Digestive Diseases and Sciences

, Volume 58, Issue 1, pp 77–87

Zinc Supplementation Modifies Tight Junctions and Alters Barrier Function of CACO-2 Human Intestinal Epithelial Layers

  • Xuexuan Wang
  • Mary Carmen Valenzano
  • Joanna M. Mercado
  • E. Peter Zurbach
  • James M. Mullin
Original Article

Abstract

Background

Zinc deficiency is known to result in epithelial barrier leak in the GI tract. Precise effects of zinc on epithelial tight junctions (TJs) are only beginning to be described and understood. Along with nutritional regimens like methionine-restriction and compounds such as berberine, quercetin, indole, glutamine and rapamycin, zinc has the potential to function as a TJ modifier and selective enhancer of epithelial barrier function.

Aims

The purpose of this study was to determine the effects of zinc-supplementation on the TJs of a well-studied in vitro GI model, CACO-2 cells.

Methods

Barrier function was assessed electrophysiologically by measuring transepithelial electrical resistance (Rt), and radiochemically, by measuring transepithelial (paracellular) diffusion of 14C-D-mannitol and 14C-polyethyleneglycol. TJ composition was studied by Western immunoblot analyses of occludin, tricellulin and claudins-1 to -5 and -7.

Results

Fifty- and 100-μM zinc concentrations (control medium is 2 μM) significantly increase Rt but simultaneously increase paracellular leak to D-mannitol. Claudins 2 and 7 are downregulated in total cell lysates, while occludin, tricellulin and claudins-1, -3, -4 and -5 are unchanged. Claudins-2 and -7 as well as tricellulin exhibit decreased cytosolic content as a result of zinc supplementation.

Conclusions

Zinc alters CACO-2 TJ composition and modifies TJ barrier function selectively. Zinc is one of a growing number of “nutraceutical” substances capable of enhancing epithelial barrier function, and may find use in countering TJ leakiness induced in various disease states.

Keywords

Zinc CACO-2 Tight junction Claudins Paracellular Nutrition Claudin Differentiation Metallothionein 

Abbreviations

TJ

Tight junction

PD

Potential difference

Iscc

Short circuit current

Rt

Transepithelial electrical resistance

MT 1/2

Metallothionein 1/2

GI

Gastrointestinal

FBS

Fetal bovine serum

PBS

Phosphate buffered saline

RDA

Recommended daily allowance

SEM

Standard error of the mean

LDH

Lactate dehydrogenase

XTT

Tetrazolium dye, sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl-5-[(phenylamino)-carbonyl]-2H-tetrazolium)

LSC

Liquid scintillation counting

PEG

Polyethyleneglycol

References

  1. 1.
    Cousins RJ, Zinc. In: Bowman BA, Russell RM, eds. Present Knowledge in Nutrition, vol 1. Washington, D.C.: ILSI Press; 2006:445–457.Google Scholar
  2. 2.
    Binnebosel M, Grommes J, Koenen B, et al. Zinc deficiency impairs wound healing of colon anastomosis in rats. Int J Colorectal Dis. 2010;25:251–257.PubMedCrossRefGoogle Scholar
  3. 3.
    Prasad AS. Impact of the discovery of human zinc deficiency on health. J Am Coll Nutr. 2009;28:257–265.PubMedGoogle Scholar
  4. 4.
    Ibs KH, Rink L. Zinc-altered immune function. J Nutr. 2003;133:1452S–1456S.PubMedGoogle Scholar
  5. 5.
    Ho E. Zinc deficiency, DNA damage and cancer risk. J Nutr Biochem. 2004;15:572–578.PubMedCrossRefGoogle Scholar
  6. 6.
    Mullin JM, Skrovanek SM, Valenzano MC. Modification of tight junction structure and permeability by nutritional means. Ann NY Acad Sci. 2009;1165:99–112.PubMedCrossRefGoogle Scholar
  7. 7.
    Amasheh S, Dullat S, Fromm M, Schulzke JD, Buhr HJ, Kroesen AJ. Inflamed pouch mucosa possesses altered tight junctions indicating recurrence of inflammatory bowel disease. Int J Colorectal Dis. 2009;24:1149–1156.PubMedCrossRefGoogle Scholar
  8. 8.
    Pearson AD, Eastham EJ, Laker MF, Craft AW, Nelson R. Intestinal permeability in children with Crohn’s disease and coeliac disease. Br Med J (Clin Res Ed). 1982;285:20–21.CrossRefGoogle Scholar
  9. 9.
    Sturniolo GC, Di Leo V, Ferronato A, D’Odorico A, D’Inca R. Zinc supplementation tightens “Leaky gut” in Crohn’s disease. Inflamm Bowel Dis. 2001;7:94–98.PubMedCrossRefGoogle Scholar
  10. 10.
    Bao S, Knoell DL. Zinc modulates cytokine-induced lung epithelial cell barrier permeability. Am J Physiol Lung Cell Mol Physiol. 2006;291:L1132–L1141.PubMedCrossRefGoogle Scholar
  11. 11.
    Rodriguez P, Darmon N, Chappuis P, et al. Intestinal paracellular permeability during malnutrition in guinea pigs: effect of high dietary zinc. Gut. 1996;39:416–422.PubMedCrossRefGoogle Scholar
  12. 12.
    Lazzerini M, Ronfani L. Oral zinc for treating diarrhoea in children. Sao Paulo Med J 2011;129:118–119.Google Scholar
  13. 13.
    Zhong W, McClain CJ, Cave M, Kang YJ, Zhou Z. The role of zinc deficiency in alcohol-induced intestinal barrier dysfunction. Am J Physiol Gastrointest Liver Physiol 2010;298:G625–G633.Google Scholar
  14. 14.
    Peterson MD, Mooseker MS. Characterization of the enterocyte-like brush border cytoskeleton of the c2bbe clones of the human intestinal cell line, CACO-2. J Cell Sci. 1992;102:581–600.PubMedGoogle Scholar
  15. 15.
    Hubatsch I, Ragnarsson EG, Artursson P. Determination of drug permeability and prediction of drug absorption in caco-2 monolayers. Nat Protoc. 2007;2:2111–2119.PubMedCrossRefGoogle Scholar
  16. 16.
    Zemann N, Zemann A, Klein P, Elmadfa I, Huettinger M. Differentiation- and polarization-dependent zinc tolerance in CACO-2 cells. Eur J Nutr. 2010;50:379–386.PubMedCrossRefGoogle Scholar
  17. 17.
    Skrovanek S, Valenzano MC, Mullin JM. Restriction of sulfur-containing amino acids alters claudin composition and improves tight junction barrier function. Am J Physiol Regul Integr Comp Physiol. 2007;293:R1046–R1055.PubMedCrossRefGoogle Scholar
  18. 18.
    Mullin JM, Marano CW, Laughlin KV, Nuciglio M, Stevenson BR, Soler P. Different size limitations for increased transepithelial paracellular solute flux across phorbol ester and tumor necrosis factor-treated epithelial cell sheets. J Cell Physiol. 1997;171:226–233.PubMedCrossRefGoogle Scholar
  19. 19.
    Burton K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J. 1956;62:315–323.PubMedGoogle Scholar
  20. 20.
    Mullin JM, Fluk L, Kleinzeller A. Basal-lateral transport and transcellular flux of methyl alpha-d-glucoside across LLC-PK1 renal epithelial cells. Biochim Biophys Acta. 1986;885:233–239.PubMedCrossRefGoogle Scholar
  21. 21.
    Rabito CA. Reassembly of the occluding junctions in a renal cell line with characteristics of proximal tubular cells. Am J Physiol. 1986;251:F978–F987.PubMedGoogle Scholar
  22. 22.
    Finamore A, Massimi M, Conti Devirgiliis L, Mengheri E. Zinc deficiency induces membrane barrier damage and increases neutrophil transmigration in CACO-2 cells. J Nutr. 2008;138:1664–1670.PubMedGoogle Scholar
  23. 23.
    Christopher P, Farrell MB, Mullin JM, Lande L, Zitin M. Epithelial barrier leak in gastrointestinal disease and multiorgan failure. J Epithelial Biol Pharmacol. 2011;5:13–18.Google Scholar
  24. 24.
    Zhong W, McClain CJ, Cave M, Kang YJ, Zhou Z. The role of zinc deficiency in alcohol-induced intestinal barrier dysfunction. Am J Physiol Gastrointest Liver Physiol. 2010;298:18.CrossRefGoogle Scholar
  25. 25.
    Zhang B, Guo Y. Supplemental zinc reduced intestinal permeability by enhancing occludin and zonula occludens protein-1 (ZO-1) expression in weaning piglets. Br J Nutr. 2009;102:687–693.PubMedCrossRefGoogle Scholar
  26. 26.
    Sturniolo GC, Fries W, Mazzon E, Di Leo V, Barollo M, D’Inca R. Effect of zinc supplementation on intestinal permeability in experimental colitis. J Lab Clin Med. 2002;139:311–315.PubMedCrossRefGoogle Scholar
  27. 27.
    Ranaldi G, Caprini V, Sambuy Y, Perozzi G, Murgia C. Intracellular zinc stores protect the intestinal epithelium from ochratoxin a toxicity. Toxicol in Vitro. 2009;23:1516–1521.PubMedCrossRefGoogle Scholar
  28. 28.
    Roselli M, Finamore A, Garaguso I, Britti MS, Mengheri E. Zinc oxide protects cultured enterocytes from the damage induced by escherichia coli. J Nutr. 2003;133:4077–4082.PubMedGoogle Scholar
  29. 29.
    Mullin JM. Editorial: Augmenting epithelial barrier function. J Epithelial Biol Pharmacol. 2012;5:10–12.CrossRefGoogle Scholar
  30. 30.
    Shen H, Qin H, Guo J. Cooperation of metallothionein and zinc transporters for regulating zinc homeostasis in human intestinal caco-2 cells. Nutr Res. 2008;28:406–413.PubMedCrossRefGoogle Scholar
  31. 31.
    Jou MY, Philipps AF, Kelleher SL, Lonnerdal B. Effects of zinc exposure on zinc transporter expression in human intestinal cells of varying maturity. J Pediatr Gastroenterol Nutr. 2010;50:587–595.PubMedCrossRefGoogle Scholar
  32. 32.
    Lal-Nag M, Morin PJ. The claudins. Genome Biol. 2009;10:235.PubMedCrossRefGoogle Scholar
  33. 33.
    Escaffit F, Boudreau F, Beaulieu JF. Differential expression of claudin-2 along the human intestine: implication of gata-4 in the maintenance of claudin-2 in differentiating cells. J Cell Physiol. 2005;203:15–26.PubMedCrossRefGoogle Scholar
  34. 34.
    Reyes JL, Lamas M, Martin D, et al. The renal segmental distribution of claudins changes with development. Kidney Int. 2002;62:476–487.PubMedCrossRefGoogle Scholar
  35. 35.
    Van Itallie CM, Anderson JM. Claudins and epithelial paracellular transport. Annu Rev Physiol. 2006;68:403–429.PubMedCrossRefGoogle Scholar
  36. 36.
    Alexandre MD, Lu Q, Chen YH. Overexpression of claudin-7 decreases the paracellular Cl conductance and increases the paracellular Na+ conductance in LLC-PK1 cells. J Cell Sci. 2005;118:2683–2693.PubMedCrossRefGoogle Scholar
  37. 37.
    Atisook K, Carlson S, Madara JL. Effects of phlorizin and sodium on glucose-elicited alterations of cell junctions in intestinal epithelia. Am J Physiol. 1990;258:C77–C85.PubMedGoogle Scholar
  38. 38.
    Ballard ST, Hunter JH, Taylor AE. Regulation of tight-junction permeability during nutrient absorption across the intestinal epithelium. Annu Rev Nutr. 1995;15:35–55.PubMedCrossRefGoogle Scholar
  39. 39.
    Gato-Pecina JJ, Ponz F. Use of the paracellular way for the intestinal absorption of sugars. Rev Esp Fisiol. 1990;46:343–352.PubMedGoogle Scholar
  40. 40.
    Sadowski DC, Meddings JB. Luminal nutrients alter tight-junction permeability in the rat jejunum: an in vivo perfusion model. Can J Physiol Pharmacol. 1993;71:835–839.PubMedCrossRefGoogle Scholar
  41. 41.
    Carr G, Wright JA, Simmons NL. Epithelial barrier resistance is increased by the divalent cation zinc in cultured MDCKII epithelial monolayers. J Membr Biol 2010;237:115–123.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Xuexuan Wang
    • 1
  • Mary Carmen Valenzano
    • 1
  • Joanna M. Mercado
    • 1
  • E. Peter Zurbach
    • 2
  • James M. Mullin
    • 1
    • 3
  1. 1.Lankenau Institute for Medical ResearchWynnewoodUSA
  2. 2.Department of ChemistrySaint Joseph’s UniversityPhiladelphiaUSA
  3. 3.Division of GastroenterologyLankenau Medical CenterWynnewoodUSA

Personalised recommendations