Glucagon Biosynthesis and Metabolism

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，在这种情况下，胰腺中产生胰岛素的β细胞几乎完全被自身免疫破坏。成功地将胰岛移植到糖尿病患者身上的尝试现在很有希望(埃德蒙顿协议)。我们假设，没有成功植入是由于胰岛中成熟的β细胞大多是有丝分裂后的情况，而成功的植入需要来自胰岛内的干细胞/祖细胞的新的β细胞的新生。因此，我们提出了一种方法，即新鲜分离的胰岛在移植前用生长因子如胰升糖素样肽-1(GLP-1)预培养数天，以扩大胰岛中的干/祖细胞数量。为了证明这种方法的合理性，我们建议将胰岛来源的干细胞/祖细胞(IPC)成功移植到用GLP-1治疗的链脲佐菌素诱导的糖尿病和NOD糖尿病小鼠中。在最初的研究中，我们成功地从人(以及大鼠和小鼠)的胰岛分离出干细胞/祖细胞，体外扩增它们，并对GLP-1做出反应，将它们转化为胰岛素产生的胰岛样簇(ILCs)。内皮祖细胞具有多潜能，因为它们分化为肝表型、神经表型、导管表型、造血表型和间质表型。胰高血糖素原基因在胰腺和肠道中都有表达。通过原高血糖素的翻译后加工机制，胰腺产生高血糖素样多肽-1(GLP-1)，在肠道中产生高血糖素样多肽-1(GLP-1)，这是一种胰岛素样激素，对胰腺的β细胞具有强大的胰岛素样、新生和细胞保护作用，对脂肪和骨骼肌起外周作用以促进葡萄糖摄取，并对肝脏产生抑制葡萄糖输出的作用。我们认为GLP-1激活胰岛β细胞上特异性cAMP偶联受体，与葡萄糖协同刺激胰岛素释放，并作用于胰岛和IPC，促进其向β细胞分化。本研究的目的是：(1)检测GLP-1促进胰岛β细胞生长、抑制细胞凋亡和刺激胰岛细胞向β细胞分化的潜在特性；(2)分离、鉴定和鉴定脂肪细胞上表达的外周GLP-1受体。我们建议从3T3-L1细胞的cDNA表达文库中克隆该受体，制备稳定表达该受体的细胞系，研究该受体与多肽激素结合的层级及其与信号转导通路的偶联，并探讨该受体在糖尿病中的潜在作用。由原高血糖素基因编码的GLP-1激素在维持血糖稳态中的重要性，以及它们与2型糖尿病发病机制的潜在相关性，为进一步了解参与GLP-1激动剂作用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.