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Effects of essential amino acids or glutamine deprivation on intestinal permeability and protein synthesis in HCT-8 cells: involvement of GCN2 and mTOR pathways

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Abstract

GCN2 and mTOR pathways are involved in the regulation of protein metabolism in response to amino acid availability in different tissues. However, regulation at intestinal level is poorly documented. The aim of the study was to evaluate the effects of a deprivation of essential amino acids (EAA) or glutamine (Gln) on these pathways in intestinal epithelial cells. Intestinal epithelial cell, HCT-8, were incubated during 6 h with 1/DMEM culture medium containing EAA, non EAA and Gln, 2/with saline as positive control of nutritional deprivation, 3/DMEM without EAA, 4/DMEM without Gln or 5/DMEM without Gln and supplemented with a glutamine synthase inhibitor (MSO, 4 mM). Intestinal permeability was evaluated by the measure of transepithelial electric resistance (TEER). Using [L-2H3]-leucine incorporation, fractional synthesis rate (FSR) was calculated from the assessed enrichment in proteins and free amino acid pool by GCMS. Expression of eiF2α (phosphorylated or not), used as marker of GCN2 pathway, and of 4E-BP1 (phosphorylated or not), used as a marker of mTOR pathway, was evaluated by immunoblot. Results were compared by ANOVA. Six-hours EAA deprivation did not significantly affect TEER and FSR but decreased p-4E-BP1 and increased p-eiF2α. In contrast, Gln deprivation decreased FSR and p-4E-BP1. MSO induced a marked decrease of TEER and FSR and an increase of p-eiF2α, whereas mTOR pathway remained activated. These results suggest that both mTOR and GCN2 pathways can mediate the limiting effects of Gln deprivation on protein synthesis according to its severity.

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Abbreviations

EAA:

Essential amino acid

eaaF:

Essential amino acid-free medium

GlnF:

Glutamine-free medium

GlnF(+):

Glutamine-free medium supplemented with glutamine synthase inhibitor

MSO:

Methionine sulfoximine

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Acknowledgments

NB was supported by a grant of the French Speaking Society of Clinical Nutrition and Metabolism (SFNEP) and Nutricia Clinical Nutrition (France).

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Authors and Affiliations

  1. ADEN EA4311, Institute for Biomedical Research and European Institute for Peptide Research (IFRMP23), Rouen University, 22 boulevard Gambetta, 76183, Rouen cedex 1, France

    Nabile Boukhettala, Sophie Claeyssens, Malik Bensifi, Juliette Abed, Alain Lavoinne, Pierre Déchelotte & Moïse Coëffier

  2. Laboratory of Medical Biochemistry, Rouen University Hospital, Rouen, France

    Sophie Claeyssens, Brigitte Maurer & Alain Lavoinne

  3. Nutrition Unit, Rouen University Hospital, Rouen, France

    Pierre Déchelotte & Moïse Coëffier

Authors
  1. Nabile Boukhettala
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  2. Sophie Claeyssens
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Correspondence to Moïse Coëffier.

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Boukhettala, N., Claeyssens, S., Bensifi, M. et al. Effects of essential amino acids or glutamine deprivation on intestinal permeability and protein synthesis in HCT-8 cells: involvement of GCN2 and mTOR pathways. Amino Acids 42, 375–383 (2012). https://doi.org/10.1007/s00726-010-0814-x

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  • DOI: https://doi.org/10.1007/s00726-010-0814-x

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