Targeting intracellular calcium leak as novel therapy for diabetes

针对细胞内钙渗漏作为糖尿病的新疗法

• 批准号: 9014187
• 负责人: Gaetano Santulli
• 金额: $ 8.82万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-14 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9014187
• 关键词: Acquaintances; Address; Adverse effects; Affect; Animal Model; Area; Award; Beta Cell; Binding; Biomedical Research; Biometry; Budgets; Calcium; Cell membrane; Cell physiology; Clinical Trials; Complex; DNA Sequence Alteration; Data; Development; Diabetes Mellitus; Disease; Drug Targeting; Education; Endoplasmic Reticulum; Environment; Experimental Designs; Extracellular Space; FKBP1B gene; Factor Analysis; Foundations; Functional disorder; Funding; Future; Genetic; Glucose; Glucose Intolerance; Goals; Grant; Heart Diseases; Homeostasis; Human; Human Resources; In Vitro; Insulin; International; Intervention; Ion Channel; Islets of Langerhans; Knock-in Mouse; Knowledge; Laboratories; Lead; Leptin; Link; Macromolecular Complexes; Mentors; Meta-Analysis; Metabolic; Metabolism; Methods; Minor; Mitochondria; Modeling; Modification; Molecular; Molecular Target; Mus; Mutate; Mutation; Myopathy; Non-Insulin-Dependent Diabetes Mellitus; Outcome; Pancreas; Patients; Phase; Phase Transition; Play; Positioning Attribute; Prevention; Principal Investigator; Production; Public Health; Reporting; Research; Role; RyR2; Ryanodine Receptors; Scientist; Signal Transduction; Syndrome; Tachycardia; Techniques; Testing; Therapeutic; Training; Universities; Ventricular Tachycardia; Work; Writing; base; biological adaptation to stress; blood glucose regulation; career; design; diabetes mellitus therapy; endoplasmic reticulum stress; falls; fitness; glucose metabolism; glucose tolerance; high throughput analysis; human subject protection; improved; in vivo; innovation; insulin secretion; insulin sensitivity; islet; meetings; mitochondrial dysfunction; mouse model; new therapeutic target; notch protein; novel; novel strategies; novel therapeutic intervention; novel therapeutics; prevent; public health relevance; release of sequestered calcium ion into cytoplasm; research study; response; responsible research conduct; skills; small molecule; therapeutic target; tool; treatment strategy; voltage; volunteer

 DESCRIPTION (provided by applicant): The proposed research study will facilitate the improvement of the education and career goals of the principal investigator (PI). Moreover, the candidate will investigate the mechanisms that underlie impaired insulin secretion in type 2 diabetes mellitus (T2DM). The candidate has set up an important educative plan for the first two years (K99 phase) of the proposed study. This plan includes also courses. In addition, the candidate will be trained in scientific and professional research skills by his mentors and other experts. The candidate will perform experiments to investigate the molecular mechanisms underlying the functional role of intracellular calcium leak in the pathophysiology of T2DM and will suggest innovative, targeted therapeutic approaches for the treatment of such a disease. Upon conclusion of the mentored phase the candidate will have established a pool of data, knowledge and scientific skills that will enhance his credentials for successful transition to independence. During the K99 phase the candidate has planned to attend courses on biostatistics, grant writing/research funding, human subject protection, and responsible conduct of research. In addition, he has set up a specific training plan with his mentors and his advisors/collaborators that will provide him with scientific knowledge on ER stress/mitochondrial fitness, β-cell dynamics, ion channels. These aspects represent useful tools that he will be able to transfer to his own laboratory. Besides the new techniques that he will acquire, the proposed project requires knowledge in areas that he has only recently been associated with, such as mitochondrial pathophysiology and metabolic signaling. Therefore, he has included in his advisory board a number of scientists with outstanding careers in these particular fields that will provide guidance of the highest possible level. All his advisors and collaborators are top-notch academic scientists (see a complete list in Personnel, in the `Budget justification' document). The candidate will also present his work at several international meetings, in order to receive input by a broad scientific audience, thereby expanding his scientific network. This will promote his transition to an independent research position and provide him a robust scientific foundation from which to apply for independent R01-level funding. The application core questions are: (1) Which is the functional role of intracellular Ca2+ leak via RyR2 in pancreatic β-cells? (2) What i the mechanism underlying impaired glucose-stimulated insulin secretion in β-cells harboring leaky RyR2 channels? (3) How can the leak be prevented in human islets from T2DM donors or animal models of diabetes? (4) How the prevention of intracellular Ca2+ leak can impact the pathophysiology of T2DM? To address these questions the candidate has designed an experimental plan with two main branches. The first falls into the K99 phase and is based on preliminary data of the current submission. This set of experiments aims to identify the mechanism that makes the Ca2+ leak via RyR2 channels in β-cells a heretofore undisclosed key player in glucose-induced insulin release. The applicant will characterize both in vivo and ex vivo the functional aspects of leaky RyR2 using knock-in mice harboring genetic mutations in RyR2 that renders the channel leaky. The same mutations characterize a rare syndrome, known as catecholaminergic polymorphic ventricular tachycardia (CPVT); intriguingly, studies provided as material supporting the present application have demonstrated that most of these patients display glucose intolerance. The candidate will verify the effects of the induction of the leak in vitro on insulin release. Moreover, he will test an orally available small molecule (Rycal) in the CPVT mouse models, to see whether such pharmacological intervention, able to prevent intracellular Ca2+ leak, can affect β-cell dynamics. The second branch of the plan falls into the R00 phase, where the candidate will determine whether the inhibition of intracellular Ca2+ leak via RyR2 improves glucose homeostasis in established models of T2DM. These models will be characterized with high throughput analysis methods and new targets may come up. Equally important, the elucidation of the mechanisms that will be investigated in the K99 phase (ER stress, mitochondria, ion channels) might also identify new targets and may suggest novel approaches for the treatment of T2DM. Ultimately, application of these strategies and interventions, as well as targeting of new factors that the analysis focused on ER Stress and Ca2+ fluxes is most likely going to indicate new directions to treat conditions of impaired glucose homeostasis. Moreover, with the scientific and professional skills acquired by the applicant during the K99 phase, the transition to independent position will be certainly facilitate. Overall, the current application will equip the candidate with novel knowledge and useful tools to continue for independent career in molecular metabolism and diabetes research. His training plan has been meticulously designed to facilitate flawless production of data in the prominent environment of Columbia University, as well as the PI's acquaintance with modern tools of biomedical research that he will transfer to his independent laboratory. Approval of his application for the K99/R00 award will result in the precise definition of novel mechanisms affecting β-cell pathophysiology, challenging current paradigms and will thereby provide potential new therapeutics for the treatment of diabetes mellitus.

 描述（由申请人提供）：拟议的研究将有助于改善主要研究者（PI）的教育和职业目标。此外，候选人将研究2型糖尿病（T2 DM）胰岛素分泌受损的机制。候选人已经为拟议研究的前两年（K99阶段）制定了重要的教育计划。该计划还包括课程。此外，候选人将接受导师和其他专家的科学和专业研究技能培训。候选人将进行实验，以研究T2 DM病理生理学中细胞内钙渗漏功能作用的分子机制，并提出治疗此类疾病的创新靶向治疗方法。在辅导阶段结束时，候选人将建立一个数据库、知识库和科学技能库，这将提高他成功过渡到独立的资格。在K99阶段，候选人计划参加生物统计学课程，赠款写作/研究资金，人类受试者保护和负责任的研究行为。此外，他还与他的导师和顾问/合作者制定了一个具体的培训计划，为他提供有关ER应激/线粒体适应性，β细胞动力学，离子通道的科学知识。这些方面代表了有用的工具，他将能够转移到他自己的实验室。除了他将获得的新技术外，拟议的项目还需要他最近才参与的领域的知识，例如线粒体病理生理学和代谢信号。因此，他在他的顾问委员会中包括了一些在这些特定领域具有杰出事业的科学家， 提供尽可能高水平的指导。他的所有顾问和合作者都是一流的学术科学家（见《人事》中的完整名单，见“预算理由”文件）。候选人还将在几次国际会议上介绍其工作，以获得广大科学受众的投入，从而扩大其科学网络。这将促进他向独立研究职位的过渡，并为他申请独立R 01级资助提供坚实的科学基础。应用的核心问题是：（1）RyR 2介导的细胞内Ca 2+渗漏在胰岛β细胞中的功能作用是什么？(2)在携带RyR 2渗漏通道的β细胞中，葡萄糖刺激的胰岛素分泌受损的潜在机制是什么？(3)如何防止来自T2 DM供体或糖尿病动物模型的人类胰岛中的渗漏？(4)预防细胞内Ca 2+渗漏如何影响T2 DM的病理生理学？为了解决这些问题，候选人设计了一个实验计划，其中有两个主要分支。第一份福尔斯报告属于K99阶段，以本次划界案的初步数据为基础。这组实验旨在鉴定使β细胞中的Ca 2+经由RyR 2通道泄漏的机制，这是迄今为止未公开的葡萄糖诱导的胰岛素释放中的关键参与者。申请人将使用在RyR 2中携带使通道渗漏的基因突变的敲入小鼠，在体内和离体表征渗漏RyR 2的功能方面。相同的突变表征了一种罕见的综合征，称为儿茶酚胺能多态性室性心动过速（CPVT）;有趣的是，作为支持本申请的材料提供的研究已经证明，这些患者中的大多数表现出葡萄糖耐受不良。候选人将验证体外诱导泄漏对胰岛素释放的影响。此外，他将在CPVT小鼠模型中测试口服小分子（Rycal），以观察这种能够防止细胞内Ca 2+泄漏的药物干预是否会影响β细胞动力学。计划的第二个分支福尔斯进入R 00阶段，在此阶段，候选人将确定通过RyR 2抑制细胞内Ca 2+泄漏是否改善了已建立的T2 DM模型中的葡萄糖稳态。这些模型将以高通量分析方法为特征，并可能出现新的目标。同样重要的是，将在K99阶段研究的机制（ER应激、线粒体、离子通道）的阐明也可能确定新的靶点，并可能提出治疗T2 DM的新方法。最终，这些策略和干预措施的应用，以及针对ER应激和Ca 2+通量分析的新因素的目标，很可能会指示治疗葡萄糖稳态受损的新方向。此外，随着申请人在K99阶段获得的科学和专业技能，过渡到独立的位置肯定会很方便。总的来说，目前的应用程序将为候选人提供新的知识和有用的工具，以继续在分子代谢和糖尿病研究的独立职业生涯。他的培训计划经过精心设计，以促进在哥伦比亚大学的突出环境中完美的数据生产，以及PI对现代生物医学研究工具的熟悉，他将转移到他的独立实验室。他的K99/R 00奖项申请获得批准将导致影响β细胞病理生理学的新机制的精确定义，挑战当前的范式，从而为糖尿病治疗提供潜在的新疗法。