Article

Signaling pathways through which insulin regulates CCAAT/enhancer binding protein (C/EBPα) phosphorylation and gene expression in 3T3-L1 adipocytes. Correlation with GLUT4 gene expression

University of Michigan, Ann Arbor, Michigan, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 11/1997; 272(41):25913-9. DOI: 10.1074/jbc.272.41.25913
Source: PubMed

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.

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    • "M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 3T3-L1 preadipocytes (NCCS, Pune, India) were cultured and differentiated as described previously (Hemati et al., 1997). Briefly, preadipocytes cells were grown for 2 days post confluence in DMEM (Invitrogen, USA) supplemented with 10% calf serum (Invitrogen, USA). "
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    ABSTRACT: Adipose tissue secretes adipokines that regulate insulin sensitivity in adipocytes and other peripheral tissues critical to glucose metabolism. Insulin resistance is associated with severe alterations in adipokines characterized by release of increased pro-inflammatory cytokines and decreased anti-inflammatory cytokines from adipose tissue. The role of Farnesoid X receptor (FXR) activation on adipokines in relation to adipose tissue inflammation and insulin resistance is not completely explored. For the first time, we have evaluated the ability of Chenodeoxycholic acid (CDCA), an endogenous FXR ligand, in restoring the disturbance in adipokine secretion and insulin resistance in palmitate treated 3T3-L1 cells and adipose tissues of High fat diet (HFD) rats. CDCA suppressed several of the tested pro-inflammatory adipokines (TNF-α, MCP-1, IL-6, Chemerin, PAI, RBP4, resistin, vaspin), and enhanced the major anti-inflammatory and insulin sensitizing adipokines (adiponectin, leptin). CDCA suppressed the activation of critical inflammatory regulators such as NF-κB and IKKβ which are activated by palmitate treatment in differentiated cells and HFD in rats. We show the altered adipokines in insulin resistance, its association with inflammatory regulators, and the role of CDCA in amelioration of insulin resistance by modulation of adipokines. Copyright © 2015. Published by Elsevier Ireland Ltd.
    Full-text · Article · Jul 2015 · Molecular and Cellular Endocrinology
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    • "3T3-L1 preadipocytes (NCCS, Pune, India) were cultured and differentiated as described previously (Hemati et al., 1997). Briefly, preadipocytes cells were grown for 2 days post confluence in DMEM (Invitrogen, USA) supplemented with 10% calf serum (Invitrogen, USA). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Adipose tissue secretes adipokines that regulate insulin sensitivity in adipocytes and other peripheral tissues critical to glucose metabolism. Insulin resistance is associated with severe alterations in adipokines characterized by release of increased pro-inflammatory cytokines and decreased anti-inflammatory cytokines from adipose tissue. The role of Farnesoid X receptor (FXR) activation on adipokines in relation to adipose tissue inflammation and insulin resistance is not completely explored. For the first time, we have evaluated the ability of Chenodeoxycholic acid (CDCA), an endogenous FXR ligand, in restoring the disturbance in adipokine secretion and insulin resistance in palmitate treated 3T3-L1 cells and adipose tissues of High fat diet (HFD) rats. CDCA suppressed several of the tested pro-inflammatory adipokines (TNF-α, MCP-1, IL-6, Chemerin, PAI, RBP4, resistin, vaspin), and enhanced the major anti-inflammatory and insulin sensitizing adipokines (adiponectin, leptin). CDCA suppressed the activation of critical inflammatory regulators such as NF-κB and IKKβ which are activated by palmitate treatment in differentiated cells and HFD in rats. We show the altered adipokines in insulin resistance, its association with inflammatory regulators, and the role of CDCA in amelioration of insulin resistance by modulation of adipokines.
    Full-text · Dataset · Jul 2015
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    • "3T3-L1 cells (ATCC, CL173, DS Pharma Biomedical Co., Osaka, Japan) were grown in Dulbecco's modified Eagle medium (DMEM; Wako Pure Chemical, Osaka, Japan) containing 10% (v/v) calf serum (BioWest). Adipocyte differentiation was done essentially as described but with minor modifications [38]. Briefly, 2 days post confluence, the medium was changed to DMEM containing 10% (v/v) fetal bovine serum (ICN Biomedicals, Aurora, OH, USA), 10 µg/ml insulin, 1 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine and 10 µM pioglitazone. "
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