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A Role for Hepatic Scavenger Receptor Class B, Type I in Decreasing High Density Lipoprotein Levels in Mice That Lack Phosphatidylethanolamine N-Methyltransferase

2008; Elsevier BV; Volume: 283; Issue: 51 Linguagem: Inglês

10.1074/jbc.m807433200

ISSN

1083-351X

Autores

Julie C. Robichaud, Gordon A. Francis, Dennis E. Vance,

Tópico(s)

Peroxisome Proliferator-Activated Receptors

Resumo

Phosphatidylethanolamine N-methyltransferase (PEMT) is a liver-specific enzyme that converts phosphatidylethanolamine to phosphatidylcholine (PC). Mice that lack PEMT have reduced plasma levels of PC and cholesterol in high density lipoproteins (HDL). We have investigated the mechanism responsible for this reduction with experiments designed to distinguish between a decreased formation of HDL particles by hepatocytes or an increased hepatic uptake of HDL lipids. Therefore, we analyzed lipid efflux to apoA-I and HDL lipid uptake using primary cultured hepatocytes isolated from Pemt+/+ and Pemt–/– mice. Hepatic levels of the ATP-binding cassette transporter A1 are not significantly different between Pemt genotypes. Moreover, hepatocytes isolated from Pemt–/– mice released cholesterol and PC into the medium as efficiently as did hepatocytes from Pemt+/+ mice. Immunoblotting of liver homogenates showed a 1.5-fold increase in the amount of the scavenger receptor, class B, type 1 (SR-BI) in Pemt–/– compared with Pemt+/+ livers. In addition, there was a 1.5-fold increase in the SR-BI-interacting protein PDZK1. Lipid uptake experiments using radiolabeled HDL particles revealed a greater uptake of [3H]cholesteryl ethers and [3H]PC by hepatocytes derived from Pemt–/– compared with Pemt+/+ mice. Furthermore, we observed an increased association of [3H]cholesteryl ethers in livers of Pemt–/– compared with Pemt+/+ mice after tail vein injection of [3H]HDL. These results strongly suggest that PEMT is involved in the regulation of plasma HDL levels in mice, mainly via HDL lipid uptake by SR-BI. Phosphatidylethanolamine N-methyltransferase (PEMT) is a liver-specific enzyme that converts phosphatidylethanolamine to phosphatidylcholine (PC). Mice that lack PEMT have reduced plasma levels of PC and cholesterol in high density lipoproteins (HDL). We have investigated the mechanism responsible for this reduction with experiments designed to distinguish between a decreased formation of HDL particles by hepatocytes or an increased hepatic uptake of HDL lipids. Therefore, we analyzed lipid efflux to apoA-I and HDL lipid uptake using primary cultured hepatocytes isolated from Pemt+/+ and Pemt–/– mice. Hepatic levels of the ATP-binding cassette transporter A1 are not significantly different between Pemt genotypes. Moreover, hepatocytes isolated from Pemt–/– mice released cholesterol and PC into the medium as efficiently as did hepatocytes from Pemt+/+ mice. Immunoblotting of liver homogenates showed a 1.5-fold increase in the amount of the scavenger receptor, class B, type 1 (SR-BI) in Pemt–/– compared with Pemt+/+ livers. In addition, there was a 1.5-fold increase in the SR-BI-interacting protein PDZK1. Lipid uptake experiments using radiolabeled HDL particles revealed a greater uptake of [3H]cholesteryl ethers and [3H]PC by hepatocytes derived from Pemt–/– compared with Pemt+/+ mice. Furthermore, we observed an increased association of [3H]cholesteryl ethers in livers of Pemt–/– compared with Pemt+/+ mice after tail vein injection of [3H]HDL. These results strongly suggest that PEMT is involved in the regulation of plasma HDL levels in mice, mainly via HDL lipid uptake by SR-BI. Phosphatidylcholine (PC) 3The abbreviations used are:PCphosphatidylcholineHDLhigh density lipoproteinsPEphosphatidylethanolaminePEMTphosphatidylethanolamine N-methyltransferaseSR-BIscavenger receptor, class B, type ITGtriacylglycerol plays a crucial role in the maintenance of membrane integrity and is involved in intracellular signal transduction. PC is also the predominant phospholipid found in plasma lipoproteins, bile and lung surfactant (1Vance J.E. Adeli K. Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes. 5th Ed. Elsevier, Amsterdam2008: 507-539Crossref Scopus (9) Google Scholar). All mammalian cells can synthesize PC from choline via the Kennedy (CDP-choline) pathway (2Kennedy E.P. Weiss S.B. J. Biol. Chem. 1956; 222: 193-214Abstract Full Text PDF PubMed Google Scholar). The rate-limiting enzyme for this synthetic route is CTP:phosphocholine cytidylyltransferase (3Vance D.E. Choy P.C. Trends Biochem. Sci. 1979; 4: 145-148Abstract Full Text PDF Scopus (110) Google Scholar). In the liver, an alternative pathway contributes to the biosynthesis of PC. This pathway involves three successive methylations of phosphatidylethanolamine (PE) catalyzed by PE N-methyltransferase (PEMT) and represents one-third of hepatic PC production (4Bremer J. Greenberg D.M. Biochim. Biophys. Acta. 1960; 37: 173-175Crossref PubMed Scopus (102) Google Scholar, 5Vance D.E. Ridgway N.D. Prog. Lipid Res. 1988; 27: 61-79Crossref PubMed Scopus (199) Google Scholar). The liver-specific expression of this enzyme has suggested several roles for PEMT-derived PC in hepatic metabolism, including its involvement in lipoprotein metabolism (5Vance D.E. Ridgway N.D. Prog. Lipid Res. 1988; 27: 61-79Crossref PubMed Scopus (199) Google Scholar, 6Vance J.E. Nguyen T. Vance D.E. Biochim. Biophys. Acta. 1986; 875: 501-509Crossref PubMed Scopus (26) Google Scholar, 7Vance J.E. Vance D.E. J. Biol. Chem. 1986; 261: 4486-4491Abstract Full Text PDF PubMed Google Scholar, 8Vance J.E. Vance D.E. Annu. Rev. Nutr. 1990; 10: 337-356Crossref PubMed Scopus (55) Google Scholar) and bile secretion (9Noga A.A. Vance D.E. J. Lipid Res. 2003; 44: 1998-2005Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 10Agellon L.B. Walkey C.J. Vance D.E. Kuipers F. Verkade H.J. Hepatology. 1999; 30: 725-729Crossref PubMed Scopus (26) Google Scholar). In 1997, a mouse lacking PEMT was generated to gain further insight into the function of this enzyme. When fed a chow diet, Pemt–/– mice appear normal, and the production of PC from the Kennedy pathway is increased by ∼50! to compensate for the absence of the PEMT activity (11Walkey C.J. Donohue L.R. Bronson R. Agellon L.B. Vance D.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12880-12885Crossref PubMed Scopus (139) Google Scholar). Indeed, hepatic levels of PC do not change significantly between the Pemt+/+ and Pemt–/– mice. However, when Pemt–/– mice are fed a choline-deficient diet to attenuate the CDP-choline pathway, severe liver damage occurred within 3 days (12Walkey C.J. Yu L. Agellon L.B. Vance D.E. J. Biol. Chem. 1998; 273: 27043-27046Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). The liver failure can be reversed if choline is fed to the mice prior to the third day of the choline-deficient diet (13Waite K.A. Cabilio N.R. Vance D.E. J. Nutr. 2002; 132: 68-71Crossref PubMed Scopus (64) Google Scholar). Interestingly, neither dietary dimethylethanolamine (14Waite K.A. Vance D.E. Biochim. Biophys. Acta. 2004; 1636: 175-182Crossref PubMed Scopus (11) Google Scholar) nor propanolamine (15Li Z. Vance D.E. Biochim. Biophys. Acta. 2007; 1771: 486-490Crossref PubMed Scopus (5) Google Scholar) can substitute for dietary choline when fed to Pemt–/– mice, indicating a striking specificity for PC in liver function. It is believed that the PEMT pathway survived throughout evolution to provide PC in situations where choline intake is insufficient, such as pregnancy/lactation or starvation (12Walkey C.J. Yu L. Agellon L.B. Vance D.E. J. Biol. Chem. 1998; 273: 27043-27046Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). phosphatidylcholine high density lipoproteins phosphatidylethanolamine phosphatidylethanolamine N-methyltransferase scavenger receptor, class B, type I triacylglycerol More recently, the role of PEMT in very low density lipoprotein metabolism was examined. PEMT deficiency in male mice challenged with a high fat/high cholesterol diet resulted in a 50! decrease in apoB100, PC and triacylglycerol (TG) secretion from the liver (9Noga A.A. Vance D.E. J. Lipid Res. 2003; 44: 1998-2005Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). These observations are in agreement with earlier work in which levels of secreted apoB100 and lipids (PC and TG) by hepatocytes from Pemt–/– mice were 50–70! lower than from Pemt+/+ mice (16Noga A.A. Zhao Y. Vance D.E. J. Biol. Chem. 2002; 277: 42358-42365Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). This defect in very low density lipoprotein secretion in the absence of PEMT activity was only evident in males fed an HF/HC diet, showing a gender- and diet-specific alteration of lipoprotein metabolism in Pemt–/– mice (16Noga A.A. Zhao Y. Vance D.E. J. Biol. Chem. 2002; 277: 42358-42365Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Several adaptive mechanisms might be involved in regulating PC homeostasis in Pemt–/– mice, such as redistribution of cellular PC pools (17Li Z. Agellon L.B. Vance D.E. J. Biol. Chem. 2007; 282: 10283-10289Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), reduction in PC degradation, and/or regulation of lipid flux in and out of hepatocytes to maintain PC levels constant and ensure optimal liver functions. In this respect, a significant reduction of cholesterol and PC in plasma HDL is observed in both genders of Pemt–/– mice fed an HF/HC diet. Surprisingly, these lower HDL levels were also detected in female Pemt–/– mice fed a chow diet (18Noga A.A. Vance D.E. J. Biol. Chem. 2003; 278: 21851-21859Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). This decrease in plasma HDL lipids is intriguing, particularly since a role for PEMT in HDL metabolism has not been previously addressed. In the last decade, many studies have focused on HDL formation, maturation, and delivery of its associated lipids to the liver. This reverse cholesterol transport (19Glomset J.A. J. Lipid Res. 1968; 9: 155-167Abstract Full Text PDF PubMed Google Scholar, 20Fielding P.E. Fielding C.J. Biochemistry. 1995; 34: 14288-14292Crossref PubMed Scopus (258) Google Scholar, 21Fielding C.J. Havel R.J. Arch. Pathol. Lab. Med. 1977; 101: 225-229PubMed Google Scholar) is believed to be atheroprotective, since HDL mediates the transport of excess cholesterol from peripheral tissues back to the liver for conversion into bile acids and/or for excretion into bile (22Kozarsky K.F. Donahee M.H. Rigotti A. Iqbal S.N. Edelman E.R. Krieger M. Nature. 1997; 387: 414-417Crossref PubMed Scopus (628) Google Scholar, 23Glass C. Pittman R.C. Weinstein D.B. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5435-5439Crossref PubMed Scopus (423) Google Scholar, 24Cohen D.E. Curr. Opin. Lipidol. 1999; 10: 295-302Crossref PubMed Scopus (41) Google Scholar). The generation of HDL particles from poorly lipidated apoA-I involves the efflux of cellular lipids and relies on the activity of ABCA1 (ATP-binding cassette transporter A1). In addition, partially lipidated “nascent” HDL can acquire unesterified cholesterol in an ABCA1-independent manner via other ABC transporters, such as ABCG1 or ABCG4, or via a passive diffusion process (25Oram J.F. Heinecke J.W. Physiol. Rev. 2005; 85: 1343-1372Crossref PubMed Scopus (423) Google Scholar, 26Yokoyama S. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 20-27Crossref PubMed Scopus (106) Google Scholar, 27Fielding C.J. Fielding P.E. Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins, and Membranes. 5th Ed. Elsevier, Amsterdam2008: 533-553Crossref Scopus (14) Google Scholar). Because hepatocytes secrete apoA-I, express ABCA1 on their cell surface, and can generate HDL by both ABCA1-dependent and -independent pathways (28Kiss R.S. McManus D.C. Franklin V. Tan W.L. McKenzie A. Chimini G. Marcel Y.L. J. Biol. Chem. 2003; 278: 10119-10127Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 29Sahoo D. Trischuk T.C. Chan T. Drover V.A. Ho S. Chimini G. Agellon L.B. Agnihotri R. Francis G.A. Lehner R. J. Lipid Res. 2004; 45: 1122-1131Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), we considered the possibility that ABCA1-mediated efflux of PC and cholesterol from PEMT-deficient hepatocytes might be decreased as a mechanism for maintaining PC homeostasis in the liver. It is also now well established that lipids associated with HDL can be delivered to steroidogenic tissues (adrenals, ovary, or testes) and to the liver via the scavenger receptor class B, type I (SR-BI) (30Schneider W.J. Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins, and Membranes. 5th Ed. Elsevier, Amsterdam2008: 555-578Crossref Scopus (3) Google Scholar). This HDL receptor is mainly expressed on the cell surface of hepatocytes and steroidogenic cells and is involved in the selective uptake of lipids from plasma lipoproteins, without concomitant degradation of the whole lipoprotein particle (31Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2006) Google Scholar). Thus, cholesteryl esters and other lipids are segregated from apoA-I, which is recycled back to the plasma compartment, whereas lipids are thought to be targeted to the canalicular membrane for biliary secretion. Although SR-BI has also been described as a promoter of cholesterol efflux (32Ji Y. Jian B. Wang N. Sun Y. Moya M.L. Phillips M.C. Rothblat G.H. Swaney J.B. Tall A.R. J. Biol. Chem. 1997; 272: 20982-20985Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar), the absence of a cholesterol efflux defect from SR-BI-deficient mouse macrophages to HDL suggests SR-BI has antiatherosclerotic effects for reasons other than cholesterol efflux (33Zhang W. Yancey P.G. Su Y.R. Babaev V.R. Zhang Y. Fazio S. Linton M.F. Circulation. 2003; 108: 2258-2263Crossref PubMed Scopus (173) Google Scholar). Currently, SR-BI is believed to play a crucial role in the final step of reverse cholesterol transport (i.e. the selective uptake and sorting of plasma HDL lipids) (32Ji Y. Jian B. Wang N. Sun Y. Moya M.L. Phillips M.C. Rothblat G.H. Swaney J.B. Tall A.R. J. Biol. Chem. 1997; 272: 20982-20985Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar, 34Jian B. de la Llera-Moya M. Ji Y. Wang N. Phillips M.C. Swaney J.B. Tall A.R. Rothblat G.H. J. Biol. Chem. 1998; 273: 5599-5606Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 35Gu X. Kozarsky K. Krieger M. J. Biol. Chem. 2000; 275: 29993-30001Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Thus, another explanation for the reduced levels of plasma HDL in Pemt–/– mice might be a compensatory up-regulation of selective lipid uptake from HDL by hepatocytes in an attempt to maintain PC levels. Thus, using Pemt–/– mice as a model, we have studied the hepatic contribution of PC and cholesterol to HDL formation and subsequent lipid uptake by the liver under conditions for which PC biosynthesis was challenged. The results show that in livers and hepatocytes from Pemt–/– mice, compared with Pemt+/+ mice, the expression of SR-BI is increased by 50!, and the uptake of cholesterol is enhanced. Materials—HDL (d = 1.07–1.21 g/ml) were obtained by ultracentrifugation of pooled plasma of healthy male and female volunteers (36Chung B.H. Wilkinson T. Geer J.C. Segrest J.P. J. Lipid Res. 1980; 21: 284-291Abstract Full Text PDF PubMed Google Scholar). The protein fraction of HDL was obtained by delipidation of HDL, and purified apoA-I was obtained by chromatography on DEAE-cellulose (37Yokoyama S. Tajima S. Yamamoto A. J. Biochem. (Tokyo). 1982; 91: 1267-1272Crossref PubMed Scopus (73) Google Scholar). Laboratory rodent chow diet 5001 for Pemt+/+ and Pemt–/– mice was from PMI Nutrition International (St. Louis, MO). Collagen-coated dishes for hepatocyte culture were from BD Biosciences (Mississauga, Canada). [3H]Glycerol, [3H]choline, and [3H]cholesteryl oleoyl ether were from Amersham Biosciences. [35S]EasyTag™ EXPRESS35S Protein Labeling Mix (PerkinElmer Life Sciences) was used for the metabolic labeling of SR-BI. Collagenase for liver perfusion was from Sigma. Silica gel G60 plates for thin layer chromatography (TLC Silica Gel 60) were from Merck. The PC standard was purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol, cholesteryl oleate ester standards, 6-amino-n-hexanoic acid, and dodecyl maltoside were obtained from Sigma. The BCA protein kit, including the bovine serum albumin standard, was obtained from Pierce. Rabbit polyclonal anti-mouse SR-BI (NB 400-101), goat polyclonal anti-SR-BI (NB 400-131), rabbit polyclonal anti-PDZK1 (NB 400-149), and rabbit polyclonal anti-human ABCA1 antibodies were obtained from Novus Biologicals (Littleton, CO). Rabbit polyclonal anti-human apoA-I antibodies were from Biodesigns (Kennebunk, ME). Rabbit polyclonal anti-rat protein-disulfide isomerase antibody was from StressGen (Victoria, Canada). Goat anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase was from Pierce, and the enhanced chemiluminescence detection system was from Amersham Biosciences (Buckinghamshire, UK). Cholesterol and choline-containing phospholipids were analyzed by fast protein liquid chromatography using the Infinity Cholesterol reagent from ThermoDMA (Calgary, Canada) and Phospholipids B kit from Wako Diagnostics (Richmond, VA), respectively. All other chemicals and reagents were from standard commercial sources. Reagents for quantitative PCR analysis of mRNA levels were purchased from Invitrogen. The molecular weight markers for the blue native PAGE were from Amersham Biosciences. Care and Feeding of Mice—Pemt+/+ and Pemt–/– mice had a mixed genetic background of 129/J and C57BL/6 (11Walkey C.J. Donohue L.R. Bronson R. Agellon L.B. Vance D.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12880-12885Crossref PubMed Scopus (139) Google Scholar) and were maintained via homozygous breeding in a reversed 12-h light/dark cycle. All mice were females 10–14 weeks old. Similar experiments were performed using male mice with comparable outcomes. Since the difference in HDL between the genotypes was greater with females than males (18Noga A.A. Vance D.E. J. Biol. Chem. 2003; 278: 21851-21859Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), the studies concentrated on female mice. Hepatocyte Isolation and Culture—Primary hepatocytes were isolated from Pemt+/+ and Pemt–/– mice after liver perfusion with collagenase (16Noga A.A. Zhao Y. Vance D.E. J. Biol. Chem. 2002; 277: 42358-42365Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Cells (2 × 106 cells/collagen-coated 60-mm dish) were cultured in Dulbecco's modified Eagle's medium containing 15! fetal bovine serum. Lipid Mobilization Assays—Hepatocytes from Pemt+/+ and Pemt–/– mice were incubated for 16 h at 37 °C in the presence of 5 μCi of [3H]glycerol, 1 mm glycerol with or without 20 μg/ml apoA-I. The media were collected, cells were scraped into phosphate-buffered saline, and lipids were extracted with CHCl3/CH3OH (2:1) and then washed with (CH3OH/H2O/CHCl3/CH3COOH (480:470:30:9.6, v/v/v/v) (38Folch J. Lees M. Sloane-Stanley G.H. J. Biol. Chem. 1959; 226: 495-509Google Scholar). The solvents were evaporated under nitrogen, and lipids were dissolved in CHCl3. Phospholipids were separated by thin layer chromatography using a developing solvent of CHCl3/CH3OH/CH3COOH/H2O (25:15:4:2, v/v/v/v) until the solvent reached halfway up the plate. The solvent was evaporated from the plates, and the neutral lipids were separated using heptane/diisopropyl ether/acetic acid (60:40:4, v/v/v), which developed to the top of the plate. Phospholipids were visualized with iodine vapor, and the band corresponding to [3H]PC was scraped into scintillation vials for measurement of radioactivity. The efflux of PC to apoA-I was expressed as the percentage of total [3H]PC (in cells and media) recovered in the culture media. Neutral lipids were visualized after reaction with sulfuric acid. Cholesterol mass was measured by gas-liquid chromatography using 5α-cholestane as a standard. Radiolabeling of Phosphatidylcholine—Two dishes of McArdle 7777 rat hepatoma cells (2.5 × 106 cells/60-mm dish) were incubated for 12 h at 37 °C with 50 μCi of [3H]choline. Cells were scraped into phosphate-buffered saline, and lipids were extracted in CHCl3/CH3OH (2:1). Phospholipids were separated by thin layer chromatography in the solvent system CHCl3/CH3OH/CH3COOH/H2O (25:15:4:2, v/v/v/v) and stained with iodine vapor. [3H]PC was scraped from the plate, and CHCl3/CH3OH was added. After centrifugation at 600 × g for 5 min, the supernatant containing [3H]PC was collected. Analysis of Plasma Lipoprotein Lipid Profiles—Lipoproteins in 12 μl of plasma were separated isocratically on a 30 × 1-cm Superose 6 gel filtration fast protein liquid chromatography column (Amersham Biosciences). Eluted fractions were mixed with Infinity cholesterol reagent (ThermoDMA), and cholesterol content was measured at 37 °C by spectrophotometric detection at 500 nm. Choline-containing phospholipids were measured postcolumn using the Phospholipids B kit from Wako Diagnostics. Isolation and Radiolabeling of HDL—Radiolabeled cholesteryl oleoyl ether (50 μCi) and PC (50 μCi) were sonicated in a 37 °C water bath in 1 ml of phosphate-buffered saline for 10 min. The donor liposomes were incubated with 25 mg of HDL and cholesteryl ester transfer protein; fresh lipoprotein-deficient human plasma (d > 1.21 g/ml) was heated at 60 °C to inactivate lecithin:cholesterol acyltransferase and was used as a source of cholesteryl ester transfer protein (39Rodrigueza W.V. Thuahnai S.T. Temel R.E. Lund-Katz S. Phillips M.C. Williams D.L. J. Biol. Chem. 1999; 274: 20344-20350Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). The lipoprotein preparation was mixed at 37 °C for 48 h on an orbital shaker at 200 rpm, and HDLs were reisolated by sequential centrifugations (36Chung B.H. Wilkinson T. Geer J.C. Segrest J.P. J. Lipid Res. 1980; 21: 284-291Abstract Full Text PDF PubMed Google Scholar). The [3H]HDL was allowed to percolate through a heparin-Sepharose column to remove apoE/apoB-containing lipoproteins. After extensive dialysis, the protein concentration was determined (40Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216428) Google Scholar). [3H]HDL was kept at 4 °C under nitrogen. Lipid Uptake by Hepatocytes—Hepatocytes from Pemt+/+ and Pemt–/– mice were incubated 1 h at 37°C in Dulbecco's modified Eagle's medium containing 75 μg of protein/ml of [3H]HDL. The radioactive media were removed, and then cells were washed and incubated with 50 μg of protein/ml of unlabeled HDL to eliminate nonspecific binding of HDL to cellular membranes. Cells were scraped, and the lipids were extracted and separated by thin layer chromatography as described above. Radioactivity associated with cell extracts (as [3H]PC and [3H]cholesteryl ether) was measured with a liquid scintillation counter. In Vivo Lipid Uptake Assays—Pemt+/+ and Pemt–/– mice were fasted overnight, after which they were injected via the tail vein with 200 μl of [3H]HDL (∼500 μg of [3H]HDL protein containing 2000 dpm/μg of [3H]cholesteryl ether and 750 dpm/μg of [3H]PC). 1 h after the injection, the mice were sacrificed, the livers were removed and homogenized, lipids were extracted, and the associated radioactivity was measured. Immunoblot Analyses—Equal amounts of proteins (from cellular extracts and from liver homogenates) were separated by electrophoresis on polyacrylamide gels (containing 0.1! SDS) (6! gel for ABCA1 analysis, 12.5! gel for SR-BI, and 10! for PDZK1), and the resolved proteins were transferred onto a nitrocellulose membrane. ABCA1 (1:250 dilution), SR-BI (1:5,000 dilution), and PDZK1 (1:3,000 dilution) were detected using specific primary antibodies. For each immunoblot, protein-disfulfide isomerase (1:2,000 dilution) was used as a loading control. The apoA-I immunoblot (1:10,000 dilution) was performed with fresh plasma, and the membrane was stained with Coomassie Blue to confirm equal loading into each lane of the gel. Densitometric analysis of the blots was performed using Quantity One software. 35S Metabolic Labeling of SR-BI in Primary Hepatocytes—Freshly isolated hepatocytes were incubated at 37 °C for 1 h in serum-free medium (Dulbecco's modified Eagle's medium) and then in methionine-free medium. 100 μCi of [35S]methionine/cysteine cell labeling mix (PerkinElmer Life Sciences) was added to each dish. The pulse period allowed radiolabeling of newly synthesized proteins for 2 h before the 35S-containing medium was removed. Chase periods ranged from 0 to 12 h. Following the pulse-chase experiment, cells were washed with PBS and lysed in immunoprecipitation lysis buffer (0.63 m Tris-HCl, pH 7.4, 0.75 m NaCl, 25 mm EDTA, 5 mm phenylmethylsulfonyl fluoride, and 5! Triton) containing protease inhibitors. Protein concentration was determined, and equal aliquots of 100 μg of cellular protein were prepared for immunoprecipitation of SR-BI. Samples were incubated overnight at 4 °C with the anti-SR-BI antibody (NB400-101) to immunoprecipitate SR-BI (1:100). Protein-Sepharose A was added to the samples, and SR-BI was pelleted by centrifugation at 2000 rpm for 3 min and washing thoroughly in between (with buffer containing 0.01 m Tris-HCl, pH 7.4, 2 mm EDTA, 0.1! Triton, and 0.1! SDS). Reducing sample buffer (6 mm Tris-HCl, pH 7.4, 10! glycerol, 2! SDS, and 8 m urea) was added directly to the samples. After boiling for 20 min, samples were loaded onto a 12! polyacrylamide gel. Immunoprecipitated proteins were visualized by immunoblotting with the goat polyclonal anti-SR-BI antibody (NB 400-131). The electrochemiluminescent signal (from the immunoblot analysis) was allowed to dissipate for 2 days before the membranes were exposed at –80 °C during 6–8 weeks for the 35S signal (autoradiograph). Preparation of mRNA Extracts for Quantitative PCR—Livers from Pemt+/+ and Pemt–/– female mice (∼100 μg) were homogenized in TRIzol solution, and RNA was precipitated with isopropyl alcohol according to the manufacturer. The isopropyl alcohol was removed, and 75! ethanol (in water containing diethylpyrocarbonate) was added. RNA concentration was determined by measuring the absorbance, and samples were then kept at –80 °C in diethylpyrocarbonate-water. cDNA preparations were synthesized with Superscript III polymerase (Invitrogen). Quantitative PCR Analysis of Hepatic SR-BI mRNA Levels—SR-BI cDNA was amplified using murine SR-BI-specific primers (forward, 5′-TGG CAT TCA GAG CAG TGT AAC-3′; reverse, 5′-CCG TTG GCA AAC AGA GTA TC-3′), the Platinium quantitative PCR Supermix-UDG (Invitrogen), and SyBR Green (Invitrogen). Cyclophilin cDNA was also amplified as the housekeeper gene using specific primers (forward, 5′-TCC AAA GAC AGC AGA AAA CTT TCG-3′; reverse, 5′-TCT TCT TGC TGG TCT TGC CAT TCC-3′). The data were analyzed with the Pfaffl mathematical method. Blue Native Polyacrylamide Gel Electrophoresis of Hepatic SR-BI—Livers from fasted Pemt+/+ and Pemt–/– mice (n > 6 mice of each genotype) were collected, washed in phosphate-buffered saline, and then flash-frozen in liquid nitrogen. Samples were homogenized in a lysis buffer containing freshly added protease inhibitors (20 mm Tris-HCl, pH 7.5, 2 mm MgCl2, 250 mm sucrose, 1 mm phenylmethylsulfonyl fluoride, and 1! dodecyl maltoside) and then briefly sonicated. Liver homogenates were incubated at 4 °C for 30 min with 1:1 solubilization buffer (40 mm Hepes-KOH, pH 7, 2 mm magnesium acetate, and 2! digitonin). After a 20-min centrifugation (20,000 × g at 4 °C), detergent-insoluble material was removed, and 50 μg of samples were combined to blue native PAGE sample buffer 5× (50 mm bis-Tris-HCl, pH 7, 50! glycerol, 25 mg/ml Coomassie Blue G250, and 375 mm 6-amino-n-hexanoic acid). The electrophoresis was performed in nonreducing conditions (SDS-free buffers and equipment) on a 4–20! gradient polyacrylamide gel, and the immunoblot analysis of SR-BI oligomeric forms was performed as described above. For all statistical analyses, Pemt+/+ mice were compared with Pemt–/– animals from the same gender using Student's t test. Plasma HDL Lipids Are Decreased by PEMT Deficiency—A significant reduction of plasma lipid levels was previously observed in chow-fed Pemt–/– mice compared with Pemt+/+ mice (18Noga A.A. Vance D.E. J. Biol. Chem. 2003; 278: 21851-21859Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). To investigate the mechanism responsible for this decrease, we first analyzed the lipoprotein profiles of plasma from mice of the two genotypes by fast protein liquid chromatography. The mice had been fasted overnight prior to the analysis. Both cholesterol (Fig. 1A) and choline-containing phospholipids (Fig. 1B) were lower in plasma of mice lacking PEMT compared with mice with PEMT. ABCA1-mediated Lipid Efflux Is Not Decreased by the Absence of PEMT—We next evaluated the role of PEMT-derived PC in HDL formation. Primary cultured hepatocytes, obtained from Pemt+/+ and Pemt–/– mice, were incubated in the presence or absence of poorly lipidated apoA-I (20 μg protein/ml), an extracellular acceptor. The efflux of cholesterol and PC from Pemt+/+ and Pemt–/– hepatocytes was linear with time for up to 24 h and with the amount of apoA-I (up to 200 μg/ml) (data not shown). The release of cholesterol from Pemt+/+ and Pemt–/– hepatocytes into the medium was determined after a 16-h incubation with or without apoA-I. The amount of cholesterol in the culture medium was analyzed by gas-liquid chromatography (Fig. 2A). The difference in the amount of cholesterol recovered in the medium of Pemt+/+ and of Pemt–/– hepatocytes did not reach statistical significance. The efflux of PC from Pemt+/+ and Pemt–/– hepatocytes was assessed after radiolabeling of cellular phospholipids with [3H]glycerol (Fig. 2B). The release of [3H]PC from Pemt–/– hepatocytes was independent of Pemt genotype and was surprisingly not stimulated by apoA-I. The Amount of Hepatic ABCA1 Is No

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