title: Modification of Akt2 by 4-hydroxynonenal inhibits insulin-dependent Akt signaling in HepG2 cells.
abstract: The production of reactive aldehydes such as 4-hydroxy-2-nonenal (4-HNE) is a key component of the pathogenesis in a spectrum of hepatic diseases involving oxidative stress such as alcoholic liver disease (ALD). One consequence of ALD is increased insulin resistance in hepatocytes. To understand the effects of 4-HNE on insulin signaling in liver cells, we employed a model using hepatocellular carcinoma cell line HepG2. Previously, we have demonstrated an increase in the level of Akt phosphorylation is mediated by 4-HNE inhibition of PTEN, a direct regulator of Akt. In this work, we evaluated the effects of 4-HNE on insulin-dependent stimulation of the Akt2 pathway. We demonstrate that 4-HNE selectively leads to phosphorylation of Akt2. Although Akt2 is phosphorylated following 4-HNE treatment, the level of downstream phosphorylation of Akt substrates such as GSK3β and MDM2 is significantly decreased. Pretreatment with 4-HNE prevented insulin-dependent Akt signaling and decreased intracellular Akt activity by 87%. Using biotin hydrazide capture, it was confirmed that 4-HNE treatment of cells resulted in carbonylation of Akt2, which was not observed in untreated control cells. Using a synthetic GSK3α/β peptide as a substrate, treatment of recombinant human myristoylated Akt2 (rAkt2) with 20 or 40 μM 4-HNE inhibited rAkt2 activity by 30 or 85%, respectively. Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF) identified Michael addition adducts of 4-HNE with His196, His267, and Cys311 of rAkt2. Computation-based molecular modeling analysis of 4-HNE adducted to His196 and Cys311 of Akt2 suggests inhibition of GSK3β peptide binding by 4-HNE in the Akt2 substrate binding pocket. The inhibition of Akt by 4-HNE provides a novel mechanism for increased insulin resistance in ALD. These data provide a potential mechanism of dysregulation of Akt2 during events associated with sustained hepatocellular oxidative stress.
authors: Department of Pharmaceutical Sciences, University of Colorado-Denver, Aurora, Colorado 80045, United States.
authors: C T
authors: CT
authors: Shearn
authors: K S
authors: KS
authors: Fritz
authors: P
authors: P
authors: Reigan
authors: Dennis R
authors: DR
authors: Petersen
mesh_terms: D000447:Aldehydes / Q000494:pharmacology*; D000595:Amino Acid Sequence; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology*; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D056945:Hep G2 Cells; D006801:Humans; D007333:Insulin Resistance / Q000502:physiology*; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
chemical_list: D000447:Aldehydes; D010455:Peptides; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; C494921:AKT2 protein, human; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3; C027576:4-hydroxy-2-nonenal