1 Department of Nutrition, Hôpital Sainte-Justine and University of Montreal, Montreal QC H3T 1C5, Canada
2 Group on the Functional Development and Physiopathology of the Digestive Tract, Canadian Institute of Health Research and Department of Cellular Biology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke QC J1H 5N4, Canada
3 Department of Biochemistry, Hôpital Sainte-Justine and University of Montreal, Montreal QC H3T 1C5, Canada
4 Department of Biological Sciences, Université du Québec, Montréal QC H3C 3P8, Canada
5 Departments of Pathology and Cell Biology, Hôpital Sainte-Justine and University of Montreal, Montreal QC H3T 1C5, Canada
* Author for correspondence (e-mail: levye{at}justine.umontreal.ca)
Accepted 5 September 2003
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Summary |
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Key words: SR-BI, Enterocyte, Cholesterol transport, Caveolin-1, Malabsorption, Atherosclerosis
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Introduction |
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The abnormalities of intestinal cholesterol transport can contribute to pathologic processes, including fat malabsorption or atherosclerosis, and emphasize the importance of the small gut in cholesterol homeostasis (Davidson and Magun, 1993; Levy and Roy, 1989
; Levy, 1992
; Thomson and Dietschy, 1981
). Notably, a positive relationship has been established between cholesterol absorption magnitude, on the one hand, and plasma cholesterol levels and coronary disease, on the other hand (Turley et al., 1994
; Kesaniemi and Miettinen, 1987
; McMurry et al., 1985
). Similarly, increased dietary fat and cholesterol intake have been tightly linked to the elevated incidence of other diseases, such as cancer, diabetes and obesity (Hannah and Howard, 1994
; Vessby, 1995
). Even if there is considerable interest in lowering cholesterol absorption efficiency and, thereby, reducing morbidity and mortality, efforts are unfortunately hampered due to our incomplete understanding of the transport mechanisms. In fact, cholesterol uptake, the first critical limiting step in cholesterol absorption, and the mechanisms regulating net cholesterol translocation from the intestinal lumen into the enterocyte remain for the most part unclear.
Several lines of evidence allow investigators to call into question the traditionally proposed passive diffusion model of cholesterol incorporation into absorptive cells (Salen et al., 1970). There is now more and more support for an active transport process mediated by protein(s) in the enterocyte brush border membrane, since (a) cholesterol uptake appears to be a saturable process that is sensitive to protease treatment (Bhattacharyya and Connor, 1974
); (b) potent saponins, such as pamaqueside, are effective in inhibiting cholesterol transport at concentrations below doses required to complex cholesterol in a 1:1 molar ratio, possibly by interfering directly with a putative cholesterol transporter molecule in the intestine (Salisbury et al., 1995
; Morehouse et al., 1999
); (c) the poor intestinal absorption of closely related plant sterol molecules suggests discrimination in absorption (Salen et al., 1970
); (d) patients with sitosterolemia, a rare inherited disorder, lose the ability to discriminate between plant sterols and cholesterol, resulting in the accumulation of sistosterol in plasma and tissues (Bhattacharyya and Connor, 1974
), and (e) the heterogeneity of cholesterol absorption efficiency in animal species and humans remains an enigma (Bhattacharyya and Eggen, 1980
; Kushwaha et al., 1993
; St Clair et al., 1981
; Turley et al., 1997
; Shwarz et al., 1998), which suggests a genetic component to the transport process.
Despite the above observations, the exact identity of the putative cholesterol transporter remains elusive. A few recent studies have proposed the scavenger receptor Class B, Type I (SR-BI) as a candidate protein for the uptake of dietary cholesterol (Cai et al., 2001; Hauser et al., 1998
; Altmann et al., 2002
). SR-BI is a cell surface receptor that binds high-density lipoproteins (HDL) particles and mediates the selective uptake of HDL-cholesteryl ester in many tissues (Acton et al., 1996
). Immunocytochemical analysis of SR-BI indicates that it is expressed most abundantly in the liver and steroidogenic cells of the adrenal gland and ovary. Although emerging information indicated the location of SR- BI in intestinal cells, its physiologic significance is unclear and its implication in cholesterol absorption remains controversial. For example, SR-BI was not identified in the intestinal mucosa (Acton et al., 1996
) and its absence in knockout mice did not affect intestinal cholesterol transport (Mardones et al., 2001
). Although emerging information indicates the location of SR-BI in intestinal epithelial cells, its physiologic significance remains unclear. Additionally, despite information concerning the location in both the yolk sac and placenta, no thorough studies are available on the developing human gastrointestinal tract. Yet our exhaustive work has shown that very early in gestation, the small intestine exhibits the capacity to absorb lipids, elaborates most of the major lipoprotein classes and efficiently exports these lipoproteins from the intestinal cells (Levy et al., 1992
; Levy et al., 1996
; Levy et al., 2001a
).
In the present study, we planned to investigate the ontogeny of SR-BI expression developing human intestine and detect whether regional differences exist among the duodenum, jejunum and ileum. We also examined SR-BI content in the colon that has shown great ability to elaborate lipids and lipoproteins during the gestational period (Levy et al., 1996). We further investigated whether SR-BI concentrates in caveolæ and colocalizes with caveolin-1 as it has been observed in murine adrenocortical cells (Babitt et al., 1997
), in which caveolin-1 regulates SR-BI-mediated selective HDL-cholesteryl ester uptake. Finally, we tested the possibility that SR-BI mediates dietary cholesterol absorption and other lipid classes. To tackle these issues, we used human small gut tissues at different periods of gestation, immunofluorescence, electronic microscopic techniques coupled to protein A-gold, the successful maintenance of human fetal intestine tissues in serum-free organ culture, as well as Caco-2 cell line that was genetically manipulated to inactivate SR-BI gene expression.
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Materials and Methods |
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Indirect immunofluorescence
The preparation and embedding of specimens for cryosectioning were performed as described previously (Levy et al., 2001b) using optimum cutting temperature embedding compounds (Tissue Tek, Miles Laboratories, Elkhat, IN). Cryosections (2-3 µm thick) cut on a Jung Frigocut 2800N Cryostat (Leica Canada, Saint-Laurent, Québec) were fixed on a glass slide with acetone-chloroform (1:1) for 5 minutes at 4°C, then blocked 30 minutes with fish gelatin 0.1% in phosphate buffer containing 0.8% Bovine Serum Albumin (BSA) at room temperature. The staining procedure using antibodies and fluorescence was performed at room temperature in humid chambers. Sections were incubated for 60 minutes with polyclonal antibodies (Novus Biologicals, Littleton, CO): scavenger receptor BI (SR-BI) 1:500 and caveolin-1:300 diluted in PBS. Fluorescein isothiocyanate-conjugated sheep anti-rabbit IgG (Chemicon International, Temecula, CA) was used as secondary antibody at the working dilution of 1:75 and added for 45 minutes. After extensive washing with PBS, sections were then contrasted with 0.01% Evans blue in PBS, mounted in glycerol-PBS (9:1) containing 1% paraphenylenediamine and viewed with a Reichert Polyvar Microscope equipped for epifluorescence (Leica Canada, Saint-Laurent, Québec). Finally, the primary antibodies were omitted or replaced by non-immune rabbit serum at 1:700 dilution and all the control experiments confirmed the specificity of the results.
Tissue preparation for electron microscopy
Intestinal specimens were fixed by immersion in 1% glutaraldehyde, 0.1 M phosphate-buffered saline (pH 7.4) for 2 hours at 4°C and embedded in Lowicryl K4M at -20°C according to our previously described procedures (Bendayan, 1984). Tissue blocks were examined by light microscopy to select well-oriented villous tips. Thin sections (60-80 nm) of the different tissue blocks were mounted on nickel grids with a carbon-coated Parlodion film and processed for immunocytochemistry.
Immunocytochemical labelling
Protein A-gold immunocytochemical techniques were employed to detect the presence of SR-BI and caveolin-1 in intestinal tissue as we have described previously (Levy et al., 2002). Briefly, the tissue sections were washed initially in distilled water, incubated for 5 minutes on a drop of PBS containing 1% ovalbumin, and transferred subsequently to a drop of the PBS-diluted antibody (see below). After incubation (90 min) at room temperature, the grids were rinsed with PBS to remove unbound antibodies. They were transferred to the PBS-ovalbumin (3 minutes) and incubated on a drop of protein A-gold (pH 7.2) for 30 minutes at room temperature. The tissue sections were then thoroughly washed with PBS, rinsed with distilled water and dried. Sections were stained with uranyl acetate and lead citrate before examination with a Philips 410 electron microscope. Polyclonal antibodies were used at various dilutions (SR-BI 1/100 and caveolin-1 1/10, 1/50, 1/100, 1/1000) in combination with protein A-gold complexes, which were prepared using 10 or 5 nm gold particles according to our established techniques (Levy et al., 2002
). Control experiments were performed to assess the specificity of the results. Excess purified SR-BI (tenfold) was added to the antibody solution. Incubation with this solution was followed by the protein A-gold complex. Pre-immune rabbit serum (diluted 1:10) was used on tissue sections before incubation with protein A-gold complex. Incubations were also performed with the protein A-gold complex alone, omitting the antibody step to test for non-specific adsorption of the protein A-gold complex to tissue sections.
Double-labeling technique
To simultaneously reveal the existence of SR-BI and caveolin-1 within the cellular compartments, the double-labeling technique was applied. The tissue sections were labeled concomitantly for SR-BI and caveolin-1. The two-phase labeling technique (Bendayan, 1982; Bendayan, 1995
) was applied to avoid any cross-reaction between reagents. The small protein A-gold complex (5 nm) was used for the first labeling protocol, and the larger (10 nm) protein A-gold complex was used for the second. This protocol allows for the simultaneous visualization of two antigens (SR-BI and caveolin-1) in the same tissue section. Anti-scavenger BI and caveolin-1 polyclonal antibodies were from Novus Biologicals. They were purified on a sepharose column and non-immune rabbit IgG was utilized as a negative control.
Cell culture
Caco-2 cells (American Type Culture Collection, Rockville, MD) were grown at 37°C with 5% CO2 in MEM (Gibco-BRL, Grand Island, NY) containing 1% penicillin/streptomycin and 1% MEM nonessential amino acids (GIBCO BRL) and supplemented with 10% decomplemented fetal bovine serum (FBS; Flow, McLean, VA). Caco-2 cells (passages 30-40) were maintained in T-75 cm2 flasks (Corning Glass Works, Corning, NY). Cultures were split (1:6) when they reached 70-90% confluence, using 0.05% trypsin-0.5 mM EDTA (GIBCO BRL). For individual experiments, cells were plated at a density of 1x106 cells/well on 24.5 mm polycarbonate transwell filter inserts with 0.4 µm pores (Costar, Cambridge, MA) in MEM (as described above) supplemented with 5% FBS. The inserts were placed into six-well culture plates and cultured for 20 days, a period at which the Caco-2 cells are highly differentiated and appropriate for lipid metabolism (Marcil et al., 2002; Courtois et al., 2000
).
Preparation of stable transformants expressing various levels of Apo SR-BI
To obtain Caco-2 cells deficient in SR-BI expression, two vectors were created, one pZeoSV containing the full human SR-BI cDNA and another containing a cDNA, inserted in the antisense orientation. The two different constructs and the expression vector without insert were separately transfected to Caco-2 cells using SuperfectTM Transfection Reagent (Quiagen) after a 72 h incubation period, Zeocine-resistant cell lines were selected and isolated from the pools with cloning cylinders and then propagated to study cells in appearance and in growth rate.
SR-BI neutralization by antibody
SR-BI antibody was obtained by Novus. A titration experiment was performed to determine the optimal antibody concentration to inhibit SR-BI activity using Caco-2 cells expressing SR-BI. It shows that a 1:1000 dilution of immune serum was sufficient to maximally reduce cholesterol transport. This concentration equaled 10-20 µg/ml protein A-purified IgG.
Western blots
To assess the presence of SR-BI and caveolin-1 and evaluate their mass, intestinal tissue was homogenized and adequately prepared for western blotting as described previously (Levy et al., 2001a). Proteins were denatured in sample buffer containing SDS and ß-mercaptoethanol, separated on a 4-20% gradient SDS/PAGE, and electroblotted onto nitrocellulose membranes. Nonspecific binding sites of the membranes were blocked using defeated milk proteins followed by the addition of primary antibodies directed against SR-BI and caveolin-1. The relative amount of primary antibody was detected with species-specific horseradish peroxidase-conjugated secondary antibody. Blots were developed and the mass of SR-BI and caveolin-1 was quantitated using an HP Scanjet scanner equipped with a transparency adapter and software.
[14C]-cholesterol absorption
To study cholesterol uptake, 10 µCi [14C]-cholesterol, 10 µCi [14C]-cholesterol ester or 10 µCi phosphatidylcholine was added as a mixed bile salt micelle (6.6 mmol/L sodium taurocholate, 1 mmol/L oleic acid, 0.5 mmol/L monoolein, 0.1 mmol/L cholesterol and 0.6 mmol/L phosphatidylcholine). Caco-2 cells were incubated at 37°C for 8-24 hours.
Statistical analysis
Data from the experiments were analyzed by using a Student's t-test. Reported values are expressed as mean ± s.e. Statistical significance was accepted at P<0.05.
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Results and Discussion |
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To establish the expression of SR-BI in the different regions of the gut and to determine their distribution along the crypt-villus axis, indirect immunofluorescence was carried out in developing as well as in adult human intestinal tissues. Figs 1, 2, 3, 4 illustrate the presence and localization of SR-BI protein along the crypt-villus axis in the human fetal and adult small and large intestine. Overall, this first series of studies supports SR-BI expression in the developing gut. Immunofluorescent staining was detected as early as the week 14 of gestation in both the small and large intestine (Figs 1 and 2) and its pattern of distribution was similar to the apparent profile observed at 20 weeks of gestation (Fig. 3) and in the adult condition (Fig. 4). Immune fluorescence staining was noted in the columnar epithelial cells of the small intestinal segments, i.e. duodenum, jejunum and ileum. Cytoplasmic immunofluorescence staining was visualized in most of the absorptive cells located in the crypt-villus axis, but also in the microvascular endothelial cells of the lamina propria (Fig. 1). However, only in few experiments, more immunofluorescence was noticeable in the columnar epithelial cells of the villi with a lower intensity in crypt cells. In the colon mucosa, all the epithelial cells lining the crypt and the surface area of the mucosa expressed SR-BI protein, which was mainly localized in the apical cytoplasm of the colonocytes. Taking into account the qualitative nature of this technique, it was not possible to speculate on quantitative aspects relative to SR-BI signals in the different human gut segments.
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Detection of SR-BI by immunoelectron microscopy approach
In an effort to better understand the cellular localization of SR-BI, Protein A-gold immunocytochemical techniques were applied on thin sections incubated with specific antibodies to disclose SR-BI in human fetal intestine. Electron microscopic immunocytochemical studies revealed significant immunogold labelling in the luminal region of enterocytes, particularly associated with the apical plasma membrane lining the microvilli (Fig. 5A,B,C). The labelling of SR-BI by gold particles was also present in endosomal invaginations and vesicles. Within the cell, the labelling, although of lower intensity, was present in the rough endoplasmic reticulum, the Golgi apparatus and the basolateral membrane (Fig. 5D). Under the control conditions tested, labelling was markedly reduced or eliminated, demonstrating its specificity (Data not shown).
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Immunoblotting evaluation of SR-BI ontogeny and distribution along the intestine
Experiments were conducted to study the distribution of SR-BI along the intestine. Samples of the different regions of the intestine containing equal quantities of protein were electrophoresed on an SDS-polyacrylamide gel (Fig. 6). An immunoblot of these samples showed immunoreactive bands corresponding to SR-BI. Densitometric estimation of the SR-BI visualized on the immunoblot showed that the jejunum, ileum, proximal colon and distal colon contained 76.2%, 57.1%, 16.7% and 9.5% of SR-BI amounts measured in the duodenum (Fig. 6). We also explored the maturation aspect of SR-BI in each region of the human small and large intestine using equal protein amount within each intestinal segment only. An increase in SR-BI was noted only in the duodenal tissue as a function of fetal age (Fig. 7). The duodenum exhibited a significant progressive rise in SR-BI protein content: 35.5% at the end of week 15 and 59.3% at the end of the week 17 compared with the value (100%) noted at the end of week 20. No marked ontogenic differences in SR-BI protein expression were recorded in the other intestinal segments except for the distal colon where a decline was apparent at the end of week 17. On the whole, SR-BI content appeared predominant in the duodenum, whereas it seemed most impoverished in the colon, thus indicating an increasing distal-to-proximal gradient. This is in accordance with our previous findings, which documented the colon's reduced capacity of the colon to synthesize lipids and assemble lipoproteins as compared with the small intestine during development (Levy et al., 1996; Loirdighi et al., 1997
). Additionally, our data clearly demonstrated that only the duodenum is endowed with a developmental SR-BI expression profile, whereas SR-BI protein expression was stable throughout the other gestational ages considered in our studies.
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Co-expression of scavenger receptor-BI and caveolin-1
In this report we have addressed three additional questions: Do intestinal fetal tissues express caveolin-1 and contain caveolae? Does SR-BI concentrate in caveolæ and colocalize with caveolin-1? Is this co-localization associated with enhanced cholesterol uptake? Babitt et al., demonstrated that SR-BI colocalized with caveolin-1, a constituent of caveolae in Chinese hamster ovary cells or in murine adrenocortical Y1-BS1 cells (Babitt et al., 1997). Other studies on macrophage cell lines documented an association between SR-BI and caveolin-1 (Matveev et al., 1999
). The localization of SR-BI in caveolin-1 enriched membrane domains on the cell surface may have implications for the mechanism of SR-BI-mediated selective lipid uptake by cells. Numerous investigators have suggested that caveolae may serve as sites of cholesterol efflux from cells (Fielding and Fielding, 1995
) where caveolin-1 appears to be a cholesterol binding protein (Murata et al., 1995
; Li et al., 1996
) involved in intracellular cholesterol transport (Smart et al., 1994
; Smart et al., 1996
). The concentration of SR-BI in caveolae may bring the receptor into proximity with caveolin-1 or other resident proteins that may be critical for direct lipid transfer into the cell. Furthermore, the ability of caveolae to internalize molecules may be essential for efficient lipid transport. Here we have used immunochemical and electrophoretic methods to study the subcellular localization of caveolin-1. The assessment of intestinal tissues by immunofluorescent techniques at various ages of gestation and in the postnatal period could not reveal caveolin-1 in the enterocyte (Fig. 8). Staining either in the fetal or adult small intestine and colon did not reveal any signal of caveolin-1 at the epithelial level. However, intense staining was detected in the sub-epithelial layer, i.e. the endothelial cells of the blood vessels and the smooth-muscle cells of the muscula layer. Because of the potential association or physical interaction of SR-BI and caveolin-1, we reasoned that the immunoprecipitation of one of the two proteins would drag the other. For this purpose, intestinal epithelial cells (detached from the duodenum) or Caco-2 cells were homogenized in a nondenaturing buffer and immunoprecipitation was also carried out with an anti-SR-BI antibody under nondenaturing conditions. The immunoprecipitates were run onto SDS-PAGE and transferred to a nitrocellulose membrane (Fig. 9). Immunoblotting the membrane with anti-SR-BI (a-SR-BI) and anti-caveolin-1 (a-cav-1) antibodies confirmed the presence of SR-BI, but could not detect any traces of caveolin-1. Thus, the immunoprecipitation procedure refutes an interaction between SR-BI and caveolin-1. Furthermore, combined high-resolution immunoelectron microscopy with specific polyclonal antibodies could not disclose the presence of caveolin-1 (data not shown). Repeated manipulations and double-labelling techniques revealed morphologically identifiable plasma membrane invaginations and endosomes in the apical side of the enterocyte, which contained SR-BI without caveolin-1 (data not shown). Altogether, our findings indicate that SR-BI localizes in microvilli, plasma membrane invaginations and endosomes, which do not exhibit caveolin-1. If our observations garner little supporting evidence for the cooperation between caveolin-1 and SR-BI, they at least suggest that endosomes may function as signalling platforms capable of delivering endocytosed molecules to specific organelles. However, further work is needed to establish the precise molecular mechanisms that couple SR-BI-mediated ligand uptake and intracellular destination in the enterocyte. Interestingly, a recent investigation has shown that SR-BI-stimulated cholesterol efflux or selective uptake is not affected by caveolin-1 expression in Fisher rat thyroid cells or human embryonic kidney cells (Wang et al., 2003
). Instead, SR-BI-mediated cholesterol uptake may occur selectively in rats, without a specific requirement for caveolin-1 or invaginated caveolae (Briand et al., 2003
).
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Role of SR-BI in cholesterol absorption
As stated above, the molecular mechanisms of cholesterol absorption in the intestine are poorly understood. With the goal of defining whether SR-BI plays a role in this process, two strategies were devised. We first used the organ culture system, allowing the morphological and physiological preservation of the cultured fetal tissues to study lipid transport and lipoprotein assembly (Levy et al., 1992; Levy et al., 2001a
; Loirdighi et al., 1997
). The incubation of intestinal explants with SR-BI antibody partially inhibited (34%) the uptake of micellar free [14C]-cholesterol (results not shown). The same amount of antibody had no effect on the uptake of labelled cholesteryl esters or phospholipids. Of note were also the preferential uptake of micellar cholesterol (compared with cholesterol bound to albumin), the efficient cholesterol absorption with intact micelles (without missing components) and the saturated cholesterol transport process as a function of cholesterol concentration (data not shown). Secondly, we utilized the Caco-2 cell line, an excellent in vitro model for the investigation of intestinal lipoprotein metabolism (Levy et al., 1995
). In order to determine the importance of SR-BI in intestinal cholesterol transport, Caco-2 cells were transfected with a constitutive expression vector (pZeoSV) containing the human SR-BI cDNA inserted in an antisense orientation for SR-BI gene inactivation. Control Caco-2 cells were transfected with the vector pZeoSV without insert. Cellular clones were obtained from each pool of transformants, and their level of SR-BI was determined by immunoblotting. As observed in Fig. 10, stable transformants generated by this constitutive antisense RNA technology were obtained and they expressed lower levels of SR-BI than control Caco-2 cells. Densitometric analysis of these clones, taking the control Caco-2 cells (infected with vector without insert) SR-BI level as 100%, revealed that the corresponding experimental cells expressed 40, 60 and 80% the control level. This genetic manipulation did not significantly modify cell growth and differentiation, as observed in Fig. 11, by the incorporation of [3H]-thymidine, sucrase activity and monolayer resistance. We confirmed the reduction of endogenous SR-BI protein expression in Caco-2 intestinal cells with EM immunogold microscopy. Indeed, the use of antisense led to an evident decrease in protein A-gold labelling over apical plasma membrane as well as over the basolateral membrane in transfected (Fig. 12B) compared with control (Fig. 12A) Caco-2 cells. The quantitative evaluation of gold particles revealed less labeling density in transfected Caco-2 cells (Table 1). Our next aim was to define whether the changes of SR-BI levels in Caco-2 cells would affect their ability to capture labelled cholesterol. As illustrated in Fig. 13A, the disruption of SR-BI in Caco-2 cells resulted in decreased [14C]-free cholesterol uptake. Under identical conditions, no changes were noted in phospholipid and cholesteryl ester uptake (Fig. 13A,B). Our findings are consistent with in vitro studies that showed that SR-BI can facilitate the cellular uptake of nonlipoprotein unesterified cholesterol in a hamster ovary cell line (Bruneau et al., 2003
). However, our observations are divergent from the results reported by Mardones et al. (Mardones et al., 2001
). These investigators addressed the role of SR-BI in SR-BI knockout mice and concluded that SR-BI is not essential for intestinal cholesterol absorption. Nevertheless, they suggested that in vivo SR-BI-independent mechanisms must have been able to efficiently compensate for the loss of SR-BI expression in the mutant mice subjected to a cholesterol-enriched diet. Furthermore, whereas there was a tight coupling between faulty free cholesterol transport and SR-BI disruption, the SR-BI receptor level was not linked to a variation in the uptake of other lipid classes. As SR-BI antisense expression specifically reduced endogenous SR-BI protein expression in Caco-2 cells, and this decrease correlated with a significant decline in protein A-gold labelling and cholesterol uptake, we propose that SR-BI functionally mediates alimentary cholesterol uptake in the intestine.
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Over the last decade, our knowledge of the biosynthetic events essential for the formation and secretion of lipoproteins has significantly increased in the human gut in general and in the developing gut in particular. It has been established that the fetal small intestine exhibits very early the capacity to absorb lipids, elaborate most of the major lipoprotein classes and efficiently export these lipid vehicles from the intestinal cells. The present work allows us to demonstrate an additional protein component in the apical membrane of the human fetal and adult SR-BI intestine.
SR-BI was detected in all segments of the small and large intestine. However, Western blot analyses revealed that SR-BI is preponderantly expressed in the duodenum where cholesterol absorption is optimal. Immunofluorescence experiments showed that SR-BI is found not only in the villus, but also in the crypt cells. Based on gene expression, it seems that SR-BI mRNA is mostly expressed in differentiated Caco-2 cells. The observed relation between SR-BI expression and cholesterol uptake supports the hypothesis that SR-BI transports alimentary free cholesterol, and not phospholipids and cholesteryl esters. It seems, therefore, that carboxyl ester lipase, a lipolytic enzyme synthesized in the acinar cells of the pancreas (Bruneau et al., 2003), is needed for hydrolysis of cholesteryl ester, thereby facilitating free cholesterol transfer to SR-BI. This is particularly important given the absence of caveolin-1 in our studies, which normally binds to cholesterol and is involved in intracellular cholesterol trafficking.
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Acknowledgments |
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References |
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