Glucagon Biosynthesis and Metabolism

在美国有1700万人患有糖尿病，其中100万人患有1型（青少年）糖尿病，其中胰腺中产生胰岛素的β细胞几乎被自身免疫完全破坏。成功地将胰岛移植到糖尿病受试者身上的尝试现在有了希望（Edmunton Protocol）。我们假设，缺乏成功的移植是由于胰岛中的成熟β细胞大多数是有丝分裂后的情况，并且成功的移植需要来自胰岛内的干细胞/祖细胞的新β细胞的新生。因此，我们提出了一种方法，其中新鲜分离的胰岛与生长因子如胰高血糖素样肽-1（GLP-1）预培养数天，以在移植前扩增胰岛中的干/祖细胞群。为了证明这种方法的合理性，我们建议证明成功植入胰岛源性干/祖细胞（IPC）移植到链脲佐菌素诱导的糖尿病和NOD糖尿病小鼠GLP-1治疗。在最初的研究中，我们已经成功地从人（和大鼠和小鼠）胰岛中分离出干细胞/祖细胞，体外扩增它们，并将它们转化为胰岛素产生胰岛样簇（ILC），以响应GLP-1。IPC是多潜能的，因为它们分化成肝、神经、导管、造血和间充质表型。胰高血糖素原基因在胰腺和肠中都表达。通过胰高血糖素原的替代性翻译后加工机制，胰腺产生胰高血糖素，并在肠中产生胰高血糖素样肽-1（GLP-1），这是一种肠降血糖素激素，对胰腺β细胞具有强效促胰岛素、新生和细胞保护作用，对脂肪和骨骼肌具有外周作用以增强葡萄糖摄取，对肝脏具有外周作用以抑制葡萄糖输出。我们提出GLP-1激活胰腺β细胞上的特异性cAMP偶联受体，并与葡萄糖协同刺激胰岛素释放，作用于胰岛和IPC，以促进其分化为β细胞。其目的是：（1）检查GLP-1促进胰腺β细胞生长和抑制其凋亡以及刺激胰腺IPC分化为β细胞的潜在特性;（2）分离、鉴定和表征脂肪细胞上表达的外周GLP-1受体。我们建议从3 T3-L1细胞cDNA表达文库中克隆该受体，制备稳定表达该受体的细胞系，表征肽激素结合的层次结构和与信号转导途径的偶联，并研究该受体在糖尿病中的潜在作用。胰高血糖素原基因编码的GLP-1激素在维持葡萄糖稳态中的重要性及其与2型糖尿病发病机制的潜在相关性，为更多地了解GLP-1激动剂作用中涉及的控制因素提供了动力。

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.