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Amino acid deprivation disrupts barrier function and induces protective autophagy in intestinal porcine epithelial cells

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Abstract

The integrity of intestinal barrier is essential for the absorption of nutrients and health in humans and animals. Dysfunction of the mucosal barrier is associated with increased gut permeability and development of various gastrointestinal diseases. Aside from serving as substrates for protein biosynthesis, amino acids also maintain the health of intestinal mucosal barrier. However, the underlying mechanisms remain unclear. We aimed to determine the effect and mechanism of non-essential amino acid (NEAA) deprivation on intestinal tight junction permeability using porcine intestinal epithelial cells as a model. We found that NEAA deprivation led to an impairment of barrier function as evidenced by increased permeability, decreased trans-epithelial resistance, and decreased expression of tight junction proteins claudin-1 and ZO-1. Importantly, NEAA deprivation induced both apoptosis and autophagy as shown by caspase-3 activation, and poly ADP-ribose polymerase cleavage; and LC3II lipidation and p62 degradation, hallmarks of apoptosis and autophagy, respectively. Importantly, we showed that the autophagy induced by NEAA deprivation counteracts apoptosis. Abrogation of autophagy by 3-methyladenine enhanced NEAA deprivation-induced barrier dysfunction and apoptosis; whereas, activation of autophagy by rapamycin partially rescued NEAA deprivation-induced barrier dysfunction and apoptosis. Taken together, our results demonstrate a critical role of NEAA on the mucosal integrity by regulating cell death and survival signaling pathways.

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Abbreviations

FBS:

Fetal bovine serum

FITC:

Fluorescein isothiocyanate

IPEC:

Intestinal porcine epithelial cells

MTT:

4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide

NEAA:

Non-essential amino acids

mTORC:

Mammalian target of rapamycin complex

PARP:

Poly ADP-ribose polymerase

PI3K:

Phosphatidylinositol 3-kinase

PKB/Akt:

Protein kinase B

PMSF:

Phenylmethylsulfonyl fluoride

TER:

Trans-epithelial electrical resistance

ZO-1:

Zonula occludens protein 1

References

  • Arrieta MC, Bistritz L, Meddings JB (2006) Alterations in intestinal permeability. Gut 55:1512–1520

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Bach SP, Renehan AG, Potten CS (2000) Stem cells: the intestinal stem cell as a paradigm. Carcinogenesis 21:469–476

    Article  CAS  PubMed  Google Scholar 

  • Blikslager AT, Moeser AJ, Gookin JL, Jones SL, Odle J (2007) Restoration of barrier function in injured intestinal mucosa. Physiol Rev 87:545–564

    Article  CAS  PubMed  Google Scholar 

  • Bojarski C, Bendfeldt K, Gitter AH, Mankertz J, Fromm M, Wagner S et al (2000) Apoptosis and intestinal barrier function. Ann N Y Acad Sci 915:270–274

    Article  CAS  PubMed  Google Scholar 

  • Camilleri M, Madsen K, Spiller R, Greenwood-Van Meerveld B, Verne GN (2012) Intestinal barrier function in health and gastrointestinal disease. Neurogastroenterol Motil 24:503–512

    Article  CAS  PubMed  Google Scholar 

  • DeMarco VG, Li N, Thomas J, West CM, Neu J (2003) Glutamine and barrier function in cultured Caco-2 epithelial cell monolayers. J Nutr 133:2176–2179

    CAS  PubMed  Google Scholar 

  • Demehri FR, Barrett M, Ralls MW, Miyasaka EA, Feng Y, Teitelbaum DH (2013) Intestinal epithelial cell apoptosis and loss of barrier function in the setting of altered microbiota with enteral nutrient deprivation. Front Cell Infect Microbiol 3:105

    Article  PubMed Central  PubMed  Google Scholar 

  • Fimia GM, Kroemer G, Piacentini M (2013) Molecular mechanisms of selective autophagy. Cell Death Differ 20:1–2

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Findley MK, Koval M (2009) Regulation and roles for claudin-family tight junction proteins. IUBMB Life 61:431–437

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Furuse M, Tsukita S (2006) Claudins in occluding junctions of humans and flies. Trends Cell Biol 16:181–188

    Article  CAS  PubMed  Google Scholar 

  • Gitter AH, Bendfeldt K, Schulzke JD, Fromm M (2000) Leaks in the epithelial barrier caused by spontaneous and TNF-alpha-induced single-cell apoptosis. FASEB J 14:1749–1753

    Article  CAS  PubMed  Google Scholar 

  • Groschwitz KR, Hogan SP (2009) Intestinal barrier function: molecular regulation and disease pathogenesis. J Allergy Clin Immunol 124:3–20 quiz 21–22

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Haynes TE, Li P, Li X, Shimotori K, Sato H, Flynn NE et al (2009) l-Glutamine or l-alanyl-l-glutamine prevents oxidant- or endotoxin-induced death of neonatal enterocytes. Amino Acids 37:131–142

    Article  CAS  PubMed  Google Scholar 

  • Jacobi SK, Odle J (2012) Nutritional factors influencing intestinal health of the neonate. Adv Nutr 3:687–696

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kroemer G, Marino G, Levine B (2010) Autophagy and the integrated stress response. Mol Cell 40:280–293

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Larson SD, Li J, Chung DH, Evers BM (2007) Molecular mechanisms contributing to glutamine-mediated intestinal cell survival. Am J Physiol Gastrointest Liver Physiol 293:G1262–G1271

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Le Bacquer O, Laboisse C, Darmaun D (2003) Glutamine preserves protein synthesis and paracellular permeability in Caco-2 cells submitted to “luminal fasting”. Am J Physiol Gastrointest Liver Physiol 285:G128–G136

    Article  PubMed  Google Scholar 

  • Levine B, Kroemer G (2008) Autophagy in the pathogenesis of disease. Cell 132:27–42

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Li N, Neu J (2009) Glutamine deprivation alters intestinal tight junctions via a PI3-K/Akt mediated pathway in Caco-2 cells. J Nutr 139:710–714

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Li N, Lewis P, Samuelson D, Liboni K, Neu J (2004) Glutamine regulates Caco-2 cell tight junction proteins. Am J Physiol Gastrointest Liver Physiol 287:G726–G733

    Article  CAS  PubMed  Google Scholar 

  • Marino G, Niso-Santano M, Baehrecke EH, Kroemer G (2014) Self-consumption: the interplay of autophagy and apoptosis. Nat Rev Mol Cell Biol 15:81–94

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Martinet W, De Meyer GR, Herman AG, Kockx MM (2005) Amino acid deprivation induces both apoptosis and autophagy in murine C2C12 muscle cells. Biotechnol Lett 27:1157–1163

    Article  CAS  PubMed  Google Scholar 

  • Mates JM, Segura JA, Alonso FJ, Marquez J (2006) Pathways from glutamine to apoptosis. Front Biosci 11:3164–3180

    Article  CAS  PubMed  Google Scholar 

  • Meadows AL, Kong B, Berdichevsky M, Roy S, Rosiva R, Blanch HW et al (2008) Metabolic and morphological differences between rapidly proliferating cancerous and normal breast epithelial cells. Biotechnol Prog 24:334–341

    Article  CAS  PubMed  Google Scholar 

  • Mizushima N, Komatsu M (2011) Autophagy: renovation of cells and tissues. Cell 147:728–741

    Article  CAS  PubMed  Google Scholar 

  • Mizushima N, Levine B, Cuervo AM, Klionsky DJ (2008) Autophagy fights disease through cellular self-digestion. Nature 451:1069–1075

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Nicklin P, Bergman P, Zhang B, Triantafellow E, Wang H, Nyfeler B et al (2009) Bidirectional transport of amino acids regulates mTOR and autophagy. Cell 136:521–534

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Noth R, Hasler R, Stuber E, Ellrichmann M, Schafer H, Geismann C et al (2013) Oral glutamine supplementation improves intestinal permeability dysfunction in a murine acute graft-vs.-host disease model. Am J Physiol Gastrointest Liver Physiol 304:G646–G654

    Article  CAS  PubMed  Google Scholar 

  • Oswald IP (2006) Role of intestinal epithelial cells in the innate immune defence of the pig intestine. Vet Res 37:359–368

    Article  CAS  PubMed  Google Scholar 

  • Paquette JC, Guerin PJ, Gauthier ER (2005) Rapid induction of the intrinsic apoptotic pathway by l-glutamine starvation. J Cell Physiol 202:912–921

    Article  CAS  PubMed  Google Scholar 

  • Parlesak A, Schafer C, Schutz T, Bode JC, Bode C (2000) Increased intestinal permeability to macromolecules and endotoxemia in patients with chronic alcohol abuse in different stages of alcohol-induced liver disease. J Hepatol 32:742–747

    Article  CAS  PubMed  Google Scholar 

  • Patel KK, Miyoshi H, Beatty WL, Head RD, Malvin NP, Cadwell K et al (2013) Autophagy proteins control goblet cell function by potentiating reactive oxygen species production. EMBO J 32:3130–3144

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Radtke F, Clevers H (2005) Self-renewal and cancer of the gut: two sides of a coin. Science 307:1904–1909

    Article  CAS  PubMed  Google Scholar 

  • Schneeberger EE, Lynch RD (2004) The tight junction: a multifunctional complex. Am J Physiol Cell Physiol 286:C1213–C1228

    Article  CAS  PubMed  Google Scholar 

  • Schulzke JD, Bojarski C, Zeissig S, Heller F, Gitter AH, Fromm M (2006) Disrupted barrier function through epithelial cell apoptosis. Ann N Y Acad Sci 1072:288–299

    Article  CAS  PubMed  Google Scholar 

  • Singletary K, Milner J (2008) Diet, autophagy, and cancer: a review. Cancer Epidemiol Biomark Prev 17:1596–1610

    Article  CAS  Google Scholar 

  • Strater J, Wellisch I, Riedl S, Walczak H, Koretz K, Tandara A et al (1997) CD95 (APO-1/Fas)-mediated apoptosis in colon epithelial cells: a possible role in ulcerative colitis. Gastroenterology 113:160–167

    Article  CAS  PubMed  Google Scholar 

  • Takayama C, Mukaizawa F, Fujita T, Ogawara K, Higaki K, Kimura T (2009) Amino acids suppress apoptosis induced by sodium laurate, an absorption enhancer. J Pharm Sci 98:4629–4638

    Article  CAS  PubMed  Google Scholar 

  • Tato I, Bartrons R, Ventura F, Rosa JL (2011) Amino acids activate mammalian target of rapamycin complex 2 (mTORC2) via PI3K/Akt signaling. J Biol Chem 286:6128–6142

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ulluwishewa D, Anderson RC, McNabb WC, Moughan PJ, Wells JM, Roy NC (2011) Regulation of tight junction permeability by intestinal bacteria and dietary components. J Nutr 141:769–776

    Article  CAS  PubMed  Google Scholar 

  • Wang J, Wu Z, Li D, Li N, Dindot SV, Satterfield MC et al (2012) Nutrition, epigenetics, and metabolic syndrome. Antioxid Redox Signal 17:282–301

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Wang B, Wu G, Zhou Z, Dai Z, Sun Y, Ji Y, Li W, Wang W, Liu C, Han F et al (2014) Glutamine and intestinal barrier function. Amino Acids. doi:10.1007/s00726-014-1773-4

  • Wittkopf N, Gunther C, Martini E, Waldner M, Amann KU, Neurath MF et al (2012) Lack of intestinal epithelial atg7 affects paneth cell granule formation but does not compromise immune homeostasis in the gut. Clin Dev Immunol 2012:278059

    Article  PubMed Central  PubMed  Google Scholar 

  • Xi P, Jiang Z, Dai Z, Li X, Yao K, Zheng C et al (2012) Regulation of protein turnover by l-glutamine in porcine intestinal epithelial cells. J Nutr Biochem 23:1012–1017

    Article  CAS  PubMed  Google Scholar 

  • Zeissig S, Bojarski C, Buergel N, Mankertz J, Zeitz M, Fromm M et al (2004) Downregulation of epithelial apoptosis and barrier repair in active Crohn’s disease by tumour necrosis factor alpha antibody treatment. Gut 53:1295–1302

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by National Key Basic Research Program (2013CB127302), the Natural Science Foundation of China (31172217, 31272450, and 31272451), the Chinese Universities Scientific Fund (2013RC002), the Program for New Century Excellent Talents in University (NCET-12-0522), and the Program for Beijing Municipal Excellent Talents.

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The authors declare no conflicts of interest.

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Correspondence to Zhenlong Wu.

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Yang, Y., Li, W., Sun, Y. et al. Amino acid deprivation disrupts barrier function and induces protective autophagy in intestinal porcine epithelial cells. Amino Acids 47, 2177–2184 (2015). https://doi.org/10.1007/s00726-014-1844-6

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