Hydrogen peroxide permeability of cellular membranes in insulin-producing cells.

Hydrogen peroxide (HO) 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 HO diffusion through subcellular membranes employing the HO biosensor HyPer in insulin-producing RINm5F cells. Plasma- and ER-membrane-bound HyPer sensors facing the cytosolic compartment reacted twice as fast to HO compared to sensors expressed in peroxisomes and mitochondria. Overexpression of the HO-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 HO, while the saturated fatty acid palmitic acid accelerated its reaction time to HO in this organelle. The results show that the plasma-, peroxisomal, and mitochondrial membrane of insulin-producing RINm5F cells are permeable for HO. Nonetheless, the organelle membranes retard HO diffusion due to a barrier function of the lipid membrane, as documented by retarded reaction times of the intraorganellar sensors. Accelerated decomposition of HO 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.