Glycogen measurement

The snap frozen hindlimb muscle tissues from mice in cold stress experiments were used for quantitative glycogen content analysis. Tissue samples were homogenized with 0.9N perchloric acid (244252, Sigma-Aldrich, St. Louis, MO, USA) in a mixer at 2000 rpm for 30 s. Then, 50 μL of perchloric acid homogenate was mixed thoroughly with 25 µL of 1M potassium bicarbonate (237205, Sigma-Aldrich, St. Louis, MO, USA). Fresh 4 mg/mL amyloglucosidase (10115, Sigma-Aldrich, St. Louis, MO, USA) in acetate buffer was added to 125 µL of the homogenate mixture, and the final solution was incubated at 56 °C for 4 h. After that, the whole mixture was centrifuged for 1 min at 13,200 rpm, and the supernatant was used to measure the glucose concentration by using a glucose assay kit (GAGO-20, Sigma-Aldrich, St. Louis, MO, USA). The glycogen content was determined by differences in glucose concentrations before and after amyloglucosidase treatment to exclude free glucose from glycogen in skeletal muscle. For the cellular glycogen content assay, a glycogen colorimetric assay kit (K646-100, Biovision, Milpitas, CA, USA) was used according to the manufacturer’s protocol. In detail, cell pellets were homogenized in distilled water on ice. Homogenates were boiled for 10 min to inactivate enzymes and centrifuged at 18,000 × g for 10 min. Finally, the supernatant was used to measure the cellular glycogen content according to the protocol provided by the manufacturer.