Insulin-like Growth factor-I Treatment Research Articles

Effects of growth hormone and insulin-like growth factor-I singly and in combination on in vivo capacity of urea synthesis, gene expression of urea cycle enzymes, and organ nitrogen contents in rats.

Improvement of nitrogen balance is desirable in patients with acute or chronic illness. Both growth hormone (GH) and insulin-like growth factor-I (IGF-I) are promising anabolic agents, and their combined administration has been shown to reverse catabolism more efficiently than each of the peptides alone. This is believed to be mediated primarily through increased peripheral protein synthesis, whereas little attention has focused on a possible participation of amino acid metabolism in the liver. Four groups of rats were given: 1) placebo; 2) GH (200 micrograms/d); 3) IGF-I (300 micrograms/d); and 4) both GH and IGF-I. After 3 days, the maximum capacity of urea-nitrogen synthesis was determined by saturating infusion of alanine (n = 8 in each group), together with measurements of liver messenger RNA (mRNA) levels for urea cycle enzymes (n = 5 in each group) and N-contents of muscles, heart, and kidney. Basal plasma alpha-amino acid concentrations were similar in all groups. The capacity of urea-N synthesis [mumol/(min x 100 g body weight)] was reduced in a stepwise manner (placebo: 8.25 +/- 1.2; GH treatment: 6.52 +/- 0.8; IGF-I treatment: 5.5 +/- 0.6; and GH/IGF-I: 4.22 +/- 1.6 [P < .001 by ANOVA]), each step being lower than the former. Serum IGF-I increased stepwise from placebo (699 +/- 40 to 1,579 +/- 96 micrograms/L in the combined GH/IGF-I group), and was correlated negatively with the capacity of urea-nitrogen synthesis (P < .01). mRNA levels for urea cycle enzymes in the liver decreased after GH and IGF-I treatment, and the effect was more pronounced after the combined treatment in which the rate-limiting enzyme, argininosuccinate synthetase, was halved. Nitrogen contents of organs increased after both GH and IGF-I treatment, and even more so after the combination treatment, reaching an increase of 30% (P < .05). Data suggest that GH and IGF-I singly and, even more so in combination, additively inhibit urea synthesis. This is supposed to favor protein buildup in organs. We speculate that this inhibitory effect on the capacity of urea synthesis is caused by a decreased translation rate of the urea cycle enzymes caused by GH and IGF-I's down-regulatory effect on urea cycle enzyme gene transcription. The findings may indicate a novel mechanism of the protein anabolic action of GH and IGF-I.

Growth factors and myelin regeneration in multiple sclerosis.

Insulin-like growth factor-I (IGF-I), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and ciliary neurotrophic factor (CNTF) are multifunctional growth factors which are found in the CNS. Oligodendroglia are the cells that form and maintain myelin sheaths and many in vitro experiments have shown that these growth factors promote the proliferation, differentiation and survival of cells in the oligodendroglial lineage. Since myelin breakdown is often severe in multiple sclerosis (MS), the possibility of growth factor use in the treatment of MS has been considered and recently, IGF-I treatment has been shown to reduce lesion severity and promote myelin regeneration in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. This review briefly summarizes the structural characteristics of these growth factors and the actions which might help reduce oligodendrocyte-myelin sheath injury in MS and promote myelin regeneration.

Insulin-like growth factor I (IGF-I) induces unique effects in the cytoskeleton of cultured rat glomerular mesangial cells.

Resident glomerular mesangial cells (MCs) have complex cytoskeletal organizations that maintain functional and structural integrity. The ability of cells to replicate, coordinate movement, change shape, and interact with contiguous cells or extracellular matrix depends on cytoskeletal organization. MCs synthesize insulin-like growth factor (IGF-I), express IGF-I receptors, and respond to IGF-I with increased proliferation. We noted that IGF-I treatment of mesangial cells was associated with a change in morphology. Therefore, these studies were undertaken to define specific IGF-I-mediated changes in cytoskeletal protein organization. Rat MCs were propagated from birth in culture without supplemental insulin. Quiescent, subconfluent cultures were treated with IGF-I (100 nM) for 1 hr. Rearrangements in f-actin, alpha-smooth muscle actin, beta-actin, vimentin, and vinculin were seen by fluorescence microscopy. As the cytoskeleton rearranged, alpha-smooth muscle actin dissociated from the f-actin bundles and beta-actin became polymerized under the leading lamellar edge. Ultrastructural changes were consistent with increased membrane turnover and metabolic activity. The normally sessile mesangial cell was induced by IGF-I to express a wound-healing phenotype characterized by movement and increased pinocytosis. These changes are different from those induced by insulin and have important implications for mesangial cell function.

Insulin-like growth factor-I treatment reduces immune cell responses in acute non-demyelinative experimental autoimmune encephalomyelitis.

To test the effects of insulin-like growth factor-I (IGF-I) on clinical deficits, lesion severity, and immune cell response in acute, non-demyelinative experimental autoimmune encephalomyelitis (EAE), we induced EAE in Lewis rats by passive transfer of an MBP-reactive T lymphocyte line. Four days after receiving 5 x 10(5) MBPL-1 T cells intravenously, ten pairs of rats had the same mild degree of tail and hind limb weakness. Ten were given 300 micrograms IGF-I i.v. twice daily for 6 days, and the other 10 received the same volume of 0.89% NaCl. Pairs of rats were sacrificed after 4 days and 6 days of IGF-I and placebo treatment and spinal cord sections were processed for immunostaining, in situ hybridization, and morphological examination. IGF-I treatment decreased clinical deficits, lesion numbers, and lesion areas significantly. Numbers of CD4-positive T cells, alpha/beta TCR-positive cells, and ED-1-positive macrophages were also significantly reduced by IGF-I treatment. Similar reductions were found in our second trial, when 11 days of placebo and IGF-I injections began the day after transfer. No demyelination was observed in either toluidine blue-stained semithin sections or sections immunostained with an antibody raised against myelin basic protein (MBP). We conclude that IGF-I-induced reductions in immune cell responses can occur in the absence of demyelination and are of major importance in decreasing clinical deficits and lesion severity in EAE. If IGF-I has similar effects in multiple sclerosis, we think that it will be useful therapeutically.

Systemic infusion of IGF-I or LR(3)IGF-I stimulates visceral organ growth and proliferation of gut tissues in suckling rats.

This study describes developmental changes in gastrointestinal response to insulin-like growth factor I (IGF-I) peptide administration. Neonatal rats were infused with IGF-I or long [Arg3]IGF-I (LR(3)IGF-I) for 6.5 days starting on day 6 or 12 postpartum. Peptides were delivered by mini osmotic pumps at 0, 2, 5, or 12.5 microg x g(-1) x day(-1). IGF-I infusion increased plasma IGF-I levels in both age groups but stimulated body weight gain only in the older rats. Infusion of LR(3)IGF-I did not change plasma IGF-I levels. Both peptides enhanced expression of IGF-binding proteins (IGFBP) 1 and 2 and induced IGFBP-3 in the older rats. For both age groups, weights of the kidney and spleen increased by up to 85 and 76%, respectively. IGF-I treatment also stimulated gut weight and length by up to 60 and 32%, respectively, but dose dependency was observed only in the older rats. LR(3)IGF-I was more potent for all growth parameters in both age groups. Histological observations included thickening of the mucosa and muscularis externa after infusion of IGF-I peptides. Thymidine labeling in the younger rats indicated that proliferative activity increased proportionately with crypt cell growth. These results show that IGF-I peptides selectively stimulate growth of gastrointestinal tissues in suckling rats and that the proximal gut was the most peptide-responsive region.

Differential Regulation of Insulin-like Growth Factor-I (IGF-I) Receptor Gene Expression by IGF-I and Basic Fibroblastic Growth Factor

Insulin-like growth factor-I receptor (IGF-IR) gene expression is regulated by various stimuli, including hormones, growth factors, and nutritional status. We have investigated the molecular mechanism by which two growth factors, insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF) regulate IGF-IR gene expression. bFGF increases the endogenous IGF-IR mRNA levels and IGF-IR promoter activity. This effect is mediated by a region of the IGF-IR promoter located between nucleotides -476 and -188 in the 5'-flanking region. In contrast, IGF-I decreases the IGF-IR mRNA levels. IGF-I down-regulates IGF-IR transcriptional activity as deduced from experiments in which the levels of pre-mRNA and mRNA were measured. IGF-I reduced pre-mRNA and mRNA levels in parallel, while the mRNA stability was found to be unchanged by IGF-I treatment. While these results strongly suggest an effect of IGF-I on IGF-IR transcriptional activity, no specific IGF-I response element was demonstrated in the 5'-untranslated region or 5'-flanking region studied. Thus, bFGF and IGF-I have differential effects on IGF-IR gene transcription, with the IGF-I response region as yet unidentified.

Regimens of IGF-I Treatment in Fetal Pancreas Transplantation

Transplantation of fetal pancreas (FP) is a potential treatment for diabetes mellitus. FP has remarkable proliferative capacity and may be induced to expand sufficiently to provide a functional β cell mass in adult recipients. We have demonstrated that local delivery of recombinant insulin-like growth factor-I (IGF-I) onto FP isografts is sufficient to reverse streptozotocin-induced diabetes in animals receiving as few as 4 fetal pancreata. In this current study we investigated other regimens of IGF-I delivery in an attempt to define whether its effects on FP requiredlocaldelivery or whether other, more clinically feasible, forms of treatment would suffice. In our model, diabetic Lewis rats received isografts of 16 FP into the anterior thighintramuscular(IM) site. In the group of FP recipients treated with vehicle alone, no animals converted to euglycemia (0/8). When the IM site waspretreatedlocally with 14 days of continuous IGF-I administration (69 μg/kg per day) prior to FP transplantation, 100% of the recipients (10/10) became euglycemic with a mean interval from transplant to euglycemia of 35 ± 15 days (P< 0.001 when compared to vehicle alone). No significant advantage over the vehicle alone group was gained either when the FP tissue was cultured for 48 hr in the presence of IGF-I (100 μg/ml) and then implanted (27% conversion to euglycemia, 3/11) or when FP isografts were treated with continuoussubcutaneousdelivery of IGF-I (69 μg/kg per day over 14 days) distant from the transplant site (0% conversion to euglycemia, 0/6). IGF-I increased the rate of conversion to euglycemia either when deliveredlocallyto FP isografts or when delivered to the transplant bedpriorto transplantation. This suggests an active role of the IGF-I-treated transplant bed in the success of FP transplantation.

Transcriptional Activation of the Porcine P450 11A Insulin-like Growth Factor Response Element in MCF-7 Breast Cancer Cells

Insulin-like growth factor-I (IGF-I) stimulates the growth of MCF-7 breast cancer cells. This study determined the transcriptional activity of an IGF-I-responsive region (IGFRE) of porcine P450 11A (P450scc) after transfection into MCF-7 cells. IGF-I induced transcriptional activity of a porcine P450scc core promoter luciferase construct containing the IGFRE transfected in MCF-7 cells. Electrophoretic mobility shift assay with nuclear protein extract from MCF-7 cells showed two transcription factors binding to the IGFRE. Supershift assay determined that one transcription factor was Sp1. Electrophoretic mobility shift assay and transfection experiments with selected mutations to the IGFRE found that binding of both transcription factors was necessary to confer an IGF-I response. The binding activity of both transcription factors was increased with IGF-I treatment. In conclusion, MCF-7 cells contain Sp1 and another unknown transcription factor, P2, that bind to a known IGFRE (porcine P450scc) and induce reporter gene transcriptional activity with IGF-I treatment. Because Sp1 is a ubiquitous transcription factor, determining the identity of P2 may lead to cell-specific methods to impair breast cancer cell growth as mediated by IGF-I.

Insulin-like growth factor I inhibits degradation and improves retention of protein in hindlimb muscle of lambs.

We infused recombinant human insulin-like growth factor I (IGF-I) for 4 h at 12.3 micrograms.h-1.kg live weight-1 directly into the left femoral artery and measured the rates of synthesis, degradation, and gain of protein by the treated and contralateral limbs of well-fed (n = 8), feed-restricted (n = 10), and fasted (n = 9) castrated male lambs. Reducing feed intake decreased net protein gain of hindlimb muscle, reduced hindlimb glucose uptake, and lowered arterial plasma concentrations of IGF-I, insulin, glucose, phenylalanine, tyrosine, and isoleucine. The effect of nutrition on IGF-binding proteins (IGFBP) was generally small; IGFBP-2 was more abundant in fasted lambs. Infusion of IGF-I into the left femoral artery increased plasma levels of IGF-I 2- to 4-fold in the left femoral vein and by 1.5- to 3-fold in the artery and right femoral vein. In the treated limb, IGF-I reduced protein degradation, increased protein gain, and increased glucose uptake without altering blood flow or oxygen uptake, regardless of feed intake. Systemically, IGF-I reduced plasma insulin, phenylalanine, tyrosine, isoleucine, and leucine in all nutrition groups. Plasma IGFBP-3 was increased by 4 h of IGF-I treatment in fasted but not in fed lambs. In fed but not fasted lambs, IGF-I increased blood glucose concentration. Effects of IGF-I on protein metabolism in the contralateral limb were affected by nutrition, generally more so in fasted than in unrestricted fed lambs.

Ras proteins mediate induction of uncoupling protein, IGF-I, and IGF-I receptor in rat fetal brown adipocyte cell lines.

Since Ras proteins are essential intermediates of some insulin-like growth factor I (IGF-I)/insulin signaling pathways, we examined whether Ras proteins mediate the IGF-I-induced uncoupling protein expression. Additionally, the role of Ras proteins on IGF-I and IGF-I receptor expression was studied. IGF-I treatment of fetal brown adipocytes cotransfected with inducible gene constructs of SV40 large T antigen (SV40LTag) and a transforming ras gene induced uncoupling protein expression (UCP) in the absence of expression of the transfected genes. The expression of the dexamethasone-inducible transforming ras gene alone or in combination with the Zn-inducible SV40LTag mimicked the IGF-I effect inducing UCP expression and IGF-I did not induce it further. However, the expression of the Zn-inducible SV40LTag did not increase UCP expression in the absence of IGF-I. Expression of the transfected ras oncogene also induced IGF-I and IGF-I receptor mRNAs, whereas expression of SV40LTag did not increase them. Specific IGF-I binding was also specifically increased by expression of the transfected ras oncogene but was not affected by expression of the SV40LTag construct. These results indicate that Ras proteins mediate the IGF-I-induced effect on UCP expression and play a role in the expression of IGF-I and IGF-I receptor. Therefore, an IGF-I autocrine/paracrine loop might be implicated in the process of thermogenic differentiation of brown adipose tissue by a new mechanism unlike that induced by norepinephrine.

Insulin-like growth factor-I decreases serum lipoprotein(a) during long-term treatment of patients with Laron syndrome

An increased circulating level of lipoprotein(a) [Lp(a)] is a well-recognized risk factor for coronary artery disease. While much remains to be understood about its regulation and physiological functions, we explored the effect of recombinant insulin-like growth factor-I (IGF-I) administration on circulating Lp(a) levels in 10 Laron syndrome (LS) patients (five children and five adults) with inherited IGF-I deficiency. There was no relationship between pretreatment or posttreatment Lp(a) levels and age and sex of the patients. With IGF-I treatment for 6 to 12 months, there was a significant reduction in Lp(a) (65.7% ± 15.5%, P < .0001) from the pretreatment level of 76 ± 45 mg/L to the posttreatment level of 29 ± 26 mg/L. This decrease was dosage-dependent on the IGF-I administered ( r = .685, F = 0.708, P = .029) and correlated more strongly with the dosage ratio of the end to the beginning of treatment ( r = .78, F = 12.23, P = .008). The higher the IGF-I dose and the higher the dose ratio, the greater the Lp(a) decrease and the lower the Lp(a) at the end of treatment. In conclusion, we observed a dose-dependent relationship between IGF-I administration and Lp(a) reduction in patients with LS. Further studies are needed to elucidate the mechanism of the effect, but our findings suggest a possible metabolic link between these two and shed more light on the regulation of apolipoprotein (a) [apo(a)] expression. It could also open an avenue for additional therapeutic usage of IGF-I.

Involvement of Raf-1 kinase and protein kinase C zeta in insulin-like growth factor I-induced brown adipocyte mitogenic signaling cascades: inhibition by cyclic adenosine 3',5'-monophosphate.

In the present study we have examined the signaling cascades involved in insulin-like growth factor I (IGF-I)-induced mitogenesis in fetal rat brown adipocyte primary cultures, a model that constitutively expresses a high number of IGF-I receptors, where IGF-I is a complete mitogen at physiological concentrations. IGF-I rapidly stimulated beta-chain IGF-I receptor autophosphorylation, which peaked at a physiological/mitogenic concentration (1.4 nM) and also stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). Tyrosine-phosphorylated IRS-1 bound and subsequently activated phosphatidylinositol 3-kinase by 3.5-fold, whereas the tyrosine-phosphorylated IGF-I receptor was not directly associated with the p85 subunit of the phosphatidylinositol 3-kinase. Moreover, mitogenic concentrations of IGF-I enhanced glucose transport by 2.5-fold. In addition, tyrosine phosphorylation of the 46- and 52-kDa SHC proteins was high in the basal state and doubled after IGF-I treatment, whereas IGF-I enhanced by 4-fold tyrosine phosphorylation of the 66-kDa SHC band. Furthermore, a 2-fold increase in the Ras. GTP active form was induced upon IGF-I stimulation. Downstream from Ras, IGF-I increased both Raf kinase and protein kinase C (PKC) zeta activities by 3.5-fold. (Bu)2cAMP, an inhibitor of IGF-I-induced mitogenesis in fetal brown adipocyte primary cultures, did not block the very early steps of the IGF-I-induced mitogenic cascade, such as IGF-I receptor autophosphorylation, IRS-1 or SHC tyrosine phosphorylation, and Ras activation to its GTP active form. However, (Bu)2cAMP disrupted IGF-I-Raf and IGF-I-PKC zeta signaling pathways by preventing IGF-I-induced Raf-1 kinase and PKC zeta enzymatic activities, respectively. Our results show the first characterization in situ of an IGF-I mitogenic signaling cascade that downstream Ras diverges to the nucleus through two different serine/threonine kinases (Raf-1 kinase and PKC zeta) in mammalian fetal primary cells under physiological conditions. Both kinases represent a point of regulation primarily described for IGF-I-induced, cAMP-inhibited mitogenic pathways.

Prolonged treatment with recombinant insulin-like growth factor-I in children with growth hormone insensitivity syndrome--a clinical research center study. GHIS Collaborative Group.

Eight children with GH insensitivity syndrome, five with GH receptor deficiency (Laron syndrome) and three with growth-attenuating antibodies to GH, were treated with recombinant human insulin-like growth factor I (IGF-I) for 24 months (one was treated for 36 months). Their ages at the beginning of therapy ranged from 2-11 yr. The dose of IGF-I ranged between 80-120 micrograms/kg, given sc twice daily. During the first year of treatment, height velocity (HV) improved in each patient (mean pretreatment HV, 4.0 cm/yr; mean of first year, 9.3 cm/yr). HV declined by 33% during the second year (mean HV, 6.2 cm/yr). The third year HV of the one patient so treated was approximately the same as that in the second year. The mean SD score HV before therapy was -2.4 and improved to +3.8 and +0.5 after 1 and 2 yr of therapy, respectively. Increased HV was accompanied by weight gain. IGF-I-related hypoglycemia occurred infrequently and only early in treatment. No adverse changes in biochemical profile were observed. Bone age did not advance more rapidly than chronological age (mean change in bone age, 2.1 yr; mean change in chronological age, 2.2 yr). The growth of the spleen and kidneys (determined by ultrasound) was rapid in the first year of therapy. In the second year, spleen growth slowed to a normal rate in most patients. Kidney growth, however, remained relatively rapid. These results indicate that IGF-I stimulates statural growth for at least 2 yr and confirms that this peptide has the capacity to act through endocrine mechanisms. Prolonged treatment of GH insensitivity syndrome patients shows promise. The stimulation of growth by IGF-I treatment over years needs to be documented, and patients need to be monitored for side-effects.

Insulin-like growth factor I (IGF-I) delays the onset of diabetes in non-obese diabetic (NOD) mice

It has been shown that prophylactic exogenous insulin treatment prevents the development of insulin-dependent diabetes mellitus (IDDM) in animal models and humans. In this study, we examined whether the development of diabetes and insulitis in female non-obese diabetic (NOD) mice could be affected by prophylactic administration of insulin-like growth factor I (IGF-I), which shares structural homology with insulin and has insulin-like metabolic effects. Two experiments which differed in duration and dosage of IGF-I treatment were carried out. In the first experiment, animals were treated from 4 to 9 weeks of age with IGF-I (17.9 nmol/day at 4–5 weeks of age and 35.9 nmol/day at 6–9 weeks of age) and observed up to 34 weeks of age. In the second experiment, the animals were treated from 4 to 34 weeks of age with IGF-I (1.79 nmol/day at 4–5 weeks of age, 3.59 nmol/day at 6–9 weeks of age, and 5.38 nmol/day at 10–34 weeks of age). The former treatment could significantly delay the onset of diabetes ( P < 0.05) and decrease the insulitis score at 10 weeks of age ( P < 0.01). On the other hand, the latter treatment did not affect the incidence of diabetes, the age at onset or the insulitis score. Our results suggest that the IGF-I treatment at the early age may provide protection against autoimmune β-cell destruction in NOD mice.

Enhanced expression of vascular endothelial growth factor in human SaOS-2 osteoblast-like cells and murine osteoblasts induced by insulin-like growth factor I.

Formation of new capillaries, a critical component of tissue growth and repair, is a recognized process in the development, formation, and remodeling of bone. Vascular endothelial growth factor (VEGF), a potent angiogenic factor with specific mitogenic actions on endothelial cells, is produced in a regulated manner by many cell types, including osteoblasts. The aim of the present investigation was to test the hypothesis that insulin-like growth factor I (IGF-I), a known osteogenic factor, modulates VEGF expression in osteoblasts. In human SaOS-2 osteoblast-like cells, 10 nM IGF-I increased the abundance of VEGF messenger RNA (mRNA) by 4-fold above the control value at 2h, and the elevated levels of mRNA returned to near basal by 8 h. IGF-I stimulated VEGF mRNA levels at IGF-I concentrations as low as 1-2 nM. The stability of VEGF mRNA was not increased after IGF-I treatment, and actinomycin D abrogated the enhanced expression of VEGF mRNA by IGF-I, indicating that the action of IGF-I was probably mediated by a transcriptional mechanism. The induction of VEGF mRNA by IGF-I in SaOS-2 cells was associated with an increase in immunoreactive VEGF protein, as detected by immunoblot analysis. IGF-I also increased the expression of VEGF mRNA in primary murine osteoblasts, which confirmed that the actions of IGF-I were not unique to SaOS-2 cells. We conclude that IGF-I enhances osteoblast synthesis of VEGF, which may then act locally on endothelium to stimulate angiogenesis, an essential component of bone growth and remodeling.

Growth hormone and insulin-like growth factor-I and mesangial matrix in uremic rats.

Combined growth hormone (GH) and insulin-like growth factor-I (IGF-I) therapy has been advocated for clinical use to minimize the diabetogenic effect of GH and enhance their anabolic effects. However, GH has been shown to accelerate the development of glomerular sclerosis in experimental animals and IGF-I mediates the renal effects of GH. The purpose of this study was therefore to examine morphometrically the effects of GH (1 mg intraperitoneally three times a week), IGF-I (50 micrograms/kg body weight subcutaneously twice a day), and combined GH/IGF-I treatments in vivo on mesangial matrix at 3-20 days after 5/6 nephrectomy in 140- to 150-g rats. There were no significant changes in growth and renal function after GH and/or IGF-I treatment. The effects of GH and IGF-I on glomerular size were additive, which were more prominent in juxtamedullary glomeruli. GH induced proportional increases in mesangial area (MA) and glomerular area (GA), whereas IGF-I induced a similar increase in GA without a corresponding change in MA. When compared with GH treatment alone, combined GH/ IGF-I treatment resulted in a lesser degree of mesangial expansion despite an enhanced glomerular size. While additional studies are needed to examine the long-term effects of these findings, our results suggest a potentially beneficial effect of combined GH/IGF-I therapy during uremia.

The Proto-oncogene Product c-Crk Associates with Insulin Receptor Substrate-1 and 4PS: MODULATION BY INSULIN GROWTH FACTOR-I (IGF) AND ENHANCED IGF-I SIGNALING

The Crk proto-oncogene product is an SH2 and SH3 domain-containing adaptor protein which we have previously shown to become rapidly tyrosine phosphorylated in response to stimulation with insulin-like growth factor I (IGF-I) in NIH-3T3 cells. In order to further characterize the role of Crk in the IGF-I signaling pathway, NIH-3T3 and 293 cells were stably transfected with an expression vector containing the Crk cDNA. The various resultant 3T3-Crk clones expressed Crk at approximately 2-15-fold higher levels than parental 3T3 cells. In 3T3-Crk cells, Crk immunoreactivity was detected in insulin receptor substrate-1 (IRS-1) immunoprecipitates. Stimulation with IGF-I resulted in a dissociation of Crk protein from IRS-1. In contrast, the association of the related adaptor protein Grb2 with IRS-1 was enhanced by IGF-I stimulation. Similar results were obtained in stably transfected 293-Crk cells, which express both IRS-1 and the IRS-1-related signaling protein 4PS. In these cells, IRS-1 and 4PS both associated with Crk, and this association was also decreased by IGF-I treatment, whereas the association of Grb2 with IRS-1 and 4PS was enhanced by IGF-I. Overexpression of Crk also enhanced IGF-I-induced mitogenesis of NIH-3T3 cells, as measured by [3H]thymidine incorporation. The levels of IGF-I-induced mitogenesis were proportional to the level of Crk expression. These results suggest that Crk is a positive effector of IGF-I signaling, and may mediate its effects via interaction with IRS-1 and/or 4PS.

Ligand occupancy of the alpha-V-beta3 integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth factor.

Smooth muscle cells (SMCs) have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the mechanism mediating this response has not been determined. The migration rates of porcine and human vascular SMCs were assessed in a monolayer wounding assay. IGF-I and IGF-II induced increases of 141% and 97%, respectively, in the number of cells that migrated in 4 days. The presence of 0.2% fetal bovine serum in the culture medium was necessary for the IGFs to stimulate migration over uncoated plastic surfaces. However, if vitronectin was used as the substratum, IGF-I stimulated migration by 162% even in the absence of serum. To determine the role of integrins in mediating this migration, SMC surface proteins were labeled with 125I and immunoprecipitated with specific anti-integrin antibodies. Integrins containing alpha-V (vitronectin receptor), alpha5 (fibronectin receptor), and alpha3 (collagen/laminin receptor) subunits were the most abundant. IGF-I treatment caused a 73% reduction in alpha5-integrin subunit protein and a 25% increase in alpha-V subunit. More importantly, ligand binding of alpha-V-beta3 was increased by 2.4-fold. We therefore examined whether the function of the alpha-V-beta3 integrin was important for IGF-I-mediated migration. The disintegrin kistrin was shown by affinity crosslinking to specifically bind with high affinity to alpha-V-beta3 and not to alpha5-beta1 or other abundant integrins. The related disintegrin echistatin specifically inhibited 125I-labeled kistrin binding to alpha-V-beta3, while a structurally distinct disintegrin, decorsin, had 1000-fold lower affinity. The addition of increasing concentrations of either kistrin or echistatin inhibited IGF-I-induced migration, whereas decorsin had a minimal effect. The potency of these disintegrins in inhibiting IGF-I-induced migration paralleled their apparent affinity for the alpha-V integrin. Furthermore, an alpha-V-beta3 blocking antibody inhibited SMC migration by 80%. In summary, vitronectin receptor activation is a necessary component of IGF-I-mediated stimulation of smooth muscle migration, and alpha-V-beta3 integrin antagonists appear to be important reagents for modulating this process.

Treatment with insulin-like growth factor I alters capillary permeability in skin and retina

Treatment with insulin-like growth factor I (IGF-I) is accompanied by mild generalized and reversible edema. These changes may be due to increased capillary permeability. Therefore, we studied the effects of subcutaneous IGF-I treatment in healthy subjects on capillary permeability of the skin and the retina. Eight healthy subjects were treated with saline or recombinant human IGF-I (rhIGF-I) (10 μg · kg−1 · h−1 s.c.) in a randomized crossover fashion. Transcapillary diffusion of sodium fluorescein (NaF) was quantitated by video densitometry in the skin in all subjects on the 4th treatment day. In seven subjects, plasma penetration of NaF at the blood-retinal barrier was investigated using vitreous fluorometry (VF) on days 3 and 5. Fluorescent light intensities of the skin and plasma penetration determined by VF were significantly higher during the IGF-I treatment as compared with those during the control situation. In conclusion, IGF-I treatment at the above dose is accompanied by increased transcapillary diffusion of NaF in skin and in retinal vessels.

Responses of young energy-restricted sheep to chronically administered insulin-like growth factor I (IGF-I): evidence that IGF-I suppresses the hepatic growth hormone receptor.

We have shown previously that chronic administration (8 weeks) of insulin-like growth factor-I (IGF-I) has little growth-promoting effect in well fed sheep. The aim of this study was to investigate the anabolic effects of IGF-I in energy-restricted conditions in which circulating concentrations of IGF-I in control animals were expected to be low. Young castrate male sheep were offered chaffed lucerne at a rate equivalent to 110% maintenance and were treated by sc injection three times per day for either 8 or 12 weeks with recombinant human IGF-I (150 micrograms/kg live wt x day) or saline in a 2 x 2 factorial design (eight animals per cell). IGF-I treatment significantly increased plasma IGF-I concentrations, but reduced plasma concentrations of IGF-II, GH, urea, and creatinine. Treatment with IGF-I also decreased (P < 0.1) GH secretion in response to a GRF load, but significantly (P < 0.05) increased the nonesterified fatty acid response to an epinephrine load. The reduction in circulating GH levels was accompanied by a suppression of [125I]oGH binding to hepatic microsomal membranes. This effect, if apparent in other tissues, may act as a feedback mechanism to limit the local synthesis of IGF-I and could explain why IGF-I treatment had little effect on the growth rate of the sheep, although it did increase nitrogen digestibility of the feed consumed and decreased the fat content of the hind leg. It also differentially promoted the growth of the spleen, thymus, and mandibular salivary gland and increased blood counts of eosinophils. It is concluded that IGF-I does not have marked effects on growth rate or body composition in sheep fed a near-maintenance diet. Possible reasons include the associated suppression of GH secretion and action, which limits the ability of treated animals to repartition absorbed nutrients.