Signaling pathways through which insulin regulates CCAAT/enhancer binding protein alpha (C/EBPalpha) phosphorylation and gene expression in 3T3-L1 adipocytes. Correlation with GLUT4 gene expression.
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.
Hemati
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N
Department of Physiology, The University of Michigan Medical Center, Ann Arbor, Michigan 48109-0622, USA.
Ross
S E
SE
Erickson
R L
RL
Groblewski
G E
GE
MacDougald
O A
OA
D016475:3T3 Cells; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005786:Gene Expression Regulation; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000502:physiology*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000235:genetics* / Q000378:metabolism; D009124:Muscle Proteins; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011090:Polyenes / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism; D015398:Signal Transduction; D020123:Sirolimus; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D009687:Nuclear Proteins; D011090:Polyenes; D012333:RNA, Messenger; C495693:Slc2a4 protein, mouse; D014157:Transcription Factors; D007334:Insulin-Like Growth Factor I; D019869:Phosphatidylinositol 3-Kinases; D011972:Receptor, Insulin; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020123:Sirolimus