Defining alpha-cell PC1/3 expression regulation for type 2 diabetes

项目摘要 胰高血糖素样肽-1（GLP-1）通过增强葡萄糖刺激的胰岛素分泌增强胰岛功能 然而，GLP-1增强GSIS的机制仍不完全 定义了在经典模型中，GLP-1由肠道L细胞分泌，以响应营养素的摄入 刺激β细胞GLP-1受体（GLP-1 R）以增强GSIS。这种模式目前受到质疑，因为短 GLP-1的半衰期及其快速降解限制了GLP-1如何介导远距离免疫效应。 靶点，如胰腺β细胞。在解释这些局限性时，有一种新兴的假设表明， 由α细胞局部产生的GLP-1以旁分泌方式作用于邻近的β细胞以刺激GSIS， 这最终促进了β的降低。胰高血糖素原在肠道和α细胞中表达， 形成GLP-1或反调节激素胰高血糖素，这取决于激素原转化酶（PC） 类型存在。以前认为，胰高血糖素原的组织特异性加工是由于胰高血糖素原的组织特异性加工导致的。 PC 1/3和PC 2的差异表达，但在啮齿动物模型和人类中的几项研究表明，α- 细胞可以产生活性GLP-1并表达PC 1/3。然而，α细胞PC 1/3表达的机制 规则是未知的。确定增加α细胞PC 1/3表达以增加GLP-1的机制 以胰高血糖素为代价的生产将提供用于调节血液的强有力的治疗方式 T2 DM患者的血糖浓度。我们已经证明，β细胞GLP-1 R信号传导增加， α-细胞PC 1/3和GLP-1表达。我的初步数据表明，胰岛素可能作为潜在的中间体， 通过β细胞GLP-1 R信号传导改变α细胞胰高血糖素原加工。具体来说，我假设β细胞 GLP-1 R信号通过α细胞胰岛素受体（IR）依赖性和 独立的道路。在目标1中，我将评估α细胞IR信号传导的作用，以确定胰岛素是否是 通过测量葡萄糖调节、GSIS和PC 1/3改变α细胞分泌表型所必需的 α细胞特异性Irα-cell +/+和Irα-cell -/-小鼠在有和无外源性GLP-1刺激下的表达。在Aim中 2，我将确定β细胞GLP-1 R信号通过以下途径增加α细胞PC 1/3表达： 来自诱导型β细胞特异性Glp-1 r β-细胞+/+和Glp-1 r-细胞-/-小鼠的胰岛的单细胞RNA测序，以及 β细胞特异性GLP-1 R敲除的人胰岛。总之，这些研究将确定 调节α细胞PC 1/3表达和GLP-1产生的途径;因此，能够靶向α细胞 分泌表型用于治疗T2 DM。

PROJECT SUMMARY Glucagon-like peptide-1 (GLP-1) enhances islet function by potentiating glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells; however, the mechanisms by which GLP-1 potentiates GSIS remain incompletely defined. In the classic model, GLP-1 secreted by the intestinal L cells in response to the ingestion of nutrients stimulates the β-cell GLP-1 receptor (GLP-1R) to enhance GSIS. This model is currently in question, as the short half-life of GLP-1 and its rapid degradation present limitations as to how GLP-1 can mediate effects in distant targets, such as pancreatic β-cells. In explanation of such limitations, there is an emerging hypothesis suggesting that GLP-1 locally produced by α-cells acts in a paracrine manner on neighboring β-cells to stimulate GSIS, which ultimately promotes lowering of glycemia. Proglucagon is expressed in the gut and α-cells and is cleaved to form GLP-1 or the counter-regulatory hormone, glucagon, depending on the prohormone convertase (PC) type present. It was previously thought that the tissue-specific processing of proglucagon was due to the differential expression of PC1/3 and PC2, but several studies in rodent models and humans have shown that α- cells can produce active GLP-1 and express PC1/3. However, the mechanisms by which α-cell PC1/3 expression is regulated are unknown. Identifying a mechanism to increase α-cell PC1/3 expression to increase GLP-1 production at the expense of glucagon will provide a powerful therapeutic modality for the regulation of blood glucose concentrations in patients with T2DM. We have shown that increased β-cell GLP-1R signaling increases α-cell PC1/3 and GLP-1 expression. My preliminary data suggest that insulin may serve as potential intermediate by which β-cell GLP-1R signaling alters α-cell proglucagon processing. Specifically, I hypothesize that β-cell GLP-1R signaling increases α-cell PC1/3 expression through both α-cell insulin receptor (IR) dependent and independent pathways. In Aim 1, I will assess the role of α-cell IR signaling to determine whether insulin is necessary to alter the secretory phenotype of α-cells by measuring glucose regulation, GSIS, and PC1/3 expression in α-cell-specific Irα-cell +/+ and Irα-cell -/- mice with and without stimulation by exogenous GLP-1. In Aim 2, I will determine the pathways through which β-cell GLP-1R signaling increases α-cell PC1/3 expression by single-cell RNA-sequencing of islets from inducible β-cell-specific Glp-1rβ-cell+/+ and Glp-1r-cell-/- mice, as well as human islets with β-cell-specific GLP-1R knockout. Together, these studies will define a link between the pathways regulating α-cell PC1/3 expression and GLP-1 production; thus, enabling targeting of the α-cell secretory phenotype for the treatment of T2DM.