Introduction

The prevalence of obesity is increasing rapidly in most countries, which may be associated with a metabolic syndrome.^1,2 Although obesity occurs less frequently in Japanese compared with various ethnic populations,^3,4 the recent changes in Japanese social environment and lifestyle have caused an increase in the prevalence of obesity and diabetes.^5,6

Overeating and physiological inactivity in combination with genetic factors are the major causes for the development of obesity in humans. Adipose tissue, which plays a crucial role in regulating the storage and mobilization of energy, has been the focus of efforts to identify candidate genes for obesity.⁷ One such gene is the ₃-adrenergic receptor (₃AR), which is the main receptor involved in the regulation of thermogenesis and lipolysis in the adipose tissue.^8,9 Stimulation of the ₃AR by -adrenergic agonist activates adenylate cyclase, which increases intracellular concentrations of cyclic AMP and results in increased lipolysis in the white adipose tissue and thermogenesis in the brown adipose tissue.^10,11,12

The base mutation predicts a replacement of tryptophan by arginine at position 64, which is located in the first intracellular loop of the ₃AR and is thought to be important for binding to noradrenaline and coupling to Gs proteins in adipose cells.⁸ The ₃AR mutation may lead to a decrease in thermogenesis and lipolysis in brown and white adipose tissue.¹⁰ There is some evidence that the genetic variation in the ₃AR may predispose subjects to abdominal obesity and insulin resistance by decreasing energy expenditure.^8,13,14 Experimental data also indicate that the Trp64Arg mutation is associated with impaired lipolysis in adipose tissues. These results led to the 'thrifty genotype' hypothesis,¹⁵ which stated that obese persons with Trp64Arg mutation of the ₃AR gene had lower daily energy expenditure, altered lipolysis and increased abdominal obesity.

About half of the obesity studies of Trp64Arg mutation of the ₃AR gene have shown an association with being overweight,^{8,14,16,17,18} whereas the other half have failed to demonstrate a link to excess body fat.^19,20,21 Even meta-analysis showed divergent results with regard to the importance of Trp64Arg mutation of the ₃AR gene.^9,22,23 We have shown that Trp64Arg mutation did not affect body mass index (BMI) and weightgain in a period of 15 y in Japanese workers.²⁴ Further, it is important to clarify the effect of Trp64Arg mutation on obesity-related phenotypes for developing an individualized prevention strategy.

Divergent results among investigators may be partially attributed to random sampling variations, cohort differences in ethnicity, sex, age and degree of obesity. Therefore, an interventional study is suitable for overcoming the confounding factors of cross-sectional nature in most studies. Our first major objective in the present investigation was to assess Trp64Arg polymorphism of the ₃AR gene on obesity-related phenotypes. This included total and visceral adipose tissue loss, insulin sensitivity and cardiovascular disease risk factors, as well as energy intake and expenditure in response to the weight-loss program. The second major objective in the present investigation was to develop a feasible, cost-effective behavioral program for obesity prevention.

Subjects and methods

Subjects

A total of 85 clinically healthy women (age: 54.37.9 y, 35–69 y) in Shimane Prefecture, Japan, who are interested in the prevention of overweight and obesity-associated diseases, voluntarily participated in the present study as a behavioral program for obesity prevention in 2000 and 2001. They were recruited with an advertisement in a local newspaper. None of them were receiving cardiovascular drugs or hormonal treatment. The ethics committee of Shimane Medical University approved all study protocols, and all the subjects gave written informed consent.

We chose 76 middle-aged women with a BMI over 21.0kg/m² for subjects of the present behavioral interventional study.^25,26 The first reason for this selection was that most Japanese women become obese during the climacteric period and have a higher prevalence of obesity-related diseases with over 21.0 kg/m² of BMI.⁶ The second reason was that women were thought to be easier than men for controlling their body weight independently, because most women can prepare food by themselves in order to improve their diet and lifestyle, thus complying with the behavioral intervention. The BMI range of the participants was 21.0–33.0 (24.52.1 kg/m²). Out of 76 participants, 24 women (31.6%) with 25.0–29.9 kg/m² of BMI were categorized as overweight, and three women (3.9%) with over 30 kg/m² of BMI were categorized as obese.²

Women with higher BMI were positively related with body weight, weight gain from age 20 y, body fat, waist circumference, hip circumference, waist/hip ratio, skin fold at the arm, resting energy expenditure (REE), systolic and diastolic blood pressure, insulin, HOMA-IR and leptin, but were negatively related with energy intake/kg weight and REE/kg weight (Table 1).

Table 1 - Correlation coefficients between BMI and baseline parameters.

Full table

They underwent a behavioral weight-loss program for 3 months including diet, exercise and supportive group therapy.^27,28 The objectives of the intervention were to lose 1.0 kg of weight, reduce metabolic measurement values by 10% in dietary caloric intake and keep taking over 7000 steps a day. The behavioral program for obesity was based on learning principles, such as recognizing participant's health risks related to obesity, studying healthy life-skills, setting specific behavioral goals, modifying behavior determinants and reinforcing desired behavior. The approach began with a health check to recognize their situation regarding obese and obesity-associated diseases. The second step offered intensive, professional, individual information to improve their unhealthy lifestyle, that is, overeating and insufficient physiological activity, and the self-monitoring ability of participants, that is, weekly checking of weight, daily estimating caloric intake and monitoring the number of steps. Participants were determined to achieve their specific behavioral and anthropometric goals, as related to their individual lifestyles and measured physiological data. During the 3-month program, participants were encouraged to record daily the results of their self-determined target, by counting the number of steps using a pedometer (HJ-002, Omron Co. Ltd, Tokyo, Japan), and the body weight once every week. Group programs provided local contact with specialists and social support to convince them that they could personally control their health condition and their environment.

Diet

Information on the participant's daily diet was obtained using an established self-administered quantitative food frequency questionnaire²⁹ before and after the behavioral intervention. Trained nutritionists asked each participant their weekly frequency of food intake for the recent month. The investigation sheet was made for Japanese people by the Working Committee for Health Guideline of the Japanese Ministry of Health and Welfare. The sheet investigates the average intake and frequency of intake for 1 month regarding 'the 30 kinds of food groups', which include soup, meat, seafood, vegetables, grains, potato, egg, beans including tofu, fruits, milk, cheese, desserts, snacks, pickles, seasonings and alcohol. The average intake at one time was confirmed by showing examples of food models or photographs. Average daily nutrient intake for the recent month was calculated using the standard food composition tables for Japan.³⁰ Dietary advice was provided to change eating habits for the better before the intervention.

Physical activity

Habitual physical activity was assessed before and after the intervention by using the questionnaire sheets. This questionnaire was subdivided into physical activity at work, sports during leisure time and physical activity excluding sports during leisure time. Participants counted the number of steps using a pedometer for 1 week before the intervention and during the program.

Anthropometric measurements

Anthropometric evaluation was performed before and after the intervention. After an overnight fast, body weight was measured to an accuracy of 0.2 kg with a standard scale, dressed only with very light clothing, and height was measured to an accuracy of 0.5 cm using a height bar fixed on the wall, with subjects standing with back, buttocks and heels pressed together against the wall. Bioelectrical impedance was measured using a leg-to-leg version of a bioimpedance analyzer (ModelBF-631, Tanita, Tokyo, Japan).³¹ Waist circumference was measured to the nearest 0.1 cm at the narrowest part of the torso as seen from the anterior aspect. Hip circumference was measured to the nearest 0.1 cm at the point of maximum extension of the buttocks. The measurement was repeated for waist and hip circumferences, and a third measurement was made if the difference between the first two readings was more than 0.5 cm. Skin-fold thickness measurements were made with an Eiyoken-type caliper (Meiosha, Tokyo, Japan) calibrated to exert a constant pressure of 10 g/ml on the right side of the body at the triceps.^32,33 BMI was computed as weight (kg) divided by squared height (m²).

REE was measured only before the intervention by an indirect calorimeter after a 12-h overnight fast. After a 15-min resting period, expired gas collection was achieved through a mouthpiece covering the nose for 3 min during the entire sampling period. Oxygen concentration was determined by nondispersive infrared analysis (VMB-002N, VINE, Tokyo, Japan). The calculated energy equivalent of oxygen volume was 4.825 kcal/l.³⁴ Blood pressure was measured twice using a standard mercury manometer, with the participants seated.

Blood samples

Venous blood was collected from the antecubital vein after a 12-h overnight fast. Blood samples were collected into two tubes containing silicone or EDTA-Na (1 mg/ml), and plasma was separated by a 10-min centrifugation at 3000 rpm. Serum was separated by a 5-min centrifugation at 3000 rpm, and then frozen at -80°C until the following measurement within 3 months.

Concentrations of total cholesterol, high-density lipo-protein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglyceride were determined using an enzymatic assay kit (Kyowa Chemical, Japan for TC and TG; Daiichi Chemical for LDL-C and HDL-C) placed on an Autoanalyzer 7350 (Hitachi, Japan). Levels of nonesterified fatty acid (NEFA), phospholipid and glucose were measured using an enzymatic assay kit (Wako Pure Chemical, Japan). Concentrations of insulin and leptin were measured by ELISA (Insulin-EIA test (Wako, Osaka, Japan) and the Human Leptin ELISA kit (Cayman Chemical, Ann Arbor, MI, USA). Homoeostasis model assessment (HOMA-IR) was calculated by the following formula: fasting serum insulin (U/ml) fasting plasma glucose (mg/dl)/405.³⁵

DNA analysis

Genomic DNA was prepared from leukocytes using a DNA Extractor WB Kit (Wako Pure Chemical, Japan). Amplification of DNA by polymerase chain reaction was carried out using 100 ng of genomic DNA as template with the primers (upstream: 5'-CGCCCAATACCGCCAACAC-3', downstream: 5'-CCACCAGGAGTCCCATCACC-3') under the same conditions used in the Kodowaki et al.¹⁷ The amplified fragments of 210 bp were digested with BstNI and analyzed by 3% agarose-gel electrophoresis. The appearance of a 161 bp fragment, instead of 99 bp and 62 bp fragments, indicated the presence of Trp64Arg mutation in the ₃AR gene.

Statistical analyses

Participants were divided into wild type and Trp64Arg mutants (Trp64Arg homozygotes and Trp64Arg heterozygotes). Analysis of data was done with SPSS statistical analysis software (Version 10, SPSS Inc., Chicago, IL, USA). Results are expressed as meanS.D. Comparisons between two genotypes were performed by Student's t-test to assess the differences in anthropometric and metabolic parameters by polymorphism in the ₃AR gene at the baseline. To assess the relation between weight loss and changes of energy intake and physical activity during intervention, Pearson's correlation coefficients were calculated. To evaluate whether the ₃AR gene had altered values from the intervention, we used a paired Student's t-test. Multiple linear regression analysis was conducted on 76 subjects to investigate whether weight loss was independently related to the ₃AR gene polymorphism, except for the effect of a decrease in energy intake and an increase of physical activity. Unless otherwise noted, a nominal two-sided P-value of less than 0.05 was used to assess significance.

Results

Overall response to the behavioral intervention

Response to the behavioral intervention for obesity in all participants is shown in Table 2. The intervention induced significant decreases in intake of energy (7.2%, P=0.001), protein (7.0%, P<0.001) and fat (12.1%, P<0.001), but the change in carbohydrate intake was not significant (4.2%, P0.05). Habitual physical activity was not changed before and after the intervention, except for walking. Number of steps per day increased significantly after intervention from 6487 steps to 9830 steps, which is approximately a 52% increase (P<0.001).

Table 2 - Response to behavioral intervention in all participants.

Full table

The intervention resulted in significant decreases in body weight (60.78.6 and 60.28.4 kg, P=0.005) and BMI (P=0.006). A total of 47 women (61.8%) reduced their body weight, and the range of changes in body weight was 2.2 to -4.0 kg. Significant changes were shown regarding waist circumference (P=0.009), hip circumference (P<0.001), skin fold at arm (P=0.035), systolic blood pressure (P=0.031) and diastolic blood pressure (P<0.001) between before and after the intervention, respectively. Waist/hip ratio did not decrease by the intervention, due to the same degree of decreases in waist and hip circumferences. Body fat altered significantly from 31.0 to 32.1% during the intervention (P<0.001), in spite of decreases in the measured values of body weight, BMI, waist circumference, hip circumference and skin fold at the arm (Table 2).

Significant differences of biochemical parameters were found in HDL-C (P<0.001), LDL-C/HDL-C (P<0.001) and phospholipid (P<0.001). There were no significant changes in glucose, insulin and HOMA-IR during the intervention (Table 2).

Comparison of baseline variables between wild type and Trp64Arg mutation

The frequency of the Trp64Arg allele was 19.7% (30/152), with the frequency of heterozygote and homozygote carriers of the mutation being 31.6% (24/76) and 3.9% (3/76), respectively. The subjects showed no statistically significant difference with respect to energy intake, protein intake, fat intake and carbohydrate intake, number of steps and REE between wild type and Trp64Arg mutants at the baseline. No significant difference in BMI, body weight, body fat, waist/hip ratio, skin fold at the arm and changes of body weight from 20 y old at baseline was found between subjects with and without the mutation, respectively (Table 3).

Table 3 - Response to behavioral intervention by 3-adrenergic receptor gene polymorphism.

Full table

No significant difference was found in total cholesterol, HDL-C, NEFA, phospholipid, glucose, insulin and HOMA-IR between subjects with and without the mutation, respectively. Trp64Arg mutants had lower concentrations of triglyceride and higher concentrations of plasma leptin compared with wild type, but they were not statistically significant differences (Table 3).

Comparison of responses to the intervention between wild type and Trp64Arg mutation

There were significant differences of behavioral changes in energy intake (P=0.016), protein intake (P=0.001), fat intake (P=0.001) and number of steps (P<0.001) in wild type, and energy intake (P=0.018), fat intake (P=0.002) and number of steps (P<0.001) in Trp64Arg mutants from the intervention (Table 3). However, there were no significant differences of a changed rate of intake of energy, protein and fat between wild type and Trp64Arg mutants during the intervention.

The intervention yielded a reduced body weight in 69 and 48%, and yielded a significant difference in weight loss (-0.740.15 and -0.011.17 kg, P=0.035) for women with wild type and Trp64Arg mutants, respectively. Significant differences were found in waist circumference (P=0.018), hip circumference (P<0.001), systolic blood pressure (P=0.026) and diastolic blood pressure (P<0.001), before and after the intervention of wild type, respectively. However, women with the mutation did not show significant changes in body weight, BMI, waist circumference, but showed significant differences in hip circumference (P=0.037) by the intervention (Table 3).

Significant differences of biochemical parameters were found in HDL-C (P<0.001), LDL-C/HDL-C (P=0.001) and phospholipid (P=0.005) in wild type, and in LDL-C (P=0.003), HDL-C (P=0.001), LDL-C/HDL-C (P<0.001) and phospholipid (P=0.019) in Trp64Arg mutants. There were no significant changes in glucose, insulin and HOMA-IR of wild type and Trp64Arg mutants from the intervention (Table 3).

The relation between weight loss and change in energy intake or change in number of steps due to the intervention is shown in Figure 1. Pearson's correlation coefficient between weight loss and change in energy intake was 0.436 (P=0.002), and the coefficiency between weight loss and change in number of steps showed a significant value of -0.502 (P<0.001) in the wild type. On the contrary, women with the Trp64Arg mutation did not show a significant association between weight loss and change in energy intake or change in number of steps after the intervention.

Figure 1.

Correlation between weight loss and changes in energy intake or number of steps Pearson's correlation coefficients associated with weight loss and p values were expressed.

Full figure and legend (50K)

Multiple linear regression analysis revealed that ₃AR gene polymorphism was independently related to weight loss (P=0.029). Change in energy intake (P=0.003) and change in the number of steps (P=0.002) were significantly associated with weight loss in all participants. Change in energy intake (P=0.004) and change in the number of steps (P=0.001) were significantly related to weight loss in the wild type independently. Conversely, in Trp64Arg mutants, change in energy intake and change in number of steps were not significantly associated with weight loss (Table 4).

Table 4 - Multivariable linear regression with weight-loss by the intervention.

Full table

Discussion

The present intervention for 3 months yielded 0.5 kg of weight loss, and 62% of the participants reduced their body weight. Further, blood pressure, HDL-C, LDL-C/HDL-C and phospholipids improved significantly, despite limited weight-loss. The National Heart, Lung and Blood Institute in the USA recommends that the initial goal of weight-loss therapy be to reduce body weight by approximately 10% from baseline through 6 months.²⁵ In the present study, we recommended that participants achieve 1.0 kg of weight loss and improvement of metabolic measurements.

Our results appear to reflect an adequate improvement in metabolic levels; however, we consider the results unsatisfactory insofar as the anthropometric parameters. One reason is that, while the number of steps satisfactorily increased to 7000 steps/day during this program, the intake of energy, especially carbohydrates, failed to achieve our target of a 10% reduction. In the light of the fact that some of our participants did not accomplish this weight-loss target, we likely need to strengthen participants' ability for self-evaluation and motivation for a more effective daily life and reduction of carbohydrate ingestion for future programs.

As another point of consideration, it has been known that a short-term reduction outcome is not necessarily maintained for a long term. The present study was a short-term weight-loss program of 3 months; however, as our method was based on the participants' greater awareness and autonomous improvement of behavior, such newly developed behavior may be more readily continued after the study program in comparison to one that incorporated strict compliance with rules and a complicated prescription of behavioristic modification.

The frequency of the Trp64Arg allele was 0.20 in the present subjects. With the frequency of this mutation also being reported to be 0.18–0.23 in other areas in Japan,^{17,18,20,21,22} the mutation is considered to be more common in Japanese than in Caucasians, African Americans and Mexican-Americans, but less common than in Pima Indians.^8,12,13,14 Anthropometric parameters did not differ significantly between women with and without the mutation at the baseline of this study, which was the same result as in our previous investigation. Meta-analysis demonstrated that, across the population, subjects with Trp64Arg polymorphism of the ₃AR gene had only a slight average BMI increase of 0.3 kg/m² compared to those with wild type.⁹ Therefore, the reasons for inconsistent findings regarding association between the mutation and obesity are assumed to be the weak influence of the ₃AR gene mutation on the pathogenesis of obesity, and/or masking effects of obesity.³⁶

The difficulty of weight loss in the Trp64Arg mutation may be more apparent by the uniqueness of our method based on relatively small changes of diet and exercise. This may be due to a weak influence of the ₃AR gene on obesity⁹ rather than overeating and insufficient physical activity. Our present study results indicate that the intervention caused greater weight loss and decreases in waist and hip circumferences in wild type in spite of smaller changes in diet and exercise, somewhat in contrast to previous investigations.^18,37,38 However, significant differences in nutritional intake and physical activity were not seen between wild type and Trp64Arg mutants by the intervention. Therefore, although our guidance for each participant's compliance (to control diet and physical exercise) was subjective in nature, we can say that both groups responded well to our intervention.

Experiments performed with a selective ₃AR agonist in vitro suggest that Trp64Arg mutation is associated with impaired lipolysis in adipose tissue from the intra-abdominal cavity.^9,12 Epidemiological studies using computed tomography images revealed that Trp64Arg mutants of postmenopausal obese Caucasian women had smaller reductions in intra-abdominal fat compared with wild type and suggested their limited capacity to lose visceral adipose tissue in response to prolonged caloric restriction.³⁹ This hypothesis was supported by epidemiological investigations of Pima Indians⁸ and Japanese,¹⁸ in which the Trp64Arg mutants tended to have a lower metabolic rate.

On the other hand, in the present study, there was no significant difference in REE between Trp64Arg mutants and wild type at the baseline using indirect calorimetry. These results should be interpreted with caution, because the sample size was small. Tchernof et al⁴⁰ also reported that, after adjusting factors for fat mass, fat-free mass and age, they found a lower energy expenditure and fat oxidation in Trp64Arg mutants among never-obese Caucasian women compared with obese women. Their results were contrary to the 'thrifty genotype' hypothesis¹⁵ that already obese individuals with Trp64Arg mutation of the ₃AR gene have lower daily energy expenditure, altered lipolysis and increased abdominal obesity.^11,12,18 It is possible that the effects of the mutation may be specific to a certain type of obesity, age and sex, as previously suggested.⁴¹ Since the increase in fat mass and fat-free mass is accompanied by an enhanced rate of NEFA release into circulation, which contributes to stimulate fat oxidation,^42,43 the reason for no difference or decreased energy expenditure in Trp64Arg mutation appears to be because of enhanced fat oxidation in the obese state itself.³⁶

Body fat increased significantly during our intervention in participants without Trp64Arg mutation, in spite of significant decreases in weight, waist circumference, hip circumference and skin fold at the arm. Bioelectrical impedance analysis has been reported to accurately assess body composition in weight-stable subjects,⁴⁴ however, it may not be possible to use in all studies to evaluate changes of fat mass after diet and exercise.^31,45 The present results indicate that bioelectrical impedance analysis may not be a useful tool in measuring changes in body composition.

In summary, the results of the present study support the view that Trp64Arg mutation of the ₃AR gene is associated with difficulty in weight loss, without lowering energy expenditure. This agrees with the fact that Trp64Arg polymorphism plays an important role in the development of obesity. Nonetheless, the influence of Trp64Arg polymorphism on BMI seems to be weak, with only a barely statistically significant difference shown by meta-analysis.^9,22,23 Therefore, the effect of Trp64Arg polymorphism on obesity might become unclear with a more intensive degree of lifestyle-related behavioral intervention for weight loss. For these reasons, we recommend further investigation of various degrees of calorie intake-restricted diet and exercise, for greater understanding of the relation between Trp64Arg polymorphism and hypoenergetic weight-loss intervention.

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Acknowledgements

This study was supported in part by Grants-in-Aid (12670355, 14570334 to KS, 13680173 to AN) for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and Grants from the Japanese Ministry of Health, Labor and Welfare (to YY).