Proteomics analysis for the novel liver specific transcription factors and clarification of their control signals

胰岛素是维持葡萄糖稳态所必需的。胰岛素与胰岛素受体结合并激活其内源性酪氨酸激酶，酪氨酸激酶进而磷酸化胰岛素受体底物（IRSs），并传递各种下游信号。肝脏是胰岛素的主要靶器官之一，胰岛素受体在肝脏中表达丰富。提示胰岛素受体的表达受组织特异性机制的调控。然而，细节仍不确定。另一方面，已知amp活化蛋白激酶（AMPK）在肝脏葡萄糖和脂质代谢的调节中起重要作用。本研究的目的是研究AMPK对肝细胞中胰岛素受体的影响，肝细胞是AMPK和胰岛素的主要靶器官，而胰岛素受体在胰岛素的作用中起着关键作用。我们研究了AMPK激活剂5-氨基咪唑-4-carboxamide-1-β- d -核糖呋喃苷（AICAR）对人肝癌HepG2细胞中胰岛素受体表达的影响。AICAR治疗48小时显著降低HepG2细胞中胰岛素受体蛋白的表达，并呈剂量依赖性。另一方面，AICAR在3T3-L1脂肪细胞和CHO细胞中均未观察到抑制作用。胰岛素受体mRNA的表达也随AICAR治疗呈剂量依赖性显著降低。此外，胰岛素受体基因启动子的转录活性也在AICAR治疗下下调。AICAR抑制剂阻断了AICAR下调胰岛素受体蛋白、mRNA和启动子活性的作用。通过对胰岛素受体基因启动子缺失突变体的研究，我们发现在胰岛素受体基因ATG密码子上游0.6 kb范围内，存在与aicar诱导的下调相关的顺式元件。我们在人胰岛素受体基因启动子中找到了5个一致的胰岛素反应元件（IRE-1 ~ 5）序列。转录因子Foxol与IRE-4和IRE-5结合，IRE-4和IRE-5存在于AICAR反应区0.6 kb范围内，并且与AICAR处理后的IRE-4和IRE-5的结合减少。本研究首次证明AMPK激活至少部分通过下调基因转录降低胰岛素受体的表达，并且这种作用可能在肝细胞中是特异性的。此外，Foxol还参与了肝细胞中胰岛素受体基因的转录调控。少

Insulin is essential for maintaining glucose homeostasis. Insulin binds to the insulin receptor and activates its endogenous tyrosine kinase, which in turn phosphorylates insulin receptor substrates (IRSs), and transmits various downstream signals. The liver is one of the major target organs of insulin in which the expression of the insulin receptor is abundant. It is suggested that the expression of insulin receptor is regulated by tissue specific mechanism. However, the detail is still uncertain. On the other hand, AMP-activated protein kinase (AMPK) has been known to be important in the regulation of glucose and lipid metabolism in liver. The aim of this study is to investigate the effect of AMPK on the insulin receptor, which has a key role in the insulin action, in liver cells that is the major target organ of both AMPK and insulin. We investigated the effect of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), which is an activator of AMPK, on the expression of insulin … More receptor in a human hepatoma cell line, HepG2 cells. AICAR treatment for 48 hours significantly decreased the expression of insulin receptor protein in a dose-dependent manner in HepG2 cells. On the other hand, the inhibitory effect of AICAR was not observed in either 3T3-L1 adipocytes or CHO cells. The expression of insulin receptor mRNA was also significantly decreased with AICAR treatment in a dose-dependent manner. In addition, transcriptional activity of the insulin receptor gene promoter was also down-regulated with AICAR treatment. The inhibitors of AICAR blocked the effects of AICAR on the down-regulation of insulin receptor protein, mRNA and promoter activity. According to the investigation with a deletion mutant of the insulin receptor gene promoter, it was suggested that cis-elements responsible for the AICAR-induced down-regulation existed within 0.6 kb upstream from the ATG codon in the insulin receptor gene. We have found five consensus sequences of insulin response element (IRE-1〜5) in the human insulin receptor gene promoter. A transcription factor Foxol was suggested to bind to IRE-4 and IRE-5, which exist within 0.6 kb of AICAR response region, and the binding to each IREs was decreased with AICAR treatment. This study demonstrated for the first time that AMPK activation reduced the expression of insulin receptor, at least in part, by a down-regulation of the gene transcription, and that this effect may be specific in liver cells. In addition, it was suggested that Foxol was involved in the transcriptional regulation of insulin receptor gene in liver cells. Less