Dissertations / Theses on the topic 'Bitter taster receptor'

In humans, taste is one of the five senses, and helps in the recognition of nutritionally important and potentially harmful substances. It triggers innate behaviour to accept or reject food. Humans can sense five basic tastes, which are sweet, umami, bitter, salt and sour. The receptors that mediate bitter, sweet and umami tastes belong to the G protein-coupled receptor (GPCR) superfamily. A group of three receptors sense sweet and umami tastes, whereas bitter taste is sensed by 25 bitter taste receptors (referred as T2Rs). T2Rs are activated by structurally diverse natural and synthetic bitter compounds. Many common pharmaceutical compounds are bitter in taste and these are effective ligands for T2Rs. Recent finding of T2Rs in extra-oral tissues suggests these receptors are also involved in various physiological and pathophysiological processes. To understand the structure and function of these receptors, studies directed at elucidating their mechanisms of activation, and identification of novel ligands including bitter blockers (antagonists and inverse agonists), are required. To obtain mechanistic insights into the role of the highly conserved, and receptor specific residues, two bitter taste receptors (T2R1 and T2R4) were targeted. In this study, a combination of molecular, biochemical and pharmacological approaches were used to identify the amino acids and motifs, important for T2Rs to switch from inactive to active state. A hydrogen-bonding network between transmembrane (TM) helices 1-2-7 was identified as important for T2R activation. Alanine-scan mutagenesis of intracellular loops (ICLs) 2 and 3 identified T2R regions important for G protein binding, and receptor activation. A pharmacological method was developed, to screen potential bitter blockers for T2Rs. Using this method, three novel bitter blockers, which include two natural antagonists and one synthetic inverse agonist for T2R4, were discovered. The role of expression tags in enhancing T2R4 expression was also pursued. T2R4 expression on the cell surface was increased 2.5 fold, when its N-terminus was tagged with rhodopsin N-terminal 33 residues (Rho33- T2R4 chimera). In conclusion, work carried out provides novel insights into the mechanisms of T2R activation, and in the discovery of bitter blockers for T2R4.

L'obésité constitue l'un des principaux problèmes de santé publique en ce début du 21ème siècle. Sa prévalence augmente régulièrement, en particulier chez les enfants. Ce constat n'est pas anodin car l'obésité est généralement associée à diverses pathologies graves (diabète de type 2, hypertension et cancer,…). Ainsi, des investigations sur les mécanismes impliqués dans la perception gustative des lipides alimentaires pourraient éclairer leurs rôles dans l’incidence de l’obésité.Plusieurs études ont démontré le rôle des endocannabinoïdes et des aliments amers dans l’obésité. Ainsi, nous avons étudié l’interaction (cross-talk) des récepteurs cannabinoïdes et du goût amer avec le goût lipidique. Cette thèse comporte ainsi deux volets : les récepteurs cannabinoïdes (CB1R), le goût amer et leurs interactions avec les récepteurs lipidiques.Dans la première partie, nous avons étudié le rôle régulateur de CB1R. Dans la présente étude, des tests comportementaux sur des souris CB1R-/- et des souris de type sauvage (WT) ont montré que l'invalidation du gène Cb1r était associée à une faible préférence pour les solutions contenant de l'huile de colza ou un acide gras à longue chaîne (AGLC) tel que l’acide linoléique (LA). L'administration de rimonabant, un agoniste-inverse de CB1R, chez la souris a également entraîné une faible préférence pour les acides gras alimentaires. Aucune différence dans l'expression des protéines CD36 et GPR120 n'a été observée dans les cellules des papilles gustatives des souris WT et CB1R-/-. La signalisation calcique via CD36 dans les cellules des papilles gustatives des souris CB1R-/- diminue de façon significative par rapport à celle observée dans les cellules gustatives des souris WT. Les cellules des papilles gustatives des souris CB1R-/- présentent également une diminution significative de l'ARNm de Pro-glucagon et de Glp-1r et un faible niveau basal de GLP-1. Nous rapportons que CB1R est impliqué dans la perception du goût du gras via la signalisation calcique et la sécrétion de GLP-1.Dans la seconde partie, nous avons d’abord caractérisé le phénotype de cellules fongiformes humaines (HTC-8). En effet, le projet de ma thèse comprend la caractérisation à l’échelle moléculaire des récepteurs amers et lipidiques et leur cross-talk dans ces cellules (collaboration BRAIN, Allemagne). Nous avons démontré que les cellules HTC-8 expriment PLCβ2 et l’α-gustducin à l’échelle des ARNm et des protéines. Elles expriment également TAS2R16 et TAS2R38 et ces mêmes cellules co-expriment CD36 et GPR120. Puis, nous avons étudié la signalisation via ces récepteurs en utilisant l’acide linoléique, un agoniste de CD36 et GPR120, la sinigrin, agoniste de TAS2R16 et TAS2R38, la salicin, agoniste du récepteur TAS2R16 et le phénylthiocarbamide, agoniste du récepteur TAS2R38. De plus, les études du signal calcique ont démontré que la signalisation en aval du goût gras partage une voie commune avec la signalisation en aval du goût amer, mettant en évidence un cross-talk entre ces deux modalités gustatives.Bien que nous ayons montré le cross-talk entre les modalités gustatives amère et lipidique, il nous reste à étudier ces phénomènes à l’échelle de l’organisme. Ces résultats, d’ores et déjà, montrent que le goût amer et le récepteur cannabinoïde-1 sont liés à la sensibilité au goût du gras et doivent être pris en compte pour la gestion de l'obésité
Obesity is one of the major public health problems at the beginning of the 21st century. Its prevalence is increasing steadily, especially among children. This observation is not insignificant because obesity is generally associated with various serious pathologies (type 2 diabetes, hypertension and cancer, etc.). Thus, investigations into the mechanisms involved in the taste perception of dietary lipids could shed light on their roles in the incidence of obesity.Several studies have demonstrated the role of endocannabinoids and bitter foods in obesity. Thus, we studied the cross-talk of cannabinoid receptors and bitter taste with lipid taste. This thesis has two components: cannabinoid receptors (CB1R), bitter taste and their interactions with lipid receptors.In the first part, we studied the regulatory role of CB1R. In the present study, behavioral tests on CB1R-/- mice and wild-type (WT) mice showed that the invalidation of the Cb1r gene was associated with a low preference for solutions containing rapeseed oil or a long chain fatty acid (LCFA) such as linoleic acid (LA). Administration of rimonabant, a CB1R inverse agonist, in mice also resulted in a low preference for dietary fatty acids. No differences in the expression of CD36 and GPR120 proteins were observed in the taste buds cells of the WT and CB1R-/- mice. Calcium signaling via CD36 in the taste bud cells of CB1R-/- mice decreased significantly compared with those observed in the taste cells of WT mice. The taste bud cells of CB1R-/- mice also show a significant decrease in Pro-glucagon and Glp-1r mRNA and a low basal level of GLP-1. We report that CB1R is involved in the perception of fat taste via calcium signaling and secretion of GLP-1.In the second part, we first characterized the phenotype of human fungiform cells (HTC-8). Indeed, the project of my thesis includes the characterization on the molecular scale of bitter and lipid receptors and their cross-talk in these cells (collaboration BRAIN, Germany). We have demonstrated that HTC-8 cells express PLCβ2 and α-gustducin at the mRNA and protein level. They also express TAS2R16 and TAS2R38 and these same cells co-express CD36 and GPR120. Then, we studied signaling via these receptors using linoleic acid, a CD36 and GPR120 agonist, sinigrin, TAS2R16 agonist and TAS2R38, salicin, TAS2R16 receptor agonist, and phenylthiocarbamide, TAS2R38 receptor agonist. In addition, calcium signal studies have shown that downstream fatty signaling shares a common path with downstream bitter taste signaling, highlighting a cross-talk between these two taste modalities.Although we have shown the cross-talk between bitter and lipid taste modalities, we still have to study these phenomena at the level of the organism. These results, already, show that the bitter taste and the cannabinoid-1 receptor are related to the taste sensitivity of fat and must be taken into account for the management of obesity

El sistema enteroendocrí es troba al tracte gastrointestinal i controla la gana i l’activitat pancreàtica endocrina, entre altres funcions. Els compostos bioactius que estimulen aquest sistema són candidats terapèutics per tractar patologies relacionades amb aquestes funcions. Prèviament s’ha identificat que un extracte de proantocianidines de llavors de raïm (GSPE) és antidiabètic per les seves capacitats de millora de la funció de les cèl·lules i la seva capacitat saciant, com a conseqüència en part de l’activació del sistema enteroendocrí. El nostre grup ha relacionat les secrecions enteroendocrines induïdes per polifenols amb l'estimulació de receptors del gust amarg (TAS2R) in vitro, però si això es reflecteix en una ingesta alterada encara és deconegut. Per això, és necessari comprendre millor aquest sistema per poder desenvolupar millors estratègies terapèutiques. Aquesta tesi aborda si les secrecions d’intererohormones induïdes per GSPE modulen la producció de glucagó pancreàtic i si aquestes secrecions es regulen mitjançant l’estimulació específica de TAS2R que condueix a un control diferencial de la ingesta d’aliments. Aquesta hipòtesi s’ha avaluat amb estudis in vivo en rates i estudis ex vivo en mostres intestinals. Hem identificat que el glucagó és més sensible que la insulina a GSPE, fet que es correlaciona amb una secreció il·leal de GLP1 millorada.
El sistema enteroendocrino se encuentra en el tracto gastrointestinal y controla el apetito y la actividad pancreática endocrina, entre otras funciones. Los compuestos bioactivos que estimulan este sistema son candidatos terapéuticos para tratar patologías relacionadas con estas funciones. Previamente se identificó que un extracto de proantocianidinas de semillas de uva (GSPE) es antidiabético por sus capacidades de mejora de la función de las células y su capacidad saciante, como consecuencia en parte activar del sistema enteroendocrino. Nuestro grupo relacionó las secreciones enteroendocrinas inducidas por polifenoles con la estimulación de receptores del gusto amargo (TAS2R) in vitro, pero si esto se refleja en una ingesta alterada aún se desconoce. Por esto, es necesario comprender mejor este sistema para poder desarrollar mejores estrategias terapéuticas. Esta tesis aborda si las secreciones enteroendocrinas inducidas por GSPE modulan la producción de glucagón pancreático y si estas se regulan mediante la estimulación específica de TAS2R que conduce a un control diferencial de la ingesta. Esta hipótesis se ha evaluado con estudios in vivo en ratas y estudios ex vivo en muestras intestinales. Hemos identificado que el glucagón es más sensible que la insulina a GSPE, lo que se correlaciona con una secreción ileal de GLP1 mejorada.
The enteroendocrine system is located in the gastrointestinal tract and controls appetite and endocrine pancreatic activity, among other functions. Thus, bioactive compounds that stimulate the enteroendocrine system are therapeutic candidates for treating pathologies related to these functions. Previous research has identified a grape-seed proanthocyanidin extract (GSPE) as antidiabetic for its -cell function enhancement abilities and its appetitesuppressing activity at least partly through activating the enteroendocrine system. Moreover, our group has linked the polyphenol-induced enteroendocrine secretions to the stimulation of some bitter taste receptors (TAS2R) in vitro, but whether it results in an altered food intake has not been studied yet. Since little is known of the mechanisms used by polyphenols to stimulate secretory mechanisms of the enteroendocrine system, there is a need to fully comprehend this system to specifically target it with a therapeutic strategy. For this reason, this thesis addressed whether GSPE-induced enterohormone secretions modulate pancreatic glucagon production, and whether these secretions are regulated through the specific stimulation of TAS2R leading to a differential control of food intake. This hypothesis was assessed with in vivo studies in rats and ex vivo studies in intestinal samples.

T2R receptors are the classical bitter taste receptors which detect and transduce bitter taste in a subset of taste receptor cells (TRCs). The TRPM5-dependent T2Rs are G-protein coupled receptors (GPCRs) and are linked to G protein, gustducin to initiate an intracellular signaling cascade for the transduction of bitter tastants. Nicotine is bitter. However, at present the transduction mechanisms for the detection of nicotine in are poorly understood. Previous studies from our laboratory using TRPM5 knockout (KO) mice demonstrated that the T2R pathway is insufficient in explaining the taste perception of nicotine. TRPM5 KO mice elicited chorda tympani (CT) taste nerve responses to nicotine, albeit significantly smaller than the wild type (WT) mice and still responded to nicotine as an aversive stimulus. Following addition of mecamylamine (Mec), a non-specific blocker of neuronal nicotinic acetylcholine receptors (nAChRs), CT responses to nicotine were partially inhibited in both WT and TRPM5 KO mice. Mec also decreases the aversive response to nicotine in both WT and TRPM5 KO mice. These studies led to the hypothesis that both a TRPM5-independent and TRPM5-dependent pathways are responsible for the detection and transduction of the bitter taste of nicotine in TRCs. The TRPM5-independent pathway most likely utilizes the nAChRs expressed in TRCs and function as bitter taste receptors for nicotine. We have subsequently demonstrated the expression of nAChRs in a subset of TRPM5-positive TRCs. However, this mechanism is not well understood in other cell types, particularly undifferentiated epithelial cells, such as HEK293T cells. The specific aims of this project were: (i) To identify which components of T2R-dependent taste reception as well as components of nAChRs are expressed in HEK293T cells; (ii) To determine if HEK293T cells co-express these components; (iii) To identify if exposure to nicotine modulates the expression of T2R and nAChR dependent components in HEK293T cells; (iv) To determine if TRCs express functional nAChR ion channels; and (v) To determine if nAChRs are involved in the release of neuropeptides, such as brain-derived neurotrophic factor (BDNF) in HEK293T cells. The data obtained in HEK293T cells was compared with parallel studies on adult cultured human fungiform taste cells (HBO) done independently by Dr. Jie Qian, a postdoctoral fellow in Dr. Vijay Lyall’s lab. The results of combined studies on HBO and HEK293T cells indicates that TRPM5-positive cells also co-express ionotropic nAChRs, comprising a and β subunits. The nAChRs are capable of forming ion pores and when stimulated by nicotine and create a parallel TRPM5-independent pathway for the detection of nicotine. Using molecular and immunocytochemical techniques, our results demonstrate that mRNAs and proteins for bitter taste receptors and downstream intracellular signaling components as well as subunits necessary for the formation of nAChRs are expressed in HBO and HEK293T cells. Results demonstrated that TRPM5-positive HEK293T cells co-expressed nAChR subunits throughout the entire population. Nicotine increased the influx of Ca2+ in a dose dependent manner, which was somewhat reduced by the addition of TRPM5 blocker, triphenylphosphine oxide (TPPO). Both mRNA and protein expression were altered in a biphasic pattern with a maximum increased observed at 0.5 µM nicotine with a decrease in expression at higher concentrations. The synthesis of neurotrophic factor BDNF, required for maturation of taste bud cells and their innervating nerves, increased in HEK293T cells exposed to nicotine, however, nicotine did not trigger the release of BDNF. These results were then compared and contrasted with HBO cells to better understand the comparative effects of nicotine on both undifferentiated and differentiated cells. The data on HBO cells is presented in the Appendix.

Grapefruit juice is an excellent source of many nutrients and phytochemicals that contribute to a healthy diet. Currently, there is an increasing interest in grapefruit products because consumption appears to be associated with a reduced risk of certain chronic diseases, such as obesity, diabetes, cancers and cardiovascular disease. The consumption of grapefruit (Citrus paradisi Macfadyen) however remains low in South Africa as some individuals like grapefruit and others do not and the reason/s for this variation is not clear. Taste, aroma and colour are important fruit product quality factors that influence consumer preferences. Perception of grapefruit flavour does not depend on only one individual sense, but is the result of multisensory integration of unimodal signals. Where there is a mixture of appearance, taste and aroma signals, cross-modal sensory interaction occurs which may potentially change the intensity and character of flavour perception. Sensory perception is interpreted differently across individuals. The main objective of the study was to determine the effect of varying the bitterness, sweetness, colour and aroma intensities of a grapefruit-like model beverage on the perception of sensory properties and consumer liking of the beverages with the aim of giving guidance to breeders on selection and improvement of grapefruit traits to optimize hedonic value. The second objective of this study was to determine the effects of sensitivity to bitter taste [as determined through 6-propylthiouracil (PROP) taster classification] and genetic variation in TAS2R38 and TAS2R19 SNP genotypes on hedonic rating of the flavour of grapefruit-like beverages differing in bitter/sweet taste intensity. A factorial design was used to formulate 36 grapefruit-like beverages with deflavoured clarified apple juice as base and modification of bitter taste (3 levels), sweet taste (3 levels), aroma intensity (2 levels) and colour (red or yellow). Descriptive analysis was used to describe the sensory profiles of the 36 beverages. Hedonic rating of colour, aroma and flavour of the 12 most diverse beverages from the design was measured with a consumer panel. Sensitivity to bitter taste of 96 young African females (18-24 years) was measure and the respondents classified into PROP taster groups. DNA was extracted from the saliva of the participants for genotyping of TAS2R38 and TAS2R19 bitter receptor genes. The subjects also rated the flavour of grapefruit-like beverages differing in bitter taste intensity for hedonic value. The results showed that varying the bitterness, sweetness, colour and aroma intensity of the grapefruit-like model beverage have an effect on the sensory properties and consumer liking of the beverages. The concentration of naringin in the grapefruit-like beverage increased the bitter taste, aftertaste and grapefruit flavour intensity of the drink. Consumers preferred grapefruit-like beverages with a red colour and low bitterness. Sensitivity to the bitterness of grapefruit beverages and whether there is an association between genetics of bitter taste perception and liking of grapefruit were further explored. The results then showed that respondents’ sensitivity to bitter taste, as well as genetic variation in TAS2R38 and TAS2R19 (single SNP genotypes) are partly responsible for the lower liking of grapefruit model beverages with higher naringin (more bitterness) concentration. In this study, sensitivity of respondents to bitter taste (PROP status) has been linked to preference for red coloured grapefruit beverages, grapefruit beverages with low bitterness/high sweetness and grapefruit-like beverages with low intensity of grapefruit aroma. This is the first study to report on consumers’ perception and acceptance of grapefruit-like model beverages that vary in taste, colour and aroma sensory properties. People differ genetically in bitter taste sensitivity and this research demonstrated the role of some genetic variables (notably rs10772420 of the TAS2R19 SNP genotype and both rs713598 and rs1726866 of the TAS2R38 SNP genotypes). It is the first study showing the effect of TAS2R38 SNP genotypes on grapefruit liking. It is also the first study to determine the effect of PROP taster status, perception of grapefruit beverage characteristics (e.g. bitterness level, colour type and aroma level) and variation in TAS2R38 and TAS2R19 SNP genotypes on hedonic ratings for colour, aroma and flavour of grapefruit-like beverages in a group of South African females. So far populations from Africa have been under represented in similar studies. Most studies where a link between rs10772420 and lower bitterness perception and greater liking for unsweetened grapefruit juice was established, included only Caucasians. Studying the role of genetic differences in sensitivity to PROP bitterness (e.g. in taster status) in modulating multisensory grapefruit flavour perception is needed to determine why the liking for grapefruit varies between individuals. The findings of this study can help researchers and breeders to change properties and traits in grapefruit varieties, can assist product formulators and quality assurance staff to optimize the flavour of grapefruit products for consumer acceptance and to make the generic product more acceptable to a larger portion of the South African population. However, the sample of respondents used in this research represents only a small portion of the South African population and therefore cannot be extrapolated to represent the population. The insights gained from this subgroup may be used to enhance the acceptance of grapefruit products for the larger population.
Thesis (PhD)--University of Pretoria, 2019.
Food Science
PhD
Unrestricted

Die Wahrnehmung von Geschmacksempfindungen beruht auf dem Zusammenspiel verschiedener Sinneseindrücke wie Schmecken, Riechen und Tasten. Diese Komplexität der gustatorischen Wahrnehmung erschwert die Beantwortung der Frage wie Geschmacksinformationen vom Mund ins Gehirn weitergeleitet, prozessiert und kodiert werden. Die Analysen zur neuronalen Prozessierung von Geschmacksinformationen erfolgten zumeist mit Bitterstimuli am Mausmodell. Zwar ist bekannt, dass das Genom der Maus für 35 funktionelle Bitterrezeptoren kodiert, jedoch war nur für zwei unter ihnen ein Ligand ermittelt worden. Um eine bessere Grundlage für tierexperimentelle Arbeiten zu schaffen, wurden 16 der 35 Bitterrezeptoren der Maus heterolog in HEK293T-Zellen exprimiert und in Calcium-Imaging-Experimenten funktionell charakterisiert. Die Daten belegen, dass das Funktionsspektrum der Bitterrezeptoren der Maus im Vergleich zum Menschen enger ist und widerlegen damit die Aussage, dass humane und murine orthologe Rezeptoren durch das gleiche Ligandenspektrum angesprochen werden. Die Interpretation von tierexperimentellen Daten und die Übertragbarkeit auf den Menschen werden folglich nicht nur durch die Komplexität des Geschmacks, sondern auch durch Speziesunterschiede verkompliziert. Die Komplexität des Geschmacks beruht u. a. auf der Tatsache, dass Geschmacksstoffe selten isoliert auftreten und daher eine Vielzahl an Informationen kodiert werden muss. Um solche geschmacksstoffassoziierten Stimuli in der Analyse der gustatorischen Kommunikationsbahnen auszuschließen, sollten Opsine, die durch Licht spezifischer Wellenlänge angeregt werden können, für die selektive Ersetzung von Geschmacksrezeptoren genutzt werden. Um die Funktionalität dieser angestrebten Knockout-Knockin-Modelle zu evaluieren, die eine Kopplung von Opsinen mit dem geschmacksspezifischen G-Protein Gustducin voraussetzte, wurden Oozyten vom Krallenfrosch Xenopus laevis mit dem Zwei-Elektroden-Spannungsklemm-Verfahren hinsichtlich dieser Interaktion analysiert. Der positiven Bewertung dieser Kopplung folgte die Erzeugung von drei Mauslinien, die in der kodierenden Region eines spezifischen Geschmacksrezeptors (Tas1r1, Tas1r2, Tas2r114) Photorezeptoren exprimierten. Durch RT-PCR-, In-situ-Hybridisierungs- und immunhistochemische Experimente konnte der erfolgreiche Knockout der Rezeptorgene und der Knockin der Opsine belegt werden. Der Nachweis der Funktionalität der Opsine im gustatorischen System wird Gegenstand zukünftiger Analysen sein. Bei erfolgreichem Beleg der Lichtempfindlichkeit von Geschmacksrezeptorzellen dieser Mausmodelle wäre ein System geschaffen, dass es ermöglichen würde, gustatorische neuronale Netzwerke und Hirnareale zu identifizieren, die auf einen reinen geschmacks- und qualitätsspezifischen Stimulus zurückzuführen wären.
Taste impression is based on the interaction of taste, smell and touch. To evaluate the nutritious content of food mammals possess five distinct taste qualities: sweet, bitter, umami (taste of amino acids), sour and salty. For bitter, sweet, and umami compounds taste signaling is initiated by binding of tastants to G protein-coupled receptors. The interactions of taste stimuli, usually watersoluble chemicals, with their cognate receptors lead to the activation of the G protein gustducin, which, in turn, initiates a signal resulting in the activation of gustatory afferents. However, details of gustatory signal transmission and processing as well as neural coding are only incompletely understood. This is partly due to the property of some tastants to elicit several sensations simultaneously, unspecific effects caused by the temperature, viscosity, osmolarity, and pH of the solvents, as well as by mechanical stimulation of the tongue during stimulus application. The analysis of gustatory processing of taste information are mainly based on mouse models after stimulation with bitter taste stimuli. Even though it is known that the mouse genome codes for 35 bitter taste receptor genes only few of them had been analysed so far. For better understanding and interpretation of animal experiments 16 mouse bitter receptors had been analysed by Calcium Imaging experiments with HEK293T cells. The data reveal that mouse bitter taste receptors are more narrow tuned than human bitter taste receptors, proving that the ligand spectra of murine and human orthologous receptors are not complient. In order to avoid the disturbing effects of solvents and stimulus application on the analysis of gustatory information transfer and processing, I employ an optogenetical approach to address this problem. For this purpose I generated three strains of gene-targeted mice in which the coding regions of the genes for the umami receptor subunit Tas1r1, the sweet receptor subunit Tas1r2 or the bitter taste receptor Tas2r114 have been replaced by the coding sequences of different opsins (photoreceptors of visual transduction) that are sensitive to light of various wavelengths. In these animals I should be able to activate sweet, bitter, or umami signalling by light avoiding any solvent effects. In initial experiments of this project I demonstrated that the various visual opsins indeed functionally couple to taste signal transduction pathway in oocyte expression system, generating basic knowledge and foundation for the generation of the gene-targeted animals. The knockout-knockin strategies have been successfully realized in the case of all three mouse models, revealed by RT-PCR, in situ hybridization and immunohistochemical analysis of taste papillae. All data confirm that the particular taste receptors have been replaced by the different opsins in taste cells. Further analysis concerning the functional consequences of opsin knockin and taste receptor knockout are part of prospective work.

Chez les animaux et en particulier les insectes, l’alimentation comprend une phase d’examen sensoriel qui précède l’ingestion, afin notamment d’éviter d’ingérer des substances toxiques. Cette détection fait intervenir des cellules spécialisées dans la détection de telles molécules, cellules qui sont généralement qualifiées de sensibles aux goûts « amers ». A l’aide d’observations électrophysiologiques et comportementales, nous avons abordé comment un insecte modèle, la drosophile, était capable de détecter des substances potentiellement toxiques mélangées à des sucres à l’aide de ses neurones gustatifs. Dans une première partie, nous avons étudié la détection de la L-canavanine, qui est un acide aminé non protéique. Cette molécule est toxique pour l’homme comme pour les animaux car elle est confondue par le métabolisme avec un acide aminé, la L-arginine, et intégrée à sa place dans les protéines. En utilisant des constructions génétiques et en particulier le système UAS-Gal4, nous avons montré que la Lcanavanine est détectée par des cellules gustatives qui expriment une protéine réceptrice GR66a, qui est impliquée dans la détection de nombreuses substances amères. Nous avons également montré que, contrairement à la caféine, la détection de L-canavanine nécessite des protéines Gαo fonctionnelles. Nous avons ensuite étudié les interactions sucré-amer. Dans un premier travail, nous avons montré que l’addition de Lcanavanine une solution sucrée n’altérait pas la détection des sucres, contrairement à la strychnine qui peut complètement supprimer la détection du sucre dans les cellules gustatives. Grâce à des ablations spécifiques des cellules détectant l’amer, nous avons pu montrer que cette inhibition était une propriété intrinsèque des cellules sensibles aux. sucres. Les cellules sensibles aux sucres auraient donc des sites récepteurs non identifiés, sensibles à certains ligands amers. Nous avons également abordé des interactions inverses, à savoir l’inhibition de la détection de substances amères par des sucres, en confrontant 4 substances amères (denatonium, berberine, caféine, umbelliferone) à 12 sucres. Les observations que nous avons réalisées montrent que certains sucres exercent un effet inhibiteur sur la détection des molécules amères testées. En utilisant des outils génétiques permettant l’ablation des cellules sensibles aux sucres, nous avons montré que cette inhibition est une propriété intrinsèque des cellules sensibles à l’amer. Cependant, cet effet inhibiteur est loin d’être aussi efficace que l’inhibition des substances amères sur la détection des sucres. Dans une dernière partie, nous avons évalué la modulation de la détection gustative à l’aide d’analogues d’une neuro-hormone, la leucokinine, connue pour ses effets sur la diurèse. Lorsqu’elle est mélangée à une solution sucrée, ces analogues inhibent la détection des sucres par les sensilles gustatives, à la fois chez le moustique Aedes aegypti et chez la drosophile. La détection de substances « amères » par les cellules gustatives de drosophiles implique donc deux voies de codage : l’une, spécifique, concerne des cellules dédiées à la détection des substances amères ; l’autre, moins spécifique, affecte les cellules dédiées à la détection des sucres. De manière réciproque, ces cellules dédiées à la détection des molécules sont affectées par la présence de ligands sucrés. Le codage des informations gustatives à la périphérie est donc un phénomène plus complexe qui nécessite d’étudier plus précisément la détection de composés en mélanges
In most animals including insects, ingestion is preceded by a close examination of the food, for example in order to detect the presence of potentially noxious chemicals. This detection involves specialized gustatory cells, which are generally described as sensitive to “bitter” tastes. Using electrophysiology and behavioral observations, we studied how a model insect, Drosophila melanogaster, can detect potentially toxic substances (described here as “bitter”) when mixed with sugar molecules, with their gustatory neurons. In a first part, we studied how L-canavanine is detected. Lcanavanine is a pseudo amino acid, which is confounded with L-arginine by the metabolism. Proteins which include Lcanavanine are non-functional and this compound is toxic for animals including insects. Using genetic constructions based on the UAS-Gal4 expression system, we showed that Lcanavanine is detected by gustatory cells expressing a receptor protein, GR66a, which is specific to most cells capable of detecting bitter substances. We also showed that, contrary to caffeine, the detection of L-canavanine requires functional Gαo proteins. Then, we studied some aspects of the detection of mixtures of sweet and bitter molecules. In a first approach, we contributed to establish that L-canavanine does not impact sugar detection, while other chemicals like strychnine completely inhibit sugar detection. By using the UAS-Gal4 system to ablate bitter-sensitive cells, we could demonstrate that such inhibition is a specific property of sugar- sensitive cells. These cells should have thus receptors for bitter substances which have not been identified yet. We also examined the reverse interaction, which is a possible role of sweet molecules to inhibit the detection of bitter substances. We examined the detection of denatonium, berberine, caffeine and umbelliferone in the presence of 12 different sugars, using behavioral and electrophysiology observations. By using genetic construction to ablate sugar-sensitive cells, we found that the sugar inhibitory action is not due to the presence of sugar-sensitive cells. It should be noted, however that in our experimental conditions, this inhibitory action is less efficient than the inhibition of bitter upon sugar detection. In a last part, we examined the modulation of gustatory perception by analogs of leucokinine, which is a neuropeptide involved in the diuresis of insects. We show that these analogs, when mixed with sugars in solution, can inhibit sugar detection by gustatory sensilla, both in Aedes aegypti mosquitoes and in Drosophila. The detection of bitter molecules by gustatory neurons in Drosophila thus involves two main coding channels: one is specific, and involves gustatory cells dedicated to the detection of bitter molecules; the second one, less specific, is affecting cells which are dedicated to the detection of sugar molecules. Gustatory coding is thus a more complex phenomenon than previously thought on the basis of examining responses to single molecules, thus urging to study the responses of gustatory receptors to more complex and natural mixtures

INTRODUÇÃO: A obesidade infantil é um importante problema de saúde pública e apresenta impacto direto na qualidade de vida das crianças e adolescentes, bem como no desenvolvimento futuro de doenças crônicas. O padrão alimentar rico em gordura e açúcar, e com baixo aporte de fibra dietética, vitaminas e minerais é reconhecido como fator de risco para o surgimento da obesidade, no entanto os fatores que contribuem para a preferência por alimentos ricos nestes nutrientes não são bem estabelecidos. O sabor dos alimentos é reconhecido como um importante preditor das escolhas alimentares, e os polimorfismos nos genes que codificam os receptores do sabor podem explicar a variabilidade da preferência e consumo alimentar na população. OBJETIVO: Avaliar a influência de polimorfismos de genes de receptores de sabor gorduroso (CD36), doce (TAS1R2) e amargo (TAS2R38) no consumo alimentar e no perfil metabólico de crianças e adolescentes obesos. MÉTODOS: Estudo transversal com 668 crianças e adolescentes obesos e um grupo controle de 135 crianças eutróficas, de ambos os gêneros. Foi realizado o estudo molecular dos polimorfismos de nucleotídeo único (SNPs) rs1761667 e rs1527483 do CD36, rs9701796 e rs35874116 do TAS1R2, e rs1726866 e rs713598 do TAS2R38, bem como análise do consumo alimentar e perfil metabólico. RESULTADOS: Em relação ao CD36, o alelo A do rs1761667 relacionou-se com menor consumo de lipídios totais, gorduras poli e monoinsaturadas, consumo de alimentos de sabor gorduroso, ingestão de óleos vegetais e açúcares totais em obesos. O alelo A do rs1527483 associou-se com menor percentil de pressão arterial diastólica, menor massa gorda e maior massa livre de gordura em obesos. Quanto ao gene TAS1R2, a variante rs9701796 teve maior risco metabólico segundo a razão circunferência da cintura-estatura (RCE), bem como relação com maior consumo de achocolatado em pó em obesos. Já a variante rs35874116 mostrou relação com a menor ingestão de fibras dietéticas em obesos. No TAS2R38, o alelo G do rs1726866 foi associado com menor consumo de gorduras monoinsaturadas e maior consumo de açúcares totais, em obesos. O alelo G do rs713598 mostrou relação com maior consumo de carboidratos, consumo de alimentos de sabor doce, refrigerantes e menor ingestão de fibras pelos indivíduos eutróficos. CONCLUSÃO: Não houve relação entre genótipos e risco de obesidade. Os achados mostram a associação entre polimorfismos dos genes de receptores de sabor com o consumo alimentar, indicando diferenças entre obesos e magros, e alelos de proteção e de risco cardiometabólico, respectivamente dos genes CD36 e TAS1R2
BACKGROUND: Childhood obesity is a major public health problem and it has a direct impact on the quality of life of children and adolescents, as well as the future risk for development of chronic diseases. The dietary pattern rich in fats and sugars associated to the low intake of dietary fibers, vitamins and minerals is widespread for the rise of obesity. However the factors that contribute to the preference for foods rich in these nutrients are not well established. Taste is recognized as an important predictor of food choices, and polymorphisms in genes encoding its receptors may explain the variability of taste preference and food intake on population. OBJECTIVE: To evaluate the influence of polymorphisms of fat (CD36), sweet (TAS1R2) and bitter (TAS2R38) taste receptor genes in diet and metabolic profile in obese children and adolescents. METHODS: Cross-sectional study with 668 obese children and adolescents and a control group of 135 normal-weight children. The molecular study was made for single nucleotide polymorphisms (SNPs) rs1761667 and rs1527483 of CD36, rs9701796 and rs35874116 of TAS1R2, rs1726866 and rs713598 of TAS2R38, and the analysis of food intake and metabolic profile. RESULTS: In relation to CD36, the A allele of rs1761667 was associated with lower intake of total fat, poly and monounsaturated fats, consumption of fatty flavor food, intake of vegetable oils and total sugars in obese. The A allele of rs1527483 was associated with lower percentile of diastolic blood pressure, lower fat mass and increased fat-free mass in obese. Regarding TAS1R2 gene, the variant rs9701796 was associated to increased metabolic risk according to waist-height ratio, as well as with higher consumption of chocolate powder in obese. The variant rs35874116 showed a lower intake of dietary fiber. In TAS2R38, the G allele of rs1726866 was associated with a lower intake of monounsaturated fat and a higher intake of total sugars in obese. The G allele of rs713598 was related to the higher carbohydrate intake, consumption of sweet tasting food, soda drinks and less fiber intake by normal weight children. CONCLUSION: There was no relationship between genotypes and risk of obesity. The findings show the association between polymorphisms of taste receptor genes with dietary intake, indicating differences between obese and lean children, as well as the protective and risk alleles for cardiometabolic risk in CD36 and TAS1R2, respectively

This thesis includes a series of clinical studies, focussing on the pivotal role of gastrointestinal (GI) function, particularly gastric emptying and GI hormones (e.g. glucagon-like peptide-1 (GLP-1)), in the regulation of postprandial glycaemia, energy expenditure and energy intake in both health and type 2 diabetes (T2D). The key themes relate to evaluation of: 1) gastric emptying of solid and liquid meals in healthy individuals and subjects with T2D, 2) the bidirectional relationship between gastric emptying and postprandial secretion of GLP-1, 3) the role of endogenous GLP-1 signalling in the regulation of postprandial glycaemia and energy expenditure in T2D, and 4) effects of intestinal bitter taste signalling on GI hormone secretion, gastric emptying, postprandial blood glucose and energy intake in health and T2D. Gastric emptying is a major determinant of the blood glucose response to dietary carbohydrate in both health and diabetes. The interaction of luminal nutrients and bioactive compounds with the intestines gives rise to the secretion of numerous GI hormones. Of particular importance to glycaemic control in T2D is the so-called incretin hormone, GLP-1, which has the capacity to stimulate insulin, suppress glucagon secretion and energy intake and slow gastric emptying. In T2D, gastric emptying is frequently abnormal, but may be either delayed, unchanged or accelerated. This discrepancy has reflected the substantial heterogeneity in subject characteristics (e.g. age, duration of diabetes, glycaemic status, pharmacotherapy and presence or absence of diabetic complications) of cohorts studied and the test meals employed (e.g. emptying of solid and liquid test meals is frequently disconcordant). The study reported in Chapter 4 evaluated gastric emptying of a semisolid high carbohydrate meal in a group of community-based individuals with relatively well-controlled T2D (HbA1c ≤ 7.9%), managed by diet or metformin monotherapy, in comparison with a cohort of age- and body mass index (BMI)-matched healthy subjects, and a group of healthy young subjects. The study described in Chapter 5, evaluated the gastric emptying of an oral glucose drink in two groups of community-based individuals with relatively well- (HbA1c ≤ 7.9%) and poorly- (HbA1c ≥ 9%) controlled T2D managed by diet or metformin alone, together with young and older subjects without diabetes. There is a complex bidirectional relationship between gastric emptying and the secretion of GLP-1 after a meal. In a given individual, the magnitude of GLP-1 secretion is related to the rate of nutrient delivery into the small intestine (i.e. gastric emptying); conversely, GLP-1 signalling slows gastric emptying. Gastric emptying exhibits a relatively modest intra-individual, but substantial inter-individual, variation. It remains unknown whether the latter reflects the differences in the ‘intestinal sensitivity’ to nutrients and hence secretion of GLP-1. In Chapter 6, the relationship between gastric emptying and the postprandial GLP-1 response was evaluated in subjects with T2D, the inter- and intra-individual variations in plasma GLP-1 response to enteral nutrient infusions were evaluated in health and T2D, and the relationship between gastric emptying of a glucose drink and the responsiveness of GLP-1 to intestinal glucose was further evaluated in subjects with and without T2D. Subsequent to its secretion, GLP-1 is rapidly degraded by the enzyme, dipeptidyl peptidase 4 (DPP-4). DPP-4 inhibitors are therefore a logical treatment option to augment intact GLP-1 levels for glycaemic control in T2D. In healthy humans, a single dose of DPP-4 inhibitor was shown to lower the blood glucose response to fat and increase energy expenditure and the thermic effect of feeding; the latter would favour a reduction in body weight with sustained use of DPP-4 inhibitors. The fact that DPP-4 inhibitors are weight neutral in subjects with T2D suggests that the effect of DPP-4 inhibition on energy expenditure may be compromised in this disorder. The study reported in Chapter 7, therefore, evaluated the effect of DPP-4 inhibition on the glycaemic and energy expenditure responses to an intraduodenal fat in subjects with T2D, including the role of endogenous GLP-1, assessed using the GLP-1 receptor antagonist, exendin (9-39). There is emerging evidence from preclinical studies suggesting that stimulation of GI bitter taste receptors (BTRs) has the potential to reduce postprandial glycaemia and suppress energy intake by modulating the secretion of GI hormones and slowing gastric emptying. The study reported in Chapter 8 evaluates the effects of a non-nutritive bitter taste compound, denatonium benzoate (DB), encapsulated for oral administration, on gastric emptying, postprandial glycaemia and energy intake in subjects with T2D. In Chapter 9, the effects of DB and a bitter tasting bile acid, taurocholic acid, administered via rectal perfusion, on GLP-1 and peptide YY secretion were evaluated in the presence or absence of a BTR antagonist, probenecid, in healthy humans.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2021

Bitter taste signaling in humans is mediated by a group of 25 bitter receptors (T2Rs) that belong to the G-protein coupled receptor (GPCR) family. Previously, several bitter peptides were isolated and characterized from bitter tasting food protein derived extracts, such as pea protein and soya bean extracts. However, their molecular targets in humans were poorly characterized. In this study, we tested the ability of the bitter tasting tri- and di-peptides to activate the human bitter receptor, T2R1. Using a heterologous expression system, T2R1 gene was transiently expressed in C6-glioma cells and changes in intracellular calcium were measured following addition of the peptides. We found that the bitter tasting tri-peptides are more potent in activating T2R1 than the di-peptides tested. Furthermore, to elucidate the potential ligand binding pocket of T2R1 we used homology molecular modeling. The ligand binding pocket in T2R1 is present on the extracellular surface of the receptor, and is formed by the transmembrane helices 1, 2, 3 and 7 and with extracellular loops 1 and 2.

Bitter taste is one of the five basic taste modalities, and is mediated by 25 bitter taste receptors (T2Rs) in humans. How these few receptors recognize a wide range of structurally diverse bitter compounds is not known. To address this question, structural and functional studies on T2Rs are necessary. Quinine is a natural alkaloid and one of the most intense bitter tasting compounds. Previously it was shown that quinine activates T2R4, however, whether T2R4 has only one binding site for quinine, and the amino acids on the receptor involved in binding to quinine remain to be determined. In this study, the ligand binding pocket on T2R4 for quinine was characterized using a combination of approaches. These included molecular model guided site-directed mutagenesis, characterization of the expression of the mutants by flow cytometry, and functional characterization by cell based calcium imaging. Twelve mutations were made in T2R4 and their expression and function were characterized. Results show that the ligand binding pocket of T2R4 for quinine is situated on the extracellular side, and is formed by the residues present on the transmembrane regions TM3, TM4, and extracellular loop regions ECL2 and TM6-ECL3-TM7 interface. Further, this study identified the following amino acids : A90, F91, Y155 N173, T174, Y258 and K270 to play an important role in quinine binding to T2R4. The detailed study of residues interacting with ligand will help in understanding how various ligands interact with T2Rs, and facilitate the pharmacological characterization of potent antagonists or bitter taste blockers. The characterization of novel ligands, including bitter taste blockers will help in dissecting the signaling mechanism(s) of T2Rs, and help in the development of novel therapeutic tools for the food and drug industry.

Bitter taste receptors (TR2) expression and functionality was recently reported in the rat choroid plexus (CP). CP epithelial cells establish a major brain barrier, the blood-cerebrospinal fluid barrier (BCSFB). Given their capacity to bind a large array of chemical compounds, we hypothesised that TR2 might be involved in monitoring the composition of blood and cerebrospinal fluid. Brain barriers play a critical role in the protection of the central nervous system (CNS) by hindering the access of toxic substances to the brain. Consequently, many drugs targeting neurological disorders are impaired to cross these barriers. This is explained through the expression of several membrane transporters in brain barriers cells that efflux drugs, thus impairing drug cell accumulation in the brain. A wide range of compounds that bind to TR2 show neuroprotective and anti-tumoral properties. However, their low bioavailability in the CNS restrains its therapeutic application. Additionally, bitter compounds might interact with transporters that are also found in brain barriers. Therefore, bitter compounds might be effluxed which explains their low bioavailability but can also regulate the action of these transporters in order to increase their or other drugs’ intracellular accumulation. Considering that bitter compounds are TR2 agonists it is possible that TR2 play an important role on the bioactive effects of bitter compounds in the CNS, as reported in other tissues. The main goal of this doctoral thesis was to analyse the expression and function of the bitter signalling pathway in the human BCSFB. Additionally, the role of human TR2 (TAS2Rs) as modulator of specific neuroactive bitter compounds on ABC transporters function and activity at the BCSFB was also evaluated. The first research work presented showed the expression of 13 TAS2Rs as well as of downstream effector proteins of the taste signalling pathway in the human BCSFB. Moreover, we demonstrated that TAS2R14 and TAS2R39 are functional in a human cell model of the BCSFB and respond to bitter compounds quercetin and chloramphenicol, respectively. The second research work evaluated resveratrol transport across the BCSFB and the involvement of TAS2Rs. Results showed that resveratrol is able to cross the BCSFB from blood to cerebrospinal fluid in a dependent manner of TAS2R14 expression at CP epithelial cells. Further, efflux transporters ABCC1, ABCC4 and ABCG2, which are expressed at CP epithelial cells, transport resveratrol. Additionally, resveratrol upregulated ABCG2 expression and regulated ABCC4 and ABCG2 efflux activity in TAS2R14 dependent way. In conclusion, the results obtained during this project demonstrate that TAS2Rs are expressed and functional at the human BCSFB and support their participation in the monitorization of chemical composition of the surrounding fluids. Furthermore, the major achievements of this thesis strongly support the role of BCSFB in the regulation of the transport of molecules into the brain. In the future, it is necessary to further exploit the role of other TAS2Rs as mediators of the effects of bitter compounds in the brain, as well as in the regulation of transport and detoxifying systems at the BCSFB. The knowledge hereby created has far-reaching potential for improving the challenging task of delivering therapeutic drugs into the CNS.
(TR2) foi demonstrada recentemente nas células epiteliais do plexo coróide (PC) de rato. As células do PC formam a barreira sangue-líquido cefalorraquidiano, uma das principais barreiras cerebrais. A presença de TR2 no PC sugere que estes recetores possam estar envolvidos na monitorização da composição química do sangue e do líquido cefalorraquidiano. As barreiras do cérebro desempenham um papel crucial na proteção do sistema nervoso central (SNC) impedindo o acesso de substâncias nocivas ao cérebro. Consequentemente, muitos fármacos direcionados para o tratamento de patologias do SNC não conseguem ultrapassar estas barreiras. Isto deve-se, em grande parte, à presença de diversos transportadores nas células que constituem estas barreiras, os quais transportam os fármacos para fora das células e, portanto, impedem a sua acumulação nas células alvo. Diversos compostos amargos, ligandos dos TR2, possuem propriedades anti-tumorais e de neuroprotecção. Contudo, a biodisponibilidade destes compostos é, normalmente, muito baixa o que dificulta a sua aplicação terapêutica. Adicionalmente, sabe-se que estes compostos interagem com transportadores membranares nas células das barreiras do cérebro. Isto sugere que os compostos amargos com potencial terapêutico sejam transportados para fora das células, o que explica a sua baixa biodisponibilidade, mas também que podem regular a ação dos transportadores de membrana o que poderá contribuir para uma maior acumulação intracelular dos compostos. Uma vez que estes compostos amargos são agonistas dos TR2, é possível que estes tenham um papel crucial na regulação da biodisponibilidade desses compostos ao nível do SNC, tal como reportado em alguns órgãos. Como tal, o trabalho desenvolvido nesta tese de doutoramento teve como principal objetivo a análise da expressão e da função da via de sinalização gustativa do amargo na barreira sangue-líquido cefalorraquidiano humana. Adicionalmente, foi estudado o papel dos TR2 no transporte do composto resveratrol ao nível da barreira sangue-líquido cefalorraquidiano humana. Na primeira parte do trabalho, foi possível confirmar a expressão de 13 TAS2Rs e das proteínas efetoras da via de transdução de sinal gustativa num modelo humano da barreira sangue-líquido cefalorraquidiano. Além disto, foi também demonstrada a funcionalidade dos TAS2R14 e 39, em resposta aos compostos quercetina e cloranfenicol, respetivamente. Na segunda parte do trabalho, analisámos o transporte do composto amargo, resveratrol, num modelo in vitro da barreira sangue-líquido cefalorraquidiano humana, e avaliámos a possível envolvência dos TAS2Rs, que ligam o resveratrol, nesse transporte. Deste trabalho concluiu-se que o resveratrol atravessa a barreira sangue-líquido cefalorraquidiano na direção sangue - líquido cefalorraquidiano (basolateral – apical), de forma dependente do TAS2R14. Observámos também que os transportadores de efluxo ABCC1, ABCC4 e ABCG2 presentes nas células epiteliais do CP transportam o resveratrol, e que este aumenta a expressão do ABCG2 e modula a sua função, bem como a do ABCC4, de forma dependente do TAS2R14. Em suma, os resultados obtidos durante o desenvolvimento deste projeto permitem afirmar que os TAS2Rs são expressos e estão funcionais na barreira sangue-líquido cefalorraquidiano humana, podendo participar na monitorização da composição química dos fluidos que a circundam. Adicionalmente, reforçam o papel crucial que esta barreira desempenha na regulação do transporte de substâncias para o cérebro. No futuro, será importante continuar a explorar o papel de outros TAS2Rs após ativação pelos seus ligandos no cérebro, assim como, na regulação dos mecanismos de transporte e, também de destoxificação existentes na barreira sangue-líquido cefalorraquidiano. Este conhecimento irá certamente contribuir para uma melhoria dos processos terapêuticos utilizados para entrega de fármacos ao SNC.

碩士
國立屏東科技大學
生物科技系
106
Exopolysaccharides (EPS) from Bacillus amyloliquefaciens have been demonstarted to have a hypoglycemic effect. Meanwhile, the EPS solution tastes bitter, while the activation of bitter taste receptors has been suggested to be associated with the regulation of blood glucose. Therefore, the purpose of this study is to characterize whether the hypoglycemic effect of EPS is associated with the activation of bitter taste receptors (TAS2R). NCI-H716, and IEC-18 cells were used as a model of the intestinal enteroendocrine cells, and that of intestinal epithelial cells, respectively. When NCI-H716 cells were treated with EPS, an increase in the intracellular Ca2+ concentration was observed. The secretion of GLP-1 from NCI-H716 cells was also promoted after EPS treatment. Meanwhile, when IEC-18 cells were treated with EPS, glucose consumption of the cells was obviously enhanced, and AKT, a signaling factor in the insulin-signaling pathway, was activated in the cells. These results supported that EPS might activate TAS2R in intestinal enteroendocrine cells, leading to the secretion of GLP-1, a hormone promoting the secretion of insulin. Meanwhile, EPS activated effectors in the insulin-signaling pathway and increased the glucose consumption of intestinal epithelial cells. These effects of EPS on the intestinal cells likely play roles in the hypoglycemic function of EPS. Keywords : Exopolysaccharides, Bacillus amyloliquefaciens, hypoglycemic effect, bitter taste receptor, intestinal cells