Glycogen Assay.

Tissues were homogenized with a bead homogenizer (MS-100R; TOMY) in ice-cold 6% perchloric acid (PA) containing 1 mM EDTA. The glycogen contained in 100-µL aliquots of homogenate was hydrolyzed to glucose by incubation for 3 h at 37 °C with 1 mL 0.2 M sodium acetate, 20 µL 1.0 M KHCO₃, and 20 U/mL amyloglucosidase. Addition of 0.5 mL PA stopped the reaction. After centrifugation (14,000 × g for 10 min at 4 °C), the supernatant was neutralized with a solution consisting of 3 M KOH, 0.3 M imidazole, and 0.4 M KCl. The sample was then centrifuged (16,000 × g for 10 min at 4 °C) and the supernatant was assayed for glucose content. Nonhydrolyzed samples were used to measure endogenous glucose levels; these samples were homogenized and centrifuged (14,000 × g for 10 min at 4 °C) and the pH of the supernatants was adjusted to 6 to 8 with KOH solution. The neutralized samples were mixed thoroughly, centrifuged (16,000 × g for 10 min at 4 °C), and assayed for endogenous glucose levels. The glucose assay was performed in 96-well plates using a coupled-enzyme assay method modified from previous studies (13, 14).