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

Abstract

Objectives: 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.

Methods: Isoforms were studied via RT-qPCR, immunoblotting and immunofluorescence in rat islets and beta cell lines.

Results: 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.

Conclusion: 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.