Cytokines dysregulate intracellular calcium and cause beta-cell death in diabetes

细胞因子失调细胞内钙并导致糖尿病中的β细胞死亡

基本信息

批准号：
7691970
负责人：
Jai Parkash
金额：
$ 10.8万
依托单位：
FLORIDA INTERNATIONAL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7691970
关键词：
Adolescent Agreement American Antibodies Apoptosis Apoptotic Applications Grants Attenuated Beta Cell Buffers Calcium Candidate Disease Gene Caspase Cause of Death Cell Cycle Regulation Cell Death Cell Nucleus Cell physiology Cells Cessation of life Complex Confocal Microscopy Credentialing Cytoplasm Cytosol DNA Binding Dependence Development Diabetes Mellitus Disease Early treatment Environment Enzyme-Linked Immunosorbent Assay Experimental Designs Fluorescence Microscopy Fluorescence Resonance Energy Transfer Functional disorder Funding Fura-2 Genes Genetic Transcription Glycoproteins Goals Grant Growth Factor Homeostasis Human Hyperglycemia IGF1 gene Immunofluorescence Immunologic Immunofluorescence Microscopy Inflammatory Injection of therapeutic agent Insulin Insulin Resistance Insulin-Dependent Diabetes Mellitus Intervention Islet Cell Journals LAMP-2 Label Lysosomes MAPK14 gene MAPK8 gene Measures Mediator of activation protein Messenger RNA Metabolic Micro Array Data Mitochondria Mitogen-Activated Protein Kinases Molecular N-terminal NF-Kappa B p65 NF-kappa B Pancreas Peer Review Phosphotransferases Physiological Play Productivity Proteins Protocols documentation Publications Publishing RNA Splicing Research Research Personnel Role Secure Signal Transduction Suggestion Supplementation TNF gene Testing Therapeutic Time Tyrosine Vascular Cell Adhesion Molecule-1 activating transcription factor base caspase-3 cytochrome c cytokine design fluorescence imaging improved innovation inorganic phosphate insight insulin secretion interest islet meetings novel novel strategies prevent programs public health relevance ras-Related G-Proteins ratiometric receptor success type I and type II diabetes

项目摘要

DESCRIPTION (provided by applicant): Our long-term goal is to characterize the changes in intracellular calcium ([Ca2+]i) homeostasis as a mediator of cytokine-induced b-cell dysfunction in diabetes. We have sufficient and compelling evidence providing the rationale for our testable hypothesis that "TNF-a-induced dysregulation of intracellular calcium, [Ca2+]i, activates NF-kB transcription that in turn leads to b-cell apoptosis." The focus of this proposal is on [Ca2+]i dysregulation as a mediator of cytokine-induced NF-kB activation that leads to b-cell death. Three specific aims are: Aim 1: Determine the activation of NF-kB caused by cytokine-induced [Ca2+]i dysregulation. In RIN cells, human b-cells, and human islets, we will study TNF-a-induced (a) dysregulation of [Ca2+]i by measuring [Ca2+]i and [Ca2+]i buffering capacity using ratiometric fluorescence imaging with Fura2, (b) degradation of the IkBa subunit by using a FunctionELISA, (c) translocation of NF-kB from cytoplasm to nucleus by immunofluorescence, and (d) NF-kB-dependent transcription by measuring the DNA binding activity of NF-kB using ELISA. Aim 2: Characterization of candidate gene screen of TNF-a regulated genes in b-cells. The levels of mRNA, isolated from control and TNF-a-treated human b-cells and RIN cells, encoding a panel of six TNF-a-induced differentially expressed genes thought to participate in b-cell apoptosis will be measured by semiquantitative multiplex PCR. We will show how these six TNF-a-induced differentially expressed genes contribute to b-cell function in human b-cells. We will measure the levels of phosphorylated JNK and phosphorylated-p38 in control and TNF-a-treated RIN cells and human b-cells in presence of added insulin growth factor-1 (IGF-1) to test our hypothesis that "TNF-a-induced activation of p38 and c-JUN N- terminal MAP kinases in b-cells that leads to b-cells death could be attenuated by IGF1". Aim 3: Determine the execution of b-cell death by mitochondria-induced caspase activation. We hypothesize that "TNF-a- induced co-localization of Bax with Bcl-2 in mitochondria in b-cells will result in release of mitochondrial cytochrome c, which, when liberated to the cytosol, will activate caspase-3 and execute b-cell death". In RIN cells and human b-cells, we will study the TNF-a-induced: (a) changes in subcellular localizations of Bcl-2 and Bax and the spatial interaction between these two proteins by using fluorescent-labeled antibodies in conjunction with fluorescence resonance energy transfer (FRET) protocols; (b) release of cytochrome c by using immunofluorescence microscopy; (c) activation of caspase 3 by using immunofluorescence microscopy, and (d) changes in cell replication, apoptotic rates, and insulin secretion. The results of the proposed studies under the three AIMS should provide novel mechanistic insights into the cellular signals and interplay of downstream effectors in b-cell death or survival due to [Ca2+]i dysregulation in type 1 and type 2 diabetes. Our experimental design will identify critical target molecules and cellular steps for designing efficacious therapeutic strategies and interventions for juvenile and late onset diabetes. PUBLIC HEALTH RELEVANCE: Diabetes, a complex and incurable disease is managed by insulin supplementation. Our innovative studies will provide insights into molecular and physiological mechanisms that cause the death of insulin secreting pancreatic cells, and novel strategies to prevent beta cell death that culminates in diabetes type 1 and 2 in millions of Americans to avert their dependence on daily injections of insulin.

描述（由申请人提供）：我们的长期目标是表征细胞内钙 ([Ca2+]i) 稳态的变化作为糖尿病中细胞因子诱导的 B 细胞功能障碍的介质。我们有足够且令人信服的证据为我们可检验的假设提供理论依据，即“TNF-a 诱导的细胞内钙 [Ca2+]i 失调，激活 NF-kB 转录，进而导致 b 细胞凋亡。”该提案的重点是 [Ca2+]i 失调作为细胞因子诱导的 NF-kB 激活的介质，从而导致 b 细胞死亡。三个具体目标是：目标 1：确定由细胞因子诱导的 [Ca2+]i 失调引起的 NF-kB 激活。在 RIN 细胞、人类 b 细胞和人类胰岛中，我们将通过使用 Fura2 比率荧光成像测量 [Ca2+]i 和 [Ca2+]i 缓冲能力来研究 TNF-a 诱导的 (a) [Ca2+]i 失调， (b) 使用 FunctionELISA 降解 IkBa 亚基，(c) 通过免疫荧光将 NF-kB 从细胞质易位到细胞核，以及 (d) NF-kB 依赖性转录通过使用 ELISA 测量 NF-kB 的 DNA 结合活性。目标 2：b 细胞中 TNF-a 调节基因的候选基因筛选特征。从对照和 TNF-a 处理的人 b 细胞和 RIN 细胞中分离出的 mRNA 水平，编码一组 6 个 TNF-a 诱导的差异表达基因，被认为参与 b 细胞凋亡，将通过半定量多重 PCR 测量。我们将展示这六个 TNF-a 诱导的差异表达基因如何促进人类 B 细胞中的 B 细胞功能。我们将在添加胰岛素生长因子-1 (IGF-1) 的情况下测量对照和 TNF-a 处理的 RIN 细胞和人 b 细胞中磷酸化 JNK 和磷酸化 p38 的水平，以检验我们的假设：“TNF-a -b 细胞中 p38 和 c-JUN N 端 MAP 激酶的诱导激活（导致 b 细胞死亡）可被 IGF1 减弱”。目标 3：确定线粒体诱导的 caspase 激活对 B 细胞死亡的执行。我们假设“TNF-a 诱导的 Bax 与 Bcl-2 在 b 细胞线粒体中的共定位将导致线粒体细胞色素 c 的释放，当释放到细胞质时，将激活 caspase-3 并执行 b-细胞死亡”。在 RIN 细胞和人类 b 细胞中，我们将通过使用荧光标记抗体与荧光结合来研究 TNF-a 诱导的：(a) Bcl-2 和 Bax 的亚细胞定位变化以及这两种蛋白质之间的空间相互作用共振能量转移（FRET）协议； (b) 使用免疫荧光显微镜观察细胞色素c的释放； (c) 使用免疫荧光显微镜激活 caspase 3，(d) 细胞复制、凋亡率和胰岛素分泌的变化。三个 AIMS 下拟议研究的结果应该为 1 型和 2 型糖尿病中由于 [Ca2+]i 失调而引起的 b 细胞死亡或存活中的细胞信号和下游效应器的相互作用提供新的机制见解。我们的实验设计将确定关键靶分子和细胞步骤，以设计针对青少年和迟发性糖尿病的有效治疗策略和干预措施。公众健康相关性：糖尿病是一种复杂且无法治愈的疾病，可以通过补充胰岛素来控制。我们的创新研究将深入了解导致胰岛素分泌胰腺细胞死亡的分子和生理机制，并提供新策略来预防 β 细胞死亡，从而避免数百万美国人患上 1 型和 2 型糖尿病，从而避免他们对每日注射胰岛素的依赖。