{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,4,18]],"date-time":"2026-04-18T03:17:52Z","timestamp":1776482272021,"version":"3.51.2"},"posted":{"date-parts":[[2025,9,26]]},"group-title":"Pathology","reference-count":26,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,9,26]],"date-time":"2025-09-26T00:00:00Z","timestamp":1758844800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001665","name":"Agence Nationale de la Recherche","doi-asserted-by":"crossref","award":["ANR-21-CE14-0088-01"],"award-info":[{"award-number":["ANR-21-CE14-0088-01"]}],"id":[{"id":"10.13039\/501100001665","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001665","name":"Agence Nationale de la Recherche","doi-asserted-by":"crossref","award":["ANR-23-IAHU-0008"],"award-info":[{"award-number":["ANR-23-IAHU-0008"]}],"id":[{"id":"10.13039\/501100001665","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100006108","name":"National Center for Advancing Translational Sciences","doi-asserted-by":"crossref","award":["5UL1TR001442"],"award-info":[{"award-number":["5UL1TR001442"]}],"id":[{"id":"10.13039\/100006108","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000062","name":"The National Institute of Diabetes and Digestive and Kidney Diseases","doi-asserted-by":"crossref","award":["U01DK061734"],"award-info":[{"award-number":["U01DK061734"]}],"id":[{"id":"10.13039\/100000062","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000062","name":"The National Institute of Diabetes and Digestive and Kidney Diseases","doi-asserted-by":"crossref","award":["U01DK130190"],"award-info":[{"award-number":["U01DK130190"]}],"id":[{"id":"10.13039\/100000062","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000062","name":"The National Institute of Diabetes and Digestive and Kidney Diseases","doi-asserted-by":"crossref","award":["R01DK106419"],"award-info":[{"award-number":["R01DK106419"]}],"id":[{"id":"10.13039\/100000062","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000062","name":"The National Institute of Diabetes and Digestive and Kidney Diseases","doi-asserted-by":"crossref","award":["R01DK121378"],"award-info":[{"award-number":["R01DK121378"]}],"id":[{"id":"10.13039\/100000062","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000062","name":"The National Institute of Diabetes and Digestive and Kidney Diseases","doi-asserted-by":"crossref","award":["R01DK124318"],"award-info":[{"award-number":["R01DK124318"]}],"id":[{"id":"10.13039\/100000062","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000062","name":"The National Institute of Diabetes and Digestive and Kidney Diseases","doi-asserted-by":"crossref","award":["P30DK120515"],"award-info":[{"award-number":["P30DK120515"]}],"id":[{"id":"10.13039\/100000062","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000050","name":"National Heart, Lung and Blood Institute","doi-asserted-by":"crossref","award":["P01HL147835"],"award-info":[{"award-number":["P01HL147835"]}],"id":[{"id":"10.13039\/100000050","id-type":"DOI","asserted-by":"crossref"}]},{"name":"John C Martin Foundation","award":["RP124"],"award-info":[{"award-number":["RP124"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,9,26]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:sec>\n                  <jats:title>Background &amp; Aims<\/jats:title>\n                  <jats:p>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.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>\n                    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\n                    <jats:italic>in vitro<\/jats:italic>\n                    using lipid labeling, MAM structural\/functional assays, and palmitate-induced respiration.\n                    <jats:italic>In vivo<\/jats:italic>\n                    effects were tested in mice fed with candidate AAAs, and MAM involvement was confirmed by expressing a specific organelle linker\n                    <jats:italic>in vitro<\/jats:italic>\n                    and\n                    <jats:italic>in vivo<\/jats:italic>\n                    .\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>\n                    N-acetyl-phenylalanine (NAPA) was strongly associated with hepatic steatosis and correlated with specific gut microbes.\n                    <jats:italic>In vitro<\/jats:italic>\n                    , 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\n                    <jats:italic>in vitro<\/jats:italic>\n                    and\n                    <jats:italic>in vivo<\/jats:italic>\n                    . Additionally, other phenylalanine- and tyrosine-derived AAAs reproduced NAPA\u2019s effects, suggesting a class-dependent mechanism.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion<\/jats:title>\n                  <jats:p>NAPA emerges as a MASLD-promoting metabolite, contributing to hepatic steatosis by disrupting ER-mitochondria calcium coupling and mitochondrial lipid oxidation.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Lay Summary<\/jats:title>\n                  <jats:p>The gut-liver axis is a key component of the development of MASLD, and circulating gut-derived metabolites, notably AAAs derived from phenylalanine and tyrosine metabolism, have been associated with MASLD. However, the specific causal mechanisms of these AAA metabolites in MASLD development remain unexplored. Here, we identified NAPA, a gut microbiome linked metabolite elevated in MASLD patients, as a causal driver of hepatic steatosis both in vitro and in vivo. Mechanistically, NAPA alters ER-mitochondria calcium coupling leading to reduced mitochondrial lipid oxidation, highlighting a new mechanism with potential therapeutic implications.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>HIGHLIGHTS<\/jats:title>\n                  <jats:list list-type=\"simple\">\n                    <jats:list-item>\n                      <jats:p>- Circulating NAPA levels are increased in MASLD patients and correlate with hepatic steatosis.<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>- NAPA levels result from a complex host-microbiota interplay<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>- NAPA induces lipid accumulation by dampening ER-mitochondria calcium coupling and mitochondrial lipid oxidation.<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>- NAPA disrupts MAMs by a LAT1-mediated electrogenic mechanism.<\/jats:p>\n                    <\/jats:list-item>\n                    <jats:list-item>\n                      <jats:p>- Other Phe- and Tyr-mediated metabolites have the same pro-steatogenic effect than NAPA pointing to a class-dependent effect.<\/jats:p>\n                    <\/jats:list-item>\n                  <\/jats:list>\n                <\/jats:sec>","DOI":"10.1101\/2025.09.24.678188","type":"posted-content","created":{"date-parts":[[2025,9,26]],"date-time":"2025-09-26T22:50:11Z","timestamp":1758927011000},"source":"Crossref","is-referenced-by-count":2,"title":["N-acetyl-phenylalanine induces hepatic steatosis in MASLD by disrupting ER-mitochondria calcium coupling and mitochondrial lipid oxidation"],"prefix":"10.64898","author":[{"given":"R\u00e9my","family":"Lefebvre","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Th\u00e9o","family":"Rousseaux","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nadia","family":"Bendridi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"St\u00e9phanie","family":"Chanon","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Delphine","family":"Arquier","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexandre","family":"Humbert","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicolas","family":"Bertocchini","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bruno","family":"Pillot","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Emmanuelle","family":"Meugnier","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aur\u00e9lie","family":"Vieille-Marchiset","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Margaux","family":"Nawrot","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Claudie","family":"Pinteur","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sophie","family":"Ayciriex","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rohit","family":"Loomba","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jennifer","family":"Rieusset","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8089-2907","authenticated-orcid":false,"given":"Cyrielle","family":"Caussy","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2025092918551366000_2025.09.24.678188v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-019-09455-9"},{"key":"2025092918551366000_2025.09.24.678188v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-018-0061-3"},{"key":"2025092918551366000_2025.09.24.678188v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.cmet.2017.04.001"},{"key":"2025092918551366000_2025.09.24.678188v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.cmet.2020.06.005"},{"key":"2025092918551366000_2025.09.24.678188v1.5","doi-asserted-by":"crossref","first-page":"296","DOI":"10.1016\/j.cgh.2018.08.065","article-title":"Emerging Role of the Gut Microbiome in Nonalcoholic Fatty Liver Disease: From Composition to Function","volume":"17","year":"2019","journal-title":"Clin Gastroenterol Hepatol"},{"key":"2025092918551366000_2025.09.24.678188v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/s41575-018-0031-8"},{"key":"2025092918551366000_2025.09.24.678188v1.7","doi-asserted-by":"publisher","DOI":"10.1016\/j.jhep.2019.10.003"},{"key":"2025092918551366000_2025.09.24.678188v1.8","doi-asserted-by":"crossref","first-page":"918","DOI":"10.1002\/hep.29892","article-title":"Link between gut-microbiome derived metabolite and shared gene-effects with hepatic steatosis and fibrosis in NAFLD","volume":"68","year":"2018","journal-title":"Hepatology"},{"key":"2025092918551366000_2025.09.24.678188v1.9","doi-asserted-by":"crossref","unstructured":"McGlinchey AJ , Govaere O , Geng D , Ratziu V , Allison M , Bousier J , et al. Metabolic signatures across the full spectrum of non-alcoholic fatty liver disease. JHEP Rep 2022;4:100477.","DOI":"10.1016\/j.jhepr.2022.100477"},{"key":"2025092918551366000_2025.09.24.678188v1.10","doi-asserted-by":"publisher","DOI":"10.1016\/j.biocel.2017.12.019"},{"key":"2025092918551366000_2025.09.24.678188v1.11","doi-asserted-by":"crossref","unstructured":"Humbert A , Lefebvre R , Nawrot M , Caussy C , Rieusset J . Calcium signalling in hepatic metabolism: Health and diseases. Cell Calcium 2023;114:102780.","DOI":"10.1016\/j.ceca.2023.102780"},{"key":"2025092918551366000_2025.09.24.678188v1.12","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1016\/j.tem.2016.09.002","article-title":"Mitochondria-Associated Membranes Response to Nutrient Availability and Role in Metabolic Diseases","volume":"28","year":"2017","journal-title":"Trends Endocrinol Metab"},{"key":"2025092918551366000_2025.09.24.678188v1.13","doi-asserted-by":"crossref","first-page":"614","DOI":"10.1007\/s00125-015-3829-8","article-title":"Disruption of calcium transfer from ER to mitochondria links alterations of mitochondria-associated ER membrane integrity to hepatic insulin resistance","volume":"59","year":"2016","journal-title":"Diabetologia"},{"key":"2025092918551366000_2025.09.24.678188v1.14","doi-asserted-by":"publisher","DOI":"10.1016\/j.jhep.2022.03.017"},{"key":"2025092918551366000_2025.09.24.678188v1.15","doi-asserted-by":"publisher","DOI":"10.1093\/jmcb\/mjw004"},{"key":"2025092918551366000_2025.09.24.678188v1.16","doi-asserted-by":"publisher","DOI":"10.2337\/db17-0316"},{"key":"2025092918551366000_2025.09.24.678188v1.17","doi-asserted-by":"publisher","DOI":"10.2337\/db13-1751"},{"key":"2025092918551366000_2025.09.24.678188v1.18","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-021-03707-9"},{"key":"2025092918551366000_2025.09.24.678188v1.19","doi-asserted-by":"crossref","unstructured":"Tubbs E , Rieusset J . Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells. J Vis Exp 2016.","DOI":"10.3791\/54899-v"},{"key":"2025092918551366000_2025.09.24.678188v1.20","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2010.06.007"},{"key":"2025092918551366000_2025.09.24.678188v1.21","doi-asserted-by":"publisher","DOI":"10.1152\/ajpendo.00026.2021"},{"key":"2025092918551366000_2025.09.24.678188v1.22","doi-asserted-by":"publisher","DOI":"10.1152\/ajpgi.00387.2010"},{"key":"2025092918551366000_2025.09.24.678188v1.23","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1016\/j.jphs.2020.06.006","article-title":"JPH203, a newly developed anti-cancer drug, shows a preincubation inhibitory effect on L-type amino acid transporter 1 function","volume":"144","year":"2020","journal-title":"J Pharmacol Sci"},{"key":"2025092918551366000_2025.09.24.678188v1.24","doi-asserted-by":"crossref","unstructured":"Zarei I , Koistinen VM , Kokla M , Klavus A , Babu AF , Lehtonen M , et al. Tissue-wide metabolomics reveals wide impact of gut microbiota on mice metabolite composition. Sci Rep 2022;12:15018.","DOI":"10.1038\/s41598-022-19327-w"},{"key":"2025092918551366000_2025.09.24.678188v1.25","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-022-33050-0"},{"key":"2025092918551366000_2025.09.24.678188v1.26","doi-asserted-by":"publisher","DOI":"10.1042\/bj20030405"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.09.24.678188","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T09:40:28Z","timestamp":1768470028000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.09.24.678188"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,9,26]]},"references-count":26,"URL":"https:\/\/doi.org\/10.1101\/2025.09.24.678188","relation":{},"subject":[],"published":{"date-parts":[[2025,9,26]]},"subtype":"preprint"}}