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Background: The role of IGF-I/IGFBP's system in the pathogenesis of diabetic vascular complications is widely discussed in the literature. We studied the influence of this system on microvasculature in patients with type 1 diabetes with respect to the effect of IGFBP-1., Methods and Results: 17 patients with type 1 diabetes were included in the study. We examined IGF-I, IGFBP-1 and IGFBP-3 serum levels, parameters of compensation of diabetes and basic values of lipid metabolism. The function of microvasculature was examined using the laser-Doppler system Periflux. We didn't found any relation between the total IGF-I serum levels, parameters of lipid metabolism or level of diabetes compensation and the degree of impairment of the function of microcirculation. IGFBP-1 serum levels positively correlated with the peak perfusion in thermal hyperaemia (r = 0.39; p < 0.03). IGFBP-3 serum levels did not affect the function of microcirculation., Conclusion: We can conclude that the activity of IGF-I/IGFBP's system belongs to factors contributing to the development of diabetic microangiopathy in type 1 diabetes. IGFBP-1 as the modulator of IGF-I activity plays probably protective role against the progression of microangiopathy. These results correspond with observations of some other authors.

Insulin-like growth factor-binding protein-1 (IGFBP-1) binds to insulin-like growth factors (IGFs) and has been shown to inhibit or stimulate cellular responses to IGF-I in vitro. This capacity of IGFBP-1 to inhibit or stimulate IGF-I actions correlates with its ability to form stable high molecular weight multimers. Since the ability of some proteins to polymerize is dependent upon transglutamination, we determined if tissue transglutaminase could catalyze this reaction and the effect of polymerization of IGFBP-1 upon IGF-I action. Following incubation with pure tissue transglutaminase (Tg), IGFBP-1 formed covalently linked multimers that were stable during SDS-polyacrylamide gel electrophoresis using reducing conditions. Dephosphorylated IGFBP-1 polymerized more rapidly and to a greater extent compared with native (phosphorylated) IGFBP-1. Exposure to IGF-I stimulated transglutamination of IGFBP-1 in vitro. An IGFBP-1 mutant in which Gln(66)-Gln(67) had been altered to Ala(66)-Ala(67) (Q66A/Q67A) was relatively resistant to polymerization by Tg compared with native IGFBP-1. Tg localized in fibroblast membranes was also shown to catalyze the formation of native IGFBP-1 multimers, however, Q66A/Q67A IGFBP-1 failed to polymerize. Although the mutant IGFBP-1 potently inhibited IGF-I stimulated protein synthesis in pSMC cultures, the same concentration of native IGFBP-1 had no inhibitory effect. The addition of higher concentrations of native IGFBP-1 did inhibit the protein synthesis response, and this degree of inhibition correlated with the amount of monomeric IGFBP-1 that was present. In conclusion, IGFBP-1 is a substrate for tissue transglutaminase and Tg leads to the formation of high molecular weight covalently linked multimers. Polymerization is an important post-translational modification of IGFBP-1 that regulates cellular responses to IGF-I.

Insulin-like growth factor (IGF)-binding protein (IGFBP)-related proteins (IGFBP-rPs) are newly described cysteine-rich proteins that share significant aminoterminal structural similarity with the conventional IGFBPs and are involved in a diversity of biological functions, including growth regulation. IGFBP-rP1 (MAC25/Angiomodulin/prostacyclin-stimulating factor) is a potential tumor-suppressor gene that is differentially expressed in meningiomas, mammary and prostatic cancers, compared with their malignant counterparts. We have previously shown that IGFBP-rP1 is preferentially produced by primary cultures of human prostate epithelial cells (HPECs) and by poorly tumorigenic P69SV40T cells, compared with the cancerous prostatic LNCaP, DU145, PC-3, and M12 cells. We now show that IGFBP-rP1 increases during senescence of HPEC. IGFBP-rP2 (also known as connective tissue growth factor), a downstream effector of transforming growth factor (TGF)-beta and modulator of growth for both fibroblasts and endothelial cells, was detected in most of the normal and malignant prostatic epithelial cells tested, with a marked up-regulation of IGFBP-rP2 during senescence of HPEC. Moreover, IGFBP-rP2 noticeably increased in response to TGF-beta1 and all-trans retinoic acid (atRA) in HPEC and PC-3 cells, and it decreased in response to IGF-I in HPEC. IGFBP-rP3 [nephroblastoma overexpressed (NOV)], the protein product of the NOV protooncogene, was not detected in HPEC but was expressed in the tumorigenic DU145 and PC-3 cells. It was also synthesized by the SV40-T antigen-transformed P69 and malignant M12 cells, where it was down-regulated by atRA. These observations suggest biological roles of IGFBP-rPs in the human prostate. IGFBP-rP1 and IGFBP-rP2 are likely to negatively regulate growth, because they seem to increase during senescence of the prostate epithelium and in response to growth inhibitors (TGF-beta1 and atRA). Although the data collected on IGFBP-rP3 in prostate are modest, its role as a growth stimulator and/or protooncogene is supported by its preferential expression in cancerous cells and its down-regulation by atRA.

Unlabelled: The aim of the present study was to investigate whether the diurnal variability of B-Glucose is dependent on GH, IGF-I and IGFBP-1 levels, apart from insulin, and if there is any difference between Tanner stages 3 and 5. Five boys in Tanner stage 3 and 6 boys in stage 5 with type 1 diabetes were included. Blood was continuously collected from a cubital vein for 24 h. S-Insulin, S-GH, S-IGF-I and S-IGFBP-1 were analysed. B-Glucose was analysed hourly at bedside. One week before and 1 wk after the 24-h study period the participants performed self-monitoring of blood glucose (SMBG) during normal physiologic conditions. In the 24-h profile of B-Glucose, insulin, IGFBP-1 and GH, we found a significant positive correlation between B-Glucose and log IGFBP-1 (r = 0.5, p = 0.005) and an inverse correlation to insulin (r = -0.5, p = 0.004) but no correlation to logGH (r = -0.04, p = 0.831). In multiple regression analysis, B-Glucose was still significantly correlated to log IGFBP-1, when adjusting for insulin and GH, in Tanner stage 5. We found a difference between Tanner stages 3 and 5 in the variability of B-Glucose over a longer period during normal daily activity (p = 0.02), but not over the 24-h study period., Conclusion: We have demonstrated in type 1 diabetes adolescent boys a relationship between simultaneously measured blood-glucose and IGFBP-1 levels independent of the insulin and GH levels, suggesting that the free fraction of IGF-I influences the glucose metabolism.

Insulin-like growth factor-1 (IGFBP-1) is particularly important in human female reproductive physiology, where it is involved with other factors in a complex system which regulates menstrual cycles, puberty, ovulation, decidualization, implantation and fetal growth. This has implications for clinical obstetrics and gynaecology, where there is evidence for a pathophysiological role for IGFBP-1 in pre-eclampsia, intrauterine growth restriction, polycystic ovarian syndrome and trophoblast and endometrial neoplasms.

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.

In the past few years, both laboratory investigations and population studies have provided strong circumstantial evidence that insulin-like growth factor (IGF) physiology influences cancer risk. In contrast to the influence of germ line mutations that are rare but are associated with very high risks, the impact of inter-individual variability in IGF physiology on risk appears to be modest but to effect a relatively high percentage of the population. Although this field of investigation is young, attention is already being given to the possibility that it may be relevant to clinical assessment of risk and/or to the identification of novel prevention strategies and intermediate endpoints. This review summarises key results in this field and provides a hypothesis concerning the mechanism by which IGF physiology influences risk of common epithelial cancers including those of breast, prostate, lung and colon.

The insulin/IGF binding protein-1 (IGFBP-1) axis is important in coordinating insulin- and IGF-mediated regulation of glucose metabolism and glycemia. Dysregulation of the axis may play a role in the pathophysiology of disorders of insulin deficiency and resistance. We have investigated this hypothesis by generating transgenic mice that overexpress hIGFBP-1. To study the axis in its true physiological context, we used a human (h) IGFBP-1 cosmid clone so that transgene expression is responsive to normal hormonal stimuli. hIGFBP-1 mRNA is expressed in a tissue-specific fashion, and measurement of serum protein levels by specific immunoassay indicates normal physiological regulation in response to fasting/feeding and appropriate post-translational modification as indicated by the detection of phosphorylated and nonphosphorylated isoforms of the protein. The hypoglycemic response to exogenous IGF-I is attenuated in transgenic mice. Transgenic mice exhibit an enhanced insulin secretory response to a glucose challenge, although basal and stimulated blood glucose levels are similar to controls. There is a sexual dimorphism in phenotypic expression: male transgenic mice had higher stimulated glucose and insulin levels than did females. Transgenic mice exhibit fasting hyperglycemia and hyperinsulinemia and glucose intolerance in later life, indicating an age-related decline in glucocompetence. These findings demonstrate the importance of the normal inverse relationship between serum insulin and IGFBP-1 levels in glucoregulation and that sustained dysregulation of the insulin/IGF-I/IGFBP-1 axis is associated with impaired glucose tolerance and abnormalities of insulin action.

Skeletal myogenic cells respond to the insulin-like growth factors (IGF-I and IGF-II) by differentiating or proliferating, which are mutually exclusive pathways. What determines which of these responses to IGF skeletal myoblast undergo is unclear. IGF-binding protein-related protein 1 (IGFBP-rP1) is a secreted protein with close homology to the IGF-binding proteins (IGFBPs) in the N-terminal region. IGFBP-rP1, previously called mac25 and IGFBP-7, is highly expressed in C2 skeletal myoblasts during the proliferative phase, but is down-regulated during myoblast differentiation. To determine the role of IGFBP-rP1 in myogenesis, IGFBP-rP1 was overexpressed in C2 myoblasts using a retroviral vector. Western blots indicated that the resulting C2-rP1 myoblasts secreted approximately 27-fold higher levels of IGFBP-rP1 than control C2-LX myoblasts that were transduced with a control vector (LXSN). Compared with C2-LX myoblasts, the differentiation responses of C2-rP1 myoblasts to IGF-I, IGF-II, insulin, and des(1-3)IGF-I were significantly reduced (P < 0.05). However, proliferation responses of C2-rP1 and C2-LX myoblasts to these same factors were not significantly different. Exposure of control C2-LX myoblasts to factors secreted by C2-rP1 myoblasts using a transwell coculture system reduced C2-LX myoblast differentiation significantly (P < 0.05). Experiments with the mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 suggested that IGFBP-rP1 inhibits a MAPK-dependent differentiation pathway. In confirmation of this idea, levels of phosphorylated extracellular signal-regulated kinase-2 (a MAPK) were reduced in C2-rP1 myoblasts compared with those in C2-LX myoblasts. These findings indicate that IGFBP-rP1 may function as an autocrine/paracrine factor that specifies the proliferative response to the IGFs in myogenesis.

Articular chondrocytes maintain cartilage throughout life by replacing lost or damaged matrix with freshly synthesized material. Synthesis activity is regulated, rapidly increasing to well above basal levels in response to cartilage injury. Such responses suggest that synthesis activity is linked to the rate of matrix loss by endogenous "damage control" mechanisms. As a major stimulator of matrix synthesis in cartilage, insulin-like growth factor I (IGF-I) is likely to play a role in such mechanisms. Although IGF-I is nearly ubiquitous, its bioavailability in cartilage is controlled by IGF-I binding proteins (IGFBPs) secreted by chondrocytes. IGFBPs are part of a complex system, termed the IGF-I axis, that tightly regulates IGF-I activities. For the most part, IGFBPs block IGF-I activity by sequestering IGF-I from its cell surface receptor. We recently found that the expression of one binding protein, IGFBP-3, increases with chondrocyte age, paralleling an age-related decline in synthesis activity. In addition, IGFBP-3 is overexpressed in osteoarthritic cartilage, leading to metabolic disturbances that contribute to cartilage degeneration. These observations indicate that IGFBP-3 plays a crucial role in regulating matrix synthesis in cartilage, and suggest that cartilage damage control mechanisms may fail due to age-related changes in IGFBP-3 expression or distribution. Our investigation of this hypothesis began with immunolocalization studies to determine the tissue distribution of IGFBP-3 in human cartilage. We found that IGFBP-3 accumulated around chondrocytes in the pericellular/territorial matrix, where it co-localized with fibronectin, but not with the other matrix proteins tenascin-C and type VI collagen. This result suggested that the IGFBP-3 distribution is determined by binding to fibronectin. Binding studies using purified proteins demonstrated that IGFBP-3 does in fact bind to fibronectin, but not to tenascin-C or type VI collagen. Finally, we investigated the metabolic effects of fibronectin and IGFBP-3 in a chondrocyte culture system. These experiments showed that fibronectin enhanced the inhibitory effect that low concentrations of IGFBP-3 had on matrix synthesis. Taken together, these observations confirm that IGFBP-3-fibronectin interactions affect the IGF-I axis, and they indicate that IGF-I is stored in the chondrocyte territorial matrix through binding to a complex of IGFBP-3 and intact fibronectin. This arrangement may play an important role in cartilage damage control mechanisms. The local increase in matrix synthesis following injury could result from damage-induced IGF-I release from such pools. An age-related failure to organize this system may contribute to degenerative disease.

Insulin-like growth factor-II (IGF-II) and IGF binding protein-1 (IGFBP-1) appear to play an important role in paracrine interactions at the maternal-fetal interface in human pregnancy. Patterns of expression of IGF-II and IGFBP-1 at the decidual-trophoblast interface suggest paracrine interactions occur between the IGF-II-expressing invading cytotrophoblast and maternal decidua-derived IGFBP-1. Autocrine/paracrine actions of trophoblast-derived IGF-II may be important in invasion, and for both trophoblast and decidual function. The actions of IGFBP-1 in binding IGF, and as an integrin ligand, suggest it may have multiple roles in the interactions between the invading trophoblast and the maternal decidua. Abundant decidual IGFBP-1 may interact with the IGF-II-expressing, protease-secreting trophoblast to modulate invasion. In-vitro studies of trophoblast-decidual cell interactions in invasion, and clinical observations in a gestational disorder with shallow placental invasion such as pre-eclampsia, have provided new insights into the possible role(s) of IGFBP-1 in trophoblast invasion. The precise mechanisms underlying IGF and IGFBP-1 action at the decidual-trophoblast interface remain to be elucidated. The potential predictive value of serum IGFBP-1 concentrations in pre-eclampsia also remains to be established.

One of the most remarkable processes associated with the establishment of pregnancy in the primate is the process of decidualization. This transformation of a stromal fibroblast to a fully differentiated decidual cell is required for implantation and embryo survival in early pregnancy. Although the morphological and biochemical characteristics of the primate decidual cell have been extensively studied, the precise cellular, biochemical and molecular signals required for this transformation have yet to be elucidated. During decidualization, stromal cells first proliferate and then differentiate. Based on our extensive in-vivo and ongoing in-vitro studies, we have suggested that the process of decidualization in the baboon can be divided into two distinct phases. The initial proliferative phase is characterized by the expression of the cytoskeletal protein alpha smooth muscle actin (alphaSMA) in the stromal fibroblasts and is independent of the presence of the conceptus. The second phase of differentiation is characterized by the expression of insulin-like growth factor binding protein-1 (IGFBP-1) and the down-regulation of alphaSMA in the decidualized stromal fibroblast. The expression of IGFBP-1 is dependent on the presence of the conceptus in vivo and is regulated by hormones and cAMP in vitro. We have postulated that, during the initial phase of stromal cell differentiation, alphaSMA expression is regulated by the interaction between stromal cell integrins with the secreted extracellular matrix proteins (ECM). In response to pregnancy a trophoblast 'factor', mediated by cAMP signal transduction, induces IGFBP-1 expression in decidualizing stromal fibroblasts. This induction of IGFBP-1 is associated with the disappearance of alphaSMA and de-novo protein synthesis. Our comparative studies suggest that the process of decidualization in the human and baboon involve similar mechanisms. However, the metabolic pathways required for decidualization in the two species appear to differ in their degree of sensitivity to external stimuli. This review focuses on the cellular events that may potentially regulate decidualization in the primate and its role in regulating trophoblast migration.

This study was performed to investigate the hypothesis that insulin-like growth factor binding protein-1 (IGFBP-1) is involved in the pathogenesis of trophoblast invasion and impaired placentation in human pregnancy. The role of total and non-phosphorylated IGFBP-1 in women with fetal growth restriction and in high risk pregnancies identified by uterine artery Doppler ultrasound screening was examined. This was a prospective study of women booked for antenatal care having second trimester anomaly scans and Doppler screening between 22-26 weeks gestation. Women were divided into three groups and compared: normal uterine artery Doppler and normal fetal growth (control group, n = 10); abnormal Doppler and normal fetal growth [bilateral uterine artery notches (BN; n = 16); abnormal Doppler and intrauterine growth restriction (IUGR; n = 8)]. Maternal serum was collected, stored and assayed simultaneously for total and non-phosphorylated IGFBP-1. There was elevated total and non-phosphorylated IGFBP-1 (mean 44.99 +/- 12.19 and 29.61 +/- 10.38 microg/l respectively) in the IUGR group compared with controls (mean 17.96 +/- 3.24 and 12.18 +/- 1.55 microg/l, P < 0.05). This finding suggests that the various IGFBP-1 isoforms, the degree of phosphorylation and the ratios of these different forms locally may be important during trophoblast invasion and may be implicated in clinical manifestations of impaired placentation later in the second trimester.

Breast cancer is associated with endogenous hormone levels, but the exact relation and underlying mechanisms remain unclear. Data from several recent epidemiologic studies suggest that a woman who experiences preeclampsia in her own pregnancy, or who was herself born to a preeclamptic pregnancy, is at reduced risk for breast cancer later in life. This paper reviews the evidence for a connection between preeclampsia and breast cancer risk, and discusses the hormonal mechanisms that might explain this association. Preeclampsia is characterized by reduced levels of estrogens and insulin-like growth factor-1, and by elevated levels of progesterone, androgens, human chorionic gonadotropin, IGF-1 binding protein, corticotropin-releasing factor, cortisol, and insulin. These factors may act both individually and synergistically to decrease breast cancer risk. The occurrence of preeclampsia during a woman's pregnancy may reflect an underlying hormonal profile that both predisposes her to preeclampsia and reduces her risk for breast cancer. In addition, the major hormonal alterations associated with preeclampsia during gestation may have lasting effects on subsequent breast cancer risk. Finally, the hormonal and nutritional environment of the womb, for which preeclampsia is a marker, may play an important role in programming lifelong risk for breast cancer in the female offspring.

Insulin-like growth factors and their binding proteins are key regulators of fetal and maternal tissue growth and development during human pregnancy. Insulin-like growth factors, particularly IGF-II, are produced in abundance by the trophoblast cells of the placenta, whereas one of the insulin-like growth factor binding proteins, IGFBP-1, is the major secretory product of the maternal decidualized endometrium. This spatial (and temporal expression) of the insulin-like growth factor axis infers a sophisticated paracrine regulatory mechanism for controlling insulin-like growth factor function. This paper reviews the potential roles of IGFBP-1 in human pregnancy by examining its effects on growth, metabolism and migration at the maternal-fetal interface and how these might be influenced by autocrine-paracrine post-translational modifications.

Premature cranial suture fusion, or craniosynostosis, can result in gross aberrations of craniofacial growth. The biology underlying cranial suture fusion remains poorly understood. Previous studies of the Sprague-Dawley rat posterior frontal suture, which fuses at between 12 and 20 days, have suggested that the regional dura mater beneath the cranial suture directs the overlying suture's fusion. To address the dura-suture paracrine signaling that results in osteogenic differentiation and suture fusion, the authors investigated the possible role of insulin-like growth factors (IGF) I and II. The authors studied the temporal and spatial patterns of the expression of IGF-I and IGF-II mRNA and IGF-I peptide and osteocalcin (bone morphogenetic protein-4) protein in fusing posterior frontal rat sutures, and they compared them with patent coronal (control) sutures. Ten Sprague-Dawley rats were studied at the following time points: 16, 18, and 20 days of gestation and 2, 5, 10, 15, 20, 30, 50, and 80 days after birth (n = 110). Posterior frontal and coronal (patent, control) sutures were analyzed for IGF-I and IGF-II mRNA expression by in situ hybridization by using 35S-labeled IGF-I and IGF-II antisense riboprobes. Levels of IGF-I and IGF-II mRNA were quantified by counting the number of autoradiograph signals per cell. IGF-I and osteocalcin immunoreactivity were identified by avidin-biotin peroxidase immunohistochemistry. IGF-I and IGF-II mRNA were expressed in dural cells beneath fusing sutures, and the relative mRNA abundance increased between 2 and 10 days before initiation of fusion. Subsequently, IGF-I and IGF-II mRNA were detected in the suture connective tissue cells at 15 and 20 days during the time of active fusion. In contrast, within large osteoblasts of the osteogenic front, the expression of IGF-I and IGF-II mRNA was minimal. However, IGF-I peptide and osteocalcin protein were intensely immunoreactive within these osteoblasts at 15 days (during the period of suture fusion). These data suggest that the dura-suture interaction may be signaled in a paracrine fashion by dura-derived growth factors, such as IGF-I and IGF-II. These peptides, in turn, stimulate nearby osteoblasts to produce bone-promoting growth factors, such as osteocalcin.

Protein kinase B (PKB) is a member of the second messenger subfamily of protein kinases. The three isoforms of PKB identified have an amino-terminal pleckstrin homology domain, a central kinase domain, and a carboxy-terminal regulatory domain. PKB is the major downstream target of receptor tyrosine kinases that signal via the phosphoinositide (PI) 3-kinase. The crucial role of lipid second messengers in PKB activation has been dissected through the use of the PI 3-kinase-specific inhibitors wortmannin and LY294002. Receptor-activated PI 3-kinase synthesises the lipid second messenger PI-3,4,5-trisphosphate, leading to the recruitment of PKB to the membrane. Membrane attachment of PKB is mediated by its pleckstrin homology domain binding to PI-3,4,5-trisphosphate or PI-3,4-bisphosphate with high affinity. Activation of PKB alpha and beta is then achieved at the plasma membrane by phosphorylation of Thr308/309 in the A-loop of the kinase domain and Ser473/474 in the carboxy-terminal regulatory region, respectively. The upstream kinase that phosphorylates PKB on Thr308, termed PI-dependent protein kinase-1, has been identified and extensively characterised. A candidate for the Ser473/474 kinase, termed the integrin-linked kinase, has been identified recently. Activated PKB is implicated in glucose metabolism, transcriptional control, and in the regulation of apoptosis in many different cell types. Stimulation of PKB activity protects cells from apoptosis by phosphorylation and inactivation of the pro-apoptotic protein BAD. These results could explain why PKB is overexpressed in some ovarian, breast, and pancreatic carcinomas.

Background/aims: Partial hepatectomy results in activation of genes in the residual liver tissue which serve to restore glucose homeostasis and regenerate liver mass. Expression of insulin-like growth factor binding protein-1 (IGFBP-1) is up-regulated following partial hepatectomy and IGFBP-1 can modulate both the metabolic and mitogenic effects of insulin-like growth factor-1 (IGF-I). The aim of the study was to compare the effects of partial hepatectomy on blood glucose levels and hepatic regeneration in wild-type and transgenic mice which constitutively overexpress IGFBP-1., Methods: Hepatic IGFBP-1 mRNA, blood glucose concentrations, liver mass and hepatic DNA synthesis were compared in sham-operated and partially hepatectomized transgenic and wild-type mice., Results: Hepatic IGFBP-1 mRNA was higher in sham-operated transgenic than wild-type mice, but in both groups of mice, partial hepatectomy was associated with a significant rise in IGFBP-1 mRNA. The absolute decline in blood glucose levels following partial hepatectomy was greater in transgenic mice. Basal DNA synthesis and the response to IGF-I in isolated hepatocytes from both groups of mice were similar, and DNA synthesis in the regenerating liver in vivo was not significantly different in transgenic as compared to wild-type mice: 449.3 +/- 63.9 vs. 321.6 +/- 52.3 cpm/microgram DNA. Hepatic regeneration as measured by liver weight after hepatectomy was not different between transgenic and wild-type mice., Conclusions: Constitutive overexpression of IGFBP-1 does not enhance hepatic regeneration and does not prevent the decline in blood glucose following partial hepatectomy.

Insulin-like growth factor-I (IGF-I) plays an important role in the stimulation of postnatal brain growth. In transgenic (Tg) mice, IGF-I overexpression stimulates postnatal brain growth, whereas decreased IGF-I availability caused by ectopic brain expression of IGF binding protein-1 [(IGFBP-1), an inhibitor of IGF-I action] retards postnatal brain growth. Because undernutrition during early postnatal development profoundly retards growth and maturation of the brain in rodents, we sought to determine the influence of IGF-I on undernutrition-induced brain growth retardation. Caloric restriction was imposed on IGF-I Tg mice, IGFBP-1 Tg mice, and their non-Tg littermates by separating half of each litter from their dams during the suckling period, postnatal d 1 to 21. Undernutrition reduced the brain growth of each group of mice, but the growth of undernourished IGF-I Tg mice was comparable to that of well-fed control mice (increased 4.13- and 4.22-fold, respectively) and greater than that of undernourished control mice (increased 3.45-fold), whereas undernourished IGFBP-1 Tg mice exhibited less growth (increased 3.15-fold) than undernourished control mice. When the effects of undernutrition were examined in specific brain regions of each group, the same pattern was observed, and IGF-I was found to be more effective in preserving the growth of the regions with the highest transgene expression (cerebral cortex, hippocampus, and diencephalon). Despite undernutrition, IGF-I transgene expression stimulated overgrowth of these regions as well as that of the posterior medial barrel subfield, a somatosensory area of the cerebral cortex in which IGF-I may be especially important in development. These data indicate that IGF-I can ameliorate the brain growth retardation caused by undernutrition imposed during development, although it is unclear whether IGF-I directly opposes the impact of undernutrition or acts independently of nutritional status. Nonetheless, these findings raise the possibility that the relatively high IGF-I expression during early postnatal life may be responsible for sparing the brain from the full impact of undernutrition during this time in development.

Objective: The present study was undertaken to examine the association of a glucose-stimulated insulin response with the fasting insulin-like growth factor-binding protein (IGFBP)-1 concentration in prepubertal obese children., Subjects and Methods: The fasting levels of serum insulin and IGFBP-1 were measured in 17 obese and 16 control children. Furthermore, we performed an oral glucose tolerance test in obese children and examined the association of the area under the curve (AUC) for insulin with the fasting IGFBP-1 level., Results: The mean serum level of IGFBP-1 was significantly lower in obese children (41.0 +/- 4.8 micrograms/l. P < 0.005) than in controls (91.2 +/- 9.9 micrograms/l). Although there was an inverse relationship between the fasting levels of serum insulin and IGFBP-1 in all subjects (r = -0.42, P < 0.05), no significant correlation between these two parameters was observed in the obese group alone. In obese children, the fasting IGFBP-1 level correlated inversely with AUC-insulin (r = -0.70, P < 0.005), whereas there was no significant relationship between the fasting insulin level and AUC-insulin., Conclusion: The present study suggests that the serum level of IGFBP-1 may be an early predictor of insulin resistance in prepubertal obesity.

The insulinlike growth factor (IGF) family is believed to be important in endometrial development during the menstrual cycle and in the process of implantation. The mitogenic, differentiative, and antiapoptotic properties of the IGFs and their binding proteins, as well as their spatial and temporal expression in cycling endometrium, suggest that they may participate in endometrial growth, differentiation, apoptosis, and perhaps angiogenesis. IGFBP proteases, which increase IGF bioavailability, have been localized to endometrial stromal cells and to the human cytotrophoblast and likely play important roles in endometrial, decidual, and trophoblast physiology. IGFBP-1 is a major protein product of nonpregnant endometrium during the mid-late secretory phase and occurs in abundance in decidua. Its roles as an IGF-binding protein and as a trophoblast integrin ligand suggest that it may have multiple roles in endometrial development and in interactions between the decidua and the invading trophoblast. Recent evidence suggests that it may have a role in the process of shallow implantation in the clinical disorder of preclampsia. In contrast to knowledge about the roles of IGF peptides, IGFBP proteases, and IGFBPs in normal endometrial development and early human pregnancy, little information is available regarding this family in abnormal endometrial development, in occult endometrial defects, and in uterine receptivity and nonreceptivity.

Trophoblast growth and invasion of the uterus are tightly regulated by locally produced factors. Since insulin-like growth factor (IGF)-II is produced by the invasive human extravillous trophoblast (EVT) cells and IGF-binding protein (IGFBP)-1 by the decidual cells in situ that are in proximity to each other, we examined the possible influence of these molecules on proliferation, migration, and invasiveness of first-trimester EVT cells in culture. These EVT cell functions were respectively measured by 3H-TdR uptake, in vitro migration, and invasion assays. Secretion of invasion-associated enzymes was assessed by zymography, and IGF-binding moieties on the EVT cell were examined by affinity cross-linking. Proliferation of serum-starved EVT cells was stimulated by addition of serum but unaffected by a wide range of IGF-I, IGF-II, and IGFBP-1 concentrations. IGF-II and IGFBP-1 or their combination stimulated EVT cell invasiveness and migration, when assays were conducted in serum-reduced media. Affinity cross-linking studies failed to detect the type 1 IGF receptor, although several IGF-II-specific and IGF-II-preferring binding molecules including type 2 IGF receptor were identified on the EVT cell surface. IGF-II enhancement of invasion was unaffected in the presence of IGF-1 receptor-blocking antibody and IGF-1 failed to influence EVT cell invasion, indicating that type 1 IGF receptor was not responsible for the IGF-II effects. Secretion of gelatinases or plasminogen activators was unaltered by IGF-II or IGFBP-1. We conclude that trophoblast-derived IGF-II and decidua-derived IGFBP-1 provide autocrine/paracrine enhancement of trophoblast invasiveness largely by stimulating migration, an essential step in invasion., (Copyright 1998 Academic Press.)

Thirty-seven children with intrauterine growth retardation (IUGR) were enrolled in a 3-mo longitudinal study. Weight, length, and knee-heel length (by knemometry) were measured at birth and at 7, 14, 30, 60, and 90 d. GH, IGF-I, IGF binding protein (BP)-3, IGFBP-1, and C-peptide were measured at birth and at 2 mo. IGFBP-3 Western immunoblotting and proteolytic activity assay were also performed. Twenty-five newborns with birth weight appropriate for gestational age were chosen as controls. At birth IUGR newborns showed levels of GH and IGFBP-1 significantly higher, and IGF-I, IGFBP-3, and C-peptide significantly lower than control subjects. At 2 mo GH and IGFBP-1 levels decreased, whereas IGF-I, IGFBP-3, and C-peptide rose, attaining the concentrations found in control subjects at birth. Baseline peptide levels as well as their 2-mo variations did not correlate with the gain in weight, supine length, and knee-heel length recorded at 3 mo. Fourteen of nineteen IUGR cord blood samples showed the presence of the intact approximately 42-39-kD IGFBP-3 doublet and the major approximately 29-kD fragment. At 2 mo the IGFBP-3 band pattern was characterized by the predominance of a approximately 18-kD fragment in 6 of 19 tested IUGR infants. The incubation of 2-mo IUGR samples with normal adult serum induced the appearance of the approximately 18-kD band, which was not modified by the addition of EDTA. These results suggest that: 1) the IGF-related growth-promoting mechanism is impaired in IUGR children at birth but is fully restored at 2 mo; 2) the cord blood levels of GH, IGF-I, IGFBP-3, IGFBP-1, and C-peptide are not predictive of the weight and length gain during the first 3 mo of life; 3) IUGR children have at least two different IGFBP-3 proteases, one cation-dependent protease that is present at birth and able to yield the major approximately 29-kD IGFBP-3 fragment and a second one, with a different activation timing, which exhibits cation independence and induces the formation of a approximately 18-kD IGFBP-3 form.

Insulin-like growth factors (IGFs) I and II stimulate growth and expression of specific genes through binding to cell membrane receptors. IGF binding proteins also bind IGF-I with higher affinity than the receptor. They are found in the circulation and tissues and can modulate IGF actions. Human IGFBP-1 is phosphorylated on serine residues, which increases its affinity for IGF-I. An acidic, presumably phosphorylated, form of human IGFBP-1 inhibits IGF-I-stimulated DNA synthesis in cultured cells, while a less acidic, unphosphorylated form potentiates this function. Phosphorylation of human IGFBP-3, however, does not affect its affinity for IGF-I. Previously we found that multiple forms of rat IGFBP-1 are obtained by anion-exchange chromatography, raising the possibility that it also is phosphorylated, which led us to examine its properties. Phosphopeptide analysis of 32P-labeled, immunoprecipitated rat IGFBP-1 synthesized by H-4-II-EC3 rat hepatoma cells indicated that it is phosphorylated on two sites that were deduced to be ser107 and ser132 in the central nonconserved domain. Dephosphorylation of purified phosphorylated rat IGFBP-1 did not affect its affinity for IGF-I or its specific binding activity, and the dephosphorylated form inhibited DNA synthesis in 3T3 cells. Incubation of cells labeled with radioactive proline in the presence of monensin and brefeldin A, which inhibit secretion at different sites, led to intracellular accumulation of the least phosphorylated form of rat IGFBP-1, but prevented further phosphorylation. The results suggested that phosphorylation occurs at two sites in cells, the cis-Golgi and the trans-Golgi network. In summary, these studies have shown that rat IGFBP-1 is phosphorylated on two sites by reactions that occur in different secretory organelles and that similar to human IGFBP-3, but unlike human IGFBP-1, phosphorylation does not affect its affinity for IGF-I., (Copyright 1998 Academic Press.)

Purpose: Our purpose was to analyze potential interactions between the embryo and the maternal endometrial interface in vivo by analyzing immunolocalization of insulin-like growth factor-binding proteins (IGFBPs) -1, -2, and -3 in implantation sites of the mouse., Methods: Six-week-old B6D2F1 female mice underwent superovulation followed by mating and sacrifice at timed intervals. Formalin-fixed paraffin-embedded tissue was used for avidin-biotin immunocytochemical localization of IGFBPs utilizing standard methodology., Results: Immunostaining at 1.5 days post coitum revealed light staining in the epithelial glandular cells and faint staining in decidual stroma for both IGFBP-1 and IGFBP-2. At 7.5-10.5 days post coitum, there was moderate-dense immunostaining in the decidualized stromal cells at the implantation site for all three IGFBPs, whereas light immunostaining was seen in nonimplantation site decidua., Conclusions: Compartmentalization of immunostaining for IGFBP-1, -2, and -3 within decidualized stroma suggests that these proteins may be regulated by trophoblastic and/or embryonic signals.

IGFBP-1 is elevated in fetuses with long-term, chronic hypoxia and intrauterine growth restriction. We investigated the hypothesis that hypoxia regulates IGFBP-1 in the human fetus in vivo and IGFBP-1 gene expression and protein in vitro. Umbilical artery IGFBP-1 levels (mean +/- SEM) from term babies with respiratory acidosis (acute hypoxia), normal babies, and those with mixed respiratory/metabolic acidosis (more profound and prolonged hypoxia) were measured using an immunoradiometric assay. IGFBP-1 levels were similar in normal (n = 12) and acutely hypoxic (n = 6) babies (189.1 +/- 71.8 vs. 175.8 +/- 45.9 ng /ml, respectively, P = 0.789). However, with more profound and prolonged hypoxia (n = 19), IGFBP-1 levels were markedly elevated (470.6 +/- 80.0 ng /ml, P = 0.044). To investigate IGFBP-1 regulation by hypoxia in vitro, HepG2 cells were incubated under hypoxia (pO2 = 2%) and normoxia (pO2 = 20%). IGFBP-1 protein and mRNA increased 8- and 12-fold, respectively, under hypoxic conditions. Hypoxia did not affect protein or mRNA levels of IGFBP-2 or -4. IGFBP-5 and -6 mRNAs, undetectable in control cells, were not induced by hypoxia, whereas minimally expressed IGFBP-3 mRNA increased twofold. Investigation into IGFBP-1 gene structure revealed three potential consensus sequences for the hypoxia response element (HRE) in the first intron. To investigate functionality, a 372-bp fragment of IGFBP-1 intron 1, containing putative HREs, was placed 5' to a heterologous hsp70 promoter in a plasmid using luciferase as a reporter gene. Under hypoxia, reporter gene activity increased up to 30-fold. Mutations in the middle HRE abolished reporter activity in response to hypoxia, suggesting that this HRE is functional in the IGFBP-1 hypoxia response. Cotransfection of HRE reporter genes with a constitutively expressing hypoxia-inducible factor 1 plasmid in HepG2 cells resulted in a fourfold induction of reporter activity, suggesting a role for hypoxia-inducible factor 1 in hypoxia induction of IGFBP-1 gene expression. These data support the hypothesis that hypoxia regulation of IGFBP-1 may be a mechanism operating in the human fetus to restrict insulin-like growth factor-mediated growth in utero under conditions of chronic hypoxia and limited substrate availability.

In order to investigate the regulatory role of only one endometrial cell type on trophoblastic invasion, we explored the effects of culture medium conditioned by in vitro decidualised stromal cells (DCM) and of insulin-like growth factor binding protein-1 (IGFBP-1, the main secretory product of decidual cells) on the trophoblastic secretion of gelatinases and tissue inhibitor of metalloproteinases (TIMP-1). First trimester cytotrophoblastic cells (CTB) were obtained from abortions and cultured in vitro in presence or absence of DCM or IGFBP-1. Secreted gelatinases were analysed in the culture supernatants by zymography and by measurements of the total gelatinolytic activity. Tissue inhibitor of metalloproteinases (TIMP-1) was measured by a commercially available immunoassay. DCM inhibited the total gelatinolytic activity of CTB but increased trophoblastic MMP-9 and TIMP-1. In contrast, IGFBP-1 increased the total gelatinolytic activity and TIMP-1 and had no effect on MMP-2 and MMP-9. We conclude that a factor secreted by decidual cells (possibly TGFbeta) inhibits the total gelatinolytic activity of trophoblast by increasing TIMP-1 but other factors, as yet unidentified, increase MMP-2 and MMP-9 to an extent which does not shift the equilibrium between the gelatinases and TIMP-1 in favour of the gelatinases. In contrast to DCM, IGFBP-1 increases the total gelatinolytic activity probably by stimulating another gelatinase (stromelysin-1?) as MMP-2 and MMP 9 are unchanged by IGFBP-1. The possibility of an integrin mediated effect of IGFBP-1 on CTB is discussed.

Insulin-like growth factors, IGF-I, IGF-II, and IGF binding protein (IGFBP-1) appear to play major roles in endometrial development during the menstrual cycle and in the process of implantation. The mitogenic, differentiative, and anti-apoptotic properties of these growth factors, as well as their spatial and temporal expression in cycling endometrium, suggest that they may participate in endometrial growth, differentiation, inhibition of apoptosis, and perhaps angiogenesis. IGFBP-1 is a major protein product of non-pregnant endometrium during the mid-late secretory phase and occurs in abundance in decidua. Its roles as an IGF-binding protein and as a trophoblast integrin ligand suggest that it may have multiple roles in endometrial development and in interactions between the decidua and the invading trophoblast. Precise elucidation of the mechanisms underlying IGF and IGFBP-1 action at the decidual-trophoblast interface in early pregnancy awaits further investigation. The future also awaits elucidation of the potential predictive utility of IGFBP-1 in serum and in decidua in, for example, pre-eclampsia and perhaps implantation failure.

Insulin-like growth factor (IGF)-I is a potent anabolic agent that plays an important role in regulating muscle protein balance. Alterations in one or more of the various components of the IGF system may be in part responsible for the muscle wasting that accompanies chronic alcohol consumption. The purpose of the present study was to characterize changes in the growth hormone-IGF axis produced by chronic alcohol consumption in rats. After 8 weeks of alcohol feeding, the IGF-I concentration was decreased in plasma (31%) as well as in the liver and skeletal muscle (40-50%), compared with pair-fed control animals. In addition, alcohol consumption decreased IGF-I mRNA abundance in liver and muscle (approximately 50%). IGF-I content in duodenum and kidney, however, was not altered by alcohol feeding. Concomitantly, the relative concentration of IGF binding protein (IGFBP)-1 was increased in plasma, liver, and muscle of alcohol-fed rats, compared with control values. In contrast, no changes in the plasma concentrations of IGFBP-2, -3, or -4 were detected in alcohol-fed rats at this time point Previous studies have indicated that elevations in glucocorticoids or decreases in insulin or growth hormone might be responsible for the decrease in IGF-I and/or the increase in IGFBP-1 in other catabolic conditions. However, there was no difference in the plasma concentrations of these hormones between alcohol-fed and control animals in this study. These data indicate that chronic alcohol feeding in rats decreases IGF-I and increases IGFBP-1 in the circulation and in skeletal muscle and that these changes appear to be independent of changes in classical hormonal regulators of the IGF system. The observed alterations in the IGF system are consistent with a reduction in the anabolic actions of IGF-I induced by chronic alcohol consumption.

Insulin-like growth factor binding protein-1 (IGFBP-1) can bind to the alpha5beta1 integrin and stimulate cellular migration in Chinese hamster ovary (CHO) cells. IGFBP-1 is a major product of the endometrium of pregnancy (decidua), and may interact with invading cytotrophoblasts expressing alpha5beta1 integrin to modulate their invasion. The present study investigated IGFBP-1 interaction with cytotrophoblast alpha5beta1 integrin, and its effects on trophoblast attachment to fibronectin and invasion into decidualized endometrial stromal cell multilayers. IGFBP-1 incubated with cytotrophoblast extracts was co-precipitated by an antibody to the alpha5 integrin subunit. Up to 55% of radiolabeled IGFBP-1 bound to cytotrophoblasts was displaced by excess non-radioactive IGFBP-1, but not by IGFBP-3. Cytotrophoblast attachment to fibronectin was inhibited by an RGD-containing octapeptide, by antibodies to the alpha5 subunit or the alpha5beta1 heterodimer, and by IGFBP-1. Cytotrophoblasts showed limited invasion into endometrial stromal multilayers decidualized in vitro secreting abundant IGFBP-1, but invaded multilayers when IGFBP-1 production was inhibited by insulin. Invasion into insulin-treated multilayers was prevented by addition of exogenous IGFBP-1 but not by IGFBP-3. These findings suggest IGFBP-1 may modulate trophoblast invasiveness.

Insulin-like growth factor binding protein 1 is the predominant insulin-like growth factor binding protein in amniotic fluid. It is produced by the decidua and by fetal tissues, and it is thought to play an important role in fetal growth. We have measured this protein in 58 samples of amniotic fluid, from 13 to 19 gestational weeks, and found a highly significant negative correlation with fetal weight at birth. We conclude that the level of insulin-like growth factor binding protein 1 in amniotic fluid at midpregnancy is a good marker of fetal growth failure.

In 1988, insulin-like growth factor-binding protein-1 (IGFBP-1) became the first characterized member of a group of structurally related soluble proteins which specifically bind and modulate the actions of the IGFs. Since then, a wealth of information has accumulated regarding the physiology of this dynamic serum protein. In this review, we update our 1993 summary (Lee PDK et al. Proc Soc Exp Biol Med 204:4-29) of the status of IGFBP-1 research. The IGFBP-1 protein sequence contains 12 N-terminal and 6 C-terminal cysteine residues which are conserved in other mammalian IGFBP-1 sequences and amongst other IGFBPs; both of the cysteine-rich regions are required for optimal IGF binding. The nonconserved IGFBP-1 midregion may act as both a hinge which defines ligand binding characteristics and as a specific target for protease activity. Integrin-binding and phosphorylation sites within the IGFBP-1 sequence have functional significance in vitro, but their physiologic relevance in vivo have not been defined. The human IGFBP-1 and IGFBP-3 genes are contiguous and located in close proximity to the homeobox A (HOXA) gene cluster on chromosome 7. The other IGFBP genes, located on chromosomes 2, 12, and 17, are also associated with HOX clusters, suggesting evolutionary linkage of the IGFBP and HOX gene families. Similarities between the hIGFBP-1 and phosphoenolpyruvate kinase (PEPCK) promoters, including regions conferring insulin, glucocorticoid, and cyclic adenosine-monophosphate responses, are consistent with our previous hypothesis that IGFBP-1 is involved in regulation of glucose metabolism. The tissue-specific patterns of IGFBP-1 gene expression in liver, kidney, decidua, and ovary may be due to stimulation of IGFBP-1 transcription by hepatic nuclear factor 1 (HNF1) proteins. Clinical and basic studies of IGFBP-1 physiology have been aided by several recently developed assay methods. Numerous investigations have confirmed that insulin, via inhibition of IGFBP-1 transcription, is the primary determinant of IGFBP-1 expression both in vitro and in vivo. IGF-I and IGF-II also have specific inhibitory effects on IGFBP-1 expression. Glucocorticoids and cAMP stimulate IGFBP-1 transcription, but these effects are observed only in conditions of low or absent insulin effect. Other stimulants of IGFBP-1 expression include thyroid hormones and epidermal growth factor. Phorbol ester stimulation of IGFBP-1 expression can supersede the effects of insulin in vitro;however, the mechanism and in vivo correlates of this effect have not been determined. Cytokines and, perhaps, growth hormones may affect IGFBP-1 expression, perhaps by altering the regulatory actions of insulin; this effect may have important clinical relevance. IGFBP-1 expression is upregulated in liver and (nonhuman) kidney during postinjury regeneration. The IGF-inhibitory actions of IGFBP-1 has been confirmed by numerous in vitro studies and several in vivo animal investigations, including administration of recombinant IGFBP-1 and IGFBP-1 transgenic models. IGFBP-1 has been shown to inhibit somatic linear growth, weight gain, tissue growth, and glucose metabolism. Moreover, IGFBP-1 appears to be a primary determinant of free IGF-I levels in serum. Excess levels of IGFBP-1 may contribute to growth failure in intrauterine growth restriction and in pediatric chronic renal failure, while low IGFBP-1 levels are associated with obesity and with cardiovascular risk factors in insulin resistance syndromes. Serum IGFBP-1 measurements may be useful biochemical marker in these pathologic conditions. IGFBP-1 is expressed in decidualized stromal cells of the uterine endometrium and in ovarian granulosa cells. IGFBP-1, together with IGFs, insulin, ovarian steroids, cytokines, and other factors, is involved in a complex system which regulates menstrual cycles, ovulation, decidualization, blastocyst implantation, and fetal growth. (ABSTRACT TRUNCATED)

In this report, comparisons between molecular affinities and cellular proliferation activities have been made for insulin-like growth factor-I (IGF-I) and two IGF-I fusion proteins in order to evaluate fusion proteins as tools for receptor binding studies. Binding affinities and growth promoting effects of the N-terminal fusion Z-IGF-I and the C-terminal fusion IGF-I-Z, and native recombinant human IGF-I, were analyzed. Binding kinetic properties of the three IGF-I variants were analyzed using BIAcore kinetic interaction analysis testing for binding to both human IGF binding protein 1 (IGFBP-1) and a soluble form of the human IGF type I receptor extracellular domains (sIGF-IR). The growth promoting effects on SaOS-2 human osteosarcoma cells of the different fusion proteins were analyzed. A comparison of receptor binding affinities and growth promoting effects shows that the fusion protein receptor affinity does not correlate with proliferative potential. The IGF-I-Z fusion, with the lowest receptor affinity, shows similar proliferative potential to native IGF-I. However, the Z-IGF-I fusion protein, with twice the receptor affinity of IGF-I-Z, displays only about 70% of the IGF-I-Z growth promoting activity. Both IGF-I fusion proteins possess similar affinity to IGFBP-1. These results indicate that determinants other than the receptor affinity could be involved in the regulation of IGF-I proliferative action. This study demonstrates that ligand fusion proteins may be useful to study mechanisms of ligand induced receptor activation.

IGFBP-1 is a major protein product of nonpregnant endometrium during the mid-late secretory phase and occurs in abundance in decidua. Its roles as an IGF-binding protein and as a trophoblast integrin ligand suggest that it may have multiple roles in endometrial development and in interactions between the decidua and the invading trophoblast. IGFBP-1 in vaginal/cervical secretions has already had clinical application as a predictor of premature rupture of fetal membranes. The future awaits elucidation of the potential utility of IGFBP-1 in serum and in decidua in predicting fetal growth restriction and preeclampsia and perhaps implantation failure.

Insulin-like growth factors (IGF-I, IGF-II) are important regulators of cell division and differentiation. In their free form, IGFs form a complex with specific binding proteins (IGFBPs), six of which have now been characterized. These IGFBPs differ in their ability to bind IGF-I and/or IGF-II, and, depending on their location and metabolic circumstances, they inhibit or augment IGF action to varying extents. New findings have changed our understanding of IGFBPs, which are now considered to be major regulators of IGF action and IGF transport proteins between compartments. Recently, the IGF-independent action of IGFBPs was discovered. Levels of IGFBP-3, for instance, are important indicators of states of altered growth hormone secretion and action, as well as being important during treatment with growth hormone or IGF-I. IGFBP-1 and -2 levels reflect changes related to nutrition, insulin secretion, foetal development and malignant state. The unravelling of the intricate interactions of IGFs, IGFBPs and their targets is bound to influence our understanding of the physiology and development of biological systems. Likewise, the possibility of regulating the IGFBP system is likely to open up new fields in the treatment of diseases in the future.

Transgenic mice that overexpress insulin-like growth factor-binding protein-1 (IGFBP-1) demonstrate a reduced litter size compared to nontransgenic, wild-type mice derived from the same genetic background. To determine the mechanism underlying this phenomenon, we examined the number of ovulatory follicles per cycle in naturally mated transgenic and wild-type mice by counting the corpora lutea and the blastocysts harvested by uterine flushing. In addition, we investigated the effects of insulin-like growth factor-I (IGF-I) on blastocyst DNA synthesis and examined the effects of a transgenic maternal environment on fetal outcome by cross transfer of blastocysts. Significantly fewer corpora lutea were observed in ovaries from transgenic vs. wild-type mice (7.6 +/- 1.8 vs. 11.8 +/- 1.5), and fewer blastocysts were harvested from transgenic mice compared to wild-type mice (7.6 +/- 2.3 vs. 10.1 +/- 2.0). DNA content and basal DNA synthesis were similar in blastocysts from nontransgenic and transgenic mice. However, unlike wild-type blastocysts, transgenic blastocysts did not respond to IGF-I with an increase in DNA synthesis. To determine the effects of maternal environment on fetal outcome, a mixture of equal numbers of transgenic and nontransgenic blastocysts was transferred into foster mothers. The ratio of transgenic to nontransgenic pups was not significantly different from the theoretically predicted value of 1. However, the litter size was significantly reduced in wild-type compared to transgenic foster mothers. These data suggest that the reduced fecundity is due to reduced ovulation and blastocyst number. Furthermore, expression of the transgene in neither the blastocyst nor the maternal tissues had any significant negative effect on implantation or fetal wastage.

Transgenic mice that overexpressed IGFBP-1 are hyperinsulinemic in the first week of life and gradually develop fasting hyperglycemia. In adult transgenic mice, the hypoglycemic response to IGF-I but not insulin or des (1-3) IGF-I was attenuated (P < 0.05) compared with wild-type mice. Furthermore, in isolated adipocytes from transgenic mice, the stimulatory effect of IGF-I but not insulin on 2-deoxy-[3H]-glucose uptake was reduced (P < 0.02). In contrast, in isolated soleus muscle, the effects of both IGF-I and insulin on 2-deoxy-3H-glucose uptake and on [3H]-glucose incorporation into glycogen were significantly reduced compared to wild-type mice. The decline in specific activity of the 2-deoxy-3H-glucose, a measure of glucose appearance in the circulation, was more marked in transgenic animals (P < 0.05). In addition, tissue uptake of glucose was significantly higher in diaphragm, heart, intestine, liver, soleus muscle, and adipose tissue from fasting transgenic mice. Plasma concentrations of alanine, lysine, and methionine were also elevated in transgenic mice. These data suggest that overexpression of IGFBP-1 attenuates the hypoglycemic effect of endogenous IGF-I, which is initially compensated for by enhanced pancreatic insulin production. However, in adult mice pancreatic insulin content is reduced, insulin resistance is demonstrable in skeletal muscle and fasting hyperglycemia develops.

The IGFs are essential stimulators of fetal and postnatal growth. Their signals are mediated predominantly by the type I IGF receptor, but another as yet unidentified receptor seems to mediate some actions, especially some of those for IGF-II. During organogenesis, the actions of IGF-I seem to be local (autocrine/paracrine), because it, as well as IGFBPs and IGF receptors, are expressed coordinately in a highly tissue and developmentally specific fashion. IGF-I, however, most likely assumes endocrine functions during postnatal life and provides signals for GH and nutritional status. On the other hand, IGF-II may have only local growth-promoting functions, and its most essential actions may be restricted to in utero life. Both IGFs may be involved in regenerative organ growth, and their uncontrolled expression, especially that of IGF-II, may be involved in the formation of some tumors. The previous conclusions are drawn mainly from in vitro and animal experiments. Although they are generally consistent with data in humans, it is most likely incorrect to extrapolate to man all of the conclusions drawn from the experiments in mice. Differences in placentation are so marked between mice and humans that an exclusive role for IGF-II in human placental growth seems unlikely. On the other hand, it is reasonable to conclude that both IGFs are important stimulants of normal human fetal growth, that IGF-I is a more potent stimulator of human postnatal growth, and that the type I IGF receptor mediates most IGF actions in humans. The fact that no human growth disorders have been linked convincingly to lack of function mutations in IGF or IGF receptor genes suggests that such defects are lethal.

Over the past few years, there has been an explosion of data in the scientific literature regarding the various components of the IGF axis. IGFBPs and related molecules are now believed to be critical elements in numerous cellular processes and key factors in several disease states related to abnormal tissue and somatic growth. Recently, the BP-Prs were included in this complex system, and their importance is being unraveled. The upcoming years will undoubtedly bring even more information on the molecular biology of these key cellular regulators. These discoveries are likely to lead to better understanding of growth and cellular regulation and to development of novel therapeutic approaches to a variety of diseases.

Co-culturing embryos on helper cells can mimic the in-vivo environment, thereby enhancing embryo development in vitro. Insulin-like growth factors (IGF) and their binding proteins (IGFBP) also enhance embryo development. To investigate the kinds of IGFBP produced by various cell monolayers and the effects of IGFBP-3 on mouse embryo co-culture systems, 2-cell ICR mouse embryos were cultured in either human tubal fluid medium alone or in the presence of Vero cells, human oviductal cells or endometrial cells. The helper cells were analysed immunohistochemically to investigate the types of IGFBP produced by various cell monolayers. The concentrations of IGF-I and IGFBP-3 in media obtained from the culture of embryos alone, cells alone or cells plus embryos were determined by radioimmunoassays. On day 7, more blastocysts hatched in the co-culture groups (73% in the Vero cell group, 76% in the endometrial cell group and 74% in the oviductal cell group) than in the control group (43%) (P < 0.0001). The results of immunohistochemistry revealed that (i) all three cell groups produced a lot of IGFBP-1, -2 and -3, but only a little of IGFBP-4 and -5; and (ii) IGFBP-1, -2, and -3 were present in blastocysts in either the presence or absence of helper cells. The IGF-I secreted by cell monolayers or embryos was undetectable (detection limit 0.83 microg/l). The IGFBP-3 concentrations in media obtained from co-cultured embryos and cells were significantly higher than in media without embryos (median values in oviductal cell culture medium, 165 versus 127 microg/l, P = 0.04; median values in endometrial cell culture medium, 277.5 versus 183.5 microg/1, P = 0. 0002; median values in Vero cell culture medium, 219 versus 120 microg/l, P = 0.011). Although IGFBP-3 concentration in the medium that contained embryos alone was undetectable by radioimmunoassay (detection limit 1.1 microg/l), immunohistochemistry demonstrated the presence of IGFBP-3 in the embryos. Co-culture in systems in which there was an increased production of IGFBP-3 led to an improved development of mouse embryos. IGFBP can improve the binding of IGF to cell surface receptors of target tissue, and thus enhance the effect of limited IGF concentrations in promoting embryo development in a co-culture system. We conclude that Vero cells, human endometrial cells and oviductal cells produce IGFBP-1, -2, -3, -4 and -5. IGFBP-3 may play a role in embryotrophic potential by either regulating the action of IGF or directly enhancing embryo development.

A sensitive RIA was used to examine regulation of IGFBP-1 in H4IIE rat hepatoma cells. IGFBP-1 was stimulated up to tenfold by dexamethasone and corticosterone, and this stimulation was abolished by RU486. The effect of dexamethasone increased with time in culture. Phorbol 12-myristate 13-acetate (PMA) stimulated IGFBP-1 up to fourfold with a maximal effect in short-term culture. Dexamethasone and PMA were additive in stimulating IGFBP-1. Under basal conditions IGFBP-1 production was linearly related to cell density: however, stimulation by dexamethasone was greatest in confluent cells, and PMA had a greater effect in sparse cultures. Insulin inhibited IGFBP-1 up to 80%, and this effect diminished with time in culture but was unaffected by cell density. Dexamethasone was stimulatory in the presence of a maximal inhibitory concentration of insulin, and insulin was inhibitory in the presence of maximal dexamethasone from 3-48 h in culture, regardless of cell density. PMA abolished the inhibitory action of insulin on IGFBP-1 secretion and mRNA expression during incubation periods of less than 4 h and not during longer incubations. PMA did not influence the stability of IGFBP-1 mRNA. We conclude that, in rat H4IIE cells, dexamethasone and PMA stimulate IGFBP-1 by independent mechanisms and speculate that when protein kinase C is activated the inhibitory action of insulin is blocked.

Circulating insulin-like growth factors (IGFs) represent an important pool of potential hypoglycemic activity, which is largely inhibited by their sequestration in a heterotrimeric complex comprising growth factor, IGF-binding protein-3 (IGFBP-3), and acid-labile subunit (ALS). Less than 1% of total IGFs circulate in the free form, yet even this amount might contribute significantly to circulating insulin-like activity. The ternary binding protein complex appears to inhibit insulin-like activity of bound IGFs by preventing their egress from the circulation. Although the integrity of this complex might be affected by limited proteolysis of IGFBP-3 in pregnancy and catabolic conditions, the evidence that this increases IGF bioavailability, and thus hypoglycemic potential, is as yet unclear. However, in patients with IGF-II-secreting tumors, hypoglycemia may result from a failure of the ternary complex to adequately sequester the IGFs. Improvement in complex formation, by treatment with corticosteroids or growth hormone, alleviates the hypoglycemia, even if (as seen with growth hormone treatment) IGF-II hypersecretion persists. In these patients, blood glucose levels are inversely correlated with IGFBP-2 levels, suggesting that this protein might play a part in transporting IGFs to their target tissues. Conversely, ALS levels correlate positively with blood glucose, emphasizing the importance of the ternary complex in preventing hypoglycemia. Unlike the other IGF-binding proteins, IGFBP-1 is acutely regulated in the circulation, in a manner consistent with its acting as a glucose counterregulator. It might act in this way by inhibiting the activity of free IGFs in the circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

IGFBP-1 secretion in humans is regulated by insulin and the counter-regulatory hormones with a high production rate and rapid turnover. The IGFBP-1 fasting levels are both genetically and environmentally determined. Serum IGFBP-1 levels may be regulated by so far unknown factors during certain conditions. The fasting IGFBP-1 level can be used as a marker of diurnal IGFBP-1 and insulin secretion.

Three hemizygous transgenic (Tg) mouse lines were generated with a fusion gene composed of the mouse metallothionein promoter (mMT-1) and a full length human insulin-like growth factor binding protein-1 (hIGFBP-1) cDNA that was truncated in its 3' untranslated (3'UT) region. The transgene was ectopically expressed in the brain of each line and resulted in postnatal brain-growth retardation that was manifested by 2 weeks of age. Despite the expression of the transgene in multiple other tissues and high serum hIGFBP-1 concentrations in two of the three lines, studies designed to detect alterations in somatic growth, in reproduction and in glucose metabolism revealed few other abnormalities. Unexpectedly, however, we found that the regulation of the transgene shared characteristics with that of the native gene, despite the fact that it lacked the endogenous gene's 5' regulatory region, as well as most of its 3' UT region. Our studies suggest that factors controlling mRNA stability are important to regulation of both the native and transgene, and that an AU-rich element 17 base pairs (bp) from the end of coding sequence is responsible for the instability of the transgene and in part for instability of the endogenous gene.

To provide further insight into the function of the IGFBPs, transgenic (Tg) mice which overexpressed IGFBP-1 and IGFBP-3 were generated. In this report we have compared the phenotypic manifestations observed in these Tg mice. The IGFBP-1 Tg mice were significantly smaller at birth, birth weight and gained less weight in the postnatal period. Organ weight was proportionately reduced relative to body weight in most organs. However the brain was markedly smaller in IGFBP-1 Tg mice. Mean plasma levels of Tg-derived IGFBP-1 ranged from 8 to 80 ng ml-1 in the different groups of IGFBP-1 Tg mice. In addition homozygous mice also demonstrated fasting hyperglycemia, impaired glucose tolerance and reduced fecundity. Two of the seven IGFBP-3 founders had measurable levels of hlGFBP-3 in the circulation and were bred to homozygosity. Maximal plasma levels of transgene-derived IGFBP-3 were 72-198 ng ml-1. Transgene expression was detected in the kidney, small intestine and colon by Northern blot analysis. The birth weight, litter size and body weight of IGFBP-3 Tg mice were not significantly different from wild-type mice. However, the spleen, liver and heart of IGFBP-3 Tg mice derived from both founders were significantly heavier compared with organs from wild-type mice. The relative weight of other organs such as the brain, kidney and lungs were similar to wild-type mice. From these data, we conclude that over expression of IGFBP-1 results in inhibition of IGF action and in profound impairment of brain development, modest inhibition of fetal and postnatal growth and inhibition of the metabolic effects of the IGFs. In contrast, modest over-expression of hlGFBP-3 has little effect other than some selective organomegaly.