© 1996 American Heart Association, Inc.
Articles |
Ethanol-Induced Redistribution of Cholesteryl Ester Transfer Protein (CETP) Between Lipoproteins
From the Department of Internal Medicine and Biocenter Oulu, University of Oulu, Finland.
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Abstract Since alcohol drinking reduces the concentration and activity of plasma cholesteryl ester transfer protein (CETP), we investigated the effects of alcohol on its synthesis and secretion by perfusing rabbit livers for 4 hours in the absence or presence of ethanol. The quantity of CETP mRNA in the perfused livers did not differ between the control and ethanol (25 mmol/L or 50 mmol/L) perfusions. CETP activity was determined by incubating [3H]cholesteryl ester–labeled human LDL and unlabeled human HDL with the perfusion medium after removing the endogenous VLDL (secreted by the perfused liver) by ultracentrifugation. CETP activity in the perfusion medium increased at a linear rate that was not affected by ethanol. When the VLDL was removed by precipitation with polyethylene glycol or a heparin-Sepharose column instead of ultracentrifugation, practically no CETP activity was detected in the ethanol perfusions, whereas these procedures did not affect CETP activity in the control perfusions. Inhibition of ethanol oxidation by 4-methylpyrazole resulted in CETP activity similar to that of the controls. We conclude that ethanol does not affect the synthesis or secretion of CETP, but its oxidation may alter the distribution of CETP in lipoproteins. CETP seems to be present in VLDL as well as in HDL, and since VLDL is more rapidly catabolized than HDL, this may explain the low plasma CETP concentration associated with alcohol consumption.
Key Words: cholesteryl ester transfer protein • ethanol • lipoproteins • VLDL • rabbit liver perfusion
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Introduction |
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Plasma cholesteryl ester transfer protein (CETP) is a hydrophobic glycoprotein that mediates the transfer and exchange of cholesteryl esters (CE) between plasma lipoproteins.1 2 3 Since it provides a mechanism for returning plasma CE to the liver, it may have an antiatherogenic function. On the other hand, recent research suggests that CETP could contribute to atherogenesis.4 5 6 Plasma CETP activity and the tissue distribution of CETP mRNA vary between species. Mice and rats lack CETP activity, whereas it is relatively high in humans and rabbits.7 CETP mRNA is expressed in several human tissues, including the liver and adipose tissue,8 9 whereas it has been found mainly in the liver in rabbits.10
Little is known at present about the regulation of plasma CETP. Diet-induced hypercholesterolemia has been found in animal experiments to be associated with increased plasma CETP concentration and activity and with increased liver CETP mRNA, suggesting an increase in the synthesis of CETP.11 12 13 14 CETP gene expression may also be sensitive to other factors, since its activity and concentration are altered in a variety of conditions. CETP activity is low in hypothyroidism15 and elevated in hypertriglyceridemia16 and hypercholesterolemia,17 as well as in patients with coronary artery disease5 and insulin-dependent diabetes.18 In addition, it may be altered by lipid-lowering drugs.19 20 21 22 23
Alcohol intake and the risk of coronary artery disease are inversely correlated, but the exact mechanisms for this association are still unknown.24 The protective effects of alcohol may partly be explained by increased plasma HDL cholesterol and alterations in other plasma lipoproteins.25 26 27 In addition, CETP, which is one of the factors regulating HDL cholesterol levels, might mediate at least part of the cardioprotective role of alcohol.
We have recently shown that alcohol consumption markedly reduces both the concentration and activity of CETP.28 29 30 Ethanol could lower plasma CETP levels by reducing its synthesis or secretion or by enhancing its catabolism. To explore these possibilities, we perfused rabbit livers in a recirculating system in the presence of ethanol or 4-methylpyrazole (an inhibitor of alcohol dehydrogenase) and determined the CETP activity in the perfusion medium and CETP mRNA in the perfused liver. The results show that ethanol does not reduce the synthesis or secretion of CETP in the perfused rabbit liver, but it may alter the distribution of CETP among lipoproteins.
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Animals
Chinchilla rabbits weighing 4.0±0.9 kg (mean±SD, n=32) were fed a standard chow (Rabbit Maintenance Diet, Special Diets Services,) and used for liver perfusion after an 18-hour fast. A blood sample was taken from the ear vein for lipid and CETP analyses before anesthesia.
Liver Perfusion
The liver perfusions were carried out
essentially as described
earlier.31 32 The rabbits were anesthetized with
an intramuscular injection of 20 mg/kg ketamine (Ketalar,
Parke-Davis) and 250 µg/kg medetomidin (Domitor, Farmos Group Ltd).
The portal vein and inferior vena cava were cannulated, and
the liver was washed with a once-through perfusion of 583±174 mL
(n=32) Krebs-Ringer bicarbonate buffer containing 10 mmol/L glucose, pH
7.4, at 37°C to remove any residual blood. It was then perfused with
the Krebs-Ringer buffer containing washed human erythrocytes at a
hematocrit of 20%, amino acids corresponding to a minimum essential
medium amino acid solution with L-glutamine (catalog No. 043-01135,
GIBCO), 50 IU/mL G-penicillin, and 0.05 mg/mL streptomycin. The
perfusion was carried out in a recirculating system with a total volume
of 300 mL at a flow rate of 0.8±0.2
mL·min-1·g-1
of liver for 4 hours. Both the flushing medium and the recirculating
medium were saturated with carbogen (95% oxygen and 5% carbon
dioxide) using a "lung" made of thin-walled Silastic
tubing.33
A recirculating system was chosen for two reasons. First, in a nonrecirculating system, the amount of medium for a 4-hour perfusion would have been large, therefore excluding the use of erythrocytes. A medium without erythrocytes would not have maintained the viability of the liver. Second, the small amount of CETP secreted would have been difficult to detect in the medium of a nonrecirculating system.
Fifteen of the liver perfusions were performed in situ, with care taken to ligate all the vessels supplying blood to the liver. In the remaining 17 perfusions, the liver was completely freed from the surrounding tissues, and the perfusion took place in a temperature-controlled cabinet (37°C). The stricter research protocol was used to ensure that the CETP detected in the perfusion medium was secreted only by the liver. Since no differences were observed in the results obtained by the two protocols, the data were combined.
To assess the viability of the perfused liver, bile flow was measured by cannulating the bile duct and collecting the bile (0.46±0.19 µL·min-1·g-1 of liver, n=32). Samples of the perfusate were taken at 30-minute intervals for the analyses of CETP activity. The pH of the perfusion medium was monitored and was found to decrease slightly during the 4-hour perfusion (average of <0.2 pH units). At the end of the perfusion, the liver was weighed and a piece excised for the CETP mRNA analyses. This piece was immediately frozen in liquid N2 and stored at -70°C.
The control (n=8) and ethanol perfusions were performed similarly without ethanol for the first 60 minutes. After 60 minutes, ethanol was added to the perfusion medium, to a final concentration of 25 mmol/L (n=6) or 50 mmol/L (n=8), and the concentration was maintained by adding additional ethanol at a rate of 0.96±0.26 µL·h-1·g-1 of liver. These concentrations of ethanol (25 mmol/L=1.15 g/L and 50 mmol/L=2.30 g/L) were chosen because they are comparable with those in humans consuming alcohol. 4-Methylpyrazole (Sigma catalog No. M-1387), an inhibitor of alcohol dehydrogenase, was added to some perfusions, to a final concentration of 0.5 mmol/L, between 20 minutes and 60 minutes after the initiation of the recirculating perfusion, while ethanol was added, to a final concentration of 50 mmol/L, at 60 minutes, with no additions of ethanol thereafter.
Isolation of Lipoproteins
The samples of the perfusion medium
were centrifuged at
800g and 4°C for 15 minutes to separate the erythrocytes.
VLDL (d<1.006 g/mL) was isolated by spinning the perfusate
samples and the rabbit plasma in a Kontron TFT 45.6 rotor at
114 000g (35 000 rpm) and 15°C for 18 hours. The samples
were stored at -70°C.
Human LDL and HDL were isolated from the plasma of healthy volunteers by sequential ultracentrifugation34 and used for the measurement of CETP activity. The plasma was adjusted to a density of 1.019 g/mL by the addition of NaCl/NaBr solution. After centrifugation in a Beckman Ti60 rotor at 247 000g (59 000 rpm) and 15°C for 18 hours, the top fraction containing VLDL and IDL was removed. The density was increased to 1.063 g/mL, and LDL (d=1.019 to 1.063 g/mL) was isolated. The density was then adjusted to 1.090 g/mL to remove apolipoprotein (apo) B–containing particles such as lipoprotein(a) and light HDL particles, which float to the surface. The density of the infranatant was raised to 1.210 g/mL to isolate HDL (d=1.090 to 1.210 g/mL) by means of a 48-hour centrifugation. Finally, LDL and HDL were washed in a Kontron TFT 45.6 rotor at 114 000g and 15°C, LDL at a density of 1.070 g/mL (for 18 hours) and HDL at a density of 1.210 g/mL (for 48 hours). The LDL and HDL fractions were dialyzed overnight against 0.15 mol/L NaCl/1 mmol/L EDTA, pH 7.4, and stored at 4°C.
Chemical Analyses
The HDL cholesterol concentration in the
plasma was
analyzed by mixing 1 mL of the VLDL-free fraction (isolated by
ultracentrifugation, as described above) with 25
µL of 2.8% (wt/vol) heparin and 25 µL of 2 mol/L manganese
chloride and centrifuging at 1000g and 4°C for 30 minutes.
Aliquots of the supernatant were taken for cholesterol
analysis. The LDL cholesterol concentration was
calculated by subtracting the HDL value from that for the total
VLDL-free fraction.
The concentrations of total cholesterol, free cholesterol, and triglycerides in the plasma, perfusion medium, and lipoprotein fractions were determined by enzymatic colorimetric methods (kits from Boehringer Diagnostica, Mannheim GmbH, catalog Nos. 236691, 310328, and 701912, respectively) using a Kone Specific analyzer (Kone Specific, Selective Chemistry Analyzer, Kone Instruments).
Ethanol concentrations in the perfusate samples were determined using a Test-Combination Blood Alcohol kit (Boehringer Diagnostica, Mannheim GmbH, catalog No. 123960), and protein concentrations in the liver samples were determined by the method of Lowry et al.35
Since swelling of the liver during the perfusion could affect the results based on weighing the liver at the end of the perfusion experiment, the concentration of perfusion medium in the liver was estimated by measuring the hemoglobin concentration in the frozen tissue sample of the perfused liver, as described previously.36 Since no significant differences in hemoglobin concentration were observed between the perfusion categories, the results expressed here are based on the wet weight of the liver.
Determination of CETP mRNA
The quantities of CETP and
-actin mRNA in the perfused
rabbit liver tissue were determined by both the Northern blot and the
quantitative dot blot method. Total cellular RNA was extracted from the
rabbit liver tissue (
500 mg) by the guanidine thiocyanate method,
with centrifugation through a CsCl
gradient,37 and quantified at A260 nm by
spectrophotometry. The A260:A280 ratios were
between 1.6 and 1.9, and the yield of total cellular RNA was 1.1 to 3.6
mg/g of liver tissue. The integrity and quality of all RNA samples were
determined by electrophoresis on agarose gel before the Northern blot
and dot blot analyses.
For the quantitative dot blot method, four dilutions of the total cellular RNA sample (2.5, 5, 10, and 20 µg in the same volume of water) were denatured with formaldehyde and applied to a nylon membrane (Hybond-N, Amersham). For the Northern blot method,38 20 µg of the total RNA dissolved in water was denatured at 65°C for 5 minutes, fractionated by electrophoresis through a 1% agarose gel, and transferred to a nylon membrane by pressure (PosiBlot Pressure Blotter, Stratagene).
Both the dot blot and Northern blot membranes were baked
at 80°C for
2 hours and prehybridized in a hybridization solution containing 0.2
mmol/L dextran sulfate (mol wt 500 000), 1 mol/L NaCl, 1% SDS, and
0.1 mg/mL denatured salmon sperm DNA.39 The membranes were
then hybridized overnight with the 32P-labeled human CETP
cDNA probe at 65°C in the hybridization solution. The probe was
labeled by the "random-primed" method.40 Human
CETP cDNA can be used to determine rabbit CETP mRNA
levels.12 After hybridization, the membranes were rinsed
quickly with 2x standard saline phosphate EDTA (SSPE), containing 300
mmol/L NaCl, 20 mmol/L sodium phosphate, and 2 mmol/L EDTA, pH 7.4, and
then washed for 30 minutes with 2x SSPE at 65°C and 15 minutes with
1x SSPE at 65°C. The membranes were exposed to Kodak X-AR films for
24 hours at -70°C using intensifier screens. Before
rehybridization, the membranes were washed with 0.1% standard saline
citrate and 0.1% SDS and reexposed to check the result of the washing
procedure. Reprobing was performed with 32P-labeled human
-actin cDNA probe in the same manner as described above.
The
proportions of CETP mRNA and -actin mRNA on the
autoradiographs were determined by densitometric scanning (300 A
Computing Densitometer and Image Quant Software v3.0 Fast Scan,
Molecular Dynamics). In the dot blot method, the lower concentration
range of the total RNA yielded a linear intensity curve by scanning
densitometry, and the measured intensities of the 2.5-µg and 5-µg
dots were used as estimates of the mRNA levels. The results were
expressed as the ratio CETP mRNA:-actin mRNA.
Determination of CETP Activity
The activity of CETP was
determined as described
earlier28 29 41 by incubating the sample
containing CETP
with labeled human LDL and unlabeled human HDL and by detecting the
transfer of radioactive cholesteryl esters. Human LDL was labeled with
[1,2(n)-3H]cholesteryl oleate.28 Samples of
the perfusion medium (45 µL) were incubated at 37°C for 16 hours
with labeled human LDL (125 nmol of total cholesterol),
unlabeled human HDL (50 nmol of total cholesterol), 0.35
µmol of 5,5'-dithio-bis(2-nitrobenzoic acid) (an
inhibitor of lecithin/cholesterol
acyltransferase), and 8.75 µmol of phosphate buffer, pH 7.4, in a
total volume of 175 µL. The tubes were then cooled on crushed ice,
and LDL was precipitated by adding 75 µL of bovine serum
albumin (80 g/L) and 25 µL of unlabeled human LDL (1 µmol
of total cholesterol) and also 27.5 µL of an equivolume
mixture of 2 mol/L MgCl2 and 20 g/L dextran sulfate (mol wt
40 000) after vortex mixing. The tubes were centrifuged at
1000g and 4°C for 20 minutes, and a 100-µL aliquot of
the supernatant was counted in 2 mL of OptiPhase "HiSafe" 3
scintillation cocktail (manufactured for LKB Wallac by FSA Laboratory
Supplies) in a scintillation counter (1209 RackBeta Liquid
Scintillation Counter, LKB Wallac). CETP activity was calculated as
nanomoles of cholesteryl esters exchanged per hour of incubation
per milliliter of sample, and then multiplied by the total volume of
the perfusion medium (in milliliters) and divided by the wet weight of
the liver (in grams). CETP activity was expressed in nanomoles per
hour per gram of liver.
CETP activity was determined in the perfusate samples containing endogenous lipoproteins secreted by the perfused liver (mainly VLDL) and after the removal of VLDL from the perfusion medium by various methods. First, VLDL was isolated by ultracentrifugation, and the VLDL-free infranatant was used as a source of CE transfer activity in the CETP assay. Second, the apoB-containing lipoproteins were precipitated by adding 200 µL of polyethylene glycol (95 g/L, mol wt 20 000) to 100 µL of the perfusate sample and centrifuging the mixture at 1000g and 4°C for 20 minutes. Third, VLDL was removed from the perfusion medium on a heparin-Sepharose column (HiTrap Heparin Sepharose affinity column, Pharmacia, code No. 17-0406-01). The column was equilibrated with 0.01 mol/L phosphate buffer, pH 7.0, which was used to elute the VLDL-free fraction of the perfusion medium. The CETP activities of the VLDL-free samples were then determined by CETP assay.
In some perfusions, CETP activities were analyzed immediately after the perfusion experiment (before freezing), and the activities were not significantly different from those analyzed after a short storage at -70°C. In addition, CETP activities were analyzed after storage for more than 11 months. The CETP activities were unchanged both in the samples with endogenous VLDL present and in those from which VLDL was removed by ultracentrifugation (data not shown), suggesting that the results obtained were not likely owing to storage.
Human LDL and HDL were used in the determination of CETP activity, since rabbit plasma contains less LDL and HDL and the use of rabbit lipoproteins would have been impractical for all the CETP assays. For comparison, however, we isolated LDL and HDL from rabbit plasma and used the rabbit fractions to determine CETP activity in some of the perfusate samples. The results correlated well with those analyzed with human LDL and HDL (r=.97, P<.001; n=11).
The distribution of CETP was investigated in the perfusion medium of control and ethanol perfusions by gel filtration, as described earlier.42
Statistical Analysis
The statistical analyses were carried
out with the SAS
package (SAS Institute Inc). The results are expressed as mean±SD
unless otherwise indicated. The statistical significances of
differences between the means were assessed using an analysis
of variance (Scheffé's test).
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Results |
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Characteristics of the Rabbits and Rabbit Liver Perfusions
The synthesis and secretion of CETP were investigated by perfusing the livers of 32 rabbits in a recirculating system for 4 hours. The rabbits used in the various categories of perfusion were similar with respect to weight, plasma CETP activity, and plasma cholesterol and triglyceride concentrations as determined before the perfusion experiment (Table 1
).
The wet weights of the perfused livers, flow rates of the perfusion
medium, and bile flow during perfusion were also similar in all the
perfusions, as were the protein concentrations of the perfused livers
(Table 2). The amount of medium in the perfused liver
was likewise constant, indicating that the substances added to
the medium (ethanol or 4-methylpyrazole) did not cause any difference
in the degree of liver sinusoid dilatation during the perfusion (data
not shown).
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The accumulation of cholesterol and
triglycerides in the perfusion medium is shown in Fig 1. At the
end of the perfusion, the
triglyceride concentration tended to be slightly higher in
the perfusions with ethanol (50 mmol/L) and slightly lower in the
perfusions with 4-methylpyrazole than in the control perfusions. The
differences between the perfusions were, however, not significant when
the final time points or the areas under the curves were compared by
analysis of variance. The accumulation of VLDL
triglycerides in the medium was also similar between the
perfusions (data not shown).
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, n=8); perfusions with ethanol at a concentration
of
25 mmol/L (
, n=6); perfusions with ethanol at a concentration
of 50 mmol/L (
, n=8); perfusions with 4-methylpyrazole at a
concentration of 0.5 mmol/L (
, n=5); and perfusions with
4-methylpyrazole and ethanol (a single addition, to a concentration of
50 mmol/L) (
, n=5). Values are given as means. SEMs are shown
only for the last data points for the sake of clarity.The mean ethanol concentration of the medium was 32.9±6.5 mmol/L in the perfusions in which ethanol was added at a concentration of 25 mmol/L (n=6) and 48.7±13.2 mmol/L with a concentration of 50 mmol/L (n=8). The concentration of ethanol in the 4-methylpyrazole perfusions was 32.2±12.5 mmol/L (n=5) 30 minutes after the single addition of ethanol and 19.1±2.4 mmol/L (n=5) at the end of the perfusion. The calculated rates of ethanol oxidation were 86±8 and 70±23 µmol·h-1·g-1 of liver in the 25-mmol/L and 50-mmol/L ethanol perfusions, respectively.
CETP mRNA Level in the Perfused Rabbit Liver
The quantity of
CETP mRNA in the perfused rabbit liver, determined
by the Northern blot method and the dot blot method and expressed as
CETP mRNA:-actin mRNA ratio, did not differ between the control
and ethanol-treated livers (Table 3), suggesting
that the synthesis of CETP in the perfused rabbit liver is not reduced
by the addition of ethanol to the perfusion medium. The CETP mRNA level
was similarly not affected by 4-methylpyrazole (Table 3). An
example of
the specificity of the Northern blot in Fig 2 shows the
position of the CETP mRNA with respect to the positions of molecular
weight markers.
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Effect of Ethanol on CETP Activity
CETP activity was
determined in the perfusion medium after the
removal by ultracentrifugation of
endogenous VLDL secreted by the perfused liver. CETP
dissociates from lipoprotein particles during
ultracentrifugation.43 44 45 Fig
3A
shows that CETP activity increased linearly at
approximately the same rate in the control and ethanol perfusions, the
rates being 1.0±0.1, 1.3±0.1, and 0.7±0.1
nmol·h-1·g-1
of liver per hour of perfusion in the control (n=8), ethanol 25-mmol/L
(n=6), and ethanol 50-mmol/L (n=8) perfusions, respectively. The
areas
under the curves did not differ between the control and ethanol
perfusions, indicating that the secretion of CETP is not reduced by
ethanol.
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If the newly synthesized VLDL was not removed from the
perfusion medium
before the CETP assay, the opposite results were obtained; ie, ethanol
reduced the appearance of CETP activity during the perfusion (the areas
under the curves, calculated from 1 hour to 4 hours, differed between
the control and ethanol 50-mmol/L perfusions, P<.05, Fig
3B
). In contrast, the accumulation of CETP activity in the
whole medium
of the control perfusions was no different from that in the VLDL-free
medium. Taken together, these observations lead us to hypothesize that
CETP may be present in the ethanol perfusion, mainly in the VLDL
particles as opposed to the HDL particles, and that in the incubation
with the medium from the ethanol perfusions, CETP seems to transfer CE
from labeled LDL to the endogenous VLDL rather than the
unlabeled HDL. Since the apoB-containing lipoproteins are precipitated
after incubation, almost no radioactivity is detected in the
supernatant in the ethanol perfusions.
Binding of CETP to Lipoproteins
To test whether CETP is
associated with VLDL in the medium of the
ethanol perfusions, VLDL was removed from the perfusion medium by two
methods. When the VLDL in the perfusion medium was precipitated with
polyethylene glycol, practically no CETP activity was detected in the
VLDL-free supernatant in the ethanol perfusions, whereas the CETP
activity remained unchanged in the control perfusions (the areas under
the curves, calculated from 1 hour to 4 hours, differed between the
control and ethanol 50-mmol/L perfusions, P<.01, Fig
4A). Furthermore, when VLDL was removed from the
perfusion medium (4-hour sample) with a heparin-Sepharose column and
the VLDL-free eluate was used in the CETP assay, CETP activity was not
affected in the control perfusions, whereas the majority of the CETP
activity tended to be abolished in the ethanol perfusions (Fig
4B). The
distribution of CETP was also investigated by gel filtration in the
perfusion medium of two control and two ethanol 50-mmol/L perfusions
(4-hour samples). Slightly more CETP was found to be associated with
VLDL in the ethanol perfusions (34.4% and 35.3%) than in the control
perfusions (29.4% and 29.9%), although this does not fully explain
the disappearance of CETP in the above-mentioned experiments.
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Effect of 4-Methylpyrazole on CETP Activity
To study whether
the effects of ethanol on CETP-lipoprotein
interaction are mediated via products of ethanol oxidation, alcohol
dehydrogenase was inhibited by adding 4-methylpyrazole to the
perfusion.46 CETP activity in the perfusions with
4-methylpyrazole and ethanol was similar to that in the control
perfusions when measured in the ultracentrifugally separated VLDL-free
perfusion medium (Fig 3C). When CETP activity was determined
with
endogenous VLDL present, 4-methylpyrazole abolished the
ethanol effect on the accumulation of CETP activity in the medium (Fig
3D, compare Fig 3B). Furthermore, when VLDL was
removed from the
perfusion medium on a heparin-Sepharose column (Fig 4B) or by
precipitation with polyethylene glycol (data not shown) and the
VLDL-free fraction was used in the CETP assay, CETP activity was not
reduced by ethanol in the presence of 4-methylpyrazole. This suggests
that the oxidation of ethanol and not ethanol itself is the cause for
the altered affinity of CETP for VLDL in the ethanol perfusions.
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Discussion |
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Our previous finding that plasma CETP activity is reduced by alcohol drinking28 30 led us to examine the effect of alcohol on the synthesis and secretion of CETP by use of a rabbit liver perfusion model. The results show that CETP activity in the medium of the perfused rabbit liver increases at a linear rate, as has been found earlier.47 48 We demonstrate here that the addition of ethanol does not affect the appearance of CETP activity in the perfusion medium. Since the CETP mRNA level in the perfused liver was also unaffected by ethanol, we conclude that ethanol does not affect the synthesis or secretion of CETP in the perfused rabbit liver. In addition, our results suggest that the oxidation of ethanol may alter the distribution of CETP in lipoproteins.
CETP activity was measured here after removing the endogenous VLDL (secreted by the perfused liver) from the medium by ultracentrifugation. CETP activity accumulated in the medium at the same rate in the control and ethanol perfusions. In the experiment in which the endogenous VLDL was not removed from the perfusion medium before the CETP assay, we observed, surprisingly, low CE transfer activity in the ethanol perfusion but not in the control one. This finding led us to propose that ethanol may cause a redistribution of CETP between the lipoproteins. An altered affinity of CETP for lipoproteins in association with the conditions of the CETP assay offers an explanation for the low CE transfer activity in the ethanol perfusions found in this experiment. The CETP assay involved incubation of the perfusion medium with labeled human LDL and unlabeled human HDL and measurement of the transfer of radioactive cholesteryl esters from LDL to HDL, and it is possible that during the incubation of the medium of the ethanol perfusions with endogenous VLDL present, cholesteryl esters may be transferred from the labeled LDL to the VLDL rather than to the HDL. After the incubation, the apoB-containing lipoproteins (and also the endogenous VLDL) were precipitated, and thus only a minor amount of radioactivity was detected in the supernatant. In contrast, practically the same CETP activity was observed in the medium of the control perfusions (with endogenous VLDL present) as in the VLDL-free medium. The finding that the CETP activity in the ultracentrifugally isolated VLDL-free perfusion medium of the ethanol perfusions was as high as in that of the controls is explained by the dissociation of CETP from lipoproteins during ultracentrifugation.43 44 45
The hypothesis that CETP is present mainly in VLDL in the perfusion medium of ethanol perfusions was investigated further by removing VLDL from the perfusion medium by different methods and using the VLDL-free perfusion medium for the measurement of CETP activity. When VLDL was removed by precipitation with polyethylene glycol or on a heparin-Sepharose column, CETP activity remained unchanged in the control perfusions, whereas almost no CETP activity was detected in the VLDL-free samples in the ethanol perfusions. The isolation of VLDL by gel filtration also suggested that more CETP is associated with VLDL in ethanol perfusions.
The aggregation of lipoproteins by polyethylene glycol is influenced by the concentration of the polyethylene glycol, the concentration of the lipoproteins, the surface charge of the lipoproteins, and the presence of other proteins.49 50 The separation of lipoproteins by a heparin-Sepharose column is affected by the properties of the column and the salt concentration of the buffer.51 52 We cannot on the basis of the present results exclude the possibility that apoE-containing HDL was coprecipitated by the polyethylene glycol or bound to the heparin-Sepharose column in addition to VLDL.
The effect of ethanol metabolism was investigated by adding 4-methylpyrazole during the perfusion to inhibit alcohol dehydrogenase. In the presence of 4-methylpyrazole, CETP activity (in the perfusion medium with endogenous VLDL present) decreased slightly immediately after the addition of ethanol but increased to the control level by the end of the perfusion. Despite this dip, which may be due to incomplete inhibition of ethanol oxidation, the area under the curve was not significantly different from that of the controls. The ability of 4-methylpyrazole to block the effect of ethanol on the redistribution of CETP suggests that the altered affinity of the latter for lipoproteins is a consequence of ethanol oxidation.
The distribution of CETP among plasma lipoproteins is still controversial. Some researchers have found an association of CETP with HDL,43 45 53 others with all the major lipoprotein classes.44 54 The majority of the plasma CETP activity has been located in HDL3,43 with lipoproteins containing apoA-I but no apoA-II,45 or in HDL3, VHDL, and to a lesser extent HDL2, but not in VLDL or LDL.53 Others, however, have detected binding of CETP to both Sepharose-bound VLDL and LDL, in addition to HDL.44 54 CETP has been observed in rabbits to be associated with small HDL particles.55
We have shown previously that plasma CETP concentrations are lower in alcohol abusers than in control subjects and that plasma CETP concentration and activity have a good positive correlation.29 In addition, we observed the same CETP concentration in the heparin-Mn2+ supernatant as in the plasma of the control subjects (after the precipitation of VLDL and LDL from the plasma with heparin–manganese chloride), whereas the alcohol abusers had lower CETP concentrations in the heparin-Mn2+ supernatant than in the plasma. This suggests that CETP is also more prevalently distributed among apoB-containing lipoproteins in alcohol abusers than in control subjects in vivo. In the present experiment in which rabbit livers were perfused in the presence of ethanol, almost no CETP activity was detected in the VLDL-free perfusion medium after the removal of VLDL by precipitation with polyethylene glycol or on a heparin-Sepharose column, but the activity was as high in the ethanol perfusions as in the control perfusions when measured in the VLDL-free perfusion medium after ultracentrifugation (during which CETP is dissociated from the lipoproteins), suggesting that CETP is present in the ethanol perfusions mainly in VLDL particles. CETP may be present in VLDL as well as in HDL in the plasma of alcohol drinkers, and since VLDL is more rapidly catabolized than HDL,56 57 this may explain the low plasma CETP concentration associated with alcohol consumption.
In conclusion, the results demonstrate that ethanol does not reduce the synthesis or secretion of CETP in the perfused rabbit liver. In addition, they suggest that ethanol may affect CETP/lipoprotein interaction. There are several possible explanations of why the CETP produced during ethanol oxidation in the perfused liver could bind VLDL more effectively than HDL. The oxidation of ethanol could result in the modification of CETP and/or lipoproteins, the induction of some cofactor(s) affecting the affinity of CETP for lipoproteins, or the stabilization of CETP/VLDL binding. Additional research is needed to determine the mechanism by which ethanol alters the CETP/lipoprotein interaction.
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Acknowledgments |
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This work was supported by grants from the Finnish Foundation for Alcohol Studies, the Finnish Foundation for Cardiovascular Research, the Medical Council of the Academy of Finland, the Research and Science Foundation of Farmos, the Yrjö Jahnsson Foundation, and the Deaconess Institute of Oulu. We are very grateful to Dr Jorma Pudas, Chief Veterinarian at the Laboratory Animal Center of the University of Oulu, Veikko Lähteenmäki, and Tapio Rautio for their help. We also acknowledge the excellent technical assistance received from Tuulikki Haataja, Saija Kortetjärvi, Sari Liikanen, Anna-Riitta Malinen, and Eila Saarikoski.
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Footnotes |
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Reprint requests to Markku J. Savolainen, MD, PhD, Department of Internal Medicine, University of Oulu, Kajaanintie 50, FIN-90220 Oulu, Finland. E-mail markku.savolainen@oulu.fi.
Received October 9, 1995; accepted November 20, 1995.
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