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Vincent Frochot,
Malik Alqub,
Anne-Laure Cattin, Véronique Carrière,
Anne Houllier,
Floriane Baraille,
Laurence Barbot,
Susan Saint-Just,
Agnès Ribeiro,
Michel Lacasa,
Philippe Cardot,
Jean Chambaz,
Monique Rousset,
Jean-Marc Lacorte
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ABSTRACT: 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.
AJP Gastrointestinal and Liver Physiology 03/2012; 302(11):G1253-63. · 3.43 Impact Factor
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ABSTRACT: 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.
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.
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.
PLoS ONE 02/2009; 4(1):e4278. · 4.09 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 06/2005; 280(20):20094-101. · 4.77 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 05/2005; 280(20):20094-20101. · 4.77 Impact Factor
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ABSTRACT: 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.
Journal of Biological Chemistry 03/2005; 280(7):5406-13. · 4.77 Impact Factor
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ABSTRACT: Aminopeptidase B (Ap-B), a ubiquitous enzyme, catalyses the amino-terminal cleavage of basic residues of peptide or protein substrates, indicating a role in precursor processing. The physiological function of Ap-B still remains an open question, even though its activity suggests that it could be involved in inflammatory processes and proliferation of tumor cells. This study was conducted to determine the expression of Ap-B in the developing and adult retina as a path to envisage physiological roles of Ap-B. RT-PCR and in situ hybridization were used to detect expression of Ap-B mRNA and activity tests, Western blotting and immunofluorescence microscopy were performed to identify and localize the enzyme in the rat retina. These biochemical and morphological methods show that Ap-B is expressed in the retina from embryo to adult. Expression level is restricted to specific layers (pigmented epithelium, outer and inner plexiform layers and ganglion cell layer) and is developmentally regulated. Moreover, a preliminary analysis indicates that Ap-B, the glucose transporter GLUT3 and choline acetyltransferase (ChAT) share a similar expression pattern in retina. Altogether, Ap-B appears predominantly expressed in neuronal cells lying in retinal layers containing neuritic extensions and synaptic junctions. Such expression is up-regulated during ontogenesis allowing to hypothesized that Ap-B participates in processes accompanying retinal neuronal cell differentiation.
Experimental Eye Research 12/2004; 79(5):639-48. · 3.26 Impact Factor
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ABSTRACT: Our purpose was to analyze whether postmitotic Caco-2 colon cancer cells, although they express most of the differentiation characteristics of terminally differentiated intestinal epithelial cells, still maintain, unlike normal cells, a proliferation potential. Experiments were performed with clone TC7 of the Caco-2 cell line. Dividing TC7 cells are undifferentiated and express detectable levels of thymidylate synthase (TS) and cytochrome P450 1A1 (CYP1A1) mRNAs. When reaching confluence TS and CYP1A1 are downregulated, mitosis is no longer detectable, and differentiation takes place, as demonstrated by appearance and increasing levels of differentiation-associated marker mRNAs (e.g., sucrase-isomaltase (SI), dipeptidylpeptidase-IV (DPP-IV) or GLUT5), increasing activities of sucrase and DPP-IV, and increasing expression, on immunofluorescence analysis, of SI on the surface of the cell layer. Trypsinization and seeding of late postconfluent cells (day 30) expressing complete differentiation results within 24 h in upregulation of TS and CYP1A1, a concomitant and dramatic disappearance of differentiation marker mRNAs associated with a decrease in sucrase and DPP-IV activities, and delayed resumption of cell division. This is followed, after the cells have reached confluence again, by downregulation of TS and CYP1A1 and resumption of cell differentiation. The ability of differentiated cells to dedifferentiate was further confirmed by wounding the cell layer of late postconfluent differentiated cultures: within 24 h following the wound, cells migrate from the wound edge and dedifferentiate, as demonstrated by transmission electron microscopy and disappearance of SI from the cell surface of migrating cells. Late postconfluent differentiated cells were tumorigenic in nude mice. These results raise the question of the validity of the concept of differentiation therapy when applied to colon cancer cells.
Experimental and Molecular Pathology 09/2000; · 2.42 Impact Factor
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Guillemette Huet,
Sylviane Hennebicq-Reig,
Carmen de Bolos,
Fausto Ulloa,
Thécla Lesuffleur,
Alain Barbat, Véronique Carrière,
Isabelle Kim,
Francisco X. Real,
Philippe Delannoy,
Alain Zweibaum
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ABSTRACT: Exposure for 24 h of mucus-secreting HT-29 cells to the sugar analogue GalNAc-α-O-benzyl results in inhibition of Galβ1-3GalNAc:α2,3-sialyltransferase, reduced mucin sialylation, and inhibition of their
secretion (Huet, G., I. Kim, C. de Bolos, J.M. Loguidice, O. Moreau, B. Hémon, C. Richet, P. Delannoy, F.X. Real., and P.
Degand. 1995. J. Cell Sci. 108:1275–1285). To determine the effects of prolonged inhibition of sialylation, differentiated HT-29 populations were grown
under permanent exposure to GalNAc-α-O-benzyl. This results in not only inhibition of mucus secretion, but also in a dramatic swelling of the cells and the accumulation
in intracytoplasmic vesicles of brush border–associated glycoproteins like dipeptidylpeptidase-IV, the mucin-like glycoprotein
MUC1, and carcinoembryonic antigen which are no longer expressed at the apical membrane. The block occurs beyond the cis-Golgi as substantiated by endoglycosidase treatment and biosynthesis analysis. In contrast, the polarized expression of
the basolateral glycoprotein GP 120 is not modified. Underlying these effects we found that (a) like in mucins, NeuAcα2-3Gal-R is expressed in the terminal position of the oligosaccharide species associated with the
apical, but not the basolateral glycoproteins of the cells, and (b) treatment with GalNAc-α-O-benzyl results in an impairment of their sialylation. These effects are reversible upon removal of the drug. It is suggested
that α2-3 sialylation is involved in apical targeting of brush border membrane glycoproteins and mucus secretion in HT-29
cells.
The Journal of Cell Biology 06/1998; 141(6):1311-1322. · 10.26 Impact Factor
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ABSTRACT: CYP2E1 is involved in the activation of various carcinogens, including N-nitrosamines, which are believed to be important in human carcinogenesis. Humans exhibit wide interindividual variability in levels of CYP2E1 mRNA and protein, which might explain interindividual differences in susceptibility to carcinogens activated by CYP2E1. Such variability could be due either to genetic polymorphisms observed in the CYP2E1 gene (Rsa I in the 5 '-flanking region, Dra I in intron 6 and Taq I in intron 7) or to varying inducibility by xenobiotics. The aim of the present study was to establish whether, in a Caucasian population (n=93), there existed a relationship between allelic forms of the CYP2E1 gene and the phenotype determined in vitro by hepatic ability to 6-hydroxylate chlorzoxazone. Rates of chlorzoxazone- 6-hydroxylation were significantly correlated with levels of immunochemically measured CYP2E1 (p<0.001). CYP1A2,2C8,2C9,2C18,2D6,3A4 and 3A5 did not appear to be significantly involved in chlorzoxazone metabolism, whereas the participation of CYP1A1 could not be excluded. Frequencies of the rare alleles for the three polymorphism sites were 2.2% for Rsa 1,7.5% for Dra I and 8.5% for Taq I. Despite substantial interindividual variations in chlorzoxazone hydroxylase activity, no relationship between any of the three polymorphisms and CYP2E1 activity was established. Therefore, in humans, interindividual variability in CYP2E1 levels is probably due to differing induction levels as a result of environmental factors, or to genetic factors other than those studied in this work.
(C) Lippincott-Raven Publishers.
Pharmacogenetics and Genomics 05/1996; 6(3). · 3.48 Impact Factor
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ABSTRACT: Our purpose was to investigate whether inducers of cytochrome P450 1A1 (CYP1A1), which cause a decreased expression in Caco-2 cells, at both the mRNA and protein levels, of membrane proteins associated with the uptake and transport of hexoses, would also affect the expression of γ-glutamyltranspeptidase (γGT) (EC 2.3.2.2). In Caco-2 clonal TC7 cells grown under standard conditions (25 mM glucose), exposure to β-naphthoflavone (β-NF), 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin, and 3-methylcholanthrene resulted in increased glucose consumption and decreased γGT activity in cells grown to confluence, i.e. when the differentiation is optimum. γGT activity was further analyzed during the time course of differentiation of TC7 cells treated or not with β-naphthoflavone: while γGT activity in untreated cells showed a 10-fold increase from the exponential phase of growth until late postconfluence, γGT activity in β-NF-treated cells, although increasing by 4-fold, remained at a much lower level (<25%). This decreased activity of γGT was associated with a decreased level of γGT mRNA. This inhibiting effect was not dependent on the CYP1A1 activity, as it also occurred in the presence of CYP1A1 inhibitors such as α-naphthoflavone, 8-methoxypsoralen or ellipticin. It was however dependent on glucose supply as it was not observed when the cells were cultured in low glucose (1 mM). These results raise the question of whether, in Caco-2 cells, CYP1A1 inducers or the signal transduction system which controls CYP1A1 are involved in the regulation of the expression of γGT through a mechanism involving glucose metabolism.
Biochemical Pharmacology.
