Hydrogen peroxide permeability of cellular membranes in insulin-producing cells

Hydrogen peroxide (H₂O₂) plays a central role in redox signalling and in oxidative stress-mediated cell death. It is generated through multiple mechanisms at various intracellular sites. Due to its chemical stability it can reach distant sites of action. However, its hydrophilicity can hamper lipid membrane passage. We therefore studied the kinetics of H₂O₂ diffusion through subcellular membranes employing the H₂O₂ biosensor HyPer in insulin-producing RINm5F cells. Plasma- and ER-membrane-bound HyPer sensors facing the cytosolic compartment reacted twice as fast to H₂O₂ compared to sensors expressed in peroxisomes and mitochondria. Overexpression of the H₂O₂-inactivating enzyme catalase in the ER-lumen and in the peroxisomes retarded the reaction time of HyPer, both localised within the peroxisomes as well as at the cytosolic surface of the ER. The unsaturated fatty acid oleic acid did not affect the reaction of the peroxisomal HyPer sensor to H₂O₂, while the saturated fatty acid palmitic acid accelerated its reaction time to H₂O₂ in this organelle. The results show that the plasma-, peroxisomal, and mitochondrial membrane of insulin-producing RINm5F cells are permeable for H₂O₂. Nonetheless, the organelle membranes retard H₂O₂ diffusion due to a barrier function of the lipid membrane, as documented by retarded reaction times of the intraorganellar sensors. Accelerated decomposition of H₂O₂ by catalase, expressed in the peroxisomes or the ER, further retarded the HyPer sensor reaction time. The results show that redox signalling and oxidative stress-mediated toxicity are crucially dependent on physicochemical membrane properties and antioxidative defence mechanisms in health and disease.