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Effects of chronic alcohol treatment on the synthesis, sialylation, and disposition of nascent apolipoprotein E by peritoneal macrophages of rats^1,2,3

¹ From the Department of Surgery, The Johns Hopkins University School of Medicine, Baltimore; the Department of Medicine, George Washington University Medical Center, Washington, DC; and the Department of Veterans Affairs Medical Center, Washington, DC.

² Supported by NIH grant AA08149 and by grant BP-PG/95/1/111 from BioProbes, Inc, Gaithersburg, MD. Financial assistance to JS was provided by John Harmon, Department of Surgery, Veterans Administration Medical Center, Washington, DC.

³ Address reprint requests to MR Lakshman, DVA Medical Center (151T), Lipid Research Laboratory, 50 Irving Street, NW, Washington, DC 20422. E-mail: rlax{at}erols.com.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Background: Plasma apolipoprotein (apo) E, a sialoprotein, plays an important role in reverse cholesterol transport. Previously, we showed that chronic alcohol consumption impairs glycosylation of apo E in rat liver. Peritoneal macrophages are another significant apo E synthesis site.

Objective: The main purpose of this study was to determine the effects of chronic alcohol feeding of rats on the synthesis, sialylation, and sialic acid content of macrophage apo E and its ability to bind to the HDL₃ molecule in vitro.

Design: Rats were fed an alcoholic diet or an isoenergetic control diet for 8 wk, after which peritoneal macrophages isolated from them were cultured and analyzed for apo E metabolism.

Results: Macrophages from alcohol-fed rats accumulated 33.3% more (P < 0.05) cholesterol than did those from control rats when incubated with acetylated LDL. These macrophages showed a 51–57% lower relative sialylation rate of apo E (P < 0.001) but no significant difference in relative protein synthetic rate. The sialic acid content of the intracellular and secreted forms of apo E was reduced by 41.8% (P < 0.001) and 50.3% (P < 0.001), respectively, with chronic alcohol treatment. Secretion of newly synthesized apo E was impaired by 53.7% (P < 0.001) and 26.1% (P < 0.001) in the absence and presence of HDL in the medium, respectively. Macrophages of alcohol-treated rats secreted apo E with 47.6–67.2% lower (P < 0.001) HDL₃ binding ability; binding ability was restored completely by resialylation of the desialylated apo E.

Conclusion: In rats, an alcohol-mediated decrease in sialylation rate resulting in loss of sialic acid residues in apo E impairs the ability of apo E to bind to HDL and consequently in defective reverse cholesterol transport.

Key Words: Chronic ethanol feeding • macrophages • apolipoprotein E • glycosylation • sialylation • sialic acid content • high-density lipoproteins • HDL • reverse cholesterol transport • binding • atherosclerosis • rats • alcohol • coronary heart disease

	INTRODUCTION

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The influence of alcohol on development of coronary heart disease (CHD) is complex and may be beneficial or harmful depending on the amount of alcohol consumed. The benefits and risks of moderate alcohol consumption in relation to the development of CHD have been reviewed (1). Thus, compared with abstinence, moderate alcohol intake may be beneficial, whereas heavy intake is harmful. The risks of coronary events as a result of heavy drinking have been shown (2).

Epidemiologic evidence has established the protective role of HDLs against atherosclerosis. Plasma HDL-cholesterol concentration is inversely correlated with the incidence of CHD (3). Changes in plasma lipids and lipoproteins associated with alcohol-induced toxicity that result in altered synthesis and metabolism of lipoproteins both in liver and peripheral tissues were reviewed earlier (4, 5).

Plasma apolipoprotein (apo) E is a component of several plasma lipoproteins, including chylomicrons, VLDLs, intermediate-density lipoproteins, and subfractions of LDL and HDL. Basu et al (6) established the role of apo E in the reverse cholesterol transport process. Furthermore, they showed that cholesterol-loaded mouse macrophages and human monocytes are capable of synthesizing and secreting apo E. Plasma apo E is an O-type sialoglycoprotein that has different isoforms depending on the presence of different amounts of sialic acid on its carbohydrate chain (7). Sialic acid residues in apo E, synthesized and secreted by macrophages, may be responsible for the structural conformity and stability of apo E, a prerequisite for the attachment of apo E to phospholipids to form lamellar discs for secretion, as well as recognition of apo E by HDL₃.

We showed earlier that chronic alcohol intake impairs both glycosylation and secretion of apo E from the liver in rats (8). Furthermore, we identified specific glycosylation sites in rat liver that are susceptible to attack by alcohol (5). We also showed that although the total plasma apo E concentration did not change with chronic alcohol intake, the proportion of apo E associated with HDL decreased with a concomitant increase in VLDL-associated apo E (9). Because macrophages are another source of plasma apo E, it was pertinent to determine whether the synthesis and glycosylation of apo E was also affected in this tissue and to test whether the association of apo E with plasma HDL₃ was altered as a result of chronic alcohol exposure. Therefore, we pursued studies that explored whether chronic alcohol exposure has any influence on 1) the synthesis, sialylation, and secretion of apo E by macrophages; 2) any alterations in the sialic acid content of intracellular and secreted apo E synthesized by macrophages; and 3) any impairment in the functional ability of secreted apo E to integrate within the HDL₃ molecule.

	MATERIALS AND METHODS

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Materials
L-[U-¹⁴C]Leucine (specific activity: 11.1 MBq/mmol, or 300 mCi/mmol) and [6-³H]N-acetyl-D-mannosamine (ManNAc; specific activity: 262.7 GBq/mmol, or 7.1 Ci/mmol) were purchased from American Radiolabeled Chemicals, Inc (St Louis). Antiserum to rat apo E was developed in New Zealand white rabbits in our laboratory and was proven to be monospecific, as evidenced by Ouchterlony analysis (10). CNBr-activated Sepharose 4B used as a matrix for the preparation of an anti-apo E affinity column was purchased from Pharmacia (Uppsala, Sweden). Other chemicals, including authentic standards, were from Sigma Chemical Co (St Louis). Dulbecco Minimum Essential Medium (DMEM, henceforth referred to as the medium) for cell culture was purchased from Gibco BRL (Rockville, MD). Hydrofluor scintillation fluid was purchased from National Diagnostics (Manville, NJ). Organic solvents were purchased from Fisher Scientific (Columbia, MD) and were of analytic grade. All separation and purification columns were purchased from Amersham Pharmacia Biotech, Inc (Piscataway, NJ).

Experimental animals
The use of animals involved in this study was approved by the Institutional Animal Care and Use Committee of the Veterans Administration Medical Center, Washington, DC. Weanling, male Wistar rats (Charles River, Wilmington, MA) were maintained with a standard, unpurified diet until they reached a body weight of 150 g. They were then divided into 2 groups and pair fed the control or alcohol diet for 8 wk. The diets were isoenergetic and were formulated according to the modified method reported by Lieber and DeCarli (11). Accordingly, 40% of the total energy of the alcohol diet was from fat, 20% from protein, 36% from alcohol, and 4% from carbohydrate. The control diet had an equal amount of dextrimaltose in place of alcohol.

Cell culture
Rat peritoneal macrophages were collected from animals of the control and alcohol groups after an intraperitoneal injection of 5.0 mL sterile saline solution. All subsequent procedures were carried out under sterile conditions, as described previously (12). Briefly, cells were plated and incubated at 37°C in a humidified incubator in an atmosphere of 5% CO₂ until they reached 90% confluency (6 x 10⁶ adherent cells/dish in 72 h; nonadherent cells were washed away). All subsequent experiments were carried out with these cultures of confluent cells.

Cholesterol loading of macrophages
The cells in culture were loaded with cholesterol by an overnight incubation with acetylated rat LDL + VLDL fraction (henceforth referred to as the LDL fraction) at 100 mg/L in medium containing 10% fetal bovine serum (FBS). All experimental incubations were carried out at 37°C in a humidified atmosphere containing 5% CO₂.

Experiment 1: effects of chronic alcohol intake on apo E synthesis and glycosylation
The cholesterol-loaded rat peritoneal macrophages were incubated in the absence (nonstimulated group) and presence (stimulated group) of rat HDL₃ in leucine-free medium without FBS. L-[U-¹⁴C]Leucine (74 GBq, or 2 µCi/dish), [6-³H]ManNAc (37 GBq, or 1 µCi/dish), and unlabeled L-leucine (25 µmol/L) were added to a total of 5 mL medium added to nonstimulated or stimulated rat peritoneal macrophages (6 x 10⁶ adherent cells) from the control and alcohol groups. The culture dishes were placed in a shaker (on low) inside an incubator for 60 min. The reaction contents were then immediately transferred into tubes and centrifuged at 500 x g for 5 min, and the medium was discarded. The cells were resuspended and washed 3 times with 10 mmol phosphate-buffered saline (PBS)/L and processed for subcellular fractionation. A 3% (wt:vol) homogenate of macrophage cells was made in sodium phosphate buffer (200 mmol/L, pH 7.0, containing 10 mmol EDTA/L, 1% trasylol, and 1% leupeptin) with 500 mmol sucrose/L. A known amount of homogenate was processed for the isolation of microsomes and a Golgi apparatus–rich fraction (henceforth referred to as the Golgi fraction) as described previously (6, 9). The whole cell homogenate, microsomes, and the Golgi fractions were then analyzed for incorporation of labeled leucine and labeled ManNAc into immunoprecipitable apo E according to our published method (6). The total protein content of the fractions was determined by using the method of Lowry et al (13).

Experiment 2: effects of chronic alcohol intake on kinetics of apo E secretion
Four sets each of peritoneal macrophage cultures (6 x 10⁶ adherent cells) from rats of the control and alcohol groups, overloaded with cholesterol and maintained on nonstimulated and stimulated media in a total volume of 5 mL as described above, were incubated for 1 h with L-[U-¹⁴C]leucine (74 GBq) and unlabeled L-leucine (25 µmol/L) in a total of 5 mL. The medium was completely removed and replaced with fresh medium with and without HDL₃ but containing no FBS, and the incubations were continued. At the end of 1.5, 3.0, 4.5, and 6 h, the incubation medium from one set each of the nonstimulated and stimulated control and alcohol groups was transferred into ice-cold tubes and centrifuged at 500 x g for 5 min at 4°C. After centrifugation, the cells were washed 3 times with ice-cold 10 mmol PBS/L. The medium collected from each reaction sample was analyzed individually for incorporation of labeled leucine into immunoprecipitable apo E according to our published method (6). A known volume of the pooled medium (secreted apo E fraction) and the whole cell homogenate (3%, wt:vol, intracellular apo E fraction) was passed through an anti-rat apo E Sepharose 4B column to purify apo E as described below. The authenticity of the eluted pure apo E from the column was checked by Ouchterlony analysis. Pure apo E fractions, obtained thus from the intracellular and secretory fractions of the control and alcohol groups, were processed for enzymatic hydrolysis by using exo--sialidase (neuraminidase; EC 3.2.1.18; 14). Finally, the sialic acid content of the apo E fractions was determined according to the published protocol of Warren (15).

Experiment 3: effects of chronic alcohol intake on the binding characteristics of secreted apo E to HDL₃ in vitro
Five hundred micrograms each of the pure apo E fractions obtained from the secreted medium of the control and alcohol-treated groups (experiment 2) was delipidated (chloroform: methanol, 1:1, by vol) and processed for complete desialylation by using neuraminidase as described below. Additionally, known amounts of the purified, intracellular and secreted apo E fractions from the control and alcohol-treated groups (500 µg) were processed for the resialylation reaction by using ß-galactosamide -2,6-sialyltransferase (sialyltransferase; EC 2.4.99.1) as described below. The sialic acid content was determined in these apo E fractions after enzymatic hydrolysis. Furthermore, the unmodified, desialylated, and resialylated apo E fractions of the control and alcohol-treated groups were labeled with ¹²⁵I according to the method of Bolton and Hunter (16). Thus, the labeled apo E (146200 cpm/mg protein) of the control compared with the alcohol-treated rats was found to have molar ratios of sialic acid to protein of 0/0, 0.96/1.93, and 1.90/1.90, respectively, in its deglycosylated, unmodified, and reglycosylated form. The purity and authenticity of labeled unmodified, desialylated, and resialylated apo E were assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Ouchterlony analysis and subjected to binding studies.

Desialylation and resialylation of secreted apo E
The enzymes used in these reactions have repeatedly shown no proteolytic activity. Both the desialylation and resialylation incubations were carried out at 37°C for 4 h in 0.5 mL of 100 mmol cacodylate buffer/L (pH 6.0) containing 500 µg apo E protein and 10 µg of either neuraminidase (desialylation reaction) or sialyltransferase (resialylation reaction). After the reaction, the content was passed through an anti-apo E affinity column to recover pure apo E. The purity of apo E was checked by SDS-PAGE. Aliquots of pure apo E were processed for protein analysis and determination of sialic acid in all fractions.

Preparation of the apo E–lipid complex
For the preparation of the apo E–lipid complex, the published protocol of Milla and Hirschberg (17) was followed, with slight modifications. Briefly, egg yolk phosphatidylcholine (1500 µmol; Sigma Chemical Co) and cholesterol (500 µmol; Sigma Chemical Co) in hexane were dried on the walls of a 10-mL glass ampoule with nitrogen gas and lyophilized overnight. The dried lipid was resuspended in 1 mL dimethylsulfoxide to obtain a clear solution. Dialyzed samples of unmodified, desialylated, and resialylated labeled apo E (200 µmol in 0.1 mL) in 200 mmol phosphate buffer/L, pH 7.4, isolated from the secreted medium of the alcohol and control macrophages, were individually mixed with lipids in dimethylsulfoxide by gentle stirring. The mixtures were then quickly frozen in a dry ice–acetone bath and allowed to thaw at room temperature (15 min). The cycle was repeated 5 times. After the fifth thawing, the mixtures were sonicated for 10 s, applied to a Sephadex G-15 column, and eluted with phosphate buffer (20 mmol/L, pH 7.4). Fractions of 2.0 mL were collected. An aliquot of the fractionated apo E–lipid complex was characterized by SDS-PAGE and its protein, sialic acid, and lipid contents were determined. The apo E–lipid complex fraction was eluted in the void volume and was used for binding studies.

Experiment 1: binding of labeled apo E–lipid complex to HDL₃
Rat plasma HDL₃ fractions were isolated from control rats and characterized for their protein and lipid contents. Triplicate samples of unmodified, desialylated, and resialylated labeled apo E–lipid complex (200 µg protein) isolated from the control and alcohol-treated groups were added individually to 5 sets of HDL₃ fractions (500 µg protein) and incubated at 37°C. The reaction was carried out for 15, 30, 60, 90, and 120 min. The reaction mixture was subjected to dextran sulfate precipitation for HDL₂ isolation according to the method described by Gavish et al (18). The isolated HDL₂ precipitate was washed twice with saline solution and dissolved in sodium citrate buffer (200 mmol/L, pH 8.3). An aliquot was taken for SDS-PAGE and lipid analysis to verify the purity and authenticity of the HDL₂ subfraction. The solution containing HDL₂ was dialyzed against saline solution for 24 h and reprecipitated with dextran sulfate. The precipitate was vacuum-dried and the radioactivity of bound [¹²⁵I]apo E in HDL was determined in an AutoGamma 5000 series spectrometer (Packard Instruments, Downers Grove, IL). The results are expressed as nmol bound apo E/µmol HDL.

Statistical analysis
All the data were analyzed to evaluate the interaction between alcohol and HDL stimulation; two-factor analysis of variance (ANOVA) was used, and unless there was a significant interaction between the 2, only the main effects are presented. Wherever appropriate, the results obtained for the control and alcohol groups were compared by using Student's two-tailed t test (19).

	RESULTS

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Uptake of acetylated LDL by alcohol-treated and control rat peritoneal macrophages
When freshly isolated peritoneal macrophages from control and alcohol-treated rats were incubated with increasing concentrations of acetylated LDL, the cellular uptake of LDL cholesterol was linear throughout the 6 h of incubation in both groups. Although the uptake rate by alcohol-treated macrophages was 20% slower (P < 0.05) than that of the control macrophages (Figure 1), the cholesterol concentration in the peritoneal macrophages from alcohol-treated rats was 33.3% higher (P < 0.05) than that in macrophages from control rats after 24 h of incubation.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Mean (±SE) cholesterol content of 6 separate peritoneal macrophage colonies isolated, respectively, from the 6 rats in each group. Peritoneal macrophages were isolated from rats fed control and alcohol liquid diets for 8 wk. Cells were plated and isolated as described in Methods. Six sets (6 x 106 adherent cells/dish) of these isolated macrophages from control and alcohol groups were then incubated with acetylated LDL and followed up to 24 h in a medium containing 10% fetal bovine serum. The cholesterol-laden macrophages were then processed for their lipid fractionation, and cholesterol content was determined. The rate of LDL uptake by alcohol-treated macrophages was significantly different from that of the control macrophages, P < 0.05.

Effect of chronic alcohol treatment on protein content of rat peritoneal macrophages
The protein content of macrophages was 21.4% greater in the whole cells and 29.2% greater in the Golgi fractions from the alcohol-treated group than in those from the control group, whereas there was no significant difference in protein contents at the microsomal level (Table 1).

View larger version (19K): [in this window] [in a new window]   FIGURE 2. Mean (±SE) binding of labeled apolipoprotein (apo) E to HDL in 6 separate peritoneal macrophage colonies isolated, respectively, from 6 rats in each group. Peritoneal macrophages were isolated from control and alcohol-treated rats and cultured as described in Figure 1 and Methods. The isolated macrophages were then incubated with acetylated LDL for 24 h in a medium containing 10% fetal bovine serum, resulting in an accumulation of cholesterol. The cholesterol-laden macrophages were then maintained in a medium free of fetal bovine serum in the absence and presence of HDL. Labeled leucine was added to the medium (for 60 min), then the medium was replaced with no labeled leucine. The secretion of labeled apo E was then followed up to 6 h as described in Methods. Apo E in the secreted medium was immunoprecipitated and radioactivity was determined. Values were adjusted for nonspecific precipitation (<5%). There was no significant difference between treatment groups in the effects of HDL stimulation.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. Mean (±SE) binding of labeled apolipoprotein (apo) E to HDL3 in 6 separate peritoneal macrophage colonies isolated, respectively, from 6 rats in each group. Peritoneal macrophages were isolated from control and alcohol-treated rats and cultured as described in Figure 1 and Methods. These cultures were maintained in medium in the presence of 500 µg HDL3 but no fetal bovine serum. The medium was collected after 6 h of incubation and the secreted apo E was purified by using an anti-rat apo E Sepharose 4B immunoaffinity column (Pharmacia, Uppsala, Sweden), and then characterized by electrophoresis. The pure protein was iodinated as described in Methods and iodinated apo E was then incubated with HDL3 fractions isolated from control rats. After incubation, the HDL2 fraction was isolated from the medium and the radioactivity of the bound labeled apo E in the HDL2 fraction was determined as described in Methods. HDL3 binding ability of apo E from alcohol-treated rats was significantly different from that of apo E from control rats, P < 0.001.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Mean (±SE) binding of labeled apolipoprotein (apo) E to HDL2 in peritoneal macrophage colonies isolated from control and alcohol-treated groups of rats and cultured as described in Figure 1 and Methods, in the presence of 500 µg HDL but no fetal bovine serum. The medium was collected after 6 h of incubation and the secreted apo E was purified as described in Figure 3. The purified apo E was further treated with neuraminidase (exo--sialidase; EC 3.2.1.18) or sialyltransferase (ß-galactosamide -2,6-sialyltranferase; EC 2.4.99.1) to completely desialylate or resialylate the protein. The modified and reconstituted apo E was characterized by electrophoresis. The pure protein was iodinated as described in Methods and the iodinated desialylated or resialylated apo E was then incubated with HDL3 fractions isolated from rats in the control group. After a specific incubation, the HDL2 fraction was isolated from the medium and the radioactivity of the bound labeled apo E in HDL2 fraction was determined as described in methods. The HDL3 binding capacity of apo E from the alcohol-treated group was reduced by 27.6–52.6% with complete removal of the sialic acid residues (P < 0.001) and that of the apo E from the control group was reduced by 47.65–67.25% (P < 0.001).

	DISCUSSION

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In agreement with previous reports (29, 30), peritoneal macrophages of both the alcohol-treated and the control rats showed increased uptake and increased content of cellular cholesterol when incubated with acetylated LDL (Figure 1). However, we found that macrophages from chronically alcohol-fed rats had a lower rate of LDL-cholesterol uptake than did those isolated from control rats. This may be attributed to a possible alcohol-mediated reduction in the number of receptor binding sites or alterations in the binding affinity of these receptors for acetylated LDL. These aspects remain to be determined. Accumulation of 33% more cholesterol after 24 h of incubation in macrophages from the alcohol group than from the control group could be attributed to lower efflux rate of cellular cholesterol in the alcohol group than in the control group.

Plasma apo E is an O-type sialoglycoprotein (7, 31). The liver appears to be the main site of apo E synthesis, although most peripheral tissues, including macrophages, can synthesize apo E (6). The mechanism and involvement of subcellular organelles, particularly the Golgi apparatus and microsomes, in the glycosylation process were reviewed previously (32). Chronic alcohol treatment markedly inhibited the relative sialylation rate of apo E synthesized in rat peritoneal macrophages without affecting the relative apo E protein synthetic rate (Table 2). This defect in apo E sialylation may have been a result of damaged glycosylation machinery in peritoneal macrophages of rats chronically pretreated with alcohol. Chronic alcohol treatment of rats might have severely affected the activities of sialyltransferase, neuraminidase, or both. We showed earlier (5) that chronic alcohol treatment in rats has deleterious effects on these 2 enzymes in rat liver and impairs both glycosylation and secretion of apo E from the liver (5, 8).

Our pulse-chase secretion kinetics studies showed that the presence of HDL₃ accelerates apo E secretion in cholesterol-laden macrophages (Figure 2). This agrees with the results of studies reported previously by Dory (33) and Brown and Goldstein's group (6). The diminished rate of apo E secretion by macrophages chronically pretreated with alcohol may be attributed to alcohol-mediated defective sialylation of apo E by macrophages, leading to alterations in its conformation. Another possibility could be an alcohol-induced, defective response of the macrophages to HDL₃ stimulation. It has been suggested that stimulation of apo E secretion by macrophages is mediated by an HDL-receptor interaction. Chronic exposure to alcohol might impair the receptor interaction and lead to diminished apo E secretion. In either case, the functional implications of this defect would ultimately be nonavailability of apo E to secreted cholesterol for its clearance.

The glycosylation machinery in the macrophage system has not been delineated. Whether sialylation of apo E is necessary for the secretion of apo E by macrophages remains to be explored further. A high degree of sialylation in secretory apo E may be essential for its stable molecular conformation and attachment to phospholipid discs. Sialic acid residues of apo E may also play a role in its recognition by the HDL₃ molecule. This would explain the loss of binding affinity of desialylated apo E to HDL₃. It is only after HDL₃ incorporates apo E along with secreted cholesterol that it transforms into HDL₂, a form that is recognized by the liver for further degradation. Integration of newly secreted apo E into the HDL₃ molecule is a physiologic process that converts HDL₃ into HDL_2, which is then taken up by the liver for cholesterol degradation. Sialic acid residues of secreted apo E may play a crucial role in the integration of apo E into HDL₃ or its recognition by surface receptors in the liver. We speculate that a specific molecular conformation of apo E may be necessary for both integration of apo E into the HDL particle and its recognition by surface receptors. Sialic acid residues may impart a specific conformation to the apo E molecule regardless of whether it is free apo E or in the bound form within the HDL particle. More efficient and specific recognition of apo E may be dependent on a particular conformation of apo E that is determined by its sialic acid content.

The results of this study suggest that depletion of sialic acid residues from newly secreted apo E, caused by chronic exposure of macrophages to alcohol, resulted in the partial inability of apo E to bind to the HDL₃ molecule. This is supported by our findings that complete depletion of sialic acid residues from secreted apo E from both control and alcohol-pretreated macrophages dramatically impaired apo E integration into the HDL particle (Figure 4). Furthermore, resialylation of apo E could completely restore the ability of apo E to integrate within the HDL molecule (Figure 4). It is intriguing to find that resialylation of apo E secreted even by alcohol-pretreated macrophages resulted in complete restoration of the ability of apo E to integrate within the HDL particle. We therefore logically conclude that sialic acid residues are absolutely necessary to provide a specific and final molecular conformation to apo E so that it can integrate within the HDL particle. Further studies are necessary to examine the functional implications of this defect in apo E glycosylation in macrophages induced by chronic alcohol exposure.

On the basis of the results of this study and foregoing discussions, we conclude that the defects in apo E glycosylation caused by alcohol exposure may impair the ability of apo E to bind to the HDL particle. This defect may ultimately result in impaired cholesterol clearance. These alterations may in turn lead to the accumulation of cholesterol and thus increase the risk of cardiovascular diseases. This may explain, at least in part, the occurrence of strokes in alcoholic persons despite high plasma HDL-cholesterol concentrations.

	ACKNOWLEDGMENTS

 
We sincerely thank Qing Hong Liu for her technical assistance.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Criqui MH. Moderate drinking: benefits and risks. In: Zakhari S, Wassef M, eds. NIAAA research monograph 31: alcohol and the cardiovascular system. Bethesda, MD: National Institutes of Health, 1996:117–26.
2. Hennekens CH. Alcohol and risk of coronary events. In: Zakhari S, Wassef M, eds. NIAAA research monograph 31: alcohol and the cardiovascular system. Bethesda, MD: National Institutes of Health, 1996:15–24.
3. Kannel WB. High-density lipoproteins: epidemiologic profile and risks of coronary artery disease. Am J Cardiol 1983;52:9B–12B.[Medline]
4. Lieber CS, Savolainen M. Alcohol and lipids. Alcohol Clin Exp Res 1984;8:409–23.[Medline]
5. Ghosh P, Liu QH, Lakshman MR. Long-term alcohol exposure impairs glycosylation of both N- and O-glycosylated proteins in rat liver. Metabolism 1995;44:890–8.[Medline]
6. Basu SK, Ho YK, Brown MS, Bilheimer DW, Anderson RG, Goldstein JL. Biochemical and genetic studies of the apoprotein E secreted by mouse macrophages and human monocytes. J Biol Chem 1982;257:9788–95.[Free Full Text]
7. Zannis VI, Breslow JL. Human very low-density lipoprotein apolipoprotein E isoprotein polymorphism is explained by genetic variation and posttranslational modification. Biochemistry 1981;20: 1033–41.[Medline]
8. Ghosh P, Chirtel SJ, Lakshman MR. Effect of chronic alcohol on apolipoprotein (apo) E synthesis and glycosylation in rats. Alcohol Clin Exp Res 1991;15:725–9.[Medline]
9. Lakshman MR, Chirtel SJ, Chambers LC. Roles of 3-fatty acids and chronic alcohol in the regulation of plasma and liver lipids and plasma apoproteins A₁ and E in rats. J Nutr 1988;118:1299–303.
10. Kabat EA, Mayer MM. Experimental immunochemistry. 2nd ed. Springfield, IL: Charles C Thomas, 1961.
11. Lieber CS, DeCarli LM. The feeding of alcohol liquid diets: two decades of applications and 1982 update. Alcohol Clin Exp Res 1982;6:523–31.[Medline]
12. Dory L. Synthesis and secretion of apoE in thioglycolate-elicited mouse peritoneal macrophages: effect of cholesterol efflux. J Lipid Res 1989;30:809–16.[Abstract]
13. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with Folin-phenol reagent. J Biol Chem 1951;193:265–75.[Free Full Text]
14. Miyagi T, Sagawa J, Konno K, Tsuiki S. Immunological discrimination of intralysosomal, cytosolic, and two membrane sialidases present in rat tissues. J Biochem (Tokyo) 1990;107:794–8.[Abstract/Free Full Text]
15. Warren L. The thiobarbituric acid assay of sialic acids. J Biol Chem 1959;234:1971–5.[Free Full Text]
16. Bolton AE, Hunter WM. The labeling of proteins to high specific radioactivities by conjugation to an ¹²⁵I-containing acylating agent. Biochem J 1973;133:529–39.[Medline]
17. Milla ME, Hirschberg CB. Reconstitution of Golgi vesicle CMP-sialic acid and adenosine 3'- phosphate 5'-phosphosulfate transport into proteoliposomes. Proc Natl Acad Sci U S A 1989;86:1786–90.[Abstract/Free Full Text]
18. Gavish D, Oschry Y, Eisenberg S. In vivo conversion of human HDL₃ to HDL₂ and apoE-rich HDL₁ in the rat: effects of lipid transfer protein. J Lipid Res 1987;28:257–67.[Abstract]
19. Sokal RR, Rohlf SJ. Biometry. San Francisco: WH Freeman Publishing Company, 1969.
20. Hamilton RL. Hepatic secretion and metabolism of high density lipoproteins. In: Dietschy J, Gotto A, Ontko JA, eds. Disturbances in lipid and lipoprotein metabolism. Bethesda, MD: American Physiological Society, 1978:155–71.
21. Glomset JA, Norum KR. The metabolic role of lecithin:cholesterol acyltransferase: perspectives from pathology. Adv Lipid Res 1973; 11:1–65.
22. Basu SK, Brown MS, Ho YK, Havel RJ, Goldstein JL. Mouse macrophages synthesize and secrete a protein resembling apoE. Proc Natl Acad Sci U S A 1981;78:7545–9.[Abstract/Free Full Text]
23. Goldstein JL, Brown MS. The metabolic basis for inherited disease. In: Stanby JB, Wyngarden JB, Fredrickson DS, Goldstein JL, Brown MS, eds. Familial hypercholesterolemia. New York: McGraw-Hill, 1983;5:672–85.
24. Goldstein JL, Ho YK, Basu SK, Brown MS. Binding site on macrophage that mediates uptake and degradation of acetylated LDL producing massive cholesterol deposition. Proc Natl Acad Sci U S A 1976;76:333–7.
25. Brown MS, Ho YK, Goldstein JL. Cholesterol ester cycle in macrophage foam cells. J Biol Chem 1980;255:9344–52.[Free Full Text]
26. Ho YK, Brown MS, Goldstein JL. Hydrolysis and excretion of cytosol cholesterol esters by macrophages: stimulation by HDL and other agents. J Lipid Res 1980;21:391–8.[Abstract]
27. Zannis VI, McPherson J, Goldberger G, Karathanasis SK, Breslow JL. Synthesis, intracellular processing, and signal peptide of human apolipoprotein E. J Biol Chem 1984;259:5495–9.[Abstract/Free Full Text]
28. Basu SK, Goldstein JL, Brown MS. Independent pathways for the secretion of cholesterol and apoE by macrophages. Science 1983;219:871–3.[Abstract/Free Full Text]
29. Goldstein JL, Ho YK, Basu SK, Brown MS. Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proc Natl Acad Sci U S A 1979;76:333–7.[Abstract/Free Full Text]
30. Brown MS, Goldstein JL, Krieger M, Ho YK, Anderson RG. Reversible accumulation of cholesteryl esters in macrophages incubated with acetylated lipoproteins. J Cell Biol 1979;82:597–613.[Abstract/Free Full Text]
31. Zannis VI, McPherson J, Goldberger G, Karathanasis SK, Breslow JL. Synthesis, intracellular processing, and signal peptide of human apolipoprotein E. J Biol Chem 1984;259:5495–9.
32. Higgins JA, Hutson JL. The roles of Golgi and endoplasmic reticulum in the synthesis and assembly of lipoprotein lipids in rat hepatocytes. J Lipid Res 1984;25:1295–305.[Abstract]
33. Dory L. Regulation of apolipoprotein E secretion by high-density lipoprotein 3 in mouse macrophages. J Lipid Res 1991;32:783–92.[Abstract]

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