Defining alpha-cell proglucagon processing for type 2 diabetes treatment

项目概要：我们建议使用最先进的小鼠模型，转基因人类胰岛 和独特的单细胞RNA-seq技术，以产生对胰岛调节的新见解， 该功能将作为开发2型糖尿病（T2 DM）新疗法的基础。的 胰高血糖素原衍生肽，胰高血糖素和胰高血糖素样肽-1（GLP-1），增强葡萄糖刺激的 胰岛素分泌（GSIS）。然而，与GLP-1不同，胰高血糖素促进肝脏葡萄糖产生，并且是一种代谢酶。 较弱的GSIS诱导剂。我们确定了一种将α细胞从产生胰高血糖素转变为产生胰高血糖素的途径 产生GLP-1。我们建议定义这一途径，以建立一个新的模型来描述GLP-1的功能， 以确定增加α细胞GLP-1产生的药物靶点。胰高血糖素原在肠L细胞中表达， 胰岛α细胞，并根据存在的激素原转化酶（PC）而被差异切割。 典型地，PC 1/3（Pcsk 1）在L细胞中表达以产生GLP-1，而PC 2（Pcsk 2）在L细胞中以α表达。 细胞产生胰高血糖素。然而，我们的数据显示，β细胞GLP-1受体（GLP-1 R）信号转导 增加α细胞GLP-1和Pcsk 1表达。我们假设β细胞GLP-1 R信号传导增加 α细胞GLP-1产生以在旁分泌正反馈回路中增加GSIS。我们将追求三个目标 为了定义涉及的α小区到β小区（目标1和2）和β小区到α小区（目标3）串扰， 我们的β细胞GLP-1 R功能模型鉴定了提高α细胞GLP-1：胰高血糖素水平的新靶点， T2 DM治疗。在目标1中，我们将定义α细胞对β细胞GLP-1 R功能的贡献， 在高脂肪饮食喂养的小鼠中对内源性GLP-1的应答。为此，我们将评估葡萄糖调节， 低脂肪饲料喂养或高脂肪饲料喂养的β细胞GLP-1 R野生型和基因敲除小鼠中的GSIS，伴或不伴α 细胞消融，有或没有L细胞衍生的GLP-1刺激。在目标2中，我们将定义alpha的贡献 细胞转化为β细胞GLP-1 R的功能响应于外源性GLP-1。为此，我们将评估葡萄糖 β细胞GLP-1 R野生型和基因敲除小鼠中的GSIS，伴或不伴α细胞消融，伴 或未用GLP-1 R激动剂治疗。在目标3中，我们将确定β细胞GLP-1 R的影响 α细胞命运的信号我们发现β细胞GLP-1 R信号增加Pcsk 1的表达， 其他β细胞特异性基因在人类α细胞，并延伸到以前的工作表明，α细胞， 表达GLP-1的细胞是不成熟的。因此，我们假设β细胞GLP-1 R信号传导调节α细胞 转录程序以促进α细胞转化为β样细胞。为了验证这一假设， 我们将在小鼠中进行IHC、谱系追踪和条件培养基研究。在 同时，我们将使用我们开发的高灵敏度单细胞RNA-seq平台来评估标记物， 小鼠和人类胰岛上的胰岛细胞身份和转录组。这些数据将有助于开发 增加用于T2 DM治疗的α细胞GLP-1产生的疗法。

PROJECT SUMMARY: We propose to use state-of-the-art mouse modeling, genetically modified human islets and a unique single cell RNA-seq technique to generate new insights into the regulation of pancreatic islet function that will serve as the basis for developing new therapeutics for type 2 diabetes mellitus (T2DM). The proglucagon-derived peptides, glucagon and glucagon-like peptide-1 (GLP-1), potentiate glucose-stimulated insulin secretion (GSIS). However, unlike GLP-1, glucagon promotes hepatic glucose production and is a weaker inducer of GSIS. We identified a pathway that shifts the alpha cell from producing glucagon to producing GLP-1. We propose to define this pathway to establish a new model describing GLP-1 function and to identify drug targets that increase alpha cell GLP-1 production. Proglucagon is expressed in gut L cells and islet alpha cells and is differentially cleaved depending on the prohormone convertase (PC) present. Canonically, PC1/3 (Pcsk1) is expressed in L cells to produce GLP-1 and PC2 (Pcsk2) is expressed in alpha cells to produce glucagon. However, our data reveal that beta cell GLP-1 receptor (GLP-1R) signaling increases alpha cell GLP-1 and Pcsk1 expression. We hypothesize that beta cell GLP-1R signaling increases alpha cell GLP-1 production to augment GSIS in a paracrine positive feedback loop. We will pursue three aims to define the alpha cell to beta cell (aims 1 and 2) and the beta cell to alpha cell (aim 3) cross-talk involved in our model of beta cell GLP-1R function to identify novel targets that raise alpha cell GLP-1:glucagon levels for T2DM treatment. In aim 1, we will define the contribution of alpha cells to beta cell GLP-1R function in response to endogenous GLP-1 in high fat diet-fed mice. To this end, we will assess glucose regulation and GSIS in low fat diet-fed or high fat diet-fed beta cell GLP-1R wild-type and knockout mice, with or without alpha cell ablation, with or without L cell-derived GLP-1 stimulation. In aim 2, we will define the contribution of alpha cells to beta cell GLP-1R function in response to exogenous GLP-1. To this end, we will assess glucose regulation and GSIS in beta cell GLP-1R wild-type and knockout mice, with or without alpha cell ablation, with or without treatment with a GLP-1R agonist. In aim 3, we will determine the impact of beta cell GLP-1R signaling on alpha cell fate. We show that beta cell GLP-1R signaling increases the expression of Pcsk1 and other beta cell-specific genes in human alpha cells and extend upon previous work showing that alpha cells that express GLP-1 are immature. Thus, we hypothesize that beta cell GLP-1R signaling regulates alpha cell transcriptional programs to promote conversion of alpha cells into beta-like cells. To test this hypothesis and identify putative mediators we will perform IHC, lineage tracing and conditioned media studies in mice. In parallel, we will use a highly sensitive single cell RNA-seq platform that we have developed to assess markers of islet cell identity and transcriptome on mouse and human islets. These data will enable the development of therapies that increase alpha cell GLP-1 production for T2DM treatment.