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A comprehensive set of serum markers of collagen turnover and growth was investigated in a longitudinal study of short children during growth induced by growth hormone (hGH) treatment. The study comprised 18 prepubertal children with short stature who had no other current illness or continuous medication. The growth rates and endogenous GH secretions covered a continuum from subnormal to normal. Before treatment, the concentrations of carboxyterminal propeptide of type I procollagen (PICP), reflecting type I collagen formation, of carboxyterminal telopeptide of type I collagen (ICTP), a degradation product of type I collagen, of amino-terminal propeptide of type III procollagen (PIIINP), a marker for type III collagen formation, of alkaline phosphatase (AP), and of insulin-like growth factor binding protein-3 (IGFBP-3) were within the lower limits of normal. The median IGF-I concentration was lower than the reference. One week after the start of treatment, the serum concentrations of ICTP, PIIINP, and osteocalcin (OC), and the increments in ICTP, PIIINP, and IGF binding protein-3 (IGFBP-3) correlated with the subsequent height velocity. During the 12-month treatment, all markers were higher than those of age-matched references, but only the three collagen markers paralleled the changes in height velocity. In molar concentrations, ICTP increased less than PICP. Throughout the study period, the serum level of ICTP correlated with that of PIIINP, but not with that of PICP. The findings suggest that during hGH treatment, linear body growth is closely associated with collagen formation and degradation.

Human colon cancer cell lines COLO205, HT29 and SW620 are known to secrete insulin-like growth factor II (IGF-II) and its modulatory binding proteins (IGFBPs). We have characterised the sensitivity of these cell lines to exogenous IGF-I and have examined the effects of their autocrine IGFBPs on these responses. Cells cultured in serum-free medium were treated with 1-100 ng/ml IGF-I, or des(1,3)IGF-I, a truncated IGF-I with low affinity for IGFBPs. DNA synthesis was determined by 24 h incorporation of 3H-thymidine. Experiments were repeated in the presence of 24 h cell-conditioned media containing endogenous IGFBPs. In all 3 cell lines, cell-conditioned media reduced sensitivity to IGF-I but not to des(1,3)IGF-I suggesting that IGFBPs in the cell-conditioned media of colon cells inhibit IGF-I action. IGFBPs in the cell layer and 24 h cell-conditioned media were identified by Western ligand and antibody analyses. IGFBP-4 was secreted by all cell lines and IGFBP-2 from the COLO205 and SW620 cells lines but not the HT29 cells. No IGFBP-3 was secreted by any of the cell lines but IGFBP-3 was found in the cell layer in all of the cell lines. When endogenous secreted IGFBPs were removed, cell lines were consistently more sensitive to IGF-I than des(1,3)IGF-I suggesting that IGFBP-3 associated with the cell layer enhances responses to IGF-I. This is in contrast to the effects of the secreted IGFBPs. Differential modulating actions of IGFBPs may be important in regulating colon cell turnover.

Insulin-like growth factors (IGFs) interact with specific cell surface receptors to mediate cell growth. Intracellular effects of the IGFs are mediated by activation of secondary messenger molecules. One of these proteins, insulin receptor substrate-1 (IRS-1), is phosphorylated after type I IGF receptor activation and has a major role in IGF signaling. Receptor activation also is influenced by high-affinity IGF binding proteins (IGFBPs). In serum, IGFBP-3 is the predominant species. The role of IGFBP-3 in the regulation of breast cancer cell growth is unclear; both growth inhibition and stimulation have been documented in tissue culture systems. To investigate the influence of IGFBP-3 and IRS-1 in breast cancer, we measured levels of these proteins by ELISA and immunoblotting in 195 node-negative primary human breast cancers and compared their levels with known prognostic factors and disease-free survival (DFS). IGFBP-3 levels correlated positively with tumor size (r = 0.27, P < 0.0001) and negatively with estrogen receptor (r = -0.35, P < 0. 0001) and progesterone receptor (r = -0.16, P = 0.021). In contrast, IRS-1 did not correlate with prognostic factors, but higher levels of IRS-1 predicted worse DFS for the subset of patients with tumors

To assess the role of insulin-like growth factors (IGFs) in growth and transformation of normal (myometrium) and tumorous smooth muscle cell (SMC) tissues, in situ hybridization (ISH) analysis for insulin-like growth factor I and II (IGF-I and IGF-II) mRNAs was combined with detection of IGF peptides, their receptors and IGF binding protein-3 (IGFBP-3). mRNAs for both IGFs were detected in smooth muscle cells in normal, benign and malignant SMC tissues, together with the IGF peptides, both IGF receptors and IGFBP-3. This suggests an autocrine role for both IGFs. Leiomyomas had higher IGF-I peptide levels and higher levels of type I IGF receptors than myometrium, supporting the idea that IGFs play a role in the growth and transformation of these tumours. Low-grade leiomyosarcomas contained more IGF-II mRNAs than myometrium and leiomyoma, fewer type II IGF/mannose 6-phosphate receptors and less IGFBP-3 than myometrium and, in addition, fewer IGF-I mRNAs and type I IGF receptors than leiomyoma. Intermediate- and high-grade leiomyosarcomas had intermediate levels of IGF-II mRNAs and peptide, ranging between those in myometrium and low-grade leiomyosarcomas. Thus, growth and transformation of leiomyosarcomas may be regulated by IGF-II, although more markedly in low-grade than in high-grade leiomyosarcomas. In conclusion, the various categories of SMC tissues are associated with a distinct expression pattern of the IGF system. This suggests that each category of SMC tumours arises as a distinct entity and that there is no progression of transformation in these tissues.

Women who have palpable breast cysts with intracystic Na/K > 3 may have a lower risk of developing breast cancer than those with intracystic Na/K < 3. In this study significantly higher concentrations of insulin-like growth factor-binding protein-3 (IGFBP-3), insulin-like growth factors I and II (IGF-I, IGF-II) and transforming growth factor-beta 2 (TGF-beta2) were found in the Na/K > 3 sub-group. No difference was found in transforming-growth factor-beta 1 (TGF-beta1) levels between the two sub-groups of breast cysts. A positive correlation was obtained for IGFBP-3 and TGF-beta1 in the Na/ K > 3 sub-group consistent with reports that TGF-beta1 may regulate the production of IGFBP-3. Equimolar amounts of total IGFs and IGFBP-3 in breast cyst fluid imply that most, if not all, of these IGFs are protein-bound. The significantly higher concentrations of TGF-beta2 in the Na/K > 3 sub-group may partly explain the lower risk of breast cancer in this group of women.

Insulin-like growth factor-I (IGF-I) and alpha 2-macroglobulin (alpha 2-M) are believed to be involved in the development of germ cells. IGF-I is mainly controlled by concentrations of human growth hormone (HGH), influences cell proliferation and differentiation and its action is mediated by insulin-like growth factor-binding proteins (IGFBP), placental protein 14 (PP14) and prostate-specific antigen (PSA). alpha 2-M acts as a broad spectrum proteinase inhibitor and a binding protein for many cytokines and hormones, e.g. inhibin and activin. This study was designed to identify concentrations of these molecules in seminal plasma in normal semen samples of healthy men, correlations with semen quality, the relationship of IGF-I and alpha 2-M with factors affecting male fertility, and whether vasectomy influences the concentrations of these molecules. Concentrations of IGF-I and alpha 2-M in human seminal plasma were related to semen quality, basal concentrations of HGH, testosterone, IGFBP-3, soluble fibronectin receptor (sFNR), PSA and PP14 in seminal plasma and to serum concentrations of luteinizing hormone (LH) and follicle stimulating hormone (FSH). Commercially available assays were used to analyse 69 semen samples of various quality and 11 post-vasectomy samples. IGF-I concentrations in seminal plasma were significantly correlated with the percentage of morphologically normal spermatozoa (r = 0.748, P = 0.00001) and sperm concentration (r = 0.301, P = 0.011), but negatively correlated with serum FSH (r = -0.506, P = 0.00006) and PSA in seminal plasma (r = -0.388, P = 0.00009). Total alpha 2-M was significantly correlated with sperm count (r = 0.423, P = 0.0005), percentage of progressively motile spermatozoa (r = 0.444, P = 0.00019), quality of motility (sperm motile efficiency, r = 802, P = 0.00001) and straight line velocity (r = 0.411, P = 0.0013). Correlation between the sperm concentration and HGH in seminal plasma was weak (r = 0.287, P = 0.015). Vasectomy reduced the concentration of total alpha 2-M (P = 0.00008) and HGH (P = 0.0068) in the seminal plasma; IGF-I was also reduced after vasectomy when the total ejaculate amount was considered. Thus IGF-I and alpha 2-M are significant for the germ cell development: IGF-I in the maturation of spermatozoa and alpha 2-M in progressive motility.

The insulin-like growth factor (IGF) system is involved in the growth of uterine leiomyomas (L), as these tumors have higher IGF-II messenger ribonucleic acid levels, type I IGF receptor levels, and IGF-I peptide concentrations than myometrium (M). Furthermore, cultured L smooth muscle cells (SMC) respond with greater efficiency to IGF-I than M SMC. Here we investigate a possible modulating role of the binding proteins for the IGFs (IGFBPs) on the actions of IGFs. IGFBP-3 is the most predominant IGFBP in conditioned medium from SMC, with levels ranging from 13-288 ng/mL. Incubation of SMC cultures with IGF-I and the IGF-I analogs long-R3IGF-I and des(1-3)-IGF-I, which have decreased affinity for IGFBPs, revealed a facilitating effect of IGFBPs on the growth-stimulating activity of a high concentration of IGF-I in cell lines with high IGFBP-3 levels. Both a decreased level of IGFBP-3 and a low concentration of the growth factors added were a disadvantage for the facilitating effect. In M and L tissue sections, IGFBP-3 was found exclusively bound to the constituting cells, not in the extracellular matrix. This suggests that a negative modulating role of IGFBP-3 due to sequestration of IGF-I, as occurs in culture medium, is less relevant in vivo. In leiomyosarcoma sections, IGFBP-3 levels are decreased, indicating a decreasing, role for this binding protein in malignant smooth muscle tissues.

The objectives were to investigate whether insulin-dependent diabetes mellitus disrupts production of estradiol and activity of the insulin-like growth factor (IGF)-I system in individual ovarian follicles during the preovulatory period of the estrous cycle. Diabetes mellitus was induced with streptozocin (150 mg/kg) in seven cyclic gilts at 180 +/- 5 days of age. On Day 12 of the estrous cycle, insulin replacement therapy was withdrawn from three gilts and continued in four; four gilts served as normal controls. After ovary removal on Day 18, all follicles > or = 3 mm diameter were dissected free and cultured for 6 h in the presence of 280 ng testosterone for assessment of estradiol and IGF-I production and binding protein activity. Treatments did not affect corpora lutea number (15.4 +/- 0.8) or serum estradiol (5.8 +/- 0.8 pg/ml) on Day 18. There were no differences for any measure of follicular development between normal and insulin-treated diabetic gilts. Untreated diabetic gilts, compared to normal and insulin-treated diabetic gilts, had fewer total visible follicles (22.7 vs. 61.3 and 63.3; SEM = 8; p < 0.01) and reduced follicular diameter (3.4 vs. 4.4 and 4.2 mm; SEM = 0.3; p < 0.0001), respectively. Untreated diabetic gilts had a greater percentage of macroscopically atretic follicles than normal and insulin-treated diabetic gilts (75% vs. 47% and 36%; SEM = 10; p < 0.05). Untreated diabetes mellitus lowered estradiol (p < 0.01); however, effects of treatment on estradiol production were not significant when diameter was part of statistical models. When contents of IGF-I in follicular fluid and conditioned medium were summed after 6 h of culture, untreated diabetic pigs had lower IGF-I at all follicle diameters than pigs in the other treatments (p < 0.05). IGF binding protein (BP) activity was affected by diabetes mellitus, with untreated diabetic pigs having greater IGFBP-1 activity in medium and with both diabetic groups having greater IGFBP-2 activity in follicular fluid (p < 0.05). Activity of IGFBP-1 predominated in conditioned medium, and IGFBP-2 activity predominated in follicular fluid. IGFBP-3 was decreased in follicular fluid of atretic follicles and in medium of atretic follicles in all except the insulin-treated diabetic gilts; in these gilts it was increased in atretic follicles (treatment by atresia interaction; p < 0.05). In conclusion, estradiol was most related to size of the follicle; however, lowering of IGF-I regardless of follicle diameter and alterations in IGFBP activity suggest that diabetes affects IGF-I and its binding proteins differently from estradiol production. These alterations may explain reduced follicular growth and increased follicular atresia in diabetic pigs.

Estradiol (E2)-active (Day 5 [D5]), transitional E2-active (D8), atretic (D12 - 1), and E2-sustained dominant follicles (DF, D12 + 1) and associated subordinate follicles (SF) were obtained through use of an experimental model described here. The ovary bearing the DF was surgically removed by colpotomy, and individual follicles were utilized to study changes in concentrations of insulin like growth factor-I (IGF-I) and -II (IGF-II) and changes in amounts and proportions of the different IGF-binding proteins (IGFBP) present in follicular fluid (FF). The ratio of FF E2 to progesterone (EPR) was utilized to classify follicles into E2 active (EPR > 1) and E2 inactive (EPR < 1). The IGF-I and IGF-II concentrations in FF were similar among experimental groups and between DF and SF. Six different molecular mass bands (49, 43, 35, 30, 28, and 22 kDa) were detected by ligand blot in FF of DF and SF. Immunoprecipitation analysis identified four IGFBPs (-2, -3, -4, and -5) in FF. The 35-kDa band corresponded to IGFBP-2, the 49- and 43-kDa bands to IGFBP-3, the 28- and 22-kDa bands to IGFBP-4, and the 30-kDa band to IGFBP-5. No IGFBP-6 was found by immunoprecipitation. Absolute amounts and proportions of low molecular mass IGFBPs (-2, -4, and -5) were increased with atresia of the DF and in SF compared to E2-active DF. Conversely, although absolute amounts of IGFBP-3 remained unchanged, their proportion in FF decreased in SF compared to DF. The ratio of IGF-I to IGFBPs decreased with atresia of the DF, possibly leaving less bioavailable IGF-I to increase FSH action at the level of the follicle.

Serum levels of carboxyterminal propeptide of type I procollagen (PICP) and aminoterminal propeptide of type III procollagen (PIIINP) can be used as markers of bone formation and the evaluation of children with growth disorders. We measured the serum levels of these collagens by radioimmunoassay (RIA), insulin-like growth factor I (IGF-I) (by immunoradiometric assay) and insulin-like growth factor binding protein-3 (IGFBP-3) (by RIA) levels in 24 children aged between 4 and 14 years with chronic renal failure (CRF) (n = 12 dialysis, n = 12 non-dialysis) and 12 age-matched healthy controls, to find out whether these parameters have a prognostic or therapeutic value on monitoring of growth retardation in CRF. Mean serum IGFBP-3 and PIIINP levels in the dialysis patients were higher than the control group, the difference between the groups was statistically significant (P < 0.05). It seemed that the pubertal stage of the patients did not affect the levels of PICP, PIIINP, and IGFBP-3 while serum IGF-I levels in pubertal patients were significantly higher than those in prepubertal patients (P < 0.001). There was no significant correlation between PICP and PIIINP in any patients. Neither PICP nor PIIINP correlated with height z-score, bone age, IGF-I, or IGFBP-3. It was concluded that the increased serum IGFBP-3 and PIIINP levels in chronic renal disease are poor prognostic indicators leading to progressive renal failure and end-stage renal disease (ESRD). Further investigations into the effects of these collagens on growth failure associated with CRF are needed.

As far as we know, there is no available information about ontogenic changes of tissue concentrations of IGF-I and II and IGFBPs in large mammals. Serum, liver and kidney levels of IGFs and IGFBPs were examined in fetuses at 90 and 110 days of gestation and in pigs at 1d, 3 wk, 3 mo and 6 mo of age. Ontogeny of mRNA levels of IGFs, IGF type I and type II receptors (IGFI-R and IGFII-R), IGFBP-1 and -3 (IGFBPs) and growth hormone receptor (GHR) were also examined by Northern blot analysis in liver, kidney and skeletal muscle of pig. Serum IGF-I, IGF-II and IGFBP-3 concentrations were low during the fetal life and increased after birth. The highest level of IGF-II mRNA was found in fetuses for all studied tissues. In the liver, IGF-I mRNA level and its protein content peaked at 3 wk of age. The highest IGF-II concentration was found at 1d and 3 wk of age. The IGFII-R mRNA remained at a constant level during the whole development period. The most abundant IGFBP-1 mRNA and its protein content were found at birth. The level of IGFBP-2 was high during fetal and early postnatal life. The IGFBP-3 content was relatively low in fetuses and reached the highest level after 3 wk of age. In the kidney, IGFs, IGFBP-3, IGFI-R and IGFII-R as well as GHR mRNA levels were relatively high during the fetal and early postnatal life. The IGFs concentrations were the highest in newborns. In the skeletal muscle, IGFs, IGFBP-3 and IGFI-R mRNA levels decreased with advancing age. During the postnatal life, the high IGFs concentrations in the liver and the kidney correspond to fast growth periods of these organs.

Objective: To identify differences in levels of insulin-like growth factor (IGF) and IGF binding proteins (IG-FBPs) between 30 patients with arthritis (14 with rheumatoid arthritis [RA], 16 with osteoarthritis [OA]) and 11 normal control subjects. IGF and IGFBP levels were correlated to the disease activity marker C-reactive protein (CRP) to determine whether they were disease related. We also examined the degree of proteolytic modification of the IGFBPs., Methods: Radioimmunoassays were used for measuring IGF and IGFBP-3 levels; CRP was measured by enzyme-linked immunosorbent assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting, chemiluminescence, and autoradiography were used for visualizing binding proteins., Results: There was a significant increase in synovial fluid levels of both IGF-1 and IGFBP-3 in both RA and OA. This resulted in an elevated IGFBP-3 to IGF molar ratio of 1.49 in the OA group and 1.47 in the RA group, compared with 0.86 in the normal control group (P = 0.0002 for both). A significantly lower degree of IGFBP-3 proteolysis was also seen in the synovial fluids from the patients compared with the controls. There were significant correlations between the CRP level and levels of IGF-1, IGF-2, and IGFBP-3 in the RA patients (r = 0.62-0.898, P = 0.04-0.0007)., Conclusion: There was significant local disruption of the IGF system in patients with arthritis. This may result in a lower amount of IGF that is able to bind to IGF receptors in the arthritic joint. Levels of IGF-1 IGF-2, and IGFBP-3 all correlated with the CRP level in patients with RA, which indicates the possibility that the IGF system is involved in the disease process.

Insulin-like growth factors (IGFs) are potent mitogens for lung cancer cells. Their proliferative activity is influenced by their binding proteins (IGFBPs). We report here on the regulatory effects of IGF-I and IGF-II on the production and release of IGFBPs by nonsmall cell lung cancer cell lines (NSCLC). The nine NSCLC cell lines used in this study showed messenger ribonucleic acid (mRNA) expression of all six IGFBPs known, as determined by PCR, and protein secretion of IGFBP-1, -2, -3, -4, and -5, as analyzed by Western immunoblots. The addition of IGFs to a serum-free medium showed divergent effects on IGFBP-3 and IGFBP-4 levels in a conditioned medium (CM). IGF-I and IGF-II, but not insulin, led to a much higher concentration of IGFBP-3 in the CM of all tested NSCLC cell lines, whereas the level of immunologically detected membrane-associated IGFBP-3 was decreased. Furthermore, Northern analysis of mRNA isolated from A549 revealed that IGFBP-3 specific mRNA was not changed by IGF-I or IGF-II, suggesting that the IGF-induced effects on IGFBP-3 depend on the release of cell-associated IGFBP-3. In contrast, IGFBP-4 levels were diminished by increasing concentrations of IGFs in the CM of the NSCLCs A549, NCI-H157, and U1752, with no response to insulin or the IGF-I analog, whereas IGFBP-4-specific mRNA was not changed by IGF-I or IGF-II, as determined by Northern analysis. The same effects were seen in a cell-free system after incubation of the CM with IGFs. The decrease in IGFBP-4 concentrations was prevented by coincubation of the CM with the IGFs and either ethylenediamine tetraacetate or 1,10-phenanthrolene, but not with other protease inhibitors. We suggest that IGFs may either activate an IGFBP-4-specific metalloprotease present in NSCLC CM or that the binding of IGFs to IGFBP-4 may enhance the susceptibility of IGFBP-4 to proteolytic degradation. Based on these data, we present evidence that IGFs may regulate their own availability both by releasing IGFBP-3 from cell membrances and through proteolytic degradation of IGFBP-4.

Objectives of this study were to determine if concentrations of steroids, insulin-like growth factor -I (IGF-I), and IGF binding proteins (IGFBP) in follicular fluid and numbers of LH and IGF-I receptors change during growth of the dominant follicle. Ovarian follicular development was monitored daily via ultrasound in lactating Holstein cows. Animals underwent bilateral ovariectomy when the dominant follicle was first identified (days 4-6; estrus = day 0; early; n = 5) or when it stopped growing (days 8-12; late; n = 8). All follicles were classified as dominant (DF), large (LG; > = 6 mm in diameter, excluding DF) or small (SM; < 6 mm), follicular fluid was aspirated, and theca and granulosa cells were collected. Levels of IGFBP-2, assessed via ligand blotting, were greater (P < 0.05) in LG and SM follicles compared with DF in early cows. Levels of IGFBP-3 in follicular fluid were unaffected by follicle class. Numbers of specific 125I-hCG/LH binding sites in thecal cells were greater (P < 0.01) in DF compared with LG and SM follicles of both early and late cows. Numbers of specific 125I-hCG/LH binding sites in granulosa cells were similar for follicle sizes in early cows, but, in late cows, were greater (P < 0.01) in DF compared with SM follicles and were severalfold greater (P < 0.01) in late DF compared with early DF. Numbers of receptors for IGF-I in thecal cells were 2-fold greater (P < 0.05) in DP and LG compared with SM in late cows. Numbers of IGF-I receptors in granulosa cells were unaffected by size or growth of follicles, but were severalfold greater than in theca cells. Concentrations of estradiol were severalfold greater (P < 0.01) in DF compared with LG and SM in both early and late cows. Concentrations of androstenedione in early cows were greater (P < 0.05) in DF and SM compared with LG follicles. Concentrations of progesterone and IGF-I did not differ (P > 0.10) among follicle classes, but both were greater (P < 0.10) in late LG compared with early LG follicles. Concentrations of IGF-II in follicular fluid did not differ (P > 0.10) between early and late cows but were greater (P < 0.10) in SM than DF or LG follicles. We conclude that low amounts of IGFBP-2 and increased thecal binding sites for hCG/LH appear to be related to establishment of the dominant follicle during the first follicular wave in cattle exhibiting regular estrous cycles during late lactation.

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

To evaluate the relative usefulness of insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) in screening for GH status, GH stimulation (arginine-insulin/L-DOPA) tests and overnight GH studies (every 20 min sampling) were performed in 104 healthy short children (32 girls), aged 3-16 yr (height, -1.8 or more SD). IGFBP-3 had no advantage over IGF-I in screening sensitivity or specificity. IGF-I correlated with mean nighttime GH. Both IGF-I and IGFBP-3 correlated with peak stimulated GH. To identify more than 90% of children with GH deficiency (GHD) and borderline GHD, the mean values for age for IGF-I and IGFBP-3 were required as the cut-off criterion. However, at this criterion, 70% or more of idiopathic short stature (ISS) children would have to undergo testing to identify 90% of GHD or borderline GHD. More stringent criteria (-1.0, -1.64, and -2.0 SD) were more specific, but lost sensitivity. A practical application is suggested. Screening use of IGF-I with criterion of -1.0 SD would identify a subgroup that includes 88% of GHD, 71% of borderline GHD, and 46% of ISS. Both IGF-I and IGF-BP-3 higher than -1.0 SD would accurately identify 68% of ISS as not needing GH testing. Evaluation of growth velocity would identify the remaining children requiring definitive testing. Thus, combined screening for GHD using both IGF-I and IGFBP-3 has no better sensitivity than either test alone. However, such combined screening will improve the specificity and thus decrease the number of normal but short children who might otherwise undergo unnecessary testing.

Objective: To measure insulin-like growth factor I (IGF-I) and its binding proteins (IGFBP) in human synovial fluid (SF), as IGF-I resistance in the chondrocyte was implicated in the pathogenesis of inflammatory arthritis., Methods: Ten patients with inflammatory arthritis and 7 controls were evaluated. IGF-I and IGFBP-3 were measured by radioimmunoassay while all IGFBP were evaluated by Western ligand blotting., Results: Serum IGF-I did not differ between patients and controls (22.3 +/- 2.9 vs 22.0 +/- 3.0 nmol/l), while significantly higher values were found in inflammatory SF compared with noninflammatory SF (14.7 +/- 1.5 vs 10.2 +/- 1.2 nmol/l; p = 0.05). IGFBP-3 and IGFBP-4 were the most prominent bands found in inflammatory SF by Western ligand blotting. The 24 kDa IGFBP-4 band appeared faintly in 2 noninflammatory effusions and was nearly absent in others. IGFBP-3 levels in sera from the 2 groups did not differ (2.9 +/- 0.3 vs 2.5 +/- 0.3 mg/l), but were significantly higher in inflammatory compared with noninflammatory effusions (1.5 +/- 0.2 vs 0.9 +/- 0.2 mg/l; p = 0.05)., Conclusion: The pattern of IGFBP in SF differs in inflammatory versus noninflammatory joints.

Objective: Little information is available regarding the regulation of serum acid-labile subunit (ALS) in human disease. We have studied alterations in serum ALS of the insulin-like growth factor (IGF) ternary complex in children with untreated insulin-dependent diabetes mellitus (IDDM) and subjects with severe burns before and after insulin therapy. In addition, we have investigated the effect of insulin plus GH on serum ALS in burn patients., Design: Serum samples were obtained from children with newly diagnosed and untreated IDDM before the initiation of insulin therapy and 1 month thereafter. Serum samples were also obtained from adult patients with severe burns who were on a continuous infusion of a carbohydrate-rich enteral diet via nasogastric and duodenal catheters under basal conditions, after a 1-week period of continuous insulin infusion, and after an additional week of insulin plus recombinant GH., Patients: Twenty children and adolescents with untreated IDDM, aged 1.2-16 years, and 6 young adult patients with severe burns aged 17-28 years were studied longitudinally. Control sera were obtained from age, sex and pubertal status matched subjects (for children with IDDM) and from fed healthy adults., Measurements: Serum insulin, GH, cortisol and IGF-I were measured by radioimmunoassay, and serum ALS levels were assessed by Western immunoblot before and after treatment periods., Results: Serum ALS levels were lower in untreated children with IDDM (69 +/- 6% of control children). Insulin therapy significantly increased serum ALS (79 +/- 5%, P < 0.05) in these children. Patients with severe burns also had lower serum ALS levels (79 +/- 10% of control adults). After one week of insulin therapy serum ALS levels increased to 90 +/- 15% of control values (P < 0.05). Addition of GH to insulin therapy for another week did not significantly further increase serum ALS levels (95 +/- 27%). Serum IGF-I concentrations increased nearly 2.5-fold in diabetic subjects and fourfold in burn subjects at the end of the study periods. There were no proteolytic fragments of ALS in the sera studied. The deglycosylation pattern of ALS did not differ between diabetic and control sera., Conclusion: Serum ALS levels were diminished in children with untreated IDDM and were partially restored after the initiation of insulin therapy. Serum ALS levels were also diminished in patients with severe burn injury and restored by insulin treatment. Addition of GH to insulin therapy did not significantly increase serum ALS levels over levels obtained during insulin therapy alone. These decreases in serum ALS were smaller than the decrease in serum IGF-I concentrations in both conditions, suggesting that IGF-I is the limiting factor for the ternary complex formation in the catabolic states. Insulin may regulate circulating ALS levels in catabolic states and helps to restore the IGF system.

Background: The insulin-like growth factor (IGF)-binding proteins (IGFBPs) regulate the actions of the IGFs by influencing interactions between the IGFs and the IGF receptors. IGFBP-3, one of the six known species of IGFBPs, is the predominant IGFBP in serum and is expressed by breast cancer cells. Compared with estrogen receptor (ER)-positive samples, ER-negative breast cancer cell lines and tumors express higher levels of IGFBP-3. Therefore, expression of IGFBP-3 may be relevant in breast cancer biology, although it is unknown whether IGFBP-3 levels correlate with other breast cancer prognostic factors besides ER status. It is also not known how different methods used to measure IGFBP-3 in breast cancer correlate., Purpose: We measured IGFBP-3 messenger RNA (mRNA) and protein levels in breast tumors by different methods to test how these methods compare and to investigate the relationship between IGFBP-3 and breast cancer prognostic factors., Methods: We analyzed 40 human breast tumors and examined IGFBP-3 expression by ligand blot analysis, immunoblot analysis, immunoradiometric assay (IRMA), and ribonuclease protection assay. Another set of 40 breast tumors, selected according to ER and progesterone receptor (PR) status, S phase, and ploidy, was analyzed by IRMA., Results: In 26 (65%) of 40 samples in which RNA could be isolated, IGFBP-3 mRNA levels correlated with IGFBP-3 levels measured by IRMA (two-sided; P = .0001) but not with IGFBP-3 levels measured by ligand blot or immunoblot. Protein levels were highly correlated among all protein assays. Because the IRMA was more sensitive and accurate than the ligand blot and immunoblot assays, we used IRMA to examine IGFBP-3 levels in an additional 20 primary breast tumors with poor prognostic features (ER and PR negativity, high S phase, and aneuploidy) and in 20 tumors with good prognostic factors (opposite features). IGFBP-3 levels were threefold higher in tumors with poor prognostic features (mean +/- standard deviation = 32.8 +/- 25.2 versus 11.8 +/- 9.7 ng/mg; two-sided; P = .003)., Conclusions: These findings suggest that in human breast cancer, IGFBP-3 mRNA and protein levels are correlated and higher levels of IGFBP-3 are detectable in tumors with poor prognostic features., Implications: IGFBP-3 may be involved in the regulation of breast cancer cell growth.

Expression of insulin-like growth factor-I (IGF-I), its receptor and IGF-binding proteins (IGFBPs) by ovarian cancer cells and its mitogenic effect on these cells in vitro, suggest that IGF-I may have a role in regulation of human ovarian cancer. We have recently shown IGFBP-2 to be markedly elevated in malignant ovarian cyst fluid in vivo. To identify the origin of increased IGFBP-2 in these cyst fluids, the gene expression and protein content of IGFBP-2 were investigated in 14 malignant and four benign epithelial ovarian neoplasms. IGFBP-2 mRNA was detected in all ovarian specimens and was 2- to 30-fold higher in malignant than in benign neoplasms. Within the malignant tissues IGFBP-2 mRNA levels correlated with the aggressiveness of the tumour and were higher in invasive tumours than in those with borderline pathology. Southern blot analysis revealed no amplification of IGFBP-2 gene in the DNA samples from ovarian tumours regardless of their nature. IGFBP-2 was the major binding protein in tissue extracts, as measured by both Western ligand blotting and immunoblotting, and was significantly higher in malignant than in benign neoplasms. These findings were further supported by immunohistochemical detection of IGFBP-2 in tumour sections. Our data suggest that increased local production by the tumour in vivo is responsible for the increased IGFBP-2 levels in the cyst fluid bathing the ovarian malignancy. This may represent an autocrine regulatory mechanism for IGF-I proliferative effect of ovarian cancer.

Retinoic acid (RA) is a potent in vitro inhibitor of cell proliferation in various malignant cell lines. The exact mechanisms of its actions, however, are not fully understood. To further elucidate the nature of this inhibition, we investigated the effects of RA in an estrogen receptor-negative human breast cancer cell line, MDA-MB-231. RA (0.01-5 microM) significantly inhibited MDA-MB-231 cell growth by 35-40% as compared with untreated controls. Similar growth inhibitory actions were observed when cells were treated with transforming growth factor beta2 (TGF-beta2), another factor with antiproliferative actions in breast cancer cells. Both RA and TGF-beta2 increased the levels of insulin-like growth factor binding protein (IGFBP) 3 (2-3-fold) and mRNA (1.5-2-fold), whereas IGFBP-4 levels remained essentially unchanged. The direct involvement of IGFBP-3 in cell growth inhibition was further confirmed by its action on cell growth: exogenous IGFBP-3 directly and significantly inhibited MDA-MB-231 cell number by 40%. These results provided circumstantial evidence that IGFBP-3 may mediate RA and TGF-beta2 growth inhibitory actions in human breast cancer cells. To test this hypothesis, we used an antisense IGFBP-3 oligodeoxynucleotide (ODN) which specifically inhibits IGFBP-3 expression. The antisense IGBP-3 ODN dramatically blocked both RA- and TGF-beta2-induced increases in IGFBP-3 protein (90%) and mRNA levels (90%). This effect was not observed when RA- or TGF-beta2-exposed cells were treated with sense IGFBP-3 ODN. Moreover, antisense ODN did not significantly affect IGFBP-4 protein or mRNA levels, strongly supporting the specificity of the antisense IGFBP-3 ODN effect on IGFBP-3 mRNA. This specific effect of antisense IGFBP-3 ODN on IGFBP-3 protein and mRNA levels was accompanied by significant attenuation of the inhibition of cell proliferation attained with RA or TGF-beta2 (approximately 40% of either RA- or TGF-beta2-induced inhibition). The control sense IGFBP-3 ODN did not reduce the growth inhibition observed with either RA or TGF-beta2. These results indicate that IGFBP-3 is an important mediator of RA- and TGF-beta2-induced cell growth inhibition in human breast cancer cells.

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) modulate the activity of IGFs. In vitro human prostate epithelial cells secrete IGFBP-2 and -3. In vivo IGFBP-2 is increased, and IGFBP-3 is decreased in the serum of patients with prostate cancer. Immunohistochemistry and in situ hybridization were performed to compare the expression of IGFBP-2 and -3 in vivo in prostate tissue containing benign epithelium, high grade prostate intraepithelial neoplasia (PIN), and adenocarcinoma. Immunoreactivity and messenger ribonucleic acid (mRNA) hybridization signals for IGFBP-2 and -3 were localized to epithelial cells. IGFBP-2 immunostaining intensity was significantly increased in PIN regions compared to that in normal epithelium and was further increased in malignant cells. IGFBP-2 mRNA was also significantly increased in PIN and cancer cells. IGFBP-3 immunoreactivity was significantly increased in PIN regions compared to normal epithelium; however, IGFBP-3 protein was significantly decreased in malignant cells. IGFBP-3 mRNA remained virtually unchanged in benign epithelium, PIN, and adenocarcinoma cells. These results demonstrate that increased IGFBP-2 protein in PIN and malignant cells is probably due to increased mRNA expression. However, levels of IGFBP-3 protein may be due to pre- and/or posttranslational mechanisms, including proteolysis. The changes in IGFBP-2 and -3 protein levels in prostatic tissue are in agreement with serum changes reported in patients with prostate cancer.

Background: Evidence has been accumulating that in many tumors, insulin-like growth factors (IGFs) promote cancer cell growth in an autocrine/paracrine manner via the IGF-I receptor. In an effort to understand the role of IGFs in prostate cancer cell growth, we characterized the IGF system components produced by human prostatic cancer cell-lines, LNCaP, DU145, and PC-3, grown in serum-free medium., Methods: IGFs, their receptors, and IGF binding proteins (IGFBPs) produced by the three human prostate cell lines were characterized by reverse transcriptase-polymerase chain reaction (RT-PCR), radioimmunoassay (RIA), Western ligand blot, Western immunoblot, and Northern blot analyses., Results: mRNA for IGF-II and receptors for IGF-I and IGF-II were detected in all three cell-lines by RT-PCR. In contrast to the published study, only LNCaP cells expressed a trace amount of IGF-I mRNA. RIA on conditioned media collected from these cells revealed that all three cell-lines produced measurable IGF-II but not IGF-I. Western Ligand blot, Western immunoblot, and Northern blot analyses revealed that LNCaP, DU145, and PC-3 cells expressed IGFBP-2, IGFBP-2/-3/-4/-6, and IGFBP-2/-3/-4/-5/-6, respectively. IGF-II stimulated [3H]thymidine incorporation into DNA in DU145 and PC-3 cells significantly although the effect was small. DNA synthesis in PC-3 cells but not in LNCaP and DU145 cells was significantly inhibited by the IGF-I receptor-specific monoclonal antibody., Conclusion: Theses results suggest potentially important roles of IGFs and IGFBPs in prostate cancer cell growth, and that in particular, IGF-II may play a critical role in prostate cancer cell growth.

The insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) in human synovial fluid play an important role in maintaining articular cartilage metabolism. In this study we measured the concentrations of IGF-I, IGF-II, and IGFBP-3 in normal human synovial fluid by RIA and characterized the IGFBPs by Western ligand blot (WLB), Western immunoblot, and immunoprecipitation. We also extended the study and compared normal synovial fluid to synovial fluids from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). The concentrations of IGF-I, IGF-II, and IGFBP-3 in normal synovial fluid were 19 +/- 3 (mean +/- SE), 194 +/- 14, and 349 +/- 65 ng/mL, respectively. In synovial fluid of patients with OA, IGF-I levels were elevated, whereas IGF-II was decreased, and the IGFBP-3 level was similar to the control value. In patients with RA, both IGF-I and IGFBP-3 were elevated, whereas IGF-II remained unchanged. WLB and immunoprecipitation of normal synovial fluid revealed IGFBP-1 (26-29 kDa), IGFBP-2 (32 kDa), IGFBP-3 (42- to 39-kDa doublet), and IGFBP-4 (24 kDa); the IGFBP-3 doublet was very faint. In RA synovial fluid, all IGFBPs were dramatically increased, whereas little change was seen in the synovial fluid of OA. Western immunoblot against IGFBP-3 revealed a prominent 30-kDa immunoreactive fragment of IGFBP-3 in synovial fluids of normal adults as well as in those of RA and OA patients. This was concurrent with detectable IGFBP-3 protease activity, which was characterized to be of the metallo- and serine protease family. Thus, in normal synovial fluid, there is a balance of circulating IGF, IGFBP, and proteases to modulate the bioactivity of IGF. In pathological states, the increased IGF-I concentrations were accompanied by an increase in IGFBP-I concentrations were accompanied by an increase in IGFBP-3 levels in synovial fluid. These findings suggest that alteration of the IGF and IGFBP axis in pathological states may be important for understanding the underlying pathophysiology of disordered articular growth and metabolism.

We compared the effect of administration of recombinant human insulin-like growth factor-1 (rhIGF-1) alone or the rhIGF-1/IGF binding protein-3 (IGFBP-3) complex on osteopenia in rats. Female Sprague-Dawley rats (8 months old) underwent combined ovariectomy and bilateral sciatic neurectomy (OVX-NX) or sham operation only. After 2 months, the OVX-NX animals were injected subcutaneously with rhIGF-1 alone or with rhIGF-1A IGFBP-3 equimolar complex for 4 weeks. The IGF-1 contents and dose were 0.3 mg/kg of body weight (BW) three times/week, 3 mg/kg of BW once/week, or 3 mg/kg of BW three times /week. At the end of the experiment, the 4th and 5th lumbar vertebrae (L4, L5) and the proximal tibiae were removed after tetracycline labeling, and histomorphometrical analyses were performed on undecalcified sections using Villanueva's staining. The cancellous bone volume at L5 significantly increased by thickening of the trabecular width in rats treated with the complex. However, the increase in the values at the proximal tibia was not significant. The bone formation rates (BFR/BS) in the lumbar vertebrae of rats treated with the complex three times a week at doses of 0.3 mg/kg of BW and 3 mg/kg of BW were both significantly increased but the parameter increase was less marked with the dose of 3 mg/kg of BW once/week. The BFR/BS did not increase significantly in animals treated with IGF-1 alone. These findings clearly demonstrated that the effect of systemically administered rhIGF-1 on bone formation was markedly potentiated when combined with IGFBP-3 in estrogen deficiency combined with reduced activity. The action of IGF-1 was less potent on the bone in paralyzed limbs. The action of rhIGF-1/IGFBP-3 on trabecular bone appeared to depend not only on the dose but also on the frequency of administration and the parts of the skeleton in rats.

The effect of hormonal therapy on enhancing recovery from neonatal malnutrition was assessed in rats. Malnutrition was induced by maternal food restriction (60% control intake) during lactation. On d 16 postpartum, restricted pups were refed by cross-fostering to control dams and were subcutaneously administered growth hormone (GH), insulin-like growth factor-I (IGF-I), GH + IGF-I or saline. At d 21, pups were weaned and continued hormonal treatments until d 39 postpartum. By d 39, body weight of GH (96% control) and GH + IGF-I- (98%) treated animals were normal, whereas IGF-I (88%) and saline- (85%) treated animals were still stunted. Hormone effectiveness was age dependent, with growth rates of GH + IGF-I-, IGF-I- and GH-treated pups being greatest between d 16 and 30, d 16 and 21 and after d 30, respectively. Protein accretion by d 39 was higher (P < 0.01) in GH and GH + IGF-I groups than saline- or IGF-I-treated groups. On d 39, serum IGF-I concentrations were as follows: GH + IGF-I > IGF-I > GH = saline (placebo), indicating that an elevated serum IGF-I was not required for GH-stimulated growth. Of the three hormonal treatments, GH + IGF-I therapy was most efficacious at promoting rapid and complete body weight recovery and supporting lean body mass accretion.

Objective: Elevated serum IGF-I and IGF binding protein-3 (IGFBP-3) levels have been found in patients with active acromegaly. We have studied the relative diagnostic merits of measurements of IGFBP-3 compared with IGF-I as a parameter of disease activity in these patients., Design/patients: Thirty untreated patients with acromegaly were compared with 30 healthy adults., Measurements: Twenty-four-hour sampling for serum GH in patients with acromegaly, serum IGF-I and IGFBP-3., Results: Mean IGF-I levels were 22.0 nmol/l (range 6.5-38.4) in the healthy adults and 118.7 nmol/l (range 67.7-206.0) in patients with acromegaly. Mean IGFBP-3 levels were 3.5 mg/l (range 2.1-4.8) in controls and 5.4 mg/l (range 4.2-6.6) in patients with acromegaly. Mean IGF-I/IGFBP-3 ratios were 6.5 nmol/mg (range 1.9-14.5) in the healthy adults and 22.0 nmol/mg (range 14.3-32.7) in patients with acromegaly. There was a considerable overlap for IGFBP-3 levels but not for IGF-I levels, between normals and acromegalics. The IGF-I/IGFBP-3 ratio also showed overlap between normals and acromegalics. There was a significant correlation between the mean 24-hour GH and IGFBP-3 levels (P = 0.036) and between the IGF-I and IGFBP-3 levels (P < 0.002) in acromegaly. In patients with acromegaly, the IGFBP-3 levels showed a decrement, but the IGF-I/IGFBP-3 ratio did not change significantly with age., Conclusions: IGFBP-2 has no additional discriminatory value over IGF-1 measurements for the assessment of clinical activity in acromegaly. In acromegaly, IGFBP-3 decreases with increasing age. In acromegaly, IGFBP-3 levels significantly correlate with mean 24-hour GH levels and IGF-I levels.

Despite extensive investigation of the insulin-like growth factor (IGF)/IGF-binding protein (IGFBP) system in the circulation and body fluids, there is no information on this in interstitial fluid. We have compared the IGF/IGFBP system in the circulation with that in fluid obtained from blisters artificially raised by negative pressure in 10 healthy volunteers. IGFBP-1, -2, -3, and -4 were all found in blister fluid, but in concentrations much lower than those in matched serum. The IGF-I, IGF-II, and IGFBP-3 levels measured by RIA were 18%, 14%, and 16% of those in serum, respectively. Fast protein liquid chromoatography showed that both IGF-I and IGFBP-3 in 150- and 50-kilodalton complexes were approximately 13% and 37%, respectively, of the corresponding peaks found in matched serum. Compared to that in serum, the IGFBP-3 in the blister fluid was predominantly in a modified 29-kilodalton form, and there was increased activity of an IGFBP-3 protease. Therefore, although IGF concentrations are much lower in interstitial fluid than in the circulation, a greater proportion of this IGF is in forms more readily available for interaction with tissue receptors. The blister fluid appears to represent physiological interstitial fluid and may provide a model for studying the physiology and pathophysiology of growth factors in the interstitial environment.