Chronic administration of diethylnitrosamine to induce hepatocarcinogenesis and to evaluate its synergistic effect with other hepatotoxins in mice.

Hepatocellular carcinoma (HCC) arises after a long period of exposition to etiological factors that might be either independent or collectively contributing. Several rodent models resemble human HCC; however, the major limitation of these models is the lack of chronic injury that reproducibly mimics the molecular alterations as it occurs in humans. Thus, we hypothesized that chronic administration of different DEN treatments identifies the best-fit dose to induce the HCC and/or to determine whether small DEN doses act synergistically with other known hepatotoxins to induce HCC in mice. C57BL/6 J male mice were intraperitoneally injected twice a week for 6 weeks with different DEN doses ranging from 2.5 to 40 mg/kg body weight; then, selected doses (2.5, 5 and 20 mg/kg) for 6, 10, 14, and 18 weeks. We demonstrated that DEN at 20 mg/kg promoted reactive oxygen species and 4-hydroxynonenal production, cell proliferation inflammatory infiltrate, and fibrosis, which in turn induced liver cancer by week 18. These parameters were established by evaluating histopathological changes, HCC markers such as glutathione S-transferase placental-1 (Gstp1), Cytokeratin-19 (Ck19) and prostaglandin reductase-1 (Ptgr1); that of Cyp2e1, a DEN metabolizing enzyme; and the expression of the proliferation marker Ki67. While DEN at 2.5 and 5 mg/kg increased Gstp1 and Ck19, DEN at 20 mg/kg decreased them and Cyp2e1 expression and activity. In summary, our results demonstrate that DEN chronically administrated at 20 mg/kg induces the HCC, while DEN at 2.5 and 5 mg/kg could be useful in elucidating its synergistic effect with other hepatotoxic agents in mice.

Rebaudioside A administration prevents experimental liver fibrosis: an in vivo and in vitro study of the mechanisms of action involved.

Rebaudioside A (Reb A) is a diterpenoid isolated from the leaves of Stevia rebaudiana (Bertoni) that has been shown to possess pharmacological activity, including anti-inflammatory and antioxidant properties. However, the ability of Reb A to prevent liver injury has not been evaluated. Therefore, we aimed to study the potential of Reb A (20 mg/kg; two times daily intraperitoneally) to prevent liver injury induced by thioacetamide (TAA) administration (200 mg/kg; three times per week intraperitoneally). In addition, cocultures were incubated with either lipopolysaccharide or ethanol. Antifibrotic, antioxidant and immunological responses were evaluated. Chronic TAA administration produced considerable liver damage and distorted the liver parenchyma with the presence of prominent thick bands of collagen. In addition, TAA upregulated the expression of α-smooth muscle actin, transforming growth factor-ß1, metalloproteinases 9, 2 and 13, and nuclear factor kappaB and downregulated nuclear erythroid factor 2. Reb A administration prevented all of these changes. In cocultured cells, Reb A prevented the upregulation of genes implicated in fibrotic and inflammatory processes when cells were exposed to ethanol and lipopolysaccharide. Altogether, our results suggest that Reb A prevents liver damage by blocking oxidative processes via upregulation of nuclear erythroid factor 2, exerts immunomodulatory effects by downregulating the nuclear factor-κB system and acts as an antifibrotic agent by maintaining collagen content.

Adverse signaling of scavenger receptor class B1 and PGC1s in alcoholic hepatosteatosis and steatohepatitis and protection by betaine in rat.

Because scavenger receptor class B type 1 is the cholesterol uptake liver receptor, whereas peroxisome proliferator-activated receptor γ coactivator-1ß (PGC-1ß) and PGC-1α are critical for lipid synthesis and degradation, we investigated the roles of these signaling molecules in the actions of ethanol-polyunsaturated fatty acids and betaine on hepatosteatosis and steatohepatitis. Ethanol-polyunsaturated fatty acid treatment caused the following: i) hepatosteatosis, as evidenced by increased liver cholesterol and triglycerides, lipid score, and decreased serum adiponectin; ii) marked inhibition of scavenger receptor class B type 1 glycosylation, its plasma membrane localization, and its hepatic cholesterol uptake function; and iii) moderate steatohepatitis, as evidenced by histopathological characteristics, increased liver tumor necrosis factor α and IL-6, decreased glutathione, and elevated serum alanine aminotransferase. These actions of ethanol involved up-regulated PGC-1ß, sterol regulatory element-binding proteins 1c and 2, acetyl-CoA carboxylase, and HMG-CoA reductase mRNAs/proteins and inactive non-phosphorylated AMP kinase; and down-regulated silence regulator gene 1 and PGC-1α mRNA/proteins and hepatic fatty acid oxidation. Betaine markedly blunted all these actions of ethanol on hepatosteatosis and steatohepatitis. Therefore, we conclude that ethanol-mediated impaired post-translational modification, trafficking, and function of scavenger receptor class B type 1 may account for alcoholic hyperlipidemia. Up-regulation of PGC-1ß and lipid synthetic genes and down-regulation of silence regulator gene 1, PGC-1α, adiponectin, and lipid degradation genes account for alcoholic hepatosteatosis. Induction of proinflammatory cytokines and depletion of endogenous antioxidant, glutathione, account for alcoholic steatohepatitis. We suggest betaine as a potential therapeutic agent because it effectively protects against adverse actions of ethanol.

Mechanisms of action of acetaldehyde in the up-regulation of the human α2(I) collagen gene in hepatic stellate cells: key roles of Ski, SMAD3, SMAD4, and SMAD7.

Alcohol-induced liver fibrosis and eventually cirrhosis is a leading cause of death. Acetaldehyde, the first metabolite of ethanol, up-regulates expression of the human α2(I) collagen gene (COL1A2). Early acetaldehyde-mediated effects involve phosphorylation and nuclear translocation of SMAD3/4-containing complexes that bind to COL1A2 promoter to induce fibrogenesis. We used human and mouse hepatic stellate cells to elucidate the mechanisms whereby acetaldehyde up-regulates COL1A2 by modulating the role of Ski and the expression of SMADs 3, 4, and 7. Acetaldehyde induced up-regulation of COL1A2 by 3.5-fold, with concomitant increases in the mRNA (threefold) and protein (4.2- and 3.5-fold) levels of SMAD3 and SMAD4, respectively. It also caused a 60% decrease in SMAD7 expression. Ski, a member of the Ski/Sno oncogene family, is colocalized in the nucleus with SMAD4. Acetaldehyde induces translocation of Ski and SMAD4 to the cytoplasm, where Ski undergoes proteasomal degradation, as confirmed by the ability of the proteasomal inhibitor lactacystin to blunt up-regulation of acetaldehyde-dependent COL1A2, but not of the nonspecific fibronectin gene (FN1). We conclude that acetaldehyde up-regulates COL1A2 by enhancing expression of the transactivators SMAD3 and SMAD4 while inhibiting the repressor SMAD7, along with promoting Ski translocation from the nucleus to cytoplasm. We speculate that drugs that prevent proteasomal degradation of repressors targeting COL1A2 may have antifibrogenic properties.

CYP2E1, oxidative stress, post-translational modifications and lipid metabolism.

Chronic 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 VLDL assembly and intracellular lipid and lipoprotein transport, which in turn is responsible for alcoholic hepatosteatosis and ALD. The mechanism of ethanol action involves thedepletion of a unique RNA binding protein that specifically interacts with its 3'-UTR region of ST6Gal1 mRNA resulting in its destabilization and consequent appearance of asialoconjugates as alcohol biomarkers. With respect to ETOH effects on Cardio-Vascular Diseases, we conclude that CYP2E1 and ETOH mediated oxidative stress significantly down regulates not only the hepatic PON1 gene expression, but also serum PON1 and HCTLase activities accompanied by depletion of hepatic GSH, the endogenous antioxidant. These results strongly implicate the susceptibility of PON1 to increased ROS production. In contrast, betaine seems to be both hepatoprotective and atheroprotective by reducing hepatosteatosis and restoring not only liver GSH that quenches free radicals, but also the antiatherogenic PON1 gene expression and activity.

TGF-ß1 up-regulates the expression of PDGF-ß receptor mRNA and induces a delayed PI3K-, AKT-, and p70(S6K) -dependent proliferative response in activated hepatic stellate cells.

BACKGROUND: Transforming growth factor beta 1 (TGF-ß1) is a pleiotropic cytokine that activates hepatic stellate cell (HSC) proliferation, but inhibits parenchymal cell proliferation. Therefore, we hypothesize that TGF-ß1 regulates HSC proliferation and elucidated its molecular action. METHODS: In order to elucidate the molecular mechanism whereby TGF-ß1 up-regulates platelet derived growth factor beta (PDGF-ß) receptor mRNA and induces a delayed proliferation of HSC, we used proliferation and apoptosis assays as well as RT-PCR, Western blot analysis, immunostaining, and flow cytometry in mouse and rat HSC. RESULTS: We show that TGF-ß1 markedly induces the proliferation of mouse HSC in culture with concomitant 2.1-fold (p < 0.001) stimulation in [(3) H]-thymidine incorporation into cellular DNA. This induction is maximal between 24 and 36 hours postcytokine exposure that is triggered by 7.6-fold (p < 0.001) up-regulation of PDGF-ß receptor mRNA and associated increase in PDGF-ß receptor protein after 48 hours. TGF-ß1-dependent HSC proliferation is mimicked by H2 O2 that is inhibited by catalase, implying that TGF-ß1 action is mediated via reactive oxygen species. HSC proliferation is blunted by PDGF-ß receptor-neutralizing antibody as well as by specific inhibitors of PI3 kinase (PI3K), AKT, and p70(S6K) , indicating that the action of TGF-ß1 involves the activation of PDGF-ß receptor via the PI3K/AKT/p70(S6K) signaling pathway. TGF-ß1 also induces a reorganization of actin and myosin filaments and cell morphology leading to the formation of palisades although their myosin and actin contents remained constant. These findings suggest that TGF-ß1-mediated oxidative stress causes the transdifferentiation of HSC and primes them for extracellular matrix (ECM) deposition and scar contraction. CONCLUSIONS: We conclude that liver injury up-regulates TGF-ß1 that inhibits parenchymal cell proliferation, but stimulates HSC proliferation leading to the production of ECM and type I collagen resulting in fibrosis.

Flightless-I is a potential biomarker for the early detection of alcoholic liver disease.

Alcoholic liver disease (ALD) is closely linked to oxidative stress induction. Antioxidant enzymes balance oxidative stress and function as intermediary signaling regulators. Nucleoredoxin (NXN), an antioxidant enzyme, regulates physiological processes through redox-sensitive interactions. NXN interacts with myeloid differentiation primary response gene-88 (MYD88) and flightless-I (FLII) to regulate toll-like receptor 4 (TLR4)/MYD88 pathway activation, but FLII also regulates key cell processes and is secreted into the bloodstream. However, the effects of chronic ethanol consumption recapitulated by either ethanol alone or in combination with lipopolysaccharides (LPS), as a two-hit ALD model, on FLII/NXN/MYD88 complex and FLII secretion have not been explored yet. In this study, we have demonstrated that ethanol feeding increased FLII protein levels, its nuclear translocation and plasma secretion, and modified its tissue distribution both in vivo and in vitro ALD models. Ethanol increased MYD88/FLII interaction ratio, and decreased NXN/MYD88 interaction ratio but this was partially reverted by two-hit model. While ethanol and two-hit model increased MYD88/TLR4 interaction ratio, two-hit model significantly decreased FLII nuclear translocation and its plasma secretion. Ethanol and LPS provoked similar effects in vitro; however, NXN overexpression partially reverted these alterations, and ethanol alone increased FLII secretion into culture medium. In summary, by analyzing the response of FLII/NXN/MYD88 complex during ALD early progression both in vivo and in vitro, we have discovered that the effects of chronic ethanol consumption disrupt this complex and identified FLII as a candidate non-invasive plasma biomarker for the early detection of ALD.

Betaine protects chronic alcohol and omega-3 PUFA-mediated down-regulations of PON1 gene, serum PON1 and homocysteine thiolactonase activities with restoration of liver GSH.

BACKGROUND: Paraoxonase (PON1) is an antioxidant enzyme that prevents LDL oxidation as well as detoxifies homocysteine thiolactone (HCTL), both of which can cause atherosclerosis. Chronic alcohol (ETOH) and high omega-3 polyunsaturated fatty acids (omega-3 PUFA) consumption may affect PON1 status presumably via reactive oxygen species by depleting liver glutathione (GSH), whereas betaine may counter their effects. Therefore, we investigated the influence of ETOH, omega-3 PUFA, and betaine on liver GSH, PON1 expression, lipid score, as well as serum PON1 and HCTLase activities. METHODS: Experimental rats belonging to various dietary groups were pair-fed with Lieber-DeCarli low (2.8% the dietary calories as omega3-fatty acids) and high (13.8% the dietary calories as omega3-fatty acids) menhaden fish alcohol-liquid diets with and without betaine (10 g/l diet) for 8 weeks after which liver PON1 mRNA, GSH, lipid score, and serum PON1, HCTLase, and ALT activities were measured. RESULTS: High omega-3 PUFA decreased liver PON1 mRNA expression, serum PON1, and HCTLase activity by 23% (p < 0.01), 20% (p < 0.05), and 28% (p < 0.05), respectively compared to the low omega-3 PUFA group. ETOH decreased PON1 mRNA expression by 25 and 30% (p < 0.01) with concomitant 27% (p < 0.05) and 38% (p < 0.01), decrease in liver GSH levels in low and high omega-3 PUFA groups, respectively. Correspondingly, serum PON1 activity decreased by 23% (p < 0.05) and 58% (p < 0.01) while serum HCTLase activity decreased by 25% (p < 0.05) and 59% (p < 0.01) in the low and high omega-3 PUFA ETOH groups, respectively. Betaine restored liver PON1 mRNA expressions in low and high omega-3 PUFA ETOH groups with parallel restorations of PON1 activity and liver GSH. Concomitantly, betaine reduced hepatosteatosis accompanied by alleviation of liver injury caused by chronic alcohol and high omega-3 PUFA. CONCLUSIONS: Based on these results, we conclude that dietary betaine not only atheroprotective by restoring liver GSH that quenches free radicals, but also may alleviate liver injury by reducing hepatosteatosis.

Nucleoredoxin interaction with flightless-I/actin complex is differentially altered in alcoholic liver disease.

Alcoholic liver disease (ALD) may be attributed to multiple hits driving several alterations. The aim of this work was to determine whether nucleoredoxin (NXN) interacts with flightless-I (FLII)/actin complex and how this ternary complex is altered during ALD progression induced by different ALD models. ALD was recapitulated in C57BL/6J female mice by the well-known ALD Lieber-DeCarli model, and by an in vitro human co-culture system overexpressing NXN. The effects of ethanol and low doses of lipopolysaccharides (LPS) and diethylnitrosamine (DEN) were also evaluated in vivo as a first approach of an ALD multi-hit protocol. We demonstrated that NXN interacts with FLII/actin complex. This complex was differentially altered in ALD in vivo and in vitro, and NXN overexpression partially reverted this alteration. We also showed that ethanol, LPS and DEN synergistically induced liver structural disarrangement, steatosis and inflammatory infiltration accompanied by increased levels of proliferation (Ki67), ethanol metabolism (CYP2E1), hepatocarcinogenesis (GSTP1) and LPS-inducible (MYD88 and TLR4) markers. In summary, we provide evidence showing that NXN/FLII/actin complex is involved in ALD progression and that NXN might be involved in the regulation of FLII/actin-dependent cellular functions. Moreover, we present a promising first approach of a multi-hit protocol to better recapitulate ALD pathogenesis.

Stevioside inhibits experimental fibrosis by down-regulating profibrotic Smad pathways and blocking hepatic stellate cell activation.

Liver cirrhosis is associated with increased morbidity and mortality with important health and social consequences; however, an effective treatment has not been found yet. Previous reports have shown some beneficial effects of stevioside (SVT) in different diseases, but the ability of SVT to inhibit liver cirrhosis has not been reported. Therefore, we studied the potential of this diterpenoid to inhibit liver cirrhosis induced by thioacetamide, a model that shares many similarities with the human disease, and investigated the possible underlying molecular mechanism using in vivo and in vitro approaches. Cirrhosis was induced in male Wistar rats by chronic thioacetamide administration (200 mg/kg) intraperitoneally three times per week. Rats received saline or SVT (20 mg/kg) two times daily intraperitoneally. In addition, co-cultures were incubated with either lipopolysaccharide or ethanol. Liver fibrosis, hepatic stellate cells activation, metalloproteinases activity, canonical and non-canonical Smads pathway and expression of several profibrogenic genes were evaluated. Thioacetamide activated hepatic stellate cells and distorted the liver parenchyma with the presence of abundant thick bands of collagen. In addition, thioacetamide up-regulated the protein expression of α-smooth muscle actin, transforming growth factor-ß1, metalloproteinases-9,-2 and -13 and overstimulate the canonical and non-canonical Smad pathways. SVT administration inhibited all of these changes. In vitro, SVT inhibited the up-regulation of several genes implicated in cirrhosis when cells were exposed to lipopolysaccharides or ethanol. We conclude that SVT inhibited liver damage by blocking hepatic stellate cells activation, down-regulating canonical and non-canonical profibrotic Smad pathways.

Antioxidant and immunomodulatory activity induced by stevioside in liver damage: In vivo, in vitro and in silico assays.

AIMS: Stevioside is a diterpenoid obtained from the leaves of Stevia rebaudiana (Bertoni) that exhibits antioxidant, antifibrotic, antiglycemic and anticancer properties. Therefore, we aimed to study whether stevioside has beneficial effects in liver injury induced by long-term thioacetamide (TAA) administration and investigated the possible underlying molecular mechanism using in vivo, in vitro and in silico approaches. MAIN METHODS: Liver injury was induced in male Wistar rats by TAA administration (200 mg/kg), intraperitoneally, three times per week. Rats received saline or stevioside (20 mg/kg) twice daily intraperitoneally. In addition, cocultures were incubated with either lipopolysaccharide or ethanol. Liver injury, antioxidant and immunological responses were evaluated. KEY FINDINGS: Chronic TAA administration induced significant liver damage. In addition, TAA upregulated the protein expression of nuclear factor (NF)-κB, thus increasing the expression of proinflammatory cytokines and decreasing the antioxidant capacity of the liver through downregulation of nuclear erythroid factor 2 (Nrf2). Notably, stevioside administration prevented all of these changes. In vitro, stevioside prevented the upregulation of several genes implicated in liver inflammation when cocultured cells were incubated with lipopolysaccharide or ethanol. In silico assays using tumor necrosis factor receptor (TNFR)-1 and Toll-like receptor (TLR)-4-MD2 demonstrated that stevioside docks with TNFR1 and TLR4-MD2, thus promoting an antagonistic action against this proinflammatory mediator. SIGNIFICANCE: Collectively, these data suggest that stevioside prevented liver damage through antioxidant activity by upregulating Nrf2 and immunomodulatory activity by blocking NF-κB signaling.

Thymosin ß4 Prevents Oxidative Stress, Inflammation, and Fibrosis in Ethanol- and LPS-Induced Liver Injury in Mice.

Thymosin beta 4 (Tß4), an actin-sequestering protein, is involved in tissue development and regeneration. It prevents inflammation and fibrosis in several tissues. We investigated the role of Tß4 in chronic ethanol- and acute lipopolysaccharide- (LPS-) induced mouse liver injury. C57BL/6 mice were fed 5% ethanol in liquid diet for 4 weeks plus binge ethanol (5 g/kg, gavage) with or without LPS (2 mg/kg, intraperitoneal) for 6 hours. Tß4 (1 mg/kg, intraperitoneal) was administered for 1 week. We demonstrated that Tß4 prevented ethanol- and LPS-mediated increase in liver injury markers as well as changes in liver pathology. It also prevented ethanol- and LPS-mediated increase in oxidative stress by decreasing ROS and lipid peroxidation and increasing the antioxidants, reduced glutathione and manganese-dependent superoxide dismutase. It also prevented the activation of nuclear factor kappa B by blocking the phosphorylation of the inhibitory protein, IκB, thereby prevented proinflammatory cytokine production. Moreover, Tß4 prevented fibrogenesis by suppressing the epigenetic repressor, methyl-CpG-binding protein 2, that coordinately reversed the expression of peroxisome proliferator-activated receptor-γ and downregulated fibrogenic genes, platelet-derived growth factor-ß receptor, α-smooth muscle actin, collagen 1, and fibronectin, resulting in reduced fibrosis. Our data suggest that Tß4 has antioxidant, anti-inflammatory, and antifibrotic potential during alcoholic liver injury.

Ethanol targets nucleoredoxin/dishevelled interactions and stimulates phosphatidylinositol 4-phosphate production in vivo and in vitro.

Nucleoredoxin (NXN) is a redox-regulating protein potentially targeted by reactive oxygen species (ROS). It regulates molecular pathways that participate in several key cellular processes. However, the role of NXN in the alcohol liver disease (ALD) redox regulation has not been fully understood. Here, we investigated the effects of ethanol and ethanol plus lipopolysaccharide, a two-hit liver injury model (Ethanol/LPS), on NXN/dishevelled (DVL) interaction and on DVL-dependent phosphoinositides production both in mouse liver and in a co-culture system consisting of human hepatic stellate cells (HSC) and ethanol metabolizing-VL17A human hepatocyte cells. Ethanol and two-hit model increased Nxn protein and mRNA expression, and 4-hydroxynonenal adducts. Two-hit model promoted Nxn nuclear translocation and Dvl/Phosphatidylinositol 4-kinase type-IIα (Pi4k2a) interaction ratio but surprisingly decreased Dvl protein and mRNA levels and reverted ethanol-induced Nxn/Dvl and Dvl/frizzled (Fzd) interaction ratios. Ethanol resulted in a significant increase of Dvl protein and mRNA expression, and decreased Nxn/Dvl interaction ratio but promoted the interaction of Dvl with Fzd and Pi4k2a; formation of this complex induced phosphatidylinositol 4-phosphate [PI(4)P] production. Ethanol and LPS treatments provoked similar alterations on NXN/DVL interaction and its downstream effect in HSC/VL17A co-culture system. Interestingly, ROS and glutathione levels as well as most of ethanol-induced alterations were modified by NXN overexpression in the co-culture system. In conclusion, two-hit model of ethanol exposure disrupts NXN/DVL homeostatic status to allow DVL/FZD/PI4K2A complex formation and stimulates PI(4)P production. These results provide a new mechanism showing that NXN also participates in the regulation of phosphoinositides production that is altered by ethanol during alcoholic liver disease progression.

Reverse cholesterol transport is regulated by varying fatty acyl chain saturation and sphingomyelin content in reconstituted high-density lipoproteins.

Because phospholipid composition of high-density lipoprotein (HDL) plays a vital role in its reverse cholesterol transport (RCT) function, we studied RCT in vitro (uptake and efflux) with reconstituted HDLs (rHDLs) containing phosphatidylcholine (PC) with fatty acids of increasing saturation levels (stearic, oleic, linoleic, linolenic) and without or with sphingomyelin (SM). Uptake significantly increased from basal value when the PC component included up to 50 mol % of oleic or linolenic acid, but did not change with linoleic acid. Increasing oleic and linoleic acids to 100 mol % significantly decreased uptake, but increasing linolenic acid to the same value did not affect it. Sphingomyelin in rHDL significantly decreased uptake, but only with PC-containing unsaturated fatty acids, and not with saturated fatty acid. Efflux was not affected in a dose-dependent manner when oleic or linoleic acid content was increased, but was significantly increased with levels of linolenic acid up to 25 mol % in PC, and was dramatically lowered with higher levels. Sphingomyelin in rHDL (PC/SM, 20:80, mol/mol) significantly increased efflux only with oleic or linoleic acid-containing rHDLs, compared with efflux without SM. In conclusion, enrichment of PC component up to 25 mol % as linolenic acid has a beneficial effect on RCT, whereas a higher percentage of it or other unsaturated fatty acids seems to be detrimental. In addition, high SM content decreases uptake with rHDL-containing unsaturated fatty acids, whereas it increases efflux for rHDL-containing oleic or linoleic acid. These results show for the first time the importance of SM in RCT in a well-defined in vitro system.

Liver Galbeta1,4GlcNAc alpha2,6-sialyltransferase is down-regulated in human alcoholics: possible cause for the appearance of asialoconjugates.

Galbetal,4GlcNAc alpha2,6-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.

Long-term ethanol consumption impairs reverse cholesterol transport function of high-density lipoproteins by depleting high-density lipoprotein sphingomyelin both in rats and in humans.

Moderate alcohol consumption has been linked to lower incidence of coronary artery disease due to increased plasma high-density lipoprotein (HDL), whereas heavy drinking has the opposite effect. Because of the crucial role of HDL in reverse cholesterol transport and positive correlation of HDL sphingomyelin (SM) content with cholesterol efflux, we have compared HDL SM content with its reverse cholesterol transport capacity both in rats fed ethanol on long-term basis and alcoholic individuals. In rats, SM HDL content was decreased in the ethanol group (-15.4%, P < .01) with a concomitant efflux decrease (-21.0%, P < .01) compared to that in controls. Similarly, HDL from the ethanol group, when compared with HDL from the control group, exhibited 13.8% (P < .05) less cholesterol uptake with control-group hepatocytes and 35.0% (P < .05) less cholesterol uptake with ethanol-group hepatocytes. Conversely, hepatocytes from the ethanol group, when compared with hepatocytes from the control group, exhibited 31.0% (P < .01) less cholesterol uptake with control-group HDL and 48.0% (P < .01) less with ethanol-group HDL. In humans, SM content in plasma HDL was also decreased in chronically alcoholic individuals without liver disease (-51.5%, P < .01) and in chronically alcoholic individuals with liver disease (-51.3%, P < .01), compared with nondrinkers. Concomitantly, in alcoholic individuals without liver disease, both efflux and uptake were decreased by 83.0% and 54.0% (P < .01), respectively, and in chronically alcoholic individuals with liver disease by 84.0% and 61.0% (P < .01), respectively, compared with nondrinkers. Based on these findings, we conclude that long-term ethanol consumption significantly impairs not only cholesterol efflux function of HDL by decreasing its SM content but also cholesterol uptake by affecting presumably hepatocyte receptors for HDL.

Chronic ethanol consumption down-regulates CMP-NeuAc:GM3 alpha 2,8-sialyltransferase (ST8Sia-1) gene in the rat brain.

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:GM(3) alpha2,8-sialyltransferase (ST8Sia-1) and Gal-beta1,3GalNAc alpha2,3-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.

Effects of chronic ethanol administration on the activities and relative synthetic rates of myelin and synaptosomal plasma membrane-associated sialidase in the rat brain.

In an attempt to understand the possible mechanism of chronic ethanol-induced generation of asialoconjugates in the brain and consequent behavioral abnormalities, we have studied the effects of chronic ethanol feeding to rats on the plasma membrane sialidase status in the various subcellular fractions of the brain. We determined sialidase activity using 3H-monosialoganglioside (3H-GM3), 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (4-MU-NeuAC) substrates and Amplex Red (Sialidase) kit. We determined the plasma membrane sialidase protein by Western blot using the anti-plasma membrane sialidase. We also determined its relative synthetic rate (RSR) by the 60 min incorporation of intracranially infused [35S]-methionine (50 microCi/100 g) into immunoprecipitable plasma membrane sialidase. Chronic ethanol administration stimulated the sialidase activity in the total brain homogenate as well as the myelin and synaptosomal membrane fractions, respectively, in all the three experimental models. Chronic ethanol also increased the concentration of the rat brain plasma membrane sialidase protein relative to that of glyceraldehyde-3-phosphate dehydrogenase by 2.4-, 1.62- and 1.51-fold in the total brain homogenate, myelin and synaptosomal membrane fractions, respectively. These increases in plasma membrane sialidase activity and its protein content were due to concomitant increases in their relative synthetic rates by 115% (p < 0.01) and 72% (p < 0.01) in the myelin and synaptosomal membrane fractions, respectively. Thus, our studies clearly show that chronic ethanol induced deglycosylation of brain gangliosides is in part, due to specific up-regulation of plasma membrane sialidase in the myelin and synaptosomal membrane fractions of the brain. This increase in plasma membrane sialidase may be responsible for chronic-ethanol-induced physiological and neurological impairment in the brain, presumably due to deglycosylation of gangliosides that are essential for crucial cellular and metabolic activities.

Chronic ethanol consumption upregulates the cytosolic and plasma membrane sialidase genes, but down regulates lysosomal membrane sialidase gene in rat liver.

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 down regulated by chronic ethanol, may account for the appearance of asialoconjugates in alcoholics.

Inverse correlation of serum paraoxonase and homocysteine thiolactonase activities and antioxidant capacity of high-density lipoprotein with the severity of cardiovascular disease in persons with type 2 diabetes mellitus.

Atherosclerotic risk is increased in diabetes partly because of increased plasma levels of the oxidized low-density lipoprotein and homocysteine, 2 independent and important cardiovascular disease (CVD) risk factors. Paraoxonase (PON) is a multifunctional antioxidant enzyme component of high-density lipoprotein (HDL), which can protect against low-density lipoprotein (LDL) oxidation. It also exhibits homocysteine thiolactonase (HCTL) activity that detoxifies homocysteine thiolactone, which can damage proteins by homocysteinylation of the lysine residues, thus leading to atherosclerosis. We conducted a cross-sectional study to correlate PON-1, HCTL activities, and the lag time of LDL oxidation in 15 healthy control subjects and in 55 subjects with type 2 diabetes mellitus with different degrees of CVD. Compared with healthy controls and diabetic subjects without evidence of overt CVD, we not only found 47% (P < .005) decrease in PON-1 activity, but also for the first time, 30% (P = .019) decrease in HCTL activity in subjects with a prior coronary artery bypass surgery. There was corresponding decreased effectiveness of HDLs from diabetic groups (with and without CVD) in protecting against LDL oxidation. Moreover, the PON-1 activity was significantly inversely correlated to the extent of intracoronary lesions determined at catheterization (ie, a high Gensini score). These decreases in PON-1 and HCTL activity were not due to any bias in preferential distribution of low-activity QQ homozygotes in the diabetic groups compared with the control group because QQ allele was equally distributed in all the experimental groups, whereas RR allele tended to increase in the diabetic subjects with coronary artery bypass surgery compared with the other groups. Therefore, clinical intervention to restore the impaired antiatherogenic activities of HDL should be considered an important goal in the treatment of persons with diabetes.