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 (HepGZ). When HepGZ cells were incubated with [3H]cholesteryl ester-labeled HDLB in the presence of increasing concentrations of CETP there was a progressive increase in cell-associ- ated 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 associa- tion or degradation. Enhanced uptake of HDL cholesteryl ester was shown for the d > 1.21 g/ml fraction of human plasma, partially purified CETP, and CETP purified to homogeneity, but not for the d 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 chloro- quine. CETP enhanced uptake

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 (HepGZ). When HepGZ cells were incubated with [3H]cholesteryl ester-labeled HDLB 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 > 1.21 g/ml fraction of human plasma, partially purified CETP, and CETP purified to homogeneity, but not for the d 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 r3H]CE in cultured smooth muscle cells and to a lesser extent in fibroblasts but did not significantly influence uptake in endothelial cells or 5774 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.
In several species, including humans, plasma cholesteryl esters are synthesized within high density lipoproteins (HDL)' as a result of the activity of the enzyme lecithincholesterol acyltransferase (1). HDL-cholesteryl esters may be transferrred to less dense triglyceride-rich lipoproteins (chylomicrons and very low density lipoproteins) by a cholesteryl ester transfer protein (CETP) (2,3). Since the remnants of triglyceride-rich lipoproteins are taken up by specific receptors in the liver @), the CETP potentially provides a mechanism for the transfer of cholesteryl ester from plasma to liver. In contrast to this indirect route of catabolism, HDLcholesteryl esters are also thought to be taken up directly by certain tissues in a process which is selective for the cholesteryl esters. Thus, a dispropo~~onate uptake of HDL cholesteryl ester compared to apoprotein A-I has been shown in rat ovary, adrenal, and liver, and also in hepatocyte cultures (5, 6).
In the present investigation we have examined whether cholesteryl ester transfer protein can play a role in the direct transfer of HDL-CE into model liver cells, thereby providing an additional potential pathway by which CETP may influence HDL catabolism. A human liver tumor cell line (HepG2) served as a model for studying the effect of cholesteryl ester transfer protein on cellular HDL cholesteryl ester uptake.

EXPERIMENTAL PROCEDURES
~a € e r i a~ ~e l~-H u m a n hepatoma cell line (HepG2) cells were kindly provided by Drs. Knowles, Howe, and Aden of the Wistar Institute. The 5774 macrophage-like cell line was obtained from Jay Unkeless (Rockefeller University). Porcine aortic endothelial cells (sixth passage) and rabbit aortic smooth muscle cells (third passage) were kindly provided by Dr. Ken Pomerantz, Columbia University. Fibroblasts were from neonatal human foreskin (sixth-eighth passage). All cells were frozen in liquid nitrogen and thawed rapidly prior to use. For each experiment the cells were plated in 16 X 35-mm plastic Petri dishes in Dulbecco's modified Eagle's medium ( D~E M ) containing 10% heat-inactivated fetal bovine serum, 100 unitslml penicillin, 100 pg/ml streptomycin, 292 pg/ml L-glutamine. Plates were incubated at 37 "C in an atmosphere containing 8% Cop, 92% air.
HepG2 cells were routinely split 1:6 every 4-5 days. At the time experiments were performed the cells appeared confluent.

L i~p r o~i n s and Cholesteryl Ester Transfer
Prote~n-~uman HDL, (1.125-1.21 g/mlf containing radiolabeled cholesteryl esters was prepared as described previously (7). The HDL contained 99% of radioactivity in cholesteryl esters and 1% in cholesterol. Compositional analysis showed that the labeled HDL contained 55% protein, 20% phospholipid, 18% cholesteryl ester, 4% cholesterol, and 3% triglyceride and was therefore similar in composition to HDLB. Also, the radiolabeled preparation co-eluted with HDL, on a 100-cm column of 6% agarose, indicating a similar particle size. In some instances cold HDL, was added to the radiolabeled HDL to achieve the desired specific activity, 2660 cpm/pg of CE. Human HDLB was radiolabeled with lzSI by the iodine monochloride method of Mac-Farlane (8). Approximately 4% of the radiolabel was associated with purified about 500-fold from pooled blood-bank plasma through the lipids. Human plasma cholesteryl ester transfer protein was routinely carboxymethyl cellulose step (9). In selected experiments this CETP preparation was further purified to homogeneity by incubation with a synthetic lipid emulsion containing egg phosphatidylcholine, triolein, and oleic acid. The mixture was subjected to chromato~aphy on a Sepharose 4B column, and the active CETP was then obtained from the emulsion following delipidation with ethanol/ether. Based on activity, the homogeneous CETP was purified 55,000-fold relative to the plasma d > 1.21 g/ml fraction.' Human and rat lipoprotein-poor plasma fractions were isolated by preparative ultracentrifugation of human and rat plasma at d 1.21 g/ Hesler, C., Swenson, T. L., and  Dulbecco's modified Eagle's medium, penicillin (10,000 units/ml), streptomycin (10,000 pg/ml), glutamine (200 mM), and trypsin were from Gibco. Fetal bovine serum was obtained from MA Bioproducts. Bovine serum albumin (essentially fatty acid-free) and chloroquine were obtained from Sigma. NaIz5I (in NaOH) (508 mCi/ml), was purchased from ICN Biochemicals Inc. 3H-Labeled cholesterol (23.7 Ci/mmol) and 3H-labeled cholesteryl hexadecyl ether (46.8 Ci/mmol) were from New England Nuclear.

Methods
Cell Association Assays-At the time of the experiments the cells were incubated at 37 "C with 1 ml of DMEM containing 0.1% BSA (essentially fatty acid-free) and lz5I-HDL3 or [,H]CE-labeled HDL3. Incubations were performed with or without added cholesteryl ester transfer protein. Cholesteryl ester transfer protein was dialyzed against DMEM before being added to the incubation medium. At the end of the incubation the dishes were placed on ice and the medium was removed. The monolayers were then washed as described by Tabas and Tall (11): three rapid (< 1 min) washes in ice-cold 0.05 M Tris-HCL, pH 7.4, containing 0.15 M NaCl and 0.2% BSA (TBS-A), three 10-min washes in TBS-A, and finally three rapid washes in TBS. The monolayer was then dissolved in 1 ml of 0.1% sodium dodecyl sulfate, an aliquot removed for protein assay (Lowry method (12)), and the radioactivity associated with the cells determined. The average protein contents per dish were: HepG2 cells, 0.65 mg; 5774 macrophages, 0.8 mg; endothelial cells, 0.22 mg; smooth muscle cells, 0.27 mg; and fibroblasts, 0.26 mg. To ensure that cholesteryl ester transfer activity did not diminish during the incubation period, CE transfer activity was measured in the medium at the end of the 18-h incubations. The activity in medium of incubations performed in the presence of CETP was compared to that in medium from incubations performed without CETP. For all cell systems studied we demonstrated at least constant or slightly increased cholesteryl ester transfer activity in the medium throughout the incubation period. In experiments in which the amount of cell membrane-bound HDL-CE was determined, the cells were treated, at the end of the 18-h incubation, with trypsin at varying concentrations between 0.05 and 1.0% for 5 min. At these trypsin concentrations cell membranes remained intact, as demonstrated by a negative trypan blue stain at termination of the 5-min incubations.
To determine the amount of Iz5I-labeled HDL bound to the cell surface following the nine sequential washes (see above) monolayers were treated with 0.05% trypsin and incubated for 5 min at 37 "C. After 5 min 1 ml of DMEM with 10% fetal bovine serum was added to inhibit further proteolysis. The cell suspension was then centrifuged (1000 X g) for 10 min at 4 "C. The radioactivity in the supernatant was the trypsin-releasable value. The cell pellet was washed with TBS-A and respun at 1000 X g for 10 min. The supernatant was discarded and the pellet counted (trypsin-resistant value). To determine whether the Iz5I-HDL trypsin-resistant radioactivity represents cellular uptake of Iz5I-HDL by the cells, displacement experiments were carried out in which excess unlabeled HDL (1 mg of protein/ ml) was added to the cells at the end of the 18-h incubation. Cells were washed rapidly three times with TBS and incubated for 3 h at 37 "C with excess unlabeled HDL.
Degradation of '"I-HDL was determined in the medium, at the end of the 18-h incubations, after precipitation of protein with trichloroacetic acid (13).
Measurement of Radiolabeled Free Cholesterol and Cholesteryl Esters in Cells-Following incubations with [3H]CE-labeled HDL3 the medium was removed and the cells were washed as described above. After the last wash, cells were scraped with a rubber policeman, suspended in TBS, and spun at 1000 rpm for 10 min, and then the supernant was discarded. Cellular lipids were extracted by the Folch method (14), and the extracts were applied to thin layer chromatography plates and analyzed in a solvent system of hexane/ether/acetic acid (70:301). Free [3H]cholesterol and [3H]cholesteryl ester, identified using reference standards, were scraped off the plates, and their radioactivity was determined in a liquid scintillation counter.

T o assess the role of CETP in the uptake of HDL cholesteryl ester by HepG2 cells, cells were incubated in medium containing [3H]CE-labeled HDL in the
presence of increasing concentrations of partially purified CETP (Fig. 1A). With increasing CETP mass, there was a progressive increase in the cellular uptake of HDL-CE up to 2.8 times that of HDL-CE uptake without CETP ( Fig. l A , closed circles). To see whether CETP caused a similar increase in cellular uptake of HDL protein, we incubated lZ5I-HDL with HepG2 cells in the presence of increasing concentrations of CETP. Cell uptake of HDL protein was calculated as the amount of '"I-HDL that remained associated with the cells plus the amount of lZ5I-HDL degraded during the incubation period as calculated from the amount of trichloroacetic acid-soluble radioactivity in the medium, i.e. cell association and degradation. As shown in Fig. lA (open circles), the presence of CETP did not result in a significant enhancement of cellular HDL protein uptake.
In six different experiments similar responses were obtained: mean cellular HDL-CE uptake was 1.1 f 0.16% of the total added radioactivity without CETP and increased progressively to 2.9 f 0.36% in the presence of CETP (210 pg/ml). Uptake of HDL protein was 1.1 f 0.09% without CETP and 1.1% k 0.05% in the presence of CETP. Thus, in the absence of CETP, uptake of HDL protein and HDL-CE were in similar into Cells proportion to the original HDL. However, in the presence of CETP, there was a selective enhancement of cellular HDL cholesteryl ester uptake compared to HDL protein uptake. Further experiments were conducted to determine whether the stimulated uptake of HDL-CE was a specific property of CETP. CETP was purified to homogeneity as described under "Methods." The effect of CETP on cellular uptake of HDL-CE was also shown by the purified homogenous CETP with enhanced cellular HDL-CE uptake at increasing CETP concentrations (Fig. 1B). Based on specific activity (CE transferred/mg of protein in an HDL-LDL exchange assay) the homogenous CETP was purified approximately 350-fold compared to the partially purified CM52 preparation. The fact that a similar increase in specific activity was found for the cellular uptake of HDL-CE (cf. Fig. 1, A and B ) suggests that the effect of the partially purified fraction on cellular uptake of CE can be entirely accounted for by its content of CETP. This was confirmed in an experiment where immunoprecipitation of the partially purified fraction with CETP-specific IgG2 completely abolished its ability to stimulate cellular uptake of HDL-CE, whereas non-immune IgG did not.
To prove further that CETP activity was responsible for the selective uptake of HDL-CE, we took advantage of the fact that human d > 1.21 g/ml lipoprotein-poor plasma shows CE transfer activity, whereas rat plasma is devoid of such activity. Therefore, we compared the effect of human and rat lipoprotein-poor plasma on the cellular uptake of HDL cholesteryl ester (Fig. 2). Cellular [3H]CE-labeled HDL uptake increased as a function of increasing concentrations of human lipoprotein-poor plasma (closed circles). By contrast, the rat d > 1.21 g/ml plasma fraction did not enhance HDL-CE uptake by HepG2 cells (open circles).
To characterize further the effect of CETP on the uptake of HDL cholesteryl ester, we studied the time course of the cellular uptake of HDL-CE (Fig. 3). The effect of CETP on HDL-CE uptake was clearly apparent after 6 h of incubation and increased throughout the length of incubations. The effect of increasing HDL-CE mass on the CETP enhancement of cellular HDL-CE uptake was also examined. Incubations were performed with increasing concentrations of [3H]CE-labeled HDL but constant CETP concentrations (Fig. 4). With increasing HDL mass there was an increase in both the basal and CETP-stimulated uptake of HDL cholesteryl ester. However, the increment in CE uptake specifically due to CETP (i.e. the difference of the two curves in Fig. 4)   cating that, under these conditions, the CETP-specific uptake was saturated at relatively low concentrations of HDL.
Further experiments were conducted to see whether facilitated CE transfer could be observed at physiological levels of HDL and transfer activity. In the earlier experiments ( Fig.   2), it was noted that the effect of the human d > 1.21 fraction reached a maximum value at 7.2 mg of protein, at which point the ratio of HDL-CE/d > 1.21 protein approximates the physiological value. In a further experiment the concentrations of both HDL and d > 1.21 fraction were increased, using a fixed, physiological ratio of HDL/d > 1.21 fraction (Fig. 5).
There was a continuous increase in both the basal and facilitated transfer of HDL-CE into the cells, with the -fold increase mediated by the d > 1.21 fraction approximately constant. Thus, the enhanced transfer of HDL-CE was observed with physiological concentrations of HDL and d > 1.21 protein in the medium.
HepG2 cells have the capacity to synthesize and secrete apoB-containing lipoproteins (15). Therefore the observed effect of CETP on cellular HDL-CE uptake might actually occur through CETP-mediated transfer of CE from HDL to apoB-containing lipoproteins and subsequent cellular uptake of these lipoproteins. Sodium dodecyl sulfate-gel analysis of the apoproteins isolated from the d < 1.063 fraction of 4 ml of 24-h conditioned medium showed the presence of several micrograms of apoB-100; by contrast, the d 1.063-1.210 g/ml The protein concentration of d > 1.21 fraction used at each HDL3 concentration was such that the mass ratio of d > 1.21 fraction protein:HDL,-CE was 173. Both the [3H]HDL3 and d > 1.21 fractions were dialyzed extensively against DMEM before they were added to the cells. Cellular HDL3-CE uptake was expressed as counts/min per mg of cell protein X lo+. Results represent the mean of triplicate experiments +-S.E. fraction did not contain detectable apoB (not illustrated). We showed that, when HepG2 cells are incubated in medium containing both HDL and CETP, the CETP can mediate the transfer of cholesteryl ester from HDL to the d < 1.063 g/ml fraction, which contains the apoB-containing lipoproteins. In incubations performed without CETP, 0.7 k 0.05% of the total radiolabeled CE in the incubation (480,000 cpm/well) transferred from HDL to the d < 1.063 g/ml fraction and, in the presence of CETP, the transferred radiolabeled CE was increased to 1.8 & 0.03% of total radioactivity. In order to evaluate the possibility that the CETP-stimulated cellular uptake of HDL-CE occurs via the uptake of these apoBcontaining lipoproteins, an aliquot of the d < 1.063 g/ml fraction (containing 5,000 cpm) was isolated from the medium after an 18 h incubation in the presence of CETP and reincubated for 18 h with new HepG2 cells. There was no detectable cellular uptake of radioactivity (ie. less than 0.1% of incubated counts/min). Thus, although CETP did enhance transfer of CE to d < 1.063 g/ml lipoproteins in the medium, cellular uptake of CE present in d < 1.063 g/ml lipoproteins is unlikely to account for the CETP-stimulated uptake of HDL cholesteryl ester. T o evaluate further a possible role of the LDL receptor in the CETP-mediated uptake of HDL-CE, HepG2 cells were grown in medium containing LDL or lipoprotein-deficient serum for 24 h, the media was removed, and then the cells were incubated with HDL k CETP. LDL preincubation resulted in an 82% decrease in subsequent lZ5I-LDL degradation by the cells. For cells preincubated in lipoprotein-deficient serum the percent uptake of the HDL-CE was 1.1% (HDL) and 2.2% (HDL + CETP), whereas the corresponding values were 0.6 and 1.1% for cells preincubated with LDL. Thus, the preincubation with LDL decreased the absolute values of HDL-CE uptake but did not influence the

Cholesteryl ester transfer activity (countslmin transferred) in HepG2 medium
CE transfer activity was assayed in conditioned and unconditioned medium at the end of 18-h incubations performed in the presence of [3H]CE-labeled HDL with (+CETP) or without (-CETP) added CETP. CETP activity was assayed by adding exogenous LDL (0.1 mg of protein) to the medium, followed by incubation at 37 "C for 16 h, precipitation of apoB-containing lipoproteins with heparin/MnC12, then determination of radioactivity remaining in the supernatant. The value obtained by subtracting the measured radioactivity (counts/min) in the supernatant in each incubation condition studied from the radioactivity (counts/min) in the supernatant of unconditioned media without CETP constituted CE transfer activity. Unconditioned medium without added CETP had no transfer activity. Results are the mean of two parallel experiments and varied by less than 5%. -fold increase due to CETP. These results suggest that cellular cholesterol stores may influence HDL-CE uptake, but do not indicate a specific role for the LDL receptor in the CETPmediated component of uptake.
Recent experiments from this laboratory3 indicate that cholesteryl ester transfer activity accumulates in a timedependent fashion in the medium of cultured HepG2 cells, suggesting synthesis of CETP by these cells. To compare the amount of CE transfer activity added to cells with that secreted by the cells during an 18-h incubation, the CE transfer activity was measured in cell-conditioned or unconditioned medium with and without addition of exogenous CETP (Table I). The results show 20-30% higher activity for the cell-conditioned medium with or without added CETP, indicating accumulation of a small amount of endogenous CE transfer activity in the medium. It is possible that this endogenous CE transfer activity contributes to the uptake of HDL cholesteryl ester; however, it is a relatively small amount of activity compared to that added with exogenous CETP.
Further experiments were performed to elucidate the mechanism of cellular HDL-CE uptake. As noted above, CETP had no effect on total cellular uptake of lz5J-HDL. Furthermore, CETP was also found to have no effect on lZ5I-HDL cell association (0.13% of total added radioactivity in the presence of CETP as compared to 0.12% without CETP) or on the fraction of the cell-associated radioactivity that was displaceable by cold HDL (40.8% in the presence of CETP and 40.3% in control experiments without CETP). Of the cell-associated protein radioactivity a major fraction (about 55%) was trypsin-releasable. By contrast, in another experiment it was found that only 6-9% of CETP-stimulated [3H] cholesteryl ester uptake was trypsin-releasable, indicating internalization of HDL-CE.
Since the major fraction of cell-associated HDL-CE is internalized by the cell, the intracellular fate of cholesteryl ester was studied. Following cellular uptake cell lipids were extracted and separated by thin layer chromatography. In the presence of CETP the amount of cell-associated radioactivity of both free cholesterol and CE was augmented 2-3-fold. Since more than 99% of the label in HDL was in cholesteryl ester and since CETP does not promote transfer of unesterified cholesterol, these results suggest hydrolysis of CE following entry into the cell. This was confirmed by the addition of 25 FM chloroquine, which inhibited the degradation of HDL cholesteryl ester to free cholesterol (Fig. 6). These results indicate that HDL cholesteryl esters entering the cell under  (Table 11). Despite the presence of an acyl-CoA:cholesterol acyltransferase inhibitor, cellular cholesteryl ester radioactivity was markedly increased. Therefore, the accumulating cholesteryl ester was directly derived from HDL and had not been derived from reesterification of [3H]cholesterol by acyl-CoAcholesterol acyltransferase.
To verify further that CETP-mediated enhancement of HDL-CE uptake was indeed an effect on neutral lipid transfer, we studied HDL cholesteryl ether uptake by HepG2 cells under the influence of CETP (cholesteryl ethers are not susceptible to degradation by hydrolases, and are taken up by cells as intact molecules). HepGZ cells were incubated in DMEM, 0.1% BSA containing [3H]CE-labeled HDL alone or in the presence of CETP. Following an 18-h incubation cell-associated [3H]cholesteryl ether was 1.40-fold higher in the presence of CETP as compared to control. In a simultaneous experiment the cell-associated [3H]cholesteryl ester increased 1.49-fold, at 18 h, in the presence of CETP. Thus, CETP caused stimulation of both cholesteryl ether and cholesteryl ester uptake. The relatively small effect observed was due to the lower specific activity of the partially purified CETP preparation used in this experiment.
In order to determine whether CETP enhancement of HDL cholesteryl ester uptake is specific for HepG2 cells we evaluated the effect of CETP on HDL-CE uptake by other cell types (Fig. 7). CETP enhanced uptake of HDL-CE by smooth muscle cells; HDL-CE uptake increased to levels that were 2.6 times the control. CETP also enhanced HDL-CE uptake by fibroblasts but to a lesser extent than in smooth muscle cells. In endothelial cells and 5774 macrophages there was no significant change in cellular HDL-CE uptake with increasing CETP concentrations, indicating that CETP does not facilitate uptake of HDL-CE in these cell types.

DISCUSSION
The human plasma CETP has been well documented to facilitate both the exchange and net transfer of CE and triglyceride between plasma lipoproteins. Stein et al. (16) have recently shown that CETP can remove cholesteryl esters from lipoproteins bound to cell surface locations and can also remove CE from intracellular locations but only following treatment that permeabilizes the cells. We present evidence for a function of CETP that has not been previously described.
Our observations indicate that CETP can promote the transfer of cholesteryl ester from HDL into intact HepGZ and smooth muscle cells. The radiolabeled cholesteryl esters are internalized and undergo lysosomal degradation.
Based on the kinetics of lipid exchange between the lipoproteins, two models for CETP-dependent CE transfer have been suggested. A ping-pong model proposes that CETP acts as a carrier of CE between donor and acceptor lipoproteins (17). Alternatively, CETP may enhance the exchange of lipids during formation of a ternary collision complex consisting of donor and acceptor lipoprotein and CETP (18). Either model could potentially explain the effect of CETP on cellular HDL-CE uptake. However, the failure of CETP to promote cell association or degradation of HDL protein suggests that CETP is not acting by enhancing binding or fusion of HDL with the cell surface. The saturation of CETP-dependent CE uptake by increasing donor (HDL) concentration is typical of the kinetics of carrier-mediated lipid transfer (17). If the transfer into the cell is carrier-mediated, then the findings of the present study imply the existence of a cell surface binding site that can be recognized by CETP. CETP binds readily to phospholipid surfaces in emulsion, lipoproteins, and vesicles, especially in the presence of an increased negative charge (7, 19,20), suggesting that CETP might also bind to the lipids of the plasma membrane. Another possibility is that CETP binds to a cell surface receptor. However, a specific role of the LDL receptor in mediating the CETP-dependent uptake seems unlikely, since down-regulation of the LDL receptor did not alter the -fold stimulation of uptake due to CETP. An analogy to the present results is suggested by the previous descriptions of lipoprotein lipase-enhanced cellular uptake of CE from liposomes or lipoproteins (21). Both the lipase and CETP molecules may have binding sites for neutral lipid which allow them to act as carriers of CE. CETP increased the uptake of HDL-CE but did not affect the cell association or degradation of HDL protein. Thus, CETP does not enhance binding or internalization of whole HDL particles. These findings are reminiscent of previous studies in which it had been shown that, in selected tissues, there is a disproportionate uptake of HDL-CE compared to HDL protein (5,6,22). HDL-CE uptake into rat adrenal cells was not changed during metabolic inhibition of sucrose pinocytotic processes, implying that the disproportionate uptake of HDL-CE in excess of HDL apoA-I probably does not involve whole particle uptake through receptor-mediated endocytosis (23). Although these studies have mostly been performed in the rat, which lacks cholesteryl ester transfer activity, selective uptake of HDL-CE has also been shown in perfused rabbit liver ( 2 2 ) , which in other studies has been shown to accumu1at.e cholesteryl ester transfer activity (24).
The CETP promotes neutral lipid transfer between lipoproteins by facilitating a cholesteryl ester-triglyceride heteroexchange process (25). The phenomenon of CETP-dependent cellular uptake of HDL-CE might also involve lipid exchange. Because the amount of CE taken up by HepG2 cells is in the range of 1.5-1.6 ,ug of CE/mg of cell protein, it was not possible to document net mass changes. Currently, there is a paucity of data to show that, upon entry into cells, HDL-derived CE affects cellular metabolism. However, we have shown that internalized HDL-CE undergoes hydrolysis via a lysosomal pathway. The observed increase in cellular free cholesterol suggests that HDL-derived CE has the potential to influence cellular cholesterol metabolism. HDL-CE uptake does promote cellular prostanoid synthesis, in part by transfer of cholesteryl arachidonate from HDL to cellular lipid pools containing arachidonate (10). The CETP has been found to increase markedly the HDL-induced prost,anoid release by smooth muscle cells partly as a result of increased incorporation of HDL-CE-derived arachidonate into prostanoids.* These experiments clearly show that fatty acid derived from CETP-induced CE entry can influence cellular metabolism and strongly imply that the facilitated entry of HDL-CE into cells is not a simple lipid exchange process.

data.
HDL-CE is the cellular specificity. We have observed CETP enhancement of HDL-CE uptake in HepG2 cells, a human tumor cell line, in rabbit smooth muscle cells, and to a lesser extent in human fibroblasts. No effect of CETP on HDL-CE uptake was noted in 5774 macrophages or porcine endothelial cells. Specificity is not related to HDL binding, as endothelial cells have been shown to bind IWI-HDL3 to a higher degree than either smooth muscle cells or fibroblasts (11) and, similarly, we have also observed that, in the absence of CETP, HDL-CE cell association per mg of cell protein was highest for endothelial cells. CETP augmentation of HDL-CE uptake could be related to cell surface characteristics that enable the cell membrane to bind CETP or to the existence of intracellular lipid pools that provide lipid for CETP-mediated exchange processes. The physiological significance of the CETP-dependent uptake of HDL-CE is unknown. As these experiments were conducted in a model cell system, definite conclusions relating to in vivo conditions cannot be made. However, the CETP did enhance cellular HDL-CE uptake at a physiological ratio of HDL-CE to human d > 1.21 g/ml fraction (Fig. 2) and at physiological concentrations of HDL and d > 1.21 fraction (Fig. 5). Since the d > 1.21 fraction shows similar CE transfer activity to whole p l a~m a ,~ this result indicates that the effect was observed at physiological levels of CE transfer activity and HDL. In those species that possess plasma cholesteryl ester transfer activity, the CETP-mediated selective uptake of HDL-CE might constitute a pathway for direct incorporation of HDL-CE, exclusive of other HDL components, into the liver or other tissues.