饮食诱导肥胖（DIO）模型（文献参考）

小鼠腹腔注射胰岛素后的血糖反应检测。禁食 4–6 小时；按 1–1.2 U/kg 给予胰岛素（源方案以 100 U/mL 原液经冷 PBS 两步稀释，注射体积按体重计算）；注射前及注射后 15、30、60、90、120 分钟测定血糖。不同组别与样本量按具体研究设计确定，本方案不预设实验分组。

以血糖浓度对时间作图得到完整血糖-时间曲线，并可计算曲线下面积（AUC）反映葡萄糖清除程度；当各受试对象基线血糖不同时应比较高于基线的幅度或曲线下面积，而非仅比较单一终点值。该检测反映葡萄糖负荷后的整体清除，不等同于胰岛素作用的直接测量。

基线与注射后 15、30、60、90、120 分钟血糖时间序列；可计算血糖下降幅度或曲线下面积。结果反映外源胰岛素负荷后的整体血糖反应，不单独等同于组织特异性胰岛素敏感性。

提前一周单笼饲养 实验前一周将小鼠改为单笼饲养。 称量体重 逐只称量小鼠体重，用于按体重计算给糖体积。 禁食6小时 禁食六小时（06:00:00）。 测定基线血糖 禁食结束后，先用血糖仪测定血糖作为基线。 腹腔注射葡萄糖 按2 g/kg体重腹腔注射葡萄糖：将2 g D-葡萄糖溶于10 ml生理盐水配成20%葡萄糖贮备液，按每克体重10 ul给予。 系列采血测定血糖 于注射葡萄糖后15、30、45、60、90和120分钟采集血样，用血糖仪测定血糖。

禁食 Fasting begins first thing in the morning (around 9am). Fast mice for 4-6 hours before ITT begins. Transfer mice to clean cage and wire top. Keep water bottles during the fasting period. 1. If using High Fat diet (HFD), save food to give back at the end of the ITT. 1. Mark tails with a sharpie for easier identification during ITT. 胰岛素溶液配制 Stock 1: 50ul of 100U/ml Humulin R into 4950ul of cold sterile 1X PBS. Invert solution 3-4 times to mix. Stock 2: 1ml of Stock 1 into 9ml of cold sterile 1X PBS. Invert solution 3-4 times to mix. Keep on ice until use 胰岛素负荷 Weigh mouse to obtain body weight for dose calculations. Calculate bolus dose of insulin (**1-1.2 units/kg**). Weight of mouse (g) x 10 = ul insulin stock 2 solution 胰岛素负荷 Prepare syringes using 1cc syringe and 26G needle. Load all syringes with calculated insulin dose and position in front of home cage. 胰岛素负荷 Prior to the delivery of the insulin solution, a time “zero” blood glucose level must be measured. Restrain mouse and clean tail with 70% ethanol/gauze. Extract a small amount of blood from the tail vein onto One Touch Ultra blood glucose strips used with the One Touch Ultra 2 blood glucose monitoring system. 胰岛素负荷 Administer the correct dose of Insulin solution using an IP (intraperitoneal) injection method. - Restrain the mouse. Then place the mouse is in dorsal recumbency with the head down allowing the viscera to move cranially. - Using the bend of the knee and the ventral abdominal midline as landmarks, the needle is inserted half way between the midline and lateral side of the animal at the natural bend of the knee. Insert the needle at a 30 to 45 degree angle into either the left or right lower abdominal quadrant. - Prior to injection, aspirate to make sure the needle has not penetrated a blood vessel, the intestines or the urinary bladder. When aspirating the syringe, you should see an air bubble in the hub of the needle, and not any form of fluid. - Inject and return mouse to cage. 胰岛素负荷 Begin timing from the point of successfully delivering the insulin solution. 胰岛素负荷 Test and record the blood glucose level at 15, 30, 60, 90, and 120 minutes after the insulin injection. Record each value and the condition of the animal on the Insulin Tolerance Test record sheet. 胰岛素负荷 Return food and environmental enrichments to cages.