2.7. Insulin and glucagon secretion assay in isolated islets

Islets were isolated from C57Bl/6NTac mice (Taconic, Denmark) with liberase digestion and allowed to recover overnight in RPMI 1640 medium with 10% FBS, penicillin–streptomycin, and 11 mmol/L glucose. Islet perifusion method was described previously [17]. Human islets were acquired from the Islet Transplant Center (University of Chicago, Chicago, IL). After culturing overnight in RPMI 1640 medium with 11 mM glucose, isolated islets were pre-incubated at 37 °C in freshly prepared Krebs–Ringer Bicarbonate (KRB) buffer without glucose for 2 h followed by another 2 h of incubation in KRB buffer with 2 or 16 mM glucose. For glucagon secretion, batches of 10 isolated islets were equilibrated in KRB buffer at 2 mmol/L glucose for 45 min at 37 °C and then transitioned in medium with presence or absence of C22. Glucose was increased in a stepwise manner. Supernatants were collected and assayed for hormone content.