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Original Article |
Correspondence to: Ko Willems van Dijk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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To investigate the quantitative requirement for apolipoprotein (apo) E in the clearance of lipoproteins via the non-low density lipoprotein (LDL) receptor mediated pathway, human APOE was overexpressed at various levels in the livers of mice deficient for both the endogenous Apoe and Ldlr genes (Apoe -/- · Ldlr -/-) using adenovirus-mediated gene transfer. We found that a low level of APOE expression, that was capable of reducing the hyperlipidemia in Apoe -/- mice, did not result in a reduction of the hyperlipidemia in Apoe -/- · Ldlr -/- mice. Surpisingly, a very high level of APOE expression also did not result in a reduction of hypercholesterolemia in Apoe -/- · Ldlr -/- mice, despite very high levels of circulating apoE (>160 mg/dl). Only a moderately high level of APOE expression resulted in a reduction of serum cholesterol level (from 35.2 ± 6.7 to 14.6 ± 2.3 mmol/l) and the disappearance of VLDL from the serum. Moreover, the very high level of APOE expression resulted in a severe hypertriglyceridemia in Apoe -/- · Ldlr -/- mice and not Apoe -/- mice (25.7 ± 8.9 and 2.2 ± 1.8 mmol/l, respectively). This hypertriglyceridemia was associated with an APOE-induced increase in the VLDL triglyceride production rate and an inhibition of VLDL-triglyceride lipolysis.
We conclude from these data that, for efficient clearance, the non-LDL receptor-mediated pathway requires a higher level of APOE expression as compared to the LDL receptor, but is more sensitive to an APOE-induced increase in VLDL production and inhibition of VLDL-triglyceride lipolysis.—van Dijk, K. W., B. J. M. Van Vlijmen, H. B. van't Hof, A. van der Zee, S. Santamarina-Fojo, T. J. C. van Berkel, L. M. Havekes, and M. H. Hofker. In LDL receptor-deficient mice, catabolism of remnant lipoproteins requires a high level of apoE but is inhibited by excess APOE. J. Lipid Res. 1999. 40: 336–344.
Supplementary key words: adenovirus-mediated gene transfer, LDL receptor-related protein, hypertriglyceridemia, VLDL-triglyceride lipolysis, hepatic VLDL triglyceride production
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Apolipoprotein (apo) E plays a key role in the metabolism of chylomicron and very low density lipoprotein (VLDL) remnants by functioning as a ligand for receptor-mediated uptake of these lipoproteins by the liver (for review, see ref. (1)). The important role of apoE was recognized from studies on patients with type III hyperlipidemia, which is characterized by serum accumulation of chylomicron and VLDL remnants and is associated with mutations in the APOE gene (for reviews, see refs. (2), (3)). Additional evidence was provided by the analysis of Apoe-deficient mice, which demonstrate massive accumulation of lipoprotein remnants (4) (5) (6). One important receptor involved in remnant lipoprotein uptake is the LDL receptor, an apoB/E specific receptor which can be responsible for up to 75% of total remnant uptake (7) (8) (9) (10). In the absence of the LDL receptor, the LDL receptor-related protein (LRP) is thought to provide the backup mechanism (11) (12) (13). This was demonstrated by the accumulation of chylomicron and VLDL remnants in the serum of LDL receptor-deficient mice injected with an adenovirus carrying the receptor-associated protein (RAP), a potent inhibitor of LRP ligand interaction (14) (15). More recently, similar observations have been made after liver specific inactivation of the LRP gene (16). Additional liver recognition sites for remnant lipoproteins have been postulated, including proteoglycans (17), the remnant receptor (18), and the lipolysis-stimulated receptor (19), although their significance remains to be elucidated.
At present it is unclear which factor(s) determine the specificity of remnants for the different receptor systems in vivo. Data from rat liver perfusion studies suggest that the size of the remnant particle is an important determinant in receptor recognition (20). This has been confirmed in vivo using apoE-enriched artificial `neo-chylomicrons' (21). In addition to size, the apoE content of a remnant particle influences receptor specificity. From ligand-blotting experiments it was concluded that binding of ß-VLDL to the LRP requires apoE enrichment, despite the already abundant presence of apoE on these particles (22). To account for these observations, in vivo uptake of remnants via the LRP has been postulated to occur after enrichment with apoE that is bound to cell surface heparan sulfate proteoglycans (HSPG) in the space of Disse (the so-called secretion re-capture process (23) (24) (25)).
To further investigate the quantitative requirement of apoE in the clearance of lipoproteins via the non-LDL receptor-mediated pathway in vivo, human APOE was over-expressed at various levels in the livers of mice deficient for both the endogenous Apoe and Ldlr genes (Apoe -/- · Ldlr -/-) using adenovirus-mediated gene transfer. We have found that in the absence of the LDL receptor, low APOE expression was not sufficient for remnant removal. High APOE expression was needed for removal of remnants via the non-LDL receptor-mediated route, but this effect was counteracted by the APOE-induced increase in liver VLDL-triglyceride production and inhibition of LPL-mediated VLDL-triglyceride lipolysis. Thus, the APOE expression level is an important factor determining serum lipid levels when LDL receptor-mediated clearance is disturbed.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Generation and analysis of transgenic mice
ApoE-deficient (Apoe -/-) mice were created as described previously (6). LDL receptor-deficient (Ldlr -/-) mice (26) were purchased from the Jackson Laboratory (Bar Harbor, ME). Apoe -/- mice were cross-bred with Ldlr -/- mice to obtain mice that lack both the apoE and LDL receptor gene (Apoe -/- · Ldlr -/- mice). The resulting breeding offspring was analyzed for the endogenous Apoe -/- and Ldlr -/- genotype through tail-tip DNA analysis, as described earlier (6) (26). C57Black/6J/Icco mice were purchased from the Broekman Institute, Someren, The Netherlands.
For experiments, female mice 8–12 weeks of age were included. Mice were housed under standard conditions in conventional cages and given free access to food (i.e., a standard chow diet) and water.
Adenovirus transfections
The generation of the recombinant adenoviral vectors expressing APOE (Ad-APOE) under control of the CMV promotor has been described (27). The recombinant adenoviral vector expressing LacZ (Ad-LacZ) under control of the CMV promotor was kindly provided by Dr. T. Willnow and Dr. J. Herz (15). The recombinant adenovirus was propagated and titrated on the Ad5 E1-transformed human embryonic kidney cell line 911 as described (28). For storage, the virus was supplemented with mouse serum albumin (0.2%) and glycerol (10%), and aliquots were flash-frozen in liquid N2, and stored at -80°C. Routine virus titers of the stocks varied from 1 to 5 x 1010/ml.
For in vivo adenovirus transfection, on day zero, 0.5 x 109 - 2.0 x 109 plaque-forming units in a total volume of 200 µl (diluted with PBS) were injected into the tail vein of female mice. Fasted blood samples were drawn from the tail vein of fasted mice at 5, 8, 11, and 14 days after virus injection.
Lipid, lipoprotein, and apoE measurements
Adenovirus-injected mice were fasted from 9 AM to 1 PM and approximately 100 µl of blood was obtained from each individual mouse through tail-bleeding. Total serum cholesterol and triglyceride levels (without measuring free glycerol) were measured enzymatically using commercially available kits: #236691 (Boehringer Mannheim, Mannheim, Germany) and #337-B (Sigma Chemical Co., St. Louis, MO).
For determination of the serum lipoprotein distribution, some 100 µl of pooled serum was subjected to density gradient ultracentrifugation according to Redgrave, Roberts, and West (29). After ultracentrifugation, the volume was fractionated in fractions of 0.5 ml and density was measured using a DMA 602M densitometer (Paar, Germany). Fractions with density d < 1.006, 1.006 –1.019, 1.019–1.063, and 1.063–1.120 g/ml correspond to VLDL, IDL, LDL, and HDL, respectively. After dialysis against PBS, containing 10 µM EDTA (pH 7.4), lipoprotein fractions were analyzed enzymatically for cholesterol and triglyceride content, using kits #236691 and #701904 (Boehringer Mannheim, Germany), respectively.
Human apoE concentrations were measured by sandwich ELISA as described previously (30).
Characterization of VLDL
Total and free cholesterol, triglyceride (without glycerol), and phospholipid content of the VLDL (d < 1.006 g/ml) fraction were measured enzymatically, using commercially available kits (#236691 and #310328: Boehringer Mannheim, Mannheim, Germany; #337-B:Sigma Chemical Co., St. Louis, MO; and 990-54009: Wako Chemicals GmbH, Neuss, Germany, respectively). VLDL protein was determined using the method of Lowry et al. (31). Human apoE levels were measured by sandwich ELISA as described previously (30). Particle size of the d < 1.006 g/ml lipoproteins was determined by photon correlation spectroscopy using a Malvern 4700C system (Malvern Instruments, UK).
Immunohistochemical procedures for light microscopy
Fasted adenovirus-injected animals were anesthetized with 7.5 ml/kg body weight of a mixture of Hypnorm (Janssen-Cilag, Animal Health, Sandilon, UK), Ketamin (Nimatek®, Eurovet, Bladel, The Netherlands), Thalamonal (Janssen-Cilag bv, Tilburg, The Netherlands). Livers were perfused through the portal vein with ice-cold saline (flow rate 1 ml/min for 3 min), followed by 5 min perfusion with ice-cold 2% paraformaldehyde in PBS (pH 7.4). Paraformaldehyde-fixed, paraffin-embedded 3-µm sections were subjected to immunohistochemistry. For apoE immunostaining, two primary antibodies were used: rabbit anti-mouse apoE polyclonal antibody (kind gift from Dr. P. Groot, SmithKline Beecham Pharmaceuticals, Harlow, UK) and a rabbit polyclonal antibody anti-human apoE. Both antisera were diluted to 1:1000 with PBS, containing 0.1% Tween 20 and 2% normal goat serum. The sections were incubated with the respective antibodies overnight at 4°C. After washing with PBS/0.1% Tween 20, the sections were incubated for 2 h at room temperature with peroxidase-conjugated goat anti-rabbit IgG (Nordic Immunology, Tilburg, The Netherlands) diluted 1:500 with PBS, containing 2% Tween 20 and 2% normal goat serum. Finally, peroxidase detection was performed using diaminobenzidine as a substrate.
Assay of lipolysis with lipoprotein lipase in solution
Lipolysis assays were performed at 37°C in 0.1 M Tris(hydroxymethyl)-aminiomethane (Tris) buffer, pH 8.5, for 5 min with bovine lipoprotein lipase (LPL; 0.2 U, Sigma, St. Louis, MO) in the presence of 2% (w/v) albumin (essentially FFA-free). The reaction was stopped by the addition of 50 mM KH2PO4, 0.1% Triton-X100, pH 6.9, and placed on ice. To obtain a time zero control, the reaction was prevented by adding Triton prior to the addition of LPL and samples were placed on ice. Free fatty acids (FFA) were measured enzymatically with a NeFa-C kit (Wako Chemicals GmbH, Neuss, Germany). The rate of FFA release by 0.2 U LPL was linear for 5 min as used in this assay. The assay was performed at VLDL-triglyceride concentration of 0.2 mmol/l with duplication of FFA determination.
In vivo hepatic VLDL-triglyceride production
After a 4-h fasting period, mice were injected intravenously with Triton WR1339 (500 mg/kg body weight) (32) using 15% (wt/vol) Triton solution in 0.9% NaCl. At 1, 10, and 20 min after injection, blood samples were drawn and analyzed for triglycerides as described above. Production of hepatic triglyceride was calculated from the slope of the curve and expressed as mmol/h per kg body weight.
Statistical analysis
Differences in lipid parameters of comparable groups after different treatment regimens or of the same group of mice at different time points were evaluated using the GraphPad Prism software (San Diego, CA). As the measured parameters showed non-Gaussian distributions, the non-parametric Mann-Whitney rank sum test was used. P values less than 0.05 were regarded as significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Serum lipid, apoE, and lipoprotein levels after adenovirus-mediated gene transfer of APOE in Apoe -/- and Apoe -/-? Ldlr -/- mice
To determine the role of apoE in the clearance of lipoproteins by the liver in the absence of the LDL receptor, APOE was over-expressed at different levels in Apoe -/- · Ldlr -/- mice using adenovirus-mediated gene transfer. Apoe -/- mice were included for comparison. To obtain different APOE expression levels, 5 x 108 PFU (low dose) and 2 x 109 PFU (high dose) of adenovirus carrying the human APOE3 cDNA driven by the CMV promotor (Ad-APOE) were injected into the tail vein of Apoe -/- and Apoe -/- · Ldlr -/- mice. As a control, similar groups of mice were injected with a high dose of an adenovirus carrying the gene encoding ß-galactosidase driven by the CMV promotor (Ad-LacZ). The mice were followed for up to 14 days.
As can be seen in Figure 1 A, both the low and high dose of Ad-APOE resulted in complete rescue of hypercholesterolemia of Apoe -/- mice on day 5 after injection, whereas a high dose of Ad-LacZ had no effect. From day 5 onwards, concurrent with the decrease of serum apoE level, serum cholesterol level gradually increased (Figure 1A and Figure E). A mild hypertriglyceridemia was evident on day 5 in the Apoe -/- mice injected with the highest dose of Ad-APOE (Figure 1C).
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In contrast, neither the low dose nor the high dose Ad-APOE had any effect on serum cholesterol level in Apoe -/- · Ldlr -/- mice on day 5 after injection (Figure 1B), despite appropriate APOE expression (serum apoE levels of 4.0 ± 3.8 and 166 ± 67.7 mg/dl for low and high dose, respectively; Figure 1F). However, on day 8, when adenovirus-mediated APOE expression was reduced, in Apoe -/- · Ldlr -/- mice injected with the high dose of Ad-APOE (serum apoE level of 10.5 ± 8.2 mg/dl; Figure 1F), serum cholesterol was significantly (P < 0.05) lowered (from 32.2 ± 7.5 to 14.6 ± 2.5 mmol/l; Figure 1B), approaching the serum cholesterol level of Apoe +/+ Ldlr -/- mice (8–10 mmol/l). At the peak of APOE expression on day 5, the high dose of Ad-APOE resulted in severe hypertriglyceridemia (serum triglyceride of 25.7 ± 9.8 mmol/l, Figure 1D). The low dose of Ad-APOE did not result in a reduction of serum cholesterol at any time point evaluated, nor did it affect serum triglyceride levels. Thus, reduction of hypercholesterolemia in Apoe -/- · Ldlr -/- mice was only evident within a certain window of APOE expression and a high serum apoE level was associated with hypertriglyceridemia.
The lipoprotein fractions of Apoe -/- mice and Apoe -/- · Ldlr -/- mice 5 and 8 days after adenovirus injection were analyzed by ultracentrifugation density gradient fractionation ( Figure 2). In Apoe -/- mice injected with Ad-LacZ, a prominent peak is present in the VLDL-sized region on day 5, whereas in Apoe -/- mice injected with the high dose of Ad-APOE, lipoproteins from the VLDL-sized range had completely disappeared both on day 5 and day 8 (Figure 2A). These latter two ultracentrifugation profiles were identical to those of non-treated wild-type mice (data not shown). In Apoe -/- · Ldlr -/- mice injected with the high dose of Ad-APOE, a large peak was present in the VLDL-sized region on day 5, similar to the Ad-LacZ-injected mice (Figure 2B), the only difference being the appearance of an HDL-sized peak in the Ad-APOE-injected group. On day 8, IDL/LDL peaks were still present, whereas the VLDL peak had completely disappeared from the Apoe -/- · Ldlr -/- mice injected with the high dose of Ad-APOE, indicating rescue of the Apoe -/- · Ldlr -/- phenotype towards that of a Ldlr -/- mouse (26).
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Characterization of the d < 1.006 lipoproteins after adenovirus-mediated APOE gene transfer
To further investigate the basis for the difference in serum VLDL levels after Ad-APOE administration, the apoE and lipid content of d < 1.006 lipoproteins (VLDL) were determined on day 5 and day 8 after injection of the high dose Ad-APOE ( Table 1). The main difference in the composition of the VLDL between the different mice and time points was in the apoE and triglyceride content. Circulating VLDL in Apoe -/- mice that had received a high dose of Ad-APOE contained 6-fold more apoE on day 5 as compared to day 8 (7.4 and 1.3 µg/mg protein, respectively). In addition, the VLDL from Apoe -/- mice given the high dose of Ad-APOE was enriched in triglycerides on day 5 as compared to day 8 (6.5 and 0.7 µmol/mg protein, respectively).
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On day 5, circulating VLDL in Apoe -/- · Ldlr -/- mice that had received a high dose of Ad-APOE also contained much more apoE as compared to VLDL circulating on day 8 (114.1 and 5.5 µg/mg protein, respectively). On day 5 the apoE level of Apoe -/- · Ldlr -/- mice was dramatically higher than that of Apoe -/- mice. Apparently, this difference can be attributed to the absence of the LDL receptor. As was the case for the Apoe -/- mice, the VLDL from Apoe -/- · Ldlr -/- mice given the high dose of Ad-APOE was enriched in triglycerides on day 5 as compared to day 8 (respectively, 6.8 and 1.2 µmol/mg protein). The accumulation of the apoE and triglyceride-rich VLDL on day 5 in the Apoe -/- · Ldlr -/- mice clearly indicated a disturbance of the VLDL metabolism that apparently could not be alleviated via the non-LDL receptor-mediated pathway.
Immunohistochemical localization of apoE in the liver after adenovirus-mediated APOE gene transfer
To confirm that adenovirus-derived apoE was available for the secretion–recapture process (22) (23) (24) both on day 5 and day 8 after injection of the high dose Ad-APOE in Apoe -/- · Ldlr -/- mice, immunohistochemical localization studies of apoE in perfusion-fixed livers were performed. As can be seen in Figure 3, apoE was clearly present in the regions lining the sinusoids in Apoe -/- · Ldlr -/- mice both on day 5 (Figure 3C) and on day 8 (Figure 3D) after injection of the high dose Ad-APOE. On day 8, apoE staining was clearly much less intense than on day 5, although at this time point efficient VLDL clearance did occur. The staining pattern of human apoE largely resembled that of mouse apoE in Ldlr -/- mice (Figure 3A). Apoe -/- · Ldlr -/-mice not injected with Ad-APOE were negative for human apoE (Figure 3B).
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Analysis of the disturbance in triglyceride metabolism after adenovirus-mediated gene transfer of APOE
Five days after injection of a high dose of Ad-APOE, serum triglyceride levels in both Apoe -/- mice and Apoe -/- · Ldlr -/- mice were significantly increased (Figure 1C, Figure 1D). Specifically, in the Apoe -/- · Ldlr -/- mice, there was a correlation in individual mice between serum apoE level and serum triglyceride level. This observation was extended by injecting three additional groups of 6 –7 mice each with, respectively, 0.75 x 109, 1.0 x 109, and 1.25 x 109 PFU of Ad-APOE. The correlations between serum apoE level and serum triglyceride level and between serum apoE level and cholesterol level are shown in Figure 4 (on day 5 after Ad-APOE injection). The strong positive correlation between serum apoE and serum triglyceride (r = 0.96, P < 0.0001; Figure 4A) and not serum cholesterol level (r = 0.37, P = 0.05, Figure 4B) is indicative of an apoE-related effect on the triglyceride metabolism. This could be the result of an increased VLDL production rate correlated with increased APOE expression or the result of an inhibition of triglyceride lipolysis mediated by the effect of excess apoE on LPL.
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Production of VLDL-triglycerides was measured by determining the triglyceride secretion rate after Triton WR1339 injection on day 5 in wild-type C57Black/6J mice injected with a high dose of Ad-APOE or a high dose of Ad-LacZ ( Figure 5). C57Black/6J mice injected with a high dose of Ad-APOE displayed a 4-fold higher VLDL-triglyceride production rate as compared to similar mice injected with a high dose of Ad-LacZ (0.200 ± 0.082 versus 0.053 ± 0.005 mmol/kg per h, P < 0.01). On day 8, VLDL-triglyceride production rate has normalized in the C57Black/6J mice injected with the high dose of Ad-APOE as compared with the mice injected with the high dose of Ad-LacZ (0.056 ± 0.004 and 0.063 ± 0.011 mmol/kg per h, respectively).
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To determine VLDL triglyceride lipolysis rate, VLDL (d < 1.006 lipoproteins) were isolated from serum of Apoe -/- · Ldlr -/- mice 5 and 8 days after injection of a high dose of Ad-APOE. The VLDL were characterized and the triglyceride lipolysis rate was determined in vitro in a solution assay using purified bovine LPL ( Table 2). The apoE-rich VLDL on day 5 after adenovirus injection were poor substrates for LPL in this assay as compared with the VLDL isolated on day 8 (8.9 ± 4.9 and 24.2 ± 6.6 µmol FFA/U · min). Thus, both increased VLDL production and inhibited VLDL triglyceride lipolysis contribute to the effect of high APOE expression on serum triglyceride levels.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we have demonstrated that clearance of remnant lipoproteins via the non-LDL receptor-mediated pathway required more apoE as compared to remnant clearance in the presence of the LDL receptor. This was illustrated by the fact that a low dose of Ad-APOE had no effect on serum lipid levels in Apoe -/- · Ldlr -/- mice while an identical dose completely rescued the hypercholesterolemia of Apoe -/- mice. A relatively high level of APOE expression in Ldlr -/- · Apoe -/- mice, such as on day 8 after a high dose of Ad-APOE, did result in a reduction of the serum cholesterol level to approximately the level of Apoe +/+ · Ldlr -/- mice.
We (33) (34) and others (14) (15) (35) (36) have shown that, in mice, the non-LDL receptor-mediated pathway for lipoprotein clearance is RAP sensitive, and most likely involves the LRP. This has recently been confirmed by liver specific inactivation of the LRP in Ldlr -/- mice (16). In vitro studies have demonstrated that the LRP requires abundant apoE on the particle for binding (22). Our present observation that in vivo the non-LDL receptor-mediated pathway required a high level of apoE for efficient clearance is in line with the LRP being the most likely candidate for mediating clearance in the absence of the LDL receptor.
To facilitate liver uptake of remnants via the LRP in vivo, it has been postulated that lipoproteins are enriched with apoE in the space of Disse, the so-called secretion–recapture process (23) (24) (25). Our immunohistochemistry studies indicated that in Apoe -/- · Ldlr -/- mice on day 5 after injection of a high dose of Ad-APOE, apoE was abundantly present in the space of Disse and on the lipoprotein particles. In addition, clearance studies with 
2-macroglobulin indicated that the LRP is active in Apoe -/- · Ldlr -/- mice (data not shown) and thus all the presumed conditions for uptake of remnant lipoproteins via a secretion–recapture process were fulfilled. However, in contrast to Apoe -/- mice, the Apoe -/- · Ldlr -/- mice did not show a reduction in serum cholesterol level on day 5 after injection of a high dose of Ad-APOE. Moreover, the Apoe -/- · Ldlr -/- mice showed a significantly more pronounced hypertriglyceridemia on day 5 than the Apoe -/- mice. These results indicate that at high expression levels of APOE, VLDL is poorly cleared via the non-LDL receptor-mediated pathway, due to apoE-induced changes in VLDL composition.
The high dose of Ad-APOE induced hypertriglyceridemia in both Apoe -/- mice and Apoe -/- · Ldlr -/- mice, albeit to a different extent. The latter mice showed a strong positive correlation between serum apoE and triglyceride levels (Figure 4A), indicative of an apoE-mediated effect on triglyceride production and/or lipolysis. Production of VLDL triglyceride was shown to be dramatically increased on day 5 after administration of the high dose of Ad-APOE to C57/Bl6 mice (Figure 5) and also Apoe -/- mice (data not shown), whereas it was normalized on day 8, both compared to a similar dose of Ad-LacZ (Figure 5). These data indicate that APOE expression, especially at high levels, results in an increase in VLDL-triglyceride production. This is in line with our recent in vitro and in vivo observations in apoE-deficient hepatocytes and Apoe -/- mice, showing that lack of APOE expression resulted in a reduced VLDL triglyceride production rate (37).
The rate of VLDL triglyceride lipolysis was inversely correlated with the apoE content of the particle (Table 2). Several variant forms of human APOE, including APOE2 (146) and APOE3Leiden, have been described that have a direct negative effect on the rate of LPL-mediated triglyceride lipolysis (38) (39) (40). In line with previously reported data (41) (42) (43), our in vivo data show that also the most common APOE variant, APOE3, when present at high levels on the VLDL particle, can also have a detrimental effect on VLDL-triglyceride lipolysis by LPL. The mechanism underlying the apoE-mediated inhibition of lipolysis has not been investigated. One likely explanation is that excess apoE on the remnant particles results in reduced accessibility of LPL to the core triglycerides of the lipoprotein particle. Alternatively, high levels of APOE could result in the displacement of apoC2, the essential cofactor for LPL-mediated triglyceride lipolysis (44) (45), from the lipoprotein particle (40).
The increase in triglyceride content of the particle was associated with almost doubling of the VLDL particle size (from 38.8 ± 2.1 to 69.3 ± 24.0 nm, Table 2) on day 5 after the high dose Ad-APOE in Ldlr -/- · Apoe -/- mice. Whether the size of the particles during the hypertriglyceridemia limited the uptake via non-LDL receptor clearance routes remains to be established. This could be either at the level of direct affinity for the binding site or at the level of physically approaching the binding site, i.e., passage through the hepatic fenestrae. However, the latter has been estimated to be restricting only at sizes beyond 100 nm, and the VLDL particles at day 5 are below this size.
APOE is considered a potential candidate gene for the treatment of recessive type III hyperlipidemia, such as associated with the common APOE2(Arg158–Cys) variant. Interestingly, the level of APOE2(Arg158–Cys) expression in transgenic mice is a determining factor resulting in either hypolipidemia or hyperlipidemia (46), and hypolipidemia is converted to hyperlipidemia by the absence of the LDL receptor (47). The in vivo demonstration that different levels of APOE3 expression can also have either a hypo- or hyperlipidemic effect has direct implications for the application of apoE as a therapeutic agent. For gene therapeutic approaches aimed at obtaining liver expression of APOE, care should be taken to optimize APOE expression levels, at which clearance of VLDL is enhanced but VLDL production and lipolysis are not affected. Our data indicate that, especially when LDL receptor mediated clearance is sub-optimal, APOE expression level requires optimization. In addition, it can be speculated that variation of APOE gene expression level in the population can have a large impact on individual serum lipid levels, not only through a well-known beneficial effect on lipoprotein clearance, but also through a disadvantageous effect on VLDL production and VLDL triglyceride lipolysis.
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FOOTNOTES |
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1 These authors contributed equally to this study. 
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ACKNOWLEDGMENTS |
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We gratefully acknowledge the technical assistance of J. van der Boom, and the input of Dr. M. Gijbels with the interpretation of the histological data. This research was supported by the Netherlands Heart Foundation (projects M93.001 and 96.178). Financial contribution of the specific RTD Programme of the European commission (BIOMED-2, #BMH4-CT96-0898) is gratefully acknowledged. Dr. Marten Hofker is an Established Investigator of the Netherlands Heart Foundation.
Manuscript received June 25, 1998; and in revised form September 28, 1998.
Abbreviations: apo, apolipoprotein; VLDL, very low density lipoproteins; IDL, intermediate density lipoproteins; LDL, low density lipoproteins; HDL, high density lipoproteins; LDLR, LDL receptor; LRP, LDL receptor-related protein; RAP, receptor-associated protein; HSPG, heparan sulphate proteoglycans; Ad, adenoviral vector; CMV, cytomegalovirus
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REFERENCES |
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K. C-W. Yu, Y. Jiang, W. Chen, and A. D. Cooper Rapid initial removal of chylomicron remnants by the mouse liver does not require hepatically localized apolipoprotein E J. Lipid Res., November 1, 2000; 41(11): 1715 - 1727. [Abstract] [Full Text]
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 P. C. N. Rensen, M. C. Jong, L. C. van Vark, H. van der Boom, W. L. Hendriks, T. J. C. van Berkel, E. A. L. Biessen, and L. M. Havekes Apolipoprotein E Is Resistant to Intracellular Degradation in Vitro and in Vivo. EVIDENCE FOR RETROENDOCYTOSIS J. Biol. Chem., March 17, 2000; 275(12): 8564 - 8571. [Abstract] [Full Text] [PDF]
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A. R Mensenkamp, M. C Jong, H. van Goor, M. J. A. van Luyn, V. Bloks, R. Havinga, P. J. Voshol, M. H. Hofker, K. W. van Dijk, L. M. Havekes, et al. Apolipoprotein E Participates in the Regulation of Very Low Density Lipoprotein-Triglyceride Secretion by the Liver J. Biol. Chem., December 10, 1999; 274(50): 35711 - 35718. [Abstract] [Full Text] [PDF]
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B. J. M. van Vlijmen, A. Rohlmann, S. T. Page, A. Bensadoun, I. S. T. Bos, T. J. C. van Berkel, L. M. Havekes, and J. Herz An Extrahepatic Receptor-associated Protein-sensitive Mechanism Is Involved in the Metabolism of Triglyceride-rich Lipoproteins J. Biol. Chem., December 3, 1999; 274(49): 35219 - 35226. [Abstract] [Full Text] [PDF]
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K. W. van Dijk, B. J.M. van Vlijmen, M. P.J. de Winther, B. van ’t Hof, A. van der Zee, H. van der Boom, L. M. Havekes, and M. H. Hofker Hyperlipidemia of ApoE2(Arg158-Cys) and ApoE3-Leiden Transgenic Mice Is Modulated Predominantly by LDL Receptor Expression Arterioscler. Thromb. Vasc. Biol., December 1, 1999; 19(12): 2945 - 2951. [Abstract] [Full Text] [PDF]
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