The Circadian Clock Mutation Promotes Intestinal Dysbiosis.

BACKGROUND: Circadian rhythm disruption is a prevalent feature of modern day society that is associated with an increase in pro-inflammatory diseases, and there is a clear need for a better understanding of the mechanism(s) underlying this phenomenon. We have previously demonstrated that both environmental and genetic circadian rhythm disruption causes intestinal hyperpermeability and exacerbates alcohol-induced intestinal hyperpermeability and liver pathology. The intestinal microbiota can influence intestinal barrier integrity and impact immune system function; thus, in this study, we sought to determine whether genetic alteration of the core circadian clock gene, Clock, altered the intestinal microbiota community. METHODS: Male Clock(Δ19) -mutant mice (mice homozygous for a dominant-negative-mutant allele) or littermate wild-type mice were fed 1 of 3 experimental diets: (i) a standard chow diet, (ii) an alcohol-containing diet, or (iii) an alcohol-control diet in which the alcohol calories were replaced with dextrose. Stool microbiota was assessed with 16S ribosomal RNA gene amplicon sequencing. RESULTS: The fecal microbial community of Clock-mutant mice had lower taxonomic diversity, relative to wild-type mice, and the Clock(Δ19) mutation was associated with intestinal dysbiosis when mice were fed either the alcohol-containing or the control diet. We found that alcohol consumption significantly altered the intestinal microbiota in both wild-type and Clock-mutant mice. CONCLUSIONS: Our data support a model by which circadian rhythm disruption by the Clock(Δ19) mutation perturbs normal intestinal microbial communities, and this trend was exacerbated in the context of a secondary dietary intestinal stressor.

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

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 this 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 Clock(Δ19) 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 5 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 3 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 Clock(Δ19) 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 Clock(Δ19) mutation and identify specific expression profiles that may contribute to tissue-specific vulnerability to alcohol-induced injury in the brain, colon, and liver.

Analysis of miRNA expression profiles in the liver of Clock Δ19 mutant mice.

The circadian clock controls the physiological functions of many tissues including the liver via an autoregulatory transcriptional-translational feedback loop, of which CLOCK is a core positive component. In addition, many studies have indicated that microRNAs (miRNAs) regulate liver function. However, how CLOCK-regulated miRNAs are linked to liver function remains largely unknown. In this study, miRNAs expression profiles were performed in the liver of Clock Δ19 mutant mice. Compared to wild type mice, totals of 61 and 57 putative CLOCK-regulated miRNAs were differentially expressed (fold change absolute value ≥2) at zeitgeber time 2 and zeitgeber time 14, respectively. According to the pathway analyses, the target genes of differentially expressed miRNAs were mainly involved in pathways in cancer, the PI3K-Akt signaling pathway and the MAPK signaling pathway. Protein-protein interaction analyses revealed that the hub genes were primarily associated with pathway in cancer and circadian rhythms. Expression validation showed that while the expression levels of miR-195 and miR-340 were up-regulated, the rhythms of these two miRNAs were always maintained. The expression level of nr1d2 mRNA was down-regulated. We identified a number of prospective CLOCK-regulated miRNAs that play roles in the various physiological processes of the liver, providing a reference to better understanding the potential regulatory mechanisms in the liver.

Functional annotation of extensively and divergently expressed miRNAs in suprachiasmatic nucleus of ClockΔ19 mutant mice.

Circadian locomotor output cycles kaput protein (CLOCK) is a core transcription factor of complex integrated feedback loops in mammalian circadian clock. More genes have been reported to be regulated by CLOCK, however little is known about the role of CLOCK-mediated miRNAs. To dissect this, we used microarray analysis to measure miRNAs expression in suprachiasmatic nuclei (SCN) of wild-type (WT) and ClockΔ19 mutant mice at two different time points. We found that miRNAs regulation in two time points was extensive (nearly 75% of the miRNAs expressed at each time point), and very little overlap, with only six miRNAs in common. Besides this, the predicted CLOCK regulated miRNAs at two time points participated in extremely diverse pathways. We validated nine miRNAs (miR-125a-3p, miR-144, miR-199a-5p, miR-199b*, miR-200a, miR-200b, miR-203, miR-449a, and miR-96), which were involved in the same signaling pathway-hippo signaling pathway. The rhythms of these miRNAs showed a broad distribution of phase, amplitude, and waveform in Clock mutation. And further analysis indicated that there may be three models of miRNA-mediated circadian rhythms and hippo signaling pathway. MiRNA, the small player, may play a hub role in connecting circadian rhythms and other pathways via its multiple target genes networks.

Expression profiles and functional annotation analysis of mRNAs in suprachiasmatic nucleus of Clock mutant mice.

The core circadian clock gene, Clock, is a positive component of the transcription/translation feedback loop in the master pacemaker suprachiasmatic nucleus (SCN) in mammals. The robust daytime peak of some clock genes in the wild-type SCN is absent in Clock mutant mice. However, very little is known about the impact of Clock mutation on the expression of other functional genes in SCN. Here, we performed cDNA microarray and found 799 differentially expressed genes (DEGs) at zeitgeber time 2 (ZT2) and 1289 DEGs at ZT14 in SCN of Clock△19/△19 mutant mice. KEGG pathway analysis showed that the changed mRNAs were highly associated with hedgehog signaling pathway, retinol metabolism, allograft rejection, drug metabolism, hematopoietic cell lineage and neuroactive ligand-receptor interaction. The top 14 and 71 hub genes were identified from the protein-protein interaction (PPI) network at ZT2 and ZT14, respectively. The sub-networks revealed hub genes were involved in olfactory transduction and neuroactive ligand-receptor interaction pathways. These results demonstrate the Clock△19/△19 mutation alters the expression of various genes involved in a wide spectrum of biological function in mouse SCN, which are helpful for better understanding the function of Clock and potential regulatory mechanisms.

Potent synchronization of peripheral circadian clocks by glucocorticoid injections in PER2::LUC-Clock/Clock mice.

In mammals, the central clock (the suprachiasmatic nuclei, SCN) is entrained mainly by the light-dark cycle, whereas peripheral clocks in the peripheral tissues are entrained/synchronized by multiple factors, including feeding patterns and endocrine hormones such as glucocorticoids. Clock-mutant mice (Clock/Clock), which have a mutation in a core clock gene, show potent phase resetting in response to light pulses compared with wild-type (WT) mice, owing to the damped and flexible oscillator in the SCN. However, the phase resetting of the peripheral clocks in Clock/Clock mice has not been elucidated. Here, we characterized the peripheral clock gene synchronization in Clock/Clock mice by daily injections of a synthetic glucocorticoid (dexamethasone, DEX) by monitoring in vivo PER2::LUCIFERASE bioluminescence. Compared with WT mice, the Clock/Clock mice showed significantly decreased bioluminescence and peripheral clock rhythms with decreased amplitudes and delayed phases. In addition, the DEX injections increased the amplitudes and advanced the phases. In order to examine the robustness of the internal oscillator, T-cycle experiments involving DEX stimulations with 24- or 30-h intervals were performed. The Clock/Clock mice synchronized to the 30-h T-cycle stimulation, which suggested that the peripheral clocks in the Clock/Clock mice had increased synchronizing ability upon DEX stimulation, to that of circadian and hour-glass type oscillations, because of weak internal clock oscillators.