Absence Of Insulin-like Growth factor-I Research Articles

The Supportive Role of Insulin-Like Growth Factor-I in the Differentiation of Murine Mesenchymal Stem Cells into Corneal-Like Cells.

This study was focused on characterizing the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into corneal-like cells. Mouse MSCs were isolated from the bone marrow, grown in cell culture for 3 weeks, and purified using a magnetic activated cell sorter. Purified MSCs were cultured with an extract prepared from excised corneas and in the presence or absence of insulin-like growth factor-I (IGF-I). Analysis by quantitative real-time polymerase chain reaction showed that the expression of corneal specific markers, such as cytokeratin 12 (K12), keratocan, and lumican, was already induced after a 3-day cultivation and gradually increased during the 10-day incubation of MSCs with the extract. The presence of IGF-I significantly increased differentiation. Immunofluorescence analysis of differentiated MSCs showed positive results for the K12 protein. The morphology of the differentiated cells and the expression of cell surface markers CD45, CD11b, CD73, CD44, and CD105 were comparable in the control and differentiated MSCs. Proliferative activity was even higher in differentiated cells than in untreated MSCs. Both untreated and differentiated MSCs inhibited the production of interleukin-2 and interferon-γ in spleen cells stimulated with Concanavalin A. The results thus show that MSCs cultured in the presence of corneal extract and IGF-I efficiently differentiate into corneal-like cells. The differentiated cells possess characteristics of corneal epithelial cells and keratocytes, while at the same time maintaining MSC properties.

Comparación de las acciones osteogénicas de la proteína relacionada con la parathormona (PTHrP) en modelos de ratón diabético y con déficit del factor de crecimiento similar a la insulina tipo I (IGF-I)

Work scholarship from the SEIOMM to attend the 35 Congress of the ASBMR (Baltimore, 2013). Summary Diabetes mellitus (DM) is a metabolic pathology characterised by chronic hyperglycemia due to a deficit in the production and/or action of insulin. DM, above all type I, is commonly associated with osteopenia/osteoporosis and with an increased risk of fractures. Insulin-like growth factor-I (IGF-I), a factor abundant in the bone matrix which plays a significant role in the development and maintenance of bone mass, diminishes with DM. Parathyroid hormone-related protein (PTHrP), a modulator of growth and osteoblast function, acts on osteoprogenitors, promoting osteoblast differentiation and bone regeneration. Its expression is reduced in the presence of DM. In this work we have evaluated and compared the osteogenic actions of PTHrP in mouse models with type 1 DM and IGF-I deficiency. Diabetic mice by injection of streptozotocin had a reduction in bone mass in the long bones associated with an increase in oxidised proteins and a reduction in the expression of genes related to the Wnt pathway and of β-catenin protein, as well as alterations in vertebral trabecular bone. In the mouse model with IGF-I deficit our results indicate the presence of osteopenia both in the femur (associated with an inhibition of the Wnt pathway) and the spine (L1-L5). Our findings demonstrate that the administration of PTHrP, predominantly through its N-terminal domain, modulates the canonical Wnt pathway in relation to its osteogenic actions in a diabetic situation and also, in part, in the absence of IGF-I.

Treatment with N- and C-Terminal Peptides of Parathyroid Hormone-Related Protein Partly Compensate the Skeletal Abnormalities in IGF-I Deficient Mice

Insulin-like growth factor-I (IGF-I) deficiency causes growth delay, and IGF-I has been shown to partially mediate bone anabolism by parathyroid hormone (PTH). PTH-related protein (PTHrP) is abundant in bone, and has osteogenic features by poorly defined mechanisms. We here examined the capacity of PTHrP (1–36) and PTHrP (107–111) (osteostatin) to reverse the skeletal alterations associated with IGF-I deficiency. Igf1-null mice and their wild type littermates were treated with each PTHrP peptide (80 µg/Kg/every other day/2 weeks; 2 males and 4 females for each genotype) or saline vehicle (3 males and 3 females for each genotype). We found that treatment with either PTHrP peptide ameliorated trabecular structure in the femur in both genotypes. However, these peptides were ineffective in normalizing the altered cortical structure at this bone site in Igf1-null mice. An aberrant gene expression of factors associated with osteoblast differentiation and function, namely runx2, osteoprotegerin/receptor activator of NF-κB ligand ratio, Wnt3a , cyclin D1, connexin 43, catalase and Gadd45, as well as in osteocyte sclerostin, was found in the long bones of Igf1-null mice. These mice also displayed a lower amount of trabecular osteoblasts and osteoclasts in the tibial metaphysis than those in wild type mice. These alterations in Igf1-null mice were only partially corrected by each PTHrP peptide treatment. The skeletal expression of Igf2, Igf1 receptor and Irs2 was increased in Igf1-null mice, and this compensatory profile was further improved by treatment with each PTHrP peptide related to ERK1/2 and FoxM1 activation. In vitro, PTHrP (1–36) and osteostatin were effective in promoting bone marrow stromal cell mineralization in normal mice but not in IGF-I-deficient mice. Collectively, these findings indicate that PTHrP (1–36) and osteostatin can exert several osteogenic actions even in the absence of IGF-I in the mouse bone.

Observations on the effects of Suppressor of Cytokine Signaling 7 (SOCS7) knockdown in breast cancer cells: their in vitro response to Insulin Like Growth Factor I (IGF-I)

Suppressor of cytokine signaling 7 (SOCS7) is a member of the SOCS family and is known to interact with phospholipase Cγ-1 (PLCγ-1), one of the insulin-like growth factor-I (IGF-I) receptor downstream molecules. In this study, we sought to observe the effect of knocking down SOCS7 gene on breast cancer cells in vitro growth and migration and to elucidate whether this involves IGF-I-PLCγ1 route using the PLCγ-1 blocker U73122. Suitable breast cancer cells (MCF7 and MDA-MB-231) were transfected with anti-SOCS7 ribozymal transgene, to create sub-lines with SOCS7 knockdown verified by RT-PCR. The growth and migration of the cells were evaluated in the presence or absence of IGF-I and PLCγ-1 inhibitor using growth assay, scratch-wound and electrical cell impedance sensing (ECIS) migration assays. IGF-I treatment produced more pronounced influence on MCF7 growth and migration and on MDA-MB-231 migration when SOCS7 gene was knocked down in both lines (p < 0.05). The absence of IGF-I-induced growth response in MDA-MB-231 could be due to the intrinsic characteristics of these cells. PLCγ-1 pharmacological inhibition during their in vitro migration seemed to only occur when SOCS7 gene was knocked down. To the best of our knowledge, this is the first report of the SOCS7 regulatory role in IGF-I induced in vitro functions in ER-positive and ER-negative breast cancer cells. IGF-I treatment and SOCS7 loss have synergistically resulted in increased growth and migration of MCF7 and in increased migration of MDA-MB-231 cells. The migratory effects could be due to a precise anti-PLCγ-1 role.

Abstract P1-04-03: Knocking down Suppressor of Cytokine Signaling 7 in breast cancer: The role in Insulin-like Growth Factor - I/Phospholipase Cγ-1 signaling

Abstract BACKGROUND: Suppressor of Cytokine Signaling 7 (SOCS7) is a member of the SOCS family and is known to interact with Phospholipase Cγ-1 (PLCγ-1), one of the Insulin like Growth Factor - I (IGF-I) receptor downstream molecules. In the present study, we sought to examine the effect of knocking down SOCS7 gene on breast cancer cells growth and migration and to elucidate whether this has involved IGF-I — PLCγ-1 signaling using the PLCγ-1 blocker U73122. METHODS: Suitable breast cancer cells (MCF7 and MDA-MB-231) were transfected with anti-SOCS7 ribozymal transgene, to create sublines with SOCS7 knockdown that was verified by RT-PCR. The growth and migration of the cells were evaluated in the presence or absence of IGF-I and PLCγ-1 inhibitor using in vitro growth assay and Electrical Cell Impedance Sensing (ECIS) migration assay. RESULTS: MCF7ΔSOCS7 and MDA-MB-231ΔSOCS7 (SOCS7 knockdown) were constructed. Both sublines showed a higher rate of growth compared to control cells with and without IGF-I stimulation. U73122 treatment did not appear to change this growth outcome. Using ECIS migration assay, it was shown that knocking down SOCS7 had a significant positive effect on the migration of MCF7 and MDA-MB-231 cells, and that both IGF-I treatment and SOCS7 knockdown had a synergistic positive influence on their migration (p &amp;lt; 0.05). We further demonstrated that the impact of U73122 on the IGF-I migratory effect was dependent upon SOCS7 knockdown as it has significantly blocked the stimulatory effect of IGF-I on MCF7ΔSOCS7 and MDA-MB-231ΔSOCS7 migration but not that of the control cells. While SOCS7 has acted to control the IGF-I effect, it appeared to cancel the inhibitory function of U73122, indicating a specific anti - PLCγ-1 role for SOCS7 in IGF-I induced breast cancer cellular migration. CONCLUSION: SOCS7 loss resulted in increased growth and migration of breast cancer cells and this had a synergistic effect on their response to IGF-I. This role could be related to its interaction with PLCγ-1 during the cellular migration but not the growth. It may be possible that SOCS7 acts through different mechanism to control the cellular growth. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-04-03.

β-Arrestin 2-mediated heterologous desensitization of IGF-IR by prolonged exposure of SH-SY5Y neuroblastoma cells to a mu opioid agonist

Prolonged (12 h) exposure of SH-SY5Y neuroblastoma cells to the mu-opioid receptor (MOPr) agonist [D-Ala 2,N-Me-Phe 4,Gly 5-ol]-enkephalin (DAMGO) causes homologous desensitization as well as heterologous desensitization of the extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation induced by insulin-like growth factor (IGF)-I. Brief (15 min) but not prolonged exposure to DAMGO transregulates the insulin-like growth factor-I (IGF-I) receptor, as evidenced by its phosphorylation in the absence of IGF-I. Silencing of β-arrestin 2 uncouples the crosstalk between the two receptors, thus maintaining IGF-I-mediated receptor phosphorylation and ERK 1/2 activation even after prolonged DAMGO exposure. Furthermore, MOPr-induced activation of IGF-I receptor requires the tyrosine kinase c-Src.

Abstract 3078: PHLPP1 phosphatase dephosphorylates Akt and inhibits tumorogenesis in the orthotopic model of pancreatic cancer

Abstract Background &amp; Aims: Akt kinase is a major prosurvival factor in pancreatic cancer (PaCa). Akt activity is regulated by its phosphorylation, which is controlled by kinases and phosphatases. Whereas kinases involved in Akt phosphorylation are well characterized, little is known about phosphatases that inactivate Akt. Here we investigated the expression of recently discovered serine phosphatase PHLPP1 in human and mouse pancreatic adenocarcinomas; its role in the regulation of Akt activity and PaCa cell death; and the effect of PHLPP1 overexpression on tumorigenesis in the nude mouse orthotopic model of PaCa. Methods: Human pancreatic adenocarcinoma MiaPaCa-2 and PANC-1 cells were cultured in the presence or absence of insulin-like growth factor-I (IGF-I) or serum (FBS). We measured apoptosis by DNA fragmentation and AnnexinV staining (flow cytometry); Akt activity, by Akt phosphorylation at Ser473; and phosphatase activity in PHLPP1 immunoprecipitate. For transient overexpression PHLPP plasmid were transfected using Amaxa nucleofector. MiaPaCa-2 cells transduced with PHLPP1 expressing lentivirus (as well as control virus) were applied in the orthotopic model of PaCa. PDXCre Kras p53 mice were used as a transgenic pancreatic cancer model. Results: Overexpression of PHLPP1 in PaCa cells reduced, whereas its’ knock-down with siRNA increased Akt phosphorylation. Accordingly, PHLPP1 overexpression increased, whereas its’ knock-down decreased apoptosis in PaCa cells. PHLPP1 stimulated death of PaCa cells through inhibiting Akt. Immunofluorescence showed that PHLPP 1 expression in human and mouse pancreatic adenocarcinomas was significantly lower than in normal pancreas. PHLPP1 activity in PaCa cells was strongly inhibited by NADPH oxidase Nox4, which is highly expressed in pancreatic cancer and is co-localized with PHLPP1. Furthermore, the activity of PHLPP1 in PaCa cells was inhibited by the growth factor IGF-I, which is abundant in pancreatic tumors. This inactivation is at least partly due to ROS since inhibition of IGF-I induced ROS production increased PHLPP1 activity in vivo and peroxide greatly inhbited PHLPP1 activity in vitro. PHLPP 1 overexpression greatly delayed subcutaneous tumor induction and decreased by 2.75± 0.8 (n=6) times tumor volume in orthotopic pancreatic cancer model. Conclusion: The data indicate that phosphatase PHLPP1 is a key negative regulator of Akt in PaCa cells. The level and activity of PHLPP1 drastically decreased in pancreatic cancer resulting in high Akt phosphorylation and the inhibition of PaCa cell death. Overexpression of PHLPP1 inhibits tumorogenesis in the orthotopic model of pancreatic cancer demonstrating that PHLPP1 is a promising target for treatment of pancreatic adenocarcinoma. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3078.

RNA Microarray Analysis in Prenatal Mouse Cochlea Reveals Novel IGF-I Target Genes: Implication of MEF2 and FOXM1 Transcription Factors

BackgroundInsulin-like growth factor-I (IGF-I) provides pivotal cell survival and differentiation signals during inner ear development throughout evolution. Homozygous mutations of human IGF1 cause syndromic sensorineural deafness, decreased intrauterine and postnatal growth rates, and mental retardation. In the mouse, deficits in IGF-I result in profound hearing loss associated with reduced survival, differentiation and maturation of auditory neurons. Nevertheless, little is known about the molecular basis of IGF-I activity in hearing and deafness.Methodology/Principal FindingsA combination of quantitative RT-PCR, subcellular fractionation and Western blotting, along with in situ hybridization studies show IGF-I and its high affinity receptor to be strongly expressed in the embryonic and postnatal mouse cochlea. The expression of both proteins decreases after birth and in the cochlea of E18.5 embryonic Igf1−/− null mice, the balance of the main IGF related signalling pathways is altered, with lower activation of Akt and ERK1/2 and stronger activation of p38 kinase. By comparing the Igf1−/− and Igf1+/+ transcriptomes in E18.5 mouse cochleae using RNA microchips and validating their results, we demonstrate the up-regulation of the FoxM1 transcription factor and the misexpression of the neural progenitor transcription factors Six6 and Mash1 associated with the loss of IGF-I. Parallel, in silico promoter analysis of the genes modulated in conjunction with the loss of IGF-I revealed the possible involvement of MEF2 in cochlear development. E18.5 Igf1+/+ mouse auditory ganglion neurons showed intense MEF2A and MEF2D nuclear staining and MEF2A was also evident in the organ of Corti. At P15, MEF2A and MEF2D expression were shown in neurons and sensory cells. In the absence of IGF-I, nuclear levels of MEF2 were diminished, indicating less transcriptional MEF2 activity. By contrast, there was an increase in the nuclear accumulation of FoxM1 and a corresponding decrease in the nuclear cyclin-dependent kinase inhibitor p27Kip1.Conclusions/SignificanceWe have defined the spatiotemporal expression of elements involved in IGF signalling during inner ear development and reveal novel regulatory mechanisms that are modulated by IGF-I in promoting sensory cell and neural survival and differentiation. These data will help us to understand the molecular bases of human sensorineural deafness associated to deficits in IGF-I.

RACK1-mediated Integration of Adhesion and Insulin-like Growth Factor I (IGF-I) Signaling and Cell Migration Are Defective in Cells Expressing an IGF-I Receptor Mutated at Tyrosines 1250 and 1251

The scaffolding protein receptor for activated C kinase (RACK1) has been proposed to mediate the integration of insulin-like growth factor I receptor (IGF-IR) and adhesion signaling. Here we investigated the mechanism of this integration of signaling, by using an IGF-IR mutant (Y1250F/Y1251F) that is deficient in anti-apoptotic and transforming function. RACK1 was found to associate with the IGF-IR only in adherent cells and did not associate with the IGF-IR in nonadherent cells, lymphocytic cells, or cells expressing the Y1250F/Y1251F mutant. In R- cells transiently expressing the Y1250F/Y1251F mutant RACK1 became constitutively associated with beta1 integrin and did not associate with Shc, Src, or Shp2. This was accompanied by the loss of formation of a complex containing the IGF-IR, RACK1, and beta1 integrin; loss of migratory capacity; enhanced Src and FAK activity; enhanced Akt phosphorylation; and decreased p38 mitogen-activated protein kinase activity. Shc was not phosphorylated in response to IGF-I in cells expressing the Y1250F/Y1251F mutant and remained associated with protein phosphatase 2A. Similar alterations in signaling were observed in cells that were stimulated with IGF-I in nonadherent cultures. Our data suggest that disruption of RACK1 scaffolding function in cells expressing the Y1250F/Y1251F mutant results in the loss of adhesion signals that are necessary to regulate Akt activity and to promote turnover of focal adhesions and cell migration.

Effect of insulin-like growth factor-I during preantral follicular culture on steroidogenesis, in vitro oocyte maturation, and embryo development in mice.

Insulin-like growth factor-I (IGF-I) is involved in the regulation of ovarian follicular development and has been shown to potentiate the FSH responsiveness of granulosa cells from preantral follicles. The aim of the present study was to investigate the effect of IGF-I during preantral follicular culture on steroidogenesis, subsequent oocyte maturation, fertilization, and embryo development in mice. Preantral follicles were isolated mechanically and cultured for 12 days in a simplified culture medium supplemented with 1% fetal calf serum, recombinant human FSH, transferrin, and selenium. In these conditions, follicles were able to grow and produce oocytes that could be matured and fertilized. The first experiment analyzed the effect of different concentrations of IGF-I (0, 10, 50, or 100 ng/ml) added to the culture medium on the follicular survival, steroidogenesis, and the oocyte maturation process. The presence of IGF-I during follicular growth increased the secretion of estradiol but had no effect on the subsequent oocyte survival and maturation rates. In the second experiment, IGF-I (0 or 50 ng/ml) was added to the culture medium during follicular growth, oocyte maturation, or both, and subsequent oocyte fertilization and embryo development rates were evaluated. Oocyte fertilization rates were comparable in the presence or absence of IGF-I. However, the blastocyst development rate was enhanced after follicular culture in the presence of IGF-I. Moreover, the total cell number of the blastocysts observed after differential labeling staining was also higher when follicles were cultured or matured in the presence of IGF-I.

Inhibitory effect of IGF-I on induced apoptosis in mouse preimplantation embryos cultured in vitro

Insulin-like growth factor I (IGF-I) has been shown to promote mammalian early embryo development. Increased cell division or decreased cell death have been proposed as two main possible mechanisms in its effect. Here we examine the nature of this promoting effect in a model situation. Camptothecin (0.01 μg/ml) and actimomycin D (0.005 μg/ml) were used to induce apoptosis. Four-cell mouse embryos were cultured in vitro to blastocyst stage in the temporary (15 h) presence or absence of apoptotic inductors and in the permanent presence or absence of IGF-I (100 ng/ml). Embryos were assessed by morphological triple staining (Hoechst 33342, propidium iodide, Calcein AM) and comet assay on Day 5, 120 h after administration of hCG. The number of nuclei, the blastocyst formation, the proportion of embryos containing fragmented DNA and the percentage of apoptotic and secondary necrotic nuclei were assessed. Both inductors of apoptosis significantly increased the percentage of apoptotic and secondary necrotic cells and reduced total cell counts (camptothecin, P>0.001; actinomycin D, P>0.001). When IGF-I was added to the culture medium in the presence of an apoptosis inductor, apoptosis incidence was significantly decreased ( P<0.001). The addition of IGF-I into control samples also decreased the percentage of apoptotic and secondary necrotic cells. In contrast, IGF-I addition had no significant influence on embryo development ( P>0.05). Our data suggest a primary role for IGF-I as an apoptotic survival factor in mouse preimplantation embryos in specific conditions.

Calphostin C as a rapid and strong inducer of apoptosis in human coronary artery smooth muscle cells

Vascular smooth muscle cells (VSMCs) play a major role in the development of atherosclerotic and restenotic lesions. The apoptotic process has been implicated in the development of this pathology. In this study, we characterized the induction of apoptosis by calphostin C (CC), a protein kinase C (PKC) inhibitor, in primary human coronary artery smooth muscle cells in the presence and absence of insulin-like growth factor-I (IGF-I). Additionally, we investigated the signal transduction pathways important for IGF-I mediated protection. Calphostin C induced apoptosis, as measured by terminal deoxy-UTP nick-end labeling (TUNEL), in a time- and dose-dependent manner, approaching 20% within 6 h of 50 nM calphostin C treatment. The amount of apoptosis increased to 44.58±8.08%, 47.54±1.66% and 78.1±11.9% after 8, 10 and 12 h of treatment, respectively ( p<0.01 vs. control). IGF-I offered significant protection ( p<0.05) at 8 and 10 h of treatment (60.6% and 52.5% protection, respectively). DNA ELISA confirmed the apoptotic effect of calphostin C and the protective effect of IGF-I. After 6 h of calphostin C treatment, DNA ELISA revealed 11.20±1.53 fold greater apoptosis as compared to baseline values. IGF-I treatment offered a level of protection of 46.6% as measured by DNA ELISA ( p=0.06). Apoptosis was further qualitatively confirmed by time-lapse video microscopy and scanning electron microscopy. Interestingly, inhibitors of phosphatidylinositol-3-kinase (PI-3-K), p38 and extracellular regulated kinase (ERK) activation significantly ( p<0.05 vs. calphostin C only treatment) increased apoptosis when used in conjunction with calphostin C. Inhibitors of phospatidylinositol-3-kinase and ERK activation reversed IGF-I protection. However, the p38 inhibitor SB203580 failed to reverse IGF-I protection. This study characterized an apoptotic system for human coronary artery smooth muscle cells offering a rapid and strong induction of programmed cell death (PCD) that remains responsive to the survival effects of IGF-I. Studies utilizing this system may prove useful in understanding the apoptotic response of VSMCs in the arterial wall.

In vitro development of equine nuclear transfer embryos: effects of oocyte maturation media and amino acid composition during embryo culture.

This study was conducted to evaluate the effects of insulin-like growth factor I (IGF-I) and other media factors during oocyte maturation, and the presence of different compositions of amino acids in embryo culture medium, on the development of equine embryos. Oocytes recovered from slaughterhouse-derived ovaries were matured in vitro for 24 h and those with a polar body were subjected to intracytoplasmic sperm injection (ICSI) or nuclear transfer with adult fibroblasts (NT). For ICSI embryos, there were no significant differences in rates of morphological cleavage, cleavage with normal nuclei or average nucleus number at 96 h post-ICSI between the absence and presence of IGF-I in maturation medium, or between embryos cultured in G1.2 or a modified CZB medium (CZB-C). Embryos produced by interspecies NT (equine donor cells into bovine cytoplasts) also showed no difference in cleavage rate or average nucleus number whether cultured in G1.2 or in CZB-C. The rates of cleavage, cleavage with normal nuclei and average nucleus number of equine NT embryos were not significantly different among oocytes matured in M199 with FSH in the presence or absence of IGF-I, or in EMMI medium, which contains IGF-I, epidermal growth factor, steroid hormones, FSH and LH. There were no differences in development of equine NT embryos cultured in any of three amino acid treatments (with or without non-essential amino acids, or containing taurine, hypotaurine and cysteine only). The cleavage rate and average nucleus number of parthenogenetically activated oocytes (treated similarly to NT oocytes but not enucleated or subjected to donor cell injection) were significantly (p < 0.05) higher than those for NT embryos. These results indicate that the presence of IGF-I or of EMMI medium during in vitro maturation of equine oocytes does not have a beneficial effect on their developmental competence as assessed at 96 h. Presence or absence of non-essential amino acids in embryo culture medium does not affect development of NT embryos within the first 96 h of culture. Factors associated with enucleation or nuclear transfer decrease the developmental competence of equine NT embryos. CZB-C medium may be used for culture of equine embryos with results similar to those obtained with G1.2 medium, thus providing a base medium that may be modified for further study of culture requirements of equine embryos.

Puberty is delayed in male growth hormone receptor gene-disrupted mice.

The role of insulin-like growth factor-I (IGF-I) in the initiation of puberty and testicular function is poorly understood. Growth hormone (GH) receptor (R) gene-disrupted mice or GHR gene "knockouts" (GHR-KO) are GH resistant and IGF-I deficient. To assess whether the age of sexual maturation is affected by the absence of IGF-I, various parameters of sexual development including testicular and accessory reproductive organ weights, balanopreputial separation, germ cell development, and intratesticular testosterone levels were determined in normal and GHR-KO mice between the ages of 25 and 60 days. In addition, at 36 days of age, the testosterone response to luteinizing hormone (LH) treatment was assessed in these mice. The results indicate that the balanopreputial separation was delayed 5 days, and a significant increase in the weights of the seminal vesicles (SV) occurred later in GHR-KO mice than in normal animals (between 30 and 35 days and between 35 and 40 days, respectively). Also, the weights of testes and epididymii were significantly reduced in GHR-KO mice. The intratesticular testosterone levels and the testosterone response to LH treatment were attenuated in GHR gene-disrupted mice. Furthermore, elongated spermatids appeared later in the testes of GHR-KO mice than in the testes of normal mice. These results suggest that the absence of IGF-I secretion delays the normal course of sexual maturation in male GHR-KO mice, indicating that IGF-I plays an important role in the initiation of puberty in male mice.

IGF-I promotes a shift in metabolic flux in vascular smooth muscle cells.

13C-nuclear magnetic resonance (NMR) spectroscopy was used to test our hypothesis that insulin-like growth factor I (IGF-I) stimulates glucose flux into both nonoxidative and oxidative pathways in vascular smooth muscle cells (VSMC). Rat VSMC were exposed to uniformly labeled [13C]glucose ([U-13C]glucose; 5.5 mM) and [3-13C]pyruvate (1 mM) in the presence and absence of IGF-I (100 ng/ml). IGF-I increased glucose flux through glycolysis and the tricarboxylic acid (TCA) cycle as well as total anaplerotic flux into the TCA cycle. Previous work in our laboratory identified an increase in GLUT1 content and glucose metabolism in neointimal VSMC that was sufficient to promote proliferation and inhibit apoptosis. To test whether IGF-I could potentiate the GLUT1-induced increased flux in the neointima, we utilized VSMC harboring constitutive overexpression of GLUT1. Indeed, IGF-I markedly potentiated the GLUT1-induced increase in glucose flux through glycolysis and the TCA cycle. Taken together, these findings demonstrate that upregulation of glucose transport through either IGF-I or increased GLUT1 content stimulates glucose flux through both nonoxidative and oxidative pathways in VSMC.

Myelination Is Altered in Insulin-Like Growth Factor-I Null Mutant Mice

Increasing evidence indicates that insulin-like growth factor-I (IGF-I) has an important role in oligodendrocyte development. In this study, we examined myelination during postnatal development in IGF-I knock-out (KO) mice by assessing myelin staining, the expression of myelin basic protein (MBP) and proteolipid protein (PLP), two major myelin-specific proteins, and the number of oligodendrocytes and their precursors. For comparison, we also measured the expression of median subunit of the neuron-specific intermediate filament, M-neurofilament (M-NF), to obtain an index of the effects of IGF-I deficiency on neurons. We found that myelin staining, MBP and PLP expression, and the percentage of oligodendrocytes and their precursors are significantly reduced in all brain regions of developing IGF-I KO mice but are similar to controls in adult IGF-I KO mice. In contrast, the abundance of M-NF was decreased in both the developing and adult brain of IGF-I KO mice. We also found that IGF-II protein abundance is increased in the brains of IGF-I KO mice. Our data indicate, therefore, that myelination during early development is altered in the absence of IGF-I by mechanisms that involve a reduction in oligodendrocyte proliferation and development. Although neuronal actions cannot be excluded in the myelin normalization, the reduced axonal growth suggested by the reduced M-NF expression makes a role for neuronal factors less compelling. These data suggest that IGF-I plays a significant role in myelination during normal early development and that IGF-II can compensate in part for IGF-I actions on myelination.

Myelination is altered in insulin-like growth factor-I null mutant mice.

Increasing evidence indicates that insulin-like growth factor-I (IGF-I) has an important role in oligodendrocyte development. In this study, we examined myelination during postnatal development in IGF-I knock-out (KO) mice by assessing myelin staining, the expression of myelin basic protein (MBP) and proteolipid protein (PLP), two major myelin-specific proteins, and the number of oligodendrocytes and their precursors. For comparison, we also measured the expression of median subunit of the neuron-specific intermediate filament, M-neurofilament (M-NF), to obtain an index of the effects of IGF-I deficiency on neurons. We found that myelin staining, MBP and PLP expression, and the percentage of oligodendrocytes and their precursors are significantly reduced in all brain regions of developing IGF-I KO mice but are similar to controls in adult IGF-I KO mice. In contrast, the abundance of M-NF was decreased in both the developing and adult brain of IGF-I KO mice. We also found that IGF-II protein abundance is increased in the brains of IGF-I KO mice. Our data indicate, therefore, that myelination during early development is altered in the absence of IGF-I by mechanisms that involve a reduction in oligodendrocyte proliferation and development. Although neuronal actions cannot be excluded in the myelin normalization, the reduced axonal growth suggested by the reduced M-NF expression makes a role for neuronal factors less compelling. These data suggest that IGF-I plays a significant role in myelination during normal early development and that IGF-II can compensate in part for IGF-I actions on myelination.

Stem cell factor and insulin-like growth factor-I stimulate luteinizing hormone-independent differentiation of rat ovarian theca cells.

The signal initiating ovarian theca cell (TC) differentiation is gonadotropin independent because theca precursor cells do not contain LH receptors. Previously we demonstrated that preantral follicles produce paracrine TC differentiating factors that promote androgen production by an LH-independent mechanism. This study tested the effects of two granulosa cell-produced peptides, insulin-like growth factor-I (IGF-I) and stem cell factor (SCF), on TC differentiation and androgen production. Neutralizing antibodies to either IGF-I or SCF blocked the stimulatory effects of follicle-conditioned medium on TC precursor differentiation more than 90%. The TC isolated from the ovaries of hypophysectomized immature rats by percoll gradient centrifugation were cultured (48 h) with and without SCF (0-100 ng/ml) and IGF-I (0-100 ng/ml) to test their effects on TC differentiation. Androsterone in the medium was measured by RIA. Luteinizing hormone receptor, steroidogenesis acute regulatory protein (StAR), CYP11A, CYP17, and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) mRNAs were measured by specific reverse transcriptase polymerase chain reaction assays. Stem cell factor or IGF-I alone did not stimulate androsterone production but in combination caused a concentration-dependent increase in androsterone levels. Maximum androsterone levels were less than those stimulated by LH (0.1 ng/ml) alone. Although IGF-I synergistically augmented LH stimulation of androsterone production, SCF did not alter LH-stimulated androsterone production in the presence or absence of IGF-I. Stem cell factor alone had no effect on LH receptor, StAR, CYP11A, and 3beta-HSD mRNA expression but decreased CYP17 mRNA levels. Insulin-like growth factor-I alone had no effect on StAR or CYP17 mRNA expression but increased LH receptor, CYP11A, and 3beta-HSD mRNA levels. In combination, SCF plus IGF-I increased the expression of all five mRNAs. These data support the conclusion that IGF-I and SCF are important regulators of TC differentiation.

Insulin-like growth factor-I (IGF-I) stimulates the development of cultured rat pre-antral follicles.

The aim of the present study is to investigate the effect of insulin-like growth factor (IGF-I) on the development of cultured rat pre-antral follicles. Pre-antral follicles with a diameter between 140 and 160 microm are mechanically isolated from ovaries of 10-day-old rats and cultured in groups for 6 days in FSH and insulin-containing serum-free medium in the absence or presence of IGF-I at concentrations of 1, 10, and 100 ng/ml, respectively. DNA content of the follicles before and after culture is measured to evaluate whether a possible growth is due to proliferation of follicular cells and the ultrastructure of the cultured follicles is studied with transmission electron microscopy to evaluate the quality of follicles cultured under different conditions. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) is used to assess the gene expression of IGF-I and type I IGF receptor in pre-antral follicles. When follicles were cultured in medium containing IGF-I at a concentration of 1 and 10 ng/ml, the increase in follicle diameter after a 6-day culture differs significantly from that in the absence of IGF-I. The ultrastructure of follicles cultured in IGF-I-containing medium is better sustained when compared to that of follicles cultured in the absence of IGF-I. Especially, theca cells, which are undergoing degeneration under controlled culture condition, demonstrate normal cellular ultrastructure with some characteristics of steroid-secreting cells in the presence of IGF-I. Moreover, in oocytes of follicles cultured in the presence of IGF-I, cortical granules are observed distributed along the ooplasma membrane whereas cortical granules are hardly present in follicles cultured without IGF-I. With RT-PCR, the presence of the mRNA of IGF-I and type I IGF receptor is demonstrated in both the somatic follicular cells and the oocytes. Taken together, these results suggest that IGF-I, in the presence of follicle-stimulating hormone (FSH), enhances rat pre-antral follicle development in vitro and supports the morphology of cultured pre-antral follicles. The stimulatory effect of exogenous IGF-I on the development of pre-antral follicle together with the gene expression of both IGF-I and type I IGF receptor in pre-antral follicles suggests the involvement of IGF-I in early folliculogenesis and its actions are likely mediated by the putative type I IGF receptor, via both endocrine and paracrine/autocrine pathways.

Stimulatory effect of zinc and growth factor on bone protein component in newborn rats: enhancement with zinc and insulin-like growth factor-I.

The effect of zinc and growth factor on bone protein component in newborn rats was investigated. The characterization of protein component in the femoral-diaphyseal and metaphyseal tissue of newborn rats (3-35 days old) was examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The diaphyseal and metaphyseal tissues of 7 days-old rats were cultured for 24 or 48 h in a medium containing either vehicle, zinc sulfate (10-4 M) or dipicolinate (10-3 M), a chelator of zinc ion, in the presence or absence of insulin-like growth factor-I (IGF-I; 10-8 M) or transforming growth factor-beta (TFG-beta; 10-10 M) with an effective concentration. Many cellular protein molecules were present in the diaphyseal and metaphyseal tissues; potent bands were seen in protein molecules with about 66 and 46 kDa. Protein molecule of about 66 kDa was greatly released in the medium cultured with the diaphyseal and metaphyseal tissues. This protein in the medium was increased by culture with zinc, IGF-I or TGF-beta. Total protein content in the medium cultured with the diaphyseal and metaphyseal tissues was significantly increased in the presence of zinc, IGF-I or TGF-beta. The IGF-I-increased medium protein content was significantly enhanced by zinc. This enhancing effect was not seen in TGF-beta. Alkaline phosphatase activity and deoxyribonucleic acid (DNA) content in the diaphyseal and metaphyseal tissues was significantly increased by culture with zinc, IGF-I or TGF-beta. The effect of IGF-I was significantly enhanced by zinc, while it was not found in TGF-beta. The effect of IGF-I or TGF-beta in increasing the bone components was seen in the presence of dipicolinate. This study demonstrates that zinc, like IGF-I and TGF-beta, can increase protein components in the femoral-diaphyseal and metaphyseal tissues of new-born rats. Zinc may especially play a role in bone growth in collaboration with IGF-I.