ObjectiveWhether and how the PI3K-AKT pathway, a central node of metabolic homeostasis, is responsible for high-fat-induced non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) remain a mystery. Characterisation of AKT regulation in this setting will provide new strategies to combat HCC.DesignMetabolite library screening disclosed that palmitic acid (PA) could activate AKT. In vivo and in vitro palmitoylation assay were employed to detect AKT palmitoylation. Diverse cell and mouse models, including generation ofAKT1^C77SandAKT1^C224Sknock-in cells,Zdhhc17andZdhhc24knockout mice andAkt1^C224Sknock-in mice were employed. Human liver tissues from patients with NASH and HCC, hydrodynamic transfection mouse model, high-fat/high-cholesterol diet (HFHCD)-induced NASH/HCC mouse model and high-fat and methionine/choline-deficient diet (HFMCD)-induced NASH mouse model were also further explored for our mechanism studies.ResultsBy screening a metabolite library, PA has been defined to activate AKT by promoting its palmitoyl modification, an essential step for growth factor-induced AKT activation. Biologically, a high-fat diet could promote AKT kinase activity, thereby promoting NASH and liver cancer. Mechanistically, palmitoyl binding anchors AKT to the cell membrane in a PIP3-independent manner, in part by preventing AKT from assembling into an inactive polymer. The palmitoyltransferases ZDHHC17/24 were characterised to palmitoylate AKT to exert oncogenic effects. Interestingly, the anti-obesity drug orlistat or specific penetrating peptides can effectively attenuate AKT palmitoylation and activation by restricting PA synthesis or repressing AKT modification, respectively, thereby antagonising liver tumorigenesis.ConclusionsOur findings elucidate a novel fine-tuned regulation of AKT by PA-ZDHHC17/24-mediated palmitoylation, and highlight tumour therapeutic strategies by taking PA-restricted diets, limiting PA synthesis, or directly targeting AKT palmitoylation.