Cholesteryl Ester Transfer Protein Directly Mediates Selective Uptake of High Density Lipoprotein Cholesteryl Esters by the Liver
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动脉硬化血栓血管生物学 2005年第10期
From Lipoprotein and Atherosclerosis Research Group & Division of Cardiology, University of Ottawa Heart Institute, Ottawa, Canada.
Address correspondence to Ruth McPherson, University of Ottawa Heart Institute, H453, 40 Ruskin St, Ottawa, ON Canada K1Y 4W7. E-mail rmcpherson@ottawaheart.ca
Abstract
Objective— To determine whether cholesteryl ester transfer protein (CETP) directly mediates selective uptake of high-density lipoprotein (HDL)-cholesteryl ester (CE) by hepatocytes and to quantify the effects of the CETP inhibitor, torcetrapib, on this process.
Methods and Results— Using adenovirus-mediated CETP (ad-CETP) expression in primary mouse hepatocytes from either wild-type, low-density lipoprotein (LDL) receptor–/– or SR-BI–/– mice, we demonstrate that CETP enhances the selective accumulation of HDL-derived 3H-CE independently of known lipoprotein receptors. Addition of torcetrapib to the media did not impair the ability of cell-associated CETP to enhance CE uptake but reduced the ability of exogenously added CETP to increase selective uptake by up to 80%. When mice were infected with ad-CETP or ad-Luciferase and treated with daily intravenous injections of torcetrapib or vehicle, hepatic CETP expression resulted in a 50% decrease in HDL cholesterol in vehicle-treated animals versus a 33% decrease in HDL cholesterol in mice treated with torcetrapib.
Conclusions— CETP mediates selective uptake of HDL-CE by hepatocytes by both torcetrapib-sensitive (exogenous CETP) and torcetrapib-insensitive (cell-associated CETP) mechanisms. Hepatic expression of CETP in vivo results in a marked decrease in cholesterol in particles in the HDL density range, consistent with a physiological role for hepatocyte CETP in selective uptake.
These studies demonstrate that cholesteryl ester transfer protein (CETP) mediates the selective uptake of IIDL-derived cholesteryl esters by hepatocytes both in vitro and in vivo by a mechanism that is independent of other lipoprotein receptors and which is only partially sensitive to inhibition by torcetrapib.
Key Words: CETP ? selective uptake ? torcetrapib ? lipoproteins ? HDL
Introduction
Cholesteryl ester transfer protein (CETP) is a hydrophobic glycoprotein that plays a central role in human high-density lipoprotein (HDL) metabolism. CETP mRNA is predominantly expressed in the liver, spleen, and adipose tissue in humans and is secreted to a variable extent from each of these tissues into plasma, where it has an established role in mediating neutral lipid transport between lipoproteins.1 The overall role of CETP in atherosclerosis is unclear. CETP functions in the regulation of cholesteryl ester (CE) trafficking in plasma. Cholesterol extracted by efflux from peripheral tissues is esterified within HDL by lecithin:cholesterol acyltransferase (LCAT). This CE can be subsequently transferred by CETP to apolipoprotein B (apoB)-containing lipoproteins.2 Under conditions of efficient hepatic apoB lipoprotein clearance, CETP may promote cholesterol transport from HDL to the liver. However, high plasma concentrations of CETP are associated with low HDL cholesterol and this observation has led to the development of CETP inhibitors as a potential therapy to reduce atherosclerosis.3,4
HDL-CE is also directly returned to the liver by a process known as selective uptake. This involves the reversible incorporation of HDL-derived CE into a plasma membrane pool followed by transfer of the lipid to an inaccessible pool by mechanisms not involving coated pit-mediated endocytosis.5 SR-BI has been shown to be the primary receptor responsible for hepatic HDL-cholesterol clearance in the mouse,6,7 a species that intrinsically lacks CETP.8 The human homologue of SR-BI, CLA-1, was cloned several years ago9 but has not yet been established as the primary receptor for HDL-CE selective uptake in humans. Genetic CETP deficiency in humans results in a marked increase in plasma concentrations of HDL-CE,10 suggesting a limited role for CLA-1 or a requirement for CETP in this process. Our laboratory has demonstrated a novel role for CETP in mediating the selective acquisition of CE from HDL by human adipocytes11 by a very high-efficiency transport process with a Tmax of 68 ng/HDL protein/mg cell protein/hour and km of 1.3 μg/mL HDL3.12
These findings led us to determine whether CETP also plays a direct role in selective uptake of HDL-CE by the liver, an important final step in reverse cholesterol transport, and to further investigate the mechanisms by which CETP functions to promote selective uptake. CETP was previously ascribed a role in the remodeling of CE-rich HDL, making it an optimal ligand for selective uptake by SR-BI/CLA-1.13 Although Granot et al reported that CETP mediates the selective uptake of HDL-CE by HepG2 cells,14 these observations were later attributed to the established function of CETP in mediating the transfer of HDL-CE to newly secreted apo B lipoproteins, followed by their internalization by the low-density lipoprotein (LDL) receptor (LDLr).15
Importantly, we show here that CETP directly mediates selective uptake of HDL-derived CE by hepatocytes, a cell of fundamental importance in HDL metabolism and that this effect occurs independently of other established lipoprotein receptors. We also demonstrate that hepatic expression of CETP in vivo results in a marked decrease in cholesterol in particles in the HDL density range consistent with a physiological role for hepatocyte CETP in selective uptake. We have explored the effects of the CETP inhibitor, torcetrapib, on this process and demonstrate that the effects of CETP in promoting hepatocyte selective uptake are partially impeded by this agent. These studies provide new and important insights into the mechanisms of CETP-mediated selective uptake and the role of CETP in cholesterol trafficking to the liver.
Methods
Materials
All common reagents of analytical grade were from Fisher (Fair Lawn, NJ) or Sigma (St Louis, Mo). Recombinant CETP (rCETP) was purchased from Cardiovascular Targets, Inc (New York, NY). Torcetrapib (CP-529,414) was kindly provided by Pfizer Inc (Groton, Conn).
Animals and Primary Hepatocyte Cultures
Retired breeders of the C57Bl6J (wild-type) mouse strain were from Charles River (Wilmington, Mass). Mice do not express CETP, making them an ideal model for these studies. As indicated, certain experiments were performed using primary hepatocytes from either SR-B1 or LDLr-deficient mice. SR-B1–deficient (strain B6.129S2-Scarb1tm1Kri)) and LDLr-deficient mice (strain B6.129S7-Ldlrtm1Her) were from Jackson Laboratory (Bar Harbor, Me). Mice were maintained on a normal chow diet. Primary hepatocytes were prepared from mice according to established protocols.16,17 Six hours after the initial plating, the cells were washed in William’s medium without fetal bovine serum and fresh media. For experiments using adenovirus-mediated expression of CETP, CETP adenovirus (ad-CETP) or luciferase adenovirus (ad-Luc) (control) was added to the wells at a concentration of 2.175x107 plaque forming units (PFU)/well (75 multiplicity of infection). The infected cells were left to express the transgene for 36 to 48 hours before the assay. Otherwise, cells were used 12 to 36 hours after initial wash.
Generation of CETP Adenovirus
Human CETP, in the pCMV5 mammalian expression vector, was subcloned into the pShuttle-CMV vector using standard molecular biology techniques; this vector was used for subsequent generation of the adenovirus DNA construct via homologous recombination with the pAd-Easy1 adenovirus DNA vector. Adenovirus was then generated, scaled-up, purified, and measured according to the manufacturer’s instructions (Qbiogene).
Lipoprotein Purification and Labeling
Total HDL was purified from normolipemic plasma from a single donor by density gradient ultracentrifugation,18 followed by dialysis 3 times against 4 L of nitrogen-sparged phosphate-buffered saline, pH 7.4, with 2 grams of Chelex (BioRad). The lipoproteins were labeled with 3H-cholesteryl oleate using a modified protocol as described by Reaven.19 Human apolipoprotein (apo) A-1 was labeled with 125I as described.20 3H-CE HDL was then incubated with 125I-labeled apoA-1 or cold apoA-1 for 24 hours at 37°C before re-isolation to obtain an HDL particle-labeled in the core with 3H-CE and on the protein moiety with either cold or 125I-apoA-1. The charge and size of the labeled HDL particles were verified by Lipogel analysis.
Selective Uptake Assay
Experiments were performed essentially as outlined in detail previously.20 Concentrations are stated in the Figures and legends, and the cells were incubated at 37°C for 8 hours, unless otherwise indicated. The cell associations of 3H-CE or 125I-apoA-1 or degradation of 125I-apoA-1 are measured in units of the amount of these labels contained in 1 ng (protein content) of HDL and corrected for mg of cell protein in each well. Measured this way, an equivalent amount of 3H-CE and 125I-apoA-1 labels represents holoparticle uptake and any additional 3H-CE cell association is attributed to selective uptake.
In Vivo Experiments
Experiments were performed in 6- to 8-week-old C57Bl6J (wild-type) mice (Charles River). In initial experiments, mice were subjected to tail vein injection of either ad-Luc or ad-CETP (1.25x108 PFU in 100 μL). Fasting plasma cholesterol was measured at baseline, 3, 6, and 10 days after injection. Animals were euthanized at 10 days and primary hepatocytes isolated for determination of CETP protein expression and 3H-CE-HDL selective uptake assays. In subsequent experiments, mice were injected with 2.5x108 PFU in 100 μL of ad-Luc or ad-CETP on day 0 and then received daily tail vein injections of 100 μL of Intralipid containing either vehicle (DMSO) or torcetrapib (0.125 mmol/L; such that the final concentration in the mouse, assuming a 3-mL total blood volume, would be 4 μmol/L). Fasting plasma cholesterol was measured and plasma CETP expression assessed by Western blot at baseline and days 2, 4, 5, and 7 after injection. On day 7, animals were exsanguinated by cardiac puncture and sera stored at 4°C before discontinuous density gradient ultracentrifugation (18 hours at 45 000 rpm).
Statistical Analysis
Results are expressed as the mean±standard error of the mean. Where indicated, the statistical significance was determined using the unpaired t test and expressed as a 2-tailed P value.
Note that further methodological details for all procedures are provided in supplementary information online (see http://atvb.ahajournals.org).
Results
Infection of Mouse Primary Hepatocytes With ad-CETP Promotes Selective Uptake of HDL-Derived CE
Western blot analysis of wild-type murine hepatocytes infected in culture with adenovirus-CETP (ad-CETP) or adenovirus-luciferase (ad-Luc) control is shown in Figure 1A, indicating CETP protein expression in cell lysates from CETP-transfected hepatocytes in contrast to control. For these experiments, to specifically address the effects of cell associated CETP as opposed to CETP secreted into the media, we used a low viral titer of ad-CETP. Under these conditions, hepatocyte secretion of CETP was negligible as demonstrated by absence of CETP in the media by Western blot after immunoprecipitation with TP2 anti-CETP monoclonal antibody (mAb) at the end of the 8-hour time course and no detectable transfer of 3H-CE from HDL to LDL in an in vitro transfer assay (data not shown). As expected in luciferase-transfected hepatocytes, 3H-CE cellular incorporation was several-fold greater than that of 125I-apo A-I, consistent with SR-BI–mediated selective uptake of HDL-CE by primary hepatocytes (Figure 1B). However, even in the absence of detectable CE transfer activity in the media, adenovirus-mediated expression of CETP in mouse primary hepatocytes resulted in a 50% increase in 3H-CE cell association as compared with ad-Luc control. These studies demonstrate that cell associated CETP can directly mediate the selective acquisition of HDL-derived CE by hepatocytes. This effect began to saturate at 50 μg/mL of HDL and therefore further experiments shown used this concentration of HDL. The cell-association of 125I-apoA-1 or the cell-mediated degradation of 125I-apoA-1 was unchanged with ad-CETP expression compared with ad-Luc control cells, indicating that although CETP increases CE uptake, it does not alter HDL particle uptake.
Figure 1. CETP mediates the selective uptake of HDL-derived CE in isolated mouse hepatocytes. A, Western blot of wild-type hepatocytes transfected with ad-CETP versus ad-Luc, showing cell lysate expression of CETP in comparison to human plasma. At this level of expression, no CETP was detectable in the media by Western blot (immunoprecipitation) or by CE transfer assay. B, Effects of endogenously expressed CETP on selective uptake. Hepatocytes were incubated at 37°C for 8 hours with increasing concentrations of 3H-CE HDL or 125I-apoA-1 HDL. Cellular incorporation of 3H-CE in cells infected with ad-CETP (black triangles) was increased 2-fold compared with cells infected with ad-Luc (white triangles). No differences were noted in 125I-apoA-I cell incorporation with ad-CETP (black circles) or ad-Luc (white circles) or in 125I-apoA-I degradation with ad-CETP (black squares) versus ad-Luc (white squares). *2-tailed P<0.007 compared with luciferase at all HDL concentrations. C, Effects of exogenously added recombinant CETP (rCETP) on selective uptake. Hepatocytes were incubated at 37°C for 8 hours with 3H-CE HDL or 125I-apoA-1 HDL (50 μg/mL), with or without the addition of CETP (0.48 μg/mL). Exogenous CETP increased 3H-CE incorporation into mouse hepatocytes by 6-fold but did not alter 125I-apoA-I cell association or degradation. *2 tailed P<0.0001.
Effects of Exogenous CETP on Selective Uptake by Murine Hepatocytes
In other experiments, we determined the ability of exogenously added recombinant CETP (rCETP) to mediate the selective uptake of HDL-derived CE. As shown in Figure 1C, rCETP, which was added to the media at a concentration of 0.48 μg/mL, increased the selective uptake of HDL-derived CE by 5-fold.
Effects of Endogenously Expressed, Cell-Associated CETP on Selective Uptake
To determine the effects of low-level in vivo hepatocyte CETP expression on selective uptake, mice were injected with 1.25x108 PFU of either ad-Luc or ad-CETP (n=4 for each) and euthanized on day10. There was no detectable CETP activity in the plasma of the ad-CETP–treated mice indicating no significant hepatocyte secretion of CETP at this level of ad-CETP infection. Plasma total cholesterol was significantly lower in mice infected with ad-CETP (P=0.01) (Figure 2A) because of a selective decrease in cholesterol in particles in the HDL density range (data not shown). In primary hepatocytes isolated from ad-CETP infected mice, CETP was detectable by Western blot of the cell lysate (Figure 2B) and 3H-CE-HDL selective uptake was increased by 20% (P=0.01) as compared with hepatocytes from ad-Luc–infected mice (Figure 2C).
Figure 2. Effects of endogenously expressed, cell-associated CETP on selective uptake. Mice were injected with 1.25x108 PFU of either ad-luciferase or ad-CETP (n=4 for each) and euthanized on day 10. A, Plasma total cholesterol was significantly lower in mice infected with ad-CETP; *2-tailed P=0.01. B, Western blot of the cell lysate from isolated hepatocytes from ad-CETP infected mice indicating CETP expression. C, Effects of endogenously expressed CETP on selective uptake. Primary hepatocytes isolated from mice 10 days after ad-Luc or ad-CETP infection were incubated at 37°C for 8 hours with 3H-CE HDL or 125I-apoA-1 HDL (50 μg/mL). Cellular incorporation of 3H-CE in hepatocytes from ad-CETP infected mice (black triangles) was increased 20% as compared with hepatocytes from ad-Luc infected mice (white triangles). **2-tailed P=0.01. No differences were noted in 125I-apoA-I cell incorporation with ad-CETP (black circles) or ad-Luc (white circles) or in 125I-apoA-I degradation with ad-CETP (solid squares) vs ad-Luc (open squares).
Effects of the CETP Inhibitor, Torcetrapib, on Selective Uptake Mediated by Exogenous Versus Cell-Associated CETP
When primary mouse hepatocytes were infected with a low viral titer of ad-CETP, CETP was detectable in cell lysates but was not in the medium. As shown in Figure 3A, under these conditions, addition of increasing concentrations of the CETP inhibitor, torcetrapib, to the media did not impair the ability of endogenously expressed cell-associated CETP to enhance selective uptake. In contrast, when rCETP(0.48 μg/mL) was added to the media, an 8-fold increase in 3H-CE incorporation into mouse hepatocytes was demonstrated (Figure 3B). Under these conditions, incubation of cells with increasing concentrations of torcetrapib reduced the ability of exogenously added CETP to enhance 3H-CE uptake by 80%. However, torcetrapib did not completely prevent selective uptake mediated by exogenously added CETP. Even in the presence of 25 μmol/L torcetrapib, exogenous CETP at a concentration of 0.48 μg/mL increased 3H-CE uptake by 2-fold (Figure 3B). In additional experiments, identical results were obtained when rCETP, 3H-CE HDL, and torcetrapib were pre-incubated for 2 hours before being added to the primary hepatocytes (data not shown). Thus, when added to cell culture media, CETP appears to increase selective uptake of HDL-CE by hepatocytes by 2 distinct pathways: one representing a process that is inhibitable by torcetrapib and another by a mechanism that is not accessible to inhibition by torcetrapib. In other studies, we incubated 0.48 μg/mL rCETP with primary murine hepatocytes and determined that 20% becomes cell associated after 2 hours at 37°C and this may represent the torcetrapib-insensitive fraction.
Figure 3. Effects of torcetrapib on selective uptake of HDL-CE mediated by cell associated or exogenously added CETP. A, In hepatocytes transfected with a low viral titer of ad-CETP, CETP was detectable by Western blot in cell lysates but was not in the medium (data not shown). Ad-CETP or ad-Luc transfected hepatocytes were incubated at 37°C for 8 hours with 3H-CE HDL or 125I-apoA-1 HDL (50 μg/mL) with or without the addition of 5 to 500 nmol of torcetrapib to the media. *2-tailed P<0.05 compared with control. B, Hepatocytes were incubated at 37°C for 8 hours with 3H-CE HDL or 125I-apoA-1 HDL (50 μg/mL), with or without the addition of CETP (0.48 μg/mL) and 0 to 25 μmol/L torcetrapib. The 2-tailed P<0.04 for all rCETP values compared with control.
Effects of Torcetrapib on Reversible and Irreversible Components of Selective Uptake Mediated by Exogenous Versus Cell-Associated CETP
To further investigate the mechanism by which exogenous and cell-associated CETP mediate selective uptake, we determined the reversible and irreversible phases of CE-uptake at 2 and 4 hours. As shown in Figure 4A, when primary hepatocytes were incubated with 0.48 μg/mL of rCETP, entry of 3H-CE into a reversible compartment was completely inhibited by torcetrapib, whereas accumulation of 3H-CE in the irreversible compartment was partially but not completely inhibited by torcetrapib (Figure 4B). In contrast, when this experiment was performed using hepatocytes infected with a low level of ad-CETP, to achieve CETP expression in the cell without detectable CETP secretion into the media, there was no effect of torcetrapib on entry of 3H-CE into either the reversible or irreversible compartments (Figure 4C), further demonstrating that selective uptake mediated by cell-associated CETP is not susceptible to inhibition by torcetrapib.
Figure 4. Effects of torcetrapib on reversible and irreversible components of selective uptake mediated by exogenous versus cell-associated CETP. A and B, Hepatocytes were preincubated with 50 μg/mL 3H-CE-HDL for the indicated times and then incubated for 4 hours with 400 μg/mL cold HDL. Cells were treated with either vehicle (DMSO) or torcetrapib (1 μmol/L). Removal of 3H-CE label from the reversible phase is represented by the media counts in (A). The irreversible phase was determined by measuring the cell-associated counts after 4 washes with Hank balanced salt solution and is represented by the cell counts in (B). C, The reversible and irreversible components of CETP selective uptake in mouse hepatocytes infected with a low level of ad-CETP to achieve cellular CETP expression without detectable CETP secretion into the media. Cells were treated as mentioned. #P=0.007; *P<0.0001; **P=0.005.
CETP-Mediated Selective Uptake Is Independent of Other Classical Pathways
To determine whether the portion of the CETP-mediated selective uptake pathway, which is sensitive to the CETP inhibitor, consists of CE transferred to apo B lipoproteins, a process known to be inhibited by torcetrapib,21 we performed CETP-mediated selective uptake in hepatocytes from LDLr-deficient mice. Importantly, absence of the LDLr did not attenuate the ability of CETP to mediate selective uptake and once again 3H-CE uptake was only partially inhibited by torcetrapib (Figure 5A). Thus, the torcetrapib inhibitable component of selective uptake does not require transfer of HDL-CE to apo B lipoproteins and subsequent clearance by the LDLr.
Figure 5. Role of lipoprotein receptors in the CETP-mediated cellular incorporation of 3H-CE from HDL. 3H-CE HDL (50 μg/mL) with or without the addition of CETP (0.48 μg/mL) and 0 to 5 μmol/L torcetrapib was incubated with hepatocytes prepared from LDLr null mice (A), SR-BI–/– mice (B) or from wild-type mice and incubated with receptor-associated protein (30 μg/mL) (C) or from wild-type mice and incubated with heparin (100 U/mL) (D). The 2-tailed P<0.05 for all CETP samples compared with control.
Additional studies were performed in hepatocytes from SR-B1–deficient mice. As expected, the overall levels of HDL-CE uptake were lower in hepatocytes from mice lacking SR-B1. However, exogenous CETP (0.48 μg/mL) enhanced hepatocyte incorporation of 3H-CE to a similar extent irrespective of SR-BI expression. Thus, the effect of CETP on hepatocyte selective uptake is not contingent on the presence of SR-BI (Figure 5B).
To exclude a role for other members of the LDLr gene family, we incubated hepatocytes with and without receptor-associated protein to block the members of the LDLr family through competitive binding. Incubation with receptor-associated protein (RAP) did not affect CETP-mediated selective uptake and both torcetrapib-sensitive and insensitive pathways were again observed (Figure 5C).
Finally, studies were performed in the presence of heparin, which disrupts binding of hepatic lipase to the heparin sulfate proteoglycan matrix.22 Heparin treatment did not reduce the ability of exogenous CETP to enhance hepatocyte accumulation of 3H-CE or alter the torcetrapib-sensitive or insensitive components of CETP-mediated selective uptake, ruling out a requirement for hepatic lipase in this process (Figure 5D).
CETP Mediates Hepatocyte Selective Uptake In Vivo
These studies clearly demonstrated that CETP mediates selective uptake of HDL-CE by primary hepatocytes in culture by a mechanism that does not require transfer of HDL-derived CETP to apoB lipoproteins or participation of SR-BI. To confirm the functional importance of CETP in hepatocyte selective uptake of HDL-CE in vivo, we determined changes in plasma total cholesterol and lipoprotein subfractions 7 days after tail vein injection of ad-CETP or ad-Luc. As shown in Figure 6A, hepatic expression of CETP at levels sufficient to elicit CETP secretion into plasma, in a species lacking endogenous CETP, reduced plasma total cholesterol by 50% relative to luciferase control. Importantly, this decrease was entirely because of a decrease in cholesterol in particles in the HDL density range (Figure 6B), consistent with an acute effect of hepatic CETP expression on selective uptake in vivo. Because pharmacological inhibitors of CETP activity increase HDL cholesterol and are likely to gain broad clinical use, studies were also performed to determine the effects of concomitant administration of the CETP inhibitor, torcetrapib, on CETP-mediated selective uptake in vivo. Torcetrapib is poorly absorbed by the oral route in rodents and thus mice received daily tail vein injections of vehicle (DMSO) or torcetrapib (0.125 mmol/L) in 100 L Intralipid. Assuming a 3-mL total blood volume, the final plasma concentration in the mouse would be 4 μmol/L. Consistent with the results of the ex vivo experiments, torcetrapib was found to partially attenuate the ability of adenovirus driven hepatocyte expressed CETP to enhance selective uptake as indicated by decreases in cholesterol in lipoprotein particles of the HDL density range (Figure 6A and 6B). Overall, ad-CETP expression acutely reduced total cholesterol (mainly in particles in the HDL density range) by 50% in mice not treated with torcetrapib and by 33% in animals subjected to concomitant torcetrapib treatment.
Figure 6. Effects of hepatic expression of CETP on selective uptake in vivo. Mice injected via tail vein with 2.5x108 PFU adenovirus and treated with daily intravenous injections of Intralipid-containing vehicle (DMSO) or torcetrapib for 7 days. A, Fasting plasma total cholesterol from day 7 after injection. Data are presented as a % of the day 0 total plasma cholesterol measurements. *2-tailed P<0.001 compared with luciferase controls; ** 2-tailed P=0.02 comparing vehicle and torcetrapib treated ad-CETP mice. B, Total cholesterol in lipoprotein fractions separated by discontinuous density gradient ultracentrifugation. The 2-tailed P values for ad-CETP mice compared with ad-Luc mice at the peak corresponding to HDL are <0.0001. The 2-tailed P value comparing vehicle and torcetrapib ad-CETP mice=0.002.
Discussion
We have shown for the first time to our knowledge that hepatocyte CETP mediates the selective uptake of HDL-derived CE by a mechanism that does not involve SR-BI and which does not require transfer of CE to apoB–containing lipoproteins and subsequent uptake via a member of the LDLr gene family. Importantly, we also demonstrate that hepatic expression of CETP in vivo results in significant remodeling of plasma HDL, consistent with a major physiological role for hepatocyte CETP as a receptor mediating selective uptake, analogous to that of the well-established HDL receptor, SR-BI. These findings have significant implications for the role of CETP in the final steps of reverse cholesterol transport. Although active debate on the role of CETP in modifying coronary heart disease risk continues,23 we propose that very high plasma concentrations of CETP have unfavorable effects on plasma lipids and possibly on atherogenesis, whereas complete CETP deficiency may not be of universal benefit, consistent with a direct role for CETP in mediating the hepatic clearance of HDL-CE.
Using the CETP inhibitor, torcetrapib, as a tool, we have made important observations relevant to the distinct mechanisms by which exogenous and cell associated CETP mediate selective uptake. To determine the effects of cell-associated CETP on selective uptake, we first used low titers of adenovirus to express CETP in isolated primary murine hepatocytes. Under these conditions, CETP expression in cell lysates was confirmed by Western blot but there was no detectable CETP protein or CETP transfer activity in the medium. Low levels of endogenously expressed CETP increased selective uptake of HDL-CE by 50% and this effect was not inhibited by torcetrapib. In this experiment, the maximum concentration of torcetrapib used was equivalent to that reported to completely prevent CE transfer.21 In contrast, when rCETP was added to the media at a concentration of 0.48 μg/mL, which is much lower than the mean plasma CETP concentration in healthy individuals (1.8 μg/mL)24 a 5- to 10-fold increase in 3H-CE incorporation into mouse hepatocytes was noted. Incubation of cells with increasing concentrations of torcetrapib reduced the ability of exogenous CETP to enhance 3H-CE uptake by 80%. However, torcetrapib did not completely prevent selective uptake and even in the presence of 25 μmol/L torcetrapib, exogenous CETP increased 3H-CE incorporation by 2-fold. This torcetrapib-insensitive fraction of selective uptake does not appear to be caused by inaccessibility of CETP to torcetrapib because preincubation of HDL and CETP with torcetrapib for 2 hours before the assay failed to reduce the fraction of selective uptake not inhibitable by torcetrapib. Thus, exogenous CETP appears to increase selective uptake of HDL-CE by hepatocytes by 2 distinct pathways. Only one of these is sensitive to torcetrapib but, as we have shown, is not contingent on transfer of CE to apo B lipoproteins and does not require the participation of SR-BI.
Selective uptake of HDL-derived CE is characterized by its entry into reversible and irreversible compartments.5,20 The reversible compartment is proposed to include CE that has entered the plasma membrane and remains accessible to extraction by extracellular, unlabeled HDL.20 This reversible pool attains a plateau value after 2 hours, possibly because the plasma membrane has a limited capacity to store CE or because the incorporation of CE into the plasma membrane is in equilibrium with the transfer out of this compartment. The plasma membrane CE is subsequently transferred to an irreversible compartment as it is internalized and becomes inaccessible to extraction by extracellular, unlabeled HDL.25 Torcetrapib has been shown to increase the association of CETP with its lipoprotein substrates creating a nonfunctional complex,21 which would be expected to impair the ability of CETP to shuttle CE directly to the plasma membrane and this could account for the torcetrapib-sensitive component of CETP mediated selective uptake. In contrast, the torcetrapib-insensitive pathway may represent CETP, which has become cell-associated. Neither the reversible nor irreversible phases of selective uptake mediated by cell-associated CETP were affected by torcetrapib.
Based on these studies in hepatocytes and our previously published studies in adipocytes,11 we propose 2 pathways to explain CETP-mediated selective uptake of CE. First, CETP may mediate selective uptake by shuttling CE directly from HDL to the plasma membrane. This process may or may not require the participation of another protein on the cell surface but clearly does not require SR-BI, members of the LDLr family, or an intact heparin sulfate proteoglycan matrix. As we have proposed previously, it is possible that CETP may transfer lipids to particular membrane structures such as microvilli or protrusions that have a high curvature and therefore may, at a molecular level, be comparable to a lipoprotein.11,26 Secondly, cell-associated CETP appears to mediate selective uptake by a mechanism that does not involve CETP-mediated shuttling of CE from HDL to the plasma membrane, but rather may represent direct interaction of HDL with CETP on the cell surface. It is possible that cell-associated CETP may mediate the transient fusion of the HDL amphipathic coat with the membrane outer leaflet, allowing CE movement into the membrane, without HDL particle uptake. CETP contains a C-terminal peptide that has a tilted orientation relative to the lipid–water interface, and this peptide has fusogenic properties similar to those of viral fusion peptides in a lipid-mixing assay.27 CETP has been shown to mediate lipoprotein fusion under certain circumstances.28 Further studies will be required to dissect the molecular mechanisms of CETP-mediated selective uptake and the relative importance of these 2 pathways under different circumstances.
Pharmacological modalities that increase HDL cholesterol include inhibition of CETP activity.29 Relevant to the potential clinical utility of torcetrapib, we also sought to determine whether this agent significantly impeded the ability of hepatocyte CETP to mediate selective uptake in vivo. When CETP was expressed in mouse liver by tail vein injection of a moderate viral titer of ad-CETP, we noted an acute and substantial decrease in cholesterol carried by particles in the HDL density range. This decrease was only partially attenuated by concomitant torcetrapib treatment (–33% versus –50% decrease in total cholesterol). Thus, under the conditions of these experiments, hepatocyte expressed CETP mediates selective uptake in vivo with moderate efficiency even in the presence of plasma concentrations of torcetrapib (4 μmol/L) that are several-fold higher than those achieved with clinically effective doses of this agent (0.1 to 1 μmol/L).21,30,31 Interestingly, the composition of HDL in humans treated with torcetrapib differs from that associated with homozygous CETP deficiency. In torcetrapib-treated subjects, there was not a significant increase in the CE content of HDL in contrast to that noted in subjects who are CETP-deficient.21 This is consistent with our studies demonstrating that torcetrapib has relatively moderate effects on CETP-mediated selective uptake of HDL-CE in vivo, suggesting that this novel process may in fact occur in humans although its overall importance remains to be determined. The implication of these findings is that the potentially atherogenic function of CETP (transfer of neutral lipids between lipoproteins) may be selectively inhibited by torcetrapib without severely compromising the potentially anti-atherogenic function of CETP (selective uptake).
As our results indicate, CETP can function directly to mediate selective uptake of HDL-derived CE by primary murine hepatocytes, as we have previously shown for human adipocytes. Unlike humans and several other species, mice do not normally express CETP and exhibit low plasma concentrations of apo B–containing lipoproteins. Thus, the role of CETP in hepatocyte selective uptake in humans and the effect of torcetrapib on this process remain to be addressed. The ongoing controversy regarding the role of CETP in atherosclerosis may be the reflection of a molecule with multiple functions. CETP promotes the CE-enrichment of apo B lipoproteins, which may result in a pro-atherogenic state when LDL receptors are down regulated. However, by facilitating selective uptake of HDL-CE, CETP may accelerate hepatic cholesterol clearance, mitigating against atherosclerosis.
Acknowledgments
Supported by the Canadian Institutes of Health Research (CIHR) MOP-44360 (R.M.) and CIHR/Canada Graduate Scholarship (A.G.). We are grateful to Pfizer Inc, Groton, CT, for provision of torcetrapib for use in these studies.
References
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Address correspondence to Ruth McPherson, University of Ottawa Heart Institute, H453, 40 Ruskin St, Ottawa, ON Canada K1Y 4W7. E-mail rmcpherson@ottawaheart.ca
Abstract
Objective— To determine whether cholesteryl ester transfer protein (CETP) directly mediates selective uptake of high-density lipoprotein (HDL)-cholesteryl ester (CE) by hepatocytes and to quantify the effects of the CETP inhibitor, torcetrapib, on this process.
Methods and Results— Using adenovirus-mediated CETP (ad-CETP) expression in primary mouse hepatocytes from either wild-type, low-density lipoprotein (LDL) receptor–/– or SR-BI–/– mice, we demonstrate that CETP enhances the selective accumulation of HDL-derived 3H-CE independently of known lipoprotein receptors. Addition of torcetrapib to the media did not impair the ability of cell-associated CETP to enhance CE uptake but reduced the ability of exogenously added CETP to increase selective uptake by up to 80%. When mice were infected with ad-CETP or ad-Luciferase and treated with daily intravenous injections of torcetrapib or vehicle, hepatic CETP expression resulted in a 50% decrease in HDL cholesterol in vehicle-treated animals versus a 33% decrease in HDL cholesterol in mice treated with torcetrapib.
Conclusions— CETP mediates selective uptake of HDL-CE by hepatocytes by both torcetrapib-sensitive (exogenous CETP) and torcetrapib-insensitive (cell-associated CETP) mechanisms. Hepatic expression of CETP in vivo results in a marked decrease in cholesterol in particles in the HDL density range, consistent with a physiological role for hepatocyte CETP in selective uptake.
These studies demonstrate that cholesteryl ester transfer protein (CETP) mediates the selective uptake of IIDL-derived cholesteryl esters by hepatocytes both in vitro and in vivo by a mechanism that is independent of other lipoprotein receptors and which is only partially sensitive to inhibition by torcetrapib.
Key Words: CETP ? selective uptake ? torcetrapib ? lipoproteins ? HDL
Introduction
Cholesteryl ester transfer protein (CETP) is a hydrophobic glycoprotein that plays a central role in human high-density lipoprotein (HDL) metabolism. CETP mRNA is predominantly expressed in the liver, spleen, and adipose tissue in humans and is secreted to a variable extent from each of these tissues into plasma, where it has an established role in mediating neutral lipid transport between lipoproteins.1 The overall role of CETP in atherosclerosis is unclear. CETP functions in the regulation of cholesteryl ester (CE) trafficking in plasma. Cholesterol extracted by efflux from peripheral tissues is esterified within HDL by lecithin:cholesterol acyltransferase (LCAT). This CE can be subsequently transferred by CETP to apolipoprotein B (apoB)-containing lipoproteins.2 Under conditions of efficient hepatic apoB lipoprotein clearance, CETP may promote cholesterol transport from HDL to the liver. However, high plasma concentrations of CETP are associated with low HDL cholesterol and this observation has led to the development of CETP inhibitors as a potential therapy to reduce atherosclerosis.3,4
HDL-CE is also directly returned to the liver by a process known as selective uptake. This involves the reversible incorporation of HDL-derived CE into a plasma membrane pool followed by transfer of the lipid to an inaccessible pool by mechanisms not involving coated pit-mediated endocytosis.5 SR-BI has been shown to be the primary receptor responsible for hepatic HDL-cholesterol clearance in the mouse,6,7 a species that intrinsically lacks CETP.8 The human homologue of SR-BI, CLA-1, was cloned several years ago9 but has not yet been established as the primary receptor for HDL-CE selective uptake in humans. Genetic CETP deficiency in humans results in a marked increase in plasma concentrations of HDL-CE,10 suggesting a limited role for CLA-1 or a requirement for CETP in this process. Our laboratory has demonstrated a novel role for CETP in mediating the selective acquisition of CE from HDL by human adipocytes11 by a very high-efficiency transport process with a Tmax of 68 ng/HDL protein/mg cell protein/hour and km of 1.3 μg/mL HDL3.12
These findings led us to determine whether CETP also plays a direct role in selective uptake of HDL-CE by the liver, an important final step in reverse cholesterol transport, and to further investigate the mechanisms by which CETP functions to promote selective uptake. CETP was previously ascribed a role in the remodeling of CE-rich HDL, making it an optimal ligand for selective uptake by SR-BI/CLA-1.13 Although Granot et al reported that CETP mediates the selective uptake of HDL-CE by HepG2 cells,14 these observations were later attributed to the established function of CETP in mediating the transfer of HDL-CE to newly secreted apo B lipoproteins, followed by their internalization by the low-density lipoprotein (LDL) receptor (LDLr).15
Importantly, we show here that CETP directly mediates selective uptake of HDL-derived CE by hepatocytes, a cell of fundamental importance in HDL metabolism and that this effect occurs independently of other established lipoprotein receptors. We also demonstrate that hepatic expression of CETP in vivo results in a marked decrease in cholesterol in particles in the HDL density range consistent with a physiological role for hepatocyte CETP in selective uptake. We have explored the effects of the CETP inhibitor, torcetrapib, on this process and demonstrate that the effects of CETP in promoting hepatocyte selective uptake are partially impeded by this agent. These studies provide new and important insights into the mechanisms of CETP-mediated selective uptake and the role of CETP in cholesterol trafficking to the liver.
Methods
Materials
All common reagents of analytical grade were from Fisher (Fair Lawn, NJ) or Sigma (St Louis, Mo). Recombinant CETP (rCETP) was purchased from Cardiovascular Targets, Inc (New York, NY). Torcetrapib (CP-529,414) was kindly provided by Pfizer Inc (Groton, Conn).
Animals and Primary Hepatocyte Cultures
Retired breeders of the C57Bl6J (wild-type) mouse strain were from Charles River (Wilmington, Mass). Mice do not express CETP, making them an ideal model for these studies. As indicated, certain experiments were performed using primary hepatocytes from either SR-B1 or LDLr-deficient mice. SR-B1–deficient (strain B6.129S2-Scarb1tm1Kri)) and LDLr-deficient mice (strain B6.129S7-Ldlrtm1Her) were from Jackson Laboratory (Bar Harbor, Me). Mice were maintained on a normal chow diet. Primary hepatocytes were prepared from mice according to established protocols.16,17 Six hours after the initial plating, the cells were washed in William’s medium without fetal bovine serum and fresh media. For experiments using adenovirus-mediated expression of CETP, CETP adenovirus (ad-CETP) or luciferase adenovirus (ad-Luc) (control) was added to the wells at a concentration of 2.175x107 plaque forming units (PFU)/well (75 multiplicity of infection). The infected cells were left to express the transgene for 36 to 48 hours before the assay. Otherwise, cells were used 12 to 36 hours after initial wash.
Generation of CETP Adenovirus
Human CETP, in the pCMV5 mammalian expression vector, was subcloned into the pShuttle-CMV vector using standard molecular biology techniques; this vector was used for subsequent generation of the adenovirus DNA construct via homologous recombination with the pAd-Easy1 adenovirus DNA vector. Adenovirus was then generated, scaled-up, purified, and measured according to the manufacturer’s instructions (Qbiogene).
Lipoprotein Purification and Labeling
Total HDL was purified from normolipemic plasma from a single donor by density gradient ultracentrifugation,18 followed by dialysis 3 times against 4 L of nitrogen-sparged phosphate-buffered saline, pH 7.4, with 2 grams of Chelex (BioRad). The lipoproteins were labeled with 3H-cholesteryl oleate using a modified protocol as described by Reaven.19 Human apolipoprotein (apo) A-1 was labeled with 125I as described.20 3H-CE HDL was then incubated with 125I-labeled apoA-1 or cold apoA-1 for 24 hours at 37°C before re-isolation to obtain an HDL particle-labeled in the core with 3H-CE and on the protein moiety with either cold or 125I-apoA-1. The charge and size of the labeled HDL particles were verified by Lipogel analysis.
Selective Uptake Assay
Experiments were performed essentially as outlined in detail previously.20 Concentrations are stated in the Figures and legends, and the cells were incubated at 37°C for 8 hours, unless otherwise indicated. The cell associations of 3H-CE or 125I-apoA-1 or degradation of 125I-apoA-1 are measured in units of the amount of these labels contained in 1 ng (protein content) of HDL and corrected for mg of cell protein in each well. Measured this way, an equivalent amount of 3H-CE and 125I-apoA-1 labels represents holoparticle uptake and any additional 3H-CE cell association is attributed to selective uptake.
In Vivo Experiments
Experiments were performed in 6- to 8-week-old C57Bl6J (wild-type) mice (Charles River). In initial experiments, mice were subjected to tail vein injection of either ad-Luc or ad-CETP (1.25x108 PFU in 100 μL). Fasting plasma cholesterol was measured at baseline, 3, 6, and 10 days after injection. Animals were euthanized at 10 days and primary hepatocytes isolated for determination of CETP protein expression and 3H-CE-HDL selective uptake assays. In subsequent experiments, mice were injected with 2.5x108 PFU in 100 μL of ad-Luc or ad-CETP on day 0 and then received daily tail vein injections of 100 μL of Intralipid containing either vehicle (DMSO) or torcetrapib (0.125 mmol/L; such that the final concentration in the mouse, assuming a 3-mL total blood volume, would be 4 μmol/L). Fasting plasma cholesterol was measured and plasma CETP expression assessed by Western blot at baseline and days 2, 4, 5, and 7 after injection. On day 7, animals were exsanguinated by cardiac puncture and sera stored at 4°C before discontinuous density gradient ultracentrifugation (18 hours at 45 000 rpm).
Statistical Analysis
Results are expressed as the mean±standard error of the mean. Where indicated, the statistical significance was determined using the unpaired t test and expressed as a 2-tailed P value.
Note that further methodological details for all procedures are provided in supplementary information online (see http://atvb.ahajournals.org).
Results
Infection of Mouse Primary Hepatocytes With ad-CETP Promotes Selective Uptake of HDL-Derived CE
Western blot analysis of wild-type murine hepatocytes infected in culture with adenovirus-CETP (ad-CETP) or adenovirus-luciferase (ad-Luc) control is shown in Figure 1A, indicating CETP protein expression in cell lysates from CETP-transfected hepatocytes in contrast to control. For these experiments, to specifically address the effects of cell associated CETP as opposed to CETP secreted into the media, we used a low viral titer of ad-CETP. Under these conditions, hepatocyte secretion of CETP was negligible as demonstrated by absence of CETP in the media by Western blot after immunoprecipitation with TP2 anti-CETP monoclonal antibody (mAb) at the end of the 8-hour time course and no detectable transfer of 3H-CE from HDL to LDL in an in vitro transfer assay (data not shown). As expected in luciferase-transfected hepatocytes, 3H-CE cellular incorporation was several-fold greater than that of 125I-apo A-I, consistent with SR-BI–mediated selective uptake of HDL-CE by primary hepatocytes (Figure 1B). However, even in the absence of detectable CE transfer activity in the media, adenovirus-mediated expression of CETP in mouse primary hepatocytes resulted in a 50% increase in 3H-CE cell association as compared with ad-Luc control. These studies demonstrate that cell associated CETP can directly mediate the selective acquisition of HDL-derived CE by hepatocytes. This effect began to saturate at 50 μg/mL of HDL and therefore further experiments shown used this concentration of HDL. The cell-association of 125I-apoA-1 or the cell-mediated degradation of 125I-apoA-1 was unchanged with ad-CETP expression compared with ad-Luc control cells, indicating that although CETP increases CE uptake, it does not alter HDL particle uptake.
Figure 1. CETP mediates the selective uptake of HDL-derived CE in isolated mouse hepatocytes. A, Western blot of wild-type hepatocytes transfected with ad-CETP versus ad-Luc, showing cell lysate expression of CETP in comparison to human plasma. At this level of expression, no CETP was detectable in the media by Western blot (immunoprecipitation) or by CE transfer assay. B, Effects of endogenously expressed CETP on selective uptake. Hepatocytes were incubated at 37°C for 8 hours with increasing concentrations of 3H-CE HDL or 125I-apoA-1 HDL. Cellular incorporation of 3H-CE in cells infected with ad-CETP (black triangles) was increased 2-fold compared with cells infected with ad-Luc (white triangles). No differences were noted in 125I-apoA-I cell incorporation with ad-CETP (black circles) or ad-Luc (white circles) or in 125I-apoA-I degradation with ad-CETP (black squares) versus ad-Luc (white squares). *2-tailed P<0.007 compared with luciferase at all HDL concentrations. C, Effects of exogenously added recombinant CETP (rCETP) on selective uptake. Hepatocytes were incubated at 37°C for 8 hours with 3H-CE HDL or 125I-apoA-1 HDL (50 μg/mL), with or without the addition of CETP (0.48 μg/mL). Exogenous CETP increased 3H-CE incorporation into mouse hepatocytes by 6-fold but did not alter 125I-apoA-I cell association or degradation. *2 tailed P<0.0001.
Effects of Exogenous CETP on Selective Uptake by Murine Hepatocytes
In other experiments, we determined the ability of exogenously added recombinant CETP (rCETP) to mediate the selective uptake of HDL-derived CE. As shown in Figure 1C, rCETP, which was added to the media at a concentration of 0.48 μg/mL, increased the selective uptake of HDL-derived CE by 5-fold.
Effects of Endogenously Expressed, Cell-Associated CETP on Selective Uptake
To determine the effects of low-level in vivo hepatocyte CETP expression on selective uptake, mice were injected with 1.25x108 PFU of either ad-Luc or ad-CETP (n=4 for each) and euthanized on day10. There was no detectable CETP activity in the plasma of the ad-CETP–treated mice indicating no significant hepatocyte secretion of CETP at this level of ad-CETP infection. Plasma total cholesterol was significantly lower in mice infected with ad-CETP (P=0.01) (Figure 2A) because of a selective decrease in cholesterol in particles in the HDL density range (data not shown). In primary hepatocytes isolated from ad-CETP infected mice, CETP was detectable by Western blot of the cell lysate (Figure 2B) and 3H-CE-HDL selective uptake was increased by 20% (P=0.01) as compared with hepatocytes from ad-Luc–infected mice (Figure 2C).
Figure 2. Effects of endogenously expressed, cell-associated CETP on selective uptake. Mice were injected with 1.25x108 PFU of either ad-luciferase or ad-CETP (n=4 for each) and euthanized on day 10. A, Plasma total cholesterol was significantly lower in mice infected with ad-CETP; *2-tailed P=0.01. B, Western blot of the cell lysate from isolated hepatocytes from ad-CETP infected mice indicating CETP expression. C, Effects of endogenously expressed CETP on selective uptake. Primary hepatocytes isolated from mice 10 days after ad-Luc or ad-CETP infection were incubated at 37°C for 8 hours with 3H-CE HDL or 125I-apoA-1 HDL (50 μg/mL). Cellular incorporation of 3H-CE in hepatocytes from ad-CETP infected mice (black triangles) was increased 20% as compared with hepatocytes from ad-Luc infected mice (white triangles). **2-tailed P=0.01. No differences were noted in 125I-apoA-I cell incorporation with ad-CETP (black circles) or ad-Luc (white circles) or in 125I-apoA-I degradation with ad-CETP (solid squares) vs ad-Luc (open squares).
Effects of the CETP Inhibitor, Torcetrapib, on Selective Uptake Mediated by Exogenous Versus Cell-Associated CETP
When primary mouse hepatocytes were infected with a low viral titer of ad-CETP, CETP was detectable in cell lysates but was not in the medium. As shown in Figure 3A, under these conditions, addition of increasing concentrations of the CETP inhibitor, torcetrapib, to the media did not impair the ability of endogenously expressed cell-associated CETP to enhance selective uptake. In contrast, when rCETP(0.48 μg/mL) was added to the media, an 8-fold increase in 3H-CE incorporation into mouse hepatocytes was demonstrated (Figure 3B). Under these conditions, incubation of cells with increasing concentrations of torcetrapib reduced the ability of exogenously added CETP to enhance 3H-CE uptake by 80%. However, torcetrapib did not completely prevent selective uptake mediated by exogenously added CETP. Even in the presence of 25 μmol/L torcetrapib, exogenous CETP at a concentration of 0.48 μg/mL increased 3H-CE uptake by 2-fold (Figure 3B). In additional experiments, identical results were obtained when rCETP, 3H-CE HDL, and torcetrapib were pre-incubated for 2 hours before being added to the primary hepatocytes (data not shown). Thus, when added to cell culture media, CETP appears to increase selective uptake of HDL-CE by hepatocytes by 2 distinct pathways: one representing a process that is inhibitable by torcetrapib and another by a mechanism that is not accessible to inhibition by torcetrapib. In other studies, we incubated 0.48 μg/mL rCETP with primary murine hepatocytes and determined that 20% becomes cell associated after 2 hours at 37°C and this may represent the torcetrapib-insensitive fraction.
Figure 3. Effects of torcetrapib on selective uptake of HDL-CE mediated by cell associated or exogenously added CETP. A, In hepatocytes transfected with a low viral titer of ad-CETP, CETP was detectable by Western blot in cell lysates but was not in the medium (data not shown). Ad-CETP or ad-Luc transfected hepatocytes were incubated at 37°C for 8 hours with 3H-CE HDL or 125I-apoA-1 HDL (50 μg/mL) with or without the addition of 5 to 500 nmol of torcetrapib to the media. *2-tailed P<0.05 compared with control. B, Hepatocytes were incubated at 37°C for 8 hours with 3H-CE HDL or 125I-apoA-1 HDL (50 μg/mL), with or without the addition of CETP (0.48 μg/mL) and 0 to 25 μmol/L torcetrapib. The 2-tailed P<0.04 for all rCETP values compared with control.
Effects of Torcetrapib on Reversible and Irreversible Components of Selective Uptake Mediated by Exogenous Versus Cell-Associated CETP
To further investigate the mechanism by which exogenous and cell-associated CETP mediate selective uptake, we determined the reversible and irreversible phases of CE-uptake at 2 and 4 hours. As shown in Figure 4A, when primary hepatocytes were incubated with 0.48 μg/mL of rCETP, entry of 3H-CE into a reversible compartment was completely inhibited by torcetrapib, whereas accumulation of 3H-CE in the irreversible compartment was partially but not completely inhibited by torcetrapib (Figure 4B). In contrast, when this experiment was performed using hepatocytes infected with a low level of ad-CETP, to achieve CETP expression in the cell without detectable CETP secretion into the media, there was no effect of torcetrapib on entry of 3H-CE into either the reversible or irreversible compartments (Figure 4C), further demonstrating that selective uptake mediated by cell-associated CETP is not susceptible to inhibition by torcetrapib.
Figure 4. Effects of torcetrapib on reversible and irreversible components of selective uptake mediated by exogenous versus cell-associated CETP. A and B, Hepatocytes were preincubated with 50 μg/mL 3H-CE-HDL for the indicated times and then incubated for 4 hours with 400 μg/mL cold HDL. Cells were treated with either vehicle (DMSO) or torcetrapib (1 μmol/L). Removal of 3H-CE label from the reversible phase is represented by the media counts in (A). The irreversible phase was determined by measuring the cell-associated counts after 4 washes with Hank balanced salt solution and is represented by the cell counts in (B). C, The reversible and irreversible components of CETP selective uptake in mouse hepatocytes infected with a low level of ad-CETP to achieve cellular CETP expression without detectable CETP secretion into the media. Cells were treated as mentioned. #P=0.007; *P<0.0001; **P=0.005.
CETP-Mediated Selective Uptake Is Independent of Other Classical Pathways
To determine whether the portion of the CETP-mediated selective uptake pathway, which is sensitive to the CETP inhibitor, consists of CE transferred to apo B lipoproteins, a process known to be inhibited by torcetrapib,21 we performed CETP-mediated selective uptake in hepatocytes from LDLr-deficient mice. Importantly, absence of the LDLr did not attenuate the ability of CETP to mediate selective uptake and once again 3H-CE uptake was only partially inhibited by torcetrapib (Figure 5A). Thus, the torcetrapib inhibitable component of selective uptake does not require transfer of HDL-CE to apo B lipoproteins and subsequent clearance by the LDLr.
Figure 5. Role of lipoprotein receptors in the CETP-mediated cellular incorporation of 3H-CE from HDL. 3H-CE HDL (50 μg/mL) with or without the addition of CETP (0.48 μg/mL) and 0 to 5 μmol/L torcetrapib was incubated with hepatocytes prepared from LDLr null mice (A), SR-BI–/– mice (B) or from wild-type mice and incubated with receptor-associated protein (30 μg/mL) (C) or from wild-type mice and incubated with heparin (100 U/mL) (D). The 2-tailed P<0.05 for all CETP samples compared with control.
Additional studies were performed in hepatocytes from SR-B1–deficient mice. As expected, the overall levels of HDL-CE uptake were lower in hepatocytes from mice lacking SR-B1. However, exogenous CETP (0.48 μg/mL) enhanced hepatocyte incorporation of 3H-CE to a similar extent irrespective of SR-BI expression. Thus, the effect of CETP on hepatocyte selective uptake is not contingent on the presence of SR-BI (Figure 5B).
To exclude a role for other members of the LDLr gene family, we incubated hepatocytes with and without receptor-associated protein to block the members of the LDLr family through competitive binding. Incubation with receptor-associated protein (RAP) did not affect CETP-mediated selective uptake and both torcetrapib-sensitive and insensitive pathways were again observed (Figure 5C).
Finally, studies were performed in the presence of heparin, which disrupts binding of hepatic lipase to the heparin sulfate proteoglycan matrix.22 Heparin treatment did not reduce the ability of exogenous CETP to enhance hepatocyte accumulation of 3H-CE or alter the torcetrapib-sensitive or insensitive components of CETP-mediated selective uptake, ruling out a requirement for hepatic lipase in this process (Figure 5D).
CETP Mediates Hepatocyte Selective Uptake In Vivo
These studies clearly demonstrated that CETP mediates selective uptake of HDL-CE by primary hepatocytes in culture by a mechanism that does not require transfer of HDL-derived CETP to apoB lipoproteins or participation of SR-BI. To confirm the functional importance of CETP in hepatocyte selective uptake of HDL-CE in vivo, we determined changes in plasma total cholesterol and lipoprotein subfractions 7 days after tail vein injection of ad-CETP or ad-Luc. As shown in Figure 6A, hepatic expression of CETP at levels sufficient to elicit CETP secretion into plasma, in a species lacking endogenous CETP, reduced plasma total cholesterol by 50% relative to luciferase control. Importantly, this decrease was entirely because of a decrease in cholesterol in particles in the HDL density range (Figure 6B), consistent with an acute effect of hepatic CETP expression on selective uptake in vivo. Because pharmacological inhibitors of CETP activity increase HDL cholesterol and are likely to gain broad clinical use, studies were also performed to determine the effects of concomitant administration of the CETP inhibitor, torcetrapib, on CETP-mediated selective uptake in vivo. Torcetrapib is poorly absorbed by the oral route in rodents and thus mice received daily tail vein injections of vehicle (DMSO) or torcetrapib (0.125 mmol/L) in 100 L Intralipid. Assuming a 3-mL total blood volume, the final plasma concentration in the mouse would be 4 μmol/L. Consistent with the results of the ex vivo experiments, torcetrapib was found to partially attenuate the ability of adenovirus driven hepatocyte expressed CETP to enhance selective uptake as indicated by decreases in cholesterol in lipoprotein particles of the HDL density range (Figure 6A and 6B). Overall, ad-CETP expression acutely reduced total cholesterol (mainly in particles in the HDL density range) by 50% in mice not treated with torcetrapib and by 33% in animals subjected to concomitant torcetrapib treatment.
Figure 6. Effects of hepatic expression of CETP on selective uptake in vivo. Mice injected via tail vein with 2.5x108 PFU adenovirus and treated with daily intravenous injections of Intralipid-containing vehicle (DMSO) or torcetrapib for 7 days. A, Fasting plasma total cholesterol from day 7 after injection. Data are presented as a % of the day 0 total plasma cholesterol measurements. *2-tailed P<0.001 compared with luciferase controls; ** 2-tailed P=0.02 comparing vehicle and torcetrapib treated ad-CETP mice. B, Total cholesterol in lipoprotein fractions separated by discontinuous density gradient ultracentrifugation. The 2-tailed P values for ad-CETP mice compared with ad-Luc mice at the peak corresponding to HDL are <0.0001. The 2-tailed P value comparing vehicle and torcetrapib ad-CETP mice=0.002.
Discussion
We have shown for the first time to our knowledge that hepatocyte CETP mediates the selective uptake of HDL-derived CE by a mechanism that does not involve SR-BI and which does not require transfer of CE to apoB–containing lipoproteins and subsequent uptake via a member of the LDLr gene family. Importantly, we also demonstrate that hepatic expression of CETP in vivo results in significant remodeling of plasma HDL, consistent with a major physiological role for hepatocyte CETP as a receptor mediating selective uptake, analogous to that of the well-established HDL receptor, SR-BI. These findings have significant implications for the role of CETP in the final steps of reverse cholesterol transport. Although active debate on the role of CETP in modifying coronary heart disease risk continues,23 we propose that very high plasma concentrations of CETP have unfavorable effects on plasma lipids and possibly on atherogenesis, whereas complete CETP deficiency may not be of universal benefit, consistent with a direct role for CETP in mediating the hepatic clearance of HDL-CE.
Using the CETP inhibitor, torcetrapib, as a tool, we have made important observations relevant to the distinct mechanisms by which exogenous and cell associated CETP mediate selective uptake. To determine the effects of cell-associated CETP on selective uptake, we first used low titers of adenovirus to express CETP in isolated primary murine hepatocytes. Under these conditions, CETP expression in cell lysates was confirmed by Western blot but there was no detectable CETP protein or CETP transfer activity in the medium. Low levels of endogenously expressed CETP increased selective uptake of HDL-CE by 50% and this effect was not inhibited by torcetrapib. In this experiment, the maximum concentration of torcetrapib used was equivalent to that reported to completely prevent CE transfer.21 In contrast, when rCETP was added to the media at a concentration of 0.48 μg/mL, which is much lower than the mean plasma CETP concentration in healthy individuals (1.8 μg/mL)24 a 5- to 10-fold increase in 3H-CE incorporation into mouse hepatocytes was noted. Incubation of cells with increasing concentrations of torcetrapib reduced the ability of exogenous CETP to enhance 3H-CE uptake by 80%. However, torcetrapib did not completely prevent selective uptake and even in the presence of 25 μmol/L torcetrapib, exogenous CETP increased 3H-CE incorporation by 2-fold. This torcetrapib-insensitive fraction of selective uptake does not appear to be caused by inaccessibility of CETP to torcetrapib because preincubation of HDL and CETP with torcetrapib for 2 hours before the assay failed to reduce the fraction of selective uptake not inhibitable by torcetrapib. Thus, exogenous CETP appears to increase selective uptake of HDL-CE by hepatocytes by 2 distinct pathways. Only one of these is sensitive to torcetrapib but, as we have shown, is not contingent on transfer of CE to apo B lipoproteins and does not require the participation of SR-BI.
Selective uptake of HDL-derived CE is characterized by its entry into reversible and irreversible compartments.5,20 The reversible compartment is proposed to include CE that has entered the plasma membrane and remains accessible to extraction by extracellular, unlabeled HDL.20 This reversible pool attains a plateau value after 2 hours, possibly because the plasma membrane has a limited capacity to store CE or because the incorporation of CE into the plasma membrane is in equilibrium with the transfer out of this compartment. The plasma membrane CE is subsequently transferred to an irreversible compartment as it is internalized and becomes inaccessible to extraction by extracellular, unlabeled HDL.25 Torcetrapib has been shown to increase the association of CETP with its lipoprotein substrates creating a nonfunctional complex,21 which would be expected to impair the ability of CETP to shuttle CE directly to the plasma membrane and this could account for the torcetrapib-sensitive component of CETP mediated selective uptake. In contrast, the torcetrapib-insensitive pathway may represent CETP, which has become cell-associated. Neither the reversible nor irreversible phases of selective uptake mediated by cell-associated CETP were affected by torcetrapib.
Based on these studies in hepatocytes and our previously published studies in adipocytes,11 we propose 2 pathways to explain CETP-mediated selective uptake of CE. First, CETP may mediate selective uptake by shuttling CE directly from HDL to the plasma membrane. This process may or may not require the participation of another protein on the cell surface but clearly does not require SR-BI, members of the LDLr family, or an intact heparin sulfate proteoglycan matrix. As we have proposed previously, it is possible that CETP may transfer lipids to particular membrane structures such as microvilli or protrusions that have a high curvature and therefore may, at a molecular level, be comparable to a lipoprotein.11,26 Secondly, cell-associated CETP appears to mediate selective uptake by a mechanism that does not involve CETP-mediated shuttling of CE from HDL to the plasma membrane, but rather may represent direct interaction of HDL with CETP on the cell surface. It is possible that cell-associated CETP may mediate the transient fusion of the HDL amphipathic coat with the membrane outer leaflet, allowing CE movement into the membrane, without HDL particle uptake. CETP contains a C-terminal peptide that has a tilted orientation relative to the lipid–water interface, and this peptide has fusogenic properties similar to those of viral fusion peptides in a lipid-mixing assay.27 CETP has been shown to mediate lipoprotein fusion under certain circumstances.28 Further studies will be required to dissect the molecular mechanisms of CETP-mediated selective uptake and the relative importance of these 2 pathways under different circumstances.
Pharmacological modalities that increase HDL cholesterol include inhibition of CETP activity.29 Relevant to the potential clinical utility of torcetrapib, we also sought to determine whether this agent significantly impeded the ability of hepatocyte CETP to mediate selective uptake in vivo. When CETP was expressed in mouse liver by tail vein injection of a moderate viral titer of ad-CETP, we noted an acute and substantial decrease in cholesterol carried by particles in the HDL density range. This decrease was only partially attenuated by concomitant torcetrapib treatment (–33% versus –50% decrease in total cholesterol). Thus, under the conditions of these experiments, hepatocyte expressed CETP mediates selective uptake in vivo with moderate efficiency even in the presence of plasma concentrations of torcetrapib (4 μmol/L) that are several-fold higher than those achieved with clinically effective doses of this agent (0.1 to 1 μmol/L).21,30,31 Interestingly, the composition of HDL in humans treated with torcetrapib differs from that associated with homozygous CETP deficiency. In torcetrapib-treated subjects, there was not a significant increase in the CE content of HDL in contrast to that noted in subjects who are CETP-deficient.21 This is consistent with our studies demonstrating that torcetrapib has relatively moderate effects on CETP-mediated selective uptake of HDL-CE in vivo, suggesting that this novel process may in fact occur in humans although its overall importance remains to be determined. The implication of these findings is that the potentially atherogenic function of CETP (transfer of neutral lipids between lipoproteins) may be selectively inhibited by torcetrapib without severely compromising the potentially anti-atherogenic function of CETP (selective uptake).
As our results indicate, CETP can function directly to mediate selective uptake of HDL-derived CE by primary murine hepatocytes, as we have previously shown for human adipocytes. Unlike humans and several other species, mice do not normally express CETP and exhibit low plasma concentrations of apo B–containing lipoproteins. Thus, the role of CETP in hepatocyte selective uptake in humans and the effect of torcetrapib on this process remain to be addressed. The ongoing controversy regarding the role of CETP in atherosclerosis may be the reflection of a molecule with multiple functions. CETP promotes the CE-enrichment of apo B lipoproteins, which may result in a pro-atherogenic state when LDL receptors are down regulated. However, by facilitating selective uptake of HDL-CE, CETP may accelerate hepatic cholesterol clearance, mitigating against atherosclerosis.
Acknowledgments
Supported by the Canadian Institutes of Health Research (CIHR) MOP-44360 (R.M.) and CIHR/Canada Graduate Scholarship (A.G.). We are grateful to Pfizer Inc, Groton, CT, for provision of torcetrapib for use in these studies.
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