Mode of action of Akkermansia muciniphila in the intestinal dialogue: role of extracellular proteins, metabolites and cell envelope components.

Akkermansia muciniphila is a promising next-generation beneficial microbe due to its natural presence in the mucus layer of the gut, its symbiotic ability to degrade mucus, and its capacity to improve the intestinal barrier function. A. muciniphila is able to counteract weight gain and immuno-metabolic disturbances in several animal models. Many of these disorders, including obesity and auto-immune diseases, have been associated with decreased gut barrier function and consequent increased inflammation. Since A. muciniphila was found to normalize these changes and strengthen the gut barrier function, it is hypothesized that other beneficial effects of A. muciniphila might be caused by this restoration. In search for A. muciniphila's mode of action in enhancing the gut barrier function and promoting health, we reasoned that secreted components or cell envelope components of A. muciniphila are interesting candidates as they can potentially reach and interact with the epithelial barrier. In this review, we focus on the potential mechanisms through which A. muciniphila can exert its beneficial effects on the host by the production of extracellular and secreted proteins, metabolites and cell envelope components. These products have been studied in isolation for their structure, signaling capacity, and in some cases, also for their effects in preclinical models. This includes the protein known as Amuc_1100, which we here rename as pilus-associated signaling (PAS) protein , the P9 protein encoded by Amuc_1631, the short-chain fatty acids acetate and propionate, and cell envelope components, such as phosphatidylethanolamine and peptidoglycan.

Chronic jetlag reprograms gene expression in the colonic smooth muscle layer inducing diurnal rhythmicity in the effect of bile acids on colonic contractility.

BACKGROUND: Secondary bile acids entrain peripheral circadian clocks and inhibit colonic motility via the bile acid receptor GPBAR1. We aimed to investigate whether chronodisruption affected the rhythm in serum bile acid levels and whether this was associated with alterations in clock gene and Gpbar1 mRNA expression in the colonic smooth muscle layer. We hypothesized that this in turn may affect the rhythm in the inhibitory effect of secondary bile acids on colonic contractility. METHODS: Mice were exposed to 4 weeks of chronic jetlag induction. The expression of Gpbar1 and clock genes was measured in colonic smooth muscle tissue using RT-qPCR over 24 h (4 h time interval). The effect of secondary bile acids on electrical field-induced neural contractions was measured isometrically in colonic smooth muscle strips. KEY RESULTS: Chronic jetlag abolished the rhythmicity in serum bile acid levels. This was associated with a phase-shift in diurnal clock gene mRNA fluctuations in smooth muscle tissue. Chronic jetlag induced a rhythm in Gpbar1 expression in the colonic smooth muscle layer. In parallel, a rhythm was induced in the inhibitory effect of taurodeoxycholic acid (TDCA), but not deoxycholic acid, on neural colonic contractions that peaked together with Gpbar1 expression. CONCLUSIONS & INFERENCES: Chronodisruption abolished the rhythm in bile acid levels which might contribute to a shift in smooth muscle clock gene expression. Our findings suggest that chronodisruption caused a transcriptional reprogramming in the colonic smooth layer thereby inducing a rhythm in the expression of Gpbar1 and in the inhibitory effect of TDCA on colonic contractility.

Human intestinal bitter taste receptors regulate innate immune responses and metabolic regulators in obesity.

Bitter taste receptors (taste 2 receptors, TAS2Rs) serve as warning sensors in the lingual system against the ingestion of potentially poisonous food. Here, we investigated the functional role of TAS2Rs in the human gut and focused on their potential to trigger an additional host defense pathway in the intestine. Human jejunal crypts, especially those from individuals with obesity, responded to bitter agonists by inducing the release of antimicrobial peptides (α-defensin 5 and regenerating islet-derived protein 3 α [REG3A]) but also regulated the expression of other innate immune factors (mucins, chemokines) that affected E. coli growth. We found that the effect of aloin on E. coli growth and on the release of the mucus glycoprotein CLCA1, identified via proteomics, was affected by TAS2R43 deletion polymorphisms and thus confirmed a role for TAS2R43. RNA-Seq revealed that denatonium benzoate induced an NRF2-mediated nutrient stress response and an unfolded protein response that increased the expression of the mitokine GDF15 but also ADM2 and LDLR, genes that are involved in anorectic signaling and lipid homeostasis. In conclusion, TAS2Rs in the intestine constitute a promising target for treating diseases that involve disturbances in the innate immune system and body weight control. TAS2R polymorphisms may be valuable genetic markers to predict therapeutic responses.

Circadian clocks in the digestive system.

Many molecular, physiological and behavioural processes display distinct 24-hour rhythms that are directed by the circadian system. The master clock, located in the suprachiasmatic nucleus region of the hypothalamus, is synchronized or entrained by the light-dark cycle and, in turn, synchronizes clocks present in peripheral tissues and organs. Other environmental cues, most importantly feeding time, also synchronize peripheral clocks. In this way, the circadian system can prepare the body for predictable environmental changes such as the availability of nutrients during the normal feeding period. This Review summarizes existing knowledge about the diurnal regulation of gastrointestinal processes by circadian clocks present in the digestive tract and its accessory organs. The circadian control of gastrointestinal digestion, motility, hormones and barrier function as well as of the gut microbiota are discussed. An overview is given of the interplay between different circadian clocks in the digestive system that regulate glucose homeostasis and lipid and bile acid metabolism. Additionally, the bidirectional interaction between the master clock and peripheral clocks in the digestive system, encompassing different entraining factors, is described. Finally, the possible behavioural adjustments or pharmacological strategies for the prevention and treatment of the adverse effects of chronodisruption are outlined.

Chronodisruption by chronic jetlag impacts metabolic and gastrointestinal homeostasis in male mice.

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.

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.

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.

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.

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

SCOPE: The satiation properties of proteins involve effects on gut peptide release and gastrointestinal motility which may be altered during obesity. This study compares 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. METHODS AND RESULTS: Basal ghrelin release, measured from mucosal segments, is maximal in the fundus and decreased distally. Obesity selectively impaires the stimulatory effect of a casein hydrolyaste on ghrelin release in the fundus but does not affect its inhibitory effect in the small intestine (SI). The SM contractions induced by a casein hydrolysate and AAs are stronger in strips from the SI than from the fundus but are reduced in the stomach of obese patients. The region-dependent expression of AA-TASRs in the mucosa and SM layer is affected by obesity. Most of the AA-induced responses are reduced by the umami antagonist, lactisole. l-Met-induced responses involve bitter taste receptors. CONCLUSION: Region-specific targeting of AA taste receptors on both enteroendocrine and SM cells with specific AA-enriched diets might be a useful strategy to combat obesity as well as hypomotility disorders.