The Effect of Eicosapentaenoic Acid on Hepatic Metabolism and Intracellular Lipid Droplet Dynamics in An in Vitro Model of Hepatic Steatosis

Background Intra-hepatocyte triglyceride accumulation is the hallmark of non-alcoholic fatty liver disease (NAFLD). Little is known of the role of lipid droplets in the pathogenesis of this increasingly common condition. Eicosapentaenoic acid (EPA), an omega-3 fatty acid, has been suggested as a potential treatment for NAFLD. Within this study we explore different energy substrates with and without EPA and evaluate biochemical markers alongside lipid droplet characteristics. Methods EPA was added to C3A cells incubated in standard media, oleate (which mimics simple steatosis) or lactate, pyruvate, octanoate and ammonia (LPON), mimicking pathophysiology of NAFLD. At 72 hours, glucose, ketone bodies and various markers of liver function were assessed and triglyceride content was determined using confocal images of cells stained with BODIPY 493/503 and DAPI. Z stacks of confocal images were analysed using Volocity 3D image analysis and Visual Basic software to estimate the physical characteristics of lipid droplets. Liver function was assessed by measuring AST, albumin and LDH. Results Albumin levels increased in control and oleate and LDH showed EPA did not affect viability in concentrations up to 250µm. There was no effect to AST with addition of EPA. Glucose and ketone bodies were reduced in LPON treated cells in a dose dependent manner. EPA reduced triglyceride concentrations and lipid droplets per nucleus in untreated cells compared with control, however, there was no significant difference to total triglyceride concentration of cells treated with oleate + EPA or LPON + EPA. Lipid droplet volume increased in both fat loaded models with the addition of EPA, but untreated control remained the same. Surface area of lipid droplets was higher in oleate model compared to LPON. Conclusion The LPON + EPA model shows reduction of glucose and ketone bodies in a dose dependent manner indicating EPA may be useful as an adjunct to treatment of type 2 diabetes. Accumulation of lipid droplets at different rates and physical characteristics compared to oleate may reflect differences in FFA metabolism or their incorporation into the cell and lipid droplet membrane.