Oxidative inhibition of AMPKα2 by 4-hydroxynonenal in human atherosclerotic lesions.

BACKGROUND: AMP-activated protein kinase (AMPK) is a critical regulator of endothelial function, oxidative stress, and inflammation. Lipid peroxidation products such as 4-hydroxynonenal (4-HNE) accumulate under conditions of oxidative stress and are implicated in vascular dysfunction. However, whether 4-HNE directly reacts with AMPK and alters its activity remains unclear. OBJECTIVES: To explore the effects of 4-HNE on the LKB1-AMPK axis, with a focus on AMPKα1 and AMPKα2 in different cell types (HUVECs, H9c2 cells), a cell-free system, and in vivo (Akita mice), and to understand the underlying mechanisms in human atherosclerotic plaques. METHODS: In HUVECs and H9c2 cells, cells were treated with 4-HNE, and protein levels were detected by Western blots. AMPKα2 activity was measured after enrichment via immunoprecipitation. Cell viability was also examined. In a cell-free system, recombinant AMPK was incubated with 4-HNE. In Akita mice, AMPKα2 activity was measured in aortas after tempol treatment. In human atherosclerotic plaques, immunohistochemistry, co - immunoprecipitation, and mass spectrometry were employed. RESULTS: In HUVECs, low 4-HNE concentrations (1-15 μM) didn't change LKB1 levels, but higher ones (20-25 μM) reduced them. 4-HNE had a variable impact on AMPKα1 levels, while significantly inhibiting AMPKα2 activity in a dose - and time-dependent manner. In H9c2 cells, 4-HNE (10 μM) selectively decreased AMPKα2 levels. In the cell-free system, 4-HNE dose-dependently inhibited AMPKα2 activity. In Akita mice, oxidative stress reduced aortic AMPKα2 activity, reversed by Tempol. In human plaques, 4-HNE levels correlated with plaque severity and AMPKα2 inhibition. Cys174 and Cys200 were key for 4-HNE-mediated AMPKα2 inactivation 4-HNE modified AMPKα2 at Cys174 and Cys200, and antioxidants blocked 4-HNE-induced inhibition. Finally, pretreatment with N-ethylmaleimide, a cysteine blocker, abolished the binding of 4-HNE and AMPKα2, supporting a primary role for cysteine modification in the binding of 4-HNE with AMPKα2. CONCLUSIONS: We conclude that 4-HNE inhibits AMPKα2 activity through oxidative modification of specific cysteine residues in human atherosclerosis.