Metabolic and Liver Assessments in Mice

The day before sacrificing, the mice were fasted overnight and underwent a glucose tolerance test (GTT) and insulin tolerance test (ITT). Glucose solution (2 g/kg) and insulin (0.75 U/kg) were administered by intraperitoneal injection. Tail blood was collected at 0, 15, 30, 60 and 120 min after glucose administration, and glucose concentrations were measured using a glucometer and test strips (ACCU-CHEK Performa; Roche, United States). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were determined by using standard kits purchased from Cusabio (Wuhan, China). Hepatic, adipose or serum lipids including triglyceride (TG), free fatty acid (FFA) and cholesterol, were determined by commercial lipids assay kits (abcam, Cambridge, MA, United States) according to the manufacturer’s protocols. Four-hydroxynonenal (4-HNE) level in the liver was measured by colorimetric assay using an ELISA kit (Cusabio, China). Formalin-fixed mouse liver and epididymal white adipose tissues were embedded in paraffin and sliced at 4.5 μm. Hematoxylin and eosin (H&E) staining was used for tissue morphology assessment. Pathology was scored in a blinded manner. Scoring ranges were as follows: Degree of steatosis (0–3), lobular inflammation (0–3), hepatocyte ballooning (0–2). In addition, the NAFLD activity score (NAS) was scored according to the non-alcoholic steatohepatitis (NASH) clinical research network scoring system (Kleiner et al., 2005 (link)). Hepatic lipids were visualized by Oil Red O (ORO) stain (Servicebio, Wuhan, China). Image Pro plus 6.0 was used for ORO staining quantification. Neutrophil infiltration in the liver was assessed by a naphthol AS-D chloroacetate (CAE) kit (Sigma-Aldrich, United States) according to the manufacturer’s directions. Apoptosis was detected by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL; EMD Millipore, Billerica, MA). CAE and TUNEL positive cells were counted by Image J software (NIH, United States) and expressed as the ratio of positive cells per 1,000 hepatocytes.