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.