Expression, localization and regulation of NADPH oxidases in pancreatic beta cells

Reactive oxygen species (ROS) are short-lived and act in a site-specific manner, underscoring the importance of identifying the subcellular localization of their sources. ROS-generating NADPH oxidases (NOX) regulate pancreatic beta cell (dys)function. However, their subcellular localization and cytokine-mediated regulation in these cells remain largely unknown. We characterized the expression, subcellular localization and time-dependent cytokine-induced regulation of NOX isoforms in beta cells. Isoforms were studied via RT-qPCR, immunoblotting and immunofluorescence in rat islets and beta cell lines. Beta cells express DUOX1 and DUOX2 proteins and Duoxa2 transcripts; lacking Duoxa1 expression. In INS-1E cells, NOX1 and DUOX1 localize in the endoplasmic reticulum (ER); DUOX2 in insulin vesicles; and NOX2 and NOX4 in vesicles, ER and plasma membrane. In INS-1E, cytokines increased expression of Nox1 and Duox1 at 4-8 h (returning to baseline at 16 h) and Nox2 and p47phox at 8 h (persisting until 24 h). Duox(a)2, p67phox and p40phox were downregulated and DUOX1 upregulated at 16-24 h. The absence of Duoxa1 in beta cells might lead to DUOX1 mismatching, impairing its trafficking and activity. NOXs in beta cells are diverse in subcellular localization and cytokine-induced regulation, suggesting their isoform-specific involvement in beta cell function, stress and apoptosis.

活性氧簇（Reactive oxygen species, ROS）寿命短暂且具有位点特异性的作用方式，这凸显了确定其来源亚细胞定位的重要性。产生活性氧的烟酰胺腺嘌呤二核苷酸磷酸氧化酶（NADPH oxidases, NOX）可调控胰岛β细胞的功能与功能异常。然而，这类酶在胰岛β细胞中的亚细胞定位以及细胞因子介导的调控机制，目前仍知之甚少。本研究对胰岛β细胞中NOX亚型的表达特征、亚细胞定位以及细胞因子诱导的动态调控过程进行了系统表征。研究采用实时定量聚合酶链式反应（RT-qPCR）、免疫印迹法以及免疫荧光法，对大鼠胰岛与β细胞系中的NOX亚型展开分析。胰岛β细胞可表达双氧化酶1（DUOX1）、双氧化酶2（DUOX2）蛋白以及双氧化酶辅助因子2（Duoxa2）转录本，但不表达双氧化酶辅助因子1（Duoxa1）。在INS-1E细胞中，NOX1与DUOX1定位于内质网（endoplasmic reticulum, ER）；DUOX2定位于胰岛素囊泡；而NOX2与NOX4则分布于囊泡、内质网与质膜中。在INS-1E细胞中，细胞因子可在4至8小时内上调Nox1与Duox1的表达水平，该表达水平在16小时时恢复至基线；并在8小时上调Nox2与p47phox的表达，该上调效应持续至24小时。而Duox(a)2、p67phox与p40phox的表达则被下调，DUOX1的表达在16至24小时期间出现上调。胰岛β细胞中缺乏Duoxa1，可能会导致DUOX1的配对异常，进而损害其转运过程与催化活性。胰岛β细胞中的NOX亚型在亚细胞定位与细胞因子诱导的调控模式上存在显著多样性，这提示不同的NOX亚型会以亚型特异性的方式参与胰岛β细胞的功能调控、应激反应以及凋亡过程。