Insulin tolerance test (ITT)

Male Wistar rats (8–9 weeks old) (Charles River, France) were allowed free access to standard food and water ad libitum until experiments were performed. For experimental procedures rats were fasted for 5–6 h. Anesthesia was performed by intraperitoneal injection of sodium pentobarbital (60 mg/kg body weight) and the experiments were carried out after loss of corneal and pedal reflexes. The previously mentioned insulin stocks (4 μmol/l) were subcutaneously injected at 0.5 ml/kg body weight into the rats' hind back, and blood samples were collected from the tail at 0, 10, 20, 30, 40 and 60 min for measurement of serum glucose and insulin. Rats were sacrificed by terminal anesthesia with sodium pentobarbital.

This study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Andalusian Center of Molecular Biology and Regenerative Medicine (Protocol number: 06-10-14-138). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.