Antonescu C N, Huang C, Niu W, Liu Z, Eyers P A, Heidenreich K A, Bilan P J, Klip A
Programme in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
Endocrinology. 2005 Sep;146(9):3773-81. doi: 10.1210/en.2005-0404. Epub 2005 Jun 9.
Insulin increases glucose uptake through translocation of the glucose transporter GLUT4 to the plasma membrane. We previously showed that insulin activates p38MAPK, and inhibitors of p38MAPKalpha and p38MAPKbeta (e.g. SB203580) reduce insulin-stimulated glucose uptake without affecting GLUT4 translocation. This observation suggested that insulin may increase GLUT4 activity via p38alpha and/or p38beta. Here we further explore the possible participation of p38MAPK through a combination of molecular strategies. SB203580 reduced insulin stimulation of glucose uptake in L6 myotubes overexpressing an SB203580-resistant p38alpha (drug-resistant p38alpha) but barely affected phosphorylation of the p38 substrate MAPK-activated protein kinase-2. Expression of dominant-negative p38alpha or p38beta reduced p38MAPK phosphorylation by 70% but had no effect on insulin-stimulated glucose uptake. Gene silencing via isoform-specific small interfering RNAs reduced expression of p38alpha or p38beta by 60-70% without diminishing insulin-stimulated glucose uptake. SB203580 reduced photoaffinity labeling of GLUT4 by bio-LC-ATB-BMPA only in the insulin-stimulated state. Unless low levels of p38MAPK suffice to regulate glucose uptake, these results suggest that the inhibition of insulin-stimulated glucose transport by SB203580 is likely not mediated by p38MAPK. Instead, changes experienced by insulin-stimulated GLUT4 make it susceptible to inhibition by SB203580.
胰岛素通过将葡萄糖转运蛋白GLUT4转运至质膜来增加葡萄糖摄取。我们之前发现胰岛素可激活p38丝裂原活化蛋白激酶（p38MAPK），并且p38MAPKα和p38MAPKβ的抑制剂（如SB203580）可降低胰岛素刺激的葡萄糖摄取，而不影响GLUT4的转运。这一观察结果提示胰岛素可能通过p38α和/或p38β增加GLUT4的活性。在此，我们通过多种分子策略进一步探究p38MAPK可能的参与情况。SB203580可降低过表达对SB203580耐药的p38α（耐药p38α）的L6肌管中胰岛素对葡萄糖摄取的刺激作用，但对p38底物丝裂原活化蛋白激酶激活的蛋白激酶-2的磷酸化几乎没有影响。显性负性p38α或p38β的表达可使p38MAPK磷酸化降低70%，但对胰岛素刺激的葡萄糖摄取没有影响。通过亚型特异性小干扰RNA进行基因沉默可使p38α或p38β的表达降低60 - 70%，而不减少胰岛素刺激的葡萄糖摄取。SB203580仅在胰岛素刺激状态下降低了生物素化的LC-ATB-BMPA对GLUT4的光亲和标记。除非低水平的p38MAPK足以调节葡萄糖摄取，否则这些结果表明SB203580对胰岛素刺激的葡萄糖转运的抑制作用可能不是由p38MAPK介导的。相反，胰岛素刺激的GLUT4所经历的变化使其易受SB203580的抑制。
