An anti-αVß3 antibody inhibits coronary artery atherosclerosis in diabetic pigs.

BACKGROUND AND AIMS: Diabetes is a major risk factor for the development of atherosclerosis. Hyperglycemia stimulates vascular smooth muscle cells (VSMC) to secrete ligands that bind to the αVß3 integrin, a receptor that regulates VSMC proliferation and migration. This study determined whether an antibody that had previously been shown to block αVß3 activation and to inhibit VSMC proliferation and migration in vitro, inhibited the development of atherosclerosis in diabetic pigs. METHODS: Twenty diabetic pigs were maintained on a high fat diet for 22 weeks. Ten received injections of anti-ß3 F(ab)2 and ten received control F(ab)2 for 18 weeks. RESULTS: The active antibody group showed reduction of atherosclerosis of 91 ± 9% in the left main, 71 ± 11%, in left anterior descending, 80 ± 10.2% in circumflex, and 76 ± 25% in right coronary artery, (p < 0.01 compared to lesions areas from corresponding control treated arteries). There were significant reductions in both cell number and extracellular matrix. Histologic analysis showed neointimal hyperplasia with macrophage infiltration, calcifications and cholesterol clefts. Antibody treatment significantly reduced number of macrophages contained within lesions, suggesting that this change contributed to the decrease in lesion cellularity. Analysis of the biochemical changes within the femoral arteries that received the active antibody showed a 46 ± 12% (p < 0.05) reduction in the tyrosine phosphorylation of the ß3 subunit of αVß3 and a 40 ± 14% (p < 0.05) reduction in MAP kinase activation. CONCLUSIONS: Blocking ligand binding to the αVß3 integrin inhibits its activation and attenuates increased VSMC proliferation that is induced by chronic hyperglycemia. These changes result in significant decreases in atherosclerotic lesion size in the coronary arteries. The results suggest that this approach may have efficacy in treating the proliferative phase of atherosclerosis in patients with diabetes.

Complement 1s is the serine protease that cleaves IGFBP-5 in human osteoarthritic joint fluid.

UNLABELLED: Insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) are trophic factors for cartilage and have been shown to be chondroprotective in animal models of osteoarthritis (OA). IGFBP-5 is degraded in joint fluid and inhibition of IGFBP-5 degradation has been shown to enhance the trophic effects of IGF-I. OBJECTIVE: To determine the identity of IGFBP-5 protease activity in human OA joint fluid. METHOD: OA joint fluid was purified and the purified material was analyzed by IGFBP-5 zymography. RESULTS: Both crude joint fluid and purified material contained a single band of proteolytic activity that cleaved IGFBP-5. Immunoblotting of joint fluid for complement 1s (C1s) showed a band that had the same Mr estimate, e.g., 88 kDa. In gel tryptic digestion and subsequent peptide analysis by LC-MS/MS showed that the band contained human C1s. A panel of protease inhibitors was tested for their ability to inhibit IGFBP-5 cleavage by the purified protease. Three serine protease inhibitors, FUT175 and CP-143217 and CB-349547 had IC50's between 1 and 6 microM. Two other serine protease inhibitors had intermediate activity (e.g., IC50's 20-40 microM) and MMP inhibitors had no detectible activity at concentrations up to 300 microM. CONCLUSION: Human OA fluid contains a serine protease that cleaves IGFBP-5. Zymography, immunoblotting and LC-MS/MS analysis indicate that C1s is the protease that accounts for this activity.

Gender-specific changes in bone turnover and skeletal architecture in igfbp-2-null mice.

IGF-binding protein-2 (IGFBP-2) is a 36-kDa protein that binds to the IGFs with high affinity. To determine its role in bone turnover, we compared Igfbp2(-/-) mice with Igfbp2(+/+) colony controls. Igfbp2(-/-) males had shorter femurs and were heavier than controls but were not insulin resistant. Serum IGF-I levels in Igfbp2(-/-) mice were 10% higher than Igfbp2(+/+) controls at 8 wk of age; in males, this was accompanied by a 3-fold increase in hepatic Igfbp3 and Igfbp5 mRNA transcripts compared with Igfbp2(+/+) controls. The skeletal phenotype of the Igfbp2(-/-) mice was gender and compartment specific; Igfbp2(-/-) females had increased cortical thickness with a greater periosteal circumference compared with controls, whereas male Igfbp2(-/-) males had reduced cortical bone area and a 20% reduction in the trabecular bone volume fraction due to thinner trabeculae than Igfbp2(+/+) controls. Serum osteocalcin levels were reduced by nearly 40% in Igfbp2(-/-) males, and in vitro, both CFU-ALP(+) preosteoblasts, and tartrate-resistant acid phosphatase-positive osteoclasts were significantly less abundant than in Igfbp2(+/+) male mice. Histomorphometry confirmed fewer osteoblasts and osteoclasts per bone perimeter and reduced bone formation in the Igfbp2(-/-) males. Lysates from both osteoblasts and osteoclasts in the Igfbp2(-/-) males had phosphatase and tensin homolog (PTEN) levels that were significantly higher than Igfbp2(+/+) controls and were suppressed by addition of exogenous IGFBP-2. In summary, there are gender- and compartment-specific changes in Igfbp2(-/-) mice. IGFBP-2 may regulate bone turnover in both an IGF-I-dependent and -independent manner.

Protease-resistant insulin-like growth factor (IGF)-binding protein-4 inhibits IGF-I actions and neointimal expansion in a porcine model of neointimal hyperplasia.

IGF-I has been shown to play a role in the progression of atherosclerosis in experimental animal models. IGF-binding protein-4 (IGFBP-4) binds to IGF-I and prevents its association with receptors. Overexpression of a protease-resistant form of IGFBP-4 has been shown to inhibit the ability of IGF-I to stimulate normal smooth muscle cell growth in mice. Based on these observations, we prepared a protease-resistant form of IGFBP-4 and infused it into hypercholesterolemic pigs. Infusion of the protease-resistant mutant inhibited lesion development by 53.3 +/- 6.1% (n = 6; P < 0.01). Control vessels that received an equimolar concentration of IGF-I and the protease-resistant IGFBP-4 showed no reduction in lesion size compared with control lesions that were infused with vehicle. Infusion of a nonmutated form of IGFBP-4 did not significantly inhibit lesion development. Proliferating cell nuclear antigen analysis showed that the mutant IGFBP-4 appeared to inhibit cell proliferation. The area occupied by extracellular matrix was also reduced proportionally compared with total lesion area. Immunoblotting revealed that the mutant IGFBP-4 remained intact, whereas the wild-type IGFBP-4 that was infused was proteolytically cleaved. Further analysis of the lesions revealed that a marker protein, IGFBP-5, whose synthesis is stimulated by IGF-I, was decreased in the lesions that received the protease-resistant, IGFBP-4 mutant, whereas there was no change in lesions that received wild-type IGFBP-4 or the mutant protein plus IGF-I. These findings clearly illustrate that infusion of protease-resistant IGFBP-4 into the perilesion environment results in inhibition of cell proliferation and attenuation of the development of neointima. The findings support the hypothesis that inhibiting IGFBP-4 proteolysis in the lesion microenvironment could be an effective means for regulating neointimal expansion.

Effects of combined recombinant insulin-like growth factor (IGF)-I and IGF binding protein-3 in type 2 diabetic patients on glycemic control and distribution of IGF-I and IGF-II among serum binding protein complexes.

CONTEXT: Administration of recombinant human IGF-I (rhIGF-I)/recombinant human IGF binding protein-3 (rhIGFBP-3) to patients with type 2 diabetes improves blood glucose and enhances insulin sensitivity. The changes in various components of the IGF system that occur in response to rhIGF-I/rhIGFBP-3 as well as the minimum effective dose have not been determined. OBJECTIVES: The aim was to determine the dose of rhIGF-I/rh-IGFBP-3 necessary to achieve a significant decrease in glucose and to determine the changes that occur in the IGF-II and acid labile subunit in response to treatment. DESIGN: A total of 39 insulin-requiring type 2 diabetics were randomized to placebo or one of six groups that received different dosages of rhIGF-I/rhIGFBP-3. After 3 d in which insulin doses were adjusted to improve glucose control, a variable insulin dosage regimen was continued, and either placebo or one of six dosages (0.125-2.0 mg/kg.d) of rhIGF-I/rhIGFBP-3 was administered for 7 d. All subjects were hospitalized, and dietary intake as well as insulin dosage were controlled with instructions to treat to normal range targets. RESULTS: Fasting glucose was reduced in the groups that received either 1 (32 +/- 5% reduction) or 2 mg/kg.d (40 +/- 6% reduction) of the complex. Mean daily glucose (four determinations) was reduced by 26 +/- 4% in the 1 mg/kg group and by 33 +/- 5% in the 2 mg/kg group compared with 18 +/- 4% in the placebo group. Total serum IGF-I increased between 2.0 +/- 0.3- and 5.7 +/- 1.3-fold by d 8. IGFBP-3 concentrations increased significantly only in the 2 mg/kg group. IGF-II concentrations declined to values that were between 27 +/- 4% and 64 +/- 7% below baseline. Acid labile subunit concentrations declined significantly in the three highest dose groups. The sum of the IGF-I + IGF-II concentrations was significantly increased at the two highest dosages. There were very few drug-associated adverse events reported in this study with the exception of hypoglycemia, which occurred in 15 subjects who had received rhIGF-I/rhIGFBP-3 treatment. CONCLUSIONS: Administration of rhIGF-I/rhIGFBP-3 resulted in a redistribution of the amount of IGF-I and IGF-II that bound to IGFBP-3. Fasting and mean daily blood glucose were reduced significantly in the two highest dosage groups. The results suggest that both the total concentration of IGF-I as well as its distribution in blood may determine the extent to which insulin sensitivity is enhanced.

Recombinant, nonglycosylated human insulin-like growth factor-binding protein-3 (IGFBP-3) is degraded preferentially after administration to type II diabetics, resulting in increased endogenous glycosylated IGFBP-3.

CONTEXT: Administration of IGF-binding protein-3 (IGFBP-3) with IGF-I stabilizes IGF-I concentrations and prolongs its half-life. One determinant of IGFBP-3 stability is proteolysis. Normal subjects have minimal IGFBP-3 protease activity; however, with pregnancy, acute catabolic illness, or diabetes, IGFBP-3 protease activity is increased. OBJECTIVE: This study was conducted to determine the degree of proteolysis that occurs in glycosylated, endogenous serum IGFBP-3 and nonglycosylated IGFBP-3 after administration of an IGF-I/IGFBP-3 combination to patients with diabetes. DESIGN: Thirty-two patients received either 1 (n = 8) or 2 (n = 24) mg/kg x d IGF-I/IGFBP-3 by bolus s.c. injection (n = 16) or continuous s.c. infusion (n = 16). RESULTS: When nonglycosylated IGFBP-3 was given, the abundance of both glycosylated and nonglycosylated forms of IGFBP-3 in serum was increased. Incubation of nonglycosylated IGFBP-3 with diabetic serum in vitro resulted in more rapid degradation compared with glycosylated IGFBP-3. When the serum obtained from subjects who had received nonglycosylated IGFBP-3 was analyzed, significant differences in the stability of glycosylated and nonglycosylated IGFBP-3 were present. The addition of increasing concentrations of nonglycosylated IGFBP-3 to diabetic serum resulted in a dose-dependent increase in the abundance of endogenous, glycosylated IGFBP-3. Administration of IGF-I and nonglycosylated IGFBP-3 for 2 wk to 32 subjects increased glycosylated IGF-I/IGFBP-3 by 20-40%. The increases were the greatest in the groups that received IGFBP-3 by infusion (e.g. 31% and 40%). CONCLUSIONS: After administration to diabetics, nonglycosylated IGFBP-3 is degraded more rapidly than glycosylated IGFBP-3. By acting as a preferential substrate for the IGFBP-3 protease, nonglycosylated IGFBP-3 protects endogenous, glycosylated IGFBP-3 from degradation, allowing total IGFBP-3 concentrations to increase.

Rh/IGF-I/rhIGFBP-3 administration to patients with type 2 diabetes mellitus reduces insulin requirements while also lowering fasting glucose.

Administration of insulin-like growth factor-I to patients with diabetes enhances insulin action and reduces the degree of hyperglycemia but it is associated with a high rate of adverse events. Infusion of the combination of rhIGFBP-3 (the principal binding protein for IGF-I in plasma) with rhIGF-I to patients with type I diabetes improved insulin sensitivity and was associated with a low incidence in side effects. In this study, 52 patients with insulin-treated type 2 diabetes received recombinant human IGF-I plus rhIGFBP-3 in one of four dosage regimens for 14 days. The four groups were: (1) continuous subcutaneous infusion of 2 mg/kg/day; (2) the same 2 mg/kg dose infused subcutaneously over 6 h between 2000 and 0200 h; (3) 1 mg/kg twice a day by bolus subcutaneous injection; (4) a single bedtime subcutaneous injection of 1 mg/kg. Across these four groups rhIGF-I/rhIGFBP-3 decreased insulin requirements between 54% and 82%. Fasting glucose decreased by 32-37%. Mean daily blood glucose (4 determinations per day) declined in all 4 groups (range 9-23% decrease). Frequent sampling for total IGF-I, free IGF-I and IGFBP-3 was performed on days 0,1,7,14 and 15. The peak total IGF-I values were increased to 4.0-4.8-fold at 16-24 h. For free IGF-I the increase varied between 7.1 and 8.2-fold and peak values were attained at 16-20 h after administration. Both the time to maximum concentration (Tmax) and the maximum free IGF-I levels (Cmax) on day 1 for all groups were substantially less than previously published studies, wherein lower doses of rhIGF-I were given without IGFBP-3. The improvement in glucose values and the degree of reduction in insulin requirement were the greatest in groups 2 and 3 and the patients in those groups had the highest free IGF-I levels. The frequency of side effects such as edema, jaw pain and arthralgias was 4% which is less than that has been reported in previous studies wherein IGF-I was administered without IGFBP-3. We conclude that rhIGF-I/rhIGFBP-3 significantly lowers insulin requirements yet improves glucose values and these changes may reflect improvement in insulin sensitivity. Coadministration of IGFBP-3 with IGF-I produces lower free IGF-I (Tmax and Cmax) levels compared to administration of IGF-I alone and is associated with relatively low incidence of side effects during 2 weeks of administration.

Expression and characterization of a serine protease that preferentially cleaves insulin-like growth factor binding protein-5.

Insulin-like growth factor binding proteins (IGFBPs) play important roles in regulating the functions of insulin-like growth factors (IGFs). Because IGFBPs have very high affinity for IGF-I and IGF-II, they can regulate the amount of each growth factor that is able to bind to cell surface receptors, therefore, factors that alter IGFBP affinity have the capacity to regulate IGF actions. Protease activities that are present in cell culture systems and physiologic fluids have been shown to degrade IGFBP-5. Previously, a region of sequence in a serine protease was identified that was homologous with the N-terminal 90 amino acids of members of the IGFBP family and with members of the CCN family of proteins. In a prior study, the protease was expressed in human kidney cultured cells and the cell culture supernatants were shown to cleave IGFBP-5, however, it is unknown whether the purified protease would cleave IGFBP-5 and whether it would also cleave other specific forms of IGFBPs. In this study, we expressed this protease in an insect cell expression system, purified it to homogeneity and tested its capacity to cleave IGFBP-5. The expressed protease preferentially cleaved IGFBP-5, and it had minimal activity toward other forms of IGFBPs. The proteolytic activity of this IGFBPase is inhibited by serine protease inhibitors including PMSF and 3,4-dichloroisocoumarin, as well as by divalent metal ions such as, Zn and Cu. Mutation of the active site serine resulted in a major reduction in IGFBP-5 cleavage. The protease binds to heparin and its ability to degrade IGFBP-5 is blocked in the presence of heparin. Inhibition of the activity of the protease following its secretion by B104 cells resulted in inhibition of IGFBP-5 proteolysis and IGF-I stimulation of protein synthesis. Northern blotting revealed that the transcript was expressed in multiple human tissues, including placenta, uterus, prostate, testis, spinal cord, brain, liver, small intestine, thyroid, and spleen. The highest expression was in uterus and placenta, suggesting a possible role of sex steroids in regulating its expression. Understanding the mechanism of how cleavage of IGFBP-5 by this protease alters its activity will help to further our understanding of the biologic actions of the IGFs.

Interaction between insulin-like growth factor-I receptor and alphaVbeta3 integrin linked signaling pathways: cellular responses to changes in multiple signaling inputs.

Integrins are heterodimeric transmembrane proteins that mediate cell attachment to extracellular matrix, migration, division, and inhibition of apoptosis. Because growth factors are also important for these processes, there has been interest in cooperative signaling between growth factor receptors and integrins. IGF-I is an important growth factor for vascular cells. One integrin, alphaVbeta3, that is expressed in smooth muscle cells modulates IGF-I actions. Ligand occupancy of alphaVbeta3 is required for IGF-I to stimulate cell migration and division. Src homology 2 containing tyrosine phosphatase (SHP-2) is a tyrosine phosphatase whose recruitment to signaling molecules is stimulated by growth factors including IGF-I. If alphaVbeta3 ligand occupancy is inhibited, there is no recruitment of SHP-2 to alphaVbeta3 and its transfer to downstream signaling molecules is blocked. Ligand occupancy of alphaVbeta3 stimulates tyrosine phosphorylation of the beta3-subunit, resulting in recruitment of SHP-2. This transfer is mediated by an insulin receptor substrate-1-related protein termed DOK-1. Subsequently, SHP-2 is transferred to another transmembrane protein, SHPS-1. This transfer requires IGF-I receptor-mediated tyrosine phosphorylation of SHPS-1, which contains two YXXL motifs that mediate SHP-2 binding. The transfer of SHP-2 to SHPS-1 is also required for recruitment of Shc to SHPS-1. Ligand occupancy of alphaVbeta3 results in sustained Shc phosphorylation and enhanced Shc recruitment. Shc activation results in induction of MAPK. Inhibition of the Shc/SHPS-1 complex formation results in failure to achieve sustained MAPK activation and an attenuated mitogenic response. Thus, within the vessel wall, a mechanism exists whereby ligand occupancy of the alphaVbeta3 integrin is required for assembly of a multicomponent membrane signaling complex that is necessary for cells to respond optimally to IGF-I.

Glucosamine induces resistance to insulin-like growth factor I (IGF-I) and insulin in Hep G2 cell cultures: biological significance of IGF-I/insulin hybrid receptors.

IGF-I stimulates insulin-like actions directly through its receptor, and it also enhances sensitivity to insulin-mediated effects in vivo. These studies were undertaken to analyze the role of IGF-I, insulin, and insulin/IGF-I hybrid receptors (HRs) in mediating IGF-I and insulin signaling in cells that had been made insulin-resistant by treatment with glucosamine. Human HepG2 cells, which express IGF-I receptors, insulin receptors (IRs), and IGF-I/insulin HRs, were exposed to 20 mM glucosamine; and the effects of IGF-I and insulin in stimulating glycogen synthesis were determined. An overnight exposure to glucosamine markedly attenuated the effects of insulin and IGF-I in stimulating glycogen synthesis. To determine which receptors were mediating this effect, the ability of insulin and IGF-I to stimulate phosphorylation of their respective receptors was analyzed. An 18-h exposure to glucosamine (20 mM) caused a 75% reduction in the ability of IGF-I to phosphorylate its receptor but no change in receptor abundance. Glucosamine also caused a major reduction in insulin-stimulated receptor phosphorylation, although, unlike IGF-I, there was also a 50% reduction in IR abundance. Exposure to glucosamine also resulted in a reduction in the ability of IGF-I or insulin to stimulate phosphorylation of insulin IGF-I/HRs. The combination of insulin plus IGF-I was a more potent stimulus of HR phosphorylation than either agent alone, and this combination was also more potent in partially reversing the inhibitory effect of glucosamine. Taken together, these findings indicate that glucosamine induces a loss of sensitivity to stimulation of insulin, IGF-I, or HR tyrosine kinase activity by insulin or IGF-I. Although insulin is able to partially reverse the effect of glucosamine on IR phosphorylation, it has a very minimal effect on glucosamine-induced inhibition of HR phosphorylation. However, the combination of IGF-I and insulin induces a major increase in HR phosphorylation, even in the presence of glucosamine, suggesting that it is improving the sensitivity of the HR to insulin activation.

A non-IGF binding mutant of IGFBP-3 modulates cell function in breast epithelial cells.

We demonstrated previously that IGFBP-3 alone had no effect on cell death, but dramatically modulated apoptosis in Hs578T IGF non-responsive cells. We investigated whether a non-IGF binding mutant of IGFBP-3 retained its intrinsic actions in this cell line, prior to investigating its actions in IGF-responsive cells (MCF-7 and MCF-10A). In the Hs578T cells, the ceramide analogue, C2-induced apoptosis, non-glycosylated, glycosylated or mutant IGFBP-3 alone had no effect but on co-incubation with C2, all forms of IGFBP-3 markedly accentuated triggered apoptosis. In MCF-7 cells, IGFBP-3 was unable to modulate C2-induced death. In the MCF-10A cells, IGFBP-3 acted as a potent survival factor. IGFBP-3 also affected cell growth in the MCF-10A cells (inhibiting at low doses but increasing growth at higher concentrations). These actions of IGFBP-3 in the MCF-10A cells were independent of IGF-1. IGFBP-3 has differential IGF-independent effects on cell death and growth in normal breast and breast cancer cells.

Effects of a non-IGF binding mutant of IGFBP-5 on cell death in human breast cancer cells.

We have demonstrated previously that IGFBP-5 alone had no effect on cell death but modulated ceramide-induced apoptosis in Hs578T IGF non-responsive cells. To investigate if IGFBP-5 maintains its intrinsic ability to modulate apoptosis in IGF-responsive cells, we used a non-IGF binding mutant of IGFBP-5. In Hs578T cells, non-glycosylated, glycosylated or mutant IGFBP-5 alone each had no effect on cell death, whereas all forms inhibited ceramide-induced apoptosis. In IGF-responsive MCF-7 cells, each wild type form reduced ceramide-induced cell death but mutant IGFBP-5 was without effect. In the presence of mutant IGFBP-5, however, IGF-I no longer conferred survival and in the presence of wild type IGFBP-5, long R3 IGF-I was also unable to confer survival. In summary, all forms of IGFBP-5 modulated ceramide-induced apoptosis in Hs578T cells. In MCF-7 cells, IGF-I-induced survival could be facilitated by IGFBP-5, but also blocked by IGFBP-5 if association with IGFBP-5 was prevented.

Long-term treatment of acromegaly with pegvisomant, a growth hormone receptor antagonist.

BACKGROUND: Pegvisomant is a new growth hormone receptor antagonist that improves symptoms and normalises insulin-like growth factor-1 (IGF-1) in a high proportion of patients with acromegaly treated for up to 12 weeks. We assessed the effects of pegvisomant in 160 patients with acromegaly treated for an average of 425 days. METHODS: Treatment efficacy was assessed by measuring changes in tumour volume by magnetic resonance imaging, and serum growth hormone and IGF-1 concentrations in 152 patients who received pegvisomant by daily subcutaneous injection for up to 18 months. The safety analysis included 160 patients some of whom received weekly injections and are excluded from the efficacy analysis. FINDINGS: Mean serum IGF-1 concentrations fell by at least 50%: 467 mg/L (SE 24), 526 mg/L (29), and 523 mg/L (40) in patients treated for 6, 12 and 18 months, respectively (p<0.001), whereas growth hormone increased by 12.5 mg/L (2.1), 12.5 mg/L (3.0), and 14.2 mg/L (5.7) (p<0.001). Of the patients treated for 12 months or more, 87 of 90 (97%) achieved a normal serum IGF-1 concentration. In patients withdrawn from pegvisomant (n=45), serum growth hormone concentrations were 8.0 mg/L (2.5) at baseline, rose to 15.2 mg/L (2.4) on drug, and fell back within 30 days of withdrawal to 8.3 mg/L (2.7). Antibodies to growth hormone were detected in 27 (16.9%) of patients, but no tachyphylaxis was seen. Serum insulin and glucose concentrations were significantly decreased (p<0.05). Two patients experienced progressive growth of their pituitary tumours, and two other patients had increased alanine and asparate aminotransferase concentrations requiring withdrawal from treatment. Mean pituitary tumour volume in 131 patients followed for a mean of 11.46 months (0.70) decreased by 0.033 cm(3) (0.057; p=0.353). INTERPRETATION: Pegvisomant is an effective medical treatment for acromegaly.

Use of mutagenesis to probe IGF-binding protein structure/function relationships.

The IGF-binding proteins (IGFBPs) are multifunctional proteins that modulate IGF actions. To determine whether specific domains within these proteins account for specific functions, we and other laboratories have used in vitro mutagenesis. Prior experiments that used a variety of techniques had identified discrete regions within each protein that were proposed to account for specific functions. Alterations of these regions by substituting charged residues with neutral residues or hydrophobic residues with nonhydrophobic residues as well as domain swapping, i.e., substituting a domain from one specific form of IGFBP for the homologous domain in another form, has resulted in the elucidation of the functions of many of these specific sequences. Because the areas of protein sequence that are altered involve a limited number of amino acids, they generally do not alter the conformation of the entire protein; therefore, these specific substitutions can often be correlated with the functional changes that occur after mutagenesis. Mutants have been particularly useful for performing functional analyses in which the purified mutant protein is added to a biological test system. In some cases it has been possible to overexpress the mutagenized protein and determine whether the constitutively synthesized, mutant form of IGFBP has altered functional activity. These results have revealed that discrete regions of IGFBP sequence can mediate important and specific functional properties of these proteins.

Commercial assays available for insulin-like growth factor I and their use in diagnosing growth hormone deficiency.

Radioimmunoassays of insulin-like growth factor I (IGF-I) are commonly used for screening adults and children for growth hormone (GH) deficiency or excess. There are, however, many problems with such assays. Attempts to resolve these problems have focused on methods of separating IGF-I from its binding proteins, and on reducing inter- and intra-assay variability. In particular, the collection of sufficient high-quality normative data is a major difficulty in many laboratories. Clinical evaluation of assays is also problematic. IGF-I levels vary with age after puberty, and this is complicated by the maintenance of IGF-binding protein 3 levels by IGF-II. Generally, studies have shown that IGF-I is sensitive and specific for the diagnosis of acromegaly, but screening for GH deficiency (GHD) is less precise. The most commonly used commercial assays are immunoradiometric (IRMA) sandwich assays, using antibodies specific to IGF-I. IRMA assays are quick and accurate, and the two-site antibody reactivity produces a high degree of specificity. Additional techniques such as acid-ethanol extraction or saturation with IGF-II can improve reliability. More recently, the introduction of chemiluminescence has provided enhanced speed and sensitivity. The clinical use of these assays has provided a wealth of information regarding the diagnosis of GHD, and it may be possible to reduce the number of patients who require provocative GH testing. IGF-I assays are also of great use in monitoring GH replacement therapy. Despite the problems, IGF-I measurement is currently the best indirect method available for screening and monitoring patients with GHD.

Relation between serum insulinlike growth factor-1, insulinlike growth factor binding protein-2, and insulinlike growth factor binding protein-3 and nutritional intake in premature infants with bronchopulmonary dysplasia.

BACKGROUND: The usefulness of serum insulinlike growth factor (IGF)-system-peptide measurement to assess the adequacy of nutritional intake in premature infants with chronic lung disease bronchopulmonary dysplasia (BPD) was assessed. METHODS: Twenty-nine premature infants had serial measurements taken of their serum IGF-1, insulinlike growth factor binding protein (IGFBP)-2, and IGFBP-3 concentrations between 2 and 6 weeks of age. Regression analyses were used to examine the relation between nutritional parameters and IGF-1, IGFBP-2, and IGFBP-3 concentrations in premature infants with and without BPD. RESULTS: The group of infants with BPD (n = 12) did not differ from infants without BPD (n = 17) in gestational age or weight at entry, but gained less weight during the study period. In infants without BPD, IGF-1 correlated positively with protein intake (r = 0.50) and caloric intake (r = 0.41) over the 3 days before sample collection and with weight change over the previous week (r = 0.46). In contrast, infants with BPD showed a significant correlation between IGF-1 and weight change (r = 0.54) only. There was a significant negative correlation between IGFBP-2 and protein intake in infants without BPD (r = -0.50) and in infants with BPD (r = -0.41). Negative correlations between IGFBP-2 and both weight change (r = -0.64) and caloric intake (r = -0.43) over the previous week were found only in the group of infants without BPD. IGFBP-3 correlated positively with weight changes and protein intake in both groups but correlated with caloric intake only in the group without BPD. Multiple regression analyses were used to determine significant independent variables associated with IGF-1, IGFBP-2, and IGFBP-3. In infants without BPD, significant independent predictors of IGFBP-2 were 7-day weight change and 2-day protein intake; 3-day caloric intake was the only significant independent predictor for IGFBP-3. For infants with BPD, 3-day weight gain was the only independent variable associated with serum IGF-1. Protein intake in the week before sample collection was an independent predictor of IGFBP-2 and 3-day weight change and 2-day protein intake were independent predictors of IGFBP-3. CONCLUSIONS: These results confirm that changes in serum IGF-1, IGFBP-2, and IGFBP-3 reflect the nutritional status of premature infants and demonstrate that the relation between these proteins and nutritional intake differs in premature infants with and without BPD. Refinement of these observations by future studies may permit a more accurate determination of the protein and caloric intake sufficient for growth and repair after injury in premature infants with lung disease.

Mutagenesis of basic amino acids in the carboxyl-terminal region of insulin-like growth factor binding protein-5 affects acid-labile subunit binding.

Like insulin-like growth factor binding protein-3 (IGFBP-3), IGFBP-5 was recently shown to form ternary complexes with insulin-like growth factor (IGF) and the acid-labile subunit (ALS). Previous studies using IGFBP-5/IGFBP-6 chimeric proteins have identified major and minor ALS binding sites in the carboxyl-terminal and central regions, respectively of IGFBP-5. We now report that ALS binds to IGFBP-3 (K(a) = 1.1 +/- 0.1 liters/nmol) and IGFBP-5 (K(a) = 1.8 +/- 0.5 liters/nmol) with similar binding affinities. Using site-specific mutants, we have identified residues K(211)/R(214)/K(217)/R(218) within the carboxyl-terminal region of IGFBP-5 as being essential for ALS binding. Mutation of K(134)R(136) or K(138)K(139) in the central region of IGFBP-5 resulted in a small decrease in ALS binding.

Structural analysis of the role of the beta 3 subunit of the alpha V beta 3 integrin in IGF-I signaling.

The disintegrin echistatin inhibits ligand occupancy of the alpha V beta 3 integrin and reduces Insulin-like growth factor I (IGF-I) stimulated migration, DNA synthesis, and receptor autophosphorylation in smooth muscle cells. This suggests that ligand occupancy of the alpha V beta 3 receptor is required for full activation of the IGF-I receptor. Transfection of the full-length beta 3 subunit into CHO cells that have no endogenous beta 3 and do not migrate in response to IGF-I was sufficient for IGF-I to stimulate migration of these anchorage dependent cells. In contrast, transfection of either of two truncated mutant forms of beta 3 (terminating at W(715) or E(731)) or a mutant with substitutions for Tyr(747) Tyr(759) (YY) into either CHO or into porcine smooth muscle cells did not restore the capacity of these cells to migrate across a surface in response to IGF-I. This effect was not due to loss of IGF-I receptor autophosphorylation since the response of the receptor to IGF-I was similar in cells expressing either the full-length or any of the mutant forms of the beta 3 subunit. Echistatin reduced IGF-I receptor phosphorylation in cells expressing the full-length or the YY mutant forms of beta 3 subunit, but it had no effect in cells expressing either of two truncated forms of beta 3. A cell-permeable peptide homologous to the C-terminal region of the beta 3 subunit (amino acids 747-762) reduced IGF-I stimulated migration and receptor autophosphorylation of non-transfected porcine smooth muscle cells. These results demonstrate that the full-length beta 3 with intact tyrosines at positions 747 and 759 is required for CHO cells to migrate in response to IGF-I. Furthermore, a region of critical amino acids between residues 742-762 is required for echistatin to induce its regulatory effect on receptor phosphorylation. Since the IGF-I receptor does not bind to alpha V beta 3 the results suggest that specific but distinct regions of the beta 3 subunit interact with intermediary proteins to facilitate IGF-I stimulated cell migration and echistatin induced inhibition of IGF-I signal transduction.

Physiological differences in insulin-like growth factor binding protein-1 (IGFBP-1) phosphorylation in IGFBP-1 transgenic mice.

Insulin-like growth factor binding protein (IGFBP)-1 has been shown to alter cellular responses to insulin-like growth factor 1 (IGF-1). Human IGFBP-1 undergoes serine phosphorylation, and this enhances both its affinity for IGF-1 by six- to eightfold and its capacity to inhibit IGF-1 actions. To investigate the physiological role of IGFBP-1 in vivo, transgenic mice have been generated using either the human IGFBP-1 or rat IGFBP-1 transgene. Both lines of mice expressed high concentrations of IGFBP-1 in serum and tissues; however, human IGFBP-1 transgenic mice did not show glucose intolerance and exhibited no significant intrauterine growth retardation, whereas rat IGFBP-1 transgenic mice showed fasting hyperglycemia and intrauterine growth restriction. The aim of this study was to investigate the physiological differences in the phosphorylation state of human IGFBP-1 and rat IGFBP-1 in these transgenic mice. The phosphorylation status of IGFBP-1 in transgenic mouse serum was analyzed by nondenaturing PAGE. Almost all of the IGFBP-1 in serum from the human IGFBP-1 transgenic mice was present as a nonphosphorylated form. Most of the rat IGFBP-1 in the serum of the mice expressing the rat IGFBP-1 was phosphorylated. Immunoprecipitation showed that mouse hepatoma (Hepa 1-6) cells (exposed to [32P]H3PO4) secrete 32P-labeled IGFBP-1. When the human IGFBP-1 transgene was transfected into Hepa 1-6 cells, all of the IGFBP-1 was secreted in the nonphosphorylated form. However, when the rat IGFBP-1 transgene was transfected into these cells, phosphorylated forms of IGFBP-1 were secreted. To confirm this result, the mouse hepatoma cell protein kinase was partially purified. This kinase activity phosphorylated mouse and rat IGFBP-1 in vitro, but it did not phosphorylate human IGFBP-1. Scatchard analysis showed that the affinity of phosphorylated rat IGFBP-1 for IGF-1 was 3.9-fold higher than that of nonphosphorylated human IGFBP-1. We conclude that the mouse IGFBP-1 kinase activity cannot phosphorylate human IGFBP-1, whereas it can phosphorylate rat IGFBP-1. The phosphorylation state of human IGFBP-1 may account for part of the phenotypic differences noted in the two studies of transgenic mice, and it is an important determinant of the capacity of human IGFBP-1 to inhibit IGF-1 actions in vivo.