doi: 10.1161/hq0801.093864
© 2001 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Mice Expressing Only the Mutant APOE3Leiden Gene Show Impaired VLDL Secretion
From the Groningen University Institute for Drug Exploration (A.R.M., J.F.W.B., H.W., R.H., F.K.), Center for Liver, Digestive, and Metabolic Diseases, Faculty of Medical Sciences and University Hospital Groningen, Groningen, the Netherlands; the Gaubius Laboratory TNO-PG (B.T., L.M.H.), Leiden, the Netherlands; and the Departments of Human Genetics (K.W.v.D.) and General Internal Medicine and Cardiology (L.M.H.), Leiden University, Leiden, the Netherlands.
Correspondence to Folkert Kuipers, PhD, Groningen University Institute for Drug Exploration, Center for Liver, Digestive, and Metabolic Diseases, University Hospital Groningen, CMC IV, Room Y2115, Hanzeplein 1, 9713 GZ Groningen, Netherlands. E-mail f.kuipers{at}med.rug.nl
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Abstract |
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Abstract—— Apolipoprotein E (apoE)-deficient mice develop hepatic steatosis and show impaired very low density lipoprotein (VLDL)-triglyceride (TG) secretion. These effects are normalized on the introduction of the human APOE3 gene. To assess whether this apoE effect is isoform specific, we studied hepatic lipid metabolism in mice expressing either APOE3 or the mutant APOE3Leiden on apoe-/- or apoe+/- backgrounds. The transgenes were expressed mainly in periportal hepatocytes, as revealed by in situ hybridization. Mice expressing APOE3Leiden, on the apoe-/- and apoe+/- backgrounds, had fatty livers, which were absent in APOE3/apoe-/- mice. APOE3Leiden/apoe-/- mice showed a strongly reduced VLDL-TG secretion compared with APOE3/apoe-/- mice (48±14 versus 82±10 µmol/kg per hour, respectively). The presence of a single mouse apoe allele increased VLDL-TG secretion in APOE3Leiden/apoe+/- mice (121±43 µ mol/kg per hour) compared with APOE3Leiden/apoe-/- mice. These results show that APOE3Leiden does not prevent development of a fatty liver and does not normalize VLDL-TG secretion in mice with an apoE-deficient background. The presence of a single mouse apoe allele is sufficient to normalize the APOE3Leiden-associated reduction of VLDL-TG secretion but does not prevent steatosis. We conclude that apoE-mediated stimulation of VLDL secretion is isoform specific.
Key Words: very low density lipoproteins • apolipoprotein E • lipoprotein assembly • lipoprotein secretion • steatosis
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Introduction |
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Apolipoprotein E is a constituent of VLDLs, chylomicrons, and HDLs and is essential for receptor-mediated uptake of remnant lipoproteins.1 ApoE deficiency in mice leads to elevated plasma cholesterol levels that are due to the accumulation of remnant lipoproteins,2–4 and apoE deficiency is associated with the development of atherosclerosis.3 In addition, these mice develop a fatty liver when fed normal chow5 and show a decreased VLDL-triglyceride (TG) secretion.4 Recently, we demonstrated that the introduction of human APOE3 in apoE-deficient mice increases VLDL-TG secretion.6 A role of apoE in control of VLDL-TG secretion in animals and cultured cells has also recently been confirmed by other groups.7–9 It has recently been shown that humans with the APOE2/E2 genotype have a decreased VLDL secretion compared with secretion in subjects with the APOE3/E3 genotype.10 In addition, it has been reported that VLDL-apoB secretion is decreased by 30% in humans with the APOE3/E4 genotype compared with those with the APOE3/E3 genotype.11
In humans, the mutant APOE3Leiden isoform is associated with a dominantly inherited form of familial dysbetalipoproteinemia.12–14 The APOE3Leiden gene contains a tandem repeat of codons 120 to 126, yielding a protein of 306 amino acids.12,13 Transgenic mice expressing APOE3Leiden develop hyperlipidemia because of defective binding of E3Leiden-containing remnant lipoproteins to the LDL receptor and to the LDL receptor–related protein and are susceptible to diet-induced atherosclerosis.15 We have shown recently that mice overexpressing APOE3Leiden in the presence of human APOC-I and the endogenous mouse apoe gene display hepatic lipid accumulation and a mildly decreased VLDL-TG secretion.16 Interpretation of these results is hampered by the presence of the APOC-I and apoe genes. To directly establish the effects of APOE3Leiden and the possible compensatory effects of mouse apoe on VLDL-TG secretion and the development of hepatic steatosis, we used mice expressing APOE3Leiden on an apoE-deficient (apoe-/-) or on an apoE-heterozygous (apoe+/-) background. Our data show that expression of only the APOE3Leiden gene is associated with a reduced VLDL-TG secretion and increased hepatic fat content compared with expression of only the human APOE3 gene. In the presence of a single endogenous apoe allele, VLDL-TG secretion in APOE3Leiden-expressing mice is normalized, whereas the dominant effect of APOE3Leiden with respect to the development of steatosis is not overruled.
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Methods |
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Animals
Transgenic mice expressing human APOE3 were generated according to Hogan et al.17 Transgenic offspring were identified by polymerase chain reaction (PCR) analysis and Southern blot analysis on genomic tail–derived DNA. One strain, expressing high levels of human APOE3 in the liver, was subsequently bred with C57BL/6J mice. APOE3Leiden transgenic mice were created as described previously.18,19 The construct used contained the APOE3Leiden gene starting from position -650 before exon 1 to 1.4 kb after the polyA site18 and the so-called hepatic control region.20 The human APOE3 or APOE3Leiden gene was introduced into apoE-deficient mice by crossbreeding the respective APOE transgenic mice with apoe-/- mice as described previously.6,19 To obtain APOE3Leiden/apoe+/- mice, APOE3Leiden/apoe-/- mice were crossbred with C57BL/6J mice. The resulting offspring were analyzed for the presence of human APOE by ELISA and the mouse apoe genotype through tail-tip DNA analysis, as described earlier.21 APOE expression in the liver was measured by Northern blot as described previously.6 Bands were semiquantified by using the computer program Image Master 1D Elite 3.0.
Mice were housed in a light- and temperature-controlled environment. Food and tap water were available ad libitum, and mice were fed a commercial laboratory chow (RMH-B, Hope Farms BV). The animals received humane care, and experimental protocols complied with local guidelines for the use of experimental animals.
Plasma and Liver Tissue Sampling
Groups of mice (n=5) were fasted overnight and anesthetized with halothane. A large blood sample for determination of plasma lipids and VLDL isolation was collected by cardiac puncture. Subsequently, the liver was quickly removed, weighed, and frozen in separate portions for RNA isolation and lipid analysis, respectively. Parts of the liver were stored in paraformaldehyde or slowly frozen in isopentane and used for microscopic examination and in situ hybridization.
In Situ Hybridization
Frozen liver sections (6 µm) were fixed with 8% buffered formalin for 24 hours at 37°C, followed by washing with PBS treated with diethyl pyrocarbonate (DEPC)/PBS twice, 2 times with DEPC/H2O, and 1 time with ethanol, and were subsequently dried by air. Sections were stored at -20°C. Fragments of APOE cDNA were obtained from a pUC-18 vector. The fragments were excised and placed into a pGEM-3zf(+) vector containing a T7 and Sp6 promoter region. To produce digoxigenin-labeled RNA, sense and antisense APOE cDNA was transcribed in vitro with T7 or Sp6 RNA polymerase, respectively, in the presence of digoxigenin-UTP according to the manufacturer’s instructions (Roche Diagnostics). Before hybridization, sections were treated with 0.25 µg/mL proteinase K for 30 minutes at 37°C. After overnight hybridization (1 ng probe per microliter) at 55°C, the sections were treated with RNase (Sigma Chemical Co). The hybridized digoxigenin-labeled probes were immunodetected with anti-digoxigenin Fab fragments conjugated to alkaline phosphatase, and the bound conjugate was visualized with nitro blue tetrazolium/bromo-4-chloro-3-indolyl phosphate (Roche Diagnostics).
Hepatic Lipid Analysis
Livers were homogenized as described previously.6 Hepatic concentrations of triglycerides and free and total cholesterol were measured by using commercial kits (Roche Diagnostics) after lipid extraction according to Bligh and Dyer22 and by resolving the lipid in 2% Triton X-100 in water. Phospholipid content of the liver tissue was determined according to Böttcher et al23 after lipid extraction.
Ketone Bodies
Five mice per group were fasted for 24 hours. Blood was taken by cardiac puncture, and 250 µL of 18% perchloric acid was added to 500 µL whole blood, mixed thoroughly, and kept on ice for at least 10 minutes. The assay was performed as described previously.24
In Vivo VLDL-TG Production Rate
The hepatic VLDL-TG production rate in APOE3Leiden/apoe-/-, APOE3Leiden/apoe+/-, and APOE3/apoe-/- mice (n=5) was determined as reported previously.4,6 In short, mice were fasted 9 hours before the experiment, and 12.5 mg Triton WR-1339 in 100 µL PBS was injected via the penile vein. Tail blood samples were taken under light halothane anesthesia before and at 1, 2, and 3 hours after injection of Triton WR-1339 for TG measurements.
VLDL Isolation, Size Determination, and ApoB Production Measurements
VLDL/IDL was isolated as previously reported.6,25 ApoB concentration was determined by applying VLDL samples to SDS-PAGE in parallel with 4 human LDL-apoB standards.25 Band intensities were determined after silver staining by using an image densitometer (Imagemaster, Amersham Pharmacia Biotech) and comparison with standards, as described previously.6,25 TG and cholesterol contents were determined as in plasma. Phospholipids were determined by using a commercial available kit (WAKO Chemicals).
Plasma Lipids and ApoE
Plasma TG, free fatty acid, free cholesterol, and total cholesterol levels were determined by using commercially available kits (Roche Diagnostics). ApoE concentrations were determined by ELISA as described previously.21 Lipoproteins were separated by using fast protein liquid chromatography on a Pharmacia Superose 6B column.
Hepatic RNA Isolation
mRNA from 
30 mg liver tissue was isolated with the Trizol method (GIBCO) followed by the SV Total RNA Isolation System (Promega RNA) to remove potential DNA contamination.
Reverse Transcription–PCR
Immediately after RNA isolation, 4.5 µg of RNA was used for cDNA synthesis according to manufacturer’s instructions (Roche Diagnostics). PCR results were normalized to 18S RNA levels determined by competitive PCR. PCR was performed in 50 µL cDNA preparations by using primers for the genes for ribosomal 18S RNA,26 diacylglycerol acyltransferase (dgat, GenBank accession No. AF078752), microsomal triglyceride transfer protein (mttp, GenBank accession No. L47970), and fatty acid synthase (fas, GenBank accession No. X13135).
Miscellaneous
Protein concentrations were determined according to the method of Lowry et al27 with BSA used as a standard.
Statistical Analysis
Analysis of data from the 3 groups was performed by ANOVA, followed by Student-Newman-Keuls t test post hoc analysis.
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Results |
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Expression Patterns of APOE3 and APOE3Leiden Transgenes
Steady-state mRNA levels of APOE3Leiden were comparable in APOE3Leiden/apoe-/- mice and APOE3Leiden/apoe+/- mice, as shown by Northern blot analysis (Figure 1). Expression of APOE3 in APOE3/apoe-/- mice appeared to be somewhat lower than APOE3Leiden expression in APOE3Leiden mice. In situ hybridization (Figure 2) revealed that the transgenes were expressed mainly in periportal (zone 1) hepatocytes in APOE3Leiden/apoe-/- (Figure 2A), APOE3Leiden/apoe+/- (Figure 2B), and APOE3/apoe-/- (Figure 2C) mice. This distribution pattern is similar to that of endogenous apoe in rat liver, as described by Massimi et al.28
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Hepatic Lipid Content
The TG content of livers from APOE3Leiden/apoe-/- mice was higher than that of livers from APOE3/apoe-/- mice (Table 1). The TG content in livers from APOE3Leiden/apoe+/- mice tended to be higher than that of livers from APOE3/apoe-/- mice, but this difference did not reach statistical significance because of the relatively large standard deviation. Free cholesterol and cholesteryl ester contents were increased in the APOE3Leiden/apoe-/- mice compared with the other groups. Figure 3 shows the oil red O staining for neutral lipids in frozen liver sections of APOE3Leiden/apoe-/- (Figure 3A), APOE3/apoe-/- (Figure 3B), and APOE3Leiden/apoe+/- (Figure 3C) mice. Livers from mice expressing only APOE3Leiden showed lipid staining along the entire lobule, but it was most prominent in the perivenous areas (zone 3) surrounding the central vein, as described previously for apoE-deficient mice.6 Livers from APOE3Leiden/apoe+/- mice showed staining in perivenous areas exclusively, whereas staining in livers from APOE3/apoe-/- mice was virtually absent.
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To test whether these lipid depositions were due to a disturbed fatty acid ß-oxidation, plasma levels of 3-hydroxybutyric acid were measured after a 24-hour fast. The fasting concentrations of this ketone body were 0.845±0.163, 0.792±0.244, and 0.872± 1.062 mmol/L for APOE3Leiden/apoe-/-, APOE3/apoe-/-, and APOE3Leiden/apoe+/- mice, respectively. Corresponding values for fasting free fatty acid concentrations in plasma were 577±80, 577±104, and 515±157 µmol/L, respectively. No significant differences were observed between the groups, indicating that disturbed ß-oxidation is unlikely the cause of fat accumulation in APOE3Leiden-expressing mice.29
Plasma Lipids
Cholesterol levels were comparable between the groups and similar to those reported previously for wild-type C57BL/6J mice fed chow.4,6 TG levels in APOE3Leiden/apoe+/- mice were increased compared with those in APOE3Leiden/apoe-/- and APOE3/apoe-/- mice (1.3±0.5 versus 0.4±0.2 and 0.5±0.1 mmol/L, respectively; P<0.05). Figure 4 shows TG (Figure 4A) and cholesterol (Figure 4B) profiles after fast protein liquid chromatography separation of plasma lipoproteins. Differences in plasma TG were mainly due changes in TG contents of the VLDL-sized fractions, as shown in Figure 4A. Whereas plasma cholesterol levels were similar in all groups, there was a clear shift from LDL/IDL-sized fractions to HDL-sized fractions in the presence of mouse apoe gene, as shown in Figure 4B. APOE3Leiden concentration in plasma of the APOE3Leiden-expressing mice was higher than that of APOE3 in the APOE3/apoe-/- mice (0.34±0.05 versus 0.020±0.003 mg/dL, respectively; P<0.05). The presence of a single apoe allele resulted in a 10-fold increase of plasma APOE3Leiden concentration (3.64±0.49 mg/dL).
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VLDL Production Rate
The VLDL-TG production rate in APOE3Leiden/apoe-/- mice, as measured after injection of Triton WR-1339, was strongly reduced compared with that in APOE3/apoe-/- mice. The presence of a single mouse apoe allele clearly increased the VLDL-TG production in APOE3Leiden-expressing mice compared with that in APOE3-expressing mice (Table 2) and in wild-type (C57BL/6J) mice.6 The apoB-100 production rate was decreased in APOE3Leiden/apoe-/- mice compared with APOE3/apoe-/- mice, whereas apoB-48 production was not affected. ApoB-100 and apoB-48 production rates in APOE3Leiden/apoe+/- mice were similar to the rates in APOE3/apoe-/- mice. The pool size of apoB-100 in APOE3Leiden/apoe+/- mice was strongly elevated compared with both other strains, in accordance with elevated TG content of VLDL-sized fractions.
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VLDL Composition
VLDL particle composition was determined by measuring lipid composition of VLDL before and 4 hours after Triton WR-1339 injection. Subtraction of these values provided an estimate of the nascent VLDL composition. VLDL composition was similar in all strains (see online Figure I, which can be accessed at http://www.atvb.ahajournals.org). Therefore, differences in secretion of VLDL-TG are mainly due to changes in the number of apoB-100–containing lipoproteins. VLDL composition in plasma before Triton WR-1339 injection reflects the average composition of nascent and partially hydrolyzed particles. In these particles, the TG content of mice expressing APOE3Leiden/apoe+/- was 66±3% compared with 32±6% and 31±3% for mice expressing APOE3Leiden/apoe-/- and APOE3/apoe-/-, respectively. This increased TG content is probably due to a decreased lipolysis rate.
mRNA levels
Steady-state mRNA levels of selected enzymes involved in lipogenesis, TG formation, and VLDL assembly in liver were estimated after an overnight fast by semiquantitative reverse transcription–PCR, including fatty acid synthase (fas), diacylglycerol acyltransferase (dgat), and microsomal triglyceride transfer protein (mttp). Expression of all genes was similar between the groups when corrected for ribosomal 18S RNA, indicating that differences in VLDL-TG production between the strains are not due to altered expression of these genes (data not shown).
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Discussion |
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Recent studies by us4–6 and by others7–9 have demonstrated that apoE is involved in the regulation of hepatic VLDL-TG secretion. The present study shows that the mutant APOE3Leiden isoform, compared with APOE3, does not stimulate VLDL-TG secretion by the apoE-deficient mouse liver. To evaluate the effects of APOE3Leiden on VLDL production, we made use of transgenic APOE3Leiden-expressing mice, either without endogenous mouse apoe19 (APOE3Leiden/apoe-/-) or with a single allele of the mouse apoe gene (APOE3Leiden/apoe+/-). As a control, we used apoe-/- mice expressing the human APOE3 gene. We have previously shown that these mice display a of VLDL-TG secretion rate similar to that observed in wild-type mice.6
In situ hybridization showed that transgene expression in APOE3Leiden- and APOE3-expressing mice is mainly in periportal hepatocytes (zone 1). This distribution pattern is in accordance with a recent report by Massimi et al28 showing that in rat liver, expression of endogenous apoe is also confined to zone 1. The construct used to create the transgenic mice contained the hepatic control region18,20 and the upstream region before exon 1, starting at location -650. This region contains a number of potential binding sites for transcription factors.30–32 It is very likely that a transcription factor(s) regulating hepatic localization of gene expression is localized in this region. To the best of our knowledge, however, these transcription factors have not yet been identified. In view of the putative role of apoE in VLDL-TG regulation, this observation supports the view that VLDL secretion is mainly in zone 1,33 and not in zone 3, as suggested by others.34
We have previously shown that apoE-deficient mice4,6 and APOE3Leiden/APOC-I mice16 develop hepatic steatosis, with fat accumulating most abundantly in perivenously localized hepatocytes (zone 3). In the present study, this pattern of fat distribution is also observed for mice expressing APOE3Leiden. This phenomenon is probably partly explained by the impaired VLDL-TG secretion in apoE-deficient and APOE3Leiden-expressing mice, because no indications for altered hepatic TG synthesis in these mice were found.4 However, mice expressing APOE3Leiden in the presence of apoe also develop a fatty liver, whereas the VLDL secretion is similar or even higher than that in mice expressing APOE3. The observations that synthesis of apoE and probably VLDL secretion are predominantly localized in zone 1 but that TG accumulation is most apparent in zone 3 might indicate a dual role for apoE in the development of a fatty liver. We speculate that in the presence of functional apoE, most TGs are removed from the circulation in the periportal zone of the liver (zone 1), where the LDL receptor–related protein is localized.35 Without functional apoE, uptake in zone 1 is reduced. Consequently, zone 3 hepatocytes face high concentrations of lipoproteins and may take them up by mechanisms not yet known. Because VLDL secretion is mainly in zone 1, zone 3 cells might not be able to handle these excess lipids. In the presence of a single functional endogenous apoe allele, lipid staining is largely absent in zone 1, probably because VLDL secretion proceeds at a normal rate. Because removal of lipoproteins from the circulation is delayed because of the nonfunctional APOE3Leiden (see below), this may lead to accumulation of TGs in zone 3 cells.
Fatty liver rapidly develops in situations of defective fatty acid ß-oxidation.36 Because apoE localizes to peroxisomes,6,37 it had to be excluded that the absence of endogenous apoE and/or the presence of dysfunctional apoE3Leiden might result in a decreased ß-oxidation, contributing to the development of steatosis. All strains showed similarly increased concentrations of fatty acids and 3-hydroxybutyric acid after 24 hours of starvation, indicating that ß-oxidation is most probably not different in the strains used.
In the presence of a single mouse apoe allele, steady-state plasma concentrations of TGs, APOE, and VLDL-apoB were increased in APOE3Leiden transgenic mice compared with APOE3/apoe-/- mice. This is likely caused by a delayed lipoprotein clearance that is due to the presence of APOE3Leiden in high concentrations and a consequently decreased hydrolysis of VLDL-TG. Jong et al38 have shown that high concentrations of apoE inhibit lipolysis, probably by steric hindrance of lipoprotein binding to heparan sulfate proteoglycans. Reduced lipolysis of TG-rich particles in APOE3Leiden transgenic mice in the presence of mouse apoe18 is probably due to the lower binding affinity of APOE3Leiden to heparan sulfate proteoglycans. In addition, increased levels of APOE3Leiden compared with APOE might also be due to the somewhat higher gene expression of APOE3Leiden in these mice. The difference in plasma APOE3Leiden levels between both APOE3Leiden-expressing strains is likely due to the increased VLDL secretion in APOE3Leiden/apoe+/- mice.
VLDL-TG and apoB-100 secretion were markedly impaired in APOE3Leiden/apoe-/- mice compared with APOE3/apoe-/- mice, and secretion rates of the former were similar to those observed in apoe-/- mice.4 In the presence of APOE3Leiden and a single endogenous apoe allele, however, VLDL-TG and apoB-100 secretion was similar to that observed in APOE3/apoe-/- mice. Inasmuch as apoB-48 secretion remained constant between the 3 strains of mice, our data indicate that the additional TGs secreted in the presence of a functional apoE (mouse apoe or human APOE) occurred mainly through a modulation of the production of apoB-100–containing particles. Inasmuch as VLDL lipid composition was similar between all strains of mice, this indicates that the decreased VLDL-TG secretion in APOE3Leiden/apoe-/- is mainly due to a decrease in number of particles secreted.
Other groups have recently shown that overexpression of APOE2 or APOE4 by adenovirus-mediated gene transfer in apoE-deficient mice leads to an increased VLDL-TG secretion.39,40 These isoforms differ only in a single amino acid from APOE3. Because APOE3Leiden is not able to increase VLDL-TG secretion, our results indicate that incorrect folding of the APOE molecule caused by the introduction of a tandem repeat of 7 amino acids is associated with impaired VLDL secretion. This suggests that this site might influence the lipid-transporting capacity of apoE. Until now, it was known that expression of APOE3Leiden results in impaired receptor-mediated uptake19 and impaired hydrolysis of TG-rich lipoproteins,18 contributing to development of atherosclerosis in humans14 and mice.41 The present study shows that APOE3Leiden, in contrast to APOE3, is unable to deter the development hepatic steatosis associated with apoE deficiency16 and to normalize impaired VLDL secretion, which has become a characteristic hallmark of this condition.4,6
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Acknowledgments |
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This study was supported by a grant from the Netherlands Heart Foundation (No. 96-011).
Received February 14, 2001; accepted May 23, 2001.
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References |
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|---|
1. Mahley RW. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science. 1988; 240: 622–630.
2. Quarfordt SH, Oswald B, Landis B, Xu HS, Zhang SH, Maeda N. In vivo cholesterol kinetics in apolipoprotein E-deficient and control mice. J Lipid Res. 1995; 36: 1227–1235.[Abstract]
3.
Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science. 1992; 258: 468–471.
4. Kuipers F, Lin Y, Havinga R, Bloks V, Verkade HJ, Jong MC, Hofker MH, Moshage H, van Vlijmen BJM, Vonk RJ, et al. Impaired production of very low density lipid proteins by apolipoprotein E-deficient mouse hepatocytes in primary culture. J Clin Invest. 1997; 100: 2915–2922.[Medline] [Order article via Infotrieve]
5. Kuipers F, van Ree JM, Hofker MH, Wolters H, in ’t Veld G, Havinga R, Vonk RJ, Princen HMG, Havekes LM. Altered lipid metabolism in apolipoprotein E-deficient mice does not affect cholesterol balance across the liver. Hepatology. 1996; 24: 241–247.[Medline] [Order article via Infotrieve]
6.
Mensenkamp AR, Jong MC, van Goor H, van Luyn MJA, Bloks V, Havinga R, Voshol PJ, Hofker MH, Willems van Dijk K, Havekes LM, et al. Apolipoprotein E participates in the regulation of very low density lipoprotein-triglyceride secretion by the liver. J Biol Chem. 1999; 274: 35711–35718.
7.
Huang Y, Ji ZS, Brecht WJ, Rall SC Jr, Taylor JM, Mahley RW. Overexpression of apolipoprotein E3 in transgenic rabbits causes combined hyperlipidemia by stimulating hepatic VLDL production and impairing VLDL lipolysis. Arterioscler Thromb Vasc Biol. 1999; 19: 2952–2959.
8.
Huang Y, Liu XQ, Rall SC Jr, Taylor JM, von Eckardstein A, Assmann G, Mahley RW. Overexpression and accumulation of apolipoprotein E as a cause of hypertriglyceridemia. J Biol Chem. 1998; 273: 26388–26393.
9.
Maugeais C, Tietge UJF, Tsukamoto K, Glick JM, Rader DJ. Hepatic apolipoprotein E expression promotes very low density lipoprotein-apolipoprotein B production in vivo in mice. J Lipid Res. 2000; 41: 1673–1679.
10. Demant T, Bedford D, Packard CJ, Shepherd J. Influence of apolipoprotein E polymorphism on apolipoprotein B-100 metabolism in normolipidemic subjects. J Clin Invest. 1991; 88: 1490–1501.
11.
Welty FK, Lichtenstein AH, Barrett PH, Jenner JL, Dolnikowski GG, Schaefer EJ. Effects of apoE genotype on apoB-48 and apoB-100 kinetics with stable isotopes in humans. Arterioscler Thromb Vasc Biol. 2000; 20: 1807–1810.
12. van den Maagdenberg AMJM, de Knijff P, Stalenhoef AF, Gevers Leuven JA, Havekes LM, Frants RR. Apolipoprotein E3*Leiden allele results from a partial gene duplication in exon 4. Biochem Biophys Res Commun. 1989; 165: 851–857.[Medline] [Order article via Infotrieve]
13. Wardell MR, Weisgraber KH, Havekes LM, Rall SC Jr. Apolipoprotein E3-Leiden contains a seven-amino acid insertion that is a tandem repeat of residues 121–127. J Biol Chem. 1989; 264: 339–348.
14. de Knijff P, van den Maagdenberg AMJM, Stalenhoef AF, Gevers Leuven JA, Demacker PN, Kuyt LP, Frants RR, Havekes LM. Familial dysbetalipoproteinemia associated with apolipoprotein E*3Leiden in an extended multigeneration pedigree. J Clin Invest. 1991; 88: 643–655.
15. van Vlijmen BJM, van den Maagdenberg AMJM, Gijbels MJJ, van der Boom H, HogenEsch H, Frants RR, Hofker MH, Havekes LM. Diet-induced hyperlipoproteinemia and atherosclerosis in apolipoprotein E3-Leiden transgenic mice. J Clin Invest. 1994; 93: 1403–1410.
16. Mensenkamp AR, van Luyn MJA, van Goor H, Bloks V, Apostel F, Greeve J, Hofker MH, Jong MC, van Vlijmen BJM, Havekes LM, et al. Hepatic lipid accumulation, altered very low density lipoprotein formation and apolipoprotein E deposition in apolipoprotein E3-Leiden transgenic mice. J Hepatol. 2000; 33: 189–198.[Medline] [Order article via Infotrieve]
17. Hogan B, Constatini F, Lacey E. Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 1986.
18.
Jong MC, Dahlmans VEH, van Gorp PJ, Breuer ML, Mol MJTM, van der Zee A, Frants RR, Hofker MH, Havekes LM. Both lipolysis and hepatic uptake of VLDL are impaired in transgenic mice coexpressing human apolipoprotein E*3Leiden and human apolipoprotein C1. Arterioscler Thromb Vasc Biol. 1996; 16: 934–940.
19.
van Vlijmen BJM, Willems van Dijk K, van ‘t Hof HB, van Gorp PJ, Zee Avd, van der Boom H, Breuer ML, Hofker MH, Havekes LM. In the absence of endogenous mouse apolipoprotein E, apolipoprotein E*2 (Arg-158
Cys) transgenic mice develop more severe hyperlipoproteinemia than apolipoprotein E*3-Leiden transgenic mice. J Biol Chem. 1996; 271: 30595–30602.
20.
Dang Q, Walker D, Taylor S. Structure of the hepatic control region of the human apolipoprotein E/C-I gene locus. J Biol Chem. 1995; 270: 22577–22585.
21. van Ree JM, van den Broek WJAA, Dahlmans VEH, Groot PHE, Vidgeon-Hart M, Frants RR, Wierenga B, Havekes LM, Hofker MH. Diet-induced hypercholesterolemia and atherosclerosis in heterozygous apolipoprotein E-deficient mice. Atherosclerosis. 1994; 111: 25–37.[Medline] [Order article via Infotrieve]
22. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959; 37: 911–917.
23. Böttcher CFJ, van Gent CM, Pries C. A rapid and sensitive sub-micro-phosphorus determination. Anal Chim Acta. 1961; 24: 203–204.
24. Williamson DH, Mellanby J. D-(-)-3-Hydroxybutyrat.In: Bergmeyer HU, ed. Methoden der enzymatischen analyse. Weinheim: Verlag Chemie; 1970: 1772–1779.
25. Li X, Catalina F, Grundy SM, Patel S. Method to measure apolipoprotein B-48 and B-100 secretion rates in an individual mouse: evidence for a very rapid turnover of VLDL and preferential removal of B-48-relative to B-100-containing lipoproteins. J Lipid Res. 1996; 37: 210–220.[Abstract]
26.
Bloks V, Plösch T, van Goor H, Roelofsen H, Baller J, Havinga R, Verkade HJ, van Tol A, Jansen PLM, Kuipers F. Hyperlipidemia and atherosclerosis associated with liver disease in ferrochelatase deficient mice. J Lipid Res. 2001; 42: 41–50.
27.
Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with Folin reagent. J Biol Chem. 1951; 193: 265–275.
28. Massimi M, Lear SR, Williams DL, Jones AL, Erickson SK. Differential expression of apolipoprotein E messenger RNA within the rat lobule determined by in situ hybridization. Hepatology. 1999; 29: 1549–1555.[Medline] [Order article via Infotrieve]
29.
Leone TC, Weinheimer CJ, Kelly DP. A critical role for the peroxisome proliferator-activated receptor alpha (PPARalpha) in the cellular fasting response: the PPARalpha-null mouse as a model of fatty acid oxidation disorders. Proc Natl Acad Sci U S A. 1999; 96: 7473–7478.
30.
Salero E, Perez-Sen R, Aruga J, Gimenez C, Zafra F. Transcription factors Zic1 and Zic2 bind and transactivate the apolipoprotein E gene promoter. J Biol Chem. 2001; 276: 1881–1888.
31.
Berg DT, Calnek DS, Grinnell BW. The human apolipoprotein E gene is negatively regulated in human liver HepG2 cells by the transcription factor BEF-1. J Biol Chem. 1995; 270: 15447–15450.
32.
Chang DJ, Paik YK, Leren TP, Walker DW, Howlett GJ, Taylor JM. Characterization of a human apolipoprotein E gene enhancer element and its associated protein factors. J Biol Chem. 1990; 265: 9496–9504.
33. Franke H, Potratz I, Dargel R. Zonal differences in lipoprotein formation in the thioacetamide-induced micronodular-cirrhotic rat liver. Exp Toxicol Pathol. 1994; 46: 503–511.[Medline] [Order article via Infotrieve]
34. Guzmán M, Castro J. Zonation of fatty acid metabolism in rat liver. Biochem J. 1989; 264: 107–113.[Medline] [Order article via Infotrieve]
35. Voorschuur AH, Kuiper J, Neelissen JA, Boers W, Van Berkel TJ. Different zonal distribution of the asialoglycoprotein receptor, the alpha 2-macroglobulin receptor/low-density-lipoprotein receptor-related protein and the lipoprotein-remnant receptor of rat liver parenchymal cells. Biochem J. 1994; 303: 809–816.
36. Pessayre D, Mansouri A, Haouzi D, Fromenty B. Hepatotoxicity due to mitochondrial dysfunction. Cell Biol Toxicol. 1999; 15: 367–373.[Medline] [Order article via Infotrieve]
37. Hamilton RL, Wong JS, Guo LSS, Krisans S, Havel RJ. Apolipoprotein E localization in rat hepatocytes by immunogold labeling of cryothin sections. J Lipid Res. 1990; 31: 1589–1603.[Abstract]
38. Jong MC, Dahlmans VEH, Hofker MH, Havekes LM. Nascent very-low-density lipoprotein triacylglycerol hydrolysis by lipoprotein lipase is inhibited by apolipoprotein E in a dose-dependent manner. Biochem J. 1997; 328: 745–750.
39.
Tsukamoto K, Maugeais C, Glick JM, Rader DJ. Markedly increased secretion of VLDL triglycerides induced by gene transfer of apolipoprotein E isoforms in apoE-deficient mice. J Lipid Res. 2000; 41: 253–259.
40.
de Beer F, Willems van Dijk K, Jong MC, van Vark LC, van Der ZA, Hofker MH, Fallaux FJ, Hoeben RC, Smelt AH, Havekes LM. Apolipoprotein E2 (Lys146Gln) causes hypertriglyceridemia due to an apolipoprotein E variant-specific inhibition of lipolysis of very low density lipoproteins-triglycerides. Arterioscler Thromb Vasc Biol. 2000; 20: 1800–1806.
41.
van den Maagdenberg AMJM, Hofker MH, Krimpenfort PJA, de Bruijn IH, van Vlijmen BJM, van der Boom H, Havekes LM, Frants RR. Transgenic mice carrying the apolipoprotein E3-Leiden gene exhibit hyperlipoproteinemia. J Biol Chem. 1993; 268: 10540–10545.
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