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Papers In Press, published online ahead of print October 1, 2007
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Journal of Lipid Research, Vol. 48, 2193-2211, October 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology
* Department of Physiology and Pharmacology, Texas A&M University, Texas Veterinary Medical Center, College Station, TX 77843-4466
Department of Pathobiology, Texas A&M University, TVMC, College Station, TX 77843-4467
Department of Pathology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7525
The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of one figure and five tables. 
Published, JLR Papers in Press, July 3, 2007.
1 To whom correspondence should be addressed. e-mail: fschroeder{at}cvm.tamu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Supplementary key words sterol carrier protein • SR-B1 • reverse cholesterol transport • ABCA1
Abbreviations: apoA-I, apolipoprotein A-I; cis-parinaric acid, 9Z,11E,13E,15Z-octatetradecanoic acid; ConA, concanavalin A; DBI, double bond index; DHE, dehydroergosterol; DPH, 1,6-diphenyl-1,3,5-hexatriene; DPH-Pro, 3(1,6-diphenyl-1,3,5-hexatrienyl)-propionic acid; DPH-TMA, 1,6-diphyenyl-1,3,5-hexatrienyl-trimethylammonium; eNOS, endothelial nitic oxide synthase; L-FABP, liver fatty acid binding protein; NBD-stearic acid, 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-octadecanoic acid; PA, phosphatidic acid; PC, choline glycerophospholipid; PE, ethanolamine glycerophospholipid; P-gp, P-glycoprotein; PI, phosphatidylinositol; PS, phosphatidylserine; RCT, reverse cholesterol transport; SCP-2, sterol carrier protein-2; SM, sphingomyelin; SR-B1, scavenger receptor class B type I
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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First, peripheral cells (fibroblasts, muscle, and endothelial cells) are rich in both lipid rafts and caveolae (a subset of lipid rafts rich in several components of the RCT pathway) but are relatively poor in intracellular cholesterol binding proteins such as sterol carrier protein-2 (SCP-2) that facilitate cholesterol retention rather than efflux (reviewed in Refs. 1, 2). Cholesterol effluxes from peripheral cells to HDL by a process facilitated by: i) the HDL receptor scavenger receptor class B type I (SR-B1) (3); ii) ABCA1, a protein that mediates cholesterol and phosphatidylcholine desorption from the plasma membrane to apolipoprotein A-I (apoA-I) in the plasma, thereby leading to the formation of HDL (4); and iii) P-glycoprotein (P-gp), a protein thought to translocate cholesterol from the cytofacial to the exofacial leaflet of the plasma membrane, facilitating cholesterol efflux to HDL (5).
Second, although HDL transports cholesterol and cholesteryl ester through the vasculature to liver hepatocytes for uptake, it is unclear at present whether this process is mediated through putative lipid rafts in the hepatocyte plasma membrane. In addition, although liver expresses SR-B1, ABCA1, and P-gp, it is not known whether: i) hepatocyte plasma membranes contain lipid rafts; ii) caveolin-1, highly expressed in liver nonparenchymal cells (e.g., endothelial, stellate, and Kupffer cells), is present in the putative hepatocyte lipid rafts; or iii) proteins essential for HDL-mediated cholesterol/cholesteryl ester transport (SR-B1, ABCA1, and P-gp) are present in the putative hepatocyte lipid rafts.
Third, hepatocytes are essentially deficient in caveolin-1 (6–8) but rich in cholesterol binding/transport proteins, including SCP-2 [also liver fatty acid binding protein (L-FABP)], which enhance cholesterol uptake, intracellular retention (esterification), and transport for secretion into bile (reviewed in Ref. 1). Because HDL-mediated cholesterol transport is bidirectional, hepatocytes contain a complement of proteins that not only facilitate efficient cholesterol transport into hepatocytes but also prevent retrograde efflux of cholesterol out of the hepatocyte. Recent real-time fluorescence imaging of sterol transport (9) as well as other studies indicate that hepatocyte intracellular cholesterol transport for secretion into bile is protein-mediated rather than vesicular (reviewed in Ref. 1). Because caveolin-1 is very low/absent in hepatocytes, two other cholesterol binding proteins have been proposed as serving in intracellular cholesterol transport: SCP-2 and L-FABP (reviewed in Ref. 1). In vitro and intact cell studies show that SCP-2 enhances sterol transfer from the plasma membrane to the endoplasmic reticulum and to other intracellular membranes, whereas L-FABP more weakly enhances sterol transfer (reviewed in Ref. 10).
The purpose of the present investigation was to demonstrate: i) the existence of lipid rafts in hepatocyte plasma membranes; ii) the compartmentalization of RCT proteins therein; iii) the unique lipid distribution and structural properties of hepatocyte lipid rafts; iv) how these properties of hepatocyte lipid rafts differ from those of nonraft domains; and v) whether the cholesterol binding/transfer protein SCP-2 differentially regulates intrinsic properties of hepatocyte lipid rafts. These issues were addressed by the successful application of an affinity chromatography method (11–14) used for the first time on mouse hepatocyte primary cell cultures to simultaneously isolate lipid rafts and nonrafts from purified plasma membranes (derived from wild-type and SCP-2/SCP-x gene-ablated mice) without the use of detergents or high-pH carbonate buffers. In addition, the data showed for the first time that, despite the near absence of caveolin-1 in liver, lipid rafts comprise a substantial portion (nearly one-third based on protein) of the hepatocyte plasma membrane, are enriched in key proteins mediating RCT, and exhibit unique lipid composition and structure compared with nonrafts, and these properties were regulated in part by the expression of SCP-2.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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were from Sigma (St. Louis, MO). Rabbit anti-CXCR4 was purchased from Abcam (Cambridge, MA). Rabbit anti-MDR (P-gp) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Lowicryl HM20 resin and goat anti-rabbit IgG conjugated to 15 nm gold were from Electron Microscopy Sciences (Fort Washington, PA). 1,6-Diphenyl-1,3,5-hexatriene (DPH), 1,6-diphyenyl-1,3,5-hexatrienyl-trimethylammonium (DPH-TMA), 3(1,6-diphenyl-1,3,5-hexatrienyl)-propionic acid (DPH-Pro), 9Z,11E,13E,15Z-octatetradecanoic acid (cis-parinaric acid), and 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]octadecanoic acid (NBD-stearic acid) were from Molecular Probes (Eugene, OR). Dehydroergosterol (DHE) was synthesized in our laboratory as described previously (16). All reagents and solvents used were of the highest grade available and were cell culture tested.
Animals
All animal protocols were approved by the Institutional Animal Care and Use Committee at Texas A&M University. Male and female (6 weeks old, 20–30 g) inbred C57BL/6NCr mice were obtained from the National Cancer Institute (Frederick Cancer Research and Developmental Center, Frederick, MD). SCP-2/SCP-x gene-ablated mice were generated as described below. All mice were kept under a 12 h light/dark cycle in a temperature-controlled facility (25°C) with access to food (standard rodent chow mix, 5% fat calories) and water ad libitum. Animals in the facility were monitored quarterly for infectious diseases.
Generation of SCP-2/SCP-x gene-ablated mice
SCP-2/SCP-x gene-ablated mice were generated by targeted disruption of the SCP-2 gene through homologous recombination. The targeting construct was designed to replace exon 16 with the neomycin cassette of the pUnivec-HPRT vector (a generous gift from Dr. Ramiro Ramírez-Solis, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX). Briefly, a genomic 129/sv Lambda FIX® library (Stratagene, La Jolla, CA) was screened with a 150 bp DNA fragment containing exon 16 of the SCP-2/SCP-x gene. Positive clones were confirmed by extensive restriction mapping and sequence analysis. Two consecutive genomic DNA fragments, a 2.0 kb PstI clone (containing intronic sequences upstream of exon 16) and a 4.5 kb PstI clone (containing intronic sequences immediately upstream of exon 16, exon 16 itself, and intronic sequences downstream of exon 16) formed the backbone of the targeting construct. The 3' homology arm was generated by ligating a blunt-ended 2.6 kb XbaI fragment from the 4.5 kb PstI clone into Litmus 39 vector (New England Biolabs, Ipswich, MA) predigested with EcoRV. This plasmid was digested with SacI/PstI and ligated with an 875 bp fragment from the 4.5 kb PstI clone digested with SacI/PstI. The resulting vector was digested with BamHI/HindIII to give a 2,824 bp fragment that was ligated into the pGEM-3Zf vector (Promega, Madison, WI) predigested with BamHI/HindIII. Digestion with EcoRI/HindIII released the 3' homology region (with an incorporated KpnI site later used for Southern blot screening). The 3' homology fragment was blunt-ended with T4 DNA polymerase and ligated into the pUnivec-HPRT vector (predigested with NotI and blunted with T4 DNA ligase) to yield an intermediate targeting construct named pUni+3' vector. The 5' homology region was prepared by digesting the 4.5 kb PstI clone with AccI/PstI to isolate a 2,562 bp fragment that was then ligated to an AccI/PstI fragment isolated from the 2.0 kb PstI clone. Digestion with BamHI/AccI released the 5' homology fragment. The targeting construct was completed by blunt-end ligation of the 5' arm into the intermediate construct pUni+3' vector (predigested with NheI and then blunted with T4 DNA polymerase).
Once complete, the targeting construct was opened with ClaI and electroporated into a 129/Ola-derived embryonic stem cell line E14 maintained on feeder layers. After selection with G418 (200 µg/ml) and gancyclovir (2 µM), DNA was isolated from surviving clones, digested with KpnI, and screened by Southern blot analysis according to standard protocols. Using a 200 bp 5' probe external to the targeting construct, targeted clones were identified by the presence of a 6 kb band indicating that exon 16 was replaced with the neomycin cassette of pUnivec-HPRT vector. Three positive clones were expanded and injected into C57BL/6NCr blastocysts to create chimeric mice by standard procedures. Three male chimeras were identified by coat color and bred to C57BL/6NCr females to determine germline transmission of the targeted allele. Tail DNA from mixed coat-colored (agouti) F1 offspring was screened by PCR to verify the genotype of each animal. Initial characterization of the SCP-2/SCP-x gene ablation by Western analysis was performed on F2 SCP-2–/– homozygous mice generated by interbreeding heterozygous F1 animals. The heterozygous F1 animals were backcrossed to a N6 C57BL/6NCr background before interbreeding to produce the SCP-2/SCP-x null mice used in this work.
Hepatocyte isolation and culture
Primary cultured hepatocytes derived from the livers of 12 week old male SCP-2/SCP-x null and wild-type mice were isolated as described earlier (17). Primary hepatocytes cultured for 
4 days retain viability and function, as indicated by their undiminished ability to synthesize and secrete albumin (17), synthesize and secrete apolipoproteins (apoA-I, apoE1, and apoB), synthesize L-FABP, secrete lipoproteins (VLDL and HDL), take up fatty acid, transport fatty acid intracellularly, and oxidize fatty acid (17, 18). Therefore, all experiments were performed with hepatocytes maintained in culture for <2 days, well within the range established for retaining function and viability.
Lipid raft and nonraft isolation from primary cultured hepatocytes
Lipid rafts and nonrafts were isolated from mouse hepatocyte primary cell cultures by a nondetergent, affinity chromatography method developed previously and applied to other cell types (11–14). In brief, for each preparation, hepatocytes (7 x 106 cells/dish), cultured in 150 mm x 25 mm Corning tissue culture dishes (30 dishes; Corning and Costar, Corning, NY), were washed two times with chilled PBS (4°C) and then scraped into PBS containing protease inhibitor. The hepatocytes were then sedimented at 1,000 g for 5 min (JA-12 conical rotor and Avanti J-25 centrifuge; Beckman Instruments, Fullerton, CA) at 4°C followed by resuspension in 12 ml of buffer A (0.25 M sucrose and 5 mM Tris-HCl, pH 7.8, 4°C) and disruption with 40 p.s.i. of N2 for 13 min in an N2 Bomb Cell Disrupter (Parr Instrument Co., Moline, IL). The postnuclear supernatant was collected after centrifugation, placed on top of 30% Percoll in sucrose/Tris (4°C), and centrifuged at 70,000 g for 30 min (SW40Ti rotor and XL90 ultracentrifuge; Beckman Instruments) at 4°C as described previously (12). The plasma membrane-enriched fraction, located at the interphase between the Percoll and sample layers, was identified by screening all of the visible protein bands separated by the Percoll gradient for the presence of plasma membrane protein markers (transferrin receptor, SR-B1, and Na+K+-ATPase). Once collected, the enriched plasma membrane fraction was sonicated by three brief 15 s pulses over 3 min with a Fisher 550 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA) to break up large sheets of plasma membrane. As shown previously by electron microscopy, the disrupted plasma membranes appeared as vesicles (average diameter of 99 nm) or as smaller, broken structures (14). The sonicated plasma membrane fraction was then mixed with ConA Sepharose resin (100 ml) in buffer X (10 mM HEPES, pH 7.4, 140 mM KCl, 1 mM MgCl2, and 1 mM MnCl2), incubated under bubbling N2 for 5 min at 4°C to allow thorough mixing and maximum interface between sample and resin, and then incubated for an additional 15 min without N2 bubbling.
The solution was poured into a column and left to stand for another 15 min at 4°C. The column was eluted under bubbling N2 at a rate of 2 ml/min, and 10 ml fractions were collected until the absorbance at 280 nm was close to zero. Based on the enrichment of nonraft marker proteins (CXCR4) and the absence/reduced level of lipid raft markers (flotillin-1, SR-B1, Gq, and GM1), the eluent was designated as "nonraft." ConA-adherent fractions were then eluted under bubbling N2 with buffer Y (buffer X plus 0.75 M -methylmannoside) and were collected at 4°C in 10 ml fractions at 2 ml/min until the absorbance at 280 nm reached a plateau. Based on the enrichment of lipid raft markers (flotillin-1, Gq, SR-B1, and GM1) and the absence/reduced level of the nonraft marker (CXCR4), the fraction eluting with buffer Y was designated as "lipid raft." Fractions rich in lipid raft markers and nonraft markers were pooled separately and concentrated with Amicon Ultra-15 centrifuge filters (Ultracel 10K; Millipore Corp., Billerica, MA) as indicated by the manufacturer.
The method described above using affinity chromatography to resolve nonrafts and lipid rafts from purified plasma membranes avoided the use of detergents or high-pH carbonate buffers and was the method of choice in the present work for the following reasons. i) The affinity chromatography method is the only method that simultaneously isolates lipid rafts and nonrafts from purified plasma membranes (1, 11–14). ii) Direct comparison of the properties of lipid rafts isolated by this affinity chromatography method versus the classic Triton X-100 detergent method showed that the affinity chromatography produced lipid rafts that structurally and functionally most closely resembled those of intact cells (13). iii) Lipid rafts isolated by the affinity chromatography method exhibited the least amount of nonraft and intracellular contaminants. Mass spectrometry-based proteomics (19) and Western blotting (13, 14) demonstrate that detergent-resistant membranes may contain 30–60% cross-contaminating nonraft and intracellular proteins. Lipid rafts isolated through the use of high-pH bicarbonate buffer may also be highly impure, as shown by MS-based proteomics demonstrating the presence of 75% cross-contaminating nonraft and intracellular proteins (19). iv) There is increasing concern that detergent-resistant membranes may not necessarily equate with lipid rafts, quantitatively reflect the proportion of plasma membrane occurring as lipid rafts, or function as lipid rafts (reviewed in Refs. 1, 13, 20, 21).
Western blot analysis of lipid raft and nonraft fractions
To determine the relative purity of the plasma membrane nonraft and lipid raft fractions eluted from the ConA affinity column, aliquots of each fraction were separated by SDS-PAGE and analyzed by Western blotting (22) to detect relative expression levels of caveolin-1, flotillin, SR-B1, SCP-2, transferrin receptor, CXCR4, eNOS, Gq, ABCA1, and P-gp. Briefly, 10 µg of protein (homogenate, plasma membrane, lipid raft, and nonraft) was loaded onto Tricine gels (12%). Gels were run on a Mini-Protean II cell (Bio-Rad Laboratories, Hercules, CA) system at 100 V constant voltage for 
1.5–2 h (30 mA per gel initially). Proteins were electrophoretically transferred to nitrocellulose membranes (Bio-Rad) by applying a 100 V constant voltage for 2 h. After transfer, the blots were blocked in 3% gelatin in TBST (10 mM Tris-HCl, pH 8, 100 mM NaCl, and 0.05% Tween-20) for 1 h at room temperature. The blots were then washed twice with TBST and incubated overnight at room temperature with the respective polyclonal rabbit primary antibodies at dilutions of 1:250 (anti-flotillin, anti-ABCA1, anti-P-gp, and anti-SR-B1), 1:500 (anti-SCP-2, anti-caveolin-1, anti-eNOS, and anti-CXCR4), or 1:1,000 (anti-Gq and anti-transferrin receptor) in 1% gelatin in TBST. The blot was then washed three times with TBST and incubated for 2 h at room temperature with the appropriate secondary antibody (alkaline phosphatase conjugates of goat anti-rabbit IgG) diluted 1:4,500 in 1% gelatin TBST.
Next, the blot was washed three times with TBST and bands of interest were visualized by development with Sigma Fast 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolim tablets (Sigma) according to the manufacturer's protocol. Images of each blot were acquired using a single-chip charge-coupled device video camera and a computer workstation (IS-500 system; Alpha Innotech, San Leandro, CA). Densitometric analysis of image files was then performed (mean eight bit gray scale density) using NIH Image, available by anonymous FTP from zippy.nimh.nih.gov, to obtain relative levels of the respective proteins detected in each fraction. SCP-2 levels were obtained by comparison with standard curves of known amounts of pure SCP-2 on the same blot. After densitometric analysis of both the standard curve and unknowns on Western blots, image intensities in unknowns were quantitated by comparison with an SCP-2 standard curve within the linear range. For those proteins for which no source of pure protein was available (caveolin-1, flotillin, SR-B1, transferrin receptor, CXCR4, eNOS, Gq, ABCA1, and P-gp), relative protein levels were quantified as described above and expressed as integrated density values.
Immunoelectron microscopy
Small segments of liver tissue from a female wild-type mouse were fixed by immersion in alcohol-free 4% formaldehyde and 0.1% glutaraldehyde in 0.1 M Na phosphate buffer (pH 7.4) for 20 h at 4°C. The tissue segments were washed in 0.1 M Na phosphate buffer, dehydrated in an ascending ethanol gradient at progressively lower temperatures (23), infiltrated overnight at –50°C with Lowicryl HM20 acrylic resin, and polymerized by UV light. Ultrathin sections (60–80 nm) of liver were mounted on Formvar/carbon-coated nickel grids and immunostained by procedures recommended in the immunogold reagent product literature (Aurion, Wageningen, The Netherlands) with several modifications. Briefly, grids were incubated for 20 min in blocking solution of 1% acetylated BSA (Aurion) in 20 mM Tris-buffered saline and 0.05% Tween-20 (TBS-Tween), pH 7.35, and rinsed in TBS-Tween. Grids were incubated in rabbit anti-caveolin-1 antiserum diluted 1:50 in TBS-Tween with 0.1% BSA for 18 h at 4°C. After washing six times with TBS-Tween and 0.1% BSA, the grids were incubated for 2 h at 24°C with goat anti-rabbit IgG conjugated to 15 nm gold (Aurion) diluted 1:75 in TBS-Tween and 0.1% BSA. Unbound antibodies were removed from the sections by washing six times with TBS-Tween and 0.1% BSA, then the grids were rinsed with deionized water and poststained very briefly with aqueous uranyl acetate and Reynold's lead citrate. Immunogold labeling patterns on cells were examined at 80 kV with a Zeiss 10c transmission EM (Carl Zeiss Microimaging, Inc., Thornwood, NY).
Lipid extraction and analysis
Ganglioside GM1 levels were determined by a dot-blot technique as described elsewhere (12). Lipids from homogenate, lipid raft, and nonraft fractions were resolved into individual lipid classes as described (12). In brief, samples were extracted with n-hexane-2-propanol (3:2, v/v), spotted onto Silica Gel G TLC plates, and developed in petroleum ether-diethyl ether-methanol-acetic acid (90:7:2:0.5, v/v/v/v) (24). Neutral lipid content including total cholesterol, FFA, and cholesteryl ester was determined by the method of Marzo et al. (25). Total phospholipids, which included choline glycerophospholipid (PC), sphingomyelin (SM), ethanolamine glycerophospholipid (PE), phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylserine (PS), were scraped from the Silica Gel G TLC plates and eluted using chloroform/methanol/HCl (100:50:0.375, v/v/v). Samples were dried under N2 and resuspended in chloroform. Half of each sample was used for phospholipid fatty acid mass analysis and half was applied to Silica Gel 60 TLC plates to resolve individual phospholipids using chloroform-methanol-water-acetic acid (150:112.5:6:10.5, v/v/v/v). Phospholipid spots were visualized by iodine vapor and identified by comparison with known standards (26). Individual phospholipids were quantitated densitometrically as described (27) and confirmed by phosphorus estimation (28).
Both methods yielded similar quantity and percentage distribution of phospholipid classes. In brief, quantitative analysis was performed by analyzing spot intensity (as for proteins in Western blotting) and compared with linear phospholipid standard curves generated with the same TLC plate to determine phospholipid mass (27). Phospholipid identity was verified by comparison with known phospholipid standards and by lipid phosphorus assay (28). In several fractions, two SM bands were separated on the TLC plate, as reported by other investigators (29). The upper band (SM2) was differentiated from PC (next in order of resolution from the bottom of the TLC plate) by comparison with known phospholipid standards, by lipid phosphorus estimation (28), and was confirmed by GC-MS, noting the differences in the phospholipid fatty acid composition of SM versus PC. The SM fraction was composed of little to no PUFAs and exhibited a prevalence of 20:0, 22:0, 24:0, and 24:1 versus the PC fraction, with substantial levels of PUFAs, including 18:2, 20:4, and 22:6 (29). When comparing SM2 versus SM1, the longer chain fatty acids partitioned more into SM2, including 22:0, 24:0, and 24:1, whereas SM1 was composed of mostly 16:0, 18:0, 18:1, and 20:0 (29). Protein concentration was determined by the method of Bradford (30) from the dried protein extract residue digested overnight in 0.2 M KOH. Lipids were stored under an atmosphere of N2 to limit oxidation, and all glassware was washed with sulfuric acid-chromate before use.
Phospholipid fatty acid distribution
To obtain the fatty acid composition of each of the major individual phospholipid classes (PC, SM, PS, PI, and PE/PA) in lipid rafts and nonrafts, each phospholipid class was transesterified by acid to convert the phospholipid acyl chains to fatty acid methyl esters. Individual fatty acid methyl ester species were resolved according to chain length and unsaturation by GC-MS using an RTX-2330 capillary column (0.25 mm i.d. x 30 m; Restek, West Chester, PA) on a Thermo-Finnigan Trace DSQ single quadruple mass spectrometer (Thermo Electron Corp., Austin, TX) with electron impact and chemical ionization sources. Injector and detector temperatures were set at 240°C, with a temperature program of 100°C for 1 min, 10°C/min to 140°C, then 2°C/min to 220°C, hold for 1 min, then ramp 20°C/min to 240°C. Individual peaks were identified by comparison with known fatty acid methyl ester standards (Nu-Chek Prep) and referenced against a set concentration of 15:0 added before analysis. Sample identity was confirmed by GC-MS using the Trace DSQ single quadruple in chemical ionization mode.
Structure of hepatocyte plasma membrane lipid raft and nonraft domains
Fluorescent probes (DHE, cis-parinaric acid, NBD-stearic acid, DPH, DPH-TMA, and DPH-Pro), prepared as stock solutions in anhydrous ethanol with 2% (w/v) butylated hydroxytoluene added as an antioxidant, were allowed to incorporate into lipid rafts and nonrafts. Briefly, aliquots of lipid raft or nonraft fractions (35 µg of protein per 2 ml of 10 nM PIPES buffer, pH 7.4) were incubated at 37 ± 0.4°C with a small amount of fluorescent probe (protein-fluorophore ratio = 1,000 µg of protein to 1 µg of fluorophore) added from a concentrated stock solution in ethanol. The final ethanol concentration was <25 mM, well below that which would perturb membrane structure, lipid distribution, or fluidity (reviewed in Refs. 12, 13, 31), induce artifacts (sterol self-aggregation/crystallization) in membrane fractions (32), or disrupt protein-lipid interactions (33). Maximal probe incorporation was ensured by incubation for 30 min at 37°C. Steady-state fluorescence polarization was acquired with a PC1 spectrofluorometer in the T-format using photon-counting electronics (ISS Instruments, Inc., Champaign, IL) as described previously (13, 31). Polarization data were corrected for residual light scatter by converting polarization to anisotropy (r = 2P/3P) and subtracting residual fluorescence anisotropy from all measurements. To avoid inner filter effects, the absorbance of each sample (fluorescent probe + sample) at the wavelength of excitation for each probe was maintained at <0.15.
The following fluorescent lipidic probe molecules were chosen based on their ability to preferentially report on select aspects of membrane structure: i) DHE polarization, to probe the relative fluidity of sterol in lipid rafts and nonrafts (13, 31, 32); ii) DPH polarization, to probe for the presence of intermediate liquid-ordered phase lipids (reviewed in Ref. 34); iii) cis-parinaric acid and NBD-stearic acid polarization, to probe the acyl chain fluidity of naturally occurring and synthetic fatty acids, respectively (reviewed in Ref. 12); and iv) DPH-TMA and DPH-Pro polarization, to selectively probe the fluidity of the hydrophobic acyl chain region of lipids in the outer (exofacial) and inner (cytofacial) leaflets of plasma membranes, respectively, (i.e., transbilayer fluidity gradient). The leaflet selectivity of each of the probes (i.e., transbilayer distribution) within isolated plasma membrane fragments was verified by leaflet-selective quenching studies performed as reviewed in Refs. 35, 36 and detailed in the following individually cited papers showing that the inner and outer leaflets of isolated plasma membrane vesicles/fragments differ in fluidity: i) leaflet-selective quenching of DPH (nonselectively partitions into both leaflets) (37–39); ii) studies with right side-out and inside-out plasma membranes (38); iii) studies with the leaflet-selective DPH derivatives DPH-TMA and DPH-Pro (39), which are anchored closer to the membrane surface than DPH (40); iv) spin-label probes and purified right side-out and inside-out oriented plasma membranes (41); and v) model membrane studies of fluidity of outer versus inner leaflet phospholipid mixtures (42). Based on the these previous findings, fluorescence polarization of the leaflet-selective DPH derivatives DPH-TMA and DPH-Pro were used to measure the transbilayer fluidity gradient in hepatocyte plasma membrane lipid rafts and nonrafts exactly as described earlier (13, 39). These two plasma membrane vesicle fractions resolved by ConA affinity chromatography are both oriented right side out (11). Although the mechanisms whereby the DPH-TMA and DPH-Pro translocate and distribute selectively across the membrane are not known, it is established that zwitterionic phospholipids (PC and SM) and anionic phospholipids (PS) are enriched in the exofacial and cytofacial leaflets, respectively, as a result of the action of phospholipid flippases and/or scramblases present in the plasma membrane (43, 44). Thus, based on the similarities of charged polar head groups, it may be postulated that DPH-TMA (a zwitterionic polar head group) and DPH-Pro (an anionic polar head group) are translocated by plasma membrane flippases and/or scramblases to distribute similarly to their zwitterionic and anionic phospholipid counterparts. Finally, in support of the finding that leaflet-selective DPH-TMA and DPH-Pro show a more rigid cytofacial leaflet and a more fluid exofacial leaflet (39), studies with a nonflipping ESR probe inserted into right side-out and inside-out oriented plasma membranes confirm the more rigid cytofacial leaflet and more fluid exofacial leaflet (41).
Statistical analysis
All data values are expressed as means ± SEM (n = 3–7). To obtain statistical differences between values, ANOVA combined with the Newman-Keuls multiple comparisons test (GraphPad Prism, San Diego, CA) was performed. Differences at the P < 0.05 level were considered statistically significant.
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RESULTS |
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-methylmannoside). Enrichment of marker proteins and lipids in the ConA nonadherent and adherent fractions isolated from peripheral cell types (fibroblasts and MDCK cells) previously established these fractions to be enriched in nonrafts and lipid rafts, respectively (1, 12, 13).
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0.04, n = 6) at the expense of the nonraft fraction.
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(47)]. Although the transferrin receptor (also known as CD71) is an excellent protein marker for nonrafts in endothelial cells as well as many different cell lines and primary cells (45, 48, 49), in this work transferrin was not detectable in the nonadherent fraction isolated from primary mouse hepatocytes (data not shown). However, when the nonadherent fraction was tested for another nonraft protein marker, CXCR4, levels were 10.8-fold greater in the ConA nonadherent fraction compared with the adherent fraction (Fig. 3D). To distinguish whether the nonadherent fraction was deficient in lipid raft protein markers, Western blotting of known lipid raft protein markers (flotillin and Gq) was also performed. The nonadherent fraction was essentially devoid of flotillin (Fig. 3B, hatched bar) and Gq (Fig. 3C, hatched bar) compared with either the cell homogenate (Fig. 3, open bars) or the ConA adherent fraction (Fig. 3, closed bars). In contrast, Western blot analysis of the ConA adherent fraction showed >9-fold enrichment in flotillin (Fig. 3B) and 14-fold enrichment in Gq (Fig. 3C) compared with the nonadherent fraction. In SCP-2/SCP-x gene-ablated mice, with the exception of SCP-2 being absent, the pattern of protein markers for lipid rafts was similar to that in wild-type mice (i.e., enrichment of flotillin and Gq in lipid rafts and enrichment of CXCR4 in nonrafts). In summary, the Western blotting of protein markers confirmed that the ConA nonadherent and adherent fractions were enriched in nonrafts and lipid rafts, respectively, and expression of SCP-2/SCP-x significantly altered the relative proportion of lipid rafts versus nonrafts in the plasma membrane of primary hepatocytes.
Distribution of lipid markers in the ConA nonadherent and adherent fractions
To further confirm that the ConA nonadherent and adherent fractions from hepatocyte plasma membranes represented nonraft- and lipid raft-enriched fractions, lipid analyses for marker lipids were performed as described in Materials and Methods. Lipid rafts from peripheral cells are known to be enriched in total lipid, cholesterol, phospholipid, and sphingolipids (GM1 and SM) (reviewed in Ref. 13). In this work, the adherent fraction was lipid-rich, as shown by the >3-fold higher total lipid/protein compared with the ConA nonadherent fraction (Fig. 4A
). Examination of individual lipid classes from lipid rafts of wild-type mice revealed that the adherent fraction had several-fold more ganglioside GM1 (Fig. 4B), SM (Fig. 4C), total cholesterol (Fig. 4D), and total phospholipids (Fig. 4E) compared with the ConA nonadherent fraction or the cell homogenate. The molar ratio of cholesterol-phospholipid (C/P) was >2-fold higher in the adherent fraction compared with the nonadherent fraction (Fig. 4F).
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When phospholipid composition was expressed as a percentage, SCP-2/SCP-x gene ablation increased the percentage of SM in lipid rafts while decreasing the percentage of PE versus that in lipid rafts from wild-type hepatocytes (Fig. 5C). In nonraft fractions, lack of SCP-2/SCP-x expression did not significantly affect either the mass (Fig. 5B) or the percentage composition (Fig. 5C) of any of the phospholipid classes examined, with the exception of PI, whose percentage (Fig. 5C) but not mass (Fig. 5B) was increased somewhat. Finally, the overall ratio of anionic to neutral zwitterionic phospholipids was not changed in SCP-2/SCP-x null lipid rafts but increased by 2.4-fold in nonrafts (Fig. 5D) compared with the corresponding fractions from wild-type hepatocytes. In summary, the lipid marker distribution confirmed that the ConA nonadherent and adherent fractions were enriched in nonrafts and lipid rafts, respectively. In wild-type hepatocytes, lipid rafts were enriched in total lipids, total cholesterol, total phospholipids, and cholesterol-to-phospholipid ratio, as well as by mass in polar lipid classes (GM1, PC, SM, PE, PS, and PI), compared with nonrafts. SCP-2/SCP-x gene ablation significantly increased the total lipid content, total phospholipid, and select phospholipid classes (SM, PC, and PI) in lipid rafts. Furthermore, SCP-2 gene ablation abolished the difference in the C/P ratio in lipid rafts versus nonrafts by increasing the content of total phospholipid in lipid rafts and decreasing the content of total phospholipid in nonrafts, without altering cholesterol mass.
Select distribution of proteins involved in HDL-mediated cholesterol uptake/efflux in lipid rafts
To determine whether key proteins involved in RCT (ABCA1, P-gp, SR-B1, SCP-2, and caveolin-1) were compartmentalized in lipid rafts, Western blotting for the respective proteins was performed. Compared with the nonraft fraction of plasma membranes from hepatocytes, the lipid raft fraction was highly enriched by >100-fold in SCP-2 (Fig. 6A
, E), 6.6-fold in ABCA1 (Fig. 6B, F), 7.4-fold in P-gp (Fig. 6C, G), and 11-fold in SR-B1 (Fig. 6D, H). The presence of caveolin-1 was not detected in lipid rafts or nonrafts (Fig. 7A
, lanes 2 and 3, respectively). Indeed, although Western blotting detected caveolin-1 in liver homogenates (Fig. 7A, lane 1), the concomitant presence of high levels of eNOS therein (Fig. 7B, lane 2) suggested that caveolin-1 may reside in nonhepatic cells (endothelial, stellate, and Kupffer cells) rather than in hepatoctyes. Consistent with this possibility, eNOS (Fig. 7B, lane 3), the transferrin receptor [protein marker present in nonrafts of other cell types (45, 48, 49)], in addition to caveolin-1 (Fig. 7, lanes 2, 3) were absent in lipid raft and nonraft fractions isolated from hepatocyte cells. Because liver contains not only hepatocytes but also a variety of other cells (endothelial, stellate, and Kupffer cells), the latter being enriched in caveolin-1 (50, 51), antigenic sites of caveolin-1 were also localized on thin sections of liver with 15 nm gold particles using immunogold electron microscopy. Multiple sites of caveolin-1 in endothelial cells (Fig. 7C, arrowheads) and other nonparenchymal cell types (data not shown) were observed. In contrast, hepatocytes (Fig. 7D) did not show staining for caveolin-1 above random background levels. These results were consistent with the absence of caveolin-1 from hepatocytes both in mouse hepatocyte primary cell cultures and in vivo.
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Distribution of phospholipid polyunsaturated fatty acids that regulate the distribution of proteins to lipid rafts
Select phospholipid classes were next examined. SM of both lipid rafts and nonrafts was relatively enriched in saturated (16:0, 18:0, 20:0, 22:0, and 24:0) and monounsaturated (16:1, 18:1, 20:1, 22:1, and 24:1) fatty acids while being relatively poor in polyunsaturated fatty acids (see supplementary Table I) compared with other phospholipid classes (see supplementary Tables II–IV). For more ready comparison, the individual groups of saturated (Sat), unsaturated (Unsat), monounsaturated, and polyunsaturated fatty acids were summed and the ratios of Sat/Unsat and MUFA/Sat and the double bond index (DBI) were calculated and collated in Table 1
. Thus, the DBI (defined as the summation of the mole fraction times the number of double bonds for each fatty acid) was 2- to 5-fold lower in SM (Table 1) than in other phospholipid classes (Tables 2
–4
). In SM from wild-type hepatoctyes, the Sat/Unsat ratio was significantly higher than in nonrafts (Table 1). SCP-2/SCP-x gene ablation abolished this difference (Table 1). Esterified fatty acids detected in the lipid raft PS class from wild-type hepatoctyes also differed significantly from nonrafts (see supplementary Table II). In wild-type hepatocyte lipid rafts, the PUFAs were 2-fold lower, giving rise to a higher Sat/Unsat ratio and a lower DBI compared with the nonraft fraction (Table 2). SCP-2/SCP-x gene ablation increased PUFA and DBI while decreasing the Sat/Unsat ratio in lipid rafts compared with lipid rafts from wild-type hepatocytes (Table 2). Concomitantly, SCP-2/SCP-x gene ablation increased MUFA and the MUFA/Sat ratio in nonrafts compared with nonrafts from wild-type hepatocytes (Table 2). Lastly, SCP-2/SCP-x gene ablation did not significantly alter any of the fatty acid parameters in raft versus nonraft fractions from SCP-2/SCP-x gene-ablated hepatocytes (Table 2).
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For PI, the esterified fatty acids in lipid rafts from wild-type hepatoctyes also differed significantly from nonrafts (see supplementary Table IV). As shown in Table 4, the wild-type lipid raft fraction had lower MUFA, MUFA/Sat ratio, and DBI values, whereas the Sat/Unsat ratio was higher compared with the nonraft fraction. SCP-2/SCP-x gene ablation did not alter fatty acid parameters in lipid rafts compared with lipid rafts from wild-type hepatocytes (Table 4). Likewise, SCP-2/SCP-x gene ablation did not alter these parameters in nonrafts compared with nonrafts from wild-type hepatocytes (Table 4). However, lipid rafts from SCP-2/SCP-x gene-ablated hepatocytes exhibited increased saturated fatty acids and Sat/Unsat ratio concomitant with decreased unsaturated fatty acids, MUFA, PUFA, and MUFA/Sat ratio compared with nonrafts from SCP-2/SCP-x gene-ablated hepatocytes (Table 4).
Esterified fatty acids detected in the lipid raft PE class from wild-type hepatoctyes also differed significantly from nonrafts (see supplementary Table V). As summarized in Table 5 , in wild-type hepatocytes compared with the nonraft fraction, the lipid raft fraction had higher saturated fatty acids and Sat/Unsat ratio but lower unsaturated fatty acids, PUFA, MUFA, and DBI. SCP-2/SCP-x gene ablation had no effect on fatty acid parameters in lipid rafts compared with lipid rafts from wild-type hepatocytes (Table 5). Likewise, SCP-2/SCP-x gene ablation had no effect on these parameters in nonrafts compared with nonrafts from wild-type hepatocytes (Table 5). However, lipid rafts from SCP-2/SCP-x gene-ablated hepatocytes exhibited increased saturated fatty acids and Sat/Unsat ratio concomitant with decreased unsaturated fatty acids, MUFA, and DBI compared with nonrafts from SCP-2/SCP-x gene-ablated hepatocytes (Table 5). Thus, phospholipids in lipid rafts from wild-type hepatocytes had a higher ratio of saturated to unsaturated fatty acids versus that in nonrafts. SCP-2/SCP-x gene ablation significantly increased the Sat/Unsat ratio in PE, did not change this ratio in SM, PC, or PI, and decreased this ratio in PS from lipid rafts compared with lipid rafts from wild-type hepatocytes.
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Fluorescence polarization of cis-parinaric acid and NBD-stearic acid to probe fatty acyl chain structure in lipid rafts and nonrafts
Long (i.e., 18 carbon) straight-chain fatty acids such as the synthetic NBD-stearic acid and the naturally occurring kinked-chain cis-parinaric acid probe the acyl chain microenvironment of membrane lipid rafts and nonrafts (1, 13, 31). Fluorescence polarization of NBD-stearic acid and cis-parinaric acid were both higher in lipid rafts than in nonrafts (Table 6). SCP-2/SCP-x gene ablation significantly increased the fluorescence polarization of both NBD-stearic acid and cis-parinaric acid in lipid rafts but not in nonrafts (Table 6). Thus, the acyl chain microenvironments sensed by NBD-stearic acid and cis-parinaric acid were significantly more rigid (i.e., higher polarization) in lipid rafts than in nonraft microdomains. SCP-2/SCP-x gene ablation significantly rigidified the lipid raft microenvironments, as indicated by increased polarization of these probes.
Fluorescence polarization of leaflet-selective probes (DPH-TMA and DPH-Pro) in lipid rafts and nonrafts
Fluorescence polarization of DPH-TMA was significantly lower than that of the inner leaflet-selective probe DPH-Pro in both lipid rafts and nonrafts (Table 6). Polarization of the outer leaflet-selective probe DPH-TMA was significantly higher in lipid rafts than in nonrafts (Table 6). SCP-2/SCP-x gene ablation increased the polarization of DPH-TMA in lipid rafts as well as nonrafts (Table 6). In contrast, SCP-2/SCP-x gene ablation decreased the polarization of DPH-Pro in lipid rafts as well as nonrafts (Table 6). Thus, these data showed that the inner leaflet of lipid rafts and nonrafts was significantly more ordered (higher polarization) than was the outer leaflet. SCP-2/SCP-x gene ablation increased the polarization of the outer leaflet-selective probe TMA-DPH but decreased that of the inner leaflet-selective probe DPH-Pro in both membrane fractions, such that the ratio of DPH-TMA to DPH-Pro polarization was increased from 0.80 to 1.00 in lipid rafts and from 0.87 to 1.04 in nonrafts. Thus, by increasing the order (DPH-TMA polarization) in the outer leaflet while concomitantly decreasing the order (DPH-Pro) in the inner leaflet, SCP-2/SCP-x gene ablation effectively abolished the normal transbilayer fluidity difference in both rafts and nonrafts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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First, lipid rafts exist in hepatocyte plasma membranes, representing 33% of total plasma membrane protein, countering the common misconception that has equated the almost negligible levels of caveolin-1 in liver with a paucity of raft domains. It should be noted that basically similar amounts of lipid rafts are isolated by affinity chromatography from purified plasma membranes of peripheral-type cells such as fibroblasts (12).
Second, key protein components of RCT, including SR-B1, ABCA1, and P-gp, were highly enriched in hepatocyte lipid raft domains, but caveolin-1 was not detected in purified lipid rafts or nonrafts. Thus, lipid rafts from hepatocyte plasma membranes differed significantly from those of peripheral cells. Because caveolin-1 correlates with enhanced cholesterol efflux and the inhibition of HDL-cholesteryl ester uptake from peripheral cells (reviewed in Ref. 1), lack of caveolin-1 in hepatocytes suggests that hepatocytes are thereby optimally positioned to facilitate HDL-cholesterol/cholesteryl ester uptake rather than efflux. For example, model membrane studies show that both spontaneous and SCP-2-mediated cholesterol transfer is slower from more rigid compared with fluid membranes (reviewed in Ref. 53). Because lipid rafts are more rigid than nonrafts, it might be expected that sterol transfer would be faster from nonrafts than from rafts. Based on these and other considerations, it has been postulated that the majority of exported cholesterol is not raft-associated (reviewed in Ref. 54). On the contrary, in vitro studies show that both spontaneous and SCP-2-mediated cholesterol transfer are nearly 1 order of magnitude faster from lipid rafts (enriched in RCT proteins such as SR-B1 and caveolin-1) than nonrafts isolated from plasma membranes of cultured liver cell fibroblasts (12). Lipid rafts from cultured primary hepatocytes are also enriched in RCT proteins such as SR-B1, ABCA1, and P-gp, which are known to confer enhanced dynamics to cholesterol because: i) SR-B1 (55) and caveolin-1 (56) directly bind cholesterol; ii) ABCA1 (57), SR-B1 (58), P-gp (59), and ABCG1 (54, 60) induce the formation of a mobile cholesterol pool/domain; iii) ABCA1 (61) and P-gp (59) both enhance transbilayer cholesterol migration from the cytofacial to the exofacial leaflet; and iv) SCP-2 overexpression enhances cholesterol uptake/esterification (62–64), whereas SCP-2/SCP-x gene ablation reduces hepatic cholesterol accumulation (65). Thus, by localizing SR-B1, ABCA1, and P-gp in lipid rafts as well as expressing high levels of SCP-2, liver hepatocytes are optimally positioned to mediate the final steps of RCT.
With regard to compensatory changes in these and other RCT proteins in response to SCP-2/SCP-x gene ablation, plasma membrane transporters (SR-B1, ABCA1, and P-gp) were not upregulated, but at least one intracellular transporter (L-FABP) was upregulated, concomitant with hypersecretion of bile acids and cholesterol (65, 66). Conversely, L-FABP gene ablation decreases biliary cholesterol (67). It should also be noted that potential compensation for SCP-2/SCP-x gene ablation by the upregulation of L-FABP (65) described above may also contribute in part to altered C/P ratios and the modest changes in the fatty acid profiles of phospholipids in lipid rafts. L-FABP is known to stimulate the rate-limiting step in microsomal phospholipid synth
