Introduction and Objective: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a major global health challenge with limited effective pharmacological therapies. Although lysine lactylation has emerged as an important regulator of metabolic homeostasis, whether lactylation of the key glycolytic enzyme phosphofructokinase liver type (PFKL) drives MASLD progression and the underlying mechanisms remain obscure.Methods: Quantitative lactylome and proteomic analyses were performed in HFD-fed mice. Hepatic lactate levels and global Kla were assessed in human and mouse MASLD samples. PFKL lactylation sites and enzymatic activity were analyzed in vitro assays. C57BL/6J mice were injected with AAV9-TBG-PFKL or AAV9-TBG-PFKL-K677R, followed by metabolic phenotyping. Mass spectrometry, co-IP, and ubiquitination assays were conducted to investigate the mechanisms of TRIM25-mediated PFKL degradation.Results: Hepatic lactate levels and global Kla were markedly elevated in patients with MASLD and in HFD- or MCD-fed mice, correlating with disease progression. Integrated lactylome and proteomic analyses identified PFKL as a key glycolytic regulator with increased expression and lactylation at lysine 677, dynamically regulated by PCAF. K677 lactylation enhanced PFKL enzymatic activity and protein stability by attenuating its interaction with the E3 ligase TRIM25, thereby preventing proteasomal degradation. Liver-specific expression of delactylation-mimic PFKL mutants conferred resistance to HFD-induced steatosis, insulin resistance, inflammation, and apoptosis. Elevated PFKL abundance promoted glycolytic flux and lactate accumulation, forming a positive feedback loop that sustained metabolic reprogramming and hepatic dysfunction. Disruption of this loop by LDHA knockdown or pharmacological inhibition effectively ameliorated MASLD progression.Conclusion: These findings reveal a lactylation-dependent mechanism driving MASLD progression and demonstrate that targeting PFKL lactylation may have therapeutic potential.
Y. Guo: None. H. Ren: None. G. Yuan: None.
National Natural Science Foundation of China (No. 82270855, No. 81974121, No. 82470867, No. 82470908 and No. 82100922)
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