Polymorphisms in the leptin and leptin receptor genes in relation to resting metabolic rate and respiratory quotient in the Québec Family Study

Abstract

Background:

Leptin (LEP) is an endocrine hormone that participates in many metabolic pathways, including those associated with the central regulation of energy homeostasis.

Objective:

We examined the associations between polymorphisms in the LEP and leptin receptor (LEPR) genes and resting metabolic rate (RMR) and respiratory quotient (RQ) in the Quebec Family Study.

Methods and subjects:

Three polymorphisms in LEPR (K109R, Q223R and K656N) and one in LEP (19A>G) were genotyped in 678 subjects. RMR, RQ at rest and RQ while sitting, standing and walking at 4.5 km/h (RQ45) were adjusted for age, sex, fat mass and fat-free mass.

Results:

RQ45 was associated with the LEPR-K109R (P=0.004) and Q223R (P=0.03) polymorphisms, and RMR showed association with the LEPR-K656N polymorphism (P=0.006). For the LEP-19A>G polymorphism, no significant associations were observed. However, LEP-A19A homozygotes who were carriers of the LEPR N656 allele had a significantly lower RQ45 compared to other genotype combinations (P for interaction=0.003).

Conclusion:

These findings suggest that DNA sequence variation in the LEPR gene contributes to human variation in RMR and in the relative rates of substrate oxidation during low-intensity exercise in steady state but not in a resting state.

Introduction

Leptin is an endocrine hormone that is highly conserved among different species, including man.1 Leptin participates in many metabolic pathways.2 One of its key roles is that of communicating to the brain information on long-term energy stores. Leptin is primarily released from the adipocytes at levels approximately proportional to the body fat content and signals to the brain in proportion to its plasma concentration. The primary site of leptin action is the hypothalamus where it triggers a cascade of neuroendocrine responses that result in the inhibition of orexigenic peptides, while stimulating anorexigenic peptides.2 The ob/ob mice, which are leptin deficient due to a point mutation in the LEP gene, are characterized by early-onset obesity, hyperphagia, low body core temperature, reduced energy expenditure and insulin resistance.1 Administration of leptin to the ob/ob mice induces weight loss through inhibition of food intake and stimulation of energy expenditure.3, 4, 5

In humans, only a few patients carrying a mutation in the LEP gene have been described.6, 7, 8 All had extremely low serum leptin levels and exhibited early-onset morbid obesity with hyperphagia. Subcutaneous administration of recombinant human leptin for up to 4 years had major and sustained beneficial effects on the multiple phenotypic abnormalities associated with the congenital human leptin deficiency.9 Findings from some previous studies in humans have supported the relationship between leptin and energy expenditure,10, 11, 12 although others failed to find such a relationship.13, 14 In addition to its central effects on metabolism, leptin has been shown to act on peripheral tissues. Leptin induces lipolysis in adipocytes without increasing the release of free fatty acids and increases lipid oxidation in Zucker rats and ob/ob mice.5 Studies in humans have confirmed this relationship, as elevated leptin concentrations were shown to be related to increased fat oxidation and decreased respiratory quotient (RQ) and carbohydrate oxidation.15, 16, 17 Sequence variations in the LEP gene have been associated with body weight, BMI (kg/m2) and obesity, although not all studies have been able to confirm this relationship.18 No significant associations between LEP polymorphisms and energy metabolism or substrate oxidation have been reported.

The leptin receptor is expressed primarily in the brain, mainly in the choroids plexus and hypothalamic regions, but is also widely distributed in peripheral tissues.19, 20 The leptin receptor gene (LEPR) was originally cloned from the mouse choroids plexus.20 The db mutation generates a canonical splice donor site that prevents the translation of a terminal exon encoding a STAT-activating motif.19 Phenotypically, the db/db mice are almost similar to the ob/ob mice. Different mutations in the LEPR gene have also been identified in two rat models for obesity, the Zucker and Koletsky rats.21, 22, 23, 24 Sequence variations in the LEPR gene in humans were associated with substrate oxidation in obese women11 and 24 h energy expenditure in nondiabetic Pima Indians.25 A genome-wide linkage scan in Pima Indians for energy metabolism phenotypes showed evidence for a quantitative trait locus for 24 h RQ close to the LEPR gene (1p31–p21).26

In this study, we tested whether sequence variations in the LEPR and LEP genes contribute to the variation in resting metabolic rate (RMR) and RQ in the Quebec Family Study (QFS). RMR and RQ are considered as intermediate phenotypes of energy balance and body composition.

Materials and methods

Study population

The aims and design of the cohort have been described previously.27 In the present study, based on Phase 2 of the QFS project, 678 individuals from 189 French–Canadian families living in and around the greater Québec City area are included (Table 1). Informed written consent was obtained from all subjects and the QFS project had been approved by the Medical Ethics Committee of Laval University.

Table 1 Descriptive characteristics of subjects

Phenotypes

RMR and RQ at rest were measured using a ventilated hood and an open-circuit indirect calorimeter, as described previously.28 Measurements were made early in the morning, after an overnight fast, while participants were lying quietly in a semireclined position. Respiratory exchange data from the final 10 min of the 30-min data collection period were used to calculate RMR and RQ. Immediately after resting, RQ was measured in steady state while participants were consecutively sitting (RQsit), standing (RQstand) and walking at 4.5 km/h on a treadmill (RQ45). For each condition, respiratory exchange ratios were collected during 8 min. Gas samples were assayed with a zirconia cell O2 analyzer (Amatek CD-3A, Thermox Instruments Division, Pittsburgh, PA, USA) and an infrared CO2 analyzer (Amatek S-3A). Analyzers were calibrated before each test using gases of known percentages of O2 and CO2. RMR is expressed in kilocalories per minute of energy expenditure, whereas RQ is simply the ratio of CO2 produced to O2 consumed.

Measurements of fat mass (FM) and fat-free mass (FFM) were obtained from underwater weighing using the conversion factor of Siri,29 as described previously.30 Body density measurements were made according to the procedures of Behnke and Wilmore,31 whereas residual lung volume was determined using the helium dilution technique.32

Genotypes

Genomic DNA was prepared from permanent lymphoblastoid cells by proteinase K and phenol/chloroform technique. DNA was dialysed four times against TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) for 6 h at 4°C and precipitated. We genotyped one polymorphism in the LEP gene and three in the LEPR gene.33, 34 Polymerase chain reaction (PCR) was performed using 100 ng genomic DNA, 30 pmol of each primer, 250 μ M dNTPs and 1 U Taq polymerase in PCR buffer (Boehringer Mannheim, Mannheim, Germany) with 1.5 mM MgCl2 for a final volume of 20 μl. PCR cycles consisted of 30–35 cycles at 94°C for 30 s, annealing at 55–58°C for 30 s and extension at 72°C for 30 s (Perkin-Elmer 9600). PCR products were digested for 12 h at 37°C with 10 U of HaeIII, 20 U of MspI, 10 U of MvnI and 2 U of NspBII restriction enzymes for the K109R (dbSNP database: rs1137100), Q223R (dbSNP database: rs12131454) and K656N (dbSNP database: rs8179183) LEPR polymorphisms and the 19A>G LEP polymorphism (dbSNP database: rs2167270), respectively.

Statistical analyses

All the analyses were performed with the SAS Statistical Software (SAS Institute Inc., Cary, NC, USA). A likelihood ratio test was performed to assess whether the observed genotype frequencies were in Hardy–Weinberg equilibrium. The pairwise linkage disequilibrium (LD) between the SNPs was assessed using the ldmax program, implemented in the GOLD software package,35 which uses the expectation–maximization algorithm of Slatkin and Excoffier.36

Associations between the gene markers and the phenotypes were analyzed using the MIXED model procedure, comparing the three genotypes. RMR and RQ measurements were adjusted for sex, age, age2, age3, FM and FFM, as these covariates contributed independently to the variance of these phenotypes, by including them into the model. Subjects were also categorized into carriers and noncarriers of the rare allele, to test for dominant effects. Nonindependence among family members was adjusted for by using a ‘sandwich estimator’, which asymptotically yields the same parameter estimates as ordinary least-squares or regression methods, but the standard errors and resulting hypothesis tests are adjusted for the dependencies.37, 38 The method is general, assuming the same degree of dependency among all members within a family. Since the K109R and Q223R polymorphisms were in moderate LD, we tested for the independent contribution of the K109R variant by including the Q223R variant in the model and vice versa.

Haplotypes were estimated using the best option of the MERLIN haplotype function, which provides haplotypes according to the most likely pattern of gene flow.39 Associations between haplotypes and phenotypes were performed using the MIXED model procedure. Only the subjects with unambiguous haplotypes were included in the analyses.

Possible gene-by-gene (including interactions between variants within the LEPR gene), sex-by-gene, BMI-by-gene (<30 vs 30 kg/m2), generation-by-gene, diet-by-gene (energy percentage of macronutrients), smoking status-by-gene and physical activity-by-gene interactions were tested with the MIXED model procedure by including main effects and interaction terms in the same model.

Results

The phenotypic characteristics of the 678 subjects are shown in Table 1. Women had a significantly (P<0.0001) lower RMR compared to men and offspring had a higher RMR compared to parents (P=0.01 in men, P=0.0004 in women). Mothers had a significantly (P=0.04) lower RQ at rest and while standing, but a higher (P=0.007) RQ while walking compared to the fathers. Offspring had a lower RQ45 compared to parents (P=0.05 for men, P=0.007 for women).

The allele frequencies of the common alleles were 0.64 for the LEP 19A>G polymorphism and 0.70, 0.55 and 0.79 for the K109R, Q223R and K656N LEPR polymorphisms, respectively. All genotype frequencies were in Hardy–Weinberg equilibrium. The three polymorphisms in the LEPR were in LD. The disequilibrium was stronger between K109R and Q223R (r2=0.40, D′=0.88) than between K109R and K656N (r2=0.07, D′=0.85) or between Q223R and K656N (r2=0.19, D′=0.88).

RQ45 showed significant associations with the K109R and the Q223R LEPR polymorphisms. The K109K homozygotes as well as the Q223Q homozygotes had significantly lower RQ45 than the other two genotypes. When analyzed by carrier status (K/K vs R-carrier for K109R and Q/Q vs R-carrier for Q223R), the associations were even stronger; P=0.0008 and 0.006 respectively. Obesity (BMI <30 vs 30 kg/m2) modified the association between RQ45 and the K109R LEPR polymorphism (P for interaction=0.0017); a significant association was observed only in the nonobese subjects (Table 2).

Table 2 Associations between leptin (LEP) and leptin receptor (LEPR) gene polymorphisms and resting metabolic rate (RMR) and respiratory quotient (RQ) measures

Figure 1 summarizes the associations between the K109R LEPR polymorphism and the four RQ measurements for all (A) and for nonobese (B) subjects. Especially for RQ in the nonobese subjects, the R109R homozygotes seem to respond differently to increasing activity than K-carriers. The K-carriers showed a steep decrease in RQ from lying to sitting, which remained low while standing and walking. The R109R homozygotes showed a stepwise reduction in RQ, which was less pronounced compared to the K-carriers.

Figure 1
figure1

Associations between the K109R LEPR polymorphism and RQ at rest and RQ while sitting, standing and walking at 4.5 km/h for all (a) and nonobese (b) subjects (P-values indicate results of the association analyses).

The K109R and Q223R polymorphisms are in moderate LD. Therefore, we tested for the independent contribution of the K109R variant by including the Q223R polymorphism in the model. The association between K109R and RQ45 remained significant (P=0.03 for all subjects, P=0.008 for nonobese subjects).

The association between RQ while walking and the Q223R LEPR polymorphism was more pronounced in the offspring (generation-by-gene interaction P=0.005), especially when Q223Q homozygotes were compared to R-carriers (P=0.0005) (Table 2).

The association between the Q223R variant and RQ45, independent from the K109R variant, was only significant in the offspring (P=0.04) but not in the total population (P=0.84).

RQ at rest showed no association with any of the three LEPR polymorphisms.

RMR was significantly associated with the K656N LEPR polymorphism, with the N656N homozygotes having a higher RMR compared to the other two genotypes (Table 2). The BMI-by-gene interaction was not significant (P=0.6).

The LEPR haplotypes could be unambiguously determined for 629 (92.8%) of all the subjects (Table 3). The LEPR haplotypes did not provide any additional information beyond the single markers. Example given, haplotypes containing the R-allele of the K109R variant showed higher RQ45 values as compared to haplotypes containing the K-allele of the K109R variant, regardless of the other alleles in the haplotype. This suggests that the results were driven by the presence of the K109R SNP, whereas the effect of the Q223R variant was secondary.

Table 3 Frequencies of LEPR haplotypes (K109R, Q223R and K656N) based on 629 subjects

We found no association with the LEP 19A>G polymorphism. However, a significant interaction (P=0.003) was observed between the LEP 19A>G and LEPR K656N polymorphism for RQ45 (Figure 2). Homozygotes for LEP A19A who were carriers of the LEPR N656 allele had a significantly lower RQ45 compared to the other genotype combinations. Interactions with dietary composition, tobacco use or physical activity level were not observed.

Figure 2
figure2

Interaction for RQ while walking at 4.5 km/h between the 19A>G LEP and K656N LEPR polymorphisms.

Discussion

Our results support the hypothesis that sequence variation in the LEPR gene is associated with RMR and RQ in humans. Significant associations were found between the K109R and Q223R LEPR polymorphisms and RQ45, whereas the K656N LEPR polymorphism showed an association with RMR. The 19A>G polymorphism in the LEP gene had no effect on RQ or RMR. However, a significant interaction was observed between 19A>G LEP and K656N LEPR polymorphism.

The 19A>G LEP polymorphism is located in the first noncoding exon of the gene.34, 40 Two of the LEPR polymorphisms result in a nonconservative change and are therefore the most likely to have functional consequences: glutamine to arginine at codon 223 (Q223R) in exon 6 and lysine to asparagine at codon 656 (K656N) in exon 14.33, 41 However, it is not known what the exact functional implications of the variant are on the LEPR mRNA and protein. The K109R LEPR polymorphisms resulted in a conservative change: lysine to arginine at codon 109 in exon 4.33, 41 Although the 19A>G LEP and the K109R LEPR polymorphism may not have functional implications, it is possible that they are in LD with other functional mutations.

In the present study, the K109R LEPR polymorphism was significantly associated with RQ45. K109K homozygotes had significantly lower RQ compared to the R109-allele carriers. In nonobese subjects, tendencies towards a lower RQ in the K109K homozygotes were also observed while sitting and standing (Figure 1). While there is no difference in RQ at rest between the three genotypes in nonobese subjects, slight increases in exercise intensity seem to increase fat oxidation (lower RQ) in the K109-allele carriers, but much less so in the R109R homozygotes. This suggests that the R109R homozygotes have a less responsive substrate oxidation phenotype that may impact on everyday activities. No other studies have reported results on associations between this K109R LEPR variant and RMR or RQ.

In young adult Pima Indians, the Q223R LEPR polymorphisms was associated with 24 h energy expenditure and physical activity level, but not with 24 h RQ.25 Homozygotes for the R223 allele had a lower 24 h energy expenditure and physical activity level. Although this LEPR variant did not seem to have an effect on whole body lipid oxidation, the R223R homozygotes had a higher subcutaneous abdominal adipocyte size, which has been shown to be related with excessive fat storage.42 In the present study, no association between Q223R and RMR was found. However, carriers of the R223 allele had a significantly higher RQ45, which was more marked in the young adult offspring. In the HERITAGE Family Study, carriers of the R-allele had a significantly higher BMI43 than Q223Q homozygotes, and in two Caucasian populations, the prevalence of the R223 allele was higher among overweight and obese than among nonoverweight adults.44, 45 In a study with postmenopausal White women, the Q223R LEPR polymorphism was found to be associated with a lower binding capacity of leptin to the soluble form of the receptor in plasma, which was accompanied by a higher BMI, FM and plasma leptin concentration.46 In contrast, postmenopausal overweight and obese women who were R223-allele carriers had less abdominal fat.47

In a group of overweight and obese women, a significant association was found between the K656N LEPR polymorphism and substrate oxidation, especially after a glucose load.11 Carbohydrate oxidation in the K656K homozygotes was 15% higher than in the N656-allele carriers, and fat oxidation was 44% lower.11 In the present study, the K656-allele carriers showed a significantly lower RMR, but no significant association was found between the K656N polymorphism and substrate oxidation rates. However, a significant interaction between 19A>G LEP and K656N LEPR was observed for RQ45. Homozygotes for the A19A LEP variant who are carriers of the N656 allele exhibited a higher fat oxidation while walking (lower RQ45). The lower RQ might suggest that this genotype combination results in a more efficient leptin–leptin receptor signaling compared to the other genotypes.

We found no associations between the 19A>G LEP variant and RMR or RQ, which is in agreement with findings in an Italian48 and a Finnish40 obese population. Obese individuals who carry the A19 allele showed significantly higher leptin concentrations compared to obese patients who were G19G homozygotes.34 However, these results were not confirmed by others.40 A recent study provided strong evidence for association between five new SNPs in the 5′ region of the LEP gene and BMI.49 These SNPs are predicted to modify transcription-factor binding sites. Therefore, we will consider genotyping these SNPs in the current population to study their association with obesity-related phenotypes as well as with RMR and RQ.

Interactions with obesity and generation were observed for the LEPR variants. The effects of the K109R LEPR variant on RQ45 and of the K656K LEPR variant on RMR were significant in the nonobese, but absent in the obese subjects. An age effect was found for the Q223R LEPR variant on RQ45, with a stronger association in the offspring. Dietary composition, as assessed by energy percentage from fat, carbohydrates or protein, or physical activity levels did not modify the effect of the LEP and LEPR polymorphisms on RQ and RMR.

The mechanism by which leptin exerts its effects on energy expenditure and substrate oxidation is complex and several pathways have been defined. Leptin stimulates sympathetic outflow, and hypoleptinemia or leptin resistance may lead to reduced activity of the sympathetic nervous system and thus to reduced RMR and increased RQ.50 Further, animal studies suggested that the pro-opiomelanocortin pathway might play an important role.51 Administration of leptin to the ob/ob mice not only resulted in an increase in the central melanocyte-stimulating hormone (MSH) levels but also in the peripheral MSH levels. Increased MSH levels result in a central inhibition of appetite mediated through melanocortin-4 receptors (MC4-R) expressing neurons, whereas peripheral cells, especially adipocytes expressing melanocortin receptors, mediate increased metabolic rate and lipolytic activity.51 Although two of the polymorphisms (Q223R, K656N) result in a nonconservative change and, therefore, may have functional consequences, the current study cannot provide conclusive evidence that these variants are causal.

In conclusion, these findings suggest that DNA sequence variations in the LEPR gene contribute to variation in RMR and in the relative rates of substrate oxidation during low-intensity exercise in steady state. N656N LEPR homozygotes had a higher RMR, and K109K LEPR homozygotes as well as Q223Q LEPR homozygotes had a lower RQ45 compared to the other genotypes of the respective LEPR gene variants. No association was found between the LEPR gene variants and substrate oxidation at rest. Sequence variation in the LEP showed an association with substrate oxidation while walking, but only in interaction with an LEPR gene variant.

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Acknowledgements

The Québec Family Study was supported by multiple grants from the Medical Research Council of Canada and the Canadian Institutes for Health Research (PG-11811, MT-13960 and GR-15187). R Loos is supported by a postdoctoral fellowship from the American Heart Association; Southeast affiliate (no. 0325355B). C Bouchard is partially supported by the George A Bray Chair in Nutrition.

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Correspondence to C Bouchard.

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Loos, R., Rankinen, T., Chagnon, Y. et al. Polymorphisms in the leptin and leptin receptor genes in relation to resting metabolic rate and respiratory quotient in the Québec Family Study. Int J Obes 30, 183–190 (2006). https://doi.org/10.1038/sj.ijo.0803127

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Keywords

  • leptin
  • leptin receptor
  • resting metabolic rate
  • respiratory quotient

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