Insulin Tolerance Test and Hyperinsulinemic-euglycemic Clamp
胰岛素耐受试验和高胰岛素-正血糖钳夹试验   

引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Nature Medicine
Dec 2012

 

Abstract

The two tests are used to evaluate in vivo sensitivity to insulin in mouse. The hypoerinsulinemic-euglycemic clamp provides information about the sensitivity to insulin in liver and other metabolically relevant tissues.

Keywords: Insulin (胰岛素), Diabetes (型糖尿病), Mouse (鼠标), Glucose (葡萄糖)

Materials and Reagents

  1. Human insulin (Eli Lilly, Indianapolis, IN)
  2. [3-3H] glucose (Perkin Elmer, catalog number: NET331A250UC )
  3. 2-deoxy-D-[1-14C] glucose (2-[14C]DG) (PerkinElmer, catalog number: NET328250UC )

Equipment

  1. Contour blood glucometer (Bayer)

Procedure

  1. C57BL/6J mice were fasted for 6 h and then injected intraperitoneally with insulin (1 U per kg of body weight), and blood glucose concentrations were monitored over time using a Contour blood glucometer on a drop of blood from the tip of the tail.
  2. Mice were cannulated in the lateral cerebral ventricle and catheterized in the right internal jugular vein for the hyperinsulinemic-euglycemic clamp (Figure 1) (Thrivikraman et al., 2002). Intravenous infusion of [3-3H] glucose (5 μCi bolus, 0.05 μCi/min) was used.
  3. Human insulin (16 mU/kg) was injected intravenously as a bolus, followed by continuous infusion at 2.5 mU/kg/min.
  4. Tail blood glucose was measured by glucometer at 10 min intervals, and 20% glucose was infused to maintain blood glucose at euglycemic levels (120 to 140 mg/dl of plasma glucose levels).
  5. After steady state had been maintained for 1 h, the glucose uptake in various tissues was determined by injecting 2-deoxy-D-[1-14C] glucose (2-[14C]DG) (10 mCi) 45 min before the end of clamps (the catheter was used for the injection). During the final 50 min of basal and clamp infusions, 20 μl blood samples were collected at 10 min intervals for measurement of [3H] glucose, [3H] H2O and 2-[14C]DG from the tail vein. Samples were stored in -20 °C.


    Figure 1. Right internal jugular vein catheterization. A catheter is placed in the right jugular vein for the infusion of glucose and insulin.

Acknowledgments

This protocol has been adapted from our previously published paper: Paschos et al. (2012). The work during the development of the protocol was supported by the US National Institutes of Health (NIH) grant RO1 HL097800.

References

  1. Paschos, G. K., Ibrahim, S., Song, W. L., Kunieda, T., Grant, G., Reyes, T. M., Bradfield, C. A., Vaughan, C. H., Eiden, M., Masoodi, M., Griffin, J. L., Wang, F., Lawson, J. A. and Fitzgerald, G. A. (2012). Obesity in mice with adipocyte-specific deletion of clock component Arntl. Nat Med 18(12): 1768-1777.
  2. Thrivikraman, K. V., Huot, R. L. and Plotsky, P. M. (2002). Jugular vein catheterization for repeated blood sampling in the unrestrained conscious rat. Brain Res Brain Res Protoc 10(2): 84-94.

简介

这两个测试用于评价小鼠体内胰岛素的敏感性。 低胰岛素血糖正常血糖钳提供了关于肝脏和其他代谢相关组织中胰岛素敏感性的信息。

关键字:胰岛素, 型糖尿病, 鼠标, 葡萄糖

材料和试剂

  1. 人胰岛素(Eli Lilly,Indianapolis,IN)
  2. [3-葡萄糖]葡萄糖(Perkin Elmer,目录号:NET331A250UC)
  3. 2-脱氧-D- [1-支链14 C]葡萄糖(2 - [14 C] DG)(PerkinElmer,目录号:NET328250UC)

设备

  1. 轮廓血糖仪(Bayer)

程序

  1. C57BL/6J小鼠禁食6小时,然后腹膜内注射胰岛素(1U/kg体重),并且使用Contour血糖仪在一滴来自尾部的一滴血液上随时间监测血糖浓度。
  2. 在侧脑室脑室中插入小鼠,并在右颈内静脉中导管插入用于高胰岛素 - 正常血糖钳夹(图1)(Thrivikraman等人,2002)。 使用静脉内输注[3- [3 H] 3 H]葡萄糖(5μCi推注,0.05μCi/min)。
  3. 将人胰岛素(16mU/kg)作为推注静脉内注射,然后以2.5mU/kg/min连续输注。
  4. 通过血糖仪以10分钟间隔测量尾血糖,并注入20%葡萄糖以维持血糖为正常血糖水平(120至140mg/dl血浆葡萄糖水平)。
  5. 在稳定状态保持1小时后,通过将2-脱氧-D- [1- [14 C]葡萄糖(2- [14 C] - 葡萄糖)在导管结束之前(导管用于注射)45分钟(10分钟)(10mCi)。在基础和夹钳输注的最后50分钟期间,以10分钟的间隔收集20μl血液样品以测量[3 H]葡萄糖,[3 H] H [来自尾静脉的2 H和2 - [sup 14 C] DG。将样品储存在-20℃下。


    图1.右颈内静脉导管插入术。 将导管置于右颈静脉中,用于输注葡萄糖和胰岛素。

致谢

该协议已经改编自我们以前发表的论文:Paschos等人(2012)。方案开发期间的工作由美国国立卫生研究院(NIH)拨款RO1 HL097800支持。

参考文献

  1. Paschos,GK,Ibrahim,S.,Song,WL,Kunieda,T.,Grant,G.,Reyes,TM,Bradfield,CA,Vaughan,CH,Eiden,M.,Masoodi,M.,Griffin,JL,Wang ,F.,Lawson,JA和Fitzgerald,GA(2012)。脂肪细胞 - 具体删除时钟分量Arntl。 18(12):1768-1777。
  2. Thrivikraman,K.V.,Huot,R.L.and Plotsky,P.M。(2002)。 颈静脉导管插入术,用于在无限制意识大鼠中重复取血。大脑 Res Brain Res Protoc 10(2):84-94。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Paschos, G. K. and FitzGerald, G. A. (2013). Insulin Tolerance Test and Hyperinsulinemic-euglycemic Clamp. Bio-protocol 3(10): e776. DOI: 10.21769/BioProtoc.776.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。