Regulation of islet gap junction coupling and function under inflammatory conditions

炎症条件下胰岛间隙连接耦合和功能的调节

• 批准号: 8834068
• 负责人: Nikki L. Farnsworth
• 金额: $ 5.33万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-05 至 2017-09-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8834068
• 关键词: Agonist; Beta Cell; Blood Glucose; Calcium; Calcium Signaling; Cell Line; Cell Survival; Cell membrane; Cells; Chemicals; Coupling; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Dependence; Development; Diabetes Mellitus; Diagnosis; Disease; Disease Progression; Event; Fluorescence Microscopy; Fluorescent Probes; Functional disorder; Future; Gap Junctions; Genetic; Glucose Intolerance; Graft Survival; Human; In Vitro; Incidence; Individual; Inflammatory; Insulin; Investigation; Islets of Langerhans; Knock-out; Knockout Mice; Label; Lead; Life; Measures; Mediating; Mediator of activation protein; Membrane; Membrane Protein Traffic; Metabolic; Microscopy; Mitochondria; Mus; NADH; Nitric Oxide; Operative Surgical Procedures; Oxidation-Reduction; Pathogenesis; Pathway Analysis; Pathway interactions; Patients; Phase; Phosphorylation; Physiologic pulse; Physiological; Prediabetes syndrome; Protein Kinase C; Regulation; Regulatory Pathway; Research; Research Training; Role; Series; Signal Pathway; Signal Transduction; Stress; Techniques; Testing; Tissues; Transplantation; Work; blood glucose regulation; connexin 36; cytokine; gap junction channel; graft function; improved; inhibitor/antagonist; insulin secretion; islet; mouse model; prevent; protective effect; public health relevance; research study; response; trafficking; type I and type II diabetes

DESCRIPTION (provided by applicant): The islets of Langerhans are responsible for maintaining blood glucose levels through insulin secretion. In ?- cells, insulin release is initiatd by metabolic and electrical events, in particular an influx of calcium into the cell following membrane depolarization. Connexin36 (Cx36) gap junctions provide electrical coupling between ? -cells, which coordinates intracellular calcium signaling across the islet to coordinate insulin release. Knockout of islet Cx36 gap junctions disrupts this coordination and elevates [Ca2+]i, leading to reduced first phase insulin secretion and diminished second phase pulses, similar to that seen in pre-diabetes. Pro- inflammatory cytokines have been implicated as mediators of ? -cell dysfunction in early diabetes and in islet transplant surgery. Preliminary data has shown that exposure of ? -cells to low levels of pro-inflammatory cytokines, similar to those seen in early diabetes alters calcium signaling and impairs insulin secretion dynamics. Taken together, this suggests a role for gap junction coupling in contributing to islet dysfunction during the pathogenesis of diabetes. We hypothesize that pro-inflammatory cytokines decrease gap junction coupling in islets via nitric oxide mediated channel closure, leading to islet dysfunction and increased calcium mediated mitochondrial stress, while GLP-1R mediated increases in cAMP will increase gap junction coupling through Epac2 and PKA regulatory pathways. To test this, we propose to do a series of in vitro studies, where isolated mouse and human islets, islets with a genetic knockout of Cx36, or islets treated with Cx36 inhibitors/activators, will be culture with varying levels of pro-inflammatory cytokines. Islet function will be assessed through calcium signaling, insulin dynamics, Cx36 gap junction coupling and mitochondrial function, including the use of advanced microscopy approaches. The role of nitric oxide and cAMP on gap junction coupling will be determined through culture with chemical inhibitors/agonists of the proposed mediators and analysis of Cx36 phosphorylation, trafficking and membrane plaque formation. The effects of pro- inflammatory cytokines on gap junction coupling in the islet could represent a previously unknown mechanism of islet dysfunction contributing to the pathogenesis of diabetes. Understanding the mechanisms involved in regulating gap junctions could help to identify targets for potential therapies in Type 1 and Type 2 Diabetes as well as ways to improve graft survival and function in islet transplant surgeries.

描述（由申请人提供）：Langerhans的胰岛负责通过胰岛素分泌维持血糖水平。在？ - 细胞中，胰岛素释放是由代谢和电动事件引起的，特别是在膜去极化后钙流入细胞中。 connexin36（CX36）间隙连接提供之间的电耦合？ - 细胞，该细胞在胰岛跨胰岛上协调胰岛素释放的细胞内钙信号传导。胰岛CX36间隙连接的敲除可破坏这种协调并提高[Ca2+] I，导致第一相胰岛素分泌减少并减少第二相脉冲，类似于糖尿病前期的糖尿病。促炎性细胞因子已被牵涉到介体的介体？ - 早期糖尿病和胰岛移植手术中的细胞功能障碍。初步数据表明 的接触 ？ - 促炎性细胞因子的细胞与早期糖尿病的促炎细胞因子相似，会改变钙信号传导并损害胰岛素分泌动力学。综上所述，这表明在糖尿病发病机理期间，间隙连接耦合在胰岛功能障碍中起作用。我们假设促炎细胞因子通过一氧化氮介导的通道闭合减少胰岛中的间隙连接耦合，从而导致胰岛功能障碍 并增加了钙介导的线粒体应激，而GLP-1R介导的cAMP增加将增加通过EPAC2和PKA调节途径的间隙连接耦合。为了测试这一点，我们建议进行一系列体外研究，其中分离的小鼠和人类胰岛，具有CX36的基因敲除或用CX36抑制剂/激活剂处理的胰岛的胰岛将具有不同水平的促炎细胞因子的培养。胰岛功能将通过钙信号传导，胰岛素动力学，CX36间隙连接耦合和线粒体功能进行评估，包括使用高级显微镜方法。一氧化氮和cAMP在间隙连接耦合方面的作用将通过拟议介体的化学抑制剂/激动剂进行培养以及CX36磷酸化，运输和膜斑块形成的分析来确定。胰岛炎性细胞因子对胰岛间隙连接耦合的影响可能代表了先前未知的胰岛功能障碍机制，导致糖尿病的发病机理。了解调节间隙连接所涉及的机制可以帮助鉴定1型和2型糖尿病中潜在疗法的靶标，以及改善胰岛移植手术中移植物存活和功能的方法。