Regulation of Islet Beta-Cell Function via Islet-Derived VGF Peptides

描述(申请人提供)：研究建议2型糖尿病(T2D)的定义是高血糖，由外周胰岛素抵抗和胰岛细胞胰岛素释放不足引起。胰岛细胞质量和功能减少已被认为是胰岛素抵抗向T2D发展的关键事件。确定调节胰岛细胞功能的药物对于开发新的糖尿病治疗方法至关重要。我们的研究集中在分泌的激素VGF(与血管内皮生长因子无关，非首字母缩写)，我们发现它控制胰岛细胞生物学的重要方面-胰岛素的分泌和生存。VGF表达于神经内分泌组织(下丘脑、海马体)、支配肠道和脂肪的外周神经元以及胰岛细胞。原VGF被激素处理酶PC1/3和PC2差异地切割；VGF多肽储存在致密的核心颗粒中，并通过调节的分泌途径分泌。在小鼠模型中靶向删除VGF已证明VGF通过中枢神经系统调节能量平衡；然而，VGF在胰岛表达的生物学意义尚不清楚。我的研究证明了一种VGF多肽TLQP-21的药理作用，它是一种新型的胰岛细胞促分泌剂，能够促进T2D啮齿动物模型中的细胞存活。为了评估内源性胰岛表达的VGF的生理功能，我们发现siRNA介导的抑制胰岛素瘤细胞中的VGF强烈地减少了燃料和非燃料刺激的胰岛素分泌。这项建议的中心主题是通过动物模型研究胰岛来源的VGF多肽在调节细胞胰岛素分泌方面的生理作用。这项建议的主要目的是：1)充分研究VGF介导的胰岛细胞调节机制 2)检测关键的VGF多肽(TLQP-21)缺失对胰岛功能和存活的影响；3)检测VGF有条件的细胞缺失对啮齿动物模型中葡萄糖稳态的影响。在特定的目标1中，我们将利用基因表达分析和超微结构检查来确定VGF siRNA抑制后观察到的胰岛素分泌不足的机制。此外，我们还将确定正常的胰岛细胞胰岛素分泌功能所需的血管生长因子多肽(S)。在特定的目标2中，我们将使用人源化的小鼠模型，在该模型中，全长的人VGF或C端截短缺少TLQP-21肽的人VGF的序列已经取代了内源基因(敲入)上的小鼠基因。研究将分为葡萄糖稳态的整体动物测量和胰岛细胞功能和存活率的单独研究。在特定的目标3中，我们将产生一种新的试剂--有条件的细胞特异性删除VGF，使用VGF等位基因和他莫昔芬诱导的细胞特异性CRE驱动器，MIP-CRE/ERT。利用这个模型，我们将确定细胞表达的VGF丢失对胰岛素抵抗小鼠的胰岛功能和葡萄糖稳态的影响。这项提案中概述的研究将为研究胰岛细胞中产生的VGF多肽的生物学意义提供新的见解。对VGF功能的发现可能转化为一种新的治疗策略，用于改善T2D患者的血糖控制和胰岛功能。候选人我的实验室的长期愿景是将我在分子和细胞生物学方面的专业知识与动物生理学结合起来，解决胰岛细胞生物学领域的关键问题。我的直接职业目标是获得一份工作 在一家大型医学(糖尿病)研究机构担任终身教职助理教授。这项建议中描述的研究将提供关键的培训机会，使我能够竞争性地申请教员职位和后续的资助机构。我的导师克里斯托弗·纽加德博士和他的实验室已经成功地开创性地使用腺病毒基因传递工具来研究对胰岛细胞功能调节至关重要的基因和途径。这个 Newgard实验室还强调使用啮齿动物模型(遗传、饮食诱导和化学诱导)在整个动物葡萄糖稳态代谢调节的背景下研究肥胖、胰岛素抵抗和胰岛细胞功能障碍的各个方面。这里概述的建议利用了斯特德曼中心的许多培训机会，但对我来说，它继续进入新的研究领域。特别有用的是，我的研究将使用最先进的基因可诱导小鼠模型来探索条件VGF消融对胰岛细胞的影响。在这一领域，我以前的经验有限，Deborah Muoio博士(共同导师)慷慨地为我提供了在这一重要的胰岛研究领域培训我的专业知识。此外，随着我深入研究细胞功能的基本组成部分(即胰岛素分泌)，我的模型中胰岛素缺陷的超微结构特征将对我作为胰岛细胞生物学家的知识和技能产生重大影响。Newgard博士和Muoio博士完全致力于帮助我实现我的长期目标，允许我独立设计这个方案，并将这个项目带到下一个级别的独立教员职位。因此，通过这个奖项，我将获得培训，并产生必要的关键试剂，以启动我的独立研究计划，并获得终身教职助理教授的职位。

DESCRIPTION (provided by applicant): Research Proposal Type 2 diabetes (T2D) is defined by hyperglycemia, caused by peripheral insulin resistance and insufficient release of insulin from the islet ¿-cell. Reduced islet ¿-cell mass and function have been identified as pivotal events in the progression of insulin resistance toward T2D. Identifying agents regulating islet ¿-cell function are critical for developing new diabetes therapies. Our studies focus on the secreted hormone VGF (unrelated to VEGF, and non-acronymic), which we show to control important aspects of islet ¿-cell biology-insulin secretion and survival. VGF is expressed in neuroendocrine tissues (hypothalamus, hippocampus), peripheral neurons innervating the gut and adipose, and pancreatic islet cells. Pro-VGF is differentially cleaved by the hormone processing enzymes PC1/3 and PC2; VGF peptides are stored in dense core granules and secreted via the regulated secretory pathway. Targeted deletion of VGF in mouse models has demonstrated VGF regulates energy balance via the CNS; however, the biological significance of VGF expression within pancreatic islets remains unknown. My studies have documented the pharmacological actions of one VGF peptide, TLQP-21, as a novel islet ¿-cell secretagogue able to promote the survival of ¿-cells in a rodent model of T2D. To assess the physiological function of endogenous islet expressed VGF, we show that siRNA- mediated suppression of VGF in insulinoma cells strongly reduces both fuel and non-fuel stimulated insulin secretion. The central theme of this proposal is to examine the physiological role of islet-derived VGF peptides in regulating insulin secretion from the ¿-cell using animal models. The major goals of this proposal are: 1) to fully investigate the mechanism of VGF-mediated regulation of islet ¿-cell function; and 2) to examine the impact of loss of a key VGF peptide (TLQP-21) on islet function and survival; and 3) to examine the impact of conditional, ¿-cell deletion of VGF on glucose homeostasis in rodent models. In Specific Aim 1, we will determine the mechanism of the insulin secretion deficiency observed upon VGF siRNA suppression utilizing gene expression analyses and ultrastructural examinations. In addition, we will identify which VGF peptide(s) is required for normal islet ¿-cell insulin secretory function. In Specific Aim 2, we will use a humanized mouse model in which either the sequence for either full length human VGF or human VGF with a C-terminal truncation lacking the TLQP-21 peptide has replaced the mouse gene at the endogenous locus (knock-in). Studies will be divided between whole animal measurements of glucose homeostasis and isolated islet studies of ¿-cell function and survival. In Specific Aim 3, we will generate a novel reagent -a conditional ¿-cell specific deletion of VGF using a floxed VGF allele and a tamoxifen inducible, ¿-cell specific Cre driver, MIP-Cre/ERT. Using this model we will determine the impact of loss of ¿-cell expressed VGF on islet function and glucose homeostasis in a mouse model of insulin resistance. The studies outlined in this proposal will provide novel insight into the biological significance of VGF peptides produced within islet ¿-cells. Discoveries into VGF function could translate into a novel treatment strategy for improving glucose control and islet function in T2D. Candidate My long-term vision for my lab is to integrate my expertise in molecular and cell biology with animal physiology to tackle key questions in the field of islet cell biology. My immediate career objective is to obtain a position as a tenure-track assistant professor at a major medical (diabetes) research institution. The studies described in this proposal will provide key training opportunities to afford me a competitive application for faculty position and subsequent funding agencies. My mentor, Dr. Christopher Newgard and his lab have successfully pioneered the use of adenoviral gene delivery tools to study genes and pathways important for the regulation of islet cell function. The Newgard lab also emphasizes the use of rodent models (genetic, diet-induced, and chemically-induced) to study aspects of obesity, insulin resistance, and islet ¿-cell dysfunction in the context of whole animal metabolic regulation of glucose homeostasis. The proposal outlined here has taken advantage of many of the Stedman Center's training opportunities, yet continues to move forward into new areas of research for me. Particularly useful, will be my studies using state-of-the-art genetically inducible mouse model to explore the impact of conditional VGF ablation in the islet ¿-cell. In this area my prior experience has been limited and Dr. Deborah Muoio (co-mentor) has graciously offered her expertise in training me in this vital area of islet research. Furthermore, as my studies delve deeper into fundamental components of ¿-cell function (i.e. insulin secretion) ultrastructural characterization of insulin defects in my model va the Duke Electron Microscopy Core facility will have great impact on my knowledge and skill set as an islet cell biologist. Drs. Newgard and Muoio are fully committed to helping me achieve my long-term goals, allowing me to independently design this proposal and carry this project with me to the next level of an independent faculty position. Thus, through this award, I will gain training and generate crucial reagents necessary to launch my independent research program and obtain a tenure-track assistant professorship.