Triglyceride assay kit

Mice serum samples were collected from eyeballs and clotted for 30 min at room temperature before centrifuging at 3000 g for 10 min. Serum levels of insulin and leptin were determined using enzyme immunoassay kits from Millipore and R&D Systems, respectively, according to the manufacturers’ instructions. Serum-free fatty acid levels were determined by the ACS-ACOD method (Wako, Cat^# 294–63601). Liver triglyceride was extracted by trichloromethane, and measured using a Triglyceride Assay Kit by the GPODAOS method as suggested by the manufacturer (Wako, Cat^# 290–63701).

Around 75 mg of liver tissue (exact weight was recorded and used to normalize lipid amount) was homogenized with Homogenizer (Thermo Fisher Scientific) in 1 mL 50 mM NaCl on ice, following the addition of 5 mL of chloroform/methanol mixture (chloroform : methanol = 2:1). The homogenized tissue solution was vortexed for 10 seconds and centrifuged at 16,000 × g for 10 minutes. The aqueous phase was carefully removed, and the left oleic phase was mixed with 1.5 mL of methanol. The resulting mixture was vortexed for 10 seconds and centrifuged at 16,000 X g for 10 minutes. The lipid extract (top phase) was carefully moved to a new tube and mixed with 75 μL of 10% Triton X100 in acetone, and dried in fume hood. The resulting pellet was used for cholesterol or triglyceride determination with cholesterol assay kit (Wako, Richmond, VA) or Triglyceride assay kit (Wako, Richmond, VA) according to manufacture instructions.