Glucagon

Blood samples collected retro-orbitally were left to clot for 30 min at room temperature in non-coated polypropylene tubes. Samples were then centrifuged at 10,000× g for 5 min at 4 °C, and the resulting serum supernatant was aliquoted and stored at −80 °C. Hormone and lipid concentrations were assessed in these aliquots using leptin (Abcam, Cambridge, UK), glucagon, and ultra-sensitive mouse insulin (Crystal Chem, Elk Grove Village, IL, USA) ELISA kits, and NEFA-C (Wako Pure Chemical Industries, Osaka, Japan) and TAG (Abcam) colorimetric assay kits.

All blood samples were collected in EDTA-coated capillary microvette tubes, and plasma was isolated after centrifugation (13,523g, 10 minutes, 4°C). During metabolic tolerance tests, blood was taken via tail vein. For terminal studies, mice were sacrificed by CO₂ inhalation, and blood was obtained by CP. For measurement of plasma active GLP-1 (Meso Scale Diagnostics) and active GIP (Crystal Chem), blood was mixed with 10% TED (vol/vol) and plasma stored at –80°C until further analysis. Plasma insulin (Alpco Diagnostics) and glucagon (Crystal Chem) levels were determined as per manufacturer’s instructions. Analysis for plasma ALT, AST, alkaline phosphatase, TG, cholesterol, LDL, and HDL was performed by the Pathology core at The Centre for Phenogenomics. The Beckman Coulter AU480 clinical chemistry analyzer was used in combination with appropriate reagents (ALT, AST, TG, cholesterol, LDL, and HDL), calibrators (Beckman Coulter Lyophilized Chemistry Calibrator levels 1 and 2), and quality control materials (Bio-Rad Liquid Assayed Multiqual levels 1 and 3). DPP4 activity was assessed using fluorometric assay (substrate: 10 mM H-Gly-Pro-AMC HBr, Bachem catalog I-1225; standard: AMC, Bachem catalog Q-1025). DPP4 protein level was measured using DPPIV/CD26 DuoSet ELISA kit (DY954; R&D Systems, Bio-Techne) following the manufacturer’s instructions.