Mamatha Garige

Quercetin up-regulates paraoxonase 1 gene expression with concomitant protection against LDL oxidation

Paraoxonase 1 (PON1) protects the oxidative modification of low-density lipoprotein (LDL) and is a major anti-atherosclerotic protein component of high-density lipoprotein (HDL). Quercetin, a ubiquitous plant flavonoid, has been shown to have a number of bioactivities and may offer a variety of potential therapeutic uses. We explored the roles of quercetin in the regulation of PON1 expression, serum and liver activity and protective capacity of HDL against LDL oxidation in rats. Compared to the pair-fed control group, feeding quercetin (10 mg/L) in the liquid diet for 4 weeks increased (a) hepatic expression of PON1 by 35% (p<0.01), (b) serum and liver PON1 activities by 29% (p<0.05) and 57% (p<0.01), respectively, and (c) serum homocysteine thiolactonase (HCTL) activity by 23% (p<0.05). Correspondingly, the lag time of low-density lipoprotein (LDL) oxidation was increased by >3-fold (p<0.001) with plasma HDL from quercetin-fed group compared to the HDL from control group. Our data suggest that quercetin has antiatherogenic effect by up regulating PON1 gene expression and its protective capacity against LDL oxidation.

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.

Is alcohol beneficial or harmful for cardioprotection

While the effects of chronic ethanol consumption on liver have been well studied and documented, its effect on the cardiovascular system is bimodal. Thus, moderate drinking in many population studies is related to lower prevalence of coronary artery disease (CAD). In contrast, heavy drinking correlates with higher prevalence of CAD. In several other studies of cardiovascular mortalities, abstainers and heavy drinkers are at higher risk than light or moderate drinkers. The composite of this disparate relation in several population studies of cardiovascular mortality has been a “U-” or “J-”shaped curve. Apart from its ability to eliminate cholesterol from the intima of the arteries by reverse cholesterol transport, another major mechanism by which HDL may have this cardioprotective property is by virtue of the ability of its component enzyme paraoxonase1 (PON1) to inhibit LDL oxidation and/or inactivate OxLDL. Therefore, PON1 plays a central role in the disposal of OxLDL and thus is antiatherogenic. Furthermore, PON1 is a multifunctional antioxidant enzyme that can also detoxify the homocysteine metabolite, homocysteine thiolactone (HTL), which can pathologically cause protein damage by homocysteinylation of the lysine residues, thereby leading to atherosclerosis. We demonstrated that moderate alcohol up regulates liver PON1 gene expression and serum activity, whereas heavy alcohol consumption had the opposite effects in both animal models and in humans. The increase in PON1 activity in light drinkers was not due to preferential distribution of high PON1 genotype in this group. It is well known that wine consumption in several countries shows a remarkable inverse correlation to local rates of CAD mortality. Significantly, apart from its alcohol content, red wine also has polyphenols such as quercetin and resveratrol that are also known to have cardioprotective effects. We have shown that quercetin also up regulates PON1 gene in rats and in human liver cells. The action of quercetin seems to be mediated via the active form of the nuclear lipogenic transcription factor, sterol-regulatory element-binding protein 2 (SREBP2) that is translocated from endoplasmic reticulum to the nucleus. However, the mechanism of action of ethanol-mediated up-regulation of PON1 gene remains to be elucidated. We conclude that both moderate ethanol and quercetin, the two major components of red wine, exhibit cardioprotective properties via the up-regulation of the antiatherogenic gene PON1.

Quercetin and ethanol attenuate the progression of atherosclerotic plaques with concomitant up regulation of paraoxonase1 (PON1) gene expression and PON1 activity in LDLR-/- mice.

BACKGROUND As moderate wine drinking is atheroprotective, it is clinically relevant to elucidate its possible mechanism/s of action/s. Our objective is to demonstrate the potential benefits of the wine components, quercetin and ethanol, on the development of aortic plaques with parallel changes in antiatherogenic factors. METHODS AND RESULTS The effects of quercetin and ethanol on the development of aortic atherosclerotic lesions, liver PON1 gene expression, and serum PON1 activity were measured in LDLR(-/-) mice on an atherogenic diet for 4 and 8 weeks. Depending on the duration and dosage of these modulators, 12.5 to 25 mg/dl quercetin (12.5Q to 25Q) and 18 to 25% ethanol, the magnitude of decreases in aortic lesions caused by moderate ethanol and quercetin ranged from 20 to 70% (p < 0.05 to p < 0.001) based on ultrasound biomicroscopy (UBM) analyses, and from 18 to 61% (p < 0.05 to p < 0.001) based on morphometric analyses. The composite plot of all the UBM and morphometric data showed significant correlation between these 2 methods (p = 0.0001, Pearson r = 0.79 for 4-week treatment; p = 0.000004, Pearson r = 0.84 for 8-week treatment). Concomitantly, 4-week treatments with 12.5Q and 18% ethanol up regulated liver PON1 mRNA by 41% (p < 0.05) and 37% (p < 0.05), respectively, accompanied by 92% (p < 0.001) and 61% (p < 0.001) increases in serum PON1 activity, respectively. The corresponding values after 8-week treatment with 12.5Q and 18% ethanol were 23% (p < 0.05) and 40% (p < 0.02) with respect to the up regulation of liver PON1 mRNA expression, while the stimulations of serum PON1 activity were 75% (p < 0.001) and 90% (p < 0.001), respectively. CONCLUSIONS Based on these findings, we conclude that quercetin and moderate ethanol significantly inhibit the progression of atherosclerosis by up regulating the hepatic expression of the antiatherogenic gene, PON1, with concomitant increased serum 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.

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.

Chronic ethanol consumption down-regulates CMP-NeuAc:GM3 α2,8-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.