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.

胰岛素刺激的有缺陷的葡萄糖吸收是胰岛素抵抗（IR）和2型糖尿病（T2D）的标志。胰岛素通过诱导葡萄糖转运蛋白glut4从细胞内存储车的迁移到细胞表面，从而促进葡萄糖摄取。为了为IR和T2D开发有效和安全的治疗方法，至关重要的是要在分子水平上全面了解胰岛素刺激的GLUT4胞吐作用。 GLUT4胞吐作用 - Glut4蔬菜与质膜的融合 - 需要膜锚定的贪食片，固体SM蛋白和C2核因子DOC2B。在我们先前的研究中，我们首次使用定义的组件重新生成了体外的GLUT4蔬菜融合，从而克服了常规方法的局限性，并使我们能够从根本上新的角度攻击问题。使用这种独特的重构系统，我们发现了SM蛋白Munc18c在SNARE拉链中的刺激功能，并且DOC2B在Glut4蔬菜融合中的膜重新塑造作用。在我们的初步研究中，我们大大扩展了重组实验，并发现了GLUT4蔬菜融合蛋白的新分子功能。此外，我们的CRISPR遗传分析揭示了Munc18b是参与GLUT4胞吐作用的另一种SM蛋白。基于这些主要进步，我们将首先定义Munc18b和Munc18c控制Glut4囊泡融合的详细分子机制。接下来，我们将建立DOC2B如何与MUNC18B和MUNC18C合作以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中分离的培养细胞系和原代组织来验证脂肪细胞和肌肉细胞中重建研究的发现。最后，我们将研究在抗皮下腹部脂肪活检中分离出的胰岛素脂肪细胞中glut4囊泡融合蛋白是否改变。这项拟议的研究的完成将填补我们对GLUT4外囊肿途径的了解。我们的发现还将阐明IR和T2D的发病机理，并有助于发展新型治疗干预策略。