RESUMO
Obesity and its metabolic complications are characterized by subclinical systemic and tissue inflammation. In rodent models of obesity, inflammation and metabolic impairments are linked with intestinal barrier damage. However, whether intestinal permeability is altered in human obesity remains to be investigated. In a cohort of 122 severely obese and non-obese patients, we analyzed intestinal barrier function combining in vivo and ex vivo investigations. We found tight junction impairments in the jejunal epithelium of obese patients, evidenced by a reduction of occludin and tricellulin. Serum levels of zonulin and LPS binding protein, two markers usually associated with intestinal barrier alterations, were also increased in obese patients. Intestinal permeability per se was assessed in vivo by quantification of urinary lactitol/mannitol (L/M) and measured directly ex vivo on jejunal samples in Ussing chambers. In the fasting condition, L/M ratio and jejunal permeability were not significantly different between obese and non-obese patients, but high jejunal permeability to small molecules (0.4 kDa) was associated with systemic inflammation within the obese cohort. Altogether, these results suggest that intestinal barrier function is subtly compromised in obese patients. We thus tested whether this barrier impairment could be exacerbated by dietary lipids. To this end, we challenged jejunal samples with lipid micelles and showed that a single exposure increased permeability to macromolecules (4 kDa). Jejunal permeability after the lipid load was two-fold higher in obese patients compared to non-obese controls and correlated with systemic and intestinal inflammation. Moreover, lipid-induced permeability was an explicative variable of type 2 diabetes. In conclusion, intestinal barrier defects are present in human severe obesity and exacerbated by a lipid challenge. This paves the way to the development of novel therapeutic approaches to modulate intestinal barrier function or personalize nutrition therapy to decrease lipid-induced jejunal leakage in metabolic diseases. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Inflamação/metabolismo , Absorção Intestinal/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Lipídeos/administração & dosagem , Obesidade/metabolismo , Proteínas de Fase Aguda , Adulto , Idoso , Células CACO-2 , Proteínas de Transporte/sangue , Estudos de Casos e Controles , Toxina da Cólera/sangue , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Haptoglobinas , Humanos , Inflamação/complicações , Inflamação/fisiopatologia , Jejuno/metabolismo , Jejuno/fisiopatologia , Proteína 2 com Domínio MARVEL/metabolismo , Masculino , Glicoproteínas de Membrana/sangue , Micelas , Pessoa de Meia-Idade , Obesidade/complicações , Obesidade/fisiopatologia , Ocludina/metabolismo , Permeabilidade , Precursores de Proteínas , Junções Íntimas/metabolismo , Adulto JovemRESUMO
Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics.
Assuntos
Colesterol/metabolismo , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Lisofosfolipídeos/metabolismo , Microdomínios da Membrana/metabolismo , Receptores Depuradores Classe B/metabolismo , Esfingomielinas/metabolismo , Células CACO-2 , Humanos , Gotículas Lipídicas/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The worldwide prevalence of metabolic diseases is increasing, and there are global recommendations to limit consumption of certain nutrients, especially saturated lipids. Insulin resistance, a common trait occurring in obesity and type 2 diabetes, is associated with intestinal lipoprotein overproduction. However, the mechanisms by which the intestine develops insulin resistance in response to lipid overload remain unknown. Here, we show that insulin inhibits triglyceride secretion and intestinal microsomal triglyceride transfer protein expression in vivo in healthy mice force-fed monounsaturated fatty acid-rich olive oil but not in mice force-fed saturated fatty acid-rich palm oil. Moreover, when mouse intestine and human Caco-2/TC7 enterocytes were treated with the saturated fatty acid, palmitic acid, the insulin-signaling pathway was impaired. We show that palmitic acid or palm oil increases ceramide production in intestinal cells and that treatment with a ceramide analogue partially reproduces the effects of palmitic acid on insulin signaling. In Caco-2/TC7 enterocytes, ceramide effects on insulin-dependent AKT phosphorylation are mediated by protein kinase C but not by protein phosphatase 2A. Finally, inhibiting de novo ceramide synthesis improves the response of palmitic acid-treated Caco-2/TC7 enterocytes to insulin. These results demonstrate that a palmitic acid-ceramide pathway accounts for impaired intestinal insulin sensitivity, which occurs within several hours following initial lipid exposure.
Assuntos
Ceramidas/biossíntese , Enterócitos/metabolismo , Insulina/metabolismo , Mucosa Intestinal/metabolismo , Ácido Palmítico/farmacologia , Transdução de Sinais , Animais , Células CACO-2 , Humanos , Camundongos , Óleo de Palmeira , Ácido Palmítico/metabolismo , Fosforilação/efeitos dos fármacos , Óleos de Plantas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
BACKGROUND: Orodental diseases include several clinically and genetically heterogeneous disorders that can present in isolation or as part of a genetic syndrome. Due to the vast number of genes implicated in these disorders, establishing a molecular diagnosis can be challenging. We aimed to develop a targeted next-generation sequencing (NGS) assay to diagnose mutations and potentially identify novel genes mutated in this group of disorders. METHODS: We designed an NGS gene panel that targets 585 known and candidate genes in orodental disease. We screened a cohort of 101 unrelated patients without a molecular diagnosis referred to the Reference Centre for Oro-Dental Manifestations of Rare Diseases, Strasbourg, France, for a variety of orodental disorders including isolated and syndromic amelogenesis imperfecta (AI), isolated and syndromic selective tooth agenesis (STHAG), isolated and syndromic dentinogenesis imperfecta, isolated dentin dysplasia, otodental dysplasia and primary failure of tooth eruption. RESULTS: We discovered 21 novel pathogenic variants and identified the causative mutation in 39 unrelated patients in known genes (overall diagnostic rate: 39%). Among the largest subcohorts of patients with isolated AI (50 unrelated patients) and isolated STHAG (21 unrelated patients), we had a definitive diagnosis in 14 (27%) and 15 cases (71%), respectively. Surprisingly, COL17A1 mutations accounted for the majority of autosomal-dominant AI cases. CONCLUSIONS: We have developed a novel targeted NGS assay for the efficient molecular diagnosis of a wide variety of orodental diseases. Furthermore, our panel will contribute to better understanding the contribution of these genes to orodental disease. TRIAL REGISTRATION NUMBERS: NCT01746121 and NCT02397824.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Anormalidades Dentárias/genética , Amelogênese Imperfeita/genética , Autoantígenos/genética , Deleção Cromossômica , Transtornos Cromossômicos/genética , Cromossomos Humanos Par 11/genética , Estudos de Coortes , Coloboma/genética , Displasia da Dentina/genética , França , Perda Auditiva Neurossensorial/genética , Humanos , Colágenos não Fibrilares/genética , Reprodutibilidade dos Testes , Colágeno Tipo XVIIRESUMO
Dental caries is a multifactorial condition that remains a major public health issue in high income countries. The prevalence of dental caries in children has markedly declined in most countries over the past 30 years. However, the disease continues to affect a vulnerable population defined as a high-risk group. As many public health policies are inefficient in dealing with this underprivileged group, it is necessary to find other strategies to decrease the incidence and the burden of dental caries. Defining dental caries as a chronic disease enables us to develop the concept of 'therapeutic patient education.' It is meant to train patients to self-manage or adapt treatment to their particular chronic disease and to cope with new processes and skills. The purpose of this paper is to propose a new approach to dental caries, in particular to early childhood caries. That should decrease the gravity and prevalence of the disease in this specific population. As a result, this new approach could increase the quality of life of many children both in terms of function and aesthetics.
Assuntos
Cárie Dentária , Educação de Pacientes como Assunto/ética , Odontopediatria/ética , Criança , Doença Crônica/epidemiologia , Doença Crônica/terapia , Cárie Dentária/epidemiologia , Cárie Dentária/terapia , Humanos , Populações Vulneráveis/estatística & dados numéricosRESUMO
BACKGROUND & AIMS: Cell adhesion is one function regulated by cellular prion protein (PrP(c)), a ubiquitous, glycosylphosphatidylinositol-anchored glycoprotein. PrP(c) is located in cell-cell junctions and interacts with desmosome proteins in the intestinal epithelium. We investigated its role in intestinal barrier function. METHODS: We analyzed permeability and structure of cell-cell junctions in intestine tissues from PrP(c) knockout (PrP(c-/-)) and wild-type mice. PrP(c) expression was knocked down in cultured human Caco-2/TC7 enterocytes using small hairpin RNAs. We analyzed colon samples from 24 patients with inflammatory bowel disease (IBD). RESULTS: Intestine tissues from PrP(c-/-) mice had greater paracellular permeability than from wild-type mice (105.9 ± 13.4 vs 59.6 ± 10.1 mg/mL fluorescein isothiocyanate-dextran flux; P < .05) and impaired intercellular junctions. PrP(c-/-) mice did not develop spontaneous disease but were more sensitive than wild-type mice to induction of colitis with dextran sulfate (32% mortality vs 4%, respectively; P = .0033). Such barrier defects were observed also in Caco-2/TC7 enterocytes following PrP(c) knockdown; the cells had increased paracellular permeability (1.5-fold over 48 hours; P < .001) and reduced transepithelial electrical resistance (281.1 ± 4.9 vs 370.6 ± 5.7 Ω.cm(2); P < .001). Monolayer shape and cell-cell junctions were altered in cultures of PrP(c) knockdown cells; levels of E-cadherin, desmoplakin, plakoglobin, claudin-4, occludin, zonula occludens 1, and tricellulin were decreased at cell contacts. Cell shape and junctions were restored on PrP(c) re-expression. Levels of PrP(c) were decreased at cell-cell junctions in colonic epithelia from patients with Crohn's disease or ulcerative colitis. CONCLUSIONS: PrP(c) regulates intestinal epithelial cell-cell junctions and barrier function. Its localization is altered in colonic epithelia from patients with IBD, supporting the concept that disrupted barrier function contributes to this disorder.
Assuntos
Doenças Inflamatórias Intestinais/metabolismo , Junções Intercelulares/metabolismo , Mucosa Intestinal/metabolismo , Proteínas PrPC/metabolismo , Animais , Permeabilidade da Membrana Celular/fisiologia , Células Cultivadas , Colo/metabolismo , Enterócitos/metabolismo , Humanos , Camundongos , Camundongos KnockoutRESUMO
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.
Assuntos
Gorduras na Dieta/metabolismo , Ácidos Graxos/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Absorção Intestinal/genética , Mucosa Intestinal/metabolismo , Animais , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Proteínas de Transporte de Ácido Graxo/genética , Proteínas de Transporte de Ácido Graxo/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Polietilenoglicóis/farmacologia , Período Pós-Prandial/fisiologiaRESUMO
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.
Assuntos
Diferenciação Celular , Citosol/metabolismo , Enterócitos/citologia , Enterócitos/metabolismo , Lipídeos/isolamento & purificação , Proteoma/metabolismo , Animais , Células CACO-2 , Células Cultivadas , Células HeLa , Humanos , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de ÓrgãosRESUMO
Intestinal mucins are very high molecular weight glycoproteins secreted by goblet cells lining the crypt and the surface of the colonic mucosa. Profound alterations of mucin O-glycans are observed in diseases such as cancer and inflammation, modifying the function of the cell and its antigenic and adhesive properties. Based on immunohistochemical studies, certain cancer- and inflammation- associated glycans have been defined as oncofetal antigens. However, little or no chemical analysis has allowed the structural elucidation of O-glycans expressed on human fetal mucins. In this paper, mucins were isolated from different regions of the normal human intestine (ileum, right, transverse and left colon) of eight fetuses with A, B or O blood group. After alkaline borohydride treatment, the released oligosaccharides were investigated by nanoESI Q-TOF MS/MS (electrospray ionization quadrupole time-of-flight tandem mass spectrometry). More than 117 different glycans were identified, mainly based on core 2 structures. Some core 1, 3 and 4 oligosaccharides were also found. Most of the structures were acidic with NeuAc residues mainly alpha2-6 linked to the N-acetylgalactosaminitol and sulphate residues 3-linked to galactose or 6-linked to GlcNAc. In contrast to adult human intestinal mucins, Sda/Cad determinants were not expressed on fetal mucin O-glycans and the presence of an acidic gradient along the intestinal tract was not observed. Similar patterns of glycosylation were found in each part of the intestine and the level of expression of the major oligosaccharides was in the same order of magnitude. This study could help determining new oncofetal antigens, which can be exploited for the diagnosis or the treatment of intestinal diseases.
Assuntos
Feto/metabolismo , Mucosa Intestinal/metabolismo , Mucinas/metabolismo , Polissacarídeos/metabolismo , Adulto , Antígenos de Grupos Sanguíneos/metabolismo , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Feminino , Idade Gestacional , Glicosilação , Humanos , Masculino , Dados de Sequência Molecular , Monossacarídeos/química , Ácido N-Acetilneuramínico/metabolismo , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Polissacarídeos/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
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.
Assuntos
Adaptação Fisiológica , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Glucose/farmacologia , Lipoproteínas/química , Lipoproteínas/metabolismo , Triglicerídeos/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Apolipoproteínas B/metabolismo , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Retículo Endoplasmático/ultraestrutura , Enterócitos/citologia , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Enterócitos/ultraestrutura , Glicogênio/metabolismo , Humanos , Lipoproteínas/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Verapamil/farmacologiaRESUMO
Enterocytes are highly polarized cells that transfer nutrients across the intestinal epithelium from the apical to the basolateral pole. Apolipoprotein B (apoB) is a secretory protein that plays a key role in the transepithelial transport of dietary fatty acids as triacylglycerol. The evaluation of the control of apoB traffic by lipids is therefore of particular interest. To get a dynamic insight into this process, we used the enterocytic Caco-2 cells cultured on microporous filters, a system in which the apical and basal compartments can be delimited. Combining biochemical and morphological approaches, our results showed that, besides their role in protection from degradation, lipids control the intracellular traffic of apoB in enterocytes. A supply of fatty acids and cholesterol is sufficient for the export of apoB from the endoplasmic reticulum and its post-Golgi traffic up to the apical brush-border domain, where it remains until an apical supply of complex lipid micelles signals its chase down to the basolateral secretory domain. This downward traffic of apoB involves a microtubule-dependent process. Our results demonstrate an enterocyte-specific bidirectional process for the lipid-dependent traffic of a secretory protein.
Assuntos
Apolipoproteínas B/metabolismo , Colesterol/biossíntese , Enterócitos/metabolismo , Ácidos Graxos/biossíntese , Complexo de Golgi/metabolismo , Animais , Transporte Biológico/fisiologia , Células CACO-2 , Diferenciação Celular , Membrana Celular/metabolismo , Polaridade Celular , Retículo Endoplasmático/metabolismo , Humanos , Microscopia Confocal , Microscopia EletrônicaRESUMO
The cellular prion protein PrP(c) plays important roles in proliferation, cell death and survival, differentiation and adhesion. The participation of PrP(c) in tumor growth and metastasis was pointed out, but the underlying mechanisms were not deciphered completely. In the constantly renewing intestinal epithelium, our group demonstrated a dual localization of PrP(c), which is targeted to cell-cell junctions in interaction with Src kinase and desmosomal proteins in differentiated enterocytes, but is predominantly nuclear in dividing cells. While the role of PrP(c) in the dynamics of intercellular junctions was confirmed in other biological systems, we unraveled its function in the nucleus only recently. We identified several nuclear PrP(c) partners, which comprise γ-catenin, one of its desmosomal partners, ß-catenin and TCF7L2, the main effectors of the canonical Wnt pathway, and YAP, one effector of the Hippo pathway. PrP(c) up-regulates the activity of the ß-catenin/TCF7L2 complex and its invalidation impairs the proliferation of intestinal progenitors. We discuss how PrP(c) could participate to oncogenic processes through its interaction with Wnt and Hippo pathway effectors, which are controlled by cell-cell junctions and Src family kinases and dysregulated during tumorigenesis. This highlights new potential mechanisms that connect PrP(c) expression and subcellular redistribution to cancer.
Assuntos
Núcleo Celular/patologia , Junções Intercelulares/patologia , Neoplasias/patologia , Proteínas PrPC/metabolismo , Transdução de Sinais , Animais , Núcleo Celular/metabolismo , Proliferação de Células , Desmossomos/metabolismo , Desmossomos/patologia , Transição Epitelial-Mesenquimal , Via de Sinalização Hippo , Humanos , Junções Intercelulares/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Neoplasias/metabolismo , Proteínas PrPC/análise , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Via de Sinalização WntRESUMO
The increasing incidence of obesity and associated metabolic complications is a worldwide public health issue. The role of the gut in the pathophysiology of obesity, with an important part for microbiota, is becoming obvious. In rodent models of diet-induced obesity, the modifications of gut microbiota are associated with an alteration of the intestinal permeability increasing the passage of food or bacterial antigens, which contribute to low-grade inflammation and insulin resistance. In human obesity, intestinal permeability modification, and its role in the crosstalk between gut microbiota changes and inflammation at systemic and tissular levels, are still poorly documented. Hence, further characterization of the triggering mechanisms of such inflammatory responses in obese subjects could enable the development of personalized intervention strategies that will help to reduce the risk of obesity-associated diseases.
Assuntos
Disbiose/complicações , Microbioma Gastrointestinal/fisiologia , Inflamação/etiologia , Mucosa Intestinal/metabolismo , Obesidade/etiologia , Animais , Disbiose/imunologia , Disbiose/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/microbiologia , Mucosa Intestinal/microbiologia , Intestinos/imunologia , Intestinos/microbiologia , Obesidade/imunologia , Obesidade/metabolismo , Obesidade/microbiologia , PermeabilidadeRESUMO
BACKGROUND & AIM: Despite extensive study of the contribution of cell death and apoptosis to radiation-induced acute intestinal injury, our knowledge of the signaling mechanisms involved in epithelial barrier dysfunction remains inadequate. Because PrP(c) plays a key role in intestinal homeostasis by renewing epithelia, we sought to study its role in epithelial barrier function after irradiation. DESIGN: Histology, morphometry and plasma FD-4 levels were used to examine ileal architecture, wound healing, and intestinal leakage in PrP(c)-deficient (KO) and wild-type (WT) mice after total-body irradiation. Impairment of the PrP(c) Src pathway after irradiation was explored by immunofluorescence and confocal microscopy, with Caco-2/Tc7 cells. Lastly, dasatinib treatment was used to switch off the Src pathway in vitro and in vivo. RESULTS: The decrease in radiation-induced lethality, improved intestinal wound healing, and reduced intestinal leakage promoted by PrP(c) deficiency demonstrate its involvement in acute intestinal damage. Irradiation of Cacao2/Tc7 cells induced PrP(c) to target the nuclei associated with Src activation. Finally, the protective effect triggered by dasatinib confirmed Src involvement in radiation-induced acute intestinal toxicity. CONCLUSION: Our data are the first to show a role for the PrP(c)-Src pathway in acute intestinal response to radiation injury and offer a novel therapeutic opportunity.
Assuntos
Dasatinibe/uso terapêutico , Intestinos/efeitos da radiação , Proteínas Priônicas/deficiência , Lesões por Radiação/prevenção & controle , Quinases da Família src/antagonistas & inibidores , Animais , Proteína Tirosina Quinase CSK , Células CACO-2 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Priônicas/fisiologia , Irradiação Corporal Total , Quinases da Família src/fisiologiaRESUMO
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.
Assuntos
Células CACO-2/fisiologia , Diferenciação Celular/efeitos dos fármacos , Meios de Cultura/química , Fosfatase Alcalina/análise , Análise de Variância , Biomarcadores/análise , Células CACO-2/efeitos dos fármacos , Células CACO-2/enzimologia , Células Cultivadas , Impedância Elétrica , Humanos , Manitol/metabolismo , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Intestine contributes to energy homeostasis through the absorption, metabolism, and transfer of nutrients to the organism. We demonstrated previously that hepatocyte nuclear receptor-4α (HNF-4α) controls intestinal epithelium homeostasis and intestinal absorption of dietary lipids. HNF-4γ, the other HNF-4 form highly expressed in intestine, is much less studied. In HNF-4γ knockout mice, we detect an exaggerated insulin peak and improvement in glucose tolerance during oral but not intraperitoneal glucose tolerance tests, highlighting the involvement of intestine. Moreover, the enteroendocrine L-type cell lineage is modified, as assessed by the increased expression of transcription factors Isl1, Foxa1/2, and Hnf4a, leading to an increase of both GLP-1-positive cell number and basal and stimulated GLP-1 plasma levels potentiating the glucose-stimulated insulin secretion. Using the GLP-1 antagonist exendin (9-39), we demonstrate a direct effect of GLP-1 on improved glucose tolerance. GLP-1 exerts a trophic effect on pancreatic ß-cells, and we report an increase of the ß-cell fraction correlated with an augmented number of proliferative islet cells and with resistance to streptozotocin-induced diabetes. In conclusion, the loss of HNF-4γ improves glucose homeostasis through a modulation of the enteroendocrine cell lineage.
Assuntos
Glicemia/metabolismo , Linhagem da Célula/fisiologia , Células Enteroendócrinas/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Insulina/sangue , Mucosa Intestinal/metabolismo , Animais , Células Enteroendócrinas/citologia , Teste de Tolerância a Glucose , Fator 4 Nuclear de Hepatócito/genética , Homeostase/fisiologia , Camundongos , Camundongos KnockoutRESUMO
We reported previously that the cellular prion protein (PrP(c)) is a component of desmosomes and contributes to the intestinal barrier function. We demonstrated also the presence of PrP(c) in the nucleus of proliferating intestinal epithelial cells. Here we sought to decipher the function of this nuclear pool. In human intestinal cancer cells Caco-2/TC7 and SW480 and normal crypt-like HIEC-6 cells, PrP(c) interacts, in cytoplasm and nucleus, with γ-catenin, one of its desmosomal partners, and with ß-catenin and TCF7L2, effectors of the canonical Wnt pathway. PrP(c) up-regulates the transcriptional activity of the ß-catenin/TCF7L2 complex, whereas γ-catenin down-regulates it. Silencing of PrP(c) results in the modulation of several Wnt target gene expressions in human cells, with different effects depending on their Wnt signaling status, and in mouse intestinal crypt cells in vivo. PrP(c) also interacts with the Hippo pathway effector YAP, suggesting that it may contribute to the regulation of gene transcription beyond the ß-catenin/TCF7L2 complex. Finally, we demonstrate that PrP(c) is required for proper formation of intestinal organoids, indicating that it contributes to proliferation and survival of intestinal progenitors. In conclusion, PrP(c) must be considered as a new modulator of the Wnt signaling pathway in proliferating intestinal epithelial cells.
Assuntos
Mucosa Intestinal/metabolismo , Proteínas PrPC/metabolismo , Via de Sinalização Wnt , Animais , Células COS , Células CACO-2 , Cateninas/metabolismo , Proliferação de Células/genética , Chlorocebus aethiops , Regulação para Baixo , Células Epiteliais/metabolismo , Humanos , Mucosa Intestinal/citologia , Camundongos , Camundongos Endogâmicos C57BL , Príons/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Regulação para Cima , beta Catenina/metabolismoRESUMO
The purpose of this study was to update the most commonly used tables of dental eruption (emergence), which are more than 50 years old and do not distinguish between ethnic and socio-economic groups. Clinical emergence data was collected for a modern sample of 574 French children and adolescents, aged 5.5 years to 15 years (294 girls and 280 boys). With respect to the present sample, the commonly used eruption tables of Hurme were accurate for most aspects of mandibular emergence, but were seriously flawed for the maxillary arch. Emergence of all permanent maxillary teeth varied by at least 3 months from Hurme's means; lateral incisors, canines, and second molars erupted earlier, but both premolars emerged later. Compared to similar French data collected in 1958, there appears to be a secular trend for later eruption of the maxillary premolars and earlier emergence of permanent second molars. These changes may reflect an evolutionary reduction in the size of the maxilla, a progressive decrease in genetic control of permanent canines, as well as first and second premolars, and/or progress in dental preventive measures to conserve primary molars. From a clinical perspective, appropriate dental emergence data are essential for effectively intercepting developmental malocclusions in children and adolescents.
Assuntos
Erupção Dentária/fisiologia , Adolescente , Fatores Etários , Dente Pré-Molar/fisiologia , Criança , Pré-Escolar , Estudos Transversais , Dente Canino/fisiologia , Feminino , França , Humanos , Incisivo/fisiologia , Masculino , Mandíbula , Maxila/anatomia & histologia , Dente Molar/fisiologia , Valores de Referência , Fatores Sexuais , Erupção Dentária/genéticaRESUMO
Enterocytes, the intestinal absorptive cells, have to deal with massive alimentary lipids upon food consumption. They orchestrate complex lipid-trafficking events that lead to the secretion of triglyceride-rich lipoproteins and/or the intracellular transient storage of lipids as lipid droplets (LDs). LDs originate from the endoplasmic reticulum (ER) membrane and are mainly composed of a triglyceride (TG) and cholesterol-ester core surrounded by a phospholipid and cholesterol monolayer and specific coat proteins. The pivotal role of LDs in cellular lipid homeostasis is clearly established, but processes regulating LD dynamics in enterocytes are poorly understood. Here we show that delivery of alimentary lipid micelles to polarized human enterocytes induces an immediate autophagic response, accompanied by phosphatidylinositol-3-phosphate appearance at the ER membrane. We observe a specific and rapid capture of newly synthesized LD at the ER membrane by nascent autophagosomal structures. By combining pharmacological and genetic approaches, we demonstrate that autophagy is a key player in TG targeting to lysosomes. Our results highlight the yet-unraveled role of autophagy in the regulation of TG distribution, trafficking, and turnover in human enterocytes.
Assuntos
Enterócitos/metabolismo , Membranas Intracelulares/metabolismo , Metabolismo dos Lipídeos , Fagossomos/fisiologia , Animais , Apolipoproteína A-I/metabolismo , Autofagia , Transporte Biológico , Células CACO-2 , Núcleo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Cinética , Lipoproteínas HDL/metabolismo , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia de Fluorescência , Fosfatos de Fosfatidilinositol/metabolismoRESUMO
In enterocytes, the dynamic accumulation and depletion of triacylglycerol (TAG) in lipid droplets (LD) during fat absorption suggests that cytosolic LD-associated TAG contribute to TAG-rich lipoprotein (TRL) production. To get insight into the mechanisms controlling the storage/secretion balance of TAG, we used as a tool hepatitis C virus core protein, which localizes onto LDs, and thus may modify their protein coat and decrease TRL secretion. We compared the proteome of LD fractions isolated from Caco-2/TC7 enterocytes expressing or not hepatitis C virus core protein by a differential proteomic approach (isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with liquid chromatography and tandem mass spectrometry). We identified 42 proteins, 21 being involved in lipid metabolism. Perilipin-2/ADRP, which is suggested to stabilize long term-stored TAG, was enriched in LD fractions isolated from Caco-2/TC7 expressing core protein while perilipin-3/TIP47, which is involved in LD synthesis from newly synthesized TAG, was decreased. Endoplasmic reticulum-associated proteins were strongly decreased, suggesting reduced interactions between LD and endoplasmic reticulum, where TRL assembly occurs. For the first time, we show that 17ß-hydroxysteroid dehydrogenase 2 (DHB2), which catalyzes the conversion of 17-keto to 17 ß-hydroxysteroids and which was the most highly enriched protein in core expressing cells, is localized to LD and interferes with TAG secretion, probably through its capacity to inactivate testosterone. Overall, we identified potential new players of lipid droplet dynamics, which may be involved in the balance between lipid storage and secretion, and may be altered in enterocytes in pathological conditions such as insulin resistance, type II diabetes and obesity.