Cellular distribution and ontogeny of insulin-like growth factors (IGFs) and IGF binding protein messenger RNAs and peptides in developing rat pancreas

To determine the role of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) in the development of the pancreas, and specifically of the islets of Langerhans, we have examined the cellular distribution and developmental changes in the expression of IGFs and IGFBPs in the pancreas of the fetal and neonatal rat between 19.5 days of gestation and postnatal day 28. This represents a period of substantial growth and restructuring of the beta cell component in islets of this species. IGF-I, IGF-II, and IGFBPs-1 to -6 mRNAs were localized by in situ hybridization, and peptides by immunohistochemistry, in histological sections. IGF-II mRNA was highly expressed in islet cells and some ductal epithelial cells in late fetal and early neonatal life, but was barely detectable by postnatal day 28. IGF-II peptide showed a similar distribution. IGF-I mRNA was barely detected in the fetus or neonate and was localized predominantly in the ductal and acinar tissues after postnatal day 7. IGF-I immunoreactivity was associated with some islet cells in the fetus and neonate, suggesting an endocrine rather than a paracrine source. We performed co-localization studies to assess whether the distribution of IGFs within the pancreas might be due to a sequestration by locally produced IGFBPs. The presence of mRNAs for both IGFBPs-1 and -2 was minimal in the pancreas prior to postnatal day 7, although subsequently IGFBP-1 mRNA was seen in islet cells, while IGFBP-2 mRNA was localized in both islets and acinar tissues. In contrast, both IGFBPs-1 and -2 immunoreactivities were identified in islets from late fetal life, suggesting a circulatory source for these IGFBPs during early pancreatic development. IGFBPs-3 to -5 mRNAs and immunoreactivities were identified within islet cells throughout fetal and neonatal life, with IGFBPs-3 and -5 being mainly associated with the alpha cell-rich islet mantle. The results show a compartmentalization of IGFs within pancreatic tissue, reflecting both paracrine and endocrine sources. The localization and action of IGFs in pancreas likely involves sequestration and distribution by endogenous as well as circulating IGFBPs.     

Inhibition of estrogen-induced sexual receptivity by androgens: role of the androgen receptor

The human IGFBP family consists of at least seven proteins, designated as IGFBP-1, -2, -3, -4, -5, -6, and-7. IGFBPs 1-6 bind IGF-I and IGF-II with high affinity whereas IGFBP-7, a newly identified IGFBP, binds IGFs with lower affinity and constitutes a low-affinity member of the IGFBP family. IGFBPs serve to transport the IGFs, prolong their half-lives, and modulate their biological action. At the cellular level, IGFBPs can either potentiate or inhibit the mitogenic effects of IGFs, depending upon cell types and IGFBP species (IGF-dependent action of IGFBPs). However, recent studies have indicated that IGFBPs, especially IGFBP-3, potently inhibit breast cancer cell growth in an IGF-independent manner. The IGF-independent action of IGFBP-3 requires interaction with cell-surface association proteins, presumably putative IGFBP-3 specific receptors, and is responsible for growth inhibitory action of the known growth suppressing factors such as TGF-beta, retinoic acid, and antiestrogens in breast cancer cells. Thus, IGFBP-3 appears to be a major factor in a negative control system involved in regulating human breast cancer cell growth in vitro. IGFBP-7, representing a low affinity IGFBP, appears to function as an IGF-independent cell growth regulator in breast cancer cells. Overall structural similarity between IGFBP-7 and classical high affinity IGFBPs 1-6 suggests that the mechanisms of action and signaling pathways used by IGFBP-7 may provide insight into the IGF-independent actions of the high affinity IGFBPs. A fuller understanding of the IGF-independent action of IGFBPs will allow us to understand how the growth of neoplastic cells can be modulated by the IGF/IGFBP system, and how other growth factors or pharmacological agents can interface with this system.     

Effects of undernutrition and diabetes on serum and liver mRNA expression of IGFs and their binding proteins during rat development

The purpose of the present study was to investigate the influence of nutrients and insulin on IGFs and their binding proteins (IGFBPs) during the fetal and neonatal periods of three rat populations: (a) rats undernourished by a 35% reduction in the diet from day 16 of gestation, (b) streptozotocin-induced diabetic rats from the same day, or 4 days after birth, and (c) control rats. Fetuses from the diabetic population showed a decrease in insulinemia at 19 and 21 days, along with an increase in glycemia at all stages. Neither glycemia nor insulinemia changed in the fetuses of undernourished mothers, but body weight was decreased at birth. Serum IGF-II decreased at 18 and 19 days of gestation in fetuses from undernourished mother, and increased at 18, 19 and 21 days in fetuses from diabetic mothers. Serum IGFBPs of low molecular weight (IGFBP-1 and IGFBP-2) increased in the three fetal populations studied, although no changes in serum IGFBPs were found from the effect of undernutrition or diabetes, but fetal liver IGFBP-1 mRNA expression was found to decreased in undernourished and diabetic animals as compared with controls. In neonatal rats, body weight, insulinemia and serum GH decreased in both undernourished and diabetic rats vs controls, while glycemia decreased in the undernourished and increased in the diabetic group. Serum IGF-II decreased only in diabetic rats and serum IGF-I decreased in both groups. The neonatal serum 30 kDa complex (IGFBP-1 and -2) also increased in undernutrition and diabetes parallel to the expression of mRNA. But, taken together, the changes in IGFBP peptide levels and liver mRNA expression strongly suggest that the 30 kDa complex seems to be composed mostly of IGFBP-1 in the diabetic group and of both IGFBP-1 and -2 in the undernourished animals. The studies of liver mRNA expression of IGFs and IGFBPs confirm the different metabolic control mechanism for the availability of IGFs by the IGFBPs, depending on the animal's maturity. The different adaptation shown by the diabetic neonatal population was confirmed by correlation studies between body weight, glycemia, insulinemia, IGF-I and IGFBPs. The different mechanism of adaptation in diabetic vs undernourished rats seems to be probably due to the decisive role played by hyperglycemia in the diabetic population, and also shows the crucial influence of nutritional status on IGFs and IGFBPs.     

Insulin-like growth factor binding protein production in bovine retinal endothelial cells

Retinopathy is the most frequent microangiopathic complication in diabetes. Many circulating hormones and locally produced mitogenic factors have been involved. Bovine retinal endothelial cells (BRECs) were cultured to investigate if insulin, insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), and a chronic high-glucose condition could control endothelial cell growth. Specific IGF-I receptors with two binding sites with high (Kd 0.03 nmol/L) and low (Kd 1.3 nmol/L) affinity were found when analyzing families of displacement curves between IGF-I versus IGF-I and IGF-I versus insulin. However, IGFs failed to be mitogenic factors in these cells. This could be explained by an inhibitory effect due to the presence of specific IGFBPs with a molecular weight between 24 and 43 kd. Using Western blot and immunoblot analysis, Northern blot study, and specific radioimmunoassay (RIA), these IGFBPs have been identified as IGFBP-3, -2, -5, and -4. Insulin, which does not bind to IGFBPs, was a potent mitogenic factor in these cells at a high concentration (10 nmol/L), suggesting a cross-reaction to IGF-I receptor. These IGFBPs, except the 24-kd form (IGFBP-4), were modulated by both IGF-I and IGF-II, with a maximum effect at 100 and 10 nmol/L, respectively. This regulation on IGFBPs was IGF-I receptor-independent. In fact, (1) IGFBP mRNA levels were not modified after stimulation with 100 nmol/L IGF-I, (2) 100 nmol/L IGF plus an equimolar concentration of alpha IR3 did not affect IGFBP production, (3) Des(1-3)IGF-I had no effect on IGFBP modulation, whereas at 10 nmol/L it enhanced BREC thymidine cell incorporation, and (4) 100 nmol/L insulin, which at this concentration can cross-react with the IGF-I receptor, did not modify the IGFBP pattern. Chronic exposure (4 weeks) of BRECs to 25 mmol/L glucose had no effect on cell growth. However, after 3 weeks, we observed a decreased IGFBP detection, and addition of 100 nmol/L IGF-I did not change IGFBP levels and did not modify cell growth. Conversely, BRECs grown in regular medium for 4 weeks showed increased IGFBP production. In conclusion, we showed that conditions mimicking hyperinsulinemia, rather than high levels of IGFs, could regulate BREC growth and that the IGF-I analog, Des(1-3), even with reduced affinity for IGFBPs but in part capable of binding to IGFBP-3, significantly stimulated BRECs growth only at 10 nmol/L. IGF actions are modulated by locally produced endothelial IGFBPs, and in turn, these endothelial IGFBPs are regulated, via in IGF-I receptor-independent mechanism, by the presence of IGFs. The autoregulatory IGF system together with the direct glucose modulation of IGFBPs could contribute in diabetic subjects to the retinal endothelial cell growth and metabolism through local changes in IGF bioavailability.     

A novel method for the extraction of sheep insulin-like growth factors-I and -II from plasma prior to radioimmunoassay

The measurement of insulin-like growth factors (IGFs) in plasma is complicated by the presence of high-affinity IGF-binding proteins (IGFBPs). Consequently, the IGFBPs need to be removed or their IGF-binding effects need to be neutralized prior to assaying samples for IGFs. It was observed that IGFs but not IGFBPs from sheep plasma bind to the size-exclusion gel Sephacryl S-100 HR at a low pH and that the IGFs can subsequently be eluted off at a neutral pH. From this observation a convenient method was developed for the extraction from plasma of ovine (o) IGF-I and -II free from detectable IGFBPs with close to 100% recovery. When assayed in homologous sheep IGF-I and -II radioimmunoassays the Sephacryl-extracted plasma samples gave dose-response curves which were parallel to purified oIGFs. Furthermore, the results obtained by Sephacryl extraction were highly correlated with those found by the established Sephadex G-75 extraction procedure.     

Expression of insulin-like growth factor binding protein-4 and -5 mRNAs in adult rat forebrain

Accumulating evidence indicates that the insulin-like growth factors (IGFs) can act as neurotrophic factors. A family of at least six IGF binding proteins (IGFBPs) has been characterized. The IGFBPs prolong the half-life of IGFs in plasma and may modulate IGF action in a cell- or tissue-specific fashion. Two recently characterized IGFBPs, IGFBP-4 and -5, have been shown by northern blot hybridization to be expressed in rat brain, but their cellular sites of synthesis are poorly characterized. Because IGFBP-4 and IGFBP-5 could potentially modulate IGF actions in the brain, we used in situ hybridization histochemistry and 35S-labeled IGFBP-4 and IGFBP-5 riboprobes to localize sites of IGFBP-4 and -5 mRNA expression in adult rat brain. The two IGFBP mRNAs are abundantly expressed within discrete regions of brain. The expression patterns of the two genes are largely nonoverlapping. Notably, IGFBP-4 mRNA is highly expressed within hippocampal and cortical areas, whereas IGFBP-5 mRNA is not detected above background in these areas. Within the hippocampus, abundant IGFBP-4 mRNA expression is detected in pyramidal neurons of the subfields of Ammon's horn and the subiculum and in the granule cell layer of the anterior hippocampal continuation. In the cortex, IGFBP-4 mRNA is widely expressed in most areas and layers. In contrast, IGFBP-5, but not IGFBP-4, mRNA is detected within thalamic nuclei, leptomeninges, and perivascular sheaths. The distinct expression patterns of IGFBP-4 and -5 mRNAs within the brain suggest that these IGFBPs may modulate paracrine/autocrine actions of the IGFs in discrete brain regions or compartmentalization of the IGFs within the brain.     

Expression of insulin-like growth factor-II and IGF-binding protein-1 mRNAs in term rhesus monkey placenta: comparison with human placenta

The patterns of expression insulin-like growth factor-II (IGF-II) and IGF-binding protein-1 (IGFBP-1) mRNAs were compared between term human and rhesus monkey placenta using in situ hybridization histochemistry. Since IGFs and IGFBPs are paracrine factors, the identification of the sites of synthesis of the IGFs and their binding proteins indicate the potential sites of biological action. In both species, IGF-II mRNA was found in highest abundance in the extravillous cytotrophoblasts. The major difference was observed in placental villi. In the human placenta, IGF-II mRNA was expressed in the chorionic mesoderm of the placental villi, whereas, in the rhesus placenta, it was expressed in the syncytiotrophoblasts and not in the chorionic mesoderm. In both species, IGFBP-1 mRNA was expressed only in the decidua. Therefore, the pattern of expression of IGFBP-1 mRNA in the maternal decidua is similar between rhesus monkey and human placenta, but that of IGF-II mRNA in the fetal placental villi is different. These data suggest that the IGF-II-IGFBP-1 interaction in the paracrine regulation of placental growth and/or function in the rhesus monkey and human placentae may have similarities and differences.     

Inhibition of insulin receptor activation by insulin-like growth factor binding proteins

The insulin-like growth factors (IGFs) are transported by a family of high-affinity binding proteins (IGFBPs) that protect IGFs from degradation, limit their binding to IGF receptors, and modulate IGF actions. The six classical IGFBPs have been believed to have no affinity for insulin. We now demonstrate that IGFBP-7/mac25, a newly identified member of the IGFBP superfamily that binds IGFs specifically with low affinity is a high-affinity insulin binding protein. IGFBP-7 blocks insulin binding to the insulin receptor and thereby inhibiting the earliest steps in insulin action, such as autophosphorylation of the insulin receptor beta subunit and phosphorylation of IRS-1, indicating that IGFBP-7 is a functional insulin-binding protein. The affinity of other IGFBPs for insulin can be enhanced by modifications that disrupt disulfide bonds or remove the conserved COOH terminus. Like IGFBP-7, an NH2-terminal fragment of IGFBP-3 (IGFBP-3((1-87))), also binds insulin with high affinity and blocks insulin action. IGFBPs with enhanced affinity for insulin might contribute to the insulin resistance of pregnancy, type II diabetes mellitus, and other pathological conditions.     

Proteolysis of insulin-like growth factors (IGF) and IGF binding proteins by cathepsin D

Various proteinases have been postulated to function in limited proteolysis of insulin-like growth factor binding proteins (IGFBPs) contributing to the regulation of IGF bioavailability. In this study, we report on the in vitro degradation of IGFs and IGFBPs by the purified acidic aspartylprotease cathepsin D that has been shown to proteolyze IGFBP-3. Recombinant human [125I] IGFBP-1 to -5 were processed by cathepsin D to fragments of defined sizes in a concentration dependent manner, whereas IGFBP-6 was not degraded. Ligand blotting revealed that none of the IGFBP-1 or -3 fragments formed by cathepsin D retain their ability to bind IGF. By N-terminal sequence analysis of nonglycosylated IGFBP-3 fragments produced by cathepsin D, at least four different cleavage sites were identified. Some of these cleavage sites were identical or differed by one amino acid from sites used by other IGFBP proteases described. The IGFBP-3 and -4 cleavage sites produced by cathepsin D are located in the nonconserved central region. IGF-I and -II, but not the unrelated platelet-derived growth factor BB, were degraded by cathepsin D in a time and concentration-dependent manner. We speculate that the major functional site of cathepsin D is intracellular and may be involved 1) in the selected clearance either of IGFBP or IGFs via different endocytic pathways or 2) in the general lysosomal inactivation of the IGF system.     

Insulin-like growth factor-binding proteins (IGFBPs) and their regulatory dynamics

The IGFBPs are a family of homologous proteins that have co-evolved with the IGFs and that confer upon the IGF regulatory system both functional and tissue specificity. IGFBPs are not merely carrier proteins for IGFs, but hold a central position in IGF ligand-receptor interactions through influences on both the bioavailability and distribution of IGFs in the extracellular environment. In addition, IGFBPs appear to have intrinsic biological activity independent of IGFs. The current status of research on IGFBPs is reviewed herein. Following a brief introduction to the entire IGF/IGFBP system, separate sections for each of the six cloned mammalian IGFBPs, the most extensive for IGFBP3, cover selected topics that emphasize the dynamics of IGFBPs--that is, their regulation in cells, their functionally important post-translational modifications, and their interactions in the cellular microenvironment--and how these dynamics influence physiological function.     

Insulin-like growth factor binding protein-1 at mouse neuromuscular synapses

The insulin-like growth factor (IGF) signaling system includes the growth factors and their cell surface receptors, along with circulating IGF binding proteins (IGFBPs) that may alter and modulate the action of these neurotrophic hormones. These IGFBPs, along with IGFs and receptors, have been detected in various tissues including the brain. In this study, using polyclonal antibody to human IGFBP-1 or bovine IGFBP-2, we found that mouse muscle extracts contain similar-sized proteins that cross-react with these antibodies on Western immunoblots. After establishing that these antibodies reacted with the homologous murine IGFBPs, we performed immunocytochemistry to demonstrate the localization of IGFBP-1 at the neuromuscular junction, a model nicotinic, cholinergic synapse, as well as within intramuscular nerves. IGFBP-2, a distinct macromolecule, is present on the surface of muscle fibers and is not present within synapses or nerves.     

Peptide growth factors regulate insulin-like growth factor binding protein production by fetal rat lung fibroblasts

Insulin-like growth factor (IGF) binding proteins (IGFBPs) are expressed in fetal lung and may provide important post-translational regulation of IGF-induced mitogenesis during lung organogenesis. Because of the observation that growth factors can control cell growth through regulation of IGFBPs, we examined IGFBP production by fetal lung fibroblasts following stimulation by peptide growth factors important for fetal lung growth and development. Fetal lung fibroblasts were cultured in serum-free medium supplemented with various growth factors for up to 48 h, and IGFBPs in conditioned medium (CM) were analyzed by ligand blot and immunoblot techniques. Accumulation of CM IGFBP-3 was increased and IGFBP-2 decreased by incubation with either keratinocyte growth factor (KGF) or epidermal growth factor (EGF). The effect of these factors on IGFBP-3 accumulation increased with time but the effects of KGF on CM IGFBP-2 decreased over 48 h of incubation. CM IGFBP-4 was increased by 24 and 48 h incubation with basic fibroblast growth factor (bFGF; 2.1- and 2.7-fold increases at 24 and 48 h, respectively) and platelet-derived growth factor-BB (PDGF-BB; 4.2- and 14.9-fold increases at 24 and 48 h, respectively), and 48 h incubation with EGF (6.3-fold increase). In 48-h coincubation experiments, EGF in combination with PDGF-BB or with bFGF, and bFGF in combination with PDGF-BB, resulted in IGFBP-4 accumulations twice that expected from a summation of the effects of either growth factor alone (IGFBP-4 increased 9.8-, 4.0-, and 1.8-fold by PDGF-BB, EGF, and bFGF, respectively; and 27.1-, 37.3-, and 13.0-fold by PDGF-BB plus EGF, PDGF-BB plus bFGF, and EGF plus bFGF, respectively). These results suggest synergistic effects of these growth factors on IGFBP-4 accumulation in fetal lung fibroblast CM. Because IGFBPs are known to regulate DNA synthesis, we speculate that peptide growth factors may alter cell proliferation in fetal lung, in part through their effect on IGFBPs.     

Prothymosin alpha 1 modulates lymphokine-activated killer cell activity and IL-2 production by peripheral blood lymphocytes from melanoma patients in vitro

Several distinct insulin-like growth factor binding proteins (IGFBPs) are present in tissues and fluids of the developing and adult eye. However, the mechanism(s) involved in the regulation of ocular IGFBP levels is unknown. We have now identified an endogenous factor in vitreous and aqueous humors that, when activated by sodium dodecyl sulfate (SDS), abolishes the capacity of specific low molecular weight IGFBPs (i.e. 24-30 kDa) to bind IGF as assessed by western ligand blotting. In contrast, IGF binding to the 46 and 32 kDa IGFBPs (IGFBP-3 and IGFBP-2 respectively) is not affected by the SDS-activated inhibitory factor (IF). Maximal activation of the IF occurs at an SDS concentration of approximately 0.015%. Incubations in the presence of the serine-proteinase inhibitor aprotinin result in marked inhibition of IF activity. Preliminary characterization by ultrafiltration suggests that the IF is large (< 100 kDa) and/or that it is present in a complex. The finding of a factor, most likely a serine proteinase, that specifically abolishes IGF binding to low molecular weight IGFBPs suggests a mechanism for regulating the levels of these IGFBPs and thus the functional activities of IGFs in ocular fluids under normal and/or pathological conditions.     

Insulin-like growth factor binding proteins localize to discrete cell culture compartments in periosteal and osteoblast cultures from fetal rat bone

Insulin-like growth factor (IGF)-I and IGF-II are expressed at biologically effective levels by bone cells. Their stability and activity are modulated by coexpression of IGF binding proteins (IGFBPs). Secreted IGFBPs may partition to soluble, cell-associated, and matrix-bound compartments. Extracellular localization may sequester, store, or present IGFs to appropriate receptors. Of the six IGFBPs known, rat osteoblasts synthesize all but IGFBP-1. Of these, IGFBP-3, -4, and -5 mRNAs are induced by an increase in cAMP. Little is known about extracellular IGFBP localization in bone and nothing about IGFBP expression by nonosteoblastic periosteal bone cells. We compared basal IGFBP expression in periosteal and osteoblast bone cell cultures and assessed the effects of changes in cAMP-dependent protein kinase A or protein kinase C. Basal IGFBP gene expression differed principally in that more IGFBP-2 and -5 occurred in osteoblast cultures, and more IGFBP-3 and -6 occurred in periosteal cultures. An increase in cAMP enhanced IGFBP-3, -4, and -5 mRNAand accordingly increased soluble IGFBP-3, -4, and -5 and matrix-bound IGFBP-3 and -5 in both bone cell populations. In contrast, protein kinase C activators suppressed IGFBP-5 mRNA, and its basal protein levels remained very low. We also detected low Mr bands reactive with antisera to IGFBP-2, -3, and -5, suggesting proteolytic processing or degradation. Our studies reveal that various bone cell populations secrete and bind IGFBPs in selective ways. Importantly, inhibitory IGFBP-4 does not significantly accumulate in cell-associated compartments, even though its secretion is enhanced by cAMP. Because IGFBPs bind IGFs less tightly in cell-bound compartments, they may prolong anabolic effects by agents that increase bone cell cAMP.     

NMDA induces NO release from primary cell cultures of human fetal cerebral cortex

In order to investigate the role of insulin-like growth factors (IGFs) in human ovulation, we evaluated the concentrations of IGF-binding proteins (IGFBPs) in human follicular fluid (FF). The concentrations of IGFBP-1 in the FFs of 15 women undergoing in vitro fertilization and embryo transfer were measured and related to those of 17beta-estradiol (E2), progesterone and androstenedione in the FFs. IGFBP-1 levels in the FFs were positively correlated with those of E2 and progesterone. No correlation was found between the IGFBP-1 and androstenedione levels in FFs. The concentrations of IGFBP-1 were significantly increased in the FFs which contained mature oocytes compared with those of immature oocytes, whereas IGFBP-3 in FFs tended to decrease as oocytes matured. It is suggested that IGFs may play important roles in human preovulatory processes, and that IGFBP-1 may be a valuable biochemical marker in the evaluation of oocytes.     

Structure of the IGF-binding domain of the insulin-like growth factor-binding protein-5 (IGFBP-5): implications for IGF and IGF-I receptor interactions

Binding proteins for insulin-like growth factors (IGFs) IGF-I and IGF-II, known as IGFBPs, control the distribution, function and activity of IGFs in various cell tissues and body fluids. Insulin-like growth factor-binding protein-5 (IGFBP-5) is known to modulate the stimulatory effects of IGFs and is the major IGF-binding protein in bone tissue. We have expressed two N-terminal fragments of IGFBP-5 in Escherichia coli; the first encodes the N-terminal domain of the protein (residues 1-104) and the second, mini-IGFBP-5, comprises residues Ala40 to Ile92. We show that the entire IGFBP-5 protein contains only one high-affinity binding site for IGFs, located in mini-IGFBP-5. The solution structure of mini-IGFBP-5, determined by nuclear magnetic resonance spectroscopy, discloses a rigid, globular structure that consists of a centrally located three-stranded anti-parallel beta-sheet. Its scaffold is stabilized further by two inside packed disulfide bridges. The binding to IGFs, which is in the nanomolar range, involves conserved Leu and Val residues localized in a hydrophobic patch on the surface of the IGFBP-5 protein. Remarkably, the IGF-I receptor binding assays of IGFBP-5 showed that IGFBP-5 inhibits the binding of IGFs to the IGF-I receptor, resulting in reduction of receptor stimulation and autophosphorylation. Compared with the full-length IGFBP-5, the smaller N-terminal fragments were less efficient inhibitors of the IGF-I receptor binding of IGFs.     

Circulating and ovarian IGF binding proteins: potential roles in normo-ovulatory cycles and in polycystic ovarian syndrome

IGFs function as co-gonadotropins in the ovary, facilitating steroidogenesis and follicle growth. IGFBP-1 to -5 are expressed in human ovary and mostly inhibit IGF action in in vitro ovarian cell culture systems. In the clinical disorder of polycystic ovarian syndrome (PCOS), which is characterized by hyperandrogenemia, polycystic ovaries and anovulation, follicles have a higher androgen: estradiol (A : E2) content and growth is arrested at the small antral stage. In the PCOS follicle, follicle stimulating hormone (FSH) and IGF levels are in the physiologic range, and even in the face of abundant androstenedione (AD) substrate, aromatase activity and E2 production are low. When PCOS granulosa are removed from their ovarian environment, they respond normally or hyperrespond to FSH. It has been postulated that an inhibitor of IGF's synergistic actions with FSH on aromatase activity may be one (or more) of the IGFBPs, which contributes to the arrested state of follicular development commonly observed in this disorder. High levels of IGFBP-2 and IGFBP-4 are present in follicular fluid (FF) from androgen-dominant follicles (FFa) from normally cycling women and in women with PCOS. This is in marked contrast to the near absence of these IGFBPs in estrogen-dominant FF (FFe), determined by Western ligand blotting. Regulation of granulosa-derived IGFBPs is effected by gonadotropins and insulin-like peptides. In addition, an IGFBP-4 metallo-serine protease is present in FFe, but not in FFa in ovaries from normally cycling women and those with PCOS, although the IGFBP-4 protease is present in PCOS follicles hyperstimulated for in vitro fertilization. Recent studies demonstrate that IGF-II in FFe is higher than in FFa' whereas IGF-I, IGFBP-3 and IGFBP-1 levels do not differ, underscoring the importance of local IGF-II production by the granulosa and the importance of IGFBP-4 and IGFBP-2 in regulation of IGF-II action within the follicle during its developmental pathway as an E2- or A-dominant follicle. In the androgen-treated female-to-male transsexual (TSX) model for PCOS, IGF-I, IGF-II, IGFBP-3 and IGFBP-1 levels do not differ.     

Pregnancy-specific alterations in the expression of the insulin-like growth factor system during early placental development in the ewe

The placenta is recognized as an important determinant of fetal growth rate, yet the factors regulating its proliferation remain poorly understood. Components of the insulin-like growth factor (IGF) system were localized in the ovine uterus using in situ hybridization between days 13-55 of gestation, the period of implantation and placentome formation. IGF-II messenger RNA (mRNA) expression was intense in the fetal mesoderm, particularly at the tips of the invading placentome villi. Moderate levels of IGF-II mRNA were also observed in the maternal caruncular stroma. In contrast, expression of IGF-1 mRNA was low (compared to estrous levels) and ubiquitous decreasing as gestation advanced. IGF-binding protein-2 (IGFBP-2 mRNA was not detected until day 29 of gestation, when it appeared restricted to the dense caruncular-like stroma lining the luminal epithelium, colocalized with IGFBP-4. High concentrations of IGFBP-4 mRNA expression were also found in the placentome capsule. IGFBP-3 mRNA expression was intense in the luminal epithelium between days 13-15 of gestation. Subsequently, levels in this region dropped significantly (P < 0.001). IGFBP-3 mRNA expression was also high in the maternal placentome villi, where photographic emulsions localized expression to blood vessel walls. Peak expression of IGF type 1 receptor (IGF-1R) mRNA was found in the deep uterine glands, with intermediate expression in the superficial uterine glands. Moderate expression of IGF-1R mRNA was initially recorded in caruncular stroma, but levels in this region decreased significantly (P < 0.001) to below the detection limit of the technique after interdigitation by the fetal allantochorion. Furthermore, IGF-1R mRNA could not be detected in any fetal placentome tissue. This study, therefore, has established the pattern of expression of the IGFs, IGF-1R, and three of the IGFBPs during establishment of the ovine placenta. It will form the basis for future work to investigate how this system is regulated and to determine the role of the IGFs in placental development.     

Skeletal muscle cell-derived insulin-like growth factor (IGF) binding proteins inhibit IGF-I-induced myogenesis in rat L6E9 cells

The insulin-like growth factors (IGFs) stimulate the differentiation of skeletal muscle cells. IGF binding proteins (IGFBPs), which are expressed by skeletal muscle cells, may enhance or inhibit IGF actions. To explore the role of skeletal muscle-derived IGFBPs in IGF-induced myogenesis, we compared the differentiation-inducing effects of IGF-I and des(1-3)IGF-I in rat L6E9 skeletal myoblasts. Des(1-3)IGF-I is a naturally occurring IGF-I analog with markedly reduced affinity for IGFBPs but with an affinity for the IGF-I receptor that is comparable to that for native IGF-I. We find that rat L6E9 cells produce principally IGFBP-4 and BP-6, with a minor component of IGFBP-5. Both IGFBP-4 and BP-6 accumulate during differentiation and increase further in response to IGF-I or des(1-3)IGF-I treatment. We find that an IGF-I analog with reduced affinity for IGFBPs is significantly more potent than native IGF-I in stimulating myogenesis (as assessed by myogenin messenger RNA abundance and muscle creatine kinase activity), indicating that IGFBPs expressed by skeletal muscle cells inhibit differentiation induced by IGF-I. In view of the relative abundance of IGFBP-4, its relatively high affinity for IGF-I and the low affinity of IGFBP-6 for IGF-I, it is likely that the inhibitory effect of rat skeletal muscle-derived IGFBPs on IGF-I-induced myogenesis is mediated principally by IGFBP-4.