A previously unknown putative polypeptide hormone identified as islet amyloid polypeptide (IAPP) was recently purified from islet amyloid (IA) of cats and humans with TYPE 2 diabetes mellitus (DM). In that IAPP -derived amyloid represents the most characteristic islet lesion in this from of DM in into the development of DM in cats and humans, information concerning the production and function of IAPP may provide new insights into the development of DM associated with aging. Our broad, long term objectives are to characterize the production and function of the IAPP-precursor, and to further elucidate the relationship of IAPP to DM. These objectives will be achieved through a series of experiments designed to: 1. Compare the primary chemical structure of IA-derived IAPP from multiple diabetic cats (by HPLC and amino acid sequence analysis) with that of IAPP or an IAPP precursor isolated (by affinity column separation) from pancreas or serum of normal cats. Specifically, we wish to investigate the possibility that certain structural aberrations in IAPP of diabetics are linked to transformation into amyloid fibrils; 2. Compare IAPP-precursor production in cats with and without DM (i.e., normal and normogylycemic cats with impaired glucose tolerance (IGT). Cats will be categorized by evaluations of fasting blood glucose, RIA insulin/glucagon levels and urinalysis prior to IV glucose tolerance test. LAPP-precursor production will be evaluated by immunocytochemical analysis of pancreatic B cells and by determination of serum IAPP using RIA. Pancreases will be histologically (Congo red) evaluated-red) evaluated to determine if LA deposits in nondiabetic cats are early reflections of IGT or a """"""""prediabetic"""""""" state; 3. Elucidate the molecular biology of IAPP production in normal islets, and also in IGT and DM. LAPP cDNA will be obtained from a lambda gt11 library (obtained after isolation of islet mRNA) using oligonucleotide probes and monospecific polyclonal antibodies. IAPP expression in normal IGT and DM cats will be examined by in situ hybridization, in vitro translation, and Northern blotting; 4. Evaluate the effects of synthetic human IAPP and synthetic fragments of IAPP on the secretion of pancreatic islet hormones (i.e. insulin, glucagon, somatostatin, and pancreatic polypeptide) using direct perfusion of rat and cat pancreas. In addition, we will evaluate rat and cat pancreas and receptor binding assays;. 5. Chemically characterize IA from degus (Octodon degus), employing extraction and purification methods used by us for cat and human IA. Amyloid samples will be purified by size exclusion and revered phase HPLC for subsequent amino acid sequencing.
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