Article

Role of intestinal circadian genes in alcohol-induced gut leakiness.

Department of Medicine, Division of Digestive Diseases and Nutrition, Rush University Medical Center, 1725 W. Harrison, Chicago, IL 60612, USA.
Alcoholism Clinical and Experimental Research (Impact Factor: 3.42). 04/2011; 35(7):1305-14. DOI: 10.1111/j.1530-0277.2011.01466.x
Source: PubMed

ABSTRACT Several studies have indicated that endotoxemia is the required co-factor for alcoholic steatohepatitis (ASH) that is seen in only about 30% of alcoholics. Recent studies have shown that gut leakiness that occurs in a subset of alcoholics is the primary cause of endotoxemia in ASH. The reasons for this differential susceptibility are not known. Since disruption of circadian rhythms occurs in some alcoholics and circadian genes control the expression of several genes that are involved in regulation of intestinal permeability, we hypothesized that alcohol induces intestinal hyperpermeability by stimulating expression of circadian clock gene proteins in the intestinal epithelial cells.
We used Caco-2 monolayers grown on culture inserts as an in vitro model of intestinal permeability and performed Western blotting, permeability, and siRNA inhibition studies to examine the role of Clock and Per2 circadian genes in alcohol-induced hyperpermeability. We also measured PER2 protein levels in intestinal mucosa of alcohol-fed rats with intestinal hyperpermeability.
Alcohol, as low as 0.2%, induced time dependent increases in both Caco-2 cell monolayer permeability and in CLOCK and PER2 proteins. SiRNA specific inhibition of either Clock or Per2 significantly inhibited alcohol-induced monolayer hyperpermeability. Alcohol-fed rats with increased total gut permeability, assessed by urinary sucralose, also had significantly higher levels of PER2 protein in their duodenum and proximal colon than control rats.
Our studies: (i) demonstrate a novel mechanism for alcohol-induced intestinal hyperpermeability through stimulation of intestinal circadian clock gene expression, and (ii) provide direct evidence for a central role of circadian genes in regulation of intestinal permeability.

0 Bookmarks
 · 
267 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Disruption of circadian rhythms is a risk factor for several human gastrointestinal (GI) diseases, ranging from diarrhea to ulcers to cancer. 4-dimensional tissue culture models that faithfully mimic the circadian clock of the GI epithelium would provide an invaluable tool to understand circadian regulation of GI health and disease. We hypothesized that rhythmicity of a key circadian component, PERIOD2 (PER2), would diminish along a continuum from ex vivo intestine, organoids (epithelial "miniguts"), and nontransformed (MSIE) and transformed (Caco-2) intestinal epithelial cells. Here we show that bioluminescent jejunal explants from PERIOD2::LUCIFERASE (PER2::LUC) mice display robust circadian rhythms for >72 hours post-excision. Circadian rhythms in primary or passaged PER2::LUC jejunal organoids are similarly robust, synchronize with serum shock, and persist beyond 2 weeks in culture. Remarkably, unshocked organoids autonomously synchronize rhythms within 12 hours of recording. The onset of this autonomous synchronization is slowed by >2 hours in the presence of glucocorticoid receptor antagonist RU486 (20 μM). Doubling standard concentrations of organoid growth factors EGF, Noggin, and R-spondin enhances PER2 oscillations, whereas subtraction of these factors individually at 24 hours following serum shock produced no detectable effects. Growth factor pulses induce modest phase delays in unshocked, but not serum-shocked, organoids. Circadian oscillations of PER2::LUC bioluminescence align with Per2 mRNA expression by qPCR. Concordant findings of robust circadian rhythms in bioluminescent jejunal explants and organoids provide further evidence for a peripheral clock intrinsic to the intestinal epithelium. The rhythmic and organotypic features of organoids should offer unprecedented advantages as a resource for elucidating the role of circadian rhythms in GI stem cell dynamics, epithelial homeostasis, and disease.
    Disease Models and Mechanisms 07/2014; · 4.96 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Evidence indicates that ethanol-induced intestinal barrier dysfunction and subsequent endotoxemia plays a key role in the pathogenesis of alcoholic liver disease. Recently, it has been demonstrated that ethanol induces RhoA kinase activation in intestinal epithelium, thereby disrupting barrier integrity. In this study, role of a rise in intracellular calcium concentration ([Ca(2+)]i) in ethanol-induced Rho-associated coiled-coil forming kinase (Rho/ROCK) activation and barrier disruption was investigated in Caco-2 cell monolayers. Treatment of Caco-2 monolayers with 40 mmol/L ethanol induced [Ca(2+)]i release as indicated by increased relative fluorescent units of Fluo-3 from 0.06±0.02 to 2.27±1.96 (P < 0.0001). Pretreatment with 1,2-Bis(2-Aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) completely, whereas the inositol 1, 4, 5-triphosphate receptor (IP3R)-antagonist, Xestospongin C, partially inhibited the ethanol-induced [Ca(2+)]i release (from 2.27 ± 1.96 to 0.03 ± 0.01; P < 0.0001), and (from 2.27 ± 1.96 to 1.19 ±1.80; P < 0.001), respectively. The rise in [Ca(2+)]i was paralleled with increased intestinal permeability, which could be attenuated by either BAPTA-AM or Xestospongin C. Furthermore, ethanol induced Rho/ROCK activation, as indicated by increased phosphorylation of myosin binding subunit, which could be prevented either by BAPTA, Xestospongin C, or the specific Rho/ROCK inhibitor Y27632. Finally, inhibition of Rho/ROCK kinase by Y27632 ameliorated the ethanol-induced redistribution of ZO-1, adherens junction proteins including E-cadherin, and β-catenin, and also disorganization of F-actin. These findings suggest that ethanol-induced [Ca(2+)]i release, mediated by stimulating IP3R-gated Ca(2+) channel, activates Rho/ROCK in Caco-2 cells, and thereby contributing to ethanol-induced intestinal barrier dysfunction.
    AJP Gastrointestinal and Liver Physiology 02/2014; · 3.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: In association with sleep/wake and fasting/feeding cycles, organisms experience dramatic oscillations in energetic demands and nutrient supply. It is therefore not surprising that various metabolic parameters, ranging from the activity status of molecular energy sensors to circulating nutrient levels, oscillate in time-of-day-dependent manners. It has become increasingly clear that rhythms in metabolic processes are not simply in response to daily environmental/behavioral influences, but are driven in part by cell autonomous circadian clocks. By synchronizing the cell with its environment, clocks modulate a host of metabolic processes in a temporally appropriate manner. The purpose of this article is to review current understanding of the interplay between circadian clocks and metabolism, in addition to the pathophysiologic consequences of disruption of this molecular mechanism, in terms of cardiometabolic disease development.
    The Journal of endocrinology. 06/2014;

Full-text (2 Sources)

Download
57 Downloads
Available from
May 28, 2014