Blood was centrifuged (10,000 g for 10 min at 4°C), and plasma was stored at −20°C. Serum β hydroxybutyrate (BHB; Stanbio Laboratory), nonesterified fatty acids (NEFA; Wako Life Sciences), and lactate (Sigma-Aldrich) were determined using commercially available kits. Serum insulin (Crystal Chem, 90080) was quantified by ELISA. Tissue and serum triglycerides (TG; Stanbio Laboratory) and protein (Thermo Scientific) were quantified by commercial kit.
For glycogen measurements, liver tissue (30–50 mg) and dilutions of glycogen type III obtained from rabbit liver (Sigma-Aldrich) were homogenized in 0.03 N HCl. An aliquot of the homogenate was mixed with 1.25 N HCl and heated for 1 h at 95°C. Samples were centrifuged at 18,400 g, and 10 µL of supernatant was mixed with 1 mL of glucose oxidase reagent (Stanbio Laboratory). After a short incubation at 37°C, the absorbance was read at 505 nm.
For glycogen measurements, liver tissue (30–50 mg) and dilutions of glycogen type III obtained from rabbit liver (Sigma-Aldrich) were homogenized in 0.03 N HCl. An aliquot of the homogenate was mixed with 1.25 N HCl and heated for 1 h at 95°C. Samples were centrifuged at 18,400 g, and 10 µL of supernatant was mixed with 1 mL of glucose oxidase reagent (Stanbio Laboratory). After a short incubation at 37°C, the absorbance was read at 505 nm.