Mice were culled by cervical dislocation. Islets were isolated after pancreatic distension with collagenase essentially as previously described (55). Islets were allowed to recover from digestion for 24 h (RC-fed mice) or 48 h (HFD-fed mice) in RPMI medium (GIBCO) supplemented with 10% (vol/vol) fetal bovine serum, 1% (wt/vol) penicillin, 1% (wt/vol) streptomycin, 11.1 mM glucose, and 2 mM l-glutamine. Insulin secretion was measured from duplicate batches of 10 islets incubated in 0.5 ml of Krebs-Ringer medium [130 mM NaCl, 3.6 mM KCl, 1.5 mM CaCl2, 0.5 mM MgSO4, 0.5 mM NaH2PO4, 2 mM NaHCO3, 10 mM HEPES, and 0.1% (wt/vol) BSA, pH 7.4] containing 3 or 16.7 mM glucose or 20 mM KCl and 3 mM glucose as indicated, and shaking at 37°C for 30 min. Total insulin was extracted into 0.5 ml of acidified ethanol [75% (vol/vol) ethanol, 1.5% (vol/vol) 1 M HCl and 0.1% (vol/vol) Triton X-100]. For continuous measurements of secretion, insulin samples from 50 perifused islets were collected using a custom-built device and a perifusion rate of 500 μl/min at 37°C as described previously (10). Secreted and total insulin concentrations were measured using a homogeneous time-resolved fluorescence-based (HTRF) insulin assay (CisBio, Codolet, France) in a PHERAstar reader (BMG Labtech), according to the manufacturer's instructions.