Twenty-four C57BL/6 male mice (age of five weeks; body weight of 12±2 gram) were obtained from the animal care center of Pasteur institute of Iran and kept under controlled conditions (at 22 °C ± 2 °C with a humidity of 55% ± 10% and a 12-hour light/dark cycle). The mice had normal rodent chow ad libitum with free access to water. After a one-week acclimation, the mice were randomly assigned to three groups: 1) control (C, n=7), 2) High fat diet-induced obese (HFD-obese, n=7), and 3) High-fat diet with exercise training (HFD-Ex, n=7) (Fig.1).
The subjects of the HFD-Ob group were fed a high-fat diet (HFD) for 14 weeks. The mice of the HFD-Ex group were submitted to continuous running on a treadmill for eight weeks along with feeding HFD. After the experiment, mice were sacrificed, and epididymal fat pads were collected (Fig.1).
The mice of the HFD-Ob group were fed a high-fat diet (HFD) for 14 weeks from 6 to 20-week old. The HFD contained 45% kcal from fat, 40% carbohydrate, 20% protein (total calorie 4.6 kcal/g) . The mice in the control group were fed with normal diet (15 kcal% fat, 60 kcal% carbohydrates, and 25 kcal% protein; total calorie 3.5 kcal/g) . The diets were obtained from the research and development unit of Behparvar Animal Feed Company at Karaj -Iran.
Body weights were registered weekly during the experiment using electronic digital weighing scales (ENTRIS 3202-1S S, Artorius, Germany). The 24-h food intake was determined weekly throughout the study.
Before the exercise training experiment, the mice of the HFD-Ex group were acclimatized to running on a treadmill. At 12 weeks of age, they were trained during the dark cycle by continuous running on a rodent treadmill (Andisheh-Sanat, made in Iran) at 0% inclination. The protocol was five sessions per week for seven weeks. Each training session consisted of continuous running for 30 minutes at 60-70% vVO2peak, preceded and followed by 5 min of warming up and cooling down at 50% vVO2 peak. The control-group mice were placed on a non-moving treadmill to be exposed to the same potential environment and handle stress.
vVO2 peak test. At the onset of the training and the end of each one-week training period, the mice were subjected to a graded progressive exercise test on the treadmill (at 0% inclination) to determine the vVO2 peak . The test consisted of two-min stages with alternating increases in speed (increments of two m/min). The vVO2 peak was reached when the animal can not keep running at a new speed for 10 s . The obtained vVO2peak values were utilized to define the intensity of future training programs.
Tissue and blood collection
The mice were anesthetized by intraperitoneal injection of a mixture of xylazine (10 mg/kg) and ketamine (100 mg/kg)  at the end of the experiment.
The blood samples (5-10 ml) were obtained from a cardiac puncture using a heparinized syringe and put immediately into glass tubes with EDTA. Plasma was obtained from blood by centrifugation (10 min, 3000 rpm, 4°C) and frozen (-78oc) for future measurement. The epididymal fat pads were quickly dissected and immediately frozen in liquid nitrogen and stored at −78oC until assessment. The mice were sacrificed 48 h after the last training session to eliminate the acute effects of exercise.
Measurement in Plasma
Fasting plasma glucose was assessed by a glucose oxidase method using a biochemistry Auto-Analyzer (Hitachi 902, Boehringer manneheim, Germany). Lipid profiles including non-esterified fatty acids (NEFA), total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) concentrations were enzymatically determined by commercially available kits (ParsAzmon, Iran) using Auto Analyzer according to the manufacturer's instructions.
Plasma insulin concentrations were measured by ELISA technique via enzyme immunoassay (EIA) kit (10-1247-01; Mercodia AB, Uppsala, Sweden) and ELISA microplate reader (Awareness, Model stat fax 2000).
Gene expression (mRNA) evaluation
RNA Extraction. Approximately 50 mg of adipose tissue samples were homogenized in 1 ml of TRIzol reagent (Thermo fisher Scientific, USA). The total RNA was isolated using TRIzol under the manufacturer's instructions (Cat. No. 15596026). RNA concentration and purity were assessed (OD: 260/280) using spectrophotometers (Ultrospec 3000, Pharmacia Biotech, Sweden).
cDNA Synthesis. First-strand complementary DNA (cDNA) was synthesized from 1 μg of RNA using Invitrogen SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, USA), according to the manufacturer's instructions (Cat. No. 18-064-014).
Primers: Primers were designed based on previous studies, then verified using primer-blast/NCBI and Oligo Analyzer (1.02), and synthesized by SinaClon (Iran). The characteristics of the used primers have shown in Supplemental Table 1.
Real-time PCR. The mRNA contents were determined by real-time PCR performed using the Rotor-Gene 6000 (Corbett, Australia ) with SYBR Green RT-qPCR Kit (QR0100-Sigma-Aldrich- Germany) and specific synthesis primers, according to the manufacturer's instructions (Cat. No. 1907/2006). The thermal cycling profile was 95°C for 10 min, followed by 40 cycles at 95°C for 10 s and 55°C to 58°C for 30 s to 40 s according to annealing temperature.
Quantification of mRNA. The efficiency of PCR product and primers were determined by LinRegPCR software (Version 2020.0), and the melting curve confirmed PCR products' specificity. The relative gene expression was quantified by Pffafl method using Genex software (7.0).
Western blot determination
Tissue preparation: Frozen adipose sample was powdered by glass mortar and pestle in liquid nitrogen. Approximately 100 mg of the powder was homogenized in RIPA lysis buffer (Sigma-Aldrich; German). The homogenate was centrifuged (Rotina 380 R; HETTICH; German) at 4°C for 15 min at 13000 × g (3000 g, 15 min, 4°C). The supernatant fraction was collected, and the fat cake was discarded.
Determination of total Protein. Total protein was determined via Bradford technique by Bradford-protein assay kit (ZellBio; German), in which bovine serum albumin was applied as a standard. In the method, protein concentration was assessed using spectrophotometers (in absorbance at 595 nm) (Ultrospec 3000, Pharmacia Biotech, Sweden).
SDS-PAGE: After preparation of SDS-polyacrylamide gel (4% and 12%), 30 μg sample was boiled for 5 minutes and loaded onto the gel. Proteins were separated using electrophoresis (Bio-Rad, Hercules, CA) at 110 V for 60 min.
Electroblotting: Proteins were then transferred from the gel onto nitrocellulose polyvinylidene difluoride (PVDF) membranes (Sigma-Aldrich) by electroblotting (Bio-Rad, Hercules, CA) at 80 V for 180 min.
Block of nonspecific proteins. The membranes were blocked for 60 min at room temperature with 5% non-fat milk in TBST solution (Tris buffer saline containing 0.1% Tween 20).
Incubation with antibody. The membranes were incubated with (1 μg/ml) primary antibody against ATG5, ATG7, LC3A/B, SQSTM, LAMP2, CTSB, CTSL, and Β-ACTIN (as control) (diluted 1:500 or 1:1000 in blocking solution, in accordance manufacturer's instructions) by an overnight period at 4°C. After incubation by the primary antibody, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (1:1000) for two hours at room temperature. The details of used antibodies have shown in Supplemental Table 2.
Quantification of bands. Bands were visualized by Gel Doc device (Uvidoc, England) using Enhanced Chemiluminescent (ECL) detection method (Sigma-Aldrich; Germany). The bands were quantified using ImageJ software (15.1, NIH, USA). Each band was normalized with Β-ACTIN as a control.
Data are expressed as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was used for intergroup comparisons. Data were then compared between groups using the Tukey post hoc test. A P value of less than 0.05 (p ≤ 0.05) was considered statistically significant. SPSS 21.0 (SPSS Inc., Chicago IL, USA) and Prism 8 (GraphPad, USA) software were used to analyze data and design graphs, respectively.