AF6 promotes lipid droplet fusion and aggravates MASH via C/EBPß–CIDEA axis

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease characterized by steatosis, inflammation, and fibrosis. However, the mechanisms linking lipid droplet (LD) metabolism to MASH progression remain poorly understood. AF6 is a polarity protein known to regulate metabolic homeostasis, but its role in the development of MASH remains largely undefined. Here, we identify AF6 as a critical regulator of hepatic lipid accumulation and inflammation in MASH. Hepatic AF6 expression is elevated in MASH patients and diet-induced mouse models. Liver-specific deletion of AF6 attenuated steatosis, inflammation, and fibrosis in both high-fat, high-cholesterol (HFHC) and choline-deficient, amino acid-defined high-fat diet (CDA-HFD)-induced MASH models. Mechanistically, lipid overload promotes nuclear translocation of AF6, which interacts with the transcription factor C/EBPß and enhances its nuclear accumulation. This interaction increases transcription of the lipid droplet fusion protein CIDEA, promoting LD growth and lipid deposition. AF6 also exacerbates MASH by enhancing C/EBPß-mediated proinflammatory signaling. Restoration of CIDEA expression in AF6-deficient mice reverses the protective effects. In human liver biopsies and iPSC-derived liver organoids, the AF6–C/EBPß–CIDEA axis is upregulated and correlates with disease severity. Importantly, a CRISPR-Cas9–based lipid nanoparticle (LNP) therapy targeting AF6 effectively reduced lipid accumulation, inflammation, and fibrosis in MASH mice and organoids. AF6 promotes MASH progression by facilitating C/EBPß nuclear localization and transcriptional activation of CIDEA. Targeting the AF6–C/EBPß–CIDEA axis represents a promising therapeutic strategy for treating MASH. Overall design: Liver-specific AF6 knockout mice (AF6fl/fl; AlbCre) and control littermates (AF6fl/fl) were used for in vivo studies. At 8 weeks of age, male mice were randomly assigned to receive HFHC diet containing 14% protein, 42% fat, 44% carbohydrates, and 0.2% cholesterol for 16 weeks to induce MASH. After the treatment, liver tissue samples from the mice were collected for RNA-seq sequencing analysis.