REGULATION OF PEPCK EXPRESSION BY THYROID HORMONE

甲状腺激素(T3)对葡萄糖的代谢有深远的影响 和肝脏中的脂类。磷酸烯醇式丙酮酸羧基激酶(PEPCK) 催化糖异生的关键启动步骤，并受 激素，包括主要在转录水平上的T3。PEPCK 基因为检测T3的作用提供了一个极好的模型，因为T3 至少通过两种机制调节PEPCK基因的表达。第一, T3通过与T3结合直接刺激PEPCK转录 主要诱导剂--高血糖素(CAMP)对受体的诱导 PEPCK基因的表达。这项研究的目的是分析这些 T3和GT的转录调控机制； 第一个目标是表征tr与tre在 PEPCK基因的启动子。PEPCK-TrE中的关键核苷酸 结合tr和刺激转录所必需的 将会被确认。TR与PEPCK-Tre的绑定将是 用甲基化干扰试验和凝胶迁移率试验进行评价。 将确定tR是否作为杂二聚体与PEPCK-tre结合 与肝脏核因子有关。第二个目标是确定推定的 与TR异源二聚体结合PEPCK的核蛋白- Tre.将形成异二聚体的维甲酸X受体(RXRpha) 将对其异二聚化能力进行评估 并刺激PEPCK基因的转录。绑定属性 RXRpha的水平将与肝细胞核因子的水平进行比较。这个 第三个目标是描述tr和肝脏之间的相互作用。 与PEPCK中第二个位点结合的特异性转录因子 推动者。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载体将被导入HepG2细胞 编码TrR和C/EBP的哺乳动物表达载体。那只猫 基因提供了一个标记物来表明这些蛋白质对 转录起源于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 PEPCK转录的诱导。最终目标是确定是否 动物的甲状腺状况影响转录因子的结合 PEPCK的发起人。核蛋白将从 甲减和甲亢大鼠，与PEPCK启动子结合将 用DNase I足迹和凝胶迁移率分析进行评估。 这项提议的重点是荷尔蒙的分子机制 调节肝脏葡萄糖生产中的一个关键酶的转录。 对这些机制的阐明将促进对 受T3和cAMP影响的其他酶的转录调控。 这样的理解可以为普通人带来临床上的好处 甲状腺功能减退、甲亢和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.