Blood sampling and biochemical assays
CLOT
To measure serum blood markers such as insulin, triacylglycerol (TAG), and non-esterified fatty acids (NEFAs), samples were allowed to clot for 30 min at room temperature and then centrifuged at 3000 rpm for 10 min. The serum sample was dispensed into plain microtubes and stored at −80 °C until the assay was performed. For plasma glucose measurements, venous blood samples were collected in tubes containing sodium fluoride-ethylenediaminetetraacetic acid. For the measurement of plasma gastric inhibitory polypeptide (GIP) and glucagon-like peptide 1 (GLP-1), venous blood samples were collected in tubes containing a DPP-4 inhibitor and protease inhibitor cocktail (BD, Tokyo, Japan). Thereafter, both samples were immediately centrifuged and stored at −80 °C until analysis. Enzymatic colorimetric assays were used to measure the plasma concentrations of glucose (GLU-HK (M); Shino-test Corporation, Kanagawa, Japan), serum TAG (Pure Auto S TAG-N; Sekisui Medical Company Limited, Tokyo, Japan), and serum NEFAs (Wako Pure Chemical Industries Limited). Enzyme-linked immunosorbent assays were used to measure plasma concentrations of insulin (Mercodia Insulin ELISA; Mercodia AB, Uppsala, Sweden), active GIP, and active GLP-1 (Yanaihara Institute, Inc. ELISA; Yanaihara Institute, Inc. Shizuoka, Japan).