Microsomal Triglyceride Transfer Protein Enhances Cellular Cholesteryl Esterification by Relieving Product Inhibition^*

EXPERIMENTAL PROCEDURES

Animals—Mttp^tm2Sgy Ldlr^tm1Her Apob^tm2Sgy Tg(Mx1-cre)-1Cgn/J 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 poly(I:C) on alternate days. Control mice received phosphate-buffered saline (PBS) injections. Food was withdrawn 16 h before the mice were killed. 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 with ice-cold PBS, cut into small pieces, and used for lipid extraction or measurement of different protein activities. Lipids were extracted from tissue homogenates following the method of Bligh and Dyer (40). Triglyceride (Infinity triglyceride, catalog number TR22421) levels in the tissues were determined using commercial kits (Thermo Trace Ltd., Melbourne, Australia). Total and free cholesterol levels were measured using kits from Wako Chemicals GmbH. 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 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 from 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 K₂HPO₄ buffer (pH 7.4) containing 0.3 m sucrose, 1 mm EDTA, 50 mm KF, 50 mm KCl, and 5 mm dithiothreitol 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 recentrifuged for 120 min at 50,000 rpm in an SW55 rotor (Beckman Instruments) at 4 °C. The microsomal pellet was resuspended 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-CoA into cholesteryl esters was determined using 100 μg of microsomal protein and [³H]cholesterol (0.2 μCi, 40–60 Ci/mmol; PerkinElmer Life Sciences) in an ACAT assay buffer of 0.25 m sucrose, 100 mm Tris-HCl, and 1 mm EDTA (pH 7.5) in a final volume of 200 μl. The mixture was incubated for 30 min 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 method of Bligh and Dyer (40) and separated by TLC. Bands corresponding to free and esterified cholesterol were quantified by scintillation counting. In some experiments, incorporation of [¹⁴C]palmitoyl-CoA (0.1 μCi, 40–60 Ci/mmol; PerkinElmer Life Sciences) 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 and 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-CoA 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 min on ice using a Model 550 Sonic Dismembrator (Fisher) 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 of microsomal protein with increasing concentrations of cholesteryl palmitate suspension for 30 min in a total volume of 1 ml of ACAT assay buffer. After incubation, microsomes were washed and repelleted for 120 min at 50,000 rpm in an SW55 rotor at 4 °C. Microsomes were resuspended in 1 ml of ACAT 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 of 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/reaction).

mRNA Quantification and Primers—Total RNA was isolated using TRIzol™ (Invitrogen). The purity and integrity of RNA were assessed by the A₂₆₀/A₂₈₀ ratio and 1% agarose gel electrophoresis, respectively. Only RNAs with ratios of >1.7 were used for cDNA synthesis. The first strand cDNA was synthesized using an Omniscript RT kit (Qiagen Inc.). Briefly, 2 μg of total RNA, 1 μm random primers (Invitrogen), 0.5 mm dNTP solution, and 0.5 units/μl Omniscript reverse transcriptase were incubated at 37 °C for 1 h in 20 μl of reverse transcription buffer, and the reaction was terminated by incubation at 95 °C for 5 min. Each quantitative reverse transcription-PCR was performed in 20 μl consisting of 5 μl of cDNA sample (1:100 dilution of the first strand cDNA sample) and 15 μl of PCR Master Mix solution containing 1× PCR buffer, 6 mm MgCl₂, 200 nm primer pair, 0.025 units/μl HotStar Gold™ DNA polymerase, 200 μm dNTP solution, and 0.3 μl SYBR Green I solution (qPCR™ core kit for SYBR Green I, Eurogentec). PCR was performed by incubating the reaction mixture first for 10 min at 95 °C, followed by 40 cycles of 15-s incubations at 95 °C and 1 min at 60 °C in an ABI Prism 7000 sequence detection system (Applied Biosystems). The data were analyzed using the ΔΔC_T method according to the manufacturer's instructions and are presented as arbitrary units. The primers used in this study were designed with PrimerExpress 3.0 software (Applied Biosystems) and are presented in Table 1.

Studies with Cells—Caco-2 human colon carcinoma cells (American Type Culture Collection, Manassas, VA) were differentiated in Transwells as described (46–49). Differentiated Caco-2 cells were then pulsed with either [³H]oleic acid (15–60 Ci/mmol; PerkinElmer Life Sciences) or [³H]cholesterol at 1 μCi/ml for 17 h in 10% fetal bovine serum (FBS) on the apical side and serum-free medium on the basolateral side, washed, and chased with serum-free medium added to the apical side containing 1.6 mm oleic acid (OA) and 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 the ACAT inhibitor 148817 in dimethyl sulfoxide were added to the apical side to study their effects on cholesteryl esterification either alone or in combination. Dimethyl sulfoxide levels were ≤0.1%. The basolateral medium was collected and used to measure apoB and apoAI as described previously (50, 51).

HepG2 cells grown in Dulbecco's modified Eagle's medium (Mediatech, Inc.) containing 10% FBS supplemented with l-glutamine and antibiotics were plated (400,000 cells/well) in 6-well plates; labeled after 2 days (∼70–80% confluence) with 1 μCi/ml [³H]cholesterol for 17 h in Dulbecco's modified Eagle's medium supplemented with 10% FBS; washed; and then chased in serum-free medium containing OA·bovine serum albumin (BSA) complexes in the absence or presence of different MTP inhibitors (1 μm) for 24 h. The levels of apoB were measured in the medium 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 TLC.

AC29 cells (52) stably transfected with either ACAT1 or ACAT2 (14) and grown in Ham's F-12 medium (Mediatech, Inc.) containing 10% FBS supplemented with l-glutamine and antibiotics were treated with trypsin and seeded in 6-well plates (400,000 cells/well). After 2 days, cells were labeled with [³H]cholesterol for 6 h in 10% FBS and chased in serum-free medium 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 6-well plates (400,000 cells/well). Transfections with human MTP expression plasmids (5 μg/well) were performed using FuGENE 6 transfection reagent (Roche Applied Science) 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 expression plasmids (1.5 μg/flask) using FuGENE 6 in T175 flasks (7.2 × 10⁶ cells). After 8 h, cells were detached by trypsin treatment, seeded in 6-well plates, and transfected with different apoB expression plasmids (1.0 μg/well). At 40 h post-transfection, cells were pulsed with [³H]cholesterol in medium containing 10% FBS for 6 h and then chased with 1 ml of lipid-containing (0.2 mm OA complexed with 0.5% BSA) serum-free medium. Following an additional 18 h of incubation, the medium was collected; protease inhibitors (Sigma) were added; the samples were centrifuged at 2500 rpm for 10 min at 4 °C to pellet cell debris; and the apoB contents were measured in the supernatants by enzyme-linked immunosorbent assay (50, 51). Lipids were extracted from the medium and cells and separated by TLC to determine free and esterified cholesterol levels.

Secretion of [³H]Cholesterol by Primary Enterocytes—Primary enterocytes from PBS- and poly(I: C)-injected mice (47, 53, 54) were suspended in 4 ml of Dulbecco's modified Eagle's medium containing 1 μCi/ml [³H]cholesterol and incubated at 37 °C for 1 h with micelles containing 1.2 mm OA and 0.5 mm taurocholate. After 2 h, enterocytes were centrifuged, and supernatants were used to determine radioactivity. The conditioned medium was also subjected to density gradient ultracentrifugation. Fractions were collected, and radioactivity was counted. ApoB-containing lipoproteins (fractions 1 and 2) and non-apoB-containing lipoproteins (fractions 9 and 10) were pooled and used for lipid extraction. Free and esterified cholesterol were separated by TLC and quantified by scintillation counting. Cell pellets were dissolved in 1 ml of 0.1 n NaOH and used to quantify protein concentrations with Coomassie Blue (Pierce).

Statistical Analyses—Data are presented as the means ± S.D. Unless noted otherwise, n = 3 for each group or condition. Statistical significance (p < 0.05) was determined using Student's t test (GraphPad Prism).

RESULTS

MTP Gene Deletion Lowers Plasma and Tissue Triglyceride Levels—We used Mttp^tm2Sgy Ldlr^tm1Her Apob^tm2Sgy 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 poly(I:C) injections in the liver, spleen, and intestine (30, 55). There was ∼80–85% reduction in MTP activity in the intestines and livers of mice injected with poly(I:C) compared with control mice (Fig. 1, A and B), indicating successful MTP gene deletion. Tissue triglyceride levels were increased by ∼8-fold in the liver (Fig. 1C) and by ∼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 due mainly to ∼85 and 79% reductions, respectively, in non-high density lipoprotein (HDL) apoB lipoproteins (Fig. 1, G and H). There was no significant change in HDL cholesterol levels (Fig. 1I), whereas HDL triglyceride levels showed a small but significant decrease (Fig. 1J). Plasma alanine aminotransferase and aspartate aminotransferase were significantly increased in poly(I:C)-injected animals (Fig. 1, K and L). 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 livers of mice injected with poly(I:C) (Fig. 2B). Similar to triglyceride levels (Fig. 1, C and D), we expected that cholesteryl ester levels would increase in the intestines and livers of mttp-deficient mice. But, unexpectedly, there were 60 and 90% reductions 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 the 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 hydroxymethylglutaryl-CoA reductase and increased ABCA1 mRNA levels in the liver (Fig. 2H). Changes in hydroxymethylglutaryl-CoA reductase 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 is for the synthesis of apoB lipoproteins (26, 27) and CD1d (31, 32). We therefore measured the hepatic steady-state mRNA levels of MTP, ACAT1, and ACAT2 in PBS- and poly(I:C)-injected mice. As expected, poly(I:C) injections reduced MTP mRNA levels in the liver (Fig. 3A), consistent with changes in MTP activity in the liver (Fig. 1A). However, poly(I:C) injections 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 poly(I:C)-injected mice. Poly(I:C) 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 lane) did not recognize mouse ACAT1. Therefore, deletion of mttp does not reduce ACAT1 and ACAT2 expression.

We next measured cholesteryl ester synthesis using [³H]cholesterol and hepatic microsomes isolated from mice injected with PBS or poly(I:C) in the presence or absence of purified MTP (Fig. 3C). The addition of purified MTP did not affect cholesteryl ester synthesis by microsomes isolated from control PBS-injected mice (Fig. 3C, PBS bars). Microsomes isolated from poly(I:C)-injected animals showed a 96% decrease in ACAT activity compared with microsomes isolated from PBS-injected animals (Fig. 3C, open bars). This could be due to a deficiency in either ACAT enzymes or MTP activity. However, as ACAT levels were not changed, we reasoned that supplementation of these microsomes with purified MTP might enhance cholesterol esterification. Indeed, the addition of purified MTP to these microsomes restored cholesteryl ester synthesis (Fig. 3C, closed 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, PBS bars), whereas MTP gene deletion decreased the esterification of cholesterol (open bars), and the addition of purified MTP to these microsomes restored cholesteryl ester synthesis (closed 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. 3, G–I). Synthesis of cholesterol esters was significantly higher under these conditions (Fig. 3G, PBS bars) compared with conditions when no additional cholesterol was provided (Fig. 3D). Cholesteryl ester synthesis by microsomes isolated from poly(I: C)-injected mice was significantly lower (Fig. 3G, open bars), and this deficiency could be corrected by the addition of purified MTP (closed 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 the 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, Bristol-Myers Squibb Co.). As expected, all MTP inhibitors reduced apoB 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 show 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. 4, J and K). However, incubation of these cells with the ACAT inhibitor 148817 (kindly provided by Dr. Steve Sturley, Columbia University) reduced radiolabeled cholesteryl esters by 83–87% (Fig. 4, J and K). These studies demonstrate that MTP inhibitors do not affect ACAT activities.

The genetic ablation and chemical inhibition studies described above clearly show 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. 4, L–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, respectively, 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 3–3.5-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 the 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 undifferentiated Caco-2 cells. Expression of MTP increased triglyceride transfer activity by 8–9-fold (Fig. 5C) and cellular cholesteryl ester levels by 50% (Fig. 5D) 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 the differences in these cell lines, we hypothesized 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 AC29-ACAT2 cells were transfected with expression plasmids for either human apoB17 or apoB48 with and without human MTP expression plasmids. ApoB17 did not require lipidation by MTP and could be secreted in the absence of MTP (Fig. 6, A and B), consistent with several published studies (57, 58). However, apoB48 secretion required MTP and was not secreted in the absence of MTP (Fig. 6, A and B, open bars). As expected, there was a significant increase in the secretion of apoB48 when the cells were cotransfected with MTP (Fig. 6, A and B, closed bars). Expression of apoB17 or apoB48 without MTP did not affect cellular (Fig. 6, C and D, open bars) or secreted (Fig. 6, E and F, open bars) cholesteryl ester levels. Cells transfected with MTP alone showed reduced cellular cholesteryl ester levels (Fig. 6, C and D, Control bars), as observed before (Fig. 5B). Cells expressing MTP and apoB17 synthesized and secreted similar amounts of cholesteryl esters compared with control cells (Fig. 6, C–F, ApoB17 bars). However, cells coexpressing both MTP and apoB48 showed a 17% increase in cellular cholesteryl ester levels (Fig. 6, C and D, ApoB48 bars) and an 80% increase in secreted cholesteryl ester levels (Fig. 6, E and F, ApoB48 bars). 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 bars). 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 whether 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 poly(I: C)-injected mice and radiolabeled them with cholesterol. Enterocytes were then chased with OA-containing medium to induce the synthesis and secretion of larger apoB lipoproteins. Determination of radioactivity in the medium showed a 42% decrease in the secretion of cholesterol by enterocytes isolated from poly(I:C)-injected mice compared with PBS-injected mice (Fig. 7A). Density gradient ultracentrifugation revealed that this decrease was due mainly to reduction in cholesterol secretion with apoB lipoproteins (fractions 1 and 2) (Fig. 7B). There was no significant change in the secretion of cholesterol with non-apoB lipoproteins (fractions 9 and 10) (Fig. 7B). Analysis of free and esterified cholesterol in the pooled apoB (fractions 1 and 2) and non-apoB (fractions 9 and 10) lipoproteins showed that deletion of MTP decreased both free (Fig. 7C, first two bars) and esterified (Fig. 7D, first two 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 poly(I:C) (Fig. 7C, second two bars). Non-apoB lipoproteins had very small amounts of esterified cholesterol associated with them, and deletion of MTP decreased this secretion (Fig. 7D, second two bars). 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 poly(I:C) 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 poly(I:C)-injected animals without any significant change in ACAT2 protein (Fig. 8B). Microsomes isolated from poly(I:C)-injected animals synthesized 63% less cholesteryl esters compared with those isolated from PBS-injected animals (Fig. 8C, open bars). The addition of purified MTP to intestinal microsomes obtained from poly(I:C)-injected mice increased ACAT activity by 51% (Fig. 8C, pIpC group, compare hatched and open bars). However, supplementation of purified MTP and LDL increased cholesterol ester synthesis by 2-fold compared with no additions (Fig. 8C, pIpC group, compare closed and open bars). 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). The 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 poly(I:C)-injected mice synthesized 45% of cholesteryl esters compared with those isolated from enterocytes of PBS-injected animals (Fig. 8D, open bars). The addition of MTP and MTP + LDL increased this synthesis to 80 and 98%, respectively, compared with normal synthesis observed in enterocytes from PBS-injected animals (Fig. 8D, compare open bar in PBS group with hatched and closed bars in pIpC group). Therefore, we conclude that MTP deletion significantly reduces cholesteryl ester synthesis in microsomes isolated from hepatocytes and enterocytes and that this reduction could be corrected by providing purified MTP and LDL.

MTP Modulates Cholesteryl Esterification by Relieving Product inhibition—On the basis of 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 whether 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. Next, we studied the effect of cholesteryl palmitate enrichment of microsomes on cholesteryl ester synthesis in the presence and absence of MTP and LDL (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 unenriched microsomes (Fig. 9C, 0 μm, compare open (control) and closed (+MTP) circles), but cholesteryl ester synthesis 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, closed diamonds), which was inhibited by increasing concentrations of cholesteryl palmitate (closed 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 did not decrease the 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 important, 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 coexpression of MTP with apoB48, which can assemble lipoproteins, but not with apoB17, which is unable to assemble lipoproteins. However, this synthesis was inhibited in the presence of higher amounts of cholesteryl esters. The transfer of cholesteryl esters to apoB lipoproteins by MTP enhanced cholesterol ester synthesis. Thus, the synthesis of cholesterol esters is inhibited by product accumulation, and MTP enhances their biosynthesis by transferring them to apoB lipoproteins.

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, the fatty acid composition of membrane phospholipids modulates the 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 of 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 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. In addition, uncontrolled biosynthesis of cholesterol esters 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 present mainly 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 acyltransferase enzyme in the plasma. It is unknown whether this reaction is also inhibited by product assimilation. It is interesting to note that plasma contains cholesteryl ester transfer proteins that remove cholesteryl esters from HDL and deposit them into apoB lipoproteins. We speculate that cholesterol ester synthesis by lecithin:cholesterol acyltransferase is also inhibited by product accumulation and that 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, by either 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 MTP 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 hydroxymethylglutaryl-CoA reductase. MTP expression is enhanced when cellular sterol levels increase, indicating that cells use MTP to reduce cellular cholesterol levels. Thus, MTP is a sterol-response gene coordinated to reduce cellular free cholesterol levels.

In summary, we have shown 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.