Multiple, Independently Regulated Pathways of Cholesterol Transport across the Intestinal Epithelial Cells
2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês
10.1074/jbc.m301177200
ISSN1083-351X
AutoresJahangir Iqbal, Kamran Anwar, M. Mahmood Hussain,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoThe 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. 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. Due to a significant positive correlation between cholesterol absorption and plasma cholesterol levels (1Kesaniemi Y.A. Miettinen T.A. Eur. J. Clin. Invest. 1987; 17: 391-395Crossref PubMed Scopus (161) Google Scholar, 2Wilson M.D. Rudel L.L. J. Lipid Res. 1994; 35: 943-955Abstract Full Text PDF PubMed Google Scholar, 3McGill Jr., H.C. Am. J. Clin. Nutr. 1979; 32: 2664-2702Crossref PubMed Scopus (116) Google Scholar), 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 (2Wilson M.D. Rudel L.L. J. Lipid Res. 1994; 35: 943-955Abstract Full Text PDF PubMed Google Scholar). 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 (4Phan C.T. Tso P. Front Biosci. 2001; 6: D299-D319Crossref PubMed Google Scholar, 5Tso P. Karlstad M.D. Bistrian B.R. DeMichele S.J. Am. J. Physiol. 1995; 268: G568-G577Crossref PubMed Google Scholar, 6Carey M.C. Small D.M. Bliss C.M. Annu. Rev. Physiol. 1983; 45: 651-677Crossref PubMed Scopus (643) Google Scholar). 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 (2Wilson M.D. Rudel L.L. J. Lipid Res. 1994; 35: 943-955Abstract Full Text PDF PubMed Google Scholar, 4Phan C.T. Tso P. Front Biosci. 2001; 6: D299-D319Crossref PubMed Google Scholar, 5Tso P. Karlstad M.D. Bistrian B.R. DeMichele S.J. Am. J. Physiol. 1995; 268: G568-G577Crossref PubMed Google Scholar, 6Carey M.C. Small D.M. Bliss C.M. Annu. Rev. Physiol. 1983; 45: 651-677Crossref PubMed Scopus (643) Google Scholar). Currently, it is believed that the uptake of cholesterol by the enterocytes is the rate-limiting step in cholesterol absorption (7Dawson P.A. Rudel L.L. Curr. Opin. Lipidol. 1999; 10: 315-320Crossref PubMed Scopus (96) Google Scholar). This is supported by the observation that a man who ate 25 eggs a day did not develop hypercholesterolemia because he absorbed less cholesterol (8Kern Jr., F. N. Engl. J. Med. 1991; 324: 896-899Crossref PubMed Scopus (104) Google Scholar). After uptake, enterocytes esterify cholesterol, a process mediated by acyl-coenzyme A:cholesterol acyltransferases (9Chang T.Y. Chang C.C. Cheng D. Annu. Rev. Biochem. 1997; 66: 613-638Crossref PubMed Scopus (441) Google Scholar, 10Chang T.Y. Chang C.C. Lin S. Yu C. Li B.L. Miyazaki A. Curr. Opin. Lipidol. 2001; 12: 289-296Crossref PubMed Scopus (211) Google Scholar), 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 (2Wilson M.D. Rudel L.L. J. Lipid Res. 1994; 35: 943-955Abstract Full Text PDF PubMed Google Scholar). 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 (11Chen H. Born E. Mathur S.N. Johlin Jr., F.C. Field F.J. Biochem. J. 1992; 286: 771-777Crossref PubMed Scopus (65) Google Scholar, 12Field F.J. Born E. Mathur S.N. J. Lipid Res. 1995; 36: 2651-2660Abstract Full Text PDF PubMed Google Scholar) 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 (13Field F.J. Born E. Chen H. Murthy S. Mathur S.N. J. Lipid Res. 1995; 36: 1533-1543Abstract Full Text PDF PubMed Google Scholar), 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. Materials—Antibodies used for the determination of apoB 1The 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. have been described (14Hussain M.M. Zhao Y. Kancha R.K. Blackhart B.D. Yao Z. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 485-494Crossref PubMed Scopus (63) Google Scholar, 15Bakillah A. Zhou Z. Luchoomun J. Hussain M.M. Lipids. 1997; 32: 1113-1118Crossref PubMed Scopus (38) Google Scholar). 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 × 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 (10× 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 (16Redinger R.N. Small D.M. Arch. Intern. Med. 1972; 130: 618-630Crossref PubMed Scopus (104) Google Scholar). 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 × 105 cells/cm2. To induce differentiation of these cells, media were changed every other day for 21 days (17Luchoomun J. Hussain M.M. J. Biol. Chem. 1999; 274: 19565-19572Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 18Nayak N. Harrison E.H. Hussain M.M. J. Lipid Res. 2001; 42: 272-280Abstract Full Text Full Text PDF PubMed Google Scholar, 19During A. Hussain M.M. Morel D.W. Harrison E.H. J. Lipid Res. 2002; 43: 1086-1095Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). 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 (17Luchoomun J. Hussain M.M. J. Biol. Chem. 1999; 274: 19565-19572Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). 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) (17Luchoomun J. Hussain M.M. J. Biol. Chem. 1999; 274: 19565-19572Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). 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 (20Wang N. Silver D.L. Thiele C. Tall A.R. J. Biol. Chem. 2001; 276: 23742-23747Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar, 21Murthy S. Born E. Mathur S.N. Field F.J. J. Lipid Res. 2002; 43: 1054-1064Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) 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 (21Murthy S. Born E. Mathur S.N. Field F.J. J. Lipid Res. 2002; 43: 1054-1064Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 22Ohama T. Hirano K. Zhang Z. Aoki R. Tsujii K. Nakagawa-Toyama Y. Tsukamoto K. Ikegami C. Matsuyama A. Ishigami M. Sakai N. Hiraoka H. Ueda K. Yamashita S. Matsuzawa Y. Biochem. Biophys. Res. Commun. 2002; 296: 625-630Crossref PubMed Scopus (58) Google Scholar), 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 (23Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43084) Google Scholar). 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 (24Weiser M.M. J. Biol. Chem. 1973; 248: 2536-2541Abstract Full Text PDF PubMed Google Scholar) as elaborated by Pinkus (25Pinkus L.M. Methods Enzymol. 1981; 77: 154-162Crossref PubMed Scopus (58) Google Scholar) and Cartwright and Higgins (26Cartwright I.J. Higgins J.A. J. Lipid Res. 1999; 40: 1357-1365Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly, proximal 1/3rd to ½ 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 (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217370) Google Scholar). 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 (14Hussain M.M. Zhao Y. Kancha R.K. Blackhart B.D. Yao Z. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 485-494Crossref PubMed Scopus (63) Google Scholar, 15Bakillah A. Zhou Z. Luchoomun J. Hussain M.M. Lipids. 1997; 32: 1113-1118Crossref PubMed Scopus (38) Google Scholar). 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 (17Luchoomun J. Hussain M.M. J. Biol. Chem. 1999; 274: 19565-19572Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 18Nayak N. Harrison E.H. Hussain M.M. J. Lipid Res. 2001; 42: 272-280Abstract Full Text Full Text PDF PubMed Google Scholar, 19During A. Hussain M.M. Morel D.W. Harrison E.H. J. Lipid Res. 2002; 43: 1086-1095Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). 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. 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. 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. 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.Table IEffect of oleic acid and BSM200150 on the secretion of total apoB, apoA1, and cholesterol by differentiated Caco-2 cellsTCOA:TCOA:TC + BMS200150ApoB (ng·well-1)421.1 ± 20.7691.3 ± 57.2ap < 0.03 when compared with TC.271.9 ± 8.9bp < 0.01 when compared with OA:TC and p < 0.02 when compared with control.ApoA1 (μg·well-1)11.3 ± 0.710.3 ± 0.911.9 ± 0.6Cholesterol (dpm·10-3·well-1)58 ± 0.582.2 ± 1.9cp < 0.04 compared with TC.54.9 ± 2.6dp < 0.01 compared with OA:TC.a p < 0.03 when compared with TC.b p < 0.01 when compared with OA:TC and p < 0.02 when compared with control.c p < 0.04 compared with TC.d p < 0.01 compared with OA:TC. Open table in a new tab 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 (18Nayak N. Harrison E.H. Hussain M.M. J. Lipid Res. 2001; 42: 272-280Abstract Full Text Full Text PDF PubMed Google Scholar). 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 (17Luchoomun J. Hussain M.M. J. Biol. Chem. 1999; 274: 19565-19572Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 18Nayak N. Harrison E.H. Hussain M.M. J. Lipid Res. 2001; 42: 272-280Abstract Full Text Full Text PDF PubMed Google Scholar). 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 wit
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