Identification of cAMP kinase as a direct target of reactive aldehydes in a murine model of alcoholic liver disease
Colin T. Shearn, David J. Orlicky, Dennis R. Petersen;
Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO
Background: The production of reactive aldehydes such as 4-hydroxy-2-nonenal (4-HNE) is a key component of the patho-genesis in alcoholic liver disease (ALD). One consequence of ALD is altered cAMP kinase (AMPK) signaling resulting in dys-regulation of β-oxidation. Recently, we determined that in conjunction with a high fat diet, chronic ethanol consumption increased phosphorylation of AMPK but did not result in into significant changes in AMPK-dependent phosphorylation of Acetyl CoA Carboxylase (ACC) (Shearn et. alJour. Nutr. Bioch. 2013). The aim of the present study was to understand the effects of increased oxidative stress on AMPK signaling and activity in vitro as well as in a murine model of chronic ethanol consumption. Methods: Using recombinant protein, an in vitro human hepatocyte HepaRG cell culture system or a murine model of ALD, the direct effects of lipid peroxidation were examined with respect to AMPK phosphorylation, carbonyla-tion, enzymatic activity and phosphorylation of ACC. Results: In HepaRG cells, incubation with increasing concentrations of 4-HNE resulted in decreased phosphorylation of AMPK and ACC. Pretreatment of 4-HNE inhibited both hydrogen peroxide and adiponectin induced phosphorylation of AMPK and subsequent phosphorylation of ACC. Using biotin hydrazide capture, it was confirmed that exposure of HepaRG cells to 4-HNE resulted in carbonylation of AMPKα/β which was not observed in untreated control cells. Based on this data, mass spectral analysis of 4-HNE treated recombinant AMPKα identified Michael addition adducts of 4-HNE on Cys130, Cys174, Cys227 and Cys309. Global computational based molecular modeling analysis of AMPK following 4-HNE modification revealed no significant changes in secondary or tertiary structure. Molecular modeling analysis of 4-HNE adducted to Cys174 AMPKα suggest inhibition of AMPK activity by steric hindrance of the active site pocket. Using a murine model of alcoholic liver disease, chronic ethanol consumption resulted in an increase in carbonylated AMPKα despite increased phosphorylation of Thr172AMPK. There was no significant change in phosphorylation of ACC. Conclusions: Combined these data demonstrate for the first time that AMPK is a direct target of reactive aldehyde during conditions of increased oxidative stress in the liver. The inhibition of AMPK by reactive aldehydes provides a novel mechanism for defective AMPK signaling and β-oxidation in ALD. This work was funded by NIH 5F32 AA018613-03 (C.T.S.) and 5R37 AA009300-16 (D.R.P.).
The following people have nothing to disclose: Colin T. Shearn, David J. Orlicky, Dennis R. Petersen