Wu X H, Chen X, Zhang S L, Pang L, To C, Wang T T, Hohman T C, Filep J G, Chan J S
Research Centre, University of Montreal, Montreal, Quebec, H1T 2MA, Canada.
J Renin Angiotensin Aldosterone Syst. 2000 Jun;1(2):166-74. doi: 10.3317/jraas.2000.021.
To investigate the molecular mechanism(s) of insulin action on the expression of the angiotensinogen (ANG) gene in kidney proximal tubular cells, we constructed a fusion gene, pOGH (hANG N-1064/+27), containing the 5'-flanking regulatory sequence of the human ANG gene fused with the human growth hormone (hGH) gene as a reporter and stably integrated the fusion gene into the opossum kidney (OK) cell genomes. The level of expression of pOGH (hANG N-1064/+27) was quantified by the amount of immunoreactive hGH secreted into the medium. The addition of a high level of D(+)-glucose (25 mM) or phorbol 12-myristate 13-acetate (PMA, 10(-7) M) stimulated the expression of the fusion gene in OK cells. The stimulatory effect of glucose (25 mM) was blocked by insulin and tolrestat (an inhibitor of aldose reductase). Tolrestat also inhibited the increase of cellular DAG and PKC activity stimulated by 25 mM glucose. While insulin did not affect the cellular DAG and PKC activity, it did block the stimulatory effect of high glucose (25 mM) and PMA on the expression of the fusion gene. Finally, PD98059 (an inhibitor of mitogen-activated protein kinase kinase (MEK)) enhanced the stimulatory effect of high levels of glucose and blocked the inhibitory effect of insulin on the expression of the fusion gene as well as on the phosphorylation of MEK and mitogen-activated protein kinase (MAPK). In contrast, Wortmannin (an inhibitor of phosphatidylinositol-3-kinase) did not block the inhibitory effect of insulin on the ANG gene expression. These studies demonstrate that the action of insulin, blocking the stimulatory effect of a high level of D(+)-glucose (25 mM) on the ANG gene expression is mediated, at least in part, via the 5'-flanking region of the ANG gene and MAPK signal transduction pathway.
为了研究胰岛素对肾近端小管细胞血管紧张素原（ANG）基因表达作用的分子机制，我们构建了一个融合基因pOGH（hANG N - 1064 / +27），它包含人ANG基因的5'侧翼调控序列与人生长激素（hGH）基因融合作为报告基因，并将该融合基因稳定整合到负鼠肾（OK）细胞基因组中。通过分泌到培养基中的免疫反应性hGH量来定量pOGH（hANG N - 1064 / +27）的表达水平。添加高水平的D（+）-葡萄糖（25 mM）或佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯（PMA，10^(-7) M）可刺激OK细胞中融合基因的表达。葡萄糖（25 mM）的刺激作用被胰岛素和托瑞司他（醛糖还原酶抑制剂）阻断。托瑞司他也抑制了25 mM葡萄糖刺激引起的细胞二酰甘油（DAG）和蛋白激酶C（PKC）活性的增加。虽然胰岛素不影响细胞DAG和PKC活性，但它确实阻断了高糖（25 mM）和PMA对融合基因表达的刺激作用。最后，PD98059（丝裂原活化蛋白激酶激酶（MEK）抑制剂）增强了高水平葡萄糖的刺激作用，并阻断了胰岛素对融合基因表达以及对MEK和丝裂原活化蛋白激酶（MAPK）磷酸化的抑制作用。相反，渥曼青霉素（磷脂酰肌醇-3-激酶抑制剂）不阻断胰岛素对ANG基因表达的抑制作用。这些研究表明，胰岛素阻断高水平D（+）-葡萄糖（25 mM）对ANG基因表达的刺激作用，至少部分是通过ANG基因的5'侧翼区域和MAPK信号转导途径介导的。
