Phosphorylation state of the GLUT4 isoform of the glucose transporter in subfractions of the rat adipose cell: effects of insulin, adenosine, and isoproterenol.

Abstract

The acute effects of insulin, adenosine, and isoproterenol on the activity, subcellular distribution, and phosphorylation state of the GLUT4 glucose transporter isoform were investigated in rat adipocytes under conditions carefully controlled to monitor changes in cAMP-dependent protein kinase (A-kinase) activity. In contrast to GLUT1, which has not been shown to be phosphorylated even when cells are exposed to any of the above agents, GLUT4 was partially phosphorylated (0.1-0.2 mol/mol) when the activity of the A-kinase was suppressed, and remained unchanged in response to insulin. Isoproterenol elicited a 64% inhibition of insulinstimulated glucose transport activity in the absence, but not the presence, of adenosine receptor agonists. However, in either the presence or the absence of agonists, A-kinase was activated as assessed by examining the phosphorylation of the major adipocyte A-kinase substrate, perilipin. Similarly, under either condition, phosphorylation of GLUT4 was enhanced 1.4-fold in the intracellular membranes, but no significant change was observed in the plasma membrane. In the absence of adenosine receptor agonists, isoproterenol exerted a small (14%) but significant inhibition of the insulin-induced translocation of GLUT4 but had no effect on the translocation of GLUT1. Thus, changes in the phosphorylation state and/or subcellular distribution of GLUT4 cannot account for the inhibition of insulin-stimulated glucose activity induced by isoproterenol.Insulin stimulates glucose transport activity in rat adipose cells primarily by inducing the translocation of the transporter isoforms GLUT1 and GLUT4 from an intracellular location to the plasma membrane (1-4). GLUT1 is widely distributed whereas GLUT4, the predominant form in adipose cells (4-6), is found only in tissues where insulin regulates glucose transport activity (i.e., white and brown adipose tissue, heart, and skeletal muscle; for review see refs. 7 and 8). Hereafter, insulin-stimulated glucose transport activity will be abbreviated as transport activity. A second level of transport regulation is exerted by agents that modulate adenylyl cyclase and lipolysis (9-11). Lipolytic agents (isoproterenol, glucagon, and corticotropin) inhibit transport activity, but only in the absence of antilipolytic agents (adenosine, nicotinic acid, and prostaglandin E1). Further, Smith et al. (12) and Kuroda et al. (13) demonstrated that both the transport inhibition by lipolytic agents and the augmentation by antilipolytic agents did not result from changes in transporter location.An unresolved issue is whether the changes in transport activity mediated by these various agents are related to the changes in cAMP. Currently, two distinct mechanisms have been proposed. Kuroda et al. (13) demonstrated an apparent dissociation between isoproterenol-mediated stimulation of cAMP-dependent protein kinase (A-kinase) and inhibition of transport activity, suggesting that changes in A-kinasemediated phosphorylation are not responsible for transport inhibi...

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