JP2004275001A - New method for screening - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for screening an agonist/an antagonist. <P>SOLUTION: The method, or the like, for screening a compound varying binding properties of a G-protein-coupled receptor protein or its salt to a fatty acid or an eicosanoid characterized as follows are provided. (1) The G protein-coupled receptor protein containing an amino acid sequence which is the same or substantially the same as an amino acid sequence represented by a specific sequence or its salt and (2) the fatty acid or the eicosanoid are used. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【０１０３】

【０１０４】
また、本明細書中で繁用される置換基、保護基および試薬を下記の記号で表記する。

【０１０５】
本明細書の配列表の配列番号は、以下の配列を示す。
配列番号：１
本発明のマウス脾臓由来ＧＰＲ４０のアミノ酸配列を示す。
配列番号：２
本発明のマウス脾臓由来ＧＰＲ４０をコードするｃＤＮＡの塩基配列を示す。
配列番号：３
本発明のラット脾臓由来ＧＰＲ４０のアミノ酸配列を示す。
配列番号：４
本発明のラット脾臓由来ＧＰＲ４０をコードするｃＤＮＡの塩基配列を示す。
配列番号：５
本発明のヒト由来ＧＰＲ４０のアミノ酸配列を示す。
配列番号：６
本発明のヒト由来ＧＰＲ４０をコードするｃＤＮＡの塩基配列を示す。
配列番号：７
以下の実施例２におけるＰＣＲ反応で使用したプライマー１の塩基配列を示す。
配列番号：８
以下の実施例２におけるＰＣＲ反応で使用したプライマー２の塩基配列を示す。
配列番号：９
以下の実施例３におけるＰＣＲ反応で使用したプライマー３の塩基配列を示す。
配列番号：１０
以下の実施例４におけるＰＣＲ反応で使用したプライマー４の塩基配列を示す。
配列番号：１１
以下の実施例４におけるＴａｑＭａｎ ＰＣＲ反応で使用したプライマーの塩基配列を示す。
配列番号：１２
以下の実施例４におけるＴａｑＭａｎ ＰＣＲ反応で使用したプローブの塩基配列を示す。
配列番号：１３
以下の実施例４におけるＴａｑＭａｎ ＰＣＲ反応で使用したプライマーの塩基配列を示す。
配列番号：１４
以下の実施例４におけるＴａｑＭａｎ ＰＣＲ反応で使用したプライマーの塩基配列を示す。
配列番号：１５
以下の実施例４におけるＴａｑＭａｎ ＰＣＲ反応で使用したプローブの塩基配列を示す。
配列番号：１６
以下の実施例４におけるＴａｑＭａｎ ＰＣＲ反応で使用したプライマーの塩基配列を示す。
配列番号：１７
本発明のカニクイザル由来ＧＰＲ４０のアミノ酸配列を示す。
配列番号：１８
本発明のカニクイザル由来ＧＰＲ４０をコードするｃＤＮＡの塩基配列を示す。
配列番号：１９
以下の実施例５におけるＰＣＲ反応で使用したプライマーの塩基配列を示す。
配列番号：２０
以下の実施例５におけるＰＣＲ反応で使用したプライマーの塩基配列を示す。
配列番号：２１
以下の実施例５におけるＰＣＲ反応で使用したプローブの塩基配列を示す。
配列番号：２２
以下の実施例５におけるＰＣＲ反応で使用したプライマーの塩基配列を示す。
配列番号：２３
以下の実施例７におけるＰＣＲ反応で使用したプライマーの塩基配列を示す。
配列番号：２４
以下の実施例７におけるＰＣＲ反応で使用したプライマーの塩基配列を示す。
配列番号：２５
以下の実施例７におけるＰＣＲ反応で使用したプローブの塩基配列を示す。
配列番号：２６
以下の実施例８におけるＰＣＲ反応で使用したプライマーの塩基配列を示す。
配列番号：２７
以下の実施例８におけるＰＣＲ反応で使用したプローブの塩基配列を示す。
配列番号：２８
以下の実施例９におけるＰＣＲ反応で使用したプライマーの塩基配列を示す。
配列番号：２９
本発明のハムスター由来ＧＰＲ４０のアミノ酸配列を示す。
配列番号：３０
本発明のハムスター由来ＧＰＲ４０をコードするｃＤＮＡの塩基配列を示す。
配列番号：３１
以下の実施例９におけるＰＣＲ反応で使用したプライマー１の塩基配列を示す。
配列番号：３２
以下の実施例９におけるＰＣＲ反応で使用したプライマー２の塩基配列を示す。
配列番号：３３
本発明のヒト由来ＧＰＲ４０のＣ末端ペプチドのアミノ酸配列を示す。
配列番号：３４
ｓｉＲＮＡ ｍ４０ｉ１０３のセンス鎖の塩基配列を示す。
配列番号：３５
ｓｉＲＮＡ ｍ４０ｉ１０３のアンチセンス鎖の塩基配列を示す。
配列番号：３６
ｓｉＲＮＡ ｍ４０ｉ２５６のセンス鎖の塩基配列を示す。
配列番号：３７
ｓｉＲＮＡ ｍ４０ｉ１０３のアンチセンス鎖の塩基配列を示す。
配列番号：３８
以下の実施例２０におけるＰＣＲ反応で使用したプライマーの塩基配列を示す。
配列番号：３９
以下の実施例２０におけるＰＣＲ反応で使用したプライマーの塩基配列を示す。
配列番号：４０
以下の参考例１におけるＰＣＲ反応で使用したセンス鎖プライマーの塩基配列を示す。
配列番号：４１
以下の参考例１におけるＰＣＲ反応で使用したアンチセンス鎖プライマーの塩基配列を示す。
【０１０６】
後述の実施例２で得られた形質転換体Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＴＯＰ１０／Ｚｅｒｏ Ｂｌｕｎｔ−ｍＧＰＲ４０は２００２年３月１８日から茨城県つくば市東１丁目１番地１ 中央第６（郵便番号３０５−８５６６）の独立行政法人産業技術総合研究所 特許生物寄託センターに寄託番号ＦＥＲＭ ＢＰ−７９６７として、２００２年２月１４日から大阪府大阪市淀川区十三本町２−１７−８５（郵便番号５３２−８６８６）の財団法人・発酵研究所（ＩＦＯ）に寄託番号ＩＦＯ １６７６２として寄託されている。
後述の実施例３で得られた形質転換体Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＪＭ１０９／ｐＣＲ２．１−ｒＧＰＲ４０は２００２年３月１８日から独立行政法人産業技術総合研究所 特許生物寄託センターに寄託番号ＦＥＲＭ ＢＰ−７９６８として、２００２年２月１４日から財団法人・発酵研究所（ＩＦＯ）に寄託番号ＩＦＯ １６７６３として寄託されている。
後述の実施例５で得られた形質転換体Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＪＭ１０９／ｐＣＲ２．１−ｍｏｎｋｅｙ ＧＰＲ４０は２００２年７月２３日から独立行政法人産業技術総合研究所 特許生物寄託センターに寄託番号ＦＥＲＭ ＢＰ−８１２５として寄託されている。
後述の実施例９で得られた形質転換体Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＪＭ１０９／ｐＴＡ ｈａｍｓｔａｒＧＰＲ４０は、形質転換体Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＪＭ１０９／ｐＴＡｈａｍｓｔｅｒＧＰＲ４０として、２００２年１２月１１日から独立行政法人産業技術総合研究所 特許生物寄託センターに寄託番号ＦＥＲＭ ＢＰ−８２５８として寄託されている。
後述の実施例２１で得られた形質転換体Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＤＨ５α／ｐＧＴ−ＧＰＲ４０は２００２年１２月１１日から独立行政法人産業技術総合研究所 特許生物寄託センターに寄託番号ＦＥＲＭ ＢＰ−８２５９として寄託されている。
【０１０７】
【実施例】
以下に参考例および実施例を示して、本発明をより詳細に説明するが、これらは本発明の範囲を限定するものではない。なお、大腸菌を用いての遺伝子は、モレキュラー・クローニング（Ｍｏｌｅｃｕｌａｒ ｃｌｏｎｉｎｇ）に記載されている方法に従った。
【０１０８】
参考例１ ヒトＧＰＲ４０の発現ベクターの構築
ヒトＧＰＲ４０をコードするＤＮＡ断片は以下のようなＰＣＲ法により取得した。すなわち、５’＞ＣＧＴＣＧＡＣＣＣＧＧＣＧＧＣＣＣＣＡＴＧＧＡＣＣＴＧＣＣＣＣＣＧ＜３’で示されるオリゴＤＮＡ（配列番号：４０）をセンス鎖プライマーとして、５’＞ＣＡＴＣＧＡＴＴＡＧＣＡＧＴＧＧＣＧＴＴＡＣＴＴＣＴＧＧＧＡＣＴＴ＜３’で示されるオリゴＤＮＡ（配列番号：４１）をアンチセンス鎖プライマーとして、各々２０ｐｍｏｌ、１０×Ａｄｖａｎｔａｇｅ（登録商標）２ ＰＣＲ Ｂｕｆｆｅｒ（ＣＬＯＮＴＥＣＨ）５μｌ、５０×ｄＮＴＰ ｍｉｘ（ＣＬＯＮＴＥＣＨ）１μｌ、５０×Ａｄｖａｎｔａｇｅ ２ Ｐｏｌｙｍｅｒａｓｅ Ｍｉｘ（ＣＬＯＮＴＥＣＨ）１μｌ、鋳型ＤＮＡとしてヒト膵臓ｃＤＮＡ液（ＣＬＯＮＴＥＣＨ）１μｌを含む混合液５０μｌを調製し、サーマルサイクラー（ＧｅｎｅＡｍｐ（登録商標） ＰＣＲ ｓｙｓｔｅｍ ｍｏｄｅｌ９７００（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ））を用いて９６℃、１分、続いて９６℃、３０秒→６１℃、３０秒→７２℃、１２０秒を３５サイクル繰り返し、さらに７２℃、１０分で伸長反応させるプログラムでＰＣＲ反応を行った。反応終了液をアガロースゲル電気泳動することにより単一のプロダクトを得、ＴＡクローニングキット（Ｉｎｖｉｔｒｏｇｅｎ）を用いてクローニングし、遺伝子配列を確認した。ＰＣＲエラーがないクローンについて、制限酵素ＳａｌＩ（宝酒造）、ＣｌａＩ（宝酒造）で二重消化した後、アガロースゲル電気泳動して、単一のプロダクトを切り出した。得られた断片（約１ｋｂ）をｐＡＫＫＯ−１１１ベクターに導入し、ＣＨＯ細胞のトランスフェクション用に用いた。
【０１０９】
【実施例１】ヒト由来ＧＰＲ４０に対する脂肪酸の反応性の確認
ＣＨＯ−Ｋ１細胞株は、特に記載が無い限り１０％ 牛胎児血清（Ｉｎｖｉｔｒｏｇｅｎ）を含むハムＦ−１２培地（Ｉｎｖｉｔｒｏｇｅｎ）を用いて培養した。トランスフェクションを行う前日に１０ｃｍ^２あたり４．５ｘ１０^５個の細胞を播き、５％ＣＯ_２濃度に調整されたＣＯ_２培養器にて３７℃で１５時間以上培養した。トランスフェクションはＬｉｐｏｆｅｃｔａｍｉｎｅ試薬（Ｉｎｖｉｔｒｏｇｅｎ）を用い、試薬添付の方法に準じて操作を行った。培養器に６−ｗｅｌｌプレートを使用する場合は、以下のように行った。まず、１．５ｍｌ容チューブを２本用意し、それぞれにＯｐｔｉ−ＭＥＭ−Ｉ培地（Ｉｎｖｉｔｒｏｇｅｎ）を１００μｌ分注した。次に、片方のチューブに発現ベクターを１μｇ、もう片方にＬｉｐｏｆｅｃｔａｍｉｎｅ試薬を６μｌ添加後、両者を混合し、２０分間室温に静置した。この溶液にＯｐｔｉ−ＭＥＭ−Ｉ培地を８００μｌ加えたトランスフェクション用混合液を、あらかじめＯｐｔｉ−ＭＥＭ−Ｉ培地を用いて洗浄したＣＨＯ−Ｋ１細胞に添加後、ＣＯ_２培養器にて６時間培養した。培養後の細胞は、ＰＢＳ（Ｉｎｖｉｔｒｏｇｅｎ）を用いてリンスした後、０．０５％ トリプシン・ＥＤＴＡ溶液（Ｉｎｖｉｔｒｏｇｅｎ）を用いて剥がし、遠心操作にて回収した。得られた細胞の数を測定し、培地２００μｌあたり５ｘ１０^４個の細胞が含まれるように希釈し、Ｂｌａｃｋ ｗａｌｌｅｄ ９６−ｗｅｌｌ ｐｌａｔｅ（Ｃｏｓｔａｒ）に１穴あたり２００μｌずつ分注後、ＣＯ_２培養器にて一晩培養した。上記トランスフェクション操作にて一過性に受容体を発現したＣＨＯ−Ｋ１細胞に各種試験サンプルを添加し、この際の細胞内カルシウム濃度の変動をＦＬＩＰＲ（Ｍｏｌｅｃｕｌａｒ Ｄｅｖｉｃｅ）を用いて測定した。ＦＬＩＰＲにて細胞内カルシウム濃度の変動を測定するために、以下の前処置を施した。まず、細胞に蛍光色素Ｆｌｕｏ−３ＡＭ（ＤＯＪＩＮ）を添加するため、あるいはＦＬＩＰＲアッセイを行う直前に細胞を洗浄するためのアッセイバッファーを作成した。ＨＢＳＳ（Ｉｎｖｉｔｒｏｇｅｎ）１０００ｍｌに１Ｍ ＨＥＰＥＳ（ｐＨ７．４）（ＤＯＪＩＮ）２０ｍｌを加えた溶液（以下、ＨＢＳＳ／ＨＥＰＥＳ溶液）に、プロべネシド（Ｓｉｇｍａ）７１０ｍｇを１ＮＮａＯＨ ５ｍｌに溶解後さらにＨＢＳＳ／ＨＥＰＥＳ溶液５ｍｌを加え混合した溶液１０ｍｌを添加し、この溶液をアッセイバッファーとした。次にＦｌｕｏ−３ＡＭ ５０μｇを２１μｌ ＤＭＳＯ（ＤＯＪＩＮ）に溶解し、さらに等量の２０％プルロン酸（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ）を加え混合後、１０５μｌの牛胎児血清を添加した１０．６ｍｌのアッセイバッファーに加え、蛍光色素溶液を調製した。トランスフェクション処理を施したＣＨＯ−Ｋ１細胞の培地を除き、直ちに蛍光色素溶液を１穴あたり１００μｌずつ分注後、ＣＯ_２培養器にて１時間培養し、細胞に蛍光色素を取り込ませた。培養後の細胞は上記のアッセイバッファーを用いて洗浄した後、ＦＬＩＰＲにセットした。また、受容体発現ＣＨＯ−Ｋ１細胞に添加する試験サンプルはアッセイバッファーを用いて調製し、同時にＦＬＩＰＲにセットした。以上の前処置を施した後、ＦＬＩＰＲにて各種試験サンプル添加後の細胞内カルシウム濃度の変動を測定した。その結果、ファルネシン酸（ｆａｒｎｅｓｏｉｃ ａｃｉｄ），５．８．１１−ｅｉｃｏｓａｔｒｉｙｎｏｉｃ ａｃｉｄ，５．８．１１．１４−ｅｉｃｏｓａｔｅｔｒａｙｎｏｉｃ ａｃｉｄ，オレイン酸（ｏｌｅｉｃ ａｃｉｄ），リノール酸（ｌｉｎｏｌｅｉｃ ａｃｉｄ），リノレン酸（ｌｉｎｏｌｅｎｉｃ ａｃｉｄ），アラキドン酸（ａｒａｃｈｉｄｏｎｉｃ ａｃｉｄ），エイコサペンタエン酸（ｅｉｃｏｓａｐｅｎｔａｅｎｏｉｃ ａｃｉｄ， ＥＰＡ），エイコサジエン酸（ｅｉｃｏｓａｄｉｅｎｏｉｃ ａｃｉｄ）、エイコサトリエン酸（ｅｉｃｏｓａｔｒｉｅｎｏｉｃ ａｃｉｄ）、ドコサヘキサエン酸（ｄｏｃｏｓａｈｅｘａｅｎｏｉｃ ａｃｉｄ， ＤＨＡ）、ドコサトリエン酸（ｄｏｃｏｓａｔｒｉｅｎｏｉｃ ａｃｉｄ）、アドレン酸（ａｄｒｅｎｉｃ ａｃｉｄ）、ラウリン酸（ｌａｕｒｉｃ ａｃｉｄ）等を１０^−５Ｍ〜１０^−６Ｍ加えたときに、ＧＰＲ４０受容体を発現するＣＨＯ−Ｋ１細胞が特異的に応答（細胞内カルシウム濃度の上昇）することが分かった〔図１〜１３〕。コントロールの発現ベクターのみを導入したＣＨＯ−Ｋ１細胞では、このような応答は見られなかった。すなわち、ＧＰＲ４０の内因性リガンドが脂肪酸であることが明らかになった。
【０１１０】
【実施例２】マウス脾臓由来のＧＰＲ４０をコードするｃＤＮＡのクローニングとその塩基配列の決定
マウス脾臓ｃＤＮＡ（Ｍａｒａｔｈｏｎ−ＲｅａｄｙＴＭ ｃＤＮＡ；Ｃｌｏｎｔｅｃｈ社）を鋳型として、２個のプライマー、プライマー１（配列番号：７）及びプライマー２（配列番号：８）を用いてＰＣＲを行なった。ＰＣＲにはＰｙｒｏｂｅｓｔ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ（宝酒造）を用い、▲１▼９８℃・１分の後、▲２▼９８℃・１０秒、５５℃・３０秒、７２℃・６０秒を４０回の後、▲３▼７２℃・２分の伸長反応を行なった。反応後、増幅産物をＺｅｒｏ Ｂｌｕｎｔ ＰＣＲクローニングキット（Ｉｎｖｉｔｒｏｇｅｎ社）の処方にしたがってプラスミドベクターｐＣＲ−Ｂｌｕｎｔ（Ｉｎｖｉｔｒｏｇｅｎ社）にクローニングした。これを大腸菌ＴＯＰ１０（Ｉｎｖｉｔｒｏｇｅｎ社）に導入して、プラスミドを持つクローンをｋａｎａｍｙｃｉｎｅを含むＬＢ寒天培地中で選択した。個々のクローンの塩基配列を解析した結果、新規Ｇタンパク共役型受容体タンパクをコードするｃＤＮＡ配列（配列番号：２）を得た。このｃＤＮＡより導き出されるアミノ酸配列（配列番号：１）を含有する新規タンパクをｍＧＰＲ４０と命名した。また形質転換体を大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）ＴＯＰ１０／Ｚｅｒｏ Ｂｌｕｎｔ−ｍＧＰＲ４０と命名した。
【０１１１】
【実施例３】ラット脾臓由来のＧＰＲ４０をコードするｃＤＮＡのクローニングとその塩基配列の決定
ラット脾臓ｃＤＮＡ（Ｍａｒａｔｈｏｎ−ＲｅａｄｙＴＭ ｃＤＮＡ；Ｃｌｏｎｔｅｃｈ 社）を鋳型として、２個のプライマー、プライマー３（配列番号：９）及びプライマー４（配列番号：１０）を用いてＰＣＲを行なった。ＰＣＲには Ａｄｖａｎｔａｇｅ ２ Ｐｏｌｙｍｅｒａｓｅ ｍｉｘ（Ｃｌｏｎｔｅｃｈ）を用い、▲１▼９６℃・１分、▲２▼９６℃・１０秒、７２℃・２分を５回、▲３▼９６℃・１０秒、７０℃・２分を２５回の後、７２℃・５分の伸長反応を行なった。反応後、増幅産物をＴＯＰＯ ＴＡ Ｃｌｏｎｉｎｇ Ｋｉｔ（Ｉｎｖｉｔｒｏｇｅｎ社）の処方にしたがってプラスミドベクターｐＣＲ２．１ＴＯＰＯ（Ｉｎｖｉｔｒｏｇｅｎ社）にクローニングした。これを大腸菌ＪＭ１０９（宝酒造）に導入して、プラスミドを持つクローンをａｍｐｉｃｉｌｉｎを含むＬＢ寒天培地中で選択した。個々のクローンの塩基配列を解析した結果、新規Ｇタンパク共役型受容体タンパクをコードするｃＤＮＡ配列（配列番号：４）を得た。このｃＤＮＡより導き出されるアミノ酸配列（配列番号：３）を含有する新規タンパク質をｒＧＰＲ４０と命名した。また形質転換体を大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）ＪＭ１０９／ｐＣＲ２．１−ｒＧＰＲ４０と命名した。
【０１１２】
【実施例４】発現分布
（１）細胞および培地
ＮＩＨ−３Ｔ３およびＢ１０４細胞はＡＴＣＣから購入した。マウス膵臓β細胞株ＭＩＮ６は文献（Ｊｕｎ−ｉｃｈｉ Ｍｉｙａｚａｋｉ ｅｔ ａｌ． Ｅｎｄｏｃｒｉｎｏｌｏｇｙ， Ｖｏｌ．１２７， Ｎｏ．１， ｐ１２６−１３２）記載のものを使用した。それぞれの細胞は１０％ＦＣＳを含むＤＭＥＭ培地（Ｉｎｖｉｔｒｏｇｅｎ社）でプレコンフルエントになるまで培養した。
（２）ＲＮＡの抽出およびｃＤＮＡ合成
ヒトおよびマウスの組織における発現分布に用いたｃＤＮＡは、ヒトおよびマウスの各種組織由来のｐｏｌｙＡ＋ＲＮＡ（クロンテック社）１μｇからランダムプライマーを用いて逆転写反応した。逆転写酵素ＳｕｐｅｒＳｃｒｉｐｔＩＩ（ＧＩＢＣＯ ＢＲＬ社）を使用し、添付のプロトコールにしたがって反応させ、エタノール沈殿してＴＥ １００μｌに溶解した。
マウスの細胞由来のｃＤＮＡは、細胞をＴｒｙｐｓｉｎ−ＥＤＴＡではがし細胞数を測定した後、ＲＮｅａｓｙ ｍｉｎｉ ＫＩＴ（ＱＩＡＧＥＮ社）のマニュアルに従ってｔｏｔａｌ ＲＮＡを抽出精製した。抽出したＲＮＡ １μｇはＳｕｐｅｒＳｃｒｉｐｔ ＩＩ （Ｉｎｖｉｔｒｏｇｅｎ社）のマニュアルに従ってランダムを用いてｆｉｒｓｔ ｓｔｒａｎｄ ｃＤＮＡを合成後エタノール沈殿してＴＥ １０μｌに溶解した。
（３）ＴａｑＭａｎ を用いた定量
組織由来ｃＤＮＡ（５ｎｇ ＲＮＡ相当）および細胞株由来ｃＤＮＡ（２５ ｎｇ ＲＮＡ相当）に対し、増幅反応試薬ＴａｑＭａｎ（商標）Ｕｎｉｖｅｒｓａｌ ＰＣＲ Ｍａｓｔｅｒ Ｍｉｘ（アプライドバイオシステムズジャパン株式会社）、ＧＰＲ４０検出用ＴａｑＭａｎ（商標）Ｐｒｏｂｅ Ｋｉｔ （配列：１１〜１６、アプライドバイオシステムズジャパン株式会社）を用い、１５μｌの合計反応液量に調製後反応を行った。各プライマー、プローブの最終濃度はマニュアルに従った。
ＴａｑＭａｎ（商標）ＰＣＲは、ＡＢＩ ＰＲＩＳＭ（商標）７９００ＨＴ配列検出システム（アプライドバイオシステムズジャパン株式会社）で行い、使用した温度周期はＴａｑＭａｎ（商標）Ｕｎｉｖｅｒｓａｌ ＰＣＲ Ｍａｓｔｅｒ Ｍｉｘ（アプライドバイオシステムズジャパン株式会社）のマニュアルに従った。
増幅生成物の定量的ＴａｑＭａｎ解析は、７９００ＨＴ ＳＤＳソフトウェア（アプライドバイオシステムズジャパン株式会社）を用いて行った。コピー数の算出に用いた検量線は、増幅領域全長を含む濃度既知のｃＤＮＡ 断片（ヒトＧＰＲ４０）またはＰｌａｓｍｉｄ ＤＮＡ（マウス ＧＰＲ４０）を用いた１０^７コピー／ｗｅｌｌから１０^２コピー／ｗｅｌｌまでの対数６点におけるＣ_Ｔ値から作成した。
ヒト各種組織でのＧＰＲ４０ ｍＲＮＡの発現分布を図１４に、マウス各種細胞でのＧＰＲ４０ｍＲＮＡの発現分布を図１５に示す。ヒト組織では、膵臓、肺、海馬、視床下部、脊髄に相対的に高い発現が認められた。マウスでは膵臓癌由来の細胞に極めて高い発現が認められた。
【０１１３】
【実施例５】カニクイザル由来のＧＰＲ４０をコードするｃＤＮＡのクローニングとその塩基配列の決定
カニクイザルＤＮＡを鋳型として、プライマー（配列番号：１９）及びプライマー２（配列番号：２０）を用いてＰＣＲを行なった。ＰＣＲには Ｐｙｒｏｂｅｓｔ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ （ＴＡＫＡＲＡ）を用い、▲１▼９５℃・１分、▲２▼９５℃・１０秒、５８℃・２０秒、７２℃・１分３０秒を４０回の後、７２℃・７分の伸長反応を行なった。反応後、増幅産物を１／ ５０希釈したものを鋳型として、プライマー（配列番号：２１）及びプライマー２（配列番号：２２）を用いてｎｅｓｔｅｄ ＰＣＲを行なった。反応後、増幅産物をＴＯＰＯ ＴＡ Ｃｌｏｎｉｎｇ Ｋｉｔ（Ｉｎｖｉｔｒｏｇｅｎ社）の処方にしたがってプラスミドベクターｐＣＲ２．１ＴＯＰＯ（Ｉｎｖｉｔｒｏｇｅｎ社）にクローニングした。これを大腸菌ＪＭ１０９（宝酒造）に導入して、プラスミドを持つクローンをａｍｐｉｃｉｌｉｎを含むＬＢ寒天培地中で選択した。個々のクローンの塩基配列を解析した結果、新規Ｇタンパク共役型受容体タンパクをコードするｃＤＮＡ配列（配列番号：１８）を得た。このｃＤＮＡより導き出されるアミノ酸配列（配列番号：１７）を含有する新規タンパクをｍｏｎｋｅｙ ＧＰＲ４０と命名した。また形質転換体を大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）ＪＭ１０９／ｐＣＲ２．１−ｍｏｎｋｅｙ ＧＰＲ４０と命名した。
【０１１４】
【実施例６】マウスインスリノーマＭＩＮ６細胞における遊離脂肪酸のインスリン分泌促進作用
ＭＩＮ６細胞は特に記載が無い限り１５％ＦＣＳ（Ｔｒａｃｅ Ｓｃｉｅｎｔｉｆｉｃ Ｌｔｄ．）、５５μＭ ２−メルカプトエタノール、１００Ｕ／ｍｌ ペニシリン、および１００μｇ／ｍｌ ストレプトマイシンを含むＤＭＥＭ（高グルコース、Ｉｎｖｉｔｒｏｇｅｎ）を用いて培養した。９６ウェルプレートに１ウェル当り１０^５個のＭＩＮ６細胞を播き、５％ＣＯ_２濃度に調整されたＣＯ_２培養器にて３７℃で３日間培養した。培地を１０％ＦＣＳ（Ｔｒａｃｅ Ｓｃｉｅｎｔｉｆｉｃ Ｌｔｄ．）、５．５ｍＭ グルコース、１００Ｕ／ｍｌペニシリン、および１００μｇ／ｍｌ ストレプトマイシンを含むＲＰＭＩ１６４０（グルコース不含、Ｉｎｖｉｔｒｏｇｅｎ）に交換しさらに２４時間培養した。培地を吸引により除去した後、１０％ＦＣＳ（Ｔｒａｃｅ Ｓｃｉｅｎｔｉｆｉｃ Ｌｔｄ．）、１１ｍＭ グルコース、１００Ｕ／ｍｌペニシリン、および１００μｇ／ｍｌ ストレプトマイシンを含むＲＰＭＩ１６４０（グルコース不含、Ｉｎｖｉｔｒｏｇｅｎ）で希釈した遊離脂肪酸−牛血清アルブミン（ＢＳＡ）混合液（４：１、モル比）を添加し、５％ＣＯ_２濃度に調整されたＣＯ_２培養器にて３７℃で９０分間（または６０分間）反応させた。反応後の９６ウェルプレートを１５００ｒｐｍ、５分間遠心した後、培養上清を回収した。この培養上清液中に分泌されたインスリン量をラットインスリンＲＩＡシステム（アマシャムファルマシア）を用いるラジオイムノアッセイ法（ＲＩＡ）にて測定した。その結果、パルミチン酸（Ｐａｌｍｉｔｉｃ ａｃｉｄ） （図１６、図１７）、γ―リノレン酸（γ−ｌｉｎｏｌｅｎｉｃ ａｃｉｄ）（図１６）、およびオレイン酸（ｏｌｅｉｃ ａｃｉｄ）（図１７）などの遊離脂肪酸を３００μＭ〜１０００μＭ加えたときに、ＭＩＮ６細胞によるインスリン分泌が促進することが分かった。すなわち、遊離脂肪酸がマウスインスリノーマＭＩＮ６細胞におけるインスリン分泌を促進させる作用を有することが明らかになった。ＭＩＮ６細胞は、実施例４（図１５）で記載している通りＧＰＲ４０を特異的に非常に多く発現していることから、本反応は、添加した脂肪酸がＧＰＲ４０を介してインスリン分泌を促進するものと判断される。
【０１１５】
【実施例７】ラット膵臓ランゲルハンス島におけるＧＰＲ４０の発現
ラット膵臓からのランゲルハンス島の単離は以下のようにして行った。ウィスターラット（雄性、８週齢）を断頭、脱血した後、ハサミで切除して開腹させ、総胆管の十二指腸側出口をクランプで結束した。次に総胆管の肝臓側に注射針で穴を開け、カニューレを挿入した。あらかじめＨＢＳＳに溶解し、氷冷させておいた１ｍｇ／ｍｌ コラゲナーゼ ＸＩ（シグマ、Ｃ−７６５７）溶液の５ｍｌをカニューレより注入した。膵臓を他の組織から切り離し、３７℃水浴中にて３分間振とうした。１０ｍｌのＨＢＳＳを添加して５〜６回ピペッティングした後、１０ｍｌのＨＢＳＳを添加して２回ピペッティングした。２０ｍｌのＨＢＳＳを添加し、４分間静置した後、上清をすて、再度１０ｍｌのＨＢＳＳを添加して２回ピペッティングした。さらに１０ｍｌのＨＢＳＳを添加して２回ピペッティングした後、２０ｍｌのＨＢＳＳを添加し、３分間静置した。上清を捨て、適当量のＨＢＳＳを添加して懸濁したものを底を黒く塗ったシャーレに移した。別のシャーレに１０％ＦＣＳ（Ｔｒａｃｅ Ｓｃｉｅｎｔｉｆｉｃ Ｌｔｄ．）、５．５ｍＭ グルコース、１００Ｕ／ｍｌペニシリン、および１００μｇ／ｍｌ ストレプトマイシンを含むＲＰＭＩ１６４０（グルコース不含、Ｉｎｖｉｔｒｏｇｅｎ）を入れ、底を黒く塗ったシャーレより実体顕微鏡下にて選んだランゲルハンス島をピペットチップを用いてこのシャーレに移した。
膵臓全体および上記で得られたランゲルハンス島をＩｓｏｇｅｎ（ニッポンジーン社）を加えてホモジナイズし、マニュアルにしたがってｔｏｔａｌＲＮＡを調製した。得られたｔｏｔａｌＲＮＡ１μｇからランダムプライマー、逆転写酵素としてＳｕｐｅｒＳｃｒｉｐｔＩＩ逆転写酵素（ＧＩＢＣＯ ＢＲＬ社）を用い、添付のマニュアルに従って４２℃で反応を行い、反応終了後にエタノール沈殿して蒸留水４０μｌに溶解した。ＧＰＲ４０ｍＲＮＡの発現量の定量はＳｅｑｕｅｎｃｅ Ｄｅｔｅｃｔｉｏｎ Ｓｙｓｔｅｍ Ｐｒｉｓｍ ７７００（ＡＢＩ社）を用いて行った。目的遺伝子の増幅と検出のためのプライマーとして５’−ＣＡＣＡＧＣＴＣＴＣＣＴＴＣＧＣＴＣＴＣＴＡＴ−３’（配列番号：２３），５’−ＣＡＧＴＴＴＣＧＣＧＴＧＧＧＡＣＡＣＴ−３’（配列番号：２４） およびＴａｑＭａｎ ｐｒｏｂｅとして５’−（Ｆａｍ）ＴＣＡＧＣＣＴＴＴＧＣＡＣＴＡＧＧＣＴＴＴＣＣＡＴＴＧＡＡＣ−（Ｔａｍｒａ）−３’（配列番号：２５）を使用した。ＲＴ−ＰＣＲ反応液はＴａｑＭａｎＵｎｉｖｅｒｓａｌ ＰＣＲ Ｍａｓｔｅｒ Ｍｉｘ（ＰＥ Ｂｉｏｓｙｓｔｅｍｓ社）１２．５μｌに、それぞれ１００μＭのプライマー溶液を０．２２５μｌ、５μＭのＴａｑＭａｎ ｐｒｏｂｅを１．２５μｌ、および上記で調製したｃＤＮＡ溶液を１μｌ加え、蒸留水で総反応液量を２５μｌとした。ＰＣＲ反応は５０℃ ２分、９５℃ １０分の後、９５℃ １５秒、６０℃ １分のサイクルを４０回繰り返した。その結果単離したランゲルハンス島ではＧＰＲ４０が高発現していることが確認された（図１８）。
【０１１６】
【実施例８】カニクイザル膵臓ランゲルハンス島におけるＧＰＲ４０の発現
カニクイザル膵臓からのランゲルハンス島の単離は以下のようにして行った。カニクイザル（雌性、体重３ｋｇ）の両脇および大腿部をメスで切開し、放血した後、ハサミで切除して開腹させ、総胆管の十二指腸側出口および肝臓側出口をクランプで結束した。次に総胆管の肝臓側に注射針で穴を開け、カニューレを挿入した。あらかじめＨＢＳＳに溶解し、氷冷させておいた１ｍｇ／ｍｌ コラゲナーゼ Ｖ（シグマ、Ｃ−９２６３）溶液の２５ｍｌをカニューレより注入した。膵臓を他の組織から切り離し、ハサミで５ｍｍ四方程度になるまで細かく切った後、３７℃水浴中にて２０分間振とうした。数十ｍｌのＨＢＳＳの添加とピペッティングを数回繰り返した後、４分間静置し、上清をすてた。沈澱物に再度、あらかじめＨＢＳＳに溶解し、氷冷させておいた１．７ｍｇ／ｍｌ コラゲナーゼ ＸＩ（シグマ、Ｃ−７６５７）溶液の１５ｍｌを添加して３７℃水浴中にて１５分間振とうした。数十ｍｌのＨＢＳＳの添加とピペッティングを繰り返した後、３分間静置し、上清をすてた。沈澱物に適当量のＨＢＳＳを添加し、懸濁したものを底を黒く塗ったシャーレに移した。別のシャーレにＨＢＳＳを入れ、底を黒く塗ったシャーレより実体顕微鏡下にて選んだランゲルハンス島をピペットチップを用いてこのシャーレに移した。
膵臓全体および上記で得られたランゲルハンス島から実施例７と同様の方法でｔｏｔａｌＲＮＡを調製、ｃＤＮＡを合成して、Ｓｅｑｕｅｎｃｅ Ｄｅｔｅｃｔｉｏｎ Ｓｙｓｔｅｍ Ｐｒｉｓｍ ７７００によりＧＰＲ４０ ｍＲＮＡの発現量を求めた。ただしサル型のＧＰＲ４０ｍＲＮＡの増幅と検出のため、プライマーとして５’−ＧＣＣＣＧＣＴＴＣＡＧＣＣＴＣＴＣＴ−３’（配列番号：２６）， ５’−ＧＡＧＧＣＡＧＣＣＣＡＣＧＴＡＧＣＡ−３’（配列番号：２７） およびＴａｑＭａｎ ｐｒｏｂｅとして５’−（Ｆａｍ）ＣＴＧＣＴＴＴＴＴＴＴＣＣＴＧＣＣＣＴＴＧＧＣＣ−（Ｔａｍｒａ）−３’（配列番号：２８）を使用した。その結果、霊長類においてもランゲルハンス島でＧＰＲ４０が高発現していることが確認された（図１９）。
【０１１７】
【実施例９】ハムスター由来のＧＰＲ４０をコードするｃＤＮＡのクローニングとその塩基配列の決定
ハムスター細胞株ＨＩＴ−Ｔ１５ｃＤＮＡを鋳型として、プライマー１（配列番号：３１）及びプライマー２（配列番号：３２）を用いてＰＣＲを行なった。ＰＣＲには Ｋｌｅｎｔａｑ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（クローンテック）を用い、▲１▼９５℃・２分、▲２▼９８℃・１０秒、６３℃・２０秒、７２℃・１分を３５回の後、７２℃・７分の伸長反応を行なった。反応後、増幅産物をＴＯＰＯ ＴＡ Ｃｌｏｎｉｎｇ Ｋｉｔ（Ｉｎｖｉｔｒｏｇｅｎ社）の処方にしたがってプラスミドベクターｐＣＲ２．１ＴＯＰＯ（Ｉｎｖｉｔｒｏｇｅｎ社）にクローニングした。これを大腸菌ＪＭ１０９（宝酒造）に導入して、プラスミドを持つクローンをａｍｐｉｃｉｌｉｎを含むＬＢ寒天培地中で選択した。個々のクローンの塩基配列を解析した結果、新規Ｇタンパク共役型受容体タンパクをコードするｃＤＮＡ配列（配列番号：３０）を得た。このｃＤＮＡより導き出されるアミノ酸配列（配列番号：２９）を含有する新規タンパク質をｈａｍｓｔａｒＧＰＲ４０と命名した。また、形質転換体を大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）ＪＭ１０９／ｐＴＡ ｈａｍｓｔａｒＧＰＲ４０と命名した。
【０１１８】
【実施例１０】ヒトＧＰＲ４０のＣ端ペプチドを含む免疫原の作製および免疫
ヒトＧＰＲ４０のＣ端ペプチド、Ｃｙｓ Ａｌａ Ａｌａ Ａｒｇ Ｔｈｒ Ｇｌｎ Ｇｌｙ Ｇｌｙ Ｌｙｓ Ｓｅｒ Ｇｌｎ Ｌｙｓ（配列番号：３３；公知手法に準じて合成した）とＢＳＡとの複合体を作製し免疫原とした。すなわち、ＢＳＡ ２０ｍｇを、０．１Ｍリン酸緩衝液（ｐＨ６．７）２．０ｍｌに溶解させ、Ｎ−（γ−マレイミドブチリロキシ）サクシニミド（ＧＭＢＳ）２．８ｍｇを含むＤＭＳＯ溶液２００μｌと混合し、室温で３０分反応させた。２ｍＭＥＤＴＡを含む０．１Ｍリン酸緩衝液（ｐＨ６．５）で平衡化したセファデックスＧ−２５カラムで過剰のＧＭＢＳ試薬を除去した後、マレイミド基の導入されたＢＳＡ５ｍｇと０．１ｍｌの水に溶解させたＣ端ペプチド０．８４ｍｇとを混合し、４℃で１夜反応させた。反応後、生理食塩水に対し、４℃で２日間透析した。８週齢のＢＡＬＢ／Ｃ雌マウスに、上記、Ｃ端ペプチド−ＢＳＡ複合体、約０．０５ｍｇ／匹をアジュバントとともに皮下免疫した。２週間後さらに２週間後同量の免疫原をアジュバントとともに追加免疫し、その１週間後に採血した。
【０１１９】
【実施例１１】西洋ワサビパーオキシダーゼ（ＨＲＰ）標識化Ｃ端ペプチドの作製
上記のＣ端ペプチド（配列番号：３３）とＨＲＰ（酵素免疫測定法用、ベーリンガーマンハイム社製）とを架橋し、酵素免疫測定法（ＥＩＡ）の標識体とした。すなわち、ＨＲＰ ２３ｍｇを２．３ｍｌの０．１Ｍ リン酸緩衝液、ｐＨ６．７に溶解させ、ＧＭＢＳ １．６ｍｇを含むＤＭＦ溶液０．２３ｍ１と混合し、室温で３０分間反応させたのち、２ｍＭ ＥＤＴＡを含む０．１Ｍ リン酸緩衝液、ｐＨ６．５で平衡化させたセファデックスＧ−２５カラムで分画した。このようにして作製したマレイミド基の導入されたＨＲＰ ２．３ｍｇとＣ鎖Ｎ端ペプチド１ｍｇとを混合し、４℃で１日反応させた。反応後、０．１Ｍ リン酸緩衝液、ｐＨ６．５で平衡化させたウルトロゲルＡｃＡ５４（ファルマシア社製）カラムで分画し、ＨＲＰ標識化Ｃ端ペプチドを得た。
【０１２０】
【実施例１２】Ｃ端ペプチドを免疫したマウスの抗血清中の抗体価の測定
マウス抗血清中の抗体価を以下の方法により測定した。抗マウスイムノグロブリン抗体結合マイクロプレートを作製するため、まずヤギ抗マウスイムノグロブリン抗体（ＩｇＧ画分、カッペル社製）を０．１ｍｇ／ｍｌ含む０．１Ｍ 炭酸緩衝液、ｐＨ９．６溶液を９６ウェルマイクロプレートに０．１ｍ１ずつ分注し、４℃で２４時間放置した。次に、プレートをリン酸緩衝生理食塩水（ＰＢＳ、ｐＨ７．４）で洗浄したのち、ウェルの余剰の結合部位をふさぐため２５％ブロックエース（雪印乳業社製）および０．０５％ ＮａＮ_３を含むＰＢＳ、ｐＨ７．２を０．３ｍ１ずつ分注し、４℃で少なくとも２４時間処理した。
上記、抗マウスイムノグロブリン抗体結合マイクロプレートの各ウエルにバッファーＥＣ［０．２％ＢＳＡ、０．４Ｍ ＮａＣｌ、０．４％ブロックエース、０．０５％ ＣＨＡＰＳ〔３−〔（コラミドプロピル）ジメチルアンモニオ〕プロパンスルホン酸〕、２ｍＭ ＥＤＴＡおよび０．０５％ ＮａＮ_３を含む０．０２Ｍ リン酸緩衝液、ｐＨ７．０］で希釈したマウス抗血清０．１４ｍｌを加え４℃で１６時間反応させた。次に、該プレートをＰＢＳ、ｐＨ７．４で洗浄したのち、バッファーＣ［１％ＢＳＡ、０．４Ｍ ＮａＣｌ、および２ｍＭ ＥＤＴＡを含む０．０２Ｍ リン酸緩衝液、ＰＨ７．０］で１０００倍に希釈した上記実施例で作製したＨＲＰ標識Ｃ端ペプチド０．１ｍｌを加え４℃で１日反応させた。次に、該プレートをＰＢＳ、ｐＨ７．４で洗浄したのち、固相上の酵素活性をＴＭＢマイクロウェルパーオキシダーゼ基質システム（ＫＩＲＫＥＧＡＡＲＤ＆ＰＥＲＲＹ ＬＡＢ．フナコシ薬品取り扱い）０．１ｍｌを加え室温で２０分間反応させることにより測定した。反応を１Ｍリン酸０．１ｍｌを加え停止させたのち、４５０ｎｍの吸光度（Ａｂｓ．４５０）をプレートリーダー（ＭＴＰ−１２０、コロナ社製またはＭｕｌｔｉｓｋａｎ Ａｓｃｅｎｔ、Ｌａｂｓｙｓｔｅｍｓ社製）で測定した。免疫したマウス全てにＣ端ペプチドに対する抗体価の上昇が認められた。
【０１２１】
【実施例１３】抗Ｃ端ペプチドモノクローナル抗体の作製
実施例１２において比較的高い抗体価を示したマウスに対してＢＳＡとして０．０８〜０．１ｍｇのＣ端ペプチド−ＢＳＡを静脈内に投与することにより最終免疫を行なった。最終免疫３日後のマウスから脾臓を摘出し、脾臓細胞浮遊液を得た。細胞融合に用いる細胞として、ＢＡＬＢ／Ｃマウス由来ミエローマ細胞Ｐ３−Ｘ６３．Ａｇ ８．Ｕ１（Ｐ３Ｕ１）を用いた〔カレント・トピックス・イン・マイクロバイオロジー・アンド・イムノロジー、８１、１（１９７８）〕。細胞融合は、原法〔ネイチャー、２５６、４９５（１９７５）〕に準じて行なった。すなわち、脾臓細胞およびＰ３Ｕ１をそれぞれ血清を含有しない培地で３度洗浄し、脾臓紬砲とＰ３Ｕ１数の比率を６．６：１になるよう混合して、７５０回転で１５分間遠心を行なって細胞を沈澱させた。上清を充分に除去した後、沈殿を軽くほぐし、４５％ポリエチレン・グリコール（ＰＥＧ）６０００（コッホライト社製）を０．３ｍ１加え、３７℃温水槽中で７分間静置して融合を行なった。融合後細胞に徐々にＭＥＭを添加し、合計１５ｍｌのＭＥＭを加えた後７５０回転１５分間遠心して上清を除去した。この細胞沈殿物を１０％牛胎児血清を含有するＧＩＴメデイウム（和光純薬）（ＧＩＴ−１０％ＦＣＳ）にＰ３Ｕ１が１ｍ １当リ２×１０^５個になるように浮遊し、９６穴マルチディッシュ（リンブロ社製）に１ウェル０．１ｍｌずつ１９２ウェルに播種した。播種後、細胞を３７℃で５％炭酸ガスインキュベーター中で培養した。２４時間後ＨＡＴ（ヒポキサンチン１×１０^−４Ｍ、アミノプテリン４×１０^−７Ｍ、チミジン１．６×１０^−３Ｍ）を含んだＧＩＴ−１０％ＦＣＳ培地（ＨＡＴ培地）を１ウェル当リ０．１ｍｌずつ添加することにより、ＨＡＴ選択培養を開始した。ＨＡＴ選択培養は、培養開始５、８日後に旧液を０．１ｍｌ捨てたあと、０．１ｍｌのＨＡＴ培地を添加することにより継続し、細胞融合後９日目に上清を採取した。
培養上清中の抗体価は、実施例に記載の方法に準じて実施した。すなわち、抗マウスイムノグロブリン抗体結合マイクロプレートの各ウエルに培養上清０．０７ｍｌおよびバッファーＥＣ０．０７ｍｌを加え４℃で１夜反応させた後、バッファーＣで１０００倍に希釈したＨＲＰ標識化Ａ鎖Ｎ端ペプチド０．１ｍｌを非標識Ｃ端ペプチド０．００２ｍＭの存在下あるいは非存在下室温で７時間反応させた。該プレートをＰＢＳで洗浄したのち、実施例１３に記載の方法に従い固相上の酵素活性を測定した。その結果、１６８ウェルの中から特異的な抗体価の認められたウェルを選択し、ハイブリドーマを凍結保存した。さらにウェルのハイブリドーマについては、希釈法によるクローニングに付した。クローニング後、培養上清中の抗体価を同様の方法に従って測定した。
陽性クローンの中から、抗Ｃ端ペプチドモノクローナル抗体産生ハイブリドーマを選択した。
【０１２２】
【実施例１４】モノクローナル抗体の精製
ハイブリドーマからのモノクローナル抗体をプロテイン−Ａカラムにより精製した。即ち、ハイブリドーマ上清約２５ｍｌを等量の結合緩衝液（３．５Ｍ ＮａＣｌ、０．０５％ ＮａＮ_３を含む１．５Ｍ グリシン、ｐＨ９．０）で希釈したのち、あらかじめ結合緩衝液で平衡化したリコンビナントプロテイン−Ａ−アガロース（Ｒｅｐｌｉｇｅｎ社製）カラムに供し、特異抗体を溶離緩衝液（０．０５％ ＮａＮ_３を含む０．１Ｍ クエン酸緩衝液、ｐＨ３．０）で溶出した。溶出液はＰＢＳ、ｐＨ７．４に対して４℃、２日間透析したのち、０．２２μｍのフィルター（ミリポア社製）により除菌濾過し４℃あるいは−８０℃で保存した。
【０１２３】
【実施例１５】Ｃ端ペプチドを免疫したウサギの抗血清中の抗体価の測定とポリクローナル抗体の調整
ウサギ抗血清中の抗体価を以下の方法により測定した。抗ウサギイムノグロブリン抗体結合マイクロプレートを作製するため、まずヤギ抗ウサギイムノグロブリン抗体（ＩｇＧ画分、カッペル社製）を０．１ｍｇ／ｍｌ含む０．１Ｍ 炭酸緩衝液、ｐＨ９．６溶液を９６ウェルマイクロプレートに０．１ｍ１ずつ分注し、４℃で２４時間放置した。次に、プレートをリン酸緩衝生理食塩水（ＰＢＳ、ｐＨ７．４）で洗浄したのち、ウェルの余剰の結合部位をふさぐため２５％ブロックエース（雪印乳業社製）および０．０５％ ＮａＮ_３を含むＰＢＳ、ｐＨ７．２を０．３ｍ１ずつ分注し、４℃で少なくとも２４時間処理した。上記、抗ウサギイムノグロブリン抗体結合マイクロプレートの各ウエルにバッファーＥＣ ［０．２％ ＢＳＡ、０．４Ｍ ＮａＣｌ、０．４％ブロックエース、０．０５％ ＣＨＡＰＳ〔３−〔（コラミドプロピル）ジメチルアンモニオ〕プロパンスルホン酸〕、２ｍＭ ＥＤＴＡおよび０．０５％ ＮａＮ_３を含む０．０２Ｍ リン酸緩衝液、ｐＨ７．０］で希釈したウサギ抗血清０．１４ｍｌを加え４℃で１６時間反応させた。次に、該プレートをＰＢＳ、ｐＨ７．４で洗浄したのち、バッファーＣ［１％ ＢＳＡ、０．４Ｍ ＮａＣｌ、および２ｍＭ ＥＤＴＡを含む０．０２ Ｍリン酸緩衝液、ＰＨ７．０］で１０００倍に希釈した上記実施例で作製したＨＲＰ標識Ｃ端ペプチド０．１ｍｌを加え４℃で１日反応させた。次に、該プレートをＰＢＳ、ｐＨ７．４で洗浄したのち、固相上の酵素活性をＴＭＢマイクロウェルパーオキシダーゼ基質システム（ＫＩＲＫＥＧＡＡＲＤ＆ＰＥＲＲＹ ＬＡＢ．フナコシ薬品取り扱い）０．１ｍｌを加え室温で２０分間反応させることにより測定した。反応を１Ｍリン酸０．１ｍｌを加え停止させたのち、４５０ｎｍの吸光度（Ａｂｓ．４５０）をプレートリーダー（ＭＴＰ−１２０、コロナ社製またはＭｕｌｔｉｓｋａｎ Ａｓｃｅｎｔ、Ｌａｂｓｙｓｔｅｍｓ社製）で測定した。免疫したウサギ三匹中二匹にＣ端ペプチドに対する抗体価の上昇が認められた。さらに抗体価の上昇したウサギの血清を採取し、定法に従い抗原精製を行い、ポリクローナル抗体を得た。
【０１２４】
【実施例１６】細胞内カルシウム濃度変化を指標とした本レセプターに対するアゴニストおよびアンタゴニストのスクリーニング方法の設定
ＧＰＲ４０に対するアゴニストおよびアンタゴニストの探索を行うための系を設定するために、アゴニストとして同定されたパルミチン酸を用いて、アッセイ系を設定した。
参考例１で作製したヒトＧＰＲ４０発現ベクターを用いて自体公知の方法で作製したヒトＧＰＲ４０を発現させたＣＨＯ細胞株（ＣＨＯ−ｈＧＰＲ４０ Ｎｏ．１０４）を３ｘ１０^４個／１００μｌの細胞が含まれるように希釈し、Ｂｌａｃｋ ｗａｌｌｅｄ ９６−ｗｅｌｌ ｐｌａｔｅ（Ｃｏｓｔａｒ）に１穴あたり１００μｌずつ分注後、ＣＯ_２培養器にて一晩培養した。細胞内カルシウム濃度の変動をＦＬＩＰＲ（Ｍｏｌｅｃｕｌａｒ Ｄｅｖｉｃｅ）を用いて測定した。方法を以下に記載した。
Ｆｌｕｏ−３ＡＭ（ＤＯＪＩＮ） ５０μｇを２１μｌ ＤＭＳＯ（ＤＯＪＩＮ）に溶解し、さらに等量の２０％プルロン酸（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ）を加え混合後、１０５μｌの牛胎児血清を添加した１０．６ｍｌのアッセイバッファー［ＨＢＳＳ（Ｉｎｖｉｔｒｏｇｅｎ）に １Ｍ ＨＥＰＥＳ（ｐＨ７．４）（ＤＯＪＩＮ）を２０ｍｌ添加し、プロべネシド（Ｓｉｇｍａ）７１０ｍｇを１Ｎ ＮａＯＨ ５ｍｌに溶解後さらに上記のＨＢＳＳ／ＨＥＰＥＳ溶液５ｍｌを加え混合した溶液１０ｍｌを添加し調製する。］に加え、蛍光色素溶液を調製した。細胞プレートの培地を除き、直ちに蛍光色素溶液を１穴あたり１００μｌずつ分注後、ＣＯ_２培養器にて１時間培養し、細胞に蛍光色素を取り込ませた。培養後の細胞は上記のアッセイバッファーを用いて洗浄した。細胞に添加するパルミチン酸はアッセイバッファーを用いて各々の濃度に希釈し、プレートに分注した。アンタゴニスト測定用に１２μＭγリノレン酸溶液（反応時の終濃度で３μＭ）をプレートに分注し、同時にＦＬＩＰＲにセットした。以上の前処置を施した後、ＦＬＩＰＲにてパルミチン酸添加後の細胞内カルシウム濃度の変動を測定しアゴニスト作用を、続いてγリノレン酸を添加してアンタゴニスト作用の検討を行った。パルミチン酸はアゴニストであるために、パルミチン酸を用いた実験ではアンタゴニストの評価は成立しないが、アンタゴニスト作用のみもつ化合物を添加した場合には、γリノレン酸の反応を抑制する活性が観測されるはずである。反応開始３０秒後の蛍光強度値の変化を用いた用量反応曲線より、ＥＣ_５０値を算出した。
【０１２５】
【実施例１７】脂肪酸添加によるｈＧＰＲ４０発現ＣＨＯ細胞でのＭＡＰキナーゼ活性化
参考例１で作製したヒトＧＰＲ４０発現ベクターを用いて自体公知の方法で作製したヒトＧＰＲ４０発現ＣＨＯ細胞（ＣＨＯ−ｈＧＰＲ４０ ＮＯ．１０４）またはＣＨＯ−ｍｏｃｋ細胞を３ｘ１０^５／ｗｅｌｌの濃度で６ウェルプレートに撒いて、血清低濃度培地（核酸不含ＭＥＭα培地に０．５％の透析ウシ胎児血清を添加したもの）にて一晩培養し、さらに無血清培地（核酸不含ＭＥＭα培地）に交換して一晩培養した。新しい無血清培地に交換して４時間培養後、１０μＭの各種脂肪酸を添加した。１０分間インキュベーションしたのち、サンプルバッファー（ＴＥＦＣＯ社）で細胞を溶解・抽出しＳＤＳ−ＰＡＧＥによって分離を行った。その後ＰｈｏｓｐｈｏＰｌｕｓ ｐ４４／４２ ＭＡＰ ｋｉｎａｓｅ（Ｔｈｒ２０２／Ｔｙｒ２０４） Ａｎｔｉｂｏｄｙ Ｋｉｔ（Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ Ｔｅｃｈｎｏｌｏｇｙ，Ｉｎｃ）を用いたウエスタンブロッテイングを行った。その結果、図２０に示す通り、ヒトＧＰＲ４０発現ＣＨＯ細胞でのみ、脂肪酸添加後にＭＡＰキナーゼのリン酸化によって示される当該タンパク質の活性化が起こることが分かった。
【０１２６】
【実施例１８】マウスＧＰＲ４０の配列に特異的なｓｉＲＮＡ導入によるマウスＧＰＲ４０−ＧＦＰ融合タンパク質の発現抑制
自体公知の方法にて作製した、マウスＧＰＲ４０とＧＦＰの融合タンパク質発現ＣＨＯ細胞を３ｘ１０^４／ｗｅｌｌにて９６ウェルプレートにまいて１日培養した。ＨＶＪ Ｅｎｖｅｌｏｐｅ ＶＥＣＴＯＲ ＫＩＴ ＧｅｎｏｍＯＮＥ^ＴＭ（石原産業）を用いて、Ｅｌｂａｓｉｒらの報告（Ｎａｔｕｒｅ ４１１（６８３６），４９４−４９８（２００１））に従って作製した種々の配列のｓｉＲＮＡ（Ｄｈａｒｍａｃｏｎ社）（２．８６ｐｍｏｌ／０．５μｌあるいは８．５７ｐｍｏｌ／１．５μｌの濃度）を細胞に導入後さらに１日培養した。マウスＧＰＲ４０−ＧＦＰの発現量の検出は以下に示す通りのエンザイムイムノアッセイにて行った。培養上清を捨ててＨＢＳＳ（インビトロゲン）にて洗浄後、０．０１％グルタルアルデヒド（和光純薬）で５分間固定し、２％ＢＳＡを含むＰＢＳ（宝酒造）にてブロッキングした。５００倍希釈した抗ＧＦＰモノクローナル抗体３Ｅ６（ニッポンジーン）を添加後室温で２時間インキュベーションした後、洗浄し５００倍希釈したＨＲＰ標識化抗マウスＩｇＧ抗体（ＩＣＮ）を添加し、室温で２時間インキュベーションした。洗浄後、ＴＭＢマイクロウェルペルオキシダーゼ基質（フナコシ）を添加し、３０分間インキュベーション後、硫酸を添加して発色反応を停止させ、４５０ｎｍで吸光度を測定した。その結果、図２１に示す通り、マウスＧＰＲ４０−ＧＦＰ発現ＣＨＯ細胞に対し、マウスＧＰＲ４０特異的なｓｉＲＮＡであるｍ４０ｉ１０３（センス鎖は配列番号：３４、アンチセンス鎖は配列番号：３５）、ｍ４０ｉ２５６（センス鎖は配列番号：３６、アンチセンス鎖は配列番号：３７）を添加することによりＧＰＲ４０−ＧＦＰの発現量の低下が認められた。このことからｍ４０ｉ１０３、ｍ４０ｉ２５６がマウスＧＰＲ４０発現を特異的に抑制させることが分かった。ｍ４０ｉ１０３およびｍ４０ｉ２５６の配列を図２２に示す。
【０１２７】
【実施例１９】ヒトＧＰＲ４０を膵臓β細胞特異的に発現するマウス作製用ＤＮＡ断片の構築
ヒトＧＰＲ４０をマウス膵臓β細胞で特異的に発現させるためにマウスインスリンＩＩプロモーターに下流に、ｐｏｌｙ（Ａ）＋付加シグナルを含むヒトＧＰＲ４０遺伝子を導入したＤＮＡ断片を作製した。
マウスゲノムからＰＣＲによってクロ−ニングしたマウスインスリンＩＩプロモ−タ−（ＭｌｕＩ−ＳａｌＩ断片：６７３ｂｐ）の下流に、同じくヒトゲノムからＰＣＲによってクロ−ニングした、開始コドンからｐｏｌｙ（Ａ）＋付加シグナルの下流まで含むヒトＧＰＲ４０遺伝子（ＳａｌＩ−ＳｐｅＩ断片：２２５６ｂｐ）断片を挿入し、マウスインスリンＩＩプロモ−タ−の制御下にヒトＧＰＲ４０遺伝子を発現する発現ユニットを作製した。このようにして得られた発現ユニットをＭｌｕＩ、ＳｐｅＩの二重消化により２９２８ｂｐ断片を切り出し、Ｃ５７ＢＬ／６Ｊマウスの受精卵にマイクロインジェクションするＤＮＡ断片とした。
【０１２８】
【実施例２０】マウスＧＰＲ４０遺伝子のクローニング
マウスＧＰＲ４０ ＯＲＦを含む領域を増幅できるプライマ−（配列番号：３８、配列番号：３９）を用いてＰＣＲによってマウス１２９ＳｖＪＢＡＣゲノムライブラリー（Ｉｎｖｉｔｒｏｇｅｎ社）のスクリ−ニングを行い、３８４コロニ−が混ざった状態の独立な３つのポジティブクロ−ンを得た。この３８４コロニ−が混ざった状態の独立な３つのポジティブクロ−ンを、上記の配列番号：３８および配列番号：３９によるＰＣＲで得られたＤＮＡ断片（１．６ｋｂｐ）をプロ−ブにしてコロニ−ハイブリダイゼーションを行い、最終的に３つのポジティブなシングルクロ−ンを単離した。
このクロ−ンが保持するＢＡＣ ＤＮＡを常法により単離した後、ＧＰＲ４０遺伝子が存在していると思われる領域約２５ｋｂについてシークエンサー（パーキンエルマー社）を用いて塩基配列を決定し、この塩基配列情報をもとにして制限酵素地図を作製した（図２３）。
【０１２９】
【実施例２１】ＧＰＲ４０遺伝子ターゲッティングベクターの構築
マウスＧＰＲ４０遺伝子ターゲッティングベクターの構築は、基本ベクタ−ｐＧＴ−Ｎ−２８（Ｎｅｗ Ｅｎｇｌａｎｄ Ｂｉｏｌａｂｓ社）にＧＰＲ４０遺伝子の５’上流側のア−ムとして
【実施例２０】で得られた１１１５１ｂｐ ＢａｍＨＩ断片、３’下流側のア−ムとして３４５８ｂｐ Ｅｃｌ１３６ＩＩ（ＳａｃＩ）断片を導入し、ポジティブセレクションマ−カ−にネオマイシン耐性遺伝子、ネガティブセレクションマ−カ−に単純ヘルペスウイルスチミジンキナ−ゼ遺伝子を持つマウスＧＰＲ４０遺伝子ターゲッティングベクターｐＧＴ−ＧＰＲ４０を構築した（図２３）。これを大腸菌ＤＨ５αに導入して、形質転換体として大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）ＤＨ５α／ｐＧＴ−ＧＰＲ４０を得た。
【０１３０】
【実施例２２】ヒト由来ＧＰＲ４０に対するエイコサノイドの反応性の確認
参考例１で作製したヒトＧＰＲ４０発現ベクターを用いて自体公知の方法で作製したヒトＧＰＲ４０を発現させたＣＨＯ細胞株（ＣＨＯ−ｈＧＰＲ４０ Ｎｏ．１０４）は特に記載が無い限り１０％ 透析牛胎児血清（Ｉｎｖｉｔｒｏｇｅｎ）を含むＭＥＭα培地（Ｉｎｖｉｔｒｏｇｅｎ）を用いて培養した。培養細胞は、ＰＢＳ（Ｉｎｖｉｔｒｏｇｅｎ）を用いてリンスした後、０．０５％ トリプシン・ＥＤＴＡ溶液（Ｉｎｖｉｔｒｏｇｅｎ）を用いて剥がし、遠心操作にて回収した。得られた細胞の数を測定し、培地１００μｌあたり３ｘ１０^４個の細胞が含まれるように希釈し、Ｂｌａｃｋ ｗａｌｌｅｄ ９６−ｗｅｌｌ ｐｌａｔｅ（Ｃｏｓｔａｒ）に１穴あたり１００μｌずつ分注後、ＣＯ_２培養器にて一晩培養した。上記ＣＨＯ細胞株に各種試験サンプルを添加し、この際の細胞内カルシウム濃度の変動をＦＬＩＰＲ（Ｍｏｌｅｃｕｌａｒ Ｄｅｖｉｃｅ）を用いて測定した。ＦＬＩＰＲにて細胞内カルシウム濃度の変動を測定するために、以下の前処置を施した。まず、細胞に蛍光色素Ｆｌｕｏ３−ＡＭ（ＤＯＪＩＮ）を添加するため、あるいはＦＬＩＰＲアッセイを行う直前に細胞を洗浄するためのアッセイバッファーを作成した。ＨＢＳＳ（Ｉｎｖｉｔｒｏｇｅｎ）１０００ｍｌに１Ｍ ＨＥＰＥＳ（ｐＨ７．４）（ＤＯＪＩＮ）２０ｍｌを加えた溶液（以下、ＨＢＳＳ／ＨＥＰＥＳ溶液）に、プロベネシド（Ｓｉｇｍａ）７１０ｍｇを１Ｎ ＮａＯＨ ５ｍｌに溶解後さらにＨＢＳＳ／ＨＥＰＥＳ溶液５ｍｌを加え混合した溶液１０ｍｌを添加し、この溶液をアッセイバッファーとした。次にＦｌｕｏ３−ＡＭ ５０μｇを２１μｌ ＤＭＳＯ（ＤＯＪＩＮ）に溶解し、さらに等量の２０％プルロン酸（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ）を加え混合後、１０５μｌの牛胎児血清を添加した１０．６ｍｌのアッセイバッファーに加え、蛍光色素溶液を調製した。培養ＣＨＯ細胞株の培地を除き、直ちに蛍光色素溶液を１穴あたり１００μｌずつ分注後、ＣＯ_２培養器にて１時間培養し、細胞に蛍光色素を取り込ませた。培養後の細胞は上記のアッセイバッファーを用いて洗浄した後、ＦＬＩＰＲにセットした。また、ＣＨＯ細胞株に添加する試験サンプルはアッセイバッファーを用いて調製し、同時にＦＬＩＰＲにセットした。以上の前処置を施した後、ＦＬＩＰＲにて各種試験サンプル添加後の細胞内カルシウム濃度の変動を測定した。その結果、（±）１４，１５−ジハイドロキシ−５Ｚ，８Ｚ，１１Ｚ−エイコサトリエン酸（（±）１４，１５−ｄｉｈｙｄｒｏｘｙ−５Ｚ，８Ｚ，１１Ｚ−ｅｉｃｏｓａｔｒｉｅｎｏｉｃ ａｃｉｄ， １４，１５−ＤＨＴ）、（±）５（６）−エポキシ−８Ｚ，１１Ｚ，１４Ｚ−エイコサトリエン酸（（±）５（６）−ｅｐｏｘｙ−８Ｚ，１１Ｚ，１４Ｚ−ｅｉｃｏｓａｔｒｉｅｎｏｉｃ ａｃｉｄ， ５，６−ＥＥＴ）、（±）８（９）−エポキシ−５Ｚ，１１Ｚ，１４Ｚ−エイコサトリエン酸（（±）８（９）−ｅｐｏｘｙ−５Ｚ，１１Ｚ，１４Ｚ−ｅｉｃｏｓａｔｒｉｅｎｏｉｃ ａｃｉｄ， ８，９−ＥＥＴ）、（±）１１（１２）−エポキシ−５Ｚ，８Ｚ，１４Ｚ−エイコサトリエン酸（（±）１１（１２）−ｅｐｏｘｙ−５Ｚ，８Ｚ，１４Ｚ−ｅｉｃｏｓａｔｒｉｅｎｏｉｃ ａｃｉｄ， １１，１２−ＥＥＴ）、（±）１４（１５）−エポキシ−５Ｚ，８Ｚ，１１Ｚ−エイコサトリエン酸（（±）１４（１５）−ｅｐｏｘｙ−５Ｚ，８Ｚ，１１Ｚ−ｅｉｃｏｓａｔｒｉｅｎｏｉｃ ａｃｉｄ，１４，１５−ＥＥＴ）を１０^−５Ｍ〜１０^−６Ｍ加えたときに、ＧＰＲ４０受容体を発現するＣＨＯ細胞株が特異的に応答（細胞内カルシウム濃度の上昇）することが分かった。コントロールの発現ベクターのみを導入したＣＨＯ細胞株（Ｍｏｃｋ細胞株）では、このような応答は見られなかった。従ってＧＰＲ４０は、これらの化合物にも反応することが判明した。
【０１３１】
【実施例２３】ｉｎ ｓｉｔｕハイブリダイゼーション法によるＧＰＲ４０ｍＲＮＡのｉｓｌｅｔにおける発現分布の検討
Ｗｉｓｔａｒラットを屠殺し、胆管十二指腸開口部を結索したのち胆管から１ｍｇ／ｍｌの冷コラゲナーゼ／ＨＢＳＳを膵臓に注入した。コラゲナーゼの注入された膵臓を切り離してコニカルチューブに移し、３７℃で浸透しながら１０分間インキュベートした。ＨＢＳＳを加え、ピペッティングにより組織をよく分散させたのち、３〜４分の静置によりｉｓｌｅｔを含む組織を沈殿させ、上清を除いた。この操作を２回繰り返した後、ＨＢＳＳに再懸濁し、実体顕微鏡下でｉｓｌｅｔをピックアップした。以上のようにして単離したｉｓｌｅｔをＯ・Ｃ・Ｔコンパウンド中で液体窒素により凍結し、−８０℃で保存した。
ＧＰＲ４０アンチセンス、センスプローブは以下の方法で調製した。まず、プラスミドベクターｐＣＲＩＩ ＴＯＰＯ（インビトロジェン社）に自体公知の方法でラット型ＧＰＲ４０ｃＤＮＡを挿入した。このｃＤＮＡを、Ｍ１３プライマー（インビトロジェン社）・Ａｄｖａｎｔａｇｅ ＧＣ２ポリメラーゼ（クロンテック社）を用いたＰＣＲにて増幅・直鎖化し、エタノール沈殿法により精製した。このｃＤＮＡから、ＤＩＧ ＲＮＡ Ｌａｂｅｌｌｉｎｇ ＫＩＴ（ＳＰ６／Ｔ７）（ロッシュ社）にてＳＰ６もしくはＴ７によるｉｎ ｖｉｔｒｏ ｔｒａｎｓｃｒｉｐｔｉｏｎを行い（４０μｌスケール）、生成されたＤＩＧラベルリボプローブをさらに４０ｍＭ ＮａＨＣＯ_３，６０ｍＭＮａ_２ＣＯ_３ ｐＨ１０．２により４００ｂｐにアルカリ加水分解した後エタノール沈殿により精製した。精製したプローブは大塚精製水１００μｌに溶解した。ｃＤＮＡの挿入方向から、ＳＰ６により生成されたＤＩＧラベルリボプローブがアンチセンスプローブ、Ｔ７により生成されたＤＩＧラベルリボプローブがセンスプローブであった。
Ｉｎ ｓｉｔｕハイブリダイゼーションには単離ｉｓｌｅｔの新鮮凍結切片を使用した。まず前述した単離ｉｓｌｅｔをクライオスタットＣＭ３０５０（ライカ社）を用いてシランコートスライド上に１６μｍの厚さで薄切した。切片を４％パラホルムアルデヒドを含むＰＢＳにて１０分間固定した後、ＰＢＳにてよく洗い、０．２５％無水酢酸を含む０．１Ｍトリエタノールアミン（ｐＨ ８．０）による処理（室温・１０分）にてアセチル化を行った。Ｈｙｂｒｉｄｉｚａｔｉｏｎ反応には、ｈｙｂｒｉｄｉｚａｔｉｏｎ ｂｕｆｆｅｒ（５０％ホルムアミド，１０ｍＭ Ｔｒｉｓ−ＨＣｌ ｐＨ７．５，１×Ｄｅｎｈａｒｄｔ’ｓ ｓｏｌｕｔｉｏｎ，２００μｇ／ｍｌ ｔＲＮＡ，１０％デキストラン硫酸，６００ｍＭ ＮａＣｌ，０．２５％ ＳＤＳ，１ｍＭ ＥＤＴＡ）でアンチセンスプローブもしくはセンスプローブを１０００倍に希釈し、８５℃で１０分変性した後、切片に加え５０℃で１２時間以上反応させた。引き続き、非特異的にｈｙｂｒｉｄｉｚａｔｉｏｎしたプローブを洗浄するため以下の操作を行った。１）２×ＳＳＣ（ＳＳＣ；１×ＳＳＣ＝１５０ｍＭ ＮａＣｌ，１５ｍＭクエン酸ナトリウム，ｐＨ７．０）／５０％ホルムアミド処理（６０℃・３０分 １回）、２）２×ＳＳＣ処理（６０℃・１５分 １回）、３）０．１×ＳＳＣ処理（６０℃・１５分 ２回）。以上の操作を行った後、ＤＩＧラベルプローブを検出するための免疫組織化学を行った。まず、ＤＩＧ−１（１００ｍＭ Ｔｒｉｓ−ＨＣｌ ｐＨ ７．５，１５０ｍＭ ＮａＣｌ，０．１％ Ｔｗｅｅｎ２０）で洗浄した後、１．５％ Ｂｌｏｃｋｉｎｇ ｒｅａｇｅｎｔ（ロッシュ社）を含むＤＩＧ−１による処理（３７℃・１時間）にて非特異反応のブロッキングを行い、ａｎｔｉ−ＤＩＧ ｆａｂ−ｆｒａｇｍｅｎｔ ａｎｔｉｂｏｄｙ ｃｏｎｊｕｇａｔｅｄ ｗｉｔｈ ａｌｋａｌｉｎｅ ｐｈｏｓｐｈａｔａｓｅ（ロッシュ社）を含むＤＩＧ−１（１：１０００）を室温で１時間反応させた。ＤＩＧ−１で十分洗浄した後、ＤＩＧ−３（１００ｍＭ Ｔｒｉｓ−ＨＣｌ ｐＨ ９．５，１００ｍＭ ＮａＣｌ，５０ｍＭ ＭｇＣｌ_２）でリンスし、０．１８ｍｇ／ｍｌ ５−ｂｒｏｍｏ−４−ｃｈｌｏｒｏ−３−ｉｎｄｏｌｙｌ−ｐｈｏｓｐｈａｔｅ（ＢＣＩＰ）を含むジメチルホルムアミド、および ０．３４ｍｇ／ｍｌ４−ｎｉｔｒｏｂｌｕｅ ｔｅｔｒａｚｏｌｉｕｍ（ＮＢＴ）を含む７０％ジメチルホルムアミド、およびｐｏｌｙｖｉｎｙｌａｌｃｈｏｌを、ＤＩＧ−３ １０ｍｌにつきそれぞれ０．３５ｍｌ、０．４５ｍｌ、３％加えた溶液によって、室温にて発色反応を行った。適時に発色をＰＢＳ洗浄により止めた後、インスリン・グルカゴン免疫組織化学の操作に移行した。
まず、１％ＢＳＡを含むＰＢＳにより非特異反応のブロッキングを行い、ウサギ抗インスリン血清（Ｈ−８６：Ｓａｎｔａ Ｃｒｕｚ，１／２００）および抗グルカゴンモノクローナル抗体（Ｋ７９ｂＢ１０：ＳＩＧＭＡ，１／８０００）を含む０．５％ＢＳＡ／ＰＢＳで１２時間インキュベートした。ＰＢＳで洗浄後、１０μｇ／ｍｌ Ａｌｅｘａ４８８−ｃｏｎｊｕｇａｔｅｄ ｇｏａｔ ａｎｔｉ−ｍｏｕｓｅ ＩｇＧ（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ社）および１０μｇ／ｍｌ Ａｌｅｘａ ５９４−ｃｏｎｊｕｇａｔｅｄ ｇｏａｔ ａｎｔｉ−ｒａｂｂｉｔ ＩｇＧ（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅ社）を含む０．５％ＢＳＡ／ＰＢＳと室温で２時間反応させた。ＰＢＳで洗浄後、９０％グリセロールを含むＰＢＳで封入し、光学顕微鏡、および蛍光顕微鏡で観察した。
ＧＰＲ４０アンチセンスプローブにより、単離ｉｓｌｅｔにおいて陽性シグナルが観察された。陽性シグナルはｉｓｌｅｔ全体に散見された。これらの領域においてセンスプローブによる発色は検出されなかった。一方、蛍光顕微鏡観察では、既知の通りｉｓｌｅｔ周辺部の細胞にグルカゴン免疫陽性シグナル、その他の部分にインスリン免疫陽性シグナルが観察された。ＧＰＲ４０アンチセンスプローブによるシグナルとインスリン・グルカゴンの免疫陽性シグナルを比較すると、ＧＰＲ４０のシグナルはインスリン免疫陽性細胞に観察されたが、グルカゴン免疫陽性細胞ではクリアなＧＰＲ４０のシグナルは観察されなかった。以上の結果から、ｉｓｌｅｔにおいてＧＰＲ４０ｍＲＮＡはβ細胞に特異的に発現している可能性が示唆された。
【０１３２】
【実施例２４】脂肪酸添加によるＭＩＮ６細胞でのＭＡＰキナーゼ活性化
培地は４．５ｇ／ｌのグルコースを含むＤｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ（ＤＭＥＭ、インビトロゲン社）に１５％ウシ胎児血清（ＴｈｅｒｍｏＴｒａｃｅ社）、５．５μＭ ２−メルカプトエタノール（インビトロゲン社）、２０ｍＭ ＨＥＰＥＳ ｐＨ７．３、１００Ｕ／ｍｌペニシリン、１００μｇ／ｍｌストレプトマイシンを添加したものを用いた。マウス膵臓β細胞株ＭＩＮ６細胞を２ｘ１０^５／ｗｅｌｌの濃度で１２ウェルプレートにまいて一晩培養し、翌朝培地を交換してさらに一晩培養した。翌朝、再び培地を交換して３時間培養後、２２ｍＭグルコースを含むＫＲＢＨにて１時間プレインキュベーションを行った。各種脂肪酸は１０μＭの濃度でＫＲＢＨ（２２ｍＭグルコース）に溶かしてＭＩＮ６細胞に添加し、２．５分間（あるいは５分間）インキュベーションした。ＭＡＰＫ阻害剤（ＰＤ９８０５９）は５０μＭの濃度でプレインキュベーション・インキュベーションの際にＫＲＢＨに添加した。サンプルバッファー（ＴＥＦＣＯ社）で細胞を溶解・抽出後、ＳＤＳ−ＰＡＧＥにより分離し、ＰｈｏｓｐｈｏＰｌｕｓ ｐ４４／４２ ＭＡＰ ｋｉｎａｓｅ（Ｔｈｒ２０２／Ｔｙｒ２０４）Ａｎｔｉｂｏｄｙ Ｋｉｔ（Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ Ｔｅｃｈｎｏｌｏｇｙ，Ｉｎｃ）を用いたウエスタンブロッテイングを行った。その結果、図２４に示す通り、ＧＰＲ４０発現ＣＨＯ細胞を用いたＦＬＩＰＲアッセイでアゴニスト活性を有する脂肪酸（レーン２，３，４）を添加したときのみＭＡＰキナーゼのリン酸化によって示される当該タンパク質の活性化が起こること、また、ＰＤ９８０５９を添加することでＭＡＰＫの活性化はほぼ抑えられることが分かった。ＧＰＲ４０発現ＣＨＯ細胞同様にアゴニスト活性を示す脂肪酸でＭＩＮ６においてもＭＡＰＫ活性化が起こることから、脂肪酸のＭＩＮ６刺激の少なくとも一部にはＧＰＲ４０が関与していることが強く支持された。
【０１３３】
【実施例２５】各種脂肪酸によるＭＩＮ６からのインスリン分泌促進
フラスコ内で培養したマウス膵臓β細胞株ＭＩＮ６を、２．５ｍＭのＥＤＴＡを含むＰＢＳを用いてはがした後、９６ウェルプレートに撒いて２日間培養した。培地は実施例２４と同じ４．５ｇ／ｌのグルコースを含むＤｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ（ＤＭＥＭ、インビトロゲン社）に１５％ウシ胎児血清（ＴｈｅｒｍｏＴｒａｃｅ社）、５．５μＭ ２−メルカプトエタノール（インビトロゲン社）、２０ｍＭ ＨＥＰＥＳ ｐＨ７．３、１００Ｕ／ｍｌペニシリン、１００μｇ／ｍｌストレプトマイシンを添加したものを用いた。細胞を改変したＫｒｅｂｓ−Ｒｉｎｇｅｒ ｂｉｃａｒｂｏｎａｔｅ ｂｕｆｆｅｒ（ＫＲＢＨ、１１６ｍＭ ＮａＣｌ、４．７ｍＭ ＫＣｌ、１．２ｍＭ ＫＨ_２ＰＯ_４、１．２ｍＭ ＭｇＳＯ_４、２．５ｍＭ ＣａＣｌ_２、２５ｍＭ ＮａＨＣＯ_３、２４ｍＭ ＨＥＰＥＳ ｐＨ７．３）で２回洗浄した後、３７℃５％ＣＯ_２条件下で３０分間プレインキュベーションした。上記のＫＲＢＨに２２ｍＭグルコースを添加したバッファーで希釈・調製した脂肪酸を、プレインキュベーション後の細胞に添加し、３７℃５％ＣＯ_２条件下で９０分間培養した。培養後、細胞の上清を回収し、凍結保存した。上清中のインスリン含量は市販のインスリンイムノアッセイキット（アマシャムファルマシア社）で測定した。その結果、図２５に示す通り、ＧＰＲ４０発現ＣＨＯ細胞を用いた細胞内カルシウムイオン動員アッセイにおいて顕著な活性を示した脂肪酸である、オレイン酸（ｏｌｅｉｃ ａｃｉｄ）、リノール酸（ｌｉｎｏｌｅｉｃ ａｃｉｄ）、α−リノレン酸（α−ｌｉｎｏｌｅｎｉｃ ａｃｉｄ）、γ−リノレン酸（γ−ｌｉｎｏｌｅｎｉｃ ａｃｉｄ）、アラキドン酸（ａｒａｃｈｉｄｏｎｉｃ ａｃｉｄ）、ＤＨＡにより有意なインスリン分泌上昇活性が認められた。一方、ＧＰＲ４０にアゴニスト活性を示さないリノール酸メチル（ｍｅｔｈｙｌ ｌｉｎｏｌａｔｅ＝ｌｉｎｏｌｅｉｃ ｍｅｔｈｙｌ）や酪酸（Ｂｕｔｙｒｉｃａｃｉｄ）は有意なインスリン分泌上昇活性を示さなかった。ＧＰＲ４０アゴニスト活性とインスリン分泌上昇活性がほぼ相関することから、ＧＰＲ４０が脂肪酸によるインスリン分泌上昇作用の少なくとも一部を担っていることが証明された。
【０１３４】
【実施例２６】ＧＰＲ４０を介したインスリン分泌上昇活性のグルコース依存性
実施例２５の方法に基づき、脂肪酸添加時のグルコース濃度の影響について検討した。脂肪酸添加時のＫＲＢＨに添加するグルコース濃度を０、５．５、１１、２２ｍＭに変えて検討した結果、図２６に示す通り、オレイン酸（ｏｌｅｉｃａｃｉｄ）、リノール酸（ｌｉｎｏｌｅｉｃ ａｃｉｄ）によるインスリン分泌上昇活性は１１ｍＭ以上の高グルコース濃度条件下で顕著であることが分かった。このことから、ＧＰＲ４０にアゴニスト活性を示す脂肪酸によるインスリン分泌上昇作用は、高血糖条件下でのインスリン分泌上昇作用・血糖効果作用が期待された。
【０１３５】
【実施例２７】ｓｉＲＮＡ導入ＭＩＮ６における、脂肪酸によるインスリン分泌上昇活性の抑制効果
実施例１８においてＧＰＲ４０特異的ｓｉＲＮＡであるｍ４０ｉ１０３がマウスＧＰＲ４０の発現を顕著に抑制させることが確認済である。実施例１８の方法に基づきＨＶＪ Ｅｎｖｅｌｏｐｅ ＶＥＣＴＯＲ ＫＩＴ ＧｅｎｏｍＯＮＥを用いてｍ４０ｉ１０３を導入させたＭＩＮ６細胞におけるインスリン分泌上昇活性について検討した。実施例２５の方法により脂肪酸によるインスリン分泌上昇活性について調べた結果、図２７に示す通り、ｍ４０ｉ１０３導入ＭＩＮ６ではリノール酸（ｌｉｎｏｌｅｉｃ ａｃｉｄ）、γ−リノレン酸（γ−ｌｉｎｏｌｅｎｉｃ ａｃｉｄ）によるインスリン分泌上昇活性が認められなくなった。一方、ランダム配列ｓｉＲＮＡであるＳｃｒａｍｂｌｅ ＩＩ ｄｕｐｌｅｘ ｓｉＲＮＡを導入したＭＩＮ６では上記脂肪酸によるインスリン分泌上昇活性が保持されていることが示された。この結果から、ＧＰＲ４０が、脂肪酸によるインスリン分泌上昇機構の少なくとも一部を担っていることが確認された。
【０１３６】
【実施例２８】ラット膵臓ランゲルハンス島初代培養における脂肪酸のインスリン分泌活性
８週齢の雄Ｗｉｓｔｅｒ ラット（Ｃｈａｒｌｅｓ Ｒｉｖｅｒ Ｊａｐａｎ）の膵臓からコラゲナーゼ消化法によりランゲルハンス島を単離した。６匹分のおよそ１０００個の単離ランゲルハンス島をＫＲＢＨ（２．８ｍＭ Ｇｌｕｃｏｓｅ）で２回洗浄し、そのまま３７℃、３０分間、５％ＣＯ_２存在下でプレインキュベーションした。遠沈後、上清を捨て再びＫＲＢＨ（２．８ｍＭ Ｇｌｕｃｏｓｅ）に１４０個／ｍＬの濃度で均一に懸濁し、９６穴プレートに１００μＬずつ分注した。各種脂肪酸サンプルのリノール酸（ｌｉｎｏｌｅｉｃ ａｃｉｄ）、γ−リノレン酸（γ−Ｌｉｎｏｌｅｎｉｃ ａｃｉｄ）、リノール酸メチル（ｍｅｔｈｙｌ ｌｉｎｏｌａｔｅ）（それぞれ最終３０μＭもしくは３μＭ）を含むＫＲＢＨ（最終１６．５ｍＭ Ｇｌｕｃｏｓｅ）を１００μＬずつ加え、３７℃、９０分間、５％ＣＯ_２存在下でインキュベートした。上清中のインスリン含量は市販のイムノアッセイキット（アマシャム）にて測定した。その結果、図２８に示す通りＧＰＲ４０にアゴニスト活性を示す脂肪酸で処理することによりラット膵臓ランゲルハンス島においてもインスリンの分泌促進活性が確認された。実施例２５〜２７で検討したβ細胞株ＭＩＮ６のみならず正常膵臓ランゲルハンス島においてもＧＰＲ４０がインスリン分泌に関与していることが確認された。
【０１３７】
【実施例２９】ＧＰＲ４０を介したインスリン分泌におけるＣａ^２＋イオンの必要性
実施例２５の方法に基づき、細胞外液中のＣａ^２＋イオンの必要性について検討した。プレインキュベーションおよびサンプルとのインキュベーションにおいて、Ｃａ^２＋イオンを抜き、さらにＣａ^２＋イオンをキレートする試薬であるＥＧＴＡを０．１ｍＭ添加した場合の脂肪酸によるインスリン分泌上昇活性を調べた。その結果、図２９に示す通り、通常のバッファーでは認められるオレイン酸（ｏｌｅｉｃ ａｃｉｄ）、リノール酸（ｌｉｎｏｌｅｉｃ ａｃｉｄ）、γ−リノレン酸（γ−ｌｉｎｏｌｅｎｉｃ ａｃｉｄ）によるインスリン分泌上昇活性がＣａ^２＋イオン不含・０．１ｍＭ ＥＧＴＡ含有バッファーでは認められないことが確かめられた。このことから、脂肪酸によるＧＰＲ４０を介したインスリン分泌上昇活性にはＣａ^２＋イオンが不可欠であることが確認された。
【０１３８】
【実施例３０】ＧＰＲ４０を介したインスリン分泌における各種阻害剤の効果
実施例２５の方法に基づき、Ｃａ^２＋チャネルブロッカー（ニフェジピン、Ｎｉｆｅｄｉｐｉｎｅ）およびＭＡＰキナーゼ阻害剤（ＰＤ９８０５９）の影響について検討した。プレインキュベーションおよびサンプルとのインキュベーションにおいて、Ｎｉｆｅｄｉｐｉｎｅ（１０μＭ）或いはＰＤ９８０５９（５０μＭ）を添加した場合の脂肪酸によるインスリン分泌上昇活性を調べた。その結果、図３０に示す通り、通常のバッファーでは認められるオレイン酸（ｏｌｅｉｃａｃｉｄ）、リノール酸（ｌｉｎｏｌｅｉｃ ａｃｉｄ）によるインスリン分泌上昇活性がニフェジピン含有バッファーでは認められなかった。これは実施例２９でＣａ^２＋イオンを除去した際に得られた結果とも符合し、脂肪酸によるＧＰＲ４０を介したインスリン分泌上昇活性にはＣａ^２＋イオンが不可欠であることが再び確認された。また、ＰＤ９８０５９含有バッファーでは通常のバッファーと同様の結果が得られたことから、ＭＡＰキナーゼが活性化されていない状態でも脂肪酸によるインスリン分泌上昇活性は起こる、つまり、脂肪酸刺激によるＭＡＰキナーゼの活性化は、インスリン分泌上昇活性に必ずしも必要でないことが分かった。ＧＰＲ４０はインスリン分泌のみならず、β細胞の増殖などにも機能を発揮する可能性が考えられた。
【０１３９】
【実施例３１】ＧＰＲ４０を介したインスリン分泌におけるＢＳＡの影響
脂肪酸はアルブミンに結合する性質が知られている。実施例２５の方法に基づき、ウシ血清アルブミン（ＢＳＡ）が存在した場合の影響について検討した。プレインキュベーションおよびサンプルとのインキュベーションにおいて、バッファー中に０．１％ＢＳＡを添加し、脂肪酸によるインスリン分泌上昇活性を調べた。その結果、図３１示す通り、通常のバッファーでは認められるオレイン酸（ｏｌｅｉｃ ａｃｉｄ）、リノール酸（ｌｉｎｏｌｅｉｃ ａｃｉｄ）によるインスリン分泌上昇活性が、０．１％ ｆａｔｔｙ ａｃｉｄ−ｆｒｅｅ ＢＳＡ含有バッファーではほぼ抑制された。インスリン分泌上昇活性はＢＳＡに結合している脂肪酸よりはむしろ結合していない状態の脂肪酸により引き起こされることが分かった。ＢＳＡに結合するという脂肪酸特有の性質が反応性に反映された結果といえる。
【０１４０】
【実施例３２】ヒト、マウスおよびラット由来ＧＰＲ４０に対する脂肪酸のＥＣ_５０値の決定
ＥＣ_５０値の決定にはヒト、マウスおよびラット由来ＧＰＲ４０を安定発現したＣＨＯ細胞株を用いた。特に記載が無い限りこれらのＣＨＯ細胞株は１０％牛胎児血清（Ｉｎｖｉｔｒｏｇｅｎ）を含むα−ＭＥＭ培地（Ｉｎｖｉｔｒｏｇｅｎ）を用いて培養した。
アッセイ前日に、ほぼコンフルエントになるまで培養した細胞を、ＰＢＳ（Ｉｎｖｉｔｒｏｇｅｎ）を用いてリンスした後、０．０５％Ｔｒｙｐｓｉｎ・ＥＤＴＡ溶液（Ｉｎｖｉｔｒｏｇｅｎ）を用いて剥がし、遠心操作にて回収した。得られた細胞の数を測定し、培地１ｍＬあたり３ｘ１０^５個の細胞が含まれるように希釈し、Ｂｌａｃｋ ｗｅｌｌｅｄ ９６−ｗｅｌｌ ｐｌａｔｅ（ｃｏｓｔｅｒ）に１穴あたり１００μＬずつ分注後、ＣＯ_２培養器にて一晩培養した。このように調整したＣＨＯ細胞に各種試験サンプルを添加し、この際の細胞内カルシウム濃度の変動をＦＬＩＰＲ（Ｍｏｌｅｃｕｌａｒ Ｄｅｖｉｃｅ）を用いて測定した。ＦＬＩＰＲにて細胞内カルシウム濃度の変動を測定するために、以下の前処置を施した。
まず、細胞に蛍光色素Ｆｌｕｏ３−ＡＭ（ＤＯＪＩＮ）を添加するため、あるいはＦＬＩＰＲアッセイを行う直前に細胞を洗浄するためのアッセイバッファーを作成した。ＨＢＳＳ（Ｉｎｖｉｔｒｏｇｅｎ）１０００ｍＬに１Ｍ ＨＥＰＥＳ（ｐＨ ７．４）（ＤＯＪＩＮ）２０ｍＬを加えた溶液（以下、ＨＢＳＳ／ＨＥＰＥＳ溶液）に、プロべネシド（Ｓｉｇｍａ）７１０ｍｇを１Ｎ ＮａＯＨ ５ｍＬに溶解後、さらにＨＢＳＳ／ＨＥＰＥＳ溶液５ｍＬを加え混合した溶液１０ｍＬを添加し、この溶液をアッセイバッファーとした。次にＦｌｕｏ３−ＡＭ５０μｇを２１μＬ ＤＭＳＯ（Ｗａｋｏ）に溶解し、さらに等量の２０％プルロン酸（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ）を加え混合後、１０５μＬの牛胎児血清を添加した１０．６ｍＬのアッセイバッファーに加え、蛍光色素溶液を調製した。アッセイ前日にＢｌａｃｋ ｗｅｌｌｅｄ ９６−ｗｅｌｌ ｐｌａｔｅにまきなおしたＣＨＯ細胞の培地を除き、直ちに蛍光色素溶液を１穴あたり１００μＬずつ分注後、ＣＯ_２培養器にて１時間培養し、細胞に蛍光色素を取り込ませた。培養後の細胞は上記のアッセイバッファーを用いて洗浄した後、ＦＬＩＰＲにセットした。また、試験サンプルはアッセイバッファーを用いて調製し、終濃度が３０，１０，３，１，０．３μＭになるように希釈し、同時にＦＬＩＰＲにセットした。以上の前処置を施した後、ＦＬＩＰＲにて各種試験サンプル添加後の細胞内カルシウム濃度の変動を測定した。そしてそれらの結果より、各脂肪酸での容量反応曲線を作成し、ＥＣ_５０値を算出した。その結果を表１に示した。
【表１】

【０１４１】
【実施例３３】ヒトおよびマウスＧＰＲ４０発現ＣＨＯ細胞のｃＡＭＰ産生に対する脂肪酸の影響
ヒトおよびマウスＧＰＲ４０発現ＣＨＯ細胞を、１×１０^５／ｗｅｌｌの濃度で ９６−ｗｅｌｌ Ｐｌａｔｅで２０時間培養した。細胞は１００μｌのＡｓｓａｙ Ｂｕｆｆｅｒ（０．１ｍＭ ＩＢＭＸ（和光純薬）および０．１ｍＭ Ｒｏ−２０−１７２４（Ｂｉｏｍｏｌ）を含むＤＭＥＭ（Ｉｎｖｉｔｒｏｇｅｎ））で２回洗浄後、２μＭのフォルスコリン（Ｆｏｒｓｋｏｌｉｎ、和光純薬）を含むまたは含まないＡｓｓａｙ Ｂｕｆｆｅｒに溶解したＤＨＡ、リノール酸およびγ−リノレン酸（ともにＳＩＧＭＡ）を加えて３７℃で１０分間反応した。反応後、ｃＡＭＰ Ｓｃｒｅｅｎ（ＡＢＩ）の処方に従って細胞を溶解し、細胞内のｃＡＭＰ量を測定した。その結果、フォルスコリンを含まない条件では、各脂肪酸はヒトおよびマウスのＧＰＲ４０発現ＣＨＯ細胞に対してｃＡＭＰ産生活性は示さなかった。一方、フォルスコリン存在下では、ヒトＧＰＲ４０発現ＣＨＯ細胞に対しては弱いｃＡＭＰ産生抑制活性を示したが、マウスＧＰＲ４０発現ＣＨＯ細胞に対しては活性を示さなかった（図３２）。
【０１４２】
【実施例３４】ＭＩＮ６を用いたＦＬＩＰＲアッセイ
フラスコ内で培養したマウス膵臓β細胞株ＭＩＮ６を、２．５ｍＭのＥＤＴＡを含むＰＢＳを用いてはがした後、培地１ｍＬあたり３ｘ１０^５個の細胞が含まれるように希釈し、Ｂｌａｃｋ ｗｅｌｌｅｄ ９６−ｗｅｌｌ ｐｌａｔｅ（ｃｏｓｔｅｒ）に１穴あたり１００μＬずつ分注後、ＣＯ_２培養器にて２日間培養した。培地は４．５ｇ／ｌのグルコースを含むＤｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ（ＤＭＥＭ、インビトロゲン社）に１５％ウシ胎児血清（ＴｈｅｒｍｏＴｒａｃｅ社）、５．５μＭ ２−メルカプトエタノール（インビトロゲン社）、２０ｍＭ ＨＥＰＥＳ ｐＨ７．３、１００Ｕ／ｍｌペニシリン、１００μｇ／ｍｌストレプトマイシンを添加したものを用いた。このように調整したＭＩＮ６細胞に各種脂肪酸（１０μＭ）を添加し、この際の細胞内カルシウム濃度の変動をＦＬＩＰＲ（Ｍｏｌｅｃｕｌａｒ Ｄｅｖｉｃｅ）を用いて測定した。ＦＬＩＰＲにて細胞内カルシウム濃度の変動を測定するための方法は、実施例２４に記載の方法に従った。その結果、ＧＰＲ４０のリガンドであるγ−リノレン酸（γ−ｌｉｎｏｌｅｎｉｃ ａｃｉｄ）、リノール酸（ｌｉｎｏｌｅｉｃ ａｃｉｄ）、オレイン酸（ｏｌｅｉｃ ａｃｉｄ）、アラキドン酸（ａｒａｃｈｉｄｏｎｉｃ ａｃｉｄ）、ドコサヘキサエン酸（ＤＨＡ）の添加で、ＭＩＮ６の細胞内Ｃａ^２＋濃度が上昇することが確認された。一方、ＧＰＲ４０のリガンドではないリノール酸メチルや酪酸ではこのような活性は見られなかった（図３３）。
【０１４３】
【実施例３５】ＧＰＲ４０に対するアゴニストおよびアンタゴニストのスクリーニング方法の設定およびアゴニスト候補を選択する基準
（１）細胞内カルシウム濃度変化を指標としたＧＰＲ４０に対するアゴニストおよびアンタゴニストのスクリーニング方法の設定
ＧＰＲ４０に対するアゴニストおよびアンタゴニストの探索を行うための系を設定するために、アゴニストとして同定されたパルミチン酸を用いて、アッセイ系を設定した。
参考例１で作製したヒトＧＰＲ４０発現ベクターを用いて自体公知の方法で作製したヒトＧＰＲ４０を発現させたＣＨＯ細胞株（ＣＨＯ−ｈＧＰＲ４０ Ｎｏ．１０４）を３ｘ１０^４個／１００μｌの細胞が含まれるように希釈し、Ｂｌａｃｋ ｗａｌｌｅｄ ９６−ｗｅｌｌ ｐｌａｔｅ（Ｃｏｓｔａｒ）に１穴あたり１００μｌずつ分注後、ＣＯ_２培養器にて一晩培養した。細胞内カルシウム濃度の変動をＦＬＩＰＲ（Ｍｏｌｅｃｕｌａｒ Ｄｅｖｉｃｅ）を用いて測定した。方法を以下に記載した。
Ｆｌｕｏ−３ＡＭ（ＤＯＪＩＮ）５０μｇを２１μｌ ＤＭＳＯ（ＤＯＪＩＮ）に溶解し、さらに等量の２０％プルロン酸（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ）を加え混合後、１０５μｌの牛胎児血清を添加した１０．６ｍｌのアッセイバッファー［ＨＢＳＳ（Ｉｎｖｉｔｒｏｇｅｎ）に１Ｍ ＨＥＰＥＳ（ｐＨ７．４）（ＤＯＪＩＮ）を２０ｍｌ添加し、プロべネシド（Ｓｉｇｍａ）７１０ｍｇを１Ｎ ＮａＯＨ ５ｍｌに溶解後さらに上記のＨＢＳＳ／ＨＥＰＥＳ溶液５ｍｌを加え混合した溶液１０ｍｌを添加し調製する。］に加え、蛍光色素溶液を調製した。細胞プレートの培地を除き、直ちに蛍光色素溶液を１穴あたり１００μｌずつ分注後、ＣＯ_２培養器にて３７℃で１時間培養し、細胞に蛍光色素を取り込ませた。培養後の細胞は上記のアッセイバッファーを用いて洗浄した。細胞に添加するパルミチン酸はアッセイバッファーを用いて各々の濃度に希釈し、プレートに分注した。アンタゴニスト測定用に１２μＭ γ−リノレン酸溶液（反応時の終濃度で３μＭ）をプレートに分注し、同時にＦＬＩＰＲにセットした。以上の前処置を施した後、ＦＬＩＰＲにてパルミチン酸添加後の細胞内カルシウム濃度の変動を測定しアゴニスト作用を、続いてγ−リノレン酸を添加してアンタゴニスト作用の検討を行った。パルミチン酸はアゴニストであるために、パルミチン酸を用いた実験ではアンタゴニストの評価は成立しないが、アンタゴニスト作用のみもつ化合物を添加した場合には、後で加えるγ−リノレン酸の反応を抑制する活性が観測される。反応開始３０秒後もしくは４０秒後の蛍光強度値の変化を用いた用量反応曲線より、ＥＣ_５０値を算出した。
（２）ＦＬＩＰＲアッセイの結果からアゴニスト候補を選択する基準
アゴニスト候補を選出するための被検体化合物は、予めその濃度が１０ｍＭとなるようにＤＭＳＯ（Ｗａｋｏ）で希釈し、測定時に上記のアッセイバッファーで希釈して使用した。そして、それらの被検体化合物を用いて上記と同様の方法で、参考例１で作製したヒトＧＰＲ４０発現ベクターを用いて自体公知の方法で作製したヒトＧＰＲ４０を発現させたＣＨＯ細胞株（ＣＨＯ−ｈＧＰＲ４０ Ｎｏ．１０４）、ヒトヒスタミンＨ１受容体発現ベクターを用いて自体公知の方法で作製したヒトヒスタミンＨ１受容体を発現させたＣＨＯ細胞株（ＣＨＯ−Ｈ１）、およびＭｏｃｋのＣＨＯ細胞株に対する反応開始３０秒もしくは４０秒後の蛍光強度値を測定した。さらに、３０μＭ γ−リノレン酸のＣＨＯ−ｈＧＰＲ４０に対する蛍光強度を１００％としたときの相対値を算出し、その値がＣＨＯ−ｈＧＰＲ４０に対し５０もしくは１００％以上であり、かつヒトヒスタミンＨ１受容体およびＭｏｃｋのＣＨＯ細胞株の両方が２５％以下を満たす被検体化合物をヒトＧＰＲ４０特異的アゴニスト候補とした。
【０１４４】
【実施例３６】マウスＧＰＲ４０の配列に特異的なｓｉＲＮＡ導入によるマウスＧＰＲ４０ｍＲＮＡの発現抑制
ＭＩＮ６細胞を２４ウェルプレートに撒いて一日培養した後、実施例１８の方法に基づきＨＶＪ Ｅｎｖｅｌｏｐｅ ＶＥＣＴＯＲ ＫＩＴ ＧｅｎｏｍＯＮＥを用いてｍ４０ｉ１０３またはランダム配列ｓｉＲＮＡであるＳｃｒａｍｂｌｅ ＩＩ ｄｕｐｌｅｘ ｓｉＲＮＡを導入した。２日培養後、同細胞のｔｏｔａｌＲＮＡを実施例７と同様にＩｓｏｇｅｎ（ニッポンジーン社）を用い、付属のマニュアルにしたがって調製した。得られたｔｏｔａｌＲＮＡ １μｇに対し１００ｎｇランダムヘキサマ−をプライマ−として、ＳｕｐｅｒＳｃｒｉｐｔＩＩ ｒｅｖｅｒｓｅ ｔｒａｎｓｃｒｉｐｔａｓｅ（インビトロジェン社）を用いてｃＤＮＡを合成後、４０μｌＴＥ ｂｕｆｆｅｒ（ｐＨ８．０）に溶解した。マウスＧＰＲ４０およびマウスＧＡＰＤＨ ｍＲＮＡの発現量は実施例４に記載した方法で行った。その結果、図３４に示したように、ｍ４０ｉ１０３を導入させたＭＩＮ６胞では、ランダム配列のｓｉＲＮＡを導入したＭＩＮ６細胞よりＧＰＲ４０ｍＲＮＡの発現が低下していることが明らかとなった。
【０１４５】
【発明の効果】
本発明のＧＰＲ４０、その部分ペプチドまたはその塩、または本発明のＧＰＲ４０もしくはその部分ペプチドをコードするＤＮＡは、糖尿病などの予防・治療剤として有用である。
本発明のＧＰＲ４０、その部分ペプチドまたはその塩とリガンドである脂肪酸とを用いることによって、脂肪酸と本発明のＧＲＰ４０、その部分ペプチドまたはその塩との結合性を変化させる化合物を効率良くスクリーニングすることができる。
【０１４６】
【配列表フリーテキスト】

【０１４７】
【配列表】

【図面の簡単な説明】
【図１】ファルネシン酸（ｆａｒｎｅｓｏｉｃ ａｃｉｄ）を添加したときの細胞内Ｃａ^２＋濃度の変化を調べた結果を示す。縦軸は細胞内Ｃａ^２＋濃度を示す蛍光強度、横軸はサンプル添加後の時間経過を示す。○はＧＰＲ４０発現ＣＨＯ−Ｋ１細胞、△はコントロールのＣＨＯ−Ｋ１細胞を示す。
【図２】５．８．１１−ｅｉｃｏｓａｔｒｉｙｎｏｉｃ ａｃｉｄを添加したときの細胞内Ｃａ^２＋濃度の変化を調べた結果を示す。縦軸は細胞内Ｃａ^２＋濃度を示す蛍光強度、横軸はサンプル添加後の時間経過を示す。○はＧＰＲ４０発現ＣＨＯ−Ｋ１細胞、△はコントロールのＣＨＯ−Ｋ１細胞を示す。
【図３】５．８．１１．１４−ｅｉｃｏｓａｔｅｔｒａｙｎｏｉｃ ａｃｉｄを添加したときの細胞内Ｃａ^２＋濃度の変化を調べた結果を示す。縦軸は細胞内Ｃａ^２＋濃度を示す蛍光強度、横軸はサンプル添加後の時間経過を示す。○はＧＰＲ４０発現ＣＨＯ−Ｋ１細胞、△はコントロールのＣＨＯ−Ｋ１細胞を示す。
【図４】リノール酸（ｌｉｎｏｌｅｉｃ ａｃｉｄ）を添加したときの細胞内Ｃａ^２＋濃度の変化を調べた結果を示す。縦軸は細胞内Ｃａ^２＋濃度を示す蛍光強度、横軸はサンプル添加後の時間経過を示す。○はＧＰＲ４０発現ＣＨＯ−Ｋ１細胞、△はコントロールのＣＨＯ−Ｋ１細胞を示す。
【図５】リノレン酸（ｌｉｎｏｌｅｎｉｃ ａｃｉｄ）を添加したときの細胞内Ｃａ^２＋濃度の変化を調べた結果を示す。縦軸は細胞内Ｃａ^２＋濃度を示す蛍光強度、横軸はサンプル添加後の時間経過を示す。○はＧＰＲ４０発現ＣＨＯ−Ｋ１細胞、△はコントロールのＣＨＯ−Ｋ１細胞を示す。
【図６】アラキドン酸（ａｒａｃｈｉｄｏｎｉｃ ａｃｉｄ）を添加したときの細胞内Ｃａ^２＋濃度の変化を調べた結果を示す。縦軸は細胞内Ｃａ^２＋濃度を示す蛍光強度、横軸はサンプル添加後の時間経過を示す。○はＧＰＲ４０発現ＣＨＯ−Ｋ１細胞、△はコントロールのＣＨＯ−Ｋ１細胞を示す。
【図７】エイコサペンタエン酸（ｅｉｃｏｓａｐｅｎｔａｅｎｏｉｃ ａｃｉｄ， ＥＰＡ）を添加したときの細胞内Ｃａ^２＋濃度の変化を調べた結果を示す。縦軸は細胞内Ｃａ^２＋濃度を示す蛍光強度、横軸はサンプル添加後の時間経過を示す。○はＧＰＲ４０発現ＣＨＯ−Ｋ１細胞、△はコントロールのＣＨＯ−Ｋ１細胞を示す。
【図８】エイコサジエン酸（ｅｉｃｏｓａｄｉｅｎｏｉｃ ａｃｉｄ）を添加したときの細胞内Ｃａ^２＋濃度の変化を調べた結果を示す。縦軸は細胞内Ｃａ^２＋濃度を示す蛍光強度、横軸はサンプル添加後の時間経過を示す。○はＧＰＲ４０発現ＣＨＯ−Ｋ１細胞、△はコントロールのＣＨＯ−Ｋ１細胞を示す。
【図９】エイコサトリエン酸（ｅｉｃｏｓａｔｒｉｅｎｏｉｃ ａｃｉｄ）を添加したときの細胞内Ｃａ^２＋濃度の変化を調べた結果を示す。縦軸は細胞内Ｃａ^２＋濃度を示す蛍光強度、横軸はサンプル添加後の時間経過を示す。○はＧＰＲ４０発現ＣＨＯ−Ｋ１細胞、△はコントロールのＣＨＯ−Ｋ１細胞を示す。
【図１０】ドコサヘキサエン酸（ｄｏｃｏｓａｈｅｘａｅｎｏｉｃ ａｃｉｄ，ＤＨＡ）を添加したときの細胞内Ｃａ^２＋濃度の変化を調べた結果を示す。縦軸は細胞内Ｃａ^２＋濃度を示す蛍光強度、横軸はサンプル添加後の時間経過を示す。○はＧＰＲ４０発現ＣＨＯ−Ｋ１細胞、△はコントロールのＣＨＯ−Ｋ１細胞を示す。
【図１１】ドコサトリエン酸（ｄｏｃｏｓａｔｒｉｅｎｏｉｃ ａｃｉｄ）を添加したときの細胞内Ｃａ^２＋濃度の変化を調べた結果を示す。縦軸は細胞内Ｃａ^２＋濃度を示す蛍光強度、横軸はサンプル添加後の時間経過を示す。○はＧＰＲ４０発現ＣＨＯ−Ｋ１細胞、△はコントロールのＣＨＯ−Ｋ１細胞を示す。
【図１２】アドレン酸（ａｄｒｅｎｉｃ ａｃｉｄ）を添加したときの細胞内Ｃａ^２＋濃度の変化を調べた結果を示す。縦軸は細胞内Ｃａ^２＋濃度を示す蛍光強度、横軸はサンプル添加後の時間経過を示す。○はＧＰＲ４０発現ＣＨＯ−Ｋ１細胞、△はコントロールのＣＨＯ−Ｋ１細胞を示す。
【図１３】ラウリン酸（ｌａｕｒｉｃ ａｃｉｄ）を添加したときの細胞内Ｃａ^２＋濃度の変化を調べた結果を示す。縦軸は細胞内Ｃａ^２＋濃度を示す蛍光強度、横軸はサンプル添加後の時間経過を示す。○はＧＰＲ４０発現ＣＨＯ−Ｋ１細胞、△はコントロールのＣＨＯ−Ｋ１細胞を示す。
【図１４】ヒト各種組織でのＧＰＲ４０ｍＲＮＡの発現分布を示す。横軸はｐｏｌｙ（Ａ）^＋ＲＮＡ（ｎｇ）当たりのコピー数を示す。
【図１５】マウス各種細胞でのＧＰＲ４０ｍＲＮＡの発現分布を示す。Ｍｉｎ−６はＭＩＮ６細胞を、ＮＩＨ ｌｏｇはＮＩＨ／３Ｔ３の対数増殖期を、ＮｉＨｃｏｎｆはＮＩＨ／３Ｔ３の定常期を、Ｂ１０４ ｌｏｇはがん遺伝子ｎｅｕで形質転換させたＮＩＨ／３Ｔ３の対数増殖期を、Ｂ１０４ ｃｏｎｆはがん遺伝子ｎｅｕで形質転換させたＮＩＨ／３Ｔ３の定常期を、ｍＬｉｖｅｒはマウス肝臓を、ｍＳｐｌｅｅｎマウス脾臓 を示す。横軸は総ＲＮＡ（２５ｎｇ）当たりのコピー数を示す。（＋）は逆転写酵素添加を、（−）は逆転写酵素非添加を示す。
【図１６】ＭＩＮ６細胞をパルミチン酸、γ−リノレン酸またはポジティブコントロールであるグリベンクラミドで９０分間刺激したときのインスリン分泌の変化を調べた結果を示す。横軸は添加した化合物とその濃度（μＭ）を示す。縦軸はインスリン分泌濃度（ｎｇ／ｍｌ）を示す。
【図１７】ＭＩＮ６細胞をパルミチン酸、オレイン酸またはポジティブコントロールであるグリベンクラミドで６０分間刺激したときのインスリン分泌の変化を調べた結果を示す。横軸は添加した化合物とその濃度（μＭ）を示す。縦軸はインスリン分泌濃度（ｎｇ／ｍｌ）を示す。
【図１８】ラット膵臓およびランゲルハンス島におけるＧＰＲ４０ ｍＲＮＡの発現量をＲＴ−ＰＣＲ法によって求めた結果を示す。膵臓全体は２匹の平均値で示した。
【図１９】カニクイザル膵臓およびランゲルハンス島におけるＧＰＲ４０ ｍＲＮＡの発現量をＲＴ−ＰＣＲ法によって求めた結果を示す。膵臓全体は２匹の平均値で示した。
【図２０】脂肪酸によるＣＨＯ−ｈＧＰＲ４０＃１０４でのＭＡＰキナーゼ活性化の検出結果を示す。γ−ｌｉｎｏはγ−リノレン酸を、Ｏｌｅはオレイン酸を、ＤＨＡは４Ｚ，７Ｚ，１０Ｚ，１３Ｚ，１６Ｚ，１９Ｚ−ドコサヘキサエン酸を、Ｍｅｔｈｙｌはリノール酸メチルを、Ｃ２は酢酸を示す。ＣＨＯ−ｍｏｃｋはヒトＧＰＲ４０を発現していないＣＨＯ細胞を、ＣＨＯ−ｈＧＰＲ４０＃１０４はヒトＧＰＲ４０を発現しているＣＨＯ細胞株を示す。ｐ４４はｐ４４ＭＡＰキナーゼのバンドを、ｐ４２はｐ４２ＭＡＰキナーゼのバンドを示す。
【図２１】マウスＧＰＲ４０の配列に特異的なｓｉＲＮＡ導入によるマウスＧＰＲ４０−ＧＦＰ融合タンパク質の発現抑制を調べた結果を示す。ＭｏｃｋはＧＰＲ４０−ＧＦＰを導入していないコントロール細胞を示す。ＮｏＴＦはトランスフェクションを行わなかった細胞、Ｎｏ ｓｉはｓｉＲＮＡを含まないトランスフェクションを示す。ｍ４０ｉ１０３およびｍ４０ｉ２５６はマウスＧＰＲ４０配列に特異的なｓｉＲＮＡを、ｓｉ ｓｃｒａｍｂｌｅ、ｓｉ ＧＬ２はネガティブコントロールとして添加したＧＰＲ４０の配列とは全く相同性を持たないｓｉＲＮＡを示す。縦軸はＧＰＲ４０の発現量（４５０ｎｍでの吸光度）を示す。Ｎ＝３で行った実験の平均値＋標準偏差で表した。
【図２２】ｍ４０ｉ１０３およびｍ４０ｉ２５６の配列を示す。
【図２３】ＧＰＲ４０遺伝子ターゲッティングベクターの構築図を示す。
【図２４】脂肪酸によるＭＩＮ６でのＭＡＰキナーゼ活性化の検出結果を示す。レーン１はコントロールを、レーン２はオレイン酸（ｏｌｅｉｃ ａｃｉｄ）添加、レーン３はリノール酸（ｌｉｎｏｌｅｉｃ ａｃｉｄ）添加、レーン４はγ−リノレン酸（γ−ｌｉｎｏｌｅｎｉｃ ａｃｉｄ）添加、レーン５はリノール酸メチル（ｍｅｔｈｙｌ ｌｉｎｏｌａｔｅ）添加、レーン６はリノール酸（ｌｉｎｏｌｅｉｃ ａｃｉｄ）添加、レーン７はリノール酸（ｌｉｎｏｌｅｉｃ ａｃｉｄ）＋ＰＤ９８０５９（５０μＭ）添加を示す。レーン１〜５は２．５分間、レーン６〜７は５分間インキュベートした結果である。各脂肪酸の濃度は１０μＭである。ｐ４４はｐ４４ＭＡＰキナーゼのバンドを、ｐ４２はｐ４２ＭＡＰキナーゼのバンドを示す。
【図２５】２２ｍＭのグルコースを含むＫＲＢＨで調製した各種脂肪酸によるＭＩＮ６からのインスリン分泌促進作用を示す。横軸のＣｏｎｃｅｎｔｒａｔｉｏｎｓは添加した脂肪酸の濃度（μＭ）を示す。Ｂａｓｅは無添加、Ｏｌｅｉｃはオレイン酸添加、Ｌｉｎｏｌｅｉｃはリノール酸添加、α−Ｌｉｎｏｌｅｎｉｃはα−リノレン酸添加、γ−Ｌｉｎｏｌｅｎｉｃはγ−リノレン酸添加、Ａｒａｃｈｉｄｏｎｉｃはアラキドン酸添加、ＤＨＡはドコサヘキサエン酸添加、Ｍｅｔｈｙｌ ｌｉｎｏｌａｔｅはリノール酸メチル添加、Ｂｕｔｙｒｉｃは酪酸添加の場合を示す。縦軸はインスリン濃度（ｎｇ／ｍｌ）を示す。値は平均＋標準誤差（ｎ＝４）を示す。＊＊はｐ＜０．０１を、＊はｐ＜０．０５（Ｓｔｕｄｅｎｔ’ｓ ｔ ｔｅｓｔ）を示す。
【図２６】脂肪酸によるＭＩＮ６からのインスリン分泌促進活性のグルコース濃度依存性を示す。横軸は添加した脂肪酸（１０μＭ）の種類を示す。Ｂａｓｅは無添加、Ｏｌｅｉｃはオレイン酸添加、Ｌｉｎｏｌｅｉｃはリノール酸添加、Ｍｅｔｈｙｌはリノール酸メチル添加、Ｂｕｔｙｒｉｃは酪酸添加の場合を示す。Ｇｌｃはグルコースを示す。縦軸はインスリン濃度（ｎｇ／ｍｌ）を示す。値は平均＋標準誤差（ｎ＝４）を示す。＊＊はｐ＜０．０１を、＊はｐ＜０．０５（Ｓｔｕｄｅｎｔ’ｓ ｔ ｔｅｓｔ）を示す。
【図２７】ｓｉＲＮＡ導入ＭＩＮ６における、脂肪酸によるインスリン分泌上昇活性の抑制効果を示す。横軸は添加した脂肪酸の濃度（μＭ）を示す。Ｂａｓｅは無添加、Ｌｉｎｏｌｅｉｃはリノール酸添加、Ｇａｍｍａ−Ｌｉｎｏｌｅｎｉｃはγ−リノレン酸添加、Ｍｅｔｈｙｌ ｌｉｎｏｌａｔｅはリノール酸メチル添加の場合を示す。縦軸はインスリン分泌量を示し、Ｂａｓｅに対する％値で表した。□カラムはｍ４０ｉ１０３導入、■カラムはＳｃｒａｍｂｌｅ ＩＩ ｄｕｐｌｅｘ ｓｉＲＮＡ導入の場合をします。値は平均＋標準誤差（ｎ＝４）を示す。＊＊はｐ＜０．０１（Ｓｔｕｄｅｎｔ’ｓ ｔ ｔｅｓｔ ｖｓ Ｂａｓｅ）を示す。
【図２８】脂肪酸によるラット膵臓ランゲルハンス島からのインスリン分泌促進作用を示す。横軸は添加した脂肪酸の濃度（μＭ）を示す。Ｂａｓｅは無添加、Ｌｉｎｏｌｅｉｃはリノール酸、γ−Ｌｉｎｏｌｅｎｉｃはγ−リノレン酸、Ｍｅｔｈｙｌ ｌｉｎｏｌａｔｅはリノール酸メチルを示す。縦軸はｉｓｌｅｔ当たりのインスリン分泌量（ｎｇ）を示す。値はｎ＝３およびｎ＝４で計２回行った実験の総和で、平均＋標準誤差を示す。＊＊はｐ＜０．０１を、＊はｐ＜０．０５（Ｓｔｕｄｅｎｔ’ｓ ｔ ｔｅｓｔ）を示す。
【図２９】脂肪酸によるＭＩＮ６からのインスリン分泌促進におけるＣａ^２＋イオンの必要性を調べた結果を示す。横軸は添加した脂肪酸を示す。Ｂａｓｅは無添加、Ｏｌｅｉｃはオレイン酸、Ｌｉｎｏｌｅｉｃはリノール酸、γ−Ｌｉｎｏｌｅｎｉｃはγ−リノレン酸、Ｍｅｔｈｙｌ ｌｉｎｏｌａｔｅはリノール酸メチルを示す。左の群は２２ｍＭのグルコースを含むＫＲＢＨで調製した脂肪酸（１０μＭ）を添加した場合、ＥＧＴＡ／Ｃａ^２＋ｆｒｅｅで示される右の群は２２ｍＭグルコースを含むＫＲＢＨでＣａ^２＋イオンを除き０．１ｍＭ ＥＧＴＡを添加したＫＲＢＨで調製した脂肪酸（１０μＭ）を添加した場合を示す。縦軸はインスリン分泌量（ｎｇ／ｍｌ）を示す。値は平均＋標準誤差（ｎ＝４）を示す。＊＊はｐ＜０．０１（Ｓｔｕｄｅｎｔ’ｓ ｔ ｔｅｓｔ ｖｓ Ｂａｓｅ）を示す。
【図３０】脂肪酸によるＭＩＮ６からのインスリン分泌促進における各種阻害剤の効果を示す。横軸は添加した脂肪酸を示す。Ｂａｓｅは無添加、Ｏｌｅｉｃはオレイン酸、Ｌｉｎｏｌｅｉｃはリノール酸、Ｍｅｔｈｙｌ ｌｉｎｏｌａｔｅはリノール酸メチルを示す。Ｎｏ ｉｎｈｉｂｉｔｏｒ群は２２ｍＭグルコース含有ＫＲＢＨで調製した脂肪酸（１０μＭ）を、Ｎｉｆｅｄｉｐｉｎｅ群は２２ｍＭグルコース含有ＫＲＢＨにニフェジピン（Ｎｉｆｅｄｉｐｉｎｅ）１０μＭを添加したバッファーで調製した脂肪酸（１０μＭ）を、ＰＤ９８０５９群は２２ｍＭグルコース含有ＫＲＢＨにＰＤ９８０５９（５０μＭ）を添加したバッファーで調製した脂肪酸（１０μＭ）を添加した場合を示す。縦軸はインスリン分泌量（ｎｇ／ｍｌ）を示す。値は平均＋標準誤差（ｎ＝４）を示す。＊＊はｐ＜０．０１（Ｓｔｕｄｅｎｔ’ｓ ｔ ｔｅｓｔ ｖｓ Ｂａｓｅ）を示す。
【図３１】脂肪酸によるＭＩＮ６からのインスリン分泌促進におけるＢＳＡの影響を示す。横軸は添加した脂肪酸（１０μＭ）を示す。Ｂａｓｅは無添加、Ｏｌｅｉｃはオレイン酸、Ｌｉｎｏｌｅｉｃはリノール酸、Ｍｅｔｈｙｌ ｌｉｎｏｌａｔｅはリノール酸メチルを示す。Ｎｏ ＢＳＡはバッファーにＢＳＡを添加しない条件を、０．１％ ＢＳＡは０．１％ ＢＳＡを添加した条件下を示す。縦軸はインスリン分泌量（ｎｇ／ｍｌ）を示す。値は平均＋標準誤差（ｎ＝４）を示す。＊＊はｐ＜０．０１（Ｓｔｕｄｅｎｔ’ｓ ｔ ｔｅｓｔ ｖｓ Ｂａｓｅ）を示す。
【図３２】ヒトＧＰＲ４０発現ＣＨＯ 細胞（ＣＨＯ−ｈＧＰＲ４０；左）およびマウスＧＰＲ４０発現ＣＨＯ細胞（ＣＨＯ−ｍＧＰＲ４０；右）のｃＡＭＰ産生に対する各種脂肪酸の影響を示す。横軸のＦＦＡ ｃｏｎｃｅｎｔｒａｔｉｏｎｓ（μＭ）は添加した脂肪酸の濃度を示す。縦軸は９６−ｗｅｌｌプレート１ｗｅｌｌ当りのｃＡＭＰ量（ｐｍｏｌ／ｗｅｌｌ）を示す。実線は２μＭ フォルスコリン存在下を、点線はフォルスコリン非存在下を示す。●はドコサヘキサエン酸（ＤＨＡ）、◇はリノール酸、□はγ−リノレン酸をそれぞれ添加した場合を示す。＊はｐ＜０．０５（Ｓｔｕｄｅｎｔ’ｓ ｔ ｔｅｓｔ）を示す。
【図３３】ＭＩＮ６細胞に各種脂肪酸を添加したときの細胞内Ｃａ^２＋濃度の変化を調べた結果を示す。縦軸は細胞内Ｃａ^２＋濃度を示す蛍光強度を示す。横軸はサンプル添加後の時間経過を示す。□はγ−リノレン酸（γ−ｌｉｎｏｌｅｎｉｃ ａｃｉｄ）、◇はリノール酸（ｌｉｎｏｌｅｉｃ ａｃｉｄ）、■はリノール酸メチル（ｍｅｔｈｙｌ ｌｉｎｏｌａｔｅ）、△はオレイン酸（ｏｌｅｉｃ ａｃｉｄ）、▲はアラキドン酸（ａｒａｃｈｉｄｏｎｉｃ ａｃｉｄ）、●はドコサヘキサエン酸（ＤＨＡ）、○は酪酸（ｂｕｔｙｒｉｃ ａｃｉｄ）をそれぞれ添加した場合を示す。
【図３４】マウスＧＰＲ４０の配列に特異的なｓｉＲＮＡ導入によるマウスＧＰＲ４０ｍＲＮＡの発現抑制を示す。横軸のｍ４０ｉ１０３はマウスＧＰＲ４０に特異的なｓｉＲＮＡを、またＳｃｒａｍｂｌｅは対照となるランダム配列ｓｉＲＮＡのＳｃｒａｍｂｌｅ ＩＩ ｄｕｐｌｅｘ ｓｉＲＮＡを示す。縦軸のｍＲＮＡの発現量（ＧＲＰ４０／ＧＡＰＤＨ）はＧＰＲ４０の発現量のＧＡＰＤＨに対する相対値（ｎ＝２＋ＳＥＭ）を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel G protein-coupled receptor protein (GPR40) or its salt derived from rat, mouse, monkey and hamster, a DNA encoding the GPR40 and uses thereof, and uses of GPR40 including human-derived GPR40.
[0002]
[Prior art]
Many physiologically active substances such as hormones and neurotransmitters regulate the functions of living organisms through specific receptor proteins present in cell membranes. Many of these receptor proteins perform intracellular signal transduction through activation of a conjugated guanine nucleotide-binding protein (hereinafter sometimes abbreviated as G protein), and have seven transmembrane regions. Because of their common structure, they are collectively referred to as G protein-coupled receptor proteins or seven-transmembrane receptor proteins (7TMR).
G protein-coupled receptor proteins are present on the surface of various functional cells of living cells and organs, and are physiologically targeted as molecules that regulate the functions of those cells and organs, such as hormones, neurotransmitters and bioactive substances. Plays an important role. The receptor protein transmits a signal into a cell through binding to a physiologically active substance, and this signal causes various reactions such as activation and suppression of the cell.
To clarify the relationship between substances that regulate complex functions in cells and organs of various organisms and their specific receptor proteins, especially G protein-coupled receptor proteins, it is necessary to clarify the functions of cells and organs of various organisms. And provide a very important means for drug development closely related to their functions.
[0003]
For example, in various organs of a living body, physiological functions are regulated under the regulation by many hormones, hormone-like substances, neurotransmitters or physiologically active substances. In particular, physiologically active substances are present at various sites in a living body, and regulate their physiological functions through their corresponding receptor proteins. There are many unknown hormones, neurotransmitters and other physiologically active substances in the living body, and many of the structures of their receptor proteins have not yet been reported. Furthermore, it is often unknown whether subtypes exist in known receptor proteins.
It is a very important tool for drug development to clarify the relationship between a substance that regulates a complex function in a living body and its specific receptor protein. In addition, in order to efficiently screen for agonists and antagonists for receptor proteins and to develop pharmaceuticals, it is necessary to elucidate the functions of receptor protein genes expressed in vivo and to express them in an appropriate expression system. Was needed.
In recent years, as a means for analyzing a gene expressed in a living body, studies for randomly analyzing the sequence of cDNA have been actively conducted, and the fragment sequence of the cDNA obtained in this manner is expressed in an Expressed Sequence Tag (EST). ) Is registered in the database and published. However, many ESTs have only sequence information, and it is difficult to estimate their functions.
The amino acid sequence of human-derived GPR40 and the DNA encoding it have been reported (Patent Document 1 and Non-Patent Document 2).
In addition, it has been reported that the ligand of human-derived GPR40 is a fatty acid (Patent Document 2).
[0004]
[Patent Document 1]
WO2000-22129
[0005]
Biochem Biophys Res Commun. 1997, Oct 20; 239 (2): 543-547.
[0006]
WO02 / 057783
[0007]
[Problems to be solved by the invention]
Conventionally, substances that inhibit the binding between a G protein-coupled receptor protein and a physiologically active substance (that is, a ligand) and substances that bind to cause the same signal transduction as a physiologically active substance (that is, a ligand) include these receptor proteins. Have been utilized as specific drugs or antagonists of the biological functions as specific antagonists or agonists. Therefore, it is important to newly find a G protein-coupled receptor protein which is not only important in physiological expression in vivo but also a target for drug development and to clone its gene (eg, cDNA). This is a very important means for finding specific ligands, agonists and antagonists of G protein-coupled receptor proteins.
However, not all G protein-coupled receptor proteins have been found. At present, unknown G protein-coupled receptor proteins and so-called orphan receptor proteins for which no corresponding ligand has been identified have been identified. There are many such proteins, and there is an eager need to search for new G protein-coupled receptor proteins and elucidate their functions.
G protein-coupled receptor proteins are useful for searching for new physiologically active substances (that is, ligands), and for searching for agonists or antagonists to the receptor protein, using the signal transduction action as an index. On the other hand, even if a physiological ligand is not found, it is also possible to produce an agonist or antagonist for the receptor protein by analyzing the physiological action of the receptor protein from an inactivation experiment (knockout animal) of the receptor protein. It is possible. These ligands, agonists or antagonists to the receptor protein can be expected to be used as preventive / therapeutic or diagnostic agents for diseases associated with dysfunction of G protein-coupled receptor proteins.
Furthermore, a decrease or increase in the function of a G protein-coupled receptor protein in a living body based on a gene mutation thereof often causes some disease. In this case, not only administration of an antagonist or agonist to the receptor protein, but also gene therapy by introducing the receptor gene into a living body (or a specific organ) or introducing an antisense nucleic acid against the receptor gene. It can also be applied. In this case, the nucleotide sequence of the receptor protein is indispensable information for examining the presence or absence of a deletion or mutation in the gene, and the gene of the receptor protein is used to prevent diseases associated with dysfunction of the receptor protein. / It can also be applied to therapeutic and diagnostic agents.
The present invention provides a novel G protein-coupled receptor protein useful as described above. That is, a novel G protein-coupled receptor protein or a partial peptide thereof or a salt thereof, and a polynucleotide (DNA, RNA, and a derivative thereof) containing a polynucleotide (DNA, RNA and a derivative thereof) encoding the G protein-coupled receptor protein or a partial peptide thereof RNA and derivatives thereof), a recombinant vector containing the polynucleotide, a transformant carrying the recombinant vector, a method for producing the G protein-coupled receptor protein or a salt thereof, the G protein-coupled receptor protein or Antibody against the partial peptide or its salt, compound that changes the expression level of the G protein-coupled receptor protein, method for determining a ligand for the G protein-coupled receptor protein, binding between the ligand and the G protein-coupled receptor protein Changed For screening a compound (antagonist, agonist) or a salt thereof, a kit for screening, a compound capable of changing the binding between a ligand obtainable by using the screening method or screening kit and the G protein-coupled receptor protein (antagonist) , An agonist) or a salt thereof, and a compound (antagonist, agonist) that changes the binding property of a ligand to the G protein-coupled receptor protein or a compound or a salt thereof that changes the expression level of the G protein-coupled receptor protein Medical supplies and the like.
[0008]
[Means for Solving the Problems]
The present inventors have conducted extensive studies and found that the ligand of human GPR40 is a fatty acid or eicosanoid. Furthermore, DNA encoding GPR40 derived from rat, mouse and hamster was successfully cloned. Furthermore, the fact that GPR40 is highly expressed in pancreatic islets of Langerhans, and that fatty acids or eicosanoids bind to GPR40, ²⁺ It has been found that an increase in concentration and suppression of cAMP production occur, and that free fatty acids promote insulin secretion from MIN6 cells. Furthermore, a monoclonal antibody against the C-terminal peptide of human GPR40 and an siRNA against mouse GPR40 were obtained. The present inventors have further studied based on these findings, and have completed the present invention.
[0009]
That is, the present invention
(1) a G protein-coupled type comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 17 or SEQ ID NO: 29 Receptor protein or a salt thereof,
(2) a G protein-coupled receptor protein or a salt thereof comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3,
(3) a G protein-coupled receptor protein consisting of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3, or a salt thereof;
(4) a G protein-coupled receptor protein consisting of the amino acid sequence represented by SEQ ID NO: 17 or a salt thereof;
(5) a G protein-coupled receptor protein consisting of the amino acid sequence represented by SEQ ID NO: 29 or a salt thereof;
(6) a partial peptide of the G protein-coupled receptor protein according to (1) or a salt thereof;
(7) a polynucleotide comprising a polynucleotide encoding the G protein-coupled receptor protein according to (1),
(8) a DNA encoding the G protein-coupled receptor protein according to (3),
(9) DNA encoding the G protein-coupled receptor protein according to (4),
(10) DNA encoding the G protein-coupled receptor protein according to (5),
(11) a DNA consisting of the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4,
(12) a DNA consisting of the base sequence represented by SEQ ID NO: 18,
(13) a DNA consisting of the base sequence represented by SEQ ID NO: 30,
(14) a recombinant vector containing the polynucleotide according to (7),
(15) a transformant transformed with the recombinant vector according to (14),
(16) The G protein-coupled receptor according to (1), wherein the transformant according to (15) is cultured to produce the G protein-coupled receptor protein or salt thereof according to (1). A method for producing a protein or a salt thereof,
(17) a medicine comprising the G protein-coupled receptor protein according to (1) or the partial peptide according to (6) or a salt thereof;
(18) a medicine comprising the polynucleotide according to (7),
(19) a diagnostic agent comprising the polynucleotide of (7) above,
(20) an antibody against the G protein-coupled receptor protein according to (1) or the partial peptide according to (6) or a salt thereof;
(21) the antibody according to (20), which is a monoclonal antibody that recognizes the C-terminal peptide of the G protein-coupled receptor protein according to (1) or a salt thereof;
(22) the antibody of the above (20), which is a monoclonal antibody recognizing the peptide represented by SEQ ID NO: 33 or a salt thereof;
(23) the antibody according to (20), which is a neutralizing antibody that inactivates signal transduction of the G protein-coupled receptor protein according to (1);
(24) a diagnostic agent comprising the antibody of (20) above,
(25) a medicine comprising the antibody according to (20),
(26) a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide of (7) or a part thereof,
(27) a diagnostic agent comprising the polynucleotide according to the above (26),
(28) a medicine comprising the polynucleotide according to the above (26),
(29) an siRNA against the polynucleotide according to (7),
(30) the siRNA according to (29), which is an siRNA comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 34 and an antisense strand consisting of the base sequence represented by SEQ ID NO: 35;
(31) the siRNA according to the above (29), which is an siRNA comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 36 and an antisense strand consisting of the base sequence represented by SEQ ID NO: 37;
(32) a diagnostic agent comprising the siRNA of the above (29),
(33) a medicament comprising the siRNA of the above (29),
(34) a G protein-coupled form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Pancreatic function regulator comprising a receptor protein or a partial peptide thereof or a salt thereof,
(35) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease containing receptor protein or partial peptide or salt thereof , Arthropathy, osteopenia, arteriosclerosis, thrombotic disease, indigestion or memory-learning disorders,
(36) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 An insulin secretagogue or a hypoglycemic agent comprising a receptor protein or a partial peptide thereof or a salt thereof,
(37) a G protein-coupled form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Pancreatic β-cell protective agent comprising a receptor protein or a partial peptide thereof or a salt thereof,
(38) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Pancreatic function regulator comprising a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(39) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia comprising a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof , Sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis, thrombotic disease, dyspepsia or memory learning prevention / treatment agent,
(40) a G protein-coupled form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 An insulin secretagogue or hypoglycemic agent comprising a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(41) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Pancreatic β-cell protective agent comprising a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(42) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia comprising a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof , Sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis, thrombotic disease, dyspepsia, memory and learning disorders, obesity, hyperlipidemia, hypoglycemia, hypertension, edema, insulin resistance syndrome, Diagnostics for stable diabetes, lipoatrophy, insulin allergy, insulinoma, lipotoxicity or cancer,
(43) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Diabetes comprising antibodies to the receptor protein or its partial peptide or a salt thereof, glucose intolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, Skin disease, arthropathy, osteopenia, arteriosclerosis, thrombotic disease, indigestion, memory and learning disorders, obesity, hyperlipidemia, hypoglycemia, hypertension, edema, insulin resistance syndrome, unstable diabetes, lipoatrophy Diagnostics for insulin allergy, insulinoma, lipotoxicity or cancer,
(44) a G protein-coupled form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Pancreatic function modulator comprising an antibody against a receptor protein or a partial peptide thereof or a salt thereof,
(45) a G protein-coupled form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, diabetic retinopathy, edema, comprising an antibody against the receptor protein or its partial peptide or a salt thereof Insulin resistance syndrome, unstable diabetes, lipodystrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity or cancer prevention and treatment,
(46) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Pancreatic function regulator comprising a polynucleotide comprising a polynucleotide comprising a polynucleotide encoding a receptor protein or a polynucleotide encoding a partial peptide thereof or a polynucleotide comprising a part thereof,
(47) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Obesity, hyperlipidemia, type 2 diabetes, low-grade obesity comprising a polynucleotide comprising a nucleotide sequence complementary to a polynucleotide comprising a polynucleotide encoding a receptor protein or a partial peptide thereof or a polynucleotide comprising a part thereof Glycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance syndrome, unstable diabetes, lipoatrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity or cancer Prophylactic and therapeutic agents,
(48) (1) G protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Screening for a compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and a fatty acid or a salt thereof, characterized by using a conjugated receptor protein, a partial peptide thereof or a salt thereof, and (2) a fatty acid or a salt thereof. Method,
(49) (1) G protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 (2) a compound or a salt thereof, which comprises a receptor protein or a salt thereof and (2) a compound capable of changing the binding property between the receptor protein or a salt thereof and a fatty acid or a salt thereof; Screening kit,
(50) Fatty acid or a salt thereof, which can be obtained by using the screening method described in (48) or the screening kit described in (49), and SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: No .: 17 or a compound having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 29, or a compound thereof, which alters the binding to a G protein-coupled receptor protein or a salt thereof;
(51) The compound according to the above (50), which is an agonist, or a salt thereof,
(52) the compound of the above-mentioned (50), which is an antagonist, or a salt thereof;
(53) a medicament comprising the compound according to (50) or a salt thereof,
(54) having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 with a fatty acid or a salt thereof A pancreatic function regulator comprising a compound that changes the binding property to a G protein-coupled receptor protein or a salt thereof, or a salt thereof;
(55) a medicament comprising the agonist according to the above (51),
(56) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Diabetes, glucose intolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease, arthropathy containing an agonist for a protein or a salt thereof , Osteopenia, arteriosclerosis, thrombotic disease, indigestion or memory learning prevention / treatment agent,
(57) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 An insulin secretagogue or a hypoglycemic agent comprising an agonist for the protein or a salt thereof,
(58) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Pancreatic β-cell protective agent comprising an agonist for a protein or a salt thereof,
(59) a medicament comprising the antagonist according to (52),
(60) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance syndrome, comprising an antagonist to the protein or a salt thereof, Prevention and treatment of unstable diabetes, lipoatrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity or cancer,
(61) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A compound or a salt thereof having an activity of regulating pancreatic function by changing the expression level of the G protein-coupled receptor protein, which comprises using a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof. Screening method,
(62) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 For screening a compound having a function of regulating pancreatic function by altering the expression level of the G protein-coupled receptor protein containing a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof, or a salt thereof kit,
(63) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: obtainable by using the screening method described in (61) or the screening kit described in (62). A compound or a salt thereof having an activity of regulating pancreatic function by changing the expression level of a G protein-coupled receptor protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 29,
(64) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: obtainable by using the screening method described in (61) or the screening kit described in (62). A compound or a salt thereof having an action of increasing the expression level of a G protein-coupled receptor protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 29, and regulating pancreatic function,
(65) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: obtainable using the screening method described in (61) or the screening kit described in (62). A compound or a salt thereof having an action of reducing the expression level of a G protein-coupled receptor protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 29, and regulating pancreatic function,
(66) a G protein-coupled form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Pancreatic function regulator comprising a compound having a function of regulating pancreatic function or a salt thereof, which changes the expression level of a receptor protein or a partial peptide thereof,
(67) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Increases the expression level of the receptor protein or its partial peptide, and comprises a compound having a function of regulating pancreatic function or a salt thereof, diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, Preventive / therapeutic agent for diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis, thrombotic disease, dyspepsia or memory learning disorder,
(68) a G protein-coupled form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 An insulin secretagogue or a hypoglycemic agent, comprising a compound having a function of regulating pancreatic function or a salt thereof, which increases the expression level of a receptor protein or a partial peptide thereof,
(69) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Pancreatic β-cell protective agent comprising a compound having a function of regulating pancreatic function or a salt thereof, which increases the expression level of a receptor protein or a partial peptide thereof,
(70) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic nerve containing a compound having a function of regulating pancreatic function by reducing the expression level of receptor protein or its partial peptide Disorders, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance syndrome, unstable diabetes, lipodystrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity or cancer prevention / treatment,
(71) A test compound comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29. Intracellular Ca when contacted with cells containing protein-coupled receptor protein ²⁺ A method for determining a ligand for the receptor protein or a salt thereof, which comprises measuring a concentration increasing activity or an intracellular cAMP inhibitory activity,
(72) a ligand obtained by the determination method according to (71),
(73) (1) G protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Coupling type receptor protein, its partial peptide or its salt and (2) (1) fatty acid or its salt or (2) compound or its salt which changes the binding property between said receptor protein or its salt and fatty acid or its salt are used. A method for screening an agonist or antagonist for the receptor protein or a salt thereof,
(74) (1) (1) A labeled fatty acid or a salt thereof, or (2) a labeled compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and a fatty acid or a salt thereof, And (2) the binding between the test compound and (1) the labeled fatty acid or its salt or (2) the receptor protein or its salt and the fatty acid or its salt. (1) labeled fatty acid or salt thereof or (2) receptor protein or salt thereof and fatty acid when a labeled compound or its salt to be changed is brought into contact with the receptor protein, its partial peptide or its salt. Or a labeled compound or a salt thereof that changes the binding property to the receptor protein, the receptor protein, a partial peptide thereof, or the like. The screening method of the above (73), wherein the measuring the amount of binding between a salt thereof,
(75) (1) (1) A labeled fatty acid or a salt thereof, or (2) a labeled compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and a fatty acid or a salt thereof, And (2) binding of a test compound and (1) a labeled fatty acid or a salt thereof, or (2) a receptor protein or a salt thereof to a fatty acid or a salt thereof. (1) labeled fatty acid or a salt thereof or (2) the receptor protein or a salt thereof when a labeled compound or a salt thereof that changes the property is brought into contact with a cell containing the receptor protein or a cell membrane fraction thereof. A labeled compound or a salt thereof that changes the binding property between the salt and a fatty acid or a salt thereof, and a cell containing the receptor protein; Or screening method of the above (73), wherein the measuring the amount of binding between the cell membrane fraction,
(76) (1) (1) a fatty acid or a salt thereof, or (2) a compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and a fatty acid or a salt thereof, or a cell containing the receptor protein or a salt thereof. (2) a test compound and (1) a fatty acid or a salt thereof, or (2) a compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and a fatty acid or a salt thereof. Intracellular Ca when contacted with a cell containing the receptor protein or a cell membrane fraction thereof ²⁺ The screening method according to the above (73), wherein the concentration increasing activity or the intracellular cAMP production inhibitory activity is measured.
(77) (1) a G protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Conjugated receptor protein, partial peptide or salt thereof, and (2) (1) fatty acid or salt thereof or (2) compound or salt thereof which changes the binding property between the receptor protein or salt and fatty acid or salt thereof A screening kit for an agonist or antagonist to the receptor protein or a salt thereof,
(78) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: which can be obtained using the screening method described in (73) or the screening kit described in (77). : An agonist to a G protein-coupled receptor protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 29;
(79) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: which can be obtained using the screening method described in (73) or the screening kit described in (77). : An antagonist to a G protein-coupled receptor protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 29;
(80) a medicament comprising the agonist of (78),
(81) a medicament comprising the antagonist according to the above (79),
(82) A test compound wherein the test compound contains an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29. The screening method according to any one of (74) to (76), which is a compound designed to bind to the ligand binding pocket based on the atomic coordinates of the active site of the protein-coupled receptor protein or a salt thereof and the position of the ligand binding pocket. ,
(83) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 (1) Pancreatic function regulator, (2) diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, high fat, characterized by using a protein or a salt thereof. Preventive / therapeutic agent for hematosis, sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis, thrombotic disease, dyspepsia or memory learning disorder, (3) insulin secretagogue, (4) hypoglycemic agent , (5) pancreatic β-cell protective agent or (6) obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance Syndrome, not How to verify constant diabetes, fatty atrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, that preventive or therapeutic drug for fat toxicity or cancer to bind to the receptor protein or a salt thereof,
(84) a G protein-coupled receptor comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 (1) Diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin characterized by using a protein or a salt thereof. Disease, arthropathy, osteopenia, arteriosclerosis, thrombotic disease, prophylactic / therapeutic agent for dyspepsia or memory learning disorder, (2) insulin secretagogue, (3) hypoglycemic agent, or (4) protection of pancreatic β-cell Is an agonist for the receptor protein or a salt thereof,
(85) a G protein-coupled receptor comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance syndrome characterized by using a protein or a salt thereof A method for confirming that a preventive / therapeutic agent for unstable diabetes, lipoatrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity or cancer is an antagonist to the receptor protein or a salt thereof,
(86) The above-mentioned method characterized in that, when each drug is brought into contact with the receptor protein, its partial peptide or its salt, the amount of each drug bound to the receptor protein, its partial peptide or its salt is measured. 83) The confirmation method described in (85),
(87) (1) (1) A labeled fatty acid or a salt thereof, or (2) a labeled compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and a fatty acid or a salt thereof, And (2) each drug and (1) a labeled fatty acid or a salt thereof or (2) a labeled compound or a salt thereof with the receptor protein or a portion thereof. The amount of binding between (1) the labeled fatty acid or its salt or (2) the labeled compound or its salt, and the receptor protein, its partial peptide or its salt when contacted with the peptide or its salt. The confirmation method according to any one of (83) to (85), wherein the measurement is performed.
(88) (1) (1) A labeled fatty acid or a salt thereof, or (2) a labeled compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and a fatty acid or a salt thereof, And (2) each drug and (1) a labeled fatty acid or a salt thereof or (2) the labeled compound or a salt thereof with the receptor protein. (1) a labeled fatty acid or a salt thereof or (2) a cell or a cell membrane fraction thereof containing the receptor protein and the labeled compound when the cell is contacted with the cell or the cell membrane fraction thereof. (83) to (85), wherein the amount of binding to the component is measured;
(89) (1) (1) A fatty acid or a salt thereof, or (2) a compound or a salt thereof that alters the binding property between the receptor protein or a salt thereof and a fatty acid or a salt thereof, or a cell containing the receptor protein or a salt thereof. When brought into contact with the cell membrane fraction, (2) each drug and (1) a fatty acid or a salt thereof or (2) a compound or a salt thereof that changes the binding property between the receptor protein or the salt thereof and the fatty acid or the salt thereof Intracellular Ca when contacted with a cell containing the receptor protein or a cell membrane fraction thereof ²⁺ The confirmation method according to any one of (83) to (85), wherein the concentration-increasing activity or the intracellular cAMP production inhibitory activity is measured.
(90) a G protein-coupled receptor comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 (1) a pancreatic function regulator, (2) diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic. Retinopathy, hyperlipidemia, sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis, thrombotic disease, indigestion or memory-learning preventive / therapeutic agent, (3) insulin secretagogue, ▲ 4) hypoglycemic drug, (5) pancreatic beta cell protective drug or (6) obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, Edema, ins Kit for confirming that a preventive or therapeutic agent for insulin resistance syndrome, unstable diabetes, lipoatrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity or cancer binds to the receptor protein or a salt thereof ,
(91) a G protein-coupled receptor comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 (1) pancreatic function modulator, which is an agonist or antagonist for protein, (2) diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sex Drugs for preventing and treating dysfunction, skin diseases, arthropathy, osteopenia, arteriosclerosis, thrombotic diseases, dyspepsia or memory learning disorders, (3) insulin secretagogues, (4) hypoglycemic drugs, (5) Pancreatic β cell protective drug or (6) obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance Group, unstable diabetes, fatty atrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity or an agent for preventing or treating cancer,
(92) (1) {1} containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Or an agonist or antagonist to G protein-coupled receptor protein, or (2) identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Or a compound or a salt thereof that changes the expression level of a G protein-coupled receptor protein or a partial peptide thereof containing substantially the same amino acid sequence;
(2) (1) Pancreatic function modulator, (2) Diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease , Arthropathy, osteopenia, arteriosclerosis, thrombotic disease, indigestion or memory and learning disorder prevention / treatment, (3) insulin secretagogue, (4) hypoglycemia, (5) pancreatic beta cell protective drug Or (6) obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance syndrome, unstable diabetes, lipoatrophy, A medicament comprising a combination of insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity or cancer prevention / treatment,
(93) a non-human mammal having an exogenous DNA encoding the G protein-coupled receptor protein according to (1) or a mutant DNA thereof,
(94) the animal according to (93), wherein the non-human mammal is a rodent;
(95) The animal according to the above (94), wherein the rodent is a mouse or a rat,
(96) a non-human mammalian embryonic stem cell in which DNA encoding the G protein-coupled receptor protein according to (1) is inactivated,
(97) The embryonic stem cell according to (96), wherein the DNA is inactivated by introducing a reporter gene,
(98) The embryonic stem cell according to (96), which is neomycin-resistant,
(99) the embryonic stem cell according to (96), wherein the non-human mammal is a rodent;
(100) The embryonic stem cell according to (99), wherein the rodent is a mouse,
(101) a non-human mammal deficient in DNA expression, wherein the DNA encoding the G protein-coupled receptor protein according to (1) is inactivated;
(102) The non-human mammal according to (101), wherein the DNA is inactivated by introducing a reporter gene, and the reporter gene can be expressed under the control of a promoter for the DNA.
(103) the non-human mammal according to the above (101), wherein the non-human mammal is a rodent;
(104) The non-human mammal according to (103), wherein the rodent is a mouse,
(105) A test compound is administered to the animal according to (102), and expression of the reporter gene is detected, whereby the promoter activity against the DNA encoding the G protein-coupled receptor protein according to (1) is determined. A method for screening for a compound that promotes or inhibits or a salt thereof,
(106) (1) a G protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A method for screening a compound or a salt thereof that changes the binding property between the receptor protein or its salt and eicosanoid, characterized by using a conjugated receptor protein, its partial peptide or its salt, and (2) eicosanoid;
(107) (1) a G protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A kit for screening a compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and eicosanoid, which comprises a conjugated receptor protein, a partial peptide or a salt thereof, and (2) an eicosanoid;
(108) Eicosanoid and SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17, which can be obtained using the screening method described in (106) or the screening kit described in (107). A compound having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 29, or a compound capable of changing the binding to a G protein-coupled receptor protein or a salt thereof, or a salt thereof;
(109) The compound according to the above (108) or a salt thereof, which is an agonist,
(110) The compound or a salt thereof according to the above (108), which is an antagonist,
(111) a medicament comprising the compound according to (108) or a salt thereof,
(112) G protein conjugate containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 with eicosanoid Pancreatic function modulator comprising a compound or a salt thereof that changes the binding property to a type receptor protein or a salt thereof,
(113) a medicament comprising the agonist according to the above (109),
(114) a medicament comprising the antagonist according to the above (110),
(115) (1) G protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Characterized by using a conjugated receptor protein, a partial peptide thereof or a salt thereof, and (2) (1) eicosanoid or (2) a compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and eicosanoid. A method for screening for an agonist or antagonist for the receptor protein or a salt thereof,
(116) (1) (1) A labeled eicosanoid or (2) a labeled compound or a salt thereof which changes the binding property between the receptor protein or a salt thereof and an eicosanoid is converted to the receptor protein, a partial peptide thereof or a compound thereof. When brought into contact with a salt, (2) a test compound and (1) a labeled eicosanoid or (2) a labeled compound or a salt thereof that changes the binding property between the receptor protein or its salt and eicosanoid are added to the test compound. (1) a labeled eicosanoid or (2) a labeled compound or a salt thereof that changes the binding property between the receptor protein or its salt and eicosanoid when contacted with a receptor protein, its partial peptide or its salt. , The binding amount to the receptor protein, its partial peptide or its salt. The screening method of the above (115), wherein the constant-,
(117) (1) (1) A labeled eicosanoid or (2) a cell containing the receptor protein or a salt thereof, which changes the binding property between the receptor protein or a salt thereof and an eicosanoid, or a cell containing the receptor protein or a salt thereof. When contacted with a cell membrane fraction, (2) a test compound and (1) a labeled eicosanoid or (2) a labeled compound or a salt thereof that changes the binding property of eicosanoid to the receptor protein or a salt thereof and eicosanoid are used. (1) a labeled eicosanoid or (2) a labeled compound that changes the binding between an eicosanoid and the receptor protein or a salt thereof when the cell is contacted with a cell containing the receptor protein or a cell membrane fraction thereof. Or a salt thereof and a cell containing the receptor protein or a cell membrane fraction thereof. The screening method of the above (115), wherein the measuring the amount of binding,
(118) (1) (1) Eikosanoid or (2) A compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and eicosanoid is brought into contact with a cell containing the receptor protein or a cell membrane fraction thereof. And (2) a test compound and (1) an eicosanoid or (2) a compound or a salt thereof that alters the binding property of eicosanoid to the receptor protein or a salt thereof and an eicosanoid, to a cell containing the receptor protein or a cell membrane fraction thereof. Minutes, the intracellular Ca ²⁺ The screening method according to the above (115), wherein the activity of increasing the concentration or the activity of suppressing intracellular cAMP production is measured.
(119) (1) a G protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 It is characterized by containing a conjugated receptor protein, a partial peptide thereof or a salt thereof, and (2) (1) eicosanoid or (2) a compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and eicosanoid. A kit for screening an agonist or antagonist for the receptor protein or a salt thereof,
(120) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: which can be obtained using the screening method according to (115) or the screening kit according to (119). : An agonist to a G protein-coupled receptor protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 29;
(121) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: which can be obtained using the screening method described in (115) or the screening kit described in (119). : An antagonist to a G protein-coupled receptor protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 29;
(122) a medicament comprising the agonist according to the above (120),
(123) a medicament comprising the antagonist according to the above (121),
(124) A test compound wherein the test compound contains an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29. The screening method according to any one of the above (116) to (118), which is a compound designed to bind to the ligand binding pocket based on the atomic coordinates of the active site of the protein-coupled receptor protein or a salt thereof and the position of the ligand binding pocket. ,
(125) For mammals,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(3) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Agonists for proteins, or
(4) a G protein-coupled form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A method for regulating pancreatic function, comprising administering an effective amount of a compound or a salt thereof that increases the expression level of a receptor protein or a partial peptide thereof,
(126) For mammals,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(3) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Agonists for proteins, or
(4) a G protein-coupled form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Diabetes, glucose intolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic nephropathy, diabetic retina characterized by administering an effective amount of a compound or a salt thereof that increases the expression level of a receptor protein or a partial peptide thereof. Disease, hyperlipidemia, sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis, thrombotic disease, dyspepsia or memory learning prevention and treatment method,
(127) For mammals,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(3) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Agonists for proteins, or
(4) a G protein-coupled form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A method for promoting insulin secretion or a method for decreasing blood glucose, which comprises administering an effective amount of a compound or a salt thereof that increases the expression level of a receptor protein or a partial peptide thereof,
(128) For mammals,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(3) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Agonists for proteins, or
(4) a G protein-coupled form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Pancreatic β-cell protective agent, characterized by administering an effective amount of a compound or a salt thereof that increases the expression level of a receptor protein or a partial peptide thereof,
(129) For mammals,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 An antibody against a receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide comprising a nucleotide sequence complementary to a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof or a part thereof,
(3) a G protein-conjugated form containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 SiRNA against a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(4) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Antagonists to proteins, or
(5) a G protein-coupled form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A method for regulating pancreatic function, comprising administering an effective amount of a compound or a salt thereof that reduces the expression level of a receptor protein or a partial peptide thereof,
(130) For mammals,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 An antibody against a receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide comprising a nucleotide sequence complementary to a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof or a part thereof,
(3) a G protein-conjugated form containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 SiRNA against a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(4) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Antagonists to proteins, or
(5) a G protein-coupled form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetes characterized by administering an effective amount of a compound or a salt thereof that reduces the expression level of a receptor protein or a partial peptide thereof. Method for preventing / treating nephropathy, diabetic retinopathy, edema, insulin resistance syndrome, unstable diabetes, lipoatrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity or cancer,
(131) for producing a pancreatic function regulator,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(3) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Agonists for proteins, or
(4) a G protein-coupled form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Use of a compound or a salt thereof that increases the expression level of a receptor protein or a partial peptide thereof,
(132) diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis, For producing a prophylactic / therapeutic agent for thrombotic disease, dyspepsia or memory learning disorder,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(3) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Agonists for proteins, or
(4) a G protein-coupled form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Use of a compound or a salt thereof that increases the expression level of a receptor protein or a partial peptide thereof,
(133) for producing an insulin secretagogue or a hypoglycemic agent,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(3) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Agonists for proteins, or
(4) a G protein-coupled form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Use of a compound or a salt thereof that increases the expression level of a receptor protein or a partial peptide thereof,
(134) for producing a pancreatic β-cell protective agent,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(3) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Agonists for proteins, or
(4) a G protein-coupled form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Use of a compound or a salt thereof that increases the expression level of a receptor protein or a partial peptide thereof,
(135) for producing a pancreatic function regulator,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 An antibody against a receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide comprising a nucleotide sequence complementary to a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof or a part thereof,
(3) a G protein-conjugated form containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 SiRNA against a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(4) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Antagonists to proteins, or
(5) a G protein-coupled form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Use of a compound or a salt thereof that reduces the expression level of a receptor protein or a partial peptide thereof, and
(136) Obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance syndrome, unstable diabetes, lipoatrophy, insulin For producing a preventive or therapeutic agent for allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity or cancer,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 An antibody against a receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide comprising a nucleotide sequence complementary to a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof or a part thereof,
(3) a G protein-conjugated form containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 SiRNA against a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(4) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Antagonists to proteins, or
(5) a G protein-coupled form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 It is intended to provide a use of a compound or a salt thereof for reducing the expression level of a receptor protein or a partial peptide thereof.
[0010]
Further, the present invention provides
(137) the protein is a) an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 17 or SEQ ID NO: 29, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 17 or SEQ ID NO: : 1 or 2 or more (preferably about 1 to 30, more preferably about 1 to 10, and still more preferably several (1 to 5)) amino acids in the amino acid sequence represented by 29 are deleted The amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 17 or SEQ ID NO: 29, one or more (preferably about 1 to 30, more preferably 1 to 30) An amino acid sequence to which about 10 to 10, more preferably several (1 to 5) amino acids have been added; c) the amino acid represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 17 or SEQ ID NO: 29 An amino acid in which one or more (preferably about 1 to 30, more preferably about 1 to 10, and still more preferably several (1 to 5)) amino acids in the sequence have been substituted with another amino acid The protein according to the above (1), which is a protein comprising a sequence or d) an amino acid sequence obtained by combining them;
(138) (i) G comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 17 or SEQ ID NO: 29 A protein-coupled receptor protein (hereinafter abbreviated as GPR40), a partial peptide or a salt thereof, and a fatty acid or a salt thereof, and (ii) GPR40, a partial peptide or a salt thereof, a fatty acid or a salt thereof. The screening method according to the above (48), wherein the comparison is performed with the case where the salt and the test compound are brought into contact with each other;
(139) (i) When the labeled fatty acid or its salt is brought into contact with GPR40, its partial peptide or its salt, and (ii) When the labeled fatty acid or its salt and the test compound are brought into contact with GPR40, its partial peptide or its salt. The screening method according to (48), wherein the amount of binding of the labeled fatty acid or its salt to GPR40, its partial peptide or its salt in the case of contacting is measured and compared,
(140) When (i) a labeled fatty acid or a salt thereof is brought into contact with a cell containing GPR40, and (ii) when a labeled fatty acid or a salt thereof and a test compound are brought into contact with a cell containing GPR40, The screening method according to the above (48), wherein the amount of binding of the labeled fatty acid or a salt thereof to the cell is measured and compared;
(141) (i) a case where a labeled fatty acid or a salt thereof is brought into contact with a membrane fraction of a GPR40-containing cell; The method of (48), wherein the amount of the labeled fatty acid or salt thereof bound to the membrane fraction of the cell is measured and compared when contacted with
(142) (i) The labeled fatty acid or a salt thereof was expressed on the cell membrane of the transformant by culturing a transformant transformed with a recombinant vector containing a DNA containing a DNA encoding GPR40. The binding amount of the labeled fatty acid or salt thereof to GPR40 when contacted with GPR40 and (ii) when the labeled fatty acid or salt thereof and the test compound were contacted with GPR40 expressed on the cell membrane of the transformant were determined. Measuring and comparing, the screening method according to the above (48),
(143) When (i) a compound activating GPR40 is brought into contact with a cell containing GPR40, and (ii) when a compound activating GPR40 and a test compound are brought into contact with a cell containing GPR40, The screening method according to (48), wherein the cell stimulating activity mediated by GPR40 is measured and compared.
(144) A compound activating GPR40 is contacted with GPR40 expressed on the cell membrane of the transformant by culturing a transformant transformed with a recombinant vector containing DNA containing DNA encoding GPR40. Measuring and comparing the GPR40-mediated cell stimulating activity between the case where the compound is activated and the case where the compound that activates GPR40 and the test compound are brought into contact with GPR40 expressed on the cell membrane of the transformant. (48) The screening method according to the above,
(145) The screening method according to the above (143) or (144), wherein the compound activating GPR40 is a fatty acid or a salt thereof.
(146) The screening kit according to the above (49), which comprises a cell containing GPR40 or a membrane fraction thereof.
(147) The method according to the above (1), wherein the transformant obtained by culturing the transformant transformed with the recombinant vector containing the DNA containing the DNA encoding GRP40 contains GPR40 expressed on the cell membrane of the transformant ( 49) The screening kit according to the above,
(148) The case where (i) GPR40, its partial peptide or its salt is brought into contact with eicosanoid, and (ii) the case where GPR40, its partial peptide or its salt is brought into contact with eicosanoid and its test compound. The screening method according to (106), wherein the comparison is performed.
(149) When (i) the labeled eicosanoid is brought into contact with GPR40, its partial peptide or its salt, and (ii) when the labeled eicosanoid and the test compound are brought into contact with GPR40, its partial peptide or its salt, The screening method according to the above (106), wherein the amount of the bound eicosanoid to GPR40, its partial peptide or its salt is measured and compared;
(150) Labeled eicosanoid cells in the case where (i) the labeled eicosanoid is brought into contact with cells containing GPR40, and (ii) the case where the labeled eicosanoid and the test compound are brought into contact with cells containing GPR40. The screening method according to the above (106), wherein the amount of binding to
(151) (i) When the labeled eicosanoid is brought into contact with the membrane fraction of cells containing GPR40, and (ii) When the labeled eicosanoid and the test compound are brought into contact with the membrane fraction of cells containing GPR40. The method of (106), wherein the amount of the bound eicosanoid bound to the membrane fraction of the cell is measured and compared.
(152) (i) Contacting the labeled eicosanoid with GPR40 expressed on the cell membrane of the transformant by culturing a transformant transformed with a recombinant vector containing a DNA containing a DNA encoding GPR40. And (ii) measuring the amount of binding of the labeled eicosanoid to GPR40 when the labeled eicosanoid and the test compound are brought into contact with GPR40 expressed on the cell membrane of the transformant, and comparing the measured amounts. The screening method according to the above (106),
(153) When (i) GPR40-activating compound is brought into contact with cells containing GPR40, and (ii) When GPR40-activating compound and a test compound are brought into contact with GPR40-containing cells, The screening method according to (106), wherein the cell stimulating activity mediated by GPR40 is measured and compared.
(154) A compound activating GPR40 is brought into contact with GPR40 expressed on the cell membrane of the transformant by culturing a transformant transformed with a recombinant vector containing DNA containing DNA encoding GPR40. Measuring and comparing the GPR40-mediated cell stimulating activity between the case where the compound is activated and the case where the compound that activates GPR40 and the test compound are brought into contact with GPR40 expressed on the cell membrane of the transformant. (106) The screening method according to the above,
(155) The screening method according to (153) or (154), wherein the compound activating GPR40 is eicosanoid.
(156) The screening kit according to (107), further comprising a cell containing GPR40 or a membrane fraction thereof, and
(157) The method according to the above (1), wherein the transformant obtained by culturing the transformant transformed with the recombinant vector containing the DNA containing the DNA encoding GRP40 contains GPR40 expressed on the cell membrane of the transformant ( 107) The screening kit and the like described in the above.
[0011]
BEST MODE FOR CARRYING OUT THE INVENTION
The G protein-coupled receptor protein (GRP40) of the present invention is identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Is a receptor protein containing the amino acid sequence of
GPR40 can be used, for example, in all cells of humans and mammals (eg, guinea pig, rat, mouse, rabbit, pig, sheep, cow, monkey, etc.) (eg, spleen cells, nerve cells, glial cells, pancreatic β cells, pancreatic Langerhans). Islets, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, Mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, Or progenitor cells of these cells, stem cells or cancer cells, etc.), blood cells, or any tissue in which these cells are present, for example, the brain, various parts of the brain (eg, olfactory bulb) Abdominal nucleus, basal sphere, hippocampus, thalamus, hypothalamus, subthalamic nucleus, cerebral cortex, medulla oblongata, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, brain stain, substantia nigra), spinal cord , Pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, It may be a protein derived from peripheral blood, peripheral blood cells, prostate, testis, testis, ovary, placenta, uterus, bone, joint, skeletal muscle, or the like, or may be a synthetic protein. In particular, GPR40 is highly expressed in pancreatic islets of Langerhans.
[0012]
Examples of the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 include, for example, SEQ ID NO: 1, SEQ ID NO: 3, an amino acid sequence having about 85% or more, preferably 90% or more, more preferably about 95% or more homology with the amino acid sequence represented by SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29. Can be
Examples of the protein of the present invention having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 include, for example, a sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, having substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, A protein having substantially the same activity as the protein consisting of the amino acid sequence represented by SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 is preferred.
Examples of substantially the same activity include a ligand binding activity and a signal transduction action. Substantially the same means that their activities are the same in nature. Therefore, the activities such as ligand binding activity and signal information transmission activity are equivalent (eg, about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably about 0.5 to 2 times). However, quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.
The activity such as the ligand binding activity and the signal information transduction activity can be measured according to a method known per se, for example, according to a screening method described later.
GPR40 includes a) one or two or more (preferably 1 to 3) amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29; An amino acid sequence in which about 30 amino acids have been deleted, more preferably about 1 to 10, and still more preferably several (1 to 5) amino acids; b) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 One or two or more (preferably about 1 to 30, more preferably about 1 to 10, more preferably several (1 to 5) amino acid sequences represented by SEQ ID NO: 17 or SEQ ID NO: 29. C) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 (preferably Is Contains about 30 to 30 amino acids, more preferably about 1 to 10 amino acids, and still more preferably several (1 to 5) amino acids substituted with another amino acid, or d) an amino acid sequence combining them. Proteins that can be used are also used.
[0013]
GPR40 in this specification has an N-terminus (amino terminus) at the left end and a C-terminus (carboxyl terminus) at the right end, according to the convention of peptide labeling. GPR40 including the amino acid sequence represented by SEQ ID NO: 1 has a carboxyl group (—COOH), a carboxylate (—COO) ⁻ ), Amide (-CONH ₂ )) Or an ester (—COOR).
Here, R in the ester is, for example, C, such as methyl, ethyl, n-propyl, isopropyl or n-butyl. _1-6 Alkyl groups such as C, cyclopentyl, cyclohexyl, etc. _3-8 Cycloalkyl groups such as phenyl, α-naphthyl and the like; _6-12 Aryl groups, for example phenyl-C such as benzyl, phenethyl, etc. _1-2 An alkyl group or α-naphthyl-C such as α-naphthylmethyl _1-2 C such as an alkyl group _7-14 In addition to aralkyl groups, pivaloyloxymethyl groups commonly used as oral esters are used.
When GPR40 has a carboxyl group (or carboxylate) other than at the C-terminus, a carboxyl group amidated or esterified is also included in the GPR40 of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
Further, GPR40 has an amino group at the N-terminal methionine residue in the above-mentioned protein, in which a protecting group (eg, formyl group, C _2-6 C such as alkanoyl group _1-6 Acyl group), a glutamyl group formed by cleavage of the N-terminal side in vivo and pyroglutamine oxidation, a substituent on the side chain of an amino acid in the molecule (for example, -OH, -SH, An amino group, an imidazole group, an indole group, a guanidino group, etc. are suitable protecting groups (for example, formyl group, C _2-6 C such as alkanoyl group _1-6 (Eg, an acyl group), or a complex protein such as a so-called glycoprotein to which a sugar chain is bound.
Specific examples of GPR40 of the present invention include, for example, mouse-derived GPR40 containing the amino acid sequence represented by SEQ ID NO: 1, rat GPR40 containing the amino acid sequence represented by SEQ ID NO: 3, and SEQ ID NO: 5. GPR40 derived from human containing the amino acid sequence represented by SEQ ID NO: 17, GPR40 derived from cynomolgus monkey containing the amino acid sequence represented by SEQ ID NO: 17, GPR40 derived from hamster containing the amino acid sequence represented by SEQ ID NO: 29, and the like are used. Among them, mouse-derived GPR40, rat-derived GPR40, cynomolgus monkey-derived GPR40 and hamster-derived GPR40 are novel proteins. Human-derived GPR40 is described in WO2000-22129, Biochem Biophys Res Commun. 1997, Oct 20; 239 (2): 543-547.
[0014]
The partial peptide of GPR40 (hereinafter sometimes abbreviated as a partial peptide) may be any peptide as long as it is a partial peptide of GPR40 described above. And a site having substantially the same receptor binding activity.
Specifically, the partial peptide of GPR40 having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 is extracellular in hydrophobic plot analysis. A peptide containing a portion that has been analyzed to be a region (hydrophilic site). Further, a peptide partially containing a hydrophobic (Hydrophobic) site can also be used. A peptide containing individual domains may be used, but a peptide containing a plurality of domains at the same time may be used.
The number of amino acids in the partial peptide of the present invention is preferably a peptide having an amino acid sequence of at least 20 or more, preferably 50 or more, more preferably 100 or more among the constituent amino acid sequences of the above-described receptor protein of the present invention. .
A substantially identical amino acid sequence refers to an amino acid sequence having about 85% or more, preferably about 90% or more, more preferably about 95% or more homology with these amino acid sequences.
Here, “substantially the same receptor activity” has the same meaning as described above. "Substantially the same receptor activity" can be measured in the same manner as described above.
Further, the partial peptide of the present invention has one or two or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the amino acid sequence, or One or more (preferably about 1 to 20, more preferably about 1 to 10, and still more preferably several (1 to 5)) amino acids are added to the amino acid sequence, or the amino acid One or more (preferably about 1 to 10, more preferably several, and still more preferably about 1 to 5) amino acids in the sequence may be substituted with another amino acid.
In the partial peptide of the present invention, the C-terminus has a carboxyl group (—COOH), a carboxylate (—COO) ⁻ ), Amide (-CONH ₂ )) Or an ester (—COOR). When the partial peptide of the present invention has a carboxyl group (or carboxylate) other than the C-terminal, those in which the carboxyl group is amidated or esterified are also included in the partial peptide of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
Further, the partial peptide of the present invention includes, as in GPR40 described above, one in which the amino group of the methionine residue at the N-terminus is protected by a protecting group, and Gln formed by cleavage of the N-terminal side in vivo is pyroglutamic acid. Also included are those in which the substituent on the side chain of the amino acid in the molecule is protected by an appropriate protecting group, or those in which a sugar chain is bonded, such as a so-called glycopeptide.
Examples of the salt of GPR40 or a partial peptide thereof of the present invention include a physiologically acceptable salt with an acid or a base, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.
The GPR40 of the present invention or a salt thereof can be produced from the above-mentioned human or mammalian cell or tissue by a method for purifying a receptor protein known per se, or contains a DNA encoding the GPR40 of the present invention described later. It can also be produced by culturing a transformant. Further, the protein can also be produced according to the protein synthesis method described later or according thereto.
When producing from human or mammalian tissues or cells, after homogenizing human or mammalian tissues or cells, extraction is performed with an acid or the like, and the extract is subjected to chromatography such as reverse phase chromatography or ion exchange chromatography. Can be purified and isolated.
[0015]
For the synthesis of GPR40 or its partial peptide, its salt or its amide in the present invention, a commercially available resin for protein synthesis can be usually used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydroxymethylmethyl. Phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. it can. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods. At the end of the reaction, the protein is cleaved from the resin, and at the same time, various protecting groups are removed. Further, an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain a target protein or its amide.
Regarding the condensation of the above protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, the protected amino acid is directly added to the resin together with a racemization inhibitor additive (eg, HOBt, HOOBt), or the protected amino acid is previously activated as a symmetric acid anhydride or a HOBt ester or a HOOBt ester. After that, it can be added to the resin.
[0016]
The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, and dimethyl sulfoxide. Sulfoxides, ethers such as pyridine, dioxane, and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof are used. The reaction temperature is appropriately selected from a range known to be usable for a protein bond forming reaction, and is usually appropriately selected from a range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When a sufficient condensation cannot be obtained by repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.
[0017]
Examples of the protecting group for the amino group of the raw material include, for example, Z, Boc, tert-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like.
The carboxyl group may be, for example, a linear, branched or cyclic alkyl ester such as an alkyl esterified ester (eg, methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl). ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonylhydrazide, tertiary butoxy It can be protected by carbonylhydrazide, tritylhydrazide and the like.
The hydroxyl group of serine can be protected, for example, by esterification or etherification. As a group suitable for this esterification, for example, a lower alkanoyl group such as an acetyl group, an aroyl group such as a benzoyl group, and a group derived from carbonic acid such as a benzyloxycarbonyl group and an ethoxycarbonyl group are used. Examples of a group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, tert-butyl and the like are used.
As the imidazole protecting group for histidine, for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
[0018]
Examples of the activated carboxyl group of the raw material include, for example, a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, Cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and an ester with HOBt). As the activated amino group of the raw material, for example, a corresponding phosphoric amide is used.
As a method for removing (eliminating) the protecting group, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, trifluoromethane, etc. Acid treatment with dichloromethane, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the above acid treatment is generally performed at a temperature of about -20 ° C to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethylsulfide, 1,4 -Addition of a cation scavenger such as butanedithiol, 1,2-ethanedithiol and the like is effective. Further, the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.
[0019]
The protection of the functional group that should not be involved in the reaction of the raw materials, the protective group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
As another method for obtaining an amide form of a protein, for example, after amidating and protecting the α-carboxyl group of the carboxy terminal amino acid, a peptide (protein) chain is extended to a desired chain length on the amino group side, and A protein from which only the protecting group for the N-terminal α-amino group of the peptide chain has been removed and a protein from which only the protecting group for the carboxyl group at the C-terminal has been removed, and these proteins are mixed in the above-mentioned mixed solvent. To condense. Details of the condensation reaction are the same as described above. After purifying the protected protein obtained by the condensation, all the protecting groups are removed by the above-mentioned method, and a desired crude protein can be obtained. The crude protein is purified by various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein.
In order to obtain an ester of a protein, for example, after condensing the α-carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, an ester of the desired protein is obtained in the same manner as the amide of the protein be able to.
[0020]
The partial peptide of GPR40 of the present invention or a salt thereof can be produced according to a peptide synthesis method known per se, or by cleaving GPR40 of the present invention with an appropriate peptidase. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the target peptide can be produced by condensing a partial peptide or amino acid capable of constituting the GPR40 of the present invention with the remaining portion, and removing the protective group when the product has a protective group. Known condensation methods and elimination of protecting groups include, for example, the methods described in the following a) to e).
a) M. Bodanszky and M.A. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
b) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
c) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd. (1975)
d) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977)
e) Supervision of Haruaki Yajima, Development of Continuing Drugs Volume 14 Peptide Synthesis Hirokawa Shoten
After the reaction, the partial peptide of the present invention can be purified and isolated by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method, and conversely, when it is obtained by a salt, it can be converted to a free form by a known method. Can be.
[0021]
The polynucleotide encoding GPR40 of the present invention may be any polynucleotide containing the above-described nucleotide sequence (DNA or RNA, preferably DNA) encoding GPR40 of the present invention. The polynucleotide is DNA such as DNA or mRNA encoding GPR40 of the present invention, and may be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA or DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand).
Using the GPR40-encoding polynucleotide of the present invention, for example, the GPR40 mRNA of the present invention can be purified by the method described in the well-known experimental medicine special edition “New PCR and its Applications” 15 (7), 1997 or a method analogous thereto. It can be quantified.
The DNA encoding GPR40 of the present invention may be any of a genomic DNA, a genomic DNA library, the above-described cell / tissue-derived cDNA, the above-described cell / tissue-derived cDNA library, and synthetic DNA. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, amplification can be directly performed by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the above-described cells / tissues.
Specifically, the DNA encoding GPR40 of the present invention contains, for example, the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 18 or SEQ ID NO: 30 A base sequence which hybridizes with DNA or the base sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 18 or SEQ ID NO: 30 under high stringent conditions; No. 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, substantially the same activity as GPR40 consisting of the amino acid sequence (eg, ligand binding activity, signal transduction action, etc.) ) May be any DNA as long as it encodes a receptor protein having
Examples of DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 18 or SEQ ID NO: 30 include, for example, SEQ ID NO: 2, SEQ ID NO: 4, sequence DNA containing a nucleotide sequence having about 85% or more, preferably about 90% or more, more preferably about 95% or more homology with the nucleotide sequence represented by SEQ ID NO: 6, SEQ ID NO: 18 or SEQ ID NO: 30, Is used.
[0022]
Hybridization can be performed according to a method known per se or a method analogous thereto, for example, a method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). . When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be carried out under high stringent conditions.
The high stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C, preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
More specifically, as the DNA encoding mouse GPR40 containing the amino acid sequence represented by SEQ ID NO: 1, a DNA containing the base sequence represented by SEQ ID NO: 2 or the like is used.
As the DNA encoding the mouse GPR40 containing the amino acid sequence represented by SEQ ID NO: 3, a DNA containing the base sequence represented by SEQ ID NO: 4 or the like is used.
As a DNA encoding mouse GPR40 containing the amino acid sequence represented by SEQ ID NO: 5, a DNA containing the base sequence represented by SEQ ID NO: 6 or the like is used.
As the DNA encoding the cynomolgus monkey GPR40 containing the amino acid sequence represented by SEQ ID NO: 17, a DNA containing the base sequence represented by SEQ ID NO: 18 or the like is used.
As the DNA encoding the hamster GPR40 containing the amino acid sequence represented by SEQ ID NO: 29, a DNA containing the base sequence represented by SEQ ID NO: 30 is used.
[0023]
The polynucleotide containing a part of the base sequence of the DNA encoding GPR40 of the present invention or a part of the base sequence complementary to the DNA includes the following DNAs encoding the partial peptide of the present invention. It is used to include not only RNA but also RNA.
According to the present invention, an antisense polynucleotide (nucleic acid) capable of inhibiting replication or expression of a GPR gene is designed based on cloned or determined nucleotide sequence information of DNA encoding GPR40, Can be synthesized. Such a polynucleotide (nucleic acid) can hybridize to the RNA of the GPR40 gene, inhibit the synthesis or function of the RNA, or inhibit the expression of the GPR40 gene through interaction with a GPR40-related RNA. It can be adjusted and controlled. Polynucleotides complementary to selected sequences of GPR-related RNA, and polynucleotides capable of specifically hybridizing to GPR-related RNA, are useful in regulating and controlling GPR40 gene expression in vivo and in vitro. It is useful, and is useful for treating or diagnosing diseases and the like. The term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. "Corresponding" between a nucleotide, base sequence or nucleic acid and a peptide (protein) usually refers to the amino acid of the peptide (protein) as directed by the nucleotide (nucleic acid) sequence or its complement. . 5′-end hairpin loop, 5′-end 6-base pair repeat, 5′-end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation initiation codon, 3′-end untranslated region, 3 ′ end of GPR40 gene The terminal palindrome region and the 3′-end hairpin loop can be selected as preferred regions of interest, but any region within the GPR40 gene can be selected as a target.
[0024]
The relationship between the target nucleic acid and the polynucleotide that is complementary to at least a part of the target region and can hybridize can be said to be “antisense” with the target. Antisense polynucleotides include polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides containing D-ribose, and other types of polynuclei that are N-glycosides of purine or pyrimidine bases. Other polymers with nucleotides or non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds, provided that the polymers are found in DNA or RNA Base pairs and nucleotides having a configuration that allows base attachment). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and also DNA: RNA hybrids, and can be unmodified polynucleotides (or unmodified oligonucleotides), or even known. Such as labeled, capped, methylated, one or more naturally-occurring nucleotides replaced by analogs, intramolecular nucleotides Modified, such as those having uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged or sulfur-containing bonds (eg, phosphorothioates, phosphorodithioates, etc.) ), Such as proteins (nucleases, nuclease inhibitors, toxic , Antibodies, signal peptides, poly-L-lysine, etc.) or sugars (for example, monosaccharides) having side chain groups, interacting compounds (for example, acridine, psoralen, etc.), chelates Those containing compounds (eg, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those having modified bonds (eg, α-anomeric nucleic acids, etc.) It may be. Here, "nucleoside", "nucleotide", and "nucleic acid" may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced by halogens, aliphatic groups, etc., or by functional groups such as ethers, amines, etc. It may have been converted.
[0025]
The antisense polynucleotide (nucleic acid) of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those that are resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to make the antisense nucleic acid more cell permeable, to have a greater affinity for the target sense strand, and to be more toxic if it is toxic. Minimize the toxicity of sense nucleic acids.
Thus, many modifications are known in the art, for example, as described in Kawakami et al. , Pharm Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; T. Crooke et al. ed. , Antisense Research and Applications, CRC Press, 1993.
The antisense nucleic acid of the present invention may contain altered or modified sugars, bases or bonds, may be provided in a special form such as liposomes or microspheres, may be applied by gene therapy, It could be provided in an added form. Thus, in the form of addition, polycations such as polylysine which acts to neutralize the charge of the phosphate skeleton, lipids which enhance the interaction with the cell membrane or increase the uptake of nucleic acids ( For example, crude water-based substances such as phospholipid and cholesterol can be used. Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond. Other groups include cap groups specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid for preventing degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
The inhibitory activity of an antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of a G protein-coupled receptor protein. it can. The nucleic acid can be applied to cells by various methods known per se.
The siRNA against the polynucleotide of the present invention is a double-stranded RNA containing a part of RNA encoding GPR40 and RNA complementary thereto. Specifically, the siRNA comprises a sense strand consisting of the base sequence represented by SEQ ID NO: 34 and an antisense strand consisting of the base sequence represented by SEQ ID NO: 35, and comprises a base sequence represented by SEQ ID NO: 36 An siRNA composed of an antisense strand consisting of a sense strand and a base sequence represented by SEQ ID NO: 37 (FIG. 22) or the like is used.
The siRNA can be designed and manufactured based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).
A ribozyme containing a part of the RNA encoding GPR40 is designed based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, p. 221, 2001). Can be manufactured. For example, it can be produced by substituting a part of the sequence of a known ribozyme with a part of RNA encoding GPR40. Examples of a part of the RNA encoding GPR40 include a sequence near a consensus sequence NUX (where N represents all bases and X represents a base other than G) that can be cleaved by a known ribozyme.
[0026]
The DNA encoding the partial peptide of the present invention may be any DNA containing the above-described nucleotide sequence encoding the partial peptide of the present invention. In addition, any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells / tissues, cDNA library derived from the above-described cells / tissues, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can be directly amplified by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR) using an mRNA fraction prepared from the above-described cells / tissues.
Specifically, the DNA encoding the partial peptide of the present invention includes, for example, (1) a base represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 18 or SEQ ID NO: 30 A DNA having a partial base sequence of the DNA having the sequence, or (2) a high stringency with the base sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 18 or SEQ ID NO: 30 GPR40 having a nucleotide sequence that hybridizes under the conditions and having substantially the same amino acid sequence as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29. For example, a DNA having a partial base sequence of a DNA encoding a receptor protein having an activity (eg, ligand binding activity, signal information transmitting action, etc.) is used.
Examples of DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 18 or SEQ ID NO: 30 include, for example, SEQ ID NO: 2, SEQ ID NO: 4, sequence DNA containing a nucleotide sequence having about 85% or more, preferably about 90% or more, more preferably about 95% or more homology with the nucleotide sequence represented by SEQ ID NO: 6, SEQ ID NO: 18 or SEQ ID NO: 30, Is used.
[0027]
As a means for cloning DNA that completely encodes GPR40 of the present invention or its partial peptide (hereinafter sometimes abbreviated as GPR40 of the present invention), a synthetic DNA primer having a partial base sequence of GPR40 of the present invention is used. Or amplified by a PCR method, or selected by hybridization with a DNA incorporated into an appropriate vector and labeled with a DNA fragment encoding a partial or entire region of the GPR40 of the present invention or a synthetic DNA. it can. Hybridization can be performed, for example, according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.
[0028]
Conversion of the DNA base sequence can be performed by PCR or a known kit, for example, Mutan. ^TM -Super Express Km (Takara Shuzo Co., Ltd.), Mutan ^TM Using -K (Takara Shuzo Co., Ltd.) or the like, it can be carried out according to a method known per se such as the ODA-LA PCR method, the Gapped Duplex method, the Kunkel method, or a method analogous thereto.
The cloned DNA encoding GPR40 can be used as it is depending on the purpose, or can be used after digesting with a restriction enzyme or adding a linker, if desired. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation termination codon at the 3' end. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
For the GPR40 expression vector of the present invention, for example, (a) a target DNA fragment is cut out from the DNA encoding the GPR40 of the present invention, and (b) the DNA fragment is ligated downstream of a promoter in an appropriate expression vector. It can be manufactured by the following.
[0029]
Examples of the vector include plasmids derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), plasmids derived from yeast (eg, pSH19, pSH15), λ phage, etc. In addition to animal viruses such as bacteriophage, retrovirus, vaccinia virus, and baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, and the like are used.
The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when an animal cell is used as a host, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
Among them, it is preferable to use the CMV promoter, SRα promoter and the like. When the host is Escherichia, trp promoter, lac promoter, recA promoter, λP _L When the host is a bacterium belonging to the genus Bacillus, an SPO1 promoter, an SPO2 promoter, a penP promoter, and the like are preferable. When the host is a yeast, a PHO5 promoter, a PGK promoter, a GAP promoter, an ADH promoter, and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.
[0030]
In addition to the above, an expression vector containing an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like may be used, if desired. it can. Examples of the selectable marker include a dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance] and an ampicillin resistance gene (hereinafter Amp). ^r Neomycin resistance gene (hereinafter Neo). ^r G418 resistance). In particular, CHO (dhfr ⁻ ) When the dhfr gene is used as a selection marker using cells, the target gene can be selected using a thymidine-free medium.
If necessary, a signal sequence suitable for the host is added to the N-terminal side of the receptor protein of the present invention. When the host is a bacterium belonging to the genus Escherichia, a PhoA signal sequence, an OmpA signal sequence, or the like is used. When the host is a bacterium belonging to the genus Bacillus, an α-amylase signal sequence, a subtilisin signal sequence, or the like is used. In some cases, MFα signal sequence, SUC2 signal sequence, etc., and when the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule, signal sequence, etc. can be used.
Using the vector containing the DNA encoding GPR40 of the present invention thus constructed, a transformant can be produced.
[0031]
As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used.
Specific examples of the genus Escherichia include Escherichia coli K12 DH1 [Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular Biology (Journal of Molecular Biology)]. 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics (Ge) netics), Vol. 39, 440 (1954)].
Examples of Bacillus spp. Include Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)]. )] Is used.
Examples of yeast include, for example, Saccharomyces cerevisiae AH22, AH22R ⁻ , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris and the like.
[0032]
When the virus is AcNPV, for example, when the virus is AcNPV, a cell line derived from a larva of night roth moth (Spodoptera frugiperda cell; Sf cell), MG1 cell derived from the midgut of Trichoplusia ni, and Hig derived from Trichoplusia ni egg. ^TM Cells, cells derived from Mamestra brassicae, cells derived from Estigmena acrea, and the like are used. When the virus is BmNPV, a cell line derived from silkworm (Bombyx mori N; BmN cell) or the like is used. As the Sf cells, for example, Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL, et al., In Vivo, 13, 213-217, (1977)) and the like are used. .
As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), and dhfr gene-deficient Chinese hamster cells CHO (hereinafter CHO (dhfr). ⁻ ) Cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, and the like.
[0033]
In order to transform Escherichia sp., For example, Procesings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 69, 2110 ( 1972) and Gene, Vol. 17, 107 (1982).
Transformation of Bacillus spp. Can be performed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 111 (1979).
To transform yeast, for example, Methods in Enzymology, 194, 182-187 (1991), Processings of the National Academy of Sciences of Science -The USA (Proc. Natl. Acad. Sci. USA), Vol. 75, 1929 (1978).
Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
To transform animal cells, for example, see Cell Engineering Separate Volume 8, New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha) and Virology, Vol. 52, 456 (1973).
Thus, a transformant transformed with the expression vector containing the DNA encoding GPR40 is obtained.
When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for the culturing, and among them, a carbon source necessary for growth of the transformant, Nitrogen sources, inorganic substances, etc. are contained. As a carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc.As a nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract and the like Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5 to 8.
[0034]
Examples of a medium for culturing Escherichia include, for example, an M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics]. , 431-433, Cold Spring Harbor Laboratory, New York 1972]. If necessary, an agent such as 3β-indolyl acrylic acid can be added in order to make the promoter work efficiently.
When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added.
When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring may be added.
When culturing a transformant in which the host is yeast, for example, Burkholder's minimum medium [Bostian, K. et al. L. Procurings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 77, 4505 (1980)] and 0.5% casamino acid. SD medium containing [Bitter, G .; A. Processing of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 81, 5330 (1984)]. Preferably, the pH of the medium is adjusted to about 5-8. The cultivation is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and if necessary, aeration or stirring is added.
[0035]
When culturing an insect cell or a transformant whose host is an insect, the culture medium is immobilized as Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)). For example, those to which an additive such as 10% bovine serum or the like is appropriately added are used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The cultivation is usually performed at about 27 ° C. for about 3 to 5 days, and if necessary, aeration and / or agitation are added.
When culturing a transformant in which the host is an animal cell, examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium. [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], 199 medium. [Proceding of the Society for the Biological Medicine, Vol. 73, 1 (1950) [Proceding of the Society for the Biological Medicine] ], Such as is used. Preferably, the pH is between about 6 and 8. The cultivation is usually carried out at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and if necessary, aeration or stirring is added.
As described above, the GPR40 of the present invention can be produced in the cells, in the cell membrane, or outside the cells of the transformant.
[0036]
The GPR40 of the present invention can be separated and purified from the culture by, for example, the following method.
When extracting the GPR40 of the present invention from cultured cells or cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasonic wave, lysozyme, and / or freeze-thawing. After the cells or cells are destroyed by the method described above, a method of obtaining a crude extract of GPR40 by centrifugation or filtration is used as appropriate. In a buffer, a protein denaturant such as urea or guanidine hydrochloride, or Triton X-100 is used. ^TM And the like. When GPR40 is secreted into the culture solution, after the culture is completed, the bacterial cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected.
The purification of GPR40 contained in the culture supernatant or extract obtained in this manner can be performed by appropriately combining known separation and purification methods. These known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight. Method using difference in charge, method using charge difference such as ion exchange chromatography, method using specific novelty such as affinity chromatography, use of difference in hydrophobicity such as reversed-phase high-performance liquid chromatography And a method utilizing the difference in isoelectric points such as isoelectric focusing.
[0037]
When GPR40 thus obtained is obtained in a free form, it can be converted to a salt by a method known per se or a method analogous thereto, and conversely, when GPR40 is obtained as a salt, a method known per se or analogous thereto Can be converted to a free form or another salt.
GPR40 produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
The activity of the thus produced GPR40 of the present invention can be measured by a binding experiment with a labeled ligand, an enzyme immunoassay using a specific antibody, or the like.
[0038]
The antibody against GPR40 of the present invention may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the GPR40 of the present invention.
The antibody against GPR40 of the present invention can be produced by using the GPR40 of the present invention as an antigen according to a method for producing an antibody or antiserum known per se.
[0039]
[Preparation of monoclonal antibody]
(A) Preparation of monoclonal antibody-producing cells
The GPR40 of the present invention is administered to a mammal at a site capable of producing an antibody by administration itself or together with a carrier or a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep and goats, and mice and rats are preferably used.
When producing monoclonal antibody-producing cells, a warm-blooded animal immunized with the antigen, for example, an individual having an antibody titer was selected from a mouse, and the spleen or lymph node was collected 2 to 5 days after the final immunization. By fusing the contained antibody-producing cells with myeloma cells, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting a labeled receptor protein described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
Examples of myeloma cells include NS-1, P3U1, SP2 / 0 and the like, and P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. Incubation at about 20 to 40 ° C., preferably about 30 to 37 ° C. for about 1 to 10 minutes allows efficient cell fusion.
[0040]
Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which the antigen of the receptor protein is directly or adsorbed together with a carrier, and then radioactively. A method of detecting a monoclonal antibody bound to a solid phase by adding an anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used when the cell used for cell fusion is a mouse) or protein A labeled with a substance or enzyme, A method in which a hybridoma culture supernatant is added to a solid phase to which an anti-immunoglobulin antibody or protein A is adsorbed, a receptor protein labeled with a radioactive substance or an enzyme is added, and a monoclonal antibody bound to the solid phase is detected. .
The selection of the monoclonal antibody can be carried out according to a method known per se or a method analogous thereto. Usually, it can be carried out in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium can be used as long as the hybridoma can grow. For example, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium (Wako Pure Chemical Industries, Ltd.) containing 1 to 10% fetal bovine serum, or serum-free for hybridoma culture A medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used. The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
[0041]
(B) Purification of monoclonal antibody
Monoclonal antibodies can be separated and purified by immunoglobulin separation and purification methods [eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchangers (eg, DEAE) adsorption / desorption method, ultracentrifugation method, gel filtration method, specific purification method in which an antibody alone is collected using an antigen-binding solid phase or an active adsorbent such as protein A or protein G, and the bond is dissociated to obtain the antibody. Can be performed according to
[0042]
(Preparation of polyclonal antibody)
The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, a complex of an immunizing antigen (GPR40 antigen) and a carrier protein is formed, a mammal is immunized in the same manner as in the above-described method for producing a monoclonal antibody, and the GPR40 antibody-containing substance of the present invention is collected from the immunized animal. The antibody can be produced by separating and purifying the antibody.
Regarding a complex of an immunizing antigen and a carrier protein used to immunize a mammal, the type of the carrier protein and the mixing ratio of the carrier and the hapten should be such that the antibody can be efficiently produced against the hapten immunized by crosslinking the carrier. Any kind may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, keyhole limpet hemocyanin, etc. may be used in a weight ratio of about 0.1 to 20 with respect to 1 hapten. Preferably, a method of coupling at a ratio of about 1 to 5 is used.
Various coupling agents can be used for coupling the hapten and the carrier. For example, glutaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.
The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration can usually be performed once every about 2 to 6 weeks, for a total of about 3 to 10 times.
The polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of the mammal immunized by the above method.
The polyclonal antibody titer in the antiserum can be measured in the same manner as the measurement of the antibody titer in the serum described above. The separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-mentioned separation and purification of the monoclonal antibody.
Specifically, as the antibody against GPR40 of the present invention, for example, an antibody (particularly, a monoclonal antibody) that recognizes the C-terminal peptide of GPR40 of the present invention or a salt thereof is preferable. More specifically, a monoclonal antibody that recognizes the peptide represented by SEQ ID NO: 33, which is a C-terminal peptide of human GPR40, or a salt thereof is used.
[0043]
One of the ligands of GPR40 of the present invention is a fatty acid or a salt thereof, for example, an unsaturated fatty acid, a saturated fatty acid or a salt thereof present in food or in a living body. Among them, unsaturated fatty acids and saturated fatty acids having about 10 to 30 carbon atoms or salts thereof are preferably used. Specifically, farnesic acid (farnesic acid), 5.8.11-eicosatrynoic acid, 5.8.11.14-eicosatetraynonic acid, oleic acid (oleic acid), linoleic acid (linoleic acid), and linolenic acid (linolenic acid) acid), α-linolenic acid, γ-linolenic acid, arachidonic acid, eicosapentaenoic acid (EPA), eicosaicoadienic acid Satrienoic acid (eicosatrienoic acid), docosahexaenoic acid (docosahh) xaenoic acid, DHA), docosatriene acid (docosatrienoic acid), adrenic acid (adrenic acid), lauric acid (lauric acid), palmitic acid (palmitic acid), or a salt thereof and the like are used.
As the salt of a fatty acid, a salt with an acid (eg, an inorganic acid, an organic acid, etc.) or a base (eg, an alkali metal such as sodium or potassium; an alkaline earth metal such as calcium) is used, and a base is particularly preferable. .
Hereinafter, in the present specification, a fatty acid or a salt thereof is simply abbreviated as “fatty acid”.
Further, one of the ligands of GPR40 of the present invention is an eicosanoid, for example, (±) 14,15-dihydroxy-5Z, 8Z, 11Z-eicosatrienoic acid ((±) 14,15-dihydroxy-5Z, 8Z, 11Z-eicosatrienoic acid, 14,15-DHT), (±) 5 (6) -epoxy-8Z, 11Z, 14Z-eicosatrienoic acid ((±) 5 (6) -epoxy-8Z, 11Z, 14Z -Eicosatrienoic acid, 5,6-EET), (±) 8 (9) -epoxy-5Z, 11Z, 14Z-eicosatrienoic acid ((±) 8 (9) -epoxy-5Z, 11Z, 14Z-eicosatrionic acid , 8,9-EET), (±) 11 (12) -epoxy-5Z, 8Z, 14Z-E Cosatrienoic acid ((±) 11 (12) -epoxy-5Z, 8Z, 14Z-eicosatorenoic acid, 11,12-EET), (±) 14 (15) -epoxy-5Z, 8Z, 11Z-eicosatrienoic acid ( (±) 14 (15) -epoxy-5Z, 8Z, 11Z-eicosatrianic acid, 14, 15-EET) or the like is used.
In addition, GPR40 of the present invention is highly expressed especially in pancreatic islets of Langerhans.
Therefore, the GRP40 of the present invention, the DNA encoding GPR40 (hereinafter sometimes abbreviated as the DNA of the present invention), the antibody against GPR40 (hereinafter sometimes abbreviated as the antibody of the present invention) and the DNA of the present invention may be used. Antisense DNA (hereinafter sometimes abbreviated as the antisense DNA of the present invention) and siRNA against the polynucleotide of the present invention (hereinafter sometimes abbreviated as the siRNA of the present invention) have the following uses. I have.
[0044]
(1) The preventive and / or therapeutic agent for a disease associated with dysfunction of GPR40 of the present invention
The DNA encoding a) GPR40 of the present invention or b) GPR40 can be used as a medicament such as an agent for preventing and / or treating a disease associated with dysfunction of GPR40 of the present invention.
For example, when the GPR40 of the present invention is decreased in the living body, and there is a patient who cannot expect the physiological action of the fatty acid or eicosanoid as a ligand (deficiency of GPR40), a) the GPR40 of the present invention is added to the patient. B) (a) administering the GPR40 encoding DNA of the present invention to the patient and expressing it, or (ii) encoding the GPR40 of the present invention in target cells. After inserting and expressing the DNA, the amount of GPR40 in the patient's body can be increased by, for example, transplanting the cells into the patient, and the effect of the ligand can be sufficiently exerted. That is, the DNA encoding the GPR40 of the present invention is a safe and low-toxic agent for preventing and / or treating a disease associated with dysfunction of the GPR40 of the present invention, a pancreatic function regulator (eg, a pancreatic function improving agent), insulin It is useful as a secretagogue, a hypoglycemic agent, a pancreatic β-cell protective agent, and the like.
Specifically, GPR40 of the present invention or DNA of the present invention may be used, for example, for diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual function It can be used as a prophylactic / therapeutic agent for disorders, skin diseases, arthrosis, osteopenia, arteriosclerosis, thrombotic diseases, dyspepsia, memory learning disorders and the like. Diabetes includes insulin dependent (type I) diabetes, non-insulin dependent (type II) diabetes and the like.
When the GPR40 of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated according to conventional means.
On the other hand, when the DNA of the present invention is used as the above-mentioned prophylactic / therapeutic agent, the DNA of the present invention is used alone or after insertion into an appropriate vector such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector, and the like. It can be implemented according to the means. The DNA of the present invention can be administered as it is or together with an auxiliary agent for promoting uptake, using a gene gun or a catheter such as a hydrogel catheter.
For example, a) GPR40 of the present invention or b) DNA of the present invention may be orally administered as tablets, capsules, elixirs, microcapsules, etc., if necessary, or water or other pharmaceutical agents. It can be used parenterally in the form of an injection, such as a sterile solution with an acceptable solution, or a suspension. For example, a) GPR40 of the present invention or b) Preparation of the DNA of the present invention which is generally recognized with known carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, etc. which are physiologically recognized. By mixing in the unit dosage form required. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
[0045]
Examples of additives that can be incorporated into tablets, capsules, and the like include binders such as gelatin, corn starch, tragacanth, and acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Useful bulking agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry and the like are used. When the unit dosage form is a capsule, a liquid carrier such as oils and fats can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80) ^TM , HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
[0046]
The prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled in a suitable ampoule.
The preparations obtained in this way are safe and have low toxicity, and should be administered to humans and mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Can be.
The dose of the GPR40 of the present invention varies depending on the administration subject, target organ, condition, administration method and the like. In the case of oral administration, for example, in a diabetic patient (60 kg), for example, about 0 kg per day is generally used. 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, condition, administration method, and the like. For example, usually in the form of an injection, for example, in a diabetic patient (as 60 kg), It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the amount can be administered in terms of 60 kg.
The dose of the DNA of the present invention varies depending on the administration subject, target organ, condition, administration method, and the like. However, in the case of oral administration, for example, in a diabetic patient (60 kg), it is generally about 0 per day. 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, condition, administration method, and the like. For example, usually in the form of an injection, for example, in a diabetic patient (as 60 kg), It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the amount can be administered in terms of 60 kg.
[0047]
(2) Gene diagnostic agent
The DNA, antisense DNA and siRNA of the present invention can be used as a probe to produce the GPR40 of the present invention in humans or mammals (eg, rat, mouse, rabbit, sheep, pig, cow, cat, dog, monkey, etc.). Or, abnormalities (genetic abnormalities) of DNA or mRNA encoding the partial peptide thereof can be detected, for example, damage, mutation or decreased expression of the DNA or mRNA, increase or excessive expression of the DNA or mRNA, etc. It is useful as a gene diagnostic agent for.
The above-mentioned genetic diagnosis using the DNA, antisense DNA or siRNA of the present invention includes, for example, Northern hybridization and PCR-SSCP method known per se (Genomics, 5, 874-879 (1989), Proceedings of the National Academy of Sciences of the United States of America, Vol. 86, pp. 2786-27, pp. 2789-27, etc. (Procedings of the National Academy of Sciences of USA), Procedings of the National Academy of Sciences of the United States of America. Can be implemented.
For example, when a decrease in the expression of GPR40 of the present invention is detected by Northern hybridization, for example, it is diagnosed that the disease is highly likely to be a disease associated with dysfunction of GPR40 of the present invention or is likely to be affected in the future. be able to.
When overexpression of the GPR40 of the present invention is detected by Northern hybridization, for example, it is diagnosed that the disease is highly likely to be caused by the overexpression of the GPR40 of the present invention or is likely to be affected in the future. be able to.
The diseases associated with the dysfunction of GPR40 of the present invention include diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, and skin diseases , Arthropathy, osteopenia, arteriosclerosis, thrombotic diseases, dyspepsia, memory learning disorders and the like. Diabetes includes insulin dependent (type I) diabetes, non-insulin dependent (type II) diabetes and the like.
Diseases caused by overexpression of GPR40 of the present invention include, for example, obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, Insulin resistance syndrome, unstable diabetes, lipoatrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity, cancer and the like.
[0048]
(3) A medicine containing a compound that changes the expression level of GPR40 of the present invention
By using the DNA of the present invention as a probe, it can be used for screening for a compound that changes the expression level of GPR40 of the present invention.
That is, the present invention includes, for example, the present invention contained in (i) a) blood of a non-human mammal, b) a specific organ, c) a tissue or cell isolated from an organ, or (ii) a transformant or the like. The present invention provides a method for screening a compound that changes the expression level of GPR40 of the present invention by measuring the amount of GPR40 mRNA.
The measurement of the mRNA amount of GPR40 of the present invention is specifically performed as follows.
(I) Normal or disease model non-human mammals (eg, mice, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc., more specifically, dementia rats, obese mice, arteriosclerotic rabbits, cancer-bearing A drug (eg, an anti-dementia drug, a blood pressure lowering drug, an anti-cancer drug, an anti-obesity drug, etc.) or a physical stress (eg, waterlogging stress, electric shock, light / dark, low temperature, etc.), etc. After the passage, blood or a specific organ (eg, brain, liver, kidney, etc.), or a tissue or cell isolated from the organ is obtained.
The GPR40 mRNA of the present invention contained in the obtained cells can be quantified by, for example, extracting mRNA from cells or the like by a usual method and using, for example, a technique such as TaqMan PCR, and by a method known per se. Analysis can also be performed by performing Northern blot.
(Ii) A transformant expressing the GPR40 of the present invention is prepared according to the above method, and the mRNA of the GPR40 of the present invention contained in the transformant can be quantified and analyzed in the same manner.
[0049]
The screening for a compound that changes the expression level of GPR40 of the present invention comprises:
(I) A given time (30 minutes to 24 hours, preferably 30 minutes to 12 hours, more preferably 1 hour) before giving a drug or physical stress to a normal or disease model non-human mammal Before to 6 hours before) or after a certain time (after 30 minutes to 3 days, preferably after 1 hour to 2 days, more preferably after 1 hour to 24 hours), or at the same time as the drug or physical stress. And after a certain period of time after administration (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), the amount of GPR40 mRNA of the present invention contained in the cells Can be performed by quantifying and analyzing
(Ii) When culturing the transformant according to a conventional method, the test compound is mixed in the medium, and after culturing for a certain period of time (1 day to 7 days, preferably 1 day to 3 days, more preferably 2 days to 3 days) Days after), the amount of the GPR40 mRNA of the present invention contained in the transformant can be quantified and analyzed.
[0050]
The compound or a salt thereof obtained by using the screening method of the present invention is a compound having an action of changing the expression level of GPR40 of the present invention, and specifically, (a) increasing the expression level of GPR40 of the present invention. By causing GPR40-mediated cell stimulating activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ Release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuations, intracellular protein phosphorylation, c-fos activation, pH reduction, etc. (B) A compound that reduces the expression of GPR40 of the present invention to thereby reduce the cell stimulating activity.
Such compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, and the like, and these compounds may be novel compounds or known compounds.
The ligand of GPR40 of the present invention is a fatty acid or eicosanoid as described above. Therefore, the compound that alters the expression level of GPR40 of the present invention obtained by the above screening method can be used as a prophylactic and / or therapeutic agent for a disease associated with dysfunction of GPR40 of the present invention.
Specifically, the compound of the present invention that increases the expression level of GPR40 and enhances the cell stimulating activity is a safe and low-toxic prophylactic / therapeutic agent for diseases related to dysfunction of GPR40 of the present invention, Agents (eg, pancreatic function improving agents), insulin secretagogues, hypoglycemic agents, and pancreatic β-cell protective agents.
The compound of the present invention which reduces the expression level of GPR40 and attenuates the cell stimulating activity is a safe and low toxic preventive / therapeutic agent for a disease caused by excessive expression of GPR40, a pancreatic function regulator (eg, pancreas) It is useful as a function improving agent), an insulin secretion inhibitor, and a blood sugar increasing agent.
The diseases associated with the dysfunction of GPR40 of the present invention include diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, and skin diseases , Arthropathy, osteopenia, arteriosclerosis, thrombotic diseases, dyspepsia, memory learning disorders and the like. Diabetes includes insulin dependent (type I) diabetes, non-insulin dependent (type II) diabetes and the like.
Diseases caused by overexpression of GPR40 of the present invention include, for example, obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, Insulin resistance syndrome, unstable diabetes, lipoatrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity, cancer and the like.
Examples of the salt of the compound include salts with physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals such as sodium and potassium; alkaline earth metals such as calcium). Preference is given to the acid addition salts used, in particular physiologically acceptable acid addition salts. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, For example, salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like are used.
Hereinafter, the same salt is used.
[0051]
When a compound or a salt thereof obtained by using the screening method of the present invention is used as a pharmaceutical composition, it can be formulated according to a conventional method.
For example, the compound may be a sugar-coated tablet, capsule, elixir, microcapsule, or the like, as needed, orally, or a sterile solution of water or other pharmaceutically acceptable liquid, Alternatively, they can be used parenterally in the form of injections such as suspensions. For example, the compound may be mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like, in a unit dosage form required for generally accepted pharmaceutical practice. Can be manufactured by The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
Examples of additives that can be incorporated into tablets, capsules, and the like include binders such as gelatin, corn starch, tragacanth, and acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Useful bulking agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry and the like are used. When the unit dosage form is a capsule, a liquid carrier such as oils and fats can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80) ^TM , HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
[0052]
The prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled in a suitable ampoule.
The preparations obtained in this way are safe and have low toxicity, and should be administered to humans and mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Can be.
The dose of the compound or a salt thereof varies depending on the administration subject, target organ, condition, administration method and the like. In the case of oral administration, for example, in a diabetic patient (60 kg), for example, about 0.1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, condition, administration method, and the like. For example, usually in the form of an injection, for example, in a diabetic patient (as 60 kg), It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the amount can be administered in terms of 60 kg.
[0053]
(4) Quantitative method and diagnostic method of GPR40 of the present invention
Since the antibody of the present invention can specifically recognize the GPR40 of the present invention, it can be used for quantification of GPR40 in a test solution, particularly for quantification by sandwich immunoassay.
That is, the present invention
(I) reacting the antibody of the present invention with a test solution and labeled GPR40 competitively, and measuring the ratio of the labeled GPR40 bound to the antibody in the test solution. GPR40 quantification method, and
(Ii) After reacting a test solution with the antibody of the present invention immobilized on a carrier and another labeled antibody of the present invention simultaneously or continuously, the activity of the labeling agent on the insolubilized carrier is measured. And a method for quantifying GPR40 in a test solution.
[0054]
In the quantitative method (ii), it is preferable that one antibody is an antibody that recognizes the N-terminal of GPR40 and the other antibody is an antibody that reacts with the C-terminal of GPR40.
In addition, GPR40 can be quantified using a monoclonal antibody against GPR40, and can also be detected by tissue staining or the like. For these purposes, the antibody molecule itself may be used, and the F (ab ') ₂ , Fab ′ or Fab fraction may be used.
The method for quantifying GPR40 using the antibody of the present invention is not particularly limited. Any measurement method may be used as long as it is detected by physical or physical means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, a competitive method, an immunometric method, and a sandwich method are preferably used, but in terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later.
[0055]
As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] is used. As the above enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, a luminol derivative, luciferin, lucigenin and the like are used. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing GPR40 or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the labeling agent, the amount of GPR40 of the present invention in the test solution can be determined. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above. In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving the measurement sensitivity and the like. You may.
[0056]
In the method for measuring GPR40 by the sandwich method of the present invention, as the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction, antibodies having different GPR40 binding sites are preferably used. That is, when the antibody used in the primary reaction and the secondary reaction recognizes the C-terminal of GPR40, for example, the antibody used in the primary reaction is preferably the C-terminal. In addition, for example, an antibody that recognizes the N-terminal is used.
The monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, a nephrometry, or the like.
In the competition method, after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the amount of any of B and F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as the antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, or a solid phase antibody is used as the first antibody. As the first antibody, a soluble antibody is used, and an immobilization method using an immobilized antibody as the second antibody is used.
In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction against a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. And an excess amount of the labeled antibody, and then immobilized antigen is added to bind unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution.
In nephelometry, the amount of insoluble precipitates generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.
In applying these individual immunoassays to the quantification method of the present invention, no special conditions, operations, and the like need to be set. The measurement system of GPR40 of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, books, and the like.
For example, Hiroe Irie, "Radio Immunoassay" (Kodansha, published in 1974), Hiroshi Irie, "Radio Immunoassay" (Kodansha, published in 1974), Eiji Ishikawa et al., "Enzyme Immunoassay" (Medical Shoin, Showa Ed. Ishikawa et al., “Enzyme Immunoassay” (2nd edition) (Issue Shoin, 1982), Eiji Ishikawa et al. “Enzyme Immunoassay” (Third Edition), 3rd edition (Medical Shoin, Showa) 62)), "Methods in ENZYMOLOGY" Vol. 70 (Immunochemical Techniques (Part A)), ibid., Vol. 73 (Immunochemical Technologies (Part B)), ibid., Vol. 74 (Immunochemical Technologies (Part C)), ibid., Vol. 84 (Immunochemical Technologies (Part D: Selected Immunoassays)), ibid., Vol. 92 (Immunochemical Technologies (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid., Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (all published by Academic Press) can be referred to.
As described above, the GPR40 of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
[0057]
Furthermore, when a decrease in the concentration of GPR40 is detected by quantifying the concentration of GPR40 using the antibody of the present invention, for example, the disease may be associated with dysfunction of GPR40 or may be affected in the future. It can be diagnosed as high.
When an increase in the concentration of GPR40 is detected, for example, it can be diagnosed that the disease is caused by overexpression of GPR40 or that the disease is likely to be caused in the future.
The diseases associated with the dysfunction of GPR40 of the present invention include diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, and skin diseases , Arthropathy, osteopenia, arteriosclerosis, thrombotic diseases, dyspepsia, memory learning disorders and the like. Diabetes includes insulin dependent (type I) diabetes, non-insulin dependent (type II) diabetes and the like.
Diseases caused by overexpression of GPR40 of the present invention include, for example, obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, Insulin resistance syndrome, unstable diabetes, lipoatrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity, cancer and the like.
[0058]
(5) Method for determining ligand other than fatty acid or eicosanoid for GPR40
The binding of fatty acids or eicosanoids to GPR40 results in intracellular Ca ²⁺ GPR40 is useful as a reagent for searching for or determining ligands other than fatty acids or eicosanoids for GPR40 using this intracellular signal as an index, since the concentration of is increased and suppression of intracellular cAMP production is observed.
That is, the present invention relates to GPR40-mediated intracellular Ca when a test compound is brought into contact with cells containing GPR40. ²⁺ Provided is a method for determining a ligand for GPR40, which comprises measuring a concentration increasing activity or an intracellular cAMP production inhibitory activity.
Test compounds include known ligands (eg, angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, PACAP (eg, PACAP27, PACAP38), secretin, Glucagon, calcitonin, adrenomedullin, somatostatin, GHRH, CRF, ACTH, GRP, PTH, VIP (Vasoactive Intestinal and Related Polypeptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene relayed peptide) ), Leukotriene, pancreastatin, prostaglandin, thromboxane, adenosine, adrenaline Chemokine superfamily (eg, CXC chemokine subfamily such as IL-8, GROα, GROβ, GROγ, NAP-2, ENA-78, GCP-2, PF4, IP-10, Mig, PBSF / SDF-1; MCAF / MCP-1, MCP-2, MCP-3, MCP-4, eotaxin, RANTES, MIP-1α, MIP-1β, HCC-1, MIP-3α / LARC, MIP-3β / ELC, I-309, TARC , MIPF-1, MIPF-2 / eotaxin-2, MDC, DC-CK1 / PARC, CC chemokine subfamily such as SLC; C chemokine subfamily such as lymphotoactin; CX3C chemokine subfamily such as fractionalkine, etc.), endothelin, entero Gust Histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA), sphingosine 1-phosphate, etc.), as well as, for example, humans or mammals (eg, mice, rats, pigs, Tissue extracts of bovine, ovine, monkey, etc.), cell culture supernatants, low molecular weight synthetic compounds and the like are used. For example, the tissue extract, cell culture supernatant or the like is added to the receptor protein of the present invention, and fractionation is performed while measuring cell stimulating activity and the like to finally obtain a single ligand.
[0059]
Specifically, the ligand determination method of the present invention comprises constructing an expression system for recombinant GRP40, and using a receptor binding assay system using the expression system to achieve intracellular Ca through GPR40. ²⁺ This is a method for determining a compound having a concentration increasing activity or an intracellular cAMP production inhibitory activity or a salt thereof.
More specifically, the present invention provides the following determination method.
(1) Intracellular Ca when a test compound is brought into contact with cells containing GPR40 ²⁺ A method for determining a ligand for GPR40, which comprises measuring a concentration increasing activity or an intracellular cAMP production inhibitory activity, and
(2) GPR40-mediated intracellular Ca when a test compound is brought into contact with a receptor protein expressed on a cell membrane by culturing a transformant containing DNA encoding GPR40. ²⁺ Provided is a method for determining a ligand for GPR40, which comprises measuring a concentration increasing activity or an intracellular cAMP production inhibitory activity.
In particular, it is preferable to perform the above test after confirming that the test compound binds to GPR40.
[0060]
When cells containing GPR40 are used in the ligand determination method of the present invention, the cells may be immobilized with glutaraldehyde, formalin, or the like. The immobilization method can be performed according to a known method.
The membrane fraction of cells containing GPR40 refers to a fraction containing a large amount of cell membrane obtained by crushing the cells and then using a known method. As a method for crushing the cells, a method of crushing the cells with a Potter-Elvehjem type homogenizer, crushing with a Waring blender or a polytron (manufactured by Kinematica), crushing with an ultrasonic wave, or ejecting the cells from a thin nozzle while applying pressure by a French press or the like. Crushing and the like. For fractionation of cell membranes, fractionation by centrifugal force such as fractionation centrifugation or density gradient centrifugation is mainly used. For example, the cell lysate is centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (usually about 1 minute to 10 minutes), and the supernatant is further centrifuged at a high speed (15000 rpm to 30000 rpm) for usually 30 minutes to 2 hours. The precipitate is the membrane fraction. The membrane fraction is rich in the expressed GPR40 and membrane components such as cell-derived phospholipids and membrane proteins.
[0061]
The amount of GPR40 in cells containing GPR40 and its cell membrane fraction is 10 per cell. ³ -10 ⁸ Preferably a molecule ⁵ -10 ⁷ Preferably it is a molecule. The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which makes it possible not only to construct a highly sensitive screening system, but also to measure a large number of samples in the same lot. .
In order to carry out the method for determining a ligand of the present invention, intracellular Ca through GPR40 is required. ²⁺ The concentration increasing activity or the intracellular cAMP production inhibitory activity can be measured using a known method or a commercially available measurement kit. Specifically, first, cells containing GPR40 are cultured in a multiwell plate or the like. Prior to ligand determination, replace the cells with a fresh medium or an appropriate buffer that is not toxic to cells, add test compounds, etc., incubate for a certain period of time, and then extract cells or collect supernatant to generate The quantified product is quantified according to each method. A substance (for example, Ca ²⁺ , CAMP, etc.) when the assay is difficult due to a degrading enzyme contained in the cells, the assay may be performed by adding an inhibitor against the degrading enzyme. In addition, the activity such as cAMP production suppression can be detected as a production suppression effect on cells whose basic production amount has been increased by forskolin or the like.
The kit for ligand determination of the present invention contains cells containing GPR40 or a cell membrane fraction thereof.
Since the ligand for GPR40 determined in this way binds to GPR40 and regulates its physiological function, it can be used as a prophylactic / therapeutic agent for diseases related to GPR40 function.
[0062]
(6) A screening method for a compound (agonist, antagonist, etc.) of the present invention that alters the binding between GPR40 and a ligand, and a medicament containing the compound of the present invention that alters the binding between GPR40 and a ligand
By using the GPR40 of the present invention or constructing a recombinant GPR40 expression system and using a receptor binding assay system using the expression system, the binding between the fatty acid or eicosanoid as a ligand and the GPR40 of the present invention can be achieved. (Eg, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc.) or salts thereof can be efficiently screened.
Such compounds include (a) cell stimulating activities (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ Release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuations, intracellular protein phosphorylation, c-fos activation, pH reduction, etc. Compounds (so-called agonists to GPR40 of the present invention), (b) compounds not having the cell stimulating activity (so-called antagonists to GPR40 of the present invention), and (c) enhancing the binding force between ligand and GPR40 of the present invention. Or (d) a compound that decreases the binding force between the ligand and GPR40 of the present invention.
That is, the present invention is characterized by comparing (i) the case where the GPR40 of the present invention is brought into contact with a ligand and (ii) the case where the GPR40 of the present invention is brought into contact with a ligand and a test compound. A method for screening a compound or a salt thereof that alters the binding property between a ligand and GPR40 of the present invention is provided.
The screening method of the present invention is characterized in that, for example, the amount of ligand binding to GPR40, the cell stimulating activity and the like in the cases (i) and (ii) are measured and compared.
[0063]
More specifically, the present invention provides
a) The amount of binding of the labeled ligand to the GPR40 was measured when the labeled ligand was brought into contact with the GPR40 of the present invention and when the labeled ligand and the test compound were brought into contact with the GPR40 of the present invention, and the results were compared. A method for screening a compound or a salt thereof that alters the binding property between a ligand and GPR40 of the present invention,
b) The case where the labeled ligand is brought into contact with the cell containing the GPR40 of the present invention or the membrane fraction of the cell, and the case where the labeled ligand and the test compound are contacted with the cell containing the GPR40 of the present invention or the membrane fraction of the cell Or a salt thereof, which changes the binding between the ligand and GPR40 of the present invention, wherein the amount of binding of the labeled ligand to the cell or the membrane fraction is measured and compared when contacted with the GPR40 of the present invention. Screening method,
c) When the labeled ligand is brought into contact with GPR40 expressed on the cell membrane by culturing the transformant containing the DNA of the present invention, and when the labeled ligand and the test compound are brought into contact with the DNA containing the DNA of the present invention. When the transformant is cultured and brought into contact with the GPR40 of the present invention expressed on the cell membrane, the amount of the labeled ligand bound to the GPR40 is measured and compared with the ligand and the GPR40 of the present invention. A method for screening a compound or a salt thereof that changes the binding property of
[0064]
d) A compound that activates GPR40 of the present invention (for example, a ligand for GPR40 of the present invention) and a compound that activates GPR40 of the present invention, GPR40-mediated cell stimulating activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ Release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuations, intracellular protein phosphorylation, c-fos activation, pH reduction, etc. A method for screening a compound or a salt thereof that alters the binding property between the ligand and GPR40 of the present invention, which is measured and compared, and
e) A compound that activates GPR40 of the present invention (eg, a ligand for GPR40 of the present invention) is brought into contact with GPR40 of the present invention expressed on the cell membrane by culturing a transformant containing the DNA of the present invention. Receptor protein when the compound activating GPR40 of the present invention and a test compound are brought into contact with GPR40 of the present invention expressed on a cell membrane by culturing a transformant containing the DNA of the present invention. Cell stimulating activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ Release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuations, intracellular protein phosphorylation, c-fos activation, pH reduction, etc. A method for screening a compound or a salt thereof that changes the binding property between a ligand and GPR40 of the present invention, which is characterized by measuring and comparing the ligand, is provided.
As the ligand, the above-mentioned fatty acid or eicosanoid is used.
Further, as the ligand, a compound or a salt thereof that changes the binding property between fatty acid or eicosanoid and GPR40 can also be used. The compound or a salt thereof that changes the binding property between the fatty acid or eicosanoid and GPR40 can be obtained, for example, by using the fatty acid or eicosanoid as a ligand and performing the scooning method of the present invention described later.
As the compound or a salt thereof that changes the binding property between fatty acid or eicosanoid and GPR40, a low-molecular-weight synthetic compound is preferable, and any novel or known compound may be used. In particular, when screening for a GPR40 antagonist, it is preferable to use a low-molecular-weight synthetic compound having agonist activity instead of a natural ligand.
Low-molecular-weight synthetic compounds are suitable for screening because they are easier to label than natural ligands.
[0065]
The specific description of the screening method of the present invention is as follows.
First, as the GPR40 of the present invention used in the screening method of the present invention, any GPR40 containing the above-described GPR40 of the present invention may be used. Are preferred. However, since it is particularly difficult to obtain human-derived organs, human-derived GPR40 and the like, which are expressed in large amounts using recombinants, are suitable for screening.
[0066]
The above-mentioned method is used for producing the GPR40 of the present invention, but it is preferably carried out by expressing the DNA of the present invention in mammalian cells or insect cells. A complementary DNA is used as the DNA fragment encoding the target protein portion, but is not necessarily limited thereto. For example, a gene fragment or a synthetic DNA may be used. In order to introduce the DNA fragment encoding GPR40 of the present invention into host animal cells and express them efficiently, the DNA fragment is converted into a nuclear polyhedrosis virus belonging to a baculovirus using an insect as a host. It is preferable to incorporate the promoter into the downstream of the polyhedrin promoter of NPV), the promoter derived from SV40, the retrovirus promoter, the metallothionein promoter, the human heat shock promoter, the cytomegalovirus promoter, the SRα promoter and the like. The amount and quality of the expressed receptor protein can be examined by a method known per se. For example, the literature [Nambi, P .; Et al., The Journal of Biological Chemistry (J. Biol. Chem.), 267, 19555-19559, 1992].
Therefore, in the screening method of the present invention, the GPR40 containing the GPR40 of the present invention may be GPR40 purified according to a method known per se, or a cell containing the GPR40 may be used. A membrane fraction of cells containing GPR40 may be used.
[0067]
When cells containing the GPR40 of the present invention are used in the screening method of the present invention, the cells may be immobilized with glutaraldehyde, formalin, or the like. The immobilization method can be performed according to a method known per se.
The cell containing the GPR40 of the present invention refers to a host cell that has expressed the GPR40, and the host cell is preferably Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, and the like.
The cell membrane fraction refers to a fraction abundant in cell membrane obtained by disrupting cells and then obtained by a method known per se. As a method for crushing the cells, a method of crushing the cells with a Potter-Elvehjem type homogenizer, crushing with a Waring blender or a polytron (manufactured by Kinematica), crushing with an ultrasonic wave, or squirting the cells from a thin nozzle while applying pressure by a French press or the like. Crushing and the like. For fractionation of cell membranes, fractionation by centrifugal force such as fractionation centrifugation or density gradient centrifugation is mainly used. For example, the cell lysate is centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (usually about 1 minute to 10 minutes), and the supernatant is further centrifuged at a high speed (15000 rpm to 30000 rpm) for usually 30 minutes to 2 hours. The precipitate is the membrane fraction. The membrane fraction is rich in the expressed GPR40 and membrane components such as cell-derived phospholipids and membrane proteins.
The amount of GPR40 in the cells containing GPR40 and in the membrane fraction is 10 per cell. ³ -10 ⁸ Preferably a molecule ⁵ -10 ⁷ Preferably it is a molecule. The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which makes it possible not only to construct a highly sensitive screening system, but also to measure a large number of samples in the same lot. .
[0068]
In order to carry out the above-mentioned a) to c) for screening a compound that changes the binding property of the ligand to GPR40 of the present invention, for example, an appropriate GPR40 fraction and a labeled ligand are required.
As the GPR40 fraction, a natural GPR40 fraction or a recombinant GPR40 fraction having an activity equivalent thereto is desirable. Here, “equivalent activity” refers to equivalent ligand binding activity, signal transduction action, and the like.
As the labeled ligand, a labeled ligand, a labeled ligand analog compound, or the like is used. For example, [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] or the like.
Specifically, to screen for a compound that alters the binding between a ligand and GPR40 of the present invention, cells containing the GPR40 of the present invention or a membrane fraction of the cells are suspended in a buffer suitable for screening. Prepare a GPR40 standard by turbidity. The buffer may be any buffer, such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a buffer such as Tris-HCl buffer, which does not inhibit the binding between the ligand and GPR40. Further, in order to reduce non-specific binding, CHAPS, Tween-80 are used. ^TM Surfactants such as (Kao-Atlas), digitonin and deoxycholate can also be added to the buffer. Furthermore, a protease inhibitor such as PMSF, leupeptin, E-64 (manufactured by Peptide Research Laboratories) and pepstatin can be added for the purpose of suppressing the degradation of the receptor protein or ligand by the protease. A fixed amount (5000 cpm to 500,000 cpm) of a labeled ligand is added to 0.01 ml to 10 ml of the receptor protein solution, and 10 ^-4 M-10 ^-10 M test compounds are allowed to coexist. A reaction tube containing a large excess of unlabeled ligand is also prepared to determine the amount of non-specific binding (NSB). The reaction is carried out at about 0-50 ° C, preferably about 4-37 ° C, for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours. After the reaction, the mixture is filtered with a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter or a γ-counter. Count when there is no antagonist (B ₀ ) Minus the amount of non-specific binding (NSB) (B ₀ -NSB) as 100%, a test compound having a specific binding amount (B-NSB) of, for example, 50% or less can be selected as a candidate substance having competitive inhibitory ability.
[0069]
In order to carry out the above-mentioned methods d) to e) of screening for a compound that changes the binding property between the ligand and GPR40 of the present invention, for example, GPR40-mediated cell stimulating activity (for example, arachidonic acid release, acetylcholine release) , Intracellular Ca ²⁺ In particular, the activity of promoting or inhibiting release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, fluctuation of cell membrane potential, phosphorylation of intracellular protein, activation of c-fos, decrease of pH, etc. Intracellular Ca ²⁺ Concentration increasing activity, intracellular cAMP production inhibitory activity) can be measured using a known method or a commercially available measurement kit.
Specifically, cells containing the GPR40 of the present invention are first cultured in a multiwell plate or the like. Before performing screening, the cells were exchanged with a fresh medium or an appropriate buffer that does not show toxicity to cells, and a test compound was added and incubated for a certain period of time. The product is quantified according to the respective method. A substance (for example, Ca ²⁺ , CAMP, etc.) when the assay is difficult due to a degrading enzyme contained in the cells, the assay may be performed by adding an inhibitor against the degrading enzyme. In addition, the activity such as cAMP production suppression can be detected as a production suppression effect on cells whose basic production amount has been increased by forskolin or the like.
In order to perform screening by measuring the cell stimulating activity, cells expressing appropriate GPR40 are required. As the cells expressing the GPR40 of the present invention, a cell line having the natural GPR40 of the present invention, a cell line expressing the above-mentioned recombinant GPR40 and the like are desirable.
Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound.
As the test compound, a compound designed to bind to the ligand binding pocket based on the atomic coordinates of the active site of GPR40 and the position of the ligand binding pocket is preferably used. The measurement of the atomic coordinates of the active site of GPR40 and the position of the ligand binding pocket can be performed by a known method or a method analogous thereto.
A specific method for evaluating whether the compound that changes the binding property between the ligand and GPR40 of the present invention is an agonist or an antagonist may be according to the following (i) or (ii).
(I) After performing the screening method of the above a) to c) to obtain a compound or a salt thereof that changes (particularly, inhibits the binding) between the ligand and GPR40 of the present invention, and then obtains the compound or a salt thereof Is determined whether it has the above-mentioned cell stimulating activity. Specifically, it can be confirmed using the above-described method for screening an agonist for GPR40 of the present invention, wherein the compound having the cell stimulating activity or a salt thereof is an agonist and the compound having no cell stimulating activity or a compound thereof Salts are antagonists.
(Ii) (a) A test compound is brought into contact with cells containing the GPR40 of the present invention, and the above-mentioned cell stimulating activity is measured. A test compound having cell stimulating activity is an agonist for GPR40 of the present invention.
(B) When a compound (for example, a ligand) that activates GPR40 of the present invention is brought into contact with cells containing the GPR40 of the present invention, a compound that activates GPR40 of the present invention and a test compound are compared with the GPR40 of the present invention. The cell stimulating activity when contacted with cells containing is measured and compared. A test compound capable of reducing the cell stimulating activity of the compound activating GPR40 of the present invention is an antagonist to GPR40 of the present invention.
More specifically, the evaluation criteria described in Example 35 can be used.
[0070]
The screening kit for a compound or a salt thereof that alters the binding property between the ligand and the GPR40 of the present invention comprises a GPR40 of the present invention, a cell containing the GPR40 of the present invention, or a membrane fraction of a cell containing the GPR40 of the present invention. And the like.
Examples of the screening kit of the present invention include the following.
1. Screening reagent
a) Measurement buffer and washing buffer
Hanks' Balanced Salt Solution (manufactured by Gibco) plus 0.05% bovine serum albumin (manufactured by Sigma).
The solution may be sterilized by filtration through a 0.45 μm filter and stored at 4 ° C., or may be prepared at use.
b) GPR40 standard
CHO cells expressing the GPR40 of the present invention were placed in a 12-well plate at 5 × 10 5 ⁵ Passage at 37 ° C, 5% CO ₂ , Cultured at 95% air for 2 days.
c) Labeled ligand
Commercially available [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S]
The aqueous solution is stored at 4 ° C. or −20 ° C., and diluted to 1 μM with a measuring buffer before use.
d) Ligand standard solution
The ligand is dissolved in PBS containing 0.1% bovine serum albumin (manufactured by Sigma) to a concentration of 1 mM and stored at -20 ° C.
[0071]
2. Measurement method
a) GPR40-expressing CHO cells of the present invention cultured in a 12-well tissue culture plate are washed twice with 1 ml of the measurement buffer, and 490 μl of the measurement buffer is added to each well.
b) 10 ^-3 -10 ^-10 After adding 5 μl of the M test compound solution, 5 μl of the labeled ligand is added, and reacted at room temperature for 1 hour. To determine the amount of non-specific binding, 10 ^-3 Add 5 μl of M ligand.
c) Remove the reaction solution and wash three times with 1 ml of washing buffer. The labeled ligand bound to the cells is dissolved in 0.2N NaOH-1% SDS, and mixed with 4 ml of liquid scintillator A (manufactured by Wako Pure Chemical Industries, Ltd.).
d) Radioactivity is measured using a liquid scintillation counter (manufactured by Beckman), and Percent Maximum Binding (PMB) is determined by the following equation.
PMB = [(B-NSB) / (B ₀ −NSB)] × 100
PMB: Percent Maximum Binding
B: Value when the sample was added
NSB: Non-specific Binding (Non-specific binding amount)
B ₀ : Maximum binding amount
[0072]
The compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a compound having an action of changing the binding property between the ligand and GPR40 of the present invention. Specifically, (A) G protein A compound having a cell stimulating activity via a coupled receptor protein (a so-called agonist to GPR40 of the present invention), (b) a compound having no such cell stimulating activity (a so-called antagonist to GPR40 of the present invention), (c) a ligand And (d) a compound that decreases the binding force between the ligand and the G protein-coupled GPR40 of the present invention.
Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, and a fermentation product. These compounds may be a novel compound or a known compound.
The compound may be a compound designed based on the atomic coordinates of the active site of GPR40 and the position of the ligand binding pocket.
Since the agonist for GPR40 of the present invention has the same action as the physiological activity of the fatty acid or eicosanoid which is the ligand for GPR40 of the present invention, it is a safe and low toxic drug according to the physiological activity of the fatty acid or eicosanoid. Useful as
Since the antagonist to GPR40 of the present invention can suppress the physiological activity of fatty acid or eicosanoid which is the ligand for GPR40 of the present invention, it is useful as a safe and low toxic drug for suppressing the physiological activity of fatty acid or eicosanoid. It is.
Since the compound that enhances the binding force between the ligand and the G protein-coupled GPR40 of the present invention can enhance the physiological activity of the ligand for GPR40 of the present invention, it is safe and safe depending on the physiological activity of the fatty acid or eicosanoid. It is useful as a low toxic drug.
Since the compound that decreases the binding force between the ligand and the G protein-coupled GPR40 of the present invention can reduce the physiological activity of the ligand for GPR40 of the present invention, it is safe to suppress the physiological activity of fatty acids or eicosanoids. And is useful as a low-toxicity drug.
Specifically, a compound or a salt thereof that enhances the binding force between (1) an agonist for GPR40 or (2) a ligand obtained using the screening method or the screening kit of the present invention and a G protein-coupled GPR40 of the present invention. For example, diabetes, glucose intolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis It is useful as a prophylactic / therapeutic agent for thrombotic diseases, dyspepsia, memory learning disorders, etc., pancreatic function regulators (eg, pancreatic function improving agents), insulin secretagogues, hypoglycemic agents, pancreatic β-cell protective agents is there. Diabetes includes insulin dependent (type I) diabetes, non-insulin dependent (type II) diabetes and the like.
A compound or a salt thereof that reduces the binding force between (1) an antagonist to GPR40 or (2) a ligand obtained by using the screening method or the screening kit of the present invention and a G protein-coupled GPR40 of the present invention is, for example, obese. , Hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance syndrome, unstable diabetes, lipoatrophy, insulin allergy, insulinoma, It is useful as a prophylactic / therapeutic agent for diseases such as arteriosclerosis, thrombotic disease, lipotoxicity, and cancer, a pancreatic function regulator (eg, a pancreatic function improving agent), an insulin secretion inhibitor, and a blood sugar increasing agent.
[0073]
Compounds or salts thereof obtained by using the screening method or the screening kit of the present invention are compounds or salts thereof that alter the expression level of GPR40 described later, other drugs against the above-mentioned diseases, other therapeutic agents for diabetes, diabetes, To be used in combination with drugs such as sexual complications, hyperlipidemia, antihypertensives, antiobesity agents, diuretics, chemotherapeutics, and immunotherapy (hereinafter sometimes abbreviated as concomitant drugs) Can be. At this time, the administration time of the compound or its salt obtained by using the screening method or the screening kit of the present invention and the concomitant drug is not limited, and these may be administered simultaneously to the subject to be administered, or with a time lag. May be administered. The dose of the concomitant drug can be appropriately selected based on the clinically used dose. In addition, the compounding ratio of the compound or its salt obtained by using the screening method or the screening kit of the present invention and the concomitant drug can be appropriately selected depending on the administration subject, administration route, target disease, symptom, combination and the like. For example, when the administration subject is a human, for example, 0.01 to 100 parts by weight of the concomitant drug may be used for 1 part by weight of the agonist.
Other antidiabetic agents include insulin preparations (eg, animal insulin preparations extracted from bovine and porcine pancreas; human insulin preparations genetically synthesized using Escherichia coli and yeast; insulin zinc; protamine insulin zinc; insulin) Or an insulin sensitizer (eg, pioglitazone hydrochloride, troglitazone, rosiglitazone or its maleate, JTT-501, MCC-555, YM-440, GI-262570) , KRP-297, FK-614, CS-011, etc.), α-glucosidase inhibitors (eg, voglibose, acarbose, miglitol, emiglitate, etc.), biguanides (eg, phenformin, metformin, buformin, etc.), sulfonylureas (eg, For example, Rubutamide, glibenclamide, gliclazide, chlorpropamide, tolazamide, acetohexamide, glyclopyramide, glimepiride and the like and other insulin secretagogues (eg, repaglinide, senaglinide, mitiglinide or its calcium salt hydrate, GLP-1, nateglinide) Etc.), dipeptidyl peptidase IV inhibitors (eg, NVP-DPP-278, PT-100, P32 / 98 etc.), β3 agonists (eg, CL-316243, SR-58611-A, UL-TG-307, AJ) -9677, AZ40140, etc.), amylin agonists (eg, pramlintide etc.), phosphotyrosine phosphatase inhibitors (eg, vanadic acid etc.), gluconeogenesis inhibitors (eg, glycogen phosphorylase inhibitors, glucose-6-phosphatase inhibitors, Rukagon antagonists etc.), SGLT (sodium-glucose cotransporter) inhibitors (e.g., T-1095 etc.) and the like.
Examples of therapeutic agents for diabetic complications include aldose reductase inhibitors (eg, tolrestat, epalrestat, zenarestat, zopolrestat, fidarestat (SNK-860), minalrestat (ARI-509), CT-112, etc.) Trophic factors (eg, NGF, NT-3, protein kinase C (PKC) inhibitors (eg, LY-333531, etc.)), AGE inhibitors (eg, ALT-945, pimagedin, pyratoxatin, N-phenacylthiazo) Lium bromide (ALT-766, EXO-226, etc.), active oxygen scavengers (eg, thioctic acid), cerebral vasodilators (eg, thioprid, etc.) and the like.
[0074]
Examples of anti-hyperlipidemic agents include statin compounds that are cholesterol synthesis inhibitors (eg, pravastatin, simvastatin, lovastatin, atorvastatin, fluvastatin, cerivastatin or salts thereof (eg, sodium salt, etc.)), squalene synthase inhibitor And fibrate compounds having a triglyceride lowering action (eg, bezafibrate, clofibrate, simfibrate, clinofibrate, etc.).
Antihypertensives include angiotensin converting enzyme inhibitors (eg, captopril, enalapril, delapril, etc.), angiotensin II antagonists (eg, losartan, candesartan, cilexetil, etc.), calcium antagonists (eg, manidipine, nifedipine, amlodipine, efonidipine, Nicardipine), clonidine and the like.
Examples of anti-obesity agents include central anti-obesity agents (eg, dexfenfluamine, fenfluramine, phentermine, sibutramine, ampepramone, dexamphetamine, mazindol, phenylpropanolamine, clobenzolex, etc.), pancreatic lipase inhibition Drugs (eg, orlistat, ATL-962, etc.), β3 agonists (eg, CL-316243, SR-58611-A, UL-TG-307, AJ-9677, AZ40140, etc.), peptidic appetite suppressants (eg, leptin) , CNTF (ciliary neurotrophic factor), etc.), cholecystokinin agonists (eg, lynch tryp, FPL-15849, etc.) and the like.
Examples of the diuretic include xanthine derivatives (eg, sodium theobromine salicylate, calcium theobromine salicylate, etc.), thiazide-based preparations (eg, ethiazide, cyclopentiazide, trichloromethiazide, hydrochlorothiazide, hydroflumethiazide, benzylhydrochlorothiazide, penflutizide, polythiazide, Methiclothiazide), anti-aldosterone preparations (eg, spironolactone, triamterene, etc.), carbonic anhydrase inhibitors (eg, acetazolamide, etc.), chlorobenzenesulfonamide preparations (eg, chlorthalidone, mefluside, indapamide, etc.), azosemide, isosorbide, Examples include ethacrynic acid, piretanide, bumetanide, furosemide and the like.
[0075]
Examples of chemotherapeutic agents include alkylating agents (eg, cyclophosphamide, ifosfamide, etc.), antimetabolites (eg, methotrexate, 5-fluorouracil, etc.), anticancer antibiotics (eg, mitomycin, adriamycin, etc.), Examples include plant-derived anticancer agents (eg, vincristine, vindesine, taxol, etc.), cisplatin, carboplatin, etopoxide and the like. Among them, a 5-fluorouracil derivative such as furturon or neofurturon is preferred.
Examples of the immunotherapeutic agent include microorganisms or bacterial components (eg, muramyl dipeptide derivatives, picibanil, etc.), polysaccharides having immunopotentiating activity (eg, lentinan, schizophyllan, krestin, etc.), cytokines obtained by genetic engineering techniques ( Examples include interferon, interleukin (IL), etc., and colony stimulating factors (eg, granulocyte colony stimulating factor, erythropoietin, etc.), among which IL-1, IL-2, IL-12 and the like are preferable.
Furthermore, drugs that have been shown to have an effect of improving cachexia in animal models and clinically, namely, cyclooxygenase inhibitors (eg, indomethacin etc.) [Cancer Research, Vol. 49, pp. 5935-5939, 1989 ], Progesterone derivatives (eg, megesterol acetate) [Journal of Clinical Oncology, Vol. 12, 213-225, 1994], carbohydrate steroids (eg, dexamethasone, etc.), metoclopramide Drugs, tetrahydrocannabinol drugs (the literature is the same as above), fat metabolism improvers (eg, eicosapentaenoic acid, etc.) [British Journal of Cancer (British Journal) f Cancer), 68, 314-318, 1993], antibodies against growth hormone, IGF-1, or TNF-α, LIF, IL-6, oncostatin M, which are factors that induce cachexia, and the like. Can also be used in combination with the formulation of the present invention.
Furthermore, glycation inhibitors (eg, ALT-711 etc.), nerve regeneration promoters (eg, Y-128, VX853, prosaptide, etc.), antidepressants (eg, desipramine, amitriptyline, imipramine), antiepileptic drugs (eg, Lamotrigine), antiarrhythmic drugs (eg, mexiletine), acetylcholine receptor ligands (eg, ABT-594), endothelin receptor antagonists (eg, ABT-627), monoamine uptake inhibitors (eg, tramadol), narcotic analgesics Drug (eg, morphine), GABA receptor agonist (eg, gabapentin), α2 receptor agonist (eg, clonidine), local analgesic (eg, capsaicin), anxiolytic (eg, benzothiazepine), phosphodiesterase Inhibitors (eg, sildenafil), dopamine receptor agonists (eg, apomorphine) It can be used in combination with the formulation.
[0076]
When the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned pharmaceutical composition, it can be formulated according to a conventional method.
For example, the compound may be a sugar-coated tablet, capsule, elixir, microcapsule, or the like, as needed, orally, or a sterile solution of water or other pharmaceutically acceptable liquid, Alternatively, they can be used parenterally in the form of injections such as suspensions. For example, the compound may be mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like, in a unit dosage form required for generally accepted pharmaceutical practice. Can be manufactured by The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
Examples of additives that can be incorporated into tablets, capsules, and the like include binders such as gelatin, corn starch, tragacanth, and acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Useful bulking agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry and the like are used. When the unit dosage form is a capsule, a liquid carrier such as oils and fats can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80) ^TM , HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
[0077]
The prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled in a suitable ampoule.
The preparations obtained in this way are safe and have low toxicity, and should be administered to humans and mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Can be.
The dose of the compound or a salt thereof varies depending on the administration subject, target organ, condition, administration method and the like. In the case of oral administration, for example, in a diabetic patient (60 kg), for example, about 0.1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, condition, administration method, and the like. For example, usually in the form of an injection, for example, in a diabetic patient (as 60 kg), It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the amount can be administered in terms of 60 kg.
[0078]
(7) Elucidation of the mechanism of action of various drugs
By using the GPR40 of the present invention, it is possible to confirm whether or not various drugs exert a pharmacological effect via the GPR40.
That is, the present invention
(1) GPR40 is used, (1) pancreatic function modulator (eg, pancreatic function improving drug), (2) diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy , Diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis, thrombotic disease, indigestion or memory learning prevention / treatment agent, (3) promotion of insulin secretion Drugs, (4) hypoglycemic drugs, (5) pancreatic beta cell protective drugs or (6) obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic The prevention or treatment of retinopathy, edema, insulin resistance syndrome, unstable diabetes, lipoatrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity or cancer binds to the receptor protein or a salt thereof. Sure How to,
(2) Use of GPR40, (1) diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin Disease, arthropathy, osteopenia, arteriosclerosis, thrombotic disease, prophylactic / therapeutic agent for dyspepsia or memory learning disorder, (2) insulin secretagogue, (3) hypoglycemic agent, or (4) protection of pancreatic β-cell Is an agonist for the receptor protein or a salt thereof,
(3) using GPR40, (1) obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin A drug for preventing or treating resistance syndrome, unstable diabetes, lipoatrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity or cancer, (2) an insulin secretion inhibitor or (3) a blood glucose increasing drug A method for confirming that the protein or its salt is an antagonist,
(4) The screening method according to any one of (1) to (3), wherein the amount of binding between each drug and GPR40 when each drug is brought into contact with GPR40 is measured.
This confirmation method can be carried out by using the above-mentioned drug in place of the test compound in the above-mentioned method for screening a compound that changes the binding property between a ligand and GPR40.
Further, the kit for a confirmation method of the present invention is a kit for screening a compound that changes the binding property between the ligand and GPR40, which contains the above drug in place of the test compound.
As described above, by using the confirmation method of the present invention, it is possible to confirm that various drugs that are commercially available or in development are exhibiting pharmacological effects via GPR40.
[0079]
(8) A medicine containing a compound that changes the amount of GPR40 of the present invention or a partial peptide thereof in a cell membrane
Since the antibody of the present invention can specifically recognize the GPR40 of the present invention, it can be used for screening a compound that changes the amount of the GPR40 of the present invention in a cell membrane.
That is, the present invention, for example,
(I) a) the blood of a non-human mammal, b) a specific organ, c) the tissue or cells isolated from the organ are destroyed, and then the cell membrane fraction is isolated, and the present invention contained in the cell membrane fraction is isolated. A method for screening a compound that changes the amount of GPR40 of the present invention in a cell membrane by quantifying GPR40,
(Ii) The amount of the GPR40 of the present invention in the cell membrane by disrupting the transformant or the like expressing the GPR40 of the present invention, isolating the cell membrane fraction, and quantifying the GPR40 of the present invention contained in the cell membrane fraction. Screening method for a compound that changes
(Iii) a) the blood of a non-human mammal, b) a specific organ, c) a tissue or a cell isolated from the organ, and then use the immunostaining method to obtain the receptor on the cell surface. Provided is a method for screening a compound that changes the amount of GPR40 of the present invention in a cell membrane by confirming the protein on the cell membrane by quantifying the degree of staining of the protein.
(Iv) After transforming the GPR40-expressing transformant of the present invention or the like as a section, the degree of staining of the receptor protein on the cell surface is quantified by using an immunostaining method. A method for screening a compound that changes the amount of GPR40 of the present invention in a cell membrane by confirming a protein is provided.
[0080]
The GPR40 of the present invention contained in the cell membrane fraction is specifically quantified as follows.
(I) Normal or disease model non-human mammals (eg, mice, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc., more specifically, dementia rats, obese mice, arteriosclerotic rabbits, cancer-bearing A drug (eg, an anti-dementia drug, a blood pressure lowering drug, an anti-cancer drug, an anti-obesity drug, etc.) or a physical stress (eg, waterlogging stress, electric shock, light / dark, low temperature, etc.), etc. After the passage, blood or a specific organ (eg, brain, liver, kidney, etc.), or a tissue or cell isolated from the organ is obtained. The obtained organ, tissue or cell is suspended in, for example, an appropriate buffer (eg, Tris-HCl buffer, phosphate buffer, Hepes buffer, etc.), and the organ, tissue or cell is destroyed. Activator (eg, Triton X100 ^TM , Tween 20 ^TM And the like, and further using a method such as centrifugation, filtration, or column fractionation to obtain a cell membrane fraction.
The cell membrane fraction refers to a fraction abundant in cell membrane obtained by disrupting cells and then obtained by a method known per se. As a method for crushing the cells, a method of crushing the cells with a Potter-Elvehjem type homogenizer, crushing with a Waring blender or a polytron (manufactured by Kinematica), crushing with an ultrasonic wave, or squirting the cells from a thin nozzle while applying pressure by a French press or the like. Crushing and the like. For fractionation of cell membranes, fractionation by centrifugal force such as fractionation centrifugation or density gradient centrifugation is mainly used. For example, the cell lysate is centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (usually about 1 minute to 10 minutes), and the supernatant is further centrifuged at a high speed (15000 rpm to 30000 rpm) for usually 30 minutes to 2 hours. The precipitate is the membrane fraction. The membrane fraction is rich in the expressed GPR40 and membrane components such as cell-derived phospholipids and membrane proteins.
GPR40 of the present invention contained in the cell membrane fraction can be quantified by, for example, a sandwich immunoassay using the antibody of the present invention, Western blot analysis, or the like.
Such a sandwich immunoassay can be performed in the same manner as described above, and the Western blot can be performed by a means known per se.
(Ii) A transformant expressing the GPR40 of the present invention is prepared according to the above method, and the GPR40 of the present invention contained in the cell membrane fraction can be quantified.
[0081]
Screening for a compound that alters the amount of GPR40 of the present invention in the cell membrane comprises:
(I) A given time (30 minutes to 24 hours, preferably 30 minutes to 12 hours, more preferably 1 hour) before giving a drug or physical stress to a normal or disease model non-human mammal Before to 6 hours before) or after a certain time (after 30 minutes to 3 days, preferably after 1 hour to 2 days, more preferably after 1 hour to 24 hours), or at the same time as the drug or physical stress. Is administered, and after a lapse of a certain time after the administration (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), the amount of the GPR40 of the present invention in the cell membrane is determined. Can be done by
(Ii) When culturing the transformant according to a conventional method, the test compound is mixed in the medium, and after culturing for a certain period of time (1 day to 7 days, preferably 1 day to 3 days, more preferably 2 days to 3 days) Days later), by quantifying the amount of GPR40 of the present invention in the cell membrane.
The GPR40 of the present invention contained in the cell membrane fraction is specifically confirmed as follows.
(Iii) Normal or disease model non-human mammals (eg, mice, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc., more specifically, dementia rats, obese mice, arteriosclerotic rabbits, cancer-bearing A drug (eg, an anti-dementia drug, a blood pressure lowering drug, an anti-cancer drug, an anti-obesity drug, etc.) or a physical stress (eg, waterlogging stress, electric shock, light / dark, low temperature, etc.), etc. After the passage, blood or a specific organ (eg, brain, liver, kidney, etc.), or a tissue or cell isolated from the organ is obtained. The obtained organ, tissue or cell is cut into a tissue section according to a conventional method, and immunostaining is performed using the antibody of the present invention. By quantifying the degree of staining of the receptor protein on the cell surface, and confirming the protein on the cell membrane, the amount of GPR40 of the present invention in the cell membrane can be quantitatively or qualitatively confirmed. .
(Iv) The same can be confirmed by using a transformant expressing the GPR40 of the present invention or the like and performing the same procedure.
[0082]
The compound obtained by using the screening method of the present invention or a salt thereof is a compound having an action of changing the amount of the GPR40 of the present invention in the cell membrane, and specifically, (a) the amount of the GPR40 of the present invention in the cell membrane To increase cell-stimulating activity through G protein-coupled receptor protein (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ Release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuations, intracellular protein phosphorylation, c-fos activation, pH reduction, etc. (B) A compound that attenuates the cell stimulating activity by decreasing the amount of GPR40 of the present invention in the cell membrane.
Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, and a fermentation product. These compounds may be a novel compound or a known compound.
The compound of the present invention which enhances the cell stimulating activity by increasing the amount of GPR40 of the present invention in the cell membrane is a safe and low-toxic preventive / therapeutic agent for a disease associated with dysfunction of GPR40 of the present invention, a pancreatic function regulator ( For example, it is useful as an agent for improving pancreatic function), an insulin secretagogue, a hypoglycemic agent, and a pancreatic β-cell protective agent.
The compound of the present invention which reduces the cell stimulating activity by decreasing the amount of GPR40 of the present invention in the cell membrane is a safe and low toxic prophylactic / therapeutic agent for a disease caused by overexpression of GPR40 of the present invention, a pancreatic function regulator ( For example, it is useful as an agent for improving pancreatic function), an insulin secretion inhibitor, and a blood sugar increasing agent.
Specifically, the compound or a salt thereof that increases the amount of GPR40 of the present invention includes, for example, diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, and hyperlipidemia. It can be used as a prophylactic / therapeutic agent for diseases such as dysfunction, sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis, thrombotic disease, dyspepsia, and memory learning disorder. Diabetes includes insulin dependent (type I) diabetes, non-insulin dependent (type II) diabetes and the like.
Compounds or salts thereof that decrease the amount of GPR40 of the present invention include, for example, obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema It can be used as a prophylactic / therapeutic agent for diseases such as insulin resistance syndrome, unstable diabetes, lipoatrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity, and cancer.
Further, it can be used in combination with the above-mentioned concomitant drug.
When a compound or a salt thereof obtained by using the screening method of the present invention is used as a pharmaceutical composition, it can be formulated according to a conventional method.
For example, the compound may be a sugar-coated tablet, capsule, elixir, microcapsule, or the like, as needed, orally, or a sterile solution of water or other pharmaceutically acceptable liquid, Alternatively, they can be used parenterally in the form of injections such as suspensions. For example, the compound may be mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like, in a unit dosage form required for generally accepted pharmaceutical practice. Can be manufactured by The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
[0083]
Examples of additives that can be incorporated into tablets, capsules, and the like include binders such as gelatin, corn starch, tragacanth, and acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Useful bulking agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry and the like are used. When the unit dosage form is a capsule, a liquid carrier such as oils and fats can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80) ^TM , HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
The prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled in a suitable ampoule.
The preparations obtained in this way are safe and have low toxicity, and should be administered to humans and mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Can be.
The dose of the compound or a salt thereof varies depending on the administration subject, target organ, condition, administration method and the like. In the case of oral administration, for example, in a diabetic patient (60 kg), for example, about 0.1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, condition, administration method, and the like. For example, usually in the form of an injection, for example, in a diabetic patient (as 60 kg), It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the amount can be administered in terms of 60 kg.
[0084]
(9) A medicine comprising the antibody against GPR40 of the present invention
The neutralizing activity of the antibody against GPR40 of the present invention means an activity of inactivating a signal transduction function involving GPR40. Therefore, when the antibody has a neutralizing activity, signal transduction involving the GPR40, for example, a cell stimulating activity via the GPR40 (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ Release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuations, intracellular protein phosphorylation, c-fos activation, pH reduction, etc. Can be inactivated.
Therefore, the neutralizing antibody against GPR40 of the present invention can be used for diseases caused by overexpression of GPR40 (eg, obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy) Prophylactic / therapeutic agents for diabetic retinopathy, edema, insulin resistance syndrome, unstable diabetes, lipoatrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic diseases, lipotoxicity, cancer, etc., pancreatic function regulators (eg , A pancreatic function improving agent), an insulin secretion inhibitor, and a blood sugar increasing agent.
Further, it can be used in combination with the above-mentioned concomitant drug.
[0085]
(10) A medicine comprising the antisense DNA or siRNA of the present invention
The antisense DNA or siRNA of the present invention may be used for diseases caused by overexpression of GPR40 (eg, obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetes) Agents for the prevention and treatment of retinopathy, edema, insulin resistance syndrome, unstable diabetes, lipoatrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic diseases, lipotoxicity, cancer, etc., pancreatic function regulators (eg, pancreas) Function improving agent), an insulin secretion inhibitor, and a blood sugar increasing agent.
Further, it can be used in combination with the above-mentioned concomitant drug.
For example, when using the antisense DNA or siRNA, the antisense DNA or siRNA is used alone or inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like, followed by a conventional method. be able to. The antisense DNA can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and administered with a gene gun or a catheter such as a hydrogel catheter.
Furthermore, the antisense DNA can also be used as a diagnostic oligonucleotide probe for examining the presence of the DNA of the present invention in tissues or cells and the state of expression thereof.
[0086]
(11) Preparation of the DNA-introduced animal of the present invention
The present invention relates to a non-human mammal having a foreign DNA of the present invention (hereinafter abbreviated as the foreign DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the foreign mutant DNA of the present invention). provide.
That is, the present invention
[1] a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof,
[2] the animal of [1], wherein the non-human mammal is a rodent;
[3] the animal of [2], wherein the rodent is a mouse or a rat, and
[4] It is to provide a recombinant vector containing the exogenous DNA of the present invention or its mutant DNA and capable of being expressed in mammals.
Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA-transferred animal of the present invention) can be used for non-fertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like. And preferably at the stage of embryonic development in non-human mammal development (more preferably at the stage of single cells or fertilized eggs and generally before the 8-cell stage), the calcium phosphate method, the electric pulse method, the lipofection method, the agglutination method, It can be produced by transferring a target DNA by a microinjection method, a particle gun method, a DEAE-dextran method, or the like. In addition, the exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like by the DNA transfer method, and can be used for cell culture, tissue culture, and the like. The DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned germ cells with a cell fusion method known per se.
As the non-human mammal, for example, cow, pig, sheep, goat, rabbit, dog, cat, guinea pig, hamster, mouse, rat and the like are used. Among them, rodents, which are relatively short in ontogeny and biological cycle in terms of preparation of disease animal model systems, and are easy to breed, especially mice (for example, hybrids such as C57BL / 6 strain and DBA2 strain as pure strains) B6C3F as a system ₁ System, BDF ₁ System, B6D2F ₁ Strain, BALB / c strain, ICR strain, etc.) or rat (eg, Wistar, SD etc.) is preferred.
"Mammals" in a recombinant vector that can be expressed in mammals include humans in addition to the above-mentioned non-human mammals.
[0087]
The exogenous DNA of the present invention refers not to the DNA of the present invention originally possessed by non-human mammals, but to the DNA of the present invention once isolated and extracted from the mammal.
As the mutant DNA of the present invention, those having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, base addition, deletion, substitution with another base, etc. The resulting DNA and the like are used, and also include abnormal DNA.
The abnormal DNA refers to a DNA that expresses the abnormal GPR40 of the present invention, and for example, a DNA that expresses GPR40 that suppresses the function of the normal GPR40 of the present invention is used.
The exogenous DNA of the present invention may be derived from a mammal that is the same or different from the animal of interest. In transferring the DNA of the present invention to a target animal, it is generally advantageous to use the DNA as a DNA construct linked downstream of a promoter capable of being expressed in animal cells. For example, when the human DNA of the present invention is transferred, DNA derived from various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology thereto is expressed. Highly expressing the DNA of the present invention by microinjecting a DNA construct (eg, a vector, etc.) in which the human DNA of the present invention is bound downstream of various promoters that can be made into a fertilized egg of a target mammal, for example, a mouse fertilized egg. DNA-transferring mammals can be created.
[0088]
Examples of the expression vector of GPR40 of the present invention include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as phage λ, retroviruses such as Moloney leukemia virus, animal viruses such as vaccinia virus or baculovirus. Are used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast is preferably used.
Examples of the promoter for controlling the DNA expression include, but are not limited to, promoters for DNA derived from (1) viruses (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, polio virus, etc.); ▼ Promoters derived from various mammals (human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.), for example, albumin, insulin II, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acid Protein, glutathione S-transferase, platelet-derived growth factor β, keratins K1, K10 and K14, collagen type I and type II, cyclic AMP-dependent protein kinase βI subunit, dystrophy Fin, tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (commonly abbreviated as Tie2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament light chain, metallothionein I and IIA, metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TPO), peptide chain elongation factor 1α (EF-1α), β actin , Α and β myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle α A Chin, pre-pro-enkephalin A, such as a promoter, such as vasopressin is used. Among them, a cytomegalovirus promoter capable of high expression throughout the body, a human peptide chain elongation factor 1α (EF-1α) promoter, human and chicken β-actin promoters and the like are preferable.
The vector preferably has a sequence (generally called a terminator) that terminates transcription of the messenger RNA of interest in a DNA-transferred mammal. A simian virus SV40 terminator or the like is preferably used.
[0089]
In addition, a splicing signal of each DNA, an enhancer region, a part of an intron of a eukaryotic DNA, and the like are placed 5 ′ upstream of the promoter region, between the promoter region and the translation region, or between the translation region for the purpose of further expressing the desired foreign DNA. It is also possible to connect 3 ′ downstream of the above depending on the purpose.
The normal translation region of GPR40 of the present invention can be obtained from liver, kidney, thyroid cells, fibroblast-derived DNA from humans or various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) and commercially available DNAs. From a variety of genomic DNA libraries, or as a starting material, a complementary DNA prepared by known methods from RNA derived from liver, kidney, thyroid cells, and fibroblasts. In addition, a translation region in which a normal GPR40 translation region obtained from the above cells or tissues is mutated by a point mutagenesis method can be prepared from the exogenous abnormal DNA.
The translation region can be prepared as a DNA construct that can be expressed in a transposed animal by a conventional DNA engineering technique in which it is ligated downstream of the above-mentioned promoter and, if desired, upstream of the transcription termination site.
Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all the progeny of the produced animal retain the exogenous DNA of the present invention in all of the germinal cells and somatic cells. means. The progeny of such animals that have inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of their germinal and somatic cells.
The non-human mammal to which the exogenous normal DNA of the present invention has been transferred can be subcultured in a normal breeding environment as a DNA-carrying animal by confirming that the exogenous DNA is stably maintained by mating. .
Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal. Excessive presence of the exogenous DNA of the present invention in germ cells of a produced animal after DNA transfer means that all offspring of the produced animal have an excessive amount of the exogenous DNA of the present invention in all of their germ cells and somatic cells. Means Progeny of such animals that have inherited the exogenous DNA of the present invention have an excess of the exogenous DNA of the present invention in all of their germinal and somatic cells.
By obtaining a homozygous animal having the introduced DNA on both homologous chromosomes and mating the male and female animals, all offspring can be bred to have the DNA in excess.
[0090]
The non-human mammal having the normal DNA of the present invention, in which the normal DNA of the present invention is highly expressed, promotes the function of the endogenous normal DNA to ultimately cause the hyperactivity of GPR40 of the present invention. Occasionally, it can be used as a disease model animal. For example, using the normal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of hyperactivity of GPR40 of the present invention and the disease associated with GPR40 of the present invention, and to examine a method for treating these diseases. It is possible.
In addition, since the mammal to which the exogenous normal DNA of the present invention has been transferred has an increased symptom of the released GPR40 of the present invention, it can be used for a screening test for a therapeutic agent for a disease associated with the GPR40 of the present invention. is there.
On the other hand, a non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by mating. Furthermore, the target foreign DNA can be incorporated into the above-mentioned plasmid and used as a raw material. The DNA construct with the promoter can be prepared by a usual DNA engineering technique. The transfer of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of its germ cells and somatic cells. The progeny of such animals that have inherited the exogenous DNA of the present invention have the abnormal DNA of the present invention in all of their germinal and somatic cells. A homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing these male and female animals, it is possible to breed so that all progeny have the DNA.
[0091]
The non-human mammal having the abnormal DNA of the present invention, in which the abnormal DNA of the present invention is highly expressed, inhibits the function of the endogenous normal DNA, and finally, the functionally inactive form of GPR40 of the present invention. It can be refractory and can be used as a model animal for the disease. For example, using the abnormal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of GPR40 function-inactive refractory disease of the present invention and to examine a method for treating this disease.
In addition, as a specific possibility, the abnormal DNA highly expressing animal of the present invention has a function of inhibiting the normal GPR40 function (dominant negative action) by the abnormal GPR40 of the present invention in the inactive refractory GPR40 function of the present invention. A model to elucidate.
In addition, since the mammal to which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the released GPR40 of the present invention, it is also used for a therapeutic drug screening test for GPR40 or a functionally inactive refractory disease of the present invention. It is possible.
In addition, other possible uses of the above two kinds of DNA transgenic animals of the present invention include, for example,
(1) Use as a cell source for tissue culture,
(2) GPR40 specifically expressed or activated by GPR40 of the present invention by directly analyzing DNA or RNA in the tissue of the DNA-transferred animal of the present invention, or by analyzing GPR40 tissue expressed by DNA. Analysis of the relationship with
{Circle around (3)} The cells of a tissue having DNA are cultured by standard tissue culture techniques, and these are used to study the function of cells from tissues that are generally difficult to culture.
(4) screening for a drug that enhances the function of the cell by using the cell described in (3) above, and
{Circle around (5)} Isolation and purification of the mutant GPR40 of the present invention and production of its antibody can be considered.
Furthermore, using the DNA-transferred animal of the present invention, it is possible to examine the clinical symptoms of a disease related to the GPR40 of the present invention, including the inactive refractory of GPR40 of the present invention, and the GPR40 of the present invention. More detailed pathological findings in each organ of the disease model related to the disease can be obtained, which can contribute to the development of a new treatment method, and further to the research and treatment of a secondary disease caused by the disease.
In addition, it is possible to take out each organ from the DNA-transferred animal of the present invention, cut it into small pieces, and then use a protease such as trypsin to obtain the released DNA-transferred cells, culture them, or systematize the cultured cells. . Furthermore, it is possible to examine the specificity of the GPR40-producing cell of the present invention, its relationship with apoptosis, differentiation or proliferation, or its signal transduction mechanism, and its abnormalities. It is an effective research material for
Further, in order to develop a therapeutic agent for a GPR40-related disease of the present invention, including a GPR40 functionally inactive refractory disease, using the DNA-transferred animal of the present invention, It is possible to provide an effective and rapid screening method for a therapeutic agent for the disease by using a method or the like. Further, using the transgenic animal of the present invention or the exogenous DNA expression vector of the present invention, it is possible to examine and develop a DNA treatment method for a disease associated with GPR40 of the present invention.
[0092]
(12) Knockout animal
The present invention provides a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated, and a non-human mammal deficient in expression of the DNA of the present invention.
That is, the present invention
[1] a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated,
[2] the embryonic stem cell according to [1], wherein the DNA is inactivated by introducing a reporter gene (eg, a β-galactosidase gene derived from Escherichia coli);
[3] the embryonic stem cell according to [1], which is neomycin-resistant;
[4] the embryonic stem cell according to [1], wherein the non-human mammal is a rodent;
[5] the embryonic stem cell of [4], wherein the rodent is a mouse;
[6] a non-human mammal deficient in expression of the DNA of the present invention, wherein the DNA is inactivated;
[7] The DNA is inactivated by introducing a reporter gene (eg, a β-galactosidase gene derived from Escherichia coli), and the reporter gene can be expressed under the control of a promoter for the DNA of the present invention [6]. The non-human mammal according to the item,
[8] the non-human mammal according to [6], wherein the non-human mammal is a rodent;
[9] the non-human mammal of [8], wherein the rodent is a mouse, and
[10] A method for screening a compound or a salt thereof that promotes or inhibits the promoter activity of the DNA of the present invention, which comprises administering a test compound to the animal according to [7] and detecting the expression of a reporter gene. I will provide a.
A non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is a DNA which is artificially mutated to the DNA of the present invention possessed by the non-human mammal to suppress the DNA expression ability, or By substantially eliminating the activity of the GPR40 of the present invention encoded by the DNA, the DNA does not substantially have the ability to express the GPR40 of the present invention (hereinafter, may be referred to as the knockout DNA of the present invention in some cases). ) Non-human mammalian embryonic stem cells (hereinafter abbreviated as ES cells).
As the non-human mammal, the same one as described above is used.
The method of artificially mutating the DNA of the present invention can be carried out, for example, by deleting a part or all of the DNA sequence and inserting or substituting another DNA by a genetic engineering technique. With these mutations, the knockout DNA of the present invention may be prepared, for example, by shifting the codon reading frame or disrupting the function of the promoter or exon.
[0093]
Specific examples of the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated (hereinafter abbreviated as the DNA-inactivated ES cells of the present invention or the knockout ES cells of the present invention) include, for example, The DNA of the present invention possessed by a non-human mammal is isolated, and its exon portion is a drug resistance gene represented by a neomycin resistance gene or a hygromycin resistance gene, or lacZ (β-galactosidase gene), cat (chloramphenicol acetyl). A reporter gene or the like typified by a transferase gene) to disrupt the exon function, or insert a DNA sequence that terminates gene transcription into an intron between exons (for example, a polyA addition signal); Inability to synthesize complete messenger RNA Thus, a DNA strand having a DNA sequence constructed so as to eventually destroy the gene (hereinafter abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, homologous recombination, and the resulting ES cells PCR using a DNA sequence on or near the DNA of the present invention as a probe and a DNA sequence on a targeting vector and a DNA sequence in a neighboring region other than the DNA of the present invention used for the preparation of the targeting vector as a primer And selecting the knockout ES cells of the present invention.
As the ES cells from which the DNA of the present invention is inactivated by the homologous recombination method or the like, for example, those already established as described above may be used, or according to the known Evans and Kaufma method. It may be a newly established one. For example, in the case of mouse ES cells, currently, 129-line ES cells are generally used. For example, for the purpose of obtaining ES cells in which BDF is clearly known, for example, BDF in which the number of eggs collected by C57BL / 6 mice or C57BL / 6 has been improved by hybridization with DBA / 2 ₁ Mouse (F of C57BL / 6 and DBA / 2) ₁ ) Can also be used favorably. BDF ₁ In addition to the advantage that the number of eggs collected and the eggs are robust, the mice have C57BL / 6 mice as a background, and thus, the ES cells obtained using the C57BL / 6 mice were used to produce C57BL / 6 mice. / 6 mice can be advantageously used in that the genetic background can be replaced by C57BL / 6 mice by backcrossing.
In addition, when ES cells are established, blastocysts 3.5 days after fertilization are generally used. In addition, a large number of blastocysts can be efficiently obtained by collecting embryos at the 8-cell stage and culturing them until blastocysts are used. Early embryos can be obtained.
Although either male or female ES cells may be used, male ES cells are generally more convenient for producing a germline chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce cumbersome culture labor.
[0094]
As a method for determining the sex of ES cells, for example, a method of amplifying and detecting a gene in a sex-determining region on the Y chromosome by a PCR method can be mentioned. If this method is used, it is conventionally necessary to perform about 10 karyotype analyses. ⁶ Since the number of ES cells is required, the number of ES cells of about 1 colony (approximately 50) is sufficient, so that primary selection of ES cells in the early stage of culture can be performed by discriminating between male and female. In addition, by enabling selection of male cells at an early stage, labor in the initial stage of culture can be significantly reduced.
The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes in the obtained ES cells is desirably 100% of the normal number. However, when it is difficult due to physical operations at the time of establishment, after knocking out the gene of the ES cells, the cells are knocked out of normal cells (for example, chromosomes in mice). It is desirable to clone again into cells (number 2n = 40).
The embryonic stem cell line obtained in this manner usually has very good growth properties, but it must be carefully subcultured because it tends to lose its ontogenic ability. For example, on a suitable feeder cell such as STO fibroblast in the presence of LIF (1 to 10,000 U / ml) in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen, 5% The cells are cultured by a method such as culturing at about 37 ° C. in carbon dioxide gas and 90% air. At the time of subculture, for example, trypsin / EDTA solution (usually 0.001 to 0.5% trypsin / 0.1 to 5 mM EDTA, Preferably, a method is used in which cells are converted into single cells by treatment with about 0.1% trypsin / 1 mM EDTA and seeded on newly prepared feeder cells. Such subculture is usually performed every 1 to 3 days. At this time, it is desirable to observe the cells and, if any morphologically abnormal cells are found, discard the cultured cells.
ES cells are differentiated into various types of cells, such as parietal muscle, visceral muscle, and cardiac muscle, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions. [M. J. Evans and M.E. H. Kaufman, Nature, 292, 154, 1981; R. Martin Proc. Of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; C. Doetschman, et al., Journal of Embryology and Experimental Morphology, Vol. 87, p. 27, 1985], that the DNA-deficient cell of the present invention obtained by differentiating the ES cell of the present invention is characterized by in vitro The present invention is useful in examining the GPR40 of the present invention or the cell biology of the GPR40 of the present invention.
[0095]
The non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level.
As the non-human mammal, those similar to the aforementioned can be used.
The non-human mammal deficient in expression of the DNA of the present invention is, for example, a DNA in which the targeting vector prepared as described above is introduced into mouse embryonic stem cells or mouse egg cells, and the DNA of the targeting vector is inactivated by the introduction. The DNA of the present invention can be knocked out by homologous recombination in which the sequence replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by homologous recombination.
Cells in which the DNA of the present invention has been knocked out can be used as a DNA sequence on a Southern hybridization analysis or targeting vector using the DNA sequence on or near the DNA of the present invention as a probe, and the DNA of the present invention derived from the mouse used for the targeting vector. The determination can be made by analysis by a PCR method using a DNA sequence of a nearby region other than DNA as a primer. When non-human mammalian embryonic stem cells are used, a cell line in which the DNA of the present invention has been inactivated by gene homologous recombination is cloned, and the cells are cultured at an appropriate time, for example, at the 8-cell stage of non-human mammalian cells. The chimeric embryo is injected into an animal embryo or a blastocyst, and the produced chimeric embryo is transplanted into the uterus of the pseudopregnant non-human mammal. The produced animal is a chimeric animal composed of both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention.
When a part of the germ cells of the chimeric animal has a mutated DNA locus of the present invention, all tissues are artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the added DNA locus of the present invention, for example, by judging coat color or the like. The individual thus obtained is usually an individual lacking the heterogeneous expression of the GPR40 of the present invention, and mated with an individual lacking the heterogeneous expression of the GPR40 of the present invention, and homozygously expressing the GPR40 of the present invention from their offspring. Defective individuals can be obtained.
[0096]
When an egg cell is used, for example, a transgenic non-human mammal having a targeting vector introduced into a chromosome can be obtained by injecting a DNA solution into a nucleus of an egg by a microinjection method. Compared to mammals, they can be obtained by selecting those having a mutation in the DNA locus of the present invention by homologous recombination.
In this manner, the individual in which the DNA of the present invention has been knocked out can be bred in an ordinary breeding environment after confirming that the DNA has been knocked out in the animal individual obtained by mating.
Furthermore, the germline can be obtained and maintained in accordance with a conventional method. That is, by crossing male and female animals having the inactivated DNA, a homozygous animal having the inactivated DNA on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by breeding the mother animal in such a manner that there are one normal animal and one homozygote. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.
The non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are extremely useful for producing a non-human mammal deficient in expressing the DNA of the present invention.
In addition, since the non-human mammal deficient in expressing the DNA of the present invention lacks various biological activities that can be induced by the GPR40 of the present invention, a model of a disease caused by inactivation of the biological activity of the GPR40 of the present invention. Therefore, it is useful for investigating the cause of these diseases and studying treatment methods.
[0097]
(12a) A method for screening a compound having a therapeutic / preventive effect on diseases caused by DNA deficiency or damage of the present invention
The non-human mammal deficient in expression of the DNA of the present invention can be used for screening for a compound having a therapeutic / preventive effect against diseases caused by deficiency or damage of the DNA of the present invention.
That is, the present invention is characterized by administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention, and observing and measuring a change in the animal. Provided is a method for screening a compound or a salt thereof having a therapeutic / prophylactic effect against a disease.
Examples of the non-human mammal deficient in expressing DNA of the present invention used in the screening method include the same ones as described above.
Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma, and these compounds are novel compounds. Or a known compound.
Specifically, a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with an untreated control animal, and tested using changes in each organ, tissue, disease symptoms, etc. of the animal as an index. The therapeutic / preventive effects of the compounds can be tested.
As a method of treating a test animal with a test compound, for example, oral administration, intravenous injection and the like are used, and it can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like. The dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.
[0098]
In the screening method, when a test compound is administered to a test animal and the blood glucose level of the test animal decreases by about 10% or more, preferably about 30% or more, more preferably about 50% or more, the test compound Can be selected as compounds having a therapeutic / preventive effect on the above diseases.
The compound obtained by using the screening method is a compound selected from the above-described test compounds, and is a disease (eg, diabetes, impaired glucose tolerance, ketosis, acidosis, diabetes) caused by deficiency or damage of GPR40 of the present invention. Sexual neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis, thrombotic disease, dyspepsia, memory learning disorders, etc.) And low-toxic therapeutic / prophylactic agents, pancreatic function regulators (eg, pancreatic function improving agents), insulin secretagogues, hypoglycemic agents, pancreatic β-cell protective agents and the like. Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.
The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals; For example, a salt with an alkaline earth metal such as calcium) is used, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, For example, salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like are used.
A drug containing the compound obtained by the screening method or a salt thereof can be produced in the same manner as the above-mentioned drug containing a compound that alters the binding property between GPR40 and a ligand of the present invention.
Since the preparation thus obtained is safe and low toxic, it can be used, for example, in humans or mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). Can be administered.
The dose of the compound or a salt thereof varies depending on the target disease, the subject of administration, the administration route, and the like. For example, when the compound is orally administered, generally a diabetic patient (with a body weight of 60 kg) consumes one day. About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease and the like. For example, the compound is usually administered in the form of an injection to a diabetic patient (with a body weight of 60 kg). In this case, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound per day by intravenous injection. In the case of other animals, the amount can be administered in terms of 60 kg.
[0099]
(12b) A method for screening a compound that promotes or inhibits the activity of a promoter for DNA of the present invention
The present invention provides a compound which promotes or inhibits the activity of a promoter for the DNA of the present invention, which comprises administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention, and detecting the expression of a reporter gene, or a compound thereof. A method for screening a salt is provided.
In the above-described screening method, the non-human mammal deficient in expressing DNA of the present invention may be a non-human mammal deficient in expressing DNA of the present invention in which the DNA of the present invention is inactivated by introducing a reporter gene, A reporter gene that can be expressed under the control of a promoter for the DNA of the present invention is used.
Examples of the test compound include the same compounds as described above.
As the reporter gene, the same one as described above is used, and a β-galactosidase gene (lacZ), a soluble alkaline phosphatase gene, a luciferase gene and the like are preferable.
In a non-human mammal deficient in expression of the DNA of the present invention in which the DNA of the present invention is replaced with a reporter gene, since the reporter gene is under the control of the promoter for the DNA of the present invention, the expression of the substance encoded by the reporter gene is traced. By doing so, the activity of the promoter can be detected.
For example, when a part of the DNA region encoding the GPR40 of the present invention is replaced with a β-galactosidase gene (lacZ) derived from Escherichia coli, the tissue that expresses the GPR40 of the present invention should be used instead of the GPR40 of the present invention. Β-galactosidase is expressed. Therefore, for example, the present invention can be easily performed by staining with a reagent serving as a substrate for β-galactosidase such as 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal). Of the GPR40 in an animal body can be observed. Specifically, the GPR40-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-gal at room temperature or around 37 ° C. After reacting for about 30 minutes to 1 hour, the β-galactosidase reaction may be stopped by washing the tissue specimen with a 1 mM EDTA / PBS solution, and coloration may be observed. Further, mRNA encoding lacZ may be detected according to a conventional method.
The compound or a salt thereof obtained by using the above-mentioned screening method is a compound selected from the above-mentioned test compounds, and is a compound that promotes or inhibits the promoter activity for the DNA of the present invention.
The compound obtained by the screening method may form a salt, and as the salt of the compound, the same salt as described above is used.
[0100]
The compound or its salt that promotes the promoter activity of the DNA of the present invention can promote the expression of the GPR40 of the present invention and promote the function of the GPR40, and for example, is associated with the dysfunction of the GPR40 of the present invention. It is useful as a medicament such as a prophylactic / therapeutic agent for diseases and the like, a pancreatic function regulator (eg, a pancreatic function improving agent), an insulin secretagogue, a hypoglycemic agent, a pancreatic β-cell protective agent and the like.
The compound or a salt thereof that inhibits the promoter activity of the DNA of the present invention can inhibit the expression of the GPR40 of the present invention and inhibit the function of the GPR40. For example, it is related to the overexpression of the GPR40 of the present invention. It is useful as a medicament such as a prophylactic / therapeutic agent for a disease, a pancreatic function regulator (eg, a pancreatic function improving agent), an insulin secretion inhibitor, a blood glucose increasing agent, and the like.
The diseases associated with the dysfunction of GPR40 of the present invention include diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, and skin diseases , Arthropathy, osteopenia, arteriosclerosis, thrombotic diseases, dyspepsia, memory learning disorders and the like. Diabetes includes insulin dependent (type I) diabetes, non-insulin dependent (type II) diabetes and the like.
Diseases caused by overexpression of GPR40 of the present invention include, for example, obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, Insulin resistance syndrome, unstable diabetes, lipoatrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity, cancer and the like.
Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.
[0101]
A drug containing a compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing a compound that changes the binding property between GPR40 or a salt thereof and a ligand of the present invention.
Since the preparation thus obtained is safe and low toxic, it can be used, for example, in humans or mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). Can be administered.
The dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like. For example, when a compound that promotes or inhibits the promoter activity of the DNA of the present invention is orally administered, in general, diabetes In a patient (assuming a body weight of 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound is administered per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, a compound that promotes the promoter activity for DNA of the present invention is usually administered in the form of an injection in the form of an injection. When administered to a patient (assuming a body weight of 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound is administered by intravenous injection per day. It is convenient to do so. In the case of other animals, the amount can be administered in terms of 60 kg.
As described above, the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening for a compound or a salt thereof that promotes or inhibits the activity of the promoter for the DNA of the present invention, It can greatly contribute to investigating the cause of various diseases caused by the disease or to develop preventive and therapeutic drugs.
In addition, using a DNA containing the promoter region of GPR40 of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (gene-transferred animal). Once created, the GPR40 can be specifically synthesized and its action in living organisms can be examined. Furthermore, if a suitable reporter gene is linked to the above-mentioned promoter portion and a cell line that expresses the reporter gene is established, a low-molecular compound having the action of specifically promoting or suppressing the production ability of GPR40 itself of the present invention in the body. Can be used as a search system.
[0102]
In the present specification and drawings, bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or by abbreviations commonly used in the art, and examples thereof are described below. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified.

[0103]

[0104]
Further, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.

[0105]
The sequence numbers in the sequence listing in the present specification indicate the following sequences.
SEQ ID NO: 1
1 shows the amino acid sequence of mouse spleen-derived GPR40 of the present invention.
SEQ ID NO: 2
1 shows the nucleotide sequence of cDNA encoding GPR40 derived from mouse spleen of the present invention.
SEQ ID NO: 3
1 shows the amino acid sequence of rat spleen-derived GPR40 of the present invention.
SEQ ID NO: 4
1 shows the nucleotide sequence of cDNA encoding rat spleen-derived GPR40 of the present invention.
SEQ ID NO: 5
1 shows the amino acid sequence of human-derived GPR40 of the present invention.
SEQ ID NO: 6
1 shows the nucleotide sequence of cDNA encoding human-derived GPR40 of the present invention.
SEQ ID NO: 7
The base sequence of the primer 1 used in the PCR reaction in Example 2 below is shown.
SEQ ID NO: 8
The base sequence of the primer 2 used in the PCR reaction in Example 2 below is shown.
SEQ ID NO: 9
The base sequence of primer 3 used in the PCR reaction in Example 3 below is shown.
SEQ ID NO: 10
The base sequence of primer 4 used in the PCR reaction in Example 4 below is shown.
SEQ ID NO: 11
The base sequence of the primer used in the TaqMan PCR reaction in Example 4 below is shown.
SEQ ID NO: 12
The base sequence of the probe used in the TaqMan PCR reaction in Example 4 below is shown.
SEQ ID NO: 13
The base sequence of the primer used in the TaqMan PCR reaction in Example 4 below is shown.
SEQ ID NO: 14
The base sequence of the primer used in the TaqMan PCR reaction in Example 4 below is shown.
SEQ ID NO: 15
The base sequence of the probe used in the TaqMan PCR reaction in Example 4 below is shown.
SEQ ID NO: 16
The base sequence of the primer used in the TaqMan PCR reaction in Example 4 below is shown.
SEQ ID NO: 17
1 shows the amino acid sequence of cynomolgus monkey-derived GPR40 of the present invention.
SEQ ID NO: 18
1 shows the nucleotide sequence of cDNA encoding GPR40 derived from cynomolgus monkey of the present invention.
SEQ ID NO: 19
The base sequence of the primer used in the PCR reaction in Example 5 below is shown.
SEQ ID NO: 20
The base sequence of the primer used in the PCR reaction in Example 5 below is shown.
SEQ ID NO: 21
The base sequence of the probe used in the PCR reaction in Example 5 below is shown.
SEQ ID NO: 22
The base sequence of the primer used in the PCR reaction in Example 5 below is shown.
SEQ ID NO: 23
The base sequence of the primer used in the PCR reaction in Example 7 below is shown.
SEQ ID NO: 24
The base sequence of the primer used in the PCR reaction in Example 7 below is shown.
SEQ ID NO: 25
The base sequence of the probe used in the PCR reaction in Example 7 below is shown.
SEQ ID NO: 26
The base sequence of the primer used in the PCR reaction in Example 8 below is shown.
SEQ ID NO: 27
The base sequence of the probe used in the PCR reaction in Example 8 below is shown.
SEQ ID NO: 28
The following shows the nucleotide sequences of the primers used in the PCR reaction in Example 9.
SEQ ID NO: 29
1 shows the amino acid sequence of hamster-derived GPR40 of the present invention.
SEQ ID NO: 30
1 shows the nucleotide sequence of cDNA encoding hamster-derived GPR40 of the present invention.
SEQ ID NO: 31
The base sequence of the primer 1 used in the PCR reaction in Example 9 below is shown.
SEQ ID NO: 32
The base sequence of the primer 2 used in the PCR reaction in Example 9 below is shown.
SEQ ID NO: 33
1 shows the amino acid sequence of the C-terminal peptide of human-derived GPR40 of the present invention.
SEQ ID NO: 34
2 shows the nucleotide sequence of the sense strand of siRNA m40i103.
SEQ ID NO: 35
The base sequence of the antisense strand of siRNA m40i103 is shown.
SEQ ID NO: 36
The nucleotide sequence of the sense strand of siRNA m40i256 is shown.
SEQ ID NO: 37
The base sequence of the antisense strand of siRNA m40i103 is shown.
SEQ ID NO: 38
The following shows the nucleotide sequences of the primers used in the PCR reaction in Example 20.
SEQ ID NO: 39
The following shows the nucleotide sequences of the primers used in the PCR reaction in Example 20.
SEQ ID NO: 40
The base sequence of the sense strand primer used in the PCR reaction in Reference Example 1 below is shown.
SEQ ID NO: 41
The base sequence of the antisense strand primer used in the PCR reaction in Reference Example 1 below is shown.
[0106]
The transformant Escherichia coli TOP10 / Zero Blunt-mGPR40 obtained in Example 2 to be described later is an independent administration of 1-1, Higashi 1-1, Tsukuba City, Ibaraki Prefecture since March 18, 2002 (zip code 305-8566). Incorporated by the National Institute of Advanced Industrial Science and Technology under the deposit number FERM BP-7796 at the Patent Organism Depositary Center, 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka-shi from February 14, 2002 (postal code 532-8686). -Deposited with the Fermentation Research Institute (IFO) under the deposit number IFO 16762.
The transformant Escherichia coli JM109 / pCR2.1-rGPR40 obtained in Example 3 described below was deposited on March 18, 2002 with the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary under the deposit number FERM BP-7968. It has been deposited with the Fermentation Research Institute (IFO) as a deposit number IFO 16763 since February 14, 2002.
The transformant Escherichia coli JM109 / pCR2.1-monkey GPR40 obtained in Example 5 described below was deposited on July 23, 2002 with the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary under the deposit number FERM BP-8125. Has been deposited.
The transformant Escherichia coli JM109 / pTA hamstar GPR40 obtained in Example 9 described below was transformed into a transformant Escherichia coli JM109 / pTAhamster GPR40 from December 11, 2002 to the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, Japan. Deposit number FERM BP-8258.
The transformant Escherichia coli DH5α / pGT-GPR40 obtained in Example 21 described later was deposited on December 11, 2002 with the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary under the deposit number FERM BP-8259. I have.
[0107]
【Example】
Hereinafter, the present invention will be described in more detail with reference to Reference Examples and Examples, but these examples do not limit the scope of the present invention. In addition, the gene using Escherichia coli followed the method described in Molecular cloning (Molecular cloning).
[0108]
Reference Example 1 Construction of Human GPR40 Expression Vector
A DNA fragment encoding human GPR40 was obtained by the following PCR method. That is, an oligo DNA (SEQ ID NO: 40) represented by 5 '> CGTCGACCCGGCGGCCCCCATGGACCTGCCCCCG <3' is used as a sense strand primer, and an oligo DNA (SEQ ID NO: 41) represented by 5 '> CATCGATTAGCAGTGGCGTTACTTCTGGGACTT <3' is used as an antisense strand primer. 20 μmol each, 10 × Advantage (registered trademark) 2 PCR Buffer (CLONTECH) 5 μl, 50 × dNTP mix (CLONTECH) 1 μl, 50 × Advantage 2 Polymerase Mix (CLONTECH) 1 μl, Human pancreatic cDNA cDNA (μL) as template DNA Is prepared, and a thermal cycler (GeneAmp (registered trademark)) is prepared. Using PCR system model 9700 (Applied Biosystems), 96 ° C, 1 minute, then 96 ° C, 30 seconds → 61 ° C, 30 seconds → 72 ° C, 120 seconds were repeated 35 cycles, and further extended at 72 ° C, 10 minutes. A PCR reaction was performed using a program for causing the PCR reaction. The reaction completed solution was subjected to agarose gel electrophoresis to obtain a single product, which was cloned using a TA cloning kit (Invitrogen) to confirm the gene sequence. A clone having no PCR error was double digested with restriction enzymes SalI (Takara Shuzo) and ClaI (Takara Shuzo), and then a single product was cut out by agarose gel electrophoresis. The obtained fragment (about 1 kb) was introduced into a pAKKO-111 vector and used for transfection of CHO cells.
[0109]
Example 1 Confirmation of Reactivity of Fatty Acid to Human GPR40
The CHO-K1 cell line was cultured using a Ham F-12 medium (Invitrogen) containing 10% fetal calf serum (Invitrogen) unless otherwise specified. 10cm the day before transfection ² 4.5x10 per ⁵ Seeded cells and 5% CO ₂ CO adjusted to concentration ₂ The cells were cultured in an incubator at 37 ° C. for 15 hours or more. Transfection was performed using Lipofectamine reagent (Invitrogen) according to the method attached to the reagent. When a 6-well plate was used for the incubator, the procedure was as follows. First, two 1.5 ml tubes were prepared, and 100 μl of Opti-MEM-I medium (Invitrogen) was dispensed to each tube. Next, 1 μg of the expression vector was added to one tube and 6 μl of the Lipofectamine reagent was added to the other tube, and the two were mixed and allowed to stand at room temperature for 20 minutes. A transfection mixture obtained by adding 800 μl of Opti-MEM-I medium to this solution was added to CHO-K1 cells previously washed using Opti-MEM-I medium, and then CO2 was added. ₂ The cells were cultured in an incubator for 6 hours. The cells after the culture were rinsed using PBS (Invitrogen), peeled off using a 0.05% trypsin / EDTA solution (Invitrogen), and collected by centrifugation. The number of cells obtained was measured and 5 × 10 ⁴ And diluted into Black walled 96-well plate (Costar) by 200 μl per well. ₂ The cells were cultured overnight in an incubator. Various test samples were added to the CHO-K1 cells transiently expressing the receptor by the above transfection operation, and the change in intracellular calcium concentration at this time was measured using FLIPR (Molecular Device). In order to measure the fluctuation of intracellular calcium concentration by FLIPR, the following pretreatment was performed. First, an assay buffer was prepared for adding the fluorescent dye Fluo-3AM (DOJIN) to the cells or washing the cells immediately before performing the FLIPR assay. In a solution (hereinafter, HBSS / HEPES solution) obtained by adding 20 ml of 1M HEPES (pH 7.4) (DOJIN) to 1000 ml of HBSS (Invitrogen), 710 mg of probenecid (Sigma) is dissolved in 5 ml of 1N NaOH, and then 5 ml of an HBSS / HEPES solution. Was added and 10 ml of the mixed solution was added, and this solution was used as an assay buffer. Next, 50 μg of Fluo-3AM was dissolved in 21 μl of DMSO (DOJIN), an equal amount of 20% pluronic acid (Molecular Probes) was added and mixed, and then added to 10.6 ml of assay buffer containing 105 μl of fetal calf serum. A fluorescent dye solution was prepared. The medium of the transfected CHO-K1 cells was removed, and the fluorochrome solution was immediately dispensed at 100 μl per well. ₂ The cells were cultured in an incubator for 1 hour, and the fluorescent dye was incorporated into the cells. The cells after the culture were washed with the above-mentioned assay buffer, and then set on the FLIPR. In addition, a test sample to be added to the receptor-expressing CHO-K1 cells was prepared using an assay buffer, and simultaneously set in the FLIPR. After the above pretreatment, the fluctuation of intracellular calcium concentration after addition of various test samples was measured by FLIPR. As a result, farnesic acid, 5.8.11-eicosatrinoic acid, 5.8.11.14-eicosatetranoic acid, oleic acid, linoleic acid, linolenic acid, and linolenic acid. , Arachidonic acid, eicosapentaenoic acid, EPA, eicosadienoic acid, eicosaenoic acid, eicosatoic acid, docosahexaenoic acid, docosahexaic acid Ad Phosphate (adrenic acid), lauric acid (lauric acid) or the like 10 ^-5 M-10 ^-6 When M was added, it was found that CHO-K1 cells expressing the GPR40 receptor specifically respond (increase in intracellular calcium concentration) [FIGS. 1 to 13]. No such response was observed in CHO-K1 cells into which only the control expression vector was introduced. That is, it was revealed that the endogenous ligand of GPR40 is a fatty acid.
[0110]
Example 2 Cloning of cDNA encoding GPR40 from mouse spleen and determination of its nucleotide sequence
Using mouse spleen cDNA (Marathon-Ready ™ cDNA; Clontech) as a template, PCR was performed using two primers, primer 1 (SEQ ID NO: 7) and primer 2 (SEQ ID NO: 8). Pyrobest DNA Polymerase (Takara Shuzo) was used for PCR, (1) after 98 ° C for 1 minute, (2) after 40 times of 98 ° C for 10 seconds, 55 ° C for 30 seconds, 72 ° C for 60 seconds, and 3) An extension reaction was performed at 72 ° C. for 2 minutes. After the reaction, the amplification product was cloned into a plasmid vector pCR-Blunt (Invitrogen) according to the prescription of Zero Blunt PCR Cloning Kit (Invitrogen). This was introduced into Escherichia coli TOP10 (Invitrogen), and a clone having the plasmid was selected in an LB agar medium containing kanamycine. As a result of analyzing the nucleotide sequence of each clone, a cDNA sequence (SEQ ID NO: 2) encoding a novel G protein-coupled receptor protein was obtained. A novel protein containing the amino acid sequence (SEQ ID NO: 1) derived from this cDNA was named mGPR40. The transformant was named Escherichia coli TOP10 / Zero Blunt-mGPR40.
[0111]
Example 3 Cloning of cDNA encoding GPR40 from rat spleen and determination of its nucleotide sequence
Using rat spleen cDNA (Marathon-Ready ™ cDNA; Clontech) as a template, PCR was performed using two primers, primer 3 (SEQ ID NO: 9) and primer 4 (SEQ ID NO: 10). The Advantage 2 Polymerase mix (Clontech) was used for PCR, (1) 96 ° C for 1 minute, (2) 96 ° C for 10 seconds, 72 ° C for 2 minutes 5 times, (3) 96 ° C for 10 seconds, 70 After 25 times at 2 ° C. for 2 minutes, an extension reaction was performed at 72 ° C. for 5 minutes. After the reaction, the amplification product was cloned into a plasmid vector pCR2.1TOPO (Invitrogen) according to the prescription of TOPO TA Cloning Kit (Invitrogen). This was introduced into Escherichia coli JM109 (Takara Shuzo), and a clone having the plasmid was selected in an LB agar medium containing ampicillin. As a result of analyzing the nucleotide sequence of each clone, a cDNA sequence (SEQ ID NO: 4) encoding a novel G protein-coupled receptor protein was obtained. A novel protein containing the amino acid sequence (SEQ ID NO: 3) derived from this cDNA was named rGPR40. The transformant was named Escherichia coli JM109 / pCR2.1-rGPR40.
[0112]
Example 4 Expression Distribution
(1) Cells and medium
NIH-3T3 and B104 cells were purchased from ATCC. As the mouse pancreatic β cell line MIN6, the one described in the literature (Jun-ichi Miyazaki et al. Endocrinology, Vol. 127, No. 1, p126-132) was used. Each cell was cultured in DMEM medium (Invitrogen) containing 10% FCS until it became pre-confluent.
(2) RNA extraction and cDNA synthesis
The cDNA used for expression distribution in human and mouse tissues was subjected to a reverse transcription reaction from 1 μg of polyA + RNA (Clontech) derived from various human and mouse tissues using random primers. Using reverse transcriptase SuperScript II (GIBCO BRL), the reaction was carried out according to the attached protocol, and ethanol was precipitated and dissolved in 100 μl of TE.
For the mouse-derived cDNA, the total RNA was extracted and purified in accordance with the manual of RNeasy mini KIT (QIAGEN) after measuring the number of cells by peeling the cells with Trypsin-EDTA. 1 μg of the extracted RNA was randomly synthesized according to the manual of SuperScript II (Invitrogen) to synthesize first strand cDNA, followed by ethanol precipitation and dissolution in 10 μl of TE.
(3) Quantification using TaqMan
For tissue-derived cDNA (equivalent to 5 ng RNA) and cell line-derived cDNA (equivalent to 25 ng RNA), an amplification reaction reagent TaqMan (trademark) Universal PCR Master Mix (Applied Biosystems Japan, Inc.), TaqMan (trademark) Probe for GPR40 detection Using Kit (sequence: 11 to 16, Applied Biosystems Japan Co., Ltd.), the reaction was performed after preparing a total reaction volume of 15 μl. The final concentration of each primer and probe was in accordance with the manual.
TaqMan ™ PCR was performed on an ABI PRISM ™ 7900HT sequence detection system (Applied Biosystems Japan, Inc.) and the temperature cycle used was TaqMan ™ Universal PCR Master Mix (Applied Biosystems Japan, Inc.) manual. Followed.
Quantitative TaqMan analysis of the amplification products was performed using 7900HT SDS software (Applied Biosystems Japan). The calibration curve used for calculating the copy number was determined using a cDNA fragment (human GPR40) of known concentration or Plasmid DNA (mouse GPR40) containing the entire length of the amplified region. ⁷ From copy / well to 10 ² C in logarithmic 6 points to copy / well _T Created from values.
FIG. 14 shows the distribution of GPR40 mRNA expression in various human tissues, and FIG. 15 shows the distribution of GPR40 mRNA expression in various mouse cells. In human tissues, relatively high expression was observed in the pancreas, lung, hippocampus, hypothalamus, and spinal cord. In mice, extremely high expression was observed in cells derived from pancreatic cancer.
[0113]
Example 5 Cloning of cDNA encoding GPR40 from cynomolgus monkey and determination of its nucleotide sequence
Using cynomolgus DNA as a template, PCR was performed using primer (SEQ ID NO: 19) and primer 2 (SEQ ID NO: 20). For PCR, Pyrobest DNA Polymerase (TAKARA) was used. (1) 95 ° C for 1 minute, (2) 95 ° C for 10 seconds, 58 ° C for 20 seconds, 72 ° C for 1 minute and 30 seconds, 72 times An extension reaction was performed at 7 ° C. for 7 minutes. After the reaction, nested PCR was performed using the primer (SEQ ID NO: 21) and the primer 2 (SEQ ID NO: 22) with the amplification product diluted 1/50 as a template. After the reaction, the amplification product was cloned into a plasmid vector pCR2.1TOPO (Invitrogen) according to the prescription of TOPO TA Cloning Kit (Invitrogen). This was introduced into Escherichia coli JM109 (Takara Shuzo), and a clone having the plasmid was selected in an LB agar medium containing ampicillin. As a result of analyzing the nucleotide sequence of each clone, a cDNA sequence (SEQ ID NO: 18) encoding a novel G protein-coupled receptor protein was obtained. A novel protein containing the amino acid sequence (SEQ ID NO: 17) derived from this cDNA was named monkey GPR40. The transformant was named Escherichia coli JM109 / pCR2.1-monkey GPR40.
[0114]
Example 6 Insulin secretion promoting effect of free fatty acids in mouse insulinoma MIN6 cells
MIN6 cells were cultured using DMEM (high glucose, Invitrogen) containing 15% FCS (Trace Scientific Ltd.), 55 μM 2-mercaptoethanol, 100 U / ml penicillin, and 100 μg / ml streptomycin unless otherwise specified. 10 per well in a 96-well plate ⁵ MIN6 cells and 5% CO ₂ CO adjusted to concentration ₂ The cells were cultured in an incubator at 37 ° C. for 3 days. The medium was replaced with RPMI 1640 (without glucose, Invitrogen) containing 10% FCS (Trace Scientific Ltd.), 5.5 mM glucose, 100 U / ml penicillin, and 100 μg / ml streptomycin, and further cultured for 24 hours. After removing the medium by suction, free fatty acid-bovine serum albumin diluted with RPMI 1640 (without glucose, Invitrogen) containing 10% FCS (Trace Scientific Ltd.), 11 mM glucose, 100 U / ml penicillin, and 100 μg / ml streptomycin (BSA) mixture (4: 1, molar ratio) and 5% CO ₂ CO adjusted to concentration ₂ The reaction was performed at 37 ° C. for 90 minutes (or 60 minutes) in an incubator. After the 96-well plate after the reaction was centrifuged at 1500 rpm for 5 minutes, the culture supernatant was recovered. The amount of insulin secreted into the culture supernatant was measured by a radioimmunoassay (RIA) using a rat insulin RIA system (Amersham Pharmacia). As a result, free fatty acids such as palmitic acid (Palmic acid) (FIGS. 16 and 17), γ-linolenic acid (FIG. 16), and oleic acid (oleic acid) (FIG. 17) were reduced to 300 μM or less. It was found that when 1000 μM was added, insulin secretion by MIN6 cells was promoted. That is, it was revealed that free fatty acids have an action of promoting insulin secretion in mouse insulinoma MIN6 cells. Since MIN6 cells specifically and extremely express GPR40 as described in Example 4 (FIG. 15), this reaction was performed in such a manner that the added fatty acid promotes insulin secretion via GPR40. Is determined.
[0115]
Example 7 Expression of GPR40 in rat pancreatic islets of Langerhans
Isolation of islets of Langerhans from rat pancreas was performed as follows. Wistar rats (male, 8 weeks of age) were decapitated, bled, excised with scissors and laparotomized, and ligated with a clamp at the duodenal exit of the common bile duct. Next, a hole was made in the liver side of the common bile duct with a syringe needle, and a cannula was inserted. 5 ml of a 1 mg / ml collagenase XI (Sigma, C-7657) solution previously dissolved in HBSS and cooled on ice was injected from the cannula. The pancreas was separated from other tissues and shaken in a 37 ° C water bath for 3 minutes. After adding 10 ml of HBSS and pipetting 5 to 6 times, 10 ml of HBSS was added and pipetting twice. After adding 20 ml of HBSS and allowing to stand for 4 minutes, the supernatant was discarded, 10 ml of HBSS was added again, and pipetting was performed twice. After further adding 10 ml of HBSS and pipetting twice, 20 ml of HBSS was added and allowed to stand for 3 minutes. The supernatant was discarded, and a suspension prepared by adding an appropriate amount of HBSS was transferred to a petri dish with a black bottom. In another Petri dish, RPMI1640 (without glucose, Invitrogen) containing 10% FCS (Trace Scientific Ltd.), 5.5 mM glucose, 100 U / ml penicillin, and 100 μg / ml streptomycin was put, and the petri dish with a black bottom was used. The islets of Langerhans selected under a microscope were transferred to this dish using a pipette tip.
The whole pancreas and the islets of Langerhans obtained above were homogenized by adding Isogen (Nippon Gene), and total RNA was prepared according to the manual. Using 1 μg of the obtained total RNA, a random primer and SuperScript II reverse transcriptase (GIBCO BRL) as a reverse transcriptase were used to carry out a reaction at 42 ° C. according to the attached manual. After the reaction was completed, ethanol was precipitated and dissolved in 40 μl of distilled water. Quantification of the expression level of GPR40 mRNA was performed using Sequence Detection System Prism 7700 (ABI). 5′-CACACGCTCTCCTTCGCTCTCTAT-3 ′ (SEQ ID NO: 23), 5′-CAGTTTCGCGTGGGGACACT-3 ′ (SEQ ID NO: 24) as primers for amplification and detection of the target gene and 5 ′-(Fam) TCAGCCTTTGCACTAGGTAC-TCAGCTACTCTACT as a TaqMan probe (Tamra) -3 ′ (SEQ ID NO: 25) was used. The RT-PCR reaction solution was added to 12.5 μl of TaqManUniversal PCR Master Mix (PE Biosystems), 0.225 μl of 100 μM primer solution, 1.25 μl of 5 μM TaqMan probe, and 1 μl of the above-prepared cDNA solution, respectively. The total reaction volume was adjusted to 25 μl with distilled water. In the PCR reaction, a cycle of 50 ° C. for 2 minutes and 95 ° C. for 10 minutes, followed by a cycle of 95 ° C. for 15 seconds and 60 ° C. for 1 minute was repeated 40 times. As a result, it was confirmed that GPR40 was highly expressed in the isolated islets of Langerhans (FIG. 18).
[0116]
Example 8 Expression of GPR40 in cynomolgus monkey pancreatic islets of Langerhans
Isolation of islets of Langerhans from cynomolgus monkey pancreas was performed as follows. Both sides and thighs of a cynomolgus monkey (female, weighing 3 kg) were incised with a scalpel, exsanguinated, excised with scissors and laparotomized, and the duodenal and liver outlets of the common bile duct were bound with a clamp. Next, a hole was made in the liver side of the common bile duct with a syringe needle, and a cannula was inserted. 25 ml of a 1 mg / ml collagenase V (Sigma, C-9263) solution previously dissolved in HBSS and ice-cooled was injected through a cannula. The pancreas was cut off from other tissues, cut into small pieces of about 5 mm square with scissors, and shaken in a 37 ° C water bath for 20 minutes. The addition of several tens of ml of HBSS and pipetting were repeated several times, and the mixture was allowed to stand for 4 minutes and the supernatant was discarded. To the precipitate, 15 ml of 1.7 mg / ml collagenase XI (Sigma, C-7657) solution previously dissolved in HBSS and cooled on ice was added again, and the mixture was shaken in a 37 ° C water bath for 15 minutes. After repeatedly adding several tens of ml of HBSS and pipetting, the mixture was allowed to stand for 3 minutes and the supernatant was discarded. An appropriate amount of HBSS was added to the precipitate, and the suspension was transferred to a petri dish with a black bottom. HBSS was put in another petri dish, and the islets of Langerhans selected from the petri dish with a black bottom under a stereoscopic microscope were transferred to this petri dish using a pipette tip.
Total RNA was prepared from the whole pancreas and the islets of Langerhans obtained as described above in the same manner as in Example 7, cDNA was synthesized, and the expression level of GPR40 mRNA was determined by Sequence Detection System Prism 7700. However, for amplification and detection of monkey GPR40 mRNA, 5′-GCCCGCTTCAGCCTCTCT-3 ′ (SEQ ID NO: 26), 5′-GAGGCAGCCCCACGGTAGCA-3 ′ (SEQ ID NO: 27) as primers and 5 ′-(Fam) as TaqMan probe ) CTGCTTTTTTTCCTCGCCCTTTGCC- (Tamra) -3 '(SEQ ID NO: 28) was used. As a result, it was confirmed that GPR40 was also highly expressed in the islets of Langerhans in primates (FIG. 19).
[0117]
Example 9 Cloning of cDNA encoding GPR40 from hamster and determination of its nucleotide sequence
Using the hamster cell line HIT-T15 cDNA as a template, PCR was performed using primer 1 (SEQ ID NO: 31) and primer 2 (SEQ ID NO: 32). Klentaq DNA Polymerase (Clontech) was used for PCR. (1) 95 ° C. for 2 minutes, (2) 98 ° C. for 10 seconds, 63 ° C. for 20 seconds, 72 ° C. for 1 minute 35 times, and then to 72 ° C. -A 7-minute extension reaction was performed. After the reaction, the amplification product was cloned into a plasmid vector pCR2.1TOPO (Invitrogen) according to the prescription of TOPO TA Cloning Kit (Invitrogen). This was introduced into Escherichia coli JM109 (Takara Shuzo), and a clone having the plasmid was selected in an LB agar medium containing ampicillin. As a result of analyzing the nucleotide sequence of each clone, a cDNA sequence (SEQ ID NO: 30) encoding a novel G protein-coupled receptor protein was obtained. A novel protein containing an amino acid sequence (SEQ ID NO: 29) derived from this cDNA was named hamstar GPR40. The transformant was named Escherichia coli JM109 / pTA hamstar GPR40.
[0118]
Example 10 Preparation and immunization of an immunogen containing the C-terminal peptide of human GPR40
A complex of the C-terminal peptide of human GPR40, Cys Ala Ala Arg Thr Gln Gly Gly Lys Ser Gln Lys (SEQ ID NO: 33; synthesized according to a known method), and BSA was prepared and used as an immunogen. That is, 20 mg of BSA was dissolved in 2.0 ml of a 0.1 M phosphate buffer (pH 6.7) and mixed with 200 μl of a DMSO solution containing 2.8 mg of N- (γ-maleimidobutyryloxy) succinimide (GMBS), The reaction was performed at room temperature for 30 minutes. Excess GMBS reagent was removed with a Sephadex G-25 column equilibrated with 0.1 M phosphate buffer (pH 6.5) containing 2 mM EDTA, and dissolved in 5 mg of maleimide-introduced BSA and 0.1 ml of water. 0.84 mg of the C-terminal peptide was mixed and allowed to react at 4 ° C. overnight. After the reaction, the solution was dialyzed against physiological saline at 4 ° C. for 2 days. Eight-week-old BALB / C female mice were immunized subcutaneously with the C-terminal peptide-BSA complex, about 0.05 mg / animal, together with an adjuvant. Two weeks later, two weeks later, the same amount of immunogen was boosted with an adjuvant, and one week later, blood was collected.
[0119]
Example 11 Preparation of Horseradish Peroxidase (HRP) Labeled C-terminal Peptide
The above C-terminal peptide (SEQ ID NO: 33) and HRP (for enzyme immunoassay, manufactured by Boehringer Mannheim) were cross-linked to give a label for enzyme immunoassay (EIA). That is, 23 mg of HRP was dissolved in 2.3 ml of 0.1 M phosphate buffer, pH 6.7, mixed with 0.23 ml of a DMF solution containing 1.6 mg of GMBS, reacted at room temperature for 30 minutes, and then reacted with 2 mM EDTA. Was fractionated on a Sephadex G-25 column equilibrated with 0.1 M phosphate buffer, pH 6.5. 2.3 mg of the maleimide group-introduced HRP thus prepared and 1 mg of the C-chain N-terminal peptide were mixed and reacted at 4 ° C. for 1 day. After the reaction, fractionation was carried out using an Ultrogel AcA54 (manufactured by Pharmacia) column equilibrated with 0.1 M phosphate buffer, pH 6.5, to obtain an HRP-labeled C-terminal peptide.
[0120]
Example 12 Measurement of Antibody Titer in Antisera of Mice Immunized with C-Terminal Peptide
The antibody titer in the mouse antiserum was measured by the following method. To prepare an anti-mouse immunoglobulin antibody-bound microplate, first, a 96-well 0.1 M carbonate buffer, pH 9.6 solution containing 0.1 mg / ml goat anti-mouse immunoglobulin antibody (IgG fraction, manufactured by Kappel) was added. 0.1 ml was dispensed into the microplate and left at 4 ° C. for 24 hours. Next, the plate was washed with phosphate buffered saline (PBS, pH 7.4), and then 25% Block Ace (Snow Brand Milk Products) and 0.05% NaN were used to block excess binding sites in the wells. ₃ , PBS, pH 7.2, was dispensed in 0.3 ml portions and treated at 4 ° C for at least 24 hours.
Buffer EC [0.2% BSA, 0.4M NaCl, 0.4% Block Ace, 0.05% CHAPS [3-[(colamidopropyl) dimethyl] was added to each well of the anti-mouse immunoglobulin antibody-binding microplate. Ammonio] propanesulfonic acid], 2 mM EDTA and 0.05% NaN ₃ And 0.14 ml of mouse antiserum diluted with 0.02 M phosphate buffer, pH 7.0], and reacted at 4 ° C. for 16 hours. Next, the plate is washed with PBS, pH 7.4, and then diluted 1000-fold with buffer C [0.02 M phosphate buffer containing 1% BSA, 0.4 M NaCl, and 2 mM EDTA, PH 7.0]. 0.1 ml of the HRP-labeled C-terminal peptide prepared in the above example was added and reacted at 4 ° C. for 1 day. Next, the plate is washed with PBS, pH 7.4, and the enzymatic activity on the solid phase is measured by adding 0.1 ml of TMB microwell peroxidase substrate system (KIRKEGARD & PERRY LAB., Funakoshi Chemicals) for 20 minutes at room temperature. Was measured by After the reaction was stopped by adding 0.1 ml of 1M phosphoric acid, the absorbance at 450 nm (Abs. 450) was measured with a plate reader (MTP-120, manufactured by Corona or Multiscan Ascent, Labsystems). An increase in the antibody titer against the C-terminal peptide was observed in all of the immunized mice.
[0121]
Example 13 Preparation of Anti-C-terminal Peptide Monoclonal Antibody
The mice that showed a relatively high antibody titer in Example 12 were finally immunized by intravenously administering 0.08 to 0.1 mg of C-terminal peptide-BSA as BSA. Three days after the final immunization, the spleen was removed from the mouse to obtain a spleen cell suspension. As cells used for cell fusion, BALB / C mouse-derived myeloma cells P3-X63. Ag8. U1 (P3U1) was used [Current Topics in Microbiology and Immunology, 81, 1 (1978)]. Cell fusion was carried out according to the original method [Nature, 256, 495 (1975)]. That is, the spleen cells and P3U1 were each washed three times with a serum-free medium, mixed at a ratio of spleen gun and P3U1 of 6.6: 1, and centrifuged at 750 rpm for 15 minutes. Was precipitated. After sufficiently removing the supernatant, the precipitate was loosened gently, 0.3 ml of 45% polyethylene glycol (PEG) 6000 (manufactured by Kochlite) was added, and the mixture was allowed to stand still in a 37 ° C. warm water bath for 7 minutes to perform fusion. Was. After the fusion, MEM was gradually added to the cells, and a total of 15 ml of MEM was added, followed by centrifugation at 750 rpm for 15 minutes to remove the supernatant. This cell sediment was added to a GIT medium (Wako Pure Chemical Industries) (GIT-10% FCS) containing 10% fetal calf serum at a rate of 2 × 10 3 P1U1 per 1 m 2. ⁵ The cells were individually suspended and seeded on a 192 well in a 96-well multi-dish (manufactured by Limbro) in 0.1 ml per well. After seeding, the cells were cultured at 37 ° C. in a 5% CO 2 incubator. HAT after 24 hours (hypoxanthine 1 × 10 ^-4 M, aminopterin 4 × 10 ^-7 M, thymidine 1.6 × 10 ^-3 HAT selective culture was started by adding 0.1 ml per well of a GIT-10% FCS medium (HAT medium) containing M). The HAT selective culture was continued by discarding 0.1 ml of the old solution 5 or 8 days after the start of the culture and then adding 0.1 ml of HAT medium, and collecting the supernatant 9 days after the cell fusion.
The antibody titer in the culture supernatant was determined according to the method described in the Examples. That is, 0.07 ml of culture supernatant and 0.07 ml of buffer EC were added to each well of the anti-mouse immunoglobulin antibody-binding microplate, reacted at 4 ° C. overnight, and then HRP-labeled A chain diluted 1000-fold with buffer C. 0.1 ml of the N-terminal peptide was reacted for 7 hours at room temperature in the presence or absence of 0.002 mM of the unlabeled C-terminal peptide. After washing the plate with PBS, the enzyme activity on the solid phase was measured according to the method described in Example 13. As a result, wells having specific antibody titers were selected from 168 wells, and the hybridomas were cryopreserved. Further, the hybridoma in the well was subjected to cloning by a dilution method. After cloning, the antibody titer in the culture supernatant was measured according to the same method.
Hybridomas producing an anti-C-terminal peptide monoclonal antibody were selected from the positive clones.
[0122]
Example 14 Purification of Monoclonal Antibody
Monoclonal antibodies from the hybridomas were purified on a protein-A column. That is, about 25 ml of the hybridoma supernatant was added to an equal volume of a binding buffer (3.5 M NaCl, 0.05% NaN). ₃ And then applied to a recombinant protein-A-agarose (manufactured by Repligen) column previously equilibrated with a binding buffer to elute the specific antibody with an elution buffer (0.05% NaN ₃ And 0.1 M citrate buffer solution (pH 3.0). The eluate was dialyzed against PBS, pH 7.4 at 4 ° C. for 2 days, sterilized and filtered through a 0.22 μm filter (Millipore), and stored at 4 ° C. or −80 ° C.
[0123]
[Example 15] Measurement of antibody titer in antiserum of rabbit immunized with C-terminal peptide and preparation of polyclonal antibody
The antibody titer in the rabbit antiserum was measured by the following method. To prepare an anti-rabbit immunoglobulin antibody-bound microplate, first, a 0.1 M carbonate buffer, pH 9.6 solution containing 0.1 mg / ml of a goat anti-rabbit immunoglobulin antibody (IgG fraction, manufactured by Kappel) was added to a 96-well plate. 0.1 ml was dispensed into the microplate and left at 4 ° C. for 24 hours. Next, the plate was washed with phosphate buffered saline (PBS, pH 7.4), and then 25% Block Ace (Snow Brand Milk Products) and 0.05% NaN were used to block excess binding sites in the wells. ₃ , PBS, pH 7.2, was dispensed in 0.3 ml portions and treated at 4 ° C for at least 24 hours. Buffer EC [0.2% BSA, 0.4M NaCl, 0.4% Block Ace, 0.05% CHAPS [3-[(colamidopropyl) dimethyl] was added to each well of the anti-rabbit immunoglobulin antibody-bound microplate. Ammonio] propanesulfonic acid], 2 mM EDTA and 0.05% NaN ₃ And 0.14 ml of rabbit antiserum diluted with 0.02 M phosphate buffer, pH 7.0], and reacted at 4 ° C. for 16 hours. Next, the plate was washed with PBS, pH 7.4, and then diluted 1000-fold with buffer C [0.02 M phosphate buffer containing 1% BSA, 0.4 M NaCl, and 2 mM EDTA, PH 7.0]. 0.1 ml of the diluted HRP-labeled C-terminal peptide prepared in the above example was added and reacted at 4 ° C. for 1 day. Next, the plate is washed with PBS, pH 7.4, and the enzymatic activity on the solid phase is measured by adding 0.1 ml of TMB microwell peroxidase substrate system (KIRKEGARD & PERRY LAB., Funakoshi Chemicals) for 20 minutes at room temperature. Was measured by After the reaction was stopped by adding 0.1 ml of 1M phosphoric acid, the absorbance at 450 nm (Abs. 450) was measured with a plate reader (MTP-120, manufactured by Corona or Multiscan Ascent, Labsystems). Two out of three immunized rabbits showed an increase in the antibody titer against the C-terminal peptide. Further, the serum of a rabbit with an increased antibody titer was collected and subjected to antigen purification according to a standard method to obtain a polyclonal antibody.
[0124]
Example 16 Setting of a Screening Method for Agonists and Antagonists to the Receptor Using Change in Intracellular Calcium as an Index
To set up a system to search for agonists and antagonists to GPR40, an assay system was set up using palmitic acid identified as an agonist.
A CHO cell line (CHO-hGPR40 No. 104) expressing human GPR40 prepared by a method known per se using the human GPR40 expression vector prepared in Reference Example 1 was 3 × 10 6 ⁴ Per 100 μl of cells, and dispensed into a Black walled 96-well plate (Costar) at 100 μl per well. ₂ The cells were cultured overnight in an incubator. The fluctuation of the intracellular calcium concentration was measured using FLIPR (Molecular Device). The method is described below.
50 μg of Fluo-3AM (DOJIN) was dissolved in 21 μl of DMSO (DOJIN), and an equal amount of 20% pluronic acid (Molecular Probes) was added and mixed. After that, 10.6 ml of an assay buffer [HBSS] added with 105 μl of fetal bovine serum was added. (Invitrogen), 20 ml of 1 M HEPES (pH 7.4) (DOJIN) was added, 710 mg of probenecid (Sigma) was dissolved in 5 ml of 1N NaOH, and then 5 ml of the above HBSS / HEPES solution was added, and 10 ml of the mixed solution was added. Prepare. ] To prepare a fluorescent dye solution. Immediately after removing the medium from the cell plate, the fluorescent dye solution was dispensed in a volume of 100 μl per well. ₂ The cells were cultured in an incubator for 1 hour, and the fluorescent dye was incorporated into the cells. The cells after the culture were washed using the above-mentioned assay buffer. Palmitic acid to be added to the cells was diluted to each concentration using an assay buffer, and dispensed to the plate. A 12 μM γ-linolenic acid solution (final concentration at the time of reaction was 3 μM) was dispensed to the plate for antagonist measurement, and simultaneously set on the FLIPR. After the above pretreatment, the fluctuation of intracellular calcium concentration after addition of palmitic acid was measured by FLIPR, and an agonist effect was examined, and then an antagonist effect was examined by adding γ-linolenic acid. Since palmitic acid is an agonist, it cannot be evaluated in an experiment using palmitic acid.However, when a compound having only an antagonistic effect is added, the activity of suppressing the reaction of γ-linolenic acid should be observed. It is. From the dose-response curve using the change in the fluorescence intensity value 30 seconds after the start of the reaction, EC ₅₀ Values were calculated.
[0125]
Example 17 Activation of MAP Kinase in hGPR40-Expressing CHO Cells by Addition of Fatty Acid
A human GPR40-expressing CHO cell (CHO-hGPR40 No. 104) or a CHO-mock cell prepared by a method known per se using the human GPR40 expression vector prepared in Reference Example 1 was 3 × 10 4 ⁵ / Well in a 6-well plate, and cultured overnight in a serum-low concentration medium (MEMα medium without nucleic acid supplemented with 0.5% dialyzed fetal bovine serum). (MEM-free medium) and cultured overnight. After replacing with a fresh serum-free medium and culturing for 4 hours, 10 μM of various fatty acids were added. After incubation for 10 minutes, the cells were lysed and extracted with a sample buffer (TEFCO) and separated by SDS-PAGE. Thereafter, Western blotting using PhosphoPlus p44 / 42 MAP kinase (Thr202 / Tyr204) Antibody Kit (Cell Signaling Technology, Inc) was performed. As a result, as shown in FIG. 20, it was found that the activation of the protein indicated by the phosphorylation of MAP kinase occurs only after the addition of the fatty acid in the CHO cells expressing human GPR40.
[0126]
Example 18 Inhibition of Expression of Mouse GPR40-GFP Fusion Protein by Introducing siRNA Specific for Mouse GPR40 Sequence
CHO cells expressing a fusion protein of mouse GPR40 and GFP, prepared by a method known ⁴ / Well and spread in a 96-well plate for 1 day. HVJ Envelope VECTOR KIT GenomONE ^TM Using (Ishihara Sangyo), siRNAs of various sequences (Dharmacon) (2.86 pmol / 0.5 μl or 8.57 pmol) prepared according to the report of Elbasir et al. (Nature 411 (6836), 494-498 (2001)). /1.5 μl) was introduced into the cells and cultured for another day. The expression level of mouse GPR40-GFP was detected by an enzyme immunoassay as shown below. The culture supernatant was discarded, washed with HBSS (Invitrogen), fixed with 0.01% glutaraldehyde (Wako Pure Chemical) for 5 minutes, and blocked with PBS containing 2% BSA (Takara Shuzo). After adding 500-fold diluted anti-GFP monoclonal antibody 3E6 (Nippon Gene), the mixture was incubated at room temperature for 2 hours, then washed and diluted 500-fold with HRP-labeled anti-mouse IgG antibody (ICN), and incubated at room temperature for 2 hours. After washing, a TMB microwell peroxidase substrate (Funakoshi) was added. After incubation for 30 minutes, the color reaction was stopped by adding sulfuric acid, and the absorbance was measured at 450 nm. As a result, as shown in FIG. 21, mouse GPR40-specific siRNAs m40i103 (sense strand: SEQ ID NO: 34, antisense strand: SEQ ID NO: 35) and m40i256 (sense The addition of SEQ ID NO: 36 for the strand and SEQ ID NO: 37 for the antisense strand resulted in a decrease in the expression level of GPR40-GFP. This indicates that m40i103 and m40i256 specifically suppress mouse GPR40 expression. The sequences of m40i103 and m40i256 are shown in FIG.
[0127]
Example 19: Construction of DNA fragment for mouse production expressing human GPR40 specifically in pancreatic β cells
To specifically express human GPR40 in mouse pancreatic β cells, a DNA fragment was prepared by introducing a human GPR40 gene containing a poly (A) + additional signal downstream of the mouse insulin II promoter.
Downstream of the mouse insulin II promoter (MluI-SalI fragment: 673 bp) cloned by PCR from the mouse genome, and downstream of the poly (A) + addition signal from the start codon, also cloned by PCR from the human genome. A fragment of the human GPR40 gene (SalI-SpeI fragment: 2256 bp) was inserted to produce an expression unit for expressing the human GPR40 gene under the control of a mouse insulin II promoter. The thus obtained expression unit was cut out of a 2928 bp fragment by double digestion with MluI and SpeI to obtain a DNA fragment to be microinjected into fertilized eggs of C57BL / 6J mice.
[0128]
Example 20 Cloning of Mouse GPR40 Gene
A mouse 129SvJBAC genomic library (Invitrogen) was screened by PCR using a primer (SEQ ID NO: 38, SEQ ID NO: 39) capable of amplifying a region containing the mouse GPR40 ORF, and a state in which 384 colonies were mixed 3 independent positive clones were obtained. The three independent positive clones mixed with 384 colonies were probed with the DNA fragment (1.6 kbp) obtained by PCR using the above-mentioned SEQ ID NO: 38 and SEQ ID NO: 39 to colony. -Hybridization was performed and finally three positive single clones were isolated.
After isolating the BAC DNA carried by this clone by a conventional method, the nucleotide sequence of about 25 kb in which the GPR40 gene is thought to be located was determined using a sequencer (Perkin Elmer), and the nucleotide sequence was determined. A restriction map was prepared based on the information (FIG. 23).
[0129]
Example 21 Construction of GPR40 Gene Targeting Vector
The construction of the mouse GPR40 gene targeting vector is performed by adding a 5 'upstream arm of the GPR40 gene to the basic vector -pGT-N-28 (New England Biolabs).
The 11151 bp BamHI fragment obtained in Example 20 was introduced with a 3458 bp Ecl136II (SacI) fragment as the 3 'downstream arm, and the neomycin resistance gene and the negative selection marker were introduced into a positive selection marker and a negative selection marker, respectively. A mouse GPR40 gene targeting vector pGT-GPR40 having the herpes simplex virus thymidine kinase gene was constructed (FIG. 23). This was introduced into Escherichia coli DH5α to obtain Escherichia coli DH5α / pGT-GPR40 as a transformant.
[0130]
Example 22 Confirmation of Reactivity of Eicosanoid to Human GPR40
A CHO cell line expressing human GPR40 (CHO-hGPR40 No. 104), which was prepared by a method known per se using the human GPR40 expression vector prepared in Reference Example 1, was 10% dialyzed fetal bovine serum (unless otherwise specified). The cells were cultured using a MEMα medium (Invitrogen) containing Invitrogen. The cultured cells were rinsed using PBS (Invitrogen), peeled off using a 0.05% trypsin / EDTA solution (Invitrogen), and collected by centrifugation. The number of cells obtained was measured and 3 × 10 ⁴ And diluted into a Black walled 96-well plate (Costar) by 100 μl per well. ₂ The cells were cultured overnight in an incubator. Various test samples were added to the CHO cell line, and the change in intracellular calcium concentration at this time was measured using FLIPR (Molecular Device). In order to measure the fluctuation of intracellular calcium concentration by FLIPR, the following pretreatment was performed. First, an assay buffer was prepared for adding the fluorescent dye Fluo3-AM (DOJIN) to the cells or washing the cells immediately before performing the FLIPR assay. To a solution (hereinafter, HBSS / HEPES solution) obtained by adding 20 ml of 1M HEPES (pH 7.4) (DOJIN) to 1000 ml of HBSS (Invitrogen), 710 mg of probenecid (Sigma) is dissolved in 5 ml of 1N NaOH, and 5 ml of the HBSS / HEPES solution is further dissolved. 10 ml of the mixed solution was added, and this solution was used as an assay buffer. Next, 50 μg of Fluo3-AM was dissolved in 21 μl of DMSO (DOJIN), an equal amount of 20% pluronic acid (Molecular Probes) was added and mixed, and then added to 10.6 ml of assay buffer to which 105 μl of fetal calf serum had been added. A fluorescent dye solution was prepared. Immediately after removing the culture medium of the cultured CHO cell line, 100 μl per well of the fluorescent dye solution was dispensed, ₂ The cells were cultured in an incubator for 1 hour, and the fluorescent dye was incorporated into the cells. The cells after the culture were washed with the above-mentioned assay buffer, and then set on the FLIPR. In addition, a test sample to be added to the CHO cell line was prepared using an assay buffer, and simultaneously set in the FLIPR. After the above pretreatment, the fluctuation of intracellular calcium concentration after addition of various test samples was measured by FLIPR. As a result, (±) 14,15-dihydroxy-5Z, 8Z, 11Z-eicosatrienoic acid ((±) 14,15-dihydroxy-5Z, 8Z, 11Z-eicosatrienoic acid, 14,15-DHT), ( ±) 5 (6) -epoxy-8Z, 11Z, 14Z-eicosatrienoic acid ((±) 5 (6) -epoxy-8Z, 11Z, 14Z-eicosatrienoic acid, 5,6-EET), (±) 8 (9) -Epoxy-5Z, 11Z, 14Z-eicosatrienoic acid ((±) 8 (9) -epoxy-5Z, 11Z, 14Z-eicosatrienoic acid, 8,9-EET), (±) 11 (12) -Epoxy-5Z, 8Z, 14Z-eicosatrienoic acid ((±) 11 (12) -epoxy-5Z, 8Z, 14 Z-eicosatrienoic acid, 11,12-EET), (±) 14 (15) -epoxy-5Z, 8Z, 11Z-eicosatrienoic acid ((±) 14 (15) -epoxy-5Z, 8Z, 11Z-eicosatrianic) acid, 14, 15-EET) ^-5 M-10 ^-6 It was found that when M was added, the CHO cell line expressing the GPR40 receptor responded specifically (increased intracellular calcium concentration). Such a response was not observed in the CHO cell line (Mock cell line) into which only the control expression vector was introduced. Therefore, GPR40 was also found to react with these compounds.
[0131]
[Example 23] Examination of expression distribution of GPR40 mRNA in islet by in situ hybridization
Wistar rats were sacrificed, and after ligation of the bile duct duodenal orifice, 1 mg / ml cold collagenase / HBSS was injected into the pancreas from the bile duct. The pancreas into which collagenase had been injected was cut off, transferred to a conical tube, and incubated at 37 ° C. for 10 minutes while penetrating. After adding HBSS and dispersing the tissue well by pipetting, the tissue containing islet was precipitated by leaving still for 3 to 4 minutes, and the supernatant was removed. After repeating this operation twice, the cells were resuspended in HBSS and the islet was picked up under a stereoscopic microscope. The islet isolated as described above was frozen with liquid nitrogen in an OCT compound and stored at -80 ° C.
GPR40 antisense and sense probes were prepared by the following method. First, rat GPR40 cDNA was inserted into a plasmid vector pCRII TOPO (Invitrogen) by a method known per se. This cDNA was amplified and linearized by PCR using M13 primer (Invitrogen) and Advantage GC2 polymerase (Clontech), and purified by ethanol precipitation. From this cDNA, in vitro transcription with SP6 or T7 (40 μl scale) was performed using DIG RNA Labeling KIT (SP6 / T7) (Roche), and the generated DIG-labeled riboprobe was further added to 40 mM NaHCO3. ₃ , 60 mM Na ₂ CO ₃ After alkaline hydrolysis to 400 bp with pH 10.2, purification was performed by ethanol precipitation. The purified probe was dissolved in 100 μl of Otsuka purified water. From the cDNA insertion direction, the DIG-labeled riboprobe generated by SP6 was an antisense probe, and the DIG-labeled riboprobe generated by T7 was a sense probe.
Fresh frozen sections of the isolated islet were used for in situ hybridization. First, the above-mentioned isolated islet was sliced at a thickness of 16 μm on a silane-coated slide using a cryostat CM3050 (Leica). The sections were fixed in PBS containing 4% paraformaldehyde for 10 minutes, washed well with PBS, and treated with 0.1 M triethanolamine (pH 8.0) containing 0.25% acetic anhydride (room temperature, 10 minutes) ) For acetylation. The hybridization reaction was carried out in a hybridization buffer (50% formamide, 10 mM Tris-HCl pH 7.5, 1 × Denhardt's solution, 200 μg / ml tRNA, 10% dextran sulfate, 600 mM NaCl, 0.25% SDS, 1 mM EDTA). The antisense probe or the sense probe was diluted 1000-fold, denatured at 85 ° C. for 10 minutes, added to a section, and allowed to react at 50 ° C. for 12 hours or more. Subsequently, the following operation was performed to wash the non-specifically hybridized probe. 1) 2 × SSC (SSC; 1 × SSC = 150 mM NaCl, 15 mM sodium citrate, pH 7.0) / 50% formamide treatment (60 ° C., 30 minutes once), 2) 2 × SSC treatment (60 ° C., 15) 1 minute), 3) 0.1 × SSC treatment (60 ° C., twice for 15 minutes). After performing the above operations, immunohistochemistry for detecting the DIG label probe was performed. First, after washing with DIG-1 (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween 20), treatment with DIG-1 containing 1.5% Blocking reagent (Roche) (37 ° C. Non-specific reaction was blocked for 1 hour), and DIG-1 (1: 1000) containing anti-DIG fab-fragment anti-conjugated with alkaline phosphatase (Roche) was reacted at room temperature for 1 hour. After sufficiently washing with DIG-1, DIG-3 (100 mM Tris-HCl pH 9.5, 100 mM NaCl, 50 mM MgCl ₂ ), Dimethylformamide containing 0.18 mg / ml 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), and 70% dimethylformamide containing 0.34 mg / ml 4-nitroblue tetrazolium (NBT), A color development reaction was carried out at room temperature with a solution in which 0.35 ml, 0.45 ml and 3% of polyvinylalchol were added to 10 ml of DIG-3, respectively. After the color development was appropriately stopped by washing with PBS, the procedure was shifted to insulin glucagon immunohistochemistry.
First, non-specific reaction was blocked with PBS containing 1% BSA, and a sample containing rabbit anti-insulin serum (H-86: Santa Cruz, 1/200) and anti-glucagon monoclonal antibody (K79bB10: SIGMA, 1/8000) was used. Incubated with 0.5% BSA / PBS for 12 hours. After washing with PBS, 10 μg / ml Alexa 488-conjugated goat anti-mouse IgG (Molecular Probes) and 10 μg / ml Alexa 594-conjugated goat anti-rabbit IgG (including room temperature PBS and 0.5% PBS / MolbeBS) containing Alexa 594-conjugated goat anti-rabbit IgG For 2 hours. After washing with PBS, the cells were sealed with PBS containing 90% glycerol and observed with an optical microscope and a fluorescence microscope.
With the GPR40 antisense probe, a positive signal was observed in the isolated islet. Positive signals were scattered throughout the islet. No color development by the sense probe was detected in these regions. On the other hand, by fluorescence microscopy, a glucagon immunopositive signal was observed in cells around the islet and an insulin immunopositive signal was observed in other parts, as is known. Comparing the signal obtained by the GPR40 antisense probe with the insulin glucagon immunopositive signal, a GPR40 signal was observed in insulin immunopositive cells, but a clear GPR40 signal was not observed in glucagon immunopositive cells. These results suggested that GPR40 mRNA may be specifically expressed in β cells in islet.
[0132]
Example 24 Activation of MAP Kinase in MIN6 Cells by Addition of Fatty Acid
The medium was 15% fetal bovine serum (ThermoTrace), 5.5 μM 2-mercaptoethanol (Invitrogen), 20 mM HEPES in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) containing 4.5 g / l glucose. A solution to which pH 7.3, 100 U / ml penicillin and 100 μg / ml streptomycin was added was used. 2 x 10 mouse pancreatic β cell line MIN6 cells ⁵ The cells were cultured overnight at a concentration of / well in a 12-well plate, and the medium was exchanged the next morning and further cultured overnight. The next morning, the medium was changed again and the cells were cultured for 3 hours, and then pre-incubated for 1 hour with KRBH containing 22 mM glucose. Various fatty acids were dissolved in KRBH (22 mM glucose) at a concentration of 10 μM, added to MIN6 cells, and incubated for 2.5 minutes (or 5 minutes). The MAPK inhibitor (PD98059) was added to KRBH at a concentration of 50 μM during the pre-incubation incubation. After lysing and extracting the cells with a sample buffer (TEFCO), the cells were separated by SDS-PAGE, and the cells were separated using PhosphoPlus p44 / 42 MAP kinase (Thr202 / Tyr204) Antibody Kit (Cell Signaling Technology, Inc.). . As a result, as shown in FIG. 24, in the FLIPR assay using GPR40-expressing CHO cells, activation of the protein indicated by phosphorylation of MAP kinase only when a fatty acid having an agonist activity (lanes 2, 3, 4) was added. It was found that MAPK activation was almost suppressed by adding PD98059. Similar to GPR40-expressing CHO cells, MAPK activation occurs in MIN6 with a fatty acid that exhibits agonist activity, indicating that GPR40 is involved in at least part of MIN6 stimulation of fatty acid.
[0133]
Example 25 Promotion of insulin secretion from MIN6 by various fatty acids
The mouse pancreatic β-cell line MIN6 cultured in the flask was peeled off using PBS containing 2.5 mM EDTA, and then spread on a 96-well plate and cultured for 2 days. The medium was 15% fetal bovine serum (ThermoTrace) in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) containing 4.5 g / l glucose as in Example 24, and 5.5 μM 2-mercaptoethanol (Invitrogen). And 20 mM HEPES pH 7.3, 100 U / ml penicillin, and 100 μg / ml streptomycin. Krebs-Ringer bicarbonate buffer (KRBH, 116 mM NaCl, 4.7 mM KCl, 1.2 mM KH) obtained by modifying cells ₂ PO ₄ , 1.2 mM MgSO ₄ , 2.5 mM CaCl ₂ , 25 mM NaHCO ₃ , 24 mM HEPES pH 7.3), and then washed at 37 ° C. with 5% CO 2. ₂ Pre-incubated for 30 minutes under conditions. The above-mentioned fatty acid diluted and prepared with the buffer obtained by adding 22 mM glucose to KRBH is added to the cells after the pre-incubation, and the cells are added at 37 ° C. and 5% CO 2. ₂ The cells were cultured under the conditions for 90 minutes. After the culture, the cell supernatant was collected and stored frozen. The insulin content in the supernatant was measured using a commercially available insulin immunoassay kit (Amersham Pharmacia). As a result, as shown in FIG. 25, oleic acid (oleic acid), linoleic acid (linoleic acid), and α-linolenic, which are fatty acids that exhibited remarkable activity in an intracellular calcium ion mobilization assay using GPR40-expressing CHO cells. Acid (α-linolenic acid), γ-linolenic acid (γ-linolenic acid), arachidonic acid (arachidonic acid), and DHA showed significant insulin secretion increasing activity. On the other hand, methyl linoleate (linoleic methyl) and butyric acid (Butyricacid), which do not show an agonist activity in GPR40, did not show significant insulin secretion increasing activity. Since the GPR40 agonist activity and the insulin secretion increasing activity are almost correlated, it was proved that GPR40 plays at least a part of the insulin secretion increasing effect of fatty acids.
[0134]
Example 26 Glucose Dependence of GPR40-mediated Insulin Secretion Elevation Activity
Based on the method of Example 25, the effect of the glucose concentration when adding a fatty acid was examined. As a result of examining the concentration of glucose added to KRBH at the time of adding fatty acids to 0, 5.5, 11, and 22 mM, as shown in FIG. 26, the activity of increasing insulin secretion by oleic acid and linoleic acid was shown as shown in FIG. Was found to be significant under conditions of high glucose concentration of 11 mM or more. From this, it was expected that the insulin secretion-increasing action by a fatty acid having an agonist activity on GPR40 would be an insulin secretion-increasing action / blood-sugar effect under high blood sugar conditions.
[0135]
Example 27 Inhibitory Effect of Fatty Acid on Insulin Secretion Elevating Activity of siRNA-Introduced MIN6
In Example 18, it was confirmed that m40i103, which is a GPR40-specific siRNA, significantly suppresses the expression of mouse GPR40. Based on the method of Example 18, the activity of increasing insulin secretion in MIN6 cells transfected with m40i103 was examined using HVJ Envelope VECTOR KIT GenomONE. As a result of examining the insulin secretion-increasing activity of the fatty acid by the method of Example 25, as shown in FIG. 27, the m40i103-introduced MIN6 showed the insulin secretion-increasing activity of linoleic acid and γ-linolenic acid. No longer allowed. On the other hand, it was shown that MIN6 into which Scramble II duplex siRNA, which is a random sequence siRNA, was introduced retains the insulin secretion increasing activity of the above fatty acids. From these results, it was confirmed that GPR40 plays at least a part of the mechanism of increasing insulin secretion by fatty acids.
[0136]
Example 28 Insulin secretion activity of fatty acids in primary culture of rat pancreatic islets of Langerhans
Langerhans islets were isolated from the pancreas of 8-week-old male Wister rats (Charles River Japan) by collagenase digestion. Approximately 1000 isolated islets of Langerhans for 6 animals were washed twice with KRBH (2.8 mM Glucose), and then directly at 37 ° C. for 30 minutes with 5% CO 2. ₂ Preincubated in the presence. After centrifugation, the supernatant was discarded, the suspension was again suspended in KRBH (2.8 mM Glucose) at a concentration of 140 cells / mL, and 100 μL was dispensed into a 96-well plate. 100 μL of KRBH (final 16.5 mM Glucose) containing linoleic acid (γ-linolenic acid), methyl linoleate (methyl linoleate) (final 30 μM or 3 μM each) of various fatty acid samples is added. , 37 ° C, 90 minutes, 5% CO ₂ Incubated in the presence. The insulin content in the supernatant was measured using a commercially available immunoassay kit (Amersham). As a result, as shown in FIG. 28, the insulin secretagogue-promoting activity was also confirmed in rat pancreatic islets of Langerhans by treating GPR40 with a fatty acid having an agonistic activity. It was confirmed that GPR40 is involved in insulin secretion not only in the β cell line MIN6 examined in Examples 25 to 27 but also in the normal pancreatic islets of Langerhans.
[0137]
Example 29 Ca in insulin secretion via GPR40 ²⁺ The need for ions
Based on the method of Example 25, ²⁺ The necessity of ions was discussed. During preincubation and incubation with the sample, Ca ²⁺ Remove the ions and further Ca ²⁺ The insulin secretion increasing activity of fatty acids when EGTA, a reagent for chelating ions, was added at 0.1 mM was examined. As a result, as shown in FIG. 29, the insulin secretion increasing activity due to oleic acid (oleic acid), linoleic acid, and γ-linolenic acid (γ-linolenic acid) observed in a normal buffer was Ca. ²⁺ It was confirmed that this was not observed in the ion-free buffer containing 0.1 mM EGTA. From this fact, the activity of GPR40-mediated insulin secretion increase by fatty acids was Ca ²⁺ It has been determined that ions are essential.
[0138]
Example 30 Effect of various inhibitors on GPR40-mediated insulin secretion
According to the method of Example 25, Ca ²⁺ The effects of channel blockers (Nifedipine, Nifedipine) and MAP kinase inhibitor (PD98059) were examined. In preincubation and incubation with the sample, the insulin secretion increasing activity of fatty acids when Nifedipine (10 μM) or PD98059 (50 μM) was added was examined. As a result, as shown in FIG. 30, the activity of increasing insulin secretion due to oleic acid and linoleic acid, which was observed in the normal buffer, was not observed in the nifedipine-containing buffer. This is the case in Example 29 ²⁺ Consistent with the results obtained when the ions were removed, fatty acid-induced insulin secretion-enhancing activity via GPR40 was Ca ²⁺ It was once again confirmed that ions were essential. In addition, since the same results were obtained with the buffer containing PD98059 as in a normal buffer, the activity of increasing insulin secretion by fatty acids occurred even when MAP kinase was not activated. In other words, activation of MAP kinase by fatty acid stimulation was not achieved. It was found that the activity was not necessarily required for insulin secretion increasing activity. It was considered that GPR40 may exert a function not only on insulin secretion but also on proliferation of β cells.
[0139]
Example 31 Effect of BSA on GPR40-mediated insulin secretion
Fatty acids are known to bind to albumin. Based on the method of Example 25, the effect of the presence of bovine serum albumin (BSA) was examined. During preincubation and incubation with the sample, 0.1% BSA was added to the buffer, and the activity of increasing insulin secretion by fatty acids was examined. As a result, as shown in FIG. 31, the activity of increasing insulin secretion due to oleic acid and linoleic acid, which was observed in the normal buffer, was almost suppressed in the buffer containing 0.1% fatty acid-free BSA. . It has been found that insulin secretion-enhancing activity is caused by unbound fatty acids rather than BSA-bound fatty acids. It can be said that the characteristic of the fatty acid that binds to BSA is reflected in the reactivity.
[0140]
Example 32 EC of fatty acid for GPR40 derived from human, mouse and rat ₅₀ Determination of value
EC ₅₀ For determination of the value, a CHO cell line stably expressing human, mouse and rat-derived GPR40 was used. Unless otherwise specified, these CHO cell lines were cultured using α-MEM medium (Invitrogen) containing 10% fetal calf serum (Invitrogen).
On the day before the assay, the cells cultured until they were almost confluent were rinsed with PBS (Invitrogen), peeled off with a 0.05% Trypsin / EDTA solution (Invitrogen), and collected by centrifugation. The number of cells obtained was measured, and 3 × 10 ⁵ And diluted into a Black welled 96-well plate (coster) by 100 μL per well. ₂ The cells were cultured overnight in an incubator. Various test samples were added to the CHO cells thus adjusted, and the change in intracellular calcium concentration at this time was measured using FLIPR (Molecular Device). In order to measure the fluctuation of intracellular calcium concentration by FLIPR, the following pretreatment was performed.
First, an assay buffer was prepared for adding the fluorescent dye Fluo3-AM (DOJIN) to the cells or washing the cells immediately before performing the FLIPR assay. After dissolving 710 mg of probenecid (Sigma) in 5 mL of 1N NaOH, a solution obtained by adding 20 mL of 1M HEPES (pH 7.4) (DOJIN) to 1000 mL of HBSS (Invitrogen), and further dissolving the same in 5 mL of 1N NaOH. 10 mL of a mixed solution was added to 5 mL of the HEPES solution, and this solution was used as an assay buffer. Next, 50 μg of Fluo3-AM was dissolved in 21 μL of DMSO (Wako), an equal amount of 20% pluronic acid (Molecular Probes) was added and mixed, and the mixture was added to 10.6 mL of assay buffer containing 105 μL of fetal calf serum, followed by fluorescence. A dye solution was prepared. On the day before the assay, the medium of the CHO cells replated on the Black welled 96-well plate was removed, and the fluorescent dye solution was immediately dispensed at 100 μL per well. ₂ The cells were cultured in an incubator for 1 hour, and the fluorescent dye was incorporated into the cells. The cells after the culture were washed with the above-mentioned assay buffer, and then set on the FLIPR. A test sample was prepared using an assay buffer, diluted to a final concentration of 30, 10, 3, 1, 0.3 μM, and simultaneously set in the FLIPR. After the above pretreatment, the fluctuation of intracellular calcium concentration after addition of various test samples was measured by FLIPR. From these results, a volume response curve was created for each fatty acid, and EC ₅₀ Values were calculated. The results are shown in Table 1.
[Table 1]

[0141]
Example 33 Effect of fatty acids on cAMP production in human and mouse GPR40 expressing CHO cells
CHO cells expressing human and mouse GPR40 were expressed in 1 × 10 ⁵ The cells were cultured at a concentration of / well on a 96-well plate for 20 hours. The cells were washed twice with 100 μl of Assay Buffer (DMEM (Invitrogen) containing 0.1 mM IBMX (Wako Pure Chemical) and 0.1 mM Ro-20-1724 (Biomol)), and then washed with 2 μM Forskolin (Wako Pure). DHA, linoleic acid and γ-linolenic acid (both SIGMA) dissolved in Assay Buffer with or without the drug) were added and reacted at 37 ° C. for 10 minutes. After the reaction, the cells were lysed according to the prescription of cAMP Screen (ABI), and the amount of cAMP in the cells was measured. As a result, under the condition without forskolin, each fatty acid did not show cAMP producing activity on human and mouse GPR40-expressing CHO cells. On the other hand, in the presence of forskolin, it exhibited weak cAMP production inhibitory activity on human GPR40-expressing CHO cells, but did not show activity on mouse GPR40-expressing CHO cells (FIG. 32).
[0142]
Example 34 FLIPR Assay Using MIN6
After the mouse pancreatic β cell line MIN6 cultured in the flask was peeled off using PBS containing 2.5 mM EDTA, 3 × 10 6 cells / mL of the medium were removed. ⁵ And diluted into a Black welled 96-well plate (coster) by 100 μL per well. ₂ The cells were cultured in an incubator for 2 days. The medium was 15% fetal bovine serum (ThermoTrace), 5.5 μM 2-mercaptoethanol (Invitrogen), 20 mM HEPES in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) containing 4.5 g / l glucose. A solution to which pH 7.3, 100 U / ml penicillin and 100 μg / ml streptomycin was added was used. Various fatty acids (10 μM) were added to the MIN6 cells thus adjusted, and the change in intracellular calcium concentration at this time was measured using FLIPR (Molecular Device). The method for measuring the fluctuation of intracellular calcium concentration by FLIPR followed the method described in Example 24. As a result, the addition of γ-linolenic acid (γ-linolenic acid), linoleic acid (linoleic acid), oleic acid (oleic acid), arachidonic acid (arachidonic acid), and docosahexaenoic acid (DHA), which are ligands of GPR40, resulted in MIN6. Intracellular Ca ²⁺ It was confirmed that the concentration increased. On the other hand, methyllinoleate and butyric acid, which are not ligands for GPR40, did not show such activity (FIG. 33).
[0143]
Example 35 Setting of Screening Method for Agonists and Antagonists to GPR40 and Criteria for Selecting Candidate Agonists
(1) Setting of screening method for agonist and antagonist for GPR40 using change in intracellular calcium concentration as an index
To set up a system to search for agonists and antagonists to GPR40, an assay system was set up using palmitic acid identified as an agonist.
A CHO cell line (CHO-hGPR40 No. 104) expressing human GPR40 prepared by a method known per se using the human GPR40 expression vector prepared in Reference Example 1 was 3 × 10 6 ⁴ Per 100 μl of cells, and dispensed into a Black walled 96-well plate (Costar) at 100 μl per well. ₂ The cells were cultured overnight in an incubator. The fluctuation of the intracellular calcium concentration was measured using FLIPR (Molecular Device). The method is described below.
50 μg of Fluo-3AM (DOJIN) was dissolved in 21 μl of DMSO (DOJIN), and an equal volume of 20% pluronic acid (Molecular Probes) was added and mixed. After that, 10.6 ml of assay buffer [HBSS] added with 105 μl of fetal bovine serum 20 ml of 1M HEPES (pH 7.4) (DOJIN) was added to (Invitrogen), 710 mg of probenecid (Sigma) was dissolved in 5 ml of 1N NaOH, and then 5 ml of the above HBSS / HEPES solution was added. Prepare. ] To prepare a fluorescent dye solution. Immediately after removing the medium from the cell plate, the fluorescent dye solution was dispensed in a volume of 100 μl per well. ₂ The cells were cultured at 37 ° C. for 1 hour in an incubator, and the fluorescent dye was incorporated into the cells. The cells after the culture were washed using the above-mentioned assay buffer. Palmitic acid to be added to the cells was diluted to each concentration using an assay buffer, and dispensed to the plate. A 12 μM γ-linolenic acid solution (final concentration at the time of reaction: 3 μM) was dispensed to the plate for antagonist measurement, and simultaneously set on the FLIPR. After the above pretreatment, the change in intracellular calcium concentration after addition of palmitic acid was measured by FLIPR, and the agonist effect was examined, and then the antagonist effect was examined by adding γ-linolenic acid. Since palmitic acid is an agonist, the evaluation of an antagonist is not established in experiments using palmitic acid.However, when a compound having only an antagonistic action is added, the activity of suppressing the reaction of γ-linolenic acid added later has an effect. Observed. From the dose-response curve using the change in the fluorescence intensity value 30 seconds or 40 seconds after the start of the reaction, EC ₅₀ Values were calculated.
(2) Criteria for selecting an agonist candidate from the results of the FLIPR assay
A test compound for selecting an agonist candidate was diluted with DMSO (Wako) in advance so that its concentration was 10 mM, and diluted with the above assay buffer at the time of measurement. A CHO cell line (CHO-hGPR40) expressing human GPR40 prepared by a method known per se using the human GPR40 expression vector prepared in Reference Example 1 in the same manner as described above using these test compounds. No. 104), CHO cell line expressing human histamine H1 receptor prepared by a method known per se using a human histamine H1 receptor expression vector (CHO-H1), and Mock's reaction with CHO cell line 30 The fluorescence intensity value after 2 or 40 seconds was measured. Furthermore, the relative value of the fluorescence intensity of 30 μM γ-linolenic acid with respect to CHO-hGPR40 was calculated as 100%, the value was 50 or 100% or more with respect to CHO-hGPR40, and the human histamine H1 receptor and A test compound in which both of the Mock CHO cell lines satisfy 25% or less was determined as a candidate human GPR40-specific agonist.
[0144]
Example 36 Inhibition of Mouse GPR40 mRNA Expression by Introducing siRNA Specific for Mouse GPR40 Sequence
After MIN6 cells were seeded on a 24-well plate and cultured for one day, m40i103 or Scramble II duplex siRNA which was a random sequence siRNA was introduced using HVJ Envelope VECTOR KIT GenomONE based on the method of Example 18. After culturing for 2 days, total RNA of the cells was prepared in the same manner as in Example 7 using Isogen (Nippon Gene) according to the attached manual. CDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen) using 100 ng random hexamer as a primer for 1 μg of the obtained total RNA, and then dissolved in 40 μl TE buffer (pH 8.0). The expression levels of mouse GPR40 and mouse GAPDH mRNA were determined by the method described in Example 4. As a result, as shown in FIG. 34, it was revealed that the expression of GPR40 mRNA in MIN6 cells into which m40i103 had been introduced was lower than that in MIN6 cells into which siRNA having a random sequence had been introduced.
[0145]
【The invention's effect】
The GPR40 of the present invention, its partial peptide or a salt thereof, or the DNA encoding the GPR40 or its partial peptide of the present invention is useful as an agent for preventing or treating diabetes and the like.
By using the GPR40 of the present invention, its partial peptide or its salt and a fatty acid which is a ligand, it is possible to efficiently screen for a compound that changes the binding property between the fatty acid and the GRP40 of the present invention, its partial peptide or its salt. it can.
[0146]
[Sequence List Free Text]

[0147]
[Sequence list]

[Brief description of the drawings]
FIG. 1. Intracellular Ca upon addition of farnesic acid ²⁺ The result of having examined the change of density is shown. The vertical axis is intracellular Ca ²⁺ The fluorescence intensity indicating the concentration, and the horizontal axis indicates the lapse of time after the addition of the sample. ○ indicates GPR40-expressing CHO-K1 cells, and △ indicates control CHO-K1 cells.
FIG. 2: Intracellular Ca upon addition of 5.8.11-eicosatrynoic acid ²⁺ The result of having examined the change of density is shown. The vertical axis is intracellular Ca ²⁺ The fluorescence intensity indicating the concentration, and the horizontal axis indicates the lapse of time after addition of the sample. ○ indicates GPR40-expressing CHO-K1 cells, and △ indicates control CHO-K1 cells.
FIG. 3. Intracellular Ca upon addition of 5.8.11.14-eicosatraynoic acid ²⁺ The result of having examined the change of density is shown. The vertical axis is intracellular Ca ²⁺ The fluorescence intensity indicating the concentration, and the horizontal axis indicates the lapse of time after addition of the sample. ○ indicates GPR40-expressing CHO-K1 cells, and △ indicates control CHO-K1 cells.
FIG. 4. Intracellular Ca when linoleic acid is added ²⁺ The result of having examined the change of density is shown. The vertical axis is intracellular Ca ²⁺ The fluorescence intensity indicating the concentration, and the horizontal axis indicates the lapse of time after addition of the sample. ○ indicates GPR40-expressing CHO-K1 cells, and △ indicates control CHO-K1 cells.
FIG. 5. Intracellular Ca upon addition of linolenic acid ²⁺ The result of having examined the change of density is shown. The vertical axis is intracellular Ca ²⁺ The fluorescence intensity indicating the concentration, and the horizontal axis indicates the lapse of time after the addition of the sample. ○ indicates GPR40-expressing CHO-K1 cells, and △ indicates control CHO-K1 cells.
FIG. 6. Intracellular Ca upon addition of arachidonic acid ²⁺ The result of having examined the change of density is shown. The vertical axis is intracellular Ca ²⁺ The fluorescence intensity indicating the concentration, and the horizontal axis indicates the lapse of time after addition of the sample. ○ indicates GPR40-expressing CHO-K1 cells, and △ indicates control CHO-K1 cells.
FIG. 7: Intracellular Ca when eicosapentaenoic acid (EPA) is added ²⁺ The result of having examined the change of density is shown. The vertical axis is intracellular Ca ²⁺ The fluorescence intensity indicating the concentration, and the horizontal axis indicates the lapse of time after the addition of the sample. ○ indicates GPR40-expressing CHO-K1 cells, and △ indicates control CHO-K1 cells.
FIG. 8. Intracellular Ca when eicosadienic acid is added. ²⁺ The result of having examined the change of density is shown. The vertical axis is intracellular Ca ²⁺ The fluorescence intensity indicating the concentration, and the horizontal axis indicates the lapse of time after the addition of the sample. ○ indicates GPR40-expressing CHO-K1 cells, and △ indicates control CHO-K1 cells.
FIG. 9: Intracellular Ca when eicosatrienoic acid is added ²⁺ The result of having examined the change of density is shown. The vertical axis is intracellular Ca ²⁺ The fluorescence intensity indicating the concentration, and the horizontal axis indicates the lapse of time after the addition of the sample. ○ indicates GPR40-expressing CHO-K1 cells, and △ indicates control CHO-K1 cells.
FIG. 10. Intracellular Ca when docosahexaenoic acid (DHA) was added. ²⁺ The result of having examined the change of density is shown. The vertical axis is intracellular Ca ²⁺ The fluorescence intensity indicating the concentration, and the horizontal axis indicates the lapse of time after the addition of the sample. ○ indicates GPR40-expressing CHO-K1 cells, and △ indicates control CHO-K1 cells.
FIG. 11. Intracellular Ca when docosatrienoic acid was added. ²⁺ The result of having examined the change of density is shown. The vertical axis is intracellular Ca ²⁺ The fluorescence intensity indicating the concentration, and the horizontal axis indicates the lapse of time after addition of the sample. ○ indicates GPR40-expressing CHO-K1 cells, and △ indicates control CHO-K1 cells.
FIG. 12 shows intracellular Ca when adrenic acid is added. ²⁺ The result of having examined the change of density is shown. The vertical axis is intracellular Ca ²⁺ The fluorescence intensity indicating the concentration, and the horizontal axis indicates the lapse of time after the addition of the sample. ○ indicates GPR40-expressing CHO-K1 cells, and △ indicates control CHO-K1 cells.
FIG. 13. Intracellular Ca when lauric acid was added. ²⁺ The result of having examined the change of density is shown. The vertical axis is intracellular Ca ²⁺ The fluorescence intensity indicating the concentration, and the horizontal axis indicates the lapse of time after addition of the sample. ○ indicates GPR40-expressing CHO-K1 cells, and △ indicates control CHO-K1 cells.
FIG. 14 shows the expression distribution of GPR40 mRNA in various human tissues. The horizontal axis is poly (A) ⁺ Shows the number of copies per RNA (ng).
FIG. 15 shows the distribution of GPR40 mRNA expression in various mouse cells. Min-6 indicates MIN6 cells, NIH log indicates the logarithmic growth phase of NIH / 3T3, NiHconf indicates the stationary phase of NIH / 3T3, and B104 log indicates the logarithmic growth phase of NIH / 3T3 transformed with the oncogene neu. , B104 conf indicates the stationary phase of NIH / 3T3 transformed with the oncogene neu, mLliver indicates the mouse liver, and mSplen mouse spleen. The horizontal axis indicates the number of copies per total RNA (25 ng). (+) Indicates reverse transcriptase addition, and (-) indicates no reverse transcriptase addition.
FIG. 16 shows the results of examining changes in insulin secretion when MIN6 cells were stimulated with palmitic acid, γ-linolenic acid, or glibenclamide as a positive control for 90 minutes. The horizontal axis shows the added compound and its concentration (μM). The vertical axis indicates the insulin secretion concentration (ng / ml).
FIG. 17 shows the results of examining changes in insulin secretion when MIN6 cells were stimulated with palmitic acid, oleic acid, or glibenclamide as a positive control for 60 minutes. The horizontal axis shows the added compound and its concentration (μM). The vertical axis indicates the insulin secretion concentration (ng / ml).
FIG. 18 shows the results obtained by determining the expression level of GPR40 mRNA in rat pancreas and islets of Langerhans by RT-PCR. The whole pancreas was indicated by the average of two animals.
FIG. 19 shows the results of determining the expression level of GPR40 mRNA in cynomolgus monkey pancreas and islets of Langerhans by RT-PCR. The whole pancreas was indicated by the average of two animals.
FIG. 20 shows the results of detecting MAP kinase activation in CHO-hGPR40 # 104 by fatty acids. γ-lino indicates γ-linolenic acid, Ole indicates oleic acid, DHA indicates 4Z, 7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenoic acid, methyl indicates methyl linoleate, and C2 indicates acetic acid. CHO-mock indicates CHO cells not expressing human GPR40, and CHO-hGPR40 # 104 indicates a CHO cell line expressing human GPR40. p44 indicates the band of p44 MAP kinase, and p42 indicates the band of p42 MAP kinase.
FIG. 21 shows the results of examining the suppression of mouse GPR40-GFP fusion protein expression by introducing siRNA specific to the mouse GPR40 sequence. Mock indicates control cells into which GPR40-GFP has not been introduced. NoTF indicates cells without transfection, and Nosi indicates transfection without siRNA. m40i103 and m40i256 indicate siRNAs specific to the mouse GPR40 sequence, and si scramble and si GL2 indicate siRNAs having no homology to the sequence of GPR40 added as a negative control. The vertical axis indicates the expression level of GPR40 (absorbance at 450 nm). It was expressed as the average of the experiments performed with N = 3 + standard deviation.
FIG. 22 shows the sequences of m40i103 and m40i256.
FIG. 23 shows a construction diagram of a GPR40 gene targeting vector.
FIG. 24 shows the results of detecting MAP kinase activation at MIN6 by fatty acids. Lane 1 is a control, lane 2 is oleic acid added, lane 3 is linoleic acid added, lane 4 is γ-linolenic acid added, lane 5 is methyl linoleate (methyl linoleic acid). Methyl linoleate, lane 6 shows the addition of linoleic acid, and lane 7 shows the addition of linoleic acid + PD98059 (50 μM). Lanes 1 to 5 are the results of incubation for 2.5 minutes, and lanes 6 to 7 are the results of incubation for 5 minutes. The concentration of each fatty acid is 10 μM. p44 indicates the band of p44 MAP kinase, and p42 indicates the band of p42 MAP kinase.
FIG. 25 shows the action of various fatty acids prepared with KRBH containing 22 mM glucose to promote insulin secretion from MIN6. Concentrations on the horizontal axis indicate the concentration (μM) of the added fatty acid. Base was not added, Oleic added oleic acid, Linoleic added linoleic acid, α-Linolenic added α-linolenic acid, γ-Linolenic added γ-linolenic acid, Arachidonic added arachidonic acid, DHA added docosahexaenoic acid, Methyl Linoleate shows the case where methyl linoleate is added, and Butyric shows the case where butyric acid is added. The vertical axis indicates the insulin concentration (ng / ml). Values represent mean + standard error (n = 4). ** indicates p <0.01, and * indicates p <0.05 (Student's t test).
FIG. 26 shows the glucose concentration dependence of the insulin secretion promoting activity from MIN6 by fatty acids. The horizontal axis shows the type of the added fatty acid (10 μM). Base shows no addition, Oleic shows oleic acid addition, Linoleic shows linoleic acid addition, Methyl shows methyl linoleate addition, and Butyric shows butyric acid addition. Glc indicates glucose. The vertical axis indicates the insulin concentration (ng / ml). Values represent mean + standard error (n = 4). ** indicates p <0.01, and * indicates p <0.05 (Student's t test).
FIG. 27 shows the inhibitory effect of a fatty acid on the activity of increasing insulin secretion by siRNA-introduced MIN6. The horizontal axis shows the concentration (μM) of the added fatty acid. Base shows no addition, Linoleic shows linoleic acid addition, Gamma-Linolenic shows γ-linolenic acid addition, and Methyl linoleate shows methyl linoleate addition. The vertical axis indicates the amount of insulin secretion and is expressed as a% value relative to Base. □ Column is for m40i103 introduction, ■ Column is for Scramble II duplex siRNA introduction. Values represent mean + standard error (n = 4). ** indicates p <0.01 (Student's t test vs. Base).
FIG. 28 shows the action of fatty acids to promote insulin secretion from rat pancreatic islets of Langerhans. The horizontal axis shows the concentration (μM) of the added fatty acid. Base indicates no addition, Linoleic indicates linoleic acid, γ-Linolenic indicates γ-linolenic acid, and methyl linoleate indicates methyl linoleate. The vertical axis shows the amount of insulin secreted per islet (ng). The value is the sum of experiments performed twice in total at n = 3 and n = 4, and indicates the average + standard error. ** indicates p <0.01, and * indicates p <0.05 (Student's t test).
FIG. 29. Ca in the promotion of insulin secretion from MIN6 by fatty acids ²⁺ The result of having investigated the necessity of ion is shown. The horizontal axis indicates the added fatty acids. Base indicates no addition, Oleic indicates oleic acid, Linoleic indicates linoleic acid, γ-Linolenic indicates γ-linolenic acid, and methyl linoleate indicates methyl linoleate. The group on the left shows EGTA / Ca when fatty acids (10 μM) prepared with KRBH containing 22 mM glucose were added. ²⁺ The right group indicated by free is KRBH containing 22 mM glucose and Ca ²⁺ The case where a fatty acid (10 μM) prepared with KRBH to which 0.1 mM EGTA is added and ions are added is shown. The vertical axis indicates the amount of insulin secretion (ng / ml). Values represent mean + standard error (n = 4). ** indicates p <0.01 (Student's t test vs. Base).
FIG. 30 shows the effects of various inhibitors on the promotion of insulin secretion from MIN6 by fatty acids. The horizontal axis indicates the added fatty acids. Base indicates no addition, Oleic indicates oleic acid, Linoleic indicates linoleic acid, and methyl linoleate indicates methyl linoleate. The No inhibitor group was a fatty acid (10 μM) prepared with KRBH containing 22 mM glucose, the Nifedipine group was a fatty acid (10 μM) prepared with a buffer prepared by adding 10 μM nifedipine to KRBH containing 22 mM glucose, and the PD98059 group was a KRBH containing 22 mM glucose. Shows a case where a fatty acid (10 μM) prepared with a buffer to which PD98059 (50 μM) was added was added. The vertical axis indicates the amount of insulin secretion (ng / ml). Values represent mean + standard error (n = 4). ** indicates p <0.01 (Student's t test vs. Base).
FIG. 31 shows the effect of BSA on the promotion of insulin secretion from MIN6 by fatty acids. The horizontal axis indicates the added fatty acid (10 μM). Base indicates no addition, Oleic indicates oleic acid, Linoleic indicates linoleic acid, and methyl linoleate indicates methyl linoleate. No BSA indicates the condition without BSA added to the buffer, and 0.1% BSA indicates the condition with 0.1% BSA added. The vertical axis indicates the amount of insulin secretion (ng / ml). Values represent mean + standard error (n = 4). ** indicates p <0.01 (Student's t test vs. Base).
FIG. 32 shows the effect of various fatty acids on cAMP production in human GPR40-expressing CHO cells (CHO-hGPR40; left) and mouse GPR40-expressing CHO cells (CHO-mGPR40; right). FFA concentrations (μM) on the horizontal axis indicate the concentration of the added fatty acid. The vertical axis indicates the amount of cAMP (pmol / well) per well of a 96-well plate. The solid line indicates the presence of 2 μM forskolin, and the dotted line indicates the absence of forskolin. ● shows the case where docosahexaenoic acid (DHA), ◇ shows the case where linoleic acid, and □ shows the case where γ-linolenic acid is added. * Indicates p <0.05 (Student's t test).
FIG. 33 shows intracellular Ca when various fatty acids are added to MIN6 cells. ²⁺ The result of having examined the change of density is shown. The vertical axis is intracellular Ca ²⁺ The fluorescence intensity indicating the concentration is shown. The horizontal axis shows the time elapsed after the addition of the sample. □ is γ-linolenic acid, ◇ is linoleic acid, ■ is methyl linoleate, △ is oleic acid, ▲ is arachidonic acid, ● shows the case where docosahexaenoic acid (DHA) was added, and ○ shows the case where butyric acid (butyric acid) was added.
FIG. 34 shows suppression of mouse GPR40 mRNA expression by introducing siRNA specific to the sequence of mouse GPR40. M40i103 on the horizontal axis represents siRNA specific to mouse GPR40, and Scramble represents Scramble II duplex siRNA of a random sequence siRNA serving as a control. The mRNA expression level (GRP40 / GAPDH) on the vertical axis indicates the relative value of the expression level of GPR40 to GAPDH (n = 2 + SEM).

Claims (136)

A G protein-coupled receptor protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 17 or SEQ ID NO: 29, or Its salt. A G protein-coupled receptor protein or a salt thereof, comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3. A G protein-coupled receptor protein consisting of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3, or a salt thereof. A G protein-coupled receptor protein consisting of the amino acid sequence represented by SEQ ID NO: 17, or a salt thereof. A G protein-coupled receptor protein consisting of the amino acid sequence represented by SEQ ID NO: 29, or a salt thereof. A partial peptide of the G protein-coupled receptor protein according to claim 1, or a salt thereof. A polynucleotide comprising a polynucleotide encoding the G protein-coupled receptor protein according to claim 1. A DNA encoding the G protein-coupled receptor protein according to claim 3. A DNA encoding the G protein-coupled receptor protein according to claim 4. A DNA encoding the G protein-coupled receptor protein according to claim 5. DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4. DNA consisting of the nucleotide sequence represented by SEQ ID NO: 18. A DNA consisting of the base sequence represented by SEQ ID NO: 30. A recombinant vector containing the polynucleotide according to claim 7. A transformant transformed with the recombinant vector according to claim 14. A method for producing a G protein-coupled receptor protein or a salt thereof according to claim 1, wherein the transformant according to claim 15 is cultured to produce the G protein-coupled receptor protein or a salt thereof according to claim 1. Law. A pharmaceutical comprising the G protein-coupled receptor protein according to claim 1 or the partial peptide according to claim 6 or a salt thereof. A medicament comprising the polynucleotide according to claim 7. A diagnostic agent comprising the polynucleotide according to claim 7. An antibody against the G protein-coupled receptor protein according to claim 1, or the partial peptide according to claim 6, or a salt thereof. 21. The antibody according to claim 20, which is a monoclonal antibody that recognizes the C-terminal peptide of the G protein-coupled receptor protein according to claim 1 or a salt thereof. 21. The antibody according to claim 20, which is a monoclonal antibody that recognizes the peptide represented by SEQ ID NO: 33 or a salt thereof. The antibody according to claim 20, which is a neutralizing antibody that inactivates signal transduction of the G protein-coupled receptor protein according to claim 1. A diagnostic agent comprising the antibody according to claim 20. A medicament comprising the antibody according to claim 20. A polynucleotide comprising a nucleotide sequence complementary to the polynucleotide according to claim 7 or a part thereof. A diagnostic agent comprising the polynucleotide according to claim 26. A medicament comprising the polynucleotide according to claim 26. An siRNA against the polynucleotide according to claim 7. 30. The siRNA according to claim 29, which is an siRNA comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 34 and an antisense strand consisting of the base sequence represented by SEQ ID NO: 35. 30. The siRNA according to claim 29, which is an siRNA comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 36 and an antisense strand consisting of the base sequence represented by SEQ ID NO: 37. A diagnostic agent comprising the siRNA according to claim 29. A medicament comprising the siRNA according to claim 29. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29; A pancreatic function regulator comprising the partial peptide or a salt thereof. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29; Diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease, arthropathy containing its partial peptide or its salt For preventing, treating osteopenia, arteriosclerosis, thrombotic diseases, dyspepsia or memory learning disorders. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29; An insulin secretagogue or a hypoglycemic agent comprising the partial peptide or a salt thereof. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29; A pancreatic β-cell protective agent comprising the partial peptide or a salt thereof. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or A pancreatic function regulator comprising a polynucleotide containing a polynucleotide encoding the partial peptide. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or Diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual function comprising a polynucleotide containing a polynucleotide encoding the partial peptide An agent for preventing or treating disorders, skin diseases, arthrosis, osteopenia, arteriosclerosis, thrombotic diseases, dyspepsia or memory learning disorders. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or An insulin secretagogue or a hypoglycemic agent comprising a polynucleotide containing a polynucleotide encoding the partial peptide. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or A pancreatic β-cell protective agent comprising a polynucleotide containing a polynucleotide encoding the partial peptide. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or Diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual function comprising a polynucleotide containing a polynucleotide encoding the partial peptide Disorders, skin disorders, arthrosis, osteopenia, arteriosclerosis, thrombotic disorders, indigestion, memory and learning disorders, obesity, hyperlipidemia, hypoglycemia, hypertension, edema, insulin resistance syndrome, unstable diabetes, Diagnostic agent for lipoatrophy, insulin allergy, insulinoma, lipotoxicity or cancer. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29; Diabetes comprising an antibody against the partial peptide or a salt thereof, glucose intolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease, Arthropathy, osteopenia, arteriosclerosis, thrombotic disorders, indigestion, memory and learning disorders, obesity, hypoglycemia, hypertension, edema, insulin resistance syndrome, unstable diabetes, lipoatrophy, insulin allergy, insulinoma, lipotoxicity Or cancer diagnostics. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29; An agent for regulating pancreatic function, comprising an antibody against the partial peptide or a salt thereof. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29; Obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance comprising an antibody against the partial peptide or a salt thereof An agent for preventing or treating syndrome, unstable diabetes, lipoatrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity or cancer. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or A pancreatic function regulator comprising a polynucleotide comprising a nucleotide sequence complementary to a polynucleotide comprising a polynucleotide encoding the partial peptide or a part thereof. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or Obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, comprising a polynucleotide comprising a polynucleotide comprising a polynucleotide encoding a partial peptide thereof or a polynucleotide comprising a part thereof. Prevention of hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance syndrome, unstable diabetes, lipoatrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity or cancer Therapeutic agent. (1) G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A method for screening a compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and a fatty acid or a salt thereof, comprising using a protein, a partial peptide thereof or a salt thereof and (2) a fatty acid or a salt thereof. (1) G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A kit for screening a compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and a fatty acid or a salt thereof, comprising a protein, a partial peptide thereof or a salt thereof, and (2) a fatty acid or a salt thereof. . A fatty acid or a salt thereof, which can be obtained by using the screening method according to claim 48 or the screening kit according to claim 49, and SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO. : A compound or a salt thereof having an amino acid sequence identical or substantially identical to the amino acid sequence represented by 29 and capable of changing the binding to a G protein-coupled receptor protein or a salt thereof. 51. The compound or a salt thereof according to claim 50, which is an agonist. 51. The compound or a salt thereof according to claim 50, which is an antagonist. A medicament comprising the compound according to claim 50 or a salt thereof. G protein conjugate containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 with a fatty acid or a salt thereof An agent for regulating pancreatic function, comprising a compound that changes the binding to a type receptor protein or a salt thereof or a salt thereof. A medicament comprising the agonist according to claim 51. A G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or a G protein-coupled receptor protein thereof Diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease, arthropathy, bone loss containing an agonist for salt For the prevention and treatment of diseases, arteriosclerosis, thrombotic diseases, dyspepsia or memory learning disorders. A G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or a G protein-coupled receptor protein thereof An insulin secretagogue or a hypoglycemic agent comprising an agonist for a salt. A G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or a G protein-coupled receptor protein thereof A pancreatic β-cell protective agent comprising an agonist for a salt. A medicament comprising the antagonist of claim 52. A G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or a G protein-coupled receptor protein thereof Obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance syndrome, unstable diabetes containing an antagonist to salt , Lipoatrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity or cancer. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or A method for screening a compound or a salt thereof, which has an action of changing the expression level of the G protein-coupled receptor protein and regulating pancreatic function, which comprises using a polynucleotide containing a polynucleotide encoding the partial peptide. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or A screening kit for a compound or a salt thereof, which has an action of changing the expression level of the G protein-coupled receptor protein containing a polynucleotide containing a polynucleotide encoding the partial peptide and regulating pancreatic function. It is represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, which can be obtained using the screening method according to claim 61 or the screening kit according to claim 62. A compound having a function of regulating pancreatic function by changing the expression level of a G protein-coupled receptor protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence, or a salt thereof. It is represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, which can be obtained using the screening method according to claim 61 or the screening kit according to claim 62. A compound or a salt thereof, which has an action of increasing the expression level of a G protein-coupled receptor protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence and regulating pancreatic function. It is represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, which can be obtained using the screening method according to claim 61 or the screening kit according to claim 62. A compound or a salt thereof having an activity of regulating pancreatic function by decreasing the expression level of a G protein-coupled receptor protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or An agent for regulating pancreatic function, comprising a compound having a function of regulating the expression of a partial peptide and regulating pancreatic function or a salt thereof. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or Diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic nephropathy, comprising a compound having a function of regulating the pancreatic function by increasing the expression level of the partial peptide Agent for the treatment of hypertension, hyperlipidemia, sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis, thrombotic disease, dyspepsia or memory learning disorder. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or An insulin secretagogue or a hypoglycemic agent comprising a compound having a function of increasing the expression level of the partial peptide and regulating pancreatic function or a salt thereof. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or A pancreatic β-cell protective agent comprising a compound having a function of increasing the expression level of the partial peptide and regulating pancreatic function or a salt thereof. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or Obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, hypertension, diabetic neuropathy, diabetes containing a compound having a function of regulating the pancreatic function by reducing the expression level of the partial peptide A preventive / therapeutic agent for nephropathy, diabetic retinopathy, edema, insulin resistance syndrome, unstable diabetes, lipoatrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity or cancer. A G protein-conjugated test compound comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A method for determining a ligand for the receptor protein or a salt thereof, comprising measuring an intracellular Ca ²⁺ concentration increasing activity or an intracellular cAMP inhibitory activity when brought into contact with a cell containing the receptor protein. A ligand obtainable by the method of claim 71. (1) G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A protein, a partial peptide thereof or a salt thereof, and (2) (1) a fatty acid or a salt thereof, or (2) a compound or a salt thereof which changes the binding property between the receptor protein or a salt thereof and a fatty acid or a salt thereof. A screening method for an agonist or antagonist to the receptor protein or a salt thereof. (1) (1) A labeled fatty acid or a salt thereof, or (2) a labeled compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and a fatty acid or a salt thereof is added to the receptor protein or a portion thereof. (2) a test compound and (1) a labeled fatty acid or a salt thereof, or (2) a label that changes the binding property between the receptor protein or a salt thereof and a fatty acid or a salt thereof. (1) labeled fatty acid or salt thereof, or (2) the receptor protein or salt thereof and fatty acid or salt thereof when the compound or its salt is brought into contact with the receptor protein, its partial peptide or its salt. A labeled compound or a salt thereof that changes the binding property to the receptor protein, its partial peptide or its The screening method of claim 73, wherein the measuring the amount of binding between. (1) (1) A labeled fatty acid or a salt thereof, or (2) a labeled compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and a fatty acid or a salt thereof contains the receptor protein. (2) Changes in the binding between the test compound and (1) the labeled fatty acid or its salt or (2) the receptor protein or its salt and the fatty acid or its salt when brought into contact with the cell or its cell membrane fraction. (1) labeled fatty acid or its salt or (2) the receptor protein or its salt when the labeled compound or its salt to be contacted is brought into contact with the cell containing the receptor protein or its cell membrane fraction. A labeled compound or a salt thereof that changes the binding property to a fatty acid or a salt thereof, and a cell or a cell containing the receptor protein. The screening method of claim 73, wherein that of measuring the binding amount of the cell membrane fraction. (1) A cell or a cell membrane fraction thereof containing the receptor protein or a salt thereof or a compound or a salt thereof which changes the binding property between the receptor protein or the salt thereof and the fatty acid or the salt thereof. And (2) a test compound and (1) a fatty acid or a salt thereof, or (2) a compound or a salt thereof that alters the binding property between the receptor protein or a salt thereof and a fatty acid or a salt thereof. 74. The screening method according to claim 73, wherein an intracellular Ca ²⁺ concentration increasing activity or an intracellular cAMP production inhibitory activity when the cell is contacted with a protein-containing cell or a cell membrane fraction thereof is measured. (1) G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A protein, a partial peptide thereof or a salt thereof, and (2) (1) a fatty acid or a salt thereof or (2) a compound or a salt thereof which changes the binding property between the receptor protein or a salt thereof and a fatty acid or a salt thereof. A kit for screening an agonist or antagonist for the receptor protein or a salt thereof, which is characterized by the following. SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, which can be obtained using the screening method according to claim 73 or the screening kit according to claim 77. An agonist for a G protein-coupled receptor protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence. SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, which can be obtained using the screening method according to claim 73 or the screening kit according to claim 77. An antagonist to a G protein-coupled receptor protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence. A medicament comprising the agonist according to claim 78. A medicament comprising the antagonist of claim 79. A G protein-coupled form in which the test compound contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 77. The screening method according to any one of claims 74 to 76, wherein the compound is designed to bind to the ligand binding pocket based on the atomic coordinates of the active site of the receptor protein or a salt thereof and the position of the ligand binding pocket. A G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or a G protein-coupled receptor protein thereof (1) a pancreatic function regulator, (2) diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, characterized by using a salt. Drugs for preventing and treating sexual dysfunction, skin diseases, arthrosis, osteopenia, arteriosclerosis, thrombotic diseases, dyspepsia or memory learning disorders, (3) insulin secretagogues, (4) hypoglycemic drugs, (5) ▼ pancreatic β-cell protective agent or ▲ 6 ▼ obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance syndrome, Stable diabetes Method to check fatty atrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, that preventive or therapeutic drug for fat toxicity or cancer binds to the receptor protein or a salt thereof. A G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or a G protein-coupled receptor protein thereof (1) Diabetes, glucose intolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease, joint characterized by using a salt And prophylactic / therapeutic agent for osteopenia, osteopenia, arteriosclerosis, thrombotic disease, indigestion or memory learning disorder, (2) insulin secretagogue, (3) hypoglycemic agent, or (4) protection of pancreatic β-cells. A method for confirming that it is an agonist for a protein or a salt thereof. A G protein-coupled receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, or a G protein-coupled receptor protein thereof Obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance syndrome, unstable using salt A method for confirming that a drug for preventing or treating diabetes, lipoatrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity or cancer is an antagonist to the receptor protein or a salt thereof. 86. The method according to claim 83, further comprising measuring the amount of each drug and the receptor protein, its partial peptide or its salt when the drug is brought into contact with the receptor protein, its partial peptide or its salt. How to check the description. (1) (1) A labeled fatty acid or a salt thereof, or (2) a labeled compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and a fatty acid or a salt thereof is added to the receptor protein or a portion thereof. (2) each drug and (1) a labeled fatty acid or a salt thereof or (2) a labeled compound or a salt thereof with the receptor protein, a partial peptide thereof or a peptide thereof. (1) Measuring the amount of binding between (1) the labeled fatty acid or its salt or (2) the labeled compound or its salt and the receptor protein, its partial peptide or its salt when brought into contact with a salt. The confirmation method according to claim 83, wherein: (1) (1) A labeled fatty acid or a salt thereof, or (2) a labeled compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and a fatty acid or a salt thereof contains the receptor protein. A cell containing the receptor protein when (2) each drug and (1) a labeled fatty acid or a salt thereof or (2) the labeled compound or a salt thereof are contacted with a cell or a cell membrane fraction thereof; Or (1) when a labeled fatty acid or a salt thereof or (2) the labeled compound or a salt thereof is contacted with a cell containing the receptor protein or a cell membrane fraction thereof when the cell is contacted with the cell membrane fraction thereof. The confirmation method according to claim 83, wherein the amount of binding is measured. (1) A cell or a cell membrane fraction thereof containing the receptor protein or a salt thereof or a compound or a salt thereof which changes the binding property between the receptor protein or the salt thereof and the fatty acid or the salt thereof. And (2) each drug and (1) a fatty acid or a salt thereof, or (2) a compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and a fatty acid or a salt thereof, with the receptor. The confirmation method according to claim 83, wherein the intracellular Ca ²⁺ concentration increasing activity or the intracellular cAMP production inhibitory activity when contacting with a protein-containing cell or a cell membrane fraction thereof is measured. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29; (1) a pancreatic function modulator, (2) diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, comprising a partial peptide or a salt thereof. Hyperlipidemia, sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis, thrombotic disease, indigestion or memory learning prevention / treatment agent, (3) insulin secretagogue, (4) blood glucose Hypotensive drug, (5) pancreatic beta cell protective drug or (6) obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance Syndrome, unstable diabetes, fatty atrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, a kit for the prophylaxis or treatment of lipotoxicity or cancer to confirm that binding to the receptor protein or a salt thereof. Agonist for a G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Or an antagonist, (1) a pancreatic function modulator, (2) diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, Prevention and treatment of skin diseases, arthropathy, osteopenia, arteriosclerosis, thrombotic diseases, dyspepsia or memory learning disorders, (3) insulin secretagogues, (4) hypoglycemic drugs, (5) pancreatic β cells Protective drugs or (6) obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance syndrome, Constant diabetes, fatty atrophy, insulin allergy, insulinoma, arteriosclerosis, thrombotic disease, lipotoxicity or prophylactic or therapeutic drug for cancer. (1) {circle around (1)} G protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Agonist or antagonist to the coupled receptor protein, or (2) identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A compound that changes the expression level of a G protein-coupled receptor protein or a partial peptide thereof containing the same amino acid sequence or a salt thereof, and (2) (1) a pancreatic function regulator, (2) diabetes, glucose intolerance, Ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease, arthrosis, Prophylactic / therapeutic agent for hypotension, arteriosclerosis, thrombotic disease, dyspepsia or memory learning disorder, (3) insulin secretagogue, (4) hypoglycemic agent, (5) pancreatic β-cell protective agent or (6) obesity , Hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance syndrome, unstable diabetes, lipoatrophy, insulin allergy, insulinoma, A medicine comprising a combination of a prophylactic / therapeutic agent for arteriosclerosis, thrombotic disease, lipotoxicity or cancer. A non-human mammal having an exogenous DNA encoding the G protein-coupled receptor protein according to claim 1 or a mutant DNA thereof. 94. The animal of claim 93, wherein the non-human mammal is a rodent. The animal according to claim 94, wherein the rodent is a mouse or a rat. A non-human mammalian embryonic stem cell in which DNA encoding the G protein-coupled receptor protein according to claim 1 has been inactivated, 97. The embryonic stem cell according to claim 96, wherein the DNA is inactivated by introducing a reporter gene. 97. The embryonic stem cell of claim 96, which is neomycin resistant. 97. The embryonic stem cell according to claim 96, wherein the non-human mammal is a rodent. 100. The embryonic stem cell according to claim 99, wherein the rodent is a mouse. A non-human mammal deficient in expression of a DNA, wherein the DNA encoding the G protein-coupled receptor protein according to claim 1 is inactivated. 103. The non-human mammal of claim 101, wherein said DNA is inactivated by introducing a reporter gene, and said reporter gene can be expressed under the control of a promoter for said DNA. 102. The non-human mammal of claim 101, wherein the non-human mammal is a rodent. 104. The non-human mammal of claim 103, wherein the rodent is a mouse. 103. A compound that promotes or inhibits promoter activity on a DNA encoding a G protein-coupled receptor protein according to claim 1, wherein a test compound is administered to the animal according to claim 102, and expression of the reporter gene is detected. Or a method of screening for a salt thereof. (1) G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A method for screening a compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and eicosanoid, which comprises using a protein, a partial peptide thereof or a salt thereof and (2) an eicosanoid. (1) G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A kit for screening a compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and eicosanoid, which comprises a protein, a partial peptide thereof or a salt thereof, and (2) an eicosanoid. An eicosanoid, which can be obtained by using the screening method according to claim 106 or the screening kit according to claim 107, comprising SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29. A compound or a salt thereof which changes the binding property to a G protein-coupled receptor protein or a salt thereof, which has the same or substantially the same amino acid sequence as the represented amino acid sequence. 109. The compound or a salt thereof according to claim 108, which is an agonist. 109. The compound or a salt thereof according to claim 108, which is an antagonist. A medicament comprising the compound according to claim 108 or a salt thereof. A G protein-coupled receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 with eicosanoid Alternatively, a pancreatic function-regulating agent comprising a compound that changes the binding property to a salt thereof or a salt thereof. A medicament comprising the agonist according to claim 109. A medicament comprising the antagonist according to claim 110. (1) G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A protein, a partial peptide thereof, or a salt thereof, and (2) (1) an eicosanoid or (2) a compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and an eicosanoid, or the salt thereof. Or a method of screening for an agonist or antagonist to a salt thereof. (1) Contacting (1) a labeled eicosanoid or (2) a labeled compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and eicosanoid with the receptor protein, a partial peptide or a salt thereof. And (2) a test compound and (1) a labeled eicosanoid or (2) a labeled compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and an eicosanoid, the receptor protein, (1) a labeled eicosanoid or (2) a labeled compound or salt thereof that changes the binding property between the receptor protein or its salt and eicosanoid when contacted with the partial peptide or its salt, and the receptor The amount of binding to protein, its partial peptide or its salt can be measured. The screening method of claim 115, wherein. (1) a cell containing the receptor protein or a cell membrane fraction thereof, comprising: (1) a labeled eicosanoid or (2) a labeled compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and an eicosanoid. And (2) a test compound and (1) a labeled eicosanoid or (2) a labeled compound or a salt thereof that alters the binding property of eicosanoid to the receptor protein or its salt with the receptor. (1) a labeled eicosanoid or (2) a labeled compound or a salt thereof that changes the binding between an eicosanoid and the receptor protein or a salt thereof when brought into contact with a protein-containing cell or a cell membrane fraction thereof. And the amount of binding between the cells containing the receptor protein and the cell membrane fraction thereof The screening method of claim 115, wherein the measuring. (1) a case where a compound or a salt thereof that changes the binding between eicosanoid or (1) eicosanoid or (2) the receptor protein or a salt thereof and eicosanoid is brought into contact with a cell containing the receptor protein or a cell membrane fraction thereof; (2) contacting a test compound and (1) eicosanoid or (2) a compound or a salt thereof that alters the binding between eicosanoid and the receptor protein or a salt thereof to a cell containing the receptor protein or a cell membrane fraction thereof 115. The screening method according to claim 115, wherein the intracellular Ca ²⁺ concentration increasing activity or the intracellular cAMP production inhibitory activity in the case where the reaction is performed is measured. (1) G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A receptor comprising a protein, a partial peptide thereof or a salt thereof, and (2) (1) an eicosanoid or (2) a compound or a salt thereof that changes the binding property between the receptor protein or a salt thereof and an eicosanoid. A kit for screening an agonist or antagonist for a protein or a salt thereof. It is represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, which can be obtained using the screening method according to claim 115 or the screening kit according to claim 119. An agonist for a G protein-coupled receptor protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence. It is represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29, which can be obtained using the screening method according to claim 115 or the screening kit according to claim 119. An antagonist to a G protein-coupled receptor protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence. A medicament comprising the agonist according to claim 120. A medicament comprising the antagonist of claim 121. A G protein-coupled form in which the test compound contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 118. The screening method according to claim 116 to 118, wherein the compound is designed to bind to the ligand binding pocket based on the atomic coordinates of the active site of the receptor protein or a salt thereof and the position of the ligand binding pocket. For mammals,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(3) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 An agonist to the protein, or (4) containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A method for regulating pancreatic function, which comprises administering an effective amount of a compound or a salt thereof that increases the expression level of a G protein-coupled receptor protein or a partial peptide thereof. For mammals,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(3) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 An agonist to the protein, or (4) containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Diabetes, glucose intolerance, ketosis, acidosis, diabetic neuropathy, diabetic kidney, comprising administering an effective amount of a compound or a salt thereof that increases the expression level of a G protein-coupled receptor protein or a partial peptide thereof. Disease, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis, thrombotic disease, indigestion Prevention and treatment methods of learning and memory disorders. For mammals,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(3) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 An agonist to the protein, or (4) containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A method of promoting insulin secretion or lowering blood glucose, which comprises administering an effective amount of a compound or a salt thereof that increases the expression level of a G protein-coupled receptor protein or a partial peptide thereof. For mammals,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(3) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 An agonist to the protein, or (4) containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A pancreatic β-cell protective agent, which comprises administering an effective amount of a compound or a salt thereof that increases the expression level of a G protein-coupled receptor protein or a partial peptide thereof. For mammals,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 An antibody against a receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide comprising a nucleotide sequence complementary to a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof or a part thereof,
(3) a G protein-conjugated form containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 SiRNA against a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(4) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Contains an antagonist to the protein or (5) an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A method for regulating pancreatic function, which comprises administering an effective amount of a compound or a salt thereof that decreases the expression level of a G protein-coupled receptor protein or a partial peptide thereof. For mammals,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 An antibody against a receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide comprising a nucleotide sequence complementary to a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof or a part thereof,
(3) a G protein-conjugated form containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 SiRNA against a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(4) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Contains an antagonist to the protein or (5) an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetes, which comprises administering an effective amount of a compound or a salt thereof that reduces the expression level of a G protein-coupled receptor protein or a partial peptide thereof. Sexual neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance syndrome, unstable diabetes, lipoatrophy, insulin allergy, Surinoma, arteriosclerosis, thrombotic disease, lipotoxicity or prophylaxis or treatment method for cancer. For producing a pancreatic function regulator,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(3) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 An agonist to the protein, or (4) containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Use of a compound or a salt thereof that increases the expression level of a G protein-coupled receptor protein or a partial peptide thereof. Diabetes, impaired glucose tolerance, ketosis, acidosis, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperlipidemia, sexual dysfunction, skin disease, arthropathy, osteopenia, arteriosclerosis, thrombotic disease For the manufacture of a prophylactic / therapeutic agent for indigestion or memory learning disorder,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(3) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 An agonist to the protein, or (4) containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Use of a compound or a salt thereof that increases the expression level of a G protein-coupled receptor protein or a partial peptide thereof. For producing an insulin secretagogue or a hypoglycemic agent,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(3) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 An agonist to the protein, or (4) containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Use of a compound or a salt thereof that increases the expression level of a G protein-coupled receptor protein or a partial peptide thereof. For producing a pancreatic β-cell protective agent,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(3) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 An agonist to the protein, or (4) containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Use of a compound or a salt thereof that increases the expression level of a G protein-coupled receptor protein or a partial peptide thereof. For producing a pancreatic function regulator,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 An antibody against a receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide comprising a nucleotide sequence complementary to a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof or a part thereof,
(3) a G protein-conjugated form containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 SiRNA against a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(4) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Contains an antagonist to the protein or (5) an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Use of a compound or a salt thereof that reduces the expression level of a G protein-coupled receptor protein or a partial peptide thereof. Obesity, hyperlipidemia, type 2 diabetes, hypoglycemia, hypertension, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, edema, insulin resistance syndrome, unstable diabetes, lipoatrophy, insulin allergy, insulinoma For the manufacture of a prophylactic or therapeutic agent for arteriosclerosis, thrombotic disease, lipotoxicity or cancer,
(1) G protein-coupled type containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 An antibody against a receptor protein or a partial peptide thereof or a salt thereof,
(2) a G protein-conjugated form containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 A polynucleotide comprising a nucleotide sequence complementary to a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof or a part thereof,
(3) a G protein-conjugated form containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 SiRNA against a polynucleotide containing a polynucleotide encoding a receptor protein or a partial peptide thereof,
(4) a G protein-coupled receptor containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Contains an antagonist to the protein or (5) an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 29 Use of a compound or a salt thereof that reduces the expression level of a G protein-coupled receptor protein or a partial peptide thereof.

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