Active glp 1

GLP-1 and PYY secretion from intact colon was investigated in hFFA2-DREADD-HA, CRE-MINUS, and wild-type mice as previously described in detail (Bolognini et al., 2019 (link)). The entire colon was removed and placed in an purpose built organ bath (3 ml) perfused with carbogenated (95% O₂–5% CO₂) Krebs solution (composition, mM: NaCl 120; KCl 5.9; NaH₂PO₄ 1.2; MgSO₄ 1.2; NaHCO₃ 15.4; CaCl₂ 2.5; glucose 11.5) (34°C). The colon was attached from either end to an inlet and outlet port, allowing intraluminal perfusion (10 ml/hr) of vehicle or test ligands using a syringe pump (Sigma-Aldrich).
GLP-1 and PYY secretion was assessed by perfusing either Krebs or MOMBA (0.1 mM) through the lumen. To further assess the specificity of MOMBA, MOMBA was also applied in the presence of the human FFA2-specific antagonist CATPB (10 μM). In this case, CATPB was also applied 15 min before the coapplication of MOMBA (0.1 mM) and CATPB (10 μM). During intraluminal perfusion, supernatants were collected every 5 min. Total PYY (Phoenix) and active GLP-1 (Millipore) concentration was measured by ELISA.

Plasma was used for biochemical measurements with reagents from Randox Laboratories Ltd. (County Antrim, UK). Plasma Glucose was measured by Glucose Oxidase method, Total Cholesterol by Cholesterol Oxidase method, Total Triglycerides by Lipase/GPO-PAP method. Glycated hemoglobin (HbA1c) was measured by HPLC (D10 Hemoglobin analyzer, Bio-Rad, Hercules, CA, USA). Plasma Leptin (RayBiotech, Norcross, GA, USA), Insulin (Merck Millipore, MA, USA), DPP4 (R&D Systems, MN, USA), and GLP-1(Active) (Merck Millipore, MA, USA) levels were measured by ELISA. Homeostatic model assessment (HOMA2) designed by Diabetes Trials Unit, The Oxford Center for Diabetes, Endocrinology, and Metabolism was used to estimate Insulin resistance (HOMA2 IR) from all fasting venous samples.