Regulation by vitamin E of the scavenger receptor BI in rat liver and HepG2 cells

The scavenger receptor class B type I (SR-BI) mediates the selective uptake of cholesterol and cholesteryl ester (CE) from high density lipoprotein (HDL) into cells. The high expression in liver and steroidogenic tissues is compatible with a role of SR-BI in reverse cholesterol transport and steroid hormone synthesis. Ways of regulation thus far described include induction by trophic hormones via cAMP-activated protein kinase A (PKA) and the effects of cellular and plasma cholesterol. Here we show that vitamin E (vitE) has a major effect on the expression of SR-BI in rat liver and in a human hepatoma-derived cell line, HepG2. Feeding rats a vitE-depleted diet resulted in an 11-fold increase in the SR-BI protein level in liver tissue. This effect was readily reversed by feeding a vitE-enriched chow. In HepG2 cells, the expression of the human SR-BI homolog was reduced when the vitE content was increased by incubating the cells with vitE-loaded HDL or with phosphatidylcholine/vitE vesicles. The downregulation of human SR-BI (hSR-BI) was accompanied by a reduced level of protein kinase C (PKC) in the particulate cell fraction, and PKC inhibition decreased the expression of hSR-BI and the uptake of vitE and cholesterol from HDL. Our results are consistent with the view that the cellular level of vitE exerts a tight control over the expression of SR-BI. Furthermore, the inhibitory effect of vitE on PKC seems to be involved in the signaling pathway. —Witt, W., I. Kolleck, H. Fechner, P. Sinha, and B. Rüstow. Regulation by vitamin E of the scavenger receptor BI in rat liver and HepG2 cells. J. Lipid Res. 2000. 41: 2009–2016. The scavenger receptor class B type I (SR-BI) mediates the selective uptake of cholesterol and cholesteryl ester (CE) from high density lipoprotein (HDL) into cells. The high expression in liver and steroidogenic tissues is compatible with a role of SR-BI in reverse cholesterol transport and steroid hormone synthesis. Ways of regulation thus far described include induction by trophic hormones via cAMP-activated protein kinase A (PKA) and the effects of cellular and plasma cholesterol. Here we show that vitamin E (vitE) has a major effect on the expression of SR-BI in rat liver and in a human hepatoma-derived cell line, HepG2. Feeding rats a vitE-depleted diet resulted in an 11-fold increase in the SR-BI protein level in liver tissue. This effect was readily reversed by feeding a vitE-enriched chow. In HepG2 cells, the expression of the human SR-BI homolog was reduced when the vitE content was increased by incubating the cells with vitE-loaded HDL or with phosphatidylcholine/vitE vesicles. The downregulation of human SR-BI (hSR-BI) was accompanied by a reduced level of protein kinase C (PKC) in the particulate cell fraction, and PKC inhibition decreased the expression of hSR-BI and the uptake of vitE and cholesterol from HDL. Our results are consistent with the view that the cellular level of vitE exerts a tight control over the expression of SR-BI. Furthermore, the inhibitory effect of vitE on PKC seems to be involved in the signaling pathway. —Witt, W., I. Kolleck, H. Fechner, P. Sinha, and B. Rüstow. Regulation by vitamin E of the scavenger receptor BI in rat liver and HepG2 cells. J. Lipid Res. 2000. 41: 2009–2016. The almost exclusive mechanism by which mammals eliminate cholesterol from the body is by the formation of bile in the liver. High density lipoprotein (HDL) exerts a major role in the uptake of cholesterol from peripheral organs and the delivery of cholesteryl ester (CE) to the liver, a process called reverse cholesterol transport. On the basis of kinetic data concerning the interaction of HDL with the surface of various cell types, specific receptors were postulated. The scavenger receptor class B type I (SR-BI) in rodents is the first biochemically well-characterized receptor for HDL, but affinity for other typical scavenger receptor ligands was also observed. SR-BI mediates the selective exchange of cholesterol between HDL and plasma membranes, meaning that only the lipid is transported; the HDL particles are not internalized into cells. Evidence is accumulating that the human homolog hSR-BI, also called CLA-1, exerts the same function. The rapidly growing literature dealing with these receptors has been thoroughly summarized (1Fidge N.H. High density lipoprotein receptors, binding proteins, and ligands.J. Lipid Res. 1999; 40: 187-201Google Scholar, 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: 523-558Google Scholar, 3Williams D.L. Connelly M.A. Temel R.E. Swarnakar S. Phillips M.C. de la Llera-Moya M. Rothblatt G.H. Scavenger receptor BI and cholesterol trafficking.Curr. Opin. Lipidol. 1999; 10: 329-339Google Scholar). SR-BI has been shown to be abundant in steroidogenic glands, in accordance with a central role in supplying cholesterol for steroid hormone synthesis. The level of SR-BI in liver is lower, but because of its mass the liver contains the major part of total body SR-BI. Kinetic analysis revealed that the mechanism by which the liver takes up CE from HDL is fully saturated at physiological concentrations of plasma CE (4Spady D.K. Woollett L.A. Meidell R.S Hobbs H.H. Kinetic characteristics and regulation of HDL cholesteryl ester and apolipoprotein transport in the apoA-I-/- mouse.J. Lipid Res. 1998; 39: 1483-1492Google Scholar). Therefore, changes in SR-BI expression in this organ should have a major impact on cholesterol homeostasis. Investigations have shown that the expression of SR-BI is influenced by several factors. In steroidogenic tissues, trophic hormones induce the selective uptake mechanism for CE and cholesterol via SR-BI (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: 523-558Google Scholar, 3Williams D.L. Connelly M.A. Temel R.E. Swarnakar S. Phillips M.C. de la Llera-Moya M. Rothblatt G.H. Scavenger receptor BI and cholesterol trafficking.Curr. Opin. Lipidol. 1999; 10: 329-339Google Scholar), and a feedback inhibition by steroid hormones was observed (5Rigotti A. Edelman E.R. Seifert P. Iqbal S.N. DeMattos R.B. Temel R.E. Krieger M. Williams D.L. Regulation by adrenocorticotropic hormone of the in vivo expression of scavenger receptor class B type I (SR-BI), a high density lipoprotein receptor, in steroidogenic cells of the murine adrenal gland.J. Biol. Chem. 1996; 271: 33545-33549Google Scholar). In parallel, the level of SR-BI in the hormone-producing cells was found to adapt to the supply with cholesterol (6Wang N. Weng W. Breslow J.L. Tall A.R. Scavenger receptor BI (SR-BI) is up-regulated in adrenal gland in apolipoprotein A-I and hepatic lipase knock-out mice as a response to depletion of cholesterol stores. In vivo evidence that SR-BI is a functional high density lipoprotein receptor under feedback control.J. Biol. Chem. 1996; 271: 21001-21004Google Scholar, 7Reaven E. Nomoto A. Leers-Sucheta S. Temel R. Williams D.L. Azhar S. Expression and microvillar localization of scavenger receptor, class B, type I (a high density lipoprotein receptor) in luteinized and hormone-desensitized rat ovarian models.Endocrinology. 1998; 139: 2847-2856Google Scholar). Some of these studies led to strikingly conflicting results. Wang et al. (6Wang N. Weng W. Breslow J.L. Tall A.R. Scavenger receptor BI (SR-BI) is up-regulated in adrenal gland in apolipoprotein A-I and hepatic lipase knock-out mice as a response to depletion of cholesterol stores. In vivo evidence that SR-BI is a functional high density lipoprotein receptor under feedback control.J. Biol. Chem. 1996; 271: 21001-21004Google Scholar) found an increased expression of SR-BI in the adrenals of apolipoprotein A-I (apoA-I) knockout mice, whereas Spady et al. (4Spady D.K. Woollett L.A. Meidell R.S Hobbs H.H. Kinetic characteristics and regulation of HDL cholesteryl ester and apolipoprotein transport in the apoA-I-/- mouse.J. Lipid Res. 1998; 39: 1483-1492Google Scholar) detected no effects on SR-BI expression in exactly the same mouse model. In spite of the central role of the liver in reverse cholesterol transport, information about the regulation of SR-BI expression is scarce. Hepatic SR-BI in rats is susceptible to downregulation by high doses of estrogens (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: 523-558Google Scholar, 8Landschulz K.T. Pathak R.K. Rigotti A. Krieger M. Hobbs H.H. Regulation of scavenger receptor, class B, type I, a high density lipoprotein receptor, in liver and steroidogenic tissues of the rat.J. Clin. Invest. 1996; 98: 984-995Google Scholar, 9Fluiter K. van der Westhuijzen D.R. van Berkel T.J.C. In vivo regulation of scavenger receptor BI and the selective uptake of high density lipoprotein cholesteryl esters in rat liver parenchymal and Kupffer cells.J. Biol. Chem. 1998; 273: 8434-8438Google Scholar). Only a few reports deal with the regulatory effects of cholesterol and other plasma lipids and lipoproteins on SR-BI in this organ, and the results are contradictory. Feeding a high cholesterol diet to rats led to a pronounced downregulation of SR-BI in liver parenchyma cells (9Fluiter K. van der Westhuijzen D.R. van Berkel T.J.C. In vivo regulation of scavenger receptor BI and the selective uptake of high density lipoprotein cholesteryl esters in rat liver parenchymal and Kupffer cells.J. Biol. Chem. 1998; 273: 8434-8438Google Scholar), whereas Spady et al. (4Spady D.K. Woollett L.A. Meidell R.S Hobbs H.H. Kinetic characteristics and regulation of HDL cholesteryl ester and apolipoprotein transport in the apoA-I-/- mouse.J. Lipid Res. 1998; 39: 1483-1492Google Scholar) found no change in SR-BI expression in apoA-I knockout mice compared with the wild type, even though these mice lacked normal HDL. Furthermore, a diet rich in polyunsaturated fatty acids increased the level of cholesterol in the liver but not in plasma and induced a moderate (50%) upregulation of hepatic SR-BI expression (10Spady D.K. Kearney D.M. Hobbs H.H. Polyunsaturated fatty acids up-regulate hepatic scavenger receptor B1 (SR-BI) expression and HDL cholesteryl ester uptake in the hamster.J. Lipid Res. 1999; 40: 1384-1394Google Scholar). Several other factors with a significant effect on the expression of SR-BI were described, including the expression of hepatic lipase (6Wang N. Weng W. Breslow J.L. Tall A.R. Scavenger receptor BI (SR-BI) is up-regulated in adrenal gland in apolipoprotein A-I and hepatic lipase knock-out mice as a response to depletion of cholesterol stores. In vivo evidence that SR-BI is a functional high density lipoprotein receptor under feedback control.J. Biol. Chem. 1996; 271: 21001-21004Google Scholar, 11Vieira van Bruggen D. Kalkman I. van Gent T. van Tol A. Jansen H. Induction of adrenal scavenger receptor BI and increased high density lipoprotein-cholesteryl ether uptake by in vivo inhibition of hepatic lipase.J. Biol. Chem. 1998; 273: 32038-32041Google Scholar) and of apoE as well as the proinflammatory stimuli bacterial lipopolysaccharide, interferon γ, and tumor necrosis factor α (12Buechler C. Ritter M. Quoc C.D. Agildere A. Schmitz G. Lipopolysaccharide inhibits the expression of the scavenger receptor Cla-1 in human monocytes and macrophages.Biochem. Biophys. Res. Commun. 1999; 262: 251-254Google Scholar). At least in steroidogenic tissues, the trophic hormone-dependent release of cAMP as second messenger and the activation of protein kinase A (PKA) were identified as components of the signaling pathway for the regulation of SR-BI expression (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: 523-558Google Scholar, 3Williams D.L. Connelly M.A. Temel R.E. Swarnakar S. Phillips M.C. de la Llera-Moya M. Rothblatt G.H. Scavenger receptor BI and cholesterol trafficking.Curr. Opin. Lipidol. 1999; 10: 329-339Google Scholar). Steroidogenic factor 1 (SF-1) has a binding sequence in the SR-BI promoter, and convincing evidence was presented that SF-1 regulates the transcription in vivo (13Cao G. Zhao L. Stangl H. Hasegawa T. Richardson J.A. Parker K.L. Hobbs H.H. Developmental and hormonal regulation of murine scavenger receptor, class B, type 1.Mol. Endocrinol. 1999; 13: 1460-1473Google Scholar). Several lines of evidence, however, have demonstrated that this scheme does not adequately describe the regulatory mechanism in all tissues. The transcription factor SF-1 was not found in liver (10Spady D.K. Kearney D.M. Hobbs H.H. Polyunsaturated fatty acids up-regulate hepatic scavenger receptor B1 (SR-BI) expression and HDL cholesteryl ester uptake in the hamster.J. Lipid Res. 1999; 40: 1384-1394Google Scholar), and several reports clearly showed that posttranscriptional events take part in the regulation of SR-BI expression (8Landschulz K.T. Pathak R.K. Rigotti A. Krieger M. Hobbs H.H. Regulation of scavenger receptor, class B, type I, a high density lipoprotein receptor, in liver and steroidogenic tissues of the rat.J. Clin. Invest. 1996; 98: 984-995Google Scholar, 12Buechler C. Ritter M. Quoc C.D. Agildere A. Schmitz G. Lipopolysaccharide inhibits the expression of the scavenger receptor Cla-1 in human monocytes and macrophages.Biochem. Biophys. Res. Commun. 1999; 262: 251-254Google Scholar, 14Arai 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: 12050-12055Google Scholar). Taken together, the regulatory mechanism at least in non-steroidogenic tissues is far from clear. The expression is obviously affected by a multitude of factors and, because interactions among these factors cannot be excluded, some of the described effects may be secondary. This article aims at determining the effects of vitamin E (vitE) on the expression of SR-BI in the liver and the resulting changes in cholesterol metabolism. A striking upregulation of SR-BI was observed on vitE depletion of liver tissue in a rat dietary model, indicating that the supply of organs with vitE is a major, and thus far not recognized, factor in the control of SR-BI expression. The vitE effect was also observed with hSR-BI in the human liver-derived tumor cell line HepG2. In addition, evidence is presented that PKC is involved in the signaling pathway. The preparation of affinity-purified rabbit anti-SR-BI polyclonal antibodies (immunization with amino acids 495–509) is described elsewhere (15Kolleck I. Schlame M. Fechner H. Looman A.C. Wissel H. Rüstow B. HDL is the major source of vitamin E for type II pneumocytes.Free Radic. Biol. Med. 1999; 27: 882-890Google Scholar). Poyclonal antibodies to SR-BI with similar properties were purchased from Novus Biologicals (Littleton, CO), and donkey peroxidase-conjugated anti-rabbit IgG was from Pierce (Rockford, IL). Polyclonal anti-PKC antibodies were obtained from Calbiochem (Bad Soden, Germany). Other chemicals were obtained from the following sources, or as indicated in text: protease inhibitors, PKC inhibitors and activators, buffer substances, detergents, and lipids, Sigma (Dreieich, Germany); [1α,2α(n)-3H]cholesterol (47.0 Ci/mmol), Amersham Pharmacia Biotech (Freiburg, Germany); dl-α-tocopherol (Serva, Heidelberg, Germany); cholesterol test kit (Merck, Darmstadt, Germany); cell culture media and fetal calf serum (GIBCO, Karlsruhe, Germany); peroxidase/chemiluminescence detection kit and protease inhibitor tablets (Complete Mini; Boehringer, Mannheim, Germany). Standard pelleted rat diet and vitE-depleted and vitE-supplemented pellets were obtained from Altromin (Lage, Germany), and HepG2 cells (ATCC HB 8065) were obtained from the German Collection of Microorganisms and Cell Culture (Braunschweig, Germany). Male Wistar rats (80–90 g) from a local animal facility received the standard pelleted rat diet ad libitum for 38–40 days (control group). In parallel, a second group was fed the same diet but depleted of vitE (α-tocopherol content not detectable). Some of these rats were subsequently fastened for 24 h followed by feeding a vitE-supplemented diet (400 mg of α-tocopherol per kg) ad libitum for 48 h. HepG2 cells were grown in culture flasks in RPMI medium with 25 mM glucose supplemented with 10% fetal calf serum, streptomycin (50 μg/ml), and 2 mM glutamine, in 5% CO2 at 37°C. The medium was changed every 2–3 days. For modifying the vitE content, about 0.5 × 106 cells were transferred to 10-cm petri dishes. The cells were grown for 1 day in RPMI medium and for 2 days in the lipoprotein-depleted medium Seromed-BMS (Biochrom KG, Berlin, Germany), supplemented with trypsin inhibitor (1 mg/ml) from soy bean, to reach about 80% confluence. The cells were washed three times in BMS medium before human HDL, to a final concentration of 34.7 μg of protein (18 μg total HDL cholesterol) per ml of BMS medium, was added, either in native form (α-tocoperol content, 6.2 μg/mg protein) or enriched with α-tocopherol to a concentration of 19.3 μg/mg protein. The cells were then incubated for another 16 h. HDL enriched in vitE was prepared as described (15Kolleck I. Schlame M. Fechner H. Looman A.C. Wissel H. Rüstow B. HDL is the major source of vitamin E for type II pneumocytes.Free Radic. Biol. Med. 1999; 27: 882-890Google Scholar). Alternatively, the cells were incubated under the same conditions with mixed egg yolk phosphatidylcholine (PC)/α-tocopherol vesicles in two concentrations, 50 μg of α-tocopherol plus 100 μg of PC per ml of medium and 250 μg of α-tocopherol plus 500 μg of PC per ml of medium. The lipids were emulsified by sonication (5 strokes, 3 s each, at 200 W; Labsonic, Braun, Melsungen, Germany) with cooling on ice. Cells were finally washed and scraped from the dishes in ice-cold phosphate-buffered saline (PBS). Human HDL was enriched in vitE (α-tocopherol) or labeled with [3H]cholesterol as outlined in previous articles (15Kolleck I. Schlame M. Fechner H. Looman A.C. Wissel H. Rüstow B. HDL is the major source of vitamin E for type II pneumocytes.Free Radic. Biol. Med. 1999; 27: 882-890Google Scholar, 16Guthmann F. Harrach-Ruprecht B. Looman A.C. Stevens P.A. Robenek H. Rüstow B. Interaction of lipoproteins with type II pneumocytes in vitro: morphological studies, uptake kinetics and secretion rate of cholesterol.Eur. J. Cell Biol. 1997; 74: 197-207Google Scholar). HepG2 cells were grown in 10-cm culture dishes to 80% confluence before they were incubated at 37°C for 1 or 2 h with 10 ml of HDL solution (15 μg of protein per ml) in RPMI medium. The vitE content of enriched HDL was 13.9 μg/mg protein, and the cholesterol-labeled HDL comprised 427 μg of cholesterol per mg protein (4.5 μCi/mg cholesterol). To stop the uptake, the cells were washed twice with PBS. The cells were then scraped from the plates in 2 ml of N-2-hydroxylethylpiperazine-N′-2-ethanesulfonic acid (HEPES)/sucrose buffer (see below) and homogenized by sonication (40 strokes, 1 s each, 70 W). Aliquots were removed for liquid scintillation counting in 4 ml of scintillation cocktail (Wallac Optiphase HiSafe; Fisher Chemicals, Loughborough, UK) and for the determination of vitE. Rats were anesthetized with pentobarbital (30 mg/100 g) and killed by bleeding before the livers were excised. The tissue was sliced and homogenized in ice-cold buffer [0.25 M sucrose, 10 mM HEPES/NaOH (pH 7.5), 2 mM phenylmethylsulfonyl fluoride (PMSF), leupeptin (2.5 μg/ml), aprotinin (5 μg/ml)] by 15 strokes in a glass/Teflon Potter device at 1,400 rpm. The complete membrane fraction was prepared according to Fisher et al. (17Fisher A.B. Dodia C. Chandler A. Kleinzeller A. Transport of choline by plasma membrane vesicles from lung derived epithelial cells.Am. J. Physiol. 1992; 263: C1250-C1257Google Scholar). The membrane pellet was extracted for proteins in Dulbecco's PBS without Ca2+ and Mg2+, comprising 1% (w/v) octyl-β-d-glucopyranoside, 2.5 mM ethylenediaminetetraacetic acid (EDTA), and the protease inhibitors as described above. These extracts were routinely subjected to electrophoresis and quantifi cation of SR-BI. Basically the same results were obtained with the crude liver homogenate, but the quantitative evaluation was difficult because of overloading of gels. Freshly harvested HepG2 cells (about 5 × 106) were collected by centrifugation at 150 g. The cell pellet was resuspended in homogenization buffer [20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM ethyleneglycol-bis(β-aminoethyl ether)-N,N,N,N-tetraacetic acid, 250 mM saccharose, 1 mM dithiothreitol, 1 mM PMSF, aprotinin (10 μg/ml), leupeptin (10 μg/ml), and a Boehringer protease inhibitor tablet/10 ml] according to Gobran and Rooney (18Gobran L.I. Rooney S.A. Surfactant secretagogue activation of protein kinase C in cultured rat type II cells.Am J. Physiol. 1999; 277: L251-L256Google Scholar), and the cells were lysed by sonication (Sonoplus HD60, 2 × 20 s; Bandelin Electronics, Berlin, Germany). The particulate fraction was collected by centrifugation at 100,000 g for 1 h. Proteins in the pellets were solubilized in homogenization buffer comprising 1% (w/v) Triton X-100 for 2 h on ice with occasional vortexing. Nondissolved material was removed by centrifugation at 10,000 g. All steps were carried out at 0–4°C. The supernatants were subjected to electrophoresis for analysis of PKC and SR-BI by immunoblotting. The procedures for separating proteins by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 8% gels, for electroblotting of the bands to nitrocellulose, and for visualization of the PKC and SR-BI bands using rabbit anti-PKC and anti-SR-BI antibodies and peroxidase-conjugated second antibodies have been outlined in previous articles (15Kolleck I. Schlame M. Fechner H. Looman A.C. Wissel H. Rüstow B. HDL is the major source of vitamin E for type II pneumocytes.Free Radic. Biol. Med. 1999; 27: 882-890Google Scholar, 19Witt W. Kolleck I. Rüstow B. Identification of high density lipoprotein-binding proteins, including a glycosyl phosphatidylinositol-anchored membrane dipeptidase, in rat lung and type II pneumocytes.Am. J. Respir. Cell Mol. Biol. 2000; 22: 739-746Google Scholar). The PKC antibodies were used at a dilution of 1:250. The peroxidase/chemiluminescence-produced bands on X-Omat films (Eastman Kodak, Rochester, NY) were quantified by scanning, using densitometers with automatic calibration [Image Master DTS (Pharmacia, Uppsala, Sweden) and GS-710 Imaging Densitometer (Bio-Rad, Hercules, CA)]. The separation of rat plasma lipoprotein fractions for analytical purposes was carried out on agarose gels, using an electrophoresis analyzer (Rapid EP; Helena Laboratories, Sunderland, UK). Plasma (2 μl) was applied to the gels and separated at pH 7.0 according to the recommendations of the manufacturer. Cholesterol in the lipoprotein fractions was determined by scanning after staining by an enzymatic procedure with nitrotetrazolium blue as substrate and cholesterol esterase, cholesterol dehydrogenase, and diaphorase as auxiliary enzymes. The procedures to determine cholesterol and vitE in plasma, liver tissue, HepG2 cells, and rat diets, were carried out as previously described (15Kolleck I. Schlame M. Fechner H. Looman A.C. Wissel H. Rüstow B. HDL is the major source of vitamin E for type II pneumocytes.Free Radic. Biol. Med. 1999; 27: 882-890Google Scholar, 19Witt W. Kolleck I. Rüstow B. Identification of high density lipoprotein-binding proteins, including a glycosyl phosphatidylinositol-anchored membrane dipeptidase, in rat lung and type II pneumocytes.Am. J. Respir. Cell Mol. Biol. 2000; 22: 739-746Google Scholar). Bile acids were measured with a commercial test kit (Merck, Darmstadt, Germany). Northern blot analysis was carried out with an SR-BI single-stranded DNA probe as previously reported (15Kolleck I. Schlame M. Fechner H. Looman A.C. Wissel H. Rüstow B. HDL is the major source of vitamin E for type II pneumocytes.Free Radic. Biol. Med. 1999; 27: 882-890Google Scholar). Radioactive signals were quantified by means of a GS-250 Molecular Imager (Bio-Rad) with a β-actin probe as internal standard. Protein was determined by the bicinchoninic acid procedure, using a test kit (Pierce, Rockford, IL), with bovine serum albumin as standard. Human HDL was prepared from the plasma of healthy, normolipidemic volunteers by KBr density gradient centrifugation and subsequent dialysis against Tris-buffered saline as described elsewhere (15Kolleck I. Schlame M. Fechner H. Looman A.C. Wissel H. Rüstow B. HDL is the major source of vitamin E for type II pneumocytes.Free Radic. Biol. Med. 1999; 27: 882-890Google Scholar). The in vivo effect of vitE on the expression of SR-BI in rat liver was investigated by feeding a vitE-depleted diet over 38–40 days. A control group of rats received the standard rat chow while a third group was treated in the same way as the first group, but the rats were fed a α-tocopherol-enriched chow for 2 days before the animals were killed. The treatment with the depleted food reduced the level of vitE in plasma to 20% (Table 1) and in the liver to 29% (Table 2) of the value in controls, while feeding the enriched diet over 2 days led to a massive accumulation of vitE in the liver (Table 2). The alimentary vitE status did not significantly affect the cholesterol level in plasma and liver (Tables TABLE 1., TABLE 2.) and the cholesterol distribution among the lipoprotein classes (Table 1).TABLE 1.Plasma lipid constituents and the distribution of total cholesterol among lipoproteins as affected by vitE depletion over 38 days and subsequent refeeding for 2 daysTreatment of RatsvitECholesterolBile AcidsHDLLDLLDLμg/mlμg/mlmM% of total lipoprotein-cholesterolControl8.0 ± 1.1460 ± 50ND73 ± 420 ± 47 ± 1vitE depletion1.6 ± 0.3aSignificantly different from controls, P < 0.005.610 ± 1509.3 ± 2.073 ± 318 ± 79 ± 7vitE refeeding19.0 ± 5.0bSignificantly different from controls, P< 0.05.490 ± 303.8 ± 0.881 ± 612 ± 65 ± 3Data represent means ± SD, n = 3. ND, Not detectable.a Significantly different from controls, P < 0.005.b Significantly different from controls, P< 0.05. Open table in a new tab TABLE 2.Effect of dietary vitE on lipid constituents in rat liver tissueTreatment of RatsVitamin ECholesterolBile Acidsμg/mg proteinμmol/mg proteinControl0.101 ± 0.0116.4 ± 0.50.8 ± 0.1vitE depletion0.029 ± 0.006aSignificantly different versus controls, P < 0.005.6.2 ± 0.30.9 ± 0.2vitE refeeding2.163 ± 0.507aSignificantly different versus controls, P < 0.005.7.6 ± 0.50.6 ± 0.2Each value represents the means ± SD for data obtained from three animals.a Significantly different versus controls, P < 0.005. Open table in a new tab Data represent means ± SD, n = 3. ND, Not detectable. Each value represents the means ± SD for data obtained from three animals. In contrast, the vitE depletion caused a strong (11-fold) increase in the expression of the SR-BI protein. The effect was partly reversed by refeeding the vitamin for 2 days, showing that the expression of SR-BI rapidly responded to the vitE content of liver tissue (Fig. 1). The constant level of the SR-BI messenger RNA (Table 3) indicates that the effect of vitE is mediated on the posttranscriptional level.TABLE 3.Effect of dietary vitE regimens on SR-B1 mRNA level in rat liver tissueTreatment of RatsSR-B1 mRNAarbitrary unitsControl diet0.82 ± 0.11vitE depletion0.70 ± 0.06vitE refeeding0.77 ± 0.33Values are expressed as means ± SD (n = 4). Open table in a new tab Values are expressed as means ± SD (n = 4). In our dietary rat model, a significant change in cholesterol in liver tissue and plasma was not observed (Tables TABLE 1., TABLE 2.), but the level of bile acids in the plasma was markedly increased in vitE-depleted rats (Table 1). Obviously, the induction of SR-BI favors cholesterol uptake in the liver, but the capacity of the degradation pathway to bile acids is sufficient to prevent the accumulation in liver tissue. The expression of SR-BI may also be influenced by steroid hormones, for example, by high doses of estrogens in the liver (8Landschulz K.T. Pathak R.K. Rigotti A. Krieger M. Hobbs H.H. Regulation of scavenger receptor, class B, type I, a high density lipoprotein receptor, in liver and steroidogenic tissues of the rat.J. Clin. Invest. 1996; 98: 984-995Google Scholar, 9Fluiter K. van der Westhuijzen D.R. van Berkel T.J.C. In vivo regulation of scavenger receptor BI and the selective uptake of high density lipoprotein cholesteryl esters in rat liver parenchymal and Kupffer cells.J. Biol. Chem. 1998; 273: 8434-8438Google Scholar). To exclude hormone effects, the level of cortisol, testosterone, β-estradiol, and progesterone in plasma was determined. No significant differences were found among vitE-deficient and refed rats as well as controls (results not shown). Furthermore, the low and constant level of the indicator enzymes aspartate and alanine aminotransferase, indicates that the liver tissue was not damaged by vitE depletion (results not shown). The change in vitE level in vivo requires lengthy feeding regimens that may lead to largely unknown adaptation processes. In addition, the various cell types in an organ sample may respond in different ways to regulatory factors (9Fluiter K. van der Westhuijzen D.R. van Berkel T.J.C. In vivo regulation of scavenger receptor BI and the se