ApoB-containing lipoproteins in apoE-deficient mice are not metabolized by the class B scavenger receptor BI
2004; Elsevier BV; Volume: 45; Issue: 2 Linguagem: Inglês
10.1194/jlr.m300319-jlr200
ISSN1539-7262
AutoresNancy R. Webb, Maria C. de Beer, Frederick C. de Beer, Deneys R. van der Westhuyzen,
Tópico(s)Lipoproteins and Cardiovascular Health
ResumoThe scavenger receptor class B type I (SR-BI) recognizes a broad variety of lipoprotein ligands, including HDL, LDL, and oxidized LDL. In this study, we investigated whether SR-BI plays a role in the metabolism of cholesterol-rich lipoprotein remnants that accumulate in apolipoprotein E (apoE)−/− mice. These particles have an unusual apolipoprotein composition compared with conventional VLDL and LDL, containing mostly apoB-48 as well as substantial amounts of apoA-I and apoA-IV. To study SR-BI activity in vivo, the receptor was overexpressed in apoE−/− mice by adenoviral vector-mediated gene transfer. An ∼10-fold increase in liver SR-BI expression resulted in no detectable alterations in VLDL-sized particles and a modest depletion of cholesterol in intermediate density lipoprotein/LDL-sized lipoprotein particles. This decrease was not attributable to altered secretion of apoB-containing lipoproteins in SR-BI-overexpressing mice. To directly assess whether SR-BI metabolizes apoE−/− mouse lipoprotein remnants, in vitro assays were performed in both CHO cells and primary hepatocytes expressing high levels of SR-BI. This analysis showed a remarkable deficiency of these particles to serve as substrates for selective lipid uptake, despite high-affinity, high-capacity binding to SR-BI.Taken together, these data establish that SR-BI does not play a direct role in the metabolism of apoE−/− mouse lipoprotein remnants. The scavenger receptor class B type I (SR-BI) recognizes a broad variety of lipoprotein ligands, including HDL, LDL, and oxidized LDL. In this study, we investigated whether SR-BI plays a role in the metabolism of cholesterol-rich lipoprotein remnants that accumulate in apolipoprotein E (apoE)−/− mice. These particles have an unusual apolipoprotein composition compared with conventional VLDL and LDL, containing mostly apoB-48 as well as substantial amounts of apoA-I and apoA-IV. To study SR-BI activity in vivo, the receptor was overexpressed in apoE−/− mice by adenoviral vector-mediated gene transfer. An ∼10-fold increase in liver SR-BI expression resulted in no detectable alterations in VLDL-sized particles and a modest depletion of cholesterol in intermediate density lipoprotein/LDL-sized lipoprotein particles. This decrease was not attributable to altered secretion of apoB-containing lipoproteins in SR-BI-overexpressing mice. To directly assess whether SR-BI metabolizes apoE−/− mouse lipoprotein remnants, in vitro assays were performed in both CHO cells and primary hepatocytes expressing high levels of SR-BI. This analysis showed a remarkable deficiency of these particles to serve as substrates for selective lipid uptake, despite high-affinity, high-capacity binding to SR-BI. Taken together, these data establish that SR-BI does not play a direct role in the metabolism of apoE−/− mouse lipoprotein remnants. The scavenger receptor class B type I (SR-BI) is an HDL receptor that mediates selective cholesteryl ester (CE) uptake, a process by which CEs from the core of the HDL particle are transferred to the cell without intracellular accumulation of the protein moiety (1Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor.Science. 1996; 271: 518-520Crossref PubMed Scopus (2011) Google Scholar). In the liver, SR-BI carries out a critical step in the reverse cholesterol transport pathway, whereby HDL-cholesterol is delivered to the liver for excretion in the bile [as reviewed in ref. (2Krieger M. Charting the fate of the "good cholesterol": identification and characterization of the high-density lipoprotein receptor SR-BI.Annu. Rev. Biochem. 1999; 68: 523-558Crossref PubMed Scopus (460) Google Scholar)]. In addition to binding HDL, SR-BI also binds LDL, VLDL, and oxidized LDL (3Acton S.L. Scherer P.E. Lodish H.F. Krieger M. Expression cloning of SR-BI, a CD36-related class B scavenger receptor.J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar, 4Calvo D. Gomez-Coronado D. Lasuncion M.A. Vega M.A. CLA-1 is an 85-kD plasma membrane glycoprotein that acts as a high-affinity receptor for both native (HDL, LDL, and VLDL) and modified (OxLDL and AcLDL) lipoproteins.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2341-2349Crossref PubMed Scopus (214) Google Scholar). The ability of SR-BI to mediate selective lipid uptake from mouse and human LDL has been investigated (5Swarnakar S. Temel R.E. Connelly M.A. Azhar S. Williams D.L. Scavenger receptor class B, type I, mediates selective uptake of low density lipoprotein cholesteryl ester.J. Biol. Chem. 1999; 274: 29733-29739Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 6Webb N.R. de Beer M.C. Yu J. Kindy M.S. Daugherty A. van der Westhuyzen D.R. de Beer F.C. Overexpression of SR-BI by adenoviral vector promotes clearance of apoA-I, but not apoB, in human apoB transgenic mice.J. Lipid Res. 2002; 43: 1421-1428Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). These studies have shown that although SR-BI mediates selective lipid uptake from LDL, the amount of CE transferred to cells relative to the amount of LDL-CE bound to the cell surface is significantly lower compared with that of HDL-CE. In addition, studies in human apolipoprotein B (apoB) transgenic mice with adenoviral vector overexpression of SR-BI indicate that this receptor does not contribute significantly to LDL metabolism in vivo (6Webb N.R. de Beer M.C. Yu J. Kindy M.S. Daugherty A. van der Westhuyzen D.R. de Beer F.C. Overexpression of SR-BI by adenoviral vector promotes clearance of apoA-I, but not apoB, in human apoB transgenic mice.J. Lipid Res. 2002; 43: 1421-1428Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). In this study, we investigated the role of SR-BI in the metabolism of non-HDL lipoproteins in apoE−/− mice. Mice lacking apoE accumulate cholesterol-rich lipoprotein remnants and develop atherosclerosis spontaneously on a low-fat diet and in an accelerated manner on a high-fat, high-cholesterol diet (7Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E.Science. 1992; 258: 468-471Crossref PubMed Scopus (1849) Google Scholar, 8Plump A.S. Smith J.D. Hayek T. Aalto-Setala K. Walsh A. Verstuyft J.G. Rubin E.M. Breslow J.L. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells.Cell. 1992; 71: 343-353Abstract Full Text PDF PubMed Scopus (1878) Google Scholar). SR-BI deficiency in apoE−/− mice results in even higher concentrations of plasma total cholesterol as well as dramatically accelerated atherosclerosis (9Trigatti B. Rayburn H. Vinals M. Braun A. Miettinen H. Penman M. Hertz M. Schrenzel M. Amigo L. Rigotti A. Krieger M. Influence of the high density lipoprotein receptor SR-BI on reproductive and cardiovascular pathophysiology.Proc. Natl. Acad. Sci. USA. 1999; 96: 9322-9327Crossref PubMed Scopus (445) Google Scholar, 10Braun A. Trigatti B.L. Post M.J. Sato K. Simons M. Edelberg J.M. Rosenberg R.D. Schrenzel M. Krieger M. Loss of SR-BI expression leads to the early onset of occlusive atherosclerotic coronary artery disease, spontaneous myocardial infarctions, severe cardiac dysfunction, and premature death in apolipoprotein E-deficient mice.Circ. Res. 2002; 90: 270-276Crossref PubMed Scopus (429) Google Scholar). The increased cholesterol, which is mainly distributed to VLDL-sized particles, appears to be at least partly attributable to the accumulation of abnormally large HDL-like particles containing apoA-I and lacking apoB (9Trigatti B. Rayburn H. Vinals M. Braun A. Miettinen H. Penman M. Hertz M. Schrenzel M. Amigo L. Rigotti A. Krieger M. Influence of the high density lipoprotein receptor SR-BI on reproductive and cardiovascular pathophysiology.Proc. Natl. Acad. Sci. USA. 1999; 96: 9322-9327Crossref PubMed Scopus (445) Google Scholar). In the case of attenuated SR-BI expression (brought about by a mutation in the SR-BI promoter), lipoprotein profiles in apoE−/− mice remain unchanged (11Arai T. Rinninger F. Varban L. Fairchild-Huntress V. Liang C.P. Chen W. Seo T. Deckelbaum R. Huszar D. Tall A.R. Decreased selective uptake of high density lipoprotein cholesteryl esters in apolipoprotein E knock-out mice.Proc. Natl. Acad. Sci. USA. 1999; 96: 12050-12055Crossref PubMed Scopus (74) Google Scholar). To date, there are no published reports directly assessing whether SR-BI mediates the uptake of apoE−/− lipoprotein remnants. Studies of the metabolism of apoB-containing lipoproteins in apoE−/− mice with respect to SR-BI may be particularly relevant in light of the unusual apolipoprotein composition of these particles (7Zhang S.H. Reddick R.L. Piedrahita J.A. Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E.Science. 1992; 258: 468-471Crossref PubMed Scopus (1849) Google Scholar). Both the VLDL and LDL density remnants in apoE−/− mice contain apoB-48 as the major apoB species as well as substantial amounts of apoA-I and apoA-IV. Given the important, but not exclusive, role of apoA-I in selective CE uptake (12Williams D.L. de la Llera-Moya M. Thuahnai S.T. Lund-Katz S. Connelly M.A. Azhar S. Anantharamaiah G.M. Phillips M.C. Binding and cross-linking studies show that scavenger receptor BI interacts with multiple sites in apolipoprotein A-I and identify the class A amphipathic α-helix as a recognition motif.J. Biol. Chem. 2000; 275: 18897-18904Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 13de Beer M.C. Durbin D.M. Cai L. Jonas A. de Beer F.C. van der Westhuyzen D.R. Apolipoprotein A-I conformation markedly influences HDL interaction with scavenger receptor BI.J. Lipid Res. 2001; 42: 309-313Abstract Full Text Full Text PDF PubMed Google Scholar, 14Temel R.E. Walzem R.L. Banka C.L. Williams D.L. Apolipoprotein A-I is necessary for the in vivo formation of high density lipoprotein competent for scavenger receptor BI-mediated cholesteryl ester-selective uptake.J. Biol. Chem. 2002; 277: 26565-26572Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), this apolipoprotein could impart a different characteristic to apoE−/− mouse LDL compared with LDL derived from normal human plasma or apoE+/+ mice. To assess whether SR-BI plays a role in the metabolism of lipoprotein remnants that accumulate in apoE−/− mice, we analyzed plasma lipoproteins before and after acute overexpression of this receptor by adenoviral vector. In addition, the ability of SR-BI to metabolize these particles was directly measured in assays in vitro using transfected CHO cells and primary hepatocytes from apoE−/− mice. LDL receptor-deficient (LDLR−/−) and apoE−/− mice (C57BL/6 background) were obtained from Jackson Laboratories (Bar Harbor, ME). The animals were housed in a pathogen-free facility with equal light/dark periods and free access to water and regular rodent chow, unless otherwise indicated. All procedures were approved by the Veterans Administration Medical Center Institutional Animal Use and Care Committee. The production of a replication-defective adenoviral vector expressing mouse SR-BI (AdSR-BI) has been described (15Webb N.R. Connell P.M. Graf G.A. Smart E.J. de Villiers W.J.S. de Beer F.C. van der Westhuyzen D.R. SR-BII, an isoform of the scavenger receptor BI containing an alternate cytoplasmic tail, mediates lipid transfer between high density lipoprotein and cells.J. Biol. Chem. 1998; 273: 15241-15248Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Adnull is a recombinant adenovirus containing no transgene. Mice weighing at least 25 g were injected into the tail vein with the indicated dose of either AdSR-BI or Adnull in 100 μl of PBS. Plasma was collected from mice after a 10 h fast. Aliquots (200 μl) were separated by size exclusion chromatography with a Superose 6 column (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ). The column was eluted at a flow rate of 0.5 ml/min in buffer containing 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, and 0.01% sodium azide. The cholesterol content of fractions (0.5 ml) and plasma was determined enzymatically (Wako Chemicals). Triglyceride analysis was performed using enzymatic assay kits (Sigma Chemical Co., St. Louis, MO). The preparation of anti-mouse SR-BI495 and immunoblot analysis of mouse liver have been described (15Webb N.R. Connell P.M. Graf G.A. Smart E.J. de Villiers W.J.S. de Beer F.C. van der Westhuyzen D.R. SR-BII, an isoform of the scavenger receptor BI containing an alternate cytoplasmic tail, mediates lipid transfer between high density lipoprotein and cells.J. Biol. Chem. 1998; 273: 15241-15248Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). VLDL (d = 1.006–1.019 g/ml), LDL (d = 1.019–1.063 g/ml), and HDL (d = 1.063–1.21 g/ml) were isolated from fresh mouse or human plasma by density gradient ultracentrifugation as described previously (16Strachan A.F. de Beer F.C. van der Westhuyzen D.R. Coetzee G.A. Identification of three isoform patterns of human serum amyloid A protein.Biochem. J. 1988; 250: 203-207Crossref PubMed Scopus (78) Google Scholar). All isolated fractions were dialyzed against 150 mM NaCl and 0.01% EDTA, pH 7.4, sterile filtered, and stored under nitrogen gas at 4°C. Protein concentrations were determined by the method of Lowry et al. (17Lowry O.H. Rosebrough N.J. Farr A.L. Randall B.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Lipoprotein fractions were traced with nonhydrolyzable, intracellularly trapped 1α,2α(n)-[3H]cholesteryl ether (CEt) according to the methods of Gwynne and Mahaffee (18Gwynne J.T. Mahaffee D.D. Rat adrenal uptake and metabolism of high density lipoprotein cholesteryl ester.J. Biol. Chem. 1989; 264: 8141-8150Abstract Full Text PDF PubMed Google Scholar). Apolipoproteins were radioiodinated by the iodine monochloride method (19Bilheimer D.W. Eisenberg S. Levy R.I. The metabolism of very low density lipoproteins.Biochim. Biophys. Acta. 1972; 260: 212-221Crossref PubMed Scopus (1185) Google Scholar). The production and maintenance of a CHO cell line stably transfected with murine SR-BI cDNA (CHO-SRBI) was described previously (15Webb N.R. Connell P.M. Graf G.A. Smart E.J. de Villiers W.J.S. de Beer F.C. van der Westhuyzen D.R. SR-BII, an isoform of the scavenger receptor BI containing an alternate cytoplasmic tail, mediates lipid transfer between high density lipoprotein and cells.J. Biol. Chem. 1998; 273: 15241-15248Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). This line, derived from CHO ldlA (clone 7) cells, is deficient in the LDLR (20Kingsley D.M. Krieger M. Receptor-mediated endocytosis of low density lipoprotein: somatic cell mutants define multiple genes required for expression of surface-receptor activity.Proc. Natl. Acad. Sci. USA. 1984; 81: 5454-5458Crossref PubMed Scopus (132) Google Scholar). Cells were seeded in six-well plates 48 h before assays (2.5 × 105 cells per well). Cell-association assays were performed at 37°C in Ham's F-12 medium containing 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, 0.5% essentially fatty acid-free BSA, and radiolabeled lipoprotein. After incubating for the indicated times, unbound ligand was removed from cells by washing three times with 50 mM Tris-HCl buffer, pH 7.4, containing 150 mM NaCl and 0.2% fatty acid-free BSA, followed by two washes with 50 mM Tris-HCl and 150 mM NaCl, pH 7.4. All washes were performed at 4°C with prechilled solutions. Cells were solubilized in 0.1 N NaOH for 60 min at room temperature before protein and radioactivity determinations. For apolipoprotein degradation assays, cell-free supernatants were analyzed for trichloroacetic acid-soluble, noniodide radioactivity (21Bierman E.L. Stein O. Stein Y. Lipoprotein uptake and metabolism by rat aortic smooth muscle cells in tissue culture.Circ. Res. 1974; 35: 136-150Crossref PubMed Scopus (307) Google Scholar). To allow for a direct comparison of different radiolabeled tracers, 3H uptake was expressed as apparent uptake of lipoprotein protein, assuming whole particle uptake (22Glass C. Pittman R.C. Weinstein D.W. Steinberg D. Dissociation of tissue uptake of cholesterol ester from that of ApoA-I of rat plasma high density lipoprotein: selective delivery of cholesterol ester to liver, adrenal, and gonad.Proc. Natl. Acad. Sci. USA. 1983; 80: 5435-5439Crossref PubMed Scopus (423) Google Scholar). Binding parameters were calculated using GraphPad Prism 3.0. Primary hepatocytes were isolated from apoE−/− mice 3 days after treatment with 1 × 1011 particles of AdSR-BI or Adnull by collagenase perfusion (fraction IV, 1 mg/ml in Hanks balanced salt solution supplemented with 10 mM HEPES, pH 7.4, 0.5 μM pig insulin, 5.6 mM glucose, and 1.3 mM CaCl2) and repeated low-speed centrifugations (60 g). Immediately after harvesting, cells were suspended in prewarmed Williams' E medium (Life Technologies) containing 0.5% BSA (2.5 × 106 cells/ml). Selective uptake experiments were performed in 12-well cluster dishes containing 1 × 106 cells. Cells were incubated at 37°C for 1 h with 125I- and 3H-labeled lipoproteins with slow shaking. After the incubation period, cells were washed once with Williams' E medium containing 0.5% BSA and then twice with Williams' E medium at 4°C. Cells were solubilized in 0.1 N NaOH for 20 min at room temperature before protein and radioactivity determinations. The viability of cells used in the experiments was greater than 90%. The effect of SR-BI overexpression on hepatic triglyceride and apoB secretion was assessed using a previously described method (23Li X. Catalina F. Grundy S.M. Patel S. Method to measure apolipoprotein B-48 and B-100 secretion rates in an individual mouse: evidence for a very rapid turnover of VLDL and preferential removal of B-48- relative to B-100-containing lipoproteins.J. Lipid Res. 1996; 37: 210-220Abstract Full Text PDF PubMed Google Scholar). Age-matched male apoE−/− mice were injected in the tail vein with either 5 × 1010 or 1.5 × 1011 particles of AdSR-BI or Adnull. Three days after injection of viral vectors, mice were placed on a fat-free diet 4 h before intravenous injection of 20 mg of Triton WR1339 (tyloxapol; Sigma) in 100 μl. Blood was drawn from the retro-orbital sinus just before and 1, 3, and 5 h after injection. Plasma triglyceride concentrations were quantified as described above. Plasma apoB content was assessed by immunoblot analysis using rabbit anti-mouse apoB (Biodesign International). Statistical analysis was performed using two-tailed Student's t-test for unpaired data. Triglyceride-rich lipoprotein particles (chylomicrons and VLDL) are hydrolyzed in the peripheral circulation by lipoprotein lipase to form cholesterol-enriched lipoprotein remnants. Normally, these lipoprotein remnants are efficiently cleared from the plasma by the liver. Mice deficient in apoE accumulate large amounts of cholesterol-rich lipoprotein remnants, demonstrating the importance of apoE in the clearance of these particles. The apolipoprotein composition of lipoprotein remnants in apoE−/− mice is markedly distinct from the light lipoprotein fractions isolated from human or LDLR−/− mouse plasma (Fig. 1). Whereas human and LDLR−/− mouse VLDL (d = 1.006–1.019 g/ml) and LDL (d = 1.019–1.063 g/ml) contain apoB-100 as the major apolipoprotein, the VLDL and LDL fractions from apoE−/− mice contain primarily apoB-48. ApoE−/− mouse VLDL and LDL also contain large amounts of apoA-I and apoA-IV, both of which are present in negligible amounts in human and LDLR−/− mouse VLDL and LDL. SR-BI is a class B scavenger receptor that binds a broad variety of lipoprotein ligands. The distinctive apolipoprotein composition of VLDL and LDL in apoE−/− mice prompted us to investigate whether SR-BI plays a role in the metabolism of these lipoprotein remnants. To assess SR-BI activity toward these lipoproteins in vivo, the receptor was overexpressed in livers of apoE−/− mice using a replication-deficient adenoviral vector, AdSR-BI (15Webb N.R. Connell P.M. Graf G.A. Smart E.J. de Villiers W.J.S. de Beer F.C. van der Westhuyzen D.R. SR-BII, an isoform of the scavenger receptor BI containing an alternate cytoplasmic tail, mediates lipid transfer between high density lipoprotein and cells.J. Biol. Chem. 1998; 273: 15241-15248Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Mice were infused with a dose of AdSR-BI (3 × 1010 particles) that resulted in a 5- to 10-fold increase in hepatic SR-BI expression (Fig. 2A). Three days after AdSR-BI infusion, mice showed a nonsignificant decrease in plasma total cholesterol concentration (190 ± 30.3 mg/dl; n = 4) compared with mice treated with a control adenovirus (290 ± 108 mg/dl; n = 4). Plasma triglyceride concentrations in AdSR-BI-treated mice (57.0 ± 8.3 mg/dl; n = 4) were similar to those of controls (55.5 ± 28 mg/dl; n = 4). Analysis of plasma lipoproteins by size exclusion chromatography revealed a depletion of LDL-cholesterol in AdSR-BI-treated mice but no alteration in VLDL-cholesterol (Fig. 2B). To assess the possibility that SR-BI may directly metabolize apoB-containing lipoproteins that accumulate in apoE−/− mice, we performed in vitro selective lipid uptake assays using CHO cells stably transfected with mouse SR-BI (15Webb N.R. Connell P.M. Graf G.A. Smart E.J. de Villiers W.J.S. de Beer F.C. van der Westhuyzen D.R. SR-BII, an isoform of the scavenger receptor BI containing an alternate cytoplasmic tail, mediates lipid transfer between high density lipoprotein and cells.J. Biol. Chem. 1998; 273: 15241-15248Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). This allowed us to quantify SR-BI-dependent LDL uptake, which is defined as the difference between the association of SR-BI-transfected and control nontransfected cells. For these studies, lipoprotein particles were separated by sequential density ultracentrifugation into two density ranges conventionally defined as VLDL and LDL. These lipoprotein fractions correspond to the fractions analyzed by SDS-PAGE in Fig. 1. For comparison, HDL isolated from C57BL/6 mouse plasma was also analyzed. SR-BI-transfected cells exhibited SR-BI-dependent 125I-lipoprotein cell association for apoE−/− VLDL and LDL as well as normal mouse HDL (Fig. 3A, C, E, hatched symbols). Very little SR-BI-dependent 125I-labeled degradation products were detected in the medium during the 2 h incubation period for any of the mouse ligands (representing less than 10% of SR-BI-dependent 125I cell association), indicating that these lipoprotein particles were not internalized and degraded by SR-BI-expressing cells (data not shown). To determine whether SR-BI mediates selective lipid uptake from apoE−/− remnant lipoproteins, the amount of cell-associated VLDL and LDL traced with nonhydrolyzable [3H]CEt was quantified. To allow for a direct assessment of selective lipid uptake, [3H]CEt cell association was expressed as the amount of apparent lipoprotein protein uptake by the cells, assuming whole particle uptake (22Glass C. Pittman R.C. Weinstein D.W. Steinberg D. Dissociation of tissue uptake of cholesterol ester from that of ApoA-I of rat plasma high density lipoprotein: selective delivery of cholesterol ester to liver, adrenal, and gonad.Proc. Natl. Acad. Sci. USA. 1983; 80: 5435-5439Crossref PubMed Scopus (423) Google Scholar). Incubation of untransfected CHO cells with [3H]VLDL resulted in the association of increasing amounts of 3H label during the 2 h incubation period that was approximately 2-fold greater than the amount that could be accounted for by 125I-VLDL cell association (Fig. 3A, B, open symbols). This result indicates SR-BI-independent selective lipid uptake from this lipoprotein fraction. Although CHO-SRBI cells showed a greater than 2-fold increase in the amount of cell-associated [3H]VLDL compared with control CHO cells (Fig. 3B, closed symbols), the amount of SR-BI-dependent [3H]VLDL cell association was far less than the corresponding amount of SR-BI-dependent 125I-VLDL cell association (Fig. 3A, B, hatched symbols). Thus, binding of apoE−/− VLDL to the transfected cells did not lead to selective lipid uptake by SR-BI. A lack of SR-BI-dependent selective lipid uptake was also observed from apoE−/− mouse LDL (Fig. 3C, D). Control CHO cells took up approximately 3.5-fold more [3H]LDL over the 2 h incubation period than 125I-LDL, indicating SR-BI-independent selective lipid uptake. During the same incubation period, CHO-SRBI cells accumulated 30% more [3H]LDL than 125I-LDL. However, the SR-BI-dependent component of [3H]LDL uptake in the transfected cells (Fig. 3D) was more than offset by the SR-BI-dependent component of 125I-LDL binding (Fig. 3C). These data indicate that LDL isolated from apoE−/− mice, like VLDL, does not serve as a substrate for SR-BI-mediated selective lipid uptake in CHO-SRBI cells. Thus, SR-BI activity toward apoB-containing lipoproteins that accumulate in apoE−/− mice contrasts markedly with its activity toward normal mouse HDL. As depicted in Fig. 3E, F, CHO-SRBI cells accumulated ∼4-fold more [3H]HDL in 2 h compared with control CHO cells, and this 3H uptake appeared to be selective, because SR-BI-dependent [3H]HDL association exceeded SR-BI-dependent 125I-HDL association more than 12-fold in the transfected cells (Fig. 3E, F; note difference in scales). We reported previously that SR-BI mediates selective lipid uptake from LDLR−/− mouse LDL (6Webb N.R. de Beer M.C. Yu J. Kindy M.S. Daugherty A. van der Westhuyzen D.R. de Beer F.C. Overexpression of SR-BI by adenoviral vector promotes clearance of apoA-I, but not apoB, in human apoB transgenic mice.J. Lipid Res. 2002; 43: 1421-1428Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Thus, the current data indicate that the interaction of SR-BI with LDL isolated from apoE−/− and LDLR−/− mice is distinct. To confirm this result directly, selective lipid uptake experiments were performed in vitro using doubly radiolabeled LDL (d = 1.019–1.063 g/ml) isolated from either LDLR−/− or apoE−/− mouse plasma. The lipoprotein fractions analyzed in these experiments correspond to the LDLR−/− and apoE−/− LDL shown in Fig. 1. As depicted in Fig. 4A, CHO-SRBI cells exhibited high-affinity, SR-BI-dependent 125I-lipoprotein cell association for both mouse ligands (apoE−/− LDL: apparent Kd = 4,600 ± 700 ng/ml, Bmax = 3,300 ± 130 ng/mg cell protein; LDLR−/− LDL: apparent Kd = 5,200 ± 600 ng/ml, Bmax = 1,600 ± 50 ng/mg cell protein). Very few SR-BI-dependent 125I-labeled degradation products were detected in the medium during the 2 h incubation period for either ligand (<7% of SR-BI-dependent 125I cell association). To assess the extent to which SR-BI mediates selective lipid uptake from the respective LDLs, [3H]CEt accumulation in cells was quantified (Fig. 4B). In the case of LDLR−/− LDL, approximately 1.2- to 1.8-fold more [3H]CEt was associated with cells in an SR-BI-dependent manner compared with 125I, indicating a modest amount of selective lipid uptake from this lipoprotein ligand. This result is in agreement with earlier reports, in which it has been shown using similar in vitro assays that the amount of CE taken up by SR-BI from human and mouse LDL represents only a fraction of the amount of LDL-CE bound to the cell surface (5Swarnakar S. Temel R.E. Connelly M.A. Azhar S. Williams D.L. Scavenger receptor class B, type I, mediates selective uptake of low density lipoprotein cholesteryl ester.J. Biol. Chem. 1999; 274: 29733-29739Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 6Webb N.R. de Beer M.C. Yu J. Kindy M.S. Daugherty A. van der Westhuyzen D.R. de Beer F.C. Overexpression of SR-BI by adenoviral vector promotes clearance of apoA-I, but not apoB, in human apoB transgenic mice.J. Lipid Res. 2002; 43: 1421-1428Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). However, in the case of apoE−/− LDL, there was no evidence of SR-BI-dependent selective lipid uptake. We conclude from these in vitro studies that, unlike normal human and mouse LDLs, SR-BI expressed in transfected CHO cells does not mediate the metabolism of apoB-containing lipoproteins from apoE−/− mice. The results depicted in Figs. 3 and 4 clearly demonstrate that SR-BI expressed in transfected CHO cells does not mediate selective lipid uptake from apoB-containing lipoproteins from apoE−/− mice. To address the possibility that SR-BI expressed in a more physiological setting could promote the metabolism of apoE−/− lipoprotein remnants, selective lipid uptake assays were carried out using primary hepatocytes isolated from apoE−/− mice. For these experiments, hepatocytes were isolated from apoE−/− mice at 3 days after infusion of 1 × 1011 particles of AdSR-BI or the control virus, Adnull. Quantitative immunoblot analysis showed that SR-BI expression was ∼60-fold higher in hepatocytes isolated from AdSR-BI-treated mice compared with control mice (data not shown). As depicted in Fig. 5A, cells isolated from Adnull-treated mice exhibited selective lipid uptake when incubated with doubly radiolabeled normal mouse HDL. SR-BI overexpression enhanced selective lipid uptake 2- to 4-fold for the range of HDL concentrations tested. In contrast, in the case of [125I,3H]apoE−/− mouse LDL, primary hepatocytes isolated from Adnull-treated mice did not accumulate [3H]LDL in excess of the amount of cell-associated 125I-LDL, indicating that this ligand is not a substrate for selective lipid uptake by these cells (Fig. 5B). Overexpression of SR-BI in hepatocytes resulted in a 3- to 4-fold increase in both cell-associated 125I-LDL and [3H]LDL. Thus, alth
Referência(s)