Enzymatic kit

On day 56 of the study, rats were euthanized by cardiocentesis exsanguination under carbon dioxide anesthesia, followed by decapitation. The liver was collected from each rat, rinsed in 0.15 M sodium chloride, minced, frozen in liquid nitrogen, and stored at -80°C until analysis. The rat livers were analyzed for total lipid concentrations via a wet tissue lipid extraction procedure.^{10 (link)} The extracted liver lipids were analyzed for total triacylglycerol concentrations by utilizing a commercially available enzymatic kit (Pointe Scientific Inc., Canton, MI, USA), following solubilization with Triton X-100.^{11 (link)}The carcasses, minus skin, feet, head, tail, and entrails, were collected for compositional assays and stored at -20°C until analysis. Rat carcasses were weighed and homogenized. Carcasses were analyzed for dry matter by placing one gram of sample in an oven at 100°C for 24 hours. Protein content of the carcasses was assessed via a micro-Kjeldahl procedure to determine nitrogen content.¹² Total protein was calculated by using a factor of 6.25 times the nitrogen content. Carcass lipids were analyzed by using a 2:1 chloroform:methanol (v:v) modified Folch procedure.^{13 (link)}

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 (link)].