Wortmannin-sensitive pathway is required for insulin-stimulated phosphorylation of inhibitor κBα

The aim of this study was to examine the signaling pathways by which insulin promotes activation of nuclear factor KB (NFKB) through the regulation of inhibitor KBalpha (IKBalpha). We show here that although insulin increased KB-dependent reporter gene expression and augmented nuclear translocation of the p65/RelA subunit of NFKB and its DNA binding, it was able to induce a time-dependent accumulation of phosphorylated and ubiquitinated IKBalpha without its proteolytic degradation. In contrast, cell stimulation with the cytokine TNFalpha allowed activation of NFKB through phosphorylation, ubiquitination, and subsequent degradation of IKBalpha. Immunofluorescence studies revealed the presence of a large pool of phosphorylated IKBalpha in the nucleus of unstimulated and insulin-treated cells. IKB kinase alpha and beta, central players in the phosphorylation of IKBalpha, were rapidly induced following exposure to TNFalpha but not insulin. Furthermore, insulin-stimulated IKBalpha phosphorylation did not depend on activation of the Ras/ERK cascade. Expression of a dominant-negative mutant of Akt1 or class I PI3K inhibited the insulin stimulation of PI3K/Akt1 signaling without affecting phosphorylation of IKBalpha. Interestingly, the PI3K inhibitors wortmannin and LY294002 blocked insulin-stimulated class I PI3K-dependent events at much lower doses than that required to inhibit phosphorylation of IKBalpha. These data demonstrate that insulin regulates IKBalpha function through a distinct low-affinity wortmannin-sensitive pathway.