Background Non-alcoholic fatty liver disease (NAFLD) is a common cause of liver disease worldwide and is a growing epidemic. A high ratio of omega-6 fatty acids to omega-3 fatty acids in the diet has been implicated in the development of NAFLD. However, the inflicted cellular pathology remains unknown. A high ratio may promote lipogenic pathways and contribute to ROS-mediated damage, perhaps leading to mitochondrial dysfunction. Therefore, these parameters were investigated to understand their contribution to NAFLD development. Aim To examine the effect of increasing ratios of omega-6:3 fatty acids on mitochondrial function and lipid metabolism mediators. Methods HepG2-derived VL-17A cells were treated with normal (1:1, 4:1) and high (15:1, 25:1) ratios of omega-6: omega-3 fatty acids (arachidonic (AA): docosahexaenoic (DHA)) at various time points. Mitochondrial activity and function was examined via MTT assay and Seahorse XF24 analyzer, respectively. Triglyceride accumulation was determined by using EnzyChrom™ and levels of ROS were measured by fluorescence intensity. Protein expression of the mediators of lipogenic, lipolytic and endocannabinoid pathways was assessed by Western blotting. Results High AA:DHA ratio decreased mitochondrial activity (p<0.01; upto 80%) and promoted intracellular triglyceride accumulation (p<0.05; 40-70%). Mechanistically, it altered the mediators of lipid metabolism; increased the expression of stearoyl-CoA desaturase (p<0.05; 22-35%), decreased the expression of peroxisome proliferator-activated receptor-alpha (p<0.05; 30-40%) and increased the expression of cannabinoid receptor 1 (p<0.05; 31%). Furthermore, the high ratio increased ROS production (p<0.01; 74-115%) and reduced mitochondrial respiratory functions such as basal and maximal respiration, ATP production, spare respiratory capacity and proton leak (p<0.01; 35-68%). Conclusion High AA:DHA ratio induced triglyceride accumulation, increased oxidative stress and disrupted mitochondrial functions. Stimulation of lipogenic and steroidal transcription factors may partly mediate these effects and contribute to NAFLD development. |