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Am J Physiol Gastrointest Liver Physiol 289: G1067-G1074, 2005. First published August 11, 2005; doi:10.1152/ajpgi.00194.2005
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LIVER AND BILIARY TRACT

Phosphatidylcholine transfer protein regulates size and hepatic uptake of high-density lipoproteins

Michele K. Wu1 and David E. Cohen1,2

Departments of 1Biochemistry and 2Medicine, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York

Submitted 29 April 2005 ; accepted in final form 8 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Phosphatidylcholine transfer protein (PC-TP) is a steroidogenic acute regulatory-related transfer domain protein that is enriched in liver cytosol and binds phosphatidylcholines with high specificity. In tissue culture systems, PC-TP promotes ATP-binding cassette protein A1-mediated efflux of cholesterol and phosphatidylcholine molecules as nascent pre-{beta}-high-density lipoprotein (HDL) particles. Here, we explored a role for PC-TP in HDL metabolism in vivo utilizing 8-wk-old male Pctp–/– and wild-type littermate C57BL/6J mice that were fed for 7 days with either chow or a high-fat/high-cholesterol diet. In chow-fed mice, neither plasma cholesterol concentrations nor the concentrations and compositions of plasma phospholipids were influenced by PC-TP expression. However, in Pctp–/– mice, there was an accumulation of small {alpha}-migrating HDL particles. This occurred without changes in hepatic expression of ATP-binding cassette protein A1 or in proteins that regulate the intravascular metabolism and clearance of HDL particles. In Pctp–/– mice fed the high-fat/high-cholesterol diet, HDL particle sizes were normalized, whereas plasma cholesterol and phospholipid concentrations were increased compared with wild-type mice. In the absence of upregulation of hepatic ATP-binding cassette protein A1, reduced HDL uptake from plasma into livers of Pctp–/– mice contributed to higher plasma lipid concentrations. These data indicate that PC-TP is not essential for the enrichment of HDL with phosphatidylcholines but that it does modulate particle size and rates of hepatic clearance.

steroidogenic acute regulatory-related transfer domain; phospholipid; cholesterol; plasma; liver

REVERSE CHOLESTEROL TRANSPORT is the process by which excess cholesterol in peripheral tissues is returned to the liver for biliary elimination, principally by high-density lipoprotein (HDL) particles in plasma (33). Formation of HDL begins when apolipoprotein A-I interacts with ATP-binding cassette protein A1 (Abca1) on hepatocyte sinusoidal membranes (3, 20, 34). This promotes the efflux of phospholipid and unesterified cholesterol to form pre-{beta}-HDL particles that mature within the circulation due to the activities of a number of intravascular enzymes (17, 33). Once enriched with cholesterol from peripheral tissues, HDL particles return to the liver and interact with scavenger receptor class B type I (SR-BI), which mediates the selective uptake into hepatocytes of lipids but not apolipoproteins.

The phospholipid composition of HDL particles formed by Abca1/apolipoprotein A-I-mediated lipid efflux from cells comprises >80% phosphatidylcholines (15, 43). This differs substantially from hepatocellular membranes, which contain heterogeneous mixtures of phospholipid classes, including phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, phosphatidylserines, and sphingomyelins (8–10). Phosphatidylcholine transfer protein (PC-TP) is a highly specific phosphatidylcholine-binding protein that is enriched in liver cytosol (25, 37). PC-TP is a member of the steroidogenic acute regulatory (StAR)-related transfer (START) domain-containing superfamily of proteins (22, 35) and has also been designated StarD2 (30). Recent studies (1, 2) in tissue culture from our laboratory have demonstrated that apolipoprotein A-I-mediated cholesterol and phospholipid efflux increase in proportion to cellular PC-TP expression, suggesting a role for this protein in the formation of HDL particles.

Using PC-TP-deficient (Pctp–/–) mice, we have provided evidence for its role in maintaining hepatic cholesterol homeostasis (38) as well as the capacity of the liver to upregulate biliary lipid secretion in response to a high-fat/high-cholesterol diet (39). Here, we utilized Pctp–/– mice to explore the function of PC-TP in HDL metabolism in vivo. In chow-fed mice, the absence of PC-TP did not affect plasma cholesterol concentrations or the concentrations and compositions of plasma phospholipids. However, complementary techniques demonstrated an accumulation of small {alpha}-migrating HDL particles in plasma of Pctp–/– mice. When mice were challenged with the high-fat/high-cholesterol diet, the absence of PC-TP was associated with increased plasma concentrations of cholesterol and phospholipids, which could be attributed to decreased hepatic uptake. These data suggest that PC-TP may play an important role in reverse cholesterol transport.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials

[1,2(n)-3H]cholesteryl oleyl ether and [125I]NaI (100 mCi/ml) were both purchased from Amersham Biosciences (Piscataway, NJ). All general chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated.

Animals and Experimental Design

Pctp–/– and wild-type littermate control mice on a C57BL/6J genetic background were bred and maintained as described elsewhere (39). Male 6- to 8-wk-old mice were fed for 7 days with either a chow diet consisting of 4% fat and <0.02% cholesterol (LabDiet 5001, PMI Nutrition; Brentwood, MO) or a high-fat/high-cholesterol diet that consisted of 15% fat, 1.25% cholesterol, and 0.5% sodium cholate (Harlan Teklad; Madison, WI), which we previously referred to as a lithogenic diet (38, 39). In earlier studies, this period of feeding was found to be suitable for eliciting phenotypic differences between Pctp–/– and wild-type mice in biliary lipid secretion (39) and hepatic cholesterol metabolism (38). On the day of an experiment, mice were fasted for 3 h and then anesthetized with intraperitoneal injections of 87 mg/kg body wt ketamine (Fort Dodge Animal Health; Fort Dodge, IA) and 13 mg/kg body wt xylazine (Lloyd Laboratories; Shenandoah, IA). This period of fasting was chosen to be consistent with previous studies of the hepatobiliary phenotype of Pctp–/– mice (38, 39) and may not have been sufficient to completely avoid the postprandial state. At 9 AM, blood was collected by cardiac puncture. Blood samples were anticoagulated by the addition of EDTA. Plasma was separated by centrifugation and stored at –80°C. This procedure was conducted with the approval of the Institutional Animal Care and Use Committee.

Hepatic uptake of HDL. Subfraction 2 of human HDL (HDL2) was isolated by buoyant density centrifugation (29). Rates of clearance from plasma were measured using HDL2 particles radiolabeled with [3H]cholesteryl oleyl ether (2.22 MBq) and 125I (28). Briefly, 3H was incorporated by incubating [1,2(n)-3H]cholesteryl oleyl ether-egg phosphatidylcholine vesicles prepared by sonication together with recombinant cholesteryl ester transfer protein (Cardiovascular Targets; New York, NY) overnight at 37°C. Proteins in HDL2 particles were then labeled with 125I using IODO-GEN tubes (Pierce; Rockford, IL) according to the manufacturer’s protocol. After being anesthetized with intraperitoneal injections of 87 mg/kg body wt ketamine (Fort Dodge Animal Health) and 13 mg/kg body wt of xylazine (Lloyd Laboratories), mice were injected via the femoral veins with 5 µg protein of radiolabeled HDL2. Blood was collected by retroorbital bleeding periodically, and mice were then killed 24 h after the injection. During these experiments, mice were provided access to food and water. Blood and livers were harvested. 125I was quantified in plasma and liver samples by gamma counting. For quantification of 3H by liquid scintillation counting, [3H]cholesteryl oleyl ether in plasma and liver samples was extracted into chloroform, dried under a stream of nitrogen, and then resuspended in Ecoscint H (National Diagnostics; Atlanta, GA). The disappearance of HDL2 from plasma was calculated by subtracting values for 3H and 125I obtained from 30 min to 24 h from values obtained 10 min after injection, which was considered to represent 0 min. Fractional catabolic rates as well as production rates of HDL-cholesterol, which represent the product of fractional catabolic rate and circulating mass of HDL-cholesterol, respectively, were calculated as described by Qin et al. (23).

Analytic Techniques

Plasma lipid concentrations. Plasma total phospholipid, cholesterol, and triglyceride concentrations were determined enzymatically using reagents from Wako Chemicals (Richmond, VA), Sigma, and Roche (Indianapolis, IN), respectively. Phospholipid classes and acyl chain compositions were quantified off-site by Lipomics Technologies (West Sacramento, CA) as previously described (39).

Fast protein liquid chromatography. Equal volumes of plasma were pooled, and lipoproteins were fractionated by fast protein liquid chromatography (FPLC) (12). Phospholipid concentrations of individual fractions were quantified using an inorganic phosphorous technique (12). Cholesterol concentrations in fractions were determined enzymatically (12).

Discontinuous gradient density ultracentrifugation. Equal volumes of pooled plasma (n = 5/group) were fractionated by discontinuous gradient density ultracentrifugation (19). Briefly, potassium bromide (1 g) and sucrose (50 mg) were added to plasma samples (0.5 ml), which were then adjusted to a final volume of 3 ml with 1 mM EDTA in 12-ml ultracentrifuge tubes (Beckman; Fullerton, CA). Solutions of varied density ({rho}) were layered on top as follows: {rho} = 1.21 g/ml KBr (2 ml), {rho} = 1.08 g/ml KBr (3 ml), and {rho} = 1.00 g/ml KBr (3 ml). Samples were then centrifuged at 150,000 g for 18 h at 4°C in a Beckman SW40 rotor. Aliquots (1 ml) were sequentially removed from the top of each tube so that individual samples were separated into fractions of increasing density, as determined gravimetrically. Fractions were desalted into 150 µl of Tris-buffered saline using Centricon 100 spin columns (Millipore; Bedford, MA). Cholesterol and triglyceride concentrations of fractions were measured enzymatically, and phospholipid concentrations were measured using the inorganic phosphorus procedure. Apolipoprotein contents were determined by Western blot analysis, as described below. Isolated lipoprotein particles were further characterized by agarose gel electrophoresis using precast gels (Beckman) (1). Briefly, equal volumes of each fraction (10 µl) were electrophoresed for 30 min at 100 V. Gels were then fixed in buffer containing 60% ethanol and 10% glacial acetic acid for 5 min. Lipids were visualized by staining gels with Sudan black for 30 min, followed by drying at 55°C.

Activity of plasma phospholipid transfer protein. Activity of plasma phospholipid transfer protein (PLTP) was measured using a kit from Cardiovascular Targets. Briefly, donor particles (3 µl) with quenched fluorescent phospholipids were mixed with 5 µl plasma and 50 µl acceptor particles in 42 µl of buffer (10 mM Tris, 150 mM NaCl, and 2 mM EDTA; pH 7.4). Fluorescence intensity, indicating transfer of phospholipid molecules from donor to acceptor particles, was measured immediately using an excitation wavelength of 465 nm and an emission wavelength of 535 nm. After samples were incubated for 15 min at 37°C, fluorescence was measured again. PLTP activity was calculated by subtracting initial from final fluorescence.

Western blot analysis. Protein expression in liver homogenates was determined by Western blot analysis using polyclonal rabbit anti-mouse antibodies to SR-BI and Abca1 (Novus Biologicals; Littleton, CO). Blots were stripped and reprobed with {beta}-actin antibody (Sigma) to control for differences in protein loading. Contents of apolipoproteins (apolipoproteins A-I and E) in plasma and in fractions from density gradient ultracentrifugation were determined using appropriate antibodies (Biodesign; Saco, ME). Detection was by enhanced chemiluminescence.

Northern blot analysis. Hepatic expression of Abca1 mRNA was quantified by Northern blot analysis using a cDNA probe generously provided by Dr. Nan Wang (Columbia University, New York, NY) and a cDNA encoding mouse {beta}-actin (La Jolla, CA). cDNAs were radiolabeled with [{alpha}-32P]dCTP (Perkin-Elmer Life Sciences; Torrance, CA) using a random primer kit (Invitrogen). After hybridization, blots were subjected to autoradiography and quantified by densitometry using a FluorChem 8900 Imaging system (Alpha Innotech; San Leandro, CA). Abca1 expression was normalized to {beta}-actin to account for differences in mRNA loading.

Quantitative real-time PCR. Hepatic mRNA expression of hepatic lipase and endothelial lipase was determined by quantitative real-time PCR (18). Briefly, total RNA was prepared from the mouse liver, followed by first-strand cDNA synthesis (Applied Biosciences; Foster City, CA). Gene-specific primers were designed for hepatic lipase (forward 5'-GCTTCAGCCAAGGTCTATGC-3' and reverse 5'-TAGGCTCTACCGGCTTCTCA-3') and endothelial lipase (forward 5'-CTTCCAGTGCACAGACTCCA-3' and reverse 5'-GGGTGTCCCCACTGTTATTG-3') using Primer3 software (26). PCR was performed on a LightCycler (Roche Applied Science) using SYBR green for DNA detection and cyclophilin for normalization.

Statistical Analysis

Data are expressed as means ± SE. Differences between experimental groups were determined using Student’s t-test assuming equal variance. Differences were considered to be significant for two-tailed P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Figure 1 displays the plasma lipid concentrations in Pctp–/– and wild-type mice. Cholesterol and phospholipid concentrations in chow-fed Pctp–/– mice were not different from those in wild-type controls, and triglyceride concentrations were slightly lower (Fig. 1A). Upon challenge with the high-fat/high-cholesterol diet (Fig. 1B), plasma cholesterol and phospholipid concentrations increased, whereas triglycerides decreased, in both Pctp–/– and wild-type mice. Compared with high-fat/high-cholesterol-fed wild-type mice, cholesterol, phospholipid, and triglyceride concentrations were higher in Pctp–/– mice fed the high-fat/high-cholesterol diet.



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  		Fig. 1. Influence of phosphatidylcholine transfer protein (PC-TP) on steady-state plasma lipid concentrations. Plasma concentrations of cholesterol, phospholipids, and triglycerides were determined for wild-type (solid bars) and Pctp–/– (open bars) mice fed either chow (A; wild type: n = 9; Pctp–/–: n = 10) or a high-fat/high-cholesterol diet (B; wild type: n = 8; Pctp–/–: n = 10) for 7 days. Error bars represent SE. *P < 0.05, wild-type vs. Pctp–/– mice; {dagger}P < 0.05, chow-fed vs. high-fat/high-cholesterol-fed mice.

 
In mice fed chow, the absence of PC-TP expression had no impact on the distribution of the major phospholipid classes (wild type: 62, 7, 17, and 14% and Pctp–/–: 60, 8, 17, and 15% phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and lysophosphatidylcholine, respectively) in total plasma samples. The same was true after mice were fed the high-fat/high-cholesterol diet (wild type: 63, 6, 11, and 20% and Pctp–/–: 59, 8, 15, and 18% phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and lysophosphatidylcholine, respectively). The most abundant fatty acid species in each phospholipid class were 16:0, 18:0, and 18:2n6, and there were no differences between Pctp–/– and wild-type mice irrespective of diet (data not shown).

To examine the influence of PC-TP on the distribution of plasma lipids among lipoprotein particles, we used complementary separation techniques. Figure 2 displays FPLC profiles of plasma from Pctp–/– and wild-type mice. Whereas the cholesterol contained in very-low-density (VLDL) and low-density lipoproteins (LDL) plus the cholesteryl ester-rich HDL subfraction, HDL1, eluted at similar volumes in chow-fed mice of both genotypes (Fig. 2A), the peak of HDL-cholesterol eluted at higher volumes in Pctp–/– than wild-type mice. Although the data dispersion was greater, HDL phospholipids in chow-fed Pctp–/– mice also appeared to be skewed toward higher elution volumes (Fig. 2C) in the absence of changes in the elution volumes of VLDL and LDL/HDL1. When fed the high-fat/high-cholesterol diet, the distributions of elution volumes for both the HDL-cholesterol and phospholipid peaks in Pctp–/– mice appeared more similar to high-fat/high-cholesterol-fed wild-type mice (Fig. 2, B and D) than to their chow-fed counterparts. Not shown in Fig. 2 are data for triglycerides for chow and high-fat/high-cholesterol-fed mice, which eluted at the void volume in each experiment and were not influenced by PC-TP expression.



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  		Fig. 2. Influence of PC-TP on lipoprotein sizes. Equal volumes of plasma were pooled and fractionated by fast protein liquid chromotography (FPLC). Concentrations of cholesterol (A and B) and phospholipid (C and D) were determined in fractions for wild-type (solid diamonds) and Pctp–/– (open circles) mice fed either chow (A and C; wild type: n = 9; Pctp–/–: n = 10) or a high-fat/high-cholesterol diet (B and D; wild type: n = 8; Pctp–/–: n = 10) for 7 days. Distributions of lipids among very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL)/subfraction 1 of high-density lipoprotein (HDL1), and HDL (12) are indicated. Because of unequal loading of the FPLC column with up to 200 µl of pooled mouse plasma, differences in magnitudes of peaks did not necessarily vary in proportion to plasma lipid concentration in Fig. 1. Data are representative of 2 independent experiments.

 
To confirm and extend the FPLC findings, plasma samples were fractionated according to density. Figure 3 shows the lipid compositions of the fractions obtained by discontinuous density gradient ultracentrifugation. In plasma of chow-fed wild-type mice, the major peaks of cholesterol (Fig. 3A) and phospholipid (Fig. 3C) were each positioned at a density of 1.092 g/ml. Whereas a minor peak of cholesterol and phospholipid sedimented at a similar density (1.075 g/ml) in plasma of chow-fed Pctp–/– mice (Fig. 3, A and C), the major peaks of cholesterol and phospholipid were positioned at a higher density of 1.139 g/ml. By contrast, the major cholesterol and phospholipid peaks sedimented at identical densities of 1.100 g/ml when mice of both genotypes were fed the high-fat/high-cholesterol diet (Fig. 3, B and D). Triglycerides were concentrated in lower density fractions and were not influenced by genotype for mice fed either chow or the high-fat/high-cholesterol diet (data not shown).



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  		Fig. 3. Influence of PC-TP expression on lipoprotein densities. Equal volumes of plasma (n = 5 mice/group) were pooled and subjected to density gradient ultracentrifugation. Concentrations of cholesterol (A and B) and phospholipid (C and D) were determined in fractions prepared from plasma of wild-type (solid diamonds) and Pctp–/– (open circles) mice fed either chow (A and C) or a high-fat/high-cholesterol diet (B and D) for 7 days. Data are representative of 2 independent experiments.

 
To assess the distribution of apolipoproteins, fractions from density gradients were subjected to agarose gel electrophoresis and Western blot analysis (Fig. 4). Consistent with their densities, particles with pre-{beta}-mobility (VLDL) and {beta}-mobility (LDL) were present in fractions 1–4 in both wild-type and Pctp–/– mice fed chow. Fractions 5–7 contained {alpha}-migrating particles, consistent with HDL, and traces of pre-{beta}-migrating HDL particles were detected in higher density fractions 8 and 9. The peaks of lipid concentration differed in wild-type and Pctp–/– mice. Peak lipid concentrations in chow-fed wild-type mice were concentrated in fraction 6 (density 1.092 g/ml), in agreement with the peaks of cholesterol and phospholipid shown in Fig. 3, A and C. In chow-fed Pctp–/– mice, the altered distribution of cholesterol and phospholipids was reflected by the major lipid band at fraction 7 (density 1.139 g/ml), with a smaller peak at fraction 5 (density 1.075 g/ml). In chow-fed wild-type mice, Western blot analyses demonstrated that apolipoproteins A-I and E were concentrated in fraction 6, consistent with the lipid staining of the agarose gels. Both of these apolipoproteins mirrored the altered distribution of lipids between fractions 5 and 7 in Pctp–/– mice fed chow. The absence of PC-TP expression did not influence plasma total concentrations of apolipoproteins A-I or E, and densitometric analyses of Western blots revealed that the distributions of apolipoprotein A-I among HDL particles were similar in wild-type mice and Pctp–/– mice. Because peak 6 (wild-type mice) and peak 7 (Pctp–/– mice) contained similar amounts of phospholipids (Fig. 3A), this indicated that the apolipoprotein A-I-to-phospholipid ratios of HDL particles were also similar. When mice were fed the high-fat/high-cholesterol diet, the differences in lipid and apolipoprotein distributions between wild-type and Pctp–/– mice were no longer apparent.



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  		Fig. 4. Electrophoretic mobilities and apolipoprotein contents of lipoproteins in Pctp–/– and wild-type mice. Wild-type and Pctp–/– mice were fed either chow or a high-fat/high-cholesterol diet for 7 days. Equal volumes of fractions obtained from discontinuous density ultracentrifugation (see Fig. 3) were subjected to agarose gel electrophoresis and then stained for lipids. Contents of apolipoprotein (apo)A-I and apoE were determined by Western blot analyses after equal loading of fractions on polyacrylamide gels. Data are representative of 2 independent experiments.

 
Notwithstanding the absence of a measurable increase in the apolipoprotein A-I-to-phospholipid ratio, data from FPLC and density gradient ultracentrifugation indicated that HDL particles in chow-fed Pctp–/– mice were smaller and more dense than in wild-type mice. Because Abca1 in the liver is a key determinant of plasma HDL formation (3), we analyzed hepatic Abca1 expression. PC-TP expression did not influence either expression of Abca1 protein (Fig. 5A) or mRNA (data not shown). PLTP is a circulating plasma enzyme that is unrelated to PC-TP and plays an important role in the maturation of HDL particles by transferring phospholipids from the surface coat of remnant particles to HDL (13). We explored whether the effects of PC-TP expression on HDL size may have been due to compensatory regulation of PLTP. As shown in Fig. 5B, there was no difference in PLTP activity in Pctp–/– compared with wild-type mice. In addition to PLTP, vascular lipases are key determinants of plasma HDL composition and particle size. Endothelial lipase and, to a lesser degree, hepatic lipase display phospholipase activities, which play important roles in HDL metabolism (17). However, as shown in Fig. 5C, PC-TP expression did not influence mRNA expression of either lipase.



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  		Fig. 5. PC-TP does not influence ATP-binding cassette protein A1 (Abca1) expression in the liver, plasma phospholipid transfer protein (PLTP) activity, or mRNA expression of hepatic and endothelial lipase. A: Western blot analysis of hepatic Abca1 expression using {beta}-actin to control for unequal loading. B: PLTP activities in plasma (n = 5 mice/group) were determined for wild-type (solid bars) and Pctp–/– (open bars) mice. C: relative levels of mRNA expression of hepatic and endothelial lipase (wild type: n = 6; Pctp–/–: n = 7) in livers of wild-type (solid bars) and Pctp–/– (open bars) mice were determined by quantitative real-time PCR using cyclophilin as an invariant control. Relative expression was set at 100% for wild-type mice. Error bars represent SE.

 
In plasma, steady-state concentrations of lipoproteins represent a balance of production and clearance, principally by the liver. To evaluate hepatic HDL uptake in the absence of PC-TP, we measured the disappearance from plasma and the hepatic appearance of HDL2 particles containing a 3H-labeled nonhydrolyzable cholesteryl ester analog and 125I-labeled protein (Fig. 6). This assessed SR-BI-mediated selective lipid uptake by the liver (29). We observed no difference in the disappearance of [3H]cholesteryl oleyl ether from chow-fed mice (Fig. 6A) and no difference in uptake into the liver (Fig. 6C). By contrast, in high-fat/high-cholesterol-fed mice, the rate of [3H]cholesteryl oleyl ether clearance from plasma was reduced (Fig. 6B). This was associated with a marked reduction of hepatic cholesteryl ester uptake in Pctp–/– mice after they were fed the high-fat/high-cholesterol diet (Fig. 6D). Not shown are data for the disappearance of 125I-labeled HDL protein, which did not differ for Pctp–/– versus wild-type mice fed either chow or the high-fat/high-cholesterol diet.



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  		Fig. 6. Influence of PC-TP on HDL clearance from plasma. HDL clearance from plasma was determined for wild-type (solid diamonds) and Pctp–/– mice (open circles) fed chow (A and C; wild type: n = 5; Pctp–/–: n = 4) or a high-fat/high-cholesterol diet (B and D; wild type: n = 7; Pctp–/–: n = 6) for 7 days. The percentage of [3H]cholesteryl oleyl ether taken up into the liver was determined for wild-type (solid bars) and Pctp–/– (open bars) mice. Insets show representative Western blots of scavenger receptor class B type I (SR-BI) expression using {beta}-actin to control for unequal loading. Error bars represent SE. *P < 0.05, wild-type vs. Pctp–/– mice.

 
As shown in Fig. 1B, the plasma total cholesterol concentration in Pctp–/– mice fed the high-fat/high-cholesterol diet was 20% higher than that in wild-type littermate controls. Fractionation by both FPLC (Fig. 2B) and density gradient ultracentrifugation (Fig. 3B) indicated that the excess cholesterol was distributed approximately equally among the lipoprotein fractions. To quantify HDL clearance and take into account the increased plasma HDL-cholesterol concentration in high-fat/high-cholesterol diet-fed Pctp–/– mice, we calculated the fractional catabolic rates as well as the production rates of HDL-cholesterol using the mean values shown in Fig. 6, A and B (23). The fractional catabolic rates of Pctp–/– and wild-type mice fed chow were similar (wild type: 0.13 pools/h and Pctp–/–: 0.19 pools/h). In wild-type mice fed the high-fat/high-cholesterol diet, the fractional catabolic rate was slightly increased (0.19 pools/h) but was reduced substantially in Pctp–/– mice to 0.03 pools/h. HDL production rates, which were only modestly influenced by PC-TP expression in chow-fed mice (wild type: 4.8 mg/h and Pctp–/–: 7.0 mg/h) also decreased sharply in high-fat/high-cholesterol-fed Pctp–/– mice (wild type: 7.0 mg/h and Pctp–/–: 1.6 mg/h).

To determine whether decreased hepatic uptake in Pctp–/– mice fed the high-fat/high-cholesterol diet could be attributed to changes in receptor expression, we measured SR-BI expression in the liver by Western blot analysis (Fig. 6, C and D, insets). The absence of PC-TP did not affect SR-BI expression in mice from either genetic background fed chow or the high-fat/high-cholesterol diet.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The hypothesis that PC-TP plays a role in regulating HDL metabolism derives from several observations in the literature. Phospholipids contained in nascent pre-{beta}-HDL particles formed by interactions of lipid-poor apolipoprotein A-I with the plasma membranes of cells in tissue culture are highly enriched (>80%) with phosphatidylcholines (15, 43), as are HDL particles newly synthesized by isolated perfused rat livers (24). PC-TP, which binds phosphatidylcholines with high specificity (37), is expressed in both hepatocytes (36, 37) and macrophages (2). Suggestive of a role in HDL formation, we have demonstrated that PC-TP promotes apolipoprotein A-I mediated phospholipid and cholesterol efflux using Chinese hamster ovary (CHO) cells (1) as well as mouse peritoneal macrophages from wild-type and Pctp–/– mice (2).

This study was designed to determine whether Pctp–/– mice exhibit phenotypes that are consistent with a role for PC-TP in HDL metabolism in vivo. The main findings were that, although phosphatidylcholine concentrations were not decreased in plasma of Pctp–/– mice fed a chow diet, there was an accumulation of small {alpha}-migrating HDL particles. When mice were challenged with dietary cholesterol in the form of a high-fat/high-cholesterol diet, increased steady-state HDL-cholesterol concentrations were associated with decreased rates of hepatic clearance.

There are two primary sources of phospholipid molecules that comprise HDL particles. During HDL formation, phospholipids and cholesterol become incorporated together with apolipoprotein A-I into pre-{beta}-HDL particles due to the activity of Abca1 (21). The other main source is the phospholipid surface coat of remnant VLDL and chylomicrons, which are transferred to HDL particles in plasma by the activity of PLTP (13, 32).

Apolipoprotein A-I-mediated cellular efflux of phospholipids via Abca1 is highly selective for phosphatidylcholines (7, 15, 43). The high degree of specificity of PC-TP for binding phosphatidylcholines (37), taken together with our observation that overexpression of PC-TP in CHO cells enhances apolipoprotein A-I-mediated pre-{beta}-HDL formation, led us to suggest that PC-TP in liver cytosol might play a critical role in delivering phosphatidylcholines to the plasma membrane for incorporation by Abca1 into pre-{beta}-HDL particles (1). However, the absence of PC-TP in mice had no affect on hepatic Abca1 mRNA or protein expression, and we observed no difference in plasma total phospholipid concentrations in chow-fed Pctp–/– versus wild-type animals. Moreover, the proportions of plasma phosphatidylcholines did not differ, and there were no changes in molecular species. Taken together, these observations argue against an essential role for PC-TP in the formation of pre-{beta}-HDL particles in vivo.

Interestingly, both FLPC and discontinuous gradient density ultracentrifugation revealed that HDL cholesterol and phospholipids in plasma of Pctp–/– mice were contained in smaller, denser particles, which exhibited {alpha}-mobility by agarose gel electrophoresis. These findings are consistent with size measurements of pre-{beta}-HDL that were formed by exposing PC-TP-transfected CHO cells to apolipoprotein A-I (1). FPLC-determined particle sizes were increased in proportion to cellular expression of PC-TP (1), which suggested that PC-TP in vivo might enrich HDL particles with phospholipids, possibly by enhancing hepatocellular trafficking of phosphatidylcholines to the plasma membrane (1, 40). In the current study, we would have expected that the ratio of apolipoprotein A-I to phospholipid to be increased in the denser HDL particles that were observed in Pctp–/– mice, even in the absence of changes in Abca1 expression. Acknowledging limitations in sensitivity of an analysis based on densitometry, it is possible that an increase in ratio that was sufficient to alter HDL density was not detected in our experiments.

Without intrinsic selectivity for phosphatidylcholines (11), PLTP in plasma plays a key role in enriching HDL particles with phospholipids from the surface coat of remnant particles (13). Indeed, the majority of phospholipid molecules contained in mature HDL particles are attributable to the activity of PLTP (14). We observed no PC-TP-dependent differences in PLTP activity in vitro. However, it is possible that the smaller HDL particles in plasma of Pctp–/– mice were less optimal substrates for PLTP in vivo. It has been shown that isolated mature HDL particles typically display {alpha}-mobility on agarose gels, whereas subclasses of HDL with a range of electrophoretic mobilities from pre-{beta} to {alpha} coexist in the plasma (4). These subclasses have varied surface charges (31), which differentially modulate PLTP activity (6). On this basis, HDL particles formed in vivo in the absence of PC-TP may have been suboptimal PLTP substrates and thereby may have failed to acquire a full complement of phospholipid molecules in chow-fed mice. Normalization of HDL size in high-fat/high-cholesterol-fed Pctp–/– mice may have been due to the high abundance of remnant particles in plasma. This would have served to increase the gradient of phospholipids between the surface coats of the remnant particles and HDL, thereby promoting PLTP-mediated transfer of phospholipids to the small HDL particles by mass action. Whereas hepatic and endothelial lipases have substantial phospholipase activity and participate in intravascular metabolism of HDL particles (17), we did not detect differences in mRNA expression between Pctp–/– and wild-type mice.

In chow-fed mice, hepatic uptake of human HDL2 was not impaired in Pctp–/– mice, suggesting that PC-TP played no role in determining steady-state plasma lipid concentrations. However, an important limitation of the present study is that we utilized purified human HDL2 to measure selective lipid uptake by the liver. Whereas this experimental design was sufficient to exclude an effect of PC-TP expression on the function of SR-BI (29), it did not take into consideration that the small HDL particles present in Pctp–/– mice might themselves constitute suboptimal SR-BI ligands. To evaluate this possibility will require a systematic comparison of the influence of PC-TP expression on hepatic uptake of HDL particles purified from plasma of both Pctp–/– and wild-type mice.

After being fed the high-fat/high-cholesterol diet, plasma steady-state cholesterol and phospholipid concentrations in Pctp–/– mice were higher than in wild-type mice. This occurred in the absence of an increase in Abca1 mRNA or protein expression levels, suggesting that increases in the rate of pre-{beta}-HDL formation did not contribute to plasma cholesterol concentrations. Rather, we observed a decrease in the rate at which HDL2-derived cholesterol disappeared from plasma and appeared in the liver, as evidenced by a markedly reduced fractional catabolic rate. The absence of changes in hepatic clearance of HDL2 protein is consistent with a defect in SR-BI-mediated selective lipid uptake (5) in high-fat/high-cholesterol-fed Pctp–/– mice. Interestingly, the decrease in selective uptake was not associated with reduced overall SR-BI protein expression. This might be explained by alterations in hepatocellular protein trafficking of SR-BI, which plays a key role in the regulation of function (16, 27) and was not assessed in our experiments. In this connection, we have provided evidence that reduced biliary lipid secretion in high-fat/high-cholesterol-fed C57BL/6J Pctp–/– mice was attributable to defective vesicular trafficking of ATP-binding cassette transporters to the canalicular plasma membrane (39). An alternative explanation for decreased selective uptake is that excess accumulation of unesterified cholesterol in hepatocellular membranes of high-fat/high-cholesterol-fed Pctp–/– mice (38) may have led to impaired function of SR-BI, which resides in fluid microdomains within the plasma membrane (5).

Whereas reduced selective uptake of HDL lipids provides a plausible explanation for increased plasma lipid concentrations in mice lacking PC-TP, there was a corresponding decrease in the HDL production rate. This calculated value incorporates rates of HDL formation together with intravascular metabolism (23). Considering that triglyceride-rich lipoproteins in plasma may modulate HDL metabolism by a number of mechanisms (17), the influence of PC-TP expression on plasma triglyceride concentrations may have also contributed to determining the HDL production rate. Whereas the present study did not address the pathophysiology of increased plasma triglyceride concentrations in high-fat/high-cholesterol-fed Pctp–/– mice, Yao and Vance (41, 42) have demonstrated that VLDL production is critically dependent on the hepatocellular supply of phosphatidylcholines. It is therefore possible that PC-TP in the liver could play a regulatory role in the formation of VLDL particles.

In summary, we provided evidence in vivo that PC-TP participates in HDL metabolism. Taken together with previous observations of impaired biliary lipid secretion in response to dietary cholesterol (39) and altered hepatic cholesterol (38) in Pctp–/– mice, the present findings support a physiological role of PC-TP in reverse cholesterol transport.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-56626 and DK-48873, an Established Investigator Award from the American Heart Association, and an International HDL Research Awards Program grant (to D. E. Cohen). M. K. Wu is the recipient of an American Liver Foundation Student Research Fellowship.


   ACKNOWLEDGMENTS
 
The authors thank Drs. Philippe Frank, Suheeta Roy, David Silver, Xian-Chen Jiang, and Keishi Kanno for helpful discussions and Earl Macatangay for editorial assistance.

Present address of M. K. Wu: Div. of Gastroenterology, Dept. of Medicine, Brigham and Women’s Hospital, Thorn 1405, 75 Francis St., Boston, MA 02115.


   FOOTNOTES
 

Present address for reprint requests and other correspondence of D. E. Cohen: Gastroenterology Div., Dept. of Medicine, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115 (e-mail: dcohen{at}partners.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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