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.     

Two families of candidate taste receptors in fishes

Vertebrates receive tastants, such as sugars, amino acids, and nucleotides, via taste bud cells in epithelial tissues. In mammals, two families of G protein-coupled receptors for tastants are expressed in taste bud cells-T1Rs for sweet tastants and umami tastants (l-amino acids) and T2Rs for bitter tastants. Here, we report two families of candidate taste receptors in fish species, fish T1Rs and T2Rs, which show significant identity to mammalian T1Rs and T2Rs, respectively. Fish T1Rs consist of three types: fish T1R1 and T1R3 that show the highest degrees of identity to mammalian T1R1 and T1R3, respectively, and fish T1R2 that shows almost equivalent identity to both mammalian T1R1 and T1R2. Unlike mammalian T1R2, fish T1R2 consists of two or three members in each species. We also identified two fish T2Rs that show low degrees of identity to mammalian T2Rs. In situ hybridization experiments revealed that fish T1R and T2R genes were expressed specifically in taste bud cells, but not in olfactory receptor cells. Fish T1R1 and T1R2 genes were expressed in different subsets of taste bud cells, and fish T1R3 gene was co-expressed with either fish T1R1 or T1R2 gene as in the case of mammals. There were also a significant number of cells expressing fish T1R2 genes only. Fish T2R genes were expressed in different cells from those expressing fish T1R genes. These results suggest that vertebrates commonly have two kinds of taste signaling pathways that are defined by the types of taste receptors expressed in taste receptor cells.     

Functional decline of sweet taste sensitivity of colobine monkeys

For many primates, sweet taste is palatable and is an indicator that the food contains carbohydrates, such as sugars and starches, as energy sources. However, we have found that Asian colobine monkeys (lutungs and langurs) have low sensitivity to various natural sugars. Sweet tastes are recognized when compounds bind to the sweet taste receptor TAS1R2/TAS1R3 in the oral cavity; accordingly, we conducted a functional assay using a heterologous expression system to evaluate the responses of Javan lutung (Trachypithecus auratus) TAS1R2/TAS1R3 to various natural sugars. We found that Javan lutung TAS1R2/TAS1R3 did not respond to natural sugars such as sucrose and maltose. We also conducted a behavioral experiment using the silvery lutung (Trachypithecus cristatus) and Hanuman langur (Semnopithecus entellus) by measuring the consumption of sugar-flavored jellies. Consistent with the functional assay results for TAS1R2/TAS1R3, these Asian colobine monkeys showed no preference for sucrose or maltose jellies. These results demonstrate that sweet taste sensitivity to natural sugars is low in Asian colobine monkeys, and this may be related to the specific feeding habits of colobine monkeys.     

The taste system in fishes and the effects of environmental variables

The adaptability of the taste system in fish has led to a large variety in taste bud morphology, abundance and distribution, as well as in taste physiology characteristics in closely related species with different modes of life and feeding ecology. However, the modifications evoked in the sense of taste, or gustation, particularly during ontogeny when fishes are subject to different environmental variables, remain poorly studied. This review paper focusses on current knowledge to show how plastic and resistant the taste system in fishes is to various external factors, linked to other sensory inputs and shifts in physiological state of individuals. Ambient water temperature is fundamental to many aspects of fish biology and taste preferences are stable to many substances, however, the taste-cell turnover rate strongly depends on water temperature. Taste preferences are stable within water salinity, which gives rise to the possibility that the taste system in anadromous and catadromous fishes will only change minimally after their migration to a new environment. Food-taste selectivity is linked to fish diet and to individual feeding experience as well as the motivation to feed evoked by attractive (water extracts of food) and repellent (alarm pheromone) odours. In contrast, starvation leads to loss of aversion to many deterrent substances, which explains the consumption by starving fishes of new objects, previously refused or just occasionally consumed. Food hardness can significantly modify the final feeding decision to swallow or to reject a grasped and highly palatable food item. Heavy metals, detergents, aromatic hydrocarbons and other water contaminants have the strongest and quickest negative effects on structure and function of taste system in fish and depress taste perception and ability of fishes to respond adequately to taste stimuli after short exposures. Owing to phenotypic plasticity, the taste system can proliferate and partially restore the ability of fishes to respond to food odour after a complete loss of olfaction. In general, the taste system, especially its functionality, is regarded as stable over the life of a fish despite any alteration in their environment and such resistance is vital for maintaining physiological homeostasis.     

Gut T1R3 sweet taste receptors do not mediate sucrose-conditioned flavor preferences in mice

Most mammals prefer the sweet taste of sugars, which is mediated by the heterodimeric T1R2+T1R3 taste receptor. Sugar appetite is also enhanced by the post-oral reinforcing actions of the nutrient in the gut. Here, we examined the contribution of gut T1R3 (either alone or as part of the T1R3+T1R3 receptor) to post-oral sugar reinforcement using a flavor-conditioning paradigm. We trained mice to associate consumption of a flavored solution (CS+) with intragastric (IG) infusions of a sweetener, and a different flavored solution (CS-) with IG infusions of water (23 h/day); then, we measured preference in a CS+ vs. CS- choice test. In experiment 1, we predicted that if activation of gut T1R3 mediates sugar reinforcement, then IG infusions of a nutritive (sucrose) or nonnutritive (sucralose) ligand for this receptor should condition a preference for the CS+ in B6 wild-type (WT) mice. While the mice that received IG sucrose infusions developed a strong preference for the CS+, those that received IG sucralose infusions developed a weak avoidance of the CS+. In experiment 2, we used T1R3 knockout (KO) mice to examine the necessity of gut T1R2+T1R3 receptors for conditioned flavor preferences. If intact gut T1R3 (or T1R2+T1R3) receptors are necessary for flavor-sugar conditioning, then T1R3 KO mice should not develop a sugar-conditioned flavor preference. We found that T1R3 KO mice, like WT mice, acquired a strong preference for the CS+ paired with IG sucrose infusions. The KO mice were also like WT mice in avoiding a CS+ flavor paired with IG sucralose infusions These findings provide clear evidence that gut T1R3 receptors are not necessary for sugar-conditioned flavor preferences or sucralose-induced flavor avoidance in mice.     

Preference conditioning produced by opioid active and inactive isomers of levorphanol and morphine in rat

Using taste and place preference conditioning, the present study examined the motivational properties produced in adult rats by systemic administration of (-) and (+) morphine, levorphanol, and dextrorphan. Conditioned place preference was stereospecific; it was only produced by the opioid receptor active isomers, levorphanol and (-) morphine. Similarly, a conditioned taste preference produced by a low dose of morphine was only seen with the active isomer. Conditioned taste aversion, however, was produced in a comparable dose range by both the active and the inactive isomers. In addition injections of inactive isomers also produced tolerance to the taste aversion produced by (-) morphine. Therefore, administration of both opioid active and inactive isomers of opioid agonists are unconditioned stimuli for the production of preference behaviors. In addition, it was concluded that the appetitive reinforcing properties of these drugs, seen as taste and place preferences, appear to require activation of specific opioid receptors, whereas the aversive effects, seen as taste aversion may also involve other mechanisms.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Lineage-specific expansions and contractions of the bitter taste receptor gene repertoire in vertebrates

The sense of bitter taste plays a critical role in how organisms avoid generally bitter toxic and harmful substances. Previous studies revealed that there were 25 intact bitter taste receptor (T2R) genes in humans and 34 in mice. However, because the recent chicken genome project reported only three T2R genes, it appears that extensive gene expansions occurred in the lineage leading to mammals or extensive gene contractions occurred in the lineage leading to birds. Here, I examined the T2R gene repertoire in placental mammals (dogs, Canis familiaris; and cows, Bos taurus), marsupials (opossums, Monodelphis domestica), amphibians (frogs, Xenopus tropicalis), and fishes (zebrafishes, Danio rerio; and pufferfishes, Takifugu rubripes) to investigate the birth-and-death process of T2R genes throughout vertebrate evolution. I show that (1) the first extensive gene expansions occurred before the divergence of mammals from reptiles/birds but after the divergence of amniotes (reptiles/birds/mammals) from amphibians, (2) subsequent gene expansions continuously took place in the ancestral mammalian lineage and the lineage leading to amphibians, as evidenced by the presence of 15, 18, 26, and 49 intact T2R genes in the dog, cow, opossum, and frog genome, respectively, and (3) contractions of the gene repertoire happened in the lineage leading to chickens. Thus, continuous gene expansions have shaped the T2R repertoire in mammals, but the contractions subsequent to the first round of expansions have made the chicken T2R repertoire narrow. These dramatic changes in the repertoire size might reflect the daily intake of foods from an external environment as a driving force of evolution.     

The chemosensory support of feeding behavior in precocial and altricial mammals during ontogeny]

The development of the structure and function of chemosensory apparatus of the tongue of mature and immature mammals was studied by scanning electron microscopy and in behavioral tests. Heterochronic development of receptors structures of dorsal surface of the tongue was established. At birth the chemoreceptors of body and root of the tongue were relatively mature in morphological aspects (the number of taste buds with pore). Taste pores in buds of anterior part of the tongue were found immediately at birth in mature and at the third week of postnatal period in immature animals. Behavioral tests found functional immaturity of chemosensory structures. Immature pups were able to recognize flavoured solutions only on the 7-10 day of age, and preference-aversion reactions of mature pups appeared to the third day of age. The data obtained are discussed in the respect of different ways of adaptation to food contacts with environment of the pups of mature and immature animals.     

Radiation-induced taste aversion: effects of radiation exposure level and the exposure-taste interval

Radiation-induced taste aversion has been suggested to possibly play a role in the dietary difficulties observed in some radiotherapy patients. In rats, these aversions can still be formed even when the radiation exposure precedes the taste experience by several hours. This study was conducted to examine whether increasing the radiation exposure level could extend the range of the exposure-taste interval that would still support the formation of a taste aversion. Separate groups of rats received either a 100 or 300 R gamma-ray exposure followed 1, 3, 6, or 24 h later by a 10-min saccharin (0.1% w/v) presentation. A control group received a sham exposure followed 1 h later by a 10-min saccharin presentation. Twenty-four hours following the saccharin presentation all rats received a series of twelve 23-h two-bottle preference tests between saccharin and water. The results indicated that the duration of the exposure-taste interval plays an increasingly more important role in determining the initial extent of the aversion as the dose decreases. The course of recovery from taste aversion seems more affected by dose than by the temporal parameters of the conditioning trial.     

Behavioral comparisons of the tastes of L-alanine and monosodium glutamate in rats

Recent research has implicated T1R1/T1R3 as the primary taste receptor in mammals for detecting L-amino acids, including L-monosodium glutamate (MSG) and L-alanine. Previous behavioral studies with rodents found only minimal evidence that these two substances share perceptual qualities, but those studies did not control for the taste of sodium associated with MSG. This study used several behavioral methods to compare the perceptual qualities of MSG and L-alanine in rats, using amiloride (a sodium channel blocker) to reduce the sodium component of MSG taste. Detection thresholds of L-alanine in rats ranged between 0.4 and 2.5 mM, with or without amiloride added, which are similar to threshold estimates for MSG. Conditioned taste aversion (CTA) found that rats showed strong cross-generalization of CTA between MSG and L-alanine when mixed with amiloride, indicating the two substances have similar perceptual qualities. Discrimination methods showed that rats easily discriminated between L-alanine and MSG unless the cue function of sodium was reduced. The discrimination became significantly more difficult at concentrations < 100 mM when amiloride was added to all stimuli and became even more difficult when NaCl was also added to L-alanine solutions to match the sodium concentrations of MSG. These results indicate that, perceptually, MSG and L-alanine have quite similar taste qualities and support the hypothesis that these two L-amino acids activate a common taste receptor. The differences in perceptual qualities also suggest separate afferent processing of one or both substances may also be involved.     

Identification of a novel member of the T1R family of putative taste receptors

In the gustatory system, the recognition of sugars, amino acids and bitter-tasting compounds is the function of specialized G protein-coupled receptors. Recently, two members of novel subfamily of G protein-coupled receptors were proposed to function as taste receptors based on their specific expression in taste receptor cells. Here, we report the identification of a third member, T1R3, of this family of receptors. T1R3 maps near the telomere of mouse chromosome 4 rendering it a candidate for the Sac locus, a primary determinant of sweet preference in mice. Consistent with its candidacy for the Sac locus, T1R3 displays taste receptor cell-specific expression. In addition, taster and non-taster strains of mouse harbor different alleles of T1R3.     

Functional expression of mammalian bitter taste receptors in Caenorhabditis elegans

Bitter taste has evolved as a central warning signal against the ingestion of potentially toxic substances appearing in the environment. The molecular events in the perception of bitter taste start with the binding of specific water-soluble molecules to G protein-coupled receptors (GPCR) called T2Rs and expressed at the surface of taste receptor cells. The functional characterisation of T2R receptors is far from been completed due to the difficulty to functionally express them in heterologous systems. Taking advantage of the parallelisms between the Caenorhabditis elegans (C. elegans) and mammalian GPCR signalling pathways, we developed a C. elegans-based expression system to express functional human and rodent GPCRs of the T2R family. We generated transgenic worms expressing T2Rs in ASI chemosensory neurons and performed behavioural assays using a variety of bitter tastants. As a proof of the concept, we generated transgenic worms expressing human T2R4 or its mouse ortholog T2R8 receptors, which respond to two bitter tastants previously characterised as their functional ligands, 6-n-propyl-2-thiouracil and denatoniun. As expected, expression of human T2R4 or its mouse ortholog T2R8 in ASI neurons counteracted the water-soluble avoidance to 6-n-propyl-2-thiouracil and denatoniun observed in control wild-type worms. The expression in ASI neurons of human T2R16, the ligand of which, phenyl-beta-d-glucopyranoside, belong to a chemically different group of bitter tastants, also counteracted the water-soluble avoidance to this compound observed in wild-type worms. These results indicate that C. elegans is a suitable heterologous expression system to express functional T2Rs providing a tool to efficiently search for specific taste receptor ligands and to extend our understanding of the molecular basis of gustation.     

Tests of adipsia and conditioned taste aversion following the intrahypothalamic injection of amylin.

Intrahypothalamic injection of amylin (AMY) was shown to reduce the intake of rat chow and water for 8 and 4 h, respectively, in schedule-fed rats. Amylin also reduced water intake to a much lesser degree in 24-h water-deprived rats. A test of the ability of AMY to form a conditioned taste aversion yielded no change in saccharin preference, as compared to controls treated with vehicle. These results suggest that although AMY has adipsic effects, the reduction in water is not of sufficient magnitude to cause the anorexia. In addition, the failure of AMY to support a conditioned taste aversion suggests that AMY does not cause anorexia by inducing malaise. Therefore, in addition to other metabolic effects, AMY may be involved in the control of food and water intake.     

Central mechanisms of roles of taste in reward and eating

Taste is unique among sensory systems in its innate association with mechanisms of reward and aversion in addition to its recognition of quality, e.g., sucrose is sweet and preferable, and quinine is bitter and aversive. Taste information is sent to the reward system and feeding center via the prefrontal cortices such as the mediodorsal and ventrolateral prefrontal cortices in rodents and the orbitofrontal cortex in primates. The amygdala, which receives taste inputs, also influences reward and feeding. In terms of neuroactive substances, palatability is closely related to benzodiazepine derivatives and beta-endorphin, both of which facilitate consumption of food and fluid. The reward system contains the ventral tegmental area, nucleus accumbens and ventral pallidum and finally sends information to the lateral hypothalamic area, the feeding center. The dopaminergic system originating from the ventral tegmental area mediates the motivation to consume palatable food. The actual ingestive behavior is promoted by the orexigenic neuropeptides from the hypothalamus. Even palatable food can become aversive and avoided as a consequence of a postingestional unpleasant experience such as malaise. The neural mechanisms of this conditioned taste aversion will also be elucidated.     

Nerve and behavioral responses of mice to various umami substances

Food contains various taste substances. Among them, umami substances play an important role with regard to the perception of the taste of food, but, few studies have examined the taste characteristics of representative umami substances other than monosodium L-glutamate (MSG). By conducting mouse behavioral studies (the 48-h 2-bottle preference test and the conditioned taste aversion test) and assessing gustatory nerve responses, we investigated the taste characteristics of unique umami substances, including sodium succinate, L-theanine, betaine, and the enantiomer of MSG, D-MSG. Furthermore, we examined the synergy of umami with inosine 5'-monophoshate (IMP). In the case of the mice, sodium succinate had an umami taste and showed strong synergy with IMP. L-theanine showed synergy with IMP but did not have an umami taste without IMP. In contrast, betaine did not have an umami taste or synergy with IMP. D-MSG might have weak synergy with IMP.     

Lineage-specific loss of function of bitter taste receptor genes in humans and nonhuman primates

Since the process of becoming dead genes or pseudogenes (pseudogenization) is irreversible and can occur rather rapidly under certain environmental circumstances, it is one plausible determinant for characterizing species specificity. To test this evolutionary hypothesis, we analyzed the tempo and mode of duplication and pseudogenization of bitter taste receptor (T2R) genes in humans as well as in 12 nonhuman primates. The results show that primates have accumulated more pseudogenes than mice after their separation from the common ancestor and that lineage-specific pseudogenization becomes more conspicuous in humans than in nonhuman primates. Although positive selection has operated on some amino acids in extracellular domains, functional constraints against T2R genes are more relaxed in primates than in mice and this trend has culminated in the rapid deterioration of the bitter-tasting capability in humans. Since T2R molecules play an important role in avoiding generally bitter toxic and harmful substances, substantial modification of the T2R gene repertoire is likely to reflect different responses to changes in the environment and to result from species-specific food preference during primate evolution.     

Effects of the long-term anosmia combined with vision deprivation on the taste sensitivity and feeding behavior of the rainbow trout <Emphasis Type="Italic">Parasalmo</Emphasis> (=<Emphasis Type="Italic">Oncorhynchus</Emphasis>) <Emphasis Type="Italic">mykiss</Emphasis>

We compared taste preferences, taste sensitivity, and behavior in testing food objects in the group of intact and two groups of sensory deprived rainbow trout Oncorhynchus mykiss yearlings. We demonstrated that long-term anosmia (for 9 months), as well as anosmia (for 9 months) combined with enucleation (object vision deprivation for 4 months), does not change the taste preference of fish for the agar pellets containing amino acids (L-alanine, L-proline, L-histidine, or glycine; 0.1 M). For all groups of fish, the threshold L-alanine concentration in pellets that caused a significant increase in consumption is 0.01 M. We showed that sensory deprived fish change their behavior of gustatory testing, namely, the rate of repeated snaps decreases as well as the pellet retention time in the mouth cavity. These results demonstrate that long-term anosmia combined with a partial vision deprivation does not significantly change the taste preferences and sensitivity in the fish that have no external taste buds. However, the observed reduction in the testing time of food objects and other changes in fish feeding behavior may suggest some functional alterations in the intraoral sensory systems (gustatory and/or tactile).     

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.     

Avoidance of potentially harmful food cannot be socially transmitted between rats

The social transmission of food preferences(STFP) is a behavioural task of olfactory memory, in which an observer rat learns safe food odours from a demonstrator rat, and shows preference for this odour in a subsequent choice test. However, previous studies have failed to detect the transmission of information about food of potential danger and food aversion using STFP test. In this study, we tested how demonstrators' health affects the exchange of odour information and whether observers can learn danger information from an unhealthy demonstrator. As expected, the observer rat formed an odour preference after interacting with a demonstrator rat that had just eaten food containing a new odour, however, odour preference rather than aversion was also formed after interacting with a demonstrator rat injected with LiCl(used to induce gastric malaise). Furthermore, anaesthetized demonstrator rats and half-anaesthetized demonstrator rats, which showed obvious motor deficits suggesting an unhealthy state, also socially transmitted food preferences to observers. These results suggest that the social transmission of food preferences task is independent of a demonstrators' health, and that information about dangerous foods cannot be transmitted using this behavioural task.