The general objective of this project is to define molecular mechanisms involved in the regulation of metabolism by insulin. Insulin lowers blood glucose by stimulating the uptake and storage of glucose as glycogen. The stimulation of glycogen synthesis occurs within min and results from activation of both glucose transport and glycogen synthase. The signal transduction pathways involved in these important responses to insulin have not been determined. Aim 1 is to identify elements in these pathways. Microinjection will be used to introduce candidate signaling molecules (or activators/inhibitors) into cardiac myocytes or 3T3-L1 adipocytes, thereby rapidly changing the intracellular concentration/activities of potential mediators of insulin action. Ultrasensitive microanalytical methods will then be used to directly measure changes in the injected cells. This system, which is already fully operational, provides a means to assess the acute effects of almost any soluble substance on glucose transport and glycogen synthase activities. No other system has this capability. In principle, it should be possible to determine whether known signaling intermediates are involved in the two actions of insulin that are most important in decreasing blood glucose. It is likely that many elements in the pathways involved in the regulation of metabolism by insulin have not been identified. For example, the functions of relatively few of the many proteins that are Ser/Thr phosphorylated in response to the hormone are known. This represents a significant gap in our understanding of insulin action, as at least some of the proteins must represent important downstream targets of insulin-stimulated kinases. Aim 2 is to address this deficit by cloning cDNA encoding isp62 and pp170, two insulin-stimulated phosphoproteins that have been partially characterized in our laboratory. By determining homologies with proteins of known function, or by identifying proteins with which isp62 and pp170 interact, we hope to determine the function of the phosphoproteins. Our recent use of this strategy led to the discovery of PHAS-I, a novel regulator of translation initiation that is involved in the actions of insulin and growth factors on stimulating protein synthesis in a variety of cell types. Aim 3 is to investigate the role of the glycogen-bound form of Type I protein phosphatase, PP1G, in insulin and epinephrine action in skeletal muscle. It is widely believed that insulin activates glycogen synthase via a pathway involving sequential activation of MAP kinase, Rsk-2 (ribosomal protein 56 kinase-2) and PP1G. However, our findings indicate that activation of MAP kinase and Rsk-2 is neither necessary nor sufficient to activate synthase in adipocytes. Experiments are proposed to determine whether the MAP kinase pathway is involved in synthase activation in skeletal muscle, the major site of insulin-stimulated glycogen deposition. Subaims are to determine whether insulin promotes dephosphorylation of the activating site in PP1G; in rat skeletal muscle and to investigate the interactions between insulin and epinephrine on the phosphorylation of PP1G.