Dietary intake of plant sterols stably increases plant sterol levels in the murine brain
2012; Elsevier BV; Volume: 53; Issue: 4 Linguagem: Inglês
10.1194/jlr.m017244
ISSN1539-7262
AutoresTim Vanmierlo, Oliver Weingärtner, Susanne van der Pol, Constanze Husche, Anja Kerksiek, Silvia Friedrichs, Eric J.G. Sijbrands, Harry Steinbusch, Marcus O.W. Grimm, Tobias Hartmann, Ulrich Laufs, Michael Böhm, Helga E. de Vries, Monique Mulder, Dieter Lütjohann,
Tópico(s)Biochemical Analysis and Sensing Techniques
ResumoPlant sterols such as sitosterol and campesterol are frequently administered as cholesterol-lowering supplements in food. Recently, it has been shown in mice that, in contrast to the structurally related cholesterol, circulating plant sterols can enter the brain. We questioned whether the accumulation of plant sterols in murine brain is reversible. After being fed a plant sterol ester-enriched diet for 6 weeks, C57BL/6NCrl mice displayed significantly increased concentrations of plant sterols in serum, liver, and brain by 2- to 3-fold. Blocking intestinal sterol uptake for the next 6 months while feeding the mice with a plant stanol ester-enriched diet resulted in strongly decreased plant sterol levels in serum and liver, without affecting brain plant sterol levels. Relative to plasma concentrations, brain levels of campesterol were higher than sitosterol, suggesting that campesterol traverses the blood-brain barrier more efficiently. In vitro experiments with brain endothelial cell cultures showed that campesterol crossed the blood-brain barrier more efficiently than sitosterol. We conclude that, over a 6-month period, plant sterol accumulation in murine brain is virtually irreversible. Plant sterols such as sitosterol and campesterol are frequently administered as cholesterol-lowering supplements in food. Recently, it has been shown in mice that, in contrast to the structurally related cholesterol, circulating plant sterols can enter the brain. We questioned whether the accumulation of plant sterols in murine brain is reversible. After being fed a plant sterol ester-enriched diet for 6 weeks, C57BL/6NCrl mice displayed significantly increased concentrations of plant sterols in serum, liver, and brain by 2- to 3-fold. Blocking intestinal sterol uptake for the next 6 months while feeding the mice with a plant stanol ester-enriched diet resulted in strongly decreased plant sterol levels in serum and liver, without affecting brain plant sterol levels. Relative to plasma concentrations, brain levels of campesterol were higher than sitosterol, suggesting that campesterol traverses the blood-brain barrier more efficiently. In vitro experiments with brain endothelial cell cultures showed that campesterol crossed the blood-brain barrier more efficiently than sitosterol. We conclude that, over a 6-month period, plant sterol accumulation in murine brain is virtually irreversible. Plant sterols differ structurally from cholesterol by an additional methyl or ethyl group at C24 and/or a double bond at C22 (Δ22). The most prevalent plant sterols are campesterol (methyl group at C24), sitosterol (ethyl group at C24), brassicasterol (methyl group at C24, Δ22), and stigmasterol (ethyl group at C24, Δ22) (1Pollak O.J. Kritchevsky D. Sitosterol.Monogr. Atheroscler. 1981; 10: 1-219PubMed Google Scholar, 2Salen G. Ahrens Jr, E.H. Grundy S.M. Metabolism of beta-sitosterol in man.J. Clin. Invest. 1970; 49: 952-967Crossref PubMed Scopus (386) Google Scholar). In contrast to cholesterol, these sterols are exclusively derived from the diet and cannot be synthesized endogenously in mammals. A high plant sterol intake (2–2.5 g/day) leads to reduced total and LDL-cholesterol (∼12%) in the circulation (3Law M. Plant sterol and stanol margarines and health.BMJ. 2000; 320: 861-864Crossref PubMed Google Scholar, 4Lees A.M. Mok H.Y. Lees R.S. McCluskey M.A. Grundy S.M. 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C57BL/6NCrl male mice were purchased from Charles River Laboratories GmbH (Sulzfeld, Germany). At 12 weeks (time point 1), mice (n = 29) were randomly divided into two groups (Fig. 1). One group was fed a 2% (w/w) plant sterol ester (PSE)-enriched diet (60% sitosterol, 30% campesterol, and 10% stigmasterol; n = 14), and the other group was fed a control diet (containing 0.015% (w/w) plant sterol esters: 65% sitosterol, 30% campesterol, and 5% stigmasterol). Both diets contained 0.0015% (w/w) plant stanol esters (55% sitostanol and 45% campestanol; n = 15). After 6 weeks of diet, six animals per group were euthanized (time point 2). Next, the remaining animals in the group previously fed a PSE-enriched diet (n = 8) were fed a 1.5% (w/w) plant stanol ester (PSA)-enriched diet (85% sitostanol, 15% campestanol). The PSA-enriched diet was administered to inhibit intestinal sterol uptake and to almost completely deplete the serum of plant sterols. The diet of the remaining animals in the control group (n = 9) remained unchanged for 6 months (time point 3; T3). All animals were euthanized at the end of the experiment with an overdose of ketamine (1 g/kg body weight) and xylazine (100 mg/kg/body weight). Liver and brain hemispheres were snap frozen in liquid nitrogen and stored at −80°C before further analysis. Blood was allowed to coagulate at room temperature for 1 h. Serum was obtained by centrifugation at 4°C for 10 min at 200 g. All dietary supplemented sterols and stanols (Raisio Research Laboratories, Finland) were mixed in standard rodent chow (Ssniff Spezialdiäten GmbH, Soest, Germany). Dietary sterol concentrations were verified by GC-MS. d6-Sitosterol/d6-campesterol (55–45%) (Sugaris GmbH, Münster, Germany), d6-cholesterol (99.0%) (CDN isotopes, Pointe-Claire, Quebec, Canada), and epi-coprostanol (99.9%) (Sigma-Aldrich Chemie, Steinheim, Germany) were quantified and tested for purity against 5α-cholestane (1 µg/µl) (Serva Electrophoresis GmbH, Heidelberg, Germany) using gas chromatography-flame ionization detection and GC-MS scan methods. Sterols were dissolved in 100% ethanol to obtain 10 mM stock solutions. Human astrocytoma CCF-STTG1 and human neuroblastoma SHSY5Y cell lines, purchased from the European Collection of Cell Cultures (CACC, Salisbury, UK), were cultured in 10% FCS (EU approved origin; Gibco, Invitrogen GmbH, Karlsruhe, Germany) containing 1:1 DMEM and Ham's F-12 Nutrient mixture (DMEM/F-12) (Gibco, Invitrogen GmbH, Karlsruhe, Germany) including 100 units penicillin and 100 µg streptomycin per milliliter (pen/strep from Gibco, Invitrogen GmbH, Karlsruhe, Germany). Immortalized human adult brain endothelial cells (hCMEC/D3 cells) were obtained from Weksler et al. (24Weksler B.B. Subileau E.A. Perriere N. Charneau P. Holloway K. Leveque M. Tricoire-Leignel H. Nicotra A. Bourdoulous S. Turowski P. et al.Blood-brain barrier-specific properties of a human adult brain endothelial cell line.FASEB J. 2005; 19: 1872-1874Crossref PubMed Scopus (1034) Google Scholar). OLN-93 cells (oligodendroglioma cells derived from primary rat brain glial cultures) were kindly provided by Dr. Richter-Landsberg (25Richter-Landsberg C. Heinrich M. OLN-93: a new permanent oligodendroglia cell line derived from primary rat brain glial cultures.J. Neurosci. Res. 1996; 45: 161-173Crossref PubMed Scopus (198) Google Scholar). Primary astrocytes were isolated from human brain as described (26Van Doorn R. Van Horssen J. Verzijl D. Witte M. Ronken E. Van Het Hof B. Lakeman K. Dijkstra C.D. Van Der Valk P. Reijerkerk A. et al.Sphingosine 1-phosphate receptor 1 and 3 are upregulated in multiple sclerosis lesions.Glia. 2010; 58: 1465-1476Crossref PubMed Scopus (159) Google Scholar) and applied in the experiments between passage 25 and 30. The human brain endothelial cell line (hCMEC/D3) was used as an in vitro BBB model (24Weksler B.B. Subileau E.A. Perriere N. Charneau P. Holloway K. Leveque M. Tricoire-Leignel H. Nicotra A. Bourdoulous S. Turowski P. et al.Blood-brain barrier-specific properties of a human adult brain endothelial cell line.FASEB J. 2005; 19: 1872-1874Crossref PubMed Scopus (1034) Google Scholar). hCMEC/D3 cells display predominant features of BBB endothelial cells, including reduced paracellular permeability. hCMEC/D3 were seeded on collagen-coated Costar Transwell filters (pore size 0.4 µm; Corning Incorporated) in growth medium containing 2.5% FCS and were cultured for 4 days (200 µl medium in the apical compartment, 800 µl in the basolateral compartment) (24Weksler B.B. Subileau E.A. Perriere N. Charneau P. Holloway K. Leveque M. Tricoire-Leignel H. Nicotra A. Bourdoulous S. Turowski P. et al.Blood-brain barrier-specific properties of a human adult brain endothelial cell line.FASEB J. 2005; 19: 1872-1874Crossref PubMed Scopus (1034) Google Scholar). Subsequently, 15 µM d6-sitosterol/d6-campesterol or d6-cholesterol was added to the apical compartment (blood side) of the Transwell filter setup and incubation was continued for 24 h. Cellular sterol uptake (%) was calculated as [(d6-sterols in endothelial cells) + (d6-sterols at brain side)]/[(d6-sterols at blood side) + (d6-sterols at brain side) + (d6-sterols in endothelial cells)] × 100%. Basolateral flux (%) was calculated as [d6-sterols at brain side]/[(d6-sterols at brain side) + (d6-sterols in endothelial cells)] × 100%. D6-sterols present in astrocytes were included in the "brain side" for calculations. Astrocyte-conditioned medium has been shown to enhance maturation of endothelial cells, further strengthening endothelial tight junctions and restricting the permeability of the endothelial monolayer, thus improving the BBB model (27Prat A. Biernacki K. Wosik K. Antel J.P. Glial cell influence on the human blood-brain barrier.Glia. 2001; 36: 145-155Crossref PubMed Scopus (276) Google Scholar). Therefore, the trans-endothelial flow experiment was repeated with primary astrocytes cultured to confluency at the bottom of the basolateral compartment in the endothelial-Transwell setup. D6-sterols were measured after sterol extraction by applying GC-MS analysis. To show that addition of sterols did not influence cellular permeability, permeability was measured as described previously (27Prat A. Biernacki K. Wosik K. Antel J.P. Glial cell influence on the human blood-brain barrier.Glia. 2001; 36: 145-155Crossref PubMed Scopus (276) Google Scholar). In short, after 23 h of culturing and 1 h before harvesting of the cells, FITC-coupled dextran (150 kDa, 1 µg/µl in culture medium; Sigma-Aldrich) was added to the apical compartment as described previously (28Schreibelt G. Kooij G. Reijerkerk A. van Doorn R. Gringhuis S.I. van der Pol S. Weksler B.B. Romero I.A. Couraud P.O. Piontek J. et al.Reactive oxygen species alter brain endothelial tight junction dynamics via RhoA, PI3 kinase, and PKB signaling.FASEB J. 2007; 21: 3666-3676Crossref PubMed Scopus (276) Google Scholar). One hour later, a 10 µl sample was collected from the apical and basolateral compartment for measurement of fluorescence intensity using a FLUOstar Galaxy microplate reader (BMG Labtechnologies; excitation 485 nm and emission 520 nm, at gain 64). The average 1 h flux of FITC-dextran through the endothelial monolayer was calculated, and extremes were excluded from further analyses based on Dixon's principles of exclusion for extreme values (29Dixon W.J. Analyses of extreme values.Ann. Math. Stat. 1959; 21: 488-506Crossref Google Scholar, 30Dixon W.J. Ratios involving extreme values.Ann. Math. Stat. 1959; 22: 68-78Crossref Google Scholar). Viability of the hCMEC/D3 cells, determined by water-soluble tetrazolium assay, was found to be unaltered after incubation with different concentrations of d6-sitosterol/d6-campesterol or d6-cholesterol (0, 5, 10, 15, 20, 25, and 30 µM) after different incubation times (6, 24, and 48 h) (data not shown). CCF-STTG1 (astrocytes), SHSY5Y (neurons), and OLN-93 (oligodendrocytes) cells were cultured in DMEM/F12 medium (Gibco, Invitrogen GmbH, Karlsruhe, Germany) supplemented with 10% FCS until 90% confluency. After washing cells with PBS, cells were incubated with medium containing 0.5% FCS and 15 µM d6-cholesterol or d6-sitosterol/d6-campesterol in the presence of 1 µM T0901317, a synthetic Liver X Receptor-agonist that is known to enhance cholesterol efflux from cells (31Maxwell K.N. Soccio R.E. Duncan E.M. Sehayek E. Breslow J.L. Novel putative SREBP and LXR target genes identified by microarray analysis in liver of cholesterol-fed mice.J. Lipid Res. 2003; 44: 2109-2119Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). After 24 h, the medium was discarded. Cells were washed thrice with PBS. Next, serum-free medium containing 25 µg/ml HDL (isolated as described previously[(32Redgrave T.G. Roberts D.C. West C.E. Separation of plasma lipoproteins by density-gradient ultracentrifugation.Anal. Biochem. 1975; 65: 42-49Crossref PubMed Scopus (870) Google Scholar] from pooled serum of four healthy individuals) as a sterol-acceptor or vehicle control (0.2% BSA in PBS) was added, and cells were incubated 6 h in the presence of 1 µM T0901317. Medium and cells were collected separately and analyzed by GC-MS for d6-cholesterol and d6-sitosterol/d6-campesterol content, respectively. SHSY5Y cells were cultured in T-75 flasks until confluency in medium containing 10% FCS. Cells were then incubated in medium containing 0.5% FCS and 15 µM of d6-sitosterol/d6-campesterol, d6-cholesterol (10 mM stock), or vehicle (100% ethanol) for 24 h. Lipid rafts or DRMs were isolated as described previously (33Yoon I.S. Chen E. Busse T. Repetto E. Lakshmana M.K. Koo E.H. Kang D.E. Low-density lipoprotein receptor-related protein promotes amyloid precursor protein trafficking to lipid rafts in the endocytic pathway.FASEB J. 2007; 21: 2742-2752Crossref PubMed Scopus (63) Google Scholar). In brief, cells were washed twice with ice-cold PBS. Next, cells were scraped in 1.5 ml ice-cold TNE buffer (25 mM Tris, pH 7.4, 150 mM NaCl, and 2 mM EDTA) containing complete protease inhibitor mix (Roche Diagnostics GmbH, Mannheim, Germany) and disrupted by 15 strokes through a 25-G needle. Nuclei were precipitated by low-speed centrifugation (20 min, 1,000 g, 4°C) and discarded. Membranes were precipitated from the supernatant by high-speed centrifugation (90 min, 17,000 g, 4°C). The membrane pellet was dissolved in 1.5 ml 1% CHAPSO (Roche Diagnostics GmbH, Mannheim, Germany) containing complete protease inhibitor mix and incubated on ice for 30 min. The CHAPSO cell extracts were mixed with 2.5 ml TNE buffer containing 72% sucrose to yield a final concentration of 45% (w/v) sucrose and placed at the bottom of 12 ml Beckman ultracentrifuge tubes. TNE buffer (4 ml) containing 35% and 5% sucrose were layered on top of the CHAPSO cell extracts. The samples were spun at 4°C for 19 h at 40,000 rpm in a SW41 TI rotor (Beckman Coulter GmbH, Krefeld, Germany). Fractions (1 ml) were collected from top to bottom to yield a total of 12 fractions. The low buoyant DRM fraction was positioned at the interface between the 35% and 5% sucrose as amorphous white material visible to the naked eye. It was collected in fractions 4 and 5. DRMs (density: 1.09–1.13 g/cm334Nebl T. Pestonjamasp K.N. Leszyk J.D. Crowley J.L. Oh S.W. Luna E.J. Proteomic analysis of a detergent-resistant membrane skeleton from neutrophil plasma membranes.J. Biol. Chem. 2002; 277: 43399-43409Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar]) were confirmed by densitometry using a DMA48 density meter (Anton Paar GmbH, Graz, Austria) and by flotillin-1 expression on Western blotting, and cholesterol content was determined by GC/MS (see Fig. 4E, F). The nonDRM fractions were confirmed to be in fractions 8–10. All separate fractions were subjected to sterol extraction preceding GC-MS analysis. Snap frozen wet mouse brain hemispheres were weighed (mg) before homogenization as described previously (35Parkin E.T. Hussain I. Karran E.H. Turner A.J. Hooper N.M. Characterization of detergent-insoluble complexes containing the familial Alzheimer's disease-associated presenilins.J. Neurochem. 1999; 72: 1534-1543Crossref PubMed Scopus (64) Google Scholar), and 1.5 ml homogenization buffer (150 mM NaCl, 20 mM Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA, and 20% (v/v) glycerol, pH 7.4) was added to each hemisphere (36Parkin E.T. Turner A.J. Hooper N.M. Amyloid precursor protein, although partially detergent-insoluble in mouse cerebral cortex, behaves as an atypical lipid raft protein.Biochem. J. 1999; 344: 23-30Crossref PubMed Scopus (102) Google Scholar). Two cycles of 30 s at 6,500 rpm in the Precellys24 homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) were run, separated by 5 min of cooling on ice. Total homogenate was used to measure protein concentrations (Biorad DC protein assay kit). After homogenization, the samples were diluted 1:4 in homogenization buffer and centrifuged at 5,000 g for 20 min. The pellet was discarded, and the supernatant was centrifuged for 90 min at 17,000 g. The resultant membrane pellet was resuspended in 1.5 ml TNE buffer containing 1% CHAPSO (Roche Diagnostics GmbH, Mannheim, Germany) and incubated on ice for 30 min. DRMs were isolated by density gradient centrifugation and characterized as described above for the confluent cell lines. Wet weights of hemispheres and protein concentrations of brain homogenates were determined to relate sterol content to wet weight or protein concentration. Sterol contents of fractions generated from brain homogenates, total brain homogenates, cell lysates, fractions generated by sucrose gradient ultracentrifugation, total medium, and cell lysates from the efflux experiment were determined by GC-MS as previously described (37Lütjohann D. Brzezinka A. Barth E. Abramowski D. Staufenbiel M. von Bergmann K. Beyreuther K. Multhaup G. Bayer T.A. Profile of cholesterol-related sterols in aged amyloid precursor protein transgenic mouse brain.J. 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Membrane fraction sterol concentrations were compared by two-way ANOVA with fraction and treatment as variable factors (post hoc Bonferroni's Multiple Comparison Test). Significance levels were determined on #,§,* P < 0.05, ##,§§,** P < 0.01, or ###,§§§,*** P < 0.001. C57BL/6NCrl mice were fed a normal chow diet (NC) (n = 15) or were given a 2% PSE-enriched diet (n = 14) for 6 weeks (Fig. 1). In line with several previous reports (13Jansen P.J. Lutjohann D. Abildayeva K. Vanmierlo T. Plosch T. Plat J. von Bergmann K. Groen A.K. Ramaekers F.C.S. Kuipers F. et al.Dietary plant sterols accumulate in the brain.Biochim. Biophys. Acta. 2006; 1761: 445-453Crossref PubMed Scopus (80) Google Scholar, 39Fricke C.B. Schroder M. Poulsen M. von Bergmann K. Wester I. Knudsen I. Mortensen A. Lutjohann D. Increased plant sterol and stanol levels in brain of Watanabe rabbits fed rapeseed oil derived plant sterol or stanol esters.Br. J. Nutr. 2007; 98: 890-899Crossref PubMed Scopus (22) Google Scholar), the PSE-fed group displayed significantly increased sitosterol and campesterol concentrations in serum, liver, and brain in comparison to the NC group (Fig. 2A–C). When related to their serum concentrations, the enrichment of campesterol in the brain was 1.2-fold higher than sitosterol. In the PSE-fed group, feeding the mice a 1.5% PSA-enriched diet for 6 months significantly reduced sitosterol and campesterol concentrations in serum (sitosterol: P < 0.001; campesterol: P < 0.001 [Fig. 2A]) and liver (sitosterol: P < 0.001; campesterol: P < 0.001 [Fig. 2B]). However, brain plant sterol concentrations remained unchanged despite almost complete depletion of circulating plant sterols (Fig. 2C). Brain cholesterol and sitostanol were not affected by the different diets, whereas brain campestanol increased 2-fold after administration of the PSA diet, albeit remaining 300-fold lower in concentration compared with campesterol (data not shown). Also relative to the cerebral cholesterol content, the amount of cerebral plant sterols did not decrease in the PSE-fed group after administration of the PSA diet for 6 months {[campesterol (ng)/cholesterol (µg)]: 2.36 ± 0.18 (PSE) vs. 2.65 ± 0.31 (PSA), n.s. and [sitosterol (ng)/cholesterol (µg)]: 0.58 ± 0.03 (PSE) vs. 0.53 ± 0.11 (PSA), n.s.}. Animals fed a NC diet displayed a modest increase in the campesterol to cholesterol ratio and a comparable sitosterol to cholesterol ratio over 6 months {[campesterol (ng)/cholesterol (µg)]: 1.10 ± 0.05 (NC 6 weeks) vs. 1.30 ± 0.05 (NC 6 months), P < 0.05 and [sitosterol (ng)/cholesterol (µg)]: 0.34 ± 0.01 (NC 6 weeks) vs. 0.31 ± 0.05 (NC 6 months), n.s.}. Together, these results strongly suggest that plant sterols enter the brain depending on the concentration in the serum and accumulate stably in the brain despite the fact that plant sterol concentrations in serum were strongly reduced during the 6 month PSA diet. To examine whether sitosterol, campesterol, and/or cholesterol traverse the endothelial cell layer of the BBB or accumulate within these cells, d6-sitosterol/d6-campesterol or d6-cholesterol were added to the apical side of a polarized confluent human brain endothelial hCMEC/D3 monolayer in an in vitro Transwell® setup (Fig. 3A). After 24 h, the internalization of all sterols was comparable (Fig. 3B). However, the amount of d6-cholesterol in the basolateral compartment (0.97 ± 0.10% of uptake) was approximately five times higher than d6-sitosterol and d6-campesterol (Fig. 3C). Despite a comparable apical uptake of d6-sitosterol and d6-campesterol, d6-campesterol was 1.4-fold higher in the basolateral compartment compared with d6-sitosterol (0.22 ± 0.04% vs. 0.15 ± 0.03%; P = 0.016; Fig. 3B). Using endothelial monolayers cultured in the presence of astrocytes, we found that the absolute amount of d6-sterols was increased about 2-fold in the basolateral compartment (Fig. 3D). One third of the d6-sterols in the lower compartment was incorporated by the astrocytes (d6-cholesterol: 40 ± 7%; d6-campesterol: 34 ± 6%; and d6-sitosterol: 29 ± 6%). These data suggest that p
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