Kusum K. Kharbanda

S-adenosyl-L-methionine co-administration prevents the ethanol-elicited dissociation of hepatic mitochondrial ribosomes in male rats

BACKGROUND Chronic ethanol feeding to male rats has been shown to result in decreased mitochondrial translation, depressed respiratory complex levels and mitochondrial respiration rates. In addition, ethanol consumption has been shown to result in an increased dissociation of mitoribosomes. S-adenosyl-L-methionine (SAM) is required for the assembly and subsequent stability of mitoribosomes and is depleted during chronic ethanol feeding. The ability of dietary SAM co-administration to prevent these ethanol-elicited lesions was investigated. METHODS Male Sprague-Dawley rats were fed a nutritionally adequate liquid diet with ethanol comprising 36% of the calories according to a pair-fed design for 28 days. For some animals, SAM was supplemented in the diet at 200 mg/l. Liver mitochondria were prepared and mitoribosomes isolated. Respiration rates, ATP levels, respiratory complex levels, and the extent of mitoribosome dissociation were determined. RESULTS Twenty-eight days of ethanol feeding were found to result in decreased SAM content, depressed respiration, and increased mitoribosome dissociation. No changes in mitochondrial protein content; levels of respiratory complexes I, III, and V; complex I activities; and ATP levels were detected. Co-administration of SAM in the diet was found to prevent ethanol-induced SAM depletion, respiration decreases and mitoribosome dissociation. CONCLUSIONS Taken together, these findings suggest (1) that mitoribosome dissociation precedes respiratory complex depressions in alcoholic animals and (2) that dietary supplementation of SAM prevents some of the early mitochondrial lesions associated with chronic ethanol consumption.

Inhibition of markers of hepatic stellate cell activation by the hormone relaxin.

Hepatic fibrosis results from excess extracellular matrix produced primarily by hepatic stellate cells (HSC). In response to injury, HSC differentiate to a myofibroblastic phenotype expressing smooth muscle actin and fibrillar collagens. Relaxin is a polypeptide hormone shown to have antifibrotic effects in fibrosis models. In this study, activated HSC from rat liver were treated with relaxin to determine if relaxin can reverse markers of HSC activation. Relaxin treatment resulted in a decrease in the expression of smooth muscle actin, but had no effect on cell proliferation rate. The levels of total collagen and type I collagen were reduced, while the synthesis of new collagen was inhibited. Furthermore, relaxin caused an increase in the expression and secretion of rodent interstitial collagenase (MMP-13), but there was no effect on the gelatinases MMP-2 or MMP-9. Relaxin also increased secretion of TIMP-1 and TIMP-2. The effective concentration of relaxin to induce these effects was consistent with action through the relaxin receptor. In conclusion, relaxin reversed markers of the activated phenotype of HSC including the production of fibrillar collagen. At the same time, the activity of a fibrillar collagenase was increased. These data suggest that relaxin not only inhibits HSC properties that contribute to the progression of hepatic fibrosis, but also promotes the clearance of fibrillar collagen. Therefore, relaxin may be a useful approach in the treatment of hepatic fibrosis.

Chronic ethanol consumption increases homocysteine accumulation in hepatocytes

Results of previous studies have shown that chronic ethanol administration impairs methionine synthetase activity and decreases S-adenosylmethionine levels in the liver, indicating interference with homocysteine remethylation. The purpose of the present study was to investigate the effects of chronic ethanol feeding on the accumulation of homocysteine (Hcy), a potentially toxic agent. The research was divided into two experiments. In Experiment A, hepatocytes were isolated from pair-fed control and ethanol-fed rats after 2 weeks of feeding, and the release of Hcy into the medium was determined. Hepatocytes obtained from ethanol-fed rats released twice as much Hcy into the medium as did those obtained from controls. When hepatocytes were challenged by a methionine load, a marked increase in Hcy generation was observed, and the increase was further enhanced in hepatocytes obtained from ethanol-fed rats. In Experiment B, hepatocytes were isolated from pair-fed control and ethanol-fed rats after 4 weeks of feeding (the feeding time required for significant formation of alcoholic fatty liver in rats). In this experiment, similar results were obtained with Hcy generation as in Experiment A. In Experiment B, supplementation of the incubation medium with betaine prevented the increase in generation of Hcy by methionine-treated control cells as well as the generation of Hcy by cells of ethanol-treated rats. These results indicate that betaine may have the potential as a therapeutic agent against toxic Hcy formation.

Hybrid malondialdehyde and acetaldehyde protein adducts form in the lungs of mice exposed to alcohol and cigarette smoke

BACKGROUND Most alcohol abusers smoke cigarettes and approximately half of all cigarette smokers consume alcohol. However, no animal models of cigarette and alcohol co-exposure exist to examine reactive aldehydes in the lungs. Cigarette smoking results in elevated lung acetaldehyde (AA) and malondialdehyde (MDA) levels. Likewise, alcohol metabolism produces AA via the action of alcohol dehydrogenase and MDA via lipid peroxidation. A high concentration of AA and MDA form stable hybrid protein adducts known as malondialdehyde-acetaldehyde (MAA) adducts. We hypothesized that chronic cigarette smoke and alcohol exposure in an in vivo mouse model would result in the in vivo formation of MAA adducts. METHODS We fed C57BL/6 mice ad libitum ethanol (20%) in drinking water and exposed them to whole-body cigarette smoke 2 h/d, 5 d/wk for 6 weeks. Bronchoalveolar lavage fluid and lung homogenates were assayed for AA, MDA, and MAA adduct concentrations. MAA-adducted proteins were identified by Western blot and ELISA. RESULTS Smoke and alcohol exposure alone elevated both AA and MDA, but only the combination of smoke+alcohol generated protein-adducting concentrations of AA and MDA. MAA-adducted protein (~500 ng/ml) was significantly elevated in the smoke+alcohol-exposed mice. Of the 5 MAA-adducted proteins identified by Western blot, 1 protein band immunoprecipitated with antibodies to surfactant protein D. Similar to in vitro PKC stimulation by purified MAA-adducted protein, protein kinase C (PKC) epsilon was activated only in tracheal epithelial extracts from smoke- and alcohol-exposed mice. CONCLUSIONS These data demonstrate that only the combination of cigarette smoke exposure and alcohol feeding in mice results in the generation of significant AA and MDA concentrations, the formation of MAA-adducted protein, and the activation of airway epithelial PKC epsilon in the lung.

Ethanol consumption reduces the proteolytic capacity and protease activities of hepatic lysosomes

Chronic ethanol consumption causes decreased hepatic protein degradation, resulting in protein accumulation within hepatocytes. In this investigation, we sought to determine whether chronic ethanol feeding alters the degradative capacity and protease activities of isolated hepatic lysosomes. Male Sprague-Dawley-derived rats were fed a liquid diet containing either ethanol (36% of calories) or isocaloric maltose-dextrin for 1-5 wk. Hepatic lysosomes were isolated by differential centrifugation and purified through Percoll gradients. Lysosomes obtained from livers of ethanol-fed rats degraded both endogenous protein substrates and the exogenously added radioactive substrate, 125I-RNase A, 26-42% more slowly than lysosomes from pair fed controls. The ethanol-elicited reduction in proteolytic capacity appeared to result in part, from a deficiency of the lysosomal cathepsins B, L, and H. Compared with controls, the specific activities of these enzymes were 31-45% lower in lysosomes from ethanol-fed rats. Immunoblot analyses also revealed that the intralysosomal as well as the intracellular content of cathepsin B was significantly lower in ethanol-fed rats. In contrast, ethanol consumption did not affect the cellular quantity of cathepsin L but lowered its amount in isolated lysosomes. Our findings suggest that chronic ethanol consumption causes a deficiency in lysosomal cathepsins by altering their biosynthesis and/or their trafficking into lysosomes.

Betaine administration corrects ethanol-induced defective VLDL secretion

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.

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 (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.

Malondialdehyde–acetaldehyde–protein adducts increase secretion of chemokines by rat hepatic stellate cells

Findings obtained from our recent studies have demonstrated that malondialdehyde, a product of lipid peroxidation, and acetaldehyde can react together with proteins in a synergistic manner and form hybrid protein conjugates, which have been designated as malondialdehyde-acetaldehyde (MAA)-protein adducts. These adducts have been detected in livers of ethanol-fed rats and are immunogenic because significant increases in circulating antibody titers against MAA-adducted proteins have been observed in ethanol-fed rats and more recently in human alcoholics. Although immunological factors may tend to perpetuate liver injury, little is known about the direct functional consequences of MAA-adducted proteins on the different cellular populations of the liver. Hepatic stellate cells (HSCs) have been shown to be pivotal in the pathogenesis of fibrosis and in the amplification and self-perpetuation of the inflammatory process. The present study was conducted to determine the effects of MAA-adducted proteins on the function of HSCs. Rat HSCs were exposed to various amounts of MAA-protein adducts and their unmodified controls, and the secretion of two chemokines, monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-2, that are involved in the chemotaxis of monocytes/macrophages and neutrophils, respectively, was determined. We observed that bovine serum albumin-MAA induced a dose- and time-dependent increase in the secretion of both of these chemokines. These findings indicate that MAA-adducted proteins may play a role in the modulation of the hepatic inflammatory response and could contribute to the pathogenesis of alcoholic liver disease.

Regulation of FOXO3 by phosphorylation and methylation in hepatitis C virus infection and alcohol exposure.

Hepatitis C virus (HCV) infection produces chronic liver injury that is significantly exacerbated by alcohol consumption. While multiple mechanisms contribute to this synergy, a viral‐induced loss of antioxidant responses has been shown to play an important role. This study examined the effects of HCV infection and alcohol on the regulation of the transcription factor FOXO3, an important regulator of Mn‐superoxide dismutase (SOD2) expression, a tumor suppressor, and a component of the hepatic antioxidant response system. FOXO3 was activated by either HCV or alcohol alone but suppressed by the combination. To understand this paradoxical result, we applied a capillary isoelectric focusing (IEF) method to determine the pattern of FOXO3 posttranslational modifications (PTMs) induced by HCV and alcohol. We observed the presence of multiple different nuclear and cytosolic species of FOXO3 and used antiphosphoserine, acetyl‐lysine, methylarginine, and ubiquitin antibodies to identify the PTM patterns present in each species. HCV caused multiple changes including phosphorylation of FOXO3 at S‐574, a novel c‐Jun N‐terminal kinase (JNK) site, which promoted nuclear translocation and transcription. Ethanol suppressed arginine‐methylation of FOXO3 promoting nuclear export and degradation of the JNK phosphorylated form. Human liver biopsy samples showed the presence of the HCV‐specific form of FOXO3 in HCV‐infected livers but not in normal liver or nonalcoholic steatohepatitis. Conclusion: The development of this novel IEF method for the simultaneous quantification of differently modified FOXO3 species allowed us to demonstrate how HCV and alcohol combine to modify a complex pattern of FOXO3 PTMs that contribute to pathogenesis. This approach will allow further dissection of the role of protein PTMs in viral liver disease. (Hepatology 2014;58:58–70)

Wilson's disease: changes in methionine metabolism and inflammation affect global DNA methylation in early liver disease.

Hepatic methionine metabolism may play an essential role in regulating methylation status and liver injury in Wilsons disease (WD) through the inhibition of S‐adenosylhomocysteine hydrolase (SAHH) by copper (Cu) and the consequent accumulation of S‐adenosylhomocysteine (SAH). We studied the transcript levels of selected genes related to liver injury, levels of SAHH, SAH, DNA methyltransferases genes (Dnmt1, Dnmt3a, Dnmt3b), and global DNA methylation in the tx‐j mouse (tx‐j), an animal model of WD. Findings were compared to those in control C3H mice, and in response to Cu chelation by penicillamine (PCA) and dietary supplementation of the methyl donor betaine to modulate inflammatory and methylation status. Transcript levels of selected genes related to endoplasmic reticulum stress, lipid synthesis, and fatty acid oxidation were down‐regulated at baseline in tx‐j mice, further down‐regulated in response to PCA, and showed little to no response to betaine. Hepatic Sahh transcript and protein levels were reduced in tx‐j mice with consequent increase of SAH levels. Hepatic Cu accumulation was associated with inflammation, as indicated by histopathology and elevated serum alanine aminotransferase (ALT) and liver tumor necrosis factor alpha (Tnf‐α) levels. Dnmt3b was down‐regulated in tx‐j mice together with global DNA hypomethylation. PCA treatment of tx‐j mice reduced Tnf‐α and ALT levels, betaine treatment increased S‐adenosylmethionine and up‐regulated Dnmt3b levels, and both treatments restored global DNA methylation levels. Conclusion: Reduced hepatic Sahh expression was associated with increased liver SAH levels in the tx‐j model of WD, with consequent global DNA hypomethylation. Increased global DNA methylation was achieved by reducing inflammation by Cu chelation or by providing methyl groups. We propose that increased SAH levels and inflammation affect widespread epigenetic regulation of gene expression in WD. (HEPATOLOGY 2013)