The augmentation of insulin-like growth factor-I messenger ribonucleic acid in cultured rat hepatocytes: activation of protein kinase-A and -C is necessary, but not sufficient.

Abstract

In previous studies it was shown that bovine GH (bGH) and glucagon, when individually added to primary rat hepatocyte cultures, modestly stimulated IGF-I mRNA levels 1.8- to 2.5-fold, but when combined, synergized to stimulate IGF-I mRNA levels by 10- to 12-fold. In the present study we have explored further the mechanism of this effect in primary rat hepatocyte cultures. Like glucagon, the addition of 3-isobutyl-1-methylxanthine (100 microM) or (Bu)2cAMP (150 microM) augmented IGF-I mRNA levels 1.8- to 2.0-fold, but when combined with bGH (50 ng/ml), they augmented levels up to 12-fold. The half-life of IGF-I mRNA, determined by incubating hepatocytes with actinomycin-D was 12 h. Although bGH did not affect the decay rate, glucagon (100 ng/ml) or (Bu)2cAMP (100 microM) reduced the rate of loss by about 70%. 4 beta-Phorbol 12 beta-myristate 13 alpha-acetate minimally stimulated IGF-I mRNA levels 1.2- to 1.4-fold, but displayed no synergism when added with bGH, glucagon, or (Bu)2cAMP. The stimulatory effect of bGH plus glucagon was inhibited 80% after preincubation with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (10 microM) for 24 h. The addition of staurosporine, sphingosine, or H-7 [1-(5-isoquinolinyl sulfonyl)2-methyl piperazine] inhibited the stimulatory effect of bGH plus glucagon on hepatocyte IGF-I mRNA by 80%, 90%, and 85%, respectively. Preincubation with cycloheximide (10 micrograms/ml) blocked the synergistic effect of bGH plus either glucagon or (Bu)2cAMP by 65-80%. The effect of glucagon, mediated via the activation of adenylate cyclase, involves in part the posttranscriptional stabilization of IGF-I mRNA levels. The effect of GH, mediated in part by the activation of protein kinase-C, appears to be at the level of transcription. The synergistic augmentation of hepatocyte IGF-I mRNA levels by GH and glucagon involves the activation of PKA and PKC, but also appears to require the synthesis of one or more protein(s).