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  • 1
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    Berkeley, Calif., etc. : Periodicals Archive Online (PAO)
    Pacific Historical Review. 62:3 (1993:Aug.) 273 
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  • 2
    ISSN: 1573-4935
    Keywords: Insulin ; L6 myoblast ; protein synthesis ; protein kinase C ; arachidonate ; DAG
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Insulin stimulated protein synthesis in L6 myoblasts but did not increase the labelling of DAG or the release of phosphocholine from phosphatidylcholine. The DAG lipase inhibitor, RHC 80267, more than doubled the amount of label appearing in DAG but did not stimulate protein synthesis. Even in the presence of the DAG lipase inhibitor insulin failed to have any effect on DAG labelling, and conversely RHC 80267 did not modify the insulin-induced increase in protein synthesis. These results suggest that endogenous DAG production is not involved in the stimulation of protein synthesis by insulin. However, exogenous diacylglycerols (1-oleoyl-2-acetyl glycerol and 1-stearoyl-2-arachidonoyl glycerol) both stimulated protein synthesis in L6 myoblasts. The efficacy of the former (arachidonatefree) DAG suggested that their action was by activation of protein kinase C rather than by arachidonate release and prostaglandin formation. Ibuprofen, an inhibitor of cyclo-oxygenase failed to block the effects of insulin whereas a second cyclo-oxygenase inhibitor, indomethacin had only a partial inhibitory effect. The protein kinase C (PKC) inhibitor, RO-31-8220, totally blocked the effect of insulin. Since indomethacin is also recognised to inhibit phospholipase A2, the data suggests that insulin acts on protein synthesis in myoblasts by arachidonate activation of PKC.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1573-4935
    Keywords: insulin ; L6 myoblast ; translation ; protein kinase C ; cyclooxygenase ; mitogen activated protein kinase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Insulin stimulated a concentration-dependent increase in protein synthesis in L6 myoblasts which was significant at 1 nM. This response was not prevented by the transcription inhibitor, actinomycin D. The protein kinase C (PKC) inhibitor, Ro-31-8220, and downregulation of PKC by prolonged incubation of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), had no effect on the ability of insulin to stimulate protein synthesis whilst completely blocking the response to TPA. In contrast, insulin failed to enhance protein synthesis significantly in the presence of either ibuprofen, a selective cyclooxygenase inhibitor or rapamycin, an inhibitor of the 70 kDa S6 kinase. When cell extracts were prepared and assayed for total myelin basic protein kinase activity, a stimulatory effect of insulin was not observed until the concentration approached 100-fold (i.e. 100 nM) that required to elicit increases in protein synthesis. Upon fractionation on a Mono-Q column, 100 nM insulin increased the activity of 3 peaks which phosphorylated myelin basic protein. Two of these peaks were identified as the 42 and 44 kDa forms of Mitogen Activated Protein (MAP) kinase by immunoblotting. In contrast, 1 nM insulin had no effect on the activity of these peaks. The data suggest that physiologically relevant concentrations of insulin do not stimulate translation in L6 cells through either PKC or the 42/44 kDa isoforms of MAP kinase and that this response is, at least in part, mediated through the activation of the 70 kDa S6 kinase by cyclooxygenase metabolites.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In C2C12 myoblasts, 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated a phospholipase D (PLD) to degrade phosphatidylcholine (PC) as measured by the release of choline and an increase in the formation of phosphatidic acid (PA) (or phosphatidylbutanol [PtdBuOH] in the presence of 0.5% butanol). Exogenous PLD also stimulated choline release, PA and PtdBuOH formation. The protein kinase C (PKC) inhibitor, Ro-31-8220, and PKC downregulation significantly inhibited the effects of TPA but Ro-31-8220 had no effect on PLD action. Neither basic Fibroblast Growth Factor (bFGF) or Epidermal Growth Factor (EGF) increased PLD activity. All agonists stimulated protein synthesis during both a 90 min and a 6 hr incubation and increased RNA accretion after 6 hr. The response at 90 min was not inhibited by the transcription inhibitor, actinomycin D. Ro-31-8220 and PKC downregulation significantly inhibited all the effects of TPA. In contrast, Ro-31-8220 significantly inhibited the increase in RNA accretion elicited by PLD but had no effect on the ability of agonists other than TPA to enhance protein synthesis. All agonists also stimulated thymidine incorporation into DNA. The effects of EGF, bFGF, and PLD were rapid and transient whereas that of TPA was delayed and sustained. Ro-31-8220 and PKC downregulation significantly inhibited the response due to TPA. Furthermore, Ro-31-8220 also significantly inhibited the effects elicited by EGF and PLD but not that induced by bFGF. In differentiated myotubes, TPA and PLD, but not bFGF or EGF, again stimulated choline release and PtdBuOH formation. However, all agents failed to stimulate protein synthesis and RNA accretion. The data demonstrate the presence in C2C12 myoblasts, but not differentiated myotubes, of both a PLD-dependent and PLD-independent pathway(s) leading to the stimulation of protein synthesis, RNA accretion, and DNA synthesis. © 1995 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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