Somatostatin (S) is being increasingly recognized as a multi-gene family of peptides with 2 principal bioactive member S-14 and S- 28, widely distributed in many tissues and subserving important regulatory functions on many body processes including the control of islet hormone secretion and nutrient metabolism. S-14 and S- 28 have evolved from separate genes in fish to an apparent single mammalian gene. The long-term aim of this proposal is to narrow the gap in the understanding of the separate evolutionary origins, cellular expression, mechanism of action, and functional roles within the islet of the somatostatin genes and their products S-14 and S-28. Using an in vitro model of cultured rat islet cells, sequence specific antisera, chromatography (affinity, gel, HPLC), the precise biosynthetic relationships between pro-S and its cleavage products will be determined by pulse-chase studies. The sub-cellular sites of processsing of pro-S will be determined by combined pulse-chase and autoradiography, and EM immunocytochemistry with antibodies against pro-S, S-14 and S- 28. Factors regulating somatostatin gene function in islet will be identified by measuring somatostatin mRNA with a sensitive cDNA hybridization assay. The level of reguation of somatostain synthesis will be determined by direct study of seretagogue induced effect on in vitro transcription rates and on mRNA stability. Using cDNA probes against rat, catfish (CF) S-22 and anglerfish (AF) S-28 genes, somatostatin gene expression in the developing rat pancreas will be elucidated. The developmental profile of islet somatostatin mRNA will be compared with that of islet mRNAs for insulin, glucagon and pancreatic polypeptide determined simultaneously by Northern Blot analysis and by in situ hybridization. Phylogenetic studies will be undertaken to identify the fish somatostatin genes and thecorresponding peptide products in the frog. EM immunochemistry will be used to determine whether subpopulations of islet D cells or D cell granules for S-14 and S-28 exist. An insulin selective role of S-28 (acting via the blood) and a glucagon selective (paracrine) role of S-14 will be defined by immunoneutralization experiments with S- 14 and S-28 antisera. A search will be made for specific receptors for CF S-22 and AF S-28 distinct from the mammalian S-14 receptor by direct binding studies. The somatostatin receptor will be purified for partial sequence analysis after solubilization with the help of approaches (additions of phospholipids, and G-proteins) to restore binding activity. Finally the profile of somatostatin mRNA and that of other islet hormone mRNAs will be characterized in streptozotocin and BB diabetic rats and in obob mice.
