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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2817-2824.)
© 1997 American Heart Association, Inc.

Articles

Genetic Factors Precipitating Type III Hyperlipoproteinemia in Hypolipidemic Transgenic Mice Expressing Human Apolipoprotein E2

Yadong Huang; Stanley C. Rall, Jr; ; Robert W. Mahley

From the Gladstone Institute of Cardiovascular Disease (Y.H., S.C.R., R.W.M.), the Cardiovascular Research Institute (Y.H., R.W.M.), and the Departments of Pathology and Medicine (R.W.M.), University of California, San Francisco.

Correspondence to Robert W. Mahley, MD, PhD, Gladstone Institute of Cardiovascular Disease, PO Box 419100, San Francisco, CA 94141-9100.

	Abstract

Top Abstract Introduction Methods Results and Discussion References

 
Abstract Several factors are hypothesized to precipitate or exacerbate type III hyperlipoproteinemia (HLP) in humans. Among such factors are those that directly overload remnant lipoprotein production or disrupt removal pathways, including an increased ratio of apolipoprotein (apo) E2 to normal apoE, overproduction of apoB-containing lipoproteins, and decreased LDL receptor activity. Hypolipidemic apoE2-transgenic mice bred onto an apoE-null background had dramatically higher plasma total cholesterol (192±26 mg/dL for males, 203±40 mg/dL for females) and triglyceride (295±51 mg/dL for males, 277±58 mg/dL for females) levels than apoE2 mice with endogenous mouse apoE. Thus, eliminating normal apoE in the presence of apoE2 (thereby increasing the relative abundance of the defective ligand) can convert a hypolipidemic to a hyperlipidemic phenotype. Hypolipidemic apoE2 transgenic mice overexpressing human apoB had moderate remnant accumulation compared with apoE2-only or apoB-only transgenic mice, indicating that overproduction of apoB-containing lipoproteins in the presence of apoE2 can augment remnant production. Hypolipidemic apoE2 transgenic mice bred onto an LDL receptor–null background had markedly higher plasma total cholesterol (288±51 mg/dL for males, 298±73 mg/dL for females) and triglyceride (356±72 mg/dL for males, 317±88 mg/dL for females) levels than apoE2-only mice, and remnant accumulation increased even in apoE2 mice with a heterozygous LDL receptor–knockout background (compared with apoE2-only mice), suggesting that reducing or eliminating a major receptor-mediated remnant-removal pathway in the presence of apoE2 can also precipitate a hyperlipidemic phenotype. In all cases where either lipoprotein remnant production or removal pathways were severely stressed, increased remnant accumulation was apparent. As judged by the chemical characteristics of the remnant lipoproteins, the lipoprotein phenotype was quite similar to that of human type III HLP, especially in the apoE2-expressing mice with no endogenous apoE or LDL receptors, and thus these mice represent improved models of the disorder.

Key Words: type III hyperlipoproteinemia • LDL receptor • apolipoprotein B • apolipoprotein E • transgenic mice

	Introduction

Top Abstract Introduction Methods Results and Discussion References

 
Type III hyperlipoproteinemia is a genetic disorder of lipid metabolism in humans that predisposes affected subjects to the premature development of atherosclerosis.^{1 2 3} It is characterized by both hypercholesterolemia and hypertriglyceridemia due to the accumulation of remnant lipoproteins, namely, ß-migrating VLDL (ß-VLDL), in plasma.^{1 4} Most commonly, type III HLP occurs in homozygous carriers of apoE2, which has cysteine at residues 112 and 158 and which binds defectively to the LDL receptor (<2% of normal apoE3-binding activity).^{1 5 6} Type III HLP associated with apoE2 homozygosity appears to be transmitted recessively; essentially no one with the apoE3/2 phenotype develops type III HLP,^{7 8} and even among apoE2/2 subjects, <10% have hyperlipidemia.¹ Most subjects homozygous for apoE2 have hypocholesterolemia caused by low levels of LDL cholesterol and, to a lesser extent, by low levels of HDL cholesterol, although they all have detectable levels of ß-VLDL.^{9 10} These observations indicate that other genetic or environmental factors are necessary for expression of the hyperlipidemia in apoE2/2 subjects.⁷

ApoE knockout mice have been produced whose lipoprotein profile shows some similarities to human type III HLP, including hypercholesterolemia, accumulation of ß-VLDL in plasma, and development of spontaneous atherosclerosis.^{11 12} However, these mice also display some substantial differences from human type III HLP, including the absence of significant hypertriglyceridemia and an atypical ß-VLDL chemical composition. To develop an animal model that better represents the type III HLP phenotype, we and others recently produced human apoE2 transgenic mice.^{13 14} Although expression of low levels of apoE2 in transgenic mice did not significantly change plasma lipid levels,^{13 14} intermediate and high expression of apoE2 resulted in hypolipidemia and hyperlipidemia, respectively.¹³ The hypolipidemic apoE2 transgenic mice provide the opportunity to study genetic or environmental factors that precipitate or exacerbate type III HLP. In the present study, to investigate the role of the amount of defective ligand (apoE2) relative to normal apoE required for remnant lipoprotein accumulation, we crossed hypolipidemic apoE2 transgenic mice with homozygous apoE knockout mice to assess the confounding influence of endogenous mouse apoE on the development of the type III HLP phenotype in mice. We also investigated the role of the overproduction of apoB-containing lipoproteins (a possible secondary factor in type III HLP) by crossing hypolipidemic apoE2 transgenic mice with mice overexpressing human apoB.

Another secondary factor implicated in the exacerbation of type III HLP is low LDL receptor activity.^{1 15} Normally, the LDL receptor removes LDL, cholesterol-rich IDL, and chylomicron remnants from the plasma and thereby regulates plasma cholesterol levels.^{5 16 17} Genetic defects in the LDL receptor lead to hypercholesterolemia in humans with FH¹⁸ and in WHHL rabbits,¹⁹ causing massively elevated LDL levels as well as somewhat elevated IDL levels.^{18 19} Recently, LDL receptor knockout mice have been produced in which plasma IDL and LDL levels are markedly elevated.^{20 21} Tracer studies with radiolabeled lipoproteins in these mice, FH patients, and WHHL rabbits showed retarded clearance of both VLDL and IDL,^{20 22 23} which suggests that the LDL receptor is important for clearing remnant lipoproteins from the plasma. However, such remnants accumulate much less in FH patients,¹⁸ WHHL rabbits,¹⁹ and LDL receptor knockout mice^{20 21} than in type III HLP patients with defective apoE,¹ indicating that the liver can clear remnant lipoproteins when the LDL receptor is absent. This backup clearance system appears to be mediated by HSPG and the LRP, both of which bind apoE-enriched remnant lipoproteins.^{24 25 26 27 28} Thus, two receptor pathways are responsible for remnant clearance: the LDL receptor pathway and the HSPG/LRP pathway. To assess the impact of the LDL receptor pathway and LDL receptor levels on the type III HLP phenotype, we crossed hypolipidemic apoE2 transgenic mice with homozygous LDL receptor knockout mice.

Here we report that any further stress on the lipoprotein remnant production/removal pathways in the presence of apoE2—specifically, reduction or elimination of normal apoE, overproduction of apoB-containing lipoproteins, or reduction or elimination of the LDL receptor—is sufficient to confer on hypolipidemic apoE2 transgenic mice a lipoprotein phenotype that is very similar to human type III HLP.

	Methods

Top Abstract Introduction Methods Results and Discussion References

 
Materials
The Superose 6 column purchased from Pharmacia was used on a Pharmacia fast protein liquid chromatography system. The Centricon concentration filters were from Amicon. Cholesterol and triglyceride standards were from Abbott and Boehringer, respectively. The automated system for lipid analysis (Kinetic Microplate Reader) was from Molecular Devices. All the reagents for agarose gel electrophoresis were from Ciba-Corning. The ECL chemiluminescence detection kit for Western blots was purchased from Amersham Life Science.

Preparation of Transgenic Mice
Hemizygous human apoE2 transgenic mice (ICR strain) were produced previously in our laboratory.¹³ Hemizygous human apoB transgenic mice (C57BL/6 strain) were provided by Dr S. Young (Gladstone Institute of Cardiovascular Disease, San Francisco, Calif).^{29 30} Homozygous apoE knockout (mE^–/–) and homozygous LDL receptor knockout (LDLR^–/–) mice (C57BL/6 strain) were purchased from Jackson Laboratories, Bar Harbor, Me.

Female hypolipidemic apoE2 mice (apoE2, 23 to 26 mg/dL) were crossbred with male mice that had each of the backgrounds described above. The outcomes of the apoE2 crosses with apoB mice were ascertained by detection of the proteins in plasma and yielded all four genotypes, ie, mice with neither human protein, only apoE2, only apoB, or both. The presence of the human apoE2 and apoB transgenes was detected by immunoblotting 1 µL of plasma with human-specific anti-apoE and anti-apoB polyclonal antisera.^{30 31} In the Western blot assay, human apoE2 was semiquantified by comparing the densitometric readings of the sample bands with those of purified human apoE2 standards. In some cases, apoE2 mice were first crossed with either mE^–/– or LDLR^–/– mice to yield obligate heterozygotes for either mouse apoE (mE^+/–) or the LDL receptor (LDLR^+/–). Those heterozygous mice also expressing apoE2 (hE2^+/0,mE^+/– or hE2^+/0,LDLR^+/–) were then crossed again with mE^–/– or LDLR^–/– mice, respectively, to yield the four genotypes listed in Table 1 for each of these crosses. Deficiency of mouse apoE was established by Western blotting with mouse-specific anti-apoE antiserum (provided by Dr J. Borén, Gladstone Institute of Cardiovascular Disease). The LDL receptor deficiency was assessed by polymerase chain reaction with specific primers designed to identify both the altered and the unaltered gene sequences. All experiments were performed under protocols approved by the Committee on Animal Research, University of California, San Francisco.

View this table: [in this window] [in a new window]   Table 1. Plasma Lipid Levels of Various ApoE2 Transgenic Mice (in mg/dL±SD)

Lipoprotein Separation and Analysis
Blood was collected from the tails of 6- to 10-week-old mice that were fed a normal chow diet and had been fasted for 5 hours starting at 10 AM. EDTA was used as an anticoagulant (final concentration, 10 mmol/L). Plasma was obtained by centrifugation at 14 000 rpm in a microfuge (Brinkmann Instruments) for 10 minutes at 4°C, and samples were stored for no more than 2 days at 4°C in the presence of 1 mmol/L PMSF as a protease inhibitor. Lipoproteins in 100 µL of plasma were separated by chromatography on a Superose 6 column as described.^{13 31 32}

The major lipoprotein classes eluted from the Superose 6 column were pooled and concentrated with Centricon filters [fractions 16 to 18, VLDL; fractions 19 to 22, IDL; fractions 23 to 27, LDL and a subclass of HDL (HDL₁); and fractions 28 to 33, HDL]. For agarose gel electrophoresis, 2-µL aliquots of concentrated lipoproteins were fractionated on precast 1% agarose gels for 45 minutes at 90 V. The gels were dried and stained with fat red 7B. For analysis of their chemical composition, VLDLs (d<1.006 g/mL) were isolated from plasma by ultracentrifugation at 98 000 rpm for 2 hours at 4°C in a Beckman TL100 ultracentrifuge.³³ Apolipoproteins were separated on 3% to 20% polyacrylamide-SDS gradient gels and detected by Coomassie staining. The ratios of apoE2 to mouse apoE and of apoB48 to apoB100 were calculated after the gels were scanned.

Cholesterol and triglycerides in total plasma and in chromatographic fractions were measured by an enzymatic colorimetric method adapted for use with a microplate reader.^{32 34} The cholesteryl ester content of VLDL and IDL was determined by subtracting the free cholesterol from the total cholesterol value. The HDL cholesterol concentrations were measured after precipitation of apoB-containing lipoproteins by heparin/MnCl₂ (Wako Pure Chemical Industries).

	Results and Discussion

Top Abstract Introduction Methods Results and Discussion References

 
Increase in the Relative Abundance of Defective Human ApoE2 Converts Hypolipidemic ApoE2 Transgenic Mice to a Type III HLP Phenotype
In the present study, apoE2/ICR mice were backcrossed with the C57BL/6 strain to obtain F1 apoE2/ICRxC57BL/6 mice. In agreement with our previous results,¹³ intermediate levels of human apoE2 expression (19 to 29 mg/dL) in both hemizygous male and female transgenic mice on this genetic background resulted in a significant decrease in both total cholesterol and HDL cholesterol [hE2^+/0 (hypo-); Table 1]. In contrast, high levels of apoE2 expression (51 to 68 mg/dL) in both male and female transgenics significantly increased total cholesterol, even in the presence of endogenous mouse apoE, whereas HDL cholesterol remained in the normal range [hE2^+/0 (hyper-); Table 1]. The VLDL and IDL levels, representing ß-VLDL, also increased substantially (data not shown; see Reference 13¹³ ). In the VLDL fraction, the ratios of total cholesterol to triglycerides and of apoB48 to apoB100 were higher in both the hE2^+/0 (hypo-) and hE2^+/0 (hyper-) mice than in nontransgenic mice (Table 2), suggesting some accumulation of lipoprotein remnants.

View this table: [in this window] [in a new window]   Table 2. Characteristics of VLDL (d<1.006 g/mL) From ApoE2 Transgenic Mice

To evaluate further the effect of the relative abundance of apoE2 on the lipoprotein profile, endogenous mouse apoE was reduced or eliminated by crossing the hypolipidemic apoE2 mice with homozygous apoE-null mice (see "Methods"). All four expected genotypes were obtained from the second cross (hE2 TgxmE^–/–; Table 1). As reported previously by others,^{11 12} both male and female homozygous mouse apoE knockout mice (hE2^0/0,mE^–/–; Table 1) had a dramatic increase in total cholesterol, a significant decrease in HDL cholesterol, and a slight increase in triglycerides. Almost all of the increased cholesterol was associated with the accumulation of VLDL and IDL (Fig 1C), both of which were ß-migrating on agarose gels (Fig 2). The elimination of one mouse apoE allele (hE2^0/0,mE^+/–) did not significantly alter plasma lipid levels (Table 1) or lipoproteins (Fig 1A versus Fig 3A). However, apoE2-expressing mice with one endogenous apoE allele (hE2^+/0,mE^+/–) had slightly higher total cholesterol and significantly higher triglyceride levels than hE2^+/0 (hypo-) mice (Table 1). In hE2^+/0,mE^+/– mice, the cholesterol-rich VLDL and IDL (fractions 16 to 22) were slightly higher than in hE2^0/0,mE^+/– mice (Fig 1B versus Fig 1A). The HDL cholesterol levels in hE2^+/0,mE^+/– mice were similar to those in hE2^+/0 (hypo-) mice (Fig 1B versus Fig 3B) but lower than those in the hE2^0/0,mE^+/– mice (Fig 1B versus Fig 1A).

View larger version (36K): [in this window] [in a new window]   Figure 1. Superose 6 chromatography of 100 µL of mouse plasma. The plasma total cholesterol (TC) and triglyceride (TG) distributions from individual male mice were analyzed as described in "Methods." Each panel is one representative profile of several analyzed in each group of mice. Note the scale differences in panels C and D versus panels A and B. The units for apoE2, TC, and TG are mg/dL.

View larger version (43K): [in this window] [in a new window]   Figure 2. Agarose gel electrophoresis of lipoprotein fractions from various male apoE2 transgenic mice. The VLDLs are Superose 6 fractions 16-18, and IDLs are fractions 19-22. The origin and migration positions of -migrating (HDL), pre–ß-migrating (VLDL), and ß-migrating (LDL) lipoproteins are indicated.

View larger version (36K): [in this window] [in a new window]   Figure 3. Superose 6 chromatography of 100 µL of mouse plasma. The plasma total cholesterol (TC) and triglyceride (TG) distributions from individual nontransgenic or transgenic male mice were analyzed as described in "Methods." Each panel is one representative profile of several analyzed in each group of mice. The units for apoE2, TC, and TG are mg/dL.

On the other hand, apoE2 expression in hE2^+/0,mE^–/– mice (Table 1) markedly increased both total cholesterol (fourfold) and triglyceride (eightfold) levels compared with those in hE2^+/0 (hypo-) mice. These increases were due exclusively to the accumulation of remnant lipoproteins, including VLDL and IDL, which increased 10-fold (Fig 1D). The ratios of total cholesterol to triglycerides and of apoB48 to apoB100 in the VLDL were significantly higher (indicating remnant lipoprotein accumulation) in the hE2^+/0,mE^–/– mice than in the hE2^+/0 (hypo-) mice (Table 2). Both the VLDL and IDL were ß-migrating on agarose gels (Fig 2). The low HDL cholesterol level seen in the hE2^+/0 (hypo-) mice was maintained in the hE2^+/0,mE^–/– mice (Table 1 and Fig 1D).

Furthermore, hE2^+/0,mE^–/– mice had significantly lower total cholesterol but substantially higher triglyceride levels than the hE2^0/0,mE^–/– mice (Table 1). In the hE2^+/0,mE^–/– mice, the remnant lipoproteins (VLDL and IDL) were more triglyceride rich, the LDL cholesterol levels were lower, and the HDL cholesterol was further decreased (Fig 1D versus Fig 1C). In addition, plasma apoE2 concentrations increased in both the hE2^+/0,mE^+/– and hE2^+/0,mE^–/– mice, indicating enhanced accumulation of apoE2 in plasma when endogenous apoE expression was reduced or eliminated (Table 1).

These results demonstrate that the ratio of apoE2 to normal mouse apoE is an important determinant of both the accumulation of remnant lipoproteins and the expression of hyperlipidemia in the hypolipidemic apoE2 mice. The extent of remnant accumulation parallels the hE2/mE ratio: hE2^+/0,mE^–/–>hE2^+/0,mE^+/–>hE2^+/0 (hypo-). In the hE2^+/0 (hypo-) and hE2^+/0,mE^+/– mice, the presence of normal mouse apoE on the remnant particles in most circumstances may allow normal or nearly normal clearance of remnant lipoproteins from the plasma via the LDL receptor and HSPG/LRP pathways, as in humans heterozygous for apoE2 (ie, apoE3/2). Conversely, in the hE2^+/0,mE^–/– mice, as in humans homozygous for apoE2, the absence of mouse apoE greatly retards the clearance of remnants, resulting in hyperlipidemia. Although plasma total cholesterol levels in hE2^+/0,mE^–/– mice were significantly higher than those in nontransgenic or hE2^+/0 (hypo-) mice, they were about half of those in hE2^0/0,mE^–/– mice, which suggests that having receptor binding–defective apoE2 is better than having no apoE at all, as far as plasma cholesterol metabolism is concerned. The apoE-null mice that express considerably lower levels of apoE2¹⁴ than our mice have less of a reduction in plasma cholesterol (about one third versus one half), suggesting that the apoE2 expression level is an important determinant of total cholesterol and that, at least up to the levels assessed so far, it correlates inversely with plasma cholesterol when no endogenous mouse apoE is present.

The significant increase in triglycerides in the hE2^+/0,mE^–/– mice yields a profile more like human type III HLP, in which plasma cholesterol and triglyceride levels are approximately equally elevated,¹ in contrast to the very low triglyceride levels and the highly cholesterol-enriched remnants in the apoE-null mice^{11 12} and in the very low-expressing apoE2 mice lacking mouse apoE.¹⁴ Our hE2^+/0,mE^–/– mice expressing about 42 mg/dL of apoE2 have higher triglyceride levels (277 mg/dL) than the comparable mice of van Vlijmen et al,¹⁴ expressing 9 mg/dL of apoE2 (210 mg/dL of triglyceride) but have a lower cholesterol/triglyceride ratio in the remnant lipoproteins [0.8 (Table 2) versus 1.7], reflecting both the higher cholesterol and lower triglyceride levels in the mice of van Vlijmen et al.¹⁴ ApoE2 is associated with elevated plasma triglyceride levels in humans^{35 36} and may be associated with defective lipolytic processing of the remnants.³⁷ The fact that the ß-VLDL from the hE2^+/0,mE^–/– mice have a higher triglyceride content than those from either the hE2^0/0,mE^–/– mice or the lower-expressing apoE2 mice of van Vlijmen et al¹⁴ supports this possibility and indicates that having apoE2 is worse than having no apoE at all, as far as plasma triglyceride metabolism is concerned.

ApoB Overexpression Induces Remnant Accumulation in Hypolipidemic ApoE2 Transgenic Mice
To determine whether apoB overproduction in combination with defective apoE2 expression would convert the hypolipidemic pattern to one characterized by remnant lipoprotein accumulation, we crossbred hE2^+/0 (hypo-) mice with hemizygous human apoB transgenic mice (hB^+/0; human apoB plasma concentration, 50 mg/dL).³⁰ The resulting hE2^0/0,hB^+/0 mice had substantially higher total cholesterol and triglyceride levels than nontransgenic mice (hE2 TgxhB Tg; Table 1). In the hE2^0/0,hB^+/0 mice, LDLs were significantly increased and triglyceride enriched (Fig 3C).³⁰ As suggested in preliminary studies,¹³ simultaneous overexpression of human apoB and apoE2 (hE2^+/0,hB^+/0; Table 1) significantly increased total cholesterol and triglycerides in both males and females (with triglycerides being higher than total cholesterol) compared with the levels in hE2^+/0 (hypo-) mice. Almost all of the increased cholesterol was associated with the accumulation of large apoB-containing lipoproteins, ie, increased VLDL and IDL (Fig 3D versus Fig 3B). The HDL cholesterol levels in the double-transgenic mice were unchanged from those in hE2^+/0 (hypo-) mice (Fig 3D versus Fig 3B). On the other hand, the LDL cholesterol level was significantly lower in the hE2^+/0,hB^+/0 mice than in the hE2^0/0,hB^+/0 mice, while LDL triglycerides remained unchanged (Fig 3D versus Fig 3C). Thus, the lipoprotein profile in the double-transgenic mice was converted from the apoB overexpression pattern characterized by an increase primarily in LDL to one in which ß-VLDL remnants predominated (Fig 3D versus Fig 3C). Overexpression of apoB in hypolipidemic apoE2 mice also increased the ratios of total cholesterol to triglycerides and of apoB48 to apoB100 in VLDL, which now resembled the ß-VLDL seen in hyperlipidemic apoE2 mice [hE2^+/0 (hyper-); Table 2]. Furthermore, the lipoproteins in the VLDL and IDL fractions of the hE2^+/0,hB^+/0 mice possessed ß-electrophoretic mobility (Fig 2), whereas the VLDL in the hE2^0/0,hB^+/0 mice had pre–ß-mobility (data not shown). These results indicate that apoB overproduction in hypolipidemic apoE2 mice increased remnant lipoprotein levels, though not to the same extent as in apoE or LDL receptor knockout mice. This could mean that remnant production stresses are less important than remnant removal stresses in determining remnant accumulation, but the results might also be influenced by other conditions peculiar to mice, such as apoB editing in the liver and the lack of cholesteryl ester transfer protein.

The fact that overproduction of apoB results in an apoE2-induced accumulation of remnant lipoproteins provides direct evidence that increased biosynthesis of apoB-containing lipoproteins contributes to remnant accumulation in type III HLP patients.¹ Increased apoB production in the transgenic mice would certainly be expected to stimulate hepatic lipoprotein production,²⁹ which in turn would further stress the catabolic pathway for VLDL, IDL, and remnant lipoprotein metabolism, which had already been disturbed by the presence of the defective ligand, apoE2. Evidence supporting this kind of gene interaction comes from a clinical study of a large family from Seattle,³⁸ in which the occurrence of the genes for familial combined hyperlipidemia (probably caused by VLDL overproduction) and for dysbetalipoproteinemia (ie, the apoE2/2 phenotype) led to the clinical expression of type III HLP. In addition, apoE2 appears to impair the final lipolytic processing of IDL to LDL^{37 39} and possibly of VLDL to IDL, which is reflected in the higher triglyceride levels associated with the remnant lipoproteins in the hE^+/0,hB^+/0 mice than in nontransgenic and hypolipidemic hE2^+/0 transgenic mice (Table 1 and Fig 3).

Low LDL Receptor Number Precipitates Type III HLP in Hypolipidemic ApoE2 Transgenic Mice
It has been hypothesized that the level of LDL receptor expression in the liver is crucial in determining remnant lipoprotein accumulation in the plasma¹ and that low LDL receptor activity precipitates expression of type III HLP in humans homozygous for apoE2.¹⁵ To test this hypothesis, we reduced or eliminated the LDL receptors by crossing the hypolipidemic apoE2-transgenic mice with LDL receptor knockout mice (see "Methods"). All four expected genotypes were obtained from the second cross (hE2 TgxLDLR^–/–; Table 1). As reported previously,²⁰ heterozygous or homozygous knockout of the LDL receptor in both male and female mice significantly increased total cholesterol (hE2^0/0,LDLR^+/– and hE2^0/0,LDLR^–/–; Table 1), which was exclusively due to an increase in LDL cholesterol (Fig 4A and 4C). ApoE2 expression in heterozygous LDL receptor knockout mice (hE2^+/0,LDLR^+/–; Table 1) slightly increased total cholesterol (statistically significant for females) and significantly increased triglycerides in both males and females compared with the respective levels in hE2^+/0 (hypo-) mice. The hE2^+/0,LDLR^+/– mice had significantly higher VLDL and IDL levels than the hE2^+/0 (hypo-) transgenics; they also had higher VLDL and IDL and lower LDL cholesterol levels than hE2^0/0,LDLR^+/– mice (Fig 4B versus Fig 4A). The HDL cholesterol levels in the hE2^+/0,LDLR^+/– mice were unchanged from those in the hE2^+/0 (hypo-) transgenics, ie, remained lower than normal.

View larger version (30K): [in this window] [in a new window]   Figure 4. Superose 6 chromatography of 100 µL of mouse plasma. The plasma total cholesterol (TC) and triglyceride (TG) distributions from individual male mice were analyzed as described in "Methods." Each panel is one representative profile of several analyzed in each group of mice. Note the scale difference in panel D. The units for apoE2, TC, and TG are mg/dL.

However, apoE2 expression on a homozygous LDL receptor knockout background (hE2^+/0,LDLR^–/–; Table 1) increased total cholesterol about sixfold and triglycerides about 10-fold compared with the levels in hE2^+/0 (hypo-) mice. Remnant accumulation in the VLDL and IDL fractions of the hE2^+/0,LDLR^–/– mice was very pronounced (Fig 4D), being about sixfold higher than in the hE2^+/0,LDLR^+/– mice (Fig 4B). The ratios of total cholesterol to triglycerides and of apoB48 to apoB100 in the VLDL fraction increased markedly (indicating remnant lipoprotein accumulation) compared with the ratios in hE2^+/0 (hypo-) mice (Table 2). Both the VLDL and IDL were ß-migrating on agarose gels (Fig 2). Again, the LDL cholesterol levels were low (compare Fig 4D to 4C), while the HDL cholesterol levels were unchanged from those in hE2^+/0 (hypo-) mice (Table 1). Moreover, as in apoE2 transgenics on a mouse apoE knockout background, plasma apoE2 concentrations increased in both heterozygous and homozygous LDL receptor knockout backgrounds (Table 1), again indicating enhanced accumulation of apoE2-containing lipoproteins in plasma.

These results indicate that a deficiency or absence of the LDL receptors can precipitate type III HLP in the presence of apoE2, a conclusion also reached in a study of human subjects that indicated the same gene-gene interaction.¹⁵ These results suggest that the level of LDL receptors plays an important role in remnant clearance, even in the presence of LDL receptor binding–defective apoE2. It should be recalled that while apoE2 has <2% of apoE3's normal receptor binding,^{1 6} this level is comparable to that of apoB in LDL binding to the LDL receptors (the affinity of apoE3 binding to the LDL receptors is 40-fold higher than that of apoB-containing LDL).⁴⁰ In addition, since the LDL receptor knockout alone selectively increases LDL cholesterol (see Fig 4C and Reference 20²⁰ ), a significant accumulation of cholesterol- and triglyceride-enriched remnant lipoproteins in the hE2^+/0,LDLR^–/– mice may reflect not only disturbed remnant clearance but also an apoE2-induced block in the lipolytic conversion of VLDL and/or IDL to LDL, as suggested previously for apoE2/2 subjects.^{1 7 9 37} Decreased LDL cholesterol levels in the hE2^+/0,LDLR^–/– mice are consistent with this notion.

The dramatic effect of the gene-gene interaction between the LDL receptor and a specific apolipoprotein has also been demonstrated in LDL receptor knockout mice overexpressing apoCIII.⁴¹ In those mice, reducing or eliminating LDL receptors exacerbated the hypertriglyceridemia induced by an apoCIII transgene and yielded a phenotype reminiscent of familial combined hyperlipidemia, with an increase in all apoB-containing lipoproteins. There are both similarities and differences between those mice and our mice. For example, the elimination of a major lipoprotein removal pathway, in addition to the presence of a deleterious apoprotein (apoCIII or apoE), leads to increases in VLDL and IDL in both situations, while it increases in the apoCIII mice of Masucci-Magoulas et al.⁴¹

Although the LDL receptor is clearly important in remnant clearance,^{1 16 42} it is also clear that a second pathway, most likely the HSPG/LRP pathway, plays a role in remnant binding and uptake by the liver.^{24 25 26 43 44 45 46} Remnant clearance by either pathway requires functional apoE.^{1 17 43 47} In this study, by comparison with the much higher total cholesterol and remnant accumulation in the apoE-null mice, the lower total cholesterol levels in the hE2^+/0,LDLR^–/– mice may reflect the ability of apoE2 to mediate at least some remnant clearance, presumably via the HSPG/LRP pathway, although this pathway appears to be less efficient than the LDL receptor pathway. ApoE2 binds to the HSPG/LRP with 50% to 90% of the binding activity of apoE3.^{25 48} Furthermore, we have previously shown that the HSPG/LRP pathway is readily saturated⁴⁹ and suggested that a variety of factors, such as low LDL receptor expression, could readily overwhelm HSPG sequestration and LRP-mediated uptake in remnant clearance.⁴⁶

The HDL-lowering effect of apoE2 in hE2^+/0 (hypo-) mice is intriguing (Table 1). We speculated previously that apoE2 might interact with endogenous mouse apoE to disrupt the formation of HDL or alter the lipoproteins in such a way that they are rapidly cleared.¹³ However, the fact that the HDL remained unchanged (markedly reduced) in the hE2^+/0,mE^–/– mice compared with the hE2^+/0 (hypo-) mice excludes this possibility. Furthermore, the HDL levels in hE2^+/0,LDLR^–/– mice were unchanged from those in the hE2^+/0 (hypo-) transgenics, indicating that the LDL receptor is not involved in the HDL-lowering effect of apoE2. It is possible that apoE2 enrichment of HDL may target them to the HSPG/LRP pathway or alternate clearance mechanisms. Modulation of HDL levels in these and other transgenic animal models remains to be clearly understood.

In summary, our results indicate that increasing the ratio of variant apoE2 to normal apoE or eliminating all normal apoE, increasing apoB production, or decreasing or eliminating LDL receptors can confer a type III HLP phenotype on hypolipidemic apoE2 transgenic mice. It is especially apparent that further stress on remnant removal mechanisms in mice already expressing defective apoE2 is sufficient to precipitate or exacerbate the hyperlipidemic phenotype. The lipoproteins in these animal models are very similar to those in humans with type III HLP, as judged by the cholesterol and triglyceride content of the ß-VLDLs and other chemical characteristics. Thus, the mice generated in the current study are significantly improved models of type III HLP and have helped to elucidate the mechanisms responsible for the precipitation of overt type III HLP in the hypolipidemic apoE2 expressers.

	Selected Abbreviations and Acronyms

 

apo = apolipoprotein FH = familial hypercholesterolemia HLP = hyperlipoproteinemia HSPG = heparan sulfate proteoglycan(s) IDL = intermediate density lipoprotein LRP = LDL receptor–related protein VLDL = very low density lipoprotein(s) WHHL = Watanabe heritable hyperlipidemic

	Acknowledgments

 
This work was supported in part by program project grant HL47660 from the National Institutes of Health, Bethesda, Md. (principal investigator, T.L. Innerarity, PhD). We thank X.Q. Liu for excellent technique assistance, K.H. Weisgraber for providing apoE and apoE antisera, P. Chin for microinjection of the DNA construct, J.M. Taylor for his helpful comments, S. Richmond for manuscript preparation, J. Carroll and A. Corder for graphics, and G. Howard and S. Ordway for editorial assistance.

Received May 7, 1997; accepted August 4, 1997.

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