Influence of Adiponectin Gene Polymorphism SNP276 (G/T) on Adiponectin in Response to Exercise Training

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1 Endocrine Journal 2007, 54 (6), Influence of Adiponectin Gene Polymorphism SNP276 (G/T) on Adiponectin in Response to Exercise Training HU HUANG, KAORUKO TADA IIDA, HARUKA MURAKAMI, YOKO SAITO, TAKESHI OTSUKI*, MOTOYUKI IEMITSU*, SEIJI MAEDA*, HIROHITO SONE**, SHINYA KUNO AND RYUICHI AJISAKA Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennodai, Tsukuba-shi, Ibaraki , Japan *Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tennodai, Tsukuba-shi, Ibaraki , Japan **Faculty of Human Life and Environmental Sciences, Ochanomizu University, Ohtsuka, Bunkyo-ku, Tokyo, , Japan Abstract. Adiponectin is an adipocytokine that is involved in insulin sensitivity. The adiponectin gene contains a single nucleotide polymorphism (SNP) at position 276 (G/T). The GG genotype of SNP276 (G/T) is associated with lower plasma adiponectin levels and a higher insulin resistance index. Therefore, we examined the influence of SNP276 (G/T) on the plasma level of adiponectin in response to exercise training. Thirty healthy Japanese (M12/F18; 56 to 79 years old) performed both resistance and endurance training, 5 times a week for 6 months. The work rate per kg of weight at doubleproduct break-point (DPBP) was measured. Blood samples were obtained before and after the experiment. Plasma concentrations of adiponectin, HbA1c, insulin, glucose, total, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) cholesterol, and triglyceride were measured. Genotypes of SNP276 were specified. Student s t-test for paired values and unpaired values was used. After the 6-month training period, the work rate per kg of weight at DPBP and the plasma HDL-cholesterol level were significantly improved (P<0.05), while no change was observed in the total plasma adiponectin level. However, the plasma adiponectin level in those with the GT + TT genotype had significantly increased (P<0.05). Additionally, the degree of the decrease in the HOMA-R level was significantly greater in the subjects with the GT + TT genotype than those with the GG genotype (p<0.05). Our results suggest that subjects with the genotype GT + TT at SNP276 (G/T) have a greater adiponectin-related response to exercise training than those with the GG genotype. Key words: Adiponectin, Genotype, Insulin sensitivity, Exercise training (Endocrine Journal 54: , 2007) ADIPONECTIN, an adipocyte-derived cytokine, is the most abundant gene product in adipose tissue and is present at high levels in plasma [1, 2]. It is thought to play an important role in the metabolism of both glucose and lipids [3]. Unlike other adipocytokines, adiponectin is found at low concentration in patients with obesity [4], type 2 diabetes [5], and coronary artery disease [6]. The plasma adiponectin concentration is correlated with insulin sensitivity and can be used to predict the development of type 2 diabetes [4, 7, 8]. Aerobic exercise is a generally accepted therapeutic Received: August 8, 2006 Accepted: August 3, 2007 Correspondence to: Hu HUANG, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennodai, Tsukuba-shi, Ibaraki , Japan strategy for type 2 diabetes because it not only has beneficial effects on the glycemic profile but also reduces metabolic risk factors for cardiovascular diseases including insulin resistance [9]. Given that exercise training potentially improves insulin sensitivity via a number of mechanisms, it can be speculated that adiponectin is involved in the regulation of insulin sensitivity through exercise training. To date, studies have reported different effects of exercise on circulating adiponectin levels; that is, no change [10 13], an increase [14], or a decrease [15]. There is now a growing realization that the physical response to a particular environmental stimulus such as exercise may be mediated by individual genetic variability [16]. Therefore, it is important to consider that the effect of exercise training on adiponectin may vary between individuals and may be influenced not only by environmental conditions,

2 880 HUANG et al. exercise intensity, and gender difference, but also by genetic background. Adiponectin is encoded by adipocyte-c1q and collagen domain-containing (ACDC), which is located on chromosome 3 at q27 [17, 18]. Studies of mutations in ACDC have revealed 16 single nucleotide polymorphisms (SNPs) [19]. Among them, SNP276 (G/T) has been reported to be associated with type 2 diabetes, circulating adiponectin levels, and insulin resistance in a Japanese population [20]. In the present study, we investigated the association of the SNP276 (G/T) polymorphism of the adiponectin gene (ACDC) with the response to exercise training in healthy middle-aged or elderly Japanese subjects. Subjects Materials and Methods The subjects were 30 healthy middle-aged or elderly Japanese (M12/F18; 56 to 79 years old) having a normal fasting glucose level (<110 mg/dl), normal total cholesterol level (<220 mg/dl), and normal BMI level (<24 kg/m 2 ) before the exercise training program started. They had not taken any prescription medicines for diabetes mellitus, hyperlipidemia, hypertension, or other conditions. The study was approved by the Ethics Committees of the Institute of Health and Sport Sciences and the Institute of Clinical Medicine of the University of Tsukuba. It conformed to the principles outlined in the Helsinki Declaration, and written informed consent was obtained from all subjects before their inclusion in the study. Exercise tolerance test Before and after exercise training, all subjects participated in an exercise tolerance test using a cycle ergometer (232CXL; CW, Tokyo, Japan) to measure the double-product (DP) of heart rate and systolic blood pressure with monitoring via an automated monitor (DPBP measurement system; Kyokko Bussan, Tokyo, Japan). The double-product break-point (DPBP) was determined at a point where there was a clear and sustained increase in lactate threshold and ventilatory threshold. Thus, the DPBP is considered an indicator of endurance. Additionally, the work rate per kg of weight at DPBP was also measured. This non-invasive and less onerous method of measuring DPBP is useful in elderly people and cardiac patients [21, 22]. Exercise programs All subjects participated in a resistance and endurance exercise session conducted 5 times a week for 6 months. The training program consisted of stretching to warm up, endurance training, resistance training, and stretching to cool down. The endurance training involved exercise on a cycle ergometer (30 min) at 80% of the work rate of each DPBP. The resistance training comprised 3 sets of squats, trunk curls, back extensions, leg extensions, hip extensions, and leg curl exercises at the subject s own weight (10 repetitions). Since weight reduction was not the aim of this study, no specific dietary interventions were included. Plasma adiponectin concentration Before and after 6-month exercise training, at the morning after an overnight fast, blood sample were obtained. The plasma adiponectin concentration was measured with a commercially available enzymelinked immunosorbent assay (ELISA) kit (Otsuka Pharmaceuticals; Tokyo, Japan). Physical and metabolic characteristics Blood samples were obtained in the morning after an overnight fast. Physical and metabolic characteristics such as body weight, systolic and diastolic blood pressure, heart rate, and plasma fasting total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglyceride, HbA1c, glucose, and insulin levels were assessed. Obesity was assessed in terms of body mass index (BMI = body weight [kg]/height [m] 2 ) and insulin resistance with the homeostasis model assessment-insulin resistance index (HOMA-R = fasting plasma glucose concentration [mmol/l] fasting plasma insulin concentration [µu/ ml]/22.5). Genotyping Genotyping was performed with genomic DNA extracted from peripheral blood leukocytes using a nucleic acid isolation system (KURABO, NA3000; Osaka, Japan). A 518-bp fragment of the adiponectin gene

3 ADIPONECTIN GENOTYPE AND EXERCISE TRAINING 881 encompassing the site of the polymorphism at 276 (G/T) was generated from genomic DNA by polymerase chain reaction (PCR) using the primers 5'- AGAAAGCAGCTCCTAGAAGT-3' (forward) and 5'- GGCACCATCTACACTCATCC-3' (reverse). The reaction profile was as follows: 35 cycles of denaturation at 96 C for 30 sec, annealing at 53 C for 30 sec, and extension at 60 C for 30 sec. The PCR products were genotyped by direct sequencing using the primer 5'- ATGGCTGACAGTGCACATGT-3' with an ABI Prism 3100-Avent Genetic Analyzer (Applied Biosystems, Foster City, CA, U.S.A.). Statistical analysis All data are expressed as the mean ± SD. Statistical analyses were performed with the StatView software (SAS Institute Inc., Cary, NC, U.S.A.). Differences between genotypes were tested with the student unpaired t-test. Differences before versus after exercise training were tested with the paired t-test. A p value less than 0.05 indicated statistical significance. The allelic distribution of SNP276 (G/T) of adiponectin was verified using Hardy-Weinberg equilibrium. Results Table 1 shows the genotypic and allelic distribution of the SNP276 (G/T) polymorphism of the adiponectin gene (ACDC). No significant deviations from Hardy- Weinberg equilibrium were observed for this locus. Table 2 shows the physical and metabolic characteristics of the subjects before and after the 6-month intervention. The work rate per kg of weight at DPBP was significantly high after the 6-month training period (1.06 ± 0.18 watt/kg to 1.14 ± 0.19 watt/kg; p = ). Systolic blood pressure (125.8 ± 16.5 mmhg to ± 15.9 mmhg; p = ), diastolic blood pressure (77.6 ± 8.1 mmhg to 76.9 ± 6.9 mmhg; p = ), and heart rate (62 ± 8 BPM to 60 ± 8 BPM; p = ) were not significantly changed compared with the baseline. Plasma HDL cholesterol level was significantly high after the 6 months of exercise (55 ± 12 mg/dl to 58 ± 11 mg/dl; p = ) compared with the baseline. Plasma HbA1c level was lower after the 6 months (4.95 ± 0.28% to 4.90 ± 0.22%; p = ). Plasma adiponectin concentration was not significantly changed Table 1. Genotypic and allelic distribution of SNP276 GG GT + TT Total N (M/F) 18 (7/11) 12 (5/7) 30 Genotype frequency 60% 40% 100% Allele frequency 80% (G allele) 20% (T allele) 100% GG: GG genotype at SNP276 (G/T) of the adiponectin gene (ACDC), GT + TT: GT + TT genotype at SNP276 (G/T) of the adiponectin gene (ACDC). ACDC: adipose-c1q and collagen domain-containing. M: Male, F: Female. Table 2. Physical and metabolic characteristics of the subjects before and after exercise training Pre Post Weight (kg) 52.8 ± ± 6.7 BMI (kg/m 2 ) 21.8 ± ± 2.2 Work rate (watt/kg) 1.06 ± ± 0.19* Systolic blood pressure (mmhg) ± ± 15.9 Diastolic blood pressure (mmhg) 77.6 ± ± 6.9 Heart rate (BPM) 62 ± 8 60 ± 8 HbA1c (%) 4.95 ± ± 0.22 Insulin (µm/ml) 4.3 ± ± 2.2 Glucose (mg/dl) 96.0 ± ± 8.1 HOMA-R 1.0 ± ± 0.6 Total cholesterol (mg/dl) 193 ± ± 27 HDL cholesterol (mg/dl) 55 ± ± 11* LDL cholesterol (mg/dl) 116 ± ± 26 Triglyceride (mg/dl) 89 ± ± 52 Adiponectin (µg/ml) 10.8 ± ± 4.2 Blood analyses were performed and HOMA-R was measured following the 6-month training program. HOMA-R of each subject was calculated from fasting plasma glucose (FPG) levels and insulin (FPI) levels (HOMA-R = FPG [mmol/l] FPI [µu/ml]/ 22.5). Body mass index (BMI) was calculated as body weight [kg]/height [m] 2. A paired Student s t-test was used to evaluate the effects of exercise training on the physical and metabolic parameters. Values are means ± SD, * p<0.05 vs. pretest. (10.8 ± 3.6 µg/ml to 11.6 ± 4.2 µg/ml; p = ). Table 3 shows the physical and metabolic characteristics of the subjects before and after exercise training according to the SNP276 (G/T) genotype of the adiponectin gene (ACDC). The work rate per kg of weight at DPBP was not significantly different between the GG genotype and the other genotypes before (GG: 1.05 ± 0.17 watt/kg vs. GT + TT: 1.07 ± 0.19 watt/kg; p = ) or after (GG: 1.11 ± 0.17 watt/kg vs. GG + GT: 1.18 ± 0.22 watt/kg; p = ) the 6- month training period. The plasma adiponectin concentration was not significantly different between the

4 882 HUANG et al. Table 3. Physical and metabolic characteristics of the subjects before and after exercise training according to the adiponectin genotypes at the position of SNP276 (G/T) GG GT + TT P value N (M/F) 18 (7/11) 12 (5/7) Work rate (watt/kg) Work rate (watt/kg) Pre 1.05 ± ± Work rate (watt/kg) Post 1.11 ± ± Change in Work rate (watt/kg) 0.06 ± ± BMI (kg/m 2 ) BMI (kg/m 2 ) Pre 21.9 ± ± BMI (kg/m 2 ) Post 22.1 ± ± Change in BMI (kg/m 2 ) 0.3 ± ± HbA1c (%) HbA1c (%) Pre 5.01 ± ± HbA1c (%) Post 4.92 ± ± Change in HbA1c (%) 0.09 ± ± HOMA-R HOMA-R Pre 1.03 ± ± HOMA-R Post 1.24 ± ± Change in HOMA-R 0.22 ± ± * HDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Pre 57 ± ± HDL cholesterol (mg/dl) Post 59 ± ± Change in HDL cholesterol (mg/dl) 2.5 ± ± Adiponectin (µg/ml) Adiponectin (µg/ml) Pre 11.6 ± ± Adiponectin (µg/ml) Post 11.8 ± ± Change in Adiponectin (µg/ml) 0.1 ± ± * Blood analyses were performed and HOMA-R was measured before and after the 6-month training period. HOMA-R of each subject was calculated from fasting plasma glucose (FPG) levels and insulin (FPI) levels (HOMA-R = FPG [mmol/l] FPI [µu/ml]/22.5). Body mass index (BMI) was calculated as body weight [kg]/height [m] 2. An unpaired Student s t-test was used to evaluate the effects of exercise training on the physical and metabolic parameters. Values are means ± SD, * p<0.05 vs. GG genotype of SNP276 (G/T). GG: GG genotype at SNP276 (G/T) of the adiponectin gene (ACDC), GT + TT: GT + TT genotype at SNP276 (G/T) of the adiponectin gene (ACDC), ACDC: adipose-c1q and collagen domain-containing. M: Male, F: Female. GG genotype and the other genotypes at baseline (p = ). However, the degree of the increase in this parameter was significantly greater in the subjects with the GT + TT genotype than those with the GG genotype after the 6 months of exercise (GT + TT: 2.1 ± 2.8 µg/ml vs. GG: 0.1 ± 2.3 µg/ml; p = ). Additionally, the plasma adiponectin concentration was unchanged after the 6-month period in those with the GG genotype of SNP276 (G/T). In contrast, in those with the GT + TT genotype of SNP276 (G/T), the plasma adiponectin concentration increased significantly (p = ) (Fig. 1). The degree of the decrease in the HOMA-R levels was significantly greater in the subjects with the GT + TT genotype than those with the GG genotype (GT + TT: 0.16 ± 0.50 vs. GG: 0.22 ± 0.31; p = ) (Table 3). Discussion Peripheral insulin resistance is a primary disorder in both obesity and non insulin-dependent diabetes mellitus (NIDDM) [23]. Adiponectin is exclusively and abundantly expressed in white adipose tissue. In animals and humans plasma adiponectin concentrations are low in a state of insulin resistance [4, 5]. Previous studies have shown that thiazolidinediones increase the serum adiponectin concentration by increasing the level of transcription of the adiponectin gene [24, 25].

5 ADIPONECTIN GENOTYPE AND EXERCISE TRAINING 883 Fig. 1. Plasma adiponectin concentrations before and after the 6-month training period were evaluated with a paired Student s t-test. Values are means ± SD, * p<0.05 vs. pretest. GG: GG genotype at SNP276 (G/T) of the adiponectin gene (ACDC), GT + TT: GT + TT genotype at SNP276 (G/T) of the adiponectin gene (ACDC), ACDC: adipose-c1q and collagen domain-containing. Also, the association between adiponectin gene polymorphisms and the risk of type 2 diabetes has been examined in many studies [19, 20, 26, 27]. Therefore, it is important to investigate whether such polymorphisms affect the response to some interventions. Kang et al. showed that the degree of the increase in serum adiponectin levels after rosiglitazone treatment was less in type 2 diabetes patients with the GG genotype of SNP276 (G/T) than in subjects with the other genotypes of SNP276 (G/T) in a Korean population [28]. Exercise training is also a common treatment for insulin resistance [29]; however, there are numerous reports that neither acute exercise nor exercise training changed circulating adiponectin levels [10 13]. It is conceivable that the effect of exercise training on adiponectin concentrations depends not only on environmental conditions, exercise intensity, and gender difference, but also on genetic background. For this reason, the present prospective intervention study was performed to evaluate how responses to exercise training vary with adiponectin gene polymorphisms. Our data suggest that there is no association between SNP276 (G/T) genotypes and the plasma adiponectin concentration before exercise training in healthy middle-aged or elderly Japanese. This result is consistent with that of Hara et al. [20]. The work rate per kg of weight at DPBP did not differ between the GG genotype and the other genotypes of SNP276 (G/T) before and after the 6-month training period; in other words, the effects of exercise training were similar regardless of genotype. Interestingly, the subjects with the genotype GT + TT at SNP276 (G/T) had a greater response to the 6 months of training. That is, a significant increase in the plasma adiponectin concentration was observed in the subjects with GT + TT at SNP276 (G/T), but not in the subjects with the GG genotype. It was also observed that the degree of the decrease in HOMA-R levels was significantly larger in the subjects with GT + TT than those with GG. Although the plasma adiponectin concentration was not significantly different between the GG genotype and the other genotypes of SNP276 (G/T) at baseline, there were differences between each genotype of SNP276 (G/T) after the 6 months of exercise. A difference in either the transcriptional activity or the mrna stability of adiponectin according to the adiponectin genotype could be responsible for the increase in the plasma adiponectin concentration in those with the GT + TT genotype compared to those with the GG genotype. The increased adiponectin levels in subjects with the GG + TT genotype may result in increased insulin sensitivity, which in turn would contribute to a significant decrease in HOMA-R levels. Recent reports show that neither acute exercise nor exercise training changes circulating adiponectin levels despite that exercise improves insulin sensitivity [10 12]. Moreover, individual variations in adiponectin levels before and after an 8-week training program have been reported [10]. Thus, in this study, we focused on the allelic frequency of the SNPs of adiponectin. It is reported that 53% of French Caucasians [30] and over 60% of Japanese [20] have the GG genotype of SNP276 (G/T) which is associated with type 2 diabetes. Therefore, it is not difficult to imagine that the 60% of subjects whose plasma adiponectin levels were unchanged after the training program affected the final result. Our data clearly show that the subjects with GT + TT at SNP276 (G/T) had significantly increased plasma adiponectin levels after exercise training; however, overall, after the 6-month training period, the plasma adiponectin concentration was not significantly changed compared with the baseline. This suggests that not only the secretion of adiponectin but also the response to exercise training varies with genotype. SNP276 (G/T) is located in intron 2 of the adiponectin gene (ACDC). Intronic SNPs can affect the expression of a gene via unknown mechanisms. There may be a specific linkage structure or gene-environmental

6 884 HUANG et al. interaction. SNP45 (T/G) located in exon 2 of the adiponectin gene (ACDC) may inactivate the gene or affect the concentration of adiponectin by influencing the pre-mrna splicing or the stability of the mrna [31], and it is reported that SNP45 (T/G) has a linkage with SNP276 (G/T), and a haplotype with GG/GG at SNP45 (T/G) and SNP276 (G/T) was less responsive to rosiglitazone treatment than other haplotypes [28]. One recent report also found that SNP (C/G) and SNP (G/A) in the promoter region of the adiponectin gene were associated with plasma adiponectin levels and insulin sensitivity [32]. These SNPs may be linked with SNP276 (G/T) and affect plasma adiponectin levels at the transcriptional step in response to an interventional stimulus such as exercise training. Alternatively, it may be related to another functional locus via a linkage disequilibrium which has yet to be identified. There are reports of susceptibility loci for type 2 diabetes [33] and quantitative trait loci for insulin resistance [34] being mapped to 3q27, where the adiponectin gene is located. Based on these results, adiponectin is one of the positional candidate genes in this region, the existence of which might explain this unknown linkage. Further study is needed to clarify the relationship between this unknown linkage and the observed association with the adiponectin gene. Conclusion In conclusion, this study suggests that subjects with the GT + TT genotype at SNP276 (G/T) of the adiponectin gene (ACDC) are more likely to respond to exercise training than those with the GG genotype. It was found that variations in the adiponectin gene could influence the effects of exercise training on the plasma adiponectin level and HOMA-R level. These findings may be clinically relevant in the prediction of subjects who will best respond to exercise therapy. However, further investigations are needed to fully elucidate the functional mechanism of this polymorphism. References 1. Maeda K, Okubo K, Shimomura I, Funahashi T, Matsuzawa Y, Matsubara K (1996) cdna cloning and expression of a novel adipose specific collagen-like factor, apm1 (Adipose Most abundant Gene transcript 1). Biochem Biophys Res Commun 221: Arita Y, Kihara S, Ouchi N, Takahashi M, Maeda K, Miyagawa J, Hotta K, Shimomura I, Nakamura T, Miyaoka K, Kuriyama H, Nishida M, Yamashita S, Okubo K, Matsubara K, Muraguchi M, Ohmoto Y, Funahashi T, Matsuzawa Y (1999) Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res Commun 257: Maeda N, Shimomura I, Kishida K, Nishizawa H, Matsuda M, Nagaretani H, Furuyama M, Kondo H, Takahashi M, Arita Y, Komuro R, Ouchi N, Kihara S, Tochino Y, Okutomi K, Horie M, Takada S, Ayoyama T, Funahashi T, Matsuzawa Y (2002) Diet-induced insulin resistance in mice lacking adiponectin/acrp30. Nat Med 8: Weyer C, Funahashi T, Tanaka S, Hotta K, Matsuzawa Y, Pratley RE, Tataranni PA (2001) Hypoadiponectinemia in obesity and type 2 diabetes: close association with insulin resistance and hyperinsulinemia. J Clin Endocrinol Metab 86: Hotta K, Funahashi T, Arita Y, Takahashi M, Matsuda M, Okamoto Y, Iwahashi H, Kuriyama H, Ouchi N, Maeda K, Nishida M, Kihara S, Sakai N, Nakajima T, Hasegawa K, Muraguchi M, Ohmoto Y, Nakamura T, Yamashita S, Hanafusa T, Matsuzawa Y (2000) Plasma concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients. Arterioscler Thromb Vasc Biol 20: Kumada M, Kihara S, Sumitsuji S, Kawamoto T, Matsumoto S, Ouchi N, Arita Y, Okamoto Y, Shimomura I, Hiraoka H, Nakamura T, Funahashi T, Matsuzawa Y (2003) Association of hypoadiponectinemia with coronary artery disease in men. Arterioscler Thromb Vasc Biol 23: Ryan AS, Berman DM, Nicklas BJ, Sinha M, Gingerich RL, Meneilly GS, Egan JM, Elahi D (2003) Plasma adiponectin and leptin levels, body composition, and glucose utilization in adult women with wide ranges of age and obesity. Diabetes Care 26: Fernandez-Real JM, Lopez-Bermejo A, Casamitjana R, Ricart W (2003) Novel interactions of adiponectin with the endocrine system and inflammatory parameters. J Clin Endocrinol Metab 88: Young JC (1995) Exercise prescription for individuals with metabolic disorders. Practical considerations. Sports medicine19: Boudou P, Sobngwi E, Mauvais-Jarvis F, Vexiau P, Gautier JF (2003) Absence of exercise-induced variations in adiponectin levels despite decreased abdominal adiposity and improved insulin sensitivity in type 2 di-

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