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Clinical Studies |
Third Department of Internal Medicine (T.N., M.Y., S.K., M.N., N.K., T.A., N.N.), National Defense Medical College, Tokorozawa, Saitama; and the Department of Internal Medicine, Self-Defense Force Central Hospital (A.A., H.Y.), Setagaya, Tokyo, Japan
Address all correspondence and requests for reprints to: Dr. Terumasa Nagase, Third Department of Internal Medicine, National Defense Medical College, Tokorozawa, Saitama, Japan.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The ß3-adrenergic receptor (ß3AR), expressed mainly in brown and white adipose tissues in rodents (4, 5, 6) and in visceral fat in humans (7), is involved in the regulation of lipolysis and thermogenesis (8, 9). Recent studies in Pima Indians, Finns, and Caucasians have shown that a missense mutation in the ß3AR gene, resulting in the replacement of tryptophan by arginine at position 64 (Trp64Arg), is associated with earlier onset of noninsulin-dependent diabetes mellitus (NIDDM), decreased resting metabolic rate, insulin resistance, and weight gain (10, 11, 12). These observations suggest that the genotype of ß3AR is one of the genetic determinants of body weight and a potential predisposing factor to NIDDM. However, none of the previous studies have examined in detail the relation between the mutation and body weight gain or BMI changes during adult life.
The present study was conducted to address the question of whether and how the ß3AR genotype affected BMI changes from young adulthood to middle age. For this purpose we chose a group of subjects who were homogeneous concerning race, sex, age, and occupation and had longitudinal records of body weight. As it is well known that BMI increases with age, and the age-related changes in BMI differ between males and females (13), we thought the current protocol was better than commonly used ones that included subjects of both sexes over a wide age range with different social backgrounds. We also examined whether the prevalence of the mutation differed between NIDDM and normal populations.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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One hundred and eighty-six Japanese men, consecutively admitted to the Self-Defense Force (SDF) Central Hospital between November 1995 and April 1996, were examined to determine the prevalence of the Trp64Arg mutation in an unselected and unrelated population and to assess the possible influence of the mutation on body weight and BMI. They were relatively homogeneous group of subjects, born between 1939 and 1944, who had worked as SDF personnel for 29–38 yr and been examined at 51–56 yr of age during their checkup before retirement. Most of them had records of body weight measured yearly for more than 25 yr from their enlistment until retirement. Of them, 13 had been diagnosed as having NIDDM and was receiving treatment at the time of the study. In the rest of the subjects, a 75-g oral glucose tolerance test was performed after an overnight fast, and the classification of NIDDM or impaired glucose tolerance was made according to the criteria of the WHO. For comparison of allelic frequency between normal and NIDDM populations, the ß3AR mutation was also examined in another group of 100 subjects (55 males and 45 females) randomly selected from patients admitted to the National Defense Medical College Hospital for treatment of NIDDM. They were a heterogeneous group of subjects, aged 21–85 yr (mean ± SD, 59 ± 13), unemployed or working at various occupations. Informed consent was obtained from all participants of this study.
Detection of the Trp64Arg mutation by restriction fragment length polymorphism analysis
Genomic DNA was isolated from peripheral lymphocytes by established methods. The PCR was carried out as described previously (14), using the pair of primers, 5'-CGCCCAATACCGCCAACAC and 5'-CCACCAGGAGTCCCATCACC. (11). The amplified PCR products were incubated for 4 h at 37 C with the addition of 6 U MvaI (Takara, Siga, Japan), a restriction enzyme specific for the sequence CC(A/T)GG. In some experiments, another restriction enzyme, BstOI (Promega, Madison, WI), was used, under incubation conditions previously described (11), to confirm the findings by MvaI. The digested samples were separated by electrophoresis through 3% agarose gel (NuSieve CTG Agarose, FMC Corp., Rockland, ME), stained with ethidium bromide, and visualized under ultraviolet light.
Analyses of body weight, BMI, and questionnaires
The BMI for each person at each year was calculated using the individual physical record and was used as an indicator of relative weight. A coefficient of variation of BMI for each subject was calculated as an indicator of the magnitude of body weight fluctuation or change as described by Lissner et al. (15), using BMI values at 5-yr intervals from age 25–50 yr. The mean BMI and body weight as a group were also calculated at 5-yr intervals. In addition, the body size of our subjects was compared with that of the age-matched general population of Japanese men using the data published by the Ministry of Health and Welfare of Japan (16). Questionnaires filled out by the subjects were analyzed to determine the possible influence of smoking on body weight and to know how the subjects themselves evaluated their tendency to gain weight.
Statistical analysis
All data are expressed as the mean ± SEM
unless otherwise stated. Statistical analysis was made using a software
(StatView II statistical package, Abacus Concepts, Berkeley, CA)
compatible with the Macintosh computer. Differences among group means
were tested by ANOVA or unpaired t test. The
2 test was used to compare frequencies.
P < 0.05 indicated statistical significance.
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Results |
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In 186 SDF men, 6 (3%) were homozygous for the
Trp64Arg mutation, 67 (36%) were heterozygous, and
113 (61%) had no mutation. Among the three genotypes there were no
significant differences in height, body weight, or BMI, although their
mean values were highest in Trp64Arg homozygotes (Table 1
). The prevalence of NIDDM was also not different among
the groups (Table 1). The frequency of the mutant allele was lower in
NIDDM (0.12; n = 26) than in non-NIDDM (0.23; n = 160)
groups, but the difference was insignificant (P =
0.07). The allelic frequency of Trp64Arg in 186 SDF men as
a whole (0.21) was comparable to that in 100 patients with NIDDM
(0.23), in which the genotype distribution (3 homozygotes for
Trp64Arg mutation, 40 heterozygotes, and 57 normal
homozygotes) was also similar to that in 186 SDF men.
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Relation of the mutation to obesity was examined in 173 SDF men, excluding 13 with prior diagnosis and treatment of NIDDM. When they were divided into 3 groups based on BMI at the time of study according to the method described by Kadowaki et al. (17), the frequency of the Trp64Arg allele was not different among obese (BMI, >26.4; n = 36), intermediate (BMI, 22.0–26.4; n = 104), and nonobese (BMI, <22; n = 33) groups (0.21, 0.22, and 0.26, respectively; P = 0.77).
Longitudinal changes in body weight and BMI were analyzed in 128 SDF
men with complete physical records and without prior diagnosis of
NIDDM. The genotype distribution of this subgroup was almost identical
to that of the original 186 men, and individual genotypes showed
characteristics very similar to those listed in Table 1
(data not
shown). Their mean height (166.9 cm), weight (67.0 kg), and BMI (24.0)
at age 50 yr were slightly greater than those calculated from the
census data of age-matched Japanese men (164.2 cm, 63.3 kg, and 23.5,
respectively) (16), indicating that these subjects were slightly bigger
than their contemporaries. However, the mean increment in BMI of 128
men from 25–50 yr of age was similar to that calculated from the
census data (1.8 vs. 2.0) (16). When the age-related changes
in BMI were compared among 3 genotypes, there were no significant
differences in BMI at each time point from age 25–50 yr (Fig. 1). The mean coefficients of variation of BMI were also
not different among Trp64Arg homozygotes, heterozygotes,
and normal homozygotes (3.3 ± 1.1%, 4.3 ± 0.3%, and
5.0 ± 0.3%, respectively; P = 0.09).
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, the weight
change from age 25 to 50 yr and the maximal weight difference, defined
as the maximal weight minus the weight at age 25 yr, were greater in
normal subjects than in the mutation group. Consistent with these data,
more subjects in normal group evaluated themselves as having a tendency
to weight gain than those in mutation group (54% vs. 33%).
Smoking status was not different between the two groups.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Kadowaki et al. reported that the frequency of the mutant allele was significantly higher in obese subjects than in nonobese subjects (17). Using the same criteria for obesity and nonobesity as Kadowaki et al., we found no difference in the frequency of the Trp64Arg allele between obese and nonobese groups. The discrepancy could be explained partly by the differences in age distribution and sex of the subjects. It is well known that BMI increases with age, and the age-related changes in BMI differ between males and females (13). Furthermore, it is shown in Japanese census data that age-matched body size is quite different among different generations. For example, the mean height and weight of 25-yr-old men increased by approximately 8.6 cm and 10.7 kg over the past 30 yr (16). Taking these findings together, it is possible that BMI-related analyses gave different results between our study conducted solely on men with a narrow age range or the study by Yoshida et al. of women alone (19) and other studies that included both sexes with a wider age range.
Contrary to the finding of Clément et al. in morbidly obese French subjects (12), we found that body weight gain during adulthood was greater in subjects with normal allele than in those with Trp64Arg mutation. We have no logical explanation for our observation. However, the disagreement in the results between the two studies can be explained by the difference in study populations, as our study has not included morbidly obese persons, who are very rare in Japan. Other possible explanations include racial, ethnic, and lifestyle differences between French and Japanese subjects. Alternatively, the seemingly opposite results can best be explained by the limitations inherent in any association study that uses polymorphism of one gene in the analysis of complex disorders such as obesity.
In addition to sex and age homogeneity, our subjects were characterized by a relatively homogeneous lifestyle. They stayed in the same job for more than 29 yr. The majority of them were exposed to job-related physical exercises, probably more than the average Japanese man of the same generation, although the age-related increase in BMI was comparable to that of the average Japanese man. Considering the fact that body weight is influenced by environmental as well as genetic factors, our findings suggest that the influence of the ß3AR mutation on body weight could be masked by a healthy lifestyle, including increased physical activity. Alternatively, the mutation of ß3AR, at least in its heterozygous form, had a negligible effect on body weight in a group of men with a relatively homogeneous environmental background.
The present study clearly demonstrated that heterozygous mutation of ß3AR was not associated with obesity or the tendency to gain weight during adulthood. However, our results do not rule out the possible effect of homozygous mutation on BMI because the small number of Trp64Arg homozygotes may have made it difficult for any statistical analysis to achieve significance. We found that Trp64Arg homozygous men were slightly taller and heavier than normal homozygous men at age 25 yr as well as at 53 yr. This observation suggests that the homozygous mutation could affect the growth and body weight gain during childhood and adolescence rather than during adulthood. Recent findings by Urhammer et al. in young healthy Danes (mean age, 25 yr) supported this possibility, although the results obtained from only 3 homozygotes of 380 subjects must be interpreted with caution. They demonstrated that homozygous, but not heterozygous, mutation of ß3AR was associated with obesity and greater weight gain during childhood to adolescence (18).
The role of the Trp64Arg mutation in the pathogenesis of obesity is still conjectural, and the biochemical consequences of the ß3AR mutation have not been well established. A recent study showed that the Trp64Arg mutant receptor was pharmacologically and functionally indistinguishable from the wild-type ß3AR receptor when expressed in Chinese hamster ovary cells (22). On the other hand, Walston et al. (10) reported that Trp64Arg homozygotes and heterozygotes, respectively, expended an average of 82 and 36 Cal/day less than normal homozygotes due to a decreased resting metabolic rate. It remains to be determined, however, whether the energy saving of up to 80 Cal/day in subjects with the Trp64Arg mutation should cause significant weight gain in a society in which people generally consume more calories than necessary. It seems more likely that the ß3AR mutation exerts an energy-saving effect during limited food intake. With this regard, it was reported that obese women with the Trp64Arg mutation were more resistant to weight loss during a low calorie diet than those with a normal genotype (19).
In summary, the present study showed that the Trp64Arg mutation of ß3AR, either homozygous or heterozygous, was not a major contributing factor to obesity in Japanese men. However, there remains the possibility that the mutation might affect body weight in women, who were not included in the current study. In this connection, the findings in ß3AR knockout mice are of interest. It was shown that the increase in body fat was greater in female than in male knockout mice (23). Future study will be necessary to test the possible interactions between gender and ß3AR genotype in humans.
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Acknowledgments |
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Received July 23, 1996.
Revised November 18, 1996.
Accepted December 9, 1996.
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References |
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