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

• 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
• 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

 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（由应用程序提供）：R03应用中提出的研究将集中于胰腺蛋白Clec16a中胰腺蛋白耦合受体的胰高血糖素样肽1受体（GLP1R）（GLP1R）（GLP1R），一种G蛋白耦合受体。 GLP1R激动剂是根据糖尿病对ß细胞的直接影响的糖尿病的有价值的治疗剂。但是，这些效应通常是通过使GLP1R依赖性信号传导的机制自限制的。因此，减慢配体结合GLP1RS的内溶性转移的策略可能是有益的，可以扩展GLP1R激动剂的活性。 CLEC16A先前已被证明是ß细胞中自噬的关键调节剂，但是内溶性蛋白质通常可以在自噬和内体贩运中扮演互补的角色。实际上，我们的初步研究表明，CLEC16A调节胰腺β-细胞中内吞流体相示踪剂的运输，并且还被蛋白激酶A（PKA）磷酸化，以响应GLP1R激动剂。此外，我们发现使用GLP1R激动剂的急性治疗显着增强了胰腺特异性CLEC16A敲除胰岛中的胰岛素释放。因此，我们假设CLEC16A通过在配体结合GLP1受体的内溶性降解中的直接作用来负调节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。在用条件CLEC16A LOXP等位基因以及在用GLP1R-Aganists处理后，使用条件CLEC16A LOXP等位基因以及在孤立的胰岛中利用ß细胞特异性功能丧失实验。实验将集中于确定葡萄糖耐受性和胰岛素分泌，以及与ß细胞增殖和/或生存变化有关的ß细胞质量的形态分析。具体目标II。研究CLEC16A依赖性 GLP1R内溶性贩运和退化的调节。实验将通过活细胞成像测定法确定CLEC16A通过内溶性途径通过内溶性途径的转换中的作用。此外，将确定CLEC16A磷酸化在调节溶酶体处GLP1R的贩运/降解的调节作用。通过完成这些目标中描述的工作，我们将确定CLEC16A和内溶性途径GLP1R降解的作用和功能机制。我们将确定抑制内体途径的效用，以减轻EX-4介导的信号传导的断裂以改善ß细胞功能，这可以为增强糖尿病患者增强GLP1R激动剂治疗的新途径。此外，这些研究可能建议使用新的基于GLP1的方法来治疗CLEC16A多态性的糖尿病患者。