New cDNAs and proteins phosphorylated by insulin using proteome analysis

胰岛素通过刺激葡萄糖摄取和糖原合成，在葡萄糖稳态调节中发挥重要作用。丝氨酸/苏氨酸蛋白激酶Akt已被提出在几个过程中介导胰岛素信号传导。然而，目前尚不清楚Akt是否参与胰岛素刺激的葡萄糖摄取，以及Akt的哪些亚型是否负责每种胰岛素作用。我们证实，使用腺病毒表达载体表达的组成型活性Akt促进葡萄糖转运蛋白4（GLUT 4）易位到质膜，2-脱氧葡萄糖（2-DG）的摄取和糖原合成在中国仓鼠卵巢（CHO）细胞和3 T3-L1脂肪细胞。通过腺病毒表达显性阴性Akt或引入合成的21-mer短干扰RNA（siRNA）抑制Akt显著降低胰岛素刺激的GLUT 4易位、2-DG摄取和糖原合成。同种型特异性siRNA的实验显示，Akt 2和Akt 1在较小程度上在两种细胞系中胰岛素刺激的GLUT 4易位和2-DG摄取中具有重要作用，而Akt 1和Akt 2同样有助于胰岛素刺激的糖原合成。这些数据表明Akt在胰岛素刺激的葡萄糖摄取中的先决条件作用和Akt亚型之间的不同功能。

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 if Akt is involved in insulin-stimulated glucose uptake, and whether 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 (CHO) 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 (siRNA) against Akt markedly reduced insulin-stimulated GLUT4 translocation, 2-DG uptake and glycogen synthesis. Experiments with isoform-specific siRNA revealed that Akt2, and Akt1 to lesser extent, has an essential role in insulin-stimulated GLUT4 translocation and 2-DG uptake in both cell lines, while 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.