This project is centered on the action of insulin and concerns mechanisms of both neural and hormonal control. Experiments are proposed to determine why motor innervation is required for stimulation of glycogen synthesis by insulin in skeletal muscle. Particular objectives are to identify the molecular change(s) in muscle that result in both the appearance of the hormonal response after innervation and the loss of the response following denervation. Other experiments are designed to determine if motor neurons maintain the ability of muscle to respond to insulin by releasing trophic substances or by promoting muscle activity. The stimulation of glycogen synthesis by insulin almost certainly involves activation of glycogen synthase. This enzyme can be phosphorylated in vitro in at least 7 nonidentical sites per subunit, and considerable selectivities among the sites are displayed by several different protein kinases--including cAMP-dependent, cAMP-independent, and calcium-sensitive kinases. Some specificity may be observed with different phosphoprotein phosphatases. Therefore, identification of the sites on glycogen synthase that are dephosphorylated as a result of insulin action would provide clues concerning which kinases and phosphatases mediate the hormone's action. This will be accomplished following immunopurification of 32P-labeled glycogen synthase from hormonally-treated muscle after incubation of the tissue with [32P]phosphate. The 32P-labeling procedure will also allow measurements of the rate of turnover of phosphate in the different sites. From these measurements it should be possible to determine directly if insulin activates glycogen synthase by decreasing protein kinase activity or by increasing phosphoprotein phosphatase activity.
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