ENO3 regulates ferroptosis by interaction with PKM2 to promote the progression of metabolic dysfunction-associated steatotic liver disease.

Abstract

Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent metabolic disorder characterized by excessive lipid accumulation in the liver. The glycolytic enzyme enolase 3 (ENO3) is reported to be most significantly elevated in the analysis of MASLD-related sequencing results based on the GEO database. However, the specific mechanism by which ENO3 regulates MASLD is not fully understood.

Objective: To investigate the role and possible molecular mechanism of ENO3 in MASLD.

Methods: The expression of ENO3 and PKM2 in the liver tissues of control and MASLD rats was detected by immunohistochemistry and western blot. In vitro studies involved treating THLE-2 cells with free fatty acids (FFA) and Ferrostatin-1 (Fer-1), as well as manipulating ENO3 expression via small interfering RNA (siRNA) and overexpression plasmids, and manipulating PKM2 expression via siRNA. Fat accumulation was assessed using Oil Red O staining and measurements of intracellular total cholesterol (TC) and triglycerides (TG). Ferroptosis markers, including SLC7A11, GPX4, Fe²⁺, and malondialdehyde (MDA), were evaluated. Protein-protein interactions between ENO3 and PKM2 were examined using co-immunoprecipitation (Co-IP) and immunofluorescence.

Results: MASLD liver tissues exhibited significantly higher levels of ENO3 and PKM2. Silencing ENO3 in FFA-treated THLE-2 cells reduced fat accumulation, downregulated PKM2 expression, and decreased ferroptosis markers. Conversely, ENO3 overexpression promoted fat accumulation and ferroptosis, which were mitigated by Fer-1 or si-PKM2. Co-IP and immunofluorescence confirmed the physical interaction and co-localization of ENO3 and PKM2 in THLE-2 cells.

Conclusions: ENO3 interacted with PKM2 to regulate ferroptosis and further promoted the progression of MASLD.