| 摘要: | PGC-1 轉錄活化輔助因子(coactivators)家族包含有PGC-1α、PGC-1β以及PRC 三個成員，他們廣泛的表現在各種高耗能的代謝器官，如棕色脂肪，心臟以及骨骼肌等，並且調控這些器官的代謝。 PRC 主要表現於骨骼肌，但PGC-1α及PGC-1β主要表現於棕色脂肪及心臟。由於這三個成員在這些高耗能的代謝器官的表現量並不相同，因此他們可能在不同的組織中扮演不同的的代謝功能。在肌肉中，PGC-1α主要表現於慢速收縮的肌纖維中。當PGC-1α高量表現於肌肉中時，可以使肌肉的代謝從無氧代謝，轉而變成有氧代謝。肌肉的組成，也從快速收縮的肌肉轉變為慢速收縮的肌肉。 PGC-1α在骨骼肌中的表現主要受到MEF2、FoxO、CREB 以及MyoD 等轉錄因子的調控，這些轉錄因子，分別反應不同的荷爾蒙以及生理刺激對PGC-1α的轉錄調控，以加強PGC-1 的表現從而增進肌肉細胞的有氧代謝。 臨床的研究結果顯示，第二型糖尿病人的肌肉亦會減少有氧代謝，但增加無氧代謝的比率。部份研究亦顯示在第二型糖尿病人的肌肉中的 PGC-1α表現量減少。 但PGC-1α的減少是否可導致第二型糖尿病，目前則尚無定論。至於對其他兩個成員在第二型糖尿病肌肉中扮演的角色的研究則更是闕如。 本研究希望能增進對PGC-1 家族在正常以及第二型糖尿肌肉中的表現之瞭解，並且希望能藉由 shRNA 的方法在肌原細胞中，把每一PGC-1 的成員的表現量減少，以便瞭解各個成員間特有的功能。 本研究亦希望能增加對PGC-1 家族在肌肉中轉錄的調控，因此將會分析各個PGC-1 成員的啟動子，以及他們在肌肉中受到轉錄因子的調控，因此本研究的主要目標有以下數項: 1．分析PGC-1 家族在快、慢收縮肌原細胞分化過程中的表現。 2．分析PGC-1 家族在正常以及糖尿病老鼠肌肉中的表現。 3．利用shRNA 將PGC-1 家族在肌原細胞中表現減少。 4．分析每一個shRNA 穩定表現株的肌肉分化特性，以及糖尿病相關的特性。 5．分析PGC-1 家族的啟動子在各種肌肉中的活度。 6．尋找各個啟動子中主要的控制序列。 7．分析主要控制序列上的轉錄因子間的交互作用。 8．分析主要控制序列上的轉錄因子間的交互作用是否受到糖尿病的影響。PGC-1 coactivator family contains PGC-1α, PGC-1β, and PRC 3 members. They are highly expressed in tissues with high energy metabolism requirement, such as heart, skeletal muscle, and brown fat tissue. But their relative expression levels among these tissues are not identical. For example, PRC is most highly expressed in skeletal muscle, but PGC-1α and PGC-1β are most highly expressed in brown fat tissue. The differential expression pattern of this gene family implies that they might be implicated in different physiological processes, rather than playing redundant roles. Manipulation of its expression, either in a cell or in an animal, has shown that over-expression of PGC-1αcan promote the switch from anaerobic glycolysis to oxidative metabolism and thus increases the ir energy output. In muscle, PGC-1 αis preferentially expressed in slow-twitch fibers. In transgenic mice as the expression of PGC-1αis driven by muscle creatine kinase (MCK) promoter, the putative fast-twitch fibers are converted into slow-twitch fibers, which are characterized by the expression of genes of oxidative metabolism. The proximal region of the PGC-1a is conserved among all mammals examined, and is targeted by MyoD, FoxO, and CREB. Two MEF2-binding sites were identified further upstream (around -2901 and -1539 of mouse PGC-1 gene), and their cross-talk with proximal factors has not been examined either. Whether the expression of PGC-1β and PRC also has a fiber-type preference is currently unknown. The regulation of their promoter in muscle is also largely unknown. In obesity and type 2 diabetes (T2D) patients, their skeletal muscle with a phenotype characteristic of reduced oxidative enzyme activity, increased glycolytic activity, and increased lipid content has been observed. These metabolic characteristics are related to insulin resistance of skeletal muscle and are factors potentially related to muscle fiber type. Recent clinic data suggest that PGC-1αmight be involved in the etiology of type 2 diabetes, because its expression was reduced in skeletal muscle of diabetes patients, although its role is still not clear. Observations regarding the involvement of the other two members in the development of T2DM are now still lacking. This study is aiming to increase the understanding of the transcriptional regulation of PGC-1 expression and their involvement in the etiology of T2D in muscle. Therefore, the specific aims of this study are: 1. Examining the isoform-specific expression pattern of PGC-1 coactivators during myogenesis in cultured fast- and slow-twitch types of myoblasts in vitro. 2. Examining the isoform-specific expression pattern of PGC-1 coactivators in various types of skeletal muscle of normal and diabetes mice. 3. Isoform-specific knock down of PGC-1 coactivator expression in myoblasts using shRNA. 4. Observing and characterizing the myogenic and diabetes related phenotypes of knock down stable clones of each isoform. 5. Characterizing the promoter of each PGC-1 coactivators in cultured fast- and slow-twitch types of myoblasts in vitro. 6. Defining the major cis-elements of the PGC-1 family promoters controlling their expression 7. Analyzing the interactions between cis-element-binding factors on PGC-1 promoters. 8. Analyzing the interactions between cis-element-binding factors on PGC-1 promoters under the context of T2D skeletal muscle. 研究期間：9808 ~ 9907 |
