2506-P: NAT10-ac4C-PPAR Axis Reprograms Hepatic Macrophage Metabolism and Polarization in Metabolic Fatty Liver Disease

Introduction and Objective: N4-acetylcytidine (ac4C) is an emerging epitranscriptomic RNA modification, but its role in hepatic macrophages in metabolic dysfunction-associated fatty liver disease (MAFLD) is unknown. NAT10 is the sole known ac4C writer. We examined whether NAT10-mediated ac4C modification of peroxisome proliferator-activated receptor (PPAR) family mRNAs regulates hepatic macrophage polarization, lipid metabolism and inflammation in MAFLD. Methods: In high-fat diet (HFD)-fed mice, ac4C abundance and NAT10 expression were assessed in Kupffer cells. A macrophage-targeted AAV-NAT10 approach was used to evaluate the effects of macrophage-specific NAT10 overexpression on hepatic steatosis and inflammation. In vitro, primary macrophages were subjected to NAT10 overexpression or knockdown, fatty acid plus IL-4 stimulation, followed by assessment of macrophage phenotype and mitochondrial function. NAT10-dependent ac4C-modified transcripts were identified by integrated acRIP-seq and RNA-seq, and ac4C modification and stability of PPAR mRNAs were validated. Results: HFD feeding markedly reduced ac4C levels and NAT10 expression in Kupffer cells. Macrophage-specific NAT10 overexpression attenuated hepatic lipid accumulation and improved metabolic parameters in vivo. In fatty acid-treated macrophages, NAT10 enhanced fatty acid oxidation and oxidative phosphorylation, increased M2 markers, and suppressed pro-inflammatory M1 cytokines. PPAR family transcripts were major NAT10-dependent ac4C targets; NAT10 increased ac4C modification, mRNA stability, and protein expression of PPARs, whereas NAT10 knockdown had opposite effects. Pharmacologic PPAR inhibition partially reversed NAT10-driven metabolic reprogramming and M2 polarization. Conclusion: NAT10-ac4C signaling is disrupted in Kupffer cells during MAFLD. By stabilizing PPAR mRNAs, NAT10 promotes a shift toward an FAO/OXPHOS-dependent M2 reparative macrophage phenotype, reducing hepatic steatosis and inflammation. Disclosure F. Shen: None. G. Wei: None. W. Cao: None. F. Ma: None. Y. Wang: None. J. Yang: None.