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

描述(申请人提供)：我们已经确定了一种新的氧化还原控制的胞浆-质膜回路，依赖于质膜氧化还原酶NQO1和胞浆NADPH调节酶NAD激酶，它调节胰岛β细胞的NAD(P)H/NADP比率、活性氧中间产物(ROI)水平和葡萄糖刺激的胰岛素分泌(GSIS)。我们的初步数据显示了这些通路对β细胞功能、存活和GSIS的关键作用。此外，NQO1还参与了依赖葡萄糖的苯醌氧化还原循环过程，调节了胞内NADH/NAD比值，增强了GSIS。NAD激酶是一种新的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激酶是细胞正常功能的关键，但尚未在胰岛β细胞中得到表征。我们将使用shRNA和腺病毒载体的过度表达或敲除策略以及NQO1基因敲除的胰岛来分析这些成分在β细胞中介代谢中的作用。我们将利用定制的集成电化学-共聚焦成像平台同时研究胰岛代谢、呼吸和信号转导，分析NQO1和NAD激酶在β细胞或胰岛对葡萄糖或其他刺激剂反应中的作用。这些研究将更深入地了解氧化还原信号调节胰岛素分泌的机制，并为2型糖尿病治疗药物的开发确定新的靶点。

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.