Dysregulation of energy homeostasis in mice overexpressing insulin-like growth factor-binding protein 6 in the brain.

AIMS/HYPOTHESIS: IGFs, IGF receptors and IGF binding proteins (IGFBPs) are widely expressed in the central nervous system. To investigate the physiological significance of IGFBP-6 in the brain we established two transgenic mouse lines overexpressing human (h)-IGFBP-6 under the control of glial fibrillary acidic protein promoter. Increasing evidence suggests that insulin/IGF signalling pathways could be implicated in the neuroendocrine regulation of energy homeostasis. We explored the impact of brain IGFBP-6 overexpression on the regulation of food intake and energy balance. METHODS: Transgenic mice were fed either a control diet or a high-fat diet for up to 3 months. Glucose and insulin tolerance tests were carried out before and after the diet period. Plasma parameters (insulin, leptin, glucose, NEFAs and triglycerides) were measured, and uncoupling protein 1 (UCP-1) expression was quantified in brown adipose tissue. Oxygen consumption was also measured in both groups. RESULTS: The transgenic mice fed a high-fat diet for 3 months developed obesity, showing increases in plasma leptin, glucose and insulin levels and mild insulin resistance. As compared with wild-type mice, no significant differences were found in the quantity of food intake. However, UCP-1 expression was down-regulated in the brown adipose tissue of the transgenic mice. CONCLUSIONS/INTERPRETATION: Our results show that brain IGFBP-6 has an impact on the regulation of energy homeostasis. These transgenic h-IGFBP-6 mice may be considered a new tool for studies of the involvement of the brain IGF system in metabolism control and obesity.

Bone formation in the context of growth retardation induced by hIGFBP-1 overexpression in transgenic mice.

In humans, intrauterine growth retardation (hIUGR) is correlated with an overexpression of insulin-like growth factor binding protein 1 (IGFBP-1). The affected children also present a delay in bone mineralization. In this study, transgenic 12-day-old mutant mice overexpressing human IGFBP-1 hepatospecifically showed a severe growth retardation. Alcian blue and alizarin red S staining of the skeleton revealed mineralization defects at the posterior level of the skull (delayed suture closure) and in appendicular and axial skeleton. Furthermore, microradiographic analysis showed a reduced bone density in the same areas. Thus, overexpressing of hIGFBP-1 demonstrates early postnatal life growth retardation and a delay in mineralization in transgenic mutant mice. These data show the involvement of the IGF/IGFBP system and more particularly IGFBP-1 in the biomineralization process.

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.

Overexpression of human insulin-like growth factor binding protein-1 in the mouse leads to nephron deficit.

IGFs and their binding proteins are important regulators of fetal development. We have previously reported that overexpression of the human IGF binding protein-1 in mice is associated with glomerulosclerosis. The aim of this study was to investigate whether, in that model, decreased bioavailability of IGFs also affected nephrogenesis. When the mothers expressed human IGF binding protein-1, pups were growth retarded and had a reduced number of nephrons. Even nontransgenic pups born to heterozygous mothers had a nephron reduction, indicating that renal hypoplasia was secondary to fetal growth retardation. When the transgene was expressed only in the fetus, pups had a normal birth weight and the kidney was normal at birth, as indicated by histologic studies. However, a significant reduction in the nephron number was observed at 3 mo of age. Because nephrogenesis continues for a few days after birth in the mouse, this indicated that human IGF binding protein-1 overexpression altered postnatal nephrogenesis. In addition, exogenously added IGF-II, but not IGF-I, was effective in stimulating in vitro nephrogenesis. Together these elements suggest that reduced amounts of circulating IGFs, presumably IGF-II, impair kidney development.

Impaired brain development and hydrocephalus in a line of transgenic mice with liver-specific expression of human insulin-like growth factor binding protein-1.

Insulin-like growth factors (IGFs) produced in the brain are known to participate in brain development via activation of the type 1 IGF receptor. IGF binding proteins (IGFBPs) modulate the cellular action of IGFs and some are expressed in the fetal brain. Under normal conditions IGFBP-1 is not one of these, but IGFBP-1 expression obtained via transgenesis using ubiquitous promoters affects brain development. In earlier work, we established a model of transgenic mouse in which liver-specific IGFBP-1 expression begins during fetal life. The repercussions of this IGFBP-1 over-expression include reproductive defects, ante- and perinatal mortality and post-natal growth retardation, the extent of which is related to the degree of transgene expression. Unexpectedly, during the first 2 months of postnatal life, there were some cases of head enlargement revealing hydrocephalus among homozygotes, frequently associated with motor disorders. Brain sections showed dilatation of the lateral ventricles in 10 out of 15 homozygotes examined. Histologically, dilatation was evident in four out of nine heterozygotes. Brain weight in transgenics was relatively less reduced than the weights of other organs. Hence, brain weight/body weight ratios were normal in heterozygotes and on average higher than normal in homozygotes. The width of the cerebral cortex was reduced in homozygotes, with disorganized neuronal layers. The corpus callosum was underdeveloped, particularly in homozygotes. The area of the hippocampus was reduced in homozygotes and one-third of the heterozygotes, with a short and thick dentate gyrus in the former. Similar anomalies have been reported in mice with disruption of the igf-I gene and in a model of transgenic mice over-expressing IGFBP-1 in all tissues, including the brain. Hydrocephalus was not mentioned in these reports, raising the possibility that insertional mutagenesis may have been involved in our mice. Nevertheless, our observations indicate that hepatic over-expression of IGFBP-1 may have endocrine effects on brain development.

Glomerulosclerosis in mice transgenic for human insulin-like growth factor-binding protein-1.

BACKGROUND: The growth hormone (GH)/insulin-like growth factor (IGF) system is thought to participate in the glomerulosclerosis process. Because IGF-binding proteins (IGFBPs) modulate IGF actions and hence GH secretion, this study assessed whether mice transgenic for human IGFBP-1 have altered susceptibility to glomerulosclerosis. METHODS: A line of transgenic mice that express human IGFBP-1 mRNA in the liver under the control of the alpha1-antitrypsin promoter has been obtained, and morphological changes in the kidney tissue were assessed. Glomerulosclerosis was identified using light microscopy, light microscopic morphometry, and electron microscopy. Extracellular matrix components were analyzed by immunohistochemistry. RESULTS: There was a marked increase in mesangial extracellular matrix area in homozygous transgenic mice at three months of age as compared with heterozygous transgenic mice and nontransgenic littermates. These changes were not associated with alterations in glomerular volume or cellularity. The expansion of extracellular matrix area was related to a marked increase in laminin and type IV collagen and to the appearance of type I collagen. CONCLUSIONS: These observations indicate that the enhanced expression of IGFBP-1 may result in the development of glomerulosclerosis without glomerular hypertrophy. The changes are potentially related to a decrease in IGF-I availability and/or to an IGF-I-independent role of IGFBP-1.

Liver-specific expression of human insulin-like growth factor binding protein-1 in transgenic mice: repercussions on reproduction, ante- and perinatal mortality and postnatal growth.

Study of the in vivo functions of the insulin-like growth factor binding proteins (IGFBPs) is complicated by their variety (six molecular species) and the differences in their expression related to tissue of origin and stage of development. To investigate the physiological role of IGFBP-1 in the bloodstream, we induced hepatic overexpression of IGFBP-1 in transgenic mice, placing human IGFBP-1 (hIGFBP-1) cDNA under the control of the alpha1-antitrypsin promoter so as to obtain liver-specific expression. Five transgenic founder mice were raised, only two of which (lines 124 and 149) produced transgenic offspring. Northern blotting revealed transgene expression exclusively in the liver during fetal life and unchanged through to adulthood, whereas expression of the endogenous gene was undetectable beyond 10-15 days postnatally. hIGFBP-1 was detected by western immunoblotting in the plasma of transgenic mice and IRMAs yielded mean concentrations of 2.41 +/- 0.33 ng/ml and 13.69 +/- 1.42 ng/ml in homozygous animals of lines 124 and 149, respectively. In the latter, IGFBP-1 levels were distinctly higher than in heterozygotes (2.99 +/- 0.39 ng/ml), P < 0.0001. These levels remained stable in each given animal and did not change with age. Plasma concentrations of IGF-I measured in line 149 exhibited the well-known profile of an increase from birth up to puberty. Values for heterozygotes were similar to those for wild-type mice, with adult levels (544 +/- 98 ng/ml) slightly below those of controls (630 +/- 56 ng/ml), P < 0.05. In homozygotes they were distinctly lower, with adult levels of 370 +/- 75 ng/ml, P = 0.001. In heterozygous and homozygous adults, there was a negative correlation between IGF-I and IGFBP-1 concentrations (r = 0.8, P < 0.0001), suggesting a link between transgene expression and IGF-I levels. Study of body weight gain in line 149 revealed growth retardation within the first weeks after birth, which was marked in homozygous males and females (P < 0.001) but also present in heterozygous males (P = 0.002), indicating some relationship with transgene expression. In addition, body weight in adult mice was negatively correlated to plasma concentrations of IGFBP-1 (r = 0.7, P < 0.0001). Reproductive function also appeared to be severely affected, especially in homozygous females: mating that failed to result in pregnancy in half of the homozygous females crossed with nontransgenic males, suggestive of impaired fertilization or implantation; interrupted or prolonged pregnancies with fetal and neonatal death. Litter size was reduced in transgenic females (by about half in homozygotes) and in nontransgenic females mated with homozygous males, resulting from pre- or neonatal mortality. Moreover, deaths occurred within the first 5 days of life, with an incidence of approximately 50% in the litters of homozygous females, 12-18% among heterozygotes mated with nontransgenic or heterozygous males, respectively, and 30% among those mated with homozygous males. These results, suggesting that fetal transgene expression largely accounted for ante- and perinatal mortality, were confirmed by the predominance of homozygotes among those that could be analyzed genetically. Similarly impaired reproductive function was seen in line 124, but to a lesser degree. Although the mechanisms responsible for these disorders remain to be determined, our results indicate that permanent and uncontrolled hepatic expression of IGFBP-1, even at low levels, affects fertility in females and both ante- and postnatal development.

Parental allele specific methylation of the human insulin-like growth factor II gene and Beckwith-Wiedemann syndrome.

In an attempt to elucidate the role of methylation in parental imprinting at the IGF-II gene locus, for which imprinting has already been described in the mouse, we undertook an allele specific methylation study of the human IGF-II gene (mapped to 11p15.5) in a control population and in patients with Beckwith-Wiedemann syndrome. In control leucocyte DNA (16 unrelated adults and eight families), the maternal allele of the IGF-II gene was specifically hypomethylated, whereas no such allele specific methylation was found for either the insulin or the calcitonin genes which are located in 11p15.5 and 11p15.1, respectively. Furthermore, the IGF-II gene specific hypomethylation was localised on the 5' portion of exon 9. In the patients with Beckwith-Wiedemann syndrome in which the IGF-II gene is thought to be involved and where paternal isodisomy has been described, hypomethylation of the maternal allele was conserved in leucocyte DNA, but abnormal methylation was detected in malformed tissues where the paternal allele was also demethylated. Some specific mechanism linked to methylation therefore seems to be involved in the pathogenesis of Beckwith-Wiedemann syndrome.

Abnormalities of insulin-like growth factor (IGF-I and IGF-II) genes in human tumor tissue.

Recent findings have indicated that Insulin-like growth factors (IGF-I and IGF-II) may play a role in neoplasia. Expression of their genes, which are highly complex structures, is tissue-specific and developmentally regulated. The purpose of the present study was to determine whether a relationship exists between tumorigenesis and the structure and expression of IGF genes. The structures of the IGF-I and IGF-II genes were investigated in 40 tumors by Southern blot analysis but no obvious re-arrangements (such as amplification or deletion) were observed in any of the tissues investigated. DNA methylation was also studied, using the enzyme Avall. The extent of DNA methylation of the IGF genes was highly variable in most of the tumors, as was the level of mRNA expression. A relationship could be detected between IGF over-expression and gene demethylation in tumors associated with hypoglycemia and in certain hepatocarcinomas. Loss of heterozygosity has been reported in the 11p15 region of some childhood tumors. The present findings provide further evidence of this loss of heterozygosity for the IGF-II gene and show an imbalance in the leukocyte alleles in several childhood tumors. Likewise, an imbalance in the alleles was noted in several adult tumors, including hepatocarcinomas and breast cancers. This suggests that in certain adult tumors alterations of the IGF-II gene may be associated with tumorigenesis, but in other tumors another mechanism may be involved.

Loss of heterozygosity in non-tumoral tissue in two children with Beckwith-Wiedemann syndrome.

The Insulin-like growth factor II (IGF-II) gene has been analysed by Southern blotting in healthy and tumor tissue in two children with Beckwith-Wiedemann syndrome, using the enzymes, AvaII and SacI. Loss of heterozygosity was determined not only in the DNA of a nephroblastoma, but also in the healthy kidney, leukocytes and fragments of tongue removed for obstructive macroglossia. Allele loss therefore seems to be sufficient to provoke tumorigenesis in some tissues, but not in others.

Expression of insulin-like growth factor II and receptors for insulin-like growth factor II, insulin-like growth factor I and insulin in isolated and cultured rat hepatocytes.

Insulin-like growth factor II is believed to play an important role in fetal growth and development. The insulin-like growth factor II gene expression is tissue specific and developmentally regulated. We have previously shown an enhanced level of insulin-like growth factor II messenger RNA and protein in human hepatocellular carcinomas. This led to the suggestion that hepatocytes might be involved in insulin-like growth factor II expression. However, previous studies based on in situ hybridization only showed insulin-like growth factor II messenger RNA in liver sinusoidal cells. This paper reports on the analysis of the expression of insulin-like growth factor II and insulin-like growth factor II, insulin-like growth factor I and insulin receptor messenger RNAs in vivo in isolated rat hepatocytes at various stages of development and in vitro in adult rat hepatocytes primary culture. Our study indicates that isolated rat hepatocytes synthesize insulin-like growth factor II messenger RNA with a switch between fetal and adult messenger RNA profiles occurring 21 days after birth. In addition, adult rat hepatocytes in culture expressed insulin-like growth factor II messenger RNA and protein. Insulin-like growth factor II, insulin-like growth factor I and insulin receptor messenger RNAs were also detected. Therefore these results are consistent with the hypothesis that insulin-like growth factor II acts as an autocrine growth factor for hepatocytes in addition to having a paracrine effect. They also indicate that primary culture of hepatocytes is a good model for further studies on insulin-like growth factor II gene regulation.

[Pathology of the growth and abnormalities of IGF genes]. / Pathologie de la croissance et anomalies des gènes des IGF.

Insulin like growth factor I (IGF I) and IGF II have both metabolic and growth promoting properties, both display endocrine and autocrine-paracrine effects. In growth stature disorders without GH deficiency (constitutionally short stature = CSS, pygmy) no IGF I gene deletion was detectable by Southern blotting. In CSS the incidence of RFLP for Hind III located near exon 5 was similar to that in the control population, the incidence of the polymorphic (13 kb) EcoRV (located near exon 1) was significantly lower. Cosegregation analysis of these RFLP in families of CSS were uniformative for IGF I gene, but family linkage studies by others demonstrated co-segragation of the IGF I gene region and hypochondroplasia. In pygmy, DNA amplification around exon 1 and direct sequencing revealed polymorphic sites. One of them is located in the consensus sequence upstream from the translational start site in Eucaryotic mRNA. IGF II RNA messenger hyperexpression was noted in many human tumours with genomic abnormalities: IGF II gene hypomethylation, and loss of allele.

Expression of insulin-like growth factor II (IGF-II) in human primary liver cancer: mRNA and protein analysis.

Insulin-like growth factor II (IGF-II) is a polypeptide growth factor thought to be involved in fetal tissue development. We previously showed an increased expression of IGF-II mRNA in human primary liver cancer. The present investigation was undertaken to characterize the overexpressed IGF-II transcripts and to determine whether they are translated into protein. Two cDNAs with distinct 5' untranslated regions, corresponding to IGF-II transcripts expressed in fetal liver, were isolated from a primary liver cancer. Complete nucleotide sequence analysis showed an identical open reading frame of 540 bp, encoding a predicted polypeptide identical to the IGF-II isolated from serum. An increased synthesis of IGF-II protein was demonstrated by a protein-binding assay in tumorous liver samples, the highest levels being found in primary liver cancers with the highest IGF-II steady state level. By contrast, serum IGF-II content was low in most of primary liver cancer cases analyzed. Altogether, the results indicate reexpression of IGF-II both at the mRNA and protein levels in primary liver cancer. This finding is consistent with IGF-II being a marker of liver cell differentiation. In addition, this growth factor might be involved in liver cancer progression by an autocrine and/or paracrine mechanism.

Insulin-like growth factor-I gene analysis in subjects with constitutionally variant stature.

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.

Molecular forms of serum insulin-like growth factor (IGF)-binding proteins in man: relationships with growth hormone and IGFs and physiological significance.

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)

Comparative assessment of ACTH and lipotropin plasma levels in the diagnosis and follow-up of patients with Cushing's syndrome: a study of 210 cases.

PURPOSE: A series of 210 patients with Cushing's syndrome was evaluated at a single center to assess the relative values of adrenocorticotropic hormone (ACTH) and lipotropin (LPH) plasma levels in the etiologic diagnosis of Cushing's syndrome and in the follow-up of treated Cushing's diseases. PATIENTS AND METHODS: These patients included 149 patients with Cushing's diseases, 20 with adrenal tumors, and 41 with ectopic ACTH/LPH syndromes. Hormone levels were measured before therapy and during the follow-up of treated Cushing's diseases. RESULTS: ACTH and LPH plasma levels were moderately elevated in Cushing's diseases, low or undetectable in adrenal tumors, and highly elevated in ectopic ACTH/LPH syndromes, but the overlap between the three etiologic groups was less for LPH than for ACTH. LPH appeared to be as sensitive as ACTH in evaluating the outcome of trans-sphenoidal surgery and in detecting the occurrence of Nelson's syndrome after bilateral adrenalectomy. CONCLUSION: Therefore, plasma LPH determinations provide a better index than ACTH measurements, probably for technical reasons as well as because of the greater stability of LPH in blood.

Differential expression of insulin-like growth factor II mRNA in human primary liver cancers, benign liver tumors, and liver cirrhosis.

We investigated insulin-like growth factor II (IGF-II) mRNA in three groups of human liver samples including primary liver cancers, benign liver tumors and cirrhosis; indeed these pathological conditions would allow us to distinguish between different steps in liver carcinogenesis. A 40- to 100-fold increase in IGF-II mRNA was shown in 9/40 of the liver cancer samples as compared to normal adult liver. RNA blot analysis using both IGF-II cDNA and oligonucleotide probes showed the reexpression of two fetal (6 and 5 kilobases) IGF-II transcripts in primary liver cancers and in some cirrhotic adjacent tissues; these included all the samples with enhanced IGF-II expression. By contrast the adult (5.3 kilobases) IGF-II transcript was identified in most of the benign liver tumors and liver cirrhosis; in addition, in some of these samples, the 5-kilobase fetal transcript was also detected. The increase of IGF-II mRNA in some liver cancers is consistent with an autocrine mechanism conferring a selective growth advantage to tumorous liver cells. Furthermore, these results indicate a differential expression of IGF-II transcripts in nonmalignant hepatocyte proliferation (benign liver tumors and cirrhosis) as compared to liver cancer. Finally this study suggests that, in liver cirrhosis and in some benign liver tumors, premalignant proliferative states might be identified by the presence of IGF-II fetal transcripts.

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)

Heterogeneity of insulin-like growth factor binding proteins between structure and affinity. 2. Forms released by human and rat liver in culture.

Since the liver is considered to be the major source both of circulating insulin-like growth factors (IGFs) and of their specific binding proteins (BPs), human and rat liver explants were cultured in serum-free medium with a view to characterizing the binding proteins released into the medium and to comparing them with serum binding proteins. In the culture media, as in the serum, IGFs are associated with their binding proteins in the form of complexes. In gel filtration experiments the liver IGF-BP complexes eluted as a single, homogeneous peak with a relative molecular mass of about 40,000, which is similar to that of the 'small' complex of serum. Their sedimentation coefficient, estimated from sucrose gradient centrifugation, was 2.9 S. Polyacrylamide gel electrophoresis (SDS-PAGE) of human liver culture media, in which the binding proteins were cross-linked to 125I-labelled IGF I revealed molecular heterogeneity. Three specific bands corresponding to Mr 46,000, 40,000 and 37,000 were observed, which resemble those of the serum small complex, but none of the higher-Mr bands seen in serum. SDS-PAGE followed by transfer onto nitrocellulose and incubation with 125I-labelled IGF I (western blot) led to the identification in human liver culture media of five molecular forms of binding protein with Mr of 41,500, 38,500, 34,000, 30,000 and 24,000, identical to those seen in serum. The relative concentrations of the 41,500 and 38,500-Mr forms varied from one medium to another, but the 34,000 and 30,000-Mr forms were regularly more abundant in the liver culture media than in normal serum. The binding proteins produced by the liver therefore represent the native forms in the circulation (although this does not exclude other sources). The absence of high-Mr IGF-BP complexes in the liver culture media, and yet the presence of the 41,500 and 38,500-Mr forms, which are the only binding units of the serum 'large' complex (150,000 Mr), indicates that these two binding proteins are capable of binding IGFs to form 'monomeric' IGF-BP complexes. Western-blot analysis of rat binding proteins revealed a certain analogy with the human proteins, three forms having their Mr between 43,000 and 39,000 and three between 32,000 and 24,000. Liver binding proteins in human adults and foetuses were found to be identical, whereas in the case of serum the 41,500 and 38,500-Mr forms were more abundant in the adult and the 34,000 and 30,000-Mr forms more abundant in the foetus.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of an insulin-like growth factor-binding protein in human cerebrospinal fluid with a selective affinity for IGF-II.

Human cerebrospinal fluid (CSF) has been found to contain several different molecular forms of IGF-specific binding proteins (BPs). Qualitatively, they are similar to those present in serum, although their relative proportions are very different, as well as to those present in the culture media of brain tissue from which these BPs presumably arise. One particular form of BP is predominant in CSF. It has an Mr of 34,000, as estimated by SDS-polyacrylamide gel electrophoresis followed by transfer onto nitrocellulose, and an isoelectric point around 5.0 based on chromatofocusing. It has a selective affinity for IGF-II (approximately 4 X 10(10) M-1) as shown by competitive binding experiments in which biosynthetic IGF-I was about 40-times less potent than native IGF-II in displacing 125I-labelled IGF-II. These findings are in agreement with the preponderance of IGF-II in nervous tissue and in CSF and suggest that this BP plays an important role in the interaction of IGF-II with its target cells.