Effect of Resistance and Endurance Training on Gene Expression of Adiponectin and its Receptors (AdipoR1 and AdipoR2) in Testicles and Serum Levels of Sex Hormones in Diabetic Rats

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adiponectin levels and AdipoR 1 gene rats, and also increased the serum testosterone levels and LH in diabetic rats. Resistance and endurance training can improve expression of adiponectin and AdipoR 1 gene by increasing the serum testosterone and LH levels in type 2 diabetic rats.

Background
Diabetes is a disorder in the carbohydrates, fats and proteins metabolism that blood glucose increases in conditions of resistance to insulin or insulin de ciency (1). Type 2 diabetes or also known as non-insulin dependent diabetes (NIDDM) with 90% of cases being the most frequent type of diabetes (2). Type 2 diabetes by increasing blood glucose levels can cause physiological and functional disorders in various body tissues, along with behavioral and sexual disorders (3). Approximately 90 percent of diabetic men suffer from their sexual ability reduction and fertility disability (4). Diabetes mellitus caused reduced infertility by altering the the cells and the hormones structure involved in the spermatogenesis process (5). A great deal of research has been accomplished on the association between diabetes and male fertility, which reported lower levels of LH, FSH, testosterone and reducing the sperm parameters and spermatogenesis quality (6).
Adiponectin is a protein hormone secreted mainly by the white adipose tissue (7)that plays a signi cant role in glucose and lipid metabolism (8).Adiponectin has 2 receptors Adiponectin-receptor1 (AdipoR 1 ), which is abundantly expressed in skeletal muscle cells, Adiponectin-receptor2 (AdipoR 2 ) that is expressed in the liver cells (9). Adiponectin and its receptors are found in organs associated with the male reproductive system like the pituitary, hypothalamus and testicles. Adiponectin is expressed by leydig cells, spermatozoa and epididymis in the testicles (10).
Few studies have indicated that adiponectin is expressed in the spermatogenesis and sperm maturation at the testis level. Adiponectin reduces the release of gonadotropin releasing hormone (GnRH) from the hypothalamus neurons, and afterward prevents LH from the pituitary releasing and it has no effect on FSH. On the other hand, this hormone activates its receptors in the testicular tissue, which results in phosphorylation of the active protein kinase AMP (AMPK) and also activating the peroxisome proliferator activated receptor alpha (PPAR-α), which inhibits testosterone secretion by leydig cells (11).
Suitable diet and exercise are considered as the diabetes care basis. The exercise effect on male fertility depends on volume and severity (12). Diabetic patients' exercise could reduce blood glucose levels by increasing the protein of the GLUT 4 vector and the insulin receptor substrate, and also by muscle mass increasing. The exercise effect on the amount of adiponectin has not yet been fully elucidated because of the lack of consistent results in various studies (13). This study purpose was to investigate the resistance and endurance training effect on the adiponectin genes expression and its receptors and sex hormones in type 2 diabetic rats.

Experimental animals and protocols
Research and animal care were approved by the Ethics Committee of Arak University of Medical Sciences. In vivo experiments were performed on 10 week's old adult male Wister rats with weight between 200-250 g (Pasteur, Iran). These rats were obtained from Baqiyatallah University of Medical.
Sciences Animals were housed under the standard conditions (24∘ C, cycles of 12 h light/12 h, in darkness) with free access to water and food supplies. 48 animals were divided into following groups by random, before performing the operation procedure (8 rats in each group): healthy control, diabetic control, healthy resistiance training, healthy endurance training, diabetic endurance training, diabetic resistance training.
Diabetic type 2 procedure Streptozotocin (STZ) (Sigma Chemical Co) was dissolved in 0.1 M citrate buffer (pH 4.5) and Nicotinamide (Sigma Chemical Co) was dissolved in normal physiological saline. Type 2 diabetes mellitus was induced in overnight fasted rats by injecting a single intraperitoneal (i.p) of 120 mg/kg Nicotinamide, by passing 15 min from the i.p. administration of 65 mg/kg of STZ. Hyperglycemia was con rmed by the elevated glucose levels in blood, determined at 72 h. The animals with a blood glucose concentration above 250 mg/dl were used for this study. Furthermore, the healthy control rats were intraperitoneally injected with normal saline at a dose of 1 cc, in order to be at the condition as same as diabetic groups (14).

Practice protocol
The exercise program included two types during10 week of endurance training and resistance training.

Endurance training
Diabetic and healthy with endurance training: The endurance training was accomplished on a rodent motor-driven treadmill at a 0˚ slope. The rats exercised for 5 day/week for duration of 10 weeks. Training blocks contained 3 phases of familiarization, overload, and nally preservation and stabilization of exercise intensity. Accordingly, in the familiarization phase ( rst week), the rats ran at treadmill with the speed of 8 m/min for 10-15 min every day. After that, during overload phase (second to fourth weeks), the rats initially ran at treadmill speed of 27 m/min for 20 min, and then the time of exercise increased (2 min in each session) gradually during 3 weeks until reaching to 60 minutes. Finally, in the preservation and stabilization stage of exercise intensity, the rats did the aerobic exercise for duration of 7 weeks with a speed of 27 m/min for 60 min. Each exercise session began with 5 min warming up (16 m/min), and 5 min was allocated to cooling down (16 m/min and gradual reduction of intensity to the least amount) (15).

Resistance training
Diabetic and control with resistance training: at the First stage, the rats were familiarized with Vertical ladder (Build by researcher) and learnt how to climb stairs. At this stage, animals were trained for 8 weeks, 5 sessions in each week and 3 sets per session, each one of them with 4 times climbing a special ladder up to one-meter-high and comprising 26 steps. Also, 1-minute rest was considered for animals between each set. At the second stage: From the the rst week beginning, 30% of the weight of the animal was connected to the tail of the animal each week, until reaching 200% of the weight of the animal in the last week (16).

Blood measurement
By passing 24 hours from the last exercise session, all of the rats were killed by intraperitoneally injecting a combination of ketamine (70 mg/kg) and xylazine (4 mg/kg). Their blood samples were collected by cardiac puncture (5 cc) and centrifuged at 3500 rpm for 10 min, and the serum samples were stored for future analysis at -70˚c. Testosterone, LH and FSH serum levels were assayed using a variety of kits with

Real time polymerase chain reaction
After sampling, total RNA was isolated by the use of RNX-Plus reagent (Yektatajhiz, Iran) in terms of the manufacturer's instructions. The concentration of RNA was measured spectrophotometrically at 260 nm wavelength using spectrophotometer (Eppendorf, Germany). after that, 3 µgr of total RNA was reverse Tran scripted into complementary DNA (cDNA) using Revert Aid ™ First Strand cDNA Synthesis Kit (parstous, Iran), with respect to manufacturer's protocols. Relative gene expression was measured by quantitative real time PCR by the use of SYBR green DNA PCR Master Mix (sina colon, Iran) and Life Cycler 96 system (Roche Diagnostics Gmblt, Germany). The PCR was performed in total volume of 20µl containing 1µl of cDNA template, 0.2µl of each of the primers, 10µl of SYBR green Master Mix and 7µl of nuclease-free distilled water. Moreover, each sample was loaded in duplicate.
The sequences of primers that was used for ampli cations are summarized in Table 1. The PCR conditions were 95 °C for 10 min followed by 45 cycles at 95 °C for 15 sec, 60 °C for 25 sec and 72 °C for 30 sec, respectively. Furthermore, melt curve analysis was performed after each run-in order to check the non-speci c PCR pro-ducts and primer dimers presence. The relative mRNA expression was determined using the 2 −ΔΔCT method and β-actin as an internal control as shown in Table 1.

Endurance and Resistance Training decrease bloodglucose in diabetic rats
The results demonstrated that in diabetic and diabetic rats with endurance training and resistance training, pre-exercise fasting blood glucose levels are signi cantly increased in comparison with the healthy control group (p = 0.01) ( Table 2). Endurance and resistance training in healthy and diabetic groups have signi cantly reduced post-exercise blood glucose levels in comparison with the diabetic control group (Table 2). This study indicates that 10-week endurance and resistance training is an effective way for reducing the level of blood glucose in diabetic rats.

The alteration expression of adiponectin and AdipoR 1 gene in Diabetic groups
Data Analysis demonstrated that in the rats of different groups, the relative expression of adiponectin gene was signi cantly increased in healthy control group compared to the diabetic control group (p = 0.02) (Fig. 1). The relative expression of AdipoR 1 gene in the diabetic control group was signi cantly increased compared to the healthy control group (p = 0.03), while the diabetic endurance with training group showed a signi cant reduction compared to the healthy control group and the other group (p = 0.02) (Fig. 2). There was no signi cant difference in the relative expression of AdipoR 1 gene in comparison with healthy control group (Fig. 2). Therefore, it appears that both methods of training can reduce the relative expression of adiponectin and AdipoR 1 gene in diabetic rats with endurance training and diabetic training with resistance training, in comparison with diabetic control group.

No change in the expression of the AdipoR 2 gene in Diabetic groups
Diabetes did not alter the expression of AdipoR 2 gene in testicular tissue compared to the healthy group.
Data Analysis demonstrated that in comparison with different groups of rats, the relative expression of AdipoR 2 gene expression was signi cantly increased in healthy endurance training and healthy resistance training groups (p = 0.03) (Fig. 3). Endurance diabetic training group and diabetic resistance training in comparison with the healthy control group showed a signi cant decrease (p = 0.02). The relative expression of AdipoR 2 gene expression in diabetic rats of control group showed a signi cant reduction compared to diabetic rats of the resistance training group. The relative expression of AdipoR 2 gene expression in comparison between diabetic rats with resistance training, the diabetic control group showed a signi cant reduction. However, in resistance training group the relative expression of this gene increased compared to the control group (Fig. 3).

The alteration level of hormonal
The results of the study indicated that the means of serum testosterone and LH concentrations signi cantly decreased in diabetic control group compared to healthy control group (p = 0.01) & (p = 0.01).
The data also revealed that the means of serum testosterone and LH concentrations of diabetic endurance training group were signi cantly greater than those of diabetic control group were (p = .01) & (p = 0.02). In the other words, it was thought that endurance training has the ability of compensating the destructive effects of diabetes on sperm parameters through increasing the levels of testosterone and LH serum (Table 3).

Discussion
Diabetes with in the amount of hormones involved in the process of spermatogenesis and changing the Structural of the cellular organs, disrupting the process of spermatogenesis and ultimately decreasing fertility (17). Many studies have demonstrated that diabetes result in a reduction in GnRH, LH, FSH, and throughout the effect on hypothalamic-pituitary-gonadal axis affects fertility (13). Our results indicated that the levels of LH and testosteronein the serum of diabetic rats decreased. The levels of FSH hormone also decreased in diabetes, however this reduction was not signi cant.
The adiponectin system (Adiponectin, AdipoR 1 and AdipoR 2 ) was expressed in organs associated with the male reproductive system (hypothalamus, pituitary and testis) in rat and human (11). Unlike other adipocytokines, Plasma adiponectin levels concentration in type 2 diabetes decreased. It makes a negative correlation between the concentration of adiponectin with insulin resistance and blood glucose concentration (18). Also, AdipoR 1 and AdipoR 2 decrease in insulin resistance were associated with obesity and diabetes (19). The level of testicular adiponectin in mRNA was different from that of circulating Blood level (20). Data analysis also indicated that the expression of the adiponectin and AdipoR1 gene in the testicular tissue increased, in spite of a reduction in their serum level in diabetes. Signi cant differences in AdipoR 2 gene is not evident.
As the studies indicate, adiponectin plays important roles in glucose homeostasis and lipid metabolism (21). The activation of receptors by adiponectin results in the activation of signaling intermediates and pathways such as peroxisome proliferator-activated receptor alpha (PPAR-α), activated protein kinase (AMPK) and mitogen-activated proteinkinase (MAPK). Searching for the binding of adiponectin to its receptorin skeletal muscles, the enzyme AMPK, has been phosphorylated and activated. After that, AMPK reduces the enzymes involved in the gluconeogenesis preventing from the liver glucose production. Indeed, adiponectin, by its insulin-like effect, boosts glucose uptake and oxidation of fatty acids, resulting in a reduction in triglyceride accumulation in the liver and adipose tissue, that preventing from the spread of insulin resistance and also metabolic abnormalities improving (10).
The hypothalamic-pituitary-gonadal axis is central for the mammalian reproductive system. Adiponectin releases gonadotropin-releasing hormones (GnRH) from hypothalamic neurons, and subsequently prevents the LH releasing from the pituitary and testosterone at the testicular level, while its effect is not on FSH anymore. In the testis, AMPK, MAPK, and PPAR-α signaling pathway have been indicated to be functional and involved in the steroid genesis regulation. Adiponectin activates the receptors in the testicular tissue, which results in AMPK phosphorylation and activation of PPAR-α, which inhibits testosterone secretion by leydig cells (11).
It can be claimed that adiponectin plays an inhibitory role in the reproductive system. Findings also suggest that testicular adiponectin may act as a paracrine/autocrine factor that could regulate various functions of testicular cells. The results of our research indicate that the expression rate of adiponectin and its receptor gene has increased blood glucose levels in testicular tissue. That is caused by the inhibitory role of adiponectin on the production of sex hormones of testosterone and LH. It has declined and nally reduced the spermatogenesis quality. Caminos et al. (2008) said that the expression of the adiponectin gene, AdipoR 1 and AdipoR 2 from the cells of the lyding was accomplished and adiponectin signi cantly inhibited the secretion of the gonadotropin-based baseline levels of testosterone secretion in the testes (9). Ocon et al. (2008) reported amounts the AdipoR 1 and AdipoR 2 mRNA in the testicles of adult chicks were much greater than immature sexually birds (22). Although investigations indicated that the results vary in different animal species and most of these studies are conducted on non-diabetic animals, so the type of study may also be different.
Recent studies have demonstrated that glycemic control may play a key role in reducing the diabetes mellitus effects on infertility (23). Exercise (endurance and resistance training) have the capability of reducing blood glucose, improve these fertility (24). Exercise in people with diabetes can lower the blood glucose levels and circulating adiponectin by increasing the protein of the glucose transport type4 (GLUT 4 ) vector following AMPK activationand the insulin receptor substrate along with increasing muscle mass (25). On the other hand, exercise can improve the endocrine glands performance, which also showed that the results of endurance and resistance training of testosterone and LH in diabetic rats increased, which could compensate the destructive effects of diabetes (17). The present study indicates that 10-week endurance and resistance training is an effective way could reduce blood glucose and increase sex hormones in diabetic rats.
Many studies have reported a lack of change, increase, or reduction of adiponectin after different treatment protocols (26). Garekani et al. (2011) investigated the effect of volume and intensity of exercise on the concentration of plasma adiponectin, liver, muscle and adipose tissue in rats. The results of this study were suggest that the mechanism of adiponectin expression in different tissues is not similar. They also reported that after training (in high and medium volume exercises), adiponectin gene expression was not increased in muscle and liver, but increased in adipose tissue (27).
Most studies have indicated that both endurance and resistance training can increase the total testosterone level, free testosterone and LH. Tremblay et al. (2005) reported that 40 and 80 minutes of endurance activity would increase the serum levels of free testosterone, total, and LH (28). Kraemer et al.
(2005) also examined the responsiveness and hormonal function of the men's activity and resistance training program, and found that resistance exercises result in a signi cant increasing in serum testosterone level (29). Perhaps one of the possible mechanisms is that by increasing the serum levels of testosterone, the expression of the adiponectin gene in the testicle decreases. Hope to be considered in future studies.
Considering the limitations of these studies on the mechanism and the function of the testis in diabetic patients, this type of study can provide a pathway for further investigation and recognition of the exercise effect on the mechanism of testicular activity in diabetic rats, and its effect on the infertility improvement in these rats.

Conclusion
The induction of diabetes by reducing sex hormones can interfere with reducing the relative expression of adiponectin and AdipoR 1 gene. On the other hand, it appears that endurance and resistance training could be effective in increasing the levels of LH and testosterone in the testes by reducing blood glucose, the relative expression of adiponectin gene and its receptors, which can be considered as a strategy for preventing from diabetes mellitus in the rat. Therefore, adiponectin plays an important role in secret sex hormones that can have an effect on fertility in trained diabetic rats.   One-way ANOVA was used to compare the relative expression of adiponectin gene in the study groups.

Abbreviations
The sign (*) indicates a signi cant difference compared to the normal group and the sign (#) that shows a signi cant difference compared to the diabetic group. Data was reported as mean ± SD (p <0.05). TA Endurance training and Resistance training. Abbreviations: A: Endurance training and TR: Resistance training.

Figure 2
One-way ANOVA analysis was used to compare the relative expression of AdipoR1 gene in the study groups. Sign (*) showed a signi cant difference compared to normal group. And the sign (#) indicates a signi cant difference compared to the diabetic group. Data was reported as mean ± SD (p <0.05). TA Endurance training and TR Resistance training. Abbreviations: A: Endurance training and TR: Resistance training.