The present proposal has four major objectives that are based on the following considerations: A. Genetically endowed with the capacity to respond to the prevailing glucose level by altering insulin secretory rates, the pancreatic beta-cell plays a preeminent role in the maintenance of glucose homeostasis. B. Phospholipase C (PLC)/protein kinase C (PKC) activation regulates insulin secretory responses to glucose. C. Pronounced species differences exist in the expression levels of several PLC isozymes and this appears responsible for the profound differences in glucose-stimulated insulin secretion that exist between rat and mouse islets. D. Muscarinic cholinergic receptor knockouts afford us the opportunity to establish how signaling via PLC activation contributes to biphasic insulin secretion, time-dependent potentiation and time-dependent suppression of secretion and the in vivo consequences of these alterations. E. Genetic manipulation of the proteins that control insulin signaling have led to a reevaluation of the autocrine effects of insulin and/or insulin-like growth factor I (IGF-I) on the beta-cell. F. Disrupted insulin secretion culminates in diabetes. These are the Specific Aims. 1. Using the rat insulin II promoter to achieve tissue-specific beta-cell expression, transgenic mice overexpressing PLCbeta1 and PLCdelta1 will be generated. Will the overexpression of these isozymes convert the minimal mouse islet glucose response to the exuberant biphasic insulin secretory response that characterizes glucose-stimulated rat or human islets? 2. Coupled to PLCbeta1 via a muscarinic receptor, cholinergic stimulation of mouse islets compensates for their minimal in vivo and in vitro responses to glucose stimulation. Muscarinic (M1 and M3) receptor knockouts have been generated. How do these alterations change the constellation of effects that cholinergic stimulation exerts on the beta-cell? Are there compensatory changes in PLC isozyme expression and how do these animals respond to diets designed to induce obesity? 3. Antisense oligonucleotides will be employed to determine the relationship between reductions in PKCalpha expression in islets and their secretory responsiveness. 4. IGF-I inhibits insulin secretion, presumably by the activation of phosphatidyinositol 3-kinase (PI3k), and reduced expression of the p85alpha regulatory subunit of PI3K is associated with augmented glucose-induced secretion. Can we identify the biochemical pathways sensitive to IGF-I signaling in the beta-cell? Considering the fact that inhibitors of PI3K are as potent as the oral secretagogues utilized to treat Type II diabetes, potentially new therapeutic targets may be identified. These aims will be accomplished by using sound principles of experimentation and employing isolated islets that retain their in vivo sensitivity to glucose.
NIH ExPorter
NIH ExPorter