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Effect of Vigorous Aerobic Exercise on Serum Levels of SIRT 1 , FGF 21 and Fetuin A in Women with Type II Diabetes

Background and Objectives: Sirtuin (SIRT), Fibroblast Growth Factor 21 (FGF 21 ) and Fetuin A are proteins that cause a wide range of metabolic disorders such as type 2 diabetes mellitus (T2DM). On the other hand, regular physical activity is known to play a key role in prevention and management of T2DM. Thus, this study investigated the effect of vigorous aerobic exercise on serum levels of metabolic parameters including SIRT 1 , FGF 21 and Fetuin A in women with T2DM. Methods: The study was performed on 28 randomly selected women with T2DM who were divided into an exercise group and a control group. The training intervention consisted eight weeks of vigorous aerobic exercise (three times a week at 70-80% of maximum heart rate). The serum levels of SIRT 1 , FGF 21 and Fetuin A were evaluated before the first session and 48 hours after the last session. Paired sample t-test and independent t-test were used to analyze within and between group differences, respectively. All statistical analyses were performed in SPSS (version 19) at significance of 0.05. Results: The eight-week aerobic training caused a significant reduction in body weight, body mass index, insulin resistance, low-density lipoprotein, fasting blood sugar, triglycerides and Fetuin A of women with T2DM. In addition, it caused a significant increase in SIRT1 and FGF 21 levels. There was no significant difference in the level of high-density lipoprotein and cholesterol between the two groups. Conclusion: As a non-pharmacological therapy, regular aerobic exercise might improve the metabolic parameters, SIRT1, FGF21, and Fetuin A in women with T2DM.

have been inconsistent. Some studies reported an increase in serum level of FGF 21 following physical activity (12,13), while in a study by Besse-Patin et al., eight weeks of endurance training in obese men did not affect serum level of FGF 21 (14). On the other hand, Yang et al. reported that serum level of FGF 21 decreases in obese women after three months of physical activity in obese women (15). In study of Malin et al., a reduction in serum level of Fetuin A was observed in obese adult men and women following a course of endurance training (16). Another study demonstrated that six weeks of aerobic exercise does not significantly change serum level of Fetuin A in obese women (17). Considering the lack of sufficient data regarding the effects of physical activity on serum level of SIRT 1 in humans and the increasing prevalence of diabetes, we conducted this study to investigate the effect of eight weeks of high intensity aerobic training on metabolic parameters and serum levels of SIRT 1 , FGF 21 , and Fetuin A in women with type 2 diabetes.

MATERIAL AND METHODS
This semi-experimental study (clinical trial registration number: IRCT20171104037225N1) was carried out on two groups (14 participants in each group) with a pretest and posttest design and a control group. The study population consisted of the females with type 2 diabetes who were referred to the Diabetes Center in Sari (Iran) for treatment. Inclusion criteria included age range between 40-50 years old, fasting blood sugar of >126 mg/dl, history of type two diabetes for at least three years with A1C of more than 6.5%, no history of insulin injection, no history of regular physical activity in the past six months, and no history of hepatic and chronic heart disease requiring treatment. Consent was obtained from all participants before taking part in the study. They were asked not to perform any other type of activities Exclusion criteria included fasting blood sugar of >300 mg/dl, any cardiovascular, liver or kidney diseases, intermittent attendance to the training sessions, and unwillingness to continue participating in the study. Exercise intervention was based on the American Diabetes Association

INTRODUCTION
Reduced physical activity and sedentary lifestyle habits increase the incidence of chronic diseases, such as diabetes mellitus (DM). DM is a complex chronic disease associated with hyperglycemia due to deficiency in insulin secretion or action or both (1). According to statistical reports in 2011, the global prevalence of DM was 8% in adults and is estimated to reach 10% by 2030 (2). Proteins involved in the metabolic pathways such as sirtuin 1 (SIRT 1 ) and some hepatokines such as fibroblast growth factor 21 (FGF 21 ) and Fetuin A have been known to play a key role in type 2 diabetes. Diabetics have decreased level of SIRT 1 and FGF 21 and increased level of Fetuin A. SIRT 1 is a protein that acts as nicotinamide adenine dinucleotide (NAD)-dependent deacetylase (3) and an important regulator of nutritional homeostasis in several metabolic tissues. SIRT 1 positively regulates the secretion of insulin in betapancreatic cells and protects cells from oxidative stress and inflammation (1). It also plays a positive role in insulin signaling pathway in muscle and adipose tissue and its expression is associated with improved insulin sensitivity (4). SIRT 1 increases lipolysis, inhibits lipogenesis, and plays a role in the reverse cholesterol transport (5). FGF 21 is a protein involved in regulation of glucose and lipid metabolism. It is mainly expressed in metabolic tissues, such as the liver and muscles (6). Systemic induction of FGF 21 prevents obesity and decreases hyperglycemia and insulin resistance. It also leads to weight loss and decreases triglyceride (TG) and lowdensity lipoprotein (LDL) levels (7). Fetuin A is also a glycoprotein produced by the liver (6), which is associated with metabolic syndrome, type 2 diabetes and cardiovascular disease. It promotes insulin resistance by inhibiting phosphorylation insulin receptor (8) and is involved in development of atherosclerosis in diabetic patients (9). Exercise and regular physical activity have beneficial health effects, especially on chronic diseases such as diabetes. Various studies have been conducted on the effect of physical activity on SIRT 1 . Sarga et al. reported an increase in SIRT 1 level of mice following endurance training (10). It has been shown that long-term exercise exerts cardio protective effects via activation of SIRT 1 (11). The results of studies on the effects of training on FGF 21 measured with enzyme-linked immunosorbent assay (ELISA). Descriptive statistics including mean ± standard deviation (SD) were used to describe the data. Kolmogorov Smirnov test and Levene's test were applied to determine normality of data distribution and homogeneity of variances. Paired sample t-test and independent t-test were performed in order to analyze differences within and between groups, respectively. All statistical analyses were performed in SPSS (version 19) at significant of 0.05.

RESULTS
Before the intervention, there was no significant difference between the control and the exercise groups. The eight weeks of highintensity aerobic exercise caused a significant decrease in body weight, BMI, A1C level, insulin resistance, low-density lipoprotein (LDL) level, fasting glucose level, TG, and Fetuin A levels. However, a non-significant decrease was also noted in the cholesterol level of the participants in the exercise group. Moreover, there was a statistically significant increase in serum levels of SIRT 1 and FGF 21 (P=0.001) and a non-significant increase in level of high density lipoprotein (HDL) (Table1).
recommendations and included three sessions of high intensity exercise per week at 70-80% of maximum heart rate (18). The initial training duration was 25 minutes, which was increased by one minute after each training session, and reached 43 minutes at the end of week six. The training intensity started at 65% of the maximum heart rate and increased by 5% every week until reaching 80% of maximum heart rate. The exercise program was performed for eight weeks. Height and weight of the participants were measured when they wearing light clothing and without shoes. Body mass index (BMI) was calculated by dividing body weight (Kg) by height squared (m 2 ). Insulin resistance was evaluated using the following formula: HOMA-IR = [(fasting insulin (μU/ml) × fasting glucose (mmol/l)] ÷ 22.5 Metabolic parameters and serum levels of SIRT 1 , FGF 21 , and Fetuin A were evaluated before the first session and 48 hours after the last session. Venous blood samples were taken between 8:00-10:00 AM following 12 hours of overnight fasting. Serum levels of metabolic parameters were measured by an auto analyzer and serum levels of SIRT 1 (Bio Vision, USA), FGF 21 (26). FGF 21 increases glucose uptake in adipose tissue independent of insulin (7), which acts through the expression of glucose transport and regulation of lipolysis (27). FGF 21 increases the mitochondrial oxidative capacity in adipose tissue, which is manifested by increasing oxygen consumption and induction of key metabolic genes (28). In other tissues, such as the liver and pancreas, FGF 21 also helps maintain homeostasis (6). AMP-activated protein kinase (AMPK) is activated by reduction of ATP/AMP ratio, phosphocreatine (CP), and glycogen levels. On the other hand, the CP and glycogen depletion is influenced by exercise intensity (29). The activation of AMPK (e.g., by exercise) triggers an increase in the NAD + /NADH ratio, which activates SIRT 1 (30). Increased level of AMPK increases NAD + intracellular levels, which in turn activates SIRT 1 (31). SIRT 1 also increases fat oxidation and glucose uptake and mitochondrial biogenesis (32). In addition to lipolysis, FGF 21 mediated activation of the AMPK-SIRT 1 is accompanied by decreased expression of lipogenic genes involved in fatty acid synthesis (5,28).
In the present study, eight weeks of aerobic training significantly reduced the serum levels of Another study on nondiabetic obese women did not find any significant changes in Fetuin A levels after six weeks of training (17). These inconsistencies could be attributed to the differences in the type of exercise protocol, duration, and intensity of exercise. Fetuin A is linked to the insulin receptor thereby inhibiting the autophosphorylation of tyrosine kinase and

DISCUSSION
In this study, we examined the effects of eight weeks of high-intensity aerobic exercise on metabolic parameters and serum levels of SIRT 1 , FGF 21 , and Fetuin A in women with type 2 diabetes. The exercise intervention caused a significant decrease in the level of fasting blood glucose, A1C, insulin resistance, triglyceride, LDL, and Fetuin A compared to the control group. We also noted a significant increase in serum levels of SIRT 1 and FGF 21 in the exercise group. Dyslipidemia and cardiovascular disease are common among patients with DM. Our results showed that regular physical activity can improve cardiovascular risk factors, which was supported by other studies (1,19). The expression and activity of the SIRT 1 are decreased in DM (1). In this study, the eightweek intense aerobic training increased the serum levels of SIRT 1 and FGF 21 in diabetic women. Previous studies have not extensively investigated the effect of physical activity on serum level of SIRT 1 . Saremi et al. reported a significant increase in serum level of SIRT 1 after two months of exercise (1). In another study, Chi Huang et al. found that swimming increased SIRT 1 level in gastrocnemius and soleus muscle of mice (20). Similarly, Causu et al. reported that six weeks of training increased SIRT 1 levels in healthy mice (21). However, a study by Marton et al. reported that physical activity had no effect on serum levels of SIRT 1 (22). In our study, the eightweek high-intensity aerobic exercise increased serum levels of FGF 21 , which was in consistent with the results of Yang et al. (15), Farzanegi (23), and Abbasi et al. (24). Farzanegi stated that eight weeks of regular physical activity in women with type 2 diabetes brought about a significant increase in serum levels of FGF 21 . In addition, the study of Abbasi et al. on obese men demonstrated an increase in serum levels of FGF 21 in response to aerobic training. Yang et al. also observed an increase in serum FGF 21 levels after 12 weeks of combined exercise training in obese women. Contradictory to the results of these studies, a study claimed that plasma level of FGF 21 was reduced following three weeks of intertropical training (25). It seems that the positive effects of regular physical exercise on the metabolic parameters of diabetics may partially be exerted through proteins associated with mitochondrial biogenesis and glucose regulation such as 4/ Effect of Vigorous Aerobic. . . effects on metabolic parameters in the body through activation of the AMPK-SIRT 1 pathway and improvement of insulin sensitivity and associated pathways.

ACKNOWLEDGMENTS
This article has been derived from a thesis approved by the Islamic Azad University of Sari. The authors gratefully acknowledge cooperation of all participants in the study.

CONFLICT OF INTEREST
All authors declare no conflicts of interest.
reducing insulin signaling in skeletal muscle and adipose tissue (8). Fetuin A also stimulates release of cytokines and reduces adiponectin release, which in turn leads to insulin resistance (36). Stimulation of lipogenesis is another consequence of increased Fetuin A level (37). It seems that regular exercise can increase FGF 21 and SIRT 1 and decrease Fetuin A levels.

CONCLUSION
As a non-pharmacological therapy, physical activity may exert some beneficial