Philippe Marmillot

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.

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.

Alcohol and molecular regulation of protein glycosylation and function

Chronic alcohol exposure leads to the appearance of carbohydrate-deficient transferrin (CDT), a N-glycosylated protein and sialic acid-deficient apolipoprotein E (apoE), an O-glycosylated protein. We show that chronic ethanol treatment destabilizes sialyltransferase (ST) mRNA resulting in a concomitant decreased steady-state level of ST mRNA. As a result, alcohol markedly decreases the hepatic synthetic rate of ST. This leads to impaired sialylation of transferrin and apoE. Consequently, apoE content in plasma high-density lipoproteins (HDL) is decreased. ApoE plays a significant role in the delivery of HDL cholesterol to the liver via apo B/E receptor, a process called reverse cholesterol transport (RCT). Desialylation of apoE results in its decreased association with HDL. Thus, the dissociation constant of HDL for binding to sialo-apoE is 90 +/- 35 nM, whereas that for desialo-apoE is 1010 +/- 250 nM. More importantly, the uptake of labeled cholesterol by human HepG2 cells is decreased by 30-40% from reconstituted HDL particles (rHDL)-containing desialo-apoE compared to rHDL with sialo-apoE. We conclude that chronic alcohol exposure down-regulates the expression of sialyltransferase genes resulting in impaired sialylation of apoE. This leads to its decreased binding to plasma HDL and thereby, impairs the RCT function of HDL.

Effect of dietary omega-3 fatty acids and chronic ethanol consumption on reverse cholesterol transport in rats.

We previously showed that chronic ethanol feeding leads to a decrease of apolipoprotein E (apoE) in high-density lipoprotein (HDL), whereas supplementing this diet with 2.8% of total dietary calories as omega3-fatty acids (omega3FAs) restores HDL-apoE to the control values. Since HDL containing apoE plays a major role in reverse cholesterol transport (RCT), we measured the effects chronic ethanol intake and omega3-FAs on RCT in the present study. Four groups of rats, control normal fat (CN), alcohol-normal fat (AN), control omega3FA fat (CF), and alcohol-omega3FA fat (AF), were fed their respective diets for 8 weeks, after which hepatocytes and HDLs from each group were evaluated for RCT capacity (cholesterol efflux from macrophages and uptake by liver cells). Compared with the control diet (CN), chronic ethanol (AN) feeding inhibited the cholesterol efflux capacity of HDL by 21% (P < .01), whereas omega3FA feeding (2.8% of total dietary calories) stimulated this capacity by 79% (P < .01) and 25% (P < .01) in CF and AF rats, respectively. With respect to cholesterol uptake by the liver, there were no significant 3-way or 4-way interactions between the 4 factors, HDL-alcohol, HDL-fish oil, hepatocyte-alcohol, and hepatocyte-fish oil. The main effects for HDL-alcohol, HDL-fish oil, and hepatocyte-alcohol were all highly significant (P = .0001, .0001, and .007, respectively). There was a significant HDL-alcohol and HDL-fish oil interaction (P = .0001). Hepatocyte-alcohol was not a factor in any 2-way interactions. Our study indicates no evidence of an interaction between the effects of omega3FAs and the effects of alcohol on hepatocytes in terms of RCT function. Thus, feeding as little as 2.8% of the total dietary calories as omega3FA not only restored the impaired RCT function of HDL caused by chronic ethanol intake, but also enhanced by severalfold the ability of HDL to promote RCT even in normal animals.

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.

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.

Taurocholate stimulates the absorption and biotransformation of beta-carotene in intact and lymph duct-cannulated ferrets

We have determined the influence of dietary taurocholate and beta-carotene on the absorption and biotransformation of newly administered beta-14C]carotene. Male ferrets were fed the control or beta-carotene diet (0.05% beta carotene wt/wt) with and without taurocholate (1% wt/wt) for four weeks, and then the absorption and biotransformation of newly administered beta-[14C]carotene was measured after eight hours in intact or thoracic lymph duct-cannulated animals. Percent recover of beta-[14C]carotene in the liver was increased 3.6-fold (p < 0.05) in the taurocholate-fed ferrets regardless of whether they were fed the control or beta-carotene diet. Percent recovery of labeled vitamin A in the liver was also increased by the same magnitude (p < 0.05). These results were confirmed in thoracic lymph duct-cannulated ferrets. The recoveries of beta-carotene label in the lymph were comparable to the corresponding values in livers of intact animals. The recovery of beta-carotene label in the liver was 50% (p M 0.05) higher in beta-carotene-fed than in control animals. Taurocholate stimulates intestinal absorption of newly administered beta [14C]carotene and its metabolic conversion to 14C-labeled vitamin A (retinol + retinyl ester) 3.6-fold. Beta-Carotene absorption is as efficient in thoracic lymph duct-cannulated ferrets as in intact animals. Prior beta-carotene feeding also stimulates the absorption of newly administered beta-carotene by 50%.