AQP8 is a crucial HO transporter in insulin-producing RINm5F cells.

Peroxiporins are distinct aquaporins (AQP) which, beside water, also facilitate the bidirectional transport of hydrogen peroxide (HO) across cellular membranes. HO serves as the major reactive oxygen species that mediates essential cell signaling events. In pancreatic β-cells, HO 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 HO 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 HO specific biosensor HyPer together with endogenous generation of HO using d-amino acid oxidase, live cell imaging revealed enhanced HO 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 HO sensor HyPer7.2 clearly unveiled AQP8 as a HO transporter in RINm5F cells. In summary, these studies establish that AQP8 is an important HO pore in insulin-producing RINm5F cells involved in the transport of HO through the mitochondria and cell membrane and may help to explain the HO transport and toxicity in pancreatic β-cells.