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.

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.

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.

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.

Oral expressions and functional analyses of the extracellular calcium-sensing receptor (CaSR) in chicken.

In vertebrates, the extracellular calcium-sensing receptor (CaSR) plays a key role in calcium homeostasis by sensing slight changes in extracellular Ca2+. CaSR is also expressed in mammals including rodent taste cells and is involved in sensing kokumi, a rich, savory quality that enhances the intensities of salty, sweet, and umami tastes. In this study, we focused on chicken CaSR (cCaSR) since calcium is an essential nutrient that is necessary for making eggshell and for the extremely rapid initial growth of bones. First we confirmed that cCaSR is expressed in taste cells. Next we cloned the cCaSR gene from kidney and transiently transfected human embryonic kidney 293 T (HEK293T) cells with the recombinant cCaSR, or empty vector and looked for the agonists and allosteric modulators (including kokumi substances) of cCaSR by Ca2+ imaging. We found that cCaSR was activated by extracellular Ca2+ and Mg2+ in a dose dependent manner. Several L-amino acids and kokumi substances such as glutathione enhanced the response of cCaSR. In addition, NPS2143 as a negative allosteric modulator of human CaSR negatively modulated the response of cCaSR. These results suggest that cCaSR can sense extracellular Ca2+ and Mg2+ as well as positive and negative allosteric modulators. Taken together, the results imply that CaSR might be a multifunctional receptor for calcium, amino acids, and kokumi substances in chicken. The present finding that functional CaSR is expressed in the chicken oral tissues will allow us to further elucidate the physiological role of CaSR in the chickens' taste sense, and to create new feeds that will contribute to the poultry industry.

Fatty Acid Taste Receptor GPR120 Activation by Arachidonic Acid, Eicosapentaenoic Acid, and Docosahexaenoic Acid in Chickens.

It has been reported that the supplementation of chicken diet with polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) affects the qualities of eggs and meat. Previous studies have shown that a functional fatty acid taste receptor, G protein-coupled receptor 120 (GPR120), is broadly expressed in chicken oral and gastrointestinal tissues, and chickens have a gustatory perception of oleic acid, which is a chicken GPR120 agonist. The aim of this study was to elucidate the role of chicken GPR120 in response to PUFAs in chicken diets. Ca2+ imaging analyses revealed that chicken GPR120 was activated by AA, EPA, and DHA in a concentration-dependent manner. These results suggest that chickens can detect PUFAs via GPR120 in the oral and gastrointestinal tissues, implying that chickens have a gustatory perception of PUFAs.

Identification of Ligands for Chicken Transient Receptor Potential Ankyrin 1 Channel and Chemosensory Perception of Herbal Compounds in Chickens.

The pungency induced by spices and herbs plays an important role in food choice and appetite, and it is suggested that adding spices and herbs to feed as natural alternatives to antibiotics has beneficial effects in poultry farming. However, our knowledge of the chemosensory perception of herbal compounds in chickens is limited. Transient receptor potential ankyrin 1 (TRPA1) is involved in the sensory perception of various herbal compounds. Here, we performed calcium imaging and electrophysiological analyses using cells transiently expressing chicken TRPA1 (cTRPA1) and identified two novel cTRPA1 ligands-eugenol and thymol. In a behavioral assay, chickens responded to cTRPA1 ligands, including eugenol, thymol, cinnamaldehyde, carvacrol, and allyl isothiocyanate. These results provide evidence that chickens have a functional TRPA1 channel and chemosensory perception of various herbal compounds.

Behavioral responses to sweet compounds via T1R2-independent pathways in chickens.

Elucidating the taste sensing systems in chickens will enhance our understanding of poultry nutrition and improve the feeding strategies used in poultry farming. It is known that chickens lack the sweet taste receptor subunit, taste receptor type 1 member 2 (T1R2), in their genome. Thus, the present study investigated T1R2-independent sweet-sensing pathways in chickens. RT-PCR analysis revealed that glucose transporters known to play an important role in T1R2-independent sweet sensing in mammals-namely sodium-glucose cotransporter 1 (SGLT1) and ATP-gated K+ channel subunits-are expressed in the palate, the main taste organ in chickens. In behavioral tests, chickens slightly preferred glucose, galactose, sucrose, maltose, lactose, and stevioside, while high doses of sucrose and fructose were rejected. Chickens did not show any preference for noncaloric sweeteners or sugar alcohol, such as acesulfame K, aspartame, saccharin, sucralose, or sorbitol. The preference for galactose was inhibited by an inhibitor of SGLT1 in a dose-dependent manner. In addition, we found that glucagon-like peptide 1 (GLP-1) and mRNA of the GLP-1 receptor, which are involved specifically in sweet transmission in mice, are also present in the oral tissues of chickens. The present results imply that chickens can sense various sweet compounds via T1R2-independent pathways in oral tissues.

Dietary Alaska Pollack Protein Induces Acute and Sustainable Skeletal Muscle Hypertrophy in Rats.

Our previous studies suggested that Alaska pollack protein (APP) intake increases skeletal muscle mass and that it may cause a slow-to-fast shift in muscle fiber type in rats fed a high-fat diet after 56 days of feeding. In this study, we explored whether dietary APP induces acute and sustainable skeletal muscle hypertrophy in rats fed a normal-fat diet. Male 5-week-old Sprague-Dawley rats were divided into four groups and fed a purified ingredient-based high-fat diet or a purified ingredient-based normal-fat diet with casein or APP, containing the same amount of crude protein. Dietary APP significantly increased gastrocnemius muscle mass (105~110%) after 2, 7 days of feeding, regardless of dietary fat content. Rats were separated into two groups and fed a normal-fat diet with casein or APP. Dietary APP significantly increased gastrocnemius muscle mass (110%) after 56 days of feeding. Dietary APP significantly increased the cross-sectional area of the gastrocnemius skeletal muscle and collagen-rich connective tissue after 7 days of feeding. It decreased the gene expression of Mstn /Myostatin, Trim63/MuRF1, and Fbxo32/atrogin-1, but not other gene expression, such as serum IGF-1 after 7 days of feeding. No differences were observed between casein and APP groups with respect to the percentage of Type I, Type IIA, and Type IIX or IIB fibers, as determined by myosin ATPase staining after 7 days of feeding. In the similar experiment, the puromycin-labeled peptides were not different between dietary casein and APP after 2 days of feeding. These results demonstrate that APP induces acute and sustainable skeletal muscle hypertrophy in rats, regardless of dietary fat content. Dietary APP, as a daily protein source, may be an approach for maintaining or increasing muscle mass.

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.

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.

The umami receptor T1R1-T1R3 heterodimer is rarely formed in chickens.

The characterization of molecular mechanisms underlying the taste-sensing system of chickens will add to our understanding of their feeding behaviors in poultry farming. In the mammalian taste system, the heterodimer of taste receptor type 1 members 1/3 (T1R1/T1R3) functions as an umami (amino acid) taste receptor. Here, we analyzed the expression patterns of T1R1 and T1R3 in the taste cells of chickens, labeled by the molecular markers for chicken taste buds (vimentin and α-gustducin). We observed that α-gustducin was expressed in some of the chicken T1R3-positive taste bud cells but rarely expressed in the T1R1-positive and T2R7-positive taste bud cells. These results raise the possibility that there is another second messenger signaling system in chicken taste sensory cells. We also observed that T1R3 and α-gustducin were expressed mostly in the vimentin-positive taste bud cells, whereas T1R1 and bitter taste receptor (i.e., taste receptor type 2 member 7, T2R7) were expressed largely in the vimentin-negative taste bud cells in chickens. In addition, we observed that T1R1 and T1R3 were co-expressed in about 5% of chickens' taste bud cells, which express T1R1 or T1R3. These results suggest that the heterodimer of T1R1 and T1R3 is rarely formed in chickens' taste bud cells, and they provide comparative insights into the expressional regulation of taste receptors in the taste bud cells of vertebrates.

Evolvement of taste sensitivity and taste buds in chickens during selective breeding.

Chickens have been reported to have a low taste bud count and thus low taste acuity. However, more recent studies indicate that the earlier reported count of chicken taste buds may have been significantly underestimated. To answer the question of whether the taste sensing system in broiler chickens evolved during the breeding selection over the past decades, we compared the taste sensitivity to bitter and taste buds between a meat-type control strain - the 1955 Athens Canadian Random Bred (ACRB), and a modern high-yielding broiler strain - the 2012 Cobb 500. The behavioral tests showed that the ACRB did not avoid bitter taste solutions of quinine hydrochloride (QHCl) at the examined concentrations (0.5, 1, 2, and 4 mM) (P > 0.05), while the Cobb 500 significantly avoided both the 2 mM and 4 mM QHCl solutions (P < 0.01). The labeling of chicken taste buds using the molecular marker Vimentin revealed that Cobb 500 chickens had a slightly higher number (P < 0.1), but lower density of taste bud clusters in the palate (P < 0.01) and the base of the oral cavity (P < 0.05) compared to the ACRB. We also found that a single amino acid change occurred in the bitter taste receptor T2R7. However, the functional analyses using HEK293T cells transiently expressing T2R7 revealed that the functions of T2R7 were comparable between the two strains. Taken together, our results demonstrated that taste sensitivities could be affected by the selection of the broiler chickens. The modern high-yielding broilers, which have massive feed intake and appetite, had a higher sensitivity to bitter taste stimuli than the meat-type chicken strain which was established decades ago. This evolvement of taste sensitivities may be associated with the alterations of an upper level of taste system, rather than the peripheral taste system, including distribution of taste buds and functions of taste receptors.

Oleuropein suppresses mitochondrial reactive oxygen species generation possibly via an activation of transient receptor potential V1 and sirtuin-1 in cultured chicken muscle cells.

This study investigated the intracellular mechanism governing the effects of oleuropein (OLE), a phenolic compound of Olea europaea, on mRNA expression of avian uncoupling protein (avUCP) and mitochondrial biogenesis-related factors, and reactive oxygen species (mitROS) generation in a primary cultured chicken muscle cells. The OLE-treated cells exhibited increases in Avucp and ATP5a1z expression and a decrease in mitROS generation (p < 0.05), while the effects was canceled by sirtuin-1 (SIRT1) or transient receptor potential vanilloid 1 (TRPV1) inhibitors, EX-527 or BCTC, respectively. Intracellular Ca2+ concentration was significantly increased by OLE, while the induction was canceled by BCTC. The study also found that TRPV1 was expressed in the cell membrane and endoplasmic reticulum (ER), and Ca2+ could be released from ER in the OLE-treated cells. The OLE-treated cells exhibited increases in the phosphorylation ratio of AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) protein content. EX-527 and BCTC inhibitors canceled the effects of OLE on p-AMPK ratio and PGC-1α content, while EX-527 SIRT did not change PGC-1α content. The results suggest that the OLE effects may be due to Ca2+ release, possibly from TRPV1 at ER, and increased p-AMPK ratio, followed by SIRT1 activation and PGC-1α protein expression.

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.

Research Note: Behavioral preference and conditioned taste aversion to oleic acid solution in chickens.

A functional fatty acid taste receptor, GPR120, is present in chicken oral tissues, and chickens show a preference for lipid in feed. However, it remains unclear whether chickens can detect fatty acids. To address this issue, we adopted 2 behavioral paradigms: a one-bowl drinking test to evaluate the preference for oleic acid solution and a conditioned taste aversion test to investigate the role of gustation in chickens' ability to detect oleic acid. In the one-bowl drinking test, chickens did not show any preference for solution containing 0.001, 0.01, 0.03, 0.1, or 30 mmol/L oleic acid although 30 mmol/L oleic acid was enough to fully activate GPR120, confirmed by Ca2+ imaging. On the other hand, chickens conditioned to avoid 30 mmol/L oleic acid solution also learned to avoid the solution. These results suggested that chickens have a gustatory perception of oleic acid solution but do not have a preference for it. The present results support the idea that chickens prefer lipid in feed, not only by a postingestive effect but also by sensing the taste of fatty acid.

Bitter Taste Receptor Antagonists Inhibit the Bitter taste of Canola Meal Extract in Chickens.

Canola meal (CM) is a commonly used feedstuff; however, it is known to be bitter, and chickens have a low preference for it. The purpose of this study was to seek clarity regarding the taste quality of CM and find methods to increase the preference for CM by chickens. We examined whether CM activates the bitter taste receptors in chickens, whether chickens show aversive responses to CM, and whether an antagonist for bitter taste receptors inhibits the bitterness of CM. Using the Ca2+ imaging technique, we showed that CM contains bitter compounds, which activate the bitter taste receptors in chickens. Further, we showed that 6-methoxyflavanone (6-meth), an antagonist for the bitter taste receptors in chickens, inhibits the activation of these receptors by CM extract. Although chickens showed a low preference for the solution of the CM extract, their preference was improved by adding 6-meth in behavioral tests. These results suggest that the preference for CM could be improved by inhibiting the bitter taste receptors in chickens.

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.