Use of RNA interference-mediated gene silencing and adenoviral overexpression to elucidate the roles of AKT/protein kinase B isoforms in insulin actions
Use of RNA interference-mediated gene silencing and adenoviral overexpression to elucidate the roles of AKT/protein kinase B isoforms in insulin actions
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DOI：
10.1074/jbc.m302094200
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发表时间：
2003-07-25
影响因子：
4.8
通讯作者：
Ebina, Y
中科院分区：
文献类型：
--
作者：
Katome, T;Obata, T;Ebina, Y
胰岛素通过刺激葡萄糖摄取和糖原合成，在调节葡萄糖动态平衡方面发挥核心作用。丝氨酸/苏氨酸蛋白激酶Akt被认为在几个过程中介导胰岛素信号传导。然而，目前尚不清楚Akt是否参与了胰岛素刺激的葡萄糖摄取，以及Akt的哪些亚型负责每种胰岛素的作用。我们证实，在中国仓鼠卵巢细胞和3T3-L1脂肪细胞中，使用腺病毒表达载体表达具有组成活性的Akt可以促进葡萄糖转运蛋白4(GLUT4)向质膜的转位、2-脱氧葡萄糖(2-DG)的摄取和糖原的合成。通过腺病毒表达显性的阴性Akt或通过引入合成的针对Akt的21聚体短干扰RNA来抑制Akt显著减少胰岛素刺激的GLUT4转位、2-DG摄取和糖原合成。用异构体特异的短干扰RNA进行的实验表明，Akt2和Akt1在胰岛素刺激的GLUT4转位和2-DG摄取方面发挥了重要作用，而Akt1和Akt2对胰岛素刺激的糖原合成起到了同等的作用。这些数据表明Akt在胰岛素刺激的葡萄糖摄取中起着先决条件的作用，并在Akt亚型之间具有不同的功能。
Insulin plays a central role in the regulation of glucose homeostasis in part by stimulating glucose uptake and glycogen synthesis. The serine/threonine protein kinase Akt has been proposed to mediate insulin signaling in several processes. However, it is unclear whether Akt is involved in insulin-stimulated glucose uptake and which isoforms of Akt are responsible for each insulin action. We confirmed that expression of a constitutively active Akt, using an adenoviral expression vector, promoted translocation of glucose transporter 4 (GLUT4) to plasma membrane, 2-deoxyglucose (2-DG) uptake, and glycogen synthesis in both Chinese hamster ovary cells and 3T3-L1 adipocytes. Inhibition of Akt either by adenoviral expression of a dominant negative Akt or by the introduction of synthetic 21-mer short interference RNA against Akt markedly reduced insulin-stimulated GLUT4 translocation, 2-DG uptake, and glycogen synthesis. Experiments with isoform-specific short interference RNA revealed that Akt2, and Akt1 to a lesser extent, has an essential role in insulin-stimulated GLUT4 translocation and 2-DG uptake in both cell lines, whereas Akt1 and Akt2 contribute equally to insulin-stimulated glycogen synthesis. These data suggest a prerequisite role of Akt in insulin-stimulated glucose uptake and distinct functions among Akt isoforms.