2.2. Animals and experimental design.
The study was performed in agreement with the ‘Guide of the Care and Use of Laboratory animals’ as promulgated by the National Institute of Health and all procedures were approved by the Ethics Committee of Laboratory Animal of the University of Granada (Spain) (Ref. No. 28/03/2016/030). Eight-week-old male C57BL/6 mice obtained from Janvier labs (St. Berthevin, Cedex, France) were housed in makrolon cages, maintained under controlled light-dark cycle (12 h light/dark cycle), temperature and relative humidity (22 ± 1ºC, 55 ± 10%) and provided with a free access to tap water. Mice were fed with either a standard chow diet (13% calories from fat, 20% calories from protein and 67 % calories from carbohydrate; Global diet 2014; Harlan Laboratories, Barcelona, Spain) or a high fat diet (HFD) (59% calories from fat, 13% calories from protein and 28% calories from carbohydrate; Purified diet 230 HF; Scientific Animal Food & Engineering, Augy, France). They were randomly divided in seven groups (n=8): control diet, HFD and five HFD-treated groups. Mice were daily treated by oral gavage with melatonin (15mg/kg), agomelatine (10, 25 and 50 mg/kg) or metformin (250 mg/kg) dissolved in water for five weeks (more detail in supplementary). Animal body weight and food and water intake were regularly controlled, and feed efficiency was calculated as the ratio of body weight gain (g) to caloric intake (kcal). One week before the sacrifice, a glucose tolerance test was performed. At the end of the experiment, mice were fasted overnight, a blood sample was collected by cardiac puncture under isoflurane anaesthesia and then sacrificed by cervical dislocation (Figure 1).