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Consequently, there is an urgent need to discover new pharmacological interventions and to investigate novel therapeutic targets for MASLD. Melatonin, known for its multifaceted biological functions, has shown therapeutic potential for the treatment of MASLD. However, the underlying mechanisms remain unclear, particularly since the direct targets of melatonin remain poorly understood. In our study, we found that melatonin significantly improved various indicators in a mouse MASLD model and protected palmitic acid-induced mouse hepatocytes from lipid accumulation. We successfully identified and validated the mitochondrial trifunctional enzyme \u03b1-subunit HADHA as a binding target for melatonin using the cellular thermal shift assay (CETSA). This interaction enhanced the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1\u03b1), which subsequently promotes mitochondrial biogenesis and accelerates lipid metabolism. In addition, melatonin reduced lipid accumulation and ameliorated MASLD through its regulatory effect on key proteins involved in fatty acid metabolism, including acyl coenzyme A oxidase 1 (ACOX1), cluster of differentiation 36 (CD36), and fatty acid synthase (FASN). Importantly, these beneficial effects were diminished when HADHA was knocked down. In conclusion, our study suggests that melatonin ameliorates MASLD through HADHA-mediated regulation of mitochondrial biogenesis and lipid oxidation, highlighting its potential as a promising therapeutic agent for MASLD. These results offer novel insights into the role of melatonin in the treatment of MASLD.<\/jats:p>","DOI":"10.1186\/s43556-026-00461-0","type":"journal-article","created":{"date-parts":[[2026,5,12]],"date-time":"2026-05-12T06:43:13Z","timestamp":1778568193000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Melatonin targets mitochondrial trifunctional enzyme HADHA to improve lipid metabolism in metabolic dysfunction-associated steatotic liver disease"],"prefix":"10.1186","volume":"7","author":[{"given":"Yongping","family":"Zhu","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yanqing","family":"Liu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rui","family":"Liu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Junzhe","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuqing","family":"Meng","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Li","family":"Liu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xin","family":"Liu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dandan","family":"Liu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Liwei","family":"Gu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Linying","family":"Zhong","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xianyu","family":"Xu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ying","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jinyan","family":"Xu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lingyun","family":"Dai","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shengnan","family":"Shen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0575-0105","authenticated-orcid":false,"given":"Jigang","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2026,5,12]]},"reference":[{"key":"461_CR1","doi-asserted-by":"publisher","first-page":"82","DOI":"10.1016\/j.metabol.2018.11.014","volume":"92","author":"SA Polyzos","year":"2019","unstructured":"Polyzos SA, Kountouras J, Mantzoros CS. 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