Aging Clin Exp Res DOI 10.1007/s40520-014-0213-3
ORIGINAL ARTICLE
Impact of UCP2 polymorphism on long-term exercise-mediated changes in adipocytokines and markers of metabolic syndrome Kang-Il Lim • Yun-A Shin
Received: 21 August 2013 / Accepted: 4 March 2014 Ó Springer International Publishing Switzerland 2014
Abstract Background and aims Variations in genes involved in energy expenditure affect aerobic exercise efficiency, but it remains unclear whether the effect of aerobic exercise on adipocytokines is modified by the obesity-associated genotypes in the uncoupling protein 2 gene (UCP2). The purpose of this study was to assess whether genetic variation in UCP2 may affect exercise-mediated changes in adipocytokines and markers of metabolic syndrome in postmenopausal obese women. Methods Forty-two sedentary postmenopausal obese women (age 52.74 ± 6.39 years) participated in this study. Participants were encouraged to train for 3 days a week, for 6 months, for 60 min per session of treadmill walking/ running at 60 % V O2 R. Subjects were genotyped for the 45-bp insertion/deletion (I/D) polymorphism in the 30 -untranslated region (UTR) of UCP2. Results Among the subjects, 23 (57.1 %) and 19 (42.9 %), were deletion homozygotes (DD) and ID heterozygotes,
respectively. For DD homozygotes, body weight, body mass index (BMI), % body fat, and waist circumference, and body weight, BMI, and waist circumference of ID heterozygotes, were significantly decreased after the exercise program. There were no significant changes in metabolic markers in individuals with the ID genotype, whereas insulin and HOMA-IR in individuals with the DD genotype were significantly decreased after the exercise program. In DD homozygotes, but not in ID heterozygotes, adiponectin was significantly increased, and leptin, TNF-a, and IL-6 were significantly decreased after exercise training. Conclusions Exercise-mediated changes in insulin resistance and adiponectin levels may be affected by genotypes in the 30 UTR I/D polymorphism in UCP2 in postmenopausal obese women. Keywords Adipocytokines Exercise Metabolic syndrome UCP2
Introduction K.-I. Lim Institute of Exercise Physiology, School of Kinesiology, Yeungnam University, 280 Daehak-Ro, Gyeongsan, Gyeongbuk 712-749, Republic of Korea e-mail: [email protected] Y.-A. Shin (&) Department of Prescription and Rehabilitation of Exercise, College of Sports Science, Dankook University, San 29, Anseo-dong, Cheonan-si, Chungnam 330-714, Republic of Korea e-mail: [email protected] Y.-A. Shin Department of Kinesiologic Medical Science, Graduate, Dankook University, San 29, Anseo-dong, Cheonan-si, Chungnam 330-714, Republic of Korea
Regular exercise is a key component in the prevention and treatment of metabolic disorders, such as hypertension, diabetes, and obesity. However, the effects of exercise on metabolic abnormalities associated with obesity vary from person to person, and these individual variations may be caused not only by specific environmental conditions, but also by genetics [1]. Thus, it is necessary to assess whether changes in energy metabolism and intracellular energy charge occurs in adipocytes during treatments that affect adiposity, insulin resistance, and other components of the metabolic syndrome. Unlike other adipocytokines, adiponectin suppresses the expression of adhesion molecules in endothelial cells and
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protects against metabolic syndrome, cardiovascular disease (CVD), and inflammation. Moreover, low levels of adiponectin are associated with chronic inflammatory conditions, such as insulin resistance and obesity. Recently, a review [2] suggested that training at an adequate duration and intensity to produce substantive changes in fitness levels and in body composition may decrease circulating leptin levels and raise circulating adiponectin levels. Uncoupling protein 2 (UCP2), a member of the uncoupling protein family, is thought to play a role in proton transfer in the mitochondrial membrane, without producing ATP, as well as in modulating ATP synthesis. The level of this protein is up-regulated in white adipose tissue [3] and the protein has been correlated with energy expenditure in humans [4]. In particular, a 45-bp insertion/deletion (I/D) in the 30 -untranslated region (UTR) of exon 8 has been implicated in obesity, metabolic rate, and exercise efficiency [5, 6]. It has also been reported that UCP2 expression is affected by physical stimulation [7]. Cortright et al. [8] found that UCP2 expression levels in mouse skeletal muscles are elevated after acute exercise. Moreover, Marti and his colleagues [9] reported that the UCP2 insertion allele is associated with physical activity. Thus, the genotypes of genes involved in energy expenditure also affect the efficiency of aerobic exercise [7], but it remains unclear whether the effect of aerobic exercise training could be modified by genotypes in specific genes. Therefore, it could be speculated that the UCP2 genotypes associated with obesity may affect the secretion of biologically active substances from adipose tissue (e.g., leptin and other adipocytokines) and could hence affect systemic insulin sensitivity during exercise training. Although most studies have reported association of UCP2 genotypes to increased body weight and energy expenditure [10, 11], the physiological role of the UCP2 insertion allele following exercise training has not yet been studied. Furthermore, the effect of exercise training on the relationship between adipocytokines and UCP2 genotypes has not been investigated. Therefore, the aims of this study were to compare markers of the metabolic syndrome and levels of adipocytokines according to UCP2 genotypes, both before and after exercise, and assess whether genetic variations in UCP2 can affect exercise-mediated changes in adipocytokines and metabolic syndrome markers in postmenopausal obese women.
Materials and methods Subjects The present study was approved by the Institutional Ethnics Committee of Physical Education of Dankook
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University. Informed consent was obtained from all subjects prior to their participation in this study. Eighty-five women were recruited from among volunteers in Seoul City. Questionnaires were used to determine recent participation in exercise, menopausal states, lifestyle, and disease states. Exclusion criteria were as follows: any specific diet, cigarette smoking (either currently or in the previous 6 months), use of anti-inflammatory drugs or steroids, surgery (in the previous 3 months), and metabolic disorders, such as myocardial infarction, stroke or hypertension, hypercholesterolemia, musculoskeletal injury, or current illness. Eventually, 42 sedentary healthy middle-aged women (52.74 ± 6.39 years) participated in this study. The subjects were postmenopausal, obese women according to body mass index (BMI C25 kg/m2) and body % fat (C30 %). None of these participants had taken part in any regular aerobic exercise over the previous 6 months. Anthropometric measurements Clinical characteristics (height, body weight, waist circumference, % body fat, and blood pressure) were measured using a standard protocol. Standing height was measured on a portable stationmaster to an accuracy of ±0.5 cm. The weight and percentage of body fat were measured using an Inbody 7.0 (Biospace, Korea) to the nearest 0.1 kg. BMI was calculated as weight (kg)/height (m2). Preliminary testing
Prior to the exercise trials, V O2 max was measured to establish the exercise intensity for each individual. Subjects were familiarized with treadmill running and were informed of what was required of them about the experiment. They then completed a graded treadmill exercise test to determine their individual V O2 max, adhering to the Bruce protocol. Aerobic exercise training Aerobic training sessions were supervised by three experienced physical education instructors. Participants were encouraged to train for 3 days a week, for 6 months; each exercise session consisted of a brief warm-up period, followed by 45 min per session of treadmill walking/running at 60 % V O2 R, followed by a brief cool-down period. The training program started at 40 % O R and gradually V 2 increased to 60 % V O2 R by week 12. After 12 weeks of training, the exercise intensity for each participant was
Aging Clin Exp Res
examined and adjusted to their new measured V O2 max, and was then maintained at 60 % V O2 R until the end of training. Chemical analysis Blood samples were obtained in the morning after overnight fasting and were stored at -80 °C for subsequent assays. Total cholesterol (TC), triglyceride (TG), and high-density lipoprotein (HDL) cholesterol levels were measured by enzymatic procedures using a chemistry analyzer (Hitachi 747; Tokyo, Japan), with a coefficient of variation (CV), of 1.9–2.2, 5.7–6.3, and 2.5 %, respectively. Plasma glucose levels and serum insulin were measured using a commercially available kit (glucose hexokinase kit, ADVIA 1650, Bayer, Japan; radioimmunoassay RIA kit, Linco Research, St. Charles, MO, USA), with a CV of 1.1 and 7.8–9.3 %, respectively. Insulin sensitivity was assessed using the homeostasis model assessment of insulin resistance index (HOMA-IR) (fasting glucose level [mmol/L] 9 fasting plasma insulin level [lU/mL]/22.5) [12]. Serum adiponectin was measured using a human adiponectin ELISA kit (BioVender, Laboratory Medicine; Brno, Czech Republic), with a CV of 4.8 %. Serum leptin was measured using the RIA method (Radio Immunoassay, Cosmic Corp, Tokyo, Japan), with a CV of 4.2–4.9 %), and IL-6 and TNF-a levels were measured by ELISA (Biosource International; Camarillo, CA, USA) with a CV of 2–4.2 and 12.6–16.9 %, respectively. Genomic DNA was extracted from peripheral blood leukocytes of all subjects, and the 30 -UTR I/D in UCP2 genotyped by polymerase chain reaction (PCR) using the primers: sense 50 -CAGTGAGGGAAGTGGGAGG-30 and antisense 50 -GGGGCAGGACGAAGATTC-30 . PCR conditions involved initial denaturation at 94 °C for 3 min, followed by 35 cycles each consisting of 30 s at 94 °C, 25 s at 56 °C, and 30 s at 72 °C, and a final extension step Table 1 Changes in clinical characteristics before and after a 6-month exercise program according to genotypes at the UCP2 insertion/deletion (I/D) polymorphism
Variables Body weight (kg) BMI (kg/m2) Body Fat (%) Waist Circumference (cm) SBP (mmHg) DBP (mmHg)
Data are the mean ± SD
at 72 °C for 7 min. The size of the PCR products were then assessed by electrophoresis in 2 % agarose gels to identify individuals homozygous for the deletion (DD), heterozygous for the ID, or homozygous for the insertion (II). Data analysis Results were expressed as mean ± standard deviation (SD), and statistical analyses were performed using SPSS for PC (version 12.5 for Windows; SPSS, Inc.; Chicago, IL, USA). Paired t test was used to determine significance differences within a group, and comparisons between groups were analyzed using the independent t-test. Conformity of the genotype distribution to Hardy–Weinberg was calculated using the v2 test. A P value of \0.05 was accepted as indicating statistical significance.
Results UCP2 DD and ID genotypes were present in 23 (57.1 %) and 19 (42.9 %) subjects, respectively. However, the II genotype was not present in any of the subjects. No significant deviation from Hardy–Weinberg equilibrium was observed. The clinical characteristics of subjects after the 6-month exercise program are shown in Table 1, according to the UCP2 I/D polymorphism genotypes. There was no significant difference at baseline between the UCP2 I/D genotypes for any of the variables (Table 1). In individuals with the DD genotype, body weight (P \ 0.001), BMI (P \ 0.001), % body fat (P \ 0.05), and waist circumstance (P \ 0.001) significantly decreased after the 6-month exercise program. In individuals with the ID genotype, body weight (P \ 0.01), BMI (P \ 0.01), and waist circumference (P \ 0.001) were significantly decreased after the exercise program.
Genotype (n)
Pre
Post
P value
DD (23)
69.58 ± 10.08
68.01 ± 9.88
0.001
ID (19)
67.95 ± 8.21
65.92 ± 8.10
0.003
DD (23)
28.46 ± 3.64
27.82 ± 3.53
0.001
ID (19)
27.68 ± 2.82
26.85 ± 2.82
0.003
DD (23)
35.35 ± 3.44
34.11 ± 4.12
0.014
ID (19)
35.76 ± 4.91
35.84 ± 4.44
0.823
DD (23)
83.43 ± 7.87
77.87 ± 7.45
\0.001 \0.001
ID (19)
83.26 ± 7.02
77.63 ± 8.41
DD (23)
124.17 ± 15.28
124.74 ± 13.99
0.849
ID (19)
123.00 ± 10.21
123.53 ± 11.96
0.794
DD (23)
85.48 ± 11.01
83.87 ± 10.27
0.383
ID (19)
83.11 ± 9.17
84.16 ± 8.59
0.494
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Aging Clin Exp Res Table 2 Changes in metabolic characteristics before and after a 6-month exercise program according to genotypes at the UCP2 insertion/deletion (I/D) polymorphism
Variables TC (mmol/L) TG (mmol/L) HDL-C (mmol/L) Glucose (mmol/L) Insulin (pmol/L) HOMA-IR
Data are the mean ± SD
Genotype (n)
Pre
DD (23)
203.78 ± 48.66
213.70 ± 45.28
0.124
ID (19)
191.11 ± 44.33
205.47 ± 41.36
0.076
P value
DD (23)
155.00 ± 73.83
138.52 ± 55.88
0.155
ID (19)
140.79 ± 90.86
137.21 ± 75.16
0.871
DD (23)
45.48 ± 8.81
45.70 ± 7.86
0.879
ID (19)
49.53 ± 14.17
49.16 ± 10.70
0.922
DD (23)
89.16 ± 8.95
88.11 ± 7.96
0.472
ID (19)
90.00 ± 5.44
91.70 ± 9.11
0.312
DD (23)
3.84 ± 3.26
2.69 ± 2.02
0.026
ID (19)
4.11 ± 3.09
3.63 ± 2.52
0.387
DD (23)
1.57 ± 1.45
1.08 ± 0.82
0.036
ID (19)
1.67 ± 1.33
1.52 ± 1.11
0.528
The metabolic characteristics of individuals after the 6-month exercise program is shown in Table 2 according to the UCP2 I/D genotypes. There were no significant changes in metabolic markers in individuals with the ID genotype, whereas insulin (P \ 0.05) and HOMA-IR (P \ 0.05) were significantly decreased in individuals with the DD genotype after the exercise program. However, there were no significant differences in TC, TG, HDL-C, and FBG levels between individuals with the DD and ID genotype at baseline as well as after exercise training. Figure 1 shows the changes in adipocytokines after the 6-month exercise training according to genotypes at the UCP2 I/D polymorphism. After aerobic training, in individuals with the DD genotype, adiponectin (9.67 ± 2.01 vs. 11.43 ± 2.75 lg/ml; P \ 0.05) was significantly increased, whereas leptin (12.22 ± 2.75 vs. 11.05 ± 2.73 lg/ml; P \ 0.01), TNF-a (3.68 ± 0.92 vs. 1.96 ± 1.05 mg/ml; P \ 0.05), and IL-6 (3.99 ± 0.91 vs. 2.01 ± 0.95 pg/ml; P \ 0.05) were significantly decreased. In individuals with the ID genotype, TNF-a (3.01 ± 0.98 vs. 1.41 ± 1.12 mg/ml; P \ 0.05) was significantly decreased after exercise training. However, no significant differences were found for any adipocytokine levels between individuals with the DD and ID genotypes, both at baseline as well as after exercise training.
Discussion In the present study, we demonstrate for the first time that genetic variation in UCP2 affects exercise-mediated changes in insulin resistance and adipocytokine levels. Recently, Lee et al. [13] reported that this polymorphism was significantly associated with BMI and waist circumference even though no significant association of this UCP2 polymorphism with the risk of metabolic syndrome was detected. Our results, in contrast to prior findings, indicated
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Post
that UCP2 genotype did not have any effect on any metabolic risk factor at base-line or after 6 months of exercise training. This discrepancy may be explained partly by differences in the initial levels of metabolic risk factors and age-dependent UCP expression. In other words, the absence of training effects and the difference between UCP genotypes might be because most subjects involved in the present study had normal levels of TC, TG, HDL-C, and FBG. In addition, the UCP2 genotypes had lower effects on UCP2 expression in adult-onset obesity than in childhood-onset obesity, because of the confounding effects of various environmental and lifestyle factors [14]. Moreover, we could not exclude the possibility that the number of subjects in each group was too small to detect significant differences. The effect of the two observed UCP2 genotypes on energy expenditure at rest or during exercise was not detected in the present study. Although our results suggest that exercise-induced changes in metabolic risk factors may not be affected by variations in UCP2 I/D genotypes, further investigations are needed to fully elucidate the effect of UCP2 variation on metabolic risk factors during aerobic exercise in patients with various chronic conditions, including metabolic syndrome, type 2 diabetes, and CVD. In our study, the effect of exercise on adipocytokines and insulin sensitivity was distinct between individuals carrying the two observed UCP2 I/D genotypes. We observed that aerobic exercise, along with improvements in insulin level and insulin resistance (HOMA-IR), induced an increase in adiponectin levels, and a decrease in leptin, TNF-a, and IL-6 levels in individuals carrying the DD genotype. In individuals carrying the ID genotype, however, there were no significant changes in insulin sensitivity and adiponectin levels, despite a significant decrease in the TNF-a level. These results suggest that the DD genotype may be more effective than the ID genotype in mediating a response to exercise training.
Aging Clin Exp Res
Fig. 1 Changes in adipocytokine levels after a 6-month exercise training program, according to genotypes at the UCP2 I/D polymorphism. Adiponectin levels in individuals with the DD genotype were significantly increased. Leptin levels in individuals with the DD genotype were significantly decreased. IL-6 levels in individuals with the DD genotype were significantly decreased. TNF-a levels in both
individuals with the DD or the ID genotypes were significantly decreased after the exercise program. However, there were no differences between these levels in individuals carrying either of these genotypes. Square, before exercise training. Filled square, after the 6-month exercise training program. Asterisk, significant difference before and after the 6-month exercise training program (P \ 0.05)
Marti et al. [9] reported that II homozygotes may have an increased risk of obesity, independent of exercise levels, and suggested that mRNA processing or the stability of the transcript may be influenced by the alleles present in the 30 -UTR of the gene. Lee et al. [6] indicated that the ID genotype was significantly associated with BMI and waist circumference, as well as with a higher risk of developing obesity. Moreover, Walder et al. [15] found that individuals with the ID genotype had higher metabolic rates at rest than those with the II genotype, and that the latter individuals also had a greater BMI than did those with the ID genotype, particularly in subjects aged above 45 years. Thus, it seems that the UCP2 insertion allele is a strong contributing factor to obesity-associated metabolic abnormalities. Although we could not investigate the difference between the effects of the ID and II genotypes, due to a
lack of II homozygotes in the present study, we assume that the DD genotype, rather than the II and ID genotypes of the UCP2 I/D polymorphism, is related to metabolic rate and exercise efficiency. Chevillotte et al. [16] previously indicated that UCP2 levels in adipose tissue are positive inducers of the gene encoding adiponectin and, therefore, may favor insulin sensitivity. Moreover, it has been suggested that the adipocyte-derived hormone adiponectin elicits protective functions against fatty liver disease and hepatic injuries, at least in part by stimulating the expression of UCP2 [16, 17]. Even though the function of UCP2 is thought to be related to secretion of adipocytokines from adipose tissue and to insulin sensitivity, it is difficult to explain the physiological effect of exercise training on the UCP2 insertion allele. However, our study showed that the DD
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genotype, but not the ID genotype, was associated with exercise-mediated improvement in insulin sensitivity and adipocytokine levels. It is possible that variants in UCP2 may also influence improvement in insulin resistance and adiponectin resistance by exercise. Furthermore, individuals with the insertion allele in UCP2 may experience less effect of exercise on insulin resistance and adiponectin resistance. Taken together, our results suggest the possibility that the effects of exercise on adipocytokine levels and insulin sensitivity could differ depending on the genotypes at the I/D polymorphism in UCP2 and that the changes in body composition and insulin resistance following exercise could be linked to the effect of the I/D genotype on mRNA expression. In conclusion, we here demonstrated that the beneficial actions of physical exercise on the suppression of production of inflammatory markers, such as TNF-a, and enhancement of the anti-inflammatory index, such as adiponectin levels, may depend on the genotype at the I/D polymorphism in UCP2. To the best of our knowledge, this is the first study investigating the changes in adipocytokine levels after exercise training in the context of UCP2 genotypes. Although the biological function on UCP2 and the relationship with adipocytokines are not fully understudied, we concluded that exercise-induced changes in adipocytokine levels and insulin sensitivity may be associated with UCP2 genotypes. In particular, the ID genotype at the UCP2 30 UTR I/D polymorphism may be related to insulin resistance and adiponectin resistance. Acknowledgments This work was supported by the Dankook University Research fund of 2013 (BK21?). Conflict of interest On behalf of all authors, the corresponding author states that there is no conflict of interest.
References 1. Kahara T, Takamura T, Hayakawa T, Nagai Y, Yamaguchi H, Katsuki T, Katsuki K, Katsuki M, Kobayashi K (2002) Prediction of exercise-mediated changes in metabolic markers by gene polymorphism. Diabetes Res Clin Pract 57(2):105–110 2. Bouassida A, Chamari K, Zaouali M, Feki Y, Zbidi A, Tabka Z (2010) Review on leptin and adiponectin responses and adaptations to acute and chronic exercise. Br J Sports Med 44(9):620–630 3. Haper M (1997) Obesity research continues to spring leaks. Clin Invest Med 20:239–244
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4. Schrauwen P, Troost FJ, Xia J, Ravussin E, Saris WH (1999) Skeletal muscle UCP2 and UCP3 expression in trained and untrained male subjects. Int J Obes Relat Metab Disord 23(9):966–972 5. Nordfors L, Heimbu¨rger O, Lo¨nnqvist F, Lindholm B, Helmrich J, Schalling M, Stenvinkel P (2000) Fat tissue accumulation during peritoneal dialysis is associated with a polymorphism in uncoupling protein 2. Kidney Int 57(4):1713–1719 6. Lee YH, Kim W, Yu BC, Park BL, Kim LH, Shin HD (2008) Association of the ins/del polymorphisms of uncoupling protein 2 (UCP2) with BMI in a Korean population. Biochem Biophys Res Commun 371(4):767–771 7. Morita E, Taniguchi H, Sakaue M (2009) Trp64Arg polymorphism in beta3-adrenergic receptor gene is associated with decreased fat oxidation both in resting and aerobic exercise in the Japanese male. Exp Diabetes Res 605(139):1–5 8. Cortright RN, Zheng D, Jones JP, Fluckey JD, DiCarlo SE, et al (1999) Regulation of skeletal muscle UCP2 and UCP-3 gene expression by exercise and denervation. Am J Physiol 276(1Pt1):E217–221 9. Marti A, Corbala´n MS, Forga L, Martinez-Gonza´lez MA, Martinez JA (2004) Higher obesity risk associated with the exon-8 insertion of the UCP2 gene in a Spanish case-control study. Nutrition 20(6):498–501 10. Yanovski JA, Diament AL, Sovik KN, Nguyen TT, Li H, Sebring NG, Warden CH (2000) Associations between uncoupling protein 2, body composition, and resting energy expenditure in lean and obese African American, white, and Asian children. Am J Clin Nutr 71(6):1405–1420 11. Ricquier D (2005) Respiration uncoupling and metabolism in the control of energy expenditure. Proc Nutr Soc 64(1):47–52 12. Matthew DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC (1985) Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28:412–419 13. Lee HJ, Ryu HJ, Shin HD, Park BL, Kim JY, Cho YM, Park KS, Song J, Oh B (2008) Associations between polymorphisms in the mitochondrial uncoupling proteins (UCPs) with T2DM. Clin Chim Acta 398(1–2):27–33 14. Evans D, Minouchehr S, Hagemann G, Mann WA, Wendt D, Wolf A, Beisiegel U (2000) Frequency of and interaction between polymorphisms in the beta3-adrenergic receptor and in uncoupling proteins 1 and 2 and obesity in Germans. Int J Obes Relat Metab Disord 24(10):1239–1245 15. Walder K, Norman RA, Hanson RL, Schrauwen P, Neverova M et al (1998) Association between uncoupling protein polymorphisms (UCP2-UCP3) and energy metabolism/obesity in Pima Indians. Hum Mol Genet 7(9):1431–1435 16. Chevillotte E, Giralt M, Miroux B, Ricquier D, Villarroya F (2007) Uncoupling protein-2 controls adiponectin gene expression in adipose tissue through the modulation of reactive oxygen species production. Diabetes 56(4):1042–1050 17. Zhou M, Xu A, Tam PK, Lam KS, Chan L, Hoo RL, Liu J, Chow KH, Wang Y (2008) Mitochondrial dysfunction contributes to the increased vulnerabilities of adiponectin knockout mice to liver injury. Hepatology 48(4):1087–1096
ORIGINAL ARTICLE
Impact of UCP2 polymorphism on long-term exercise-mediated changes in adipocytokines and markers of metabolic syndrome Kang-Il Lim • Yun-A Shin
Received: 21 August 2013 / Accepted: 4 March 2014 Ó Springer International Publishing Switzerland 2014
Abstract Background and aims Variations in genes involved in energy expenditure affect aerobic exercise efficiency, but it remains unclear whether the effect of aerobic exercise on adipocytokines is modified by the obesity-associated genotypes in the uncoupling protein 2 gene (UCP2). The purpose of this study was to assess whether genetic variation in UCP2 may affect exercise-mediated changes in adipocytokines and markers of metabolic syndrome in postmenopausal obese women. Methods Forty-two sedentary postmenopausal obese women (age 52.74 ± 6.39 years) participated in this study. Participants were encouraged to train for 3 days a week, for 6 months, for 60 min per session of treadmill walking/ running at 60 % V O2 R. Subjects were genotyped for the 45-bp insertion/deletion (I/D) polymorphism in the 30 -untranslated region (UTR) of UCP2. Results Among the subjects, 23 (57.1 %) and 19 (42.9 %), were deletion homozygotes (DD) and ID heterozygotes,
respectively. For DD homozygotes, body weight, body mass index (BMI), % body fat, and waist circumference, and body weight, BMI, and waist circumference of ID heterozygotes, were significantly decreased after the exercise program. There were no significant changes in metabolic markers in individuals with the ID genotype, whereas insulin and HOMA-IR in individuals with the DD genotype were significantly decreased after the exercise program. In DD homozygotes, but not in ID heterozygotes, adiponectin was significantly increased, and leptin, TNF-a, and IL-6 were significantly decreased after exercise training. Conclusions Exercise-mediated changes in insulin resistance and adiponectin levels may be affected by genotypes in the 30 UTR I/D polymorphism in UCP2 in postmenopausal obese women. Keywords Adipocytokines Exercise Metabolic syndrome UCP2
Introduction K.-I. Lim Institute of Exercise Physiology, School of Kinesiology, Yeungnam University, 280 Daehak-Ro, Gyeongsan, Gyeongbuk 712-749, Republic of Korea e-mail: [email protected] Y.-A. Shin (&) Department of Prescription and Rehabilitation of Exercise, College of Sports Science, Dankook University, San 29, Anseo-dong, Cheonan-si, Chungnam 330-714, Republic of Korea e-mail: [email protected] Y.-A. Shin Department of Kinesiologic Medical Science, Graduate, Dankook University, San 29, Anseo-dong, Cheonan-si, Chungnam 330-714, Republic of Korea
Regular exercise is a key component in the prevention and treatment of metabolic disorders, such as hypertension, diabetes, and obesity. However, the effects of exercise on metabolic abnormalities associated with obesity vary from person to person, and these individual variations may be caused not only by specific environmental conditions, but also by genetics [1]. Thus, it is necessary to assess whether changes in energy metabolism and intracellular energy charge occurs in adipocytes during treatments that affect adiposity, insulin resistance, and other components of the metabolic syndrome. Unlike other adipocytokines, adiponectin suppresses the expression of adhesion molecules in endothelial cells and
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Aging Clin Exp Res
protects against metabolic syndrome, cardiovascular disease (CVD), and inflammation. Moreover, low levels of adiponectin are associated with chronic inflammatory conditions, such as insulin resistance and obesity. Recently, a review [2] suggested that training at an adequate duration and intensity to produce substantive changes in fitness levels and in body composition may decrease circulating leptin levels and raise circulating adiponectin levels. Uncoupling protein 2 (UCP2), a member of the uncoupling protein family, is thought to play a role in proton transfer in the mitochondrial membrane, without producing ATP, as well as in modulating ATP synthesis. The level of this protein is up-regulated in white adipose tissue [3] and the protein has been correlated with energy expenditure in humans [4]. In particular, a 45-bp insertion/deletion (I/D) in the 30 -untranslated region (UTR) of exon 8 has been implicated in obesity, metabolic rate, and exercise efficiency [5, 6]. It has also been reported that UCP2 expression is affected by physical stimulation [7]. Cortright et al. [8] found that UCP2 expression levels in mouse skeletal muscles are elevated after acute exercise. Moreover, Marti and his colleagues [9] reported that the UCP2 insertion allele is associated with physical activity. Thus, the genotypes of genes involved in energy expenditure also affect the efficiency of aerobic exercise [7], but it remains unclear whether the effect of aerobic exercise training could be modified by genotypes in specific genes. Therefore, it could be speculated that the UCP2 genotypes associated with obesity may affect the secretion of biologically active substances from adipose tissue (e.g., leptin and other adipocytokines) and could hence affect systemic insulin sensitivity during exercise training. Although most studies have reported association of UCP2 genotypes to increased body weight and energy expenditure [10, 11], the physiological role of the UCP2 insertion allele following exercise training has not yet been studied. Furthermore, the effect of exercise training on the relationship between adipocytokines and UCP2 genotypes has not been investigated. Therefore, the aims of this study were to compare markers of the metabolic syndrome and levels of adipocytokines according to UCP2 genotypes, both before and after exercise, and assess whether genetic variations in UCP2 can affect exercise-mediated changes in adipocytokines and metabolic syndrome markers in postmenopausal obese women.
Materials and methods Subjects The present study was approved by the Institutional Ethnics Committee of Physical Education of Dankook
123
University. Informed consent was obtained from all subjects prior to their participation in this study. Eighty-five women were recruited from among volunteers in Seoul City. Questionnaires were used to determine recent participation in exercise, menopausal states, lifestyle, and disease states. Exclusion criteria were as follows: any specific diet, cigarette smoking (either currently or in the previous 6 months), use of anti-inflammatory drugs or steroids, surgery (in the previous 3 months), and metabolic disorders, such as myocardial infarction, stroke or hypertension, hypercholesterolemia, musculoskeletal injury, or current illness. Eventually, 42 sedentary healthy middle-aged women (52.74 ± 6.39 years) participated in this study. The subjects were postmenopausal, obese women according to body mass index (BMI C25 kg/m2) and body % fat (C30 %). None of these participants had taken part in any regular aerobic exercise over the previous 6 months. Anthropometric measurements Clinical characteristics (height, body weight, waist circumference, % body fat, and blood pressure) were measured using a standard protocol. Standing height was measured on a portable stationmaster to an accuracy of ±0.5 cm. The weight and percentage of body fat were measured using an Inbody 7.0 (Biospace, Korea) to the nearest 0.1 kg. BMI was calculated as weight (kg)/height (m2). Preliminary testing
Prior to the exercise trials, V O2 max was measured to establish the exercise intensity for each individual. Subjects were familiarized with treadmill running and were informed of what was required of them about the experiment. They then completed a graded treadmill exercise test to determine their individual V O2 max, adhering to the Bruce protocol. Aerobic exercise training Aerobic training sessions were supervised by three experienced physical education instructors. Participants were encouraged to train for 3 days a week, for 6 months; each exercise session consisted of a brief warm-up period, followed by 45 min per session of treadmill walking/running at 60 % V O2 R, followed by a brief cool-down period. The training program started at 40 % O R and gradually V 2 increased to 60 % V O2 R by week 12. After 12 weeks of training, the exercise intensity for each participant was
Aging Clin Exp Res
examined and adjusted to their new measured V O2 max, and was then maintained at 60 % V O2 R until the end of training. Chemical analysis Blood samples were obtained in the morning after overnight fasting and were stored at -80 °C for subsequent assays. Total cholesterol (TC), triglyceride (TG), and high-density lipoprotein (HDL) cholesterol levels were measured by enzymatic procedures using a chemistry analyzer (Hitachi 747; Tokyo, Japan), with a coefficient of variation (CV), of 1.9–2.2, 5.7–6.3, and 2.5 %, respectively. Plasma glucose levels and serum insulin were measured using a commercially available kit (glucose hexokinase kit, ADVIA 1650, Bayer, Japan; radioimmunoassay RIA kit, Linco Research, St. Charles, MO, USA), with a CV of 1.1 and 7.8–9.3 %, respectively. Insulin sensitivity was assessed using the homeostasis model assessment of insulin resistance index (HOMA-IR) (fasting glucose level [mmol/L] 9 fasting plasma insulin level [lU/mL]/22.5) [12]. Serum adiponectin was measured using a human adiponectin ELISA kit (BioVender, Laboratory Medicine; Brno, Czech Republic), with a CV of 4.8 %. Serum leptin was measured using the RIA method (Radio Immunoassay, Cosmic Corp, Tokyo, Japan), with a CV of 4.2–4.9 %), and IL-6 and TNF-a levels were measured by ELISA (Biosource International; Camarillo, CA, USA) with a CV of 2–4.2 and 12.6–16.9 %, respectively. Genomic DNA was extracted from peripheral blood leukocytes of all subjects, and the 30 -UTR I/D in UCP2 genotyped by polymerase chain reaction (PCR) using the primers: sense 50 -CAGTGAGGGAAGTGGGAGG-30 and antisense 50 -GGGGCAGGACGAAGATTC-30 . PCR conditions involved initial denaturation at 94 °C for 3 min, followed by 35 cycles each consisting of 30 s at 94 °C, 25 s at 56 °C, and 30 s at 72 °C, and a final extension step Table 1 Changes in clinical characteristics before and after a 6-month exercise program according to genotypes at the UCP2 insertion/deletion (I/D) polymorphism
Variables Body weight (kg) BMI (kg/m2) Body Fat (%) Waist Circumference (cm) SBP (mmHg) DBP (mmHg)
Data are the mean ± SD
at 72 °C for 7 min. The size of the PCR products were then assessed by electrophoresis in 2 % agarose gels to identify individuals homozygous for the deletion (DD), heterozygous for the ID, or homozygous for the insertion (II). Data analysis Results were expressed as mean ± standard deviation (SD), and statistical analyses were performed using SPSS for PC (version 12.5 for Windows; SPSS, Inc.; Chicago, IL, USA). Paired t test was used to determine significance differences within a group, and comparisons between groups were analyzed using the independent t-test. Conformity of the genotype distribution to Hardy–Weinberg was calculated using the v2 test. A P value of \0.05 was accepted as indicating statistical significance.
Results UCP2 DD and ID genotypes were present in 23 (57.1 %) and 19 (42.9 %) subjects, respectively. However, the II genotype was not present in any of the subjects. No significant deviation from Hardy–Weinberg equilibrium was observed. The clinical characteristics of subjects after the 6-month exercise program are shown in Table 1, according to the UCP2 I/D polymorphism genotypes. There was no significant difference at baseline between the UCP2 I/D genotypes for any of the variables (Table 1). In individuals with the DD genotype, body weight (P \ 0.001), BMI (P \ 0.001), % body fat (P \ 0.05), and waist circumstance (P \ 0.001) significantly decreased after the 6-month exercise program. In individuals with the ID genotype, body weight (P \ 0.01), BMI (P \ 0.01), and waist circumference (P \ 0.001) were significantly decreased after the exercise program.
Genotype (n)
Pre
Post
P value
DD (23)
69.58 ± 10.08
68.01 ± 9.88
0.001
ID (19)
67.95 ± 8.21
65.92 ± 8.10
0.003
DD (23)
28.46 ± 3.64
27.82 ± 3.53
0.001
ID (19)
27.68 ± 2.82
26.85 ± 2.82
0.003
DD (23)
35.35 ± 3.44
34.11 ± 4.12
0.014
ID (19)
35.76 ± 4.91
35.84 ± 4.44
0.823
DD (23)
83.43 ± 7.87
77.87 ± 7.45
\0.001 \0.001
ID (19)
83.26 ± 7.02
77.63 ± 8.41
DD (23)
124.17 ± 15.28
124.74 ± 13.99
0.849
ID (19)
123.00 ± 10.21
123.53 ± 11.96
0.794
DD (23)
85.48 ± 11.01
83.87 ± 10.27
0.383
ID (19)
83.11 ± 9.17
84.16 ± 8.59
0.494
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Aging Clin Exp Res Table 2 Changes in metabolic characteristics before and after a 6-month exercise program according to genotypes at the UCP2 insertion/deletion (I/D) polymorphism
Variables TC (mmol/L) TG (mmol/L) HDL-C (mmol/L) Glucose (mmol/L) Insulin (pmol/L) HOMA-IR
Data are the mean ± SD
Genotype (n)
Pre
DD (23)
203.78 ± 48.66
213.70 ± 45.28
0.124
ID (19)
191.11 ± 44.33
205.47 ± 41.36
0.076
P value
DD (23)
155.00 ± 73.83
138.52 ± 55.88
0.155
ID (19)
140.79 ± 90.86
137.21 ± 75.16
0.871
DD (23)
45.48 ± 8.81
45.70 ± 7.86
0.879
ID (19)
49.53 ± 14.17
49.16 ± 10.70
0.922
DD (23)
89.16 ± 8.95
88.11 ± 7.96
0.472
ID (19)
90.00 ± 5.44
91.70 ± 9.11
0.312
DD (23)
3.84 ± 3.26
2.69 ± 2.02
0.026
ID (19)
4.11 ± 3.09
3.63 ± 2.52
0.387
DD (23)
1.57 ± 1.45
1.08 ± 0.82
0.036
ID (19)
1.67 ± 1.33
1.52 ± 1.11
0.528
The metabolic characteristics of individuals after the 6-month exercise program is shown in Table 2 according to the UCP2 I/D genotypes. There were no significant changes in metabolic markers in individuals with the ID genotype, whereas insulin (P \ 0.05) and HOMA-IR (P \ 0.05) were significantly decreased in individuals with the DD genotype after the exercise program. However, there were no significant differences in TC, TG, HDL-C, and FBG levels between individuals with the DD and ID genotype at baseline as well as after exercise training. Figure 1 shows the changes in adipocytokines after the 6-month exercise training according to genotypes at the UCP2 I/D polymorphism. After aerobic training, in individuals with the DD genotype, adiponectin (9.67 ± 2.01 vs. 11.43 ± 2.75 lg/ml; P \ 0.05) was significantly increased, whereas leptin (12.22 ± 2.75 vs. 11.05 ± 2.73 lg/ml; P \ 0.01), TNF-a (3.68 ± 0.92 vs. 1.96 ± 1.05 mg/ml; P \ 0.05), and IL-6 (3.99 ± 0.91 vs. 2.01 ± 0.95 pg/ml; P \ 0.05) were significantly decreased. In individuals with the ID genotype, TNF-a (3.01 ± 0.98 vs. 1.41 ± 1.12 mg/ml; P \ 0.05) was significantly decreased after exercise training. However, no significant differences were found for any adipocytokine levels between individuals with the DD and ID genotypes, both at baseline as well as after exercise training.
Discussion In the present study, we demonstrate for the first time that genetic variation in UCP2 affects exercise-mediated changes in insulin resistance and adipocytokine levels. Recently, Lee et al. [13] reported that this polymorphism was significantly associated with BMI and waist circumference even though no significant association of this UCP2 polymorphism with the risk of metabolic syndrome was detected. Our results, in contrast to prior findings, indicated
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Post
that UCP2 genotype did not have any effect on any metabolic risk factor at base-line or after 6 months of exercise training. This discrepancy may be explained partly by differences in the initial levels of metabolic risk factors and age-dependent UCP expression. In other words, the absence of training effects and the difference between UCP genotypes might be because most subjects involved in the present study had normal levels of TC, TG, HDL-C, and FBG. In addition, the UCP2 genotypes had lower effects on UCP2 expression in adult-onset obesity than in childhood-onset obesity, because of the confounding effects of various environmental and lifestyle factors [14]. Moreover, we could not exclude the possibility that the number of subjects in each group was too small to detect significant differences. The effect of the two observed UCP2 genotypes on energy expenditure at rest or during exercise was not detected in the present study. Although our results suggest that exercise-induced changes in metabolic risk factors may not be affected by variations in UCP2 I/D genotypes, further investigations are needed to fully elucidate the effect of UCP2 variation on metabolic risk factors during aerobic exercise in patients with various chronic conditions, including metabolic syndrome, type 2 diabetes, and CVD. In our study, the effect of exercise on adipocytokines and insulin sensitivity was distinct between individuals carrying the two observed UCP2 I/D genotypes. We observed that aerobic exercise, along with improvements in insulin level and insulin resistance (HOMA-IR), induced an increase in adiponectin levels, and a decrease in leptin, TNF-a, and IL-6 levels in individuals carrying the DD genotype. In individuals carrying the ID genotype, however, there were no significant changes in insulin sensitivity and adiponectin levels, despite a significant decrease in the TNF-a level. These results suggest that the DD genotype may be more effective than the ID genotype in mediating a response to exercise training.
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Fig. 1 Changes in adipocytokine levels after a 6-month exercise training program, according to genotypes at the UCP2 I/D polymorphism. Adiponectin levels in individuals with the DD genotype were significantly increased. Leptin levels in individuals with the DD genotype were significantly decreased. IL-6 levels in individuals with the DD genotype were significantly decreased. TNF-a levels in both
individuals with the DD or the ID genotypes were significantly decreased after the exercise program. However, there were no differences between these levels in individuals carrying either of these genotypes. Square, before exercise training. Filled square, after the 6-month exercise training program. Asterisk, significant difference before and after the 6-month exercise training program (P \ 0.05)
Marti et al. [9] reported that II homozygotes may have an increased risk of obesity, independent of exercise levels, and suggested that mRNA processing or the stability of the transcript may be influenced by the alleles present in the 30 -UTR of the gene. Lee et al. [6] indicated that the ID genotype was significantly associated with BMI and waist circumference, as well as with a higher risk of developing obesity. Moreover, Walder et al. [15] found that individuals with the ID genotype had higher metabolic rates at rest than those with the II genotype, and that the latter individuals also had a greater BMI than did those with the ID genotype, particularly in subjects aged above 45 years. Thus, it seems that the UCP2 insertion allele is a strong contributing factor to obesity-associated metabolic abnormalities. Although we could not investigate the difference between the effects of the ID and II genotypes, due to a
lack of II homozygotes in the present study, we assume that the DD genotype, rather than the II and ID genotypes of the UCP2 I/D polymorphism, is related to metabolic rate and exercise efficiency. Chevillotte et al. [16] previously indicated that UCP2 levels in adipose tissue are positive inducers of the gene encoding adiponectin and, therefore, may favor insulin sensitivity. Moreover, it has been suggested that the adipocyte-derived hormone adiponectin elicits protective functions against fatty liver disease and hepatic injuries, at least in part by stimulating the expression of UCP2 [16, 17]. Even though the function of UCP2 is thought to be related to secretion of adipocytokines from adipose tissue and to insulin sensitivity, it is difficult to explain the physiological effect of exercise training on the UCP2 insertion allele. However, our study showed that the DD
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genotype, but not the ID genotype, was associated with exercise-mediated improvement in insulin sensitivity and adipocytokine levels. It is possible that variants in UCP2 may also influence improvement in insulin resistance and adiponectin resistance by exercise. Furthermore, individuals with the insertion allele in UCP2 may experience less effect of exercise on insulin resistance and adiponectin resistance. Taken together, our results suggest the possibility that the effects of exercise on adipocytokine levels and insulin sensitivity could differ depending on the genotypes at the I/D polymorphism in UCP2 and that the changes in body composition and insulin resistance following exercise could be linked to the effect of the I/D genotype on mRNA expression. In conclusion, we here demonstrated that the beneficial actions of physical exercise on the suppression of production of inflammatory markers, such as TNF-a, and enhancement of the anti-inflammatory index, such as adiponectin levels, may depend on the genotype at the I/D polymorphism in UCP2. To the best of our knowledge, this is the first study investigating the changes in adipocytokine levels after exercise training in the context of UCP2 genotypes. Although the biological function on UCP2 and the relationship with adipocytokines are not fully understudied, we concluded that exercise-induced changes in adipocytokine levels and insulin sensitivity may be associated with UCP2 genotypes. In particular, the ID genotype at the UCP2 30 UTR I/D polymorphism may be related to insulin resistance and adiponectin resistance. Acknowledgments This work was supported by the Dankook University Research fund of 2013 (BK21?). Conflict of interest On behalf of all authors, the corresponding author states that there is no conflict of interest.
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