Colorimetric assay

After 24 or 32 weeks on the respective diets, the fasting serum glucose levels were determined by colorimetric assay (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and insulin levels were assessed using a commercial ELISA kit (Mercodia AB, Uppsala, Sweden), according to the manufacturer’s instructions. The HOMA-IR index was calculated using the following formula: HOMA-IR = [fasting glucose levels (mmol/L)] × [fasting serum insulin (mU/L)]/22.5. The glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed following intragastric glucose administration and intraperitoneal insulin injection, respectively, after overnight starvation of the rats. Blood samples were collected from the tail vein at 0, 15, 30, 60 and 120 min, and the glucose levels were measured with a Glucose Meter (Roche Diagnostics, Shanghai, China).

After the interventions were carried out, all rats were subjected to OGTT. During OGTT, the rats were fasted for 12 h before 2 g/kg of glucose was administrated. Tail vein blood samples were collected from each rat at 0, 60, and 120 min after the intragastric administration to detect the blood glucose levels using a glucose monitor (Roche, German).
After the experiment was completed, all animals were anesthetized by intraperitoneal injection of 50 mg/kg pentobarbital sodium. Abdominal aorta blood samples were then collected using heparin sodium anticoagulant tubes. Subsequently, the blood samples were centrifuged at 3000 rpm for 10 min to separate plasma. A part of the separated plasma was used to measure the levels of lipids, while the remaining part of the separated plasma was rapidly frozen under liquid nitrogen and stored at −80°C for proteomic analysis.
The concentration of plasma insulin was measured using a Rat/Mouse Insulin ELISA kit (Millipore, German). For the detection of triglyceride (TG) content and total cholesterol (T-CHO), a colorimetric assay (Jiancheng, Nanjing, China) was used. A double reagent direct method (Jiancheng, Nanjing, China) was used to detect the level of low-density lipoprotein (LDL).

At 4 weeks after BDL, under anesthesia, blood samples were collected from the abdominal aorta and centrifuged at 3000 rpm for 15 min to obtain serum. Serum concentrations of triglycerides (TG), total bile acids (TBA), alanine aminotransferase (ALT), and aspartate aminotransaminase (AST) were determined using a microplate (Reitman-Frankel colorimetric assay) according to the manufacturer’s protocol (Nanjing Jiancheng Corp, NanJing, China).