A novel mechanism of insulin sensitivity regulation by post-translational mechanisms

相扑结合酶Ubc9已被证明可上调L6成肌细胞中的GLUT4，尽管作用机制尚不清楚。在此，我们通过腺病毒载体介导的Ubc9在3T3-L1脂肪细胞中的过表达和通过siRNA介导的Ubc9基因沉默来研究Ubc9在GLUT4转运和亚细胞靶向中的生理意义。Ubc9的过表达抑制了GLUT4的降解，促进了它对GLUT4储存室(GSC)的靶向，导致GLUT4水平增加，胰岛素应答的GLUT4转位和葡萄糖转运。长期(6小时以上)胰岛素刺激可导致GLUT4表达下调40-50%，该作用可被溶酶体抑制剂抑制，并与GLUT4选择性降低有关，而Ubc9的过表达可拮抗胰岛素的这些作用。相反，用siRNA沉默Ubc9基因显著加速了胰岛素诱导的GLUT4下调，而过表达催化失活突变体Ubc9-C93A则增加了GLUT4和胰岛素刺激的葡萄糖转运，达到与野生型Ubc9相当的水平。这些结果表明，Ubc9通过抑制溶酶体分选上调GLUT4，促进GLUT4靶向GSC，其机制与其催化活性无关。因此，Ubc9在脂肪细胞表达和维持胰岛素敏感的葡萄糖运输系统中起着不可或缺的作用。

SUMO conjugating enzyme Ubc9 has been shown to upregulate GLUT4 in L6 myoblasts although the mechanism of action is undefined. Here we investigated the physiological significance of Ubc9 in GLUT4 turnover and subcellular targeting by adenovirus vector-mediated overexpression and by siRNA-mediated gene silencing of Ubc9 in 3T3-L1 adipocytes. Overexpression of Ubc9 resulted in inhibition of GLUT4 degradation and promotion of its targeting to the GLUT4 storage compartment (GSC), leading to an increase in GLUT4 level and insulin-responsive GLUT4 translocation and glucose transport. While long-term (6 hours and more) insulin stimulation caused GLUT4 downregulation by 40-50%, which was inhibited with lysosomal inhibitors and was associated with a selective reduction in GLUT4 in GSC, overexpression of Ubc9 antagonized these effects of insulin. By contrast, Ubc9 gene silencing with siRNA markedly accelerated the insulin-induced GLUT4 downregulation, whereas overexpression of the catalytically inactive mutant Ubc9-C93A increased GLUT4 and insulin-stimulated glucose transport to the level comparable to that with wild-type Ubc9. These results suggest that Ubc9 upregulates GLUT4 by inhibition of lysosomal sorting and promotes GLUT4 targeting to GSC by a mechanism unrelated to its catalytic activity. Thus, Ubc9 plays a indispensable role in expression and maintenance of the insulin-sensitive glucose transport system in adipocytes.