The expression pattern of an insulin-like growth factor (IGF)-binding protein gene is distinct from IGF-II in the midgestational rat embryo.

Insulin-like growth factor-II (IGF-II), the predominant form of IGF in fetal and neonatal serum and tissues, is found in vivo complexed with IGF-binding proteins. One of these binding proteins, IGFBP-2, is present at high levels in fetal rat plasma and binds both IGF-I and IGF-II with high affinity. We here have used in situ hybridization to compare the distribution of IGFBP-2 mRNA with that of IGF-II mRNA in embryonic day 13.5-15 rat embryos. The spatial patterns of IGF-II and IGFBP-2 expression in the fetal trunk were distinct and, in general, nonoverlapping. Most mesoderm derivatives that express IGF-II at high levels contained little, if any, IGFBP-2 mRNA. Instead, IGFBP-2 mRNA was expressed at high levels in many cell types derived from ectoderm and endoderm. The expression of IGFBP-2 mRNA in the central nervous system (CNS) during this developmental period was examined in particular detail. The three most prominent sites of IGFBP-2 expression in the CNS were comprised of cells with nonneuronal phenotypes: 1) the epithelium of the choroid plexus, a tissue that produces cerebrospinal fluid; 2) the floor plate, an area that can guide axonal outgrowth from commissural neurons of the spinal cord in vitro; and 3) the infundibulum, the progenitor of the posterior pituitary that is believed to influence differentiation of the adjacent intermediate pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of the IGFBP-2 gene in post-implantation rat embryos.

The insulin-like growth factors (IGFs) stimulate mitogenesis in a variety of cell types both in vitro and in vivo. These effects are mediated by both IGF receptors and a family of IGF binding proteins (IGFBPs), which are found complexed with the IGFs in serum and tissue fluids. Here we compare the sites of expression during early rat embryogenesis of the genes encoding the RGD-containing IGF binding protein IGFBP-2 and IGF-II. At all ages from early post-implantation through mid-gestation, the expression of IGFBP-2 was highly complementary to IGF-II. IGFBP-2 mRNA was detected throughout the epiblast of the egg cylinder as early as e7, when IGF-II expression was restricted to trophectoderm and other extraembryonic cells. As gastrulation proceeded, IGFBP-2 expression ceased as IGF-II expression began in the newly formed embryonic and extra-embryonic mesoderm, but was retained in other epiblast derivatives including the surface ectoderm and neuroectoderm, throughout its rostral-caudal extent. By e10-e11, IGFBP-2 expression in neuroectoderm was restricted to the rostral brain of the primary neural tube and was found in the new population of neuroepithelium formed in the tail bud during secondary neurulation. IGFBP-2 expression remained high in the ventricular layer of the rostral brain into mid-gestation ages but decreased or disappeared as cells entered the mantle layer and began to express the neurofilament-related gene alpha-internexin. IGFBP-2 mRNA was abundant in surface ectoderm, particularly that of the branchial arches, and all ectodermal placodes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-like growth factor family and combined antisense approach in therapy of lung carcinoma

BACKGROUND: Perturbation in a level of any peptide from insulin-like growth factor (IGF) family (ligands, receptors, and binding proteins) seems to be implicated in lung cancer formation; IGF ligands and IGF-I receptor through their mitogenic and anti-apoptotic action, and the mannose 6-phosphate/insulin-like growth factor II receptor (M6-P/IGF-IIR) possibly as a tumor suppressor. MATERIALS AND METHODS: To determine the identity, role, and mutual relationship of IGFs in lung cancer growth and maintenance, we examined IGF's gene (by RT-PCR) and protein (by immunohistochemistry) expression in 69 human lung carcinoma tissues. We also examined IGF-I receptor numbers (Scatchard analysis) and IGF-II production and release (by Western blot) in IGF-II/IGF-IR mRNA positive and negative lung carcinomas. Finally, the potential role of IGF-IR and IGF-II as growth promoting factors in lung cancer was studied using antisense oligodeoxynucleotides that specifically inhibit IGF-IR and IGF-II mRNA. RESULTS: Thirty-two tumors were positive for IGF-I, 39 for IGF-II, 48 for IGF-IR, and 35 for IGFBP-4 mRNA. Seventeen tumors were concomitantly positive for all four IGFs, whereas 34 were positive for IGF-II, IGF-IR, and IGFBP-4 mRNA. An elevated amount of IGF-II peptide was secreted into the growth medium of cell cultures established from five different IGF-II/IGF-IR mRNA positive lung cancer tissues. The cells also expressed elevated numbers of IGF-IR. Nine IGF-II-negative and 19 IGF-II-positive lung cancers of different stages were selected, and M6-P/ IGF-II receptor was determined immunohistochemically. Most of the IGF-II-negative tumors were strongly positive for M6-P/IGF-IIR. IGF-II-positive tumors were mostly negative for M6-P/IGF-II receptors. Antisense oligodeoxynucleotides to IGF-II significantly inhibited, by 25-60%, the in vitro growth of all six lung cancer cell lines. However, the best results (growth inhibition of up to 80%) were achieved with concomitant antisense treatment (to IGF-IR and IGF-II). CONCLUSION: Our data suggest that lung cancer cells produce IGF-IR and IGF-II, which in turn stimulates their proliferation by autocrine mechanism. Cancer cell proliferation can be abrogated or alleviated by blocking the mRNA activity of these genes indicating that an antisense approach may represent an effective and practical cancer gene therapy strategy.     

Expression of insulin-like growth factor-II and insulin-like growth factor binding proteins during Caco-2 cell proliferation and differentiation

The components of the insulin-like growth factor (IGF) axis and their roles in regulating proliferation and differentiation of the human colon adenocarcinoma cell line, Caco-2, have been investigated. Caco-2 cells proliferated in serum-free medium at 75% the rate observed in medium containing 10% fetal bovine serum. IGF-I (10 nM) increased Caco-2 cell growth in serum-free medium, but not to the rate seen with serum. Multiple IGF-II mRNA species were produced by Caco-2 cells, but IGF-I mRNA was undetectable. Secretion of radioimmunoassayable IGF-II corresponded with steady-state levels of IGF-II mRNA, neither of which was observed to change markedly over the course of 16 days of Caco-2 cell differentiation. Levels of sucrase-isomaltase mRNA, a marker for enterocytic differentiation, increased 12-fold between days 5 and 16 of culture. Northern blotting of total RNA and ligand blot and immunoblot analyses of serum-free conditioned medium revealed that Caco-2 cells produce several IGF binding proteins (IGFBPs), including IGFBP-2, -3, and -4, as well as a 31,000 M, species that was not identified. The pattern of IGFBP secretion changed dramatically during Caco-2 cell differentiation: IGFBP-3 and IGFBP-2 increased 8.5-fold and 5-fold, respectively, whereas IGFBP-4 and the 31,000 M, species decreased 43% and 90%. Caco-2 cell clones stably transfected with a human IGFBP-4 cDNA construct exhibited a 60% increase in steady-state level of IGFBP-4 mRNA, and secreted twice as much IGFBP-4 protein as controls. Moreover, IGFBP-4-overexpressing cells proliferated at only 25% the rate of control cells in serum-free medium, in conjunction with a 70% increase in expression of sucrase-isomaltase. In summary, these studies indicate that a complex IGF axis is involved in autocrine regulation of Caco-2 cell proliferation and differentiation. © 1996 Wiley-Liss, Inc.     

Expression of mRNAs encoding insulin-like growth factor (IGF) ligands, IGF receptors and IGF binding proteins during follicular growth and atresia in the ovine ovary throughout the oestrous cycle

The components of the insulin-like growth factor (IGF) system appear to be involved in the regulation of ovarian follicular growth and atresia in sheep. However, previous studies have only investigated a select few components of the system. The aim of the present study was to investigate the expression of mRNA encoding all of the components of the sheep IGF system among follicles of varying size and health status throughout the oestrous cycle using sheep-specific ribonucleotide probes and in situ hybridisation. For all IGF components, gene expression was unaffected by stage of oestrous cycle. IGF-I mRNA expression in all classes of follicle was generally low throughout the oestrous cycle, while IGFBP-1 mRNA expression could not be demonstrated at all. In contrast, there was relatively intense follicular expression of mRNAs encoding all remaining IGF system components. For IGF-II, both IGF receptors and IGFBP-2, -3, -4, -5, and -6, gene expression decreased as follicles increased in diameter (P < 0.01). IGF-II, type I IGF-R and IGFBP-2, -3, -4, and -6 mRNA expression significantly decreased as follicles progressed from healthy to atretic status (P < 0.01), whereas gene expression for type II IGF-R and IGFBP-5 was greater in atretic follicles (P < 0.01). This study demonstrates the spatial patterns of follicular gene expression for all of the IGF system components in cycling sheep for the first time. These results further highlight the potential functional role of IGF-II, in contrast to IGF-I, in the autocrine and/or paracrine regulation of follicle growth in sheep.     

Insulin-like growth factor II binding and action in human fetal fibroblasts

To investigate the role of insulin-like growth factor II (IGF-II) in human prenatal growth, IGF-II binding and biological action were studied in four lines of fetal and three lines of postnatal human fibroblasts. Specific binding of IGF-II was similar in both groups: 15.7% and 14.9% for fetal and postnatal fibroblasts, respectively. This was 5-10 times the amount of IGF-I binding found in these cells. IGF-I and IGF-II caused dose-dependent increases in [14C]aminoisobutyric acid (AIB) uptake. IGF-II was sevenfold less potent than IGF-I in stimulating this metabolic response in both fetal and postnatal fibroblasts. The maximal effect of IGF-II in stimulating [14C]AIB uptake approach that of IGF-I. Similar results were obtained when IGF-I and IGF-II stimulation of [3H]thymidine incorporation was compared in fetal and postnatal fibroblasts. Incubation in the presence of alpha IR-3, a monoclonal antibody to the type I IGF receptor, inhibited the ability of both IGF-I and IGF-II to stimulate [14C]AIB uptake and [3H]thymidine incorporation in fetal and postnatal cells. A monoclonal antibody to the insulin receptor did not affect IGF action. These data indicate that IGF-II is a potent metabolic and mitogenic stimulus for human fetal fibroblasts. However, despite the presence of abundant type II IGF receptors on both fetal and postnatal human fibroblasts, IGF-II stimulation of amino acid transport and DNA synthesis appears to be mediated through the type I rather than through its own type II IGF receptor.     

An in situ hybridization study of the insulin-like growth factor system in developing condylar cartilage of the fetal mouse mandible

The objective of this study was to investigate the involvement of the insulin-like growth factor (IGF) system in the developing mandibular condylar cartilage and temporomandibular joint (TMJ). Fetal mice at embryonic day (E) 13.0-18.5 were used for in situ hybridization studies using [35S]-labeled RNA probes for IGF-I, IGF-II, IGF-I receptor (-IR), and IGF binding proteins (-BPs). At E13.0, IGF-I and IGF-II mRNA were expressed in the mesenchyme around the mandibular bone, but IGF-IR mRNA was not expressed within the bone. At E14.0, IGF-I and IGF-II mRNA were expressed in the outer layer of the condylar anlage, and IGF-IR mRNA was first detected within the condylar anlage, suggesting that the presence of IGF-IR mRNA in an IGF-rich environment triggers the initial formation of the condylar cartilage. IGFBP-4 mRNA was expressed in the anlagen of the articular disc and lower joint cavity from E15.0 to 18.5. When the upper joint cavity was formed at E18.5, IGFBP-4 mRNA expression was reduced in the fibrous mesenchymal tissue facing the upper joint cavity. Enhanced IGFBP-2 mRNA expression was first recognized in the anlagen of both the articular disc and lower joint cavity at E16.0 and continued expression in these tissues as well as in the fibrous mesenchymal tissue facing the upper joint cavity was observed at E18.5. IGFBP-5 mRNA was continuously expressed in the outer layer of the perichondrium/fibrous cell layer in the developing mandibular condyle. These findings suggest that the IGF system is involved in the formation of the condylar cartilage as well as in the TMJ.     

Alteration of the insulin-like growth factor axis during in vitro differentiation of the human osteosarcoma cell line HOS 58

The insulin-like growth factors I and II (IGF-I, IGF-II), their receptors, and high affinity binding proteins (IGFBPs) represent a family of cellular modulators that play essential roles in the development and differentiation of cells and tissues including the skeleton. Recently, the human osteosarcoma cell line HOS 58 cells were used as an in vitro model of osteoblast differentiation characterized by (i) a rapid proliferation rate in low-density cells that decreased continuously with time of culture and (ii) an increasing secretion of matrix proteins during their in vitro differentiation. In the present paper, HOS 58 cells with low cell density at early time points of the in vitro differentiation (i) displayed a low expression of IGF-I and -II; (ii) synthesized low levels of IGFBP-2, -3, -4, and -5, but (iii) showed high expression levels of both the type I and II IGF receptors. During the in vitro differentiation of HOS 58 cells, IGF-I and -II expressions increased continuously in parallel with an upregulation of IGFBP-2, -3, -4, and -5 whereas the IGF-I receptor and IGF-II/M6P receptor mRNA were downregulated. In conclusion, the high proliferative activity in low cell density HOS 58 cells was associated with high mRNA levels of the IGF-IR, but low concentrations of IGFBP-2. The rate of proliferation of HOS 58 cells continuously decreased during cultivation in parallel with a decline in IGF-IR expression, but increase of mitoinhibitory IGFBP-2. These data are indicative for a role of the IGF axis during the in vitro differentiation of HOS 58 cells.     

Distribution and possible function of an adrenomedullin-like peptide in the developing chick limb bud

Adrenomedullin (AM) is a multifunctional peptide that exhibits discrete domains of expression during mouse embryogenesis consistent with a role in regulating growth and differentiation during morphogenesis. Here we report that AM immunoreactivity is present at high levels throughout the apical ectodermal ridge (AER) of the chick limb bud as the AER is directing the outgrowth and patterning of underlying limb mesoderm. Immunostaining is particularly strong along the surfaces of the contiguous cells of the AER. AM immunoreactivity attenuates as the AER regresses and is absent from the distal apical ectoderm of stage 20 limbless mutant limb buds which fail to develop an AER. To explore the possible role of AM in AER activity, we examined the effect of exogenous AM and an AM inhibitor on the in vitro morphogenesis of limb mesoderm, cultured in the presence and absence of the AER. Although exogenous AM cannot substitute for the AER in promoting outgrowth of limb mesoderm in vitro, a specific AM antagonist, AM(22-52), impairs the outgrowth and proliferation of limb mesoderm cultured in the presence of the AER. This is consistent with the possibility that inhibition of endogenous AM activity in the AER impairs the ability of the AER to promote limb morphogenesis. Taken together, these studies suggest that an AM-like molecule may function in an autocrine fashion to regulate some aspect of AER activity.     

A role for LH in the regulation of expression of mRNAs encoding components of the insulin-like growth factor (IGF) system in the ovine corpus luteum

Evidence suggests the insulin-like growth factor (IGF) system may be involved in luteal maintenance and regression. However, previous studies have only investigated a few components of the system, primarily in bovine and non-ruminant species. The present study investigated gene expression for the components of the IGF system in ovine corpora lutea (CL) at various key stages of the oestrous cycle (Experiment 1), and the possible regulatory effects of LH on IGF gene expression in ovine CL using a GnRH antagonist model system (Experiment 2). Experiment 1 revealed that IGF-I (P<0.001), type I (P=0.008) and II (P=0.005) IGF-Rs and IGFBP-5 (P<0.05) mRNA levels were significantly elevated in early regressing CL. In contrast, IGF-II levels were high in CL but did not vary throughout the oestrous cycle, while IGFBP-2, -3, -4 and -6 mRNA levels were highest throughout the luteal phase but lower in regressing CL (P<0.05). IGFBP-1 mRNA could not be detected in any CL. Abrogation of LH action following GnRH antagonist administration (Experiment 2) resulted in a significant increase in expression for IGF-I (P<0.001), type II IGF-R (P=0.004) and IGFBP-5 (P<0.05) after only 12h, but these increases were transient. IGF-II, type I IGF-R and IGFBP-2, -3, -4 and -6 mRNA levels remained unaffected by GnRH antagonist treatment. These data highlight the role that LH plays in regulating IGF-I gene expression and lends further support that IGF-I may be a key luteotrophic factor in sheep.     

Tissue-specific expression of messenger ribonucleic acids for insulin-like growth factors and insulin-like growth factor-binding proteins during perinatal development of the rat uterus

Insulin-like growth factor (IGF)-I and IGF-II play a number of important roles in growth and differentiation, and IGF-binding proteins (IGFBPs) modulate IGF biological activity. IGF-I has been shown previously to be essential for normal uterine development. Therefore, we used in situ hybridization assays to characterize the unique tissue- and developmental stage-specific pattern of expression for each IGF and IGFBP gene in the rat uterus during perinatal development (gestational day [GD]-20 to postnatal day [PND]-24). IGF-I and IGFBP-1 mRNAs were expressed in all uterine tissues throughout this period. IGFBP-3 mRNA was not detectable at GD-20 but became detectable beginning at PND-5, and the signal intensity appeared to increase during stromal and muscle development. IGFBP-4 mRNA was abundant throughout perinatal development in the myometrium and in the stroma, particularly near the luminal epithelium. IGFBP-5 mRNA was abundantly expressed in myometrium throughout perinatal development. IGFBP-6 mRNA was detected throughout perinatal development in both the stroma and myometrium in a diffuse expression pattern. IGF-II and IGFBP-2 mRNAs were not detected in perinatal uteri. Our results suggest that coordinated temporal and spatial expression of IGF-I and its binding proteins (IGFBP-1,-3,-4,-5, and -6) could play important roles in perinatal rodent uterine development.     

Inhibitory insulin-like growth factor-binding protein: cloning, complete sequence, and physiological regulation.

In this study we report the preparation of a human osteosarcoma cell cDNA library and describe the isolation and sequence determination of a clone encoding the complete sequence of a novel human insulin-like growth factor (IGF)-binding protein (hIGFBP-4). Previous work indicated that hIGFBP-4 is the predominant IGFBP expressed by human osteoblast-like cells, and that IGFBP-4 binds and inhibits the mitogenic activities of IGF-I and IGF-II. Sequence determination revealed that hIGFBP-4 is a unique gene product with significant amino- and carboxy-terminal sequence similarity to three other known IGFBPs. Identical alignment of 18 cysteines in IGFBP-4 and the three other IGFBPs is a key structural feature of this protein family. In vitro studies of human osteoblast-like cells suggest that PTH regulates the expression of hIGFBP-4 and that the PTH effect is mediated through a cAMP mechanism. hIGFBP-4 mRNA was also expressed in skin fibroblasts, and thus, this inhibitory IGFBP could be an important physiological regulator of IGF actions in bone cells and other cell types as well.     

Effect of a high maternal dietary intake during mid-gestation on components of the utero-placental insulin-like growth factor (IGF) system in adolescent sheep with retarded placental development

The aim of the present study was to investigate the effects of administering a high plane diet during early to mid-gestation on the uterine and placental insulin-like growth factor (IGF) system and on systemic IGF-I concentrations in pregnant adolescent ewes with restricted placental growth. Embryos recovered from superovulated ewes inseminated by a single sire were transferred in singleton to the uterus of adolescent recipients. After transfer ewes were offered a high (H) or moderate (M) amount of a complete diet calculated to promote rapid or normal maternal growth rates, respectively. Five ewes from each group were switched from either M to H or H to M diets at day 52 of gestation. Maternal and fetal blood samples and placental tissues were collected from all animals at day 104. Ewes on the high plane diet from mid-gestation (HH, MH groups) had restricted placental mass (P < 0.01) and tended to have smaller fetuses. This was associated with increased maternal plasma IGF-I concentrations (P < 0.001). The pattern of expression of components of the IGF system in the uterus and placenta was studied by in situ hybridization. IGF-I mRNA concentrations were below the limit of detection. IGF-II mRNA expression was high in the fetal mesoderm and present in maternal stroma, but was not influenced by nutritional treatment. In contrast, IGF binding protein 1 (IGFBP-1) mRNA expression was higher (P < 0.05) and IGFBP-3 mRNA expression was lower (P < 0.05) in the endometrial glands of ewes in HH and MH groups. In the fetal trophoblast, IGFBP-3 mRNA expression was higher in the MH group. Type 1 IGF receptor expression was increased (P < 0. 01) in the luminal epithelium of the HM group and IGFBP-2 mRNA expression was highest in the placentome capsule of ewes in the HH group. Together, these results indicate that reprogramming of the uterine and placental IGF axis by maternal nutrition could contribute to placental growth retardation in growing adolescent sheep.     

Expression profile of IGF system during lung injury and recovery in rats exposed to hyperoxia: a possible role of IGF-1 in alveolar epithelial cell proliferation and differentiation

Although several studies have shown that an induction of insulin-like growth factor (IGF) components occurs during hyperoxia-mediated lung injury, the role of these components in tissue repair is not well known. The present study aimed to elucidate the role of IGF system components in normal tissue remodeling. We used a rat model of lung injury and remodeling by exposing rats to > 95% oxygen for 48 h and allowing them to recover in room air for up to 7 days. The mRNA expression of IGF-I, IGF-II, and IGF-1 receptor (IGF-1R) increased during injury. However, the protein levels of these components remained elevated until day 3 of the recovery and were highly abundant in alveolar type II cells. Among IGF binding proteins (IGFBPs), IGFBP-5 mRNA expression increased during injury and at all the recovery time points. IGFBP-2 and -3 mRNA were also elevated during injury phase. In an in vitro model of cell differentiation, the expression of IGF-I and IGF-II increased during trans-differentiation of alveolar epithelial type II cells into type-I like cells. The addition of anti-IGF-1R and anti-IGF-I antibodies inhibited the cell proliferation and trans-differentiation to some extent, as evident by cell morphology and the expression of type I and type II cell markers. These findings demonstrate that the IGF signaling pathway plays a critical role in proliferation and differentiation of alveolar epithelium during tissue remodeling.     

Role of insulin-like growth factors and the type I insulin-like growth factor receptor in the estrogen-stimulated proliferation of human breast cancer cells

Estrogen sensitizes the MCF-7 estrogen-responsive breast cancer cell line to the mitogenic effect of insulin and the insulin-like growth factors (IGFs). This sensitization is specific for estrogen and occurs at physiological concentrations of estradiol. Dose-response experiments with insulin, IGF-I, and IGF-II suggested that the sensitization is mediated through the type I IGF receptor. Binding experiments with 125I-IGF-I and hybridization of a type I IGF receptor probe to RNA showed that the levels of the type I IGF receptor and its mRNA are increased 7- and 6.5-fold, respectively, by estradiol. IGF-I and estradiol had similar synergistic effects on other estrogen-responsive breast cancer cell lines, but IGF-I alone increased the proliferation of the MDA MB-231 cell line which is not responsive to estrogens. These experiments suggest that an important mechanism by which estrogens stimulate the proliferation of hormone-dependent breast cancer cells involves sensitization to the proliferative effects of IGFs and that this may involve regulation of the type I IGF receptor.     

Expression of insulin-like growth factor binding protein-4, insulin-like growth factor-I receptor, and insulin-like growth factor-I in the mouse uterus throughout the estrous cycle

Recent evidence suggests that a regulated insulin-like growth factor (IGF) system mediates the effects of estrogen, promoting the proliferation and differentiation of specific uterine cell types throughout the estrous cycle and during gestation in the rodent. Previous studies have shown that IGFs are differentially expressed in the mouse uterus during the periimplantation period. In the current study, we examined the expression of IGF binding protein-4 (IGFBP-4), IGF-I receptor (IGF-IR), and IGF-I in the mouse uterus throughout the estrous cycle. Ligand blot analysis was conducted on uterine homogenates using [125I]IGF-I. IGFBP-4 was detected in all uterine homogenates, varying in intensity throughout the estrous cycle. In situ hybridization studies at metestrus and diestrus demonstrated an intense IGFBP-4 mRNA signal in antimesometrial stromal cells between the luminal epithelium and the myometrium, but at proestrus and estrus, no IGFBP-4 signal was detected. No IGF-I mRNA was detected at any stage of the estrous cycle by in situ hybridization. However, by RT-PCR analysis, IGF-I mRNA was detected at all stages of the estrous cycle. RT-PCR analysis also showed IGF-IR mRNA throughout the estrous cycle. Using immunohistochemistry, IGF-IR immunostaining was detected throughout the estrous cycle and on days 2-7 of gestation, but was restricted to the glandular epithelium. These results suggest that uterine IGFBP-4 expression may not be dependent on uterine IGF-I expression. They also suggest that IGFBP-4 may play a role in uterine physiology independent of the inhibition of IGF-I action, and that IGF-IR is constitutively expressed in the mouse uterus.     

Insulin-like growth factor binding protein (IGFBP)-3-bound IGF-I and IGFBP-3-bound IGF-II in growth hormone deficiency

In blood, circulating IGFs are bound to six high-affinity IGFBPs, which modulate IGF delivery to target cells. Serum IGFs and IGFBP-3, the main carrier of IGFs, are upregulated by GH. The functional role of serum IGFBP-3-bound IGFs is not well understood, but they constitute the main reservoir of IGFs in the circulation. We have used an equation derived from the law of mass action to estimate serum IGFBP-3-bound IGF-I and IGFBP-3-bound IGF-II, as well as serum free IGF-I and free IGF-II, in 129 control children and adolescents (48 girls and 81 boys) and in 13 patients with GHD. Levels of serum total IGF-I, total IGF-II, IGFBP-1, IGFBP-2 and IGFBP-3 were determined experimentally, while those of IGFBP-4, IGFBP-5 and IGFPB-6, as well as the 12 affinity constants of association of the two IGFs with the six IGFBPs, were taken from published values. A correction for in vivo proteolysis of serum IGFBP-3 was also considered. In controls, serum total IGF-I, total IGF-II, IGFBP-3, IGFBP-3-bound IGF-I, IGFBP-3-bound IGF-II and free IGF-I increased linearly with age, from less than 1 to 15 years, in the two sexes. The concentrations of serum free IGF-I and free IGF-II were approximately two orders of magnitude below published values, as well as below the affinity constant of association of IGF-I with the type-1 IGF receptor. Therefore, it is unlikely that these levels can interact with the receptor. In the 13 patients with GHD, mean (+/- SD) SDS of serum IGFBP-3-bound IGF-I was -2.89 +/- 0.97. It was significantly lower than serum total IGF-I, free IGF-I or IGFBP-3 SDSs (-2.35 +/- 0.83, -1.12 +/- 0.78 and -2.55 +/- 1.07, respectively, p = 0.0001). The mean SDS of serum total IGF-II, IGFBP-3-bound IGF-II and free IGF-II were -1.25 +/- 0.68, -2.03 +/- 0.87 and 0.59 +/- 1.10, respectively, in GHD. In control subjects, 89.8 +/- 4.47% of serum total IGF-I and 77.3 +/- 9.4% of serum total IGF-II were bound to serum IGFBP-3. In patients with GHD, the mean serum IGFBP-3-bound IGF-I and IGFBP-3-bound IGF-II were 8.63 +/- 8. 53 and 19.1 +/- 14.7% below the respective means of control subjects (p < 0.02). In conclusion, in GHD there was a relative change in the distribution of serum IGFs among IGFBPs, due to the combined effects of the decrease in both total IGF-I and IGFBP-3. As a result, serum IGFBP-3-bound IGF-I and IGFBP-3 bound IGF-II, the main reservoirs of serum IGFs, were severely affected. This suggests that the decrease in serum IGFPB-3-bound IGF-I and IGFBP-3-bound IGF-II might have a negative effect for growth promotion and other biological effects of IGF-I and IGF-II. Finally, the estimation of serum IGFBP-3-bound IGF-I, or the percentage of total IGF-I and IGF-II bound to IGFBP-3, might be useful markers in the diagnosis of GHD.