Food intake measurement

Male C57black6/J mice aged 8–10 weeks were maintained in a 12/12 hrs light/dark cycle. For ICV injection in mice, a gauge guide cannula (type; OM205-113, diameter; 0.2 mm; Unique medical, Tokyo, Japan) was placed stereotaxically into 3V, at 1.5 mm caudal to the bregma in the midline and 5 mm below the surface of the skull, under anesthesia with tribromoethanol (200 mg/kg). Mouse was allowed to recover from the operation for 1 week while they were habituated to handling. On the day of experiments, food was removed form cages at 16:00. At 19:30, mouse received an injection of 150 μg mouse purified MMW-adiponectin⁴⁶ without or with 100 μg glucose dissolved in vehicle (sterile saline; 0.9% NaCl). The food was returned to cages, and food intake at 0.5, 1, 2, 3, 6, 16 hr were measured. At the end of the experiments, sections of the hypothalamus were histologically examined to verify the position of the cannulas. The animal protocols for this study were approved by Jichi Medical University Institute of Animal Care and Use Committee., and all experiments were carried out in accordance with the approved protocols.