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J. Biol. Chem., Vol. 278, Issue 34, 31610-31620, August 22, 2003
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Multiple, Independently Regulated Pathways of Cholesterol Transport across the Intestinal Epithelial Cells*
From the Departments of Anatomy and Cell Biology, and Pediatrics, State University of New York Downstate Medical Center, Brooklyn, New York 11203
Received for publication, February 3, 2003 , and in revised form, May 19, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The present study provides a new understanding about the mechanisms involved in cholesterol absorption by the intestinal cells. Contrary to general belief, our data show that newly absorbed cholesterol is neither immediately available for secretion with apoB lipoproteins nor exclusively secreted as part of chylomicrons. Based on our data, cholesterol transport by enterocytes can be broadly classified into two independently modulated, apoB-dependent and -independent, pathways. Cholesterol secretion by the apoB-dependent pathway is induced by oleic acid, is repressed by microsomal triglyceride transfer protein inhibitors, and occurs only with larger apoB-containing lipoproteins. ApoB-independent pathways do not require microsomal triglyceride transfer protein and involve efflux mediated by ABCA1, high density lipoprotein assembly, and possibly other unknown mechanisms. There are at least two different metabolic pools of cholesterol. The newly absorbed and pre-absorbed cholesterol are preferentially secreted via apoB-independent and apoB-dependent pathways, respectively. In contrast to compartmentalization for secretion, these two metabolic pools are equally accessible for cellular esterification. The esterified cholesterol is mainly secreted by the apoB-dependent pathway, whereas both the pathways are involved in the secretion of free cholesterol. Thus, enterocytes transport exogenous cholesterol by several independently regulated pathways raising the possibility that targeting of apoB-independent pathways may result in selective inhibition of cholesterol transport without affecting triglyceride transport.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Due to a significant positive correlation between cholesterol absorption and plasma cholesterol levels (1–3), cholesterol absorption has been the subject of intense research. Cholesterol absorption is defined as the transfer of cholesterol from the intestinal lumen to the mesenteric or thoracic lymph duct (2). Early studies resulted in the identification and characterization of enzymes that hydrolyze cholesterol esters in the intestinal lumen, and an appreciation of the role bile acids play in the solubilization and cellular uptake of cholesterol (4–6). From these studies, it was established that the dietary cholesterol esters are hydrolyzed in the intestinal lumen, and free cholesterol is solubilized in the bile salt micelles and is taken up by intestinal epithelial cells (2, 4–6). Currently, it is believed that the uptake of cholesterol by the enterocytes is the rate-limiting step in cholesterol absorption (7). This is supported by the observation that a man who ate 25 eggs a day did not develop hypercholesterolemia because he absorbed less cholesterol (8). After uptake, enterocytes esterify cholesterol, a process mediated by acyl-coenzyme A:cholesterol acyltransferases (9, 10), and package it into chylomicrons for basolateral secretion into the mesenteric lymph.
The major sources of cholesterol for absorption are of exogenous and endogenous origins. The exogenous source of cholesterol is food, whereas endogenous cholesterol is derived from bile, desquamated cells, and biosynthesis. Endogenous cholesterol, especially biliary cholesterol, enters the intestinal lumen in association with bile salts and is immediately available for absorption. On the other hand, there is a time delay for the absorption of dietary cholesterol because it needs to be solubilized in bile salt micelles. The differences observed in the metabolism of biliary and dietary cholesterol have been explained based on the differences in the solubilization and uptake (2). It is possible that after entering the cells these two pools are handled differently.
The intracellular mechanisms for cholesterol packaging in chylomicrons are poorly understood because most of the animal studies involve measurement of radiolabeled cholesterol in the feces and thoracic duct lymph or blood. Recent advances in cellular models to study intestinal lipid absorption have resulted in newer attempts toward the understanding of cholesterol absorption by enterocytes. In this regards, Field and co-workers (11, 12) have shown that Caco-2 cells preferentially secrete plasma membrane cholesterol along with triglyceride-rich lipoproteins and that this secretion is modulated by sphingomyelinase. The plasma membrane cholesterol is transported to the endoplasmic reticulum by intracellular vesicles, a process stimulated by oleic acid (13), and is replenished by cholesterol obtained from luminal absorption or cellular biosynthesis. We studied the mechanisms involved in the transport of cholesterol, and we observed that intestinal cells compartmentalize absorbed cholesterol and secrete by several mechanisms.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Materials—Antibodies used for the determination of apoB1 have been described (14, 15). Monoclonal anti-apoA-1 antibody, 4H1, was from Dr. Yves Marcel of the University of Ottawa Heart Institute. Polyclonal anti-apoA-1 antibodies were from Roche Applied Science. Glyburide, 9-cis-retinoic acid, 22-hydroxycholesterol, oleic acid (OA), monopalmitoylglycerol, sodium cholate, sodium deoxycholate, and taurocholate (TC) were from Sigma. Phosphatidylcholine was from Avanti Lipids (Alabaster, AL). Microsomal triglyceride transfer activity (MTP) inhibitor, BMS200150, was a kind gift from Dr. Haris Jamil of the Bristol-Myers Squibb Co. To prepare OA:TC (20 x 1.6:0.5 mM) stocks, 97.4 mg of sodium oleate were added to 10 ml of 10 mM TC solution, mixed by swirling, and incubated at 37 °C until a clear solution was obtained. This stock was filtered (0.2 µm) and stored (–20 °C) in 1-ml batches until use. To prepare micelles for incubation with primary rat enterocytes, lipids were dissolved in chloroform/methanol, 1:1 (v/v), and were dried under nitrogen. Appropriate volumes of DMEM were added to obtain micelles containing 1.4 mM sodium cholate, 1.5 mM sodium deoxycholate, 1.7 mM phosphatidylcholine, 2.2 mM oleic acid, and 1.9 mM monopalmitoylglycerol (10x micelle stock) and mixed. The suspension was sonicated using probe sonicator (550 Sonic Dismembrator, continuous pulse, output setting of 3, 3 min). These micelles (1/10th volume) were then added to enterocyte suspension to obtain physiologic concentrations of micelles (16).
Studies with Cells—Caco-2 (human colon carcinoma) cells obtained from the American Type Culture Collection (Manassas, VA) were cultured (75-cm2 flasks, Corning Glassworks, Corning, NY) in Dulbecco's modified Eagle's medium containing high glucose supplemented with L-glutamine and antibiotic/antimycotic mixture (DMEM) and 20% fetal bovine serum (FBS). For experiments, cells from 70 to 80% confluent flasks were seeded on polycarbonate micropore membrane inserts (Transwells®, 6-well plate, 24 mm diameter, 3 µm pore size, Corning Costar Corp., Cambridge, MA) at a density of 1 x 105 cells/cm2. To induce differentiation of these cells, media were changed every other day for 21 days (17–19). Experiments were conducted using two different pulse and pulse-chase labeling protocols. For the pulse-labeling experiments, cells received 2 ml of DMEM containing 20% FBS, 5 µCi/well [3H]cholesterol (PerkinElmer Life Sciences), OA:TC (1.6:0.5 mM) on the apical side and 2 ml of medium with or without 1% BSA on the basolateral side for 17 h. For pulse-chase studies, cells were labeled with cholesterol for 17 h as described above with two exceptions; the OA:TC was omitted from the apical media, and the basolateral side received DMEM + 20% FBS. After pulse labeling, cells were thoroughly washed and incubated with DMEM containing 20% FBS and OA:TC (1.6:0.5 mM) on the apical side and DMEM ± 1% BSA on the basolateral side. Basolateral conditioned media (1.6 ml) were subjected to sequential density gradient ultracentrifugation (see below), and the rest was used for the measurement of apolipoproteins and radiolabeled cholesterol.
Density Gradient Ultracentrifugation—Variable amounts of the conditioned media were brought to 4 ml with 1.006 g/ml density solution in SW41 ultracentrifuge tubes (17). To adjust the density to 1.12 g/ml, 0.565 g of KBr were added and dissolved by repeated pipetting. Media were sequentially overlaid with 3 ml each of 1.063 and 1.019 g/ml, and 2 ml of 1.006 g/ml density solutions. The tubes were subjected to ultracentrifugation (SW41 rotor, 40,000 rpm, 33 min, 15 °C), and the top 1 ml was aspirated. This was called fraction 1 and represents large chylomicrons (Sf >400) (17). The gradients were then overlaid with 1 ml of 1.006 g/ml solution and centrifuged (40,000 rpm, 3 h and 30 min, 15 °C), and the top 1 ml was collected (fraction 2, small chylomicrons). The tubes were replenished with 1.006 g/ml solutions and centrifuged (40,000 rpm, 15 °C, 17 h), and the top 1 ml was collected by aspiration. This fraction 3 represents the VLDL size particles. The rest of the contents were fractionated into 1.5-ml fractions numbered 4–10. ApoB and radioactivity were measured in each fraction in triplicate.
Secretion of Cholesterol with ApoB Lipoproteins—In most of the experiments, pulse-chase protocol was used to study the secretion of cholesterol with apoB lipoproteins. After 17 h of pulse labeling with [3H]cholesterol, differentiated Caco-2 cells were extensively washed with DMEM. Cells were then incubated for another 24 h in the presence of OA:TC to induce secretion of cholesterol with chylomicrons. To inhibit the secretion of cholesterol with lipoproteins, we used MTP inhibitor (BMS200150, 10 µM) along with OA:TC. Basolateral conditioned media were subjected to density gradient ultracentrifugation.
Secretion of ApoB-free Cholesterol—Inhibition of apoB-free cholesterol secretion was studied by the addition of 1 mM glyburide (20, 21) on the basolateral side during pulse and pulse-chase labeling experiments. Higher concentrations of glyburide were not used because of its insolubility in media. To induce ABCA1 expression (21, 22), Caco-2 cells were treated with DMEM + 20% FBS + 1 µM 9-cis-retinoic acid (RA) + 25 µM 22-hydroxycholesterol (22(OH)C) along with [3H]cholesterol for 17 h. The basolateral side received DMEM + 20% FBS. Cells were washed and then received serum-free DMEM + RA + 22(OH)C on the apical side and serum-free DMEM on the basolateral side. After 8 h, cells were washed and supplemented with DMEM + 1% BSA + RA + 22(OH)C on the apical side and with DMEM + 1% BSA ± 1 mM glyburide on the basolateral side. Basolateral media were collected after 17 h and used for radioactive measurements and density gradient ultracentrifugation. After rinsing cells with PBS, lipids were extracted with isopropyl alcohol.
Extraction and Analysis of Free and Esterified Cholesterol—Lipids were extracted from the media and fractions according to the method of Bligh and Dyer (23). To 1 ml of media or fractions was added 3.75 ml of chloroform:methanol (1:2, v/v), mixed, and incubated at room temperature with intermittent mixing. After 15 min, 1.25 ml of chloroform was added, mixed, and incubated for 1 min. Subsequently, 1.25 ml of water was added; contents thoroughly mixed and centrifuged (10 min, 5,000 rpm), and the lower phase was collected with a Pasteur pipette. The leftover upper phase was extracted again with 1.25 ml of chloroform and combined with the earlier extract. Organic extracts from cells, media, and fractions were dried under nitrogen, dissolved in 100 µl of chloroform, and separated by thin layer chromatography on PE SIL G Silica gels (Whatman) using hexane:diethyl ether:glacial acetic acid (82:17:2, v/v/v). Bands corresponding to free cholesterol and cholesteryl esters were visualized after iodine exposure, scraped, and counted by liquid scintillation counting.
Studies with Rat Primary Enterocytes—Rat enterocytes were
isolated using EDTA treatment by the method of Weiser
(24) as elaborated by Pinkus
(25) and Cartwright and
Higgins (26). Briefly,
proximal 1/3rd to 
portions of the intestines were collected from
anesthetized rats, and the luminal contents were emptied and washed with 115
mM NaCl, 5.4 mM KCl, 0.96 mM
NaH2PO4, 26.19 mM NaHCO3, 5.5
mM glucose buffer, pH 7.4, gassed for 20 min with 95%
O2, 5% CO2. One end of the intestines was then tied and
filled with 67.5 mM NaCl, 1.5 mM KCl, 0.96 mM
NaH2PO4, 26.19 mM NaHCO3, 27
mM sodium citrate, 5.5 mM glucose buffer, pH 7.4,
saturated with 95% O2,5%CO2. The intestines were then
incubated in a bath containing oxygenated 0.9% saline at 37 °C with
constant shaking. After 10–15 min, the luminal solution was discarded
and filled with 115 mM NaCl, 5.4 mM KCl, 0.96
mM NaH2PO4, 26.19 mM
NaHCO3, 1.5 mM EDTA, 5.5 mM glucose, 0.5
mM dithiothreitol buffer, pH 7.4, bubbled with 95% O2,
5% CO2 and bathed in saline as described above. After 15 min the
luminal contents were collected and centrifuged (1,500 rpm, 5 min, room
temperature), and pellets were resuspended in DMEM saturated with 95%
O2, 5% CO2. Isolated enterocytes were incubated with 1
µCi of [3H]cholesterol at 37 °C with constant shaking, and
cell suspensions were gassed with 95% O2, 5% CO2 at
15-min intervals. After 1 h, enterocytes were centrifuged at 3,000 rpm for 5
min, and the cell pellets were washed with media. After washing, cells were
chased for 2 h with DMEM containing 0.14 sodium cholate, 0.15 mM
sodium deoxycholate, 0.17 mM phosphatidylcholine, 0.22
mM oleic acid, and 0.19 mM monopalmitoylglycerol
micelles in the presence and absence of 10 µM BMS200150. At the
end of the incubation, enterocytes were centrifuged (3,000 rpm, 5 min), and
supernatants were used for density gradient ultracentrifugation.
Other Methods—Protein was measured by the method of Bradford (27). ApoB and apoA-I were quantified in the conditioned media and in different density gradient fractions using a sandwich enzyme-linked immunosorbent assay as described previously (14, 15).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Effect of Oleic Acid Supplementation on the Cellular Accumulation and Secretion of Cholesterol—It is generally believed that dietary cholesterol enters the body as part of chylomicrons synthesized by the intestinal cells. We have shown that supplementation of high concentrations of oleic acid (OA) induces chylomicron assembly and secretion by differentiated Caco-2 cells (17–19). To study the effect of OA on the rate of cellular accumulation of cholesterol, differentiated Caco-2 cells were supplemented with [3H]cholesterol in the presence of either taurocholate (TC) or OA:TC on the apical side (Fig. 1A). There were no significant differences in the cellular accumulation of cholesterol under both experimental conditions. We then studied the secretion of radiolabeled cholesterol by these cells (Fig. 1B). Because OA increases secretion of larger apoB-containing lipoproteins by differentiated Caco-2 cells, we were expecting that OA treatment would increase cholesterol secretion. Unexpectedly, OA:TC neither changed the rates nor the amounts of cholesterol secreted compared with TC-treated cells (Fig. 1B). These studies indicate that OA supplementation does not affect cellular uptake and secretion of cholesterol.
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Consideration was given to the possibility that OA might have increased the
secretion of lipoprotein-associated cholesterol. To test this hypothesis,
cholesterol secretion by Caco-2 cells was studied under pulse labeling and
pulse-chase labeling protocols described under "Experimental
Procedures." Secretion of cholesterol with chylomicrons was evaluated by
density gradient ultracentrifugation and compared with the flotation
properties of cholesterol in non-conditioned media
(Fig. 2). Cholesterol was
present at a d > 1.12 g/ml when centrifuged in non-conditioned
media containing 1% BSA (Fig.
2A). During continuous pulse, the majority of the
cholesterol was in the same fractions as in the non-conditioned media (compare
Fig. 2, A with
B). A small amount of cholesterol (
2% of total
secreted) was present in fractions 1–3 that correspond to d
< 1.006 g/ml. In contrast to pulse labeling, during the chase of the
pulse-chase protocol significantly higher amounts of cholesterol (40% of total
secreted) were in larger lipoproteins (Fig.
2C, fractions 1–3). These studies
indicated that during continuous pulse, the majority of the secreted
cholesterol was in the bottom fractions, whereas during pulse-chase protocol
significant amounts of cholesterol were in the top fractions that contained
larger lipoproteins.
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In control experiments performed with [3H]mannitol, we
determined that the increased amounts of cholesterol in the bottom fractions
were not due to paracellular leakage (data not shown). To determine whether
the large amounts of cholesterol present unassociated with lipoproteins was
due to the presence of BSA in the conditioned media, we repeated these
experiments in the presence of serum-free media (SFM) on the basolateral side.
Cholesterol was mainly present at a density of 1.02 to 1.06 g/ml when
centrifuged in SFM (Fig.
2D, fractions 5–7). During continuous
pulse the majority of the secreted cholesterol was in the same fractions (1.02
< d > 1.06 g/ml) as it was in the non-conditioned media
(compare Fig. 2, D with
E). In addition, two more peaks (fractions 1–3 and
9 and 10) corresponding to d < 1.006 g/ml and d > 1.12
g/ml were evident (Fig.
2E). During pulse-chase
(Fig. 2F), cholesterol
was in two peaks (fractions 1–3 and 9 and 10) corresponding to
d < 1.006 g/ml and d > 1.12 g/ml. Now 51% of total
secreted cholesterol was with larger lipoproteins. These studies extended
earlier observations that during pulse labeling the majority of the secreted
cholesterol was not associated with larger lipoproteins, but it was associated
with these lipoproteins during the chase.
To determine whether cholesterol secretion in d > 1.12 g/ml fraction was unique to differentiated Caco-2 cells, we extended these studies to rat primary enterocytes (Fig. 3). First, we determined the flotation properties of cholesterol given to cells. As shown in Fig. 3A, free cholesterol was mainly present in fractions 4–6. Next, rat primary enterocytes were incubated with cholesterol for 1 h, washed, and then incubated with OA to induce chylomicron assembly and secretion. After 2 h, the conditioned media were subjected to density gradient ultracentrifugation (Fig. 3B). The secreted cholesterol was distributed in fractions 1–4 and 9 and 10. About 51% of the total secreted cholesterol was present in the top four fractions, and the bottom fractions contained 30% of secreted cholesterol. These studies suggest that intestinal cells secrete cholesterol in two forms that can be separated based on their flotation properties.
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Modulation of Lipoprotein-associated Cholesterol Secretion— Studies described in Figs. 2 and 3 indicated that during pulse-chase experiments higher amounts of cholesterol were secreted with lipoproteins. To determine whether OA-induced chylomicron assembly is required for cholesterol secretion during pulse-chase protocol, differentiated Caco-2 cells were pulse-labeled with [3H]cholesterol for 17 h and then chased with TC, OA:TC, or OA:TC + BMS200150 for 24 h. ApoB, apoAI, and cholesterol levels were measured in the basolateral media. OA:TC treatment specifically increased apoB secretion by 64% without affecting apoAI secretion compared with TC treatment (Table I). BMS200150 decreased the secretion of total apoB by 61% but had no effect on apoAI secretion (Table I). Analysis of cholesterol revealed that OA:TC treatment increased the secretion of cholesterol during chase by 41%. BMS200150 obliterated the OA-induced cholesterol secretion. These studies indicate that OA treatment increases apoB and cholesterol secretion during chase but has no effect on apoAI secretion. The increases in apoB and cholesterol secretion are abolished by the inhibition of MTP.
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Density gradient ultracentrifugation analysis
(Fig. 4A) showed that
TC-treated cells did not secrete apoB as chylomicrons
(Fig. 4A,
fractions 1 and 2). Instead the majority of apoB was in
smaller VLDL/intermediate density lipoprotein/low density lipoprotein-size
lipoproteins (fractions 3–6) as has been described previously
(18). OA treatment increased
(9-fold) the secretion of apoB as part of large and small chylomicrons and
VLDL (Fig. 4A,
fractions 1–3), consistent with our earlier studies
(17,
18). Now, fractions 1–3
contained 70% of secreted apoB. In the presence of the MTP inhibitor,
secretion of apoB with large and small chylomicrons was completely inhibited.
Instead, apoB was mainly present in smaller lipoproteins
(Fig. 4A,
fractions 3–6). Next, we looked at the distribution of secreted
cholesterol in different lipoprotein fractions
(Fig. 4B). There was
no cholesterol in larger lipoprotein fractions in the conditioned media of
TC-treated cells (Fig.
4B, fractions 1 and 2). Note that these
cells secrete apoB as small lipoproteins (fractions 3–6), and these
fractions also did not contain any appreciable amounts of cholesterol. The
majority of the secreted cholesterol was in the bottom fraction that had no
apoB (Fig. 4B,
fraction 10). OA treatment drastically increased the secretion of
cholesterol in larger apoB lipoproteins
(Fig. 4B,
fractions 1 and 2), but this treatment had no effect on the
amounts of cholesterol secreted unassociated with lipoproteins
(Fig. 4B,
fractions 9 and 10). In BMS200150-treated cells, concomitant
with decreased apoB secretion in larger lipoproteins, there was a significant
decrease in the secretion of cholesterol in lipoprotein fractions
(Fig. 4B,
fractions 1 and 2). Note that BMS200150 had no effect on the
secretion of apoB-free cholesterol (Fig.
4B, fraction 10). These studies showed that when
apoB was secreted as part of smaller lipoproteins, cholesterol was not
associated with these particles. Thus, it appears that OA-induced assembly of
larger lipoproteins is essential for the secretion of cholesterol with apoB
lipoproteins.
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Next, we studied the modulation of cholesterol secretion in rat primary enterocytes (Fig. 4C). When enterocytes were not supplemented with lipid micelles, cholesterol was present in two peaks (fractions 3 and 4 and fractions 9 and 10). Incubation of enterocytes with lipid micelles increased the amounts of cholesterol in the first peak (fractions 3 and 4). In addition, significant amounts of cholesterol were also present in fractions 1 and 2 corresponding to large and small chylomicrons. In the presence of BMS200150, the secretion of cholesterol in fractions 1 and 2 was completely inhibited, and the amounts of cholesterol in fractions 3 and 4 were significantly decreased. Note that these manipulations had no effect on the amounts of cholesterol present in fractions 9 and 10. We conclude that the assembly of larger lipoproteins is necessary for the secretion of cholesterol with lipoproteins, and this secretion is inhibited in the presence of MTP inhibitors. In contrast, modulation of lipoprotein has no effect on the cholesterol secretion in apoB-free fractions.
Efflux Mechanisms Contribute to ApoB-free Cholesterol
Secretion—The presence of apoB-free cholesterol in the basolateral
media was intriguing. To determine whether cholesterol efflux contributes to
the secretion of cholesterol independent of lipoprotein secretion, we labeled
differentiated Caco-2 cells for 17 h and chased in the presence and absence of
an efflux inhibitor glyburide
(20,
21) along with OA:TC to induce
chylomicron assembly. Glyburide (1 mM) decreased cholesterol
secretion by 27% (Fig.
5A) but had no effect on cell viability as determined by
[3H]adenine release
(28,
29) in parallel dishes (data
not shown). Density gradient ultracentrifugation analysis showed that
glyburide decreased cholesterol in the apoB-free fractions by 33% without
affecting the amounts of cholesterol secreted with apoB lipoproteins
(Fig. 5B). Glyburide
also inhibited cholesterol secretion during the pulse-labeling protocol (data
not shown). To confirm further the role of cholesterol efflux, we induced
cholesterol efflux pathway by providing LXR/RXR agonists,
22-hydroxycholesterol, and retinoic acid, which have been shown to increase
the expression of ABCA1 in Caco-2 cells
(21,
22). Treatment of
differentiated Caco-2 cells with retinoic acid and 22-hydroxycholesterol
significantly increased (25%) the amounts of cholesterol secreted, and
this increase was completely obliterated in the presence of glyburide
(Fig. 5C). Density
gradient ultracentrifugation analysis revealed that LXR/RXR agonists increased
apoB-free cholesterol secretion by 62%, and this increase was abolished in the
presence of glyburide (Fig.
5D, fraction 10). Note that there is no
cholesterol in apoB-lipoproteins because these cells did not receive OA:TC.
These studies indicated that efflux mechanisms might contribute
(25–30%) to apoB-free cholesterol secretion by Caco-2 cells.
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Pre-absorbed Cholesterol Is Preferentially Used for Secretion with Chylomicrons—In order to comprehend reasons for the secretion of cholesterol with chylomicrons during pulse-chase protocol, we studied the kinetics of secretion of two different cholesterol pools, newly absorbed and pre-absorbed. To monitor newly absorbed cholesterol, cells were supplemented with radiolabeled cholesterol for the indicated time points. To study the metabolism of pre-absorbed cholesterol, cells were first labeled with [3H]cholesterol for 17 h and chased for indicated time points (Fig. 6). Fig. 6A shows changes in the newly and pre-absorbed cellular pools of cholesterol. As expected, the cellular accumulation of newly absorbed cholesterol increased with time due to uptake (Fig. 6A). On the other hand, the pre-absorbed cellular cholesterol pool slightly decreased with time. Analysis of the basolateral media revealed that the amounts of newly absorbed cholesterol were significantly higher than those of the pre-absorbed pools at all time points (Fig. 6B). The preferential secretion of newly absorbed cholesterol was especially significant at early time points. For example, at 1 h, cellular levels of pre-absorbed cholesterol were 5.6-fold higher than the newly absorbed cholesterol (Fig. 6A). Yet the basolateral media contained 2-fold higher amounts of newly absorbed cholesterol compared with pre-absorbed cholesterol (Fig. 6B). The cellular ratio of pre- to newly absorbed cholesterol decreased with time, most likely due to increased uptake of radiolabeled cholesterol (Fig. 6C). The secreted media contained 2–3-fold higher amounts of newly absorbed cholesterol, and the ratio between pre- and newly absorbed cholesterol was always <1 (Fig. 6C). These data suggest that differentiated Caco-2 cells predominantly secrete newly absorbed cholesterol, and this secretion is independent of the amounts present in the cellular pool.
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Next we determined the pools of cholesterol secreted with apoB
(Fig. 6D) and without
apoB (Fig. 6E). ApoB
lipoproteins contained 2–8-fold higher amounts of pre-absorbed
cholesterol than newly absorbed cholesterol at all time points
(Fig. 6D). In
contrast, apoB-free fractions contained equal amounts of newly absorbed and
pre-absorbed cholesterol up to 2 h (Fig.
6E). This balance shifted in favor of newly absorbed
cholesterol with time, and the amounts of newly absorbed cholesterol were
4-fold higher at 24 h (Fig.
6E). The pre- to newly absorbed cholesterol ratio in apoB
lipoproteins was 3 in the first 2 h
(Fig. 6F), increased
to 7.5-fold at 4 and 6 h, and returned to 3 at 8–24 h. In
apoB-free fractions this ratio was 0.3 and decreased to 0.1 at 24 h. From
these studies we conclude that the pre-absorbed cholesterol was preferentially
secreted with apoB lipoproteins, and the newly absorbed cholesterol was mainly
secreted unassociated with these lipoproteins.
Secretion of Free and Esterified Cholesterol—So far, we
studied the transport of total cholesterol across the differentiated Caco-2
cells. It is known that cholesterol exists in un-esterified (free) and
esterified forms. To learn about differences in the transport of free and
esterified cholesterol, we first studied their distribution in different
lipoprotein fractions. Lipoprotein analysis revealed that TC-treated cells
secreted free cholesterol mainly in apoB-free fractions
(Fig. 7A,
fractions 9 and 10). In these fractions, esterified
cholesterol constituted 3–4% of the total cholesterol
(Fig. 7B). OA
increased the amounts of free (Fig.
7A) and esterified cholesterol
(Fig. 7B) in apoB
lipoproteins (fractions 1–2, large and small chylomicrons) but had no
effect on the free and esterified cholesterol present in the bottom apoB-free
fractions 9 and 10. In lipoproteins, esterified cholesterol constituted
35% of the total cholesterol. BMS200150 completely abolished the secretion
of free (Fig. 7A) and
esterified cholesterol (Fig.
7B) in apoB lipoproteins but had no effect on the
secretion of free and esterified cholesterol in apoB-free fractions 9 and 10.
Modulation of efflux by LXR/RXR agonists only affected free cholesterol in
bottom fractions (data not shown). These studies highlighted the differential
modulation of free and esterified cholesterol secretion by apoB-independent
and apoB-dependent pathways.
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To identify cholesterol pools used for cellular esterification, we measured the amounts of pre- and newly absorbed free and esterified cholesterol in cells at different time points (Fig. 8). Similar to increases observed for total cellular cholesterol (Fig. 6A), the amounts of newly absorbed free cholesterol increased with time (Fig. 8A). On the other hand, the amounts of pre-absorbed free cholesterol slightly decreased with time in cells. Next, we looked for the esterification of different cholesterol pools (Fig. 8B). The esterification of both the newly and pre-absorbed cholesterol increased with time (Fig. 8B). However, the rate of esterification of pre-absorbed cholesterol appeared to be faster than that of newly absorbed cholesterol. Quantification of the cellular esterified cholesterol revealed that about 8% of the pre-absorbed cholesterol was in the esterified form at 1 h, and this increased to 19% at 24 h (Fig. 8C). At 1 h, 3% of the newly absorbed cholesterol was in esterified form and increased to 5% at 24 h. These studies indicate that cellular esterification mechanisms do not discriminate between pre-absorbed and newly absorbed cholesterol pools and that both the pools are accessible for esterification.
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Next, we identified different metabolic pools of free and esterified
cholesterol in apoB lipoproteins and apoB-free fractions at 24 h. The amounts
of pre-absorbed free cholesterol in lipoprotein fractions were about 2-fold
higher than those of newly absorbed cholesterol
(Fig. 8D). In
contrast, apoB-free fractions contained 8–10-fold higher amounts of
newly absorbed free cholesterol (Fig.
8D). Quantification of cholesterol esters in different
fractions showed that cholesterol esters derived from pre-absorbed cholesterol
pool were 2-fold higher than those derived from newly absorbed cholesterol
in apoB lipoproteins (Fig.
8E). In apoB-free fractions, the amounts of cholesterol
esters derived from newly absorbed cholesterol were higher
(Fig. 8E). These
studies indicate that apoB-free fractions contain free and esterified
cholesterol mainly derived from the newly absorbed cholesterol pool. In
contrast, apoB lipoproteins derive their free and esterified cholesterol
predominantly from the pre-absorbed cholesterol pool. Next, we quantified the
percent distribution of esterified cholesterol in different fractions
(Fig. 8F). In apoB
lipoproteins, 35% of both newly and pre-absorbed cholesterol was in the
esterified form. In contrast, apoB-free fractions contained only 4–8% of
esterified cholesterol. These studies indicate that majority of the free
cholesterol is secreted independent of apoB, whereas cholesterol esters are
secreted mainly associated with lipoproteins.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cholesterol Transport by Intestinal Cells—The studies reported here provide a new understanding about the mechanisms involved in the transport of cholesterol by intestinal cells. Most of the peripheral cells receive cholesterol by receptor-mediated endocytosis of lipoproteins and remove excess cholesterol by efflux pathways. Intestinal cells are unique because, in addition to receptor-mediated endocytosis from the basolateral side (30), these cells take up cholesterol from the intestinal lumen and secrete cholesterol as part of chylomicrons. Contrary to the general belief, the data presented here indicate that newly absorbed cholesterol is neither immediately available for secretion with apoB lipoproteins nor exclusively secreted as part of chylomicrons. Based on our data, cholesterol transport by enterocytes can be broadly classified into two independently modulated apoB-dependent and apoB-independent pathways. These pathways show differential specificity toward the free and esterified cholesterol. The apoB-dependent pathway transports the esterified cholesterol, whereas both pathways secrete free cholesterol.
Although it is generally believed that cholesterol is mainly transported as
part of chylomicrons, there are reports in the literature describing the
presence of cholesterol in lymph lipoproteins other than chylomicrons. Riley
et al. (31) have
shown that in normal chow-fed rats d < 1.006 and d >
1.006 g/ml lymph lipoproteins carry 92 and 8% of cholesterol, respectively.
The transport of cholesterol in d > 1.006 g/ml lipoproteins
increased to 13% in olive oil-fed rats and to 42% in cholesterol-fed rats.
They concluded that increases in d > 1.006 g/ml lipoproteins may
be due to impairment of the assembly of d < 1.006 g/ml lymph
lipoproteins by high cholesterol diet
(31). Klein and Rudel
(32,
33) have shown that
cholesterol is present in both d < 1.006 and d > 1.006
g/ml lipoprotein fractions in non-human primates. They showed that 7 and 20%
of the exogenous and endogenous cholesterol is transported on d >
1.006 g/ml fractions. In d > 1.006 g/ml lipoproteins, cholesterol
was present in region III lymph lipoproteins that resembled HDL and did not
contain apoB (33). They
thought that these lipoproteins might have been derived from plasma by
filtration (32,
33). Beaumier-Gallon et
al. (34) reported that
7% of the newly absorbed cholesterol was not associated with apoB48
triglyceride-rich lipoproteins in healthy human beings. We interpret these
studies to support our hypothesis that intestine secretes cholesterol using
both apoB-dependent and apoB-independent pathways. Certainly, the
apoB-dependent pathway contributes significantly to cholesterol transport
during the postprandial state. The apoB-independent pathways may be more
important during very early phases of absorption, in the fasting state, and
during high cholesterol diet.
It is known that cholesterol absorption is severely impaired in the absence of apoB lipoprotein assembly and secretion. In two abetalipoproteinemia subjects with defective microsomal triglyceride transfer protein, cholesterol absorption was found to be <3% of the normal individuals (35). In intestine-specific apoB knockout mice, Young et al. (36) have reported no cholesterol absorption. These data are usually interpreted to suggest that apoB lipoprotein assembly is the only mechanism present for cholesterol transport across the intestinal epithelial cells. Nonetheless, there could be other explanations for the very low to no cholesterol absorption in the absence of apoB lipoprotein assembly. We have eliminated the possibility that MTP may be required for apoB-independent secretion pathway; inhibition of apoB lipoprotein assembly by MTP antagonists has no effect on apoB-independent cholesterol secretion (Figs. 3 and 6). It is possible that apoB-independent secretion is secondarily affected by the pathologic situations arising due to the excess accumulation of lipids in the absence of apoB lipoprotein assembly and secretion. Lipid droplets may extract cholesterol from cellular membranes and deplete its availability for apoB-independent secretory pathways.
Secretion of Cholesterol with ApoB Lipoproteins—Characteristics of cholesterol secretion with apoB lipoproteins are very different from that of triglyceride transport. We have shown that newly synthesized triglycerides are primarily secreted with chylomicrons in Caco-2 cells (17). In contrast, cholesterol taken up by these cells is not immediately available for secretion along with chylomicrons (Figs. 2, 3, 4). Instead, enterocytes sequester cholesterol away from the site of chylomicron assembly. Subsequently, these cells secrete small portions of cholesterol with chylomicrons. The mechanisms of immediate sequestration and targeted secretion of cholesterol can explain in vivo studies demonstrating cholesterol secretion into the blood over a long period after its consumption in periodic rhythmicity that follows feeding and chylomicron secretion (34). Thus, a significant time lag occurs from the time of cellular cholesterol uptake to its secretion with apoB lipoproteins.
It is known that differentiated Caco-2 cells secrete two types of apoB-containing particles (17, 18). In the absence of fatty acids, apoB is secreted as smaller LDL-size particles by Caco-2 cells, whereas it is secreted on larger lipoproteins after the supplementation of free fatty acids. These data were interpreted to suggest that apoB lipoproteins are synthesized as smaller primordial lipoprotein particles that are converted to larger nascent lipoproteins by core expansion (37–39). Here we show that smaller apoB lipoproteins do not contain detectable levels of cholesterol. Instead, cholesterol is secreted with larger apoB lipoproteins indicating that their assembly is essential for apoB-dependent transport pathway. To explain the preferential secretion with larger lipoproteins, we propose that cholesterol is added during the core expansion of primordial lipoproteins into larger nascent lipoproteins.
Secretion of Cholesterol by ApoB-independent Pathways—We
consistently observed that cholesterol was present in the non-apoB fractions
(Figs. 2, fractions
5–8). The apoB-independent cholesterol secretion was not due to
paracellular leakage (data not shown) and was observed in serum-free media
(Fig. 2) and in media
containing either BSA (Fig. 2)
or FBS (data not shown). Other investigators have observed cholesterol in
apoB-free lipoproteins in Caco-2 cell culture media
(12) and in the lymph of
monkeys (32,
33) and rats
(31). In isolated human
chylomicrons, 7% of cholesterol was in apoB48-free fractions
(34). Based on these studies,
we propose that apoB-independent pathways contribute to cholesterol transport
across the intestinal epithelial cells.
One possible mechanism for apoB-independent transport of cholesterol is the
efflux pathway. Cholesterol efflux mediated by ABCA1 is implicated in
apoB-free cholesterol secretion because of its inhibition by glyburide and
up-regulation by LXR/RXR agonists (Fig.
5). In our studies, this process contributed to 25–30%
of cholesterol secretion. Although the data about cholesterol absorption have
been equivocal in knockout mice, cholesterol absorption is decreased by
10–15% in some ABCA1 knockout studies
(40,
41) and increased in other
studies (42). In birds, 79% of
the cholesterol is absorbed by the ABCA1-mediated efflux pathway
(43). Thus, we suggest that
cholesterol efflux pathway in enterocytes can actually contribute to the
absorption of cholesterol. Note that efflux pathways are implicated in reverse
cholesterol transfer for excretion out of the body.
Another pathway that could contribute to apoB-independent cholesterol transport is the intracellular assembly and secretion of HDL by the intestine (33, 44–46). ApoAI deficiency has been correlated with decreased total and HDL cholesterol in mice and men (47, 48). This is generally attributed to the ability of HDL to transport cholesterol in the plasma. Careful studies are needed to correlate cholesterol absorption with apoAI genotype.
Efflux from the apical side of enterocytes to the intestinal lumen has been suggested as a mechanism for the decreased cholesterol absorption (49). Recent studies have shown that LXR/RXR agonists do not affect efflux from the apical side to bile salt micelles and other acceptors (21, 22). We have also made similar observations (data not shown). In contrast, our data and that of others (21, 22) indicate that efflux may actually play a role in the import of cholesterol from the intestinal lumen to the circulation.
Modulation of apoB-independent cholesterol secretion may be a useful target to lower plasma cholesterol levels. A favorite approach to lower plasma cholesterol is to inhibit assembly and secretion of apoB lipoproteins. For this purpose, various MTP inhibitors are being evaluated (50). Our data indicate that a better strategy to control plasma cholesterol might be to inhibit the apoB-independent cholesterol transport pathway. Although this strategy is expected to result in moderate inhibition of cholesterol absorption, it would avoid complications related to triglyceride accumulation that arise as a consequence of the inhibition of apoB lipoprotein secretion.
Two Different Metabolic Pools of Cholesterol and Their Differential Secretion—Newly absorbed and pre-absorbed pools may represent dietary and biliary cholesterol, respectively. We observed that these two metabolic pools of cholesterol are differentially targeted for secretion. The newly absorbed and pre-absorbed cholesterols are preferentially secreted via apoB-independent and apoB-dependent pathways, respectively. The preferential secretion of newly absorbed cholesterol by an apoB-independent pathway is in agreement with the studies of Klein and Rudel (32, 33) who showed that greater proportions of exogenous cholesterol were in d > 1.006 g/ml lymph lipoproteins. The differential secretion of two metabolic pools probably reflects the compartmentalization of cholesterol in different intracellular organelles. The newly absorbed cholesterol is probably incorporated into the plasma membrane as suggested by Field et al. (12). This pool is available for efflux in the early periods from both the apical and basolateral sides. However, it becomes resistant to efflux with time because it is transported to other intracellular organelles. Subsequently, it becomes available for secretion with apoB-containing lipoproteins.
In contrast to the differential secretion, these two metabolic pools are equally accessible for esterification. This is in agreement with studies demonstrating no effect on the esterification of plasma membrane cholesterol by newly synthesized and absorbed cholesterol (51). The efficient cholesterol esterification may be due to the presence of at least two enzymes that might have access to different cholesterol pools simultaneously (9, 10).
In summary, we have shown that cholesterol transport by enterocytes is a highly regulated process involving multiple mechanisms. ApoB-dependent pathways mainly transport cholesterol esters and pre-absorbed free cholesterol. It is induced by oleic acid and inhibited by MTP inhibitors. Cholesterol secretion by this pathway is dependent on the assembly of larger lipoproteins indicating that cholesterol is probably added to these lipoproteins at later stages of assembly. ApoB-independent pathways are primarily involved in the secretion of newly absorbed cholesterol and may play a role in the homeostasis of cellular free cholesterol. We have identified that ABCA1-mediated efflux contributes to this pathway. It is likely that additional mechanisms also contribute to apoB-independent cholesterol secretion. These pathways may serve as new targets to lower plasma cholesterol levels.
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FOOTNOTES |
|---|
* This work was supported by National Institutes of Health Grants DK46900 and HL64272 and an Established Investigator award by the American Heart Association (to M. M. H.). 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.
To whom correspondence should be addressed: Depts. of Anatomy and Cell
Biology, and Pediatrics, State University of New York Downstate Medical
Center, 450 Clarkson Ave., Box 5, Brooklyn, NY 11203. Fax: 718-270-2462;
E-mail:
mahmood.hussain{at}downstate.edu.
1 The abbreviations used are: apoB, apolipoprotein B; BSA, bovine serum
albumin; DMEM, Dulbecco's modified Eagle's medium containing high glucose
supplemented with L-glutamine and antibiotic/antimycotic mixture;
FBS, fetal bovine serum; MTP, microsomal triglyceride transfer protein; OA,
oleic acid; RA, retinoic acid; SFM, serum-free media; TC, taurocholate;
22(OH)C, 22-hydroxycholesterol; LXR, ligand X receptor; RXR, retinoid X
receptor; VLDL, very low density lipoprotein; LDL, low density
lipoprotein.
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