Glycogen Content Quantification in Muscle

For the biochemical analysis, the TA and TB muscles were rapidly dissected, frozen in liquid nitrogen and stored at − 80 °C until processing. The tissues were homogenized in phosphate buffer containing protease inhibitors (Roche, Mannheim, Germany) using Precellys® (Ozyme, Montigny Le Bretonneux, France). The homogenates were centrifuged at 13,000 rpm for 10 min at 4 °C, and the resulting supernatant was used for the biochemical measurement of the glycogen content as described elsewhere [28 (link)]. Briefly, the tissue extracts were boiled for 3 min and incubated at 54 °C for 1 h with or without 5 U/mL Aspergillus niger amylo-α-1,4-α-1,6 glucosidase (Roche, Mannheim, Germany), which converts glycogen to glucose. The samples were centrifuged, and the glucose level in the supernatant was determined using an AmplexRed® Glucose Assay Kit (A22189, Invitrogen, Cergy-Pontoise, France) per the manufacturer’s instructions.