PMID- 33892285 OWN - NLM STAT- MEDLINE DCOM- 20210705 LR - 20220531 IS - 2213-2317 (Electronic) IS - 2213-2317 (Linking) VI - 43 DP - 2021 Jul TI - AQP8 is a crucial H(2)O(2) transporter in insulin-producing RINm5F cells. PG - 101962 LID - S2213-2317(21)00110-5 [pii] LID - 10.1016/j.redox.2021.101962 [doi] LID - 101962 AB - 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 beta-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 upward arrow) 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 upward arrow cells underlining the importance of AQP8 in the regulation of H(2)O(2) homeostasis in pancreatic beta-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 beta-cells. CI - Copyright (c) 2021 The Authors. Published by Elsevier B.V. All rights reserved. FAU - Kruger, Christina AU - Kruger C AD - Institute of Clinical Biochemistry, Hannover Medical School, 30623, Hannover, Germany. FAU - Waldeck-Weiermair, Markus AU - Waldeck-Weiermair M AD - Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 02115, USA. FAU - Kaynert, Jonas AU - Kaynert J AD - Institute of Clinical Biochemistry, Hannover Medical School, 30623, Hannover, Germany. FAU - Pokrant, Thomas AU - Pokrant T AD - Institute of Clinical Biochemistry, Hannover Medical School, 30623, Hannover, Germany. FAU - Komaragiri, Yesaswini AU - Komaragiri Y AD - Zentrum fur Innovationskompetenz: Humorale Immunreaktion bei Kardiovaskularen Erkrankungen, Universitat Greifswald, 17489, Greifswald, Germany; Deutsches Zentrum fur Herz-Kreislauf-Forschung e. V., Standort Greifswald, Universitatsmedizin Greifswald, Fleischmannstr. 42, 17489, Greifswald, Germany. FAU - Otto, Oliver AU - Otto O AD - Zentrum fur Innovationskompetenz: Humorale Immunreaktion bei Kardiovaskularen Erkrankungen, Universitat Greifswald, 17489, Greifswald, Germany; Deutsches Zentrum fur Herz-Kreislauf-Forschung e. V., Standort Greifswald, Universitatsmedizin Greifswald, Fleischmannstr. 42, 17489, Greifswald, Germany. FAU - Michel, Thomas AU - Michel T AD - Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 02115, USA. FAU - Elsner, Matthias AU - Elsner M AD - Institute of Clinical Biochemistry, Hannover Medical School, 30623, Hannover, Germany. Electronic address: elsner.matthias@mh-hannover.de. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20210401 PL - Netherlands TA - Redox Biol JT - Redox biology JID - 101605639 RN - 0 (Aquaporins) RN - 0 (Insulins) RN - 0 (Reactive Oxygen Species) RN - 0 (aquaporin 8) RN - BBX060AN9V (Hydrogen Peroxide) SB - IM MH - Animals MH - *Aquaporins MH - Cell Membrane/metabolism MH - Hydrogen Peroxide/metabolism MH - *Insulins MH - Rats MH - Reactive Oxygen Species/metabolism PMC - PMC8082690 OTO - NOTNLM OT - Aquaporin-8 OT - Diabetes OT - HyPer sensor OT - Hydrogen peroxide OT - Peroxiporin OT - d-amino acid oxidase OT - beta-cell COIS- The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Oliver Otto is co-founder of Zellmechanik Dresden GmbH distributing Real-Time Deformability Cytometry. The other authors declare no conflict of interest. EDAT- 2021/04/24 06:00 MHDA- 2021/07/06 06:00 PMCR- 2021/04/01 CRDT- 2021/04/23 20:20 PHST- 2020/12/06 00:00 [received] PHST- 2021/03/28 00:00 [revised] PHST- 2021/03/28 00:00 [accepted] PHST- 2021/04/24 06:00 [pubmed] PHST- 2021/07/06 06:00 [medline] PHST- 2021/04/23 20:20 [entrez] PHST- 2021/04/01 00:00 [pmc-release] AID - S2213-2317(21)00110-5 [pii] AID - 101962 [pii] AID - 10.1016/j.redox.2021.101962 [doi] PST - ppublish SO - Redox Biol. 2021 Jul;43:101962. doi: 10.1016/j.redox.2021.101962. Epub 2021 Apr 1.