K753 100

AST activity was used to calculate glutamate deamination at OD₄₅₀. One hundred milligrams of liver tissue homogenate was placed in 500 μL AST assay buffer and all subsequent steps were performed according to the manufacturer’s instructions (K753-100, Biovision). Serial glutamate dilutions (0 nmol, 2 nmol, 4 nmol, 6 nmol, 8 nmol, and 10 nmol in 50 μL assay buffer) were used to plot the standard curve. The 50 μL samples were incubated at 28 °C and analysed in a VERSAmax microplate reader (Molecular Devices LLC) at 450 nm. AST activity (mU mg⁻¹) = B/((T₂ − T₁)·V)/g wet wt, where B is the amount of glutamate in nmol calculated from the standard curve, T₁ is the time of the first reading (in min), and T₂ is the time of the second reading (in min).

To determine triglyceride, cholesterol, lactate and uric acid levels in blood, serum was collected from each mouse group. Total cholesterol and uric acid were analyzed using kits (TR13421 and TR24321) obtained from Thermo Fisher Scientific (Waltham, MA, USA). Triglycerides were measured with a Serum Triglyceride Determination Kit (TR0100, Sigma-Aldrich, St. Louis, MO, USA) and lactate measured with a colorimetric/fluorometric kit (K607–100, Biovision, Milpitas, CA, USA). Liver transaminase activity (AST/ALT) was analyzed using colorimetric kits (K753-100 and K752-100, Biovision). To determine serum biochemistry and liver transaminase levels, standard curves for each respective kit were prepared and samples were analyzed according to manufacturer’s instructions using the SpectraMax i3x (Molecular Devices, Sunnyvale, CA, USA).

Blood glucose levels were measured at indicated time points using an Accu-Chek (Roche, Basel, Switzerland). Serum levels of glucagon (#48-GLUHU-E01) and insulin (#80-INSMS-E01) were measured by specific assay kits (both from ALPCO, Salem, NH, USA). Serum levels of free fatty acids were determined using a colorimetric assay kit (#MAK044, Sigma-Aldrich). Serum level of FGF21 was detected by a FGF21 ELSIA kit (#212160, Abcam). Tail-tip blood and medium βOHB levels were measured using the CareSens Dual monitoring system (i-SENS, Seoul, Korea). Hepatic βOHB levels were measured with a βOHB colorimetric assay kit (#700190, Cayman Chemical, Ann Arbor, MI, USA). Liver and serum TG levels were determined using a TG assay kit (#AM157S-K, Asan Pharmaceutical, Seoul, Korea). To quantify liver TG, liver tissues (100 mg) were homogenized and extracted in a 1-ml mixture of 5% Triton X-100 (Sigma-Aldrich) in PBS. Serum levels of ALT and AST were analyzed using specific kits (#K753-100 and K752-100, Biovision, Milpitas, CA, USA).