Extraction and nutritional/hormonal regulation of tissue insulin-like growth factor 1 activity

Studies of insulin-like growth factor 1 (IGF-1) mRNA translation products suggest synthesis as a high Mr precursor, larger than circulating forms. To search for a precursor, we characterized IGF-1 immunoreactivity and IGF bioactivity in extracts from the liver and other body tissues. Sequential extraction with neutral followed by acid buffer was superior to extraction with acid/ethanol or acid alone in yield of immunoreactivity and specific activity. Extracts of normal rat liver exhibited both immuno- and bioactivity parallel to that of recombinant IGF-1 and serum IGFs over a 25-fold concentration range. Based on immunoreactivity, the liver of a 134-g rat appears to contain 1.2 micrograms of IGF-1 equivalents, 50% of the 2.45 micrograms in the circulation. Diaphragm, spleen, and kidney contained no significant IGF bioactivity and 8, 17, and 32% of the IGF-1 immunoreactivity of normal liver, respectively. Although serum IGFs were found at 7.5 kDa after size exclusion chromatography at pH 3, hepatic extracts contained a predominant peak of immuno- and bioactivity of apparent molecular mass of 30-35 kDa; both sizes were present in liver perfusates. Both immunoaffinity chromatography followed by Western blotting and IGF-binding protein affinity chromatography followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two predominant species, at 18-19 and 12 kDa. The 18-19-kDa species is consistent with the apparent size of the glycosylated propeptide encoded by IGF-1A mRNA, while the 12-kDa species may be nonglycosylated propeptide. Extract activity was pituitary-dependent; the livers of hypophysectomized rats contained 15.4 and 48.8% of normal immuno- and bioactivity, respectively. During fasting and refeeding of rats, fluctuations in hepatic extract IGF-1 immunoreactivity generally paralleled changes in serum IGF-1 (r = 0.93, p less than 0.001). These studies demonstrate that the liver contains a pituitary- and nutrition-dependent, high Mr form of IGF-1 with immunological and biological properties similar to circulating IGF-1. Processing of this 18-19-kDa molecule through a 12-kDa intermediate may contribute IGF-1 to the circulation.