Genetic variation of umami taste genes in Koreans

Umami is a pleasant savoury taste imparted by glutamate, a type of amino acid, and ribonucleotides, including inosinate and guanylate, which occur naturally in many foods including meat, fish, vegetables and dairy products. A heterodimer of TAS1R1 and TAS1R3 is known to function as umami taste receptor in humans. To address the association between genetic polymorphism of TAS1R1 / TAS1R3 genes and individual difference in umami taste sensitivity, we have examined the entire coding region of these genes using PCR-mediated direct sequencing analysis. A total of 11 SNPs were identified from 98 unrelated Korean individuals and were in Hardy-Weinberg Equilibrium. Four out of 11 SNPs were found in the exons and two of them were nonsynonymous ones. These coding SNPs (cSNPs), p.A372T in TAS1R1 and p.C757R in TAS1R3 genes, were common in Koreans, so these will be useful resource for further studies to find a functional allele for individual variation to umami taste sensitivity in our population.     

Behavioral evidence for a role of alpha-gustducin in glutamate taste

The taste perception of monosodium glutamate (MSG) is termed 'umami'. Two putative taste receptors for glutamate have been identified, a truncated form of mGluR4 (taste-mGluR4) and the presumed heterodimer T1R1 + T1R3. Both receptors respond to glutamate when expressed in heterologous cells, but the G protein involved is not known. Galpha-Gustducin mediates the transduction of several bitter and sweet compounds; however, its role in umami has not been determined. We used standard two-bottle preference tests on alpha-gustducin knockout (KO) and wildtype (WT) mice to compare preferences for ascending concentrations of MSG and MSG + 5'-inosine monophosphate (IMP). A Latin Square was used to assign the order of tastants presented to each mouse. Statistical comparisons between KO and WT mice revealed that whereas WT mice preferred solutions of MSG and MSG + IMP over water, KO mice showed little preference for these stimuli. Denatonium and sucrose served as control stimuli and, as shown previously, WT mice prefered sucrose and avoided denatonium significantly more than did KO mice. Na?ve mice were also tested, and while prior exposure to taste stimuli influenced the magnitude of the preferences, experience did not change the overall pattern of intake. These data suggest that alpha-gustducin plays a role in glutamate taste.     

Variation in the human TAS1R taste receptor genes

We have performed a comprehensive evaluation of single-nucleotide polymorphisms (SNPs) and haplotypes in the human TAS1R gene family, which encodes receptors for sweet and umami tastes. Complete DNA sequences of TAS1R1-, TAS1R2-, and TAS1R3-coding regions, obtained from 88 individuals of African, Asian, European, and Native American origin, revealed substantial coding and noncoding diversity: polymorphisms are common in these genes, and polymorphic sites and SNP frequencies vary widely in human populations. The genes TAS1R1 and TAS1R3, which encode proteins that act as a dimer to form the umami (glutamate) taste receptor, showed less variation than the TAS1R2 gene, which acts as a dimer with TAS1R3 to form the sweet taste receptor. The TAS1R3 gene, which encodes a subunit common to both the sweet and umami receptors, was the most conserved. Evolutionary genetic analysis indicates that these variants have come to their current frequencies under natural selection during population growth and support the view that the coding sequence variants affect receptor function. We propose that human populations likely vary little with respect to umami perception, which is controlled by one major form of the receptor that is optimized for detecting glutamate but may vary much more with respect to sweet perception.     

A regulatory gene network related to the porcine umami taste receptor (TAS1R1/TAS1R3)

Taste perception plays an important role in the mediation of food choices in mammals. The first porcine taste receptor genes identified, sequenced and characterized, TAS1R1 and TAS1R3, were related to the dimeric receptor for umami taste. However, little is known about their regulatory network. The objective of this study was to unfold the genetic network involved in porcine umami taste perception. We performed a meta‐analysis of 20 gene expression studies spanning 480 porcine microarray chips and screened 328 taste‐related genes by selective mining steps among the available 12 320 genes. A porcine umami taste‐specific regulatory network was constructed based on the normalized coexpression data of the 328 genes across 27 tissues. From the network, we revealed the ‘taste module’ and identified a coexpression cluster for the umami taste according to the first connector with the TAS1R1/TAS1R3 genes. Our findings identify several taste‐related regulatory genes and extend previous genetic background of porcine umami taste.     

Taste preference synergy between glutamate receptor agonists and inosine monophosphate in rats

Monosodium glutamate (MSG) elicits a taste called umami and interacts synergistically with nucleotide monophosphates such as 5'-inosine monophosphate (IMP) to potentiate this taste intensity. Indeed, the synergistic interaction of nucleotide monophosphates and MSG is a hallmark of umami. We examined interactions between MSG and other taste stimuli, including IMP, by measuring the lick rates of non-deprived rats during 30 s trials. To control for non-linear psychophysical functions, the concentration of one taste stimulus in a binary mixture was systematically increased while the concentration of the second taste stimulus was decreased (stimulus substitution method). Synergy between two stimuli was detected if the lick rate for a binary mixture exceeded that expected from the sum of the lick rates for each stimulus alone. In initial experiments, taste synergy was observed when rats were presented with mixtures of MSG and IMP but not with mixtures of MSG and sucrose. In subsequent experiments, glutamate receptor agonists other than MSG were presented with IMP to test for taste synergy. No evidence of synergy was seen when rats were presented with mixtures of IMP and kainic acid or IMP and N:-methyl-D-aspartate. However, taste synergy between IMP and L-AP4, a potent agonist at mGluR4 receptors, was observed. These results suggest that a metabotropic glutamate receptor similar to mGluR4 may be involved in the taste synergy that characterizes umami.     

Umami changes intracellular Ca2+ levels using intracellular and extracellular sources in mouse taste receptor cells

Recently, candidates for umami receptors have been identified in taste cells, but the precise transduction mechanisms of the downstream receptor remain unknown. To investigate how intracellular Ca(2+) increases in the umami transduction pathway, we measured changes in intracellular Ca(2+) levels in response to umami stimuli monosodium glutamate (MSG), IMP, and MSG + IMP in mouse taste receptor cells (TRCs) by Ca(2+) imaging. Even when extracellular Ca(2+) was absent, 1/3 of umami-responsive TRCs exhibited increased intracellular Ca(2+) levels. When intracellular Ca(2+) was depleted, half of the TRCs retained their response to umami. These results suggest that umami-responsive TRCs increase their intracellular Ca(2+) levels through two pathways: by releasing Ca(2+) from intracellular stores and by an influx of Ca(2+) from extracellular sources. We conclude that the Ca(2+) influx from extracellular source might play an important role in the synergistic effect between MSG and IMP.     

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.     

Binding of glutamate to the umami receptor

The umami taste receptor is a heterodimer composed of two members of the T1R taste receptor family: T1R1 and T1R3. It detects glutamate in humans, and is a more general amino acid detector in other species. We have constructed homology models of the ligand binding domains of the human umami receptor (based on crystallographic structures of the metabotropic glutamate receptor of the central nervous system). We have carried out molecular dynamics simulations of the ligand binding domains, and we find that the likely conformation is that T1R1 receptor protein exists in the closed conformation, and T1R3 receptor in the open conformation in the heterodimer. Further, we have identified the important binding interactions and have made an estimate of the relative free energies associated with the two glutamate binding sites.     

Gustatory neural responses to umami taste stimuli in C57BL/6ByJ and 129P3/J mice

In long-term two-bottle tests, mice from the C57BL/6ByJ (B6) strain drink more monosodium L-glutamate (MSG) and inosine-5'-monophosphate (IMP) compared with mice from the 129P3/J (129) strain. The goal of this study was to assess the role of afferent gustatory input in these strain differences. We measured integrated responses of the mouse chorda tympani and glossopharyngeal nerves to lingual application of compounds that evoke umami taste in humans: MSG, monoammonium L-glutamate (NH(4) glutamate), IMP and guanosine-5'-monophosphate (GMP) and also to other taste stimuli. Chorda tympani responses to MSG and NH(4) glutamate were similar in B6 and 129 mice. Chorda tympani responses to IMP and GMP were lower in B6 than in 129 mice. Responses to umami stimuli in the glossopharyngeal nerve did not differ between the B6 and 129 strains. Responses to MSG, IMP and GMP were not affected by sodium present in these compounds because B6 and 129 mice had similar neural taste responses to NaCl. This study has demonstrated that the increased ingestive responses to the umami stimuli in B6 mice are accompanied by either unchanged or decreased neural responses to these stimuli. Lack of support for the role of the chorda tympani or glossopharyngeal nerves in the enhanced consumption of MSG and IMP by B6 mice suggests that it is due to some other factors. Although results of our previous study suggest that postingestive effects of MSG can affect its intake, contribution of other gustatory components (e.g. greater superficial petrosal nerve or central gustatory processing) to the strain differences in consumption of umami compounds also cannot be excluded. Strain differences in gustatory neural responses to nucleotides but not glutamate suggest that these compounds may activate distinct taste transduction mechanisms.     

Synergistic responses of the chorda tympani to mixtures of umami and sweet substances in rats

It has been known that umami substances such as monosodium L-glutamate (MSG) and 5'-inosine monophosphate (IMP) elicit a unique taste called 'umami' in humans. One of the characteristics of the umami taste is synergism: the synergistic enhancement of the magnitude of response produced by the addition of 5'-ribonucleotides to MSG. In addition to this well-documented synergism, we report here for the first time on another type of synergism between a glutamate receptor agonist, L-AP4, and sweet substances, by analyzing the chorda tympani responses in rats. The results are as follows: (i) when L-AP4 was mixed with one of the sweet substances, such as sucrose, glucose, fructose and maltose, large synergistic responses were observed. (ii) These synergistic responses, except to L-AP4 + sucrose, were not suppressed by sweet taste suppressants, gurmarin and pronase E. (iii) These synergistic responses were not suppressed by either metabotropic or ionotropic glutamate receptor antagonists. (iv) Fibers that responded well to the binary mixtures of L-AP4 and sweet substances also responded well to NaCl and HCl, but very weakly to sucrose. These findings are different from the characteristics of synergism between glutamate and IMP. The multiple transduction mechanisms for the umami taste in rat taste cells are discussed.     

L-Amino Acids Elicit Diverse Response Patterns in Taste Sensory Cells: A Role for Multiple Receptors

Umami, the fifth basic taste, is elicited by the L-amino acid, glutamate. A unique characteristic of umami taste is the response potentiation by 5’ ribonucleotide monophosphates, which are also capable of eliciting an umami taste. Initial reports using human embryonic kidney (HEK) cells suggested that there is one broadly tuned receptor heterodimer, T1r1+T1r3, which detects L-glutamate and all other L-amino acids. However, there is growing evidence that multiple receptors detect glutamate in the oral cavity. While much is understood about glutamate transduction, the mechanisms for detecting the tastes of other L-amino acids are less well understood. We used calcium imaging of isolated taste sensory cells and taste cell clusters from the circumvallate and foliate papillae of C57BL/6J and T1r3 knockout mice to determine if other receptors might also be involved in detection of L-amino acids. Ratiometric imaging with Fura-2 was used to study calcium responses to monopotassium L-glutamate, L-serine, L-arginine, and L-glutamine, with and without inosine 5’ monophosphate (IMP). The results of these experiments showed that the response patterns elicited by L-amino acids varied significantly across taste sensory cells. L-amino acids other than glutamate also elicited synergistic responses in a subset of taste sensory cells. Along with its role in synergism, IMP alone elicited a response in a large number of taste sensory cells. Our data indicate that synergistic and non-synergistic responses to L-amino acids and IMP are mediated by multiple receptors or possibly a receptor complex.     

Human genetic polymorphisms in T1R1 and T1R3 taste receptor subunits affect their function

Umami is the typical taste induced by monosodium glutamate (MSG), which is thought to be detected by the heterodimeric G protein-coupled receptor, T1R1 and T1R3. Previously, we showed that MSG detection thresholds differ substantially between individuals and we further showed that nontaster and hypotaster subjects are associated with nonsynonymous single polymorphisms occurring in the T1R1 and T1R3 genes. Here, we show using functional expression that both amino acid substitutions (A110V and R507Q) in the N-terminal ligand-binding domain of T1R1 and the 2 other ones (F749S and R757C), located in the transmembrane domain of T1R3, severely impair in vitro T1R1/T1R3 response to MSG. A molecular model of the ligand-binding region of T1R1/T1R3 provides a mechanistic explanation supporting functional expression data. The data presented here support causal relations between the genotype and previous in vivo psychophysical studies in human evaluating sensitivity to MSG.     

Differentiated adaptive evolution,episodic relaxation of selective constraints,and pseudogenization of umami and sweet taste genes <Emphasis Type="Italic">TAS1Rs</Emphasis> in catarrhine primates

Background

Umami and sweet tastes are two important basic taste perceptions that allow animals to recognize diets with nutritious carbohydrates and proteins, respectively. Until recently, analyses of umami and sweet taste were performed on various domestic and wild animals. While most of these studies focused on the pseudogenization of taste genes, which occur mostly in carnivores and species with absolute feeding specialization, omnivores and herbivores were more or less neglected. Catarrhine primates are a group of herbivorous animals (feeding mostly on plants) with significant divergence in dietary preference, especially the specialized folivorous Colobinae. Here, we conducted the most comprehensive investigation to date of selection pressure on sweet and umami taste genes (TAS1Rs) in catarrhine primates to test whether specific adaptive evolution occurred during their diversification, in association with particular plant diets.

Results

We documented significant relaxation of selective constraints on sweet taste gene TAS1R2 in the ancestral branch of Colobinae, which might correlate with their unique ingestion and digestion of leaves. Additionally, we identified positive selection acting on Cercopithecidae lineages for the umami taste gene TAS1R1, on the Cercopithecinae and extant Colobinae and Hylobatidae lineages for TAS1R2, and on Macaca lineages for TAS1R3. Our research further identified several site mutations in Cercopithecidae, Colobinae and Pygathrix, which were detected by previous studies altering the sensitivity of receptors. The positively selected sites were located mostly on the extra-cellular region of TAS1Rs. Among these positively selected sites, two vital sites for TAS1R1 and four vital sites for TAS1R2 in extra-cellular region were identified as being responsible for the binding of certain sweet and umami taste molecules through molecular modelling and docking.

Conclusions

Our results suggest that episodic and differentiated adaptive evolution of TAS1Rs pervasively occurred in catarrhine primates, most concentrated upon the extra-cellular region of TAS1Rs.

     

An analysis of 5'-inosine and 5'-guanosine monophosphate taste in rats

Inosine monophosphate (IMP) and guanosine monophosphate (GMP) elicit an umami taste in humans and synergistically increase the intensity of the umami taste of monosodium glutamate (MSG). Conditioned taste aversion (CTA) studies in rodents indicate that these nucleotides and MSG elicit quite similar tastes, but recent physiological evidence suggests that these nucleotides and MSG may not activate the same population of taste receptors and therefore may not elicit identical taste qualities. This study reports the findings of several behavioral experiments with rats that compared the taste properties of IMP and GMP with each other and with those of MSG. Well-trained rats were able to detect both nucleotides at nanomolar concentrations, but they did not respond to either nucleotide in two-bottle preference tests or brief-access CTA tests at concentrations less than 0.5 mM. Discrimination experiments found that the tastes of these nucleotides could not be discriminated from each other, but both could be discriminated from MSG, even when the taste of Na(+) was controlled. Overall, these experiments indicate the taste properties of the two 5'-ribonucleotides are quite similar to each other, and even though they may elicit an umami sensation, these sensations are not identical to the taste of MSG.     

Studies on after-taste of various taste stimuli in humans

This series of experiments was performed in order to evaluatethe physiological characteristics and patterns of after-tasteof various taste substances. The durations of after-taste followingmonosodium glutamate (MSG), inosine-5'-monophosphate (IMP) andguanosine-5'-monophosphate (GMP) (umami substances) were longerthan those for sucrose, NaCl, tartaric acid and quinine-HCIat concentrations corresponding to the recognition threshold.The periods of after-taste of solutions of MSG and IMP, andMSG and GMP, were longer than those for the single componentsolutions. Most subjects recognized sucrose as sweet, NaCl assalty, tartaric acid as sour and quinine-HCI as bitter, bothin terms of immediate taste and after-taste. According to thepatterns of after-taste for umami substances, the subjects weredivided into three groups. In group A, umami (appealing, savorytaste in Japanese cuisine) was the main quality of the after-tastesensation; in group B, an indefinite, equivocal taste was thecharacteristic quality of the aftertaste; and no differencewas reported in group C between the immediate taste and after-taste.These results suggest that the characteristics of after-tastefor MSG, IMP and GMP are different from those of the four basictastes.     

Sucrose and monosodium glutamate taste thresholds and discrimination ability of T1R3 knockout mice

Molecular and behavioral studies have identified heterodimers of the T1R family as receptors for detecting the tastes of sweet (T1R2 + T1R3) and umami (T1R1 + T1R3). However, behavioral studies have reported conflicting findings with T1R3 knockout (KO) mice. One study showed a complete or nearly complete loss of preference for sweet and umami substances by KO mice, whereas KO mice in another study showed only a partial reduction in preferences for sucrose and monosodium glutamate (MSG), the prototypical umami substance. The present experiments used psychophysical methods to assess how sensitive T1R1-KO mice are to sucrose and MSG and discrimination methods to determine if these mice could distinguish between the tastes of sucrose and MSG. Detection thresholds of T1R3-KO mice and wild-type (WT) C57Bl mice were nearly identical for sucrose and MSG. Mice of both genotypes were easily able to discriminate between the tastes of sucrose and MSG. When amiloride (a sodium channel blocker) was added to all solutions to reduce the taste of Na+, discrimination accuracy of both genotypes of mice decreased but more so for the T1R3-KO mice than the WT mice. However, even when the sodium taste of MSG was neutralized, both genotypes could still discriminate between the two substances well above chance performance. These results suggest that sucrose and MSG can be detected by taste receptors other than T1R2 + T1R3 and T1R1 + T1R3 and that the conflicts between the previous studies may have been due to the methodological limitations.     

Interaction Between Umami Peptide and Taste Receptor T1R1/T1R3

The umami taste receptor is a heterodimer composed of two members of the T1R taste receptor family: T1R1 and T1R3. The homology models of the ligand binding domains of the human umami receptor have been constructed based on crystallographic structures of the taste receptor of the central nervous system. Furthermore, the molecular simulations of the ligand binding domain show that the likely conformation was that T1R1 protein exists in the closed conformation, and T1R3 in the open conformation in the heterodimer. The molecular docking study of T1R1 and T1R3 in complex with four peptides, including Lys–Gly–Asp–Glu–Ser–Leu–Leu–Ala, Ser–Glu–Glu, G1u–Ser, and Asp–Glu–Ser, displayed that the amino acid residue of SER146 and Glu277 in T1R3 may play great roles in the synergism of umami taste. This docking result further validated the robustness of the model. In the paper, binding of umami peptide and the T1R1/T1R3 receptor was first described and the interaction is the base of umami activity theory.     

Design,synthesis, and taste evaluation of a high-intensity umami-imparting oxazole-based compound

Umami taste is imparted predominantly by monosodium glutamate (MSG) and 5′-ribonucleotides. Recently, several different classes of hydrophobic umami-imparting compounds, the structures of which are quite different from MSG, have been reported. To obtain a novel umami-imparting compound, N-cinnamoyl phenethylamine was chosen as the lead compound, and a rational structure-optimization study was conducted on the basis of the pharmacophore model of previously reported compounds. The extremely potent umami-imparting compound 2-[[[2-[(1E)-2-(1,3-benzodioxol-5-yl)ethenyl]-4-oxazolyle]methoxy]methyl]pyridine, which exhibits 27,000 times the umami taste of MSG, was found. Its terminal pyridine residue and linear structure are suggested to be responsible for its strong activity. The time taken to reach maximum taste intensity exhibited by it, as determined by the time-intensity method, is 22.0 s, whereas the maximum taste intensity of MSG occurs immediately. This distinct difference in the time-course taste profile may be due to the hydrophobicity and strong receptor affinity of the new compound.     

Obese Women Have Lower Monosodium Glutamate Taste Sensitivity and Prefer Higher Concentrations Than Do Normal‐weight Women

The goal of this study was to determine whether obese women exhibit altered umami and sweet taste perception compared to normal‐weight women. A total of 57 subjects (23 obese and 34 normal weight) participated in a 2‐day study separated by 1 week. Half of the women in each group were evaluated using monosodium glutamate (MSG; prototypical umami stimulus) on the first test day and sucrose on the second test day; the order was reversed for the remaining women. We used two‐alternative forced‐choice staircase procedures to measure taste detection thresholds, forced‐choice tracking technique to measure preferences, the general Labeled Magnitude Scale (gLMS) to measure perceived intensity of suprathreshold concentrations, and a triangle test to measure discrimination between 29 mmol/l MSG and 29 mmol/l NaCl. Obese women required higher MSG concentrations to detect a taste and preferred significantly higher MSG concentrations in a soup‐like vehicle. However, their perception of MSG at suprathreshold concentrations, their ability to discriminate MSG from salt, and their preference for sucrose were similar to that observed in normal‐weight women. Regardless of their body weight category, 28% of the women did not discriminate 29 mmol/l MSG from 29 mmol/l NaCl (nondiscriminators). Surprisingly, we found that, relative to discriminators, nondiscriminators perceived less savoriness when tasting suprathreshold MSG concentrations and less sweetness from suprathreshold sucrose concentrations but had similar MSG and sucrose detection thresholds. Taken together, these data suggest that body weight is related to some components of umami taste and that different mechanisms are involved in the perception of threshold and suprathreshold MSG concentrations.     

A Comparison of Collection Techniques for Gene Expression Analysis of Human Oral Taste Tissue

Variability in human taste perception is associated with both genetic and environmental factors. The influence of taste receptor expression on this variability is unknown, in part, due to the difficulty in obtaining human oral tissue that enables quantitative expression measures of taste genes. In a comparison of six current techniques (Oragene RNeasy Kit, Isohelix swab, Livibrush cytobrush, tongue saliva, cheek saliva collection, and fungiform papillae biopsy), we identify the fungiform papillae biopsy is the optimal sampling technique to analyse human taste gene expression. The fungiform papillae biopsy resulted in the highest RNA integrity, enabling amplification of all the assessed taste receptor genes (TAS1R1, TAS1R2, TAS1R3, SCNN1A and CD36) and taste tissue marker genes (NCAM1, GNAT3 and PLCβ2). Furthermore, quantitative expression was observed in a subset of taste genes assessed from the saliva collection techniques (cheek saliva, tongue saliva and Oragene RNA kit). These saliva collection techniques may be useful as a non-invasive alternative sampling technique to the fungiform papillae biopsy. Identification of the fungiform papillae biopsy as the optimal collection method will facilitate further research into understanding the effect of gene expression on variability in human taste perception.