REGULATION OF PEPCK EXPRESSION BY THYROID HORMONE

甲状腺激素（T3）对葡萄糖代谢有重要影响 和肝脏中的脂质。 磷酸烯醇式丙酮酸羧激酶 催化胚胎发生的关键起始步骤，并受 激素，包括T3主要在转录水平。 PEPCK 基因提供了一个很好的模型来检查T3的作用，因为T3 通过至少两种机制调节PEPCK基因表达。 第一、 T3通过与PEPCK的结合直接刺激PEPCK的转录。 胰高血糖素（cAMP）是主要的诱导剂， PEPCK基因的表达。 本研究的目的是分析这些 T3的转录调控机制> 第一个目的是表征TR与TRE的结合， PEPCK基因的启动子。 PEPCK-TRE中的关键核苷酸 需要结合TR和刺激转录 将被识别。 TR与PEPCK-TRE的结合将是 用甲基化干扰测定和凝胶迁移率测定进行评价。 将确定TR是否作为异二聚体与PEPCK-TRE结合 肝细胞核因子 第二个目的是确定假定的 与TR异源二聚化以结合PEPCK的核蛋白- TRE。 将形成异二聚体的视黄酸X受体（RXR α） 将评估其与TR异二聚化的能力， TR并刺激PEPCK基因的转录。 的结合特性发生 将RXR α与肝核因子进行比较。 的 第三个目的是表征TR和肝脏之间的相互作用- 与PEPCK中的第二位点结合的特异性转录因子 启动子 这些实验将测试肝脏之间的相互作用， TR和CCAAT增强子结合蛋白（C/EBP）。 C/EBP有助于 PEPCK基因的肝脏特异性表达和cAMP反应性。 PEPCK-CAT载体将与以下一起沿着引入HepG2细胞中： 编码TR和C/EBP的哺乳动物表达载体。 猫 基因提供了一个标记，以表明这些蛋白质的影响， 转录起始于PEPCK启动子。 这些研究将 表征参与T3和cAMP的蛋白质之间的串扰 诱导PEPck转录。 最终目的是确定 动物的甲状腺状态影响转录因子的结合 PEPck启动子。 核蛋白质将从 甲状腺功能减退和甲状腺功能亢进大鼠，并结合PEPCK启动子将 用DNA酶I足迹法和凝胶迁移率测定法进行评价。 这项建议的重点是激素的分子机制， 调节肝脏葡萄糖产生中关键酶的转录。 阐明这些机制将促进对 受T3和cAMP影响的其他酶的转录调节。 这样的理解可能会导致临床利益的共同 甲状腺功能减退和甲状腺功能亢进以及糖尿病的内分泌紊乱。

Thyroid hormone (T3) has profound effects on the metabolism of glucose and lipids in the liver. Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the key initiating step in gluconeogenesis and is regulated by hormones, including T3 primarily at the transcriptional level. The PEPCK gene provides an excellent model for examining T3 action because T3 modulates PEPCK gene expression through at least two mechanisms. First, T3 directly stimulates PEPCK transcription through the binding of the T3 receptor (TR) induction by glucagon (cAMP), which is the major inducer of PEPCK gene expression. The goals of this study are to analyze these mechanisms of transcriptional regulation by T3> The first aim is to characterize the binding of the TR to the TRE in the promoter of the PEPCK gene. The key nucleotides in the PEPCK-TRE required for the binding of the TR and the stimulation of transcription will be identified. The binding of the TR to the PEPCK-TRE will be evaluated with methylation interference assays and gel mobility assays. It will be determined if the TR binds to the PEPCK-TRE as a heterodimer with a liver nuclear factor. The second aim is to identify the putative nuclear protein that heterodimerizes with the TR to bind to the PEPCK- TRE. The retinoic X receptor (RXRalpha) which will form heterodimerizes with the TR will be evaluated for its ability to heterodimerize with the TR and stimulate transcription of the PEPCK gene. The binding properties of RXRalpha will be compared with those of the liver nuclear factor. The third aim is to characterize the interactions between the TR and a liver- specific transcription factor bound to a second site in the PEPCK promoter. These experiments will test for liver interactions between the TR and CCAAT enhancer binding protein (C/EBP). C/EBP contributes to the liver-specific expression and cAMP responsiveness of the PEPCK gene. PEPCK-CAT vectors will be introduced into HepG2 cells along with mammalian expression vectors encoding either the TR and C/EBP. The CAT gene provides a marker to indicate the effects of these proteins on transcription originating from the PEPCK promoter. These studies will characterize the cross-talk between proteins involved in the T3 and cAMP induction of PEPck transcription. The final aim is to determine if the thyroid status of the animal affects the binding of transcription factors to the PEPck promoter. Nuclear proteins will be isolated from hypothyroid and hyperthyroid rats, and binding to the PEPCK promoter will be evaluated with Dnase I footprinting and gel mobility assays. This proposal focuses on the molecular mechanisms by which hormones regulate the transcription of a key enzyme in hepatic glucose production. Elucidation of these mechanisms will promote understanding of the transcriptional regulation of other enzymes affected by T3 and cAMP. Such understanding could lead to clinical benefits for the common endocrine disorders of hypo-and hyperthyroidism and diabetes.