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The pancreatic ATP-sensitive potassium (KATP) channels couple glucose metabolism to excitability of the pancreatic β-cells to regulate insulin secretion. The channel subunits, Kir6.2 and SUR1, are encoded by the KCNJ11 and ABCC8 genes respectively. Genetic polymorphisms in these genes, which reduce channel activity, cause congenital hyperinsulinism (CHI) characterized by insulin hyper-secretion and hypoglycemia. The hERG (human ether-a-go-go related gene) potassium channels,encoded by the KCNH2 gene, contribute to the rapidly activating delayed rectifier K+ current (IKr), which is responsible for rapid repolarisation of the cardiac action potential. Decreased hERG channel function causes the Long QT syndrome 2 (LQTS2) and life threatening cardiac arrhythmias. Several mutations in these two clinically important potassium ion channels alter their surface density leading to disease. Therefore, it is of fundamental importance to investigate the trafficking mechanisms that regulate the surface density of these channels. Techniques in cell biology, molecular biology and biochemistry were employed to identify the molecular basis of Sar1-GTPase dependent ER exit of the KATP and hERG channels in COPII vesicles. Blocking the cargo binding sites on the Sec24 protein of the COPII coat with membrane-permeable synthetic peptides prevented ER exit of both these channels. While the diacidic 280DLE282 sequence on the Kir6.2 subunit of KATP channels was found to be the ER exit motif required for entry of the channels into COPII vesicles at the ER exit sites, such a motif was found to be absent on hERG Cterminus. Further, endocytic trafficking mechanism of hERG channels was studied in recombinant (HEK MSRII and HeLa) and native (neonatal rat cardiac myocytes) systems using cell biological and pharmacological tools. hERG channels were found to be internalised by a dynamin-independent, raft-mediated, and ARF6-dependent pathway. A prolonged block of this pathway revealed that the channels could also undergo internalisation by an alternate dynamin-mediated pathway. Internalised hERG channels were found to recycle back to the cell surface and undergo lysosomal degradation. Degradation of the channels was enhanced when Rab11a-GTPase function was disrupted leading to reduced surface density indicating that recycling is crucial to maintain cell surface density of the channels. Thus this study investigated and compared the previously unknown mechanisms of biosynthetic and endosomal trafficking of the KATP and hERG potassium channels with a conclusion that these processes play an important role in maintaining surface density and thereby in the function of these channels in physiological and patho-physiological conditions.

Glucose homeostasis depends on the ability of insulin to stimulate glucose uptake into both muscle and adipose tissue by promoting the translocation of glucose transporters (GLUT4) from intracellular sites to the plasma membrane (PM). In individuals with Type 2 diabetes the ability of insulin to stimulate glucose transport is impaired. The incidence of Type 2 diabetes is increasing worldwide, highlighting the need to understand the molecular basis of insulin-stimulated glucose uptake. GLUT4 translocation is a specialised example of vesicular trafficking. Within the context of vesicle trafficking, all eukaryotic cells contain a common set of conserved components responsible for the execution of membrane fusion. Central to this machinery are members of the SNARE (soluble NSF attachment protein receptor) family of proteins. The process of SNARE-mediated membrane fusion needs to be tightly regulated and the SNARE proteins are partially responsible for the specificity in communication between eukaryotic subcellular organelles. Other proteins such as the Sec1p/ Munc18 (SM) proteins were shown to be essential for SNARE-mediated membrane fusion. Several methods were used to test the ability of SNARE proteins to drive membrane fusion, and one of the most important methods described to date is the in vitro fusion assay used in this study. The first topic addressed in this thesis was related to the molecular interactions between the regulatory SM protein Munc18c and the SNARE proteins VAMP2 and syntaxin 4. The use of pull-down assays revealed the novel fact that Munc18c interacts not only with the t-SNARE syntaxin 4 but also with the v-SNARE VAMP2 via its SNARE motif. The SM:v-SNARE interaction was disrupted by the presence of syntaxin 4 revealing that these two SNARE proteins compete for binding to Munc18c. Next, the role of Munc18c in membrane fusion driven by four different versions of syntaxin 4 plus SNAP23 and VAMP2 liposomes, was investigated using the well-characterised in vitro fusion assay. Results suggested that Munc18c negatively regulates SNARE- mediated membrane fusion by inhibiting the formation of SNARE complexes. Interestingly, deletion of the first 36 amino acids of syntaxin 4 was not sufficient to suppress Munc18c negative regulation of fusion indicating that this inhibition might involve other interactions apart from the short N-terminal peptide of syntaxin 4. Finally, the role of phosphorylation in SNARE complex formation was assessed using several techniques such as site-directed mutagenesis, pull-down assays and radiolabelling studies. Data obtained revealed that both syntaxin 4 and Munc18c become phosphorylated in vitro by a recombinant cytoplasmic insulin receptor kinase (CIRK). Munc18c phosphorylated by CIRK was unable to bind syntaxin 4 in vitro. Furthermore, phosphomimetic mutations were also introduced on both proteins and pull-down assays indicated that phosphorylated Munc18c is unable to interact with both syntaxin 4 and VAMP2, whereas phosphomimetic mutations in syntaxin 4 did not affect the interaction with its cognate SNARE proteins and Munc18c. These results were very useful to further understand and confirm the importance of phosphorylation in SNARE complex formation. Collectively, these data suggest that Munc18c acts through different modes of interaction with its cognate SNARE proteins, and support a model in which Munc18c negatively regulates SNARE complex formation. However, this regulation might also be dependent on other factors such as phosphorylation upon insulin signalling.

The molecular mechanism of glucose regulated insulin secretion, which is essential for maintaining normal blood glucose levels, is not fully understood. The disruption of insulin secretion is characteristic of type 2 diabetes, a disease which affects 300 million people globally, and is strongly associated with obesity. The role of plasma membrane K⁺ATP channels in insulin secretion has been well established, however their intracellular localisation and role in organellar cation homeostasis has been debated. The aim of this study was to verify the subcellular presence, and location of the K⁺ATP channel subunits Kir6.2 and SURl in pancreatic β-cells, which may help to elucidate the hill mechanisms underlying insulin secretion .K⁺ATP channel subunits were localised in MIN6 pancreatic β-cells lets, using immunogold staining in conjunction with Transmission Microscopy (TEM). Kir6.2 and SURl were successfully localised to the insulin containing vesicles, but not to mitochondria. Further staining was also attributed to other cellular organelles such as the endoplasmic reticulum (ER) and Golgi apparatus.

Insulin is the principal anabolic hormone and is responsible for a vast array of cellular and physiological processes. One of the most fundamental of insulin's actions is the maintenance of glucose homeostasis, primarily ahieved by the selective targeting of the GLUT4 (glucose tansporter 4) vesicle from intracellular stores to the plasma membrane. Insulin triggered signalling cascades activate kinases, notably PKCζ, which influence proteins required for GLUT4 vesicle targeting to the plasma membrane. These vesicle targeting proteins include syntaxin4, VAMP2 and muncl8c. Muncl8c is thought to preclude interaction between VAMP2, present in the GLUT4 vesicle, and syntaxin4 which is found in the plasma membrane, thus preventing GLUT4 vesicle exocytosis in the basal state. Previously, insulin action was thought to release muncl8c from syntaxin4, thus enabling GLUT4 vesicle docking at the plasma membrane. 80K-H forms a complex with PKCζ and muncl8c in response to insulin, thus acting as a bridge to enable the cell signalling to affect the GLUT4 vesicle trafficking machinery. In this thesis, a number of in vitro and in vivo techniques, including fluorescence correlation spectroscopy (FCS), were used to determine the nature and role of the protein:protein interactions in GLUT4 vesicle trafficking. The results show that insulin activated PKCζ acts on 80K-H to release it from a membrane tether. This un-tethering of 80K-H causes a repositioning of muncl8c on syntaxin4. It is this repositioning of muncl8c, rather than the release of this protein from syntaxin4, that enables VAMP2 to bind syntaxin4 thus facilitating the docking of the GLUT4 vesicle at the plasma membrane. This is the first time that it has been shown that muncl8c isn't merely released from syntaxin4 in response to insulin. These results further elucidate the molecular mechanisms of insulin action, which will hopefully lead to treatments for diseases such as Type II diabetes in the future.

The pancreatic beta-cell rapidly releases insulin in response to glucose and in order to maintain insulin stores within the beta-cell, there is a rapid and specific increase in proinsulin (PI) synthesis (10-20 fold within 60min). This rapid increase in PI synthesis is mediated entirely at the post-transcriptional level, likely through an increase in the rate of protein synthesis. As the regulation of glucose-stimulated proinsulin synthesis is poorly understood, the first objective of this thesis was to investigate the mechanism of glucose-regulated proinsulin synthesis in pancreatic beta-cells. This thesis shows that contrary to previous reports, there is no pool of 'free' preproinsulin (PPI) mRNA at low glucose concentrations and that glucose does not stimulate the rate of de novo initiation. However, glucose does stimulate the recruitment of ribosomes onto ribosome-associated PPI mRNA, indicative of an increase in the rate of initiation of translation. Additionally, glucose stimulates the recruitment of PPI, PC2 and CPH mRNAs to the ER. Moreover, PPI mRNA is preferentially recruited to the ER over the other secretory protein mRNAs. This preferential recruitment of PPI mRNA may be an important mechanism for the specific up-regulation of PI synthesis at high glucose concentrations. Indeed, data presented here shows that the recruitment of PPI mRNA to the ER plays an important role in glucose stimulated PI synthesis. In addition to an increase in PI synthesis, glucose stimulates a rapid increase in the synthesis of more than 260 unidentified beta-cell proteins. It is likely that this rapid up- regulation of beta-cell proteins is important in beta-cell function. Therefore, microarray analysis of polysomal mRNAs was used to identify beta-cell proteins that were translationally regulated in response to increases in glucose concentration. Interestingly, the majority of polysomal mRNAs whose levels changed between low and high glucose encoded proteins important in transcription and oxidative stress.

Transcription factors are vitally important to the developmental biology of all life. They also define many of the developmental stages of organs within the body. Their expression allows for the activation of developmentally important cues and signalling cascades, through activation of specific subsets of genes. When transcription factor function is disrupted, it can have serious repercussions on the development and function of organ systems. This dysfunction, in many cases, leads to the onset of diseases such as cancer and diabetes. The general objectives of this thesis were to describe the role and function of several transcription factors vitally important in the development and maintenance of the pancreas through three lines of research. Firstly, neurogenin3 (ngn3) is vitally important to the development of the islets of Langerhans. Knocking out this gene prevents mice from developing endocrine cells of the pancreas (islets of Langerhans). This thesis set out to label the ngn3 positive precursor cell population in developing mice, and characterise their phenotype. Detailed immunohistochemistry and fluorescence activated cell sorting coupled with reverse transcriptase (RT)-PCR were employed to fully characterise the EGFP positive cell population at embryonic day (E)15.5 and E l8.5. EGFP positive cells were able to be isolated at both E l5.5 and E l8.5 and were found to co-express endocrine markers both in immunohistochemical and RT-PCR based assays. Marking cells that are developmentally important to pancreas function, and being able to identify their phenotype, may provide clues to markers that could be exploited as targets for future diabetes therapies. Secondly, pancreatic duodenal homeobox-1 (PDX-1) is another critically important transcription factor in both pancreas development and in maintaining the insulin expression of the beta cells in the islets of Langerhans. Preventing PDX-1 function causes severe agenesis of the pancreas, and mutations in the gene cause one form of diabetes. This thesis describes the production of a super-activated form of the PDX-1 protein using the transactivation domain VP 16. The main objective of this work was to drive PDX-1 function in a non-beta cell. Using chromatin immunoprecipitation (ChIP) assays, the PDX-1 VP 16 protein appeared to increase the acetylation state of the insulin promoter in the mouse non-beta cell line (alpha-TCI.6), particularly through altering the acetylation state of the insulin 2 promoter. The use of the PDX-1VP16 protein could provide a potential starting point for research into driving differentiation of stem cells to a pancreatic phenotype. Finally, there are four transcription factors that are known to drive expression of the insulin gene synergistically. They are the homeodomain protein PDX-1, the leucine zipper protein MafA, and the basic helix loop helix heterodimer beta2/E47. All of these proteins have binding sites within 300 bp of the transcriptional start site of the insulin gene. Through the use of ChIP assays this thesis shows, for the first time, the ordered cyclical binding of these four transcription factors on the insulin 2 gene promoter in the mouse beta cell line MIN-6. Understanding how the transcription factors that drive insulin expression interact on the promoter in vivo could be used for future studies to understand the process of insulin gene transcription both in healthy and diabetic patients. This may then shed light on the processes that cause the perturbations in insulin transcription seen with certain forms of diabetes.

The hypothesis that the adipocyte hormone leptin has modulatory effects on the secretion of insulin in the presence of glucose by the islets of Langerhans of normal male rats, and which was postulated to be inhibitory, was investigated. Evidence obtained indicated that the modulatory effect was mediated through the possible combination of neural and endocrine parameters. A previously unreported humoral connection between the intestine and the pancreas was demonstrated, by which incretins could potentially mediate their effects on the production of insulin and other endocrine functions. In the study, an ex vivo perfusion of the pancreas was used, in a purposely designed perfusion system with a surgery platform, and an artificial perfusion medium (KRBB medium) with combinations of concentrations of 4, 8, 12 & 16mM glucose and leptin at 0, 5, 10, 15 & 20ng/ml, in the aim of developing an index for the modulatory effect of leptin on insulin secretion. Insulin output was measured by radioimmunoassay and the results were statistically assessed and analyzed. It was confirmed that insulin secretion from the ex-vivo perfused pancreas in response to glucose stimulation follows a bi-phasic secretion pattern. Leptin reduced insulin secretion at 8 and 12 mmol/L stimulatory concentrations of glucose but either stimulated or had no effect at 4 and 16 mmol/L glucose. A U-shaped dose-response relationship was observed when the range of leptin and glucose concentrations of 5, 10, 15 and 20 ng/ml and 4, 8, 12 and 16 mmol/L respectively were examined, where minimal suppressions were observed at low and high concentrations of both leptin and glucose. In addition, leptin showed distinct suppressive effects on phase one and phase two concentrations depending on the glucose concentration used. This suggested the presence of two or more interdependent intracellular cascades responsible for the secretion and synthesis of insulin in the B-cells of the pancreas, through which leptin exerts its effects on both phases. The presence of various potential incretins together with the newly reported humoral connection between the duodenum and the pancreas implicates other factors controlling insulin secretion and the manner by which leptin modulates insulin secretion. It was concluded that leptin possesses both an inhibitory and a stimulatory effect on insulin secretion and synthesis in a dose-dependent manner and in the presence of different glucose concentrations. This modulatory effect may depend on the presence of certain incretins, possibly including Glucagon Like Peptide (GLP-1), where the common intracellular pathways present potential common sites of action for the inhibitory cascade of leptin.

Complications associated with type 1 diabetes mellitus (T1DM) are a major health care problem worldwide. Although injectable insulin permits a relatively normal lifestyle, it falls a long way short of the perfect treatment for T1DM─it is not a cure. Pancreatic islet transplantation has proved a promising treatment for T1DA, however, progress has been limited due to a shortage in the supply of cadaveric pancreases. An exciting alternative to this is stem cell therapy, whereby β cells would be generated in vitro as an unlimited supply for transportation. In order to achieve these ambitious goals, an in depth understanding of human pancreas development is highly desirable. The results presented in this thesis aim to provide some of this information. They are also complimented by initial strategies for human fetal pancreas culture, which is aimed at proliferating and differentiating early epithelial cells into functional β cells. The rationale underlying this work is presented at the end of chapter 1 and each chapter of results contains an abstract summarising its data.

The work presented in this thesis provides evidence that a maternal low protein (LP) diet during gestation and lactation periods in rats 'programs' pancreatic β-cell glucokinase (GK) the glucose sensor and glucose induced insulin secretion response in newborn, suckling and adult offspring. Pregnant female rats were divided into 3 groups, group A (20%) was kept on a normal protein diet, group B on a LP (6[percent]) diet during pregnancy and lactation and group C on a LP diet during pregnancy and a normal protein diet during lactation. The glucose- induced total insulin secretion response and peak insulin secretion was markedly reduced in group B newborn and 3-week old suckling pups compared with group A controls which is a result of poor nutrition. The insulin secretion also showed an altered secretory response in group B adults and group C 3- week old and adult stage pups compared with group A. The GK protein levels were reduced in newborn, 3-week old pups and adult rats from both group B and C. The GK enzyme exhibited interesting changes in enzyme activity to convert glucose to glucose-6-phosphate particularly in the kinetic reaction parameters pertaining to the enzymes affinity for glucose (Km values) and maximal reaction velocity (Vmax). Poor nutrition reduced GK protein enzyme activity and Km values. The prenatal and postnatal LP diet appears to have a permanent programming effect by increasing the GK affinity for glucose and decreasing the reaction velocity indicating that the critical period of programming of GK's function is after birth during the postnatal weaning period since the adult offspring of group B when fed a normal protein diet showed no reversal in the Km values of the enzyme. Similar experiments on adult offspring of group C which were fed a normal protein diet during weaning and after showed normalisation of the GK Km values for glucose but still exhibited a permanent reduction in Vmax. In conclusion low protein diets during both pregnancy and weaning have immediate and sustained irreversible effects on glucose homeostatic mechanisms of a mother's offspring. Fetal and early life infantile nutrition programs pancreatic β-cell function, with poor nutrition predisposing to diabetes in later life.