MICROSOMAL TRIGLYCERIDE TRANSFER PROTEIN ENHANCES CELLULAR CHOLESTERYL

MICROSOMAL TRIGLYCERIDE TRANSFER PROTEIN ENHANCES CELLULAR CHOLESTERYL ESTERIFICATION BY RELIEVING PRODUCT INHIBITION

Jahangir Iqbal*, Lawrence L. Rudel†, M. Mahmood Hussain*

From the Department of Anatomy and Cell Biology, and Pediatrics*, State University of New York Downstate Medical Center, Brooklyn, NY 11203; Departments of Pathology and

Biochemistry†, Wake Forest University School of Medicine, Winston-Salem, NC 27157 Running head: MTP deficiency increases cellular free cholesterol

Address correspondence to M. Mahmood Hussain E-mail: [email protected], Phone: 718-270-4790, Fax: 718-270-2462

Cholesteryl ester synthesis by the acyl-CoA:cholesterol acyltransferase enzymes, ACAT1 and ACAT2 is, in part, a cellular homeostatic mechanism to avoid toxicity associated with high free cholesterol levels. In hepatocytes and enterocytes, cholesteryl esters are secreted as part of apoB-lipoproteins, whose assembly is critically dependent on microsomal triglyceride transfer protein (MTP). Conditional genetic ablation of MTP reduces cholesteryl esters and enhances free cholesterol in the liver and intestine without diminishing ACAT1 and ACAT2 mRNA levels. As expected, increases in hepatic free cholesterol are associated with decreases in 3-hydroxy-3-methyl-glutaryl-CoA reductase and increases in ATP binding cassette transporter 1 mRNA levels. Chemical inhibition of MTP also decreases esterification of cholesterol in Caco-2 and HepG2 cells. Conversely, co-expression of MTP and apoB in AC29 cells stably transfected with ACAT1 and ACAT2 increases cholesteryl ester synthesis. Liver and enterocyte microsomes from MTP-deficient animals synthesize lesser amounts of cholesteryl esters in vitro, but addition of purified MTP and LDL corrects this deficiency. Enrichment of microsomes with cholesteryl esters also inhibits cholesterol ester synthesis. Thus,

MTP enhances cellular cholesterol esterification by removing cholesteryl esters from their site of synthesis and depositing them into nascent apoB-lipoproteins. Therefore, MTP plays a novel role in regulating cholesteryl ester biosynthesis in cells that produce lipoproteins. We speculate that non-lipoprotein producing cells may use different mechanisms to alleviate product inhibition and modulate cholesteryl ester biosynthesis.

High concentrations of free cholesterol are toxic to cells (1). Therefore, cells maintain exquisite control over free cholesterol levels via a complex set of homeostatic mechanisms (2) involving cholesteryl esterification, cholesterol biosynthesis, and receptor-mediated endocytosis of cholesterol-rich, low-density

lipoproteins (LDL). Excess free cholesterol down-regulates cholesterol biosynthesis and receptor-mediated endocytosis of lipoproteins but stimulates cholesteryl esterification resulting in storage of excess cholesterol in the form of cytosolic-neutral lipid droplets. Cholesteryl esterification

involves covalent attachment of fatty acids to the 3-position hydroxyl group of cholesterol by acyl-coA:cholesterol acyltransferase (ACAT) enzymes [for reviews, (3-7)]. Chang and associates cloned

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the first enzyme involved in cholesterol esterification using an expression cloning strategy (8;9), and another has since been cloned (10-12). These two enzymes (9-13), ACAT1 and ACAT2, mediate cellular cholesteryl esterification activity (3;5;7). Both proteins carry out similar enzymatic reactions, but have different tissue distributions. ACAT1 is present in a variety of tissues (14-17), whereas ACAT2 expression is restricted to enterocytes and hepatocytes (14;17-19). Gene deletion experiments in mice indicate that ACAT1 maintains cholesterol levels in cell membranes, whereas ACAT2 is responsible for cholesteryl ester secretion with apoB-lipoproteins. Ablation of ACAT1 results in less atherosclerosis and no esters in brain, skin, adrenal glands, or macrophages, whereas the absence of ACAT2 results in decreased cholesteryl esters in hepatocytes, enterocytes and apoB-lipoproteins [for reviews, (3-7)].

Both ACAT1 and ACAT2 are integral membrane proteins with multiple transmembrane domains (20-22) and reside in the endoplasmic reticulum (ER). Cholesteryl esters formed by ACAT partition into the ER membrane, but the mechanism of transporting these esters from the ER to cytosolic lipid droplets is unknown. FIT1 and FIT2 may play a role in lipid droplet biogenesis (23). Nonetheless, in the liver and intestine, some cholesteryl esters are incorporated into apoB-lipoproteins by an ER-resident chaperone, the microsomal triglyceride transfer protein (MTP). MTP is absolutely required for lipoprotein assembly and secretion [reviewed in (24-27)], and MTP deficiency in microsomes of abetalipoproteinemia patients results in the absence of apoB-lipoproteins in the plasma (28). Furthermore, there is a significant correlation between the diurnal expression of MTP and daily variations in plasma

apoB-lipoproteins (29). In addition, MTP regulates the biosynthesis of CD1d, a glycolipid antigen-presenting molecule (30-32). Besides its role in the biosynthesis of apoB-lipoproteins and CD1d, MTP facilitates the transfer of triglycerides into the ER lumen. MTP activity is required for the accumulation of triglyceride, but not phosphatidylcholine, within the ER lumen (33;34). Inhibition of MTP leads to the accumulation of triglycerides in cells (35-38). However, very little attention has been paid to understand the role of MTP in cellular cholesterol homeostasis. Here, we describe a novel role of MTP in the control of cholesteryl ester biosynthesis.

Experimental Procedures

Animals: Mttp Ldlr Apob Tg(Mx1-cre)1Cgn/J

tm2Sgy tm1Her tm2Sgy

mice (stock number 004192) described by Lieu et al (39) were obtained from the Jackson Laboratory and kept on a 07:00-19:00 h lighting schedule. All animals had free access to water and standard laboratory chow. To induce mttp gene deletion, mice were injected (200 µl) three times intraperitoneally with 500 µg of polyinosinic-polycytidylic ribonucleic acid (pIpC) on alternate days. Control mice received PBS injections. Food was withdrawn 16 h before the sacrifice of the mice. On the day of the experiment, mice were anesthetized and blood was collected from the heart. Liver and proximal intestine segments (~2-3 cm) were collected, washed in ice-cold PBS, cut into small pieces, and used for lipid extraction or to measure different protein activities. Lipids were extracted from tissue homogenates following the Bligh and Dyer method (40). Triglyceride (Infinity Triglyceride, TR22421) levels in the tissues were determined using commercial kits (Thermo Trace, Melbourne, Australia). Total and free cholesterol levels were measured using kits

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from Wako Chemicals (Germany). Esterified cholesterol was measured by subtracting the free cholesterol from the total cholesterol.

Determination of MTP activity in tissues: After extensive washes with ice-cold PBS, small pieces (0.1 g) of liver and ~ 1-cm segments of proximal small intestine were homogenized with 1 ml of ice-cold 1 mM Tris-HCl, pH 7.6, 1 mM EGTA, and 1 mM MgCl2 buffer in a glass homogenizer. The homogenates were centrifuged (SW55 Ti rotor, 50,000 rpm, 10 °C, 1 h), and supernatants were used for an MTP assay as described (41;42) using a kit (Chylos, Inc.).

ACAT activity: Microsomes were prepared as described by Cheema et al. (43). Briefly, liver pieces (50-100 mg), jejunal segments (1-2 cm), or enterocytes prepared from the whole jejunum were homogenized in 1 ml of ice-cold 0.1 M K2HP04 buffer, pH 7.4, containing 0.3 M sucrose, 1 mM EDTA, 50 mM KF, 50 mM KC1, and 5 mM DTT using a 2-ml capacity Potter-Elvehjem glass-Teflon homogenizer. Homogenates were centrifuged for 20 min at 10,000 rpm at 4 °C to remove cell debris, and the supernatants were re-centrifuged for 120 min at 50,000 rpm in a SW-55 rotor (Beckman Instruments) at 4 °C. The microsomal pellet was re-suspended in 1 ml of the same buffer and several aliquots were rapidly frozen in liquid nitrogen and stored at -80 °C until assayed. ACAT activity assays using

purified microsomes were performed by the method of Erickson et al (44). Briefly, the incorporation of palmitoyl coenzyme A into cholesteryl esters was determined using 100 µg of microsomal protein and [3H]cholesterol (0.2 µCi, 40-60 Ci/mmol, PerkinElmer) in an ACAT assay buffer of 0.25 M sucrose, 100 mM Tris-HCl, 1 mM EDTA, pH 7.5, in a final volume of 200 µl. The mixture was incubated for 30 minutes at 37 °C and the activity was stopped by the

addition of 1 ml of chloroform-methanol 2:1. Lipids were extracted from the reaction mixture by the Bligh-Dyer method (40) and separated by thin layer chromatography (TLC). Bands corresponding to free and esterified cholesterol were quantified by scintillation counting. In some experiments, incorporation of [14C]pamitoyl coenzyme A (0.1 µCi, 40-60 Ci/mmol, PerkinElmer) into cholesteryl esters was determined using 100 µg of microsomal protein and 20 µg of

exogenous cholesterol or 50 nmol of free cholesterol in cyclodextran (45). At the end of the reaction, lipids were extracted, separated by TLC, and the amounts of the radioactivity in cholesteryl esters were quantified using a phosphorimager.

Product inhibition of ACAT enzyme was studied by incubating palmitoyl coenzyme A with 100 µg of microsomal protein, [3H]cholesterol, and increasing concentrations of cholesteryl palmitate. Cholesteryl palmitate was prepared by dissolving 62.5 mg of cholesteryl palmitate in 1 ml of chloroform:methanol (2:1), evaporating the solvent under nitrogen, suspending the powder in 10 ml of ACAT assay buffer, and sonicating for 30 minutes on ice using a Sonic Dismembrator 550 (Fisher Scientific) set at speed 3. After sonication, cholesterol levels were measured in the suspension and diluted to obtain a final concentration of 1 mM. The sonication resulted in very small amounts of cholesterol esters suspended in buffer and we did not check for stability of the preparation. These suspensions were used immediately with no further characterization. In some experiments, enrichment of microsomes with cholesteryl esters was achieved by incubating 100 mg microsomal protein with increasing concentrations of cholesteryl palmitate suspension for 30 min in a total volume of 1 ml of ACAT activity assay buffer. After incubation, microsomes were washed and re-pelleted for 120 min at

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50,000 rpm in a SW-55 rotor at 4 °C. Microsomes were re-suspended in 1 ml of assay buffer and 100 µl was used to extract lipids. Total cholesterol was measured in the lipid extracts with a cholesterol kit. Aliquots of the enriched microsomes (100 µg protein, ~1.2 µl) were also used to measure ACAT activity in the presence or absence of MTP and/or LDL (1 µg of protein per reaction).

mRNA quantification and primers: Total RNA was isolated using TriZolTM (Invitrogen). The purity and integrity of RNA was assessed by the A260/A280 ratio and 1% agarose gel electrophoresis, respectively. Only RNAs with ratios more than 1.7 were used for cDNA synthesis. The first strand cDNA was synthesized using Omniscript RT (Qiagen) kit. Briefly, 2 µg of total RNA, 1 µM random primers (Invitrogen), 0.5 mM dNTP solution, and 0.5 U/µl Omniscript Reverse Transcriptase were incubated at 37 °C for 1 h in 20 µl RT buffer and the reaction was terminated by incubating at 95 °C for 5 minutes. Each quantitative RT-PCR reaction was performed in 20 µl consisting of 5 µl cDNA sample (1:100 dilution of the first strand cDNA sample) and 15 µl of PCR master mix solution containing 1X PCR reaction buffer, 6 mM MgCl2, 200 nM primer pair, 0.025 U/µl HotStar GoldTM DNA polymerase, 200 µM dNTP solution, and 0.3 µl SYBR Green I solution (qPCRTM Core Kit for SYBR Green I, Eurogentec). The PCR was performed by incubating the reaction mixture first for 10 min at 95 °C, followed by 40 cycles of 15 sec incubations at 95 °C and 1 min at 60 °C in the ABI 7000 SDS PCR machine. The data were analyzed using the ∆∆CT method according to manufacturer’s instructions and presented as arbitrary units. The primers used in this study were designed with PrimerExpress 3.0 software (Applied Biosystems, CA) and are presented as Table 1.

Studies with cells: Caco-2 (human colon carcinoma) cells obtained from the American Type Culture Collection (Manassas, VA) were differentiated in Transwells as described (46-49). Differentiated Caco-2 cells were then pulsed with 1 µCi/ml of either [3H]oleic acid (15-60 Ci/mmol, PerkinElmer) or [3H]cholesterol for 17 h in 10% FBS on the apical side and serum free media on the basolateral side, washed, and then chased with serum-free media added to the apical side containing 1.6 mM oleic acid (OA):0.5 mM taurocholate in the absence or presence of the MTP inhibitor, BMS200150 (10 µM), for 24 h. In some experiments, different concentrations of the MTP inhibitor, BMS197636, or ACAT inhibitor, 148817, in dimethylsulfoxide (DMSO) were added to the apical side for studying their effects on cholesteryl esterification either alone or in combination. DMSO levels were less than or equal to 0.1%. Basolateral media were collected and used to measure apoB and apoAI as described previously (50;51).

HepG2 cells grown in Dulbecco's modified Eagle's medium (CellGrow) containing 10% fetal bovine serum (FBS) supplemented with L-glutamine and

antibiotics were plated (400,000 cells/well) in six-well plates and after two days (~70-80% confluence) were labeled with 1 µCi/ml of [3H]cholesterol for 17 h in DMEM supplemented with 10% FBS, washed and then chased in serum free media containing OA:BSA complexes in the absence or presence of different MTP inhibitors (1 µM) for 24 h. ApoB levels were measured in the media to determine the effects of these inhibitors on its secretion. Lipids were extracted from the cells and used to quantify cholesteryl esters after their separation by thin layer chromatography (TLC).

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AC29 cells (52) stably transfected with either ACAT1 or ACAT2 (14) grown in Ham’s F12 media (CellGrow) containing

10% FBS supplemented with L-glutamine and antibiotics were treated with trypsin and seeded in six-well plates (400,000 cells/well). After two days, cells were labeled with [3H]cholesterol for 6 h in 10% FBS and chased in serum free media containing OA (0.2 mM):BSA (0.5% w/v) complexes in the absence or presence of either the MTP inhibitor, BMS197636 (1 µM), or the ACAT inhibitor, 148817 (1 µM), for 17 h. Lipids were extracted and used to quantify cholesteryl esters.

Overexpression of apoB and MTP in different cell lines: Undifferentiated Caco-2 cells were treated with trypsin and seeded in six-well plates (400,000 cells/well). Transfections with human MTP expression plasmids (5 µg/well) were performed using the FuGENE 6 transfection reagent (Roche Applied Sciences) according to the manufacturer's instructions. AC29 cells stably transfected with either ACAT1 or ACAT2 were used for sequential transfections. First, cells were transfected with human MTP-expressing plasmids (1.5 µg/flask) using FuGENE 6 in T175 flasks (7.2 x 106 cells). After 8 h, cells were detached by trypsin treatment, seeded in six-well plates, and transfected with 1.0 µg/well of different apoB-expression plasmids. At 40 h post-transfection, cells were pulsed with [3H]cholesterol in media containing 10% FBS for 6 h and then chased with 1 ml of lipid-containing (0.2 mM oleic acid complexed with 0.5% BSA), serum free medium. Following an additional 18 h of incubation, the media were collected, protease inhibitors (Sigma) were added, the samples were centrifuged (2,500 rpm, 4 °C, 10 min) to pellet cell debris, and the apoB contents were measured in the supernatants by ELISA (50;51). Lipids were extracted from media and cells and separated on TLC

to determine free and esterified cholesterol levels.

Secretion of [3H]Cholesterol by Primary Enterocytes: Primary enterocytes from PBS and pIpC injected mice (47;53;54) were suspended in 4 ml of DMEM containing 1 µCi/ml of [3H]cholesterol and incubated at 37 °C for 1 hr with micelles containing 1.2 mM oleic acid and 0.5 mM taurocholate. After 2 hr, enterocytes were centrifuged and supernatants were used to determine radioactivity. Conditioned media were also subjected to density gradient ultracentrifugation. Fractions were collected and radioactivity was counted. ApoB containing lipoproteins (Fractions 1-2) and non-apoB containing lipoproteins (Fractions 9-10) were pooled and used for lipid extraction. Free and esterified cholesterol were separated on TLC and quantified by scintillation counter. Cell pellets were dissolved in 1 ml of 0.1 N NaOH and used to quantify protein concentrations using Coomassie reagent (Pierce Chemical Company). Statistical analyses: Data are presented as mean ± S.D. Unless noted otherwise, n = 3 for each group or condition. Statistical significance (P<0.05) was determined using the Student’s t-test (GraphPad Prism).

Results

MTP gene deletion lowers plasma and tissue triglyceride levels: We used Mttp Ldlr Apob Tg(Mx1-cre)1Cgn/J


mice (39) to study the effect of mttp gene deletion on plasma and tissue lipid levels. These mice express Cre recombinase under the control of Mx1 promoter, which is induced after pIpC injections in the liver, spleen and intestine (30;55). There was ~ 80-85% reduction in MTP activity in the intestine and liver of mice injected with pIpC compared to control

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mice (Fig. 1A, 1B), indicating successful MTP gene deletion. Tissue triglyceride levels were increased by ~ 8-fold in the liver (Fig. 1C) and ~ 3-fold in the intestine (Fig. 1D). Plasma total cholesterol (Fig. 1E) and triglycerides (Fig. 1F) were decreased by 66% and 62%, respectively, in agreement with the studies of Lieu et al (39). The decrease in plasma cholesterol and triglyceride was mainly due to ~ 85% and 79%, respectively, reductions in non-HDL apoB-lipoproteins (Fig. 1G, 1H). There was no significant change in HDL cholesterol levels (Fig. 1I), whereas HDL triglyceride showed a small but significant decrease (Fig. 1J). Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly increased in pIpC-injected animals (Fig. 1K, 1L). Therefore, deletion of the mttp gene increases tissue triglyceride, decreases lipids in apoB-lipoproteins, and elevates liver enzymes in the plasma.

MTP gene deletion increases cellular free cholesterol: Next, we concentrated on the tissue cholesterol levels. There was no change in the levels of total cholesterol in the intestine (Fig. 2A), but total cholesterol increased by 52% in the liver of mice injected with pIpC (Fig. 2B). Similar to triglycerides (Fig. 1C, 1D), we expected that cholesteryl ester levels would increase in the intestine and liver of mttp-deficient mice. But, unexpectedly, there was a 60% and 90% reduction in esterified cholesterol in the intestine (Fig. 2C) and liver (Fig. 2D), respectively, and free cholesterol increased by 29% and 132% in the intestine (Fig. 2E) and liver (Fig. 2F), respectively. Changes in cellular free cholesterol levels affect genes involved in cholesterol homeostasis. Therefore, we measured changes in mRNA levels of different candidate genes in the intestine (Fig. 2G) and liver (Fig. 2H). As expected, increased free cholesterol levels were associated with decreased HMG-coA reductase (HMGR) and increased ABCA1

mRNA levels in the liver (Fig. 2H). Changes in HMGR in the intestine were not significant, perhaps because of lower increases in free cholesterol in this tissue. Intestinal ABCA1 mRNA levels were lower. In contrast, genes involved in fatty acid metabolism, FAS and ACC, were not changed. Thus, hepatic MTP ablation results in the accumulation of free cholesterol and alterations in sterol response genes.

MTP gene deletion attenuates cellular cholesterol esterification: To explain the decreases in tissue cholesteryl ester levels, we hypothesized that MTP may be a chaperone for the enzymes involved in the biosynthesis of cholesteryl esters, as it does for the synthesis of apoB-lipoproteins (26;27) and CD1d (31;32). We therefore measured hepatic steady state mRNA levels of MTP, ACAT1, and ACAT2 in PBS and pIpC injected mice. As expected, pIpC injections reduced MTP mRNA levels in the liver (Fig. 3A), consistent with changes in MTP activity in the liver (Fig. 1A). pIpC injections, however, increased hepatic ACAT1 mRNA levels but did not affect ACAT2 mRNA levels (Fig. 3A). At this time, we have no explanation for the increases in ACAT1 mRNA levels in pIpC-injected mice. pIpC injection did not increase ACAT1 mRNA in C57Bl/6J mice (data not shown). Increases in ACAT1 mRNA may be an adaptive response to increases in cellular free cholesterol levels. Changes in mRNA levels were further confirmed by Western blot analysis (Fig. 3B). MTP protein was significantly reduced, but there was no significant change in ACAT2 protein levels. Changes in ACAT1 protein levels could not be quantified because our antibody that recognizes human ACAT1 (Fig. 3B, AC29 cell, ACAT1) did not recognize mouse ACAT1. Therefore, deletion of mttp does not reduce ACAT1 and ACAT2 expression.

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We next measured cholesteryl ester synthesis using [3H]-cholesterol and hepatic microsomes isolated from mice injected with PBS or pIpC in the presence or absence of purified MTP (Fig. 3C). Addition of purified MTP did not affect cholesteryl ester synthesis by microsomes isolated from control PBS injected mice (Fig. 3C, first pair of bars). Microsomes isolated from pIpC-injected animals showed a 96% decrease in ACAT activity compared to microsomes isolated from PBS injected animals (Fig. 3C, open bars). This could be either due to a deficiency in ACAT enzymes or MTP activity. However, as ACAT levels were not changed, we reasoned that supplementation of purified MTP to these microsomes might enhance cholesterol esterification. Indeed, addition of purified MTP to these microsomes restored cholesteryl ester synthesis (Fig. 3C, solid bars). We found similar results by measuring microsomal cholesteryl esterification with radiolabeled palmitoyl-coA (Fig. 3D). Supplementation of control microsomes with purified MTP did not affect cholesteryl ester synthesis (Fig. 3D, first pair of bars), whereas MTP gene deletion decreased the esterification of cholesterol (Fig. 3D, open bars) and addition of purified MTP to these microsomes restored cholesteryl ester synthesis (Fig. 3D, solid bars). MTP gene deletion or supplementation with purified MTP did not affect triglyceride (Fig. 3E) or phospholipid (Fig. 3F) synthesis by hepatic microsomes. To address the possibility that cholesterol in the membrane might be limiting, cholesterol ester synthesis was studied by providing cholesterol as part of cyclodextran complexes (Fig. 3G-I). Synthesis of cholesterol esters was significantly higher under these conditions (Fig. 3G, first pair of bars) compared with conditions when no additional cholesterol was provided (Fig. 3D). Cholesteryl ester synthesis by microsomes isolated from pIpC-injected

mice was significantly lower (Fig. 3G, open bars), and this deficiency could be corrected by the addition of purified MTP (Fig. 3G, solid bars). Under the same conditions, triglyceride (Fig. 3H) and phospholipid (Fig. 3I) synthesis was unaffected by changes in MTP levels. Thus, MTP gene deletion specifically decreases cholesteryl ester synthesis, which can be restored by the addition of purified MTP. We conclude that MTP is not required for the biosynthesis of enzymes involved in cholesteryl ester synthesis but instead modulates the cholesteryl esterification reaction by a hitherto unrecognized mechanism.

MTP and ACAT antagonists additively inhibit cellular cholesteryl ester levels: mttp deletion alters cellular lipid homeostasis (56). To eliminate mechanisms involving homeostatic changes due to long-term mttp deletion, we investigated the effect of acute inhibition of MTP activity using specific chemical antagonists on cellular cholesteryl ester synthesis (Fig. 4). As expected, MTP inhibitors decreased apoB secretion (Fig. 4A), had no effect on apoAI secretion (Fig. 4B), decreased triglyceride secretion (Fig. 4C), increased cellular triglycerides (Fig. 4D), and decreased cholesteryl ester secretion (Fig. 4E) in differentiated Caco-2 cells. Instead of an expected increase in cellular radiolabeled cholesteryl esters due to their decreased secretion, we observed a 60% reduction in their cellular levels (Fig. 4F). We could not measure changes in cholesterol mass in these cell culture studies due to their low concentrations. To rule out the possibility that the effect on cellular cholesterol esterification was unique to the inhibitor and Caco-2 cells, we labeled HepG2 cells with [3H]cholesterol and then treated them with different MTP inhibitors (1 µM) kindly provided by Drs. Haris Jamil and David Gordon of the Bristol-Myers Squibb. As expected, all MTP inhibitors reduced apoB

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secretion (Fig. 4G) and had no effect on apoAI secretion (Fig. 4H). Next, we studied the effect of these inhibitors on cellular cholesteryl ester synthesis. Newly synthesized cellular cholesteryl ester levels were significantly reduced in cells treated with different antagonists (Fig. 4I). These studies showed that chemical inhibition of MTP also leads to decreased cholesteryl ester synthesis in HepG2 cells.

To confirm that MTP antagonists specifically inhibit MTP without inhibiting ACAT enzymes, we studied the effect of MTP antagonists in AC29 cells, which do not express MTP but stably express human ACAT1 or ACAT2 (14). In these cells, the MTP inhibitor, BMS197636, did not affect cellular cholesteryl ester synthesis (Fig. 4J and 4K). However, incubation of these cells with the ACAT inhibitor, 148817, kindly provided by Dr. Steve Sturley of Columbia University, reduced radiolabeled cholesteryl esters by 83-87% (Fig. 4J, 4K). These studies demonstrate that MTP inhibitors do not affect ACAT activities.

The genetic ablation and chemical inhibition studies described above clearly showed that MTP affects cholesteryl esterification without affecting ACAT activity. Thus, we hypothesized that MTP (BMS197636) and ACAT (148817) antagonists would additively inhibit cellular esterification. To test this hypothesis, Caco-2 cells were treated with different amounts of these inhibitors, alone or in combination (Fig. 4L-N). Increasing concentrations of BMS197636 decreased apoB secretion (Fig. 4L), whereas the ACAT inhibitor had no effect on apoB secretion. Combined treatment was similar to the MTP inhibitor alone. Neither inhibitor affected ApoAI secretion (Fig. 4M). Both antagonists individually inhibited cellular esterification of [3H]cholesterol to similar extents (25% ± 2% and 32% ± 3% inhibition at 1 µM) (Fig.

4N), and combined reduced cholesteryl ester synthesis by 45% ± 4% (Fig. 4N). Thus, MTP and ACAT act in tandem to facilitate cellular cholesteryl esterification.

MTP and apoB are required for optimal synthesis of cholesteryl esters: We next hypothesized that MTP overexpression would increase cellular cholesteryl esterification. To test this hypothesis, we expressed human MTP in AC29-ACAT1 and AC29-ACAT2 cells. These cells were generated by stable transfection of AC29 cells, which lack cholesteryl ester synthesizing enzymes (52), with human ACAT1 and ACAT2 enzymes (14). As expected, MTP expression increased triglyceride transfer activity by 3- to 3.5-fold over the background levels in both cell lines (Fig. 5A). We then measured the esterification of [3H]cholesterol in these cell lines. Instead of an expected increase in the levels of cholesteryl esters, we consistently found lower cholesteryl ester levels in these cells (Fig. 5B). To explain these unanticipated results, we repeated similar experiments in non-differentiated Caco-2 cells. Expression of MTP increased triglyceride transfer activity by 8 to 9-fold (Fig. 5C) and cellular cholesteryl ester levels (Fig. 5D) by 50% in these cells. These studies show that MTP expression increases cellular cholesteryl esterification in Caco-2 cells, but not in AC29 cells expressing ACAT enzymes.

To explain differences in these cell lines, we hypothesize that AC29 cells lack a factor required for maximal cellular esterification that is present in Caco-2 cells. One major difference between these two cell lines is that Caco-2 cells synthesize and secrete apoB-lipoproteins, whereas AC29 cells do not. Thus, we asked whether apoB is also required for enhanced cellular cholesteryl esterification. AC29-ACAT1 or ACAT2 cells were transfected with

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expression plasmids for either human apoB17 or apoB48, with or without human MTP expression plasmids. ApoB17 does not require lipidation by MTP and can be secreted in the absence of MTP (Fig. 6A, 6B), consistent with several published studies (57;58). However, apoB48 secretion requires lipidation by MTP and is not secreted in the absence of MTP (Fig. 6A, 6B, open bars). As expected, there was a significant increase in the secretion of apoB48 when the cells were co-transfected with MTP (Fig. 6A, 6B, closed bars). Expression of apoB17 or apoB48 without MTP did not affect cellular (Fig. 6C, 6D, open bars) or secreted (Fig. 6E, 6F, open bars) cholesteryl ester levels. Cells transfected with MTP alone showed reduced cellular cholesteryl ester levels (Fig. 6C, 6D, control), as observed before (Fig. 5B). Cells expressing MTP and apoB17 synthesized and secreted similar amounts of cholesteryl esters as control cells (Fig. 6C-F, apoB17). However, cells co-expressing both MTP and apoB48 showed a 17% increase in cellular cholesteryl esters (Fig. 6C, 6D, apoB48) and 80% increase in secreted cholesteryl ester levels (Fig. 6E, 6F, apoB48). These studies indicate that both MTP and apoB-lipoprotein assembly are required for optimal cellular cholesteryl ester synthesis.

This finding was confirmed by studying the effect of apoB and MTP on in vitro cholesteryl esterification using microsomes isolated from ACAT2 cells (Fig. 6G). Microsomes incubated with purified MTP alone showed no change in cholesteryl ester synthesis (Fig. 6G, control). Similarly, incubation of microsomes with human HDL in the presence or absence of purified MTP also did not change cholesteryl ester synthesis. However, incubation of microsomes with LDL in the presence of purified MTP significantly increased cholesteryl esterification (Fig. 6G). These studies show that the transfer of

cholesteryl esters by MTP to apoB lipoproteins enhances cholesteryl ester biosynthesis.

MTP deletion decreases secretion of cholesteryl esters by enterocytes: Deletion of MTP is known to reduce secretion of apoB-containing lipoproteins. We have previously shown that cholesterol in the intestine is secreted by apoB-dependent and apoB-independent pathways (47;53;54). We wondered if deletion of MTP in the intestine would increase secretion of cholesteryl esters by the apoB-independent pathway. To test this possibility, we isolated primary enterocytes from PBS and pIpC injected mice and radiolabeled them with cholesterol. Enterocytes were then chased with oleic acid-containing media to induce the synthesis and secretion of larger apoB-lipoproteins. Determination of radioactivity in the media showed a 42% decrease in the secretion of cholesterol by enterocytes isolated from pIpC-injected mice as compared to PBS injected mice (Fig. 7A). Density gradient ultracentrifugation revealed that this decrease was mainly due to reduction in cholesterol secretion with apoB-lipoproteins (Fractions 1-2, Fig. 7B). There was no significant change in the secretion of cholesterol with non-apoB lipoproteins (Fractions 9-10, Fig. 7B). Analysis of free and esterified cholesterol in the pooled apoB (fractions 1-2) and non-apoB (Fractions 9-10) lipoproteins showed that deletion of MTP decreased both free (Fig. 7C, first pair of bars) and esterified (Fig. 7D, first pair of bars) cholesterol in apoB-containing lipoproteins. The secretion of free cholesterol with non-apoB lipoproteins was similar in enterocytes isolated from mice injected with PBS and pIpC (Fig. 7C, second pair of bars). Non-apoB lipoproteins had very small amounts of esterified cholesterol associated with them and deletion of MTP decreased this secretion (Fig. 7D, second pair of bars).

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Therefore, MTP deletion decreases secretion of free and esterified cholesterol with apoB-containing lipoproteins.

Cholesteryl ester synthesis in intestinal microsomes is also dependent on MTP and apoB: To determine whether MTP deletion decreases cholesteryl ester synthesis in the intestine, we measured the steady state mRNA levels of MTP, ACAT1, and ACAT2. Similar to the liver (Fig. 3), injection of pIpC significantly reduced MTP mRNA, increased ACAT1 mRNA, and did not affect ACAT2 mRNA in the intestine (Fig. 8A). Western blot analysis showed that MTP protein was reduced in the intestines of pIpC injected animals without any significant change in ACAT2 protein (Fig. 8B). Microsomes isolated from pIpC-injected animals synthesized 63% less cholesteryl esters than PBS injected animals (Fig. 8C, open bars). Addition of purified MTP to intestinal microsomes obtained from pIpC-injected mice increased ACAT activity by 51% (Fig. 8C, compare thatched bar with open bar in pIpC group). However, supplementation of purified MTP and LDL increased cholesterol ester synthesis by 2-fold compared with no additions (Fig. 8C, compare solid bar with open bar in pIpC group). These studies indicate that ablation of MTP in the intestine decreases cholesteryl ester synthesis. This decrease can be partially ameliorated by providing MTP and LDL. However, the restoration of cholesterol ester biosynthesis was less than that observed in liver homogenates (Fig. 3). Liver consists mainly of hepatocytes. In contrast, intestinal homogenates contain significant amounts of smooth muscle cells in addition to enterocytes. Therefore we performed these experiments using microsomes prepared from partially purified enterocytes. Microsomes isolated from enterocytes of pIpC-injected mice synthesized 45% of cholesteryl esters compared to those synthesized by

microsomes isolated from enterocytes of PBS injected animals (Fig. 8D, open bars). Addition of MTP and MTP + LDL increased this synthesis to 80% and 98%, respectively, compared to normal synthesis observed in enterocytes from PBS injected animals (Fig. 8D, compare open bar in PBS group with hatched and solid bars in pIpC group). Therefore, we conclude that MTP deletion significantly reduces cholesteryl ester synthesis in microsomes isolated from hepatocytes and enterocytes and this reduction could be corrected by providing purified MTP and LDL.

MTP modulates cholesteryl esterification by relieving product inhibition: Based on the above data, we hypothesized that cholesteryl esters inhibit ACAT activity, and that MTP relieves the product inhibition by transferring them to apoB-containing lipoproteins. To test this hypothesis, we studied the effect of increasing cholesteryl palmitate on the esterification of [3H]-cholesterol by liver microsomes. At 0.5 µM cholesteryl palmitate, cholesteryl ester synthesis was inhibited by 32% (Fig. 9A). However, it was not clear whether cholesteryl palmitate was inhibiting cholesteryl ester synthesis by partitioning into the membrane. To determine if microsomal membrane enrichment with cholesteryl palmitate was necessary for decreased cholesteryl ester synthesis, we incubated liver microsomes with different concentrations of cholesteryl palmitate and measured cholesterol enrichment. Incubation of microsomes with increasing amounts of cholesteryl palmitate dose-dependently enriched cholesterol up to 1.25 µM cholesteryl palmitate (Fig. 9B), when the signal became saturated. Thereafter, there was no significant increase in the enrichment. Next, we studied the effect of cholesteryl palmitate enrichment of microsomes on cholesteryl ester synthesis in the presence and absence of MTP and LDL

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(Fig. 9C). Esterification of cholesterol decreased with increased enrichment of microsomes with cholesteryl palmitate (Fig. 9C, open circles). As seen before (Fig. 3), MTP did not affect cholesteryl ester synthesis in un-enriched microsomes (Fig. 9C, 0 µM, compare control with +MTP) but was reduced with increasing concentrations of cholesteryl palmitate. In these experiments and those presented in Fig. 3, small endogenous apoB-lipoproteins present as contaminants in microsomal preparations are perhaps sufficient to provide basal cholesterol ester synthesis. However, supplementation with LDL increased cholesteryl ester synthesis by 35% (Fig. 9C, 0 µM, open diamonds) and this synthesis was reduced after incubation with increasing amounts of cholesteryl palmitate. Treatment with both MTP and LDL significantly increased cholesteryl ester synthesis by 69% (Fig. 9C, 0 µM, solid diamonds), which could also be inhibited by cholesteryl palmitate (Fig. 9C, solid diamonds). These studies indicate that enrichment of microsomes with cholesteryl palmitate inhibits cholesteryl ester synthesis. MTP transfers cholesteryl esters from microsomal membranes to apoB-lipoproteins to relieve product inhibition and enhance cholesteryl ester synthesis.

Discussion

These studies indicate that reductions in MTP activity decrease cholesteryl ester synthesis and increase free cholesterol levels in hepatic and intestinal cells. These changes were not related to alterations in cholesterol esterifying enzymes. Microsomes isolated from MTP-deficient animals synthesized reduced amounts of cholesteryl esters, and providing purified MTP ameliorated this deficiency. Furthermore, expression of MTP and apoB along with esterifying enzymes in cells that do not express these proteins enhanced cholesteryl ester synthesis. The

enrichment of membranes with cholesteryl esters inhibits cholesteryl ester synthesis, and transfer of cholesteryl esters to apoB-lipoproteins by MTP augments cholesteryl ester synthesis. Therefore, these data indicate, for the first time, that cholesteryl ester biosynthesis is inhibited by product accumulation, and that MTP circumvents this inhibition by transferring the products to nascent apoB-lipoproteins.

MTP inhibition or genetic ablation leads to cellular accumulation of triglycerides (35;59-61). It has been assumed that cells accumulate cholesteryl esters along with triglycerides. Surprisingly, we found that decreases in MTP activity reduce cholesteryl esters in hepatic and intestinal cells and enhance free cholesterol levels. MTP inhibition or genetic ablation does not decrease mRNA or protein levels of ACAT2. Microsomal cholesteryl ester biosynthesis was, however, severely curtailed when MTP activity was reduced in the liver and intestinal cells. More importantly, cholesteryl ester synthesis could be restored in these microsomes by the supplementation of purified MTP. Therefore, MTP modulates cholesteryl ester synthesis by mechanisms other than transcriptional or translational control of enzyme biosynthesis.

Cellular cholesteryl ester biosynthesis was enhanced by the co-expression of MTP with apoB48, which can assemble lipoproteins, but not with apoB17, which is unable to assemble lipoproteins. However, this synthesis is inhibited in the presence of higher amounts of cholesteryl esters. The transfer of cholesteryl esters to apoB-lipoproteins by MTP enhances cholesterol ester synthesis. Thus, cholesterol ester synthesis is inhibited by product accumulation, and MTP enhances their biosynthesis by transferring them to apoB-lipoproteins.

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Both substrates of the cholesteryl esterification reaction, fatty acids and free cholesterol, enhance ACAT activities (4;62). For example, increases in fatty acyl-CoA concentrations promote cholesteryl ester synthesis (62). Different fatty acids affect the synthesis of cholesteryl esters by ACAT1 and ACAT2 (63). Furthermore, fatty acid composition of membrane phospholipids modulates esterification reaction (4). Free cholesterol acts as an allosteric activator of the esterification reaction (4). Cholesteryl ester synthesis is minimal when free cholesterol levels are low, and high free cholesterol concentrations significantly enhance ACAT activities.

In contrast to the substrate effects, modulation of the esterification reaction by its products has garnered little attention. Enrichment of membranes with cholesteryl esters inhibited the esterification reaction (Fig. 9). Cholesterol esters may act as allosteric inhibitors, a scenario opposite to the allosteric activation by free cholesterol. Both free and esterified cholesterol may compete for the same site on the enzyme. Alternatively, free and esterified cholesterol may bind to independent sites, causing conformational changes in enzymes leading to activation and suppression, respectively, of cholesterol ester biosynthesis.

MTP inhibition studies provide a biochemical explanation for the regulation of cholesteryl ester synthesis in cells that synthesize lipoproteins such as enterocytes and hepatocytes. We believe that the inhibition of cholesteryl ester synthesis by cholesteryl esters may not be specific to lipoprotein producing cells because non-lipoprotein-producing cells also accumulate significant amounts of cholesterol esters when exposed to cholesterol-enriched lipoproteins. In non-lipoprotein producing cells, cholesteryl esters may be moved from the ER membrane to cytosolic lipid droplets

by a different mechanism, because there is no evidence that a neutral lipid transfer protein, such as MTP, exists in the cytosol. Accumulation of neutral lipids in microsomes may facilitate the budding of neutral-lipid rich lipid droplets (64). Furthermore, membrane proteins (65) can augment such a process. For example, viral evasins, US2 and US11, may play a role in the formation of lipid droplets involving “bicellar” structures (65). FIT1 and FIT2 also affect lipid droplet formation (23). Mechanisms involved in the removal of cholesteryl esters from ER membranes in non-lipoprotein producing cells require further exploration.

The inhibition of cholesteryl ester synthesis by its products might prevent excess accumulation of neutral lipids in ER membranes. Neutral lipids have limited solubility in a phospholipid bilayer. And uncontrolled cholesterol ester biosynthesis may disrupt bilayer organization. Therefore, their transfer to apoB-lipoproteins or deposition into lipid droplets might be critical for microsomal membrane and cellular integrity.

ACATs and cholesterol ester hydrolase control cellular cholesterol ester homeostasis. Cholesterol ester hydrolase is mainly present in lipid droplets and is critical for the efflux of free cholesterol (66). Our studies indicate that MTP also plays a critical role in cellular cholesterol ester homeostasis by removing the cholesterol esters from their site of synthesis, depositing them into apoB-lipoproteins for secretion, and enhancing their biosynthesis.

In addition to cellular synthesis, cholesterol esters are also synthesized by the lecithin:cholesterol acyl transferase enzyme in the plasma. It is unknown whether this reaction is also inhibited by product assimilation. It is interesting to note that

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plasma contains cholesteryl ester transfer proteins that removes cholesteryl esters from HDL and deposits them into apoB-lipoproteins. We speculate that cholesterol ester synthesis by lecithin:cholesterol acyl transferase is also inhibited by product accumulation and cholesteryl ester transfer protein may relieve this inhibition by transferring cholesteryl esters to apoB-lipoproteins.

Pharmacologic interventions to reduce MTP result in hepatosteatosis and enhanced plasma concentrations of liver enzymes (35;61). The mechanistic studies described here indicate that the toxicity associated with blocking MTP activity might result from the accumulation of free cholesterol. Experiments can now be designed to assess the consequences of simultaneous lowering of free cholesterol, either by reducing cholesterol biosynthesis or enhancing its efflux, on the efficacy of MTP antagonists in decreasing plasma lipid levels.

The present data also provide an explanation for the observed relationship between cellular cholesterol levels and MTP expression. The MTP promoter contains sterol response elements that promote MTP expression when cellular sterols are

increased and suppress its expression when their availability is reduced (67;68). Animal studies show that a high cholesterol diet increases hepatic MTP levels (69). The regulation of MTP by sterols is similar to proteins involved in cholesterol esterification, ACAT2, and cholesterol efflux, ABCA1, but opposite of sterol-responsive genes, such as the LDL receptor and HMG CoA reductase. MTP expression is enhanced when cellular sterols levels increase, indicating that cells use MTP to reduce cellular cholesterol levels. Thus, MTP is a sterol-responsive gene coordinated to reduce cellular free cholesterol levels.

In summary, we show that genetic ablation or chemical inhibition of MTP increases cellular free cholesterol. Cholesteryl ester biosynthesis is regulated by feedback inhibition mechanisms involving reaction products. MTP alleviates this product inhibition by removing cholesteryl esters from their site of synthesis and depositing them into apoB-lipoproteins. These studies suggest that other mechanisms may exist to control cholesterol ester biosynthesis in cells that do not produce lipoproteins. Furthermore, they advocate for new strategies to avoid toxicities associated with therapies involving MTP inhibition.

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FOOTNOTES Acknowledgements: This work was supported in part by NIH grants DK46700 to MMH and HL-49373 to LLR.

Abbreviations used: low density lipoproteins (LDL); acyl-coA:cholesterol acyltransferase (ACAT); endoplasmic reticulum (ER); microsomal triglyceride transfer protein (MTP); oleic acid (OA); polyinosinic-polycytidylic ribonucleic acid (pIpC); thin layer chromatography (TLC); dimethyl sulfoxide (DMSO)

Keywords: Cholesteryl ester; free cholesterol; lipids; MTP; ACAT1; ACAT2; apoB; triglycerides;

FIGURE LEGENDS

Figure 1. Effect of mttp gene deletion on plasma lipids and lipoproteins: Conditional MTP deletion was achieved by injecting (n=3) pIpC three times in Mttp Ldlr Apob Tg(Mx1-cre)1Cgn/J


mice on alternate days. Control mice (n=3) received PBS injections. Liver (A) and intestinal (B) tissues were used to measure MTP activity. In addition, triglyceride levels were measured in the liver (C) and intestine (D). Plasma samples were then assayed for total (E, F), non-HDL apoB-lipoproteins (G, H), and HDL (I, J) cholesterol and triglyceride levels. Plasma was also used to measure alanine aminotransferase (K) and aspartate aminotransferase (L) activities. Each measurement was performed in triplicate. The data are representative of 3 independent experiments. The asterisks show statistically significant differences between PBS and pIpC injected mice. * P<0.05, *** P<0.001.

Figure 2. Intestinal and hepatic cholesterol levels in conditional MTP knockout mice. Samples of liver and intestine from PBS and pIpC injected animals were used to measure total cholesterol (Panels A and B), cholesteryl ester (Panels C and D), and free cholesterol (Panels E and F) mass, respectively. Intestine (G) and liver (H) total RNA were used to quantify expression of the indicated genes. The asterisks represent statistically significant differences between PBS and pIpC injected mice. * P<0.05, ** P<0.01, *** P<0.001. The data are representative of 3 independent experiments.

Figure 3. Hepatic expression of MTP and ACAT in conditional MTP knockout mice and the effect of purified MTP on microsomal cholesterol esterification. Panel A: Mttp Ldlr Apob Tg(Mx1-cre)1Cgn/J

tm2Sgy

tm1Her tm2Sgy mice (n=3) were injected three times with PBS or pIpC and total RNA was isolated from the liver as described in Experimental Procedures and used for quantitative RT-PCR of candidate genes as well as ARPp0. The ratio of candidate gene to ARPp0 in one PBS injected mice was used for normalization. Panel B: Hepatic microsomes were isolated and used to determine MTP, ACAT1, ACAT2 and PDI protein levels by Western blotting. Cellular homogenates from AC29 cells stably transfected with human ACAT1 or ACAT2 were used as a positive control for ACAT1 and ACAT2 protein expression. Panels C-I: For in vitro cholesteryl ester synthesis, 100 µg of hepatic microsomal proteins from PBS or pIpC

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injected mice were incubated in triplicate with [3H]cholesterol (0.2 µCi/reaction) and palmitoyl CoA (5 µM) (Panel C), or [14C]palmitoyl CoA (0.1 µCi/reaction) and cholesterol (0.1 mg/reaction) (Panels D-F) in 100 µl buffer for 10 min at 37 °C (open bars). The assay was also performed using free cholesterol in hydroxy-propyl β-cyclodextran and [14C]palmitoyl CoA (0.1 µCi/reaction, Panels G-I). To study the effect of MTP (filled bars), reaction mixtures were carried out in the presence (+MTP) or absence (-MTP) of purified bovine MTP (1 µg/assay). Reactions were stopped by the addition of 1 ml of chloroform:methanol (2:1), lipids were extracted and subjected to thin layer chromatography. Bands corresponding to [3H]-labeled free and esterified cholesterol (Panel C) or [14C]-labeled esterified cholesterol (Panel D and G), triacylglycerol (Panel E and H) and phospholipids (Panel F and I) were identified and quantified in a liquid scintillation counter. The data are representative of 2 independent experiments. The asterisks represent statistically significant differences between PBS and pIpC injected mice. * P<0.05, ** P<0.01, *** P<0.001.

Figure 4. Chemical inhibition of MTP decreases cholesteryl esters in cultured cells. Panels A-F: Differentiated Caco-2 cells were pulsed from the apical side with 1 µCi/ml of [3H]oleic acid for 17 h, washed, and then chased for 24 h with OA:taurocholate (1.6:0.5 mM) in the presence or absence of BMS200150 (10 µM). Basolateral media were used to measure apoB (Panel A) and apoAI (Panel B). Media and cells were used to extract lipids and to quantify secreted and cellular [3H]triglyceride (Panels C and D) and cholesteryl esters (Panels E and F), respectively. Each experiment was performed in triplicate and data are representative of 2 independent experiments. Panels G-I: HepG2 cells were labeled with 1 µCi/ml of [3H]cholesterol for 17 h, washed and then chased with OA:BSA complexes with or without 1 µM BMS200150, BMS192951, or BMS197636 for 24 h. ApoB and apoAI levels were measured in the media to determine the effects of these inhibitors on secretion (Panels G and H). Lipids were extracted from the cells and used to quantify radiolabeled cholesteryl esters (Panel I). Panels J-K: AC29 cells stably transfected with either ACAT1 or ACAT2 were labeled with [3H]cholesterol and chased with OA (0.2 mM):BSA (0.5%) complexes with or without BMS197636 (1 µM), or 148817 (1 µM) for 17 h. Lipids were extracted from the cells and used to quantify [3H]cholesteryl esters (Panels J and K). Panels L-N: Differentiated Caco-2 cells were pulsed with 1 µCi/ml of [3H]cholesterol for 17 h, washed and then chased with OA:taurocholate (1.6:0.5 mM) in the presence or absence of 1, 5, or 10 µM of 148817, BMS197636, or a combination of both inhibitors. Basolateral media were used to measure apoB (Panel L) and apoAI (Panel M). Lipids were extracted from cells and used to quantify [3H]cholesteryl esters (Panel N). Results are plotted as percent of control. * P<0.05, ** P<0.01, *** P<0.001. Each experiment was done in triplicate and data are representative of 3 independent experiments.

Figure 5. Overexpression of MTP is not sufficient to increase cholesteryl ester levels. Panels A-D: AC29 cells, stably transfected with either ACAT1 or ACAT2 (Panels A, B), and undifferentiated Caco-2 cells (Panels C, D) were incubated in triplicate with plasmids expressing (+MTP) or not (-MTP) expressing human MTP for 62 h and were used to measure MTP activity (Panels A and C, respectively). In parallel dishes, cells were also labeled with radiolabel cholesterol for 6 h in the presence of OA (0.2 mM):BSA (0.5%) complexes. Lipids were isolated

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from cells and used to measure radiolabel cholesteryl esters (Panels B and D, respectively). The data are representative of 3 independent experiments.

Figure 6. Expression of MTP and apoB is required for optimum cholesteryl ester synthesis. Panels A-F: AC29 cells, stably transfected with either ACAT1 or ACAT2, were transfected without (-MTP) or with (+MTP) plasmids containing human MTP cDNA in 75-mm2 flasks. After 8 h, cells from each flask were plated on 6-well plates and transfected without or with plasmids expressing apoB17 or apoB48 for 64 h in triplicate. Cells were labeled with [3H]cholesterol for 6 h and chased for 17 h in the presence of OA:BSA complexes. Conditioned media were used to measure secreted apoB (Panels A-B) and [3H]cholesteryl ester (Panels E-F). Cells were used for [3H]cholesteryl ester (Panels C-D) measurements. Panel G: Microsomes prepared from AC29-ACAT2 cells were used in triplicate to measure in vitro cholesteryl ester synthesis in the presence or absence of purified MTP using HDL or LDL in the reaction. The data are representative of 2 independent experiments. The asterisks represent statistically significant differences between -MTP and +MTP conditions. * P<0.05, ** P<0.01, *** P<0.001.

Figure 7. Conditional deletion of MTP decreases secretion of cholesteryl esters with apoB containing lipoproteins. Enterocytes were isolated from PBS and pIpC injected mice, pulsed for 1 h with 1 µCi/ml [3H]cholesterol, and chased with 1.2 mM oleic acid-containing micelles for 2 h. Conditioned media were used to measure radioactivity (Panel A) or for density gradient ultracentrifugation (Panel B). Fractions 1-2 (apoB lipoproteins) and 9-10 (non-apoB lipoproteins) from density gradient ultracentrifugation were pooled to extract lipids. Free (Panel C) and esterified cholesterol (Panel D) were separated by thin layer chromatography and counted in a scintillation counter. The asterisks represent statistically significant differences between PBS (open bars) and pIpC (solid bars) mice. ** P<0.01, *** P<0.001.

Figure 8. Intestinal expression of MTP and ACAT in conditional MTP knockout mice and effect of purified MTP and LDL on microsomal cholesterol esterification. Panel A: Mttp Ldlr Apob Tg(Mx1-cre)1Cgn/Jtm2Sgy tm1Her tm2Sgy mice (n=3) were injected three times with PBS or pIpC, and total RNA was isolated from the jejunum and used for quantitative RT-PCR of candidate genes as well as ARPp0. The ratio of candidate genes to ARPp0 in one PBS injected mice was used for normalization. Panel B: Intestinal microsomes were isolated and used to determine expression of MTP, ACAT2, and PDI by Western blotting. Bands were quantified, normalized to PDI, and plotted as bar graphs. The asterisks represent statistically significant differences between PBS (open bars) and pIpC (solid bars) injected mice. *** P<0.001. Panels C-D: In vitro cholesteryl ester synthesis was determined in the presence or absence of MTP or MTP + LDL using intestinal (Panel C, n=6) or enterocyte (Panel D, n=3) microsomal proteins from PBS and pIpC injected mice. The asterisks represent statistically significant differences between control (open bars) MTP (hatched bars) or MTP + LDL (solid bars). *** P<0.001.

Figure 9. Cholesteryl esters inhibit cholesteryl ester synthesis and this inhibition is alleviated by MTP. Panel A: Mice liver microsomes (100 µg protein) were incubated in

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triplicate with [3H]cholesterol (0.2 µCi/reaction), palmitoyl coA (5 µM) and increasing amounts [0-500 µM] of cholesteryl palmitate in 100 µl of assay buffer for 10 min at 37 °C. Reactions were stopped by the addition of 1 ml of chloroform:methanol (2:1), and lipids were extracted and subjected to thin layer chromatography. Bands corresponding to [3H]-labeled free and esterified cholesterol were identified and quantified in a liquid scintillation counter. The data are representative of 2 independent experiments. Panel B: In an independent experiment, liver microsomes were pre-incubated in triplicate with different concentrations of cholesteryl palmitate for 30 min, centrifuged, and re-suspended in assay buffer. Aliquots of re-suspended microsomes were used to determine total cholesterol. The data are representative of 2 independent experiments. Panel C: Microsomes were enriched with cholesteryl esters as described in Panel B and used in triplicate for the determination of cholesteryl ester synthesis in the presence of either 1 µg of MTP (solid circles) or LDL (open diamonds) or a combination of both (solid diamonds).

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Iqbal et al. Figure 1

22

PBS pIpC0

60

120

180

240

300

***

Tota

l Pla

sma

Cho

lest

erol

(mg/

dl)

PBS pIpC0

25

50

75

100

125

***

Tota

l Pla

sma

Trig

lyce

ride

(mg/

dl)

PBS pIpC0

45

90

135

180

225

***

Non

-HD

L C

hole

ster

ol(m

g/dl

)

PBS pIpC0

15

30

45

60

75

***Non

-HD

L Tr

igly

ceri

de(m

g/dl

)

PBS pIpC0

12

24

36

48

60

HD

L C

hole

ster

ol(m

g/dl

)

PBS pIpC0

7

14

21

28

35

*

HD

L Tr

igly

cerid

e(m

g/dl

)

PBS pIpC0

50

100

150

200

250

***

Plas

ma

ALT

(IU/L

)

PBS pIpC0

180

360

540

720

900

***

Plas

ma

AST

(IU/L

)

PBS pIpC0

60

120

180

240

300

***Hepa

tic M

TP A

ctiv

ity(%

Tra

nsfe

r/m

g/h)

PBS pIpC0

130

260

390

520

650

***Inte

stin

al M

TP A

ctiv

ity(%

Tra

nsfe

r/m

g/h)

PBS pIpC0

80

160

240

320

400***

Hep

atic

Trig

lyce

ride

(mg/

g of

pro

tein

)

PBS pIpC0

60

120

180

240

300***

Inte

stin

al T

rigly

cerid

e(m

g/g

of p

rote

in)

A B

C D

E F

G H

I J

K L


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PBS pIpC0

5

10

15

20

25

Tota

l Cho

lest

erol

(mg/

g of

pro

tein

)

PBS pIpC0

6

12

18

24

30**

Tota

l Cho

lest

erol

(mg/

g of

pro

tein

)

PBS pIpC0

4

8

12

16

20*

Free

Cho

lest

erol

(mg/

g of

pro

tein

)

PBS pIpC0

6

12

18

24

30***

Free

Cho

lest

erol

(mg/

g of

pro

tein

)

PBS pIpC0

2

4

6

8

10

**

Este

rifie

d C

hole

ster

ol(m

g/g

of p

rote

in)

PBS pIpC0

2

4

6

8

10

***Este

rifie

d C

hole

ster

ol(m

g/g

of p

rote

in)

HMGR ABCA1 FAS ACC0.0

0.5

1.0

1.5

2.0

2.5

*

pIpCPBS

mRN

A le

vels

(fol

d ch

ange

)

HMGR ABCA1 FAS ACC0.0

0.6

1.2

1.8

2.4

3.0

***pIpCPBS

**

mRN

A le

vels

(fol

d ch

ange

)

A B

C D

E F

G H

Intestine Liver

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[14C]-Palmitoyl coA

PBS pIpC0

2000

4000

6000

8000

10000+ MTP- MTP

Phos

phol

ipid

s(d

pm)

[14C]-Palmitoyl coA

PBS pIpC0

900

1800

2700

3600

4500+ MTP- MTP

Tria

glyc

erid

e(d

pm)

[14C]-Palmitoyl coA

PBS pIpC0

150

300

450

600 + MTP- MTP

***

Cho

lest

eryl

Est

ers

(dpm

)

[14C]-Palmitoyl coA

PBS pIpC0

250

500

750

1000 + MTP- MTP

Trig

lyce

ride

(dpm

)

[14C]-Palmitoyl coA

PBS pIpC0

2500

5000

7500

10000 + MTP- MTP

Phos

phol

ipid

s(d

pm)

D

MTP ACAT1 ACAT20.0

0.6

1.2

1.8

2.4

3.0

***

pIpC

***

PBS

mRN

A(f

old

chan

ge)

[3H]-Cholesterol

PBS pIpC0

30

60

90

120

150+ MTP- MTP

***

Cho

lest

eryl

Est

ers

(% o

f con

trol

)

[14C]-Palmitoyl coA

PBS pIpC0

11000

22000

33000

44000

55000+ MTP- MTP

***

Cho

lest

eryl

Est

ers

(dpm

)

A B C

E F

G H I

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Caco-2 Cells

Control BMS2001500

200

400

600

800

1000

***Secr

eted

Apo

B(n

g/w

ell)

Caco-2 Cells

Control BMS2001500

3000

6000

9000

12000

15000

Secr

eted

Apo

AI(n

g/w

ell)

Caco-2 Cells

Control BMS2001500

25

50

75

100

125

***

Secr

eted

Tri

glyc

erid

e(%

of c

ontr

ol)

Caco-2 Cells

Control BMS2001500

32

64

96

128

160***

Cel

lula

r Trig

lyce

ride

(% o

f con

trol

)

Caco-2 Cells

Control BMS2001500

25

50

75

100

125

***

Secr

eted

Cho

lest

eryl

Este

rs (%

of c

ontr

ol)

Caco-2 Cells

Control BMS2001500

25

50

75

100

125

***

Cel

lula

r Cho

lest

eryl

Este

rs (%

of c

ontr

ol)

HepG2 Cells

Contro

l

BMS2001

50

BMS1929

51

BMS1976

360

25

50

75

100

125

** ******

Cel

lula

r Cho

lest

eryl

Este

rs (%

of c

ontr

ol)

HepG2 Cells

Contro

l

BMS2001

50

BMS1929

51

BMS1976

360

500

1000

1500

2000

2500

*****

***Secr

eted

Apo

B(n

g/w

ell)

AC29-ACAT1 Cells

Control BMS197636 1488170

25

50

75

100

125

***Cel

lula

r Cho

lest

eryl

Este

rs (%

of c

ontr

ol)

AC29-ACAT2 Cells

Control BMS197636 1488170

25

50

75

100

125

***

Cel

lula

r Cho

lest

eryl

Este

rs (%

of c

ontr

ol)

HepG2 Cells

Contro

l

BMS2001

50

BMS1929

51

BMS1976

360

10000

20000

30000

40000

50000

Secr

eted

Apo

A1

(ng/

wel

l)

Caco-2 Cells

0 2 4 6 8 100

30

60

90

120

150148817BMS197636BMS + 148817

Inhibitors [µ M]

Secr

eted

Apo

B(%

of C

ontr

ol)

Caco-2 Cells

0 2 4 6 8 100

40

80

120

160

200 148817BMS197636BMS + 148817

Inhibitors [µ M]

Secr

eted

Apo

AI

(% o

f Con

trol

)

Caco-2 Cells

0 2 4 6 8 100

15

30

45

60

75

148817BMS197636BMS + 148817

Inhibitors [µ M]

Cel

lula

r Cho

lest

eryl

Este

rs (%

Inhi

bitio

n)

A B C

D E F

G H I

J K

L M N

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AC29 Cells

ACAT1 ACAT20

30

60

90

120

150+ MTP- MTP

****

MTP

Act

ivity

(% T

rans

fer/

mg/

h)

AC29 Cells

ACAT1 ACAT20

25

50

75

100

125+ MTP- MTP

*** ***

Cho

lest

eryl

Est

ers

(% o

f con

trol

)

Caco-2 Cells

-MTP +MTP0

60

120

180

240

300***

MTP

Act

ivity

(% T

rans

fer/

mg/

h)

Caco-2 Cells

-MTP +MTP0

35

70

105

140

175***

Cho

lest

eryl

Est

ers

(% o

f con

trol

)

BA

DC

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AC29-ACAT2 Cells

Conrol ApoB17 ApoB4875

110

145

180

215

250+ MTP- MTP

**

Secr

eted

Cho

lest

eryl

Este

rs (%

of c

ontr

ol)

AC29-ACAT1 Cells


110

145

180

215

250 - MTP+ MTP

Secr

eted

Cho

lest

eryl

Este

rs (%

of c

ontr

ol)

***

AC29-ACAT2 Cells


40

80

120

160

200+ MTP- MTP

***

Apo

B S

ecre

tion

(ng/

wel

l)

AC29-ACAT1 Cells


40

80

120

160

200- MTP+ MTP

***

Apo

B S

ecre

tion

(ng/

wel

l)

AC29-ACAT2 Cells


90

105

120

135+ MTP- MTP

*

*C

ellu

lar C

hole

ster

ylEs

ters

(% o

f con

trol

)

AC29-ACAT1 Cells


90

105

120

135- MTP+ MTP

Cel

lula

r Cho

lest

eryl

Este

rs (%

of c

ontr

ol)

*

Microsomal

Control HDL LDL0

40

80

120

160

200+ MTP- MTP

**

Cho

lest

eryl

Est

ers

(% o

f con

trol

)

BA

D

E F

C

G

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0 1 2 3 4 5 6 7 8 9 100

9000

18000

27000

36000

45000PBSpIpC

*** ***

Fractions

Tota

l [3 H

]Cho

lest

erol

(dpm

/mg)

PBS pIpC0

40000

80000

120000

160000

200000

**

Secr

eted

Tot

al[3 H

]Cho

lest

erol

(dpm

/mg)

apoB lipoproteins Non-apoB lipoproteins0

8000

16000

24000

32000

40000PBSpIpC

Free

[3 H]C

hole

ster

ol(d

pm/m

g)

***apoB lipoproteins Non-apoB lipoproteins

0

2000

4000

6000

8000

10000PBSpIpC

Este

rifie

d [3 H

]Cho

lest

erol

(dpm

/mg)

*** ***

A B

C D

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Enterocyte Microsomes

PBS pIpC0

30

60

90

120

150+ MTPControl

***+ MTP + LDL

***

***

Cho

lest

eryl

Est

ers

(% o

f con

trol

)MTP ACAT2

0.0

0.2

0.4

0.6

0.8

1.0

pIpC

***

PBS

Rel

ativ

e C

hang

e(c

ompa

red

to P

DI)

MTP ACAT1 ACAT20.0

0.5

1.0

1.5

2.0

2.5

*pIpC

***

PBS

mRN

A(f

old

chan

ge)

Intestinal Microsomes

PBS pIpC0

30

60

90

120

150+ MTPControl

***

+ MTP + LDL

***

Cho

lest

eryl

Est

ers

(% o

f con

trol

)

A B

C D

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-10 -9 -8 -7 -6 -5 -4 -350

62

74

86

98

110

Log [Cholesteryl Palmitate], M

Cho

lest

eryl

Est

ers

(% o

f Con

trol

)

0 4 8 12 16 2030

33

36

39

42

45

Cholesteryl Palmitate [µM]

Mic

roso

mal

Cho

lest

erol

(µg/

mg

prot

ein)

0 4 8 12 16 2050

75

100

125

150

175+ MTPControl

+ LDL+ MTP+LDL

Cholesteryl Palmitate [µM]

Cho

lest

eryl

Est

ers

(% o

f con

trol

)

A

B

C

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Table 1. Mouse primer pairs used for quantitative PCR studies.

Gene ID 5’ primer 3’ primer

ARPp0 GTCCAACTACTTCCTCAAGATCATCCA ACATGCGGATCTGCTGCAT

MTP CACACAACTGGCCTCTCATTAAAT TGCCCCCATCAAGAAACACT

ACAT1 TGGGTGCCACTTCGATGACT TGAGTGCACACCCACCATTG

ACAT2 GGTGGAACTATGTGGCCAAGA CCAGGATGAAGCAGGCATAGA

HMGR CTTGTGGAATGCCTTGTGATTG AGCCGAAGCAGCACATGAT

ABCA1 TTGGCGCTCAACTTTTACGAA GAGCGAATGTCCTTCCCCA

FAS GGAAGGCTGGGCTCTATGG GGCGTCGAACTTGGAGAGATC

CPT1 CACGAAGAGCTAAACTTG TATGAGAGGGTGCTGCTT

ACC TGGATCCGCTTACAGAGACTTTT GCCGGAGCATCTCATTCG

SCD1 CTACAAGCCTGGCCTCCTGC GGACCCCAGGGAAACCAGGA

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Jahangir Iqbal, Lawrence L. Rudel and M. Mahmood Hussainrelieving product inhibition

Microsomal triglyceride transfer protein enhances cellular cholesteryl esterification by

published online May 22, 2008J. Biol. Chem.

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