Identification of 5' AMP-activated kinase as a target of reactive aldehydes during chronic ingestion of high concentrations of ethanol.
The production of reactive aldehydes including 4-hydroxy-2-nonenal (4-HNE) is a key component of the pathogenesis in a spectrum of chronic inflammatory hepatic diseases including alcoholic liver disease (ALD). One consequence of ALD is increased oxidative stress and altered β-oxidation in hepatocytes. A major regulator of β-oxidation is 5' AMP protein kinase (AMPK). In an in vitro cellular model, we identified AMPK as a direct target of 4-HNE adduction resulting in inhibition of both H2O2 and 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR)-induced downstream signaling. By employing biotin hydrazide capture, it was confirmed that 4-HNE treatment of cells resulted in carbonylation of AMPKα/β, which was not observed in untreated cells. Using a murine model of alcoholic liver disease, treatment with high concentrations of ethanol resulted in an increase in phosphorylated as well as carbonylated AMPKα. Despite increased AMPK phosphorylation, there was no significant change in phosphorylation of acetyl CoA carboxylase. Mass spectrometry identified Michael addition adducts of 4-HNE on Cys(130), Cys(174), Cys(227), and Cys(304) on recombinant AMPKα and Cys(225) on recombinant AMPKβ. Molecular modeling analysis of identified 4-HNE adducts on AMPKα suggest that inhibition of AMPK occurs by steric hindrance of the active site pocket and by inhibition of hydrogen peroxide induced oxidation. The observed inhibition of AMPK by 4-HNE provides a novel mechanism for altered β-oxidation in ALD, and these data demonstrate for the first time that AMPK is subject to regulation by reactive aldehydes in vivo.
From the Department of Pharmaceutical Sciences and.
Colin T
CT
Shearn
From the Department of Pharmaceutical Sciences and.
Donald S
DS
Backos
Department of Pathology, University of Colorado Denver-Anschutz Medical Campus, Aurora, Colorado 80045 and.
David J
DJ
Orlicky
Department of Pathobiology, Cleveland Clinic, Cleveland, Ohio 44195.
Rebecca L
RL
Smathers-McCullough
From the Department of Pharmaceutical Sciences and Dennis.Petersen@ucdenver.edu.
Dennis R
DR
Petersen
D055372:AMP-Activated Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D002492:Central Nervous System Depressants / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004195:Disease Models, Animal; D000431:Ethanol / Q000494:pharmacology*; D005234:Fatty Liver / Q000201:enzymology*; D056945:Hep G2 Cells; D006801:Humans; D015227:Lipid Peroxidation / Q000187:drug effects / Q000502:physiology; D008108:Liver Diseases, Alcoholic / Q000201:enzymology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008956:Models, Chemical; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D050050:Protein Carbonylation / Q000187:drug effects / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
D000447:Aldehydes; D002492:Central Nervous System Depressants; D015853:Cysteine Proteinase Inhibitors; D000431:Ethanol; D055372:AMP-Activated Protein Kinases; C027576:4-hydroxy-2-nonenal
AMP-activated Kinase (AMPK); Alcohol; Lipid Peroxidation; Liver; Oxidative Stress