Insulin-like growth factor type-1 receptor down-regulation associated with dwarfism in Holstein calves

Perturbations in endocrine functions can impact normal growth. Endocrine traits were studied in three dwarf calves exhibiting retarded but proportionate growth and four phenotypically normal half-siblings, sired by the same bull, and four unrelated control calves. Plasma 3,5,3'-triiodothyronine and thyroxine concentrations in dwarfs and half-siblings were in the physiological range and responded normally to injected thyroid-releasing hormone. Plasma glucagon concentrations were different (dwarfs, controls>half-siblings; P<0.05). Plasma growth hormone (GH), insulin-like growth factor-1 (IGF-1) and insulin concentrations in the three groups during an 8-h period were similar, but integrated GH concentrations (areas under concentration curves) were different (dwarfs>controls, P<0.02; half-siblings>controls, P=0.08). Responses of GH to xylazine and to a GH-releasing-factor analogue were similar in dwarfs and half-siblings. Relative gene expression of IGF-1, IGF-2, GH receptor (GHR), insulin receptor, IGF-1 type-1 and -2 receptors (IGF-1R, IGF-2R), and IGF binding proteins were measured in liver and anconeus muscle. GHR mRNA levels were different in liver (dwarfs

Subcellular distribution of the insulin-like growth factor (IGF) binding proteins (IGFBPs) 2 and 3 in articular chondrocytes

The insulin-like growth factor (IGF) is a major anabolic regulator in articular cartilage. The IGF-binding proteins (IGFBPs) are increased during osteoarthritis (OA), but the function of the later proteins remains unknown. In general, the IGFBPs are pluripotential effectors capable of IGF regulation and of acting on their own to control key cell functions, including survival and proliferation. The independent functions are often associated with their cell location, and therefore this study explores the distribution of IGFBP-2 and IGFBP-3 in articular chondrocytes. Immunohistochemistry was used to localize IGFBP-2 in normal human articular cartilage. Bovine chondrocytes were used for subcellular fractionation (hypotonic cell lysis) under nonreducing conditions and nuclear purification (centrifugation on sucrose cushions). Cell fraction markers and IGFBPs were assayed in the subcellular fractions by Western immunoblot. The IHC results showed association of IGFBP-2 with chondrocytes, but not with the nuclei. Subcellular fractionation of isolated chondrocytes yielded intact nuclei as assessed at the light microscopic level; the nuclear marker histone H1 was exclusively associated with this fraction. More than 90% of the cytoplasmic marker GAPDH and all the detectable IGFBP-2 were in the cytoplasmic fraction. Immunoreactive IGFBP-3 was found in the cytoplasmic and peri-nuclear/nuclear fractions. Chondrocytes contain intracellular IGFBP-2 and IGFBP-3 but only IGFBP-3 is associated with nuclei. This suggests the hypothesis that the actions of these IGFBPs in articular cartilage extend beyond the classic modulation of IGF receptor action.

Effect of maternal cold exposure and nutrient restriction on insulin-like growth factor sensitivity in adipose tissue of newborn sheep

Adipose tissue mass in the newborn is determined in part by insulin-like growth factor (IGF)s, which are dependent on the maternal nutritional and metabolic environment during late gestation. The present study was designed to determine whether maternal cold exposure (CE) commencing in mid gestation could modulate some of the adaptive effects of nutrient restriction in late gestation on adipose tissue endocrine sensitivity in the resulting offspring. Twenty eight pregnant sheep were entered into the study and were either shorn, i.e. cold exposed, from 70 days gestation (term = 147 days), or remained unshorn, and were fed either their total calculated metabolisable energy (ME) requirements for body weight and pregnancy from 110 days gestation or 50% of this amount (n=7 per group). Adipose tissue was sampled from the offspring at one day of age and the mRNA abundance for IGF-I, II their receptors (R) and GH secretagogue receptor-1a (GHSR-1a) were determined. CE mothers produced larger offspring with more perirenal adipose tissue, an adaptation prevented by maternal nutrient restriction. Nutrient restriction in unshorn mothers increased IGF-I and IIR mRNA abundance. The mRNA abundances for IGF-I, II and IIR in adipose tissue were reduced by CE, adaptations independent of maternal food intake, whereas CE plus nutrient restriction increased GHSR-1a mRNA. In conclusion, maternal nutrient restriction with or without CE has very different effects on IGF sensitivity of adipose tissue and may act to ensure adequate fat stores are present in the newborn in the face of very different maternal endocrine and metabolic environments.

Ontogeny and nutritional programming of the hepatic growth hormone-insulin-like growth factor-prolactin axis in the sheep

The liver is an important metabolic and endocrine organ in the fetus but the extent to which its hormone receptor (R) sensitivity is developmentally regulated in early life is not fully established. We, therefore, examined developmental changes in mRNA abundance for the growth hormone (GH) and prolactin (PRL) receptors (R) plus insulin-like growth factor (IGF)-I and –II and their receptors. Fetal and postnatal sheep were sampled at either 80, or 140 days gestation, 1, 30 days or six months of age. The effect of maternal nutrient restriction between early to mid (i.e. 28 to 80 days gestation, the time of early liver growth) gestation on gene expression was also examined in the fetus and juvenile offspring. Gene expression for the GHR, PRLR and IGF-IR increased through gestation peaking at birth, whereas IGF-I was maximal near to term. In contrast, IGF-II mRNA decreased between mid and late gestation to increase after birth whereas IGF-IIR remained unchanged. A substantial decline in mRNA abundance for GHR, PRLR and IGF-IR then occurred up to six months. Maternal nutrient restriction reduced GHR and IGF-IIR mRNA abundance in the fetus, but caused a precocious increase in the PRLR. Gene expression for IGF-I and –II were increased in juvenile offspring born to nutrient restricted mothers. In conclusion, there are marked differences in the developmental ontogeny and nutritional programming of specific hormones and their receptors involved in hepatic growth and development in the fetus. These could contribute to changes in liver function during adult life.

Characterization of the molecular mechanisms mediated by the insulin-like growth factor-2 mRNA-binding proteins and Ephrin receptor A2 (EPHA2) signaling in the malignancy of CIC-DUX4 sarcomas

Ewing sarcoma (EWS) and CIC-DUX4 sarcoma (CDS) are pediatric fusion gene-driven tumors of mesenchymal origin characterized by an extremely stable genome and limited clinical solutions. Post-transcriptional regulatory mechanisms are crucial for understanding the development of this class of tumors. RNA binding proteins (RBPs) play a crucial role in the aggressiveness of these tumors. Numerous RBP families are dysregulated in cancer, including IGF2BPs. Among these, IGF2BP3 is a negative prognostic factor in EWS because it promotes cell growth, chemoresistence, and induces the metastatic process. Based on preliminary RNA sequencing data from clinical samples of EWS vs CDS patients, three major axes that are more expressed in CDS have been identified, two of which are dissected in this PhD work. The first involves the transcription factor HMGA2, IGF2BP2-3, and IGF2; the other involves the ephrin receptor system, particularly EphA2. EphA2 is involved in numerous cellular functions during embryonic stages, and its increased expression in adult tissues is often associated with pathological conditions. In tumors, its role is controversial because it can be associated with both pro- and anti-tumoral mechanisms. In EWS, it has been shown to play a role in promoting cell migration and neoangiogenesis. Our study has confirmed that the HMGA2/IGF2BPs/IGF2 axis contributes to CDS malignancy, and Akt hyperactivation has a strong impact on migration. Using loss/gain of function models for EphA2, we confirmed that it is a substrate of Akt, and Akt hyperactivation in CDS triggers ligand-independent activation of EphA2 through phosphorylation of S897. Moreover, the combination of Trabectedin and NVP/BEZ235 partially inhibits Akt/mTOR activation, resulting in reduced tumor growth in vivo. Inhibition of EphA2 through ALWII 41_27 significantly reduces migration in vitro. The project aim is the identification of target molecules in CDS that can distinguish it from EWS and thus develop new targeted therapeutic strategies.

Evaluation of the insulin-like growth factors (IGF) IGF-I and IGF binding protein 3 in patients at high risk for breast cancer

Our data suggest that serum concentrations of insulin-like growth factor I and insulin-like growth factor binding protein 3 do not correlate with breast cancer development. (Fertil Steril (R) 2011;95:2753-5. (C)2011 by American Society for Reproductive Medicine.)

Can the Insulin Sensitivity Index (ISI) in Association with Insulin-like Growth Factor Binding Protein-1 Identify Insulin Resistance Early in Overweight Children?

Association between insulin resistance (IR) and non-alcoholic fatty liver disease (NAFLD) has been reported. This prompted us to evaluate the power of the insulin sensitivity index (ISI) in association with IGFBP-1 to identify IR early in obese children/adolescents. OGTT was performed in 34 obese/overweight children/adolescents. Glucose, insulin and IGFBP-1 were measured in serum samples and ISI was calculated. Considering the presence of three or more risk factors for IR as a criterion for IR, ISI <4.6 showed 87.5% sensitivity and 94.5% specificity in diagnosing IR. IGFBP-1 was lower in the group with ISI <4.6 (p <0.01). In this group, three patients had higher than expected IGFBP-1, suggesting hepatic IR, while three patients with ISI >4.6 showed very low IGFBP-1 levels. Conclusion: ISI <4.6 is a good indicator of early peripheral IR and, associated with IGFBP-1, can identify increased risk of hepatic IR. Low IGFBP-1 levels among non-IR children may indicate increased portal insulin levels.

Insulin-like growth factor-1 protects preimplantation embryos from anti-developmental actions of menadione

Menadione is a naphthoquinone used as a vitamin K source in animal feed that can generate reactive oxygen species (ROS) and cause apoptosis. Here, we examined whether menadione reduces development of preimplantation bovine embryos in a ROS-dependent process and tested the hypothesis that actions of menadione would be reduced by insulin-like growth factor-1 (IGF-1). Menadione caused a concentration-dependent decrease in the proportion of embryos that became blastocysts. All concentrations tested (1, 2.5, and 5.0 mu M) inhibited development. Treatment with 100 ng/ml IGF-1 reduced the magnitude of the anti-developmental effects of the two lowest menadione concentrations. Menadione also caused a concentration-dependent increase in the percent of cells positive for the TUNEL reaction. The response was lower for IGF-1-treated embryos. The effects of menadione were mediated by ROS because (1) the anti-developmental effect of menadione was blocked by the antioxidants dithiothreitol and Trolox and (2) menadione caused an increase in ROS generation. Treatment with IGF-1 did not reduce ROS formation in menadione-treated embryos. In conclusion, concentrations of menadione as low as 1.0 mu M can compromise development of bovine preimplantation embryos to the blastocyst stage of development in a ROS-dependent mechanism. Anti-developmental actions of menadione can be blocked by IGF-1 through effects downstream of ROS generation.

Glutamine Stimulates Biosynthesis and Secretion of Insulin-like Growth Factor 2 (IGF2), an Autocrine Regulator of Beta Cell Mass and Function.

IGF2 is an autocrine ligand for the beta cell IGF1R receptor and GLP-1 increases the activity of this autocrine loop by enhancing IGF1R expression, a mechanism that mediates the trophic effects of GLP-1 on beta cell mass and function. Here, we investigated the regulation of IGF2 biosynthesis and secretion. We showed that glutamine rapidly and strongly induced IGF2 mRNA translation using reporter constructs transduced in MIN6 cells and primary islet cells. This was followed by rapid secretion of IGF2 via the regulated pathway, as revealed by the presence of mature IGF2 in insulin granule fractions and by inhibition of secretion by nimodipine and diazoxide. When maximally stimulated by glutamine, the amount of secreted IGF2 rapidly exceeded its initial intracellular pool and tolbutamide, and high K(+) increased IGF2 secretion only marginally. This indicates that the intracellular pool of IGF2 is small and that sustained secretion requires de novo synthesis. The stimulatory effect of glutamine necessitates its metabolism but not mTOR activation. Finally, exposure of insulinomas or beta cells to glutamine induced Akt phosphorylation, an effect that was dependent on IGF2 secretion, and reduced cytokine-induced apoptosis. Thus, glutamine controls the activity of the beta cell IGF2/IGF1R autocrine loop by increasing the biosynthesis and secretion of IGF2. This autocrine loop can thus integrate changes in feeding and metabolic state to adapt beta cell mass and function.

PPARγ induces growth inhibition and apoptosis through upregulation of insulin-like growth factor-binding protein-3 in gastric cancer cells

Peroxisome proliferator activator receptor-gamma (PPARγ) is a ligand-activated transcriptional factor involved in the carcinogenesis of various cancers. Insulin-like growth factor-binding protein-3 (IGFBP-3) is a tumor suppressor gene that has anti-apoptotic activity. The purpose of this study was to investigate the anticancer mechanism of PPARγ with respect to IGFBP-3. PPARγ was overexpressed in SNU-668 gastric cancer cells using an adenovirus gene transfer system. The cells in which PPARγ was overexpressed exhibited growth inhibition, induction of apoptosis, and a significant increase in IGFBP-3 expression. We investigated the underlying molecular mechanisms of PPARγ in SNU-668 cells using an IGFBP-3 promoter/luciferase reporter system. Luciferase activity was increased up to 15-fold in PPARγ transfected cells, suggesting that PPARγ may directly interact with IGFBP-3 promoter to induce its expression. Deletion analysis of the IGFBP-3 promoter showed that luciferase activity was markedly reduced in cells without putative p53-binding sites (-Δ1755, -Δ1795). This suggests that the critical PPARγ-response region is located within the p53-binding region of the IGFBP-3 promoter. We further demonstrated an increase in PPARγ-induced luciferase activity even in cells treated with siRNA to silence p53 expression. Taken together, these data suggest that PPARγ exhibits its anticancer effect by increasing IGFBP-3 expression, and that IGFBP-3 is a significant tumor suppressor.

Modulation of Endothelin-1 and Insulin-like Growth Factor Type 1-induced Signaling by Curcumin in A-10 Vascular Smooth Muscle Cells

Les maladies cardio-vasculaires (MCV), telles que l’hypertension et l’athérosclérose, s’accompagnent de modifications structurales et fonctionnelles au niveau vasculaire. Un fonctionnement aberrant de la migration, l’hypertrophie et la prolifération des cellules musculaires lisses vasculaires (CMLV) sont des évènements cellulaires à l’origine de ces changements. L’endothéline-1 (ET-1) contribue à la pathogénèse des anomalies vasculaires, notamment via l’activation des protéines MAPK et PI3-K/PKB, des composantes clés impliquées dans les voies prolifératives et de croissance cellulaires. Il a été suggéré que le stress oxydant jouerait un rôle intermédiaire dans les effets pathophysiologiques vasculaires de l’ET-1. En conséquence, une modulation de la signalisation induite par l’ET-1 peut servir comme éventuelle stratégie thérapeutique contre le développement des MCV. Il apparaît de nos jours un regain d’intérêt dans l’utilisation des agents phyto-chimiques pour traiter plusieurs maladies. La curcumine, constituant essentiel de l’épice curcuma, est dotée de plusieurs propriétés biologiques parmi lesquelles des propriétés anti-oxydantes, anti-prolifératrices et cardio-protectrices. Cependant, les mécanismes moléculaires de son effet cardio-protecteur demeurent obscurs. Dans cette optique, l’objectif de cette étude a été d’examiner l’efficacité de la curcumine à inhiber la signalisation induite par l’ET-1 dans les CMLV. La curcumine a inhibé la phosphorylation des protéines IGF-1R, PKB, c-Raf et ERK1/2, induite par l’ET-1 et l’IGF-1. De plus, la curcumine a inhibé l’expression du facteur de transcription Egr-1 induite par l’ET-1 et l’IGF-1, dans les CMLV. Ces résultats suggèrent que la capacité de la curcumine à atténuer ces voies de signalisation serait un mécanisme d’action potentiel de ses effets protecteurs au niveau cardiovasculaire.

Avaliação de IGF-1 (Insulin-like growth factor-1), IGFBP-1 e IGFBP-3 (Insulin-like to growth binding protein-1 e 3) no fluído folicular de pacientes infertéis com endometriose

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Association of an insulin-like growth factor 1 gene microsatellite with phenotypic variation and estimated breeding values of growth traits in Canchim cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)