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: 3.21). 04/2011; 35(7):1305-14. DOI: 10.1111/j.1530-0277.2011.01466.x
Source: PubMed


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.

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    • "To determine if these effects were also observed in vivo intestinal tissue from rats chronically administered alcohol (6 g/kg/day, 10 weeks) and that exhibited intestinal dysbiosis, intestinal hyperpermeability, endotoxemia , and alcoholic steatohepatitis [18] [79] were evaluated for levels of circadian proteins. Evaluation of PER2 protein levels in the duodenum and colon intestinal tissue from these alcohol-fed and control rats show a significant alcohol-induced elevation in intestinal PER2 protein [35]. Thus, both the Caco-2 and in vivo data support that alcohol stimulation of the circadian clock protein PER2 occurs at a time when intestinal permeability is elevated and may be an important factor in alcohol-induced gut leakiness. "
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    ABSTRACT: Chronic alcohol use can result in many pathological effects including alcoholic liver disease (ALD). While alcohol is necessary for the development of ALD, only 20-30% of alcoholics develop alcoholic steatohepatitis (ASH) with progressive liver disease leading to cirrhosis and liver failure (ALD). This suggests that while chronic alcohol consumption is necessary it is not sufficient to induce clinically relevant liver damage in the absence of a secondary risk factor. Studies in rodent models and alcoholic patients show that increased intestinal permeability to microbial products like endotoxin play a critical role in promoting liver inflammation in ALD pathogenesis. Therefore identifying mechanisms of alcohol-induced intestinal permeability is important in identifying mechanisms of ALD and for designing new avenues for therapy. Cyp2e1 is a cytochrome P450 enzyme that metabolizes alcohol has been shown to be upregulated by chronic alcohol use and to be a major source of oxidative stress and liver injury in alcoholics and in animal and in vitro models of chronic alcohol use. Because Cyp2e1 is also expressed in the intestine and is upregulated by chronic alcohol use, we hypothesized it could play a role in alcohol-induced intestinal hyperpermeability. Our in vitro studies with intestinal Caco-2 cells and in mice fed alcohol showed that circadian clock proteins CLOCK and PER2 are required for alcohol-induced permeability. We also showed that alcohol increases Cyp2e1 protein and activity but not mRNA in Caco-2 cells and that an inhibitor of oxidative stress or siRNA knockdown of Cyp2e1 prevents the increase in CLOCK or PER2 proteins and prevents alcohol-induced hyperpermeability. With our collaborators we have also shown that Cyp2e1 knockout mice are resistant to alcohol-induced gut leakiness and liver inflammation. Taken together our data support a novel Cyp2e1-circadian clock protein mechanism for alcohol-induced gut leakiness that could provide new avenues for therapy of ALD. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.
    10/2014; 3C:40-46. DOI:10.1016/j.redox.2014.10.002
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    • "Progress in understanding the mechanisms by which circadian rhythms influence GI health and disease has lagged, in part, due to a paucity of in vitro models with appropriate phenotypes and robust circadian rhythms (Hughes et al., 2009). For example, Caco-2 cells are a commonly studied human colorectal adenocarcinoma cell line that has recently been shown to exhibit circadian oscillations of circadian component PERIOD2 (PER2) (Ballesta et al., 2011; Swanson et al., 2011); however, the limited capacity of Caco-2 cells for differentiation and their dampened circadian oscillations over time might hinder extrapolation of in vitro results to functional GI tissue (Hughes et al., 2009). To our knowledge, a tissue culture system that overcomes these limitations has yet to be described. "
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    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; 7(9). DOI:10.1242/dmm.014399 · 4.97 Impact Factor
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    • "Second, the primary mechanism of DSS-induced colitis in rodents is impaired intestinal barrier integrity, and we have shown that circadian disruption exacerbates colitis in DSS-treated mice [9]. Third, our recent study [22] implicates circadian clock genes in the regulation of intestinal epithelial barrier integrity in vitro (i.e., Caco-2 monolayers, a human intestinal epithelial cell line used to model barrier function): siRNA knockdown of the canonical circadian genes Clock and Per2 blocks alcohol-induced increases in Caco-2 layer permeability [22]. However, direct in vivo evidence to support the hypothesis that disruption of circadian homeostasis alters intestinal barrier function is lacking. "
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    ABSTRACT: The circadian clock orchestrates temporal patterns of physiology and behavior relative to the environmental light:dark cycle by generating and organizing transcriptional and biochemical rhythms in cells and tissues throughout the body. Circadian clock genes have been shown to regulate the physiology and function of the gastrointestinal tract. Disruption of the intestinal epithelial barrier enables the translocation of proinflammatory bacterial products, such as endotoxin, across the intestinal wall and into systemic circulation; a process that has been linked to pathologic inflammatory states associated with metabolic, hepatic, cardiovascular and neurodegenerative diseases - many of which are commonly reported in shift workers. Here we report, for the first time, that circadian disorganization, using independent genetic and environmental strategies, increases permeability of the intestinal epithelial barrier (i.e., gut leakiness) in mice. Utilizing chronic alcohol consumption as a well-established model of induced intestinal hyperpermeability, we also found that both genetic and environmental circadian disruption promote alcohol-induced gut leakiness, endotoxemia and steatohepatitis, possibly through a mechanism involving the tight junction protein occludin. Circadian organization thus appears critical for the maintenance of intestinal barrier integrity, especially in the context of injurious agents, such as alcohol. Circadian disruption may therefore represent a previously unrecognized risk factor underlying the susceptibility to or development of alcoholic liver disease, as well as other conditions associated with intestinal hyperpermeability and an endotoxin-triggered inflammatory state.
    PLoS ONE 06/2013; 8(6):e67102. DOI:10.1371/journal.pone.0067102 · 3.23 Impact Factor
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