Cytosolic and Plasma Membrane Circuitry of Beta Cell Redox Control: Role in Survival, Oxidative Defense and Insulin Secretion

描述（由申请人提供）：我们已经确定了一种新型的氧化还原控制细胞质膜回路，依赖于质膜氧化还原酶 NQO1 和胞质 NADPH 调节酶 NAD 激酶，其调节 NAD(P)H/NADP 比率、活性氧中间体 (ROI) 水平和胰腺 β 细胞的葡萄糖刺激胰岛素分泌 (GSIS)。我们的初步数据证明了这些途径对于 β 细胞功能、存活和 GSIS 的关键作用。此外，NQO1 参与醌氧化还原循环的葡萄糖依赖性过程，调节胞质 NADH/NAD 比率并增强 GSIS。 NAD 激酶是一种新型 NADPH 调节酶，负责 NADPH 的从头产生，并参与保护 β 细胞免受氧化应激。 NQO1 和 NAD 激酶对于正常细胞功能至关重要，但尚未在胰腺 β 细胞中进行表征。我们将使用 shRNA 和腺病毒载体策略以及 NQO1 敲除胰岛进行过度表达或敲低，以分析这些成分在 β 细胞中间代谢中的作用。我们将使用定制的集成电化学共聚焦成像平台来分析 NQO1 和 NAD 激酶对 β 细胞或胰岛对葡萄糖或其他刺激剂的反应的作用，以同时研究胰岛代谢、呼吸和信号传导。这些研究将更深入地了解氧化还原信号在调节胰岛素分泌中的机制，并确定开发 2 型糖尿病治疗药物的新靶点。

DESCRIPTION (provided by applicant): We have identified a novel cytosolic-plasma membrane circuit of redox control, dependent on plasma membrane oxidoreductase NQO1 and the cytosolic NADPH-regulating enzyme NAD kinase, which regulates NAD(P)H/NADP+ ratio, level of reactive oxygen intermediates (ROI) and glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells. Our Preliminary Data demonstrate the critical role of these pathways for beta cell function, survival, and GSIS. Furthermore, NQO1 participates in the glucose-dependent process of quinone redox cycling, which regulates cytosolic NADH/NAD+ ratio and enhances GSIS. NAD kinase, a novel NADPH- regulating enzyme, is responsible for the de novo production of NADPH and participates in the protection of beta cells from oxidative stress. NQO1 and NAD kinase are critical for normal cell function, and have not been characterized in pancreatic beta cells. We will employ over- expression or knockdown using shRNA and adenoviral vectors strategies as well as NQO1 knockout pancreatic islets to analyze the role of these components in beta cell intermediary metabolism. We will analyze the role of NQO1 and NAD kinase on the response of beta cells or islets to glucose or other stimulatory agents using a custom-made integrated electrochemical- confocal imaging platform to simultaneously study islet metabolism, respiration, and signaling. These studies will provide greater insight into the mechanism of redox signaling in regulating insulin secretion and identify novel targets for the development of therapeutics for type 2 diabetes.