A role for lipid rafts in the protection afforded by docosahexaenoic acid against ethanol toxicity in primary rat hepatocytes

Abstract

Previously, we demonstrated that eicosapentaenoic acid enhanced ethanol-induced oxidative stress and cell death in primary rat hepatocytes via an increase in membrane fluidity and lipid raft clustering. In this context, another n-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA), was tested with a special emphasis on physical and chemical alteration of lipid rafts. Pretreatment of hepatocytes with DHA reduced significantly ethanol-induced oxidative stress and cell death. DHA protection could be related to an alteration of lipid rafts. Indeed, rafts exhibited a marked increase in membrane fluidity and packing defects leading to the exclusion of a raft protein marker, flotillin. Furthermore, DHA strongly inhibited disulfide bridge formation, even in control cells, thus suggesting a disruption of protein-protein interactions inside lipid rafts. This particular spatial organization of lipid rafts due to DHA subsequently prevented the ethanol-induced lipid raft clustering. Such a prevention was then responsible for the inhibition of phospholipase C-γ translocation into rafts, and consequently of both lysosome accumulation and elevation in cellular low-molecular-weight iron content, a prooxidant factor. In total, the present study suggests that DHA supplementation could represent a new preventive approach for patients with alcoholic liver disease based upon modulation of the membrane structures.

Keywords: 4-hydroxynonenal; DHA; Docosahexaenoic acid; EPA; EPR; Ethanol; GM1; GPI; H(2)FDA; HNE; LMW iron; Lipid peroxidation; Lipid rafts; MAL-6; O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride; O-PFB; Oxidative stress; PFBHA·HCl; PLCγ1; ROS; dihydrofluorescein diacetate; docosahexaenoic acid; eicosapentaenoic acid; electron paramagnetic resonance; glycosylphosphatidylinositol; low-molecular-weight iron; monosialotetrahexosyl ganglioside; n-3 PUFA; n-3 Polyunsaturated fatty acids; n-3 polyunsaturated fatty acid; pentafluorobenzyl-oxime; phospholipase C γ1; reactive oxygen species, GSH: reduced glutathione; tetramethyl-4-maleimidopiperidine-1-oxyl.