Modulation of Sweet Taste by Umami Compounds via Sweet Taste Receptor Subunit hT1R2

Although the five basic taste qualities—sweet, sour, bitter, salty and umami—can be recognized by the respective gustatory system, interactions between these taste qualities are often experienced when food is consumed. Specifically, the umami taste has been investigated in terms of whether it enhances or reduces the other taste modalities. These studies, however, are based on individual perception and not on a molecular level. In this study we investigated umami-sweet taste interactions using umami compounds including monosodium glutamate (MSG), 5’-mononucleotides and glutamyl-dipeptides, glutamate-glutamate (Glu-Glu) and glutamate-aspartic acid (Glu-Asp), in human sweet taste receptor hT1R2/hT1R3-expressing cells. The sensitivity of sucrose to hT1R2/hT1R3 was significantly attenuated by MSG and umami active peptides but not by umami active nucleotides. Inhibition of sweet receptor activation by MSG and glutamyl peptides is obvious when sweet receptors are activated by sweeteners that target the extracellular domain (ECD) of T1R2, such as sucrose and acesulfame K, but not by cyclamate, which interact with the T1R3 transmembrane domain (TMD). Application of umami compounds with lactisole, inhibitory drugs that target T1R3, exerted a more severe inhibitory effect. The inhibition was also observed with F778A sweet receptor mutant, which have the defect in function of T1R3 TMD. These results suggest that umami peptides affect sweet taste receptors and this interaction prevents sweet receptor agonists from binding to the T1R2 ECD in an allosteric manner, not to the T1R3. This is the first report to define the interaction between umami and sweet taste receptors.     

The receptors for mammalian sweet and umami taste

Sweet and umami (the taste of monosodium glutamate) are the main attractive taste modalities in humans. T1Rs are candidate mammalian taste receptors that combine to assemble two heteromeric G-protein-coupled receptor complexes: T1R1+3, an umami sensor, and T1R2+3, a sweet receptor. We now report the behavioral and physiological characterization of T1R1, T1R2, and T1R3 knockout mice. We demonstrate that sweet and umami taste are strictly dependent on T1R-receptors, and show that selective elimination of T1R-subunits differentially abolishes detection and perception of these two taste modalities. To examine the basis of sweet tastant recognition and coding, we engineered animals expressing either the human T1R2-receptor (hT1R2), or a modified opioid-receptor (RASSL) in sweet cells. Expression of hT1R2 in mice generates animals with humanized sweet taste preferences, while expression of RASSL drives strong attraction to a synthetic opiate, demonstrating that sweet cells trigger dedicated behavioral outputs, but their tastant selectivity is determined by the nature of the receptors.     

Amiloride reduces the sweet taste intensity by inhibiting the human sweet taste receptor

In mammals, sweet taste perception is mediated by the heterodimeric G-protein-coupled receptor, T1R2/T1R3. An interesting characteristic of this sweet taste receptor is that it has multiple ligand binding sites. Although there have been several studies on agonists of sweet taste receptors, little is known about antagonists of these receptors. In this study, we constructed a cell line stably expressing the human sweet taste receptor (hT1R2/hT1R3) and a functional chimeric G-protein (hGα16gust44) using the Flp-In system for measuring the antagonistic activity against the receptor. This constructed cell line responded quite intensely and frequently to the compounds applied for activation of hT1R2/hT1R3. In the presence of 3 mM amiloride, the responses to sweet tastants such as sugar, artificial sweetener, and sweet protein were significantly reduced. The inhibitory activity of amiloride toward 1 mM aspartame was observed in a dose-dependent manner with an IC₅₀ value of 0.87 mM. Our analysis of a cell line expressing hT1R3 mutants (hT1R3-A733V or hT1R3-F778A) made us to conclude that the target site of amiloride is distinct from that of lactisole, a known sweet taste inhibitor. Our results strongly indicate that amiloride reduces the sweet taste intensity by inhibiting the human sweet taste receptor and also that this receptor has multiple inhibitor binding sites.     

Modulation of T1R chemosensory receptors for sweet nutrients - new paradigms in metabolic regulation

Recent studies have demonstrated that the sweet-sensing receptors T1R2/3, thought to be "taste receptors" specifically expressed in lingual system, are also expressed and involved in the chemo-detection of sweetening molecules circulating in other organs. Researches that focus on their roles in intestinal absorption, metabolic regulation and glucose homeostasis, in particular, are increasing. Indeed, the sweet-sensing receptor could provide a new therapeutic target for certain metabolic disorders and diseases like obesity and diabetes. If the natural and artificial sweeteners agonists are diverse and well known, the "anti-sweeteners" antagonistic molecules are a class of compounds that received very little attention until now. Their potential roles and pharmacological relevance outside the taste system are discussed. Moreover, the recent finding that 2 major classes of compounds belonging respectively to the fields of medicine (fibrates) and agriculture (phenoxy-herbicides) are potent inhibitors of human T1R3 receptor is reported, raising new questions about their potential impact on human metabolism.     

Taste-modifying sweet protein, neoculin, is received at human T1R3 amino terminal domain

This study examines taste reception of neoculin, a Curculigo latifolia sweet protein with taste-modifying activity which converts sourness to sweetness. Neoculin tastes sweet to humans, but not to mice, and is received by the human sweet taste receptor hT1R2-hT1R3. In the present study with calcium imaging analysis of HEK cells expressing human and mouse T1Rs, we demonstrated that hT1R3 is required for the reception of neoculin. Further experiments using human/mouse chimeric T1R3s revealed that the extracellular amino terminal domain (ATD) of hT1R3 is essential for the reception of neoculin. Although T1R2-T1R3 is known to have multiple potential ligand-binding sites to receive a wide variety of sweeteners, the present study is apparently the first to identify the ATD of hT1R3 as a new sweetener-binding region.     

Distinct contributions of T1R2 and T1R3 taste receptor subunits to the detection of sweet stimuli

Animals utilize hundreds of distinct G protein-coupled receptor (GPCR)-type chemosensory receptors to detect a diverse array of chemical signals in their environment, including odors, pheromones, and tastants. However, the molecular mechanisms by which these receptors selectively interact with their cognate ligands remain poorly understood. There is growing evidence that many chemosensory receptors exist in multimeric complexes, though little is known about the relative contributions of individual subunits to receptor functions. Here, we report that each of the two subunits in the heteromeric T1R2:T1R3 sweet taste receptor binds sweet stimuli though with distinct affinities and conformational changes. Furthermore, ligand affinities for T1R3 are drastically reduced by the introduction of a single amino acid change associated with decreased sweet taste sensitivity in behaving mice. Thus, individual T1R subunits increase the receptive range of the sweet taste receptor, offering a functional mechanism for phenotypic variations in sweet taste.     

Artificial Sweeteners Stimulate Adipogenesis and Suppress Lipolysis Independently of Sweet Taste Receptors

G protein-coupled receptors mediate responses to a myriad of ligands, some of which regulate adipocyte differentiation and metabolism. The sweet taste receptors T1R2 and T1R3 are G protein-coupled receptors that function as carbohydrate sensors in taste buds, gut, and pancreas. Here we report that sweet taste receptors T1R2 and T1R3 are expressed throughout adipogenesis and in adipose tissues. Treatment of mouse and human precursor cells with artificial sweeteners, saccharin and acesulfame potassium, enhanced adipogenesis. Saccharin treatment of 3T3-L1 cells and primary mesenchymal stem cells rapidly stimulated phosphorylation of Akt and downstream targets with functions in adipogenesis such as cAMP-response element-binding protein and FOXO1; however, increased expression of peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α was not observed until relatively late in differentiation. Saccharin-stimulated Akt phosphorylation at Thr-308 occurred within 5 min, was phosphatidylinositol 3-kinase-dependent, and occurred in the presence of high concentrations of insulin and dexamethasone; phosphorylation of Ser-473 occurred more gradually. Surprisingly, neither saccharin-stimulated adipogenesis nor Thr-308 phosphorylation was dependent on expression of T1R2 and/or T1R3, although Ser-473 phosphorylation was impaired in T1R2/T1R3 double knock-out precursors. In mature adipocytes, artificial sweetener treatment suppressed lipolysis even in the presence of forskolin, and lipolytic responses were correlated with phosphorylation of hormone-sensitive lipase. Suppression of lipolysis by saccharin in adipocytes was also independent of T1R2 and T1R3. These results suggest that some artificial sweeteners have previously uncharacterized metabolic effects on adipocyte differentiation and metabolism and that effects of artificial sweeteners on adipose tissue biology may be largely independent of the classical sweet taste receptors, T1R2 and T1R3.     

Capsaicin receptors are colocalized with sweet/bitter receptors in the taste sensing cells of circumvallate papillae

We examined co-localization of vanilloid receptor (VR1) with sweet receptors T1R2, T1R3, or bitter receptor T2R6 in taste receptor cells of rat circumvallate papillae. Tissue sections of rat circumvallate papillae were doubly reacted with anti-VR1 antibodies and anti-T1R2, anti-T1R3 or anti-T2R6 antibodies, using double-immunofluorescence histochemistry technique. Localizations of VR1, T1Rs and T2R6 in the vallate taste cells containing α-gustducin were also examined. VR1 immunoreactivities (-ir) were observed in subsets of taste cells in the circumvallate papillae, and 96–99% of the vallate taste cells exhibiting T1R2-, T1R3- or T2R6-ir co-exhibited VR1-ir. Approximately half of T2R6-ir cells (~49%), and 50–58% of T1Rs-ir cells, co-exhibited α-gustducin-ir in the vallate taste buds. About 58% of VR1-ir cells in the vallate exhibited α-gustducin-ir as well. Results support the idea that capsaicin may interact with the transduction pathways of sweet and bitter taste stimuli, possibly in mediation of its receptor VR1 localized in taste receptor cells. Additionally, the partial co-localization of α-gustducin with VR1 suggests that a tentative modulatory function of capsaicin in sweet and bitter transductions in the rat circumvallate comprises of both α-gustducin-mediated and non-mediated transduction pathways.     

The human bitter taste receptor, hTAS2R16, discriminates slight differences in the configuration of disaccharides

Sweetness and bitterness are key determinants of food acceptance and rejection, respectively. Sugars, such as sucrose and fructose, are generally recognized as sweet. However, not all sugars are sweet, and even anomers may have quite different tastes. For example, gentiobiose is bitter, whereas its anomer, isomaltose, is sweet. Despite this unique sensory character, the molecular basis of the bitterness of gentiobiose remains to be clarified. In this study, we used calcium imaging analysis of human embryonic kidney 293T cells that heterologously expressed human taste receptors to demonstrate that gentiobiose activated hTAS2R16, a bitter taste receptor, but not hT1R2/hT1R3, a sweet taste receptor. In contrast, isomaltose activated hT1R2/hT1R3. As a result, these anomers elicit different taste sensations. Mutational analysis of hTAS2R16 also indicated that gentiobiose and β-d-glucopyranosides, such as salicin share a common binding site of hTAS2R16.     

The liaison of sweet and savory

The sense of taste provides humans with necessary information about the composition and quality of food. For humans, five basic tastes are readily distinguishable and include sweet, bitter, salty, sour, and savory (or umami). Although each of these qualities has individualized transduction pathways, sweet and umami tastes are believed to share a common receptor element, the T1R3 receptor subunit. The two G-protein-coupled heteromer receptors that comprise an umami stimulus receptor (T1R1-T1R3) and a sweetener receptor (T1R2-T1R3) constitute a potential link between these two qualities of perception. While the role of the individual monomers in each human heteromer has been examined in vitro, very little is known of the implication of this research for human perception, or specifically, how sweet and savory taste perceptions may be connected. Using a psychophysical approach, we demonstrate that lactisole, a potent sweetness inhibitor that binds in vitro to hT1R3, also inhibits a significant portion of the perception of umami taste from monosodium glutamate. Following the molecular logic put forward by Xu et al. (2004, Proc. Natl Acad. Sci. USA, 101, 14258-14263), our psychophysical data support the in vitro hypothesis that the shared T1R3 monomer moderates the activation of both T1R2 and T1R1 in humans and impairs suprathreshold perception, respectively, of sweetness and, to a lesser degree, umaminess in the presence of lactisole.     

Sweet taste receptor gene variation and aspartame taste in primates and other species

Aspartame is a sweetener added to foods and beverages as a low-calorie sugar replacement. Unlike sugars, which are apparently perceived as sweet and desirable by a range of mammals, the ability to taste aspartame varies, with humans, apes, and Old World monkeys perceiving aspartame as sweet but not other primate species. To investigate whether the ability to perceive the sweetness of aspartame correlates with variations in the DNA sequence of the genes encoding sweet taste receptor proteins, T1R2 and T1R3, we sequenced these genes in 9 aspartame taster and nontaster primate species. We then compared these sequences with sequences of their orthologs in 4 other nontasters species. We identified 9 variant sites in the gene encoding T1R2 and 32 variant sites in the gene encoding T1R3 that distinguish aspartame tasters and nontasters. Molecular docking of aspartame to computer-generated models of the T1R2 + T1R3 receptor dimer suggests that species variation at a secondary, allosteric binding site in the T1R2 protein is the most likely origin of differences in perception of the sweetness of aspartame. These results identified a previously unknown site of aspartame interaction with the sweet receptor and suggest that the ability to taste aspartame might have developed during evolution to exploit a specialized food niche.     

Mammalian sweet taste receptors

The sense of taste provides animals with valuable information about the quality and nutritional value of food. Previously, we identified a large family of mammalian taste receptors involved in bitter taste perception (the T2Rs). We now report the characterization of mammalian sweet taste receptors. First, transgenic rescue experiments prove that the Sac locus encodes T1R3, a member of the T1R family of candidate taste receptors. Second, using a heterologous expression system, we demonstrate that T1R2 and T1R3 combine to function as a sweet receptor, recognizing sweet-tasting molecules as diverse as sucrose, saccharin, dulcin, and acesulfame-K. Finally, we present a detailed analysis of the patterns of expression of T1Rs and T2Rs, thus providing a view of the representation of sweet and bitter taste at the periphery.     

The anatomy of mammalian sweet taste receptors

All sweet‐tasting compounds are detected by a single G‐protein coupled receptor (GPCR), the heterodimer T1R2‐T1R3, for which no experimental structure is available. The sweet taste receptor is a class C GPCR, and the recently published crystallographic structures of metabotropic glutamate receptor (mGluR) 1 and 5 provide a significant step forward for understanding structure‐function relationships within this family. In this article, we recapitulate more than 600 single point site‐directed mutations and available structural data to obtain a critical alignment of the sweet taste receptor sequences with respect to other class C GPCRs. Using this alignment, a homology 3D‐model of the human sweet taste receptor is built and analyzed to dissect out the role of key residues involved in ligand binding and those responsible for receptor activation. Proteins 2017; 85:332–341. © 2016 Wiley Periodicals, Inc.     

Alteration of sweet taste in high-fat diet induced obese rats after 4 weeks treatment with exenatide

Exenatide, a glucagon-like peptide-1 (GLP-1) receptor agonist, is effective in inducing weight loss. The exact mechanisms are not fully understood. Reduced appetite and food intake may play important roles. Sweet taste contributes to food palatability, which promotes appetite. Interestingly, GLP-1 and its receptor are expressed in the taste buds of rodents and their interaction has an effect on mediating sweet taste sensitivity. Our aim was to investigate whether sweet taste will be changed after long term treatment with exenatide. The results showed that high-fat diet induced obese rats (HF-C) presented metabolic disorders in food intake, body weight, blood glucose and lipid metabolism compared with long term exenatide treated obese rats (EX) and normal chow fed control rats (NC). Meanwhile, greater preference for sweet taste was observed in HF-C rats but not in EX rats. Compared with NC rats, brain activities induced by sweet taste stimulation were stronger in HF-C rats, however these stronger activities were not found in EX rats. We further found reduced sweet taste receptor T1R3 in circumvallte taste buds of HF-C rats, while GLP-1 was increased. Besides, serum leptin was evaluated in HF-C rats with decreased leptin receptor expressed in taste buds. These changes were not observed in EX rats, which suggest them to be the underlying hormone and molecular mechanisms responsible for alterations in sweet taste of HF-C rats and EX rats. In summary, our results suggest that long term treatment with exenatide could benefit dietary obese rats partially by reversing sweet taste changes.     

The mechanism of interaction of sweet proteins with the T1R2-T1R3 receptor: evidence from the solution structure of G16A-MNEI

The mechanism by which sweet proteins elicit a response on the T1R2-T1R3 sweet taste receptor is still mostly unknown but has been so far related to the presence of "sweet fingers" on the protein surface able to interact with the same mechanism as that of low molecular mass sweeteners. In the search for the identification of sweet fingers, we have solved the solution structure of G16A MNEI, a structural mutant that shows a reduction of one order of magnitude in sweetness with respect to its parent protein, MNEI, a single-chain monellin. Comparison of the structures of wild-type monellin and its G16A mutant shows that the mutation does not affect the structure of potential glucophores but produces a distortion of the surface owing to the partial relative displacement of elements of secondary structure. These results show conclusively that sweet proteins do not possess a sweet finger and strongly support the hypothesis that the mechanism of interaction of sweet-tasting proteins with the recently identified T1R2-T1R3 GPC receptor is different from that of low molecular mass sweeteners.     

The history of sweet taste: not exactly a piece of cake

Understanding the molecular bases of sweet taste is of crucial importance not only in biotechnology but also for its medical implications, since an increasing number of people is affected by food-related diseases like, diabetes, hyperlipemia, caries, that are more or less directly linked to the secondary effects of sugar intake. Despite the interest paid to the field, it is only through the recent identification and functional expression of the receptor for sweet taste that new perspectives have been opened, drastically changing our approach to the development of new sweeteners. We shall give an overview of the field starting from the early days up to discussing the newest developments. After a review of early models of the active site, the mechanisms of interaction of small and macromolecular sweet molecules will be examined in the light of accurate modeling of the sweet taste receptor. The analysis of the homology models of all possible dimers allowed by combinations of the human T1R2 and T1R3 sequences of the sweet receptor and the closed (A) and open (B) conformations of the mGluR1 glutamate receptor shows that only 'type B' sites, either T1R2(B) and T1R3(B), can host the majority of small molecular weight sweeteners. Simultaneous binding to the A and B sites is not possible with two large sweeteners but is possible with a small molecule in site A and a large one in site B. This observation accounted for the first time for the peculiar phenomenon of synergy between some sweeteners. In addition to these two sites, the models showed an external binding site that can host sweet proteins.     

Molecular Mechanisms for Sweet-suppressing Effect of Gymnemic Acids

Gymnemic acids are triterpene glycosides that selectively suppress taste responses to various sweet substances in humans but not in mice. This sweet-suppressing effect of gymnemic acids is diminished by rinsing the tongue with γ-cyclodextrin (γ-CD). However, little is known about the molecular mechanisms underlying the sweet-suppressing effect of gymnemic acids and the interaction between gymnemic acids versus sweet taste receptor and/or γ-CD. To investigate whether gymnemic acids directly interact with human (h) sweet receptor hT1R2 + hT1R3, we used the sweet receptor T1R2 + T1R3 assay in transiently transfected HEK293 cells. Similar to previous studies in humans and mice, gymnemic acids (100 μg/ml) inhibited the [Ca²⁺]_i responses to sweet compounds in HEK293 cells heterologously expressing hT1R2 + hT1R3 but not in those expressing the mouse (m) sweet receptor mT1R2 + mT1R3. The effect of gymnemic acids rapidly disappeared after rinsing the HEK293 cells with γ-CD. Using mixed species pairings of human and mouse sweet receptor subunits and chimeras, we determined that the transmembrane domain of hT1R3 was mainly required for the sweet-suppressing effect of gymnemic acids. Directed mutagenesis in the transmembrane domain of hT1R3 revealed that the interaction site for gymnemic acids shared the amino acid residues that determined the sensitivity to another sweet antagonist, lactisole. Glucuronic acid, which is the common structure of gymnemic acids, also reduced sensitivity to sweet compounds. In our models, gymnemic acids were predicted to dock to a binding pocket within the transmembrane domain of hT1R3.     

Molecular mechanisms underlying the reception and transmission of sour taste information

Taste enables organisms to determine the properties of ingested substances by conveying information regarding the five basic taste modalities: sweet, salty, sour, bitter, and umami. The sweet, salty, and umami taste modalities convey the carbohydrate, electrolyte, and glutamate content of food, indicating its desirability and stimulating appetitive responses. The sour and bitter modalities convey the acidity of food and the presence of potential toxins, respectively, stimulating aversive responses to such tastes. In recent years, the receptors mediating sweet, bitter, and umami tastes have been identified as members of the T1R and T2R G-protein-coupled receptor families; however, the molecular mechanisms underlying sour taste detection have yet to be clearly elucidated. This review covers the molecular mechanisms proposed to mediate the detection and transmission of sour stimuli, focusing on polycystic kidney disease 1-like 3 (Pkd1l3), Pkd2l1, and carbonic anhydrase 4 (Car4).     

Taste receptor T1R3 is an essential molecule for the cellular recognition of the disaccharide trehalose

Recently, a sweet taste receptor family, the T1R family, that recognizes some carbohydrates including sucrose was identified. Although the T1R3 molecule is known to participate in heterodimers that are used as sweet- and umami-tasting receptors, there is no evidence that T1R3 alone recognizes similar ligands. We demonstrate for the first time that the candidate sweet taste receptor T1R3 is essential for the recognition and response to the disaccharide trehalose. Our system is a valuable tool not only for understanding the relationship between sweeteners and their receptors but also for exploring the diversities of their receptors, resulting in the design of new high-potency sweeteners.