Regulation of Glucose Homeostasis by Munc18 Proteins

Munc18 蛋白对葡萄糖稳态的调节

基本信息

批准号：
8061679
负责人：
Debbie C Thurmond
金额：
$ 31.62万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-07-01 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8061679
关键词：
Address Adipocytes Adipose tissue Advanced Development Beta Cell Binding Biochemical Biological Biological Assay Cell Fractionation Cell membrane Cells Coin Complex Coupled Cytoplasmic Granules Cytoskeleton Data Defect Development Diabetes Mellitus Disease Dissection Dissociation Docking Euglycemic Clamping Event Exhibits Exocytosis F-Actin Failure Functional disorder Funding GLUT4 gene Glucose Glucose Clamp Glucose tolerance test Goals Grant Homeostasis Human Imagery Insulin Insulin Receptor Insulin Resistance Islets of Langerhans Knock-out Knockout Mice Knowledge Link Location Mediating Microscopy Microtubules Molecular Munc18c protein Mus Muscle Muscle Fibers Non-Insulin-Dependent Diabetes Mellitus Obesity Patients Phase Phosphorylation Phosphorylation Site Physiological Positioning Attribute Predisposition Process Protein Tyrosine Kinase Proteins Publishing Regulation Reporting Research Rodent Model Role SNAP receptor Single Nucleotide Polymorphism Skeletal Muscle Small Interfering RNA Stimulus Structure-Activity Relationship Testing Therapeutic Therapeutic Intervention Tissues Tyrosine Tyrosine Phosphorylation Vesicle Work base blood glucose regulation c new cell type diabetic early onset glucose uptake glycosylation improved in vivo insulin granule insulin secretion insulin sensitivity islet mouse model multitask novel novel therapeutics preference prevent protein complex protein protein interaction public health relevance response scaffold syntaxin syntaxin 1 syntaxin 4 syntaxin binding protein 1 target SNARE proteins tissue processing treatment strategy

项目摘要

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.

描述（由申请人提供）：葡萄糖稳态是通过胰岛的胰岛素分泌和骨骼肌和脂肪组织的胰岛素作用-葡萄糖摄取的协调作用来实现的。这两个过程均由 SNARE 介导的胞吐作用事件控制，当这些事件出错时，2 型糖尿病就会随之而来。 t-SNARE 蛋白 Syntaxin 4 及其调节结合伴侣 Munc18c 现在被认为是这两个过程的胞吐机制的重要组成部分，尽管详细的分子机制仍未解决。这里的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，本文描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“转换机制”的开始，涉及刺激诱导的Munc18c磷酸化及其随后与新伙伴Doc2b结合的偏好。本申请的目的是描绘 Munc18 和 Doc2b 蛋白促进胰岛素分泌和胰岛素反应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 Munc18c 作为关键分子开关，短暂激活 Syntaxin 4 并以刺激诱导的方式促进囊泡递送事件。新的发现支持了这一点，这些发现表明：a) 在称为“突触蛋白激活”的胞吐作用的新对接前步骤中对 Munc18c 的要求； b) 候选Munc18c修饰因子，以催化其存在于脂肪细胞和β细胞中的酪氨酸磷酸化； c) 参与细胞骨架重塑和颗粒递送/定位的新 Munc18c 结合因子。该假设将在三个具体目标上进行检验： 1) 确定Munc18c-Doc2b相互作用在调节GLUT4易位和全身葡萄糖稳态中的要求； 2) 描绘Munc18-Doc2b蛋白复合物在双相胰岛素分泌中的作用； 3) 阐明 Munc18c 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作用的机制。研究将使用 Doc2b 和 Munc18 敲除小鼠进行全身稳态分析和组织分析，以了解对双相胰岛素释放（胰岛灌注）和骨骼肌葡萄糖摄取（GLUT4 易位）的组织特异性影响。 MIN6 β 细胞和 3T3L1 脂肪细胞将用于对蛋白质相互作用的空间和时间变化进行分子/生化解剖，并使用显微镜和生化亚细胞分级分析对颗粒/囊泡胞吐作用事件的动态变化进行可视化。了解 Munc18c 如何调节这些胞吐事件将标志着在可能同时调节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方面取得进展。公众健康相关性：2 型糖尿病被称为“二次打击”疾病；一种“打击”是骨骼肌和脂肪组织的葡萄糖清除功能障碍，另一种“打击”是胰岛β细胞的胰岛素分泌功能障碍。据报道，在患有糖尿病的人类肌肉和胰岛中，Munc18c 蛋白及其结合伴侣的丰度和功能异常，因此有可能介导串扰并成为这两种“打击”的基础。我们使用新型啮齿动物模型进行的研究支持了 Munc18c 的重要且必需的作用，现在迫切需要阐明 Munc18c 在骨骼肌、脂肪和胰岛这些过程中涉及的详细分子机制。基于 Munc18c 的机制的保护对于开发新的治疗策略具有巨大的潜力，这些策略可以同时应对这种疾病的两个“打击”，从而改善糖尿病患者的生活。