Mice were fasted overnight with free access to water. A fasting (time 0) blood sample (∼50 μl) was collected from the tail vein into a lithium-heparin-lined Microvette (Starstedt, Leicester, UK) before administering of glucose (3 g/kg body wt) via ip injection. Blood samples were then collected at 15 and 30 min after injection. Glycemia was also measured at these time points. Plasma was collected by centrifuging samples at 2,000 g for 10 min at 4°C. Plasma insulin was measured using an ultrasensitive mouse insulin ELISA (Crystal Chem, IL). For random-fed insulin/proinsulin ratio measurements, a blood sample was collected into a lithium-heparin-lined Microvette from the tail vein and the aorta immediately after culling via cervical dislocation. Samples were kept on ice at all times to prevent degradation of proinsulin, and plasma was collected as described above. Insulin was measured as described above, and proinsulin was measured using a Rat/Mouse Proinsulin ELISA (Mercodia, Uppsala, Sweden).