Calcium Signaling in Pancreatic Beta Cell Endoplasmic Reticulum

胰腺β细胞内质网中的钙信号传导

• 批准号: 7080765
• 负责人: MICHAEL WILLIAM ROE
• 金额: $ 29.13万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-10 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7080765
• 关键词: RNA interference; adenosinetriphosphatase; calcium channel; calcium flux; calcium ion; confocal scanning microscopy; cytoplasm; disease /disorder model; endoplasmic reticulum; free radicals; gene expression; genetically modified animals; glucose; hormone regulation /control mechanism; inositol phosphates; insulin; laboratory mouse; mitochondria; noninsulin dependent diabetes mellitus; pancreatic islet function; receptor expression; stromal cells; voltage gated channel

DESCRIPTION (provided by applicant): The overall objectives of this proposal are to better understand the regulation of insulin secretion by free calcium ions (Ca2+) and the role of abnormal Ca2+ signaling in the pathophysiology of Type 2 diabetes mellitus (T2DM). Glucose stimulation evokes changes in intracellular Ca2+ ([Ca2+]c) in islets and insulin- secreting cell lines that temporally correlate with insulin secretion. Although much is known about the contribution of Ca2+ influx through voltage-gated Ca2+ channels to B-cell insulin secretion, our knowledge of the contribution of endoplasmic reticulum (ER) Ca2+ stores is limited. This is in part due to few reports of direct measurements of secretagogue effects on [Ca2+]ER and incomplete understanding of the mechanisms that regulate ER Ca2+ stores in B-cells. The proposed experiments will focus on defining these mechanisms. Preliminary studies suggest that sarcoendoplasmic reticulum Ca2+-ATPase (SERCA), a key regulator of ER Ca2+ homeostasis, is impaired in islets from the db/db mouse model of T2DM. This raises the intriguing possibility that loss of B-cell function in T2DM is related to defects in ER Ca2+ signaling. We will employ a combination of biosensor technology, RNA silencing, novel transgenic and imaging approaches to identify and characterize underlying regulatory mechanisms. The proposed experiments will [1] identify spatial and temporal interplay between cytoplasmic and ER Ca2+ signaling in single cells; [2] determine kinetics of signals that affect ER Ca2+ mobilization; [3] define novel mechanisms that regulate ER Ca2+ store refilling; [4] define the role of mitochondria in regulation of ER Ca2+ homeostasis; [5] determine whether stromal interaction molecule (STIM) couples ER Ca2+ levels with store-operated Ca2+ entry (SOCE); and [6] define the role of ER Ca2+ signaling defects in B-cell dysfunction associated with diabetes. The following hypotheses will be tested: [1] glucose stimulates ER Ca2+ signaling; [2] glucose-induced [Ca2+]c and [Ca2+]ER oscillations are temporally and causally interrelated; [3] STIM1 is expressed in B-cells and essential for SOCE; [4] mitochondria and plasma membrane-related Ca2+-ATPase-1 (Pmr-1) regulate ER Ca2+ homeostasis; [5] B-cell dysfunction in T2DM is due to defects in ER Ca2+ homeostasis consequent to decreased expression of SERCA and STIM. We will test these hypotheses in MIN6 B-cells, islets and primary B-cells from transgenic biosensor mice and db/db mice. Identification and characterization of the signaling pathways that control ER Ca2+ is a necessary step in advancing our knowledge of the molecular mechanisms regulating B-cell Ca2+ signal transduction and function. The experiments will contribute new information essential to improve understanding of the pathogenesis of T2DM, and facilitate development of novel strategies used in the preservation and maintenance of B-cell function in patients with diabetes.

描述（由申请人提供）：该提案的总体目标是更好地了解游离钙离子（CA2+）对胰岛素分泌的调节，以及异常Ca2+信号传导在2型糖尿病型糖尿病（T2DM）的病理生理学中的作用。葡萄糖刺激引起了胰岛和胰岛素分泌细胞系中细胞内Ca2+（[Ca2+] C）的变化，这些细胞系与胰岛素分泌时间相关。尽管对CA2+涌入通过电压门控Ca2+通道对B细胞胰岛素分泌的贡献知之甚少，但我们对内质网（ER）Ca2+商店的贡献的了解有限。这部分是由于对[Ca2+] ER的促进作用的直接测量以及对调节B细胞中ER Ca2+存储的机制的不完全理解的直接测量的报道。提出的实验将集中于定义这些机制。初步研究表明，肉壳质网Ca2+ -ATPase（SERCA）是ER Ca2+稳态的关键调节剂，在T2DM的DB/DB小鼠模型中受到了损害。这提出了一个有趣的可能性，即T2DM中B细胞函数的丧失与ER Ca2+信号传导中的缺陷有关。我们将采用生物传感器技术，RNA沉默，新颖的转基因和成像方法的结合来识别和表征潜在的调节机制。提出的实验将[1]识别单个细胞中细胞质和ER Ca2+信号之间的空间和时间相互作用； [2]确定影响ER CA2+动员的信号的动力学； [3]定义调节ER Ca2+储存补充的新型机制； [4]定义线粒体在ER Ca2+稳态调节中的作用； [5]确定基质相互作用分子（Stim）是否具有储存的Ca2+进入（SOCE）的Ca2+水平； [6]定义了ER Ca2+信号传导缺陷在与糖尿病相关的B细胞功能障碍中的作用。将测试以下假设：[1]葡萄糖刺激ER CA2+信号传导； [2]葡萄糖诱导的[Ca2+] C和[Ca2+] ER振荡在时间上和因果相互关联。 [3] STIM1在B细胞中表达，对于SOCE必不可少。 [4]线粒体和质膜相关的Ca2+ -ATPase-1（PMR-1）调节ER Ca2+稳态； [5] T2DM中的B细胞功能障碍是由于ER Ca2+稳态中的缺陷导致SERCA和Stim的表达降低。我们将在转基因生物传感器小鼠和DB/DB小鼠的MIN6 B细胞，胰岛和原代B细胞中检验这些假设。控制ER Ca2+的信号通路的识别和表征是促进我们对调节B细胞CA2+信号转导和功能的分子机制的了解的必要步骤。该实验将为提高对T2DM发病机理的理解至关重要，并有助于开发用于保存和维持糖尿病患者B-Cell功能的新型策略。