Polymorphisms in the leptin receptor gene, body composition and fat distribution in overweight and obese women

Abstract

OBJECTIVE: Leptin is an adipocyte-secreted hormone involved in body weight regulation, acting through the leptin receptor, localised centrally in the hypothalamus as well as peripherally, amongst others on adipose tissue. The aim of this study was to evaluate whether polymorphisms in the leptin receptor (LEPR) gene were related to obesity and body fat distribution phenotypes, such as waist and hip circumferences and the amount of visceral and subcutaneous fat.

METHODS: Three known LEPR polymorphisms, Lys109Arg, Gln223Arg and Lys656Asn, were typed on genomic DNA of 280 overweight and obese women (body mass index (BMI)>25), aged 18–60 y. General linear model (GLM) analyses were performed in 198 pre- and 82 postmenopausal women, adjusting the data for age and menopausal state, plus fat mass for the fat distribution phenotypes.

RESULTS: No associations were found between the LEPR polymorphisms and BMI or fat mass. In postmenopausal women, carriers of the Asn656 allele had increased hip circumference (P=0.03), total abdominal fat (P=0.03) and subcutaneous fat (P=0.04) measured by CT scan. Total abdominal fat was also higher in Gln223Gln homozygotes (P=0.04). Also in postmenopausal women, leptin levels were higher in Lys109Lys homozygotes (P=0.02).

CONCLUSION: In conclusion, polymorphisms in the leptin receptor gene are associated with levels of abdominal fat in postmenopausal overweight women. Since body fat distribution variables were adjusted for fat mass, these results suggest that DNA sequence variations in the leptin receptor gene play a role in fat topography and may be involved in the predisposition to abdominal obesity.

Introduction

Leptin, a hormone primarily secreted by the adipocytes, was identified in 1994,1 and has been shown to play an important role in body weight regulation. In humans, leptin (LEP) gene expression is increased in adipose tissue of obese subjects2 and blood leptin levels are higher in most of these individuals.3 A strong correlation is found between leptin concentrations and total body fat.3 Therefore, a state of leptin resistance has been proposed as a potential mechanism for the development of human obesity, in analogy with the insulin resistance seen in type 2 diabetes. A single-gene mutation animal model providing support for this notion is the db/db mouse, in which a genetic defect in the leptin receptor causes leptin resistance and leads to a phenotype similar to the ob/ob mouse, characterised by obesity and insulin resistance, but with high levels of circulating leptin.4,5,6,7 Moreover, leptin resistance has recently been described in a polygenic rodent model, Psammomys obesus.8

The leptin receptor (LEPR) is expressed primarily in the brain, mainly in the choroid plexus and hypothalamic regions such as arcuate nucleus, paraventricular nucleus, ventromedial hypothalamic nucleus and mediolateral hypothalamus,4,9,10,11 but is also widely distributed in peripheral tissues including lung, kidneys, spleen, testes, uterus, lymph nodes, liver, pancreas and adipocytes.4,7,9,12,13,14

Three sisters with LEPR deficiency were recently shown to be characterised by severe early-onset obesity with several metabolic disturbances.15 This is, however, a very rare condition and in most obese humans, no such mutation can be found.10,16 In contrast, several common polymorphisms of the LEPR gene have been uncovered.10,16,17,18,19,20 None of them seems to have a major effect on obesity.10,16,18,20,21,22,23 In the Québec Family Study, significant linkages were observed between LEPR markers and several adiposity and body composition variables, the strongest result being observed between Gln223Arg and fat mass.24 LEPR markers have also been associated with obesity-related phenotypes in Pima Indians,17 in Danish subjects25 and in French Caucasians.21

We have typed three LEPR polymorphisms, all three located in the extracellular binding domain of the receptor, in overweight and obese women, to evaluate whether these gene variants are associated with weight, body composition and fat distribution. The DNA sequence variants resulted in two non-conservative changes (changes in charge): glutamine to arginine at codon 223 (Gln223Arg) in exon 6 as reported previously10 and lysine to asparagine at codon 656 (Lys656Asn) in exon 14; and a conservative change: lysine to arginine at codon 109 (Lys109Arg) in exon 4, both as described earlier.19

Methods

Subjects

Subjects were 280 Caucasian women visiting the outpatient obesity clinic at the University Hospital of Antwerp, who were overweight (25 ≤body mass index (BMI)<30 kg/m2) or obese (BMI≥30 kg/m2). Most subjects were sedentary. Patients with known diabetes were excluded from the study. Subjects defined as being diabetic or with impaired glucose tolerance, based on fasting and 2 h post-glucose load glucose levels using the WHO-criteria,26 were also excluded. Other exclusion criteria were pregnancy, the use of antidepressants or medication known to influence glucose tolerance, appetite or metabolic rate, kidney or liver disease, and specific endocrine diseases such as Cushing's disease or hypothyroidism (TSH-levels above 4 µU/l). All subjects were clinically examined by a physician and shown to be in general good health. The study protocol was approved by the Ethical Committee of the University Hospital Antwerp and subjects gave informed consent.

Anthropometry

All measurements were performed in the morning with the patients in a fasting state. Height was measured to the nearest 0.5 cm, body weight was measured with a digital scale to the nearest 0.1 kg; BMI was calculated as weight (in kg) over height (in m)2. Waist circumference was measured at mid-level between the lower rib margin and the iliac crest, and hip circumference at the level of the trochanter major. The waist-to-hip ratio (WHR) was calculated from these two circumference values. Fat mass (in kg), percentage body fat and fat free mass (in kg) were assessed by bio-impedance (using a BIA-101, RJL Systems, Detroit) as described by Lukaski27 and calculated with the formula of Deurenberg.28 A computerised tomography (CT) scan was performed at the L4-L5 level to determine visceral, subcutaneous and total abdominal fat areas, according to the technique described by Van der Kooy et al.29

Blood sampling

A fasting blood sample was drawn to measure leptin levels in 80 subjects. A serum sample was frozen and stored at −80°C until analysis in batch. Serum leptin levels were measured using a radioimmunoassay, as described previously.30

Molecular analysis

Genomic DNA was extracted from whole blood by a method adapted from Miller et al.31 Human leucocyte nuclei were precipitated, washed and lysed in SDS and pronase (Boehringer Mannheim). Proteins were precipitated by addition of a saturated NaCl solution, and DNA was subsequently precipitated with ethanol and dissolved in a Tris-EDTA solution (pH 7.5). The three restriction fragment length polymorphisms (RFLPs) used have been described elsewhere.10,19 Polymerase chain reaction (PCR) was performed using 100 ng of genomic DNA, 300 nM of each primer, 200 µM dNTPs and 0.5 U Taq polymerase in PCR buffer (Pharmacia Biotech, Baie d'Urfé, Canada) in a total volume of 20 µl. PCR consisted of one cycle of denaturation for 3 min at 95°C, annealing for 1 min at 55°C, and extension for 1 min at 72°C, followed by 40 cycles at 95°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 30 s, and a final extension at 72°C for 10 min (GeneAmp 9600 Thermocycler, Perkin Elmer, Foster City, CA, USA). PCR products were digested overnight at 37°C with 5 U of HaeIII, MspI or BstUI restriction enzymes (New England Biolabs, Mississauga, Canada) for the Lys109Arg, Glu223Arg and Lys656Asn polymorphisms, respectively, and DNA fragments were separated on 3% agarose gel electrophoresis.

Statistical analysis

The normality of the phenotypic distribution was verified for each variable individually, and phenotypes that were not distributed normally were log-transformed, in order to normalise them. A GLM (general linear model) procedure was used to test for differences between the different genotypes at each polymorphism, and results were considered significant if P<0.05. Analyses were performed on phenotypes adjusted for age, menopausal state and fat mass as covariates in the model, except for BMI, fat mass and fat-free mass which were only adjusted for age and menopausal state. Subjects were divided in two groups according to menopausal state: 198 women were premenopausal and 82 postmenopausal. If no clinical data on menopausal state were available, subjects were defined as postmenopausal when age was above 55, or when follicle stimulating hormone (FSH)≥15 U/L (n=3). The interaction between menopausal state and genotype was tested with an interaction term in the model. When this interaction term was significant (P<0.05), the analyses were repeated for pre- and postmenopausal women separately. Since frequencies of the homozygotes for the variant alleles were low in each subgroup, analyses were performed by carrier status, ie carriers of the rare allele (heterozygotes plus homozygotes together) were compared to the noncarriers.

Nine phenotypes were tested for three markers, for a total of 27 tests performed. The Bonferroni-corrected α-level for multiple testing would thus be 0.0019. However, if we apply this correction very strictly the risk of type II error will be increased, and thereby valuable information may be lost. Since the purpose of this study is to explore possible associations between LEPR gene variants and obesity phenotypes, we have chosen to report all results with unadjusted P-values, and take the issue into consideration in the interpretation of the results.

All statistical analyses were performed with the SAS statistical software package (version 6.12). Linkage disequilibrium among the alleles of the three LEPR polymorphisms was tested with the EH program (Columbia University, NY).32

Results

Subject characteristics

The mean age of the subjects was 39±11 (mean±s.d.) y (range 18–60 y). Anthropometric and metabolic characteristics are shown in Table 1. All women were overweight or obese, with a mean BMI of 34.8±5.4, and a range from 25.9 to 53.6 kg/m2. Pre- and postmenopausal women did not differ in body weight, but postmenopausal women had more visceral fat and a larger waist circumference. Metabolic abnormalities which are known to be associated with abdominal fat were also significantly higher in postmenopausal women.

Table 1 Anthropometric and metabolic characteristics of the study subjects

Genotype and allele frequencies

The genotype and allele frequencies for the three LEPR polymorphisms are presented in Table 2. The genotypes at each of the three markers were in Hardy–Weinberg equilibrium.

Table 2 Genotypic and allelic frequencies at the three different polymorphisms of the LEPR gene

The three polymorphisms were in linkage disequilibrium (P<0.001). All 10 subjects who were homozygotes for the Asn variant allele at the Lys656Asn polymorphism were also homozygotes for the wild-type allele at the two other polymorphisms; subjects homozygous for the Arg allele at the Lys109Arg polymorphism were all homozygotes for the Lys allele at the Lys656Asn polymorphism; homozygotes for the Gln allele at the Gln223Arg polymorphism were either homozygotes or heterozygotes for the Lys allele of the Lys109Arg polymorphism, but none of them was homozygote for the variant allele.

Associations of LEPR polymorphisms with leptin levels

Leptin levels were measured in 57 pre- and 23 postmenopausal women. No significant differences across genotypes were observed in premenopausal women (Table 3). In the group of postmenopausal women only, an association with the Lys109Arg polymorphism was found (P=0.02), the Lys109Lys homozygotes showing higher leptin levels compared to the Arg carriers (a mean of 20 ng/dl more), despite the fact that the former had a significantly lower BMI (30.8±1.9 compared to 36.6±2.0, P=0.04).

Table 3 Associations between LEPR genotypes and antropometric phenotypes in obese and overweight women

Associations of LEPR polymorphisms with anthropometric variables

No associations were found between any of the three LEPR polymorphisms and body weight, BMI or fat mass (Table 3). A significant association was found for the hip circumference with Lys656Asn (P=0.03), and for total abdominal fat with Gln223Arg (P=0.04).

A significant interaction effect between menopausal state and the Lys109Arg genotype was seen for BMI (P=0.01), and with the Lys656Asn genotype for subcutaneous and total abdominal fat (both P=0.03). These phenotypes were further analysed in pre- and postmenopausal women separately, and significant results were only found in postmenopausal women (Table 4). In this group, a trend (P=0.08) for a somewhat (2.3 kg/m2) higher BMI was seen in Arg109 carriers. Also in postmenopausal women only, significant associations with Lys656Asn were found for the hip circumference (P=0.04), subcutaneous fat (P=0.03) and total abdominal fat (P=0.04), measured by CT-scan and adjusted for variability in fat mass. Carriers of the Asn656 variant allele showed a 3 cm larger hip circumference, and respectively 58 and 59 cm2 larger subcutaneous and total abdominal fat areas than the Lys656Lys homozygotes. An association (P=0.03) was also found between total abdominal fat and Gln223Arg in postmenopausal women, with the Gln223Gln homozygotes having on average 64 cm2 more total abdominal fat than the Arg223 allele carriers. A similar trend was seen for subcutaneous fat (P=0.08).

Table 4 Significant differences between LEPR genotypes in antropometric phenotypes in pre- and postmenopausal women separately

Discussion

In this study, we evaluated whether polymorphisms in the LEPR gene were associated with obesity phenotypes, especially with body composition and abdominal fat level, adjusted for total body fat. To this end, three LEPR polymorphisms were typed in a group of overweight and obese women, with a wide range of BMI, in whom extensive anthropometry was performed, including intra-abdominal fat distribution by CT-scan.

The genotype and allele frequencies were comparable to those reported previously in obese as well as in normal-weight Caucasian subjects.18,20,24,33,34 A linkage disequilibrium was observed between the three markers which had been previously reported for Gln223Arg and Lys656Asn,22 but not for Lys109Arg.

Previous reports on these LEPR polymorphisms have not found any association between them and BMI10,20 or percentage of body fat.17 In one study, a small effect on BMI was reported for the Lys656Asn polymorphism, but this was found only in a lean population, and was not observed in obese subjects.20 In Pima indians, three other polymorphisms in the LEPR gene were found to be associated with differences in percentage body fat, but no associations with body fat were found for the LEPR polymorphisms studied here.17 In the Québec Family Study, linkage was observed between Gln223Arg and fat mass, BMI, skinfold measurements and fat-free mass.24 However, no significant associations were found between the three polymorphisms and body composition phenotypes after adjusting for age, sex and height, except for a weak association between fat-free mass and Lys656Asn in obese females.24 In the Heritage Family Study, linkage was observed for Lys109Arg with BMI and fat mass, and an association with BMI and fat mass was reported for the Gln223Arg, but only in Caucasian males (parental generation).33 Preliminary results of a meta-analysis of nine different studies, with a total of 3269 subjects, males and females, showed no significant association of any of these three LEPR genotypes with BMI or waist circumference, after controlling for age and sex.34

In the present study, we also could not find any associations between the LEPR markers and BMI and body fat. However, some trends were observed for body fat distribution phenotypes. Associations between Lys656Asn and hip circumference, total abdominal and subcutaneous fat, and between Gln223Arg and abdominal fat were observed in postmenopausal women. These results were obtained after adjustment for fat mass. It should be noted that waist circumference reflects primarily total abdominal fat, both visceral and subcutaneous, while hip circumference gives an estimate of gluteal subcutaneous fat, but also of muscle mass.

An association between blood leptin levels and subcutaneous fat distribution was reported previously, a phenomenon that is thought to be related to the higher leptin secretion by subcutaneous fat compared to omental fat.35 In this study, we also find an association of this subcutaneous fat depot with some polymorphisms in the leptin receptor gene, at least in obese postmenopausal women. It is known that leptin receptors are present on adipose tissue, and it was suggested that these could mediate an autocrine or paracrine function of leptin. The association which we found with subcutaneous fat suggests that leptin is not only secreted by these cells, but that it could also affect fat accumulation in these cells acting through the leptin receptor. Moreover, variants of the LEPR gene may predispose to an increased abdominal fat accumulation over time.

We are aware that since our population is limited to overweight and obese subjects, we have restricted the range of BMI and other obesity-related variables. This reduction in range has potentially decreased our ability to identify LEPR variant effects as no comparison could be undertaken between normal-weight and obese women. Nevertheless, the range of BMI is still large in this group of women, with values from 25.5 to 53.6.

Leptin levels have been shown to vary widely for a given degree of adiposity, and it has been proposed that they could partly be modulated by genetic factors. An effect of genetic polymorphisms on leptin levels was shown previously for polymorphisms in LEP, PPARγ, and POMC genes.36,37,38 Previous studies found associations between leptin levels and the LEPR Lys109Arg polymorphism39 or with the Gln223Arg polymorphism,40,41 but none of these was independent of BMI. We find an association between blood leptin levels and the Lys109Arg polymorphism in the LEPR gene in postmenopausal women, with significantly higher leptin levels in the Lys109Lys homozygotes, despite a lower body weight, BMI and fat mass in this subgroup. However, this postmenopausal group of women in whom leptin levels were measured was very small, so that no conclusions can be drawn from this.

When testing for differences between carriers of the rare allele and non carriers instead of comparing all three genotypes, recessive effects will be missed particularly if the number of homozygotes for the rare allele is small. Under the conditions of this study, only additive and dominant effects of the rare alleles are likely to be detected. Because of the limited sample size and especially the number of homozygotes for the less frequently occurring variants, we however had no choice but to perform the analyses as performed.

In addition, it is important to remember that the P-values were not adjusted for multiple testing. Bonferroni adjusted P-levels would have been as low as 0.0019 compared to 0.05. However, we have elected to report differences significant at 0.05 in order to make sure that all potentially useful leads are identified for subsequent studies. This has led us to the observation that LEPR sequence variants may be involved in the predisposition to become abdominally obese.

In conclusion, frequently occuring genetic polymorphisms in the leptin receptor gene are not associated with total adiposity, but seem to be associated mainly with subcutaneous fat accumulation in postmenopausal overweight and obese women when data are adjusted for total body fat level. These observations suggest that mutations in the leptin receptor gene play a role in subcutaneous abdominal fat accumulation in women who have an excess amount of body fat.

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Acknowledgements

The authors wish to acknowledge the collaboration of Jan Vertommen and co-workers at the Laboratory of Endocrinology (Professor I De Leeuw) at the University of Antwerp for DNA-extraction and handling of the samples, and to Lin Gan, M.D., of the Physical Activity Sciences Laboratory, Laval University, Québec, for his contribution to the DNA studies. The genetic studies of C Bouchard and his colleagues were funded by the Medical Research Council of Canada (PG-11811, MT-13960 and GR-15187). C Bouchard is currently partially supported by the George A Bray Chair in Nutrition.

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Wauters, M., Mertens, I., Chagnon, M. et al. Polymorphisms in the leptin receptor gene, body composition and fat distribution in overweight and obese women. Int J Obes 25, 714–720 (2001). https://doi.org/10.1038/sj.ijo.0801609

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Keywords

  • leptin receptor
  • obesity
  • body composition
  • abdominal fat distribution
  • leptin
  • genetic polymorphisms

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