Mechanisms of GLUT4 Exocytosis

GLUT4胞吐作用的机制

• 批准号: 10379955
• 负责人: Jingshi Shen
• 金额: $ 37.81万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10379955
• 关键词: Address; Adipocytes; Biopsy; Blood Glucose; C2 Domain; Cell Line; Cell membrane; Cell surface; Chimeric Proteins; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Development; Disease; Exocytosis; GLUT 4 protein; Genetically Engineered Mouse; Glean; Glucagon; Glucose Transporter; Goals; Human; Impairment; In Vitro; Insulin; Insulin Resistance; Knowledge; Light; Link; Maintenance; Mediating; Membrane; Membrane Fusion; Molecular; Molecular Conformation; Munc18c protein; Muscle Cells; Non-Insulin-Dependent Diabetes Mellitus; Pathogenesis; Pathway interactions; Play; Pluripotent Stem Cells; Reaction; Research; Role; SNAP receptor; System; Therapeutic Intervention; Tissues; Vesicle; Work; abdominal fat; adipocyte differentiation; base; blood glucose regulation; experimental study; genetic analysis; glucagon-like peptide 1; glucose uptake; insight; insulin secretion; novel therapeutic intervention; reconstitution; subcutaneous; syntaxin; syntaxin binding protein 1; target SNARE proteins; time use

Defective insulin-stimulated glucose uptake is a hallmark of insulin resistance (IR) and type 2 diabetes (T2D). Insulin promotes glucose uptake by triggering the relocation of the glucose transporter GLUT4 from intracellular storage vesicles to the cell surface through exocytosis. To develop effective and safe treatments for IR and T2D, it is crucial to gain a comprehensive understanding of insulin-stimulated GLUT4 exocytosis at the molecular level. GLUT4 exocytosis – the fusion of GLUT4 vesicles with the plasma membrane – requires the membrane-anchored SNAREs, the soluble SM proteins, and the C2-domain factor Doc2b. In our previous research, we reconstituted GLUT4 vesicle fusion in vitro, for the first time, using defined components, which overcame the limitations of conventional approaches and enabled us to attack the problem from a fundamentally new angle. Using this unique reconstitution system, we discovered a stimulatory function of the SM protein Munc18c in SNARE zippering and a membrane-remodeling role of Doc2b in GLUT4 vesicle fusion. In our preliminary studies, we substantially expanded our reconstitution experiments and uncovered new molecular functions of GLUT4 vesicle fusion proteins. In addition, our CRISPR genetic analyses revealed Munc18b as another SM protein involved in GLUT4 exocytosis. Based on these major advances, we will first define the detailed molecular mechanisms by which Munc18b and Munc18c control GLUT4 vesicle fusion using our reconstitution system. Next, we will establish how Doc2b cooperates with Munc18b and Munc18c to control each stage of the membrane fusion reaction. We will then validate the findings of the reconstitution studies in adipocytes and muscle cells, using both cultured cell lines and primary tissues isolated from genetically engineered mice. Finally, we will examine whether and how GLUT4 vesicle fusion proteins are altered in insulin-resistant human adipocytes isolated from biopsies of subcutaneous abdominal fat. Completion of this proposed research will fill major gaps in our knowledge of the GLUT4 exocytic pathway. Our findings will also shed light upon the pathogenesis of IR and T2D, and will facilitate the development of novel strategies for therapeutic intervention.

胰岛素刺激的葡萄糖摄取缺陷是胰岛素抵抗（IR）和2型糖尿病（T2 D）的标志。胰岛素通过胞吐作用触发葡萄糖转运蛋白GLUT 4从细胞内储存囊泡重新定位到细胞表面来促进葡萄糖摄取。为了开发IR和T2 D的有效和安全的治疗方法，在分子水平上全面了解胰岛素刺激的GLUT 4胞吐作用至关重要。GLUT 4胞吐-GLUT 4囊泡与质膜的融合-需要膜锚定的SNARE、可溶性SM蛋白和C2结构域因子Doc 2b。在我们之前的研究中，我们首次使用定义的组件在体外重建了GLUT 4囊泡融合，这克服了传统方法的局限性，使我们能够从一个全新的角度解决问题。使用这种独特的重建系统，我们发现了SM蛋白Munc 18 c在SNARE拉链中的刺激功能和Doc 2b在GLUT 4囊泡融合中的膜重塑作用。在我们的初步研究中，我们大大扩展了我们的重建实验，并发现了新的GLUT 4囊泡融合蛋白的分子功能。此外，我们的CRISPR遗传分析显示Munc 18 b是另一种参与GLUT 4胞吐的SM蛋白。基于这些重大进展，我们将首先定义详细的分子机制，Munc 18 b和Munc 18 c控制GLUT 4囊泡融合使用我们的重建系统。接下来，我们将确定Doc 2b如何与Munc 18 b和Munc 18 c合作来控制膜融合反应的每个阶段。然后，我们将使用从基因工程小鼠分离的培养细胞系和原代组织，验证脂肪细胞和肌肉细胞重建研究的结果。最后，我们将研究是否以及如何改变GLUT 4囊泡融合蛋白在胰岛素抵抗的人脂肪细胞分离皮下腹部脂肪活检。这项研究的完成将填补我们对GLUT 4胞吐途径的认识中的主要空白。我们的研究结果也将阐明IR和T2 D的发病机制，并将促进新的治疗干预策略的发展。