Insulin-like growth factor binding protein-6 inhibits neuroblastoma cell proliferation and tumour development.

In neuroblastoma cells, survival and proliferation are dependent upon the insulin-like growth factor (IGF) system. IGFs actively participate in cell growth, whereas IGFBP-6, is associated with the arrest of growth. With a view to blocking IGF-II action, we produced recombinant human IGFBP-6 capable of binding IGFs with affinities between 1.23 and 6.36 x 10(9) M(-1). Ex vivo mitogenic activities were tested on two human neuroblastoma cell lines, in which 100 ng/ml IGFBP-6 completely abolished the effects of both endogenous and exogenous IGF-II. In vivo, nude mice previously injected with neuroblastoma cells were submitted to either 15 daily injections of 4-20 microg IGFBP-6 or implantation of mini-pumps diffusing 20-100 microg IGFBP-6 over 2 weeks. The result was an average 18% reduction in the incidence and development of tumours. Delivery of the IGFBP-6 via mini-pumps also delayed tumour appearance by 6-15 days. Our results therefore show the involvement of IGFBP-6 in neuroblastoma cell growth, both ex vivo in terms of proliferation and in vivo in terms of tumour development.

Use of the ligand immunofunctional assay for human insulin-like growth factor ((IGF) binding protein-3 (IGFBP-3) to analyze IGFBP-3 proteolysis and igf-i bioavailability in healthy adults, GH-deficient and acromegalic patients, and diabetics.

The ligand immunofunctional assay for plasma insulin-like growth factor (IGF) binding protein (IGFBP)-3 developed in our laboratory provides for specific measurement of intact, as opposed to proteolyzed, IGFBP-3. IGFBP-bound IGFs are dissociated and separated by acid pH ultrafiltration; thereafter, intact and proteolyzed IGFBP-3 are captured by a monoclonal antibody in a solid-phase assay and incubated with (125)I-IGF-I, which detects the intact protein but not its proteolytic fragments. This assay was combined with assays for IGF-I (RIA of the ultrafiltrate) and total IGFBP-3 (immunoradiometric assay) to quantify the percentage of proteolyzed IGFBP-3 (percent proteolyzed IGFBP-3) and to calculate the IGF-I/intact IGFBP-3 ratio as an index of the fraction of exchangeable IGF-I bound to IGFBP-3. This fraction represents most of the IGF-I that is bioavailable. Because GH and insulin control the hepatic production and plasma concentrations of IGF-I and IGFBP-3, we set out to determine whether variations in the secretion of the two hormones are involved in the regulation of IGFBP-3 proteolysis. The study included adult populations of 36 healthy subjects, 23 hypopituitary patients untreated with GH, 43 acromegalics (13 untreated), 42 insulin-treated type 1 diabetics [insulin-dependent diabetes mellitus (IDDM)] patients, and 50 type 2 diabetics [non-IDDM (NIDDM)] patients, 22 of whom were insulin-treated and the remaining 28 treated with sulfonylurea and/or metformin). Unlike IGF-I and (to a lesser extent) total IGFBP-3 levels, which decline with age, percent proteolyzed IGFBP-3 seemed relatively stable. In healthy adults, the mean +/- SEM was 29.4 +/- 1.9 for subjects less than 45 yr old and was slightly (but not significantly) lower, 25.7 +/- 3, for those of more than 45 yr. There was no difference between male and female subjects. In GH-deficient patients, despite severely depressed IGF-I levels, percent proteolyzed IGFBP-3 and IGF-I/intact IGFBP-3 ratios were within the normal range. Among acromegalics, percent proteolyzed IGFBP-3 was elevated: 36.6 +/- 3.3 for patients of less than 45 yr, 33.3 +/- 3.2 for patients of more than 45 yr (P = 0.02 vs. healthy subjects). Consequently, the effects of excessive IGF-I synthesis are exacerbated by the enlarged exchangeable fraction of IGFBP-3-bound IGF-I. There was no significant difference in percent proteolyzed IGFBP-3 between GH-deficient patients before and after GH treatment or between treated and untreated acromegalics. In IDDM patients, the means for percent proteolyzed IGFBP-3 were higher than those in healthy adults: 36.7 +/- 3.7 (P = 0.03) and 31.3 +/- 3.3 for subjects of less than 45 and more than 45 yr, respectively. In NIDDM patients, all of whom were more than 45 yr old, the means were 35.2 +/- 2.5 (P = 0.02) for insulin-treated patients and 33 +/- 2.5 for the group treated orally. Among the diabetics, increased IGFBP-3 proteolysis resulted in an IGF-I/intact IGFBP-3 ratio that was normal for IDDM patients of less than 45 yr and above normal (P = 0.01) for the others. Percentage proteolyzed IGFBP-3 and the IGF-I/intact IGFBP-3 ratio were inversely related to body mass index in IDDM patients (r = -0.42, P = 0.008; and r = -0.31, P = 0.05, respectively) and to percentage glycosylated hemoglobin in all insulin-treated diabetics (r = -0.25, P = 0.05; and r = -0.33, P = 0.008, respectively). There was also an inverse relationship between IGF-I/intact IGFBP-3 ratios and IGFBP-1 levels in healthy adults (r = -0.39, P = 0.03) and orally treated NIDDM patients (r = -0.37, P = 0.05). Percentage proteolyzed IGFBP-3 was positively correlated to total IGFBP-3 in healthy adults (r = 0.65, P = 0.0001) and in all the groups of patients. It was negatively correlated to IGF-I/total IGFBP-3 in healthy subjects (r = -0.40, P = 0.02) and diabetics (r = -0.30, P = 0.005). This suggests an autoregulatory mechanism controlling the bioavailability of IGFBP-3-bound IGF-I in the 140-kDa complexes. In the pathological conditions studied here, regulation of IGF-I bioavailability by limited proteolysis of IGFBP-3 contributes toward an appropriate adaptation to insulin deficiency and/or resistance but not to disturbances of GH secretion.

Measurement of intact insulin-like growth factor-binding protein-3 in human plasma using a ligand immunofunctional assay.

Limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) is a fundamental mechanism in the regulation of IGF-I bioavailability in the bloodstream. Its measurement by Western immunoblotting provides only semiquantitative estimation. We have developed a ligand immunofunctional assay (LIFA) for quantifying human (h) intact IGFBP-3 in biological fluids. IGFBP-bound IGFs are dissociated and separated by acid pH ultrafiltration, and a monoclonal antibody specific to the first 160 amino acids of IGFBP-3 is used to capture hIGFBP-3 in a solid-phase assay. The complex is then incubated with (125)I-IGF-I, which binds to intact IGFBP-3 but not to its proteolytic fragments. Binding specificity was demonstrated in competition experiments with unlabeled IGF. Nonspecific binding was 1.4%. The fragments comprising residues 1-160 and 1-95 of recombinant hIGFBP-3 [corresponding to the major proteolytic fragments of approximately 30 kDa and (glycosylated) 20 or (nonglycosylated) 16 kDa detected in serum by Western immunoblotting, respectively] fail to bind (125)I-IGF-I when complexed with the monoclonal antibody. Similarly, no binding of (125)I-IGF-I was obtained in the LIFA when applied to plasmas from pregnant women during the final 3 months of pregnancy, where the characteristic 42- to 39-kDa doublet of intact IGFBP-3 is undetectable. The standard curve was established using a pool of plasmas (EDTA) from healthy adults, for which standardization with glycosylated recombinant hIGFBP-3 yielded an intact IGFBP-3 content of 2 microg/mL. The dynamic range of the LIFA was 0.50-3.75 microL equivalent of the plasma pool in a total volume of 300 microL per assay tube, with a sensitivity threshold of approximately 1 ng intact IGFBP-3. Unknown plasma samples were studied at three concentrations. Intra- and interassay variations were 3.6% and 4%, respectively. In 31 healthy adults, the mean plasma concentration of intact IGFBP-3 was 2.24 +/- 0.08 (SEM) mg/L, and that of total IGFBP-3 measured by immunoradiometric assay was 3.27 +/- 0.14 mg/L. The calculated mean proportion of proteolysed IGFBP-3 was 29.4 +/- 1.9%. In these subjects, a close correlation was found between intact and total IGFBP-3 (r = 0.71, P = 0.0001). The LIFA for IGFBP-3, therefore, provides accurate and sensitive measurement of intact IGFBP-3, the form with the functional capacity to sequester IGF-I in the bloodstream by association with the acid-labile subunit in 140-kDa complexes. In combination with total IGFBP-3 and IGF-I assays, the LIFA opens new perspectives in investigating the regulation of IGFBP-3 proteolysis and IGF-I bioavailability in man.

Growth retardation in constitutionally short children is related both to low serum levels of insulin-like growth factor-I and to its reduced bioavailability.

Measurements of serum levels of insulin-like growth factor (IGF)-I, IGF-II and IGF binding protein (IGFBP)-1 have been carried out in conjunction with Western ligand blot analysis of serum IGFBPs in 39 constitutionally short children and adolescents and compared with those of 27 age-matched normal subjects (and also with 23 hypopituitary patients). Estimated amounts of the two forms of IGFBP-3 (42 and 39 kDa) and of IGFBP-2 (34 kDa) were obtained by laser densitometry scanning. Mean serum levels of IGF-I were decreased by 46% +/- 5% in short, compared to normal, prepubertal children (P < 0.01) and reduced slightly, but not significantly, in short pubertal children. IGFBP-1 levels decreased with age in short children, as they did in normals, but average values were significantly higher in short children (P < 0.001). There was also a tendency for higher IGFBP-2 levels in short prepubertal and pubertal children. IGFBP-3 bands were of equal intensity in short and normal subjects. Physiologically, IGFBP-3 undergoes limited proteolysis which results in facilitated dissociation of the IGFs, particularly IGF-I, and an increase in their turnover. Western immunoblotting detects proteolytic fragments of IGFBP-3 (the major one being of 30 kDa) that are not detected by ligand blotting. The ratio of proteolysed to total IGFBP-3 in short prepubertal children (36.8% +/- 2.6%) was significantly lower (P < 0.01) than in normal prepubertal subjects (60.6% +/- 8.9%). This lesser proteolysis of IGFBP-3 would explain the excessive levels of IGFBP-3 (detected by ligand blotting) relative to IGF levels in short children. These results suggest that growth retardation in short children involves IGF-I deficiency resulting from both decreased IGF-I synthesis and lesser bioavailability of the circulating IGF-I bound to IGFBP-3. High IGFBP-1 levels may also contribute towards limiting the availability of IGF-I to its target cells.

A proteolytic fragment of insulin-like growth factor (IGF) binding protein-3 that fails to bind IGFs inhibits the mitogenic effects of IGF-I and insulin.

Limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) is increasingly becoming recognized as an essential mechanism in the regulation of insulin-like growth factor (IGF) bioavailability, both in the bloodstream and at cellular level. Plasmin generated on contact with various cell types provokes proteolytic cleavages that are similar to those induced in vivo by (as yet unidentified) IGFBP-3 proteases. Experimental conditions were determined to achieve plasmin-induced limited proteolysis of recombinant human nonglycosylated IGFBP-3. Two major fragments of 22/25 kilodaltons (kDa) and one of 16 kDa were identified by Western immunoblotting and isolated by reverse-phase chromatography. The 22/25-kDa fragments correspond to the major approximately 30-kDa glycosylated fragment of IGFBP-3 in serum and the 16-kDa fragment, to one of the same size, that is nonglycosylated. Western ligand blot analysis, affinity cross-linking, and competitive binding experiments using radiolabeled IGF and unlabeled IGF-I or -II showed that in the high performance liquid chromatography eluate containing the 16-kDa fragment, all affinity for IGFs had been lost, whereas the affinity of the 22/25-kDa fragments was considerably reduced. Scatchard analysis of the data indicated a 20-fold loss of affinity for IGF-II and an 50-fold loss for IGF-I compared with that of recombinant human IGFBP-3. In a chick embryo fibroblast assay in which DNA synthesis was stimulated both by IGF-I and by insulin (at 100-fold concentrations, so that interaction with the Type 1 IGF receptor would occur), IGFBP-3 was found to inhibit IGF-I-induced stimulation almost totally. It had no effect on stimulation by insulin, which has no affinity for the IGFBPs. With the 22/25-kDa fragments, barely 50% inhibition of IGF-I stimulation was achieved and no inhibition of insulin stimulation. Unexpectedly, with the fraction containing the 16-kDa fragment (despite the total lack of affinity for IGF-I), IGF-I-induced stimulation was inhibited to nearly the same extent as with intact IGFBP-3. In addition, insulin-induced stimulation was inhibited with similar potency. IGFBP-3 proteolysis therefore generates two types of fragment with different activities. One has weak affinity for IGF-I and is only a weak antagonist of IGF action. The other lacks affinity for the IGFs, but nevertheless inhibits IGF-stimulated mitogenesis, thus acting by a mechanism that is independent of the IGFs.

Isolation and characterization of proteolytic fragments of insulin-like growth factor-binding protein-3.

Limited proteolysis of insulin-like growth factor-binding protein-3 (IGFBP-3) is a normal process in the regulation of insulin-like growth factor (IGF) activity, which we have reproduced in vitro using plasmin and recombinant human non-glycosylated IGFBP-3 in order to isolate and characterize the fragments obtained. Two major fragments of 22-25 and 16 kD were purified by RP-HPLC. The 22- to 25-kD fragment had severely reduced affinity for IGF-I, compared with intact IGFBP-3. It weakly inhibited cell proliferation stimulated by IGF-I and had no effect on insulin-induced stimulation. The 16-kD fragment, which had lost all affinity for IGFs, unexpectedly proved to be a potent inhibitor of both IGF-I-induced and insulin-induced cell growth. This proteolytic fragment of IGFBP-3 therefore exhibits intrinsic inhibitory activity.

A proteolytic fragment of insulin-like growth factor (IGF) binding protein-3 that fails to bind IGF is a cell growth inhibitor.

Limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) is now recognized as a normal process in the regulation of insulin-like growth factor (IGF) activity, its major effect being to increase IGF bioavialability. In order to characterize the proteolytic fragments of IGFBP-3, we reproduced this proteolysis in vitro using plasmin which provokes cleavages that are similar to those induced in vivo by (unidentified) specific IGFBP-3 proteases. Two major peaks were purified by RP-HPLC. One contained a 16 kDa fragment and the other comprised two fragments of 22 and 25 kDa. Competitive binding experiments showed that the 16 kDa material had no affinity for IGFs. The 22-25 kDa fragments had considerably reduced affinity, particularly for IGF-I. In a chick embryo fibroblast assay where DNA synthesis was stimulated by IGF-I or insulin, the 22-25 kDa fragments weakly inhibited IGF-I-induced cell proliferation and had no effect on stimulation by insulin. The 16 kDa fragment unexpectedly proved to be a potent inhibitor of both IGF- and insulin-induced cell growth. This proteolytic fragment of IGFBP-3 therefore exhibits intrinsic inhibitory activity.

Protease-induced alteration of insulin-like growth factor binding protein-3 as detected by radioimmunoassay. Agreement with ligand blotting data.

Structural alteration of insulin-like growth factor binding protein-3 (IGFBP-3) resulting from limited proteolysis by one or more serine proteases in vivo was first described in the serum of pregnant women and in certain pathological conditions. Western immunoblotting has since been employed to detect the phenomenon in normal serum, using a polyclonal antibody raised against recombinant human IGFBP-3 and a highly sensitive technique of visualization by chemiluminescence. The major proteolytic fragment of 30 kDa, which fails to be detected in native serum by ligand blotting owing to its weak affinity for IGFs, has proved clearly visible in all serum samples tested, sometimes accompanied by smaller fragments of 20 and 16 kDa. Among the serum samples analysed, increasing proportions of proteolysed IGFBP-3 were found in the following order: acromegalic patients, normal subjects, GH-deficient patients, pregnant women. In RIAs done with the same antibody, many of the serum samples yielded dose-response curves which were not parallel with standard curves, with lower gradients. In the samples where measurements were possible, apparent IGFBP-3 levels proved lower in pregnant women (2.28 +/- 0.23 mg/l, mean +/- SEM) than in normal adults (4.26 +/- 0.33 mg/l, P < 0.001). These observations, which contradict earlier reports of higher levels in pregnant women, suggest that the 30 kDa proteolytic fragment has a weaker affinity for the antibody than the intact IGFBP-3 (which in ligand- and immunoblotting appears as a characteristic 42-39 kDa doublet and which is barely or not detectable in pregnancy serum).(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor binding protein-3 is functionally altered in pregnancy plasma.

In a variety of physio-pathological conditions, but also in the normal state, calcium-dependent serine protease(s) partially proteolyze proportions of circulating insulin-like growth factor binding protein-3 (IGFBP-3). This occurs without disrupting the 150-kilodalton (kDa) complexes in which IGFBP-3 carries of most of the IGF-I and IGF-II in serum. In this work we show that the 150-kDa complex is functionally altered during pregnancy, which is when the largest proportion of IGFBP-3 is proteolyzed. After preincubation at 4 C with [125I]IGF-I or -II with or without 1 microgram unlabeled IGF-I or -II, pooled plasma samples were gel filtered at pH 7.4. Larger proportions of labeled IGFs were found in the 150-kDa complexes of the third trimester pregnancy plasma pool than in those of the normal plasma pool, suggesting increased binding capacity. Nevertheless, competitive binding experiments using [125I]IGF-I and -II and IGFBP-3 preparations from each of the plasma pools showed that the competitive potencies of IGF-II and especially IGF-I were lower in pregnancy IGFBP-3 than in normal IGFBP-3. Scatchard analysis revealed a 2-fold loss of affinity for IGF-II and a 10-fold loss for IGF-I compared with that for normal plasma IGFBP-3. In studies at 37 C of the kinetics of dissociation of [125I]IGF-I and -II bound to IGFBP-3, IGF-II was dissociated 6 times faster, and IGF-I 10 times faster from pregnancy plasma IGFBP-3 than from normal plasma IGFBP-3. After incubation of individual plasma samples at 37 C and gel filtration at room temperature (in the presence of EDTA), IGFs were assayed in the three circulating pools (150-kDa and 40-kDa complexes and free IGFs), revealing a redistribution of pregnancy plasma IGFs. The proportion of total IGF-I in free form was nearly three times greater in pregnancy than in normal plasma (11.4% vs. 4.1%, P < 0.005), whereas that of free IGF-II was slightly smaller (1.5% vs. 2%). In the 150-kDa complexes, the proportion of total IGF-I was significantly smaller in pregnancy than in normal plasma, and that of IGF-II was greater. In the 40-kDa complexes, the proportion of total IGF-II was significantly smaller. The mean ratios of molar concentrations of free IGF-I/IGF-II were 0.43 in normal plasma and 2.23 in pregnancy plasma (P < 0.005).(ABSTRACT TRUNCATED AT 400 WORDS)

Two insulin-like growth factor (IGF)-binding proteins are responsible for the selective affinity for IGF-II of cerebrospinal fluid binding proteins.

We have studied the relationships between the structure and affinity of two insulin-like growth factor-binding proteins (IGFBPs) purified from human cerebrospinal fluid (CSF). Competitive binding studies were performed using preparations of human recombinant IGF (rhIGF-I, rhIGF-II, and their labeled homologs) and the truncated variant form of IGF-I, rh-Des-(1-3)-IGF-I. One of these BPs, which is the most consistently detected in CSF, corresponds to IGFBP-2. The other is a new form whose N-terminal sequence we reported earlier, which we call the 32-30K BP on the basis of its electrophoretic migration. Comparisons were made with an IGFBP-1 preparation purified from amniotic fluid and with two BPs purified from human serum, which are homologous to the CSF BPs. The CSF BPs have particularly strong affinities for IGF-II. The estimated affinity constants (Ka) were 2 X 10(10) M-1 for IGFBP-2 and 10(11) M-1 for the 32-30K BP. These affinities were 15-20 and 70 times stronger than the respective affinities for IGF-I. The affinity of the 32-30K BP is the strongest among the BPs identified to date. The two BPs isolated from serum, which correspond to the 32-30K CSF BP and IGFBP-2, had affinities for IGF-II and IGF-I similar to those of the CSF BPs. IGFBP-1 had nearly identical affinities for the two IGFs of approximately 10(10) M-1. Des-(1-3)-IGF-I failed to bind to the CSF BPs, but bound to IGFBP-1, although with a 40-fold weaker affinity than IGF-I. From our data it would seem that IGFBP-1 has two classes of IGF-binding site, one of high and one of low (less than 10(9) M-1) affinity for both IGFs. The other two BPs, by contrast, each possess a predominant class of high affinity binding site for IGF-II. A second class of lower affinity (greater than 10(9) M-1) sites bind both IGF-I and IGF-II. In the case of the 32-30K BP, these preferentially bind IGF-II; in the case of IGFBP-2, their binding of the two IGFs is similar. These different types of binding site may play an important role in controlling the bioavailability of IGF-I and IGF-II.(ABSTRACT TRUNCATED AT 400 WORDS)

Serum insulin-like growth factors and insulin-like growth factor binding proteins in the human fetus. Relationships with growth in normal subjects and in subjects with intrauterine growth retardation.

IGF-I, IGF-II, and their binding proteins (BP) were studied in sera obtained by direct puncture of umbilical cords in utero between 20 and 37 wk of gestation in 103 normal fetuses and in 16 fetuses with intrauterine growth retardation, as well as in the cord blood of 37 normal newborns of 38- to 42-wk pregnancies. In normal fetuses, IGF-I levels were approximately 50 ng/mL and IGF-II levels approximately 350 ng/mL up to the 33rd wk of pregnancy. Thereafter, both increased to reach values two to three times higher at term. Correlations were found between fetal placental lactogen levels and those of IGF-I and IGF-II, which is consistent with the hypothesis that placental lactogen is involved in the regulation of IGF synthesis in the fetus. With weight (either measured at birth or deduced from echographical data) as index of fetal size, IGF-I levels were significantly (p less than 0.001) higher in fetuses with weights above the mean for gestational age than in fetuses with weights below the mean, whereas IGF-II levels were similar in the two groups. Similarly, IGF-I (but not IGF-II) levels in fetuses with intrauterine growth retardation were significantly lower than those in normal fetuses of the same age (p less than 0.01). These findings suggest that, during the latter months of intrauterine life, IGF-I (but not IGF-II) is involved in the control of fetal size. Total fetal BP concentrations were approximately 1/3 those of adults. The fetal electrophoretic profile obtained by Western-ligand blotting bore a strong resemblance to that of subjects with growth hormone deficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence of enzymatic degradation of insulin-like growth factor-binding proteins in the 150K complex during pregnancy.

Western ligand blot analysis of the different molecular forms of insulin-like growth factor-binding protein IGF-BP) in serum and plasma samples from 89 pregnant women has revealed a marked decrease, after the second month of pregnancy, in the 41.5 and 38.5K species (which are the binding units of the 150K complex) as well as in the 24K form. There was also a slight decrease in the 34K form, the 30K form was unaffected, and additional 21.5 and 20K bands appeared. Cross-linking experiments demonstrated the disapperance of a 49K band which is characteristic of the 150K complex. The alterations of the electrophoretic profile of the BPs were accompanied by a decrease in binding activity of up to 90%. Gel filtration at pH 7.4 confirmed that the decrease was essentially attributable to changes in the 150K complex BPs: 1) material eluting in the 150K zone contained only one third of the binding activity, as opposed to three quarters in reference material; 2) radiocompetition experiments illustrated the loss of affinity for IGF-I and IGF-II of the BPs extracted from the 150K complex; 3) ligand blot analysis revealed, in contrast with the virtual disappearance of the 41.5 and 38.5K forms, the appearance of a broad indistinct band at 30K and additional bands at 21.5 and 20K. With immunoblotting, the anti-IGF-BP-3 antibody, which specifically recognizes the 41.5 and 38.5K species, cross-reacted with this 30K material. The alterations of the BPs appeared to be enzymatic. When pregnancy serum was mixed with reference serum, the 41.5, 38.5, and 24K forms contributed by the reference serum were markedly reduced after 30 min of incubation at 37 C. However, these alterations could be prevented by incubation at either 0 or at 37 C in the presence of EDTA or aprotinin and could be curbed in the presence of high concentrations of phenylmethylsulfonylfluoride. Unmixed reference serum incubated at 37 C yielded an unchanged BP profile. Incubation of pregnancy serum with hypopituitary serum, which has elevated levels of the 34 and 30K BPs, resulted in a marked decrease in the 41.5 and 38.5K forms, a slight alteration of the 34K form, and no change in the 30K form. These findings suggest that during pregnancy, enzymatic (probably protease) activity either appears or is significantly increased in the circulation, which specifically degrades some of the IGF-BPs.(ABSTRACT TRUNCATED AT 400 WORDS)

Heterogeneity of insulin-like growth factor binding proteins and relationships between structure and affinity. 1. Circulating forms in man.

Circulating insulin-like growth factors (IGFs) are bound to specific, high-affinity binding proteins (BPs), and form complexes with relative molecular masses of about 150,000 ('large' complex) and 40,000 ('small' complex). The large complex appears to be under growth-hormone control and its proportions vary with those of the IGFs. Molecular heterogeneity among the binding proteins was revealed by polyacrylamide gel electrophoresis (SDS-PAGE) of serum in which they were cross-linked to 125I-labelled IGF I or II. Out of the six specific bands observed, of 150,000, 120,000, 49,000, 40,000 and 37,000 Mr, the last three appeared in both complexes, whereas the first three were visible only in the large complex. Some or all of the 49,000-37,000-Mr species may constitute the subunits of 150,000-Mr and/or 120,000-Mr IGF-BP complexes. With electrophoresis followed by transfer onto nitrocellulose and incubation with either 125I-labelled IGF I or II (western blot), the different binding proteins were identified per se. There were five molecular forms with Mr of 41,500, 38,500, 34,000, 30,000 and 24,000. In normal serum the 41,500 and 38,500-Mr forms were the major binding proteins. They appeared in both complexes, but were predominant in the large complex where they constitute the elementary binding units. These two proteins therefore bind to IGFs to form both 'monomeric' IGF-BP and 'oligomeric' (IGF-BP)n complexes. The 34,000, 30,000 and 24,000-Mr forms, by contrast, were visible only in the small complex. Different mechanisms appear to regulate the different binding proteins: in acromegalic serum the 41,500 and 38,500-Mr forms were augmented and the 34,000-Mr form diminished, whereas in hypopituitary serum the reverse was true and, in addition, the 30,000-Mr form was augmented. With chromatofocusing, the 34,000, 30,000 and 24,000-Mr forms eluted in three peaks between pH 6.0 and 4.0, whereas the 41,500 and 38,500-Mr forms eluted throughout the gradient, principally at pH 7.5 and 7.0. Competitive binding studies, done on binding proteins separated either by chromatofocusing or by SDS-PAGE and transfer onto nitrocellulose, revealed different affinities for the IGFs among the different molecular forms. The 41,500 and 24,000-Mr binding proteins preferentially bound IGF I and the 38,500, 34,000 and 30,000-Mr proteins preferentially bound IGF II. Our findings demonstrate the molecular heterogeneity of the binding proteins and the existence of a relationship between their structure and their affinities for the IGFs.(ABSTRACT TRUNCATED AT 400 WORDS)

Specific assay for insulin-like growth factor (IGF) II using the IGF binding proteins extracted from human cerebrospinal fluid.

A protein-binding assay for insulin-like growth factor II (IGF II) is described. The assay uses IGF binding proteins extracted from human cerebrospinal fluid which have selective affinity for IGF II. IGF I was 9 times less potent than IGF II in displacing [125I]IGF II, and when mixtures of the IGFs were assayed at IGF I/IGF II ratios of 2, 5, and 10, interference from IGF I in the assay was 0%, 5%, and 9%, respectively. Given the serum concentrations of IGF I and IGF II estimated by RIA and by this protein-binding assay, IGF I can be said to have had no cross-reaction when IGF II was assayed in human serum and at most 5% cross-reaction in the case of rat serum. After separation of IGFs from their binding proteins by acidic gel filtration, serum IGF II levels (mean +/- SE) measured by this method were 1322 +/- 66 ng/ml in normal adults, 500 +/- 65 ng/ml in patients with total GH deficiency, 1327 +/- 69 ng/ml in untreated acromegalic patients, and 1817 +/- 145 ng/ml in uremic patients undergoing chronic hemodialysis. In postpubertal young rats, the mean serum IGF II level was 43 +/- 2.6 ng/ml and after hypophysectomy it was 16 +/- 2.4 ng/ml. Although the IGF II levels in man and in the rat were different, they appeared to be similarly GH dependent, although less so than IGF I. In view of the sensitivity (0.03 ng IGF II) and the specificity of this assay, the small quantities of cerebrospinal fluid required (1 mu leq/assay tube) and its applicability for IGF II measurement in several species, the use of this assay for measuring IGF II in a variety of biological media can be envisaged.

Somatomedin (insulin-like growth factors)-binding proteins. Molecular forms and regulation.

The insulin-like growth factor (IGF)-binding proteins present in the human serum and various biological media have been characterized by several methods: gel filtration, sucrose gradient sedimentation, polyacrylamide gel electrophoresis and chromatofocusing. Several forms have been identified with molecular weights of approximately 42,000, 39,000, 34,000, 30,000 and 24,000 daltons. Results of competitive binding studies suggest that the different forms of binding proteins have different affinities for IGF-I and IGF-II. The influence of various hormones and pathophysiological conditions on the biosynthesis of the binding proteins has been investigated.

Triiodothyronine stimulates the production of insulin-like growth factor (IGF) by fetal hypothalamus cells cultured in serum-free medium.

Grown in serum-free medium, dissociated cells from fetal mouse hypothalami release insulin-like growth factors (IGFs) and their binding proteins (IGF BPs) into the culture medium. Addition of triiodothyronine (10-12-10-8 M), which enhances neuron maturation, resulted in a significant increase in IGF concentration. By contrast, there was no significant effect on IGF BP. These results suggest a role for thyroid hormone in the control of IGF biosynthesis in nerve cells.

Preferential measurement of insulin-like growth factor (IGF) I-related peptides in serum with the aid of IGF-binding proteins (IGF BPs) produced by rat liver in culture. Estimation of serum IGF BP levels.

A protein-binding assay for insulin-like growth factor (IGF) was developed which preferentially measures IGF I-related peptides in serum. Binding proteins (BPs) extracted from the culture media of livers from approximately 4-week-old rats were used in the assay, with pure IGF I as tracer and a partially purified IGF preparation as standard. Serum samples were gel filtered in acetic acid to separate the IGFs from their BPs. IGFs could be detected with this assay in extracts corresponding to as little as 0.2 microliter normal serum. The affinity of these liver BPs was greatest for IGF I. IGF II, somatomedin A, and multiplication-stimulating activity were found to be 2, 3, and 10 times less potent, respectively, than IGF I in displacing [125I]IGF I. There was no cross-reaction with insulin and proinsulin, structurally the most closely related peptides. There was a highly significant correlation (r = 0.98, P less than 0.001) between IGF values obtained from simultaneous assays, using either 1) somatomedin C/IGF I antibodies, or 2) the liver BPs, for acromegalic, normal, and hypopituitary serum extracts. Nevertheless, the protein-binding assay yielded values 1.7-fold those of the RIA. The BPs therefore do not possess the specificity of the antibodies for IGF I, but they do have the advantage of being less species specific in that they permit measurement of IGFs in the rat (among others) with the same sensitivity as in man. The validity of the assay was demonstrated both by the studies of IGF levels as a function of age, which yielded a profile characteristic of IGF I, and by the GH-dependence of the IGF levels measured. Mean IGF levels (+/- SEM) were the following: 1.03 +/- 0.03 U/ml in normal adults; 2.62 +/- 0.10 in acromegalic patients; 0.19 +/- 0.01 in patients with total GH deficiency; 0.58 +/- 0.04 in patients with partial GH deficiency (the reference serum being assigned a potency of 1 U IGF/ml). Human GH administration (6 mg/m2, im) to untreated hypopituitary patients on average provoked a 2-fold increase in IGF levels within 24 h. In hypophysectomized rats there was a close relation between the IGF level attained and the dose of GH administered (P less than 0.001). IGF BPs were titrated by incubating different concentrations of the serum extracts with [125I]IGF I and comparing these with a BP preparation obtained from the reference serum used in the IGF assay and arbitrarily assigned a value of 1 U IGF BP/ml.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for production by the liver of two IGF binding proteins with similar molecular weights but different affinities for IGF I and IGF II. Their relations with serum and cerebrospinal fluid IGF binding proteins.

Studies of the competitive binding of IGF I and IGF II and 125I-labelled IGF I and IGF II to the specific binding proteins (BPs) produced by the liver in culture suggest that two BPs exist, one with a selective affinity for IGF I and the other with a selective affinity for IGF II. The ratio of the former BP to the latter appeared to be higher in the culture medium of young rat livers and of fetal human livers than that in the culture medium of adult human livers. The two BPs form complexes with their IGFs with the same apparent molecular weight of approximately 40K. With gel filtration of adult human serum both types of BP eluted with the greater than 100K and the 40-70K molecular weight material. The BP with the selective affinity for IGF II predominated in the 40-70K material as well as in the cerebrospinal fluid of adult human subjects.