This program project continues its investigation of the cell and molecular biology of the four neuroendocrine cell types comprising the pancreatic islets, so as to clarify their inter-relationships and distinguishing features. Of particular relevance to the mechanism of type I diabetes are the unique qualities of the insulin producing beta cells, which are selectively destroyed, leaving the alpha, delta, and PP cells intact. An exciting candidate is the 65kd isoform of glutamic acid decarboxylase, which was discovered by Baekkeskov et al. in the context of this program project to be the '64kd' beta cell autoantigen in type I diabetes. GAD65 has been and will continue to be a major focus of this interactive program, with all three projects planning studies on the biology of GAD65. Our plans are: Project 1 (Hanahan) will produce mice carrying gene knockouts of GAD65 and betacellulin, a TGF-alpha family member expressed in beta cells, to assess the roles of these genes in embryogenesis and in islet development and function. The oncogene SV40 Tag will be integrated into the genes for somatostatin and pancreatic polypeptide, and gene-targeted mice produced, from which delta and PP cell tumors and tumor cell lines will be derived, to complete the set of representative islet cell lines. A cell type (mPAC) associated with juvenile islets will be investigated both for its potential as an islet progenitor, and as a target for pancreatic ductal adenocarcinoma. Project 2 (Baekkeskov) will characterize the biosynthetic pathway of GAD65, which is membrane anchored, and identify the sequences that elicit its subcellular localization to synaptic like micro-vesicles. Tansgenic mice over-expressing human GAD65 will be investigated to learn more about the normal functions of GAD in beta cells, and its ability to disrupt beta cell function through improper expression. Similarly, the GAD65 knockout mice will be studied to ascertain the physiological consequences of a failure to produce this enzyme. The capability of GAD to act as an autoantigen in beta cells of transgenic mice will be investigated, which should contribute to our understanding of its roles in the human autoimmune disease. Project 3 (German/Rutter) will investigate transcriptional regulation of genes implicated in islet cell function and disease, including the islet hormones, glucokinase, islet amyloid, and GAD65, using cell lines representative of differentiated and 'progenitor' islet cell types. The goal is to define the key transcriptional regulatory proteins that elicit cell type specificity among the closely inter-related alpha, beta, delta, and PP cells, with the expectation that this information will help explain the unique susceptibility of b cells to immune destruction. Having identified candidates for key islet regulatory factors, gene knockout mice will be produced to assess the importance of these factors for embryogenesis in general, and islet cell development and function in particular.
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