[Show abstract][Hide abstract] ABSTRACT: Hepatocyte nuclear factor 4 alpha (HNF4α) is a transcription factor involved in the regulation of serum glucose and lipid levels. Several HNF4A gene variants have been associated with the risk of developing type 2 diabetes mellitus. However, no study has yet explored its association with insulin resistance and the cardiometabolic risk in children. We aimed to investigate the relationship between HNF4A genetic variants and the presence of metabolic syndrome (MetS) and metabolic parameters in a pediatric population.
Our study included 1,749 French-Canadians aged 9, 13 and 16 years and evaluated 24 HNF4A polymorphisms that were previously identified by sequencing.
Analyses revealed that, after correction for multiple testing, one SNP (rs736824; P<0.022) and two haplotypes (P1 promoter haplotype rs6130608-rs2425637; P<0.032 and intronic haplotype rs736824-rs745975-rs3212183; P<0.025) were associated with the risk of MetS. Additionally, a significant association was found between rs3212172 and apolipoprotein B levels (coefficient: -0.14 ± 0.05; P<0.022). These polymorphisms are located in HNF4A P1 promoter or in intronic regions.
Our study demonstrates that HNF4α genetic variants are associated with the MetS and metabolic parameters in French Canadian children and adolescents. This study, the first exploring the relation between HNF4A genetic variants and MetS and metabolic variables in a pediatric cohort, suggests that HNF4α could represent an early marker for the risk of developing type 2 diabetes mellitus.
PLoS ONE 02/2015; 10(2):e0117238. DOI:10.1371/journal.pone.0117238 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objectives:
The proprotein convertase subtillisin/kexin type 9 (PCSK9) regulates cholesterol metabolism via degradation of low-density lipoprotein receptor (LDLr). Although PCSK9 is abundantly expressed in the intestine, limited data are available on its functions. The present study aims at determining whether PCSK9 plays important roles in cholesterol homeostasis and lipid transport in the gut.
Methods and results:
Caco-2/15 cells were used allowing the exploration of the PCSK9 secretory route through the apical and basolateral compartments corresponding to intestinal lumen and serosal circulation, respectively. The output of PCSK9 occurred through the basolateral membrane, a site characterized by the location of LDLr. Co-immunoprecipitation studies indicated an association between PCSK9 and LDLr. Addition of purified recombinant wild type and D374Y gain-of function PCSK9 proteins to the basolateral medium was followed by a decrease in LDLr concomitantly with the accumulation of both forms of PCSK9. Furthermore, the latter caused a significant enhancement in cholesterol uptake also evidenced by a raised protein expression of cholesterol transporters NPC1L1 and CD36 without changes in SR-BI, ABCA1, and ABCG5/G8. Moreover, exogenous PCSK9 altered the activity of HMG-CoA reductase and acylcoenzyme A: cholesterol acyltransferase, and was able to enhance chylomicron secretion by positively modulating lipids and apolipoprotein B-48 biogenesis. Importantly, PCSK9 silencing led to opposite findings, which validate our data on the role of PCSK9 in lipid transport and metabolism. Moreover, PCSK9-mediated changes persisted despite LDLr knockdown.
These findings indicate that, in addition to its effect on LDLr, PCSK9 modulates cholesterol transport and metabolism, as well as production of apo B-containing lipoproteins in intestinal cells.
[Show abstract][Hide abstract] ABSTRACT: The use of the anti-inflammatory drug indomethacin (INDO) in preterm infants has been associated with an increased risk of developing enteropathies. In this study, we have investigated the direct impact of INDO on the human mid-gestation intestinal transcriptome using serum-free organ culture. After determining the optimal dose of 1μM of INDO (90% inhibition of intestinal prostaglandin E2 production and range of circulating levels in treated preterm babies), global gene expression profiles were determined using Illumina bead chip microarrays in both small and large intestines after 48h of INDO treatment. Using Ingenuity Pathway Analysis software, we identified critical metabolic pathways that were significantly altered by INDO in both intestinal segments including inflammation and also glycolysis, oxidative phosphorylation and free radical scavenging/oxidoreductase activity, which were confirmed by qPCR at the level of individual genes. Taken together, these data revealed that INDO directly exerts multiple detrimental effects on the immature human intestine.
[Show abstract][Hide abstract] ABSTRACT: Hepatocyte nuclear 4 alpha (HNF4α), involved in glucose and lipid metabolism, has been linked to intestinal inflammation and abnormal mucosal permeability. Moreover, in a genome-wide association study, the HNF4A locus has been associated with ulcerative colitis. The objective of our study was to evaluate the association between HNF4α genetic variants and Crohn's disease (CD) in two distinct Canadian pediatric cohorts. The sequencing of the HNF4A gene in 40 French Canadian patients led to the identification of 27 single nucleotide polymorphism (SNP)s with a minor allele frequency >5%. To assess the impact of these SNPs on disease susceptibility, we first conducted a case-control discovery study on 358 subjects with CD and 542 controls. We then carried out a replication study in a separate cohort of 416 cases and 1208 controls. In the discovery cohort, the genotyping of the identified SNPs revealed that six were significantly associated with CD. Among them, rs1884613 was replicated in the second CD cohort (odds ratio (OR): 1.33; P<0.012) and this association remained significant when both cohorts were combined and after correction for multiple testing (OR: 1.39; P<0.004). An 8-marker P2 promoter haplotype containing rs1884613 was also found associated with CD (P<2.09 × 10(-4) for combined cohorts). This is the first report showing that the HNF4A locus may be a common genetic determinant of childhood-onset CD. These findings highlight the importance of the intestinal epithelium and oxidative protection in the pathogenesis of CD.
Genes and immunity 08/2012; 13(7):556-65. DOI:10.1038/gene.2012.37 · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The inflammatory response of the preterm infants' intestine underlines its inability to respond to hemodynamic stress, microbes, and nutrients. Recent evidence suggests that exogenous epidermal growth factor (EGF) exerts a therapeutic influence on neonatal enteropathies. However, the molecular mechanisms underlying the beneficial effects of EGF remain to be clarified. The purpose of this study was to evaluate the impact of EGF on the gene expression profiles of the developing human small and large intestine at midgestation in serum-free organ cultures using microarrays. The gene expression profiles of cultured human fetal ileal and colonic explants were investigated in the absence or presence of a physiological concentration of 50 ng/ml EGF for 48 h. Data were analyzed with the Ingenuity Pathway Analysis (IPA) software and confirmed by qPCR. We found a total of 6,474 differentially expressed genes in the two segments in response to EGF. IPA functional analysis revealed that in addition to differentially modulating distinct cellular, molecular, and physiological functions in the small and large intestine, EGF regulated the inflammatory response in both intestinal segments in a distinct manner. For instance, several intestinal-derived chemokines such as CCL2, CCL25, CXCL5, and CXCL10 were found to be differentially regulated by EGF in the immature ileum and colon. The findings showing the anti-inflammatory influence of exogenous EGF suggests a mechanistic basis for the beneficial effects of EGF on neonatal enteropathies. These results reinforce growing evidence that by midgestation, the human small intestine and colon rely on specific and distinct regulatory pathways.
[Show abstract][Hide abstract] ABSTRACT: The intestinal epithelium is a highly dynamic tissue undergoing constant and rapid renewal. It consists of a functional villus compartment responsible for terminal digestion and nutrient absorption and a progenitor cell compartment located in the crypts that produce new cells. The mechanisms regulating cell proliferation in the crypt, their migration, and differentiation are still incompletely understood. Until recently, normal human intestinal cell models allowing the study of these mechanisms have been lacking. In our laboratory, using fetal human intestines obtained at mid-gestation, we have generated the first normal human intestinal epithelial crypt-like (HIEC) cell line and villus-like primary cultures of differentiated enterocytes (PCDE). In this chapter, we provide a detailed description of the methodologies used to generate and characterize these normal intestinal crypt and villus cell models.
[Show abstract][Hide abstract] ABSTRACT: Our understanding of gastric epithelial physiology in man is limited by the absence of normal or appropriate cancer cell lines that could serve as an in vitro model. Research mostly relied on primary culture of gastric epithelial cells of animal species, enriched with surface mucous cells, and devoid of glandular zymogenic chief cells. We successfully applied a new nonenzymatic procedure using Matrisperse Cell Recovery Solution to dissociate the entire epithelium from human fetal stomach. Cultures were generated by seeding multicellular aggregates prepared by mechanical fragmentation. We further demonstrate that this simple and convenient technique allows for the maintenance of heterogenous gastric epithelial primary cultures on plastic without a biological matrix as well as the persistence of viable chief cells able to synthesize and secrete gastric digestive enzymes, i.e., pepsinogen and gastric lipase. In wounding experiments, epithelial restitution occurred in serum-reduced conditions and was modulated by exogenous agents. This culture system is thus representative of the foveolus-gland axis and offers new perspectives to establish the influence of individual growth factors and extracellular matrix components as well as their combinatory effects on gastric epithelium homeostasis.
[Show abstract][Hide abstract] ABSTRACT: The occurrence of many neonatal inflammatory intestinal diseases in preterm infants highlights the susceptibility of the immature intestine to responding inadequately to nutrients and microbes. A better understanding of functional intestinal development is essential for the design of optimal treatments ensuring survival and growth of premature infants. The purpose of this study was to evaluate the gene expression profiles of the human ileum and colon at mid-gestation because these 2 segments are considered to be similar at this stage and are the sites of the most frequent pathologies in preterm infants.
We compared the gene-expression profiles of human fetal small and large intestines using a cDNA microarray and analyzed the data with Ingenuity Pathway Analysis software.
We found that a significant proportion of the genes was differentially expressed in the 2 segments. Gene cluster analysis revealed an even higher level of transcriptional dissimilarity at the functional level. For instance, segment-specific/overexpressed gene clusters in the ileum included genes involved with amino acid, vitamin, and mineral metabolism, reflecting the higher level of maturity of the small intestine as compared with the colon in which genes involved with cell cycle, cell death, and cell signaling were the predominant clusters of genes expressed.
Functional clustering analysis of the differentially expressed genes revealed important functional differences between the 2 segments and a relative immaturity of the colon, suggesting that already at mid-gestation, the 2 intestinal segments should be considered as 2 distinct organs.
Journal of pediatric gastroenterology and nutrition 06/2011; 52(6):670-8. DOI:10.1097/MPG.0b013e3182078370 · 2.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hepatocyte nuclear factor 4α (HNF4α) is a nuclear transcription factor mainly expressed in the liver, intestine, kidney, and pancreas. Many of its hepatic and pancreatic functions have been described, but limited information is available on its role in the gastrointestinal tract. The objectives of this study were to evaluate the anti-inflammatory and antioxidant functions of HNF4α as well as its implication in intestinal lipid transport and metabolism. To this end, the HNF4A gene was knocked down by transfecting Caco-2 cells with a pGFP-V-RS lentiviral vector containing an shRNA against HNF4α. Inactivation of HNF4α in Caco-2 cells resulted in the following: (a) an increase in oxidative stress as demonstrated by the levels of malondialdehyde and conjugated dienes; (b) a reduction in secondary endogenous antioxidants (catalase, glutathione peroxidase, and heme oxygenase-1); (c) a lower protein expression of nuclear factor erythroid 2-related factor that controls the antioxidant response elements-regulated antioxidant enzymes; (d) an accentuation of cellular inflammatory activation as shown by levels of nuclear factor-κB, interleukin-6, interleukin-8, and leukotriene B4; (e) a decrease in the output of high density lipoproteins and of their anti-inflammatory and anti-oxidative components apolipoproteins (apo) A-I and A-IV; (f) a diminution in cellular lipid transport revealed by a lower cellular secretion of chylomicrons and their apoB-48 moiety; and (g) alterations in the transcription factors sterol regulatory element-binding protein 2, peroxisome proliferator-activated receptor α, and liver X receptor α and β. In conclusion, HNF4α appears to play a key role in intestinal lipid metabolism as well as intestinal anti-oxidative and anti-inflammatory defense mechanisms.
[Show abstract][Hide abstract] ABSTRACT: Hepatocyte nuclear factor 4α (HNF4α) is a nuclear transcription factor mainly expressed in the liver, intestine, kidney, and
pancreas. Many of its hepatic and pancreatic functions have been described, but limited information is available on its role
in the gastrointestinal tract. The objectives of this study were to evaluate the anti-inflammatory and antioxidant functions
of HNF4α as well as its implication in intestinal lipid transport and metabolism. To this end, the HNF4A gene was knocked down by transfecting Caco-2 cells with a pGFP-V-RS lentiviral vector containing an shRNA against HNF4α.
Inactivation of HNF4α in Caco-2 cells resulted in the following: (a) an increase in oxidative stress as demonstrated by the levels of malondialdehyde and conjugated dienes; (b) a reduction in secondary endogenous antioxidants (catalase, glutathione peroxidase, and heme oxygenase-1); (c) a lower protein expression of nuclear factor erythroid 2-related factor that controls the antioxidant response elements-regulated
antioxidant enzymes; (d) an accentuation of cellular inflammatory activation as shown by levels of nuclear factor-κB, interleukin-6, interleukin-8,
and leukotriene B4; (e) a decrease in the output of high density lipoproteins and of their anti-inflammatory and anti-oxidative components apolipoproteins
(apo) A-I and A-IV; (f) a diminution in cellular lipid transport revealed by a lower cellular secretion of chylomicrons and their apoB-48 moiety;
and (g) alterations in the transcription factors sterol regulatory element-binding protein 2, peroxisome proliferator-activated
receptor α, and liver X receptor α and β. In conclusion, HNF4α appears to play a key role in intestinal lipid metabolism as
well as intestinal anti-oxidative and anti-inflammatory defense mechanisms.
Journal of Biological Chemistry 12/2010; 285(52):40448-40460. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although mitochondrial dysfunction and oxidative stress are central mechanisms in various pathological conditions, they have not been extensively studied in the gastrointestinal tract, which is known to be constantly exposed to luminal oxidants from ingested foods. Key among these is the simultaneous consumption of iron salts and ascorbic acid, which can cause oxidative damage to biomolecules.
The objective of the present work was to evaluate how iron-ascorbate (FE/ASC)-mediated lipid peroxidation affects mitochondrion functioning in Caco-2/15 cells. Our results show that treatment of Caco-2/15 cells with FE/ASC (0.2 mM/2 mM) (1) increased malondialdehyde levels assessed by HPLC; (2) reduced ATP production noted by luminescence assay; (3) provoked dysregulation of mitochondrial calcium homeostasis as evidenced by confocal fluorescence microscopy; (4) upregulated the protein expression of cytochrome C and apoptotic inducing factor, indicating exaggerated apoptosis; (5) affected mitochondrial respiratory chain complexes I, II, III and IV; (6) elicited mtDNA lesions as illustrated by the raised levels of 8-OHdG; (7) lowered DNA glycosylase, one of the first lines of defense against 8-OHdG mutagenicity; and (8) altered the gene expression and protein mass of mitochondrial transcription factors (mtTFA, mtTFB1, mtTFB2) without any effects on RNA Polymerase. The presence of the powerful antioxidant BHT (50 microM) prevented the occurrence of oxidative stress and most of the mitochondrial abnormalities.
Collectively, our findings indicate that acute exposure of Caco-2/15 cells to FE/ASC-catalyzed peroxidation produces harmful effects on mitochondrial functions and DNA integrity, which are abrogated by the powerful exogenous BHT antioxidant. Functional derangements of mitochondria may have implications in oxidative stress-related disorders such as inflammatory bowel diseases.
PLoS ONE 07/2010; 5(7):e11817. DOI:10.1371/journal.pone.0011817 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although intestinal (I) and liver (L) fatty acid binding proteins (FABP) have been widely studied, the physiological significance of the presence of the two FABP forms (I- and L-FABP) in absorptive cells remains unknown as do the differences related to their distribution along the crypt-villus axis, regional expression, ontogeny and regulation in the human intestine. Our morphological experiments supported the expression of I- and L-FABP as early as 13 weeks of gestation. Whereas cytoplasmic immunofluorescence staining of L-FABP was barely detectable in the lower half of the villus and in the crypt epithelial cells, I-FABP was visualized in epithelial cells of the crypt-villus axis in all intestinal segments until the adult period in which the staining was maximized in the upper part of the villus. Immunoelectron microscopy revealed more intense labeling of L-FABP compared with I-FABP, accompanied with a heterogeneous distribution in the cytoplasm, microvilli and basolateral membranes. By western blot analysis, I- and L-FABP at 15 weeks of gestation appeared predominant in jejunum compared with duodenum, ileum, proximal and distal colon. Exploration of the maturation aspect documented a rise in L-FABP in adult tissues. Permanent transfections of Caco-2 cells with I-FABP cDNA resulted in decreased lipid export, apolipoprotein (apo) biogenesis and chylomicron secretion. Additionally, supplementation of Caco-2 with insulin, hydrocortisone and epidermal growth factor differentially modulated the expression of I- and L-FABP, apo B-48 and microsomal triglyceride transfer protein (MTP), emphasizing that these key proteins do not exhibit a parallel modulation. Overall, our findings indicate that the two FABPs display differences in localization, regulation and developmental pattern.
[Show abstract][Hide abstract] ABSTRACT: The integrin alpha6 subunit exists as two different variants, termed alpha6A and alpha6B. These two variants have been shown to harbor potentially distinct biochemical properties but little is known about their cellular function. The aim of this work was to characterize the expression of the integrin alpha6A and B variants in relation to cell proliferation and differentiation in the human small intestinal epithelium. The results showed distinct expression patterns for the two variants along the crypt-villus axis. Indeed, proliferative cells of the crypt were found to predominantly express alpha6A, while differentiated enterocytes and Paneth cells expressed the alpha6B variant. A similar relationship was observed in intestinal cell models by competitive RT-PCR. Further studies in the Caco-2 cell model showed that manipulating the cellular balance of the two alpha6 variants can influence transcriptional activities related to cell proliferation but not differentiation. This suggests that differential expression of the alpha6 subunits is involved in the intestinal epithelial cell renewal process. Further studies will be needed to substantiate this hypothesis.
[Show abstract][Hide abstract] ABSTRACT: The morphological development of the gastrointestinal tract (GI), in laboratory animals as well as in humans, has been well described since more than 100 years. However, even though its functional development and regulatory mechanisms are pretty well understood, our knowledge of the human GI functions originated primarily from studies on rat and mouse. Because of clear differences in genetic make up, development rates and sequences, as well as physiological differences, extrapolations of animal data to the human must be made with caution. A reliable organ culture technique in which the morphological as well as physiological parameters are well maintained has been set up. This technique allows studies of basic physiological functions such as gene expression, localization of specific cell markers, numerous digestive enzymatic activities, and lipid and lipoprotein processing. Furthermore, it also permits to determine and characterize the biological actions of potential regulators such as growth factors and hormones. Finally, the establishment of human intestinal epithelial cell lines allows the validation and the characterization of the molecular mechanisms involved in the specific regulatory pathways of the human GI development.
[Show abstract][Hide abstract] ABSTRACT: Mechanisms that specifically modulate cell spreading and/or cell migration following epithelial wounding are poorly understood. Using micro-wounded human gastric epithelial monolayers, we show herein that EGF and TGFalpha maximally increase spreading of epithelial sheets under a cell proliferation-independent mechanism. Treatment of confluent HGE-17 cells with the phosphatidylinositol 3-kinase inhibitor, LY294002, and the epidermal growth factor receptor inhibitor, PD153035, strongly reduced basal and TGFalpha-stimulated cell spreading. While pharmacological inhibition of pp60src-kinase activity also attenuated basal epithelial spreading, addition of the mTOR/p70S6K inhibitor rapamycin or a specific siRNA targeting ILK sequence did not affect the kinetic rates of wound closure. Epithelial wound healing was initiated by actin purse-string contraction followed by lamellae formation. Conversely, disruption of actin and tubulin stability with cytochalasin D and nocodazole, respectively, inhibited epithelial sheet spreading. Finally, antibodies directed against the alpha3 integrin subunit, but not against the alpha6 or alpha2 subunits, attenuated epithelial sheet spreading as well as lamellae formation. In conclusion, the current investigation establishes that EGF/TGFalpha and the alpha3beta1 integrin, pp60c-src, EGFR and PI3K pathways are mainly associated with the cell spreading of the restitution process during healing of human gastric epithelial wounds.
[Show abstract][Hide abstract] ABSTRACT: The role of intestinal fatty acid binding protein (I-FABP) in lipid metabolism remains elusive. To address this issue, normal human intestinal epithelial cells (HIEC-6) were transfected with cDNA to overexpress I-FABP and compared with cells treated with empty pQCXIP vector. I-FABP overexpression stimulated mitochondrial [U-14C]oleate oxidation to CO2 and acid-soluble metabolites via mechanisms including the upregulation of protein expression and the activity of carnitine palmitoyltransferase 1, a critical enzyme controlling the entry of fatty acid (FA) into mitochondria, and increased activity of 3-hydroxyacyl-CoA dehydrogenase, a mitochondrial beta-oxidation enzyme. On the other hand, the gene and protein expression of the key enzymes FA synthase and acetyl-coenzyme A carboxylase 2 was decreased, suggesting diminished lipogenesis. Furthermore, I-FABP overexpression caused a decline in [14C]free cholesterol (CHOL) incorporation. Accordingly, a significant lessening was observed in the gene expression of Niemann Pick C1-Like 1, a mediator of CHOL uptake, along with an increase in the transcripts and protein content of ABCA1 and ABCG5/ABCG8, acting as CHOL efflux pumps. Furthermore, I-FABP overexpression resulted in increased levels of mRNA, protein mass, and activity of HMG-CoA reductase, the rate-limiting step in CHOL synthesis. Scrutiny of the nuclear receptors revealed augmented peroxisome proliferator-activated receptor alpha,gamma and reduced liver X receptor-alpha in HIEC-6 overexpressing I-FABP. Finally, I-FABP overexpression did not influence acyl-coenzyme A oxidase 1, which catalyzes the first rate-limiting step in peroxisomal FA beta-oxidation. Overall, our data suggest that I-FABP may influence mitochondrial FA oxidation and CHOL transport by regulating gene expression and interaction with nuclear receptors.
The Journal of Lipid Research 06/2008; 49(5):961-72. DOI:10.1194/jlr.M700363-JLR200 · 4.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: IntroductionDevelopment of the Gastrointestinal MucosaGastric Cell Proliferation and DifferentiationIntestinal Cell Proliferation and DifferentiationBiomedical Applications
Cell Signaling and Growth Factors in Development: From Molecules to Organogenesis, 03/2008: pages 755 - 790; , ISBN: 9783527619689
[Show abstract][Hide abstract] ABSTRACT: Restitution is a crucial event during the healing of superficial injury of the gastric mucosa involving epithelial cell sheet movement into the damaged area. We demonstrated that growth factors promote the restitution of human gastric epithelial cells. However, the intracellular signaling pathways that transmit extracellular cues as well as regulate basal and growth factor-stimulated gastric epithelial cell migration are still unclear. Herein, confluent human gastric epithelial cell monolayers (HGE-17) or primary cultures of gastric epithelial cells were wounded with a razor blade and the migration response was analyzed in presence or absence of TGFalpha or of pharmacological inhibitors of signaling proteins. Kinase activation profile analysis and phase-contrast microscopy were also performed in parallel. We report that ERK1/2 and Akt activities are rapidly stimulated following wounding of HGE-17 cells. Treatment of confluent HGE-17 cells or primary cultures of gastric epithelial cells with the phosphatidylinositol 3-kinase inhibitor LY294002, but not the MEK1 inhibitor, PD98059, significantly inhibits basal and TGFalpha-induced migration following wounding. Conversely, treatment of wounded HGE-17 cells with phosphatidylinositol(3,4,5)-triphosphate is sufficient to stimulate basal cell migration by 235%. In addition, pp60c-src kinase activity and tyrosine phosphorylation of epidermal growth factor receptors (EGFR) are also rapidly enhanced after wounding and pharmacological inhibition of both these activities strongly attenuates basal and TGFalpha-induced migration as well as Akt phosphorylation levels. In conclusion, the present results indicate that EGFR-dependent PI3K activation promotes restitution of wounded human gastric epithelial monolayers.