Endosomal regulation of GLP-1 receptor function in beta cells by Clec16a

Clec16a 对 β 细胞中 GLP-1 受体功能的内体调节

基本信息

批准号：
9086362
负责人：
Scott Soleimanpour
金额：
$ 7.75万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-01 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9086362
关键词：
Acute Agonist Alleles Autophagocytosis Beta Cell Biological Assay CREB1 gene Cell Line Cell Proliferation Cell physiology Cells Confocal Microscopy Cyclic AMP Cyclic AMP-Dependent Protein Kinases Data Diabetes Mellitus Endocytosis Endosomes Evaluation G-Protein-Coupled Receptors GCG gene Generations Genes Genetic Polymorphism Glucose Glucose Intolerance Goals Health Human Insulin Knock-out Label Life Ligand Binding Ligands Liquid substance Lysosomes Mediating Methods Mission Modeling Molecular Mus Outcome Pancreas Pathway interactions Patients Pharmaceutical Preparations Phase Phosphorylation Phosphorylation Site Physiologic pulse Physiological Play Post-Translational Protein Processing Proteins Receptor Signaling Regulation Research Role Signal Transduction Site-Directed Mutagenesis Structure of beta Cell of islet Testing Therapeutic Agents Tracer Work base blood glucose regulation diabetic patient exenatide glucagon-like peptide glucagon-like peptide 1 glucose tolerance improved improved functioning in vivo insulin secretion islet live cell imaging loss of function migration mouse model receptor receptor function research study response trafficking uptake

项目摘要

DESCRIPTION (provided by applicant): The research proposed in this R03 application will focus on the endosomal turnover of the glucagon- like peptide 1 receptor (GLP1R), a G-protein coupled receptor, in pancreatic ß-cells by the endolysosomal protein Clec16a. GLP1R agonists are valuable therapeutic agents for diabetes based upon their direct effects on ß-cells; however, these effects are often self-limited by mechanisms to inactivate GLP1R-dependent signaling. Strategies to slow endolysosomal turnover of ligand bound GLP1Rs could therefore be beneficial to extend the activity of GLP1R agonists. Clec16a has been previously shown to be a key regulator of autophagy in ß-cells, but endolysosomal proteins can often play complementary roles in both autophagy and endosomal trafficking. Indeed, our preliminary studies reveal that Clec16a regulates trafficking of endocytic fluid-phase tracers in pancreatic ß-cells and is also phosphorylated by protein kinase A (PKA) in response to GLP1R agonist treatment. Furthermore, we find that acute treatment with GLP1R agonists markedly enhances insulin release in pancreas-specific Clec16a knockout islets. Therefore, we hypothesize that Clec16a negatively regulates GLP1R signaling through a direct role in the endolysosomal degradation of ligand bound GLP1 receptors. Specific Aim I. Determine the physiologic impact of loss of Clec16a on GLP1R signaling in vivo. This aim will utilize ß-cell specific loss-of-function experiments in vivo using a conditional Clec16a loxP allele, as well as in isolated islets, following treatment with GLP1R-agonists. Experiments will focus on determinations of glucose tolerance and insulin secretion, as well as morphologic analysis of ß-cell mass related to changes in ß-cell proliferation and/or survival. Specific Aim II. To investigate Clec16a-dependent regulation of GLP1R endolysosomal trafficking and degradation. Experiments will determine the role of Clec16a in turnover of ligand-bound GLP1Rs through the endolysosomal pathway by live cell imaging assays. In addition, the regulatory role of PKA-dependent phosphorylation of Clec16a to regulate trafficking/degradation of GLP1R at the lysosome will be determined. By completing the work described in these aims, we will establish the role and functional mechanism for GLP1R-degradation by Clec16a and the endolysosomal pathway. We will determine the utility of inhibition of the endosomal pathway to relieve the breaks on Ex-4 mediated signaling to improve ß-cell function, which could open a new pathway to enhance GLP1R-agonist therapy for patients with diabetes. Furthermore, these studies may suggest new GLP1-based approaches to treat diabetic patients with CLEC16A polymorphisms.

描述（由申请人提供）：本 R03 申请中提出的研究将重点关注胰高血糖素样肽 1 受体 (GLP1R)（一种 G 蛋白偶联受体）在胰腺 β 细胞中通过内溶酶体蛋白 Clec16a 进行的内体周转。 GLP1R 激动剂是有价值的糖尿病治疗药物，因为它们对 β 细胞具有直接作用，但这些作用通常受到机制的自我限制；因此，减缓配体结合 GLP1R 的内溶酶体周转的策略可能有利于延长 GLP1R 激动剂的活性，此前已证明 Clec16a 是 β 细胞自噬的关键调节因子。事实上，我们的初步研究表明 Clec16a 调节内吞转运。胰腺 ß 细胞中的液相示踪剂，也被蛋白激酶 A (PKA) 磷酸化以响应 GLP1R 激动剂治疗。此外，我们发现用 GLP1R 激动剂进行急性治疗可显着增强胰腺特异性 Clec16a 敲除胰岛中的胰岛素释放。，我们见证了 Clec16a 通过直接作用于配体结合的 GLP1 受体的内溶酶体降解，负向调节 GLP1R 信号传导。具体目标 I. 确定 Clec16a 丢失对体内 GLP1R 信号传导的生理影响该目标将利用条件性 Clec16a loxP 等位基因以及治疗后的分离胰岛进行体内 β 细胞特异性功能丧失实验。实验将集中于葡萄糖耐量和胰岛素分泌的测定，以及与β细胞质量变化相关的形态学分析。 ß-细胞增殖和/或存活。研究 Clec16a 依赖性。 GLP1R 内溶酶体运输和降解的调节实验将通过活细胞成像测定确定 Clec16a 在配体结合 GLP1R 通过内溶酶体途径的周转中的作用。通过完成这些目标中描述的工作，我们将确定 GLP1R 在溶酶体中的作用和功能机制。 Clec16a 和内溶酶体途径对 GLP1R 的降解我们将确定抑制内体途径的效用，以缓解 Ex-4 介导的信号传导中断，从而改善 ß 细胞功能，这可能会开辟一条增强 GLP1R 激动剂治疗的新途径。此外，这些研究可能提出基于 GLP1 的新方法来治疗具有 CLEC16A 多态性的糖尿病患者。