Animals and grouping
Forty-eight Sprague-Dawley (SD) male rats aged 7 weeks (170–190 g body weight) were purchased from Beijing Vital River laboratory Animal Technology Co. Ltd., and housed under standard specific pathogen free (SPF) conditions in a temperature and humility-controlled room on a 12-h light:12-h dark cycle. Food and water were freely available throughout the study. The animal protocol was approved and all the experimental procedures were supervised by the Ethics Committee of Shanghai University of Sport (No. 2018002). After acclimating to laboratory conditions for 3 days, rats were randomly and evenly divided into six groups: control (C), AR antagonist flutamide embedded (F), resistance training (R), resistance training plus flutamide (R + F), endurance exercise (E) and endurance exercise plus flutamide (E + F).
Flutamide treatment
Rats in F group, E + F group and R + F group were anesthetized and embedded with flutamide releasing pellet (50 mg/pellet, evenly releases for 21 days, Innovative Research of America, USA.) prior to exercise intervention. Briefly, after anesthesia, an almost 0.5 cm long incision was cut on the neck, and pellet was implanted into the incision site, then gently sutured the incision. Rats in C group also underwent the surgery but no pellet embedded.
Exercise protocol
As shown in Fig. 1, after one-day adaptation to endurance or resistance training, rats in E and E + F groups participated in a moderate intensity aerobic exercise on a treadmill at a speed of 20 m/min for 1 hour each time, while the rats in R and R + F groups underwent resistance training, which comprises three sets of ladder climbing with progressively incremental load. Briefly, rats start climbing at the bottom of the ladder (1 m in height, 2 cm distance from each stairs and a slope of 85°) with progressively increased load on tail (the initial weight attached to tail was 40% of body weight, and increased by 10% every 2 days until reached 120% of body weight). Every set consists of four repetitions and three sets (20 s interval period between sets) in each time. Resistance or endurance training lasts for 3 weeks and 6 days per week.
Detection of serum testosterone and IGF-1 by ELISA
Serum testosterone and IGF-1 levels of rats were measured by ELISA according to manufacturer’s instructions. The intra- and inter-assay coefficients of variation (CV) were less than 2.9 and 6.8% for detecting testosterone (KGE010, R&D systems, Minnesota Minneapolis, USA) as well as 4.1 and 4.3% for IGF-1 kit (MG100, R&D systems, Minnesota Minneapolis, USA).
Quantitative real-time PCR
Rats were anaesthetized at 36 h after the last exercise, gastrocnemius and soleus were collected and frozen in liquid nitrogen then stored at − 80 °C until analyzed or immediately processed as the following process. Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA), the first strand cDNA was synthesized by Revert Aid First Stand cDNA Synthesis Kit (Thermo Scientific, MA, USA) in accordance with the manufacturer’s instructions. Primers for amplification genes of IGF-1R (sense: 5′-AAA CGC TGA CCT CTG TTA CCT-3′, antisense: 5′-ACG CCT TTG TAG TAG TAG TGT CG-3′), mTOR (sense: 5′-GAT ACG CCG TCA TTC CTC-3′, antisense: 5′-TGC TCA AAC ACC TCC ACC-3′) and GAPDH (sense: 5′-GCT GAG TAT GTC GTG GAG-3′, antisense: 5′-TCT TCT GAG TGG CAG TGA T-3′) were synthesized by Sangon Biotech Co, Ltd. Shanghai. 50 ng of cDNA templates were added into FastStart universal SYBR Green Master (Roche company, Switzerland) to amplify the genes above, and the amplification conditions for these genes were the same: 10 min denaturation at 95 °C followed by 40 cycles of 15 s denaturation at 95 °C, 60 s annealing and elongation at 60 °C. The mRNA values of IGF-1R and mTOR of samples were corrected by that of the internal control of GAPDH and shown as the ratios of target genes to GAPDH.
Western blot
About 50 mg of gastrocnemius and soleus were cut into pieces and homogenized after adding 500 μL of radioimmunoprecipitation assay (RIPA) buffer containing phenylmethanesulfonyl fluoride (PMSF) (Beyotime Biotechnology, China) (RIPA:PMSF = 100:1) to extract total protein. After sonication on the ice, lysates were centrifuged at 14000 rpm for 20 min at 4 °C. Then supernatants were collected and protein concentration was determined by BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Extracts (50 μg) of gastrocnemius or soleus was fractionated on 10% SDS-PAGE gels, then electro-transferred onto nitrocellulose membranes. After blocking with 5% nonfat milk for 2 h, the nitrocellulose membranes were incubated overnight at 4 °C with primary antibodies against AR and IGF-1R (both 1:500, Santa Cruz Biotechnology, CA, USA), PI3K, Akt, mTOR, p-PI3K and p-Akt (all 1:1000, Cell Signaling Technology, MA, USA). After washing three times with Tris-buffered saline with 0.1% Tween 20 (TBST), each time for 5 min, the bands were incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (Santa Cruz Biotechnology, CA, USA) for 1 h at room temperature. Then, the bands were washed again as described above, developed with Immbilon Western chemiluminescent HRP substrate (Millipore, MA, USA), and visualized by automatic chemiluminescence image analysis system (Tanon Biotechnology, Shanghai, China). The density of blot was determined using Bio-image software (Tanon Biotechnology, Shanghai, China) and normalized against GAPDH.
Statistical analysis
Statistical analysis of data was performed using SPSS for Windows 19.0 software package (IBM Corporation, Armonk, NY, USA). All data were presented as Mean ± SD. Statistical difference for C, F, R and R + F groups, as well as the C, F, E and E + F groups were determined by one-way analysis of variance (ANOVA) and post hoc comparison using least significant difference (LSD)-t test. The level of statistical significance was set as p < 0.05.