1 Department of Biochemistry and 2 Department of Cardiology and Medicine, Royal Perth Hospital, Perth, Western Australia 6001, Australia
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ABSTRACT |
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Compared with apolipoprotein E3 (apoE3), apoE2 is less effective in mediating the binding of lipoproteins to the low-density lipoprotein (LDL) receptor. The influence of the E4 isoform, which is associated with adverse effects on plasma lipids and coronary heart disease, is less clear. We compared the ability of very low density lipoprotein (VLDL) and LDL from paired E4/4 and E3/3 subjects to compete against 125I-labeled LDL for binding with the LDL receptor on cultured fibroblasts and Hep G2 cells. The concentrations of VLDL or LDL required to inhibit binding of 125I-LDL by 50% (IC50, µg apoB/ml) were determined, and results were assessed in terms of an IC50 ratio, E4/4 IC50 relative to E3/3 IC50, to reduce the influence of interassay variability. In Hep G2 cells, E4/4 VLDL was more effective than E3/3 VLDL in competing for the LDL receptor, the IC50 ratio being lower than unity (0.73 ± 0.31, P < 0.05, two-tailed t-test). IC50 values themselves were marginally lower in E4/4 than E3/3 subjects (3.7 ± 1.3 vs. 6.1 ± 3.7, P < 0.08). However, there was no difference between E4/4 and E3/3 VLDL in competing for the LDL receptor on fibroblasts or between E4/4 and E3/3 LDL in competing for the LDL receptor on either cell type. These results suggest that inheritance of apoE4 is associated with an increased affinity of VLDL particles for LDL receptors on hepatocytes and may partly explain the influence of the E4 isoform on lipid metabolism.
apolipoprotein E genotype; fibroblasts; Hep G2 cells
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INTRODUCTION |
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THERE ARE THREE COMMON polymorphisms of apolipoprotein
E: E2, E3, and E4. Compared with the most common E3 isoform, carriers of the E2 isoform have lower plasma cholesterols, whereas the opposite
holds for carriers of the E4 isoform (4). Genetic variation in apoE
also influences plasma apoE concentrations, E2 > E3 > E4 (4), and
the plasma clearance of apoE, E4 > E3 > E2 (12, 13), of
chylomicron remnants, E4 E3 > E2 (3, 25), and of very low density
lipoprotein (VLDL) and low-density lipoprotein (LDL) (8). The evidence
on how the E2 isoform influences lipid metabolism indicates that,
compared with the E3 isoform, E2 is a poor ligand for the LDL receptor
when incorporated into phospholipid complexes (26) and is less
effective in mediating binding of lipoproteins to cell surface
proteoglycans and to LDL-receptor-related protein (17, 26). There is
little comparable information on the E4 isoform despite its importance
as a risk factor in coronary artery disease (27). As indicated above,
the influence of the E4 isoform on plasma cholesterol and lipoprotein
clearance is opposite to that of the E2 isoform, suggesting that apoE4
might be associated with increased LDL-receptor binding. Although
apoE4- and apoE3-containing synthetic phospholipid complexes were found to compete similarly against LDL for the LDL receptor (26), these
synthetic complexes might not reflect the binding of native lipoproteins. We decided therefore to test the hypothesis that inheritance of the E4 isoform increases the affinity of VLDL and LDL
particles to the LDL receptor. The lipoproteins were isolated from
apoE4 and -E3 homozygous subjects, and their affinity for the LDL
receptor was studied in cultured fibroblasts and Hep G2 cells.
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MATERIALS AND METHODS |
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Study subjects. Binding studies were
carried out to determine the LDL-receptor affinities of VLDL and LDL
isolated from eight pairs of E3/3 and E4/4 subjects who were matched as
closely as possible in regard to age and serum lipid levels (Table
1). The E3/3 and E4/4 subjects studied were
aged 45 ± 7 (SD) and 42 ± 6 yr, respectively, and were
Caucasian males who were not on any lipid-lowering
therapy. The study was approved by the Ethics Committee of
Royal Perth Hospital.
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Blood collection and lipid and apolipoprotein
analyses. After an overnight fast, blood was collected
for isolation of LDL and VLDL, extraction of genomic DNA, and
estimation of serum lipids. Serum total and high-density lipoprotein
(HDL) cholesterol was measured enzymatically (Miles, Tarrytown, NY),
with HDL cholesterol measured after precipitation of -lipoproteins
with dextran sulfate. LDL and VLDL protein was measured by the Lowry
method as modified for lipoproteins (21). Plasma and LDL apolipoprotein
B (apoB) were measured by an immunonephelometric method on the Behring nephelometer, and VLDL apoB was estimated as isopropanol-insoluble VLDL
protein (16). ApoE genotyping was carried out on genomic DNA with the
method of Hixson and Vernier (14).
Isolation of lipoproteins. Blood was collected into 0.15% EDTA (final) and immediately centrifuged. Phenylmethylsulfonyl fluoride, gentamycin, and aprotinin were then added to final plasma concentrations of 1 mmol/l, 80 mg/l, and 10,000 U/l, respectively (9). LDL was isolated by equilibrium density gradient ultracentrifugation as we have previously described (20), dialyzed at 4°C against 250 volumes of 150 mmol/l NaCl, 10 mmol/l phosphate (PBS) containing 0.01% EDTA for 24 h, passed through a 0.45-µm filter, and stored under nitrogen at 4°C. VLDL was isolated by flotation ultracentrifugation at a density of 1.006 g/ml. Briefly, 5.5 ml of plasma was overlaid with 2.5 ml of a NaCl solution of density 1.006 g/ml containing 0.01% EDTA and was centrifuged in a Ti80 rotor at 50,000 rpm for 18 h at 20°C. VLDL was then removed from the top 1.0 ml. To ensure minimal contamination by lipoproteins of higher density, the VLDL was washed by a further centrifugation under the same conditions, dialyzed, and stored as described for LDL. In some experiments, VLDL-free plasma (VFP) comprised of the infranatant from the first centrifugation was also collected, dialyzed, and stored as described for LDL and VLDL.
Cell culture. Monolayer cultures of foreskin fibroblasts from a healthy male and Hep G2 cells were maintained in 75-cm2 flasks with Dulbecco's modified Eagle's medium (DMEM, GIBCO, Grand Island, NY) supplemented with 10% fetal calf serum (GIBCO). For binding experiments, cells were grown to 75% confluence in 24-well plates, washed twice with PBS, and incubated in DMEM supplemented with lipoprotein-deficient serum (LPDS) for 24-48 h to upregulate LDL receptors before binding studies. LPDS was prepared from fetal calf serum as described by Goldstein et al. (11).
Radioiodination of LDL. LDL was labeled with 125I by the iodine monochloride method as modified for lipoproteins by Bilheimer et al. (2), and then it was immediately dialyzed and filtered as described above, stored at 4°C, and further dialyzed for 24 h just before binding studies. Studies on eight 125I-LDL preparations showed that 92 ± 3% of the radioactivity was associated with apoB, 98 ± 2% with protein, and 2 ± 1% with lipid as determined on isopropanol and trichloroacetic acid precipitates and chloroform extracts, respectively, of the radioiodinated LDL.
Binding studies. Conditions for
binding studies were as previously described (21). Cells were
preincubated in assay buffer consisting of Eagle's minimal essential
medium (ICN Biomedicals, Aurora, OH) containing 2.5 mmol/l HEPES, 42 mmol/l NaHCO3, and 4 mg/ml BSA (pH
7.4) for 20 min at 4°C. For each E4/4 and E3/3 pair studied, LDL
from the E3/3 subject was radioiodinated as described previously (sp.
act. 164 ± 48 counts · min1 · ng
apoB
1,
n = 8) and added to the medium at a
final concentration of 10 µg apoB/ml, simultaneously with a range of
concentrations of unlabeled test VLDL or LDL and incubated for 2 h at
4°C. Cells were then washed five times with ice-cold assay buffer
and twice with ice-cold PBS containing 1.8 mmol/l
CaCl2 and 0.8 mmol/l
MgSO4 and then were solubilized by
a 10-min incubation in 0.2 mol/l NaOH, and an aliquot was taken for
gamma counting.
Data analysis and statistics. The concentrations of VLDL or LDL required to inhibit binding of 125I-LDL to the LDL receptor by 50% (IC50) were determined with a four-parameter logistic fitting equation on the RiaCalc program (LKB Wallac, Turku, Finland). Concentrations of VLDL and LDL are expressed in terms of their apoB content. Whereas individual IC50 values are reported, the ratios of the IC50 values for the pairs of E4/4 and E3/3 subjects are also reported and are more relevant for statistical purposes because this method of expression reduces the influence of interassay variability. Values are expressed as means ± SD, and differences in values were compared with a two-tailed Student's t-test unless otherwise indicated.
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RESULTS |
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Table 1 shows the fasting serum lipid levels of the E4/4 and E3/3 subjects studied. Fasting serum cholesterol and triglycerides were above recommended levels but were within one standard deviation of the mean for male subjects of a similar age as reported for the Australian population (1989 National Heart Foundation of Australia Risk Factor Survey) and were similar for E3/3 and E4/4 subjects.
Direct binding studies on radiolabeled LDL from these subjects showed
high-affinity binding in both of the cell types studied. For the LDL of
eight E3/3 and eight E4/4 subjects, we found an overall dissociation
constant
(Kd)
of 4.7 ± 1.4 nmol/l in fibroblasts and for six E3/3 and six E4/4
subjects, 22 ± 10 nmol/l in Hep G2 cells. We also found that VLDL
could compete against LDL for this high-affinity binding. Figure
1 shows typical results for VLDL from a
pair of E4/4 and E3/3 subjects competing against
125I-LDL for binding to Hep G2
cells. Individual IC50 values and IC50 ratios in paired E4/4 and
E3/3 subjects in both fibroblasts and Hep G2 cells are presented in
Table 2.
IC50 values are expressed, as
outlined in the METHODS, in terms of
micrograms apoB per milliliter. In Hep G2 cells, E4/4 VLDL was more
effective than E3/3 VLDL in competing for the LDL receptor, the
IC50 ratio being significantly lower than unity (0.73 ± 0.31, P < 0.05). IC50 values themselves were marginally lower in E4/4 than E3/3 subjects (3.7 ± 1.3 vs. 6.1 ± 3.7, P < 0.08). These
significance values would change to P < 0.02 and P < 0.05, respectively,
with one-tailed t-tests based on our
initial hypothesis. In contrast to results on Hep G2 cells, the data do
not support a difference in the affinity of E4/4 and E3/3 VLDL for the
LDL receptors on fibroblasts.
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The composition of the VLDLs used in these binding studies is shown in
Table 3. There was no significant
difference in the lipid composition of VLDL from the E4/4 and E3/3
subjects and no relationship between the triglyceride or cholesterol
content, or triglyceride-to-cholesterol ratio, of the VLDLs and their
corresponding IC50 values or
IC50 ratios for studies in either
cell type.
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Competitive studies on LDL from E3/3 and E4/4 subjects were also
carried out, and the results are presented in Table
4. In summary, those results showed that
there was not a significant difference between the affinity of LDL
particles from E4/4 and E3/3 subjects in either Hep G2 cells or
fibroblasts. To determine whether the method of preparation of LDL
could have affected receptor affinity and abolished an E
isoform-dependent effect, we compared LDL to homologous VFP in ability
to compete against 125I-LDL for
receptor binding. The results, summarized in Table
5, showed that there was no difference
between the IC50 values of these
two preparations in fibroblasts. In Hep G2 cells, VFP was marginally a
better competitor than LDL, but there was no difference between E4/4
and E3/3 subjects.
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DISCUSSION |
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The important new finding is that inheritance of the E4 isoform is associated with an increased affinity of VLDL particles for the LDL receptor on cultured liver cells. It has previously been shown that apoE can mediate the binding of large VLDL [Svedberg units (Sf > 100)] from hypertriglyceridemic subjects and small VLDL (Sf range 20-60) from normal subjects to the LDL receptor (7, 10). We found that VLDL from normal subjects competes against LDL for binding to high-affinity binding sites, that is, specifically the binding to LDL receptors. On the basis of the competitive binding studies, we also found that homozygosity for the E4 isoform increased the affinity of the VLDL of the subject for the LDL receptor on Hep G2 cells. The binding of VLDL was expressed relative to apoB to reflect affinity in terms of lipoprotein particle numbers because each VLDL particle contains only a single apoB molecule. VLDL apoE was not quantitated, and the increased affinity of VLDL from E4 homozygous subjects may be due to the extra positive charge conferred by arginine at amino acid 112 in apoE4 (26) or to apoE content. Concerning other VLDL components, there was no significant difference in the triglyceride or cholesterol content of VLDL from the E4/4 and E3/3 subjects, and there was no correlation between the triglyceride or cholesterol content, or triglyceride-to-cholesterol ratio, of the VLDLs and the binding results.
There are few studies in the literature with approaches comparable to ours. Recently, Bohnet et al. (5), studying direct binding, showed that the affinity of 125I-labeled VLDL for Hep G2 cells was higher for VLDL from subjects without an E2 allele (E4/4, E4/3, E3/3) than those with an E2 allele. However, no difference could be discerned, after adjustment for apoB content as in our study, between the affinities of VLDL from E4/4, E4/3, or E3/3 subjects. This contrasts with the competitive binding studies presented here, which were used in preference to direct binding studies because they permit comparison of relatively unaltered VLDLs, whereas direct binding studies require the use of harsh reagents, such as iodine-monochloride, and exposure to a strong radiation source (125I), which has been shown to change lipoprotein composition and cell binding (19). Indeed, in direct binding studies on the VLDL of six E4/4 and six E3/3 subjects (not shown), we also did not find any difference in affinity between E4/4 and E3/3 subjects in binding to Hep G2 cells at 4°C (Kd values: 22 ± 11 vs. 21 ± 10 nM, respectively). A further advantage of the competition studies is that they allow comparison of VLDLs in their ability to inhibit high-affinity binding of 125I-LDL to LDL receptors; that is, the binding studied is specifically that to LDL receptors, whereas, conceptually, direct binding of VLDL may also reflect binding to other VLDL or apoE receptors. That the binding of 125I-LDL was specific for the LDL receptor is based on the following considerations. First, binding of 125I-LDL to the cells could be downregulated by incubation of the cells in LDL-containing nutrient medium, that is, medium containing unmodified fetal calf serum instead of lipoprotein-depleted fetal calf serum (data not shown). Second, the binding was of high affinity and yielded Scatchard plots consistent with the presence of one major binding site for LDL. In addition, whereas other receptors, such as the scavenger receptor, are thought capable of binding oxidized or chemically modified LDL, the LDL receptor is the only receptor yet described capable of binding unmodified LDL.
In contrast to our competitive binding results on Hep G2 cells, there was little difference between the affinity of E4/4 and E3/3 VLDL for the LDL receptor of cultured fibroblasts. This may be related to differences in binding characteristics between the LDL receptor of fibroblasts and of Hep G2 cells because differences between these cells have been clearly documented. For example, the LDL receptor on Hep G2 cells and on liver membranes has a lower affinity for LDL than that on fibroblasts, as reported by us and others (15, 21, 24). Furthermore, whereas the uptake of hypertriglyceridemic LDL by cultured fibroblasts was found to be lower than that of normal LDL, the reverse was observed for Hep G2 cells (23).
Few studies have examined the role of apoE in binding of LDL to the LDL
receptor, possibly because the concentration of apoE in LDL is low.
However, Chappell et al. (6) showed that apoE mediates the binding of
large LDL to the LDL receptor, and indirect evidence from the study of
a subject homozygous for the apoB
Arg3500Gln mutation
suggests that apoE can mediate LDL binding (22). There is also evidence
of apoE genotype dependence; Gregg and Brewer (12) found a lower plasma
clearance rate for the LDL of E2 homozygotes, and a preliminary report
by Arnold et al. (1) suggests that LDL from E2/2 subjects binds with
lower affinity to the LDL receptor. We did not find a difference in
binding affinity of LDL from E4/4 and E3/3 subjects, and the concordant
result on VFP, comprised primarily of LDL as the major LDL-receptor
binding species, suggests that this was not due to an artifact produced
by LDL isolation.
However, that the E4 isoform increases binding of VLDL to the LDL receptors of hepatocytes has implications for the metabolism of LDL as well as for VLDL. The elevated LDL plasma concentration of E4/4 subjects has been related by Demant et al. (8) to increased production and decreased catabolism; it was postulated that the latter may be related to the increased intestinal cholesterol absorption (18) and chylomicron remnant clearance in E4/4 subjects (25), which, by increasing the hepatic cholesterol pool, downregulates LDL-receptor expression and hence hepatic receptor-mediated clearance of LDL (8). Perhaps increased delivery of lipids by E4/4 VLDL also augments the hepatic cholesterol pool. Additionally, VLDL may retard hepatic uptake of LDL more in E4/4 subjects by greater competition with LDL for uptake.
In conclusion, homozygosity for apoE4 increases binding of VLDL to LDL receptors on cultured hepatic cells; this might partly explain the variations in lipoprotein metabolism and the propensity to atherosclerosis associated with the E4 genotype.
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ACKNOWLEDGEMENTS |
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This study was supported by the Medical Research Fund of Western Australia, Len Buckeridge, and the Royal Perth Hospital Medical Research Foundation.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: C. D. S. Mamotte, Dept. of Biochemistry, Royal Perth Hospital, GPO Box X2213, Perth, WA 6001, Australia.
Received 19 June 1998; accepted in final form 10 November 1998.
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