Evidence from clinical and experimental studies indicates that elevated intestinal permeability to endotoxins and the resulting endotoxemia play a crucial role in the pathogenesis of alcoholic liver disease. Our studies conducted so far have shown that acetaldehyde, the metabolic product of ethanol, disrupts the intestinal epithelial barrier function and increases the permeability to endotoxins. The mechanism of this acetaldehyde-induced disruption of epithelial barrier function involves inhibition of a protein tyrosine phosphatase, PTP1B, tyrosine phosphorylation of junctional proteins, disruption of the interactions among the junctional proteins (that determine the barrier function), and loss of integrity of the junctional complexes. Furthermore, our studies demonstrated that epidermal growth factor (EGF) and L-glutamine prevent acetaldehyde-mediated increase in permeability to endotoxins by a PLC?, PKC?, PKC?I and calcium-dependent mechanism. Our preliminary studies indicate that acetaldehyde induces translocation of PP2A leading to dephosphorylation of occludin and claudin-4, ethanol amplifies the effect of acetaldehyde by a Src kinase and MLCK-dependent mechanism and that probiotic, L. plantarum prevents acetaldehyde- induced barrier disruption. On the basis of these results it is further hypothesized that: a) ethanol metabolism and gut microflora play crucial roles in ethanol-induced intestinal barrier dysfunction, b) PP2A-dependent dephosphorylation of occludin and Cldn-4 is involved in acetaldehyde-induced disruption of intestinal barrier function, c) ethanol synergizes acetaldehyde-induced barrier disruption by c-Src-mediated MLCK activation, and d) probiotic, L. plantarum, prevents ethanol and acetaldehyde-induced disruption of barrier function by a EGF receptor, p38MAPK and Rac1- dependent mechanism. Using a cell culture model of the intestinal epithelium and human colonic biopsies we will determine that: 1) ADH1B and ALDH2 modulate ethanol-induced disruption of TJs and barrier function. 2) ALDH2 deficient mice are more sensitive to ethanol-induced barrier dysfunction. 3) Gut microflora play a role in ethanol metabolism and ethanol-induced disruption of TJs. 4) Acetaldehyde-induced PP2A methylation and translocation leads to dephosphorylation of TJ proteins and disruption of barrier function. 5) Dephosphorylation of occludin and Cldn-4 on specific Ser and Thr residues is associated with acetaldehyde-induced disruption of TJs and barrier dysfunction. 6) PP2A translocation plays a role in acetaldehyde-induced TJ disruption in mouse intestine. 7) Ethanol-mediated c-Src activation synergizes acetaldehyde-induced TJ disruption. 8) MLCK mediates synergization of acetaldehyde-induced TJ disruption by ethanol. 9) Ethanol sensitizes mouse colon for acetaldehyde-induced barrier dysfunction by a c-Src and MLCK- dependent mechanism. 10) L. plantarum prevents ethanol/acetaldehyde-induced disruption of junctions by p38MAPK-dependent mechanism. 11) Rac1 activation and stabilization of actomyosin ring are involved in the L. plantarum-mediated prevention of ethanol/acetaldehyde-induced tight junction disruption. 12) L. plantarum ameliorates ethanol/acetaldehyde-induced intestinal barrier dysfunction in mice and human colonic mucosa. The outcome of these studies has a direct relevance to our understanding of the pathogenesis of alcoholic liver and pancreatic diseases, and has the potential to contribute to the future development of new therapeutic strategies.

Public Health Relevance

On the basis of our research during the past several years we hypothesized that ethanol metabolism by gut microflora into acetaldehyde disrupts intestinal epithelial barrier function by inducing phosphorylation of proteins of intercellular junctions, and the probiotic L. plantarum prevents such cellular damage by acetaldehyde. We propose to conduct studies to uncover the cellular and molecular mechanisms involved in these processes and determine the protective role of a probiotic in alleviating the alcohol- induced tissue injury. The outcome of these studies is expected to provide knowledge to develop new therapies in the treatment of alcoholic liver disease and alcohol-induced tissue injury in pancreas and lung.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA012307-13
Application #
8306354
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Gao, Peter
Project Start
2000-05-01
Project End
2015-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
13
Fiscal Year
2012
Total Cost
$337,862
Indirect Cost
$109,577
Name
University of Tennessee Health Science Center
Department
Physiology
Type
Schools of Medicine
DUNS #
941884009
City
Memphis
State
TN
Country
United States
Zip Code
38163
Rao, R K; Samak, G (2013) Protection and Restitution of Gut Barrier by Probiotics: Nutritional and Clinical Implications. Curr Nutr Food Sci 9:99-107
Aggarwal, Sudhir; Suzuki, Takuya; Taylor, William L et al. (2011) Contrasting effects of ERK on tight junction integrity in differentiated and under-differentiated Caco-2 cell monolayers. Biochem J 433:51-63
Samak, Geetha; Narayanan, Damodaran; Jaggar, Jonathan H et al. (2011) CaV1.3 channels and intracellular calcium mediate osmotic stress-induced N-terminal c-Jun kinase activation and disruption of tight junctions in Caco-2 CELL MONOLAYERS. J Biol Chem 286:30232-43
Samak, G; Aggarwal, S; Rao, R K (2011) ERK is involved in EGF-mediated protection of tight junctions, but not adherens junctions, in acetaldehyde-treated Caco-2 cell monolayers. Am J Physiol Gastrointest Liver Physiol 301:G50-9
Guntaka, Srikar R; Samak, Geetha; Seth, Ankur et al. (2011) Epidermal growth factor protects the apical junctional complexes from hydrogen peroxide in bile duct epithelium. Lab Invest 91:1396-409
Jain, Suneet; Suzuki, Takuya; Seth, Ankur et al. (2011) Protein kinase Cýý phosphorylates occludin and promotes assembly of epithelial tight junctions. Biochem J 437:289-99
Basuroy, Shyamali; Dunagan, Mitzi; Sheth, Parimal et al. (2010) Hydrogen peroxide activates focal adhesion kinase and c-Src by a phosphatidylinositol 3 kinase-dependent mechanism and promotes cell migration in Caco-2 cell monolayers. Am J Physiol Gastrointest Liver Physiol 299:G186-95
Rao, Radhakrishna (2009) Endotoxemia and gut barrier dysfunction in alcoholic liver disease. Hepatology 50:638-44
Rao, Radhakrishna (2009) Occludin phosphorylation in regulation of epithelial tight junctions. Ann N Y Acad Sci 1165:62-8
Sheth, Parimal; Samak, Geetha; Shull, J Andrew et al. (2009) Protein phosphatase 2A plays a role in hydrogen peroxide-induced disruption of tight junctions in Caco-2 cell monolayers. Biochem J 421:59-70

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