ABSTRACT Background Hepatic fibrosis represents a critical pathological process associated with substantial morbidity and mortality. Despite significant advances in understanding fibrogenesis, the molecular mechanisms driving fibrosis progression remain incompletely defined, and effective anti‐fibrotic therapies are still limited. This study aims to investigate the role of threonine tyrosine kinase (TTK) in hepatic fibrosis and uncover the underlying mechanisms involved. Methods Murine fibrosis models, HSC‐specific TTK knockout mice, and TTK inhibitors were employed to investigate the role of TTK in hepatic fibrosis. Fibrosis was evaluated by histological and biochemical assessments. Mix1 homeobox‐like 1 (MIXL1) was screened from RNA‐seq analysis and analysed by dual‐luciferase reporter assay. Mass spectrometry and Co‐immunoprecipitation identified TTK binding proteins. Metabolomics analysis and Seahorse analyser were performed to characterize glutamate metabolism and ATP production in HSC. Results TTK expression was elevated in patients with fibrosis and in CCl 4 —and TAA—induced fibrosis models. TTK silencing attenuated liver fibrosis in vitro and in vivo. Whereas TTK overexpression markedly exacerbated TGFβ‐induced fibrotic responses. Mechanistic investigations revealed TTK modulated glutamine catabolism in HSC. TTK inhibition impaired the integration of glutamate into the tricarboxylic acid cycle, thereby reducing ATP production required for HSC activation. Specifically, TTK upregulated transcription factor MIXL1 and transcriptionally activated GLS1, which was ultimately involved in HSC glutamine catabolism. Furthermore, TTK‐phosphorylated PKM2 translocated into the nucleus and phosphorylated histone H3 at threonine 11 (H3T11), facilitating MIXL1‐mediated transcriptional activation of GLS1. Conclusions These findings revealed a novel mechanism underlying the upregulation of GLS1 mediated by transcriptional control and highlighted TTK as a critical metabolic regulator in HSC activation and hepatic fibrosis.
