Reagents and materials
Long-acting glucagon-like peptide-1 analogue polyethylene glycol losenatide (PEX-168) was provided by Hausen Pharmaceutical Co., Ltd (Jiangsu,China), lot number: H20190024, specification: 0.5 ML (0.1 mg) per bottle, stored at 4~8℃, Elisa kit was purchased from Liko Biotechnology Co., Ltd (Nanjing,China), precision electronic balance was purchased from Shuangjie Electronics Co., Ltd (Jiangsu,China). Automatic biochemical detector (Hitachi, Japan). Glucometer (Roche, Germany).
The basic diet and high-fat diet were purchased from Yangzhou University College of Veterinary Medicine (Jiangsu,China) and Medison Biomedical Co., Ltd (Jiangsu,China) respectively. The basic diet was composed of 22.8% protein, 13.8% fat, and 63.4% carbohydrate, the high-fat diet was composed of 26.2% protein, 34.9% fat, and 26.3% carbohydrate. The caloric density was 3656 kcal/kg for the basic diet and 5240 kcal/kg for the high-fat diet.
Animals
Animal model and test methods
Thirty six-week-old SPF-grade male C57BL/6 mice were purchased from the College of Agriculture, Yangzhou University, and housed in cages at room temperature between 18-24°C. They were first fed normal chow for one week for acclimation and exposed to light for 12 H daily, with unlimited access to chow and water. This experiment was approved by the Animal Ethics Committee of Yangzhou University. Starting from the age of seven weeks, they were randomly divided into five groups, and one group (ND group, n=6) was randomly selected separate from the five groups as a control group and continued to be fed with ordinary feed, and the rest of the groups were fed high-fat feed to establish an obesity model until the weight of the obesity model group exceeded 20% of the control group, and there was no difference in body weight between the obesity groups, meanings that the model is successful.
After successful modeling, the four obesity model groups were randomly divided into three intervention groups: low dose (0.03 mg/kg) group (LD group, n=6), medium dose (0.1 mg/kg) group (MD group, n=6), high dose (0.33 mg/kg) group (HD group, n=6) and a control group (HF group, n=6).
After grouping, the three intervention groups and the control group transitioned from a high-fat diet to a normal diet after one week, and the intervention groups were injected intraperitoneally with different doses of PEX-168, according to their specific subgroup, once a week at 8 a.m. on Tuesday; the HF and ND groups were injected intraperitoneally with the same volume of saline. Before the intervention, the mean FBG of mice in the ND group was 7.08 mmol/L and that of obese mice in the four groups was 7.89 mmol/L. The FBG was within the normal range, indicating that the mice were all nondiabetic simple obese mice. The body weight and food intake of the mice were measured regularly every day. At the end of the animal experiment, all mice were fasted overnight for 12 h, blood was collected from the eyes, and the supernatant was collected after centrifugation at 5000 r/min for 15 min.
Indicators and measurement
(1) General condition: daily observation of the mice's activities, including observation of their appearance, urine, stool, activity, gait, spirit, appetite, and any abnormal conditions.
(2) Determination of body weight, food intake and blood glucose: The body weight of mice was measured regularly every day, the appropriate amount of food was placed in the enclosures and weighed before putting in, and the weight of the remaining feed was weighed the next day (the large pieces of residue in the cage box of each group were weighed and recorded, and the residue crumbs were ignored), and the daily food intake was calculated. Mice were fasted for 12 h each week without the restriction of water intake, and their fasting blood glucose was measured regularly by tail-tip cutting the next morning for 12 weeks.
(3) Serum biochemical analysis: At the end of 12 weeks intervention, all mice were fasted overnight for 12H, and they were executed by cervical dislocation after eye blood collection, and the supernatant was preserved after centrifugation at 5000r/min for 15 min. The levels of FBG, INS and CRP were detected, and the level of Homa-IR was calculated according to the steady-state model method formula: Homa-IR index = FBG (mmol/L) x INS (mIU/L) /22.5. The serum chemerin and omentin levels were determined in mice according to the kit instructions.
Statistical analysis
Analysis was performed using SPSS 22.0. The measurement data were presented as the means ± standard deviation (x ± s); statistical comparisons among groups were performed using one-way analysis of variance (ANOVA) and t-tests; Pearson correlation analysis was used for correlation; P < 0.05 was considered statistically significant.