Measurement of Serum Insulin, Cytokine, and Lipid Levels of Mice

At 21 days after rFGF21 treatments, blood samples from the three groups of mice were collected via cardiac puncture. After clotting at room temperature for 1 h, blood samples were centrifuged at 3000 rpm/min for 15 min. Serum samples were transferred to new tubes and stored at −80 °C. Serum insulin, IL-1β, and TNFα concentrations were measured using a mouse insulin ELISA kit (Crystal Chem, USA), a mouse IL- 1β ELISA kit (Thermo Fisher Scientific, USA), and a mouse TNFα ELISA kit (Life Technologies, USA), respectively, according to the manufacturers’ protocols. Serum total cholesterol (TC), triacylglycerol (TG), and high-density lipoprotein (HDL) concentrations were measured using an enzymatic kit (Pointe Scientific, USA) according to the manufacturers’ protocols. Serum low-density lipoprotein (LDL) concentration was calculated using the formula LDL-c = TC-c − HDLc − (TG-c/5) as previously described [45].