AQP8 is a crucial H2O2 transporter in insulin-producing RINm5F cells

Abstract

Peroxiporins are distinct aquaporins (AQP) which, beside water, also facilitate the bidirectional transport of hydrogen peroxide (H₂O₂) across cellular membranes. H₂O₂ serves as the major reactive oxygen species that mediates essential cell signaling events. In pancreatic β-cells, H₂O₂ has been associated with the regulation of cell growth but in excess it leads to failure of insulin secretion, making it important for diabetes mellitus (DM) pathogenesis. In the present study, the role of aquaporin-8 (AQP8) as a peroxiporin was investigated in RINm5F cells. The role of AQP8 was studied in an insulin-producing cell model, on the basis of stable AQP8 overexpression (AQP8↑) and CRISPR/Cas9-mediated AQP8 knockdown (KD). A complete AQP8 knock-out was found to result in cell death, however we demonstrate that mild lentiviral re-expression through a Tet-On-regulated genetically modified AQP8 leads to cell survival, enabling functional characterization. Proliferation and insulin content were found to be increased in AQP8↑ cells underlining the importance of AQP8 in the regulation of H₂O₂ homeostasis in pancreatic β-cells. Colocalization analyses of V5-tagged AQP8 proteins based on confocal microscopic imaging revealed its membrane targeting to both the mitochondria and the plasma membrane, but not to the ER, the Golgi apparatus, insulin vesicles, or peroxisomes. By using the fluorescence H₂O₂ specific biosensor HyPer together with endogenous generation of H₂O₂ using d-amino acid oxidase, live cell imaging revealed enhanced H₂O₂ flux to the same subcellular regions in AQP8 overexpressing cells pointing to its importance in the development of type-1 DM. Moreover, the novel ultrasensitive H₂O₂ sensor HyPer7.2 clearly unveiled AQP8 as a H₂O₂ transporter in RINm5F cells. In summary, these studies establish that AQP8 is an important H₂O₂ pore in insulin-producing RINm5F cells involved in the transport of H₂O₂ through the mitochondria and cell membrane and may help to explain the H₂O₂ transport and toxicity in pancreatic β-cells.

Keywords: Aquaporin-8; Diabetes; HyPer sensor; Hydrogen peroxide; Peroxiporin; d-amino acid oxidase; β-cell.