Role of C/EBPalpha phosphorylation in regulation of gene expression and metabolism.

Obesity is associated with hyperglycemia, dyslipidemia, hypertension, and insulin resistance, conditions that contribute to the metabolic syndrome, which is associated with an increased risk for cardiovascular disease and type 2 diabetes. The recognition that adipose tissues are not passive storage vessels for energy, but rather active endocrine organs that influence glucose tolerance and insulin sensitivity has spurred investigations into the development, metabolism, and endocrine properties of adipocytes. CCAAT/enhancer-binding protein alpha (C/EBPalpha) is critical for establishing insulin-stimulated glucose uptake in adipocytes. How this function of C/EBPalpha is regulated has yet to be determined. Phosphorylation sites in the transactivation domains of C/EBPalpha have been characterized, and recent research interest has focused on how S21 phosphorylation regulates C/EBPalpha activity in hematopoietic and hepatic cell lines. To better understand the role of S21 phosphorylation on C/EBPalpha function during adipogenesis, we developed constructs in which this serine was mutated to alanine or aspartate to model dephosphorylation or phosphorylation, respectively. In two cell lines deficient in endogenous C/EBPalpha, enforced expression of phosphorylation-resistant (S21 to alanine) S21A-C/EBPalpha resulted in normal lipid accumulation and expression of many adipogenic markers. However, S21A-C/EBPalpha had impaired ability to transactivate the GLUT4 promoter, and adipocytes expressing S21A-C/EBPalpha had diminished GLUT4 and adiponectin expression and reduced insulin-stimulated glucose uptake. No alterations in insulin signaling or GLUT4 vesicle trafficking were identified in S21A-C/EBPalpha adipocytes, and enforced expression of exogenous GLUT4 reconstituted insulin-stimulated glucose uptake, suggesting the primary defect was deficient GLUT4. Furthermore, mouse models in which the endogenous C/EBPalpha locus was mutated to encode S21A-C/EBPalpha displayed no differences in total body mass, but male S2IA knock-in mice had adipose tissue with reduced expression of GLUT4 and adiponectin protein, and were glucose intolerant. S21A mice also displayed elevated fasted and fed blood glucose levels, perhaps as a result of increased hepatic glucose production secondary to increased hepatic mRNA expression of the gluconeogenic enzymes, PEPCK and glucose-6-phophatase. Together, these results suggest phosphorylation of C/EBPalpha on S21 has an important role in regulating protein function both in vitro and in vivo. Further research on this mouse model may give insights into mechanisms whereby S21 phosphorylation of C/EBPalpha alters its ability to regulate aspects of adipocyte and hepatic metabolism, and hence, contribute to the coordination of whole-body glucose homeostasis.Ph.D.Biological SciencesMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/127004/2/3304936.pd