly associated with anthropometric indices of central adiposity independently from total body mass, thus, suggesting that this variant or a polymorphism in linkage disequilibrium with it may affect body fat distribution in men.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The study was supported in part by funds from MURST (Italian Ministry of University and of Scientific and Technological Research COFIN 1998). Dr. Cappuccio is a member of the St. George's Cardiovascular Research Group.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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