Tumour protein D52 is hypothesised to be involved in regulated secretion in the pancreatic acinar cell, indicated by rapid phosphorylation in response to secretagogue stimulation. The phosphorylation status of TPD52 in response to glucose stimulation in the pancreatic β cell was analysed by an <i>in vitro</i> method involving the incorporation of  ³²P-ATP into the TPD52 protein. Limitations of the system prevented the full confirmation of the rapid phosphorylation of TPD52 in response to glucose stimulation, however preliminary data suggested this was likely to occur. Bio-informatic phosphorylation site prediction was used and we hypothesised that CaMKII was the key enzyme phosphorylating TPD52 in the β cell at the serine phosphorylation site within the motif SPTFKsFEEKV. Proteomic analysis of the phosphorylated TPD52 isoform confirmed the novel identification of the motif phosphorylated by CaMKII. To determine the effect of TPD52 on regulated secretion we reduced TPD52 RNA levels using vector based siRNA. Two stable knockdown cell lines were created  showing a 50 % and 80 % reduction in RNA levels. No difference was observed in glucose stimulated insulin secretion between TPD52 knockdown and control cells. Patch clamp experiments also showed no significant difference in capacitance changes following depolarisation between knockdown and control clones. Quantification of insulin granule number and size from TEM pictures confirmed a high level of inter-clonal variation. Focus shifted to explore the previously published protein interaction between TPD52 and MAL2 as both proteins have been identified within the β cell. The original interaction between MAL2 and TPD52 was confirmed; however, when natural isoforms with shorter N-terminal regions were substituted for TPD52 and MAL2 the interaction was diminished slightly. This suggests that the N-terminal regions are not integral to the interaction between the proteins, but are perhaps required for stability of the complex.

Thesis (Ph.D.)--Aberdeen University, 2008. / Title from web page (viewed on July 14, 2009). Includes bibliographical references.

Embryonic stem (ES) cells represent a possible source of islet tissue for the treatment of diabetes.  Achieving this goal will require a detailed understanding of how the transcription factor cascade initiated by the homeodomain transcription factor Pdx1 culminates in pancreatic beta-cell development.  Here we describe a genetic approach that enables fine control of Pdx1 transcriptional activity during endoderm differentiation of mouse ES cell.  By activating an exogenous Pdx1VP16 protein in populations of cells enriched in definitive endoderm we show a distinct lineage-dependent requirement for this transcription factor’s activity.  mimicking the natural biphasic pattern of Pdx1 expression was necessary to induce an endocrine pancreas-like cell phenotype, in which 30% of the cells were beta-cell-like.  Cell markers consistent with the different beta-cell differentiation stages appeared in a sequential order following the natural pattern of pancreatic development.  Furthermore, the differential beta-like cells secreted C-peptide (insulin) in response to KC1 and IBMX, suggesting that following a natural path of development in vitro represents the best approach to generate functional pancreatic cells.  Together these results reveal for the first time a significant effect of the timed expression of Pdx1 on the non-beta cells in the developing endocrine pancreas.  Collectively, we show that this method of <i>in vitro</i> differentiation provides a template for inducing and studying ES cell differentiation into insulin-secreting cells.

High circulating glucose and non-esterified free fatty acid (NEFA) levels can cause pancreatic β-cell failure. The molecular mechanisms of this β-cell glucolipotoxicity are yet to be established conclusively. In this thesis by exploring mitochondrial energy metabolism in INS-1E insulinoma cells and isolated pancreatic islets, a role of mitochondria in pancreatic β-cell glucolipotoxicity is uncovered. It is reported that prolonged palmitate exposure at high glucose attenuates glucose-stimulated mitochondrial respiration which is coupled to ADP phosphorylation. These mitochondrial defects coincide with an increased level of mitochondrial reactive oxygen species (ROS), impaired glucose-stimulated insulin secretion (GSIS) and decreased cell viability. Palmitoleate, on the other hand, does not affect mitochondrial ROS levels or cell viability and protects against the adverse effects of palmitate on these phenotypes. Interestingly, palmitoleate does not significantly protect against mitochondrial respiratory or insulin secretion defects and in pancreatic islets tends to limit these functions on its own. Furthermore, strong evidence suggests that glucolipotoxic-induced ROS are of a mitochondrial origin and these ROS are somehow linked with NEFA-induced loss in cell viability. To explore the mechanism of glucolipotxic-induced mitochondrial ROS and associated cell loss, uncoupling protein-2 (UCP2) protein levels and activity were probed in NEFA exposed INS-1E cells. It is concluded that UCP2 neither mediates palmitate-induced mitochondrial ROS production and the related cell loss, nor protects against these deleterious effects. Instead, UCP2 dampens palmitoleate protection against palmitate toxicity. Collectively, these data shed important new light on the area of glucolipotoxicity in pancreatic β-cells and provide novel insights into the pathogenesis of Type 2 diabetes.