Q 1025

For metabolic studies, blood was taken via the tail vein into heparin-coated capillary tubes. For DPP-4 activity assays, blood was collected before and 30 minutes or 1 hour after olive oil gavage, and activity was assessed using a fluorometric assay (substrate: 10 mM H-Gly-Pro-AMC HBr [Bachem, I-1225], standard: AMC [Bachem, Q-1025]). For the measurement of active GLP-1 (Mesoscale, 150JVC-1), blood was taken before treatment (t –30 minutes) and 10 minutes after olive oil gavage and mixed with 10% TED (5000 KIU/mL Trasylol, 1.2/mL mg/mL EDTA, and 0.1 nmol/L Diprotin A). For TG measurements, blood was taken before and 1, 2, and 3 hours after olive oil gavage. Plasma was isolated and stored at –80°C until analysis.
At the end of the study, mice were sacrificed by CO₂ inhalation, blood was obtained by cardiac puncture, and plasma was stored at –80°C. Tissues for analysis were snap frozen in liquid nitrogen and stored at –80°C.

DPP4 activity levels were measured in 10 μl of mouse or human plasma or 25 μl of mouse tissue protein extract (undiluted for bone marrow and diluted 1/10 to 1/50 for other tissues) via fluorometric assay (AMC standard (Bachem #Q1025), H-Gly-Pro-AMC HBr substrate (Bachem #I-1225). DPP4 activity in tissue was normalised to total protein content.

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.