Clinical Features Associated With the Homozygous Trp64Arg
Mutation of the b3-Adrenergic Receptor
No Evidence for Its Association With Obesity in Japanese
Liming Sun, Shun Ishibashi, Jun-ichi Osuga, Kenji Harada, Ken Ohashi, Takanari Gotoda,
Yoshihiro Fukuo, Yoshio Yazaki, Nobuhiro Yamada
Abstract—To characterize the clinical features associated with the Trp64Arg mutation of the b3-adrenergic receptor(b3-AR), the effects of this mutation, in particular the homozygous state (Arg/Arg), on obesity, blood pressure, andplasma lipoproteins were investigated in 2 populations: subjects residing on a small isolated island (group 1; n5746)
and patients residing in Tokyo who attend a clinic for metabolic diseases (group 2; n5371). The allelic frequency of
the Trp64Arg mutation was 23.4% in group 1 and 18.3% in group 2. No significant difference in the body mass index
was observed between subjects with 3 different genotypes in each group. There was a trend that the Arg/Arg had higher
systolic blood pressure than the Trp/Trp in both groups, but the differences were not statistically significant. The plasma
LDL cholesterol levels were significantly lower in Arg/Arg than in Trp/Trp in men from the group 1 cohort (2.8260.84
versus 3.1960.7 mmol/L, P,0.05). These results suggest that the homozygous Trp64Arg mutation is not a major
contributing factor for obesity, but potentially contributed to higher systolic blood pressure and low plasma levels of
LDL cholesterol in Japanese men. (Arterioscler Thromb Vasc Biol. 1998;18:941-946.)
Key Words: b3-adrenergic receptor n obesity n hypertension n diabetes n cholesterol
populations, including Japanese,13–18 Danes,19 and Australians.20 However, the role of this mutation in the pathogenesisof obesity is still controversial, and contradictory results havebeen reported with respect to the effects of the Trp64Arg
mutation on obesity.21–25
Phenotypic expression of certain mutations can be more
remarkable in a homozygous state than in a heterozygous
state. In this regard, it is noteworthy that several studies haveshown reduced resting metabolic rates,12 hyperinsulinemia
with obesity,13 and reduced insulin sensitivity9 in subjects
homozygous for the Trp64Arg mutation. But other studies
have shown lack of association between the homozygous
mutation and obesity.24,25 A limitation of these studies, however, is that relatively few individuals homozygous for the
Trp64Arg allele were studied.
In the current study, we attempted to more completely
describe parameters associated with the Trp64Arg mutation of
the b3-AR gene and also test the hypothesis that the mutationaccounts for obesity. We investigated the association of thismutation with multiple clinical parameters, including bloodpressure, plasma lipoproteins, and liver function, in an
isolated population living on a small island in Japan and inpatients residing in Tokyo who attend a clinic for metabolic
diseases that collectively contained a total of 67 subjects
homozygous for the Trp64Arg mutation.

O

besity is associated with various diseases, such as
hypertension, glucose intolerance, and hyperlipidemia,
which collectively contribute to the development of ischemic
heart disease.1 Both genetic and environmental factors are
involved in the development of human obesity.2 The role ofgenetic factors has long been enigmatic, until recent discovery of molecules that govern the metabolism of adipocytes.3
b3-AR1 is one of these molecules.4,5 In conjunction with
b1-AR and b2-AR, accumulating evidence supports an important role for the b3-AR in mediating adrenergic regulationof the metabolism of adipose tissues. b3-AR is expressed inbrown adipose tissue of neonates and visceral adipocytes inadults,6 where it regulates thermogenesis and lipid mobilization. Furthermore, the b3-AR has been shown to be widely
expressed in other tissue, such as heart7 and gastrointestinal
tract.8 Targeted disruption of this receptor gene in mice
resulted in modest obesity, supporting its critical role in thedevelopment of obesity.9
In 1995, a missense mutation that replaces tryptophan witharginine (Trp64Arg) of the human b3-AR gene was reported
to be associated with increased capacity to gain weight in the
French population,10 the features of insulin resistance syndrome in Finns,11 and the earlier onset of NIDDM in obese
Pima Indians,12 even in a heterozygous state. These observations were supported by subsequent reports in various ethnic

Received July 8, 1997; revision accepted January 5, 1998.
From the Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo; and the Second Department of Internal Medicine, Facultyof Medicine, Nippon Medical School (Y.F.), Japan.
Correspondences to Shun Ishibashi, MD, The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7–3-1 Hongo,
Bunkyo-ku, Tokyo 113, Japan.
E-mail ishibash-tky@umin.u-tokyo.ac.jp© 1998 American Heart Association, Inc.

941

942

Homozygous Trp64Arg Mutation and Obesity
LDL-C was calculated using the Friedewald equation. Urinalysis
also was performed.

Selected Abbreviations and Acronyms
AR 5 adrenergic receptor
BMI 5 body mass index
DBP 5 diastolic blood pressure
GOT 5 glutamic oxaloacetic transaminase
GPT 5 glutamic pyruvic transaminase
g-GTP 5 g-glutamyltransferase
HDL-C 5 HDL-cholesterol
LDL-C 5 LDL-cholesterol
NIDDM 5 non–insulin-dependent diabetes mellitus
PCR 5 polymerase chain reaction
SBP 5 systolic blood pressure
TC 5 total cholesterol
TG 5 triglyceride
TP 5 total protein

DNA Analysis
Genomic DNA was extracted from whole blood. PCR was used toamplify a genomic DNA fragment containing codon 64 of the b3-AR
in a volume of 25 mL containing 50 ng of genomic DNA; 10 pmol
each of the primers up (59-CGCCCAATACCGCCAACAC-39) anddown (59-CCACCAGGAGTCCCATCACC-39); 2.5 mL of 10%
DMSO, 103 PCR buffer (supplied by Perkin-Elmer Cetus), 103
BSA, 0.4 mL of 2.5 mmol/L dNTP, and 0.625 U of Taq polymerase,
essentially according to Widén et al.11 The PCR reactions began withdenaturation at 93°C for 2 minutes, annealing at 60°C for 2 minutes,
and extension at 65°C for 5 minutes, followed by 39 cycles ofdenaturation at 94°C for 30 seconds, annealing at 60°C for 30
seconds, and extension at 65°C for 2 minutes, with a final extension
at 72°C for 10 minutes.
The amplified PCR products were digested with 5 U of BstNI andwere separated by electrophoresis through 3% agarose gel. DNA wasvisualized by staining with ethidium bromide.

Methods
Subjects
The first cohort (group 1) consisted of participants in a regular healthcheck survey that live on a small isolated island with approximately
10 000 inhabitants located 290 km from the Japanese mainland.26 A
total of 746 subjects (224 men and 522 women, aged 30 to 89 years;
mean age 61.5611.33 years) were randomly recruited.
The second cohort (group 2) was recruited from patients attendinga clinic for metabolic diseases at Tokyo University Hospital. The 371
subjects chosen (180 men and 191 women, aged 21 to 88 years;
mean age 58.6611.4 years) suffered from diabetes and/or hyperlipidemia. Of these 371, 107 (28.8%) were treated with oral hypoglycemic drugs, 230 (62%) were treated with hypolipemic drugs, and 91(24.5%) were treated with antihypertensives.
The subjects’ age, sex, accompanying diseases, and medications
were collected, and height (m), weight (kg), and blood pressure were
measured. BMI was calculated as kg/(m)2. Severe and moderate
obesity were defined as BMI.28 and BMI524 to 28, respectively.
Blood pressure (SBP/DBP) was determined based on the average of
2 or more readings obtained on 2 or more visits. ECGs were recorded
and the ischemic changes were defined as follows: ECG evidence
representing previous myocardial infarction, such as abnormal Q
wave, or ischemic changes, such as depression of ST-T. Patients withliver cirrhosis were excluded from the present study.

All data are expressed as mean6SD. ANOVA, Student’s t, KruskalWallis, Mann-Whitney U, and x2 tests were performed to estimate
the effects of each genotype on quantitative variables and qualitative
variables, respectively (Statview 2 statistical package). Statisticalsignificance was established at the P,0.05 level.

Results
Genotype Frequencies
Table 1 illustrates the number of subjects homozygous(Arg/Arg), heterozygous (Trp/Arg), and wild type (Trp/Trp)
for the Trp64Arg allele in groups 1 and 2. The 3 genotypes
were distributed in both study groups in accordance with
Hardy-Weinberg equilibrium. The calculated frequency of
the Trp64Arg allele was 23.4% for group 1 and 18.3% forgroup 2. No significant difference in the allelic frequencies
was observed between the 2 study groups, indicating thatthese 2 populations represent the general Japanese population
with respect to the b3-AR locus.

Body Mass Index

Laboratory Tests
Blood samples were obtained from all subjects after a 12-hour fast.
Serum concentrations of TC, TG, HDL-C, TP, GOT, GPT, g-GTP,
and choline esterase were measured using conventional methods.

TABLE 1.

Statistical Analysis

Univariate analyses in each study group did not reveal any
difference in age and BMI between Arg/Arg, Trp/Arg, and
Trp/Trp subjects in both men and women (Table 1). Allelic

Clinical Characteristics of Subjects According to Trp64Arg Mutation of b3-AR Gene
Men

Group 1, nAge, yWomen

Arg/Arg

Trp/Arg

Trp/Trp

22

77

125

P

Arg/Arg

Trp/Arg

Trp/Trp

29

169

324

0.11

60.8611.8

61.5610.6

61.4611.4

P

63.6612.6

63.2612.2

60.0611.3

2

0.96

BMI, kg/(m)23.462.7

23.863.2

24.463.4

0.28

24.163.4

23.763.5

23.663.4

0.75

SBP, mm Hg

1496231426191416190.161376181396201396200.91

DBP, mm Hg

83612

82611

83611

0.99

7869

80611

80611

0.49

Group 2, n

8

49

121

8

54

127

Age, y58.5611.1

55.5612.5

56.0612.3

0.82

63.167.6

60.9610.5

61.169.2

BMI, kg/(m)2

23.062.5

23.763.8

23.162.7

0.63

23.063.5

23.063.1

22.663.0

0.96

SBP, mm Hg

1316221236171226140.31126616

128619124616

0.24

DBP, mm Hg

8364

76613

7669

0.17

7767

79610

7567

0.05

Data are mean6SD, analyzed by ANOVA.

0.82

Sun et alJune 1998

943

TABLE 2. Body Mass Index and Blood Pressure Analyses ofCombined Populations (Groups 1 and 2)
P

Arg/Arg

Trp/Arg

Trp/Trp

Men

30

127

247

Women

37

222

247

Total

67

349

702

Men

23.562.7

23.863.4

23.863.1

0.87

Women

23.963.4

23.663.4

23.363.5

0.45

Total

23.763.1

23.663.4

23.563.4

0.71

Men

144.3618.2*

134.8620.5

131.7619.3

0.0026

Women

134.7618.2

136.2620.3

134.2620.1

0.48

Total

138.8621.5

135.7620.3

133.1619.8

0.025

Men

82.9610.4

79.861279.4610.3

0.28

Women

77.468.1

79.4610.6

78.8610.3

0.50

Total

80.4610.6

79.5611.1

79.0610.3

0.49

NBMI, kg/(m)2

SBP, mm Hg

DBP, mm Hg

Statistical analyses were performed on the combined population consistingof groups 1 and 2. Data are mean6SD, analyzed by ANOVA.
*Statistically significant difference at P,0.05 vs Trp/Trp genotype by Fisher
test.

frequency in subjects with severe obesity (BMI.28) was not
significantly different from that in moderately obese (BMI 24
to 28) or nonobese people (BMI,24). The Figure comparesthe distribution of BMI among the 3 genotypes in the total(top) men (middle), and women (bottom) from group 1. Male
Arg/Arg were found more frequently in subjects with BMI
between 26 and 28 than in subjects in any other BMI range,
although this difference was not statistically significant.
Furthermore, no significant difference in allelic frequency
was detected between any BMI quartile either in men,
women, or the total (data not shown). Notably, the BMI of all
51 Arg/Arg was ,35. In group 2, no significant correlation
was detected between BMI and the Trp64Arg mutation (Table1). Furthermore, analysis of the combined populations ofgroups 1 and 2 revealed no significant correlation between
BMI and the Trp64Arg mutation (Table 2).

the Arg/Arg to have higher SBP than the Trp/Trp in each
population, but the difference did not reach statistical significance by univariate analyses (Table 1). In group 2, there wasalso a trend for the Arg/Arg to have higher SBP than the
Trp/Trp in men, although the difference was not statisticallysignificant (Table 1). The number of patients under treatment
for hypertension was 5 (31.2%) in the Arg/Arg, 24 (23.2%) in
the Trp/Arg, and 64 (24.7%) in the Trp/Trp, respectively, but
the difference was not statistically significant.
Next, the 2 populations were analyzed in combination, thus
yielding 30 Arg/Arg men. The Arg/Arg had significantly
higher SBP than the Trp/Arg and Trp/Trp (Table 2). No
differences were observed in women. Because blood pressure
is influenced by several factors, multivariate analyses using
age, sex, BMI, group, and the genotype of b3-AR as
explanatory factors were performed to determine whether theeffects of the b3-AR mutation on elevated SBP is independent of other confounding factors. As expected, age, BMI,
and group were strongly associated with elevated SBP(P,0.0001). The effects of the b3-AR genotype were nolonger significant (P50.18). When only men were analyzed,
a weak association between the b3-AR mutation and SBP
was found (P50.067).

Blood Pressure

Incidence of NIDDM and Abnormal ECG

The mean SBP of group 2 was significantly lower than that ofgroup 1 in any given genotype and sex (Table 1). Since thedifference remained even after eliminating patients treatedwith antihypertensive drugs, the lower SBP may be ascribedto either adherence to low-salt diets or the difference in othergenetic and/or environmental factors. There was a trend for

No difference in the incidence of NIDDM was observed
between the Arg/Arg (3.9%), Trp/Arg (6.9%), and Trp/Trp
subjects (4.9%) in group 1. The allelic frequency (18.6%) in
the diabetic patients in the group 1 cohort was surprisingly
similar to the value in group 2 (18.1%), of which 216 (58%)
were diabetic.

Distribution of allelic frequencies for Trp64Arg mutation of b3-AR
gene according to BMI in total (All), men, and women. Homozygotes and heterozygotes for the Trp64Arg mutation are indicatedas solid and hatched bars, respectively.

944

Homozygous Trp64Arg Mutation and Obesity
TABLE 3.

Biochemical Characteristics of Subjects According to Trp64Arg Mutation of b3-AR Gene
Men

Women

Trp64Arg

Arg/Arg

Trp/Arg

Trp/TrpGroup 1, n

22

77

125

TC, mmol/L

4.7061.03*

5.0560.83

5.2060.79

TG, mmol/L

1.2160.62

1.4360.88

1.5661.26

HDL-C, mmol/L

1.3360.32

1.4060.37

LDL-C, mmol/L

2.8260.84*

2.9960.78

P

P

Arg/Arg

Trp/Arg

Trp/Trp

29

169

324

0.03

5.3360.95

5.2760.83

5.2660.82

0.91

0.35

1.3860.57

1.1960.56

1.2460.72

0.29

1.3060.37

0.18

1.2960.33

1.3960.31

1.4060.31

0.22

3.1960.70

0.04

3.4060.93

3.3460.76

3.2960.72

0.67

TP, g/L

7764*

7464

7564

0.01

7665

7565

7565

0.29

GOT, U

25 (21–38)

23 (20–28)

25 (21.8–30)

0.16

21 (19–24.3)

20 (18–25)

21 (18–25)

0.45

GPT, U

22 (16–34)

21 (17–32.3)

21 (15–28)

0.76

15 (12–21.3)

14 (11–20)

15 (12–21)

0.31

g-GTP, U

37 (20–68)

28 (18–42)

30 (20–53)

0.31

12 (9–15)

12 (9–18.5)

14 (9–22.3)

0.15Group 2, n

8

49

121

8

54

127

TC, mmol/L

5.2561.0

5.3461.1

5.561.2

0.61

5.7360.81

6.0760.96

5.8560.83

0.25

TG, mmol/L

1.6060.94

1.8061.18

1.9961.79

0.67

1.4860.66

1.6060.82

1.3760.89

0.25

HDL-C, mmol/L

1.3960.47

1.460.39

1.4760.47

0.59

1.660.23

1.7560.51

1.6960.54

0.38

LDL-C, mmol/L

3.1461.1

3.1661.03

3.0761.16

0.89

3.3960.68

3.760.94

3.5160.89

0.38

TP, g/L

7467

7565

7665

0.18

7664

7665

7664

0.94

GOT, U23 (21–30)

21.5 (18–26.5)

22 (18–27)

0.46

23.5 (19.5–26)

19 (16–24)

0.10

GPT, U

23 (14–35.5)

19 (12–29.5)

20 (14–29.5)

0.63

20 (12.5–29.5)

17.5 (12–24)

14 (11–18)

0.02

g-GTP, U

47.5 (34–87)

39 (26–61)

41 (25.3–71)

0.71

36.5 (24–101)

24.5 (17–37.5)

23 (17–32)

0.05

20 (17.5–26.5)

The results are represented by mean6SD for nonskewed data (TC, TG, HDL-C, LDL-C, and TP) and median (25th to 75th percentile) for skewed
data (GOT, GPT, and g-GTP). ANOVA and Kruskal-Wallis were performed for the nonskewed and skewed data, respectively.
*Statisticlly significant difference at P,0.05 vs Trp/Trp genotype by Fisher test.

The prevalence of abnormal ECG changes indicative ofischemic heart disease was similar among the 3 genotypes ingroup 1.

Biochemical Data

Since the results of liver function tests exhibited highly
skewed distribution, nonparametric tests were employed forstatistical analyses. We found a statistically significant association between the plasma levels of g-GTP and the Trp64Arg
mutation in women from the group 2 cohort (Table 3).

Plasma levels of TC and LDL-C were significantly lower inthe Arg/Arg than those in the Trp/Trp in men, but not in
women, from group 1 cohort by ANOVA (Table 3). To
examine whether other factors confounded the results, we
eliminated the subjects treated with hypolipemic drugs, yielding 21 Arg/Arg, 64 Trp/Arg, and 110 Trp/Trp men available
for the analyses. In these subjects not treated with hypolipemic drugs, the TC and LDL-C levels of the Arg/Arg men
were still significantly lower than those of Trp/Trp men. To
determine whether the effects of the b3-AR genotype are
independent of other potential confounding factors, multivariate analyses were performed using age, BMI, and the
genotype of b3-AR as explanatory factors. BMI, but not age,
was strongly associated with plasma LDL-C levels(P,0.003). The effects of the b3-AR mutation remained
significant (P,0.05). In the group 2 cohort, no significant
differences were observed in the plasma levels of either TC,
TG, HDL-C, or LDL-C between different genotypes, probably because the effects of the b3-AR mutation were severely
confounded by associating hyperlipidemia and hypolipemic
medication, which were prevalent in this particular
population.
In the group 1 cohort, the plasma level of TP was
significantly higher in the Arg/Arg than in the Trp/Arg and
Trp/Trp in men, but not in women, by ANOVA (Table 3).

In both group 1 and 2 populations, the frequency of the
Trp64Arg mutation is similar to that reported in other Japanese
populations.13–17,24,25 In addition, although condensation of the
His101Gln mutation in the a-tocopherol transfer protein gene
was found in the same population, most likely due to afounder effect,26 these results indicate that Japanese appear to
be relatively homogeneous in terms of the mutation in theb3-AR gene.
Since the original discovery of the Trp64Arg mutation of
the b3-AR gene, numerous studies have addressed the problem concerning the association between this variant andobesity, NIDDM, and/or insulin resistance.10 –25 Overall, theeffects of this mutation in heterozygous form on adiposity
and/or insulin resistance, if present, appear to be small.
Several studies have reported the effects of this mutation in
the homozygous state; 3 Arg/Arg subjects exhibited progressive weight gain in young white Danish.19 However, thenumber of Arg/Arg subjects employed seems too small toperform meaningful statistical analyses. In contrast, our study
subjects contained a total of 67 Arg/Arg from 2 different
populations, rendering our comparison meaningful. No significant difference was found in mean BMI among the
subjects with 3 different genotypes in each population ana-

Discussion

Sun et allyzed separately or in combination, even in the Arg/Arg
homozygotes. In accordance with our results, several groups
have recently published results failing to support the hypothesis that the Trp64Arg mutation of the b3-AR gene isassociated with obesity.21–25
Gagnon et al22 argued that the Trp64Arg mutation of theb3-AR gene may be functionally silent based on previous
findings that the third intracellular domain (i3), but not i1,
which contains the Trp64 codon, is involved in both ligandbinding interactions and in receptor coupling to G proteins.
They proposed that this notion is supported by the fact that
cows, rats, and mice have arginine instead of tryptophan at
the same position. In fact, the Trp64Arg mutant receptor was
shown to be pharmacologically and functionally indistinguishable from the wild-type b3-AR when expressed in
Chinese hamster ovary cells.27 We are not in complete
agreement with these arguments, because the b3-AR genemutation was found to be associated with other clinicalfactors, such as SBP and some of the laboratory data. Because
obesity is a complex phenotype regulated by numerous
factors, the effects of functional defects of the b3-AR may
easily be mitigated by other confounding factors, thus resulting in apparently contradictory findings.
In contrast to previous reports showing association between the b3-AR gene mutation and early onset12 and highprevalence of NIDDM,15 we did not find an increase in theprevalence of NIDDM either in homozygotes or in heterozygotes in either study population. One limitation of the current
study is that we did not measure insulin sensitivity or fat
distribution, both of which were reported to be abnormal inpatients carrying the Trp64Arg mutation.17,19
There was a trend for the Arg/Arg men to have higher SBP
than the Trp/Trp men (Table 1), but the difference obtained
from separate analyses on whole subjects from a single cohort
did not reach the level of statistical significance. Althoughcombined analyses of group 1 and 2 revealed elevation of
SBP in the Arg/Arg men (Table 2), multivariate analyses
failed to demonstrate independent effects of the genotype ofb3-AR on SBP. Since the group 2 cohort had significantly
lower SBP than group 1, probably due to medical intervention(Table 1), the difference in the combined analysis may result
from the effects of stratification.
Several previous studies have reported associations between hypertension and the b3-AR gene mutation in bothheterozygous11,20 and homozygous forms.16 Functional b3-AR
was demonstrated to be present in cardiomyocytes isolated
from human hearts,7 and b3-AR agonists were shown to haveboth negative inotropic and positive chronotropic activities.28
Thus, impaired action of the b3-AR of the heart may
contribute to elevation of blood pressure. Alternatively,
several reports have shown that patients with the Trp64Arg
mutation of the b3-AR gene are hyperinsulinemic. Elevationof SBP may manifest as a result of insulin resistance, as
postulated by Widén et al.11 Apparently, both sex and age areimportant in the phenotypic expression of elevated SBP.
Further studies are required to clarify the precise mechanisms
at work.
We found a decrease in the plasma LDL-C levels in the
Arg/Arg men from group 1. These associations were ob-

June 1998

945

served only in men, suggesting sex-specific expression of thephenotype of the mutation. The reduced lipolysis due todefective b3-AR function limits the supply of free fatty acids
to the liver, thereby decreasing the production of VLDL,
which in turn reduces the LDL-C levels. The fact that men
have more visceral fat, the metabolism of which is under the
regulation of the b3-AR, than women, in whom subcutaneous
fat is predominant, may explain the sex difference observed.
Similar association between the decreases in the plasma TG
levels and the Trp64Arg mutation has recently been reported
by Kim-Motoyama et al.17
Higher plasma levels of TP and g-GTP in the Arg/Arg
subjects were also novel findings. This result may represent areal effect or a spurious chance statistical finding.
In conclusion, our current results did not support theoriginal hypothesis that the Trp64Arg variant of the b3-AR isa determinant of adiposity in Japanese, even in the Arg/Arg
homozygotes.

Acknowledgments
This study was supported in part by a research grant from the Tanabe
Medical Conference Foundation. We are grateful to Mihoko
Kusubae for data collection and to Yoko Iizuka, Hiroaki Yagyu,
Naoya Yahagi, Futoshi Shionoiri, Zhong Chen, and Stéphane Perrey
for helpful discussion and comments.

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