The broad and long term objectives of the research are to understand the detailed mechanisms by which glucose and other physiological secretagogues stimulate insulin release. The work will combine physiological and biochemical techniques and will be performed on beta-cell lines and rodent islets. However, the eventual goal is the description of these events in human islets. The studies will be focused on exocytosis with emphasis on the docked pool of insulin-containing granules, the rapid release of which appears to be largely responsible for the first phase of glucose-stimulated insulin release. The major aims are 1. to determine the mechanisms which control the size and releasability of the docked granule pool in beta-cells; 2. the nature of the primed (readily releasable) state of the docked granules; and 3. the effects of glucose; increased cyclic AMP and activated PKA; and activated PKC-on the docked granules. We have developed a method to study the assembly and dissociation of the core complex of SNARE proteins that we think is a marker of the docked granule pool. The core complex is defined on the basis of the interaction between the plasma membrane SNARE proteins syntaxin and SNAP-25, and the granule membrane SNARE protein synaptobrevin. Antibodies against syntaxin co- immunoprecipitate synaptobrevin and the co-immunoprecipitation indicates the presence of the core complex of docked granules. With the ability to quantify the core complex, many questions related to the stimulation, modulation and inhibition of insulin release can be answered. We will study several effects of glucose on the core complex. For examples, we will study the changes that occur in the relative size of the core complex with the time of exposure to glucose. We will study the relationship between the size of the docked pool and the rate of insulin release. We will study the composition of the core complex under different physiological conditions. In particular, we believe we have the ability to study the core complex in both the primed state (which occurs under basal conditions) and in the largely unprimed state (which occurs in the second phase of glucose-stimulated insulin secretion). We will determine what changes take place in the core complex in response to increased [Ca2+]i, to the glucose augmentation pathway(s), and to increased PKA and PKC activity.
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