Mechanisms of GLUT4 Exocytosis

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(T2D)的标志。胰岛素通过胞吐作用触发葡萄糖转运体GLUT4从细胞内的储存囊重新定位到细胞表面，从而促进葡萄糖的摄取。为了开发有效和安全的治疗IR和T2D的方法，在分子水平上全面了解胰岛素刺激的GLUT4胞吐是至关重要的。GLUT4胞吐作用--GLUT4囊泡与质膜的融合--需要膜锚定的SNARs、可溶性SM蛋白和C2结构域因子DOC2b。在我们之前的研究中，我们首次使用定义的组分在体外重建了GLUT4囊泡融合，克服了传统方法的局限性，使我们能够从一个全新的角度来解决这一问题。利用这个独特的重组系统，我们发现SM蛋白Munc18c在圈套拉链中具有刺激作用，Doc2b在GLUT4囊泡融合中具有膜重塑作用。在我们的初步研究中，我们大大扩展了我们的重组实验，并发现了GLUT4囊泡融合蛋白的新的分子功能。此外，我们的CRISPR基因分析显示，Munc18b是另一种参与GLUT4胞吐的SM蛋白。在这些主要进展的基础上，我们将首先确定Munc18b和Munc18c通过我们的重建系统控制GLUT4囊泡融合的详细分子机制。接下来，我们将确定Doc2b与Munc18b和Munc18c如何合作控制膜融合反应的每个阶段。然后，我们将使用培养的细胞系和从基因工程小鼠分离的原始组织来验证脂肪细胞和肌肉细胞重建研究的结果。最后，我们将研究从腹部皮下脂肪活检中分离的胰岛素抵抗的人类脂肪细胞中GLUT4囊泡融合蛋白是否以及如何改变。这项拟议研究的完成将填补我们对GLUT4胞外途径知识的主要空白。我们的发现也将有助于阐明IR和T2D的发病机制，并将有助于开发新的治疗干预策略。

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.