Glp 1

For measurement of glucose only, overnight-fasted C57BL/6 mice in a normal light cycle were orally dosed with vehicle or compound, followed by an oral bolus of 3 g/kg glucose at 15 minutes post compound. At 0, 20, 40, 60, and 120 minutes post glucose dose, blood glucose was assessed via glucometers. Resulting raw data was used to generate AUCs. For measurement of hormones and glucose, overnight-fasted C57BL/6 mice acclimated for two weeks to a reverse light cycle were orally dosed with vehicle or compound, followed by an oral bolus of 3 g/kg glucose at 15 minutes post compound. At 0, 2, 5, 10, 20, 40, and 60 minutes post glucose dose, blood glucose was assessed via glucometers, followed immediately by collection of plasma for active GLP-1 (Mesoscale Discovery), insulin (Mesoscale Discovery), and total PYY. Glucose tolerance tests in diet-induced obese (DIO) C57BL/6 mice were performed in overnight-fasted animals dosed either acutely or after 2-week administration with vehicle or compound 18 at 60 mg/kg, followed 30 minutes post compound by an oral bolus of 2 g/kg glucose. At 0, 15, 30, 60, and 120 minutes post glucose dose, blood glucose was assessed via glucometers. Resulting raw data was used to generate AUCs.

Animals were euthanized following overnight fasting or a 16 h fasting and 4 h refeeding protocol to allow measurement of the metabolic status and the expression of hepatic gluconeogenic enzymes under fasting and fed conditions, respectively. Fasting blood glucose was measured before administration of Euthanyl. To avoid degradation of liver proteins, a lobe of the liver was harvested and snap-frozen immediately after animals achieved a surgical plane of anesthesia without damaging the common bile duct for pancreas perfusion. Blood samples were then collected via cardiac puncture for measurement of insulin, glucagon, and glucagon-like-peptide 1 (GLP-1). DPP-IV inhibitor (10 µmol/L, Millipore, Billerica, MA, USA) was added to blood collected for GLP-1 measurement. Insulin, glucagon, and GLP-1 concentrations were determined by ELISA (Insulin, Crystal Chem Inc., Downers Grove, IL, USA; glucagon, ALPCO Diagnostics Inc., Salem, NH; GLP-1, Meso Scale Discovery, Rockville, MD, USA). The pancreas tail was clamped and collected from fasted animals and fixed in 10% Neutral Buffered Formalin for immunohistochemical (IHC) examination. Islet isolation and calculation of body mass index (BMI), insulin sensitivity index (ISI), homeostatic model assessment of insulin resistance (HOMA-IR), and the homeostatic model assessment of β-cell function (HOMA-B) were as described previously [16 (link)].