Biochemical analysis
Blood samples were collected at sacrifice from the abdominal vein by using disposable syringe and transferred to serum separation tubes (SST) and centrifuged at 3,000 rpm for 3 min using a centrifuge to separate serum from blood. The following parameters were measured in the serum using a blood-chemical analyzer (7180, HITACHI, Japan): alanine aminotransferase (ALT; JSCC (UV Kinetic)), aspartate aminotransferase (AST; JSCC (UV Kinetic)), total cholesterol (T-chol; cholesterol oxidase-HMMPS), triglycerides (TG; GPO-HMMPS glycerol blanking), LDL-cholesterol (selective protection enzymatic), HDL-cholesterol (direct), and glucose (Glu; Hexokinase-G6PDH). Serum insulin level (ng/mL) was measured by using Mouse Insulin ELISA kit, TBM TMB type (Lot No.: MIT-696, Shibayagi Co., Ltd. Japan). Serum leptin level (pg/mL) was measured using Mouse Leptin ELISA kit, TMBBM type (Lot No.: MLP-817, Shibayagi Co., Ltd. Japan). HOMA-IR was calculated as FIRI (fasting plasma insulin level) × FPG (fasting plasma glucose level) ÷ 22.5.