Nefa hr 2 r1

For metabolite levels assessment, liver samples were homogenized and deproteinized by ultrasonic disruption in 7.5 vols of ice-cooled 0.6 M perchloric acid and neutralized with 1 M potassium bicarbonate. The homogenate was centrifuged at 10,000 g for 4.5 min at 4 °C, and the supernatant was used to assess tissue metabolite levels. Similarly, plasma samples were also deproteinized (0.6 M perchloric acid), neutralized (1 M potassium bicarbonate), and supernatant was collected after being centrifuged at 13,500 g for 4.5 min at 4 °C. Glucose, lactate, triglycerides (TAG), total cholesterol, and non-esterified fatty acids (NEFA) levels were determined enzymatically using commercial kits (1001190, 1001330, 1001313 and 1001090, Spinreact, Spain, and 434–91795 NEFA-HR (2) R1, and 436–91995 NEFA-HR (2) R2, Wako Chemicals, Germany, respectively), adapting manufacturer’s instructions to a microplate format. Total α-amino acid levels were assessed through the colorimetric ninhydrin method (Moore, 1968 (link)), with alanine as standard. Liver glycogen level was assessed using the Keppler and Decker (1974) method.