Serum levels of insulin-like growth factor (IGF) and IGF binding protein in constitutionally tall children and adolescents

Serum insulin-like growth factor (IGF) and IGF binding protein (BP) levels were studied in 89 constitutionally tall children and adolescents (height greater than mean for age + 3 SD in 90% of the subjects). After separation by acidic gel filtration, the samples were assayed for IGF using a protein-binding assay (which measures mainly IGF I-related peptides) and for IGF BP by titration, in both cases using IGF I as tracer. The reference standard was a pool of normal adult serum with an assigned potency of 1 U IGF and 1 U IGF BP/ml. IGF levels increased with age in a manner similar to that in normal subjects, but at higher concentrations (P less than 0.02). The values were 0.77 +/- 0.05 (SE) U/ml in 1- to 5-yr-old children, 0.96 +/- 0.08 U/ml in 5- to 11-yr-old prepubescent children and 1.51 +/- 0.07 U/ml during puberty. BP levels developed in a different way from the above in that the increase with age was slight (0.70 +/- 0.07, 0.67 +/- 0.09, and 0.97 +/- 0.07 U/ml, respectively, for the three periods considered) and the levels were lower than those of normal subjects (P less than 0.005). After fusion of the epiphyses both IGF and BP levels were within the range of normal values. Thirteen girls underwent prolonged treatment (mean, 26 months) with ethinyl estradiol (250-300 micrograms/day). Deceleration in growth was accompanied by a progressive decrease in IGF levels throughout the period of therapy and a rise in BP levels during the first 6 months, after which they stabilized within the range of normal values. Although it is possible that excessive secretion of GH (which was not demonstrated by the usual tests) may be the cause of the elevated IGF levels during growth in these constitutionally tall subjects, it seems likely that the high IGF levels combined with the abnormally low levels of BP were responsible for their excessive growth.     

Serum levels of insulin-like growth factor (IGF) and IGF-binding protein in constitutionally short children and adolescents

Serum insulin-like growth factor (IGF) and IGF-binding protein (IGF-bp) levels were studied in 92 constitutionally short children and adolescents (height less than mean for age -2 SD) and in 13 subjects after completion of growth. IGF levels increased with age in a manner similar to that in normal subjects, but at lower levels (P less than 0.001). The values were 0.41 +/- 0.03 SEM U/ml in 1 to 5 year old children, 0.72 +/- 0.03 U/ml in 6- to 16 year old prepubescent children and 0.95 +/- 0.05 U/ml during puberty. IGF-bp levels developed in a similar way, the values being 0.45 +/- 0.06, 0.61 +/- 0.04 and 0.85 +/- 0.06 U/ml, respectively, for the three periods considered. Both IGF and IGF-bp levels in each of the three groups were significantly lower than those in normal subjects at the same stage of development. After fusion of the epiphyses, IGF and IGF-bp levels were within the normal range. A longitudinal study was undertaken in 15 subjects, showing increases in height corresponding with increases in IGF levels. For all the subjects studied during their growth period, there was a correlation between height age and IGF levels (r = 0.64, P less than 0.001). All the subjects exhibited a normal rise in GH levels following stimulation. Although the possibility of quantitative or qualitative disorders of GH biosynthesis cannot be excluded in some of the cases, our data are compatible with the hypothesis that the growth retardation observed in constitutionally short children results, at least in part, from insufficient IGF production during post-natal growth.     

Serum levels of free insulin-like growth factor (IGF)-I and IGF-binding protein-1 in prepubertal children with idiopathic short stature

The study was performed to evaluate the relationships among serum free and total insulin-like growth factor (IGF)-I, IGF-binding protein-1 (IGFBP-1), IGFBP-3, and insulin concentrations in prepubertal children with idiopathic short stature (ISS). Eighteen children with ISS and 15 age-matched controls were included in the study. All short children had a height standard deviation score of more than 2 below the mean, and maximum stimulated GH levels greater than 10 microg/l after two standard provocation tests. The serum levels of free IGF-I were significantly lower in short children (1.6 +/- 0.3 microg/l) than in the controls (2.8 +/- 0.6 microg/l, P<0.05), while total IGF-I levels were slightly, but not significantly, lower in short children than in controls. The serum levels of IGFBP-1 were significantly higher in the ISS group (124.6 +/- 5.6 microg/l) than in controls (80.0 +/- 8.7 microg/l, P < 0.0001). The fasting insulin and IGFBP-3 levels were similar in both groups. A stepwise regression analysis for all subjects revealed that IGFBP-1 is the only independent predictor of log free IGF-I (R2 = 0.23, P<0.01). The present study shows that the serum levels of free IGF-1 are significantly lower and insulin-like growth factor-binding protein-1 levels are higher in prepubertal children with idiopathic short stature, as compared with age-matched controls. The high IGFBP-1 may contribute to growth retardation in a subgroup of idiopathic short stature through a decrease in free IGF-1.     

Bioactive insulin-like growth factor (IGF) I and IGF-binding protein-1 in anorexia nervosa

CONTEXT: Regulation of IGF-I activity appears crucial in anorexia nervosa (AN) during adaptation to chronic starvation as well as during the regenerative processes on nutritional restoration. OBJECTIVE: The objective of this study was to examine the relationship between IGF-I bioactivity and IGF-binding capacity as expressed as formation of the binary complex of IGF-binding protein-1 (IGFBP-1) and IGF-I in patients with AN at different stages and with different subtypes of the disease. DESIGN: This was a longitudinal study. SETTING: The study took place at a clinical research center at a university hospital. STUDY PARTICIPANTS: We studied a total of 45 women with AN and 24 age-comparable healthy controls. MAIN OUTCOME MEASURES: IGF-I bioactivity was determined using an IGF-I receptor activation assay, and IGF-I/IGFBP-1 complex formation was determined by an assay that allows direct determination of the binary complex. RESULTS: IGF-I bioactivity was significantly decreased in serum from patients with AN. We found significant correlations between total, ultrafiltered free, and bioactive IGF-I. Despite increased IGFBP-1 concentrations, levels of IGF-I/IGFBP-1 binary complex were not significantly increased in AN. Oral contraceptives were associated with increased levels of IGF-I, IGFBP-1, and binary complex formation. Ghrelin levels were only significantly raised in those patients who had lost more than 5% of the body weight during the last 4 wk, whereas ghrelin levels in weight-stable as well as in weight-gaining patients did not significantly differ from the controls. CONCLUSIONS: Total IGF-I level is a suitable marker of IGF-I bioactivity in emaciated patients with AN irrespective of the clinical subtype and acute nutritional state.     

Testosterone and insulin-like growth factor (IGF) I interact in controlling IGF-binding protein production in androgen-responsive foreskin fibroblasts

The growth of the male external genitalia is primarily regulated by androgens. However, human genital fibroblast growth is also stimulated by insulin-like growth factor (IGF) I. In this study, we report that IGF-binding protein (IGFBP) production in human foreskin fibroblasts is regulated by androgens and IGF-I. Human foreskin fibroblasts secrete IGFBP-3, IGFBP-4, and IGFBP-5. IGF-I increased the abundance of both intact IGFBP-3 and -5 in the culture medium. Testosterone increased IGFBP-3, and the combination of IGF-I and testosterone had an additive effect. Following its secretion, IGFBP-5 was degraded, but the effect of IGF-I on IGFBP-5 peptide abundance in conditioned media did not seem to be due to inhibition of proteolysis. Testosterone had no effect on IGFBP-5 degradation. Intact IGFBP-4 was decreased by IGF-I, and the combination resulted in a similar reduction. The mechanism seemed to be decreased synthesis, since IGFBP-4 messenger RNA was also decreased. The increase in IGFBP-5 synthesis was associated with an increase in the abundance of intact IGFBP-5 in the extracellular matrix. The combination of testosterone and IGF-I resulted in a synergistic stimulation of total protein synthesis by the fibroblast cultures, suggesting that a maximum anabolic response requires both hormones. These observations suggest that combined exposure to androgen and IGF-I altered the abundance of some forms of IGFBPs and that the IGFBPs that are regulated may play a role in modulating the effects of IGF-I on the anabolic response.     

Effect of metformin on insulin-like growth factor (IGF) I and IGF-binding protein I in polycystic ovary syndrome

The objective of the present study was to investigate whether metformin affected plasma concentrations of insulin-like growth factor (IGF) I and IGF-binding protein I (IGFBP-I) in polycystic ovary syndrome (PCOS) patients. This was an open study conducted by the Department of Obstetrics and Gynecology at the University of Siena, Italy. Seventeen women with PCOS participated in the study and were administered metformin at a dose of 500 mg three times a day. Treatment was continued for 30-32 days, after which the pretreatment evaluation was repeated. Plasma concentrations of LH, FSH, estradiol, free testosterone, IGF-I, IGFBP-I, sex hormone-binding globulin, and insulin were evaluated. Metformin led to a significant reduction in areas under the insulin curves (9310 +/- 1509 vs. 6520 +/-1108 mU/mL x min; P < 0.05) and was associated with a decrease in plasma free testosterone levels (12.7 +/- 1.7 vs. 10.3 +/- 2 pg/mL; P < 0.05) and an increase in plasma sex hormone-binding globulin concentrations (62 +/- 8 vs. 94 +/- 13 nmol/L; P < 0.05). A nonsignificant increase in plasma IGF-I levels was observed after metformin (276 +/-48 vs. 291 +/- 71 mcg/L), with a significant increase in plasma IGFBP-I levels (0.56 +/- 0.2 vs. 0.98 +/- 0.38 mcg/L; P < 0.05). The IGF-I/IGFBP-I ratio was significantly lower (492.8 +/- 117 vs. 296.9 +/- 82; P < 0.05) at the end of therapy than before treatment. In conclusion, it seems to be appropriate to intervene with an insulin-sensitizing agent such as metformin in an attempt to break the pathogenetic link between hyperinsulinemia and hormonal perturbations in PCOS.     

Associations of insulin-like growth factor (IGF)-I and IGF-binding protein-3 with HIV disease progression in women

BACKGROUND: The insulin-like growth factor (IGF) axis has been hypothesized to influence the rate of human immunodeficiency virus (HIV) disease progression. This premise is based largely on laboratory models showing that IGF-I stimulates thymic growth and increases lymphocyte numbers and that IGF-binding protein (IGFBP)-3 has an opposing effect, inhibiting hematopoietic stem cell development. METHODS: We studied 1422 HIV-infected women enrolled in a large cohort that entailed semiannual follow-up (initiated in 1994). Baseline serum samples were tested for IGF-I and IGFBP-3 to determine their associations with incident clinical acquired immunodeficiency syndrome (AIDS) and CD4+ T cell count decline prior to April 1996 (before the era of highly active antiretroviral therapy [HAART]). RESULTS: Low IGF-I levels (Ptrend= .02) and high IGFBP-3 levels (Ptrend= .02) were associated with rapid CD4+ T cell count decline. Only IGFBP-3, however, was significantly associated with AIDS incidence (hazard ratio for highest vs. lowest quartile, 2.65 [95% confidence interval, 1.30-5.42]; Ptrend= .02) in multivariable models. CONCLUSIONS: These findings suggest that serum levels of IGFBP-3 (and possibly IGF-I) are associated with the rate of HIV disease progression in women and, more broadly, that interindividual heterogeneity in the IGF axis may influence HIV pathogenesis. If correct, the IGF axis could be a target for interventions to slow HIV disease progression and extend the time before use of HAART becomes necessary.     

Effect of intraperitoneal insulin delivery on growth hormone binding protein,insulin-like growth factor (IGF)-I,and IGF-binding protein-3 in IDDM

Summary Low plasma insulin-like growth factor (IGF)-I despite high circulating growth hormone (GH) in insulin-dependent diabetes mellitus (IDDM) indicate a hepatic GH resistance. This state may be reflected by the reduction of the circulating GH binding protein (GHBP), corresponding to the extracellular domain of the GH receptor, and the reduction of insulin-like growth factor binding protein (IGFBP)-3, major IGF-I binding protein, upregulated by GH. We carried out two studies. In the first, plasma GHBP activity was compared in patients with IDDM on continuous subcutaneous insulin infusion (CSII) or on conventional therapy and in healthy subjects. In the second study, the 18 patients on CSII at baseline were then treated by continuous intraperitoneal insulin infusion with an implantable pump (CPII) and prospectively studied for GH-IGF-I axis. Although HbA_{1 c} was lower in patients on CSII than in those on conventional therapy, GHBP was similarly reduced in both when compared to control subjects (10.2 ± 0.8 and 11.6 ± 0.9 % vs 21.0 ± 1.3, p < 0.01). CPII for 12 months resulted in: a slight and transient improvement in HbA_{1 c} (Time (T)0: 7.6 ± 0.2 %, T3:7.1 ± 0.2 %, T12: 7.5 ± 0.2 %, p < 0.02), improvement in GHBP (T0: 10.2 ± 0.8 %, T12: 15.5 ± 1.5, p < 0.0001), near-normalization of IGF-I (T0: 89.4 ± 8.8 ng/ml, T12: 146.9 ± 15.6, p < 0.002) and normalization of IGFBP-3 (T0: 1974 ± 121 ng/ml, T12: 3534 ± 305, p < 0.0001). The hepatic GH resistance profile in IDDM does not seem to be related to glycaemic control, but partly to insufficient portal insulinization. Intraperitoneal insulin delivery, allowing primary portal venous absorption, may influence GH sensitivity, and improve hepatic IGF-I and IGFBP-3 generation. [Diabetologia (1996) 39: 1498–1504] Received: 23 March 1996 and in revised form: 22 July 1996     

Evaluation of diagnostic accuracy of insulin-like growth factor (IGF)-I and IGF-binding protein-3 in growth hormone-deficient children and adults using ROC plot analysis

We critically evaluated the diagnostic value of IGF-I and IGF-binding protein-3 (IGFBP-3) in GH deficiency (GHD) in children and adults using receiver operating characteristic (ROC) plot analysis. Sixty-six children (chronological age, 1.3-15 yr) were studied: 34 GHD and 32 idiopathic short stature (ISS). Ninety-two adults (chronological age, 18-70 yr) were also evaluated: 72 GHD, 34 of childhood onset (AGHD-CO), and 38 of adult onset (AGHD-AO); and 20 healthy volunteers. The SD score (SDS) for IGF-I was calculated from 596 normal subjects (212 children and 384 adults), and the SDS for IGFBP-3 was calculated from 350 normal subjects (212 children and 138 adults). The ROC plot showed that the best IGF-I SDS cut-off line was -1.65 for children [sensitivity (S), 68%; specificity (Sp), 97%, diagnostic efficiency (DEf), 81%], the cut-off line for AGHD was -1.65 for AGHD-CO (S, 91%; Sp, 100%; DEf, 94%), and the cut-off line for AGHD-AO was -1.80 (S, 81%; Sp, 100%; DEf, 88%). For IGFBP-3 SDS, the best cut-off line was -1.80 for children (S, 90%; Sp, 60%; DEf, 78%); it was -1.45 for AGHD-CO (S, 90%; Sp, 75%; DEf, 82%) and -0.90 for AGHD-AO (S, 90%; Sp, 68%; DEf, 77%). An accurate diagnosis was obtained using IGF-I SDS alone in GHD children 65%; ISS, 97%; AGHD-CO, 92%; AGHD-AO, 86%, with IGFBP-3 SDS alone in GHD children 60%; ISS, 90%; AGHD-CO, 75%; AGHD-AO, 68%. Considering both, an accurate diagnosis was obtained in GHD children 60%; ISS, 87%; AGHD-CO, 71%; AGHD-AO, 64%. In conclusion, our findings support the need to use cut-off lines expressed in SDS obtained using an appropriate statistical methodology for better characterization of the various clinical presentations. IGF-I proved to be more useful because of its good diagnostic efficiency and accuracy in both children and adults, whereas IGFBP-3 did not significantly contribute to the diagnosis of GHD.     

Increased activity of insulin-like growth factor (IGF) in osteoblastic cells in the presence of growth hormone (GH): positive correlation with the presence of the GH-induced IGF-binding protein BP-3

Insulin-like growth factors (IGFs) are bound in the circulation to specific binding proteins (BP). The predominant BP is a GH-dependent glycosylated protein of 42-49 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BP-3), whereas nonglycosylated GH-independent IGFBPs of 32 kDa and less are minor constituents. Primary cultures of rat osteoblastic cells constitutively produce IGFBP species of 32 kDa, while GH induces the accumulation of BP-3. To examine whether BP-3 could regulate the biological activity of IGF-I on osteoblasts, we compared the effects of recombinant native human IGF-I (hIGF-I) on primary cultures of osteoblasts in the presence and absence of GH. hIGF-I stimulated cell replication and alpha 1(I) collagen gene expression in a dose-dependent manner, and these effects were potentiated by the presence of GH, which increased the accumulation of BP-3. To further examine this correlation, we compared the effects of two IGF-I peptides on the osteoblastic cell line PyMS, which constitutively produces BP-3, to those in RCT-3 cells, which do not secrete this IGFBP. Using hIGF-I and [Gln3,Ala4,Tyr15,Leu16]IGF-I ([QAYL]IGF-I), a mutated IGF-I with reduced affinity to IGFBPs, we found that at equimolar concentrations hIGF-I produced higher stimulation than [QAYL]IGF-I on [3H]thymidine incorporation, cell replication, and collagen gene expression in PyMS cells. In contrast, both IGF-I peptides had similar potency in RCT-3 cells. Hence, these data show that the accumulation of BP-3 correlates with enhanced hIGF-I activity on osteoblastic cells, suggesting that BPs may act locally to augment the effects of IGF-I in bone.     

Potentiation of insulin-like growth factor (IGF) action by IGF-binding protein-3: studies of underlying mechanism.

In this study we investigated the mechanism(s) by which insulin-like growth factor-binding protein-3 (IGFBP-3) potentiates IGF-I action in cultured bovine fibroblasts. Preincubation of cells with glycosylated or nonglycosylated recombinant human IGFBP-3 enhanced responsiveness to IGF-I in a time-dependent manner. A preincubation period of at least 24 h with IGFBP-3 was required to see a significant effect. Pretreatment with IGFBP-3 for 72 h resulted in a 2- to 4-fold augmentation of IGF-I-stimulated [3H]aminoisobutyric acid uptake; IGFBP-3 had no effect on basal [3H]aminoisobutyric acid uptake. During the preincubation period, exogenous IGFBP-3 associated with the fibroblast surface and exhibited time-dependent processing to lower mol wt forms that retained the ability to bind radiolabeled IGF-I. Initial surface adherence (preincubation time of 24 h or less) was readily reversible. However, IGFBP-3, once processed, appeared to be closely associated with the cell. After 72 h of exposure to bovine fibroblasts, cell-associated IGFBP-3 had a 10-fold lower affinity for IGF-I compared to intact IGFBP-3 in solution. In addition, incubation of bovine fibroblasts with IGFBP-3 had modifying effects on type I IGF receptor-mediated signalling because 1) the bioeffectiveness of [Gln3,Ala4,Tyr15,Leu16]IGF-I and insulin, IGF-I receptor activators with little or no affinity for IGFBP-3, was potentiated by preincubation with IGFBP-3; and 2) fibroblast responsiveness to IGF-I analogs with different affinities for the type I IGF receptor was enhanced in direct relation to the ability of the peptide to bind to the receptor. There was no evidence for an increase in receptor number or affinity as a result of IGFBP-3 treatment. These data suggest that IGFBP-3 potentiation of IGF-I action in bovine fibroblasts may involve changes in IGFBP-3 and type I IGF receptor responsiveness. Thus, cell-associated IGFBP-3 may provide a mechanism for optimal presentation of IGF-I to its receptor as well as a means to heighten receptor reactivity to IGF-I and related peptides.     

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.     

Overexpression of mature insulin-like growth factor (IGF)-II leads to growth arrest in Caco-2 human colon cancer cells.

Caco-2 cells produce both mature 7500 M(r) and higher M(r) forms of IGF-II (pro IGF-II - pIGF-II) and pIGF-II production is much higher than that of mature IGF-II (mIGF-II). The present study was performed to determine whether overexpression of mIGF-II or pIGF-II stimulates Caco-2 cell growth. A pIGF-II cDNA construct that expresses IGF-II including the E-domain was prepared by cloning a 1250 bp BamH I-Apa I human prepro IGF-II cDNA fragment downstream of the CMV promoter in pcDNA3. To create a mIGF-II cDNA construct which does not express the E-domain, two stop codons were inserted right after the glutamine residue of the D-peptide by site directed mutagenesis utilizing the pIGF-II cDNA expression construct as the template. Caco-2 cells were stably transfected with the mIGF-II or pIGF-II construct. Secretion of the mature and higher M(r) forms of IGF-II into serum-free medium was higher in clones transfected with the mIGF-II or pIGF-II expression constructs compared to vector controls. Both IGF-II clonal cell lines grew faster than the control Caco-2 cells until six days of culture. However, at day 12 the final cell density of the pIGF-II expressing cells was higher than that of the mature IGF-II clones. Western blot analysis of cell lysates at day 8 through day 12 with anti-IGF-I receptor (IGF-IR) beta subunit antibody revealed that the mature IGF-IR levels were lower in both IGF-II overexpressing cell lines compared to the vector control clone. Furthermore, it was shown that at day 12 the IGF-IR levels were significantly lower in mIGF-II clones than in pIGF-II clones. These results indicate that mIGF-II is more effective in down-regulating the IGF-IR than pIGF-II. We propose that overexpression of mIGF-II causes down-regulation of the IGF-IR, leading to growth arrest of Caco-2 cells.     

Roles of insulin-like growth factor (IGF) binding proteins in regulating IGF actions

The insulin-like growth factor (IGF) system is an evolutionarily conserved signaling pathway that is composed of two IGF ligands, two IGF receptors, and six IGF binding proteins. Studies in a variety of species suggest that the IGF signaling system plays a fundamental role in regulating embryonic growth and differentiation as well as in maintaining homeostasis in the adults. In extracellular fluids, IGFs are present in a complex with an IGF-binding protein (IGFBP). These IGFBPs are traditionally thought to function as carrier proteins and regulate circulating IGF turnover, transport, and distribution. Locally expressed IGFBPs can also inhibit and/or potentiate IGF activities. Recent studies have shown that some IGFBPs, in particular IGFBP-3 and -5, possess intrinsic biological activities and can act through IGF-independent mechanisms. In this article, we provide a brief overview of our current understanding of the IGF signaling system with particular reference to IGFBPs.     

Quantitative ontogeny of murine insulin-like growth factor (IGF)-I, IGF-binding protein-3 and the IGF-related acid-labile subunit.

OBJECTIVE: The mouse serves as an important model for insulin-like (IGF)-related research. However, lack of homologous mouse assays has prevented studies of the normal ontology of the murine IGF system. Therefore, we developed and validated immunoaassays for murine IGF-I, IGFBP-3 and ALS and studied levels of these analytes in mice. METHODS: Using commercially available reagents, we developed and validated specific enzyme-linked immunosorbent assays (ELISAs) for murine IGF-I, IGFBP-3, and ALS. Levels of these analytes were measured in sera from CD-1 mice, male and female, sampled at 1, 2, 4, 8, 20 and 32 weeks of age. In addition, sera from pregnant and postpartum CD-1 mice were also studied. RESULTS: Validation of specific ELISAs for murine IGF-I, IGFBP-3 and ALS are described; all 3 assays were highly sensitive, precise and accurate. As measured by our homologous ELISA, IGF-I levels (ng/mL, mean+/-SD) in male mice were relatively low at 1 week (53+/-8), rising sharply to peak at 8 weeks of age (636+/-48), and gradually declining thereafter, reaching 395+/-64 at 32 weeks. IGF-I levels in non-pregnant female mice peaked at 4 weeks of age (548+/-77) declined at 8 weeks (417+/-61), then recovered to plateau at 539+/-78 and 535+/-88 at 20 and 32 weeks, respectively. In male mice, trends in IGFBP-3 were similar to the patterns of IGF-I. However, in non-pregnant female mice, the IGFBP-3 levels declined relatively slowly after peaking at 4-weeks of age, unlike IGF-I levels during this period. ALS levels followed the same pattern as IGF-I in both sexes. IGF-I to IGFBP-3 molar ratios (percent) were similar between sexes, rising continuously with age: approximately 30% at 1 week, 80% at 4 weeks, 135% at 32 weeks. IGF-I was reduced in 8 week old mice in mid-pregnancy (354+/-75 vs 417+/-61 in non-pregnant 8 week females), reaching a nadir in late-term (146+/-40), and only partially recovering in the postpartum period (239+/-23). IGFBP-3 was also lower in late-pregnancy (1245+/-100 vs 1925+/-439) and remained depressed postpartum. In contrast to IGF-I and IGFBP-3, ALS increased more than threefold in mid-pregnancy (12180+/-1641 vs 3741+/-910), followed by a 4-fold decrease in late-pregnancy (2964+/-489), recovering postpartum (6104+/-1178). CONCLUSIONS: We report the first ontological studies of IGF-I, IGFBP-3 and ALS in mice using newly-characterized sensitive, homologous immunoassays. Our results indicate that mice have a generally similar pattern in IGF-related axis components, with low levels early in life, increasing to peak during sexual maturation and declining thereafter. Significant gender differences in non-pregnant levels and dramatic changes during pregnancy were also found. Knowledge of the normal developmental changes in the murine IGF system and accurate tools for investigations of this system are a necessary foundation for research in this field.