Yonago Acta medica 2001;44:55–59

Association of the Trp64Arg Mutation of the β3-Adrenergic Receptor
with Diabetes Mellitus, Impaired Glucose Tolerance and Lifestyle inJapanese Workers
Hirofumi Okada, Akihiko Suyama*, Yoneatsu Osaki*, Mikizo Okamoto*,
Toshiyuki Yamamoto**, Eiji Nanba** and Takuji Kishimoto*
Second Department of Internal Medicine and *Department of Hygiene, Faculty of Medicine
and **Gene Research Center, Tottori University, Yonago 683-0826 Japan
In order to investigate whether the Trp64Arg (a missense mutation of tryptophan forarginine at position 64 codon) polymorphism of the β3-adrenergic receptor (β3-AR)
gene is related to the incidence of non-insulin-dependent diabetes mellitus (NIDDM)
and impaired glucose tolerance (IGT), a retrospective cohort study among Japanese
workers was conducted. The subjects were Japanese workers at an occupational site in
Shimane Prefecture. Informed consent was obtained from 492 workers. The baseline
data were obtained at the regular health examination in 1992 and a retrospective cohort
study was performed for analyzing the incidence of NIDDM and IGT in 1998. The
Trp64Arg polymorphism β3-AR gene for each worker was detected by the single strand
conformation polymerase analysis. Relative risks were calculated by the logistic
regression analysis. The rates of Trp64Trp (TT), Trp64Arg (TA) and Arg64Arg (AA)genotypes were 66.3%, 31.1% and 2.6%, respectively. The relative risk of (TA + AA)
against TT for the incidence of NIDDM and IGT by univariate analysis was 1.37 (95%
confidence interval, 0.69–2.72). The relative risk of (TA + AA) against TT for theincidence of NIDDM and IGT adjusted for confounders in a multiple logistic regression
model including age, gender, family history, body mass index, alcohol consumption,eating habits and exercise was 1.31 (95% confidence interval, 0.65–2.67). The present
findings suggested that a weak association between Trp64Arg polymorphism of the β3AR gene and the incidence of NIDDM and IGT.
Key words: β3-Adrenergic receptor; impaired glucose tolerance; multiple risk factors; non-insulindependent diabetes mellitus; Trp64Arg polymorphism

Several case-control studies revealed that thepolymorphism of the β3-adrenergic receptor(β3-AR) gene (Trp64Arg) is associated with thedevelopment of non-insulin-dependent diabetes
mellitus (NIDDM), insulin resistance, obesityand syndrome X (Reaven, 1988). It was also
reported that the Trp64Arg of the β3-AR gene issignificantly associated with the developmentof NIDDM and obesity in Pima Indians (Wallston
et al., 1995), Finns (Widen et al., 1995) and
French Caucasians (Clement et al., 1995). More-over, the locus of Trp64Arg mutation wasreported to be associated with the developmentof obesity and hyperinsulinemia in Japanese(Yoshida et al., 1995; Kadowaki et al., 1995;
Fujisawa et al., 1996). However, other studies
suggested that the polymorphism of the β3-AR
gene (Trp64Arg mutation) is not associatedwith the development of NIDDM and obesity(Li et al., 1996; Oksanen et al., 1996; Urhammer
et al., 1996; Nagase et al., 1997; Ueda et al.,
1997; Azuma et al., 1998). Although many

Abbreviations: β3-AR, β 3-aderenergic receptor; BMI, body mass index; CI, confidence interval; IGT,
impaired glucose tolerance; NIDDM, non-insulin-dependent diabetes mellitus; PCR, polymerase chain
reaction; RR, relative risk; Trp64Arg, a missense mutation of arginine for tryptophan at position 64 codon

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H. Okada et al.

case-control studies regarding Trp64Arg havebeen conducted, there have been few cohort
studies. Moreover, various lifestyle-related
habits such as eating and drinking habits areassociated with the development of NIDDM, in
addition to a hereditary predisposition. Therefore, we conducted a cohort study concerning
possible lifestyle habits relating NIDDM in
Japanese workers with similar occupations toclarify the genetic risk factors for NIDDM.

was amplified by the standard polymerase chain
reaction (PCR) method. PCR was performed in
total of 10 µL, containing 0.04 µg of DNA,batch of 1 µmol/L of sense and antisense primers (sense: 5'-TTC CGT GGG AGG CGG CCC
TAG-3', antisense: 5'-CAC CAC CAG GAG
TCC CAT CAC C-3'), AmpliTaq Gold 0.1 U(Applied Biosystems Japan, Inc., Tokyo, Japan),dNTPs 200 µmol/L (Applied Biosystems Japan),and 10 × buffer 1 µL recommended by manufacture’s company. After initial heat denaturation
at 95˚C for 10 min, 35 cycles of 3 steps each,consisting of heat denaturation at 95˚C for 30 s,
annealing at 65˚C for 30 s and extension at 72˚C
for 30 s, were carried out, followed by extension at 72˚C for 5 min. Allelic genotypes were
determined by single strand conformational
polymorphism analysis (Fig. 1). One microgram each of the PCR products was mixed withdenaturing solution, and denatured at 95˚C for 5
min. Subsequently, the PCR products wererapidly cooled on ice, and then electrophoresedby using mini gels containing 12% of acrylamide and 5% of glycerol, at 24˚C for 6 h. Afterelectrophoresis, bands were visualized by standard silver staining. Initially, by sequencing aftercloning into a TA vector, each band pattern was
confirmed as to whether it had a wild type alleleor polymorphism. After that, diagnosis wasmade by comparison of the band pattern withinitial data.

Subjects and Methods
The ethics committee of the Tottori University
Faculty of Medicine approved this research.
The subjects were 492 staff members at an occupational site in Shimane Prefecture above 40
years of age (301 males and 191 females) who
gave informed consent for gene examinations
during a regular health examination in 1998.
Baseline data were obtained at consultation in
1992, and a retrospective cohort study was conducted to investigate the incidence of NIDDM
and impaired glucose tolerance (IGT) over a 7-year
observation period. Those who had NIDDM or
IGT in 1992 were excluded from the study.
As possible risk factors for NIDDM and
IGT, drinking habits, frequency of exercise,eating habits between meals, food intake, midnight meals and preference of seasoning ofmeals were evaluated. As described below,
hereditary predispositions for NIDDM were
evaluated by identifying the polymorphism of
the β3-AR gene (Trp64Arg) using the genomic
DNA. According to the diagnostic criteria proposed by the American Diabetes Association,
the development of NIDDM and IGT was defined as the exhibition of fasting blood glucose
levels above 126 mg/dL at least once during the
7-year observation period. The body mass index (BMI) was calculated by dividing bodyweight (kg) by a square of height (m).
Blood samples were collected in a tube
containing EDTA-Na as an anticoagulant, andthe genomic DNA was extracted from 100 µL
of whole blood using MagExtractor, a full automatic DNA extractor (MFX-2000, Toyobo,
Osaka, Japan). Subsequently, the β3-AR gene

Lane 1 Lane 2 Lane 3 Lane 4 Lane 5

Fig. 1. Genotype determination of the β3-adrenergic
receptor with the polymerase chain reaction-single
strand conformation polymorphism analysis. Lane
1 is the plasmid of a wild type and lane 2 is thepolymorphism type. Three allelic genotypes were
determined as to whether they had a wild type allele
or a polymorphism. Lane 3 is Arg64Arg, lane 4 is
Trp64Arg and lane 5 is Trp64Trp.

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Gene polymorphism of β3-AR and DM

Table 1. Properties of cohort and baseline lifestyle statuses
Items

Trp64Trp

Trp64Arg

Arg64Arg

Sample size
326 (66.3)
Age(year)
42.33 ± 5.33
22.57 ± 2.57
BMI
(kg/m2)Presence of family history
26 ( 8.0)
Presence of drinking habits
202 (62.0)
Absence of exercise
298 (91.4)
Skipping meals
267 (81.9)
Eating between meals
219 (67.2)
Neglect the balance of meals
284 (87.1)( ), the percentage of indivisual item.
BMI, body mass index.

153 (31.1)
42.52 ± 5.12
22.94 ± 2.80
10 ( 6.5)
108 (70.6)
137 (89.5)
135 (88.2)
99 (64.7)
132 (86.3)

13 ( 2.6)
43.92 ± 6.18
22.85 ± 2.87
0 ( 0.0)
6 ( 46.2)
13 (100.0)
11 ( 84.6)
8 ( 61.5)
12 ( 92.3)

Statistical analysis was performed using
StatView-J 5.0 for windows (Abacus Concepts,
Inc., Berkeley, CA). Each value for age and BMI
was expressed as mean ± SD. Relative risks(RRs) of Trp64Arg and lifestyles for NIDDM
and IGT were calculated using the multiple logistic regression analysis.

no significant differences in age among subjects
with the respective allelic genotypes. In addition, the total mean BMI was 22.69 ± 2.65 in
1992, and there were no significant differencesin BMI by allelic genotypes. Moreover, there
were no significant differences in various
lifestyle-related items among subjects with therespective allelic genotypes.
As possible risk factors for NIDDM, thepresence or absence of Trp64Arg, BMI, age,
drinking habits, frequency of exercise, eating
habits between meals, foods intake, midnight
meals and preference of seasoning of meals
were analyzed by both univariate and multivariate logistic analyses (Table 2). Concerning
Trp64Arg, univariate analysis showed an RR of
1.37 (95% CI: 0.69–2.72), whereas multivariate analysis showed an RR of 1.31 (95% CI:
0.65–2.67). Therefore, it was considered that

Results
The frequency of Trp64Arg mutation was investigated in 492 Japanese workers using
genomic DNAs. As a result, the allelic frequency of Trp64Arg was 20.8%. The proportions ofgenotype for TT, TA and AA were 66.3%, 31.1%
and 2.6%, respectively.
As shown in Table 1, the total mean age was
42.43 ± 5.30 years old in 1992, and there wereTable 2. Relative risks of possible risk factors for NIDDM and IGT
Items

Univariate analysis
RR
95% CI

Multivariate analysis
RR
95% CI

Trp64Arg
Hetero+homo/wild
1.37
0.69–2.72
1.31
0.65–2.67
Family history
Present/absent
1.60
0.53–4.81
1.53
0.49–4.75
Age
1.06
1.00–1.12
1.06
1.00–1.12
GenderFemale/male
0.17
0.06–0.50
0.27
0.07–0.99
BMI
1.00
0.88–1.14
0.98
0.86–1.11
Presence of drinking habits Yes/no3.85
1.47–10.08
1.54
0.47–5.00
Meals
Eat 3 times a day/skip 1.49
0.67–3.40
1.33
0.56–3.16
Balance of meals
Neglect /consider
1.76
0.52–5.89
1.36
0.40–4.86
Eating between meals
Yes /no0.45
0.23–0.89
0.75
0.36–1.55
Taking exercise
Yes /no1.12
0.33–3.82
1.44
0.41–5.06
BMI, body mass index; CI, confidence interval; IGT, impaired glucose tolerance; NIDDM, non-insulin-dependent
diabetes mellitus; RR, relative risk.

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H. Okada et al.

the Trp64Arg mutation of the β 3 -AR geneslightly increases the risk of NIDDM and IGT,
although the RR was not significant. Moreover,gender (female/male) was the greatest risk for
NIDDM and IGT; i.e., univariate analysis showed an RR of 0.17 (95% CI: 0.06–0.50), while
multivariate analysis showed an RR of 0.27
(95% CI: 0.07–0.99), demonstrating significantly higher risks for NIDDM and IGT inmales than in females. Concerning drinking
habits, univariate analysis alone showed a significant RR of 3.85 (95% CI: 1.47–10.08). However, no other lifestyle-related habits showed asignificantly higher RR for NIDDM.

ly increase the risk of NIDDM and IGT, because
the propriety of this analytical model was confirmed.
However, this cohort study has some controversial issues because the amount of food ingestion including salt and sugar, and the amountof alcohol intake and exercise in individual subjects depended on subjective data based on selfassessment, and were not numerically evaluatedduring a questionnaire survey.
In the present study, none of these items
including the Trp64Arg and lifestyle-related
items such as age, family history and BMI
showed any significant association with
NIDDM and IGT, except for gender. In addition, the results of other case-control studies onallelic frequencies in Japanese were similar toours (Awata and Katayama, 1996; Nagase et al.,
1997; Ueda et al., 1997; Azuma et al., 1998).
Fujisawa et al. (1996) also reported results similar to those of previous case-control studies,
except for a slightly higher odds ratio of 1.72.
Therefore, it is considered that this cohort study
using a superior methodology reconfirmed theresults of previous case-control studies.
Currently, we have investigated a total of
492 subjects. However, further accumulationof cases and long-term follow-up may reveal
significant associations of certain lifestylerelated items with NIDDM and IGT.

Discussion
There were no significant differences in mean
age and BMI among subjects with the respective allelic genotypes in 1992. The rates of TT,
TA and AA genotypes were 66.3%, 31.1% and2.6%, respectively. These findings were compatible with Hardy-Weinberg’s law. In addition,
allelic frequencies in this study did not markedly
differ from those reported by previous studies
in Japanese.
In this cohort study, univariate analysis
showed an RR of 1.37, and multivariate analysis
showed an RR of 1.31, suggesting a possible relationship between the Trp64Arg gene and thedevelopment of NIDDM and IGT, although the
RR was not significant. Among various lifestyle-related habits, none showed any significant
association with NIDDM and IGT. This wasprobably because the relationship between
NIDDM and IGT and various lifestyle-related
habits were evaluated together with gender; i.e.,
gender is closely associated with lifestyle relating NIDDM and IGT. Therefore, we reanalyzed these habits excluding gender. As a result,drinking habits showed an RR of 3.11 (95% CI:
1.13–8.58), suggesting that drinking habits may
be a significant risk factor for NIDDM and IGT.
However, Trp64Arg showed an RR of 1.29
(95% CI: 0.64–2.62), and this level of RR was
similar to the results of analysis including gender. Therefore, the results of this cohort study
suggest that the Trp64Arg mutation may slight-

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Received December 20, 2000; accepted December 28, 2000
Corresponding author: Prof. Takuji Kishimoto

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