Aim: The microbiota shows diurnal oscillations that are synchronized by the host's circadian clock and feeding rhythms. Short-chain fatty acids (SCFAs) produced by the microbiota are possible synchronizers of peripheral circadian clocks.We aimed to investigate whether faecal SCFAs show a diurnal rhythm that regulates the rhythm of SCFA receptor expression (FFAR2/3, OLFR78, HCAR2) and SCFA-induced colonic contractility. The role of the circadian clock was studied in mice lacking the core clock gene Bmal1. Methods: Mice were sacrificed at 4-hour intervals. Faecal SCFA concentrations and SCFA receptor expression were determined. The effect of increasing concentrations of a SCFA mix on electrical field-induced neural responses in colon strips was measured isometrically. Results: Diurnal fluctuations in faecal SCFA concentrations (peak 4 hours after lights on) were observed that were in phase with the rhythm of Ffar2/3 expression in the colonic muscle layer. Olfr78 expression was not diurnal and Hcar2 was not detectable. The inhibitory effect of a SCFA mix on neural contractions in colonic smooth muscle strips showed a diurnal rhythm and oscillated in phase with faecal SCFA concentrations and Ffar2/3 expression. In contrast, neither excitatory neural responses nor acetylcholine-induced smooth muscle contractions showed a diurnal rhythm. In Bmal1 −/− mice, no fluctuations in faecal SCFA levels, Ffar3 expression and neural responses to SCFAs were observed. Conclusion: Diurnal microbial SCFA levels regulate the rhythm of Ffar3 expression in the colonic myenteric plexus, which causes rhythmicity in SCFA-induced colonic motility. Deletion of Bmal1 abolishes rhythmicity of SCFA levels and their downstream effects. K E Y W O R D Scircadian clock, colon contractility, short-chain fatty acids | INTRODUCTIONThe circadian clock coordinates the timing of physiological processes with solar time. The circadian system consists of a master clock, located in the suprachiasmatic nuclei of the anterior hypothalamus, and several peripheral clocks in different organs that are synchronized by the master clock. 1

Aim: Chronodisruption desynchronizes peripheral clocks and leads to metabolic diseases. Feeding cues are important synchronizers of peripheral clocks and influence rhythmic oscillations in intestinal microbiota and their metabolites. We investigated whether chronic jetlag, mimicking frequent time zone travelling, affected the diurnal fluctuations in faecal short-chain fatty acid (SCFA) levels, that feed back to the gut clock to regulate rhythmicity in gut function. Methods: Rhythms in faecal SCFAs levels and in the expression of clock genes and epithelial markers were measured in the colonic mucosa of control and jetlagged mice. The entraining effect of SCFAs on the rhythm in clock gene mRNA expression was studied in primary colonic crypts. The role of the circadian clock in epithelial marker expression was studied in Arntl −/− mice. Results: Chronic jetlag increased body weight gain and abolished the day/night food intake pattern which resulted in a phase-delay in the rhythm of faecal SCFAs that paralleled the shift in the expression of mucosal clock genes. This effect was mimicked by stimulation of primary colonic crypts from control mice with SCFAs. Jetlag abolished the rhythm in Tnfα, proglucagon and ghrelin expression but not in the expression of tight junction markers. Only a dampening in plasma glucagon-like peptide-1 but not in ghrelin levels was observed. Rhythms in ghrelin but not proglucagon mRNA expression were abolished in Arntl −/− mice. Conclusion:The altered food intake pattern during chronodisruption corresponds with the changes in rhythmicity of SCFA levels that entrain clock genes to affect rhythms in mRNA expression of gut epithelial markers.

The known crosstalk between short-chain fatty acids (SCFAs) and the circadian clock is tightly intertwined with feeding time. We aimed to investigate the role of the core clock gene Bmal1 and feeding time in the diurnal rhythms in plasma and caecal SCFA levels and in their effect on the release of the hunger hormone ghrelin in the stomach and colon. WT, Bmal1-/- (ad libitum fed) and night-time-restricted-fed (RF)-Bmal1-/- littermates were killed at zeitgeber time (ZT) 4 and 16. SCFA concentrations were measured by gas chromatography. To investigate the effect of SCFAs on ghrelin release, stomach and colonic full-thickness strips were incubated with Krebs or a SCFA mix mimicking plasma or caecal concentrations, after which octanoyl ghrelin release was measured by RIA. Diurnal rhythms in caecal and plasma SCFAs oscillated in phase but rhythmic changes were abolished in Bmal1-/- mice. RF of Bmal1-/- mice restored fluctuations in caecal SCFAs. Plasma SCFA concentrations failed to affect gastric ghrelin release. The effect of caecal SCFA concentrations on colonic ghrelin release was rhythmic (inhibition at ZT 4, no effect at ZT 16). In Bmal1-/- mice, the inhibitory effect of SCFAs at ZT 4 was abolished. RF Bmal1-/- mice restored the inhibitory effect and increased colonic Clock expression. To conclude, diurnal fluctuations in caecal SCFAs and the effect of SCFAs on colonic ghrelin release are regulated by feeding time, independent of the core clock gene Bmal1. However, local entrainment of other clock genes might contribute to the observed effects.

We used time-restricted feeding (TRF) to investigate whether microbial metabolites and the hunger hormone ghrelin can become the dominant entraining factor during chronic jetlag to prevent disruption of the master and peripheral clocks, in order to promote health. Therefore, hypothalamic clock gene and Agrp/Npy mRNA expression were measured in mice that were either chronically jetlagged and fed ad libitum, jetlagged and fed a TRF diet, or not jetlagged and fed a TRF diet. Fecal short-chain fatty acid (SCFA) concentrations, plasma ghrelin and corticosterone levels, and colonic clock gene mRNA expression were measured. Preventing the disruption of the food intake pattern during chronic jetlag using TRF restored the rhythmicity in hypothalamic clock gene mRNA expression of Reverbα but not of Arntl. TRF countered the changes in plasma ghrelin levels and in hypothalamic Npy mRNA expression induced by chronic jetlag, thereby reestablishing the food intake pattern. Increase in body mass induced by chronic jetlag was prevented. Alterations in diurnal fluctuations in fecal SCFAs during chronic jetlag were prevented thereby re-entraining the rhythmic expression of peripheral clock genes. In conclusion, TRF during chronodisruption re-entrains the rhythms in clock gene expression and signals from the gut that regulate food intake to normalize body homeostasis.

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