Regulation of Glucose Homeostasis by Munc18 Proteins

描述（由申请方提供）：葡萄糖稳态通过胰岛分泌胰岛素和骨骼肌和脂肪组织摄取胰岛素作用葡萄糖的协调努力实现。这两个过程都是由SNARE介导的胞吐事件控制的，当这些事件出错时，2型糖尿病就会发病。t-SNARE蛋白，Syntaxin 4，和它的调节结合伙伴，Munc 18 c，现在被认为是这两个过程的胞吐机制的重要组成部分，虽然详细的分子机制尚未解决。长期目标是了解导致2型糖尿病发病的葡萄糖稳态失调的分子、细胞和生理基础。为了实现这一目标，本文所述的已发表和初步数据揭示了β细胞和脂肪细胞中保守的详细“开关机制”的开始，涉及刺激诱导的Munc 18 c磷酸化及其随后与新伴侣Doc 2b结合的偏好。本申请的目的是描述Munc 18和Doc 2b蛋白促进胰岛素分泌和胰岛素应答细胞类型中受调节的胞吐作用以控制葡萄糖稳态的分子机制。中心假设是Munc 18 c作为关键的分子开关发挥作用，以瞬时激活Syntaxin 4并以刺激诱导的方式促进囊泡递送事件。支持这一点的是新的发现，其显示：a）在称为“突触融合蛋白激活”的胞吐作用的新的预对接步骤中需要Munc 18 c; B）候选Munc 18 c修饰因子以催化存在于脂肪细胞和β细胞中的其酪氨酸磷酸化; c）参与细胞骨架重塑和颗粒递送/定位的新的Munc 18 c结合因子。将在三个具体目标中检验这一假设： 1)确定Munc 18 c-Doc 2b相互作用在调节GLUT 4易位和全身葡萄糖稳态中的需求; 2）描述Munc 18-Doc 2b蛋白复合物在双相胰岛素分泌中的作用; 3）阐明Munc 18 c在Syntaxin 4激活和颗粒递送/动员过程中发挥作用以促进胰岛素胞吐的机制。将使用Doc 2b和Munc 18敲除小鼠完成研究，以进行体内平衡的全身分析和组织分析，以确定对双相胰岛素释放（胰岛灌流）和骨骼肌葡萄糖摄取（GLUT 4易位）的组织特异性影响。MIN 6 β细胞和3 T3 L1脂肪细胞将用于蛋白质蛋白质相互作用的空间和时间变化的分子/生化解剖，以及使用显微镜和生化亚细胞分级分析观察颗粒/囊泡胞吐事件的动态变化。获得Munc 18 c如何调节这些胞吐事件的知识将标志着朝着调节（可能同时）胰岛素分泌和胰岛素刺激的葡萄糖摄取以改善患者葡萄糖稳态的长期目标的进展。 公共卫生相关性：2型糖尿病被称为“两击”疾病;一个“击”是骨骼肌和脂肪组织的葡萄糖清除功能障碍，另一个“击”是胰岛β细胞的胰岛素分泌功能障碍。Munc 18 c蛋白及其结合伴侣的异常丰度和功能已在糖尿病患者的肌肉和胰岛中报道，因此有可能介导串扰并成为两种“命中”的基础。我们使用新型啮齿动物模型的研究支持Munc 18 c的重要和必要作用，现在必须阐明Munc 18 c在骨骼肌，脂肪和胰岛中这些过程中的详细分子机制。基于Munc 18 c的机制的保护具有开发新的治疗策略的巨大潜力，这些策略可以同时解决这种疾病的两个“打击”，以改善糖尿病患者的生活。

DESCRIPTION (provided by applicant): Glucose homeostasis is achieved by the coordinated efforts of insulin secretion from pancreatic islets and insulin action-glucose uptake by skeletal muscle and adipose tissues. Both processes are controlled by SNARE-mediated exocytosis events, and when these events go awry, Type 2 diabetes ensues. The t-SNARE protein, Syntaxin 4, and its regulatory binding partner, Munc18c, are now recognized as essential lcomponents of the exocytic mechanisms for both processes, although the detailed molecular mechanisms remain unresolved. The long term goal here is to understand the molecular, cellular and physiological basis for the dysregulation of glucose homeostasis leading to the onset of Type 2 diabetes. Towards this goal, published and preliminary data described herein reveal the beginnings of a detailed 'switch mechanism' conserved in beta cells and adipocytes involving the stimulus-induced phosphorylation of Munc18c and its subsequent preference for binding to a new partner, Doc2b. The objective of this application is to delineate the molecular mechanisms by which Munc18 and Doc2b proteins facilitate regulated exocytosis in insulinsecreting and insulin-responsive cell types to control glucose homeostasis. The central hypothesis is that Munc18c functions as the key molecular switch to transiently activate Syntaxin 4 and to promote vesicle delivery events in a stimulus-induced fashion. Supporting this are new findings which show: a) Requirement for Munc18c in a new pre-docking step of exocytosis termed 'syntaxin activation'; b) Candidate Munc18c modifying factors to catalyze its tyrosine-phosphorylation present in adipocytes and in beta cells; c) New Munc18c binding factors involved in cytoskeletal remodeling and granule delivery/positioning. This hypothesis will be tested in three specific aims: 1) Determine the requirement for Munc18c-Doc2b interaction in regulating GLUT4 translocation and whole body glucose homeostasis; 2) Delineate the role of Munc18-Doc2b protein complexes in biphasic insulin secretion; 3) Elucidate the mechanisms by which Munc18c functions in Syntaxin 4-activation and in granule delivery/mobilization processes to facilitate insulin exocytosis. Studies will be accomplished using Doc2b and Munc18 knockout mice for whole body analyses of homeostasis and tissues there from for tissue-specific effects upon biphasic insulin release (islet perifusion) and skeletal muscle glucose uptake (GLUT4 translocation). MIN6 beta cells and 3T3L1 adipocytes will be utilized for molecular/biochemical dissection of spatial and temporal changes in proteinprotein interactions coupled with visualization of dynamic changes in granule/vesicle exocytosis events using microscopy and biochemical subcellular fractionation analyses. Gaining knowledge of how Munc18c regulates these exocytic events will mark progress towards the long-term goal of modulating, perhaps simultaneously, both insulin secretion and insulin-stimulated glucose uptake in order to improve glucose homeostasis in the patient. PUBLIC HEALTH RELEVANCE: Type 2 diabetes has been coined a 'two-hit' disease; one 'hit' is dysfunction of glucose clearance by the skeletal muscle and adipose tissues, and another 'hit' is dysfunction of insulin secretion by the pancreatic islet beta cells. Aberrant abundance and function of the Munc18c protein and its binding partners have been reported in diabetic human muscle and islets and therefore have the potential to mediate cross-talk and underlie both 'hits'. Our research using novel rodent models supports an important and required role for Munc18c, and now it is imperative that the detailed molecular mechanisms involving Munc18c in these processes in skeletal muscle, adipose and islets be elucidated. Conservation of Munc18c-based mechanisms carries great potential for development of novel therapeutic strategies that could simultaneously tackle both 'hits' of this disease to improve the livelihood of people with diabetes.