JP2004065181A - Diabetes model mouse - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a model mouse of diabetes extremely close to non-fat type-2 diabetes frequent in Japanese and characterized by the neurogenic disorder of the control of insulin secretion. <P>SOLUTION: The model mouse is a descendant of a wild castaneus mouse and an inbred line C57BL/6J mouse and has the following characteristics. (A) The mouse shows high blood sugar level and/or abnormal sugar resistance compared with a normal line mouse, (B) the body weight and/or obesity degree are significantly lower than those of normal-line mouse and (C) the insulin secretion suppressing action of the intrinsic adrenalin through α2-adrenalin receptor is remarkably strong compared with the normal-line mouse. <P>COPYRIGHT: (C)2004,JPO

Description

【００８０】
（２−３）　　血糖値
ＨＮＤマウス系統の６週齢時における血糖値は、非
絶食時で３５８±３１ｍｇ／ｄｌ、絶食時で１３１±５ｍｇ／ｄｌとＢ６（非絶食時：１７６±６，絶食時：８９±３）に比べて顕著に高かった（図４）。これは、明らかに糖尿病領域を示す値である。
（２−４）　　血中インスリン濃度
７、２３、４０週齢時におけるＨＮＤマウス系統の血中インスリン濃度は、非絶食時、絶食時のいずれにおいてもＢ６マウスと違いを認めなかった（図５）。
（２−５）　　グルコース刺激インスリン分泌試験
７週齢時のマウスを用いて、糖負荷試験と同様の条件下のもとで、血中インスリン濃度を測定したところ、Ｂ６では、糖負荷後の血中インスリン濃度の上昇が認められたのに対し、ＨＮＤマウス系統では全く上昇が認められなかった（図６（ａ））。つまり、ＨＮＤマウス系統は、グルコース刺激によるインスリン分泌に明らかな障害が認められた。また、２０週齢時のＨＮＤマウス系統のマウスをＩＰＧＴＴ　１２０分値が２００ｍｇ／ｄｌを境界に、高値を示すものをｄｉａｂｅｔｅｓ群、低値を示すものをｎｏｎ−ｄｉａｂｅｔｅｓ群と分けて検討したとき、ｄｉａｂｅｔｅｓ群は血中インスリン濃度の上昇が認められず、Ｎｏｎ−ｄｉａｂｅｔｅｓ群では上昇が認められた（図６（ｂ））。このことから、グルコース刺激インスリン分泌能の障害が、ＨＮＤマウス系統の耐糖能異常の主因として考えられた。
（２−６）　　インスリン負荷試験
７、２０、４０週齢時において、インスリン負荷試験を行ったところＢ６マウスとＨＮＤマウス系統の間にインスリン負荷後の血糖低下に違いは認めなかった（図７）。従って、ＨＮＤマウス系統の末梢組織でのインスリン作用は、Ｂ６マウスのそれと違いがないと考えられた。
（２−７）　　血中脂質成分
７週齢において絶食時、非絶食時の血中脂質成分（遊離脂肪酸、中性脂肪、総コレステロール）について解析を行った。
遊離脂肪酸：
絶食時において、Ｂ６（１６７５±１８７μＥｑ／ｌ）とＨＮＤマウス系統（１６９２±１６６μＥｑ／ｌ）に違いは認められなかった。しかし、非絶食時の時Ｂ６（７９０±６２μＥｑ／ｌ）に比較して、ＨＮＤマウス系統（１０７１±８２μＥｑ／ｌ）は有意に高い値を示した（図８（ａ））。
中性脂肪：
絶食時においては、Ｂ６マウスとＨＮＤマウス系統の両マウス間で違いは認められなかった。しかし、非絶食時においては、Ｂ６マウス（１４９±１１ｍｇ／ｄｌ）に比し、ＨＮＤマウス系統（２１４±２７ｍｇ／ｄｌ）で高い値が観察された（図８（ｂ））。
総コレステロール：
絶食時においては、Ｂ６（１１６±１ｍｇ／ｄｌ）に比し、ＨＮＤマウス系統で１０５±４ｍｇ／ｄｌとわずかに低い値を示した。非絶食時においては両マウス間で違いは認められなかった（図８（ｃ））。
実施例３：単離膵島におけるインスリン分泌能の解析
実施例１で作出したＨＮＤマウス系統は、実施例２に示したとおり末梢組織でのインスリン作用は正常であった。そこで、インスリン分泌組織である膵臓のランゲルハンス島（膵島）を単離し、そのインスリン分泌量を解析した。
（１）　　方法
（１−１）　　膵臓からの膵島単離
（１−１−１）　　試薬
Ｋｒｅｂｓ−Ｒｉｎｇｅｒ　ｓｏｌｕｔｉｏｎ：
最終濃度、各々１１９ｍＭ　Ｎａｃｌ，４．７５ｍＭ　ＫＣｌ，５ｍＭ　ＮａＨＣ０_３，２．５４ｍＭ　ＣａＣｌ_２，１．２　ｍＭＭｇＳＯ_４，１．２ｍＭ　ＫＨ_２ＰＯ_４，２０ｍＭ　ＨＥＰＥＳとなるように蒸留水で調製後、ＮａＯＨでｐＨ７．４に合わせたものをｓｔｏｃｋ　ｓｏｌｕｔｉｏｎとした。必要に応じ、実験当日に３ｍＭグルコース、５ｍｇ／ｍｌ　ＢＳＡを添加しｗｏｒｋｉｎｇ　ｓｏｌｕｔｉｏｎとした。
Ｃｏｌｌａｇｅｎａｓｅ　ｓｏｌｕｔｉｏｎ：
Ｃｏｌｌａｇｅｎａｓｅ　ｔｙｐｅ　ＸＩ（ＳＩＧＭＡ）を４ｍｇ／ｍＬとなるようにＫｒｅｂｓ−Ｒｉｎｇｅｒ　ｓｏｌｕｔｉｏｎ（３ｍＭグルコース）に溶解した。
（１−１−２）　　コラゲナーゼ消化による膵島単離
マウスの膵臓をコラゲナーゼの作用により消化して、膵島と周囲の外分泌細胞とを分離した。すなわち、非絶食条件下のマウスをジエチルエーテル麻酔し、開腹後脱血により屠殺した。その後、Ｃｏｌｌａｇｅｎａｓｅ　ｓｏｌｕｔｉｏｎが膵臓全体に均一にゆきわたるように、２７Ｇ注射針を用いて、膵臓に少しずつ何度も注入した。膵臓を傷つけないように注意して摘出した後、残りのコラゲナーゼ溶液と共にバックに入れて３７℃で３分間消化反応を行った（注入に使ったもの、バックに入れたもの合わせて２．５ｍｌのコラゲナーゼ溶液を使った）。反応後の組織を５０ｍＬチューブに移し、ボルテックスミキサーで攪拌後、ガラス試験管に移し換えた。膵島は大きな細胞集団であるため溶液中では沈降するため、これを利用して外分泌細胞との分離を行った。具体的には、消化された組織をＫｒｅｂｓ−Ｒｉｎｇｅｒ　ｓｏｌｕｔｉｏｎ（３ｍＭグルコース）により懸濁し、２分間静置した。その後、下から４ｃｍ程の溶液（膵島が沈降した部分）を残し、その上の液を再び新しいＫｒｅｂｓ−Ｒｉｎｇｅｒ　ｓｏｌｕｔｉｏｎ（３ｍＭグルコース）と置き換えた。これと同様の操作を２回繰り返し行った。これにより外分泌細胞をできる限り取り除き、最後に残った下層をガラスシャーレに移した。それから、実体顕微鏡下で膵島をオートピペッターを用いてピックアップし、Ｋｒｅｂｓ−Ｒｉｎｇｅｒ　ｓｏｌｕｔｉｏｎ（３ｍＭグルコース、５ｍｇ／ｍｌ　ＢＳＡ）の入ったガラスシャーレに移し替えた。この一連の操作により、膵島を外分泌細胞から分離することができる。
（１−２）　　膵島を用いたインスリン分泌実験の方法
（１−２−１）　　Ｂａｔｃｈ　ｉｎｃｕｂａｔｉｏｎ法
単離した膵島をＫｒｅｂｓ−Ｒｉｎｇｅｒ　ｓｏｌｕｔｉｏｎ（３ｍＭグルコース、５ｍｇ／ｍｌ　ＢＳＡ）入ったガラスシャーレ上に集め、実体顕微鏡下で大きさ、形の整った膵島を選別して実験に用いた。用いる膵島の大きさが不均一にならないように実体顕微鏡下で４個ずつマッチングした。次いで、小試験管（ＩＷＡＫＩ）に０．６ｍｌのＫｒｅｂｓ−Ｒｉｎｇｅｒ　ｓｏｌｕｔｉｏｎ（３ｍＭグルコース、５ｍｇ／ｍｌ　ＢＳＡ）を注入し、その試験管１本あたり４個となるように膵島を添加し、実験に用いた。３７℃のインキュベーターで１時間プレインキュベートした後、溶液をできるだけ除去した。そして、様々な試薬（グルコース，ＧＬＰ−１，ＧＩＰ、グルカゴン、アドレナリン、ソマトスタチン、クロニジン）をＫｒｅｂｓ−Ｒｉｎｇｅｒ　ｓｏｌｕｔｉｏｎ（５ｍｇ／ｍｌ　ＢＳＡ）で調製し、これをｉｎｃｕｂａｔｉｏｎ　ｍｅｄｉｕｍとして０．６ｍｌ添加し、恒温槽で軽く振とうしながら３７℃で１時間インキューベートを行った。終了後直ちに上清を回収し、インスリン濃度をラジオイムノアッセイ（ＳｈｉｏｎｏＲＩＡ，塩野義製薬株式会社）を用いて測定した。
（１−２−２）　　グルコース刺激インスリン分泌試験
６週齢のマウスから単離した膵島を実験に使用した。前記したように、１時間のプレインキュベートの後、その溶液を３，１０，１５，２０，４０ｍＭのグルコースを含む０．６ｍｌ　ｉｎｃｕｂａｔｉｏｎ　ｍｅｄｉｕｍに置き換え、１時間インキュベートした。そして、その溶液のインスリン濃度を測定した。
（１−２−３）　　　ＧＬＰ−１，ＧＩＰ，グルカゴンによるインスリン分泌促進効果の検討
６週齢のマウスから単離した膵島を実験に使用した。前記したように、１時間のプレインキューベートの後、その溶液を１５ｍＭグルコースに加え、各々１００ｎＭのＧＬＰ−１，ＧＩＰ，グルカゴン（全てＳＩＧＭＡ）を含む０．６ｍｌ　ｉｎｃｕｂａｔｉｏｎ　ｍｅｄｉｕｍに置き換え、１時間インキュベートした。そして、その溶液のインスリン濃度を測定した。（１−２−４）　　ソマトスタチン，アドレナリン，クロニジンによるインスリン分泌抑制効果の検討
２０−３０週齢のマウスから単離した膵島を実験に使用した。前記したように、１時間のプレインキュベートの後、その溶液を２０ｍＭグルコースに加え、１，１０，１００ｎＭソマトスタチン（ｓｏｍａｔｏｓｔａｉｎ−２８、ＳＩＧＭＡ）、０．３，１，３，１０，３０ｎＭアドレナリン（［−］−ａｄｒｅｎｅｒｌｉｎｅ（＋）　ｂｉｔａｒｔａｒａｔｅ、ＳＩＧＭＡ）あるいは１，１０ｎＭクロニジン（クロニジン塩酸塩、和光純薬工業株式会社）を含む０．６ｍｌ　ｉｎｃｕｂａｔｉｏｎ　ｍｅｄｉｕｍに置き換え、１時間インキュベートした。そして、その溶液のインスリン濃度を測定した。（２）　　結果
（２−１）　　グルコース刺激インスリン分泌能
６週齢のマウスから膵島を単離して、ｂａｔｃｈ　ｉｎｃｕｂａｔｉｏｎ法によりグルコース刺激による１時間のインスリン分泌量を測定したところ、３−４０ｍＭのどのグルコース濃度においても、Ｂ６とＨＮＤマウス系統の両マウス間に違いは認められなかった（図９）。この結果から、ＨＮＤマウス系統の膵Ｂ細胞は、少なくとも単離膵島レベルではグルコースに応答するインスリン分泌能が正常に保たれていることが示唆された。
（２−２）　　ＧＬＰ−１、ＧＩＰ、グルカゴンがインスリン分泌に与える影響
ＧＬＰ−１、ＧＩＰ、グルカゴンはグルコース刺激によるインスリン分泌を促進する働きを持つホルモンである。従って、高濃度のグルコース（１５ｍＭ）存在下におけるこれらのホルモンの影響を検討した。その結果、どのホルモンを加えたときも、Ｂ６とＨＮＤマウス系統との間でインスリン分泌量に明らかな違いを認めなかった（図１０）。従って、ＨＮＤマウス系統ではこれらのインスリン分泌促進ホルモンの作用は正常に保たれており、ｉｎ　ｖｉｖｏで認められる明らかなインスリン分泌障害の要因となっていないことが示唆された。
（２−３）　　ソマトスタチン、アドレナリンがインスリン分泌に与える濃度依存的な影響
ソマトスタチン、アドレナリンはグルコース刺激によるインスリン分泌を抑制する働きを持つホルモンである。従って、高濃度のグルコース（２０ｍＭ）存在下における、これらのホルモンの濃度依存的な影響を検討した。
【００８１】
ソマトスタチンの場合、Ｂ６マウスにおいては１ｎＭ添加で約１０％の分泌抑制、１０ｎＭ添加で約３５％の抑制、１０ｎＭ添加で約５０％の抑制が見られた。これらの各濃度のソマトスタチンのインスリン分泌抑制作用は、ＨＮＤマウスにおいてもＢ６マウスと同程度に観察された（図１１−ａ）。すなわち、ソマトスタチンがインスリン分泌に与える作用に関しては、Ｂ６とＨＮＤマウス系統の両マウス間で差がないと推察された。
【００８２】
一方、アドレナリンの濃度依存的なインスリン分泌に対する影響を検討したところ、Ｂ６マウスでは０．３ｎＭ添加で約１０％、１ｎＭで約１５％、３ｎＭで約４０％、１０ｎＭで約６０％のインスリン分泌抑制が観察された。ＨＮＤマウス系統では１ｎＭおよび３ｎＭのアドレナリン添加の時に、Ｂ６マウスよりもインスリン分泌が有意に強く抑制されていた。のインスリン分泌量がＢ６マウスより有意に減少しており、３ｎＭのときも減少傾向が認められた（図１１−ｂ）。これらの結果は、ＨＮＤマウス系統の膵島はアドレナリンによるインスリン分泌の抑制がＢ６マウスよりも強く現れることを示している。そして、ｉｎ　ｖｉｖｏで認められるＨＮＤマウス系統のインスリン分泌障害の要因にアドレナリン作用が関与している可能性が示唆された。
（２−４）　　α_２−アドレナリンレセプターアゴニストがインスリン分泌に与える影響アドレナリンはインスリン分泌を抑制するが、これは膵Ｂ細胞に発現しているα_２−アドレナリンレセプターを介する作用によるものであることが知られている（ＤｉＴｕｌｌｉｏ　ＮＷ．ｅｔ　ａｌ．，　Ｊ．　Ｐｈａｒｍａｃｏｌ．　Ｅｘｐ．　Ｔｈｅｒ．　１９８４；　２２８（１）：　１６８−１７３，Ｎａｋａｉ　Ｔ．　ｅｔ　ａｌ．，　Ｊ．　Ｐｈａｒｍａｃｏｌ．　Ｅｘｐ．　Ｔｈｅｒ．　１９８１；　２１６（３９）：　６０７−６１２，Ａｎｇｅｌ　Ｉ．　ｅｔ　ａｌ．，　Ｊ．　Ｐｈａｒｍａｃｏｌ．　Ｅｘｐ．　Ｔｈｅｒ．　１９８８；　２４６（３）：１０９８−１１０３，　Ａｎｇｅｌ　Ｉ．　ａｎｄ　Ｌａｎｇｅｒ　ＳＺ．，　Ｅｕｒ．　Ｊ．　Ｐｈａｒｍａｃｏｌ．　１９８８；　１５４（２）：１９１−１９６）。従って、ＨＮＤマウス系統で認めたアドレナリン作用の増強が、α_２−アドレナリンレセプターを介した作用によるものであるかどうかの検討を行うことにした。そこで、α_２−アドレナリンレセプターの選択的なアゴニストであるクロニジンを用いて、グルコース（２０ｍＭ）刺激インスリン分泌に与える抑制効果を両マウス間で比較検討した。その結果、１ｎＭクロニジンを添加した場合、Ｂ６マウスの膵島に比較して、ＨＮＤマウス系統ではインスリン分泌量が減少している傾向が示された（図１２）。また、１０ｎＭ、クロニジン添加時には有意な減少が確認された。これらの結果から、ＨＮＤマウス系統で認められたアドレナリンによるインスリン分泌抑制効果の増強がα_２−アドレナリンレセプターを介する作用によるものであることが示唆された。
実施例４：マウス個体におけるα_２−アドレナリンレセプター作用の検討
実施例２の結果から、ＨＮＤマウス系統の耐糖能異常および高血糖がインスリン分泌障害に起因することが確認され、実施例３の結果から、インスリン分泌障害が膵Ｂ細胞のα_２−アドレナリンレセプターを介すものであることが推測された。そこで、マウス個体におけるα_２−アドレナリンレセプター作用と耐糖能異常との関係を検討した。
（１）　　方法
（１−１）　　クロニジン投与実験：血糖測定
１０−１４週齢あるいは４０週齢のマウスを用いて、絶食時あるいは非絶食時において実験を行った。まず、体重測定後６μｌの血液を尾静脈より採血した。そのすぐ後に、生理食塩水で調製したクロニジン溶液（クロニジン塩酸塩、和光純薬工業株式会社）をクロニジンの投与量がマウスの体重１ｋｇあたり、５μｇあるいは５０μｇとなるように腹腔内へ注射し（１００μｌ／１０ｇ体重）、クロニジン投与後３０分、６０分、１２０分で６μｌの血液を尾静脈より採取した。採取した血液はグルコース濃度の測定までは氷中で保存し、微量遠心機（トミー精工株式会社製　ＮＲＸ−１５０）で１５，０００ｒｐｍ、１０分間遠心して得られた血清１．５μｌを用いて、グルコース濃度を測定した。（１−２）　　ヨヒンビン投与実験：血糖測定
１１−１６週齢マウスを約１４時間絶食させ、体重を測定した後、６μｌの血液を尾静脈より採血した。そのすぐ後に、生理食塩水または生理食塩水で調製したヨヒンビン溶液（ヨヒンビン塩酸塩、ＳＩＧＭＡ）を腹腔内へ注射した（１００μｌ／１０ｇ体重）。ヨヒンビン投与後３０分で６μｌの血液を尾静脈から採取した後、ＩＰＧＴＴを行った。ＩＰＧＴＴは、グルコースの投与量がマウスの体重１Ｋｇあたり２ｇとなるように２０％のグルコース溶液を（１００μｌ／１０ｇ体重）腹腔内へ注射
し、グルコース投与後３０分、６０分、１２０分で６μｌの血液を尾静脈より採取した。採取した血液はグルコース濃度の測定までは氷中で保存し、微量遠心機（トミー精工株式会社製　ＮＲＸ−１５０）で１５，０００ｒｐｍ、１０分間遠心して得られた血清１．５μｌを用いて、グルコース渡度を測定した。
（１−３）　　ヨヒンビン投与実験：インスリン測定
１１−１６週齢マウスを約１４時間絶食させ、体重を測定した後、生理食塩水または生理食塩水で調製したヨヒンビン溶液（ヨヒンビン塩酸塩、ＳＩＧＭＡ）を腹腔内へ注射した（１００μｇ／１０ｇ体重）。ヨヒンビン投与後３０分で１６０μｌの血液を眼窩静脈から採取した。その後すぐに、グルコースの投与量がマウスの体重１Ｋｇあたり２ｇとなるように２０％のグルコース溶液を（１００μ
ｌ／１０ｇ体重）腹腔内へ注射し、グルコース投与後３０分で１６０μｌの血液を眼窩静脈より採取した。前記と同様に遠心して血清を得て、測定時まで−２０℃で保存した。血中インスリン濃度の測定は、ラジオイムノアッセイ（ＳｈｉｏｎｏＲＩＡ，塩野義製薬株式会社）を用いて行った。
（２）　　結果
（２−１）　　クロニジン投与実験の結果
絶食時あるいは非絶食時条件下において、クロニジン投与による血糖値の変化に着目して検討した。絶食時条件下において５μｇ／ｋｇ　ＢＷのクロニジンを投与した時、Ｂ６マウスでは血糖値の上昇を認めなかったのに対して、ＨＮＤマウス系統では約１２０ｍｇ／ｄｌの大きな上昇を認めた（図１３）。また、非絶食時条件下においては、Ｂ６が５０ｍｇ／ｄｌ程度の血糖値の上昇であったのに比べて、ＨＮＤマウス系統では約２２０ｍｇ／ｄｌ程度と明らかに大きな血糖値の上昇を認めた（図１４）。また、絶食時条件下において、より多量の５０μｇ／ｋｇ　ＢＷのクロニジンを投与した時も同様に、ＨＮＤマウス系統はＢ６よりも有意に大きな血糖値の上昇を認めた（図１５）。しかし、非絶食時条件下においてはこのクロニジンの投与量ではＢ６においても血糖の上昇の程度は大きく、Ｂ６とＨＮＤマウス系統で差を認めなかった（図１６）。また、５と５０μｇ／ｋｇＢＷの両濃度による血糖変化を比較したところ、Ｂ６ではクロニジンの投与量依存的に血糖値の上昇が明らかに大きくなったのに対して、ＨＮＤマウス系統では５ｍｇ／ｋｇ　ＢＷのクロニジン投与の場合でも大きな血糖上昇が観察され、５０ｍｇ／ｋｇ　ＢＷの投与を行ってもさらなる血糖値の上昇が見られなかった。すなわち、ＨＮＤマウス系統では少量のクロニジンの投与に対しても血糖値の上昇は十分におこることが示された（図１３−１６）。
（２−２）　　ヨヒンビン投与実験の結果
Ｂ６を用いた予備的な検討により、ヨヒンビンは投与量依存的に耐糖能を改善させる傾向が示された（図１７）。そして、１００μｇ／ｋｇ　ＢＷ投与では、Ｂ６においては耐糖能改善作用は見られない（図１７）。そこで、この投与量においてＢ６とＨＮＤマウス系統との間で、ヨヒンビンが耐糖能に与える影響について比較検討を行うことにした。
【００８３】
Ｂ６マウスでは、ヨヒンビン（１００μｇ／ｋｇ　ＢＷ）投与群と生理食塩水投与群においてグルコース投与後の血糖値の推移に違いは認められなかった（図１８−ａ）。一方、ＨＮＤマウス系統のヨヒンビン（１００μｇ／ｋｇ　ＢＷ）投与群では生理食塩水投与群に比較して、グルコース投与後６０、１２０分の血糖値が低下していた（図１８−ｂ）。この結果は、ＨＮＤマウス系統では、α_２−アドレナリンレセプターの阻害によって血糖の低下作用が増強したことを示している。
【００８４】
次いで、このようなヨヒンビンによる耐糖能の改善が、インスリン分泌の改善によるものかどうかを検討した。その結果、ＨＮＤマウス系統の生理食塩水投与群では、実施例２での結果同様にグルコース投与後の血中インスリン濃度の上昇が認められず、インスリン分泌障害が観察きれた（図１９−ｂ）。しかし、ヨヒンビン投与群では明確な血中インスリン濃度の上昇が確認された（図１９−ｂ）。これ警告　７らの結果から、ヨヒンビン投与によるＨＮＤマウス系統の耐糖能異常の改善は、インスリン分泌障害を改善したことによるものであることが示唆された。つまり、ＨＮＤマウス系統のインスリン分泌障害にはα_２−アドレナリンレセプター作用の増強が関与していることが示された。
【００８５】
【発明の効果】
以上詳しく説明したとおり、この出願の発明によって、日本人に多い非肥満型の２型糖尿病に極めて類似した糖尿病モデルであり、神経因性のインスリン分泌制御の障害を特徴とするマウス（ＨＮＤマウス）が提供される。これによって、２型糖尿病の病態解析や遺伝子解析が一層促進される。また、この出願の発明によって、２型糖尿病の治療薬の高精度でのスクリーニングが可能となる。さらに、このモデルマウスは７週齢前後の若齢から糖尿病を発症し、生体への長期の高血糖の作用を検出しうるという利点を有している。
【図面の簡単な説明】
【図１】（ａ）は、Ｂ６マウス（白丸）とＨＮＤマウス系統（黒四角）の体重変化を示したグラフであり、（ｂ）はＢ６マウス（白丸）とＨＮＤマウス系統（黒四角）の肥満度の変化を示したグラフである。個体数は５〜１５。数値は平均値±ＳＥＭ。＊はｕｎｐａｉｒｅｄ　ｔテスト（以下、ｔ　テストと略す）によるＢ６マウスとの差がｐ＜０．００１水準で有意であることを示す。
【図２】（ａ）は６週齢のＢ６マウス（白丸：ｎ＝９）とＨＮＤマウス系統（黒四角：ｎ＝２４）に対する耐糖能試験による血中グルコース量の変化を示したグラフである。（ｂ）は１２週齢のＢ６マウス（白丸：ｎ＝１４）とＨＮＤマウス系統（黒四角：ｎ＝２０）に対する耐糖能試験による血中グルコース量の変化を示したグラフである。（ｃ）は、２０週齢のＢ６マウス（白丸：ｎ＝１０）とＨＮＤマウス系統（黒四角：ｎ＝２４）に対する耐糖能試験による血中グルコース量の変化を示したグラフである。（ｄ）は３０週齢のＢ６マウス（白丸：ｎ＝１２）とＨＮＤマウス系統（黒四角：ｎ＝３０）に対する耐糖能試験による血中グルコース量の変化を示したグラフである。数値は平均値±ＳＥＭ。＊および＊＊はそれぞれ、ｔテストによるＢ６マウスとの差がｐ＜０．０１およびｐ＜０．００１水準で有意であることを示す。
【図３】（ａ）は６−３０週齢のＢ６マウス（ｎ＝５−１５）とＨＮＤマウス系統（ｎ＝１６−４２）の耐糖能試験におけるＡＵＣを示したグラフである。数値は平均値±ＳＥＭ。＊は　ｔテストによるＢ６マウスとの差がｐ＜０．０１水準で有意であることを示す。
【図４】（ａ）は６−４０週齢のＢ６マウス（ｎ＝５−１２）およびのＨＮＤマウス系統（ｎ＝１６−４２）の非絶食時の血中グルコース量である。（ｂ）は同じく絶食時の血中グルコース量である。数値は平均値±ＳＥＭ。＊、＊＊、＊＊＊はそれぞれ、ｔテストによるＢ６マウスとの差がｐ＜０．０５、ｐ＜０．０１およびｐ＜０．００１水準で有意であることを示す。
【図５】（ａ）は６週齢のＢ６マウス（白バー：ｎ＝５−１２）とＨＮＤマウス系統（グレーバー：ｎ＝１６−４２）の非絶食（Ｎｏｎ−ｆａｓｔｉｎｇ）および絶食時（Ｆａｓｔｉｎｇ）における血中インスリン濃度を示したグラフである。（ｂ）は２３週齢マウス、（ｃ）は４０週齢マウスの同様の結果である。数値は平均値±ＳＥＭ。統計処理はｔテストによって行った。
【図６】（ａ）は７週齢のＢ６マウス（白丸）とＨＮＤマウス系統（黒四角）に対する耐糖能試験中のグルコース刺激によるインスリン分泌量の変化を示したグラフである。（ｂ）は２３週齢のＢ６マウス（白丸）とＨＮＤマウス系統（黒四角および黒三角）の同様のインスリン分泌量の変化を示したグラフである。ＨＮＤマウス系統は２０週齢時における血糖値の２００ｍｇ／ｄｌより多いか少ないかで糖尿病群（黒四角）および非糖尿病群（黒三角）に分けた。数値は６−８個体の測定の平均値±ＳＥＭ。＊および＊＊はそれぞれ、ｔテストによるＢ６マウスとの差がｐ＜０．０５およびｐ＜０．００１水準で有意であることを示す。
【図７】（ａ）は７週齢のＢ６マウス（白丸）とＨＮＤマウス系統（黒四角）に対するインスリン負荷試験による血中グルコース量の変化を示したグラフである。（ｂ）は２０週齢マウス、（ｃ）は４０週齢マウスの同様の結果を示したグラフである。数値は平均値±ＳＥＭ。＊は、ｔテストによるＢ６マウスとの差がｐ＜０．０１水準で有意であることを示す。
【図８】（ａ）は７週齢のＢ６マウス（白バー：ｎ＝７）とＨＮＤマウス系統（グレーバー：ｎ＝５−８）の非絶食（Ｎｏｎ−ｆａｓｔｉｎｇ）および絶食時（Ｆａｓｔｉｎｇ）における血清ＮＥＦＡ量を示したグラフである。（ｂ）は血清ＴＧ量、（ｃ）は血清ＴＣ量を同様に示したグラフである。平均値±ＳＥＭ。＊は、ｔテストによるＢ６マウスとの差がｐ＜０．０５水準で有意であることを示す。
【図９】６週齢のＢ６マウス（白丸）とＨＮＤマウス系統（黒四角）のそれぞれから単離した膵島のインスリン分泌に対するグルコースの投与量依存効果を示したグラフである。数値は３−６検体の測定の平均値±ＳＥＭ。統計処理はｔテストによって行った。
【図１０】６週齢のＢ６マウス（白バー）とＨＮＤマウス系統（黒バー）のそれぞれから単離した膵島のグルコース刺激インスリン分泌量に対するＧＬＰ−１、ＧＩＰまたはグルカゴンの効果を示したグラフである。数値は３−６検体の測定の平均値±ＳＥＭ。統計処理はｔテストによって行った。
【図１１】（ａ）は２０−３０週齢のＢ６マウス（白丸）とＨＮＤマウス系統（黒四角）のそれぞれから単離した膵島のグルコース刺激インスリン分泌量に対するソマトスタチンの投与量依存効果を示したグラフである。（ｂ）はアドレナリンの同様の効果を示したグラフである。数値は、（ａ）は４−７検体、（ｂ）は５−２０検体の測定の平均値±ＳＥＭ。＊は、ｔテストによるＢ６マウスとの差がｐ＜０．０５水準で有意であることを示す。
【図１２】２０−３０週齢のＢ６マウス（白バー）とＨＮＤマウス系統（黒バー）のそれぞれから単離した膵島のグルコース刺激インスリン分泌量にα２−アドレナリンレセプターのアゴニストであるクロニジンの効果を示したグラフである。数値は４−５検体の測定の平均値±ＳＥＭ。＊は、ｔテストによるＢ６マウスとの差がｐ＜０．０５水準で有意であることを示す。
【図１３】（ａ）は１０−１４週齢のＢ６マウス（白丸：ｎ＝６）とＨＮＤマウス系統（黒四角：ｎ＝８）の絶食時血糖値に対するクロニジン（５μｇ／ｋｇ　ＢＷ）の効果の経時的変化を示したグラフである。（ｂ）は同様の効果の時間依存性変化を示したグラフである。数値は平均値±ＳＥＭ。＊および＊＊はそれぞれ、ｔテストによるＢ６マウスとの差がｐ＜０．０１およびｐ＜０．００１水準で有意であることを示す。
【図１４】（ａ）は１０−１４週齢のＢ６マウス（白丸：ｎ＝４）とＨＮＤマウス系統（黒四角：ｎ＝８）の非絶食時血糖値に対するクロニジン（５μｇ／ｋｇ　ＢＷ）の効果の経時的変化を示したグラフである。（ｂ）は同様の効果の時間依存性変化を示したグラフである。数値は平均値±ＳＥＭ。＊、＊＊および＊＊＊はそれぞれ、ｔテストによるＢ６マウスとの差がｐ＜０．０５、ｐ＜０．０１およびｐ＜０．００１水準で有意であることを示す。
【図１５】（ａ）は１０−１４週齢のＢ６マウス（白丸：ｎ＝８）とＨＮＤマウス系統（黒四角：ｎ＝１２の絶食時血糖値に対するクロニジン（５０μｇ／ｋｇ　ＢＷ）の効果の経時的変化を示したグラフである。（ｂ）は同様の効果の時間依存性変化を示したグラフである。数値は平均値±ＳＥＭ。＊および＊＊はそれぞれ、ｔテストによるＢ６マウスとの差がｐ＜０．０１およびｐ＜０．００１水準で有意であることを示す。
【図１６】（ａ）は１０−１４週齢のＢ６マウス（白丸：ｎ＝４）とＨＮＤマウス系統（黒四角：ｎ＝８）の非絶食時血糖値に対するクロニジン（５０μｇ／ｋｇ　ＢＷ）の効果の経時的変化を示したグラフである。（ｂ）は同様の効果の時間依存性変化を示したグラフである。数値は平均値±ＳＥＭ。＊はｔテストによるＢ６マウスとの差がｐ＜０．００１水準で有意であることを示す。
【図１７】２５−５０週齢のＢ６マウスの耐糖能試験における生理食塩水（ｓａｌｉｎｅ）またはヨヒンビンの投与量依存効果を示したグラフである。数値は３−４検体の測定の平均値±ＳＥＭ。＊は、ｔテストによるＢ６マウスとの差がｐ＜０．０５水準で有意であることを示す。
【図１８】（ａ）は１１−１６週齢のＢ６マウス（ｎ＝８）の耐糖能試験における生理食塩水（白丸）またはヨヒンビン（黒四角：１００μｇ／ｋｇ　ＢＷ）の投与量依存効果を示したグラフである。（ｂ）はＨＮＤマウス系統（ｎ＝１０）における同様の効果を示したグラフである。（ｃ）はＡＵＣの結果であり、白バーは生理食塩水、グレーバーはヨヒンビン、左側がＢ６マウス、右側がＨＮＤマウス系統である。数値は平均値±ＳＥＭ。＊および＊＊はそれぞれ、ｔテストによるＢ６マウスとの差がｐ＜０．０５およびｐ＜０．００１水準で有意であることを示す。
【図１９】（ａ）は１１−１６週齢のＢ６マウス（ｎ＝３−４）の耐糖能試験中のインスリン分泌量に対する、生理食塩水（白丸）またはヨヒンビン（黒四角：１００μｇ／ｋｇ　ＢＷ）の効果を示したグラフである。（ｂ）はＨＮＤマウス系統（ｎ＝３−４）における同様の効果を示したグラフである。（ｃ）は試験中の初期３０分間の血中インスリン濃度の変化であり、白バーは生理食塩水、グレーバーはヨヒンビン、左側がＢ６マウス、右側がＨＮＤマウス系統である。数値は平均値±ＳＥＭ。＊および＊＊はそれぞれ、ｔテストによるＢ６マウスとの差がｐ＜０．０５およびｐ＜０．０１水準で有意であることを示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The invention of this application relates to a diabetic model mouse, a method for producing the same, and an invention utilizing the model mouse. More specifically, the invention of this application relates to a novel model mouse that spontaneously develops type 2 diabetes that frequently occurs in Japanese people, a method for producing the model mouse, and a diabetes treatment using the model mouse or its tissues and cells. The present invention relates to a drug screening method.
[0002]
[Prior art]
Diabetes is a national illness that amounts to 13.7 million people, including 6.9 million patients and a pre-diabetes group who cannot rule out the possibility of developing diabetes. Diabetes is a disease that presents with chronic hyperglycemia and causes various complications.Many of them are less likely to cause symptoms such as "silent disease", and are often associated with vocalism, polydipsia, polyuria, and polyphagia. When symptoms appear, the condition is advanced and may already be accompanied by serious complications. And it is a disease that often has an arteriosclerotic disease in addition to typical complications such as diabetic neuropathy, diabetic nephropathy, and diabetic retinopathy. Diabetes with these dangerous complications is thought to be triggered by a decrease in insulin supply from pancreatic β cells or insulin resistance in insulin-sensitive tissues. In other words, diabetes is characterized by a decrease in insulin supply and insulin resistance, resulting in abnormal metabolism (increased blood sugar) and the onset of complications.
[0003]
As mentioned above, diabetes is caused by insufficient insulin action caused by two mechanisms of decreased insulin supply and insulin resistance, and the causes are various. Diabetes is classified according to its etiology as (1) type 1, (2) type 2, (3) due to other specific mechanisms or diseases, and (4) gestational diabetes mellitus (Katsuya Ken et al., Diabetes # 1999; # 42) (5): 385-404). (1) is defined as diabetes caused by insulin deficiency in a destructive lesion of pancreatic β-cells, which are insulin-secreting cells, and since autoantibodies to pancreatic islet antigens are often observed, It is recognized as a disease mainly involving an autoimmune mechanism. In other words, it is an autoimmune disease that causes "absolute" insulin deficiency. On the other hand, (2) is a type in which the two of insulin secretion decrease and insulin resistance in the insulin-sensitive tissue coexist at various ratios, and are characterized by a "relative" deficiency of insulin action. (3) (A) Genetic abnormalities identified as genetic factors (such as the insulin gene itself, gene abnormalities related to pancreatic β-cell function such as MODY, and abnormalities of the insulin receptor gene) Genetic abnormalities related to the insulin action transmission mechanism) and (B) Other diseases and conditions (pancreatic exocrine diseases, endocrine diseases, infectious diseases) are included, and are classified and distinguished from type 1 and type 2 diabetes ing. In addition, (4) refers to abnormalities in glucose metabolism that developed during pregnancy or were discovered for the first time, and are pathogenically based on a common pathogenesis mechanism with other types of diabetes. As described above, diabetes is classified into four types according to causes, and most of them are characterized by being susceptible to a genetic predisposition.
[0004]
Type 2 diabetes accounts for the majority of all diabetic patients and is a complex multifactorial disease that develops and progresses with a number of environmental factors such as overeating, diet, stress, and lack of exercise in addition to genetic predisposition. Recent studies on the analysis of both insulin secretion deficiency and insulin resistance, which are the causes of type 2 diabetes, have shown the molecular mechanism of glucose-dependent insulin secretion in pancreatic β-cells and intracellular information following receptors in insulin-sensitive cells. The outline of the transmission route was clarified. Further detailed mechanisms have been energetically analyzed, and not only studies in vitro and at the cell level, but also knockout (KO) mice and transgenic (Tg) created by technological advances in developmental engineering techniques. Animal models such as mice have also contributed significantly.
[0005]
For example, as a KO mouse of a molecule related to insulin biosynthesis and secretion, pancreatic β-cell type GK (glucokinase) KO mouse (Terauchi Y. et al., J. Biol. Chem. 1995; 44 (12): 1447-1457, Aizawa T. et al., Biochem. Biophys. Res. Commun. 1996; 229 (2): 460-465) and Kir6.2 (ATP sensitive K channel) KO mouse (Miki T. et al., Proc. Natl. Acad. \ Sci. \ USA. \ 1998; $ 95 (18): 10402-10406), indicating that these molecules play an important role in insulin secretion. In addition, IPF-1 {(insulin-promoter-factor1; also referred to as PDX-1)} KO mouse (Jonson J. et al., Nature 1994; 371 (6494): 606-609) shows that IPF-1 forms pancreas. Is also important.
[0006]
In addition, as KO mice relating to insulin action, insulin receptor (insulin receptor: IR) @KO mice (Accili @ RL. @ Et @ al, @ Nat. @ Getet. @ 1996; @ 12 (1): @ 106-109), skeletal muscle, pancreatic β cells, Liver and adipose tissue-specific IR @ KO mice (Wojtaszewski@J.etal, @ J. @ Clin. @ Invest. @ 1999; 104 (9): 1257-64, @Ueki Kojiro, Medical History 2000; @ 192 (5): 374-380) Insulin receptor substrate (IRS) -1,2,3 KO mouse (Tamemoto H. et al., Nature 1994; 372 (6504): 82-186, Kubota N. et al., Diabetes. 2000; 49 (11): 1880-1889, Liu SC. Et al., J. Biol. Chem. 1999; 274 (259): 18093-18099) Specific GLUT4 KO mice (Katz EB. Et al., Nature 1995; 377 (6545): 151-155, Kim JK et al., J. Clin. Invest. 2001; 108 (1): 153- 160, Abel ED. Et al., Nature 2001; 409 (6821): ２９729-733) and many other KO mice have been produced, with only the role of each protein relating to the insulin action. Rather, many findings have been obtained in considering the pathogenesis and pathophysiology of diabetes, such as organ-specific analysis of insulin resistance and the mechanism of pancreatic β-cell proliferation in the presence of insulin resistance.
[0007]
Applied research on animals developed using these developmental engineering techniques not only helps to elucidate the molecular mechanisms of insulin secretion and action, but also causes the burden of multifactorial diseases due to the load of several genetic and environmental factors. It is expected to be able to elucidate the pathology of type 2 diabetes. It is an indispensable research field because it is expected to be applied not only to elucidation of the onset and pathophysiology of diabetes but also to the development of new therapeutic methods and therapeutics including gene therapy / regenerative medicine.
[0008]
However, the contribution of rapidly increasing genetically modified diabetic animals is extremely small in terms of identifying unknown diabetes responsible genes. This is because the majority of genetically modified diabetic animal models are made with known molecules that are known to be important in the development of diabetes. Genes that have been discussed in the creation of genetically modified models account for only a few percent of all diabetes. Identification of unknown causative genes is an urgent task for elucidating the full picture of type 2 diabetes involving many genetic factors.
[0009]
An approach to identify novel causal genes in patients with type 2 diabetes includes a “candidate gene approach,” which analyzes genes that may be functionally involved in the disease, and uses random markers in sibling-affected families. There is a "random marker approach" that screens the entire genome to identify the location of genes involved in the disease. In fact, some promising genes have been identified in such genetic analysis (Froguel P. et al., N. Engl. J. Med. 1993; 328, 697-702 Hanis CL. Et al., Nat. @ Genet. @ 1996; 13 (2): @ 161-166, @ Horikawa @ Y. @ et.al., @ Nat. @ Genet. @ 2000; @ 26 (2): @ 163-175, @ Nishigori @ H. @ et.al., @ Proc. @ Natl. @ Acad. Sci. @ USA.
2001; $ 98 (2): $ 575-580).
[0010]
It is not surprising that most type 2 diabetes in humans is caused by complex effects of genetic or other factors. However, the probability of identifying each diabetic factor from diabetic cases or animals with heterogeneous genetic background is expected to be very low. Because the effects of common diabetic factors alone are very small, they are likely to mask their diabetic effects in genetically heterogeneous systems, and on the other hand, many unknown genes This is because no method has been established for analyzing the effects of genomics using a genetically heterogeneous system. Therefore, analysis using a spontaneously diabetic animal model is a powerful tool for identifying unknown causative genes. In particular, inbred model animals can play a significant role in understanding the pathophysiology of diabetes, searching for causative genes, and developing therapeutic drugs for diabetes, etc., because they can make the genetic background and external environment uniform.
[0011]
Several spontaneous type 2 diabetes model animals have been developed so far. For example, ob / ob mice and db / db mice have both been found at the Jackson Laboratory in the United States and have been widely used as models for hereditary obesity. These mice have reduced energy expenditure in addition to increased energy intake due to overeating, and have phenotypes such as hyperglycemia, hyperinsulinemia, insulin resistance, and increased weight of white adipocytes. Then, in 1994, the ob / ob responsible gene was identified by positional cloning and named leptin (Zhang Y. et al., Nature 1994; 372 (6505): 425-432). And in 1995, the leptin receptor gene was cloned and revealed to be the responsible gene for db / db mice (Tartaglia LA. Et al., Cell 1995; 83 (7): 1263-1271). At present, these mice are widely used models for the study of diabetes and obesity, including the physiological and pharmacological functions of leptin.
[0012]
GK rats (Kimura K. et al., Tohoku J. Exp. Med. 1982; 137 (4): 453- 459) are among the few non-obese type 2 diabetes models that have impaired insulin secretion and insulin resistance. It has a phenotype often seen in Japanese type 2 diabetes patients. This model has been used for research such as mapping of disease susceptibility genes and analysis of the pathology of insulin secretion deficiency.
[0013]
OLETF rats (Kawano K. et al., Diabetes 1992; 41 (11): 1422-1428), ZDF rats (Zucker LM et al., Ann. NY Acad. Sci. 1965; 131: 447) -458) is a model of type 2 diabetes exhibiting obesity and insulin resistance. Since all of them show a decrease in the amount of pancreatic β cells, they are also used in studies on the vulnerability of pancreatic β cells in the development of type 2 diabetes (Kenji Shima, Advances in Molecular Diabetes 1999; 76-82) ). In addition, Wistar fatty rats (Ikeda H. et al., Diabetes 1981; 30 (12): 1045-1050), NSY mice (Ueda H. et al., Diabetologia 1995; 38 (5): 503-508) And Kita mice (Yoshioka M. et al., Diabetes 1997; 46 (5): 887-894). These spontaneous model animals have been used for a wide range of purposes, including analysis of the pathology of type 2 diabetes, search for causative genes, therapeutic methods, and development of therapeutic drugs, and have provided effective information for clinical diabetics. .
[0014]
[Problems to be solved by the invention]
As described above, a large number of type 2 diabetes model animals have been developed so far. However, since type 2 diabetes is an overly complex multifactorial disease, the pathological condition explained by analysis using existing model animals is only a small part of type 2 diabetes. Therefore, in recent years, research on chromosome mapping of disease susceptibility genes has been actively conducted, and the development of a unique new type 2 diabetes model animal is expected to assist in the discovery of a new causative gene. It may help to understand the complex pathophysiology of diabetes.
[0015]
The invention of this application was made in view of the above circumstances, and a new diabetes model mouse spontaneously developing a disease state similar to non-obese type 2 diabetes, which is common in Japanese, The task is to provide a production method.
[0016]
Another object of the invention of the present application is to provide a method for screening a drug component using the new diabetes model mouse and its tissue and cells.
[0017]
[Means for Solving the Problems]
The inventors of the present application have conducted research based on the following working hypothesis to solve the above-mentioned problem, and completed the present invention.
[0018]
In part, type 2 diabetes model animals that have been developed to date have been phylogenetic based on phenotypic abnormalities (eg, obesity and impaired insulin secretion) that were accidentally found in experimental strains. There are many things. However, in order to create a faithful model system for each type of type 2 diabetes that is a complex multifactorial disease, it is necessary to identify individuals that can be model animals using a phenotype that is suited to each disease state as a selection index. It is necessary to fix the phenotype by crossing them. In addition, as a diabetes model animal created by performing selective mating based on a specific index as described above, a GK rat (Goto @ Y., Produced by selective mating of Wistar rats using a glucose tolerance test as a selective index). et al., Proc. Jap. Acad., 1975; 51: 80-85), and NSY mice (Ueda, H. et al.) produced by selective crossing from outbred Lcl: ICR mice using the glucose tolerance test as an index. , Diabetologia $ 1995; $ 38 (5): $ 503-508).
[0019]
The second is the diversification of selection indices. In particular, in order to create a non-obese type 2 diabetes model, which is common in Japanese, it is necessary to carry out selective mating in addition to body weight and eating and drinking behavior as indices.
[0020]
Third, conventional inbred mouse models are based on experimental inbred mice. Such inbred mice have some of the important genes involved in the spontaneous onset of type 2 diabetes in the process of their production. There is a high risk of losing. Therefore, introduction of various phenotypes of wild-type mice is extremely effective in creating a model animal that mimics the spontaneous onset of human type 2 diabetes.
[0021]
Fourth, a major factor, sensitivity to adrenaline, a major factor in the control of neuronal insulin secretion, was added to the selection index. In considering the cause of the recent rapid increase in type 2 diabetes, it is not possible to ignore the effects of neural factors such as increased stress in the modern society on the endocrine metabolic system. For this reason, it is indispensable to create a model mouse that can not only impair insulin secretion or non-obesity but also relate abnormal neuronal insulin secretion control to its pathological condition.
[0022]
This application has been completed based on the working hypothesis described above, and provides the following invention as a solution to the above-mentioned problem.
[0023]
The first invention is a progeny mouse of a wild castaneus mouse and an inbred C57BL / 6J mouse, having the following characteristics:
(A) show hyperglycemia and / or impaired glucose tolerance when compared to normal strain mice;
(B) significantly lower body weight and / or obesity as compared to normal strain mice;
(C) Endogenous adrenaline is α2-adrenergic when compared to normal strain mice
The effect of suppressing insulin secretion via putter is remarkably strong,
A diabetes model mouse characterized by having:
[0024]
The second invention is one of the preferable embodiments of the first invention, and is a diabetes model mouse in which the offspring mouse is the nth generation (n is 1 or more) of sibling mating.
[0025]
A third invention is a diabetes model mouse created by crossing the diabetes model mouse of the first invention with another diabetes model mouse.
[0026]
A fourth invention is the tissue of the above-mentioned diabetes model mouse.
[0027]
A fifth invention is a cell of the above-mentioned diabetes model mouse.
[0028]
A sixth invention is a fertilized egg of the aforementioned diabetes model mouse.
[0029]
The seventh invention includes the following steps:
(1) creating a selected basal population consisting of offspring mice obtained by crossing a plurality of wild castaneus mice with inbred C57BL / 6J mice;
(2) From the mice of the selected basic population, the following criteria:
(A) show hyperglycemia and / or impaired glucose tolerance when compared to normal strain mice;
(B) significantly lower body weight and / or obesity as compared to normal strain mice;
Selecting from the first-generation sibling mating mice obtained by mating sibling mice of individuals matching the criteria (a) and (b),
And a method for producing a diabetes model mouse.
[0030]
According to an eighth aspect, in addition to the steps (1) and (2) of the seventh aspect, the following steps are further included:
(3) selecting individuals matching the criteria (a) and (b) from the second generation siblings obtained by mating the siblings of the first generation siblings selected in the step (2); and
(4) repeating the above step (3) to obtain a sibling crossed nth generation (n is 3 or more) mouse meeting criteria (a) and (b);
A method for producing a diabetes model mouse comprising:
[0031]
The ninth invention includes the following steps:
(1) creating a selected basal population consisting of offspring mice obtained by crossing a plurality of wild castaneus mice with inbred C57BL / 6J mice;
(2) From the mice of the selected basic population, the following criteria:
(C) blood glucose levels are significantly increased after administration of α2-adrenergic receptor agonists when compared to normal strain mice;
(D) Abnormal glucose tolerance is normal after administration of α2-adrenergic receptor antagonist
Selecting from the first generation mice mated with siblings obtained by mating sibling mice of individuals matching at least one of the following criteria: (c) or (d). This is a method for producing a diabetic model mouse.
[0032]
According to a tenth aspect, in addition to the steps (1) and (2) of the ninth aspect, the following steps are further included:
(3) From the second generation mice mated with siblings of the first generation offspring mice selected in step (2), an individual matching at least one of the criteria (c) and (d) is obtained. Selecting; and
(4) Repeating the step (3) to obtain a sibling mating nth generation (n is 3 or more) mouse meeting at least one of the criteria (c) and (d). Is the way.
[0033]
The eleventh invention includes the following steps:
(1) creating a selected basal population consisting of offspring mice obtained by crossing a plurality of wild castaneus mice with inbred C57BL / 6J mice;
(2) From the mice of the selected basic population, the following criteria:
(A) show hyperglycemia and / or impaired glucose tolerance when compared to normal strain mice;
(B) significantly lower body weight and / or obesity as compared to normal strain mice;
(C) blood glucose levels are significantly increased after administration of α2-adrenergic receptor agonists when compared to normal strain mice;
(D) Abnormal glucose tolerance is normal after administration of α2-adrenergic receptor antagonist
(A) and (c); (a) and (d); (b) and (c); (b) and (d); (a), (b), and (c). (A), (b) and (d); or from a first-generation sibling mouse obtained by mating sibling mice matching (a), (b), (c) and (d), Selecting individuals that meet similar criteria,
And a method for producing a diabetes model mouse.
[0034]
According to a twelfth invention, in addition to the steps (1) and (2) of the eleventh invention, the following steps are further included:
(3) Reference (a) and (c); (a) and (d) from the sibling mated second generation mouse obtained by mating the sibling mated first generation mouse selected in step (2). (B) and (c); (b) and (d); (a), (b) and (c); (a), (b) and (d); or (a), (b), ( selecting individuals that meet c) and (d); and
(4) Repeating step (3) above, the criteria (a) and (c); (a) and (d); (b) and (c); (b) and (d); (B) and (c); (a), (b) and (d); or n-th generation of sibling crosses matching (a), (b), (c) and (d) (n is 3 or more) Getting a mouse,
A method for producing a diabetes model mouse comprising:
[0035]
A thirteenth invention is a method for screening a therapeutic agent for diabetes, comprising administering a candidate substance to the diabetic model mouse of the first, second, or third invention, and identifying a substance that improves the blood glucose level of the model mouse. It is a method characterized by doing.
[0036]
A fourteenth invention is a method for screening a therapeutic agent for diabetes, comprising administering a candidate substance to the diabetes model mouse of the first, second or third invention, and then administering an agonist of an α2-adrenergic receptor, This is a method characterized by identifying a substance that suppresses an increase in blood glucose level after administration.
[0037]
A fifteenth invention is a method for screening a therapeutic agent for diabetes, which comprises administering a candidate substance to the diabetic model mouse of the first, second or third invention, and administering an antagonist of α2-adrenergic receptor to the model mouse. The method is characterized by specifying a substance which has an improving effect at the same level as the abnormal glucose tolerance improving effect after the above.
[0038]
A sixteenth invention is a method for screening a therapeutic agent for diabetes, which comprises causing a candidate substance to act on pancreatic tissue or pancreatic β cells of the diabetes model mouse according to the first, second, or third invention, and then causing adrenaline or α2- This is a method characterized by causing an agonist of an adrenergic receptor to act, and specifying a substance that suppresses a decrease in the amount of insulin secretion after administration of the adrenaline or the agonist.
[0039]
A seventeenth invention is a method for screening a therapeutic agent for diabetes, which comprises causing a candidate substance to act on pancreatic tissue or pancreatic β cells of the diabetes model mouse of the first, second, or third invention in the presence of adrenaline. This is a method characterized by specifying a substance which causes an increase in insulin secretion level equivalent to an increase in insulin secretion level after an α2-adrenergic receptor antagonist acts on pancreatic tissue or pancreatic β cells.
[0040]
Hereinafter, embodiments of these inventions will be described in detail.
[0041]
BEST MODE FOR CARRYING OUT THE INVENTION
The diabetes model mouse of the first invention is a progeny mouse of a wild castaneus mouse and an inbred C57BL / 6J mouse, and has the following characteristics.
(A) Δ shows impaired glucose tolerance when compared to normal mice.
(B) 体重 Significantly lower body weight and obesity as compared to normal mice.
(C) Endogenous adrenaline has a stronger effect of suppressing insulin secretion via α2-adrenergic receptor than normal mice.
[0042]
The diabetes model mouse of the second invention is an nth generation (n is 1 or more) of mating siblings having the characteristics of the mouse of the first invention.
[0043]
In the following description, this diabetes model mouse may be described as an HND mouse (Horio-Niki \ Diabetic \ Mouse).
[0044]
The HND mouse of this application can be produced by the method of the seventh to twelfth inventions.
[0045]
The method of the seventh invention is a method for producing the HND mouse of the first invention, and includes the following steps (1) and (2) as essential.
Step (1):
A selection basal group consisting of offspring mice obtained by crossing a plurality of wild Castaneus mice with inbred C57BL / 6J mice is created.
Step (2):
From the mice of the selected basic population, the following criteria:
(A) show hyperglycemia and / or impaired glucose tolerance when compared to normal strain mice;
(B) From the first generation mice mated with siblings obtained by mating sibling mice of an individual that has significantly lower body weight and / or obesity degree compared to normal strain mice, the above criteria (a) and ( Individuals matching b) are selected.
[0046]
In step (1), “Wild Castaneus mice” (hereinafter, referred to as “Cas mice”) are mice distributed in Southeast Asia, and can capture and use mice that live in those areas. “C57BL / 6J mouse” (hereinafter referred to as “B6 mouse”) is a common inbred strain mouse derived from Mus @ musculus @ domesticus, and has a well-established genetic background and is continuously passaged. It is possible. This B6 mouse is one of the mice used as a control strain in the analysis of existing diabetes model mice because glucose tolerance, blood glucose level, etc. are in the normal range under normal diet. The B6 mice are readily available from experimental animal suppliers.
[0047]
When these mice are bred to obtain offspring mice of the selected basal group, either may be male or female, but it is preferable to breed the male Cas mouse and the female B6 mouse in that the subsequent breeding will obtain progeny. As the selection basic population in step (1), a progeny mouse obtained by crossing a Cas mouse and a B6 mouse can be used as it is, but a progeny mouse population obtained by the following method is preferably used. That is, a male Cas mouse and a female B6 mouse are bred, and the female offspring mouse is backcrossed to the Cas mouse. Then, the blood sugar level of the backcross generation at the time of fasting was measured, and a male with high blood sugar (specifically, a serum glucose level of 130 to 140 mg / dl or more) was selected. And the resulting offspring mice are used as a selected basal group (F0).
[0048]
The "blood glucose level", which is a selection index for the backcross generation, can be measured, for example, using a commercially available kit based on the glucose oxidase method (Glucose B Test Wako, Wako Pure Chemical Industries, Ltd.). That is, 250 μl of an enzyme solution is added to 1.5 μl of mouse serum, and the mixture is incubated at 37 ° C. for 20 minutes. Then, the absorbance at 505 nm is measured, and the glucose concentration in the serum can be determined from the absorbance of the standard solution. Further, “at fasting” means a state after fasting for 12 to 14 hours (in contrast, “non-fasting” means a state of free eating except for about 1 hour fasting).
[0049]
In step (2), a mouse individual that satisfies both the criteria (a) and (b) is selected from the mouse group of the selected basic population. In addition, these criteria can be performed at an arbitrary age of a mouse individual, but preferably at any one or two or more of the age of 6 weeks, 12 weeks, 20 weeks, and 40 weeks. To
[0050]
In the criterion (a), the “normal strain mouse” to be compared can be a mouse that does not show abnormalities in blood glucose level or insulin secretion ability without exception, but preferably is one strain of the cross. Yes, B6 mice used as a control strain in the analysis of existing diabetes model mice are used. As described above, the criterion for “hyperglycemia” may be a glucose level in serum during fasting of 130 to 140 mg / dl or more, preferably 175 mg / dl or more, and most preferably 200 mg / dl or more. . In addition, “abnormal glucose tolerance” refers to an individual having a glucose tolerance test (see Examples below) and a blood glucose level of 140 mg / dl or more, preferably 175 mg / dl or more, 120 minutes after glucose (2 g / KgBW) administration.
Is determined to be impaired glucose tolerance, most preferably an individual having a serum glucose level of 200 mg / dl or more based on the standard method for determining diabetes (Ueda et al., Diabetologia 1995; 38 (5): 503-508). Is judged to be glucose intolerance.
[0051]
In the criterion (b), the “normal strain mouse” can be the same as the criterion (a). The “body weight” can be measured, for example, by anesthetizing a mouse with ether and using a usual measuring instrument. Further, the obesity degree ((BMI: Body @ mass @ index)) is obtained by measuring the length from the tip of the nose to the anus (Anal-nasal @ length = height) using calipers after measuring the weight, and using the following formula.
[0052]
BMI (g / cm²) = Body @ weight (g) / (Anal-nasal @ length)²(Cm)²
Furthermore, “significantly lower” in the weight and / or obesity level means that the difference between a normal mouse and a progeny mouse for each value is compared with a normal significant difference test (for example, a t test). It means that there is a difference of 5% or less, preferably 1% or less, and most preferably 0.1% or less. Hereinafter, the expression “significantly” or “statistically significant” means a difference by the significance test as described above.
[0053]
By the above steps (1) and (2), a non-obese, hyperglycemic diabetic model mouse (first generation of sibling mating: F1) can be obtained. Preferably, in addition to the above steps (1) and (2), The following steps are repeated to produce an inbred model mouse (8th invention).
Step (3):
Individuals matching the criteria (a) and (b) are selected from the second generation mice (F2) obtained by mating the first generation sibling mice selected in step (2).
Step (4):
The above step (3) is repeated to obtain n-th generation (n is 3 or more) mice (Fn) of mating siblings meeting the criteria (a) and (b).
[0054]
In the present invention, the “inbred model mouse” means a progeny mouse of the third generation or more, preferably the 15th generation or more, most preferably the 20th generation or more of sibling mating. Mice of the 20th generation or more of sibling crosses have almost completely matched genetic backgrounds, and all offspring mice satisfy the above criteria (a) and (b).
[0055]
The HND mouse of the first invention can also be produced by the methods of the ninth and tenth inventions. That is, the method of the ninth invention is characterized in that the method essentially includes the same step (1) as the seventh invention and the following step (2).
Step (2):
From the mice of the selected basic population, the following criteria:
(C) agonists of α2-adrenergic receptor when compared to normal strain mice
Post-dose blood glucose levels increase significantly;
(D) Abnormal glucose tolerance is normal after administration of α2-adrenergic receptor antagonist
Become
From the first generation mice obtained by mating sibling mice of individuals matching at least one of the above, individuals matching the criteria (c) or (d) are selected.
[0056]
The criterion (c) of this step (2) is to administer an α2-adrenergic receptor agonist (for example, clonidine) at the time of fasting or non-fasting, and measure the blood glucose level 30 to 120 minutes later. Then, when the blood sugar level is statistically significantly increased as compared with the blood sugar level of a control mouse (such as a B6 mouse) that has undergone the same treatment, it is determined that the criterion (c) is satisfied. The criterion (d) is that an α2-adrenergic receptor antagonist (for example, yohimbine) is administered, the blood glucose level is measured 30 to 120 minutes later, and the blood glucose level is statistically significantly higher than that of the control (administration of physiological saline). It is determined that the case where the temperature has decreased meets the criterion (c).
[0057]
The offspring mouse (first generation sibling mating mouse) selected according to the above criteria (c) and / or (d) is described as follows: "In comparison with normal strain mice, the endogenous adrenaline secretes insulin via α2-adrenergic receptor. Are significantly stronger in suppressing the effect of insulin "(characteristic (C) of the diabetic model mouse of the first invention), which further suppresses insulin secretion, resulting in hyperglycemia and impaired glucose tolerance (characteristic (A) Mouse). In addition, this mouse is a mouse that strongly suppresses insulin secretion by an adrenergic action, which is one of the neural factors, and exhibits hyperglycemia without obesity (characteristic (B)). In the control (c), “significantly strong action” is defined as an operational definition as described above, in which the degree of increase or decrease in blood glucose level when an agonist or antagonist of α2-adrenergic receptor is administered is compared with that of a normal strain mouse. Means statistically significant.
[0058]
Then, sibling mice meeting the criteria (a) and (b) are bred from the first-generation sibling mating mice meeting the criteria (c) and / or (d) to obtain sibling second-generation mice, and furthermore, By repeating the selective breeding of the above, sibling mating (inbred) nth generation mice can be obtained (10th invention).
[0059]
Furthermore, the diabetes model mouse of the first invention can be produced by the method of the eleventh invention and the twelfth invention. That is, the method of the eleventh invention is characterized by including the same step (1) as the seventh invention and the ninth invention and the following step (2) as essential.
Step (2):
From the mice of the selected basic population, the following criteria:
(A) show hyperglycemia and / or impaired glucose tolerance when compared to normal strain mice;
(B) significantly lower body weight and / or obesity as compared to normal strain mice;
(C) blood glucose levels are significantly increased after administration of α2-adrenergic receptor agonists when compared to normal strain mice;
(D) Abnormal glucose tolerance is normal after administration of α2-adrenergic receptor antagonist
(A) and (c); (a) and (d); (b) and (c); (b) and (d); (a), (b), and (c). (A), (b) and (d); or from a first-generation sibling mouse obtained by mating sibling mice matching (a), (b), (c) and (d), Individuals meeting similar criteria are selected.
[0060]
Criteria (a) and (b) are the same as in the seventh aspect, and criteria (c) and (d) are the same as in the ninth aspect. Then, preferably, males and females of individuals meeting the criteria (a), (b), (c) and / or (d) are bred to obtain first-generation brother-sister mated mice. The diabetic model mouse of the first invention can be produced by selecting individual mice. Further, inbred model mice can be obtained by repeating the same selective breeding from progeny mice meeting the criteria (a), (b), (c) and / or (d) (twelfth invention).
[0061]
The HND mouse of the first invention or the second invention created by the above method can be further bred to another diabetes model mouse (third invention). That is, the HND mice of the first and second inventions are (A) hyperglycemic and impaired glucose tolerance, (B) non-obese, and (C) neuropathic insulin secretion abnormality. As a characteristic, by crossing this HND mouse with another diabetic model mouse (for example, a mouse showing hyperglycemia due to other factors), more severe impaired glucose tolerance and hyperglycemia develop, and complicated 2 A model mouse similar to the pathology of type diabetes can be obtained. In this case, as the “other diabetes model mouse”, a strain that develops diabetes with a single gene is preferable because the HND mouse is presumed to be involved in a plurality of diabetes-related genes. For example, among the spontaneously-occurring model mice, Ay (yellow obse) mice having insulin resistance (insulin dysfunction), ob / ob mice, Tubby mice, Fat mice, and NSY mice having deficiency in insulin secretion are preferable. Furthermore, KO (knockout) mice and TG (transgenic), which show insulin resistance and insulin secretion deficiency created by developmental engineering techniques, can be mentioned.
[0062]
The diabetes model mouse of the third invention obtained by such further crossing may be the F0 generation thereof, or may be the aforementioned criteria (a), (b), (c) and / or (d), or Inbreeding may be repeated by selecting and mating based on other criteria.
[0063]
The diabetes model mice of the first to third inventions as described above can be used for the invention of the screening methods of the thirteenth to fifteenth inventions, in addition to the pathological analysis and gene analysis of type 2 diabetes.
[0064]
A fourth invention is the tissue of the diabetes model mouse according to the first to third inventions. The “tissue” in this case is a tissue having a change (such as a change in the amount of insulin or adrenergic sensitivity) related to the pathological condition of diabetes, and means, for example, an organ, an organ, a body fluid, a DNA, a protein, or the like of a mouse. . For example, the organ is the pancreas, and the body fluid is blood or urine. These tissues can be used, for example, in the invention of the screening methods of the fourteenth and fifteenth inventions.
[0065]
A fifth invention is the cell of the diabetes model mouse according to the first to third inventions. The “cell” in this case is a cell that expresses a mutation associated with the pathology of diabetes, such as a pancreatic B cell. Such cells can also be used for the invention of the screening methods of the fourteenth and fifteenth inventions.
[0066]
A sixth invention is a fertilized egg of the diabetes model mouse of the first to third inventions. This fertilized egg is useful for stably storing and supplying the diabetes model mouse of the present invention.
[0067]
The thirteenth to fifteenth inventions are a method of screening for a component of a therapeutic drug for diabetes using the diabetes model mouse of the first to third inventions. In the method of the thirteenth invention, specifically, a candidate substance is administered to a model mouse, and a substance that improves the diabetic symptoms (eg, blood glucose level, insulin secretion amount, body weight, etc.) of the model mouse is selected. “Candidate substances” include unknown and known organic or inorganic compounds, proteins, peptides, polynucleotides, oligonucleotides, and the like. In addition, “administration” may be a method of introducing a candidate substance into a mouse body by injection, or a method in which the candidate substance is mixed with food or drinking water to be taken. In the case of a polynucleotide or an oligonucleotide (a DNA fragment or an RNA fragment), a method of gene therapy using an adenovirus or the like may be employed.
[0068]
Specifically, the method of the fourteenth invention is a method of administering a candidate substance to a diabetic model mouse, and then administering an agonist (eg, clonidine) of an α2-adrenergic receptor, and suppressing an increase in blood glucose level after administration of the agonist. It is a method of specifying. The method according to the fifteenth aspect of the present invention provides a method of administering a candidate substance to a diabetic model mouse, and reducing the blood glucose level to about the same level as the decrease in blood glucose level after administering an α2-adrenergic receptor antagonist (eg, yohimbine) to the model mouse. This is a method of identifying candidate substances to be brought. The determination of the increase or suppression of the blood sugar level in these cases can be performed in the same manner as the determination of the reference (c) in step (2) of the ninth aspect. These screening methods identify substances that have a therapeutic effect on insulin deficiency, especially under stress.
[0069]
The method of the sixteenth and seventeenth inventions is a method of screening for a component substance of a therapeutic agent for diabetes using the pancreatic tissue or pancreatic B cells of the diabetes model mouse. That is, an adrenaline or α2-adrenergic receptor agonist (for example, clonidine) is allowed to act on pancreatic tissue or cells (16th invention), or an α2-adrenergic receptor antagonist (for example, yohimbine) is caused to act on the pancreatic tissue or cells to cause insulin secretion of the tissue or cell. The target substance is specified using the change in the amount as an index. The amount of insulin secretion can be measured according to the method described in the examples below. To “act” an agonist, antagonist, or candidate substance on a tissue or cell means that the substance or substance is manipulated to cause some change in the tissue or cell (for example, a change in blood glucose level). For example, this means contacting a substance with a tissue or cell, physically introducing a substance into a tissue or cell, and the like.
[0070]
Hereinafter, the invention of this application will be described in more detail and specifically with reference to examples, but the invention of this application is not limited to the following examples.
[0071]
【Example】
Example 1: Production of a diabetic model mouse and inbreeding thereof
The diabetes model mouse of the present invention was produced and inbred.
(1) Materials and methods
(1-1) Mouse and breeding conditions
The Cas mouse used was one that was captured by Dr. Takao Namikawa (Nagoya University Faculty of Agriculture, Department of Animal Genetics Control) in 1994 near Manila, Philippines. C57BL6 / J (B6) mice were purchased from SLC Japan. All mice were bred in an environment with a temperature of 22 ± 2 ° C., a humidity of 50 ± 5%, a light-dark cycle of 12L12D (8:00 to 20:00), solid feed (Northan Lab MR breeder, Nippon Agricultural Industry Co., Ltd.) and They had free access to water. The state of fasting for 12-14 hours (fasting start time: 8:00 pm) was defined as fasting, and the state of fasting for about 1 hour from 9:00 am was defined as non-fasting.
(1-2) Measurement of blood sugar level
The measurement was performed using a commercially available kit based on the glucose oxidase method (Glucose B Test Wako, Wako Pure Chemical Industries, Ltd.). After adding 250 μl of the enzyme solution to 1.5 μl of the serum and incubating at 37 ° C. for 20 minutes, the absorbance at 505 nm was measured, and the glucose concentration in the serum was determined from the absorbance of the standard solution.
(1-3) Sugar tolerance test (IPGTT: Intraperitoneal glucose tolerance test)
The mice were fasted for about 14 hours, weighed, and 6 μl of blood was collected from the tail vein. Immediately thereafter, a 20% glucose solution was injected intraperitoneally (100 μl / 10 g body weight) so that the dose of glucose was 2 g per kg of mouse body weight, and 6 μl of glucose solution was administered 30 minutes, 60 minutes, and 120 minutes after glucose administration. Blood was collected from the tail vein. The collected blood was stored on ice until the glucose concentration was measured, and glucose was collected using 1.5 μl of serum obtained by centrifugation at 15,000 rpm for 10 minutes in a microcentrifuge (NRX-150 manufactured by Tommy Seiko Co., Ltd.). The concentration was measured.
[0072]
Judgment of glucose intolerance is based on a standard method for judging diabetes (Ueda et al., Diabetologia 1995; $ 38 (5): 503-508), and the blood glucose level at 200 minutes after administration of glucose (2 g / KgBW) is 200 mg / dl. Glucose tolerance of the above individuals
It was determined to be abnormal.
(1-4) Measurement of body weight and obesity level
After the mice were anesthetized with ether and weighed, the length from the tip of the nose to the anus (Anal-nasal length) was measured using calipers. The BMI was determined by the following equation.
[0073]
BMI (g / cm²) = Body @ weight (g) / (Anal-nasal @ length)²(Cm)²
(2) Mating and sorting
Male Cas mice and female B6 mice were crossed to obtain offspring mice. The females of the offspring mice were backcrossed to Cas mice, and blood glucose levels during fasting were measured for the resulting backcross generations (about 1000), and males having a value of 130 to 140 mg / dl or more were selected. The selected males were again bred to female B6 mice, and the obtained progeny mice were used as a selection base group (F0).
[0074]
Next, in the F0 group, individuals showing hyperglycemia at the time of feeding (non-fasting) were crossed with each other to obtain offspring mice. The progeny mice were subjected to a glucose tolerance test, and siblings were mated using abnormal glucose tolerance as a selection index to obtain first-generation siblings. Furthermore, in addition to impaired glucose tolerance, selective mating was repeated using the body weight and the degree of obesity as indices to create an inbred diabetes model mouse strain (HND mouse strain) up to the 17th generation of sibling mating. In this HND mouse strain, theoretically, 62.5% of the whole genome is considered to be derived from B6 mouse.
Example 2: Analysis of diabetes traits in mouse individuals
Diabetic traits were examined for the inbred HND mouse strain (12th generation of sibling mating) created in Example 1.
(1) Method
(1-1) @AUC (Area \ under \ the \ curve)
It is the area of a line graph in which the time of 0, 30, 60, and 120 minutes in the glucose tolerance test is plotted on the horizontal axis, and the blood glucose level at each time is plotted on the vertical axis, and was calculated by the following formula. In order to reflect all four glucose levels in the glucose tolerance test, it was used as a comprehensive evaluation of glucose tolerance.
It is assumed that the 0-minute blood sugar level = a, the 30-minute blood sugar level = b, the 60-minute blood sugar level = c, and the 120-minute blood sugar level = d.
[0075]
AUC (mg / dl × min) = 15a + 30b + 45c + 30d
(1-2) Measurement of insulin concentration
Approximately 160 μl of blood was collected from the orbital vein under non-anesthesia at the time of fasting and non-fasting, and serum was obtained by centrifugation in the same manner as in the glucose tolerance test of Example 1, and stored at −20 ° C. until measurement. Serum immunoreactive insulin (IRI) levels were measured using a radioimmunoassay (ShionoRIA, Shionogi & Co., Ltd.). 50 μl of serum¹²⁵After adding 50 μl of the insulin solution labeled with I, adding 50 μl of the insulin antiserum solution and incubating at 25 ° C. for 2 hours, adding 500 μl of the secondary antibody suspension and performing immunoprecipitation for 30 minutes at 25 ° C. Incubated. After centrifugation at 2,000 g for 10 minutes, the supernatant was removed with an aspirator, and the radioactivity remaining in the precipitate was measured with an Autowell gamma counter (Aloka ARC-380). The insulin concentration in the sample was determined using rat insulin as a standard. Insulin antibody¹²⁵Competitive reaction between I-insulin and insulin in sample binds to insulin antibody¹²⁵The amount of I-insulin decreases according to the amount of unlabeled insulin in the serum collected from the mouse. That is, the insulin concentration in the sample can be measured by measuring the radioactivity of the insulin-insulin antibody conjugate precipitated by the addition of the secondary antibody.
(1-3) Insulin secretion ability test
The mice were fasted for about 14 hours, weighed, and then 50 μl of blood was collected from the orbital vein under non-anesthesia. Immediately after that, the glucose dose was adjusted to 20 g so that the dose was 2 g per 1 kg of mouse body weight.
Was injected intraperitoneally (100 μl / 10 g body weight), and 50 μl of blood was collected from the orbital vein 10 minutes and 30 minutes after glucose administration. Then, serum was obtained by centrifugation in the same manner as described above, and stored at −20 ° C. until measurement. The insulin concentration was measured using an insulin measurement kit (Morinaga Science Laboratory Co., Ltd.) using 20 μl of serum.
(1-4) Insulin tolerance test (ITT: insulin tolerance test)
The mice were fasted for about 14 hours, weighed, and then 8 μl of blood was collected from the tail vein. A solution of human insulin (Humulin R, Shionogi & Co., Ltd.) was intraperitoneally injected so that the dose of insulin was 0.5 U / kg of mouse body weight, and 8 μl at 15, 30, and 60 minutes after insulin administration. Was collected from the tail vein. The collected blood was stored on ice until the glucose concentration was measured, and serum was collected by centrifugation in the same manner as described above, and the glucose concentration was measured using 2 μl of the serum.
(1-5) Analysis of blood lipid components
At the time of fasting at 7 weeks of age and at the time of non-fasting, neutral fat, free fatty acid, total fatty acid, and total fat were obtained using -20 ° C-preserved serum obtained by collecting blood from the orbital vein of mice under non-anesthesia as described above. The concentration of cholesterol was determined using a commercially available kit based on the enzymatic method.
[0076]
Neutral fat was measured using Triglyceride E Test Wako (Wako Pure Chemical Industries, Ltd.). After adding 2 μl of serum to 200 μl of the enzyme solution and incubating at 37 ° C. for 5 minutes, the absorbance at 600 nm was measured, and the neutral fat concentration in the serum sample was determined from the absorbance of the standard solution.
[0077]
Free fatty acids were measured using NEFAzyme-S (Eiken Chemical Co., Ltd.). After adding 5 μl of serum to 200 μl of the enzyme solution and incubating at 37 ° C. for 10 minutes, the absorbance at 555 nm was measured, and the free fatty acid concentration in the serum sample was determined from the absorbance of the standard solution.
[0078]
Cholesterol E test Wako (Wako Pure Chemical Industries, Ltd.) was used for the measurement of total cholesterol. After adding 2 μl of serum to 200 μl of the enzyme solution and incubating at 37 ° C. for 5 minutes, the absorbance at 600 nm was measured, and the total cholesterol concentration in the serum sample was determined from the absorbance of the standard solution.
(1-6) Other methods
The measurement of the degree of obesity, the blood glucose level, and the glucose tolerance test were performed in the same manner as in Example 1.
(2) Result
(2-1) Weight, obesity level
Individuals of the HND mouse strain showed significantly lower values in both body weight and obesity than the control strain B6 mouse at any of the age of 6 to 40 weeks (FIG. 1). Although it is difficult to determine obesity and non-obesity, the HND mouse strain was determined to be a non-obese strain. B6 showed an increase in body weight up to the age of 40 weeks, whereas individuals of the HND mouse strain did not show an increase in body weight from the age of 20 weeks to the age of 40 weeks. Indicated.
(2-2) Glucose tolerance brain test
As a result of the glucose tolerance test at the age of 6 weeks, the incidence of abnormal glucose tolerance of the HND mouse strain was as high as 74% despite the young age (Table 1). In addition, the HND mouse strain showed a higher value than B6 at all times except for the value 30 minutes after the glucose load (FIG. 2 (a)). Among them, the 120-minute value serving as a criterion for determination of impaired glucose tolerance was significantly higher at 287 ± 19 mg / dl in the HND mouse mouse strain compared to 122 ± 3 mg / dl of B6 mice, indicating a value in the diabetic area. I was Also, a large difference was observed for AUC (FIG. 4). As described above, in the HND mouse strain, a clear and high rate of glucose intolerance was already observed at the age of 6 weeks. Blood glucose after glucose loading was higher than that of B6 even at 12, 20, and 30 weeks of age, and a significant difference was also observed in AUC (FIGS. 2 (b)-(d), FIG. 4). The incidence of impaired glucose tolerance observed in the HND mouse strain was the highest at 80 weeks at 12 weeks of age, 63% at 20 weeks of age, and 43% at 30 weeks of age.
[0079]
[Table 1]

[0080]
(2-3) Blood sugar level
The blood glucose level of the HND mouse strain at the age of 6 weeks was non-
It was 358 ± 31 mg / dl during fasting and 131 ± 5 mg / dl during fasting, which was significantly higher than B6 (non-fasting: 176 ± 6, fasting: 89 ± 3) (FIG. 4). This is a value that clearly indicates the diabetic area.
(2-4) Blood insulin concentration
At 7, 23, and 40 weeks of age, the blood insulin concentration of the HND mouse strain did not differ from that of the B6 mouse during both non-fasting and fasting (FIG. 5).
(2-5) Glucose-stimulated insulin secretion test
When blood insulin levels were measured using mice at the age of 7 weeks under the same conditions as in the glucose tolerance test, an increase in blood insulin levels after glucose loading was observed in B6. In contrast, no increase was observed in the HND mouse strain (FIG. 6 (a)). That is, in the HND mouse strain, a clear impairment was observed in glucose-stimulated insulin secretion. In addition, when the mice of the HND mouse strain at the age of 20 weeks were examined by dividing the IPGTT 120 minute value at a boundary of 200 mg / dl, those showing high values into the diabets group and those showing low values from the non-diabetes group, No increase in blood insulin concentration was observed in the diabetes group, and an increase was observed in the non-diabetes group (FIG. 6 (b)). From this, impairment of glucose-stimulated insulin secretion ability was considered to be the main cause of impaired glucose tolerance in the HND mouse strain.
(2-6) Insulin tolerance test
At 7, 20, and 40 weeks of age, when an insulin tolerance test was performed, there was no difference in blood glucose reduction after insulin loading between the B6 mouse and the HND mouse strain (FIG. 7). Therefore, it was considered that the insulin action in the peripheral tissues of the HND mouse strain was not different from that of the B6 mouse.
(2-7) Blood lipid components
At 7 weeks of age, blood lipid components (free fatty acids, neutral fats, total cholesterol) during fasting and non-fasting were analyzed.
Free fatty acids:
Upon fasting, no difference was observed between B6 (1675 ± 187 μEq / l) and the HND mouse strain (1692 ± 166 μEq / l). However, the HND mouse strain (1071 ± 82 μEq / l) showed a significantly higher value than B6 (790 ± 62 μEq / l) at the time of non-fasting (FIG. 8 (a)).
Neutral fat:
At the time of fasting, no difference was observed between the B6 mouse and the HND mouse strain. However, at the time of non-fasting, a higher value was observed in the HND mouse strain (214 ± 27 mg / dl) than in the B6 mouse (149 ± 11 mg / dl) (FIG. 8 (b)).
Total cholesterol:
At the time of fasting, the HND mouse strain showed a slightly lower value of 105 ± 4 mg / dl compared to B6 (116 ± 1 mg / dl). At the time of non-fasting, no difference was observed between both mice (FIG. 8 (c)).
Example 3: Analysis of insulin secretion ability in isolated pancreatic islets
As shown in Example 2, the HND mouse strain created in Example 1 had normal insulin action in peripheral tissues. Thus, the pancreatic islets of Langerhans (pancreatic islets), which are insulin secreting tissues, were isolated, and the amount of insulin secreted was analyzed.
(1) Method
(1-1) Isolation of pancreatic islets from pancreas
(1-1-1) Reagent
Krebs-Ringer Solution:
Final concentrations, each of 119 mM NaCl, 4.75 mM KCl, 5 mM NaHC0₃, 2.54 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM @ KH₂PO₄, 20 mM @ HEPES, prepared with distilled water, and adjusted to pH 7.4 with NaOH to obtain a stock @ solution. If necessary, on the day of the experiment, 3 mM glucose, 5 mg / ml @BSA was added to obtain working @solution.
Collagenase @ solution:
Collagenase type XI (SIGMA) was dissolved in Krebs-Ringer solution (3 mM glucose) to a concentration of 4 mg / mL.
(1-1-2) Isolation of pancreatic islets by collagenase digestion
The mouse pancreas was digested by the action of collagenase to separate islets from surrounding exocrine cells. That is, mice under non-fasting conditions were anesthetized with diethyl ether, and sacrificed by bleeding after laparotomy. Thereafter, the collagen was injected into the pancreas little by little using a 27G injection needle so that the collagenase solution spread evenly throughout the pancreas. After the pancreas was excised carefully so as not to damage it, it was put in a bag together with the remaining collagenase solution and digested at 37 ° C. for 3 minutes (total of 2.5 ml of the one used for injection and the one put in the bag). Collagenase solution was used). The tissue after the reaction was transferred to a 50 mL tube, stirred with a vortex mixer, and then transferred to a glass test tube. Pancreatic islets are a large cell population and sediment in a solution, and thus were used to separate them from exocrine cells. Specifically, the digested tissue was suspended in Krebs-Ringer Solution (3 mM glucose) and allowed to stand for 2 minutes. Thereafter, a solution of about 4 cm from the bottom (the portion where the islets had settled) was left, and the solution above was replaced again with fresh Krebs-Ringer solution (3 mM glucose). The same operation was repeated twice. As a result, exocrine cells were removed as much as possible, and the remaining lower layer was transferred to a glass Petri dish. Then, the islets were picked up using an autopipettor under a stereoscopic microscope, and transferred to a glass petri dish containing Krebs-Ringer Solution (3 mM glucose, 5 mg / ml BSA). Through this series of operations, pancreatic islets can be separated from exocrine cells.
(1-2) Method of insulin secretion experiment using pancreatic islets
(1-2-1) Batch Incubation Method
The isolated islets were collected on a glass Petri dish containing Krebs-Ringer Solution (3 mM glucose, 5 mg / ml BSA), and the size and shape of the islets were selected under a stereoscopic microscope and used for the experiment. The four islets were matched under a stereoscopic microscope so that the size of the pancreatic islets used did not become uneven. Next, 0.6 ml of Krebs-Ringer solution (3 mM glucose, 5 mg / ml BSA) was injected into a small test tube (IWAKI), and islets were added so that the number of the test tubes became 4 per test tube. Was. After preincubation for 1 hour in a 37 ° C. incubator, the solution was removed as much as possible. Then, various reagents (glucose, GLP-1, GIP, glucagon, adrenaline, somatostatin, clonidine) were prepared with Krebs-Ringer @ solution (5 mg / ml @ BSA), and 0.6 ml of this was added as incubation @ medium. For 1 hour at 37 ° C. while shaking lightly. Immediately after completion, the supernatant was collected, and the insulin concentration was measured using a radioimmunoassay (ShionoRIA, Shionogi & Co., Ltd.).
(1-2-2) Glucose-stimulated insulin secretion test
Pancreatic islets isolated from 6 week old mice were used for the experiments. After preincubation for 1 hour as described above, the solution was replaced with 0.6 ml incubation medium containing 3,10,15,20,40 mM glucose and incubated for 1 hour. Then, the insulin concentration of the solution was measured.
(1-2-3) Examination of insulin secretion promoting effect of GLP-1, GIP and glucagon
Pancreatic islets isolated from 6 week old mice were used for the experiments. As described above, after 1 hour of pre-incubation, the solution was added to 15 mM glucose, replaced with 0.6 ml incubation medium containing 100 nM GLP-1, GIP, glucagon (all SIGMA) and incubated for 1 hour. . Then, the insulin concentration of the solution was measured. (1-2-4) Examination of the inhibitory effect of somatostatin, adrenaline and clonidine on insulin secretion
Pancreatic islets isolated from 20-30 week old mice were used for the experiments. After preincubation for 1 hour as described above, the solution was added to 20 mM glucose and 1,10,100 nM somatostain-28 (SIGMA), 0.3,1,3,10,30 nM adrenaline ([- ] -Adrenerline (+) @ bitarterate (SIGMA) or 0.6 ml of incubation medium containing 1,10 nM clonidine (clonidine hydrochloride, Wako Pure Chemical Industries, Ltd.) and incubated for 1 hour. Then, the insulin concentration of the solution was measured. (2) Result
(2-1) Glucose-stimulated insulin secretory ability
Isolation of pancreatic islets from 6-week-old mice and measurement of glucose-stimulated 1-hour insulin secretion by the batch incubation method revealed that the B6 and HND mouse strains showed no difference at any glucose concentration of 3-40 mM. No difference was observed (FIG. 9). These results suggested that the pancreatic B cells of the HND mouse strain had normal insulin secretion ability in response to glucose at least at the isolated pancreatic islet level.
(2-2) Influence of GLP-1, GIP and glucagon on insulin secretion
GLP-1, GIP, and glucagon are hormones that function to promote insulin secretion by glucose stimulation. Therefore, the effects of these hormones in the presence of a high concentration of glucose (15 mM) were examined. As a result, when any hormone was added, no clear difference was observed in the amount of insulin secreted between B6 and the HND mouse strain (FIG. 10). Therefore, it was suggested that the action of these insulin secretion-promoting hormones was kept normal in the HND mouse strain and did not cause the apparent insulin secretion disorder observed in vivo.
(2-3) Somatostatin and concentration-dependent effects of adrenaline on insulin secretion
Somatostatin and adrenaline are hormones that function to suppress glucose-stimulated insulin secretion. Therefore, the concentration-dependent effects of these hormones in the presence of a high concentration of glucose (20 mM) were examined.
[0081]
In the case of somatostatin, in B6 mice, about 10% of secretion was suppressed by adding 1 nM, about 35% was suppressed by adding 10 nM, and about 50% was suppressed by adding 10 nM. The inhibitory action of somatostatin at each of these concentrations on insulin secretion was observed in HND mice as well as in B6 mice (FIG. 11-a). That is, it was presumed that there was no difference in the effect of somatostatin on insulin secretion between the B6 and HND mouse strains.
[0082]
On the other hand, when the effect of epinephrine on insulin secretion in a concentration-dependent manner was examined, in B6 mice, insulin secretion was suppressed by about 10% at 0.3 nM addition, about 15% at 1 nM, about 40% at 3 nM, and about 60% at 10 nM. Was observed. In the HND mouse strain, the addition of 1 nM and 3 nM of adrenaline significantly inhibited insulin secretion more than in the B6 mouse. Showed significantly lower insulin secretion than B6 mice, and a decreasing tendency was observed even at 3 nM (FIG. 11-b). These results indicate that the islet of the HND mouse strain has a stronger suppression of insulin secretion by adrenaline than the B6 mouse. Then, it was suggested that adrenergic action may be involved in the factor of impaired insulin secretion of the HND mouse strain observed in vivo.
(2-4) Δα₂Effect of Adrenergic Receptor Agonist on Insulin Secretion Adrenaline suppresses insulin secretion, which is caused by α-expression expressed in pancreatic B cells.₂-It is known to be caused by an action via an adrenergic receptor (DiTullio NW. Et al., J. Pharmacol. Exp. Ther. 1984; 228 (1): 168-173, Nakai T. et al., J. Pharmacol. Exp. Ther. 1981; 216 (39): 607-612, Angel I. et al., J. Pharmacol. Exp. Ther. 1988; 246 (3): 1098-1103, Angel Ian. SZ., Eur. J. Pharmacol.ｏｌ1988; 154 (2): 191-196). Therefore, the enhancement of adrenergic action observed in the HND mouse strain was due to αα₂-To determine whether it is due to action via the adrenergic receptor. Then, α₂-Using clonidine, which is a selective agonist of the adrenergic receptor, the inhibitory effect on glucose (20 mM) -stimulated insulin secretion was compared between the two mice. As a result, when 1 nM clonidine was added, there was a tendency that the amount of insulin secretion was decreased in the HND mouse strain as compared to the islets of the B6 mouse (FIG. 12). In addition, a significant decrease was confirmed when 10 nM and clonidine were added. From these results, it was found that the enhancement of the insulin secretion inhibitory effect by adrenaline observed in the HND mouse strain was α₂-It was suggested that this was due to an action via an adrenergic receptor.
Example 4: α in mouse individuals₂-Study of adrenergic receptor action
The results of Example 2 confirmed that the impaired glucose tolerance and hyperglycemia of the HND mouse strain were caused by impaired insulin secretion. From the results of Example 3, the impaired insulin secretion was caused by α-pancreatic B-cell α.₂-It was speculated that it is through an adrenergic receptor. Therefore, α₂-The relationship between adrenergic receptor action and impaired glucose tolerance was examined.
(1) Method
(1-1) Clonidine administration experiment: blood glucose measurement
Experiments were performed using fasted or non-fasted mice using 10-14 week old or 40 week old mice. First, after measuring the body weight, 6 μl of blood was collected from the tail vein. Immediately thereafter, a clonidine solution (clonidine hydrochloride, Wako Pure Chemical Industries, Ltd.) prepared in physiological saline was injected intraperitoneally (100 μl) such that the dose of clonidine was 5 μg or 50 μg per 1 kg of mouse body weight. / 10 g body weight), and 6 μl of blood was collected from the tail vein at 30, 60, and 120 minutes after clonidine administration. The collected blood was stored on ice until the glucose concentration was measured, and the blood was centrifuged at 15,000 rpm for 10 minutes in a microcentrifuge (NRM-150, manufactured by Tommy Seiko Co., Ltd.), and 1.5 μl of the serum was used to obtain glucose. The concentration was measured. (1-2) Yohimbine administration experiment: blood glucose measurement
The 11-16 week old mice were fasted for about 14 hours, weighed, and then 6 μl of blood was collected from the tail vein. Immediately thereafter, saline or a yohimbine solution prepared with saline (yohimbine hydrochloride, SIGMA) was injected intraperitoneally (100 μl / 10 g body weight). Thirty minutes after the administration of yohimbine, 6 μl of blood was collected from the tail vein, and then IPGTT was performed. IPGTT is intraperitoneally injected with a 20% glucose solution (100 μl / 10 g body weight) such that the dose of glucose is 2 g per 1 kg body weight of mice.
Then, 30 minutes, 60 minutes, and 120 minutes after glucose administration, 6 μl of blood was collected from the tail vein. The collected blood was stored on ice until the glucose concentration was measured, and the blood was centrifuged at 15,000 rpm for 10 minutes in a microcentrifuge (NRM-150, manufactured by Tommy Seiko Co., Ltd.), and 1.5 μl of the serum was used to obtain glucose. Handing was measured.
(1-3) Yohimbine administration experiment: insulin measurement
11-16 week old mice were fasted for about 14 hours, weighed, and then injected intraperitoneally with saline or yohimbine solution (yohimbine hydrochloride, SIGMA) prepared with saline (100 μg / 10 g body weight). . Thirty minutes after the administration of yohimbine, 160 μl of blood was collected from the orbital vein. Immediately thereafter, a 20% glucose solution was added (100 μl) so that the dose of glucose was 2 g per kg of mouse body weight.
(1/10 g body weight). Intraperitoneally, and 30 minutes after glucose administration, 160 μl of blood was collected from the orbital vein. Serum was obtained by centrifugation as above and stored at -20 ° C until measurement. The measurement of the blood insulin concentration was performed using a radioimmunoassay (ShionoRIA, Shionogi & Co., Ltd.).
(2) Result
(2-1) Results of clonidine administration experiment
Under fasting or non-fasting conditions, the study focused on the change in blood glucose level due to clonidine administration. When 5 μg / kg @ BW clonidine was administered under fasting conditions, B6 mice did not show an increase in blood sugar level, whereas HND mouse strain showed a large increase of about 120 mg / dl (FIG. 13). . Under the non-fasting condition, the blood sugar level of B6 was increased by about 50 mg / dl, whereas the blood sugar level of the HND mouse strain was clearly increased by about 220 mg / dl (see FIG. 4). (FIG. 14). Similarly, when a larger amount of 50 μg / kg @ BW of clonidine was administered under the fasting condition, the HND mouse strain also showed a significantly higher blood glucose level than B6 (FIG. 15). However, under the non-fasting condition, the blood glucose level of B6 increased significantly at this dose of clonidine, and no difference was observed between B6 and the HND mouse strain (FIG. 16). In addition, when blood glucose changes at both concentrations of 5 and 50 μg / kg BW were compared, the increase in blood glucose was clearly increased in the B6 dose-dependent manner, whereas that in the HND mouse strain was 5 mg / kg BW. A large increase in blood sugar was observed even when clonidine was administered, and no further increase in blood sugar was observed even after administration of 50 mg / kg @ BW. That is, it was shown that in the HND mouse strain, the blood sugar level was sufficiently increased even with the administration of a small amount of clonidine (FIGS. 13 to 16).
(2-2) Results of the yohimbine administration experiment
Preliminary studies using B6 showed that yohimbine tended to improve glucose tolerance in a dose-dependent manner (FIG. 17). And, at 100 μg / kg @ BW administration, glucose tolerance improving effect is not seen in B6 (FIG. 17). Therefore, it was decided to compare the effects of yohimbine on glucose tolerance between B6 and the HND mouse strain at this dose.
[0083]
In B6 mice, there was no difference in the change in blood glucose level after glucose administration between the yohimbine (100 μg / kg @ BW) administration group and the physiological saline administration group (FIG. 18-a). On the other hand, in the group administered with yohimbine (100 μg / kg @ BW) of the HND mouse strain, the blood glucose level decreased 60 and 120 minutes after glucose administration as compared with the group administered with physiological saline (FIG. 18-b). This result indicates that in the HND mouse strain, α₂-Shows that the inhibitory effect of the adrenergic receptor enhanced the blood glucose lowering effect.
[0084]
Next, it was examined whether such improvement of glucose tolerance by yohimbine was due to improvement of insulin secretion. As a result, in the saline-administered group of the HND mouse strain, similarly to the result in Example 2, no increase in blood insulin concentration after glucose administration was observed, and impaired insulin secretion was observed (FIG. 19-b). . However, a clear increase in blood insulin concentration was confirmed in the yohimbine administration group (FIG. 19-b). The results of Warning # 7 suggested that the improvement of impaired glucose tolerance in the HND mouse strain by yohimbine administration was due to the improvement of impaired insulin secretion. In other words, the insulin secretion disorder of the HND mouse strain is α₂-Enhanced adrenergic receptor action was shown to be involved.
[0085]
【The invention's effect】
As described in detail above, the invention of this application is a diabetes model very similar to non-obese type 2 diabetes, which is common in Japanese, and is characterized by a neuropathic disorder of insulin secretion control (HND mouse) Is provided. Thereby, the pathological analysis and genetic analysis of type 2 diabetes are further promoted. Further, the invention of this application makes it possible to screen a therapeutic agent for type 2 diabetes with high accuracy. Furthermore, this model mouse has the advantage that it develops diabetes from a young age of about 7 weeks and can detect the effect of long-term hyperglycemia on the living body.
[Brief description of the drawings]
FIG. 1 (a) is a graph showing the change in body weight of B6 mice (open circles) and HND mouse strains (closed squares); It is the graph which showed the change of the obesity degree. The population is 5-15. Values are mean ± SEM. * Indicates that the difference from the B6 mouse by the unpaired @ t test (hereinafter abbreviated as the t @ test) is significant at the p <0.001 level.
FIG. 2 (a) is a graph showing changes in blood glucose levels in 6-week-old B6 mice (open circles: n = 9) and HND mouse strains (closed squares: n = 24) by a glucose tolerance test. . (B) is a graph showing the change in blood glucose level by a glucose tolerance test for 12-week-old B6 mice (open circles: n = 14) and HND mouse strains (closed squares: n = 20). (C) is a graph showing a change in blood glucose level by a glucose tolerance test for a 20-week-old B6 mouse (open circle: n = 10) and an HND mouse strain (closed square: n = 24). (D) is a graph showing the change in blood glucose level by a glucose tolerance test for a 30-week-old B6 mouse (open circle: n = 12) and an HND mouse strain (closed square: n = 30). Values are mean ± SEM. * And ** indicate that the difference from the B6 mouse by the t test is significant at the p <0.01 and p <0.001 levels, respectively.
FIG. 3 (a) is a graph showing AUC in a glucose tolerance test of 6-30 week old B6 mice (n = 5-15) and HND mouse strains (n = 16-42). Values are mean ± SEM. * Indicates that the difference from the B6 mouse by the Δt test is significant at the p <0.01 level.
FIG. 4 (a) shows blood glucose levels of non-fasted B6 mice (n = 5-12) and HND mouse strains (n = 16-42) of 6-40 weeks of age. (B) is the blood glucose level during fasting. Values are mean ± SEM. *, **, and *** indicate that the difference from the B6 mouse by the t test is significant at the p <0.05, p <0.01, and p <0.001 levels, respectively.
FIG. 5 (a) shows non-fasting and fasting (Fasting) of 6-week-old B6 mouse (white bar: n = 5-12) and HND mouse strain (gray bar: n = 16-42). 4 is a graph showing the blood insulin concentration in FIG. (B) shows the same results for a 23-week-old mouse, and (c) shows the same results for a 40-week-old mouse. Values are mean ± SEM. Statistical processing was performed by the t test.
FIG. 6 (a) is a graph showing changes in insulin secretion amount due to glucose stimulation during a glucose tolerance test for B6 mice (open circles) and HND mouse strains (closed squares) at 7 weeks of age. (B) is a graph showing a similar change in insulin secretion in a 23-week-old B6 mouse (open circle) and an HND mouse strain (closed square and closed triangle). The HND mouse strain was divided into a diabetic group (closed squares) and a non-diabetic group (closed triangles) depending on the blood sugar level at 20 weeks of age, which was higher or lower than 200 mg / dl. Numerical values are mean ± SEM of 6-8 individual measurements. * And ** indicate that the difference from the B6 mouse by the t-test is significant at the p <0.05 and p <0.001 levels, respectively.
FIG. 7 (a) is a graph showing changes in blood glucose levels in a B6 mouse (open circle) and a HND mouse strain (closed square) at the age of 7 weeks by an insulin tolerance test. (B) is a graph showing similar results for a 20-week-old mouse and (c) is a 40-week-old mouse. Values are mean ± SEM. * Indicates that the difference from the B6 mouse by the t test is significant at the p <0.01 level.
FIG. 8 (a) shows non-fasting and fasting (Fasting) of 7-week-old B6 mouse (white bar: n = 7) and HND mouse strain (gray bar: n = 5-8). It is the graph which showed the serum NEFA amount. (B) is a graph showing the amount of serum TG, and (c) is a graph showing the amount of serum TC. Mean ± SEM. * Indicates that the difference from the B6 mouse by the t test is significant at the p <0.05 level.
FIG. 9 is a graph showing the dose-dependent effect of glucose on insulin secretion of pancreatic islets isolated from 6-week-old B6 mice (open circles) and HND mouse strains (closed squares). The numerical values are the mean ± SEM of the measurements of 3-6 samples. Statistical processing was performed by the t test.
FIG. 10 is a graph showing the effect of GLP-1, GIP or glucagon on glucose-stimulated insulin secretion in pancreatic islets isolated from each of 6-week-old B6 mice (white bars) and HND mouse strains (black bars). is there. The numerical values are the mean ± SEM of the measurements of 3-6 samples. Statistical processing was performed by the t test.
FIG. 11 (a) shows the dose-dependent effect of somatostatin on glucose-stimulated insulin secretion in islets isolated from B6 mice (open circles) and HND mouse strains (closed squares) at the age of 20 to 30 weeks. It is a graph. (B) is a graph showing the same effect of adrenaline. The numerical values are (a) the average value of 4-7 samples, and (b) the average value of 5-20 samples ± SEM. * Indicates that the difference from the B6 mouse by the t test is significant at the p <0.05 level.
FIG. 12 shows the effect of clonidine, an agonist of the α2-adrenergic receptor, on glucose-stimulated insulin secretion in pancreatic islets isolated from B30 mice (white bars) and HND mouse strains (black bars) at the age of 20-30 weeks. It is the graph shown. The numerical values are the mean ± SEM of the measurements of 4-5 samples. * Indicates that the difference from the B6 mouse by the t test is significant at the p <0.05 level.
FIG. 13 (a) shows the effect of clonidine (5 μg / kg @ BW) on fasting blood glucose levels of 10-14 week-old B6 mice (open circles: n = 6) and HND mouse strains (closed squares: n = 8). 3 is a graph showing the change over time. (B) is a graph showing the time-dependent change of the same effect. Values are mean ± SEM. * And ** indicate that the difference from the B6 mouse by the t test is significant at the p <0.01 and p <0.001 levels, respectively.
FIG. 14 (a) shows the effect of clonidine (5 μg / kg @ BW) on the non-fasting blood glucose level of 10-14-week-old B6 mice (open circles: n = 4) and the HND mouse strain (solid squares: n = 8). It is the graph which showed the time-dependent change of the effect. (B) is a graph showing the time-dependent change of the same effect. Values are mean ± SEM. *, ** and *** indicate that the difference from the B6 mouse by the t test is significant at the p <0.05, p <0.01 and p <0.001 levels, respectively.
FIG. 15 (a) shows the effect of clonidine (50 μg / kg @ BW) on fasting blood glucose levels of 10-14-week-old B6 mice (open circles: n = 8) and HND mouse strains (closed squares: n = 12). (B) is a graph showing the time-dependent change of the same effect, and the numerical values are the mean ± SEM. The differences are significant at the p <0.01 and p <0.001 levels.
FIG. 16 (a) shows the effect of clonidine (50 μg / kg @ BW) on the non-fasting blood glucose level of B6 mice (open circles: n = 4) and HND mouse strains (closed squares: n = 8) of 10-14 weeks old. It is the graph which showed the time-dependent change of the effect. (B) is a graph showing the time-dependent change of the same effect. Values are mean ± SEM. * Indicates that the difference from the B6 mouse by the t test is significant at the p <0.001 level.
FIG. 17 is a graph showing a dose-dependent effect of saline or yohimbine in a glucose tolerance test of B6 mice of 25 to 50 weeks of age. Numerical values are the mean ± SEM of 3-4 sample measurements. * Indicates that the difference from the B6 mouse by the t test is significant at the p <0.05 level.
FIG. 18 (a) shows the dose-dependent effect of saline (open circles) or yohimbine (closed squares: 100 μg / kg @ BW) in a glucose tolerance test of 11-16 week old B6 mice (n = 8). FIG. (B) is a graph showing the same effect in the HND mouse strain (n = 10). (C) is the result of AUC, in which the white bar is saline, the gray bar is yohimbine, the left is B6 mouse, and the right is HND mouse strain. Values are mean ± SEM. * And ** indicate that the difference from the B6 mouse by the t-test is significant at the p <0.05 and p <0.001 levels, respectively.
FIG. 19 (a) shows the amount of insulin secreted from 11-6 week old B6 mice (n = 3-4) during a glucose tolerance test in saline (open circles) or yohimbine (closed squares: 100 μg / kg @ BW). 4 is a graph showing the effect of (1). (B) is a graph showing the same effect in the HND mouse strain (n = 3-4). (C) is a change in blood insulin concentration during the initial 30 minutes during the test, in which the white bar is physiological saline, the gray bar is yohimbine, the left is B6 mouse, and the right is HND mouse strain. Values are mean ± SEM. * And ** indicate that the difference from the B6 mouse by the t test is significant at the p <0.05 and p <0.01 levels, respectively.

Claims (17)

Progeny mice of wild Castaneus mice and inbred C57BL / 6J mice, having the following characteristics:
(A) show hyperglycemia and / or impaired glucose tolerance when compared to normal strain mice;
(B) significantly lower body weight and / or obesity as compared to normal strain mice;
(C) the effect of endogenous adrenaline on insulin secretion via α2-adrenergic receptor is remarkably strong when compared with normal strain mice;
A diabetes model mouse characterized by having: 2. The diabetes model mouse according to claim 1, wherein the offspring mouse is the nth generation (n is 1 or more) of mating siblings. A diabetes model mouse produced by crossing the diabetes model mouse of claim 1 or 2 with another diabetes model mouse. The tissue of the diabetes model mouse according to claim 1, 2 or 3. The cell of the diabetes model mouse according to claim 1, 2 or 3. A fertilized egg of the diabetes model mouse according to claim 1, 2 or 3. The following steps:
(1) creating a selected basal population consisting of offspring mice obtained by crossing a plurality of wild Castaneus mice with inbred C57BL / 6J mice;
(2) From the mice of the selected basic population, the following criteria:
(A) show hyperglycemia and / or impaired glucose tolerance when compared to normal strain mice;
(B) significantly lower body weight and / or obesity as compared to normal strain mice;
Selecting from the first-generation sibling mating mice obtained by mating sibling mice of individuals matching the criteria (a) and (b),
A method for producing a diabetes model mouse, comprising: Further steps:
(3) selecting individuals matching the criteria (a) and (b) from the second-generation siblings obtained by mating the siblings of the first-generation siblings selected in step (2); And (4) repeating the above step (3) to obtain n-th generation (n is 3 or more) mated siblings meeting criteria (a) and (b);
The method for producing a diabetes model mouse according to claim 7, comprising: The following steps:
(1) creating a selected basal population consisting of offspring mice obtained by crossing a plurality of wild Castaneus mice with inbred C57BL / 6J mice;
(2) From the mice of the selected basic population, the following criteria:
(C) significantly increased blood glucose levels after administration of α2-adrenergic receptor agonist when compared to normal strain mice;
(D) abnormal glucose tolerance is normalized after administration of an antagonist of α2-adrenergic receptor,
Selecting from a first-generation sibling mating mouse obtained by mating a sibling mouse of an individual matching at least one of the criteria (c) or (d),
A method for producing a diabetes model mouse, comprising: Further steps:
(3) From the second generation mice mated with the siblings of the first generation offspring mice selected in step (2), those individuals matching at least one of the criteria (c) and (d) are obtained. Selecting; and (4) repeating step (3) above to obtain sibling mating nth generation (n is 3 or more) mice meeting at least one of criteria (c) and (d);
The method for producing a diabetes model mouse according to claim 9, comprising: The following steps:
(1) creating a selected basal population consisting of offspring mice obtained by crossing a plurality of wild Castaneus mice with inbred C57BL / 6J mice;
(2) From the mice of the selected basic population, the following criteria:
(A) show hyperglycemia and / or impaired glucose tolerance when compared to normal strain mice;
(B) significantly lower body weight and / or obesity as compared to normal strain mice;
(C) significantly increased blood glucose levels after administration of α2-adrenergic receptor agonist when compared to normal strain mice;
(D) abnormal glucose tolerance is normalized after administration of an antagonist of α2-adrenergic receptor,
(A) and (c); (a) and (d); (b) and (c); (b) and (d); (a), (b) and (c); ), (B) and (d); or the same criteria as described above, from first-generation sibling mice obtained by mating sibling mice matching (a), (b), (c) and (d). Selecting individuals that match
A method for producing a diabetes model mouse, comprising: Further steps:
(3) Reference (a) and (c); (a) and (d) from sibling mated second generation mice obtained by mating sibling mice of sibling mated first generation mice selected in step (2); (B) and (c); (b) and (d); (a), (b) and (c); (a), (b) and (d); or (a), (b), ( (c) selecting individuals that meet c) and (d); and (4) repeating step (3) above to obtain criteria (a) and (c); (a) and (d); (b) and (C); (b) and (d); (a), (b) and (c); (a), (b) and (d); or (a), (b), (c) and ( obtaining an n-th generation (n is 3 or more) mouse sibling mating according to d);
The method for producing a diabetes model mouse according to claim 11, comprising: A method for screening a therapeutic agent for diabetes, which comprises administering a candidate substance to the diabetes model mouse according to claim 1, 2, or 3, and specifying a substance that improves the blood glucose level of the model mouse. A method for screening a therapeutic agent for diabetes, comprising administering a candidate substance to the diabetes model mouse according to claim 1, 2 or 3, and then administering an agonist of an α2-adrenergic receptor, thereby suppressing an increase in blood glucose level after administration of the agonist. A method comprising identifying a substance to be treated. A method for screening a therapeutic agent for diabetes, comprising administering a candidate substance to the diabetes model mouse according to claim 1, 2, or 3, and improving the abnormal glucose tolerance after administering an antagonist of an α2-adrenergic receptor to the model mouse. A method comprising identifying a substance having a similar improvement effect. A method for screening a therapeutic agent for diabetes, which comprises causing a candidate substance to act on pancreatic tissue or pancreatic β-cells of the diabetes model mouse according to claim 1, 2, or 3, and then acting adrenaline or an agonist of α2-adrenergic receptor, Alternatively, a method comprising identifying a substance that suppresses a decrease in insulin secretion after administration of an agonist. A method for screening a therapeutic agent for diabetes, comprising the step of causing a candidate substance to act on pancreatic tissue or pancreatic β-cells of the diabetes model mouse according to claim 1, 2, or 3 in the presence of adrenaline, and allowing α2- A method comprising identifying a substance which causes an increase in insulin secretion equivalent to an increase in insulin secretion after the action of an adrenergic receptor antagonist.

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