M. Raj Lakshman

Light, but not heavy alcohol drinking, stimulates paraoxonase by upregulating liver mRNA in rats and humans

Paraoxonase 1 (PON) may contribute to the cardioprotective action of high-density lipoprotein (HDL) because it inhibits low-density lipoprotein (LDL) oxidation, a prerequisite for the onset of atherosclerosis. Because light drinking and heavy drinking have diametrically opposite effects on cardioprotection, we have determined the effects of ethanol dosage on rat serum PON activity and its hepatic expression. Furthermore, we have investigated PON activity and polymorphism in human light and heavy drinkers. Our results confirm that HDL-PON inhibited LDL oxidation, destroyed oxidized LDL, and inhibited its uptake by macrophages. Light ethanol feeding caused a 20% to 25% (P <.05) increase in PON activity in both serum and liver and a 59% (P <.001) increase in the level of liver PON mRNA compared with pair-fed control rats. In contrast, heavy ethanol feeding caused a 25% (P <.05) decrease in serum and liver PON activities with a 51% (P <.01) decrease in liver PON mRNA level. Light drinkers had a 395% (P <.001) higher, whereas heavy drinkers had a 45% (P <.001) lower serum PON activity compared with nondrinkers. Significantly, the number of homozygotes versus heterozygotes with respect to high or low activity PON phenotype was similar in all the groups. Therefore, we conclude that light drinking upregulates, whereas heavy drinking downregulates PON activity and its expression, irrespective of its genetic polymorphism.

Desialylation of human apolipoprotein E decreases its binding to human high-density lipoprotein and its ability to deliver esterified cholesterol to the liver

Apolipoprotein E (apoE) plays a significant role in the delivery of high-density lipoprotein (HDL) cholesterol to the liver via the apoB/E receptor. The roles of the apoE sialylation status in its association with HDL and in the reverse cholesterol transport (RCT) function of HDL have not been well defined. Furthermore, long-term ethanol treatment impairs apoE sialylation and leads to its decreased content in HDL. Therefore, we investigated the association of either sialo apoE (SapoE) or desialo apoE (DSapoE) with HDL and its effect on the RCT function of HDL. The dextran sulfate precipitation method showed that [125I]DSapoE binding to HDL was 27.3% (P < .02) to 35.5% (P < .001) lower versus [125I]SapoE. Scatchard analysis of the specific binding data showed that [125I]SapoE had 11.2 times more affinity for HDL than [125I]DSapoE based on size-exclusion chromatography (Kd = 89.7 v 1,010 nmol/L). Similarly, [1251]HDL had 4.5 times more affinity for SapoE compared with DSapoE based on solid-phase binding (Kd = 21.9 v 104.4 nmol/L). Furthermore, esterified cholesterol uptake from reconstituted HDL particles (rHDLs) by HepG2 cells increased over basal uptake up to 153% when rHDLs contained SapoE, versus only 37% with DSapoE. Enzymatic resialylation of DSapoE completely restored its HDL-binding and RCT properties, identical to those of SapoE. It is therefore concluded that desialylation of apoE decreases its binding to plasma HDL, leading to an impaired RCT function.

Quercetin up-regulates paraoxonase 1 gene expression via sterol regulatory element binding protein 2 that translocates from the endoplasmic reticulum to the nucleus where it specifically interacts with sterol responsive element–like sequence in paraoxonase 1 promoter in HuH7 liver cells

We previously showed that quercetin expresses its antiatherogenic effects by up-regulating paraoxonase 1 (PON1) gene and high-density lipoproteins protective capacity against low-density lipoprotein oxidation. In an attempt to elucidate the mechanism of action of quercetin, we have now determined the effects of quercetin on PON1 gene expression, activity, protein level, nuclear mature sterol regulatory element binding protein 2 (SREBP2) level, and its translocation from the endoplasmic reticulum to nucleus and its interaction with PON1 promoter in human HuH7 liver cells using real-time reverse transcriptase polymerase chain reaction, spectrophotometry, immunoblot, confocal microscopy, and electrophoretic mobility shift assay techniques, respectively. Quercetin (20 micromol/L) treatment increased PON1 messenger RNA by 75% (P < .02), with a concomitant 2-fold (P < .05) increase in PON1 activity accompanied by 60% (P < .01) increase in PON1 protein level. There was parallel to the 1.5- to 2.0-fold increase (P < .05) in mature SREBP2 in the cell nuclei that was verified by increased immunolocalization of the mature SREBP2 (65-kd species) in the nuclei of quercetin-treated cells by confocal microscopy. Evaluation of the binding of biotin-labeled sterol responsive element (SRE)-like element of the PON1 promoter to the nuclear extract from the 24-hour quercetin (20 micromol/L)-treated HuH7 cells by electrophoretic mobility shift assay revealed that the SREBP2 specifically binds to the SRE-like element that was abolished by prior incubation with anti-SREBP2 or significantly decreased by 200-fold molar excess of unlabeled SRE-like sequence. Based on these results, we conclude that quercetin exhibits its antiatherogenic property by eliciting the translocation of the mature SREBP2 from endoplasmic reticulum to the nucleus, where it binds to SRE-like sequence in the PON1 promoter and up-regulates PON1 gene transcription and PON1 activity.

Betaine Protects Chronic Alcohol and ω-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.

Chronic ethanol exposure in rats affects rabs-dependent hepatic trafficking of apolipoprotein E and transferrin

Because of the important roles of rabs in protein trafficking, we tested whether chronic ethanol exposure affected the trafficking of newly synthesized apolipoprotein E (apoE) or transferrin (O-glycosylated and N-glycosylated proteins, respectively) attached to acylated or prenylated rabs. The in vivo 30-min incorporation ratios of [3H]palmitate:[35S]methionine or [3H]mevalonate:[35S]methionine (relative ratios of rabs acylation or prenylation to total protein or to immunoisolated apoE or transferrin) were measured in various hepatic subcellular organelles of 8 week-ethanol-fed (E) and pair-fed control (C) Wistar-Furth rats. With respect to total protein trafficking, ethanol increased rabs acylation ratio by 136% (P <.01), 69% (P <.05), and 64% (P <.01) in the endoplasmic reticulum (ER), Golgi light fraction (GLF), and Golgi heavy fraction (GHF), respectively, and decreased this ratio by 76% (P <.01) in carrier vesicle fraction 2 (CV2). With respect to apoE trafficking, ethanol increased rabs acylation ratio by 121% in GHF and decreased this ratio by 27% in CV2. Rabs prenylation ratio increased by 21% and 53% in GHF and CV2, respectively, and decreased by 42% in GLF. With respect to transferrin trafficking, ethanol increased rabs acylation ratio by 53% (P <.01) in GHF, with no significant effect in ER, whereas rabs prenylation ratio increased by 26% (P <.05) in ER, with no significant effect in GHF. Therefore, we conclude that ethanol-induced impaired trafficking of newly synthesized O- and N-glycosylated proteins occurs primarily in ER and Golgi and is due to altered lipidation of rabs, possibly rabs 1, 2, or 6 or combinations of these three rabs.

Characterization of Sialic Acid Index of Plasma Apolipoprotein J and Phosphatidylethanol During Alcohol Detoxification-A Pilot Study.

BACKGROUND Apolipoprotein J (ApoJ) is a component of plasma high-density lipoproteins. Previous studies have shown progressive recovery of ApoJ sialic acid content with increased duration of alcohol abstinence. Therefore, the sialic acid index of plasma apolipoprotein J (SIJ) seems to be a promising alcohol biomarker. Phosphatidylethanol (PEth) is a direct ethanol metabolite and has recently attracted attention as a biomarker of prolonged intake of higher amounts of alcohol. The aim of the pilot study was to explore sensitivity, specificity, and normalization of SIJ and PEth in comparison with traditional and emerging biomarkers. METHODS Five male alcohol-dependent patients (International Classification of Diseases 10, F 10.25) were included (median: 40 years old; Alcohol Use Disorders Identification Test value, 30; alcohol consumption in the previous 7 days, 1,680 g). SIJ, PEth, urinary ethyl glucuronide (UEtG), urinary ethyl sulfate (UEtS), and gamma glutamyl-transpeptidase (GGT) were determined at days 1, 3, 7, 10, 14, 21, and 28. RESULTS At study entry, SIJ, PEth, UEtG, and UEtS were positive in all subjects, whereas GGT and mean corpuscular volume were positive in 3 of 5 (60%) of the subjects. Individual SIJ levels increased between day 1 and 28 between 13.7 and 44.3%, respectively. For SIJ and PEth, the ANOVA (p < 0.005) showed a significant trend with the average subjects SIJ and PEth changing 1.22 and 1.02, respectively, per week. CONCLUSIONS Our preliminary data suggest that SIJ and PEth might hold potential as markers of heavy ethanol intake.

Protective Role of Dietary Curcumin in the Prevention of the Oxidative Stress Induced by Chronic Alcohol with respect to Hepatic Injury and Antiatherogenic Markers

Curcumin, an antioxidant compound found in Asian spices, was evaluated for its protective effects against ethanol-induced hepatosteatosis, liver injury, antiatherogenic markers, and antioxidant status in rats fed with Lieber-deCarli low menhaden (2.7% of total calories from ω-3 polyunsaturated fatty acids (PUFA)) and Lieber-deCarli high menhaden (13.8% of total calories from ω-3 PUFA) alcohol-liquid (5%) diets supplemented with or without curcumin (150 mg/kg/day) for 8 weeks. Treatment with curcumin protected against high ω-3 PUFA and ethanol-induced hepatosteatosis and increase in liver injury markers, alanine aminotransferase, and aspartate aminotransferase. Curcumin upregulated paraoxonase 1 (PON1) mRNA and caused significant increase in serum PON1 and homocysteine thiolactonase activities as compared to high ω-3 PUFA and ethanol group. Moreover, treatment with curcumin protected against ethanol-induced oxidative stress by increasing the antioxidant glutathione and decreasing the lipid peroxidation adduct 4-hydroxynonenal. These results strongly suggest that chronic ethanol in combination with high ω-3 PUFA exacerbated hepatosteatosis and liver injury and adversely decreases antiatherogenic markers due to increased oxidative stress and depletion of glutathione. Curcumin supplementation significantly prevented these deleterious actions of chronic ethanol and high ω-3 PUFA. Therefore, we conclude that curcumin may have therapeutic potential to protect against chronic alcohol-induced liver injury and atherosclerosis.

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-1β (PGC-1β) 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-1β, 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-1β 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.

Down-regulation of liver Galβ1, 4GlcNAc α2, 6-sialyltransferase gene by ethanol significantly correlates with alcoholic steatosis in humans

Hepatic steatosis and steatohepatitis are frequent results of long-term ethanol exposure. We have previously demonstrated that long-term ethanol down-regulates Galbetal, 4GlcNAc alpha2, 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.

Ethanol Destabilizes Liver Galβl, 4GlcNAc α2,6-Sialyltransferase, mRNA by Depleting a 3′-Untranslated Region-Specific Binding Protein

Asialoconjugates are viable biomarkers for alcohol abuse. We previously showed that chronic ethanol feeding down-regulated liver Galβl, 4GlcNAc α2,6-sialyltransferase (ST6Gal l) mRNA by destabilizing it. Since RNA-binding proteins are known to stabilize many eukaryotic mRNAs by interacting with the 3′-untranslated region (UTR), we have delineated the possible mechanism by which ethanol destabilizes ST6Gal l mRNA. Using 32P-labeled RNA probes generated from a 2.7-kb 3′-UTR of ST6Gal l mRNA, we identified a liver cytosolic 41-kDa specific binding protein that interacts with its 3′-UTR domain and protects it from degradation in normal rat liver but disappears after chronic ethanol treatment. Mapping of the binding region revealed that four RNA probes of 80-base pair (bp) length spanning the 304 bp of the 3′-UTR of ST6Gal l mRNA showed equal binding intensity. The corresponding cDNA sequences for the four 80-bp RNA probes share the 13-bp consensus sequence. Mutagenesis analysis identified that four nucleotides, AG and TC, among the consensus sequences were critical for the RNA-protein interaction. Therefore, 5′-CAGCCTCCTCCCT-3′ serves as a cis-element critically involved in this interaction. The RNA-protein complex formation progressively decreased with increasing dietary ethanol, resulting in its virtual disappearance with 36% of the dietary calories as ethanol. Concomitantly, the same ethanol diet decreased sialic acid index of plasma apolipoprotein J by 45% (p < 0.05). Thus, depletion of a binding protein that specifically interacts with its 3′-UTR region of ST6Gal l mRNA may account for its destabilization and consequent appearance of asialoconjugates as alcohol biomarkers.