2.6. Blood Analyses
high-density lipoprotein
Plasma and serum samples were collected at rest prior to the muscle biopsy collection and SS exercise bout, centrifuged and then stored at −80 °C. Serum samples were analyzed for total cholesterol, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol, and total triglycerides by a commercial laboratory. Plasma samples were collected for the determination of leptin and adiponectin.Leptin levels were measured using a human leptin ELISA kit with recombinant human leptin as a standard, according to manufacturer’s instructions (Abcam, Melbourne, VIC, Australia). Briefly, 100 µL of the plasma, diluted in sample diluent, and each standard was added to the well and incubated for 2.5 h at room temperature with gentle shaking. Following this step, the plate was incubated with 100 μL of biotinylated leptin detection antibody for 1 h, 100 μL of horseradish peroxidase (HRP)–streptavidin solution for 45 min, and 100 μL of tetramethylbenzidine (TMB) one-step substrate reagent for 30 min at room temperature in the dark with gentle shaking. Stop Solution (50 μL) was added to each well, and absorbance was read at 450 nm immediately with a SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA). The plate was washed three times between each step with 300 μL of washing buffer. A standard curve was generated, and leptin content for each sample was calculated.AMPK can be stimulated in skeletal muscle via adiponectin [