Selective Uptake Of High Density Lipoprotein Cholesteryl Esters Research Articles

Cooperation between hepatic cholesteryl ester hydrolase and scavenger receptor BI for hydrolysis of HDL-CE

Liver is the sole organ responsible for the final elimination of cholesterol from the body either as biliary cholesterol or bile acids. High density lipoprotein (HDL)-derived cholesterol is the major source of biliary sterols and represents a mechanism for the removal of cholesterol from peripheral tissues including artery wall-associated macrophage foam cells. Via selective uptake through scavenger receptor BI (SR-BI), HDL-cholesterol is thought to be directly secreted into bile, and HDL cholesteryl esters (HDL-CEs) enter the hepatic metabolic pool and need to be hydrolyzed prior to conversion to bile acids. However, the identity of hepatic CE hydrolase (CEH) as well as the role of SR-BI in bile acid synthesis remains elusive. In this study we examined the role of human hepatic CEH (CES1) in facilitating hydrolysis of SR-BI-delivered HDL-CEs. Over-expression of CEH led to increased hydrolysis of HDL-[³H]CE in primary hepatocytes and SR-BI expression was required for this process. Intracellular CEH associated with BODIPY-CE delivered by selective uptake via SR-BI. CEH and SR-BI expression enhanced the movement of [³H]label from HDL-[³H]CE to bile acids in vitro and in vivo. Taken together, these studies demonstrate that SR-BI-delivered HDL-CEs are hydrolyzed by hepatic CEH and utilized for bile acid synthesis.

High-capacity selective uptake of cholesteryl ester from native LDL during macrophage foam cell formation

Macrophage foam cells are a defining pathologic feature of atherosclerotic lesions. Recent studies have demonstrated that at high concentrations associated with hypercholesterolemia, native LDL induces macrophage lipid accumulation. LDL particles are taken up by macrophages as part of bulk fluid pinocytosis. However, the uptake and metabolism of cholesterol from native LDL during foam cell formation has not been clearly defined. Previous reports have suggested that selective cholesteryl ester (CE) uptake might contribute to cholesterol uptake from LDL independently of particle endocytosis. In this study we demonstrate that the majority of macrophage LDL-derived cholesterol is acquired by selective CE uptake in excess of LDL pinocytosis and degradation. Macrophage selective CE uptake does not saturate at high LDL concentrations and is not down-regulated during cholesterol accumulation. In contrast to CE uptake, macrophages exhibit little selective uptake of free cholesterol (FC) from LDL. Following selective uptake from LDL, CE is rapidly hydrolyzed by a novel chloroquine-sensitive pathway. FC released from LDL-derived CE hydrolysis is largely effluxed from cells but also is subject to ACAT-mediated reesterification. These results indicate that selective CE uptake plays a major role in macrophage metabolism of LDL.

Metabolism of high density lipoprotein apolipoprotein A‐I and cholesteryl ester in insulin resistant dog: a stable isotope study

In reverse cholesterol transport (RCT), hepatic Scavenger Receptor class B type I (SR-BI) plays an important role by mediating the selective uptake of high-density lipoprotein cholesteryl ester (HDL-CE). However, little is known about this antiatherogenic mechanism in insulin resistance. HDL-CE selective uptake represents the main process for HDL-CE turnover in dog, a species lacking cholesteryl ester transfer protein activity. We therefore investigate the effects of diet induced insulin resistance on RCT. Five beagle dogs, in healthy and insulin resistant states, underwent a primed constant infusion of [1,2(13)C(2)]acetate and [5,5,5-(2)H(3)]leucine, as labelled precursors of CE and apolipoprotein (apo) A-I, respectively. Data were analysed using modelling methods. HDL-apo A-I concentration did not change in insulin resistant state but apo A-I absolute production rate (APR) and fractional catabolic rate (FCR) were both higher (2.2- and 2.4-fold, respectively, p < 0.05). HDL-CE levels were lower (1.2-fold, p < 0.05). HDL-CE APR and FCR were both lower (2.3- and 2-fold, respectively, p < 0.05), as well as selective uptake (2.6-fold, p < 0.05). Lower HDL-CE selective uptake suggests that RCT is impaired in obese insulin resistant dog.

Scavenger Receptor Class B Type I Mediates the Selective Uptake of High-Density Lipoprotein–Associated Cholesteryl Ester by the Liver in Mice

High-density lipoprotein (HDL) cholesteryl esters (CE) are taken up by liver and adrenals selectively, ie, independent from particle internalization. Class B type I scavenger receptor (SR-BI) mediates this uptake in vitro. The role of SR-BI in HDL metabolism was explored in mice. Mice with a mutation in the SR-BI gene (SR-BI KO) and wild-type (WT) littermates were used. Mutants had increased HDL cholesterol. HDL was labeled with 125I (protein) and [3H] (CE). After HDL injection, blood samples were drawn and finally the mice were euthanized. In WT, the plasma decay of HDL-associated [3H] is faster compared with 125I and this represents whole-body selective CE uptake. In SR-BI KO, the decay of both tracers is similar, yielding no selective CE removal. In WT liver and adrenals, uptake of [3H] is higher than 125I, showing selective uptake. In SR-BI KO, liver uptake of [3H] and 125I are similar, proposing no selective HDL CE uptake. In SR-BI KO adrenals, selective uptake is reduced; however, even in the absence of SR-BI, this uptake is detected using WT-HDL. SR-BI mediates selective uptake of HDL CE by the liver. In adrenals, an alternative mechanism or mechanisms can play a role in selective CE uptake.

Changes in Plasma Membrane Properties and Phosphatidylcholine Subspecies of Insect Sf9 Cells Due to Expression of Scavenger Receptor Class B, Type I, and CD36

In mammalian cells scavenger receptor class B, type I (SR-BI), mediates the selective uptake of high density lipoprotein (HDL) cholesteryl ester into hepatic and steroidogenic cells. In addition, SR-BI has a variety of effects on plasma membrane properties including stimulation of the bidirectional flux of free cholesterol (FC) between cells and HDL and changes in the organization of plasma membrane FC as indicated by increased susceptibility to exogenous cholesterol oxidase. Recent studies in SR-BI-deficient mice and in SR-BI-expressing Sf9 insect cells showed that SR-BI has significant effects on plasma membrane ultrastructure. The present study was designed to test the range of SR-BI effects in Sf9 insect cells that typically have very low cholesterol content and a different phospholipid profile compared with mammalian cells. The results showed that, as in mammalian cells, SR-BI expression increased HDL cholesteryl ester selective uptake, cellular cholesterol mass, FC efflux to HDL, and the sensitivity of membrane FC to cholesterol oxidase. These activities were diminished or absent upon expression of the related scavenger receptor CD36. Thus, SR-BI has fundamental effects on cholesterol flux and membrane properties that occur in cells of evolutionarily divergent origins. Profiling of phospholipid species by electrospray ionization mass spectrometry showed that scavenger receptor expression led to the accumulation of phosphatidylcholine species with longer mono- or polyunsaturated acyl chains. These changes would be expected to decrease phosphatidylcholine/cholesterol interactions and thereby enhance cholesterol desorption from the membrane. Scavenger receptor-mediated changes in membrane phosphatidylcholine may contribute to the increased flux of cholesterol and other lipids elicited by these receptors.

A novel efflux-recapture process underlies the mechanism of high-density lipoprotein cholesteryl ester-selective uptake mediated by the low-density lipoprotein receptor-related protein.

To determine the mechanism of low-density lipoprotein (LDL) receptor-related protein (LRP)-mediated selective uptake of high-density lipoprotein (HDL)-derived cholesteryl esters (CE). Apolipoprotein E (apoE) and heparin sulfate proteoglycans are required for LRP-mediated selective uptake in adipocytes. Furthermore, 2-deoxyglucose and NaN(3) abolish this process, indicating that cellular energy is required. LRP-mediated selective uptake is also abolished by monensin or when clathrin-mediated internalization is inhibited (using hypotonic, K(+)-free medium or hyperosmolar sucrose), clearly implicating receptor endocytosis. The receptor-associated protein (RAP), an inhibitor of ligand binding to LRP, reduced the transport of CE into an intracellular compartment but not into the plasma membrane. Remarkably, the CE that is ultimately transported by LRP first enters the plasma membrane then undergoes apoE-mediated CE efflux before being recaptured and internalized by LRP. According to this "efflux-recapture" model, LRP contributes to selective uptake because it recovers CE that would normally be lost by efflux mediated by apoE. In adipocytes, the LDL receptor-related protein contributes to selective uptake when it recaptures and internalizes HDL-derived cholesteryl esters that are otherwise lost by apoE-mediated efflux. This novel "efflux-recapture" process explains some conflicting observations of selective uptake and underscores the bi-directional nature of efflux.

SR‐BI and HDL Cholesteryl Ester Metabolism

Scavenger receptor class B, type I (SR‐BI) is the receptor for high density lipoprotein (HDL) that mediates cellular uptake of HDL cholesteryl ester (CE) and is a major route for cholesterol delivery to steroidogenic pathways. SR‐BI is localized in specialized microvillar channel plasma membrane compartments that retain HDL and are sites for HDL CE selective uptake. In fact, adrenal gland microvillar channel formation is regulated by adrenocorticotropin hormone and requires SR‐BI expression. SR‐BI‐mediated uptake of HDL CE is a two‐step process requiring high affinity HDL binding followed by transfer of CE to the membrane. SR‐BI delivers HDL CE to sites in the membrane where it is readily metabolized to free cholesterol by cell type‐specific neutral CE hydrolases. The most likely candidate for the hydrolysis of HDL CE delivered via SR‐BI in the adrenal gland is hormone sensitive lipase. New data in adrenocortical cells as well as the study of a mutant SR‐BI receptor lend insight into the mechanism of cholesterol transfer from plasma HDL to the steroidogenic pathway in endocrine cells.

Enhancement of Scavenger Receptor Class B Type I-mediated Selective Cholesteryl Ester Uptake fromapoA-I−/− High Density Lipoprotein (HDL) by Apolipoprotein A-I Requires HDL Reorganization by Lecithin Cholesterol Acyltransferase

The severe depletion of cholesteryl ester (CE) in adrenocortical cells of apoA-I(-/-) mice suggests that apolipoprotein (apo) A-I plays an important role in the high density lipoprotein (HDL) CE selective uptake process mediated by scavenger receptor BI (SR-BI) in vivo. A recent study showed that apoA-I(-/-) HDL binds to SR-BI with the same affinity as apoA-I(+/+) HDL, but apoA-I(-/-) HDL has a decreased V(max) for CE transfer from the HDL particle to adrenal cells. The present study was designed to determine the basis for the reduced selective uptake of CE from apoA-I(-/-) HDL. Variations in apoA-I(-/-) HDL particle diameter, free cholesterol or phospholipid content, or the apoE or apoA-II content of apoA-I(-/-) HDL had little effect on HDL CE selective uptake into Y1-BS1 adrenal cells. Lecithin cholesterol acyltransferase treatment alone or addition of apoA-I to apoA-I(-/-) HDL alone also had little effect. However, addition of apoA-I to apoA-I(-/-) HDL in the presence of lecithin cholesterol acyltransferase reorganized the large heterogeneous apoA-I(-/-) HDL to a more discrete particle with enhanced CE selective uptake activity. These results show a unique role for apoA-I in HDL CE selective uptake that is distinct from its role as a ligand for HDL binding to SR-BI. These data suggest that the conformation of apoA-I at the HDL surface is important for the efficient transfer of CE to the cell.

Analysis of chimeric receptors shows that multiple distinct functional activities of scavenger receptor, class B, type I (SR-BI), are localized to the extracellular receptor domain.

Scavenger receptor BI (SR-BI) mediates the selective uptake of high-density lipoprotein (HDL) cholesteryl ester (CE), a process by which HDL CE is taken into the cell without degradation of the HDL particle. In addition, SR-BI stimulates the bi-directional flux of free cholesterol (FC) between cells and lipoproteins, an activity that may be responsible for net cholesterol efflux from peripheral cells as well as the rapid hepatic clearance of FC from plasma HDL. SR-BI also increases cellular cholesterol mass and alters cholesterol distribution in plasma membrane domains as judged by the enhanced sensitivity of membrane cholesterol to extracellular cholesterol oxidase. In contrast, CD36, a closely related class B scavenger receptor, has none of these activities despite binding HDL with high affinity. In the present study, analyses of chimeric SR-BI/CD36 receptors and domain-deleted SR-BI have been used to test the various domains of SR-BI for functional activities related to HDL CE selective uptake, bi-directional FC flux, and the alteration of membrane cholesterol mass and distribution. The results show that each of these activities localizes to the extracellular domain of SR-BI. The N-terminal cytoplasmic tail and transmembrane domains appear to play no role in these activities other than targeting the receptor to the plasma membrane. The C-terminal tail of SR-BI is dispensable for activity as well for targeting to the plasma membrane. Thus, multiple distinct functional activities are localized to the SR-BI extracellular domain.

Comparison of Class B Scavenger Receptors, CD36 and Scavenger Receptor BI (SR-BI), Shows That Both Receptors Mediate High Density Lipoprotein-Cholesteryl Ester Selective Uptake but SR-BI Exhibits a Unique Enhancement of Cholesteryl Ester Uptake

Scavenger receptor BI (SR-BI) mediates the selective uptake of high density lipoprotein (HDL) cholesteryl ester (CE), a process by which HDL CE is taken into the cell without internalization and degradation of the HDL particle. The biochemical mechanism by which SR-BI mediates the selective uptake of HDL CE is poorly understood. Given that CE transfer will occur to some extent from HDL to protein-free synthetic membranes, one hypothesis is that the role of SR-BI is primarily to tether HDL close to the cell surface to facilitate CE transfer from the particle to the plasma membrane. In the present study, this hypothesis was tested by comparing the selective uptake of HDL CE mediated by mouse SR-BI (mSR-BI) with that mediated by rat CD36 (rCD36), a closely related class B scavenger receptor. Both mSR-BI and rCD36 bind HDL with high affinity, and both receptors mediate HDL CE selective uptake. However, SR-BI mediates selective uptake of HDL CE with a 7-fold greater efficiency than rCD36. HDL CE selective uptake mediated by rCD36 is dependent on HDL binding to the receptor, since a mutation that blocks HDL binding also blocks HDL CE selective uptake. These data lead us to hypothesize that one component of HDL CE selective uptake is the tethering of HDL particles to the cell surface. To explore the molecular domains responsible for the greater efficiency of selective uptake by mSR-BI, we compared binding and selective uptake among mSR-BI, scavenger receptor BII, and various chimeric receptors formed from mSR-BI and rCD36. The results show that the extracellular domain of mSR-BI is essential for efficient HDL CE uptake, but the C-terminal cytoplasmic tail also has a major influence on the selective uptake process.

Expression and Microvillar Localization of Scavenger Receptor, Class B, Type I (a High Density Lipoprotein Receptor) in Luteinized and Hormone-Desensitized Rat Ovarian Models

Steroidogenic cells in rats and mice obtain most of their cholesterol for steroid production and cholesteryl ester (CE) storage via the selective uptake pathway in which high density lipoprotein CE (HDL-CE) is taken into the cell without the uptake and degradation of the HDL particle. A number of recent studies show that the scavenger receptor, class B, type I (SR-BI) can mediate HDL-CE selective uptake in cultured cells and suggest that this receptor may be responsible for HDL-CE selective uptake in steroidogenic cells in vivo. In the current study we examine the relationship between SR-BI expression and HDL-CE selective uptake in the gonadotropin-primed, luteinized rat ovary and in the ovary that is desensitized by multiple gonadotropin treatments. Results from this study demonstrate a tight association between expression of SR-BI and measurements of HDL-CE selective uptake regardless of the steroidogenic state of the ovary. Thus, in the luteinized ovary (which is actively producing progestins), HDL-CE selective uptake is high, as is the expression of SR-BI. In the desensitized ovary (where CE content is reduced by 90% and progestin production is virtually absent), HDL-CE selective uptake and SR-BI are induced 2- to 3-fold compared with those in the luteinized ovary. These data argue that SR-BI can be regulated by the cholesterol status of the luteal cell independently of gonadotropic stimulation. Immunostaining at the light microscopic level showed strong expression of SR-BI specifically on the surface of luteal cells in the luteinized and desensitized ovary. Immunolocalization at the electron microscopic level showed that SR-BI was associated with microvilli and microvillar channels of the luteal cell surface. This result supports the hypothesis that microvilli and microvillar channels represent a cell surface compartment that is specialized for the selective uptake of lipoprotein cholesterol into steroidogenic cells.

Expression and microvillar localization of scavenger receptor, class B, type I (a high density lipoprotein receptor) in luteinized and hormone-desensitized rat ovarian models.

Steroidogenic cells in rats and mice obtain most of their cholesterol for steroid production and cholesteryl ester (CE) storage via the selective uptake pathway in which high density lipoprotein CE (HDL-CE) is taken into the cell without the uptake and degradation of the HDL particle. A number of recent studies show that the scavenger receptor, class B, type I (SR-BI) can mediate HDL-CE selective uptake in cultured cells and suggest that this receptor may be responsible for HDL-CE selective uptake in steroidogenic cells in vivo. In the current study we examine the relationship between SR-BI expression and HDL-CE selective uptake in the gonadotropin-primed, luteinized rat ovary and in the ovary that is desensitized by multiple gonadotropin treatments. Results from this study demonstrate a tight association between expression of SR-BI and measurements of HDL-CE selective uptake regardless of the steroidogenic state of the ovary. Thus, in the luteinized ovary (which is actively producing progestins), HDL-CE selective uptake is high, as is the expression of SR-BI. In the desensitized ovary (where CE content is reduced by 90% and progestin production is virtually absent), HDL-CE selective uptake and SR-BI are induced 2- to 3-fold compared with those in the luteinized ovary. These data argue that SR-BI can be regulated by the cholesterol status of the luteal cell independently of gonadotropic stimulation. Immunostaining at the light microscopic level showed strong expression of SR-BI specifically on the surface of luteal cells in the luteinized and desensitized ovary. Immunolocalization at the electron microscopic level showed that SR-BI was associated with microvilli and microvillar channels of the luteal cell surface. This result supports the hypothesis that microvilli and microvillar channels represent a cell surface compartment that is specialized for the selective uptake of lipoprotein cholesterol into steroidogenic cells.

In Vivo Regulation of Scavenger Receptor BI and the Selective Uptake of High Density Lipoprotein Cholesteryl Esters in Rat Liver Parenchymal and Kupffer Cells

High density lipoprotein cholesteryl esters (HDL-CE) are selectively taken up by liver parenchymal cells without parallel apolipoprotein uptake. This selective uptake route forms an important step in the so-called reverse cholesterol transport. Scavenger receptor BI (SR-BI) is the only known HDL receptor which can mediate selective uptake of HDL-CE. In the present study we investigated its regulation in liver cells. The down-regulation of SR-BI expression in liver by 17alpha-ethinyl estradiol (EE) treatment was found by immunoblotting to be the consequence of down-regulation of SR-BI in parenchymal cells, while SR-BI expression in Kupffer cells was up-regulated. The selective uptake of HDL-CE in vivo by parenchymal and Kupffer cells was measured by labeling of HDL with [3H]CE and analysis of the cellular uptake at 10 min after injection. After EE treatment, uptake of [3H]CE-labeled HDL by parenchymal cells decreased by 85%, while Kupffer cells showed a 4-fold increase in selective uptake of [3H]CE-labeled HDL. In vitro studies with isolated parenchymal cells indicated that after EE treatment, the selective uptake of [3H]CE labeled HDL was 3-4-fold lower, indicating that the in vivo observations are also reflected in vitro. A 2-week high-cholesterol diet leads to lowering of SR-BI expression in parenchymal cells, while the expression in Kupffer cells is increased. Like EE treatment, the selective uptake of [3H]CE-labeled HDL by the two hepatic cell types in vivo correlated with the changes in expression of SR-BI. Our results thus demonstrate that within the liver, the regulation of SR-BI expression by EE treatment or a high-cholesterol diet, correlates with changes in the selective uptake of HDL-CE, supporting a function of SR-BI to mediate the selective uptake of HDL-CE in the liver parenchymal cells. The contrasting regulatory effect on parenchymal cells and Kupffer cells might indicate a different function of SR-BI in the latter cell type.

Scavenger receptor B1 (SR-B1) substrates inhibit the selective uptake of high-density-lipoprotein cholesteryl esters by rat parenchymal liver cells

High-density lipoprotein cholesteryl esters (HDL-CE) are selectively taken up by liver parenchymal cells without parallel apolipoprotein uptake, and this selective uptake route forms an important step in reverse cholesterol transport. Recent data from Acton, Rigotti, Landschulz, Xu, Hobbs and Krieger [(1996) Science 271, 518-520] provide evidence that scavenger receptor B (SR-B1) can mediate selective uptake of HDL-CE. In order to identify if selective uptake of HDL-CE by rat liver parenchymal cells can be mediated by a protein with scavenger receptor properties we performed competition experiments in vivo with substrates for scavenger receptors. Addition of either low-density lipoprotein (LDL), acetylated LDL (AcLDL) or oxidized LDL (OxLDL) only marginally (<10%) decreased the association of HDL particles to parenchymal cells as measured by 125I-labelled HDL. HDL-CE association was inhibited by AcLDL by 35%, while addition of OxLDL did inhibit HDL-CE association by 80%, thereby completely blocking the selective uptake of HDL-CE. Studies with HDL labelled with a fluorescent cholesteryl-ester analogue confirmed that OxLDL mediated complete inhibition of HDL-CE selective uptake by rat liver parenchymal cells. The inhibition of HDL-CE selective uptake by OxLDL was insensitive to the additional presence of polyinosinic acid (poly I), indicating that the inhibitory effect did not involve a poly I-sensitive site. Anionic phospholipid liposomes inhibited HDL-CE association by 40%, while neutral liposomes were ineffective. The inhibition of the selective uptake of HDL-CE in liver parenchymal cells by modified LDL, in particular OxLDL and anionic phospholipids suggests that, in liver, the SR-B1 is responsible for the efficient uptake of HDL-CE.

Selective uptake of high-density lipoprotein-associated cholesteryl esters and high-density lipoprotein particle uptake by human monocyte-macrophages

High-density lipoprotein (HDL) cholesteryl esters (CE) are taken up by many cells without parallel uptake of HDL apolipoproteins. This selective uptake of HDL CE was investigated in human monocyte-derived macrophages (HMM). HDL 3 ( d= 1.125-1.21 g/ml) was labeled in its apolipoprotein A-I moiety with 125I and in its CE moiety with [ 3H]cholesteryl oleyl ether. Cultured human monocyte-macrophages were incubated in the presence of doubly labeled HDL 3 followed by determination of tracer uptake. HMM took up HDL 3 particles as indicated by the uptake of HDL 3 apolipoproteins. Uptake of HDL 3-associated CE tracer was in significant excess of that due to HDL 3 particle uptake indicating selective uptake of CE. Increased cell cholesterol due to preincubation with acetylated low-density lipoprotein (LDL) down-regulated selective uptake by HMM. According to several experimental approaches, selective uptake of HDL 3 CE was independent from cell-secreted products, LDL receptor-mediated endocytosis or HDL 3 retroendocytosis. The intracellular catabolism of HDL 3 CE was investigated with HDL 3 labeled in its CE moiety with [ 3H]cholesteryl oleate. The lysosomal inhibitor chloroquine had no effect on CE hydrolysis indicating that CE selectively taken up is hydrolyzed independently from lysosomes. In conclusion, HMM selectively take up HDL 3-associated CE. The cellular mechanism of selective uptake is independent from endocytosis or retroendocytosis. Intracellularly, HDL 3 CE selectively taken up are catabolized independently from lysosomes.

Mechanism of the Cholesteryl Ester Transfer Protein-mediated Uptake of High Density Lipoprotein Cholesteryl Esters by Hep G2 Cells

Plasma cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters (CE) between lipoproteins and was reported to also directly mediate the uptake of high density lipoprotein (HDL) CE by human Hep G2 cells and fibroblasts. The present study investigates that uptake and its relationship to a pathway for "selective uptake" of HDL CE that does not require CETP. HDL3 labeled in both the CE and apoprotein moieties was incubated with Hep G2 cells. During 4-h incubations, CE tracer was selectively taken up from doubly labeled HDL3 in excess of apoA-I tracer, and added CETP did not modify that uptake. However, during 18-20-h incubations, CETP stimulated the uptake of CE tracer more than 4-fold without modifying the uptake of apoA-I tracer. This suggested that secreted products, perhaps lipoproteins, might be required for the CETP effect. Four inhibitors of lipoprotein uptake via low density lipoprotein (LDL) receptors (heparin, monensin, an antibody against the LDL receptor, and antibodies against the receptor binding domains of apoB and apoE) effectively blocked the CETP stimulation of CE tracer uptake. Heparin caused an increase in CE tracer in a d less than 1.063 g/ml fraction of the medium that more than accounted for the heparin blockade of CETP-stimulated CE uptake. CETP did not affect the uptake of doubly labeled HDL3 by human fibroblasts, even at twice plasma levels of activity, and heparin did not modify uptake of HDL3 tracers. Thus the CETP effect on Hep G2 cells can be accounted for by transfer of HDL CE to secreted lipoproteins which are then retaken up, and there is no evidence for a direct effect of CETP on cellular uptake of HDL CE.