Research Projects -
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Elucidation of the mechanism of taste cell shedding and its application to model mice for neurological disorders
Grant number：24K22186 2024.06 - 2027.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)
吉田 竜介, 美藤 純弘, 堀江 謙吾
Grant amount：\6370000 （ Direct expense: \4900000 、 Indirect expense：\1470000 ）
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うま味でサルコペニア克服に挑戦する
Grant number：22K19634 2022.06 - 2025.03
日本学術振興会 科学研究費助成事業 挑戦的研究(萌芽)
古株 彰一郎, 中道 敦子, 吉田 竜介, 細見 周平
Grant amount：\6370000 （ Direct expense: \4900000 、 Indirect expense：\1470000 ）
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口腔機能低下症に迷走神経刺激は有効か：脳腸連関によるニューロモジュレーション解析
Grant number：22K10246 2022.04 - 2025.03
日本学術振興会 科学研究費助成事業 基盤研究(C)
植田 紘貴, 吉田 竜介, 美藤 純弘
Grant amount：\4290000 （ Direct expense: \3300000 、 Indirect expense：\990000 ）
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single cell RNA-seqによるうま味細胞における新規機能分子探索
2022.01 - 2024.12
うま味研究会 うま味研究会助成金
Authorship：Principal investigator Grant type：Competitive
Grant amount：\1000000 （ Direct expense: \1000000 ）
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Analysis of taste function in oral-brain-gut axis using new photogenetical tool
Grant number：21K19601 2021.07 - 2024.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)
吉田 竜介, 古株 彰一郎, 高尾 知佳
Grant amount：\6370000 （ Direct expense: \4900000 、 Indirect expense：\1470000 ）
本年度は、野生型マウスおよび全身で甘味受容体コンポーネントを欠損するT1R3-KOマウスを用い、グルコースを口腔から摂取した場合と胃内投与した場合の血糖値および血中インスリン濃度の経時的変化（０～１２０分）について調べた。マウスをリック装置から溶液を摂取する様トレーニングし、グルコース摂取量が1mg/g体重となるよう2Mグルコースを摂取させた場合（口腔摂取）と同量を直接胃内投与した場合とを比較すると、血糖値のピークはいずれのマウスにおいても口腔摂取した方が早く（およそ摂取後１０分）、胃内投与した場合には遅くなっていた（およそ摂取後３０分）。血漿インスリン濃度についても同様の差が見られた。この結果から、口腔からグルコースを摂取した場合には頭相インスリン分泌が見られ、これがグルコース摂取後の血糖値変化を影響を与えるものと考えられる。また、甘味受容体T1R2/T1R3を介さない口腔からの何らかの感覚情報が重要であると考えられる。
また、光KOマウス作成について、開発済みのTet offシステムにより光活性化-Cre（PA-Cre）発現を制御するTRE-PA-Creマウスと、全身的にテトラサイクリン調節性トランス活性化因子（tTA）を発現するROSA-tTAマウスを開発し掛け合わせ、全身でPA-Creを発現するマウス（PA-Cre）を作成する予定であったが、ROSA-tTAマウスを取得することが出来なかったため、全身性PA-Creマウスはまだ作成できていない。 -
The raison d'etre of classical synapses in taste buds
Grant number：21H03106 2021.04 - 2025.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
吉田 竜介, 美藤 純弘, 樽野 陽幸, 池亀 美華
Grant amount：\17420000 （ Direct expense: \13400000 、 Indirect expense：\4020000 ）
本年度は、本研究で用いる以下の遺伝子改変マウスを作成した。(1)味蕾においてGABA合成酵素であるGAD67を欠損するマウス（K5-Cre;GAD67-flox）、(2)味蕾においてGABA合成酵素であるGAD67を欠損しGFPを発現するマウス（K5-Cre;GAD67-GFP/flox）、(3)味蕾においてシナプス関連遺伝子SNAP25を欠損するマウス（K5-Cre;SNAP25-flox）、(4)味蕾内でIII型細胞のみで発現する遺伝子を欠損するマウス２種（TypeIII-A-KO、TypeIII-B-KO）。今後、(3)または(4)かつGAD67-GFPを発現するマウス、(1)～(4)かつTRPV1を欠損するマウスを順次作成する予定である。また、K5-Creの味蕾での発現を確認するため、GAD67-GFPを発現し、K5発現細胞でtomatoの発現を誘導するマウス（K5-Cre;ROSA26-tomato;GAD67-GFP）を作成し、GAD67発現細胞（III型細胞）を含む味蕾細胞でK5-Creが発現することを確認した。さらに免疫組織化学的実験により、(2)のマウスにおいて味蕾内GFP発現細胞でGAD67発現が消失し、他の味細胞マーカー遺伝子の発現は変化しないことを確認した。
次に、これらマウスを作成するために用いた各種遺伝子改変マウス（K5-Cre、GAD67-flox、SNAP25-flox、TRPV1-KO）の味覚行動応答について短時間リック法にて調べた。その結果、これらのマウスは各種味溶液（ショ糖、キニーネ、NaCl、グルタミン酸ナトリウム、HCl、クエン酸など）に対し野生型マウスとほぼ同様の応答を示すことを確認した。ただし、TRPV1-KOマウスに関してはカプサイシンに対する忌避反応が減弱していた。 -
食品由来成分の味覚を介した全身機能に対する影響
2021.04 - 2024.05
糧食研究会 特定委託研究
Authorship：Principal investigator
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Elucidation of nutrient-specific taste cell-neuron matching mechanism
Grant number：18K19652 2018.06 - 2021.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory) Grant-in-Aid for Challenging Research (Exploratory)
Shigemura Noriatsu
Grant amount：\6370000 （ Direct expense: \4900000 、 Indirect expense：\1470000 ）
Taste quality specific information (taste discrimination) is constantly maintained, despite lifespan of taste cells is as short as about 10 days. This suggests there would be a molecular mechanisms that newborn taste cells are selectively matched with distinct taste neurons during continuous turnover. However, little is known about such taste cell-neuron matching mechanisms. In this study, to clarify this, we focused on the cell adheasion protein, Cadherin (Cdh) super-family, and examined its involvement in the matching mechanisms. Expresssion analyses including GeneChip revealed that protocadherin 20 was expressed specifically in both sweet taste cells and taste neurons, which may contribute to cell-neuron matching to generate the sweet taste infomation line.
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Grant number：18K19653 2018.06 - 2020.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory) Grant-in-Aid for Challenging Research (Exploratory)
Ninomiya Yuzo
Grant amount：\6240000 （ Direct expense: \4800000 、 Indirect expense：\1440000 ）
The present study predicted fat taste- and calorie sensing-related molecules based on comprehensive gene analysis of mouse single taste cells, and made functional analyses on their expression in taste cells and potential relations to taste nerve and behavioral responses to various taste stimuli. The results showed that, in addition to known taste information pathways for each of 5 basic tastes (sweet, sour, salt, bitter and umami), there exist two different taste neural pathways for fat perception: one is a fat taste-specific pathway derived from cells expressing fat receptors such as GPR120 etc, and the other is a calorie sensing pathway derived from cells co-expressing multiple transporters and receptors for lipids and sugars, and metabolic sensor channels (KATP). Thus, the new taste cell omics technology, which is a fusion of taste cell comprehensive gene analysis and taste response analysis, has led to the new findings and its effectiveness was suggested.
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Molecular and cellular mechanisms for modulation of sweet taste sensitivity
Grant number：18K09507 2018.04 - 2021.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
Yoshida Ryusuke
Grant amount：\4290000 （ Direct expense: \3300000 、 Indirect expense：\990000 ）
This study demonstrated that (1) sweet-sensitive taste cells (T1R3-expressing taste cells) possess leptin signaling components (STAT3, SHP2, PI3K), (2) among these components, PI3K plays a critical role in suppression of sweet responses of taste cells by leptin, and (3) leptin-induced activation of PI3K leads to production of PIP3 and phosphorylation of Akt in T1R3-expressing taste cells.
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Molecular physiological basis of modulation and repair by oxidative stress in oral/gut/ brain chemosensory-endocrine interaction for dietary regulation.
Grant number：18H02968 2018.04 - 2021.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
Ninomiya Yuzo
Grant amount：\17290000 （ Direct expense: \13300000 、 Indirect expense：\3990000 ）
Leptin and angiotensin II are hormones that regulate eating and drinking cravings in the brain, and sweet and salt taste sensitivities in the peripheral taste organ and contribute to energy and Na+ homeostasis. Also, the hormones at high concentrations are known to act as oxidative stress inducers in their target organs. This study investigated potential effects of oxidative stress on taste cells and target molecules based on analyses of mouse neural and behavioral taste responses and molecular expression. The results showed that oxidative stress agents increased sweet responses and decreased sodium responses and their targets of oxidative stress may be at intracellular region of KATP (sweet taste) known as a leptin target, and at extracellular region of ENaCs (sodium taste) known as an angiotensin II target.
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Grant number：15H02571 2015.04 - 2018.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A) Grant-in-Aid for Scientific Research (A)
NINOMIYA YUZO, Yasumatsu Keiko, Takai Shingo
Grant amount：\40820000 （ Direct expense: \31400000 、 Indirect expense：\9420000 ）
The present study investigated molecular basis of taste perception and humoral modulation of taste/hormone sensing cells in the oral-gut-brain circuit and its role in regulating food intake. The results showed that mouse sweet-sensitive cells in the oral cavity and gut possess at least two sweet reception systems; one is T1R2/T1R3 which can detect not only sugars but also artificial sweeteners and the other is T1Rs-independent sugar sensing system including glucose transporters (SGLTs/GLUTs), a metabolic sensor(KATP) and GLP-1, gut peptide hormone. In addition to GLP-1 for sweet signal transmission, CCK was shown be involved in bitter signal transmission in bitter-responsive cells. Leptin, a satiety hormone, inhibits sweet taste responses and glucose absorption via activation of KATP involved in the sugar sensing pathway of sweet-sensitive cells. This taste modulation by leptin may be involved in regulating energy homeostasis, of which the abnormality may possibly lead to the obesity.
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Coding of bitter taste information among taste bud cells
Grant number：26462815 2014.04 - 2017.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
Yoshida Ryusuke
Grant amount：\4810000 （ Direct expense: \3700000 、 Indirect expense：\1110000 ）
This study has demonstrated that (1) Bitter sensitive taste cells are heterogeneous from the point of view of their responsiveness, (2) Overall response profile of bitter taste cells to multiple bitter compounds was not significantly different between fungiform and circumvallate papillae, (3) Bitter responses of taste cells were significantly suppressed by TRPM5 inhibitor and PLC inhibitor, (4) Bitter responses of mouse taste cells were not affected by human bitter receptor antagonists, (5) CCK may be involved in peripheral bitter taste signaling. These data help understanding receptors and transduction mechanisms for bitter taste and give insights into coding of bitter taste information in the peripheral taste system.
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Grant number：26670810 2014.04 - 2016.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research Grant-in-Aid for Challenging Exploratory Research
Ninomiya Yuzo, Shigemura Noriatsu, Yoshida Ryusuke
Grant amount：\3510000 （ Direct expense: \2700000 、 Indirect expense：\810000 ）
The present study investigated molecular and neural mechanisms of detection, modulation and transmission of taste signals for fat preference. The results showed that there exists selective neural pathway for fatty acids in mice, that some of taste fibers best responding to sweet, umami or Ca are also sensitive to fatty acids, and that sweet-suppressive effect by leptin in diet-induced obese mice decreases with increasing circulating leptin which in turn reciprocally increase sweet-enhancing effect by endocannabinoids (eCBs). In another aspect, repeated stimulation with mixture of sugar and fatty acids gradually increase taste responses to the mixture. This enhancement of responses by repeated stimulations was abolished by eCB receptor blockage, suggesting involvement of eCB in the enhancement. Collectively, taste enhancement of sugar by mixing fatty acids may lead to behavioral preference for the mixture, thereby potential facilitation of development of obesity.
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The roles of GABA on development and function of taste bud cells
Grant number：23689076 2011.04 - 2014.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (A) Grant-in-Aid for Young Scientists (A)
YOSHIDA Ryusuke
Grant amount：\23530000 （ Direct expense: \18100000 、 Indirect expense：\5430000 ）
This study has demonstrated that (1) GABA may not have a pivotal role in development of taste buds or taste cells and in innervation of gustatory nerve fibers to taste buds, (2) GABA may be released from a subset of taste cells, possibly Type III cells, by depolarization of these cells, (3) intracellular Cl- concentration in taste cells may be very high therefore GABA would induce excitatory responses in these taste cells. These results suggest that GABA may play some roles in modulation of taste sensitivities in taste buds.
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Grant number：21791808 2009 - 2010
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B) Grant-in-Aid for Young Scientists (B)
YOSHIDA Ryusuke
Grant amount：\4160000 （ Direct expense: \3200000 、 Indirect expense：\960000 ）
This study has revealed (1) Endocannabinoids selectively enhance sweet taste via CB1 receptor on sweet sensitive taste cells, (2) Taste cells expressing T1R3, a sweet and umami taste receptor component, that are identified by using T1R3-GFP mice respond to various sweet and/or umami taste stimuli, (3) T1R3-GFP taste cells lacking T1R3, gustducin, or TRPM5 do not respond to taste stimuli, (4) T1R3-GFP taste cells express Gα11, 14, s, q, and i2 as Gα subunits other than gustducin.
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Discrimination of taste information among taste bud cells
Grant number：19791367 2007 - 2008
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B) Grant-in-Aid for Young Scientists (B)
YOSHIDA Ryusuke
Grant amount：\3680000 （ Direct expense: \3200000 、 Indirect expense：\480000 ）
本研究では、(1)5基本味(甘・苦・塩・酸・うま味)のうち、甘味、苦味、うま味を受容する味細胞と酸味を受容する味細胞の細胞型が異なること、
(2)塩味受容細胞はアミロライド感受性により2つのタイプが存在すること、
(3)うま味の受容細胞には甘味とうま味を受容する味細胞、うま味特異的味細胞が存在し、それぞれにうま味相乗効果の有無により更に2つのタイプに分類されることを明らかにした。これらは味情報が味細胞レベルで分別されている可能性を示唆する。 -
Grant number：18077004 2006 - 2010
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas Grant-in-Aid for Scientific Research on Priority Areas
NINOMIYA Yuzo, SHIGEMURA Noriatsu, YOSHIDA Ryusuke
Grant amount：\64400000 （ Direct expense: \64400000 ）
Potential modal-shifts of sensitivities of taste cell sensors were studied by examining cellular and behavior responses to sweet and salty stimuli under various molecular and environmental conditions. The results suggest that as comparable with the action of central nervous system, endocannabinoids, orexigenic mediators, enhanced peripheral sweet taste sensitivities, that opposes the action of leptin, an anorexigenic mediator. Species-specific sweet-suppressing effect of gurmarin occurs its binding to the mouse extracellular domain of T1r3(partially that of T1r2). In addition, molecular based modal-shift was found in sodium-salt sensor ENaC channels where an amino acid change (R616W) in αsubunit may produce variation in amiloride-sensitivity of the channel.
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Grant number：18109013 2006 - 2010
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (S) Grant-in-Aid for Scientific Research (S)
NINOMIYA Yuzo, SHIGEMURA Noriatsu, YOSHIDA Ryusuke, SANEMATSU KEISUKE
Grant amount：\112450000 （ Direct expense: \86500000 、 Indirect expense：\25950000 ）
Reception and modulation of taste signals and their roles in the regulation of food intake are investigated by examining taste cell, nerve and behavioral responses to various taste compounds. The results showed that sweet taste signal is initiated from a particular group of taste cells expressing T1r2/T1r3 sweet receptors and is transmitted to its corresponding group of nerve fibers through ATP release. Sweet taste sensitivity is inhibited by leptin, a satiety hormone, of which plasma level has a diurnal variation. Sweet taste thresholds also show diurnal variation that is parallel with that of plasma leptin levels. This synchronization, however, is not evident in obese subjects. Analyses on single nucleotide polymorphisms of umami receptor genes indicate existence of multiple receptors in umami perception in humans. These results provide new findings in the taste receptor and transduction mechanisms and new insights for potential roles of peripheral taste signal in the control for food intake, of which abnormality may possibly lead to the obesity.
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味細胞の生理機能と発現分子との連関:シングルセルRT-PCR法による解析
Grant number：17791325 2005 - 2006
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B) Grant-in-Aid for Young Scientists (B)
吉田 竜介
Grant amount：\3300000 （ Direct expense: \3300000 ）
本研究は、マウスの舌上皮・味蕾標本を用いることにより味覚受容器の構築を大きく損なうことなく細胞の生存性を高め、ルーズパッチ法により味細胞の味刺激に対する応答を記録し、その細胞における発現遺伝子をマルチプレックス・シングルセルRT-PCR法により検索することにより、受容体などの味覚関連分子の味細胞での発現と味細胞の応答特性との関連を解析することを目的とする。
マウス茸状乳頭味細胞のNaCl応答をルーズパッチ法で解析した結果、およそ半数の細胞が上皮性ナトリウムチャネル(ENaC)の阻害剤であるアミロライドに感受性があり(AS細胞)、残り半数はアミロライドに感受性を示さなかった(Al細胞)。AS細胞はNaClに特異的な応答を示し、Al細胞はNaCl以外にも電解質溶液(KCI、HCIなど)に応答を示した。アミロライドの効果は味孔側に与えられた場合に限られ、基底外側膜側に与えた場合には効果がなかった。これらの味細胞の応答特性や存在比は、鼓索神経にみられる2つのタイプ(アミロライド感受性のNタイプと非感受性のEタイプ)の線維のそれらと一致することが示唆された。シングルセルRT-PCRにより、これらの細胞でのENaCの発現を調べたところ、AS細胞ではα-ENaCの発現が検出され、Al細胞ではENaCサブユニットの発現は検出できなかったことから、AS細胞ではENaCがNaCl応答に関与している可能性が示唆される。
また、味細胞のグルタミン酸(MSG)応答を解析した結果、MSG応答細胞はサッカリンに応答する細胞、NaClに応答する細胞、MSGのみに応答する細胞に大別された。これらの応答特性の違いと、発現遺伝子との関連性については、現在の所、まだ明らかにはできていない。