Detection of LongR3 -IGF-I, Des(1-3)-IGF-I, and R3 -IGF-I using immunopurification and high resolution mass spectrometry for antidoping purposes.

Insulin-like growth factor-I (IGF-I) and its analogs LongR3 -IGF-I, Des(1-3)-IGF-I, and R3 -IGF-I are prohibited substances in sport. Although they were never approved for use in humans, they are readily available as black market products for bodybuilding and can be used to enhance physical performance. This study's aims were to validate a fast and sensitive detection method for IGF-I analogs and to evaluate their detectability after intramuscular administration in rats. The sample preparation consisted of an immunopurification on MSIA™ microcolumns using a polyclonal anti-human-IGF-I antibody. The target substances were then directly analyzed by nano-liquid chromatography coupled with high-resolution mass spectrometry. Abundant signs of lower quality, oxidized peptide forms were found in black market products, justifying the need to monitor at least both the native and mono-oxidized forms. The analytical performance of this method (linearity, carry over, detection limits, precision, specificity, recovery, and matrix effect) was studied by spiking the analogs into human serum. Following a single intramuscular administration (100 µg/kg) in rats, detection was evaluated up to 36 h after injection. While unchanged Des(1-3)-IGF-I and R3 -IGF-I were detected until 24 h after administration, LongR3 -IGF-I disappeared rapidly after 4 h. Des(1)-LongR3 -IGF-I, a new N-terminal Long-R3 -IGF-I degradation product, was detected in addition to Des(1-10)-LongR3 -IGF-I and Des(1-11)-LongR3- IGF-I: the latter was detected up to 16 h. The same products were found after in vitro incubation of the analogs in human whole blood, suggesting that observations in rats may be extrapolated to humans and that the validated method may be applicable to antidoping testing.

Disease mechanism, biomarker and therapeutics for spinal and bulbar muscular atrophy (SBMA).

Spinal and bulbar muscular atrophy (SBMA) is a hereditary neuromuscular disorder caused by CAG trinucleotide expansion in the gene encoding the androgen receptor (AR). In the central nervous system, lower motor neurons are selectively affected, whereas pathology of patients and animal models also indicates involvement of skeletal muscle including loss of fast-twitch type 2 fibres and increased slow-twitch type 1 fibres, together with a glycolytic-to-oxidative metabolic switch. Evaluation of muscle and fat using MRI, in addition to biochemical indices such as serum creatinine level, are promising biomarkers to track the disease progression. The serum level of creatinine starts to decrease before the onset of muscle weakness, followed by the emergence of hand tremor, a prodromal sign of the disease. Androgen-dependent nuclear accumulation of the polyglutamine-expanded AR is an essential step in the pathogenesis, providing therapeutic opportunities via hormonal manipulation and gene silencing with antisense oligonucleotides. Animal studies also suggest that hyperactivation of Src, alteration of autophagy and a mitochondrial deficit underlie the neuromuscular degeneration in SBMA and provide alternative therapeutic targets.

Effect of expression alteration in flanking genes on phenotypes of St8sia2-deficient mice.

ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 2 (ST8SIA2) synthesizes polysialic acid (PSA), which is essential for brain development. Although previous studies reported that St8sia2-deficient mice that have a mixed 129 and C57BL/6 (B6) genetic background showed mild and variable phenotypes, the reasons for this remain unknown. We hypothesized that this phenotypic difference is caused by diversity in the expression or function of flanking genes of St8sia2. A genomic polymorphism and gene expression analysis in the flanking region revealed reduced expression of insulin-like growth factor 1 receptor (Igf1r) on the B6 background than on that of the 129 strain. This observation, along with the finding that administration of an IGF1R agonist during pregnancy increased litter size, suggests that the decreased expression of Igf1r associated with ST8SIA2 deficiency caused lethality. This study demonstrates the importance of gene expression level in the flanking regions of a targeted null allele having an effect on phenotype.

Inhibition of activin-like kinase 4/5 attenuates cancer cachexia associated muscle wasting.

Cancer mediated activation of the ActRIIB-ALK4/5 heterodimer by myostatin is strongly associated with muscle wasting. We investigated in vitro and in vivo the efficacy of ALK4/5 receptor blockers SB431542 and GW788388 in preventing muscle wasting, and explored synergy with IGF-I analogue LONG R3 (LR3) IGF-I. In vitro, C2C12 skeletal muscle cells were treated with vehicle, SB431542, GW788388 and LR3 IGF-I. A C26-CD2F1 cachexia model was used to induce cachexia in vivo. Mice were allocated as non-tumour bearing (NTB) or C26 tumour-bearing (C26 TB) vehicle control, treated with SB431542, LR3 IGF-I, SB431542 and LR3 IGF-I, or GW788388 (intraperitoneally or orally). In vitro, differentiation index and mean nuclei count increased using SB431542, GW788388, LR3 IGF-I. In vivo, GW788388 was superior to SB431542 in limiting loss of bodyweight, grip-strength and gastrocnemius weight. and downregulated Atrogin-1 expression comparable to NTB mice. LR3 IGF-I treatment limited loss of muscle mass, but at the expense of accelerated tumour growth. In conclusion, treatment with GW788388 prevented cancer cachexia, and downregulated associated ubiquitin ligase Atrogin-1.

Comparison of the effect of growth factors on chondrogenesis of canine mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are proposed to be useful in cartilage regenerative medicine, however, canine MSCs have been reported to show poor chondrogenic capacity. Therefore, optimal conditions for chondrogenic differentiation should be determined by mimicking the developmental process. We have previously established novel and superior canine MSCs named bone marrow peri-adipocyte cells (BM-PACs) and the objective of this study was to evaluate the effects of growth factors required for in vivo chondrogenesis using canine BM-PACs. Spheroids of BM-PACs were cultured in chondrogenic medium containing 10 ng/ml transforming growth factor-ß1 (TGF-ß1) with or without 100 ng/ml bone morphogenetic protein-2 (BMP-2), 100 ng/ml growth differentiation factor-5 (GDF-5) or 100 ng/ml insulin-like growth factor-1 (IGF-1). Chondrogenic differentiation was evaluated by the quantification of glycosaminoglycan and Safranin O staining for proteoglycan production. The expression of cartilage matrix or hypertrophic gene/protein was also evaluated by qPCR and immunohistochemistry. Spheroids in all groups were strongly stained with Safranin O. Although BMP-2 significantly increased glycosaminoglycan production, Safranin O-negative outer layer was formed and the mRNA expression of COL10 relating to cartilage hypertrophy was also significantly upregulated (P<0.05). GDF-5 promoted the production of glycosaminoglycan and type II collagen without increasing COL10 mRNA expression. The supplementation of IGF-1 did not significantly affect cartilaginous and hypertrophic differentiation. Our results indicate that GDF-5 is a useful growth factor for the generation of articular cartilage from canine MSCs.

Establishing a model of primary bovine hepatocytes with responsive growth hormone receptor expression.

The liver becomes resistant to growth hormone before parturition in dairy cows (uncoupling of the somatotropic axis). However, the mechanism of growth hormone insensitivity has not been fully described. The aim of the present study was to improve a previous model of adult bovine hepatocytes in a sandwich culture system to ensure growth hormone receptor (GHR) expression. First, we modified the protocol for hepatocyte retrieval and tested the effect of short (18 min) and long (up to 30 min) warm ischemia on hepatocyte viability. Second, we used medium additives that affect GHR expression in vivo-insulin (INS), dexamethasone (DEX), both (INS+DEX), or no hormone additives (CTRL)-to ensure the functionality of hepatocytes, as measured by lactate dehydrogenase activity and urea concentration in the medium. We also used reverse transcriptase PCR of hepatocytes to evaluate expression of albumin (ALB), hepatocyte nuclear factor 4α (HNF4A), nuclear factor-κ-B-inhibitor α (NFKBIA), cytosolic phosphoenolpyruvate carboxykinase (PCK1), and vimentin (VIM) mRNA. Moreover, we analyzed the expression of GHRtot (GHR), GHR1A, insulin-like growth factor-1 (IGF1), and IGF binding protein-2 (IGFBP2) in response to exposure to media with the different compositions. Modification of the protocol (changes in rinsing and perfusion times, buffer composition, and the volume and standardization of collagenase) led to increased cell counts and cell viability. Short warm ischemia with the modified protocol significantly increased cell count (4.7 × 107 ± 1.9 × 107 vs. 3.5 × 106 ± 1.5 × 106 vital cells/g of liver) and viability (79.1 ± 8.4 vs. 37.1 ± 8.9%). Therefore, we gathered hepatocytes from the liver after short warm ischemia with the modified protocol. For these hepatocytes, lactate dehydrogenase activity was lower in media with INS and with DEX than in media with INS+DEX or CTRL; urea concentrations were highest at d 4 for INS+DEX. As well, HNF4A and ALB were more highly expressed in hepatocytes cultured with INS and INS+DEX than in those cultured with DEX or CTRL, and the substitution of DEX suppressed VIM and NFKBIA expression but increased PCK1 expression. The expression of GHR, GHR1A, and IGF1 was suppressed by dexamethasone (DEX and INS+DEX), whereas INS distinctly increased GHR, GHR1A, and IGF1 mRNA expression. Hepatocytes in a sandwich culture showed influenceable GHR expression; this study provides a model that can be used in studies examining factors that influence the expression and signal transduction of GHR in dairy cows.

Insulin-like Growth Factor 1 Analogs Clicked in the C Domain: Chemical Synthesis and Biological Activities.

Human insulin-like growth factor 1 (IGF-1) is a 70 amino acid protein hormone, with key impact on growth, development, and lifespan. The physiological and clinical importance of IGF-1 prompted challenging chemical and biological trials toward the development of its analogs as molecular tools for the IGF-1 receptor (IGF1-R) studies and as new therapeutics. Here, we report a new method for the total chemical synthesis of IGF-1 analogs, which entails the solid-phase synthesis of two IGF-1 precursor chains that is followed by the CuI-catalyzed azide-alkyne cycloaddition ligation and by biomimetic formation of a native pattern of disulfides. The connection of the two IGF-1 precursor chains by the triazole-containing moieties, and variation of its neighboring sequences (Arg36 and Arg37), was tolerated in IGF-1R binding and its activation. These new synthetic IGF-1 analogs are unique examples of disulfide bonds' rich proteins with intra main-chain triazole links. The methodology reported here also presents a convenient synthetic platform for the design and production of new analogs of this important human hormone with non-standard protein modifications.

Role of Substance P Neuropeptide in Inflammation, Wound Healing, and Tissue Homeostasis.

Substance P (SP) is an undecapeptide present in the CNS and the peripheral nervous system. SP released from the peripheral nerves exerts its biological and immunological activity via high-affinity neurokinin 1 receptor (NK1R). SP is also produced by immune cells and acts as an autocrine or paracrine fashion to regulate the function of immune cells. In addition to its proinflammatory role, SP and its metabolites in combination with insulin-like growth factor-1 are shown to promote the corneal epithelial wound healing. Recently, we showed an altered ocular surface homeostasis in unmanipulated NK1R-/- mice, suggesting the role of SP-NK1R signaling in ocular surface homeostasis under steady-state. This review summarizes the immunobiology of SP and its effect on immune cells and immunity to microbial infection. In addition, the effect of SP in inflammation, wound healing, and corneal epithelial homeostasis in the eye is discussed.

Is insulin-like growth factor 1 (IGF-1) system an attractive target inflammatory bowel diseases? Benefits and limitation of potential therapy.

Inflammatory bowel diseases (IBD) are chronic gastrointestinal disorders with unknown etiology, whose incidence dramatically increased over the past 50 years. Currently available strategies for IBD treatment, such as biological therapies, corticosteroids, and immunosuppressive agents are effective, but their side effects and economic costs cannot be ignored. Better understanding of IBD etiology and new therapeutics are thus needed. The aim of this paper is to briefly discuss IGF-1 dependent functions, with particular focus on IGF-1 use in IBD therapy. Data collection was based on records found in medical literature. Data analysis included records published between 1984 and 2014. The IGF-1 system is involved in major physiological functions, such as cell proliferation and metabolism, and growth promotion. Most importantly IGF-1 has anti-inflammatory properties and its use in IBD treatment can be recommended. However, potential IGF-1 therapy has some limitations, which include aggravation of fibrosis in Crohn's patients and facilitated transformation to malignancy. Taken into consideration their possible side effects, IGF-1 analogs and recombinants are nonetheless a promising target for IBD therapy for a specific group of patients. Further studies, at the clinical level are thus recommended.

Epidermal growth factor receptor is required for estradiol-stimulated bovine satellite cell proliferation.

The objective of this study was to assess the role of the epidermal growth factor receptor (EGFR) in estradiol-17ß (E2)-stimulated proliferation of cultured bovine satellite cells (BSCs). Treatment of BSC cultures with AG1478 (a specific inhibitor of EGFR tyrosine kinase activity) suppresses E2-stimulated BSC proliferation (P < 0.05). In addition, E2-stimulated proliferation is completely suppressed (P < 0.05) in BSCs in which EGFR expression is silenced by treatment with EGFR small interfering RNA (siRNA). These results indicate that EGFR is required for E2 to stimulate proliferation in BSC cultures. Both AG1478 treatment and EGFR silencing also suppress proliferation stimulated by LR3-IGF-1 (an IGF1 analogue that binds normally to the insulin-like growth factor receptor (IGFR)-1 but has little or no affinity for IGF binding proteins) in cultured BSCs (P < 0.05). Even though EGFR siRNA treatment has no effect on IGFR-1ß mRNA expression in cultured BSCs, IGFR-1ß protein level is substantially reduced in BSCs treated with EGFR siRNA. These data suggest that EGFR silencing results in post-transcriptional modifications that result in decreased IGFR-1ß protein levels. Although it is clear that functional EGFR is necessary for E2-stimulated proliferation of BSCs, the role of EGFR is not clear. Transactivation of EGFR may directly stimulate proliferation, or EGFR may function to maintain the level of IGFR-1ß which is necessary for E2-stimulated proliferation. It also is possible that the role of EGFR in E2-stimulated BSC proliferation may involve both of these mechanisms.

Insulin-like growth factor-I regulates LH release by modulation of kisspeptin and NMDA-mediated neurotransmission in young and middle-aged female rats.

This study investigated potential mechanisms by which age and IGF-I receptor (IGF-Ir) signaling in the neuroendocrine hypothalamus affect estradiol-positive feedback effects on GnRH neuronal activation and on kisspeptin and N-methyl-D-aspartate (NMDA)-induced LH release and on the abundance of NMDA receptor subunits Nr1 and Nr2b and Kiss1r transcript and protein in the hypothalamus of young and middle-aged female rats. We infused vehicle, IGF-I, or JB-1, a selective antagonist of IGF-Ir, into the third ventricle of ovariectomized female rats primed with estradiol or vehicle and injected with vehicle, kisspeptin (3 or 30 nmol/kg), or NMDA (15 or 30 mg/kg). Regardless of dose, NMDA and kisspeptin resulted in significantly more LH release, GnRH/c-Fos colabeling, and c-Fos immunoreative cells in young than in middle-aged females. Estradiol priming significantly increased Kiss1r, Nr1, and Nr2b receptor transcript and protein abundance in young but not middle-aged female hypothalamus. JB-1 attenuated kisspeptin and NMDA-induced LH release, numbers of GnRH/c-Fos and c-Fos cells, and Kiss1r, Nr1, and Nr2b transcript and protein abundance in young females to levels observed in middle-aged females. IGF-I significantly enhanced NMDA and kisspeptin-induced LH release in middle-aged females without increasing numbers of GnRH/c-Fos or c-Fos immunoreactive cells. IGF-I infusion in middle-aged females also increased Kiss1r, Nr1, and Nr2b protein and transcript to levels that were equivalent to young estradiol-primed females. These findings indicate that age-related changes in estradiol-regulated responsiveness to excitatory input from glutamate and kisspeptin reflect reduced IGF-Ir signaling.

Transcriptome analyses of CHO cells with the next-generation microarray CHO41K: development and validation by analysing the influence of the growth stimulating substance IGF-1 substitute LongR(3.).

The increasing importance of Chinese hamster ovary (CHO) cells for the production of pharmaceutical proteins has awakened the demand to understand the cellular metabolism of these cells. However, satisfactory gene expression studies have yet been impractical due to insufficient coverage of sequences. In this work, previously determined sequence information of CHO cells and newly derived data from 454 and Illumina sequencing was used to establish the CHO41K microarray which contains 41,304 probes. Self-hybridisation was performed for replica determination and samples were run in triplicates to increase statistical power. For determination of technical variance, confidence intervals at an M-value of ±0.6 for 95% and at ±2.3 for 99% of the probes were calculated. Intra-microarray and slide to slide variance was not detectable. In a first application, this microarray enabled an in-depth look inside the cellular transcriptome of CHO cells cultured in the presence or absence of the growth supporting substance "insulin like growth factor 1" (IGF-1) analogue LongR(3). Its effect on the cells ranged from enhanced growth to delay of cell death as well as cytoskeletal installation. Suggesting that under supplementation, a minimised cellular effort in installation of a large cytoskeleton occurs, possibly in favour of promoting faster cell division.

Critical role of a survivin/TGF-ß/mTORC1 axis in IGF-I-mediated growth of prostate epithelial cells.

Survivin is a unique member of the inhibitor of apoptosis (IAP) proteins that is overexpressed in numerous cancers through poorly defined mechanisms. One such mechanism may be through constitutive activation of the insulin-like growth factor-I (IGF-I) signaling pathway, implicated in the development and progression of prostate cancer. Using the pre-neoplastic NRP-152 rat prostate cell line as a model, we showed that IGF-I induces Survivin expression, and that silencing Survivin by lentiviral-mediated small hairpin RNA (shRNA) represses IGF-I-stimulated cell growth, implicating Survivin as a mediator of this growth response. Moreover, our data support that the induction of Survivin by IGF-I occurs through a transcriptional mechanism that is mediated in part by the PI3K/Akt/mTORC1 pathway. Use of various Survivin promoter-luciferase constructs revealed that the CDE and CHR response elements in the proximal region of the Survivin promoter are involved in this IGF-I response. Transforming growth factor (TGF-ß) signaling antagonists similarly activated the Surivin promoter and rendered cells refractory to further promoter activation by IGF-I. IGF-I suppressed levels of phospho-Smads 2 and 3 with kinetics similar to that of Survivin induction. Suppression of TGF-ß signaling, either by TGF-ß receptor kinase inhibitors or by silencing Smads 2 and 3, induced Survivin expression and promoted cell growth similar to that induced by IGF-I. TGF-ß receptor antagonists also rescued cells from down-regulation of Survivin expression and growth suppression by pharmacological inhibitors of PI3K, Akt, MEK and mTOR. Sh-RNA gene silencing studies suggest that mTORC1 induces while mTORC2 represses the expression of Survivin by IGF-I. Taken together, these results suggest that IGF-I signaling through a PI3K/Akt/mTORC1 mechanism elevates expression of Survivin and promotes growth of prostate epithelial cells by suppressing Smad-dependent autocrine TGF-ß signaling.

Insulin-like growth factor-1 and its derivatives: potential pharmaceutical application for treating neurological conditions.

Ischemic brain damage remains a major cause of disability at all ages. This review examines the efficacy, mode of action and mechanisms of insulin-like growth factor (IGF)-1 and its derivatives in animal models of acute brain injury and neurodegenerative conditions, their potential in pharmaceutical developments. IGF-1 reduces cell loss and improves long-term neurological function in animal models. IGF-1 needs to be given within a few hours of the insult. However, the therapeutic window can be extended by mild hypothermia, likely by delaying apoptosis. Nevertheless, the poor central uptake of IGF-1 and its mitogenic potential limit clinical translation. Thus, recent studies have examined related compounds. For example, intravenous infusion of the N-terminal tripeptide of IGF-1 (glycine- proline-glutamate, GPE) can alleviate brain injury and improve long-term function in rats, with a broad effective dose range and a 3-7 hour therapeutic window, but has a short half-life. G-2meth-PE(G-2mPE), a GPE analogue with a longer half-life, is also neuroprotective. GPE/G-2mPE do not interact with IGF receptors and may act by modulating postinjury inflammation, astrogliosis and vascular remodeling. Cyclo-glycyl-proline (cGP), an endogenous diketopiperazine possibly derived from GPE is also neuroprotective. An analogue, cyclo-L-glycyl-L-2-allylproline (NNZ-2591) improves long-term somatosensory-motor function and histology after ischemic injury. Treatment with NNZ-2591 after 6-hypdroxydopamine injection in adult rats improves neurogenesis and long-term motor function. Further, oral administration of NNZ-2591 also prevents scopolamine-induced acute memory impairment. These beneficial effects may mediated by improved neuroplasticity. This review is an updated version of a previous publication in Recent Pat CNS Drug Discov.

Short-term estradiol administration in aging ovariectomized rats provides lasting benefits for memory and the hippocampus: a role for insulin-like growth factor-I.

We previously demonstrated that aged ovariectomized rats that had received prior estradiol treatment in middle age exhibited enhanced spatial memory and increased levels of estrogen receptor (ER)-α in the hippocampus long after estradiol treatment was terminated. The implication for cognition of increased levels of ERα resulting from prior estradiol exposure is unknown. In the absence of estrogens, growth factors, including IGF-I, can induce ERα-mediated transcription through ligand-independent mechanisms. Our current goal was to determine whether IGF-I mediates the ability of short-term exposure to estradiol to exert long-term effects on cognition and the hippocampus of aging females. Ovariectomized middle-aged rats were implanted with estradiol or cholesterol vehicle capsules. After 40 days, all capsules were removed and drug treatments were initiated. Half of each hormone treatment group received chronic intracerebroventricular delivery of the IGF-I receptor antagonist JB1, and the other half received artificial cerebrospinal fluid vehicle. Rats were tested on a spatial memory radial-arm maze task and hippocampi were immunostained for proteins of interest by Western blotting. As expected, previous treatment with estradiol enhanced spatial memory and increased levels of ERα in the hippocampus. JB1 reversed these effects. Previous treatment with estradiol resulted in lasting increases in levels of IGF-I receptors and phosphorylation of ERK/MAPK, a downstream signaling molecule of both ERα and IGF-I receptors, and increased levels of the ERα-regulated protein, choline acetyltransferase. JB1 blocked effects on ERK/MAPK and choline acetyltransferase. Results indicate that activation of IGF-I receptors is necessary for prior estradiol exposure to exert lasting impact on the hippocampus and memory.

Molecular characterisation of long-acting insulin analogues in comparison with human insulin, IGF-1 and insulin X10.

AIMS/HYPOTHESIS: There is controversy with respect to molecular characteristics of insulin analogues. We report a series of experiments forming a comprehensive characterisation of the long acting insulin analogues, glargine and detemir, in comparison with human insulin, IGF-1, and the super-mitogenic insulin, X10. METHODS: We measured binding of ligands to membrane-bound and solubilised receptors, receptor activation and mitogenicity in a number of cell types. RESULTS: Detemir and glargine each displayed a balanced affinity for insulin receptor (IR) isoforms A and B. This was also true for X10, whereas IGF-1 had a higher affinity for IR-A than IR-B. X10 and glargine both exhibited a higher relative IGF-1R than IR binding affinity, whereas detemir displayed an IGF-1R:IR binding ratio of ≤ 1. Ligands with high relative IGF-1R affinity also had high affinity for IR/IGF-1R hybrid receptors. In general, the relative binding affinities of the analogues were reflected in their ability to phosphorylate the IR and IGF-1R. Detailed analysis revealed that X10, in contrast to the other ligands, seemed to evoke a preferential phosphorylation of juxtamembrane and kinase domain phosphorylation sites of the IR. Sustained phosphorylation was only observed from the IR after stimulation with X10, and after stimulation with IGF-1 from the IGF-1R. Both X10 and glargine showed an increased mitogenic potency compared to human insulin in cells expressing many IGF-1Rs, whereas only X10 showed increased mitogenicity in cells expressing many IRs. CONCLUSIONS: Detailed analysis of receptor binding, activation and in vitro mitogenicity indicated no molecular safety concern with detemir.

Understanding the mechanism of insulin and insulin-like growth factor (IGF) receptor activation by IGF-II.

BACKGROUND: Insulin-like growth factor-II (IGF-II) promotes cell proliferation and survival and plays an important role in normal fetal development and placental function. IGF-II binds both the insulin-like growth factor receptor (IGF-1R) and insulin receptor isoform A (IR-A) with high affinity. Interestingly both IGF-II and the IR-A are often upregulated in cancer and IGF-II acts via both receptors to promote cancer proliferation. There is relatively little known about the mechanism of ligand induced activation of the insulin (IR) and IGF-1R. The recently solved IR structure reveals a folded over dimer with two potential ligand binding pockets arising from residues on each receptor half. Site-directed mutagenesis has mapped receptor residues important for ligand binding to two separate sites within the ligand binding pocket and we have recently shown that the IGFs have two separate binding surfaces which interact with the receptor sites 1 and 2. METHODOLOGY/PRINCIPAL FINDINGS: In this study we describe a series of partial IGF-1R and IR agonists generated by mutating Glu12 of IGF-II. By comparing receptor binding affinities, abilities to induce negative cooperativity and potencies in receptor activation, we provide evidence that residue Glu12 bridges the two receptor halves leading to receptor activation. CONCLUSIONS/SIGNIFICANCE: This study provides novel insight into the mechanism of receptor binding and activation by IGF-II, which may be important for the future development of inhibitors of its action for the treatment of cancer.

Therapeutic effects of PEGylated insulin-like growth factor I in the pmn mouse model of motoneuron disease.

Based on its potent neurotrophic and myotrophic activities, insulin-like growth factor I (IGF-I) has been proposed for treatment of neuromuscular disorders such as muscular dystrophies and amyotrophic lateral sclerosis (ALS). However, the short half life in the circulation limits its use in vivo. At least in mouse models, beneficial effects are generally only observed by dosing regimens such as minipumps or gene therapy that are difficult to translate to patients. We have developed a polyethylene glycol coupled IGF-I (PEG-IGF-I) that could circumvent these problems by longer half-life, showing all features of a therapeutic agent supporting muscular and neuronal function. Here we investigated its effects in the pmn mutant mouse, a model with typical dying-back motoneuron degeneration. In vitro, PEG-IGF-I and rhIGF-I profoundly promoted survival axonal growth of wild-type as well as pmn mutant embryonic motoneurons, suggesting that PEG-IGF-I had a fully conserved neurotrophic activity via its receptor. In vivo, treatment of pmn mutant mice with PEG-IGF-I prolonged survival, protected against late stage weight loss and significantly maintained muscle force and motor coordination. Consistently, PEG-IGF-I treatment rescued facial and lumbar motoneurons from cell death and partially preserved phrenic nerve myelinated axons. The data support that PEG-IGF-I could be used for treatment of neuromuscular diseases in a clinically feasible manner.

Talla baja idiopática. Revisión y puesta al día / Idiopathic short stature. A literature review and update

Con la denominación de talla baja idiopática (TBI) se agrupan una serie de entidades clínicas de etiología desconocida que tienen en común un retraso crónico de crecimiento con talla inferior a −2 desviaciones estándar (DE), con preservación de la armonía entre los segmentos corporales y en las que, en su evolución espontánea, las expectativas de talla adulta son inferiores a −2 DE. Es un diagnóstico de exclusión que exige una evaluación clínica, bioquímica, hormonal y molecular minuciosa con el objetivo de descartar cualquier etiología conocida del retraso de crecimiento, especialmente el retraso constitucional del crecimiento y desarrollo (RCCD). La TBI es un diagnóstico frecuente entre los pacientes que consultan por retraso de crecimiento, existiendo lagunas y controversias sobre su abordaje diagnóstico y terapéutico. Este documento de consenso recoge información actualizada sobre la definición, diagnóstico y tratamiento de la TBI, y aporta datos y recomendaciones que no han sido contemplados en documentos anteriores (AU)

Idiopathic short stature (ISS) refers to all clinical conditions involving an alteration of growth (height <−2 SD) of unknown cause, with preservation of proportionality among body segments, with the expectation of adult height < −2 SDS, and in which a diagnosis of constitutional delay of growth and development has been previously ruled out. ISS is an exclusion diagnostic which requires clinical, biochemical, hormonal and molecular studies in order to rule out all known causes of growth retardation and short stature.ISS is a frequent diagnosis among children with short stature. Despite its frequency, there is still controversy on the best diagnostic and therapeutic approach when treating patients with ISS. This consensus document contains updated information on the definition, diagnosis and treatment of ISS, and provides new data and recommendations that have not been addressed in previous documents (AU)

PEGylation enhances the therapeutic potential for insulin-like growth factor I in central nervous system disorders.

OBJECTIVE: Due to its potent neurotrophic activity, insulin-like growth factor I (IGF-I) has been proposed many times for therapeutic application in disorders of the central nervous system (CNS). However, insufficient brain delivery to yield beneficial central without peripheral side effects have prevented clinical development in most instances. DESIGN: We recently reported the generation of a polyethylene-glycol modified IGF-I variant (PEG-IGF-I) with prolonged half-life and less acute side effects, but with fully maintained slow anabolic activity. Here we investigated if these beneficial properties result in improved brain availability of the drug, thereby reaching therapeutically relevant steady-state concentrations to elicit beneficial effects on neuronal function. RESULTS: After a single subcutaneous injection, PEG-IGF-I reached much higher steady-state levels in brain tissue and cerebrospinal fluid compared with IGF-I. Two weeks treatment with PEG-IGF-I was sufficient to modulate brain plasticity processes, as judged by changes in synaptic proteins and related animal behavior. Furthermore, chronic treatment of a mouse model of brain amyloidosis with PEG-IGF-I reverted deficits in insulin/IGF-I signaling, synaptic proteins and cognitive performance. CONCLUSIONS: Our data generate the therapeutic potential for PEG-IGF-I to treat CNS disorders by systemic drug application, and in addition scientifically support its application in disorders of synaptic function and neuronal development.