Calcium Signaling in Pancreatic Beta Cell Endoplasmic Reticulum

描述（由申请方提供）：本提案的总体目标是更好地了解游离钙离子（Ca 2+）对胰岛素分泌的调节以及异常Ca 2+信号传导在2型糖尿病（T2 DM）病理生理学中的作用。葡萄糖刺激引起胰岛和胰岛素分泌细胞系中细胞内Ca 2+（[Ca 2 +]c）的变化，其在时间上与胰岛素分泌相关。虽然我们对电压门控性钙通道内流对B细胞胰岛素分泌的作用了解很多，但对内质网钙库的作用了解有限。这部分是由于促分泌素对[Ca 2 +]ER的直接测量的报道很少，以及对调节B细胞中ER Ca 2+储存的机制的不完全理解。拟议的实验将侧重于确定这些机制。初步研究表明，肌内质网Ca 2 +-ATP酶（SERCA），ER Ca 2+稳态的关键调节剂，在来自T2 DM db/db小鼠模型的胰岛中受损。这提出了一个有趣的可能性，即T2 DM中B细胞功能的丧失与ER Ca 2+信号传导的缺陷有关。我们将采用生物传感器技术，RNA沉默，新的转基因和成像方法的组合，以确定和表征潜在的调控机制。这些实验将[1]确定单个细胞中胞质和ER Ca 2+信号之间的空间和时间相互作用; [2]确定影响ER Ca 2+动员的信号动力学; [3]确定调节ER Ca 2+库再填充的新机制; [4]确定线粒体在ER Ca 2+稳态调节中的作用; [5]确定线粒体在ER Ca 2+稳态调节中的作用。[5]确定基质相互作用分子（STIM）是否将ER Ca 2+水平与钙池操纵的Ca 2+内流（SOCE）偶联;[6]定义ER Ca 2+信号传导缺陷在糖尿病相关B细胞功能障碍中的作用。将检验以下假设：[1]葡萄糖刺激ER Ca 2+信号传导; [2]葡萄糖诱导的[Ca 2 +]c和[Ca 2 +]ER振荡在时间上和因果上相互关联; [3] STIM 1在B细胞中表达，并且是SOCE所必需的; [4]线粒体和质膜相关的Ca 2 +-ATP酶-1（Pmr-1）调节ER Ca 2+稳态;[5] T2 DM中的B细胞功能障碍是由于ERCa 2+稳态缺陷导致SERCA和STIM表达降低。我们将在来自转基因生物传感器小鼠和db/db小鼠的MIN 6 B细胞、胰岛和原代B细胞中测试这些假设。识别和表征控制ER Ca 2+的信号通路是推进我们对调节B细胞Ca 2+信号转导和功能的分子机制的认识的必要步骤。这些实验将为提高对T2 DM发病机制的理解提供必要的新信息，并促进开发用于保护和维持糖尿病患者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.