Humalog insulin

Mice were fasted for 5 h, then injected i. p. with Humalog insulin (Lilly; 0.75 U/kg body weight), followed 10 min later with an injection of 2-deoxyglucose (Sigma–Aldrich; 32.8 μg/g body weight). Tissues were collected 20 min after administration of 2-deoxyglucose. Tissues were lysed in 10 mM Tris–HCL, pH 8.0, by boiling for 15 min 2-deoxyglucose-6-phosphate (2DGP) was measured using a Glucose Uptake-Glo Assay Kit (Promega) following the manufacturer's instructions.

For the glucose tolerance test, mice were intraperitoneally injected with glucose at 2 g per kg body weight after 12 h of fasting. For the insulin tolerance test, mice were intraperitoneally injected with insulin at 1 IU per kg body weight (Eli Lilly and Co., Humalog Insulin) after 6 h of fasting. In all tests, tail blood glucose levels were measured with a glucometer (TheraSense Freestyle) at the indicated times (0, 15, 30, 60, 90, and 120 min) after injection.

Mice were fasted for 12 h to analyze the glucose tolerance test after an intraperitoneal injection of 2 g/kg glucose. For the insulin tolerance test, mice were intraperitoneally injected with 1 IU/kg insulin (Eli Lilly and Co., Humalog Insulin) after 6 h fasting. Blood samples from the tail veil were measured with a glucometer (TheraSense Freestyle, CA, USA) at the indicated times (0, 30, 60, 90, 120, and 180 min) after injection.

Prior to collection of fasted blood glucose and plasma for insulin measurements mice were fasted for at least seven hours. Blood glucose was measured by tail nick and using Bayer Counter Next EZ Glucose Meter. After glucose measurements blood was obtained via retro-orbital bleeding into EDTA treated tubes (ThermoFisher) by centrifugation for 15 minutes in 4 C at 2,000 x g. Insulin was measured using Insulin Mouse ELISA kit (ThermoFisher). HOMA-IR was calculated by multiplying fasted plasma insulin and fasted blood glucose and dividing the product by 22.5, the inverse of this result was taken to represent Insulin Sensitivity [76 (link)]. For GTT, ITT, or PTT, following fasting mice were i.p. challenged with 1 mg/kg glucose, or 1 U/kg Humalog insulin (Eli Lilly), or 2.5 g/kg sodium pyruvate in PBS respectively and blood glucose measured as described above.

Serum levels of albumin, creatinine, bilirubin, urea, alanine aminotransferase (ALT), cholesterol, low density lipoprotein (LDL) and high density lipoprotein (HDL) were determined using ABX Pentra (Horiba Medical). To perform GTT and ITT, mice were i.p. injected with 2 g of glucose/kg of body weight and 0.75 U insulin/kg of body weight (Eli Lilly; Humalog Insulin), respectively. In the case of GTT mice were previously fasted for 16 h. For ITT mice were not fasted and basal levels of glucose were between 80–100 mg/dL.