I. Gaffar1,2, K. Prasadan2, P. Guo2, X. Xiao2, J. Wiersch2, C. Shiota2, G. Gittes2 1University Of Pittsburgh Medical Center,General Surgery,Pittsburgh, PA, USA 2Children’s Hospital Of Pittsburgh Of UPMC,Pediatric General And Thoracic Surgery,Pittsburgh, PA, USA
Introduction: Diabetes develops as a result of insufficient insulin production. One method to replace the beta cell mass would include the transdifferentiation of other endocrine cells into beta cells. Given that endocrine cells already have the machinery to produce and release hormones, it is plausible that a cell could be forced to express another hormone. The long-acting GLP-1 agonist, Exendin-4, has insulinotrophic properties in vitro. Exendin-4 has several important benefits with reduced risk for hypoglycemia and is an approved drug for the treatment for Type 2 Diabetes. Other studies have used Exendin-4 to treat neonatal mice with intrauterine growth retardation and prevented development of adult hepatic insulin resistance. Additionally, the transcription factors Pdx1 and MafA have been demonstrated to play a key role in beta cell function and development. Our lab has previously used viral vectors to induce ectopic expression of transcription factors. In this study, we aim to show that alpha cells could become insulin producing cells.
Methods: Alpha TC cells (a transformed alpha cell line) were cultured with either Exendin-4 or an adeno-associated virus carrying Pdx1 and/or MafA. The viral vectors were constructed from recombinant rAAV plasmid vectors, pAAV-CMV-Pdx1, pAAV-CMV-MafA and pAAV-CMV-Pdx1-2A-MafA were constructed from pAAV-CMV-ZsGreen. AAV serotype 8 has been used in pancreatic cell transduction. AAV purification was performed with the rapid and simplified method.
Results: Our data show that alpha TC cells became insulin expressing cells when they were treated with a GLP-1 agonist, Exendin-4, or if they are transfected with an adeno-associated virus leading to ectopic expression of Pdx1 and MafA. We found that Exendin-4 induced alpha cell proliferation and some insulin expression, but most of the key markers of mature beta cells were not expressed. Further analysis show an increased expression of proliferation markers, but none of the mature beta cell markers were present in the cells exposed to Exendin-4. However, for the cells with forced expression of the transcription factors Pdx1 and/or MafA we saw robust expression of insulin and markers of mature beta cells.
Conclusion: In summary, our data suggests that there are two different possible mechanisms of transdifferentiation of alpha TC cells into insulin producing cells utilizing Exendin-4 to induced cell proliferation and immature beta cells, while ectopic expression of Pdx1 and MafA resulted in a more mature beta cell phenotype.