2017-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/659799摘要:肥胖與第二型糖尿病已經成為全世界主要的健康威脅,它的起因主要是長期的能量代謝不平衡引起之脂肪堆積與伴隨發生胰島素阻抗性。過氧化小體增生活化受體γ(peroxisome proliferator-activated receptor γ, PPARγ) 主表現在白色與棕色脂肪組織, 巨噬細胞, 與上皮細胞。PPARγ在調節全身能量平衡, 血糖代謝, 胰島素阻抗性, 及發炎反應有重大作用。而臨床上廣泛被使用的降血糖藥物thiazolidinedione (TZD), 被認為是透過與PPARγ 結合, 發揮降低胰島素阻抗與血糖之效果。然而TZD卻有體重增加, 水分滯留, 心臟衰竭, 骨質疏鬆的副作用, 以至於幾乎退出臨床使用, 所以目前急需研發更好的PPAR γ 配體以治療第二型糖尿病。內生性的天然PPARγ 配體迄今未被確認,吾人與其他團隊發現,多元不飽和脂肪酸的天然代謝衍生物15-keto-PGE2, 極可能是內生性的PPARγ配體, 可以在生理濃度活化PPARγ。而前列腺素還原酶2 (prostaglandin reductase 2, PTGR-2)會代謝15-keto-PGE2為無活性物質, 進而抑制PPARγ活性。吾人更闡明了PTGR-2結晶構造, 以合理化藥物設計, 發展出PTGR-2小分子抑制劑apigenin與luteonin。在細胞模式中, PTGR-2抑制劑apigenin與luteonin被證明可以增加15-keto-PGE2 濃度及並增加PPARγ活性。我們更進一步發現PTGR-2基因剔除鼠的胰島素敏感性與耐糖性大幅改善, 且體重不增反減, 亦無水份滯留之副作用。這證明調節PTGR-2活性與內生性PPARγ配體15-keto-PGE2濃度, 比起現有人工合成的PPARγ配體TZD, 為治療第二型糖尿病更好的標的:我們計劃在未來三年完成:1. 釐清PTGR-2基因剔除鼠血糖代謝與胰島素阻抗性改善以及體重減輕的機轉。(1) 以同位素葡萄糖標定之高胰島素血糖恆定鉗制術, 測定PTGR-2基因剔除鼠的葡萄糖處理率, 肝臟葡萄糖輸出, 以及各組織包含肝臟, 骨骼肌, 肝臟, 與脂肪組織的葡萄糖攝取與胰島素信息傳遞。(2) 測定PTGR-2基因剔除鼠在一般飲食與高脂高蔗糖飲食下的進食量, 腸道脂肪吸收, 代謝率, 寒冷引發之適應性產熱, 進食引發之適應性產熱, 活動量, 身體組成成份, 內分泌賀爾蒙變化, 與骨質密度, 與發炎反應變化。(3) 測定相對應組織的相關前列腺素濃度, 脂肪酸組成, 脂肪酸氧化, 脂肪溶解, 粒線體功能, 組織學變化, 與轉錄體變化。2. 比較不同細胞型態中, 15-keto-PGE2 與人工合成的配體TZD, 活化PPARγ的機轉之不同, 包含(1) 配體結合方式與所引發構型變化之差異。(2) 所徵募之共同活化子, 共同壓抑子, 與染色質修飾之差異。(3) DNA結合區域與下游活化基因差異。(4) 獨立於PPARγ活化外的可能機轉。3. 測試apogenin與luteonin對於第二型糖尿病小鼠的療效與副作用(體重, 水份滯留, 骨質密度), 毒性, 與與藥物動力學測試。4. 以高通量小分子藥物庫, 篩選更專一的小分子PTGR-2抑制劑, 分析其先導化合物調控血糖作用(體重, 水份滯留, 骨質密度), 副作用, 毒性, 與藥物動力學測試。<br> Abstract: Obesity and type 2 diabetes have become major health threat worldwide, which result form long-term imbalances in the energy metabolism, leading to fat accumulation and insulin resistance. Peroxisome- proliferator of the receptor γ (peroxisome proliferator-activated receptor γ, PPARγ) is a master regulator of energy metabolism, lipid metabolism, and glucose/insulin homeostasis mainly expressed in adipose tissue and brown, macrophages, and epithelial cells. The widely used oral ant-diabetic agents thiazolidinedione (TZD), is considered to lower glucose by binding and activating PPARγ. However, TZD therapy have significant side effect including weight gain, water retention, heart failure, and osteoporosis. Therefore, there is urgent need to develop better PPAR γ ligands to treat type 2 diabetes.Natural endogenous PPARγ ligands to date has not been confirmed, we and other teams found that derivatives of polyunsaturated fatty acids, 15-keto-PGE2, is likely to be endogenous PPARγ ligands that can activates PPARγ at physiological concentrations. The prostaglandin reductase 2 (prostaglandin reductase 2, PTGR-2) catalyzes 15-keto-PGE2 to inactive substances, thereby inhibiting PPARγ activity. We further clarified PTGR-2 crystal structure and develop small-molecule inhibitors PTGR-2 apigenin and luteonin by rational drug design. In cell modes, PTGR-2 inhibitor apigenin and luteonin been proven to increase the 15-keto-PGE2 concentration and increased PPARγ activity.We further found that PTGR-2 knockout mice are markedly more insulin-sensitive and glucose-tolerant upon high-fat high-sucrose diet with less weight gain as compared to wild-type control. No excessive fluid retention was found. This indicates that regulating PTGR-2 activity and endogenous PPARγ ligand 15-keto-PGE2 concentration is better therapeutic target than current synthetic PPARγ ligands TZD for the treatment of type 2 diabetes :We plan to be completed in the next 3years:1. Clarify the mechanism by which PTGR-2 knockout mice display more insulin sensitivity and glucose tolerance with less weight gain(1) Using isotope-labeled glucose and hyperinsulinemic euglycemic clamp technique to measure glucose disposal rate and hepatic glucose output as well as measure the glucose uptake/insulin signaling of individual tissues including liver, skeletal muscle, liver, and adipose tissue(2) Determination of diet intake, intestinal fat absorption, metabolic rate, adaptive thermogenesis caused by cold, diet-induced adaptive thermogenesis, physical activity, body composition, endocrine hormone changes, and bone density, inflammatory changes between PTGR-2 knockout mice and wild-type mice on chow or high-fat high-sucrose diet(3) Determine the concentrations of the relevant prostaglandins, fatty acid composition, fatty acid oxidation rate, lipolysis, mitochondrial function, histological changes, and transcriptional changes.2. Compare the differential mechanism by which 15-keto-PGE2 and synthetic ligands TZD activates PPARγ(1) Difference in ligand binding domain and conformational changes(2) Difference in recruitments of co-activator and co-repressor complex and chromatin modification(2) Difference in DNA-binding domain and downstream activated gene.3. Test apogenin and luteonin for their efficacy and side effects (weight, water retention, bone density) on type 2 diabetic mice and toxicology test and pharmacokinetics4. High-throughput small-molecule drug library screening to find more specific small-molecule PTGR-2 inhibitors, analyze the effect of lead compound on therapeutic effect on type 2 diabetes mice mode and side effect (body weight, water retention, one density) and toxicology test and pharmacokinetics. Structural - functional relationship (structure-activity relationship) with the application of technology transfer.前列腺素還原酶2過氧化小體增生活化受體γ第2型糖尿病降血糖藥物PTGR-2PPARγtype 2 diabetesoral anti-diabetic agentsThe Role of Prostaglandin Reductase 2 (Ptgr2) in the Pathogenesis of Type 2 Diabetes and Development of Its Small-Molecule Inhibitors for Anti-Diabetic Therapy