L-threo-C6-pyridinium-ceramide Bromide, a Novel Cationic Ceramide, Induces NADPH Oxidase Activation, Mitochondrial Dysfunction and Loss in Cell Viability in INS 832/13 β-cells

Abstract

Background: Emerging evidence indicates that exposure of isolated pancreatic β-cells to elevated glucose [glucotoxicity] or saturated fatty acids such as palmitate [lipotoxicity] or both [glucolipotoxicity] results in excessive intracellular oxidative stress mediated by phagocyte-like NADPH oxidase [Nox2]. Pharmacological evidence also implicates the intracellular generation of ceramide [CER] as one of the mediators of palmitate-induced cytotoxicity of the islet β- cell. Herein, we investigated the effects of L-threo-C6-pyridinium-ceramide bromide, a novel water soluble cationic ceramide [Ws-CER], on mitochondrial function and cell viability in insulin-secreting INS 832/13 cells. Methods: Ws-CER, was synthesized as we reported earlier. Rac1 activation was quantitated by pull-down assay. Mitochondrial membrane potential was quantitated by JC-1 staining. Nox2 subunit expression and caspase-3 activity were determined by Western blotting. Results: Our findings suggested a marked increase in the Nox2 activation [i.e., ROS generation and subunit expression and activation] in cells exposed to Ws-CER. We also noticed a significant reduction in mitochondrial membrane potential, increased in caspase-3 activity and associated loss in cell viability in Ws-CER-treated cells. Conclusion: Based on these data we conclude that ceramide-induced Nox2-mediated oxidative stress couples mitochondrial dysfunction to loss in cell viability in the pancreatic β-cell.