Making sense of what we eat: amino acid taste receptors tune ghrelin release and smooth muscle contractility

Consumption of basic nutrients, such as proteins, carbohydrates and fats, is a cornerstone for a healthy life as they provide both energy and building blocks for various functional molecules. Since the nutrient composition of a meal varies depending on food choices, a diverse spectrum in nutrient sensing pathways are present along the GI tract to control the process of food intake. This includes the coordination of gastric emptying and gut motility as well as the activation of both hormonal and neuronal signalling pathways to inform the brain about the current energy status. Overconsumption of unhealthy foods results in the onset of metabolic diseases like obesity and diabetes mellitus. Many pathways are affected but also the overexposure of the gut to nutrients may lead to disturbances or maladaptations in the communication of this sensory information throughout the body. To conquer the current obesity epidemic, new therapeutic approaches need to be explored to induce sustained body weight loss and remission of obesity-induced comorbidities. Bariatric surgery is considered as the golden standard to obtain sustained weight loss but remains an invasive technique. Therefore, research focuses on diets and/or therapeutics to restore the postprandial plasma gut hormone levels (ghrelin, GLP-1, PYY), known to be dysregulated in obesity and improved following bariatric surgery. The release of these hormones - affected by the feeding status and the macronutrient composition of a meal - is controlled by enteroendocrine cells via activation of taste-specific receptors (sweet, bitter, fatty acid and umami) similar to those present in the lingual system. Nowadays, high protein diets are a very popular weight loss strategy, especially since they induce both body weight loss and body weight maintenance in a more effective way compared to diets either low in fat or carbohydrates.These beneficial effects of dietary proteins are suggested to affect both pre-absorptive (gut motility, peptide release) and post-absorptive (diet-induced thermogenesis, gluconeogenesis, brain activation etc.) processes. Despite these favourable properties, the mechanisms involved in the effects of amino acids on gut hormone release and motility (pre-absorptive phase) are still poorly understood. In this PhD thesis we aimed to unravel the chemosensory signalling pathways involved in the effect of a casein hydrolysate and AAs on the release of the hunger hormone ghrelin in several mice models. In the next step, we translated our findings from the animal study to a human setting and evaluated whether the AA/peptide sensing mechanisms of the ghrelin cell were affected by obesity. Furthermore, we hypothesized that in addition to enteroendocrine cells also smooth muscle cells might be equipped with AA taste receptors to control gut motility. Therefore, we compared the in vitrocontractile effects and mechanism of action of AAs in the proximal gut of lean and obese subjects. In the first part of this thesis, we investigated the role of amino acid taste receptors (AA-TASRs) in the effect of AAs on ghrelin release in in vitro, ex vivoand in vivo. In a ghrelinoma cell line (MGN3-1), a dose-dependent increase in ghrelin release was observed following a 3-hour stimulation with a casein hydrolysate (peptone) (0-5%). Accordingly, thearomatic AAs (L-Phe, L-Trp), the aliphatic AA (L-Ala) and the sulphur- or hydroxyl-containing AA (L-Met, L-Thr) stereoselectively increased ghrelin release at a concentration of 10mM. At higher concentrations (20-40 mM), monosodium glutamate (MSG), a characteristic umami taste, dose-dependently enhanced octanoyl ghrelin release independent of its sodium component. All AA-TASRs were expressed on the MGN3-1 cells, but the sensing of individual AAs was found to be taste receptor-specific. Sensing of L-Phe occured via the CaSR, MSG via the TAS1R1-TAS1R3 while L-Ala and peptone acted via 2 different AA-TASRs: CaSR & TAS1R1-TAS1R3 and CaSR& GPRC6A,respectively. In agreement with the increased Ca2+release in the presence of L-Ala and MSG, both peptone- and MSG-induced ghrelin release was reduced by antagonizing the intracellular, but not extracellular, Ca2+signalling pathway. The stimulatory effect of peptone in the gastric ghrelinoma cell line could be confirmed ex vivoin mouse gastric tissue segments, while an inhibition in ghrelin release was detected following stimulation of jejunal segments. The hypothesis that reduced ghrelin release in the gut was the result of an indirect effect mediated via peptone-induced release of CCK, GLP-1 or somatostatin, all known to inhibit ghrelin release, was refuted following pretreatment with their respective receptor antagonist. In vivo, plasma ghrelin levels were reduced both upon intragastric (peptone or L-Phe) or intravenous (L-Phe) administration, indicating that AA sensing is not polarized. Thus, the in vivo/ex vivostudies demonstrated that both di-/tripeptides and specific classes of AAs have opposing effects on ghrelin release in the stomach and small intestine. Nevertheless, in the in vivosetting only an inhibition was observed upon intragastric or intravenous administration of AAs. These findings suggest that the local nutrient sensing mechanisms of the ghrelin cell are overruled in vivoby indirect mechanisms inhibiting ghrelin release. In the second part, we investigated the effect of obesity on the AA/peptide sensing mechanisms of the ghrelin cell in mucosal segments of lean and obese patients. In addition, we explored for the first time the presence and functional role of AA-TASRs in smooth muscle tissue. Basal ghrelin release and ghrelin tissue content were, in the absence of any stimulus, maximal in segments from the proximal stomach and decreased towards the small intestine in both patient populations. Nevertheless, the reduction in ghrelin tissue content along the GI tract was not reflected in a parallel decrease in the magnitude of the amount of ghrelin released. In the presence of peptone, and in line with the observations in mouse tissue segments, ghrelin release was stimulated in the stomach and reduced in the small intestine in the lean population. Strikingly, obesity impaired ghrelin release from segments of the fundus but not from the small intestine. TAS1R1, CaSR and LPAR5 were expressed in the fundus but a CaSR or TAS1R1-TAS1R3 antagonist could not block the effects of peptone on ghrelin release. The role of the oligopeptide receptor could not be investigated due to lack of a specific antagonist. Since GI motility is altered by the consumption of proteins and may affect food intake as well, the presence of AA-TASRs in GI smooth muscle tissue was evaluated too. In line with the mucosal AA-TASR expression, a subset of these receptors and their G-proteins (α-gustducin and α-transducin) was expressed in the smooth muscle layer in a region-dependent manner. Obesity mainly reduced the expression level of TAS1R1 and α-transducin. The magnitude of the smooth muscle contractions differed between AAs and was stereoselective. The contractions were stronger in smooth muscle strips from the small intestinecompared to the fundus and were mainly regulated by TAS1R1-TASR3 and extracellular Ca2+. Obesity impaired AA-induced contractions in the fundus. The bitter-tasting AA L-Met, affected gut contractility via bitter taste receptors which were previously reported to be present in the GI smooth muscle layer too. Region-specific targeting of AA-TASRs on both enteroendocrine and smooth muscle cells with specific AA-enriched diets might be a useful strategy to combat obesity as well as hypomotility disorders. Collectively, this thesis has improved our knowledge on the contribution of peptide-/AA-TASRs in both the release of ghrelin and in the induction of smooth muscle contractions along the upper GI tract during health and disease (obesity). Our findings highlight a general impairment in AA/peptide-induced ghrelin release and contractility in the fundus of obese patients. Further research is needed to understand the role of AA-TASRs in the disturbances and maladaptations that occur during disease to confirm their potential as novel therapeutic targets. In view of the multitude of AA-TASRs and the region specific effects, tailoring of treatment may be warranted.status: publishe