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• W2110593962 abstract "Apolipoprotein E (apoE) is essential for the clearance of plasma chylomicron and VLDL remnants. The human APOE locus is polymorphic and 5–10% of APOE*2 homozygotes exhibit type-III hyperlipoproteinemia (THL), while the remaining homozygotes have less than normal plasma cholesterol. In contrast, mice expressing APOE*2 in place of the mouse Apoe (Apoe2/2 mice) are markedly hyperlipoproteinemic, suggesting a species difference in lipid metabolism (e.g., editing of apolipoprotein B) enhances THL development. Since apoB-100 has an LDLR binding site absent in apoB-48, we hypothesized that the Apoe2/2 THL phenotype would improve if all Apoe2/2 VLDL contained apoB-100. To test this, we crossed Apoe2/2 mice with mice lacking the editing enzyme for apoB (Apobec−/−). Consistent with an increase in remnant clearance, Apoe2/2 · Apobec−/− mice have a significant reduction in IDL/LDL cholesterol (IDL/LDL-C) compared with Apoe2/2 mice. However, Apoe2/2 ·Apobec−/− mice have twice as much VLDL triglyceride as Apoe2/2 mice. In vitro tests show the apoB-100-containing VLDL are poorer substrates for lipoprotein lipase than apoB-48-containing VLDL.Thus, despite a lowering in IDL/LDL-C, substituting apoB-48 lipoproteins with apoB-100 lipoproteins did not improve the THL phenotype in the Apoe2/2 ·Apobec−/− mice, because apoB-48 and apoB-100 differentially influence the catabolism of lipoproteins. Apolipoprotein E (apoE) is essential for the clearance of plasma chylomicron and VLDL remnants. The human APOE locus is polymorphic and 5–10% of APOE*2 homozygotes exhibit type-III hyperlipoproteinemia (THL), while the remaining homozygotes have less than normal plasma cholesterol. In contrast, mice expressing APOE*2 in place of the mouse Apoe (Apoe2/2 mice) are markedly hyperlipoproteinemic, suggesting a species difference in lipid metabolism (e.g., editing of apolipoprotein B) enhances THL development. Since apoB-100 has an LDLR binding site absent in apoB-48, we hypothesized that the Apoe2/2 THL phenotype would improve if all Apoe2/2 VLDL contained apoB-100. To test this, we crossed Apoe2/2 mice with mice lacking the editing enzyme for apoB (Apobec−/−). Consistent with an increase in remnant clearance, Apoe2/2 · Apobec−/− mice have a significant reduction in IDL/LDL cholesterol (IDL/LDL-C) compared with Apoe2/2 mice. However, Apoe2/2 ·Apobec−/− mice have twice as much VLDL triglyceride as Apoe2/2 mice. In vitro tests show the apoB-100-containing VLDL are poorer substrates for lipoprotein lipase than apoB-48-containing VLDL. Thus, despite a lowering in IDL/LDL-C, substituting apoB-48 lipoproteins with apoB-100 lipoproteins did not improve the THL phenotype in the Apoe2/2 ·Apobec−/− mice, because apoB-48 and apoB-100 differentially influence the catabolism of lipoproteins. Apolipoprotein E (apoE) is essential for the clearance of chylomicron and VLDL remnants from the plasma. Variant apoE proteins in humans are known to cause primary dysbetalipoproteinemia [or type-III hyperlipoproteinemia (THL)], which is characterized by the accumulation of chylomicron and VLDL remnants in the plasma and a high incidence of coronary artery and peripheral vessel atherosclerosis (1Utermann G. Hees M. Steinmetz A. Polymorphism of apolipoprotein E and occurrence of dysbetalipoproteinaemia in man.Nature. 1977; 269: 604-607Google Scholar). The most common form of THL is associated with homozygosity for the APOE*2 allele, whose product, apoE–2, has <2% normal binding to the low density lipoprotein receptor (LDLR) in vitro. However, the majority of APOE*2 homozygotes typically have normal to below normal plasma levels of cholesterol and triglycerides (TG) and only 5–10% of the homozygotes develop THL. Thus, reductions in lipoprotein clearance due to hormonal, dietary, and genetic changes resulting in reduced LDLR function or capacity are thought to trigger the THL (2Mahley R.W. Rall Jr., S.C. Type III hyperlipoproteinemia (Dysbetalipoproteinemia): The Role of Apolipoprotein E in Normal and Abnormal Metabolism.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. 7th edition. The Molecular and Metabolic Bases of Inherited Disease. Volume 2. McGraw-Hill, Inc., New York, New York1995: 1953-1980Google Scholar). In support of this hypothesis, some of these THL patients have other disorders such as hyperuricemia, glucose intolerance, obesity, and hypothyroidism (3Mahley R.W. Huang Y. Rall Jr., S.C. Pathogenesis of type III hyperlipoproteinemia (dysbetalipoproteinemia). Questions, quandaries, and paradoxes.J. Lipid Res. 1999; 40: 1933-1949Google Scholar, 4Davignon J. Cohn J.S. Mabile L. Bernier L. Apolipoprotein E and atherosclerosis: insight from animal and human studies.Clin. Chim. Acta. 1999; 286: 115-143Google Scholar). Nevertheless, the basic mechanism why only 5–10% of APOE*2 homozygotes develop THL while the remaining homozygotes have below normal cholesterol levels remains unexplained. To approach this fundamental question, we previously generated mice (Apoe 2/2 mice) which solely express the APOE*2 allele under the control of the endogenous mouse Apoe promoter by using a gene-targeted replacement strategy (5Sullivan P.M. Mezdour H. Quarfordt S.H. Maeda N. Type III hyperlipoproteinemia and spontaneous atherosclerosis in mice resulting from gene replacement of mouse Apoe with human Apoe*2.J. Clin. Invest. 1998; 102: 130-135Google Scholar). Surprisingly, all the Apoe 2/2 mice, regardless of age and gender, exhibit many characteristics of THL, while mice similarly made to express only the APOE*3 allele (Apoe3/3 mice) have a normal plasma lipid profile (6Knouff C. Hinsdale M.E. Mezdour H. Altenburg M.K. Watanabe M. Quarfordt S.H. Sullivan P.M. Maeda N. Apo E structure determines VLDL clearance and atherosclerosis risk in mice.J. Clin. Invest. 1999; 103: 1579-1586Google Scholar). Dissecting the basis for the complete penetrance of THL phenotype in Apoe 2/2 mice would likely contribute to a better understanding of the mechanism of THL in humans. For example, decreased binding of the apoE-2 in the Apoe 2/2 mice to the LDLR as compared with Apoe3/3 mice is likely one component of the THL phenotype because a modest increase in expression of the LDLR can eliminate the hyperlipoproteinemia in the Apoe2/2 mice (7Knouff C. Malloy S. Wilder J. Altenburg M.K. Maeda N. Doubling expression of the LDL receptor by truncation of the 3′ UTR sequence ameliorates type III hyperlipoproteinemia in mice expressing the Human ApoE2 isoform.J. Biol. Chem. 2000; 276: 3856-3862Google Scholar). At present, it is unknown what influence, if any, the different structural protein components of the remnant lipoprotein particles have in THL. In humans, each chylomicron contains a single apoB-48, and each VLDL contains a single apoB-100. The transcripts for the apoB-48 are made by editing of nascent APOB transcripts, which introduces a translational stop codon at 48% of the full length coding sequence, as compared with the unedited transcripts which generate apoB-100 (8Powell L.M. Wallis S.C. Pease R.J. Edwards Y.H. Knott T.J. Scott J. A novel form of tissue-specific RNA processing produces apolipoprotein- B48 in intestine.Cell. 1987; 50: 831-840Google Scholar, 9Chen S.H. Habib G. Yang C.Y. Gu Z.W. Lee B.R. Weng S.A. Silberman S.R. Cai S.J. Deslypere J.P. Rosseneu M. Gotto Jr. A.M. Li W.H. Chan L. Apolipoprotein B-48 is the product of a messenger RNA with an organ-specific in-frame stop codon.Science. 1987; 238: 363-366Google Scholar). Unlike apoB-100, apoB-48 lacks LDLR binding domains, and consequently apoB-48-containing lipoproteins are totally dependent on apoE for clearance. Mice differ from humans, and produce apoB-48-containing particles from both the liver and intestine, and approximately 20–30% of apoB-containing particles in fasting mouse plasma contain apoB-48 (10Oka K. Kobayashi K. Sullivan M. Martinez J. Teng B.B. Ishimura-Oka K. Chan L. Tissue-specific inhibition of apolipoprotein B mRNA editing in the liver by adenovirus-mediated transfer of a dominant negative mutant APOBEC-1 leads to increased low density lipoprotein in mice.J. Biol. Chem. 1997; 272: 1456-1460Google Scholar, 11Higuchi K. Kitagawa K. Kogishi K. Takeda T. Developmental and age-related changes in apolipoprotein B mRNA editing in mice.J. Lipid Res. 1992; 33: 1753-1764Google Scholar). The levels of apoB-48 containing particles produced by the liver, in the fed state, are speculated to be higher since feeding increases Apob mRNA editing (11Higuchi K. Kitagawa K. Kogishi K. Takeda T. Developmental and age-related changes in apolipoprotein B mRNA editing in mice.J. Lipid Res. 1992; 33: 1753-1764Google Scholar). Since apoB-48 is dependent on apoE for clearance, the higher amount of apoB-48-containing lipoproteins in mice likely contributes to the THL phenotype in the Apoe 2/2 mice. In this paper, we hypothesized that the hyperlipidemic phenotype of the Apoe 2/2 mice would be improved if all chylomicrons and VLDL in the Apoe 2/2 mice had apoB-100 only and were cleared independently of apoE. Therefore, we made Apoe 2/2 mice that are deficient in apoB-100 mRNA editing, thus producing only apoB-100 containing VLDL and chylomicrons. Mice used in this study were generated from crosses of APOE*2 replacement mice with Apobec-1 knock out mice (Apobec−/−) kindly provided by Dr. Edward Rubin, Lawrence Berkley National Laboratory (12Morrison J.R. Paszty C. Stevens M.E. Hughes S.D. Forte T. Scott J. Rubin E.M. Apolipoprotein B RNA editing enzyme-deficient mice are viable despite alterations in lipoprotein metabolism.Proc. Natl. Acad. Sci. USA. 1996; 93: 7154-7159Google Scholar). Double homozygous Apoe2/2· Apobec−/− mice and controls (Apoe2/2) used in experiments were littermates generated from crosses of compound heterozygous mice (Apoe2/+·Apobec+/−) with Apoe2/2 mice followed by intercrossing Apoe2/2·Apobec+/− mice. They had mixed genetic background between C57BL/6 and 129. The mice were maintained on PROLAB ISOPRO RMH 3000 diet. All procedures were approved by the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee. Mice were genotyped using PCR. Reaction conditions were 94°C 1 min, followed by 45 cycles of 94°C 20 s, 60°C 30 s, 72°C 30 s, with a final 5 min at 72°C. PGKpolyA (5′GCAGCCTC TGTTCCACATACACT3′) and exon 4 (5′TTGATTCTCCTGG GCCACTG3′) primers produced an ∼350 bp fragment diagnostic for the APOE*2 locus while intron 2 (5′GCAAGAGGTGA TGGTACTCG3′) and intron 3 (5′GTCTCGGCTCTGAACTAC ATAG3′) primers amplified the wild-type locus giving an ∼600 bp product. These primers were used in a multiplex reaction. PGKpolyA (5′GCAGCCTC TGTTCCACATACACT3′) and intron 6 (5′TTCCCAGTAGCA ACAACCACAGA3′) primers amplified the Apobec−/− locus giving an ∼260 bp fragment, and the exon 6 (5′TGAGCCGACACCC CTATGTAACTCT3′) and intron 6 primers amplified the wild-type locus giving an ∼350 bp fragment. After a 5 h fast, the mice were anesthetized with avertin, and blood was collected from the retroorbital sinus into microcentrifuge tubes containing 0.007 TIU aprotinin (Sigma), 0.19 mg EDTA, and 50 μg gentamicin per 200 μl of blood. Plasma total cholesterol (TC) was determined using a diagnostic kit (Wako Chemicals Inc.) as per kit instructions. For TG measurements, a Sigma diagnostic kit was used as per kit instructions. HDL cholesterol (HDL-C) was determined as previously described (13Warnick G.R. Benderson J. Albers J.J. Dextran sulfate-Mg2+ precipitation procedure for quantitation of high- density-lipoprotein cholesterol.Clin. Chem. 1982; 28: 1379-1388Google Scholar). ApoE was measured using an ELISA with antibodies specific for human apoE as described (14Sullivan P.M. Mezdour H. Aratani Y. Knouff C. Najib J. Reddick R.L. Quarfordt S.H. Maeda N. Targeted replacement of the mouse apolipoprotein E gene with the common human APOE3 allele enhances diet-induced hypercholesterolemia and atherosclerosis.J. Biol. Chem. 1997; 272: 17972-17980Google Scholar). For gel filtration analysis with fast protein liquid chromatography (FPLC), 100 μl of pooled plasma from the indicated mice was fractionated using a Superose 6 HR1/30 column (Pharmacia Biotech Inc.). For each 0.5 ml fraction collected, TC, TG, and apoE concentrations were determined. Fasted plasma lipoproteins were fractionated by ultracentrifugation using a Beckmann TLA 120.2 rotor at 70,000 rpm for 8 h at 4°C in a Beckman Optima TLX for each fraction. Fractions collected were d = 1.006, 1.02, 1.04, 1.06, 1.08, 1.10, and 1.21 g/ml. Fractions were dialyzed in 50 mM Tris, 1 mM EDTA, and 10 mM NaCl at 4°C, and plasma equivalents were loaded on precast 4–15% acrylamide gels (BioRad) or precast 1% agarose gels (Helena Laboratories). Molecular weight standards (BioRad) were used to determine size of detected proteins. Stained gels were scanned and analyzed with NIH Image software version 1.62. Western blots were probed with a rabbit polyclonal (1:10,000) anti-mouse apoB antibody (a kind gift from Dr. Harshini DeSilva) followed by a conjugated goat anti-rabbit antibody (1:40,000) (Calbiochem). Chemiluminescence (Amersham Pharmacia Biotech) was used for detection of all secondary antibodies. Hybridization buffer was 1% nonfat dry milk in PBS. The ability of VLDL to act as a substrate for lipoprotein lipase (LpL) was measured according to van Dijik et al. (15van Dijk K.W. van Vlijmen B.J. van't Hof H.B. van der Zee A. Santamarina-Fojo S. van Berkel T.J. Havekes L.M. Hofker M.H. 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: 336-344Google Scholar) with some modifications. Plasma VLDL from individual animals was isolated by ultracentrifugation at d < 1.006 g/ml and dialyzed. Samples were diluted with reaction buffer to 100 μl aliquots containing 2, 4, 8, and 16 μg of VLDL-TG. These were incubated at 37°C for 10 min. Reaction buffer was 0.1 M Tris pH 8.5 plus 1.0% fatty acid free BSA. One hundred and fifty milliunits of bovine milk LpL (Sigma-Aldrich, diluted to 15 U/ml with 0.1 Tris, 20% glycerol v/v, pH 8.5) was added and the samples were incubated for another 10 min. The reactions were terminated by adding 50 μl of stop solution (50 mM KH2PO4, 0.1% v/v Triton X-100, ph 6.9) and placed on ice. Free fatty acids (FFA) were measured using the non-esterified free fatty acid kit (NEFA C, Wako Chemicals). Duplicate samples, without LpL, were incubated to determine background FFA release. The FFA concentration for each sample was plotted followed by curve fitting to a rectangular hyperbola using Dataraid software (written by Dr. Jolyon Jesty at the State University of New York at Stony Brook). For 8.0 μg of TG substrate, released FFA per min per unit of enzyme was calculated and then analyzed byStudent's t-test (JMP Statistical software version 4.0.3, SAS Institute Inc.). The VLDL used in the lipase assay was analyzed for phospholipid (PL), free cholesterol (FC), TC, and TG using commercial kits from Wako Chemicals (PL, FC, and TC) and Sigma-Aldrich (TG) as per kit instructions. Total protein was measured using the Bradford assay (Pierce). Cholesterol ester (CE) mass was estimated by multiplying the difference between FC and TC by a factor of 1.67 (16Maugeais C. Tietge U.J. Tsukamoto K. Glick J.M. Rader D.J. Hepatic apolipoprotein E expression promotes very low density lipoprotein-apolipoprotein B production in vivo in mice.J. Lipid Res. 2000; 41: 1673-1679Google Scholar). Percentages of TG, FC, PL, CE, and protein in the total mass were then calculated. Two-tailed Student's t-test and linear correlation were used to analyze statistical significance. The liver secretion of TG was assayed as described (17Li X. Catalina F. Grundy S.M. Patel S. Method to measure apolipoprotein B-48 and B-100 secretion rates in an individual mouse: evidence for a very rapid turnover of VLDL and preferential removal of B-48- relative to B-100-containing lipoproteins.J. Lipid Res. 1996; 37: 210-220Google Scholar). Briefly, a 10% solution of Triton WR-1339 (Tyloxapol, Sigma-Aldrich) in 0.9% saline was injected into the tail vein at a dose of 0.7 mg/g body weight (total volume injected was ∼200 μl). Approximately 75 μl of blood was collected at time 0 (i.e., before Tyloxapol injection), 30 min. (i.e., after tyloxapol injection), 60 min, 120 min, and 180 min using non-heparinized capillary tubes and was immediately added to microcentrifuge tubes containing aprotinin (Sigma-Aldrich), EDTA, and gentamicin. Plasma TG concentrations (mg/dl) were then measured and normalized for liver weight. The μmols TG secreted were then estimated for average total body plasma volume (4.5% of body weight) as previously reported in the mouse (18Wish L. Furth J. Storey R.H. Direct determination of plasma, cell, and organ-blood volumes in normal and hypervolemic mice.Proc. Soc. Exp. Biol. Med. 1950; 74: 644-648Google Scholar). These TG values (μmols/g liver weight) were then plotted against time for each mouse, and a linear curve fit was performed. The slopes from the curve fit equations were used to calculate the μmols of TG secreted per gram of liver per hour and analyzed by t-test. Plasma lipid and lipoprotein profiles in the Apoe2/2· Apobec−/− mice were significantly different from those in the Apoe 2/2 mice. Both females and males had significantly increased plasma TG (∼60% and 100%, respectively) (Table 1). In addition, the females had an ∼18% decrease in TC (P = 0.03, student's t-test) while the males had an ∼60% increase in HDL-C concentrations (P = 0.01, student's t-test)(Table 1). FPLC fractionation of the plasma followed by lipid analysis of each fraction showed the increase in TG of the Apoe 2/2·Apobec−/− mice was localized to the VLDL fractions and the decrease in TC in the females was localized to the LDL fractions (Fig. 1, left panel). A slight decrease in LDL-C was also seen in males (Fig. 1, right panel).TABLE 1Plasma lipids and apoE concentrationGenderGenotypeTCTGHDL-CApoEmg/dlFemalesApoe 2/2 (n = 14)347 ± 71165 ± 6549 ± 1033 ± 8Apoe 2/2·Apobec−/− (n = 11)285 ± 39aP = 0.03, compared to Apoe2/2 females.263 ± 48bP < 0.001, compared to Apoe2/2 females.53 ± 1436 ± 8MalesApoe 2/2 (n = 6)290 ± 40159 ± 4448 ± 2228 ± 9Apoe 2/2·Apobec−/− (n = 8)281 ± 47325 ± 56cP < 0.001, compared to Apoe2/2 males.77 ± 13dP = 0.01, compared to Apoe2/2 males.34 ± 7Data are means ± SD in mg/dl in plasma from mice fed normal chow diet with 5.0% fat. TC, total cholesterol; TG, triglyceride; HDL-C, HDL cholesterol.a P = 0.03, compared to Apoe2/2 females.b P < 0.001, compared to Apoe2/2 females.c P < 0.001, compared to Apoe2/2 males.d P = 0.01, compared to Apoe2/2 males. Open table in a new tab Data are means ± SD in mg/dl in plasma from mice fed normal chow diet with 5.0% fat. TC, total cholesterol; TG, triglyceride; HDL-C, HDL cholesterol. Plasma apoE-2 concentrations were similar between the Apoe 2/2·Apobec−/− and Apoe 2/2 mice (Table 1). However, apoE-2 distribution in FPLC fractions was very different in the Apoe 2/2·Apobec−/− mice having a reduction of ∼50–60%, particularly in the IDL/LDL fractions (Fig. 1). This apoE-2 distribution parallels the cholesterol reduction as seen in the same FPLC fractions. These findings suggest the Apoe 2/2·Apobec−/− mice have markedly less apoE bound to lipoproteins (∼50% and ∼30% less in females and males, respectively). In the males, but not in the females, apoE-2 was reduced slightly in the HDL fractions. In both male and female Apoe 2/2·Apobec−/− mice, the amount of apoE-2 associated with the VLDL fraction was similar to that in the Apoe 2/2 mice despite the larger difference in VLDL-TG. To further investigate the apolipoprotein content of plasma lipoproteins in the females, plasma from 10 mice of each genotype was pooled and fractionated by ultracentrifugation. Plasma equivalents of these fractions were separated by SDS polyacrylamide gel electrophoresis (Fig. 2A, B). The Apoe 2/2·Apobec−/− plasma had less apoE-2 in the 1.02–1.04 fractions (Fig. 2A, right), as compared with the Apoe2/2 plasma where apoE-2 was more evenly present in fractions 1.006–1.04 (Fig. 2A, left). ApoA-I was primarily associated with fractions 1.08–1.21 g/ml in the Apoe 2/2· Apobec−/− mice as compared with the Apoe 2/2 mice that had a wider distribution of apoA-I, including the 1.04 and 1.06 fractions. Gels with reduced sample volume show (Fig. 2B) about a 1:2 ratio of apoB-100:B48 in the Apoe 2/2 VLDL while no apoB-48 was detectable in the Apoe 2/2·Apobec−/− VLDL. We estimated, by scanning densitometry of the coomassie stained SDS-PAGE gel, the total amount of apoB (apoB-48 plus apoB-100) in the Apoe 2/2·Apobec−/− plasma VLDL was twice that in the Apoe 2/2 plasma VLDL. This increase in VLDL apoB was also seen in males (data not shown). In addition, the Apoe 2/2·Apobec−/− plasma had ∼18% more apoE in the 1.006 fraction but ∼26% less in the 1.02 fraction than the same fractions from Apoe2/2 plasma. Therefore, in Apoe 2/2·Apobec−/− plasma, VLDL are doubled, and the distribution of apoE and apoA-I is restricted to the VLDL fractions and HDL fractions, respectively. When these ultracentrifugation fractions were separated on agarose gels and stained with fat red 7B, we found increased staining in the 1.006 fraction and less in the 1.02 and 1.04 fractions in the Apoe 2/2·Apobec−/− mice as compared with the Apoe 2/2 mice (Fig. 2C, top). These findings confirm the FPLC distribution of high VLDL-TG levels in the Apoe 2/2·Apobec−/− plasma. Immunoblots of the ultracentrifugation fractions using a polyclonal apoB antibody showed an ∼60% decrease in apoB containing lipoproteins in the 1.06 and their absence in 1.08 fractions (Fig. 2C, bottom). Again, these findings were consistent with the FPLC analyses that showed low IDL/LDL-C levels in the Apoe 2/2·Apobec−/− mice. Phospholipid levels measurements of the ultracentrifugation fractions showed an ∼25% increased in VLDL-PL and an ∼50–70% decreased in LDL-PL (fractions 1.04–1.06) in the Apoe 2/2·Apobec−/− compared with those in the Apoe 2/2 (Fig. 2D). In summary, the Apoe 2/2·Apobec−/− mice have reduced IDL/LDL particles and increased VLDL particles as compared with the Apoe 2/2 mice. An increase in TG particle secretion could increase steady state levels of plasma TG in the Apoe 2/2·Apobec−/− mice. To test this possibility, we injected the Apoe2/2· Apobec−/− and Apoe 2/2 mice with Triton WR-1339 and measured the accumulation of plasma TG over time. As shown in Fig. 3, there was no significant difference in TG secretion rates between the Apoe2/2·Apobec−/−and Apoe 2/2 mice. Therefore, the difference in plasma TG is not due to differences in secretion rates of VLDL. The proportional masses of CE, FC, PL, TG, and protein in the Apoe 2/2·Apobec−/− VLDL (n = 5) and the Apoe 2/2 VLDL (n = 5) (Fig. 4)showed that the Apoe2/2· Apobec−/− VLDL, as compared with Apoe2/2, have significantly high TG (47.6 ± 1.6% vs. 37.4 ± 2.8%, P = 0.01) and low esterified cholesterol (18.0 ± 1.4% vs. 25.3 ± 2.4%, P = 0.03). The percent of core lipids (TG plus CE mass) were, however, about equal. On an individual animal basis, CE content and TG content were inversely correlated each other (r = −0.99). Protein content was not different (10.6 ± 0.3% vs. 10.3 ± 0.1%, P = 0.4). Because VLDL-CE in the Apoe 2/2·Apobec−/− mice is 30% lower than that in the Apoe 2/2 mice (Fig. 4), the Apoe2/2·Apobec−/− VLDL could be up to 30% larger in size, or 30% more in number, or a combination of both. However, since VLDL apoB levels are higher in the Apoe 2/2·Apobec−/− mice, as judged from PAGE gels (Fig. 2), the actual number of VLDL particles in these mice is likely increased. Thus, these data suggest that the Apoe2/2·Apobec−/− mice have twice as much TG-rich VLDL as compared with the Apoe 2/2 mice whose plasma VLDL are more enriched with CE. We examined whether the increased steady state levels of VLDL-TG in the Apoe 2/2·Apobec−/− mice was caused by a reduced lipolysis of VLDL-TG by LpL as compared with Apoe 2/2 mice. Free fatty acid released from VLDL-TG by LpL was 2.8 ± 0.5 nmol FFA/min/U in the Apoe 2/2· Apobec−/− mice (n = 5) as compared with 4.5 ± 0.8 nmol FFA/ml/U in the Apoe2/2 mice (n = 4, Fig. 5). This 62% difference was significant (P < 0.01). There is a significant correlation between the VLDL-FFA release (Fig. 5) and the PL percentage of VLDL particle mass (Fig. 4) (r = 0.69, P = 0.04). This implies that the VLDL with a lower percentage of PL release less FFA under our assay conditions. We also find that the mass percentage of PL per VLDL particle is lowest in the Apoe 2/2·Apobec−/− mice (P < 0.0001). This difference in individual VLDL-PL is probably a result of the increased VLDL-TG and not a cause of the decreased LpL lipolysis. Taken together with the fact that the TG secretion rates in the two groups of mice are the same, a significant part of the increase in steady state levels of plasma VLDL-TG in the Apoe 2/2· Apobec−/− mice results from a reduction in lipolysis of VLDL-TG. Apoe 2/2 mice expressing human apoE-2 isoform in place of mouse apoE exhibit exaggerated THL compared with the majority of humans homozygous for APOE*2 who have relatively low plasma cholesterol levels. The predominant apoB containing lipoprotein accumulating in the Apoe 2/2 mice contains apoB-48 (5Sullivan P.M. Mezdour H. Quarfordt S.H. Maeda N. Type III hyperlipoproteinemia and spontaneous atherosclerosis in mice resulting from gene replacement of mouse Apoe with human Apoe*2.J. Clin. Invest. 1998; 102: 130-135Google Scholar). In this paper, we examined whether or not this exaggerated THL phenotype is because mice produce apoB-48 containing VLDL particles from the liver. This is in contrast to human liver VLDL that are entirely apoB-100 containing lipoproteins. We hypothesized that if the Apoe 2/2 mice had only apoB-100 containing lipoprotein particles, their hyperlipoproteinemic phenotype would be improved due to increased clearance of the human-like VLDL remnants. Various other studies support this hypothesis. For example, apoB-48-containing particles are the primary circulating apoB-containing lipoprotein in apoE deficient mice (19Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E.Science. 1992; 258: 468-471Google Scholar). Changing the apoB in Apoe−/− mice to totally apoB-100 by crossing them to Apobec−/− mice or with apoB-100 only mice have shown to decrease their total plasma cholesterol by about 55% (20Nakamuta M. Taniguchi S. Ishida B.Y. Kobayashi K. Chan L. Phenotype interaction of apobec-1 and CETP, LDLR, and apoE gene expression in mice: role of apoB mRNA editing in lipoprotein phenotype expression.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 747-755Google Scholar) or 20% (21Farese Jr., R.V. Veniant M.M. Cham C.M. Flynn L.M. Pierotti V. Loring J.F. Traber M. Ruland S. Stokowski R.S. Huszar D. Young S.G. Phenotypic analysis of mice expressing exclusively apolipoprotein B48 or apolipoprotein B100.Proc. Natl. Acad. Sci. USA. 1996; 93: 6393-6398Google Scholar), respectively. These decreases were in both VLDL-C and LDL-C, and resulted from increased clearance of lipoprotein particles most likely via the apoB-100 binding to LDLR (20Nakamuta M. Taniguchi S. Ishida B.Y. Kobayashi K. Chan L. Phenotype interaction of apobec-1 and CETP, LDLR, and apoE gene expression in mice: role of apoB mRNA editing in lipoprotein phenotype expression.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 747-755Google Scholar, 21Farese Jr., R.V. Veniant M.M. Cham C.M. Flynn L.M. Pierotti V. Loring J.F. Traber M. Ruland S. Stokowski R.S. Huszar D. Young S.G. Phenotypic analysis of mice expressing exclusively apolipoprotein B48 or apolipoprotein B100.Proc. Natl. Acad. Sci. USA. 1996; 93: 6393-6398Google Scholar). Thus, it can be argued that the plasma lipid profile in the Apoe 2/2·Apobec−/− mice should improve. However, the overall THL phenotype did not improve in the Apoe 2/2·Apobec−/− mice when they produced only apoB-100-containing VLDL and chylomicrons. The exclusive presence of apoB-100 on lipoprotein particles in the Apoe2/2·Apobec−/− mice did alter the metabolism of the lipoprotein particles. Similar to the majority of human APOE*2 homozygotes without THL, female Apoe2/2· Apobec−/− mice had a small but significant reduction in IDL/LDL. The reduction in males was however not significant. It should be noted there is a sex predilection for low LDL in female APOE*2 homozygotes that also have familial hypercholesterolemia (22Ferrieres J. Sing C.F. Roy M. Davignon J. Lussier-Cacan S. Apolipoprotein E polymorphism and heterozygous familial hypercholesterolemia. Sex-specific effects.Arterioscler. Thromb. 1994; 14: 1553-1560Google Scholar). In the Apoe 2/2·Apobec−/− mice, there are two possible explanations for the reduction of cholesterol in the IDL/LDL range particles. It could directly result from increased clearance of apoB-100 lipoproteins through binding of apoB-100 to the LDLR. Furthermore, LDL clearance may be enhanced by the poor competition of apoE-2-containing remnants with apoB-100 containing LDL for the LDLR (23Woollett L.A. Osono Y. Herz J. Dietschy J.M. Apolipoprotein E competitively inhibits receptor-dependent low density lipoprotein uptake by the liver but has no effect on cholesterol absorption or synthesis in the mouse.Proc. Natl. Acad. Sci. USA. 1995; 92: 12500-12504Google Scholar) or by the up regulation of the LDLR in THL (24Davignon J. Gregg R.E. Sing C.F. Apolipoprotein E polymorphism and atherosclerosis.Arteriosclerosis. 1988; 8: 1-21Google Scholar). Alternatively, the reduced IDL/LDL-C could be from decreased conversion of VLDL particles to IDL/LDL particles. ApoB-100 associated with VLDL is not in a conformation that binds well to the LDLR. For apoB-100 to become an effective ligand, conversion of VLDL to IDL/LDL mainly" @default.
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