Radioimmunoassay for insulin-like growth factor I (IGF-I) using biosynthetic IGF-I

We produced antiserum to insulin-like growth factor I (IGF-I), and developed a specific and sensitive radioimmunoassay (RIA) for IGF-I using the biosynthetic IGF-I. This antiserum to IGF-I was specific for IGF-I; no cross-reactivities with multiplication stimulating activity, porcine insulin or human growth hormone (hGH) were detected. The sensitivity was 10-25 pg/tube with 50% displacement at 125 pg/tube. The intra- and inter-assay coefficients of variation for IGF-I were 5.4 and 9.7%, respectively. The plasma IGF-I levels as determined by RIA in normal adults (N = 46), patients with active acromegaly (N = 31), and pituitary dwarfs (N = 31) were 21.6 +/- 1.0, 157.3 +/- 17.0, and 2.5 +/- 0.3 ng/ml (Mean +/- SEM), respectively, indicating the levels were GH-dependent. The plasma IGF-I levels were significantly increased from 2.2 +/- 0.2 to 26.5 +/- 3.2 ng/ml after hGH administrations for three consecutive days in five pituitary dwarfs. The IGF-I levels were low in patients with hypothyroidism and liver cirrhosis, but were normal in patients with chronic renal failure. These data confirm previous reports and this radioimmunoassay proves useful in evaluating plasma IGF-I levels.     

Cloning of the rat insulin-like growth factor binding protein-1 gene and analysis of its 5' promoter region.

To understand specific mechanisms involved in the regulation of insulin-like growth factor binding protein-1 (IGFBP-1), an important modulator of IGF bioactivity, we cloned the rat IGFBP-1 gene and sequenced a 1.5 kb Sph1-Sph1 fragment containing 1110 bases upstream from the translation start site. Computer analysis reveals the presence of ATA, CACCC, and CCAAT elements, and putative homeodomain, AP-1, insulin and glucocorticoid response elements in the 5' promoter. Primer extension and ribonuclease protection studies reveal a single cap site in RNA from rat hepatoma cells and both control and diabetic rat liver.     

Purification and characterization of the receptor for insulin-like growth factor I

The receptor for insulin-like growth factor I (IGF-I) was purified from the rat liver cell line BRL-3A by a combination monoclonal anti-receptor antibody column and a wheat germ agglutinin column. Analyses of these receptor preparations on reduced sodium dodecyl sulfate-polyacrylamide gels yielded protein bands of Mr 136K (alpha subunit) and Mr 85K and 94K (beta subunit). These receptor preparations bound 5 times more IGF-I than insulin, and the binding of both labeled ligands was more potently inhibited by unlabeled IGF-I than by insulin. These results indicate that these receptor preparations contained predominantly the IGF-I receptor. This highly purified receptor preparation was found to possess an intrinsic kinase activity; autophosphorylation of the receptor beta subunit was stimulated by low concentrations of IGF-I (half-maximal stimulation at 0.4 nM IGF-I). Twentyfold higher concentrations of insulin were required to give comparable levels of stimulation. A monoclonal antibody that inhibits the insulin receptor kinase was found to inhibit the IGF-I receptor kinase with the same potency with which it inhibits the insulin receptor. In contrast, monoclonal antibodies to other parts of the insulin receptor only poorly recognized the IGF-I receptor. A comparison of V8 protease digests of the insulin and IGF-I receptors again revealed some similarities and also some differences in the structures of these two receptors. Thus, the IGF-I receptor is structurally, antigenically, and functionally similar to but not identical with the insulin receptor.     

Effect of insulin-like growth factor I (IGF-I) on Sertoli cell metabolism in the pubescent rat]

The influence of Insulin-like Growth Factor I (IGF-I) on some metabolic functions of Sertoli cells from peripubertal rats was investigated. Sertoli cells were isolated from the testes of 24-day-old animals and cultured at 32 degrees C in Eagle's MEM with or without 1 nM IGF-I. Sertoli cells cultured in the presence of IGF-I showed increased nuclear RNA polymerase activity (+80%) and augmented protein synthesis (+50%).     

Recombinant human insulin-like growth factor I (IGF-I): risks and benefits of normalizing blood IGF-I concentrations

Recombinant human (rh) insulin-like growth factor I (IGF-I) is being developed as a therapy for short stature caused by IGF deficiency (IGFD) and also for diabetes mellitus. To complement the human efficacy and safety data, a large amount of information is available regarding the pharmacology and toxicology of rhIGF-I in animals. This review summarizes the risks and benefits of normalizing blood IGF-I concentrations in IGFD, especially with regard to carcinogenicity, and compares and contrasts safety data for rhIGF-I, recombinant human growth hormone (rhGH), and insulin. A major difference between rhIGF-I and rhGH is that rhIGF-I (like insulin) has hypoglycaemic activity, whereas rhGH opposes insulin action and is diabetogenic. In most of their actions, GH and IGF-I are similar. IGF-I mediates most of the actions of GH, so the safety of rhGH and that of rhIGF-I also share many common features. In animals, the transgenic expression of hGH has been shown to act directly, by activating the prolactin receptor, to increase the incidence of mammary and prostate tumours. In comparison, the over-expression of IGF-I in animals or the administration of rhIGF-I does not have a carcinogenic effect. In formal toxicology and carcinogenicity studies, rhIGF-I has similar effects to insulin in that it can increase food intake, body size, and the growth rate of existing tumours. In animals and humans, IGFD has many long-term detrimental effects besides short stature: it increases the risk of diabetes, cardiovascular disease, and low bone mineral density. Therefore, a case can be made for replacement therapy with rhIGF-I to normalize blood IGF-I levels and reverse the detrimental effects of IGFD.     

Autoinhibition of the insulin-like growth factor I receptor by the juxtamembrane region

The juxtamembrane (JM) regions of several receptor tyrosine kinases are involved in autoinhibitory interactions that maintain the low basal activity of the receptors; mutations can give rise to constitutive kinase activity and signaling. In this report, we show that the JM region of the human insulin-like growth factor I receptor (IGF1R) plays a role in kinase regulation. We mutated JM residues that were conserved in this subfamily of receptor tyrosine kinases, and expressed and purified the cytoplasmic domains using the Sf9/baculovirus system. We show that a kinase-proximal mutation (Y957F) and (to a lesser extent) a mutation in the central part of the JM region (N947A) increase the autophosphorylation activity of the kinase. Steady-state kinetic measurements show the mutations cause an increase in V(max) for phosphorylation of peptide substrates. When the holoreceptors were expressed in fibroblasts derived from IGF1R-deficient mice, the Y957F mutation led to a large increase in basal and in IGF1-stimulated receptor autophosphorylation. Together, these data demonstrate that the JM region of IGF1R plays an important role in limiting the basal activity of the receptor.     

Cloning and characterization of the growth hormone-dependent insulin-like growth factor binding protein (IGFBP-3) in the rat.

We report for the first time the complete amino acid sequence for the growth hormone dependent insulin-like growth factor binding protein (IGFBP-3) in the rat. A human IGFBP-3 clone was generated using the polymerase chain reaction (PCR) and used to screen a rat liver cDNA library. cDNA clones of the rat IGFBP-3 were isolated and the full amino acid sequence deduced. The sequence begins with a putative, 26 amino acid signal peptide followed by a 265 amino acid binding protein. The amino acid sequence is over 80% homologous with the equivalent human IGFBP-3 form and shows complete conservation of 18 cysteine residues that are clustered at the amino and carboxy ends of the protein. IGFBP-3 is the binding subunit of the major circulating IGFBP in the rat, and hence the availability of precise structural data and cDNA probes provides an important opportunity for a detailed study of the control of IGFBP-3 synthesis at the level of gene expression.     

Infusion of human insulin-like growth factor I (IGF-I) into malnourished rats reduces hepatic IGF-I mRNA abundance

To determine whether the serum level of IGF-I influences its hepatic synthesis through negative feedback regulation, we infused 200 micrograms/d of human IGF-I subcutaneously into young male rats eating either an energy-restricted or ad lib diet. In energy-restricted rats, a two-fold increase in serum IGF-I concentration produced a 41% increase in growth rate at the end of one week, and a 30% decrease in steady state hepatic IGF-I mRNA and 56% drop in serum GH at the end of two weeks. In ad lib fed rats, the increased serum IGF-I concentration neither enhanced growth rate nor significantly reduced hepatic IGF-I mRNA abundance or serum GH levels. These data suggest that the abundance of hepatic IGF-I mRNA in energy-restricted rats is controlled, in part, by serum IGF-I levels via negative feedback regulation.