IGFBP-3 Fragments Research Articles

Reduced Fragmentation of IGFBP-2 and IGFBP-3 as a Potential Mechanism for Decreased Ratio of IGF-II to IGFBPs in Cerebrospinal Fluid in Response to Repeated Intrathecal Administration of Triamcinolone Acetonide in Patients With Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune disease of the brain and spinal cord causing a wide range of symptoms such as impaired walking capability, spasticity, fatigue, and pain. The insulin-like growth factor (IGF) system has regulatory functions for the induction of inflammatory pathways in experimental encephalomyelitis. We have therefore assessed expression and regulation of the IGF system on the level of IGFs and IGFBPs in serum and cerebrospinal fluid (CSF) in the course of four repeated triamcinolone acetonide (TCA) administrations in two female and four male MS patients. Sample series of 20 treatment cycles were analyzed. IGF-I and IGF-II were quantified by ELISAs, and IGFBPs were analyzed by quantitative Western ligand (qWLB) and Western immunoblotting (WIB) in order to differentiate intact and fragmented IGFBPs. The ratios of fragmented to intact IGFBP-2 and -3 were calculated in serum and CSF. Finally, the ratios of IGF-I and IGF-II to the total IGF-binding activity, quantified by qWLB, were determined as an indicator of IGF-related bioactivity. After the fourth TCA administration, the average level of IGF-I was increased in serum (p < 0.001). The increase of IGF-I concentrations in serum resulted in an increased ratio of IGF-I to IGFBPs in the circulation. By contrast in CSF, fragmentation of IGFBP-2 and IGFBP-3 and the ratio of IGF-II to intact IGFBPs were decreased at the fourth TCA administration (p < 0.01). Furthermore, reduced fragmentation of IGFBP-3 in CSF was accompanied by increased concentrations of intact IGFBP-3 (p < 0.001). We conclude that reduced fragmentation of IGFBPs and concomitant reduction of IGF-II to IGFBP ratios indicate regulation of bioactivity of IGF-II in CSF during repeated intrathecal TCA administration in MS patients.

Sex-Specific Control of Muscle Mass: Elevated IGFBP Proteolysis and Reductions of IGF-1 Levels Are Associated with Substantial Loss of Carcass Weight in Male DU6PxIGFBP-2 Transgenic Mice.

In farmed animals, carcass weight represents an important economic trait. Since we had demonstrated that IGFBP-2 represents a potent inhibitor of muscle accretion in inbred mice, we wanted to quantify the inhibitory effects of IGFBP-2 under conditions of elevated protein mass in growth selected non-inbred mice (DU6P). Therefore, we crossed male DU6P mice with female IGFBP-2 transgenic mice. Male IGFBP-2 transgenic offspring (DU6P/IGFBP-2) were characterized by more than 20% reductions of carcass mass compared to male non-transgenic littermates. The carcass mass in males was also significantly lower (p < 0.001) than in transgenic female DU6P/IGFBP-2 mice, which showed a reduction of less than 10% (p < 0.05) compared to non-transgenic female DU6P/IGFBP-2 mice. Although transgene expression was elevated in the muscle of both sexes (p < 0.001), serum levels were normal in female, but significantly reduced in male transgenic DU6P/IGFBP-2 mice (p < 0.001). In this group, also IGFBP-3 and IGFBP-4 were significantly reduced in the circulation (p < 0.01). Particularly in male transgenic mice, we were able to identify proteolytic activity against recombinant IGFBP-2 included in diluted serum. IGFBP-proteolysis in males correlated with massive reductions of IGF-1 in serum samples and the presence of elevated levels of IGFBP-2 fragments. From our data, we conclude that elevated tissue expression of IGFBP-2 is an essential effector of muscle accretion and may block more than 20% of carcass mass. However, in the circulation, intact IGFBP-2 contained no reliable biomarker content. Notably, for the estimation of breeding values in meat-producing animal species, monitoring of IGFBP-2 expression in muscle appears to be supported by the present study in a model system.

Development of a novel assay for IGFBP-2 complexed with IGF-I and-II in human serum

BackgroundInsulin-like growth factor binding-protein 2 (IGFBP-2) was originally identified as an IGF-carrier, governing IGF half-life, tissue accessibility and biological effects. Later, IGFBP-2 was discovered to possess IGF-independent effects. IGFBP-2 circulates in several forms, as free protein, complexed with IGF-I or IGF-II, or as IGFBP-2 fragments. The various IGFBP-2 forms are all included when measuring serum IGFBP-2 concentrations by immunoassay (i.e., immunoreactive (ir-)IGFBP-2). In this study, we describe a novel method to measure the amount of IGF that circulates bound to IGFBP-2. MethodIGFBP-2 was immunoprecipitated from human serum using magnetic beads, which were subsequently eluted by acidification. After neutralization, eluates were assayed for ir-IGFBP-2, IGF-I and IGF-II and compared to serum concentrations. This allowed measurement of IGFBP-2-compexed IGF-I and IGF-II, respectively. To test the method clinically, serum from 146 patients with lung cancer, 151 patients with non-cancer pulmonary diseases and 28 healthy controls were analyzed. ResultsWe immuno-precipitated 97 ± 3.3% of serum IGFBP-2 and recovered > 75% of IGFBP-2-complexed IGFs, with intra- and inter-assay coefficient of variations (CVs) averaging < 5% and < 13%, respectively. No co-precipitation with IGFBP-1, −3 or − 4 was detected.Serum levels of ir-IGFBP-2 (median [25;75%]) differed between groups (cancer patients vs. non-cancer patients vs. healthy controls): 342 [260;480] vs. 262 [189;388] vs. 190 [141;269] μg/l (p < .0001). In parallel with this, concentrations of IGF-II carried by IGFBP-2 averaged: 45.0 [33.3;52.5] vs. 34.2 [25.4;46.1] vs. 19.8 [14.1;26.0] μg/l (p < .0001), and concentrations of IGF-I 8.0 [5.2;11.8] vs. 5.4 [3.6;7.3] vs. 7.0 [3.8;13.0] μg/l (p < .0001). Thus, IGFBP-2 carried more IGF-II than IGF-I in all groups (p < .0001). When expressed relative to IGF-concentrations, IGFBP-2 carried 9.0 [5.3;15.5] % of the IGF-I and 4.8 [2.9;5.8] % of the IGF-II in serum from healthy subjects. Notably, in patients, IGFBP-2 carried relatively less IGF-I, but more IGF-II (p < .0001). ConclusionUsing our novel assay, we demonstrate: that IGFBP-2 carries ≈10% of circulating IGF-I and ≈5% of circulating IGF-II in healthy subjects; that IGF-II is the primary ligand for IGFBP-2; and that IGFBP-2 carries even more IGF-II in patients than in healthy subjects. Thus, our assay may provide information on IGFBP-2 beyond what is achievable by simply measuring ir-IGFBP-2.

Benzo[a]pyrene induced p53-mediated cell cycle arrest, DNA repair, and apoptosis pathways in Chinese rare minnow (Gobiocypris rarus).

The p53 pathways play an important role in carcinogenesis. In mammals, p53 and p53 target genes have been extensively studied, but little is known about their functions and regulation in fish. In this study, the cDNA fragments of p53 network genes, including p53, p21, mdm2, gadd45α, gadd45β, igfbp-3, and bax, were cloned from Chinese rare minnow (Gobiocypris rarus). These genes displayed high amino acid sequence identities with their zebrafish orthologs. The mRNA levels of p53 network genes and pathological changes in the liver were determined after adult rare minnow were exposed to 0.4, 2, and 10 µg/L of benzo[a]pyrene (BaP) for 28 days. The results showed that p53, p21, mdm2, gadd45α, and bax mRNA expressions in the livers from males and females were significantly upregulated compared with those of the controls (p < 0.05), but gadd45β and igfbp-3 expression was not significantly changed. Microphotographs revealed enlargement of the cell nuclei and cellular degeneration in males, while atrophy and vacuolization of hepatocytes were observed in females (10 µg/L). These results suggested that BaP induced liver DNA repair and apoptosis pathways and caused adverse pathological changes in rare minnow. The strongly responsive p53 network genes in the livers suggest that rare minnow is suitable as an experimental fish to screen environmental carcinogens. In addition, the p53 network genes in rare minnow could feasibly be used to identify the mechanism of environmental carcinogenesis. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 979-988, 2017.

ShRNA Constructs Targeting IGFBP-3 Alleviate Age Related Erectile Dysfunction in the Rat

We investigated whether injecting shRNA constructs targeting IGFBP-3 in the penis of old rats would improve erectile function. The most validated IGFBP-3 shRNA plasmid vector (pGPU6/GFP/Neo-shIGFBP-3) was prepared and injected in penile corpus cavernosum tissue. A total of 30 old (age 24 months) male Sprague Dawley® rats were randomly divided into 3 groups, including 10 each that received phosphate buffered saline only (100 μl), pGPU6/GFP/Neo-shNC (100 μg) and the most validated plasmid constructs pGPU6/GFP/Neo-shIGFBP-3 (100 μg). At 4 weeks the erectile response was measured as intracavernous pressure. The percent ofsmooth muscle in corpus cavernosum tissue was evaluated. Nitric oxide synthase activity and the cGMP concentration in penile tissue were also analyzed. IGFBP-3 was estimated in penile tissue by Western blot, real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry. pGPU6/GFP/Neo-shIGFBP-3 corrected the impaired erectile response in aged rats compared with the response in those injected with phosphate buffered saline and pGPU6/GFP/Neo-shNC (each p <0.01). The percent of cavernous smooth muscle was increased in the pGPU6/GFP/Neo-shIGFBP-3 group. Nitric oxide synthase activity and the cGMP concentration were also significantly increased in rats treated with pGPU6/GFP/Neo-shIGFBP-3. IGFBP-3 shRNA effectively reduced IGFBP-3 mRNA and protein expression in penile corpus cavernosum tissue. Decreasing IGFBP-3 expression by plasmid expressed shRNA improved erectile function in aged rats. The therapy may modulate smooth muscle integrity and increase the cGMP concentration. This may be a new direction for treating erectile dysfunction in clinical practice.

P01-23 Effects of IGFBP-1 and IGFBP-2 fragments on migration and IGF-induced proliferation of human dermal fibroblasts

P01-23 Effects of IGFBP-1 and IGFBP-2 fragments on migration and IGF-induced proliferation of human dermal fibroblasts

Interstitial fluid contains higher in vitro IGF bioactivity than serum: A study utilizing the suction blister technique

ContextCirculating insulin-like growth factors (IGFs) are bound in complexes which affect their tissue-accessibility. Interstitial fluid is in close proximity to target cells, but the IGF-system is not well-described herein. ObjectiveTo perform a thorough comparison of the IGF-system in suction blister fluid (SBF) vs. in serum, with emphasis on bioactive IGF levels. DesignEight hour study including samples collected in the fasting state (20h) and after a meal. SettingClinical research facility. ParticipantsSix healthy males (age 37.0±8.8years, BMI 22.5±1.4kg/m2) (mean±SD). Main outcome measureSerum and SBF concentrations of bioactive IGF (determined in vitro by specific IGF-I receptor (IGF-IR) phosphorylation assay), immunoreactive IGF and IGF binding protein (IGFBP) levels, Western ligand blotting (WLB) of IGFBPs and IGFBP-3 Western immunoblotting (WiB). ResultsThe ability of SBF to phosphorylate the IGF-IR in vitro was 41±27% higher than that of serum (P=0.007 by repeated measures ANOVA). By contrast, immunoreactive IGF and IGFBP-concentrations were approximately 50% lower in SBF than in serum (all P≤0.002). A marked difference in the composition of IGFBPs between serum and SBF was observed, including 3-fold elevated amounts of IGFBP-3 fragments in SBF (P<0.001). For both IGF-I, IGF-II and IGFBP-2, the effect of food intake differed between serum and SBF (all P≤0.03). ConclusionDespite lower concentrations, the in vitro IGF bioactivity was higher in SBF than in serum. This may relate to an increased enzymatic IGFBP-degradation and an altered IGFBP-composition in SBF, making more IGF-I and -II accessible to the IGF-IR. The impact of food intake on the IGF system differs between serum and interstitial fluid.

Functional and total IGFBP3 for the assessment of disorders of the GH/IGF1 axis in children with chronic kidney disease, GH deficiency, or short stature after SGA status at birth

IGFBP3 immunoreactivity may appear elevated in patients with chronic kidney disease (CKD), in part due to accumulation of low molecular fragments. The importance of these IGFBP3 variants for binding and inactivation of IGF1 and their relevance for the impaired growth of uremic children are unclear. Nevertheless, IGFBP3, measured as total (t-)IGFBP3, is frequently used as a diagnostic parameter in pediatric CKD patients. A new assay for functional (f-)IGFBP3 exclusively detects IGFBP3 capable of IGF binding. The aim of the study was to evaluate the significance of f-IGFBP3 measurements for the assessment of uremic abnormalities of the GH/IGF1 axis. Prospective cross-sectional study. t-IGFBP3, f-IGFBP3, and IGF1 were measured in pediatric CKD patients, including patients with CKD stage 3-4 not on dialysis (CKD, n=33), on dialysis treatment (DT, n=26), patients after renal transplantation (RTx, n=89), healthy children (n=29), children with GH deficiency (GHD, n=42), and small for gestational age (SGA) children (SGA, n=34). Mean t-IGFBP3 SDS was elevated in CKD, DT, and RTx children compared with controls and GHD patients (P≤0.0004). Highest values were reached in DT (P<0.0001 vs all groups). In contrast, mean f-IGFBP3 was similar in all groups (P=0.30). Pediatric CKD patients displayed elevated serum concentrations of t-IGFBP3 but not f-IGFBP3, supporting the hypothesis that IGFBP3 fragments not binding IGF1 accumulate during uremia. f-IGFBP3 is an indicator of IGFBP3 fragmentation and seems to reflect IGF1 binding in CKD better than t-IGFBP3. However, the role of f-IGFBP3 for the diagnosis of disturbances of the GH/IGF hormonal axis appears to be limited.

Isolation and Characterization of Vitreous Insulin-like Growth Factor Binding Proteins

Previous studies from this laboratory revealed that vitreous insulin-like growth factor (IGF) biological activity increases in proliferative diabetic retinopathy and that this activity is normally attenuated by IGFBPs. The goal of this study was to identify and characterize the species involved. Human and porcine vitreous, plasma, recombinant IGFBP-2, and IGFBP-3 were separated by gel electrophoresis. Functional IGFBPs were detected in Western ligand blots with biotinylated IGF-II. IGFBPs were identified using IGFBP-specific antibodies. Western ligand blots of normal vitreous and plasma detected two major proteins at ∼35 kDa and ∼29 kDa. Western blot analysis of human and porcine vitreous and plasma confirmed the identity of the ∼35-kDa band as IGFBP-2 and the ∼29-kDa band as a fragment of IGFBP-3. Western blot and Western ligand blot analyses of vitreous and plasma proteins separated by two-dimensional gel electrophoresis revealed that the IGFBP-3 fragments in vitreous and plasma have virtually identical profiles. Lyase digestion revealed that the ∼29-kDa IGFBP-3 fragment is a glycoprotein with a peptide core of ∼25 kDa. N-terminal sequence data obtained from vitreous IGFBP-3 revealed that the protein is proteolytically truncated at the C terminus. CONCLUSIONS. Normal human and porcine vitreous contain two major IGFBPs, IGFBP-2 and an ∼29-kDa fragment of IGFBP-3. Both IGFBPs retain biological activity, and IGFBP-3 has one or more glycosylation sites with a protein core of ∼25 kDa. Systematic comparisons indicate that the vitreous IGFBP-3 is similar to and perhaps identical with a previously described IGFBP-3 fragment in plasma with reduced growth factor affinities.

Lack of sex differences in the IGF–IGFBP response to ultra endurance exercise

The insulin-like growth factor (IGF)-IGF binding proteins (BP) and the pituitary-gonadal axes were investigated during ultra endurance exercise in 16 endurance-trained athletes (seven women). Median duration of the race was 6.3 days. Although food and drink were ad libitum, energy balance was negative. Blood samples were drawn before (PRE), at the end of (END) and 24 h after (POST24h) the race. Serum concentrations of total IGF-I (t-IGF-I) and free IGF-I (f-IGF-I) decreased by 33 (SD 38)% and 54 (19)%, respectively. The decrease in t-IGF-I appeared to be associated to the total energy deficit during the race. At END, the IGFBP-3 fragmentation and IGFBP-1 were increased but these changes did not predict changes in f-IGF-I. An increase in POST24h IGFBP-2 levels in women was the only sex difference. Testosterone was decreased by 67 (12)% in the men and estradiol became undetectable in the women without any detectable increase in LH and/or FSH. In conclusion ultra endurance exercise results in similar IGF-IGFBP responses in men and women reflecting a catabolic state. IGFBP-2 was the only exception, with increased levels in women after exercise. A concomitant decrease in gonadal hormones was not related to endocrine changes in the IGF-IGFBP axis but may be related to local changes in IGF-I expression.

Defined Carboxy-Terminal Fragments of Insulin-Like Growth Factor (IGF) Binding Protein-2 Exert Similar Mitogenic Activity on Cultured Rat Growth Plate Chondrocytes as IGF-I

The IGF/IGF binding protein (IGFBP) system is an important component in the hormonal regulation of longitudinal growth. Evidence from in vitro studies indicates that IGFBPs may have IGF-independent effects. We analyzed the biological activity of intact IGFBP-2 and defined carboxy-terminal IGFBP-2 fragments isolated from human hemofiltrate in two cell culture systems of the growth plate: rat growth plate chondrocytes in primary culture and the mesenchymal chondrogenic cell line RCJ3.1C5.18. The IGFBP-2 fragments IGFBP-2(167-279), IGFBP-2(167-289), and IGFBP-2(104-289) exerted a strong (2- to 3-fold) mitogenic effect on growth plate chondrocytes, which was comparable with IGF-I in equimolar concentrations (7.8 nm) but was not mediated through the type 1 IGF receptor. In a dose-response experiment, the most effective concentration of IGFBP-2(104-289) for the stimulation of cell proliferation was 10 nm. This biological activity of IGFBP-2 fragments was associated with cell membrane binding, demonstrated by Western blot analysis of fractionated cell lysates and immunohistochemistry. Whereas intact IGFBP-2 did not modulate chondrocyte proliferation, partially reduced (by dithiothreitol) full-length IGFBP-2 stimulated cell proliferation to a comparable extent (3.4-fold) as carboxy-terminal IGFBP-2 fragments. The mitogenic activity of these IGFBP-2 fragments and of partially reduced full-length IGFBP-2 was mediated through the use of the MAPK/ERK 1/2. These data imply a novel role of naturally occurring IGFBP-2 fragments for the endocrine and paracrine/autocrine regulation of longitudinal growth.

Various components of the insulin‐like growth factor system in tumor tissue, cerebrospinal fluid and peripheral blood of pediatric medulloblastoma and ependymoma patients

The insulin-like growth factor (IGF) system plays an important role in neuronal development and may contribute to the development of brain tumors. In this study, we studied mRNA expression levels of IGFs, insulin-like growth factor binding proteins (IGFBPs) and insulin-like growth factor receptors (IGFRs) in 27 pediatric medulloblastomas, 13 pediatric ependymomas and 5 control cerebella. Compared to normal cerebellum, mRNA levels of IGFBP-2 and IGFBP-3 were significantly increased in medulloblastomas and ependymomas. IGFBP-2 expression was indicative of poor prognosis in medulloblastomas, whereas IGFBP-3 mRNA levels were especially high in anaplastic ependymomas. IGFBP-5 and IGF-II mRNA levels were significantly increased in ependymomas compared to control cerebellum. Protein expression levels of IGFs and IGFBPs were analyzed in the cerebrospinal fluid (CSF) of 16 medulloblastoma, 4 ependymoma and 23 control patients by radioimmuno assay to determine whether they could be used as markers for residual disease after surgery. No aberrant CSF protein expression levels were found for ependymoma patients. In medulloblastoma patients, the IGFBP-3 protein levels were significantly higher than in ependymoma patients and controls. Moreover, enhanced levels of proteolytic fragments of IGFBP-3 were found in the CSF of medulloblastoma patients, being in concordance with a significantly increased IGFBP-3 proteolytic activity in the CSF of these patients. In conclusion, our data suggest that the IGF system is of importance in pediatric medulloblastomas and ependymomas. Larger studies should be conducted to validate the predictive values of the levels of intact IGFBP-3 and proteolytic fragments in CSF in the follow-up of medulloblastomas.

A 30-kDa fragment of insulin-like growth factor (IGF) binding protein-3 in human pregnancy serum with strongly reduced IGF-I binding.

Proteolytic cleavage of insulin-like growth factor (IGF) binding protein (IGFBP)-3 during pregnancy is likely to have both IGF-dependent and -independent effects on maternal, placental and fetal growth and metabolism. A 30-kDa proteolytic IGFBP-3 fragment was isolated from third trimester pregnancy human serum and identified by N- and C-terminal amino acid sequence analysis and mass spectrometry to correspond to residues 1-212 of the parent protein. This fragment is the dominating IGFBP-3 immunoreactive species in pregnancy serum. The 30-kDa fragment was also detected in serum of non-pregnant women where it coexists with intact IGFBP-3. Using biosensor technology, (1-212)IGFBP-3 was found to have 11-fold lower affinity for IGF-I compared to intact IGFBP-3, while a 4-fold decrease in affinity was found for IGF-II. Tests with des(1-3)IGF-I suggest fast binding of IGF-I to the N-terminal region of IGFBP-3 and similar affinity to a slow binding site in the C-terminal region.

Hormonal regulation of IGFBP‐2 proteolysis is attenuated with progression to androgen insensitivity in the LNCaP progression model

The identification of molecular determinants involved in the promotion of metastasis and development of androgen insensitive prostate cancer (AI-PCa) is necessary to discriminate aggressive from indolent disease and to identify therapeutic targets for advanced disease. Overexpression of one particular member of the insulin like growth factor (IGF) axis, IGFBP-2, is implicated in the development of AI-PCa and other cancers. Using the LNCaP human PCa progression model, we show that the AI and metastatic prostate cancer cell line C4-2B4 expresses greater amounts of secreted IGFBP-2 than the androgen sensitive (AS), non-metastatic LNCaP progenitor cell line. Further, the ability of androgens to decrease extracellular IGFBP-2 levels is attenuated in the AI and metastatic C4-2 cell line. The ability of androgen to negatively regulate extracellular IGFBP-2 levels was blocked by Casodex in a dose-dependent manner. The mechanism underlying the androgen-induced downregulation of secreted IGFBP-2 appears to involve extracellular proteolysis, resulting in the production of IGFBP-2 fragments lacking the ability to bind IGF-I and IGF-II. As C4-2 cells have an attenuated ability to proteolyze IGFBP-2 in response to androgen and C4-2B4 cells express greater amounts of IGFBP-2, our data implies that the diminished regulation of IGFBP-2 and loss of associated proteolytic fragments play a role in the increased metastatic behavior of these cells in vivo. Furthermore, our results suggest that either increased levels of intact IGFBP-2 or decreased levels of IGFBP-2 proteolytic fragments could serve as a biomarker to monitor for progression to AI-PCa.

Insulin-Like Growth Factors and Binding Proteins in Early Milk from Mothers of Preterm and Term Infants

Breast-fed preterm infants often show a better outcome, partly ascribed to the benefit of insulin-like growth factors (IGFs) and their binding proteins (IGFBP). We compared IGF-I, IGF-II, IGFBP-2 and IGFBP-3 levels, measured by radioimmunoassays in milk samples from 30 mothers of preterm (<31 weeks) and from 19 mothers of term (>37 weeks) infants at days 7 and 21 postpartum. Proteolysis of IGFBP-2 within mother’s milk and digestion of ¹²⁵I-IGF-II and ¹²⁵I-IGFBP-2 by gastric juice from neonates were assessed by electrophoretic techniques. Mean concentrations did not differ between preterm and term milk: IGF-I (2.8 ± 0.2 vs. 2.3 ± 0.1 ng/ml), IGF-II (12.0 ± 0.4 vs. 12.2 ± 0.5 ng/ml), IGFBP-3 (100.0 ± 5.1 vs. 80.0 ± 5.8 ng/ml), but did so for IGFBP-2 (3,144 ± 172 vs. 2,428 ± 188 ng/ml, p < 0.02). Immunoblots revealed 42% (p < 0.05) more IGFBP-2 fragments of 14 and 25 kDa in preterm milk. Incubation with gastric juice caused cleavage of ¹²⁵I-IGFBP-2 and partial cleavage of ¹²⁵I-IGF-II. Mutual complexation protected IGF-II and IGFBP-2 from cleavage, suggesting that both are likely to arrive in the bowel in an intact form to exert promotive effects. The results provide further evidence that IGFBP-2 and IGF-II in breast milk are relevant factors for the early development of preterm infants.

Inaccuracy of Insulin-Like Growth Factor (IGF) Binding Protein (IGFBP)-3 Assessment in the Diagnosis of Growth Hormone (GH) Deficiency from Childhood to Young Adulthood: Association to Low GH Dependency of IGF-II and Presence of Circulating IGFBP-3 18-Kilodalton Fragment

Poor sensitivity of IGF binding protein (IGFBP)-3 assessment in the work-up of GH deficiency (GHD) has been ascribed to the equal affinity of IGFBP-3 for IGF-I and IGF-II and to IGFBP-3 proteolysis. The objective of this study was to determine the IGF-II GH dependency and IGFBP-3 proteolysis in patients with GHD from childhood to young adulthood. This study was cross-sectional. This was a national multicenter study performed in university hospitals. One hundred thirty-one subjects (chronological age, 1.3-25 yr), 72 patients with GHD and 59 subjects with idiopathic short stature, were studied. IGF-I, IGF-II, and IGFBP-3 serum concentrations were measured by immunoradiometric assay. IGFBP-3 circulating forms were assessed by Western immunoblot (WIB) analysis. Main outcome measures were sensitivity and specificity of IGF-I, IGF-II, and IGFBP-3 measurements. Sensitivity and specificity of IGFBP-3 measurement were 27 and 100%, respectively. IGFBP-3 sensitivity was 46% in young adulthood. Sensitivity and specificity of IGF-I were 69 and 81%, respectively. Sensitivity and specificity of IGF-II assessment were 23 and 97%, respectively. IGFBP-3 WIB revealed the presence of the intact form and the major 29-kDa fragment in both GHD and subjects with idiopathic short stature. In patients with GHD, WIB showed the presence of an additional smaller IGFBP-3 fragment migrating at approximately 18 kDa. Our results suggest that in children and young adults with GHD, the low GH dependency of IGF-II together with IGFBP-3 proteolytic activity yielding the 18-kDa fragment concur to reduce the sensitivity of IGFBP-3 assessment, ultimately making it too inaccurate as a screening test in the work-up of GHD.

Diversity of Human Insulin-like Growth Factor (IGF) Binding Protein-2 Fragments in Plasma: Primary Structure, IGF-Binding Properties, and Disulfide Bonding Pattern

The insulin-like growth factor binding proteins (IGFBPs) play a major role in the regulation of the effects and the bioavailability of the insulin-like growth factors (IGFs). IGFs are released from IGFBP-IGF complexes by proteolysis of IGFBPs generating fragments with reduced ligand-binding properties. To identify naturally occurring fragments of IGFBP-2, a peptide library generated from human hemofiltrate was immunologically screened. Purification of immunoreactive IGFBP-2 fragments was performed by consecutive chromatographic steps. A total of 18 different IGFBP-2 fragments was isolated and characterized. The peptides exhibited different N-terminal amino acid residues that were located in the variable midregion of IGFBP-2. Four major cleavage sites were determined to be between Tyr103 and Gly104, Leu152 and Ala153, Arg156 and Glu157, and Gln165 and Met166. The resulting fragments were further processed by amino and/or carboxy peptidases and comprised 37-185 amino acid residues. Ligand blotting, solution binding assays, and BIAcore analyses revealed that all tested fragments retained low IGF-binding capacity. The most abundant fragment IGFBP-2 (167-279) showed 10% of IGF-II binding compared to recombinant human (rh)IGFBP-2. Furthermore, the disulfide bonding pattern of the C-terminal domain of rhIGFBP-2 was defined, indicating linkages between cysteine residues 191-225, 236-247, and 249-270. This study provides the most comprehensive molecular characterization of human IGFBP-2 fragments formed in vivo, exhibiting both residual IGF-binding capacities and the integrin-binding sequence.

Growth hormone/insulin-like growth factor system in children with chronic renal failure

Disturbances of the somatotropic hormone axis play an important pathogenic role in growth retardation and catabolism in children with chronic renal failure (CRF). The apparent discrepancy between normal or elevated growth hormone (GH) levels and diminished longitudinal growth in CRF has led to the concept of GH insensitivity, which is caused by multiple alterations in the distal components of the somatotropic hormone axis. Serum levels of IGF-I and IGF-II are normal in preterminal CRF, while in end-stage renal disease (ESRD) IGF-I levels are slightly decreased and IGF-II levels slightly increased. In view of the prevailing elevated GH levels in ESRD, these serum IGF-I levels appear inadequately low. Indeed, there is both clinical and experimental evidence for decreased hepatic production of IGF-I in CRF. This hepatic insensitivity to the action of GH may be partly the consequence of reduced GH receptor expression in liver tissue and partly a consequence of disturbed GH receptor signaling. The actions and metabolism of IGFs are modulated by specific high-affinity IGFBPs. CRF serum has an IGF-binding capacity that is increased by seven- to tenfold, leading to decreased IGF bioactivity of CRF serum despite normal total IGF levels. Serum levels of intact IGFBP-1, -2, -4, -6 and low molecular weight fragments of IGFBP-3 are elevated in CRF serum in relation to the degree of renal dysfunction, whereas serum levels of intact IGFBP-3 are normal. Levels of immunoreactive IGFBP-5 are not altered in CRF serum, but the majority of IGFBP-5 is fragmented. Decreased renal filtration and increased hepatic production of IGFBP-1 and -2 both contribute to high levels of serum IGFBP. Experimental and clinical evidence suggests that these excessive high-affinity IGFBPs in CRF serum inhibit IGF action in growth plate chondrocytes by competition with the type 1 IGF receptor for IGF binding. These data indicate that growth failure in CRF is mainly due to functional IGF deficiency. Combined therapy with rhGH and rhIGF-I is therefore a logical approach.

Muscle insulin-like growth factor status, body composition, and functional capacity in hemodialysis patients.

Hemodialysis (HD) patients typically have reduced muscle mass and diminished functional capacity. The role of the muscle insulin-like growth factors (IGFs), a principal anabolic system that is involved in protein synthesis and that has downregulation that is implicated in muscle loss in animal models of uremia, has previously not been assessed in vivo in HD patients. Seventeen HD patients were compared cross-sectionally with 17 age-, sex-, and body mass index-matched healthy controls. Body composition was assessed by dual energy x-ray absorptiometry and bioelectrical impedance spectrometry; functional capacity by hand grip strength, quadriceps strength, and 30-second sit-to-stand test; systemic inflammation by tumor necrosis factor-alpha (TNF-alpha) and TNF receptor 1 (TNFR1); serum and muscle IGF-I and IGFBP-3 by radioimmunoassay; and fragmentation of serum IGFBP-3 by Western immunoblotting. Appendicular lean mass was significantly decreased in HD patients compared with controls (17.6 +/- 0.9 versus 21.5 +/- 1.5 kg, P < .05), as were all measures of functional capacity (P < .01 to .001), and highly significant positive correlations between appendicular lean mass and functional capacity were evident (appendicular lean mass and hand-grip strength, quadriceps strength, 30-second sit-to-stand test, all P < .001). TNF-alpha and TNFR1 were elevated in patients (P < .001). Although serum IGF-I and IGFBP-3 levels did not differ between the groups (P = .295 and .379 respectively), fragmented IGFBP-3 levels were increased (53.1 +/- 16.0 versus 29.81 +/- 15.3%, P < .005). In contrast, muscle IGF-I was substantially diminished in the patient group (n = 7) relative to control (n = 5) levels (0.84 +/- 0.06 versus 2.78 +/- 1.80 pg/microg, P < .05). We provide evidence of reduced IGF-I in HD patients' skeletal muscle that may be a causal factor in the muscle wasting characteristic of this population. Future research should determine the exact consequences and causes of alterations to the muscle IGF system in HD patients.

Muscle insulin-like growth factor status, body composition, and functional capacity in hemodialysis patients

<h3>Background</h3> Hemodialysis (HD) patients typically have reduced muscle mass and diminished functional capacity. The role of the muscle insulin-like growth factors (IGFs), a principal anabolic system that is involved in protein synthesis and that has downregulation that is implicated in muscle loss in animal models of uremia, has previously not been assessed in vivo in HD patients. <h3>Methods</h3> Seventeen HD patients were compared cross-sectionally with 17 age-, sex-, and body mass index-matched healthy controls. Body composition was assessed by dual energy x-ray absorptiometry and bioelectrical impedance spectrometry; functional capacity by hand grip strength, quadriceps strength, and 30-second sit-to-stand test; systemic inflammation by tumor necrosis factor-α (TNF-α) and TNF receptor 1 (TNFR1); serum and muscle IGF-I and IGFBP-3 by radioimmunoassay; and fragmentation of serum IGFBP-3 by Western immunoblotting. <h3>Results</h3> Appendicular lean mass was significantly decreased in HD patients compared with controls (17.6 ± 0.9 versus 21.5 ± 1.5 kg, <i>P</i> < .05), as were all measures of functional capacity (<i>P</i> < .01 to .001), and highly significant positive correlations between appendicular lean mass and functional capacity were evident (appendicular lean mass and hand-grip strength, quadriceps strength, 30-second sit-to-stand test, all <i>P</i> < .001). TNF-α and TNFR1 were elevated in patients (<i>P</i> < .001). Although serum IGF-I and IGFBP-3 levels did not differ between the groups (<i>P</i> = .295 and .379 respectively), fragmented IGFBP-3 levels were increased (53.1 ± 16.0 versus 29.81 ± 15.3%, <i>P</i> < .005). In contrast, muscle IGF-I was substantially diminished in the patient group (n = 7) relative to control (<i>n</i> = 5) levels (0.84 ± 0.06 versus 2.78 ± 1.80 pg/μg, <i>P</i> < .05). <h3>Conclusions</h3> We provide evidence of reduced IGF-I in HD patients' skeletal muscle that may be a causal factor in the muscle wasting characteristic of this population. Future research should determine the exact consequences and causes of alterations to the muscle IGF system in HD patients.