Abnormal low density lipoprotein metabolism in apolipoprotein E deficiency

Apolipoprotein(apo) E deficiency is an inherited disease characterized by type III hyperlipoproteinemia and less than 1% normal plasma apoE concentration. The role of apoE in LDL metabolism was investigated by quantitating the metabolism of radiolabeled normal and apoE-deficient LDL in both normal and apoE-deficient subjects. ApoE deficiency resulted in an accumulation of plasma IDL, and a decreased synthesis of LDL consistent with a block in the conversion of IDL to LDL. The LDL isolated from the apoE-deficient patient was similar to normal LDL in hydrated density, size, and composition. However, the apoE-deficient LDL was kinetically abnormal with delayed catabolism in both normal subjects and the apoE-deficient patient. In addition, the catabolism of normal LDL in the apoE-deficient subject was increased. These results were interpreted as indicating that apoE is necessary for the conversion of IDL to LDL and the formation of kinetically normal LDL. The rapid catabolism of normal LDL in the apoE-deficient patient suggests an up-regulation of the hepatic LDL receptor pathway. Based on these results, apoE is proposed to play an important role in the conversion of IDL to LDL, the formation of kinetically normal LDL, and the regulation of LDL receptor function.     

Effect of apolipoprotein M on high density lipoprotein metabolism and atherosclerosis in low density lipoprotein receptor knock-out mice

To investigate the role of apoM in high density lipoprotein (HDL) metabolism and atherogenesis, we generated human apoM transgenic (apoM-Tg) and apoM-deficient (apoM(-/-)) mice. Plasma apoM was predominantly associated with 10-12-nm alpha-migrating HDL particles. Human apoM overexpression (11-fold) increased plasma cholesterol concentration by 13-22%, whereas apoM deficiency decreased it by 17-21%. The size and charge of apoA-I-containing HDL in plasma were not changed in apoM-Tg or apoM(-/-) mice. However, in plasma incubated at 37 degrees C, lecithin:cholesterol acyltransferase-dependent conversion of alpha- to pre-alpha-migrating HDL was delayed in apoM-Tg mice. Moreover, lecithin: cholesterol acyltransferase-independent generation of pre-beta-migrating apoA-I-containing particles in plasma was increased in apoM-Tg mice (4.2 +/- 1.1%, p = 0.06) and decreased in apoM(-/-) mice (0.5 +/- 0.3%, p = 0.03) versus controls (1.8 +/- 0.05%). In the setting of low density lipoprotein receptor deficiency, apoM-Tg mice with approximately 2-fold increased plasma apoM concentrations developed smaller atherosclerotic lesions than controls. The effect of apoM on atherosclerosis may be facilitated by enzymatic modulation of plasma HDL particles, increased cholesterol efflux from foam cells, and an antioxidative effect of apoM-containing HDL.     

Lecithin:cholesterol acyltransferase deficiency increases atherosclerosis in the low density lipoprotein receptor and apolipoprotein E knockout mice.

The purpose of the present study was to test the hypothesis that lecithin:cholesterol acyltransferase (LCAT) deficiency would accelerate atherosclerosis development in low density lipoprotein (LDL) receptor (LDLr-/-) and apoE (apoE-/-) knockout mice. After 16 weeks of atherogenic diet (0.1% cholesterol, 10% calories from palm oil) consumption, LDLr-/- LCAT-/- double knockout mice, compared with LDLr-/- mice, had similar plasma concentrations of free (FC), esterified (EC), and apoB lipoprotein cholesterol, increased plasma concentrations of phospholipid and triglyceride, decreased HDL cholesterol, and 2-fold more aortic FC (142 +/- 28 versus 61 +/- 20 mg/g protein) and EC (102 +/- 27 versus 61+/- 27 mg/g). ApoE-/- LCAT-/- mice fed the atherogenic diet, compared with apoE-/- mice, had higher concentrations of plasma FC, EC, apoB lipoprotein cholesterol, and phospholipid, and significantly more aortic FC (149 +/- 62 versus 109 +/- 33 mg/g) and EC (101 +/- 23 versus 69 +/- 20 mg/g) than did the apoE-/- mice. LCAT deficiency resulted in a 12-fold increase in the ratio of saturated + monounsaturated to polyunsaturated cholesteryl esters in apoB lipoproteins in LDLr-/- mice and a 3-fold increase in the apoE-/- mice compared with their counterparts with active LCAT. We conclude that LCAT deficiency in LDLr-/- and apoE-/- mice fed an atherogenic diet resulted in increased aortic cholesterol deposition, likely due to a reduction in plasma HDL, an increased saturation of cholesteryl esters in apoB lipoproteins and, in the apoE-/- background, an increased plasma concentration of apoB lipoproteins.     

High density lipoprotein metabolism in low density lipoprotein receptor-deficient mice

The LDL receptor (LDLR) and scavenger receptor class B type I (SR-BI) play physiological roles in LDL and HDL metabolism in vivo. In this study, we explored HDL metabolism in LDLR-deficient mice in comparison with WT littermates. Murine HDL was radiolabeled in the protein (¹²⁵I) and in the cholesteryl ester (CE) moiety ([³H]). The metabolism of ¹²⁵I-/[³H]HDL was investigated in plasma and in tissues of mice and in murine hepatocytes. In WT mice, liver and adrenals selectively take up HDL-associated CE ([³H]). In contrast, in LDLR^−/− mice, selective HDL CE uptake is significantly reduced in liver and adrenals. In hepatocytes isolated from LDLR^−/− mice, selective HDL CE uptake is substantially diminished compared with WT liver cells. Hepatic and adrenal protein expression of lipoprotein receptors SR-BI, cluster of differentiation 36 (CD36), and LDL receptor-related protein 1 (LRP1) was analyzed by immunoblots. The respective protein levels were identical both in hepatic and adrenal membranes prepared from WT or from LDLR^−/− mice. In summary, an LDLR deficiency substantially decreases selective HDL CE uptake by liver and adrenals. This decrease is independent from regulation of receptor proteins like SR-BI, CD36, and LRP1. Thus, LDLR expression has a substantial impact on both HDL and LDL metabolism in mice.     

Inhibition by a coantioxidant of aortic lipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E and low density lipoprotein receptor gene double knockout mice.

Antioxidants can inhibit atherosclerosis in animals, though it is not clear whether this is due to the inhibition of aortic lipoprotein lipid (per)oxidation. Coantioxidants inhibit radical-induced, tocopherol-mediated peroxidation of lipids in lipoproteins through elimination of tocopheroxyl radical. Here we tested the effect of the bisphenolic probucol metabolite and coantioxidant H 212/43 on atherogenesis in apolipoprotein E and low density lipoprotein (LDL) receptor gene double knockout (apoE-/-;LDLr-/-) mice, and how this related to aortic lipid (per)oxidation measured by specific HPLC analyses. Dietary supplementation with H 212/43 resulted in circulating drug levels of approximately 200 microM, increased plasma total cholesterol slightly and decreased plasma and aortic alpha-tocopherol significantly relative to age-matched control mice. Treatment with H 212/43 increased the antioxidant capacity of plasma, as indicated by prolonged inhibition of peroxyl radical-induced, ex vivo lipid peroxidation. Aortic tissue from control apoE-/-;LDLr-/- mice contained lipid hydro(pero)xides and substantial atherosclerotic lesions, both of which were decreased strongly by supplementation of the animals with H 212/43. The results show that a coantioxidant effectively inhibits in vivo lipid peroxidation and atherosclerosis in apoE-/-;LDLr-/- mice, consistent with though not proving a causal relationship between aortic lipoprotein lipid oxidation and atherosclerosis in this model of the disease.     

Prevention of low density lipoprotein aggregation by high density lipoprotein or apolipoprotein A-I

We have shown previously that low density lipoprotein (LDL) subjected to vortexing forms self-aggregates that are avidly phagocytosed by macrophages. That phagocytic uptake is mediated by the LDL receptor. We now show that LDL self-aggregation is strongly inhibited (80-95%) by the presence of high density lipoprotein (HDL) or apolipoprotein (apo) A-I. Another type of LDL aggregation, namely that induced by incubation of LDL with phospholipase C, was also markedly inhibited by HDL or apoA-I. The aggregation of LDL induced by vortexing was not inhibited by 2.5 M NaCl, and apoA-I was still able to block LDL aggregation at this high salt concentration, strongly suggesting hydrophobic interactions as the basis for the effect of apoA-I. The fact that apoA-I protected against LDL aggregation induced by two apparently quite different procedures suggests that the aggregation in these two cases has common features. We propose that these forms of LDL aggregation result from the exposure of hydrophobic domains normally masked in LDL and that the LDL-LDL association occurs when these domains interact. ApoA-I, because of its amphipathic character, is able to interact with the exposed hydrophobic domains of LDL and thus block the intermolecular interactions that cause aggregation.     

The lipid-associated conformation of the low density lipoprotein receptor binding domain of human apolipoprotein E

Apolipoprotein E (apoE) is a 34-kDa exchangeable apolipoprotein that regulates metabolism of plasma lipoproteins by functioning as a ligand for members of the LDL receptor family. The receptor-binding region localizes to the vicinity of residues 130-150 within its independently folded 22-kDa N-terminal domain. In the absence of lipid, this domain exists as a receptor-inactive, globular four-helix bundle. Receptor recognition properties of this domain are manifest upon lipid association, which is accompanied by a conformational change in the protein. Fluorescence resonance energy transfer has been used to monitor helix repositioning, which accompanies lipid association of the apoE N-terminal domain. Site-directed mutagenesis was used to replace naturally occurring Trp residues with phenylalanine, creating a Trp-null apoE3 N-terminal domain (residues 1-183). Subsequently, tyrosine residues in helix 2, helix 3, or helix 4 were converted to Trp, generating single Trp mutant proteins. The lone cysteine at position 112 was covalently modified with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine, which serves as an energy acceptor from excited tryptophan residues. Fluorescence resonance energy transfer analysis of apoE N-terminal domain variants in phospholipid disc complexes suggests that the helix bundle opens to adopt a partially extended conformation. A model is presented that depicts a tandem arrangement of the receptor-binding region of the protein in the disc complex, corresponding to its low density lipoprotein receptor-active conformation.     

Up-regulation of ATP binding cassette transporter A1 expression by very low density lipoprotein receptor and apolipoprotein E receptor 2

Activation of very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (apoER2) results in either pro- or anti-atherogenic effects depending on the ligand. Using reelin and apoE as ligands, we studied the impact of VLDLR- and apoER2-mediated signaling on the expression of ATP binding cassette transporter A1 (ABCA1) and cholesterol efflux using RAW264.7 cells. Treatment of these mouse macrophages with reelin or human apoE3 significantly increased ABCA1 mRNA and protein levels, and apoAI-mediated cholesterol efflux. In addition, both reelin and apoE3 significantly increased phosphorylated disabled-1 (Dab1), phosphatidylinositol 3-kinase (PI3K), protein kinase Cζ (PKCζ), and specificity protein 1 (Sp1). This reelin- or apoER2-mediated up-regulation of ABCA1 expression was suppressed by 1) knockdown of Dab1, VLDLR, and apoER2 with small interfering RNAs (siRNAs), 2) inhibition of PI3K and PKC with kinase inhibitors, 3) overexpression of kinase-dead PKCζ, and 4) inhibition of Sp1 DNA binding with mithramycin A. Activation of the Dab1-PI3K signaling pathway has been implicated in VLDLR- and apoER2-mediated cellular functions, whereas the PI3K-PKCζ-Sp1 signaling cascade has been implicated in the regulation of ABCA1 expression induced by apoE/apoB-carrying lipoproteins. Taken together, these data support a model in which activation of VLDLR and apoER2 by reelin and apoE induces ABCA1 expression and cholesterol efflux via a Dab1-PI3K-PKCζ-Sp1 signaling cascade.     

Effect of arginine 172 on the binding of apolipoprotein E to the low density lipoprotein receptor

The region of apolipoprotein E (apoE) that interacts directly with the low density lipoprotein (LDL) receptor lies in the vicinity of residues 136-150, where lysine and arginine residues are crucial for full binding activity. However, defective binding of carboxyl-terminal truncations of apoE3 has suggested that residues in the vicinity of 170-183 are also important. To characterize and define the role of this region in LDL receptor binding, we created either mutants of apoE in which this region was deleted or in which arginine residues within this region were sequentially changed to alanine. Deletion of residues 167-185 reduced binding activity (15% of apoE3), and elimination of arginines at positions 167, 172, 178, and 180 revealed that only position 172 affected binding activity (2% of apoE3). Substitution of lysine for Arg(172) reduced binding activity to 6%, indicating a specific requirement for arginine at this position. The higher binding activity of the Delta167-185 mutant relative to the Arg(172) mutant (15% versus 2%) is explained by the fact that arginine residues at positions 189 and 191 are shifted in the deletion mutant into positions equivalent to 170 and 172 in the intact protein. Mutation of these residues and modeling the region around these residues suggested that the influence of Arg(172) on receptor binding activity may be determined by its orientation at a lipid surface. Thus, the association of apoE with phospholipids allows Arg(172) to interact directly with the LDL receptor or with other residues in apoE to promote its receptor-active conformation.     

A change in apolipoprotein B expression is required for the binding of apolipoprotein E to very low density lipoprotein

Factors affecting the association of apolipoprotein E (apoE) with human plasma very low density lipoprotein (VLDL) were investigated in experiments in which the lipid content of the lipoprotein was modified either by lipid transfer in the absence of lipolysis or through the action of lipoprotein lipase. In both cases, lipoprotein particles initially containing no apoE (VLDL-E), isolated by heparin affinity chromatography, were modified until they had the same lipid composition as native apoE-containing VLDL (VLDL+E) from the same plasma. Transfer-modified lipoproteins, unlike native VLDL+E, did not bind apoE or interact with heparin. In contrast, VLDL-E, whose lipid composition was modified to the same extent by lipase, bound apoE and bound to heparin under the same conditions as native VLDL+E. A structural protein (apolipoprotein B) epitope characteristic of VLDL+E was expressed during lipolysis prior to ApoE or heparin binding. The data suggest that the reaction of apoE with VLDL-E is a two-step reaction. The appearance of apoB is modified during lipolysis, with expression of a major heparin-binding site. The modified VLDL then becomes competent to bind apoE. The lipid composition of VLDL appears not to be a major factor in the ability of VLDL to bind apoE or to bind to heparin.     

Lipid-induced extension of apolipoprotein E helix 4 correlates with low density lipoprotein receptor binding ability

Apolipoprotein E (apoE) serves as a ligand for the low density lipoprotein receptor (LDLR) only when bound to lipid. The N-terminal domain of lipid-free apoE exists as globular 4-helix bundle that is conferred with LDLR recognition ability after undergoing a lipid binding-induced conformational change. To investigate the structural basis for this phenomenon, site-directed spin label electron paramagnetic resonance spectroscopy experiments were conducted, focusing on the region near the C-terminal end of helix 4 (Ala-164). Using C112S apoE-N-terminal as template, a series of single cysteine substitution variants (at sequence positions 161, 165, 169, 173, 176, and 181) were produced, isolated, and labeled with the nitroxide probe, methane thiosulfonate. Electron paramagnetic resonance analysis revealed that lipid association induced fixed secondary structure in a region of the molecule known to exist as random coil in the lipid-free state. In a complementary approach, site-directed fluorescence analysis using an environmentally sensitive probe indicated that the lipid-induced transition of this region of the protein to alpha helix was accompanied by relocation to a more hydrophobic environment. In studies with full-length apoE single Cys variants, a similar random coil to stable backbone transition was observed, consistent with the concept that lipid interaction induced an extension of helix 4 beyond the boundary defining its lipid-free conformation. This structural transition likely represents a key conformational change necessary for manifestation of the LDLR recognition properties of apoE.     

Severe hypercholesterolemia,impaired fat tolerance,and advanced atherosclerosis in mice lacking both low density lipoprotein receptor-related protein 5 and apolipoprotein E

LDL receptor-related protein 5 (LRP5) plays multiple roles, including embryonic development and bone accrual development. Recently, we demonstrated that LRP5 is also required for normal cholesterol metabolism and glucose-induced insulin secretion. To further define the role of LRP5 in the lipoprotein metabolism, we compared plasma lipoproteins in mice lacking LRP5, apolipoprotein E (apoE), or both (apoE;LRP5 double knockout). On a normal chow diet, the apoE;LRP5 double knockout mice (older than 4 months of age) had approximately 60% higher plasma cholesterol levels compared with the age-matched apoE knockout mice. In contrast, LRP5 deficiency alone had no significant effects on the plasma cholesterol levels. High performance liquid chromatography analysis of plasma lipoproteins revealed that cholesterol levels in the very low density lipoprotein and low density lipoprotein fractions were markedly increased in the apoE;LRP5 double knockout mice. There were no apparent differences in the pattern of apoproteins between the apoE knockout mice and the apoE;LRP5 double knockout mice. The plasma clearance of intragastrically loaded triglyceride was markedly impaired by LRP5 deficiency. The atherosclerotic lesions of the apoE;LRP5 double knockout mice aged 6 months were approximately 3-fold greater than those in the age-matched apoE-knockout mice. Furthermore, histological examination revealed highly advanced atherosclerosis, with remarkable accumulation of foam cells and destruction of the internal elastic lamina in the apoE;LRP5 double knockout mice. These data suggest that LRP5 mediates both apoE-dependent and apoE-independent catabolism of plasma lipoproteins.     

Evolution of lipoprotein receptors. The chicken oocyte receptor for very low density lipoprotein and vitellogenin binds the mammalian ligand apolipoprotein E

The laying hen expresses two different lipoprotein transport receptors in cell-specific fashion. On the one hand, a 95-kDa oocyte membrane protein mediates the uptake of the major yolk precursors, very low density lipoprotein, and vitellogenin; on the other hand, somatic cells synthesize a 130-kDa receptor that is involved in the regulation of cellular cholesterol homeostasis (Hayashi, K., Nimpf, J., and Schneider, W. J. (1989) J. Biol. Chem. 264, 3131-3139). Here we show that the oocyte-specific receptor binds, in addition to the yolk precursor proteins, an apolipoprotein of mammalian origin, apolipoprotein E. Ligand blotting, a solid-phase binding assay, and antireceptor antibodies were employed to demonstrate that binding of vitellogenin, very low density lipoprotein (via apolipoprotein B), and apolipoprotein E occurs to closely related, if not identical, sites on the 95-kDa oocyte receptor. The binding properties of lipovitellin, which harbors the receptor recognition site of vitellogenin, are analogous to those of apolipoprotein E: both require association with lipid for expression of functional receptor binding. The ligand specificity of the avian oocyte lipoprotein receptor supports the hypothesis that vitellogenin, which has evolved in oviparous species, represents a counterpart to mammalian apolipoprotein E.     

Enlargement of high density lipoprotein in mice via liver X receptor activation requires apolipoprotein E and is abolished by cholesteryl ester transfer protein expression

The factors involved in the generation of larger high density lipoprotein (HDL) particles, HDL1 and HDLc, are still not well understood. Administration of a specific synthetic liver X receptor (LXR) agonist, T0901317, in mice resulted in an increase of not only HDL cholesterol but also HDL particle size (Cao, G., Beyer, T. P., Yang, X. P., Schmidt, R. J., Zhang, Y., Bensch, W. R., Kauffman, R. F., Gao, H., Ryan, T. P., Liang, Y., Eacho, P. I., and Jiang, X. C. (2002) J. Biol. Chem. 277, 39561-39565). We have investigated the roles that apoE and CETP may play in this process. We treated apoE-deficient, cholesterol ester transport protein (CETP) transgenic, and wild type mice with various doses of the LXR agonist and monitored their HDL levels. Fast protein liquid chromatography and apolipoprotein analysis revealed that in apoE knockout mouse plasma, there was neither induction of larger HDL formation nor increase of HDL cholesterol, suggesting that apoE is essential for the LXR agonist effects on HDL metabolism. In CETP transgenic mice, CETP expression completely abolished LXR agonist-mediated HDL enlargement and greatly attenuated HDL cholesterol levels. Analysis of HDL particles by electron microscope and nondenaturing gel electrophoresis revealed similar findings. In apoE-deficient mice, LXR agonist also produced a significant increase in very low density lipoprotein/low density lipoprotein cholesterol and apolipoprotein B content. Our studies provide direct evidence that apoE and CETP are intimately involved in the accumulation of the enlarged HDL (HDL1 or HDLc) particles in mice.     

The 39-kDa receptor-associated protein interacts with two members of the low density lipoprotein receptor family, alpha 2-macroglobulin receptor and glycoprotein 330.

The 39-kDa receptor-associated protein (RAP) binds to the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) and inhibits binding of ligands to this receptor. The in vivo function of RAP may be to regulate ligand binding and/or assist in the correct biosynthetic processing or trafficking of the alpha 2MR/LRP. Here we show that RAP binds another putative receptor, the kidney glycoprotein 330 (gp330). Gp330 is a high molecular weight glycoprotein that is structurally similar to both the alpha 2MR/LRP and low density lipoprotein receptor. The ability of RAP to bind to gp330 was demonstrated by ligand blotting and solid phase binding assays, which showed that RAP binds to gp330 with high affinity (Kd = 8 nM). Exploiting the interaction of gp330 and RAP, we purified gp330 by affinity chromatography with a column of RAP coupled to Sepharose. Gp330 preparations obtained by this procedure were notably more homogeneous than those obtained by conventional methods. Immunocytochemical staining of human kidney sections localized RAP to the brush-border epithelium of proximal tubules. The fact that gp330 is also primarily expressed by proximal tubule epithelial cells strengthens the likelihood that the interaction between gp330 and RAP occurs in vivo. The functional significance of RAP binding to gp330 may be to antagonize ligand binding as has been demonstrated for the alpha 2MR/LRP or to assist in the biosynthetic processing and/or trafficking of this receptor.     

Single nucleotide polymorphisms in the apolipoprotein B and low density lipoprotein receptor genes affect response to antihypertensive treatment

Background

Dyslipidemia has been associated with hypertension. The present study explored if polymorphisms in genes encoding proteins in lipid metabolism could be used as predictors for the individual response to antihypertensive treatment.

Methods

Ten single nucleotide polymorphisms (SNP) in genes related to lipid metabolism were analysed by a microarray based minisequencing system in DNA samples from ninety-seven hypertensive subjects randomised to treatment with either 150 mg of the angiotensin II type 1 receptor blocker irbesartan or 50 mg of the β₁-adrenergic receptor blocker atenolol for twelve weeks.

Results

The reduction in blood pressure was similar in both treatment groups. The SNP C711T in the apolipoprotein B gene was associated with the blood pressure response to irbesartan with an average reduction of 19 mmHg in the individuals carrying the C-allele, but not to atenolol. The C16730T polymorphism in the low density lipoprotein receptor gene predicted the change in systolic blood pressure in the atenolol group with an average reduction of 14 mmHg in the individuals carrying the C-allele.

Conclusions

Polymorphisms in genes encoding proteins in the lipid metabolism are associated with the response to antihypertensive treatment in a drug specific pattern. These results highlight the potential use of pharmacogenetics as a guide for individualised antihypertensive treatment, and also the role of lipids in blood pressure control.     

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

Apolipoprotein (apo) E stimulates the secretion of very low density lipoproteins (VLDLs) by an as yet unknown mechanism. Recently, a working mechanism for apoE was proposed (Twisk, J., Gillian-Daniel, D. L., Tebon, A., Wang, L., Barrett, P. H., and Attie, A. D. (2000) J. Clin. Invest. 105, 521-532) in which apoE prevents the inhibitory action of the low density lipoprotein receptor (LDLr) by binding to it. We have first tested whether this newly described effect of the LDLr on VLDL secretion, obtained in vitro, is also observed in vivo. In LDLr knockout mice (LDLr-/-), the production of VLDL triglycerides and apoB was 30% higher than that in controls. Also the ratio of apoB100:apoB48 secretion was increased in the LDLr-/- mice. The composition of nascent VLDL was similar in both strains. To test whether the action of apoE depends on the presence of the LDLr, VLDL production was measured in LDLr-/- and apoE-/- LDLr-/- mice. Deletion of apoE on a LDLr-/- background still caused a 50% decrease of VLDL triglycerides and apoB production. The composition of nascent VLDL was again similar for both strains. We conclude that the effect of apoE on hepatic VLDL production is independent of the presence of the LDLr.     

Hepatic apolipoprotein E expression promotes very low density lipoprotein-apolipoprotein B production in vivo in mice

In addition to its role in the uptake of apolipoprotein B (apoB)-containing lipoproteins, apoE promotes hepatic very low density lipoprotein-triglyceride (VLDL-TG) production in animal models. However, it is not known if apoE increases the amount of TG per VLDL particle or the number of VLDL particles secreted. VLDL-apoB production is a measure of the rate of VLDL particle secretion. We determined the effects of apoE deficiency and apoE overexpression on VLDL-apoB production in mice. [(35)S]methionine was injected into endogenously label VLDL-apoB and Triton WR-1339 was simultaneously injected to block the catabolism of VLDL. Compared with wild-type mice, the VLDL-apoB production rate was decreased by 33% in apoE-deficient mice. Conversely, VLDL-apoB production was increased by 48% in mice overexpressing apoE compared with controls. Nascent VLDL, obtained from post-Triton plasma, had a decreased, not increased, content of TG per apoB in the apoE-overexpressing group compared with the control group. This study demonstrates that hepatic apoE expression increases the output of VLDL triglyceride by increasing the production rate of VLDL-apoB, suggesting that hepatic apoE influences the number of VLDL particles secreted by the liver.     

Mechanisms of inhibition by apolipoprotein C of apolipoprotein E-dependent cellular metabolism of human triglyceride-rich lipoproteins through the low density lipoprotein receptor pathway

The mechanism of inhibition by apolipoprotein C of the uptake and degradation of triglyceride-rich lipoproteins from human plasma via the low density lipoprotein (LDL) receptor pathway was investigated in cultured human skin fibroblasts. Very low density lipoprotein (VLDL) density subfractions and intermediate density lipoprotein (IDL) with or without added exogenous recombinant apolipoprotein E-3 were used. Total and individual (C-I, C-II, C-III-1, and C-III-2) apoC molecules effectively inhibited apoE-3-mediated cell metabolism of the lipoproteins through the LDL receptor, with apoC-I being most effective. When the incubation was carried out with different amounts of exogenous apoE-3 and exogenous apoC, it was shown that the ratio of apoE-3 to apoC determined the uptake and degradation of VLDL. Excess apoE-3 overcame, at least in part, the inhibition by apoC. ApoC, in contrast, did not affect LDL metabolism. Neither apoA-I nor apoA-II, two apoproteins that do not readily associate with VLDL, had any effect on VLDL cell metabolism. The inhibition of VLDL and IDL metabolism cannot be fully explained by interference of association of exogenous apoE-3 with or displacement of endogenous apoE from the lipoproteins. IDL is a lipoprotein that contains both apoB-100 and apoE. By using monoclonal antibodies 4G3 and 1D7, which specifically block cell interaction by apoB-100 and apoE, respectively, it was possible to assess the effects of apoC on either apoprotein. ApoC dramatically depressed the interaction of IDL with the fibroblast receptor through apoE, but had only a moderate effect on apoB-100. The study thus demonstrates that apoC inhibits predominantly the apoE-3-dependent interaction of triglyceride-rich lipoproteins with the LDL receptor in cultured fibroblasts and that the mechanism of inhibition reflects association of apoC with the lipoproteins and specific concentration-dependent effects on apoE-3 at the lipoprotein surface.