Alcohol and Metabolic Stressors Synergistically Increase Triglyceride Content and Oxidative Stress in a MetALD Spheroid Model

Metabolic and alcohol-associated steatotic liver disease (MetALD) is more prevalent than alcohol-associated liver disease (ALD), yet differential cellular adaptations to the combination of these stressors are poorly defined. We hypothesize that ethanol and metabolic stressors, such as excess nutrients, increase lipid accumulation and oxidative stress synergistically in HepaRG spheroids.HepaRG cells that were terminally differentiated into hepatocytes were seeded at 2000 cells per well, formed spheroids over four days, and were then incubated for 10 days in the following conditions: control (C), 50mM ethanol (E10), high sugars and fats (SF10), and the combination of these factors (ESF10). Triglyceride content was determined using a luminescent assay and RT-qPCR was used to determine relative gene expression. H 2 O 2 production was determined by using an Amplex Red assay. A minimum of 3 independent experiments were completed for each group, and statistical significance was determined using a two-way ANOVA for analysis. For post-hoc tests, when n≤5, Fisher’s LSD was used, and when n>5, Tukey’s correction was used. ESF10 increased spheroid triglyceride content compared to all other groups (E10 and SF10 interaction: p<0.0001). E10 and SF10 did not increase triglyceride content compared to control. ESF10 increased H 2 O 2 production compared to C, E10, and SF10 (E10 and SF10 interaction: p<0.0001). E10 and SF10 also increased H 2 O 2 production compared to control (p=0.0317 and p=0.0087, respectively). Gene expression of both coenzyme Q10A and glutathione peroxidase 1 were decreased by SF10 (main effects of SF10: p=0.0488 and p=0.0099, respectively), and ESF10 decreased coenzyme Q10A gene expression compared to both SF10 (p=0.0384) and control (p=0.0067).Glutathione peroxidase 4 gene expression was increased by ESF10 compared to control (p=0.0242) and SF10 (p=0.0015). Glutathione reductase and glutathione synthetase gene expression was not changed in any of the groups. Our results indicate that ethanol, sugars, and fats synergistically increase lipid accumulation and oxidative stress in HepaRG spheroids. Ongoing analysis of mitochondrial functional assays and associated RNA and protein expression will reveal additional disrupted molecular pathways that correlate with these metabolic perturbations. These findings will provide evidence for pathways implicated in hepatocyte injury in MetALD and may elucidate potential therapeutic targets. (Supported by F30AA030910 and P60AA009803) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.