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

A Co-Culture-Based Multiparametric Imaging Technique to Dissect Local H2O2 Signals with Targeted HyPer7

“…Having established COMPARE IT, we next sought to visualize H 2 O 2 uptake and its diffusion in endothelial cells into cell compartments in direct comparison among neighboring cells. Former studies emphasize that the stable reactive oxygen species (ROS) H 2 O 2 can readily diffuse into cells [ 26 , 27 ] to modulate diverse redox-sensitive proteins and cell functions, thus serving as an essential signaling molecule [ 28 ]. Several studies suggest that different aquaporin isoforms facilitate H 2 O 2 diffusion across the plasma membrane and intracellular membranes of cellular organelles [ 29 , 30 , 31 ].…”

Signaling Mechanisms and Pharmacological Modulators Governing Diverse Aquaporin Functions in Human Health and Disease

“…AQP8 in the inner mitochondrial membrane was shown to confer Hg 2+ -sensitive water permeability in liver cell mitochondria [540]. In a pancreatic β-cell line, excess H 2 O 2 was linked to a failure of insulin release, potentially significant for diabetes; increased levels of AQP8 had a protective effect, proposed to result from the AQP8-mediated fluxes of H 2 O 2 across mitochondrial and plasma membranes [541]. AQP8 in the inner mitochondrial membrane of renal proximal cells increases as an adaptive response to acidosis, allowing the excretion of ammonia [542].…”

Inhibition of activin A receptor signalling attenuates age-related pathological cardiac remodelling

“…The upregulation of Srxn1 has been shown to increase the survival of cardiac progenitor cells and astrocytes by protecting them against oxidative stress ( Li et al, 2018 ; Zhou et al, 2015 ). Aqp8 expression is a marker of mitochondrial function ( Ikaga et al, 2015 ), and has been shown to be protective by transporting H 2 O 2 out of pancreatic cells in the context of diabetes mellitus ( Krüger et al, 2021 ). The downregulation of Aqp8 in Ercc1 Δ/− hearts would lead to increased oxidative stress and mitochondrial dysfunction.…”