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: 8099604
• 负责人: Jai Parkash
• 金额: $ 10.77万
• 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 2012-05-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8099604
• 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 Survival; Cell physiology; Cells; Cessation of life; Complex; Confocal Microscopy; 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; Health; 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; jun Oncogene; meetings; novel; novel strategies; prevent; programs; 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（[Ca2+] i）稳态的变化，是糖尿病中细胞因子诱导的B细胞功能障碍的介体。我们有足够且令人信服的证据为我们的可检验假设提供了理由，即“ TNF-A诱导的细胞内钙失调[Ca2+] I，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激活的介体，从而导致B细胞死亡。三个特定目的是：目标1：确定由细胞因子诱导的[Ca2+] I失调引起的NF-KB的激活。 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通过使用ELISA测量NF-KB的DNA结合活性，通过免疫荧光和（D）NF-KB依赖性转录的核。 AIM 2：B细胞中TNF-A调控基因的候选基因筛选的表征。从对照和TNF-A处理的人类B细胞和RIN细胞中分离出的mRNA水平，编码一个六个TNF-A诱导的差异表达的基因，这些基因将通过半词性多重PCR来测量。我们将展示这六个TNF-A诱导的差异表达的基因在人B细胞中有助于B细胞功能。我们将在存在胰岛素生长因子1（IGF-1）的情况下测量对照和TNF-A处理的RIN细胞和人类B细胞中磷酸化的JNK和磷酸化的P38的水平，以测试我们的假说，“ TNF-A诱导的p38和C-Jun N-终端映射Kins酶的激活可能会导致B-CINS酶的激活，从而导致B-CINS酶在b-Cils酶中的生存。 IGF1”。 AIM 3：通过线粒体诱导的caspase激活确定B细胞死亡的执行。我们假设“ TNF-A诱导的BCL-2在B细胞中的线粒体中Bcl-2的共定位将导致线粒体细胞色素C的释放，当将其解放到细胞质时，将激活caspase-3并执行B-Cell死亡”。在RIN细胞和人类B细胞中，我们将研究TNF-A引起的：（a）通过使用荧光共振能量转移（FRET）方案，通过使用荧光标记的抗体，通过使用荧光标记的抗体来改变这两种蛋白质之间的Bcl-2和Bax的亚细胞位置以及这两种蛋白质之间的空间相互作用； （b）使用免疫荧光显微镜释放细胞色素C； （c）使用免疫荧光显微镜激活caspase 3，以及（d）细胞复制，凋亡率和胰岛素分泌的变化。在三个目标下提出的研究的结果应提供对B细胞死亡中下游效应子的细胞信号和相互作用的新型机械见解，或者由于[Ca2+] I型糖尿病的[Ca2+] I失调而引起的生存。我们的实验设计将确定关键的靶标分子和细胞步骤，以设计有效的治疗策略以及针对少年和晚期糖尿病的干预措施。 公共卫生相关性：糖尿病，一种复杂而无法治愈的疾病是通过补充胰岛素来管理的。我们的创新研究将提供有关导致胰岛素分泌胰岛细胞死亡的分子和生理机制的见解，以及防止β细胞死亡的新型策略，以达到1型糖尿病和2型糖尿病的糖尿病，以避免对每日胰岛素注射的依赖。