Abstract
Treatment of 3T3-L1 adipocytes with insulin (IC50 approximately 200 pM insulin) or insulin-like growth factor-1 (IC50 approximately 200 pM IGF-1) stimulates dephosphorylation of CCAAT/enhancer binding protein alpha (C/EBPalpha), 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/EBPalpha and an additional site within p42C/EBPalpha. Consistent with insulin causing dephosphorylation of C/EBPalpha through activation of phosphatidylinositol 3-kinase, addition of phosphatidylinositol 3-kinase inhibitors (e.g. wortmannin) blocks insulin-stimulated dephosphorylation of C/EBPalpha. In the absence of insulin, wortmannin or LY294002 enhance C/EBPalpha phosphorylation. Similarly, blocking the activity of FKBP-rapamycin-associated protein with rapamycin increases phosphorylation of C/EBPalpha in the absence of insulin. Dephosphorylation of C/EBPalpha 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/EBPalpha. The dephosphorylation of C/EBPalpha by insulin, in conjunction with the insulin-dependent decline in C/EBPalpha 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/EBPalpha. However, the repression of C/EBPalpha 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/EBPalpha and GLUT4 mRNAs by insulin. A decline in C/EBPalpha mRNA and protein may not be required to suppress GLUT4 transcription because insulin also induces expression of the dominant-negative form of C/EBPbeta (liver inhibitory protein), which blocks transactivation by C/EBP transcription factors.
Highlights
Treatment of 3T3-L1 adipocytes with insulin (IC50 ϳ200 pM insulin) or insulin-like growth factor-1 (IC50 ϳ200 pM IGF-1) stimulates dephosphorylation of CCAAT/ enhancer binding protein ␣ (C/EBP␣), a transcription factor involved in preadipocyte differentiation
Insulin Regulates C/EBP␣ Dephosphorylation through Activation of the Insulin Receptor—We have shown previously that high concentrations of insulin stimulate an increase in mobility of p30C/EBP␣ and p42C/EBP␣ on SDS-PAGE (21)
To assess whether insulin regulates mobility of C/EBP␣ through activation of the insulin receptor, we examined the dependence of this effect on concentration of insulin. 3T3-L1 adipocytes were incubated with 0 –300 nM insulin prior to cell lysis
Summary
C/EBP, CCAAT/enhancer-binding protein; FRAP, FKBP-rapamycin-associated protein; GLUT4, insulin-responsive glucose transporter; IGF-1, insulin-like growth factor-1; PBS, phosphate-buffered saline; DMEM, Dulbecco’s modified Eagle’s medium; PAGE, polyacrylamide gel electrophoresis; PI, phosphatidylinositol; MAPK, mitogen-activated protein kinase; PVDF, polyvinylidene difluoride. The decline of GLUT4 following exposure of adipocytes to insulin (30) is temporally correlated with the suppression of C/EBP␣ mRNA and protein, the dephosphorylation of C/EBP␣, and the induction of LIP (21). Taken together, these data are consistent with a model in which insulin-mediated regulation of C/EBP transcription factors results in repression of GLUT4 transcription. We demonstrate that suppression of GLUT4 mRNA by insulin does not require a decline in C/EBP␣ mRNA and protein, perhaps indicating that dephosphorylation of C/EBP␣ and induction of LIP play important roles in the regulation of GLUT4 by insulin
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