Beta hydroxybutyrate assay kit

Beta-hydroxybutyrate assay kit (Abcam, Cambridge, UK) and plasma glucose assay kit (BioVision Incorporated, CA, USA) were used and the analyses performed as described by the manufacturer.

Blood glucose was measured using a glucometer (Glutest PRO R; Sanwa Kagaku Kenkyusho Co., Ltd., Aichi, Japan). Serum non-esterified fatty acid (NEFA), triglyceride (TG), and 3-hydroxybutyrate levels were determined with NEFA C-Test Wako (Wako Pure Chemical Industries, Ltd., Osaka, Japan), TG E-Test Wako (Wako Pure Chemical Industries, Ltd.), and beta Hydroxybutyrate Assay Kit (Abcam plc, Cambridge, UK), respectively. Serum alanine aminotransferase (ALT) levels were measured using Fuji Dry-chem 7000V (Fujifilm corporation, Tokyo, Japan). Urine glucose levels were analyzed with enzymatic assays in a laboratory of Oriental Yeast Co., Ltd. (Tokyo, Japan). Serum insulin and plasma glucagon levels were measured with an enzyme-linked immunosorbent assay kit (Morinaga Institute of Biological Science, Inc., Kanagawa, Japan) and Mercodia Glucagon ELISA (Mercodia AB, Uppsala, Sweden), respectively. Total lipids were extracted from the liver with chloroform and methanol (2:1 v/v), and liver TG content was assayed with TG E-Test Wako.

Blood was obtained from the retro-orbital sinus after 4 h fasting. Plasma leptin concentrations were measured by ELISA kit for rat leptin (Millipore, St. Charles, MO). Plasma glucose concentrations were measured by a glucose assay kit (Wako Pure Chemical Industries, Osaka, Japan). Plasma insulin concentrations were measured by an insulin-ELISA kit (Morinaga Institute of Biological Science, Yokohama, Japan). Plasma triglyceride (TG), nonesterified fatty acid (NEFA), and total cholesterol concentrations were measured by enzymatic kits (Triglyceride E-test Wako, NEFA C-test Wako, and Cholesterol E-test Wako, respectively; Wako Pure Chemical Industries). For measurement of plasma βHB concentrations, blood was obtained 4 h after water or BD administration. Plasma βHB concentrations were measured by beta Hydroxybutyrate Assay Kit (abcam, Cambridge, MA).