This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by de Beer, F. Articles by Havekes, L. M. Search for Related Content PubMed PubMed Citation Articles by de Beer, F. Articles by Havekes, L. M. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Medline Plus Health Information Triglycerides Related Collections Animal models of human disease Gene therapy Lipid and lipoprotein metabolism

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1800.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Apolipoprotein E2 (Lys146Gln) Causes Hypertriglyceridemia due to an Apolipoprotein E Variant–Specific Inhibition of Lipolysis of Very Low Density Lipoproteins–Triglycerides

Femke de Beer; Ko Willems van Dijk; Miek C. Jong; Leonie C. van Vark; Andre van der Zee; Marten H. Hofker; Frits J. Fallaux; Rob C. Hoeben; Augustinus H. M. Smelt; Louis M. Havekes

From TNO-Prevention and Health (F.d.B., M.C.J., L.C.v.V., L.M.H.), Gaubius Laboratory, Leiden, the Netherlands, and the Departments of Internal Medicine (F.d.B., M.C.J., L.C.v.V., A.H.M.S., L.M.H.), Human Genetics (K.W.v.D., A.v.d.Z., M.H.H.), Molecular Cell Biology (F.J.F., R.C.H.), and Cardiology (L.M.H.), Leiden University Medical Center, Leiden, the Netherlands.

Correspondence to Prof Dr L.M. Havekes, TNO-Prevention and Health, Gaubius Laboratory, Zernikedreef 9, 2333 CK Leiden, Netherlands, or PO Box 2215, 2301 CE Leiden, Netherlands. E-mail LM.Havekes{at}PG.TNO.NL

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract—The apolipoprotein E2 (Lys146Gln) variant is associated with a dominant form of familial dysbetalipoproteinemia. Heterozygous carriers of this variant have elevated levels of plasma triglycerides, cholesterol, and apolipoprotein E (apoE). It was hypothesized that the high amounts of triglycerides in the very low density lipoprotein (VLDL) fraction are due to a disturbed lipolysis of VLDL. To test this hypothesis, apoE knockout mice were injected with an adenovirus containing the human APOE*2 (Lys146Gln) gene, Ad-E2(146), under the control of the cytomegalovirus promoter. ApoE knockout mice injected with an adenovirus vector encoding human apoE3 (Ad-E3) were used as controls. Five days after adenovirus injection, plasma cholesterol levels of mice injected with a high dose of Ad-E2(146) (2x10⁹ plaque-forming units) were not changed compared with preinjection levels, whereas in the group who received a low dose of Ad-E2(146) (5x10⁸ plaque-forming units) and in the groups injected with a low or a high dose of Ad-E3, plasma cholesterol levels were decreased 5-, 6-, and 12-fold, respectively. Plasma triglycerides were not affected in mice injected with Ad-E3. In contrast, a 7-fold increase in plasma triglycerides was observed in mice injected with the low dose of Ad-E2(146) compared with mice injected with Ad-E3. Injection with the high dose of Ad-E2(146) resulted in a dramatic increase of plasma triglycerides (50-fold compared with Ad-E3 injection). In vitro lipolysis experiments showed that the lipolysis rate of VLDLs containing normal amounts of apoE2 (Lys146Gln) was decreased by 54% compared with that of VLDLs containing comparable amounts of apoE3. The in vivo VLDL-triglyceride production rate of Ad-E2(146)–injected mice was not significantly different from that of Ad-E3–injected mice. These results demonstrate that expression of apoE2 (Lys146Gln) causes hypertriglyceridemia due to an apoE variant–specific inhibition of the hydrolysis of VLDL-triglycerides.

Key Words: adenovirus-mediated gene transfer • apolipoprotein E • familial dysbetalipoproteinemia • lipolysis

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Apolipoprotein E (apoE) plays a key role in lipoprotein metabolism.¹ It is a major constituent of chylomicron and VLDL remnants and serves as a ligand for the receptor-mediated uptake of these lipoproteins by the liver.^{1 2 3} Mutations in the APOE gene can lead to an impaired clearance of remnant lipoproteins by the liver. This metabolic disorder is known as familial dysbetalipoproteinemia (FD).^{4 5} The accumulation of atherogenic remnant lipoproteins (ß-VLDL) in the plasma of FD patients is associated with an increased risk for the development of atherosclerosis.⁴

ApoE is a polymorphic protein. In humans, 3 major common isoforms are found: apoE2, apoE3, and apoE4.⁶ ApoE3 (Cys112; Arg158) is the most frequent isoform and is considered the wild type. ApoE2 (Arg158Cys) and apoE4 (Cys112Arg) differ from apoE3 by single amino acid substitutions at positions 158 and 112, respectively. More than 90% of all FD patients are homozygous carriers of the apoE2 (Arg158Cys) variant.^{5 7} In case of apoE2 homozygosity, FD is inherited in a recessive fashion. On the other hand, FD is also associated with several rare apoE variants that display a dominant inheritance pattern (for a review, see Reference ⁸ ).

In the Netherlands, 2 apoE variants with a dominant association of FD have been found in >40 carriers: apoE3-Leiden (Cys112Arg, 7 amino acid insertion) and apoE2 (Lys146Gln).^{9 10 11} The apoE2 (Lys146Gln) variant was first described by Rall et al.¹² Compared with apoE2 (Arg158Cys) homozygous FD patients and FD patients with heterozygosity for apoE3-Leiden (Cys112Arg, 7 amino acid insertion), heterozygous carriers of apoE2 (Lys146Gln) display high plasma triglyceride (TG), VLDL-TG, and apoE levels.⁹ The increased lipid levels in apoE2 (Lys146Gln) carriers are mainly associated with increased VLDL levels, whereas in apoE2 (Arg158Cys) homozygous FD patients, the increased lipid levels are also associated with elevated VLDL remnant levels.¹³

The receptor binding domain of apoE is located between amino acid residues 130 and 150. This region is important for binding to the LDL receptor.¹⁴ It has been shown that apoE2 (Lys146Gln)/phospholipid complexes display only 40% of the binding activity of wild-type apoE3/phospholipid complexes.¹² Surprisingly, VLDL isolated from heterozygous carriers of the apoE2 (Lys146Gln) variant does not show a severe binding defect to the LDL receptor.^{13 15} This suggests that the underlying mechanism leading to the development of FD in heterozygous carriers of the apoE2 (Lys146Gln) variant cannot be fully attributed to a binding defect of this apoE mutant to the LDL receptor.

In a previous study, we suggested that in APOE*2 (Lys146Gln) allele carriers, the conversion of VLDL into VLDL remnants is impaired because of an inefficient lipolysis.¹³ To investigate the effect of human apoE2 (Lys146Gln) on lipoprotein metabolism in vivo, without the confounding presence of the second normal APOE allele, and to further elucidate the mechanism behind the development of FD in subjects with this apoE variant, we used adenovirus-mediated gene transfer of apoE2 (Lys146Gln) in apoE knockout mice. We found that compared with the expression of normal apoE3, the expression of apoE2 (Lys146Gln) resulted in a marked increase in plasma TG levels that was due to an impaired lipolysis of VLDL-TGs.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Animals
Homozygous apoE knockout mice were generated previously.¹⁶ Mice were housed under standard conditions with free access to water and food. For experiments, male mice aged 7 to 20 weeks were included. All mice were fed an SRM-A diet (Hope Farms).

Adenovirus Transfections
The apoE3 and apoE2 (Lys146Gln)–expressing adenovirus vectors [Ad-E3 and Ad-E2(146), respectively] were generated according to published methods by use of an adapter plasmid and pJM17.¹⁷ The adenoviral adapter plasmid pCMV-E3 was generated from the plasmid pCMV10 that supplies the human adenovirus type 5 (Ad5) 5'-inverted terminal repeat, the Ad5 origin of replication, the Ad5 encapsidation signal, the CMV immediate-early promoter, the SV40 19S exon, the SV40 truncated intron, and a splice acceptor site. The adenoviral adapter plasmid pCMV-E2(146) was generated from the plasmid pMLP-TK that supplies a polyadenylation site and Ad5 sequences from map unit 9.2 to 16.9 to serve as a homologous recombination fragment. Both adenoviral adapter plasmids were kindly provided by Introgene BV (Leiden, the Netherlands). The human APOE*3 and APOE*2 (Lys146Gln) genomic fragments (3418 bp in size, from the MscI site in exon 2 to the EcoRI site in the 3'-flanking region) were inserted immediately downstream from the splice acceptor site. Recombinant adenovirus was generated by cotransfection of the adapter plasmids with pJM17¹⁷ into 911 cells¹⁸ by use of a calcium phosphate transfection kit (Promega). The recombinant adenovirus expressing the ß-galactosidase gene under control of the CMV promoter (Ad-LacZ) was kindly provided by Dr J. Herz (University of Texas Southwestern Medical Center, Dallas).¹⁹ The APOE-expressing vectors were expanded on 293 cells, and Ad-LacZ was expanded on 911 cells, according to previously described methods.¹⁸ Titrations were performed on 911 cells.¹⁸

For in vivo administration, the virus was purified twice via CsCl gradient centrifugation and dialyzed extensively against dialysis buffer consisting of 25 mmol/L Tris, 137 mmol/L NaCl, 5 mmol/L KCl, 0.73 mmol/L NaH₂PO₄, 0.9 mmol/L CaCl₂, and 0.5 mmol/L MgCl₂, pH 7.45. For storage, the virus was supplemented with mouse serum albumin (0.2%) and glycerol (10%), and aliquots were frozen in liquid N₂ before transfer to -80°C. Routine virus titers of stocks varied from 2x10¹⁰ to 3x10¹¹ plaque-forming units (pfu) per milliliter.

Some 7 to 9 days before adenovirus injection, baseline lipid values were measured. At day 0, mice were injected into the tail vein with 5x10⁸, 2x10⁹, or 5x10⁹ pfu of recombinant adenovirus diluted with PBS to a total volume of 200 µL. Blood samples were drawn from the tail vein or by orbital puncture of fasted mice at 4, 5, 8, and 11 days after virus injection.

Lipid and Lipoprotein Analysis
At each time point, blood was collected in heparinized capillary tubes (Hawksley & Sons Ltd) from each individual mouse through tail-bleeding, after a 4-hour fasting period. Tubes were placed on ice. Plasma was obtained by centrifugation in a hematocrit centrifuge. Plasma TG and total cholesterol (TC) levels were measured enzymatically by using commercially available kits (337-B, Sigma Chemical Co, and 236691, Boehringer-Mannheim, respectively). Plasma apoE levels were determined by ELISA, as described previously.²⁰

At 5, 8, and 11 days after adenovirus injection, mice were euthanized to collect blood by orbital puncture and to excise the livers for hepatic APOE mRNA measurements. Serum was separated from the blood cells by centrifugation at 1500g for 15 minutes at room temperature. Pooled sera were ultracentrifuged to isolate VLDL (density <1.006 g/mL). Protein content of the VLDL samples was determined by the method of Lowry et al.²¹ TG and TC concentrations of the VLDL fractions were measured as described above. Free cholesterol (FC) and phospholipid content of the VLDL fractions was measured enzymatically by using commercially available kits (310328, Boehringer-Mannheim, and 990-54009, Wako Chemicals, respectively). Esterified cholesterol content was calculated by subtracting the concentration of FC from the concentration of TC. The mass of cholesteryl ester (CE) was estimated as 1.67xmass of FC. The total lipoprotein mass (in milligrams per deciliter) was calculated as the sum of masses of FC, CE, TG, phospholipid, and protein content. VLDL apoE levels were measured by ELISA.²⁰ VLDL particle size was determined by use of a Malvern 4700 C system (Malvern Instruments). Measurements were performed at 25°C with a 90° angle between laser and detector. The number of apoE molecules per VLDL particle was calculated from the total lipoprotein mass and VLDL particle size, with the assumption that the particles were spherical in shape and that their density was 1.006 g/mL.

Human APOE mRNA Measurements
Total RNA was isolated from the livers of adenovirus-injected mice by use of the RNAzol procedure (Cinna/Biotecx). Human APOE transcript was detected by Northern blotting after electrophoresis of RNA samples (7.5 µg per lane) on a denaturing agarose gel (1% [wt/vol]) containing 7.5% formaldehyde and transferred to a nylon membrane (Hybond N, Amersham Corp). Blots were subsequently hybridized with a ³²P-labeled probe of human APOE cDNA²² and a rat GAPDH cDNA²³ in a solution containing 50% formamide. The intensity of the hybridization signal was quantified with a PhosphorImager (Molecular Dynamics), and human APOE mRNA was related to the level of GAPDH mRNA.

In Vitro Lipolysis Assay With HSPG-Bound LPL
Microtiter plates (Greiner GmbH) were coated with commercially available heparan sulfate proteoglycans (HSPG; H4777, Sigma) as described previously.²⁴ Briefly, wells were incubated with 0.5 µg HSPG in 75 µL PBS for 18 hours at 4°C. Nonspecific binding sites were blocked with PBS containing 1% (wt/vol) essentially free fatty acid (FFA)-free BSA (Sigma) for 1 hour at 37°C. Thereafter, wells were incubated with 1 U bovine lipoprotein lipase (LPL; L2254, Sigma) in 75 µL Tris-glycerol buffer (0.1 mol/L Tris and 20% [vol/vol] glycerol, pH 8.5) for 1 hour at 4°C and subsequently washed 3 times with Tris buffer (0.1 mol/L Tris, pH 8.5) to remove unbound LPL.

Lipolysis was started by adding 50 µL VLDL-TG at a final concentration of 0.2 mmol/L to the preconditioned well in the presence of 1% (wt/vol) essentially FFA-free BSA and placing the plate in a shaking incubator at 37°C. The reaction was stopped after 20 minutes by placing the plate on ice and by adding Tris buffer containing 1% (vol/vol) Triton X-100 (Merck) to the wells. FFA concentrations were determined enzymatically by use of a commercially available kit (994-75409, Wako Chemicals).

In Vivo Hepatic VLDL-TG Production
Fasted mice were injected intravenously with 500 mg of Triton WR 1339 per kilogram body weight as a 15 g/L solution in 0.9% NaCl. Plasma VLDL clearance is virtually completely inhibited under these circumstances.²⁵ Blood samples (50 µL) were withdrawn 0, 20, 40, and 60 minutes after the Triton injection, and plasma TG levels were determined and related to the body mass of the mice. The production rate of hepatic TGs was calculated from the slope of the curve and expressed as micromoles per hour per kilogram body weight.

Statistical Analyses
Results are presented as mean±SD. Mean differences between the groups were calculated by the Mann-Whitney test. Statistical analyses were performed with SPSSWIN 6.1.3 (SPSS).

	Results

Top Abstract Introduction Methods Results Discussion References

 
Plasma Lipid and ApoE Levels After Adenovirus-Mediated Gene Transfer of ApoE2 (Lys146Gln) and ApoE3
ApoE knockout mice were injected intravenously with a low or a high dose of Ad-E2(146). Littermates injected with a low or a high dose of Ad-E3 were used as control groups. Injection with adenoviruses results in transient gene expression with a peak of gene expression at day 5.¹⁹ The Figure shows the effect of the expression of apoE2 (Lys146Gln) and apoE3 on plasma cholesterol in time. Plasma cholesterol levels were decreased 5 days after injection with the low or high dose of Ad-E3 as well as with the low dose of Ad-E2(146). In contrast, the high dose of Ad-E2(146) had no significant effect up to day 8. On day 11, when adenovirus-mediated APOE expression was reduced, plasma cholesterol levels in mice injected with the high dose of Ad-E2(146) were decreased.

View larger version (19K): [in this window] [in a new window]   Figure 1. Effect of adenovirus-mediated gene transfer of apoE2 (Lys146Gln) and apoE3 on plasma cholesterol levels. Plasma cholesterol levels were measured as described in Methods. At day 0, apoE knockout mice were injected with 5x108 pfu Ad-E2(146) (n=3, •), 2x109 pfu Ad-E2(146) (n=4, ), 5x108 pfu Ad-E3 (n=5, ), or 2x109 pfu Ad-E3 (n=4, ). Values represent mean±SD. On day 5, all animals from the groups injected with the low dose of Ad-E2(146) and Ad-E3 as well as 3 animals from the group injected with the high dose of Ad-E2(146) were euthanized for determination of VLDL composition and hepatic mRNA levels.

On day 5, the difference between the high and low dose of Ad-E2(146) was most prominent. Therefore, we studied the mice at this day in more detail (Table 1). Injection of a high dose of Ad-E2(146) in mice caused a dramatic increase in plasma TG levels (up to 50-fold) compared with the plasma TG levels observed in mice before adenovirus injection and in mice who received Ad-E3. Mice injected with the low dose of Ad-E2(146) displayed a mild hypertriglyceridemia. Plasma apoE levels were significantly increased in mice injected with Ad-E2(146) compared with mice injected with Ad-E3. The increase in plasma apoE levels was associated with a concomitant increase in human hepatic APOE mRNA levels. No effect on plasma lipid levels was observed in mice that received Ad-LacZ (data not shown).

View this table: [in this window] [in a new window]   Table 1. Plasma Lipid and ApoE and Human APOE mRNA Levels in Liver Before and 5 Days After Adenovirus Injection

Characterization of VLDL After Adenovirus-Mediated Gene Transfer of ApoE2 (Lys146Gln) and ApoE3
Table 2 shows the effect of Ad-E2(146) and Ad-E3 overexpression on VLDL composition. VLDL isolated from apoE knockout mice before adenovirus injection mainly contained CE. In contrast, injection of Ad-E3 or Ad-E2(146) caused a decrease in CE content and an increase in relative TG content of the VLDL particles, as reflected by the increased TG/TC ratio. In particular, VLDL isolated from mice that received the high dose of Ad-E2(146) were large particles, which were enriched in TGs and apoE compared with the other VLDL particles.

View this table: [in this window] [in a new window]   Table 2. VLDL Composition Before and 5 Days After Adenovirus Injection

Hepatic VLDL-TG Production
As shown in Table 1, plasma TG levels of Ad-E2(146)–injected mice were increased after adenovirus injection. To evaluate whether the hypertriglyceridemia after Ad-E2(146) injection was due to an enhanced VLDL-TG production, additional experiments were performed to measure the in vivo hepatic VLDL-TG production rate in Ad-E2(146)– and Ad-E3–injected mice that displayed similar levels of hepatic APOE mRNA expression. As shown in Table 3, no significant difference in VLDL-TG production rate was observed between Ad-E2(146)– and Ad-E3–injected mice 8 days after adenovirus injection.

View this table: [in this window] [in a new window]   Table 3. Human APOE mRNA Levels and In Vivo VLDL-TG Production Rates After Adenovirus-Mediated Gene Transfer of ApoE2 (Lys146Gln) and ApoE3

Lipolysis of VLDL
It has been demonstrated that high amounts of apoE present on the surface of VLDL particles result in the inhibition of VLDL lipolysis.^{20 26 27 28} To study the apoE dose–independent effect of the apoE2 (Lys146Gln) variant on VLDL lipolysis, VLDL was isolated from Ad-E2(146)– and Ad-E3–injected mice that contained comparable and relatively low amounts of apoE per VLDL particle (Table 4). The lipolysis rate of VLDL isolated from Ad-E2(146)–injected mice was decreased by 54% (1.05±0.05 versus 2.27±0.16 µmol FFA/L per minute). Thus, inhibition of VLDL lipolysis contributes to the increased plasma TG levels of Ad-E2(146)–injected mice.

View this table: [in this window] [in a new window]   Table 4. Effect of Comparable Amounts of ApoE3 and ApoE2 (Lys146Gln) on the Hydrolysis of VLDL-TG

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The apoE2 (Lys146Gln) variant is associated with a dominant mode of inheritance of FD.^{9 11 12} The mutation is located in the LDL receptor binding domain of apoE.¹⁴ However, it was found that VLDL isolated from heterozygous carriers of the apoE2 (Lys146Gln) variant does not show a severe binding defect to the LDL receptor.^{13 15} This finding suggests that the underlying mechanism leading to the development of FD in carriers of apoE2 (Lys146Gln) cannot be fully explained by an LDL receptor binding defect. In addition to the function of apoE as a ligand for the receptor-mediated uptake of lipoproteins by the liver, recent data have shown that apoE plays an important role in hepatic VLDL-TG production. Hepatic VLDL-TG production was found to be decreased in apoE knockout mice compared with wild-type mice.²⁹ We^{27 30} and others²⁶ have shown that the hepatic VLDL-TG production increases with increasing expression levels of normal apoE3. In the present study, injection of apoE knockout mice with an adenovirus containing the human APOE*2 (Lys146Gln) gene resulted in hypertriglyceridemia. To determine whether the hypertriglyceridemia was the result of an effect on the VLDL-TG secretion rate, Triton experiments were performed in apoE knockout mice after introducing similar expression levels of APOE*3 or APOE*2 (Lys146Gln). Our in vivo data show no significant differences in the effect on the VLDL-TG production rate between Ad-E2(146)– and Ad-E3–injected mice (Table 3).

The hypertriglyceridemia observed in Ad-E2(146)–injected mice could also be caused by an impaired LPL-mediated lipolysis. Human data from our previous studies suggested that heterozygous carriers of the apoE2 (Lys146Gln) variant have an impaired conversion of VLDL into VLDL remnants.^{13 31} In humans, the effect of the apoE2 (Lys146Gln) variant on lipolysis is confounded by the presence of the second normal APOE allele. In addition, it is virtually impossible to obtain VLDL particles with equal apoE content from patients. It has previously been demonstrated that high amounts of normal apoE3 lead to the inhibition of VLDL lipolysis.^{20 26 27 28} Thus, in view of the fact that VLDL particles from mice injected with the high dose of Ad-E2(146) were very rich in apoE (Table 2), the amount of apoE protein per particle itself may be the primary cause of the impaired lipolysis in these mice. To rule out the possibility that a dose-dependent inhibition by apoE is the only cause, the effect of apoE2 (Lys146Gln) on lipolysis was studied by comparing the lipolysis rates of VLDL with comparable amounts of either apoE3 or apoE2 (Lys146Gln) on the surface (Table 4). This was achieved by injecting varying amounts of APOE adenovirus and by isolating VLDL at different time points after adenovirus injection. From these experiments, we conclude that the inhibition of lipolysis by apoE2 (Lys146Gln) indeed occurs in an apoE variant–specific manner.

It is interesting to note that VLDL isolated from mice injected with the high dose of Ad-E2(146) was not removed efficiently from the circulation by hepatic receptors, despite the presence of high amounts of apoE on the surface (Table 2). Apparently, normal hepatic clearance can only occur after the VLDL particles have been subjected to lipolysis. This is in line with our previous results showing that high amounts of TGs limit the uptake of lipoproteins by the liver via the LDL receptor and the LDL receptor–related protein.²⁷

Several dominant apoE mutations, which are located between amino acid residues 142 to 146, were found to display a defective binding to HSPG/heparin.^{32 33 34} In the present study, the effect of apoE2 (Lys146Gln) on VLDL lipolysis was studied by using an in vitro lipolysis assay with HSPG-bound LPL. Impaired VLDL lipolysis could be due to a disturbed interaction of VLDL containing the apoE2 (Lys146Gln) variant with LPL on one hand or with HSPG on the other hand. Although the lipolysis assay with HSPG-bound LPL has been shown to be a valid method to study VLDL lipolysis,^{24 31} we cannot exclude the possibility that the impaired VLDL lipolysis is caused by a defective binding of the apoE2 (Lys146Gln) variant to HSPG. However, it is important to note that the advantage of the lipolysis assay with HSPG-bound LPL is its resemblance to the in vivo situation.

An alternative explanation for the mechanism behind the inhibitory effect of the apoE2 (Lys146Gln) variant on lipolysis would be an effect of apoE2 (Lys146Gln) on the amount of apoC2 on the VLDL particles. Several studies have shown that an excess of apoE on the VLDL surface results in displacement of apoC2, the cofactor for LPL activity,^{35 36} from the lipoprotein particle.^{26 37 38} In fact, it was found that VLDL containing the apoE2 (Arg158Cys) variant exhibits impaired lipolysis because of the displacement of apoC2.³⁷ However, the mechanism of apoC2 displacement is not very likely for apoE2 (Lys146Gln) for several reasons. Isoelectric focusing followed by protein staining showed that VLDLs isolated from heterozygous carriers of the apoE2 (Lys146Gln) variant do contain apoC2 in normal quantities despite the presence of high amounts of apoE.³⁹ There is evidence that apoC2 displacement occurs only when extremely high amounts of apoE are present on the VLDL particle. Huang and colleagues^{26 37} found that the high apoE content of VLDL from apoE2 (Arg158Cys) and apoE3 transgenic mice resulted in a decrease in apoC2 content of the VLDL particles. However, in the present study, the amounts of apoE2 (Lys146Gln) on the VLDL particle were 25-fold lower, strongly suggesting that under our experimental conditions, the occurrence of apoC2 displacement is not very likely.

It has been suggested that VLDL lipolysis is also impaired in FD patients homozygous for apoE2 (Arg158Cys).^{40 41 42} ApoE2 (Arg158Cys) homozygous carriers have elevated levels of VLDL and VLDL remnants. In contrast, the increased lipid levels in apoE2 (Lys146Gln) carriers are mainly associated with increased VLDL levels,¹³ suggesting that in apoE2 (Lys146Gln) carriers, the conversion of VLDL into VLDL remnants is more severely affected. The effect of apoE2 (Arg158Cys) on VLDL lipolysis has been investigated by using VLDL with high amounts of this apoE variant on the surface.³⁷ The effect of low amounts of apoE2 (Arg158Cys) on lipolysis remains to be determined.

In the present study, we found that relatively low amounts of apoE2 (Lys146Gln) present on VLDL particles resulted in an impaired lipolysis of VLDL-TG. These data show that in addition to the known apoE dose–dependent effect, apoE2 (Lys146Gln) displays an apoE variant–specific effect on lipolysis.

	Acknowledgments

 
This study was financially supported by the Netherlands Heart Foundation (project No. 94.114) and by grants BIOMED-2 (BMH4-CT96–0898) from the specific RTD Program of the European Commission. Dr M.H. Hofker is an Established Investigator of the Netherlands Heart Foundation. The authors thank Sylvia Kamerling, Vivian Dahlmans, Linda van ‘t Hof, and Patrick van Gorp for expert technical assistance.

Received October 4, 1999; accepted March 28, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Mahley RW. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science. 1988;240:622–630.[Abstract/Free Full Text]

2. Havel RJ, Chao Y, Windler EE, Kotite L, Guo LS. Isoprotein specificity in the hepatic uptake of apolipoprotein E and the pathogenesis of familial dysbetalipoproteinemia. Proc Natl Acad Sci U S A. 1980;77:4349–4353.[Abstract/Free Full Text]

3. Sherill BC, Innerarity TL, Mahley RW. Rapid hepatic clearance of the canine lipoproteins containing only the E apoprotein by a high affinity receptor. J Biol Chem. 1980;255:1804–1807.[Abstract/Free Full Text]

4. Mahley RW, Rall SC. Type III hyperlipoproteinemia (dysbetalipoproteinemia): the role of apolipoprotein E in normal and abnormal lipoprotein metabolism. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. New York, NY: McGraw-Hill; 1995:1953–1980.

5. Mahley RW, Innerarity TL, Rall SC Jr, Weisgraber KH, Taylor JM. Apolipoprotein E: genetic variants provide insights into it structure and function. Curr Opin Lipidol. 1990;1:87–95.

6. Utermann G, Hees M, Steinmetz A. Polymorphism of apolipoprotein E and occurrence of dysbetalipoproteinemia in man. Nature. 1977;269:604–607.[Medline] [Order article via Infotrieve]

7. Utermann G, Vogelmann KH, Steinmetz A, Schoenborg W, Pruin N, Jaeschke M, Hees M, Canzler H. Polymorphism of apolipoprotein E, II: genetics of hyperlipoproteinemia type III. Clin Genet. 1979;15:37–62.[Medline] [Order article via Infotrieve]

8. de Knijff P, van den Maagdenberg AM, Frants RR, Havekes LM. Genetic heterogeneity of apolipoprotein E and its influence on plasma lipid and lipoprotein levels. Hum Mutat. 1994;4:178–194.[Medline] [Order article via Infotrieve]

9. de Knijff P, van den Maagdenberg AM, Boomsma DI, Stalenhoef AF, Smelt AH, Kastelein JJ, Marais AD, Frants RR, Havekes LM. Variable expression of familial dysbetalipoproteinemia in apolipoprotein E*2 (Lys146Gln) allele carriers. J Clin Invest. 1994;94:1252–1262.

10. de Knijff P, van den Maagdenberg AM, Stalenhoef AF, Leuven JA, Demacker PN, Kuyt LP, Frants RR, Havekes LM. Familial dysbetalipoproteinemia associated with apolipoprotein E3-Leiden in an extended multigeneration pedigree. J Clin Invest. 1991;88:643–655.

11. Smit M, de Knijff P, van der Kooij Meijs E, Groenendijk C, van den Maagdenberg AM, Gevers Leuven JA, Stalenhoef AF, Stuyt PM, Frants RR, Havekes LM. Genetic heterogeneity in familial dysbetalipoproteinemia: the E2(Lys146-Gln) variant results in a dominant mode of inheritance. J Lipid Res. 1990;31:45–53.[Abstract]

12. Rall SC, Weisgraber KH, Innerarity TL, Bersot TP, Mahley RW. Identification of a new structural variant of human apolipoprotein E, E2(Lys146-Gln), in a type III hyperlipoproteinemic subject with the E3/E2 phenotype. J Clin Invest. 1983;72:1288–1297.

13. Mulder M, van der Boom H, de Knijff P, Braam C, van den Maagdenberg A, Leuven JA, Havekes LM. Triglyceride-rich lipoproteins of subjects heterozygous for apolipoprotein E2(Lys146Gln) are inefficiently converted to cholesterol-rich lipoproteins. Atherosclerosis. 1994;108:183–192.[Medline] [Order article via Infotrieve]

14. Wilson C, Wardell MR, Weisgraber KH, Mahley RW, Agard DA. Three-dimensional structure of the LDL receptor-binding domain of human apolipoprotein E. Science. 1991;252:1817–1822.[Abstract/Free Full Text]

15. Chappell DA. High receptor binding affinity of lipoproteins in atypical dysbetalipoproteinemia (type III hyperlipoproteinemia). J Clin Invest. 1989;84:1906–1915.

16. van Ree JH, van den Broek WJ, Dahlmans VE, Groot PH, Vidgeon-Hart M, Frants RR, Wieringa 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]

17. McGrory WJ, Bautista DS, Graham FL. A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5. Virology. 1998;163:614–617.

18. Fallaux FJ, Kranenburg O, Cramer SJ, Houweling A, van Ormondt H, Hoeben RC, van der Eb AJ. Characterization of 911: a new helper cell line for the titration and propagation of early region 1-deleted adenoviral vectors. Hum Gene Ther. 1996;7:215–222.[Medline] [Order article via Infotrieve]

19. Herz J, Gerard RD. Adenovirus-mediated transfer of low density lipoprotein receptor acutely accelerates cholesterol clearance in normal mice. Proc Natl Acad Sci U S A. 1993;90:2812–2816.[Abstract/Free Full Text]

20. 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.

21. Lowry OH, Rosebrough RJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265–275.[Free Full Text]

22. Zannis VI, McPherson J, Goldberger G, Karathanasis SK, Breslow JL. Synthesis, intracellular processing, and signal peptide of human apolipoprotein E. J Biol Chem. 1984;259:5495–5499.[Abstract/Free Full Text]

23. Fort P, Marty L, Piechaczyk M, Sabrouty SE, Dani C, Jeanteur P, Blanchard JM. Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family. Nucleic Acids Res. 1985;13:1431–1442.[Abstract/Free Full Text]

24. de Man FHAF, de Beer F, van der Laarse A, Smelt AHM, Havekes LM. Lipolysis of very low density lipoproteins by heparan sulphate proteoglycan-bound lipoprotein lipase. J Lipid Res. 1997;38:2465–2472.[Abstract]

25. Aalto Setala K, Fisher EA, Chen X, Chajek Shaul T, Hayek T, Zechner R, Walsh A, Ramakrishnan R, Ginsberg HN, Breslow JL. Mechanism of hypertriglyceridemia in human apolipoprotein (apo) CIII transgenic mice: diminished very low density lipoprotein fractional catabolic rate associated with increased apo CIII and reduced apo E on the particles. J Clin Invest. 1992;90:1889–1900.

26. Huang Y, Liu XQ, Rall SC, 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.[Abstract/Free Full Text]

27. Willems van Dijk K, van Vlijmen BJM, van ‘t Hof HB, van der Zee A, Santamarina-Fojo S, van Berkel TJC, Havekes LM, Hofker MH. 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.[Abstract/Free Full Text]

28. Rensen PCN, van Berkel TJC. Apolipoprotein E effectively inhibits lipoprotein lipase-mediated lipolysis of chylomicron-like triglyceride-rich lipid emulsions in vitro and in vivo. J Biol Chem. 1996;271:14791–14799.[Abstract/Free Full Text]

29. Kuipers F, Jong MC, Lin Y, van Eck M, Havinga R, Bloks V, Verkade HJ, Hofker MH, Moshage H, van Berkel TJ, et al. Impaired secretion of very low density lipoprotein-triglycerides by apolipoprotein E-deficient mouse hepatocytes. J Clin Invest. 1997;100:2915–2922.[Medline] [Order article via Infotrieve]

30. Mensenkamp AR, Jong MC, van Goor H, van Luyn MJA, Bloks V, Havinga R, Voshol PJ, Hofker MH, Willems van Dijk K, 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.[Abstract/Free Full Text]

31. de Man FHAF, de Beer F, van der Laarse A, Smelt AHM, Gevers Leuven JA, Havekes LM. Effect of apolipoprotein E variants on lipolysis of very low density lipoproteins by heparan sulphate proteoglycan-bound lipoprotein lipase. Atherosclerosis. 1998;136:255–262.[Medline] [Order article via Infotrieve]

32. Ji ZS, Fazio S, Mahley RW. Variable heparan sulfate proteoglycan binding of apolipoprotein E variants may modulate the expression of type III hyperlipoproteinemia. J Biol Chem. 1994;269:13421–13428.[Abstract/Free Full Text]

33. Mann WA, Meyer N, Weber W, Greten H, Beisiegel U. Apolipoprotein E isoforms and rare mutations: parallel reduction in binding to cells and to heparin reflects severity of associated type III hyperlipoproteinemia. J Lipid Res. 1995;36:517–525.[Abstract]

34. Mann WA, Meyer N, Berg D, Greten H, Beisiegel U. Lipoprotein lipase compensates for the defective function of apo E variants in vitro by interacting with proteoglycans and lipoprotein receptors. Atherosclerosis. 1999;145:61–69.[Medline] [Order article via Infotrieve]

35. Havel RJ, Fielding CJ, Olivecrona T, Shore VG, Fielding PE, Egelrud T. Cofactor activity of protein components of human very low density lipoproteins in the hydrolysis of triglycerides by lipoprotein lipase from different sources. Biochemistry. 1973;12:1828–1833.[Medline] [Order article via Infotrieve]

36. LaRosa JC, Levy RI, Herbert P, Lux SE, Fredrickson DS. A specific apoprotein activator for lipoprotein lipase. Biochem Biophys Res Commun. 1970;41:57–62.[Medline] [Order article via Infotrieve]

37. Huang Y, Liu XQ, Rall SC Jr, Mahley RW. Apolipoprotein E2 reduces the low density lipoprotein level in transgenic mice by impairing lipoprotein lipase-mediated lipolysis of triglyceride-rich lipoproteins. J Biol Chem. 1998;273:17483–17490.[Abstract/Free Full Text]

38. De Silva HV, Lauer SJ, Wang J, Simonet WS, Weisgraber KH, Mahley RW, Taylor JM. Overexpression of human apolipoprotein C-III in transgenic mice results in an accumulation of apolipoprotein B48 remnants that is corrected by excess apolipoprotein E. J Biol Chem. 1994;269:2324–2334.[Abstract/Free Full Text]

39. Smit M, de Knijff P, Frants RR, Klasen EC, Havekes LM. Familial dysbetalipoproteinemic subjects with the E3/E2 phenotype exhibit an E2 isoform with only one cysteine residue. Clin Genet. 1987;32:335–341.[Medline] [Order article via Infotrieve]

40. Chait A, Hazzard WR, Albers JJ, Kushwaha RP, Brunzell JD. Impaired very low density lipoprotein and triglyceride removal in broad beta disease: comparison with endogenous hypertriglyceridemia. Metabolism. 1978;27:1055–1066.[Medline] [Order article via Infotrieve]

41. Ehnholm C, Mahley RW, Chappell DA, Weisgraber KH, Ludwig E, Witztum JL. Role of apolipoprotein E in the lipolytic conversion of ß-very low density lipoproteins to low density lipoproteins in type III hyperlipoproteinemia. Proc Natl Acad Sci U S A. 1984;81:5566–5570.[Abstract/Free Full Text]

42. Chung BH, Segrest JP. Resistance of very low density lipoprotein subpopulation from familial dysbetalipoproteinemia to in vitro lipolytic conversion to the low density lipoprotein density fraction. J Lipid Res. 1983;24:1148–1159.[Abstract]

This article has been cited by other articles:

				K. E. North, H. H. H. Goring, S. A. Cole, V. P. Diego, L. Almasy, S. Laston, T. Cantu, B. V. Howard, E. T. Lee, L. G. Best, et al. Linkage analysis of LDL cholesterol in American Indian populations: the Strong Heart Family Study J. Lipid Res., January 1, 2006; 47(1): 59 - 66. [Abstract] [Full Text] [PDF]

				G. Gerritsen, K. E. Kypreos, A. van der Zee, B. Teusink, V. I. Zannis, L. M. Havekes, and K. W. van Dijk Hyperlipidemia in APOE2 transgenic mice is ameliorated by a truncated apoE variant lacking the C-terminal domain J. Lipid Res., February 1, 2003; 44(2): 408 - 414. [Abstract] [Full Text] [PDF]

				S.C. Elbein and S.J. Hasstedt Quantitative Trait Linkage Analysis of Lipid-Related Traits in Familial Type 2 Diabetes: Evidence for Linkage of Triglyceride Levels to Chromosome 19q Diabetes, February 1, 2002; 51(2): 528 - 535. [Abstract] [Full Text] [PDF]

				A. R. Mensenkamp, B. Teusink, J. F.W. Baller, H. Wolters, R. Havinga, K. W. van Dijk, L. M. Havekes, and F. Kuipers Mice Expressing Only the Mutant APOE3Leiden Gene Show Impaired VLDL Secretion Arterioscler Thromb Vasc Biol, August 1, 2001; 21(8): 1366 - 1372. [Abstract] [Full Text] [PDF]

				B. Teusink, A. R. Mensenkamp, H. van der Boom, F. Kuipers, K. W. van Dijk, and L. M. Havekes Stimulation of the in Vivo Production of Very Low Density Lipoproteins by Apolipoprotein E Is Independent of the Presence of the Low Density Lipoprotein Receptor J. Biol. Chem., October 26, 2001; 276(44): 40693 - 40697. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by de Beer, F. Articles by Havekes, L. M. Search for Related Content PubMed PubMed Citation Articles by de Beer, F. Articles by Havekes, L. M. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Medline Plus Health Information Triglycerides Related Collections Animal models of human disease Gene therapy Lipid and lipoprotein metabolism