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).