Previous studies have shown that aging modifies taste sensitivity. However, the factors affecting the changes in taste sensitivity remain unclear. To investigate the cause of the age-related changes in taste sensitivity, we compared the peripheral taste detection systems in young and old mice. First, we examined whether taste sensitivity varied according to age using behavioral assays. We confirmed that the taste sensitivities to salty and bitter tastes decreased with aging. In other assays, the gustatory nerve responses to salty and sweet tastes increased significantly with aging, while those to bitter taste did not change. Thus, the profile of the gustatory nerve responses was inconsistent with the profile of the behavioral responses. Next, we evaluated the expressions of taste-related molecules in the taste buds. Although no apparent differences in the expressions of representative taste receptors were observed between the two age groups, the mRNA expressions of signaling effectors were slightly, but significantly, decreased in old mice. No significant differences in the turnover rates of taste bud cells were observed between the two age groups. Thus, we did not observe any large decreases in the expressions of taste-related molecules and turnover rates of taste bud cells with aging. Based on these findings, we conclude that changes in taste sensitivity with aging were not caused by aging-related degradation of peripheral taste organs. Meanwhile, the concentrations of several serum components that modify taste responses changed with age. Thus, taste signal-modifying factors such as serum components may have a contributing role in aging-related changes in taste sensitivity.

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 (hG(alpha)16gust44) 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 3mM amiloride, the responses to sweet tastants such as sugar, artificial sweetener, and sweet protein were significantly reduced. The inhibitory activity of amiloride toward 1mM aspartame was observed in a dose-dependent manner with an IC(50) 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.

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.

Nasal obstruction causes mouth breathing, and affects the growth and development of craniofacial structures, muscle function in the stomatognathic system, and the taste perceptive system. However, the detailed mechanism underlying the effects of nasal obstruction on taste perception has not been fully elucidated. In this study, we investigated this mechanism using the two-bottle taste preference test, immunohistological analysis, and quantification of the mRNA expression of taste-related molecules in the circumvallate papillae. Neonatal male Wistar rats were divided randomly into control and experimental groups. Rats in the experimental group underwent unilateral nasal obstruction by cauterization of the external nostril at the age of 8 days. Arterial oxygen saturation (SpO2) was recorded in awake rats using collar clip sensors. Taste preference for five basic taste solutions was evaluated. Immunohistochemical analysis and quantitative real-time polymerase chain reaction (RT-PCR) were conducted to evaluate the expressions of taste-related molecules in the taste cells of the circumvallate papillae. Body weights were similar between the two groups throughout the experimental period. The SpO2 in the 7- to 12-week-old rats in the experimental group was significantly lower than that in the age-matched rats in the control group. In the two-bottle taste preference test, the sensitivities to sweet taste decreased in the experimental group. The mRNA expression of T1R2, T1R3, α-gustducin, and PLCβ2 was significantly lower in the experimental group than in the control group as determined by quantitative RT-PCR, and the immunohistochemical staining for α-gustducin and PLCβ2 was less prominent. These findings suggest that nasal obstruction may affect sweet taste perception via the reduced expression of taste-related molecules in the taste cells in rat circumvallate papillae.

Taste information from type III taste cells to gustatory neurons is thought to be transmitted via synapses. However, the molecular mechanisms underlying taste transduction through this pathway have not been fully elucidated. In this study, to identify molecules that participate in synaptic taste transduction, we investigated whether complexins (Cplxs), which play roles in regulating membrane fusion in synaptic vesicle exocytosis, were expressed in taste bud cells. Among four Cplx isoforms, strong expression of Cplx2 mRNA was detected in type III taste cells. To investigate the function of CPLX2 in taste transduction, we observed taste responses in CPLX2-knockout mice. When assessed with electrophysiological and behavioral assays, taste responses to some sour stimuli in CPLX2-knockout mice were significantly lower than those in wild-type mice. These results suggested that CPLX2 participated in synaptic taste transduction from type III taste cells to gustatory neurons. A part of taste information is thought to be transmitted via synapses. However, the molecular mechanisms have not been fully elucidated. To identify molecules that participate in synaptic taste transduction, we investigated complexins (Cplxs) expression in taste bud cells. Strong expression of Cplx2 mRNA was detected in taste bud cells. Furthermore, taste responses to some sour stimuli in CPLX2- knockout mice were significantly lower than those in wild-type mice. These suggested that CPLX2 participated in synaptic taste transduction.

The gustatory system plays a critical role in sensing appetitive and aversive taste stimuli for evaluating food quality. Although taste preference is known to change depending on internal states such as hunger, a mechanistic insight remains unclear. Here, we examine the neuronal mechanisms regulating hunger-induced taste modification. Starved mice exhibit an increased preference for sweetness and tolerance for aversive taste. This hunger-induced taste modification is recapitulated by selective activation of orexigenic Agouti-related peptide (AgRP)-expressing neurons in the hypothalamus projecting to the lateral hypothalamus, but not to other regions. Glutamatergic, but not GABAergic, neurons in the lateral hypothalamus function as downstream neurons of AgRP neurons. Importantly, these neurons play a key role in modulating preferences for both appetitive and aversive tastes by using distinct pathways projecting to the lateral septum or the lateral habenula, respectively. Our results suggest that these hypothalamic circuits would be important for optimizing feeding behavior under fasting.

The gustatory system plays an important role in sensing appetitive and aversive tastes for evaluating food quality. In mice, taste signals are relayed by multiple brain regions, including the parabrachial nucleus (PBN) of the pons, before reaching the gustatory cortex via the gustatory thalamus. Recent studies show that taste information at the periphery is encoded in a labeled-line manner, such that each taste modality has its own receptors and neuronal pathway. In contrast, the molecular identity of gustatory neurons in the CNS remains unknown. Here, we show that SatB2-expressing neurons in the PBN play a pivotal role in sweet taste transduction. With cell ablation, in vivo calcium imaging, and optogenetics, we reveal that SatB2PBN neurons encode positive valance and selectively transmit sweet taste signals to the gustatory thalamus.

The polycystic kidney disease (PKD) 1L3-PKD2L1 channel is a candidate sour taste receptor expressed in mammalian taste receptor cells. Various acids are reported to activate PKD channels after the removal of the acid stimuli, but little information is available on the activation of these channels by acetic acid. It was difficult to analyze the PKD channel activation by acetic acid using Ca2+ imaging experiments because this acid induces a transient and nonspecific response in cultured cells. Here, we developed a novel method to evaluate PKD channel activation by acetic acid. Nonspecific responses were observed only over a short period after the application of acetic acid. In contrast, PKD channel activation evoked by acetic acid as well as citric acid was detected even at a later time point. This method revealed that PKD1L3-PKD2L1 channel activation by acetic acid was pH-dependent and occurred when the ambient pH was <3.1.

Taste perception plays an essential role in food selection. Umami (savory) tastes are sensed by a taste receptor complex, T1R1/T1R3, that detects proteinogenic amino acids.1 High sensitivity to l-glutamate (l-Glu) is a characteristic of human T1R1/T1R3, but the T1R1/T1R3 of other vertebrates does not consistently show this l-Glu response.1,2 Here, we demonstrate that the l-Glu sensitivity of T1R1/T1R3 is a derived state that has evolved repeatedly in large primates that rely on leaves as protein sources, after their divergence from insectivorous ancestors. Receptor expression experiments show that common amino acid substitutions at ligand binding sites that render T1R1/T1R3 sensitive to l-Glu occur independently at least three times in primate evolution. Meanwhile T1R1/T1R3 senses 5'-ribonucleotides as opposed to l-Glu in several mammalian species, including insectivorous primates. Our chemical analysis reveal that l-Glu is one of the major free amino acids in primate diets and that insects, but not leaves, contain large amounts of free 5'-ribonucleotides. Altering the ligand-binding preference of T1R1/T1R3 from 5'-ribonucleotides to l-Glu might promote leaf consumption, overcoming bitter and aversive tastes. Altogether, our results provide insight into the foraging ecology of a diverse mammalian radiation and help reveal how evolution of sensory genes facilitates invasion of new ecological niches.

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.

Some studies have reported a decline in taste sensitivities in patients with Alzheimer's disease. However, the detail remains unknown.

Umami taste perception in mammals is mediated by a heteromeric complex of two G-protein-coupled receptors, T1R1 and T1R3. T1R1/T1R3 exhibits species-dependent differences in ligand specificity; human T1R1/T1R3 specifically responds to L-Glu, whereas mouse T1R1/T1R3 responds more strongly to other L-amino acids than to L-Glu. The mechanism underlying this species difference remains unknown. In this study we analyzed chimeric human-mouse receptors and point mutants of T1R1/T1R3 and identified 12 key residues that modulate amino acid recognition in the human- and mouse-type responses in the extracellular Venus flytrap domain of T1R1. Molecular modeling revealed that the residues critical for human-type acidic amino acid recognition were located at the orthosteric ligand binding site. In contrast, all of the key residues for the mouse-type broad response were located at regions outside of both the orthosteric ligand binding site and the allosteric binding site for inosine-5'-monophosphate (IMP), a known natural umami taste enhancer. Site-directed mutagenesis demonstrated that the newly identified key residues for the mouse-type responses modulated receptor activity in a manner distinct from that of the allosteric modulation via IMP. Analyses of multiple point mutants suggested that the combination of two distinct determinants, amino acid selectivity at the orthosteric site and receptor activity modulation at the non-orthosteric sites, may mediate the ligand specificity of T1R1/T1R3. This hypothesis was supported by the results of studies using nonhuman primate T1R1 receptors. A complex molecular mechanism involving changes in the properties of both the orthosteric and non-orthosteric sites of T1R1 underlies the determination of ligand specificity in mammalian T1R1/T1R3.

To investigate aging-dependent changes in taste sensitivities, we performed behavioral tests regarding taste sensitivity among young and old SAMP1 mice. In this senescence-accelerated mice model, dramatic changes in taste sensitivities were observed at least 70 weeks old. As for in a brief access test, old mice showed significantly increased taste sensitivity to bitter, salty, sweet, and umami tastes. On the other hand, in a two-bottle test, avoidance of bitter and salty tastes increased, while preference for umami decreased with aging. To investigate the participation of peripheral taste detection systems in the observed changes, we analyzed both the expression of representative taste-related molecules and also turnover rates of taste bud cells. The mRNA expressions of the bitter taste receptor Tas2r105 and its coupled G protein gustducin were significantly decreased with aging. However, the majority of molecules tested did not show significant expression changes. In addition, no significant differences in the turnover rates of taste bud cells were observed between the two age groups. These results suggest that the changes in taste sensitivity of SAMP1 mice due to aging are caused by factors other than the deterioration of taste detection systems in the oral cavity.

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.

"Salty taste" sensation is evoked when sodium and chloride ions are present together in the oral cavity. The presence of an epithelial cation channel that receives Na+ has previously been reported. However, no molecular entity involving Cl- receptors has been elucidated. We report the strong expression of transmembrane channel-like 4 (TMC4) in the circumvallate and foliate papillae projected to the glossopharyngeal nerve, mediating a high-concentration of NaCl. Electrophysiological analysis using HEK293T cells revealed that TMC4 was a voltage-dependent Cl- channel and the consequent currents were completely inhibited by NPPB, an anion channel blocker. TMC4 allowed permeation of organic anions including gluconate, but their current amplitudes at positive potentials were less than that of Cl-. Tmc4-deficient mice showed significantly weaker glossopharyngeal nerve response to high-concentration of NaCl than the wild-type littermates. These results indicated that TMC4 is a novel chloride channel that responds to high-concentration of NaCl.

Taste-taste interactions often showed in human psychophysical studies. Considering that each tastant in foodstuffs individually stimulates its responsible gustatory systems to elicit relevant taste modalities, taste-taste interaction should be performed in taste receptor cell-based assay. While umami substances have been proposed to suppress the bitterness of various chemicals in human sensory evaluation, the bitter-umami interaction has not been explored in bitter taste receptors, TAS2Rs. We investigated umami-bitter taste interactions by presenting umami peptides with bitter substance (salicin) on Ca(2+)-flux signaling assay using hTAS2R16-expressing cells. Five representative umami peptides (Glu-Asp, Glu-Glu, Glu-Ser, Asp-Glu-Ser, and Glu-Gly-Ser) derived from soybean markedly attenuated the salicin-induced intracellular calcium influx in a time-dependent manner, respectively, while Gly-Gly, a tasteless peptide did not. The efficacies of Glu-Glu suppressing salicin-induced activation of hTAS2R16 were higher than that of probenecid, a specific antagonist of hTAS2R16. According to Ca(2+)-flux signaling assay using the mixtures of salicin and umami peptides, all five umami peptides suppressed salicin-induced intracellular calcium influx in a noncompetitive manner. These results may provide evidence that umami peptides suppress bitter taste via bitter taste receptor(s). This is the first report which defines the interaction between bitter and umami taste in taste receptor level.

G-protein-coupled receptors mediate the senses of taste, smell, and vision in mammals. Humans recognize thousands of compounds as bitter, and this response is mediated by the hTAS2R family, which is one of the G-protein-coupled receptors composed of only 25 receptors. However, structural information on these receptors is limited. To address the molecular basis of bitter tastant discrimination by the hTAS2Rs, we performed ligand docking simulation and functional analysis using a series of point mutants of hTAS2R16 to identify its binding sites. The docking simulation predicted two candidate binding structures for a salicin-hTAS2R16 complex, and at least seven amino acid residues in transmembrane 3 (TM3), TM5, and TM6 were shown to be involved in ligand recognition. We also identified the probable salicin-hTAS2R16 binding mode using a mutated receptor experiment. This study characterizes the molecular interaction between hTAS2R16 and beta-D-glucopyranoside and will also facilitate rational design of bitter blockers.

New World monkeys (NWMs) are unique in that they exhibit remarkable interspecific variation in color vision and feeding behavior, making them an excellent model for studying sensory ecology. However, it is largely unknown whether non-visual senses co-vary with feeding ecology, especially gustation, which is expected to be indispensable in food selection. Bitter taste, which is mediated by bitter taste receptors (TAS2Rs) in the tongue, helps organisms avoid ingesting potentially toxic substances in food. In this study, we compared the ligand sensitivities of the TAS2Rs of five species of NWMs by heterologous expression in HEK293T cells and calcium imaging.

Matured hop bitter acids (MHBA) are bitter acid oxides derived from hops, widely consumed as food ingredients to add bitterness and flavor in beers. Previous studies have suggested a potential gut-brain mechanism in which MHBA simulates enteroendocrine cells to produce cholecystokinin (CCK), a gastrointestinal hormone which activates autonomic nerves, resulting in body fat reduction and cognitive improvement; however, the MHBA recognition site on enteroendocrine cells has not been fully elucidated. In this study, we report that MHBA is recognized by specific human and mouse bitter taste receptors (human TAS2R1, 8, 10 and mouse Tas2r119, 130, 105) using a heterologous receptor expression system in human embryonic kidney 293T cells. In addition, knockdown of each of these receptors using siRNA transfection partially but significantly suppressed an MHBA-induced calcium response and CCK production in enteroendocrine cells. Furthermore, blocking one of the essential taste signaling components, transient receptor potential cation channel subfamily M member 5, remarkably inhibited the MHBA-induced calcium response and CCK production in enteroendocrine cells. Our results demonstrate that specific bitter taste receptor activation by MHBA drives downstream calcium response and CCK production in enteroendocrine cells. These findings reveal a mechanism by which food ingredients derived from hops in beer activate the gut-brain axis for the first time.

Brazilian green propolis is a popular health supplement because of its various biological properties. The ethanol extract of Brazilian green propolis (EEBP) is characteristic for its herb-like smell and unique pungent taste. However, the ingredients responsible for its pungency have not yet been identified. This study provides the first evidence that artepillin C is the main pungent ingredient in EEBP and that it potently activates human transient receptor potential ankyrin 1 (TRPA1) channels. EEBP was fractionated using column chromatography with a step gradient elution of an ethanol-water solution, and the fractions having the pungent taste were determined by sensory tests. HPLC analysis revealed that the pungent fraction was composed primarily of artepillin C, a prenylated derivative of cinnamic acid. Artepillin C was also identified as the pungent compound of EEBP by organoleptic examiners. Furthermore, the effects of artepillin C and other cinnamic acids found in EEBP on TRPA1 channels were examined by calcium imaging and plate reader-based assays in human TRPA1-expressing cells to investigate the molecular mechanisms underlying their pungent tastes. Artepillin C and baccharin activated the TRPA1 channel strongly, whereas drupanin caused a slight activation and p-coumaric acid showed no activation. Because the EC(50) values of artepillin C, baccharin, and allyl isothiocyanate were 1.8 µM, 15.5 µM, and 6.2 µM, respectively, artepillin C was more potent than the typical TRPA1 agonist allyl isothiocyanate. These findings strongly indicate that artepillin C is the main pungent ingredient in EEBP and stimulates a pungent taste by activating TRPA1 channels.