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.
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.
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