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This study aimed to investigate the changes of free fatty acids (FFA) on the expression of genes related to the AMP-activated protein kinase (AMPK) signaling pathway in the primary hepatocytes of laying hens. The primary hepatocytes of laying hens were treated with FFA (containing a 2:1 ratio of oleic and palmitic acids) for 24 h. FFA significantly increased lipid droplet accumulation, decreased glycogen synthesis, increased the levels of triglycerides (TG), total cholesterol (TC), reactive oxygen species (ROS), malondialdehyde (MDA), and glucose content in the supernatant (GLU) in the primary hepatocytes of laying hens, and decreased the levels of total antioxidant capacity (T-AOC) and superoxide dismutase (SOD), as well as mitochondrial membrane potential (MMP). The results of the PCR array combined with Western blotting experiments showed that the activity of AMPK was inhibited. Inhibition of AMPK signaling pathway decreases the expression of genes involved in fatty acid oxidation, increases the expression of genes involved in lipid synthesis, decreases the expression of genes involved in glycogen synthesis, increases the expression of genes involved in glycolysis, increases the expression of genes involved in oxidative stress, and increases the expression of genes involved in cell proliferation and apoptosis. Taken together, our results suggest that FFA can affect the homeostasis of the AMPK signaling pathway by altering energy metabolic homeostasis, inducing oxidative stress, and adjusting the onset of cell proliferation and apoptosis.<\/jats:p>","DOI":"10.3390\/metabo12090825","type":"journal-article","created":{"date-parts":[[2022,9,2]],"date-time":"2022-09-02T00:19:01Z","timestamp":1662077941000},"page":"825","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["Inhibition of Hepatic AMPK Pathway Contributes to Free Fatty Acids-Induced Fatty Liver Disease in Laying Hen"],"prefix":"10.3390","volume":"12","author":[{"given":"Cheng","family":"Huang","sequence":"first","affiliation":[{"name":"Jiangxi Provincial Key Laboratory for Animal Health, College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, 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China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guoliang","family":"Hu","sequence":"additional","affiliation":[{"name":"Jiangxi Provincial Key Laboratory for Animal Health, College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6041-8765","authenticated-orcid":false,"given":"Xiaoquan","family":"Guo","sequence":"additional","affiliation":[{"name":"Jiangxi Provincial Key Laboratory for Animal Health, College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,9,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1999","DOI":"10.1053\/j.gastro.2019.11.312","article-title":"MAFLD: A Consensus-Driven Proposed Nomenclature for Metabolic Associated Fatty Liver 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