Adiponectin

Blood glucose concentrations were measured using Glucocard strips (28,350, Menarini Diagnostics) and plasma triglyceride and cholesterol were evaluated using routine clinical assays. Plasma insulin (Mercodia), adiponectin (R&D Systems) and leptin (DY498, R&D Systems) levels were measured using commercially available ELISAs. The Homeostatic Model Assessment of Insulin Resistance (HOMA-IR-) index was calculated as follows: fasting glucose levels (mM) x fasting insulin levels (mU/L)/22.5. The content of triglycerides in the liver and BAT was quantified with the triglycerides FS* kit (Diasys), after preparation of liver tissue as described previously [6]. Briefly, 30 mg tissue was incubated overnight at 55°C in alcoholic KOH (2:3 60 ml EtOH 99% + 30 ml 30% KOH) until digestion was complete. The next day, 1 M MgCl₂ (1:1, vol/vol) was added to the digested samples. After 10 min of incubation on ice and subsequent centrifugation, the supernatant was aspirated and analysed using the triglycerides FS* kit (Diasys). Lactate dehydrogenase (LDH) levels were assessed using the LDH assay kit (Abcam) according to the manufacturer’s instructions.

Before the end of the study, at weeks six and twelve, the mice were fasted for six hours in the morning. Under isoflurane anesthesia, blood was collected via cardiac punction, and euthanasia was performed by cervical dislocation. Blood samples were incubated for 30 min at room temperature and centrifuged for 15 min at 1500× g, and the obtained serum was immediately frozen and stored at −80 °C. Serum cholesterol and triglyceride levels as well as alanine aminotransferase (ALT) activity were measured by fluorometric assays (Abcam, Cambridge, UK). Serum insulin (Mercodia Ltd., Uppsala, Sweden), serum amyloid A (SAA), leptin, resistin, adipsin, and adiponectin (R&D Systems Europe Ltd., Abingdon, UK) were measured by enzyme-linked immunoassays (ELISAs). The detection limits were 33 pmol/L for insulin, 62.5 ng/L for SAA, 7.8 ng/L for leptin and resistin, 375 ng/L for adipsin, and 15.6 ng/L for adiponectin.

Venous blood was obtained from the participants pre- and postexercise intervention, following 12 h of fasting (09:00 a.m.). Blood was collected in plasma-separating tubes, centrifuged at 3000 rpm for 10 min to obtain the plasma, and subsequently stored at −80 °C until analysis. BDNF (R&D Systems, Minneapolis, MN, USA), NGF (R&D Systems, Minneapolis, MN, USA), adiponectin (R&D System, Minneapolis, MN, USA), leptin (R&D System, Minneapolis, MN, USA) and GLP-1 (Cusabio, Wuhan, China) blood levels were analyzed using enzyme-linked immunosorbent assay (ELISA) kits, following the manufacturer’s instructions. Absorbance was measured using a microplate spectrophotometer (SpectraMax M2e Microplate Reader; CA, USA).

Tissues and cell lysates were prepared in ice-cold PRO-PREP (Intron Biotech) and cleared by centrifugation (12,000 rpm, 30 min, 4°C). The protein concentration in each sample was estimated using the Bradford assay. Each protein sample (25 μg) was resolved on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and transferred to a polyvinylidene fluoride membrane. The membrane was incubated for 1 h in blocking solution (5% non-fat milk in PBST) and incubated with primary antibodies for 3 h at room temperature. Primary antibodies included those against adiponectin (1:500; R&D Systems) and GAPDH (1:10,000; Fitzgerald Industries International Inc., Westborough, MA, USA). The blots were washed in PBST and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Results were visualized using an enhanced chemiluminescent system (Invitrogen, Carlsbad, CA, USA).