Green tea catechins decrease apolipoprotein B-100 secretion from HepG2 cells

To understand the hypocholesterolemic activity of green tea, our in vitro studies screened the relative efficacy of two structurally distinct green tea catechins, epicatechin (EC) and epigallocatechin gallate (EGCG), on apolipoprotein B-100 (apoB) and lipid production using a well established human hepatoma cell-line, HepG2, as the model system. This study showed that HepG2 cells pretreated with EC and EGCG for 8 h exerted a dose-dependent inhibitory effect on apoB secretion. Total protein and albumin synthesis and secretion were unaffected indicating the effects on apoB secretion to be specific. Under lipid-rich conditions, apoB secretion was markedly reduced by EGCG and to a lesser extent by EC at 50 M. Mechanistic study showed that tea catechins inhibited apoB secretion via a proteasome-independent pathway as indicated by a lack of response to N-acetyl-leucyl-leucyl-norleucinal (ALLN), a proteasome inhibitor. The effect on apoB secretion was also found to be independent of lipid biosynthesis. In summary, the data suggest that EGCG in contrast to EC is a potent inhibitor of apoB secretion. The results indicate that the gallate moiety in the catechin molecule may result in a beneficial effect on lipid metabolism in terms of apoB secretion.     

Lipid aldehyde-mediated cross-linking of apolipoprotein B-100 inhibits secretion from HepG2 cells

Hepatic oxidative stress and lipid peroxidation are common features of several prevalent disease states, including alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD), a common component of the metabolic syndrome. These conditions are characterized in part by excessive accumulation of lipids within hepatocytes, which can lead to autocatalytic degradation of cellular lipids giving rise to electrophilic end products of lipid peroxidation. The pathobiology of reactive lipid aldehydes remains poorly understood. We therefore sought to investigate the effects of 4-hydroxynonenal (4-HNE) and 4-oxononenal (4-ONE) on the transport and secretion of very low-density lipoprotein using HepG2 cells as a model hepatocyte system. Physiologically relevant concentrations of 4-HNE and 4-ONE rapidly disrupted cellular microtubules in a concentration-dependent manner. Interestingly, 4-ONE reduced apolipoprotein B-100 (ApoB) secretion while 4-HNE did not significantly impair secretion. Both 4-HNE and 4-ONE formed adducts with ApoB protein, but 4-HNE adducts were detectable as mono-adducts, while 4-ONE adducts were present as protein–protein cross-links. These results demonstrate that reactive aldehydes generated by lipid peroxidation can differ in their biological effects, and that these differences can be mechanistically explained by the structures of the protein adducts formed.     

Distribution of lipid-binding regions in human apolipoprotein B-100

The distribution of lipid-binding regions of human apolipoprotein B-100 has been investigated by recombining proteolytic fragments of B-100 with lipids and characterizing the lipid-bound fragments by peptide mapping, amino acid sequencing, and immunoblotting. Fragments of B-100 were generated by digestion of low-density lipoproteins (LDL) in the presence of sodium decyl sulfate with either Staphylococcus aureus V8 protease, pancreatic elastase, or chymotrypsin. Particles with electron microscopic appearance of native lipoproteins formed spontaneously when detergent was removed by dialysis from enzyme digests containing fragments of B-100 and endogenous lipids, or from incubation mixtures of delipidated B-100 fragments mixed with microemulsions of exogenous lipids (cholesteryl oleate and egg phosphatidylcholine). Fractionation of the recombinant particles by isopycnic or density gradient ultracentrifugation yielded complexes similar to native LDL with respect to shape, diameter, electrophoretic mobility, and surface and core compositions. Circular dichroic spectra of these particles showed helicity similar to LDL but a somewhat decreased content of beta-structure. Most of the fragments of B-100 were capable of binding to lipids; 12 were identified by direct sequence analysis and 14 by reaction with antisera against specific sequences within B-100. Our results indicate that lipid-binding regions of B-100 are widely distributed within the protein molecule and that proteolytic fragments derived from B-100 can reassociate in vitro with lipids to form LDL-like particles.     

Pulse-chase studies of the synthesis and intracellular transport of apolipoprotein B-100 in Hep G2 cells

The synthesis and secretion of apolipoprotein B-100 (apoB-100) have been studied in a human hepatoma cell line, the Hep G2 cells. The time needed for the synthesis of apoB-100 was estimated to be 14 min, which corresponds to a translation rate of approximately 6 amino acids/s. ApoB-100 was compared with albumin and alpha 2-macroglobulin as to the distribution between the membrane and the luminal content in the endoplasmic reticulum (ER) and the Golgi apparatus. The results suggested that apoB-100 approximately followed the distribution of these secretory proteins in the Golgi, while the ratios between the percent membrane-bound apoB-100 and percent membrane-bound albumin or alpha 2-macroglobulin were 3-4:1 in the ER. This may suggest that apoB-100 occurs in a membrane-associated form in ER prior to the integration in the lipoproteins. Pulse-chase studies combined with subcellular fractionation was used to investigate the kinetics for the intracellular transfer of apoB-100. A 3-min pulse of [35S]methionine was followed by an increase in apoB-100 radioactivity in the ER during the first 10-15 min of chase. The following 10-15 min of chase were characterized by linear decrease in apoB-100 radioactivity with a decay rate of approximately 6%/min. The residence kinetics for apoB-100 in the ER differed from that of transferrin and probably also from that of albumin. By comparing the time for the pulse maximum in ER with that in the denser Golgi fractions the time needed for the transfer between ER and Golgi could be estimated to be 10 min. The time needed for the secretion of newly synthesized apoB-100 was estimated to be 30 min. This indicates that the transfer of the protein through the Golgi apparatus to the extracellular space requires 20 min.     

Studies on the assembly of apo B-100-containing lipoproteins in HepG2 cells

The relationship between apoB-100 and the membrane of the endoplasmic reticulum (ER) has been studied by a combination of pulse-chase methodology and subcellular fractionation. HepG2 cells were pulse-labeled with [35S]methionine for 3 min and chased with cold methionine for periods between 0 and 20 min. ApoB-100 and albumin, present in the membrane as well as in the luminal content of the ER vesicles, were isolated after each chase period. The results indicated that apoB-100 was cotranslationally bound to the membrane of the ER, and from this membrane-bound form, was transferred to the lumen after a delay of 10-15 min. Albumin was, as could be expected for a typical secretory protein, cotranslationally sequestered in the lumen of the ER. Apo-B-100-containing lipoproteins present in the microsomal lumen were analyzed by ultracentrifugation in a sucrose gradient. ApoB-100 occurred on rounded particles in three density regions: (i) d 1.1065-1.170 g/ml (Fraction I), (ii) d 1.011-1.045 g/ml (Fraction II), and (iii) d less than 1.011 g/ml (Fraction III). Fraction I, isolated from cells cultured in the absence of oleic acid, contained a homogenous population of particles with a mean diameter of approximately 200 A. Fraction I isolated from cells cultured in the presence of oleic acid was slightly more heterogeneous and had a mean diameter of approximately 250 A. Fractions II and III had mean diameters of 300 and 500 A, respectively. Cholesterol esters and triacylglycerol were the quantitatively dominating lipid constituents of all three fractions. Pulse-chase experiments indicated that Fraction I contained the newly assembled lipoproteins. With increasing chase time, the apoB-100 radioactivity was redistributed from Fraction I to Fractions II and III, indicating that Fraction I is converted into Fractions II and III during the intracellular transfer. Particles corresponding to Fractions II and III were by far the most abundant lipoproteins found in the medium. The results presented support the possibility of a sequential assembly of apoB-100-containing lipoproteins.     

Identification of sequence homology between human plasma apolipoprotein B-100 and apolipoprotein B-48

The structural relationship between apolipoprotein B-100 (apo-B-100) and apolipoprotein B-48 (apo-B-48) has not been elucidated. A peptide fragment (MDB-18) of approximately 6 kDa was isolated from a tryptic digest of apo-B-100. The sequence of the first 22 amino acids of MDB-18 was determined by Edman degradation. A 15-residue peptide corresponding to this sequence was synthesized by the solid-phase method and was utilized to develop a sequence-specific polyclonal antibody. On immunoblot analysis, the antibody recognized both intact apo-B-100 and apo-B-48. In addition, preincubating the antibody with the synthetic peptide abolished the recognition of both apo-B-100 and apo-B-48. These data are interpreted as indicating that there is an amino acid sequence homology between apo-B-100 and apo-B-48. Since the MDB-18 peptide is located in the carboxyl region of the B-100 molecule, we propose apo-B-100 and apo-B-48 share a common carboxyl region sequence.     

Reduction of lipid hydroperoxides by apolipoprotein B-100.

We have previously isolated two proteins which can reduce phosphatidylcholine hydroperoxide (PC-OOH) from human blood plasma and identified one of the proteins as apolipoprotein A-I (Mashima, R. , et al. (1998) J. Lipid Res. 39, 1133-1140). In the present study we have identified the other protein as apolipoprotein B-100 (apo B-100) by amino acid sequence analysis of its tryptic peptides. The reactivity of lipid hydroperoxides with apo B-100 decreased in the order of PC-OOH > linoleic acid hydroperoxide > cholesteryl ester hydroperoxide under our experimental conditions. Pretreatment of apo B-100 with chloramine T, an oxidant of methionine, diminished the PC-OOH-reducing activity, indicating that some of 78 methionines are responsible for the reduction of PC-OOH. Despite the presence of 6 methionines in albumin, albumin was inactive to reduce PC-OOH. Free methionine was also inactive. These data suggest that the accessibility and binding of lipid hydroperoxides to the protein methionine residues are crucial for reduction of lipid hydroperoxides.     

Degradation of apolipoprotein B-100 in human chylomicrons

The purpose of this study was to investigate the molecular forms of apolipoprotein B (ApoB) in human chylomicrons under well-preserved conditions. To this end, plasma and serum were collected from the same normal subjects after ingestion of a fatty meal. The samples were divided into three or four aliquots before the addition of various preservative mixtures, including antibiotics, antioxidants and proteinase inhibitors. The chylomicrons were isolated immediately, and all steps were carried out at or below 4 degrees C. Changes in the molecular weight of ApoB in chylomicrons were followed by a time study using 3.3% polyacrylamide gel electrophoresis containing SDS. ApoB from chylomicrons analyzed within 5 h of blood collection showed a single band with mobility identical to that of ApoB (ApoB-100) in low-density lipoproteins. When analyzed after 1-2 days, satellite bands smaller than ApoB-100 were observed, and a very faint band with Mr 200,000 appeared, which comigrated with intestinal ApoB (ApoB-48). Upon storage, the molecular weight of ApoB was smaller in chylomicrons subjected to a higher number of reflotations than those in chylomicrons washed less frequently, suggesting that purified chylomicrons degrade faster. A longer storage time at 4 degrees C (i.e., 7 or 14 days) revealed a stepwise degradation of ApoB, yielding Mr 200,000 band as the prominent form. The degradation of ApoB-100 was slower when both proteinase inhibitors, leupeptin and epsilon-amino caproic acid, were employed, and the appearance of Mr 200,000 band was quicker when the chylomicrons were processed at higher temperature (15-25 degrees C) in the absence of a proteinase inhibitor. Immunoblotting shows that the segment removed from ApoB-100 was the carboxyl-terminal portion. These results suggest the possible presence of a proteinase(s), which copurified with chylomicrons, and which converts ApoB-100 from a large to a smaller molecular form. Although the stop codon has been discovered recently in intestinal ApoB mRNA, which explains the mechanism for direct synthesis of ApoB-48, apparently ApoB-100 is also synthesized in the intestine of all eight subjects studied here, and the ApoB-100 degrades to a form which is ApoB-48-like.     

Cytosolic components are required for proteasomal degradation of newly synthesized apolipoprotein B in permeabilized HepG2 cells.

Recent studies have proposed that post-translational degradation of apolipoprotein B100 (apoB) involves the cytosolic ubiquitin-proteasome pathway. In this study, immunocytochemistry indicated that endoplasmic reticulum (ER)-associated proteasome molecules were concentrated in perinuclear regions of digitonin-permeabilized HepG2 cells. Signals produced by antibodies that recognize both alpha- and beta-subunits of the proteasome co-localized in the ER with specific domains of apoB. The mechanism of apoB degradation in the ER by the ubiquitin-proteasome pathway was studied using pulse-chase labeling and digitonin-permeabilized cells. ApoB in permeabilized cells incubated at 37 degrees C in buffer alone was relatively stable. When permeabilized cells were incubated with both exogenous ATP and rabbit reticulocyte lysate (RRL) as a source of ubiquitin-proteasome factors, >50% of [3H]apoB was degraded in 30 min. The degradation of apoB in the intact ER of permeabilized cells was much more rapid than that of extracted [3H]apoB incubated with RRL and ATP in vitro. The degradation of apoB was reduced by clasto-lactacystin beta-lactone, a potent proteasome inhibitor, and by ubiquitin K48R mutant protein, an inhibitor of polyubiquitination. ApoB in HepG2 cells was ubiquitinated, and polyubiquitination of apoB was stimulated by incubation of permeabilized cells with RRL. These results suggest that newly synthesized apoB in the ER is accessible to the cytoplasmic ubiquitin-proteasome pathway and that factors in RRL stimulate polyubiquitination of apoB, leading to rapid degradation of apoB in permeabilized cells.     

Knockdown of apolipoprotein B, an atherogenic apolipoprotein, in HepG2 cells by lentivirus-mediated siRNA

ApoB is an important determinant of atherosclerosis susceptibility and a potential pharmaceutical target for lowering atherogenic lipoproteins. In the present study, we used a lentiviral vector to express short hairpin RNAs for inhibition of apoB production in HepG2 cells. We first demonstrated that lentivirus could efficiently deliver transgene into HepG2 cells by using GFP lentivirus. We then made three lentiviral siApoB constructs, two of which were highly efficient for silencing apoB expression in HepG2 cells. We showed that siApoB lentivirus specifically knocked down apoB but had no effects on other proteins such as apoAI and albumin. Consequently, the secretion of apoB was reduced markedly. The silencing effect of siApoB lentivirus appeared to be permanent. Knocking down apoB did not alter the expression of cytoplasmic stress proteins (HSP70 and HSP90) and their ER homologues (GRP78 and GRP94). Furthermore, neither IKKalpha and JNK nor phosphorylated IKK and JNK were increased in long-term apoB-deficient hepatocytes as compared to the control cells. Consistent with these findings, apoB-deficient hepatocytes responded to insulin to a similar extent as the control cells as determined by measuring insulin-induced phosphorylation of IRS and ERK. Our studies indicate that lentiviral siRNAs provide an excellent approach for delivering siRNA into HepG2 cells and may be used for gene therapy for hyperlipidemia.     

Structure of apolipoprotein B-100 in low density lipoproteins

There is general consensus that amphipathic alpha-helices and beta sheets represent the major lipid-associating motifs of apolipoprotein (apo)B-100. In this review, we examine the existing experimental and computational evidence for the pentapartite domain structure of apoB. In the pentapartite nomenclature presented in this review (NH(2)-betaalpha(1)-beta(1)-alpha(2)-beta(2)-alpha(3)-COOH), the original alpha(1) globular domain (Segrest, J. P. et al. 1994. Arterioscler. Thromb. 14: 1674;-1685) is expanded to include residues 1;-1,000 and renamed the betaalpha(1) domain. This change reflects the likelihood that the betaalpha(1) domain, like lamprey lipovitellin, is a globular composite of alpha-helical and beta-sheet secondary structures that participates in lipid accumulation in the co-translationally assembled prenascent triglyceride-rich lipoprotein particles. Evidence is presented that the hydrophobic faces of the amphipathic beta sheets of the beta(1) and beta(2) domains of apoB-100 are in direct contact with the neutral lipid core of apoB-containing lipoproteins and play a role in core lipid organization. Evidence is also presented that these beta sheets largely determine LDL particle diameter. Analysis of published data shows that with a reduction in particle size, there is an increase in the number of amphipathic helices of the alpha(2) and alpha(3) domains associated with the surface lipids of the LDL particle; these increases modulate the surface pressure decreases caused by a reduction in radius of curvature. The properties of the LDL receptor-binding region within the overall domain structure of apoB-100 are also discussed. Finally, recent three-dimensional models of LDL obtained by cryoelectron microscopy and X-ray crystallography are discussed. These models show three common features: a semidiscoidal shape, a surface knob with the dimensions of the betaC globular domain of lipovitellin, and planar multilayers in the lipid core that are approximately 35 A apart; the multilayers are thought to represent cholesteryl ester in the smectic phase. These models present a conundrum: are LDL particles circulating at 37 degrees C spheroidal in shape, as generally assumed, or are they semidiscoidal in shape, as suggested by the models? The limited evidence available supports a spheroidal shape.     

Expression of apolipoprotein B-100 in isolated human small intestine epithelium.

Apolipoprotein B-48 (apoB48) is synthesized in the small intestine and becomes a component of chylomicrons (CM). Apolipoprotein B-100 (apoB100) is synthesized in liver and becomes a component of both very low density lipoprotein (VLDL) and low density lipoprotein (LDL). To evaluate whether apoB100 is present in the human small intestine, we performed immunohistochemical staining using anti-apoB100 monoclonal antibody (mAb). Jejunal samples stained positive and the granular staining was noted in the supranuclear region of epithelial cells. We also identified apoB100 expression in the epithelial cells by immunoblotting and dot-blotting of PCR-amplified cDNA. In order to exclude submucosal stroma contaminated with blood, we used isolated epithelium from human small intestine obtained by a crypt isolation technique. The results indicate that not only apoB48, but also apoB100 are expressed in human small intestine epithelium. The expression of apoB100 suggests that the dietary VLDL may be synthesized in human small intestine epithelium and converted into LDL, which might play an important role in atherosclerosis.     

Major apolipoprotein B-100 mutations in lipoprotein metabolism and atherosclerosis

Apolipoprotein (apo) B-100 is a key protein compound of plasma lipid metabolism. This protein, as a sole component of LDL particles, to a great extent controls the homeostasis of LDL cholesterol in the plasma. Therefore, this protein and its structural variants play an important role in development of hyperlipidemia and atherosclerosis. Intensive research into the structure and biological functions of apoB-100 has led to identification of its complete structure as well as the responsible binding sites. With the development of the methods of molecular biology, some structural variants of the apoB-100 protein that directly affect its binding properties have been described. These are mutations leading to amino acid substitution at positions 3500 (R3500Q and R3500W) and 3531 (R3531C) that have been shown to decrease the binding affinity of apoB-100 in vitro. However, only the former mutations have been unequivocally demonstrated to cause hyperlipidemia in vivo. This minireview is aimed to discuss the impact of apoB-100 and its structural variants on plasma lipid metabolism and development of hyperlipidemia.     

Effects of different doses of atorvastatin on human apolipoprotein B-100, B-48, and A-I metabolism

Nine hypercholesterolemic and hypertriglyceridemic subjects were enrolled in a randomized, placebo-controlled, double-blind, crossover study to test the effect of atorvastatin 20 mg/day and 80 mg/day on the kinetics of apolipoprotein B-100 (apoB-100) in triglyceride-rich lipoprotein (TRL), intermediate density lipoprotein (IDL), and LDL, of apoB-48 in TRL, and of apoA-I in HDL. Compared with placebo, atorvastatin 20 mg/day was associated with significant reductions in TRL, IDL, and LDL apoB-100 pool size as a result of significant increases in fractional catabolic rate (FCR) without changes in production rate (PR). Compared with the 20 mg/day dose, atorvastatin 80 mg/day caused a further significant reduction in the LDL apoB-100 pool size as a result of a further increase in FCR. ApoB-48 pool size was reduced significantly by both atorvastatin doses, and this reduction was associated with nonsignificant increases in FCR. The lathosterol-campesterol ratio was decreased by atorvastatin treatment, and changes in this ratio were inversely correlated with changes in TRL apoB-100 and apoB-48 PR. No significant effect on apoA-I kinetics was observed at either dose of atorvastatin. Our data indicate that atorvastatin reduces apoB-100- and apoB-48-containing lipoproteins by increasing their catabolism and has a dose-dependent effect on LDL apoB-100 kinetics. Atorvastatin-mediated changes in cholesterol homeostasis may contribute to apoB PR regulation.     

Correlation between intima-to-media ratio, apolipoprotein B-100, myeloperoxidase, and hypochlorite-oxidized proteins in human atherosclerosis.

The oxidative modification of low-density lipoprotein (LDL) is thought to contribute to atherogenesis, and there is evidence that oxidants derived from myeloperoxidase (MPO) contribute to such oxidative damage. Using human iliac arteries we investigated the relationship between lesion stage indicated by the intima-to-media (I/M) ratio and the presence of apolipoprotein B-100 (apoB, a marker for LDL), MPO, and hypochlorite (HOCl)-oxidized proteins identified by immunohistochemistry in the intima, media, and adventitia. More staining for apoB, MPO, and HOCl-oxidized proteins was observed in diseased than healthy vessels. Diseased segments also stained more for the three parameters than healthy segments in the same diseased vessel, highlighting the variability that can occur within a single cross-section of a vessel. However, significant positive correlation between I/M ratio and positive staining for apoB, MPO, and HOCl-oxidized proteins in different segments of individual arteries were apparent in segments with an I/M ratio of > 1.8. Also, the overall extent of intimal staining for apoB, MPO, and HOCl-oxidized proteins increased with increasing I/M ratio. In addition, the extent of apoB staining was greater and appeared at comparatively lower I/M ratios than that of MPO and HOCl-oxidized proteins. Our results support a contribution to atherogenesis of all three parameters assessed, although MPO and HOCl-oxidized proteins appear to participate in the disease process at a later stage than apoB.     

Apolipoprotein B-100 is the major form of this apolipoprotein secreted by human intestinal Caco-2 cells

Although the discovery of stop codon has explained the mechanism for the formation of the intestinal marker, apolipoprotein B-48, the dispute regarding the presence of apolipoprotein B-100 in the intestine is still unsettled. To further investigate the characteristics of intestinal apolipoprotein B, the newly developed human colonic adenocarcinoma Caco-2 cells which express functional properties of the differentiated enterocytes, were used. SDS-polyacrylamide gel electrophoresis analyses of the intact culture medium or its lipoproteins of d less than 1.23 g/ml showed the presence of only a single protein band of apolipoprotein B-100 with no detectable apolipoprotein B-48. After immunoblotting with oligoclonal antibodies to the amino-terminal peptide of apolipoprotein B, a trace amount of apolipoprotein B-48 was observed in the isolated lipoproteins, but not in the intact culture medium. These results suggest that apolipoprotein B-100 is the major form of apolipoprotein B secreted by human intestinal cells.     

Degradation of apolipoprotein B-100 by lysosomal cysteine cathepsins

Although the degradation of cellular or endocytosed proteins comprises the normal function of lysosomal proteinases, these enzymes were also detected extracellularly during diseases such as atherosclerosis. Since lysosomal cysteine cathepsins were demonstrated to transform native LDL particles into a proatherogenic type, the following study was undertaken to characterize the modification of LDL particles and the degradation of apolipoprotein B-100 in more detail. LDL was incubated with cathepsins B, F, K, L, S, and V at pH 5.5 and under physiological conditions (pH 7.4) for 2 h to mimic conditions of limited proteolysis. Gel electrophoretic analysis of the degradation products revealed that cathepsin-mediated proteolysis of apolipoprotein B-100 is a fast process carried out by all enzymes at pH 5.5, and by cathepsin S also at pH 7.4. Electron microscopic analysis showed that cathepsin-mediated degradation of apolipoprotein B-100 rendered LDL particles fusion-competent compared to controls. N-Terminal sequencing of cathepsin cleavage fragments from apolipoprotein B-100 revealed an abundance of enzyme-specific cleavage sites located in almost all structurally and functionally essential regions. Since the cleavage sites superimpose well with results from substrate specificity studies, they might be useful for the development of cathepsin-specific inhibitors and substrates.     

Effect of apolipoprotein E genotype on apolipoprotein B-100 metabolism in normolipidemic and hyperlipidemic subjects

The effect of apolipoprotein (apo) E genotype on apoB-100 metabolism was examined in three normolipidemic apoE2/E2, five type III hyperlipidemic apoE2/E2, and five hyperlipidemic apoE3/E2 subjects using simultaneous administration of ¹³¹I-VLDL and ¹²⁵I-LDL, and multi-compartmental modeling. Compared with normolipidemic apoE2/E2 subjects, type III hyperlipidemic E2/E2 subjects had increased plasma and VLDL cholesterol, plasma and VLDL triglycerides, and VLDL and intermediate density lipoprotein (IDL) apoB concentrations (P < 0.05). These abnormalities were chiefly a consequence of decreased VLDL and IDL apoB fractional catabolic rate (FCR). Compared with hyperlipidemic E3/E2 subjects, type III hyperlipidemic E2/E2 subjects had increased IDL apoB concentration and decreased conversion of IDL to LDL particles (P < 0.05). In a pooled analysis, VLDL cholesterol was positively associated with VLDL and IDL apoB concentrations and the proportion of VLDL apoB in the slowly turning over VLDL pool, and was negatively associated with VLDL apoB FCR after adjusting for subject group. VLDL triglyceride was positively associated with VLDL apoB concentration and VLDL and IDL apoB production rates after adjusting for subject group. A defective apoE contributes to altered lipoprotein metabolism but is not sufficient to cause overt hyperlipidemia. Additional genetic mutations and environmental factors, including insulin resistance and obesity, may contribute to the development of type III hyperlipidemia.