Enzymatic assay kit

After a 4 h-fast during the daylight cycle, mice were euthanized. Blood was collected and plasma prepared. Tissues were harvested and snap-frozen in liquid nitrogen for analysis of hepatic lipids as described previously^{68 (link),69 (link)}. Plasma concentrations of total cholesterol (TC), free cholesterol (FC), and total phospholipids (PLs) were measured by using the TC (Cat. #: 999-02601), FC (Cat. #: 993-02501), and PL (Cat. #: 997-01801) enzymatic assay kits from Wako Diagnostics, Richmond, VA. Plasma concentrations of total TGs were determined by using the enzymatic assay kit from Sigma (Cat. #: F6428 and T2449). Plasma concentrations of nonesterified fatty acids (NEFAs) were determined by using HR Series NEFA-HR Reagents (Cat. #: 999-34691, 995-34791, 991-3489, 993-35191 and 276-76491, Wako Diagnostics, Richmond, VA) following the manufacturer’s instruction. Plasma activities of alanine transaminase (ALT) and aspartate transaminase (AST) were measured by using the commercial kits (Cat. #: A7526, and A7561, Point Scientific, Inc., Canton, MI). For analysis of cellular lipids, the cell pellet from a 10-mm dish with ~80% cell confluence was used. Lipids were extracted twice with two volumes of chloroform:methanol (2:1; v/v) mixture, followed by assays of lipids as described previously^{70 (link)}.

The activity of aconitase in the lung homogenate was measured using a commercially available enzymatic assay kit (Sigma Aldrich, St louis, MO).

Mice were fasted for 4 hr and received an intraperitoneal injection of insulin (1 U/kg body weight) or D-glucose (2 g/kg body weight). For insulin tolerance tests, blood samples (5 μl) were collected from the tail vein before and at 15, 30, 45, and 60 min after the bolus insulin injection. Similarly, for glucose tolerance tests, blood samples were collected from the tail vein before and at 30, 60, 90 and 120 min after the glucose bolus injection42 (link)43 (link). The levels of plasma glucose were measured using an enzymatic assay kit (Sigma, St. Louis, MO).

Plasma TNF‐α and triglycerides concentrations were determined using commercially available kits (ThermoFisher, Courtaboeuf, France). Plasma creatinine was determined spectrophotometrically using an enzymatic assay kit (Sigma‐Aldrich, L'Isle d'Abeau, France). Several parameters of the oxidative stress (protein thiol residues, total antioxidant defenses (FRAP), glutathione peroxidase activity, GSH and GSSG) were determined as already described previously (Mourmoura et al. 2013).

Serum levels of D-βHB were determined by using an enzymatic assay kit produced by Sigma-Aldrich (St. Louis, USA). The kit is designed to produce a compound whose colorimetric intensity, determined at a wavelength of 450 nm, is proportional to the concentration of D-βHB. Glucose and mineral (K⁺, Na⁺, and Ca²⁺) content in serum were determined using the analyzer Cobas 8000 manufactured by Roche Diagnostics (Mannheim, Germany) and carried out in the clinical laboratory of the university hospital in Muenster.