PROJECT SUMMARY Impaired insulin-stimulated glucose uptake is a common metabolic complication in aged and obese skeletal muscle. This insulin “resistance” increases morbidity risk and is a primary defect that underlies the etiology of type 2 diabetes. Despite the fact that the trafficking of GLUT4 to the plasma membrane is fundamental to insulin- stimulated glucose uptake, there is still much unknown about the signals that underlie GLUT4 translocation after insulin stimulation in skeletal muscle. The long-term objective of this research is to define the molecular regulation of insulin-stimulated glucose transport in skeletal muscle. Currently, phosphorylation-based signaling from the insulin receptor through phosphoinositide 3-kinase to Akt2 and Akt substrate of 160kD is considered the principal mechanism underlying insulin-stimulated GLUT4 translocation and glucose uptake. We believe, however, that lysine acetylation of proteins key to GLUT4 trafficking is necessary for insulin-stimulated glucose uptake. Accordingly, the primary objective of this application is to elucidate the importance of the acetyltransferases, p300 (E1A binding protein p300) and CBP (cAMP response element-binding protein [CREB] binding protein), to insulin-mediated GLUT4 mobilization to the plasma membrane and skeletal muscle glucose uptake. Our central hypothesis is that lysine acetylation of specific residues on distinct GLUT4 trafficking-related proteins by cytosolic p300/CBP is required for insulin-stimulated GLUT4 translocation to the plasma membrane and a resultant increase in glucose uptake by skeletal muscle; as part of this, we hypothesize that p300/CBP are regulated by Akt2. To address our hypothesis, we will measure skeletal muscle glucose uptake, cytosolic lysine acetylation, canonical insulin signaling and GLUT4 translocation in response to insulin in adult mouse skeletal muscle and muscle cells lines in which we modulate p300/CBP activity. Our model predicts that concurrent inhibition of p300 and CBP acetyltransferase activity will abrogate insulin-stimulated glucose transport, whilst p300/CBP activation will augment these measures. Specifically, Aim #1 will elucidate the importance of cytosolic versus nuclear p300/CBP and gene transcription to our model, Aim #2 will determine how p300/CBP are regulated in response to insulin stimulation and, Aim #3 will define the proteins that are acetylated by p300/CBP in response to insulin and the functional importance of the acetylation sites on target proteins to insulin-stimulated GLUT4 translocation and glucose uptake. Together, these studies will broaden our understanding of the contribution of p300/CBP and lysine acetylation to skeletal muscle glucose metabolism in response to insulin. Ultimately, we expect this knowledge to provide a new framework for developing novel therapies to modulate insulin action in skeletal muscle. By extension, such work is anticipated to advance the treatment of diseases and complication...