Chronic ethanol consumption leads to destabilization of rat liver β-galactoside α2,6-sialyltransferase mRNA

doi:10.1016/S0026-0495(99)90182-8

Metabolism

Volume 48, Issue 6, June 1999, Pages 797-803

https://doi.org/10.1016/S0026-0495(99)90182-8 Get rights and content

Abstract

Chronic ethanol consumption in rats is accompanied by decreased levels of Galβ1,4GlcNAc α2,6-sialyltransferase (2,6-ST) activity in the liver. Our previous studies have shown that there is a concomitant decrease in the levels of 2,6-ST mRNA. In this study, the alteration in the regulation of 2,6-ST expression by chronic ethanol consumption was assessed by Northern hybridization, nuclear run-on experiments, and 2,6-ST mRNA stability studies. 2,6-ST downregulation was found at 4 weeks of feeding an ethanol diet (36% of calories from ethanol) and remained up to 8 weeks. The decrease in 2,6-ST mRNA levels was found to be dose-dependent, with lower dose of ethanol (12% and 24% of total dietary calories from ethanol) being ineffective and the effects being manifested only when 36% of the dietary calories were from ethanol. The effects of chronic ethanol feeding could be completely reversed within 1 week after ethanol consumption was stopped, when 2,6-ST mRNA levels were restored to normal. The downregulation was not sensitive to actinomycin D, indicating that the regulation was not affected at the transcriptional level but at the posttranscriptional level. This was confirmed by nuclear run-on experiments showing that the rate of 2,6-ST mRNA transcription was unaffected by ethanol. Finally, mRNA stability experiments showed that the half-life of 2,6-ST mRNA was reduced 50% in ethanol-fed rat livers compared with control rat livers. Taken together, the results show that 2,6-ST mRNA is regulated at the posttranscriptional level and chronic ethanol intake downregulates 2,6-ST expression by destabilizing its mRNA.

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Alteration of protein glycosylation in liver diseases
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Citation Excerpt :
A decreased level of dolichol has been observed in rats fed ethanol [24]. The abnormal terminal sialylation can be explained by a decrease in β-galactoside α2,6 sialyltransferase (ST6GalI) mRNA and protein expression and/or an increase in hepatocyte membrane associated sialidase observed during chronic alcohol abuse [25–27]. Oxidation products of ethanol such as acetaldehyde interfere with the N-glycan biosynthesis and/or transfer by binding the involved enzymes.
Chronic liver diseases are a serious health problem worldwide. The current gold standard to assess structural liver damage is through a liver biopsy which has several disadvantages. A non-invasive, simple and non-expensive test to diagnose liver pathology would be highly desirable. Protein glycosylation has drawn the attention of many researchers in the search for an objective feature to achieve this goal. Glycosylation is a posttranslational modification of many secreted proteins and it has been known for decades that structural changes in the glycan structures of serum proteins are an indication for liver damage. The aim of this paper is to give an overview of this altered protein glycosylation in different etiologies of liver fibrosis / cirrhosis and hepatocellular carcinoma. Although individual liver diseases have their own specific markers, the same modifications seem to continuously reappear in all liver diseases: hyperfucosylation, increased branching and a bisecting N-acetylglucosamine. Analysis at mRNA and protein level of the corresponding glycosyltransferases confirm their altered status in liver pathology. The last part of this review deals with some recently developed glycomic techniques that could potentially be used in the diagnosis of liver pathology.
Down-regulation of liver Galβ1, 4GlcNAc α2, 6-sialyltransferase gene by ethanol significantly correlates with alcoholic steatosis in humans
2008, Metabolism: Clinical and Experimental
Citation Excerpt :
The key enzyme that is responsible for the terminal sialylation of various glycoproteins is Galβl, 4GlcNAc α2, 6-sialyltransferase (ST6Gal1), which mediates the addition of α2, 6-linked sialic acid to glycoproteins and glycolipids in the Golgi compartment. In our ongoing studies in both rats and humans, we have shown that ST6Gal1 messenger RNA (mRNA) expression is markedly reduced in the long-term alcohol group compared with the control group [13-15]. We have shown that the concomitant decreased hepatic ST6Gal1 activity is due to its decreased synthetic rate [6], whereas the down-regulation of ST6Gal1 mRNA is due to its decreased stability [14].
Hepatic steatosis and steatohepatitis are frequent results of long-term ethanol exposure. We have previously demonstrated that long-term ethanol down-regulates Galβl, 4GlcNAc α2, 6-sialyltransferase (ST6Gal1), leading to defective glycosylation of a number of proteins including apolipoprotein (apo) E and apo J and the appearance of asialoconjugates in the blood of continuously alcohol-fed animals as well as in human alcoholics. In the current study, we have explored the possibility of whether ethanol-induced down-regulation of ST6Gal1 could contribute toward alcoholic steatosis in human alcoholics presumably because of impaired lipid and lipoprotein transport caused by this down-regulation. Real-time quantitative polymerase chain reaction analyses of liver samples from nondrinkers, moderate drinkers, and heavy drinkers as well as from subjects with and without alcoholic liver disease revealed direct evidence that the down-regulation of ST6Gal1 may be due to ethanol per se. The ST6Gal1 messenger RNA level was reduced by as much as 70% in moderate and heavy drinkers as well as in patients with alcoholic liver disease, but was not changed in subjects with liver disease due to causes other than alcohol exposure. Biochemical and histopathologic analysis demonstrated that the liver total cholesterol was increased by more than 30% (P < .05) and 75% (P < .01), respectively, in moderate and heavy drinkers compared with nondrinkers, with even more dramatic changes in triglyceride levels. Significantly, there was a strong inverse correlation between ST6Gal1 messenger RNA level and liver lipid deposit (F = 8.68, P < .001) by statistical analysis. Thus, it is suggested that alcohol-mediated down-regulation of hepatic ST6Gal1 gene leads to defective glycosylation of lipid-carrying apolipoproteins such as apo E and apo J, resulting in defective intracellular lipid and lipoprotein transport, which in turn may contribute to alcoholic steatosis.
Liver Galβ1,4GlcNAc α2,6-sialyltransferase is down-regulated in human alcoholics: possible cause for the appearance of asialoconjugates
2007, Metabolism: Clinical and Experimental
Citation Excerpt :
Accordingly, we have demonstrated that plasma SIJ is significantly decreased by 50% (P < .001) in human long-term alcoholics of both sexes compared with nondrinkers [14]. In our rat alcohol-feeding model, ST6GalI messenger RNA (mRNA) expression is reduced by as much as 59% by long-term alcohol treatment compared with the pair-fed control group in a dose-dependent manner [15,16]. We have shown that the concomitant decreased hepatic ST6GalI activity is due to its decreased synthetic rate [17], whereas the down-regulation of ST6GalI mRNA is due to its decreased stability [16].
Galβl,4GlcNAc α2,6-sialyltransferase (ST6GalI) mediates the glycosylation of proteins and lipids to form functionally important glycoproteins and glycolipids in the Golgi compartment. Our previous work demonstrated that long-term ethanol feeding in rats caused a marked 59% decrease in ST6GalI activity as well as ST6GalI messenger RNA (mRNA) level in the liver that was due to decreased stability of the mRNA. Clinical observations show that down-regulation of ST6GalI gene and consequent impaired activity of ST6GalI seems to be the major cause for the appearance of asialoconjugates in the blood of long-term alcoholics. The plasma carbohydrate-deficient transferrin (CDT) and sialic acid index of plasma apolipoprotein J were also altered in the alcoholic group compared with the nondrinkers. We have now investigated how alcohol affects the gene regulation of ST6GalI and the possible mechanism in postmortem human liver specimens taken from nondrinkers, moderate alcohol drinkers, and heavy alcohol drinkers. Real-time polymerase chain reaction analyses of the liver RNA extract showed that ST6GalI mRNA level was progressively decreased by 49% in moderate drinkers (P < .01) and by 69% in heavy drinkers (P < .01) compared with nondrinkers. Western blot analysis showed that liver ST6GalI protein level was negligibly decreased in moderate drinkers but decreased by 30% (P < .05) in heavy drinkers compared with nondrinkers. We further demonstrated a single ST6GalI mRNA-binding protein complex in the normal human liver extract, which progressively decreased in the liver extracts of moderate and heavy alcohol drinkers. Thus, it is concluded that the appearance of asialoconjugates in alcoholics is possibly due to the down-regulation of ST6GalI gene expression.
Chronic ethanol consumption down-regulates CMP-NeuAc:GM<inf>3</inf> α2,8-sialyltransferase (ST8Sia-1) gene in the rat brain
2006, Neurochemistry International
Alcoholics have an increase in sialic acid-deficient glycoconjugates such as carbohydrate-deficient transferrin, sialic acid-deficient gangliosides and free sialic acids. The elevated presence of these asialoconjugates could be a consequence of alcohol-mediated impaired sialylation rate or due to increased desialylation rate. Chronic ethanol-induced brain abnormalities and behavioral changes could be mediated through these asialogangliosides. We have therefore determined the level of brain CMP-NeuAc:GM₃ α2,8-sialyltransferase (ST8Sia-1) and Gal-β1,3GalNAc α2,3-sialyltransferase (ST3Gal-11) messenger RNA (mRNA) and correlated with the activity of these key enzymes in male Wistar rats as a function of increasing dietary concentration of ethanol after 8 weeks of feeding. The relative level of brain synaptosomal ST8Sia-1 and ST3Gal-11 mRNA were determined by real-time quantitative polymerase chain reaction (RT-PCR). We compared the observed ST8Sia-1 gene expression with its enzymatic activity in the synaptosomal membrane fraction isolated from the rat brain in the ethanol and pair-fed control groups. The results showed that the relative level of brain ST8Sia-1 mRNA expression was down-regulated by 13% (p < 0.05) in 10.6%, by 40% (p < 0.01) in 20.8% and by 57% (p < 0.01) in the 36% ethanol-calorie groups, compared to the control (0% ethanol-calorie) group. In addition, ethanol at 36% dietary calories caused a significant 61% (p < 0.01) decrease in the brain synaptosomal ST8Sia-1 activity compared to the control group. However, ethanol (10.6, 20.8 or 36% level) did not significantly affect the relative level of brain ST3Gal-11 mRNA as compared to the control (0% ethanol-calorie) group. Thus, our findings imply that chronic ethanol exposure preferentially down-regulates brain ST8Sia-1 mRNA accompanied by a concomitant decrease in its activity in a dose-dependent manner. Therefore, the selective loss of 2,8-sialic acid residues from gangliosides might contribute towards the appearance of asialogangliosides and related brain-abnormalities associated with ethanol abuse.
Chronic ethanol consumption upregulates the cytosolic and plasma membrane sialidase genes, but downregulates lysosomal membrane sialidase gene in rat liver
2006, Metabolism: Clinical and Experimental
We have previously shown that chronic ethanol feeding stimulates liver cytosolic sialidase (CS) and plasma membrane sialidase (PMS), whereas it decreases lysosomal membrane sialidase (LMS) activities with concomitant alterations in their relative synthetic rate in rat. To understand the molecular mechanism(s) for these changes, we have evaluated the effect of ethanol administration in male Wistar rats as a function of increasing dietary ethanol concentration after 8 weeks of pair-feeding on (i) the expression of CS, PMS, and LMS genes by real-time quantitative polymerase chain reaction method; (ii) their relative transcription rates by nuclear run-on assay; and (iii) the actual amount of these sialidase proteins in the liver fractions of the respective groups by Western blot method. We have demonstrated that the animals fed with 10.6%, 20.8%, and 36% of total calories as ethanol showed a 20% (P < .05), 34% (P < .01), and 69% (P < .01) increase in CS mRNA level, and 22% (P < .05), 26% (P < .01), and 47% (P < .01) increase in PMS mRNA level, but a decrease in LMS mRNA level by 35% (P < .05), 50% (P < .01), and 80% (P < .01), respectively, as compared to controls. Western blot analyses of CS, PMS, and LMS in the liver subfractions showed that changes in protein levels of CS, LMS, and PMS were consistent with the corresponding changes in the respective mRNA levels. Thus, the upregulation of CS and PMS, but not LMS which is downregulated by chronic ethanol, may account for the appearance of asialoconjugates in alcoholics.
Chronic ethanol feeding controls the activities of various sialidases by regulating their relative synthetic rates in the rat liver
2005, Metabolism: Clinical and Experimental
We have determined the concentration effects of feeding for 8 weeks 10.8%, 21.6%, and 36% dietary ethanol calories on the activities and relative synthetic rates (RSRs) of various subcellular sialidases of rat liver. The hepatic RSRs of each species of sialidase was determined based on the ratio of 1-hour incorporation of [³⁵S]-methionine into immunoprecipitable sialidase as percent of the incorporation into total protein in each subcellular fraction. Ganglioside sialidase activities in the hepatic subcellular fractions were also determined. Ethanol feeding at 36% dietary calories caused an increase in the ganglioside sialidase activity of the plasma membrane sialidase (PMS) by 232% (P < .01) and that of cytosolic sialidase (CS) by 184% (P < .05), but decreased the lysosomal membrane sialidase (LMS) by 54% (P < .01) when compared with the control animals. The specificity of each antisialidase antibody was verified by immunoblots. The RSR of PMS was increased by 40% (P < .01), 67% (P < .01), and 220% (P < .01) in the 10.8%, 21.6%, and 36% ethanol groups, respectively. Similarly, the RSR of CS was increased by 17% (P < .01), 19% (P < .01), and 63% (P < .01), respectively, in these ethanol groups. In contrast, the RSR of LMS was inhibited by 36% (P < .01), 34% (P < .01), and 69% (P < .01), respectively, in these ethanol groups. Intralysosomal sialidase failed to hydrolyze gangliosides. Thus, PMS and CS, but not LMS or intralysosomal sialidase, may play important roles in ethanol-modulated desialylation of gangliosides and consequent liver injury and behavioral alterations.

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^☆: Supported by National Institute for Alcoholism and Alcohol Abuse Grant No. AA08149 (M.R.L.).

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Chronic ethanol consumption leads to destabilization of rat liver β-galactoside α2,6-sialyltransferase mRNA☆

Abstract

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