Insulin secretion assay

MIN6 cells were starved for 1 h in Krebs–Ringer buffer containing 2 mM glucose. After starvation, medium was exchanged for normal culture medium (glucose 23 mM) or glucose‐deprived medium (glucose 0.2 mM) to measure glucose‐stimulated insulin secretion. After 1 h, the medium was collected and the remaining cells were lysed with RIPA buffer to measure total protein content. Secreted insulin amount was measured with an ultra‐sensitive mouse insulin enzyme‐linked immunosorbent assay (ELISA) kit (Morinaga). Secreted insulin was normalized to total protein content and presented as a percentage of the non‐treated cells.