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

使用腺病毒载体进行基因转移分析胰岛素诱导的 PDE 激酶激活

基本信息

批准号：
11671122
负责人：
MAKINO Hideichi
金额：
$ 2.24万
依托单位：
Ehime University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671122/
关键词：
Insulin PDE3B adenovirus Akt PI 3-K PKB PKCλ

项目摘要

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.

磷酸二酯酶 (PDE) 3B 是参与胰岛素抗脂肪分解作用的关键酶。 PDE3B 被胰岛素磷酸化和激活，导致脂肪细胞游离脂肪酸 (FFA) 的输出减少。我们建立了 PDE 激酶的无细胞检测系统，PDE 激酶是一种磷酸化 PDE3B 的未知激酶。该系统使我们能够发现该激酶在磷脂酰肌醇 3-激酶（PI3-激酶）下游被胰岛素激活。 Akt 在一些重要的胰岛素信号通路（例如导致蛋白质合成的信号通路）中位于 PI3 激酶的下游，因此可能是激活 PDE 激酶的分子。为了检查 Akt 在胰岛素激活 PDE 激酶中的作用，我们使用腺病毒载体在 3T3-L1 脂肪细胞中建立了基因转移系统。含有 Akt 或 PI3-K 显性失活的腺病毒载体和作为对照的 β-半乳糖苷酶 (β-gal) 由神户大学的小川博士提供。去年，我们用这些腺病毒载体感染了293个细胞，并制备了高滴度病毒试剂。今年，我们将这些腺病毒载体转染到3T3-L1脂肪细胞中。我们通过蛋白质印迹检查 3T3-L1 脂肪细胞中引入的 β-gal、Akt 或 PI 3-K 基因表达，确定了每种病毒试剂的最佳滴度。我们现在正在研究 Akt 或 PI 3-K 显性失活的过度表达对胰岛素诱导的 PDE3B 激活和 PDE 激酶磷酸化的影响。这些实验将阐明 Akt 和 PI 3-K 在胰岛素诱导的 PDE 激酶激活中的作用。