Taste buds are not derived from neural crest in mouse, chicken, and zebrafish.

Our lineage tracing studies using multiple Cre mouse lines showed a concurrent labeling of abundant taste bud cells and the underlying connective tissue with a neural crest (NC) origin, warranting a further examination on the issue of whether there is an NC derivation of taste bud cells. In this study, we mapped NC cell lineages in three different models, Sox10-iCreERT2/tdT mouse, GFP+ neural fold transplantation to GFP- chickens, and Sox10-Cre/GFP-RFP zebrafish model. We found that in mice, Sox10-iCreERT2 specifically labels NC cell lineages with a single dose of tamoxifen at E7.5 and that the labeled cells were widely distributed in the connective tissue of the tongue. No labeled cells were found in taste buds or the surrounding epithelium in the postnatal mice. In the GFP+/GFP- chicken chimera model, GFP+ cells migrated extensively to the cranial region of chicken embryos ipsilateral to the surgery side but were absent in taste buds in the base of oral cavity and palate. In zebrafish, Sox10-Cre/GFP-RFP faithfully labeled known NC-derived tissues but did not label taste buds in lower jaw or the barbel. Our data, together with previous findings in axolotl, indicate that taste buds are not derived from NC cells in rodents, birds, amphibians or teleost fish.

Overlapping distributions of mammalian types I, II, and III taste cell markers in chicken taste buds.

Mammalian taste buds comprise types I, II, and III taste cells, with each type having specific characteristics: glia-like supporting cells (type I), taste receptor cells (type II), and presynaptic cells (type III). In this study, to characterize the peripheral taste-sensing systems in chickens, we analyzed the distributions of the mammalian types I, II, and III taste cell markers in chicken taste buds: glutamate-aspartate transporter (GLAST) for type I; taste receptor type 1 members 1 and 3 (T1R1 and T1R3), taste receptor type 2 member 7 (T2R7), and α-gustducin for type II; and synaptosomal protein 25 (SNAP25) and neural cell adhesion molecule (NCAM) for type III. We found that most GLAST+ taste cells expressed α-gustducin and SNAP25 and that high percentages of T1R3+ or α-gustducin+ taste cells expressed SNAP25 and NCAM. These results demonstrated a unique subset of chicken taste cells expressing multiple taste cell type marker proteins. Taken together, these results provide new insights into the taste-sensing mechanisms in vertebrate taste buds.

Intragastric administration of AMG517, a TRPV1 antagonist, enhanced activity-dependent energy metabolism via capsaicin-sensitive sensory nerves in mice.

Transient receptor potential vanilloid 1 (TRPV1), a nociceptive cation channel, is known to play roles in regulating the energy metabolism (EM) of the whole body. We previously reported that TRPV1 antagonists such as AMG517 enhanced EM in mice, however, these mechanisms remain unclear. The aim of this study was to explore the mechanisms underlying the enhancement of EM by AMG517, a selective TRPV1 antagonist, in mice. Respiratory gas analysis indicated that intragastric administration of AMG517 enhanced EM along with increasing locomotor activity in mice. Next, to clarify the possible involvement with afferent sensory nerves, including the vagus, we desensitized the capsaicin-sensitive sensory nerves of mice by systemic capsaicin treatment. In the desensitized mice, intragastric administration of AMG517 did not change EM and locomotor activity. Therefore, this study indicated that intragastric administration of AMG517 enhanced EM and increased locomotor activity via capsaicin-sensitive sensory nerves, including vagal afferents in mice.

Differences in the acidic sensitivity of transient receptor potential vanilloid 1 (TRPV1) between chickens and mice.

Chickens, one of the most important industrial animals, are a biological animal model. Here we focused on the transient receptor potential vanilloid 1 (TRPV1) to understand the pain system for acidic stimuli in chickens compared with mice. By using a whole-cell patch clamp system, we confirmed that acidic stimuli activate both chicken TRPV1 (cTRPV1) and mouse TRPV1 (mTRPV1), but the peak current of cTRPV1 is lower than that of mTRPV1, and it is difficult to desensitize cTRPV1 with an acidic stimulus compared to mTRPV1. Since the C-terminal of the calmodulin (CaM) binding site in TRPV1 was reported as one of the important structures for TRPV1 desensitization, we made chimeric cTRPV1 in which the CaM binding site of chicken is changed to that of mouse (cTRPV1-mCaM). We also compared the acidic responses of native chicken dorsal root ganglion (DRG) cells with that of mouse DRG cells. The TRPV1-mCaM results showed that the desensitization of mutant cTRPV1 was similar to that of mTRPV1, and that the basal activities of mutant cTRPV1 were significantly higher than those of cTRPV1. It was also difficult to desensitize the chicken DRG cells with an acidic stimulus, unlike the mouse DRG cells. These results suggest that there are differences in the pain transduction systems for acidic stimuli between chickens and mice that are caused by the dysfunction of the C-terminal CaM biding site of cTRPV1. These results imply that chickens repeatedly feel weak pain from an acidic stimulus, without desensitization.

Bitter taste receptor T2R7 and umami taste receptor subunit T1R1 are expressed highly in Vimentin-negative taste bud cells in chickens.

In the mammalian taste system, the taste receptor type 2 (T2R) family mediates bitter taste, and the taste receptor type 1 (T1R) family mediates sweet and umami tastes (the heterodimer of T1R2/T1R3 forms the sweet taste receptor, and the heterodimer of T1R1/T1R3 forms the umami taste receptor). In the chicken genome, bitter (T2R1, T2R2, and T2R7) and umami (T1R1 and T1R3) taste receptor genes have been found. However, the localization of these taste receptors in the taste buds of chickens has not been elucidated. In the present study, we demonstrated that the bitter taste receptor T2R7 and the umami taste receptor subunit T1R1 were expressed specifically in the taste buds of chickens labeled by Vimentin, a molecular marker for chicken taste buds. We analyzed the distributions of T2R7 and T1R1 on the oral epithelial sheets of chickens and among 3 different oral tissues of chickens: the palate, the base of the oral cavity, and the posterior tongue. We found that the distribution patterns and numbers were similar between taste bud clusters expressing these receptors and those expressing Vimentin. These results indicated broad distributions of T2R7 and T1R1 in the gustatory tissues of the chicken oral cavity. In addition, 3D-reconstructed images clearly revealed that high levels of T2R7 and T1R1 were expressed in Vimentin-negative taste bud cells. Taken together, the present results indicated the presence of bitter and umami sensing systems in the taste buds of chickens, and broad distribution of T2R7 and T1R1 in the chicken oral cavity.

Oral lipase activities and fat-taste receptors for fat-taste sensing in chickens.

It has been reported that a functional fat-taste receptor, GPR120, is present in chicken oral tissues, and that chickens can detect fat taste in a behavioral test. However, although triglycerides need to be digested to free fatty acids to be recognized by fat-taste receptors such as GPR120, it remains unknown whether lipase activities exist in chicken oral tissues. To examine this question, we first cloned another fat-taste receptor candidate gene, CD36, from the chicken palate. Then, using RT-PCR, we determined that GPR120 and CD36 were broadly expressed in chicken oral and gastrointestinal tissues. Also by RT-PCR, we confirmed that several lipase genes were expressed in both oral and gastrointestinal tissues. Finally, we analyzed the lipase activities of oral tissues by using a fluorogenic triglyceride analog as a lipase substrate. We found there are functional lipases in oral tissues as well as in the stomach and pancreas. These results suggested that chickens have a basic fat-taste reception system that incorporates a triglycerides/oral-lipases/free fatty acids/GPR120 axis and CD36 axis.

Identification of functional bitter taste receptors and their antagonist in chickens.

Elucidation of the taste sense of chickens is important not only for the development of chicken feedstuffs for the chicken industry but also to help clarify the evolution of the taste sense among animals. There are three putative chicken bitter taste receptors, chicken T2R1 (cT2R1), cT2R2 and cT2R7, which were identified using genome information and cell-based assays. Previously, we have shown that cT2R1 is a functional bitter taste receptor through both cell-based assays and behavioral tests. In this study, therefore, we focused on the sensitivities of the other two bitter receptors, cT2R2 and cT2R7, by using their agonists in behavioral tests. We tested three agonists of cT2R2 and three agonists of cT2R7. In a 10-min drinking study, the intakes of cT2R2 agonist solutions were not different from that of water. On the other hand, the intakes of cT2R7 agonist solutions were significantly lower compared to water. In addition, we constructed cT2R1-and cT2R7-expressing cells in order to search for an antagonist for these functional bitter taste receptors. By using Ca2+ imaging methods, we found that 6-methoxyflavanone (6-meth) can inhibit the activities of both cT2R1 and cT2R7. Moreover, 6-meth also inhibited the reduction of the intake of bitter solutions containing cT2R1 or cT2R7 agonists in behavioral tests. Taken together, these results suggested that cT2R7 is a functional bitter taste receptor like cT2R1, but that cT2R2 is not, and that 6-meth is an antagonist for these two functional chicken bitter taste receptors. This is the first identification of an antagonist of chicken bitter receptors.

Distribution of α-Gustducin and Vimentin in premature and mature taste buds in chickens.

The sensory organs for taste in chickens (Gallus sp.) are taste buds in the oral epithelium of the palate, base of the oral cavity, and posterior tongue. Although there is not a pan-taste cell marker that labels all chicken taste bud cells, α-Gustducin and Vimentin each label a subpopulation of taste bud cells. In the present study, we used both α-Gustducin and Vimentin to further characterize chicken taste buds at the embryonic and post-hatching stages (E17-P5). We found that both α-Gustducin and Vimentin label distinct and overlapping populations of, but not all, taste bud cells. A-Gustducin immunosignals were observed as early as E18 and were consistently distributed in early and mature taste buds in embryos and hatchlings. Vimentin immunoreactivity was initially sparse at the embryonic stages then became apparent in taste buds after hatch. In hatchlings, α-Gustducin and Vimentin immunosignals largely co-localized in taste buds. A small subset of taste bud cells were labeled by either α-Gustducin or Vimentin or were not labeled. Importantly, each of the markers was observed in all of the examined taste buds. Our data suggest that the early onset of α-Gustducin in taste buds might be important for enabling chickens to respond to taste stimuli immediately after hatch and that distinctive population of taste bud cells that are labeled by different molecular markers might represent different cell types or different phases of taste bud cells. Additionally, α-Gustducin and Vimentin can potentially be used as molecular markers of all chicken taste buds in whole mount tissue.

Expressions of multiple umami taste receptors in oral and gastrointestinal tissues, and umami taste synergism in chickens.

Umami taste is one of the five basic taste qualities, along with sweet, bitter, sour, and salty, and is elicited by some l-amino acids and their salts, including monopotassium l-glutamate (MPG). The unique characteristic of umami taste is that it is synergistically enhanced by 5'-ribonucleotides such as inosine 5'-monophosphate (IMP). Unlike the other four basic taste qualities, the presence of umami taste sense in avian species is not fully understood. In this study, we demonstrated the expression of multiple umami taste receptor candidates in oral and gastrointestinal tract tissues in chickens using RT-PCR analysis. We first showed the metabotropic glutamate receptors (mGluRs) expressed in these tissues. Furthermore, we examined the preference for umami taste in chickens, focusing on the synergistic effect of umami taste as determined by the two-feed choice test. We concluded that chickens preferred feed containing both added MPG and added IMP over feeds containing either added MPG or added IMP alone and over the control feed. These results suggest that the umami taste sense and synergism are conserved in chickens.

The role of G-protein-coupled receptor 120 in fatty acids sensing in chicken oral tissues.

Clarification of the mechanism of chickens' taste sense will provide meaningful information for creating and improving new feedstuff for chickens, because the character of taste receptors in oral tissues affects feeding behavior in animals. Although fatty acids are partly recognized via G-protein coupled receptor 120 (GPR120) for fat taste in mammalian oral tissues, the fat taste receptor of chickens has not been elucidated. Here we cloned chicken GPR120 (cGPR120) from the chicken palate, which contains taste buds. By using Ca(2+) imaging methods, we identified oleic acid and linoleic acid as cGPR120 agonists. Interestingly, in a behavioral study the chickens preferred corn oil-rich feed over mineral oil (control oil)-rich feed. Because corn oil contains high amounts of oleic acid and linoleic acid, this result was thought to be reasonable. Taken together, the present results suggest that cGPR120 is one of the functional fat taste receptors in chickens.

Bitter taste receptor T2R1 activities were compatible with behavioral sensitivity to bitterness in chickens.

Clarification of the mechanism of the sense of taste in chickens will provide information useful for creating and improving new feedstuffs for chickens, because the character of the taste receptors in oral tissues affects feeding behavior in animals. In this study, we focused on the sensitivity to bitterness in chickens. We cloned one of the bitter taste receptors, T2R1, from the chicken palate, constructed several biosensor-cells expressing chicken T2R1 (cT2R1), and determined a highly sensitive biosensor of cT2R1 among them. By using Ca(2+) imaging methods, we identified two agonists of cT2R1, dextromethorphan (Dex) and diphenidol (Dip). Dex was a new agonist of cT2R1 that was more potent than Dip. In a behavioral drinking study, the intake volumes of solutions of these compounds were significantly lower than that of water in chickens. These aversive concentrations were identical to the concentrations that could activate cT2R1 in a cell-based assay. These results suggest that the cT2R1 activities induced by these agonists are linked to behavioral sensitivity to bitterness in chickens.

Fish protein intake induces fast-muscle hypertrophy and reduces liver lipids and serum glucose levels in rats.

In our previous study, fish protein was proven to reduce serum lipids and body fat accumulation by skeletal muscle hypertrophy and enhancing basal energy expenditure in rats. In the present study, we examined the precise effects of fish protein intake on different skeletal muscle fiber types and metabolic gene expression of the muscle. Fish protein increased fast-twitch muscle weight, reduced liver triglycerides and serum glucose levels, compared with the casein diet after 6 or 8 weeks of feeding. Furthermore, fish protein upregulated the gene expressions of a fast-twitch muscle-type marker and a glucose transporter in the muscle. These results suggest that fish protein induces fast-muscle hypertrophy, and the enhancement of basal energy expenditure by muscle hypertrophy and the increase in muscle glucose uptake reduced liver lipids and serum glucose levels. The present results also imply that fish protein intake causes a slow-to-fast shift in muscle fiber type.

Supplementation with eicosapentaenoic acid-rich fish oil improves exercise economy and reduces perceived exertion during submaximal steady-state exercise in normal healthy untrained men.

Based on the effects of n-3 polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on reduction of blood viscosity, we theorized that PUFA could improve aerobic performance by increasing oxygen supply to tissues. Twenty male subjects were randomly divided into two groups (n = 10): a fish oil group (FG) and a control (CG). Maximal oxygen uptake and oxygen uptake during submaximal exercise were measured using a cycle ergometer. For 8 weeks, the FG then ingested capsules containing 3.6 g/day of EPA-rich fish oil, while the CG took 3.6 g/day of a medium-chain triglyceride. After supplementation, erythrocyte EPA and DHA in the FG were significantly increased. In the FG, a negative linear correlation was detected in the change between erythrocyte EPA and whole oxygen uptake during submaximal exercise pre- and post-supplementation. The present study showed that EPA-rich fish oil supplementation improves exercise economy in humans.

Characterization of taste sensitivities to amino acids and sugars by conditioned taste aversion learning in chickens.

Taste plays an essential role in regulating the feeding behaviors of animals. The present study aimed to characterize the taste sensory profiles of amino acids and sugars in chickens. To achieve this, we employed a conditioned taste aversion learning method, which is characterized by a specific pairing of gastrointestinal malaise and taste perception. Our findings revealed that chickens were able to learn to avoid L-Val, L-Lys, and L-His through conditioned taste aversion learning, and exhibited a strong aversion to L-Arg. These results suggest that chickens are primarily sensitive to basic amino acids, including L-Lys, which is a crucial limiting amino acid in feeds. Interstingly, this sensitivity to basic amino acids in chickens contrasts with humans, who are mainly sensitive to acidic amino acids as umami taste. Furthermore, despite the absence of a mammalian sweet taste receptor gene in the chicken genome, we demonstrated that chickens learned to avoid glucose, galactose, sucrose, and maltose by conditioned taste aversion learning. Taken together, the present study provides the idea that chickens possess a gustatory perception toward specific amino acids and sugars for the detection of beneficial nutrients in their feeds.

Eicosapentaenoic acid increases proportion of type 1 muscle fibers through PPARδ and AMPK pathways in rats.

Muscle fiber type composition (% slow-twitch and % fast-twitch fibers) is associated with metabolism, with increased slow-twitch fibers alleviating metabolic disorders. Previously, we reported that dietary fish oil intake induced a muscle fiber-type transition in a slower direction in rats. The aim of this study was to determine the functionality of eicosapentaenoic acid (EPA), a unique fatty acid in fish oil, to skeletal muscle fiber type and metabolism in rats. Here, we showed that dietary EPA promotes whole-body oxidative metabolism and improves muscle function by increasing proportion of slow-twitch type 1 fibers in rats. Transcriptomic and metabolomic analyses revealed that EPA supplementation activated the peroxisome proliferator-activated receptor δ (PPARδ) and AMP-activated protein kinase (AMPK) pathways in L6 myotube cultures, which potentially increasing slow-twitch fiber share. This highlights the role of EPA as an exercise-mimetic dietary component that improves metabolism and muscle function, with potential benefits for health and athletic performance.

Relationship between olfactory and gustatory functions: The Iwaki health promotion project 2019.

OBJECTIVE: Olfactory and gustatory functions are important sensory aspects in humans. Although they are believed to influence each other, their interrelationship is not well understood. In this study, we aimed to investigate the relationship between the olfactory and gustatory functions based on the results of a large-scale epidemiological study (Iwaki Health Promotion Project) of the general local population. METHODS: We analyzed 565 participants who underwent taste and olfactory tests in the 2019 Iwaki Project. Gustatory function was tested for four taste qualities (sweet, sour, salty, and bitter) using whole-mouth taste tests. Olfactory function was tested using the University of Pennsylvania Smell Identification Test modified for Japanese (UPSIT-J). We evaluated sex-related differences between olfactory and gustatory functions and the effects of various factors on olfactory identification using multivariate analysis. Furthermore, we compared the percentage of accurate UPSIT-J responses between the normal and hypogeusia groups. We also analyzed the effects of taste and olfactory functions on eating. RESULTS: Olfactory and gustatory functions were lower in men than in women. Among the four taste qualities, salty taste was the most closely associated with olfactory identification ability, with lower olfactory scores of salty taste in the hypogeusia group than in the normal group. Moreover, the hyposmia group had higher daily salt intake than the normal olfaction group in women. CONCLUSION: These results suggest that olfactory identification tests may be useful in predicting elevated salt cognitive thresholds, leading to a reduction in salt intake, which may contribute to hypertension prevention.

The Antiarrhythmic Drug Flecainide Enhances Aversion to HCl in Mice.

Drug-induced taste disorders reduce quality of life, but little is known about the molecular mechanisms by which drugs induce taste disturbances. In this study, we investigated the short-term and long-term effects of the antiarrhythmic drug flecainide, which is known to cause taste dysfunction. Analyses of behavioral responses (licking tests) revealed that mice given a single intraperitoneal injection of flecainide exhibited a significant reduction in preference for a sour tastant (HCl) but not for other taste solutions (NaCl, quinine, sucrose, KCl and monopotassium glutamate) when compared with controls. Mice administered a single dose of flecainide also had significantly higher taste nerve responses to HCl but not to other taste solutions. Compared with controls, mice administered flecainide once-daily for 30 d showed a reduced preference for HCl without any changes in the behavioral responses to other taste solutions. The electrophysiological experiments using HEK293T cells transiently expressing otopetrin-1 (Otop1; the mouse sour taste receptor) showed that flecainide did not alter the responses to HCl. Taken together, our results suggest that flecainide specifically enhances the response to HCl in mice during short-term and long-term administration. Although further studies will be needed to elucidate the molecular mechanisms, these findings provide new insights into the pathophysiology of drug-induced taste disorders.

The G protein-coupled receptor GPRC5C is a saccharide sensor with a novel 'off' response.

GPRC5C is an orphan G protein-coupled receptor (GPCR) that belongs to the class C GPCR family. Although GPRC5C is expressed in various organs, its function and ligand are still undetermined. We found that GPRC5C is expressed in mouse taste cells, enterocytes, and pancreatic α-cells. In functional imaging assays, HEK293 cells heterologously expressing GPRC5C and the chimeric G protein α subunit Gα16-gust44 showed robust intracellular Ca2+ increases in response to monosaccharides, disaccharides, and a sugar alcohol, but not an artificial sweetener or sweet-tasting amino acid. Notably, Ca2+ increases occurred after washout, not during stimulation. Our findings suggest that GPRC5C has receptor properties which lead to novel 'off' responses to saccharide detachment and may work as an internal or external chemosensor specifically tuned to natural sugars.

Bitter Taste Perception in Chickens.

Many behavioral studies and histological analyses of the sense of taste have been conducted in chickens, as it plays an important role in the ingestion of feed. In recent years, various taste receptors have been analyzed, and the functions of fatty acids, umami, and bitter taste receptors in chickens have become clear. In this review, the bitter taste sense in chickens, which is the taste quality by which animals reject poisons, is discussed among a variety of taste qualities. Chickens have taste buds in the palate, the base of the oral cavity, and the root of the tongue. Bitter taste receptors, taste receptor type 2 members 1, 2, and 7 (T2R1, T2R2, and T2R7) are expressed in these tissues. According to functional analyses of bitter taste receptors and behavioral studies, T2R1 and T2R7 are thought to be especially involved in the rejection of bitter compounds in chickens. Furthermore, the antagonists of these two functional bitter taste receptors were also identified, and it is expected that such antagonists will be useful in improving the taste quality of feed materials and poultry drugs that have a bitter taste. Bitter taste receptors are also expressed in extra-oral tissues, and it has been suggested that gastrointestinal bitter taste receptors may be involved in the secretion of gastrointestinal hormones and pathogen defense mechanisms. Thus, bitter taste receptors in chickens are suspected to play major roles in taste sensing and other physiological systems.

Conditioned Taste Aversion to L-Amino Acid Taste Stimuli and Oral Transcriptional Changes to Type 1 Taste Receptors T1R1 and T1R3 on Chronic Exposure to L-Alanine Solution in Chickens.

Elucidating taste sensing systems in chickens is an important step toward understanding poultry nutrition. Amino acid taste receptors, type 1 taste receptors 1 and 3 (T1R1 and T1R3, respectively), are expressed in chicken taste cells, and chicken T1R1/T1R3 is activated by L-alanine (L-Ala) and L-serine (L-Ser), but not by L-proline (L-Pro). However, it is not clear whether chickens have a gustatory perception of L-amino acids. Here, we found that chickens conditioned to avoid either L-Ala, L-Ser, or L-Pro solutions could successfully learn to avoid the corresponding L-amino acid solution in the conditioned taste aversion (CTA) test. Because CTA is a well-established learning paradigm generated specifically by pairing gustatory perception and gastrointestinal malaise, the present study suggests that chickens can sense L-amino acids by gustatory perception. In addition, we found that the expression of the T1R1 and T1R3 genes was significantly downregulated in response to chronic exposure to L-Ala solution, but not to acute oral stimulation. Taken together, the present study suggests that chickens have a gustatory perception of L-amino acids, and the expression of T1R1/T1R3 mRNAs in the oral cavity can be regulated by L-amino acid intake. Since chickens can detect L-Pro solutions, additional amino acid receptors, other than T1R1/T1R3, may be involved in L-amino acid taste detection in chickens.