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

• 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
• 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

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（[Ca 2 +]i）稳态的变化，作为糖尿病中精氨酸诱导的b细胞功能障碍的介质。我们有充分和令人信服的证据为我们的可检验假设提供了理论基础，即“TNF-α诱导的细胞内钙离子[Ca 2 +]i失调激活NF-κ B转录，进而导致b细胞凋亡。“这项建议的重点是[Ca 2 +]i失调作为一种介导的尼古丁诱导的NF-κ B激活，导致b细胞死亡。三个具体的目标是：目标1：确定激活的NF-κ B引起的精氨酸诱导的[Ca 2 +]i失调。在RIN细胞、人b细胞和人胰岛中，我们将研究TNF-α诱导的（a）[Ca 2 +]i的失调，通过使用Fura 2的比率荧光成像测量[Ca 2 +]i和[Ca 2 +]i缓冲能力，（B）通过使用FunctionELISA降解IkBa亚基，（c）通过免疫荧光从细胞质到细胞核的NF-κ B易位，和（d）通过使用ELISA测量NF-kB的DNA结合活性的NF-kB依赖性转录。目的2：筛选B细胞中TNF-α调控基因的候选基因。将通过半定量多重PCR测量从对照和TNF-α处理的人b细胞和RIN细胞分离的mRNA水平，所述mRNA编码一组被认为参与b细胞凋亡的六个TNF-α诱导的差异表达基因。我们将展示这六个TNF-α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-α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，即“TNF-α诱导的b细胞中p38和c-JUN N-末端MAP激酶的活化（导致b细胞死亡）可以被IGF-1减弱”。目的3：确定通过激活胱天蛋白酶来执行b细胞死亡。我们假设“TNF-α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，其将激活半胱天冬酶-3并执行b细胞死亡”。在RIN细胞和人b细胞中，我们将研究TNF-α诱导的：（a）Bcl-2和Bax的亚细胞定位的变化和这两种蛋白之间的空间相互作用，通过使用荧光标记抗体结合荧光共振能量转移（FRET）方案：（B）细胞色素c的释放，通过使用免疫荧光显微镜。（c）通过使用免疫荧光显微术激活半胱天冬酶3，和（d）细胞复制、凋亡率和胰岛素分泌的变化。根据三个AIMS的拟议研究的结果应提供新的机制的见解，细胞信号和下游效应器的相互作用，在b细胞死亡或生存由于1型和2型糖尿病[Ca 2 +]i失调。我们的实验设计将确定关键的靶分子和细胞步骤，为青少年和迟发性糖尿病设计有效的治疗策略和干预措施。 公共卫生相关性：糖尿病是一种复杂的不治之症，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，以及预防β细胞死亡的新策略，这种死亡最终导致数百万美国人的1型和2型糖尿病，以避免他们对每日注射胰岛素的依赖。