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Originally published In Press as doi:10.1074/jbc.M508868200 on January 18, 2006

J. Biol. Chem., Vol. 281, Issue 11, 7214-7219, March 17, 2006
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Accelerated Lipid Absorption in Mice Overexpressing Intestinal SR-BI*

Florence Bietrix{ddagger}1, Daoguang Yan{ddagger}2, Michel Nauze{ddagger}, Corinne Rolland{ddagger}, Justine Bertrand-Michel{ddagger}, Christine Coméra{ddagger}, Stephane Schaak{ddagger}, Ronald Barbaras{ddagger}, Albert K. Groen§, Bertrand Perret{ddagger}, François Tercé{ddagger}, and Xavier Collet{ddagger}3

From the {ddagger}INSERM U 563, Centre de Physiopathologie de Toulouse Purpan, Département Lipoprotéines et Médiateurs Lipidiques, IFR30 and Université Paul Sabatier, 31024 Toulouse Cedex 3, France and §Academic Medical Center, Liver Center, Meibergdreef 69-71, 1105 BK Amsterdam, The Netherlands

Received for publication, August 11, 2005 , and in revised form, January 17, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Dietary cholesterol absorption contributes to a large part of the circulating cholesterol. However, the mechanism of sterol intestinal uptake is not clearly elucidated. Scavenger receptor class B type I (SR-BI), major component in the control of cholesterol homeostasis, is expressed in the intestine, but its role in this organ remains unclear. We have generated transgenic mice overexpressing SR-BI primarily in the intestine by using the mouse SR-BI gene under the control of intestinal specific "apoC-III enhancer coupled with apoA-IV promoter." We found SR-BI overexpression with respect to the natural protein along the intestine and at the top of the villosities. After a meal containing [14C]cholesterol and [3H]triolein, SR-BI transgenic mice presented a rise in intestinal absorption of both lipids that was not due to a defect in chylomicron clearance nor to a change in the bile flow or the bile acid content. Nevertheless, SR-BI transgenic mice showed a decrease of total cholesterol but an increase of triglyceride content in plasma without any change in the high density lipoprotein apoA-I level. Thus, we described for the first time a functional role in vivo for SR-BI in cholesterol but also in triglyceride intestinal absorption.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Dyslipidemia (e.g. hyperlipidemia or dyslipoproteinemia) plays a key role in the development of obesity, cardiovascular disease, or the metabolic syndrome. Intestinal lipid absorption is an important contributor to the overall balance of lipid metabolism, but regulation at the molecular level remains largely unknown. Intestinal lipid absorption involves the luminal lipolysis of triglycerides to monoglycerides and fatty acids and of cholesterol esters to free cholesterol and fatty acids. Free cholesterol from diet or bile and the products of lipid hydrolysis interact with bile salts to form mixed micelles, which pass through an unstirred water layer covering the enterocyte surface (1). The lipid molecules are then released from micelles and enter the enterocyte where they are assembled into chylomicrons before secretion into the circulation via the lymphatic network. They are finally transported to the liver through the chylomicron-remnant pathway (2).

The mechanisms by which enterocyte lipid absorption occurs are still controversial. In addition to a simple passive diffusion, there is strong evidence for the existence of a protein-facilitated mechanism (3–6). Because of a significant positive correlation between cholesterol absorption and plasma cholesterol levels (7), cholesterol absorption has been the subject of intense research. Different proteins have successively been implicated in intestinal lipid absorption. For instance, CD 36, a multiligand scavenger receptor, has been proposed to take up monoglycerides and fatty acids (8) but could also participate in cholesterol absorption (9, 10). Recently, a newly discovered protein, NPC1L1 (the Niemann-Pick C1 Like 1), has been shown to play a major role in cholesterol absorption (11) because knock-out mice for this protein decreased intestinal cholesterol absorption by 80%. Also aminopeptidase-N has been implicated in cholesterol absorption (12).

Scavenger receptor class B type I (SR-BI)4 is also a likely element of intestinal cholesterol absorption. SR-BI is an 82-kDa membrane protein mostly expressed in liver and steroidogenic tissues. It binds native high density lipoproteins (HDL) (13), LDL, or oxidized LDL and anionic phospholipids and mediates both the selective uptake of HDL cholesteryl ester by the liver and free cholesterol efflux from cells of peripheral tissues (14). SR-BI is also expressed in enterocyte brush-border membranes mainly at the top of intestinal villosities and in the proximal part of intestine where cholesterol absorption mainly occurs (15). However, the importance of SR-BI in intestinal cholesterol absorption is under debate because the disruption of the SR-BI gene in mice does not affect cholesterol absorption (16). However, the process seems to be more complex and might depend on the combined actions of transporter proteins involved in uptake and efflux of cholesterol, which could compensate for the lack of SR-BI in the intestine.

In order to get new data on the role of SR-BI in cholesterol absorption, we have generated transgenic mice overexpressing SR-BI primarily in the intestine. The transgene contains the mouse SR-BI gene under the control of the human intestinal specific apolipoprotein (apo) C-III enhancer coupled with the apoA-IV promoter. This construct is the only promoter known to induce a decreasing expression along the gastro-colic axis and an increasing one from the crypt to the top of the villosity (17). Here we show that SR-BI intestinal overexpression increases intestinal absorption of both cholesterol and triglyceride.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of Transgenic Mice—The murine SR-BI cDNA was obtained by digestion of pRC/CMV vector by restriction enzymes HindIII and XbaI. The apolipoprotein C-III enhancer (-500/-890 pb)-apolipoprotein A-IV promoter (-700 pb) was excised from pUCSHCAT plasmids (18) with the restriction enzymes XbaI and HindIII. The construct containing the human apolipoprotein C-III enhancer-apolipoprotein A-IV promoter to the intestinal specificity and the 1.8-kb cDNA fragment of SR-BI (19) was cloned into XbaI site of the pcDNA 1.1 vector. A linearized fragment of the construct was obtained by digestion of the vector by the restriction enzymes SalI and AvrII and used to generate transgenic mice by standard procedures in a B6D2 background. The mouse genotyping was performed on DNA extracted from tail biopsies by PCR amplification using primers specific for the construct alleles as follows: primer 1, 5'-GCTAGGGCTTTGAATAACTAG-3', and primer 2, 5'-GACCAAGATGTTAGGCAGTACAAT-3'. Founder animals were backcrossed to C57/Bl6 for 12 generations to have a homogeneous background. The mice were housed at the Institute Animal Core Facility in a temperature- and humidity-controlled room with a 12-h light/dark cycle and were fed a basal chow diet. Studies were performed using 8–15-week-old SR-BI transgene versus control littermates negative for the SR-BI transgene and fasted overnight before the initiation of absorption studies. All experiments were performed with the approval of the Ethical Committee for Animal Experiments of the University of Toulouse (MP/04/11/03/05).

Plasma Lipid Analysis—Blood samples were collected from mice, and plasma HDL were isolated by LDL/VLDL precipitation with phosphotungstate/MgCl2 reagent (HDL-cholesterol; Sigma). Total cholesterol, HDL cholesterol, and total triglycerides were determined using commercial enzymatic assays (Randox) adapted for micro methods. LDL/VLDL cholesterol was determined from the difference between total and HDL cholesterol.

Plasma Lipoprotein Analysis—Lipoproteins were separated from equal pool of plasma (400 µl) of transgenic SR-BI and wild type mice by sequential density ultracentrifugation, intermediate density lipoprotein/VLDL (d = 1.006–1.019), LDL (d = 1.019–1.055), and HDL (d = 1.055–1.21). Samples were collected in equal volume, and proteins of each fraction were separated by electrophoresis on 4–16% SDS-polyacrylamide gel and stained with Coomassie Blue.

Membrane Preparation and Protein Analysis—Animals were sacrificed, and different tissues were collected and immediately frozen. Intestine was removed and flushed with cold phosphate-buffered saline (PBS) and then cut into three equal sections (proximal-medium-distal) on ice. Each section was opened, and the mucosa was scraped, immediately frozen in liquid nitrogen, and stored at -80 °C. Tissues were homogenized at 4 °C in 1.5 ml of PBS containing 0.1 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin, 1 mM iodoacetamide, 1 mM phenanthroline (from Sigma) by using a Turax blender and then 20 strokes in a Dounce homogenizer. Lysates were centrifuged at 4 °C, 250 x g, for 5 min to eliminate tissue debris, and then membrane proteins were pelleted at 4 °C, 200,000 x g for 15 min. Total membrane proteins were resuspended in PBS with the protease inhibitor mixture. Proteins were determined using Bradford reagent (Bio-Rad). In some experiments, intestinal epithelial cells were isolated and separated in a villus to crypt gradient according to Weiser (20) and directly used for Western blot analysis. Proteins from the different tissue extracts were separated on 10% SDS-polyacrylamide gel (Bio-Rad) and transferred to nitrocellulose membrane. Immunoblot analysis of SR-BI was performed by chemiluminescence detection (PerkinElmer Life Sciences) using rabbit antipeptide polyclonal antibodies against mouse SR-BI protein (dilution 1:1000, Abcam, antibody 3) and horseradish peroxidase coupled-anti rabbit polyclonal secondary antibody (Sigma).

Immunohistochemistry—In some experiments, small pieces of intestinal sections were collected separately and included in paraffin. Paraffin sections were mounted on silanized slides, and paraffin was removed with different toluene baths. Sections were regenerated by microwave in citrate buffer, pH 6, and biotin and endogenous peroxidase activity were inhibited by the use of Dako kit. Sections were then incubated for 30 min at room temperature with anti-SR-BI primary antibody (1:500, Abcam, anti-SR-BI/BII, antibody 115) diluted in Tris-buffered saline, 0.3% bovine serum albumin. After washing, sections were incubated with pig anti-goat biotinylated secondary antibody (1:100) for 30 min and diluted in Tris-buffered saline with pig serum (1:25). Sections were washed and incubated with streptavidin-peroxidase complex (Dako) for 30 min. The peroxidase activity was revealed using 3'-diaminobenzidine tetrahydrochloride as chromogen (Dako). Samples were observed under a light microscope.

Lipid Tissue Distribution—Liver and intestinal mucosa divided in three parts were homogenized in 1 ml of methanol/water (2:1, v/v), EGTA 5 mM (Sigma). Lipids were extracted according to Bligh and Dyer (21), and molecular species of neutral lipids (cholesterol, cholesteryl esters, diacylglycerols, and triacylglycerols) were quantitated by gas liquid chromatography (22).

Intestinal Absorption of Lipids—To determine dietary lipid absorption, mice were fasted overnight and then fed by gavage with a mixture of 2 µCi of [4-14C]cholesterol/1 µCi of [9,10-3H]triolein (PerkinElmer Life Sciences) and 2 g/liter cholesterol (Sigma) suspended in 100 µl of corn oil. Blood was collected hourly from the tail, and plasma was isolated for liquid scintillation counting. Six hours after gavage, mice were sacrificed. Intestines were removed, flushed with PBS, and cut in three equal parts, and the mucosa was scraped and rapidly frozen in liquid nitrogen. The gallbladder was removed, and the bile was collected for liquid scintillation counting. Livers were harvested and immediately frozen. The different samples were homogenized in methanol/water (2:1, v/v), and tissue distribution of radioactivity was determined by scintillation counting. In a series of experiments, to gain access to the total intestinal lipid uptake, plasma lipoprotein lipase was inhibited by retro-orbital injection of 100 µl of Triton WR1339/saline (1:6, v/v) prior to gavage (23).

Bile Analysis—To determine the excretion rate of cholesterol into bile, animals were anesthetized by intraperitoneal injection of Hypnorm (fentanyl/fluanisone, 1 ml/kg) and diazepam (10 mg/kg). 100 µl of medium chain triglycerides (intralipid; Sigma) containing 2 µCi of [14C]cholesterol were injected in the caudal vein; the common bile duct was ligated close to the duodenum; and then the gallbladder was punctured and its content was removed. After 30 min, newly secreted bile was collected for 90 min by gallbladder cannulation, and the associated 14C radiolabel was determined by scintillation counting. During bile collection, body temperature was stabilized using a humidified incubator.

In other experiments, bile flow and the bile acid rates were analyzed from the bile collected for 60 min via gallbladder cannulation (24). Bile flow was determined gravimetrically assuming a density of 1 g/ml for bile. Bile acids were determined by HPLC. Briefly, 2 µl of bile were diluted with 15 µl of internal standard (norcholic acid 6.7 mM; Steraloids, Wilton, NH) in methanol to precipitate proteins. Samples were centrifuged at 20,000 x g for 10 min at 4 °C, and the supernatant was further diluted in methanol (1:600). Samples (100 µl) were separated by HPLC (DIONEX Sumit) on a LiChrosorb RP-18 analytical column (5 µm particle size, 25 cm x 4.6 mm inner diameter) fitted with a C18 guard column cartridge (1 cm x 4.6 mm; Supelco) and coupled to a light scattering detector (Polymer Laboratory ELS 2100, nitrogen flow 1.2 ml/min, evaporating temperature 90 °C, and nebulizer temperature 70 °C). Separation was achieved at a flow rate of 0.7 ml/min using a ternary solvent system (25) as follows: ammonium acetate, pH 5.2 (30 mM), methanol, acetonitrile, 35:60:5 v/v/v for 25 min move to 24:53:23 in 5 min, then plateau for 20 min.


Figure 1
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  		FIGURE 1.
Tissue distribution of SR-BI in transgenic mice. A, 50 µg of membrane protein from total small intestine were analyzed by Western blotting with anti-SR-BI antibodies. W, wild type mice; T, SR-BI Tg mice. B, 100 µg of protein from homogenates of different tissues from transgenic and wild type mice were analyzed by Western blotting with anti-SR-BI antibodies. C, 50 µg of membrane protein from different intestinal segments (duodenum to colon) of wild type and transgenic mice were analyzed by Western blotting with anti-SR-BI antibodies. P, proximal; M, medium; D, distal part of the small intestine; C, colon. D, enterocytes from wild type and transgenic mice were separated into eight different fractions along the villus-crypt axis according to Weiser (20) and homogenized. 50 µg of membrane proteins were analyzed by Western blotting with anti-SR-BI antibodies.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of the Transgenic Mice Overexpressing SR-BI in the Intestine—We have generated transgenic mice overexpressing SR-BI under the control of the apolipoprotein C-III enhancer coupled to the apolipoprotein A-IV promoter, and we analyzed the presence of SR-BI in tissue extracts (Fig. 1). In control animals, SR-BI protein was classically expressed mostly in the liver among other tissues, whereas transgenic mice displayed a dramatic overexpression of a mature (82 kDa) SR-BI almost totally restricted to the intestine (Fig. 1, A and B). Some overexpression was observed in liver extracts from transgenic mice because of the activation of the promoter also in this organ (17). RNA analysis in the two tissues confirmed a 25-fold increase of SR-BI in the intestine and a 3-fold increase in the liver from transgenic mice when compared with control (not shown). The promoter induced an overexpression of SR-BI in accordance to the distribution of endogenous membrane-bound protein, i.e. decreasing from the duodenum (proximal part of the small intestine) to the colon (Fig. 1C) and increasing from the crypt to the top of the villosities (Fig. 1D) where intestinal absorption is mainly present. We confirmed the overexpression of SR-BI by immunocytochemistry on different intestinal sections (Fig. 2). We also observed the presence of the protein at the top of the villosities and on the luminal side of enterocytes in control mice (Fig. 2, B, E, and H), and overexpression was observed at the same sites in transgenic mice (Fig. 2, C, F, and I).


Figure 2
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  		FIGURE 2.
Intestinal localization of SR-BI. Sections of small intestine from wild type and transgenic mice were incubated with anti-SR-BI antibodies followed by incubation with biotinylated secondary antibodies and with the streptavidin-peroxidase complex. Peroxidase activity was revealed using 3'-diaminobenzidine tetrahydrochloride. A, D, and G, control was obtained with an incubation of wild type sections in only the secondary antibodies; B, E, and H, wild type mice; C, F, and I, SR-BI Tg mice. P, proximal; M, medium; D, colon; V, villosity; C, crypt.

 


Figure 3
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  		FIGURE 3.
Effect of intestinal overexpression of SR-BI on plasma cholesterol and triglycerides levels. Plasma was collected from 8-week-old mice. CT total cholesterol, HDL cholesterol, LDL/VLDL cholesterol, and triglycerides were determined as described under "Experimental Procedures." Wild type, closed bars; SR-BI Tg mice, open bars. Data are presented as mean ± S.E. from 10 mice in each group. Statistical significance between wild type and SR-BI Tg mice was determined by Student's t test. ***, p < 0.01; *, p < 0.05.

 
Plasma Total Cholesterol but Not Triglyceride Is Decreased in Transgenic Mice—Transgenic mice at 8 weeks were further characterized according to plasma lipid and lipoprotein contents (Fig. 3). Most interestingly, we observed a dramatic decrease (2.5-fold) in plasma total cholesterol content that was present both in HDL and LDL/VLDL fractions. By contrast, total triglycerides were mildly but significantly increased, reflecting a differential regulation between cholesterol and triglyceride in the transgenic mice. Analysis of the proteins present in lipoproteins fractions separated by density gradients from the plasma did not show any significant difference in the amount of the apolipoproteins (apoB100, apoB48, apoE, and apoA-I) present in VLDL, LDL, or HDL fractions between control and transgenic mice (Fig. 4). Thus the size of the lipoprotein particles might be different because the ratio of cholesterol to protein is decreased by more than 2 in the transgenic animals. Analysis of lipid content in tissues extracts from intestine or liver (Table 1) did not reveal any difference between control and transgenic mice for free or esterified cholesterol, triglyceride, or diglyceride, indicating that the decrease in cholesterol content observed in plasma was not linked to an accumulation in the major organs. By contrast, free cholesterol was increased by 20% in bile suggesting an increased catabolism of cholesterol in the transgenic mice. However, this was not accompanied by a change in the volume of bile in gallbladder (Table 2). In addition no significant difference in the body mass, the weight of the liver and adipose tissues (inguinal and epididymal representing the peripheral and profound white adipose tissue, respectively), was observed between wild type and SR-BI Tg animals (Table 2). The liver did not show steatosis.


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  		TABLE 1
Lipid tissue distribution in wild type and SR-BI Tg mice Liver and intestinal mucosa divided in three parts were homogenized, and then lipids were extracted and quantitated as described under "Experimental Procedures." Values are expressed as nmol/mg of tissue and shown as means ± S.E., n = 4, for each group. Bile acids were collected and lipids analyzed as described and expressed as total nanomoles in collected bile. Statistical significance between wild type and SR-BI Tg mice was determined by Student's t test.

 


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  		TABLE 2
Characterization of SR-BI Tg mice Wild type and SR-BI Tg mice were analyzed at 10 weeks of age. After weighing, animals were sacrificed, and the different organs were examined and weighed. iWAT and eWAT indicates inguinal and epididymal white adipose tissues. Values are expressed as means ± S.E., n = 5 for each group.

 
Intestinal Lipid Absorption in Transgenic Mice—To assess the importance of SR-BI intestinal overexpression on intestinal lipid absorption, control or transgenic mice were force-fed with a mixture of [14C]cholesterol and 3H-fatty acid-labeled triglycerides in corn oil, and plasma radioactivities were then determined in a time-dependent manner. Most interestingly, we observed a significant increase for both 14C (Fig. 5A) and 3H (Fig. 5B) labels in the plasma up to 4 h following gavage (corresponding to a postprandial status). This indicated a net and similar accelerated absorption of cholesterol and at least the fatty acyl part of triglyceride in response to the overexpression of SR-BI in the intestine. Moreover, when the experiment was performed after the inhibition of lipoprotein lipase (infusion with Triton WR1339), the levels of radiolabels in plasma were much higher, but the same difference between wild type and SR-BI Tg mice was observed for both 14C (Fig. 5C) and 3H (Fig. 5D) labels, indicating that increased absorption rates were not because of a defective chylomicron remnant clearance.

Radioactive cholesterol also significantly accumulated in the intestinal tissue and in the liver (Fig. 6A), indicating a redistribution of the absorbed cholesterol in the organism. Also, radioactivity related to the triglyceride fraction was increased in different organs (Fig. 6B), which was in agreement with the observed increase in plasma total triglycerides (Fig. 3).


Figure 4
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  		FIGURE 4.
Analysis of plasma apolipoprotein content. An aliquot of lipoproteins isolated by sequential density ultracentrifugation from equal amounts (400 µl) of pooled plasma from five mice were loaded onto reducing 4–16% SDS-polyacrylamide gel and stained with Coomassie Blue. Proteins were identified with respect to the migration of molecular weight standards. W, wild type mice; T, SR-BI Tg mice; IDL, intermediate density lipoprotein.

 
Bile content in the 14C label was also increased 2-fold in the transgenic animals (not shown), suggesting a stimulation of the cholesterol secretion that could be attributed to the overexpression of SR-BI in liver. To confirm this hypothesis, labeled cholesterol was directly injected in the caudal vein, and the distribution of cholesterol label was analyzed in bile and plasma (Fig. 7). A net increase of the bile to plasma cholesterol ratio was observed in transgenic mice, indicating that the radioactive cholesterol injected in blood was rapidly excreted into bile, supporting the increased secretion of cholesterol from liver of SR-BI transgenic animals.

However, to evaluate a possible role of the bile acid pool in the increased lipid absorption observed in SR-BI Tg mice, we performed gallbladder cannulation followed by bile collection up to 1 h to access the bile acid pool and bile flow delivered to the intestine. No significant difference was observed in the bile flow rate (4.2 ± 0.7 and 2.5 ± 1.1 µl/min/100 g of body weight) nor in the two major bile acids, taurocholic acid (198 ± 41 versus 134 ± 64 nmol/min/100 g of body weight) and tauromuricholic acid (166 ± 39 and 138 ± 49 nmol/min/100 g of body weight) in wild type (n = 5) and SR-BI Tg (n = 4), respectively.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We generated transgenic mice overexpressing SR-BI mainly in the small intestine using a specific part of the apoC-III/apoA-IV promoter sequence. This promoter also induced a mild overexpression in the liver as described previously (17) using chloramphenicol acetyltransferase gene reporter. Until now, this transgenic mouse strain was the only model of intestinal overexpression of SR-BI respecting the natural expression of the protein, i.e. at the apical site of enterocytes, increasing from crypt to villus and with a cephalo-caudal decrease. Thus, SR-BI is expressed where intestinal cholesterol absorption mainly occurs in SR-BI transgenic mice as in wild type littermates.


Figure 5
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  		FIGURE 5.
Lipid absorption rates in wild type and SR-BI Tg mice. Wild type (squares) and SR-BI Tg (triangles) mice were fed by stomach gavage with a 100-µl test meal containing 2 µCi of [14C]cholesterol, 1 µCi of [3H]triolein, and 2 g/liter cholesterol suspended in corn oil, without (A and B) or after (C and D) retro-orbital injection of 0.1 ml of Triton WR1339 in 0.9% NaCl (1:6, v/v). Blood samples were collected up to 4 h, and radioactivity was measured by liquid scintillation counting. Cholesterol (A and C) and triglycerides (B and D) absorption rates are presented as mean ± S.E. from six mice in each group (A and B) and mean ± S.E. from three mice (C and D). Statistical significance between wild type and SR-BI Tg mice was determined by Student's t test; *, p < 0.05; ***, p < 0.01.

 


Figure 6
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  		FIGURE 6.
Tissue distribution of 14C and 3H radiolabels in wild type and SR-BI Tg mice following gavage. On the same animals as in Fig. 5 (A and B), liver and intestinal mucosa (divided in three parts) were harvested 6 h after gavage and homogenized as described under "Experimental Procedures." Tissue distribution of [14C]cholesterol (A) and 3H radiolabel (B) were determined by scintillation counting. Wild type, closed bars; SR-BI Tg mice, open bars. Data are presented as mean ± S.E. from six mice in each group. Statistical significance between wild type and SR-BI Tg was determined by Student's t test. *, p < 0.05.

 
This study shows for the first time that SR-BI plays a role in vivo in intestinal cholesterol absorption. Indeed, the intestinal cholesterol uptake determined by radioactive acute phase measurement of plasma cholesterol was doubled 3 h after gavage in the transgenic mice. However, this is in contradiction with recent results obtained on knock-out SR-BI mice, which indicated only a minor effect of SR-BI in intestinal cholesterol uptake (16). Nevertheless, numerous studies indicated that intestinal absorption mechanisms involved the combined actions of different transporters that could compensate for the lack of SR-BI in the knock-out model, such as ABCA1 (26), ABCG-5/ABCG-8 (27), NPC1L1 (11, 28), and the aminopeptidase N (29). Moreover, the increased intestinal absorption of lipids could not be attributed to a defect in the clearance of chylomicrons in SR-BI Tg mice, because inhibition of lipoprotein lipase did not change the differences in the absorption rates between wild type and transgenic mice.


Figure 7
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  		FIGURE 7.
Contribution of hepatic SR-BI overexpression to the catabolism of cholesterol. Mice were injected intravenously with 100 µl of intralipid containing 2 µCi of [14C]cholesterol. Bile acids were collected from 30 to 120 min (see "Experimental Procedures"). 14C radiolabel excreted into bile was determined by scintillation counting. Data are presented as the percentage of 14C radiolabel in the bile per 14C radiolabel in plasma ± S.E. from 5 to 6 mice in each group. Wild type, closed bars; SR-BI Tg, open bars. Statistical significance between wild type and SR-BI Tg was determined by Student's t test. *, p < 0.05.

 
Most interestingly, in contrast to the increased intestinal cholesterol uptake, we observed an unexpected decrease of plasma cholesterol content in all lipoprotein fractions (around 50%), which resembles the major phenotype observed in animals overexpressing SR-BI in the liver (30–33). All of these studies indicate a major role for hepatic SR-BI in the clearance of HDL cholesterol as well as in the increased bile cholesterol secretion. From our transgenic model, we show the contribution of SR-BI overexpression both in the intestine and the liver. In the intestine, the observed increase of the radiolabeled cholesterol in plasma following acute phase might reflect an increased secretion of chylomicrons. Indeed, a net increase of tocopherol, a specific marker for chylomicron synthesis (34), is measured in the plasma of our transgenic mice following gavage with tocopherol (35). But the rise of intestinal cholesterol absorption is likely balanced by the moderate overexpression of SR-BI in the liver inducing an increase of cholesterol catabolism shown by the bile cholesterol secretion enhancement (Fig. 7 and Table 1) as well as an increase in catabolism of chylomicrons (36). Nevertheless, the increased intestinal uptake is not because of a simple rise in the bile flow or in the bile acid pool that remained unchanged in the wild type and SR-BI Tg mice.

Also contradictory is the status of apoA-I, the major apolipoprotein of HDL; indeed, liver SR-BI overexpression presented a decrease of cholesterol associated with a decrease of apoA-I in correlation with the expression levels of SR-BI (30–33). Surprisingly, in our transgenic model we did not observe such a diminution in plasma apoA-I that remained constant, but HDL cholesterol was decreased by 2-fold. ApoA-I can be synthesized by the liver through HDL formation or by the intestine associated with chylomicron secretion. Because up-regulation of apoA-I synthesis by the liver has not been observed in the liver SR-BI overexpressing models (31), our results suggest that the intestine could participate in the regulation of apoA-I synthesis through an increase of chylomicron secretion.

Our transgenic model also highlights a discrepancy between cholesterol and TG metabolism. Indeed, by contrast to cholesterol, the plasmatic increase of radioactivity coming from diet TG paralleled a small rise in plasma TG content. It has been already demonstrated that SR-BI was able to uptake TG in vitro using SR-BI-transfected COS cells (37), adrenal Y1 cells, and hepatic HepG2 cells (38). Our data are the first in vivo evidence for a role of SR-BI in intestinal uptake of either TG hydrolysis products or direct TG. In fact, we cannot completely exclude that some TG molecules could be directly taken up through SR-BI, although diet TG are almost completely hydrolyzed in vivo by gastric and pancreatic lipases (39). Our in vivo observations are, however, in contradiction with the recent paper by van Bennekum et al. (10), which showed that SR-BI as well as CD36 in transfected COS cells did not play any role in fatty acid uptake, although it was clearly demonstrated that CD36-deficient enterocytes accumulate lipids, reflecting abnormal lipid processing (8). One hypothesis is that SR-BI may act as a micelle sensor to favor the uptake of micellar lipids in cooperation with other transporters. Interestingly, Hansen et al. (40) showed that SR-BI was mainly localized in the microvillar membrane in a fasting state and evidenced that during absorption of dietary fat, SR-BI is endocytosed from the enterocyte brush border as a mean to regulate SR-BI availability at the surface. This original mechanism may explain contradictory results concerning the role of SR-BI because major works were done in a nonfasting state. Moreover, in macrophages, SR-BI is implicated in bidirectional fluxes of cholesterol and phospholipids (19, 41); thus, SR-BI could also modulate bidirectional fluxes of lipids in function of the lipids status of enterocytes.

All these results demonstrate that SR-BI acts in vivo as a multiligand transporter in the small intestine. SR-BI is responsible for the accelerated uptake of cholesterol and triglyceride hydrolysis products (present work) as well as nonpolar vitamins such as beta-carotene (10) and vitamin E (35). Genetic factors have been proposed to explain the human inter-individual variability in the intestinal absorption of lipids (42). SR-BI may contribute to these observations because a recently described polymorphism at the exon 1 has been implicated in a lower postprandial lipemic response (43). Thus, this receptor may represent a new target to modulate intestinal lipid absorption.


   FOOTNOTES
 
* This work was supported in part by AIC Nutrition INSERM/INRA (ER 66) and ANRS Grant R03038BS and by Genopole Toulouse Midi-Pyrénées/Platforms of Physiopathological Exploration of Genome/Experimental Histopathology Technical Plateau and Lipid Mediator Analysis Technical Facilities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by a doctoral fellowship from the French Atherosclerosis Society and the French Nutrition Society. Back

2 Supported by a postdoctoral fellowship from the French government. Back

3 To whom correspondence should be addressed: INSERM U563, CPTP Bat C, Hôpital Purpan, BP 3028 31024 Toulouse Cedex 3, France. Tel.: 33-5-61-77-94-76; Fax: 33-5-61-77-94-01; E-mail: xcollet{at}toulouse.inserm.fr.

4 The abbreviations used are: SR-BI, scavenger receptor-class B type 1; apo, apolipoprotein; HDL, high density lipoprotein; LDL, low density lipoprotein; TG, triglyceride; VLDL, very low density lipoprotein; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography; Tg, transgenic. Back


   ACKNOWLEDGMENTS
 
We thank Prof. J. Chambaz and Prof. P. Cardot for the gift of the apoCIII-apoA-IV construct and Prof. A. Tall for providing the SR-BI cDNA. We thank Florence Capilla, Veronique Roques, and Caroline Nevoit for technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Dawson, P. A., and Rudel, L. L. (1999) Curr. Opin. Lipidol. 10, 315-320[CrossRef][Medline] [Order article via Infotrieve]
  2. Ros, E. (2000) Atherosclerosis 151, 357-379[CrossRef][Medline] [Order article via Infotrieve]
  3. Compassi, S., Werder, M., Boffelli, D., Weber, F. E., Hauser, H., and Schulthess, G. (1995) Biochemistry 34, 16473-16482[CrossRef][Medline] [Order article via Infotrieve]
  4. Thurnhofer, H., and Hauser, H. (1990) Biochemistry 29, 2142-2148[CrossRef][Medline] [Order article via Infotrieve]
  5. Schulthess, G., Compassi, S., Boffelli, D., Werder, M., Weber, F. E., and Hauser, H. (1996) J. Lipid Res. 37, 2405-2419[Abstract]
  6. Hauser, H., Dyer, J. H., Nandy, A., Vega, M. A., Werder, M., Bieliauskaite, E., Weber, F. E., Compassi, S., Gemperli, A., Boffelli, D., Wehrli, E., Schulthess, G., and Phillips, M. C. (1998) Biochemistry 37, 17843-17850[CrossRef][Medline] [Order article via Infotrieve]
  7. McGill, H. C., Jr. (1979) Am. J. Clin. Nutr. 32, 2664-2702[Free Full Text]
  8. Drover, V. A., Ajmal, M., Nassir, F., Davidson, N. O., Nauli, A. M., Sahoo, D., Tso, P., and Abumrad, N. A. (2005) J. Clin. Investig. 115, 1290-1297[CrossRef][Medline] [Order article via Infotrieve]
  9. Werder, M., Han, C. H., Wehrli, E., Bimmler, D., Schulthess, G., and Hauser, H. (2001) Biochemistry 40, 11643-11650[CrossRef][Medline] [Order article via Infotrieve]
  10. van Bennekum, A., Werder, M., Thuahnai, S. T., Han, C. H., Duong, P., Williams, D. L., Wettstein, P., Schulthess, G., Phillips, M. C., and Hauser, H. (2005) Biochemistry 44, 4517-4525[CrossRef][Medline] [Order article via Infotrieve]
  11. Altmann, S. W., Davis, H. R., Jr., Zhu, L. J., Yao, X., Hoos, L. M., Tetzloff, G., Iyer, S. P., Maguire, M., Golovko, A., Zeng, M., Wang, L., Murgolo, N., and Graziano, M. P. (2004) Science 303, 1201-1204[Abstract/Free Full Text]
  12. Kramer, W., Girbig, F., Corsiero, D., Burger, K., Fahrenholz, F., Jung, C., and Muller, G. (2003) Biochim. Biophys. Acta 1633, 13-26[Medline] [Order article via Infotrieve]
  13. Acton, S. L., Rigotti, A., Landschulz, K., Xu, S., Hobbs, H. H., and Krieger, M. (1996) Science 271, 518-520[Abstract]
  14. Stangl, H., Hyatt, M., and Hobbs, H. H. (1999) J. Biol. Chem. 274, 32692-32698[Abstract/Free Full Text]
  15. Cai, S. F., Kirby, R. J., Howles, P. N., and Hui, D. Y. (2001) J. Lipid Res. 42, 902-909[Abstract/Free Full Text]
  16. Mardones, P., Quinones, V., Amigo, L., Moreno, M., Miquel, J. F., Schwarz, M., Miettinen, H. E., Trigatti, B., Krieger, M., VanPatten, S., Cohen, D. E., and Rigotti, A. (2001) J. Lipid Res. 42, 170-180[Abstract/Free Full Text]
  17. Le Beyec, J., Chauffeton, V., Kan, H. Y., Janvier, P. L., Cywiner-Golenzer, C., Chatelet, F. P., Kalopissis, A. D., Zannis, V., Chambaz, J., Pincon-Raymond, M., and Cardot, P. (1999) J. Biol. Chem. 274, 4954-4961[Abstract/Free Full Text]
  18. Ogami, K., Hadzopoulou-Cladaras, M., Cladaras, C., and Zannis, V. I. (1990) J. Biol. Chem. 265, 9808-9815[Abstract/Free Full Text]
  19. Ji, Y., Jian, B., Wang, N., Sun, Y., Moya, M. L., Phillips, M. C., Rothblat, G. H., Swaney, J. B., and Tall, A. R. (1997) J. Biol. Chem. 272, 20982-20985[Abstract/Free Full Text]
  20. Weiser, M. M. (1973) J. Biol. Chem. 248, 2536-2541[Abstract/Free Full Text]
  21. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917[Medline] [Order article via Infotrieve]
  22. Vieu, C., Jaspard, B., Barbaras, R., Manent, J., Chap, H., Perret, B., and Collet, X. (1996) J. Lipid Res. 37, 1153-1161[Abstract]
  23. Kirby, R. J., Howles, P. N., and Hui, D. Y. (2004) J. Lipid Res. 45, 89-98[Abstract/Free Full Text]
  24. Kruit, J. K., Plosch, T., Havinga, R., Boverhof, R., Groot, P. H., Groen, A. K., and Kuipers, F. (2005) Gastroenterology 128, 147-156[CrossRef][Medline] [Order article via Infotrieve]
  25. Torchia, E. C., Labonte, E. D., and Agellon, L. B. (2001) Anal. Biochem. 298, 293-298[CrossRef][Medline] [Order article via Infotrieve]
  26. Drobnik, W., Lindenthal, B., Lieser, B., Ritter, M., Christiansen Weber, T., Liebisch, G., Giesa, U., Igel, M., Borsukova, H., Buchler, C., Fung-Leung, W. P., Von Bergmann, K., and Schmitz, G. (2001) Gastroenterology 120, 1203-1211[CrossRef][Medline] [Order article via Infotrieve]
  27. Yu, L., Li-Hawkins, J., Hammer, R. E., Berge, K. E., Horton, J. D., Cohen, J. C., and Hobbs, H. H. (2002) J. Clin. Investig. 110, 671-680[CrossRef][Medline] [Order article via Infotrieve]
  28. Davis, H. R., Jr., Zhu, L. J., Hoos, L. M., Tetzloff, G., Maguire, M., Liu, J., Yao, X., Iyer, S. P., Lam, M. H., Lund, E. G., Detmers, P. A., Graziano, M. P., and Altmann, S. W. (2004) J. Biol. Chem. 279, 33586-33592[Abstract/Free Full Text]
  29. Kramer, W., Girbig, F., Corsiero, D., Pfenninger, A., Frick, W., Rhein, M., Wendler, W., Lottspeich, F., Hochleitner, E. O., Orso, E., and Schmitz, G. (0000)
  30. Ueda, Y., Royer, L., Gong, E., Zhang, J., Cooper, P. N., Francone, O., and Rubin, E. M. (1999) J. Biol. Chem. 274, 7165-7171[Abstract/Free Full Text]
  31. Wang, N., Arai, T., Ji, Y., Rinninger, F., and Tall, A. R. (1998) J. Biol. Chem. 273, 32920-32926[Abstract/Free Full Text]
  32. Ji, Y., Wang, N., Ramakrishnan, R., Sehayek, E., Huszar, D., Breslow, J. L., and Tall, A. R. (1999) J. Biol. Chem. 274, 33398-33402[Abstract/Free Full Text]
  33. Kozarsky, K. F., Donahee, M. H., Rigotti, A., Iqbal, S. N., Edelman, E. R., and Krieger, M. (1997) Nature 387, 414-417[CrossRef][Medline] [Order article via Infotrieve]
  34. Traber, M. G., Burton, G. W., Ingold, K. U., and Kayden, H. J. (1990) J. Lipid Res. 31, 675-685[Abstract]
  35. Reboul, E., Klein, A., Bietrix, F., Gleize, B., Malezet-Desmoulins, C., Schneider, M., Margotat, A., Lagrost, L., Collet, X., and Borel, P. (2006) J. Biol. Chem. 281, 4739-4745[Abstract/Free Full Text]
  36. Out, R., Kruijt, J. K., Rensen, P. C., Hildebrand, R. B., de Vos, P., Van Eck, M., and Van Berkel, T. J. (2004) J. Biol. Chem. 279, 18401-18406[Abstract/Free Full Text]
  37. Thuahnai, S. T., Lund-Katz, S., Williams, D. L., and Phillips, M. C. (2001) J. Biol. Chem. 276, 43801-43808[Abstract/Free Full Text]
  38. Greene, D. J., Skeggs, J. W., and Morton, R. E. (2001) J. Biol. Chem. 276, 4804-4811[Abstract/Free Full Text]
  39. Hui, D. Y., and Howles, P. N. (2005) Semin. Cell Dev. Biol. 16, 183-192[CrossRef][Medline] [Order article via Infotrieve]
  40. Hansen, G. H., Niels-Christiansen, L. L., Immerdal, L., and Danielsen, E. M. (2003) Gut 52, 1424-1431[Abstract/Free Full Text]
  41. de La Llera-Moya, M., Connelly, M. A., Drazul, D., Klein, S. M., Favari, E., Yancey, P. G., Williams, D. L., and Rothblat, G. H. (2001) J. Lipid Res. 42, 1969-1978[Abstract/Free Full Text]
  42. Wang, D. Q., Paigen, B., and Carey, M. C. (2001) J. Lipid Res. 42, 1820-1830[Abstract/Free Full Text]
  43. Perez-Martinez, P., Lopez-Miranda, J., Ordovas, J. M., Bellido, C., Marin, C., Gomez, P., Paniagua, J. A., Moreno, J. A., Fuentes, F., and Perez-Jimenez, F. (2004) J. Mol. Endocrinol. 32, 237-245[Abstract]

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