The aim of this study was to examine the signaling pathways by which insulin promotes activation of nuclear factor κB (NFκB) through the regulation of inhibitor κBα (IκBα). We show here that although insulin increased κB-dependent reporter gene expression and augmented nuclear translocation of the p65/RelA subunit of NFκB and its DNA binding, it was able to induce a time-dependent accumulation of phosphorylated and ubiquitinated IκBα without its proteolytic degradation. In contrast, cell stimulation with the cytokine TNFα allowed activation of NFκB through phosphorylation, ubiquitination, and subsequent degradation of IκBα. Immunofluorescence studies revealed the presence of a large pool of phosphorylated IκBα in the nucleus of unstimulated and insulin-treated cells. IκB kinase α and β, central players in the phosphorylation of IκBα, were rapidly induced following exposure to TNFα but not insulin. Furthermore, insulin-stimulated IκBα 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 IκBα. 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 IκBα. These data demonstrate that insulin regulates IκBα function through a distinct low-affinity wortmannin-sensitive pathway.
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