Lipid peroxidation and hepatic endoplasmic reticulum dysfunction in alcoholic liver disease

Hepatic oxidative stress is a well-documented result of sustained ethanol consumption and has been proposed to play a major role in alcoholic liver disease (ALD) progression. The work presented in this dissertation evaluates the pathogenic contribution of oxidative stress in a six week murine model for ALD. Ethanol-fed mice exhibited significant hepatic panlobular steatosis and elevated plasma ALT values when compared to isocaloric controls. Oxidative stress was observed in ethanol-treated mice through a significant decrease in hepatic glutathione as well as enhanced lipid peroxidation through a 2-fold increase in hepatic TSARS and increased immunohistochemical staining of reactive aldehydes 4-HNE, 4-ONE, ACR and MDA. Biotin-tagging and hydrazide chemistry coupled to highly sensitive 2D LC-MS/MS was used to identify protein targets for modification. In total, 414 proteins were identified as targets for modification, including eight site-specific modifications by 4-HNE (2), 4-ONE (4) and ACR (2). The impact of aldehyde adduction on these 414 protein targets was evaluated with the use of multiple bioinformatic analyses. Identified pathways included fatty acid metabolism, drug metabolism, oxidative phosphorylation, the TCA cycle and protein folding. Altered protein folding occurs in numerous disease states, including ALD. Identified as a potential mechanism for altered protein folding, decreases in hepatic glycosylation machinery have been observed in rodent models of ALD, but specific protein targets have not been identified. Utilizing 2D-PAGE and glycoprotein staining techniques, ethanol-fed mice displayed a global decrease in hepatic microsomal glycosylation. Protein targets were identified using LC-MS/MS, revealing the identification of 30 glycoproteins within hepatic microsomes. Previous work has identified both oxidative stress and impaired glycosylation as initiating factors for the ER stress response. Work presented herein evaluates the pathogenic contribution of the ER stress response during early-stage ALD. Immunoblot analysis of the UPR, namely the PERK, IRE1alpha, ATF6 and SREBP pathways, reveals no significant role for these signaling cascades in the etiology of hepatic steatosis associated with early stage ALD. Collectively, this work demonstrates a primary pathogenic role for oxidative stress, lipid peroxidation and ER glycosylation in the early initiating stages of ALD that precedes the involvement of the ER stress response.