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小鼠口服葡萄糖耐量试验（OGTT）

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This protocol adopts the protocols.io Oral Glucose Tolerance Test in Mouse v1. Mice are fasted for 4–6 hours, weighed, and given oral glucose at 1 g/kg (0.5 g/kg after at least 4 weeks of high-fat diet). Blood glucose is measured before and at 15, 30, 60, 90 and 120 minutes after the load, with tail-vein plasma collected at selected time points for insulin measurement. The record describes the assay procedure, not executed disease-model results.

Scope and method

Sample：mouse blood and plasma

葡萄糖耐量试验

Reagents and materials

Procedure

  1. 禁食

    Fasting begins first thing in the morning (around 9am). Fast mice for 4-6 hours before OGTT 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 OGTT. 1. mark tails with a sharpie for easier identification during OGTT.

  2. 口服葡萄糖负荷

    Weigh mouse to obtain body weight for dose calculations. Calculate bolus dose of glucose (**1g/kg**). Weight of mouse (g) x 2 = ul sterile glucose (50% dextrose) solution *If using HFD for 4 weeks or more, use 0.5g/kg (weight of mouse (g) x 1 = ul sterile glucose (50% dextrose) solution)

  3. 口服葡萄糖负荷

    Prepare syringes using 1cc syringe and 18G oral gavage needle. Load all syringes with calculated glucose dose and position in front of home cage.

  4. 口服葡萄糖负荷

    Prior to the delivery of the sterile glucose (50% dextrose) 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. Blood can be collected into microvette tubes that can be used to extract plasma which can be assayed for plasma insulin (see step 9 for plasma collection). Microvette tubes are blood collection tubes with a capillary. Tap tube until all the blood is collected in the tube. Place on ice until plasma collection.

  5. 口服葡萄糖负荷

    Administer the correct dose of sterile glucose (50% dextrose) solution using the syringe and gavage needle. - Restraint of the Mouse, avoiding an excessively tight grip of the mouse, as this will induce a panic response. Ensure that the mouse cannot bite you and cannot move its head. - Gravity alone should be used to move the cannula down the esophagus. While gliding the cannula, rotate slightly as the cannula goes past the epiglottis and into the esophagus.The mouse should swallow the cannula, and this can be observed. - Inject the fluid slowly. - The cannula is then gently removed and the animal is returned to its cage.

  6. 口服葡萄糖负荷

    Begin timing from the point of successfully delivering the sterile glucose (50% dextrose) solution.

  7. 口服葡萄糖负荷

    Test and record the blood glucose level (see step 4) at 15, 30, 60, 90 and 120 minutes after the initial glucose delivery. Record each value and the condition of the animal on the Glucose Tolerance Test record sheet. Extract a small amount of blood from the tail vein at 15, 30, 60 and 120 minutes for plasma; skip blood collection at 90 minutes to ensure not to exceed 0.007 ml blood/ g body weight.

  8. 口服葡萄糖负荷

    Return food and environmental enrichments to cages.

  9. 血浆采集

    Place tubes into pre-chilled centrifuge. Centrifguge for 2 mins at 2000 rpm. 00:02:00 4 °C

  10. 血浆采集

    Remove the callipary-lid and dispose in biohazard bin. Place the base lid and seal the microvette (click position).

  11. 血浆采集

    Centrifuge at 4°C for 10 mins at 10 000 rpm. 00:10:00 4 °C

  12. 血浆采集

    Using a pipette, transfer the plasma (top transparent layer) to a clean and labelled tube. Store plasma samples at -20°C until assayed.

  13. 血浆胰岛素测定

    Assay plasma insulin with the STELLUX Chemi Rodent Insulin ELISA kit (ALPCO #80-INSMR-CH01) according to the manufacturer's protocol. Include the kit standards and quality controls in each assay and read the plate by the kit's chemiluminescence procedure.

Quality control and readouts

禁食时间、葡萄糖剂量与给药途径在研究期间保持一致；血糖仪与试纸匹配并按厂商要求校准和保存；逐动物记录体重、时间点与读数；尾静脉采血总量不超过 0.007 mL/g 体重，源方案在 90 分钟时跳过采血以满足该限制；血浆离心与保存按方案条件执行。

基线与负荷后 15、30、60、90、120 分钟血糖时间序列；可计算血糖曲线下面积。血浆胰岛素结果按所用 ELISA 的校准与量程判读。

Study design
小鼠经口灌胃葡萄糖负荷的耐受试验。禁食 4–6 小时；普通饮食按 1 g/kg、高脂饮食 4 周以上按 0.5 g/kg 给予 50% 葡萄糖溶液；负荷前及负荷后 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。

References

  1. Nancy Smith, Mourad Ferdaoussi, Haopeng Lin, Patrick Macdonald. Oral Glucose Tolerance Test in Mouse. protocols.io. https://doi.org/10.17504/protocols.io.ujjeukn
  2. Benedé-Ubieto R, Estévez-Vázquez O, Ramadori P, Cubero FJ, Nevzorova YA. Guidelines and Considerations for Metabolic Tolerance Tests in Mice. Diabetes Metab Syndr Obes. 2020;13:439-450. PMID 32110077. doi:10.2147/DMSO.S234665
  3. ALPCO. STELLUX Chemi Rodent Insulin ELISA (Catalog #80-INSMR-CH01) instructions.

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