I am studying the effect of a protein on Insulin secretion in pancreatic Beta cells of Rattus norvegicus. To prove this effect of that protein, I have to quantify expression of two genes which affect insulin secretion pathways. Which genes do you suggest?
You can use these two gene.
Pdx1 pancreatic and duodenal homeobox 1
Ins1 insulin 1
Sercan
I would check Ins2 as well even if gene expression is not informative for insulin secretion.
As Julie said, it will be difficult to relate gene expression levels with perturbations in secretion per se, inasmuch as the secretion pathway is complex and involves many different steps and genes (glucose entry/GLUT2, Glycolysis/rate limiting Glucokinase, TCA cycle, membrane depolarization/ K+ ATP channel, Ca2+ entry-/Voltage sensitive Ca2+ channels, amplifying pathway/Glutamate dehydrogenase, exocytosis/SNARE proteins, Cell to cell communication /Cx36).
In addition, you can have a small effect on the expression levels of insulin, for instance, whithout any detectable changes in insulin secretion, as insulin content in mouse islets is 100 fold the level of secretion upon glucose stimulation. Insulin stores are huge compared to what is secreted. Also pay attention to 1st and 2nd phase of insulin secretion.
I would advise, if you really can't do insulin ELISA or RIA, to look at those different genes (also note that SNARE proteins are involved in the last step, so if you observe an impairment at this terminal step, this might imply impairment in both 1st and 2nd phase), and really pay attention to your conclusions !! you won't be able to prove any effect by looking at only 2 genes! Unless you are super lucky..
Good luck!
Thank you all for the support.
David! Actually, the protein I'm working on, is an unknown protein, and there is a little information available about it. There are some hypothesis that claim it could affect the insulin secretion. We are going to pick two genes at the begining to start the project. In this case, what should I do to choose the best genes? I really appreciate your suggestions.
If the insulin secretion is expected to be effected, it will make most sense to measure insulin secretion, as others have suggested. Measure insulin secretion in the presence of low and high glucose in the presence and absence of your test protein. If you wish to go beyond the insulin secretion in response to glucose, add arginine to see whether or not your protein specifically interferes only with glucose-stimulated insulin secretion.
You may find one of our old publication somewhat useful:
http://www.pnas.org/content/83/24/9749.full.pdf
Pegah,
You MUST convince your chief to go through insulin secretion assay!
you should work with a beta cell line that is well characterized for good glucose responsiveness, like INS1-E cells, stimulate them with 15mM glucose or with 30mM Kcl, for 1st phase release analysis (you might have a look to our previous paper: http://www.ncbi.nlm.nih.gov/pubmed/18385973, which also refer to Maechler's lab papers), with or without knock down of your protein.
Then, when you have a positive result, you can look at gene expression to find the mechanism (see my first message). It is a non sense to analyze insulin secretion with the expression levels of two genes!
If your chief really insist with this idea, then give a try with 2 of these 4 genes: Glut2, Pdx1, insulin or snap25.
i agree with pdx (pancreatic and duodensl homeobox1),ins1,glut2 ,ins2,znt2,glucokinase and can choose 2 of the ones most convenient for you.
I'd certain agree that if you are interested in understanding how your novel protein impacts insulin secretion, your first port of call should be to determine if insulin secretion is in any way altered at high & low glucose in an appropriately glucose responsive cell line (after o/exp and/or silencing your protein). Just looking at genes in the insulin secretion pathway isn't necessarily going to correlate with secretion.
INS-1E's have been mentioned, and I would second that - they are the best we've worked with for this sort of study - easy to transfect and robustly glucose responsive. To gather more information, rather than just doing a static incubation you could configure your cells as pseudoislets (3D clusters of beta cells) and perform a perifusion (if you have the appropriate kit). This method will give you information about secretion over time (i.e. is first or second phase secretion altered) and configuring beta-cell lines as pseudoislets can dramatically improve their glucose responsiveness (MIN6's may be a better choice if you go down this route). Hope this is some help, and good luck!
I agree with previous posts to check insulin secretion whether it is effected by your protein. Also wanted to mention that checking PDX1 sometimes might give interesting results! For example if you stain your pancreatic sections for PDX1 you can easily see that a lot of cells are negative for PDX1 while they are positive for insulin.
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