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 confirmed 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 finally preservation and stabilization of exercise intensity. Accordingly, in the familiarization phase (first 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 first 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 respect to their manufacturer's instructions. Testosterone (Rat ELISA Kit, Eastbiopharm Cat. No Ck-E90243, China, sensitivity: 0.25 nmol/L, Assay range: 0.5–100 nmol/L), LH (Rat ELISA Kit, Eastbiopharm Cat. No Ck-E90904, China, sensitivity: 0.11mIU/L, Assay range: 0.2-60mIU/L) and FSH (Rat ELISA Kit, Eastbiopharm Cat. NoCk-E30597, China, sensitivity: 0.12mIU/L, Assay range: 0.2-60mIU/L).
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 amplifications 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-specific 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.
Table 1
Primer sets used for amplification
| Target genes | Primers sequences (5' to 3') | TM |
| Adiponectin- FW | 5’-AGGTTGGATGGCAGGCATC − 3’ | 58.83 |
| Adiponectin-RV | 5’-GGCTCTCCTTTCCTGCCAG − 3’ | 60.98 |
| AdipoR1-FW | 5’-CTTCTACTGCTCCCCACAGC- 3’ | 61.40 |
| AdipoR1-RV | 5’-TCCCAGGAACACTCCTGCTC − 3’ | 61.40 |
| AdipoR2-FW | 5’-CCACACAACACAAGAATCCG − 3’ | 57.30 |
| AdipoR2-RV | 5’-CCCTTCTTCTTGGGAGAATGG − 3’ | 59.82 |
| β-actin -FW | 5’-TCACCCACACTGTGCCCCATCTACGA − 3’ | 67.95 |
| β-actin -RV | 5’-CAGCGGAACCGCTCATTGCCAATGG − 3’ | 67.90 |
Statistical analysis
A Shapiro-Wilk test was applied for determining the normality of measures distribution, which was found to be normally distributed. After that, a Leven test indicated that the variances were homogeneous. A one-way analysis of variance (ANOVA) was performed for determining the presence of differences amongst groups. Significant differences were quantified by the use of a post hoc test (Tukey). Moreover, data were expressed as Means±SD and significance was set at the alpha level P ≤ 0.05.