Clarke J F, Young P W, Yonezawa K, Kasuga M, Holman G D
Department of Biochemistry, University of Bath, U.K.
Biochem J. 1994 Jun 15;300 ( Pt 3)(Pt 3):631-5. doi: 10.1042/bj3000631.
Wortmannin is a potent and reversible inhibitor of insulin-stimulated PtdIns 3-kinase activity in 3T3-L1 cells (IC50 = 2.6 +/- 0.8 nM). Wortmannin inhibits the PtdIns 3-kinase activity which is precipitated with antibodies against insulin receptor substrate 1 and against the alpha-p85 subunit of PtdIns 3-kinase. These observations suggest that wortmannin inhibits at the p110 catalytic subunit of PtdIns 3-kinase. Insulin stimulation of glucose transport in permeabilized 3T3-L1 cells is also inhibited by wortmannin (IC50 = 6.4 +/- 1.4 nM). Wortmannin did not inhibit basal glucose transport activity. The close similarity of the IC50 values for wortmannin inhibition of insulin-stimulated PtdIns 3-kinase and glucose transport activities suggests that the PtdIns 3-kinase is a key intermediate in insulin signalling of glucose-transport stimulation. The wortmannin inhibitory effect on transport is associated with a reduction in the cell-surface, but not the total cellular, levels of both GLUT1 and GLUT4 glucose transporter isoforms that are accessible to the cell-impermeant photolabel, ATB-BMPA. These photolabelling results suggest that the glucose transporter translocation process is dependent upon PtdIns 3-kinase activity. The stimulatory effect of guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) on glucose transport activity in permeabilized cells is only partially blocked by concentrations of wortmannin that completely inhibit the stimulatory effect of insulin. The residual stimulatory effect of GTP gamma S that occurs in the presence of wortmannin suggests that at least part of the GTP gamma S effect is mediated at a signalling site that is downstream of the site at which wortmannin inhibits the insulin stimulation of PtdIns 3-kinase and glucose transport activities.
渥曼青霉素是3T3-L1细胞中胰岛素刺激的磷脂酰肌醇3激酶（PtdIns 3-激酶）活性的一种强效可逆抑制剂（IC50 = 2.6±0.8 nM）。渥曼青霉素抑制与抗胰岛素受体底物1抗体和抗PtdIns 3-激酶α-p85亚基抗体沉淀的PtdIns 3-激酶活性。这些观察结果表明，渥曼青霉素在PtdIns 3-激酶的p110催化亚基处起抑制作用。渥曼青霉素也抑制透化的3T3-L1细胞中胰岛素刺激的葡萄糖转运（IC50 = 6.4±1.4 nM）。渥曼青霉素不抑制基础葡萄糖转运活性。渥曼青霉素抑制胰岛素刺激的PtdIns 3-激酶和葡萄糖转运活性的IC50值非常相似，这表明PtdIns 3-激酶是胰岛素刺激葡萄糖转运信号传导中的关键中间体。渥曼青霉素对转运的抑制作用与细胞表面（而非总细胞）水平的GLUT1和GLUT4葡萄糖转运异构体的减少有关，这些异构体可被细胞不透性光标记物ATB-BMPA标记。这些光标记结果表明，葡萄糖转运体转位过程依赖于PtdIns 3-激酶活性。鸟苷5'-[γ-硫代]三磷酸（GTPγS）对透化细胞中葡萄糖转运活性的刺激作用仅被完全抑制胰岛素刺激作用的渥曼青霉素浓度部分阻断。渥曼青霉素存在时GTPγS的残余刺激作用表明，至少部分GTPγS效应是在渥曼青霉素抑制胰岛素刺激PtdIns 3-激酶和葡萄糖转运活性的位点下游的信号位点介导的。
