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Butyrate modifies intestinal barrier function in IPEC-J2 cells through a selective upregulation of tight junction proteins and activation of the Akt signaling pathway.

ABSTRACT: The intestinal epithelial barrier, composed of epithelial cells, tight junction proteins and intestinal secretions, prevents passage of luminal substances and antigens through the paracellular space. Dysfunction of the intestinal barrier integrity induced by toxins and pathogens is associated with a variety of gastrointestinal disorders and diseases. Although butyrate is known to enhance intestinal health, its role in the protection of intestinal barrier function is poorly characterized. Therefore, we investigated the effect of butyrate on intestinal epithelial integrity and tight junction permeability in a model of LPS-induced inflammation in IPEC-J2 cells. Butyrate dose-dependently reduced LPS impairment of intestinal barrier integrity and tight junction permeability, measured by trans-epithelial electrical resistance (TEER) and paracellular uptake of fluorescein isothiocyanate-dextran (FITC-dextran). Additionally, butyrate increased both mRNA expression and protein abundance of claudins-3 and 4, and influenced intracellular ATP concentration in a dose-dependent manner. Furthermore, butyrate prevented the downregulation of Akt and 4E-BP1 phosphorylation by LPS, indicating that butyrate might enhance tight junction protein abundance through mechanisms that included activation of Akt/mTOR mediated protein synthesis. The regulation of AMPK activity and intracellular ATP level by butyrate indicates that butyrate might regulate energy status of the cell, perhaps by serving as a nutrient substrate for ATP synthesis, to support intestinal epithelial barrier tight junction protein abundance. Our findings suggest that butyrate might protect epithelial cells from LPS-induced impairment of barrier integrity through an increase in the synthesis of tight junction proteins, and perhaps regulation of energy homeostasis.

SUBMITTER: Yan H

PROVIDER: S-EPMC5487041 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Butyrate modifies intestinal barrier function in IPEC-J2 cells through a selective upregulation of tight junction proteins and activation of the Akt signaling pathway.

Yan Hui H Ajuwon Kolapo M KM

PloS one 20170627 6

The intestinal epithelial barrier, composed of epithelial cells, tight junction proteins and intestinal secretions, prevents passage of luminal substances and antigens through the paracellular space. Dysfunction of the intestinal barrier integrity induced by toxins and pathogens is associated with a variety of gastrointestinal disorders and diseases. Although butyrate is known to enhance intestinal health, its role in the protection of intestinal barrier function is poorly characterized. Therefo ...[more]

PMID: 28654658

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Project description:Clostridium butyricum (CB), a probiotic, is a gram-positive obligate anaerobic bacillus with acid and heat resistant properties. Previous studies have reported that CB has beneficial effects in intestinal diseases and regulates intestinal function. The aim of the present study was to investigate the protective effects and mechanisms of CB on the intestinal barrier function. Mice were randomly divided into three experimental groups (n=15 mice/group), including control, dextran sodium sulfate (DSS) and DSS + CB. In the DSS and DSS + CB groups colitis was induced with 3% DSS dissolved in drinking water for 7 days. DSS + CB group mice were co-treated daily with 200 µl (2x108 CFU) CB solution via gavage. The intestinal mucosal barrier function in mice was assessed by measuring FITC-labeled 4-kDa dextran (molecular weight, 4,000 Da) flux and by analyzing the expression of tight junction (TJ)-related proteins using western blot analysis. In addition, the secretion levels of tumor necrosis factor-? (TNF-?), interleukin (IL)-1?, IL-6, IL-10 and IL-13, and the concentration of malondialdehyde, glutathione and superoxide dismutase were detected using ELISAs to determine inflammation and oxidative stress, respectively. The activation status of the Akt/mTOR signaling pathway was also investigated using western blot analysis. The results demonstrated that, in mice with DSS-induced colitis treatment, co-treatment with CB attenuated colitis symptoms and intestinal permeability, increased the expression levels of TJ-related proteins, decreased TNF-?, IL-1? and IL-13 secretion levels but increased those of IL-10, and reduced oxidative stress. Additionally, CB elevated the phosphorylation of Akt, mTOR and p70 ribosomal protein S6 kinase. Collectively, the present results indicated that CB protected intestinal barrier function and decreased intestinal mucosal permeability via upregulating the expression levels of TJ-related proteins in a mouse model of DSS-induced colitis. Moreover, the results suggested that the effects of CB could be mediated by the Akt/mTOR signaling pathway.

Project description:The intestinal porcine epithelial cell line IPEC-J2, cultured under the air-liquid interface (ALI) conditions, develops remarkable morphological characteristics close to intestinal epithelial cells in vivo. Improved oxygen availability has been hypothesised to be the leading cause of this morphological differentiation. We assessed oxygen availability in ALI cultures and examined the influence of this cell culture method on glycolysis and oxidative phosphorylation in IPEC-J2 using the submerged membrane culture (SMC) and ALI cultures. Furthermore, the role of HIF-1 as mediator of oxygen availability was analysed. Measurements of oxygen tension confirmed increased oxygen availability at the medium-cell interface and demonstrated reduced oxygen extraction at the basal compartment in ALI. Microarray analysis to determine changes in the genetic profile of IPEC-J2 in ALI identified 2751 modified transcripts. Further examinations of candidate genes revealed reduced levels of glycolytic enzymes hexokinase II and GAPDH, as well as lactate transporting monocarboxylate transporter 1 in ALI, whereas expression of the glucose transporter GLUT1 remained unchanged. Cytochrome c oxidase (COX) subunit 5B protein analysis was increased in ALI, although mRNA level remained at constant level. COX activity was assessed using photometric quantification and a three-fold increase was found in ALI. Quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. In order to evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenised cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In addition, HIF showed reduced mRNA levels in ALI. Furthermore, HIF-1α protein was reduced in the nuclear compartment of ALI when compared to SCM as confirmed by confocal microscopy. These results indicate a metabolic switch in IPEC-J2 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis. ALI-induced improvement of oxygen supply reduced nuclear HIF-1α, demonstrating a major change in the transcriptional response.

Dataset Information

Butyrate modifies intestinal barrier function in IPEC-J2 cells through a selective upregulation of tight junction proteins and activation of the Akt signaling pathway.

Publications

Butyrate modifies intestinal barrier function in IPEC-J2 cells through a selective upregulation of tight junction proteins and activation of the Akt signaling pathway.

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