A113 2 1

In vitro, HepG2 cells were seeded in six-well plates at a density of 5 × 10⁵ cells per well. The cells were incubated for 24 h, and then starved in serum-free MEM overnight at 37 °C. Then, cells were incubated in MEM with different treaments for 24 h. After that, the cells were rinsed with PBS and lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor (Roche, Basel, Switzerland) for 30 min. In vivo, all mice were anaesthetized and sacrificed by cervical dislocation at the ninth week of treatments. Blood samples were collected and centrifuged at 2000× g for 20 min to get the serum. Cellular and serum TG and TC were assayed using assay kits E1013 or E1015 (Applygen Technologies Inc.) according to the manufacturer’s recommended protocol. And, serum LDL-C and HDL-C were assayed using specific assay kits A113-2-1 and A112-2-1 according to the manufacturer’s instructions, respectively (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Blood glucose levels were measured using a portable glucometer (Johnson & Johnson, New Brunswick, NJ, USA). Serum amounts of fasting insulin and GHb were determined using a Mouse Insulin (INS) ELISA Kit OM451180 and a Mouse GHb ELISA Kit OM625774 from Omnimabs (Omnimabs, Alhambra, CA, USA), respectively.

Serum biochemical index triglyceride (TAG), total cholesterol (TCHO), high density lipid protein cholesterol (HDL-C), and low-density lipid protein cholesterol (LDL-C) were measured according to the relevant toolkits (A110-2-1, A111-2-1, A112-2-1, and A113-2-1, Nanjing Jiancheng Bioengineering Ltd., China) according to specification.

Blood samples were collected and left for 2–3 h at room temperature. The serum was centrifuged at 3000 rpm for 15 min, divided, and stored at −80 °C. TC, TG, LDL-C, and HDL-C levels in serum were determined using commercially available assay kits (cat: F002-1-1, F001-1-1, A113-2-1, and A112-2-1, Nanjing Jiancheng Bioengineering Institute, China).