Intracellular sorting of glucose transporter and insulin action

胰岛素能迅速刺激肌肉和脂肪组织中的葡萄糖转运活动。葡萄糖转运活性的增加主要是由于葡萄糖转运体GLUT4从细胞内转运到质膜上。我们发现，PI3激酶的p110a催化亚基的过表达和P85亚基的显性负表达分别模拟和抑制了胰岛素的这一作用，从而证明了PI3激酶的激活在胰岛素刺激葡萄糖转运过程中是至关重要的。我们还研究了PI3激酶下游的信号。胰岛素刺激使Thr308和Ser473上的AKT1磷酸化，导致各种信号的激活，包括那些与葡萄糖代谢有关的信号。为了研究PDK1在AKT1磷酸化状态中的作用，用腺病毒基因转导系统在CHO-IR细胞中表达了野生型PDK1(wt-PDK1)及其激酶失活突变体(kd-PDK1)。抗磷酸化AKT1抗体免疫印迹显示，Thr308在胰岛素刺激1min时已达到最大磷酸化，但在5min时几乎完全去磷酸化。在过量表达wt-PDK1的CHO-IR细胞中，Thr308在胰岛素刺激下的磷酸化状态在1min时显著增加，但在5min时恢复到基础水平。相反，在高表达kd-PDK1的细胞中，Thr308的胰岛素刺激的磷酸化状态即使在15min也保持不变，这表明kd-PDK1的过表达抑制了胰岛素刺激的磷酸酶活性。WE-PDK1或kd-PDK1过表达不影响胰岛素刺激的Ser473磷酸化。

Insulin rapidly stimulates glucose transport activity in muscle and adipose tissue. This increase in glucose transport activity is primarily due to translocation of glucose transporter GLUT4 from intracellular compartment to the plasma membrane. We have shown that activation of PI3 kinase is crucial for insulin-stimulation of glucose transport, based on our findings that overexpression of p110a catalytic subuniti of PI3 kinase and dominant negative p85 subunit mimic and inhibit, respectively, this insulin action. We have also studied signals downstream of PI3 kinase. Insulin stimulation phosphorylates AKt1 at both Thr308 and Ser473, leading to activation of various signals including those towards glucose metabolism. To investigate the role of PDK1 on the phosphorylation state of AKt1, the wild-type PDK1 (wt-PDK1) and its kinase dead mutant (kd-PDK1) were expressed in CHO-IR cells using adenovirus gene transduction system. Immunoblotting using antiphosphorylated AKt1 antibody revealed that Thr308 was maximally phosphorylated already at 1 min by insulin stimulation but almost completely dephosphorylated at 5 min. Insulin-stimulated phosphorylation state of Thr308 was markedly increased in CHO-IR cells overexpressing wt-PDK1 at 1 min, but it returned to the basal level at 5 min. On the contrary, insulin-stimulated phosphorylation state of Thr308 was maintained even at 15 min in cells overexpressing kd-PDK1, suggesting that kd-PDK1 overexpression inhibited insulin-stimulated phosphatase activity. Insulin-stimulated Ser473 phosphorylation was not affected by overexpression of we-PDK1 or kd-PDK1.