Insulin tolerance test (ITT)
Reagents:
- Clinical insulin (Novolin) from Novo Nordisk, 100 U/ml.
- Heparinized capillaries from Fisher, #22-362-566.
ITT is usually used to assess the insulin sensitivity in mouse. I usually fast the mice for ~ 3 hrs before experiments. 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. So they recomment 5 hrs fasting. To collect the serum, I blow the total blood into the eppendorf tube and spin down at 6000 RPM for 6 min and then remove the serum for analysis.
For the dosage, I choose 1U/kg body weight. However, for some insulin resistant animals such as obob or diet-induced obese animals, higher dosage may be required.
1. Fast the animals for ~ 3 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 ITT after my lunch.
2. Prepare the ITT. Take 10 ul insulin solution and dissolve in 10 ml distilled H2O and vortex. 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. Since you only need 2 ul serum for glucose assay. A quarter of a capillary total blood is sufficient. I keep them on ice.
4. Inject insulin of 10 X body weight (in gram) ul intraperitoneally. In order to be precise, stagger the mice by one minute since that is the time you would need to get sufficient blood for both glucose and insulin measurement. Store the whole blood on ice until you have time to spin down.
5. On time 30 min, collect blood and store on ice.
6. On time 60 min, collect blood and store on ice.
7. On time 90 min, collect blood and store on ice.
8. On time 120 min, collect blood and store on ice. 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 analysis.