THE AUGMENTATION OF INSULIN-LIKE GROWTH FACTOR-I MESSENGER-RIBONUCLEIC-ACID IN CULTURED RAT HEPATOCYTES - ACTIVATION OF PROTEIN KINASE-A AND KINASE-C IS NECESSARY, BUT NOT SUFFICIENT

In previous studies it was shown that bovine GH (bGH) and glucagon, wh en individually added to primary rat hepatocyte cultures, modestly sti mulated IGF-I mRNA levels 1.8- to 2.5-fold, but when combined, synergi zed to stimulate IGF-I mRNA levels by 10- to 12-fold. In the present s tudy we have explored further the mechanism of this effect in primary rat hepatocyte cultures. Like glucagon, the addition of 3-isobutyl-1-m ethylxanthine (100 mu M) or (Bu)(2)cAMP (150 mu M) augmented IGF-I mRN A 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, determin ed by incubating hepatocytes with actinomycin-D was 12 h. Although bGH did not affect the decay rate, glucagon (100 ng/ml) or (Bu)(2)cAMP (1 00 mu M) 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, glucago n, or (Bu)(2)cAMP. The stimulatory effect of bGH plus glucagon was inh ibited 80% after preincubation with 4 beta-phorbol 12 beta-myristate 1 3 alpha-acetate (10 mu M) for 24 h. The addition of staurosporine, sph ingosine, or H-7 [1-(5-isoquinolinyl sulfonyl)2-methyl piperazine] inh ibited the stimulatory effect of bGH plus glucagon on hepatocyte IGF-I mRNA by 80%, 90%, and 85%, respectively. Preincubation with cyclohexi mide (10 mu g/ml) blocked the synergistic effect of bGH plus either gl ucagon or (Bu)(2)cAMP by 65-80%. The effect of glucagon, mediated via the activation of adenylate cyclase, involves in part the posttranscri ptional 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 l evel of transcription. The synergistic augmentation of hepatocyte IGF- I mRNA levels by GH and glucagon involves the activation of PKA and PK C, but also appears to require the synthesis of one or more protein(s) .