Analysis of insulin-induced PDE kinase activation by gene transfer using adenovirus vector

磷酸二酯酶(PDE)3B是参与胰岛素抗脂作用的关键酶。PDE3B被磷酸化并被胰岛素激活，这导致脂肪细胞产生的游离脂肪酸(FFA)减少。我们已经建立了PDE激酶的无细胞检测系统，该系统是一种使PDE3B磷酸化的未知激酶。该系统使我们能够发现该激酶是由磷脂酰肌醇3-激酶(PI3-K)下游的胰岛素激活的。在一些重要的胰岛素信号通路中，AKT位于PI3-激酶的下游，如蛋白质合成途径，因此，AKT可能是一个激活PDE激酶的分子。为了研究Akt在胰岛素激活PDE激酶中的作用，我们利用腺病毒载体建立了3T3-L1脂肪细胞的基因转移系统。携带AKT或PI3-K显性阴性的腺病毒载体和β-半乳糖苷酶(β-GAL)作为对照，由神户大学的Ogawa博士提供。去年，我们用这些腺病毒载体感染了293细胞，并制备了高效价病毒试剂。今年，我们将这些腺病毒载体导入3T3-L1脂肪细胞。我们通过Western blotting检测导入的β-Gal、Akt或PI 3-K基因在3T3-L1脂肪细胞中的表达，确定了每种病毒试剂的最佳滴度。我们现在正在研究Akt或PI3-K显性负值的过度表达对胰岛素诱导的PDE3B激活和PDE激酶对PDE3B磷酸化的影响。这些实验将阐明Akt和PI3-K在胰岛素诱导的PDE激酶激活中的作用。

Phosphodiesterase (PDE) 3B is a key enzyme involved in anti-lipolytic action of insulin. PDE3B is phosphorylated and activated by insulin, which results in a reduced output of free fatty acids (FFA) from adipocytes. We have established cell-free assay system for PDE kinase, an unknown kinase which phosphorylates PDE3B.This system enabled us to find this kinase is activated by insulin at the downstream of phosphatidylinositol 3-kinase (PI3-kinase). Akt is located at the downstream of PI3-kinase in some of important insulin signaling pathways such as ones leading to protein synthesis, thus, could be a molecule which activates PDE kinase. To examine a role of Akt in PDE kinase activation by insulin, we established gene transfer system into 3T3-L1 adipocytes using adnovirus vectors. Adenovirus vectors containing dominant negatives for Akt or PI3-K, and β-galactosidase (β-gal) as controls were provided by Dr.Ogawa at Kobe University. Last year, we infected 293 cells with these adenovirus vectors and prepared high titer virus reagents. This year, we transfected these adenovirus vectors into 3T3-L1 adipocytes. We determined optimal titers for each virus reagents by checking either introduced β-gal, Akt, or PI 3-K gene expression in 3T3-L1 adipocytes by Western blotting. We are now examining the effect of overexpression of dominant negatives for Akt or PI 3-K on insulin-induced PDE3B activation and PDE3B phosphorylation by PDE kinase. These experiments will clarify roles of Akt and PI 3-K in insulin-induced PDE kinase activation.