The adult enhancer factor-1, a Drosophila melanogaster transcriptional repressor, modulates the promoter activity of the rat class-I alcohol dehydrogenase-encoding gene.

Expression of the Drosophila melanogaster alcohol dehydrogenase-encoding gene (ADH) in the adult fat body is controlled by the ADH adult enhancer site (AAE). The D. melanogaster transcription repressor, adult enhancer factor-1 (AEF-1), binds to AAE at a site which overlaps with a sequence recognized by the mammalian transcription factor, CCAAT/enhancer-binding protein alpha [C/EBP alpha; Falb and Maniatis, Genes Dev. 6 (1992a) 454-465]. C/EBP alpha also activates the promoter of the rat class-I ADH gene in a sequence-specific manner [Potter et al., Arch. Biochem. Biophys. 285 (1991a) 246-251]. In this study, we explored the possibility that D. melanogaster AEF-1 influences transcription of the rat class-I ADH. By DNase I footprint analysis, bacterially produced AEF-1 protects a region of DNA between nucleotides (nt) -22 and -36 of the rat class-I ADH promoter (pADH), just 5' to the binding site of C/EBP alpha, a result confirmed by the electrophoretic mobility shift assay (EMSA). Co-transfection of a rat pADH-CAT reporter construct with expression vectors containing C/EBP alpha, AEF-1, or both, indicates that AEF-1 inhibits induction of the rat pADH by C/EBP alpha. Moreover, rat liver nuclear extracts appear to contain AEF-1-like-binding activities to AAE by EMSA. These experiments suggest an evolutionarily conserved mechanism by which AEF-1 modulates expression of the D. melanogaster and rat ADH genes.

Effect of ethanol feeding on bone composition in the rat.

The effect of ethanol feeding for a period of 3 months on the mineral and collagen content of bone was determined in the rat. Ethanol feeding resulted in no changes in the density or in the concentrations of calcium, phosphorus, hydroxyproline, or nitrogen in the tibiae. Also, the serum concentrations of calcium, phosphorus, and 25-hydroxyvitamin D were unaffected by ethanol feeding. The development of a mild degree of osteomolacia was suggested, however, by decreases in EDTA-extractable mineral content and in the calcium/hydroxyproline ratio in the tibiae of the ethanol-fed as compared with the control animals. The urinary excretion of glycosaminoglycans was not changed by ethanol-feeding while the urinary excretion of peptide-bound hydroxyproline was increased. The minimal bone changes found after ethanol feeding in this study are an unlikely cause for the observed increases in the urinary excretion of hydroxyproline.

Changes in plasma amino acids during sobriety in alcoholic patients with and without liver disease.

Plasma amino acid profiles of alcoholic subjects without clinically apparent liver disease, alcoholic patients with biopsy-proven alcoholic hepatitis, and nonalcoholic controls were compared. Differences in the plasma aminograms of alcoholic subjects with and without liver disease appear to be due predominantly to differences in liver function whereas differences in plasma amino acid levels between the alcoholic subjects and nonalcoholic controls may be related to inadequate dietary-protein intake and pyridoxine deficiency in the alcoholic groups.

Endogenous ethanol production and hepatic disease following jejunoileal bypass for morbid obesity.

In this study, we sought to determine whether or not elevated levels of ethanol were present in the systemic circulation, resulting from endogenous ethanol production, which might contribute to the hepatic damage following jejunoileal bypass for morbid obesity. Venous serum samples for assay of ethanol by gas-liquid chromatography were obtained in 8 normal subjects, 9 obese patients prior to surgery, 20 obese patients 2 weeks to 40 months after jejunoileal bypass, and in 2 dogs before and after jejunoileal bypass. Ethanol was detected after jejunoileal bypass in only 7 of the 20 patients and in the 2 dogs. Serum ethanol concentrations ranged from 0.15 to 4.12 mg/100 ml with a mean of 1.18 +/- 1.59 (SD)( mg/100 ml in the 7 patients and ranged from 0.20 to 2.23 mg/100 ml in the dogs. Incubation of the contents of the bypassed intestine of a dog with dextrose resulted in the production of significant amounts of ethanol. However, there was no correlation between the presence of ethanol in the serum and liver histology, when liver biopsy was obtained, postoperatively. Since ethanol was detected in the serum in only small concentrations and in only one-third of the patients, it is unlikely that ethanol production by bacteria in the intestine is of significance in the pathogenesis of liver disease following jejunoileal bypass.

Effect of castration on the turnover of rat liver alcohol dehydrogenase.

Castration increased liver alcohol dehydrogenase activity and enzyme protein in male rats. The turnover of alcohol dehydrogenase determined from the decline in radioactivity present in immunoprecipitated enzyme after injection of NaH14CO3 was decreased after castration. The fractional rate of degradation (Kd) for the enzyme was 0.11 . day-1 in the castrated as compared with 0.13 . day-1 in the control animals (P less than 0.05). The fractional rate of synthesis (Ks) of the enzyme was not affected by castration, while the absolute rate of synthesis was increased slightly. This study shows that a decrease in the rate of degradation is the principal cause for the increase in liver alcohol dehydrogenase following castration.

Depression of alcohol dehydrogenase activity in rat hepatocyte culture by dihydrotestosterone.

Hepatocytes harvested from castrated rats retained a higher alcohol dehydrogenase (EC 1.1.1.1) activity than hepatocytes harvested from normal rats during 7 days of culture. Dihydrotestosterone (1 microM) decreased the enzyme activity, after 2 and 5 days of culture, in hepatocytes from castrated and control animals respectively. Dihydrotestosterone decreased the enzyme activity to similar values in both groups of hepatocytes by the end of 7 days of culture. Testosterone (1 microM) had no effect on the enzyme activity in normal hepatocytes and only a transitory effect in decreasing the enzyme activity in hepatocytes from castrated animals. The increases in alcohol dehydrogenase activity after castration and their suppression by dihydrotestosterone were associated with parallel changes in the rate of ethanol elimination. Additions of substrates of the malate-aspartate shuttle or dinitrophenol did not modify ethanol elimination. These observations indicate that dihydrotestosterone has a direct suppressant effect on hepatocyte alcohol dehydrogenase and that the enzyme activity is a major determinant of the rate of ethanol elimination.

Characterization of human erythrocyte aldehyde dehydrogenase.

Human erythrocyte aldehyde dehydrogenase was purified to homogeneity. The enzyme exhibited a single band of activity on starch gel electrophoresis and on isoelectric focusing. It was a tetramer with an estimated molecular weight of 230,000 daltons and an isoelectric point of 5.0. Its pH optimum of 8.5, Michaelis-Menten constant for acetaldehyde of 46 microM, and high sensitivity to noncompetitive inhibition by disulfiram resembled human liver cytosolic aldehyde dehydrogenase. Low concentrations of magnesium (5-10 microM) resulted in enhancement of erythrocyte aldehyde dehydrogenase activity, whereas higher physiological concentrations of magnesium resulted in uncompetitive inhibition of enzyme activity. Magnesium inhibited the enzyme activity by increasing the binding of NADH to the enzyme as had been found to be the case for the inhibitory effect of magnesium on the human liver cytosolic enzyme. Erythrocyte aldehyde dehydrogenase may metabolize small amounts of acetaldehyde escaping the liver during ethanol metabolism and protect extrahepatic tissues from acetaldehyde toxicity.

Influence of epinephrine on alcohol dehydrogenase activity in rat hepatocyte culture.

The effects of epinephrine on alcohol dehydrogenase activity and on rates of ethanol elimination were determined in rat hepatocyte culture. Continuous exposure of the hepatocytes to epinephrine (10 microM) in combination with dexamethasone (0.1 microM) enhanced alcohol dehydrogenase activity on days 4-7 of culture, whereas neither hormone alone had an effect. The increased alcohol dehydrogenase activity was associated with an increased rate of ethanol elimination. Acute addition of 10 microM epinephrine to hepatocytes maintained in culture with 0.1 microM dexamethasone did not change alcohol dehydrogenase activity, but resulted in an immediate marked, but transitory, increase in ethanol elimination within the first 5 min after the addition of the hormone. Prazosin, an alpha 1-adrenergic blocker, and antimycin, an inhibitor of mitochondrial respiration, were powerful inhibitors of the transient increase in ethanol elimination, whereas 4-methylpyrazole was only partially inhibitory. These observations indicate that epinephrine has a chronic effect in increasing alcohol dehydrogenase activity and ethanol elimination and, also, an acute transient effect of increasing ethanol elimination which is not limited by alcohol dehydrogenase activity.

Effect of epinephrine on ethanol metabolism by isolated rat hepatocytes.

The effect of epinephrine on ethanol metabolism was determined in isolated rat hepatocytes. Epinephrine (10 microM) enhanced an initial rapid rate of ethanol elimination observed in the first 5 min. Thereafter, between 5 and 90 min, the rate of ethanol elimination was slower and not affected by epinephrine. Epinephrine resulted in higher acetaldehyde concentrations at 2 min, but not thereafter. Acetaldehyde production in the presence and absence of epinephrine was inhibited by 4-methylpyrazole, by a low free extracellular calcium concentration, and by the alpha 1-adrenergic blocker prazosin. Ethanol alone and epinephrine alone increased oxygen consumption, but the effects were not additive. The ethanol-induced decreases in the cytosolic NAD-/NADH and NADP++NADPH ratios and in the mitochondrial NAD+/NADH ratio were delayed by the presence of epinephrine. An accelerated initial alcohol dehydrogenase activity sufficient to account for the rapid initial rate of ethanol elimination shown with epinephrine was demonstrated by coupling ethanol oxidation with lactaldehyde reduction, a system which increases the rate of dissociation of NADH from the enzyme and its oxidation back to NAD+. The findings in this study indicate that an increased reoxidation of NADH during ethanol oxidation by alcohol dehydrogenase is the basis for the rapid transient increase in ethanol elimination produced by epinephrine.

Identification of new sites of binding and activation of the murine alpha1(I) collagen promoter by CCAAT/enhancer binding protein beta.

The CCAAT/enhancer binding protein beta (C/EBPbeta) was previously shown to bind to the alpha(1)(I) collagen promoter at -365 to -335 (site 1) and to activate it. Acetaldehyde also activates the promoter, and this effect is mediated by an increase in stellate-cell C/EBPbeta protein and C/EBPbeta binding. The present study identified two additional distal sites (sites 2 and 3) of binding of C/EBPbeta, in the nuclear extracts of stellate cells, at -399 to -370 and -623 to -592 in the alpha(1)(I) collagen promoter. The C/EBPbeta protein activates the promoter at all three sites. Acetaldehyde increases C/EBPbeta binding to all three sites. Activation by acetaldehyde is abrogated in the transfected promoter mutated at either site 1 or site 3 but is not affected by mutation at site 2. Binding of the 20-kDa C/EBPbeta isoform (p20C/EBPbeta), which is eliminated by mutation at the distal site 3 of C/EBP binding, is necessary for the activation by acetaldehyde of the alpha(1)(I) collagen promoter.

Hepatic collagen proline hydroxylase activity in alcoholic liver disease.

The activity of hepatic collagen proline hydroxylase was examined in biopsy samples as a factor in collagen synthesis in 77 patients with alcoholic liver disease. The urinary excretion of peptide bound hydroxyproline was also measured in most of the patients, as an index of collagen degradation. The highest activities of collagen proline hydroxylase were found in the patients with alcoholic hepatitis. Enzyme activity was markedly increased in patients with non-specific changes on liver biopsy, whereas, patients with fatty infiltration had only mild elevations, and those with inactive cirrhosis had normal enzyme activity. Urinary hydroxyproline was elevated only in patients with alcoholic hepatitis and inactive cirrhosis. Follow-up determinations in 16 patients with alcoholic hepatitis, after 4 to 5 weeks, revealed a decrease in enzyme activity, but no change in urinary hydroxyproline. We conclude that among the types of alcohol-related liver diseases, alcoholic hepatitis is associated with the greatest turnover of hepatic collagen.

Effect of testosterone administration on rates of ethanol elimination in hypogonadal patients.

Rates of ethanol elimination were determined in four hypogonadal subjects at one week and again at eight weeks after the administration of one dose of 200 mg of testosterone cypionate (Depo-testosterone). Ethanol elimination was unchanged in two patients, slightly decreased in one, and markedly increased in one patient at eight weeks as compared to one week after testosterone administration. In the three patients with little or no change in ethanol elimination, initial high levels of plasma-free testosterone, ranging from 445.0 to 3.8 ng/dl did not decrease to abnormally low levels, but ranged between 1.6 and 7.7 ng/dl (normal, 1.20-2.10 ng/dl). In the fourth patient, an increase in ethanol elimination from 86.6 to 107.4 mg/kg body weight/hr was associated with a decrease in plasma-free testosterone from a high level of 4.7 to 0.8 ng/dl. These results indicate that pharmacological plasma concentration of testosterone do not affect the rate of ethanol elimination. A suppressing effect of testosterone on rate of ethanol elimination may occur at levels of plasma-free testosterone which extend from abnormally low up into the normal physiologic range.

Ethanol increases ADP-ribosylation of histones in rat hepatocyte nuclei.

Ethanol was shown previously to increase ADP-ribosylation of hepatocyte proteins. The purpose of this study was to determine the effect of ethanol on ADP-ribosylation of histones in hepatocyte nuclei. Freshly isolated hepatocytes were exposed to 100 mM ethanol for 2 h and ADP-ribosylated histones were separated from nonribosylated histones by phenylboronate agarose chromatography. Both histone factions were then separated into the individual histones by 12% acetic-urea-triton polyacrylamide gel electrophoresis. Ethanol did not change the amounts of outer histone H1 or amounts of core histones (H2A, H2B, H3.1, and H4) but increased the histone variants H3.2 and H3.3. The principal effect of ethanol was to increase the ADP-ribosylation of all the above histones. Exposure of hepatocytes in culture to 100 mM ethanol for 3 days did not increase the synthesis of histones as determined by the incorporation of 14C-L-lysine, but increased the ADP-ribosylation of histones, principally histone H2A, determined from the incorporation of 2, 8, 3H adenosine. These results show that ethanol increases the ADP-ribosylation of histones. This is a potential mechanism for effects of ethanol on the regulation of gene expression and cell differentiation.

Meta-analysis: vitamin D and non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent condition. Emerging evidence suggests that vitamin D may play a role in the pathogenesis of NAFLD. AIM: To review systematically the association between vitamin D levels, measured as serum 25-hydroxy vitamin D [25(OH)D], and NAFLD. METHODS: We used PubMed and EMBASE databases to identify all studies that assessed the association between vitamin D and NAFLD up until 22 April 2013, without language restrictions. We included studies that compared vitamin D levels between NAFLD cases and controls and also those that compared the odds of vitamin D deficiency by NAFLD status. Pooled standardised differences and odds ratios were calculated using an inverse variance method. RESULTS: Seventeen cross-sectional and case-control studies have evaluated the association between vitamin D and NAFLD. NAFLD was diagnosed using biopsy (4 studies), ultrasound or CT (10 studies) and liver enzymes (3 studies). Nine studies provided data for a quantitative meta-analysis. Compared to controls, NAFLD patients had 0.36 ng/mL (95% CI: 0.32, 0.40 ng/mL) lower levels of 25(OH)D and were 1.26 times more likely to be vitamin D deficient (OR 1.26, 95% CI: 1.17, 1.35). CONCLUSIONS: NAFLD patients have decreased serum 25(OH)D concentrations, suggesting that vitamin D may play a role in the development of NAFLD. The directionality of this association cannot be determined from cross-sectional studies. Demonstration of a causal role of hypovitaminosis D in NAFLD development in future studies could have important therapeutic implications.