Glucose and Insulin Tolerance Assays

Mice were fasted for 6 h before intraperitoneal (i.p.) injection of a 10% d-glucose solution (1.0 g/kg body weight)^{38 (link),46 (link)} at age indicated in the main text. Blood samples were obtained from the tail tip at the indicated times, and glucose levels were measured using a glucometer (AccuCheck II; Roche, New South Wales, Castle Hill, Australia). Serum from mice administrated with glucose was collected and stored for subsequent insulin assay using a Rat/Mouse insulin RIA kits (Linco Research, St Charles, MO, USA). Serum insulin levels at fed and fasted states of these mice were also determined using the same insulin RIA kits following the manufacturers’ instructions. ITT was carried out to determine insulin-induced hypoglycemia in mice at ages indicated in the main text. Briefly, the mice were injected with insulin (1 IU/kg body weight) intraperitoneally to induce hypoglycemia after being fasted for 6 h, then blood samples were collected from the tail tip at the indicated times, glucose levels were measured using a glucometer (Roche) as shown in the results.