Abstract
Catecholamines, acting through adrenergic receptors, play an important role in modulating the effects of insulin on glucose metabolism. Insulin activation of glycogen synthesis is mediated in part by the inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3). In this study, catecholamine regulation of GSK-3beta was investigated in Rat-1 fibroblasts stably expressing the alpha1A-adrenergic receptor. Treatment of these cells with either insulin or phenylephrine (PE), an alpha1-adrenergic receptor agonist, induced Ser-9 phosphorylation of GSK-3beta and inhibited GSK-3beta activity. Insulin-induced GSK-3beta phosphorylation is mediated by the phosphatidylinositol 3-kinase/Akt signaling pathway. PE treatment does not activate phosphatidylinositol 3-kinase or Akt (Ballou, L. M., Cross, M. E., Huang, S., McReynolds, E. M., Zhang, B. X., and Lin, R. Z. (2000) J. Biol. Chem. 275, 4803-4809), but instead inhibits insulin-induced Akt activation and GSK-3beta phosphorylation. Experiments using protein kinase C (PKC) inhibitors suggest that phorbol ester-sensitive novel PKC and Gö 6983-sensitive atypical PKC isoforms are involved in the PE-induced phosphorylation of GSK-3beta. Indeed, PE treatment of Rat-1 cells increased the activity of atypical PKCzeta, and expression of PKCzeta in COS-7 cells stimulated GSK-3beta Ser-9 phosphorylation. In addition, PE-induced GSK-3beta phosphorylation was reduced in Rat-1 cells treated with a cell-permeable PKCzeta pseudosubstrate peptide inhibitor. These results suggest that the alpha1A-adrenergic receptor regulates GSK-3beta through two signaling pathways. One pathway inhibits insulin-induced GSK-3beta phosphorylation by blocking insulin activation of Akt. The second pathway stimulates Ser-9 phosphorylation of GSK-3beta, probably via PKC.
Highlights
Catecholamines, acting through adrenergic receptors, found to play a key role in the regulation of glycogen metaboplay an important role in modulating the effects of insulin on glucose metabolism
Both isoforms were found to be phosphorylated at a single tyrosine residue in unstimulated cells, and Glycogen synthase kinase-3 (GSK-3)1 is an evolutionarily conserved signaling molecule that plays an important role in diverse biological processes, including metabolism, differentiation, and development
To determine whether Ser-9 phosphorylation correlated with a decrease in glycogen synthase kinase-3 (GSK-3) kinase activity, Rat-1 cells stably expressing both the ␣1A-adrenergic receptor and HA-tagged wild-type GSK-3 were treated for 10 min with or without PE or insulin, and GSK-3 activity was measured in HA immunoprecipitates (Fig. 2A)
Summary
Catecholamine regulation of GSK-3 was investigated in Rat-1 fibroblasts stably expressing the ␣1Aadrenergic receptor Treatment of these cells with either insulin or phenylephrine (PE), an ␣1-adrenergic receptor agonist, induced Ser-9 phosphorylation of GSK-3 and inhibited GSK-3 activity. Two isoforms of GSK-3 (GSK-3␣ and GSK-3) have been identified in mammalian tissues, and both are negatively regulated by insulin [1] Both isoforms were found to be phosphorylated at a single tyrosine residue in unstimulated cells, and Glycogen synthase kinase-3 (GSK-3) is an evolutionarily conserved signaling molecule that plays an important role in diverse biological processes, including metabolism, differentiation, and development. GSK-3 was initially identified as a kinase that phosphorylates glycogen synthase and has been dephosphorylation of GSK-3 with a protein tyrosine phosphatase in vitro inactivates the enzyme [8] These results led to the suggestion that insulin inactivates GSK-3 through tyrosine dephosphorylation. It is important to note that, in addition to ␣1-adrenergic receptors, other receptors that couple to Gq/11, including vasopressin, purinergic, and angiotensin II receptors, inhibit glycogen synthase [24, 25]
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