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 (10 mg/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 SAM : S-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.
Our previous studies, demonstrating ethanol-induced alterations in phosphatidylcholine (PC) synthesis via the phosphatidylethanolamine methyltransferase (PEMT) pathway, implicated a defect in very low-density lipoprotein (VLDL) secretion in the pathogenesis of hepatic steatosis. The objective of this study was to determine whether VLDL secretion was reduced by chronic ethanol consumption and whether betaine supplementation, that restores PEMT activity and prevents the development of alcoholic steatosis, could normalize VLDL secretion. The VLDL secretion in rats fed with control, ethanol and the betaine supplemented diets was determined using Triton WR-1339 to inhibit plasma VLDL metabolism. We observed reduced VLDL production rates in chronic alcohol-fed rats compared to control animals. Supplementation of betaine in the ethanol diet increased VLDL production rate to values significantly higher than those observed in the control diet-fed rats. To conclude, chronic ethanol consumption impairs PC generation via the PEMT pathway resulting in diminished VLDL secretion which contributes to the development of hepatic steatosis. By increasing PEMT-mediated PC generation, betaine results in increased fat export from the liver and attenuates the development of alcoholic fatty liver.
Processing of peptides for antigen presentation is catalyzed by antigen-trimming enzymes, including the proteasome and leucine aminopeptidase. Oxidative stress suppresses proteasome function. We hypothesized that in liver cells, processing of antigenic peptides is altered by ethanol metabolism. To address this issue, soluble extracts of ethanol-metabolizing VL-17A cells treated with 100 mM ethanol or left untreated were incubated with C-extended or N-extended 18-27 HBV core peptides. T he proteasome is a multicatalytic enzyme crucially important for proteolysis of oxidized and misfolded proteins, peptides and short-lived signal transduction factors. In vivo, the proteasome exists in equilibrium as 2 particles: the 20S proteasome degrades substrate proteins with no prior ubiquitylation and the 26S form degrades ubiquitylated proteins in an ATP-dependent manner.Both 20S and 26S proteasomes generate peptides for MHC class I-restricted antigen presentation. 1,2 Processing of antigens is a necessary step for recognition by cytotoxic T lymphocytes (CTLs) of virally infected cells. To be recognized by CTLs, "nonself" antigens are processed to peptides of 8-10 amino acids, which then bind to MHC class I molecules in the endoplasmic reticulum. 3 Initial processing of antigenic proteins in the cytoplasm is catalyzed by the proteasome and/or leucine aminopeptidase (LAP). 4 Both enzymes are activated by the proinflammatory cytokine interferon gamma (IFN␥), 5 which induces formation of the immunoproteasome that cleaves antigenic proteins to uniformly sized peptides for presentation.The foregoing series of events occurs after hepatitis B or C infections during which CTLs eliminate HBV-infected or HCV-infected hepatocytes are based on ability of immune system to clear infected hepatocytes. Epidemiological studies have provided supporting evidence of
A potential in vitro model for studying the mechanisms of alcohol-induced hepatocyte injury is the WIF-B cell line. It has many hepatocyte-like features, including a differentiated, polarized phenotype resulting in formation of bile canaliculi. The aim of this study was to examine the effects of ethanol treatment on this cell line. WIF-B cells were cultured up to 96 h in the absence or presence of 25 mM ethanol and subsequently were analyzed for ethanol-induced physiological and morphological changes. Initial studies revealed WIF-B cells exhibited alcohol dehydrogenase (ADH) activity, expressed cytochrome P4502E1 (CYP2E1), and efficiently metabolized ethanol in culture. This cell line also produced the ethanol metabolite acetaldehyde and exhibited low K m aldehyde dehydrogenase (ALDH) activity, comparable to hepatocytes. Ethanol treatment of the WIF-B cells for 48 h led to significant increases in the lactate/pyruvate redox ratio and cellular triglyceride levels. Ethanol treatment also significantly altered WIF-B morphology, decreasing the number of bile canaliculi, increasing the number of cells exhibiting finger-like projections, and increasing cell diameter. The ethanol-induced changes occurring in this cell line were negated by addition of the ADH inhibitor, 4-methylpyrazole (4-MP), indicating the effects were due to ethanol metabolism. In summary, the WIF-B cell line metabolizes ethanol and exhibits many ethanol-induced changes similar to those found in hepatocytes. Because of these similarities, WIF-B cells appear to be a suitable model for studying ethanol-induced hepatocyte injury.
L-Buthionine (S,R) sulfoximine, administered with ethanol, significantly depleted hepatic glutathione, compared with controls. However, despite the decrease in hepatic antioxidant levels, liver injury by ethanol was alleviated, due, in part, to a BSO-elicited acceleration of ethanol metabolism.
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