Pancreatic beta-cells secrete insulin in response to increase in blood glucose concentration with a rapid first phase and slower, sustained second phase. This secretion pattern is similar in entire pancreas, isolated islets of Langerhans and single beta-cells and it is disrupted in type 2-diabetes. Insulin stored in secretory vesicles has to undergo preparatory steps upon translocation to the plasma membrane which include docking and priming before being released by exocytosis. A better understanding of the molecules involved in these steps is required to determine the rate limiting factors for sustained secretion. Here these processes were studied in real time using total internal reflection fluorescence microscopy, which enables observation of insulin granules localized at the plasma membrane. A pool of granules morphologically docked at the plasma membrane was found to be depleted upon repeated stimulations. Recovery of the docked pool of granules took tens of minutes and became rate limiting for sustained secretion. Shorter depolarization stimuli did not deplete the docked pool and allowed rapid recovery of releasable granules. When a new granule arrived at the plasma membrane, docking was initiated by de novo formation of syntaxin/munc18 clusters at the docking site. Two-thirds of the granules which arrived at the plasma membrane failed to recruit these proteins and hence failed to dock. Priming involved recruitment of several other proteins including munc13, SNAP25 and Cav1.2 channels. Exocytosing granules were in close proximity to Ca2+ influx sites with high degree of association with Cav1.2 channels. This is because of the association of these channels to exocytosis site through syntaxin and SNAP25. During exocytosis the assembled release machinery disintegrated and the proteins at the release site dispersed. Syntaxin dispersal was initiated already during fusion pore formation rather than after release during exocytosis. This was studied using a newly developed red fluorescent probe - NPY-tdmOrange2 which was the most reliable pH sensitive red granule marker to label insulin granules. Overall these data give new insights into the molecular mechanisms involved in biphasic insulin secretion. Disturbances in the secretion at the level of granule docking and fusion may contribute to the early manifestations of type-2 diabetes.

This thesis gives insight into the way that transcription of the insulin gene is regulated by nutrients. This is achieved primarily by characterising a MAP kinase pathway which links glucose metabolism to the activation of a beta cell transcription factor IUF1. An understanding of the precise mechanisms by which nutrients control beta cell function may be invaluable for the development of artificial cell lines that can be used for gene replacement therapy. A study of the E2 element of the rat II promoter illustrated that at least three factors bound to the region. These were identified as IUF1 (complex D5), USF (complex D4) and an uncharacterised factor D3. IUF1 is a beta cell specific transcription factor that has been implicated previously in glucose responsive insulin gene transcription. IUF1 binds to the insulin promoter in response to high levels of extracellular glucose. USF has been shown to be involved in the carbohydrate responsive transcription of various hepatic genes. The recently characterised stress activated (Reactivating Kinase) MAP kinase pathway was clearly shown to be involved in mediating the link between glucose metabolism within the beta cell and the binding activity of IUF1. Phosphorylation of the factor serves to induce an alteration in protein structure, which converts the factor to an active form that shows a high affinity for its DNA binding site, thus activating transcription. The RK pathway may prove to be a crucial link between nutrient metabolism and the activity of other physiological processes.

The Type 2 Secretion System (T2SS) is responsible for the transport of toxins and enzymes across the outer membrane of many Gram-negative bacteria. A crucial component of the T2SS is a large pore, composed of a multimer of EpsD, named the secretin. This pore inserts in the outer membrane with the assistance of a pilotin (EpsS) or assembly factors (EpsAB), both of which are present within the genome of Vibrio cholerae. The goal of this study was to determine whether or not both assembly mechanisms operate on the same secretin assembly in V. cholerae. Protease deficient mutants generated from an insertion transposon library in V. cholerae epsAB were analyzed. The transposon was found to disrupt the operon encoding VC1702 and epsS. Mutant strains of V. cholerae were constructed or obtained that are deficient in epsA, epsB, epsC, and epsS. Double mutants were constructed that were deficient in epsA and epsS or epsB and epsS. These mutants were tested for assembly of the secretin and secretion of lipase, protease, and cholera toxin. The epsA and epsB mutants have slightly reduced levels of secretion and secretin assembly, while the levels in the pilotin mutant are drastically reduced. The double mutants had little to no assembly, and secretion was reduced to the levels of the control mutant epsC. In an attempt to restore function epsAB was over-expressed in all strains. It successfully complemented the epsA and epsB mutants, and restored levels of secretion to epsS levels in the double mutant, epsAS. In a similar manner to epsAB complementation, epsS was over-expressed. It was found to require the preceding gene VC1702 to complement. The operon, encoding both epsS and VC1702, could complement both epsA and epsS mutations and over-expression increased secretin assembly and secretion to levels greater than wild-type levels. Lastly, a phylogenomic analysis demonstrates that the EpsAB protein complex is found in most orders of the gamma proteobacteria and is ancestral. The pilotins appear to be a late acquisition as they are only found in the family Enterobacteriales.

A reverse-phase high pressure liquid chromatographic method for the detection and quantitation of unchanged ascorbic acid in human urine is described. Twenty-four hour ascorbate excretion profiles from nine subjects were determined. The urinary excretion profiles for orally ingested and intravenously infused ascorbic acid in these nine subjects are compared.

We investigated whether human lysosomal hydrolases, in common with secretory and plasma membrane glycoproteins, associate with the ER chaperone calnexin. Neither $ alpha$- or $ beta$-chains of $ beta$-hexosaminidase A, cathepsin D, nor the endogenous proteases cathepsins B or L associated with calnexin in COS-I cells. Hex $ alpha$-chains misfolded due to either the incorporation of azetidine-2-carboxylic acid, treatment with dithiothreitol, or the presence of a Tay-Sachs Disease mutation (leading to retention of Hex A $ alpha$-chains in the ER) also did not associate with calnexin. Chemical-crosslinking reagents or long-term labeling also failed to show a Hex A $ alpha$-chain association with calnexin. Lysosomal hydrolases also did not associate with the ER chaperone calreticulin. Surprisingly, $ alpha$-L-iduronidase and Hex A $ alpha$-chains associated with calnexin when overexpressed using a CMV promoter. The segregation of lysosomal hydrolases from secretory proteins thus occurs at an earlier stage than predicted. Hydrolase folding appears to be controlled by a pathway different from that used by secretory and plasma membrane glycoproteins.

Our study aims to investigate the role of Syntaxin-3 in glucose stimulated insulin secretion (GSIS) and how it regulates the recruitment to plasma membrane and/or exocytotic fusion of insulin granules. We examined endogenous Syn-3 function by down-regulating its expression using siRNA/lenti-shRNA, which impaired GSIS. Although Syn-3 depleted cells showed no change in the number and fusion of docked granules, there was a reduction in newcomer granules and their subsequent exocytotic fusion. We then examined the effects of overexpressing Syn-3-WT, which enhanced biphasic GSIS. Since open conformation (OF) Syn-1A was reported to enhance exocytosis by promoting SNARE complex formation, we constructed OF Syn-3. Exogenous OF Syn-3 had no effect on secretion as it is unable to be trafficked to insulin granules. Taken together, we conclude that Syn-3 facilitates mobilization of newcomer insulin granules to the plasma membrane, to contribute to both first and second phase of GSIS in pancreatic beta cells.

Our study aims to investigate the role of Syntaxin-3 in glucose stimulated insulin secretion (GSIS) and how it regulates the recruitment to plasma membrane and/or exocytotic fusion of insulin granules. We examined endogenous Syn-3 function by down-regulating its expression using siRNA/lenti-shRNA, which impaired GSIS. Although Syn-3 depleted cells showed no change in the number and fusion of docked granules, there was a reduction in newcomer granules and their subsequent exocytotic fusion. We then examined the effects of overexpressing Syn-3-WT, which enhanced biphasic GSIS. Since open conformation (OF) Syn-1A was reported to enhance exocytosis by promoting SNARE complex formation, we constructed OF Syn-3. Exogenous OF Syn-3 had no effect on secretion as it is unable to be trafficked to insulin granules. Taken together, we conclude that Syn-3 facilitates mobilization of newcomer insulin granules to the plasma membrane, to contribute to both first and second phase of GSIS in pancreatic beta cells.

Thesis (Ph.D. 1979) from the Department of Biochemistry, University of Adelaide.

Thesis (D.Phil (Zoologyy ))--University of Pretoria, 2002. / Includes abstract in English. Includes bibliographical references.

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