Kamaljit K. Chaudhry

Acetaldehyde disrupts tight junctions in Caco-2 cell monolayers by a protein phosphatase 2A-dependent mechanism

Acetaldehyde is accumulated at high concentrations in the colonic lumen following ethanol administration. Previous studies demonstrated that acetaldehyde disrupts intestinal epithelial tight junctions and increases paracellular permeability. In the present study, we investigated the role of PP2A in the acetaldehyde-induced disruption of intestinal epithelial tight junctions. Caco-2 cell monolayers were exposed to 200-600 μM acetaldehyde for varying times, and the epithelial barrier function was evaluated by measuring transepithelial electrical resistance and inulin permeability. Acetaldehyde treatment resulted in a time-dependent increase in inulin permeability and redistribution of occludin and ZO-1 from the intercellular junctions. Treatment of cells with fostriecin (a PP2A-selective inhibitor) or knockdown of PP2A by siRNA blocked acetaldehyde-induced increase in inulin permeability and redistribution of occludin and ZO-1. The effects of fostriecin and acetaldehyde were confirmed in mouse intestine ex vivo. Acetaldehyde-induced tight junction disruption and barrier dysfunction were also attenuated by a PP2A-specific inhibitory peptide, TPDYFL. Coimmunoprecipitation studies showed that acetaldehyde increased the interaction of PP2A with occludin and induced dephosphorylation of occludin on threonine residues. Fostriecin and TPDYFL significantly reduced acetaldehyde-induced threonine dephosphorylation of occludin. Acetaldehyde failed to change the level of the methylated form of PP2A-C subunit. However, genistein (a tyrosine kinase inhibitor) blocked acetaldehyde-induced association of PP2A with occludin and threonine dephosphorylation of occludin. These results demonstrate that acetaldehyde-induced disruption of tight junctions is mediated by PP2A translocation to tight junctions and dephosphorylation of occludin on threonine residues.

Glutamine supplementation attenuates ethanol-induced disruption of apical junctional complexes in colonic epithelium and ameliorates gut barrier dysfunction and fatty liver in mice

Previous in vitro studies showed that glutamine (Gln) prevents acetaldehyde-induced disruption of tight junctions and adherens junctions in Caco-2 cell monolayers and human colonic mucosa. In the present study, we evaluated the effect of Gln supplementation on ethanol-induced gut barrier dysfunction and liver injury in mice in vivo. Ethanol feeding caused a significant increase in inulin permeability in distal colon. Elevated permeability was associated with a redistribution of tight junction and adherens junction proteins and depletion of detergent-insoluble fractions of these proteins, suggesting that ethanol disrupts apical junctional complexes in colonic epithelium and increases paracellular permeability. Ethanol-induced increase in colonic mucosal permeability and disruption of junctional complexes were most severe in mice fed Gln-free diet. Gln supplementation attenuated ethanol-induced mucosal permeability and disruption of tight junctions and adherens junctions in a dose-dependent manner, indicating the potential role of Gln in nutritional intervention to alcoholic tissue injury. Gln supplementation dose-dependently elevated reduced-protein thiols in colon without affecting the level of oxidized-protein thiols. Ethanol feeding depleted reduced protein thiols and elevated oxidized protein thiols. Ethanol-induced protein thiol oxidation was most severe in mice fed with Gln-free diet and absent in mice fed with Gln-supplemented diet, suggesting that antioxidant effect is one of the likely mechanisms involved in Gln-mediated amelioration of ethanol-induced gut barrier dysfunction. Ethanol feeding elevated plasma transaminase and liver triglyceride, which was accompanied by histopathologic lesions in the liver; ethanol-induced liver damage was attenuated by Gln supplementation. These results indicate that Gln supplementation ameliorates alcohol-induced gut and liver injury.

Chronic ethanol feeding promotes azoxymethane and dextran sulfate sodium- induced colonic tumorigenesis potentially by enhancing mucosal inflammation

BackgroundAlcohol consumption is one of the major risk factors for colorectal cancer. However, the mechanism involved in this effect of alcohol is unknown.MethodsWe evaluated the effect of chronic ethanol feeding on azoxymethane and dextran sulfate sodium (AOM/DSS)-induced carcinogenesis in mouse colon. Inflammation in colonic mucosa was assessed at a precancerous stage by evaluating mucosal infiltration of neutrophils and macrophages, and analysis of cytokine and chemokine gene expression.ResultsChronic ethanol feeding significantly increased the number and size of polyps in colon of AOM/DSS treated mice. Confocal microscopic and immunoblot analyses showed a significant elevation of phospho-Smad, VEGF and HIF1α in the colonic mucosa. RT-PCR analysis at a precancerous stage indicated that ethanol significantly increases the expression of cytokines IL-1α, IL-6 and TNFα, and the chemokines CCL5/RANTES, CXCL9/MIG and CXCL10/IP-10 in the colonic mucosa of AOM/DSS treated mice. Confocal microscopy showed that ethanol feeding induces a dramatic elevation of myeloperoxidase, Gr1 and CD68-positive cells in the colonic mucosa of AOM/DSS-treated mice. Ethanol feeding enhanced AOM/DSS-induced suppression of tight junction protein expression and elevated cell proliferation marker, Ki-67 in the colonic epithelium.ConclusionThis study demonstrates that chronic ethanol feeding promotes colonic tumorigenesis potentially by enhancing inflammation and elevation of proinflammatory cytokines and chemokines.

Injectable In Situ Forming Depot Systems for Long-Acting Contraception

Up to date, no long‐acting reversible contraceptive (LARC) is developed to be injectable through needles smaller than 18 G and can also provide contraception for more than 3 months after single injection. In this study, injectable polymeric in situ forming depot (ISD) systems are developed to have injectability through 21–23 G needles, and capability of sustained release of levonorgestrel (LNG) for at least 7 months in vitro and in vivo after single subcutaneous injection in rats. The systems are polymeric solutions composed of biodegradable poly(lactide‐co‐glycolide) and poly(lactic acid) polymers dissolved in a mixture of solvents like N‐methyl‐2‐pyrrolidone and benzyl benzoate or triethyl citrate. LNG released from ISD systems successfully suppressed the estrous cycle of rats at plasma concentration above 0.35 ng mL−1. At the end of the treatment, when LNG plasma concentration drops down to be nondetectable, predictable return of fertility is observed in rats. The designed ISD systems have great potential to be further developed into robust injectable LARCs that can be injected through a 21 G or smaller needle and achieve a variety of contraception durations with high patient compliance and low cost.

Glutamine Protects GI Epithelial Tight Junctions

l-Glutamine is the most abundant amino acid in blood stream accounting for 30–35 % of the amino acid nitrogen in plasma. It was classified as a non-essential amino acid because it can be readily synthesized in the body from glutamate by glutamine synthetase, which is expressed at high levels in skeletal muscle, liver, brain and stomach tissue. Intracellular concentration of l-glutamine ranges from 2 to 20 mM, and its concentration in the extracellular fluid varies from 0.5 to 0.8 mM. Under conditions of extreme physical exertion, trauma and severe infections, the rate of utilization of glutamine is more than its rate of synthesis, resulting in a significant decline in plasma glutamine concentration. Glutamine is an essential fuel for the gastrointestinal tract. It is required for the synthesis of proteins, nucleic acids and antioxidants, such as glutathione, and involved in the maintenance of acid–base balance with the release of ammonia during its metabolism. Under conditions of reduced plasma glutamine concentration, body depends on the exogenous glutamine to meet its requirements. Therefore, l-glutamine now is reclassified as a conditionally essential amino acid.