Folate deficiency disturbs hepatic methionine metabolism and promotes liver injury in the ethanol-fed micropig

Alcoholic liver disease is associated with abnormal hepatic methionine metabolism and folate deficiency. Because folate is integral to the methionine cycle, its deficiency could promote alcoholic liver disease by enhancing ethanol-induced perturbations of hepatic methionine metabolism and DNA damage. We grouped 24 juvenile micropigs to receive folate-sufficient (FS) or folate-depleted (FD) diets or the same diets containing 40% of energy as ethanol (FSE and FDE) for 14 wk, and the significance of differences among the groups was determined by ANOVA. Plasma homocysteine levels were increased in all experimental groups from 6 wk onward and were greatest in FDE. Ethanol feeding reduced liver methionine synthase activity, S-adenosylmethionine (SAM), and glutathione, and elevated plasma malondialdehyde (MDA) and alanine transaminase. Folate deficiency decreased liver folate levels and increased global DNA hypomethylation. Ethanol feeding and folate deficiency acted together to decrease the liver SAM/S-adenosylhomocysteine (SAH) ratio and to increase liver SAH, DNA strand breaks, urinary 8-oxo-2'-deoxyguanosine [oxo(8)dG]/mg of creatinine, plasma homocysteine, and aspartate transaminase by more than 8-fold. Liver SAM correlated positively with glutathione, which correlated negatively with plasma MDA and urinary oxo(8)dG. Liver SAM/SAH correlated negatively with DNA strand breaks, which correlated with urinary oxo(8)dG. Livers from ethanol-fed animals showed increased centrilobular CYP2E1 and protein adducts with acetaldehyde and MDA. Steatohepatitis occurred in five of six pigs in FDE but not in the other groups. In summary, folate deficiency enhances perturbations in hepatic methionine metabolism and DNA damage while promoting alcoholic liver injury.     

Vitamin B12 and Folate Status in Rats after Chronic Administration of Ethanol and Acute Exposure to Nitrous Oxide

The chronic administration of ethanol or brief exposure to nitrous oxide (N2O) decreases the activity of hepatic methionine synthase and disrupts normal metabolic processes that require folate and vitamin B12. This combination of drugs has clinical relevance since alcoholic patients often require surgery and receive N2O as a component of their anesthetic. To assess this clinical problem using a rodent model, rats were given a liquid ethanol diet (35% of calories as ethanol) and control rats were pair-fed a liquid diet with carbohydrate substituting for the caloric content of ethanol. After receiving liquid diets for 6 weeks, rats were exposed to 60% N2O/40% O2 for 6 hr. Urinary excretions of formic acid and formiminoglutamic acid (FIGLU) were used as indirect markers of folate status. In both the ethanol-fed and control groups, excretion of formic acid and FIGLU markedly increased the first day after N2O and returned towards background values by the second day after N2O exposure. Ethanol treatment alone decreased methionine synthase activities in liver, but not kidney or brain. Exposure to N2O further decreased methionine synthase activities, and recovery of methionine synthase activity after N2O occurred over a period of 4 days at the same rate in both the ethanol-fed and control groups. Ethanol treatment for 6 weeks combined with acute exposure to N2O did not deplete the rats of vitamin B12 in blood, liver, kidney, or brain. We conclude that in this animal model, chronic treatment with ethanol does not markedly exacerbate the disturbances in folate/vitamin B12 metabolism caused by brief exposure to N2O.     

Dietary Betaine Promotes Generation of Hepatic S-Adenosylmethionine and Protects the Liver from Ethanol-Induced Fatty Infiltration

Previous studies have shown that ethanol feeding to rats alters methionine metabolism by decreasing the activity of methionine synthetase. This is the enzyme that converts homocysteine in the presence of vitamin B₁₂ and N⁵-methyltetrahydrofolate to methionine. The action of the ethanol results in an increase in the hepatic level of the substrate N⁵-methyltetrahydrofolate but as an adaptive mechanism, betaine homocysteine methyltransferase, is induced in order to maintain hepatic S-adenosylmethionine at normal levels. Continued ethanol feeding, beyond 2 months, however, produces depressed levels of hepatic S-adenosylmethionine. Because betaine homocysteine methyltransferase is induced in the livers of ethanolfed rats, this study was conducted to determine what effect the feeding of betaine, a substrate of betaine homocysteine methyltransferase, has on methionine metabolism in control and ethanol-fed animals. Control and ethanol-fed rats were given both betainelacking and betaine-containing liquid diets for 4 weeks, and parameters of methionine metabolism were measured. These measurements demonstrated that betaine administration doubled the hepatic levels of S-adenosylmethionine in control animals and increased by 4-fold the levels of hepatic S-adenosylmethionine in the ethanol-fed rats. The ethanol-induced infiltration of triglycerides in the liver was also reduced by the feeding of betaine to the ethanol-fed animals. These results indicate that betaine administration has the capacity to elevate hepatic S-adenosylmethionine and to prevent the ethanol-induced fatty liver.     

Comparison of Effects of Long-Term Ethanol Consumption on the Heart and Liver of the Rat

Alterations in heart and liver metabolism were determined periodically in Sprague-Dawley rats pair-fed a liquid diet (ethanol, 36% of calories) for times as long as 1 year. In liver mitochondria the rate of ATP synthesis was lowered significantly after ethanol administration for 1 month and longer feeding periods. In liver microsomes from ethanol-fed animals, ethanol oxidation and aniline hydroxylation increased 1.5- and 3.5-fold, respectively, after 1 month and remained elevated at the longer feeding intervals. Electron microscopic analyses of heart left ventricles revealed no alterations from ethanol consumption for 1 month. Alterations including disrupted mitochondrial cristae, dilatation of sarcoplasmic reticulum, and widening of the intercalated discs were observed after 6.5-month feeding periods. Myocardial concentrations of creatine, creatine phosphate, ATP, ADP, and Pi remained constant even after ethanol consumption for 9 months. After a 12-month feeding period slight changes in cardiac mitochondrial energy-linked properties were observed which were not as pronounced as those occurring in liver mitochondria. The activity and oligomycin sensitivity of the ATPase were not altered in cardiac mitochondria, whereas in liver preparations significant alterations in these properties of the ATPase were apparent after ethanol consumption for 1 month and the longer feeding periods. These observations suggest that the liver responds more quickly and dramatically to chronic ethanol consumption than does the heart.     

Chronic ethanol consumption alters rat liver plasma membranes and potentiates release of alkaline phosphatase

The aim of this study was to investigate possible mechanisms involved in the elevation of serum alkaline phosphatase activity in alcoholics. Male Sprague-Dawley rats were pair-fed nutritionally adequate liquid diets containing ethanol as 36% of energy or an isocaloric amount of carbohydrate for 4-5 wk. Serum alkaline phosphatase activity was increased moderately but significantly. Hepatocytes isolated from ethanol-fed animals exhibited pronounced morphologic alterations of their plasma membranes by scanning electron microscopy and a reduced content of alkaline phosphatase despite an increase in total liver alkaline phosphatase content. Chronic ethanol feeding also potentiated the release of alkaline phosphatase from the cells during incubation with 50 mM ethanol. Furthermore, chronic ethanol feeding resulted in reduced recovery of alkaline phosphatase in hepatic plasma membranes isolated by sucrose gradient centrifugation but did not affect the recoveries of other plasma membrane markers (5'-nucleotidase and Na+,K+-adenosine triphosphatase) nor the subcellular distribution of alkaline phosphatase in the nuclear, mitochondrial, microsomal, and cytosolic fractions. These findings suggest that the increased serum alkaline phosphatase levels observed in response to chronic ethanol feeding may be due, at least in part, to increased lability of this plasma membrane enzyme.     

Ethanol-induced changes in hepatic free radical defense mechanisms and fatty-acid composition in the miniature pig

In the miniature pig, ethanol consumption has been reported to induce alterations in hepatic antioxidant defense capacity, which could result in increased risk of peroxidative damage. However, ethanol may also induce changes in membrane fatty acid composition, which could reduce the risk of peroxidative damage. This study examined lipid peroxidation, antioxidant defense and fatty acid composition in livers from miniature pigs fed ethanol in diets containing 12% of their calories as fat for 20 mo. After 12 and 20 mo of feeding, ethanol-fed pigs had higher hepatic manganese-superoxide dismutase activity, lower hepatic copper concentrations and low hepatic copper-zinc-superoxide dismutase and glutathione peroxidase activities compared with controls. Lipid peroxidation as assessed by thiobarbituric acid reacting substance assay was lower in liver homogenate and mitochondrial and microsomal fractions from ethanol-fed pigs than in controls. The percentage contribution of highly unsaturated fatty acids to total fatty acids in liver homogenates (after 12 mo of feeding) and microsome fractions (after 20 mo of feeding) was lower in the ethanol-fed pigs than in the controls, resulting in a lower peroxidizability index. Ethanol-fed pigs had minimal or no hepatic damage as assessed by histological methods. We suggest that the relative resistance of microsomes to lipid peroxidation is due to the lower peroxidizability index in the ethanol-fed pigs and may account in part for the absence of significant histopathological findings after 20 mo of ethanol feeding.     

Effects of Ethanol on Tissue Folate Incorporation and Recovery from Folate Deficiency in Rats

Studies in folate-deficient alcoholics suggest that ethanol interferes with the recovery of folate status and the hematopoietic response to folate. Previous animal studies have suggested diverse effects of ethanol on intestinal absorption, hepatic metabolism, and urinary excretion of folate. In order to examine the effects of ethanol on folate distribution during folate deficiency, tissue incorporation of a tracer dose of folate was studied in rats chronically fed ethanol-containing and/or folate-deficient diets. Rats fed these diets were also used to study the effect of chronic ethanol consumption on the dietary reversal of folate deficiency by changing the diets (adding folate or replacing ethanol) from 12 to 16 weeks. After 16 weeks, tissue folate depletion was severe in rats fed folate-deficient diets. Plasma and whole body retention of the tracer dose of folate was decreased in folate-deficient rats consuming ethanol. In folate-deficient rats, ethanol consumption increased the incorporation of folate by the kidney and brain, but had no effect in other tissues (liver, lung, spleen, intestine, testis). In ethanol-fed folate-deficient rats that continued to consume ethanol, but with added folate in their diets, urine, plasma, liver, and kidney folate levels returned to control levels in 4 weeks. In the rats that stopped ethanol, but continued low folate diet consumption, no recovery of tissue folate levels was seen in 4 weeks. These results suggest that chronic ethanol consumption can exacerbate folate requirements by inhibiting body retention of small doses of folate. However, these effects are minor because ethanol consumption does not block recovery from folate deficiency when rats are fed sufficient amounts of folate.     

Feeding S-adenosyl-l-methionine attenuates both ethanol-induced depletion of mitochondrial glutathione and mitochondrial dysfunction in periportal and perivenous rat hepatocytes

Mitochondrial glutathione plays an important role in maintaining a functionally competent organelle. Previous studies have shown that ethanol feeding selectively depletes the mitochondrial glutathione pool, more predominantly in mitochondria from perivenous hepatocytes. Because S-adenosyl-l-methionine (SAM) is a glutathione precursor and maintains the structure and function of biological membranes, the purpose of the present study was to determine the effects of SAM on glutathione and function of perivenous (PV) and peri-portal (PP) mitochondria from chronic ethanol-fed rats. SAM administration resulted in a significant increase in the basal cytosol and mitochondrial glutathione in both PP and PV cells from both pair-fed or ethanol-fed groups. When hepatocytes from ethanol-fed rats supplemented with SAM were incubated with methionine plus serine or N-acetylcysteine, mitochondrial glutathione increased in parallel with cytosol, an effect not observed in cells from ethanol-fed rats without SAM. Feeding equimolar N-acetylcysteine raised cytosol glutathione but did not prevent the mitochondrial glutathione defect. In addition, SAM feeding resulted in significant preservation of cellular adenosine triphosphate (ATP) levels (23% to 43%), mitochondrial membrane potential (17% to 25%), and the uncoupler control ratio (UCR) of respiration (from 5.1 ± 0.7 to 7.3 ± 0.6 and 2.1 ± 0.3 to 6.1 ± 0.7) for PP and PV mitochondria, respectively. Thus, these effects of SAM suggest that it may be a useful agent to preserve the disturbed mitochondrial integrity in liver disease caused by alcoholism through maintenance of mitochondrial glutathione transport.     

Changes in the Fatty Acid Profile of Plasma and Adipose Tissue in Rats after Long-Term Ethanol Feeding

The effect of chronic ethanol feeding on the fatty acid composition of plasma and abdominal adipose tissue in rats was studied. Animals were maintained on a 30% ethanol solution in drinking water for 3 and 5 months. Control rats were given water. Caloric intake was similar in control and ethanol-fed rats at the end of the experimental period. However, a decrease in body weight was observed in rats that had consumed ethanol. Palmitoleic (16:1n7) and oleic (18:1n9) acids increased markedly, and linoleic acid (18:2n6) decreased in the plasma and in the adipose tissue of ethanol-fed rats with respect to control rats. After 3 months of ethanol ingestion, long-chain polyunsaturated fatty acids were reduced both in plasma and adipose tissue. When ethanol was administered for 5 months, only plasma long-chain polyunsaturated fatty acids of the n-3 series were decreased. This suggest that changes induced by ethanol ingestion in essential fatty acid metabolism is less pronounced when ethanol feeding is maintained for a long period of time.     

Effects of Dietary Methionine and Vitamin B12 Deficiency on Folate Metabolism

S ummary . Glutamate formimino transferase activity (GFA) and liver folic acid and vitamin B₁₂ levels were decreased in rats fed diets deficient in methionine and vitamin B₁₂. The addition of either methionine or vitamin B₁₂ resulted in a rise in GFA and in liver folate levels. However, L. casei serum folate was not affected by the dietary level of vitamin B₁₂ or methionine, but the S. faecalis serum folate decreased as the level of dietary methionine and vitamin B₁₂ was increased. The results of these studies do not appear to support the concept that vitamin B₁₂ deficiency results in a 'pile-up' of N⁵-methyl folate but suggests that it induces folate deficiency. Also the results suggest that the effect of vitamin B₁₂ on folate metabolism may be mediated via methionine metabolism.     

Zonal heterogeneity of the effects of chronic ethanol feeding on hepatic fatty acid metabolism

Periportal and perivenous hepatocytes were isolated from rats fed a high-fat, ethanol-containing diet to investigate the acinar heterogeneity of the effects of prolonged ethanol administration on lipid metabolism. Chronic feeding of ethanol caused a rather selective accumulation of triacylglycerols in the perivenous zone of the liver. In control animals the rate of lipogenesis and the activity of acetyl-CoA carboxylase were higher in perivenous than in periportal hepatocytes, whereas the rate of fatty acid oxidation and the activity of carnitine palmitoyltransferase I were higher in periportal than in perivenous cells; however, no zonation was evident for very-low-density-lipoprotein-lipid secretion. Prolonged ethanol administration abolished the zonal asymmetry of the lipogenic process and inverted the acinar distribution of the fatty acid-oxidative process (i.e., in ethanol-fed animals the rate of fatty acid oxidation and the activity of carnitine palmitoyltransferase I were higher in perivenous than in periportal hepatocytes). Moreover, chronic feeding of ethanol led to a marked and selective inhibition of very-low-density-lipoprotein-triacylglycerol secretion by the perivenous zone of the liver. Nevertheless, no zonal differences were observed between control and ethanol-fed animals with respect to the effects of acute doses of ethanol and acetaldehyde on lipid metabolism. In conclusion, our results show that chronic ethanol intake produces important alterations in the acinar distribution of the different fatty acid-metabolizing pathways.     

Chronic ethanol consumption alters the glutathione/glutathione peroxidase-1 system and protein oxidation status in rat liver

BACKGROUND: Alcohol-induced liver damage is associated with oxidative stress, which might be linked to disturbances in liver antioxidant defense mechanisms. The effect of chronic ethanol consumption on the mitochondrial and cytosolic glutathione/glutathione peroxidase-1 (GSHPx-1) system and oxidative modification of proteins was therefore studied in the rat. METHODS: Male Sprague-Dawley rats were fed liquid diets that provided 36% total calories as ethanol for at least 31 days. Pair-fed controls received isocaloric diets with ethanol calories substituted with maltose-dextrins. Mitochondrial and cytosolic fractions were prepared from livers and assayed for GSHPx-1 and glutathione reductase activities and total and oxidized concentrations of glutathione. Catalase activity was measured in the postmitochondrial supernatant. Levels of GSHPx-1, lactate dehydrogenase, and the beta subunit of the F1 portion of the ATP synthase protein were determined by western blot analysis. Concentrations of mitochondrial and cytosolic protein carbonyls were measured to assess ethanol-induced oxidation of proteins. RESULTS: Chronic ethanol consumption significantly decreased cytosolic and mitochondrial GSHPx-1 activities by 40% and 30%, respectively. Levels of GSHPx-1 protein in cytosol were unaffected by ethanol feeding, whereas there was a small decrease in GSHPx-1 protein levels in mitochondria isolated from ethanol-fed rats. Glutathione reductase activities were increased in both intracellular compartments and catalase activity was increased as a consequence of ethanol exposure. Cytosolic total glutathione was mildly decreased, whereas ethanol feeding increased mitochondrial levels of total glutathione. Chronic ethanol feeding significantly increased both cytosolic and mitochondrial concentrations of protein carbonyls by 30% and 60%, respectively. CONCLUSIONS: This study demonstrates that chronic ethanol-induced alterations in the glutathione/GSHPx-1 antioxidant system might promote oxidative modification of liver proteins, namely those of the mitochondrion, which could contribute to the adverse effects of ethanol on the liver.     

Biochemical and morphological alterations of baboon hepatic mitochondria after chronic ethanol consumption

Baboons fed ethanol (50% of total calories) chronically develop ultrastructural alterations of hepatic mitochondria. To determine whether mitochondrial functions are also altered, mitochondria were isolated from nine baboons fed ethanol chronically and their pair-fed controls. At the fatty liver stage, ADP-stimulated respiration was depressed in ethanol-fed baboons by 59.4% with glutamate, 43.2% with acetaldehyde, 45.1% with succinate and 51.1% with ascorbate as substrates. A similar decrease was noted in the ADP/O ratio (14 to 28%) and respiratory control ratio (20 to 44%) with all substrates. Similar alterations of mitochondrial functions were observed in baboons with more advanced stages of liver disease, namely fibrosis. These changes after ethanol treatment were associated with decreases in the enzyme activities of mitochondrial respiratory chain: glutamate, NADH and succinate dehydrogenase (42, 24 and 28%, respectively), glutamate-, NADH- or succinate-cytochrome c reductase (42, 27 and 32%, respectively) and cytochrome oxidase (59.6%). The content of all cytochromes was also decreased in ethanol-fed baboons, especially aa3 (57%). Moreover, [14C]leucine incorporation into mitochondrial membranes was depressed by 21% after ethanol treatment. On the other hand, glutamate dehydrogenase activities of serum and cytosol in ethanol-fed baboons were significantly higher than those in pair-fed controls. Morphologically, mitochondria of ethanol-fed baboons were larger than those of pair-fed controls. However, the mitochondrial protein content per mitochondrial DNA was unchanged. From these results, we conclude that, morphologically and functionally, hepatic mitochondria in baboons are altered by chronic ethanol consumption; it is noteworthy that these changes are fully developed already at the fatty liver stage, and that morphological alteration appears to reflect the damage of mitochondrial membranes rather than an adaptive hypertrophy.     

Long-Term Ethanol Feeding Enhances Susceptibility of the Liver to Orally Administered Lipopolysaccharides in Rats

Background Endotoxin has been implicated in the pathogenesis and progression of alcoholic liver disease. However, it is still unclear how long-term ethanol feeding affects absorption of endotoxin from the intestine and susceptibility of the liver to gut-derived endotoxin. The object of this study was to determine the effect of long-term ethanol feeding on hepatic susceptibility to orally administered endotoxin.
Methods Male Wistar rats that weighed approximately 150 g were pair-fed with an ethanol-containing liquid diet or a control diet for 35 days. In some experiments, 0, 10, or 20 mg/kg of lipopolysaccharides (LPS) was added to the liquid diet for 7 days beginning on day 29. On day 36, the animals were killed for blood biochemistry and histologic examination of the liver. We also determined plasma endotoxin levels after 20 mg/kg of LPS administration using a gastric tube. In another set of experiments, we determined intestinal permeability using FD4 (fluorescein isothiocyanate-labeled dextran with an average molecular weight of 4000 D).
Results With 10 mg/kg of LPS, serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels were significantly increased in the ethanol-fed rats but not in controls. After 20 mg/kg of LPS administration, more substantial increases in serum ALT and ALP levels were observed in ethanol-fed rats as compared with control diet-fed rats. Plasma endotoxin levels in long-term ethanol-fed rats were higher than those in control rats after intragastric administration of high-dose endotoxin (20 mg/kg). Furthermore, intestinal permeability to FD4 was increased by long-term ethanol administration.
Conclusions Long-term ethanol feeding increases intestinal permeability to and absorption of endotoxin, which can sequentially enhance hepatic susceptibility to orally administered endotoxin. This model has potential as a subclinical experimental model for the study of alcoholic liver disease.     

Deposition of Cellular Fibronectin Increases before Stellate Cell Activation in Rat Liver during Ethanol Feeding

Cellular fibronectin (cFN)—a structural extracellular matrix protein—facilitates cell adhesion, migration, and differentiation during organ development; wound healing; tissue regeneration; and fibrogenic processes. cFN is deposited early in various fibrotic diseases and seems to function as a template for deposition of other extracellular matrix proteins, such as collagen type I and laminin, in the injured area. We have compared the relative changes in cFN levels with other pathogenic markers of alcoholic liver injury over time of ethanol feeding in the rat. Male Wistar rats were allowed free access to a liquid diet containing 36% of total energy as ethanol or pair-fed an isocaloric control diet for 4, 8, and 12 weeks. Serum alanine arnino-transferase activity and total liver lipid were increased in ethanol-fed animals, compared with pair-fed controls after 4,8, and 12 weeks of feeding. Liver lipid content was higher in ethanol-fed rats as early as 4 weeks and was further increased by 12 weeks of feeding. Total fibronectin and cFN protein quantity was greater in liver from ethanol-fed rats after 8 and 12 weeks (fibronectin: 2.3-fold and 2.6-fold; cFN: 4.3-fold and 2.6-fold higher than pair-fed at 8 and 12 weeks, respectively). α-Smooth muscle actin, an indicator of hepatic stellate cell activation, was increased in the liver of ethanol-fed rats after 12 weeks of feeding (344% higher compared with pair-fed), with no differences observed at any earlier time points. In summary, increases in hepatic immunoreactive cFN content were observed subsequent to increased liver lipid concentration, but before hepatic stellate cell activation in rats fed the ethanol-based diet. These data suggest that deposition of cFN in the liver during long-term ethanol consumption may represent an early response to injury similar to that observed in other models of liver injury and wound healing.     

Effect of Dietary Ethanol and Cholesterol on Metabolic Functions of Hepatic Mitochondria and Microsomes from the Monkey, Macaca nemestrina

Monkeys (Macaca nemestrina) were divided into four groups, and each group was fed a particular diet. The variables in the diets were as follows: diet A, 0.3 mg cholesterol/kcal nutrient; diet B, 1.0 mg cholesterol/kcal nutrient; diet C, 0.3 mg cholesterol/kcal nutrient, ethanol (36% of calories); diet D, i.O mg cholesterol/ kcal nutrient, ethanol (36% of calories). Monkeys on the diets containing ethanol developed fatty liver. Mitochondria from ethanol-fed animals demonstrated significant decreases in uncoupler-stimulated, state 3, and state 4 succinate oxidation activity; respiratory control ratio; and ATP content. Liver microsomes isolated from the ethanol-fed groups demonstrated increased ethanol oxidizing activity with either NADPH or H2O2 as cosubstrate. Aniline hydroxylase and ami-nopyrine-N-demethylase activities were also elevated in ethanol-fed animals. The alterations in these functional properties were related primarily to ethanol in the diets. Cholesterol, while being less of a perturbant than ethanol, did elicit a significant decrease in cytochrome oxidase activity of mitochondria and a small but statistically significant increase in microsomal-associated ethanol oxidation activity. It appeared to potentiate the effect of ethanol in lowering mitochondrial respiratory control and ATP concentrations.