Novolin r

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).

For the insulin tolerance test (ITT), on the 11^th day of lactation, all pads were renewed to prevent mice from eating food that had spilled on the pad. After being fasted for 6 h, each dam was weighed and intraperitoneally injected with insulin (100 units/mL, Novolin R, Novo Nordisk, Denmark) at a dose of 0.75 IU/kg body weight. Before injection, the baseline blood glucose concentration was measured and recorded as the concentration at 0 min. After insulin injection, the blood glucose level in the tail vein was measured at 15, 30, 60, 90 and 120 min using a TIDEX glucose analyzer (Sankyo, Tokyo). The rate constant for each ITT (KITT) was calculated using the equation KITT (%/min) = 0.693 / t(1/2), where t(1/2) was calculated from the slope of the plasma glucose concentration 15–120 min after the administration of intravenous insulin.

After 12 and 24 weeks on different diets, mice were fasted overnight, and the blood glucose levels in the tail vein were measured using a handheld glucometer (OneTouch Ultra Easy, LifeScan). For the intraperitoneal glucose tolerance test (IPGTT), glucose concentrations were measured at 15, 30, 60, and 120 min after intraperitoneal injection of a glucose load (2 g/kg). For the intraperitoneal insulin tolerance test (IPITT), mice were fasted for 6 h, after which 0.75 U/kg body weight of insulin (Novolin R, Novo Nordisk, Copenhagen, Denmark) was injected intraperitoneally, and blood glucose levels were determined as above at 0, 15, 30, 60, and 120 min.