JP4294981B2 - Testing method for fat mass increase inhibiting ability - Google Patents

Description

＊ 参考例１の結果に基づく値
＊＊ 参考例２の結果に基づく値
また被験物質が他の公知の脂肪量増加抑制能力を有する物質である場合においても同様な傾向の結果を得た。
以上より、本酵素阻害能力を評価するための指標となる阻害率と、蓄積される脂肪量の直接的な測定に基づく結果である、後述の参考例１及び参考例２での抑制率との関係は、特定な濃度範囲においては少なくとも用量的依存性を示しており、この関係は正の相関関係であることが確認された。
【００３９】
参考例１
（１−１）脂肪組織の摘出
１４週齢のウィスター系雄ラット（日本エスエルシー社）３２匹を屠殺・開腹し、腸間膜脂肪組織を摘出した。摘出した組織を、リン酸緩衝液（０．２０ｇ／Ｌ ＫＣｌ、０．２０ｇ／Ｌ ＫＨ₂ＰＯ₄、８．００ｇ／Ｌ ＮａＣｌ、２．１６ｇ／Ｌ Ｎａ₂ＨＰＯ₄・７Ｈ₂Ｏ、１００ユニット／ｍｌペニシリン（ギブコ社）、１００μｇ／ｍｌストレプトマイシン（ギブコ社）、２５０ｎｇ／ｍｌアンフォテリシン（ギブコ社））に浸し、室温で洗浄した。
【００４０】
（１−２）脂肪組織からの脂肪細胞の調製と培養
洗浄後の腸間膜脂肪組織について次に示す処理を行った。
まず、上記（１−１）で摘出された脂肪組識を、コラゲナーゼ（タイプII又はVIII、シグマ社）、ペニシリン（ギブコ社）、ストレプトマイシン（ＧＩＢVIIIＣＯ社製）及びアンフォテリシン（ギブコ社）をそれぞれ終濃度１ｍｇ／ｍｌ、１００ユニット／ｍｌ、１００μｇ／ｍｌ及び２５０ｎｇ／ｍｌとなるように添加したダルベッコ改変イーグル培地（４．５ｇ／Ｌ Ｄ−グルコース及び５８４ｍｇ／Ｌ Ｌ−グルタミン含有、ギブコ社）約３００ｍｌ中でハサミを用いて約５ｍｍ角に細断した。次いで、これを３７℃で６０分間振とう（約１７０ｒｐｍ）し、ナイロンメッシュ（８０Ｓ［目の大きさが２５０μｍ］、三紳工業社）で濾過し、濾液（細胞懸濁液）を回収した。当該濾液を室温で１８００ｒｐｍ、５分間遠心分離した後、液層をデカンテーションにより静かに除去し、沈査を得た。この沈査を、ウシ胎児血清（以下、ＦＢＳと記す。）（ギブコ社）、アスコルビン酸（和光純薬工業社）、ペニシリン（ギブコ社）、ストレプトマイシン（ギブコ社）及びアンフォテリシン（ギブコ社）をそれぞれ終濃度１０％、２００μＭ、１００ユニット／ｍｌ、１００μｇ／ｍｌ及び２５０ｎｇ／ｍｌとなるように添加したダルベッコ改変イーグル培地（４．５ｇ／Ｌ Ｄ−グルコース及び５８４ｍｇ／Ｌ Ｌ−グルタミン含有、ギブコ社、以下、ＦＢＳ含有培地と記すこともある。）５０ｍｌに懸濁し、懸濁液をナイロンメッシュ（４２０Ｓ［目の大きさが２５μｍ］、三紳工業社）で濾過した。濾液を回収し、室温で１８００ｒｐｍ、５分間遠心分離した後、液層をデカンテーションにより静かに除去し、沈査を再度ＦＢＳ含有培地５０ｍｌに懸濁した。当該懸濁液について、遠心分離、液層除去、ＦＢＳ含有培地に懸濁するという操作を更に２回、前記と同様に行い、懸濁液（１２０ｍｌ）を調製した。当該懸濁液を細胞培養用フラスコ（接着細胞用Ｔ１５０、岩城硝子社）に３０ｍｌずつ分注し、３７℃、５％ＣＯ₂存在下で培養した。培養開始２〜３時間後に培地を除き、フラスコの器壁を１５ｍｌの前記リン酸緩衝液で洗浄した。洗液を除き、当該洗浄操作を再度行った後、リン酸緩衝液を除き、３０ｍｌのＦＢＳ含有培地をフラスコに添加し、３７℃、５％ＣＯ₂存在下で培養した。培養開始１日後に培地を除き、１５ｍｌのリン酸緩衝液でフラスコの器壁を１回洗浄した後、当該フラスコにトリプシン−エチレンジアミン四酢酸（以下、ＥＤＴＡと記す。）溶液（０．０５%トリプシン、０．５３ｍＭ ＥＤＴＡ・４Ｎａ、ギブコ社）を細胞が浸る程度に添加し、３７℃で５分放置した。これに、ＦＢＳ含有培地をトリプシン−ＥＤＴＡ溶液の約１０倍量添加し、細胞懸濁液を得た。
【００４１】
（１−３）被験物質の脂肪量増加抑制能力の測定（その１）
上記の（１−２）で腸間膜脂肪組織から調製された細胞懸濁液中の細胞数を血球計算盤を用いて測定し、１．４×１０⁵細胞／ｍｌになるようにＦＢＳ含有培地を加えることにより当該細胞懸濁液を希釈した。このようにして得られた希釈液を９６穴プレート（接着細胞培養用、岩城硝子社）に１ウェルあたり１００μｌずつ分注し、５％ＣＯ₂存在下、３７℃にて２〜３日間培養した後、９６穴プレートの各ウェルから培地を除き、１０μｇ／ｍｌインシュリン（シグマ社製）、０．２５μＭデキサメサゾン（和光純薬工業社）、０．５ｍＭ ３−イソブチル−１−メチル−キサンチン（シグマ社製）及び５μＭ １５−デオキシ−Δ^12,14−プロスタグランジンＪ₂（Ｃａｙｍａｎ社）を含むＦＢＳ含有培地１００μｌを各ウェルに添加して５％ＣＯ₂存在下、３７℃にて２日間培養した。次いで、各ウェルの培地を除き、１０μｇ／ｍｌインシュリン及び５μＭ １５−デオキシ−Δ^12,14−プロスタグランジンＪ₂を含むＦＢＳ含有培地１００μｌを各ウェルに添加した。さらに２日間、５％ＣＯ₂存在下、３７℃にて培養した後、各ウェルの培地を除き、１０μｇ／ｍｌインシュリン、５μＭ １５−デオキシ−Δ^12,14−プロスタグランジンＪ₂、被験物質５０μＭ、０．５％ＤＭＳＯ（和光純薬工業社）を含むＦＢＳ含有培地１００μｌを各ウェルに添加し、同様に培養した。尚、被験物質無添加区では、前記培地の代わりに１０μｇ／ｍｌインシュリン、５μＭ １５−デオキシ−Δ^12,14−プロスタグランジンＪ₂、０．５％ＤＭＳＯを含むＦＢＳ含有培地１００μｌを添加する以外は前記と同様な方法で培養を行なった。２日間培養した後、オイルレッド Ｏ（Ｓｕｄａｎ ＩＩ、和光純薬工業社）を用いて細胞内の脂肪を染色し、比色定量法により染色された脂肪の量を測定した。即ち、まず、細胞培養液の入った各ウェルに直接、０．０７５％オイルレッド Ｏ染色液／６０％リン酸トリエチル溶液３０μｌを添加した。室温で３０分間放置後、オイルレッド Ｏ染色液を含む培地を除き、２０％リン酸トリエチル水溶液１００μｌを各ウェルに添加した。添加後、２０％リン酸トリエチル水溶液を各ウェルから除き、新たに同水溶液１００μlを各ウェルに添加した。再度前記の操作を繰り返した後、細胞溶解液（２％ＳＤＳ、０．２Ｎ ＮａＯＨ）１００μｌを各ウェルに添加した。これを３７℃で３時間以上保温した後、各ウェルについて４９０ｎｍの波長の吸光度をプレートリーダー（Ｖｍａｘ、ベックマン社）を用いて測定した（以下、測定値１と記す。）。被験物質無添加区についても、前記と同様な方法により細胞内の脂肪を染色し、比色定量法により染色された脂肪の量を測定した（以下、測定値２と記す。）。これらの測定値から、被験物質の脂肪量増加抑制能力を抑制率として下記の式に従って算出した。
抑制率（％）＝｛（測定値２−測定値１）／測定値２｝×１００
その結果、被験物質である公知化合物 Ｎ−［２−（４−ベンジル−２−エチルフェノキシ）エチル］−５，６−ジメチル［１，２，４］トリアゾロ［１，５−ａ］ピリミジン−７−アミン（即ち、化合物Ａ）の抑制率は７２％であった。
【００４２】
参考例２ （被験物質の脂肪量増加抑制能力の測定（その２））
（２−１）脂肪組織片の調製
１４週齢のウィスター系（日本エスエルシー社）３２匹を屠殺・開腹し、腸間膜に付着した脂肪組織（即ち、腸間膜脂肪組織）を全て摘出した。また、腹側部から大腿部にかけての皮下の脂肪組織（以下、腹側部脂肪組織と記す。）を一匹あたり片側からのみ摘出した。摘出されたこれらの組織を、２ｍｌのダルベッコ改変イーグル培地（４．５ｇ／Ｌ Ｄ−グルコース及び５８４ｍｇ／Ｌ Ｌ−グルタミン含有、ギブコ社）で洗浄した後、同培地中でハサミを用いて約１ｍｍ角に細断した。
【００４３】
（２−２）被験物質の脂肪量増加抑制能力の測定（その２）
まず、４８穴プレート（接着細胞培養用、住友ベークライト社）にダルベッコ改変イーグル培地−低グルコース（１．０ｇ／Ｌ Ｄ−グルコース及び５８４ｍｇ／Ｌ Ｌ−グルタミン含有、ギブコ社）をウェル当り５００μｌずつ分注し、さらに、ＤＭＳＯに溶解させた被験物質を、当該被験物質の終濃度が５０μＭ、ＤＭＳＯの終濃度が０．５％となるように添加した。各ウェルに、前記（２−１）で調製された脂肪組織片５０〜１００ｍｇを入れ、５％ＣＯ₂存在下、３７℃で３０分間保温した。尚、被験物質無添加区では、前記の被験物質のＤＭＳＯ溶液に代えてＤＭＳＯのみを終濃度０．５％となるよう添加すること以外は前記と同様な方法を用いた。保温開始３０分後に放射性同位体標識されたグルコース溶液（Ｄ−［Ｕ−¹⁴Ｃ］グルコース、７．４ＭＢｑ／ｍｌ、アマシャム社）１５μlを各ウェルに添加し、さらに５％ＣＯ₂存在下、３７℃で７時間保温した。次に保温終了後、各ウェルの脂肪組織片をヘプタン／イソプロパノール（ヘプタン：イソプロパノール＝３：２）７５０μl中に移し、室温で約１５時間放置した。次いで前記の液から脂肪組織片を取り除き、ヘプタン及びイソプロパノールを揮発させ、得られた残査のうち６μlをクロロホルム／メタノール（クロロホルム：メタノール＝２：１）１２０μlに溶解し、当該溶解液のうち６μlを薄層クロマトグラフィー用ガラスプレート（Ｋ５シリカゲル１５０オングストローム、ワットマン社、以下、ＴＬＣプレートと記すこともある。）上にスポットした。密閉容器にヘキサン：ジエチルエーテル：酢酸（７５：２５：１）の展開溶媒を入れ、当該展開溶媒を用いて前記ＴＬＣプレートを展開した後、当該ＴＬＣプレートを室温で乾燥した。乾燥された当該ＴＬＣプレートにイメージングプレート（富士フィルム社）を４〜５時間露光させた。露光終了後、イメージングプレートをバイオイメージアナライザー（ＢＡＳ２０００、富士フィルム社）で分析し、前記ＴＬＣプレート上のトリグリセライドの展開位置に相当する部分の［^１４Ｃ］放射活性を測定した（以下、測定値５と記す。）。被験物質無添加区についても、前記と同様な方法を用いてＴＬＣプレート上のトリグリセライドの展開位置に相当する部分の［^１４Ｃ］放射活性を測定した（以下、測定値６と記す。）。これらの測定値から、下記の式に従って、被験物質の脂肪量増加抑制能力を、脂肪組織単位体積当たりの脂肪（トリグリセライド）蓄積の抑制率として算出した。
脂肪組織単位体積当たりの脂肪量増加の抑制率（％）＝｛（測定値６−測定値５）／測定値６｝×１００
その結果、被験物質である公知化合物 Ｎ−［２−（４−ベンジル−２−エチルフェノキシ）エチル］−５，６−ジメチル［１，２，４］トリアゾロ［１，５−ａ］ピリミジン−７−アミン（即ち、化合物Ａ）における抑制率は８４％であった。さらに被験物質の供試濃度を変化させながら、前記と同様な方法を用いて各供試濃度における抑制率を算出した。その結果を前者の結果と併せて表２に示した。
【００４４】
【表２】

【００４５】
実施例３ （マウス由来の本遺伝子のクローニング（その１））
マウス由来の本遺伝子（ｍＡＴ１２）を有するＤＮＡ断片をＰＣＲにより増幅するために、まずＭｏｕｓｅ Ｎｏｒｍａｌ Ａｄｉｐｏｓｅ ｃＤＮＡ (Ｂｉｏｃｈａｉｎ社)1μＬ、配列番号５で示される塩基配列からなるプライマー５ ２０ｐｍｏｌ、配列番号６で示される塩基配列からなるプライマー６ ２０ｐｍｏｌ、Ｔａｋａｒａ Ｅｘ-Ｔａｑポリメラーゼ（宝酒造社）２Ｕ、Ｔａｋａｒａ Ｅｘ-Ｔａｑポリメラーゼ添付のバッファー ５μＬ及びＴａｋａｒａ Ｅｘ-Ｔａｑポリメラーゼ添付のｄＮＴＰ ｍｉｘｔｕｒｅ（２．５ｍＭ）４μＬを含む５０μLのＰＣＲ用反応液を調製し、これをＰＣＲに供した。ＰＣＲは、まず９４℃で３０秒間、次いで６０℃で３０秒間、更に７２℃で１分間からなる保温サイクルを５０回繰り返し、最後に７２℃で５分間保温する条件にて行われた。ＰＣＲ後、アガロース電気泳動で約１．２Ｋｂｐを示すＰＣＲ産物を回収した。回収されたＰＣＲ産物をｐＴ７−Ｂｌｕｅ ｖｅｃｔｏｒ(Ｎｏｖａｇｅｎ社)にサブクローニングすることにより調製されたプラスミドでＥ．ｃｏｌｉ ＪＭ１０９株コンピテントセル（東洋紡社）を形質転換した。形質転換された細胞を５０μg/ｍＬアンピシリン含有ＬＢ培地１００ｍＬで培養することにより得られる培養菌体から前記プラスミドをＱＩＡＧＥＮ Ｐｌａｓｍｉｄ Ｍａｘｉ Ｋｉｔ(ＱＩＡＧＥＮ社)を用いて分離・精製することにより、マウス由来の本遺伝子（ｍＡＴ１２）の塩基配列を有するＤＮＡを含むプラスミドを得た。
【００４６】
実施例４ （マウス由来の本遺伝子の塩基配列の決定（その１））
実施例３で得られたＰＣＲ産物（約１．２kbp）を含むプラスミドを鋳型として、Ｔｈｅｒｍｏ Ｓｅｑｕｅｎａｓｅ IIダイ・ターミネーターキット（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ 社）及びＡＢＩ３７３ＤＮＡ配列読み取り装置（ＰＥ Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社）を用いて、サンガーの方法（Ｆ．Ｓａｎｇｅｒ、Ｓ．Ｎｉｃｋｌｅｎ、Ａ．Ｒ．ＣｏｕｌｓｏｎA著、Ｐｒｏｃｅｅｄｉｎｇｓ ｏｆ Ｎａｔｉｏｎａｌ Ａｃａｄｅｍｙ ｏｆ Ｓｃｉｅｎｃｅ Ｕ．Ｓ．Ａ．(１９７７), ７４, ５４６３−５４６７）により、配列番号２で示される塩基配列を有するＤＮＡからなるマウス由来の本遺伝子（ｍＡＴ１２）の塩基配列を決定した。
【００４７】
実施例５ （ヒト由来の本遺伝子のクローニング（その１））
ヒト由来の本遺伝子（ｈＡＴ１２）を有するＤＮＡ断片をＰＣＲにより増幅するために、まずＨｕｍａｎ Ａｄｉｐｏｃｙｔｅ Ｍａｒａｔｈｏｎ−ｒｅａｄｙ ｃＤＮＡ (ＣＬＯＮＴＥＣＨ社)1μＬ、配列番号７で示される塩基配列からなるプライマー７ ２０ｐｍｏｌ、配列番号８で示される塩基配列からなるプライマー８ ２０ｐｍｏｌ、Ｔａｋａｒａ Ｅｘ-Ｔａｑポリメラーゼ（宝酒造社）２Ｕ、Ｔａｋａｒａ Ｅｘ-Ｔａｑポリメラーゼ添付のバッファー ５μＬ及びＴａｋａｒａ Ｅｘ-Ｔａｑポリメラーゼ添付のｄＮＴＰ ｍｉｘｔｕｒｅ（２．５ｍＭ）４μＬを含む５０μLのＰＣＲ用反応液を調製し、これをＰＣＲに供した。ＰＣＲは、まず９４℃で３０秒間、次いで６０℃で３０秒間、更に７２℃で１分間からなる保温サイクルを５０回繰り返し、最後に７２℃で５分間保温する条件にて行われた。ＰＣＲ後、アガロース電気泳動で約１．２Ｋｂｐを示すＰＣＲ産物を回収した。回収されたＰＣＲ産物をｐＴ７−Ｂｌｕｅ ｖｅｃｔｏｒ(Ｎｏｖａｇｅｎ社)にサブクローニングすることにより調製されたプラスミドでＥ．ｃｏｌｉ ＪＭ１０９株コンピテントセル（東洋紡社）を形質転換した。形質転換された細胞を５０μg/ｍＬアンピシリン含有ＬＢ培地１００ｍＬで培養することにより得られる培養菌体から前記プラスミドをＱＩＡＧＥＮ Ｐｌａｓｍｉｄ Ｍａｘｉ Ｋｉｔ(ＱＩＡＧＥＮ社)を用いて分離・精製することにより、ヒト由来の本遺伝子（ｈＡＴ１２）の塩基配列を有するＤＮＡを含むプラスミドを得た。
【００４８】
実施例６ （ヒト由来の本遺伝子の塩基配列の決定（その１））
実施例５で得られたＰＣＲ産物（約１．２kbp）を含むプラスミドを鋳型として、Ｔｈｅｒｍｏ Ｓｅｑｕｅｎａｓｅ IIダイ・ターミネーターキット（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ 社）及びＡＢＩ３７３ＤＮＡ配列読み取り装置（ＰＥＡｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社）を用いて、サンガーの方法（Ｆ．Ｓａｎｇｅｒ、Ｓ．Ｎｉｃｋｌｅｎ、Ａ．Ｒ．ＣｏｕｌｓｏｎA著、Ｐｒｏｃｅｅｄｉｎｇｓ ｏｆ Ｎａｔｉｏｎａｌ Ａｃａｄｅｍｙ ｏｆ Ｓｃｉｅｎｃｅ Ｕ．Ｓ．Ａ．(１９７７), ７４, ５４６３−５４６７）により、配列番号４で示される塩基配列を有するＤＮＡからなるヒト由来の本遺伝子（ｈＡＴ１２）の塩基配列を決定した。
【００４９】
実施例７ （ラット由来の本遺伝子のクローニング及びその塩基配列の決定（その１））
実施例３及び４に記載される方法に準じて、ラット由来の本遺伝子（ｒＡＴ１２）の塩基配列を有するＤＮＡを含むプラスミドを得、さらに、配列番号１２で示される塩基配列を有するＤＮＡからなるラット由来の本遺伝子（ｒＡＴ１２）の塩基配列を決定した。
【００５０】
実施例８ （配列番号１で示されるアミノ酸配列のアミノ末端にＦＬＡＧが付与された融合蛋白質を発現するプラスミドの調製）
実施例３で分離・精製されたプラスミド０．０５μｇ、配列番号９で示される塩基配列からなるプライマー９ ２０ｐｍｏｌ及び配列番号１０で示される塩基配列からなるプライマー１０ ２０ｐｍｏｌ、Ｔａｋａｒａ ＥｘＴａｑポリメラーゼ（宝酒造社）０．５Ｕ、Ｔａｋａｒａ ＥｘＴａｑポリメラーゼ添付のバッファー ５μＬ及びＴａｋａｒａ ＥｘＴａｑポリメラーゼ添付のｄＮＴＰ ｍｉｘｔｕｒｅ（２.５ｍＭ） ４μＬ含む５０μＬのＰＣＲ用反応液を調製し、これをＰＣＲに供した。ＰＣＲは、まず９４℃を１分間、次いで６０℃を３０秒間、更に７２℃で１分間からなる保温サイクルを３０回繰り返し、最後に７２℃で5分間保温する条件にて行われた。ＰＣＲ後、アガロース電気泳動で約１．１Ｋｂｐを示すＰＣＲ産物を回収した。回収されたＰＣＲ産物をｐＣＭＶ−Ｔａｇ２Ｂ（Ｓｔｒａｔａｇｅｎｅ社）にサブクローニングすることにより調製されたプラスミドでＥ．ｃｏｌｉ ＪＭ１０９株コンピテントセル（東洋紡社）を形質転換した。形質転換された細胞を５０μg/ｍＬカナマイシン含有ＬＢ培地１００ｍＬで培養することにより得られる培養菌体から前記プラスミドをＱＩＡＧＥＮＱＩＡｆｉｌｔｅｒ Ｍｅｇａ Ｋｉｔ(ＱＩＡＧＥＮ社)を用いて分離・精製することにより、配列番号１で示されるアミノ酸配列のアミノ末端にＦＬＡＧ（一文字表記によるアミノ酸配列：ＭＤＹＫＤＤＤＤＫＳＰＧＧＳＰＧＬＱＥＦ）が付与された融合蛋白質（以下、ＦＬＡＧ融合ｍＡＴ１２蛋白質と記すこともある。）を発現するプラスミドを得た。さらに、当該プラスミドに変異がないことを実施例４に記載される方法により確認した。
【００５１】
実施例９ （本酵素を含む除核細胞抽出液の調製（その１）：ＦＬＡＧ融合ｍＡＴ１２蛋白質を含む除核細胞抽出液の調製）
実施例８で得られたＦＬＡＧ融合ｍＡＴ１２蛋白質発現用プラスミドをＬｉｐｏｆｅｃｔＡＭＩＮＥ ＰＬＵＳ試薬キット（インビトロジェン社）を用いて、当該試薬キットに添付される手順書に従って、ＣＨＯ−Ｋ１細胞（大日本製薬社）に導入した。発現用プラスミドが導入されたＣＨＯ−Ｋ１の細胞を、当該細胞の購入時に配布される手順書に従って２４時間培養した。培養後、細胞をＤ−ＰＢＳ（ギブコ社）で２回洗浄した後、セルスクレイパーによりかき集め、これを遠心分離することにより細胞を回収した。回収された細胞を５０ｍＭトリス−塩酸（ｐＨ ７．５）、０．２５Ｍスクロース、１ｍＭエチレングリコールビス２アミノエチルエーテル四酢酸（以下、ＥＧＴＡと記す。）、１ｍＭジチオスレイトール（以下、ＤＴＴと記す。）、１００分の１容量のプロテアーゼインヒビターカクテル（シグマ社）を含む緩衝液（以下、緩衝液Ａと記す。）に懸濁した。この懸濁液を超音波処理（氷冷下、５秒間×６回）することにより懸濁液中の細胞を破砕した後、得られた細胞破砕液を１０００×ｇ、３０秒間、４℃で遠心分離することにより、未破砕の細胞及び核画分を沈殿として取り除いた。得られた上清について、ウシ血清アルブミンを標準としたブラッドフォード法により蛋白質の濃度を測定し、それぞれ２μｇ／μＬとなるように緩衝液Ａを加えて希釈したものを、除核細胞抽出液とした。
【００５２】
実施例１０ （対照となる除核細胞抽出液の調製（その１））
対照（ネガティブコントロール）として、ＦＬＡＧ融合ｍＡＴ１２蛋白質発現用プラスミドの代わりにｐＣＭＶ−Ｔａｇ２Ｂ（インサートを含まないもの）を用いる以外は実施例９に記載される方法と同様の方法により、除核細胞抽出液（対照）を得た。
【００５３】
実施例１１ （本酵素の分離及び検出（その１））
実施例９及び１０で得られた除核細胞抽出液５μＬを用いて、抗ＦＬＡＧタグＭ２抗体（シグマ社）を用いたイムノブロッティングを行ったところ、対照（ネガティブコントロール）の除核細胞抽出液の場合には、何も検出されなかった。一方、ＦＬＡＧ融合ｍＡＴ１２蛋白質発現用プラスミドが導入された細胞より調製された除核細胞抽出液の場合には、約４５ｋＤａの分子量の単一バンドである蛋白質が検出された。
【００５４】
実施例１２ （モノアシルグリセロールアシルトランスフェラーゼの活性の測定（その１））
実施例９及び１０で得られた除核細胞抽出液１０μＬを１５０μＬの反応用緩衝液（２４ｍＭ トリス−塩酸（ｐＨ ７．５）、５０ｍＭ 塩化カリウム、８ｍＭ 硫酸マグネシウム、１．２５ｍｇ／ｍＬ ウシ血清アルブミン、１ｍＭ ＤＴＴ、０．５ｍＭ ２−モノオレオイルグリセロール）に懸濁した後、当該懸濁液に５μＬの９００μＭ （０．２ＭＢｑ／μＬ）［^１４Ｃ］パルミトイル−コエンザイムＡ水溶液を加えた。この混合物を室温で１０分間保温した。尚、２−モノオレオイルグリセロールを含まない以外は上記の同じ組成である反応用緩衝液を用いて上記と同様の操作を行った試験系をバックグラウンド区とした。
次に、当該混合物に３００μＬのヘプタン／２−プロパノール／水（＝８０：２０：２）を加えて撹拌し、１０分間放置した後、さらに当該混合物に２００μＬのヘプタン及び１００μＬの水を加えて撹拌した。攪拌後、当該混合物を遠心分離することにより下層を取り除いた後、残った上層を遠心エバポレータを用いて乾固した。このようにして得られた乾固物（脂肪画分）をクロロホルムに再溶解した。次にこの溶解液をシルカゲル１５０薄層クロマトグラフィー用ガラスプレート（Ｋ５シリカゲル１５０オングストローム、ワットマン社）にスポットし、ヘキサン／ジエチルエーテル／酢酸（７５：２５：１）の展開溶媒を用いて展開することにより、当該溶解液に含まれる［^１４Ｃ］１，２−ジアシルグリセロールを分離した。分離された［^１４Ｃ］１，２−ジアシルグリセロールを、バイオイメージングアナライザシステムＢＡＳ−２０００（富士フィルム社）を用いて定量した。２−モノオレオイルグリセロールを含む反応用緩衝液での測定値から、バックグラウンド区での測定値を差し引いた値を酵素活性として算出した。対照（ネガティブコントロール）のプラスミドが導入された試験系及びＦＬＡＧ融合ｍＡＴ１２蛋白質発現用プラスミドが導入された試験系において、それぞれの除核細胞抽出液中のモノアシルグリセロールアシルトランスフェラーゼの活性を表３に示した。
【００５５】
【表３】

以上より、ＦＬＡＧ融合ｍＡＴ１２蛋白質発現用プラスミドが導入された試験系では、対照（ネガティブコントロール）のプラスミドが導入された試験系と比較して、除核細胞抽出液中のモノアシルグリセロールアシルトランスフェラーゼの活性が上昇したことから、配列番号１に示したアミノ酸配列からなる蛋白質がモノアシルグリセロールアシルトランスフェラーゼの活性を示すことが確認された。
【００５６】
実施例１３ （マウス由来の本遺伝子のクローニング（その２））
マウス由来の本遺伝子（ｍＡＴ１４）を有するＤＮＡ断片をＰＣＲにより増幅するために、まずＭｏｕｓｅ Ｎｏｒｍａｌ Ａｄｉｐｏｓｅ ｃＤＮＡ (Ｂｉｏｃｈａｉｎ社)1μＬ、配列番号１９で示される塩基配列からなるプライマー１９２０ｐｍｏｌ、配列番号２０で示される塩基配列からなるプライマー２０ ２０ｐｍｏｌ、Ｔａｋａｒａ Ｅｘ-Ｔａｑポリメラーゼ（宝酒造社）２Ｕ、Ｔａｋａｒａ Ｅｘ-Ｔａｑポリメラーゼ添付のバッファー ５μＬ及びＴａｋａｒａ Ｅｘ-Ｔａｑポリメラーゼ添付のｄＮＴＰ ｍｉｘｔｕｒｅ（２．５ｍＭ）４μＬを含む５０μLのＰＣＲ用反応液を調製し、これをＰＣＲに供した。ＰＣＲは、まず９４℃で６０秒間保温した後、９４℃で３０秒間、５５℃で３０秒間、７２℃で１分間からなる保温サイクルを４０回繰り返し、最後に７２℃で４分間保温する条件にて行われた。ＰＣＲ後、アガロース電気泳動で約１．３Ｋｂｐを示すＰＣＲ産物を回収した。回収されたＰＣＲ産物をｐＴ７−Ｂｌｕｅ ｖｅｃｔｏｒ(Ｎｏｖａｇｅｎ社)にサブクローニングすることにより調製されたプラスミドでＥ．ｃｏｌｉ ＪＭ１０９株コンピテントセル（東洋紡社）を形質転換した。形質転換された細胞を５０μg/ｍＬアンピシリン含有ＬＢ培地１００ｍＬで培養することにより得られる培養菌体から前記プラスミドをＱＩＡＧＥＮ Ｐｌａｓｍｉｄ Ｍａｘｉ Ｋｉｔ(ＱＩＡＧＥＮ社)を用いて分離・精製することにより、マウス由来の本遺伝子（ｍＡＴ１４）の塩基配列を有するＤＮＡを含むプラスミドを得た。
【００５７】
実施例１４ （マウス由来の本遺伝子の塩基配列の決定（その２））
実施例１３で得られたＰＣＲ産物（約１．３kbp）を含むプラスミドを鋳型として、Ｔｈｅｒｍｏ Ｓｅｑｕｅｎａｓｅ IIダイ・ターミネーターキット（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ 社）及びＡＢＩ３７３ＤＮＡ配列読み取り装置（ＰＥ Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社）を用いて、サンガーの方法（Ｆ．Ｓａｎｇｅｒ、Ｓ．Ｎｉｃｋｌｅｎ、Ａ．Ｒ．ＣｏｕｌｓｏｎA著、Ｐｒｏｃｅｅｄｉｎｇｓ ｏｆ Ｎａｔｉｏｎａｌ Ａｃａｄｅｍｙ ｏｆ Ｓｃｉｅｎｃｅ Ｕ．Ｓ．Ａ．(１９７７), ７４, ５４６３−５４６７）により、配列番号１４で示される塩基配列を有するＤＮＡからなるマウス由来の本遺伝子（ｍＡＴ１４）の塩基配列を決定した。
【００５８】
実施例１５ （ヒト由来の本遺伝子のクローニング（その２））
ヒト由来の本遺伝子（ｈＡＴ１４）を有するＤＮＡ断片をＰＣＲにより増幅するために、まずＨｕｍａｎ Ａｄｉｐｏｃｙｔｅ Ｍａｒａｔｈｏｎ−ｒｅａｄｙ ｃＤＮＡ (ＣＬＯＮＴＥＣＨ社)1μＬ、配列番号２１で示される塩基配列からなるプライマー２１ ２０ｐｍｏｌ、配列番号２２で示される塩基配列からなるプライマー２２ ２０ｐｍｏｌ、Ｔａｋａｒａ Ｅｘ-Ｔａｑポリメラーゼ（宝酒造社）２Ｕ、Ｔａｋａｒａ Ｅｘ-Ｔａｑポリメラーゼ添付のバッファー ５μＬ及びＴａｋａｒａ Ｅｘ-Ｔａｑポリメラーゼ添付のｄＮＴＰ ｍｉｘｔｕｒｅ（２．５ｍＭ）４μＬを含む５０μLのＰＣＲ用反応液を調製し、これをＰＣＲに供した。ＰＣＲは、まず９４℃で６０秒間保温した後、９４℃で３０秒間、６０℃で３０秒間、７２℃で１分間からなる保温サイクルを４０回繰り返し、最後に７２℃で５分間保温する条件にて行われた。ＰＣＲ後、アガロース電気泳動で約１．４Ｋｂｐを示すＰＣＲ産物を回収した。回収されたＰＣＲ産物をｐＴ７−Ｂｌｕｅ ｖｅｃｔｏｒ(Ｎｏｖａｇｅｎ社)にサブクローニングすることにより調製されたプラスミドでＥ．ｃｏｌｉ ＪＭ１０９株コンピテントセル（東洋紡社）を形質転換した。形質転換された細胞を５０μg/ｍＬアンピシリン含有ＬＢ培地１００ｍＬで培養することにより得られる培養菌体から前記プラスミドをＱＩＡＧＥＮ Ｐｌａｓｍｉｄ Ｍａｘｉ Ｋｉｔ(ＱＩＡＧＥＮ社)を用いて分離・精製することにより、ヒト由来の本遺伝子（ｈＡＴ１４）の塩基配列を有するＤＮＡを含むプラスミドを得た。
【００５９】
実施例１６ （ヒト由来の本遺伝子の塩基配列の決定（その２））
実施例１５で得られたＰＣＲ産物（約１．４kbp）を含むプラスミドを鋳型として、Ｔｈｅｒｍｏ Ｓｅｑｕｅｎａｓｅ IIダイ・ターミネーターキット（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ 社）及びＡＢＩ３７３ＤＮＡ配列読み取り装置（ＰＥＡｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社）を用いて、サンガーの方法（Ｆ．Ｓａｎｇｅｒ、Ｓ．Ｎｉｃｋｌｅｎ、Ａ．Ｒ．ＣｏｕｌｓｏｎA著、Ｐｒｏｃｅｅｄｉｎｇｓ ｏｆ Ｎａｔｉｏｎａｌ Ａｃａｄｅｍｙ ｏｆ Ｓｃｉｅｎｃｅ Ｕ．Ｓ．Ａ．(１９７７), ７４, ５４６３−５４６７）により、配列番号１６で示される塩基配列を有するＤＮＡからなるヒト由来の本遺伝子（ｈＡＴ１４）の塩基配列を決定した。
【００６０】
実施例１７ （ラット由来の本遺伝子のクローニング（その２））
ラット由来の本遺伝子（ｒＡＴ１４）を有するＤＮＡ断片をＰＣＲにより増幅するために、まずＲａｔ Ａｄｉｐｏｃｙｔｅ Ｍａｒａｔｈｏｎ−ｒｅａｄｙｃＤＮＡ (ＣＬＯＮＴＥＣＨ社)1μＬ、配列番号２３で示される塩基配列からなるプライマー２３ ２０ｐｍｏｌ、配列番号２４で示される塩基配列からなるプライマー２４ ２０ｐｍｏｌ、Ｔａｋａｒａ Ｅｘ-Ｔａｑポリメラーゼ（宝酒造社）２Ｕ、Ｔａｋａｒａ Ｅｘ-Ｔａｑポリメラーゼ添付のバッファー ５μＬ及びＴａｋａｒａ Ｅｘ-Ｔａｑポリメラーゼ添付のｄＮＴＰ ｍｉｘｔｕｒｅ（２．５ｍＭ）４μＬを含む５０μLのＰＣＲ用反応液を調製し、これをＰＣＲに供した。ＰＣＲは、まず９４℃で６０秒間保温した後、９４℃で３０秒間、６０℃で３０秒間、７２℃で１分間からなる保温サイクルを４０回繰り返し、最後に７２℃で５分間保温する条件にて行われた。ＰＣＲ後、アガロース電気泳動で約１．３Ｋｂｐを示すＰＣＲ産物を回収した。回収されたＰＣＲ産物をｐＴ７−Ｂｌｕｅ ｖｅｃｔｏｒ(Ｎｏｖａｇｅｎ社)にサブクローニングすることにより調製されたプラスミドでＥ．ｃｏｌｉ ＪＭ１０９株コンピテントセル（東洋紡社）を形質転換した。形質転換された細胞を５０μg/ｍＬアンピシリン含有ＬＢ培地１００ｍＬで培養することにより得られる培養菌体から前記プラスミドをＱＩＡＧＥＮ Ｐｌａｓｍｉｄ Ｍａｘｉ Ｋｉｔ(ＱＩＡＧＥＮ社)を用いて分離・精製することにより、ラット由来の本遺伝子（ｒＡＴ１４）の塩基配列を有するＤＮＡを含むプラスミドを得た。
【００６１】
実施例１８ （ラット由来の本遺伝子の塩基配列の決定（その２））
実施例１７で得られたＰＣＲ産物（約１．３kbp）を含むプラスミドを鋳型として、Ｔｈｅｒｍｏ Ｓｅｑｕｅｎａｓｅ IIダイ・ターミネーターキット（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ 社）及びＡＢＩ３７３ＤＮＡ配列読み取り装置（ＰＥＡｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社）を用いて、サンガーの方法（Ｆ．Ｓａｎｇｅｒ、Ｓ．Ｎｉｃｋｌｅｎ、Ａ．Ｒ．ＣｏｕｌｓｏｎA著、Ｐｒｏｃｅｅｄｉｎｇｓ ｏｆ Ｎａｔｉｏｎａｌ Ａｃａｄｅｍｙ ｏｆ Ｓｃｉｅｎｃｅ Ｕ．Ｓ．Ａ．(１９７７), ７４, ５４６３−５４６７）により、配列番号１８で示される塩基配列を有するＤＮＡからなるラット由来の本遺伝子（ｒＡＴ１４）の塩基配列を決定した。
【００６２】
実施例１９ （配列番号１３で示されるアミノ酸配列のアミノ末端にＦＬＡＧが付与された融合蛋白質を発現するプラスミドの調製）
実施例１３で分離・精製されたプラスミド０．０５μｇ、配列番号２５で示される塩基配列からなるプライマー２５ ２０ｐｍｏｌ及び配列番号２６で示される塩基配列からなるプライマー２６ ２０ｐｍｏｌ、Ｔａｋａｒａ ＥｘＴａｑポリメラーゼ（宝酒造社）０．５Ｕ、Ｔａｋａｒａ ＥｘＴａｑポリメラーゼ添付のバッファー ５μＬ及びＴａｋａｒａ ＥｘＴａｑポリメラーゼ添付のｄＮＴＰ ｍｉｘｔｕｒｅ（２.５ｍＭ） ４μＬ含む５０μＬのＰＣＲ用反応液を調製し、これをＰＣＲに供した。ＰＣＲは、まず９４℃を１分間、次いで６０℃を３０秒間、更に７２℃で１分間からなる保温サイクルを３０回繰り返し、最後に７２℃で5分間保温する条件にて行われた。ＰＣＲ後、アガロース電気泳動で約１．１Ｋｂｐを示すＰＣＲ産物を回収した。回収されたＰＣＲ産物をｐＣＭＶ−Ｔａｇ２Ｂ（Ｓｔｒａｔａｇｅｎｅ社）にサブクローニングすることにより調製されたプラスミドでＥ．ｃｏｌｉ ＪＭ１０９株コンピテントセル（東洋紡社）を形質転換した。形質転換された細胞を５０μg/ｍＬカナマイシン含有ＬＢ培地５００ｍＬで培養することにより得られる培養菌体から前記プラスミドをＱＩＡＧＥＮ ＱＩＡｆｉｌｔｅｒ Ｍｅｇａ Ｋｉｔ(ＱＩＡＧＥＮ社)を用いて分離・精製することにより、配列番号１３で示されるアミノ酸配列のアミノ末端にＦＬＡＧ（一文字表記によるアミノ酸配列：ＭＤＹＫＤＤＤＤＫＳＰＧＧＳ）が付与された融合蛋白質（以下、ＦＬＡＧ融合ｍＡＴ１４蛋白質と記すこともある。）を発現するプラスミドを得た。さらに、当該プラスミドに変異がないことを実施例１４に記載される方法により確認した。
【００６３】
実施例２０ （本酵素を含む除核細胞抽出液の調製（その２）：ＦＬＡＧ融合ｍＡＴ１４蛋白質を含む除核細胞抽出液の調製）
実施例１９で得られたＦＬＡＧ融合ｍＡＴ１４蛋白質発現用プラスミドをＬｉｐｏｆｅｃｔＡＭＩＮＥ ＰＬＵＳ試薬キット（インビトロジェン社）を用いて、当該試薬キットに添付される手順書に従って、ＣＨＯ−Ｋ１細胞（大日本製薬社）に導入した。発現用プラスミドが導入されたＣＨＯ−Ｋ１の細胞を、当該細胞の購入時に配布される手順書に従って２４時間培養した。培養後、細胞をＤ−ＰＢＳ（ギブコ社）で２回洗浄した後、セルスクレイパーによりかき集め、これを遠心分離することにより細胞を回収した。回収された細胞を５０ｍＭトリス−塩酸（ｐＨ ７．５）、０．２５Ｍスクロース、１ｍＭエチレングリコールビス２アミノエチルエーテル四酢酸（以下、ＥＧＴＡと記す。）、１ｍＭジチオスレイトール（以下、ＤＴＴと記す。）、１００分の１容量のプロテアーゼインヒビターカクテル（シグマ社）を含む緩衝液（以下、緩衝液Ａと記す。）に懸濁した。この懸濁液を超音波処理（氷冷下、５秒間×６回）することにより懸濁液中の細胞を破砕した後、得られた細胞破砕液を１０００×ｇ、３０秒間、４℃で遠心分離することにより、未破砕の細胞及び核画分を沈殿として取り除いた。得られた上清について、ウシ血清アルブミンを標準としたブラッドフォード法により蛋白質の濃度を測定し、それぞれ２μｇ／μＬとなるように緩衝液Ａを加えて希釈したものを、除核細胞抽出液とした。
【００６４】
実施例２１ （対照となる除核細胞抽出液の調製（その２））
対照（ネガティブコントロール）として、ＦＬＡＧ融合ｍＡＴ１４蛋白質発現用プラスミドの代わりにｐＣＭＶ−Ｔａｇ２Ｂ（インサートを含まないもの）を用いる以外は実施例２０に記載される方法と同様の方法により、除核細胞抽出液（対照）を得た。
【００６５】
実施例２２ （本酵素の分離及び検出（その２））
実施例２０及び２１で得られた除核細胞抽出液５μＬを用いて、抗ＦＬＡＧタグＭ２抗体（シグマ社）を用いたイムノブロッティングを行ったところ、対照（ネガティブコントロール）の除核細胞抽出液の場合には、何も検出されなかった。一方、ＦＬＡＧ融合ｍＡＴ１４蛋白質発現用プラスミドが導入された細胞より調製された除核細胞抽出液の場合には、約４６ｋＤａの分子量の単一バンドである蛋白質が検出された。
【００６６】
実施例２３ （モノアシルグリセロールアシルトランスフェラーゼの活性の測定（その２））
実施例２０及び２１で得られた除核細胞抽出液１０μＬを１５０μＬの反応用緩衝液（２４ｍＭ トリス−塩酸（ｐＨ ７．５）、５０ｍＭ 塩化カリウム、８ｍＭ 硫酸マグネシウム、１．２５ｍｇ／ｍＬ ウシ血清アルブミン、１ｍＭ ＤＴＴ、０．５ｍＭ ２−モノオレオイルグリセロール）に懸濁した後、当該懸濁液に５μＬの９００μＭ （０．２ＭＢｑ／μＬ）［^１４Ｃ］パルミトイル−コエンザイムＡ水溶液を加えた。この混合物を室温で１０分間保温した。尚、２−モノオレオイルグリセロールを含まない以外は上記の同じ組成である反応用緩衝液を用いて上記と同様の操作を行った試験系をバックグラウンド区とした。
次に、当該混合物に３００μＬのヘプタン／２−プロパノール／水（＝８０：２０：２）を加えて撹拌し、１０分間放置した後、さらに当該混合物に２００μＬのヘプタン及び１００μＬの水を加えて撹拌した。攪拌後、当該混合物を遠心分離することにより下層を取り除いた後、残った上層を遠心エバポレータを用いて乾固した。このようにして得られた乾固物（脂肪画分）をクロロホルムに再溶解した。次にこの溶解液をシルカゲル１５０薄層クロマトグラフィー用ガラスプレート（Ｋ５シリカゲル１５０オングストローム、ワットマン社）にスポットし、ヘキサン／ジエチルエーテル／酢酸（７５：２５：１）の展開溶媒を用いて展開することにより、当該溶解液に含まれる［^１４Ｃ］１，２−ジアシルグリセロールを分離した。分離された［^１４Ｃ］１，２−ジアシルグリセロールを、バイオイメージングアナライザシステムＢＡＳ−２０００（富士フィルム社）を用いて定量した。２−モノオレオイルグリセロールを含む反応用緩衝液での測定値から、バックグラウンド区での測定値を差し引いた値を酵素活性として算出した。対照（ネガティブコントロール）のプラスミドが導入された試験系及びＦＬＡＧ融合ｍＡＴ１４蛋白質発現用プラスミドが導入された試験系において、それぞれの除核細胞抽出液中のモノアシルグリセロールアシルトランスフェラーゼの活性を表４に示した。
【表４】

以上より、ＦＬＡＧ融合ｍＡＴ１４蛋白質発現用プラスミドが導入された試験系では、対照（ネガティブコントロール）のプラスミドが導入された試験系と比較して、除核細胞抽出液中のモノアシルグリセロールアシルトランスフェラーゼの活性が上昇したことから、配列番号１３に示したアミノ酸配列からなる蛋白質がモノアシルグリセロールアシルトランスフェラーゼの活性を示すことが確認された。
【００６７】
実施例２４ （配列番号３および配列番号１５で示されるアミノ酸配列のアミノ末端にＦＬＡＧが付与された融合蛋白質を発現するプラスミドの調製）
実施例８と同様に、実施例５で分離・精製されたｈＡＴ−１２プラスミド、配列番号２７および２８で示される塩基配列からなるプライマーを用いたＰＣＲ法により、配列番号３で示されるアミノ酸配列のアミノ末端にＦＬＡＧが付与された融合蛋白質（以下ＦＬＡＧ融合ｈＡＴ−１２と称することもある。）を発現するプラスミドを得た。さらに、当該プラスミドに変異がないことを実施例４に記載される方法により確認した。
また、実施例８と同様に、実施例１５で分離・精製されたｈＡＴ−１４プラスミド、配列番号２９および３０で示される塩基配列からなるプライマーを用いたＰＣＲ法により、配列番号１５で示されるアミノ酸配列のアミノ末端にＦＬＡＧが付与された融合蛋白質（以下ＦＬＡＧ融合ｈＡＴ−１４と称することもある。）を発現するプラスミドを得た。さらに、当該プラスミドに変異がないことを実施例１６に記載される方法により確認した。
【００６８】
実施例２５ （本酵素を含む除核細胞抽出液の調製（その３）：ＦＬＡＧ融合ｈＡＴ１２蛋白質・ＦＬＡＧ融合ｈＡＴ１４蛋白質を含む除核細胞抽出液の調製）実施例２４で得られたＦＬＡＧ融合蛋白質発現用プラスミドを、ＣＨＯ−Ｋ１細胞に導入し、実施例９と同様の方法で、除核細胞抽出液を調製した。
【００６９】
実施例２６ （対照となる除核細胞抽出液の調製（その３））
対照（ネガティブコントロール）として、ＦＬＡＧ融合ｈＡＴ−１２蛋白質発現用プラスミド又はＦＬＡＧ融合ｈＡＴ−１４蛋白質発現プラスミドの代わりにｐＣＭＶ−Ｔａｇ２Ｂ（インサートを含まないもの）を用いる以外は実施例９に記載される方法と同様の方法により、除核細胞抽出液（対照）を得た。
【００７０】
実施例２７ （本酵素の分離及び検出（その３））
実施例２５及び２６で得られた除核細胞抽出液５μＬを用いて、抗ＦＬＡＧタグＭ２抗体（シグマ社）を用いたイムノブロッティングを行ったところ、対照（ネガティブコントロール）の除核細胞抽出液の場合には、何も検出されなかった。一方、ＦＬＡＧ融合ｈＡＴ−１２蛋白質発現用プラスミド又はＦＬＡＧ融合ｈＡＴ−１４蛋白質発現用プラスミドが導入された細胞より調製された除核細胞抽出液の場合には、それぞれ、４５ｋＤおよび４６ｋＤの分子量の単一バンドである蛋白質が検出された。
【００７１】
実施例２８ （モノアシルグリセロールアシルトランスフェラーゼの活性の測定）
実施例１２と同様の方法で、実施例２５および２６で調製した除核細胞抽出液中のモノアシルグリセロールアシルトランスフェラーゼの活性を測定した。結果を表５に示した。
【表５】

以上より、ＦＬＡＧ融合ｈＡＴ１２蛋白質発現用プラスミドおよびＦＬＡＧ融合ｈＡＴ１４蛋白質発現用プラスミドが導入された試験系では、対照（ネガティブコントロールが導入された試験系と比較して、除核細胞抽出液中のモノアシルグリセロールアシルトランスフェラーゼの活性が上昇したことから、配列番号：３および配列番号：１５に示したアミノ酸配列からなる蛋白質がモノアシルグリセロールアシルトランスフェラーゼの活性を示すことが確認された。
【００７２】
実施例２９ （モノアシルグリセロールアシルトランスフェラーゼ遺伝子の発現分布）
実施例８、１９及び２４において得られたプラスミド（ＦＬＡＧ−ｍＡＴ１２、ｍＡＴ１４、ｈＡＴ１２、ｈＡＴ１４）を、ＦＬＡＧ配列を除く遺伝子コード配列の両端で切れるように制限酵素処理した。ＤＮＡを電気泳動したゲルから目的の長さのバンドを切り出し、ＧＥＮＥＣＬＥＡＮIIキット（宝酒造社製）によりＤＮＡを精製した。このＤＮＡ約１０−１００ｎｇを鋳型にして、ランダムプライム法による^３３Ｐ−ｄＣＴＰ放射標識を行った。反応はランダムプライムキット（宝酒造社製）を用い、キット添付のプロトコールに従った。反応液から未反応のｄＮＴＰｓをスピンカラムによって除去した後、プローブの比活性を液体シンチレーションカウンターにより測定し、プローブが十分に放射標識されていることを確認した。プローブの一部を熱変性・急冷させて１本鎖にした後、８ｍｌのＤＩＧ Ｅａｓｙ−Ｈｙｂ液（ベーリンガーマンハイム社製）に添加し、これをハイブリダイズ液とした。このハイブリダイズ液中に、前もってＤＩＧ Ｅａｓｙ−Ｈｙｂ液中でプレハイブリダイズしておいた組織ＲＮＡブロットメンブレン（Ｍｏｕｓｅ Ａｄｕｌｔ Ｎｏｒｍａｌ Ｔｉｓｓｕｅ ｍＲＮＡ Ｎｏｒｔｈｅｒｎ Ｂｌｏｔ、Ｈｕｍａｎ Ａｄｕｌｔ Ｎｏｒｍａｌ Ｔｉｓｓｕｅ Ｔｏｔａｌ ＲＮＡ Ｎｏｒｔｈｅｒｎ Ｂｌｏｔ、全てＢＩＯＣＨＡＩＮ社製）を浸し、ハイブリバッグ中にシールしてハイブリダイズを開始した。ハイブリダイズ条件は、プレハイブリダイズ：５０℃、３時間以上、ハイブリダイズ：５０℃、一晩振とう、という条件で行った。ハイブリダイズ終了後、メンブレンを２×ＳＳＣ―０．１％ ＳＤＳ、室温で２回、０．１×ＳＳＣ―０．１％ ＳＤＳ、５０℃で１回、という条件で洗浄後、メンブレンをイメージングプレートに１晩暴露した。イメージングプレートをバイオイメージングアナライザシステムＢＡＳ−２５００（富士フィルム社）を用いて読み取った結果を図１、２，３，４に示す。
これより、マウスＡＴ１２およびＡＴ１４ ｍＲＮＡは、マウス肝臓および小腸において有意に発現していること、ヒトＡＴ１２およびＡＴ１４ ｍＲＮＡは、ヒト小腸において有意に発現していることが確かめられた。
以上の結果をまとめると、ＡＴ１２およびＡＴ１４は細胞内においてモノアシルグリセロールアシルトランスフェラーゼ活性を有すること、マウスにおいて肝臓および小腸で有意に発現が見られること、ヒトにおいて小腸で有意に発現が見られることから、ＡＴ１２およびＡＴ１４は肝臓および小腸においてトリグリセライド合成に関与するモノアシルグリセロールアシルトランスフェラーゼであると考えられる。
【００７３】
実施例３０ （ＦＬＡＧ融合ｍＡＴ１２蛋白質を安定して発現する細胞株の樹立）
実施例８で得られたＦＬＡＧ融合ｍＡＴ１２蛋白質発現用プラスミドをＬｉｐｏｆｅｃｔＡＭＩＮＥ ＰＬＵＳ試薬キット（インビトロジェン社）を用いて、当該試薬キットに添付される手順書に従って、ＣＨＯ−Ｋ１細胞（大日本製薬社）に導入した。発現用プラスミドが導入されたＣＨＯ−Ｋ１細胞を当該細胞の購入時に配布される手順書に従って７２時間培養した後、４００μｇ／ｍｌのＧＥＮＥＴＩＣＩＮ（ギブコ社）を含む１０％ ＦＣＳ添加Ｈａｍ’ｓ Ｆ−１２培地（以下、選択培地と記す）に交換し、適宜新しい培地に交換しながら、７日間培養を続けた。培養後、限外希釈法により、安定発現株のクローン化を行った。得られた細胞株の各々について、一部を５０ｍＭトリス−塩酸（ｐＨ ７．５）、０．２５Ｍスクロース、１ｍＭエチレングリコールビス２アミノエチルエーテル四酢酸（以下、ＥＧＴＡと記す。）、１ｍＭジチオスレイトール（以下、ＤＴＴと記す。）、１００分の１容量のプロテアーゼインヒビターカクテル（シグマ社）を含む緩衝液（以下、緩衝液Ａと記す。）に懸濁した。この懸濁液を超音波処理（氷冷下、５秒間×６回）することにより懸濁液中の細胞を破砕した後、得られた細胞破砕液を１０００×ｇ、３０秒間、４℃で遠心分離することにより、未破砕の細胞及び核画分を沈殿として取り除いた。得られた上清について、抗ＦＬＡＧタグＭ２抗体（シグマ社）を用いたイムノブロッティングを行い、約４５ｋＤａの分子量の単一バンドである蛋白質を最も多く発現している細胞株を選択し、ｍＡＴ−１２安定発現株を得た。
【００７４】
実施例３１ （ＦＬＡＧ融合ｍＡＴ１２蛋白質の精製）
実施例３０で得られたＦＬＡＧ融合ｍＡＴ１２蛋白質を安定して発現する細胞株を選択培地中で拡大培養した。培養後、細胞をＤ−ＰＢＳ（ギブコ社）で２回洗浄した後、セルスクレイパーによりかき集め、これを遠心分離することにより細胞を回収した。回収された細胞を５０ｍＭトリス−塩酸（ｐＨ ７．５）、１５０ｍＭ塩化ナトリウム、０．１％ {３−〔（３−Ｃｈｏｌａｍｉｄｏｐｒｏｐｙｌ）ｄｉｍｅｔｈｙｌａｍｍｏｎｉｏ〕−１−ｐｒｏｐａｎｅｓｕｌｆｏｎａｔｅ}（以下、 ＣＨＡＰＳと記す）、１ｍＭ ＥＧＴＡ、１ｍＭ ＤＴＴ、１００分の１容量のプロテアーゼインヒビターカクテル（シグマ社）を含む緩衝液に懸濁した。この懸濁液を超音波処理（氷冷下、６秒間×６回）することにより懸濁液中の細胞を破砕した後、得られた細胞破砕液を１５０００×ｇ、５分間、４℃で遠心分離することにより、未破砕の細胞及び核画分を沈殿として取り除き、上清を細胞粗抽出液として得た。Ａｎｔｉ−ＦＬＡＧ Ｍ２アフィニティゲル１ｍｌを５０ｍＭトリス−塩酸（ｐＨ ７．５）、１５０ｍＭ塩化ナトリウム、０．１％ ＣＨＡＰＳ（以下、緩衝液Ｂと記す。）により平衡化したカラムに、細胞粗抽出液を加え、ＦＬＡＧ融合ｍＡＴ１２蛋白質を吸着させた。１０倍容量の緩衝液Bにてカラムを洗浄した後、流出を停止し、ＦＬＡＧペプチドを含む緩衝液Ｂを添加してインキュベートした。１０分後、再び流出を開始し、ＦＬＡＧ融合ｍＡＴ１２タンパク質精製画分を得た。抗ＦＬＡＧタグＭ２抗体（シグマ社）を用いたイムノブロッティングを行い、本画分に約４５ｋＤａの分子量の単一バンドである蛋白質が含まれることを確認した。
【００７５】
実施例３２（精製ＦＬＡＧ融合ｍＡＴ１２蛋白質のモノアシルグリセロールアシルトランスフェラーゼ活性の測定）
実施例３１で得られた精製ＦＬＡＧ融合ｍＡＴ１２蛋白質精製画分１０μlを反応用緩衝液（２４ｍＭ トリス−塩酸（ｐＨ７．５）、５０ｍＭ 塩化カリウム、８ｍＭ 硫酸マグネシウム、１．２５ｍｇ／ｍｌ ウシ血清アルブミン、１ｍＭ ＤＴＴ、０．５ｍＭ ２−モノオレオイルグリセロール）１３０μlに懸濁した後、被験物質（ジメチルスルホキシド（以下、ＤＭＳＯと記す。）に溶解、終濃度１％ ＤＭＳＯとなるように添加）を添加し、混合した。この混合物に１０μＬの４５０μＭ［^１４Ｃ］パルミトイル−コエンザイムＡ水溶液を添加し、室温で２０分間保温した。尚、被験物質無添加区では、前記の被験物質のＤＭＳＯ溶液に代えてＤＭＳＯのみ１．５μｌを添加すること以外は前記と同様な方法を用いた。また、バックグラウンド区では、前記の被験物質及び２−モノオレオイルグリセロールに代えてＤＭＳＯのみを添加すること以外は、被験物質及び２−モノオレオイルグリセロール添加区と同様な方法を用いた。 ２０分間保温終了後、前記反応混合物に３００μＬのヘプタン／２−プロパノール／水（＝８０：２０：２）を添加することにより反応を停止し、さらにヘプタン２００μｌ及び水１００μｌを添加した後、懸濁した。攪拌後、当該混合物を遠心分離することにより下層を取り除いた後、残った上層を遠心エバポレータを用いて乾固した。このようにして得られた乾固物（脂肪画分）をクロロホルム／メタノール（２：１、ｖ／ｖ）３０μｌに再溶解した。次にこの溶解液をシリカゲル１５０薄層クロマトグラフィー用ガラスプレート（Ｋ５シリカゲル１５０オングストローム、ワットマン社）にスポットした。密閉容器にヘキサン／ジエチルエーテル／酢酸（７５：２５：１、ｖ／ｖ）の展開溶媒を入れ、当該展開溶媒を用いて前記ＴＬＣプレートを展開した後、当該ＴＬＣプレートを室温で乾燥した。乾燥された当該ＴＬＣプレートにイメージングプレート（富士フィルム社）を１６時間露光させた。露光終了後、イメージングプレートをバイオイメージアナライザー（ＢＡＳ２５００、富士フィルム社）で分析し、前記ＴＬＣプレート上の１，２−ジアシルグリセロールおよびトリグリセライドの展開位置に相当する部分の［^１４Ｃ］放射活性を測定した（以下、１，２−ジアシルグリセロール部分の展開位置に相当する部分の［^１４Ｃ］放射活性を測定値Ｇ、トリアシルグリセロールの展開位置に相当する部分の［^１４Ｃ］放射活性を測定値Ｈと記す。）。これらの測定値から、被検物質を添加した場合のモノアシルグリセロールアシルトランスフェラーゼの活性（測定値Ｇ＋測定値Ｈ／２、以下、活性値４と記す）を算出した。また、被験物質無添加区及びバックグラウンド区についても、前記と同様な方法を用いてＴＬＣプレート上の１，２−ジアシルグリセロールおよびトリグリセライドの展開位置に相当する部分の［^１４Ｃ］放射活性を測定した（以下、被験物質無添加区の１，２−ジアシルグリセロールの展開位置に相当する部分の［^１４Ｃ］放射活性を測定値Ｉ、被験物質無添加区のトリアシルグリセロールの展開位置に相当する部分の［^１４Ｃ］放射活性を測定値Ｊ、バックグラウンド区の１，２−ジアシルグリセロールの展開位置に相当する部分の放射活性を測定値Ｋ、バックグラウンド区のトリアシルグリセロールの展開位置に相当する部分の放射活性を測定値Ｌと記す。）。これらの測定値から、被験物質無添加区のモノアシルグリセロールアシルトランスフェラーゼの活性（測定値Ｉ＋測定値Ｊ／２、以下、活性値５と記す。）およびバックグラウンド区のモノアシルグリセロールアシルトランスフェラーゼの活性（測定値Ｋ＋測定値Ｌ／２、以下、活性値６と記す。）を算出した。得られた活性値から被験物質のモノアシルグリセロールアシルトランスフェラーゼ阻害能力を阻害率（（活性値５−活性値４）×１００／（活性値５−活性値６））として算出した。
その結果、被験物質である公知化合物 Ｎ−［２−（４−ベンジル−２−エチルフェノキシ）エチル］−５，６−ジメチル［１，２，４］トリアゾロ［１，５−ａ］ピリミジン−７−アミン（以下、化合物Ａと記すこともある。）における阻害率は１０μMで約６５％であった。さらに被験物質の供試濃度を変化させながら、前記と同様な方法を用いて各供試濃度における阻害率を算出した。その結果を表６に示した。
以上より、公知化合物ＡはｍＡＴ１２あるいはＦＬＡＧ融合ｍＡＴ１２のモノアシルグリセロールアシルトランスフェラーゼ酵素活性を効果的に阻害することがわかった。
【表６】

【００７６】
実施例３３ （ヒト肝臓細胞のトリグリセライド合成に対するモノアシルグリセロールアシルトランスフェラーゼ阻害剤の抑制効果）
ヒト肝癌由来細胞株ＨｅｐＧ２細胞（大日本製薬）を６穴プレート中でコンフルエント状態になるまで培養後、血清を含まないＤ−ＭＥＭ培地（ＧＩＢＣＯ社製、高グルコース含有）で１晩処理することによって飢餓状態にした。この細胞の培地を、終濃度１２．９μＭ［Ｕ−^１４Ｃ］Ｄ−グルコース（アマシャム社製、放射活性４ｍＣｉ／Ｌ）を含むＤ−ＭＥＭ培地（ＧＩＢＣＯ社製、低グルコース含有）に交換後、直ちに被験物質（ジメチルスルホキシド（以下、ＤＭＳＯと記す。））に溶解、終濃度０．５％ ＤＭＳＯとなるように添加）を添加して３７℃で４時間培養した。尚、被験物質無添加区では、前記の被験物質のＤＭＳＯ溶液に代えてＤＭＳＯのみ添加すること以外は前記と同様な方法を用いた。培養後の細胞をＰＢＳ緩衝液で１回洗浄後、細胞を１ｍｌのヘプタン・イソプロパノール（２：１）で１０分間処理した。細胞中のトリグリセライドを含むヘプタン・イソプロパノール液をピペットで回収し、回収液を遠心エバポレータを用いて乾固した。このようにして得られた乾固物（脂肪画分）をクロロホルム／メタノール（２：１、ｖ／ｖ）３０μｌに再溶解した。次にこの溶解液をシリカゲル１５０薄層クロマトグラフィー用ガラスプレート（Ｋ５シリカゲル１５０オングストローム、ワットマン社）にスポットした。密閉容器にヘキサン／ジエチルエーテル／酢酸（７５：２５：１、ｖ／ｖ）の展開溶媒を入れ、当該展開溶媒を用いて前記ＴＬＣプレートを展開した後、当該ＴＬＣプレートを室温で乾燥した。乾燥された当該ＴＬＣプレートにイメージングプレート（富士フィルム社）を１６時間露光させた。露光終了後、イメージングプレートをバイオイメージアナライザー（ＢＡＳ２５００、富士フィルム社）で分析し、前記ＴＬＣプレート上のトリアシルグリセロールの展開位置に相当する部分の［^１４Ｃ］放射活性を測定した。また、被験物質無添加区についても、前記と同様な方法を用いてＴＬＣプレート上のトリアシルグリセロールの展開位置に相当する部分の［^１４Ｃ］放射活性を測定した。
その結果、被験物質である公知化合物 Ｎ−［２−（４−ベンジル−２−エチルフェノキシ）エチル］−５，６−ジメチル［１，２，４］トリアゾロ［１，５−ａ］ピリミジン−７−アミン（以下、化合物Ａと記すこともある。）添加時のトリグリセライド合成量は、被検物質無添加区における合成量を１００％としたとき、１０μMで約３１％であった。さらに被験物質の供試濃度を変化させながら、前記と同様な方法を用いて各供試濃度における合成量を算出した結果を表７に示した。
この結果、公知化合物Ａはヒト肝臓細胞のトリグリセライド合成を効果的に阻害することがわかった。従って、モノアシルグリセロールアシルトランスフェラーゼ活性阻害剤は、肝臓のトリグリセライド合成を阻害することによって、リポ蛋白（ＶＬＤＬ）合成を阻害することができると考えられる。
【表７】

【００７７】
実施例３４ （マウスへの油脂投与後の血中トリグリセライド上昇に対するモノアシルグリセロール阻害剤の効果）
ＩＣＲマウス（１２週齢、オス、日本エスエルシー）８匹／群を１晩絶食後、Ｎ−［２−（４−ベンジル−２−エチルフェノキシ）エチル］−５，６−ジメチル［１，２，４］トリアゾロ［１，５−ａ］ピリミジン−７−アミン（以下、化合物Ａ）を懸濁したオリーブ油（ナカライテスク社製）:５ｍｌ／ｋｇを強制経口投与した。被検物質非処理区として、化合物Ａを含まないオリーブ油をマウス８匹に経口投与した。マウスの尾静脈から経時的に１０μＬの血液を採取し、１ｍｌのイソプロパノールに溶解した。イソプロパノール抽出液中のトリグリセライド量を、トリグリセライドテストキットワコー（和光純薬工業）を用いて測定した。化合物Ａを３０ｍｇ／ｋｇの用量で投与した時の血中トリグリセライド量の経時変化を図５に、化合物Ａの投与量を０，１０，３０ｍｇ／ｋｇに変化させた時の３時間後の血中トリグリセライド量を測定した結果を図６に示す。
この結果、公知化合物Ａを脂質と共にマウスに投与した場合、血中のトリグリセライドの上昇が効果的に阻害されることが確認できた。従って、モノアシルグリセロールアシルトランスフェラーゼ阻害剤は、小腸細胞のトリグリセライド合成を阻害することによって、小腸細胞のリポ蛋白（カイロミクロン）合成・分泌を効果的に阻害し、結果として血中トリグリセライド濃度を低下させることが可能と考えられる。
【００７８】
【発明の効果】
本発明により、簡易な脂肪量増加抑制能力の検定方法等が提供可能となった。
【００７９】
［配列表フリーテキスト］
配列番号５
ＰＣＲのために設計されたプライマー
配列番号６
ＰＣＲのために設計されたプライマー
配列番号７
ＰＣＲのために設計されたプライマー
配列番号８
ＰＣＲのために設計されたプライマー
配列番号９
ＰＣＲのために設計されたプライマー
配列番号１０
ＰＣＲのために設計されたプライマー
配列番号１９
ＰＣＲのために設計されたプライマー
配列番号２０
ＰＣＲのために設計されたプライマー
配列番号２１
ＰＣＲのために設計されたプライマー
配列番号２２
ＰＣＲのために設計されたプライマー
配列番号２３
ＰＣＲのために設計されたプライマー
配列番号２４
ＰＣＲのために設計されたプライマー
配列番号２５
ＰＣＲのために設計されたプライマー
配列番号２６
ＰＣＲのために設計されたプライマー
配列番号２７
ＰＣＲのために設計されたプライマー
配列番号２８
ＰＣＲのために設計されたプライマー
配列番号２９
ＰＣＲのために設計されたプライマー
配列番号３０
ＰＣＲのために設計されたプライマー
【００８０】
【配列表】

【図面の簡単な説明】
【図１】マウスＡＴ１２配列をプローブにして、マウスＡｄｕｌｔ Ｎｏｒｍａｌ Ｔｉｓｓｕｅ ｍＲＮＡ Ｎｏｒｔｈｅｒｎ Ｂｌｏｔに対するノーザンハイブリダイゼーションを行った。左のレーンから順に、脳、心臓、腎臓、肝臓、肺、骨格筋、皮膚、小腸、脾臓、胃、精巣、胸腺の組織ＲＮＡである。
【図２】マウスＡＴ１４配列をプローブにして、マウスＡｄｕｌｔ Ｎｏｒｍａｌ Ｔｉｓｓｕｅ ｍＲＮＡ Ｎｏｒｔｈｅｒｎ Ｂｌｏｔに対するノーザンハイブリダイゼーションを行った。左のレーンから順に、脳、心臓、腎臓、肝臓、肺、骨格筋、皮膚、小腸、脾臓、胃、精巣、胸腺の組織ＲＮＡである。
【図３】ヒトＡＴ１２配列をプローブにして、ヒトＡｄｕｌｔ Ｎｏｒｍａｌ Ｔｉｓｓｕｅ Ｔｏｔａｌ ＲＮＡ Ｎｏｒｔｈｅｒｎ Ｂｌｏｔに対するノーザンハイブリダイゼーションを行った。左のレーンから順に、食道、胃、小腸、結腸、子宮、胎盤、膀胱、脂肪の組織ＲＮＡである。図の左には、各組織の由来する検体の性別（男性＝Ｍ、女性＝Ｆ）および年齢を示す。
【図４】ヒトＡＴ１４配列をプローブにして、ヒトＡｄｕｌｔ Ｎｏｒｍａｌ Ｔｉｓｓｕｅ Ｔｏｔａｌ ＲＮＡ Ｎｏｒｔｈｅｒｎ Ｂｌｏｔに対するノーザンハイブリダイゼーションを行った。左のレーンから順に、食道、胃、小腸、結腸、子宮、胎盤、膀胱、脂肪の組織ＲＮＡである。図の左には、各組織の由来する検体の性別（男性＝Ｍ、女性＝Ｆ）および年齢を示す。
【図５】マウスを２群（８匹／群）に分け、第１群にはオリーブオイルのみ、第２群にはオリーブオイルと化合物Ａを投与した。投与直後から６時間まで１時間ごとに尾静脈から約１０μｌ採血を行い、血中のトリグリセライド量を定量した。定量結果を、縦軸に血中トリグリセライド濃度（ｍｇ／ｄＬ）、横軸に時間をプロットしたグラフで示した。
【図６】マウスを３群（８匹／群）に分け、第１群にはオリーブオイルのみ、第２群にはオリーブオイル＋化合物Ａ：１０ｍｇ／ｋｇ、第３群にはオリーブオイル＋化合物Ａ：３０ｍｇ／ｋｇを投与した。各群について投与前（０ｈｒ）および投与後３時間（３ｈｒ）に尾静脈から約１０μｌ採血を行い、血中のトリグリセライド量を定量した。測定したトリグリセライド濃度（ｍｇ／ｄＬ）を縦軸に示した。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for testing a fat mass increase inhibiting ability and the like.
[0002]
[Prior art]
Fat in the body can be broadly classified into fat accumulated in cells of body tissue and fat present in blood.
The former fat is, for example, in the body of a mammal or the like, (1) subcutaneous adipose tissue such as ventral region, thigh, buttocks or chest, (2) adipose tissue in contact with kidney or accessory testicle, mesentery It accumulates in various tissues such as adipose tissue or abdominal adipose tissue such as omental adipose tissue, (3) adipose tissue contained in the liver, or (4) intrathoracic adipose tissue. The amount of tissue by accumulation of fat in adipose tissue, particularly, for example, intraperitoneal adipose tissue (in particular, abdominal adipose tissue existing around blood vessels flowing into portal veins such as mesenteric adipose tissue and omental adipose tissue) Is closely related to the onset of metabolic diseases such as impaired glucose tolerance, diabetes, hyperlipidemia, hypertension, arteriosclerosis, and diseases such as coronary artery disease, angina pectoris, and myocardial infarction This has been clarified. For this reason, the treatment or prevention of the aforementioned diseases closely related to the accumulation of fat in the adipose tissue by suppressing the accumulation of fat in the adipose tissue, particularly, for example, the abdominal adipose tissue and suppressing the increase in the amount of adipose tissue Is possible.
[0003]
Fat in the blood is the fat synthesized in the cell and released into the blood. Specifically, it forms a complex particle (lipoprotein) with a protein core called apoprotein. It is circulating. In recent years, an increase in the amount of fat in the blood, that is, hypertriglyceridemia, is considered to be one of the risk factors for arteriosclerotic diseases such as ischemic heart disease and cerebral infarction as well as diabetes, hypertension or smoking. Became. From this, it is closely related to the increase in the amount of fat in the blood by suppressing fat synthesis in the organ responsible for fat supply to the blood, specifically the small intestine and liver, and suppressing the increase in the amount of fat in the blood. The related disease can be treated or prevented.
[0004]
Until now, several methods for finding a substance having an ability to suppress the increase in fat mass have been known (for example, Patent Document 1), but it has not always been simple and sufficient.
[0005]
Meanwhile, the following reaction:
2 or 1-monoacylglycerol + acyl-CoA
⇒ 1, 2, or 1, 3-diacylglycerol + CoA
The degree of contribution of the enzyme that catalyzes the above, ie, monoacylglycerol acyltransferase (hereinafter sometimes abbreviated as MGAT) in the accumulation of fat and the increase in blood fat mass by lipoprotein synthesis was unknown. In addition, although there have been reported examples of the sequence of monoacylglycerol acyltransferase (Non-Patent Document 1), the sequence is involved in lipoprotein synthesis and release of triglycerides into blood. The sequence of the enzyme that is hardly expressed in, and works effectively in the human body has not been known.
[Patent Document 1]
International Publication No. 00/44754 Pamphlet
[Non-Patent Document 1]
RV Farese Jr. et al., Proceedings of National Academy of Science USA, p8512-8517, 2002
[0006]
[Problems to be solved by the invention]
Therefore, it has been desired to develop a simple and excellent assay method for suppressing the increase in fat mass, which is indispensable for searching for a substance having an ability to suppress the increase in fat mass.
[0007]
[Means for Solving the Problems]
As a result of intensive studies under these circumstances, the present inventors have found that a substance having monoacylglycerol transferase (MGAT) inhibitory activity has the action of suppressing fat accumulation in adipocytes and lipoprotein synthesis in the small intestine and liver. And found to be effective as a fat mass increase inhibitor. Further, at the same time as identifying the base sequence and amino acid sequence of a novel human, mouse and rat MGAT, the substance having the MGAT inhibitory activity inhibits the enzymatic activity of MGAT comprising the sequence identified in the present invention, thereby It has been found that the increase in fat mass in cells or lipoprotein synthesis in small intestine or liver cells can be suppressed, and the present invention has been completed.
[0008]
That is, the present invention
1. A test method for the ability to suppress fat increase,
(1) a first step of measuring the activity of the monoacylglycerol acyltransferase in a contact system between the monoacylglycerol acyltransferase and the test substance; and
(2) a second step of evaluating the fat mass increase inhibiting ability of the substance based on the difference obtained by comparing the activity measured in the first step and the activity in the control;
A method for assaying the ability to inhibit fat mass increase, characterized by comprising:
2. A method for searching a substance for inhibiting increase in fat mass, comprising selecting a substance having an ability to inhibit fat mass increase based on the ability to inhibit fat mass increase evaluated by the assay method according to Item 1;
3. A substance for suppressing the increase in fat mass, comprising a substance selected by the search method according to Item 2 or a pharmaceutically acceptable salt thereof as an active ingredient;
4). A fat increase inhibitor comprising a substance having a monoacylglycerol acyltransferase inhibiting ability or a pharmaceutically acceptable salt thereof as an active ingredient;
5. Item 1. The method for assaying a fat mass increase inhibiting ability according to Item 1, wherein the monoacylglycerol acyltransferase is derived from a mammal.
6). Item 1. The method for assaying an increase in fat mass according to Item 1, wherein the monoacylglycerol acyltransferase is one of the following proteins;
<Protein group>
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1, 3 or 11
(A-1) A protein comprising the amino acid sequence represented by SEQ ID NO: 13, 15 or 17 (b) In the amino acid sequence represented by SEQ ID NO: 1, 3 or 11, one or more amino acids are deleted, added or substituted A protein having an amino acid sequence and having monoacylglycerol acyltransferase activity
(B-1) A protein comprising an amino acid sequence in which one or more amino acids are deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 13, 15 or 17, and having monoacylglycerol acyltransferase activity
(C) a protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, 3, or 11 and having monoacylglycerol acyltransferase activity
(C-1) a protein comprising a monoacylglycerol acyltransferase activity, comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 13, 15 or 17
(D) a protein comprising an amino acid sequence encoded by a DNA having the base sequence represented by SEQ ID NO: 2, 4 or 12
(D-1) a protein comprising an amino acid sequence encoded by DNA having the base sequence represented by SEQ ID NO: 14, 16, or 18
(E) consisting of an amino acid sequence encoded by a DNA having a base sequence of 80% or more of the DNA having the base sequence represented by SEQ ID NO: 2, 4 or 12, and having monoacylglycerol acyltransferase activity Protein
(E-1) a monoacylglycerol acyltransferase comprising an amino acid sequence encoded by a DNA having a base sequence of 80% or more of the DNA having the base sequence represented by SEQ ID NO: 14, 16 or 18, and Active protein
(F) a DNA comprising a base sequence complementary to the DNA having the base sequence represented by SEQ ID NO: 2, 4 or 12, and an amino acid sequence encoded by the DNA that hybridizes under stringent conditions; Protein having acylglycerol acyltransferase activity
(F-1) consisting of a DNA consisting of a base sequence complementary to the DNA having the base sequence shown in SEQ ID NO: 14, 16 or 18, and an amino acid sequence encoded by the DNA that hybridizes under stringent conditions; And monoacylglycerol acyltransferase activity protein
7). The assay method according to any one of Items 1, 5 and 6, wherein the fat mass increase inhibiting ability is a fat accumulation inhibiting ability;
8). A method for searching a fat accumulation-inhibiting substance, comprising selecting a substance having a fat accumulation inhibiting ability based on the fat accumulation inhibiting ability evaluated by the assay method according to Item 7;
9. A fat accumulation inhibitor characterized by comprising a substance selected by the search method according to Item 8 or a pharmaceutically acceptable salt thereof as an active ingredient;
10. A fat accumulation inhibitor comprising a substance having a monoacylglycerol acyltransferase inhibitory ability or a pharmaceutically acceptable salt thereof as an active ingredient;
11. Item 6. The assay method according to any one of Items 1, 5 and 6, wherein the fat mass increase inhibiting ability is lipoprotein synthesis inhibiting ability;
12 A method for searching a lipoprotein synthesis inhibitor, comprising selecting a substance having a lipoprotein synthesis inhibitory ability based on the lipoprotein synthesis inhibitory ability evaluated by the assay method according to Item 11;
13. Item 21. A lipoprotein synthesis inhibitor comprising a substance selected by the search method according to Item 12 or a pharmaceutically acceptable salt thereof as an active ingredient;
14 A lipoprotein synthesis inhibitor comprising a substance having a monoacylglycerol acyltransferase inhibiting ability or a pharmaceutically acceptable salt thereof as an active ingredient;
15. A substance having the ability to inhibit monoacylglycerol acyltransferase is N- [2- (4-benzyl-2-ethylphenoxy) ethyl] -5,6-dimethyl [1,2,4] triazolo [1,5-a] pyrimidine Item 15. The lipoprotein synthesis inhibitor according to Item 14, which is a -7-amine;
16. N- [2- (4-benzyl-2-ethylphenoxy) ethyl] -5,6-dimethyl [1,2,4] triazolo [1,5-a] pyrimidin-7-amine as an active ingredient, Monoacylglycerol acyltransferase inhibitors;
17. A protein having any of the following amino acid sequences;
<Amino acid sequence group>
(A) the amino acid sequence represented by SEQ ID NO: 1, 3 or 11
(A-1) Amino acid sequence represented by SEQ ID NO: 13, 15 or 17
(B) an amino acid sequence of a protein having a monoacylglycerol acyltransferase activity, wherein the amino acid sequence represented by SEQ ID NO: 1, 3, or 11 has one or more amino acids deleted, added or substituted;
(B-1) An amino acid sequence in which one or more amino acids are deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 13, 15 or 17, and having a monoacylglycerol acyltransferase activity Amino acid sequence
(C) an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 1, 3 or 11 and having a monoacylglycerol acyltransferase activity
(C-1) an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 13, 15 or 17 and having a monoacylglycerol acyltransferase activity
(D) an amino acid sequence encoded by DNA having the base sequence represented by SEQ ID NO: 2, 4 or 12
(D-1) Amino acid sequence encoded by DNA having the base sequence represented by SEQ ID NO: 14, 16 or 18
(E) an amino acid sequence encoded by a DNA having a base sequence of 80% or more of the DNA having the base sequence represented by SEQ ID NO: 2, 4 or 12, and having a monoacylglycerol acyltransferase activity Amino acid sequence of a protein having
(E-1) an amino acid sequence encoded by a DNA having the base sequence represented by SEQ ID NO: 14, 16 or 18 and having a base sequence having 80% or more of sequence identity, and monoacylglycerol acyl Amino acid sequence of a protein having transferase activity
(F) an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA having the base sequence represented by SEQ ID NO: 2, 4 or 12, and Amino acid sequence of a protein having monoacylglycerol acyltransferase activity
(F-1) an amino acid sequence encoded by DNA having a base sequence complementary to DNA having the base sequence represented by SEQ ID NO: 14, 16, or 18 and DNA hybridized under stringent conditions; And amino acid sequence of a protein having monoacylglycerol acyltransferase activity
18. A gene having a base sequence encoding any of the following amino acid sequences;
<Amino acid sequence group>
(A) the amino acid sequence represented by SEQ ID NO: 1, 3 or 11
(A-1) Amino acid sequence represented by SEQ ID NO: 13, 15 or 17
(B) an amino acid sequence of a protein having a monoacylglycerol acyltransferase activity, wherein the amino acid sequence represented by SEQ ID NO: 1, 3, or 11 has one or more amino acids deleted, added or substituted;
(B-1) An amino acid sequence in which one or more amino acids are deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 13, 15 or 17, and having a monoacylglycerol acyltransferase activity Amino acid sequence
(C) an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 1, 3 or 11 and having a monoacylglycerol acyltransferase activity
(C-1) an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 13, 15 or 17 and having a monoacylglycerol acyltransferase activity
(D) an amino acid sequence encoded by DNA having the base sequence represented by SEQ ID NO: 2, 4 or 12
(D-1) Amino acid sequence encoded by DNA having the base sequence represented by SEQ ID NO: 14, 16 or 18
(E) an amino acid sequence encoded by a DNA having a base sequence of 80% or more of the DNA having the base sequence represented by SEQ ID NO: 2, 4 or 12, and having a monoacylglycerol acyltransferase activity Amino acid sequence of a protein having
(E-1) an amino acid sequence encoded by a DNA having the base sequence represented by SEQ ID NO: 14, 16 or 18 and having a base sequence having 80% or more of sequence identity, and monoacylglycerol acyl Amino acid sequence of a protein having transferase activity
(F) an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA having the base sequence represented by SEQ ID NO: 2, 4 or 12, and Amino acid sequence of a protein having monoacylglycerol acyltransferase activity
(F-1) an amino acid sequence encoded by DNA having a base sequence complementary to DNA having the base sequence represented by SEQ ID NO: 14, 16, or 18 and DNA hybridized under stringent conditions; And amino acid sequence of a protein having monoacylglycerol acyltransferase activity
19. Item 18. A catalyst for the following reaction, comprising the protein according to Item 17.
2 or 1-monoacylglycerol + acyl-CoA ⇒ 1,2-diacylglycerol + CoA
;
Etc. are provided.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
The present invention is described in detail below.
As used herein, “fat” refers to fat contained in any cell, tissue, or body fluid in a mammal, and specifically refers to a glycerol fatty acid ester such as triglyceride.
The "fat mass increase inhibiting ability" is a concept including both fat accumulation inhibiting ability or fat synthesis inhibiting ability, specifically, the ability to inhibit fat accumulation in fat cells, adipose tissue or hepatocytes, or It means the ability to suppress fat synthesis in the small intestine or liver.
[0010]
The “adipose tissue” refers to any adipose tissue of mammals. Specifically, (1) subcutaneous adipose tissue such as ventral region, thigh, buttocks or chest, (2) kidney or accessory testicle Adipose tissue in contact with the abdominal cavity, mesenteric adipose tissue, or abdominal adipose tissue such as omental adipose tissue, (3) adipose tissue contained in the liver, or (4) intrathoracic adipose tissue. An adipocyte means an individual cell constituting the adipose tissue.
In adipose tissue, glycerol fatty acid esters such as triglyceride accumulate as fat.
[0011]
On the other hand, fat in blood exists in the form of lipoprotein in which a glycerol fatty acid ester such as triglyceride forms a complex with apoprotein. The lipoproteins are classified according to their density into chylomicron, VLDL (very low density lipoprotein), LDL (low density lipoprotein), HDL (high density lipoprotein) and the like.
The supply tissues of triglyceride into the blood are the small intestine and liver. The small intestine absorbs triglyceride from food, and the liver regulates the amount of triglyceride in the blood by collecting and re-secreting blood lipoproteins. In the small intestine, triglyceride in food is broken down into monoacylglycerol and fatty acid in the intestinal tract by the action of pancreatic lipase, then absorbed into small intestinal epithelial cells and re-synthesized into triglyceride in small intestinal cells. The re-synthesized triglyceride is incorporated into chylomicron, a kind of lipoprotein, in the small intestinal cells and secreted into the blood. Chylomicron is converted into particles called chylomicron remnant while supplying triglyceride to peripheral tissues, and finally absorbed into the liver. In the liver, triglyceride synthesis is performed using fatty acids in liver cells, and the synthesized triglyceride is incorporated into VLDL, which is a kind of lipoprotein, and secreted into the blood. Similar to chylomicron, VLDL circulates in the body while supplying triglyceride to peripheral tissues, and is finally collected in the liver in the form of LDL.
That is, the fat synthesis inhibiting ability preferably represents the ability to inhibit lipoprotein synthesis in the small intestine or liver.
[0012]
The monoacylglycerol acyltransferase used in the assay method of the present invention is an enzyme that catalyzes the following reaction (hereinafter sometimes referred to as the present enzyme).
2 or 1-monoacylglycerol + acyl-CoA ⇒ 1,2-diacylglycerol + CoA
[0013]
The activity of such an enzyme can be measured, for example, by the following method (for example, the method of SC Jamdar et al. (Arch. Biochem. Biophys. 1992; 296 (2): 419-25)).
An enzyme sample containing this enzyme was added to 150 μL of a reaction buffer (24 mM Tris-hydrochloric acid (pH 7.5), 50 mM potassium chloride, 8 mM magnesium sulfate, 1.25 mg / mL bovine serum albumin, 1 mM DTT, 0.5 mM 2 -Monooleoylglycerol) and 5 μL of 900 μM (0.2 MBq / μL) [ ¹⁴ C] An aqueous solution of palmitoyl-coenzyme A is added and the mixture is incubated at room temperature for 10 minutes. The reaction is stopped by adding 300 μL of heptane / 2-propanol / water (= 80: 20: 2) to the mixture and stirring. After standing for 10 minutes, 200 μL heptane and 100 μL water are further added to the mixture and stirred. Thereafter, the reaction product contained in the organic layer obtained by centrifuging the mixture is separated by thin layer chromatography, and detected and quantified using a bioimaging analyzer system BAS-2000 (Fuji Film).
[0014]
Preferred examples of the enzyme include monoacylglycerol acyltransferases derived from mammals such as humans, mice and rats. Further, for example, (a) a protein comprising the amino acid sequence shown in SEQ ID NO: 1, 3 or 11, (b1) an amino acid sequence shown in SEQ ID NO: 1 (one letter code: MAVTVEEAPWLGWIVAKALMRFAFMVANNLVAIPSYICYVIILQPLRVLDSKRFWYIEGLMYKWLLGMVASWGWYAGYTVMEWGEDIKAIAKDEAVMLVNHQATGDVCTLMMCLQDKGPVVAQMMWLMDHIFKYTNFGIVSLIHGDFFIRQGRAYRDQQLLVLKKHLEHNYRSRDRKWIVLFPEGGFLRKRRETSQAFAKKNNLPFLTHVTLPRFGATNIILKALVARQENGSPAGGDARGLECKSRGLQWIIDTTIAYPKAEPIDIQTWILGYRKPTVTHVHYRIFPIGDVPLETEDLTSWLYQRFIEKEDLLSHFYKTGAFPPPQGQKEAVCREMTLSNMWIFLIQSFAFLSGYLWYHIIQYFYHCLF) in which one or more amino acids are deleted A protein consisting of an added or substituted amino acid sequence and having monoacylglycerol acyltransferase activity; (b2) an amino acid sequence represented by SEQ ID NO: 3 (single letter code: MAITLEEAPWLGWLLVKALMRFAFMVVNNLVAIPSYICYVIILQPLRVLDSKRFWYIEGIMYKWLLGMVASWGWYAGYTVMEWGEDIKAVSKATV In EmushierukyudikeijierubuibuieikyuemuemudaburyueruemudieichiaiefukeiwaitienuefujiaibuiesuerubuieichijidiefuefuaiarukyujiaruesuwaiarudikyukyueruerueruerukeikeieichieruienuenuwaiaruesuarudiarukeidaburyuaibuieruefuPiijijiefueruarukeiaruaruitiesukyueiefueikeikeienuenueruPiefuerutienubuitieruPiaruesujieitikeiaiaieruenueierubuieikyukyukeienujiesuPieijijidieikeiierudiesukeiesukeijierukyudaburyuaiaidititiaieiwaiPikeieiiPiaidiaikyutidaburyuaierujiwaiarukeiPitibuitieichibuieichiwaiaruaiefuPiaikeidibuiPieruitididierutitidaburyueruwaikyuaruefubuiikeiidierueruesueichiefuwaiitijiEiefupiPiesukeijieichikeiieibuiesuaruiemutieruesuenuerudaburyuaiefuLIQSFAFLSGYMWYNIIQYFYHCLF), made of one or more amino acids are deleted, added or substituted in the amino acid sequence, and protein having a monoacylglycerol acyltransferase activity, the amino acid sequence (single letter code indicated by (b3) SEQ ID NO 11: MAVTVEEAPWLGWIVAKALMRFAFMVANNLVAIPSYICYAIILQPLRVLDSKRFWYIEGLMYKWLLGMVASWGWYAGYTVMEWGEDIKAIAKDEAVMLVNHQATGDVCTLMMCLQDKGPVVAQMMWLMDHIFKYTNFGIVSLIHGDFFIRQGRAYRDQQLLVLKKHLEHNYRSRDRKWIVLFPEGGFLRKRRETSQAFAKKNNLPFLTHVTLPRFRATNIILKALVARQENGSPAGGDARGLECKSRGLQWIIDTTIAYPKAEPIDIQTWILGYRKPTVTHVHYRIFPIADVPLETEDLTNWLYQRFIEKEDLLSHFYKTGAFPPPQGQKEAVSRAMTLSNVWILLIQSFAFLSGYLWYHVIQYFYHCLF ) In one or more amino acids Acid are deleted, added or substituted in the amino acid sequence, and protein having a monoacylglycerol acyltransferase activity, (b4) SEQ ID NO: 13 amino acid sequence represented by (one-letter code: MVSWKGIYFILFLFAGSFFGSIFMLGPILPLMFINLSWYRWISSRLVATWLTLPVALLETMFGVRVVITGDAFVPGERSVIIMNHRTRVDWMFLWNCLMRYSYLRVEKICLKSSLKSVPGFGWAMQVAAFIFIHRKWKDDKSHFEDMIDYFCAIHEPLQLLIFPEGTDLTENNKARSNDFAEKNGLQKYEYVLHPRTTGFTFVVDRLREGKNLDAVHDITVAYPYNIPQTEKHLLLGDFPKEIHFHVQRYPADSLPTSKEDLQLWCHRRWEEKEERLRSFYQGEKNFHFTGQSTVPPCKSELRVLVVKLLSIVYWALFCSAMCLLIYLYSPVRWYFIISIVFFVLQERIFGGLEIIELACYRFLHKHPHLNSKKNE) in which one or more amino acids A protein consisting of an amino acid sequence deleted, added or substituted and having monoacylglycerol acyltransferase activity, (b5) amino acid sequence represented by SEQ ID NO: 15 (single letter code: MVSWKGIYFILTLFWGSFFGSIFMLSPFLPLM In FVNPSWYRWINNRLVATWLTLPVALLETMFGVKVIITGDAFVPGERSVIIMNHRTRMDWMFLWNCLMRYSYLRLEKICLKASLKGVPGFGWAMQAAAYIFIHRKWKDDKSHFEDMIDYFCDIHEPLQLLIFPEGTDLTENSKSRSNAFAEKNGLQKYEYVLHPRTTGFTFVVDRLREGKNLDAVHDITVAYPHNIPQSEKHLLQGDFPREIHFHVHRYPIDTLPTSKEDLQLWCHKRWEEKEERLRSFYQGEKNFYFTGQSVIPPCKSELRVLVVKLLSILYWTLFSPAMCLLIYLYSLVKWYFIITIVIFVLQERIFGGLEIIELACYRLLHKQPHLNSKKNE), 1 or more amino acids are deleted, added or substituted in the amino acid sequence, and protein having a monoacylglycerol acyltransferase activity, (b6) the amino acid sequence shown in SEQ ID NO: 17 (single letter: EmubuiesudaburyukeijiaiwaiefuaieruefueruefueijiesuefuefujiesuaiefuemueruJipiaieruPieruemuefuaienueruesudaburyuwaiarudaburyuaiesuesuaruerubuieiemudaburyuerutieruPibuieierueruitiemuefujibuikeibuibuiaitijidieiefubuiPijiiaruesubuiaiaiemuenueichiarutiarubuididaburyuemuefuerudaburyuenushieruemuaruwaiesuwaieruarueruikeiaishierukeiesuesuerukeiesubuiPijiefujidaburyueiemukyubuieieiefuaiefuaieichiarukeidaburyukeididikeiesueichiefuidiemubuidiwaiefushieiaieichiiPierukyuerueruaiefuPiijitidierutiienuenukeieiaruesuenudiefueiikeienujierukyukeiwaiiwaibuierueichiPiarutitijiefutiefubuibuidiarueruaruiaruaruenuerudieibuieichidiaitibuieiwaiPiwaienuaiPikyutiikeieichierueruerujidiefuPikeiiaieichiefueichibuieichiaruwaiPibuiditieruPitiesukeiidierukyuerudaburyushieichikeiarudaburyuiikeiiiarueruaruesuefuwaikyujiikeienuefueichiefutijikyuesutibuiPiPishikeiesuieruarubuierubuibuikeierueruSIVYWALFCSAMCLLIYLYSPVRWY FIISIVFFVLQERIFGRLEIIELACYRFLHKHPHLNSKKNE), a protein consisting of an amino acid sequence in which one or more amino acids are deleted, added or substituted, and having monoacylglycerol acyltransferase activity, (c1) 80% or more of the amino acid sequence represented by SEQ ID NO: 1 A protein comprising an amino acid sequence having sequence identity and having monoacylglycerol acyltransferase activity; (c2) comprising an amino acid sequence having 80% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 3, and monoacyl A protein having glycerol acyltransferase activity, (c3) consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 11, and having a monoacylglycerol acyltransferase activity (C4) a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13 and having monoacylglycerol acyltransferase activity, (c5) an amino acid shown in SEQ ID NO: 15 A protein comprising an amino acid sequence having 80% or more sequence identity to the sequence and having monoacylglycerol acyltransferase activity; (c7) an amino acid sequence having 80% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 17 A protein having a monoacylglycerol acyltransferase activity, (d1) a protein comprising an amino acid sequence encoded by a DNA having a base sequence represented by SEQ ID NO: 2, and (d2) a base sequence represented by SEQ ID NO: 4 The DNA (D3) a protein comprising an amino acid sequence encoded by a DNA having a base sequence represented by SEQ ID NO: 12, and (d4) a protein comprising a base sequence represented by SEQ ID NO: 14 (D5) a protein consisting of an amino acid sequence encoded by a DNA having the base sequence shown by SEQ ID NO: 16, (d6) an amino acid encoded by a DNA having the base sequence shown by SEQ ID NO: 18 A protein comprising a sequence; (e1) a monoacylglycerol acyltransferase activity comprising an amino acid sequence encoded by a DNA having a base sequence having 80% or more of sequence identity with DNA having the base sequence represented by SEQ ID NO: 2 (E2) shown in SEQ ID NO: 4 (E3) a protein having a monoacylglycerol acyltransferase activity, consisting of an amino acid sequence encoded by a DNA having a base sequence of 80% or more and a DNA having a base sequence having a sequence identity of 80% or more, and represented by SEQ ID NO: 12 A protein consisting of an amino acid sequence encoded by a DNA having a base sequence and a DNA having a base sequence having 80% or more sequence identity, and having monoacylglycerol acyltransferase activity; (e4) a base represented by SEQ ID NO: 14 A protein consisting of an amino acid sequence encoded by a DNA having a sequence and a DNA having a base sequence having 80% or more sequence identity, and having a monoacylglycerol acyltransferase activity; (e5) a base sequence represented by SEQ ID NO: 16 Have A protein comprising an amino acid sequence encoded by a DNA having a base sequence having 80% or more sequence identity to DNA and having monoacylglycerol acyltransferase activity; (e6) a DNA having a base sequence represented by SEQ ID NO: 18 A protein having an amino acid sequence encoded by a DNA having a base sequence having 80% or more sequence identity and having monoacylglycerol acyltransferase activity, (f1) a DNA having a base sequence represented by SEQ ID NO: 2 A protein comprising a DNA consisting of a complementary base sequence and an amino acid sequence encoded by DNA that hybridizes under stringent conditions and having monoacylglycerol acyltransferase activity; (f2) a base represented by SEQ ID NO: 4 Arrangement A protein having a monoacylglycerol acyltransferase activity, comprising a DNA comprising a base sequence complementary to a DNA having a sequence, and an amino acid sequence encoded by DNA that hybridizes under stringent conditions; (f3) SEQ ID NO: A protein comprising a DNA having a base sequence complementary to the DNA having a base sequence represented by 12 and an amino acid sequence encoded by a DNA that hybridizes under stringent conditions and having monoacylglycerol acyltransferase activity; (F4) a monoacylglycerol amino acid comprising a DNA comprising a base sequence complementary to the DNA having the base sequence represented by SEQ ID NO: 14 and an amino acid sequence encoded by the DNA hybridizing under stringent conditions (F5) a DNA having a base sequence complementary to the DNA having the base sequence represented by SEQ ID NO: 16, and an amino acid sequence encoded by the DNA that hybridizes under stringent conditions And a protein having monoacylglycerol acyltransferase activity, (f6) encoded by a DNA that hybridizes under stringent conditions with a DNA having a base sequence complementary to the DNA having the base sequence represented by SEQ ID NO: 18 Preferred examples include a protein having the amino acid sequence and having monoacylglycerol acyltransferase activity.
[0015]
Here, (b), (b1), (b2), (b3), (b4), (b5). (Deletion, addition or substitution of amino acids) in (b6) and the above (c), (c1), (c2), (c3), (c4), (c5), (c6) and (e), (e e1), (e2), (e3), (e4), (e5), (e6) “80% or more sequence identity” includes, for example, SEQ ID NO: 1, 3, 11, 13, 15 or This includes processing that a protein having the amino acid sequence shown in 17 undergoes in cells, naturally occurring mutations due to species differences, individual differences, differences between tissues, etc. from which the protein is derived, and artificial amino acid mutations. .
“Deletion, addition or substitution of amino acids” in the above (b), (b1), (b2), (b3), (b4), (b5), (b6) (hereinafter collectively referred to as amino acid modification) As an example of a method for artificially carrying out (1), for example, conventional site-directed mutagenesis is performed on DNA encoding the amino acid sequence represented by SEQ ID NO: 1, 3, 11, 13, 15 or 17. And then a method for expressing the DNA by a conventional method. Here, as a site-specific mutagenesis method, for example, a method using amber mutation (gapped duplex method, Nucleic Acids Res., 12, 9441-9456 (1984)), a PCR method using a mutagenesis primer Etc. The number of amino acids modified as described above is at least one residue, specifically one or several, or more. The number of such modifications may be in a range where monoacylglycerol acyltransferase activity can be found.
Of the deletions, additions or substitutions, the modification relating to amino acid substitution is particularly preferable. The substitution is more preferably substitution with an amino acid having similar properties such as hydrophobicity, charge, pK, and structural features. Examples of such substitution include (1) glycine, alanine; (2) valine, isoleucine, leucine; (3) aspartic acid, glutamic acid, asparagine, glutamine; (4) serine, threonine; (5) lysine, arginine. And (6) substitution within the group of phenylalanine and tyrosine.
In the present invention, “sequence identity” refers to sequence identity and homology between two DNAs or two proteins. The “sequence identity” is determined by comparing two sequences that are optimally aligned over the region of the sequence to be compared. Here, the DNA or protein to be compared may have an addition or a deletion (for example, a gap) in the optimal alignment of the two sequences. Such sequence identity is calculated, for example, by creating an alignment using the ClustalW algorithm (Nucleic Acid Res., 22 (22): 4673-4680 (1994)) using Vector NTI. Can do. The sequence identity is measured using sequence analysis software, specifically Vector NTI, GENETYX, or an analysis tool provided by a public database. The public database is generally available at, for example, a homepage address http://www.ddbj.nig.ac.jp.
The sequence identity in the present invention may be 80% or more, preferably 90% or more, more preferably 95% or more.
(F), (f1). With respect to “hybridize under stringent conditions” in (f2), (f3), (f4), (f5), (f6), the hybridization used herein is, for example, Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory press (Cold Spring Harbor Laboratory press), etc. “Stringent conditions” means, for example, 6 × SSC (a solution containing 1.5M NaCl, 0.15M trisodium citrate is 10 × SSC), 45% in a solution containing 50% formamide. The conditions (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6), etc., where a hybrid is formed at 50 ° C. and then washed with 2 × SSC at 50 ° C. it can. The salt concentration in the washing step can be selected, for example, from 2 × SSC at 50 ° C. (low stringency conditions) to 0.2 × SSC at 50 ° C. (high stringency conditions). The temperature in the washing step can be selected, for example, from a temperature from room temperature (low stringency conditions) to 65 ° C. (high stringency conditions). It is also possible to change both the salt concentration and the temperature.
[0016]
The preparation method of this enzyme is demonstrated below.
When the enzyme is in the form of an enzyme sample containing the enzyme, such as a microsomal fraction, the enzyme is prepared by a normal cell fractionation method using adipose tissue containing the enzyme as a biomaterial. do it. Specific examples include the method described in the method of Wang Fang Cao et al. (Archives of Biochemistry and Biophysics, 296 (2), 419-425 (1992)).
In addition, using a conventional genetic engineering method or the like, first, a gene comprising a base sequence encoding the amino acid sequence of the present enzyme (hereinafter sometimes referred to as the present gene), for example, (I) SEQ ID NO: 1, 3 A gene having a base sequence such as a base sequence encoding the amino acid sequence represented by 11, 13, 15 or 17, (II) a base sequence represented by SEQ ID NO: 2, 4, 12, 14, 16 or 18, It is described in the usual genetic engineering methods (for example, Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition), Cold Spring Harbor Laboratory publication (Cold Spring Harbor Laboratory press), etc. In accordance with the method). Next, by using the obtained present gene, the present enzyme is produced and obtained according to a normal genetic engineering method. In this way, the present enzyme can also be prepared.
[0017]
For example, a culture obtained by preparing a plasmid capable of expressing this gene in a host cell, introducing it into the host cell, transforming it, and then culturing the transformed host cell (transformant). What is necessary is just to acquire this enzyme from a thing. Examples of the plasmid include a promoter that contains genetic information that can be replicated in a host cell, can be propagated autonomously, can be easily isolated and purified from the host cell, and can function in the host cell. Preferred examples include those obtained by introducing a gene comprising a base sequence encoding the amino acid sequence of the present enzyme into an expression vector having a detectable marker. Various types of expression vectors are commercially available. For example, vectors used for expression in mammalian cells include promoters such as SV40 virus promoter, cytomegalovirus promoter (CMV promoter), Raus Sarcoma Virus promoter (RSV promoter), β-actin gene promoter, aP2 gene promoter, etc. Expression vectors, which are commercially available from Toyobo, Takara Shuzo and others.
[0018]
Examples of host cells include prokaryotic or eukaryotic microbial cells, insect cells, or mammalian cells. For example, from the viewpoint that the original structure of the present enzyme can be expected, mammalian cells and the like can be preferably mentioned.
The plasmid obtained as described above can be introduced into the host cell by an ordinary genetic engineering method.
The transformant can be cultured by a conventional method used for culturing microorganisms, insect cells or mammalian cells. For example, in the case of mammalian cells, the cells are cultured in a medium appropriately containing a suitable carbon source, nitrogen source, micronutrients such as vitamins, and cell growth factors such as bovine serum. The culture method may be any of solid culture and liquid culture, and preferred examples include liquid culture such as aeration and agitation culture.
The acquisition of this enzyme may be carried out in combination with methods commonly used for isolation and purification of general proteins. For example, the transformants obtained by the above culture are collected by centrifugation or the like, and the transformants are crushed or dissolved, and if necessary, proteins are solubilized, and various types such as ion exchange, hydrophobicity, gel filtration, etc. What is necessary is just to refine | purify the process using a chromatography individually or in combination. In addition, affinity purification may be performed by adding an amino acid sequence suitable for affinity purification to the N-terminus or C-terminus of the enzyme by a conventional genetic engineering technique. If necessary, an operation for restoring the higher-order structure of the purified protein may be further performed.
[0019]
The assay method of the present invention comprises (1) a first step of measuring the activity of a monoacylglycerol acyltransferase in a contact system between a monoacylglycerol acyltransferase and a test substance, and (2) an activity measured by the first step. A second step of evaluating the ability of the substance to inhibit the increase in fat content based on the difference obtained by comparing the activity with the control.
In the assay method of the present invention, the fat of the test substance is calculated by calculating the inhibition rate (details will be described later), which is an index for evaluating the enzyme inhibitory ability, for example, without directly measuring the fat mass. Since the ability to suppress increase in amount can be evaluated, it is simple and optimal for primary screening and the like.
[0020]
In order to increase the sensitivity of the assay method of the present invention, for example, a divalent metal cation such as magnesium ion or dithiothreitol (hereinafter referred to as DTT) is added to the contact system in the first step. Good. For example, in the case of magnesium ions, the concentration of addition is, for example, 2 mM or more, preferably 5 mM to 10 mM. In the case of DTT, the addition concentration is, for example, 50 μM or more, preferably 0.2 mM to 2 mM. In order to increase the reactivity of the substrate, phosphatidylcholine (hereinafter also referred to as PC), phosphatidylserine (hereinafter also referred to as PS) or the like may be added to the above contact system. . For example, in the case of PC and PS, the concentration of addition is, for example, 5 μg / mL or more, preferably 10 μg / mL to 50 μg / mL.
[0021]
In the first step of the assay method of the present invention, (A) three kinds of substances, that is, two substrates (for example, 2 or 1-monoacylglycerol (preferably 2-monoacylglycerol) and acyl-CoA) and a test substance are used. A form in contact with the enzyme, and (B) a form in which one of two substrates (eg, 2 or 1-monoacylglycerol and acyl-CoA) and a test substance are in contact with the enzyme (ie, , When the test substance is handled as the other one of the two kinds of substrates).
[0022]
Furthermore, in the first step of the assay method of the present invention, the order in which the test substance and the substrate are brought into contact with the enzyme is as follows: (a) first contact the enzyme with the test substance and incubate for a certain period of time; (B) The enzyme, the test substance, and two or one substrate are contacted at the same time. (C) The enzyme is first contacted with two or one substrate. It is possible to use any form of adding a test substance after keeping it warm for a certain period of time, but the form (a) is preferred.
In the case of (a), the contact time between the present enzyme and the test substance can be, for example, 1 minute or longer, preferably 1 minute or longer and within 1 hour. As a heat retention temperature in the said contact system, 0 degreeC-70 degreeC can be raised, for example, Preferably 4 degreeC-40 degreeC can be mention | raise | lifted.
In the case of (c) above, the heat retention temperature in the contact system can be, for example, 0 ° C. to 70 ° C., preferably 4 ° C. to 40 ° C.
[0023]
The concentration of the test substance in the first step of the assay method of the present invention is, for example, 1 nM to 10 mM, preferably 0.01 μM to 5 mM. The concentration of the substrate is, for example, 1 μM to 1 mM, preferably 5 μM to 100 μM.
The form of the enzyme that is contacted with the test substance in the first step of the assay method of the present invention may be (A) a purified product, a crude product, or the like of the enzyme, or (B) contained in the cell. When the test substance is contacted with a transformed cell into which the enzyme (for example, a gene comprising a base sequence encoding the amino acid sequence of the enzyme has been introduced (hereinafter sometimes referred to as the transformed cell)) Or the like. Furthermore, the cells may be cells separated from the tissue, or may be cells in a state forming a population having the same function / morphology.
For example, in the case of (A) above, the concentration of the present enzyme to be contacted with the test substance is, for example, 1 ng / ml or more, preferably 10 ng / ml or more.
In the case of (B) above, the concentration of the enzyme to be contacted with the test substance is, for example, 1 × 10 5 as the concentration of the transformed cell. ^Three Cells / ml or more, preferably 1 × 10 ^Four More than cells / ml.
[0024]
This transformed cell can be prepared as follows.
A plasmid is prepared by inserting the gene into a vector that can be used in a cell into which the gene is introduced so as to be connected to a promoter in a form that can be expressed using a normal genetic engineering technique. The promoter used here may be any one that can function in the cell into which the present gene is introduced. When the cell is an animal cell, the SV40 virus promoter, cytomegalovirus promoter (CMV promoter), Raus Sarcoma Examples include Virus promoter (RSV promoter), β-actin gene promoter, aP2 gene promoter and the like. A commercially available vector containing such a promoter upstream of the multicloning site may be used.
The plasmid is then introduced into the cell. Examples of the introduction method into the cell include a calcium phosphate method, an electroporation introduction method, a DEAE dextran method, and a micelle formation method. The calcium phosphate method is described in Grimm, S. et al., Proc. Natl. Acad. Sci. USA, 93, 10923-10927, etc., the electroporation introduction method and the DEAE dextran method are Ting, AT et al. , EMBO J., 15, 6189-6196 etc., and the micelle formation method is a method described in Hawkins, CJ et al., Proc. Natl. Acad. Sci. USA, 93, 13786-13790 etc. Can be mentioned. When using the micelle formation method, commercially available reagents such as Lipofectamine (manufactured by Gibco) and Fugene (manufactured by Boehringer) may be used.
The transformed cells are selected by culturing the cells subjected to the plasmid introduction treatment, for example, using a selection marker gene previously contained in the vector and culturing in a medium under selection conditions corresponding to the selection marker gene. be able to. Further, selection may be continued to obtain the present transformed cell that has become a stable transformant in which this gene is introduced into the chromosome. In order to confirm that the introduced gene has been incorporated into a chromosome present in the cell, the genomic DNA of the cell is prepared according to a normal genetic engineering method and has a partial base sequence of the gene. The presence of the present gene in the genomic DNA may be detected and confirmed using a method such as PCR using DNA as a primer or Southern hybridization using a DNA having a partial base sequence of the present gene as a probe.
[0025]
As a method for measuring the activity of monoacylglycerol acyltransferase (that is, the present enzyme) in the first step of the assay method of the present invention, for example, a mixed solution containing the present enzyme, test substance and substrate is reacted for a certain period of time. Thereafter, a method for analyzing a decrease amount of a raw material such as a substrate in the reaction or a method for analyzing an increase amount of a product in the reaction can be given. These methods may be any known activity measurement method. Specific examples include the method described in Arch. Biochem. Biophys. 1992; 296 (2): 419-25 as described above.
The reaction temperature at the time of activity measurement in the first step of the assay method of the present invention may be, for example, 15 ° C to 70 ° C, preferably 20 ° C to 40 ° C. The reaction time is, for example, 1 minute or longer, preferably 15 minutes to 1 hour. The reaction pH is, for example, 6.0 to 9.5, preferably pH 7.0 to 8.0.
[0026]
For detection of raw materials or products such as substrates in enzyme reactions or measurement of their amounts, the reaction solution is separated by, for example, HPLC, thin layer chromatography, paper chromatography, etc. A method of detecting ultraviolet absorption or radioactivity (when the raw material previously labeled with a radioisotope is used) or the like, or measuring ultraviolet absorption or radioactivity or the like may be used. When it is necessary to remove the protein from the reaction solution at the time of the above analysis, the protein may be removed from the reaction solution by a method described in, for example, Methods Enzymol. 280, 211-221.
[0027]
Based on the difference obtained by comparing the activity measured as described above with the activity in the control (ie, reference substance, negative control, etc.), the substance's ability to inhibit the increase in fat mass is evaluated. That is, when the activity measured as described above is lower than the activity in the control, it may be evaluated that the substance has the ability to suppress the increase in fat mass.
Thus, the test for the ability to suppress the increase in fat content of the test substance (the assay method of the present invention) can be performed.
In the assay method of the present invention, when the measured activity and the activity in the control are compared, at least one of the two or more different substances is a substance (for example, a solvent) that does not have the ability to inhibit fat mass increase. Or a negative control such as a test system solution as a background.), The ability to inhibit the increase in fat content of the other test substance may be evaluated, Of the substances, the fat mass increase inhibiting ability of the other test substance may be evaluated based on the fat mass increase inhibiting ability of at least one substance (for example, a reference substance). Of course, both may be evaluated.
[0028]
More specifically, for example, the first step of the assay method of the present invention comprises (A) two substrates (for example, 2 or 1-monoacylglycerol (preferably 2-monoacylglycerol) and acyl-CoA). ) And the test substance are in contact with this enzyme, and when a negative control is used as a control, the inhibition rate may be determined according to the following formula.
Inhibition rate (%) = {control (negative control) value−measurement (test substance) value} × 100 / control (negative control) value
Then, the fat mass increase suppressing ability may be evaluated based on the calculated inhibition rate. That is, the inhibition rates of the various test substances calculated as described above are compared, and as a result, the test substance showing a high inhibition rate has a higher fat mass suppression than the test substance showing a low inhibition rate. What is necessary is just to evaluate having ability.
On the other hand, the first step of the assay method of the present invention comprises (A) a combination of two substrates (for example, 2 or 1-monoacylglycerol (preferably 2-monoacylglycerol) and acyl-CoA) and a test substance. When the substance is in contact with this enzyme and a substance (reference substance) that has the ability to inhibit fat mass increase is used as a control, the control (reference substance) value and the measured (test substance) value May be used to evaluate the fat mass increase inhibiting ability. In this case, if the measured (test substance) value is lower than the control (reference substance) value, the test substance is evaluated as having higher ability to suppress the increase in fat mass than the reference substance.
In addition, for example, the first step of the assay method of the present invention includes (B) one of the two substrates (for example, 2 or 1-monoacylglycerol (preferably 2-monoacylglycerol) and acyl-CoA. ) And the test substance are in the form of contact with the enzyme (that is, when the test substance is handled as the other of the two substrates) What is necessary is just to evaluate fat mass increase inhibitory ability by using a substance (reference substance) which has and comparing a control (reference substance) value and a measurement (test substance) value. In this case, if the measured (test substance) value is higher than the control (reference substance) value, the test substance is superior to the reference substance as a substrate for the enzyme. It is superior to the reference substance as a competitive inhibitor of this enzyme.
[0029]
In order to search for a fat mass increase inhibitory substance, a substance having a fat mass growth inhibitory ability may be selected based on the fat mass increase inhibitory ability evaluated by the assay method of the present invention (the present invention search method).
For example, in the case of (A) above, when a negative control is used as a control, the inhibition rate serving as an index for evaluating the ability of the test substance to inhibit the increase in fat mass is a statistically significant value. More specifically, for example, a substance having an inhibition rate of 30% or more in the above formula, more preferably a substance showing 50% or more, is selected as a substance having an ability to suppress increase in fat mass. On the other hand, in the case of (A) above, when a substance (reference substance) having the ability to suppress fat increase is used as a control, the measured (test substance) value is lower than the control (reference substance) value. Are selected as substances having the ability to suppress the increase in fat mass. Further, for example, in the case of (B) above, a substance whose measured (test substance) value is higher than the control (reference substance) value is selected as a substance having an ability to suppress increase in fat mass. The substance may be any substance such as a low molecular weight compound, a protein or a peptide as long as it has the ability to suppress the increase in fat mass.
[0030]
The substance selected by the search method of the present invention has a fat mass increase inhibitory ability and may be used as an active ingredient of a fat mass increase inhibitor.
As such a substance (that is, a substance having the ability to inhibit monoacylglycerol acyltransferase), for example, N- [2- (4-benzyl-2-ethylphenoxy) ethyl] -5,6-dimethyl [1,2, 4] Triazolo [1,5-a] pyrimidin-7-amine.
[0031]
The monoacylglycerol acyltransferase inhibitor (hereinafter referred to as “inhibitor”) selected by the search method of the present invention has the ability to suppress the increase in fat mass, as described in the Examples herein, or International Publication No. WO 00/44754. It can be confirmed by a known method such as the method described in the issue pamphlet.
Moreover, it can be confirmed by the following known methods that the inhibitor selected by the method for searching for the present invention has the ability to suppress lipoprotein synthesis.
That is, in the presence of a substance having the ability to inhibit monoacylglycerol acyltransferase (hereinafter sometimes referred to as the present inhibitor), cells having the ability to synthesize lipoproteins are cultured in a test tube, and the amount of triglyceride is measured. Compared to the amount of triglyceride in the absence of inhibitor. Examples of such cells include small intestinal cells or liver cells prepared from living tissue, cultured cells derived from small intestine or liver, or cells that have acquired lipoprotein synthesis ability by introducing a monoacylglycerol acyltransferase gene from the outside. Can do. The preparation of small intestinal cells from tissues can be performed by, for example, the method described in The Journal of Biological Chemistry, Milton M. Weiser, p2536-2541, 1973, that is, a method of suspending collected small intestinal cells in a medium, or Experimental Cell Research, Jean -The method described in Francois Beaulieu et al., P34-42, p1998, that is, a method in which the collected small intestinal cells are adhered to the plate wall and cultured. Preparation of liver cells is performed by, for example, the method described in Endocrinology, Jonathan R. Seckl et al., P4754-4761, 1995, that is, a method of preparing liver cells by treating the extracted liver with collagenase, and the obtained liver cells. Can be performed by, for example, a method of culturing by adhering to the wall of the plate device. Although cultured cells derived from the small intestine have not been established as a general cultured cell system, cells derived from human colon cancer, such as CaR-1, 2, 3 and CaCo-2 cells, are small intestinal lipoproteins. Alternatively, it can be used as a cell having synthetic ability. As cultured cells derived from the liver, many cells such as JHH-1,2,3, huH-1,2,4, and HepG2 are generally available, but usually HepG2 is often used as a human liver model.
[0032]
Examples of the method for quantifying the amount of triglyceride in the liver or small intestinal cells include a method in which fatty acid or glycerol is added to the cells and cultured for a certain period of time, and the triglyceride contained in the supernatant fraction or the cells is quantified. There are two methods for quantifying triglyceride: a method using a radiolabel and a method of directly quantifying triglyceride without using a radiolabel. Examples of the former method include the method described in Journal of Lipid Research, Joan A. Higgins et al., P1728-1739, 2000, and the method described in The Journal of Biological Chemistry, M. Mahmood Hussian et al. P19565-19572, 1999. There is a way. Examples of the latter method include culturing small intestine-like cells such as Caco2 or cultured cells of the liver in a test tube, and extracting and quantifying triglyceride from the culture supernatant using an organic solvent such as chloroform / methanol. I can do it. In any method, preferably, the culture supernatant cultured by the above method is fractionated by density, and the triglyceride contained in VLDL in the case of liver cells and chylomicron in the case of small intestinal cells is quantified. Evaluation of the ability to inhibit lipoprotein synthesis by an inhibitor can be carried out by carrying out the above-described triglyceride synthesis in contact with the inhibitor and comparing it with the ability to synthesize triglyceride without contact with the inhibitor. The VLDL and chylomicron can be isolated by methods well known to those skilled in the art such as centrifugation.
Alternatively, this inhibitor is administered to mice, etc., the blood triglyceride (= lipoprotein) level is measured, and compared with the blood triglyceride level when this inhibitor is not administered, the lipoprotein synthesis of this inhibitor The suppression ability can also be confirmed. Specifically, a method of measuring blood triglyceride concentration by lowering blood triglyceride concentration by fasting and measuring the increase in blood triglyceride concentration by lipid administration (International Journal of Obesity, K. Cianflone et al., P705-713 , 2001).
[0033]
A fat amount increase inhibitor characterized by comprising a substance having a monoacylglycerol acyltransferase inhibitory ability or a pharmaceutically acceptable salt thereof as an active ingredient (that is, the fat amount increase inhibitor of the present invention) is an effective amount thereof. Can be orally or parenterally administered to mammals such as humans. For example, when administered orally, the fat mass increase inhibitor of the present invention can be used in a normal form such as a tablet, capsule, syrup, suspension or the like. In addition, when administered parenterally, the fat mass increase inhibitor of the present invention can be used in the form of a usual solution such as a solution, emulsion, suspension or the like. Examples of the method for parenterally administering the above-mentioned form of the present invention fat increase inhibitor include injection and suppository in the rectum.
The above-mentioned appropriate dosage form contains a substance having a monoacylglycerol acyltransferase inhibiting ability or a pharmaceutically acceptable salt thereof in an acceptable normal carrier, excipient, binder, stabilizer, diluent, etc. Can be manufactured. When used in an injection form, acceptable buffering agents, solubilizing agents, isotonic agents and the like can be added.
The dose varies depending on the age, sex, weight, severity of the disease, the type of fat growth inhibitor, dosage form, etc. of the mammal to be administered. About 1 mg to about 2 g as the amount of the component, preferably about 5 mg to about 1 g as the amount of the active ingredient may be administered, and about 0.1 mg to about 500 mg as the amount of the active ingredient may be administered as an adult in the case of injection. In addition, the above-mentioned daily dose can be administered once or divided into several times.
Examples of the diseases to which the fat mass increase inhibitor of the present invention can be applied include metabolic diseases such as impaired glucose tolerance associated with insulin resistance, type II diabetes, hyperlipidemia, hypertension, arteriosclerosis, coronary artery disease, Diseases such as cardiovascular disorders such as cardiomyopathy and myocardial infarction can be listed.
[0034]
As described above, in the present invention, an increase in fat mass is achieved by administering to a living body a pharmacologically effective amount of a substance having the ability to inhibit monoacylglycerol acyltransferase to inhibit the activity of monoacylglycerol acyltransferase. Can be suppressed.
In the present invention, the use of monoacylglycerol acyltransferase as a reagent for providing an index for evaluating the ability to inhibit fat mass increase, and the reagent for providing an index for evaluating the ability to inhibit fat mass increase, Also provided is the use of monoacylglycerol acyltransferase genes. As an example of the former, use of monoacylglycerol acyltransferase related to the assay method of the present invention, the search method of the present invention and the like can be mentioned. Examples of the latter include use as a reagent that provides an index for evaluating the ability to inhibit increase in fat mass in the case of inhibiting the increase in fat mass by controlling the expression of monoacylglycerol acyltransferase. it can. More specifically, use as a primer or probe in a known method for specifically detecting a specific gene such as Northern blot method, RT-PCR method, in situ hybridization method, DNA chip and the like can be mentioned.
[0035]
【Example】
The present invention will be described in more detail below by way of examples, but the present invention is not limited to these examples.
[0036]
Example 1 (Preparation of microsomal fraction from adipose tissue (one form of enzyme sample containing this enzyme))
(1-1) Removal of adipose tissue
Thirty 14-week-old Wistar male rats (Japan SLC, Inc.) were sacrificed and opened, and adipose tissue adhering to the mesentery (hereinafter referred to as mesenteric adipose tissue) was removed. The excised tissue was treated with phosphate buffer (0.20 g / L KCl, 0.20 g / L KH). ₂ PO _Four 8.00 g / L NaCl, 2.16 g / L Na ₂ HPO _Four ・ 7H ₂ O, 100 units / ml penicillin (Gibco), 100 μg / ml streptomycin (Gibco), 250 ng / ml amphotericin (Gibco)) was immersed in about 100 ml and washed at room temperature.
(1-2) Preparation of microsomal fraction (one form of enzyme sample containing this enzyme) from adipose tissue
First, medium A (0.25 M Sucrose, 1 mM Tris-hydrochloric acid (pH 7.5), 1 mM ethylenediaminetetraacetic acid tetraacetate, which is three times the volume of the adipose tissue, was removed from the adipose tissue extracted in (1-1) above. Sodium (hereinafter referred to as EDTA · 4Na) and 1 mM dithiothreitol (hereinafter referred to as DTT) were added, and the adipose tissue was pulverized and equalized with a homogenizer (Wheaton). The pulverized and equalized adipose tissue was centrifuged at 600 g × 15 minutes at 4 ° C., and an intermediate layer containing no upper layer (Fat Cake) and lower layer (contaminants) was collected. The separated intermediate layer was further centrifuged at 16,000 g × 15 minutes at 4 ° C. The obtained supernatant was centrifuged at 105,000 g × 60 minutes at 4 ° C. to collect a precipitate (microsome fraction). The recovered precipitate was suspended in the above Medium A, and then washed by centrifuging at 105,000 g × 60 minutes at 4 ° C. The washed precipitate was resuspended in Medium A to prepare a microsomal fraction with a protein concentration of 1 mg / ml.
[0037]
Example 2 (Measurement of activity of monoacylglycerol acyltransferase in microsomal fraction (one form of enzyme sample containing the present enzyme))
In an assay solution (24 mM Tris-HCl (pH 7.5), 50 mM potassium chloride, 8 mM magnesium sulfate, 1.25 mg / ml bovine serum albumin, 1 mM DTT) 140 μl, 800 μg / ml test substance (1% dimersoxide (hereinafter referred to as DMSO) Note :) Lysis solution) 0.75 μl, 1 mg / ml microsomal fraction 5 μl was added. To this assay solution, 0.7 μl of 100 mM 2-monooleoylglycerol (Sigma) was further added and mixed. To this mixture 0.45 mM [ ¹⁴ C] Palmitoylcoenzyme A (Amersham) 10 μl (0.03 μCi / total reaction volume 150 μl) was added and incubated at room temperature for 5 minutes. In the test substance-free group, the same method as described above was used, except that 0.75 μl of only 1% DMSO was added instead of the DMSO solution of the test substance. In the background group, the same method as that for the test substance and 2-monooleoylglycerol was used except that only DMSO was added instead of the test substance and 2-monooleoylglycerol.
Next, after the incubation for 5 minutes, the reaction was stopped by adding 300 μl of isopropanol / heptane / water (80: 20: 2, v / v) to the reaction mixture, and further 200 μl of heptane and 100 μl of water were added. Suspended. The suspension was centrifuged at 3,000 rpm for 1 minute at room temperature to remove the lower layer, and then the remaining upper layer (organic solvent layer) was dried. The dried product thus obtained was suspended in 30 μl of chloroform / methanol (2: 1, v / v). Next, this suspension was spotted on a glass plate for thin layer chromatography (K5 silica gel 150 Å, Whatman, hereinafter sometimes referred to as TLC plate). A developing solvent of hexane / diethyl ether / acetic acid (75: 25: 1, v / v) was put in a sealed container, and the TLC plate was developed using the developing solvent, and then the TLC plate was dried at room temperature. The dried TLC plate was exposed to an imaging plate (Fuji Film Co.) for 16 hours. After completion of the exposure, the imaging plate was analyzed with a bio image analyzer (BAS 2500, Fuji Film), and the portion corresponding to the development position of 1,2-diacylglycerol and triglyceride on the TLC plate [ ¹⁴ C] The radioactivity was measured (hereinafter, the portion corresponding to the development position of the 1,2-diacylglycerol moiety [ ¹⁴ C] Radioactivity is measured value A, the portion corresponding to the development position of triacylglycerol [ ¹⁴ C] Radioactivity is described as measured value B. ). From these measured values, the activity of monoacylglycerol acyltransferase (measured value A + measured value B / 2, hereinafter referred to as activity value 1) when a test substance was added was calculated. For the test substance-free group and the background group, using the same method as described above, the portion corresponding to the development position of 1,2-diacylglycerol and triglyceride on the TLC plate [ ¹⁴ C] The radioactivity was measured (hereinafter, the portion corresponding to the development position of 1,2-diacylglycerol in the test substance-free group [ ¹⁴ C] Radioactivity is measured value C, and the portion corresponding to the development position of triacylglycerol in the test substance-free group [ ¹⁴ C] Radioactivity is measured value D, radioactivity of the portion corresponding to the development position of 1,2-diacylglycerol in the background section is measured value E, radiation of the portion corresponding to the development position of triacylglycerol in the background section The activity is described as measured value F. ). From these measured values, the activity of the monoacylglycerol acyltransferase in the group to which no test substance was added (measured value C + measured value D / 2, hereinafter referred to as activity value 2) and the activity of the monoacylglycerol acyltransferase in the background group. (Measurement value E + measurement value F / 2, hereinafter referred to as activity value 3) was calculated. From the obtained activity value, the monoacylglycerol acyltransferase inhibitory ability of the test substance was calculated as an inhibition rate ((activity value 2−activity value 1) × 100 / (activity value 2−activity value 3)).
As a result, the known compound N- [2- (4-benzyl-2-ethylphenoxy) ethyl] -5,6-dimethyl [1,2,4] triazolo [1,5-a] pyrimidine-7 as a test substance -Inhibition rate in amine (hereinafter also referred to as compound A) was 44 to 50%. Further, the inhibition rate at each test concentration was calculated using the same method as described above while changing the test concentration of the test substance. The results are shown in Table 1 together with the former results.
[0038]
[Table 1]

* Values based on the results of Reference Example 1
** Value based on the result of Reference Example 2
Similar results were also obtained when the test substance was another known substance capable of suppressing increase in fat mass.
From the above, the inhibition rate, which is an index for evaluating the enzyme inhibition ability, and the inhibition rate in Reference Example 1 and Reference Example 2 described later, which are results based on direct measurement of accumulated fat mass The relationship was at least dose-dependent over a specific concentration range, confirming that this relationship is a positive correlation.
[0039]
Reference example 1
(1-1) Removal of adipose tissue
Thirty-two 14-week-old Wistar male rats (Japan SLC) were sacrificed and opened, and the mesenteric adipose tissue was removed. The excised tissue was treated with phosphate buffer (0.20 g / L KCl, 0.20 g / L KH). ₂ PO _Four 8.00 g / L NaCl, 2.16 g / L Na ₂ HPO _Four ・ 7H ₂ O, 100 units / ml penicillin (Gibco), 100 μg / ml streptomycin (Gibco), 250 ng / ml amphotericin (Gibco)) and washed at room temperature.
[0040]
(1-2) Preparation and culture of adipocytes from adipose tissue
The following treatment was performed on the washed mesenteric adipose tissue.
First, the fat tissues extracted in the above (1-1) were prepared with collagenase (type II or VIII, Sigma), penicillin (Gibco), streptomycin (GIBVIIICO) and amphotericin (Gibco), respectively. In about 300 ml of Dulbecco's modified Eagle medium (containing 4.5 g / L D-glucose and 584 mg / L L-glutamine, Gibco) added to 1 mg / ml, 100 units / ml, 100 μg / ml and 250 ng / ml Then, it was cut into about 5 mm square using scissors. Next, this was shaken at 37 ° C. for 60 minutes (about 170 rpm), filtered through a nylon mesh (80S [eye size is 250 μm], Sangen Industrial Co., Ltd.), and the filtrate (cell suspension) was recovered. The filtrate was centrifuged at 1800 rpm for 5 minutes at room temperature, and then the liquid layer was gently removed by decantation to obtain a precipitate. This sedimentation was completed for fetal bovine serum (hereinafter referred to as FBS) (Gibco), ascorbic acid (Wako Pure Chemical Industries), penicillin (Gibco), streptomycin (Gibco) and amphotericin (Gibco), respectively. Dulbecco's modified Eagle's medium (contains 4.5 g / L D-glucose and 584 mg / L L-glutamine, Gibco, the following) added to concentrations of 10%, 200 μM, 100 units / ml, 100 μg / ml and 250 ng / ml The suspension was suspended in 50 ml, and the suspension was filtered through a nylon mesh (420S [eye size: 25 μm], Sangen Industrial Co., Ltd.). The filtrate was collected, centrifuged at 1800 rpm for 5 minutes at room temperature, the liquid layer was gently removed by decantation, and the precipitate was suspended again in 50 ml of FBS-containing medium. The suspension was subjected to centrifugation, liquid layer removal, and suspension in an FBS-containing medium two more times in the same manner as described above to prepare a suspension (120 ml). 30 ml of this suspension was dispensed into a cell culture flask (T150 for adherent cells, Iwaki Glass Co., Ltd.) at 37 ° C., 5% CO ₂ Cultured in the presence. Two to three hours after the start of the culture, the medium was removed, and the wall of the flask was washed with 15 ml of the phosphate buffer. After removing the washing solution and performing the washing operation again, the phosphate buffer solution was removed, and 30 ml of FBS-containing medium was added to the flask. ₂ Cultured in the presence. One day after the start of the culture, the medium was removed and the wall of the flask was washed once with 15 ml of phosphate buffer, and then the trypsin-ethylenediaminetetraacetic acid (hereinafter referred to as EDTA) solution (0.05% trypsin) was added to the flask. , 0.53 mM EDTA · 4Na, Gibco) was added to such an extent that the cells were immersed, and left at 37 ° C. for 5 minutes. To this, an FBS-containing medium was added in an amount approximately 10 times that of a trypsin-EDTA solution to obtain a cell suspension.
[0041]
(1-3) Measurement of ability to inhibit increase in fat content of test substance (Part 1)
The number of cells in the cell suspension prepared from the mesenteric adipose tissue in (1-2) above was measured using a hemocytometer, and 1.4 × 10 ^Five The cell suspension was diluted by adding FBS-containing medium to cells / ml. The diluted solution thus obtained was dispensed at 100 μl per well into a 96-well plate (for adherent cell culture, Iwaki Glass Co., Ltd.). ₂ After culturing at 37 ° C. for 2 to 3 days in the presence, the medium was removed from each well of the 96-well plate, 10 μg / ml insulin (manufactured by Sigma), 0.25 μM dexamethasone (Wako Pure Chemical Industries), 5 mM 3-isobutyl-1-methyl-xanthine (manufactured by Sigma) and 5 μM 15-deoxy-Δ ^12,14 -Prostaglandin J ₂ 100 μl of FBS-containing medium containing (Cayman) was added to each well and 5% CO ₂ In the presence, the cells were cultured at 37 ° C. for 2 days. The medium in each well was then removed and 10 μg / ml insulin and 5 μM 15-deoxy-Δ. ^12,14 -Prostaglandin J ₂ 100 μl of FBS-containing medium containing was added to each well. 2 more days, 5% CO ₂ After culturing at 37 ° C. in the presence, the medium in each well was removed, 10 μg / ml insulin, 5 μM 15-deoxy-Δ ^12,14 -Prostaglandin J ₂ 100 μl of FBS-containing medium containing 50 μM of the test substance and 0.5% DMSO (Wako Pure Chemical Industries, Ltd.) was added to each well and cultured in the same manner. In the test substance-free group, 10 μg / ml insulin, 5 μM 15-deoxy-Δ instead of the medium. ^12,14 -Prostaglandin J ₂ The culture was performed in the same manner as described above except that 100 μl of FBS-containing medium containing 0.5% DMSO was added. After culturing for 2 days, the fat in the cells was stained with Oil Red O (Sudan II, Wako Pure Chemical Industries), and the amount of the stained fat was measured by a colorimetric method. Specifically, first, 30 μl of 0.075% oil red O staining solution / 60% triethyl phosphate solution was added directly to each well containing the cell culture solution. After standing at room temperature for 30 minutes, the medium containing Oil Red O staining solution was removed, and 100 μl of 20% aqueous triethyl phosphate solution was added to each well. After the addition, 20% triethyl phosphate aqueous solution was removed from each well, and 100 μl of the same aqueous solution was newly added to each well. After repeating the above operation again, 100 μl of cell lysate (2% SDS, 0.2N NaOH) was added to each well. After incubating at 37 ° C. for 3 hours or more, the absorbance at a wavelength of 490 nm was measured for each well using a plate reader (Vmax, Beckman) (hereinafter referred to as measurement value 1). Also in the test substance-free group, intracellular fat was stained by the same method as described above, and the amount of stained fat was measured by a colorimetric method (hereinafter referred to as measured value 2). From these measured values, the fat mass increase inhibiting ability of the test substance was calculated according to the following formula as the inhibition rate.
Inhibition rate (%) = {(measured value 2−measured value 1) / measured value 2} × 100
As a result, the known compound N- [2- (4-benzyl-2-ethylphenoxy) ethyl] -5,6-dimethyl [1,2,4] triazolo [1,5-a] pyrimidine-7 as a test substance -The inhibition rate of amine (namely, compound A) was 72%.
[0042]
Reference Example 2 (Measurement of ability of test substance to suppress increase in fat mass (Part 2))
(2-1) Preparation of adipose tissue piece
Thirty-two 14-week-old Wistar strains (Japan SLC) were sacrificed and opened, and all adipose tissue adhering to the mesentery (ie, mesenteric adipose tissue) was removed. In addition, a subcutaneous adipose tissue (hereinafter referred to as abdominal adipose tissue) from the abdominal side to the thigh was extracted from only one side per animal. These excised tissues were washed with 2 ml of Dulbecco's modified Eagle medium (containing 4.5 g / L D-glucose and 584 mg / L L-glutamine, Gibco), and then scissors were used in the same medium with scissors. Shredded into corners.
[0043]
(2-2) Measurement of fat mass increase inhibiting ability of test substance (part 2)
First, Dulbecco's modified eagle medium-low glucose (containing 1.0 g / L D-glucose and 584 mg / L L-glutamine, Gibco) is dispensed into a 48-well plate (for adherent cell culture, Sumitomo Bakelite) at 500 μl per well. Further, a test substance dissolved in DMSO was added so that the final concentration of the test substance was 50 μM and the final concentration of DMSO was 0.5%. In each well, 50 to 100 mg of the adipose tissue piece prepared in the above (2-1) is placed, and 5% CO ₂ In the presence, it was kept at 37 ° C. for 30 minutes. In the test substance-free group, the same method as described above was used except that DMSO alone was added to a final concentration of 0.5% instead of the DMSO solution of the test substance. 30 minutes after the start of incubation, a radioisotope-labeled glucose solution (D- [U- ¹⁴ C] glucose, 7.4 MBq / ml, Amersham) 15 μl is added to each well and 5% CO ₂ In the presence, it was kept at 37 ° C. for 7 hours. Next, after completion of the incubation, the fat tissue pieces of each well were transferred into 750 μl of heptane / isopropanol (heptane: isopropanol = 3: 2) and left at room temperature for about 15 hours. Next, adipose tissue pieces were removed from the above solution, heptane and isopropanol were volatilized, 6 μl of the obtained residue was dissolved in 120 μl of chloroform / methanol (chloroform: methanol = 2: 1), and 6 μl of the solution was dissolved. Were spotted on a glass plate for thin layer chromatography (K5 silica gel 150 Å, Whatman, hereinafter sometimes referred to as TLC plate). A developing solvent of hexane: diethyl ether: acetic acid (75: 25: 1) was put into a sealed container, the TLC plate was developed using the developing solvent, and then the TLC plate was dried at room temperature. The dried TLC plate was exposed to an imaging plate (Fuji Film) for 4-5 hours. After completion of exposure, the imaging plate was analyzed with a bioimage analyzer (BAS2000, Fuji Film), and the portion corresponding to the development position of triglyceride on the TLC plate [ ¹⁴ C] Radioactivity was measured (hereinafter referred to as measured value 5). Also in the test substance-free group, a portion corresponding to the development position of the triglyceride on the TLC plate using the same method as described above [ ¹⁴ C] Radioactivity was measured (hereinafter referred to as measured value 6). From these measured values, according to the following formula, the fat mass increase inhibiting ability of the test substance was calculated as the inhibition rate of fat (triglyceride) accumulation per adipose tissue unit volume.
Inhibition rate of increase in fat mass per unit volume of adipose tissue (%) = {(measured value 6−measured value 5) / measured value 6} × 100
As a result, the known compound N- [2- (4-benzyl-2-ethylphenoxy) ethyl] -5,6-dimethyl [1,2,4] triazolo [1,5-a] pyrimidine-7 as a test substance -The inhibition rate in amine (namely, compound A) was 84%. Further, the inhibition rate at each test concentration was calculated using the same method as described above while changing the test concentration of the test substance. The results are shown in Table 2 together with the former results.
[0044]
[Table 2]

[0045]
Example 3 (Cloning of this gene derived from mouse (part 1))
In order to amplify a DNA fragment having this gene (mAT12) derived from mouse by PCR, first, Mouse Normal Adipose cDNA (Biochain) 1 μL, primer 5 consisting of the nucleotide sequence shown in SEQ ID NO: 5, 20 pmol shown in SEQ ID NO: 6 50 μL PCR containing 20 μmol of primer 6 consisting of a base sequence, Takara Ex-Taq polymerase (Takara Shuzo) 2 U, 5 μL of buffer attached to Takara Ex-Taq polymerase and 4 μL of dNTP mixture (2.5 mM) attached to Takara Ex-Taq polymerase A reaction solution was prepared and subjected to PCR. The PCR was carried out under the condition that the heat retention cycle consisting of 94 ° C. for 30 seconds, 60 ° C. for 30 seconds and 72 ° C. for 1 minute was repeated 50 times, and finally 72 ° C. for 5 minutes. After PCR, a PCR product showing about 1.2 Kbp was collected by agarose electrophoresis. An E. coli plasmid prepared by subcloning the recovered PCR product into pT7-Blue vector (Novagen). E. coli JM109 strain competent cell (Toyobo) was transformed. This plasmid derived from a mouse is isolated and purified from cultured cells obtained by culturing transformed cells in 100 mL of LB medium containing 50 μg / mL ampicillin using QIAGEN Plasmid Maxi Kit (QIAGEN). A plasmid containing DNA having the base sequence of the gene (mAT12) was obtained.
[0046]
Example 4 (Determination of the base sequence of this gene derived from mouse (part 1))
Using the plasmid containing the PCR product (about 1.2 kbp) obtained in Example 3 as a template, Thermo Sequenase II dye terminator kit (Amersham Pharmacia Biotech) and ABI373 DNA sequence reader (PE Applied Biosystems), The base represented by SEQ ID NO: 2 by the method of Sanger (F. Sanger, S. Nicklen, AR Coulson A, Proceedings of National Academy of Science USA (1977), 74, 5463-5467). The base sequence of this mouse-derived gene (mAT12) consisting of DNA having a sequence was determined.
[0047]
Example 5 (Cloning of this human-derived gene (1))
In order to amplify a DNA fragment having this human-derived gene (hAT12) by PCR, first, Human Adipocyte Marathon-ready cDNA (CLONTECH) 1 μL, primer 7 consisting of the base sequence shown in SEQ ID NO: 7, 20 pmol, SEQ ID NO: 8 50 μL containing 20 μmol of primer 8 consisting of the base sequence shown in FIG. 2, 5 μL of Takara Ex-Taq polymerase (Takara Shuzo) 2 U, 5 μL of buffer attached to Takara Ex-Taq polymerase, and 4 μL of dNTP mixture (2.5 mM) attached to Takara Ex-Taq polymerase A reaction solution for PCR was prepared and subjected to PCR. The PCR was carried out under the condition that the heat retention cycle consisting of 94 ° C. for 30 seconds, 60 ° C. for 30 seconds and 72 ° C. for 1 minute was repeated 50 times, and finally 72 ° C. for 5 minutes. After PCR, a PCR product showing about 1.2 Kbp was collected by agarose electrophoresis. An E. coli plasmid prepared by subcloning the recovered PCR product into pT7-Blue vector (Novagen). E. coli JM109 strain competent cell (Toyobo) was transformed. The plasmid is isolated and purified from the cultured cells obtained by culturing the transformed cells in 100 mL of LB medium containing 50 μg / mL ampicillin using QIAGEN Plasmid Maxi Kit (QIAGEN). A plasmid containing DNA having the base sequence of the gene (hAT12) was obtained.
[0048]
Example 6 (Determination of the base sequence of this human-derived gene (part 1))
Using the plasmid containing the PCR product (about 1.2 kbp) obtained in Example 5 as a template, Thermo Sequenase II die terminator kit (Amersham Pharmacia Biotech) and ABI373 DNA sequence reader (PEApplied Biosystems) (F. Sanger, S. Nicklen, AR Coulson A, Proceedings of National Academy of Science USA (1977), 74, 5463-5467). The base sequence of this human-derived gene (hAT12) consisting of DNA having
[0049]
Example 7 (Cloning of the rat-derived gene and determination of its nucleotide sequence (1))
In accordance with the method described in Examples 3 and 4, a plasmid containing DNA having the base sequence of the rat-derived gene (rAT12) was obtained, and the rat comprising the DNA having the base sequence represented by SEQ ID NO: 12 The nucleotide sequence of the derived gene (rAT12) was determined.
[0050]
Example 8 (Preparation of a plasmid expressing a fusion protein in which FLAG is added to the amino terminus of the amino acid sequence represented by SEQ ID NO: 1)
0.05 μg of the plasmid isolated and purified in Example 3, 20 pmol of primer 9 consisting of the base sequence shown in SEQ ID NO: 9 and 20 pmol of primer 10 consisting of the base sequence shown in SEQ ID NO: 10, Takara ExTaq polymerase (Takara Shuzo) 0 A reaction solution for PCR of 5 μL, 5 μL of buffer attached to Takara ExTaq polymerase and 4 μL of dNTP mixture (2.5 mM) attached to Takara ExTaq polymerase was prepared and used for PCR. The PCR was carried out under the condition that the temperature was first kept at 94 ° C. for 1 minute, then 60 ° C. for 30 seconds, and further at 72 ° C. for 1 minute 30 times, and finally at 72 ° C. for 5 minutes. After PCR, a PCR product showing about 1.1 Kbp was collected by agarose electrophoresis. A plasmid prepared by subcloning the recovered PCR product into pCMV-Tag2B (Stratagene) with E. coli. E. coli JM109 strain competent cell (Toyobo) was transformed. The plasmid is isolated and purified from cultured cells obtained by culturing transformed cells in 100 mL of LB medium containing 50 μg / mL kanamycin using QIAGENQIAfilter Mega Kit (QIAGEN), and is represented by SEQ ID NO: 1. A plasmid expressing a fusion protein to which FLAG (amino acid sequence in one letter code: MDYKDDDDKSPGGSPGLQEF) was added at the amino terminus of the amino acid sequence (hereinafter sometimes referred to as FLAG fusion mAT12 protein) was obtained. Furthermore, it was confirmed by the method described in Example 4 that there was no mutation in the plasmid.
[0051]
Example 9 (Preparation of enucleated cell extract containing this enzyme (Part 1): Preparation of enucleated cell extract containing FLAG fusion mAT12 protein)
Using the LipofectAMINE PLUS reagent kit (Invitrogen), the FLAG-fusion mAT12 protein expression plasmid obtained in Example 8 was introduced into CHO-K1 cells (Dainippon Pharmaceutical Co., Ltd.) according to the procedure attached to the reagent kit. did. The CHO-K1 cells into which the expression plasmid was introduced were cultured for 24 hours according to the protocol distributed at the time of purchase of the cells. After culturing, the cells were washed twice with D-PBS (Gibco), scraped with a cell scraper, and centrifuged to collect the cells. The collected cells were treated with 50 mM Tris-HCl (pH 7.5), 0.25 M sucrose, 1 mM ethylene glycol bis 2-aminoethyl ether tetraacetic acid (hereinafter referred to as EGTA), 1 mM dithiothreitol (hereinafter referred to as DTT). ), And suspended in a buffer solution (hereinafter referred to as Buffer A) containing 1/100 volume of protease inhibitor cocktail (Sigma). The suspension was subjected to ultrasonic treatment (under ice cooling, 5 seconds × 6 times) to disrupt the cells in the suspension, and then the resulting cell disruption solution was treated at 1000 × g for 30 seconds at 4 ° C. Centrifugation removed unbroken cells and nuclear fractions as precipitates. The obtained supernatant was measured for the protein concentration by Bradford method using bovine serum albumin as a standard, and diluted with buffer A so as to be 2 μg / μL, respectively. did.
[0052]
Example 10 (Preparation of enucleated cell extract as control (part 1))
As a control (negative control), an enucleated cell extract was obtained by the same method as described in Example 9 except that pCMV-Tag2B (without insert) was used instead of the FLAG-fusion mAT12 protein expression plasmid. (Control) was obtained.
[0053]
Example 11 (Separation and detection of this enzyme (part 1))
Immunoblotting using an anti-FLAG tag M2 antibody (Sigma) was performed using 5 μL of the enucleated cell extract obtained in Examples 9 and 10, and the control (negative control) enucleated cell extract was obtained. In case, nothing was detected. On the other hand, in the case of the enucleated cell extract prepared from the cells into which the plasmid for expressing the FLAG fusion mAT12 protein was introduced, a protein having a single band with a molecular weight of about 45 kDa was detected.
[0054]
Example 12 (Measurement of Monoacylglycerol Acyltransferase Activity (Part 1))
10 μL of the enucleated cell extract obtained in Examples 9 and 10 was added to 150 μL of a reaction buffer solution (24 mM Tris-HCl (pH 7.5), 50 mM potassium chloride, 8 mM magnesium sulfate, 1.25 mg / mL bovine serum albumin) 1 μM DTT, 0.5 mM 2-monooleoylglycerol), and then 5 μL of 900 μM (0.2 MBq / μL) [ ¹⁴ C] Palmitoyl-coenzyme A aqueous solution was added. This mixture was incubated at room temperature for 10 minutes. In addition, the test system which performed operation similar to the above using the reaction buffer solution having the same composition as described above except that 2-monooleoylglycerol was not included was used as a background section.
Next, 300 μL of heptane / 2-propanol / water (= 80: 20: 2) was added to the mixture and stirred, allowed to stand for 10 minutes, and then 200 μL of heptane and 100 μL of water were further added to the mixture and stirred. did. After stirring, the mixture was centrifuged to remove the lower layer, and the remaining upper layer was dried using a centrifugal evaporator. The dried product (fat fraction) thus obtained was redissolved in chloroform. Next, this solution is spotted on a glass plate for silica gel 150 thin layer chromatography (K5 silica gel 150 Å, Whatman) and developed using a developing solvent of hexane / diethyl ether / acetic acid (75: 25: 1). To be contained in the solution [ ¹⁴ C] 1,2-diacylglycerol was isolated. Isolated [ ¹⁴ C] 1,2-diacylglycerol was quantified using a bioimaging analyzer system BAS-2000 (Fuji Film). A value obtained by subtracting the measured value in the background section from the measured value in the reaction buffer containing 2-monooleoylglycerol was calculated as the enzyme activity. Table 3 shows the activity of monoacylglycerol acyltransferase in the enucleated cell extract in each of the test system in which the control (negative control) plasmid was introduced and the test system in which the plasmid for expressing the FLAG fusion mAT12 protein was introduced. It was.
[0055]
[Table 3]

As described above, the activity of the monoacylglycerol acyltransferase in the enucleated cell extract was higher in the test system introduced with the FLAG-fused mAT12 protein expression plasmid than in the test system introduced with the control (negative control) plasmid. As a result, the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 was confirmed to exhibit monoacylglycerol acyltransferase activity.
[0056]
Example 13 (Cloning of this gene derived from mouse (2))
In order to amplify a mouse-derived DNA fragment having this gene (mAT14) by PCR, first, Mouse Normal Adipose cDNA (Biochain) 1 μL, primer consisting of the nucleotide sequence shown in SEQ ID NO: 19, 1920 pmol, shown in SEQ ID NO: 20 For PCR of 50 μL, including 20 pmol of primer 20 consisting of base sequence, 2 U of Takara Ex-Taq polymerase (Takara Shuzo) 2 U, 5 μL of buffer attached to Takara Ex-Taq polymerase, and 4 μL of dNTP mixture (2.5 mM) attached to Takara Ex-Taq polymerase A reaction solution was prepared and subjected to PCR. In PCR, the temperature is first kept at 94 ° C. for 60 seconds, followed by 40 heat insulation cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 1 minute, and finally at 72 ° C. for 4 minutes. Was done. After PCR, a PCR product showing about 1.3 Kbp was collected by agarose electrophoresis. An E. coli plasmid prepared by subcloning the recovered PCR product into pT7-Blue vector (Novagen). E. coli JM109 strain competent cell (Toyobo) was transformed. This plasmid derived from a mouse is isolated and purified from cultured cells obtained by culturing transformed cells in 100 mL of LB medium containing 50 μg / mL ampicillin using QIAGEN Plasmid Maxi Kit (QIAGEN). A plasmid containing DNA having the base sequence of the gene (mAT14) was obtained.
[0057]
Example 14 (Determination of the base sequence of this gene derived from mouse (part 2))
Using the plasmid containing the PCR product (about 1.3 kbp) obtained in Example 13 as a template, Thermo Sequenase II dye terminator kit (Amersham Pharmacia Biotech) and ABI373 DNA sequence reader (PE Applied Biosystems), The base represented by SEQ ID NO: 14 by the method of Sanger (F. Sanger, S. Nicklen, AR Coulson A, Proceedings of National Academy of Science USA (1977), 74, 5463-5467). The base sequence of this mouse-derived gene (mAT14) consisting of DNA having the sequence was determined.
[0058]
Example 15 (Cloning of this human-derived gene (part 2))
In order to amplify a DNA fragment having this human-derived gene (hAT14) by PCR, first, Human Adipocyte Marathon-ready cDNA (CLONTECH) 1 μL, primer 21 consisting of the base sequence shown in SEQ ID NO: 21 20 pmol, SEQ ID NO: 22 50 μL containing 20 pmol of primer 22 consisting of the base sequence shown in the following, 5 μL of buffer attached to Takara Ex-Taq polymerase (Takara Shuzo) 2 U, Takara Ex-Taq polymerase, and 4 μL of dNTP mixture (2.5 mM) attached to Takara Ex-Taq polymerase A reaction solution for PCR was prepared and subjected to PCR. In PCR, the temperature is first kept at 94 ° C. for 60 seconds, followed by 40 heat insulation cycles of 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, 72 ° C. for 1 minute, and finally at 72 ° C. for 5 minutes. Was done. After PCR, a PCR product showing about 1.4 Kbp was collected by agarose electrophoresis. An E. coli plasmid prepared by subcloning the recovered PCR product into pT7-Blue vector (Novagen). E. coli JM109 strain competent cell (Toyobo) was transformed. The plasmid is isolated and purified from the cultured cells obtained by culturing the transformed cells in 100 mL of LB medium containing 50 μg / mL ampicillin using QIAGEN Plasmid Maxi Kit (QIAGEN). A plasmid containing DNA having the base sequence of the gene (hAT14) was obtained.
[0059]
Example 16 (Determination of the base sequence of this human-derived gene (part 2))
Using the plasmid containing the PCR product (about 1.4 kbp) obtained in Example 15 as a template, Thermo Sequenase II die terminator kit (Amersham Pharmacia Biotech) and ABI373 DNA sequence reader (PEApplied Biosystems) were used. (F. Sanger, S. Nicklen, AR Coulson A, Proceedings of National Academy of Science USA (1977), 74, 5463-5467). The base sequence of this human-derived gene (hAT14) consisting of DNA having the above was determined.
[0060]
Example 17 (Cloning of this gene derived from rat (part 2))
In order to amplify a DNA fragment having this rat-derived gene (rAT14) by PCR, first, Rat Adipocyte Marathon-ready cDNA (CLONTECH) 1 μL, primer 23 consisting of the nucleotide sequence shown in SEQ ID NO: 23, 20 pmol, SEQ ID NO: 24 50 μL containing 20 μmol of primer 24 consisting of the nucleotide sequence shown, 2 μL of Takara Ex-Taq polymerase (Takara Shuzo) 2 U, 5 μL of buffer attached to Takara Ex-Taq polymerase and 4 μL of dNTP mixture (2.5 mM) attached to Takara Ex-Taq polymerase A reaction solution for PCR was prepared and used for PCR. In PCR, the temperature is first kept at 94 ° C. for 60 seconds, followed by 40 heat insulation cycles of 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, 72 ° C. for 1 minute, and finally at 72 ° C. for 5 minutes. Was done. After PCR, a PCR product showing about 1.3 Kbp was collected by agarose electrophoresis. An E. coli plasmid prepared by subcloning the recovered PCR product into pT7-Blue vector (Novagen). E. coli JM109 strain competent cell (Toyobo) was transformed. By separating and purifying the plasmid from cultured cells obtained by culturing the transformed cells in 100 mL of LB medium containing 50 μg / mL ampicillin using QIAGEN Plasmid Maxi Kit (QIAGEN), a rat-derived book A plasmid containing DNA having the base sequence of the gene (rAT14) was obtained.
[0061]
Example 18 (Determination of the nucleotide sequence of the rat-derived gene (part 2))
Using the plasmid containing the PCR product (about 1.3 kbp) obtained in Example 17 as a template, Thermo Sequenase II die terminator kit (Amersham Pharmacia Biotech) and ABI373 DNA sequence reader (PEApplied Biosystems) were used. (F. Sanger, S. Nicklen, AR Coulson A, Proceedings of National Academy of Science USA (1977), 74, 5463-5467). The base sequence of this rat-derived gene (rAT14) consisting of DNA having the above was determined.
[0062]
Example 19 (Preparation of a plasmid expressing a fusion protein with FLAG added to the amino terminus of the amino acid sequence shown in SEQ ID NO: 13)
0.05 μg of the plasmid isolated and purified in Example 13, 20 pmol of primer 25 consisting of the base sequence shown in SEQ ID NO: 25 and 20 pmol of primer 26 consisting of the base sequence shown in SEQ ID NO: 26, Takara ExTaq polymerase (Takara Shuzo) 0 A reaction solution for PCR of 5 μL, 5 μL of buffer attached to Takara ExTaq polymerase and 4 μL of dNTP mixture (2.5 mM) attached to Takara ExTaq polymerase was prepared and used for PCR. The PCR was carried out under the condition that the temperature was first kept at 94 ° C. for 1 minute, then 60 ° C. for 30 seconds, and further at 72 ° C. for 1 minute 30 times, and finally at 72 ° C. for 5 minutes. After PCR, a PCR product showing about 1.1 Kbp was collected by agarose electrophoresis. A plasmid prepared by subcloning the recovered PCR product into pCMV-Tag2B (Stratagene) with E. coli. E. coli JM109 strain competent cell (Toyobo) was transformed. By isolating and purifying the plasmid from the cultured cells obtained by culturing the transformed cells in 500 mL of LB medium containing 50 μg / mL kanamycin using QIAGEN QIAfilter Mega Kit (QIAGEN), SEQ ID NO: 13 A plasmid was obtained that expresses a fusion protein to which FLAG (amino acid sequence in one letter code: MDYKDDDDKSPGS) was added to the amino terminus of the amino acid sequence shown (hereinafter sometimes referred to as FLAG fusion mAT14 protein). Furthermore, it was confirmed by the method described in Example 14 that there was no mutation in the plasmid.
[0063]
Example 20 (Preparation of enucleated cell extract containing this enzyme (part 2): Preparation of enucleated cell extract containing FLAG fusion mAT14 protein)
The FLAG fusion mAT14 protein expression plasmid obtained in Example 19 was introduced into CHO-K1 cells (Dainippon Pharmaceutical Co., Ltd.) using the LipofectAMINE PLUS reagent kit (Invitrogen) according to the procedure attached to the reagent kit. did. The CHO-K1 cells into which the expression plasmid was introduced were cultured for 24 hours according to the protocol distributed at the time of purchase of the cells. After culturing, the cells were washed twice with D-PBS (Gibco), scraped with a cell scraper, and centrifuged to collect the cells. The collected cells were treated with 50 mM Tris-HCl (pH 7.5), 0.25 M sucrose, 1 mM ethylene glycol bis 2-aminoethyl ether tetraacetic acid (hereinafter referred to as EGTA), 1 mM dithiothreitol (hereinafter referred to as DTT). ), And suspended in a buffer solution (hereinafter referred to as Buffer A) containing 1/100 volume of protease inhibitor cocktail (Sigma). The suspension was subjected to ultrasonic treatment (under ice cooling, 5 seconds × 6 times) to disrupt the cells in the suspension, and then the resulting cell disruption solution was treated at 1000 × g for 30 seconds at 4 ° C. Centrifugation removed unbroken cells and nuclear fractions as precipitates. The obtained supernatant was measured for the protein concentration by Bradford method using bovine serum albumin as a standard, and diluted with buffer A so as to be 2 μg / μL, respectively. did.
[0064]
Example 21 (Preparation of enucleated cell extract as control (part 2))
As a control (negative control), an enucleated cell extract was obtained by the same method as described in Example 20, except that pCMV-Tag2B (without insert) was used instead of the FLAG fusion mAT14 protein expression plasmid. (Control) was obtained.
[0065]
Example 22 (Separation and detection of this enzyme (part 2))
Immunoblotting using an anti-FLAG tag M2 antibody (Sigma) was performed using 5 μL of the enucleated cell extract obtained in Examples 20 and 21, and the control (negative control) enucleated cell extract was obtained. In case, nothing was detected. On the other hand, in the case of the enucleated cell extract prepared from the cells into which the plasmid for expressing the FLAG fusion mAT14 protein was introduced, a protein having a single band with a molecular weight of about 46 kDa was detected.
[0066]
Example 23 (Measurement of activity of monoacylglycerol acyltransferase (part 2))
10 μL of the enucleated cell extract obtained in Examples 20 and 21 was added to 150 μL of a reaction buffer solution (24 mM Tris-HCl (pH 7.5), 50 mM potassium chloride, 8 mM magnesium sulfate, 1.25 mg / mL bovine serum albumin) 1 μM DTT, 0.5 mM 2-monooleoylglycerol), and then 5 μL of 900 μM (0.2 MBq / μL) [ ¹⁴ C] Palmitoyl-coenzyme A aqueous solution was added. This mixture was incubated at room temperature for 10 minutes. In addition, the test system which performed operation similar to the above using the reaction buffer solution having the same composition as described above except that 2-monooleoylglycerol was not included was used as a background section.
Next, 300 μL of heptane / 2-propanol / water (= 80: 20: 2) was added to the mixture and stirred, allowed to stand for 10 minutes, and then 200 μL of heptane and 100 μL of water were further added to the mixture and stirred. did. After stirring, the mixture was centrifuged to remove the lower layer, and the remaining upper layer was dried using a centrifugal evaporator. The dried product (fat fraction) thus obtained was redissolved in chloroform. Next, this solution is spotted on a glass plate for silica gel 150 thin layer chromatography (K5 silica gel 150 Å, Whatman) and developed using a developing solvent of hexane / diethyl ether / acetic acid (75: 25: 1). To be contained in the solution [ ¹⁴ C] 1,2-diacylglycerol was isolated. Isolated [ ¹⁴ C] 1,2-diacylglycerol was quantified using a bioimaging analyzer system BAS-2000 (Fuji Film). A value obtained by subtracting the measured value in the background section from the measured value in the reaction buffer containing 2-monooleoylglycerol was calculated as the enzyme activity. Table 4 shows the activity of monoacylglycerol acyltransferase in each enucleated cell extract in the test system in which the control (negative control) plasmid was introduced and the test system in which the FLAG fusion mAT14 protein expression plasmid was introduced. It was.
[Table 4]

As described above, the activity of the monoacylglycerol acyltransferase in the enucleated cell extract was higher in the test system introduced with the FLAG fusion mAT14 protein expression plasmid than in the test system introduced with the control (negative control) plasmid. As a result, the protein consisting of the amino acid sequence shown in SEQ ID NO: 13 was confirmed to exhibit monoacylglycerol acyltransferase activity.
[0067]
Example 24 (Preparation of a plasmid expressing a fusion protein in which FLAG is added to the amino terminus of the amino acid sequences shown in SEQ ID NO: 3 and SEQ ID NO: 15)
As in Example 8, the amino acid sequence represented by SEQ ID NO: 3 was obtained by PCR using the hAT-12 plasmid separated and purified in Example 5 and primers comprising the nucleotide sequences represented by SEQ ID NOs: 27 and 28. A plasmid was obtained that expressed a fusion protein with FLAG attached to the amino terminus (hereinafter sometimes referred to as FLAG fusion hAT-12). Furthermore, it was confirmed by the method described in Example 4 that there was no mutation in the plasmid.
Similarly to Example 8, the amino acid represented by SEQ ID NO: 15 was obtained by PCR using the hAT-14 plasmid isolated and purified in Example 15 and a primer comprising the nucleotide sequences represented by SEQ ID NOs: 29 and 30. A plasmid was obtained that expressed a fusion protein in which FLAG was added to the amino terminus of the sequence (hereinafter sometimes referred to as FLAG fusion hAT-14). Furthermore, it was confirmed by the method described in Example 16 that there was no mutation in the plasmid.
[0068]
Example 25 (Preparation of enucleated cell extract containing this enzyme (part 3): Preparation of enucleated cell extract containing FLAG fusion hAT12 protein / FLAG fusion hAT14 protein) Expression of FLAG fusion protein obtained in Example 24 The plasmid for nucleation was introduced into CHO-K1 cells, and an enucleated cell extract was prepared in the same manner as in Example 9.
[0069]
Example 26 (Preparation of enucleated cell extract as control (part 3))
As a control (negative control), the method described in Example 9 except that pCMV-Tag2B (without insert) is used instead of the FLAG-fused hAT-12 protein expression plasmid or the FLAG-fused hAT-14 protein expression plasmid. In the same manner as above, an enucleated cell extract (control) was obtained.
[0070]
Example 27 (Separation and detection of this enzyme (Part 3))
Immunoblotting using an anti-FLAG tag M2 antibody (Sigma) was performed using 5 μL of the enucleated cell extract obtained in Examples 25 and 26, and the control (negative control) enucleated cell extract was obtained. In case, nothing was detected. On the other hand, in the case of enucleated cell extracts prepared from cells introduced with a FLAG-fused hAT-12 protein expression plasmid or a FLAG-fused hAT-14 protein expression plasmid, single molecular weights of 45 kD and 46 kD, respectively. A protein band was detected.
[0071]
Example 28 (Measurement of activity of monoacylglycerol acyltransferase)
In the same manner as in Example 12, the activity of monoacylglycerol acyltransferase in the enucleated cell extract prepared in Examples 25 and 26 was measured. The results are shown in Table 5.
[Table 5]

From the above, in the test system into which the FLAG-fused hAT12 protein expression plasmid and the FLAG-fused hAT14 protein expression plasmid were introduced, the control (monoacyl in the enucleated cell extract compared to the test system into which the negative control was introduced). Since the activity of glycerol acyltransferase increased, it was confirmed that the protein consisting of the amino acid sequences shown in SEQ ID NO: 3 and SEQ ID NO: 15 showed the activity of monoacylglycerol acyltransferase.
[0072]
Example 29 (Expression distribution of monoacylglycerol acyltransferase gene)
The plasmids (FLAG-mAT12, mAT14, hAT12, hAT14) obtained in Examples 8, 19 and 24 were treated with a restriction enzyme so as to be cut at both ends of the gene coding sequence excluding the FLAG sequence. A band of the desired length was cut out from the gel on which the DNA was electrophoresed, and the DNA was purified with a GENECLEAN II kit (Takara Shuzo). Using this DNA about 10-100 ng as a template, by random prime method ³³ P-dCTP radiolabeling was performed. For the reaction, a random prime kit (Takara Shuzo) was used and the protocol attached to the kit was followed. After removing unreacted dNTPs from the reaction solution with a spin column, the specific activity of the probe was measured with a liquid scintillation counter to confirm that the probe was sufficiently radiolabeled. A part of the probe was heat-denatured and rapidly cooled to a single strand, and then added to 8 ml of DIG Easy-Hyb solution (Boehringer Mannheim), which was used as a hybridization solution. In this hybridizing solution, tissue RNA blot membranes (Mouse Adult Normal Tissue mRNA Northern Blot, Human Adult Normal Tissue Total RNA North Blot, manufactured by DIG Easy-Hyb solution, all manufactured by BIO Blot, Inc.) Soaking and sealing in a hybrid bag started hybridization. Hybridization conditions were as follows: prehybridization: 50 ° C., 3 hours or more, hybridization: 50 ° C., shaken overnight. After hybridization, the membrane was washed with 2 × SSC-0.1% SDS, twice at room temperature, 0.1 × SSC-0.1% SDS, once at 50 ° C., and the membrane was then imaged on the imaging plate. Exposed overnight. The results of reading the imaging plate using a bioimaging analyzer system BAS-2500 (Fuji Film) are shown in FIGS.
From this, it was confirmed that mouse AT12 and AT14 mRNA are significantly expressed in mouse liver and small intestine, and that human AT12 and AT14 mRNA are significantly expressed in human small intestine.
To summarize the above results, AT12 and AT14 have monoacylglycerol acyltransferase activity in cells, significant expression is observed in the liver and small intestine in mice, and significant expression is observed in the small intestine in humans. AT12 and AT14 are thought to be monoacylglycerol acyltransferases involved in triglyceride synthesis in the liver and small intestine.
[0073]
Example 30 (Establishment of a cell line stably expressing the FLAG fusion mAT12 protein)
Using the LipofectAMINE PLUS reagent kit (Invitrogen), the FLAG-fusion mAT12 protein expression plasmid obtained in Example 8 was introduced into CHO-K1 cells (Dainippon Pharmaceutical Co., Ltd.) according to the procedure attached to the reagent kit. did. After culturing CHO-K1 cells introduced with the expression plasmid for 72 hours according to the procedure distributed at the time of purchase, Ham's F-12 supplemented with 10% FCS containing 400 μg / ml GENETICIN (Gibco) The medium was replaced with a medium (hereinafter referred to as a selective medium), and the culture was continued for 7 days while appropriately replacing with a new medium. After culturing, the stable expression strain was cloned by the limiting dilution method. For each of the obtained cell lines, a part was 50 mM Tris-hydrochloric acid (pH 7.5), 0.25 M sucrose, 1 mM ethylene glycol bis 2-aminoethyl ether tetraacetic acid (hereinafter referred to as EGTA), 1 mM dithiothrei. It was suspended in a buffer solution (hereinafter referred to as Buffer A) containing Toll (hereinafter referred to as DTT) and 1/100 volume of protease inhibitor cocktail (Sigma). The suspension was subjected to ultrasonic treatment (under ice cooling, 5 seconds × 6 times) to disrupt the cells in the suspension, and then the resulting cell disruption solution was treated at 1000 × g for 30 seconds at 4 ° C. Centrifugation removed unbroken cells and nuclear fractions as precipitates. The resulting supernatant was subjected to immunoblotting using an anti-FLAG tag M2 antibody (Sigma), and a cell line expressing the most protein with a single band with a molecular weight of about 45 kDa was selected. 12 stable expression strains were obtained.
[0074]
Example 31 (Purification of FLAG fusion mAT12 protein)
The cell line stably expressing the FLAG fusion mAT12 protein obtained in Example 30 was expanded in a selective medium. After culturing, the cells were washed twice with D-PBS (Gibco), scraped with a cell scraper, and centrifuged to collect the cells. The collected cells were treated with 50 mM Tris-hydrochloric acid (pH 7.5), 150 mM sodium chloride, 0.1% {3-[(3-Cholamidopropylo) dimethylamine-1-propanesulfonate} (hereinafter referred to as CHAPS), 1 mM EGTA. It was suspended in a buffer containing 1 mM DTT, 1/100 volume protease inhibitor cocktail (Sigma). This suspension was sonicated (6 seconds x 6 times under ice-cooling) to disrupt the cells in the suspension, and the resulting cell disruption solution was 15000 xg for 5 minutes at 4 ° C. By centrifuging, unbroken cells and nuclear fractions were removed as precipitates, and the supernatant was obtained as a crude cell extract. The crude cell extract was applied to a column in which 1 ml of Anti-FLAG M2 affinity gel was equilibrated with 50 mM Tris-hydrochloric acid (pH 7.5), 150 mM sodium chloride, 0.1% CHAPS (hereinafter referred to as buffer B). In addition, FLAG fusion mAT12 protein was adsorbed. After washing the column with 10 volumes of buffer B, the flow was stopped, and buffer B containing FLAG peptide was added and incubated. After 10 minutes, effluxing was started again, and a FLAG-fused mAT12 protein purified fraction was obtained. Immunoblotting using an anti-FLAG tag M2 antibody (Sigma) was performed, and it was confirmed that this fraction contained a protein having a single band with a molecular weight of about 45 kDa.
[0075]
Example 32 (Measurement of monoacylglycerol acyltransferase activity of purified FLAG-fused mAT12 protein)
10 μl of the purified FLAG fusion mAT12 protein purified fraction obtained in Example 31 was used as a reaction buffer (24 mM Tris-HCl (pH 7.5), 50 mM potassium chloride, 8 mM magnesium sulfate, 1.25 mg / ml bovine serum albumin, 1 mM. After suspending in 130 μl of DTT, 0.5 mM 2-monooleoylglycerol, a test substance (dissolved in dimethyl sulfoxide (hereinafter referred to as DMSO), added to a final concentration of 1% DMSO) was added, Mixed. To this mixture, 10 μL of 450 μM [ ¹⁴ C] An aqueous solution of palmitoyl-coenzyme A was added and kept at room temperature for 20 minutes. In the test substance-free group, the same method as described above was used, except that 1.5 μl of DMSO alone was added instead of the DMSO solution of the test substance. In the background group, the same method as that for the test substance and 2-monooleoylglycerol was used except that only DMSO was added instead of the test substance and 2-monooleoylglycerol. After the incubation for 20 minutes, the reaction was stopped by adding 300 μL of heptane / 2-propanol / water (= 80: 20: 2) to the reaction mixture, and after adding 200 μl of heptane and 100 μl of water, did. After stirring, the mixture was centrifuged to remove the lower layer, and the remaining upper layer was dried using a centrifugal evaporator. The dried product (fat fraction) thus obtained was redissolved in 30 μl of chloroform / methanol (2: 1, v / v). This solution was then spotted onto a silica gel 150 thin layer chromatography glass plate (K5 silica gel 150 Å, Whatman). A developing solvent of hexane / diethyl ether / acetic acid (75: 25: 1, v / v) was put in a sealed container, and the TLC plate was developed using the developing solvent, and then the TLC plate was dried at room temperature. The dried TLC plate was exposed to an imaging plate (Fuji Film Co.) for 16 hours. After completion of the exposure, the imaging plate was analyzed with a bio image analyzer (BAS 2500, Fuji Film Co., Ltd.), and the portion corresponding to the development position of 1,2-diacylglycerol and triglyceride on the TLC plate [ ¹⁴ C] The radioactivity was measured (hereinafter, the portion corresponding to the development position of the 1,2-diacylglycerol moiety [ ¹⁴ C] Radioactivity is measured value G, the portion corresponding to the development position of triacylglycerol [ ¹⁴ C] Radioactivity is described as measured value H. ). From these measured values, the activity of monoacylglycerol acyltransferase (measured value G + measured value H / 2, hereinafter referred to as activity value 4) when a test substance was added was calculated. In addition, for the test substance-free group and the background group, using the same method as described above, the portion corresponding to the development position of 1,2-diacylglycerol and triglyceride on the TLC plate [ ¹⁴ C] The radioactivity was measured (hereinafter, the portion corresponding to the development position of 1,2-diacylglycerol in the test substance-free group [ ¹⁴ C] Radioactivity is measured value I, and the portion corresponding to the development position of triacylglycerol in the test substance-free group [ ¹⁴ C] Radioactivity is measured value J, radioactivity of the portion corresponding to the development position of 1,2-diacylglycerol in the background section is measured value K, radiation of the portion corresponding to the development position of triacylglycerol in the background section The activity is denoted as measured value L. ). From these measured values, the activity of the monoacylglycerol acyltransferase in the group to which no test substance was added (measured value I + measured value J / 2, hereinafter referred to as activity value 5) and the activity of the monoacylglycerol acyltransferase in the background group. (Measured value K + Measured value L / 2, hereinafter referred to as activity value 6) was calculated. From the obtained activity value, the monoacylglycerol acyltransferase inhibitory ability of the test substance was calculated as an inhibition rate ((activity value 5−activity value 4) × 100 / (activity value 5−activity value 6)).
As a result, the known compound N- [2- (4-benzyl-2-ethylphenoxy) ethyl] -5,6-dimethyl [1,2,4] triazolo [1,5-a] pyrimidine-7 as a test substance -The inhibition rate in amine (hereinafter also referred to as Compound A) was about 65% at 10 μM. Further, the inhibition rate at each test concentration was calculated using the same method as described above while changing the test concentration of the test substance. The results are shown in Table 6.
From the above, it was found that the known compound A effectively inhibits the monoacylglycerol acyltransferase enzyme activity of mAT12 or FLAG fusion mAT12.
[Table 6]

[0076]
Example 33 (Inhibitory effect of monoacylglycerol acyltransferase inhibitor on triglyceride synthesis in human liver cells)
By culturing human hepatoma-derived cell line HepG2 cells (Dainippon Pharmaceutical Co., Ltd.) in a 6-well plate until confluent, and then treating with a serum-free D-MEM medium (GIBCO, high glucose content) overnight. Hungry. The cell culture medium was diluted to a final concentration of 12.9 μM [U− ¹⁴ C] The test substance (dimethyl sulfoxide (hereinafter referred to as DMSO)) was immediately replaced with a D-MEM medium (GIBCO, low glucose-containing) containing D-glucose (Amersham, radioactive 4 mCi / L). And added to a final concentration of 0.5% DMSO) and incubated at 37 ° C. for 4 hours. In the test substance-free group, the same method as described above was used except that only DMSO was added instead of the DMSO solution of the test substance. The cultured cells were washed once with PBS buffer, and the cells were treated with 1 ml of heptane / isopropanol (2: 1) for 10 minutes. A heptane-isopropanol solution containing triglyceride in the cells was collected with a pipette, and the collected solution was dried using a centrifugal evaporator. The dried product (fat fraction) thus obtained was redissolved in 30 μl of chloroform / methanol (2: 1, v / v). This solution was then spotted onto a silica gel 150 thin layer chromatography glass plate (K5 silica gel 150 Å, Whatman). A developing solvent of hexane / diethyl ether / acetic acid (75: 25: 1, v / v) was put in a sealed container, and the TLC plate was developed using the developing solvent, and then the TLC plate was dried at room temperature. The dried TLC plate was exposed to an imaging plate (Fuji Film Co.) for 16 hours. After completion of the exposure, the imaging plate was analyzed with a bio image analyzer (BAS 2500, Fuji Film Co., Ltd.), and the portion corresponding to the development position of triacylglycerol on the TLC plate [ ¹⁴ C] Radioactivity was measured. In addition, for the test substance-free group, the portion corresponding to the development position of the triacylglycerol on the TLC plate using the same method as described above [ ¹⁴ C] Radioactivity was measured.
As a result, the known compound N- [2- (4-benzyl-2-ethylphenoxy) ethyl] -5,6-dimethyl [1,2,4] triazolo [1,5-a] pyrimidine-7 as a test substance -The amount of triglyceride synthesized at the time of adding an amine (hereinafter sometimes referred to as Compound A) was about 31% at 10 µM, assuming that the amount of synthesis in the group without the test substance was 100%. Further, Table 7 shows the results of calculating the synthesis amount at each test concentration using the same method as described above while changing the test concentration of the test substance.
As a result, it was found that the known compound A effectively inhibits triglyceride synthesis in human liver cells. Therefore, it is considered that a monoacylglycerol acyltransferase activity inhibitor can inhibit lipoprotein (VLDL) synthesis by inhibiting hepatic triglyceride synthesis.
[Table 7]

[0077]
Example 34 (Effect of monoacylglycerol inhibitor on blood triglyceride elevation after fat and oil administration to mice)
8 ICR mice (12 weeks old, male, Japan SLC) / group were fasted overnight and then N- [2- (4-benzyl-2-ethylphenoxy) ethyl] -5,6-dimethyl [1,2 , 4] triazolo [1,5-a] pyrimidin-7-amine (hereinafter referred to as Compound A) suspended in olive oil (manufactured by Nacalai Tesque): 5 ml / kg was forcibly orally administered. As a test substance non-treatment group, olive oil not containing Compound A was orally administered to 8 mice. 10 μL of blood was collected over time from the tail vein of the mouse and dissolved in 1 ml of isopropanol. The amount of triglyceride in the isopropanol extract was measured using a triglyceride test kit Wako (Wako Pure Chemical Industries). FIG. 5 shows the time course of blood triglyceride when Compound A is administered at a dose of 30 mg / kg. FIG. 5 shows blood triglyceride after 3 hours when the dose of Compound A is changed to 0, 10, 30 mg / kg. The result of measuring the amount of triglyceride is shown in FIG.
As a result, it was confirmed that when known compound A was administered to mice together with lipids, the increase in blood triglyceride was effectively inhibited. Therefore, monoacylglycerol acyltransferase inhibitors effectively inhibit small intestinal cell triglyceride synthesis, thereby effectively inhibiting small intestinal cell lipoprotein (chylomicron) synthesis and secretion, resulting in a decrease in blood triglyceride concentration. It is considered possible.
[0078]
【The invention's effect】
According to the present invention, it is possible to provide a simple assay method for the ability to suppress increase in fat mass.
[0079]
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[0080]
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[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1: Northern hybridization was carried out on mouse Adult Normal Tissue mRNA Northern Blot using mouse AT12 sequence as a probe. In order from the left lane are brain, heart, kidney, liver, lung, skeletal muscle, skin, small intestine, spleen, stomach, testis, thymus tissue RNA.
[FIG. 2] Northern hybridization was carried out on mouse Adult Normal Tissue mRNA Northern Blot using the mouse AT14 sequence as a probe. In order from the left lane are brain, heart, kidney, liver, lung, skeletal muscle, skin, small intestine, spleen, stomach, testis, thymus tissue RNA.
[FIG. 3] Northern hybridization was carried out with respect to the human Normal Tissue Total RNA Northern Blot using the human AT12 sequence as a probe. In order from the left lane, there are tissue RNAs of the esophagus, stomach, small intestine, colon, uterus, placenta, bladder, and fat. The left side of the figure shows the sex (male = M, female = F) and age of the specimen from each tissue.
[FIG. 4] Northern hybridization to human Adult Normal Tissue Total RNA Northern Blot was performed using the human AT14 sequence as a probe. In order from the left lane, there are tissue RNAs of the esophagus, stomach, small intestine, colon, uterus, placenta, bladder, and fat. The left side of the figure shows the sex (male = M, female = F) and age of the specimen from each tissue.
FIG. 5: Mice were divided into 2 groups (8 animals / group). The first group received olive oil alone, and the second group received olive oil and Compound A. About 10 μl of blood was collected from the tail vein every hour from immediately after administration to 6 hours, and the amount of triglyceride in the blood was quantified. The quantification results are shown as a graph in which the vertical axis represents blood triglyceride concentration (mg / dL) and the horizontal axis represents time.
FIG. 6: Mice are divided into 3 groups (8 animals / group), the first group is olive oil only, the second group is olive oil + compound A: 10 mg / kg, the third group is olive oil + compound A: 30 mg / kg was administered. About 10 μl of blood was collected from the tail vein before administration (0 hr) and 3 hours (3 hr) after administration for each group, and the amount of triglyceride in the blood was quantified. The measured triglyceride concentration (mg / dL) is shown on the vertical axis.

Claims (6)

A test method for the ability to suppress triglyceride accumulation ,
(1) a first step of measuring the activity of the monoacylglycerol acyltransferase in a contact system between the monoacylglycerol acyltransferase and the test substance, and (2) the activity measured in the first step and the activity in the control. A second step of evaluating the triglyceride accumulation inhibiting ability of the substance based on the difference obtained by comparing,
And
The method for assaying the ability to inhibit the increase in fat mass, wherein the monoacylglycerol acyltransferase is one of the following proteins:
<Protein group>
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1, 3 or 11
(B) the amino acid sequence shown in SEQ ID NO: 1, 3 or 11, one or more amino acids are deleted, added or substituted in the amino acid sequence, and protein having a monoacylglycerol acyltransferase activity
(C) SEQ ID NO: 1, 3 or consisting of an amino acid sequence having an amino acid sequence at least 90% sequence identity represented by 11, and proteins having a monoacylglycerol acyltransferase activity
( D) a protein comprising an amino acid sequence encoded by a DNA having the base sequence represented by SEQ ID NO: 2, 4 or 12
( E) consisting of an amino acid sequence encoded by a DNA having a base sequence of 90 % or more of the DNA having the base sequence represented by SEQ ID NO: 2, 4 or 12, and having a monoacylglycerol acyltransferase activity protein having
( F) a DNA comprising a base sequence complementary to the DNA having the base sequence represented by SEQ ID NO: 2, 4 or 12, and an amino acid sequence encoded by the DNA that hybridizes under stringent conditions; protein having an acyl glycerol acyl transferase activity Method of searching for triglyceride accumulation inhibiting substance characterized by selecting a substance having Claim 1 triglyceride accumulation suppressing capability based on the assessed triglyceride accumulation inhibiting ability by test methods described. 3. The assay method according to claim 1, wherein the triglyceride accumulation inhibiting ability is a lipoprotein synthesis inhibiting ability. A method for searching a lipoprotein synthesis inhibitor, comprising selecting a substance having a lipoprotein synthesis inhibitory ability based on the lipoprotein synthesis inhibitory ability evaluated by the assay method according to claim 3 . A catalyst for the following reaction comprising a protein having the following amino acid sequence .
2 or 1-monoacylglycerol + acyl-CoA ⇒ 1,2-diacylglycerol + CoA
<Amino acid sequence group>
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1, 3 or 11
(B) the amino acid sequence shown in SEQ ID NO: 1, 3 or 11, one or more amino acids are deleted, added or substituted in the amino acid sequence, and protein having a monoacylglycerol acyltransferase activity
(C) SEQ ID NO: 1, 3 or consisting of an amino acid sequence having an amino acid sequence at least 90% sequence identity represented by 11, and proteins having a monoacylglycerol acyltransferase activity
( D) a protein comprising an amino acid sequence encoded by a DNA having the base sequence represented by SEQ ID NO: 2, 4 or 12
( E) consisting of an amino acid sequence encoded by a DNA having a base sequence of 90 % or more of the DNA having the base sequence represented by SEQ ID NO: 2, 4 or 12, and having a monoacylglycerol acyltransferase activity protein having
( F) a DNA comprising a base sequence complementary to the DNA having the base sequence represented by SEQ ID NO: 2, 4 or 12, and an amino acid sequence encoded by the DNA that hybridizes under stringent conditions; protein having an acyl glycerol acyl transferase activity A method for assaying monoacylglycerol acyltransferase inhibitory activity, comprising:
(1) a first step of measuring the monoacylglycerol acyltransferase activity in a contact system between any of the following proteins and a test substance;
(2) a second step of evaluating the monoacylglycerol acyltransferase inhibitory activity of the substance based on the difference obtained by comparing the activity measured in the first step and the activity in the control;
An assay method comprising:
<Protein group>
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1, 3 or 11
(B) a protein comprising an amino acid sequence in which one or more amino acids are deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 1, 3, or 11, and having monoacylglycerol acyltransferase activity
(C) a protein comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, 3 or 11 and having monoacylglycerol acyltransferase activity
(D) a protein comprising an amino acid sequence encoded by a DNA having the base sequence represented by SEQ ID NO: 2, 4 or 12
(E) consisting of an amino acid sequence encoded by a DNA having a base sequence of 90% or more of the DNA having the base sequence represented by SEQ ID NO: 2, 4 or 12, and having monoacylglycerol acyltransferase activity Protein
(F) a DNA comprising a base sequence complementary to the DNA having the base sequence represented by SEQ ID NO: 2, 4 or 12, and an amino acid sequence encoded by the DNA that hybridizes under stringent conditions; Protein having acylglycerol acyltransferase activity

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