Am157s k

After 9 weeks of experimental period, all the mice were subjected to fasting for 16 h and anesthetized with isoflurane. Before tissue harvest, blood was collected by cardiac puncture into serum separation tubes (BD Microtainer ® tube, 365967, BD Biosciences, Franklin Lakes, NJ, USA), clotted for 30 min at RT, and centrifuged at 21,206 × g at 4°C for 90 s to isolate the serum. The serum was stored at −70°C until further analysis. Assay kits for determining serum levels of glucose (AM201-K), triacylglycerol (TG; AM-157S-K), total cholesterol (TC; AM-157S-K), and high-density lipoprotein cholesterol (HDL-C; AM-202-K) were purchased from Asan Pharmaceutical Co. (Seoul, Korea). Serum levels were determined using each commercial kit according to the manufacturer’s instructions. Low-density lipoprotein cholesterol (LDL-C) levels were calculated using the Friedewald equation (22 (link)). After collecting the blood, epididymal adipose tissue was rapidly removed from the mice and weighed immediately. The tissues were snap-frozen in liquid nitrogen and stored at −70°C until analysis.

Triglycerides and total cholesterol were extracted from the liver tissue with chloroform-methanol (2:1, vol:vol), as previously described [21 (link)]. Briefly, chloroform-methanol was added to homogenized liver tissue, vortexed, and centrifuged; the lower phase was collected and evaporated at room temperature under a fume hood. The resulting semi-dried pellets were then dissolved in 1% Triton X-100 (VWR, USA). The hepatic and serum triglycerides (TG) and total cholesterol (TC) contents were quantified using commercial kits (Asan Pharmaceutical Co., Seoul, Korea, #AM157S-K, #AM202-K). Hepatic functional parameters such as alanine aminotransferase (ALT) and aspartate transaminase (AST) activities were measured using commercial kits (Asan Pharmaceutical Co., Seoul, Korea, #AM102-K, #AM103-K). Serum concentrations of TNF-α and IL-6 were determined using Quantikine ELISA kits (R&D Systems, Minneapolis, MN, USA, #MTA00B, #M6000B).