Insulin Tolerance Test

We followed a published and standard protocol to conduct an insulin tolerance test [ITT; (31, 32)]. Briefly, immediately before conducting the ITT, the mice were fasted for 4 h with free access to drinking water. During the test period, mice were kept in a warm environment to ensure adequate peripheral blood flow. Baseline (0 min) blood samples were collected from the dorsal pedal vein by gently pricking the conscious mice with a sterile needle. Using the droplets of blood, glucose levels were measured on a Contour Glucometer. After that mice were given intraperitoneal injections of human insulin (Humalin® R-100, Eli Lilly, Indianapolis, IN, United States) at a dose of 1 U/kg body weight using an insulin syringe. Blood samples were collected at 20, 40, 60, 80, 100, and 120 min after insulin injection for glucose determination, using the same procedure described for the 0 min sampling.