Glucose-Stimulated Insulin Secretion Assay

The islet function was evaluated using the previously reported GSIS method [33 (link)]. Following drug treatments, the islets were collected to perform the GSIS assay. In brief, 150 islet equivalents (IEQs) were initially incubated in KRBH buffer (composed of 129 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 5 mM NaHCO₃, 10 mM Hepes, and 0.1% BSA) supplemented with 2.8 mM glucose for 1 h. Subsequently, the islets were placed in a KRBH solution containing 2.8 mM glucose (low) and incubated at 37 °C for 1 h, and the supernatants were collected. Following this, the islets were washed thrice with KRBH and then incubated in KRBH solution spiked with 16.7 mM glucose (high) at 37 °C for 1 h, and the supernatants were collected again. The insulin content in the supernatant samples was measured using the Ultrasensitive Insulin ELISA kit (Mercodia, Uppsala, Sweden) following the manufacturer’s instructions. The stimulation index was calculated using the following equation: Stimulation index = C_{high glucose}/C_{low glucose.}C_high-glucose was the secreted insulin concentration of islets under high-glucose stimulation, and C_low-glucose was the secreted insulin concentration of islets under low-glucose stimulation.