Liver-specific Overexpression of Scavenger Receptor BI Decreases Levels of Very Low Density Lipoprotein ApoB, Low Density Lipoprotein ApoB, and High Density Lipoprotein in Transgenic Mice
1998; Elsevier BV; Volume: 273; Issue: 49 Linguagem: Inglês
10.1074/jbc.273.49.32920
ISSN1083-351X
AutoresNan Wang, Takeshi Arai, Yong Ji, Franz Rinninger, Alan R. Tall,
Tópico(s)Hormonal and reproductive studies
ResumoScavenger receptor BI (SR-BI) is known to mediate the selective uptake of high density lipoprotein (HDL) cholesteryl ester (CE) in liver and steroidogenic tissues. To evaluate the role of SR-BI in plasma lipoprotein metabolism, we have generated transgenic mice with liver-specific overexpression of murine SR-BI. On a chow diet SR-BI transgenic (SR-BI Tg) mice have decreased HDL-CE, apoA-I, and apoA-II levels; plasma triglycerides, low density lipoprotein (LDL) cholesterol, and very low density lipoprotein (VLDL) and LDL apoB were also decreased, compared with control mice. Turnover studies using non-degradable CE and protein labels showed markedly increased total and selective uptake of HDL-CE in the liver and increased HDL protein catabolism in both liver and kidney. To evaluate the changes in apoB further, mice were challenged with high fat, high cholesterol diets. In SR-BI Tg mice plasma apoB levels were only 3–15% of control levels, and the dietary increase in VLDL and LDL apoB was virtually abolished. These studies show that steady state overexpression of hepatic SR-BI reduces HDL levels and increases reverse cholesterol transport. They also indicate that SR-BI can play a role in the metabolism of apoB-containing lipoproteins. The dual effects of increased reverse cholesterol transport and lowering of apoB-containing lipoproteins that result from hepatic SR-BI overexpression could have anti-atherogenic consequences. Scavenger receptor BI (SR-BI) is known to mediate the selective uptake of high density lipoprotein (HDL) cholesteryl ester (CE) in liver and steroidogenic tissues. To evaluate the role of SR-BI in plasma lipoprotein metabolism, we have generated transgenic mice with liver-specific overexpression of murine SR-BI. On a chow diet SR-BI transgenic (SR-BI Tg) mice have decreased HDL-CE, apoA-I, and apoA-II levels; plasma triglycerides, low density lipoprotein (LDL) cholesterol, and very low density lipoprotein (VLDL) and LDL apoB were also decreased, compared with control mice. Turnover studies using non-degradable CE and protein labels showed markedly increased total and selective uptake of HDL-CE in the liver and increased HDL protein catabolism in both liver and kidney. To evaluate the changes in apoB further, mice were challenged with high fat, high cholesterol diets. In SR-BI Tg mice plasma apoB levels were only 3–15% of control levels, and the dietary increase in VLDL and LDL apoB was virtually abolished. These studies show that steady state overexpression of hepatic SR-BI reduces HDL levels and increases reverse cholesterol transport. They also indicate that SR-BI can play a role in the metabolism of apoB-containing lipoproteins. The dual effects of increased reverse cholesterol transport and lowering of apoB-containing lipoproteins that result from hepatic SR-BI overexpression could have anti-atherogenic consequences. free cholesterol cholesteryl ester cholesteryl oleyl ether HDL cholesteryl ester fractional catabolic rate lecithin:cholesterol acyltransferase scavenger receptor class B type I transgenic apolipoprotein A-I apolipoprotein A-II apolipoprotein B apolipoprotein E high density lipoprotein low density lipoprotein intermediate density lipoprotein very low density lipoprotein phospholipids 125I-N-methyltyramine cellobiose polyacrylamide gel electrophoresis fast protein liquid chromatography. The risk of coronary heart disease is inversely correlated with the levels of plasma high density lipoproteins (HDL)1 (1Gordon D.J. Rifkind B.M. N. Engl. J. Med. 1989; 321: 1311-1316Crossref PubMed Scopus (1412) Google Scholar, 2Tall A.R. Breslow J.L. Fuster V. Ross R. Topol E.J. Atherosclerosis and Coronary Artery Disease. Lippincott-Raven Publishers, Philadelphia1996: 105-128Google Scholar). HDL appears to transport cholesterol from peripheral tissues to the liver for catabolism and secretion (reverse cholesterol transport) (3Eisenberg S. J. Lipid Res. 1984; 25: 1017-1058Abstract Full Text PDF PubMed Google Scholar, 4Tall A.R. J. Clin. Invest. 1990; 86: 379-384Crossref PubMed Scopus (586) Google Scholar). A putative cell-surface receptor for this process has been identified (5Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2011) Google Scholar). This receptor, scavenger receptor BI (SR-BI), mediates high affinity binding of HDL and the selective uptake of HDL cholesteryl ester (CE) (5Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2011) Google Scholar), a process for delivery of cholesteryl ester into cells without degradation of HDL proteins (6Glass 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). Furthermore, SR-BI mRNA and protein levels are highest in adrenal gland, ovary, testis, and liver, tissues that display greatest selective cholesteryl ester uptake from HDL (7Cao G. Garcia C.K. Wyne K.L. Schultz R.A. Parker K.L. Hobbs H.H. J. Biol. Chem. 1997; 272: 33068-33076Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 8Wang N. Weng W. Breslow J.L. Tall A.R. J. Biol. Chem. 1996; 271: 21001-21004Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 9Landschultz K.T. Pathak R.K. Rigotti A. Krieger M. Hobbs H.H. J. Clin. Invest. 1996; 98: 984-995Crossref PubMed Scopus (470) Google Scholar). SR-BI expression in steroidogenic cells is regulated by hormones and mutations that alter cholesterol supply or metabolism in those tissues in vivo (8Wang N. Weng W. Breslow J.L. Tall A.R. J. Biol. Chem. 1996; 271: 21001-21004Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 9Landschultz K.T. Pathak R.K. Rigotti A. Krieger M. Hobbs H.H. J. Clin. Invest. 1996; 98: 984-995Crossref PubMed Scopus (470) Google Scholar, 10Rigotti A. Edelman E.R. Seifert P. Iqbal S.N. DeMattos R.B. Temel R.E. Krieger M. William D.L. J. Biol. Chem. 1996; 271: 33545-33549Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 11Ng D.S. Francone O.L. Forte T.M. Zhang J. Haghpassand M. Rubin E.M. J. Biol. Chem. 1997; 272: 15777-15781Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). More recently, strong support for the role of SR-BI in HDL metabolism has been provided by studies of mice with a targeted mutation resulting in decreased SR-BI gene expression (12Rigotti A. Trigatti B.L. Penman M. Rayburn H. Herz J. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12610-12615Crossref PubMed Scopus (761) Google Scholar, 13Varban M.L. Rinninger F. Wang N. Huntress V.F. Dunmore J.H. Fang Q. Gosselin M.L. Dixon K.L. Deeds J.D. Acton S.L. Tall A.R. Huszar D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4619-4624Crossref PubMed Scopus (269) Google Scholar). These mice demonstrate increased plasma HDL cholesterol, decreased adrenal cholesterol content (12Rigotti A. Trigatti B.L. Penman M. Rayburn H. Herz J. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12610-12615Crossref PubMed Scopus (761) Google Scholar, 13Varban M.L. Rinninger F. Wang N. Huntress V.F. Dunmore J.H. Fang Q. Gosselin M.L. Dixon K.L. Deeds J.D. Acton S.L. Tall A.R. Huszar D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4619-4624Crossref PubMed Scopus (269) Google Scholar), and decreased hepatic fractional clearance rate (FCR) for HDL-CE (13Varban M.L. Rinninger F. Wang N. Huntress V.F. Dunmore J.H. Fang Q. Gosselin M.L. Dixon K.L. Deeds J.D. Acton S.L. Tall A.R. Huszar D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4619-4624Crossref PubMed Scopus (269) Google Scholar), suggesting that SR-BI is the major molecule mediating HDL-CE-selective uptake in the liver. By contrast, adenovirus-mediated, hepatic overexpression of SR-BI in mice results in depletion of plasma HDL and an increase in biliary cholesterol concentration (14Kozarsky K.F. Donahee M.H. Rigotti A. Iqbal S.N. Edelman E.R. Krieger M. Nature. 1997; 387: 414-417Crossref PubMed Scopus (631) Google Scholar). Although these studies nicely demonstrate the effect of acute overexpression of SR-BI on HDL levels (14Kozarsky K.F. Donahee M.H. Rigotti A. Iqbal S.N. Edelman E.R. Krieger M. Nature. 1997; 387: 414-417Crossref PubMed Scopus (631) Google Scholar), they do not necessarily demonstrate plasma lipoprotein changes that would accompany steady state overexpression of SR-BI. In this paper we report an in depth study of transgenic mice with hepatic overexpression of murine SR-BI. These studies were designed to understand better the role of SR-BI in HDL metabolism and reverse cholesterol transport. During the initial characterization of these animals on a chow diet, we observed decreased LDL cholesterol and apoB levels. Whereas the mouse model studies to date have focused on HDL changes, SR-BI was originally identified as a receptor recognizing both native and modified LDL (15Acton S.L. Scherer P.E. Lodish H.F. Krieger M. J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar). Thus, further studies were performed on high fat, high cholesterol diets in order to delineate the effects of SR-BI on plasma apoB levels. A 1.5-kilobase cDNA fragment of murine SR-BI (16Ji Y. Jian B. Wang N. Sun Y. de la Llera-Moya M. 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) was cloned into the HpaI site of the pLIV-7 plasmid (17Fan J. Wang J. Bensadoun A. Lauer S.J. Dang Q. Mahley R.W. Taylor J.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8724-8728Crossref PubMed Scopus (220) Google Scholar), kindly provided by Dr. John M. Taylor (Gladstone Institute of Cardiovascular Disease, University of California, San Francisco). A linearized fragment of the construct containing the promoter, first exon, first intron, and part of the second exon of the human apoE gene, the murine SR-BI cDNA, and the polyadenylation sequence, and hepatic control region of the apoE/C-I gene locus was used to generate transgenic mice by standard procedures. Founder animals were backcrossed to C57Bl/6J mice and two transgenic mouse lines, SR-BI Tg(1) and SR-BI Tg(2), were established. Studies in this paper were performed using 8–10-week-old SR-BI Tg(1) or SR-BI Tg(2) N2 or N3 mice positive for both SR-BI transgene genotype and phenotype (decreased plasma total cholesterol) versuscontrol littermates negative for the SR-BI transgene. For studies of responses to high fat diets, mice were fed either a Western type diet containing 20% hydrogenated coconut oil and 0.15% cholesterol (Research Diets, Inc.) or a very high cholesterol diet containing 1.25% cholesterol, 7.5% cocoa butter, 7.5% casein, and 0.5% sodium cholate for 2 weeks. Total plasma cholesterol, free cholesterol, phospholipids, and triglycerides were determined using commercial enzymatic assays (Wako, Japan) (8Wang N. Weng W. Breslow J.L. Tall A.R. J. Biol. Chem. 1996; 271: 21001-21004Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Determination of plasma apoB levels was carried out using an enzyme-linked immunosorbent immunoassay with an affinity purified polyclonal antibody against murine apoB. For SDS-PAGE, VLDL (d <1.006 g/ml), IDL + LDL (d = 1.006–1.055 g/ml), and HDL (d = 1.055–1.21 g/ml) were separated by sequential density ultracentrifugation of pooled mouse plasma. In some experiments VLDL + IDL (d <1.006–1.019) and LDL (d = 1.019–1.055) were separated as indicated. Denaturing polyacrylamide gel analysis of isolated lipoproteins was performed using 4–20% SDS-PAGE gradient gels from Bio-Rad. Gels were stained with Coomassie Brilliant Blue R, and the identity of individual apolipoproteins was confirmed by Western analysis. HDL was prepared in the density range 1.063–1.21 g/ml from plasma of C57BL/6 wild type mice, dialyzed against phosphate-buffered saline containing 0.3 mm EDTA and 0.02% NaN3, and radiolabeled in the protein moiety with 125I-N-methyltyramine cellobiose (125I-NMTC) (18Pittman R.C. Taylor Jr., C.A. Methods Enzymol. 1986; 129: 612-628Crossref PubMed Scopus (58) Google Scholar), and thereafter with [3H]cholesteryl oleyl ether ([3H]CEt, Amersham Pharmacia Biotech) (19Rinninger F. Pittman R.C. J. Lipid Res. 1987; 28: 1313-1325Abstract Full Text PDF PubMed Google Scholar). [3H]CEt was introduced in a liposomal preparation and exchanged (6 h, 37 °C) into125I-NMTC-labeled HDL using purified recombinant human plasma cholesteryl ester transfer protein. The donor liposomes were separated from labeled HDL by ultracentrifugation at d= 1.063 g/ml, followed by another spin at d = 1.21 g/ml to remove cholesteryl ester transfer protein from the labeled HDL preparation. Then the doubly labeled HDL was dialyzed against phosphate-buffered saline containing 0.3 mm EDTA. Experiments to determine plasma decay of both HDL tracers and their tissue sites of uptake were carried out (19Rinninger F. Pittman R.C. J. Lipid Res. 1987; 28: 1313-1325Abstract Full Text PDF PubMed Google Scholar, 20Glass C. Pittman R.C. Civen R.C. Steinberg D. J. Biol. Chem. 1985; 260: 744-750Abstract Full Text PDF PubMed Google Scholar). Food was removed from five female control and SR-BI Tg mice 4 h before tracer injection, and animals were fasted throughout the 24-h study period but had free access to water. Doubly radiolabeled HDL was injected at 10:00 a.m. in an iliac vein, and blood samples were drawn from the tail vein of each animal at 0.08, 0.5, 2.0, 5.0, 9.0, and 24.0 h post-injection. Plasma samples were directly radioassayed for 125I and analyzed for [3H] after lipid extraction (19Rinninger F. Pittman R.C. J. Lipid Res. 1987; 28: 1313-1325Abstract Full Text PDF PubMed Google Scholar). 24 h after tracer injection the animals were anesthetized and perfused with saline (50 ml per animal), and organs were collected, weighed, homogenized, and radioassayed. Tissue content of 125I radioactivity was directly assayed and that of [3H] was analyzed after lipid extraction. Based on plasma decay of both HDL tracers, plasma FCRs were calculated using a two-compartment model (21Le N.A. Ramakrishnan R. Dell R.B. Ginsberg H.N. Brown W.V. Methods Enzymol. 1986; 129: 384-395Crossref PubMed Scopus (19) Google Scholar). Organ FCRs, representing the fraction of the plasma pool of the traced HDL component cleared per h by an organ, were calculated as the plasma FCR × fraction of total tracer (%) recovered in a specific organ (19Rinninger F. Pittman R.C. J. Lipid Res. 1987; 28: 1313-1325Abstract Full Text PDF PubMed Google Scholar, 20Glass C. Pittman R.C. Civen R.C. Steinberg D. J. Biol. Chem. 1985; 260: 744-750Abstract Full Text PDF PubMed Google Scholar). Western blot analysis for SR-BI and Southern and Northern analysis were performed as described (8Wang N. Weng W. Breslow J.L. Tall A.R. J. Biol. Chem. 1996; 271: 21001-21004Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 22Jiang X Francone O.L. Bruce C. Milne R. Mar J. Walsh A. Breslow J.L. Tall A.R. J. Clin. Invest. 1996; 98: 2373-2380Crossref PubMed Scopus (164) Google Scholar). Dot blot was carried out with a 900-base pair cDNA fragment of mouse apoB to determine hepatic apoB mRNA levels. Hepatic and adrenal cholesterol contents were measured by gas-liquid chromatography (23Plump A. Azrolan S., N. Odaka H. Wu L. Jiang X. Tall A.R. Eisenberg S. Breslow J.L. J. Lipid Res. 1997; 38: 1033-1047Abstract Full Text PDF PubMed Google Scholar). Plasma relative LCAT activity was determined by examining conversion of endogenous plasma free cholesterol to cholesteryl ester. Plasma-specific LCAT activity was determined using reconstituted discoidal HDL as exogenous substrate, which was prepared with the sodium cholate method at an initial molar ratio of 80:4:1:160, egg phosphatidylcholine:cholesterol:apoA-I:sodium cholate (34Jonas A. Methods Enzymol. 1986; 128: 553-582Crossref PubMed Scopus (296) Google Scholar). Briefly, each assay mixture contained reconstituted HDL (24 μg of apoA-I), 4% defatted bovine serum albumin, and 4 mm β-mercaptoethanol in a total volume of 0.5 ml. The reaction was initiated by adding the indicated amount of plasma and carried out at 37 °C for 20 min. LCAT activity was determined by the percentage conversion of [14C]cholesterol to CE. Two separate lines of SR-BI Tg mice, SR-BI Tg(1) and SR-BI Tg(2), were established. The SR-BI Tg(1) mice demonstrated a pattern of marked liver-specific overexpression of SR-BI mRNA (Fig. 1 A); there was no appreciable expression in the kidney (Fig. 1 A, lane 1). Western analysis showed a 12-fold increase in hepatic membrane SR-BI levels in transgenic mice (Fig. 1, B and C). Similar levels of expression were observed for both lines of SR-BI Tg mice. Analysis of plasma lipids on a chow diet revealed that female SR-BI Tg mice (both lines) had a profound 92–94% decrease of plasma total cholesterol (TC) (Table I), with decreases in both free cholesterol (FC) (∼80%) and cholesteryl ester (CE) (96%). There was also a significant but less pronounced decrease in plasma phospholipids (PL) (∼75%) and triglycerides (TG) (45–58%) (Table I). Similar results were obtained for male mice (not shown).Table IPlasma lipid concentrations of control and SR-BI Tg miceDietAnimalTCFCCEPLTGChowControl67 ± 1115 ± 552 ± 10185 ± 1877 ± 37SR-BI Tg(1)5 ± 21-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.3 ± 21-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.2 ± 11-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.47 ± 61-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.32 ± 61-bp < 0.05.SR-BI Tg(2)4 ± 21-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.2 ± 11-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.2 ± 11-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.45 ± 81-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.42 ± 161-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.WesternControl107 ± 1324 ± 582 ± 11255 ± 30148 ± 30SR-BI Tg(1)78 ± 131-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.56 ± 51-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.16 ± 71-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.213 ± 241-bp < 0.05.93 ± 371-bp < 0.05.SR-BI Tg(2)68 ± 201-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.51 ± 131-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.19 ± 81-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.211 ± 331-bp < 0.05.95 ± 271-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.Very high cholesterolControl114 ± 1141 ± 673 ± 8207 ± 4085 ± 30SR-BI Tg(1)54 ± 251-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.18 ± 81-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.24 ± 31-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.132 ± 441-bp < 0.05.61 ± 24SR-BI Tg(2)16 ± 81-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.8 ± 51-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.8 ± 41-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.82 ± 321-aStatistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.67 ± 37Plasma was collected, and lipid concentrations were determined as described under "Materials and Methods." The values are expressed as mg/dl and shown as means ± S.D., n = 7–12 female mice.1-a Statistical significance, p < 0.01 between the control and SR-BI Tg animals on the same diet as determined by two-tailed Student's t test for unpaired data.1-b p < 0.05. Open table in a new tab Plasma was collected, and lipid concentrations were determined as described under "Materials and Methods." The values are expressed as mg/dl and shown as means ± S.D., n = 7–12 female mice. When plasma was analyzed by fast protein liquid chromatography (FPLC), most of the CE and FC were in the HDL fraction in the control mice on the chow diet (Fig. 2, A and B). By contrast HDL-CE and FC were almost undetectable in SR-BI Tg mice. HDL phospholipids were also markedly decreased (Fig. 2 C). There was also a decrease in lipids in VLDL and LDL region, although these were also low in control mice. Assessment of apolipoprotein composition of centrifugally isolated lipoproteins by reducing SDS-PAGE gels revealed a marked decrease of HDL apoA-I, apoA-II, and apoE levels in SR-BI Tg(1) mice (Fig. 3 A). The VLDL and LDL apoB and apoE levels also were decreased. The results were confirmed by Western analysis using antisera specific for murine apoA-I, apoA-II, and apoE. Similar results were obtained in four separate analyses of pooled plasma from a total of 9 SR-BI Tg(1) mice and 10 control mice and were also confirmed in the SR-BI Tg(2) line (data not shown). The changes in HDL in SR-BI Tg mice resemble these occurring 3 days after adenovirus-mediated expression of SR-BI (14Kozarsky K.F. Donahee M.H. Rigotti A. Iqbal S.N. Edelman E.R. Krieger M. Nature. 1997; 387: 414-417Crossref PubMed Scopus (631) Google Scholar) where HDL turnover was evaluated using 125I and DiI labels (14Kozarsky K.F. Donahee M.H. Rigotti A. Iqbal S.N. Edelman E.R. Krieger M. Nature. 1997; 387: 414-417Crossref PubMed Scopus (631) Google Scholar). Next we carried out HDL turnover studies using non-degradable radiolabels (18Pittman R.C. Taylor Jr., C.A. Methods Enzymol. 1986; 129: 612-628Crossref PubMed Scopus (58) Google Scholar, 19Rinninger F. Pittman R.C. J. Lipid Res. 1987; 28: 1313-1325Abstract Full Text PDF PubMed Google Scholar). In the control mice, the higher rate of removal from plasma of the lipid ([3H]CEt), relative to protein (125I-NMTC), represents whole body selective uptake of HDL-CE (Fig. 4 A). There was a significantly accelerated rate of clearance for both tracers in SR-BI Tg mice. The plasma FCRs calculated from these decay curves showed a 370% increase in protein catabolism and 330% increase in lipid tracer catabolism (Fig. 4 B). The selective removal of HDL-CE from plasma, calculated as the difference between CE and protein FCRs, was increased by 260% in SR-BI Tg mice. Tissue sites of tracer uptake from doubly radiolabeled HDL were determined, and results are expressed as the organ FCRs (TableII). The liver was the predominant organ for both HDL lipid and protein catabolism (19Rinninger F. Pittman R.C. J. Lipid Res. 1987; 28: 1313-1325Abstract Full Text PDF PubMed Google Scholar, 20Glass C. Pittman R.C. Civen R.C. Steinberg D. J. Biol. Chem. 1985; 260: 744-750Abstract Full Text PDF PubMed Google Scholar). The higher liver FCR for lipid, relative to protein, indicates selective uptake of HDL [3H]CEt in the liver. In contrast, a negative value of3H minus 125I was derived for kidney FCR (TableII), indicating this organ is a major site for selective HDL protein catabolism (20Glass C. Pittman R.C. Civen R.C. Steinberg D. J. Biol. Chem. 1985; 260: 744-750Abstract Full Text PDF PubMed Google Scholar). SR-BI Tg mice showed a substantial increase in hepatic FCRs for both HDL protein (7.5-fold) and lipid (6.4-fold). The renal FCR of HDL proteins was also markedly increased (6.6-fold) in SR-BI Tg mice, whereas this organ contributed little to the clearance of HDL lipid in wild type or SR-BI Tg mice. Adrenal FCRs for HDL protein and lipid were increased 10.5-and 6.3-fold, respectively, and the adrenal-selective uptake of HDL-CE was increased 5.6-fold. This finding may have reflected an up-regulation of endogenous adrenal SR-BI expression secondary to reduced HDL levels and depletion of adrenal cholesterol stores (see below). Other organs (heart, spleen, and stomach), with minor contribution to HDL lipid and protein uptake, did not display any major changes in SR-BI Tg mice.Table IIOrgan fractional catabolic rates for125I-NMTC/[3H]CEt double-labeled HDL in miceOrganMice125I-NMTC[3H]CEt3H-125ILiverControl16.7 ± 1.554 ± 5.638 ± 5.7SR-BI Tg124 ± 18.12-aStatistical significance p < 0.01 between the control and SR-BI Tg animals as determined by two-tailed Student's t test for unpaired data.346 ± 742-aStatistical significance p < 0.01 between the control and SR-BI Tg animals as determined by two-tailed Student's t test for unpaired data.221 ± 572-aStatistical significance p < 0.01 between the control and SR-BI Tg animals as determined by two-tailed Student's t test for unpaired data.KidneyControl3.5 ± 0.40.5 ± 0.05−3.1 ± 0.4SR-BI Tg23.3 ± 1.22-aStatistical significance p < 0.01 between the control and SR-BI Tg animals as determined by two-tailed Student's t test for unpaired data.1.8 ± 0.12-aStatistical significance p < 0.01 between the control and SR-BI Tg animals as determined by two-tailed Student's t test for unpaired data.−21.5 ± 1.12-aStatistical significance p < 0.01 between the control and SR-BI Tg animals as determined by two-tailed Student's t test for unpaired data.AdrenalControl0.1 ± 0.010.4 ± 0.20.3 ± 0.2SR-BI Tg0.6 ± 0.32-aStatistical significance p < 0.01 between the control and SR-BI Tg animals as determined by two-tailed Student's t test for unpaired data.2.5 ± 0.92-aStatistical significance p < 0.01 between the control and SR-BI Tg animals as determined by two-tailed Student's t test for unpaired data.1.9 ± 0.72-aStatistical significance p < 0.01 between the control and SR-BI Tg animals as determined by two-tailed Student's t test for unpaired data.HeartControl0.2 ± 0.020.3 ± 0.030.02 ± 0.03SR-BI Tg0.6 ± 0.122-aStatistical significance p < 0.01 between the control and SR-BI Tg animals as determined by two-tailed Student's t test for unpaired data.0.3 ± 0.1−0.3 ± 0.042-aStatistical significance p < 0.01 between the control and SR-BI Tg animals as determined by two-tailed Student's t test for unpaired data.SpleenControl0.4 ± 0.040.7 ± 0.10.3 ± 0.06SR-BI Tg1.1 ± 0.062-aStatistical significance p < 0.01 between the control and SR-BI Tg animals as determined by two-tailed Student's t test for unpaired data.1.3 ± 0.12-aStatistical significance p < 0.01 between the control and SR-BI Tg animals as determined by two-tailed Student's t test for unpaired data.0.2 ± 0.2StomachControl0.2 ± 0.10.2 ± 0.10.01 ± 0.02SR-BI Tg0.4 ± 0.20.4 ± 0.12-aStatistical significance p < 0.01 between the control and SR-BI Tg animals as determined by two-tailed Student's t test for unpaired data.0.02 ± 0.2Organs were harvested from five female control and five female SR-BI Tg(1) mice 24 h after injection of labeled HDL. Organ FCRs were calculated as described under "Materials and Methods." The values are expressed as fraction of plasma pool cleared × h−1× 10−3 and shown as means ± S.D.2-a Statistical significance p < 0.01 between the control and SR-BI Tg animals as determined by two-tailed Student's t test for unpaired data. Open table in a new tab Organs were harvested from five female control and five female SR-BI Tg(1) mice 24 h after injection of labeled HDL. Organ FCRs were calculated as described under "Materials and Methods." The values are expressed as fraction of plasma pool cleared × h−1× 10−3 and shown as means ± S.D. The HDL turnover studies demonstrated that the reduced plasma HDL lipids and apolipoproteins were at least in part due to accelerated HDL catabolism in SR-BI Tg mice. We also measured hepatic apoA-I mRNA levels and found no difference between the control and SR-BI Tg animals (not shown). Hepatic free cholesterol was increased by 43% (p < 0.001) in SR-BI Tg mice (TableIII). By contrast adrenal cholesteryl ester virtually disappeared in SR-BI Tg mice, and free cholesterol was also decreased (Table III), probably reflecting the decreased plasma HDL-CE levels that result from hepatic overexpression of SR-BI (8Wang N. Weng
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