Increased methylation demand exacerbates ethanol-induced liver injury.

We previously reported that chronic ethanol intake lowers hepatocellular S-adenosylmethionine to S-adenosylhomocysteine ratio and significantly impairs many liver methylation reactions. One such reaction, catalyzed by guanidinoacetate methyltransferase (GAMT), is a major consumer of methyl groups and utilizes as much as 40% of the SAM-derived groups to convert guanidinoacetate (GAA) to creatine. The exposure to methyl-group consuming compounds has substantially increased over the past decade that puts additional stresses on the cellular methylation potential. The purpose of our study was to investigate whether increased ingestion of a methyl-group consumer (GAA) either alone or combined with ethanol intake, plays a role in the pathogenesis of liver injury. Adult male Wistar rats were pair-fed the Lieber DeCarli control or ethanol diet in the presence or absence of GAA for 2weeks. At the end of the feeding regimen, biochemical and histological analyses were conducted. We observed that 2 weeks of GAA- or ethanol-alone treatment increases hepatic triglyceride accumulation by 4.5 and 7-fold, respectively as compared with the pair-fed controls. However, supplementing GAA in the ethanol diet produced panlobular macro- and micro-vesicular steatosis, a marked decrease in the methylation potential and a 28-fold increased triglyceride accumulation. These GAA-supplemented ethanol diet-fed rats displayed inflammatory changes and significantly increased liver toxicity compared to the other groups. In conclusion, increased methylation demand superimposed on chronic ethanol consumption causes more pronounced liver injury. Thus, alcoholic patients should be cautioned for increased dietary intake of methyl-group consuming compounds even for a short period of time.

Alcohol consumption decreases rat hepatic creatine biosynthesis via altered guanidinoacetate methyltransferase activity.

BACKGROUND: We have previously shown that decreased S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) ratio generated in livers of alcohol-fed rats can impair the activities of many SAM-dependent methyltransferases. One such methyltransferase is guanidinoacetate methyltransferase (GAMT) that catalyzes the last step of creatine synthesis. As GAMT is the major utilizer of SAM, the purpose of the study was to examine the effects of ethanol (EtOH) on liver creatine levels and GAMT activity. METHODS: Male Wistar rats were pair-fed the Lieber-DeCarli control and EtOH diet for 4 to 5 weeks. At the end of the feeding regimen, the liver, kidney, and blood were removed from these rats for subsequent biochemical analyses. RESULTS: We observed ~60% decrease in creatine levels in the livers from EtOH-fed rats as compared to controls. The reduction in creatine levels correlated with lower SAM:SAH ratio observed in the livers of the EtOH-fed rats. Further, in vitro experiments with cell-free system and hepatic cells revealed it is indeed elevated SAH and lower SAM:SAH ratio that directly impairs GAMT activity and significantly reduces creatine synthesis. EtOH intake also slightly decreases the hepatocellular uptake of the creatine precursor, guanidinoacetate (GAA), and the GAMT enzyme expression that could additionally contribute to reduced liver creatine synthesis. The consequences of impaired hepatic creatine synthesis by chronic EtOH consumption include (i) increased toxicity due to GAA accumulation in the liver; (ii) reduced protection due to lower creatine levels in the liver, and (iii) reduced circulating and cardiac creatine levels. CONCLUSIONS: Chronic EtOH consumption affects the hepatic creatine biosynthetic pathway leading to detrimental consequences not only in the liver but could also affect distal organs such as the heart that depend on a steady supply of creatine from the 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.

Early growth response-1 contributes to steatosis development after acute ethanol administration.

BACKGROUND: Previous work demonstrated that the transcription factor, early growth response-1 (Egr-1), participates in the development of steatosis (fatty liver) after chronic ethanol (EtOH) administration. Here, we determined the extent to which Egr-1 is involved in fatty liver development in mice subjected to acute EtOH administration. METHODS: In acute studies, we treated both wild-type and Egr-1 null mice with either EtOH or phosphate-buffered saline (PBS) by gastric intubation. At various times after treatment, we harvested sera and livers and quantified endotoxin, indices of liver injury, steatosis, and hepatic Egr-1 content. In chronic studies, groups of mice were fed liquid diets containing either EtOH or isocaloric maltose-dextrin for 7 to 8 weeks. RESULTS: Compared with controls, acute EtOH-treated mice showed a rapid, transient elevation in serum endotoxin beginning 30 minutes after treatment. One hour postgavage, livers from EtOH-treated mice exhibited a robust elevation of both Egr-1 mRNA and protein. By 3 hours postgavage, liver triglyceride increased in EtOH-treated mice as did lipid peroxidation. Acute EtOH treatment of Egr-1-null mice showed no Egr-1 expression, but these animals still developed elevated triglycerides, although significantly lower than EtOH-fed wild-type littermates. Despite showing decreased fatty liver, EtOH-treated Egr-1 null mice exhibited greater liver injury. After chronic EtOH feeding, steatosis and liver enlargement were clearly evident, but there was no indication of elevated endotoxin. Egr-1 levels in EtOH-fed mice were equal to those of pair-fed controls. CONCLUSIONS: Acute EtOH administration induced the synthesis of Egr-1 in mouse liver. However, despite its robust increase, the transcription factor had a smaller, albeit significant, function in steatosis development after acute EtOH treatment. We propose that the rise in Egr-1 after acute EtOH is an hepatoprotective adaptation to acute liver injury from binge drinking that is triggered by EtOH metabolism and elevated levels of endotoxin.

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.

Lysosomal leakage and lack of adaptation of hepatoprotective enzyme contribute to enhanced susceptibility to ethanol-induced liver injury in female rats.

BACKGROUND: Women exhibit greater liver damage than men after chronic alcohol consumption. Similar findings are reported in animal models. Here, we determined whether differential liver injury occurred in male and female rats after feeding these animals liquid diets containing either ethanol or isocaloric dextrose with fish oil as the sole source of lipid. METHODS: Control and ethanol liquid diets containing fish oil were pair-fed to male and female rats for 8 weeks. Liver damage was evaluated by triglyceride accumulation, lipid peroxide formation, serum transaminases, histological evaluation, and the activities of selected lysosomal and hepatoprotective enzymes. RESULTS: Fatty liver was detected after ethanol feeding in both genders, but in female rats, triglyceride levels were 60% higher, lipid peroxides were 2-fold higher, and inflammatory cells were more evident than in males. A 2-fold elevation of cathepsin B in hepatic cytosol fractions, indicating lysosomal leakage, was detected in ethanol-fed female rats but no such elevation was observed in males. The basal activity of the hepatoprotective enzyme, betaine-homocysteine methyltransferase was 4-fold higher in livers of control male rats than females, and the enzyme activity was further elevated in ethanol-fed male rats but not in females. CONCLUSIONS: Thus, female rats given ethanol in a diet containing fish oil exhibited more severe liver damage than males. We propose that this difference results, in part, from a greater tendency by females to accumulate hepatic fat, thereby enhancing the potential for oxidative stress, which in turn leads to hepatic inflammation. In addition, our findings indicate that female rats have a higher susceptibility to liver damage because of a reduced capacity for hepatoprotection.

L-Buthionine (S,R) sulfoximine depletes hepatic glutathione but protects against ethanol-induced liver injury.

BACKGROUND: L-Buthionine (S,R) sulfoximine (BSO) is an inhibitor of glutathione biosynthesis and has been used as an effective means of depleting glutathione from cells and tissues. Here we investigated whether treatment with BSO enhanced ethanol-induced liver injury in mice. METHODS: Female C57Bl/6 mice were pair fed with control and ethanol-containing liquid diets in which ethanol was 29.2% of total calories. During the final 7 days of pair feeding, groups of control-fed and ethanol-fed mice were given 0, 5 or 7.6 mM BSO in the liquid diets. RESULTS: Compared with controls, ethanol given alone decreased total liver glutathione. This effect was exacerbated in mice given ethanol with 7.6 mM BSO, causing a 72% decline in hepatic glutathione. While ethanol alone caused no decrease in mitochondrial glutathione, inclusion of 7.6 mM BSO caused a 2-fold decline compared with untreated controls. L-Buthionine (S,R) sulfoximine did not affect ethanol consumption, but serum ethanol levels in BSO-treated mice were nearly 6-fold lower than in mice given ethanol alone. The latter decline in serum ethanol was associated with a significant elevation in the specific activities of cytochrome P450 2E1 and alcohol dehydrogenase in livers of BSO-treated animals. Ethanol consumption caused a 3.5-fold elevation in serum alanine aminotransferase levels but the enzyme fell to control levels when BSO was included in the diet. L-Buthionine (S,R) sulfoximine administration also attenuated ethanol-induced steatosis, prevented the leakage of lysosomal cathepsins into the cytosol, and prevented the ethanol-elicited decline in proteasome activity. CONCLUSIONS: 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.

Chronic ethanol treatment impairs Rac and Cdc42 activation in rat hepatocytes.

BACKGROUND: The effects of chronic ethanol feeding on rat hepatocytes have been shown to include impaired cell-extracellular matrix (ECM) adhesion events, such as decreased attachment and spreading as well as increased integrin-actin cytoskeleton association. These results, observed previously by this laboratory, are highly suggestive of impaired actin cytoskeleton reorganization, an event mediated by differential activation of the Rho family GTPases Rac, Cdc42, and RhoA. Therefore, the purpose of this study was to examine the effects of chronic ethanol administration on these GTPases. METHODS: Male Wistar rats were pair-fed 4 to 5 weeks with a liquid diet containing either ethanol (as 36% of total calories) or isocaloric carbohydrate. Hepatocytes were isolated and plated on collagen IV up to 24 hours. At specific times, the hepatocytes were lysed and these lysates were analyzed for RhoA, Cdc42, and Rac activation. RESULTS: In freshly isolated hepatocytes from ethanol-fed rats, the GTP-bound (active) forms of Rac and Cdc42 were significantly decreased compared with pair-fed control rats, while the GTP-bound form of RhoA was not significantly altered. These ethanol-induced impairments in Rac and Cdc42 activation persisted even after plating the hepatocytes on collagen IV. Additionally, chronic ethanol treatment did not directly affect GTP binding of Cdc42 and Rac, as incorporation of GTPgammaS was not affected. CONCLUSIONS: Chronic ethanol administration selectively impairs Rac and Cdc42 activation in rat hepatocytes. As activation of these 2 GTPases is crucial for efficient cell attachment and spreading on ECM substrates, the results from this study suggest that the ethanol-induced impairments in Rac and Cdc42 activation are responsible for the impaired hepatocyte-ECM adhesion events observed previously by our laboratory. Furthermore, these results raise the intriguing possibility that these GTPases are involved in other ethanol-induced functional impairments, such as protein trafficking and receptor-mediated endocytosis.

WIF-B cells as a model for alcohol-induced hepatocyte injury.

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.