JP2016532098A - 転写因子tsc22d4の阻害剤によるインスリン耐性の治療 - Google Patents
転写因子tsc22d4の阻害剤によるインスリン耐性の治療 Download PDFInfo
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Abstract
Description
U6プロモーターの制御下でTSC22D4又は非特異的shRNA、又はCMVプロモーターの制御下でTSC22D4 cDNAを発現しているアデノウイルスは、BLOCK-iT Adenoviral RNAi expression system(Invitrogen社、カールスルーエ、ドイツ)を使用してクローニングされた。以前記載した通り、ウィルスは、動物注入の前に塩化セシウム法により精製され、10%グリセロール含有リン酸塩緩衝生理食塩水バッファーに対して透析された(Herzig S, Hedrick S, Morantte I, Koo SH, Galimi F, Montminy M. CREB controls hepatic lipid metabolism through nuclear hormone receptor PPAR-gamma. Nature. 2003; 426: 190-193. Herzig S, Long F, Jhala US, Hedrick S, Quinn R, Bauer A, Rudolph D, Yoon C, Puigserver P, Spiegelman B, et al. CREB regulates hepatic gluconeogenesis through the coactivator PGC-1. Nature. 2001; 413: 179-183)。肝細胞特異的プロモーターの制御下でコントロール又はTSC22D4特異的miRNAをコードするAAVは、以前記載された通りに樹立された(Rose AJ, Frosig C, Kiens B, Wojtaszewski JF, Richter EA. Effect of endurance exercise training on Ca2+ calmodulin-dependent protein kinase II expression and signaling in skeletal muscle of humans. J Physiol. 2007; 583: 785-795)。
オスの8〜12週齢C57Bl/6及び10週齢db/dbマウスは、Charles River Laboratories社(ブリュッセル、ベルギー)から得られ、通常の無制限の食事で12時間の明暗周期で維持された。インスリン及びグルコース負荷試験の前に、動物は、4時間絶食された。別の方法では、動物は自由に餌を与えられ、水まで自由にアクセスできた。アデノウイルス注入については、組換えウィルスあたり1〜2 × 109プラーク形成単位(pfu)が尾静脈注射により投与された。AAV実験については、5 × 1011ウィルスが尾静脈を介して注入された。各実験では、6〜12匹の動物が同一の処理を受けて、示された通りに絶食(18時間の絶食)、ランダムな給餌又は再栄養(18時間の絶食に続く6時間の再給餌)条件の下で解析された。肝臓、精巣上体及び腹部脂肪体、及び腓腹筋を含む器官は、特定時間後に採取され、計量され、瞬間凍結され、更なる解析に使用された。全体脂肪量(Total body fat content)は、Echo MRI body composition analyzer(Echo Medical Systems社、ヒューストン、アメリカ)により決定された。動物取扱い及び実験は、NIHガイドラインに従って行われ、地方自治体により承認された。
本発明者らは、ルーワイバイパス手術(Roux en Y bypass)、スリーブ状胃切除術、選択的胆嚢摘出術(elective cholecystectomy)又は試験開腹のための開腹外科手術(open abdominal surgery)を受けたことを特徴とする66人の広範囲にわたる白色人種の肥満及び痩身の男性及び女性から得た肝臓組織サンプル中のTSC22D4 mRNA発現を調査した。経口グルコース負荷試験で、本発明者らは、各個人をタイプ2糖尿病(n=26)又は正常な糖耐性(n=40)と同定した。インスリン感受性は、記載の通り、正常血糖高インスリンクランプ法(euglycemic-hyperinsulinemic clamp method)を使用して評価された。全ての基線血液サンプルは、一晩絶食後の午前8時から10時の間に採取された。全ての研究プロトコルは、ライプツィヒ大学(363-10-13122010及び017-12-230112)の倫理委員会により承認された。全ての参加者は、研究に参加する前に、書面のインフォームドコンセントを提出した。
グルコース及びトリグリセリド(TG)の血清レベルは、自動グルコース計測器(One Touch、Lifescan社、ネッカーゲミュント、ドイツ)又は商用キット(それぞれ、Sigma社、ミュンヘン、ドイツ;RANDOX社、クラムリン、北アイルランド;WAKO社、ノイス、ドイツ)を使用して決定された。インスリンレベルは、mouse insulin enzyme-linked immunosorbent assay(Mercodia社、ウプサラ、スウェーデン)を使用して決定された。
肝臓の脂質は、以前記載された通りに抽出された(Herzig S, Hedrick S, Morantte I, Koo SH, Galimi F, Montminy M. CREB controls hepatic lipid metabolism through nuclear hormone receptor PPAR-gamma. Nature. 2003; 426: 190-193)。
肝臓組織は、Tissue Tek optimal cutting temperature compound中で包埋された(Sakura社、トーランス、アメリカ)。5 μmの凍結切片は、記載の通り、ヘマトキシリン及びエオシン又はオイルレッドOで染色された(Peet DJ, Turley SD, Ma W, Janowski BA, Lobaccaro JM, Hammer RE, Mangelsdorf DJ (1998) Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR alpha. Cell 93: 693- 704)。
全RNAは、Qiazol reagent(Qiagen社、ヒルデン、ドイツ)を使用して、ホモジナイズされたマウス肝臓又は細胞溶解物から抽出された。cDNAは、M-MuLV酵素及びオリゴdTプライマー(Fermentas社、ザンクト・レオン=ロート、ドイツ)を使用した逆転写により調製された。cDNAsは、assay-on-demandキット及びABIPRISM 7700 Sequence detector(Applied Biosystems社、ダルムシュタット、ドイツ)を使用して増幅された。RNA発現データは、TATAボックス結合タンパク質(TBP)RNAのレベルに標準化された。
タンパク質は、細胞溶解バッファー中の凍結器官サンプル又は培養肝細胞から抽出され(Rose AJ, Frosig C, Kiens B, Wojtaszewski JF, Richter EA. Effect of endurance exercise training on Ca2+ calmodulin-dependent protein kinase II expression and signaling in skeletal muscle of humans. J Physiol. 2007; 583: 785-795)、20 μgのタンパク質は、4〜12%のSDSポリアクリルアミドゲルにロードされ、ニトロセルロース膜上にブロットされた。ウェスタンブロット解析は、TSC22D4(Abcam社、ケンブリッジ、イギリス又はシグマ社、ミュンヘン、ドイツ)、AKT、p-AKT、GSK、p-GSK(Cell signaling社、ダンヴァーズ、アメリカ)又はVCP(Abcam社)に特異的な抗体を使用して、記載の通りに(Herzig et al, 2001)実施された。
shRNA実験のために、マウスTSC22D4(GCCTGGTTGGCATTGACAACACGAATG;配列番号1)をターゲットとするオリゴヌクレオチドは、アニールされ、pENTR/U6 shRNAベクター(Invitrogen社)にクローニングされた 。いかなる哺乳類遺伝子配列にも有意な相同性を有さない非特異的オリゴヌクレオチド(5'- GATCTGATCGACACTGTAATG-3' 配列番号2)は、全ての実験において非サイレンシング(non-silencing)コントロールとして使用された。miRNA実験のために、マウスTSC22D4(5'- GACAGCGATGACGATAGTGGT-3' 配列番号3)をターゲットとするオリゴヌクレオチド及び非特異的オリゴヌクレオチド(5'-AAATGTACTGCGCGTGGAGAC-3' 配列番号4)は、pdsAAV-LP1ベクターにクローニングされた。
初代マウス肝細胞は、記載の通りに(Klingmuller U, Bauer A, Bohl S, Nickel PJ, Breitkopf K, Dooley S, Zellmer S, Kern C, Merfort I, Sparna T, et al. Primary mouse hepatocytes for systems biology approaches: a standardized in vitro system for modelling of signal transduction pathways. IEE Proc Syst Biol. 2006; 153: 433-447)単離され、培養された。簡潔に述べると、オスの8〜12週齢のC57Bl/6マウスは、100 mg/kg体重の塩酸ケタミン及び5 mg/kg体重の塩酸キシラジンの腹腔内注射により麻酔をかけられた。腹腔を開いた後に、肝臓は、門脈を介して5分間HANKS I(pH 7.4に調整された1 L蒸留H2O、2.5 mM EGTA、0.1%グルコース中、8 g NaCl、0.4 g KCl、3.57 g Hepes、0.06 g Na2HPO4 × 2 H2O、0.06 g KH2PO4)で37℃で灌流され、続いて5〜7分間HANKS II(pH 7.4に調整された1 L蒸留H2O、0.1%グルコース、3 mg/mLのコラゲナーゼCLSII、5 mM CaCl2中、8 g NaCl、0.4 g KCl、3.57 g Hepes、0.06 g Na2HPO4 × 2 H2O、0.06 g KH2PO4)で、肝臓構造の崩壊が観察されるまで潅流された。肝臓カプセル(liver capsule)は除去され、細胞浮遊液は、100 μmメッシュを通して濾過された。細胞は洗浄され、続いて、細胞の生存率は、トリパンブルー染色により決定された。1,000,000生存細胞/ウェルがコラーゲンIコーティングされた6ウェルプレート上に播種された。24時間後、細胞は、100の感染の多様性(multiplicity of infection)で組換えアデノウイルスに感染させられた。刺激実験のために、初代肝細胞は、10分間100 nM/6ウェルの濃度でPBS{コントロール溶媒(control medium)}又はインスリンで処理された。細胞は、感染48時間後に回収された。
Chipシークエンシング(Chip-Sequencing)結果のKEGG-Pathway解析は、有意性によりソートされた。インスリンシグナル伝達経路は、有意に制御されると分かった(p=0.00005)。Chipシークエンシングは、Flag-TSC22D4 cDNAアデノウイルスを注入されたオスC57Bl/6マウスからの肝臓抽出物中で注入7日後に行われた。
Claims (14)
- 哺乳動物中のインスリン耐性、メタボリックシンドローム及び/又は糖尿病タイプ1若しくは2の予防、治療及び/又は制御のための、及び/又は、腫瘍疾患との関連におけるインスリン感受性のようなインスリン感受性を改善するための、
モジュレーターを同定する方法であって、以下の工程を含む:
a) TSC22D4をコードする核酸配列又はTSC22D4の遺伝子発現産物を含む生体サンプルを提供すること、
b) 前記サンプルを前記TSC22D4をコードする核酸配列又はTSC22D4の遺伝子発現産物の少なくとも1つの推定上のモジュレーターと接触させること、及び
c) 前記少なくとも1つの推定上のモジュレーターと前記TSC22D4をコードする核酸配列又はTSC22D4の遺伝子発現産物との間の結合を検出すること、及び
d) 前記TSC22D4をコードする核酸配列又はTSC22D4の遺伝子発現産物の前記モジュレーターを同定すること。 - 前記モジュレーターの存在下又は非存在下における少なくとも1つのTSC22D4活性又は発現を評価する工程を更に含み、
ここで、前記モジュレーター非存在下におけるTSC22D4の測定された活性又は発現と比較された、前記モジュレーター存在下におけるTSC22D4の測定された活性又は発現との間の低下は、前記モジュレーターがインスリン耐性、メタボリックシンドローム及び/又は糖尿病タイプ1若しくは2の予防、治療及び/又は制御において、及び/又は、腫瘍性疾患との関連におけるインスリン感受性のようなインスリン感受性を改善するために、使用されることを示す、
請求項1に記載の方法。 - 前記TSC22D4の活性又は発現が肝臓の(hepatic)活性又は発現である、請求項1又は2に記載の方法。
- インスリン耐性、メタボリックシンドローム及び/又は糖尿病タイプ1若しくは2の予防、治療、及び/又は制御における、及び/又は、腫瘍疾患との関連におけるインスリン感受性のようなインスリン感受性を改善するための、前記モジュレーターの効果を解析する工程を更に含む、請求項1〜3のいずれか1項に記載の方法。
- 前記モジュレーターは、ペプチドライブラリー分子、アプタマー、組み合わせライブラリー分子、細胞抽出物由来分子、小分子薬、細菌代謝産物、ファージディスプレイ分子、その抗体又は断片、タンパク質、タンパク質断片、ポリヌクレオチド、オリゴヌクレオチド、miRNA、siRNA又はshRNA、及びそれらの組み合わせから選択される、請求項1〜4のいずれか1項に記載の方法。
- TSC22D4の活性を評価することは、酵素活性解析、免疫測定、レポーターアッセイ及び/又はウェスタンブロットを含み、及び/又は、TSC22D4の発現を評価することは、ノーザンブロット、マイクロアレイ解析及び/又はRT-PCRを含む、請求項1〜5のいずれか1項に記載の方法。
- 前記哺乳動物由来の生体サンプル中の血糖、炎症性サイトカイン及び/又はレジスチンの循環レベルを解析する工程を更に含む、請求項1〜6のいずれか1項に記載の方法。
- 前記モジュレーターは、ペプチドライブラリー分子、アプタマー、組み合わせライブラリー分子、細胞抽出物由来分子、小分子薬、細菌代謝産物、ファージディスプレイ分子、抗体又はその断片、タンパク質、タンパク質断片、ポリヌクレオチド、オリゴヌクレオチド、miRNA、siRNA又はshRNA、及びそれらの組み合わせの群から選択される、又は、TSC22D4の発現及び/又は生物活性の阻害剤から選択される、請求項1〜7のいずれか1項に記載の方法により同定されたTSC22D4に特異的であるモジュレーター。
- 医薬組成物の生産方法であって、以下の工程を含む:
a) 任意に、請求項1〜7のいずれか1項に記載の方法を実施すること、及び
b) 同定された前記少なくとも1つのモジュレーター又は請求項8に記載のモジュレーターを少なくとも1つの薬学的に許容される賦形剤とともに調合すること。 - 前記医薬組成物は、経口的に、経直腸的に、経粘膜的に、経皮的に、経腸的に、非経口的に、筋肉内に、髄腔内に、直接脳室内に、静脈内に、腹腔内に、鼻腔内に、眼内に、又は皮下に投与される、請求項9に記載の方法により生産された医薬組成物。
- 疾患は、インスリン耐性、メタボリックシンドローム及び/又は糖尿病タイプ1若しくは2から選択される、該疾患の診断における使用のための、及び/又は該疾患の予防、制御、及び/又は治療における使用のための、及び/又は、腫瘍疾患との関連におけるインスリン感受性のようなインスリン感受性を改善するための、請求項8に記載のモジュレーター又は請求項10に記載の医薬組成物。
- インスリン耐性、メタボリックシンドローム及び/又は糖尿病から選択された疾患の検出方法であって、そのような疾患を有すると疑われた被験者から得られた生体サンプル中のTSC22D4の発現及び/又は生物活性を測定する工程を含み;
ここで健康な被験者からのサンプル中のTSC22D4の測定された活性又は発現と比較された前記サンプル中のTSC22D4の測定された活性又は発現の低下は、インスリン耐性、メタボリックシンドローム及び/又は糖尿病タイプ1若しくは2から選択された疾患についての、及び/又は、腫瘍疾患との関連におけるインスリン感受性のようなインスリン感受性を改善することについての、存在又はリスクを示す、疾患の検出方法。 - 前記検出は、請求項8に記載のモジュレーターの結合の検出を含む、請求項12に記載の方法。
- 任意に適切なバッファー及び賦形剤、及び取扱説明書とともに、請求項1〜7又は12又は13のいずれか1項に記載の方法を実施するための材料を含む、診断又は治療用キット。
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