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 metabolic dysfunction and alcohol-associated liver disease (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 wk. 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 wk. 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 chemokine (C-C motif) ligand 2 (Ccl2)/tissue inhibitor of metalloproteinase 1 (Timp1)-associated fibroinflammatory remodeling. However, ethanol selectively modified collagen-positive fibrotic remodeling and lipid-droplet architecture. At 16 wk, FFC + SW + E increased collagen area compared with FFC + SW despite lower body/liver mass and no additive aminotransferase response. Boron-dipyrromethene (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. chemokine (C-X-C motif) ligand 1 (Cxcl1) was induced by ethanol in CD mice but not uniformly upregulated on FFC. By 24 wk, fibrosis, alpha-smooth muscle actin staining, quantitative PCR (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.NEW & NOTEWORTHY This study presents a refined murine MetALD model designed to distinguish ethanol-modified features from FFC-driven metabolic steatohepatitis. Chronic ethanol did not uniformly worsen liver injury. Instead, it selectively modified disease trajectory through greater early collagen-positive fibrotic remodeling, altering lipid-droplet architecture, and context-dependent inflammatory chemokine signaling. By 24 wk, FFC and FFC/ethanol injury programs converged, supporting MetALD as a timing- and pathway-dependent modification of metabolic steatohepatitis.