Colon epithelial cell-specific Bmal1 deletion impairs bone formation in mice.

The circadian clock system regulates multiple metabolic processes, including bone metabolism. Previous studies have demonstrated that both central and peripheral circadian signaling regulate skeletal growth and homeostasis in mice. Disruption in central circadian rhythms has been associated with a decline in bone mineral density in humans and the global and osteoblast-specific disruption of clock genes in bone tissue leads to lower bone mass in mice. Gut physiology is highly sensitive to circadian disruption. Since the gut is also known to affect bone remodeling, we sought to test the hypothesis that circadian signaling disruption in colon epithelial cells affects bone. We therefore assessed structural, functional, and cellular properties of bone in 8 week old Ts4-Cre and Ts4-Cre;Bmal1fl/fl (cBmalKO) mice, where the clock gene Bmal1 is deleted in colon epithelial cells. Axial and appendicular trabecular bone volume was significantly lower in cBmalKO compared to Ts4-Cre 8-week old mice in a sex-dependent fashion, with male but not female mice showing the phenotype. Similarly, the whole bone mechanical properties were deteriorated in cBmalKO male mice. The tissue level mechanisms involved suppressed bone formation with normal resorption, as evidenced by serum markers and dynamic histomorphometry. Our studies demonstrate that colon epithelial cell-specific deletion of Bmal1 leads to failure to acquire trabecular and cortical bone in male mice.

Abnormal Eating Patterns Cause Circadian Disruption and Promote Alcohol-Associated Colon Carcinogenesis.

BACKGROUND & AIMS: Alcohol intake with circadian rhythm disruption (CRD) increases colon cancer risk. We hypothesized that eating during or around physiologic rest time, a common habit in modern society, causes CRD and investigated the mechanisms by which it promotes alcohol-associated colon carcinogenesis. METHODS: The effect of feeding time on CRD was assessed using B6 mice expressing a fusion protein of PERIOD2 and LUCIFERASE (PER2::LUC) were used to model colon polyposis and to assess the effects of feeding schedules, alcohol consumption, and prebiotic treatment on microbiota composition, short-chain fatty acid levels, colon inflammation, and cancer risk. The relationship between butyrate signaling and a proinflammatory profile was assessed by inactivating the butyrate receptor GPR109A. RESULTS: Eating at rest (wrong-time eating [WTE]) shifted the phase of the colon rhythm in PER2::LUC mice. In TS4Cre × APClox468 mice, a combination of WTE and alcohol exposure (WTE + alcohol) decreased the levels of short-chain fatty acid-producing bacteria and of butyrate, reduced colonic densities of regulatory T cells, induced a proinflammatory profile characterized by hyperpermeability and an increased mucosal T-helper cell 17/regulatory T cell ratio, and promoted colorectal cancer. Prebiotic treatment improved the mucosal inflammatory profile and attenuated inflammation and cancer. WTE + alcohol-induced polyposis was associated with increased signal transducer and activator of transcription 3 expression. Decreased butyrate signaling activated the epithelial signal transducer and activator of transcription 3 in vitro. The relationship between butyrate signaling and a proinflammatory profile was confirmed in human colorectal cancers using The Cancer Genome Atlas. CONCLUSIONS: Abnormal timing of food intake caused CRD and interacts with alcohol consumption to promote colon carcinogenesis by inducing a protumorigenic inflammatory profile driven by changes in the colon microbiota and butyrate signaling. Accession number of repository for microbiota sequence data: raw FASTQ data were deposited in the NCBI Sequence Read Archive under project PRJNA523141.

Alcohol Effects on Colon Epithelium are Time-Dependent.

BACKGROUND: Alcohol intake increases the risk of developing colon cancer. Circadian disruption promotes alcohol's effect on colon carcinogenesis through unknown mechanisms. Alcohol's metabolites induce DNA damage, an early step in carcinogenesis. We assessed the effect of time of alcohol consumption on markers of tissue damage in the colonic epithelium. METHODS: Mice were treated by alcohol or phosphate-buffered saline (PBS), at 4-hour intervals for 3 days, and their colons were analyzed for (i) proliferation (Ki67) and antiapoptosis (Bcl-2) markers, (ii) DNA damage (γ-H2AX), and (iii) the major acetaldehyde (AcH)-DNA adduct, N2 -ethylidene-dG. To model circadian disruption, mice were shifted once weekly for 12 h and then were sacrificed at 4-hour intervals. Samples of mice with a dysfunctional molecular clock were analyzed. The dynamics of DNA damage repair from AcH treatment as well as role of xeroderma pigmentosum, complementation group A (XPA) in their repair were studied in vitro. RESULTS: Proliferation and survival of colonic epithelium have daily rhythmicity. Alcohol induced colonic epithelium proliferation in a time-dependent manner, with a stronger effect during the light/rest period. Alcohol-associated DNA damage also occurred more when alcohol was given at light. Levels of DNA adduct did not vary by time, suggesting rather lower repair efficiency during the light versus dark. XPA gene expression, a key excision repair gene, was time-dependent, peaking at the beginning of the dark. XPA knockout colon epithelial cells were inefficient in repair of the DNA damage induced by alcohol's metabolite. CONCLUSIONS: Time of day of alcohol intake may be an important determinant of colon tissue damage and carcinogenicity.

Dietary Fiber Treatment Corrects the Composition of Gut Microbiota, Promotes SCFA Production, and Suppresses Colon Carcinogenesis.

Epidemiological studies propose a protective role for dietary fiber in colon cancer (CRC). One possible mechanism of fiber is its fermentation property in the gut and ability to change microbiota composition and function. Here, we investigate the role of a dietary fiber mixture in polyposis and elucidate potential mechanisms using TS4Cre×cAPCl°x468 mice. Stool microbiota profiling was performed, while functional prediction was done using PICRUSt. Stool short-chain fatty acid (SCFA) metabolites were measured. Histone acetylation and expression of SCFA butyrate receptor were assessed. We found that SCFA-producing bacteria were lower in the polyposis mice, suggesting a decline in the fermentation product of dietary fibers with polyposis. Next, a high fiber diet was given to polyposis mice, which significantly increased SCFA-producing bacteria as well as SCFA levels. This was associated with an increase in SCFA butyrate receptor and a significant decrease in polyposis. In conclusion, we found polyposis to be associated with dysbiotic microbiota characterized by a decline in SCFA-producing bacteria, which was targetable by high fiber treatment, leading to an increase in SCFA levels and amelioration of polyposis. The prebiotic activity of fiber, promoting beneficial bacteria, could be the key mechanism for the protective effects of fiber on colon carcinogenesis. SCFA-promoting fermentable fibers are a promising dietary intervention to prevent CRC.