The female, 6–8 weeks mice (5 mice/group by randomization) were used to induce obesity and non-alcoholic fatty liver disease by feeding a high fat diet (HFD). The mice in control groups were fed control diet (CD). All mice were permitted consumption of tap water and HFD or CD ad libitum. Mouse body weight and diet consumption were measured weekly. In HFD, fat calories are 60%. HFD and CD contain the same amount (20.5%) of protein. The mice were fasted overnight (15 h) and then sacrificed. Blood was collected and serum levels of triglyceride, free fatty acids, β-hydroxyl butyrate, FGF21, insulin, adiponectin, leptin, and CTRP3 were measured using commercially available kits (supplemental Table 2). The livers were rapidly excised into fragments and washed with cold saline. Liver tissues from same lobes of different mice were put in neutral Formalin solution for paraffin bedding. The other liver tissue aliquots were stored at −80°C for future assays. Liver homogenates were prepared in ice-cold 0.15 M potassium chloride (KCL) and liver triglyceride content was measured using the same assay kit as serum triglyceride.
