Type II diabetes mellitus is consistently associated with the deposition of amyloid in the islets of the pancreas. The major protein component of the amyloid of diabetes has been purified and termed Diabetes Associated peptide (DAP). This 37 amino acid peptide has been colocalized to the secretory granule of islet cells with insulin, and has a 46% identity with calcitonin gene related peptide (CGRP). A proteolytically processed tissue- specific neuropeptide. I have cloned and sequenced the rat cDNA for DAP, and like CGRP, it contains dibasic residues which would predict proteolytic processing of precursor to a mature product. By Northern analysis, the DAP mRNA is present in pancreatic islet cells. Expression of DAP in other tissues will be assessed by Northerns and used to delineate sources for studies of the structure of the protein. Insulinoma cell lines and rat pancreatic islets maintained in short term cult""""""""re will be used in immunoprecipitation studies with anti-peptide antibodies derived from the predicted proteolytic fragments of the DAP precursor. This will confirm expression and processing of the protein in these cell lines so that the kinetics of the processing can be established through pulse-chase labeling. Pharmacologic maneuvers intended to stimulate DAP expression in normal animals will be assayed by in situ hybridization and immunoprecipitation. Once defined in normal animals, genetic models of diabetes, will be used to determine if this pathophysiologic state changes the expression and processing of DAP in a presumably deleterious way.
