Insulin secretion assay

The INS-1 pancreatic β-cells in the subculture were collected and seeded at 2 × 10⁴/well in a 96-well plate and incubated for 24 h at 37℃ in a 5% CO₂ incubator to stabilize them. After cultivation, the medium was removed and the cells were washed twice with phosphate-buffered saline. To identify the mechanism of insulin secretion, INS-1 pancreatic β-cells were incubated in the presence or absence of POE under the following conditions, in the presence of various glucose concentrations: 1.1, 5.6, or 16.7 mM glucose. To measure the synergistic effect of POE on insulin secretion by INS-1 pancreatic β-cells, the cells were simultaneously incubated with L-alanine (10 mM, insulin secretion promoter), 3-isobutyl-1-methylxanthine (IBMX, 100 µM, β-cell K⁺_ATP channel blocker), or tolbutamide (200 µM, cAMP activator). To determine whether insulin secretion is dependent on the K⁺_ATP channel in INS-1 pancreatic β-cells, the cells were incubated with KCl (30 mM), verapamil (50 mM), or diazoxide (300 µM). The respective test compounds were added to Kreb's-Ringer bicarbonate (KRB buffer; 118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.18 mM KH₂PO₄, 1.18 mM MgSO₄, 25 mM NaHCO₃, 10 mM HEPES, 0.1% BSA, pH 7.4), and then cultured for 1 h. After incubation, the supernatant was collected and centrifuged to completely eliminate the cells, and the insulin secretory function was measured by means of an insulin ELISA kit (Lingo Research, MO, USA). For the experiments, all reagents were purchased from Sigma-Aldrich (Sigma, St. Louis, MO, USA).