Nefa colorimetric assay kit

Serum levels of TC, TG, HDL-C, LDL-C, nonesterified fatty acids (NEFA), alanine aminotransferase (ALT), and aspartic acid transaminase (AST) were measured following the instructions of the respective test kits (NEFA Colorimetric Assay Kit, Elabscience, Wuhan, China; the serum TC, TG, HDL-C, LDL-C, ALT and AST biochemical analysis kits, Rayto, Shenzhen, China). Briefly, all the serum samples were thawed and subjected to centrifugation for 10 min at 4 °C and 3000 rpm. A 15-µL aliquot of each serum sample was added to a Chemray800 Automatic Biochemistry Analyzer to measure the lipid profile and liver function.

Plasma levels of insulin, glucose, triglycerides, cholesterol, and non-esterified fatty acid (NEFA) were measured with the following commercial kits: ultrasensitive insulin ELISA kit (Mercodia, Uppsala, Sweden), glucose liquid (Química Analítica Aplicada SA, Tarragona, Spain), triglyceride colorimetric assay kit (Elabscience, Houston, TX, USA), cholesterol liquid kit (Química Analítica Aplicada SA, Spain), and NEFA colorimetric assay kit (Elabscience, USA), respectively. The HOMA-IR index was calculated as fasting plasma insulin (mU/L) × fasting plasma glucose (mmol/L)/22.5.
The citrate synthase activity was measured in the BAT using a colorimetric assay kit (BioVision, Milpitas, CA, USA). Hepatic glycogen was quantified according to the manufacturer’s instruction of a commercial kit (Sigma-Aldrich, USA). In addition, the lipid content was quantified in the liver after extraction with chloroform, as previously described [23 (link)]. Briefly, the tissues were homogenized in chloroform/methanol (2:1) solution. After 3 h of shaking, Milli-Q water was added, and the organic layer was separated by centrifugation (16,000× g, 20 min) and dried overnight. Triglyceride concentration was measured as described above.