Benita L. McVicker

Betaine Treatment Attenuates Chronic Ethanol-Induced Hepatic Steatosis and Alterations to the Mitochondrial Respiratory Chain Proteome

Introduction. Mitochondrial damage and disruption in oxidative phosphorylation contributes to the pathogenesis of alcoholic liver injury. Herein, we tested the hypothesis that the hepatoprotective actions of betaine against alcoholic liver injury occur at the level of the mitochondrial proteome. Methods. Male Wister rats were pair-fed control or ethanol-containing liquid diets supplemented with or without betaine (10u2009mg/mL) for 4-5 wks. Liver was examined for triglyceride accumulation, levels of methionine cycle metabolites, and alterations in mitochondrial proteins. Results. Chronic ethanol ingestion resulted in triglyceride accumulation which was attenuated in the ethanol plus betaine group. Blue native gel electrophoresis (BN-PAGE) revealed significant decreases in the content of the intact oxidative phosphorylation complexes in mitochondria from ethanol-fed animals. The alcohol-dependent loss in many of the low molecular weight oxidative phosphorylation proteins was prevented by betaine supplementation. This protection by betaine was associated with normalization of SAMu2009:u2009S-adenosylhomocysteine (SAH) ratios and the attenuation of the ethanol-induced increase in inducible nitric oxide synthase and nitric oxide generation in the liver. Discussion/Conclusion. In summary, betaine attenuates alcoholic steatosis and alterations to the oxidative phosphorylation system. Therefore, preservation of mitochondrial function may be another key molecular mechanism responsible for betaine hepatoprotection.

Ethanol selectively impairs clathrin-mediated internalization in polarized hepatic cells

Although alcoholic liver disease is clinically well-described, the molecular basis for alcohol-induced hepatotoxicity is not well understood. Previously, we determined that the clathrin-mediated internalization of asialoglycoprotein receptor was impaired in ethanol-treated WIF-B cells whereas the internalization of a glycophosphatidylinositol-anchored protein thought to be endocytosed via a caveolae/raft-mediated pathway was not changed suggesting that clathrin-mediated endocytosis is selectively impaired by ethanol. To test this possibility, we examined the internalization of a panel of proteins and compounds internalized by different mechanisms in control and ethanol-treated WIF-B cells. We determined that the internalization of markers known to be internalized via clathrin-mediated mechanisms was impaired. In contrast, the internalization of markers for caveolae/raft-mediated endocytosis, fluid phase internalization or non-vesicle-mediated uptake was not impaired in ethanol-treated cells. We further determined that clathrin heavy chain accumulated at the basolateral surface in small puncta in ethanol-treated cells while there was decreased dynamin-2 membrane association. Interestingly, the internalization of resident apical proteins that lack any known internalization signals was also disrupted by ethanol suggesting that these proteins are internalized via clathrin-mediated mechanisms. This conclusion is consistent with our findings that dominant negative dynamin-2 overexpression impaired internalization of known clathrin markers and single spanning apical residents, but not of markers of fluid phase or raft-mediated internalization. Together these results indicate that ethanol exposure selectively impairs hepatic clathrin-mediated internalization by preventing vesicle fission from the plasma membrane.

Cellular fibronectin stimulates hepatocytes to produce factors that promote alcohol-induced liver injury

AIMnTo examine the consequences of cellular fibronectin (cFn) accumulation during alcohol-induced injury, and investigate whether increased cFn could have an effect on hepatocytes (HCs) by producing factors that could contribute to alcohol-induced liver injury.nnnMETHODSnHCs were isolated from rats fed a control or ethanol liquid diet for four to six weeks. Exogenous cFn (up to 7.5 μg/mL) was added to cells cultured for 20 h, and viability (lactate dehydrogenase,LDH), apoptosis (caspase activity) and secretion of proinflammatory cytokines (tumor necrosis factor alpha, TNF-α and interleukin 6 IL-6), matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of metalloproteinases, TIMPs) was determined. Degradation of iodinated cFn was determined over a 3 h time period in the preparations.nnnRESULTSncFn degradation is impaired in HCs isolated from ethanol-fed animals, leading to its accumulation in the matrix. Addition of exogenous cFn did not affect viability of HCs from control or ethanol-fed animals, and apoptosis was affected only at the higher concentration. Secretion of MMPs, TIMPs, TNF-α and IL-6, however, was increased by exogenously added cFn, with HCs from ethanol-fed animals showing increased susceptibility compared to the controls.nnnCONCLUSIONnThese results suggest that the elevated amounts of cFn observed in alcoholic liver injury can stimulate hepatocytes to produce factors which promote further tissue damage.

Mode of Oral Ethanol Feeding Affects Liver Oxidative Stress Levelsand Methylation Status: Study on NS5A -Transgenic Mice

Background : Alcohol consumption acceleratesthe progressionand worsens the outcomes of hepatitis C viral (HCV) infection in heavyand moderate drinkers.Our aim was to investigate the effects of two modes of oral ethanol feeding on induction of oxidative stress, impaired methylation status and downstream changes in proteasome activity in livers of NS5A -transgenic (Tg) mice. Methods :Ethanol was administered eitherin water (chow fed mice given 20% ethanol in water; designated chow-EtOH) or fed in Lieber De Carli liquid diet (LCD -EtOH). Appropriate controls were used. The mechanisms of alcohol and NS5A -induced changes

Alcohol, carcinoembryonic antigen processing and colorectal liver metastases.

It is well established that alcohol consumption is related to the development of alcoholic liver disease. Additionally, it is appreciated that other major health issues are associated with alcohol abuse, including colorectal cancer (CRC) and its metastatic growth to the liver. Although a correlation exists between alcohol use and the development of diseases, the search continues for a better understanding of specific mechanisms. Concerning the role of alcohol in CRC liver metastases, recent research is aimed at characterizing the processing of carcinoembryonic antigen (CEA), a glycoprotein that is associated with and secreted by CRC cells. A positive correlation exists between serum CEA levels, liver metastasis, and alcohol consumption in CRC patients, although the mechanism is not understood. It is known that circulating CEA is processed primarily by the liver, first by nonparenchymal Kupffer cells (KCs) and secondarily, by hepatocytes via the asialoglycoprotein receptor (ASGPR). Since both KCs and hepatocytes are known to be significantly impacted by alcohol, it is hypothesized that alcohol-related effects to these liver cells will lead to altered CEA processing, including impaired asialo-CEA degradation, resulting in changes to the liver microenvironment and the metastatic potential of CRC cells. Also, it is predicted that CEA processing will affect cytokine production in the alcohol-injured liver, resulting in pro-metastatic changes such as enhanced adhesion molecule expression on the hepatic sinusoidal endothelium. This chapter examines the potential role that alcohol-induced liver cell impairments can have in the processing of CEA and associated mechanisms involved in CEA-related colorectal cancer liver metastasis.

Structure, Function and Metabolism of Hepatic and Adipose Tissue Lipid Droplets: Implications in Alcoholic Liver Disease

For more than 30 years, lipid droplets (LDs) were considered as an inert bag of lipid for storage of energy-rich fat molecules. Following a paradigm shift almost a decade ago, LDs are presently considered an active subcellular organelle especially designed for assembling, storing and subsequently supplying lipids for generating energy and membrane synthesis (and in the case of hepatocytes for VLDL secretion). LDs also play a central role in many other cellular functions such as viral assembly and protein degradation. Here, we have explored the structural and functional changes that occur in hepatic and adipose tissue LDs following chronic ethanol consumption in relation to their role in the pathogenesis of alcoholic liver injury.

90 – Alcohol and Apoptosis

This chapter describes the relationship between alcohol-induced apoptosis and liver function. Alcohol abuse and/or dependence are related to many major medical problems and the chronic consumption of ethanol has been associated with an increased risk of stroke, cancer, osteoporosis, lung infections, neurological changes, and the development of alcoholic liver disease. Although much has been learned about the medical symptoms associated with alcohol-related diseases, the search continues for a better understanding of the molecular and/or cellular mechanisms by which ethanol exerts its deleterious effects. One particular cellular event that is observed in alcohol-induced diseases that may contribute to adverse pathology is the increased production and subsequent accumulation of apoptotic cells. Apoptosis is an active and highly regulated mode of cell death, which in a healthy organ helps to maintain tissue homeostasis. However, when apoptotic death factors are inappropriately expressed because of the introduction of a pathological stimulus such as alcohol, deleterious effects to the organism may occur. Moreover, if the production of apoptotic cells becomes large enough to overwhelm the protection obtained from dead cell removal, a loss of tissue organization and organ viability may occur.