The insulin-like growth factor system in the GT1-7 GnRH neuronal cell line

Evidence suggests that insulin-like growth factors (IGFs; IGF-I and IGF-II) are involved in the regulation of reproductive function including the development of the gonadotropin-releasing hormone (GnRH) neuronal system and the modulation of GnRH secretory activities. To further characterize the regulatory role of the IGF system on GnRH neuronal function, we have examined the gene expression of IGF-I, IGF-II, IGF-I receptor (IGF-IR), and IGF-binding proteins (IGFBPs) in a GnRH neuronal cell line (GT1-7 cells). The relative effects of IGFs and insulin on GnRH secretion by these cells was also investigated. RT-PCR analysis demonstrated IGF-I, IGF-II and IGF-IR mRNAs in GT1-7 cells. The mRNAs for IGFBP-2, -3, -4, -5 and -6 but not IGFBP-1 were also detected. Immunoreactive protein bands for IGFBP-2, -4 and -5 but not for other IGFBPs were demonstrated by Western blot with IGFBP-5 appearing to be the most abundant IGFBP secreted by GT1-7 cells. IGFBP-5 production by GT1-7 cells was stimulated by both IGF-I and IGF-II in a dose-dependent manner with approximately equal potency, whereas insulin caused no significant effect. GnRH secretion by GT1-7 cells treated with IGF-I or IGF-II but not insulin showed an increase (80-100%) at 2 h of treatment followed by a decrease (46%) at 6 h that continued up to 24 h. We conclude that the expression of IGFs, IGF-IR and IGFBPs and their interactions in the regulation of GnRH secretion by GT1-7 cells as demonstrated by our study provide a basis for an autocrine regulatory role for the IGF system in GnRH neuronal secretory activities.     

Administration of human recombinant insulin-like growth factor-I to patients following major gastrointestinal surgery

OBJECTIVE: The aim was to study the pharmacokinetic parameters and biological activity of a single dose of human recombinant IGF-I (rhIGF-I) administered to patients following major gastrointestinal surgery. DESIGN: A double blind placebo controlled externally randomized study of 30 patients; the study commencing 24 hours after major colonic or gastric surgery. MEASUREMENTS: After a baseline blood sampling day, IGF-I (40 micrograms/kg by single subcutaneous dose, n = 20) or placebo (n = 10) was administered and serum and urine samples collected over the ensuing 72 hours. Serum IGF-I, IGF-II, IGF binding proteins (IGFBP-1, IGFBP-3), GH and insulin were measured by radioimmunoassay. Serum IGF bioactivity was assessed using a validated porcine cartilage bioassay. Serum and urinary electrolytes were measured by standard methodology. RESULTS: Serum immunoreactive IGF-I levels peaked at 4 hours following injection of IGF-I (1.09 +/- 0.12 U/ml mean +/- SEM), remained elevated for 15 hours and returned to basal levels by 24 hours after injection. IGF bioactivity was increased by 57% 6 hours after IGF-I injection. Mean levels of IGFBP-1 and IGFBP-3, IGF-II and GH were unaffected by IGF-I administration. Insulin levels were suppressed at 30 minutes following injection of IGF-I compared with the placebo group (16.9 +/- 3.0 mU/I vs 32.3 +/- 7.1, P = 0.02); thereafter, there were no differences in insulin levels. The mean change in serum creatinine following IGF-I (-6.3 +/- 3.0 mmol/l) was significantly different from that in the control group (+7.2 +/- 6.2, P = 0.03). Creatinine clearance rose from a mean of 71.6 +/- 7.5 ml/min to 83.2 +/- 7.6 ml/min after IGF-I treatment (P = 0.02). In the IGF treated patients, cholesterol levels consistently fell (-0.20 +/- 0.05 mmol/l); this was not observed in the placebo group (+0.20 +/- 0.14, P = 0.006). Basal serum potassium levels in the IGF treatment group (4.1 +/- 0.1 mmol/l) fell to 3.8 +/- 0.1 at 4 hours (P = 0.002) and 3.6 +/- 0.1 at 10 hours (P = 0.001) returning to a level of 4.0 +/- 0.1 (P = 0.293) at 24 hours after injection. There were no other observed differences in serum or urinary electrolytes or serum free fatty acids and triglycerides. Pharmacokinetic parameters derived from baseline adjusted IGF-I measurements revealed a slow absorption of the administered dose with a Tmax of 5.0 +/- 0.43 hours and an elimination half-life of 10.8 +/- 1.2 hours. The computed volume of distribution was 0.33 +/- 0.05 I/kg and the clearance on average 25 ml/min. CONCLUSION: A single subcutaneous dose of IGF-I normalized circulating IGF-I levels in post-operative patients, was well tolerated and without side-effects. IGF bioactivity was increased and associated with a fall in serum cholesterol, potassium and creatinine levels and a rise in creatinine clearance. Further long-term studies are now required to assess the anabolic effects of rhIGF-I in this type of patient group.     

Coordinated control of fetal gastric epithelial functions by insulin-like growth factors and their binding proteins

The influence of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) on human gastric functions are unknown. This study was undertaken to evaluate the ability of fetal gastric mucosa to produce IGFBPs and to test the effects of IGF-I, IGF-II, and synthetic truncated IGFs that do not interact with IGFBPs on epithelial cell proliferation and glandular zymogenic function. Western blots, Far Western blots, and immunohistochemistry were performed to characterize the expression of IGFBP-1 to -6 and IGF-I receptor. The effects of growth factors on DNA synthesis and lipase and pepsin activities were determined in gastric explants maintained in serum-free organ culture. All gastric epithelial cells expressed the IGF-I receptor. IGFBP-2 to -6 were produced endogenously, and they were differentially localized along the foveolus-gland axis and modulated in culture. Exogenous IGF-I and IGF-II were able to reduce lipase activity without affecting pepsin, whereas they exerted different effects on cellular proliferation: IGF-I was stimulatory and IGF-II had no influence. Illustrating the complex regulatory effect that IGFBPs exert on IGF bioactivity, both truncated IGF-I and IGF-II stimulated DNA synthesis more than IGF-I. Moreover, the striking difference in mitogenic activity between truncated and native forms of IGF-II probably reflects the abundance of IGFBP-2 and IGFBP-6, two IGF-II carriers, in the foveolus/neck region, including the proliferative compartment. This study provides new evidence for the involvement of an intragastric IGF/IGFBP system in the fine regulation of epithelial cell division and also in the control of zymogen synthesis. Moreover, the specific influence of IGF-II as a mitogen appears to be tightly regulated by IGFBP isoforms preferentially associated with this growth factor and proliferative cells.     

Insulin-like growth factor II and transforming growth factor beta 1 regulate insulin-like growth factor I secretion in mouse bone cells.

Bone cells in culture produce and respond to growth factors, suggesting that local as well as systemic factors regulate bone volume. Previous studies have shown that IGF-I is the major mitogen produced by mouse bone cells and that its production is regulated by systemic agents such as PTH and estrogen. Because IGF-II and transforming growth factor beta 1 have been shown, respectively, to increase and decrease MC3T3-E1 cell proliferation, we tested the hypothesis that these two growth factors modulate the production of IGF-I in this cell line. In order to eliminate artifacts owing to IGF binding proteins, conditioned media samples were pretreated with IGF-II before measurement of IGF-I by RIA. After 24 h treatment at a density of 2.5 x 10(4) cells/cm2, IGF-II (10 micrograms/l) induced a 2.2-fold increase compared with untreated control (9.5 +/- 1.5 vs 4.2 +/- 0.44 pg/micrograms protein, p less than 0.001), whereas transforming growth factor beta 1 (1 microgram/l) caused a 66% decrease in IGF-I production (1.5 +/- 0.3 vs 4.2 +/- 0.44 pg/micrograms protein, p less than 0.001). Both IGF-II and transforming growth factor beta 1 regulated IGF-I production in a dose-, time- and cell density-dependent manner. The lowest effective doses for IGF-II and transforming growth factor beta 1 were 1 and 0.01 microgram/l, respectively. These results support a role for IGF-II and transforming growth factor beta 1 as potent modulators of IGF-I secretion in mouse bone cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Integrin-mediated action of insulin-like growth factor binding protein-2 in tumor cells

The neoplastic production of the insulin-like growth factor binding protein (IGFBP)-2 often correlates with tumor malignancy and aggressiveness. Since IGFBP-2 contains an RGD motif in its C-terminus, it was hypothesized that this protein may act independently of IGF on tumor cells through integrins. To investigate this, integrin binding, intracellular signaling and the impact of IGFBP-2 on cell adhesion and proliferation were examined in two tumor cell lines. In tracer displacement studies, up to 30% of the added (125)I-hIGFBP-2 specifically bound to the cells. Bound (125)I-hIGFBP-2 was reversibly displaced by IGFBP-2, IGFBP-1 and RGD-(Gly-Arg-Asp)-containing peptides, but not by IGFBP-3, -4, -5, -6 and RGE-(Gly-Arg-Glu)-containing peptides. Blocking with antibodies directed against different integrins and with fibronectin demonstrated that IGFBP-2 cell surface binding is specific for alpha5beta1-integrin. Incubation of IGFBP-2 with equimolar quantities of IGF-I and IGF-II annihilated RGD-specific binding. IGFBP-2 binding at the cell surface led to dephosphorylation of the focal adhesion-kinase (FAK) of up to 37% (P<0.01), and of the p42/44 MAP-kinases of up to 40% (P<0.01). In addition, IGFBP-2 promoted de-adhesion of the cells dose-dependently by up to 30% (P<0.05), and reduced proliferation by 24% (P<0.01). Since one of the cell lines used does not express a functional IGF-I receptor, these data demonstrate that IGFBP-2 can act in an IGF-independent manner, at least in part by an interaction with alpha5beta1-integrin.     

Circulating insulin-like growth factors and related binding proteins are selectively altered in amyotrophic lateral sclerosis and multiple sclerosis

OBJECTIVE: To provide a detailed profile of the peripheral IGF system in the neurological conditions; amyotrophic lateral sclerosis (ALS), post polio syndrome (PPS) and multiple sclerosis (MS). To determine whether subsets of patients within the disease groups could be identified in whom one or more components of the IGF regulatory system are altered compared to healthy control subjects matched for age, sex and BMI. DESIGN: Three cohorts of patients were recruited, 28 with ALS, 18 with PPS and 23 with MS. Patients were individually matched to a healthy control based on sex, age (+/-3 yr), and BMI (+/-2.5 kg m(-2)). The concentration (ng/ml) of serum IGF-I, IGF-II, IGFBP-1, IGFBP-2 and IGFBP-3 and acid-labile subunit (microg/ml) was determined by IRMA. RESULTS: In ALS patients, there was an increase of 11% in [IGF(TOTAL)] (p=0.042) ([IGF(TOTAL)]=[IGF-I]+[IGF-II]) and [IGFBP-1] was decreased by 34% (p=0.050) compared to matched controls. In "surviving" ALS patients, defined as those ALS patients with long disease duration (+2 SD from the mean survival time for Irish patients post diagnosis), there was an increase in [IGF-I] 36% (p=0.032) and a large decrease in [IGFBP-1] -58% (p=0.020) compared to controls. These differences were not evident in pre-agonal ALS patients. The concentration of serum IGF-I was 38% (p=0.018), acid-labile subunit 17% (p=0.044) and IGFBP-2 43% (p=0.035) higher in MS patients compared to controls. When stratified for interferon-beta (IFN-beta) use, we observed an increase in serum [IGF-I] 52% (p=0.013) and [IGF(TOTAL)] 19% (p=0.043) in MS patients undergoing IFN-beta treatment, but MS patients not undergoing IFN-beta treatment had similar IGF and IGFBP concentration to controls. Serum [IGFBP-3] 18% (p=0.033), [IGFBP-2] 86% (p=0.015) and (acid-labile subunit) 33% (p=0.012) was also higher in IFN-beta patients compared to controls. Stratified by stage of disease the most significant increase in components of the peripheral IGF system was attributed to relapsing-remitting MS patients treated with IFN-beta. All components of the peripheral IGF system in PPS patients were similar to controls. CONCLUSIONS: The increase in circulating IGF-I and a reduction in regulatory binding protein IGFBP-1 in ALS patients with a "stable" disease profile suggest a potential change in peripheral IGF bioavailability in these subjects. In MS, we report a change in a number of components of the peripheral IGF system, the observed increase in IGF-I in patients treated with IFN-beta being of most significance as a potential therapeutic biomarker.     

Evidence for a novel insulin-like growth factor (IGF)-dependent protease regulating IGF-binding protein-4 in dermal fibroblasts.

The mechanisms by which insulin-like growth factors (IGFs) reduce IGF-binding protein-4 (IGFBP-4) levels in cellular conditioned media are poorly understood. The effect of IGFs on IGFBP-4 levels in fibroblast conditioned media is not mediated via the type 1 or type 2 cellular IGF receptors, and the IGFs exert little or no effects on IGFBP-4 messenger RNA levels in human adult fibroblasts or in rat neuroblastoma cells. To determine whether the effects of IGFs on IGFBP-4 might be exerted through alterations in IGFBP-4 degradation, we incubated cell-free, fibroblast-conditioned media from either sheep or human dermal fibroblasts with or without IGF-I, IGF-II (each 1 microgram/ml), or insulin (10 micrograms/ml) for 72 h at 37 C. Samples were then analyzed by Western ligand blot using radiolabeled IGFs and by immunoblotting using a polyclonal antisera to human IGFBP-4. In the absence of IGFs, no apparent changes in the basal concentrations of the various IGFBPs were observed. In contrast, incubation of media with IGFs caused a 70-80% reduction in levels of both sheep and human IGFBP-4, whereas incubation with insulin was without effect. Similarly, incubation of cell-free conditioned media containing recombinant human IGFBP-4 with IGF-I caused a reduction in detectable levels of the 28K protein. The decrease in IGFBP-4 levels was accompanied by the appearance of an immunoreactive approximate 17-20K fragment that did not bind radiolabeled IGFs by ligand blot. The IGF-dependent decrease in IGFBP-4 was prevented by coincubation of the media with serine protease inhibitors, EDTA, or 1,10-phenanthrolene, suggesting that IGFs may activate an IGFBP-4 specific metallo-serine protease present in fibroblast conditioned media. Alternatively, binding of IGF-I or -II to IGFBP-4 may enhance the susceptibility of IGFBP-4 to proteolytic degradation. The demonstration that IGF-I and IGF-II can promote directly the proteolytic degradation of IGFBP-4 into fragments that do not bind IGFs provides a novel mechanism by which the IGFs may increase their own availability and/or activity in biological fluids.     

Insulin-like growth factors (IGFs) reduce IGF-binding protein-4 (IGFBP-4) concentration and stimulate IGFBP-3 independently of IGF receptors in human fibroblasts and epidermal cells.

Recent studies have provided a consensus that insulin-like growth factor-I (IGF-I) stimulates IGF-binding protein-3 (IGFBP-3) in vivo and in vitro. While it also appears well established that IGFBP-1 is inversely related to insulin concentrations, evidence regarding regulation of other IGFBP is inconclusive. Using immunoprecipitation and Western ligand blot, we have characterized the IGFBPs released into conditioned medium (CM) by cells from the adult human fibroblast cell line N3652 and the human epidermal squamous cell carcinoma line SCL-1. N3652 cells expressed IGFBP-3, IGFBP-2, a 24-kilodalton (kDa) IGFBP presumed to be IGFBP-4, and IGFBPs at 30 and 28 kDa. SCL-1 expressed IGFBP-3 and a putative IGFBP-4, with intermediate bands at 34 and 30 kDa. As determined by ligand blot of CM from confluent cells 72 h after the addition of peptides to serum-free medium, IGF-I and IGF-II potently stimulated IGFBP-3 in both cell lines, but otherwise IGFBP regulation in the two cells diverged. In N3652 cells, IGFBP-3 concentrations in CM increased to 700% and 800% of basal levels in the presence of IGF-I and IGF-II (at 100 ng/ml; n = 5 experiments), respectively. IGFBP-3 was not affected by insulin up to 10 micrograms/ml. In contrast, IGFBP-4 levels were diminished 54% and 73% by 100 ng/ml IGF-I and IGF-II, respectively, with no response to insulin. In SCL-1 cells, IGF-I and IGF-II were virtually identical in stimulating a mean 200% increase in IGFBP-3 (n = 5 experiments). Insulin was less potent, but caused a significant stimulation of IGFBP-3 levels. IGF-I, IGF-II, and insulin all stimulated an approximately 50% increase in IGFBP-4 concentrations. To test the hypothesis that IGF-induced alterations in IGFBP-3 and IGFBP-4 concentrations were regulated via the type 1 IGF receptor, we attempted to block IGFBP changes with type 1 IGF receptor antibody alpha IR-3 and to induce IGFBP changes with an IGF-II analog, [Leu27]IGF-II, with little affinity for the type 1 receptor. alpha IR-3 failed to block either the IGF-induced rise in IGFBP-3 in each cell line or the decline in IGFBP-4 in N3652 CM. [Leu27]IGF-II was as potent as IGF-II or IGF-I in inducing changes in IGFBP-3 and IGFBP-4 concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of human fibroblast insulin-like growth factors binding protein (IGFBP) production by IGFBP-3.

Human neonatal fibroblasts in monolayer culture secrete a number of insulin-like growth factor binding proteins (IGFBPs), including IGFBP-3, which may alter paracrine or autocrine IGF activity. Studies in vitro have demonstrated that exogenous IGFBP-3 can both inhibit and potentiate IGF action in these cells; however, it is not known to what extent there is regulatory interaction between the IGFBPs. In this study we report that exogenous and endogenous IGFBP-3 inhibit production of an IGF inducible IGFBP. When analyzed by SDS-PAGE and [125I]IGF-II ligand blotting, human neonatal fibroblasts secrete IGFBP-3, an IGFBP of 29-31 kDa, and a 22-24 kDa IGFBP after treatment with 50 ng/ml IGF-I. When IGF-I treatment was carried out in the presence of increasing concentrations (50-1000 ng/ml) of pure human serum-derived IGFBP-3, there was a dose-dependent decrease in the 29-31 kDa protein. In the presence of excess (250 ng/ml) IGF-I, IGFBP-3 had approximately 20-fold reduced potency in inhibiting 29-31 kDa IGFBP. When endogenous production of IGFBP-3 was increased by treatment with transforming growth factor-beta 1 (TGF beta 1), there was complete inhibition of 29-31 kDa IGFBP, while at high IGF-I concentrations TGF beta 1 had 2 to 3-fold reduced potency. These results demonstrate that fibroblast IGFBP production can be altered by exogenous and endogenous IGFBP-3, and suggest the existence of regulatory interactions between fibroblast IGFBPs.     

The divergent effect of insulin-like growth factor binding protein (IGFBP) - 1 on IGF-induced Steroidogenesis in bovine adrenocortical cells is not due to its phosphorylation status.

In previous studies we have shown that insulin-like growth factor (IGF)-II stimulates basal as well as ACTH-induced cortisol secretion from bovine adrenocortical cells more potently than IGF-I. The steroidogenic effect of both, IGF-I and IGF-II, is mediated through the IGF-I receptor and modified by locally produced IGF binding proteins. Further studies demonstrate that bovine adrenocortical cells do synthesize IGFBP-1 to -4 and that their secretion is regulated differentially by IGFs and ACTH. Since IGFBP-1 selectively is induced 3- to 4- fold by ACTH, the aim of the following studies was to evaluate the effect of IGFBP-1 on IGF-induced cortisol secretion from bovine adrenocortical cells in primary culture. Coincubation of bovine adrenocortical cells with purified IGFBP-1 (30 nM) induced a time--and dose-dependent potentiating effect (38+/-2%) on IGF-I-stimulated (6.5 nM) cortisol-secretion, wheras the IGF-II induced steroidogenic effect was inhibited (40+/-3%) by IGFBP-1. Similar results were found when bovine adrenocortical cells were preincubated with IGFBP-1 before stimulation experiments with IGFs were performed. In order to evaluate the influence of different phosphorylation states of IGFBP-1 on the steroidogenic effect of IGF-I and IGF-II and on the affinity to IGFs, a highly phosphorylated (pIGFBP-1) and a dephosphorylated isoform (dIGFBP-1) of IGFBP-1 have been separated by anion exchange chromatography for further incubation experiments and binding studies. No different effects on IGF-I or IGF-II-induced steroidogenesis were observed when a highly phosphorylated IGFBP-1 isoform was used, compared to the dephosphorylated IGFBP-1 isoforms. In binding studies IGFBP-1 did show high affinity binding for IGF-I with a calculated association constant (K (a)) of 2,17 x 10 (9) M (-1). Similar association constants were calculated for dIGFBP-1 and pIGFBP-1 (1,93 x 10 (9) M (-1) and 2,67 x 10 (9) M (-1) respectively) and no difference in binding capacity was observed when IGF-II was used as ligand. IN CONCLUSION, these results demonstrate that in bovine adrenocortical cells IGFBP-1 time- and dose-dependently inhibits the steroidogenic effect of IGF-II and stimulates the effect of IGF-I. The observed modulatory effect of IGFBP-1 is independent of the mode of incubation and its phosphorylation status. The previously observed stronger steroidogenic potency of IGF-II in bovine adrenocortical cells therefore can not be explained by an interaction with IGFBP-1. The mechanisms by which IGFBP-1 divergently regulates the steroidogenic effect of IGF-I and IGF-II remain unclear at present and further investigation is necessary to elucidate the mechanisms by which IGFBP-1 modulates IGF action in the adult adrenal gland.     

Changes in systemic levels of insulin-like growth factors and their binding proteins in patients with rheumatoid arthritis

OBJECTIVE: To determine whether circulating levels of insulin-like growth factors and their binding proteins are altered in patients with adult onset rheumatoid arthritis. METHODS: Plasma-levels of insulin-like growth factor-I (IGF-I), IGF-II, IGF-binding-protein 2 (IGFBP-2), and IGFBP-3 were measured by radioimmunoassay in 53 patients with clinically active rheumatoid arthritis (RA) and in 51 control subjects. RESULTS: In RA patients plasma levels of IGF-II were lower (601 +/- 34 vs. 731 +/- 32 micrograms/L (mean +/- SEM); p = 0.005; Mann-Whitney rank sum test) than in age- and sex-matched controls (n = 30 per group). In contrast, plasma levels of IGFBP-2 (412 +/- 40 vs. 254 +/- 20 micrograms/L; p = 0.003) and IGFBP-3 were elevated in RA patients (3.34 +/- 0.19 vs. 2.87 +/- 0.21 mg/L; p = 0.019) as compared with the matched controls. The molar ratio of IGF-I to IGFBP-3 was significantly reduced in subjects with RA (0.18 +/- 0.01 vs. 0.24 +/- 0.02; p = 0.008). Furthermore, in RA patients plasma levels of IGFBP-2 were positively (r = 0.45), and levels of IGF-2 negatively (r = -0.45) correlated with circulating levels of C-reactive protein (p < 0.01 in both cases; Spearman rank correlation). CONCLUSION: Increased levels of IGFBPs in RA may result in the reduced availability of free IGFs that can bind to IGF receptors. The observed changes in the IGF system may thus participate in the catabolic processes in rheumatoid arthritis.     

Autocrine regulation of human tumor cell proliferation by insulin-like growth factor II: an in-vitro model.

We have shown that a pleomorphic cell line of abnormal human karyotype derived from a stomach carcinoma (LIM-1839) proliferates in serum-free medium, expresses insulin-like growth factor II (IGF-II) mRNA, and secretes IGF-II (up to 56 ng/ml in serum-free conditioned medium, as measured in a rat liver RRA. No detectable levels of IGF-I can be measured in serum-free conditioned medium by RIA. These cells also secrete IGF-binding proteins, detected by a charcoal adsorption assay. The release of IGF-II and IGF binding proteins into serum-free conditioned medium (1.7 pmol/10(6) cells.24 h and 0.8 pmol binding sites/10(6) cells.24 h for 3 days, respectively) is inhibited 80% by cycloheximide (10 micrograms/ml). The LIM-1839 cells have type I and type II IGF receptors, determined by affinity cross-linking and competition binding studies. These cells proliferated 1.6-fold over 4 days in serum-free medium, with fresh medium changes on days 0 and 2: their growth was inhibited 56% by 40 micrograms/ml Sm 1.2, a monoclonal antibody which recognizes IGF-I and IGF-II. The addition of 20 and 50 ng/ml multiplication stimulating activity (rat IGF-II) caused 1.8- and 1.7-fold increases in cell growth between days 0 and 4 compared to controls, while [Thr59]IGF-I, at 20 and 50 ng/ml, caused 1.6- and 2.0-fold increases. Insulin, at 2 and 10 micrograms/ml, had no significant effect. The stimulatory effects of endogenous and exogenous IGFs on LIM-1839 cell proliferation were inhibited by a monoclonal antibody to the type I IGF receptor, alpha IR-3. These results suggest that the LIM-1839 cells are biologically responsive to endogenously produced IGF-II, and may thereby provide an in vitro model for autocrine regulation of human tumor growth by IGF-II.     

Associations of childhood and adulthood height and the components of height with insulin-like growth factor levels in adulthood: a 65-year follow-up of the Boyd Orr cohort

CONTEXT: Taller individuals with longer legs have a higher risk of cancer but a lower risk of coronary heart disease. OBJECTIVE: We investigated whether childhood height and its components are associated with the IGF system in adulthood. DESIGN AND PARTICIPANTS: We analyzed data from 429 participants of the Boyd Orr cohort, for whom height measured in childhood (mean age, 7.4 yr) in 1937-1939 could be related to levels of IGF-I, IGF-II, IGF binding protein (IGFBP)-2, and IGFBP-3 in adulthood (mean age, 71.1 yr). In 385 participants, measured height in adulthood could be related to IGF levels. RESULTS: In fully adjusted models (controlling for age, sex, socioeconomic factors, lifestyle, and body mass index), childhood height and its components were not associated with adult circulating IGF-I, IGF-II, or IGFBP-2 levels. IGFBP-3 was 85.5 ng/ml higher (95% confidence interval, -11.6 to 182.5; P = 0.08) per sd increase in childhood trunk length and 83.6 ng/ml lower (95% confidence interval, -10.3 to 177.5; P = 0.08) per sd increase in childhood leg/trunk ratio. Height in adulthood was not associated with IGF-I, IGF-II, or IGFBP-3 and was inversely associated with IGFBP-2 (P = 0.05) after additionally controlling for childhood height. CONCLUSION: There was no evidence that associations of childhood height with cancer and coronary heart disease risk are mediated by IGF-I in adulthood. The anthropometric associations with IGFBP-2 and IGFBP-3 could be chance findings but warrant additional investigation. IGF levels in childhood may be more important determinants of long-term disease risk than adult levels.     

Association between insulin-like growth factor status and physical activity levels in rheumatoid arthritis

OBJECTIVE: To determine if the altered insulin-like growth factor (IGF) status in rheumatoid arthritis (RA) is due to inflammation, altered body composition, or lack of exercise. METHODS: Subjects included 73 patients with RA, 54 patients with other rheumatic diseases, both inflammatory and noninflammatory, and 28 healthy, physically active controls. Serum levels of IGF-I, IGF-II, and IGF binding protein-3 (IGFBP-3) were measured by radioimmunoassay. Body composition was estimated by bioelectrical impedance analysis, and habitual exercise level approximated by questionnaire. Statistical analysis was performed by 2 and 3 way ANOVA and moderated hierarchical regression. RESULTS: Serum IGF-I (p < 0.001), IGFBP-3 (p < 0.001), and the BP-3:total IGF molar ratio (p < 0.001) were depressed in both patient groups relative to controls. In contrast, IGF-II levels were depressed only in patients with RA (p < 0.01). Differences in the IGF proteins between patients and controls could not be attributed to inflammation. Habitual exercise level, but not body composition, was shown to be a significant predictor for IGF-I, IGFBP-3, and BP-3:total IGF molar ratio (p < 0.001). CONCLUSION: Our results indicate that the reduction in circulating IGF proteins observed in our patients is more related to their sedentary lifestyle than to the inflammatory process. This conclusion is in agreement with reports that show that highly active individuals typically exhibit higher levels of systemic IGF proteins than age matched sedentary controls.     

Post-transcriptional and post-translational regulation of insulin-like growth factor binding protein-3 and -4 by insulin-like growth factor-I in uterine myometrial cells.

Involution of the uterus induced by oestrogen depletion is associated with a decrease in uterine insulin-like growth factor (IGF)-I and an increase in IGF binding protein (IGFBP) gene expression. We examined the effects of IGF-I on primary uterine myometrial cell proliferation, and on IGFBP-3 and IGFBP-4 gene expression. IGF-I enhanced DNA synthesis in these cells. In conditioned media, IGF-I increased IGFBP-3 accumulation by release of cell associated IGFBP-3. A low dose of IGF-I increased IGFBP-4 accumulation, and a high dose caused IGFBP-4 to disappear. In cell-free conditioned media IGF-I protected IGFBP-3 and enhanced IGFBP-4 proteolysis. Co-incubation of [(125)I]-IGFBP-4 with cell-free conditioned media cleaved IGFBP-4 into 18 and 12 kDa fragments. Northern blot analysis indicated that IGF-I increased IGFBP-4 mRNA accumulation by stabilizing the mRNA while IGFBP-3 gene expression was slightly decreased. The results demonstrate that IGF-I regulates IGFBP-4 post-trancriptionally and post-translationally, whereas IGFBP-3 is only affected post-translationally. By enhancing IGFBP-4 proteolysis, increasing cell-associated IGFBP-3 and stabilizing IGFBP-3, IGF-I may initiate a mitogenic response.     

Binding properties of insulin-like growth factor binding protein-3 (IGFBP-3), IGFBP-3 N- and C-terminal fragments, and structurally related proteins mac25 and connective tissue growth factor measured using a biosensor

We measured the binding of IGF-I and IGF-II to recombinant human N-terminal [residues 1-97; recombinant human IGF-binding protein-3(1-97) (rhIGFBP-3(1-97))] and C-terminal (residues 98-264; rhIGFBP-3(98-264)) IGFBP-3 fragments and compared it with IGF binding to intact IGFBP-3 using biosensor analysis. Experiments were carried out in different configurations, either with binding protein or fragment immobilized or with IGF immobilized. These experiments showed that IGF-I and IGF-II bind to IGFBP-3 with affinities of 4-5 x 10(9) M(-1) and similar binding kinetics. The affinities of both rhIGFBP-3(1-97) and rhIGFBP-3(98-264) for IGF proteins were approximately 3 orders of magnitude less than that of full-length IGFBP-3. These results further support the concept that high affinity binding of IGF to IGF-binding proteins results from a two-site interaction of IGF with both the N- and C-terminal regions of the binding protein. Binding of insulin to IGFBP-3 and its N- and C-terminal fragments and of IGF-I and IGF-II to the structurally related proteins mac25 and connective tissue growth factor was also investigated. Weak insulin binding to full-length IGFBP-3 could be demonstrated in a few experiments, but we found that binding of IGF-I, IGF-II, and insulin to mac25 or connective tissue growth factor was below the detection limit of the biosensor instrument.     

Associations of adiposity from childhood into adulthood with insulin resistance and the insulin-like growth factor system: 65-year follow-up of the Boyd Orr Cohort

CONTEXT: One metabolic pathway through which adiposity influences disease risk may be via alterations in insulin and IGF metabolism. OBJECTIVE: Our objective was to investigate associations of adiposity at different stages of life with insulin and the IGF system. DESIGN, SETTING, AND PARTICIPANTS: The study was a 65-yr follow-up of 728 Boyd Orr cohort participants (mean age, 71 yr) originally surveyed between 1937 and 1939. MAIN OUTCOMES: Outcomes included homeostasis model assessment of insulin resistance, total IGF-I and IGF-II, IGF binding protein (IGFBP)-2, and IGFBP-3 in adulthood. RESULTS: Childhood body mass index (BMI) was weakly inversely related to adult IGF-I (coefficient per BMI sd, -3.4 ng/ml; 95% confidence interval, -7.3 to 0.5; P = 0.09). IGF-II (but not IGF-I) increased with higher current fat mass index (coefficient, 26.1 ng/ml; 95% confidence interval, 4.6 to 47.6; P = 0.02) and waist-hip ratio (30.0 ng/ml; 9.4 to 50.5; P = 0.004). IGFBP-2 decreased by 21.2% (17.2 to 24.9; P < 0.001), and homeostasis model assessment of insulin resistance increased by 38.8% (28.9 to 49.6; P < 0.001) per sd higher adult BMI. Among thin adults (BMI tertiles 1 and 2), IGFBP-2 was positively, and insulin resistance was inversely, associated with childhood BMI. CONCLUSION: There was only weak evidence that associations of childhood BMI with chronic disease risk may be mediated by adult IGF-I levels. Circulating IGFBP-2 in adulthood, a marker for insulin sensitivity, was inversely associated with current adiposity, but overweight children who became relatively lean adults were more insulin sensitive than thinner children. The findings may indicate programming of later insulin sensitivity and consequently IGFBP-2 levels in response to childhood adiposity. The role of IGF-II in obesity-related chronic diseases warrants additional investigation.     

Structural and functional evidence for the interaction of insulin-like growth factors (IGFs) and IGF binding proteins with vitronectin

Previous studies demonstrated that IGF-II binds directly to vitronectin (VN), whereas IGF-I binds poorly. However, binding of VN to integrins has been demonstrated to be essential for a range of IGF-I-stimulated biological effects, including IGF binding protein (IGFBP)-5 production, IGF type-1 receptor autophosphorylation, and cell migration. Thus, we hypothesized that a link between IGF-I and VN must occur and may be mediated through IGFBPs. This was tested using competitive binding assays with VN and (125)iodine-labeled IGFs in the absence and presence of IGFBPs. IGFBP-4, IGFBP-5, and nonglycosylated IGFBP-3 were shown to significantly enhance binding of IGF-I to VN, whereas IGFBP-2 and glycosylated IGFBP-3 had a smaller effect. Furthermore, binding studies with analogs indicate that glycosylation status and the heparin-binding domain of IGFBP-3 are important in this interaction. To examine the functional significance of IGFs binding to VN, cell migration in MCF7 cells was measured and found to be enhanced when VN was prebound to IGF-I in the presence of IGFBP-5. The effect required IGF:IGFBP:VN complex formation; this was demonstrated by use of a non-IGFBP-binding IGF-I analog. Together, these data indicate the importance of IGFBPs in modulating IGF-I binding to VN and that this binding has functional consequences in cells.     

Studies on regulation of insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-4 production in human bone cells.

Previous studies have shown that the actions of IGF-II in bone are determined not only by its concentration, but also by the concentration of IGFBP-4 as well as other IGFBPs. In this study, we sought to determine by Western ligand blotting the effects of growth hormone, IGF-I and IGF-II on the production of IGFBP-3 and IGFBP-4 in TE89 human osteosarcoma cells and in untransformed normal human bone cells derived from rib. Human growth hormone at 10 micrograms/l decreased the amount of IGFBP-4 but had no effect on the IGFBP-3 level in the conditioned medium of low density cultures of TE89 cells and human bone cells derived from rib. Human growth hormone had no effect on IGFBP-3 or IGFBP-4 levels in the conditioned medium of high density human bone cell cultures. IGF-I and IGF-II, which increased human bone cell proliferation, decreased the level of IGFBP-4 (30% of control at 100 micrograms/l IGF-I and IGF-II) but increased the level of IGFBP-3 (3-10 fold at 100 micrograms/l IGF-I and IGF-II) after 48 h of treatment in the conditioned medium of both low and high density TE89 cell cultures. Similar changes in IGFBP-3 and IGFBP-4 levels were also seen in the conditioned medium of human bone cells derived from rib after treatment with IGF-I and IGF-II. Studies to determine the underlying molecular mechanisms by which IGF-II decreased the amount of IGFBP-4 in the conditioned medium revealed that IGF-II decreased the IGFBP-4 mRNA abundance and increased the IGFBP-3 mRNA abundance in human bone cells. Based on the above findings, we conclude that the production of both IGFBP-3 and IGFBP-4 is regulated in bone cells and that local and systemic agents may modulate the responsiveness of bone cells to IGFs by regulated secretion of IGFBP-3 and IGFBP-4.