Growth hormone and amino acid supply interact synergistically to control insulin-like growth factor-I production and gene expression in cultured ovine hepatocytes

Many of the anabolic effects of growth hormone (GH) are indirect, occurring through GH-stimulated production of insulin-like growth factor-I (IGF-I) by the liver. As well as being regulated by GH, plasma IGF-I concentrations have been demonstrated to depend upon the level of dietary protein intake, with low protein diets bring associated with reduced circulatory IGF-I levels. This inhibitory effect cannot be reversed by GH injection, suggesting that liver sensitivity to GH becomes impaired.

To investigate the mechanisms through which protein supply affects GH sensitivity, primary cultures of ovine hepatocytes were grown in defined media, containing various proportions (0.2, 1.0 and 5.0) of jugular amino acid concentrations in fed sheep. Production of IGF-I by these cells was measured after 24 and 48 h in culture by radioimmunoassay. In the first 24-h period basal IGF-I production was the same in all defined media, and GH caused an approximately 2-fold increase in IGF-I release in cells grown in 1.0 x or 5.0 x amino acid media (P < 0.01). Although GH appeared to increase IGF-I release in this period for cells grown in 0.2 x amino acid media, this effect was not statistically significant. In the period from 24-48 h in defined media, both basal and GH-stimulated IGF-I production was dependent on amino acid availability (P < 0.05 and P < 0.001 respectively). Factorial analysis of variance demonstrated a strong positive interaction (P < 0.001) between the effects of amino acid availability and GH, such that GH increased IGF-I production by more than 2-fold in cells grown in 5.0 x amino acid media (P < 0.01) but had no effect on production by cells grown in 1.0 x or 0.2 x amino acid media.

Measurement of steady state concentrations of exon 1-derived IGF-I mRNAs using an RNase protection assay demonstrated that the observed effects on IGF-I peptide secretion were strongly associated with parallel effects at the RNA level.

Incorporation of S-35-methionine into cellular proteins over a 4-h period starting 20 h after transfer to defined culture media was not significantly reduced in 1.0 x compared with 5.0 x amino acid media, although rates under both of these conditions were significantly higher than those seen in 0.2 x amino acid media (P < 0.01). The lack of correspondence between the dose-dependent effects of amino acid supply on cellular protein synthesis and those on basal and GH-stimulated IGF-I production, suggests that amino acid supply modulates IGF-I production through selective mechanisms.

Steady state levels of the CCAAT/enhancer-binding protein beta (C/EBP beta) isoforms, liver-enriched activating protein (LAP) and Liver-enriched inhibitory protein (LIP) were determined by Western blotting. When levels of LAP were expressed relative to LIP levels in the same extracts, a significant decrease in the LAP:LIP ratio was observed in response to amino acid limitation (P < 0.05).

These data strengthen earlier arguments that synergistic interaction between the effects of amino acids and GH on hepatic IGF-I gene expression underlie nutrition-dependent changes in circulating IGF-I titres. The association between these effects and altered levels of C/EBP beta isoforms suggests that CCAAT/enhancer mediated control of IGF-I gene expression may be involved in this phenomenon.