Animals and maintenance
Forty male C57BL/6 (4 weeks old) mice were purchased from Samtako Bio Korea (Osan, Korea) and fed freely with standard chow and water until 30 weeks old. Three or four mice were housed in each cage and maintained under standardized conditions in an animal facility of the Dong-A University College of Medicine. Relative humidity (55 ± 5 %), temperature (22 ± 2 °C) and a 12-h dark-light cycle remained constant. The animal experiments were approved by the Dong-A University Medical School Institutional Animal Care and Use Committee (DIACUC-approval-19-11) and all procedures were conducted in accordance with the committee guidelines.
Experimental design
When the mice were 30 weeks old, mice were randomly divided into two groups to induce obesity by HFD: a normal diet (CO, n = 10) and a high-fat diet (HF, n = 30) groups. The HF group was fed with a 60% fat chow (60% lipid, 20% carbohydrate, and 20% protein), whereas the CO group was fed with standard chow (6.3% lipid, 69.4% carbohydrate, and 24.3% protein) for eight weeks. Following obesity induction period, the mice in the HF group were randomly subdivided into an HFD + sedentary (HF, n = 10), HFD + aerobic training (HFAT, n = 10), and HFD + resistance training (HFRT, n = 10) groups, and the training groups exercised for eight weeks. Body weight was measured every week until the end of the experimental period.
Training intervention
Mice in both aerobic and resistance training groups exercised with a 65 ~ 70% of exercise intensity for 8 weeks, five days per week while maintaining an HFD. In the first week, mice exercised at a low intensity for adaptation of the exercise equipment and to set their exercise intensity. Thereafter, mice exercised at a determined speed or weight for the remaining 7 weeks. Mice in the CO and HF groups were placed next to the treadmill to expose a similar stress condition when the mice in the training groups were exercising.
Mice in the HFAT group exercised 5 days per week for 8 weeks using an animal treadmill. During the four days of the first week, aerobic exercise was performed for 5 min at a speed of 5 m/min on the first day, and the exercise time was increased 5 min every day. On the 5th day of the first week, an incremental load test was performed to determine the treadmill exercise speed of the HFAT group [15]. Briefly, after 5 min warm-up at 5 m/min, the speed of the treadmill was increased by 3 m/min every 3 min at 0% grade until exhaustion. The exercise speed in the HFAT was set to 65~70% of the maximal speed based on the results of the incremental load test. Mice exercised 5 days per week, 50 minutes a day, including 10 min of warm-up and 10 min of cool-down exercise.
Mice in the HFRT group exercised 5 days per week for 8 weeks using a climbing ladder (100cm ladder with 2cm grid and 80° incline). House Chamber (L × W × H = 25 × 25 × 20cm) was set up on the top of the ladder for a 2-min rest between the trial. During the four days of the first week, exercise was performed without any resistance for adaptation of the ladder. On the 5th day of the first week, an incremental load test was conducted by referring to the previous study to determine the exercise intensity [16]. Briefly, 50% of body weight was attached to the mouse’s tail (lead), and the lead weight was progressively increased by 3 g when the mouse was a success to climb the ladder. Mice exercised 8 sets for the remaining 7 weeks with a 65~70% of the maximum successful weight. An incremental load test was conducted after 4 weeks to adjust the weight of the load.
Tissue sampling
Tissue sampling was conducted two days after the last exercise session to rule out temporary training effects. Food was removed 12 h before sacrifice. The liver samples were excised after complete anesthesia using ethyl ether. The samples were immediately frozen in liquid nitrogen and stored at -80 °C refrigerator.
Hematoxylin and eosin staining
Small pieces of liver tissue were fixed with formalin (10% neutral-buffer formalin) and embedded in paraffin. Four-micrometer sections were cut and stained using hematoxylin and eosin (H&E staining). Digital images of the slides were captured with an Aperio ScanScope (Leica Biosystems, Buffalo Grove, IL, USA).
Extraction of liver triglycerides
As previously described [13], Liver tissue (50 mg) was placed in 200 μl of ethanolic KOH (2 parts ethanol: 1 part 30% KOH) and incubated overnight at 55 °C. Subsequently, it was mixed with 50% ethanol into each tube to bring the volume to 0.5 ml and centrifuged for 5 min at 13,000 rpm. The supernatant was transferred into new tubes, and 50% ethanol was added again into each tube to bring the volume to 0.6 ml. After vortexing, 200 μl was moved to a new tube, and 215 μl of 1 M MgCl2 was added, incubated for 10 min on ice, and centrifuged for 5 min at 13,000 rpm. The supernatant was used to measure liver TG using ASAN set Triglyceride-S Reagent (Asan Pharmaceutical, Seoul, South Korea) by the enzymatic colorimetric method. Liver lysates and standards were added into the microplate and incubated at 37 °C for 10 min. Absorbance values were measured at 550 nm [13].
Protein analysis
As previously described [15], the liver tissue was lysed in 200 μl RIPA buffer. Thereafter, the tissues were homogenized and centrifuged for 30 min at 14,000 rpm. The protein concentration was measured by the BCA method. Samples of equal protein content were resolved by SDS-polyacrylamide gel electrophoresis and transferred to a membrane. The membrane was blocked with 5% skim milk in PBS. The membrane was subsequently incubated at 4 °C overnight with primary antibodies against cannabinoid receptor 1 (CB1; sc-293419), Sterol-regulatory element binding protein 1 (SREBP-1; sc-13551), fatty acid synthase (FAS, sc-74540), carnitine palmitoyltransferase1 (CPT1, sc-98834) (all from Santa Cruz Biotechnology, Dallas, TX, USA), AMP-activated protein kinase (AMPKα, #2532, Cell Signaling Technology, Danvers, MA, USA), and phosphor-AMPK (p-AMPK, #2531, Cell Signaling Technology, Danvers, MA, USA). The membrane was incubated with its secondary antibody for 1 h at room temperature. The signal was developed with an ECL solution and visualized with the ImageQuantTM LAS-4000 system (GE Healthcare, Uppsala, Sweden).
Statistical analysis
All statistical analysis was performed with the SPSS 22.0 version (SPSS Inc., Chicago, IL, USA), and the values are reported as means ± standard error. Two-way repeated measures analysis of variance (ANOVA) was used to compare the changes in body weight, and one-way ANOVA followed by a Tukey HSD post hoc test was conducted to compare the differences between groups after exercise intervention. All data have confirmed the homogeneity of the variance by Levene Statistic, and a significance level of P = 0.05 was used as a threshold for statistical significance.