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Staurosporine inhibits phorbol 12-myristate 13-acetate- and insulin-stimulated translocation of GLUT1 and GLUT4 glucose transporters in rat adipose cells.

ABSTRACT: Staurosporine, a widely used protein kinase C inhibitor, completely inhibited both phorbol 12-myristate 13-acetate (PMA)- and insulin-stimulated glucose transport activity in isolated rat adipocytes. The inhibition was non-competitive and was attributed to a blockade of the PMA- and insulin-induced translocation of both GLUT1 and GLUT4 glucose transporters. The PMA-stimulated glucose transport activity was more sensitive to inhibition by staurosporine than was insulin-stimulated transport activity (PMA, IC50 = 1.1 +/- 0.1 microM; insulin, IC50 = 6.4 +/- 0.7 microM; P < 0.05, n = 3). At 1 microM staurosporine the insulin-sensitivity was decreased, i.e. EC50 increased from 0.12 nM to 5.4 nM, but the maximum response to insulin and the time course for stimulation were unaffected. At 6 microM staurosporine the insulin-sensitivity was further decreased, the maximal stimulation was decreased by 25%, and the apparent half-time for stimulation was extended from 2.5 min in control cells to 9.4 min. Staurosporine (30 microM) was able to block insulin's ability to stimulate glucose transport, whether added before or after insulin, by a mechanism that did not alter the rate of GLUT4 internalization. In intact adipose cells, staurosporine (30 microM) induced a slight (30%) decrease in the maximal insulin-induced receptor autophosphorylation and a similar decrease in the tyrosine phosphorylation of pp60 and pp160 (insulin-receptor substrate-1: 'IRS-1'), but was without effect on insulin binding to its receptor. Conversely, staurosporine induced a concentration-dependent inhibition of the constitutively tyrosine-phosphorylated (pp120) protein and of an insulin-stimulated protein pp53 in the cytosol. The locus of staurosporine's action appears to be distal from the initial insulin-receptor signalling, at a step that regulates the specific translocation of the glucose transporters to the plasma membranes.

SUBMITTER: Nishimura H

PROVIDER: S-EPMC1137219 | biostudies-other | 1994 Aug

REPOSITORIES: biostudies-other

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Project description:Phorbol 12-myristate 13-acetate (PMA)-mediated signalling was investigated in relation to the ability of murine (CBA) bone marrow cells to form colonies in vitro. Treatment of marrow cells with PMA did not influence the 1,2-diacylglycerol or cyclic AMP concentrations, the intracellular Ca2+ concentration or phospholipase D activity. PMA increased particulate phospholipase A2 (PLA2) activity, lysophosphatidylcholine formation and arachidonic acid release from bone marrow cells; these effects were abolished when cells were pretreated with the putative PLA2 inhibitors heparin and mepacrine. While indomethacin and nordihydroguaiaretic acid inhibited either the cyclo-oxygenase or lipoxygenase pathway of arachidonic acid metabolism, as measured by their products prostaglandin E2 and leukotriene B4, they did not influence PMA-mediated PLA2 activation or translocation of protein kinase C (PKC) from the soluble to the particulate fraction. Treatment of cells with PMA increased the amounts of membrane-bound alpha, beta, delta, epsilon and zeta isoforms of PKC in bone marrow cells. Pretreatment of cells with PLA2 inhibitors reduced the amount of membrane-bound PKC-zeta in unstimulated cells and diminished PMA-induced translocation of PKC-zeta to membranes without affecting other PKC isoforms. This effect could be overcome by exogenous addition of arachidonic acid, suggesting that PKC-zeta may operate downstream of the activated PLA2. On the other hand, wortmannin, an inhibitor of phosphatidylinositol 3-kinase, did not influence the amount of PKC-zeta associated with particulate fractions in control cells and could not abolish the PMA-mediated translocation of this isoform. Short-term exposure (45 min) of bone marrow cells to PMA, phorbol 12,13-dibutyrate or arachidonic acid increased the number of colonies formed over 7 days in a methylcellulose-based culture in vitro. The effects of PMA, but not those of arachidonic acid, could be prevented by putative PLA2 inhibitors. This suggests that PMA-mediated activation of conventional PKCs and novel PKCs leads to PLA2 activation which, by releasing arachidonic acid from phospholipids, activates PKC-zeta. This signalling pathway appears to be mitogenic for bone marrow cells.

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Staurosporine inhibits phorbol 12-myristate 13-acetate- and insulin-stimulated translocation of GLUT1 and GLUT4 glucose transporters in rat adipose cells.

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