Exendin 9

Normal rat intestinal epithelial cell line IEC-6 were cultured in completed DMEM (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.) (v/v), 2 mM L-glutamine, 1% antibiotic (v/v) solution containing penicillin G (10,000 U/ml) and streptomycin (10,000 U/ml), 1.5 g/l NaHCO₃, 2 g/l HEPES and 0.01 mg/ml insulin, and maintained in a 37°C homothermal incubator with a 5% CO₂ atmosphere. IEC-6 cells were seeded into six-well plates and cultured for 48 h (to 80% confluence) in complete DMEM. Cells were then cultured in serum-free DMEM for 12 h, and then exposed to exendin-4 at different concentrations (0, 0.1, 1, 10 and 100 nM) for 12 h at 37°C to select the optimal concentration, as described previously (24 (link)). Subsequent experiments were conducted at 37°C for a number of time periods (0, 3, 6, 12 and 24 h). In certain experiments, the GLP-1R antagonist exendin-9 (10 µM; Sigma-Aldrich; Merck KGaA) was used 1 h prior to exendin-4 treatment at 37°C. The IEC-6 cells were pre-stimulated with H89 (10 µM; Beyotime Institute of Biotechnology) and SQ22536 (10 µM; Cayman Chemical Company) for 30 min at 37°C with or without exendin-4 treatment. Forskolin (5 µM; Cayman Chemical Company) was administrated for 12 h at 37°C with or without SQ22536 pre-treatment in IEC-6 cells. All experiments were performed in triplicate.