Insulin and Glucose Tolerance Tests in Rats

For ITT and GTT assays, after a 6 h fasting period, rats were injected intraperitoneally with a saline injection of insulin (0.75 U/kg BW of Novo Rapid, Novo Nordisk^®, Paço de Arcos, Portugal), or with a glucose solution (2.0 g/kg BW, Sigma-Aldrich, Merck, St. Louis, MO, USA, EUA), respectively. Blood samples were collected from the tail vein, being the first drop of blood discarded, and the second drop used to measure baseline glucose levels. Serum glucose measurements occurred at subsequent time points following glucose or insulin administration (ITT assay: 15, 30, 45, 60, 120 min; GTT assay: 15, 30, 60, 120 min) at selected time-points. The glucose levels were measured using a portable commercial glucometer kit (GlucoMen^® aero 2K, A. Menarini Diagnósticos, Paço de Arcos, Portugal). The area under curve (AUC) of ITT and of GTT curves were calculated using the trapezoidal method [18 (link),19 (link)].