The production of reactive aldehydes such as 4-hydroxynonenal (4-HNE) is a key event in the pathogenesis of alcoholic liver disease which ranges from simple steatosis to fibrosis. The lipid phosphatase PTEN plays a central role in the regulation of lipid metabolism in the liver. In the present study, the effects of chronic ethanol feeding and carbonylation on the PTEN signaling pathway were examined in a 9-week mouse feeding model for ALD. Chronic ethanol consumption resulted in altered REDOX homeostasis as evidenced by decreased GSH, decreased Trx1 and increased GST activity. Both PTEN expression and phosphorylation was significantly increased in the livers of ethanol-fed mice. Carbonylation of PTEN increased significantly in the ethanol-fed mice compared to pair-fed control animals corresponding to decreased PTEN 3-phosphatase activity. Concomitantly, increased expression of Akt2 along with increased Akt phosphorylation at residues Thr308, Thr450 and Ser473 was observed resulting in increased Akt2 activity in the ethanol-fed animals. Akt2 activation corresponded to a decrease in cytosolic SREBP and ChREBP. Subsequent LC-MS/MS analysis of 4-HNE modified recombinant human PTEN identified Michael addition adducts of 4-HNE on Cys71, Cys136, Lys147, Lys223, Cys250, Lys254, Lys313, Lys327 and Lys344. Computational based molecular modeling analysis of 4-HNE adducted to Cys71 near the active site and Lys327 in the C2 domain of PTEN suggest inhibition of enzyme catalysis via either stearic hindrance of the active site pocket or by prevention of C2 domain-dependent PTEN function. We hypothesize that 4-HNE-mediated PTEN inhibition contributes to the observed activation of Akt2 suggesting a possible novel mechanism of lipid accumulation in response to increased reactive aldehyde production during chronic ethanol administration in mice.

4-Hydroxynonenal (4-HNE) is an endogenous product of lipid peroxidation known to play a role in cellular signaling through protein modification and is a major precursor for protein carbonyl adducts found in alcoholic liver disease (ALD). In the present study, a greater than 2-fold increase in protein carbonylation of sirtuin 3 (SIRT3), a mitochondrial class III histone deacetylase, is reported in liver mitochondrial extracts of ethanol consuming mice. The consequence of this in vivo carbonylation on SIRT3 deacetylase activity is unknown. Interestingly, mitochondrial protein hyperacetylation was observed in a time-dependent increase in a model of chronic ethanol consumption; however, the underlying mechanisms for this remain unknown. Tandem mass spectrometry was used to identify and characterize the in vitro covalent modification of rSIRT3 by 4-HNE at Cys280, a critical zinc-binding residue, and the resulting inhibition of rSIRT3 activity via pathophysiologically relevant concentrations of 4-HNE. Computational-based molecular modeling simulations indicate that 4-HNE modification alters the conformation of the zinc-binding domain inducing minor changes within the active site, resulting in the allosteric inhibition of SIRT3 activity. These conformational data are supported by the calculated binding energies derived from molecular docking studies suggesting the substrate peptide of acetyl-CoA synthetase 2 (AceCS2-Kac), displays a greater affinity for native SIRT3 compared with the 4-HNE adducted protein. The results of this study characterize altered mitochondrial protein acetylation in a mouse model of chronic ethanol ingestion and thiol-specific allosteric inhibition of rSIRT3 resulting from 4-HNE adduction.

The production of reactive aldehydes such as 4-hydroxynonenal (4-HNE) is proposed to be an important factor in the etiology of alcoholic liver disease. To understand the effects of 4-HNE on homeostatic signaling pathways in hepatocytes, cellular models consisting of the human hepatocellular carcinoma cell line (HepG2) and primary rat hepatocytes were evaluated. Treatment of both HepG2 cells and primary hepatocytes with subcytotoxic concentrations of 4-HNE resulted in the activation of Akt within 30 min as demonstrated by increased phosphorylation of residues Ser473 and Thr308. Quantification and subsequent immunocytochemistry of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P 3 [rsqb ] resulted in a 6-fold increase in total PtdIns(3,4,5)P 3 and increased immunostaining at the plasma membrane after 4-HNE treatment. Cotreatment of HepG2 cells with 4-HNE and the phosphatidylinositol 3-kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (Ly294002) or the protein phosphatase 2A (PP2A) inhibitor okadaic acid revealed that the mechanism of activation of Akt is PI3K-dependent and PP2A-independent. Using biotin hydrazide detection, it was established that the incubation of HepG2 cells with 4-HNE resulted in increased carbonylation of the lipid phosphatase known as "phosphatase and tensin homolog deleted on chromosome 10" (PTEN), a key regulator of Akt activation. Activity assays both in HepG2 cells and recombinant PTEN revealed a decrease in PTEN lipid phosphatase activity after 4-HNE application. Mass spectral analysis of 4-HNE-treated recombinant PTEN detected a single 4-HNE adduct. Subsequent analysis of Akt dependent physiological consequences of 4-HNE in HepG2 cells revealed significant increases in the accumulation of neutral lipids. These results provide a potential mechanism of Akt activation and cellular consequences of 4-HNE in hepatocytes. IntroductionOxidative modification of proteins by reactive aldehydes has been implicated in an increasing number of hepatic disease states, including hepatitis C, nonalcoholic steatohepatitis, and chronic alcoholic liver disease (Paradis et al., 1997;Seki et al., 2002;Roede et al., 2008). A documented marker for increased oxidative stress in cells is the presence of elevated levels of 4-hydroxynonenal (4-HNE). Reactive aldehydes such as 4-HNE originate from peroxidation of membrane lipids, including linoleic acid (Poli et al., 2008). 4-HNE is a potent electrophile that will react with nucleophilic functional groups in DNA as well as Cys, Lys, and His residues within proteins. Proteins documented to be targets for modification by 4-HNE include protein disulfide isomerase, actin, Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.110.069534.□ S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material.ABBREVIATIONS: 4-HNE, 4-hydroxynonenal; PTEN, phosphatase and tensin homolog deleted on chromosome 10; PH, Pleckstr...

Alcoholic liver disease (ALD) is a primary cause of morbidity and mortality in the United States and constitutes a significant socioeconomic burden. Previous work has implicated oxidative stress and endoplasmic reticulum (ER) stress in the etiology of ALD; however, the complex and interrelated nature of these cellular responses presently confounds our understanding of ethanol-induced hepatopathy. In this paper, we assessed the pathological contribution of oxidative stress and ER stress in a time-course mouse model of early-stage ALD. Ethanol-treated mice exhibited significant hepatic panlobular steatosis and elevated plasma ALT values compared to isocaloric controls. Oxidative stress was observed in the ethanol-treated animals through a significant increase in hepatic TBARS and immunohistochemical staining of 4-HNE-modified proteins. Hepatic glutathione (GSH) levels were significantly decreased as a consequence of decreased CBS activity, increased GSH utilization, and increased protein glutathionylation. At the same time, immunoblot analysis of the PERK, IRE1α, ATF6, and SREBP pathways reveals no significant role for these UPR pathways in the etiology of hepatic steatosis associated with early-stage ALD. Collectively, our results indicate a primary pathogenic role for oxidative stress in the early initiating stages of ALD that precedes the involvement of the ER stress response.

SUMMARY Energetic nutrients are oxidized to sustain high intra-cellular NADPH/NADP+ ratios. NADPH-dependent reduction of thioredoxin-1 (Trx1) disulfide and glutathione disulfide by thioredoxin reductase-1 (TrxR1) and glutathione reductase (Gsr), respectively, fuels antioxidant systems and deoxyribonucleotide synthesis. Mouse livers lacking both TrxR1 and Gsr sustain these essential activities using an NADPH-independent methionine-consuming pathway; however, it remains unclear how this reducing power is distributed. Here, we show that liver-specific co-disruption of the genes encoding Trx1, TrxR1, and Gsr (triple-null) causes dramatic hepatocyte hyperproliferation. Thus, even in the absence of Trx1, methionine-fueled glutathione production supports hepatocyte S phase deoxyribonucleotide production. Also, Trx1 in the absence of TrxR1 provides a survival advantage to cells under hyperglycemic stress, suggesting that glutathione, likely via glutaredoxins, can reduce Trx1 disulfide in vivo. In triple-null livers like in many cancers, deoxyribonucleotide synthesis places a critical yet relatively low-volume demand on these reductase systems, thereby favoring high hepatocyte turnover over sustained hepatocyte integrity.