Alcohol and Metabolic Stress Synergize to Dysregulate Mitochondrial Health and Lipid Metabolism; Evidence from a Hepatocyte Spheroid Model

Abstract Metabolic dysfunction-associated steatotic liver disease and alcohol-associated liver disease frequently co-occur, manifesting as MetALD. Understanding the hepatocyte-specific effects of alcohol and metabolic stressors is critical to uncovering mechanisms of synergistic injury. This study evaluated the individual and combined effects of ethanol, sugars, and saturated/monounsaturated fats on hepatocyte lipid metabolism, oxidative stress, and mitochondrial function using a 3D human HepaRG spheroid model. HepaRG spheroids were treated with ethanol (50 mM), sugar (glucose and fructose), and fatty acids alone or in combination for 10 d. The combination of ethanol (E) and metabolic (sugar and fat, SF) stressors (ESF) synergistically increased triglyceride content and lipid droplet accumulation. ESF increased gene expression of lipid handling targets including perilipins 1 and 2, fatty acid binding protein 1, and hepatic lipase compared to controls. ESF also induced the highest rate of ROS production compared to E and SF and dysregulated antioxidant gene expression. E and SF additively impaired ATP content and ATP production linked mitochondrial respiration. Ethanol and metabolic stressors synergize to dysregulate hepatocyte lipid homeostasis and oxidative stress while additively impairing mitochondrial bioenergetics. Gene expression results suggest that lipid accumulation may be driven by altered expression of triglyceride storage and lipid handling markers rather than de novo lipogenesis. These findings highlight the importance of metabolic contributions in alcohol-induced hepatocellular dysfunction and establish HepaRG spheroids as a robust model to elucidate hepatocyte-specific responses in MetALD.