Quantifying Muscle Glycogen, LPL, and TG

The glycogen content of the muscle was measured according to the method previously described59 (link). Briefly, powdered muscle (50 mg) was hydrolyzed in 150 μl of 1 mol/L KOH by heating at 95 °C for 20 min. The samples were incubated at 37 °C for 2 h, centrifuged at 4 °C/16000 × g for 10 min, and neutralized with NaOH. The resulting free glycosyl units were determined using D-glucose as a standard, and the results are expressed as mg/g muscle tissue. The LPL activity and TG content in the skeletal muscle were measured using a Triglyceride Assay Kit and a Lipoprotein Lipase Assay Kit provided by the Nanjing Jiancheng Bioengineering Institute, China. Approximately 100 mg of frozen muscle was minced, weighed and thoroughly homogenized with 1 ml of ice-cold PBS using a mechanical tissue disrupter. After centrifugation (2500 × g at 4 °C), the supernatants were decanted and saved for assaying LPL activity, and the TG concentration was measured in triplicate at the appropriate dilutions. The LPL activity is expressed as U/mg protein of muscle tissue, and the TG content is expressed as mmol/g protein of muscle tissue. The total protein content of the supernatant was measured with the Coomassie Brilliant Blue Protein Assay Kit (Nanjing Jiancheng Institute of Bioengineering, China).