Calcium Signaling in Pancreatic Beta Cell Endoplasmic Reticulum

描述（由申请人提供）：本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+信号在2型糖尿病（T2DM）病理生理中的作用。葡萄糖刺激引起胰岛和胰岛素分泌细胞系细胞内Ca2+ ([Ca2+]c)的变化，这些变化与胰岛素分泌在时间上相关。虽然我们对Ca2+通过电压门控Ca2+通道内流对b细胞胰岛素分泌的贡献了解很多，但我们对内质网（ER） Ca2+储存的贡献了解有限。这部分是由于对[Ca2+]内质网促分泌作用的直接测量的报道很少，以及对b细胞中ER Ca2+储存的调节机制的理解不完整。拟议的实验将侧重于定义这些机制。初步研究表明，在db/db小鼠T2DM模型中，肌内质网Ca2+- atp酶（SERCA）是ER Ca2+稳态的关键调节因子，在胰岛中受损。这提出了一种有趣的可能性，即T2DM中b细胞功能的丧失与ER Ca2+信号的缺陷有关。我们将结合生物传感器技术、RNA沉默、新型转基因和成像方法来识别和表征潜在的调控机制。提出的实验将[1]确定细胞质和ER Ca2+信号传导之间的空间和时间相互作用；[2]确定影响ER Ca2+动员的信号动力学；[3]定义了调节内质网Ca2+储存再填充的新机制；[4]确定线粒体在调节ER Ca2+稳态中的作用；[5]确定基质相互作用分子（STIM）是否将ER Ca2+水平与储存操作的Ca2+进入（SOCE）耦合；和[6]定义了ER Ca2+信号缺陷在糖尿病相关的b细胞功能障碍中的作用。以下假设将被验证：[1]葡萄糖刺激内质网Ca2+信号；葡萄糖诱导的[Ca2+]c和[Ca2+]ER振荡在时间和因果关系上是相关的；[3] STIM1在b细胞中表达，对SOCE至关重要；[4]线粒体和质膜相关的Ca2+- atp酶-1 （Pmr-1）调节ER Ca2+稳态；2型糖尿病的b细胞功能障碍是由于SERCA和STIM表达减少导致内质网Ca2+稳态缺陷。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细胞中验证这些假设。识别和表征控制ER Ca2+的信号通路是提高我们对调节b细胞Ca2+信号转导和功能的分子机制的认识的必要步骤。该实验将提供新的信息，以提高对T2DM发病机制的理解，并促进开发用于保存和维持糖尿病患者b细胞功能的新策略。

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.