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苦味受容体の口腔以外の組織における発現プロファイル及び脂肪細胞における機能解析

木村, 駿介 北海道大学

2022.03.24

概要

哺乳動物には、甘味、旨味、苦味、酸味、塩味の 5 つの味、いわゆる 5 基本味が存在する。基本味のうち苦味は、Gタンパク質共役型受容体(GPCR)である苦味受容体(T2R)がリガンドとなる化合物を受容することによって感知される。T2R のリガンドには、植物由来のアルカロイドやテルペノイドなどをはじめとして毒性成分が多く知られており、動物はこうした毒性成分を口腔内の T2R で感知することで忌避行動を起こす。したがって、口腔で苦味を感じる生物学的な意義は、毒物や異物を体の中に取り込まないようにするためであると考えられている。

T2R は、口腔以外の様々な組織にも発現している。気道や消化管などの上皮性組織に発現している T2R は、口腔の T2R と同様に外来の毒物・異物を検知して排除する役割を担っている。一方で、脂肪組織や肝臓などの非上皮性組織に発現している T2R は、外来の毒物・異物に直接触れることが少ないため、口腔や上皮性組織に発現する T2R とは異なる役割を担っているのではないかと推察される。また、T2R のリガンドである食品中の苦味成分が、脂質や糖の代謝機能の調節に関わることを示す報告が複数あることも、非上皮性組織の T2R が上皮性組織とは異なる役割を担っていることを示唆している。

非上皮性組織に発現する T2R の機能を明らかにすることは、苦味の持つ新たな生理的意義を見出すための有用な手がかりとなる。また、T2R が脂質や糖の代謝を調節するための標的分子であることがわかれば、そのリガンド化合物を肥満や糖尿病の予防や改善へと応用することも期待できる。そこで、本研究では非上皮性組織の中でも脂質や糖の代謝を担う組織に発現する T2R の機能を明らかにすることを目的とした。

非上皮性組織に発現する T2R の機能について調べるためには、各種組織の T2R の発現プロファイルを明らかにする必要がある。そのため、マウス組織とマウス由来の細胞株に発現する T2R を RT-PCR 法を用いて解析した。マウス組織として、脂質代謝や糖代謝を担う 4 種類の非上皮性組織(褐色脂肪、白色脂肪、骨格筋、肝臓)に加えて小腸についても調べた。細胞株としては、白色脂肪細胞のモデルとして用いられる 3T3-L1 細胞と、骨格筋のモデルとして用いられる C2C12 細胞を解析した。その結果、Tas2r108、126、135、137、143 がマウスの組織および細胞株に共通して発現していることを明らかとした。また、DNA シーケンス解析から C57BL/6J と 3T3-L1 細胞の T2R は同一の塩基配列を有している事を確認し、3T3-L1 細胞が脂肪細胞に発現している T2R の機能解析を行うための有用なモデル細胞であることを示した。

T2R の発現レベルは細胞分化や外部環境からの刺激により変動することが知られている。各種刺激に対して T2R が応答し、遺伝子発現レベルの変化が起これば、発現している T2R を介したシグナル経路の活性化が期待できる。また、各 T2R がどのような刺激に応答するのかを明らかにすれば、それら T2R の機能についても推定することができる。細胞分化が脂肪細胞と骨格筋の T2R 発現に与える影響を 3T3-L1 細胞と C2C12 細胞を用いて解析した結果、3T3-L1 細胞では解析対象とした全ての T2R(Tas2r108、126、135、137、143)の発現が上昇し、C2C12細胞では Tas2r126 と Tas2r135 の発現が上昇した。次に分化後の 3T3-L1 脂肪細胞の T2R 発現レベルを変動させる因子を探索した結果、苦味化合物刺激や血清飢餓処理を行うことにより T2R の発現が上昇することが明らかとなった。また、マウス脂肪組織においても、苦味化合物刺激や絶食により、T2R の発現が上昇することを明らかとした。これらの結果から、脂肪組織に発現している T2R は、細胞分化、苦味化合物刺激、絶食や血清飢餓などの影響を受けて T2Rシグナリングを活性化させている可能性があると考えられた。

脂肪細胞における T2R の機能を調べるため、T2R のリガンドである苦味化合物が 3T3-L1 脂肪細胞の遺伝子発現プロファイルに与える影響について調べた。分化させた 3T3-L1 脂肪細胞を苦味化合物で 1 時間刺激したときの遺伝子発現プロファイルを RNAseq により網羅的に解析した結果、苦味化合物刺激は 3T3-L1 細胞の細胞分化に関わる転写因子の発現を上昇させることが明らかとなった。したがって、脂肪細胞において T2R は細胞分化の調節に関与しているのではないかと考えられた。T2R を過剰発現させた 3T3-L1 細胞を用いて分化に与える影響を解析した結果、T2R の過剰発現は 3T3-L1 細胞の分化を抑制することが明らかとなった。したがって、 3T3-L1 細胞に発現している T2R は細胞分化の調節に関わっていることが示された。

以上のように本研究では、脂質や糖の代謝に関わる非上皮性組織が T2R 遺伝子を発現していることを示し、そのうち脂肪細胞に発現している T2R が各種刺激に応答して発現上昇することから、脂肪細胞における T2R を介したシグナリングを活性化させている可能性を示した。さらに T2R を過剰発現させた脂肪細胞株を用いた解析により、T2R が脂肪細胞の分化調節に関わっていることを明らかにした。

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