Functional Study of Porcine Peroxisome Proliferator-Activated Receptor γ and δ

過氧化小體增生活化受體γ(peroxisome proliferator-activated receptor γ, PPARγ)為調控脂肪細胞分化和胰島素敏感性之核內型轉錄因子。同一家族的PPARδ在脂質代謝中也扮演多功能的角色，例如調節脂肪組織的脂解作用和促進骨骼肌的脂肪酸氧化作用。而活化PPARγ和PPARδ的過程需要配體的結合，多元不飽和脂肪酸(polyunsaturated fatty acids, PUFA)對活化PPARγ和PPARδ具有較高的結合能力，然而，目前仍沒有直接證據能證實PUFA透過活化體內的PPARγ和PPARδ來達到調節脂質代謝的功能。故本試驗的目的為探討豬PPARγ和PPARδ在脂質代謝的功能。驗一結果顯示豬之正常型與突變型PPARγ皆能促使肌纖維母細胞轉分化為脂肪細胞，而突變型PPARγ促進轉分化之效果顯著高於正常型，因此PPARγ之絲胺酸的存在與否顯著影響其對脂肪細胞分化的促進功能。試驗二證實豬之PPARδ於PPARγ的配體刺激下能促使肌纖維母細胞轉分化為脂肪細胞，此外，PPARδ和它的配體單獨存在下能增加內源性PPARγ的表現，進而調節脂肪細胞分化的基因。試驗三發現二十二碳六烯酸(docosahexaenoic acid, DHA)並不能直接活化豬PPARγ，但它的代謝產物具有活化豬PPARγ的能力，此結果證實DHA可能為活化豬PPARγ的配體前驅物。試驗四產製兩種轉基因小鼠分別作為研究PPARγ和PPARδ的動物模式，一種為肌肉專一表現豬PPARγ的轉基因小鼠，另一種為脂肪組織專一表現豬PPARδ的轉基因小鼠。兩種轉基因小鼠以含有高度飽合脂肪酸(13%牛油)和高度不飽和脂肪酸(13%魚油)的飼糧餵飼四個月，試驗結果顯示經魚油餵飼的PPARγ轉基因小鼠比正常小鼠更能代謝葡萄糖，魚油餵飼的PPARδ轉基因小鼠比正常小鼠有較少的脂肪堆積、血脂和血中游離脂肪酸。這些結果證實PUFA為調節體內葡萄糖和脂質代謝的有效且天然的物質，而這些影響或許是透過活化PPARγ和PPARδ。合上述結果顯示，豬PPARγ和PPARδ在脂質代謝上扮演重要的角色，轉基因動物模式提供直接的證據證實魚油的PUFA能透過與PPARγ和PPARδ的交互作用以調節體內葡萄糖代謝和脂質代謝。Peroxisome proliferator-activated receptor γ (PPARγ) is a transcription factor that promotes adipocyte differentiation and insulin sensitivity. The PPARδ are multifunctional roles in lipid metabolism, specifically on lipolysis in the adipose tissue and fatty acid oxidation in the skeletal muscle. A ligand for PPARγ and PPARδ is required to activate PPARγ and PPARδ function. Fatty acids and its derivatives have been proposed to be the endogenous ligands for PPARγ and PPARδ. Polyunsaturated fatty acids (PUFA) have good binding activities with PPARγ and PPARδ. However, no direct evidence PUFA regulate lipid metabolism through activation of PPARγ and PPARδ in vivo. The current experiment was designed to study the function of porcine PPARγ and PPARδ in lipid metabolism.e found that porcine wild-type PPARγ and mutated PPARγ (serine 112 was mutated to alanine) enhanced the conversion of myoblast cells into adipocytes and the ability to transdifferentiation was greater in cells containing the mutated PPARγ than in cells containing the wild-type PPARγ. Therefore, the existence of serine 112 in PPARγ may have a role in regulating adipocyte differentiation. In the second experiment, we demonstrated that in myoblasts transfected with porcine PPARδ, adipogenesis was promoted in the presence of a PPARγ ligand. In addition, we showed that PPARδ alone with its ligand modulated adipogenic genes via expression of endogenous PPARγ. The third experiment demonstrated that docosahexaenoic acid (DHA) was a potent ligand precursor for porcine PPARγ activation. The DHA itself did not directly activate PPARγ but waking through its metabolites. In the in vivo study, we generated two transgenic mice models to study the function of PPARγ and PPARδ. One is muscle-specific expression of porcine PPARγ (PPARγ transgenic mice), and the other is adipose tissue-specific expression of PPARδ (PPARδ transgenic mice). Transgenic mice were generated and fed with high-saturated fat (13% beef tallow) or high-unsaturated fat (13% fish oil) diets for 4 months. Compared with wild-type mice, fish oil feeding increased glucose metabolism in PPARγ transgenic mice and reduced fat deposition, plasma triacylglycerol and free fatty acids in PPARδ transgenic mice. The results indicate PUFA may serves as natural and effective regulator of glucose and lipid metabolism in vivo and these effects may generate from activation of PPARγ and PPARδ.n conclusion, we demonstrated that porcine PPARγ and PPARδ play an important role in regulating lipid metabolism. Transgenic mouse models provide direct evidences to demonstrate PUFA, especially from fish oil, regulate glucose metabolism and lipid metabolism through interaction with PPARγ and PPARδ, respectively.目錄謝 I文摘要 II文摘要 IV言 1hapter I Literatures review 3、脂質代謝 3、脂質代謝 3一）脂肪酸合成 4二）三酸甘油酯的生成 5三）三酸甘油酯的分解 6四）脂肪酸的氧化作用 7、內分泌素調節脂質代謝 8一）胰島素 8二）昇醣素 9三）腎上腺素 9、過氧化小體增生活化受體 10、過氧化小體增生活化受體家族 10、 過氧化小體增生活化受體 γ 12一）過氧化小體增生活化受體 γ的構型 12二）過氧化小體增生活化受體 γ對基因的調節 13三）過氧化小體增生活化受體 γ的基因轉殖動物模式 14. PPARγ的全身性剔除 14. 脂肪組織的PPARγ剔除 15. 肌肉組織的PPARγ剔除 15. 肝臟組織的PPARγ剔除 15四）過氧化小體增生活化受體 γ的磷酸化作用 16、過氧化小體增生活化受體 δ 17一）過氧化小體增生活化受體 δ在組織間的表現 17二）過氧化小體增生活化受體δ的功能 17. 脂肪組織 17. 肌肉組織 18. 肝臟組織 19、脂肪酸與PPAR 20、 脂肪酸與PPARγ 20、 脂肪酸與PPARδ 22、肌肉細胞和脂肪細胞分化 23、肌肉細胞和脂肪細胞系的決定 23、參與肌肉細胞分化的轉錄因子和轉分化作用 23、參與脂肪細胞分化的轉錄因子和轉分化作用 24hapter II Porcine peroxisome-proliferator-activated receptor γ induces transdifferentiation of myoblasts into adipocytes 26NTRODUCTION 26ATERIALS AND METHODS 26ESULTS 30ISCUSSION 42hapter III Ectopic expression of porcine peroxisome-proliferator-activated receptor δ regulates adipogenesis in myoblasts 45NTRODUCTION 45ATERIALS AND METHODS 45ESULTS 47ISCUSSION 61hapter IV DHA regulates adipogenic genes in myoblasts via porcine PPARγ 65NTRODUCTION 65ATERIALS AND METHODS 65ESULTS 68ISCUSSION 76hapter V The function of porcine PPARγ and dietary fish oil effect on the expression of lipid and glucose metabolism related genes 79NTRODUCTION 79ATERIALS AND METHODS 80ESULTS 82ISCUSSION 92hapter VI Porcine peroxisome proliferator-activated receptor δ mediates the lipolytic effects of dietary fish oil to reduce body fat deposition 95NTRODUCTION 95ATERIALS AND METHODS 95ESULTS 98ISCUSSION 107eneral discussion 111onclusion 113考文獻 11