Polymorphism of Trp64Arg in β 3 -adrenergic receptor gene and serum LDL-cholesterol concentrations in healthy Japanese

Abstract

Background

Polymorphism of Trp64Arg in the β₃-adrenergic receptor (β₃-AR) gene may play a critical role in lipid and lipoprotein metabolism by mediating lipolysis and thermogenesis. Since the frequency of Arg alleles of the β₃-AR gene is generally low among many populations, studies on the Arg/Arg genotype in relation to lipid and lipoprotein metabolism are required in countries such as Japan which has a relatively high frequency of the Arg allele.

Methods

We genotyped 275 clinically healthy Japanese (male/female, 134/141, mean 45.7 years) without medication for β₃-AR gene polymorphism by polymerase chain reaction–restriction fragment length polymorphism analysis, and investigated the effects of the gene polymorphism on clinical parameters including body mass index (BMI), blood pressure and serum lipid and lipoprotein concentrations.

Results

The genotype frequencies were: Trp/Trp, 68.0%; Try/Arg, 28.0% and Arg/Arg, 4.0%, with an allele frequency of 0.18 for Arg64. When subjects were divided into three groups according to the genotype, a significant increase of serum LDL-cholesterol (LDL-C) concentration in the Arg/Arg group (3.48 ± 1.59 mmol/L) was observed when compared with those of the Trp/Trp and Arg/Trp groups (3.15 ± 0.80, 3.25 ± 0.92 mmol/L, respectively). Genotype differences did not show any significant effect on other parameters. Spearman's rank correlation demonstrated a significant relationship between LDL-C concentrations and the number of Arg alleles, age and BMI. Multiple regression analysis, using LDL-C concentration as a criterion variable and some factors including β₃-AR gene polymorphism as explanatory variables, revealed that the number of Arg alleles was a significant and independent factor for LDL-C concentrations, along with age and BMI.

Conclusions

These findings suggested a role of the β₃-AR gene polymorphism in regulating lipid and lipoprotein metabolism, showing small but significant effects on elevated LDL-C values in subjects with Arg/Arg, but not Trp/Arg and Trp/Trp genotypes.

Introduction

There has been a report that obesity and insulin resistance are associated with increased serum concentrations of LDL-cholesterol (LDL-C) and triglycerides. ¹ The β₃-adrenergic receptor (β₃-AR), expressed mainly in visceral fat, can affect lipid and lipoprotein metabolism by mediating lipolysis and thermogenesis, relating to resting metabolic rate and body fat accumulation. ² Polymorphism of Trp64Arg in the β₃-AR gene is a possible genetic factor for the development of obesity and insulin resistance in several ethnic groups, whose polymorphism may be involved in the development of atherosclerosis. ^1–4

Although some relation of β₃-AR Trp64Arg polymorphism to lipid and lipoprotein metabolism (i.e. triglycerides) has been suggested ^2,4–6 , the effects of this polymorphism have still not been sufficiently understood, especially in healthy people. As the Arg allele frequency is generally low, studies with a substantial number of Arg-homozygous subjects are still needed. ⁷ The allele frequency in Japanese subjects, although lower than in Pima Indians, is higher than in other populations such as Finns and Caucasians. ³ We investigated the association of β₃-AR gene polymorphism and serum lipid and lipoprotein concentrations in healthy Japanese subjects.

Materials and methods

A total of 275 asymptomatic subjects (male/female, 134/141), aged 35–50 years (mean 45.7 ± 5.8 [SD]), were enrolled. All subjects were randomly recruited from general community volunteers and outpatients for health check-up. None of these subjects were taking any medication. The study was approved by the Ethics Committee of Tottori University and each subject gave informed consent. We measured blood pressure in the right upper arm of seated patients using a standard sphygmomanometer. Fasting serum total cholesterol and triglyceride values were measured by the enzymatic methods, and HDL-cholesterol (HDL-C) values were measured by a homogenous method. LDL-C values were determined using a homogeneous enzymatic direct method (Cholestest LDL^®-LDL assay system, Daiichi Pure Chemicals Co, Ltd, Japan). The β₃-AR gene Trp64Arg polymorphism was detected using the polymerase chain reaction (PCR)–restriction fragment length polymorphism method. ⁵ Genomic DNA was extracted from peripheral white blood cells. The PCR primers were 5′-CCAATACCGCCAACACCAGT-3′ (upstream) and 5′-AGGAGTCCCATCACCAGGTC-3′ (downstream), which flank the whole exon 1 of the β₃-AR gene. In a total reaction mixture of 25 µL, 75 ng of genomic DNA was used for PCR. The PCR amplification was performed for 30 cycles (94ºC for 30 s, 67ºC for 30 s, 72ºC for 30 s). Five microlitres of PCR product was then incubated for 1 h with 10 U of MvaI at 37ºC in a 10 µL final volume. The samples were run on a 3.0% agarose gel, stained with ethidium bromide. In the presence of the β₃-AR gene polymorphism (the T to C transversion at codon 64), the restriction site for MvaI is lost; therefore, the allele of this polymorphism corresponds to the 158 base-pair undigested band. Thus, Trp-homozygotes (Trp/Trp), heterozygotes (Trp/Arg) and Arg-homozygotes (Arg/Arg) were genotyped.

The prevalence of genotypes was compared by chi-square test. The differences among groups were analysed by analysis of variance, followed by Scheffe's post hoc test. The relation between LDL-C concentrations and influential factors such as Arg allele number, gender, age and body mass index (BMI) was examined by Spearman's rank correlation test and multiple regression analysis. P<0.05 was considered significant.

Results

The genotype frequencies of β₃-AR gene were 68.0, 28.0 and 4.0% for the Trp/Trp, Trp/Arg and Arg/Arg, respectively (Table 1). The Arg allele frequency was 0.18. Genotype frequencies were in Hardy-Weinberg equilibrium. There were no significant differences in the subjects' clinical characteristics according to genotype, except for LDL-C concentrations. Subjects carrying the Arg/Arg genotype had significantly higher LDL-C concentrations than subjects with Trp/Trp and Trp/Arg. The influence of β₃-AR gene polymorphism on clinical parameters was not different between men and women.

Table 1

Characteristics of the subjects by β₃-adrenergic receptor gene Trp64Arg polymorphism

	Genotype of Trp64Arg polymorphism
	Trp/Trp	Trp/Arg	Arg/Arg
Subject number (n)	187	78	10
Gender (male/female)	85/102	45/33	4/6
Age (years)	45.7 ± 5.8	46.0 ± 5.3	45.2 ± 3.9
Body mass index (kg/m2)	22.7 ± 2.9	22.3 ± 2.7	22.1 ± 2.5
Total cholesterol (mmol/L)	5.49 ± 0.90	5.70 ± 0.99	5.99 ± 1.32
Triglyceride (mmol/L)	1.29 ± 1.22	1.34 ± 1.38	1.45 ± 0.73
HDL-cholesterol (mmol/L)	1.80 ± 0.49	1.84 ± 0.52	1.61 ± 0.46
LDL-cholesterol (mmol/L)	3.15 ± 0.80*	3.25 ± 0.92†	3.48 ± 1.59*,†
Systolic blood pressure (mmHg)	121.6 ± 16.9	123.0 ± 23.2	117.2 ± 12.3
Diastolic blood pressure (mmHg)	75.4 ± 12.3	76.4 ± 13.6	76.3 ± 11.2

Values are presented as mean ± SD. Male gender and female gender present respective subject numbers

*^,† P < 0.05; Arg/Arg vs. Trp/Arg vs. Trp/Trp. P values were calculated using analysis of variance, followed by Scheffe's procedure among groups

Spearman's rank correlation showed that the number of Arg allele in individuals (ρ = 0.129, P = 0.04), age (ρ = 0.151, P = 0.02) and BMI (ρ = 0.294, P < 0.01) were significantly associated with LDL-C concentrations but gender was not (ρ = 0.098, P = 0.13). After adjusting for age, gender, BMI and Arg allele, Arg allele numbers were significantly and independently associated with LDL-C concentrations (β = 0.142, P = 0.02), as were age (β=0.125, P = 0.04) and BMI (β = 0.266, P < 0.01) but not gender (β = 0.065, P = 0.30).

Discussion

It has been reported that subjects with the Arg allele showed increased ^2,5 /decreased ⁶ serum triglyceride concentrations and decreased serum HDL concentrations ⁵ and that those with Arg-Arg genotype showed smaller LDL particle size. ⁴ This study raised the notion of a small but close relationship between Arg/Arg genotype and serum LDL-C concentrations. The mechanism remains unclear, but the β₃-AR polymorphism seems to have some effects on LDL-C regulation. It might be presumed that the β₃-AR gene polymorphism regulates lipolysis and energy expenditure ² , resulting in the change of LDL-C metabolism.

It was notable that subjects with Arg/Arg, but not Trp/Arg, had higher LDL-C values. We observed a relatively higher prevalence of β₃-AR Trp64Arg gene polymorphism in apparently healthy Japanese subjects, but a study limitation was that there were still small samples of Arg/Arg. In previous studies, subjects with Arg/Arg were grouped with those of Trp/Arg to mix effects of two genotypes and mask natural results. ⁷ For example, Arg/Arg contributed to the pathogenesis of obesity in a fraction of subjects, but not to a large enough degree to influence the traits of the whole population. ⁷ The effects of β₃-AR gene polymorphism on LDL-C may have previously been masked.

Another study limitation was no evaluation of menstrual states, which might somewhat affect lipid and lipoprotein regulation, among female subjects. Since the average age of menopause in Japanese women is approximately 50.5 years, postmenopausal subjects might be included in the study. Despite this, the mean age and gender ratio were not significantly different between subject groups according to genotype, and adjustment for age and gender was performed in this study.

Increased serum LDL-C value is a risk factor for atherosclerosis. ⁴ More studies into whether β₃-AR polymorphism is a genetic factor in the development of atherosclerosis are required to acquire deeper knowledge in this field.