Loss of sweet taste despite the conservation of sweet receptor genes in insectivorous bats

The evolution of taste perception is usually associated with the ecology and dietary changes of organisms. However, the association between feeding ecology and taste receptor evolution is unclear in some lineages of vertebrate animals. One example is the sweet taste receptor gene Tas1r2. Previous analysis of partial sequences has revealed that Tas1r2 has undergone equally strong purifying selection between insectivorous and frugivorous bats. To test whether the sweet taste function is also important in bats with contrasting diets, we examined the complete coding sequences of both sweet taste receptor genes (Tas1r2 and Tas1r3) in 34 representative bat species. Although these two genes are highly conserved between frugivorous and insectivorous bats at the sequence level, our behavioral experiments revealed that an insectivorous bat (Myotis ricketti) showed no preference for natural sugars, whereas the frugivorous species (Rousettus leschenaultii) showed strong preferences for sucrose and fructose. Furthermore, while both sweet taste receptor genes are expressed in the taste tissue of insectivorous and frugivorous bats, our cell-based assays revealed striking functional divergence: the sweet taste receptors of frugivorous bats are able to respond to natural sugars whereas those of insectivorous bats are not, which is consistent with the behavioral preference tests, suggesting that functional evolution of sweet taste receptors is closely related to diet. This comprehensive study suggests that using sequence conservation alone could be misleading in inferring protein and physiological function and highlights the power of combining behavioral experiments, expression analysis, and functional assays in molecular evolutionary studies.

The sense of taste plays a crucial role in selecting and ingesting food items, and is therefore of fundamental importance for the survival and growth of animals (1, 2). Among the five basic tastes (sweet, umami, bitter, salty, and sour) in mammals (3, 4), the sweet/umami and bitter tastes are mediated by Tas1rs and Tas2rs, respectively, both of which are G protein-coupled receptors (1). The receptors of the sweet and umami tastes encoded by Tas1r genes are heterodimers. Specifically, Tas1r3 can bind to Tas1r2 to form the sweet taste receptor, which responds to dietary carbohydrates; Tas1r3 can also bind to Tas1r1 to form the umami taste receptor, which recognizes dietary proteins (5–7). The bitter taste receptors, encoded by Tas2r genes, mainly detect dietary toxins (8–10).The evolution of taste perception is usually associated with the ecology and dietary changes of organisms (11–13). A well-known case is the Tas2r gene family, which shows great variation in family size among species (11). In general, a higher number of Tas2r genes are found in herbivorous and insectivorous vertebrates compared to carnivorous species, indicating that the proportion of potential dietary toxins largely shapes the Tas2r repertoire size (11, 14). Another interesting example is the convergent pseudogenization of the umami-specific taste receptor gene (Tas1r1) in the giant panda and red panda (15, 16), two species that have also evolved a specialized bamboo diet that rendered the umami taste redundant (16). Similarly, pseudogenization of the sweet-specific taste receptor gene (Tas1r2) was detected in many carnivorous mammals that swallow food whole, which may have made the sweet taste useless in food choice (12, 17).Despite that such close links were observed between diet and taste receptor evolution, mismatches between feeding ecology and taste receptor evolution have also been identified in a number of vertebrates (18, 19). Such mismatches are exemplified by the sweet taste receptor gene Tas1r2 in bats. Among all mammalian orders, Chiroptera (bats) shows the highest diversity in terms of diet, which includes insects, small vertebrates, fruits, nectar, pollen, flowers, foliage, and even blood (20); bats thus offer an excellent model system for studying molecular signatures of dietary changes (21). Despite the clear dietary differences among bats, especially in the proportion of carbohydrates, our previous study of 42 insectivorous and frugivorous bat species found no inactivating mutations in exon 6 of Tas1r2 (22). Moreover, no significant variation in selective pressure on Tas1r2 was detected between these two groups with distinct diets, leading to the suggestion that diet may not play a major role in the evolution of Tas1r2 in bats (22). However, while a pseudogene is a good indicator of loss of original function, a complete and intact gene does not necessarily reflect the full function of a protein because functional loss could also result from mutations in regulatory regions that abolish gene expression, or from missense mutations in coding regions that lead to a change in protein structure (23). In other words, one cannot directly infer functional conservation from sequence conservation alone. As a result, the correlation between diet and sweet taste receptor evolution in bats remains inconclusive.In this study, we analyzed the full-length coding sequences of both sweet taste receptor genes (Tas1r2 and Tas1r3) in 34 bat species to test whether these two genes are highly conserved at the sequence level, as previously reported (22). We performed behavioral experiments to test whether behavioral taste preference for natural sugars is similar between insectivorous and frugivorous bat species. We next conducted gene expression experiments to test whether both sweet taste receptor genes are expressed in the taste tissue. Finally, we examined the responsiveness of the sweet taste receptors of insectivorous and frugivorous bats to three natural sugars using a cell-based functional assay with a heterologous expression system.     

Glucose transporters and ATP-gated K+ (KATP) metabolic sensors are present in type 1 taste receptor 3 (T1r3)-expressing taste cells

Although the heteromeric combination of type 1 taste receptors 2 and 3 (T1r2 + T1r3) is well established as the major receptor for sugars and noncaloric sweeteners, there is also evidence of T1r-independent sweet taste in mice, particularly so for sugars. Before the molecular cloning of the T1rs, it had been proposed that sweet taste detection depended on (a) activation of sugar-gated cation channels and/or (b) sugar binding to G protein-coupled receptors to initiate second-messenger cascades. By either mechanism, sugars would elicit depolarization of sweet-responsive taste cells, which would transmit their signal to gustatory afferents. We examined the nature of T1r-independent sweet taste; our starting point was to determine if taste cells express glucose transporters (GLUTs) and metabolic sensors that serve as sugar sensors in other tissues. Using RT-PCR, quantitative PCR, in situ hybridization, and immunohistochemistry, we determined that several GLUTs (GLUT2, GLUT4, GLUT8, and GLUT9), a sodium-glucose cotransporter (SGLT1), and two components of the ATP-gated K(+) (K(ATP)) metabolic sensor [sulfonylurea receptor (SUR) 1 and potassium inwardly rectifying channel (Kir) 6.1] were expressed selectively in taste cells. Consistent with a role in sweet taste, GLUT4, SGLT1, and SUR1 were expressed preferentially in T1r3-positive taste cells. Electrophysiological recording determined that nearly 20% of the total outward current of mouse fungiform taste cells was composed of K(ATP) channels. Because the overwhelming majority of T1r3-expressing taste cells also express SUR1, and vice versa, it is likely that K(ATP) channels constitute a major portion of K(+) channels in the T1r3 subset of taste cells. Taste cell-expressed glucose sensors and K(ATP) may serve as mediators of the T1r-independent sweet taste of sugars.     

Endocannabinoids selectively enhance sweet taste

Endocannabinoids such as anandamide [N-arachidonoylethanolamine (AEA)] and 2-arachidonoyl glycerol (2-AG) are known orexigenic mediators that act via CB₁ receptors in hypothalamus and limbic forebrain to induce appetite and stimulate food intake. Circulating endocannabinoid levels inversely correlate with plasma levels of leptin, an anorexigenic mediator that reduces food intake by acting on hypothalamic receptors. Recently, taste has been found to be a peripheral target of leptin. Leptin selectively suppresses sweet taste responses in wild-type mice but not in leptin receptor-deficient db/db mice. Here, we show that endocannabinoids oppose the action of leptin to act as enhancers of sweet taste. We found that administration of AEA or 2-AG increases gustatory nerve responses to sweeteners in a concentration-dependent manner without affecting responses to salty, sour, bitter, and umami compounds. The cannabinoids increase behavioral responses to sweet-bitter mixtures and electrophysiological responses of taste receptor cells to sweet compounds. Mice genetically lacking CB₁ receptors show no enhancement by endocannnabinoids of sweet taste responses at cellular, nerve, or behavioral levels. In addition, the effects of endocannabinoids on sweet taste responses of taste cells are diminished by AM251, a CB₁ receptor antagonist, but not by AM630, a CB₂ receptor antagonist. Immunohistochemistry shows that CB₁ receptors are expressed in type II taste cells that also express the T1r3 sweet taste receptor component. Taken together, these observations suggest that the taste organ is a peripheral target of endocannabinoids. Reciprocal regulation of peripheral sweet taste reception by endocannabinoids and leptin may contribute to their opposing actions on food intake and play an important role in regulating energy homeostasis.     

Taste cell-expressed α-glucosidase enzymes contribute to gustatory responses to disaccharides

The primary sweet sensor in mammalian taste cells for sugars and noncaloric sweeteners is the heteromeric combination of type 1 taste receptors 2 and 3 (T1R2+T1R3, encoded by Tas1r2 and Tas1r3 genes). However, in the absence of T1R2+T1R3 (e.g., in Tas1r3 KO mice), animals still respond to sugars, arguing for the presence of T1R-independent detection mechanism(s). Our previous findings that several glucose transporters (GLUTs), sodium glucose cotransporter 1 (SGLT1), and the ATP-gated K⁺ (K_ATP) metabolic sensor are preferentially expressed in the same taste cells with T1R3 provides a potential explanation for the T1R-independent detection of sugars: sweet-responsive taste cells that respond to sugars and sweeteners may contain a T1R-dependent (T1R2+T1R3) sweet-sensing pathway for detecting sugars and noncaloric sweeteners, as well as a T1R-independent (GLUTs, SGLT1, K_ATP) pathway for detecting monosaccharides. However, the T1R-independent pathway would not explain responses to disaccharide and oligomeric sugars, such as sucrose, maltose, and maltotriose, which are not substrates for GLUTs or SGLT1. Using RT-PCR, quantitative PCR, in situ hybridization, and immunohistochemistry, we found that taste cells express multiple α-glycosidases (e.g., amylase and neutral α glucosidase C) and so-called intestinal “brush border” disaccharide-hydrolyzing enzymes (e.g., maltase-glucoamylase and sucrase-isomaltase). Treating the tongue with inhibitors of disaccharidases specifically decreased gustatory nerve responses to disaccharides, but not to monosaccharides or noncaloric sweeteners, indicating that lingual disaccharidases are functional. These taste cell-expressed enzymes may locally break down dietary disaccharides and starch hydrolysis products into monosaccharides that could serve as substrates for the T1R-independent sugar sensing pathways.In humans, the heteromeric combination of type 1 taste receptors 2 and 3 (T1R2+T1R3, encoded by TAS1R2 and TAS1R3) forms a sweet taste receptor responsive to sugars (e.g., glucose, fructose, sucrose), noncaloric sweeteners (e.g., aspartame, sucralose, saccharin, acesulfame K, rebaudioside A), and protein sweeteners (e.g., monellin, thaumatin, and brazzein), but not polysaccharides (1). The mouse sweet receptor (T1R2+T1R3) also responds to sugars, some of the same noncaloric sweeteners (e.g., sucralose, saccharin, acesulfame K, rebaudioside A), but not the protein sweeteners or polysaccharides. It is well established from multiple studies that T1R2+T1R3 is the major sweet taste receptor for sugars and likely the only sweet taste receptor for noncaloric sweeteners. For example, heterologous expression of human or mouse T1R2+T1R3 receptors in cultured cells recapitulates the host organism’s response to sweeteners (2–4). KO mice lacking Tas1r2 or Tas1r3 have generally diminished responses to most sweet compounds as assessed by brief access lick assays, two bottle preference tests, and gustatory nerve recordings (5, 6).However, in some studies, Tas1r3 KO mice were found to still have significant behavioral and nerve responses to glucose and other sugars (5, 7). Many quantitative trait loci other than Tas1r3 contribute to sweet taste perception in mice (8, 9). From this we inferred the presence of a sweet-sensing pathway that is independent of T1R3 (5, 7). We showed that multiple glucose transporters (GLUT2, GLUT4, GLUT8, and GLUT9), sodium glucose cotransporter 1 (SGLT1), and ATP-gated K⁺ (K_ATP) channel subunits (KIR6.2 and SUR1) are present preferentially in the Tas1r3-expressing taste cells in mouse taste buds (10). Other groups (11–13) have confirmed some of these results. We proposed that the T1R-independent sweet pathway depends on uptake of glucose into Tas1r3-expressing taste cells, followed by its metabolism to ATP, which binds to K_ATP, closing the channel and depolarizing the sweet taste cell (10). The existence of two sweet pathways, both of which detect sugars, could explain why noncaloric sweeteners are fully cross-adapted by sugars, but sugars are only partially cross-adapted by noncaloric sweeteners (14, 15).However, this proposed alternative pathway does not, on its own, explain the remaining taste responses of Tas1r3 KO mice to the disaccharides maltose (5) and sucrose (5, 7). Dietary carbohydrates are hydrolyzed into constituent monosaccharides before uptake by enterocytes. Starch is partially hydrolyzed by extracellular enzymes, first in the oral cavity by salivary amylase (AMY1), and then in the small intestine by pancreatic amylase (AMY2). The end products of amylase-catalyzed starch hydrolysis are disaccharides like maltose and higher-molecular-weight oligomers of glucose; amylase cannot generate glucose from starch. Disaccharidases localized to the apical plasma membrane of enterocytes (brush border enzymes), such as maltase-glucoamylase (MGAM), sucrase-isomaltase (SIS), lactase (LCT), and trehalase (TREH) hydrolyze the disaccharides maltose, sucrose, lactose, and trehalose, respectively, to generate monosaccharides (16–19). Here, we used PCR, in situ hybridization, and immunohistochemistry to determine that multiple sugar- and starch-hydrolyzing enzymes are expressed in taste cells. We found that Mgam, Sis, Lct, Treh, Amy1, and neutral α-glucosidase C (Ganc) are all expressed in taste cells. The majority of Tas1r3-expressing taste cells express Mgam and Sis, as we previously showed for GLUTs and K_ATP. Furthermore, inhibition of MGAM and SIS specifically decreased gustatory nerve responses to the disaccharides sucrose and maltose. Our results indicate that the actions of these orally expressed digestive enzymes may contribute to the unique sweet taste of sucrose and other sugars by generating monosaccharide substrates for the T1R-independent sweet pathway.     

Different functional roles of T1R subunits in the heteromeric taste receptors

The T1R receptors, a family of taste-specific class C G protein-coupled receptors, mediate mammalian sweet and umami tastes. The structure-function relationships of T1R receptors remain largely unknown. In this study, we demonstrate the different functional roles of T1R extracellular and transmembrane domains in ligand recognition and G protein coupling. Similar to other family C G protein-coupled receptors, the N-terminal Venus flytrap domain of T1R2 is required for recognizing sweeteners, such as aspartame and neotame. The G protein coupling requires the transmembrane domain of T1R2. Surprisingly, the C-terminal transmembrane domain of T1R3 is required for recognizing sweetener cyclamate and sweet taste inhibitor lactisole. Because T1R3 is the common subunit in the sweet taste receptor and the umami taste receptor, we tested the interaction of lactisole and cyclamate with the umami taste receptor. Lactisole inhibits the activity of the human T1R1/T1R3 receptor, and, as predicted, blocked the umami taste of l-glutamate in human taste tests. Cyclamate does not activate the T1R1/T1R3 receptor by itself, but potentiates the receptor's response to l-glutamate. Taken together, these findings demonstrate the different functional roles of T1R3 and T1R2 and the presence of multiple ligand binding sites on the sweet taste receptor.     

Cholinergic chemosensory cells in the trachea regulate breathing

In the epithelium of the lower airways, a cell type of unknown function has been termed "brush cell" because of a distinctive ultrastructural feature, an apical tuft of microvilli. Morphologically similar cells in the nose have been identified as solitary chemosensory cells responding to taste stimuli and triggering trigeminal reflexes. Here we show that brush cells of the mouse trachea express the receptors (Tas2R105, Tas2R108), the downstream signaling molecules (α-gustducin, phospholipase C(β2)) of bitter taste transduction, the synthesis and packaging machinery for acetylcholine, and are addressed by vagal sensory nerve fibers carrying nicotinic acetylcholine receptors. Tracheal application of an nAChR agonist caused a reduction in breathing frequency. Similarly, cycloheximide, a Tas2R108 agonist, evoked a drop in respiratory rate, being sensitive to nicotinic receptor blockade and epithelium removal. This identifies brush cells as cholinergic sensors of the chemical composition of the lower airway luminal microenvironment that are directly linked to the regulation of respiration.     

What Does Diabetes “Taste” Like?

The T1R2 (taste type 1 receptor, member 2)/T1R3 (taste type 1 receptor, member 3) sweet taste receptor is expressed in taste buds on the tongue, where it allows the detection of energy-rich carbohydrates of food. This single receptor responds to all compounds perceived as sweet by humans, including natural sugars and natural and artificial sweeteners. Importantly, the T1R2/T1R3 sweet taste receptor is also expressed in extra-oral tissues, including the stomach, pancreas, gut, liver, and brain. Although its physiological role remains to be established in numerous organs, T1R2/T1R3 is suspected to be involved in the regulation of metabolic processes, such as sugar sensing, glucose homeostasis, and satiety hormone release. In this review, the physiological role of the sweet taste receptor in taste perception and metabolic regulation is discussed by focusing on dysfunctions leading to diabetes. Current knowledge of T1R2/T1R3 inhibitors making this receptor a promising therapeutic target for the treatment of type 2 diabetes is also summarized and discussed.     

Detection of sweet tastants by a conserved group of insect gustatory receptors

Sweet taste cells play critical roles in food selection and feeding behaviors. Drosophila sweet neurons express eight gustatory receptors (Grs) belonging to a highly conserved clade in insects. Despite ongoing efforts, little is known about the fundamental principles that underlie how sweet tastants are detected by these receptors. Here, we provide a systematic functional analysis of Drosophila sweet receptors using the ab1C CO₂-sensing olfactory neuron as a unique in vivo decoder. We find that each of the eight receptors of this group confers sensitivity to one or more sweet tastants, indicating direct roles in ligand recognition for all sweet receptors. Receptor response profiles are validated by analysis of taste responses in corresponding Gr mutants. The response matrix shows extensive overlap in Gr–ligand interactions and loosely separates sweet receptors into two groups matching their relationships by sequence. We then show that expression of a bitter taste receptor confers sensitivity to selected aversive tastants that match the responses of the neuron that the Gr is derived from. Finally, we characterize an internal fructose-sensing receptor, Gr43a, and its ortholog in the malaria mosquito, AgGr25, in the ab1C expression system. We find that both receptors show robust responses to fructose along with a number of other sweet tastants. Our results provide a molecular basis for tastant detection by the entire repertoire of sweet taste receptors in the fly and lay the foundation for studying Grs in mosquitoes and other insects that transmit deadly diseases.The detection of high-calorie sweet compounds is a fundamental property of the taste system. In Drosophila, as in mammals, sweet tastants are detected by a distinct subpopulation of sensory cells that drives innate acceptance (1). An investigation of the mechanisms of tastant detection by sweet receptors is therefore necessary for understanding key principles of feeding behaviors.Insect gustatory receptors (Grs) belong to a novel arthropod superfamily unrelated to mammalian taste receptors (2). The Drosophila melanogaster Gr gene family encodes 68 receptors that are expressed in complex overlapping patterns in chemosensory neurons in both adult and larval stages (3–6). Expression analysis shows that individual Grs are exclusive to either sweet (attractive) or bitter (aversive) taste neurons, delineating separation of sweet and bitter taste receptors within Grs (3–5, 7). Eight Grs have been mapped to sweet taste neurons (5, 8–10), defining a distinct clade of receptors whose members are found across insects and arthropods (11). Of these, Gr5a and Gr64a are broadly required for responses to complementary subsets of sugars in labellar sweet taste neurons (9). Several lines of evidence suggest that one or more of the remaining six receptors are coexpressed with Gr5a in sweet neurons of the fly labellum (8–10, 12). Genetic analysis for some of these other Grs suggests that they are also necessary for responses to sweet tastants, although the breadth of sugar response defects can vary between different Gr mutants (12–14).Receptor expression in a heterologous context is crucial for deciphering its response properties. Our current understanding of how volatile chemicals are encoded by insect odorant receptor (Or) proteins was made possible by both cell-based and in vivo expression systems that allowed comprehensive functional analysis of Or gene repertoires in D. melanogaster (15, 16) and the malaria mosquito Anopheles gambiae (17, 18). The success of ectopic analysis of Ors is in stark contrast to similar studies for Grs, which have met with limited progress (19–22), despite over a decade of efforts. Although the antennal CO₂ receptors Gr21a and Gr63a were successfully expressed in an “empty” olfactory neuron lacking functional Ors (20, 21), attempts to express other Gr genes have been largely futile. Only few instances of functional Gr expression in tissue culture have been reported, which include Gr5a and Gr43a from Drosophila, BmGr8 and BmGr9 from the silkworm Bombyx mori, and PxutGr1 from the swallowtail butterfly Papilio xuthus (19, 22–24). In each case, expression of a single receptor was found to be sufficient to confer tastant responses. To date, however, these instances remain notable exceptions. Because Grs represent a highly divergent chemoreceptor family across insects (25), limitations in examining their functional properties embody a critical gap in the field.We recently expressed Gr64e in the Gr21a/Gr63a CO₂-sensing neuron in the olfactory system and showed that it confers sensitivity to glycerol (12). Here we investigate how tastants are detected by the entire repertoire of sweet taste receptors in Drosophila. We express each receptor individually in the ab1C neuron and examine its responses to a diagnostic panel of sweet compounds derived from the response profile of sweet taste neurons (9). We find that Gr5a and Gr64a confer complementary responses and that every other receptor is activated by one or more sweet tastants. Typically, ectopic responses of other receptors overlap with either Gr5a or Gr64a, but not with both. Ectopic responses are validated by tastant response defects in corresponding Gr mutants. We extend our study by testing a bitter receptor, Gr59c, and observe bitter responses that match previously associated ligands for Gr59c (5). We also test an internal fructose-sensing receptor, Gr43a, and its malaria mosquito ortholog, AgGr25. Both Gr43a and AgGr25 confer robust responses to fructose and some other sweet tastants. Importantly, our results show that AgGr25 can function independently, in the absence of other mosquito proteins. Together, our findings reveal tastant detection properties of Drosophila sweet taste receptors and describe and provide a tool for further analysis of insect taste receptors.     

Molecular mechanism of the sweet taste enhancers

Positive allosteric modulators of the human sweet taste receptor have been developed as a new way of reducing dietary sugar intake. Besides their potential health benefit, the sweet taste enhancers are also valuable tool molecules to study the general mechanism of positive allosteric modulations of T1R taste receptors. Using chimeric receptors, mutagenesis, and molecular modeling, we reveal how these sweet enhancers work at the molecular level. Our data argue that the sweet enhancers follow a similar mechanism as the natural umami taste enhancer molecules. Whereas the sweeteners bind to the hinge region and induce the closure of the Venus flytrap domain of T1R2, the enhancers bind close to the opening and further stabilize the closed and active conformation of the receptor.     

Olfactory expression of a single and highly variable V1r pheromone receptor-like gene in fish species

Sensory neurons expressing members of the seven-transmembrane V1r receptor superfamily allow mice to perceive pheromones. These receptors, which exhibit no sequence homology to any known protein except a weak similarity to taste receptors, have only been found in mammals. In the mouse, the V1r repertoire contains >150 members, which are expressed by neurons of the vomeronasal organ, a structure present exclusively in some tetrapod species. Here, we report the existence of a single V1r gene in multiple species of a non-terrestrial, vomeronasal organ-lacking taxon, the teleosts. In zebrafish, this V1r gene is expressed in chemosensory neurons of the olfactory rosette with a punctate distribution, strongly suggesting a role in chemodetection. This unique receptor gene exhibits a remarkably high degree of sequence variability between fish species. It likely corresponds to the original V1r present in the common ancestor of vertebrates, which led to the large and very diverse expansion of vertebrate pheromone receptor repertoires, and suggests the presence of V1rs in multiple nonmammalian phyla.     

Leptin as a modulator of sweet taste sensitivities in mice

Leptin acts as a potent inhibitory factor against obesity by regulating energy expenditure, food intake, and adiposity. The obese diabetic db/db mouse, which has defects in leptin receptor, displays enhanced neural responses and elevated behavioral preference to sweet stimuli. Here, we show the effects of leptin on the peripheral taste system. An administration of leptin into lean mice suppressed responses of peripheral taste nerves (chorda tympani and glossopharyngeal) to sweet substances (sucrose and saccharin) without affecting responses to sour, salty, and bitter substances. Whole-cell patch-clamp recordings of activities of taste receptor cells isolated from circumvallate papillae (innervated by the glossopharyngeal nerve) demonstrated that leptin activated outward K(+) currents, which resulted in hyperpolarization of taste cells. The db/db mouse with impaired leptin receptors showed no such leptin suppression. Taste tissue (circumvallate papilla) of lean mice expressed leptin-receptor mRNA and some of the taste cells exhibited immunoreactivities to antibodies of the leptin receptor. Taken together, these observations suggest that the taste organ is a peripheral target for leptin, and that leptin may be a sweet-sensing modulator (suppressor) that may take part in regulation of food intake. Defects in this leptin suppression system in db/db mice may lead to their enhanced peripheral neural responses and enhanced behavioral preferences for sweet substances.     

Characterization and solubilization of bitter-responsive receptors that couple to gustducin

The tastes of many bitter and sweet compounds are thought to be transduced via guanine nucleotide binding protein (G-protein)-coupled receptors, although the biochemical nature of these receptors is poorly understood at present. Gustducin, a taste-specific G-protein closely related to the transducins, is a key component in transducing the responses to compounds that humans equate with bitter and sweet. Rod transducin, which is also expressed in taste receptor cells, can be activated by the bitter compound denatonium in the presence of bovine taste membranes. In this paper, we show that gustducin is expressed in bovine taste tissue and that both gustducin and transducin, in the presence of bovine taste membranes, can be activated specifically by several bitter compounds, including denatonium, quinine, and strychnine. We also demonstrate that the activation in response to denatonium of gustducin by presumptive bitter-responsive receptors present in taste membranes depends on an interaction with the C terminus of gustducin and requires G-protein βγ subunits to provide the receptor-interacting heterotrimer. The taste receptor–gustducin interaction can be competitively inhibited by peptides derived from the sites of interaction of rhodopsin and transducin. Finally, as the initial step toward purifying taste receptors, we have solubilized this bitter-responsive taste receptor and maintained its biological activity.     

Transient receptor potential family members PKD1L3 and PKD2L1 form a candidate sour taste receptor

Animals use their gustatory systems to evaluate the nutritious value, toxicity, sodium content, and acidity of food. Although characterization of molecular identities that receive taste chemicals is essential, molecular receptors underlying sour taste sensation remain unclear. Here, we show that two transient receptor potential (TRP) channel members, PKD1L3 and PKD2L1, are coexpressed in a subset of taste receptor cells in specific taste areas. Cells expressing these molecules are distinct from taste cells having receptors for bitter, sweet, or umami tastants. The PKD2L1 proteins are accumulated at the taste pore region, where taste chemicals are detected. PKD1L3 and PKD2L1 proteins can interact with each other, and coexpression of the PKD1L3 and PKD2L1 is necessary for their functional cell surface expression. Finally, PKD1L3 and PKD2L1 are activated by various acids when coexpressed in heterologous cells but not by other classes of tastants. These results suggest that PKD1L3 and PKD2L1 heteromers may function as sour taste receptors.     

Requirement for an Otopetrin-like protein for acid taste in Drosophila

Receptors for bitter, sugar, and other tastes have been identified in the fruit fly Drosophila melanogaster, while a broadly tuned receptor for the taste of acid has been elusive. Previous work showed that such a receptor was unlikely to be encoded by a gene within one of the two major families of taste receptors in Drosophila, the “gustatory receptors” and “ionotropic receptors.” Here, to identify the acid taste receptor, we tested the contributions of genes encoding proteins distantly related to the mammalian Otopertrin1 (OTOP1) proton channel that functions as a sour receptor in mice. RNA interference (RNAi) knockdown or mutation by CRISPR/Cas9 of one of the genes, Otopetrin-Like A (OtopLA), but not of the others (OtopLB or OtopLC) severely impaired the behavioral rejection to a sweet solution laced with high levels of HCl or carboxylic acids and greatly reduced acid-induced action potentials measured from taste hairs. An isoform of OtopLA that we isolated from the proboscis was sufficient to restore behavioral sensitivity and acid-induced action potential firing in OtopLA mutant flies. At lower concentrations, HCl was attractive to the flies, and this attraction was abolished in the OtopLA mutant. Cell type–specific rescue experiments showed that OtopLA functions in distinct subsets of gustatory receptor neurons for repulsion and attraction to high and low levels of protons, respectively. This work highlights a functional conservation of a sensory receptor in flies and mammals and shows that the same receptor can function in both appetitive and repulsive behaviors.

Humans possess the ability to distinguish among five basic tastes: sweet, bitter, salt, sour, and umami. Interestingly, there is considerable variety in the ability of other mammals to detect these qualities. For example, cats are missing sweet taste (1) and the bottlenose dolphin only detects salt in food (2). Yet the fruit fly, Drosophila melanogaster, responds to a similar repertoire of tastes as humans. This is all the more remarkable given the very distant evolutionary relatedness and the enormous differences in the anatomy of the fly and mammalian taste organs and points to a conserved function of these taste qualities in assessing food quality.Many of the receptors involved in Drosophila taste have been defined (3, 4). Those that contribute to sweet and bitter tastes have been characterized extensively and are members of the “gustatory receptor” (GR) family (3, 4). GRs are unrelated to the G protein–coupled receptors that function in mammalian sweet and bitter taste (3). Therefore, the abilities of insects and humans to respond to similar repertoires of chemicals such as sweet and bitter tastants have emerged independently.In mice, the taste of acids depends on a proton-selective channel, Otopetrin1 (OTOP1), which is expressed in type III taste receptor cells (5–7). OTOP1 was first identified based on its essential role in the vestibular systems of the mouse and zebrafish (8–11) and was found to encode a family of proton-selective ion channels functionally conserved from worms to humans (5, 9, 12). In sea urchins, an Otop channel functions in calcifying primary mesenchymal cells by promoting the removal of protons generated during the production of CaCO₃ (13). Otop family members are structurally unrelated to other ion channels and are composed of 12 transmembrane segments (12, 14, 15), which assemble as a dimer with no obvious permeation pathway (14, 15). Flies, mice, and human genomes each contain three otop genes, although the fly genes are not direct homologs of the vertebrate genes (9).In Drosophila, low or moderate levels of some organic acids are attractive and promote feeding, while the same acids at higher concentrations repress food consumption (16, 17). This rejection contributes to survival as it discourages the animals from eating very acidic foods in the environment that can decrease lifespan. Two members of the large family of “ionotropic receptors” (IRs; IR25a and IR76b) function in GR neurons (GRNs) in the legs for sensing carboxylic acids and HCl (18). Mutation of either of these IRs disrupts the preference to lay eggs on acid-containing substrates (18). Flies prefer consuming lactic acid over water, and this preference is mildly reduced in Ir25a mutants (19).The receptors required for the gustatory rejection of noxious levels of acids have been largely enigmatic. An exception is IR7a, which is needed to suppress feeding on foods laced with acetic acid (17). IR7a is very narrowly tuned, as it does not impair the rejection of foods with HCl or any other carboxylic acid tested. This receptor acts in a subset of GRNs called B GRNs that are also activated by bitter chemicals and certain other aversive compounds (4, 17).Here, we identified a member of the family of Otop channels that in Drosophila is required for the detection of protons in food. Wild-type flies are strongly repelled by high levels of HCl and mildly attracted to a low level of HCl. We found that these responses depend on the Otopetrin-Like protein (OtopLA), which has a common evolutionarily origin with mammalian OTOP channels. By performing cell type–specific rescue experiments, we found that the strong repulsion and mild attraction to different levels of acids depends on expression of OtopLA in distinct subsets of GRNs.     

Genetic loss or pharmacological blockade of testes-expressed taste genes causes male sterility

TAS1R taste receptors and their associated heterotrimeric G protein gustducin are involved in sugar and amino acid sensing in taste cells and in the gastrointestinal tract. They are also strongly expressed in testis and sperm, but their functions in these tissues were previously unknown. Using mouse models, we show that the genetic absence of both TAS1R3, a component of sweet and amino acid taste receptors, and the gustducin α-subunit GNAT3 leads to male-specific sterility. To gain further insight into this effect, we generated a mouse model that expressed a humanized form of TAS1R3 susceptible to inhibition by the antilipid medication clofibrate. Sperm formation in animals without functional TAS1R3 and GNAT3 is compromised, with malformed and immotile sperm. Furthermore, clofibrate inhibition of humanized TAS1R3 in the genetic background of Tas1r3^−/−, Gnat3^−/− doubly null mice led to inducible male sterility. These results indicate a crucial role for these extraoral “taste” molecules in sperm development and maturation. We previously reported that blocking of human TAS1R3, but not mouse TAS1R3, can be achieved by common medications or chemicals in the environment. We hypothesize that even low levels of these compounds can lower sperm count and negatively affect human male fertility, which common mouse toxicology assays would not reveal. Conversely, we speculate that TAS1R3 and GNAT3 activators may help infertile men, particularly those that are affected by some of the mentioned inhibitors and/or are diagnosed with idiopathic infertility involving signaling pathway of these receptors.     

Leptin modulates behavioral responses to sweet substances by influencing peripheral taste structures

Leptin is a hormone that regulates body weight homeostasis mainly via the hypothalamic functional leptin receptor Ob-Rb. Recently, we proposed that the taste organ is a new peripheral target for leptin. Leptin selectively inhibits mouse taste cell responses to sweet substances and thereby may act as a sweet taste modulator. The present study further investigated leptin action on the taste system by examining expression of Ob-Rb in taste cells and behavioral responses to sweet substances in leptin-deficient ob/ob, and Ob-Rb-deficient db/db mice and their normal litter mates. RT-PCR analysis showed that Ob-Rb was expressed in taste cells in all strains tested. The db/db mice, however, had a RT-PCR product containing an abnormal db insertion that leads to an impaired shorter intracellular domain. In situ hybridization analysis showed that the hybridization signals for normal Ob-Rb mRNA were detected in taste cells in lean and ob/ob mice but not in db/db mice. Two different behavioral tests, one using sweet-bitter mixtures as taste stimuli and the other a conditioned taste aversion paradigm, demonstrated that responses to sucrose and saccharin were significantly decreased after ip injection of leptin in ob/ob and normal littermates, but not in db/db mice. These results suggest that leptin suppresses behavioral responses to sweet substances through its action on Ob-Rb in taste cells. Such taste modulation by leptin may be involved in regulation for food intake.     

Human receptors for sweet and umami taste

The three members of the T1R class of taste-specific G protein-coupled receptors have been hypothesized to function in combination as heterodimeric sweet taste receptors. Here we show that human T1R2/T1R3 recognizes diverse natural and synthetic sweeteners. In contrast, human T1R1/T1R3 responds to the umami taste stimulus l-glutamate, and this response is enhanced by 5'-ribonucleotides, a hallmark of umami taste. The ligand specificities of rat T1R2/T1R3 and T1R1/T1R3 correspond to those of their human counterparts. These findings implicate the T1Rs in umami taste and suggest that sweet and umami taste receptors share a common subunit.     

Divergence of T2R chemosensory receptor families in humans, bonobos, and chimpanzees

T2R (Tas2R) genes encode a family of G protein-coupled gustatory receptors, several involved in bitter taste perception. So far, few ligands for these receptors have been identified, and the specificity of most T2Rs is unclear. Differences between individual T2Rs result in altered taste perception in either specificity or sensitivity. All 33 human T2Rs are characterized by significant sequence homology. However, with a total of eight pseudogenes and >83 coding region single-nucleotide polymorphisms, the family displays broad diversity. The underlying variability of individual T2Rs might be the source for personalized taste perception. To test this hypothesis and also to identify T2Rs that possibly function beyond bitter taste, we compared all human T2R genes with those of the closely related primate species Pan paniscus (bonobo) and Pan troglodytes (chimpanzee). The differences identified range from large sequence alterations to nonsynonymous and synonymous changes of single base pairs. In contrast to olfactory receptors, no human-specific loss of the amount of functional genes was observed. Taken together, the results indicate ongoing evolutionary diversification of T2R receptors and a role for T2Rs in dietary adaptation and personalized food uptake.     

The retina is a target for GnRH-3 system in the European sea bass, Dicentrarchus labrax

The European sea bass expresses three GnRH (Gonadotrophin Releasing Hormone) forms that exert pleiotropic actions via several classes of receptors. The GnRH-1 form is responsible for the endogenous regulation of gonadotrophin release by the pituitary gland but the role of GnRH-2 and GnRH-3 remains unclear in fish. In a previous study performed in sea bass, we have provided evidence of direct links between the GnRH-2 cells and the pineal organ and demonstrated a functional role for GnRH-2 in the modulation of the secretory activity of this photoreceptive organ. In this study, we have investigated the possible relationship between the GnRH-3 system and the retina in the same species. Thus, using a biotinylated dextran-amine tract-tracing method, we reveal the presence of retinopetal cells in the terminal nerve of sea bass, a region that also contains GnRH-3-immunopositive cells. Moreover, GnRH-3-immunoreactive fibers were observed at the boundary between the inner nuclear and the inner plexiform layers, and also within the ganglion cell layer. These results strongly suggest that the GnRH-3 neurons located in the terminal nerve area represent the source of GnRH-3 innervation in the retina of this species. In order to clarify whether the retina is a target for GnRH, the expression pattern of GnRH receptors (dlGnRHR) was also analyzed by RT-PCR and in situ hybridization. RT-PCR revealed the retinal expression of dlGnRHR-II-2b, -1a, -1b and -1c, while in situ hybridization only showed positive signals for the receptors dlGnRHR-II-2b and -1a. Finally, double-immunohistochemistry showed that GnRH-3 projections reaching the sea bass retina end in close proximity to tyrosine hydroxylase (dopaminergic) cells, which also expressed the dlGnRHR-II-2b receptor subtype. Taken together, these results suggest an important role for GnRH-3 in the modulation of dopaminergic cell activities and retinal functions in sea bass.     

Gut-expressed gustducin and taste receptors regulate secretion of glucagon-like peptide-1

Glucagon-like peptide-1 (GLP-1), released from gut endocrine L cells in response to glucose, regulates appetite, insulin secretion, and gut motility. How glucose given orally, but not systemically, induces GLP-1 secretion is unknown. We show that human duodenal L cells express sweet taste receptors, the taste G protein gustducin, and several other taste transduction elements. Mouse intestinal L cells also express alpha-gustducin. Ingestion of glucose by alpha-gustducin null mice revealed deficiencies in secretion of GLP-1 and the regulation of plasma insulin and glucose. Isolated small bowel and intestinal villi from alpha-gustducin null mice showed markedly defective GLP-1 secretion in response to glucose. The human L cell line NCI-H716 expresses alpha-gustducin, taste receptors, and several other taste signaling elements. GLP-1 release from NCI-H716 cells was promoted by sugars and the noncaloric sweetener sucralose, and blocked by the sweet receptor antagonist lactisole or siRNA for alpha-gustducin. We conclude that L cells of the gut "taste" glucose through the same mechanisms used by taste cells of the tongue. Modulating GLP-1 secretion in gut "taste cells" may provide an important treatment for obesity, diabetes and abnormal gut motility.