Glucose-regulating Enzyme Activity Assay

Glucose-regulating enzyme activities were examined in 10% homogenates of liver tissue (0.3 g) in 0.1 M triethanolamine, 0.2 M EDTA and 2 mM dithiothreitol as described previously [46 (link)]. The homogenates were centrifuged twice at 1000× g and at 10,000× g for 15 min at 4 °C, and the supernatant was collected. For GK, the supernatant was incubated with the reaction buffer containing 50 mM Hepes-NaGT (pH 7.4), 100 mM KCl, 7.5 mM MgCl₂, 2.5 mM dithioerythritol, 10 mg/mL albumin, 10 mM glucose, and 4 units glucose-6-phosphate dehydrogenase for 10 min at 37 °C. The activity was measured at 340 nm after incubating with 5 mM ATP at 37 °C for 10 min. For G6pase, the supernatant was measured at 340 nm after incubating with the buffer containing 131.58 mM Hepes-NaGT (pH 6.5), 18 mM EDTA (pH 6.5), 265 mM glucose-6-phosphate, 0.2 M NADP⁺, 0.6 IU/mL mutarotase, and 0.6 IU/mL glucose dehydrogenase for 4 min at 37 °C. For PEPCK, the supernatant was mixed with the buffer containing 72.92 mM sodium Hepes (pH 7.0), 10 mM dithiothreitol, 500 mM NaHCO₃, 10 mM MnCl₂, 25 mM NADH, 100 mM inositol-1,4-diphosphate, 200 mM phosphoenolpyruvate, and 7.2 units malic dehydrogenase. The activity was measured at 340 nm as the reduced absorbance using a UV/Vis spectrophotometer (OPTIZEN POP, Mecasys, Daejeon, Korea). All reagents were purchased from Sigma-Aldrich (St. Louise, MO, USA).