Islet isolation and insulin secretion assay

Islets were isolated by perfusing the pancreatic duct with Collagenase P (Roche, Basel, Switzerland), followed by digestion for 12 min at 37 °C. Islets were picked manually and incubated in RPMI 1640 media overnight at 37 °C. For the insulin secretion assay, islets were incubated with HKRB buffer (135 mM NaCl, 3.6 mM KCl, 2 mM NaHCO₃, 0.5 mM NaH₂PO₄, 0.5 mM MgCl₂, 1.5 mM CaCl₂, HEPES 10 mM, BSA 0.5% (w/v)) containing 3 mM glucose for 30 min and then HKRB buffer containing glucose, KCl or glibenclamide (Sigma-Aldrich, Darmstadt, Germany) was subsequently added. After centrifugation, supernatants were collected and insulin concentrations were measured using a Mouse Insulin ELISA Kit (Morinaga Institute of Biological Science, Yokohama, Japan). Pellets were also washed with phosphate buffered saline (PBS), solubilized with lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) and subjected to insulin measurement. In the insulin secretion assay of 4-PBA treated islets, islets were incubated in RPMI media with 1 mM 4-PBA (Sigma-Aldrich) or with dimethyl sulfoxide (Wako, Osaka, Japan) overnight prior to glucose stimulation.