Activation of protein kinase B/cAkt in hepatocytes is sufficient for the induction of expression of the gene encoding glucokinase.
Activation of protein kinase B/cAkt in hepatocytes is sufficient for the induction of expression of the gene encoding glucokinase.
复制标题
DOI：
10.1042/bj3510621
复制
发表时间：
2000-11
期刊：
影响因子：
--
通讯作者：
P. Iynedjian;R. Roth;M. Fleischmann;A. Gjinovci
中科院分区：
文献类型：
--
作者：
P. Iynedjian;R. Roth;M. Fleischmann;A. Gjinovci
信号通路的抑制剂被用来剖析胰岛素对培养的大鼠肝细胞中编码葡萄糖激酶的基因表达的作用机制。Wortmannin和LY 294002完全阻止了胰岛素诱导的葡萄糖激酶mRNA的增加，这表明磷酸肌醇3-激酶模块具有关键作用。通过在原代培养的肝细胞中使用腺病毒转导表达配体诱导的蛋白激酶B（PKB，也称为cAkt）融合蛋白。这种蛋白质的PKB活性被证明是激活的，在30分钟内的4-羟基他莫昔芬，并保持高的8小时内，作为在位置473的PKB的丝氨酸磷酸化的结果，在转导的肝细胞。PKB活性的增加反映在由胰岛素蛋白（PHAS-I;也称为4 E-BP 1，用于真核起始因子4 E-结合蛋白1）调节的磷酸化的、热和酸稳定的蛋白质的过度磷酸化中，胰岛素蛋白是一种参与调节翻译起始的蛋白质。这些作用与非转导肝细胞中胰岛素诱导的内源性PKB活化和PHAS-I磷酸化相当。他莫昔芬转导肝细胞的加入导致葡萄糖激酶mRNA的诱导与胰岛素诱导的mRNA积累的动力学和幅度相似。他莫昔芬的作用依赖于刺激的PKB活性，因为它不会发生在用突变型PKB融合蛋白转导的肝细胞中，所述突变型PKB融合蛋白难以用他莫昔芬激活。这些结果表明，PKB的急性激活足以在分离的肝细胞中产生葡萄糖激酶的胰岛素样诱导。与磷酸肌醇3-激酶抑制剂的抑制作用一起，他们表明PKB的激活可能在介导胰岛素诱导葡萄糖激酶中起关键作用。此外，实验表明，PD 98059减少了一半的葡萄糖激酶mRNA的增加所带来的胰岛素，这表明有丝分裂原活化蛋白激酶级联的贡献作用。
Inhibitors of signalling pathways were used to dissect the mechanism of insulin action on expression of the gene encoding glucokinase in cultured rat hepatocytes. Wortmannin and LY 294002 completely prevented the insulin-induced increase in glucokinase mRNA seen in unhibited cells, indicating that the phosphoinositide 3-kinase module has a key role. A ligand inducible protein kinase B (PKB, also termed cAkt) fusion protein was expressed by using adenoviral transduction of hepatocytes in primary culture. The PKB activity of this protein was shown to be activated in transduced hepatocytes within 30 min of the addition of 4-hydroxytamoxifen and to stay high for 8 h, as a result of serine phosphorylation at position 473 of PKB. The increase in PKB activity was reflected in the hyperphosphorylation of phosphorylated, heat and acid stable regulated by insulin protein (PHAS-I; also termed 4E-BP1, for eukaryotic initiation factor 4E-binding protein 1), a protein involved in the regulation of translation initiation. These effects were comparable to the insulin-induced activation of endogenous PKB and phosphorylation of PHAS-I in non-transduced hepatocytes. The addition of tamoxifen to transduced hepatocytes resulted in an induction of glucokinase mRNA with kinetics and magnitude similar to those of insulin-induced mRNA accumulation. The effect of tamoxifen depended on stimulated PKB activity because it did not occur in hepatocytes that were transduced with a mutant PKB fusion protein that was refractory to activation with tamoxifen. These results establish that acute activation of PKB is sufficient to produce an insulin-like induction of glucokinase in isolated hepatocytes. Together with the inhibition by phosphoinositide 3-kinase inhibitors, they suggest that the activation of PKB might be critical in mediating the induction of glucokinase by insulin. In addition, experiments showed that PD98059 decreased by half the increase in glucokinase mRNA brought about by insulin, suggesting a contributory role of the mitogen-activated protein kinase cascade.