Glycogen Synthase from Rabbit Skeletal Muscle; Effect of Insulin on the State of phosphorylation of the Seven Phosphoserine Residues in vivo

Rabbits were starved for 24 h and injected with propranolol (2 mg/kg) with or without insulin (33 μg/kg) 15 min prior to sacrifice. In muscle extracts prepared from propranolol‐treated animals the activity ratio (± glucose 6‐phosphate) of glycogen synthase was 0.18 ± 0.02 and the K_a for glucose 6‐phosphate was 1.2 ± 0.1 mM. In (propranolol + insulin)‐treated animals the activity ratio was 0.35 ± 0.02 and the K_a for glucose 6‐phosphate was 0.60 ± 0.05 mM. Glycogen synthase purified to homogeneity from propranolol‐treated animals contained 2.74 ± 0.09 mol phosphate/mol subunit, whereas this value was 2.33 ± 0.09 mol phosphate/mol subunit from (propranolol + insulin)‐treated animals. The distribution of phosphate between the seven phosphorylation sites was elucidated. In (propranolol + insulin)‐treated animals there was a decrease of 0.4—0.45 mol phosphate/mol subunit in sites (3a + 3b + 3c), and no significant changes in any other site (1a, 1b, 2 and 5). The results demonstrate that dephosphorylation of sites (3a + 3b + 3c) is responsible for the activation of glycogen synthase observed after acute administration of insulin. Neither protein phosphatase 1 nor protein phosphatase 2A, which account for virtually all the glycogen synthase phosphatase activity in skeletal muscle extracts, were able to mimic the specific dephosphorylation of sites (3a + 3b + 3c) produced by insulin. Both enzymes dephosphorylated site 2 and sites (3a + 3b + 3c) in vitro at comparable rates. The action of insulin may therefore involve a decrease in the activity of glycogen synthase kinase 3 and the regulation of this enzyme is discussed. The failure of insulin to produce a significant dephosphorylation of the sites phosphorylated by cyclic‐AMP‐dependent protein kinase (1a, 1b and 2) demonstrates that inhibition of this protein kinase does not underlie the activation of glycogen synthase by this hormone. This conclusion is supported by measurements of the phosphorylation states of inhibitor 1, phosphorylase kinase and phosphorylase. Neither 24‐h starvation, nor alloxon‐induced diabetes, produced significant changes in the kinetic properties or phosphorylation state of glycogen synthase. Possible reasons for these findings are discussed.