SIGNALING PATHWAYS THROUGH WHICH INSULIN REGULATES CCAAT ENHANCER BINDING-PROTEIN-ALPHA (C/EBP-ALPHA) PHOSPHORYLATION AND GENE-EXPRESSION IN 3T3-L1 ADIPOCYTES - CORRELATION WITH GLUT4 GENE-EXPRESSION/

Treatment of 3T3-L1 adipocytes with insulin (IC50 similar to 200 pM in sulin) or insulin-like growth factor-1 (IC50 similar to 200 pM IGF-1) stimulates dephosphorylation of CCAAT/enhancer binding protein alpha ( C/EBP alpha), a transcription factor involved in preadipocyte differen tiation. As assessed by immunoblot analysis of one-and two-dimensional PAGE, insulin appears to dephosphorylate one site within p30C/EBP alp ha and an additional site within p42C/EBP alpha. Consistent with insul in causing dephosphorylation of C/EBP alpha through activation of phos phatidylinositol 3-kinase, addition of phosphatidylinositol 3-kinase i nhibitors (e.g. wortmannin) blocks insulin-stimulated dephosphorylatio n of C/EBP alpha. In the absence of insulin, wortmannin or LY294002 en hance C/EBP alpha phosphorylation. Similarly, blocking the activity of FKBP-rapamycin-associated protein with rapamycin increases phosphoryl ation of C/EBP alpha in the absence of insulin, Dephosphorylation of C /EBP alpha by insulin is partially blocked by rapamycin, consistent wi th a model in which activation of FKBP-rapamycin-associated protein by phosphatidylinositol 3-kinase results in dephosphorylation of C/EBP a lpha. The dephosphorylation of C/EBP alpha by insulin, in conjunction with the insulin-dependent decline in C/EBP alpha mRNA and protein, ha s been hypothesized to play a role in repression of GLUT4 transcriptio n by insulin, Consistent with this hypothesis, the decline of GLUT4 mR NA following exposure of adipocytes to insulin correlates with dephosp horylation of C/EBP alpha. However, the repression of C/EBP alpha mRNA and protein levels by insulin is blocked with an inhibitor of the mit ogen-activated protein kinase pathway without blocking the repression of GLUT4 mRNA, thus dissociating the regulation of C/EBP alpha and GLU T4 mRNAs by insulin, A decline in C/EBP alpha mRNA and protein may not be required to suppress GLUT4 transcription because insulin also indu ces expression of the dominant-negative form of C/EBP beta (liver inhi bitory protein), which blocks transactivation by C/EBP transcription f actors.