Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M502200200 on March 29, 2005

J. Biol. Chem., Vol. 280, Issue 22, 21612-21621, June 3, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/22/21612    most recent
M502200200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zheng, H.
Right arrow Articles by Marcel, Y. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zheng, H.
Right arrow Articles by Marcel, Y. L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

ApoA-I Lipidation in Primary Mouse Hepatocytes

SEPARATE CONTROLS FOR PHOSPHOLIPID AND CHOLESTEROL TRANSFERS*

Hui Zheng{ddagger}, Robert S. Kiss§, Vivian Franklin, Ming-Dong Wang, Bassam Haidar§, and Yves L. Marcel

From the Lipoprotein and Atherosclerosis Research Group, University of Ottawa Heart Institute, Departments of Biochemistry, Microbiology, and Immunology, and of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Ontario K1Y 4W7, Canada

Received for publication, February 25, 2005 , and in revised form, March 25, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The liver is the major site of both apolipoprotein A-I (apoA-I) synthesis and ATP-binding cassette transporter A1 (ABCA1) expression. Here, we compare the lipidation with cholesterol and phospholipid of newly synthesized human apoA-I (hapoA-I) using adenoviral vector-mediated endogenous expression or exogenously added hapoA-I in wild type and ABCA1-null hepatocytes. Hepatocytes were labeled with [3H]cholesterol (delivered with LDL or methyl-{beta}-cyclodextrin), [3H]mevalonate, or [3H]choline. ABCA1 deficiency decreased apoA-I phospholipidation by 80%, but acquisition of de novo synthesized and exogenous cholesterol only decreased by 40–60%. The transfer of de novo synthesized cholesterol to apoA-I was decreased at all time points, but that of exogenously delivered cholesterol was independent of ABCA1 activity at the early time points. Progesterone does not affect apoA-I synthesis or its lipidation but inhibited the early phase of apoA-I cholesterol lipidation in both wild type and ABCA1-null hepatocytes. Fast protein liquid chromatography analysis of medium lipoproteins confirmed that with ABCA1 deficiency, the proportion of secreted high density lipoprotein-associated apoA-I and cholesterol decreased by about 50%. The very low density lipoprotein (VLDL)/LDL size fraction also contained a significant level of cholesterol in ABCA1 deficiency, consistent with the result of immunoprecipitations showing the presence of lipoproteins with both apoA-I and murine apoB. ApoA-I lipidation with newly synthesized cholesterol in ABCA1-null hepatocytes was significantly decreased by brefeldin A and monensin. In conclusion, we demonstrate that: (i) whereas most hepatic phospholipidation of apoA-I is mediated by ABCA1, acquisition of cholesterol depends on active transfer from intracellular compartments by ABCA1-dependent and -independent pathways, both sensitive to progesterone and (ii) there is separate regulation of phospholipid and cholesterol lipidation of apoA-I in hepatocytes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The ATP-binding cassette transporter protein, ABCA1,1 is the major determinant of the phospholipid and cholesterol lipidation of apolipoprotein A-I that enables formation of high density lipoprotein (HDL) (1). ABCA1 has been shown to regulate lipid efflux, and a deficiency in ABCA1, as in Tangier disease, results in nearly undetectable levels of HDL (2–6). Macrophages in various tissues are profoundly affected by this disease, first suggesting that ABCA1 function in macrophages was critical for the maintenance of HDL levels in the plasma, as part of the reverse cholesterol transport. However, Haghpassand et al. (7) demonstrated that macrophages account for only 20% of the generation of circulating HDL. ABCA1 (4, 8–12) and apoA-I (13, 14) are both highly expressed in the liver and intestine, suggesting that ABCA1 contributes to the lipidation of newly secreted lipoproteins in those tissues. Basso et al. (15) used an ABCA1 adenovirus expression system to promote liver expression of ABCA1, thereby raising plasma HDL-cholesterol and promoting cholesterol efflux from primary hepatocytes, supporting the role of the liver as a major source of HDL.

As noted by Tall et al. (16), the importance of ABCA1-mediated lipidation of apoA-I in the liver challenges the concept of reverse cholesterol transport. If apoA-I secreted by hepatocytes enters the circulation in a lipidated form, the cholesterol thus exported by the liver together with apoA-I contributes to the forward cholesterol transport and must subtract from the cholesterol eventually returned to the liver and presumed to be derived from peripheral tissues. Clearly, the nature and composition of apoA-I-containing lipoproteins secreted by the hepatocytes and their quantitative contribution to the pool of circulating HDL require further studies.

Earlier studies of the synthesis and secretion of apoA-I in liver and hepatocytes provided good evidence for the intracellular and pericellular lipidation of apoA-I (17–21). Although the formation of lipoproteins with the density of HDL was not observed in well defined subcellular fractions (22), recent studies supported the notion of intracellular lipidation (23, 24). However, we have overall a limited knowledge of the process and pathways of HDL assembly in hepatocytes and of the nature of lipoproteins secreted. In primary mouse hepatocytes, we demonstrated that endogenously synthesized apoA-I acquired more phospholipid in an ABCA1-dependent manner than exogenously added apoA-I, therefore indicating that intracellular lipidation of apoA-I is greater than pericellular lipidation by the efflux process (25). We also demonstrated the existence of a limited ABCA1-independent lipidation pathway for newly synthesized apoA-I (25). Cholesterol lipidation of exogenous apoA-I in hepatocytes was investigated by Sahoo et al. (26), who showed lipidation of the exogenous apoA-I and increased formation of lipoproteins containing endogenous murine apoA-I. However, the cholesterol lipidation of endogenously synthesized apoA-I has not been evaluated.

Here, we extend our previous studies by comparing the kinetics of cholesterol and phospholipid acquisition by newly synthesized and secreted apoA-I and contrasting it with that of exogenously added apoA-I in primary hepatocytes from wild type and ABCA1-/- mice. We also compare the acquisition of cholesterol derived from exogenous sources and endogenously synthesized. The results, recently presented and published in abstract form (27), show that newly synthesized cholesterol is transferred to apoA-I during secretion in a partially ABCA1-dependent manner. A major transfer of cholesterol also occurs at the cell surface, some of which is independent of ABCA1. Surprisingly, the acquisition of cholesterol by apoA-I is less dependent on ABCA1 activity than its phospholipidation, which provides new evidence for the dissociation of phospholipid and cholesterol transfers by ABCA1. We also analyzed the cellular origin of the cholesterol transferred to apoA-I, which is mostly derived from intracellular pools and depends on active transfer.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—[1,2-3H]-Cholesterol, [5-3H(N)]mevalono-lactone-Rs, choline [methyl-3H]chloride were obtained from PerkinElmer Life Sciences. Williams' Medium E, HepatoZYME-SFM, and antibiotic-antimycotic were purchased from Invitrogen. Rabbit polyclonal anti-human apoA-I antibody was purchased from Calbiochem. Monoclonal antibodies directed against human apoA-I (a combination of 4H1 (against the extreme N terminus) and 5F6 (against the central region)) were described previously (28, 29) and biotinylated with sulfo-NHS-biotin from Pierce. Polyclonal anti-mouse apoB antibody was purchased from Biodesign International. Streptavidin-horseradish peroxidase conjugate and protein G-Sepharose were obtained from Amersham Biosciences. Progesterone (4-pregnene-3, 20-dione), brefeldin A, monensin, and other reagents were obtained from Sigma.

Animals and Primary Hepatocyte Cultures—Wild type C57BL/6J mice were purchased from Jackson Laboratories. ABCA1-deficient mice (a kind gift from Dr. Edward M. Rubin, Department of Energy Joint Genome Institute, Berkeley, CA) have been generated as follows. A 1.65-kb PCR-generated fragment containing abca1 exon 16 was inserted between the phosphoglycerate kinase (PGK) terminator of the PGKNeo cassette and the double PGKtk cassettes of pPN2T at EcoRI and BamHI sites. A 12-kb PCR-generated fragment from intron 22 to exon 30 was then inserted on the PGK promoter side of the PGKneo at blunted XhoI and NotI sites to generate the final targeting vector pAbc1SALA. The Stratagene TaqPlus long PCR system was used for amplification of 12-kb fragments. ESVJ ES cells from Genomesystems were electroporated with NotI-linearized pAbc1SALA and subjected to G418 and 2'-fluoro-2'-deoxy-1-{beta}-D-arabinofuranosyl-5-iodouracil (FIAU) selection. Correctly targeted clones were screened by PCR and then confirmed by Southern blotting. Chimeric mice were generated using standard procedures. Chimeric males were bred with C57BL/6J (Charles River Laboratories) to create mice heterozygous for the targeted allele on the 129Sv/C57 mixed background. 129SV/C57 mice have higher baseline HDL levels and are less susceptible to atherosclerosis than the C57BL/6J (30) and, as a result, provide an easier phenotype to detect differences in HDL synthesis and secretion. All mice were maintained on a normal chow diet in a 12-h light/12-h dark schedule and used between the ages of 4 and 6 months. All experiments performed were in accordance with protocols approved by the University of Ottawa Animal Care Committee. Primary hepatocytes were isolated from these mice by liver collagenase perfusion according to the established protocols (31, 32). Briefly, the cells were plated in fibronectin precoated (25 µg/well) 6-well plates at an initial density of 1.5 x 106 cells/well in Williams' Medium E containing penicillin (100 units/ml), streptomycin sulfate (100 units/ml), Fungizone (250 ng/ml), and 10% fetal bovine serum.

Cell Labeling and Adenovirus Infection—Five h following the initial seeding, the cells were washed in Williams' Medium E without serum (2 x 2 ml) and incubated with HepatoZYME-SFM containing antibiotic-antimycotic, 10 µCi/ml [3H]mevalonate, or 5 µCi/ml [3H]cholesterol delivered with low density lipoprotein (LDL) or [3H]choline. Mevalonate label is incorporated predominantly into cholesterol (33). The following day (24 h), the labeled medium was removed, and the cells were infected for 1 h with either the recombinant adenovector expressing human apoA-I (Ad5-Ad AI) or as control, adeno-luciferase (Ad5-Ad Luc) at a multiplicity of infection of 75:1 plaque-forming units/cell in Williams' Medium E without serum (28, 29). After 1 h of infection, hepatocytes were incubated for an additional 24 h with original labeling medium as described above. To label the plasma membrane cholesterol pool, hepatocytes were cooled down to 4 °C for 1 h on the 3rd day. The medium was replaced by 5 mM methyl-{beta} cyclodextrin (m{beta}CD): [3H]cholesterol (5 µCi/ml) at a molar ratio 8:1 for 3 h at 4 °C (34, 35). After washing three times, the cells were immediately used for efflux assays.

Time Course of [3H]Cholesterol and [3H]Choline-Phospholipid Efflux Assay—On the 3rd day, hepatocytes were equilibrated in non-labeled HepatoZYME-SFM for 1 h at 37 °C. Following 2 x 2 ml washes in Williams' Medium E, the cells were incubated with non-labeled HepatoZYME-SFM (1 ml/well) in the absence (endogenous apoA-I study) or presence of 5 µg of human apoA-I (hapoA-I) (exogenous-apoA-I study). The cells were returned to the 37 °C incubator (5% CO2) for the various times, and the efflux medium was subsequently collected at different time points and spun down to pellet any cell debris. All media were analyzed as described below. In some experiments, plasma membrane cholesterol was extracted by treatment with 20 mM m{beta}CD at 4 °C for 15 min. In other experiments, 10 µg/ml progesterone, an inhibitor of cholesterol transport from intracellular compartments to the plasma membrane (36–39), was added 15 min before and during the efflux period, and the media were analyzed in the same manner.

Distribution of Secreted ApoA-I in the Different Lipoprotein Fractions—The medium from five 6-well plates (30 wells) were pooled and concentrated down to 2 ml with Amicon 10K filter units. The sample was immediately loaded on calibrated Superdex 200 columns connected in series as described previously for isolation of lipoproteins from plasma samples (28). Very low density lipoprotein (VLDL)- and LDL-sized species eluted close to the void volume of the column, whereas HDL particles and smaller very high density lipoprotein (VHDL) fractions containing albumin (7.1 nm diameter) were well separated. Aliquots from each fraction were analyzed for apoA-I by immunoblot analysis following transfer to nitrocellulose with a slot blot apparatus (Bio-Rad Bio-Dot SF unit) as described previously (28). The concentration of apoA-I in the VLDL/LDL, HDL, and VHDL pools was determined by densitometric scanning (Bio-Rad software, Quantity One, version 4.11). Analysis of medium collected from hepatocytes infected with the Ad Luc was also carried out as negative control for background signal. For comparison, the relative distribution of murine apoB (apoB48 and apoB100) was also measured using the polyclonal anti-mouse apoB antibody and visualized by chemiluminescence (Pierce West Pico SuperSignal substrate, Pierce) after incubation with horseradish peroxidase-conjugated anti-rabbit IgG.

Immunoprecipitation of ApoA-I-associated Cholesterol and Choline-Phospholipids—ApoA-I from hepatocyte medium was immunoprecipitated either directly from the efflux medium or from lipoprotein fractions separated by FPLC. The immunoprecipitations were carried out with a polyclonal anti-human apoA-I antiserum from sheep followed by protein G-Sepharose. An equal volume of an anti-human apoB antiserum from sheep, which does not cross-react with murine apoB, or the efflux medium from time 0 that immunoprecipitated with anti-human apoA-I antiserum from sheep was used as a background control. The immunoprecipitates were collected as described previously (25). The cholesterol and phospholipid associated with hapoA-I were quantified by scintillation counting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
We have previously provided evidence for the phospholipidation of apoA-I during secretion and post-secretion at the cell surface and shown that most but not all (80%) of this phospholipidation is ABCA1-dependent (25). Here, our first objective was to compare the kinetics of cholesterol lipidation of apoA-I during secretion in relation to that of phospholipidation. Although [3H]choline labeling allows uniform labeling of phosphatidylcholine and sphingomyelin in cellular membranes, labeling of cholesterol pools varies with methods used to deliver the labeled cholesterol or its precursors. Therefore, we have first evaluated the distribution of label in several models of cellular cholesterol labeling.

Quantification of Different Cellular Pools of [3H]Cholesterol by Accessibility of Cholesterol to m{beta}CD Extraction at Different Temperatures—To quantify the distribution of [3H]cholesterol in the different cellular compartments as a function of the labeling method, hepatocytes were labeled with [3H]mevalonate, [3H]cholesterol delivered by LDL ([3H]cholesterol-LDL), or [3H]cholesterol delivered by m{beta}CD ([3H]cholesterol-m{beta}CD) as described under "Experimental Procedures." Using the accessibility of [3H]cholesterol to m{beta}CD extraction (20 mM) at 37 and 4 °C, we estimated the labeling of different pools: plasma membrane, defined by accessibility at 4 °C; recycling endosome compartment, represented by the difference between accessibility at 4 and 37 °C; and other intracellular pools, represented by [3H]cholesterol that is not accessible at 37 °C. Low concentrations of m{beta}CD have been used as a carrier to deliver cholesterol to the cells (40, 41), whereas higher concentrations extract cholesterol (34, 42). Table I summarized the results of this analysis of the distribution of cellular [3H]cholesterol as a function of labeling methods. m{beta}CD preferentially labeled the plasma membrane cholesterol pool, delivering only 10 and 5% of [3H]cholesterol in the recycling compartment and other intracellular pools, respectively. In contrast, newly synthesized cholesterol derived from [3H]mevalonate preferentially predominantly labeled intracellular compartments (58% of the [3H]cholesterol). [3H]Cholesterol derived from LDL labeled 61% on the plasma membrane and 39% in the intracellular compartments.


View this table:
[in this window]
[in a new window]
  		TABLE I
Quantification of different cellular pools of [3H]cholesterol in C57B control hepatocytes

 
Time Course of Acquisition of Cholesterol and Phospholipid by Newly Synthesized HapoA-I in ABCA1+/+ and ABCA1-/- Hepatocytes—To compare the kinetics of cholesterol and phospholipid acquisition by apoA-I during secretion, primary hepatocytes from ABCA1-wild type and -null mice were isolated and incubated with [3H]mevalonate or [3H]choline for labeling of endogenously synthesized cholesterol and phospholipids, respectively. hapoA-I was expressed in the cells by adenoviral-vector gene transfer for 24 h, and secretion was monitored for various times after a 1-h equilibration period in non-labeled HepatoZYME-SFM. Media collected at each time point were immunoprecipitated with anti-human apoA-I to quantify the radiolabeled lipids specifically associated with hapoA-I (Fig. 1).

In ABCA1+/+ hepatocytes, the acquisition by hapoA-I of newly synthesized phospholipids (Fig. 1A) and newly synthesized cholesterol (Fig. 1B) increased in parallel with time, indicating that newly synthesized apoA-I was secreted with both lipids, although the results did not define where apoA-I acquires these lipids, intracellularly or at the cell surface. Consistent with our previous finding, ABCA1 deficiency resulted in the loss of ~80% of phospholipid acquisition by apoA-I at both 4- and 8-h time points. In contrast, ABCA1 deficiency caused only a loss of 40 and 45% in the acquisition of newly synthesized cholesterol by apoA-I at the respective 4- and 8-h time points. The discrepancy between the rates of cholesterol and phospholipid association with apoA-I first suggested the possibility of differential access to lipid pools and second suggested the heterogeneity in labeling of cholesterol pools.

The hepatocytes were then labeled with exogenously supplied cholesterol derived from LDL and m{beta}CD as indicated previously. As we observed with de novo synthesized cholesterol, in ABCA1 wild type hepatocytes, there was a time-dependent increase of exogenously supplied cholesterol transfer to apoA-I. The data also indicated that with all labeling procedures, the acquisition of cholesterol by newly synthesized and secreted apoA-I was only decreased by 40–60% in ABCA1 deficiency (Fig. 1, B–D). However, with exogenous cholesterol labeling delivered by LDL and m{beta}CD, which preferentially label plasma membrane 61 and 85%, respectively, ABCA1 deficiency had no effect on the early time points of apoA-I cholesterol acquisition, a result not seen with mevalonate-derived cholesterol. This suggests the existence of a pool of cholesterol at the plasma membrane whose transfer to apoA-I is mostly independent of ABCA1. In addition, long term incubation showed the existence of a pathway for cholesterol acquisition by secreted apoA-I that is independent of ABCA1 activity. In these experiments, efflux from m{beta}CD-labeled cells was limited to 4 h to prevent complete redistribution of plasma membrane-delivered cholesterol.

Acquisition of Cholesterol and Phospholipid by Exogenous HapoA-I (exo-apoA-I) in ABCA1+/+ and ABCA1-/- Hepatocytes—To further evaluate the cholesterol acquisition by apoA-I after its secretion at the cell surface by interaction with the plasma membrane and the recycling compartment, apoA-I was added exogenously to the cells. As above, the hepatocytes from ABCA1+/+ and ABCA1-/- mice were labeled with [3H]mevalonate, [3H]cholesterol-LDL, [3H]cholesterol-m{beta}CD, or [3H]choline. To allow comparison with the lipidation of endogenously expressed apoA-I driven by adenovector expression, the cells were also infected with Ad Luc. Exogenous human apoA-I was added during the efflux period, and the specific association of lipids was determined by immunoprecipitation (Fig. 2). The data clearly showed that the hepatocytes also contribute to apoA-I cholesterol acquisition at the cell surface; there was a similar time-dependent transfer of cholesterol derived from all labeling methods (Fig. 2, B, C, and D) to exogenous apoA-I in wild type hepatocytes. Consistent with our previous observations (25) and in parallel to cholesterol, exogenous apoA-I similarly acquired phospholipid in a time-dependent manner in ABCA1 wild type hepatocytes (Fig. 2A).

As with newly synthesized apoA-I, ABCA1 deficiency had no effect on the early time points of exogenous apoA-I acquisition of exogenously supplied cholesterol (Fig. 2, C and D). This was not seen for endogenously synthesized cholesterol derived from mevalonate (Fig. 2B) and for endogenously synthesized phospholipid (Fig. 2A). Again, ABCA1 deficiency had significantly less effect on apoA-I lipidation by cholesterol, which decreased by 25–50% depending on labeling methods, and by phospholipid, which decreased by 75%.

Effect of ABCA1 Deficiency on Distribution of HapoA-I and Cholesterol in the Lipoprotein Fractions Separated by FPLC—To evaluate the distribution of apoA-I-containing lipoproteins secreted by the hepatocytes expressing hapoA-I, efflux media were collected at 4 h, concentrated, and fractionated by FPLC. Fractions corresponding to VLDL/LDL, HDL, and VHDL were collected based on the distribution of markers (murine apoB and human apoA-I). The distribution of immunoreactive human apoA-I in these fractions was quantified by slot blot analysis (Fig. 3A). In ABCA1+/+ hepatocytes, there was a large amount of secreted apoA-I associated with the HDL fraction, and a smaller but significant proportion was found in the VLDL/LDL size fraction. These results are consistent with previous observations in monkey hepatocytes (43). In ABCA1-/- hepatocytes, there was a significant decrease of apoA-I associated with the HDL fraction and a concomitant increase in the VHDL fraction when compared with control hepatocytes. However, large proportions of the total apoA-I were still associated with the VLDL/LDL and HDL fractions in ABCA1 deficiency. These values are higher than those we reported earlier (25), a difference related to the mice strain backgrounds.



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 1.
Comparison of acquisition of phospholipid and cholesterol by newly synthesized hapoA-I in ABCA1+/+ and ABCA1-/- hepatocytes. Cells were isolated and labeled with [3H]choline or [3H]mevalonate for labeling endogenously synthesized phospholipid or cholesterol, respectively, and [3H]cholesterol derived from LDL or [3H]cholesterol delivered by 5 mM m{beta}CD for labeling exogenously supplied cholesterol. On the 2nd day, hepatocytes were infected with Ad AI for 1 h in Williams' Medium E without serum and then switched to the original labeling media for anther 24 h. The 3rd day, after a 1-h equilibration, cells were allowed to efflux in 1 ml/well in non-labeled HepatoZYME-SFM. Media containing newly secreted apoA-I were collected at each time point and immunoprecipitated with anti-human apoA-I and then subjected to scintillation counting. All of the values shown were determined by subtracting the background value of time 0 or the value from immunoprecipitation with anti-human apoB. Newly synthesized apoA-I associated with [3H]choline-phospholipid (A), [3H]mevalonate-derived cholesterol (B), [3H]cholesterol-LDL (C), and [3H]cholesterol-m{beta}CD (D) in ABCA1-wild type (WT) hepatocytes were compared with ABCA1-null hepatocytes (KO). The results presented are representative of three independent experiments (±S.D.).

 



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 2.
Comparison of acquisition of phospholipid and cholesterol by exogenous hapoA-I in ABCA1+/+ and ABCA1-/- hepatocytes. ABCA1-wild type (WT) and -null hepatocytes (KO) were isolated and labeled as in Fig. 1. On the 2nd day, cells were infected with Ad Luc for 1 h in Williams' Medium E without serum and then switched to original labeling media for another 24 h as in Fig. 1. On the 3rd day, efflux was monitored in HepatoZYME-SFM in the presence of 5 µg of hapoA-I. Media were collected at each time point and analyzed in the same manner as in Fig. 1. Exogenous apoA-I associated with [3H]choline-phospholipid (A), [3H]mevalonate-derived cholesterol (B), [3H]cholesterol-LDL (C), and [3H]cholesterol-m{beta}CD (D) in ABCA1-wild type hepatocytes were compared with ABCA1-null hepatocytes. The results presented are representative of three independent experiments (±S.D.).

 
Quantification of [3H]cholesterol associated with immunoprecipitated apoA-I in the different lipoprotein fractions (Fig. 3B) showed that most cholesterol was found in the HDL size fraction, demonstrating the secretion of cholesterol-containing and large apoA-I-lipoproteins from wild type hepatocytes. Noteworthy was the significant proportion of labeled cholesterol associated with VHDL apoA-I in ABCA1+/+ hepatocytes, a fraction representing the small pre{beta}-apoA-I lipoproteins. Although the [3H]cholesterol content decreased significantly in both HDL and VHDL fractions in ABCA1-deficient hepatocytes, it is important to note that the HDL secreted by ABCA1-/- hepatocytes still contained a significant amount of labeled cholesterol (Fig. 3B).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 3.
Distribution of apoA-I and [3H]cholesterol in lipoprotein fractions as a function of ABCA1 activity and respective contributions of apoA-I, apoB, and apoE to the export of cholesterol. ABCA1-wild type and -null hepatocytes were isolated and labeled with [3H]cholesterol-LDL. Cells were infected with Ad AI on the 2nd day. A 4-h efflux was carried out in non-labeled HepatoZYME-SFM. A, the efflux media were pooled, concentrated down to 2 ml, and immediately loaded on calibrated Superdex 200 columns for separation of different lipoprotein fractions. Aliquots from each fraction were analyzed for apoA-I by slot blot following transfer to nitrocellulose and quantified by densitometric scanning. The absolute concentration (µg of apoA-I/ml of media) of apoA-I in the VLDL/LDL, HDL, and VHDL lipoprotein fractions is compared between ABCA1-wild type and -null hepatocytes. B, aliquots from each fraction separated by FPLC were immunoprecipitated with anti-human apoA-I and quantified by scintillation counting. The association of [3H]cholesterol with hapoA-I in the VLDL/LDL, HDL, and VHDL lipoprotein fractions (cpm/µg of apoA-I) is compared between ABCA1-wild type and -null hepatocytes. Results are presented as the mean value for three separate experiments (±S.D.). C, contribution of apoA-I, apoB, and apoE to the export of cholesterol-containing lipoproteins. Four-hour efflux media were immunoprecipitated with anti-human apoA-I and anti-murine apoB and apoE (mapoB and mapoE, respectively) and then counted. CTL, control. D, immunoblot analysis of murine apoB. Four-hour efflux media were immunoprecipitated (IP) with control, non-immune, antibody, or anti-human apoA-I antibody. As a positive control, the media were probed for secretion of mouse apoB.

 
The increase in apoA-I-associated [3H]cholesterol in the VLDL/LDL fraction in ABCA1-/-, when compared with control hepatocytes, is in agreement with the recent reports of Sahoo et al. (26) and Sniderman et al. (44), which demonstrate a regulatory pool of cholesterol in hepatocytes that is shared for the formation of both VLDL and HDL. Immunoprecipitations of the total efflux media from [3H]cholesterol-LDL-labeled hepatocytes with anti-human apoA-I, anti-murine apoB, or anti-murine apoE showed as expected that apoB-containing lipoproteins were major exporters of cholesterol followed by apoA-I and apoE (Fig. 3C). The immunoprecipitates obtained with anti-apoA-I were analyzed by slot blot assays with anti-apoB (Fig. 3D), which demonstrated the presence of murine apoB in the anti-apoA-I immunoprecipitate. This reflected the secretion of lipoproteins containing both apolipoproteins in both control and ABCA1-null hepatocytes.

Progesterone Caused Significant Inhibition of HapoA-I Cholesterol-LDL Acquisition but Not Phospholipid Acquisition in C57B Hepatocytes—The difference in kinetics of apoA-I lipidation by cholesterol derived from endogenous synthesis or from exogenous delivery suggested heterogeneity in origin and transfer from cholesterol pools. To identify the contribution of other pathways to the cholesterol lipidation of apoA-I and to define the pools of cholesterol transferred to apoA-I during secretion, we labeled the hepatocytes with either [3H]cholesterol-LDL or [3H]mevalonate or [3H]choline under conditions in which specific pathways are inhibited. Progesterone has been shown to inhibit cholesterol transport from intracellular compartments to the plasma membrane, and in particular, the transport from late endosome and/or lysosome to plasma membrane, subsequently causing cholesterol accumulation in the late endosome and/or lysosome (36, 37).

Hepatocytes were infected with Ad AI for 1 h and incubated for 24 h as indicated previously. Progesterone (10 µg/ml) was preincubated with the cells for 15 min, and then secretion was monitored for a 4-h efflux in the presence of progesterone. Western blots of cell lysates and efflux media showed that progesterone had no effect on either the expression or the secretion of apoA-I (Fig. 4, D and E).

Control hepatocytes were labeled with [3H]cholesterol-LDL or [3H]choline, infected with either Ad A-I or Ad Luc and treated with progesterone as indicated above. Four-hour efflux media were collected and immunoprecipitated for apoA-I. ApoA-I-associated cholesterol, corrected for the Ad Luc nonspecific effect, was significantly decreased after progesterone treatment, and further treatment of cells with m{beta}CD for 15 min at 4 °C caused additional significant decrease (Fig. 4A). In contrast, progesterone did not inhibit apoA-I phospholipid acquisition (Fig. 4B). Taken together, these results indicate that acquisition of cholesterol by apoA-I depends on the active transfer from intracellular compartments to plasma membrane via a process dissociable from phospholipid transport.

To test the reversibility of progesterone-induced cholesterol accumulation and subsequent inhibition of apoA-I cholesterol acquisition, hepatocytes were infected with Ad AI and treated with progesterone for 4 h, as in other experiments. When the progesterone-treated cells were washed and incubated for efflux for another 4 h in the absence of progesterone, apoA-I cholesterol acquisition was totally recovered (Fig. 4C). This result, consistent with other studies, indicates that the inhibition of cholesterol trafficking by progesterone is reversible.



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 4.
Progesterone (Prog) significantly inhibits hapoA-I cholesterol acquisition but not phospholipid acquisition in C57B hepatocytes. C57B hepatocytes were labeled with either [3H]cholesterol-LDL or [3H]choline-phospholipid and infected with Ad AI (or Ad Luc as a control (CTL)). The 4-h efflux was carried out in the presence or absence of progesterone (10 µg/ml). To determine apoA-I-specific lipid association, the background value from cells infected with Ad Luc were subtracted. In some conditions in panels A and B, cells were pretreated with 20 mM m{beta}CD at 4 °C for 15 min before the efflux. A, the effect of progesterone on apoA-I [3H]cholesterol acquisition. B, the effect of progesterone on apoA-I [3H]phospholipid acquisition. C, the reversibility of progesterone-induced inhibition of apoA-I cholesterol acquisition was analyzed by removing the progesterone and allowing cells to efflux for another 4 h. Results are presented as the mean values for three separate experiments (±S.D.). D, the effect of progesterone on newly synthesized apoA-I. Hepatocyte cell lysate was analyzed by Western blot following transfer to nitrocellulose and quantified by densitometric scanning. E, the effect of progesterone on the secretion of newly synthesized apoA-I. ApoA-I-containing media were analyzed by slot blot and quantified by densitometric scanning.

 
Progesterone Inhibited HapoA-I Cholesterol Acquisition in Both ABCA1+/+ and ABCA1-/- Hepatocytes—To define the origin of the cholesterol pool secreted with apoA-I in the ABCA1-independent pathway, we labeled both ABCA1+/+ and ABCA1-/- hepatocytes with either [3H]cholesterol-LDL or [3H]mevalonate. The cells were infected with Ad AI or Ad Luc, and the effect of progesterone on the apoA-I cholesterol acquisition was analyzed as described above. Progesterone significantly inhibited apoA-I cholesterol acquisition in both wild type and ABCA1-deficient hepatocytes, and this effect was observed both with cholesterol derived from LDL and with cholesterol derived from de novo synthesis (Fig. 5, A and B). However, it is clear that progesterone had a greater effect on newly synthesized cholesterol secretion in ABCA1-deficient hepatocytes. These results further support the existence of an ABCA1-independent but progesterone-sensitive pathway for apoA-I cholesterol acquisition in hepatocytes that is dependent on active transfer between cholesterol compartments. Again, progesterone did not affect apoA-I phospholipid acquisition in either ABCA1+/+ or ABCA1-/- hepatocytes (Fig. 5D).

To evaluate the sensitivity of early and late phases of cholesterol efflux to progesterone, we included a progesterone preincubation (1 h) immediately after the removal of the labeling medium and before the start of the efflux assay (zero time). Under these conditions, the early phase of cholesterol efflux from ABCA1-/- cells was markedly reduced by progesterone, and, most interestingly, the residual efflux (ABCA1-independent) was the same as that from control cells treated with progesterone (Fig. 5C). At 4 h, the efflux from control cells treated with progesterone, but not that from ABCA1-/- cells, increased significantly, demonstrating that ABCA1 acts as a cholesterol transporter independently of a progesterone block. At 8 h, efflux from ABCA1-/- cells started to increase, an effect that we attribute to the leakiness of the progesterone block with time. This experiment thus identifies three control levels for cholesterol efflux, one dependent on a progesterone-sensitive transport, one independent of progesterone but ABCA1-dependent, and finally, one independent of both ABCA1 and progesterone (Fig. 5C).

Both Brefeldin A and Monensin Inhibited Exo-ApoA-I Acquisition of [3H]Mevalonate in ABCA1-/- Hepatocytes—Brefeldin A (BFA), which inhibits vesicle formation by reversible disassembly of the Golgi complex, and monensin, which is an inhibitor of trans-Golgi apparatus function, have been reported to block both ABCA1-dependent efflux (45) and diffusional efflux (46). Here, we tested their effects on cholesterol acquisition by exogenous apoA-I in ABCA1-/- hepatocytes. ABCA1-/- hepatocytes were isolated and labeled with [3H]mevalonate, and on the 2nd day, they were infected with Ad Luc. Hepatocytes were preincubated with 5 µM BFA or 20 µM monensin for 15 min prior to efflux, and then efflux assays were conducted in the presence of BFA or monensin and 5 µg/ml exogenous apoA-I for 4 h. The media were collected and analyzed as described previously. The result (Fig. 6) clearly demonstrated that both BFA and monensin significantly inhibited exogenous apoA-I cholesterol acquisition in ABCA1-deficient hepatocytes. As expected from the literature, the inhibition caused by BFA was reversible.



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 5.
Progesterone (prog) inhibits hapoA-I cholesterol acquisition in both ABCA1+/+ and ABCA1-/- hepatocytes. Hepatocytes from ABCA1-wild type and -null mice were labeled with [3H]cholesterol-LDL, [3H]mevalonate, or [3H]choline and infected with either Ad AI or Ad Luc. Cells were treated with progesterone, and efflux was carried out as in Fig. 4. All values shown were determined by subtracting the background value from cells infected with Ad Luc. A, apoA-I [3H]cholesterol-LDL acquisition. CTL, control. B, apoA-I [3H]mevalonate-derived cholesterol acquisition. C, the effect of progesterone on the time course of [3H]cholesterol acquisition by newly synthesized apoA-I. Hepatocytes from ABCA1-wild type (WT) and -null (KO) mice were labeled with [3H]cholesterol-LDL as described. In cells to be treated, progesterone was added immediately after removal of the labeling medium for 1 h before the start and during the efflux assay. D, apoA-I [3H]choline-phospholipid acquisition. Results are presented as the mean values for three separate experiments (±S.D.).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown here that hepatocytes secrete lipidated apoA-I-containing lipoprotein particles that contain both phospholipids and cholesterol. One of our aims was to identify the origin of cholesterol transferred to apoA-I and understand the relationship of cholesterol and phospholipid transfers. For that purpose, a simplified model for cellular cholesterol distribution classified into plasma membrane, recycling endosome, and other intracellular pools has been estimated by differential accessibility to m{beta}CD. Although newly synthesized cholesterol predominantly associates (58%) with intracellular compartments that are not accessible to m{beta}CD at 4 °C, a majority of the exogenously delivered cholesterol remains associated with the plasma membrane (61 and 85% for [3H]cholesterol-LDL and [3H]cholesterol-m{beta}CD, respectively), in good general agreement with the data from the literature (47, 48). This allows us to infer that both newly synthesized and secreted apoA-I acquire mostly intracellular cholesterol because the endogenous apoA-I acquires more newly synthesized cholesterol than exogenously added apoA-I (compare Figs. 1B and 2B), whereas both endogenous and exogenous apoA-I acquire the same proportion of exogenously delivered cholesterol (compare Figs. 1C and 2C and Figs. 1D and 2D). Therefore, in the hepatocyte, newly synthesized apoA-I acquires cholesterol molecules during secretion, primarily intracellularly and secondarily at the cell surface. This contrasts with exogenous apoA-I, which as expected acquires cholesterol mostly at the cell surface. Thus, the same pool of cell surface cholesterol (located at the plasma membrane and the recycling endosome compartment), the efflux of which is mediated by ABCA1 (45, 49, 50), appears to be used by both endogenous and exogenous apoA-I.

In ABCA1 deficiency, the hepatocyte also secretes lipidated apoA-I particles that contain both phospholipids and cholesterol, but the proportion of lipidated apoA-I is decreased (Fig. 3). In keeping with our previous observations (25), the ABCA1-/- hepatocytes secrete apoA-I with about 85% less phospholipids (Fig. 1A) but only 40% less newly synthesized cholesterol (Fig. 1B). This surprising result is corroborated by the similar decreases observed with exogenously delivered cholesterol, which range from 50 to 60% (Fig. 1, C and D). A similar picture also emerges for the cholesterol acquisition by exogenous apoA-I (Fig. 2). We must therefore conclude that cholesterol transfer to apoA-I can occur independent of ABCA1 activity. In the absence of ABCA1, the remaining basal phospholipid transfer activity at 15% of control is sufficient to elicit 50% or more of the normal cholesterol transfer. The mechanisms for cholesterol transfer under these conditions remain unclear, but diffusional transfer and/or microsolubilization may take place, driven by the secretion and formation of an apoA-I/phospholipid complex (51–53).



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 6.
Brefeldin A and monensin (Mon) inhibit exo-apoA-I acquisition of [3H]mevalonate-cholesterol in ABCA1-/- hepatocytes. ABCA1-/- hepatocytes were labeled with [3H]mevalonate and infected with Ad Luc. Cells were preincubated with 5 µM brefeldin A or 20 µM monensin for 15 min, and a 4-h efflux was carried out in the presence of brefeldin A or monensin and 5 µg/ml apoA-I. Efflux media were collected and analyzed. Results are presented as the mean values for three separate experiments (±S.D.). CTL, control.

 
Two ABCA1-mediated transfer mechanisms have been suggested: the simultaneous or the sequential/two-step transfer of phospholipids and cholesterol to apoA-I (54–56). Our results support the latter and suggest that cholesterol transfer may be secondary to phospholipid transfer but not necessarily linked to ABCA1 activity. This is also compatible with the independent regulation of cholesterol and phospholipid efflux (57–59) as shown with stimulators and inhibitors of cholesterol efflux that have no effect on phospholipid efflux.

If both ABCA1-dependent and -independent transfer of cholesterol are occurring in excess of phospholipid transfer, what controls the availability of transferred cholesterol? Some of the cholesterol transferred by ABCA1 has been shown to be mobilized from late endosomes in a process controlled by cell surface binding of apoA-I (50) and following protein kinase C activation (59). As well, a number of cholesterol transporters, such as STAR proteins, SCP2, caveolin-1, and OSBP (60–64), have been described that can contribute to delivery in this cell surface pool. The intracellular transport of cholesterol is complex and impacts on cellular cholesterol homeostasis and export. Progesterone is known to interrupt cholesterol transport from the endoplasmic reticulum (60) and from the plasma membrane to the endoplasmic reticulum (37) and transport from late endosomes-lysosomes to the plasma membrane (36, 38). It also impairs the acquisition by newly synthesized apoA-I of exogenously derived cholesterol (65) (Fig. 4). However, the progesterone effect is partial (about 60% inhibition) and additive to the effect of m{beta}CD, which is compatible with the observation that secreted apoA-I is lipidated with cholesterol derived from both intracellular and cell surface pools. When the cells are labeled with mevalonate, progesterone inhibits cholesterol transfer to apoA-I by 80% in keeping with its block on transport of newly synthesized cholesterol from endoplasmic reticulum to plasma membrane (60, 65). Preincubation of the cells with progesterone demonstrated that apoA-I lipidation with cholesterol depended on transport events that are progesterone-sensitive and progesterone-insensitive but ABCA1-dependent (Fig. 5C).

BFA, as well as monensin, significantly affected exo-apoA-I mevalonate-derived cholesterol acquisition in ABCA1-deficient hepatocytes (Fig. 6), indicating that vesicular transport of intracellular cholesterol plays an important role in mediating the ABCA1-independent pathway for apoA-I cholesterol lipidation. Previous studies have provided evidence for different transport of cholesterol and phospholipid from the endoplasmic reticulum to the plasma membrane. Cholesterol transport is energy-dependent and temperature-sensitive and requires vesicular transport (66), whereas phospholipid transport is not mediated by vesicles and independent of energy and temperature (67). Other studies from DeGrella and Simoni (68) and Urbani and Simoni (69) also reported that ATP depletion or low temperature inhibit rapid endoplasmic reticulum to plasma membrane cholesterol transport. Therefore, it is not surprising that apoA-I acquires cholesterol and phospholipid by different mechanisms and/or pathways.

We have previously shown that when compared with wild type, ABCA1-/- hepatocytes secrete fewer but qualitatively similar apoA-I-containing lipoproteins that include large particles of about 17 nm, HDL size particles of 8–10 nm, and small particles of about 7 nm (25). Here, we demonstrate that most of the cholesterol associated with these particles resides in the HDL size fraction (Fig. 3). It follows that in the absence of ABCA1, normal apoA-I particles are formed and that the size and composition of the lipoprotein formed is dictated by apoA-I structure. This concept is in keeping with a large body of knowledge derived from in vitro experiments that linked the structure of apoA-I and other exchangeable apolipoproteins (70–77) to their ability to associate with lipids and form stable lipoproteins. The concept that apoA-I structure drives the formation of specific lipoprotein particles with a defined molar ratio of apolipoprotein and lipids differs from the recent report that ABCA1 oligomers control the formation of lipoprotein particles containing four apoA-I molecules (78). Recently, Tsujita et al. (79) reported that ABCA1-deficient mice did not produce HDL, and HepG2 cells treated with probucol secreted mostly a lipid-free form of apoA-I. However, the lack of apoA-I immunoprecipitation in this study made the definition of its degree of lipidation less accurate. An antibody selective for lipid-free apoA-I, although interaction with partially lipidated apoA-I was not excluded, was also shown to block both phospholipid and cholesterol lipidation of apoA-I. This was taken to demonstrate cell surface lipidation of apoA-I (79). However, the antibody can be endocytosed and recycled in the recycling and late endosomes. As such, it does not demonstrate exclusive lipidation at the cell surface. Our results do not eliminate the possible contribution of a secretion and recapture pathway but concur with previous studies with chicken (18–20) or rat (17) hepatocytes that demonstrated the intracellular lipidation of apoA-I and with studies with HepG2 cells (24) showing that intracellular and extracellular lipidation of apoA-I both occur to form buoyant HDL particles.

We observed that a significant amount of apoA-I was secreted in the VLDL/LDL fraction in wild type and ABCA1-/- hepatocytes (Fig. 3A), in keeping with earlier reports of apoA-I association with newly synthesized VLDL (80, 81). We corroborated these observations with immunoprecipitation of newly secreted lipoproteins with antibodies to human apoA-I and with slot blots of the immunoprecipitates with anti-murine apoB (Fig. 3C, D). Furthermore, in ABCA1 deficiency, the amount of [3H]cholesterol associated with VLDL/LDL fraction was increased when compared with control hepatocytes (Fig. 3B). HDL and apoB-containing VLDL may share the same regulatory pool of cholesterol. Previous studies have shown that inhibition of hydroxymethylglutaryl-CoA reductase also inhibited VLDL secretion in cultured hepatocytes (82, 83). Cholesterol availability thus appears to be an important determinant of VLDL secretion, and depletion of the regulatory pool of cholesterol by apoA-I made less cholesterol available for VLDL synthesis and secretion (26, 44).

The liver is the major site of both apoA-I (13, 14) and ABCA1 (4, 8–12) synthesis, making it also a major contributor of HDL cholesterol. Our studies clearly document the contribution of the hepatocytes to the secretion of cholesterol-containing apoA-I particles and thus its contribution to the genesis of the HDL cholesterol pool. This challenges and dampens the traditional notion of reverse cholesterol transport from peripheral cells to the liver via HDL (84) The knowledge of the sites and control of ABCA1 expression has already prompted the discussion of the concept of reverse cholesterol transport (16). Intriguingly, the HDL level appears to have no relation to net flux of cholesterol through HDL (85).

In conclusion, our study demonstrated that in primary mouse hepatocytes, newly synthesized apoA-I acquires cholesterol and phospholipid during secretion as well at the plasma membrane. ApoA-I cholesterol acquisition operates by both ABCA1-dependent and -independent pathways, both depending on active transfer from intracellular compartments. These transfers include both progesterone-sensitive transport and an ABCA1-dependent transport that is insensitive to progesterone. There is more cholesterol than phospholipid acquired by apoA-I independent of ABCA1 activity. Cholesterol is acquired by apoA-I, mainly from intracellular pools for endogenously expressed apoA-I and mainly at the cell surface for exogenous apoA-I. Finally, the lipidation of apoA-I with cholesterol and phospholipid is controlled through different pathways and/or occurs through independent mechanisms. Our future work will be to identify the subcellular localization of apoA-I lipidation and to evaluate the contribution of hepatic ABCA1 in apoA-I lipidation at these compartments.


   FOOTNOTES
 
* This work was supported in part by Canadian Institutes of Health Research Grant 44359 (to Y. L. M.) and Group Grant 64519 and by an academic grant in aid from Pfizer Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} The recipient of a scholarship from the Ontario Graduate Student Science and Technology Award. Back

§ Supported by postdoctoral fellowships from the Heart and Stroke Foundation of Canada. Back

To whom correspondence should be addressed: University of Ottawa Heart Institute, 40 Ruskin St., Ottawa, Ontario K1Y 4W7, Canada. Tel.: 613-761-5254; Fax: 613-761-5281; E-mail: ylmarcel{at}ottawaheart.ca.

1 The abbreviations used are: ABCA1, ATP-binding cassette transporter A1; HDL, high density lipoprotein; VHDL, very high density lipoprotein; LDL, low density lipoprotein; VLDL, very low density lipoprotein; apoA-I, apolipoprotein A-I; hapoA-I, human apoA-I; Ad AI, adeno-hapoA-I; Ad Luc, adeno-luciferase; M{beta}CD, methyl-{beta} cyclodextrin; FPLC, fast protein liquid chromatography; BFA, brefeldin A; SFM, serum-free medium; PGK, phosphoglycerate kinase. Back


   ACKNOWLEDGMENTS
 
We thank Ross W. Milne for the critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Lee, J. Y., and Parks, J. S. (2005) Curr. Opin. Lipidol. 16, 19-25[Medline] [Order article via Infotrieve]
  2. Bodzioch, M., Orsó, E., Klucken, T., Langmann, T., Böttcher, L., Diederich, W., Drobnik, W., Barlage, S., Büchler, C., Porsch-Özcürümez, M., Kaminski, W. E., Hahmann, H. W., Oette, K., Rothe, G., Aslanidis, C., Lackner, K. J., and Schmitz, G. (1999) Nat. Genet. 22, 347-351[CrossRef][Medline] [Order article via Infotrieve]
  3. Brooks-Wilson, A., Marcil, M., Clee, S. M., Zhang, L. H., Roomp, K., Van Dam, M., Yu, L., Brewer, C., Collins, J. A., Molhuizen, H. O. F., Loubser, O., Ouelette, B. F. F., Fichter, K., Ashbourne-Excoffon, K. J. D., Sensen, C. W., Scherer, S., Mott, S., Denis, M., Martindale, D., Frohlich, J., Morgan, K., Koop, B., Pimstone, S., and Kastelein, J. J. P. (1999) Nat. Genet. 22, 336-345[CrossRef][Medline] [Order article via Infotrieve]
  4. Lawn, R. M., Wade, D. P., Garvin, M. R., Wang, X. B., Schwartz, K., Porter, J. G., Seilhamer, J. J., Vaughan, A. M., and Oram, J. F. (1999) J. Clin. Investig. 104, R25-R31
  5. Remaley, A. T., Rust, S., Rosier, M., Knapper, C., Naudin, L., Broccardo, C., Peterson, K. M., Koch, C., Arnould, I., Prades, C., Duverger, N., Funke, H., Assman, G., Dinger, M., Dean, M., Chimini, G., Santamarina-Fojo, S., Fredrickson, D. S., Denefle, P., and Brewer, H. B., Jr. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12685-12690[Abstract/Free Full Text]
  6. Rust, S., Rosier, M., Funke, H., Real, J., Amoura, Z., Piette, J. C., Deleuze, J. F., Brewer, H. B., Duverger, N., Denèfle, P., and Assmann, G. (1999) Nat. Genet. 22, 352-355[CrossRef][Medline] [Order article via Infotrieve]
  7. Haghpassand, M., Bourassa, P. A., Francone, O. L., and Aiello, R. J. (2001) J. Clin. Investig. 108, 1315-1320[CrossRef][Medline] [Order article via Infotrieve]
  8. Singaraja, R. R., Bocher, V., James, E. R., Clee, S. M., Zhang, L. H., Leavitt, B. R., Tan, B., Brooks-Wilson, A., Kwok, A., Bissada, N., Yang, Y. Z., Liu, G. Q., Tafuri, S. R., Fievet, C., Wellington, C. L., Staels, B., and Hayden, M. R. (2001) J. Biol. Chem. 276, 33969-33979[Abstract/Free Full Text]
  9. Wellington, C. L., Walker, E. K., Suarez, A., Kwok, A., Bissada, N., Singaraja, R., Yang, Y. Z., Zhang, L. H., James, E., Wilson, J. E., Francone, O., McManus, B. M., and Hayden, M. R. (2002) Lab. Investig. 82, 273-283[CrossRef][Medline] [Order article via Infotrieve]
  10. Cavelier, L. B., Qiu, Y., Bielicki, J. K., Afzal, V., Cheng, J. F., and Rubin, E. M. (2001) J. Biol. Chem. 276, 18046-18051[Abstract/Free Full Text]
  11. Langmann, T., Klucken, J., Reil, M., Liebisch, G., Luciani, M. F., Chimini, G., Kaminski, W. E., and Schmitz, G. (1999) Biochem. Biophys. Res. Commun. 257, 29-33[CrossRef][Medline] [Order article via Infotrieve]
  12. Langmann, T., Mauerer, R., Zahn, A., Moehle, C., Probst, M., Stremmel, W., and Schmitz, G. (2003) Clin. Chem. 49, 230-238[Abstract/Free Full Text]
  13. Breslow, J. L. (1987) Am. Heart J. 113, 422-427[CrossRef][Medline] [Order article via Infotrieve]
  14. Breslow, J. L. (1985) Annu. Rev. Biochem. 54, 699-727[CrossRef][Medline] [Order article via Infotrieve]
  15. Basso, F., Freeman, L., Knapper, C. L., Remaley, A., Stonik, J., Neufeld, E. B., Tansey, T., Amar, M. J., Fruchart-Najib, J., Duverger, N., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2003) J. Lipid Res. 44, 296-302[Abstract/Free Full Text]
  16. Tall, A. R., Wang, N., and Mucksavage, P. (2001) J. Clin. Investig. 108, 1273-1275[CrossRef][Medline] [Order article via Infotrieve]
  17. Howell, K. E., and Palade, G. E. (1982) J. Cell Biol. 92, 833-845[Abstract/Free Full Text]
  18. Banerjee, D., and Redman, C. M. (1983) J. Cell Biol. 96, 651-660[Abstract/Free Full Text]
  19. Banerjee, D., and Redman, C. M. (1984) J. Cell Biol. 99, 1917-1926[Abstract/Free Full Text]
  20. Banerjee, D., Mukherjee, T. K., and Redman, C. M. (1985) J. Cell Biol. 101, 1219-1226[Abstract/Free Full Text]
  21. Forte, T. M., McCall, M. R., Amacher, S., Nordhausen, R. W., Vigne, J. L., and Mallory, J. B. (1990) Biochim. Biophys. Acta 1047, 11-18[Medline] [Order article via Infotrieve]
  22. Hamilton, R. L., Moorehouse, A., and Havel, R. J. (1991) J. Lipid Res. 32, 529-543[Abstract]
  23. Banerjee, D., Rodriguez, M., and Rajasekaran, A. K. (1997) Exp. Cell Res. 235, 334-344[CrossRef][Medline] [Order article via Infotrieve]
  24. Chisholm, J. W., Burleson, E. R., Shelness, G. S., and Parks, J. S. (2002) J. Lipid Res. 43, 36-44[Abstract/Free Full Text]
  25. Kiss, R. S., McManus, D. C., Franklin, V., Tan, W. L., McKenzie, A., Chimini, G., and Marcel, Y. L. (2003) J. Biol. Chem. 278, 10119-10127[Abstract/Free Full Text]
  26. Sahoo, D., Trischuk, T. C., Chan, T., Drover, V. A., Ho, S., Chimini, G., Agellon, L. B., Agnihotri, R., Francis, G. A., and Lehner, R. (2004) J. Lipid Res. 45, 1122-1131[Abstract/Free Full Text]
  27. Zheng, H., Kiss, R. S., Franklin, V., Wang, M. D., Haidar, B., and Marcel, Y. L. (2004) Circulation 110, Suppl. 3, 272 (Abstr. 13942)
  28. McManus, D. C., Scott, B. R., Frank, P. G., Franklin, V., Schultz, J. R., and Marcel, Y. L. (2000) J. Biol. Chem. 275, 5043-5051[Abstract/Free Full Text]
  29. McManus, D. C., Scott, B. R., Franklin, V., Sparks, D. L., and Marcel, Y. L. (2001) J. Biol. Chem. 276, 21292-21302[Abstract/Free Full Text]
  30. Ishimori, N., Li, R., Kelmenson, P. M., Korstanje, R., Walsh, K. A., Churchill, G. A., Forsman-Semb, K., and Paigen, B. (2004) Arterioscler. Thromb. Vasc. Biol. 24, 161-166[Abstract/Free Full Text]
  31. Thomas, S. S., Plenkiewicz, J., Ison, E. R., Bols, M., Zou, W., Szarek, W. A., and Kisilevsky, R. (1995) Biochim. Biophys. Acta 1272, 37-48[Medline] [Order article via Infotrieve]
  32. Subrahmanyan, L., and Kisilevsky, R. (1988) Scand. J. Immunol. 27, 251-260[CrossRef][Medline] [Order article via Infotrieve]
  33. Johnson, W. J., Fischer, R. T., Phillips, M. C., and Rothblat, G. H. (1995) J. Biol. Chem. 270, 25037-25046[Abstract/Free Full Text]
  34. Atger, V. M., Moya, M. D., Stoudt, G. W., Rodrigueza, W. V., Phillips, M. C., and Rothblat, G. H. (1997) J. Clin. Investig. 99, 773-780[Medline] [Order article via Infotrieve]
  35. Christian, A. E., Byun, H. S., Zhong, N., Wanunu, M., Marti, T., Fürer, A., Diederich, F., Bittman, R., and Rothblat, G. H. (1999) J. Lipid Res. 40, 1475-1482[Abstract/Free Full Text]
  36. Butler, J. D., Blanchette-Mackie, J., Goldin, E., O'Neill, R. R., Carstea, G., Roff, C. F., Patterson, M. C., Patel, S., Comly, M. E., Cooney, A., Vanier, M. T., Brady, R. O., and Pentchev, P. G. (1992) J. Biol. Chem. 267, 23797-23805[Abstract/Free Full Text]
  37. Lange, Y. (1994) J. Biol. Chem. 269, 3411-3414[Abstract/Free Full Text]
  38. Lange, Y., Ye, J., and Chin, J. (1997) J. Biol. Chem. 272, 17018-17022[Abstract/Free Full Text]
  39. Mazzone, T., Krishna, M., and Lange, Y. (1995) J. Lipid Res. 36, 544-551[Abstract]
  40. Cruz, J. C., Sugii, S., Yu, C. J., and Chang, T. Y. (2000) J. Biol. Chem. 275, 4013-4021[Abstract/Free Full Text]
  41. Lange, Y., Ye, J., Rigney, M., and Steck, T. (2000) J. Biol. Chem. 275, 17468-17475[Abstract/Free Full Text]
  42. Rodrigueza, W. V., Williams, K. J., Rothblat, G. H., and Phillips, M. C. (1997) Arterioscler. Thromb. Vasc. Biol. 17, 383-393[Abstract/Free Full Text]
  43. Castle, C. K., Pape, M. E., Marotti, K. R., and Melchior, G. W. (1991) J. Lipid Res. 32, 439-447[Abstract]
  44. Sniderman, A. D., Zhang, Z., Genest, J., and Cianflone, K. (2003) J. Lipid Res. 44, 527-532[Abstract/Free Full Text]
  45. Neufeld, E. B., Remaley, A. T., Demosky, S. J., Stonik, J. A., Cooney, A. M., Comly, M., Dwyer, N. K., Zhang, M., Blanchette-Mackie, J., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2001) J. Biol. Chem. 276, 27584-27590[Abstract/Free Full Text]
  46. Mendez, A. J. (1995) J. Biol. Chem. 270, 5891-5900[Abstract/Free Full Text]
  47. Van Meer, G. (1989) Annu. Rev. Cell Biol. 5, 247-275[CrossRef][Medline] [Order article via Infotrieve]
  48. Lange, Y., Swaisgood, M. H., Ramos, B. V., and Steck, T. L. (1989) J. Biol. Chem. 264, 3786-3793[Abstract/Free Full Text]
  49. Neufeld, E. B., Wastney, M., Patel, S., Suresh, S., Cooney, A. M., Dwyer, N. K., Roff, C. F., Ohno, K., Morris, J. A., Carstea, E. D., Incardona, J. P., Strauss, J. F., III, Vanier, M. T., Patterson, M. C., Brady, R. O., Pentchev, P. G., and Blanchette-Mackie, E. J. (1999) J. Biol. Chem. 274, 9627-9635[Abstract/Free Full Text]
  50. Neufeld, E. B., Stonik, J. A., Demosky, S. J., Jr., Knapper, C. L., Combs, C. A., Cooney, A., Comly, M., Dwyer, N., Blanchette-Mackie, J., Remaley, A. T., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2004) J. Biol. Chem. 279, 15571-15578[Abstract/Free Full Text]
  51. Johnson, W. J., Mahlberg, F. H., Rothblat, G. H., and Phillips, M. C. (1991) Biochim. Biophys. Acta 1085, 273-298[Medline] [Order article via Infotrieve]
  52. Phillips, M. C., Gillotte, K. L., Haynes, M. P., Johnson, W. J., Lund-Katz, S., and Rothblat, G. H. (1998) Atherosclerosis 137, S13-S17
  53. Rothblat, G. H., Llera-Moya, M., Atger, V., Kellner-Weibel, G., Williams, D. L., and Phillips, M. C. (1999) J. Lipid Res. 40, 781-796[Abstract/Free Full Text]
  54. Fielding, P. E., Nagao, K., Hakamata, H., Chimini, G., and Fielding, C. J. (2000) Biochemistry 39, 14113-14120[CrossRef][Medline] [Order article via Infotrieve]
  55. Wang, N., Silver, D. L., Thiele, C., and Tall, A. R. (2001) J. Biol. Chem. 276, 23742-23747[Abstract/Free Full Text]
  56. Marcil, M., Bissonnette, R., Vincent, J., Krimbou, L., and Genest, J. (2003) Circulation 107, 1366-1371[Abstract/Free Full Text]
  57. Li, Q., Komaba, A., and Yokoyama, S. (1993) Biochemistry 32, 4597-4603[CrossRef][Medline] [Order article via Infotrieve]
  58. Yamauchi, Y., Abe-Dohmae, S., and Yokoyama, S. (2002) Biochim. Biophys. Acta 1585, 1-10[Medline] [Order article via Infotrieve]
  59. Yamauchi, Y., Chang, C. C., Hayashi, M., Abe-Dohmae, S., Reid, P. C., Chang, T. Y., and Yokoyama, S. (2004) J. Lipid Res. 45, 1943-1951[Abstract/Free Full Text]
  60. Smart, E. J., Ying, Y., Donzell, W. C., and Anderson, R. G. (1996) J. Biol. Chem. 271, 29427-29435[Abstract/Free Full Text]
  61. Olkkonen, V. M. (2004) Curr. Opin. Lipidol. 15, 321-327[CrossRef][Medline] [Order article via Infotrieve]
  62. Soccio, R. E., and Breslow, J. L. (2003) J. Biol. Chem. 278, 22183-22186[Free Full Text]
  63. Stolowich, N. J., Petrescu, A. D., Huang, H., Martin, G. G., Scott, A. I., and Schroeder, F. (2002) Cell. Mol. Life Sci. 59, 193-212[CrossRef][Medline] [Order article via Infotrieve]
  64. Ridgway, N. D. (2000) Biochim. Biophys. Acta 1484, 129-141[Medline] [Order article via Infotrieve]
  65. Kojima, K., Abe-Dohmae, S., Arakawa, R., Murakami, I., Suzumori, K., and Yokoyama, S. (2001) Biochim. Biophys. Acta 1532, 173-184[Medline] [Order article via Infotrieve]
  66. Kaplan, M. R., and Simoni, R. D. (1985) J. Cell Biol. 101, 446-453[Abstract/Free Full Text]
  67. Kaplan, M. R., and Simoni, R. D. (1985) J. Cell Biol. 101, 441-445[Abstract/Free Full Text]
  68. DeGrella, R. F., and Simoni, R. D. (1982) J. Biol. Chem. 257, 14256-14262[Abstract/Free Full Text]
  69. Urbani, L., and Simoni, R. D. (1990) J. Biol. Chem. 265, 1919-1923[Abstract/Free Full Text]
  70. Calabresi, L., Meng, Q.-H., Castro, G. R., and Marcel, Y. L. (1993) Biochemistry 32, 6477-6484[CrossRef][Medline] [Order article via Infotrieve]
  71. Clay, M. A., Cehic, D. A., Pyle, D. H., Rye, K. A., and Barter, P. J. (1999) Biochem. J. 337, 445-451
  72. Davidson, W. S., Hazlett, T., Mantulin, W. W., and Jonas, A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13605-13610[Abstract/Free Full Text]
  73. Jonas, A., Wald, J. H., Toohill, K. L., Krul, E. S., and Kezdy, K. E. (1990) J. Biol. Chem. 265, 22123-22129[Abstract/Free Full Text]
  74. Sparks, D. L., Frank, P. G., Braschi, S., Neville, T. A., and Marcel, Y. L. (1999) Biochemistry 38, 1727-1735[CrossRef][Medline] [Order article via Infotrieve]
  75. Frank, P. G., and Marcel, Y. L. (2000) J. Lipid Res. 41, 853-872[Abstract/Free Full Text]
  76. Marcel, Y. L., and Kiss, R. S. (2003) Curr. Opin. Lipidol. 14, 151-157[CrossRef][Medline] [Order article via Infotrieve]
  77. Liu, L., Bortnick, A. E., Nickel, M., Dhanasekaran, P., Subbaiah, P. V., Lund-Katz, S., Rothblat, G. H., and Phillips, M. C. (2003) J. Biol. Chem. 278, 42976-42984[Abstract/Free Full Text]
  78. Denis, M., Haidar, B., Marcil, M., Bouvier, M., Krimbou, L., and Genest, J. (2004) J. Biol. Chem. 279, 41529-41536[Abstract/Free Full Text]
  79. Tsujita, M., Wu, C. A., Abe-Dohmae, S., Usui, S., Okazaki, M., and Yokoyama, S. (2005) J. Lipid Res. 46, 154-162[Abstract/Free Full Text]
  80. Swift, L. L., Valyi-Nagy, K., Rowland, C., and Harris, C. (2001) J. Lipid Res. 42, 218-224[Abstract/Free Full Text]
  81. Tarugi, P., Ballarini, G., Pinotti, B., Franchini, A., Ottaviani, E., and Calandra, S. (1998) J. Lipid Res. 39, 731-743[Abstract/Free Full Text]
  82. Khan, B. V., Fungwe, T. V., Wilcox, H. G., and Heimberg, M. (1990) Biochim. Biophys. Acta 1044, 297-304[Medline] [Order article via Infotrieve]
  83. Brown, A., Wiggins, D., and Gibbons, G. F. (1999) Biochim. Biophys. Acta 1440, 253-265[Medline] [Order article via Infotrieve]
  84. Glomset, J. A. (1973) Adv. Lipid Res. 11, 1-65[Medline] [Order article via Infotrieve]
  85. Osono, Y., Woollett, L. A., Marotti, K. R., Melchior, G. W., and Dietschy, J. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4114-4119[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Eur Heart JHome page
S. Rashid, M. Marcil, I. Ruel, and J. Genest
Identification of a novel human cellular HDL biosynthesis defect
Eur. Heart J., June 24, 2009; (2009) ehp250v1.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. L. Jacobs, S. Lingrell, Y. Zhao, G. A. Francis, and D. E. Vance
Hepatic CTP:Phosphocholine Cytidylyltransferase-{alpha} Is a Critical Predictor of Plasma High Density Lipoprotein and Very Low Density Lipoprotein
J. Biol. Chem., January 25, 2008; 283(4): 2147 - 2155.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M.-D. Wang, V. Franklin, M. Sundaram, R. S. Kiss, K. Ho, M. Gallant, and Y. L. Marcel
Differential Regulation of ATP Binding Cassette Protein A1 Expression and ApoA-I Lipidation by Niemann-Pick Type C1 in Murine Hepatocytes and Macrophages
J. Biol. Chem., August 3, 2007; 282(31): 22525 - 22533.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
M.-D. Wang, V. Franklin, and Y. L. Marcel
In Vivo Reverse Cholesterol Transport From Macrophages Lacking ABCA1 Expression Is Impaired
Arterioscler. Thromb. Vasc. Biol., August 1, 2007; 27(8): 1837 - 1842.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
M.-D. Wang, R. S. Kiss, V. Franklin, H. M. McBride, S. C. Whitman, and Y. L. Marcel
Different cellular traffic of LDL-cholesterol and acetylated LDL-cholesterol leads to distinct reverse cholesterol transport pathways
J. Lipid Res., March 1, 2007; 48(3): 633 - 645.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
E. Ikonen
Mechanisms for cellular cholesterol transport: defects and human disease.
Physiol Rev, October 1, 2006; 86(4): 1237 - 1261.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
M. Lee-Rueckert, R. Vikstedt, J. Metso, C. Ehnholm, P. T. Kovanen, and M. Jauhiainen
Absence of endogenous phospholipid transfer protein impairs ABCA1-dependent efflux of cholesterol from macrophage foam cells
J. Lipid Res., August 1, 2006; 47(8): 1725 - 1732.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
A. Kobayashi, Y. Takanezawa, T. Hirata, Y. Shimizu, K. Misasa, N. Kioka, H. Arai, K. Ueda, and M. Matsuo
Efflux of sphingomyelin, cholesterol, and phosphatidylcholine by ABCG1
J. Lipid Res., August 1, 2006; 47(8): 1791 - 1802.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Z. Liu, Y. Tang, T. Qiu, X. Cao, and T. L. Clemens
A Dishevelled-1/Smad1 Interaction Couples WNT and Bone Morphogenetic Protein Signaling Pathways in Uncommitted Bone Marrow Stromal Cells
J. Biol. Chem., June 23, 2006; 281(25): 17156 - 17163.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
S. Yokoyama
Assembly of High-Density Lipoprotein
Arterioscler. Thromb. Vasc. Biol., January 1, 2006; 26(1): 20 - 27.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Maric, R. S. Kiss, V. Franklin, and Y. L. Marcel
Intracellular Lipidation of Newly Synthesized Apolipoprotein A-I in Primary Murine Hepatocytes
J. Biol. Chem., December 2, 2005; 280(48): 39942 - 39949.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/22/21612    most recent
M502200200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zheng, H.
Right arrow Articles by Marcel, Y. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zheng, H.
Right arrow Articles by Marcel, Y. L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement