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Insulin regulation of pyruvate kinase activity in cultured rat hepatocytes, in the presence of vasopressin, ionophore A23187 or 4 beta-phorbol 12 beta-myristate 13 alpha-acetate.

ABSTRACT: The short-term interactions of insulin and vasopressin on pyruvate kinase (PK) activity were studied in primary cultures of rat hepatocytes. (1) Vasopressin inhibited PK activity by approx. 30% within 15 s, but activity returned to control values by 5 min. The transient inhibition by vasopressin was mimicked by either 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) or ionophore A23187. (2) Insulin alone transiently inhibited PK activity at 1 min, but stimulated PK activity at 5 and 15 min. (3) Insulin completely antagonized the early inhibition by vasopressin, PMA or A23187 of PK activity at 15 s. (4) Insulin inhibited PK activity in the presence of vasopressin, PMA or A23187 at 5 min. (5) 8-Bromo cyclic AMP inhibited PK activity within 15 s, and this inhibition was maintained for at least 5 min. Insulin did not antagonized the inhibition by the cyclic AMP analogue. These results show that insulin under appropriate conditions can act as an inhibitor or activator of PK.

SUBMITTER: Pittner RA

PROVIDER: S-EPMC1149207 | biostudies-other | 1988 Jun

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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Insulin regulation of pyruvate kinase activity in cultured rat hepatocytes, in the presence of vasopressin, ionophore A23187 or 4 beta-phorbol 12 beta-myristate 13 alpha-acetate.

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