Glucose-stimulated insulin secretion (GSIS) assay

The cells were seeded into 24-well plates (10, 000 cells per well) and incubated with 0.5 mM PA in the presence or absence of different doses of PCs for 24 h. Then the cells were washed and preincubated in Krebs buffer (KRB: 135 mM NaCl, 3.6 mM KCl, 0.5 mM NaH₂PO₄, 0.5 mM MgSO₄, 1.5 mM CaCl₂, 2 mM NaHCO₃, 10 mM HEPES, and 0.1% w/v BSA, pH 7.4) for 1 h at 37 °C. Then, the cells were incubated in KRB containing 5 mM glucose and, subsequently, 25 mM glucose for 1 h at 37 °C. The insulin level in the incubation media was measured using an insulin ELISA kit (Mercodia, Uppsala, Sweden) according to the manufacturer’s instructions. Cells in the plate were lysed by RIPA buffer, and the protein concentration of the lysates was detected using a BCA assay kit. The insulin secretion was normalized by cellular protein content.