Leptin receptor Gln223Arg variant is associated with a cluster of metabolic abnormalities in response to long-term overfeeding

Authors


Claude Bouchard PhD, Executive Director Pennington Biomedical Research Center 6400 Perkins Road, Baton Rouge, Louisiana 70808-4124, USA (fax: +1225 7630935; e-mail: bouchac@pbrc.edu).

Abstract

Abstract. Ukkola O, Tremblay A, Després J-P, Chagnon YC, Campfield LA, Bouchard C (Pennington Biomedical Research Center, Baton Rouge, Louisiana, USA; University of Oulu, Oulu, Finland; Laval University, Ste-Foy, Québec, Canada; and University of Colorado Health Sciences Center, Denver, Colorado, USA). Leptin receptor Gln223Arg variant is associated with a cluster of metabolic abnormalities in response to long-term overfeeding. J Intern Med 2000; 248: 435–439.

Objectives. The role of the leptin receptor (LEPR) gene Gln223Arg polymorphism on the metabolic and body composition changes in response to overfeeding was studied.

Subjects. Twelve pairs of male monozygotic twins ate a 4.2 MJ day–1 energy surplus, 6 days week–1, during a period of 100 days.

Results. Overfeeding induced a significantly greater increase in glucose (P = 0.001 for percentage change) and insulin (P = 0.038) areas under the curve during oral glucose tolerance tests (OGTTs) in the GlnGln (n = 10) than in the GlnArg/ArgArg (n = 14) subjects. In addition, the GlnGln genotype was associated with a greater increase in plasma levels of leptin (P = 0.037) and total triglycerides (P = 0.003), as well as a greater decrease in high-density lipoprotein cholesterol (P = 0.010), than for the combined GlnArg/ArgArg genotypes. Body composition changes were not different between the genotypes.

Conclusions. We conclude that the GlnGln subjects of the LEPR gene polymorphism are more susceptible to metabolic abnormalities when they are exposed to long-term positive energy balance. These findings provide new information on the genetic basis of individual differences in response to chronically elevated food intake.

Introduction

There are large interindividual differences in the response to various dietary changes. However, the genetic basis of the differences in response to alterations in food intake is largely unknown. Genes encoding proteins that are key regulators of energy balance are likely involved in the modulation of the response to environmental pressures causing weight gain. The leptin receptor gene is reasonably such a candidate gene based on its role in animal obesity models [1] and its reported involvement in the regulation of body composition in humans [2 3]. Therefore, the aim of the present study was to assess the role of the LEPR Gln223Arg polymorphism in metabolic and body composition changes observed in response to a long-term (100 days) overfeeding protocol conducted with 12 pairs of monozygotic twins.

Methods

The specific aims, study design and methodology have been described in detail previously [4]. Briefly, 24 sedentary young men [mean (± SD) age, 21 ± 2 years] who constituted 12 pairs of healthy identical twins were studied. Each man gave written consent for participation in this study, which was approved by the Laval University Medical Ethics Committee and the Office for Protection from Research Risks of the National Institutes of Health, Bethesda, Maryland. The men were housed in a closed section of a dormitory on the campus of Laval University. Each man stayed in the unit for 120 consecutive days: 14 days for baseline testing for habitual daily energy intake, 3 days for testing before the period of overfeeding, 100 days for the period of overfeeding, and 3 days for testing after the period of overfeeding. During the period of overfeeding, the men were fed a diet containing 4.2 MJ day–1 (1000 kcal) above their measured baseline energy intake, 6 days a week, for 100 days. On the seventh day of each week, they consumed their habitual daily energy intake. The food consumed each day had the following prescribed nutrient composition: 50% carbohydrate, 35% fat and 15% protein. The men ate three meals per day plus an evening snack tailored to complete the daily prescription for energy intake.

Phenotype measurements

The body mass index (BMI) was calculated as body weight (in kg) divided by height2 (m2). Body density was determined by underwater weighing, and fat mass was calculated with a standard equation [4]. On the first 2 days of the 3-day test period before the overfeeding period, blood samples were obtained in the morning after an overnight fast for the determination of total cholesterol, high-density lipoprotein (HDL) cholesterol, total triglycerides, plasma glucose and insulin concentrations. On day 2, a 75 g oral glucose tolerance test (OGTT) was performed. Blood samples were obtained every 15 min during the first hour and every 30 min during the next 2 h for determination of plasma glucose and insulin. The same measurements were repeated during the 3-day test period after the overfeeding period. Plasma lipid, glucose and insulin determinations were assayed as described earlier [5] and plasma leptin was measured with an enzyme-linked immunosorbent assay specific for the human peptide [6].

Genotype measurements

The description of the Gln223Arg polymorphism of the LEPR gene and the PCR conditions under which it was uncovered have been described earlier [2].

Statistical analysis

Differences in phenotype changes (percentage and absolute changes) between Gln223Arg genotypes were assessed by t-tests. However, since both series of results were highly concordant, the emphasis is put on percentage changes in this report. Percentage changes were calculated from individual scores. Analyses were performed both with the 24 subjects considered as unrelated persons and with the phenotype mean of each of the 12 pairs. Statistical analyses were performed using the SAS statistical package (SAS institute, Cary, NC).

Results

Changes in body weight and measures of body composition with overfeeding in the Quebec Overfeeding Study have been reported previously for the 12 pairs of twins [4] and will not be detailed here.

In the ArgArg subjects (n = 4) the phenotype changes in response to overfeeding were similar to those in the GlnArg subjects (n = 10). Thus, the ArgArg subjects were pooled together with the GlnArg subjects in all the analyses. Table 1 shows the effect of overfeeding on body composition, fasting plasma insulin, leptin and lipid levels for the two LEPR Gln223Arg genotypes. Plasma levels of fasting insulin (P = 0.002), leptin (P = 0.037) and total triglycerides (P = 0.003) increased, whilst HDL cholesterol decreased (P = 0.010), more in the GlnGln (n = 10) than in the GlnArg/ArgArg subjects. Plasma glucose and insulin as well as total areas under the curve during the OGTT before and after overfeeding are depicted in Figs 1 and 2. Overfeeding induced significantly larger increases in the OGTT glucose (P = 0.001) ( Fig. 1) and insulin (P = 0.038) ( Fig. 2) areas in the GlnGln subjects than in the GlnArg/ArgArg subjects.

Table 1.  Effect of overfeeding on body composition, plasma leptin and lipid levels in 12 pairs of male twins in relation to the Gln223Arg genotype
 GlnGln (n = 10) GlnArg/ArgArg (n = 14)
BeforeAfterChangeBeforeAfterChange
  1. Values are means (SE). BMI, body mass index; HDL, high-density lipoprotein.

  2. P-values based on 24 subjects considered as unrelated persons are as follows: *P = 0.002, **P = 0.037, ***P = 0.010, ****P = 0.003 between GlnGln and GlnArg/ArgArg.

  3. P-values when phenotype means of each pair are compared (n = 12): *P = 0.011, **P = 0.064, ***P = 0.035, ****P = 0.015 between GlnGln and GlnArg/ArgArg.

BMI (kg m–2)
19.3 (0.4)
22.2 (0.4)
   +2.9 (0.3)
 (+15%)
20.0 (0.7)
22.5 (0.6)
   +2.5 (0.1)
 (+13%)
Fat mass (kg)
8.6 (0.9)
14.7 (1.0)
   +6.1 (0.7)
 (+76%)
5.8 (1.0)
10.6 (1.2)
   +4.8 (0.4)
(+117%)
Fasting plasma insulin (pmol L–1)
49.8 (4.3)
94.6 (7.3)
  +44.8 (8.0)
(+110%) *
42.3 (1.6)
47.8 (3.2)
     5.5 (3.3)
 (+14%)
Plasma leptin (ng mL–1)
1.5 (0.5)
7.4 (2.1)
   +5.8 (1.6)
(+470%) **
 0.9 (0.1)
3.1 (0.6)
   +2.3 (0.5)
(+262%)
Total cholesterol (mmol L–1)
4.7 (0.2)
5.6 (0.4)
  +0.9 (0.3)
 (+19%)
4.3 (0.2)
4.4 (0.2)
   +0.1 (0.2)
  (+4%)
HDL cholesterol (mmol L–1)
1.2 (0.0)
1.0 (0.0)
 −0.2 (0.0)
 (−14%) ***
1.2 (0.1)
1.1 (0.0)
 −0.0 (0.0)
  (−3%)
Total triglycerides (mmol L–1)
1.3 (0.2)
2.6 (0.5)
  +1.2 (0.4)
 (+95%) ****
1.0 (0.1)
1.2 (0.1)
   +0.2 (0.1)
 (+23%)
Figure 1.

Plasma glucose levels in the GlnArg/ArgArg (a) and GlnGln (b) genotypes before and after overfeeding, as well as the changes (%) of the total glucose areas under the curve (c) during the OGTT. *P = 0.001 between the GlnArg/ArgArg and GlnGln genotypes for the glucose area changes (24 cases), and P = 0.006 when the means of each pair (n = 12) are considered.

Figure 2.

Plasma insulin levels in the GlnArg/ArgArg (a) and GlnGln (b) genotypes before and after overfeeding, as well as the changes (%) of the total insulin areas under the curve (c) during the OGTT. *P = 0.038 between the GlnArg/ArgArg and GlnGln genotypes for the insulin area changes (24 cases), and P = 0.093 when the means of each pair (n = 12) are considered.

The influence of the LEPR Gln223Arg polymorphism on changes in plasma insulin and glucose areas as well as plasma lipids remained significant after adjustment for the change in fat mass (except for HDL cholesterol, which became a non-significant trend).

Discussion

The present data indicate that overfeeding worsened glucose tolerance and induced hyperinsulinaemia and dyslipidaemia, which is consistent with insulin resistance being present in the LEPR GlnGln subjects. In contrast, the changes in metabolic parameters were minimal in the GlnArg/ArgArg subjects. Body composition changes were not different between the two classes of genotypes.

Leptin receptors are present in a number of tissues, including beta-cells of the pancreas [7], where leptin is able to suppress the secretion of insulin [8]. In the presence of leptin deficiency or resistance, there is a failure of leptin to inhibit glucose-stimulated insulin secretion, which could lead to hyperinsulinaemia and insulin resistance [9]. The fact that overfeeding induced a more marked elevation of plasma leptin and insulin concentrations in GlnGln subjects supports the hypothesis that, due to a genetic alteration in the leptin receptor, leptin might be less efficient in exerting its effects on beta-cells. However, since the GlnGln genotype was also associated with a more pronounced glucose intolerance, one could speculate that stimulated insulin secretion after the overfeeding protocol could only compensate partially for a diminished insulin action. Therefore, overfeeding could have potentially induced a relative islet cell dysfunction amongst GlnGln subjects but not in the other genotypes. In addition, the decreased HDL and increased triglyceride levels observed after overfeeding in GlnGln subjects are perhaps associated with alterations in lipoprotein lipase, which is known to be sensitive to insulin [10].

The Gln223Arg variant is located in the extracellular domain of the leptin receptor near the mutation seen in the Zucker fatty rat which results in a charge change in amino acid. The latter could have functional consequences [11]. Alternatively, the effects of the Gln223Arg mutation could be mediated through allelic association with an as yet unknown functional mutation elsewhere. Further studies are needed to test this issue.

Some earlier studies [2 3] have reported a significant effect of the LEPR Gln223Arg polymorphism on adiposity. In the present study, body composition changes were not different between the genotypes, although a trend for a greater absolute gain in fat mass amongst Gln223 homozygote subjects was seen. The genotype influence on plasma insulin and glucose areas, as well as on triglyceride changes, remained significant after adjustment for the changes in fat mass. This is consistent with the notion that the LEPR genotype effects reported herein associated with chronic overfeeding are independent of the body composition changes.

In conclusion, GlnGln subjects of the LEPR gene Gln223Arg variant are more susceptible to metabolic abnormalities than are GlnArg/ArgArg subjects in response to long-term overfeeding. When the former are exposed to environmental pressures, such as those present during chronic overfeeding, disturbances commonly seen in those prone to type 2 diabetes mellitus are observed.

Acknowledgements

We are indebted to Jacques Bouillon, Suzie Hamel, Brigitte Zément, Maryse Lebrun, Martine Marcotte, Monique Chagnon, Josée Lapointe, Henri Bessette, Gilles Bouchard and Serge Carbonneau for their contributions to this study. Gratitude is expressed to Dr A Nadeau and the staff of the Diabetes Research Unit for the glucose and insulin assays.

Special thanks go to Guy Fournier and Dr Germain Thériault for their role in the management of the study and also to Claude Leblanc for his statistical support. Supported in part by a grant (DK 34624) from the National Institutes of Health. O. Ukkola is a fellow of the Medical Council of the Academy of Finland.

Received 13 July 2000; accepted 27 July 2000.

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