Ethanol reshapes FFC-driven steatohepatitis through time-dependent fibrosis and lipid-droplet remodeling

Metabolic dysfunction and alcohol exposure drive steatotic liver disease, yet how ethanol intersects with metabolic steatohepatitis remains incompletely defined. Our iterative model examined how ethanol ingestion alters high-fat, -fructose, and -cholesterol (FFC)-driven liver injury, identifying MetALD-relevant features. A pilot study in male and female C57BL/6J mice compared chow diet (CD), FFC, CD+ethanol (E), and FFC+E on high-glucose/fructose (sugar) water (SW) at 6, 16, and 24 weeks. A redesigned study lowered glucose/fructose exposure, modified ethanol acclimation, and tested five male cohorts, CD, CD+SW, FFC+SW, CD+SW+E, FFC+SW+E, at 16 and 24 weeks. In the pilot study, FFC-dominant hepatomegaly, aminotransferase elevation, steatosis, chemokine induction, and fibrosis, without additive ethanol effects were observed. In the redesigned model, FFC drove weight gain, hepatomegaly, steatosis, aminotransferase elevation, and Ccl2/Timp1-associated fibroinflammatory remodeling. However, ethanol selectively modified collagen-positive fibrotic remodeling and lipid-droplet architecture. At 16 weeks, FFC+SW+E increased collagen area compared with FFC+SW despite lower body/liver mass and no additive aminotransferase response. BODIPY morphometry showed enlarged lipid-droplets in both FFC groups, but FFC/ethanol demonstrated reduced lipid-droplet area. In contrast, hepatic triglycerides increased with FFC but did not differ between FFC+SW and FFC+SW+E, supporting altered lipid-storage architecture rather than lipid accumulation or steatosis. Cxcl1 was induced by ethanol in CD mice but not uniformly upregulated on FFC. By 24 weeks, fibrosis, alpha smooth muscle actin staining, qPCR markers, and NanoString immune profiles converged between FFC groups. Thus, chronic ethanol in a MetALD mouse model selectively modifies collagen-positive fibrotic remodeling, lipid-storage and inflammatory features of FFC-driven liver injury.