Insulin secretion assay

For the glucose-stimulated insulin secretion (GSIS) and sulfonylurea-stimulated insulin secretion (SU-SIS) assays, MIN6 cells were plated in 48-well dishes (1.5 × 10⁴ cells/well) and exposed to PA and OA for 0, 24, or 48 h and washed out for another 24 h. After washing with Krebs–Ringer bicarbonate HEPES buffer (KRBH 135 mmol/L, NaCl 3.6 mmol/L, KCl 0.5 mmol/L, MgSO₄ 0.5 mmol/L, NaH₂PO₄ 1.5 mmol/L, CaCl₂ 2 mmol/L, NaHCO₃ 10 mmol/L, and HEPES and 0.2% BSA adjusted to pH7.4), cells were preincubated for 1 h in glucose-free KRBH without test agent for batch incubation. Cells were then stimulated with basal (2 mmol/L) and high glucose levels (20 mmol/L) and 100 μmol/L glibenclamide and glimepiride in 0.2 mL KRBH buffer for 2 h. The insulin concentrations of the supernatants and extracts were determined using an Insulin ELISA Kit (EZassay MS300, Shenzhen, China). Insulin secretion and content were normalized to total protein content.