FBP1‐Mediated Compartmentalization of Glycolytic Enzyme Complexes Suppresses PKM2 Function to Ameliorate MASLD Progression

Metabolic dysfunction-associated steatotic liver disease (MASLD) is driven by aberrant hepatocyte glycolytic reprogramming. Fructose-1,6-bisphosphatase 1 (FBP1), a key gluconeogenic enzyme, has poorly defined roles in MASLD progression. Here, we show that FBP1 is consistently downregulated during MASLD progression, with a progressive decline from MASL to MASH in clinical specimens and key preclinical models. Mechanistically, FBP1 directly interacts with glycolytic enzymes Pyruvate kinase M2 (PKM2), Enolase 1 (ENO1), and Lactate Dehydrogenase A (LDHA) to form compartmentalized complexes, suppressing PKM2 activity and restricting excessive glycolysis and lipid accumulation in hepatocytes. Domain mapping defined the precise interaction interface between FBP1 189-235 aa and PKM2 390-531 aa, and disruption of this binding abrogated FBP1's glycolysis-repressive effects in hepatocytes. In vivo, only liver-specific overexpression of enzymatically inactive but binding-competent FBP1 G260R, not its interaction-deficient mutant FBP1 G260R-Δ189-235, alleviated hepatic steatosis, inflammation, and fibrosis in diet-induced MASLD mice. This study reveals a non-canonical function of FBP1 independent of its enzymatic activity in regulating glycolytic complex assembly and identifies the FBP1-PKM2 interaction as a novel candidate intervention target for MASLD.