Physiologic Role of Type 2 Deiodinase in Skeletal Muscle

描述（由申请人提供）： 本提案中描述的工作的目标是表征2型脱碘酶（D2）在人类骨骼肌中的生理作用。D2将甲状腺素原（T4）转化为活性激素3，5，3 '-三碘甲状腺原氨酸（T3）。我们的假设是，在人类骨骼肌中，D2通过调节核T3受体（TRs）的饱和度来调节T3响应性代谢途径。这一假设是建立在脑，垂体和棕色脂肪组织的数据表明，D2催化的T4到T3的转换是核内T3的重要来源，并在文献上建立T3作为骨骼肌代谢过程的重要调节剂。我们测试这一假设的方法主要利用商业上可获得的原代人骨骼肌细胞培养（HSMM细胞），但也包括一个新的转基因“SkD 2”小鼠与条件，骨骼肌特异性表达的D2。在特定目标I中，我们将描述D2在人骨骼肌细胞中的调节，然后测试D2催化的T4至T3转化可以增加这些细胞中的核T3浓度足以改变T3响应基因的表达的假设。在特定目标II中，我们将评估T4通过D2调节这些细胞中间代谢的潜力。在Specific Aim III中，我们将通过tet-off系统创建在其骨骼肌中具有条件性表达D2的转基因小鼠。我们将使用这只小鼠来研究正常条件下和代谢应激期间骨骼肌D2的生理作用。此外，我们将与耶鲁大学小鼠代谢表型中心的Jason Kim博士合作，确定D2诱导对体内葡萄糖稳态的影响。这些研究将为候选人提供广泛的分子生物学技术经验。他将成为甲状腺激素作用的专家，同时也在能量学和代谢领域获得宝贵的曝光，使他能够继续发展成为内分泌学的独立研究者。

DESCRIPTION (provided by applicant): The goal of the work described in this proposal is to characterize the physiologic role of the type 2 deiodinase (D2) in human skeletal muscle. D2 converts the prohormone thyroxine (T4) to 3,5,3'-triiodothyronine (T3), the active hormone. Our hypothesis is that, in human skeletal muscle, D2 regulates T3- responsive metabolic pathways by modulating the saturation of nuclear T3-receptors (TRs). This hypothesis is founded on data from brain, pituitary, and brown adipose tissue showing that D2-catalyzed T4 to T3 conversion is an important source of intranuclear T3, and on the literature establishing T3 as an important regulator of metabolic processes in skeletal muscle. Our approach to test this hypothesis primarily utilizes commercially available primary human skeletal myocytes in culture (HSMM cells), but also includes a new transgenic "SkD2" mouse with conditional, skeletal muscle-specific expression of D2. In Specific Aim I, we will characterize the regulation of D2 in human skeletal myocytes, and then test the hypothesis that D2- catalyzed T4 to T3 conversion can increase nuclear T3 concentration in these cells enough to alter the expression of T3-responsive genes. In Specific Aim II, we will evaluate the potential for T4, via D2, to modulate intermediary metabolism in these cells. In Specific Aim III, we will create a transgenic mouse with conditional expression of D2 in its skeletal muscle via the tet-off system. We will use this mouse to study the physiologic role of skeletal muscle D2 under normal conditions and during periods of metabolic stress. In addition, we will determine the effect of D2 induction on in vivo glucose homeostasis in collaboration with Dr. Jason Kim of the Yale Mouse Metabolic Phenotypic Center. These studies will provide the candidate with experience in a wide range of molecular biological techniques. He will become an expert in thyroid hormone action, while also gaining valuable exposure in the areas of energetics and metabolism, positioning him well to continue his development towards becoming an independent investigator in endocrinology.