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J Biol Chem, Vol. 274, Issue 28, 19565-19572, July 9, 1999

Assembly and Secretion of Chylomicrons by Differentiated Caco-2 Cells
NASCENT TRIGLYCERIDES AND PREFORMED PHOSPHOLIPIDS ARE PREFERENTIALLY USED FOR LIPOPROTEIN ASSEMBLY^*

Jayraz Luchoomun and M. Mahmood Hussain^§

From the Department of Biochemistry, School of Medicine, MCP Hahnemann University, Philadelphia, Pennsylvania 19129

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To develop a cell culture model for chyclomicron (CM) assembly, the apical media of differentiated Caco-2 cells were supplemented with oleic acid (OA) together with either albumin or taurocholate (TC). The basolateral media were subjected to sequential density gradient ultracentrifugations to obtain large CM, small CM, and very low density lipoproteins (VLDL), and the distribution of apoB in these fractions was quantified. In the absence of OA, apoB was secreted as VLDL/LDL size particles. Addition of OA (0.8 mM) with TC, but not with albumin, resulted in the secretion of one-third of apoB as CM. Lipid analysis revealed that half of the secreted phospholipids (PL) and triglycerides (TG) were associated with CM. In CM, TG were 7-11-fold higher than PL indicating that CM were TG-rich particles. Secreted CM contained apoB100, apoB48, and other apolipoproteins. Secretion of large CM was specifically inhibited by Pluronic L81, a detergent known to inhibit CM secretion in animals. These studies demonstrate that differentiated Caco-2 cells assemble and secrete CM in a manner similar to enterocytes in vivo. Next, experiments were performed to identify the sources of lipids used for lipoprotein assembly. Cells were labeled with [³H]glycerol for 12 h, washed, and supplemented with OA, TC, and [¹⁴C] glycerol for various times to induce CM assembly and to radiolabel nascent lipids. TG and PL were extracted from cells and media and the association of preformed and nascent lipids with lipoproteins was determined. All the lipoproteins contained higher amounts of preformed PL compared with nascent PL. VLDL contained equal amounts of nascent and preformed TG, whereas CM contained higher amounts of nascent TG even when nascent TG constituted a small fraction of the total cellular pool. These studies indicate that nascent TG and preformed PL are preferentially used for CM assembly and provide a molecular explanation for the in vivo observations that the fatty acid composition of TG, but not PL, of secreted CM reflects the composition of dietary fat. It is proposed that in the intestinal cells the preformed PL from the endoplasmic reticulum bud off with apoB as primordial particles and the assembly of larger lipoproteins is dependent on the synthesis and delivery of nascent TG to these particles.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Assembly and secretion of chylomicrons (CM),¹ the very large triglyceride-rich lipoprotein particles synthesized only by intestinal epithelial cells after a fat meal, are essential for the transport of dietary fat and fat-soluble vitamins. The molecular mechanisms involved in the assembly of these lipoproteins have not been elucidated mainly due to lack of cell culture models that secrete these particles. Caco-2, human colon carcinoma, cells have been used as a model to study intestinal lipid metabolisms (for reviews, see Refs. 1-4). These cells secrete apoB-containing particles which have flotation properties similar to those of plasma LDL (reviewed in Ref. 1). However, supplementation of Caco-2 cells with oleic acid (OA) has generally been shown to result in the secretion of more VLDL-sized particles and fewer LDL size particles but not chylomicrons (for review, see Ref. 1). The reasons for the lack of CM secretion are not known. One reason could be low levels of apoB48 synthesized by these cells. In contrast to enterocytes which mainly synthesize apoB48, Caco-2 cells mainly synthesize apoB100 (2, 5-9). In a previous study, we showed that the inability of these cells to assemble CM was not due to limited synthesis of apoB48, since overexpression of apoB48 did not result in the secretion of chylomicrons (10). Furthermore, lipid absorption and transport were not impaired in mice expressing only apoB100 (11, 12). These studies indicated that lipid transport involving CM is a property of intestinal cells and is not totally dependent on apoB48 expression since apoB100 can substitute for this function. Thus, in the present study, we examined the hypothesis that the mode and amount of lipids delivered to intestinal cells may be limiting for the assembly and secretion of CM by Caco-2 cells. The hypothesis was based on the observation that intestinal cells synthesize VLDL or CM during fasting or postprandial states, respectively (13, 14). Furthermore, synthesis of these two lipoproteins is affected differently during infusion of egg phosphatidylcholine, different amounts of oleic acid, and Pluronic L81 (15-17). We provide evidence that differentiated Caco-2 cells assemble and secrete apoB-containing CM particles under optimal conditions and that preformed phospholipids and nascent triglyceride are preferentially used for the assembly of these particles.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Antibodies used for the determination of apoB have been described (18, 19). OA, taurocholate (TC), fatty acid free bovine serum albumin (BSA), and other chemicals were obtained from Sigma. Pluronic L81 was kindly provided by the BASF Corp. (Washington, NJ), and was freshly prepared in media for each experiment.

Cell Cultures-- Caco-2 cells were obtained from the American Type Culture Collection (Rockville, MD), and grown in Dulbecco's modified essential medium containing high glucose, 20% fetal bovine serum (FBS), and a 1% antibiotic:antimycotic mixture. Half of the cells from a 70-80% confluent 75-mm² flask were divided into 12 Transwells (24 mm diameter, 3 µM pore size, Corning Costar Corp., Cambridge, MA) and the medium was changed every other day for 21 days. This treatment is known to induce differentiation of Caco-2 cells into enterocyte-like cells (1, 3, 20, 21). To prepare OA:TC (20 × 1.6:0.5 mM) stocks, required amounts of OA were added to 10 mM TC solution, mixed by swirling, and incubated at 37 °C until a clear solution was obtained. To prepare OA:BSA (10 × 1.6:3.15 mM) stocks, required amounts of OA were added to 31.5 mM BSA solution, mixed by swirling, and incubated at 37 °C until a clear solution was obtained. These stocks were filtered using a 0.2-µm filter and stored at 20 °C. For experiments, cells were then incubated with 2 ml of medium containing 20% FBS, OA with either BSA or TC on the apical side of the Transwells for different times. The basolateral side received 2 ml of either serum-free medium or serum-free media containing 0.1% FBS. Basolateral conditioned media were subjected to sequential density gradient ultracentrifugation.

Density Gradient Ultracentrifugation-- Conditions were optimized to isolate large CM, small CM, and VLDL from cell culture media by sequential ultracentrifugation based on methods used to isolate these particles from lymph and plasma (22-24). In preliminary studies, it was observed that adjustment of the density of the conditioned media to 1.10 g/ml (24) followed by overlaying of d < 1.006 g/ml density solution was crucial for the reproducible separation of these lipoproteins from cell culture media. Analytical ultracentrifugation was preferred to light scattering or electron microscopy because rapidly floating units during centrifugation are more homogeneous (reviewed in Ref. 25).

To the conditioned media (4 ml) was added KBr (0.57 g) to obtain a density of 1.10 g/ml. The media was then overlaid with 3 ml each of 1.063 and 1.019 g/ml, and 2 ml of 1.006 g/ml density solutions using the Auto Density Flow (Buchler Instruments, Lenexa, KS) and subjected to sequential ultracentrifugation (24). To obtain large CM (S_f > 400), samples were ultracentrifuged (SW41 rotor, 33 min, 40,000 rpm, 15 °C) and the top 1 ml was collected. The samples in the ultracentrifuge tubes were then overlaid with 1 ml of d = 1.006 g/ml solution. Small CM (S_f 60-400, 1 ml) were obtained from the top after a second ultracentrifugation (3 h and 28 min, 40,000 rpm, 15 °C). The top 1 ml was again replenished with d = 1.006 g/ml solution and ultracentrifuged for 17 h. The first 1-ml fraction collected from the top represented VLDL (d < 1.006 g/ml, S_f 20-60). The rest of the gradient was fractionated into additional seven 1.5-ml fractions. The fractions 2-4 and 5-7 were considered intermediate density lipoprotein/LDL (d = 1.02-1.063 g/ml) and high density lipoprotein/bottom (1.063-1.1 g/ml), respectively.

Measurement of ApoB-- ApoB was quantified in the conditioned media and in different density gradient fractions using a sandwich enzyme-linked immunosorbent assay (18, 19) based on the binding of apoB to immobilized monoclonal antibody, 1D1, that recognizes an epitope in the N terminus (amino acids 474-539) of human apoB (26, 27). The recovery of apoB during ultracentrifugation ranged between 45 and 70% in earlier experiments when basolateral surfaces were exposed to serum-free media. In later experiments, addition of 0.1% FBS to serum-free conditioned media increased the recovery to 60-90% without affecting the distribution of apoB in different lipoprotein fractions. In all figures and tables, apoB (% of total secreted) present in VLDL, small CM, and large CM is shown. Differences in the amounts of apoB under various conditions were analyzed by Student's t test using Primer of Bio-statistics (McGraw-Hill Co., New York).

Radiolabeling of Cells and Protein Analysis-- Differentiated Caco-2 cells were radiolabeled under different conditions by placing [³⁵S]Met/Cys radiolabeling mixture (150 µCi/ml, NEN Life Science Products, Boston, MA) in Cys/Met-free medium containing 20% FBS at the apical side. The basolateral side received Cys/Met- and serum-free medium. After overnight incubations, basolateral conditioned media were subjected to density gradient ultracentrifugation as described above. Apolipoproteins were precipitated from different fractions (28). For this purpose, deoxycholate and trichloroacetic acid were added to the fractions at final concentrations of 0.7 mM and 1 M, respectively (28). The mixtures were incubated at 4 °C for 30 min, centrifuged, and proteins were resuspended in sample buffers by incubating at 80 °C for 10 min. Samples were applied to 3-15% polyacrylamide gradient gels, electrophoresed, exposed to a PhosphorImager screen, and individual bands were quantified.

Lipid Analyses-- Cells were labeled with either [U-¹⁴C]glycerol (147.8 mCi/mmol) or [1,2,3-³H]glycerol (200 mCi/mmol). Lipids were extracted from cells or medium with isopropyl alcohol or chloroform:methanol (2:1, v/v), respectively, and separated by thin layer chromatography on LK5D silica gels (Whatman) using petroleum ether:ethyl ether:acetic acid (90:10:1, v/v). After visualization with iodine vapor, lipid bands corresponding to triglycerides and phospholipids were scraped from the plates, and counted after adding 3 ml of scintillation fluid.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chylomicron Secretion by Differentiated Caco-2 Cells-- To induce chylomicron secretion by Caco-2 cells, we added OA complexed with either BSA or TC (Table I). BSA is usually used for the delivery of fatty acids to cultured cells, whereas TC is present in the intestinal lumen. Both of these vehicles have been used to deliver fatty acids in different studies with Caco-2 cells (6, 7, 29-32). Lower concentrations of TC were used because higher concentrations were reported to decrease apoB secretion in these cells (33). First, we studied the effect of OA supplementation on total apoB secretion. Supplementation of OA with BSA or TC increased apoB secretion by 20% (Table I). The increase in apoB secretion was less pronounced than that observed in other studies (6, 29, 30, 34) where Caco-2 cells were supplemented with OA in serum-free media. Thus, OA supplemented with serum-containing media has a modest effect on total apoB secretion.

Next, we determined the distribution of apoB in different lipoprotein fractions. When cells were incubated with either BSA or TC in the absence of OA, no apoB was present in the large and small CM density fractions. However, 5-11% of apoB was in the VLDL fraction. Supplementation of OA as the BSA complex increased the amount of apoB secreted as VLDL to 18%. Furthermore, 17% of apoB was now associated with small CM. However, no apoB was in the large CM fraction. Supplementation of OA with TC resulted in a significant increase of apoB (23-24%) in VLDL and small CM. What is more important, 10% of apoB was now associated with large CM, and approximately 35% of the total secreted apoB was found associated with small and large CM. These studies indicated that OA and TC had modest effects on the amount of apoB secreted but had a profound effect on the secretion of apoB as CM.

Next, we asked whether simultaneous incremental increases in OA and TC would increase the secretion of larger lipoproteins (Table II). Compared with TC-treated cells, inclusion of OA:TC up to 1.6:1.0 mM resulted in a ~30-40% increase in apoB secretion. At OA:TC concentrations of 6.4:4.0 mM, total apoB secreted was significantly (60%) decreased most likely due to OA toxicity since 4.0 mM TC in the presence of 1.6 mM OA had no significant effect on apoB secretion (Fig. 1B). Again, secretion of apoB as part of CM was dependent on the presence of OA (Table II). Maximum amounts of apoB in CM fractions were observed at OA:TC concentrations of 1.6:1.0 mM. Higher concentrations of OA:TC reduced the distribution of apoB into larger lipoproteins. In addition, we studied the chronic effect of OA supplementation on the secretion of apoB as CM. For this purpose, cells were exposed to OA:TC (1.6:0.5 mM) for 8 h. Next, they were fed with fresh media containing additional OA:TC and incubated for an additional 18 h. The percent of apoB secreted as CM was not affected by these treatments (data not shown, see also Fig. 2 below) which indicated that up to one-third of the total secreted apoB could be secreted as small and large CM.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effect of different concentrations of oleic acid and taurocholate on the secretion of large and small chylomicrons, and VLDL by differentiated Caco-2 cells. Differentiated Caco-2 cells (21-day-old cultures in Transwells) were incubated (14 h) in media containing 20% FBS with either TC (0.5 mM) and several concentrations of OA (Panel A) or OA (1.6 mM) and various concentrations of taurocholate (Panel B). The basolateral medium was subjected to density gradient ultracentrifugation as described under "Experimental Procedures" and the amounts of apoB in the different lipoprotein fractions were determined by enzyme-linked immunosorbent assay (18, 19). In panels A and B, average values of duplicate determinations and mean ± S.D, of triplicate determinations, respectively, are plotted. The data are representative of two independent experiments.

View larger version (68K): [in this window] [in a new window]   Fig. 2.   Apolipoproteins associated with larger lipoproteins secreted by differentiated Caco-2 cells. A, cells were incubated overnight with TC (0.5 mM); B, cells were incubated overnight with OA:TC (1.6:0.5 mM); C, cells were preincubated for 8 h with OA:TC (1.6:0.5 mM) and incubated overnight with OA:TC (1.6:0.5 mM). Differentiated Caco-2 cells were radiolabeled by providing [35S]Met/Cys labeling mixture (150 µCi/ml) to the apical side during overnight incubation. Basolateral media were subjected to differential ultracentrifugation to obtain different lipoproteins as described under "Experimental Procedures." Proteins in different fractions were precipitated using trichloroacetic acid/deoxycholate (28), dissolved in a sample buffer, electrophoretically separated on 3-15% polyacrylamide gradient gels, dried, and exposed to PhosphorImager screens.

We then determined the optimum amounts of OA required for the assembly and secretion of CM (Fig. 1A). Different concentrations of OA had differential effects on the secretion of apoB in VLDL, small CM, and large CM. Similar to results shown in Tables I and II, no apoB was observed in the large or small CM fractions in the conditioned media obtained from cells cultured in the absence of OA. However, 7% of apoB was associated with the VLDL fraction (Fig. 1A). Addition of 0.4 mM OA resulted in a significant increase (15% each) in the secretion of apoB as VLDL and small CM. Again, no apoB was found associated with large CM. Increasing the concentration of OA to 0.8 mM resulted in further increases in the secretion of apoB in the small CM and VLDL fractions (20% each). Now, however, 6% of total apoB was recovered as large CM which reached a maximum of 8% at 1.6 mM OA. The effect of different concentrations of OA was similar to that observed in vivo (35). In rats, secretion of VLDL was saturated at OA infusion rates of 60 µmol/h whereas infusion of higher amounts of OA (60 µmol/h) increased CM secretion (35). These studies indicate that 0.8 mM OA was required for the secretion of large CM by differentiated Caco-2 cells.

The importance of TC in the secretion of CM was then studied by incubating Caco-2 cells with a constant amount of OA (1.6 mM) and different concentrations of TC (Fig. 1B). Total apoB secreted was not affected by increasing concentrations of TC (data not shown). Similarly, higher concentrations of TC had no additional effect on the secretion of apoB in large CM, small CM, or VLDL fractions (Fig. 1B). Thus, the secretion of larger lipoproteins was not enhanced by higher TC concentrations.

The studies described above indicated that OA induces secretion of CM in differentiated Caco-2 cells. Next, we asked whether other fatty acids would further enhance the secretion of CM. For this purpose, cells were incubated with OA:TC and different fatty acids as described in Table III. Different fatty acids did not significantly increase total apoB secretion. Furthermore, palmitic and linoleic acids had no additional stimulatory effect on the secretion of lipoproteins by these cells.

Composition of Secreted Chylomicrons-- To determine the TG and PL contents of the secreted lipoproteins, cells were labeled with [³H]glycerol (36-38) and the distribution of these lipids in different lipoprotein fractions was determined (Table IV). Cells incubated with OA:TC secreted 58% and 13-fold higher amounts of PL and TG, respectively, compared with cells incubated with OA:BSA indicating that significantly higher amounts of TG were secreted by these cells. Most of the secreted TG were in CM. Analysis of the media revealed that cells incubated with OA:TC and OA:BSA secreted 48 and 24% of TG, and 46 and 11% of PL, respectively, as large and small CM. The amounts of core lipids were 7-12-fold higher than surface lipids indicating that these particles were TG-rich. The TG:PL ratios of 7 to 12 were the same as that observed for CM isolated from lymph during the postprandial state (25, 39-44). A relationship has been described between TG:PL ratios and the volume:surface areas of the particles (40, 42) which has been used to calculate particle diameters (41). Using the same equations, we calculated that the diameters of CM secreted by Caco-2 cells ranged between 104 and 162 nm. These values are in the range of diameters reported for lymph and plasma CM (25, 41, 42, 44-46). Thus, Caco-2 cells secrete TG-rich particles with a lipid composition and a size similar to that of lymph and plasma CM.

To determine the apolipoprotein composition of the secreted lipoproteins, cells were incubated with either TC (Fig. 2, lane A), or OA:TC (lane B) for 18 h. In addition, cells were incubated with OA:TC for 8 h followed by an additional 18 h with OA:TC (lane C). All cells received [³⁵S]Met/Cys labeling mixture during 18-h incubations. Incubation with OA resulted in moderate increases (up to 40%) in the secretion of apolipoproteins (Fig. 2, total). In the absence of OA no apolipoproteins were observed in CM. However, OA treatment had a profound effect on the distribution of apolipoproteins in these lipoproteins. For example, apoBs in CM fractions increased by 15-100-fold (Fig. 2). Similar changes were also observed for other apolipoproteins. Thus, secreted CM contained both apoB100 and apoB48 and other exchangeable apolipoproteins such as apoA-IV, apoE, apoA-I, and apoCs.

Inhibition of Chylomicron Secretion-- After establishing conditions for the induction of CM by differentiated Caco-2 cells, we studied the inhibition of chylomicron secretion by Pluronic L81. Pluronic L81 is a nonionic detergent that has been shown to inhibit CM secretion in animal studies (16, 47, 48). Inclusion of Pluronic L81 up to 8 µg/ml had no effect on the secretion of total apoB (Fig. 3A) indicating that it was not toxic to cells. Next, we studied the effect of Pluronic L81 on the distribution of apoB in different lipoprotein fractions (Fig. 3B). Pluronic L81 (1 µg/ml) significantly inhibited secretion of apoB as large CM particles but had no effect on its secretion as small CM and VLDL particles. At 2-4 µg/ml, Pluronic L81 had some inhibitory effect on the secretion of small CM. At higher concentrations (4 µg/ml), Pluronic L81 inhibited the secretion of both small CM and VLDL by 50% and 25%, respectively. Consideration was given to the possibility that Pluronic L81 might preferentially solubilize larger lipoproteins and affect the flotation of apoB as large CM. For this purpose, conditioned media from cells incubated with OA:TC was supplemented with different concentrations of Pluronic L81 (0-8 µg/ml) prior to ultracentrifugation. Different concentrations of Pluronic L81 had no effect on the distribution of apoB in lipoprotein fractions (data not shown). These studies indicated that at lower concentrations, Pluronic L81 specifically inhibits the secretion of large CM.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Inhibition of chylomicron secretion by Pluronic L81. Panel A, effect of Pluronic L81 on total apoB secretion. Cells were incubated in media containing 20% FBS and OA:TC (1.6:0.5 mM) in the presence of different indicated amounts of Pluronic L81 for 18 h. Basolateral condition media was used in triplicate to measure amounts of apoB secreted. Panel B, effect of Pluronic L81 on the secretion of different lipoproteins. Cells were incubated with different amounts of Pluronic L81 as described in Panel A. Basolateral conditioned media was subjected to density gradient ultracentrifugation and apoB was measured in individual fractions as described under "Experimental Procedures." The inhibition of apoB secretion in different lipoprotein fractions at different concentrations of Pluronic L81 is plotted.

Nascent Triglycerides and Preformed Phospholipids Are Preferentially Used for CM Assembly and Secretion-- Studies were then undertaken to identify the sources of lipids used for lipoprotein assembly by differentiated Caco-2 cells. First, studies were limited to studying the metabolism of the glycerol backbone and not the metabolism of fatty acids. Several investigators have studied metabolism of fatty acids and the effect of OA supplementation on cellular and secreted lipids (6, 8, 21, 29, 30, 34, 49). Second, to simulate postprandial conditions, cells cultured under normal conditions were challenged with OA. Third, glycerol radiolabeled with two different isotopes of similar specific activities was used for labeling and data were converted to nanomoles to compare different pools on molar basis. Fourth, cells were prelabeled for 12 h to obtain uniform labeling of the cellular pool. The experimental protocol consisted of labeling cells with [³H]glycerol for 12 h in normal media followed by an addition of fresh media containing OA:TC and [¹⁴C]glycerol for different time periods (Fig. 4). Lipids containing [³H]- and [¹⁴C]glycerol are referred to as "preformed" and "nascent" pools, respectively. The top panels in Fig. 4A represent cellular changes in TG and PL pools. Before the addition of the second label, the preformed TG were about 0.25 nmol/well (Fig. 4A, top left panel). This level of preformed TG remained constant for about 4 h after the OA challenge. Subsequently, there was an increase in the TG pool containing [³H]glycerol which reached equilibrium around 9 h. The increase in the [³H]glycerol-labeled TG could be accounted for by a decrease in preformed PL between 4 and 6 h (Fig. 4A, top right panel). It has been demonstrated that the fatty acids of cellular PL pools contribute to TG synthesis (37). The present studies indicated that the preformed TG pool was not significantly affected by an OA challenge until 4 h. After this time, there was an increase in TG containing [³H]glycerol which was probably derived from the preformed PL pool by the action of phospholipase C generating diacylglycerol intermediate. In contrast to the preformed pool, the incorporation of [¹⁴C]glycerol into nascent TG increased with time (the major increases occurred between 4 and 9 h) and reached saturation around 9 h. At this new steady state, the cellular levels of preformed and nascent TG were similar. It should be emphasized that after the OA challenge there was a significant increase (3-4-fold) in the total (preformed and nascent) cellular TG levels. Thus, addition of OA results in increased intracellular accumulation of TG.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Sources of lipids used for lipoprotein assembly by differentiated Caco-2 cells. Cells were first labeled for 12 h with [3H]glycerol (15 µCi/ml) to obtain a uniform labeling of intracellular pools. Cells were then washed and incubated with OA/TC and [14C]glycerol (1.5 µCi/ml) for various indicated times. Lipids containing [14C]glycerol thus represented nascent pools. Basolateral media were collected from individual wells at the indicated times and subjected to ultracentrifugation as described under "Experimental Procedures." Lipids were extracted from the different fractions, counted in a scintillation counter, and converted to nanomole by dividing the disintegrations/min by specific activity. A, top panels represent amounts of preformed and nascent cellular TG and PL levels at different time points, whereas the middle panels represent the amounts of these lipids secreted into the media. The bottom panels show ratios (preformed:nascent) for TG and PL present in cells and media at different time points. B, top, middle, and bottom panels show the distribution of preformed and nascent TG and PL in large CM, small CM, and VLDL, respectively.

In contrast to TG, the amounts of the preformed PL in cells were 1.25 nmol/well, a value that was almost 5-fold greater than cellular TG levels before the addition of the new label and OA:TC indicating that cellular PL pools were that much higher. It should be pointed out the ratio of five between PL and TG was the same as that observed in other studies where changes in cellular mass were measured (50). After the addition of the OA:TC, there was a slight increase in preformed PL which may be due to the utilization of precursors, such as glycerol 3-phosphate, for PL synthesis. After 4 h, there was some decrease in [³H]glycerol-containing PL most likely due to its conversion to TG since this decrease coincided with an increase in TG containing [³H]glycerol. After the addition of OA, newly available [¹⁴C]glycerol was predominantly used in the first 4 h for PL synthesis compared with TG synthesis. However, the incorporation of glycerol into PL quickly reached saturation around 4 h and was followed by a significant increase in TG synthesis. These studies indicate that supplementation of OA first results in significant increases in PL synthesis followed by a substantial increase in TG synthesis.

The middle panels in Fig. 4 show the time course of TG and PL secretion into the media. At early time points, the media predominantly contained nascent TG even though the amounts of intracellular nascent TG levels were significantly lower than the preformed pool. For example, at 4 h there were 3-fold higher amounts of preformed TG in cells and yet the secreted TG contained 2-fold higher amounts of nascent TG. Plotting the cellular preformed:nascent TG ratios revealed that at 2 h there were 5-fold greater amounts of preformed TG than the nascent TG levels (Fig. 4A, bottom left panel). This ratio decreased until 6 h mainly due to an increase in nascent TG. After 6 h, the ratio did not change reflecting equilibration of the two pools. In contrast, the secreted lipoproteins contained twice the amounts of nascent TG compared with preformed TG; the preformed:nascent TG ratio in the media remained constant (0.5) at all times. These studies indicated that a constant proportion of both of these pools were being secreted and that the amounts of nascent TG secreted were twice that of preformed TG.

Examination of PL in the medium revealed that secreted PL were predominantly derived from the preformed pool of PL. At any given time, 2-3-fold higher amounts of preformed PL were secreted compared with newly synthesized PL. The changes in the ratios of these two pools revealed that preformed PL levels in cells were always in excess compared with nascent PL. In cells, this ratio decreased from 4 to 2 during the first 6 h, and remained constant at 2 after 6 h indicating that there were 2-fold greater amounts of preformed PL than nascent PL. In contrast, the amount of preformed PL secreted was stabilized around 6 h at a ratio of 3 indicating that the secreted PL contained 3-fold greater amounts of preformed PL than nascent PL. Thus, the preformed PL pool appears to be preferred to the nascent PL pool for secretion. These studies indicate that in contrast to cellular TG levels, cellular PL levels change modestly (increase of about 30%). Furthermore, the nascent TG and preformed PL are preferentially secreted into the medium by these cells.

Fig. 4B shows the distribution of different lipid pools in secreted lipoproteins. The amounts of nascent TG secreted as large and small CM were significantly higher (2-3-fold) than preformed TG (Fig. 4B). VLDL also contained higher amounts of nascent TG; however, the differences between nascent and preformed TG pools were not as significant as in the CM. In contrast to TG, all lipoproteins contained significantly higher amounts of the preformed PL compared with nascent PL. The increased amounts of preformed PL secreted compared with nascent PL were not simply due to higher intracellular levels, because the preformed:nascent ratio in lipoproteins was higher than that present in cells. These data indicated that preformed PL and nascent TG are preferentially used for lipoprotein biosynthesis.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Assembly and Secretion of CM-- We have provided evidence for the synthesis and secretion of apoB-containing CM by differentiated Caco-2 cells based on four different criteria that are characteristic of CM assembly by enterocytes. First, synthesis was induced after providing high concentrations of OA which probably mimicked the postprandial state. Second, secreted CM were shown to have flotation properties similar to those isolated from either lymph or plasma. Third, it was demonstrated that CM were TG-rich particles based on the amounts of TG associated with these particles and the ratio between surface and core lipids (Table IV). Fourth, we studied the effect of a physiologic inhibitor of CM secretion, Pluronic L81, to document that CM secretion was similarly inhibited (Fig. 2B) in cell cultures as in animal studies (16, 47, 48).

Several studies have documented that supplementation of OA to Caco-2 cells results in the redistribution of normally secreted LDL size particles into VLDL size particles (1, 6, 7, 30). We have shown that secretion of lipoproteins can be further modulated by OA supplementation to obtain secretion of small and large CM. In some studies, OA was delivered to Caco-2 cells as part of TC micelles (21, 32, 50, 51). In these studies the concentrations of OA used were below 0.8 mM; a concentration that we found to be less than the optimum amount required for CM secretion (Fig. 1A). Thus, Caco-2 cells assemble and secrete significant amounts of CM when OA is provided under optimal conditions.

High concentrations of OA complexed to albumin have been provided to these cells in earlier studies (5-8, 29, 30, 34). Consistent with these studies, Caco-2 cells provided with OA·BSA complexes secreted VLDL but not large CM (Table I). On the other hand, OA:TC supported CM secretion. Several studies have documented that OA is taken up more efficiently by Caco-2 cells when provided with TC compared with BSA (20, 32, 49). Thus, rapid and efficient delivery of OA may be important for the assembly and secretion of large CM. Rapid and efficient delivery of OA may occur by saturable protein-mediated and non-saturable diffusion processes (for reviews, see Refs. 52 and 53).

Characteristic Features of Chylomicrons-- CM secreted by Caco-2 cells contained apoB100 and apoB48, and the apoB100:apoB48 ratio was similar in smaller and larger lipoproteins, indicating that both these apolipoproteins could be efficiently utilized for CM assembly. This is in accordance with data obtained from transgenic mice that showed that expression of apoB100 in intestinal cells results in efficient transport of dietary fat (11, 12). Thus, it appears that CM assembly is not a unique property of apoB48 but is a characteristic property of enterocytes.

CM secreted by Caco-2 cells also contained several exchangeable apolipoproteins. The apolipoprotein composition of CM (Fig. 2) secreted by Caco-2 cells was similar to that of lymph CM except for the additional presence of apoB100 and apoE. Lymph CM do not contain these two apolipoproteins because enterocytes do not synthesize them. CM appear to contain more exchangeable apolipoproteins than VLDL (Fig. 2). This probably reflects the larger surface area in CM available for their association. Thus, synthesis of individual apolipoproteins at the site of CM assembly and the surface area available on these particles may determine the association of exchangeable apolipoproteins with CM.

The small CM represent an interesting population of lipoproteins. Based on several parameters (TG:PL ratio, Pluronic L81 sensitivity), they resemble VLDL particles and can be called large VLDL. However, based on apolipoprotein composition and TG mass they resemble CM. It has been well documented that lipoproteins isolated by ultracentrifugation represent a heterogeneous population of particles of varying sizes (25, 40, 45). Thus, the different lipoprotein fractions separated as distinct populations based on flotation properties in these studies most likely represent a continuum of particles of different sizes.

Insights into Chylomicron Assembly-- The radiolabeling experiments involving glycerol provided some insights into the synthesis, intracellular storage, and secretion of lipids by Caco-2 cells (Fig. 4). When cells were provided with radiolabeled glycerol and challenged with OA under conditions that stimulate CM secretion, the Caco-2 cells first synthesized PL. PL synthesis rates quickly reached a plateau with a modest increase in cellular PL levels. This is consistent with the tight cellular regulation of PL synthesis (for reviews, see Refs. 54 and 55). Subsequently, synthesis of TG was induced leading to a 4-fold increase in the intracellular TG pool. This is entirely predictable because TG is the preferred form of stored intracellular lipids. In contrast to the type and amounts of lipids synthesized, Caco-2 cells preferentially used nascent TG and preformed PL for CM assembly. However, it is important to note that a substantial amount of preformed TG is also used for CM assembly because the preformed:nascent TG ratio is remarkably constant over time (Fig. 4). Thus, it appears that nascent PL and TG do not quickly equilibrate with other cellular pools. Instead, newly synthesized lipids are sequestered into new pools that are used for different purposes. In the current study, no attempts were made to obtain information concerning the fate of nascent PL. In the long term, this may replace the preformed PL pool used for CM assembly and secretion. In the case of TG, the newly synthesized pool appears to be used first for secretion and subsequently for storage.

It has been documented from very early feeding studies that the fatty acid composition of TG, not PL, in CM reflects dietary fatty acids (25, 39, 41, 56). Furthermore, it has been demonstrated that the contribution of endogenous fatty acids to PL was much greater than it was to TG of secreted CM, and that the overall fatty acid composition of PL was relatively constant and independent of dietary composition (39, 56). These differences have generally been attributed to differences in activities and substrate specificities of enzymes involved in TG and PL syntheses. Our observations that preformed PL and nascent TG are preferentially used for CM assembly provide a different explanation for these observations. In the postprandial state, newly absorbed fatty acids are used for TG synthesis and this TG is preferentially targeted for secretion as part of CM. In contrast, preformed cellular PL pools which might have derived their fatty acids from a previous meal, from biliary lipids, or cell debris are preferentially used for CM assembly and thus do not contain dietary fatty acids.

In the intestinal cells, assembly of VLDL and CM has been postulated to occur by two independent pathways because CM secretion is specifically inhibited by Pluronic L81 (for reviews, see Refs. 1, 15, and 57). In the present study, we observed that preformed PL are preferentially incorporated into VLDL and CM indicating that the initial step in the assembly of these particles may be similar. Thus, the first step in the assembly of both these particles may involve a detachment of preformed PL from the ER membrane in association with nascent apoB resulting in the release of a "primordial particle" into the lumen of the endoplasmic reticulum. Since, nascent PL are not preferentially incorporated in lipoproteins, it can be speculated that nascent PL are not targeted to the site of primordial lipoprotein assembly in the endoplasmic reticulum membrane and that ongoing synthesis of PL may not contribute to this step. In contrast to PL, secreted VLDL contains equal amounts of nascent and preformed TG whereas small and large CM contain increased amounts of nascent TG. Thus, in the intestinal cells, assembly of larger lipoproteins of different size may occur by a second process of "core expansion." In this process, the size of the nascent lipoproteins may be critically dependent on the synthesis, presumably at smooth endoplasmic reticulum, and delivery of nascent TG to primordial particles. Pluronic L81, at low concentrations, may be a specific inhibitor for the delivery of large boluses of TG, presumably in the form of droplets, and may not affect the delivery of smaller droplets to primordial particles. At higher concentrations (8 µg/ml) it might inhibit delivery of smaller droplets to the primordial particles. Thus, the size of the secreted particle may depend on the synthesis and delivery of nascent TG to primordial particles and the extent of core expansion.

Knowledge concerning the assembly of intestinal lipoproteins has lagged behind our understanding of the assembly of hepatic lipoprotein assembly. This is mainly due to a lack of an appropriate cell culture model (reviewed in Ref. 3). Here, we have provided a cell culture model that produces CM. We recognize the limitations of any cell culture model as a surrogate for animal intestinal lipoprotein assembly and secretion. Nonetheless, the cell culture model described in this paper may be useful in furthering our understanding of the molecular mechanisms of intestinal lipoprotein assembly, to identify factors that modulate CM assembly and secretion, and to study the effect of various drugs and dietary components on the assembly and secretion of these particles. It has already helped us in delineating the origins of TG and PL pools utilized for lipoprotein assembly (Fig. 4). Moreover, it may be useful to study in vitro absorption and transport of various lipophilic drugs under postprandial conditions.

	ACKNOWLEDGEMENTS

Technical assistance of Neeru Nayak and Eugene Jiwanmall, and critical reading of the manuscript by Drs. Julian Marsh and Michael Phillips are gratefully acknowledged.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants DK-46900 and HL-22633 and the American Heart Association (National Center).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: AtheroGenics Inc., 8995 West Side Pkwy., Alpharetta, GA 30004.

^§ Established investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Biochemistry, MCP Hahnemann University, 2900 Queen Lane, Philadelphia, PA 19129. Tel: 215-991-8497; Fax: 215-843-8849; E-mail: Hussaim@wpo.auhs.edu.

	ABBREVIATIONS

The abbreviations used are: CM, chylomicrons; apo, apolipoprotein; FBS, fetal bovine serum; BSA, bovine serum albumin; OA, oleic acid; TC, taurocholate; VLDL, very low density lipoproteins.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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