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Originally published In Press as doi:10.1074/jbc.M703330200 on May 15, 2007

J. Biol. Chem., Vol. 282, Issue 27, 19493-19501, July 6, 2007
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CD36 Is Important for Fatty Acid and Cholesterol Uptake by the Proximal but Not Distal Intestine*

Fatiha Nassir{ddagger}1, Brody Wilson{ddagger}, Xianlin Han§, Richard W. Gross§, and Nada A. Abumrad{ddagger}2

From the Department of Medicine, Divisions of {ddagger}Nutritional Science and §Bioorganic Chemistry and Molecular Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, April 20, 2007 , and in revised form, May 14, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
CD36, a membrane protein that facilitates fatty acid uptake, is highly expressed in the intestine on the luminal surface of enterocytes. Cd36 null (Cd36–/–) mice exhibit impaired chylomicron secretion but no overall lipid absorption defect. Because chylomicron production is most efficient proximally we examined whether CD36 function is important for proximal lipid absorption. CD36 levels followed a steep decreasing gradient along three equal-length, proximal to distal intestinal segments (S1–S3). Enterocytes isolated from the small intestines of Cd36–/– mice, when compared with wild type counterparts, exhibited reduced uptake of fatty acid (50%) and cholesterol (60%) in S1. The high affinity fatty acid uptake component was missing in Cd36–/– cells. Fatty acid incorporation into triglyceride and triglyceride secretion were also reduced in Cd36–/– S1 enterocytes. In vivo, proximal absorption was monitored using mass spectrometry from oleic acid enrichment of S1 lipids, 90 min (active absorption) and 5 h (steady state) after intragastric olive oil (70% triolein). Oleate enrichment was 50% reduced at 90 min in Cd36–/– tissue consistent with defective uptake whereas no differences were measured at 5 h. In Cd36–/– S1, mRNA for L-fabp, Dgat1, and apoA-IV was reduced. Protein levels for FATP4, SR-BI, and NPC1L1 were similar, whereas those for apoB48 and apoA-IV were significantly lower. A large increase in NPC1L1 was observed in Cd36–/– S2 and S3. The findings support the role of CD36 in proximal absorption of dietary fatty acid and cholesterol for optimal chylomicron formation, whereas CD36-independent mechanisms predominate in distal segments.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
CD36 or fatty acid translocase (FAT)3 is an 88-kDa transmembrane protein with broad specificity. Its ligands include long-chain fatty acids, native and oxidized lipoproteins, thrombospondin-1, collagen, amyloid beta, and malaria-infected erythrocytes, recently reviewed in Ref. 1. CD36 has been shown to bind long-chain fatty acids (2, 3) and to facilitate their transfer into the cell (4, 5). Deficiency or overexpression of the protein is associated with alterations in uptake and metabolism of long-chain fatty acids in rodents (4, 6, 7). In humans, Cd36 deficiency (8) and polymorphisms in the Cd36 gene (9) are associated with abnormalities in FA clearance (10, 11), insulin responsiveness (11, 12), and lipoprotein metabolism (13, 14). As a result CD36 has been implicated in the etiology of diabetes and atherosclerosis (14, 15).

Consistent with its role in FA uptake CD36 is very abundant in the heart, skeletal muscle, adipose tissue (16), and the capillary endothelium (17). The protein is also highly expressed in the small intestine (16, 18, 19) and localizes to the apical membrane of villi enterocytes (19). Expression levels and localization strongly suggest a function in lipid absorption but this could not be documented in previous studies by us and others (20–22). Administration of a lipid load to Cd36-deficient mice did not identify alterations in the blood appearance of intestinally derived triglycerides (20, 21). Also, using the sucrose polybehenate method (23), which evaluates overall lipid absorption from the ratio of fecal fat to a non-absorbable marker, no significant differences were observed (22). These findings contrasted with the demonstration, using the lymph fistula mouse model of 50% reductions in lipid output into the lymph of Cd36 null mice (20, 22). In addition, more of the infused lipid was retained in the intestinal mucosa and the lipoprotein particles secreted into the lymph were 35% smaller (22).

So previous in vivo findings implicated CD36 in chylomicron formation and secretion but largely ruled out its contribution to intestinal FA uptake (20–22). In this study, we re-examined the role of intestinal CD36 in lipid uptake based on the hypothesis that it may have a primary role in proximal fatty acid absorption for chylomicron formation, whereas other mechanisms would play the major role in more distal parts of the intestine.

We determined that CD36 expression measured in three equal-length segments (S1–S3, proximal to distal) was highest in S1 and lowest in S3. To relate lipid uptake to the decreasing expression gradient we isolated enterocytes from the S1–S3 intestinal segments of WT versus Cd36 null mice and examined lipid uptake and secretion. Second, using mass spectrometry, we measured in vivo, oleic acid enrichment of proximal mucosal lipids following oral administration of triolein-rich olive oil. Our findings clearly document a defect of FA and cholesterol uptake in Cd36-deficient proximal enterocytes. This is associated with significant alterations in expression of various genes involved in lipid synthesis and chylomicron production.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—[3H]Oleic acid, [3H]glucose, and deoxy-D-glucose, 2-[1,2-3H], were from American Radiolabeled Chemicals and [14C]cholesterol from PerkinElmer. Oleic acid (water soluble), triolein, cholesterol, egg phosphatidylcholine, and sodium taurocholate were from Sigma. Ficoll-Paque was from Amersham Biosciences. Silica Gel 60 plates were from Fisher Scientific. TRIzol® and SuperScript II Reverse Transcriptase were from Invitrogen, SYBR Green Master Mix was from Applied Biosystems. Monoclonal anti-CD36 was from Cascade BioScience (Winchester, MA), anti-tubulin and anti-ran were from Santa Cruz Biotechnology, Inc. Anti-SR-BI and anti-NPC1L1 antibodies were purchased from Novus Biologicals (Littleton, CO). ApoA-IV and apoB antibodies were a gift from Dr. N. O. Davidson (24, 25).

Animals—Cd36 null mice on the C57BL6 background (5) were maintained on a regular chow diet under a 12:12-h light-dark cycle, in a full-barrier facility. The mice were used at 10–14 weeks of age.

Lipid Analysis of the Proximal Intestine by Mass Spectrometry—To determine enrichment of mucosal lipid by dietary FA as an indicator of absorption, mice were given olive oil (6 µl/g) via intragastric gavage and sacrificed 90 min and 5 h later at which times the proximal intestine was harvested and flushed several times with cold saline. Tissues were homogenized, extracted by the method of Bligh and Dyer (26), and processed for shotgun lipidomics as described elsewhere (27). Shotgun lipidomics is a multistep approach to analyze individual lipids directly from a lipid extract that employs multiplex extractions, intrasource separation, and multidimensional mass spectrometry (27, 28).

Enterocyte Isolation—The intestine was divided into 3 equal-length segments (S1–S3) from proximal to distal. The segments were washed with ice-cold phosphate-buffered saline, opened longitudinally, and sliced into small pieces that were washed with Hanks' balanced salt solution containing 1% fetal bovine serum (FBS) followed by Hanks' balanced salt solution with 2% each BSA and glucose. Enterocytes were isolated by incubation (2 x 15 min) at 37 °C with shaking in Hanks' balanced salt solution supplemented with 0.5 mM dithiothreitol and 1.5 mM EDTA. The cells collected by centrifugation (1000 x g for 5 min) were suspended in the buffer appropriate for the experiment. Cell viability was determined by trypan blue exclusion and found higher than 85% for both WT and Cd36-deficient cells.

Fatty Acid Uptake Kinetics in Primary Enterocytes—The uptake buffer consisted of Krebs-Ringer Hepes (KRH, pH 7.4) containing [3H]oleate-BSA complexes at ratios of 0.7, 1.2, and 2. The reaction was started by adding cells (0.5 x 106 in 50 µlof KRH) to 50 µl of uptake buffer for 2, 4, 10, and 30 min. Uptake was stopped by adding 200 µl of ice-cold KRH and immediately overlaying the reaction mixture over 200 µl of ice-cold Ficoll-Paque diluted 4 times in phosphate-buffered saline and prepipetted in 6 x 44-mm microcentrifuge tubes. Cells were separated from medium by centrifugation (12,000 x g, 2 min). The tube bottom with the cell pellet was cut and cell radioactivity counted. Nonspecific radioactivity, determined by adding cells and label together to 200 µl of ice-cold KRH was subtracted from uptake values and the data were normalized to the number of viable cells.

Lipid Incorporation and Secretion Studies—For FA incubations, the medium contained 250 µM oleate bound to BSA (2:1) and 5 µCi/ml of [3H]oleate. For cholesterol, the medium contained 4 µmol of triolein, 0.78 µmol of cholesterol with 0.05 µCi of [14C]cholesterol, 0.78 µmol of egg phosphatidylcholine, and 5.7 µmol of sodium taurocholate (22). Recovery of label in free FA and cholesterol were measured after 30-min uptake incubations. The cells were collected by centrifugation, washed with phosphate-buffered saline, extracted for lipid, and subjected to thin layer chromatography (TLC). The developing solvent was hexane:diethyl ether:acetic acid, 75:25:1. Lipid species were visualized by staining samples and co-migrating standards with iodine vapor. The lipid bands were scraped and counted. FA incorporation into TG and TG secretion were determined from 2-h incubations, after which cells and media were harvested, lipid extracted, and subjected to TLC. Under all incubation conditions uptake did not exceed 10% of the medium substrate for FA and 3% for cholesterol.

Glucose Uptake and Metabolism—For glucose transport, proximal enterocytes were incubated (0, 5, 10, 20, and 30 min) in KRH with 0.5 mM 2-deoxyglucose (1 mM, 2 µCi/ml [3H]2-deoxyglucose), which can only be processed to deoxyglucose 6-phosphate. Uptake time courses were linear for 20 min. Incubations were stopped with cold KRH and the cells, collected by centrifugation through Ficoll, were analyzed for radioactivity. For glucose metabolism, cells were incubated with 1 mM glucose and 0.5 µCi/ml D-[14C]glucose. [14C]Glucose incorporation into glycogen and TG was determined from 90-min incubations at 37 °C (29) in the presence of 250 µl/ml of oleic acid complexed to BSA (ratio of 2).

Western Blot Analysis—Samples were homogenized in RIPA buffer with protease inhibitors and equal amounts of proteins were separated on 5–15% SDS-PAGE and reacted with the specific antibodies.

RNA Extraction and Real-time Quantitative PCR—RNA was extracted from the proximal segment (S1) using TRIzol and reverse transcription was performed using the SuperScript II First-strand Synthesis System (Invitrogen), with 1 µg of total RNA and random hexamers. Real time quantitative PCR assays were performed on an ABI Prim 7000 Sequence Detection System (Applied Biosystems) using SYBR Green PCR Master Mix. Primers used for real time PCR are presented in Table 1. The mRNA level of individual genes was quantified and normalized to 18 S mRNA. Relative mRNA abundance of individual genes was calculated as -fold change compared with levels in WT mice.


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  		TABLE 1
Oligodeoxyribonucleotide primer sequences for real-time quantitative PCR

 


Figure 1
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  		FIGURE 1.
Distribution of CD36 along the longitudinal axis of the intestine. I, intestines from WT and Cd36 null mice were separated into three equal segments from proximal to distal (S1–S3). Tissues were homogenized and equal amounts of protein were separated by SDS-PAGE and transferred to Immobilon-P membranes. CD36 was detected using anti-mouse monoclonal antibody and visualized with enhanced chemiluminesence. II, total length of the small intestine of WT and Cd36 null mice expressed per unit body weight (n = 10, **, p < 0.005). Data are means ± S.E.

 


Figure 2
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  		FIGURE 2.
Fatty acid uptake by proximal (S1) enterocytes from WT and Cd36 null mice. Time course of oleic acid uptake was determined using enterocytes from WT (filled squares) and Cd36-deficient mice (open squares). Cells were incubated with [3H]oleate for the indicated times (0–30 min) and uptake was stopped using cold KRH. Cells were collected by centrifugation through a Ficoll layer and analyzed for associated radioactivity, which was normalized to the number of viable cells. Uptake assays were determined at FA/BSA ratios of 0.7 (A), 1.2 (B), and 2.0 (C). Hanes plot of fatty acid uptake by enterocytes from WT and Cd36 null mice is shown in D. S is the concentration of unbound FA (nM) calculated according to the dissociation constants reported by Richieri et al. (52). V is the rate in nmol/min/106 cells and the x intercept represents the –Km. Data shown are as mean ± S.E. from three independent experiments (*, p < 0.05; **, p < 0.01).

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Distribution of CD36 in the Small Intestine—CD36 protein levels were examined along the longitudinal axis of the small intestine divided into three equal segments from proximal (segment 1 or S1) to mid-gut (S2) and distal (S3). CD36 was expressed in a steeply decreasing gradient from S1 to S3 (Fig. 1I). These findings are broadly consistent with published data showing levels to be the highest in the proximal intestine, duodenum, and early jejunum (18, 19). They support CD36 function in lipid absorption that is most efficient proximally. It is worth noting that the length of the Cd36–/– small intestine per unit body weight was significantly longer than that of age-matched controls (Fig. 1II). The difference was also significant (p < 0.05) even when not adjusted for the smaller (12%) body size of Cd36 null as compared with WT mice.

Fatty Acid Uptake and Processing by Primary Enterocytes—Based on the gradient of CD36 expression measured in S1–S3 (Fig. 1), it would be predicted that differences in FA uptake between WT and Cd36–/– mice would be most apparent in segment 1. Measurements of FA uptake by primary enterocytes isolated form the three different segments are shown in Figs. 2 and 3. Uptake of [3H]oleate and [3H]linoleate (data not shown) was reduced by more than 50% in cells isolated from S1 of Cd36 null as compared with WT mice (Fig. 2, A–C). To examine whether this reflected loss of a saturable component of uptake, oleate uptake rates were examined as a function of unbound FA concentration by varying the ratio of FA/BSA (0.7, 1.2, and 2). Uptake of oleate in WT enterocytes exhibited a low Km of about 5 nM (Fig. 2D) consistent with the range previously published in other CD36-expressing cells (5). No Km could be determined for the Cd36–/– enterocytes at the range of unbound FA concentrations used. In addition, oleate uptake in the proximal enterocytes was inhibited by 50% when phloretin (200 µM) was added to the uptake assay (data not shown) consistent with previous findings with other cells (30). These data supported the interpretation that the high affinity saturable component of uptake was reduced in enterocytes from the proximal intestine of Cd36–/– mice as a direct consequence of Cd36 deletion. Oleate uptake was also reduced in enterocytes from S2 but not in enterocytes from S3 (Fig. 3I). A reduction of FA incorporation into TG by S1 Cd36–/– enterocytes (Fig. 3II) was observed and paralleled by a decrease in secretion of newly made TG into the incubation medium (Fig. 3III). No such differences were observed in S2 or S3.


Figure 3
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  		FIGURE 3.
FA uptake, incorporation into cell TG, and TG secretion by S1–S3 enterocytes. Primary enterocytes were isolated from intestinal segments (S1–S3) and incubated with serum-free medium containing 250 µM oleic acid with 5 µCi of [3H]oleate/ml (FA:BSA ratio = 2). Recovery of [3H]oleate into cell FA (I) was measured at 30 min (counts/min in FA amounted to about 10% of total cell associated radioactivity). Incorporation of [3H]oleate into cell TG (II) and TG secretion into the medium (III) were measured at 2 h. Lipids were extracted from cells and media and separated by thin layer chromatography. Label in the FA and TG bands, identified from co-migrating standards was quantified by scintillation counting. Data are mean ± S.E. from **, p < 0.005, n = 4.

 
To determine whether the effect of Cd36 deficiency is specific to FA uptake, we examined glucose uptake and metabolism (Fig. 4). Glucose transport was determined using [3H]2-deoxyglucose, which is not processed beyond phosphorylation to deoxyglucose 6-phosphate. Uptake rate was increased in Cd36-deficient cells (Fig 4II) suggesting that enterocytes compensate for the reduction in FA uptake by up-regulating glucose utilization. This is similar to what is observed with other tissues such as skeletal, muscle, and heart (7). Despite the increase in uptake, glycogen synthesis was determined from incorporation of [14C]glucose and was found similar for cells from both genotypes (Fig. 4III). In contrast, glucose incorporation into triglycerides (Fig. 4IV) was significantly reduced in Cd36–/– S1 enterocytes because it would be limited by the supply of exogenous FA. Taken together, these findings document a specific defect in FA uptake in the proximal intestine of Cd36–/– mice, which is associated with reduced FA incorporation into cell TG and with diminished TG secretion.

Cholesterol Uptake—Cd36–/– mice retained more cholesterol in the intestinal lumen and exhibited a 50% reduction in cholesterol output into the lymph (22). The defect in cholesterol secretion could have reflected either impaired cholesterol uptake or normal uptake but reduced availability of FA for cholesteryl ester production. To determine whether CD36 plays a direct role in cholesterol uptake in the small intestine, enterocytes from WT and Cd36–/– mice were incubated with a micellar solution of [14C]cholesterol for 30 min (Fig. 5). Compared with WT, cholesterol uptake was reduced by more than 60% in S1 enterocytes from Cd36–/– mice. Under these conditions where no FA was provided, cholesterol esterification was undetectable. The defect was not apparent in enterocytes isolated from more distal segments S2 and S3. These findings suggest that, in addition to its role in FA uptake, CD36 contributes to proximal cholesterol absorption.

Apoprotein Levels in the Different Segments—The fatty acids and cholesterol absorbed by enterocytes are re-esterified and combined with apoproteins to produce chylomicrons that are secreted into the lymph. We examined whether the defect in fatty acid and cholesterol uptake observed in Cd36–/– intestines was associated with alterations in the levels of intestinal apoproteins. Protein content for apoB48 and apoA-IV (Fig. 6) showed a decreasing gradient from the proximal to distal segments consistent with the proximal intestine being the major site of lipid secretion. Levels of both apoproteins were significantly decreased in the intestines from Cd36–/– mice.

Analysis of Mucosal Lipids in the Proximal Intestine—Cd36–/– mice do not exhibit obvious impairment of intestinal lipid absorption when examined by the SPB method (22). However, SPB does not measure absorption rate, which appears defective based on our in vitro data. We examined whether the uptake defect could be demonstrated in vivo. For this we used the mass spectrometry technique of shotgun lipidomics (28) to compare oleic acid enrichment of mucosal lipids in proximal intestines of WT and Cd36–/– mice after gavage with olive oil, which is 70% triolein. If uptake of oleic acid is impaired proximally, its enrichment in major lipid fractions would be reduced during the absorptive phase. This was examined at 90 min (active absorption) and at 5 h (steady state) post-gavage (20). Analysis of the proximal intestine for FA composition of major lipids showed that oleate enrichment into mucosal free FA and TG was reduced by about 50% in Cd36–/– as compared with WT tissues at 90 min post-gavage with olive oil (Fig. 7, I and II). An apparent enrichment of 18:2 was observed in Cd36–/– tissues, which reflected the lower amounts of oleate present as oleate represents a large fraction of cell FA (60 and 50%, respectively, for WT and Cd36–/–). At 5 h post-gavage (data not shown), no significant differences were observed in relative oleate enrichment of mucosal lipids between WT and Cd36–/– mice reflecting the establishment of a steady state. These findings validated the in vitro data by showing a defect in proximal FA absorption by the Cd36–/– intestine. As a result, lipid absorption and the achievement of a steady state in mucosal lipids are delayed.


Figure 4
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  		FIGURE 4.
Uptake and metabolism of glucose by S1 enterocytes. I, schematic of glucose metabolism showing incorporation of label from glucose into TG via esterification of glycerol phosphate and via fatty acid synthesis. II, glucose uptake measured using the nonmetabolizable glucose analog [3H]2-deoxyglucose, which accumulates as [3H]2-deoxyglucose phosphate. Rates are shown per min per 106 cells and were obtained from 0 to 30-min time courses. Metabolism of glucose was evaluated using [14C]glucose. Panel III shows recovery of 14C label into glycogen (90 min), whereas panel IV shows [14C]glucose incorporation into triglycerides (90 min). Data in panels III and IV were normalized to milligram of cell protein. All values shown are mean ± S.E. from n = 3.

 


Figure 5
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  		FIGURE 5.
Cholesterol uptake by S1–S3 enterocytes. Primary enterocytes isolated from the intestinal segments S1–S3 were incubated with [14C]cholesterol for 30 min as described under "Experimental Procedures." Lipids were extracted from cells with chloroform:methanol and separated by TLC. Values are mean ± S.E. (*, p < 0.005, n = 3).

 
Membrane Proteins Implicated in Intestinal Lipid Absorption—We examined expression of several proteins implicated in lipid absorption in the small intestine, (FATP4) (31) for long-chain FA, SR-BI (32–34), and NPC1L1 for cholesterol (35–40). Levels were compared in S1–S3 from WT and Cd36–/– mice. As shown in Fig. 8I, FATP4 was expressed in all three segments with highest levels in S1 and S2. No differences were apparent between WT and Cd36–/– mice. NPC1L1 levels in WT mice were most abundant in S1 and decreased significantly in S2 and S3 (Fig. 8II). Levels in S1 were similar for WT and Cd36–/– mice. However, NPC1L1 levels were significantly up-regulated in CD36-deficient S2 and S3 as compared with CD36 sufficient segments (Fig. 8II). SR-BI was expressed in all three intestinal segments and its levels did not appear to be significantly altered with CD36 deficiency (Fig. 8III).

Regulation of Gene Expression—Expression of a number of genes involved in FA utilization and chylomicron formation downstream of CD36 was examined (Fig. 9). Cytosolic FA-binding proteins (FABPs) have been documented to play a role in intracellular FA trafficking (41) and co-expression of CD36 and L-FABP in proximal villi enterocytes has been documented (19). As shown in Fig. 9I, there was a significant decrease in expression of L-fabp but not of I-fabp in Cd36-deficient S1. A decrease of acyl-CoA: diacylglycerol acyltransferase 1 (Dgat1) was observed with no change in Dgat2. Other genes related to triglyceride formation included monoacylglycerol acyltransferases 2 and 3 and acyl-CoA synthases 1, 4, and 5 where no significant changes were observed (data not shown). Genes involved in lipid absorption and lipoprotein secretion included apoB and microsomal triglyceride transfer protein, which did not exhibit alterations in expression (Fig. 9II) and apoA-IV, which was markedly decreased.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies in CD36–/– mice documented an important role of intestinal CD36 in lipid secretion into the lymph but could not show an effect on overall absorption. As a result CD36 function in intestinal FA uptake was ruled out (20–22). However, it remained difficult to explain how the protein expressed on the apical surface of enterocytes while not affecting uptake at that site would affect secretion on the basolateral side. This study used both in vitro and in vivo approaches to examine the hypothesis that CD36 might function in lipid absorption primarily in the proximal intestine, the major site of chylomicron formation. The data obtained provide several novel findings. First, CD36 function in the uptake of both FA and cholesterol was documented in the proximal intestine. Second, the uptake defect is reflected in impaired lipid secretion by isolated enterocytes and in marked down-regulation of protein levels for apoB48 and apoA-IV. These data suggest that CD36 coordinates proximal absorption of FA and cholesterol for chylomicron production and they provide a likely mechanism for the defect in lymph lipid secretion previously reported in Cd36 null mice. Third, up-regulation of NPC1L1 in mid and distal segments of CD36-deficient mice was shown suggesting that the two proteins may functionally interact in cholesterol absorption. Down-regulation of FA handling proteins implicated in chylomicron production such as L-FABP and DGAT1 would suggest that these proteins are involved downstream of CD36. These findings are discussed in more detail below.


Figure 6
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  		FIGURE 6.
Distribution of apoB48 and apoA-IV in different intestinal segments from WT and Cd36 null mice. Intestinal segments (S1–S3) from 3 WT and 3 Cd36 null mice were homogenized and equal amounts of protein were separated by SDS-PAGE and transferred to Immobilon-P membranes as in the legend to Fig. 1. Immune complexes of apoB 48 (I) and apoA-IV (II) were visualized with enhanced chemiluminescence.

 


Figure 7
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  		FIGURE 7.
Proximal absorption in vivo. Analysis of proximal mucosal lipids after intragastric triolein: WT and Cd36 null mice fasted overnight were given an intragastric load of olive oil (70% triolein). The proximal intestinal segment was harvested 90 min post-gavage and mucosal lipids were extracted and analyzed by mass spectrometry as described under "Experimental Procedures." Relative enrichment of the major FA species in cell-free FA (I) and TG (II) are expressed as -fold change relative to WT mice. Data (n = 3) are mean ± S.E. (p = 0.01).

 
CD36 expression is highest in the proximal one-third of the intestine (S1) and follows a decreasing gradient down to almost undetectable levels in the most distal part (S3). Comparing Cd36-sufficient, to Cd36-deficient enterocytes isolated from segments S1–S3 documented defects in uptake of FA in S1 and S2 and in that of cholesterol in S1. This supported an important role of the protein in facilitating uptake of these nutrients in the proximal intestine. In the intestinal lumen, millimolar concentrations of FA are solubilized in bile acid micelles for absorption by enterocytes. The micelles are associated with low concentrations of free FA monomer in solution (42–44) and there is evidence that the monomer is the FA species important for enterocyte uptake (45–47). Several studies previously examined FA uptake by the intestine using everted sacs (48), brush-border membranes (49), isolated enterocytes (18, 50), or immortalized lines such as Caco-2 cells (45, 51). Uptake was shown to involve both saturable and diffusion-like components. Our uptake rate measurements were based on the initial linear phase of the time course using intact enterocytes that actively esterify exogenous FA. Although initial rates (sensitive to phloretin) were used to derive the km, we cannot rule out the possibility that the km may reflect both uptake and initial metabolism of the FA. The km value we measured (about 5 nM) is similar to values previously reported by others for uptake of FA bound to albumin by isolated jejunal mucosal cells (18, 50) or Caco-2 cells (45, 46) provided the dissociation constants for FA and albumin as determined by Richieri et al. (52) are used for calculating unbound FA. The km is also in agreement with that measured for the CD36 facilitated uptake in other cell types (reviewed in Ref. 30). The physiological relevance of the low uptake km determined in vitro is difficult to evaluate because concentrations of monomeric FA that the enterocytes are exposed to in vivo are unknown. Monomer concentration for FA in bile acid micelles have been measured in vitro using polyethylene discs as the organic phase of a partitioning system (43). The concentrations varied with total FA and solubility of the particular FA. However, the values obtained that ranged between 10–7 and 10–9 M for long-chain FA are not dissimilar from those measured for FA bound to albumin. Thus it is not unreasonable to expect that enterocytes like other cells concerned with high rates of FA processing would possess receptors with high affinity for FA.


Figure 8
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  		FIGURE 8.
Levels of membrane proteins implicated in intestinal lipid absorption. Protein levels of FATP4, NPC1L1, and SR-BI were determined in S1–S3 segments from WT and Cd36 null mice. Tissues, homogenized and processed as described in the legend to Fig. 1, were probed with specific antibodies for FATP4 (I), NPC1L1 (II), and SR-BI (III). Tubulin and ran are used as internal controls.

 


Figure 9
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  		FIGURE 9.
Gene expression for proteins involved in FA processing and lipoprotein assembly. RNAs were isolated from S1 of WT and Cd36 null mice. Relative mRNA expression was determined by real time quantitative PCR and normalized to 18 S content. Data are expressed as -fold change compared with WT. I, genes that may be involved in FA handling and TG formation downstream of CD36. II, genes involved in lipoprotein assembly. Data are mean ± S.E. (*, p < 0.05, n = 3).

 
The role of CD36 in enterocyte cholesterol uptake was surprising but not totally unexpected. CD36 has been shown to function in selective uptake of cholesterol esters from high density lipoproteins although less efficiently than SR-B1 (53). CD36 also binds cholesterol (54) and this interaction was used to purify the protein. Finally cholesterol loading induces cellular redistribution of CD36 (55) and up-regulates its expression (56). Our data would support the interpretation that the effect of Cd36 deletion on cholesterol absorption is likely direct and not secondary to the impaired FA uptake. This is based on the fact that the micelles used for cholesterol delivery did not contain FA because no lipase was added to hydrolyze the triolein. In addition the uptake radioactivity inside the cell was recovered as free cholesterol. However, indirect effects, for example, consequent to changes in membrane composition or gene expression cannot be entirely ruled out and more extensive studies are needed to better understand the role of CD36 in cholesterol absorption. There was no apparent compensation in the proximal intestine (S1) for the impaired absorption of FA and cholesterol consequent to Cd36 deletion as evidenced from the defects observed both in vitro (Figs. 2 and 4) and in vivo (Fig. 7). Consistent with this there was no alteration in S1 in expression of several proteins implicated in intestinal lipid absorption, SR-B1, NPC1L1, and FATP4.

Significant up-regulation of NPC1L1 was observed in segments 2 and 3 of Cd36 null mice. As a result, the null mouse has an altered distribution of NPC1L1 with abundant levels in the middle and distal intestine where it is normally very low (compare S2 and S3 in WT and Cd36 null). Because this protein is implicated in cholesterol absorption (35, 36, 38–40), the increase in its level in S2 and S3 may have contributed to the lack of difference in cholesterol uptake between WT and Cd36 null S2 and S3 enterocytes. Recent evidence suggests that the function of NPC1L1 in cholesterol uptake may require other membrane proteins (38). NPC1L1 co-expression with CD36 along the longitudinal axis of the small intestine and its up-regulation in Cd36-deficient tissue may have functional significance. In addition, the lack of defect in cholesterol absorption in Cd36-deficient S2 and S3 suggests that NPC1L1 in these segments may interact with other proteins, possibly SR-B1 to promote cholesterol absorption. The data suggest that functional interactions between CD36, SR-B1, and NPC1L1 may need to be explored in future studies.

The in vivo relevance of the lipid uptake defect was confirmed by measuring less oleic acid enrichment of mucosal lipids in S1 of Cd36 null mice after intragastric administration of olive oil. Because the proximal intestine is the site most active in chylomicron production, the defect in FA and cholesterol uptake would be responsible for the reduced lymph lipid secretion previously documented in Cd36 null mice (22). The uptake impairment would result in less efficiently lipidated and consequently smaller lipoprotein particles (20, 22). Thus low lipid availability such as with fasting (57) and Cd36 deficiency would result in very low density lipoprotein-sized particles. Interestingly, the drop in apoB48 and apoA-IV suggests that lipid availability regulates levels of these apoproteins. Whereas apoA-IV appears regulated via changes in its expression, the apoB48 level seems to be modulated post-transcriptionally, similar to what has been documented for apoB turnover in the liver (58).

Our findings are consistent with the existence of multiple mechanisms for lipid uptake in the intestine with the redundancy ensuring the maintenance of absorption. The relative contribution of the various mechanisms would differ markedly between the various segments with the CD36-dependent pathway being important proximally. The CD36 pathway might direct FA (and possibly cholesterol) to chylomicron formation, which may involve CD36 internalization and interaction with intracellular proteins important for lipid targeting such as the FABPs. The decrease in expression of L-fabp but not I-fabp in Cd36-deficient S1 suggests that CD36 and L-FABP are part of the same pathway. This is consistent with previous reports suggesting a role for L-FABP in chylomicron production and lipid export, whereas I-FABP is postulated to function in channeling FA to pathways needed for the constitutive needs of enterocytes (59). The decrease of Dgat1 expression with no change in Dgat2 is consistent with the previous finding that DGAT1, which is the main DGAT present in the small intestine, may be involved in TG synthesis for chylomicrons under high nutrient supply (60, 61).

In summary deletion of Cd36 impairs uptake of FA and cholesterol with the major impact at the level of the proximal intestine. The CD36 pathway is, to our knowledge, the first one to be implicated in the uptake of both FA and cholesterol in the proximal intestine and it may function to coordinate processing of both nutrients for formation of highly lipidated particles. The full physiological implications of the findings remain to be determined. It would be important to examine whether selective down-regulation of intestinal CD36, which would be accessible from the intestinal lumen, would be beneficial in terms of reducing lipid intake. Preliminary data suggest that "postingestive effects" of the delayed absorption in Cd36 null mice result in a significant reduction of fat intake.4 Furthermore, the proximal intestine contributes to regulating food intake and energy homeostasis via the release of a variety of peptide hormones (62) and testing whether this role is significantly impacted by CD36 function and the rate of lipid absorption would be warranted.

Finally, it is possible to reconcile the findings in the present study with earlier data that ruled out a role of CD36 in intestinal absorption based on the SPB method and on the rate of appearance of intestinal lipoproteins. Defects in proximal absorption as a result of Cd36 deletion are ultimately compensated for distally by CD36-independent mechanisms. As a result this is not detected by the SPB method, which evaluates net absorption from fecal lipid. In addition, SPB does not measure the rate of intestinal absorption that is the parameter affected in Cd36 deficiency. Finally, impaired blood lipoprotein removal combined with the altered lipoprotein distribution in Cd36 null mice (20, 21) invalidate estimates of absorption from appearance of intestinally derived lipids.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants DK 600220 and DK33301 and a grant from the Monsanto Co. (to N. A. A.). 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 To whom correspondence may be addressed. E-mail: fnassir{at}im.wustl.edu. 2 To whom correspondence may be addressed: Campus Box 8031, Washington University School of Medicine, St. Louis, MO 63110. E-mail: nabumrad{at}wustl.edu.

3 The abbreviations used are: CD36/FAT, fatty acid translocase; FA, fatty acid; TG, triglyceride; DGAT, diacylglycerol acyltransferase; BSA, bovine serum albumin; FATP4, fatty acid transport protein 4; SR-BI, scavenger receptor class B type I; NPC1L1, Niemann-Pick C1-like 1; ApoB, apolipoprotein B; ApoA-IV, apolipoprotein A-IV; L-FABP, liver fatty acid-binding protein; IFABP, intestinal fatty acid-binding protein; SPB, sucrose polybehenate; WT, wild type. Back

4 A. Sclafani, K. Ackroff, and N. A. Abumrad, unpublished observations. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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