Michel Binoux

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

ABSTRACT: 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 < 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 < 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. In new borns, the proportions of IGF-I and IGF-II 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.

Limited Proteolysis of Insulin-Like Growth Factor Binding Protein-3 (IGFBP-3) : A Physiological Mechanism in the Regulation of IGF Bioavailability

Enzymes are known to play a role in activating or releasing growth factors like the FGFs and TFGs from the extra-cellular matrix (1, 2). When first discovered, in vivo proteolysis of serum IGFBPs, and particularly IGFBP-3, by pregnancy-associated protease(s) was naturally suspected to be physiologically significant (3, 4). Although the structural alteration of IGFBP-3 and its loss of affinity for the IGFs were clearly demonstrated by Western ligand blotting, immunoblotting and competitive binding studies (3 – 5), some doubt was expressed as to its physiological nature in view of the harsh experimental conditions used (SDS, acidification). Both IGFBP-3 and the acid-labile (a) subunit were found in the 150-kDa material and ternary complex formation was found to be possible with acidified pregnancy serum in the presence of a-subunit (6). Actually, in our initial work on pregnancy serum, we showed that the 150-kDa complex was not disrupted in pregnancy serum analysed by gel filtration at neutral pH, although there were also small IGFBP-3 fragments in the fractions containing low molecular weight proteins (3). It therefore seemed possible that the stability of the 150-kDa complex would be diminished and the IGFBP-3 functionally altered, even if proteolysis remained limited.

Insulin-Like Growth Factor-I Gene Analysis in Subjects with Constitutionally Variant Stature

ABSTRACT: The IGF-I gene from leukocyte DNA of a control population of normal stature was studied using Southern blotting. Restriction fragment lengths for 21 enzymes were determined and three restriction fragment length polymorphisms (RFLP) were found (EcoRV, HindIII, and PvuII). In addition, the IGF-I gene of 64 constitutionally short subjects, five Pygmies, and 10 constitutionally tall subjects was analyzed. No IGF-I gene alterations were detectable by Southern blot in any of these conditions. Linkage analysis using genetic markers (RFLP) yielded results that were uninformative for five constitutionally short families investigated, owing to the limited number of RFLP and their low incidence (17% for the 5.2- kb HindIII, 5-kb PvuII RFLP alleles, and 13% for the 13- kb EcoRV RFLP allele). The EcoRV RFLP was found to map near Exon 1. The incidence of the 13-kb polymorphic allele with EcoRV proved to be lower (4%) in the group with constitutionally short stature than in controls. These results could suggest that modifications in the region of the IGF-I gene may be involved in constitutionally short subjects.

Cerebrospinal IGF Binding Proteins : Isolation and Characterization

In biological fluids, IGFs (insulin-like growth factors I and II) are associated with specific, high-affinity (≈ 1010 M-1) binding proteins (BPs) which control both their bioavailability and their action at target cell level (Review in 1). Research done in our laboratory has shown that the BPs are molecularly and functionally heterogenous. Using Western ligand blotting, we have identified five molecular forms in man, of 41.5, 38.5, 34, 30 and 24 kDa. The proportions of these forms vary in different biological fluids and culture media and their regulation and affinities for IGF-I and IGF-II are different (2–6).

Functional Alteration of the Somatotrophic Axis in Transgenic Mice with Liver-Specific Expression of Human Insulin-like Growth Factor Binding Protein-1

In earlier work, postnatal growth restriction (more marked in males) was observed in a model of transgenic mice with liver-specific expression of human IGF binding protein-1. This was associated with diminished plasma IGF-I levels, the cause of which remained unexplained. Subsequently, abnormalities of CNS development were ascertained, justifying investigation of the somatotrophic axis. Pituitary gland weight in transgenic animals was reduced proportionally to body weight. Immunohistochemical examination of the pituitaries in 3- to 4-mo-old mice revealed somatotrophs of normal size in homozygotes, but density was decreased to approximately two thirds of that in wild-type siblings (p = 0.001). The same was true of lactotrophs. The GH content of the pituitary was significantly reduced in heterozygotes (p < 0.02) and more so in homozygotes (p < 0.0003), although the GH/total protein ratio was similar to that in wild types. Pituitary perifusion experiments showed that in vitro the amounts of GH secreted under basal conditions and under GH-releasing hormone stimulation were similar in transgenic and wild-type mice. Ten days of treatment with human GH (100 μg/d) in 45-d-old transgenic and wild-type mice provoked significant weight gain (p = 0.02) in all animals, the means being 12.4% for homozygotes and 10.4% in heterozygotes, as opposed to 5.8% in wild-type mice. The increase in weight tended to correlate with an increase in plasma IGF-I. From these results, we conclude that the reduced plasma IGF-I in IGF binding protein-1 transgenic mice may result from insufficient GH production by the depressed number of somatotrophs, possibly associated with functional alteration of hypothalamic control.

Biological Actions of Proteolytic Fragments of the IGF Binding Proteins

The existence of insulin-like growth factor binding protein (IGFBP) fragments was first discerned during purification of IGFBP-1 from human placenta (1) and IGFBP-3 from human (2,3) and rat serum (4,5) and human cerebrospinal fluid (6). A truncated form of IGFBP-5 was isolated in advance of the intact protein (6,7). The amino (N)-terminal sequences of these fragments were found to correspond to forms lacking the carboxy (C)-terminal third of the molecule. However, in the case of IGFBP-2, a fragment corresponding to the C-terminus of the native binding protein was isolated from the conditioned medium of a fetal rat liver cell line (8). At the same time, it was discovered that IGFBP-3 undergoes limited proteolysis during pregnancy (9–11). The resulting functional alteration was revealed by the disappearance of the characteristic 42–39-kDa doublet in Western ligand blotting and the concomitant appearance of a 30-kDa fragment in the chromatographic fragments normally containing the 140-kDa complexes that comprise IGFBP-3, IGF-I or -II, and the acid-labile subunit (ALS). From these observations, it seemed that the fragment must play some physiological role. Further studies confirmed its weak affinities for the IGFs, which would explain their facilitated dissociation from the 140-kDa complexes and their enhanced bioavailability (12,13).

INSULIN-LIKE GROWTH FACTORS AND THEIR BINDING PROTEINS (IGF-BP) IN HUMAN FOETAL SERUM

IGF I and IGF II were measured in serum samples collected from the umbilical cord in healthy foetuses (in utero) and newborns. Results (mean tSE) were as follows :In foetuses, IGF I levels were similar to those in cases of total GH deficiency. IGF II levels were on average 6.5 times those of IGF I. No correlation was seen between IGF levels and growth parameters estimated by ultrasounds. Western blot analysis of IGF-BPs showed that foetal serum contained smaller quantities than normal adult serum of 42 and 39 K MW forms (which in the adult appear chiefly in the ~ 150 K [IGF-BP] complex) and large quantities of the 34 and 30 K forms, which have a selective affinity for IGF II and are minor forms in adults. Such a pattern occurs in GH deficiency. Conclusion : The profile of IGFs and IGF-BPs in foetal serum is similar to that seen after birth when GH control is absent.

30 SERUM IGF BINDING PROTEINS ELECTROPHORETIC PROFILES IN CHILDREN WITH COELIAC DISEASE

Subjects with impaired nutritional status are known to have high hGH levels and very low IGF I levels. With a view to studying the regulation of the different molecular forms of IGF binding proteins (BPs), Western blotting was used to analyse the sera of 19 4-to 15-year-old children with coeliac disease before and/or during treatment, and to compare their BP profiles with those of hypopituitary (n=7) and obese (n=7) children of the same age. The untreated coeliac disease patients (n=14) were all stunted in height (m = -3.6 SD) and weight (m = -2.1 SD) and had high hGH (m =28 ng/ml) and low IGF (0.30 U/ml) levels. In 12, the electrophoretic profiles closely resembled those of the hypopituitary patients with significantly decreased 42- and 39K BPs (which are the subunits of the 150 K [IGF-BP] complex) and a strongly enhanced 34 K 8P. In the treated subjects (n=7), the clear resumption of growth (+ 8.2cm in 8 months) was accompanied by a slight rise in IGF levels and a gradual change in the BP profiles which became normal in only two out of the three patients on gluten-free diet over one year. In the obese cases, negligible GH levels accompanied normal IGF levels, normal 39- and 42K, and diminished 34K BPs. These findings indicate 1) that the amount of 39- and 42K BP in the serum is more closely linked to IGF than to GH levels and 2) that their slow rise in patients on a gluten-free diet may favour growth resumption by increasing the bioavailability of IGFs.

SERUM ICF II LEVELS IN CHILDREN AND ADOLESCENTS OF CONSTITUTIONALLY VARIANT STATURE

A specific assay for IGF II using binding proteins extracted from cerebrospinal fluid (JCEM 1986) was used to investigate a population of children and adolescents of constitutionally variant stature with normal GH secretion following provocative stimuli. The results were compared with those of a control population of the same age. In all three groups of subjects (control, short and tall subjects) IGF II levels increased from infancy until puberty as it was the case for IGF I, but with a weak slope and without further significant increase of IGF II during puberty; control values were 691 ± 165 ng/ml before 5 years, 963 ± 247 ng/ml in 5-11 years (P1) and 1009 ± 182 ng/ml in pubertal subjects. The tall subjects had higher levels than controls (p < .02). Among the short subjects, only the youngest had lower levels than controls of the same age (p < .05). For the three groups of subjects taken together there was a positive correlation between IGF II levels and 1) height age (r =.50, p < .001) and 2) bono age (r = .44, p < .001). A close correlation was seen between IGF I and IGF II levels (p < .001). This is not the case in the situations where excess or deficiency of GH secretion is demonstrated.

50 BODY SIZE AND GROWTH VELOCITY PARALLEL INSULIN-LIKE GROWTH FACTOR (IGF) I LEVELS IN CHILDREN AND ADOLESCENTS WITH CONSTITUTIONAL VARIANT STATURES

In subjects with various growth rates (without any clinical or biological abnormalities) IGF I related peptides increase with age in a similar way but at different levels. In comparison with normal subjects (height = mean for age ± 1 SD), IGF levels measured by competitive protein-binding assay were found significantly higher in subjects with tall stature (height > mean for age + 2 SD) and significantly lower in subjects with short stature (height < mean for age - 2 SD). Analysis of the data altogether shows that 1) the positive correlation between IGF and height (r = 0.71, p < 0.001, n = 227) does exist without respect to age, as shown when studying the ratios of height/normal height for age vs IGF/normal IGF for age (r = 0.61, p < 0.001); 2) in subjects whose growth was followed up (tall and short stature) a positive correlation is found between the ratios of growth velocity (gv)/normal gv for age and IGF/normal IGF for age (r = 0.52, p < 0.001, n = 111). The differences in IGF levels between the three groups of subjects (which contrast with no differences in GH levels in the usual pharmacological tests) disappear after epiphyseal fusion. These results suggest that, during growth, individual differences in height and growth velocity seem to be directly related to the IGF secretory capacity. This provides further evidence for an important rôle of IGF I in the physiological control of growth.