N-acetyl-phenylalanine induces hepatic steatosis in MASLD by disrupting ER-mitochondria calcium coupling and mitochondrial lipid oxidation.

Background & Aims: The gut-liver axis and hepatic ER-mitochondria miscommunication (at contact sites called MAMs) are involved in the development of metabolic dysfunction-associated steatotic liver disease (MASLD). We investigated the role of circulating aromatic amino acids (AAA) derived from phenylalanine and tyrosine in MASLD potentially through MAM alterations. Methods: We analyzed AAA metabolomic profiles in individuals with and without MASLD and validated findings in a biopsy-proven cohort. The pro-steatogenic effect of MASLD-associated AAAs was validated in vitro using lipid labeling, MAM structural/functional assays, and palmitate-induced respiration. In vivo effects were tested in mice fed with candidate AAAs, and MAM involvement was confirmed by expressing a specific organelle linker in vitro and in vivo. Results: N-acetyl-phenylalanine (NAPA) was strongly associated with hepatic steatosis and correlated with specific gut microbes. In vitro, NAPA promoted lipid accumulation by impairing ER-mitochondria calcium exchange via a LAT1-dependent electrogenic mechanism, reducing mitochondrial lipid oxidation. Chronic NAPA administration in mice induced steatosis and MAM disruption. Notably, enhancing ER-mitochondria contacts with an organelle linker prevented NAPA-induced steatosis in vitro and in vivo. Additionally, other phenylalanine- and tyrosine-derived AAAs reproduced NAPA's effects, suggesting a class-dependent mechanism. Conclusion: NAPA emerges as a MASLD-promoting metabolite, contributing to hepatic steatosis by disrupting ER-mitochondria calcium coupling and mitochondrial lipid oxidation.