JP6920071B2 - アディポネクチン分泌向上剤 - Google Patents
アディポネクチン分泌向上剤 Download PDFInfo
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- JP6920071B2 JP6920071B2 JP2017027373A JP2017027373A JP6920071B2 JP 6920071 B2 JP6920071 B2 JP 6920071B2 JP 2017027373 A JP2017027373 A JP 2017027373A JP 2017027373 A JP2017027373 A JP 2017027373A JP 6920071 B2 JP6920071 B2 JP 6920071B2
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Description
[1] β−NMN、その薬理学的に許容される塩、及びそれらの溶媒和物から選ばれるひとつを有効成分とし、血清又は血漿中のアディポネクチン量が低下していることが確認された動物に投与されることを特徴とする、アディポネクチン分泌向上剤。
[2] 経口投与される、前記[1]の分泌向上剤。
[3] 前記[1]又は[2]の分泌向上剤を含有し、血清又は血漿中のアディポネクチン量が低下していることが確認された動物に投与されることを特徴とする、アディポネクチンの分泌量を増加させるための、健康補助食品。
[4] 前記[1]又は[2]の分泌向上剤を含有し、血清又は血漿中のアディポネクチン量が低下していることが確認された動物に投与されることを特徴とする、アディポネクチンの分泌量を増加させるための、飼料。
以降の実験に用いたCD(SD)ラット、C57BL/6Nマウス、及びC57BL/6Jマウスは、全実験期間中を通して、SPF環境下で飼育した。
経口投与は、β−NMN(オリエンタル酵母工業社製)を注射用水(大塚製薬工場社製)もしくはPBS(リン酸生理食塩水)に溶解させた溶液を、金属製胃ゾンデ(フチガミ器械社製)を取り付けたポリプロピレン製ディスポーザブル注射筒(テルモ社製)などで強制経口投与した。
腹腔内投与は、β−NMN(オリエンタル酵母工業社製)をPBS(リン酸生理食塩水)に溶解させた溶液を、Dr. Sinclair 等の方法(非特許文献2)及びDr. Imai等の方法(非特許文献3)などに従って腹腔内投与した。
以降の実験において、アディポネクチン量の測定は、以下のようにして行った。
1次抗体液としては、抗マウスアディポネクチンポリクロ―ナル抗体(オリエンタル酵母工業社製)をPBSにて10μg/mLに調製した。また、2次抗体液としては、抗マウスアディポネクチンポリクロ―ナル抗体(オリエンタル酵母工業社製)をビオチン化させた後、2μg/mLに調製した。
マウスアディポネクチン(オリエンタル酵母工業社製)を検体希釈液で45.2ng/mLに調製したものを、標準品とした。
標準品45.2ng/mLを検体希釈液で2倍段階希釈し、22.6ng/mL、11.3ng/mL、5.65ng/mL、2.83ng/mL、1.41ng/mL、0.71ng/mL、0.35ng/mLの標準液を調製した。なお、0ng/mLについては、検体希釈液を用いた。また、検体については、ラット血清は808倍、マウス血清は6161倍になるように、検体希釈液にて2段階希釈で調製した。
まず、1次抗体液を96ウェルプレートの各ウェルに100μLずつ加え、プレートシールで蓋をした後、4℃にて一晩静置した。静置後、洗浄液(0.05%Tween20含有PBS)を各ウェルに350μLずつ加えた後、そのまま除去する操作(以下、「洗浄操作」ということがある。)を3回実施した。続いて、ブロッキング液を各ウェルに300μLずつ加え、プレートシールで蓋をした後、4℃にて一晩静置し、その後、前記洗浄操作を3回実施した。
続いて、調製した標準品と検体をそれぞれ指定したウェルに100μL加え、プレートシールで蓋をした後、25℃で1時間静置して反応させた。反応終了後には、反応液を除去し、前記洗浄操作を3回実施した。続いて、2次抗体液を各ウェルに100μLずつ加え、プレートシールで蓋をした後、25℃で1時間静置して反応させた。反応終了後には、反応液を除去し、前記洗浄操作を3回実施した。その後、各ウェルに、酵素標識ストレプトアビジン(DAKO社製、#P0397)の2000倍希釈液を100μLずつ加え、プレートシールで蓋をした後、25℃で1時間静置して反応させた。反応終了後には、反応液を除去し、前記洗浄操作を3回実施した。
さらに、基質液(ベクトン・ディッキンソン社製、TMB Substrate Reagent Set、#555214)を各ウェルに100μLずつ加え、25℃で15分間反応させた。2N硫酸を各ウェルに100μLずつ加えて反応を停止させた後、プレートリーダー(BMG LABTECH社製、FLUO star OPTIMA)にて、各ウェルの正450nm/副570nmにおける吸光度を測定した。なお、ELISAにて使用するマウスアディポネクチンポリクローナル抗体はラットアディポネクチンにも交差するため、標準品としているマウスアディポネクチンの濃度に換算して測定した。
CDラット(4週齢、オス及びメス)に対して、β−NMNを28日間経口投与した。各群(n=6)の雌雄とβ−NMNの投与量を表1に示す。β−NMNの投与量が「0mg/kg/day」とは、β−NMNを含まないPBSを等量経口投与したことを意味する。第1群、第2群、第3群はオス、第4群、第5群、第6群はメスを用いた。
C57BL/6Nマウス(8月齢、メス)に対して、β−NMNを4日間経口投与した。各群(n=4)のβ−NMNの投与量を表2に示す。β−NMNの投与量が「0mg/kg/day」とは、β−NMNを含まないPBSを等量経口投与したことを意味する。
C57BL/6Jマウス(8月齢、メス)に対して、β−NMNを4日間腹腔内投与した。各群(n=3)のβ−NMNの投与量を表3に示す。β−NMNの投与量が「0mg/kg/day」とは、β−NMNを含まないPBSを等量腹腔内投与したことを意味する。
3T3−L1細胞を脂肪細胞に分化させた後、β−NMNで刺激してアディポネクチンの分泌量の変化を観察した。
まず、24ウェルプレートに、3T3−L1細胞を5×104細胞/ウェルで播き、培養培地(10%FBS(ウシ胎児血清)含有DMEM)中で37℃、5%CO2環境下で2〜3日間培養した。次いで、第1分化誘導培地(培養培地に、10μMのデキサメタゾン、5μMのIBMX(3−イソブチル−1−メチルキサンチン)、10μg/mLのインシュリンを含有させた培地)に培地交換し、37℃、5%CO2環境下で2日間培養した。続いて、第2分化誘導培地(培養培地に、10μg/mLのインシュリンを含有させた培地)に培地交換し、37℃、5%CO2環境下で2日間培養した。その後、2〜3日置きに培養培地への培地交換を2回実施し、3T3−L1細胞を成熟脂肪細胞に分化させた。
本発明者らは、NAD+生合成における律速酵素であり、肥満及び老齢のげっ歯類及びヒトの脂肪組織において減少することが知られているNAMPTが脂肪細胞特異的に欠損しているマウス(以下、「ANKOマウス」)を調べた。この結果、ANKOマウスは、脂肪組織、肝臓及び骨格筋において重度のインスリン抵抗性を示し(結果は図示せず)、また、血漿中遊離脂肪酸濃度の増加及び主要なインスリン感受性アディポカインであるアディポネクチンの血漿濃度の低下に現れる脂肪組織機能不全も示した(結果は図示せず)。さらに、ANKOマウスでは、CDK5(cyclin-dependent kinase 5)及びPPARγ(peroxisome proliferator-activated receptor gamma)(273番目のセリン)のリン酸化が増加しており、脂肪組織において肥満に関連するリン酸化PPARγの標的の遺伝子発現が減少していた(結果は図示せず)。
Claims (4)
- β−ニコチンアミドモノヌクレオチド、その薬理学的に許容される塩、及びそれらの溶媒和物から選ばれるひとつを有効成分とし、血清又は血漿中のアディポネクチン量が低下していることが確認された動物に投与されることを特徴とする、アディポネクチン分泌向上剤。
- 経口投与される、請求項1に記載の分泌向上剤。
- 請求項1又は2に記載の分泌向上剤を含有し、血清又は血漿中のアディポネクチン量が低下していることが確認された動物に投与されることを特徴とする、アディポネクチンの分泌量を増加させるための、健康補助食品。
- 請求項1又は2に記載の分泌向上剤を含有し、血清又は血漿中のアディポネクチン量が低下していることが確認された動物に投与されることを特徴とする、アディポネクチンの分泌量を増加させるための、飼料。
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