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

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

Heterogeneity of insulin-like growth factor-I affinity for the insulin-like growth factor-II receptor: comparison of natural, synthetic and recombinant DNA-derived insulin-like growth factor-I

Although insulin-like growth factors (IGF) I and II bind with high affinity to structurally discrete receptors, they bind with a lesser affinity to each other's receptor. We have evaluated the affinity of five different IGF-I preparations (three natural IGF-I preparations, one synthetic preparation, and one recombinant DNA-derived) for the IGF-II receptor in rat placental membranes, 18-54,SF cells and BRL-3A cells. In all tissues tested, the natural IGF-I preparations demonstrated an affinity for the IGF-II receptor which was 10-20% that of IGF-II. However, the recombinant and synthetic IGF-I preparations exhibited substantially lower affinities than natural IGF-I for this receptor, with only 10-25% reduction in (125-I)iodo IGF-II binding at peptide concentrations up to 400 ng/ml. Radioimmunoassay of the natural IGF-I preparations with an antibody directed against the unique C-peptide region of IGF-II demonstrated that contamination of IGF-I preparations with immunoreactive IGF-II could not exceed 5%. These results demonstrate that IGF-I purified from human plasma has a different affinity for the IGF-II receptor than does synthetic or recombinant IGF-I. Furthermore, these data are consistent with the hypothesis that IGF-I, itself, may be heterogeneous, and that subforms may vary in their affinities for the IGF receptors. Alternatively, IGF-I preparations which have been considered to be pure may be contaminated with small amounts of IGF-II, resulting in overestimation of the affinity of IGF-I for the type II IGF receptor.     

Identification of a nonpeptide ligand that releases bioactive insulin-like growth factor-I from its binding protein complex.

Insulin-like growth factor-I (IGF-I) has both metabolic and mitogenic activities mediated through interaction with the type 1 IGF receptor. The circulation of IGF-I in blood and interstitial fluid is not free but bound mostly to a family of six high affinity IGF-binding proteins, which form stable complexes with IGF and neutralize its bioactivity. Therefore, displacement of this large pool of endogenous IGF from the binding proteins could elevate "free" IGF levels to elicit beneficial effects in diabetes and other IGF-responsive diseases comparable with those produced by administration of exogenous IGF-I. We report here the identification of a nonpeptide ligand NBI-31772, which displaces IGF-I from all six IGF-binding proteins at low nanomolar concentrations from screening of the in-house chemical libraries. Furthermore, the released free IGF-I was shown to be biologically active in an in vitro bioassay. Thus, NBI-31772 could serve as a valuable lead molecule for the design of novel therapeutics to treat diabetes and other IGF-responsive diseases.     

Activation of the insulin-like growth factor-I receptor inhibits tumor necrosis factor-induced cell death

The effect of insulin-like growth factor (IGF) on tumor necrosis factor (TNF)-induced cell killing was determined for mouse BALB/c3T3 fibroblasts in vitro. Cells maintained in 0.5% fetal bovine serum (FBS) were killed by TNF within 6 h in a concentration-dependent manner, an effect that was prevented by IGF-I. TNF-induced cytotoxicity of 3T3 cells that overexpress the human IGF-I receptor (p6 cells) was prevented by IGF-I alone in the absence of serum. TNF-induced cell death was associated with the morphologic features of apoptosis and the release of low-molecular-weight DNA, both of which were prevented by IGF-I. Neither epidermal growth factor (EGF) nor platelet-derived growth factor (PDGF) protected p6 cells from TNF-induced apoptosis. The specific protective action of the IGF-I receptor was demonstrated further by the marked sensitivity to TNF of embryo fibroblasts derived from mice with targeted disruption of the IGF-I receptor (R cells) but not of fibroblasts derived from wild-type littermates or R cells transfected with the cDNA for the human IGF-I receptor. Cycloheximide or actinomycin D markedly reduced the protection offered by IGF-I. IGF-I protection of BALB/c3T3 cells persisted for up to 5 days in the presence of PDGF and EGF, whereas IGF-I lost its effectiveness after 2 days in the absence of growth factors. IGF-I did not prevent TNF-induced release of arachidonic acid. The results demonstrate a specific role for the IGF-I receptor in the protection against TNF cytotoxicity. This action of the IGF-I receptor is mediated by protective cytosolic proteins that exhibit a high rate of turnover and whose levels are regulated principally by factors within serum other than IGF-I. © 1996 Wiley-Liss, Inc.     

Effect of celiac disease and gluten-free diet on growth hormone-binding protein, insulin-like growth factor-I, and insulin-like growth factor-binding proteins

Failure to thrive is common in children with celiac disease. As alterations in the growth hormone-insulin-like growth factor I (GH-IGF-I) growth axis have been reported in these patients, we studied the behavior of growth hormone-binding proteins (GH-BPs I and II), IGF-I and its binding proteins in 14 children with celiac disease, either before or after a 6-month gluten-free diet. GH-BP II levels were significantly lower in patients during the active phase of the disease than after the diet or in comparison with control subjects, appropriate for age and sex. There was no difference in the GH-BP-I levels of patients and controls, nor did they change after the diet. Blood levels of IGF-I and IGFBP-3 were reduced before the diet in all patients while ligand blotting showed that IGFBP-2 and 1 were increased. All of these parameters normalized after the gluten-free diet. IGFBP-4 was not greatly influenced by the disease. Furthermore, we found a significant, positive correlation between GH-BP II and IGF-I or IGFBP-3 levels. The height standard deviation scores and body mass indices of the patients improved significantly after the diet. The body mass index significantly and positively correlated with GH-BP II, IGF-I or IGFBP-3 levels. In conclusion, our data show that celiac children had multiple alterations in the growth axis during the active phase of the disease which disappeared during the gluten-free diet.     

Serum insulin-like growth factor-I, insulin-like growth factor binding protein-3, and the pubertal growth spurt in the female rhesus monkey

While there is good evidence suggesting IGF-I links to pubertal development and crown-rump length growth among rhesus monkeys, linkages between IGF-I and other measures of morphological growth have not been established. In this study, the pubertal growth spurt in a number of morphological characteristics of female rhesus monkeys is related to serum endocrine status of insulin-like growth factor-I (IGF-I) and its binding protein, insulin-like growth factor binding protein-3 (IGFBP-3), to test the hypothesis that elevations in IGF-I and IGFBP-3 coincide with the time of greatest growth rate of different morphological characteristics. A longitudinal study of pubertal growth among four female rhesus monkeys was carried out across a 3-year period. Morphometric measurements included weight, crown-rump length, foot-length, and skinfolds at five sites (biceps, triceps, abdominal, subscapular, and suprailiac). These measures were taken as being representative of total mass, skeletal growth of the trunk and head, limb length, and body fatness, respectively. Measurements were carried out as closely as possible to 3-monthly, with interpolations being performed to standardise the data to exactly 3-monthly intervals for all individuals. Blood samples were taken at time of morphometry. Elevations in serum IGF-I and IGFBP-3 took place in a manner similar to that of humans, and across the period associated with onset of puberty. Mean 3-monthly gain in crown-rump length and foot length showed significant peaks across the measurement period, while mean 3-monthly gains in weight and sum of five skinfolds did not. Greatest foot length gain occurred on average between 3-3.5 years of age, while crown-rump length gain was greatest between 3.75-4 years of age. Periods of greatest gain in crown-rump length and foot length took place across the period of elevated serum IGF-I levels, which was between 3-4.5 years of age. Significant elevations in IGF-I and IGFBP-3 were not coincident with greatest gains in foot length or crown-rump length. Thus the hypothesis does not hold true for the two measures showing significant peaks in 3-monthly gain across the measurement period. The nature of the endocrine impact on macaque morphology remains unclear, although this may be fundamental to the understanding of the variation in the pubertal growth spurt and its influence on morphology at maturity both within and across primate species.     

The roles of tyrosines 24, 31, and 60 in the high affinity binding of insulin-like growth factor-I to the type 1 insulin-like growth factor receptor

A series of insulin-like growth factor I (IGF-I) structural analogs in which one or more of the three tyrosine residues were replaced with nonaromatic residues were produced and their binding properties characterized. The single point mutations, [Leu24]IGF-I, [Ala31]IGF-I, and [Leu60]IGF-I result in an 18-, 6-, or 20-fold loss in affinity, respectively, for the type 1 IGF receptor. Multiple mutations, [Ala31,Leu60]IGF-I, [Leu24, Ala31]IGF-I, [Leu24, Leu60]IGF-I, or [Leu24, Ala31, Leu60]IGF-I result in a 520-, 240-, 1200-, or greater than 1200-fold loss in affinity, respectively, at the type 1 IGF receptor. In contrast, none of the analogs display greater than a 2-fold loss in affinity for the acid-stable human serum binding proteins. At the insulin receptor, [Ala31]IGF-I and [Leu24]IGF-I are equipotent to and 5-fold less potent than IGF-I, whereas [Leu60]IGF-I and the multiple mutation analogs are inactive up to 10 microM. Analogs [Leu24]IGF-I, [Ala31]IGF-I, and [Leu24, Ala31]IGF-I are equipotent to IGF-I at the type 2 IGF receptor, whereas all analogs containing Leu60 demonstrate little measurable affinity at this receptor. Thus, Tyr24, Tyr31, and Tyr60 are involved in the high affinity binding of IGF-I to the type 1 IGF receptor, while Tyr60 is important for maintaining binding to the type 2 IGF receptor.     

The ligand binding subunit of the insulin-like growth factor 1 receptor has properties of a peripheral membrane protein

125I-insulin-like growth factor 1 was cross-linked to its receptor in human placenta microsomal membranes. The microsomes were treated with urea, with dithiothreitol or with both reagents prior to centrifugation at 100,000 X g. We found that greater than 80% of the label was membrane-associated following separate treatment with urea or dithiothreitol, but greater than 80% of the radioactivity remained in the supernatant after simultaneous exposure to both reagents. In identical experiments employing 125I-epidermal growth factor, no condition led to the release of greater than 10% of label from the membrane. We conclude that the ligand binding subunit of the insulin-like growth factor 1 receptor, like peripheral membrane proteins, lacks a membrane anchoring domain.     

Differential expression of insulin-like growth factor-I and insulin-like growth factor binding protein-1 in the diabetic rat

Summary Multiple factors contribute to the growth retardation which is a characteristic feature of uncontrolled diabetes. In this report we have examined the effects of streptozotocin-induced (STZ) diabetes on expression of insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-1 (IGFBP-1) in various tissues. As early as 7 days after STZ administration there was a modest reduction in IGF-I mRNA abundance. The reduction (10–30%) was of similar magnitude in each of the 7 tissues examined; liver, kidney, lung, diaphragm, quadraceps, heart and adipose tissue. However, the reduction achieved statistical significance only in the lung (p < 0.05) and diaphragm (p < 0.01). A further reduction in IGF-I mRNA abundance was seen in many tissues, 32 and 91 days after STZ administration. In contrast to the decrease in IGF-I mRNA, IGFBP-1 mRNA was significantly increased in the liver and kidney of diabetic rats. IGFBP-1 mRNA was detectable at only very low levels in other tissues but was increased in diabetic rats compared non-diabetic rats. In diabetic rats, a highly significant correlation (R = 0.75, p < 0.001) between hepatic IGFBP-1 mRNA and glucose was observed whereas there was no significant correlation between serum glucose and hepatic IGF-I mRNA abundance (R = 0.24, p = NS). Treatment of diabetic rats with insulin resulted in a small, non significant increase in hepatic and renal IGF-I mRNA and a significant decrease in renal IGFBP-1 mRNA abundance. The observations reported here are consistent with the hypothesis that diminished IGF-I expression and inhibition of available IGF-1 by increased levels of IGFBP-1 may explain the impaired growth seen in diabetic animals.     

A panel of monoclonal antibodies for the type I insulin-like growth factor receptor. Epitope mapping, effects on ligand binding, and biological activity.

We obtained 20 mouse monoclonal antibodies specific for human type I insulin-like growth factor (IGF) receptors, using transfected cells expressing high levels of receptors (IGF-1R/3T3 cells) as immunogen. The antibodies immunoprecipitated receptor.125I-IGF-I complexes and biosynthetically labeled receptors from IGF-1R/3T3 cells but did not react with human insulin receptors or rat type I IGF receptors. Several antibodies stimulated DNA synthesis in IGF-1R/3T3 cells, but the maximum stimulation was only 25% of that produced by IGF-I. The antibodies fell into seven groups recognizing distinct epitopes and with different effects on receptor function. All the antibodies reacted with the extracellular portion of the receptor, and epitopes were localized to specific domains by investigating their reaction with a series of chimeric IGF/insulin receptor constructs. Binding of IGF-I was inhibited up to 90% by antibody 24-60 reacting in the region 184-283, and by antibody 24-57 reacting in the region 440-586. IGF-I binding was stimulated up to 2.5-fold by antibodies 4-52 and 16-13 reacting in the region 62-184, and by antibody 26-3 reacting downstream of 283. The latter two groups of antibodies also dramatically stimulated insulin binding to intact IGF-1R/3T3 cells (by up to 50-fold), and potentiated insulin stimulation of DNA synthesis. Scatchard analysis indicated that in the presence of these antibodies, the affinity of the type I IGF receptor for insulin was comparable with that of the insulin receptor. These data indicate that regions both within and outside the cysteine-rich domain of the receptor alpha-subunit are important in determining the affinity and specificity of ligand binding. These antibodies promise to be valuable tools in resolving issues of IGF-I receptor heterogeneity and in studying the structure and function of classical type I receptors and insulin/IGF receptor hybrids.     

The binding of insulin-like growth factor II to the erythroleukemia cell line K562

The erythroleukemia cell line K562 was previously shown to have specific binding sites for insulin but not for insulin-like growth factor I (IGF-I). In this study the presence of specific receptors for insulin-like growth factor II (IGFqI) is established. Scatchard analysis of the competition curve for IGF-II disclosed a non-cooperative binding kinetic with a calculated affinity constant of 2.4×108 M^–1 and a receptor number of 4.8×l0⁴ sites/cell. IGF-I displayed 10% crossreactivity over the IGF-II receptor but insulin did not crossreact at all. Instead insulin, present in high concentrations, enhanced the binding of IGF-II. The presence of IGF II but not IGF-I receptors makes t h e K562 cell line suitable for studying properties of the type-2 receptor.     

Requirement of different signaling pathways mediated by insulin-like growth factor-I receptor for proliferation,invasion, and VPF/VEGF expression in a pancreatic carcinoma cell line

Several oncogenes and growth factors are found to be mutated and overexpressed in adenocarcinoma of the pancreas, and may correlate with its highly aggressive nature. Insulin-like growth factor (IGF-I) and its receptor (IGF-IR) are highly expressed in this tumor type. We examined the IGF-IR-mediated signaling pathways in relation to cell proliferation, invasiveness, and expression pattern of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) in the pancreatic cancer line ASPC-1. Our findings show that IGF-IR is an important growth factor receptor for cell proliferation and invasion, and VPF/VEGF expression in ASPC-1. Further experiments indicate that IGF-IR mediates different signaling pathways to execute its functions. Activation of Ras by IGF-IR was found to be required for the cell invasion. On the other hand Src activation through IGF-IR is required for the cell proliferation, invasion, and also VPF/VEGF expression. Taken together, our data indicate the importance of IGF-IR in growth and invasiveness of the pancreatic cancer cell lines and also point out the multiple signaling pathways channeled through this receptor.     

Influence of smoking on serum and milk of mothers, and their infants' serum insulin-like growth factor-I and insulin-like growth factor binding protein-3 levels

OBJECTIVE: To investigate the serum and milk in active-smoking and nonsmoking mothers, and their infants' insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) levels. DESIGN AND SETTING: A cohort study conducted at a tertiary medical center. Subjects: Forty-four mothers (age range: 21-34 years) and their newborns (7 days old) were enrolled in the study. Mothers were interviewed and classified according to their smoking status into one of two groups: the active-smoking mothers (n = 21) and the nonsmoking mothers (n = 23). RESULTS: There was no difference noted in either IGF-I, IGFBP-3 or IGF-I/IGFBP-3 ratios in serum and milk of mothers, and their infants' serum samples according to maternal smoking. CONCLUSION: This study demonstrated that maternal smoking (5-10 cigarettes/day) did not influence the maternal and infant serum levels of IGF-I and IGFBP-3 as well as the breast milk levels of these peptides.     

Effects of dexamethasone on the production of insulin-like growth factor-I and insulin-like growth factor binding proteins in primary cultures of rat hepatocytes.

The effects of dexamethasone (Dex) on insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-1 production were investigated in primary cultures of rat hepatocytes. Dex enhanced the secretion of IGFBP-1 as measured by ligand blot analysis but did not show any prominent effect on immunoreactive IGF-I secretion. EC50 of Dex on IGFBP-1 secretion was calculated to be 3 x 10(-8) M. The content of IGFBP-1 mRNA in the cells increased greatly in the presence of Dex but the IGF-I mRNA content did not change significantly under the same conditions. Insulin showed the opposite effect of Dex by decreasing the production of IGFBP-1 and the cellular content of IGFBP-1 mRNA. This effect of insulin was observed also with Dex in the medium. These results show that the gene expression of IGF-I and IGFBP-1 is differently regulated by glucocorticoids and insulin in primary cultures of rat hepatocytes. The results most possibly explain the in vivo effects of glucocorticoids and insulin in regulation of IGF-I and IGFBP-1 production by liver.     

The effects of the insulin-like growth factor-I aptamer, NBI-31772, on glucose homeostasis in the mouse

The majority of insulin-like growth factor-I (IGF-I) in the adult rodent circulation is bound to high affinity IGF binding proteins. We investigated the changes in IGF-I clearance, blood glucose and plasma insulin levels, and tissue 2-deoxyglucose uptake after intravenous administration of the IGF aptamer, NBI-31772, which selectively competes with IGF-I for binding to the IGFBPs, but has no effect at the IGF-I receptor. Clearance of 125I-IGF-I was significantly increased in NBI-31772-treated mice compared with vehicle-treated mice (t1/2 = 45.0 +/- 1.9 vs. 56.3 +/- 3.9 min, respectively; p = 0.021). However, NBI-31772 had no significant effect on glucose levels, and no insulin sparing effect was apparent neither under basal conditions nor during an intravenous glucose challenge. The decline in the specific activity after 3H-2-deoxyglucose administration was significantly less rapid in NBI-31772-treated mice compared with controls, suggesting that the IGF-I aptamer had an inhibitory effect on hepatic gluconeogenesis. In contrast, no insulin-like effect was apparent in other tissues examined. 3H-2-deoxyglucose accumulation was similar in all tissues analyzed, including skeletal muscle, which is thought to be particularly sensitive to IGF-I. These data suggest that the IGF-I aptamer affects clearance of radiolabeled IGF-I from the circulation, but has no marked effects on glucose nor insulin homeostasis. The search for hydrophilic IGF aptamers with longer duration of action that could be used in the treatment of diabetes may be rewarding.