Abstract
The transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) is required during adipogenesis for development of insulin-stimulated glucose uptake. Modes for regulating this function of C/EBPalpha have yet to be determined. Phosphorylation of C/EBPalpha 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/EBPalpha 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/EBPalpha, enforced expression of S21A-C/EBPalpha resulted in normal lipid accumulation and expression of many adipogenic markers. However, S21A-C/EBPalpha had impaired ability to activate the Glut4 promoter specifically, and S21A-C/EBPalpha 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/EBPalpha expression, and when exogenous GLUT4 expression was enforced to normalize expression in S21A-C/EBPalpha cells, insulin-responsive glucose transport was reconstituted, suggesting that the primary defect was a deficit in GLUT4 levels. Mice in which endogenous C/EBPalpha was replaced with S21A-C/EBPalpha 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/EBPalpha on Ser-21 may regulate adipocyte gene expression and whole body glucose homeostasis.
Highlights
Adipocytes were initially thought to be passive storage vessels for caloric excess, it is known that adipose tissue acts as an important metabolic and endocrine organ [1,2,3,4]
C/EBP␣ Phosphorylation and Glucose Transport in Adipocytes. These studies indicate that C/EBP␣ is required for acquisition of insulin sensitivity, PPAR␥ may be sufficient for lipid accumulation [19], indicating that each may be required for expression of a subset of adipocyte genes and that together these master regulators of adipogenesis stimulate the full complement of genes required for the adipocyte phenotype
C/EBP␣ Phosphorylation on Ser-21 Is Not Required for Adipogenic Lipid Accumulation but Is Necessary for Full Expression of GLUT4 and Adiponectin—To determine whether C/EBP␣ is phosphorylated on Ser-21 during adipogenesis, we performed a time course in which whole cell lysates were collected throughout differentiation of 3T3-L1 preadipocytes
Summary
C/EBP␣, CCAAT/enhancer-binding protein ␣; PPAR␥, peroxisome proliferator-activated receptor ␥; MEF, mouse embryonic fibroblast; PBS, phosphate-buffered saline; MAPK, mitogen-activated protein kinase; Erk, extracellular signal-regulated kinase; E-WAT, epididymal white adipose tissue; EGFP, enhanced green fluorescent protein; RT, reverse transcription. Together, these studies indicate that C/EBP␣ is required for acquisition of insulin sensitivity, PPAR␥ may be sufficient for lipid accumulation [19], indicating that each may be required for expression of a subset of adipocyte genes and that together these master regulators of adipogenesis stimulate the full complement of genes required for the adipocyte phenotype. Male S21A mice have elevated fasted and fed blood glucose levels compared with wild-type littermates and are glucose-intolerant, suggesting that phosphorylation of Ser-21 may be required to maintain glucose homeostasis
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