Human plasma cholesteryl ester transfer protein enhances the transfer of cholesteryl ester from high density lipoproteins into cultured HepG2 cells

The role of human plasma cholesteryl ester transfer protein (CETP) in the cellular uptake of high density lipoprotein (HDL) cholesteryl ester (CE) was studied in a liver tumor cell line (HepG2). When HepG2 cells were incubated with [3H]cholesteryl ester-labeled HDL3 in the presence of increasing concentrations of CETP there was a progressive increase in cell-associated radioactivity to levels that were 2.8 times control. The CETP-dependent uptake of HDL-CE was found to be saturated by increasing concentrations of both CETP and HDL. The CETP-dependent uptake of CE radioactivity increased continuously during an 18-h incubation. In contrast to the effect on cholesteryl ester, CETP failed to enhance HDL protein cell association or degradation. Enhanced uptake of HDL cholesteryl ester was shown for the d greater than 1.21 g/ml fraction of human plasma, partially purified CETP, and CETP purified to homogeneity, but not for the d greater than 1.21 g/ml fraction of rat plasma which lacks cholesteryl ester transfer activity. HDL cholesteryl ester entering the cell under the influence of CETP was largely degraded to free cholesterol by a process inhibitable by chloroquine. CETP enhanced uptake of HDL [3H]CE in cultured smooth muscle cells and to a lesser extent in fibroblasts but did not significantly influence uptake in endothelial cells or J774 macrophages. These experiments show that, in addition to its known role in enhancing the exchange of CE between lipoproteins, plasma CETP can facilitate the in vitro selective transfer of CE from HDL into certain cells.     

Effects of various non-esterified fatty acids on the transfer of cholesteryl esters from HDL to LDL induced by the cholesteryl ester transfer protein

The effects of saturated, monounsaturated and polyunsaturated non-esterified fatty acids on the rate of transfer of radiolabeled cholesteryl esters from high density lipoproteins (HDL) to low density lipoproteins (LDL), induced by the cholesteryl ester transfer protein (CETP), have been studied. Human high-density lipoproteins-subfraction 3 (HDL3) containing radiolabeled cholesteryl esters were incubated with LDL at 37 degrees C with or without CETP and in the absence or in the presence of non-esterified fatty acids. Less than 6% of the total radioactivity was recovered in the LDL fraction after incubation of HDL3, and LDL for 3 h at 37 degrees C in the absence of CETP, regardless of whether or not non-esterified fatty acids were added. The addition of CETP to the incubation mixture induced a time-dependent redistribution of radiolabeled cholesteryl esters from HDL3 to LDL. Non-esterified fatty acids were found to alter the rate of transfer of cholesteryl esters induced by CETP. While short chain saturated non-esterified fatty acids (caprylic and capric acids) had no effect on the rate of transfer of cholesteryl esters, the medium and long chain ones (lauric, myristic, palmitic and stearic acids) significantly increased the CETP-mediated transfers from HDL3 to LDL. At low concentrations, unsaturated fatty acids also stimulated the CETP-mediated redistribution of radiolabeled cholesteryl esters from HDL3 to LDL. As the concentration of either oleic, linoleic or arachidonic acids increased to higher levels, a significant proportion of fatty acids remained unassociated with lipoprotein particles. Under these circumstances the transfer process was inhibited. These results show that non-esterified fatty acids can modulate the CETP-mediated transfer of cholesteryl esters from HDL to LDL and that this effect is dependent on both the length and the degree of unsaturation of their monomeric carbon chain.     

Effect of apolipoprotein E-free high density lipoproteins on cholesterol metabolism in cultured pig hepatocytes

We studied cholesterol synthesis from [14C]acetate, cholesterol esterification from [14C]oleate, and cellular cholesterol and cholesteryl ester levels after incubating cells with apoE-free high density lipoproteins (HDL) or low density lipoproteins (LDL). LDL suppressed synthesis by up to 60%, stimulated esterification by up to 280%, and increased cell cholesteryl ester content about 4-fold. Esterification increased within 2 h, but synthesis was not suppressed until after 6 h. ApoE-free HDL suppressed esterification by about 50% within 2 h. Cholesterol synthesis was changed very little within 6 h, unless esterification was maximally suppressed; synthesis was then stimulated about 4-fold. HDL lowered cellular unesterified cholesterol by 13-20% within 2 h and promoted the removal of newly synthesized cholesterol and cholesteryl esters. These changes were transient; by 24 h, both esterification and cellular unesterified cholesterol returned to control levels, and cholesteryl esters increased 2-3-fold. HDL core lipid was taken up selectively from 125I-labeled [3H]cholesteryl ester- and ether-labeled HDL. LDL core lipid uptake was proportional to LDL apoprotein uptake. The findings suggest that 1) the cells respond initially to HDL or LDL with changes in esterification, and 2) HDL mediates both the removal of free cholesterol from the cell and the delivery of HDL cholesteryl esters to the cell.     

Serum amyloid A is a ligand for scavenger receptor class B type I and inhibits high density lipoprotein binding and selective lipid uptake

Serum amyloid A is an acute phase protein that is carried in the plasma largely as an apolipoprotein of high density lipoprotein (HDL). In this study we investigated whether SAA is a ligand for the HDL receptor, scavenger receptor class B type I (SR-BI), and how SAA may influence SR-BI-mediated HDL binding and selective cholesteryl ester uptake. Studies using Chinese hamster ovary cells expressing SR-BI showed that (125)I-labeled SAA, both in lipid-free form and in reconstituted HDL particles, functions as a high affinity ligand for SR-BI. SAA also bound with high affinity to the hepatocyte cell line, HepG2. Alexa-labeled SAA was shown by fluorescence confocal microscopy to be internalized by cells in a SR-BI-dependent manner. To assess how SAA association with HDL influences HDL interaction with SR-BI, SAA-containing HDL was isolated from mice overexpressing SAA through adenoviral gene transfer. SAA presence on HDL had little effect on HDL binding to SR-BI but decreased (30-50%) selective cholesteryl ester uptake. Lipid-free SAA, unlike lipid-free apoA-I, was an effective inhibitor of both SR-BI-dependent binding and selective cholesteryl ester uptake of HDL. We have concluded that SR-BI plays a key role in SAA metabolism through its ability to interact with and internalize SAA and, further, that SAA influences HDL cholesterol metabolism through its inhibitory effects on SR-BI-mediated selective lipid uptake.     

Interaction of plasma-derived lipid transfer protein with macrophages in culture

This study investigates the ability of human plasma-derived lipid transfer protein to facilitate lipid transfer to and from intact viable cells in culture. Mouse peritoneal macrophages or J774 macrophages were preincubated with acetylated low density lipoprotein and [3H]oleate/albumin to promote the intracellular synthesis and accumulation of cholesteryl [3H]oleate and 3H-labeled triglyceride. The addition of partially purified lipid transfer protein to cultures of lipid-loaded macrophages resulted in a time and concentration-dependent transfer of radiolabeled cholesteryl ester and triglyceride from macrophages to the medium. At 48 hr, lipid transfer protein facilitated the net transfer of 16 and 11% of cellular cholesteryl ester and triglyceride radioactivity, respectively, to the medium; transfer in the absence of the lipid transfer protein was less than 2%. The transfer of cholesteryl ester radioactivity was accompanied by a similar decrease in cellular cholesteryl ester mass indicating a net transfer event. Lipid transfer from cells was not dependent on the presence of a lipoprotein acceptor in the medium; however, low and high density lipoproteins present at 200 micrograms cholesterol/ml did significantly stimulate the transfer protein-facilitated efflux of these lipids. Lipid transfer protein did not appear capable of transferring radiolabeled lipid from low density or high density lipoprotein to macrophages. Radiolabeled cholesteryl ester and triglyceride transferred from cells to the medium by lipid transfer protein were associated with large molecular weight (greater than 2 x 10(6)) components in the medium with an average density greater than 1.21 g/ml; these lipids were not associated with lipid transfer protein itself. However, these radiolabeled lipids were readily incorporated into low or high density lipoproteins when these lipoproteins were added to the medium either during or after its incubation with cells. It is concluded that lipid transfer protein can facilitate the net efflux of cholesteryl esters from intact, living macrophages. These studies suggest a novel and potentially antiatherogenic role for lipid transfer protein.     

Human low density lipoprotein subfractions separated by gradient gel electrophoresis: composition, distribution, and alterations induced by cholesteryl ester transfer protein

Low density lipoprotein (LDL) subfractions were studied in sera from 208 normolipidemic, 22 hypercholesterolemic, and 33 hypertriglyceridemic subjects. Whole serum without preliminary ultracentrifugation was submitted to electrophoresis in a nondenaturing polyacrylamide gel. Three main LDL patterns were observed in normolipidemic sera: type 1, characterized by the presence of only one major band; type 2, characterized by the presence of two close major bands; and type 3, where LDL were more dispersed and presented at least three distinct bands. Type 1 was more frequent in men (43%) than in women (19%). The tendency for a higher potential coronary disease risk profile sera, namely higher triglyceride level, higher very low density lipoprotein + LDL fraction and lower high density lipoprotein (HDL) fraction, was type 3 less than type 2 less than type 1. The LDL patterns found in hypercholesterolemic sera were of type 1. Hypertriglyceridemic sera were characterized by the presence of a major band of small size. Separated LDL subfractions were collected by electroelution and analyzed for composition. In all subspecies, the mass ratio of core to surface components was constant as well as the molar ratio of the two lipid surface components, phospholipids and free cholesterol. Surface lipid to apolipoprotein B ratio, cholesteryl ester to triglyceride ratio, and cholesteryl ester to apoB ratio increased with particle size increment. Incubation of LDL with HDL and purified cholesteryl ester transfer protein induced a transfer of lipids, mainly cholesteryl esters and phospholipids, to LDL and an increase of the sizes of LDL subfractions. This suggests that lipid transfers from HDL to LDL might be a process of intravascular LDL remodeling and a factor of LDL polymorphism.     

Effect of lipid transfer protein on plasma lipids, apolipoproteins and metabolism of high-density lipoprotein cholesteryl ester in the rat

The role of human plasma lipid transfer protein (LTP) in lipoprotein metabolism was studied in the rat, a species without endogenous cholesteryl ester and triacylglycerol transfer activity. Partially purified human LTP was injected intravenously into rats. The plasma activity was between 1.5- and 4-fold that of human plasma during the experiments. 6 h after the injection of LTP, a significant increase in serum apoB, and no significant changes in serum total cholesterol, free cholesterol, triacylglycerols, apoA-I, apoE, or apoA-IV were noted. Cholesterol was increased in very-low density and low-density lipoproteins (VLDL and LDL) and decreased in large-sized apoE-rich HDL. ApoA-I-containing particles with a size smaller than in normal rats were present in serum of LTP-treated rats. The mean diameter of HDL particles decreased and apoE, normally present on large-sized HDL, was present on smaller sized particles. The metabolic fate of cholesteryl ester, originally associated with HDL, was studied by injection of [3H]cholesteryl linoleyl ether-labelled apoA-I-rich HDL in the absence and in the presence of LTP. The disappearance of [3H]cholesteryl linoleyl ether, injected as part of apoA-I-rich HDL, from serum was increased in the LTP-treated rats; the t1/2 changed from 3.9 to 2.2 h, resulting in an increased accumulation of [3H]cholesteryl linoleyl ether in the liver. This can be explained by the redistribution of HDL [3H]cholesteryl linoleyl ether to VLDL and LDL in the presence of LTP, leading to the combined contribution of VLDL, LDL and HDL to the hepatic uptake. The present findings show profound effects of LTP on the chemical composition of HDL subspecies, the size of HDL and on the plasma turnover and hepatic uptake of cholesteryl esters originally present in apo A-I-rich HDL.     

Low density lipoprotein uptake: holoparticle and cholesteryl ester selective uptake.

Low density lipoproteins (LDL) contain apolipoprotein B-100 and are cholesteryl ester-rich, triglyceride-poor macromolecules, arising from the lipolysis of very low density lipoproteins. This review will describe the receptors responsible for uptake of whole LDL particles (holoparticle uptake), and the selective uptake of LDL cholesteryl ester. The LDL-receptor mediates the internalization of whole LDL through an endosomal-lysosomal pathway, leading to complete degradation of LDL. Increasing LDL-receptor expression by pharmacological intervention efficiently reduces blood LDL concentrations. The lipolysis stimulated receptor and LDL-receptor related protein may also lead to complete degradation of LDL in presence of free fatty acids and apolipoprotein E- or lipase-LDL complexes, respectively. Selective uptake of LDL cholesteryl ester has been demonstrated in the liver, especially in rodents and humans. This activity brings five times more LDL cholesteryl ester than the LDL-receptor to human hepatoma cells, suggesting that it is a physiologically significant pathway. The lipoprotein binding site of HepG2 cells mediates this process and recognizes all lipoprotein classes. Scavenger receptor class B type I and CD36, which mediate the selective uptake of high density lipoprotein cholesteryl ester, are potentially involved in LDL cholesteryl ester selective uptake, since they both bind LDL with high affinity. It is not known whether they are identical to the uncloned lipoprotein binding site and if the selective uptake of LDL cholesteryl ester produces a less atherogenic particle. If this is verified, pharmacological up-regulation of LDL cholesteryl ester selective uptake may become another therapeutic approach for reducing blood LDL-cholesterol levels and the risk of atherosclerosis.     

In vitro incorporation of radiolabeled cholesteryl esters into high and low density lipoproteins

We have developed and validated a method for in vitro incorporation of radiolabeled cholesteryl esters into low density (LDL) and high density lipoproteins (HDL). Radiolabeled cholesteryl esters dissolved in absolute ethanol were mixed with LDL or HDL in the presence of lipoprotein-deficient serum (LPDS) as a source of core lipid transfer activity. The efficiency of incorporation was dependent on: a) the core lipid transfer activity and quantity of LPDS, b) the mass of added radiolabeled cholesteryl esters, c) the length of incubation, and d) the amount of acceptor lipoprotein cholesterol. The tracer incorporation was documented by repeat density gradient ultracentrifugation, agarose gel electrophoresis, and precipitation with heparin-MnCl2. The radiolabeling conditions did not affect the following properties of the lipoproteins: 1) chemical composition, 2) electrophoretic mobility on agarose gels, 3) hydrated density, 4) distribution of apoproteins on SDS gels, 5) plasma clearance rates, and 6) immunoprecipitability of HDL apoproteins A-I and A-II. Rat HDL containing radiolabeled cholesteryl esters incorporated in vitro had plasma disappearance rates identical to HDL radiolabeled in vivo.     

Selective uptake of cholesteryl esters from various classes of lipoproteins by HepG2 cells.

Selective uptake of cholesteryl esters (CE) from lipoproteins by cells has been extensively studied with high density lipoproteins (HDL). It is only recently that such a mechanism has been attributed to intermediate and low density lipoproteins (IDL and LDL). Here, we compare the association of proteins and CE from very low density lipoproteins (VLDL), IDL, LDL and HDL3 to HepG2 cells. These lipoproteins were either labelled in proteins with 125I or in CE with 3H-cholesteryl oleate. We show that, at any lipoprotein concentration, protein association to the cells is significantly smaller for IDL, LDL, and HDL3 than CE association, but not for VLDL. At a concentration of 20 microg lipoprotein/mL, these associations reveal CE-selective uptake in the order of 2-, 4-, and 11-fold for IDL, LDL, and HDL3, respectively. These studies reveal that LDL and HDL3 are good selective donors of CE to HepG2 cells, while IDL is a poor donor and VLDL is not a donor. A significant inverse correlation (r2 = 0.973) was found between the total lipid/protein ratios of the four classes of lipoproteins and the extent of CE-selective uptake by HepG2 cells. The fate of 3H-CE of the two best CE donors (LDL and HDL3) was followed in HepG2 cells after 3 h of incubation. Cells were shown to hydrolyze approximately 25% of the 3H-CE of both lipoproteins. However, when the cells were treated with 100 microM of chloroquine, a lysosomotropic agent, 85 and 40% of 3H-CE hydrolysis was lost for LDL and HDL3, respectively. The fate of LDL and HDL3-CE in HepG2 cells deficient in LDL-receptor was found to be the same, indicating that the portion of CE hydrolysis sensitive to chloroquine is not significantly linked to LDL-receptor activity. Thus, in HepG2 cells, the magnitude of CE-selective uptake is inversely correlated with the total lipid/protein ratios of the lipoproteins and CE-selective uptake from the two best CE donors (LDL and HDL3) appears to follow different pathways.     

Affinity of lipid transfer protein for lipid and lipoprotein particles as influenced by lecithin-cholesterol acyltransferase

The effects of lecithin-cholesterol acyltransferase (LCAT) on the transfer of cholesterol esters mediated by lipid transfer protein (LTP) and its affinity for lipid and lipoprotein particles were investigated. When the single bilayer vesicle preparations (containing phosphatidylcholine, cholesterol, cholesteryl ester, and apolipoprotein- (apo) A-I at the molar ratio of 90:30:1.2:0.18) or high density lipoprotein 3 (HDL3) were used as the cholesteryl ester donor and low density lipoproteins (LDL) as the acceptor, the transfer activity of LTP was enhanced by the addition of low concentrations of LCAT. In contrast, no enhancement of cholesteryl ester transfer was observed upon addition of LCAT to either the discoidal bilayer particle preparations (containing phosphatidylcholine, cholesterol, cholesteryl ester, and apo-A-I at the molar ratio of 90:30:1.2:1.0) or high density lipoprotein 2 (HDL2). Although both apo-A-I and apo-A-II promoted the transfer of cholesteryl ester from vesicles to LDL, the additional enhancement of the transfer by LCAT was observed only with the vesicles containing apo-A-I. Gel permeation chromatography of LTP/vesicle and LTP/HDL3 mixtures in the presence and absence of LCAT showed that the affinity of LTP for both the vesicles and HDL3 increased upon addition of LCAT. In contrast, neither HDL2 nor discoidal bilayer particles showed any significant enhancement of LTP binding upon addition of LCAT. By using LCAT covalently bound to Sepharose 4B, a maximal interaction between LTP and bound LCAT was shown to occur at the ionic strength of 0.16. Deviation from this ionic strength reduced the extent of the interaction. At the ionic strength of 0.01 and 0.5, the elution volume of LTP was identical to that of bovine serum albumin.     

Evaluation of cholesteryl ester transfer in the seminiferous tubule cells of immature rats in vivo and in vitro

Sertoli cells and germ cells are separated from the interstitial blood capillaries by an extracellular matrix and the peritubular cells, which constitute a barrier to the movement of plasma lipoproteins. The present study was undertaken to evaluate in vivo and in vitro the high density lipoprotein (HDL) cholesteryl ester transfer from plasma to seminiferous tubule cells in the testis of 30-day-old rats. Firstly, the transfer of HDL cholesteryl oleate from plasma to testicular compartments was evaluated and, secondly, the role of apolipoproteins A-I and E in the uptake of cholesteryl ester by Sertoli cells was investigated. At 2 h after the administration of HDL reconstituted with [3H]cholesteryl ester, dimyristoyl phosphatidylcholine and apolipoproteins, the tissue space in the interstitial cells (740 +/- 60 microliters g-1 cell protein) was fourfold higher than that in the seminiferous tubule cells (170 +/- 10 microliters g-1). Sertoli cells were isolated and incubated with [3H]cholesteryl ester HDL reconstituted with apolipoprotein A-I or E to evaluate the mechanisms of cholesteryl ester influx. At the same apolipoprotein concentration (50 micrograms apolipoprotein ml-1 medium), the uptake of [3H]cholesteryl oleate from phospholipid-apolipoprotein E vesicles was twofold higher than that with phospholipid-apolipoprotein A-I vesicles. The presence of heparin reduced the uptake of cholesteryl ester from apolipoprotein E vesicles but not with apolipoprotein A-I vesicles, indicating that uptake of apolipoprotein A-I vesicles via a secretion of apolipoprotein E by the cells themselves was not involved. These results demonstrate that plasma lipoprotein cholesterol is able to cross the testis lamina propria and that Sertoli cells take up cholesteryl ester for seminiferous tubule cell metabolism mainly via an apolipoprotein E pathway.     

Free cholesterol is a potent regulator of lipid transfer protein function

This study investigates the effect of altered lipoprotein free cholesterol (FC) content on the transfer of cholesteryl ester (CE) and triglyceride (TG) from very low- (VLDL), low- (LDL), and high-(HDL) density lipoproteins by the plasma-derived lipid transfer protein (LTP). The FC content of VLDL and HDL was selectively altered by incubating these lipoproteins with FC/phospholipid dispersions of varying composition. FC-modified lipoproteins were then equilibrated with [3H] TG, [14C]CE-labeled lipoproteins of another class to facilitate the subsequent modification of the radiolabeled donor lipoproteins. LTP was added and the extent of radiolabeled TG and CE transfer determined after 1 h. With either LDL or VLDL as lipid donor, an increase in the FC content of these lipoproteins caused a concentration-dependent inhibition (up to 50%) of CE transfer from these particles, without any significant effect on TG transfer. In contrast, with HDL as donor, increasing the HDL FC content had little effect on CE transfer from HDL, but markedly stimulated (up to 2.5-fold) the transfer of TG. This differential effect of FC on the unidirectional transfer of radiolabeled lipids from VLDL and HDL led to marked effects on LTP-facilitated net mass transfer of lipids. During long-term incubation of a constant amount of LTP with FC-modified VLDL and HDL, the extent of net mass transfer was linearly related to lipoprotein FC content; a 4-fold increase in FC content resulted in a 3-fold stimulation of the CE mass transferred to VLDL, which was coupled to an equimolar, reciprocal transfer of TG mass to HDL. Since lipid transfer between lipoproteins is integral to the process of reverse cholesterol transport, we conclude that lipoprotein FC levels are a potent, positive regulator of the pathways involved in sterol clearance. FC may modulate lipid transfer by altering the availability of CE and TG to LTP at the lipoprotein surface.     

High-density lipoprotein particle uptake and selective uptake of high-density lipoprotein-associated cholesteryl esters by J774 macrophages

High-density lipoprotein (HDL) cholesteryl esters are taken up by fibroblasts via HDL particle uptake and via selective uptake, i.e., cholesteryl ester uptake independent of HDL particle uptake. In the present study we investigated HDL selective uptake and HDL particle uptake by J774 macrophages. HDL3 (d = 1.125-1.21 g/ml) was labeled with intracellularly trapped tracers: 125I-labeled N-methyltyramine-cellobiose-apo A-I (125I-NMTC-apo A-I) to trace apolipoprotein A-I (apo A-I) and [3H]cholesteryl oleyl ether to trace cholesteryl esters. J774 macrophages, incubated at 37 degrees C in medium containing doubly labeled HDL3, took up 125I-NMTC-apo A-I, indicating HDL3 particle uptake (102.7 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein). Apparent HDL3 uptake according to the uptake of [3H]cholesteryl oleyl ether (470.4 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein) was in significant excess on 125I-NMTC-apo A-I uptake, i.e., J774 macrophages demonstrated selective uptake of HDL3 cholesteryl esters. To investigate regulation of HDL3 uptake, cell cholesterol was modified by preincubation with low-density lipoprotein (LDL) or acetylated LDL (acetyl-LDL). Afterwards, uptake of doubly labeled HDL3, LDL (apo B,E) receptor activity or cholesterol mass were determined. Preincubation with LDL or acetyl-LDL increased cell cholesterol up to approx. 3.5-fold over basal levels. Increased cell cholesterol had no effect on HDL3 particle uptake. In contrast, LDL- and acetyl-LDL-loading decreased selective uptake (apparent uptake 606 vs. 366 ng HDL3 protein/mg cell protein per 4 h in unloaded versus acetyl-LDL-loaded cells at 20 micrograms HDL3 protein/ml). In parallel with decreased selective uptake, specific 125I-LDL degradation was down-regulated. Using heparin as well as excess unlabeled LDL, it was shown that HDL3 uptake is independent of LDL (apo B,E) receptors. In summary, J774 macrophages take up HDL3 particles. In addition, J774 cells also selectively take up HDL3-associated cholesteryl esters. HDL3 selective uptake, but not HDL3 particle uptake, can be regulated.     

LDL and HDL enriched in triglyceride promote abnormal cholesterol transport

Hypertriglyceridemia induces multiple changes in lipoprotein composition. Here we investigate how one of these modifications, triglyceride (TG) enrichment, affects HDL and LDL function when this alteration occurs under conditions in which more polar components can naturally re-equilibrate. TG-enriched lipoproteins were produced by co-incubating VLDL, LDL, and HDL with cholesteryl ester (CE) transfer protein. The resulting 2.5-fold increase in TG/CE ratio did not measurably alter the apoprotein composition of LDL or HDL, or modify LDL size. HDL mean diameter increased slightly from 9.1 to 9.4 nm. Modified LDL was internalized by fibroblasts normally, but its protein was degraded much less efficiently. This likely reflects an aberrant apolipoprotein B (apoB) conformation, as suggested by its resistance to V8 protease digestion and altered LDL electrophoretic mobility. TG-enriched LDL ineffectively down-regulated cholesterol biosynthesis compared with control LDL at the same protein concentration, but was equivalent in sterol regulation when compared on a cholesterol basis. TG-enriched HDL promoted greater net cholesterol efflux from cholesterol-loaded J774 cells. However, cholesterol associated with TG-enriched HDL was inefficiently esterified by lecithin:cholesterol acyltransferase, and TG-enriched HDLs were poor donors of CE to HepG2 hepatocytes by selective uptake. We conclude that TG-enrichment, in the absence of other significant alterations in lipoprotein composition, is sufficient to alter both cholesterol delivery and removal mechanisms. Some of these abnormalities may contribute to increased coronary disease in hypertriglyceridemia.     

Ethanol induced alterations in low and high density lipoproteins

Male squirrel monkeys fed ethanol (ETOH) at variable doses were used to determine whether alcohol modifies levels of plasma low density lipoproteins (LDL) in addition to increasing high density lipoproteins (HDL). Because we earlier showed that high alcohol consumption enhances lipoprotein cholesterol synthesis, experiments were also performed to further assess whether ETOH alters lipoprotein clearance and plasma transfer processes in vivo. Monkeys were divided into three groups: Controls fed isocaloric liquid diet; and Low and High ETOH animals fed liquid diet with vodka substituted isocalorically for carbohydrate at 12 and 24 of the calories, respectively. High ETOH primates had significantly more LDL lipid and protein while serum glutamate oxaloacetate transaminase was similar for the three groups. Although removal of 3H LDL cholesteryl ester (CE) from the plasma compartment was not affected by dietary ETOH, transfer of LDL CE to HDL was impaired in the High ETOH group suggesting a mechanism for the enlarged circulating pool of LDL. Transfer of 14C HDL CE to lower density lipoproteins was similar for the three groups. However, ETOH at both doses delayed clearance of radiolabeled HDL CE from circulation. Thus besides enhancing synthesis of lipoproteins, ETOH at a moderately high dose (24% of calories) influences lipoprotein levels in primates by modifying lipid transfer processes (LDL) as well as by altering clearance (HDL) without adversely affecting liver function.     

Very low and low density lipoprotein synthesis and secretion by the human hepatoma cell line Hep-G2: effects of free fatty acid

The liver is a major source of the plasma lipoproteins; however, direct studies of the regulation of lipoprotein synthesis and secretion by human liver are lacking. Dense monolayers of Hep-G2 cells incorporated radiolabeled precursors into protein ([35S]methionine), cholesterol ([3H]mevalonate and [14C]acetate), triacylglycerol, and phospholipid ([3H]glycerol), and secreted them as lipoproteins. In the absence of free fatty acid in the media, the principal lipoprotein secretory product that accumulated had a density maximum of 1.039 g/ml, similar to serum low density lipoprotein (LDL). ApoB-100 represented greater than 95% of the radiolabeled apoprotein of these particles, with only traces of apoproteins A and E present. Inclusion of 0.8 mM oleic acid in the media resulted in a 54% reduction in radiolabeled triacylglycerol in the LDL fraction and a 324% increase in triacylglycerol in the very low density lipoprotein (VLDL) fraction. Similar changes occurred in the secretion of newly synthesized apoB-100. The VLDL contained apoB-100 as well as apoE. In the absence of exogenous free fatty acid, the radiolabeled cholesterol was recovered in both the LDL and the high density lipoprotein (HDL) regions. Oleic acid caused a 50% decrease in HDL radiolabeled cholesterol and increases of radiolabeled cholesterol in VLDL and LDL. In general, less than 15% of the radiolabeled cholesterol was esterified, despite the presence of cholesteryl ester in the cell. Incubation with oleic acid did not cause an increase in the total amount of radiolabeled lipid or protein secreted. We conclude that human liver-derived cells can secrete distinct VLDL and LDL-like particles, and the relative amounts of these lipoproteins are determined, at least in part, by the availability of free fatty acid.     

FXRalpha down-regulates LXRalpha signaling at the CETP promoter via a common element

The cholesteryl ester transfer protein (CETP), a key player in cholesterol metabolism, has been shown to promote the transfer of triglycerides from very low density lipoprotein (VLDL) and low density lipoprotein (LDL) to high density lipoprotein (HDL) in exchange for cholesterol ester. Here we demonstrate that farnesoid X receptor alpha (FXRalpha; NR1H4) down-regulates CETP expression in HepG2 cells. A FXRalpha ligand, chenodeoxycholic acid (CDCA), suppressed basal mRNA levels of the CETP gene in HepG2 cells in a dose-dependent manner. Using gel shift and chromatin immunoprecipitation (ChIP) assays, we found that FXRalpha could bind to the liver X receptor alpha (LXRalpha; NR1H3) binding site (LXRE; DR4RE) located within the CETP 5' promoter region. FXRalpha suppressed LXRalpha-induced DR4RE-luciferase activity and this effect was mediated by a binding competition between FXRalpha and LXRalpha for DR4RE. Furthermore, the addition of CDCA together with a LXRalpha ligand, GW3965, to HepG2 cells was shown to substantially decrease mRNA levels of hepatic CETP gene, which is typically induced by GW3965. Together, our data demonstrate that FXRalpha down-regulates CETP gene expression via binding to the DR4RE sequence within the CETP 5' promoter and this FXRalpha binding is essential for FXRalpha inhibition of LXRalpha-induced CETP expression.     

Postprandial chylomicrons: potent vehicles for transporting cholesterol from endogenous LDL+HDL and cell membranes to the liver via LCAT and CETP

We examined whether postprandial (PP) chylomicrons (CMs) can serve as vehicles for transporting cholesterol from endogenous cholesterol-rich lipoprotein (LDL+HDL) fractions and cell membranes to the liver via lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) activities. During incubation of fresh fasting and PP plasma containing [(3)H]cholesteryl ester (CE)-labeled LDL+HDL, both CMs and VLDL served as acceptors of [(3)H]CE or cholesterol from LDL+HDL. The presence of CMs in PP plasma suppressed the ability of VLDL to accept [(3)H]CE from LDL+HDL. In reconstituted plasma containing an equivalent amount of triglycerides from isolated VLDL or CMs, a CM particle was about 40 times more potent than a VLDL particle in accepting [(3)H]CE or cholesterol from LDL+HDLs. When incubated with red blood cells (RBCs) as a source for cell membrane cholesterol, the cholesterol content of CMs, VLDL, LDL, and HDL in PP plasma increased by 485%, 74%, 13%, and 30%, respectively, via LCAT and CETP activities. The presence of CMs in plasma suppressed the ability of endogenous lipoproteins to accept cholesterol from RBCs. Our data suggest that PP CMs may play an important role in promoting reverse cholesterol transport in vivo by serving as the preferred ultimate vehicle for transporting cholesterol released from cell membranes to the liver via LCAT and CETP.     

Structural basis of the lipid transfer mechanism of phospholipid transfer protein (PLTP)

Human phospholipid transfer protein (PLTP) mediates the transfer of phospholipids among atheroprotective high-density lipoproteins (HDL) and atherogenic low-density lipoproteins (LDL) by an unknown mechanism. Delineating this mechanism would represent the first step towards understanding PLTP-mediated lipid transfers, which may be important for treating lipoprotein abnormalities and cardiovascular disease. Here, using various electron microscopy techniques, PLTP is revealed to have a banana-shaped structure similar to cholesteryl ester transfer protein (CETP). We provide evidence that PLTP penetrates into the HDL and LDL surfaces, respectively, and then forms a ternary complex with HDL and LDL. Insights into the interaction of PLTP with lipoproteins at the molecular level provide a basis to understand the PLTP-dependent lipid transfer mechanisms for dyslipidemia treatment.