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[Bio101] Glucose Tolerance Test in Mice   

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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.
Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Zhang, P. (2011). Glucose Tolerance Test in Mice. Bio-protocol Bio101: e159. DOI: 10.21769/BioProtoc.159.
Q&A

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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, USA

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, USA

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