Abstract

This study aimed to investigate the molecular mechanisms of glucose stimulated insulin secretion (GSIS), specifically the role of microRNAs (miRNAs) in this process. TaqMan low density miRNA arrays identified a panel of 10 miRNAs down-regulated in glucose non-responsive compared to glucose responsive MIN6 cells. Functional investigations involving knockdown of mir-200a, mir-130a and mir-410 in MIN6 cells exhibiting GSIS resulted in reduced GSIS. This suggested that these three miRNAs functioned in supporting the capability of MIN6 cells to secrete insulin in response to stimulatory glucose levels i.e. they are “pro-GSIS” miRNAs, presumably downregulating “anti-GSIS” mRNAs/protein. Over-expression studies of mir-410 supported the idea that it may enhance levels of GSIS. However, the MIN6 system was somewhat unpredictable. The advent of iPS technology represented a possible route for generation of human pancreatic beta cells for the study of regulated insulin secretion. Attempts were made to generate iPS cells from the MiaPaCa2 pancreatic adenocarcinoma cell-line, normal human epidermal keratinocytes, and normal human limbal epithelial cells. Alterations in cell morphology and marker expression were observed, possibly representing partially reprogrammed cells, but fully reprogrammed iPS cells were not identified. Directed differentiation of an established iPS cell line was performed in 2D and 3D culture, with improved efficiency of definitive endoderm formation in 3D cultures (a step on the differentiation routetowards beta cells). In parallel, serum from diabetes patients was examined to identify new disease iomarkers in a collaborative clinical study with Connolly Hospital, Blanchardstown. iRNAs identified as being potentially involved in GSIS from the MIN6 study were easured in serum from type 1 and type 2 diabetes patients. Proteomic and etabolomic profiling were performed on type 1 diabetes samples. These studies emonstrated the importance of stringent criteria for ontrol sample selection, as evidenced by altered caffeine and lipid metabolism in control samples in this study.