Glucose-stimulated insulin secretion

To examine in vivo insulin secretion, blood samples were collected at 0 and 30 min after glucose injection (2 g/kg body weight). After centrifugation at 3000 g for 20 min, serum was collected. INS-1 cells or isolated islets (8 islets/group) from each age group were plated on 24-well plates and incubated with HBSS for 2h followed by stimulation with 3 or 17 mM glucose, and insulin secretion into the media was measured [16]. The amount of insulin released into the media was quantified using an insulin EIA kit (Alpco Diagnostics, Windham, NH, USA) according to the manufacturer’s instructions and was normalized to the amount of protein.