Abstract
We have investigated the effects of insulin on the phosphorylation of glycogen phosphorylase in skeletal muscle. Rat epitrochlearis muscles were incubated in vitro with 32Pi to label cellular phosphoproteins, before being treated with hormones. Phosphorylase, phosphorylase kinase, and glycogen synthase were immunoprecipitated under conditions that prevented changes in their phosphorylation states. Based on measurements of the activity ratio (-AMP/+AMP) and the 32P content of phosphorylase, 4-8% of the phosphorylase in untreated muscles appeared to be phosphorylated. Epinephrine promoted increases of approximately 4-fold in the 32P content and activity ratio. Neither these effects nor the epinephrine-stimulated increases in phosphorylation of glycogen synthase and phosphorylase kinase were attenuated by insulin. However, insulin at physiological concentrations rapidly decreased the 32P content of phosphorylase in muscles incubated without epinephrine. Results from peptide mapping experiments indicate that phosphorylase was phosphorylated at a single site in both control and insulin on phosphorylase represented a decrease in 32P of approximately 50%. By comparison, the 32P content of glycogen synthase and the beta subunit of phosphorylase kinase were decreased by only 20 and 16%, respectively; the 32P content of the kinase alpha subunit was not affected by insulin. The results provide direct evidence that insulin decreases the amount of phosphate in phosphorylase and phosphorylase kinase. These findings have important implications with respect to both the regulation of glycogen metabolism in skeletal muscle and the mechanism of insulin action.
Highlights
We have investigated the effects of insulin on the to a conversion may be followed by monitoring the increase phosphorylation of glycogen phosphorylase in skeletal in the -AMP/+AMP activity ratio.Glycogen synthase is muscle
The maximumeffect of insulinon the inactivation of phosphorylase; but in previous studies the phosphorylase representeda decrease in32Pof approx- hormone has had little, if any, effect on the phosphorylase imately 50%.By comparison, the 32Pcontent of glyco- activity ratio [10, 14, 15, 17, 18]. gen synthase and tBhseubunit of phosphorylase kinase Insulin stimulates the dephosphorylation of several intrawere decreased by only 20 and 16%,respectively; the cellular proteins, in addition to glycogen synthase [19, 20]. 32Pcontent of the kinasea subunit was not affected byThe mechanism is not yet known, but it seems clear that it insulin
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Summary
Insulin-stimulated Dephosphorylation of Phosphorylase ularly at the low activity ratios that are found in nonstimulated skeletal muscle. In previous investigations with insulin, the phosphorylation state of phosphorylasehad not been directly assessed but inferred from measurements of enzymatic activity. In the presentexperiments we have used 32Plabeling procedures to investigate the effects of insulin on the phosphorylation of phosphorylase, phosphorylase kinase,and glycogen synthase
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