In vivo insulin signaling
Reagents:
- Clinical insulin (Novolin) from Novo Nordisk, 100 U/ml.
Insulin signaling in vivo somwhow is more informative than in vitro studies. The experiment is relatively simple to carry out. You can assess insulin response in peripheral target tissues including liver, adipose tissue and skeletal muscle. I have tried this protocol for adipose tissue 15 min after insulin administration. For liver, you may need reduce the time to 5 min.
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 such as 1.5-2 U/kg body weight.
1. Fast the animals overnight. I change the bedding as well in case there are some food pieces in the cage. I usually do that in the afternoon and then perform the experiment first thing next morning.
2. Prepare the experiment. Take 10 ul insulin solution and dissolve in 10 ml distilled H2O and vortex. Bring aluminum foils labeld with names of the samples, needles, 1 ml syringes, vehicle (H2O), timer and bucket of liquid nitrogen to animal room.
3. Schedule your experiment. Since the reaction is fairly quick, you may need to stagger your animals. For an example, I inject every 3 min which is the time I need to take adipose tissues from a mouse. Like this: Inject mouse 1 at time 0, mouse 2 at time 3 min, mouse 3 at time 6 min...Take tissues of mouse 1 at time 15 min, mouse 2 at 18 min, so on and so forth.
4. In the morning, weigh the animals and label the tails using marker pen for easy identification. Inject insulin of 10 X body weight (in gram) ul intraperitoneally and finish the experiment as planned. I usually use isoflurane to quickly knockdown the animals. Depending on the regulations in your institute, you may try physical dislocation which is very fast.
5. Store the samples at -80C until analysis.
6. Refer to protein extraction method if you need to prepare protein lysates.
7. Try Western blotting for phospho-AKT, total AKT, phospho-GSK-3b, total GSK-3b, etc.