Glucagon Biosynthesis and Metabolism

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Seventeen million individuals in the USA suffer from diabetes mellitus, 1 million of whom have type 1 (juvenile) diabetes in which the insulin-producing beta-cells in the pancreas are nearly completely destroyed by autoimmunity. Attempts to successfully transplant islets to diabetic subjects now hold promise (Edmunton Protocol). We hypothesize that the lack of successful engraftment is due to the situation in which mature beta-cells in the islets are mostly post-mitotic, and that successful engraftment requires the neogenesis of new beta-cells from stem/progenitor cells that reside within the islets. Therefore, we propose an approach whereby freshly isolated islets are pre-cultured for several days with growth factors such as glucagon-like peptide-1 (GLP-1) to expand the population of stem/progenitor cells in the islets prior to their transplantation. To justify such an approach, we propose to demonstrate successful engraftment of islet-derived stem/progenitor cells (IPCs) transplanted into streptozotocin-induced diabetic and NOD diabetic mice treated with GLP-1. In initial studies, we have successfully isolated stem/progenitor cells from human (and rat, and mouse) pancreatic islets, have expanded them ex vivo, and have converted them into insulin-producing islet-like clusters, (ILCs) in response to GLP-1. The IPCs are multipotential as they differentiate into hepatic, neural, ductal, hematopoietic and mesenchymal phenotypes. The proglucagon gene is expressed in both the pancreas and the intestine. By mechanisms of alternative post-translational processing of proglucagon, the pancreas produces glucagon and in the intestine produces glucagon-like peptide-1 (GLP-1), an incretin hormone that has potent insulinotropic, neogenic and cytoprotective actions on beta-cells of the pancreas, peripheral actions on adipose and skeletal muscle to enhance glucose uptake, and on liver to inhibit glucose output. We propose that GLP-1 activates specific cAMP-coupled receptors on pancreatic beta-cells and, synergetically with glucose, stimulates insulin release and acts on islet and IPCs to promote their differentiation into beta-cells. The aims are to: (1) examine the potential properties of GLP-1 to enhance growth and to inhibit apoptosis of pancreatic beta-cells and to stimulate the differentiation of pancreatic IPCs into beta-cells; (2) isolate, identify, and characterize the peripheral GLP-1 receptor expressed on adipocytes. We propose to clone the receptor from a 3T3-L1 cell cDNA expression library, prepare stable cell lines expressing the receptor, characterize the hierarchy of peptide hormone binding and the coupling to signal transduction pathways, and investigate the potential role of the receptor in diabetes. The importance of GLP-1 hormones encoded by the proglucagon gene in the maintenance of glucose homeostasis, and their potential relevance to the pathogenesis of type 2 diabetes, provides impetus in learning more about the controlling factors involved in the actions of GLP-1 agonists.