Human IGF-I Research Articles

Evaluation of circulating insulin-like growth factor (IGF)-I and IGF-binding proteins as growth indices in rainbow trout (Oncorhynchus mykiss)

Circulating insulin-like growth factor (IGF)-I has been proposed as a growth index in several teleosts, including salmonids, and its level in circulation is stabilized by multiple IGF-binding proteins (IGFBPs). Three IGFBPs, IGFBP-2b, -1a, and -1b, are consistently detected in salmonid blood and are suggested to be indices of positive or negative growth, although their applicability to rainbow trout (Oncorhynchus mykiss) is unclear. The present study examined the usefulness of IGFBPs along with IGF-I as a physiological indicator of growth rate in rainbow trout through a rearing experiment. Two groups of underyearling rainbow trout were pit-tagged and either fed or fasted for 33 days. A third group was fasted for 22 days, followed by refeeding for 11 days. Serum IGF-I levels were reduced after fasting for 22 days, but refeeding did not retore its levels to those of the fed control. Nevertheless, there was a positive relationship between serum IGF-I levels and individual growth rates over 33 days of experimentation, confirming its validity as a growth index. Ligand blotting using labeled human IGF-I revealed two IGFBP bands at 43 and 32 kDa, which corresponded to IGFBP-2b and an unidentified form, respectively. In contrast, bands corresponding to IGFBP-1a and -1b, which usually increase after fasting, were hardly detected, even in the fasted fish. The responses of circulating IGFBP-2b to fasting and refeeding were similar to those of circulating IGF-I and positively correlated with growth rate and IGF-I levels. The intensity of the serum 32-kDa IGFBP band was higher in constantly fed fish than in the fasted fish; however, its correlation with growth rate was weaker than those of IGF-I and IGFBP-2b. The present study shows that IGF-I and IGFBP-2b can be used as growth indices for rainbow trout. In contrast, circulating IGFBP-1a and -1b may not serve as negative growth indices in rainbow trout under regular aquaculture conditions because they are rarely detected by ligand blotting or respond to fasting/refeeding.

An insulin growth factor-I/II-neutralizing monoclonal antibody in combination with epidermal growth factor receptor inhibitors potently inhibits tumor cell growth.

The insulin-like growth factors (IGFs), IGF-1 and IGF-II, which bind to the IGF receptor type 1 (IGF-1R) and the insulin receptor (IR), have been implicated in the growth, survival, and metastasis of tumor cells. We have previously identified a novel human monoclonal antibody (mAb), m708.5, which neutralizes both human IGF-I and IGF-II, and potently inhibits phosphorylation of the IGF-1R and the IR in breast cancer cells. In this study, m708.5 exhibited very strong synergy with the epidermal growth factor receptor (EGFR) inhibitor gefitinib, and synergy with chemotherapeutic agents in vitro against either neuroblastoma or breast cancer cells. In xenografted models, the combination of m708.5 and gefitinib significantly inhibited LAN-1 cell growth better than single agent alone. Taken together, these results support the clinical development of m708.5 for solid tumors with potential for synergy with chemotherapy and EGFR inhibitors.

Effects of growth hormone and cortisol administration on plasma insulin-like growth factor binding proteins in juveniles of three subspecies of masu salmon (Oncorhynchus masou)

In this study, we examined the effects of porcine growth hormone (GH) and cortisol on plasma insulin-like growth factor binding proteins (IGFBPs) in juveniles of three subspecies of Oncorhynchus masou (masu, amago, and Biwa salmon). Ligand blotting using digoxigenin-labeled human IGF-I was used to detect and semi-quantify three major circulating IGFBP bands at 41, 28, and 22 kDa, corresponding to IGFBP-2b, −1a, and -1b, respectively. GH increased plasma IGFBP-2b concentration in masu and Biwa salmon but suppressed it in amago salmon. Plasma IGFBP-2b levels were increased by cortisol in the three subspecies. Cortisol induced plasma IGFBP-1a in the three subspecies, whereas GH had a suppressive effect in masu and Biwa salmon. Sham and cortisol injections increased plasma IGFBP-1b levels after 1 day in masu and amago salmon, suggesting that IGFBP-1b is induced following exposure to stressors via cortisol. Increased IGFBP-1b levels were restored to basal levels when co-injected with GH in Biwa salmon, and the same trend was seen in masu and amago salmon. However, the suppressive effect of GH disappeared 2 days after injection in the three subspecies. Despite some differences among subspecies, the findings suggest that cortisol is a primary inducer of plasma IGFBP-1b; however, GH counteracts it in the short term. Therefore, GH has the potential to modulate the degree of increase in circulating IGFBP-1b levels during acute stress.

Production of two recombinant insulin-like growth factor binding protein-1 subtypes specific to salmonids

Salmonids have four subtypes of insulin-like growth factor binding protein (IGFBP)-1, termed -1a1, -1a2, -1b1 and 1b2, owing to teleost- and a lineage-specific whole-genome duplications. We have previously produced recombinant proteins of masu salmon IGFBP-1a1 and -1b2 and conducted functional analysis. To further characterize salmonid-specific IGFBP-1s, we cloned cDNAs encoding mature proteins of IGFBP-1a2 and -1b1 from the liver of masu salmon (Oncorhynchus masou). IGFBP-1a2 and -1b1 shared a 56% amino acid sequence homology whereas their homologies with their counterparts (i.e. -1a1 and -1b2) were 77% and 82%, respectively. We next expressed recombinant masu salmon (rs) IGFBP-1a2 and -1b1 with fusion partners thioredoxin (Trx) and a His-tag using the pET-32a(+) vector system in Escherichia coli. Trx.His.rsIGFBP-1s were detected in the insoluble faction, solubilized in a buffer containing urea, and isolated by Ni-affinity chromatography. They were refolded by dialysis and cleaved from the fusion partners by enterokinase. rsIGFBP-1a2 and -1b1 were purified by reversed-phase high performance liquid chromatography. Purified rsIGFBP-1a2 and -1b1 had the ability to bind digoxigenin-labeled human IGF-I on ligand blotting. We then examined the effects of rsIGFBP-1a1, -1a2, -1b1 and -1b2 in combination with human IGF-I on growth hormone (GH) release from cultured pituitary cells of masu salmon. IGF-I alone reduced GH release while the addition of rsIGFBP-1a1, -1b1 or -1b2, but not rsIGFBP-1a2, diminished the suppressive effect of IGF-I. Addition of rsIGFBP-1s without IGF-I had no effect on GH release. These results show that rsIGFBP-1b1, along with rsIGFBP-1a1 and -1b2, inhibits IGF-I action on the pituitary in masu salmon. The lack of the effect by rsIGFBP-1a2 suggests that salmon IGFBP-1 subtypes underwent subfunction partitioning and have different degrees of IGF-inhibitory action.

Targeting the IGF-Axis for Cancer Therapy: Development and Validation of an IGF-Trap as a Potential Drug.

The insulin-like growth factor (IGF)-axis was implicated in cancer progression and identified as a clinically important therapeutic target. Several IGF-I receptor (IGF-IR) targeting drugs including humanized monoclonal antibodies have advanced to phase II/III clinical trials, but to date, have not progressed to clinical use, due, at least in part, to interference with insulin receptor signaling and compensatory signaling by the insulin receptor (IR) isoform A that can bind IGF-II and initiate mitogenic signaling. Here we briefly review the current state of IGF-targeting biologicals, discuss some factors that may be responsible for their poor performance in the clinic and outline the stepwise bioengineering and validation of an IGF-Trap—a novel anti-cancer therapeutic that could bypass these limitations. The IGF-Trap is a heterotetramer, consisting of the entire extracellular domain of the IGF-IR fused to the Fc portion of human IgG1. It binds human IGF-I and IGF-II with a three-log higher affinity than insulin and could inhibit IGF-IR driven cellular functions such as survival, proliferation and invasion in multiple carcinoma cell models in vitro. In vivo, the IGF-Trap has favorable pharmacokinetic properties and could markedly reduce metastatic outgrowth of colon and lung carcinoma cells in the liver, outperforming IGF-IR and ligand-binding monoclonal antibodies. Moreover, IGF-Trap dose-response profiles correlate with their bio-availability profiles, as measured by the IGF kinase receptor-activation (KIRA) assay, providing a novel, surrogate biomarker for drug efficacy. Our studies identify the IGF-Trap as a potent, safe, anti-cancer therapeutic that could overcome some of the obstacles encountered by IGF-targeting biologicals that have already been evaluated in clinical settings.

Potential applications for rhIGF-I: Bone disease and IGF[sbnd

Growth hormone (GH) and insulin like growth factor-I (IGFI) are key bone trophic hormones, whose rising levels during puberty are critical for pubertal bone accrual. Conditions of GH deficiency and genetic resistance impact cortical and trabecular bone deleteriously with reduced estimates of bone strength. In humans, conditions of undernutrition (as in anorexia nervosa (AN), or subsequent to chronic illnesses) are associated with low IGF-I levels, which correlate with disease severity, and also with lower bone mineral density (BMD), impaired bone structure and lower strength estimates. In adolescents and adults with AN, studies have demonstrated a nutritionally acquired GH resistance with low IGF-I levels despite high concentrations of GH. IGF-I levels go up with increasing body weight, and are associated with rising levels of bone turnover markers. In short-term studies lasting 6–10 days, recombinant human IGF-I (rhIGF-I) administration in physiologic replacement doses normalized IGF-I levels and increased levels of bone formation markers in both adults and adolescents with AN. In a randomized controlled trial in adults with AN in which participants were randomized to one of four arms: (i) rhIGF-I with oral estrogen-progesterone (EP), (ii) rhIGF-I alone, (iii) EP alone, or (iv) neither for 9 months, a significant increase in bone formation markers was noted in the groups that received rhIGF-I, and a significant decrease in bone resorption markers in the groups that received EP. The group that received both rhIGF-I and EP had a significant increase in bone density at the spine and hip compared to the group that received neither. Side effects were minimal, with no documented fingerstick glucose of <50 mg/dl. These data thus suggest a potential role for rhIGF-I administration in optimizing bone accrual in states of undernutrition associated with low IGF-I.

Sequential measurements of IGF-I serum concentrations in adolescents with Laron syndrome treated with recombinant human IGF-I (rhIGF-I).

Background Recombinant human insulin-like growth factor 1 (rhIGF-I) has been approved as an orphan drug for the treatment of growth failure in children and adolescents with severe primary IGF-I deficiency (SPIGFD) with little pharmacokinetic data available. Therefore, sequential measurements of serum IGF-I, glucose, potassium, insulin and cortisol were performed in patients treated with rhIGF-I to evaluate their significance in safety and efficacy. Methods Repetitive blood samples were taken after meals before and 30, 60, 120, 180 and 360 min after rhIGF-I injections in two male patients with Laron syndrome at times of dose adjustments. Results Maximal IGF-I concentrations were observed 2 h after injections (495 ng/mL) and concentrations were still higher 6 h after injections than at baseline (303 ng/mL vs. 137 ng/mL). Thirteen percent of all and 33% of maximum IGF-I concentrations were greater than +2 standard deviation score (SDS) calculated for bone age (BA) (IGF-I SDS BA) rather than chronological age (CA) as BA was significantly delayed to CA by 3.2 years (p=0.0007). Height velocities correlated with individual maximum IGF-I SDS BA (ρ=0.735; p<0.0001). Serum insulin, cortisol and glucose did not correlate with IGF-I concentrations, but serum potassium showed a negative correlation (ρ=-0.364; p<0.0001) with IGF-I concentrations. Conclusions Sequential measurements of serum IGF-I, glucose and potassium in patients with Laron syndrome may aid in optimizing and individualizing rhIGF-I treatment. IGF-I concentrations should be referenced according to BA which better reflects the biological age. The inverse correlation of IGF-I and serum potassium concentrations after injections of rhIGF-I has not been reported before and warrants further consideration.

Takifugu rubripes cation independent mannose 6-phosphate receptor: Cloning, expression and functional characterization of the IGF-II binding domain

Mannose 6-phosphate/IGF-II receptor mediated lysosomal clearance of insulin-like growth factor-II is significantly associated with the evolution of placental mammals. The protein is also referred to as the IGF-II receptor. Earlier studies suggested relatively low binding affinity between the receptor and ligand in prototherian and metatherian mammals. In the present study, we cloned the IGF-II binding domain of the early vertebrate fugu fish and expressed it in bacteria. A 72000Da truncated receptor containing the IGF-II binding domain was obtained. Analysis of this protein (covering domains 11–13 of the CIMPR) for its affinity to fish and human IGF-II by ligand blot assays and ELISA showed that the expressed receptor can specifically bind to both fish and human IGF-II. Additionally, a peptide-specific antibody raised against the region of the IGF-II binding domain also was able to recognize the IGF-II binding regions of mammalian and non-mammalian cation independent MPR protein. These interactions were further characterized by Surface Plasma resonance support that the receptor binds to fish IGF-II, with a dissociation constant of 548nM. Preliminary analysis suggests that the binding mechanism as well as the affinity of the fish and human receptor for IGF-II may have varied according to different evolutionary pressures.

Human IGF-I propeptide A promotes articular chondrocyte biosynthesis and employs glycosylation-dependent heparin binding

BackgroundInsulin-like growth factor I (IGF-I) is a key regulator of chondrogenesis, but its therapeutic application to articular cartilage damage is limited by rapid elimination from the repair site. The human IGF-I gene gives rise to three IGF-I propeptides (proIGF-IA, proIGF-IB and proIGF-IC) that are cleaved to create mature IGF-I. In this study, we elucidate the processing of IGF-I precursors by articular chondrocytes, and test the hypotheses that proIGF-I isoforms bind to heparin and regulate articular chondrocyte biosynthesis. MethodsHuman IGF-I propeptides and mutants were overexpressed in bovine articular chondrocytes. IGF-I products were characterized by ELISA, western blot and FPLC using a heparin column. The biosynthetic activity of IGF-I products on articular chondrocytes was assayed for DNA and glycosaminoglycan that the cells produced. ResultsSecreted IGF-I propeptides stimulated articular chondrocyte biosynthetic activity to the same degree as mature IGF-I. Of the three IGF-I propeptides, only one, proIGF-IA, strongly bound to heparin. Interestingly, heparin binding of proIGF-IA depended on N-glycosylation at Asn92 in the EA peptide. To our knowledge, this is the first demonstration that N-glycosylation determines the binding of a heparin-binding protein to heparin. ConclusionThe biosynthetic and heparin binding abilities of proIGF-IA, coupled with its generation of IGF-I, suggest that proIGF-IA may have therapeutic value for articular cartilage repair. General significanceThese data identify human pro-insulin-like growth factor IA as a bifunctional protein. Its combined ability to bind heparin and augment chondrocyte biosynthesis makes it a promising therapeutic agent for cartilage damage due to trauma and osteoarthritis.

Production of recombinant salmon insulin-like growth factor binding protein-1 subtypes

Insulin-like growth factor (IGF)-I is a growth promoting hormone that exerts its actions through endocrine, paracrine and autocrine modes. Local IGF-I is essential for normal growth, whereas circulating IGF-I plays a crucial role in regulating the production and secretion of growth hormone (GH) by the pituitary gland. These actions of IGF-I are modulated by six insulin-like growth factor binding proteins (IGFBPs). In teleosts, two subtypes of each IGFBP are present due to an extra round of whole-genome duplication. IGFBP-1 is generally inhibitory to IGF-I action under catabolic conditions such as fasting and stress. In salmon, IGFBP-1a and -1b are two of three major circulating IGFBPs and assumed to affect growth through modulating IGF-I action. However, exact functions of salmon IGFBP-1 subtypes on growth regulation are not known due to the lack of purified or recombinant protein. We expressed recombinant salmon (rs) IGFBP-1a and -1b with a fusion protein (thioredoxin, Trx) and a His-tag using the pET-32a(+) vector expression system in Escherichia coli. Trx.His.rsIGFBP-1s were isolated by Ni-affinity chromatography, enzymatically cleaved by enterokinase to remove the fusion partners and further purified by reversed-phase HPLC. We next examined effects of rsIGFBP-1a and -1b in combination with human IGF-I on GH release from cultured masu salmon (Oncorhynchus masou) pituitary cells. Unexpectedly, IGF-I increased GH release and an addition of rsIGFBP-1a, but not rsIGFBP-1b, restored GH levels. The results suggest that IGFBP-1a can inhibit IGF-I action on the pituitary in masu salmon. Availability of recombinant salmon IGFBP-1s should facilitate further functional analyses and assay development.

Human IGF-I Eb-peptide induces cell attachment and lamellipodia outspread of metastatic breast carcinoma cells (MDA-MB-231)

Although Insulin-like growth factor (IGF-I) has been intensively studied, the functions of E-domain peptides of pro-IGF-I, however, have been overlooked. In our laboratory, several anti-cancer activities of the E-peptide of pro-IGF-I have been identified for the longest isoforms of human and rainbow trout E-peptides. These activities include dose-dependent inhibition of colony formation, inhibition of cancer cell metastasis and invasion through matrigel, suppression of cancer-induced angiogenesis, and attenuation of expression of apoptotic genes in favor of cell death. In this study, we were able to produce two-tagged recombinant human Eb-peptide (hEb) of pro-IGF-I with a purity over 99%. With its antimicrobial peptide (AMP)-like characteristics such as binding to the cytoplasmic membrane, and the affinity to the substratum of culture plate, hEb forms a layer of interface rapidly which facilitates the attachment of breast carcinoma cells, MDA-MB-231. Furthermore, the likely conformational change of homo-dimerized hEb through a single disulfide bond, as well as the ability to trigger clathrin-mediated endocytosis may play important roles for inducing lamellipodia outspread in MDA-MB-231 cells. With the highly purified hEb-peptide, not only could we study its function(s) in detail but also the minimum requirement for cancerous cells to metastasize to a suitable environment and grow.

STAT5B deficiency: Impacts on human growth and immunity

Growth hormone (GH) promotes postnatal human growth primarily by regulating insulin-like growth factor (IGF)-I production through activation of the GH receptor (GHR)-signal transducer and activator of transcription (STAT)-5B signaling cascade. The critical importance of STAT5B in human IGF-I production was confirmed with the identification of the first homozygous, autosomal recessive, STAT5B mutation in a young female patient who phenotypically resembled patients with classical growth hormone insensitivity (GHI) syndrome (Laron syndrome) due to mutations in the GHR gene, presenting with severe postnatal growth failure and marked IGF-I deficiency. Of note, the closely related STAT5A, which shares >95% amino acid identity with STAT5B, could not compensate for loss of functional STAT5B. To date, 7 homozygous, inactivating, STAT5B mutations in 10 patients have been reported. STAT5B deficient patients, unlike patients deficient in GHR, can also present with a novel, potentially fatal, primary immunodeficiency, which can manifest as chronic pulmonary disease. STAT5B deficiency may be underestimated in endocrine, immunology and pulmonary clinics.

IGF-I induces upregulation of DDR1 collagen receptor in breast cancer cells by suppressing MIR-199a-5p through the PI3K/AKT pathway.

Discoidin Domain Receptor 1 (DDR1) is a collagen receptor tyrosine-kinase that contributes to epithelial-to-mesenchymal transition and enhances cancer progression. Our previous data indicate that, in breast cancer cells, DDR1 interacts with IGF-1R and positively modulates IGF-1R expression and biological responses, suggesting that the DDR1-IGF-IR cross-talk may play an important role in cancer.In this study, we set out to evaluate whether IGF-I stimulation may affect DDR1 expression. Indeed, in breast cancer cells (MCF-7 and MDA-MB-231) IGF-I induced significant increase of DDR1 protein expression, in a time and dose dependent manner. However, we did not observe parallel changes in DDR1 mRNA. DDR1 upregulation required the activation of the PI3K/AKT pathway while the ERK1/2, the p70/mTOR and the PKC pathways were not involved. Moreover, we observed that DDR1 protein upregulation was induced by translational mechanisms involving miR-199a-5p suppression through PI3K/AKT activation. This effect was confirmed by both IGF-II produced by cancer-associated fibroblasts from human breast cancer and by stable transfection of breast cancer cells with a human IGF-II expression construct. Transfection with a constitutively active form of AKT was sufficient to decrease miR-199a-5p and upregulate DDR1. Accordingly, IGF-I-induced DDR1 upregulation was inhibited by transfection with pre-miR-199a-5p, which also impaired AKT activation and cell migration and proliferation in response to IGF-I.These results demonstrate that, in breast cancer cells, a novel pathway involving AKT/miR-199a-5p/DDR1 plays a role in modulating IGFs biological responses. Therefore, this signaling pathway may represent an important target for breast cancers with over-activation of the IGF-IR axis.

The impact of a human IGF-II analog ([Leu27]IGF-II) on fetal growth in a mouse model of fetal growth restriction.

Enhancing placental insulin-like growth factor (IGF) availability appears to be an attractive strategy for improving outcomes in fetal growth restriction (FGR). Our approach was the novel use of [Leu27]IGF-II, a human IGF-II analog that binds the IGF-II clearance receptor IGF-IIR in fetal growth-restricted (FGR) mice. We hypothesized that the impact of [Leu27]IGF-II infusion in C57BL/6J (wild-type) and endothelial nitric oxide synthase knockout (eNOS−/−; FGR) mice would be to enhance fetal growth and investigated this from mid- to late gestation; 1 mg·kg−1·day−1 [Leu27]IGF-II was delivered via a subcutaneous miniosmotic pump from E12.5 to E18.5. Fetal and placental weights recorded at E18.5 were used to generate frequency distribution curves; fetuses <5th centile were deemed growth restricted. Placentas were harvested for immunohistochemical analysis of the IGF system, and maternal serum was collected for measurement of exogenously administered IGF-II. In WT pregnancies, [Leu27]IGF-II treatment halved the number of FGR fetuses, reduced fetal(P = 0.028) and placental weight variations (P = 0.0032), and increased the numbers of pups close to the mean fetal weight (131 vs. 112 pups within 1 SD). Mixed-model analysis confirmed litter size to be negatively correlated with fetal and placental weight and showed that [Leu27]IGF-II preferentially improved fetal weight in the largest litters, as defined by number. Unidirectional 14CMeAIB transfer per gram placenta (System A amino acid transporter activity) was inversely correlated with fetal weight in [Leu27]IGF-II-treated WT animals (P < 0.01). In eNOS−/− mice, [Leu27]IGF-II reduced the number of FGR fetuses(1 vs. 5 in the untreated group). The observed reduction in FGR pup numbers in both C57 and eNOS−/− litters suggests the use of this analog as a means of standardizing and rescuing fetal growth, preferentially in the smallest offspring.

Tissue IGF-I Measured by Microdialysis Reflects Body Glucose Utilization After rhIGF-I Injection in Type 1 Diabetes.

Type 1 diabetes is associated with portal insulin deficiency and disturbances in the GH-IGF axis including low circulating IGF-I and GH hypersecretion. Whether peripheral hyperinsulinemia and GH hypersecretion, which are relevant to the development of vascular complications, result in elevated tissue IGF-I remains unknown. The purpose of this study was to determine the relationship between whole-body glucose uptake and tissue IGF-I measured by microdialysis. This was a single-blind placebo-controlled crossover study. The setting was a tertiary pediatric endocrine referral center. The participants were seven young male adults with type 1 diabetes. After an overnight fast, a 6-h lasting euglycemic clamp was performed (constant insulin infusion at 0.5 mU/kg × minute and variable glucose infusion rate [GIR]) and a subcutaneous injection of recombinant human (rh) IGF-I (120 μg/kg) or saline was given after 2 hours. In parallel, tissue IGF-I levels were determined by microdialysis (md-IGF-I). md-IGF-I levels in muscle and subcutaneous fat, and GIR were determined. md-IGF-I levels were detectable but unchanged after saline. After rhIGF-I, muscle and subcutaneous fat md-IGF-I increased during the second and third hour and then reached a plateau up to 10-fold higher than baseline (P < .001). GIR was unchanged after saline, whereas it increased 2.5-fold concomitantly with the increase in md-IGF-I (P < .0001). In contrast, serum IGF-I was increased already at 30 minutes after rhIGF-I and reached a plateau 2-fold above baseline (P < .0001). We demonstrate that md-IGF-I measurements are valid and physiologically relevant by reflecting rhIGF-I-induced glucose uptake. Future studies should be conducted to elucidate the role of local tissue IGF-I in diabetic vascular complications.

Development and characterization of an enzyme-linked DNA aptamer-magnetic bead-based assay for human IGF-I in serum

DNA aptamer sequences were selected and screened for affinity against recombinant human insulin-like growth factor-I (rhIGF-I) using an ELISA-like aptamer-based assay (ELASA). When the top four aptamers were paired against one another in all possible sandwich assay combinations using capture aptamers on magnetic beads (MBs), one sandwich combination appeared superior. This combination produced a reliably sensitive assay with a probable limit of detection<16ng/mL even in human serum. The assay did not require separation of rhIGF-I from IGF binding proteins (IGFBP) in human serum, suggesting that the capture and reporter aptamers may bind an exposed IGF-I epitope in the IGF-I–IGFBP complex.

IGF-I sensitivity in Silver–Russell syndrome with IGF2/H19 hypomethylation

BackgroundSilver–Russell syndrome (SRS) is characterized by intrauterine and postnatal growth retardation, typical facial appearance and body asymmetry. The mechanism of growth retardation is unclear. 50% of the patients have a paternal chromosome 11 epimutation — DNA hypomethylation of the imprinting center region 1 (ICR1) of the insulin-like growth factor 2 (IGF2)/H19 locus. SRS children who carry such an epimutation have increased levels of IGF-I and IGFBP-3 in relation to their stature and body weight, suggesting IGF-I resistance. No IGF-I receptor (IGF-1R) defect has been discovered. Therefore, another mechanism, probably an IGF-I post-receptor signaling defect, might be present. ObjectiveThe aim of this in-vitro study was to examine: 1) if IGF-I- and IGF-II-induced fibroblast growth is different in SRS children with IGF2/H19 hypomethylation compared to controls; and 2) whether there is IGF-I insensitivity in this subgroup of SRS children due to IGF-I post-receptor signaling defects. DesignFour SRS patients (two males, two females; 9.2 to 16.6years of age) with an IGF2/H19 hypomethylation defect and three age-matched healthy controls were included in the in-vitro study. Cultivated skin fibroblasts from the patients and the healthy controls were used for the experiments. Proliferation rates of fibroblasts were measured in the presence or absence of recombinant human IGF-I and IGF-II using the commercially available 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) test. PI3K (phosphoinositide 3-kinase) assay and NF-κB transcription factor assay were performed using ELISA in order to estimate the IGF-I-stimulated Akt phosphorylation and IκB phosphorylation, respectively. ResultsFibroblasts from SRS patients and fibroblasts from control individuals showed a comparable potential to proliferate in serum-free medium when stimulated with IGFs. No significant differences were found between both groups concerning Akt phosphorylation and IκB phosphorylation rates. ConclusionsThe results of the in-vitro study do not support the hypothesis that IGF-I/IGF-II resistance is a major pathogenetic mechanism responsible for the growth failure in the subgroup of SRS children with IGF2/H19 hypomethylation.

Quantification of intact human insulin‐like growth factor‐I in serum by nano‐ultrahigh‐performance liquid chromatography/tandem mass spectrometry

Insulin-like growth factor-I is one of the biomarkers used to detect growth hormone administration prohibited in human sport. Current testing approaches for IGF-I rely on commercial immunoassays, which may change from time to time requiring complex revalidation. Mass spectrometry (MS)-based approaches often rely on enzymatically digesting the protein and measuring specific peptide concentrations. In order to reinforce the current available methodology for IGF-I testing, a reliable and equally sensitive MS method is required for the analysis of intact protein using small sample volumes (<25 μL). IGF-I was extracted from human serum samples by a simple protein precipitation procedure. Separation was achieved via nano-ultrahigh-performance liquid chromatography and MS analysis was conducted by nano-electrospray ionisation triple-quadrupole mass spectrometry in the selected reaction monitoring mode using a stable-isotope-labelled internal standard. A six-point calibration curve ranging from 50 to 1000 ng/mL of human IGF-I in rat serum was used to establish instrument response. The method provided a limit of quantification of 50 ng/mL, with intra- and inter-day precision ≤5% and intra- and inter-day accuracy ≥95%. A quantitative method was developed for the quantification of intact IGF-I in human serum samples. The data generated provided important information for the development of a new reference method for the growth hormone biomarker test and helped create a reliable system for monitoring peptide hormones in individual athletes, a possible extension to the athlete biological passport system. Nano-electrospray has here been shown to be sufficiently robust for routine use in an analytical laboratory, allowing for the analysis of minute sample volumes.

Biochemical Markers of Insulin-Like Growth Factor-I Misuse in Athletes: The Response of Serum IGF-I, Procollagen Type III Amino-Terminal Propeptide, and the GH-2000 Score to the Administration of rhIGF-I/rhIGF Binding Protein-3 Complex

The GH-2000 and GH-2004 research groups developed a method for detecting GH misuse in athletes based on the measurement of serum IGF-I and procollagen type III amino-terminal propeptide (P-III-NP). There are reports that IGF-I is also misused by athletes, but currently there is no internationally recognized test designed to detect recombinant human IGF-I misuse. The objective of the study was to examine the response of serum IGF-I, P-III-NP, and the GH-2000 score to recombinant human (rh) IGF-I/rhIGF binding protein-3 (IGFBP-3) administration in recreational athletes. This was a randomized, double-blind, placebo-controlled rhIGF-I/rhIGFBP-3 administration study at Southampton General Hospital (Southampton, United Kingdom). Fifty-six recreational athletes (26 women, 30 men) participated in the study. Participants were randomized to treatment with low-dose (30 mg/d) or high-dose (60 mg/d) rhIGF-I/rhIGFBP-3 complex or placebo for 28 days. Blood was collected throughout the drug administration and washout periods. Serum IGF-I and P-III-NP were measured using commercial immunoassays and GH-2000 scores were calculated. IGF-I, P-III-NP, and the GH-2000 score rose in response to both low- and high-dose rhIGF-I/rhIGFBP-3 administration. The relative maximum response of IGF-I (approximately 4-fold increase in women and men) was greater than that of P-III-NP (40%-50% increase in women, 35%-50% increase in men). The GH-2000 formulae, which incorporate IGF-I and P-III-NP results, detected up to 61% of women and 80% of men in the rhIGF-I/rhIGFBP-3 groups but, using IGF-I concentrations alone, the sensitivity increased to 94% in both women and men during the administration period. The rise in P-III-NP after rhIGF-I/rhIGFBP-3 administration is small compared with that after rhGH administration. Although rhIGF-I/rhIGFBP-3 administration can be detected using the GH-2000 score method, a test based on serum IGF-I alone provides better sensitivity.

Pharmacodynamic and Antineoplastic Activity of BI 836845, a Fully Human IGF Ligand-Neutralizing Antibody, and Mechanistic Rationale for Combination with Rapamycin

Insulin-like growth factor (IGF) signaling is thought to play a role in the development and progression of multiple cancer types. To date, therapeutic strategies aimed at disrupting IGF signaling have largely focused on antibodies that target the IGF-I receptor (IGF-IR). Here, we describe the pharmacologic profile of BI 836845, a fully human monoclonal antibody that utilizes an alternative approach to IGF signaling inhibition by selectively neutralizing the bioactivity of IGF ligands. Biochemical analyses of BI 836845 demonstrated high affinity to human IGF-I and IGF-II, resulting in effective inhibition of IGF-induced activation of both IGF-IR and IR-A in vitro. Cross-reactivity to rodent IGFs has enabled rigorous assessment of the pharmacologic activity of BI 836845 in preclinical models. Pharmacodynamic studies in rats showed potent reduction of serum IGF bioactivity in the absence of metabolic adverse effects, leading to growth inhibition as evidenced by reduced body weight gain and tail length. Moreover, BI 836845 reduced the proliferation of human cell lines derived from different cancer types and enhanced the antitumor efficacy of rapamycin by blocking a rapamycin-induced increase in upstream signaling in vitro as well as in human tumor xenograft models in nude mice. Our data suggest that BI 836845 represents a potentially more effective and tolerable approach to the inhibition of IGF signaling compared with agents that target the IGF-I receptor directly, with potential for rational combinations with other targeted agents in clinical studies.