An Induction in Hepatic HDL Secretion Associated with Reduced ATPase Expression
2009; Elsevier BV; Volume: 175; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.090082
ISSN1525-2191
AutoresNihar R. Pandey, Joanna Renwick, Seham Rabaa, Ayesha Misquith, Lara Kouri, Erin Twomey, Daniel L. Sparks,
Tópico(s)Diet, Metabolism, and Disease
ResumoLinoleic acid-phospholipids stimulate high-density lipoprotein (HDL) net secretion from liver cells by blocking the endocytic recycling of apoA-I. Experiments were undertaken to determine whether apoA-I accumulation in the cell media is associated with membrane ATPase expression. Treatment of HepG2 cells with dilinoeoylphosphatidylcholine (DLPC) increased apoA-I secretion fourfold. DLPC also significantly reduced cell surface F1-ATPase expression and reduced cellular ATP binding cassette (ABC)A1 and ABCG1 protein levels by ∼50%. In addition, treatment of HepG2 cells with the ABC transporter inhibitor, glyburide, stimulated the apoA-I secretory effects of both DLPC and clofibrate. Pretreatment of HepG2 cells with compounds that increased ABC transport protein levels (TO901317, N-Acetyl-l-leucyl-l-leucyl-l-norleucinal, and resveratrol) blocked the DLPC-induced stimulation in apoA-I net secretion. Furthermore, whereas HepG2 cells normally secrete nascent preβ-HDL, DLPC treatment promoted secretion of α-migrating HDL particles. These data show that an linoleic acid-phospholipid induced stimulation in hepatic HDL secretion is related to the expression and function of membrane ATP metabolizing proteins. Linoleic acid-phospholipids stimulate high-density lipoprotein (HDL) net secretion from liver cells by blocking the endocytic recycling of apoA-I. Experiments were undertaken to determine whether apoA-I accumulation in the cell media is associated with membrane ATPase expression. Treatment of HepG2 cells with dilinoeoylphosphatidylcholine (DLPC) increased apoA-I secretion fourfold. DLPC also significantly reduced cell surface F1-ATPase expression and reduced cellular ATP binding cassette (ABC)A1 and ABCG1 protein levels by ∼50%. In addition, treatment of HepG2 cells with the ABC transporter inhibitor, glyburide, stimulated the apoA-I secretory effects of both DLPC and clofibrate. Pretreatment of HepG2 cells with compounds that increased ABC transport protein levels (TO901317, N-Acetyl-l-leucyl-l-leucyl-l-norleucinal, and resveratrol) blocked the DLPC-induced stimulation in apoA-I net secretion. Furthermore, whereas HepG2 cells normally secrete nascent preβ-HDL, DLPC treatment promoted secretion of α-migrating HDL particles. These data show that an linoleic acid-phospholipid induced stimulation in hepatic HDL secretion is related to the expression and function of membrane ATP metabolizing proteins. High-density lipoprotein (HDL) is predominantly produced in the liver in humans and is formed by the synthesis and secretion of apolipoprotein components, followed by the lipidation of these proteins with specific lipids.1Barter P Kastelein J Nunn A Hobbs R High density lipoproteins (HDLs) and atherosclerosis; the unanswered questions.Atherosclerosis. 2003; 168: 195-211Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 2Brewer Jr, HB Santamarina-Fojo S New insights into the role of the adenosine triphosphate-binding cassette transporters in high-density lipoprotein metabolism and reverse cholesterol transport.Am J Cardiol. 2003; 91: 3E-11EAbstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 3Rader DJ Molecular regulation of HDL metabolism and function: implications for novel therapies.J Clin Invest. 2006; 116: 3090-3100Crossref PubMed Scopus (464) Google Scholar HDL lipidation is believed to be regulated by the actions of the ATP-binding cassette (ABC) transporters, ABCA1 and ABCG1. ABCA1 and ABCG1 lipidate apolipoprotein (apo)A-I and convert nascent HDL particles to lipid-rich HDL.4Fielding CJ Fielding PE Cellular cholesterol efflux.Biochim Biophys Acta. 2001; 1533: 175-189Crossref PubMed Scopus (156) Google Scholar, 5Oram JF HDL Apolipoproteins and ABCA1: partners in the removal of excess cellular cholesterol.Arterioscler Thromb Vasc Biol. 2003; 23: 720-727Crossref PubMed Scopus (207) Google Scholar, 6Oram JF Vaughan AM ATP-Binding cassette cholesterol transporters and cardiovascular disease.Circ Res. 2006; 99: 1031-1043Crossref PubMed Scopus (321) Google Scholar Through this process, the ABC transport proteins have been thought to play a central role in both the production and maturation of HDL.6Oram JF Vaughan AM ATP-Binding cassette cholesterol transporters and cardiovascular disease.Circ Res. 2006; 99: 1031-1043Crossref PubMed Scopus (321) Google Scholar ABC transporter expression is regulated by the liver X receptor (LXR), and LXR agonists such as the oxysterols have been shown to increase the expression and lipid secretory activity of ABCA1.7Venkateswaran A Laffitte BA Joseph SB Mak PA Wilpitz DC Edwards PA Tontonoz P Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXR alpha.Proc Natl Acad Sci USA. 2000; 97: 12097-12102Crossref PubMed Scopus (823) Google Scholar Recent work has shown that despite the activation of ABCA1, LXR agonists such as TO901317 actually inhibit the synthesis and secretion of HDL and apoA-I by liver-derived cells.8Huuskonen J Vishnu M Chau P Fielding PE Fielding CJ Liver X receptor inhibits the synthesis and secretion of apolipoprotein A1 by human liver-derived cells.Biochemistry. 2006; 45: 15068-15074Crossref PubMed Scopus (22) Google Scholar This suggests that ABC transporter expression may not be linked to the production of HDL. In addition to lipidating apoA-I, ABC transporters play other roles in HDL metabolism. ABCA1 has been shown to interact directly with apoA-I9Wang N Silver DL Costet P Tall AR Specific binding of ApoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1.J Biol Chem. 2000; 275: 33053-33058Crossref PubMed Scopus (496) Google Scholar, 10Nieland TJ Chroni A Fitzgerald ML Maliga Z Zannis VI Kirchhausen T Krieger M Cross-inhibition of SR-BI- and ABCA1-mediated cholesterol transport by the small molecules BLT-4 and glyburide.J Lipid Res. 2004; 45: 1256-1265Crossref PubMed Scopus (80) Google Scholar and to impact the endocytic uptake and resecretion of apoA-I.11Hassan HH Denis M Lee DY Iatan I Nyholt D Ruel I Krimbou L Genest J Identification of an ABCA1-dependent phospholipid-rich plasma membrane apolipoprotein A-I binding site for nascent HDL formation: implications for current models of HDL biogenesis.J Lipid Res. 2007; 48: 2428-2442Crossref PubMed Scopus (82) Google Scholar, 12Denis M Landry YD Zha X ATP-binding cassette A1-mediated lipidation of apolipoprotein A-I occurs at the plasma membrane and not in the endocytic compartments.J Biol Chem. 2008; 283: 16178-16186Crossref PubMed Scopus (82) Google Scholar, 13Faulkner LE Panagotopulos SE Johnson JD Woollett LA Hui DY Witting SR Maiorano JN Davidson WS An analysis of the role of a retroendocytosis pathway in ABCA1-mediated cholesterol efflux from macrophages.J Lipid Res. 2008; 49: 1322-1332Crossref PubMed Scopus (54) Google Scholar, 14Hassan HH Bailey D Lee DY Iatan I Hafiane A Ruel I Krimbou L Genest J Quantitative analysis of ABCA1-dependent compartmentalization and trafficking of apolipoprotein A-I: implications for determining cellular kinetics of nascent high density lipoprotein biogenesis.J Biol Chem. 2008; 283: 11164-11175Crossref PubMed Scopus (49) Google Scholar ABCA1-dependent endocytic uptake of apoA-I has been shown to promote the lysosomal degradation of apoA-I.12Denis M Landry YD Zha X ATP-binding cassette A1-mediated lipidation of apolipoprotein A-I occurs at the plasma membrane and not in the endocytic compartments.J Biol Chem. 2008; 283: 16178-16186Crossref PubMed Scopus (82) Google Scholar, 13Faulkner LE Panagotopulos SE Johnson JD Woollett LA Hui DY Witting SR Maiorano JN Davidson WS An analysis of the role of a retroendocytosis pathway in ABCA1-mediated cholesterol efflux from macrophages.J Lipid Res. 2008; 49: 1322-1332Crossref PubMed Scopus (54) Google Scholar Therapeutic compounds that are inhibitors of ABC transporters have been shown to modestly increase plasma HDL levels.15Singh T Singh S Bhullar GS The effect of sulphonylurea therapy on serum total cholesterol and high density lipoprotein cholesterol.J Indian Med Assoc. 1992; 90: 259-261PubMed Google Scholar, 16Kerenyi Z Samer H James R Yan Y Stewart M Combination therapy with rosiglitazone and glibenclamide compared with upward titration of glibenclamide alone in patients with type 2 diabetes mellitus.Diabetes Res Clin Pract. 2004; 63: 213-223Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar Glyburide inhibits ABCA1 activity by blocking the ATPase activity of the protein.17Takahashi K Kimura Y Kioka N Matsuo M Ueda K Purification and ATPase activity of human ABCA1.J Biol Chem. 2006; 281: 10760-10768Crossref PubMed Scopus (87) Google Scholar Glyburide has also been shown to block interactions between apoA-I and ABCA19Wang N Silver DL Costet P Tall AR Specific binding of ApoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1.J Biol Chem. 2000; 275: 33053-33058Crossref PubMed Scopus (496) Google Scholar, 10Nieland TJ Chroni A Fitzgerald ML Maliga Z Zannis VI Kirchhausen T Krieger M Cross-inhibition of SR-BI- and ABCA1-mediated cholesterol transport by the small molecules BLT-4 and glyburide.J Lipid Res. 2004; 45: 1256-1265Crossref PubMed Scopus (80) Google Scholar and to block apoA-I signaling through ABCA1.18Nofer JR Remaley AT Feuerborn R Wolinnska I Engel T von EA Assmann G Apolipoprotein A-I activates Cdc42 signaling through the ABCA1 transporter.J. Lipid Res. 2006; 47: 794-803Crossref PubMed Scopus (59) Google Scholar There is evidence to suggest that addition of an ABC transporter inhibitor, such as glyburide, to a fibrate therapy, may also increase the HDL raising potential of the fibrate.19Smud R Sermukslis B Bezafibrate and fenofibrate in type II diabetics with hyperlipoproteinaemia.Curr Med Res Opin. 1987; 10: 612-624Crossref PubMed Scopus (17) Google Scholar A different membrane ATPase, F1F0-ATP synthase (F1-ATPase), has also been shown to impact cellular HDL metabolism.20Martinez LO Jacquet S Esteve JP Rolland C Cabezon E Champagne E Pineau T Georgeaud V Walker JE Terce F Collet X Perret B Barbaras R Ectopic beta-chain of ATP synthase is an apolipoprotein A-I receptor in hepatic HDL endocytosis.Nature. 2003; 421: 75-79Crossref PubMed Scopus (380) Google Scholar, 21Martinez LO Jacquet S Terce F Collet X Perret B Barbaras R New insight on the molecular mechanisms of high-density lipoprotein cellular interactions.Cell Mol Life Sci. 2004; 61: 2343-2360Crossref PubMed Scopus (47) Google Scholar, 22Jacquet S Malaval C Martinez LO Sak K Rolland C Perez C Nauze M Champagne E Terce F Gachet C Perret B Collet X Boeynaems JM Barbaras R The nucleotide receptor P2Y13 is a key regulator of hepatic high-density lipoprotein (HDL) endocytosis.Cell Mol Life Sci. 2005; 62: 2508-2515Crossref PubMed Scopus (92) Google Scholar, 23Fabre AC Vantourout P Champagne E Terce F Rolland C Perret B Collet X Barbaras R Martinez LO Cell surface adenylate kinase activity regulates the F(1)-ATPase/P2Y (13)-mediated HDL endocytosis pathway on human hepatocytes.Cell Mol Life Sci. 2006; 63: 2829-2837Crossref PubMed Scopus (67) Google Scholar Studies have shown that apoA-I can stimulate a plasma membrane bound F1-ATPase and promote the endocytic uptake of HDL through a specific plasma membrane G-protein coupled receptor, P2Y13.22Jacquet S Malaval C Martinez LO Sak K Rolland C Perez C Nauze M Champagne E Terce F Gachet C Perret B Collet X Boeynaems JM Barbaras R The nucleotide receptor P2Y13 is a key regulator of hepatic high-density lipoprotein (HDL) endocytosis.Cell Mol Life Sci. 2005; 62: 2508-2515Crossref PubMed Scopus (92) Google Scholar Inhibition of F1-ATPase with antibodies or selective inhibitors (IF1) blocks HDL endocytosis in hepatic cell culture and in vivo. Recent work suggests that niacin may act through this pathway and increase HDL secretion through reducing membrane F1-ATPase levels.24Zhang LH Kamanna VS Zhang MC Kashyap ML Niacin inhibits surface expression of ATP synthase {beta} chain in HepG2 cells: implications for raising HDL.J Lipid Res. 2008; 49: 1195-1201Crossref PubMed Scopus (88) Google Scholar Niacin reduces membrane F1-ATPase levels and inhibits the reuptake and recycling of apoA-I. Linoleic acid (LA)-phospholipids are considerably more effective at stimulating hepatic apoA-I secretion and HDL production, than niacin and the fibrate drugs.25Pandey NR Renwick J Misquith A Sokoll K Sparks DL Linoleic acid-enriched phospholipids act through peroxisome proliferator-activated receptors alpha to stimulate hepatic apolipoprotein A-I secretion.Biochemistry. 2008; 47: 1579-1587Crossref PubMed Scopus (27) Google Scholar Much like niacin, these compounds act through protein kinase C and mitogen-activated protein kinase pathways to activate a peroxisome-proliferator activator receptor (PPAR)α-dependent secretion of apoA-I. However, in contrast to the fibrate drugs, LA-phospholipids do not increase cellular apoA-I mRNA levels and instead increase apoA-I secretion by blocking the endocytic recycling of apoA-I.26Hopewell S Pandey NR Misquith A Twomey E Sparks DL Phosphatidylinositol acts through mitogen-activated protein kinase to stimulate hepatic apolipoprotein A-I secretion.Metabolism. 2008; 57: 1677-1684Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar LA-phospholipids are therefore a novel class of HDL effectors that have a similar mechanism of action to niacin and are not metabolized by the cytochrome P450 enzymes.25Pandey NR Renwick J Misquith A Sokoll K Sparks DL Linoleic acid-enriched phospholipids act through peroxisome proliferator-activated receptors alpha to stimulate hepatic apolipoprotein A-I secretion.Biochemistry. 2008; 47: 1579-1587Crossref PubMed Scopus (27) Google Scholar, 26Hopewell S Pandey NR Misquith A Twomey E Sparks DL Phosphatidylinositol acts through mitogen-activated protein kinase to stimulate hepatic apolipoprotein A-I secretion.Metabolism. 2008; 57: 1677-1684Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 27Pandey NR Sparks DL Phospholipids as cardiovascular therapeutics.Curr Opin Investig Drugs. 2008; 9: 281-285PubMed Google Scholar Experiments were undertaken to elucidate how an LA-phospholipid stimulation in apoA-I secretion may involve membrane ATPases. We show that increased apoA-I secretion is inversely related to cell membrane ATPase protein levels. Compounds that inhibit ATPase activity significantly stimulate apoA-I secretion and those that increase ABC transporter levels in HepG2 cells inhibit apoA-I secretion. The data suggests that hepatic apoA-I secretion is closely linked to the expression and function of membrane-bound ATP metabolizing proteins. Phospholipids, soy phosphatidylinositol (PI) and dilinoeoylphosphatidylcholine (DLPC) were procured from Avanti Polar Lipids Inc., Alabaster, AL. TO901317, a synthetic LXRα agonist, was obtained from Cayman Chemical, Ann Arbor, MI. Resveratrol (trans-3,5,4′-trihydroxystilbene), ALLN (N-Acetyl-l-leucyl-l-leucyl-l-norleucinal), a calpain-I inhibitor, and glyburide an ATP-sensitive potassium channel blocker, linoleic acid sodium salt, and oligomycin (a mixture of oligomycins A, B and C; approx. 65% oligomycin A) were purchased from Sigma-Aldrich, Saint Louis, MO. Mouse monoclonal anti-human apoA-I (cat#H45402M), and horse radish peroxidase-conjugated goat anti-human apoA-I (cat#A-I K45252P) were obtained from Meridian Life Science, Saco, ME. The mouse monoclonal anti-human apoA-I (5F6 and 4H1) antibodies for Western blots were kindly provided by Dr. Ives Marcel. The goat polyclonal anti-human apoA-II antibody was from Chemicon International, Billerica, MA. β-actin (cat # 4967) and anti-rabbit IgG-HRP (cat # 7074) were obtained from Cell Signaling Technology, Danvers, MA. Mouse monoclonal anti-ABCA1 IgG (cat#G090) was purchased from ABM Inc., and affinity purified peroxidase linked goat anti-mouse antibody (cat#4741806) was purchased from Kirkegaard and Perry Laboratories. Affinity purified goat polyclonal anti-ABCG1 antibody (cat # 11150) and donkey anti-goat IgG-HRP (cat# sc-2020) were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. Mouse monoclonal anti-β chain of F1F0-ATP synthase was purchased from Invitrogen, Carlsbad, CA. Unless otherwise stated, drugs and inhibitors were of analytical grade and were solubilized in dimethyl sulfoxide. HepG2 cells were cultured in normal glucose Dulbecco’s modified Eagle medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. Almost confluent cells were subjected to stimulation with or without drugs for 24 hours under serum-starved conditions, as indicated. Phospholipid vesicles in PBS (1 mg/ml) were prepared by sonication as previously described.25Pandey NR Renwick J Misquith A Sokoll K Sparks DL Linoleic acid-enriched phospholipids act through peroxisome proliferator-activated receptors alpha to stimulate hepatic apolipoprotein A-I secretion.Biochemistry. 2008; 47: 1579-1587Crossref PubMed Scopus (27) Google Scholar Briefly, phospholipids in chloroform were dried down under N2 gas and 1 ml of PBS was added by vortexing. The mix was then sonicated (Branson sonicator set at 100% duty cycle and 10% power) for 1 minute. The sonicated preparation was incubated for 30 minutes at 37°C in a water bath, and samples were resonicated for 5 minutes at 95% duty cycle and 10% power and filtered before use. Purity of all phospholipids was >99% (Avanti Polar Lipids) and was verified by high-performance liquid chromatography. Protein in conditioned medium from each stimulation was analyzed by enzyme-linked immunosorbent assay (ELISA) on a 96 well plate according to manufacturer’s instructions, with minor modifications. Briefly, the Nunc Immuno-maxisorp 96 well plates were coated overnight with a mouse anti-human apoA-I monoclonal antibody. Samples and standards were incubated in the wells for 2 hours, followed by a 1-hour incubation with a horseradish peroxidase-linked goat anti-human apoA-I antibody. K-blue Max TMB substrate was added to each well and the reaction was stopped with a 1 M/L HCl solution; and the absorbance was recorded at 450 nm. The assay conditions were optimized to minimize any apoA-I conformation interference with the apoA-I ELISA. Confluent HepG2 cells in 6 well plates were radio-labeled with 5μCi/ml [3H]-cholesterol in complete medium for 24 hours. Following incubation, cells were washed three times with pre-warmed serum free media and were treated with 12 μmol/L phospholipid vesicles in serum-free Dulbecco’s modified Eagle medium for 24 hours. Conditioned media was collected into tubes and the cells were lysed using 0.5 M/L NaOH. Pre-cleared media samples and cell lysates were mixed with the EcoLite scintillation liquid and were counted by TRI-CARB 2100TR Liquid Scintillation Analyzer. The cell lysates were quantified for total protein concentration using the bicinchoninic acid assay (as per manufacturer’s specifications). Plasma membrane proteins were biotinylated and isolated using a Cell Surface Labeling Accessory Pack (Pierce Chemical, Rockford, IL) according to manufacturer’s protocol. Briefly, cell monolayers were biotinylated with EZlink-sulfo-NHS-LC-biotin at 4°C for 30 minutes with gentle agitation. The cells were harvested after addition of Quenching solution and then washed three times with Tris-buffered saline (TBS) using centrifugation at 500 × g for 3 minutes each. Cells were then lysed using lysis buffer supplemented with protease inhibitors, followed by low-power sonication and centrifugation to disrupt the cells and were incubated on ice for 30 minutes. Cleared cell lysates were obtained for each sample as supernatant by centrifugation at 10,000 × g for 2 minutes at 4°C. Columns packed with Immobilized NeutrAvidin Gel were used to isolate labeled proteins. Finally SDS-polyacrylamide gel electrophoresis sample buffer supplemented with 50 mmol/L dithiothreitol was used to elute labeled proteins by centrifugation at 1000 × g for 2 minutes. Protein assay was performed by bicinchoninic acid method with minor modifications. Samples were first treated with sodium deoxicholate, followed by TCA precipitation. An equal amount of protein then separated on a 12% SDS-polyacrylamide electrophoresis gels, transferred to polyvinylidene difluoride membranes, analyzed by immunoblotting with ATP-synthase β antibody, and then visualized with the α-Innotech FluorChem HD Imager (Fisher Scientific). HepG2 cells were treated and plasma membranes were isolated as previously described.28Kauffmann HM Keppler D Kartenbeck J Schrenk D Induction of cMrp/cMoat gene expression by cisplatin, 2-acetylaminofluorene, or cycloheximide in rat hepatocytes.Hepatology. 1997; 26: 980-985PubMed Google Scholar, 29Draber P Draberova L Heneberg P Smid F Farghali H Draber P Preformed STAT3 transducer complexes in human HepG2 cells and rat hepatocytes.Cell Signal. 2007; 19: 2400-2412Crossref PubMed Scopus (5) Google Scholar Briefly, cells were washed twice with 0.9% sodium chloride and scraped off in lysis buffer (10 mmol/L Tris [pH 7.4], 10 mmol/L sodium chloride, 1.5 mmol/L magnesium chloride, 0.05% sodium azide, 1 mmol/L phenylmethylsulfonyl fluoride, and 1 mmol/L dithiothreitol). After homogenization by sonication, lysates were subjected to centrifugation at 1600 rpm for 2 minutes. Supernatants were then centrifuged at 100,000 × g for 30 minutes to prepare cytosolic and membrane fractions. Supernatant (cytosolic protein) and pellets (membrane protein) were then suspended in lysis buffer. Protein concentration was determined and an equal amount of protein was separated on 12% SDS-polyacrylamide gel electrophoresis and subjected to immunoblotting for ATP synthase and for peroxiredoxin-3 using specific antibodies. HepG2 cells were transiently transfected with All Stars Negative small-interfering (si)RNA or four different ABCA1-siRNA sequences (separately) from the Flexitube Gene Solution siRNA kit (Qiagen Inc., Mississauga, ON) by reverse transfection using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Complexes were prepared per manufacturer’s specifications with a Lipofectamine 2000-to-siRNA volume-to-mole ratio of 2:40 (μL to ρmol) in 200 μl of Opti-MEM I Reduced Serum Media (Invitrogen, Carlsbad, CA). HepG2 cells were trypsinized and seeded in 12-well plates at a density of 500,000 cells/well and then 200 μl of the transfection complexes were immediately added to the suspended cells. Transfection of the siRNA alone showed no adverse cytotoxic effects compared with nontransfected cells. Conditioned media was collected 48 hours post-transfection and an ELISA was performed to determine the amount of apoA-I. ABCA1 knockdown was confirmed by Western blot analysis. After incubation with drugs for the indicated times and doses, cells were washed twice with ice-cold PBS-T on ice. Cells were lysed by adding buffer (NaF 1 mmol/L, NaCl 5 mmol/L, EDTA 1 mmol/L, NP40 1 mmol/L [Roche Diagnostics, Indianapolis, IN], HEPES 10 mmol/L, pepstatin A 1 mg/ml, leupeptin 1 mg/ml, aprotinin 1 mg/ml, Na3VO4 1 mmol/L, and phenylmethylsulfonyl fluoride 1 mmol/L) and total protein was extracted. Equal amounts of cell proteins were separated by SDS-12% polyacrylamide gel electrophoresis and were analyzed by Western blot with specific antibodies to ATP synthase, ABCA1, and ABCG1. Blots for the similar experiments were also subjected to β-actin for a loading control. Band intensity was analyzed with the α-Innotech FluorChem HD Imager. Lipoprotein electrophoretic mobility was determined by apoA-I immunoblots of prepoured 0.5% agarose gels (Beckman, Paragon-Lipo). Conditioned media samples were loaded and the gel was electrophoresed with a barbital buffer in the Beckman Paragon Electrophoresis System at 100 V for 30 minutes, as previously described.30Sparks DL Phillips MC Quantitative measurement of lipoprotein surface charge by agarose gel electrophoresis.J Lipid Res. 1992; 33: 123-130Abstract Full Text PDF PubMed Google Scholar Once complete, the gel was covered in 1× Tris-Glycine transfer buffer, and then layered with a presoaked nitrocellulose membrane. The gel and membrane were covered in plastic wrap and an even pressure of 3 kg was applied. After a 4 hour transfer, the membrane was blocked in 5% skim milk or bovine serum albumin for 1 hour at room temperature and incubated with the primary apoA-I antibodies (mix of 5F6 and 4H1, 1:5000 dilution) or apoA-II antibody, overnight at 4°C. The membrane was washed three times and then incubated with secondary antibody (1:10,000 dilutions) for 1 hour at room temperature. The membrane was incubated with SuperSignal West Femto for 3 minutes and then visualized using the α-Innotech FluorChem HD Imager. Conditioned media from HepG2 cells stimulated with DLPC were electrophoresed in triplicate on a 4% to 20% Tris-Glycine Novex gel (Invitrogen, Carlsbad, CA) under non-denaturing conditions for 19 hours at 100V alongside high molecular weight native markers (Amersham, Piscataway, NJ). The gel was then soaked in 0.1% SDS for 15 minutes to give the proteins a slight negative charge in order for unidirectional transfer onto a polyvinylidene difluoride membrane for 4 hours at 125V in Tris-glycine transfer buffer containing 20% methanol. The membrane was allowed to dry at which point the molecular weight markers were outlined and the membrane was cut in three. The apoA-I membrane was blocked with 5% milk/TBS-Tween (TBST) and then probed for apoA-I using a 1:2500 dilution of the monoclonal apoA-I antibodies (4H1 and 5F6) and a 1:20,000 dilution of the goat anti-mouse IgG linked HRP secondary antibody (KPL, Gaithersburg, MD) in 1% milk/TBST. ApoA-II was probed by blocking in 1% BSA/TBST followed by incubation with 1:1000 dilution of the goat anti-apolipoprotein A-II polyclonal antibody in 1% BSA/TBST and 1:10,000 dilution of the donkey anti-goat IgG-HRP secondary antibody in 1% BSA/TBST (Santa Cruz Biotechnology, Santa Cruz, CA). Blots were developed using the West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL) on the Fluorochem AlphaImager. Densitometry profiles were obtained using the 1D-Multi application of the AlphaEaseFC software. Values are shown as Mean ± SEM for at least 3 to 4 independent experiments as indicated, and P < 0.05 was considered significant. Differences between mean values were evaluated by one-way analysis of variance on ranks by a pairwise multiple comparison using the Student-Newman-Keuls posthoc test (SigmaStat; Systat Software, Inc., San Jose, CA). Linoleic acid-enriched phospholipids were shown to stimulate hepatic apoA-I secretion in both primary hepatocytes and HepG2 cells.25Pandey NR Renwick J Misquith A Sokoll K Sparks DL Linoleic acid-enriched phospholipids act through peroxisome proliferator-activated receptors alpha to stimulate hepatic apolipoprotein A-I secretion.Biochemistry. 2008; 47: 1579-1587Crossref PubMed Scopus (27) Google Scholar To determine whether phospholipid-induced apoA-I secretion may involve ATP dependent reactions, experiments were undertaken with the ABC transporter inhibitor, glyburide. Glyburide (50 μmol/L) alone had a small stimulatory effect on apoA-I secretion (Figure 1A). However, PI and DLPC-induced apoA-I secretion was synergistically increased, by 60% and 75% respectively (Figure 1B) at 24 hours, when HepG2 cells were pre-incubated for 30 minutes with glyburide. Clofibrate (10 μmol/L) only had a mild stimulatory effect on apoA-I secretion, while clofibrate and glyburide showed significant increase in apoA-I secretion, at 33% over control (data not shown). The effect of PI and DLPC on cellular F1-ATPase expression was determined in HepG2 cells. Cells were treated with PI or DLPC for 24 hours and then membrane and cytosolic proteins were fractionated and isolated centrifugally and probed for F1-ATPase. Membrane F1-ATPase was reduced ∼40% to 60% by PI and DLPC (Figure 2A). Cytosolic F1-ATPase levels were also significantly reduced (Figure 2B). Isolated membrane proteins did not contain mitochondrial proteins, as peroxiredoxin-3 was not detectible in the membrane proteins, but present in the cytosolic fraction (Figure 2A). Similar results were obtained after biotinylation and reisolation of plasma membranes, where membrane F1-ATPase levels were reduced by 18% after DLPC treatment (Supplementary Figure S1, see http://ajp.amjpathol.org). To determine whether inhibition of ATP synthase affects hepatic apoA-I secretion, cells were pretreated with oligomycin (5 μg/ml for 30 minutes) and then with PI or DLPC for 24 hours and apoA-I secretion was measured. Oligomycin blocked both basal and phospholipid-induced apoA-I secretion (Supplementary Figure S2, see http://ajp.amjpathol.org). HepG2 cells were treated with PI or DLPC, and ABC transporter expression was measured. As shown in Figure 3, PI and DLPC reduced hepatic ABCA1 and ABCG1 protein expression. PI reduced ABCA1 and ABCG1 protein levels by 48% and 50% respectively, while DLPC reduced ABCA1 and ABCG1 × 74% and 50% respectively (Figure 3, A and B). To determine the effect of PI and DLPC on cellular cholesterol secretion, media cholesterol levels were measured after loading the HepG2 cells with 3H-cholesterol and treatment with PI or DLPC. Even though PI reduced ABCA1 and ABCG1 expression, the lipid had little effect on cellular cholesterol secretion. In contrast, DLPC significantly blocked cholesterol secretion (Figure 4). DLPC reduces the 3H-cholesterol levels in the d >1.063 lipoproteins (HDL), but had no effect on the cholesterol levels in the d <1.063 lipoproteins (Figure 4). To determine whether ABCA1 knockdown affects apoA-I secretion, HepG2 cells were treated with 2 different ABCA1-siRNA sequences and apoA-I secretion was measured. ABCA1 protein expression was reduced to below detection limits. Knockdown had minimal effect on basal apoA-I secretion but almost completely abolished DLPC-induced hep
Referência(s)