Nutrition and Somatomedin: XXIII. Molecular Regulation of IGF-I by Amino Acid Availability in Cultured Hepatocytes

The poor growth associated with protein-calorie malnutrition occurs despite circulating growth hormone levels that are normal or elevated and is thought to be mediated partly by blunted generation of insulinlike growth factor I (IGF-I) in the liver. To explore underlying mechanisms, we asked whether altered availability of amino acids could regulate hepatic IGF-I release independent of the contributions of regulatory hormones. Normal rat hepatocytes were isolated by collagenase digestion and maintained in serum-free medium with fixed concentrations of insulin and dexamethasone. Levels of immunoassayable albumin and IGF-I accumulation in daily changes of medium were sustained for 3–5 days, and all studies were performed within this period. Cellular viability and content of DNA were unaffected by deprivation of the essential amino acids lysine or tryptophan and the nonessential amino acids cysteine and/or cystine. However, deletion of tryptophan or lysine from the culture medium led to 63 and 76% declines in IGF-I release, respectively (both P < 0.001 vs. complete medium), although omission of cysteine or cysteine plus cystine produced no significant change. Over 5 days of culture, release of albumin was maintained in complete medium, but omission of tryptophan depressed albumin release over days 2—5 (P < 0.001). In complete medium, IGF-I release rose for 3 days and then declined. In tryptophan-deficient medium, IGF-I levels were comparable to control values after 24 h but did not rise at 48 h and then fell rapidly after 72 h in culture, with values significantly below levels in complete medium (all P < 0.005). Although tryptophan deprivation led to decreased release of IGF-I on day 3 of culture, release of albumin did not fall until day 4, suggesting that IGF-I is more sensitive to the presence of this essential amino acid. Because availability of tryptophan could affect formation of IGF-I via either utilization of this substrate for protein synthesis or alternate regulatory mechanisms, we searched for the basis of tryptophan effects by measuring the impact of tryptophan provision on levels of hepatic IGF-I mRNA. Omission of tryptophan led to a 50% decline in levels of IGF-I mRNA measured by dot blotting of total hepatic RNA (P < 0.05 vs. RNA from cells cultured in complete medium) but had little effect on levels of mRNA for the structural protein p-actin. This study demonstrates that amino acid availability may modulate hepatic production of IGF-I independent of the contributions of regulatory hormones, which change along with circulating metabolic fuels in the malnourished state. In addition to potential limitations on utilization of substrate, underlying mechanisms appear to involve changes in the level of hepatic mRNA, which directs synthesis of IGF-I for delivery to the circulation.