Glucose and insulin tolerance tests

Intraperitoneal glucose (IPGTT) and insulin (ITT) tolerance tests IPGTT were performed at week 36 (at the end of the study period). For the IPGTT test, overnight fasted mice were injected intraperitoneally with glucose (2 g/kg body weight) and blood samples were collected at 0, 15, 30, 60 and 120 min. To measure glucose-stimulated insulin secretion, at same time points, blood samples were collected, and plasma was isolated and stored at −80 °C for the insulin analysis. For the ITT test, after 6 h of fasting, mice were injected intraperitoneally with 0.5 units/kg body weight human insulin (Humalin; Eli Lilly), and blood samples were collected at 0, 15, 30, 60 and 120 min. Blood glucose was measured using an automatic glucometer (Accu-Chek Aviva, Roche, Indianapolis, IN, USA). Insulin was assayed by ELISA using the Mercodia kit. Values for the IPGTT and ITT tests were calculated by estimation of the area under the curve (AUC) using the trapezoid rule³². Insulin resistance was calculated using the homeostasis model assessment (HOMA)³³. The constant rate for glucose disappearance (K_ITT) was calculated from the slope of the regression line obtained with log-transformed glucose values between 0 and 60 min after the administration of insulin (linear phase of glucose decay)³⁴.