Interplay between short-chain fatty acids and the circadian clock in the rhythmicity of contractility and hormone release in the gut

Our bodies keep track of time using circadian clocks to anticipate predictable 24-h environmental changes. The circadian system and the timing of food intake dictate diurnal rhythms in the microbiota. In turn, microbial metabolites such as short-chain fatty acids (SCFAs) can entrain the circadian system. The aim of this PhD thesis was to identify the role of the circadian clock in the diurnal rhythms of SCFA concentrations and their effects on gut contractility and on the release of the hunger hormone ghrelin. In wild type mice, SCFA levels in caecum, distal colon and plasma showed diurnal rhythms. Caecal and plasma SCFAs oscillated in phase, while colonic SCFA concentrations peaked 9h later. Colonic SCFA concentrations fluctuated in phase with mRNA expression of the SCFA receptor Ffar3 in the colonic myenteric plexus. SCFAs inhibited neural contractions of colonic smooth muscle strips diurnally, and their effect peaked in the resting phase, together with SCFA levels and Ffar3 expression. The effect of SCFAs on ghrelin release from stomach and colon full-thickness strips also showed diurnal rhythmicity, with higher responses during the resting phase. However, SCFAs stimulated ghrelin release from stomach strips, while they inhibited ghrelin release from the colon. In mice lacking the core clock gene Bmal1, no fluctuations in SCFA levels, Ffar3 expression and neural responses to SCFAs were observed. In addition, the effects of SCFAs on ghrelin release were lost. Since Bmal1-/- mice eat continuously and do not show a typical day-night feeding rhythm, we studied whether this was the main cause for the loss of SCFA rhythms and of their effect on ghrelin release. Night-time restricted feeding of Bmal1-/- mice restored fluctuations in SCFA concentrations in the caecum and colon, and restored the inhibitory effect of SCFAs on colonic ghrelin release. However, local entrainment of other clock genes might contribute to the observed effects, as restricted feeding increased colonic Clock mRNA expression. To conclude, in this PhD thesis, we demonstrated that 24-h fluctuations of SCFAs act as a signal that integrates cues from the circadian clock and feeding time to regulate diurnal rhythms in SCFA receptor expression, gut contractility and ghrelin release.status: publishe

Night-time feeding of Bmal1(-/-) mice restores SCFA rhythms and their effect on ghrelin

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.status: publishe

The circadian clock regulates the diurnal levels of microbial short-chain fatty acids and their rhythmic effects on colon contractility in mice

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.status: publishe

Involvement of the GHSR in the developmental programming and metabolic disturbances induced by maternal undernutrition

The mismatch between maternal undernutrition and adequate nutrition after birth increases the risk of developing metabolic diseases. We aimed to investigate whether the hyperghrelinemia during maternal undernourishment rewires the hypothalamic development of the offspring and contributes to the conversion to an obese phenotype when fed a high-fat diet (HFD). Pregnant C57BL/6 J, wild type (WT) and ghrelin receptor (GHSR)-/- mice were assigned to either a normal nourished (NN) group, or an undernutrition (UN) (30% food restricted) group. All pups were fostered by NN Swiss mice. After weaning, pups were fed a normal diet, followed by a HFD from week 9. Plasma ghrelin levels peaked at postnatal day 15 (P15) in both C57BL/6 J UN and NN pups. Hypothalamic Ghsr mRNA expression was upregulated at P15 in UN pups compared to NN pups and inhibited agouti-related peptide (AgRP) projections. Adequate lactation increased body weight of UN WT but not of GHSR-/- pups compared to NN littermates. After weaning with a HFD, body weight and food intake was higher in WT UN pups but lower in GHSR-/- UN pups than in NN controls. The GHSR prevented a decrease in ambulatory activity and oxygen consumption in UN offspring during ad libitum feeding. Maternal undernutrition triggers developmental changes in the hypothalamus in utero which were further affected by adequate feeding after birth during the postnatal period by affecting GHSR signaling. The GHSR contributes to the hyperphagia and the increase in body weight when maternal undernutrition is followed by an obesity prone life environment.status: publishe

Obesity Impairs Oligopeptide/Amino Acid-Induced Ghrelin Release and Smooth Muscle Contractions in the Human Proximal Stomach

The satiation properties of proteins involve effects on gut peptide release and gastrointestinal motility which may be altered during obesity. This study compared the in vitro response and role of amino acid (AA) taste receptors (TASR) in the effect of AAs and a casein hydrolysate on ghrelin release and smooth muscle (SM) contractions in the proximal gut of lean and obese patients.status: publishe

Nationwide Harmonization Effort for Semi-Quantitative Reporting of SARS-CoV-2 PCR Test Results in Belgium.

From early 2020, a high demand for SARS-CoV-2 tests was driven by several testing indications, including asymptomatic cases, resulting in the massive roll-out of PCR assays to combat the pandemic. Considering the dynamic of viral shedding during the course of infection, the demand to report cycle threshold (Ct) values rapidly emerged. As Ct values can be affected by a number of factors, we considered that harmonization of semi-quantitative PCR results across laboratories would avoid potential divergent interpretations, particularly in the absence of clinical or serological information. A proposal to harmonize reporting of test results was drafted by the National Reference Centre (NRC) UZ/KU Leuven, distinguishing four categories of positivity based on RNA copies/mL. Pre-quantified control material was shipped to 124 laboratories with instructions to setup a standard curve to define thresholds per assay. For each assay, the mean Ct value and corresponding standard deviation was calculated per target gene, for the three concentrations (10, 10 and 10 copies/mL) that determine the classification. The results of 17 assays are summarized. This harmonization effort allowed to ensure that all Belgian laboratories would report positive PCR results in the same semi-quantitative manner to clinicians and to the national database which feeds contact tracing interventions