Molecular mechanisms of insulin secretion : identification of a novel glucose regulated protein

Diabetes mellitus has reached epidemic proportions and affects more than 200 million people world-wide. It is a heterogenous metabolic disorder, defined by the presence of hyperglycaemia. This study sought to use the application of proteomics to elucidate the mechanisms involved in glucose stimulated insulin secretion in MIN6 pancreatic p--cells, by determining and identifying differential expression of proteins in response to glucose stimulation (i) in a global context, and (ii) at the insulin-containing vesicle level. Optimisation of 2-dimensional gel electrophoresis (2DGE) resulted in the determination of the MIN6 p-cell proteome and the identification of 84 different proteins. Many proteins were found to be differentially expressed as a result of glucose stimulation, reflecting the enhancement of insulin exocytosis. These include the up-regulation of molecular chaperones involved in protein biosynthesis, such as T complex protein, which mediates folding of actin and tubulin, and heat shock cognate protein 70, which is involved in uncoating of clathrin coated vesicles. Proteins known to be involved in the transport of insulin- containing vesicles from the storage pool to the readily releasable pool, such as actin, tubulin B5 and tubulin alpha were also identified to be up regulated. For an increase in insulin biosynthesis to manifest itself as a rise in glucose-stimulated insulin secretion, translocation of insulin-containing vesicles from the Golgi region to the plasma membrane, and maturation of vesicles are required. Proteomic analysis of the insulin-containing vesicle was undertaken to obtain a better understanding of vesicle regulation. To date 24 proteins have been identified including the heat shock cognate protein - involved in uncoating of clathrin- coated vesicles; vacuolar ATP synthase subunits, which maintain a low intravesicular pH, important for proinsulin conversion and storage of zinc-insulin hexamers, and glucose-regulated proteins 54, 78 and 94. Several proteins not directly attached to vesicles, but involved in vesicle cycle regulation co purified with vesicles for example actin and tubulin. Both actin and tubulin form core complex with synapsin proteins, synaptotagmin and SNAP-25 during vesicle docking thus explaining their presence on insulin vesicles. Proteomic analysis of the MIN6 pancreatic p-cell also identified translationally controlled tumour protein (TCTP) to be significantly up-regulated in response to glucose stimulation, leading to further investigation into its role and function. This is the first study (i) to identify the presence of TCTP in the p-cell by proteomic analysis and (ii) to investigate the regulation and function of TCTP in the pancreatic p-cell. From the data presented TCTP has been identified as a novel glucose-regulated protein, whose regulation involves post-translational modification by both phosphorylation and glycosylation. These post-translational modifications are regulated and induced by glucose, and have been demonstrated to affect its cellular localisation, with an increase in the nuclear localisation of TCTP in response to glucose stimulation, as well as increased localisation with the cytoskeletal protein actin. Additionally, reducing TCTP protein expression using siRNA resulted in enhanced p-cell death. Preliminary studies also suggest that TCTP may be involved in the process of insulin secretion, with a significant decrease in insulin secretion observed as a result of reduced TCTP protein expression. Data presented in this study illustrate that TCTP is not only vital to p-cell survival, but potentially is a key player in GSIS, at multiple levels; from the regulation of gene transcription, to the trafficking and release of the insulin-containing vesicles.EThOS - Electronic Theses Online ServiceGBUnited Kingdo