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[Bio101] Glucose Tolerance Test in Mice
[Bio101] 小鼠体内葡萄糖耐受测试   

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Abstract

Glucose tolerance test is a standard procedure that addresses how quickly exogenous glucose can be cleared from blood. Specifically, uptake of glucose from the blood by cells is regulated by insulin. Impairment of glucose tolerance (i.e, longer time to clear given amount of glucose) indicates problems with maintenance of glucose homeostasis (insulin resistance, carbohydrate metabolism, diabetes, etc).
According to the WHO, in a standard oral glucose tolerance test (OGTT), glucose level should be below 7.8 mmol/L (140 mg/dl) at 2 h. Levels between this and 11.1 mmol/L (200 mg/dl) indicate “impaired glucose tolerance”, and any level above 11.1 mmol/L (200 mg/dl) confirms a diagnosis of diabetes.

Materials and reagents

  1. Mice (~20 C57BL/6J (B6) males of 2-3 months old)
  2. 70% ethanol
  3. Beta-D(+)-glucose (Sigma-Aldrich, catalog number: G8270 )
  4. NaCl
  5. KCl
  6. Sodium phosphate
  7. Phosphate buffered saline (PBS) (see Recipes)

Equipment

  1. ACCU-CHEK comfort curve glucometer (Roche Diagnostics, catalog number: 03537536001 ) (this product has been discontinued. Any new product of ACCU-CHEK should work fine as well)
    Such device quantifies glucose amperometrically by measuring the current produced upon oxidation of glucose to gluconic acid by glucose oxidase, or to gluconolactone by dehydrogenase.
  2. 27 gauge needle (Single-Use Needles, supplied by VWR, BD Medical, catalog number: BD305109 )
  3. Microvette CB300 Z serum separator (SARSTEDT, catalog number: 16.440.100 )
  4. Acrodisc 25 mm syringe filters w/ 0.2 μM HT Tuffryn membrane (Pall Corporation, catalog number: 4192 )

Procedure

Note: All the following experimental procedures that involve animals (rodents) should receive approval from IACUC or equivalent committee. Humane treatment of animals should be practiced all the time.
In the Cavener lab, we used glucose tolerance test extensively in the characterization of mice lacking Pek/Perk, encoding an eIF2alpha kinase that is crucial to translational control and ER homeostasis (Zhang et al., 2002).

  1. Obtain ~20 C57BL/6J (B6) males of 2-3 months old from Jackson Laboratory or Harlan Teklad and raise them on a regular diet for about 2 weeks.
    Tip 1: Males, if not from the same litter, tend to fight with each other. Raise only 3-4 animals per cage and monitor any aggressive behavior.
  2. Fast animals O/N in fresh cages with water supply for ~16 h the day before experiment.
  3. Monitor body weight as well as baseline blood glucose level for each animal.
    Tip 2: Clean tail with 70% ethanol and rub it for better circulation before making a clip with sterile scissors.
    In non-terminal procedures, collect sufficient amount of blood sample (i.e., ~30 μl) from saphenous vein for serum isolation and insulin determination by radioimmunoassay (RIA). To reduce lysis of red blood cells, make multiple punctures on the saphenous vein with a small needle and collect blood using Microvette CB300 Z serum separator.
  4. Prepare 10% glucose in 1x PBS and sterilize the solution by 0.2 μm-filtration.
  5. Prepare injection solution in a 0.5 ml eppendorf tube for each animal (2 mg glucose/g body weight). Volume (μl) = Body Weight (g) x 20. Use 20% glucose instead, should the body weight be greater than 30 g.
  6. Sterilize abdominal region of animal with 70% ethanol, and clean up with dry cotton ball. Hold the back of the animal firmly, and inject glucose intraperitoneally into recipient subject with a 27-gauge sterile needle.
    Tip3: Remove air bubble before injection and do NOT stick the needle into the body too deeply. Perform injection slowly with an angle of 30 to 45 °C to avoid subcutaneous injection. Pick the injection site away from the liver and close to the ventral axis to avoid kidney damage.
  7. Put the animal back into the cage and measure blood glucose levels at 15, 30, 60, 90 and 120 min (or 30, 60, 90, 120 and 240 min). At least 3-4 replicate animals should be used for each time-point.

Representative data



Figure 1. This figure is adapted from the original (Zhang et al., 2002).
Fasted B6 mice were injected at time zero with glucose, and blood glucose levels were monitored and normalized to the value of time zero. Each data point represents the average of three to five individual mice. The baseline value of blood glucose is typically ~100 mg/dl. Representative data are shown here.

Notes

Please note that mice of different genders, ages, and inbred backgrounds can show different responses to glucose. Certain strain-specific phenotype data can be found at http://phenome.jax.org/db/q?rtn=meas/methodologies.

Recipes

  1. PBS (pH 7.4)
    0.8% NaCl
    0.02% KCl
    12 mM sodium phosphate

Acknowledgments

This protocol was adapted from work performed by Dr. Maureen Gannon at the Vanderbilt University Medical Center. PZ was supported by a research assistantship in the Cavener lab at the Pennsylvania State University.

References

  1. Zhang, P., McGrath, B., Li, S., Frank, A., Zambito, F., Reinert, J., Gannon, M., Ma, K., McNaughton, K. and Cavener, D. R. (2002). The PERK eukaryotic initiation factor 2 alpha kinase is required for the development of the skeletal system, postnatal growth, and the function and viability of the pancreas. Mol Cell Biol 22(11): 3864-3874.

简介

葡萄糖耐量试验是解决外源葡萄糖可以从血液中清除的标准程序。 具体地,通过胰岛素调节细胞从血液摄取葡萄糖。 葡萄糖耐量的损害(即,更长的时间来清除给定量的葡萄糖)表明维持葡萄糖内稳态(胰岛素抵抗,碳水化合物代谢,糖尿病等)的问题。
根据WHO,在标准口服葡萄糖耐量试验(OGTT)中,2小时时葡萄糖水平应低于7.8 mmol/L(140 mg/dl)。 该浓度与11.1mmol/L(200mg/dl)之间的水平表示"葡萄糖耐量降低",并且高于11.1mmol/L(200mg/dl)的任何水平证实了糖尿病的诊断。

材料和试剂

  1. 小鼠(约20个C57BL/6J(B6)2-3个月大的男性)
  2. 70%乙醇
  3. β-D(+) - 葡萄糖(Sigma-Aldrich,目录号:G8270)
  4. NaCl
  5. KCl
  6. 磷酸钠
  7. 磷酸盐缓冲盐水(PBS)(见Recipes)

设备

  1. ACCU-CHEK舒张曲线血糖仪(Roche Diagnostics,目录号:03537536001)(此产品已停产,ACCU-CHEK的任何新产品也应该正常工作)
    这种装置通过测量葡萄糖由葡萄糖氧化酶氧化为葡萄糖酸或通过脱氢酶氧化为葡萄糖酸内酯时产生的电流来量化葡萄糖。
  2. 27号针(单用针,由VWR提供,BD Medical,目录号:BD305109)
  3. Microvette CB300 Z血清分离器(SARSTEDT,目录号:16.440.100)
  4. Acrodisc 25mm注射器过滤器w /0.2μMHT Tuffryn膜(Pall Corporation,目录号:4192)

程序

注意:涉及动物(啮齿类动物)的所有以下实验程序应获得IACUC或同等委员会的批准。人类对动物的治疗应该一直进行。
在Cavener实验室中,我们在缺乏pek/Perk的小鼠的表征中广泛使用葡萄糖耐量试验,编码对翻译控制和ER内环境稳定至关重要的eIF2α激酶(Zhang等人,/em,2002)。

  1. 从Jackson Laboratory或Harlan Teklad获取〜20只C57BL/6J(B6)雄性2-3个月大的男性,并定期饮食约2周。
    提示1 :男性,如果不是从同一个垃圾,往往会互相战斗。每笼只增加3-4只动物,并监测任何攻击行为
  2. 快速动物O/N在新鲜笼子里用实验前一天的水供应约16小时
  3. 监测每只动物的体重和基线血糖水平。
    提示2 :用无菌剪刀制作夹子之前,用70%乙醇清洁尾巴并擦拭,以便更好地循环。
    在非终止性程序中,从隐静脉收集足够量的血样(〜30μl),用于通过放射免疫测定(RIA)进行血清分离和胰岛素测定。为了减少红细胞的溶解,用小针在隐静脉上多次穿刺,并使用Microvette CB300 Z血清分离器收集血液。
  4. 在1×PBS中制备10%葡萄糖,并通过0.2μm过滤对溶液灭菌
  5. 在每只动物的0.5ml eppendorf管中制备注射溶液(2mg葡萄糖/g体重)。体积(μl)=体重(g)x 20.如果体重大于30g,则使用20%葡萄糖。
  6. 用70%乙醇消毒动物的腹部区域,用干棉球清理。牢牢握住动物的背部,用27号无菌针将葡萄糖腹膜内注射到受体受试者体内 Tip3 :在注射前取出气泡,并且不要将针头深入身体。 以30至45°C的角度缓慢注射,以避免皮下注射。选择注射部位远离 肝脏和靠近腹侧轴,以避免肾脏损伤
  7. 将动物放回笼中并在15,30,60,90和120分钟(或30,60,90,120和240分钟)测量血糖水平。 每个时间点应至少使用3-4个复制动物。

代表数据



图1.该图改编自原始的(Zhang等人,2002)。在时间零点用空白B6小鼠注射葡萄糖,监测血糖水平,并标准化为 时间零的值。 每个数据点代表3至5只个体小鼠的平均值。 血糖的基线值通常为〜100mg/dl。 此处显示了代表性数据。

笔记

请注意,不同性别,年龄和近交背景的小鼠可以显示不同的葡萄糖反应。 某些菌株特异性表型数据可以在 http://phenome.jax.org/db/q?rtn = meas/methodsologies

食谱

  1. PBS(pH 7.4)
    0.8%NaCl
    0.02%KCl
    12mM磷酸钠

致谢

该协议改编自范德比尔特大学医学中心的Maureen Gannon博士所做的工作。 PZ由宾夕法尼亚州立大学Cavener实验室的研究助理支持。

参考文献

  1. Zhang,P.,McGrath,B.,Li,S.,Frank,A.,Zambito,F.,Reinert,J.,Gannon,M.,Ma,K.,McNaughton,K.and Cavener,DR )。 PERK真核起始因子2α激酶是骨骼系统发育,出生后生长所必需的, 和胰腺的功能和存活力。 Mol Cell Biol 22(11):3864-3874。
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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: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhang, P. (2011). Glucose Tolerance Test in Mice. Bio-protocol Bio101: e159. DOI: 10.21769/BioProtoc.159;
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Ram Sudheer Adluri
GVK Biosciences
Hello,

Could you please let me know what would be best time point to measure GLP-1 in this model in BALB/c or C57BL/6 mice?

Thanks,
Sudheer.
4/1/2013 3:27:38 AM Reply
Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

Hi Sudheer,

We have never measured GLP-1 for our mice in these tests. Here is some reference that could be interesting to you: http://www.pnas.org/content/97/12/6874.long It seemed that this group measured GLP-1 at an early time point (15min after the administration of glucose).

Best,
Peichuan

4/2/2013 5:52:55 PM


Ram Sudheer Adluri
GVK Biosciences

Thank you somuch Peichuan...

4/3/2013 3:17:47 AM


Hello,

I performed the IPGTT with 70 Mice genetic changed mice and would like to know what are normal parameters for the glucose level at 15,30,60 and 120 minutes.

Would help me a lot!
Thanks
7/29/2012 1:46:21 AM Reply
Peichuan Zhang
Calico Life Sciences

Hello, sorry that I couldn't find the raw data as I did this work a long time ago. I contacted a former colleague and haven't got any reply yet.

I checked both my own paper and another reference http://ajpendo.physiology.org/content/295/6/E1323.full Andrikopoulos et al, Am J Physiol Endocrinol Metab (2008). I could tell from my paper that the levels should be about 320mg/dL (15min) and 350mg/dL (45min). Estimated serum glucose levels from the other paper: ~320mg/dL (15min), 300mg/dL (30min), 220mg/dL (60min), 120mg/dL (120min). Hope that these data points would serve well as references for your own work.

Best luck,
P

8/6/2012 11:12:03 PM


Hi! Thank you sir Peichuan for the fast reply. I will be using Male 6 to 8 weeks old ICR mice for my study. I have difficulty in searching for the normal fasting blood glucose range. Some journal articles just say that mice were considered normal when they have fasting bgl of <100.Thank you so much.
7/9/2012 4:06:34 AM Reply
I just want to ask if what is the normal fasting blood glucose level of mice fasted for 12 hours? Thank you.
7/7/2012 11:18:55 PM Reply
Peichuan Zhang
Calico Life Sciences

Hello,

We used both B6 black mice as well as littermates (sibling) of the PERK knockout mice as the controls. The fasting plasma glucose level was around 100 mg/dL (~5.5 mmol) or maybe even lower. I recall that the technician moved the mice into a FRESH cage with water just before he left (around 6PM), and then we perform the test next morning or around noon (~16 to 18 hours).

The PERK knockout mice had mixed genetic background of B6 and 129. Please be aware that the fasting levels might be different for mice from different backgrouns --- refer to http://ajpendo.physiology.org/content/295/6/E1323.full Andrikopoulos et al, Am J Physiol Endocrinol Metab, 2008.

Please feel free to ask us questions

Thanks,
Peichuan

7/8/2012 9:30:44 PM