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 insulin) 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 differentiation. As assessed by immunoblot analysis of one-and two-dimensional PAGE, insulin appears to dephosphorylate one site within p30C/EBP alpha and an additional site within p42C/EBP alpha. Consistent with insulin causing dephosphorylation of C/EBP alpha through activation of phosphatidylinositol 3-kinase, addition of phosphatidylinositol 3-kinase inhibitors (e.g. wortmannin) blocks insulin-stimulated dephosphorylation of C/EBP alpha. In the absence of insulin, wortmannin or LY294002 enhance C/EBP alpha phosphorylation. Similarly, blocking the activity of FKBP-rapamycin-associated protein with rapamycin increases phosphorylation of C/EBP alpha in the absence of insulin, Dephosphorylation of C/EBP alpha by insulin is partially blocked by rapamycin, consistent with a model in which activation of FKBP-rapamycin-associated protein by phosphatidylinositol 3-kinase results in dephosphorylation of C/EBP alpha. The dephosphorylation of C/EBP alpha by insulin, in conjunction with the insulin-dependent decline in C/EBP alpha mRNA and protein, has been hypothesized to play a role in repression of GLUT4 transcription by insulin, Consistent with this hypothesis, the decline of GLUT4 mRNA following exposure of adipocytes to insulin correlates with dephosphorylation of C/EBP alpha. However, the repression of C/EBP alpha mRNA and protein levels by insulin is blocked with an inhibitor of the mitogen-activated protein kinase pathway without blocking the repression of GLUT4 mRNA, thus dissociating the regulation of C/EBP alpha and GLUT4 mRNAs by insulin, A decline in C/EBP alpha mRNA and protein may not be required to suppress GLUT4 transcription because insulin also induces expression of the dominant-negative form of C/EBP beta (liver inhibitory protein), which blocks transactivation by C/EBP transcription factors.