I expected the insulin signal will be better (increase in insulin sensitivity) in the liver of the normoglycemic mice ( have normal basal blood glucose and GTT) than the hyperglycemic mice ( have high basal blood glucose and GTT). but I found that the pAKT s473 is inhibited in the normal and activated in the diabetic mice. what the explanation and what shall I do next?
Not really clear what you did experimentally. If you mean that you fasted the animals then injected with acute insulin and then harvested the livers and blotted, you might expect an increase in Ser473 in the wild type over diabetic but it all depends on the diabetes model and the basal levels of phosphorylation. In addition, it matters when the mice were last fed, their age, strain and to some extent their sex. Ser473 phosphorylation isn't "inhibited" in the normal, but may be lower than the basal level in the diabetic model, which, being hyperglycemic is likely experiencing high circulating insulin. Controls for these experiments are not trivial due to t he counter-balancing of various signals, adaptation, etc. In other words, I would characterize your system in normal mice, understand acute insulin responsiveness, work out the conditions for optimal insulin response and only then initiate the diabetic control studies. There are lots of papers on this but make sure you are comparing the right models.
Thanks James, what I did exactly: 2 groups of C57BL/6J male mice were fed HFD (60% kalo.) for 12 weeks one is exposed to intervention the other just fed restrictedly to gain the same weight as the treated group. the treated group showed significant improvement in basal glycemia and GTT compared to the control group while no difference in ITT.
At the day of sacrificing the mic, the intervention was stopped and the food was removed from the cages 2 hours from sacrificing.
liver, muscle, hypothalamus, WAT were collected and freezes directly in -80.
Protein was extracted from 100 mg of frozen liver of each mouse then blotted (western blot).
The control mice showed stronger pAKT Ser473 signal than the treated mice.
That's a long term experiment (3 months) and clearly the HFD worked. However, mice tend to eat at night and so fasting them for 2 hours in the morning could have little effect if the aim was to measure baseline activities. It is difficult to control for variations in signalling without longer term fasting (6 hr or so). Western blotting of phosphospecific residues can also be misleading as it is not trivial to determine stoichiometry. For example, if the baseline is low, then a small increase over control can look large. Ditto, 0.5 vs 0.7 (mol/mol) can appear small. The best way of dealing with this is to have a control series of samples from a cell line treated with a PI3K agonist.
Hello, induced activation of Akt should be lower than normal so if after intervention it is still lower I believe that probably the intervention did not work as expected
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Disease Models
- Animal Models
- Rodent Models
- Mouse Models