Plasma Hormone and Amino Acid Analysis

Blood was collected from the tip of the tails, and blood glucose levels were measured with an Antsense Duo Small Electrode Glucose Analyzer (Horiba, Kyoto, Japan). Blood samples were centrifuged (13,500 rpm, 10 min, 4 °C) twice, and the collected plasma samples were stored at −80 °C until the biochemical analyses. Plasma hormone levels were measured using the following assays: insulin, Mouse/Rat Insulin ELISA Kit (Morinaga Institute of Biological Science, Kanagawa, Japan); FGF21, Mouse and Rat FGF21 ELISA Kit (BioVendor Inc., Brno, Czech Republic); GCG, Glucagon ELISA Kit (Mercodia, Uppsala, Sweden); active GLP-1, GLP-1 Active Form Assay Kit (IBL, Gunma, Japan); GIP, GIP (total) ELISA kit (Millipore, Billerica, MA, USA); and corticosterone, Corticosterone Enzyme Immunoassay Kit (Arbor Assays, Ann Arbor, MI, USA). Plasma amino acid concentrations were measured by standard assays at SRL, Tokyo, Japan [21 (link)].