No abstract

ABSTRACT'. IGF-I, IGF-11, 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-I1 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-11, 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 < 0.001) higher in fetuses with weights above the mean for gestational age than in fetuses with weights below the mean, whereas IGF-I1 levels were similar in the two groups. Similarly, IGF-I (but not IGF-11) levels in fetuses with intrauterine growth retardation were significantly lower than those in normal fetuses of the same age ( p < 0.01). These findings suggest that, during the latter months of intrauterine life, IGF-I (but not IGF-11) is involved in the control of fetal size. Total fetal BP concentrations were approximately '/J those of adults. The fetal electrophoretic profile obtained by Western-ligand blotting bore a strong resemblance to that of subjects with growth hormone deficiency. In newborns, the proportions of IGF-I and IGF-I1 associated with BP to form 150-kD complexes were considerably lower than those in adults, but similar to those in hypopituitary patients. It may be deduced from these findings that during fetal life, BP synthesis is adapted to increase the bioavailability of the IGF at a time when growth is at a maximum.

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)

Insulin-like growth factor-I (IGF-I) and IGF-II are associated in the blood with specific binding proteins (BPs), forming complexes that elute in gel filtration with estimated mol wt around 40 and 150 kD. The latter appears to be under GH control. Five molecular forms of BP (41.5, 38.5, 34, 30, and 24 kD) have been identified by Western blotting using 125I-labeled IGF. All five forms are present in the smaller complexes, but only the 41.5- and 38.5-kD forms are found in the larger complexes. In this study immunoblotting showed that the 41.5- and 38.5-kD forms were recognized by antibodies directed against the GH-dependent BP purified from human plasma, and the 30-kD form was recognized by antibodies directed against the BP purified from amniotic fluid. The 34- and 24-kD forms proved to be immunologically unrelated to the other three. In sera with large quantities of the 41.5- and 38.5-kD forms, an additional band was often observed immediately ahead of the migration front of the 30 kD band. This was recognized by the anti-GH-dependent BP antibody and probably corresponds to a degradation product of the 41.5- and 38.5-kD BPs. Serum 41.5- and 38.5-kD BPs have been found to be elevated in acromegaly, where GH hypersecretion causes increased IGF-I levels, and diminished in cases of genetic or idiopathic GH deficiency and defects of the GH receptor (Laron's syndrome), where both IGF-I and IGF-II are decreased, as well as in Pygmy adults and children who have isolated IGF-I deficiency. In all of these conditions, the proportions of the 34- and 30-kD forms were inversely related to those of the 41.5- and 38.5-forms. Under treatment, the BP profiles tended to return to normal. In cases of GH deficiency caused by a tumor, the BP profiles resembled those of hypopituitary or normal serum, depending on whether IGF levels were diminished or normal. It, therefore, seems that BP synthesis is coordinated with IGF-I synthesis and may not be directly GH dependent. The results of neutral pH gel filtration analysis of hypopituitary (idiopathic and tumoral) and normal sera point to a relationship between the levels of circulating IGFs and those of the 150-kD IGF-BP complex whose binding units are the 41.5- and 38.5-kD BPs. It, therefore, seems that the 150-kD complex controls the bioavailability of IGF-I and IGF-II.(ABSTRACT TRUNCATED AT 400 WORDS)

The extent to which the association between insulin-like growth factors (IGFs) and their specific binding proteins (BPs) prevents their crossing the capillary barrier was studied by comparing their distribution in serum with that in samples of lymph collected from the lower leg of five subjects undergoing radiographical investigation of the lymphatic system. The IGF concentrations in lymph were 10-30% of the corresponding serum levels, and in each subject the ratios of IGF-I and IGF-II in the lymph to those in the serum were similar. Western blot analysis of the BPs revealed that the five molecular forms identified in serum also were present in lymph, but in significantly smaller quantities. The 41.5K and 38.5K forms, which constitute the binding units of the large complex (approximately 150K) of serum and are also capable of binding IGFs in monomeric form, were present in smaller amounts than the 34K, 30K and 24K forms, which belong specifically to the small complex (approximately 40K) of serum. The BPs extracted from lymph were similar to those of the small complex, with a preferential affinity for IGF-II and only half of the affinity for IGF-I of the BPs extracted from serum. With neutral pH gel filtration of lymph, more than 90% of IGFs and binding activity eluted with the material in the area of the 40K zone. These data indicate that the 150K IGF-BP complexes do not cross the capillary barrier, whereas the 40K complexes do. The function of the former may be to provide a reservoir and buffering action of the IGFs, whereas the latter may be involved in the transport of the IGFs to their target cells.

IGF Binding Protein-3 (IGFBP-3), the major IGF carrier in the blood, undergoes limited proteolysis which reduces its affinity for IGFs, thus facilitating dissociation. The functional effects of this at the cellular level were studied by comparing two serum pools, one from healthy adults, one from women during late pregnancy when IGFBP-3 proteolysis is increased. Sera were mixed to yield identical IGF-I and IGF-II concentrations in the two pools. Western ligand and immunoblotting gave the characteristic IGFBP patterns for the two types of serum. Both pools dose-dependently stimulated DNA synthesis in cultured chick embryo fibroblasts. Stimulation by pregnancy serum was twice that by normal serum at 0.05-0.2% concentrations (P < 0.001). In the presence of excess monoclonal anti-IGF-I and -II antibodies, stimulation by both (0.1-0.2%) pools was 70-80% reduced and residual stimulation was similar. Addition of recombinant human (rh)IGFBP-3 dose-dependently depressed both pools' activity, more so for normal serum at 25 and 50 ng/ml, equally for each at 100 ng/ml. At the latter concentration, slight proteolysis of the rhIGFBP-3 was detectable in the presence of 0.2% pregnancy serum, but at 25 ng/ml, proteolysis was absent. These results suggest that IGFs are released more readily from pregnancy serum, accounting for the weaker inhibitory effect of low rhIGFBP-3 concentrations. For identical IGF concentrations, pregnancy serum's greater biological activity therefore reflects greater IGF availability to the cells. This study demonstrates the functional consequences at cellular level of serum IGFBP-3 proteolysis, underlining its significance in regulating serum IGF bioavailability. (J. Clin.

Circulating insulin‐like growth factors (IGFs) are bound to specific, high‐affinity binding proteins (BPs), and form complexes with relative molecular masses of about 150000 (‘large’ complex) and 40000 (‘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 polydcrylamide 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 150000, 120000, 49000, 46000, 40000 and 37000 Mr, the last three appeared in both complexes, whereas the first three were visible only in the large complex. Some or all of the 49000–37000‐Mr species may constitute the subunits of 150000‐Mr and/or 120000‐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, 34000, 30000 and 24000. 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 34000, 30000 and 24000‐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 34000‐Mr form diminished, whereas in hypopituitary serum the reverse was true and, in addition, the 30000‐Mr form was augmented. With chromatofocusing, the 34000, 30000 and 24000‐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. They also suggest that, apart from their function as IGF carriers, the binding proteins also play a modulating role in the interaction between IGFs and their target cells.

Insulin-like growth factors (IGFs) in blood form two complexes with specific binding proteins (BPs): a large, growth hormone (GH)-dependent complex with restricted capillary permeability, and a smaller complex, inversely related to GH, with high turnover of its IGF pool and free capillary permeability. The distribution of BPs and of IGFs I and II between these complexes was studied in sera from healthy adults treated with IGF I or/and GH and from patients with extrapancreatic tumor hypoglycemia. Like GH, IGF I administration raises IGF I and two glycosylation variants of IGFBP-3 in the large complex, but unlike GH drastically reduces IGF II. During IGF I infusion, IGFBP-3 appears in the small complex whose IGFBP-2 and IGF I increase three-to fivefold and fivefold, respectively. GH treatment, associated with elevated insulin levels, suppresses IGFBP-2 and inhibits its increase owing to infused IGF I. The small complex of tumor sera contains increased amounts of IGFBP-2 and -3, and two-to threefold elevated IGF IL Conclusions: low GH and/or insulin during IGF I infusion and in extrapancreatic tumor hypoglycemia enhance expression of IGFBP-2 and favor partition of IGFBP-3 into the small complex. Free capillary passage and high turnover of its increased IGF I or II pools may contribute to compensate for suppressed insulin secretion during IGF I infusion or to development of tumor hypoglycemia. (J. Clin. Invest. 1990. 86:952-961.) Key words: IGF binding proteins-IGF I treatment * extrapancreatic tumor hypoglycemia Introduction Insulin-like growth factors (IGFs)' are synthesized and released by many tissues and cell types (1, 2). The liver contributes most of the IGFs in the circulation (1, 3). IGFs are always found in association with specific high-affinity binding proteins (2). As is true for IGFs, the highest concentrations of IGF