2.5. Glycogen Analysis

Glycogen was measured using the amyloglucosidase method established in a previous study (H. Hwang et al., 2014). Approximately 30 mg of liver and muscle tissue was placed on ice. We added 0.5 mL of 2 N HCl and incubated the samples for 2 h at 96 °C after adding 1.5 mL of 0.67 M NaOH. The supernatant, a 100-µL sample of glucose, was taken, and 1 mL of reaction buffer was added to it; the solution was incubated for 30 min at 37 °C. Next, the sample reactions were stopped by putting them on ice. The glucose content level was analyzed in a 96-well plate using a spectrophotometer (Multiskan Go microplate reader, ThermoFisher, Waltham, MA, USA) at an absorbance of 340 nm.