RESUMO
The importance of gut barrier integrity in intestinal homeostasis and the consequences of its alteration in the etiology of human pathologies have been subjects of exponentially growing interest during the last decade [...].
Assuntos
Mucosa Intestinal , Homeostase/fisiologia , HumanosRESUMO
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 & AIMS: Reducing postprandial triglyceridemia may be a promising strategy to lower the risk of cardiovascular disorders associated with obesity and type 2 diabetes. In enterocytes, scavenger receptor class B, type 1 (SR-B1, encoded by SCARB1) mediates lipid-micelle sensing to promote assembly and secretion of chylomicrons. The nuclear receptor subfamily 1, group H, members 2 and 3 (also known as liver X receptors [LXRs]) regulate genes involved in cholesterol and fatty acid metabolism. We aimed to determine whether intestinal LXRs regulate triglyceride absorption. METHODS: C57BL/6J mice were either fed a cholesterol-enriched diet or given synthetic LXR agonists (GW3965 or T0901317). We measured the production of chylomicrons and localized SR-B1 by immunohistochemistry. Mechanisms of postprandial triglyceridemia and SR-B1 regulation were studied in Caco-2/TC7 cells incubated with LXR agonists. RESULTS: In mice and in the Caco-2/TC7 cell line, LXR agonists caused localization of intestinal SR-B1 from apical membranes to intracellular organelles and reduced chylomicron secretion. In Caco-2/TC7 cells, LXR agonists reduced SR-B1-dependent lipidic-micelle-induced Erk phosphorylation. LXR agonists also reduced intracellular trafficking of the apical apolipoprotein B pool toward secretory compartments. LXR reduced levels of SR-B1 in Caco-2/TC7 cells via a post-transcriptional mechanism that involves microRNAs. CONCLUSION: In Caco-2/TC7 cells and mice, intestinal activation of LXR reduces the production of chylomicrons by a mechanism dependent on the apical localization of SR-B1.
Assuntos
Absorção Intestinal , Mucosa Intestinal/metabolismo , Jejuno/metabolismo , Receptores Nucleares Órfãos/metabolismo , Receptores Depuradores Classe B/metabolismo , Triglicerídeos/metabolismo , Animais , Apolipoproteína B-100/metabolismo , Apolipoproteínas B/metabolismo , Benzoatos/farmacologia , Benzilaminas/farmacologia , Células CACO-2 , Colesterol na Dieta/metabolismo , Quilomícrons/metabolismo , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Regulação para Baixo , Humanos , Hidrocarbonetos Fluorados/farmacologia , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Receptores X do Fígado , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Receptores Nucleares Órfãos/agonistas , Transporte Proteico , Interferência de RNA , Ribonuclease III/deficiência , Ribonuclease III/genética , Receptores Depuradores Classe B/deficiência , Receptores Depuradores Classe B/genética , Transdução de Sinais , Sulfonamidas/farmacologia , Transcrição Gênica , TransfecçãoRESUMO
Cytosolic lipid droplets (LDs) are observed in enterocytes of jejunum during lipid absorption. One important function of the intestine is to secrete chylomicrons, which provide dietary lipids throughout the body, from digested lipids in meals. The current hypothesis is that cytosolic LDs in enterocytes constitute a transient pool of stored lipids that provides lipids during interprandial period while lowering chylomicron production during the post-prandial phase. This smoothens the magnitude of peaks of hypertriglyceridemia. Here, we review the composition and functions of lipids and associated proteins of enterocyte LDs, the known physiological functions of LDs as well as the role of LDs in pathological processes in the context of the intestine.
Assuntos
Transporte Biológico/fisiologia , Quilomícrons/metabolismo , Enterócitos/metabolismo , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/fisiologia , Animais , Humanos , Triglicerídeos/metabolismoRESUMO
RATIONALE: Signal initiation by the high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), which is important to actions of HDL on endothelium and other processes, requires cholesterol efflux and the C-terminal transmembrane domain. The C-terminal transmembrane domain uniquely interacts with plasma membrane (PM) cholesterol. OBJECTIVE: The molecular basis and functional significance of SR-BI interaction with PM cholesterol are unknown. We tested the hypotheses that the interaction is required for SR-BI signaling, and that it enables SR-BI to serve as a PM cholesterol sensor. METHODS AND RESULTS: In studies performed in COS-M6 cells, mutation of a highly conserved C-terminal transmembrane domain glutamine to alanine (SR-BI-Q445A) decreased PM cholesterol interaction with the receptor by 71% without altering HDL binding or cholesterol uptake or efflux, and it yielded a receptor incapable of HDL-induced signaling. Signaling prompted by cholesterol efflux to methyl-ß-cyclodextrin also was prevented, indicating that PM cholesterol interaction with the receptor enables it to serve as a PM cholesterol sensor. Using SR-BI-Q445A, we further demonstrated that PM cholesterol sensing by SR-BI does not influence SR-BI-mediated reverse cholesterol transport to the liver in mice. However, the PM cholesterol sensing does underlie apolipoprotein B intracellular trafficking in response to postprandial micelles or methyl-ß-cyclodextrin in cultured enterocytes, and it is required for HDL activation of endothelial NO synthase and migration in cultured endothelial cells and HDL-induced angiogenesis in vivo. CONCLUSIONS: Through interaction with PM cholesterol, SR-BI serves as a PM cholesterol sensor, and the resulting intracellular signaling governs processes in both enterocytes and endothelial cells.
Assuntos
Membrana Celular/metabolismo , Colesterol/metabolismo , Células Endoteliais/metabolismo , Enterócitos/metabolismo , Receptores Depuradores Classe B/metabolismo , Transdução de Sinais , Alanina , Animais , Apolipoproteínas B/metabolismo , Células CACO-2 , Bovinos , Membrana Celular/efeitos dos fármacos , HDL-Colesterol/metabolismo , Células Endoteliais/efeitos dos fármacos , Enterócitos/efeitos dos fármacos , Glutamina , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/metabolismo , Masculino , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Neovascularização Fisiológica , Óxido Nítrico Sintase Tipo III/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Depuradores Classe B/química , Receptores Depuradores Classe B/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , beta-Ciclodextrinas/farmacologiaRESUMO
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
In the gut ecosystem, microorganisms regulate group behaviour and interplay with the host via a molecular system called quorum sensing (QS). The QS molecule 3-oxo-C12:2-HSL, first identified in human gut microbiota, exerts anti-inflammatory effects and could play a role in inflammatory bowel diseases where dysbiosis has been described. Our aim was to identify which signalling pathways are involved in this effect. We observed that 3-oxo-C12:2-HSL decreases expression of pro-inflammatory cytokines such as Interleukine-1ß (- 35%) and Tumor Necrosis Factor-α (TNFα) (- 40%) by stimulated immune RAW264.7 cells and decreased TNF secretion by stimulated PBMC in a dose-dependent manner, between 25 to 100 µM. Transcriptomic analysis of RAW264.7 cells exposed to 3-oxo-C12:2-HSL, in a pro-inflammatory context, highlighted JAK-STAT, NF-κB and TFN signalling pathways and we confirmed that 3-oxo-C12:2-HSL inhibited JAK1 and STAT1 phosphorylation. We also showed through a screening assay that 3-oxo-C12:2-HSL interacted with several human bitter taste receptors. Its anti-inflammatory effect involved TAS2R38 as shown by pharmacologic inhibition and led to an increase in intracellular calcium levels. We thus unravelled the involvement of several cellular pathways in the anti-inflammatory effects exerted by the QS molecule 3-oxo-C12:2-HSL.
Assuntos
Microbioma Gastrointestinal , Percepção de Quorum , 4-Butirolactona/metabolismo , Anti-Inflamatórios/metabolismo , Ecossistema , Homosserina/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Pseudomonas aeruginosa/fisiologia , PaladarRESUMO
An increased intestinal permeability has been described in many diseases including inflammatory bowel disease and metabolic disorders, and a better understanding of the contribution of intestinal barrier impairment to pathogenesis is needed. In recent years, attention has been paid to the leak pathway, which is the route of paracellular transport allowing the diffusion of macromolecules through the tight junctions of the intestinal epithelial lining. While the passage of macromolecules by this pathway is very restricted under physiological conditions, its amplification is thought to promote an excessive immune activation in the intestinal mucosa. The Ussing chambers have been widely used to measure both active and passive transepithelial fluxes in intact tissues. In this chapter we present how this simple device can be used to measure paracellular permeability to macromolecules in the mouse intestine. We propose a detailed protocol and describe how to best exploit all the possibilities of this technique, correctly interpret the results, and avoid the main pitfalls.
Assuntos
Intestinos , Animais , Colite , Mucosa Intestinal , Substâncias Macromoleculares , Camundongos , Permeabilidade , Junções ÍntimasRESUMO
Paracellular permeability of the intestinal epithelium is a feature of the intestinal barrier, which plays an important role in the physiology of gut and the whole organism. Intestinal paracellular permeability is controlled by complex processes and is involved in the passage of ions and fluids (called pore pathway) and macromolecules (called leak pathway) through tight junctions, which seal the intercellular space. Impairment of intestinal paracellular permeability is associated with several diseases. The identification of a defect in intestinal paracellular permeability may help to understand the implication of gut barrier as a cause or a consequence in human pathology. Here we describe two complementary methods to evaluate alteration of paracellular permeability in cell culture, using the human intestinal cell line Caco-2 and its clone Caco-2/TC7.
Assuntos
Enterócitos , Células CACO-2 , Permeabilidade da Membrana Celular , Celulose Oxidada , Humanos , Mucosa Intestinal/metabolismo , Permeabilidade , Junções Íntimas/metabolismoRESUMO
Bacteria are known to communicate with each other and regulate their activities in social networks by secreting and sensing signaling molecules called autoinducers, a process known as quorum sensing (QS). This is a growing area of research in which we are expanding our understanding of how bacteria collectively modify their behavior but are also involved in the crosstalk between the host and gut microbiome. This is particularly relevant in the case of pathologies associated with dysbiosis or disorders of the intestinal ecosystem. This review will examine the different QS systems and the evidence for their presence in the intestinal ecosystem. We will also provide clues on the role of QS molecules that may exert, directly or indirectly through their bacterial gossip, an influence on intestinal epithelial barrier function, intestinal inflammation, and intestinal carcinogenesis. This review aims to provide evidence on the role of QS molecules in gut physiology and the potential shared by this new player. Better understanding the impact of intestinal bacterial social networks and ultimately developing new therapeutic strategies to control intestinal disorders remains a challenge that needs to be addressed in the future.
Assuntos
Microbioma Gastrointestinal , Percepção de Quorum , Bactérias , Disbiose , Ecossistema , HumanosRESUMO
The mechanisms leading to the low-grade inflammation observed during obesity are not fully understood. Seeking the initiating events, we tested the hypothesis that the intestine could be damaged by repeated lipid supply and therefore participate in inflammation. In mice, 1-5 palm oil gavages increased intestinal permeability via decreased expression and mislocalization of junctional proteins at the cell-cell contacts; altered the intestinal bacterial species by decreasing the abundance of Akkermansia muciniphila, segmented filamentous bacteria, and Clostridium leptum; and increased inflammatory cytokine expression. This was further studied in human intestinal epithelial Caco-2/TC7 cells using the two main components of palm oil, i.e., palmitic and oleic acid. Saturated palmitic acid impaired paracellular permeability and junctional protein localization, and induced inflammatory cytokine expression in the cells, but unsaturated oleic acid did not. Inhibiting de novo ceramide synthesis prevented part of these effects. Altogether, our data show that short exposure to palm oil or palmitic acid induces intestinal dysfunctions targeting barrier integrity and inflammation. Excessive palm oil consumption could be an early player in the gut alterations observed in metabolic diseases.
Assuntos
Microbioma Gastrointestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Síndrome Metabólica/patologia , Óleo de Palmeira/efeitos adversos , Ácido Palmítico/efeitos adversos , Administração Oral , Animais , Células CACO-2 , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/imunologia , Fezes/microbiologia , Microbioma Gastrointestinal/imunologia , Humanos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Síndrome Metabólica/imunologia , Camundongos , Óleo de Palmeira/administração & dosagem , Óleo de Palmeira/química , Ácido Palmítico/administração & dosagem , Permeabilidade , Junções Íntimas/efeitos dos fármacosRESUMO
The intestine is home to the largest microbiota community of the human body and strictly regulates its barrier function. Tight junctions (TJ) are major actors of the intestinal barrier, which is impaired in inflammatory bowel disease (IBD), along with an unbalanced microbiota composition. With the aim to identify new actors involved in host-microbiota interplay in IBD, we studied N-acyl homoserine lactones (AHL), molecules of the bacterial quorum sensing, which also impact the host. We previously identified in the gut a new and prominent AHL, 3-oxo-C12:2, which is lost in IBD. We investigated how 3-oxo-C12:2 impacts the intestinal barrier function, in comparison to 3-oxo-C12, a structurally close AHL produced by the opportunistic pathogen P. aeruginosa. Using Caco-2/TC7 cells as a model of polarized enterocytes, we compared the effects on paracellular permeability and TJ integrity of these two AHL, separately or combined with pro-inflammatory cytokines, Interferon-γ and Tumor Necrosis Factor-α, known to disrupt the barrier function during IBD. While 3-oxo-C12 increased paracellular permeability and decreased occludin and tricellulin signal at bicellular and tricellular TJ, respectively, 3-oxo-C12:2 modified neither permeability nor TJ integrity. Whereas 3-oxo-C12 potentiated the hyperpermeability induced by cytokines, 3-oxo-C12:2 attenuated their deleterious effects on occludin and tricellulin, and maintained their interaction with their partner ZO-1. In addition, 3-oxo-C12:2 limited the cytokine-induced ubiquitination of occludin and tricellulin, suggesting that this AHL prevented their endocytosis. In conclusion, the role of 3-oxo-C12:2 in maintaining TJ integrity under inflammatory conditions identifies this new AHL as a potential beneficial actor of host-microbiota interactions in IBD.
Assuntos
Acil-Butirolactonas/metabolismo , Citocinas/metabolismo , Percepção de Quorum/genética , Junções Íntimas/metabolismo , HumanosRESUMO
OBJECTIVE: Obesity is characterized by systemic and low-grade tissue inflammation. In the intestine, alteration of the intestinal barrier and accumulation of inflammatory cells in the epithelium are important contributors of gut inflammation. Recent studies demonstrated the role of the aryl hydrocarbon receptor (AhR) in the maintenance of immune cells at mucosal barrier sites. A wide range of ligands of external and local origin can activate this receptor. We studied the causal relationship between AhR activation and gut inflammation in obesity. METHODS: Jejunum samples from subjects with normal weight and severe obesity were phenotyped according to T lymphocyte infiltration in the epithelium from lamina propria and assayed for the mRNA level of AhR target genes. The effect of an AhR agonist was studied in mice and Caco-2/TC7 cells. AhR target gene expression, permeability to small molecules and ions, and location of cell-cell junction proteins were recorded under conditions of altered intestinal permeability. RESULTS: We showed that a low AhR tone correlated with a high inflammatory score in the intestinal epithelium in severe human obesity. Moreover, AhR activation protected junctional complexes in the intestinal epithelium in mice challenged by an oral lipid load. AhR ligands prevented chemically induced damage to barrier integrity and cytokine expression in Caco-2/TC7 cells. The PKC and p38MAPK signaling pathways were involved in this AhR action. CONCLUSIONS: The results of these series of human, mouse, and cell culture experiments demonstrate the protective effect of AhR activation in the intestine targeting particularly tight junctions and cytokine expression. We propose that AhR constitutes a valuable target to protect intestinal functions in metabolic diseases, which can be achieved in the future via food or drug ligands.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Mucosa Intestinal/metabolismo , Obesidade/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Adiposidade/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Biomarcadores , Linhagem Celular , Comorbidade , Citocinas/metabolismo , Células Epiteliais/metabolismo , Feminino , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Jejuno/metabolismo , Metabolismo dos Lipídeos , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Biológicos , Obesidade/etiologia , Obesidade/patologia , Permeabilidade , Receptores de Hidrocarboneto Arílico/genética , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismoRESUMO
Sporozoite forms of the Plasmodium parasite, the causative agent of malaria, are transmitted by mosquitoes and first infect the liver for an initial round of replication before parasite proliferation in the blood. The molecular mechanisms involved during sporozoite invasion of hepatocytes remain poorly understood. Two receptors of the Hepatitis C virus (HCV), the tetraspanin CD81 and the scavenger receptor class B type 1 (SR-B1), play an important role during the entry of Plasmodium sporozoites into hepatocytes. In contrast to HCV entry, which requires both CD81 and SR-B1 together with additional host factors, CD81 and SR-B1 operate independently during malaria liver infection. Sporozoites from human-infecting P. falciparum and P. vivax rely respectively on CD81 or SR-B1. Rodent-infecting P. berghei can use SR-B1 to infect host cells as an alternative pathway to CD81, providing a tractable model to investigate the role of SR-B1 during Plasmodium liver infection. Here we show that mouse SR-B1 is less functional as compared to human SR-B1 during P. berghei infection. We took advantage of this functional difference to investigate the structural determinants of SR-B1 required for infection. Using a structure-guided strategy and chimeric mouse/human SR-B1 constructs, we could map the functional region of human SR-B1 within apical loops, suggesting that this region of the protein may play a crucial role for interaction of sporozoite ligands with host cells and thus the very first step of Plasmodium infection.
Assuntos
Antígenos CD36/metabolismo , Hepatócitos/metabolismo , Hepatócitos/parasitologia , Plasmodium/fisiologia , Esporozoítos/fisiologia , Sequência de Aminoácidos , Animais , Antígenos CD36/química , Humanos , Camundongos , Modelos Moleculares , Domínios Proteicos , Tetraspanina 28/metabolismoRESUMO
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
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
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.
Assuntos
Enterócitos/metabolismo , Lipídeos/química , Receptores Depuradores Classe B/metabolismo , Apolipoproteínas B/metabolismo , Bile/metabolismo , Células CACO-2 , Gorduras na Dieta/metabolismo , Humanos , Hidrólise , Metabolismo dos Lipídeos , Micelas , Modelos Biológicos , Fatores de Risco , Transdução de Sinais , Triglicerídeos/metabolismoRESUMO
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.
Assuntos
Gorduras na Dieta/farmacologia , Enterócitos/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Lipídeos/farmacologia , Células CACO-2 , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Enterócitos/citologia , Humanos , Transdução de Sinais/efeitos dos fármacosRESUMO
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.