Ek 001 21

Whole blood was collected into iced empty or heparinized tubes. Blood was centrifuged at 2000 g for 20 min at 4° C, and then plasma or serum was recentrifuged at 6000 g for 10 min at 4° C before being stored at –80° C for subsequent determination of ACTH in the plasma and corticosterone in the serum. ACTH or corticosterone concentrations of the mouse plasma or serum and Y-1 cell culture media were measured by commercially available ELISA kits (EK-001-21, Phoenix Pharmaceuticals, USA and Cayman, USA), as described by the manufacturer.

Plasma concentrations of adrenocorticotropic hormone (ACTH, EK-001-21, Phoenix Pharmaceuticals), corticosterone (YK240, Yanaihara Institute) and irisin (EK-067-16, Phoenix Pharmaceuticals) were measured by enzyme immunoassays using iMark Microplate Absorbance Reader (Bio-Rad), according to the manufacturer’s instructions. Plasma concentrations of insulin (MS303, Morinaga Institute of Biological Science), C-peptide (AKRCP-031, Shibayagi), FGF21 (MF2100, R&D Systems) and adiponectin (AKMAN-011, Shibayagi) were measured by ELISA using iMark Microplate Absorbance Reader, according to the manufacturer’s instructions. Plasma concentration of alanine (K652-100, BioVision), free fatty acids (K612-100, BioVision) and 3-hydroxybutyric acid (Ketone Test B ‘Sanwa’ Liquid, Sanwa Kagaku Kenkyusho) were measured by colorimetric assays using NanoDrop 2000c (Thermo Fisher Scientific Inc.), according to the manufacturer’s instructions.