Na+/H+ Exchanger Regulates Amino Acid-Mediated Autophagy in Intestinal Epithelial Cells

Background/Aims: Dysfunctional autophagy has been reported to be associated with aberrant intestinal metabolism. Amino acids can regulate autophagic activity in intestinal epithelial cells (IECs). Na+/H+-exchanger 3 (NHE3) has been found to participate in the absorption of amino acids in the intestine, but whether NHE3 is involved in the regulation of autophagy in IECs is unclear. Methods: In the present study, an amino acid starvation-induced autophagic model was established. Then, the effects of alanine and proline with or without the NHE inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) were evaluated. Autophagy was examined based on the microtubule-associated light chain 3 (LC3) levels, transmission electron microscopy (TEM), tandem GFP-mCherry-LC3 construct, sequestosome-1 (SQSTM1, P62) mRNA and protein levels, and autophagy-related gene (ATG) 5, 7, and 12 expression levels. The autophagic flux was evaluated as the ratio of yellow (autophagosomes) to red (autolysosomes) LC3 puncta. Results: Following amino acid starvation, we found the LC3-II and ATG expression levels were enhanced in the IEC-18 cells. An increase in the number of autophagic vacuoles was concomitantly observed by TEM and confocal microscopy. Based on the results, supplementation with either alanine or proline depressed autophagy in the IEC-18 cells. Consistent with the elevated LC3-II levels, ATG expression increased upon NHE3 inhibition. Moreover, the mCherry-GFP-LC3 autophagic puncta representing both autophagosomes and autolysosomes per cell increased after EIPA treatment. Conclusions: These results demonstrate that NHE (most likely NHE3) may participate in the amino acid regulation of autophagy in IECs, which would aid in the design of better treatments for intestinal inflammation.

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Heat induction of heat shock protein 25 requires cellular glutamine in intestinal epithelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00225.2005 ◽

2006 ◽

Vol 291 (2) ◽

pp. C290-C299 ◽

Cited By ~ 18

Author(s):

Kittiporn Phanvijhitsiri ◽

Mark W. Musch ◽

Mark J. Ropeleski ◽

Eugene B. Chang

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Heat Shock ◽

Epithelial Cells ◽

Heat Shock Protein ◽

Cellular Response ◽

Intestinal Epithelial Cells ◽

Dependent Manner ◽

Intestinal Epithelial ◽

Heat Induction

Glutamine is considered a nonessential amino acid; however, it becomes conditionally essential during critical illness when consumption exceeds production. Glutamine may modulate the heat shock/stress response, an important adaptive cellular response for survival. Glutamine increases heat induction of heat shock protein (Hsp) 25 in both intestinal epithelial cells (IEC-18) and mesenchymal NIH/3T3 cells, an effect that is neither glucose nor serum dependent. Neither arginine, histidine, proline, leucine, asparagine, nor tyrosine acts as physiological substitutes for glutamine for heat induction of Hsp25. The lack of effect of these amino acids was not caused by deficient transport, although some amino acids, including glutamate (a major direct metabolite of glutamine), were transported poorly by IEC-18 cells. Glutamate uptake could be augmented in a concentration- and time-dependent manner by increasing either media concentration and/or duration of exposure. Under these conditions, glutamate promoted heat induction of Hsp25, albeit not as efficiently as glutamine. Further evidence for the role of glutamine conversion to glutamate was obtained with the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine (DON), which inhibited the effect of glutamine on heat-induced Hsp25. DON inhibited phosphate-dependent glutaminase by 75% after 3 h, decreasing cell glutamate. Increased glutamine/glutamate conversion to glutathione was not involved, since the glutathione synthesis inhibitor, buthionine sulfoximine, did not block glutamine’s effect on heat induction of Hsp25. A large drop in ATP levels did not appear to account for the diminished Hsp25 induction during glutamine deficiency. In summary, glutamine is an important amino acid, and its requirement for heat-induced Hsp25 supports a role for glutamine supplementation to optimize cellular responses to pathophysiological stress.

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Stimulation of basic amino acid uptake by certain neutral amino acids in isolated intestinal epithelial cells

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/0005-2736(71)90308-7 ◽

1971 ◽

Vol 241 (1) ◽

pp. 102-113 ◽

Cited By ~ 26

Author(s):

Sheldon Reiser ◽

Philip A. Christiansen

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Epithelial Cells ◽

Intestinal Epithelial Cells ◽

Basic Amino Acid ◽

Amino Acid Uptake ◽

Acid Uptake ◽

Intestinal Epithelial ◽

Neutral Amino Acids ◽

Stimulation Of

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A basis for the difference in the inhibition of the uptake of various neutral amino acids by lysine in intestinal epithelial cells

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/0005-2736(72)90136-8 ◽

1972 ◽

Vol 266 (1) ◽

pp. 217-229 ◽

Cited By ~ 16

Author(s):

Sheldon Reiser ◽

Philip A. Christiansen

Keyword(s):

Amino Acids ◽

Epithelial Cells ◽

Intestinal Epithelial Cells ◽

Intestinal Epithelial ◽

Neutral Amino Acids ◽

The Difference

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Corrigendum to “Let-7b ameliorates Crohn’s disease-associated adherent-invasive E coli induced intestinal inflammation via modulating Toll-Like Receptor 4 expression in intestinal epithelial cells” [Biochem. Pharmacol. 15 (2018) 196–203]

Biochemical Pharmacology ◽

10.1016/j.bcp.2018.09.025 ◽

2018 ◽

Vol 158 ◽

pp. 35-36

Author(s):

Zhen Guo ◽

Xingchen Cai ◽

Xian Guo ◽

Yihan Xu ◽

Jianfeng Gong ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Epithelial Cells ◽

Intestinal Inflammation ◽

Intestinal Epithelial Cells ◽

Intestinal Epithelial ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

E Coli

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Unique Regulation of Enterocyte Brush Border Membrane Na-Glutamine and Na-Alanine Co-Transport by Peroxynitrite during Chronic Intestinal Inflammation

International Journal of Molecular Sciences ◽

10.3390/ijms20061504 ◽

2019 ◽

Vol 20 (6) ◽

pp. 1504 ◽

Cited By ~ 1

Author(s):

Subha Arthur ◽

Palanikumar Manoharan ◽

Shanmuga Sundaram ◽

M Rahman ◽

Balasubramanian Palaniappan ◽

...

Keyword(s):

Epithelial Cells ◽

Brush Border ◽

Brush Border Membrane ◽

Intestinal Inflammation ◽

Intestinal Epithelial Cells ◽

Rabbit Model ◽

Intestinal Epithelial ◽

Mechanism Of Inhibition ◽

Chronic Intestinal Inflammation

Na-amino acid co-transporters (NaAAcT) are uniquely affected in rabbit intestinal villus cell brush border membrane (BBM) during chronic intestinal inflammation. Specifically, Na-alanine co-transport (ASCT1) is inhibited secondary to a reduction in the affinity of the co-transporter for alanine, whereas Na-glutamine co-transport (B0AT1) is inhibited secondary to a reduction in BBM co-transporter numbers. During chronic intestinal inflammation, there is abundant production of the potent oxidant peroxynitrite (OONO). However, whether OONO mediates the unique alteration in NaAAcT in intestinal epithelial cells during chronic intestinal inflammation is unknown. In this study, ASCT1 and B0AT1 were inhibited by OONO in vitro. The mechanism of inhibition of ASCT1 by OONO was secondary to a reduction in the affinity of the co-transporter for alanine, and secondary to a reduction in the number of co-transporters for B0AT1, which were further confirmed by Western blot analyses. In conclusion, peroxynitrite inhibited both BBM ASCT1 and B0AT1 in intestinal epithelial cells but by different mechanisms. These alterations in the villus cells are similar to those seen in the rabbit model of chronic enteritis. Therefore, this study indicates that peroxynitrite may mediate the inhibition of ASCT1 and B0AT1 during inflammation, when OONO levels are known to be elevated in the mucosa.

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7 LACTOBACILLUS REUTERI SUPPRESSES PRO-INFLAMMATORY DRIVEN REACTIVE OXYGEN SPECIES IN VITRO IN HUMAN INTESTINAL EPITHELIAL CELLS AND IN VIVO IN A TNBS COLITIS MOUSE MODEL

Inflammatory Bowel Diseases ◽

10.1093/ibd/zaa010.106 ◽

2020 ◽

Vol 26 (Supplement_1) ◽

pp. S41-S41 ◽

Cited By ~ 1

Author(s):

Wenly Ruan ◽

Melinda Engevik ◽

Alexandra Chang-Graham ◽

Joseph Hyser ◽

James Versalovic

Keyword(s):

Reactive Oxygen Species ◽

Epithelial Cells ◽

Lactobacillus Reuteri ◽

Intestinal Inflammation ◽

Intestinal Epithelial Cells ◽

Oxygen Species ◽

Intestinal Epithelial ◽

Colitis Model

Abstract Background Reactive oxygen species (ROS) play a role in maintaining intestinal epithelial homeostasis and are normally kept at low levels via antioxidant compounds. Dysregulation of ROS can lead to intestinal inflammation and contribute to inflammatory bowel disease (IBD). Select gut microbes possess the enzymatic machinery to produce antioxidants whereas others can dysregulate levels of ROS. Our model microbe, Lactobacillus reuteri (ATCC PTA 6475), has been demonstrated to reduce intestinal inflammation in mice models. It contains the genes encoding two distinct GshA-like glutamylcysteine ligases. We hypothesize that L. reuteri can secrete γ-glutamylcysteine to suppress ROS, minimize NFκB activation and regulate secretion of e pithelial cytokines. Methods & Results Conditioned media from L. reuteri was analyzed via mass spectrometry to confirm the presence of γ-glutamylcysteine. All cysteine containing products including γ-glutamylcysteine were fluorescently tagged in the conditioned media and then incubated with HT29 cell monolayers as well as human jejunal enteroid (HJE) monolayers. γ-glutamylcysteine was demonstrated to enter intestinal epithelial cells based on microscopy. Next, a Thioltracker assay was used to show increased intracellular glutathione levels by L. reuteri secreted γ-glutamylcysteine. HT29 cells and HJEs were then treated with IL-1β or hydrogen peroxide, and L. reuteri metabolites as well as γ-glutamylcysteine significantly suppressed pro-inflammatory cytokine driven ROS and IL-8 production. L. reuteri secreted products also reduced activity of NFκB as determined by a luciferase reporter assay. γ-glutamylcysteine deficient mutants were generated by targeted mutagenesis of GshA genes, and these mutant L. reuteri strains had a diminished ability to suppress IL-8 production and ROS. To further test the role of L. reuteri secreted γ-glutamylcysteine in vivo, a 2,4,6-Trinitrobenzenesulfonic acid (TNBS)- induced mouse colitis model was used. Adolescent mice were orogavaged with PBS, L. reuteri, L. reuteri GshA2 mutant, or γ-glutamylcysteine for a week after which TNBS was rectally administered to induce colitis. We demonstrate that L. reuteri and γ-glutamylcysteine can suppress histologic inflammation compared to PBS control and L. reuteri GshA2 mutant groups. Conclusions Together these data indicate that L. reuteri secretes γ-glutamylcysteine which can enter the intestinal epithelial cells and modulate epithelial cytokine production. It acts via suppression of ROS and NFκB which then decreases IL-8 production. We are able to demonstrate this in vitro in both HT 29 cells and HJEs. We now also demonstrate this in vivo in a mouse colitis model. These experiments highlight a prominent role for ROS intermediates in microbiome-mammalian cell signaling processes involved in immune responses and intestinal inflammation.

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OC.11.4 GATA6 DEFICIENCY IN THE INTESTINAL EPITHELIAL CELLS EXACERBATES INTESTINAL INFLAMMATION

Digestive and Liver Disease ◽

10.1016/s1590-8658(20)30576-4 ◽

2020 ◽

Vol 52 ◽

pp. S35-S36

Author(s):

F. Laudisi ◽

G. Bevivino ◽

C. Stolfi ◽

I. Marafini ◽

E. Troncone ◽

...

Keyword(s):

Epithelial Cells ◽

Intestinal Inflammation ◽

Intestinal Epithelial Cells ◽

Intestinal Epithelial

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Succinate Activates EMT in Intestinal Epithelial Cells through SUCNR1: A Novel Protagonist in Fistula Development

Cells ◽

10.3390/cells9051104 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1104 ◽

Cited By ~ 1

Author(s):

Dolores Ortiz-Masiá ◽

Laura Gisbert-Ferrándiz ◽

Cristina Bauset ◽

Sandra Coll ◽

Céline Mamie ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Epithelial Cells ◽

Wnt Signaling ◽

Intestinal Inflammation ◽

Intestinal Epithelial Cells ◽

Fistula Tract ◽

Intestinal Epithelial ◽

Mesenchymal Transition ◽

Ht29 Cells

The pathogenesis of Crohn’s disease-associated fibrostenosis and fistulas imply the epithelial-to-mesenchymal transition (EMT) process. As succinate and its receptor (SUCNR1) are involved in intestinal inflammation and fibrosis, we investigated their relevance in EMT and Crohn’s disease (CD) fistulas. Succinate levels and SUCNR1-expression were analyzed in intestinal resections from non-Inflammatory Bowel Disease (non-IBD) subjects and CD patients with stenosing-B2 or penetrating-B3 complications and in a murine heterotopic-transplant model of intestinal fibrosis. EMT, as increased expression of Snail1, Snail2 and vimentin and reduction in E-cadherin, was analyzed in tissues and succinate-treated HT29 cells. The role played by SUCNR1 was studied by silencing its gene. Succinate levels and SUCNR1 expression are increased in B3-CD patients and correlate with EMT markers. SUCNR1 is detected in transitional cells lining the fistula tract and in surrounding mesenchymal cells. Grafts from wild type (WT) mice present increased succinate levels, SUCNR1 up-regulation and EMT activation, effects not observed in SUCNR1−/− tissues. SUCNR1 activation induces the expression of Wnt ligands, activates WNT signaling and induces a WNT-mediated EMT in HT29 cells. In conclusion, succinate and its receptor are up-regulated around CD-fistulas and activate Wnt signaling and EMT in intestinal epithelial cells. These results point to SUCNR1 as a novel pharmacological target for fistula prevention.

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