Fasting Glucose and Insulin Testing in Mice

All experiments requiring fasting conditions were performed in the afternoon, 5–6 h following the removal of food at the beginning of the light-cycle for reasons outlined elsewhere^{70 (link)}. To ensure fasting conditions, mice were transferred to clean cages containing ISO cotton padding and clean cardboard enrichment tubes. Non-terminal blood was collected via tail snip. Glucose tolerance tests (GTT) were performed following the administration of a filtered dextrose (1 g/kg body mass) solution via IP injection^{71 (link)}. Insulin tolerance tests (ITT) were performed following the administration of a filtered insulin (0.75 mU/g body mass; Novolin-R, Novo Nordisk, Bagsvaerd, DK) solution via IP injection^{72 (link)}. Blood glucose was measured immediately pre-injection (time 0) and at 15, 30, 45, 60, 90, and 120 min post-injection during the GTT and ITT. The area under curve (AUC) for each animal during both the GTT and ITT were also calculated and presented as the average for each group. Blood glucose levels were determined using Accu-Chek Aviva Plus glucometers (Roche, Basel, CH). Fasting insulin levels from blood collected at baseline and during the terminal harvest were evaluated using a Mouse Ultrasensitive Insulin ELISA from Alpco (Salem, NH, USA).