Incubation of isolated rat adipocytes with insulin, vasopressin, or oxytocin increased plasma membranebound protein kinase C (PKC) activity by 100-400%. PKC activity was assayed by a procedure that is virtually background-free, thus permitting assay of protein kinase activity in highly diluted samples of solubilized membranes. Hormonedependent increases in PKC activity were limited to plasma membranes. Stimulation of the kinase was half-maximal with 70 pM insulin, and the hormone effect was rapid. Oxytocin and vasopressin produced effects on PKC similar to insulin, but the magnitude of the vasopressin stimulation exhibited seasonal variations. Treatment of cells with phorbol 12-myristate 13-acetate (PMA) resulted in a loss of PKC activity from the cytosol and a gain in plasma membrane activity, indicative of translocation of the enzyme. With activity measurements it was not possible to determine if insulin stimulated a translocation of the kinase. However, Western blot analysis of plasma membranes with polyclonal antibodies directed against PKC suggest that at least some ofthe insulin-stimulated PKC activity resulted from enzyme translocation.hand, decreased phorbol ester binding to adipocyte plasma membrane sites, thought to represent PKC, has been observed after treatment of fat cells with insulin (27).Like phorbol esters, vasopressin and oxytocin mimic a number of insulin effects on adipocytes (for examples, see refs. 1, 28-30). However, in contrast to insulin, stimulation of phospholipid metabolism and diacylglycerol formation in adipocytes by vasopressin and oxytocin is readily detected (20,21). Moreover, PKC has been implicated in the actions of these nonapeptide hormones in a variety of cells (for examples, see refs. [31][32][33][34]. Nevertheless, other than a report on activation of hepatic PKC with vasopressin (35), there appear to be no reports that directly demonstrate increased PKC activity in isolated membranes after exposure of adipocytes or other types of cells to either vasopressin or oxytocin.In this paper, with the use of a highly sensitive protein kinase assay method (36), we report that exposure of isolated rat adipocytes to insulin, vasopressin, or oxytocin increases PKC activity in adipocyte plasma membranes.In 1966 Rodbell (1) noted that "there is some common action of oxytocin, insulin and phospholipase C," based on the observation that, when applied extracellularly, the three agents increased glucose transport and amino acid incorporation into proteins of fat cells. A possible basis for this mimicry of hormones by exogenous phospholipase C lies in the finding that many hormone receptors increase phospholipid turnover, production ofdiacylglycerol, and activation of the calcium-and phospholipid-dependent enzyme, protein kinase C (PKC) (2-4), and there is indirect evidence for PKC participation in acute metabolic effects of insulin on adipocytes and other cells. Tumor-promoting phorbol esters, which substitute for diacylglycerol in stimulating PKC (3), increase transport of glucose...
Cultured vascular smooth muscle cells derived from the spontaneously hypertensive rat (SHR) are known to replicate more rapidly than cells from the normotensive Wistar-Kyoto (WKY) rat. In this study we compared the responses of vascular smooth muscle cells from the two strains to transforming growth factor-01 (TGF-/31) and evaluated its potential to account for the different growth properties of these cells in response to a number of vascular-derived growth factors. TGF-/31 potentiated the proliferative effects of epidermal growth factor, basic fibroblast growth factor, or the different isoforms of platelet-derived growth factor on vascular smooth muscle cells from SHR but inhibited growth factorstimulated proliferation of vascular smooth muscle cells from WKY rats. These differential effects of TGF-01 on proliferation could not be attributed to alterations in the expression of the type I, II, or III TGF-/3 receptors but appeared more
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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