Parker Booren, Brigham Young University
Both Type 1 and Type 2 Diabetes result in β cell depletion. This is treated through β cell transplantation from cadaver pancreata. Currently, collecting sufficient β cells for one diabetes patient requires pancreata from multiple cadavers. Increasing functional β cell mass ex vivo by increasing insulin secretion and inducing β cell proliferation could be used to increase the transplant ready β cell number. Most of the donors will be from older age groups so it is essential that pathways increasing β cell proliferation work in aged β cells. We have shown that overexpression of the β cell transcription factor Nkx6.1 is sufficient to enhance insulin secretion (through VGF) and increase proliferation (through Nr4a1 and Nr4a3) in young (5 week) but not aged (5 month) β cells. Our data shows that Nkx6.1 is overexpressed at similar levels in both young and aged β cells, but only the young β cells proliferate and have increased insulin secretion, which corresponds with an inability to induce the target genes VGF, Nr4a1 and Nr4a3 in the aged β cells. This disruption in upregulation may be inhibiting the proliferation in aged β cells. One explanation is that there are too many cyclin-dependent kinase inhibitors (CDKI’s) in an aged β cell for it to proliferate. We performed immunofluorescent staining on young and old pancreata to visualize the presence of CDKI’s in young/aged β cells. CDKI’s do not account for all of the loss of proliferation of β cells. We therefore sought to identify the other inhibiting factors that limit proliferation. The inhibiting factors that we studied were: Nkx6.1 has a binding partner that is not expressed in aged β cells, after Nkx6.1 is transcribed, it undergoes post-translational modifications, and that the presence of epigenetic modification of promoters prevents upregulation. We present data from a Co-Immunoprecipitation (Co-IP) on β cell lysate that isolated Nkx6.1 together with its potential binding partners. We present data visualizing the Co-IP products and the results from a mass spectrometry determining the identity of any other proteins that appeared together with Nkx6.1. We also present data from mass spectrometry on Nkx6.1 samples from young and aged β cells showing the post translational modifications that take place to limit the transcription factor’s function. Finally, we present data regarding epigenetic modification at the promoters of VGF, Nr4a1 and Nr4a3 that may be inhibiting their gene expression in the aged β cells.