I have frozen mice livers, pancreas and skeletal muscle. The blood sample obtained at the time of sacrificing the animals was just enough for complete lipid profile. i need to get an idea of insulin resistance in the animals. Any ideas of anything i can do with the tissue to assess/ compare insulin resistance between groups?
Regards
Dear,
See the following paper:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC377430/pdf/JCI0007917.pdf
Dear Tania
In addition to the excellent paper by Accili et al. would recommend looking into GLUT-4 protein expression in muscle (ideally use only soleus or quadriceps and don't mix muscle groups), IRS-1/2 differential phosporylations (Ser vs Tyr) and AKT pathway in liver as well as muscle. If you have handled adipose tissue for western blotting in the past and if your models are not very fat you may also consider studying GLUT4 expression in adipose tissue.
In the past we have also used cell fractionation technique to study expression of GLUT4 and/or phosphorylated AKT in cell cytosol vs membrane (http://www.ncbi.nlm.nih.gov/pubmed/24917575).
All the best!
Kirti
Tania, if you are assessing insulin resistance, your first target should have been measurements of glucose and insulin levels. I hope you have done at least a spot readings or few before euthanizing your animals.
What strains are you using? If they are IRS1, IRS2, or Glut4 mutants, you will simply confirm what you already know, unless you have a concern as to the identity of mice strain.
Attempting to assess Glut4 in two compartments by isolating cytosol membrane from the internal compartments may give less than reliable results from frozen tissue simply because lysosome would rupture upon freezing tissue and depending on time , you will variable and ambiguous results.
It is far more useful to plan an experiment better than to look for methods of salvage, after killing an experimental animal.
Best,
Thank you Kirti and Tausif, all very good ideas. my main outcome was serum lipid profile, which used up all the sample. now that i analyse the results i am thinking it would be good if in addition i could get sort of assessment about insulin resistance. my mice strain was swiss webster albino.
Tania, you will need more serum/plasma to assay insulin. You may have to repeat the experiment, which may be good because it will provide further validation of your previous numbers on lipid profile, in addition to hormone levels.
As for being able to utilize remaining plasma, if you had some, that could have given you misleading results, if it was not stored in a way that will keep insulin from degrading, including not having frozen or frozen and thawed more than once.
Hi Tania
Since you have looked at serum lipid profile in addition to insulin signaling cascade in muscle, liver and adipose tissue you could also look at lipidomic and miRNA analysis of tissues. Completely agree with Tausif in that repeating some experiments for fresh serum is a good idea but in case you don't have that option make the most of your tissue samples. I would recommend planning out the types of analysis have available (proteomics,lipidomics, RNA expression analysis etc). In principle the insulin cascade should suffice.
Good luck!
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