The transcription factor HNF-4α: a key factor of the intestinal uptake of fatty acids in mouse.

With an excessive postprandial accumulation of intestine-derived, triglyceride-rich lipoproteins being a risk factor of cardiovascular diseases, it is essential to characterize the mechanisms controlling the intestinal absorption of dietary lipids. Our aim was to investigate the role of the transcription factor hepatocyte nuclear factor (HNF)-4α in this process. We used transgenic mice with a specific and inducible intestinal knockout of Hnf-4α gene. One hour after a lipid bolus, in the presence of the lipase inhibitor tyloxapol, lower amounts of triglycerides were found in both plasma and intestinal epithelium of the intestine-specific Hnf-4α knockout (Hnf-4α(intΔ)) mice compared with the Hnf-4α(loxP/loxP) control mice. These discrepancies were due to a net decrease of the intestinal uptake of fatty acid in Hnf-4α(intΔ) mice compared with Hnf-4α(loxP/loxP) mice, as assessed by the amount of radioactivity that was recovered in intestine and plasma after gavage with labeled triolein or oleic acid, or in intestinal epithelial cells isolated from jejunum after a supply of labeled oleic acid-containing micelles. This decreased fatty acid uptake was associated with significant lower levels of the fatty acid transport protein-4 mRNA and protein along the intestinal tract and with a lower acyl-CoA synthetase activity in Hnf-4α(intΔ) mice compared with the control mice. We conclude that the transcription factor HNF-4α is a key factor of the intestinal absorption of dietary lipids, which controls this process as early as in the initial step of fatty acid uptake by enterocytes.

The proteome of cytosolic lipid droplets isolated from differentiated Caco-2/TC7 enterocytes reveals cell-specific characteristics.

BACKGROUND INFORMATION: Intestinal absorption of alimentary lipids is a complex process ensured by enterocytes and leading to TRL [TAG (triacylglycerol)-rich lipoprotein] assembly and secretion. The accumulation of circulating intestine-derived TRL is associated with atherosclerosis, stressing the importance of the control of postprandial hypertriglyceridaemia. During the postprandial period, TAGs are also transiently stored as CLDs (cytosolic lipid droplets) in enterocytes. As a first step for determining whether CLDs could play a role in the control of enterocyte TRL secretion, we analysed the protein endowment of CLDs isolated by sucrose-gradient centrifugation from differentiated Caco-2/TC7 enterocytes, the only human model able to secrete TRL in culture and to store transiently TAGs as CLDs when supplied with lipids. Cells were analysed after a 24 h incubation with lipid micelles and thus in a state of CLD-associated TAG mobilization. RESULTS: Among the 105 proteins identified in the CLD fraction by LC-MS/MS (liquid chromatography coupled with tandem MS), 27 were directly involved in lipid metabolism pathways potentially relevant to enterocyte-specific functions. The transient feature of CLDs was consistent with the presence of proteins necessary for fatty acid activation (acyl-CoA synthetases) and for TAG hydrolysis. In differentiated Caco-2/TC7 enterocytes, we identified for the first time LPCAT2 (lysophosphatidylcholine acyltransferase 2), involved in PC (phosphatidylcholine) synthesis, and 3BHS1 (3-ß-hydroxysteroid dehydrogenase 1), involved in steroid metabolism, and confirmed their partial CLD localization by immunofluorescence. In enterocytes, LPCAT2 may provide an economical source of PC, necessary for membrane synthesis and lipoprotein assembly, from the lysoPC present in the intestinal lumen. We also identified proteins involved in lipoprotein metabolism, such as ApoA-IV (apolipoprotein A-IV), which is specifically expressed by enterocytes and has been proposed to play many functions in vivo, including the formation of lipoproteins and the control of their size. The association of ApoA-IV with CLD was confirmed by confocal and immunoelectron microscopy and validated in vivo in the jejunum of mice fed with a high-fat diet. CONCLUSIONS: We report for the first time the protein endowment of Caco-2/TC7 enterocyte CLDs. Our results suggest that their formation and mobilization may participate in the control of enterocyte TRL secretion in a cell-specific manner.

Short-term adaptation of postprandial lipoprotein secretion and intestinal gene expression to a high-fat diet.

Western diet is characterized by a hypercaloric and hyperlipidic intake, enriched in saturated fats, that is associated with the increased occurrence of metabolic diseases. To cope with this overload of dietary lipids, the intestine, which delivers dietary lipids to the body, has to adapt its capacity in lipid absorption and lipoprotein synthesis. We have studied the early effects of a high-fat diet (HFD) on intestinal lipid metabolism in mice. After 7 days of HFD, mice displayed normal fasting triglyceridemia but postprandial hypertriglyceridemia. HFD induced a decreased number of secreted chylomicrons with increased associated triglycerides. Secretion of larger chylomicrons was correlated with increased intestinal microsomal triglyceride transfer protein (MTP) content and activity. Seven days of HFD induced a repression of genes involved in fatty acid synthesis (FAS, ACC) and an increased expression of genes involved in lipoprotein assembly (apoB, MTP, and apoA-IV), suggesting a coordinated control of intestinal lipid metabolism to manage a high-fat loading. Of note, the mature form of the transcription factor SREBP-1c was increased and translocated to the nucleus, suggesting that it could be involved in the coordinated control of gene transcription. Activation of SREBP-1c was partly independent of LXR. Moreover, HFD induced hepatic insulin resistance whereas intestine remained insulin sensitive. Altogether, these results demonstrate that a short-term HFD is sufficient to impact intestinal lipid metabolism, which might participate in the development of dyslipidemia and metabolic diseases.

Sensing of dietary lipids by enterocytes: a new role for SR-BI/CLA-1.

BACKGROUND: The intestine is responsible for absorbing dietary lipids and delivering them to the organism as triglyceride-rich lipoproteins (TRL). It is important to determine how this process is regulated in enterocytes, the absorptive cells of the intestine, as prolonged postprandial hypertriglyceridemia is a known risk factor for atherosclerosis. During the postprandial period, dietary lipids, mostly triglycerides (TG) hydrolyzed by pancreatic enzymes, are combined with bile products and reach the apical membrane of enterocytes as postprandial micelles (PPM). Our aim was to determine whether these micelles induce, in enterocytes, specific early cell signaling events that could control the processes leading to TRL secretion. METHODOLOGY/PRINCIPAL FINDINGS: The effects of supplying PPM to the apex of Caco-2/TC7 enterocytes were analyzed. Micelles devoid of TG hydrolysis products, like those present in the intestinal lumen in the interprandial period, were used as controls. The apical delivery of PPM specifically induced a number of cellular events that are not induced by interprandial micelles. These early events included the trafficking of apolipoprotein B, a structural component of TRL, from apical towards secretory domains, and the rapid, dose-dependent activation of ERK and p38MAPK. PPM supply induced the scavenger receptor SR-BI/CLA-1 to cluster at the apical brush border membrane and to move from non-raft to raft domains. Competition, inhibition or knockdown of SR-BI/CLA-1 impaired the PPM-dependent apoB trafficking and ERK activation. CONCLUSIONS/SIGNIFICANCE: These results are the first evidence that enterocytes specifically sense postprandial dietary lipid-containing micelles. SR-BI/CLA-1 is involved in this process and could be a target for further study with a view to modifying intestinal TRL secretion early in the control pathway.

Epidermal growth factor receptor is involved in enterocyte anoikis through the dismantling of E-cadherin-mediated junctions.

Enterocytes of the intestinal epithelium are continually regenerated. They arise from precursor cells in crypts, migrate along villi, and finally die, 3-4 days later, when they reach the villus apex. Their death is thought to occur by anoikis, a form of apoptosis induced by cell detachment, but the mechanism of this process remains poorly understood. We have previously shown that a key event in the onset of anoikis in normal enterocytes detached from the basal lamina is the disruption of adherens junctions mediated by E-cadherin (Fouquet S, Lugo-Martinez VH, Faussat AM, Renaud F, Cardot P, Chambaz J, Pincon-Raymond M, Thenet S. J Biol Chem 279: 43061-43069, 2004). Here we have further investigated the mechanisms underlying this disassembly of the adherens junctions. We show that disruption of the junctions occurs through endocytosis of E-cadherin and that this process depends on the tyrosine-kinase activity of the epidermal growth factor receptor (EGFR). Activation of EGFR was detected in detached enterocytes before E-cadherin disappearance. Specific inhibition of EGFR by tyrphostin AG-1478 maintained E-cadherin and its cytoplasmic partners beta- and alpha-catenin at cell-cell contacts and decreased anoikis. Finally, EGFR activation was evidenced in the intestinal epithelium in vivo, in rare individual cells, which were shown to lose their interactions with the basal lamina. We conclude that EGFR is activated as enterocytes become detached from the basal lamina, and that this mechanism contributes to the disruption of E-cadherin-dependent junctions leading to anoikis. This suggests that EGFR participates in the physiological elimination of the enterocytes.

The cellular prion protein PrP(c) is involved in the proliferation of epithelial cells and in the distribution of junction-associated proteins.

BACKGROUND: The physiological function of the ubiquitous cellular prion protein, PrP(c), is still under debate. It was essentially studied in nervous system, but poorly investigated in epithelial cells. We previously reported that PrP(c) is targeted to cell-cell junctions of polarized epithelial cells, where it interacts with c-Src. METHODOLOGY/FINDINGS: We show here that, in cultured human enterocytes and in intestine in vivo, the mature PrP(c) is differentially targeted either to the nucleus in dividing cells or to cell-cell contacts in polarized/differentiated cells. By proteomic analysis, we demonstrate that the junctional PrP(c) interacts with cytoskeleton-associated proteins, such as gamma- and beta-actin, alpha-spectrin, annexin A2, and with the desmosome-associated proteins desmoglein, plakoglobin and desmoplakin. In addition, co-immunoprecipitation experiments revealed complexes associating PrP(c), desmoglein and c-Src in raft domains. Through siRNA strategy, we show that PrP(c) is necessary to complete the process of epithelial cell proliferation and for the sub-cellular distribution of proteins involved in cell architecture and junctions. Moreover, analysis of the architecture of the intestinal epithelium of PrP(c) knock-out mice revealed a net decrease in the size of desmosomal junctions and, without change in the amount of BrdU incorporation, a shortening of the length of intestinal villi. CONCLUSIONS/SIGNIFICANCE: From these results, PrP(c) could be considered as a new partner involved in the balance between proliferation and polarization/differentiation in epithelial cells.

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

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.

Apolipoprotein A-II is catabolized in the kidney as a function of its plasma concentration.

We investigated in vivo catabolism of apolipoprotein A-II (apo A-II), a major determinant of plasma HDL levels. Like apoA-I, murine apoA-II (mapoA-II) and human apoA-II (hapoA-II) were reabsorbed in the first segment of kidney proximal tubules of control and hapoA-II-transgenic mice, respectively. ApoA-II colocalized in brush border membranes with cubilin and megalin (the apoA-I receptor and coreceptor, respectively), with mapoA-I in intracellular vesicles of tubular epithelial cells, and was targeted to lysosomes, suggestive of degradation. By use of three transgenic lines with plasma hapoA-II concentrations ranging from normal to three times higher, we established an association between plasma concentration and renal catabolism of hapoA-II. HapoA-II was rapidly internalized in yolk sac epithelial cells expressing high levels of cubilin and megalin, colocalized with cubilin and megalin on the cell surface, and effectively competed with apoA-I for uptake, which was inhibitable by anti-cubilin antibodies. Kidney cortical cells that only express megalin internalized LDL but not apoA-II, apoA-I, or HDL, suggesting that megalin is not an apoA-II receptor. We show that apoA-II is efficiently reabsorbed in kidney proximal tubules in relation to its plasma concentration.

Human apolipoprotein A-II associates with triglyceride-rich lipoproteins in plasma and impairs their catabolism.

Postprandial hypertriglyceridemia and low plasma HDL levels, which are principal features of the metabolic syndrome, are displayed by transgenic mice expressing human apolipoprotein A-II (hapoA-II). In these mice, hypertriglyceridemia results from the inhibition of lipoprotein lipase and hepatic lipase activities by hapoA-II carried on VLDL. This study aimed to determine whether the association of hapoA-II with triglyceride-rich lipoproteins (TRLs) is sufficient to impair their catabolism. To measure plasma TRL residence time, intestinal TRL production was induced by a radioactive oral lipid bolus. Radioactive and total triglyceride (TG) were rapidly cleared in control mice but accumulated in plasma of transgenic mice, in relation to hapoA-II concentration. Similar plasma TG accumulations were measured in transgenic mice with or without endogenous apoA-II expression. HapoA-II (synthesized in liver) was detected in chylomicrons (produced by intestine). The association of hapoA-II with TRL in plasma was further confirmed by the absence of hapoA-II in chylomicrons and VLDL of transgenic mice injected with Triton WR 1339, which prevents apolipoprotein exchanges. We show that the association of hapoA-II with TRL occurs in the circulation and induces postprandial hypertriglyceridemia.

Preferential association of Hepatitis C virus with apolipoprotein B48-containing lipoproteins.

Hepatitis C virus (HCV) in cell culture has a density comparable to that of other members of the family Flaviviridae, whereas in vivo infectious particles are found partially in low-density fractions, associated with triacylglycerol (TG)-rich lipoproteins (TRLs). In the blood of infected patients, HCV circulates as heterogeneous particles, among which are lipo-viroparticles (LVPs), globular particles rich in TG and containing viral capsid and RNA. The dual viral and lipoprotein nature of LVPs was addressed further with respect to apolipoprotein composition and post-prandial dynamic lipid changes. The TRLs exchangeable apoE, -CII and -CIII, but not the high-density lipoprotein apoA-II, were present on LVPs, as well as the viral envelope proteins. apoB100 and -B48, the two isoforms of the non-exchangeable apoB, were represented equally on LVPs, despite the fact that apoB48 was barely detectable in the plasma of these fasting patients. This indicates that a significant fraction of plasma HCV was associated with apoB48-containing LVPs. Furthermore, LVPs were enriched dramatically and rapidly in triglycerides after a fat meal. As apoB48 is synthesized exclusively by the intestine, these data highlight the preferential association of HCV with chylomicrons, the intestine-derived TRLs. These data raise the question of the contribution of the intestine to the viral load and suggest that the virus could take advantage of TRL assembly and secretion for its own production and of TRL fate to be delivered to the liver.

Adaptation of enterocytic Caco-2 cells to glucose modulates triacylglycerol-rich lipoprotein secretion through triacylglycerol targeting into the endoplasmic reticulum lumen.

Enterocytes are responsible for the absorption of dietary lipids, which involves TRL [TG (triacylglycerol)-rich lipoprotein] assembly and secretion. In the present study, we analysed the effect on TRL secretion of Caco-2 enterocyte adaptation to a differential glucose supply. We showed that TG secretion in cells adapted to a low glucose supply for 2 weeks after confluence was double that of control cells maintained in high-glucose-containing medium, whereas the level of TG synthesis remained similar in both conditions. This increased secretion resulted mainly from an enlargement of the mean size of the secreted TRL. The increased TG availability for TRL assembly and secretion was not due to an increase in the MTP (microsomal TG transfer protein) activity that is required for lipid droplet biogenesis in the ER (endoplasmic reticulum) lumen, or to the channelling of absorbed fatty acids towards the monoacylglycerol pathway for TG synthesis. Interestingly, by electron microscopy and subcellular fractionation studies, we observed, in the low glucose condition, an increase in the TG content available for lipoprotein assembly in the ER lumen, with the cytosolic/microsomal TG levels being verapamil-sensitive. Overall, we demonstrate that Caco-2 enterocytes modulate TRL secretion through TG partitioning between the cytosol and the ER lumen according to the glucose supply. Our model will help in identifying the proteins involved in the control of the balance between TRL assembly and cytosolic lipid storage. This mechanism may be a way for enterocytes to regulate TRL secretion after a meal, and thus impact on our understanding of post-prandial hypertriglyceridaemia.

E-cadherin-dependent transcriptional control of apolipoprotein A-IV gene expression in intestinal epithelial cells: a role for the hepatic nuclear factor 4.

Cell-matrix and cell-cell adhesion play a central role in the control of cell proliferation, differentiation, and gene expression. Integrins and E-cadherin are the key components involved in these processes in epithelial cells. We recently showed that integrin-dependent adhesion to the extracellular matrix reinforces the formation of E-cadherin-actin complexes inducing the polarization of Caco-2 enterocytes and increases the expression of a marker of enterocyte differentiation, the apolipoprotein A-IV (apoA-IV) gene. By impairing or enhancing E-cadherin-dependent cell adhesion, we demonstrate in the present study its involvement in the transcriptional activation of the apoA-IV gene in Caco-2 cells. This control requires the regulatory sequence that we have previously identified as necessary and sufficient to drive and restrict apoA-IV gene expression in enterocytes in vivo. Furthermore, using chimeric E-cadherin-Fc homophilic ligand-coated surfaces, we show that a direct activation of E-cadherin triggers the transcriptional activation of the apoA-IV promoter. Finally, E-cadherin-dependent cell-cell adhesion controls the nuclear abundance of the transcription factor hepatic nuclear factor 4alpha, which is involved in the enterocyte-specific expression of apoA-IV gene. Altogether, our results suggest that E-cadherin controls enterocyte-specific expression of genes, such as the apoA-IV gene, through the control of hepatic nuclear factor 4alpha nuclear abundance.

An inter-laboratory study to evaluate the effects of medium composition on the differentiation and barrier function of Caco-2 cell lines.

Differentiated human intestinal Caco-2 cells are frequently used in toxicology and pharmacology as in vitro models for studies on intestinal barrier functions. Since several discrepancies exist among the different lines and clones of Caco-2 cells, comparison of the results obtained and optimisation of models for use for regulatory purposes are particularly difficult, especially with respect to culture conditions and morphological and biochemical parameters. An inter-laboratory study has been performed on the parental cell line and on three clonal Caco-2 cell lines, with the aim of standardising the culture conditions and identifying the best cell line with respect to parameters relevant to barrier integrity, namely, trans-epithelial electrical resistance (TEER) and mannitol passage, and of epithelial differentiation (alkaline phosphatase activity). Comparison of the cell lines maintained in traditional serum-supplemented culture medium or in defined medium, containing insulin, transferrin, selenium and lipids, showed that parameter performance was better and more reproducible with the traditional medium. The maintenance of the cell lines for 15 days in culture was found to be sufficient for the development of barrier properties, but not for full epithelial differentiation. Caco-2/TC7 cells performed better than the other three cell lines, both in terms of reproducibility and performance, exhibiting low TEER and mannitol passage, and high alkaline phosphatase activity.

Bovine prion is endocytosed by human enterocytes via the 37 kDa/67 kDa laminin receptor.

Some forms of transmissible spongiform encephalopathies result from oral infection. We have thus analyzed the early mechanisms that could account for an uptake of infectious prion particles by enterocytes, the major cell population of the intestinal epithelium. Human Caco-2/TC7 enterocytes cultured on microporous filters were incubated with different prion strains and contaminated brain homogenates in the apical compartment. Internalization of infectious particles was analyzed by Western blotting and immunofluorescence. We observed internalization by enterocytes of prion particles from bovine spongiform encephalopathy brain homogenates but not from mouse-adapted scrapie-strain brain homogenates or purified bovine spongiform encephalopathy scrapie-associated fibrils. Bovine prion particles were internalized via endocytosis within minutes of infection and were associated with subapical vesicular structures related to early endosomes. The endocytosis of the infectious bovine PrP(Sc) was reduced by preincubating the cells with an anti-LRP/LR blocking antibody, identifying the 37 kDa/67 kDa laminin receptor (LRP/LR), which is apically expressed in Caco-2/TC7 cells, as the receptor for the infectious prion protein. Altogether, our results underscore a potential role of enterocytes in the absorption of bovine prions during oral infection through specific LRP/LR-dependent endocytosis.

Intestinal apolipoprotein A-IV gene transcription is controlled by two hormone-responsive elements: a role for hepatic nuclear factor-4 isoforms.

In the small intestine, the expression of the apolipoprotein (apo) C-III and A-IV genes is restricted to the enterocytes of the villi. We have previously shown that, in transgenic mice, specific expression of the human apo C-III requires a hormone-responsive element (HRE) located in the distal region of the human apoA-IV promoter. This HRE binds the hepatic nuclear factors (HNF)-4alpha and gamma. Here, intraduodenal injections in mice and infections of human enterocytic Caco-2/TC7 cells with an adenovirus expressing a dominant-negative form of HNF-4alpha repress the expression of the apoA-IV gene, demonstrating that HNF-4 controls the apoA-IV gene expression in enterocytes. We show that HNF-4alpha and gamma functionally interact with a second HRE present in the proximal region of the human apoA-IV promoter. New sets of transgenic mice expressing mutated forms of the promoter, combined with the human apo C-III enhancer, demonstrate that, whereas a single HRE is sufficient to reproduce the physiological cephalo-caudal gradient of apoA-IV gene expression, both HREs are required for expression that is restricted to villi. The combination of multiple HREs may specifically recruit regulatory complexes associating HNF-4 and either coactivators in villi or corepressors in crypts.

Intestinal glucose-dependent expression of glucose-6-phosphatase: involvement of the aryl receptor nuclear translocator transcription factor.

Glucose-6-phosphatase (G6Pase) catalyzes the release of glucose from glucose 6-phosphate. This enzyme was mainly studied in the liver, but while detected in the small intestine little is known about the regulation of its intestinal expression. This study describes the mechanisms of the glucose-dependent regulation of G6Pase expression in intestinal cells. Results obtained in vivo and in Caco-2/TC7 enterocytes showed that glucose increases the G6Pase mRNA level. In Caco-2/TC7 cells, glucose stabilized G6Pase mRNA and activated the transcription of the gene, meaning that glucose-dependent G6Pase expression involved both transcriptional and post-transcriptional mechanisms. Reporter-gene studies showed that, although the -299/+57 region of the human G6Pase promoter was sufficient to trigger the glucose response in the hepatoma cell line HepG2, the -1157/-1133 fragment was required for maximal activation of glucose-6-phosphatase gene transcription in Caco-2/TC7 cells. This fragment binds the aryl receptor nuclear translocator (ARNT), cAMP-responsive element-binding protein, and upstream stimulatory factor transcription factors. The DNA binding activity of these transcription factors was increased in nuclear extracts of differentiated cells from the intestinal villus of mice fed sugar-rich diets as compared with mice fed a no-sugar diet. A direct implication of ARNT in the activation of G6Pase gene transcription by glucose has been observed in Caco-2/TC7 cells using RNA interference experiments. These results support a physiological role for G6Pase in the control of nutrient absorption in the small intestine.

HNF-4-dependent induction of apolipoprotein A-IV gene transcription by an apical supply of lipid micelles in intestinal cells.

Apolipoprotein (apo) A-IV, a component of triglyceride-rich lipoproteins secreted by the small intestine, has been shown to play an important role in the control of lipid homeostasis. Numerous studies have described the induction of apoA-IV gene expression by lipids, but the molecular mechanisms involved in this process remain unknown. In this study, we have demonstrated that a lipid bolus induced transcription of the apoA-IV gene in transgenic mice and that the regulatory region of the apoA-IV gene, composed of the apoC-III enhancer and the apoA-IV promoter (eC3-A4), was responsible for this induction. In enterocyte Caco-2/TC7 cells, a permanent supply of lipids at the basal pole induced expression of the apoA-IV gene both at the transcriptional level and through mRNA stabilization. ApoA-IV gene transcription and protein secretion were further induced by an apical supply of complex lipid micelles mimicking the composition of duodenal micelles, and this effect was not reproduced by apical delivery of different combinations of micelle components. Only induction of the apoA-IV gene by lipid micelles involved the participation of hepatic nuclear factor (HNF)-4, as demonstrated using a dominant negative form of this transcription factor. Accordingly, lipid micelles increased the DNA binding activity of HNF-4 on the eC3-A4 region. These results emphasize the importance of physiological delivery of dietary lipids on apoA-IV gene expression and the implication of HNF-4 in this regulation.

Lipid micelles stimulate the secretion of triglyceride-enriched apolipoprotein B48-containing lipoproteins by Caco-2 cells.

Intestinal triglyceride-rich lipoproteins (TRL) are synthesized from dietary lipids. This study was designed to evaluate the effects of lipid micelles, mimicking post-digestive duodenal micelles, on the fate of apolipoprotein B (apoB)48-containing lipoproteins by Caco-2 cells. Such micelles, consisting of oleic acid (OA), taurocholate, 2-monooleoylglycerol (2-MO), cholesterol (Chol), and L-alpha-lysophospatidylcholine, were the most efficient inducers of OA uptake and esterification. The efficiency of TG and apoB48 secretion increased specifically as a function of cell differentiation. PAGE analysis of secreted lipoproteins separated by sequential ultracentrifugation after [35S] labeling revealed differences in the secretion of apoB100- and apoB48-containing lipoproteins. In absence of micelles, apoB48 was secreted mostly in "HDL-like" particles, as observed in enterocytes in vivo. Micelle application increased 2.7-fold the secretion of apoB, resulting in 53 times more apoB48 being recovered as TG-enriched lipoproteins at d < 1.006 g/ml. Electron microscopy revealed the presence of lipid droplets in the secretory pathway and the accumulation of newly synthesized TG in cytoplasmic lipid droplets, as in enterocytes in vivo. We showed that these droplets could be used for secretion. However, apoB48 preferentially bound to newly synthesized TG in the presence of micelles, accounting in part for the functional advantage of apoB editing in the intestine. While Caco-2 cells express both apoB isoforms, our results show that the apical supply of complex lipid micelles favors the physiological route of apoB48-containing TG-enriched lipoproteins.

Apple procyanidins decrease cholesterol esterification and lipoprotein secretion in Caco-2/TC7 enterocytes.

Decrease of plasma lipid levels by polyphenols was linked to impairment of hepatic lipoprotein secretion. However, the intestine is the first epithelium that faces dietary compounds, and it contributes to lipid homeostasis by secreting triglyceride-rich lipoproteins during the postprandial state. The purpose of this study was to examine the effect of apple and wine polyphenol extracts on lipoprotein synthesis and secretion in human Caco-2/TC7 enterocytes apically supplied with complex lipid micelles. Our results clearly demonstrate that apple, but not wine, polyphenol extract dose-dependently decreases the esterification of cholesterol and the enterocyte secretion of lipoproteins. Apple polyphenols decrease apolipoprotein B (apoB) secretion by inhibiting apoB synthesis without increasing the degradation of the newly synthesized protein. Under our conditions, cholesterol uptake, apoB mRNA, and microsomal triglyceride protein activity were not modified by apple polyphenols. The main monomers present in our mixture did not interfere with the intestinal lipid metabolism. By contrast, apple procyanidins reproduced the inhibition of both cholesteryl ester synthesis and lipoprotein secretion. Overall, our results are compatible with a mechanism of action of polyphenols resulting in impaired lipid availability that could induce the inhibition of intestinal lipoprotein secretion and contribute to the hypolipidemic effect of these compounds in vivo.

Early loss of E-cadherin from cell-cell contacts is involved in the onset of Anoikis in enterocytes.

Anoikis, i.e. apoptosis induced by detachment from the extracellular matrix, is thought to be involved in the shedding of enterocytes at the tip of intestinal villi. Mechanisms controlling enterocyte survival are poorly understood. We investigated the role of E-cadherin, a key protein of cell-cell adhesion, in the control of anoikis of normal intestinal epithelial cells, by detaching murine villus epithelial cells from the underlying basement membrane while preserving cell-cell interactions. We show that upon the loss of anchorage, normal enterocytes execute a program of apoptosis within minutes, via a Bcl-2-regulated and caspase-9-dependent pathway. E-cadherin is lost early from cell-cell contacts. This process precedes the execution phase of detachment-induced apoptosis as it is only weakly modulated by Bcl-2 overexpression or caspase inhibition. E-cadherin loss, however, is efficiently prevented by lysosome and proteasome inhibitors. We also found that a blocking anti-E-cadherin antibody increases the rate of anoikis, whereas the activation of E-cadherin using E-cadherin-Fc chimera proteins reduces anoikis. In conclusion, our results stress the striking sensitivity of normal enterocytes to the loss of anchorage and the contribution of E-cadherin to the control of their survival/apoptosis balance. They open new perspectives on the key role of this protein, which is dysregulated in the intestinal epithelium in both inflammatory bowel disease and cancer.