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Am J Physiol Gastrointest Liver Physiol 295: G942-G952, 2008. First published August 28, 2008; doi:10.1152/ajpgi.90237.2008
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MUCOSAL BIOLOGY

Transcriptome response of enterocytes to dietary lipids: impact on cell architecture, signaling, and metabolism genes

Olivier Béaslas,1,2,3 François Torreilles,4 Pierre Casellas,5 Dominique Simon,5 Gérard Fabre,4 Michel Lacasa,1,2,3 François Delers,1,2,3 Jean Chambaz,1,2,3 Monique Rousset,1,2,3 and Véronique Carrière1,2,3

1Université Pierre et Marie Curie-Paris 6, Les Cordeliers UMRS 872, Paris; 2Institut National de la Santé et de la Recherche Médicale U872, Paris; 3Université Paris Descartes UMRS 872, Paris; 4Sanofi-Aventis, DMPK-S, Discovery Metabolism, Pharmacokinetics and Safety Department, Montpellier; and 5Sanofi-Aventis, Oncology Department, Montpellier, France

Submitted 13 March 2008 ; accepted in final form 25 August 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Intestine contributes to lipid homeostasis through the absorption of dietary lipids, which reach the apical pole of enterocytes as micelles. The present study aimed to identify the specific impact of these dietary lipid-containing micelles on gene expression in enterocytes. We analyzed, by microarray, the modulation of gene expression in Caco-2/TC7 cells in response to different lipid supply conditions that reproduced either the permanent presence of albumin-bound lipids at the basal pole of enterocytes or the physiological delivery, at the apical pole, of lipid micelles, which differ in their composition during the interprandial (IPM) or the postprandial (PPM) state. These different conditions led to distinct gene expression profiles. We observed that, contrary to lipids supplied at the basal pole, apical lipid micelles modulated a large number of genes. Moreover, compared with the apical supply of IPM, PPM specifically impacted 46 genes from three major cell function categories: signal transduction, lipid metabolism, and cell adhesion/architecture. Results from this first large-scale analysis underline the importance of the mode and polarity of lipid delivery on enterocyte gene expression. They demonstrate specific and coordinated transcriptional effects of dietary lipid-containing micelles that could impact the structure and polarization of enterocytes and their functions in nutrient transfer.

nutrients; alimentary lipids; mRNA; intestine

THE EXPANSION OF METABOLIC diseases (obesity, metabolic syndrome, and diabetes), and the subsequent atherosclerosis and cardiovascular diseases, are linked to the important changes in dietary habits that have occurred during the last decades, among which is an increase in fat intake (45). Although this impairment of energy homeostasis has been largely analyzed, little attention has been paid to the role that the intestine could play in this process. This organ is the first to face nutrients and may therefore contribute actively to these pathologies through the control of absorption, metabolism, and delivery of dietary lipids.

Enterocytes represent the major cell population of the intestinal epithelium. As absorptive cells (31), they ensure the delivery of dietary lipids through a highly polarized process, from absorption at the apical pole of enterocytes to secretion at their basolateral pole toward lymph then general circulation (for review, see Ref. 33). Dietary lipids, mostly triglycerides (TG), are supplied to enterocytes as complex micelles resulting from their hydrolysis by pancreatic enzymes, into fatty acids (FA) and monoglycerides (MG), and their solubilization by biliary salts and lipids in the intestinal lumen. After absorption of FA and MG, TG must be resynthesized in enterocytes and associated to the structural apolipoprotein (apo) B48 to produce chylomicrons, the intestinal form of TG-rich lipoproteins (TRL) (12, 33). Chylomicrons also carry apolipoproteins A-I and A-IV, the latter being proposed as relaying satiety signaling (48).

As highly polarized cells, enterocytes integrate specific signals originating both from luminal stimuli at their apical pole and from hormones, growth factors, and neurotransmitters at their basal pole (for review, see Ref. 36). Studies on lipid-induced signals must consider another degree of complexity resulting from the asymmetry in the composition and vehicle of supplied lipids between the apical and basal poles of enterocytes. The basolateral pole of enterocytes faces the permanent presence of circulating FA and cholesterol whereas their apical pole faces micelles made of bile lipids in the interprandial period, to which FA and MG resulting from the digestion of TG are periodically added after a meal. The way these various modes of lipid supply could differently modulate the enterocyte function has been poorly investigated. In vivo, these simultaneous stimuli are integrated and cannot be discriminated from each other. We thus developed a model for the study of the specific impact exerted by these different modes of lipid delivery in cultured Caco-2/TC7 cells, a clonal population of the human colon carcinoma-derived Caco-2 cells, which reproduces to a high degree most of the morphological and functional characteristics of enterocytes (8, 57). We previously reported that the trafficking of apoB requires cell polarization and depends on the mode of lipid delivery to the enterocytes. The presence of FA and cholesterol at the basal pole of the cells, supplied as albumin-bound lipids, promotes the targeting of apoB to the apical brush border domain. Conversely, an apical supply of dietary lipid-containing micelles specifically triggers the displacement of the apical apoB toward the secretory compartments (32). This apoB movement is specifically induced by complex micelles that mimic the composition of those found in the intestinal lumen after a lipid-rich meal (postprandial micelles, PPM), which concomitantly induce the secretion of TG as TRL (9). We have also shown that the lipid-dependent increase of apoA-IV gene expression, observed both in vivo and in Caco-2/TC7 cells, resulted from distinct mechanisms depending on the mode of lipid delivery (6). The permanent supply of lipids at the basal pole targets both transcription and mRNA stabilization, whereas an apical supply of PPM induces only apoA-IV transcription, through the hepatocyte nuclear factor HNF-4 (6), a process that was recently also reported in vivo, in swine enterocytes (25). Moreover, this PPM-dependent activation of apoA-IV gene transcription was not reproduced by the apical delivery of incomplete combinations of PPM components in Caco-2/TC7 cells. This suggested that complete PPM may induce a specific signaling pathway leading to a peculiar gene expression profile (6).

The purpose of the present study was to analyze the transcriptional variations induced by the different modes of lipid delivery to enterocytes with a particular attention to the effects of an apical supply of alimentary lipids. To achieve this, we compared the effects of an apical supply of postprandial micelles or of interprandial micelles devoid of FA and MG and of a basolateral supply of lipids on the transcriptome of Caco-2/TC7 cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Caco-2/TC7 cells (8) were seeded at 6 x 104 cells/cm2 on six-well semipermeable filters (3-µm pore size, BD Biosciences, Le Pont de Claix, France) and cultured as already reported (32). In some experiments, serum was replaced by ITS (GIBCO-BRL; 1x: 10 µg/ml insulin, 0.55 µg/ml transferrin, 6.7 ng/ml selenium) 48 h after seeding. From the confluence, serum or ITS was added only to the medium of the basal compartment to fit with the physiological asymmetry of intestinal epithelium environment in vivo. All experiments were performed after 15 days of culture.

Preparation of micelles and of lipid-supplemented media. The preparation of lipid-containing media and of lipid micelles was performed as previously described (6, 32). The composition of postprandial lipid micelles (PPM) was similar to that resulting from lipid digestion in the human duodenum lumen (0.6 mM oleic acid, 0.2 mM L-{alpha}-lysophosphatidylcholine, 0.05 mM cholesterol, 0.2 mM 2-monooleoylglycerol, 2 mM taurocholic acid). Interprandial lipid micelles (IPM) were prepared as PPM. To be close to physiological conditions, IPM contained the bile products (L-{alpha}-lysophosphatidylcholine, cholesterol, taurocholic acid) that are always found in the intestinal lumen, even in the interprandial period (39), but were devoid of the postprandial TG digestion products (oleic acid and 2-monooleoylglycerol). PPM or IPM were added to the medium of the upper compartment for the last 24 h of culture, a time necessary to obtain the maximal effect of PPM on apoA-IV gene expression (6).

In some experiments, the ITS medium of the lower compartment was supplemented with lipids (0.4 mM oleic acid, 0.2 mM palmitic acid, 0.04 mM cholesterol; ITS/L medium) combined with 1% (wt/vol) FA-free bovine serum albumin (FAF-BSA, Sigma), with a 4:1 molar ratio of FA to albumin. Caco-2/TC7 cells were cultured in the presence of ITS/L medium from the confluence.

RNA extraction, hybridization on Affymetrix chips, and microarray analysis. Total RNA was extracted by the guanidium isothiocyanate method (10). After ultracentrifugation through CsCl gradient and precipitation, RNAs were checked for purity and integrity. RNA targets (biotin-labeled RNA fragments) were produced from 5 µg of total RNAs by synthesizing double-stranded cDNA followed by in vitro transcription and fragmentation reactions. Hybridization mixture containing the cRNA fragments, probe array controls (Affymetrix), BSA, and herring sperm DNA was prepared and hybridized to Human Genome U95Av2 arrays, representing ~10,000 full-length genes (Affymetrix) at 45°C for 16 h. Each RNA preparation was hybridized to duplicate chips. After washing, bound biotin-labeled cRNA was detected with a streptavidin-phycoerythrin conjugate. Subsequent signal amplification was performed with a biotinylated anti-streptavidin antibody. Washing and staining procedures were automated by using Affymetrix fluidics station and arrays were scanned with a Hewlett-Packard GeneArray Scanner. Low-level data analysis was performed using MAS 5.0 software (Affymetrix) by running absolute analyses on each array and comparative analyses between pairs of arrays. Detailed descriptions of the analysis algorithms have been published (18, 27). All arrays were scaled to a mean gene expression intensity of 300. Starting from duplicate chips for each experimental condition, duplicate comparative analyses were run to identify differentially expressed genes (Affymetrix Change >0.997, P values <0.003) and provide estimates of the expression intensity ratios for each gene studied between the two conditions being compared. The ratios, called signal log ratios (SLR), are expressed in base 2 logarithmic scale and will be further transformed into decimal values representing the fold change between two experimental conditions for an easier reading.

For cluster analysis, only genes differentially expressed in at least one experimental condition were retained; i.e., significant modulations had to be reproduced in the two duplicate comparative analyses and the absolute value of the SLR had to be above 1 in at least one duplicate. Two-dimensional hierarchical clustering was performed on SLR values by using cosine correlation and the average linkage algorithm (UPGMA) with the GeneMaths software package (Applied Maths). Classification of genes by cell function category was based on Gene Ontology (GO) terms included in HG-U95Av2 array annotations (Affymetrix). Differentially expressed genes were analyzed according to predefined pathways and functional categories annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) (22) and GO terms (2) and according to human genetic association of complex diseases and disorders by Genetic Association Database (3) using the DAVID bioinformatics resource (http://david.abcc.ncifcrf.gov/home.jsp; Ref. 13).

The expression data have been deposited in the EBI's ArrayExpress under the accession number E-MEXP-1446.

TLDA analysis. Reverse transcription from total RNA was performed as described previously (6). Messenger RNA was quantified by the TaqMan Low Density Array (TLDA) technology (Applied Biosystems, Courtaboeuf, France). Predesigned TaqMan probe and primer sets for target genes were factory loaded into the 384 wells of TLDA configured to contain three replicates per target gene. PCRs were performed using cDNA samples corresponding to 100 ng of starting RNA and TaqMan Universal PCR Master Mix (Applied Biosystems). PCR conditions were one step of 94.5°C for 10 min followed by 40 cycles at 97°C for 30 s and 59.7°C for 1 min. Gene expression values were determined using the calculation of the relative quantitation (RQ) of target genes normalized to a calibrator corresponding to one chosen culture condition through which all other conditions were analyzed. RQ calculation was performed using SDS 2.3 software package (Applied Biosystems) and was based on the {Delta}{Delta}CT method using 18S RNA as endogenous control gene. SDS 2.3 software package calculates the error bars based on the maximum and the minimum expression levels for each gene, defining the region of expression within which the true expression level value is likely to occur with a confidence level of 95% (P < 0.05).

Lipid labeling. After 15 days of culture on filters, cells were incubated with PPM or IPM for 20 min or 24 h. At the end of the incubation period, upper and lower media were removed and cells were washed with PBS (4°C), fixed with 4% paraformaldehyde in PBS (30 min, 4°C), washed with PBS containing 0.2 M glycine (2 x 15 min, 4°C), and allowed to react 30 min with Bodipy 493/503 (Molecular Probes). Cells on filters were then washed with PBS, mounted with Fluoprep (bioMérieux, Marcy l'Etoile, France), and kept at 4°C until observation with a Zeiss LSM 510 confocal microscope.

Western blot analyses. Caco-2/TC7 cells were scraped into lysis buffer (20 mM Tris·HCl, pH 7.4, 5 mM EDTA, 0.15 M NaCl, 1% Triton X-100, 0.5% sodium deoxycholate) containing protease and phosphatase inhibitor cocktails. Western blots were performed as described (32). Rabbit polyclonal antibodies against DUSP1 (a gift from P. Lenormand), apoA-IV (a gift from A. Mazur), and annexin A13 (Abcam); mouse monoclonal antibody against CEACAM1 (Abcam); and chicken polyclonal antibodies against ADRP (USBiological) were used for Western blots. Proteins were quantified by use of Image J software.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Gene expression pattern in Caco-2/TC7 cells under different conditions of lipid supply. We first analyzed the respective influence on gene expression of lipids supplied at the apical or the basal pole of Caco-2/TC7 cells (Table 1), in conditions reproducing the physiological asymmetry of lipid supply to enterocytes. The effect of lipids supplied at the basal pole was analyzed in cells permanently cultured in the presence of defined ITS medium supplemented with albumin-bound oleic acid, palmitic acid, and cholesterol (ITS/L) or in the presence of fetal bovine serum (FBS) providing both lipids and growth factors. The effect of apically supplied lipids was analyzed after the addition of PPM or IPM micelles for 24 h (see MATERIALS AND METHODS). These different conditions were compared with cultures in a minimal serum-free/lipid-free ITS medium, which allows Caco-2/TC7 cells to grow and differentiate normally (21, 32).


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  		Table 1. Lipid supply conditions in Caco-2/TC7 cell cultures

 
The DNA microarray analysis of Caco-2/TC7 cells under these different conditions revealed a significant twofold modulation of the expression of 412 genes in at least one condition of lipid supply, compared with the ITS condition (Fig. 1A). The hierarchical clustering of modulated genes, with duplicates of each lipid supply condition grouped together, demonstrated that the two conditions of lipid supply at the basal pole (FBS and ITS/L) were closely related and clustered in a same subbranch. This observation is consistent with our previous results showing that ITS/L condition reproduced the effect of FBS supply on the targeting of apoB to the apical domain of Caco-2 cells (32) and on apoA-IV gene expression (6). The vertical cluster formed by the two conditions of lipid supply at the basal pole differed from that obtained in the two conditions of lipid supply at the apical pole as PPM or IPM. These results pointed out that the different modes of lipid delivery at the apical or basal pole of enterocytes lead to distinct gene expression profiles.


Figure 1
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  		Fig. 1. Transcriptome analysis of enterocytes according to the mode of lipid delivery. A: hierarchical clustering of the 412 genes that are modulated in the different conditions of lipid supply in Caco-2/TC7 cells. Rows represent the 412 genes with a modulation of expression ratio over a cutoff value of 2, compared with insulin/transferrin/selenium-containing medium (ITS), in at least 1 lipid supply condition. Columns represent the 8 arrays corresponding to 4 different conditions (analyzed in duplicate) of lipid supply (see Table 1) compared with ITS condition. Red and green indicate up- and downregulated expression, respectively. PPM, postprandial micelles; IPM, interprandial micelles; ITS/L, ITS medium supplemented with albumin-bound lipids; FBS, fetal bovine serum. For all genes, differences are statistically significant, compared with lipid-free ITS, in at least 1 lipid supply condition (Affymetrix Change P values <0.003). B: schematic representation of gene expression modulation under an apical or a basal supply of lipids to Caco-2/TC7 cells. The mean value of the modulation of gene expression observed in PPM and IPM conditions was calculated to obtain the apical lipid supply condition that was then compared with the values obtained in ITS/L condition corresponding to the supply of lipids at the basal pole. The apical or basal supply of lipids significantly modulated the expression of 319 genes (with at least a 1.6 factor compared with ITS condition). Among them, 118 genes were modulated only by the apical lipid micelles (#1); 29 genes were preferentially modulated by apical lipid micelles (#2, apical-to-basal ratio >1.6); 141 genes were equally modulated by both apical lipid micelles and basal lipids (#3, apical-to-basal ratio <1.6); 20 genes were preferentially modulated by basal lipids (#4, apical-to-basal ratio <0.6); 11 genes were inversely modulated by apical lipid micelles or basal lipids (#5).

 
We further analyzed the differential effects of lipids when supplied at the apical or basal pole. To achieve this, we combined the PPM and IPM conditions and calculated the mean value of the modulation of gene expression observed in these conditions to obtain values corresponding to the "apical" lipid micelle supply condition. This apical condition was then compared with the ITS/L condition. FBS condition was not included in this comparison since FBS also comprises growth factors that could modulate the expression of genes independently of lipids. We identified 319 genes that displayed, in at least one condition of lipid supply, a significant 1.6-fold modulation of their expression compared with ITS condition, i.e., in the absence of lipids. Among them, 141 genes were equally modulated by lipids when supplied at the apical or basal pole (apical-to-basal ratio <1.6). The apical supply of lipid micelles specifically (118 genes, without effect of basal lipids) or preferentially (29 genes, apical-to-basal ratio >1.6) modulated 147 genes compared with the ITS/L condition, whereas 20 genes were preferentially modulated by basal lipids (apical-to-basal ratio <0.6). Eleven genes were found inversely modulated by apical micelles vs. basal lipids (Fig. 1B).

Among the 141 genes that were equally modulated by apical lipid micelles and basal albumin-bound lipids (Fig. 1B, #3), 62 genes were upregulated (Table S1).1 Their classification, according to their cellular function and functional annotation using DAVID tools, revealed that the main cell function category grouped 10 genes involved in glucose metabolism and more particularly in glycolysis pathway (PFK, PKM2, GPI, PGK1, ALDOA, PGAM, TPI1, and LDHA). The cell adhesion/architecture and the lipid metabolism categories were also important, with an overrepresentation of genes involved in collagen synthesis, and in cholesterol and lipoprotein metabolism, respectively. Seventy-nine genes were equally downregulated in both basal and apical lipid supply conditions (Table S2). Their classification showed that they are equally distributed in several cell function categories. However, DAVID analysis revealed that several genes, from different cell function categories, were also associated with biological processes related to various stress responses (such as defense response, wounding, etc.) (Table S2).

Some genes were inversely modulated by apical micelles and basal lipids. All these genes, except for the APOD gene, which encodes a multifunction lipid carrier protein (40), were upregulated by apical lipid micelles and downregulated in the presence of basal lipids (Fig. 1B, #5 and Table 2, top). These genes were essentially from two cell function categories: the cell adhesion/architecture category, including two components of intermediate filaments (KRT8 and KRT18) and of microtubules (TUBB2A and TUBA1B), and the lipid metabolism category, with genes related to cholesterol metabolism (LDLR, INSIG1, and NSDHL genes).


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  		Table 2. Modulated genes

 
Our analysis revealed that 20 genes are preferentially modulated by basal albumin-bound lipids (Fig. 1B, #4). These genes (Table 2, middle) were equally distributed in several cell function categories and were essentially downregulated in the presence of albumin-bound lipids. Only five genes were upregulated by basal lipids: ZG16, encoding a recently discovered secreted protein involved in glycoprotein secretion (55); GABRA2, involved in the signaling effects of the major inhibitory neurotransmitter {gamma}-aminobutyric acid and associated with alcohol dependence (11); SLC36A1 and SLC1A1, implicated in amino acid transport (47); and S100G, encoding calbindin D9K, an intracellular calcium-binding protein involved in calcium homeostasis (5).

The apical lipid micelles preferentially (Fig. 1B, #2 and Table S3) or specifically (Fig. 1B, #1 and Table S4) modulated a large number of genes (29 and 118, respectively). Among genes preferentially modulated by apical lipid micelles (Table S3), the upregulated ones (14 genes) were mainly from two cell function categories corresponding to lipid metabolism and cell adhesion/architecture, whereas the amino acid metabolism and nucleus-transcription-translation categories were the most represented categories among the downregulated genes. Among the 118 genes specifically modulated by the apical lipid micelles, 83 genes were upregulated and 35 genes were downregulated (Table S4). These genes were mainly from the same four top cell function categories: cell adhesion/architecture, receptor-signal transduction, nucleus-transcription-translation, and lipid metabolism (Table 2, bottom). We identified several KEGG pathways that were enriched in the upregulated genes compared with the downregulated genes. Four KEGG pathways were related to cell-matrix and cell-cell interactions: focal adhesion, regulation of actin cytoskeleton, tight junction, and cell communication. The steroid biosynthesis KEGG pathway was also well represented.

Identification of changes in gene expression specifically induced by micellar dietary lipids. We then focused on the identification of genes specifically modulated by alimentary lipids through the comparison between the apical supply of PPM and the apical supply of IPM devoid of FA and MG. Analysis of the microarray data revealed 46 genes that were differentially modulated between PPM and IPM conditions with a significant change of more than 1.6-fold (Table 3), among which 21 genes displayed a more than twofold change. A majority of the PPM-modulated genes (33/46) was upregulated in PPM vs. IPM condition. Classification of genes by cell function showed that three top cell function categories were overrepresented: lipid metabolism, signal transduction, and cell adhesion/architecture (26, 24, and 24%, respectively).


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  		Table 3. Modulation of gene expression in PPM vs. IPM condition

 
The lipid metabolism category included 11 genes, among which 8 are upregulated in the PPM condition compared with the IPM condition (Table 3). Four genes are involved in lipoprotein homeostasis, namely APOA4, which exhibited the highest upregulation in this category (2.6- to 2.4-fold change), APOC3, MTTP, and LDR genes (Table 3). Using the Genetic Association Database through DAVID tools, the functional analysis revealed a genetic association between PCK1, APOA4, APOC3, LDLR, and MTTP and Type 2 diabetes and between APOA4, APOC3, and LDLR genes and obesity. Concerning the other genes, the cytosolic HMGCS1 and INSIG1 genes are involved in cholesterol homeostasis (16, 17), HMGCS2 in the synthesis of ketone bodies (17), and ADFP gene encodes an intrinsic protein of cytosolic lipid droplets (7). The three downregulated genes present in the lipid metabolism category were ALDH3B1, a member of a superfamily that metabolizes a wide variety of endogenous and exogenous aldehydes and ensures lipid peroxidation (30); PDTSS1, implicated in phospholipid biosynthesis (52); and AKR1C1, which plays a role in steroid hormone activation and in the biosynthesis of bile acid intermediates (38).

The signal transduction category displayed a set of 11 genes, among which 8 genes were upregulated by PPM compared with IPM (Table 3), including highly upregulated genes (>2-fold, bold in Table 3), such as DUSP1 and DUSP5, which encode phosphatases involved in the MAPK signaling pathways (23). Two G proteins were also present in this cell function category: the coagulation factor II (thrombin) receptor like 1 (F2LR1) implicated in ion transport and/or cell proliferation in intestinal cells (29), and the ras homolog gene family member B (RHOB) that is involved in cytoskeleton architecture and endocytosis traffic (14).

The third top cell function category, i.e., cell adhesion/architecture, grouped a set of nine genes, of which eight were upregulated after an apical supply of PPM compared with IPM (Table 3). Some of them are involved in cell-cell interactions such as claudins 3 and 4, which are components of tight junctions (51); syntenin, a component of adherens junctions that is involved in the traffic of plasma membrane-associated proteins (56); and CEACAM1, an adhesion molecule that is also involved in a number of distinct physiological processes (24). Other genes of this category are involved in cell-matrix interactions, such as integrin alpha 6 (28), and/or in the maintenance of cell structure, such as ectodermal-neutral cortex (53) and thymosin beta 10 (19), which are both implicated in actin dynamics. Keratin 20 is a component of intermediate filaments (54), and annexin A13 is involved in apical and basolateral vesicular transport (49).

Using BIND database through DAVID tools, the functional annotation analysis of the 46 genes differentially expressed between PPM and IPM conditions revealed that 43% (20/46, see Table 3) of these genes were putative or known targets of HNF-4 (35).

The modulation of the expression of the 21 genes differentially expressed in PPM vs. IPM conditions, with at least a twofold change in their expression (Table 3, genes in bold), were then confirmed by quantitative RT-PCR assays, via a TLDA approach. According to their role in lipid metabolism, we also included in this analysis MTTP, ADFP, and APOC3 genes that are differentially expressed in PPM vs. IPM conditions, though to a lower extent (1.8–1.7, 1.7–1.6, 1.7–1.5, respectively; Table 3). As shown in Fig. 2A, the modulation of gene expression determined by TLDA closely fits with that obtained from the initial Affymetrix DNA microarray analysis (Table 3). To further demonstrate the specific impact of PPM compared with IPM, we analyzed the expression of some proteins involved in the three main function categories. Results of this Western blot study (Fig. 2B), which concerned DUSP1 (signal transduction), CEACAM1 and annexin A13 (cell adhesion/architecture), and apoA-IV and ADRP (lipid metabolism), revealed a significant increase of the levels of these proteins in PPM condition and strengthened, at the functional level, the results obtained through the transcriptome approach. Finally, the accumulation of lipid droplets, which is known to occur very rapidly during absorption of dietary lipids (9), was compared between PPM and IPM conditions (Fig. 2C). Results show that, as soon as after 20 min, lipid droplets accumulate in the cytoplasm of PPM-treated Caco-2/TC7 cells, in both the median and basal planes, whereas very scarce lipid droplets were observed in IPM-treated cells, most probably resulting from the use of glucose in the culture medium for TG synthesis in this condition. The discrepancy between PPM and IPM conditions was still enhanced after 24 h incubation (Fig. 2C).


Figure 2
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  		Fig. 2. Comparison of the effects induced by PPM and IPM supplies in Caco-2/TC7 cells. A: validation of results from affymetrix DNA microarray. The mRNA levels of genes selected from DNA microarray analysis on the basis of at least a twofold changed expression in PPM condition vs. IPM condition, and the mRNA levels of MTTP, ADFP, and APOC3 genes were quantified by real-time PCR analysis, using the TaqMan Low Density Array (TLDA) approach. Assays were performed in triplicate and expression measurements were normalized to the 18S rRNA level. The IPM condition was defined as the calibrator condition, to which the PPM condition was compared. Open bars represent the normalized relative quantities (RQ) of target gene expression in the PPM condition compared with the IPM condition (set at 1), determined by TLDA analysis. Error bars were based on the maximum and the minimum expression levels (RQmax and RQmin) for each gene calculated by using the SDS 2.3 software package (P < 0.05 for all the selected genes). Solid bars represent the mRNA level of genes in PPM condition compared with IPM condition (set at 1), determined by Affymetrix DNA microarray analysis and performed in duplicate (Affymetrix change P < 0.003 for all the selected genes). Results are expressed as means ± SE. B: comparison of the protein levels in PPM and IPM conditions. Top: representative of Western blots (duplicate for each condition) obtained for proteins selected among the genes differentially modulated between PPM and IPM conditions (see Table 3): DUSP1 for cell signaling, CEACAM1 and annexin A13 (ANXA13) for cell adhesion/architecture, apoA-IV and ADRP for lipid metabolism. Bottom: ratio between the levels of each analyzed protein in the PPM compared with the IPM (set at 1) condition. Results are from duplicates of 2 independent experiments. **P < 0.01 from Student's t-test. C: lipid labeling in the median and basal planes of Caco-2/TC7 cells incubated for 20 min or 24 h with PPM or IPM. Bodipy was used to visualize both the cell layer, through labeling of cell membrane phospholipids, and cytoplasmic lipid droplet-associated neutral lipids. Note the higher amount of lipid droplets in the median and basal planes of the cells as soon as after 20-min incubation with PPM compared with IPM. Bar: 10 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The importance of micellar solubilization of lipid digestion products for their uptake by enterocytes is well documented (for review, see Ref. 34). However, only few studies have taken into account this physiological form of dietary lipids in the analysis of the specific molecular mechanisms involved in their polarized transfer, from the intestinal lumen to the lymph, through TG synthesis and lipoprotein assembly and secretion.

Using a transcriptomic approach in Caco-2/TC7 cells, we demonstrate for the first time that 1) the specificity of the signals induced by lipids are dependent on the polarity and the mode of their physiological delivery, since distinct gene expression profiles were obtained after their basolateral supply as albumin-bound components (ITS/L and FBS conditions) or their apical delivery as micelles in the postprandial or interprandial state (PPM and IPM conditions); and 2) micelles comprising lipid digestion products (PPM) specifically modulate the expression of genes involved in three major cell function categories, i.e., lipid metabolism, as could be expected, but also signal transduction and cell adhesion/architecture.

These results were obtained after 24-h incubation with PPM or IPM, a time that we previously reported necessary to obtain the maximum effect of PPM on apoA-IV gene expression (6). This does not preclude a specific effect of PPM on the transcription of other genes, for example those involved in signal transduction, within a much narrower time scale. Further kinetic studies on individual gene of interest would answer this question.

Among genes specifically modulated by the supply of PPM compared with IPM, the induction of apoA-IV, apoC3, two components of lipoproteins and of MTP necessary to the transfer of lipid to apoB for the stabilization of this structural component of TRL, was consistent with the induction of TRL secretion that we previously observed in Caco-2/TC7 cells after PPM supply (9). Concomitantly to TRL secretion, PPM supply was reported to induce in enterocytes, in vivo (43) and in Caco-2 cells (9), a transient storage of TG as cytosolic droplets, which can be further mobilized and redirected toward ER lumen for TRL secretion. It is thus of interest to note that PPM supply induced the expression of the PAT family protein ADRP, a major cytosolic lipid droplet-associated protein (7).

The intestinal function of nutrient absorption, which is ensured by enterocytes, closely depends on the maintenance of cell differentiation and polarity that occurs through the regulation of the expression of components of cell-matrix and cell-cell adhesion. These components are also required for multiple cellular events including endocytosis, polarized targeting of newly synthesized or recycled proteins to the apical or basolateral plasma membranes, and secretion (for review, see Ref. 1). A cross talk between cell-cell and cell-matrix adhesion pathways and intestinal lipid metabolism has been reported in Caco-2 cells, in which the integrin-dependent adhesion to the extracellular matrix strengthened the formation of E-cadherin-actin complexes and increased the expression of apoA-IV gene (44) through the control of nuclear HNF-4{alpha} abundance (37). We have previously demonstrated that the PPM-induced activation of APOA4 gene transcription was under the control of the transcription factor HNF-4 (6). In the present study, among the 46 genes that are differentially modulated by PPM compared with IPM, 20 genes are known or putative targets for HNF-4. Such an impact of HNF-4 on intestinal gene expression is poorly documented, whereas a key role of HNF-4 in hepatocyte differentiation and function has been largely demonstrated (see for review, see Ref. 41). In recent transcriptome, metabolome, and bioinformatics analyses of mouse small intestine, authors hypothesized that HNF-4 was involved in the regulation of genes involved in the differentiation process and in lipid metabolism in enterocytes (26, 46). Studies, mainly performed in the liver, have reported that FA or acyl-CoAs were able to modulate HNF-4-DNA binding activity (for review, see Ref. 41). However, we previously demonstrated in Caco-2/TC7 cells that distinct molecular mechanisms were involved in the lipid-dependent induction of apoA-IV gene expression by albumin-bound FA added at the basal pole of enterocytes or by FA-containing lipid micelles (PPM) at the apical pole. It so appears that supply of FA cannot account by itself for the HNF-4-dependent induction of lipid metabolism-related gene expression by dietary lipids (6).

We showed that one fourth of the genes that are specifically modulated by PPM compared with IPM were involved in signal transduction pathways. This observation is consistent with our hypothesis that dietary lipids under the form of lipid micelles could induce specific signaling pathways leading to the mobilization of the apical apoB and to its transfer to intracellular secretory compartments (32), a process necessary for the fusion of this structural protein to TG for the assembly of TRL. Among these genes, we observed an increased expression of RhoB, a GTPase protein involved in signaling events and participating to the cross talk between actin dynamics, cell-cell adhesion, signaling, and membrane trafficking (1, 42). Moreover, a regulatory role of Rho family protein in lipid metabolism, and particularly in cholesterol efflux, has been recently suggested (50). We also observed an increased expression of DUSP1 and DUSP5, two genes that are involved in the MAPK signaling pathway (23). Interestingly, in fructose-fed hamster, an animal model of insulin resistance characterized by an overproduction of hepatic VLDL and intestinal apoB-containing lipoproteins, Federico et al. (15) showed increased basal levels of phosphorylated ERK1/2 in enterocytes and decreased intestinal apoB48 synthesis and secretion after the inhibition of ERK pathway in vivo.

Among the genes specifically induced by PPM, we also observed a twofold increase in the expression of TM4SF1 gene, a member of transmembrane-4 superfamily, which, despite a still unknown biological function, shares topological features with the tetraspanin superfamily, which is involved in cell proliferation and adhesion as well as in protein trafficking and signal transduction (4). The potential implication of TM4SF1 in lipid metabolism is highlighted by a recent report showing that this protein is markedly expressed in large adipocytes compared with small adipocytes from human adipose tissues (20).

In conclusion, our results, which are summarized in the scheme of Fig. 3, emphasize a specific and coordinated impact of dietary lipids, when supplied under their physiological micellar form, on the expression of genes involved not only in energy metabolism but also in cell polarization, trafficking, and signaling processes in enterocytes that could enhance the vectorial nutrient transfer capacity in these intestinal absorptive cells, a process in which HNF-4 could act as a major coordinator.


Figure 3
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  		Fig. 3. Schematic representation of the specific effects of PPM on cell functions and gene expression in enterocytes. Based on the results of the comparison between PPM and IPM supply, the diagram summarizes how the primary PPM-associated dietary lipid stimulus modulates the expression of genes involved in 3 main cell functions (lipid metabolism, cell adhesion/architecture, and signal transduction) through the potential implication of HNF-4 or other signaling pathways, such as DUSP1 and DUSP5 or TM4SF1. Activation of cell function (+) or of gene expression ({oplus}) is differentially marked. The effects on gene expression are based on the present results. When already reported in enterocytes, a line illustrates the effects on cell functions. The potential impact of TM4SF1 on lipid storage, which was reported only in adipocytes, is suggested by a dotted line. The cross talk between cell adhesion/architecture and apolipoprotein (apo)A-IV through the control of nuclear HNF-4 abundance is indicated. Numbers in parentheses refer to the reference list; *putative or known HNF-4-target gene. TRL, TG-rich lipoproteins; MTP, microsomal triglyceride transfer protein; ADRP, adipose differentiation-related protein.

 

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O. Béaslas is recipient of fellowships from Ministère de l'Enseignement Supérieur et de la Recherche and from Fondation pour la Recherche Médicale.


   ACKNOWLEDGMENTS
 
We thank T. Pédron (Institut Pasteur, Institut National de la Santé et de la Recherche Médicale U786, Paris, France) and D. M. Mutch (Centre de Recherche des Cordeliers, UMRS U872 team 7, Paris, France) for helpful discussions. Anti-DUSP1 and anti-apoA-IV antibodies were kindly provided by Philippe Lenormand (Centre National de la Recherche Scientifique UMR 6543, Nice, France) and D. Mazur (Institut National de la Recherche Agronomique, Theix, France), respectively. Confocal microscopy analyses were performed using the facilities of Centre de Recherche des Cordeliers, UMR S 872.


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. Carrière, Centre de Recherche des Cordeliers UMR S 872 équipe 6, 15 rue de l'école de Médecine, 75006 Paris, France (e-mail: veronique.carriere{at}crc.jussieu.fr)

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.

1 The online version of this article contains supplemental data. Back


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