Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2016 Oct 1.
Published in final edited form as: Alcohol Clin Exp Res. 2015 Sep 2;39(10):1917–1929. doi: 10.1111/acer.12834

Chronic Alcohol Exposure and the Circadian Clock Mutation Exert Tissue-Specific Effects on Gene Expression in Mouse Hippocampus, Liver and Proximal Colon

Keith C Summa 1, Peng Jiang 1, Karrie Fitzpatrick 1, Robin M Voigt 2, Samuel J Bowers 1, Christopher B Forsyth 2,3, Martha Hotz Vitaterna 1, Ali Keshavarzian 2,3,4,5,6, Fred W Turek 1
PMCID: PMC4562391  NIHMSID: NIHMS707147  PMID: 26332085

Abstract

Background

Chronic alcohol exposure exerts numerous adverse effects, although the specific mechanisms underlying these negative effects on different tissues are not completely understood. Alcohol also affects core properties of the circadian clock system and it has been shown that disruption of circadian rhythms confers vulnerability to alcohol-induced pathology of the gastrointestinal barrier and liver. Despite these findings, little is known of the molecular interactions between alcohol and the circadian clock system, especially regarding implications for tissue-specific susceptibility to alcohol pathologies. The aim of the present study was to identify changes in expression of genes relevant to alcohol pathologies and circadian clock function in different tissues in response to chronic alcohol intake.

Methods

Wild-type and circadian ClockΔ19 mutant mice were subjected to a 10-week chronic alcohol protocol, after which hippocampal, liver and proximal colon tissues were harvested for gene expression analysis using a custom-designed multiplex magnetic bead hybridization assay that provided quantitative assessment of 80 mRNA targets of interest, including five housekeeping genes and a predetermined set of 75 genes relevant for alcohol pathology and circadian clock function.

Results

Significant alterations in expression levels attributable to genotype, alcohol and/or a genotype by alcohol interaction were observed in all three tissues, with distinct patterns of expression changes observed in each. Of particular interest was the finding that a high proportion of genes involved in inflammation and metabolism on the array was significantly affected by alcohol and the ClockΔ19 mutation in the hippocampus, suggesting a suite of molecular changes that may contribute to pathological change.

Conclusions

These results reveal the tissue-specific nature of gene expression responses to chronic alcohol exposure and the ClockΔ19 mutation, and identify specific expression profiles that may contribute to tissue-specific vulnerability to alcohol-induced injury in the brain, colon and liver.

Keywords: Circadian rhythms, Alcohol, Gene expression, Mouse models, Clock mutation

Introduction

Chronic alcohol consumption is associated with significant mortality and substantial medical, neurological and psychiatric co-morbidity, including steatohepatitis and cirrhosis, pancreatitis, cardiomyopathy, increased risk of hypertension, several cancers (e.g., head and neck, esophagus, colon, pancreas and liver), cognitive impairment, mood disorders and depression (Gao and Bataller, 2011, Kelley and Dantzer, 2011). Interestingly, not all alcoholics develop organ damage (Grant et al., 1988, Keshavarzian et al., 1999). Furthermore, of those alcoholics that do exhibit some degree of alcohol-related toxicity, not all organs are susceptible (Wang et al., 2010, Szabo and Bala, 2010). As such, we sought to understand tissue-specific responses to alcohol by examining three different tissues that are affected by alcohol: hippocampus, liver and colon. These tissues were studied because chronic alcohol exposure contributes to pathological damage and/or functional impairment in each.

The genes examined in each organ encompass mechanisms believed to contribute to the pathogenesis of alcohol-related tissue injury, including inflammation, metabolism, oxidative stress, cell-cell junction and epithelial permeability, brain-gut axis, cell division/cancer and circadian rhythms and circadian clock-controlled genes, to allow us to determine whether these organs affected by alcohol exhibit similar transcriptional profiles. Although broad associations between chronic alcohol consumption and subsequent biological responses have been made, the precise molecular mechanisms leading to alcohol-induced physiological dysfunction, tissue injury and organ dysfunction remain incompletely understood, and we sought to fill this gap in knowledge.

The circadian clock system represents a biological mechanism that may contribute to alcohol-induced tissue injury and organ damage because: (1) disruption of the circadian clock system promotes inflammation (Castanon-Cervantes et al., 2010), and (2) alcohol can impact the circadian clock in different organs (Chen et al., 2004, Spanagel et al., 2005b, Summa et al., 2013, Swanson et al., 2011). Studies using Caco-2 cells, a human epithelial colorectal adenocarcinoma cell line that serves as an in vitro model of intestinal barrier function, have demonstrated that circadian clock genes influence alcohol-induced increases in monolayer permeability: knock-down of the circadian genes Clock and Per2 prevents alcohol-induced increases in permeability, indicating that these genes respond to alcohol and impact the regulation of barrier integrity (Swanson et al., 2011). Additionally, our recent studies have shown that disruption of circadian organization in mice, using independent genetic and environmental strategies, exacerbates alcohol-induced intestinal hyperpermeability and hepatic pathology (Summa et al., 2013). Together, these results suggest complex relationships between the circadian clock system and biological responses to alcohol, however the mechanisms through which alcohol and the circadian system interact to promote tissue injury are not fully understood.

A number of studies have examined how alcohol impacts core properties of the circadian clock system, such as the free-running period, the phase of entrainment and the phase-shifting effects of exposure to light pulses (Brager et al., 2011a, Brager et al., 2010, Brager et al., 2011b, Rosenwasser et al., 2005). There has also been significant interest in identifying and cataloging diurnal and circadian gene expression rhythms, as well as identifying downstream targets and gene networks affected by the circadian clock in various mouse tissues and genetic models, albeit in the absence of alcohol (Miller et al., 2007, Panda et al., 2002, Storch et al., 2002). However, relatively little effort has been made in linking alcohol consumption and circadian rhythm disruption, especially in different genetic models of circadian disruption. Besides liver, other tissues and brain regions most vulnerable to alcohol-related damage have not been well-studied. Therefore not much is known regarding the specific molecular mechanisms underlying the impact of alcohol on circadian clock gene expression in different tissues or on the regulation of molecular/cellular pathways relevant to alcohol-related pathology in mouse models of circadian disruption.

We attempted to understand the relationships between alcohol, alcohol-related pathologies, the circadian clock, and the regulation of gene expression by examining expression patterns in the hippocampus, liver and colon of alcohol-fed or alcohol-free control diet-fed wild-type mice and circadian ClockΔ19 mutant mice (Clock mutant hereafter). Hippocampus, liver and colon were examined in the current study because chronic alcohol exposure contributes to pathological damage and/or functional impairment in each tissue. The results presented in this study were obtained from analyses of tissues collected from mice originally described in a previous publication reporting in vivo physiological measurements as well as post-mortem tissue analyses (Summa et al., 2013). Mutant mice harbor a dominant-negative mutation (Δ19) of the core circadian gene Clock (Clock mutation hereafter) (King et al., 1997a, King et al., 1997b, Vitaterna et al., 1994). Clock mutant mice were utilized as a model of circadian clock disruption to determine whether this particular genetic aberration of the clock affects the regulation of gene expression in response to chronic alcohol consumption in tissues susceptible to alcohol toxicity. These animals have been used widely to study the role of the circadian clock system in various physiological and pathophysiological processes (Naylor et al., 2000, Roybal et al., 2007, Summa et al., 2013, Turek et al., 2005).

We examined the expression of 75 genes (inflammation, metabolism, oxidative stress, cell-cell junction and epithelial permeability, brain-gut axis, cell division/cancer and circadian rhythms and circadian clock-controlled genes) and identified tissue-specific alterations in expression from each functional gene category. Distinct sets of genes were identified that were significantly affected by genotype, alcohol or genotype by alcohol interaction. Such alterations may contribute to pathological transformation in tissues affected by chronic alcohol exposure and may underlie, at least in part, tissue-specific vulnerability to alcohol toxicity and injury.

Materials and Methods

Laboratory Animals

Young adult (7-9 week old) male mice homozygous Clock mutant mice and wild-type littermate mice, obtained from the breeding colony maintained at Northwestern University, were individually housed in light-tight, ventilated cabinets and maintained on a constant 12 hours light:12 hours dark (12:12 LD) cycle at constant temperature and humidity, as described previously (Summa et al., 2013). Locomotor activity rhythms were measured as previously described using cages equipped with infrared beams that cross through the cage and record breaks in the beam as activity (Summa et al., 2013). Upon separation into individual cages, mice were assigned to either the alcohol experimental group or the alcohol-free, dextrose control group for the duration of the experimental protocol described below. Group selection was determined by randomly assigning an individual mouse of each genotype to each dietary group (i.e., alcohol or dextrose control), then matching subsequent animals by body weight such that there were no significant differences in body weight between groups at the onset of the experiment. The experimental design included four groups: 1) Wild-type dextrose diet (WTD), wild-type alcohol diet (WTA), Clock mutant dextrose diet (CD) and Clock mutant alcohol diet (CA). All studies were reviewed and approved in advance by the Institutional Animal Care and Use Committee at Northwestern University.

Experimental Protocol

The experiments utilized the Nanji liquid alcohol diet protocol as described previously (Nanji et al., 1994, Summa et al., 2013). This diet, used to study alcoholic steatohepatitis in rodents (Forsyth et al., 2011, Summa et al., 2013), is a modification of the Lieber-DiCarli rodent alcohol diet. The Nanji diet utilized in the present study is nutritionally balanced, prepared fresh daily and provided to the mice at Zeitgeber Time 4 (i.e., ZT4, four hours after light onset) in specialized individual feeding tubes that allow for daily monitoring of food intake, which was monitored by checking diet levels at light onset and light offset for one or two consecutive 24-hour periods each week of the experimental protocol. There was a two-week gradual introduction to alcohol in the diet (0-29% of total calories from alcohol), followed by eight weeks on the full alcohol-containing diet (29% of total calories from alcohol, 4.5% v/v).

All mice were euthanized by conscious decapitation and tissues were harvested at ZT6 for gene expression analyses. The hippocampus was dissected over dry ice. Isolated hippocampus, liver and proximal colon samples were immediately frozen in liquid nitrogen and stored at -80°C until RNA isolation and subsequent gene expression analysis.

The tissues were harvested for expression analysis at ZT6 because we observed that mice were reliably consuming relatively large portions of their daily diet intake during the light phase after placement of the freshly prepared diet into each animal's home cage daily at ZT4 (Supporting Online Information). Thus, collection at ZT6 was presumed to enable assessment of expression changes occurring near the onset of a predictable daily bout of drinking. In addition, we have data for serum alcohol levels at ZT6 (Summa et al., 2013) and preliminary data suggesting that intestinal permeability peaks near ZT0 (data not shown), therefore we felt that analyzing expression at ZT6 may incorporate biological responses occurring near the apparent peak in daily endotoxin levels.

Assessment and Analysis of Gene Expression

Gene expression analysis was performed at the University of Illinois-Chicago core facility using an Affymetrix (Santa Clara, CA) custom QuantiGene 80 Plex assay. Briefly, this assay provides quantitative measurement of 80 mRNA targets of interest on a single plate. The 80 genes measured in this study included five housekeeping genes and a set of 75 genes selected from nine functional categories, including inflammation, metabolism, oxidative stress, cell-junction and epithelial permeability, brain-gut axis, cell division/cancer and circadian rhythms and circadian clock-controlled genes (CCGs) (Table 1).

Table 1. Genes analyzed for expression levels using a custom-designed Affymetrix 80Plex bead chip.

The selected genes were chosen for analysis because of their classification in one of the following functional categories: circadian clock and clock-controlled genes, inflammation, metabolism, oxidative stress, cell-junction and epithelial permeability, brain-gut axis and cancer.

Gene Symbol Gene Name RefSeq No. Functional Category
Arntl aryl hydrocarbon receptor nuclear translocator-like NM_007489 clock and clock-controlled genes
Clock circadian locomotor output cycles kaput NM_007715 clock and clock-controlled genes
Creb1 cAMP responsive element binding protein 1 NM_133828 clock and clock-controlled genes
Cry1 cryptochrome 1 (photolyase-like) NM_007771 clock and clock-controlled genes
Cry2 cryptochrome 2 (photolyase-like) NM_009963 clock and clock-controlled genes
Csnk1e casein kinase 1, epsilon NM_013767 clock and clock-controlled genes
Dbp D site albumin promoter binding protein NM_016974 clock and clock-controlled genes
Hlf hepatic leukemia factor NM_172563 clock and clock-controlled genes
Hnf1a HNF1 homeobox A NM_009327 clock and clock-controlled genes
Hnf4a hepatic nuclear factor 4, alpha NM_008261 clock and clock-controlled genes
Nampt nicotinamide phosphoribosyltransferase NM_021524 clock and clock-controlled genes
Nfil3 nuclear factor, interleukin 3, regulated NM_017373 clock and clock-controlled genes
Npas2 neuronal PAS domain protein 2 NM_008719 clock and clock-controlled genes
Nr1d1 nuclear receptor subfamily 1, group D, member 1 NM_145434 clock and clock-controlled genes
Per1 period 1 NM_011065 clock and clock-controlled genes
Per2 period 2 NM_011066 clock and clock-controlled genes
Per3 period 3 NM_011067 clock and clock-controlled genes
Prkab1 protein kinase, AMP-activated, beta 1 non-catalytic subunit NM_031869 clock and clock-controlled genes
Rora RAR-related orphan receptor alpha NM_013646 clock and clock-controlled genes
Sirt1 sirtuin 1 NM_019812 clock and clock-controlled genes
Tef thyrotrophic embryonic factor NM_017376 clock and clock-controlled genes
Akp3 alkaline phosphatase 3, intestine, not Mn requiring NM_007432 inflammation
Ccl2 chemokine (C-C motif) ligand 2 NM_011333 inflammation
Cxcl1 chemokine (C-X-C motif) ligand 1 NM_008176 inflammation
Cxcl10 chemokine (C-X-C motif) ligand 10 NM_021274 inflammation
Hmgb1 high mobility group box 1 NM_010439 inflammation
Il10 interleukin 10 NM_010548 inflammation
Il17a interleukin 17A NM_010552 inflammation
Il18 interleukin 18 NM_008360 inflammation
Il1b interleukin 1 beta NM_008361 inflammation
Il6 interleukin 6 NM_031168 inflammation
Lbp lipopolysaccharide binding protein NM_008489 inflammation
Nlrp3 NLR family, pyrin domain containing 3 NM_145827 inflammation
Pde4b phosphodiesterase 4B, cAMP specific NM_019840 inflammation
Ptgs2 prostaglandin-endoperoxide synthase 2 NM_011198 inflammation
Rela v-rel reticuloendotheliosis viral oncogene homolog A (avian) NM_009045 inflammation
Tlr2 toll-like receptor 2 NM_011905 inflammation
Tlr4 toll-like receptor 4 NM_021297 inflammation
Tlr5 toll-like receptor 5 NM_016928 inflammation
Tlr9 toll-like receptor 9 NM_031178 inflammation
Tnf tumor necrosis factor NM_013693 inflammation
Adipoq adiponectin, C1Q and collagen domain containing NM_009605 metabolism
Ccrn4l CCR4 carbon catabolite repression 4-like (S. cerevisiae) NM_009834 metabolism
Cyp7a1 cytochrome P450, family 7, subfamily a, polypeptide 1 NM_007824 metabolism
Hmgcr 3-hydroxy-3-methylglutaryl-Coenzyme A reductase NM_008255 metabolism
Lep leptin NM_008493 metabolism
Nr1h3 nuclear receptor subfamily 1, group H, member 3 NM_013839 metabolism
Nr1h4 nuclear receptor subfamily 1, group H, member 4 NM_009108 metabolism
Ppara peroxisome proliferator activated receptor alpha NM_011144 metabolism
Pparg peroxisome proliferator-activated receptor gamma NM_011146 metabolism
Ppargc1a peroxisome proliferative activated receptor, gamma, coactivator 1 alpha NM_008904 metabolism
Prkcd protein kinase C, delta NM_011103 metabolism
Srebf1 sterol regulatory element binding transcription factor 1 NM_011480 metabolism
Cyp2e1 cytochrome P450, family 2, subfamily e, polypeptide 1 NM_021282 oxidative stress
Gpx2 glutathione peroxidase 2 NM_030677 oxidative stress
Nfe2l2 nuclear factor, erythroid derived 2, like 2 NM_010902 oxidative stress
Nos1 nitric oxide synthase 1, neuronal NM_008712 oxidative stress
Nos2 nitric oxide synthase 2, inducible NM_010927 oxidative stress
Nox1 NADPH oxidase 1 NM_172203 oxidative stress
Ucp2 uncoupling protein 2 (mitochondrial, proton carrier) NM_011671 oxidative stress
Cdh1 cadherin 1, type 1, E-cadherin (epithelial) NM_009864 cell-junction and epithelial permeability
Cldn1 claudin 1 NM_016674 cell-junction and epithelial permeability
Cldn2 claudin 2 NM_016675 cell-junction and epithelial permeability
Cldn4 claudin 4 NM_009903 cell-junction and epithelial permeability
Ocln occludin NM_008756 cell-junction and epithelial permeability
Tjp1 tight junction protein 1 NM_009386 cell-junction and epithelial permeability
Fos FBJ osteosarcoma oncogene NM_010234 brain-gut axis
Gfap glial fibrillary acidic protein NM_010277 brain-gut axis
Npy neuropeptide Y NM_023456 brain-gut axis
Vip vasoactive intestinal polypeptide NM_011702 brain-gut axis
Ccnd1 cyclin D1 NM_007631 cancer
Ctnnb1 catenin (cadherin associated protein), beta 1 NM_007614 cancer
Hif1a hypoxia inducible factor 1, alpha subunit NM_010431 cancer
Mmp9 matrix metallopeptidase 9 NM_013599 cancer
Tgfb1 transforming growth factor, beta 1 NM_011577 cancer
Open in a new tab

Hippocampus, liver and proximal colon samples were prepared as tissue homogenates in our laboratory using Affymetrix lysis buffer and processed according to the manufacturer's instructions. Raw intensity readings of RNA amount were obtained for subsequent statistical analyses. These values were processed and analyzed as described in the Supporting Online Information. In order to validate results obtained from the analysis of the array expression data, we performed RT-PCR on Tlr9 in the hippocampus, Tlr5 in the liver and Cdh1 in the proximal colon, as described in the Supporting Online Information.

Results

Hippocampus

In total, 56 genes exhibited significantly altered expression patterns in the hippocampus for at least one term (genotype, alcohol or genotype by alcohol interaction) among experimental groups. These genes group into four clusters (i.e., orange, blue, pink and green) based on the similarity of expression changes (Table 2, Figure 1).

Table 2. Genes that varied significantly across experimental groups in the hippocampus.

Genes are grouped into clusters, as shown in Figure 1.

Gene Symbol Functional Category 2-way ANOVA Clusters
Genotype Diet G × D interaction
Ocln cell-junction and epithelial permeability F1,14 = 6.32, P = 0.0248 n.s. n.s. orange
Il18 inflammation F1,14 = 6.52, P = 0.023 n.s. n.s. orange
Npy brain-gut axis F1,14 = 36.01, P = 3.25E-05 n.s. n.s. orange
Dbp clock and clock-controlled genes F1,14 = 311.04, P = 5.87E-11 n.s. n.s. orange
Cry2 clock and clock-controlled genes F1,14 = 125.28, P = 2.27E-08 n.s. n.s. orange
Nr1d1 clock and clock-controlled genes F1,14 = 141.13, P = 1.06E-08 n.s. n.s. orange
Per1 clock and clock-controlled genes F1,14 = 30.34, P = 7.71E-05 n.s. n.s. orange
Tef clock and clock-controlled genes F1,14 = 100.31, P = 9.17E-08 n.s. n.s. orange
Per3 clock and clock-controlled genes F1,14 = 266.57, P = 1.65E-10 n.s. n.s. orange
Per2 clock and clock-controlled genes F1,14 = 36.92, P = 2.86E-05 F1,14 = 6.75, P = 0.021 n.s. orange
Hlf clock and clock-controlled genes F1,14 = 34.36, P = 4.13E-05 n.s. F1,14 = 15.23, P = 0.0016 orange
Nampt clock and clock-controlled genes F1,14 = 13.72, P = 0.00236 n.s. n.s. orange
Hif1a cancer F1,14 = 18.82, P = 0.000681 n.s. n.s. orange
Sirt1 clock and clock-controlled genes F1,14 = 6.1, P = 0.027 n.s. n.s. orange
Hmgb1 inflammation F1,14 = 8.09, P = 0.013 n.s. n.s. orange
Rela inflammation F1,14 = 4.86, P = 0.0448 n.s. n.s. orange
Nfe2l2 oxidative stress F1,14 = 13.75, P = 0.00234 F1,14 = 18.66, P = 0.000707 n.s. orange
Clock clock and clock-controlled genes F1,14 = 15.64, P = 0.00144 F1,14 = 16.02, P = 0.00131 n.s. orange
Vip brain-gut axis n.s. n.s. F1,14 = 6.41, P = 0.0239 pink
Tlr4 inflammation n.s. F1,14 = 13.37, P = 0.00259 n.s. blue
Cxcl1 inflammation n.s. F1,14 = 10.94, P = 0.00519 n.s. blue
Ppara metabolism F1,14 = 17.61, P = 0.000897 F1,14 = 14.92, P = 0.00172 n.s. green
Creb1 clock and clock-controlled genes n.s. F1,14 = 6.69, P = 0.0216 n.s. green
Mmp9 cancer F1,14 = 12.86, P = 0.00298 n.s. n.s. green
Pparg metabolism F1,14 = 12.19, P = 0.0036 F1,14 = 10.58, P = 0.00578 n.s. green
Lep metabolism F1,14 = 12.75, P = 0.00307 F1,14 = 8.95, P = 0.00971 n.s. green
Cldn4 cell-junction and epithelial permeability F1,14 = 22.34, P = 0.000325 F1,14 = 11.38, P = 0.00455 n.s. green
Tlr9 inflammation F1,14 = 16.83, P = 0.00108 F1,14 = 13.97, P = 0.00221 n.s. green
Il17a inflammation F1,14 = 9.4, P = 0.00837 F1,14 = 6.36, P = 0.0244 n.s. green
Cxcl10 inflammation F1,14 = 19.39, P = 0.000601 F1,14 = 16.4, P = 0.00119 n.s. green
Hnf4a clock and clock-controlled genes F1,14 = 7.98, P = 0.0135 n.s. n.s. green
Nlrp3 inflammation F1,14 = 16.19, P = 0.00126 F1,14 = 6.77, P = 0.0209 n.s. green
Adipoq metabolism F1,14 = 29.65, P = 8.63E-05 F1,14 = 17.33, P = 0.000956 n.s. green
Tlr5 inflammation F1,14 = 13.7, P = 0.00237 F1,14 = 10.68, P = 0.00561 n.s. green
Akp3 inflammation F1,14 = 24.75, P = 0.000204 F1,14 = 12.93, P = 0.00293 n.s. green
Nox1 oxidative stress F1,14 = 29.38, P = 9.02E-05 F1,14 = 12.36, P = 0.00343 n.s. green
Nfil3 clock and clock-controlled genes F1,14 = 36.41, P = 3.07E-05 F1,14 = 10.29, P = 0.00632 n.s. green
Tlr2 inflammation F1,14 = 18.58, P = 0.000718 F1,14 = 8.78, P = 0.0103 n.s. green
Hnf1a clock and clock-controlled genes F1,14 = 8.04, P = 0.0132 F1,14 = 5.1, P = 0.0403 n.s. green
Il10 inflammation F1,14 = 21.07, P = 0.00042 F1,14 = 9.42, P = 0.00832 n.s. green
Nr1h4 metabolism F1,14 = 4.78, P = 0.0463 n.s. n.s. green
Fos brain-gut axis F1,14 = 8.91, P = 0.00984 F1,14 = 10.94, P = 0.00518 n.s. green
Npas2 clock and clock-controlled genes F1,14 = 5.07, P = 0.0409 n.s. n.s. green
Cyp2e1 oxidative stress F1,14 = 12.16, P = 0.00362 F1,14 = 9.26, P = 0.00877 F1,14 = 4.66, P = 0.0487 green
Tnf inflammation F1,14 = 8.02, P = 0.0133 F1,14 = 5.85, P = 0.0298 n.s. green
Il1b inflammation n.s. F1,14 = 7.2, P = 0.0179 n.s. green
Cry1 clock and clock-controlled genes n.s. F1,14 = 16.52, P = 0.00116 n.s. green
Cyp7a1 metabolism F1,14 = 5.84, P = 0.0299 F1,14 = 7.19, P = 0.0179 F1,14 = 6.71, P = 0.0214 green
Gpx2 oxidative stress n.s. F1,14 = 5.66, P = 0.0321 n.s. green
Tgfb1 cancer n.s. F1,14 = 11.5, P = 0.00439 n.s. green
Nr1h3 metabolism F1,14 = 6.01, P = 0.028 F1,14 = 32.14, P = 5.79E-05 n.s. green
Il6 inflammation n.s. F1,14 = 33.53, P = 4.68E-05 n.s. green
Srebf1 metabolism n.s. F1,14 = 7.03, P = 0.019 n.s. green
Nos2 oxidative stress F1,14 = 4.9, P = 0.0439 n.s. n.s. green
Arntl clock and clock-controlled genes F1,14 = 11.51, P = 0.00437 n.s. n.s. green
Ptgs2 inflammation F1,14 = 12.17, P = 0.00362 F1,14 = 5.06, P = 0.0412 n.s. green
Open in a new tab

Figure 1. Hippocampal gene expression levels in wild-type (WT) and Clock mutant (C) mice on either an alcohol (A) or dextrose-containing control (D) diet.

Figure 1

Open in a new tab

Expression levels of genes exhibiting significant changes in the hippocampus for at least one term (genotype, alcohol or genotype by alcohol interaction) were normalized as Z scores and plotted as a heatmap. Genes with similar expression patterns were grouped into clusters, depicted by colors on the left side of the graph. The orange cluster reflects groups of genes influenced by genotype. The blue cluster includes genes affected by diet. The pink cluster contains a single gene, Vip, that is characterized by a genotype by diet interaction. The green cluster contains genes affected by genotype as well as alcohol. Cluster color was assigned independently and randomly in each tissue, thus there is no implied association between clusters of the same color in different tissues. Gene symbols are listed on the right side. There were four experimental groups: wild-type dextrose (WTD), wild-type alcohol (WTA), Clock mutant dextrose (CD) and Clock mutant alcohol (CA). Each column represents an individual mouse, denoted by the ID number listed after the experimental group designation. Within the heatmap, green indicates increased expression and red indicates reduced expression, with color intensity reflecting the magnitude of the expression change.

Genotype

Genes in the orange cluster were predominantly influenced by genotype. This cluster includes 18 genes, the expression levels of which were lower in Clock mutants compared to wild-type. Notably, several of these genes (i.e., Per1, Per2, Per3 and Cry2, among others) are targets of the transactivating CLOCK-ARTNL dimer, perhaps revealing reduced activation of expression by the mutated Clock gene. Another gene in this cluster is Clock itself, as its expression is affected by the genotype at the Clock locus.

Diet

The blue cluster was small, containing only two genes: Tlr4 and Cxcl1. Alcohol led to increased expression of both of these genes in the hippocampus.

Genotype*Diet

The pink cluster contained a single gene, Vip, and was characterized by a significant genotype by diet interaction. The green cluster was largest in the hippocampus, consisting of 35 genes. The majority of these genes were affected by both genotype and diet. Highest expression levels were typically observed in the alcohol-fed Clock mutant mice compared to all other experimental groups. Expression of the Cyp2e1 gene, which encodes an enzyme important for the metabolism of alcohol, was influenced by a genotype by alcohol interaction, in addition to significant effects of both genotype and diet alone. This increase in Cyp2e1 may be related to alcohol-induced changes in other circadian genes, as we have previously shown in the intestine (Forsyth et al., 2011). Among the entire set of 75 genes examined, 20 were selected because of a functional role in the regulation of inflammation. Twelve of these 20 genes (60%) were significantly altered in the hippocampus and grouped together into the green cluster, with increased expression in alcohol-fed Clock mutants, suggesting a trend for enrichment of inflammation-related genes in this cluster (odds ratio=2.066, P=0.128, Fisher's exact test). A high proportion of the genes involved in metabolism grouped in the green cluster as well, with 8 of 12 selected metabolism-related genes (67%) being present in this cluster (odds ratio=2.632, P=0.115, Fisher's exact test).

Liver

Fewer genes (22 in total) significantly varied across experimental groups in the liver (Table 3), compared to 56 in the hippocampus (Table 2) and 30 in the proximal colon (Table 4). In the liver, those genes with significant expression changes grouped into five clusters (i.e., cyan, green, blue, orange and pink; Table 3, Figure 2).

Table 3. Genes that varied significantly across experimental groups in the liver.

Genes are grouped into clusters, as shown in Figure 2.

Gene Symbol Functional Category 2-way ANOVA Clusters
Genotype Diet G × D interaction
Ccnd1 cancer n.s. F1,13 = 6.16, P = 0.0275 n.s. cyan
Hmgb1 inflammation n.s. F1,13 = 4.72, P = 0.0488 n.s. cyan
Per3 clock and clock-controlled genes F1,13 = 41.56, P = 2.18E-05 n.s. n.s. green
Dbp clock and clock-controlled genes F1,13 = 76.41, P = 8.37E-07 n.s. n.s. green
Tef clock and clock-controlled genes F1,13 = 80.18, P = 6.38E-07 n.s. n.s. green
Per2 clock and clock-controlled genes F1,13 = 10.5, P = 0.00644 n.s. n.s. green
Per1 clock and clock-controlled genes F1,13 = 18.04, P = 0.000953 n.s. n.s. green
Nampt clock and clock-controlled genes F1,13 = 17.32, P = 0.00112 n.s. n.s. green
Nr1d1 clock and clock-controlled genes F1,13 = 27.48, P = 0.000159 n.s. n.s. green
Cry2 clock and clock-controlled genes F1,13 = 8.03, P = 0.0141 n.s. n.s. green
Cdh1 cell-junction and epithelial permeability F1,13 = 9.54, P = 0.00862 n.s. n.s. green
Clock clock and clock-controlled genes F1,13 = 10.05, P = 0.00738 n.s. n.s. green
Nos2 oxidative stress n.s. n.s. F1,13 = 5.76, P = 0.0321 blue
Nfil3 clock and clock-controlled genes F1,13 = 62.1, P = 2.64E-06 n.s. n.s. orange
Arntl clock and clock-controlled genes F1,13 = 25.47, P = 0.000224 n.s. n.s. orange
Cry1 clock and clock-controlled genes F1,13 = 127.62, P = 4.3E-08 n.s. n.s. orange
Pde4b inflammation F1,13 = 6.57, P = 0.0236 n.s. n.s. orange
Cldn1 cell-junction and epithelial permeability n.s. n.s. F1,13 = 6.02, P = 0.029 orange
Pparg metabolism F1,13 = 7.91, P = 0.0147 n.s. n.s. orange
Tlr5 inflammation F1,13 = 8.24, P = 0.0131 n.s. n.s. orange
Creb1 clock and clock-controlled genes F1,13 = 4.89, P = 0.0456 n.s. n.s. orange
Hnf4a clock and clock-controlled genes n.s. F1,13 = 5.17, P = 0.0406 n.s. pink
Open in a new tab

Table 4. Genes that varied significantly across experimental groups in the proximal colon.

Genes are grouped into clusters, as shown in Figure 3.

Gene Symbol Functional Category 2-way ANOVA Clusters
Genotype Diet G × D interaction
Rela inflammation F1,16 = 7.01, P = 0.0176 n.s. n.s. cyan
Vip brain-gut axis F1,16 = 6.9, P = 0.0183 n.s. n.s. cyan
Nox1 oxidative stress F1,16 = 12.18, P = 0.00303 n.s. n.s. cyan
Srebf1 metabolism F1,16 = 20.28, P = 0.000361 n.s. n.s. cyan
Mmp9 cancer F1,16 = 8.8, P = 0.00909 n.s. n.s. cyan
Tnf inflammation F1,16 = 9.83, P = 0.00639 F1,16 = 7.84, P = 0.0128 F1,16 = 4.73, P = 0.045 orange
Cxcl10 inflammation F1,16 = 5.95, P = 0.0268 F1,16 = 5.61, P = 0.0308 n.s. orange
Fos brain-gut axis F1,16 = 4.53, P = 0.0493 n.s. n.s. green
Cry1 clock and clock-controlled genes F1,16 = 16.25, P = 0.000967 n.s. n.s. green
Prkcd metabolism F1,16 = 9.03, P = 0.00838 F1,16 = 4.87, P = 0.0423 n.s. green
Hif1a cancer F1,16 = 7.75, P = 0.0133 n.s. n.s. green
Cldn1 cell-junction and epithelial permeability F1,16 = 6.26, P = 0.0235 n.s. n.s. green
Ctnnb1 cancer F1,16 = 6.03, P = 0.0259 n.s. F1,16 = 4.84, P = 0.0429 purple
Cdh1 cell-junction and epithelial permeability F1,16 = 12.03, P = 0.00316 n.s. n.s. purple
Hmgcr metabolism F1,16 = 11.48, P = 0.00375 n.s. n.s. purple
Nfil3 clock and clock-controlled genes F1,16 = 16.76, P = 0.000848 n.s. n.s. purple
Arntl clock and clock-controlled genes F1,16 = 110.22, P = 1.39E-08 n.s. n.s. purple
Hnf1a clock and clock-controlled genes n.s. n.s. F1,16 = 12.02, P = 0.00318 pink
Ocln cell-junction and epithelial permeability n.s. n.s. F1,16 = 5.51, P = 0.0321 pink
Il17a inflammation n.s. n.s. F1,16 = 5.53, P = 0.0318 yellow
Lep metabolism n.s. n.s. F1,16 = 5.22, P = 0.0363 yellow
Cxcl1 inflammation n.s. n.s. F1,16 = 4.53, P = 0.0492 yellow
Akp3 inflammation n.s. F1,16 = 8.79, P = 0.00912 n.s. yellow
Clock clock and clock-controlled genes F1,16 = 9.37, P = 0.00746 n.s. n.s. yellow
Ccl2 Inflammation n.s. n.s. F1,16 = 4.6, P = 0.0477 yellow
Per3 clock and clock-controlled genes F1,16 = 17.39, P = 0.000722 n.s. n.s. blue
Dbp clock and clock-controlled genes F1,16 = 23.98, P = 0.000161 n.s. n.s. blue
Tef clock and clock-controlled genes F1,16 = 17.54, P = 0.000696 n.s. n.s. blue
Cry2 clock and clock-controlled genes F1,16 = 18.4, P = 0.000562 n.s. n.s. blue
Nr1d1 clock and clock-controlled genes F1,16 = 28.07, P = 7.22E-05 n.s. n.s. blue
Open in a new tab

Figure 2. Liver gene expression levels in wild-type (WT) and Clock mutant (C) mice on either an alcohol (A) or dextrose-containing control (D) diet.

Figure 2

Open in a new tab

Expression levels of genes exhibiting significant changes for at least one term (genotype, alcohol or genotype by alcohol interaction) in the liver were normalized as Z scores and plotted as a heatmap. Genes with similar expression patterns were grouped into clusters, depicted by colors on the left side of the graph. The green and orange clusters include genes influenced by genotype. The pink and cyan clusters include genes affected by diet. The blue cluster contains a single gene influenced by a genotype by diet interaction (Nos2). Cluster color was assigned independently and randomly in each tissue, thus there is no implied association between clusters of the same color in different tissues. Gene symbols are listed on the right side. There were four experimental groups: wild-type dextrose (WTD), wild-type alcohol (WTA), Clock mutant dextrose (CD) and Clock mutant alcohol (CA). Each column represents an individual mouse, denoted by the ID number listed after the experimental group designation. Within the heatmap, green indicates increased expression and red indicates reduced expression, with color intensity reflecting the magnitude of the expression change.

Genotype

Genes significantly influenced by genotype grouped into two clusters, with opposing directions of impact. In the green cluster, nine circadian clock genes or CCGs (including the Pers, Tef, Dbp and Nampt), as well as one cell-cell junction related gene (Cdh1), were decreased in Clock mutant mice, while in the orange cluster four other clock genes or CCGs (including Arntl and Cry1), two inflammation genes (Pde4b and Tlr5), one metabolism gene (Pparg) and one cell-cell junction related gene (Cldn1) were increased.

Diet

Both genes in the cyan cluster and the only gene in the pink cluster were significantly affected by alcohol, although the direction of the effect differed: in the pink cluster, alcohol resulted in increased expression of the circadian clock controlled gene Hnf4a, whereas it decreased expression of the inflammation-related gene Hmgb1 and the cancer-related gene Ccnd1 (cyan cluster).

Genotype*Diet

The blue cluster contained only the oxidative stress-related gene, Nos2. Its expression was significantly affected by a genotype by alcohol interaction: expression levels increased in wild-type mice with alcohol, but decreased in alcohol-fed Clock mutants.

Proximal Colon

A total of 30 genes were significantly affected by genotype, diet and/or the interaction between the two in the proximal colon (Table 4). These genes grouped into seven clusters (i.e., cyan, orange, green, purple, pink, yellow and blue; Table 4, Figure 3), which revealed distinct effects on gene expression by alcohol and the Clock mutation.

Figure 3. Proximal colon expression levels in wild-type (WT) and Clock mutant (C) mice on either an alcohol (A) or dextrose-containing control (D) diet.

Figure 3

Open in a new tab

Expression levels of genes exhibiting significant changes for at least one term (genotype, alcohol or genotype by alcohol interaction) in the proximal colon were normalized as Z scores and plotted as a heatmap. Genes with similar expression patterns were grouped into clusters, depicted by colors on the left side of the graph. The cyan, blue, green and purple clusters contain genes influenced by genotype. The orange cluster includes genes significantly affected by diet. The pink, orange and yellow clusters contain genes impacted by a genotype by alcohol interaction. Cluster color was assigned independently and randomly in each tissue, thus there is no implied association between clusters of the same color in different tissues. Gene symbols are listed on the right side. There were four experimental groups: wild-type dextrose (WTD), wild-type alcohol (WTA), Clock mutant dextrose (CD) and Clock mutant alcohol (CA). Each column represents an individual mouse, denoted by the ID number listed after the experimental group designation. Within the heatmap, green indicates increased expression and red indicates reduced expression, with color intensity reflecting the magnitude of the expression change.

Genotype

Genes in the cyan, green and purple clusters contained high proportions of genes affected by genotype, with higher expression levels in Clock mutants, regardless of diet. These three clusters include genes belonging to several functional groups, including metabolism (Srebf1 and Prkcd), cell division/cancer (Mmp9), oxidative stress (Nox1), brain-gut axis (Vip) and cell-cell junction and epithelial permeability (Cldn1 and Cdh1). The blue cluster, which included five genes, showed a significant genotype effect in the opposite direction, with low expression levels in Clock mutants regardless of diet. All of the genes in this cluster are under direct control of the CLOCK-ARNTL transcriptional machinery.

Diet

Only four genes were significantly affected by alcohol, including two inflammation-related genes from the orange cluster (Tnf and Cxcl10), one metabolism gene from the green cluster (Prkcd) and one inflammation-related gene from the yellow cluster (Akp3).

Genotype*Diet

Several genes in the orange, pink and yellow clusters exhibited complex responses to alcohol and the Clock mutation, with a high proportion being significantly affected by a genotype by alcohol interaction. For the pink cluster, higher expression levels tended to be present in the alcohol-fed Clock mutants, whereas in the orange and yellow clusters lower levels were observed.

The two genes of the pink cluster exhibited a significant genotype by alcohol interaction. Hnf1a belongs to the circadian clock/circadian clock controlled gene category and Ocln is an epithelial permeability-related gene. Expression levels were reduced by alcohol in wild-type mice and increased by alcohol in Clock mutants.

Four of the six genes in the yellow cluster were significantly affected by a genotype by alcohol interaction. These genes belong to the functional categories of inflammation (Il17a, Cxcl1 and Ccl2) and metabolism (Lep). Generally, expression was slightly increased by alcohol in the wild-type mice but reduced by alcohol in Clock mutants. The two remaining genes in the yellow cluster include an inflammation-related gene that was significantly affected by diet (Akp3) and the circadian gene Clock, which was significantly affected by genotype, indicating cis regulation of expression at the Clock locus.

Both genes in the orange cluster, which are inflammation-related genes, were affected by genotype alone and by diet alone. In addition, the gene Tnf was affected by a genotype by alcohol interaction: expression levels were not affected by alcohol in wild-type mice, but alcohol led to reduced expression in the Clock mutants.

RT-PCR Validation

In order to validate results obtained from the analysis of the array expression data, we performed RT-PCR on Tlr9 in the hippocampus, Tlr5 in the liver and Cdh1 in the proximal colon. In each tissue, expression patterns in different groups observed with RT-PCR generally matched those detected by the array (i.e., the directions of expression changes between groups in the RT-PCR data were consistent with those observed in the analysis of the array data). Although the directions of expression patterns seen in the RT-PCR studies were qualitatively similar to those of the array, the differences between groups failed to reach statistical significance, likely due to the small number of samples available for validation studies (Supporting Information Online).

Discussion

Alterations in expression levels attributed to genotype (i.e., Clock mutant vs. wild-type), alcohol (i.e., alcohol vs. dextrose control) and/or a genotype by alcohol interaction were observed in all three tissues, with differing patterns of effect and little overlap observed in each (Figure 4). Of the 75 genes examined, 38 were significantly altered by alcohol in at least one tissue. None of these genes were altered in three tissues simultaneously, and only two were shared across more than one tissue: Tnf and Cxcl10 were both significantly affected in the hippocampus and proximal colon (Figure 4A). A total of 56 genes were significantly altered by the Clock mutation in at least one tissue. Eight genes, all of which are involved in the regulation of circadian rhythms or of rhythmic gene expression, were affected in all three tissues and 16 of the remaining 48 genes were affected in two tissues (Figure 4B). Thus, only 43% (24/56) of the genes significantly affected by the mutation were affected in multiple tissues despite the presence of the mutation in all cells and tissues of the body.

Figure 4. Alcohol (A) and the circadian ClockΔ19 mutation (B) exert tissue-specific effects on gene expression in the mouse hippocampus, liver and proximal colon.

Figure 4

Open in a new tab

(A) Venn diagram listing genes significantly affected by alcohol in the hippocampus (blue circle), liver (red circle) and proximal colon (green circle) in this study. There was very little overlap of significantly affected genes across tissues. (B) Venn diagram listing genes significantly affected by the circadian ClockΔ19 mutation in the hippocampus (blue circle), liver (red circle) and proximal colon (green circle) in this study.

Interestingly, there were no genes significantly affected by alcohol in more all three tissues simultaneously and only two were affected in more than one tissue, which suggests that tissues exhibit distinct gene expression response patterns in the presence of alcohol, at least with respect to the genes analyzed in the present study. In contrast, several genes involved or associated with circadian rhythms were significantly affected by the Clock mutation in multiple tissues, which supports a molecular circadian clock mechanism that is conserved across tissues, although we do note that the majority of genes examined did exhibit tissue-specificity. Such prominent tissue-specific responses to alcohol and, to a lesser degree, circadian disruption, at least in the case of the Clock mutation, may contribute to tissue-specific susceptibility to alcohol toxicity.

In the present study, the hippocampus contained the most genes significantly affected by alcohol, compared to liver and proximal colon, and genes in all categories (circadian rhythms, metabolism, oxidative stress, inflammation, brain-gut axis, cell division/cancer, cell-cell junction and intestinal permeability) were affected. The master circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus was not examined because SCN expression responses to alcohol have been studied previously (Filiano et al., 2013) and the primary purpose of the current study was to determine whether expression changes and/or genetic clock disruption in different tissues contribute to alcohol toxicity, not to assess the impact of alcohol on the central circadian timekeeping system. Interestingly, a large proportion of the genes (the green cluster) were significantly affected by both genotype and alcohol in the hippocampus. A high proportion of the genes involved in inflammation and metabolism present on the array were present in this cluster (60% and 67%, respectively), suggesting that the circadian clock and alcohol combine to influence the regulation of inflammatory and metabolic gene expression in the hippocampus.

Given recent evidence linking neuroinflammation to alcohol-induced cognitive dysfunction and brain damage (Kelley and Dantzer, 2011), our results suggest that disruption of circadian rhythms may contribute to the development of cognitive impairment and neurological damage associated with chronic alcoholism through up-regulation of proinflammatory and metabolic gene expression. Furthermore, the alcohol-metabolizing enzyme Cyp2e1, which contributes to the generation of reactive oxygen species (ROS), is significantly affected by a genotype by alcohol interaction, with the highest expression levels observed in Clock mutants on the alcohol-containing diet. This implies that the combination of genetic circadian disruption and chronic alcohol exposure interacts to activate Cyp2e1 expression, which may promote or exacerbate pathological transformation through increased oxidative stress burden.

The fewest number of significantly affected genes were observed in the liver, as compared to the hippocampus and proximal colon. Of these, the majority belonged to the functional group of circadian clock and clock-controlled genes, and most were significantly affected by genotype (Table 3). These results suggest a particularly strong role for the circadian clock in regulating gene expression in the liver, which is known to exhibit robust rhythms of gene expression and physiology (Hughes et al., 2009, Lamia et al., 2008). Indeed, it has been reported that diurnal rhythms of expression of core circadian clock genes and certain metabolic genes are altered in mice exposed to a chronic alcohol consumption protocol (Filiano et al., 2013).

There are several potential reasons that we did not observe greater differential expression in the liver, including: the number and specific functions of genes included in the assay, the duration of the experimental protocol, the possible effects of feeding rhythms on liver gene expression (Vollmers et al., 2009), especially considering the observed changes in diurnal activity rhythms we observed in the present study (Supporting Information), the limitation of gene expression analysis to a single time point and considerations regarding how gene expression changes translate into changes in protein level or function. Interestingly, bone marrow-derived macrophages (BMDMs) from Clock mutant mice are less responsive to LPS or TNF and exhibit reduced expression of inflammatory, metabolic and circadian genes (Bellet et al., 2013). The Clock mutation may suppress gene expression to a relatively large degree in specific cell types and tissues, perhaps explaining, at least in part, the relative lack of robust, significant changes in expression in the liver observed in this study.

The results of the current study and findings described by Bellet et al. (Bellet et al., 2013), may also explain our previously reported finding that alcohol-fed Clock mutant mice failed to exhibit significant liver inflammation or liver cell injury despite increased intestinal permeability and endotoxin levels (Summa et al., 2013). However, these potential limitations seem unlikely to completely explain the findings in the liver because of the significant interaction between alcohol and the circadian clock system in the hippocampus and intestine. To clarify these potential issues, further studies are required to more completely characterize and understand the expression changes in the liver in response to circadian disruption and alcohol.

Within the proximal colon, the majority of genes were significantly affected by genotype. Significantly affected genes belonged to each of functional categories examined (Table 4). The expression of the gene encoding the tight junction protein Ocln (occludin) was decreased by alcohol in wild-type mice, which likely contributes to increased alcohol-induced gut leakiness, as we have shown in these mice (Summa et al., 2013). Another example that may impact intestinal barrier integrity is increased expression of the gene encoding the tight junction protein Cldn1 (claudin-1), which was increased in Clock mutant mice. Cldn1 encodes a tight junction protein that has paradoxically been associated with increases in intestinal permeability (Han et al., 2003, Prasad et al., 2005, Al-Sadi and Ma, 2007), an effect that appears to be cell-type specific and sensitive to certain environmental exposures. We have previously shown that Clock mutant mice have increased intestinal permeability, and these findings suggest that decreased Ocln as well as increased Cldn1 expression may be part of the mechanism underlying this effect. Taken together, our results suggest that interactions between alcohol and the circadian clock are particularly prominent and functionally relevant within the proximal colon, which supports previous in vitro and in vivo evidence linking the circadian clock and alcohol to the regulation of intestinal barrier integrity (Summa et al., 2013, Swanson et al., 2011).

Our results reveal interesting new details regarding the complex biological effects of alcohol on different tissues; however, we recognize certain limitations inherent in our experimental design. Only a single time point (ZT6) was used for gene expression measurement, thus we were unable to determine whether or not changes in expression exist at other times in the diurnal cycle. However, the mean phase angle of entrainment to a 12:12 LD cycle in Clock mutant mice has been shown to be normal with respect to wild-type mice (Vitaterna et al., 1994, Vitaterna et al., 2006), indicating that there is no a priori rationale to test for differences in the phase angle of expression rhythms between wild-type and Clock mutants. While it has been reported that there is increased ethanol preference in a two-bottle choice paradigm and sensitivity to ethanol-induced sedation in Clock mutant mice (Ozburn et al., 2013), suggesting that the Clock mutation contributes to alcohol sensitivity and the regulation of alcohol intake. However, those studies used examined mice with a genetic background (BALB/c coisogenic) than ours (C57BL/6J coisogenic). Differences between these strains in responses to alcohol are well-known (Kakihana et al., 1966). We did not observe any differences in alcohol intake between the Clock mutants and wild-type mice in our studies (Supporting Online Information). This discrepancy may be due, at least in part, to differences in the genetic background of the mutant strains and/or to the specific diet, source of ethanol and experimental protocols used in each respective study.

Another limitation is that the analysis was restricted to the pre-selected set of 75 genes included in the assay, which may therefore overlook other expression changes. We selected these genes based on their potential importance for: (1) the mechanism(s) of alcohol-induced organ pathology, (2) answering specific questions regarding interactions between alcohol consumption and the circadian clock system, and (3) determining if alcohol and/or circadian-induced effects are universal for all tissues or are specific for a given organ or tissue, which may contribute to tissue-specific susceptibility to alcohol toxicity. Thus, our inclusion of the selected 75 genes in the three different organs was appropriate for the aims and scope of the present study. Finally, gene expression does not always directly translate into changes in protein levels or function. Thus, future studies to evaluate the functional consequences of the observed changes in gene expression will be necessary.

In summary, these results facilitate a maturing understanding of the nature of the molecular changes that occur in response to chronic alcohol consumption and disruption of circadian rhythms. In particular, they help fill the gap in our knowledge of how alcohol and circadian clock system interactions contribute to adverse pathological consequences in diverse tissues, they may explain in part why different organs have differential susceptibility to alcohol toxicity and they suggest that disruption of circadian rhythms in the context of chronic alcohol use may be a previously unrecognized, and potentially modifiable, risk factor for the development or progression of alcohol-related pathologies. While these studies utilized a genetic model of circadian disruption, we recently observed similar alcohol-induced physiological responses in the intestine of both Clock mutant and phase-shifted mice (Summa et al., 2013), the latter likely being a more accurate model for humans with disrupted circadian rhythms, suggesting that responses at the level of gene expression may be similar. Strategies designed to limit circadian misalignment or that target the circadian clock to improve organization of circadian rhythms may provide useful approaches for the prevention and/or mitigation of alcohol-related pathologies. This study provides strong scientific rationale to study the efficacy of circadian clock-directed interventions, such as chronotherapy, for the prevention and/or treatment of alcohol-related pathologies.

Supplementary Material

Supp MaterialS1

Acknowledgments

Support: This work was supported by the National Institutes of Health (award R01 AA020216 to AK and FWT, with support for CBF and RMV). KCS was supported in part by the National Center for Research Resources (NCRR) and the National Center for Advancing Translational Sciences (NCATS) at the National Institutes of Health through a Northwestern University Clinical and Translational Sciences Institute Predoctoral Training Grant (8UL1TR000150). KCS and KF also received support from a National Institutes of Health Training Grant (award T32 HL007909 to FWT).

References

  1. Al-Sadi RM, Ma TY. IL-1beta causes an increase in intestinal epithelial tight junction permeability. Journal of immunology. 2007;178:4641–4649. doi: 10.4049/jimmunol.178.7.4641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bellet MM, Deriu E, Liu JZ, Grimaldi B, Blaschitz C, Zeller M, Edwards RA, Sahar S, Dandekar S, Baldi P, George MD, Raffatellu M, Sassone-Corsi P. Circadian clock regulates the host response to Salmonella. Proceedings of the National Academy of Sciences of the United States of America. 2013;110:9897–9902. doi: 10.1073/pnas.1120636110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brager AJ, Prosser RA, Glass JD. Circadian and acamprosate modulation of elevated ethanol drinking in mPer2 clock gene mutant mice. Chronobiol Int. 2011a;28:664–672. doi: 10.3109/07420528.2011.601968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brager AJ, Ruby CL, Prosser RA, Glass JD. Chronic ethanol disrupts circadian photic entrainment and daily locomotor activity in the mouse. Alcoholism, clinical and experimental research. 2010;34:1266–1273. doi: 10.1111/j.1530-0277.2010.01204.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brager AJ, Ruby CL, Prosser RA, Glass JD. Acute ethanol disrupts photic and serotonergic circadian clock phase-resetting in the mouse. Alcoholism, clinical and experimental research. 2011b;35:1467–1474. doi: 10.1111/j.1530-0277.2011.01483.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Castanon-Cervantes O, Wu M, Ehlen JC, Paul K, Gamble KL, Johnson RL, Besing RC, Menaker M, Gewirtz AT, Davidson AJ. Dysregulation of inflammatory responses by chronic circadian disruption. Journal of immunology. 2010;185:5796–5805. doi: 10.4049/jimmunol.1001026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen CP, Kuhn P, Advis JP, Sarkar DK. Chronic ethanol consumption impairs the circadian rhythm of pro-opiomelanocortin and period genes mRNA expression in the hypothalamus of the male rat. J Neurochem. 2004;88:1547–1554. doi: 10.1046/j.1471-4159.2003.02300.x. [DOI] [PubMed] [Google Scholar]
  8. Filiano AN, Millender-Swain T, Johnson R, Jr, Young ME, Gamble KL, Bailey SM. Chronic ethanol consumption disrupts the core molecular clock and diurnal rhythms of metabolic genes in the liver without affecting the suprachiasmatic nucleus. PLoS One. 2013;8:e71684. doi: 10.1371/journal.pone.0071684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Forsyth CB, Tang Y, Shaikh M, Zhang L, Keshavarzian A. Role of snail activation in alcohol-induced iNOS-mediated disruption of intestinal epithelial cell permeability. Alcoholism, clinical and experimental research. 2011;35:1635–1643. doi: 10.1111/j.1530-0277.2011.01510.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gao B, Bataller R. Alcoholic liver disease: pathogenesis and new therapeutic targets. Gastroenterology. 2011;141:1572–1585. doi: 10.1053/j.gastro.2011.09.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grant BF, Dufour MC, Harford TC. Epidemiology of alcoholic liver disease. Seminars in liver disease. 1988;8:12–25. doi: 10.1055/s-2008-1040525. [DOI] [PubMed] [Google Scholar]
  12. Han X, Fink MP, Delude RL. Proinflammatory cytokines cause NO*-dependent and -independent changes in expression and localization of tight junction proteins in intestinal epithelial cells. Shock. 2003;19:229–237. doi: 10.1097/00024382-200303000-00006. [DOI] [PubMed] [Google Scholar]
  13. Hughes ME, DiTacchio L, Hayes KR, Vollmers C, Pulivarthy S, Baggs JE, Panda S, Hogenesch JB. Harmonics of circadian gene transcription in mammals. PLoS Genet. 2009;5:e1000442. doi: 10.1371/journal.pgen.1000442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kakihana R, Brown DR, McClearn GE, Tabershaw IR. Brain sensitivity to alcohol in inbred mouse strains. Science. 1966;154:1574–1575. doi: 10.1126/science.154.3756.1574. [DOI] [PubMed] [Google Scholar]
  15. Kelley KW, Dantzer R. Alcoholism and inflammation: neuroimmunology of behavioral and mood disorders. Brain Behav Immun. 2011;25(Suppl 1):S13–20. doi: 10.1016/j.bbi.2010.12.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Keshavarzian A, Holmes EW, Patel M, Iber F, Fields JZ, Pethkar S. Leaky gut in alcoholic cirrhosis: a possible mechanism for alcohol-induced liver damage. Am J Gastroenterol. 1999;94:200–207. doi: 10.1111/j.1572-0241.1999.00797.x. [DOI] [PubMed] [Google Scholar]
  17. King DP, Vitaterna MH, Chang AM, Dove WF, Pinto LH, Turek FW, Takahashi JS. The mouse Clock mutation behaves as an antimorph and maps within the W19H deletion, distal of Kit. Genetics. 1997a;146:1049–1060. doi: 10.1093/genetics/146.3.1049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. King DP, Zhao Y, Sangoram AM, Wilsbacher LD, Tanaka M, Antoch MP, Steeves TD, Vitaterna MH, Kornhauser JM, Lowrey PL, Turek FW, Takahashi JS. Positional cloning of the mouse circadian clock gene. Cell. 1997b;89:641–653. doi: 10.1016/s0092-8674(00)80245-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lamia KA, Storch KF, Weitz CJ. Physiological significance of a peripheral tissue circadian clock. Proceedings of the National Academy of Sciences of the United States of America. 2008;105:15172–15177. doi: 10.1073/pnas.0806717105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Miller BH, McDearmon EL, Panda S, Hayes KR, Zhang J, Andrews JL, Antoch MP, Walker JR, Esser KA, Hogenesch JB, Takahashi JS. Circadian and CLOCK-controlled regulation of the mouse transcriptome and cell proliferation. Proc Natl Acad Sci U S A. 2007;104:3342–3347. doi: 10.1073/pnas.0611724104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nanji AA, Zhao S, Sadrzadeh SM, Dannenberg AJ, Tahan SR, Waxman DJ. Markedly enhanced cytochrome P450 2E1 induction and lipid peroxidation is associated with severe liver injury in fish oil-ethanol-fed rats. Alcohol Clin Exp Res. 1994;18:1280–1285. doi: 10.1111/j.1530-0277.1994.tb00119.x. [DOI] [PubMed] [Google Scholar]
  22. Naylor E, Bergmann BM, Krauski K, Zee PC, Takahashi JS, Vitaterna MH, Turek FW. The circadian clock mutation alters sleep homeostasis in the mouse. J Neurosci. 2000;20:8138–8143. doi: 10.1523/JNEUROSCI.20-21-08138.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ozburn AR, Falcon E, Mukherjee S, Gillman A, Arey R, Spencer S, McClung CA. The role of clock in ethanol-related behaviors. Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology. 2013;38:2393–2400. doi: 10.1038/npp.2013.138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Panda S, Antoch MP, Miller BH, Su AI, Schook AB, Straume M, Schultz PG, Kay SA, Takahashi JS, Hogenesch JB. Coordinated transcription of key pathways in the mouse by the circadian clock. Cell. 2002;109:307–320. doi: 10.1016/s0092-8674(02)00722-5. [DOI] [PubMed] [Google Scholar]
  25. Prasad S, Mingrino R, Kaukinen K, Hayes KL, Powell RM, MacDonald TT, Collins JE. Inflammatory processes have differential effects on claudins 2, 3 and 4 in colonic epithelial cells. Laboratory investigation; a journal of technical methods and pathology. 2005;85:1139–1162. doi: 10.1038/labinvest.3700316. [DOI] [PubMed] [Google Scholar]
  26. Rosenwasser AM, Fecteau ME, Logan RW. Effects of ethanol intake and ethanol withdrawal on free-running circadian activity rhythms in rats. Physiol Behav. 2005;84:537–542. doi: 10.1016/j.physbeh.2005.01.016. [DOI] [PubMed] [Google Scholar]
  27. Roybal K, Theobold D, Graham A, DiNieri JA, Russo SJ, Krishnan V, Chakravarty S, Peevey J, Oehrlein N, Birnbaum S, Vitaterna MH, Orsulak P, Takahashi JS, Nestler EJ, Carlezon WA, Jr, McClung CA. Mania-like behavior induced by disruption of CLOCK. Proceedings of the National Academy of Sciences of the United States of America. 2007;104:6406–6411. doi: 10.1073/pnas.0609625104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Spanagel R, Rosenwasser AM, Schumann G, Sarkar DK. Alcohol consumption and the body's biological clock. Alcoholism, clinical and experimental research. 2005;29:1550–1557. doi: 10.1097/01.alc.0000175074.70807.fd. [DOI] [PubMed] [Google Scholar]
  29. Storch KF, Lipan O, Leykin I, Viswanathan N, Davis FC, Wong WH, Weitz CJ. Extensive and divergent circadian gene expression in liver and heart. Nature. 2002;417:78–83. doi: 10.1038/nature744. [DOI] [PubMed] [Google Scholar]
  30. Summa KC, Voigt RM, Forsyth CB, Shaikh M, Cavanaugh K, Tang Y, Vitaterna MH, Song S, Turek FW, Keshavarzian A. Disruption of the Circadian Clock in Mice Increases Intestinal Permeability and Promotes Alcohol-Induced Hepatic Pathology and Inflammation. PloS one. 2013;8:e67102. doi: 10.1371/journal.pone.0067102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Swanson G, Forsyth CB, Tang Y, Shaikh M, Zhang L, Turek FW, Keshavarzian A. Role of intestinal circadian genes in alcohol-induced gut leakiness. Alcoholism, clinical and experimental research. 2011;35:1305–1314. doi: 10.1111/j.1530-0277.2011.01466.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Szabo G, Bala S. Alcoholic liver disease and the gut-liver axis. World journal of gastroenterology: WJG. 2010;16:1321–1329. doi: 10.3748/wjg.v16.i11.1321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Turek FW, Joshu C, Kohsaka A, Lin E, Ivanova G, McDearmon E, Laposky A, Losee-Olson S, Easton A, Jensen DR, Eckel RH, Takahashi JS, Bass J. Obesity and metabolic syndrome in circadian Clock mutant mice. Science. 2005;308:1043–1045. doi: 10.1126/science.1108750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vitaterna MH, King DP, Chang AM, Kornhauser JM, Lowrey PL, McDonald JD, Dove WF, Pinto LH, Turek FW, Takahashi JS. Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior. Science. 1994;264:719–725. doi: 10.1126/science.8171325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Vitaterna MH, Ko CH, Chang AM, Buhr ED, Fruechte EM, Schook A, Antoch MP, Turek FW, Takahashi JS. The mouse Clock mutation reduces circadian pacemaker amplitude and enhances efficacy of resetting stimuli and phase-response curve amplitude. Proceedings of the National Academy of Sciences of the United States of America. 2006;103:9327–9332. doi: 10.1073/pnas.0603601103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Vollmers C, Gill S, DiTacchio L, Pulivarthy SR, Le HD, Panda S. Time of feeding and the intrinsic circadian clock drive rhythms in hepatic gene expression. Proc Natl Acad Sci U S A. 2009;106:21453–21458. doi: 10.1073/pnas.0909591106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wang HJ, Zakhari S, Jung MK. Alcohol, inflammation, and gut-liver-brain interactions in tissue damage and disease development. World journal of gastroenterology: WJG. 2010;16:1304–1313. doi: 10.3748/wjg.v16.i11.1304. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp MaterialS1
NIHMS707147-supplement-Supp_MaterialS1.docx (28.4KB, docx)

RESOURCES