Phosphorylation of CCAAT/Enhancer-binding Protein α Regulates GLUT4 Expression and Glucose Transport in Adipocytes

Abstract

The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) is required during adipogenesis for development of insulin-stimulated glucose uptake. Modes for regulating this function of C/EBPα have yet to be determined. Phosphorylation of C/EBPα on Ser-21 has been implicated in the regulation of granulopoiesis and hepatic gene expression. To explore the role of Ser-21 phosphorylation on C/EBPα function during adipogenesis, we developed constructs in which Ser-21 was mutated to alanine (S21A) to model dephosphorylation. In two cell culture models deficient in endogenous C/EBPα, enforced expression of S21A-C/EBPα resulted in normal lipid accumulation and expression of many adipogenic markers. However, S21A-C/EBPα had impaired ability to activate the Glut4 promoter specifically, and S21A-C/EBPα expression resulted in diminished GLUT4 and adiponectin expression, as well as reduced insulin-stimulated glucose uptake. No defects in insulin signaling or GLUT4 vesicle trafficking were identified with S21A-C/EBPα expression, and when exogenous GLUT4 expression was enforced to normalize expression in S21A-C/EBPα cells, insulin-responsive glucose transport was reconstituted, suggesting that the primary defect was a deficit in GLUT4 levels. Mice in which endogenous C/EBPα was replaced with S21A-C/EBPα displayed reduced GLUT4 and adiponectin protein expression in epididymal adipose tissue and increased blood glucose compared with wild-type littermates. These results suggest that phosphorylation of C/EBPα on Ser-21 may regulate adipocyte gene expression and whole body glucose homeostasis. The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) is required during adipogenesis for development of insulin-stimulated glucose uptake. Modes for regulating this function of C/EBPα have yet to be determined. Phosphorylation of C/EBPα on Ser-21 has been implicated in the regulation of granulopoiesis and hepatic gene expression. To explore the role of Ser-21 phosphorylation on C/EBPα function during adipogenesis, we developed constructs in which Ser-21 was mutated to alanine (S21A) to model dephosphorylation. In two cell culture models deficient in endogenous C/EBPα, enforced expression of S21A-C/EBPα resulted in normal lipid accumulation and expression of many adipogenic markers. However, S21A-C/EBPα had impaired ability to activate the Glut4 promoter specifically, and S21A-C/EBPα expression resulted in diminished GLUT4 and adiponectin expression, as well as reduced insulin-stimulated glucose uptake. No defects in insulin signaling or GLUT4 vesicle trafficking were identified with S21A-C/EBPα expression, and when exogenous GLUT4 expression was enforced to normalize expression in S21A-C/EBPα cells, insulin-responsive glucose transport was reconstituted, suggesting that the primary defect was a deficit in GLUT4 levels. Mice in which endogenous C/EBPα was replaced with S21A-C/EBPα displayed reduced GLUT4 and adiponectin protein expression in epididymal adipose tissue and increased blood glucose compared with wild-type littermates. These results suggest that phosphorylation of C/EBPα on Ser-21 may regulate adipocyte gene expression and whole body glucose homeostasis. Although adipocytes were initially thought to be passive storage vessels for caloric excess, it is now known that adipose tissue acts as an important metabolic and endocrine organ (1Minokoshi Y. Kahn C.R. Kahn B.B. J. Biol. 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Full Text Full Text PDF PubMed Scopus Google the C/EBPα, the expression of or in two cell deficient in C/EBPα, and in ability to adipogenesis as by and by of lipid that as of Ser-21 of C/EBPα protein In S21A-C/EBPα expression of adipocyte as and PPARγ reduced of GLUT4 and adiponectin suggest that phosphorylation of Ser-21 may regulate expression of Although we have on and the S21A-C/EBPα results were in and to wild-type However, we with be of GLUT4 and with of adipocyte is in S21A-C/EBPα during adipogenesis, were the and expression of adipocyte was by and PPARγ were increased by wild-type and S21A-C/EBPα during of Although GLUT4 and adiponectin were of in wild-type and S21A-C/EBPα cells, expression with S21A-C/EBPα and that of Ser-21 phosphorylation expression of GLUT4 and of in with expression of C/EBPα and endogenous PPARγ, and in wild-type and S21A-C/EBPα adipocytes reduced expression of GLUT4 and adiponectin in S21A-C/EBPα for a that is with insulin S. 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S. 2001; PubMed Scopus Google was increased in this was in the for reduced expression of GLUT4 by of gene constructs by wild-type C/EBPα and phosphorylation Although and activate the for C/EBPα and in and cell the Glut4 promoter is by expression of wild-type and S21A-C/EBPα in was by These suggest that phosphorylation of Ser-21 is required for of the Glut4 promoter by with S21A-C/EBPα of and on on that phosphorylation of Ser-21 is required for C/EBPα to activate expression of the glucose and a protein with insulin in two cell models for adipogenesis, we that adipocytes that S21A-C/EBPα may have impaired to Although glucose uptake was insulin-stimulated glucose uptake was in adipocytes that wild-type C/EBPα to with S21A-C/EBPα and S21A-C/EBPα have diminished glucose uptake in to insulin, is a in glucose as a of an in expression in with enforced expression of S21A-C/EBPα studies that adipocytes S21A-C/EBPα have a in glucose uptake insulin compared with and to of insulin is in in studies suggest that in S21A-C/EBPα glucose uptake in to insulin is to of insulin of insulin the for insulin-stimulated glucose uptake was suggesting that reduced expression of GLUT4 is the primary defect in S21A-C/EBPα S21A-C/EBPα many insulin to receptor and the transport of glucose the The the well with of insulin to receptor by receptor and phosphorylation of To the reduced insulin-stimulated glucose uptake in is a of insulin we expression and insulin-stimulated phosphorylation of insulin insulin receptor and the phosphorylation of which is in insulin-stimulated glucose and which is in cell and of S21A-C/EBPα in adipocytes expression insulin-stimulated phosphorylation of of of the insulin signaling phosphorylation of and was reduced in adipocytes with insulin results were in with of insulin the that expression of S21A-C/EBPα may to reduced phosphorylation of in to insulin However, S21A-C/EBPα glucose uptake to of insulin it is that this in insulin-stimulated phosphorylation is a to impaired insulin-stimulated glucose uptake in S21A-C/EBPα GLUT4 to in S21A-C/EBPα to the of GLUT4 vesicle trafficking in we that expression of and for GLUT4 vesicle was in adipocytes S21A-C/EBPα In and were reduced in adipocytes S21A-C/EBPα To GLUT4 vesicle trafficking is in adipocytes that we two we adipocytes wild-type or S21A-C/EBPα with a expression with insulin, we of in which GLUT4 to the fluorescent and cell or fluorescent results were in we and by that in to insulin, the of endogenous GLUT4 to the was to expression in wild-type or S21A-C/EBPα phosphorylation of C/EBPα on Ser-21 to GLUT4 vesicle and reduced GLUT4 in S21A-C/EBPα adipocytes the in expression of of GLUT4 in S21A-C/EBPα on insulin signaling insulin-stimulated GLUT4 vesicle transport to be in S21A-C/EBPα we developed expression constructs for GLUT4 and adiponectin to enforced expression of insulin-stimulated glucose uptake in S21A-C/EBPα suggest that constructs normalize expression of GLUT4 and adiponectin in S21A-C/EBPα adipocytes to with wild-type C/EBPα enforced expression of GLUT4 in S21A-C/EBPα adipocytes the to insulin-stimulated glucose uptake In S21A-C/EBPα adipocytes with expression of adiponectin had reduced insulin-stimulated glucose uptake compared with wild-type C/EBPα adipocytes in adiponectin expression may be a of glucose as enforced expression of GLUT4 expression of adiponectin in S21A-C/EBPα adipocytes results the that the primary defect to reduced insulin-stimulated glucose uptake in S21A-C/EBPα adipocytes is reduced GLUT4 expression. S21A-C/EBPα Mice of GLUT4 and in and and phosphorylation of Ser-21 is important for ability of C/EBPα to development and gene expression of adipose tissue in we developed in which the endogenous C/EBPα is replaced with S21A-C/EBPα or Although and wild-type had in body or epididymal white adipose tissue we that epididymal in had an in GLUT4 protein expression and in adiponectin protein expression this in GLUT4 and adiponectin protein a in C/EBPα protein expression compared with wild-type Although in blood glucose were in the to an in and blood glucose in suggesting in of C/EBPα phosphorylation on blood glucose levels. were as by the to glucose had glucose to wild-type of reduced epididymal adipose tissue expression of increased blood and diminished glucose in S21A-C/EBPα suggest that phosphorylation of C/EBPα may to of glucose a yet to be C/EBPα and PPARγ for many of the adipocyte and cell have of the function for In deficient in PPARγ, enforced expression of C/EBPα is to lipid in and expression of PPARγ lipid accumulation and the of an transport glucose in to insulin (8Wu Z. 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In the we to the ability of C/EBPα to insulin-stimulated glucose transport in adipocytes is regulated by phosphorylation on phosphorylation of C/EBPα on Ser-21 was by a to and displayed a in insulin-responsive glucose uptake In phosphorylation of C/EBPα on Ser-21 was required for expression of GLUT4 and in lipid gene expression, or insulin and this deficit in GLUT4 expression was for reduced glucose uptake. we that expression of S21A-C/EBPα a or for in in body or epididymal white adipose tissue However, expression of GLUT4 and adiponectin was reduced in and displayed and glucose role for that glucose as in to has been is the for Ser-21 phosphorylation in adipocytes S.E. MacDougald O.A. Mol. Cell. Biol. 2004; 24: PubMed Scopus Google in is thought to be for phosphorylation this MacDougald O.A. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). 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Full Text Full Text PDF PubMed Scopus Google on Ser-21 has deficient function in this in C/EBPα expression may be a of to GLUT4 adiponectin in for in of C/EBPα function of or of phosphorylation on Ser-21 may to this The of GLUT4 expression in adipocyte and whole body has been by in and by In of insulin as in and GLUT4 expression is in adipose tissue in muscle P.R. Kahn B.B. N. Engl. J. Med. 1999; 341: 248-257Crossref PubMed Scopus (1095) Google Scholar, Diabetes PubMed Scopus Google Scholar). on the insulin C/EBPα and adipocyte it is that insulin may adipocyte function regulation of C/EBPα insulin was to GLUT4 expression in adipocytes increased C/EBPα expression R. T. M. J. 2003; PubMed Google insulin which is the for this is by increased phosphorylation on Ser-21 to enhanced GLUT4 expression. In of regulation of GLUT4 expression of C/EBPα expression or in to cell have been S. R. T. M. M. PubMed Scopus Google Scholar, D. Res. PubMed Scopus Google Scholar, D. J. N. PubMed Scopus Google Scholar, J. Mol. PubMed Scopus Google Scholar). The of regulation of GLUT4 expression by transcription factors in in glucose has been M. C. Metab. 18: Full Text Full Text PDF PubMed Scopus Google Scholar). Mice in which GLUT4 has been in adipose tissue have normal and adipose impaired insulin-stimulated glucose uptake in insulin in muscle and glucose and E.D. J.K. T. Kahn B.B. Nature. 2001; PubMed Scopus Google Scholar). In GLUT4 displayed in and expression S.E. S. Kahn B.B. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google we in or expression in GLUT4 was in to normal expression of and is the that defects during adipogenesis, as to defects that may in to metabolic of GLUT4 insulin and in GLUT4 in muscle K. Kahn B.B. J. 2005; PubMed Scopus Google the important insulin-responsive in of glucose homeostasis. and displayed in blood had and blood glucose were in insulin or adiponectin adiponectin expression in of defects primary defects as a of enforced of C/EBPα on Ser-21 or a of by the of expression in have yet to be determined. In we that of the displayed phosphorylation Ser-21 S.E. MacDougald O.A. Mol. Cell. Biol. 2004; 24: PubMed Scopus Google and it has been that increased in MacDougald O.A. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and GLUT4 expression in adipose tissue with P.R. Kahn B.B. N. Engl. J. Med. 1999; 341: 248-257Crossref PubMed Scopus (1095) Google Scholar, Diabetes PubMed Scopus Google suggesting that the in glucose in may be a of C/EBPα function in the as well as adipose The role of C/EBPα in adipocyte and as well as gene expression J. J. PubMed Scopus Google that the by which C/EBPα function is by phosphorylation may into the regulation of whole body glucose homeostasis.