Relation of the size and intracellular sorting of apoB to the formation of VLDL 1 and VLDL 2

In this study, we tested the hypothesis that two separate pathways, the two-step process and an apolipoprotein B (apoB) size-dependent lipidation process, give rise to different lipoproteins. Expression of apoB-100 and C-terminally truncated forms of apoB-100 in McA-RH7777 cells demonstrated that VLDL particles can be assembled by apoB size-dependent linear lipidation, resulting in particles whose density is inversely related to the size of apoB. This lipidation results in a LDL-VLDL 2 particle containing apoB-100. VLDL 1 is assembled by the two-step process by apoB-48 and larger forms of apoB but not to any significant amount by apoB-41. The major amount of intracellular apoB-80 and apoB-100 banded with a mean density of 1.10 g/ml. Its formation was dependent on the sequence between apoB-72 and apoB-90. This dense particle, which is retained in the cell, possibly by chaperones or association with the microsomal membrane, is a precursor of secreted VLDL 1. The intracellular LDL-VLDL 2 particles formed during size-dependent lipidation appear to be the precursors of intracellular VLDL 1. We propose that the dense apoB-100 intracellular particle is converted to LDL-VLDL 2 by size-dependent lipidation. LDL-VLDL 2 is secreted or converted to VLDL 1 by the uptake of the major amount of triglycerides.     

Effect of fenofibrate and atorvastatin on VLDL apoE metabolism in men with the metabolic syndrome

We examined the effects of fenofibrate and atorvastatin on very low density lipoprotein (VLDL) apolipoprotein (apo)E metabolism in the metabolic syndrome (MetS). We studied 11 MetS men in a randomized, double-blind, crossover trial. VLDL-apoE kinetics were examined using stable isotope methods and compartmental modeling. Compared with placebo, fenofibrate (200 mg/day) and atorvastatin (40 mg/day) decreased plasma apoE concentrations (P < 0.05). Fenofibrate decreased VLDL-apoE concentration and production rate (PR) and increased VLDL-apoE fractional catabolic rate (FCR) compared with placebo (P < 0.05). Compared with placebo, atorvastatin decreased VLDL-apoE concentration and increased VLDL-apoE FCR (P < 0.05). Fenofibrate and atorvastatin had comparable effects on VLDL-apoE concentration. The increase in VLDL-apoE FCR with fenofibrate was 22% less than that with atorvastatin (P < 0.01). With fenofibrate, the change in VLDL-apoE concentration was positively correlated with change in VLDL-apoB concentration, and negatively correlated with change in VLDL-apoB FCR. In MetS, fenofibrate and atorvastatin decreased plasma apoE concentrations. Fenofibrate decreased VLDL-apoE concentration by lowering VLDL-apoE production and increasing VLDL-apoE catabolism. By contrast, atorvastatin decreased VLDL-apoE concentration chiefly by increasing VLDL-apoE catabolism. Our study provides new insights into the mechanisms of action of two different lipid-lowering therapies on VLDL-apoE metabolism in MetS.     

Secretion of triacylglycerol-poor VLDL particles from McA-RH7777 cells expressing human hepatic lipase

Hepatic lipase (HL) plays a role in the catabolism of apolipoprotein (apo)B-containing lipoproteins through its lipolytic and ligand-binding properties. We describe a potential intracellular role of HL in the assembly and secretion of VLDL. Transient or stable expression of HL in McA-RH7777 cells resulted in decreased (by 40%) incorporation of [(3)H]glycerol into cell-associated and secreted triacylglycerol (TAG) relative to control cells. However, incorporation of [(35)S]methionine/cysteine into cell and medium apoB-100 was not decreased by HL expression. The decreased (3)H-TAG synthesis/secretion in HL expressing cells was not attributable to decreased expression of genes involved in lipogenesis. Fractionation of medium revealed that the decreased [(3)H]TAG from HL expressing cells was mainly attributable to decreased VLDL. Expression of catalytically-inactive HL (HL(SG)) (Ser-145 at the catalytic site was substituted with Gly) in the cells also resulted in decreased secretion of VLDL-[(3)H]TAG. Examination of lumenal contents of microsomes showed a 40% decrease in [(3)H]TAG associated with lumenal lipid droplets in HL or HL(SG) expressing cells as compared with control. The microsomal membrane-associated [(3)H]TAG was decreased by 50% in HL expressing cells but not in HL(SG) expressing cells. Thus, expression of HL, irrespective of its lipolytic function, impairs formation of VLDL precursor [(3)H]TAG in the form of lumenal lipid droplets. These results suggest that HL expression in McA-RH7777 cells result in secretion of [(3)H]TAG-poor VLDL.     

The apoE isoform binding properties of the VLDL receptor reveal marked differences from LRP and the LDL receptor

Apolipoprotein E (apoE) associates with lipoproteins and mediates their interaction with members of the LDL receptor family. ApoE exists as three common isoforms that have important distinct functional and biological properties. Two apoE isoforms, apoE3 and apoE4, are recognized by the LDL receptor, whereas apoE2 binds poorly to this receptor and is associated with type III hyperlipidemia. In addition, the apoE4 isoform is associated with the common late-onset familial and sporadic forms of Alzheimer's disease. Although the interaction of apoE with the LDL receptor is well characterized, the specificity of other members of this receptor family for apoE is poorly understood. In the current investigation, we have characterized the binding of apoE to the VLDL receptor and the LDL receptor-related protein (LRP). Our results indicate that like the LDL receptor, LRP prefers lipid-bound forms of apoE, but in contrast to the LDL receptor, both LRP and the VLDL receptor recognize all apoE isoforms. Interestingly, the VLDL receptor does not require the association of apoE with lipid for optimal recognition and avidly binds lipid-free apoE. It is likely that this receptor-dependent specificity for various apoE isoforms and for lipid-free versus lipid-bound forms of apoE is physiologically significant and is connected to distinct functions for these receptors.     

Effects of apoA-V on HDL and VLDL metabolism in APOC3 transgenic mice

Apolipoprotein A-V (apoA-V) and apoC-III are exchangeable constituents of VLDL and HDL. ApoA-V counteracts the effect of apoC-III on triglyceride (TG) metabolism with poorly defined mechanisms. To better understand the effects of apoA-V on TG and cholesterol metabolism, we delivered apoA-V cDNA into livers of hypertriglyceridemic APOC3 transgenic mice by adenovirus-mediated gene transfer. In response to hepatic apoA-V production, plasma TG levels were reduced significantly as a result of enhanced VLDL catabolism without alternations in VLDL production. This effect was associated with reduced apoC-III content in VLDL. Increased apoA-V production also resulted in decreased apoC-III and increased apoA-I content in HDL. Furthermore, apoA-V-enriched HDL was associated with enhanced LCAT activity and increased cholesterol efflux. This effect, along with apoE enrichment in HDL, contributed to HDL core expansion and alpha-HDL formation, accounting for significant increases in both the number and size of HDL particles. As a result, apoA-V-treated APOC3 transgenic mice exhibited decreased VLDL-cholesterol and increased HDL-cholesterol levels. ApoA-V-mediated reduction of apoC-III content in VLDL represents an important mechanism by which apoA-V acts to ameliorate hypertriglyceridemia in adult APOC3 transgenic mice. In addition, increased apoA-V levels accounted for cholesterol redistribution from VLDL to larger HDL particles. These data suggest that in addition to its TG-lowering effect, apoA-V plays a significant role in modulating HDL maturation and cholesterol metabolism.     

Comparative effects of ethanol, n-propranol and isopropanol on lipid disposal by rat liver.

Besides ethanol, other aliphatic alcohols such as n-propanol and isopropanol induce a triacylglycerol (TAG) accumulation in the liver. To determine whether a common mechanism is responsible for the effects of these three alcohols on hepatic lipid metabolism, each was administered by gastric tube to female Wistar rats at the dose of 50 mmol/kg body wt. Whichever alcohol was administered, the hepatic triacylglycerol accumulation was found to be related to the duration of elevated blood alcohol concentration. After administration of n-propanol or isopropanol, the liver [14C]palmitate uptake was increased whereas hepatic palmitate oxidation to 14CO2 was impaired and palmitate esterification into TAG enhanced; these perturbations were however more discrete than after ethanol administration. In contrast to ethanol and n-propanol which, at the dose presently used, increase precursor incorporation into blood TAG, isopropanol inhibits this incorporation. Interference with the process of very low density lipoprotein (VLDL) synthesis and/or secretion, which appears only at a late stage of isopropanol intoxication, is probably responsible for the intensity and duration of the fatty liver observed after administration of this alcohol.     

Triglyceride-rich lipoprotein metabolism in unique VLDL receptor, LDL receptor, and LRP triple-deficient mice

The very low density lipoprotein receptor (VLDLR), low density lipoprotein receptor (LDLR), and low density lipoprotein receptor-related protein (LRP) are the three main apolipoprotein E-recognizing endocytic receptors involved in the clearance of triglyceride (TG)-rich lipoproteins from plasma. Whereas LDLR deficiency in mice results in the accumulation of plasma LDL-sized lipoproteins, VLDLR or LRP deficiency alone only minimally affects plasma lipoproteins. To investigate the combined effect of the absence of these receptors on TG-rich lipoprotein levels, we have generated unique VLDLR, LDLR, and LRP triple-deficient mice. Compared with wild-type mice, these mice markedly accumulated plasma lipids and lipases. These mice did not show aggravated hyperlipidemia compared with LDLR and LRP double-deficient mice, but plasma TG was increased after high-fat diet feeding. In addition, these mice showed a severely decreased postprandial TG clearance typical of VLDLR-deficient (VLDLR-/-) mice. Collectively, although VLDLR deficiency in LRP- and LDLR-/- mice does not aggravate hyperlipidemia, these triple-deficient mice represent a unique model of markedly delayed TG clearance on a hyperlipidemic background.     

Differential impact of hepatic deficiency and total body inhibition of MTP on cholesterol metabolism and RCT in mice

Because apoB-containing lipoproteins are pro-atherogenic and their secretion by liver and intestine largely depends on microsomal triglyceride transfer protein (MTP) activity, MTP inhibition strategies are actively pursued. How decreasing the secretion of apoB-containing lipoproteins affects intracellular rerouting of cholesterol is unclear. Therefore, the aim of the present study was to determine the effects of reducing either systemic or liver-specific MTP activity on cholesterol metabolism and reverse cholesterol transport (RCT) using a pharmacological MTP inhibitor or a genetic model, respectively. Plasma total cholesterol and triglyceride levels were decreased in both MTP inhibitor-treated and liver-specific MTP knockout (L-Mttp^−/−) mice (each P < 0.001). With both inhibition approaches, hepatic cholesterol as well as triglyceride content was consistently increased (each P < 0.001), while biliary cholesterol and bile acid secretion remained unchanged. A small but significant decrease in fecal bile acid excretion was observed in inhibitor-treated mice (P < 0.05), whereas fecal neutral sterol excretion was substantially increased by 75% (P < 0.001), conceivably due to decreased intestinal absorption. In contrast, in L-Mttp^−/− mice both fecal neutral sterol and bile acid excretion remained unchanged. However, while total RCT increased in inhibitor-treated mice (P < 0.01), it surprisingly decreased in L-Mttp^−/− mice (P < 0.05). These data demonstrate that: i) pharmacological MTP inhibition increases RCT, an effect that might provide additional clinical benefit of MTP inhibitors; and ii) decreasing hepatic MTP decreases RCT, pointing toward a potential contribution of hepatocyte-derived VLDLs to RCT.     

ApoA-IV modulates the secretory trafficking of apoB and the size of triglyceride-rich lipoproteins

Although the evidence linking apoA-IV expression and triglyceride (TG)-rich lipoprotein assembly and secretion is compelling, the intracellular mechanisms by which apoA-IV could modulate these processes remain poorly understood. We therefore examined the functional impact of apoA-IV expression on endogenous apoB, TG, and VLDL secretion in stably transfected McA-RH7777 rat hepatoma cells. Expression of apoA-IV modified with the endoplasmic reticulum (ER) retention signal KDEL (apoA-IV-KDEL) dramatically decreased both the rate and efficiency of endogenous apoB secretion, suggesting a presecretory interaction between apoA-IV-KDEL and apoB or apoB-containing lipoproteins. Expression of native apoA-IV using either a constitutive or tetracycline-inducible promoter delayed the initial rate of apoB secretion and reduced the final secretion efficiency by ～40%. However, whereas apoA-IV-KDEL reduced TG secretion by 75%, expression of native apoA-IV caused a 20-35% increase in TG secretion, accompanied by a ～55% increase in VLDL-associated apoB, an increase in the TG:phospholipid ratio of secreted d < 1.006 lipoproteins, and a 10.1 nm increase in peak VLDL(1) particle diameter. Native apoA-IV expression had a negligible impact on expression of the MTP gene. These data suggest that by interacting with apoB in the secretory pathway, apoA-IV alters the trafficking kinetics of apoB-containing TG-rich lipoproteins through cellular lipidation compartments, which in turn, enhances particle expansion and increases TG secretion.     

Microsomal triglyceride transfer protein expression in adipocytes: a new component in fat metabolism

Microsomal triglyceride transfer protein (MTP) is a carrier of triglyceride essential for the assembly of apolipoprotein (apo)B-containing lipoproteins by the liver and the small intestine. Its role in triglyceride transfer in tissues that do not secrete lipoproteins has not been explored. In particular, MTP would seem to be a candidate for a role in triglyceride metabolism within the adipocyte. To test this hypothesis, we probed adipocytes for the presence of MTP. Immunohistochemical and biochemical studies demonstrate MTP in adipocytes from brown and white fat depots of mice and human, as well as in 3T3-L1 cells. Confocal microscopy revealed MTP throughout 3T3 cells; however, MTP fluorescence was prominent in juxtanuclear areas. In differentiated 3T3 cells MTP fluorescence was very striking around lipid droplets. In vitro lipid transfer assays demonstrated the presence of triglyceride transfer activity within microsomal fractions isolated from rat adipose tissue. In addition, quantitative rtPCR studies showed that MTP expression in mouse white fat depots was approximately 1% of MTP expression in mouse liver. MTP mRNA in differentiated 3T3 cells was approximately 13% of liver expression. Our results provide unequivocal evidence for the presence of MTP in adipocytes and present new possibilities for defining the mechanisms by which triglyceride is stored and/or hydrolyzed and mobilized.     

Lipoprotein separation in a novel iodixanol density gradient, for composition, density, and phenotype analysis

Separation of lipoproteins by traditional sequential salt density floatation is a prolonged process ( approximately 72 h) with variable recovery, whereas iodixanol-based, self-generating density gradients provide a rapid ( approximately 4 h) alternative. A novel, three-layered iodixanol gradient was evaluated for its ability to separate lipoprotein fractions in 63 subjects with varying degrees of dyslipidemia. Lipoprotein cholesterol, triglycerides, and apolipoproteins were measured in 21 successive iodixanol density fractions. Iodixanol fractionation was compared with sequential floatation ultracentrifugation. Iodixanol gradient formation showed a coefficient of variation of 0.29% and total lipid recovery from the gradient of 95.4% for cholesterol and 84.7% for triglyceride. Recoveries for VLDL-, LDL-, and HDL-cholesterol, triglycerides, and apolipoproteins were approximately 10% higher with iodixanol compared with sequential floatation. The iodixanol gradient effectively discriminated classic lipoproteins and their subfractions, and there was evidence for improved resolution of lipoproteins with the iodixanol gradient. LDL particles subfractionated by the gradient showed good correlation between density and particle size with small, dense LDL (<25.5 nm) separated in fractions with density >1.028 g/dl. The new iodixanol density gradient enabled rapid separation with improved resolution and recovery of all lipoproteins and their subfractions, providing important information with regard to LDL phenotype from a single centrifugation step with minimal in-vitro modification of lipoproteins.     

Hepatic trans-Golgi action coordinated by the GTPase ARFRP1 is crucial for lipoprotein lipidation and assembly

The liver is a major organ in whole body lipid metabolism and malfunctioning can lead to various diseases including dyslipidemia, fatty liver disease, and type 2 diabetes. Triglycerides and cholesteryl esters are packed in the liver as very low density lipoproteins (VLDLs). Generation of these lipoproteins is initiated in the endoplasmic reticulum and further maturation likely occurs in the Golgi. ADP-ribosylation factor-related protein 1 (ARFRP1) is a small trans-Golgi-associated guanosine triphosphatase (GTPase) that regulates protein sorting and is required for chylomicron lipidation and assembly in the intestine. Here we show that the hepatocyte-specific deletion of Arfrp1 (Arfrp1^liv−/−) results in impaired VLDL lipidation leading to reduced plasma triglyceride levels in the fasted state as well as after inhibition of lipoprotein lipase activity by Triton WR-1339. In addition, the concentration of ApoC3 that comprises 40% of protein mass of secreted VLDLs is markedly reduced in the plasma of Arfrp1^liv−/− mice but accumulates in the liver accompanied by elevated triglycerides. Fractionation of Arfrp1^liv−/− liver homogenates reveals more ApoB48 and a lower concentration of triglycerides in the Golgi compartments than in the corresponding fractions from control livers. In conclusion, ARFRP1 and the Golgi apparatus play an important role in lipoprotein maturation in the liver by influencing lipidation and assembly of proteins to the lipid particles.     

Effects of part sequences of human growth hormone on in vivo hepatic glycogen metabolism in the rat

Acute effects of two part sequences of human growth hormone on the in vivo activity levels of hepatic glycogen synthase and glycogen phosphorylase were examined. The peptide corresponding to residues 6 to 13 of the hormone (hGH 6–13) decreased the percentage of phosphorylase in the active form without affecting synthase activity. This action was indirect and dependent upon insulin. The peptide hGH 177–191 decreased the level of the active form of synthase without affecting phosphorylase activity. This effect was also observed with analogous peptides containing the sequence hGH 178–191 (i.e., hGH 172–191 and hGH 178–191), whereas the peptide hGH 179–191 was inert.The onset of these effects was rapid, and maximum changes in activity were produced in 5 min by both peptides. The effect for hGH 177–191 was short-lived, and synthase activity had returned to normal levels by 15 min, whereas the action of hGH 6–13 was of longer duration and was still quite marked at 60 min. Both peptides showed a linear dependence of response to the log dose of peptide injected over the range 0.1–250 μg hGH 6–13 per kg body weight and 0.05–25 gmg hGH 177–191 per kg body weight. Hepatic 3′,5′-cyclicadenylic acid levels were not affected by either peptide. Incorporation of glycerol carbon liver glycogen was increased by hGH 6–13 and decreased by hGH 177–191. This discussed in terms of a futile cycle between glycogen and hexone phosphate in the liver, as the basis for a control mechanism for hepatic glycogen metabolism. The present observations are consistent with other in vivo and in vitro actions of these and related peptides.     

Apolipoprotein A-V association with intracellular lipid droplets

Apolipoprotein A-V (apoA-V) plays a key role in the regulation of triglyceride (TG) metabolism. Given the very low concentration of apoA-V in plasma, we hypothesized that apoA-V may influence plasma TG levels by affecting the assembly and/or secretion of apoB-containing lipoproteins. When apoA-V was overexpressed in cultured Hep3B cells, neither the amount of apoB secreted nor the density distribution of apoB-containing lipoproteins was affected. Fluorescence microscopy and cell lysate immunoprecipitation studies revealed that apoA-V is not associated with apoB intracellularly, yet immunoprecipitation of apoA-V from the cell culture medium resulted in coprecipitation of apoB. These data suggest that the apoA-V association with apoB-containing lipoproteins is a postsecretory event. Confocal fluorescence microscopy revealed the presence of apoA-V in distinct cellular structures. Based on Nile Red staining, we identified these structures to be intracellular lipid droplets. These data suggest that apoA-V has a unique association with cellular lipids and, therefore, may be involved in the storage or mobilization of intracellular lipids.     

Mechanisms of glucosamine-induced suppression of the hepatic assembly and secretion of apolipoprotein B-100-containing lipoproteins

Glucosamine-induced endoplasmic reticulum (ER) stress was recently shown to specifically reduce apolipoprotein B-100 (apoB-100) secretion by enhancing the proteasomal degradation of apoB-100. Here, we examined the mechanisms linking glucosamine-induced ER stress and apoB-lipoprotein biogenesis. Trypsin sensitivity studies suggested glucosamine-induced changes in apoB-100 conformation. Endoglycosidase H studies of newly synthesized apoB-100 revealed glucosamine induced N-linked glycosylation defects resulting in reduced apoB-100 secretion. We also examined glucosamine-induced changes in VLDL assembly and secretion. A dose-dependent (1-10 mM glucosamine) reduction was observed in VLDL-apoB-100 secretion in primary hepatocytes (24.2-67.3%) and rat McA-RH7777 cells (23.2-89.5%). Glucosamine also inhibited the assembly of larger VLDL-, LDL-, and intermediate density lipoprotein-apoB-100 but did not affect smaller HDL-sized apoB-100 particles. Glucosamine treatment during the chase period (posttranslational) led to a 24% reduction in apoB-100 secretion (P < 0.01; n = 4) and promoted post-ER apoB degradation. However, the contribution of post-ER apoB-100 degradation appeared to be quantitatively minor. Interestingly, the glucosamine-induced posttranslational reduction in apoB-100 secretion could be partially prevented by treatment with desferrioxamine or vitamin E. Together, these data suggest that cotranslational glucosamine treatment may cause defects in apoB-100 N-linked glycosylation and folding, resulting in enhanced proteasomal degradation. Posttranslationally, glucosamine may interfere with the assembly process of apoB lipoproteins, leading to post-ER degradation via nonproteasomal pathways.     

The hepatic uptake of VLDL in lrp-ldlr-/-vldlr-/- mice is regulated by LPL activity and involves proteoglycans and SR-BI

LPL activity plays an important role in preceding the VLDL remnant clearance via the three major apolipoprotein E (apoE)-recognizing receptors: the LDL receptor (LDLr), LDL receptor-related protein (LRP), and VLDL receptor (VLDLr). The aim of this study was to determine whether LPL activity is also important for VLDL remnant clearance irrespective of these receptors and to determine the mechanisms involved in the hepatic remnant uptake. Administration of an adenovirus expressing LPL (AdLPL) into lrp(-)ldlr(-/-)vldlr(-/-) mice reduced both VLDL-triglyceride (TG) and VLDL-total cholesterol (TC) levels. Conversely, inhibition of LPL by AdAPOC1 increased plasma VLDL-TG and VLDL-TC levels. Metabolic studies with radiolabeled VLDL-like emulsion particles showed that the clearance and hepatic association of their remnants positively correlated with LPL activity. This hepatic association was independent of the bridging function of LPL and HL, since heparin did not reduce the liver association. In vitro studies demonstrated that VLDL-like emulsion particles avidly bound to the cell surface of primary hepatocytes from lrp(-)ldlr(-/-)vldlr(-/-) mice, followed by slow internalization, and involved heparin-releaseable cell surface proteins as well as scavenger receptor class B type I (SR-BI). Collectively, we conclude that hepatic VLDL remnant uptake in the absence of the three classical apoE-recognizing receptors is regulated by LPL activity and involves heparan sulfate proteoglycans and SR-BI.     

Effect of high-intensity exercise and high-fat diet on lipid metabolism in the liver of rats

[Purpose]

This study investigated the effects of high-intensity exercise (Ex) and high dietary fat intake on lipid metabolism in the liver of rats.

[Methods]

Male Sprague-Dawley rats were randomly assigned to one of the four groups (n=10 per group) that were maintained on a normal diet (ND) or high-fat diet (HFD) consisting of 30% fat (w/w), with or without exercise on a treadmill at 30 m/min and 8% grade) for 4 weeks (i.e., ND, ND+Ex, HFD, and HFD+Ex groups).

[Results]

Body weight (p<.001), total plasma cholesterol (TC) (p<.001), triglyceride (TG) (p<.05), and liver TG levels (p<.05) were increased in the HFD group relative to the ND groups, and serum glucose (p<.05), insulin (p<.05), homeostatic model assessment of insulin resistance (HOMA-IR) (p<.01), and liver TG levels (p<.01) were also higher in the HFD group compared to the ND+Ex group. Plasma free fatty acid was elevated in the HFD+Ex group compared to the HFD group (p<.01). With the exception of acetyl coenzyme A carboxylase, the expression of lipid metabolism-related genes in the liver was altered in the Ex groups compared to the control group (p<.05), with genes involved in lipolysis specifically up regulated in the HFD+Ex group compared to the other groups.

[Conclusion]

Vigorous exercise may increase glucose utilization and fat oxidation by activating genes in the liver that are associated with lipid metabolism compared to that in animals consuming a HFD without exercise. Therefore, high intensity exercise can be considered to counter the adverse effects of high dietary fat intake.     

Transport of vitamin E by differentiated Caco-2 cells

In hepatocytes, vitamin E is secreted via the efflux pathway and is believed to associate with apolipoprotein B (apoB)-lipoproteins extracellularly. The molecular mechanisms involved in the uptake, intracellular trafficking, and secretion of dietary vitamin E by the intestinal cells are unknown. We observed that low concentrations of Tween-40 were better for the solubilization and delivery of vitamin E to differentiated Caco-2 cells, whereas high concentrations of Tween-40 and sera inhibited this uptake. Vitamin E uptake was initially rapid and then reached saturation. Subcellular localization revealed that vitamin E primarily accumulated in microsomal membranes. Oleic acid (OA) treatment, which induces chylomicron assembly and secretion, decreased microsomal membrane-bound vitamin E in a time-dependent manner. To study secretion, differentiated Caco-2 cells were pulse-labeled with vitamin E and chased in the presence and absence of OA. In the absence of OA, vitamin E was associated with intestinal high density lipoprotein (I-HDL), whereas OA-treated cells secreted vitamin E with I-HDL and chylomicrons. No extracellular transfer of vitamin E between these lipoproteins was observed. Glyburide, an antagonist of ABCA1, partially inhibited its secretion with I-HDL, whereas plasma HDL increased vitamin E efflux. An antagonist of microsomal triglyceride transfer protein, brefeldin A, and monensin specifically inhibited vitamin E secretion with chylomicrons. These studies indicate that vitamin E taken up by Caco-2 cells is stored in the microsomal membranes and secreted with chylomicrons and I-HDL. Transport via I-HDL might contribute to vitamin E absorption in patients with abetalipoproteinemia receiving large oral doses of the vitamin.