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Intraperitoneal glucose tolerance test (IPGTT)

Reagents:

• Glucose from Sigma, #G8270-1KG.
• Heparinized capillaries from Fisher, #22-362-566.

IPGTT is usually used to test the glucose tolerance in mouse. It is different compared to OGTT. In OGTT, the response includes the secretion of GLP-1 from gut which may augment the secretion of insulin from beta cells. In IPTGG, the gut effect is bypassed and the glucose stimulated insulin secretion is lower compared to OGTT.

I usually fast the mice for 6 hrs before experiments to make sure the gut is empty. Some groups fast the animals for 16 hrs (overnight). We think that is more of starvation instead of fasting. However, other people are arguing that there will be gastric emptying effects on glucose tolerance if only after 3 hrs fasting. During IPGTT, I also collect serum for insulin measurements in time 0, 15 and 30 min to assess the glucose stimulated insulin secretion. For that purpose, I usually collect more than 10 ul serum in time 0, 15 and 30 min. I then set aside ~ 2 ul serum for glucose measurement using glucose assay and the rest are for insulin assessment using insulin ELISA assay (from Linco, now Millipore). To collect the serum, I blow the total blood into an eppendorf tube and spin down at 6000 RPM for 6 min and then remove the serum for analysis.

For the amount of gluose, I usually use 2.5 g/kg body weight. However, a range of 1 - 2.5 g/kg has been used in various studies.

I collect tail blood from 0 min to 3 hrs since the time is necessary for glucose level back to normal. However, 2 hrs has also been widely used.

1. Fast the animals for 6 hrs. I change the bedding as well in case there are some food pieces in the cage. I usually do that in the morning and then perform IPGTT in the afternoon.

2. Prepare the IPGTT. Weigh 3.75 g glucose and dissolve in 15 ml distilled H2O completely. Bring enough eppendorf tubes, capillaries, timer, bucket of ice and iPOD to animal room.

3. Weigh the animals and label the tails using marker pen for easy identification. Collect serum for time 0. I usually draw more than a half of the capillary which should be sufficient for glucose assay and insulin ELISA assay. I then aliquot ~ 2 ul to a tube and the rest in another tube. I keep them on ice.

4. Intraperitoneal injection of glucose of 10 X body weight (in gram) ul glucose solution. In order to be precise, stagger the mice by one minute since that is the minimal time you would need to get sufficient blood for both glucose and insulin measurement.Since you may need the time 15 for glucose stimulated insulin secretion and one minute is minimally required for each mouse, the maximal number of animals you can handle is 15. Store the whole blood on ice until you have time to spin down.

5. On time 15 min, collect blood and store on ice.

6. On time 30 min, collect blood and store on ice.

7. On time 60 min, collect blood and store on ice. From this time point on, you do not have to collect too much blood since only 2 ul serum are required to measure glucose. I usually draw less than a quarter of the capillary of blood from each mouse.

8. Continue collect blood on time 120 min and 180 min. During the waiting time, you can spin down the blood and separate the serum.

9. After finishing the experiments, put the food back on for the animals!

10. Store the serum at -20C until assay.