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

Abstract

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.