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Context: Maternal lipids during pregnancy and placental growth factors are associated with excess fetal growth. However, how these factors interact to increase the risk of delivering large-for-gestational-age (LGA) neonates remains unclear. In this study, we investigated the relationship between maternal plasma triglycerides (TGs) and free fatty acids (FFAs) during pregnancy, cord blood insulin-like growth factors (IGF), and LGA., Objective: In a cell model, we studied the effect of different FAs on placental IGF-1 secretion., Methods: This cohort study included pregnant women with term pregnancy and without diabetes or hypertensive disorders in pregnancy. Maternal fasting plasma TGs and FFAs were measured in the second trimester. Cord blood IGF-1, IGF-2, and IGF binding protein-1 and protein-3 were measured at the time of delivery. A human trophoblast cell line, 3A-sub-E, was used to evaluate the effect of different FFAs on placental IGF-1 secretion., Results: We recruited 598 pregnant women-neonate pairs. Maternal plasma TG (180 mg/dL [152.5-185.5 mg/dL] vs 166 mg/dL [133-206 mg/dL], P = .04) and cord blood IGF-1 concentrations (72.7 ± 23.0 vs 54.1 ± 22.8 ng/mL, P < .001) were higher in the LGA group and were significantly associated with birth weight z score. Maternal plasma free palmitic acid (PA) and stearic acid (SA), but not oleic acid (OA) or linoleic acid (LA), were significantly associated with cord blood IGF-1 concentrations. In 3A-sub-E cells, treatment with PA, SA, and LA, but not OA, induced IGF-1 expression and secretion., Conclusion: Certain FFAs can induce placental IGF-1 secretion, which suggests a potential pathophysiology linking maternal plasma lipids and LGA., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The insulin/insulin-like signaling (IIS) pathways, including insulin-like growth factors (IGFs), vary with age. However, their association with late-life cognition and neuroimaging parameters is not well characterized., Methods: Using data from the British 1946 birth cohort, we investigated associations of IGF-I, IGF-II and IGF binding protein 3 (IGFBP-3; measured at 53 and 60-64 years of age) with cognitive performance [word-learning test (WLT) and visual letter search (VLS) at 60-64 years and 69 years of age] and cognitive state [Addenbrooke's Cognitive Exam III (ACE-III) at 69-71 years of age], and in a proportion, quantified neuroimaging measures [whole brain volume (WBV), white matter hyperintensity volume (WMHV), hippocampal volume (HV)]. Regression models included adjustments for demographic, lifestyle, and health factors., Results: Higher IGF-I and IGF-II at 53 years of age was associated with higher ACE-III scores [ß 0.07 95% confidence interval (CI) (0.02, 0.12); scoreACE-III 89.48 (88.86, 90.1), respectively). IGF-II at 53 years of age was additionally associated with higher WLT scores [scoreWLT 20 (19.35, 20.65)]. IGFBP-3 at 60 to 64 years of age was associated with favorable VLS score at 60 to 64 and 69 years of age [ß 0.07 (0.01, 0.12); ß 0.07 (0.02, 0.12), respectively], higher memory and cognitive state at 69 years of age [ß 0.07 (0.01, 0.12); ß 0.07 (0.01, 0.13), respectively], and reduced WMHV [ß -0.1 (-0.21, -0.00)]. IGF-I/IGFBP-3 at 60 to 64 years of was associated with lower VLS scores at 69 years of age [ß -0.08 (-0.15, -0.02)]., Conclusions: Increased measure in IIS parameters (IGF-I, IGF-II, and IGFBP-3) relate to better cognitive state in later life. There were apparent associations with specific cognitive domains (IGF-II relating to memory; IGFBP-3 relating to memory, processing speed, and WMHV; and IGF-I/IGFBP-3 molar ratio related to slower processing speed). IGFs and IGFBP-3 are associated with favorable cognitive function outcomes., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

Context: Daily growth hormone (GH) injections can be burdensome for patients and carers. Somapacitan is a long-acting, reversible albumin-binding GH derivative in development for once-weekly administration in patients with growth hormone deficiency (GHD)., Objective: The objective of this study is to evaluate the efficacy, safety, and tolerability of once-weekly somapacitan vs once-daily GH., Design: REAL 3 is a multicenter, randomized, controlled, double-blind (somapacitan doses), phase 2 study with a 26-week main and 26-week extension phase (NCT02616562)., Setting: This study took place at 29 sites in 11 countries., Patients: Fifty-nine GH treatment-naive prepubertal children with GHD were randomly assigned; 58 completed the trial., Interventions: Interventions comprised 3 somapacitan doses (0.04 [n = 16], 0.08 [n = 15], or 0.16 mg/kg/wk [n = 14]) and daily GH (0.034 mg/kg/d [n = 14]), administered subcutaneously., Main Outcome Measures: The primary end point was height velocity (HV) at week 26. Secondary efficacy end points included HV SD score (SDS) and insulin-like growth factor-I (IGF-I) SDS., Results: At week 26, mean (SD) annualized HV for the somapacitan groups was 8.0 (2.0), 10.9 (1.9), and 12.9 (3.5) cm/year, respectively, vs 11.4 (3.3) cm/year for daily GH; estimated treatment difference (somapacitan 0.16 mg/kg/week-daily GH): 1.7 [95% CI -0.2 to 3.6] cm/year. HV was sustained at week 52, and significantly greater with somapacitan 0.16 mg/kg/week vs daily GH. Mean (SD) change from baseline in HV SDS at week 52 was 4.72 (2.79), 6.14 (3.36), and 8.60 (3.15) for the somapacitan groups, respectively, vs 7.41 (4.08) for daily GH. Model-derived mean (SD) IGF-I SDS for the somapacitan groups was -1.62 (0.86), -1.09 (0.78), and 0.31 (1.06), respectively, vs -0.40 (1.50) observed for daily GH. Safety and tolerability were consistent with the profile of daily GH., Conclusions: In children with GHD, once-weekly somapacitan 0.16 mg/kg/week provided the closest efficacy match with similar safety and tolerability to daily GH after 26 and 52 weeks of treatment. A short visual summary of our work is available (1)., (© Endocrine Society 2020.)

Aims: To investigate circulating levels and liver gene expression of 3 hormonal pathways associated with obesity, insulin resistance, and inflammation to identify leads towards potential diagnostic markers and therapeutic targets in patients with nonalcoholic fatty liver disease (NAFLD)., Methods: We compared circulating levels of (1) proglucagon-derived hormones (glucagon-like peptide [GLP]-1, GLP-2, glicentin, oxyntomodulin, glucagon, major proglucagon fragment [MPGF]), (2) follistatins-activins (follistatin-like [FSTL]3, activin B), (3) IGF axis (insulin-like growth factor [IGF]-1, total and intact IGF binding protein [IGFBP]-3 and IGFBP-4, and pregnancy-associated plasma protein [PAPP]-A) in 2 studies: (1) 18 individuals with early stage NAFLD versus 14 controls (study 1; early NAFLD study) and in (2) 31 individuals with biopsy proven NAFLD (15 with simple steatosis [SS] and 16 with nonalcoholic steatohepatitis [NASH]), vs 50 controls (24 lean and 26 obese) (study 2). Liver gene expression was assessed in 22 subjects (12 controls, 5 NASH, 5 NASH-related cirrhosis)., Results: Patients in early stages of NAFLD demonstrate higher fasting MPGF and lower incremental increase of glicentin during oral glucose tolerance test than controls. In more advanced stages, FSTL3 levels are higher in NASH than simple steatosis and, within NAFLD patients, in those with more severe lobular and portal inflammation. The IGF-1/intact IGFBP-3 ratio is lower in patients with liver fibrosis. Genes encoding follistatin, activin A, activin B, and the IGF-1 receptor are higher in NASH., Conclusion: MPGF and glicentin may be involved in early stages of NAFLD, whereas FSTL3 and IGF-1/intact IGFBP3 in the progression to NASH and liver fibrosis respectively, suggesting potential as diagnostic markers or therapeutic targets., (© Endocrine Society 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: Insulin-like growth factor-I (IGF-I) has structural and functional similarities to insulin and may play a role in glucose homeostasis, along with insulin-like growth factor binding protein-3 (IGFBP-3), which binds the majority of circulating IGF-I., Objective: To assess whether IGF-I and IGFBP-3 are associated with a higher risk of incident diabetes in older adults., Design: Participants in the Cardiovascular Health Study (n = 3133), a cohort of adults aged ≥65 years, were observed for 16 years (n = 3133) for the development of incident diabetes. Statistical models were fit separately for men and women because of interactions with sex (P interaction: IGF-I, 0.02; IGFBP-3, 0.009) and were adjusted for relevant covariates., Setting: General community., Participants: Older adults who were nondiabetic at baseline and who did not develop diabetes within the first year of follow-up., Interventions: Not applicable., Main Outcome Measure: Incident diabetes as measured by fasting plasma glucose (FPG) ≥126 mg/dL, non-FPG ≥200 mg/dL, use of pharmacological treatment of diabetes, or existence of two or more inpatient or three or more outpatient or (at least one inpatient and at least one outpatient) Centers for Medicare & Medicaid Services claims with the diagnostic International Classification of Diseases, Ninth Revision, Clinical Modification code of 250.xx., Results: In women, higher IGFBP-3 (hazard ratio tertile 3 vs tertile 1 = 2.30; 95% confidence interval, 1.55 to 3.40; P trend < 0.0001) was significantly associated with incident diabetes. Total IGF-I was not significantly associated with incident diabetes. In men, neither IGF-I nor IGFBP-3 was significantly associated with incident diabetes., Conclusions: We confirmed a previously reported association between circulating IGFBP-3 and diabetes risk in the older adult population, establishing that this association is present among women but could not be shown to be associated in men., (Copyright © 2017 Endocrine Society)

Background: The variation in laboratory measurements represents a challenge in clinical practice and epidemiological research. The use of different analytical platforms might have led to different results, which were often discussed in the interpretation of conflicting results. We aim to study the impact of two different IGF-I and IGF binding protein 3 assays on published epidemiological studies., Methods: We compared epidemiological results based on the previous gold standard Nichols Advantage, which is no longer available, with these based on the IDS-iSYS assay. The latter follows the recently proposed Keswick criteria. We reinvestigated published association studies between IGF-I or IGF binding protein 3 and anthropometry, subclinical cardiovascular diseases including intima-media thickness or left ventricular mass index, and hard end points like mortality and single-nucleotide polymorphisms of our genome-wide association study in the Study of Health in Pomerania., Results: We demonstrated that there are significant differences in the associations of IGF-I measured by the Nichols or IDS-iSYS assay and subclinical outcomes including intima-media thickness and left ventricular mass index. However, concerning hard outcomes like mortality or single-nucleotide polymorphisms, our analyses revealed similar results with comparable regression estimates., Conclusion: With our study we queried not only the accuracy of measurement but also the effect of different methods on study results. The establishment of laboratory standards like the Keswick criteria should be enforced to allow reliable comparisons of different methods and thus clinical and epidemiological studies. Single-center studies have to be interpreted carefully. Moreover, to assure the reliability of studies, their results should be replicated in a meta-analysis, and a generated hypothesis by epidemiology should be proven by intervention studies.

Context: Obesity is associated with decreased spontaneous and stimulated GH secretion, but the effect of body mass index (BMI) on results of GH stimulation testing in children with short stature is not known., Objective: The aim of the study was to determine the impact of BMI on peak GH to provocative testing in children with short stature., Design, Setting, and Participants: This was a retrospective review of provocative GH testing performed in 116 children 2-18 yr old in the ambulatory clinic of the Pediatric Endocrinology Unit at the Massachusetts General Hospital from 2004-2008., Main Outcome Measures: The main outcome measure was peak stimulated GH. Height, weight, IGF-I, and IGF-binding protein 3 were also measured., Results: In univariate regression analysis, BMI sd score (BMI SDS) was inversely associated with natural log (ln) peak GH to provocative testing (P = 0.002), whereas height SDS, ln IGF-I, and IGF-binding protein 3 were not significantly associated with ln peak GH. After controlling for age, gender, BMI, and pubertal status, BMI (P = 0.002) remained independently associated with ln peak GH. BMI SDS significantly influenced the likelihood of diagnosis of GH deficiency using peak GH cutoffs of 10, 7, and 5 microg/liter., Conclusion: In children with short stature, BMI affects peak stimulated GH and should be considered when interpreting GH testing. Higher BMI SDS, even within the normal range, may lead to overdiagnosis of GH deficiency.

Background: There is strong evidence that IGF-I and IGF binding protein 3 (IGFBP-3), as central mediators of endocrine and finally metabolic or anabolic effects of GH, were associated with increased lung size in acromegaly or a decrease of respiratory muscle pressures in patients with GH deficiency. The aim of the present study was to further clarify the impact of IGF-I and IGFBP-3 levels on lung volumes and respiratory pressures in a general adult population., Material and Methods: From the Study of Health in Pomerania, 1326 subjects aged 25 to 85 yr participated in standardized pulmonary function testing. IGF-I and IGFBP-3 levels were measured with the Immulite 2500 system. Multivariable linear regression analyses adjusted for age, sex, body mass index, physical activity, and smoking were performed., Results: In men, positive linear associations between IGF-I and IGF-I/IGFBP-3 ratio with forced expiratory volume in 1 sec (FEV1) as well as with forced vital capacity (FVC) were detected across all ages, whereas in women this positive association was only detectable above 50 yr. Furthermore, the analyses indicated positive linear relations of IGF-I/IGFBP-3 ratio with FEV1 and FVC, respectively. No significant relations between IGF-I or IGFBP-3 and maximal inspiratory pressure was detectable in both sexes., Conclusion: In conclusion, higher IGF-I levels were associated with higher lung volumes in men, whereas in women this association was only detectable in subjects older than 50 yr. Higher IGF-I values were not associated with increased respiratory muscle strength measured as maximal inspiratory pressure.

Posttranslational modifications, particularly proteolysis, may play a significant role in the regulation of insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) physiology, and thus, measurement of modified variants of IGFBP-3 and/or their combination ratios may have important research and diagnostic relevance. Based on evaluation of a panel of monoclonal and polyclonal IGFBP-3 antibodies, we constructed three new enzyme-linked immunosorbent assays (ELISAs) using a common capture and polyclonal (ELISA-3) or monoclonal (ELISA-1 and -2) detection antibodies and evaluated them in a two-step colorimetric procedure. Evaluation of ELISA-1-3 demonstrated detection limit, dynamic range, overall precision, and recovery of the added IGFBP-3 to be generally less than 0.04 microg/L, 2-100 microg/L, less than 10%, and 91-113%, respectively. IGF-I and -II, and IGFBP-1, -2, -4, -5, and -6 did not interfere. In normal adult sera (n = 26), seminal plasma (n = 14), pregnancy sera (n = 30), and amniotic fluid (n = 30), ELISA-1-3 detected significantly different IGFBP-3 levels (by up to 6-fold, on the average), whereas levels in seminal plasma determined by ELISA-1 were undetectable. Comparison of the values obtained vs. corresponding levels by an established method (Diagnostic Systems Laboratories, Inc., active IGFBP-3 ELISA) were similarly sample dependent and, on the average, varied by up to 19-fold. Only ELISA-3 compared well with the Diagnostic Systems Laboratories, Inc., IGFBP-3 ELISA when samples from normal adults were analyzed. The observed variability could not be totally explained by 50% lower reactivity of ELISA-1-3 for glycosylated IGFBP-3 vs. the nonglycosylated form, and changes in phosphorylation had no effect on immunoreactivity. Evaluation of IGFBP-3 after proteolysis by seminal plasma, plasmin, or thrombin suggested recognition of intact IGFBP-3 by ELISA-1, whereas ELISA-3 appeared to measure intact and proteolyzed IGFBP-3 (total IGFBP-3) with similar potency. In contrast, levels determined by ELISA-2 increased severalfold, indicating preferential recognition of IGFBP-3 fragments. We propose that immunoassay capable of differential determination of IGFBP-3 variants may help better define the physiological importance and potential clinical value of IGFBP-3 measurements.

Measurements of insulin-like growth factor I(IGF-I), IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS) are important in assessing the GH-IGF axis. As nearly all IGF-I, IGFBP-3, and ALS circulate in a GH-dependent ternary protein complex, direct determination of the complex may be of significant analytical and clinical importance. We evaluated a panel of monoclonal antibodies (mAb) to human IGFBP-3 and classified them into four groups (G-1 to G-4). G-1 antibodies recognized epitopes that mapped at or near IGFBP-3 ligand (IGF)-binding site. This region overlapped with the G-2 defined region, which, in turn, overlapped with G-3 epitopes defined by one antibody (mAb 3). Only G-1 and G-3 antibodies paired without interference. mAb 9 recognized a conformational epitope (G-4), and mAb 10 was nonreactive. In pairwise mixed antibody evaluation, mAbs in G-2 and G-3 showed simultaneous binding to serum IGFBP-3 complexes in combination with an anti-IGF-I or an anti-ALS antibody. On this basis, two novel enzyme-linked immunosorbent assays (ELISAs) involving IGFBP-3/IGF-I (ELISA-1) and IGFBP-3/ALS (ELISA-2) recognition partners were developed, both demonstrating acceptable analytical performance characteristics. IGFBP-3 complexes measured by ELISA-1 and -2 in samples from normal individuals and subjects with GH deficiency, acromegaly, and GH receptor deficiency more tightly correlated with IGF-I, IGFBP-3, and ALS than IGF-II. ELISA-1 determinations were comparatively more age dependent and, in comparison to ELISA-2, showed better discriminations among the various sample groups, particularly among GH receptor deficiency, normal, and GH deficiency subjects. The development of IGFBP-3 complex ELISAs may simplify diagnostic applications and facilitate investigations of the physiological relevance of the ternary complex formation.

To investigate cortical bone composition and the role of the insulin-like growth factor (IGF) system in active acromegaly, iliac crest bone biopsies were obtained from 15 patients (3 women and 12 men), aged 21-64 yr (mean, 45.6 yr), and 25 age- and sex-matched controls (8 women and 17 men), aged 22-66 yr (mean, 44.6 yr). Levels of IGF-I, IGF-II, IGF-binding protein-3 (IGFBP-3), IGFBP-5, and total protein were determined in extracts obtained after ethylenediamine tetraacetate and guanidine hydrochloride extraction. Osteocalcin and calcium were determined in extracts after HCl hydrolysis. Cortical bone contents of IGF-I, IGF-II, and IGFBP-5 were significantly elevated in the acromegalic patients compared with control values [91% (P < 0.001), 44% (P < 0.04), and 115% (P < 0.004), respectively]. There was no significant difference in IGFBP-3, osteocalcin, protein, and calcium between patients and controls. This study suggests that the increased levels of growth factors in cortical bone from acromegalics is a reflection of local production, secondary to a chronic systemic excess of GH and IGF-I.

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.

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.

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.

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.

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.

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.