Insulin-like Growth Factor Binding Protein-3 mRNA Research Articles

Runx1 promotes angiogenesis by downregulation of insulin-like growth factor-binding protein-3

Mouse embryos lacking the Runx1 transcription factor exhibit an angiogenic defect accompanied by the absence of hematopoietic stem cells (HSCs). To ask whether Runx1 plays a direct role in angiogenesis, we established a novel endothelial progenitor cell line, designated AEL-DeltaR1, from the aorta-gonad-mesonephros (AGM) region of Runx1-null mouse. We introduced Runx1 cDNA into AEL-DeltaR1 cells under the doxycycline-inducible promoter. The ability of AEL-DeltaR1 cells to form vascular networks on matrigel was highly enhanced by the restored expression of Runx1. By molecular comparison of mRNAs in AEL-DeltaR1 cells before and after the induction of Runx1, we found that mRNA expression of insulin-like growth factor-binding protein 3 (IGFBP-3) is downregulated by Runx1. Gel retardation and reporter assays revealed that Runx1 binds to the promoter region of mouse IGFBP-3 gene and represses its transcription. When IGFBP-3 was exogenously added in the matrigel assay, the angiogenesis-enhancing activity of Runx1 was suppressed in a dose-dependent manner. These results demonstrate that Runx1 is directly involved in angiogenesis by repression of IGFBP-3 mRNA expression.

Suppressor of Cytokine Signaling-2 Deficiency Induces Molecular and Metabolic Changes that Partially Overlap with Growth Hormone-Dependent Effects

Suppressor of cytokine signaling-2 (SOCS2)-deficient (SOCS2-/-) mice grow significantly larger than their littermates, suggesting that SOCS2 is important in the negative regulation of the actions of GH and/or IGF-I. The aim of this study was to identify genes and metabolic parameters that might contribute to the SOCS2-/- phenotype. We demonstrate that although SOCS2 deficiency induces significant changes in hepatic gene expression, only a fraction of these overlap with known GH-induced effects in the liver, suggesting that SOCS2 might be an important regulator of other growth factors and cytokines acting on the liver. However, an important role of GH and IGF-I in the phenotype of these animals was demonstrated by an overexpression of IGF-binding protein-3 mRNA in the liver and increased levels of circulating IGF-binding protein-3. Other GH-like effects included diminished serum triglycerides and down-regulation of lipoprotein lipase in adipose tissue. Interestingly, SOCS2-/- mice did not differ from their wild-type littermates in glucose or insulin tolerance tests, which is in contrast with the known diabetogenic effects of GH. Furthermore, there was no evidence of impaired insulin signaling in primary hepatocytes isolated from SOCS2-/- mice. Moreover, increased expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha mRNA was detected in skeletal muscle, which might contribute to normal glycemic control despite the apparent overactivity of the GH/IGF-I axis. Our data indicate that SOCS2 deficiency partially mimics a state of increased GH activity, but also results in changes that cannot be related to known GH effects.

Cellular localisation of insulin-like growth factor binding protein-2 (IGFBP-2) during development of the marine fish, Sparus aurata

The spatial localisation of insulin-like growth-factor-binding protein-2 (IGFBP-2) and its mRNA was investigated during larval and post-larval developmental stages of the gilthead seabream ( Sparus aurata) by immunohistochemistry and in situ hybridisation with specific antisera and riboprobes. During larval development, immunoreactivity was found in skin, muscle, gills, pharynx, intestine, liver and olfactory epithelium. After metamorphosis, immunoreactivity was found in the oesophageal epithelium (the strongest reaction) and in red skeletal muscle, heart muscle, the thymus and the epithelium of renal tubules. In the adult, immunostaining with IGFBP-2 antibody was also found in the saccus vasculosus, ovary and testis. IGFBP-2 mRNA was detected by in situ hybridisation mainly in the intestine, skeletal musculature and ovary. These results show that IGFBP-2 protein and mRNA are expressed in a variety of seabream tissues, suggesting that IGFBP-2 regulates the actions of IGFs on these tissues during development and growth.

2,3,7,8-Tetrachlorodibenzo-p-dioxin Induces Insulin-Like Growth Factor Binding Protein-1 Gene Expression and Counteracts the Negative Effect of Insulin

Recent epidemiological studies have revealed a possible correlation between exposure to high levels of dioxins or dioxin-like compounds and diabetes. Yet the interaction between insulin and dioxin actions remains elusive. We studied the regulation of insulin-like growth factor binding protein-1 (IGFBP-1), a protein involved in glucose homeostasis and whose expression is down-regulated by insulin. We showed that 2,3,7,8-tetrachorodibenzo-p-dioxin (TCDD) specifically induced IGFBP-1 mRNA in human hepatocytes and HepG2 human hepatoma cells (2.5- and 8-fold, respectively). Cellular and secreted IGFBP-1 protein levels were also up-regulated. Transfection and reporter assays showed that the IGFBP-1 promoter was activated by TCDD and that this activation was dependent on the integrity of a proximal xenobiotic-responsive element (XRE). This XRE, located near the insulin-glucocorticoid regulatory region, binds the aryl-hydrocarbon receptor. In agreement with previous studies, the IGFBP-1 promoter was down-regulated by insulin (50%); we show here that although TCDD activated the IGFBP-1 promoter 5- to 6-fold, the combination of TCDD and insulin led to an expression level of IGFBP-1 that was higher than basal level (2- to 3-fold activation). Similar regulations were observed for the endogenous IGFBP-1 mRNA. These data suggest that the xenobiotic-hormonal regulatory region of the IGFBP-1 promoter mediates an up-regulation of IGFBP-1 expression by TCDD even in the presence of insulin. Because IGFBP-1 modulates blood glucose levels, the up-regulation of IGFBP-1 by dioxins might account for the disruptive effects of these pollutants on glucose metabolism.

3,5-diiodothyronine Mimics the Effect of Triiodothyronine on Insulin-like Growth Factor Binding Protein-4 Expression in Cultured Rat Hepatocytes

We have previously demonstrated that triiodothyronine (T(3)) stimulates hepatic IGFBP-4 expression in rats. Since there is evidence that some of the genes whose expression is regulated by T(3) are also sensitive to 3,5-diiodothyronine (T(2)), we used the adult rat hepatocyte model in primary cultures directly exposed to T(2) to evaluate insulin-like growth factor binding protein-4 (IGFBP-4) expression by Northern and Ligand blot analyses in this study. Our results demonstrate that T(2), like T(3), is able to enhance IGFBP-4 mRNA and protein after 12-24 h of incubation. The potency of the two iodothyronines is comparable as judged by dose-dependence experiments. The T(2)-induced IGFBP-4 increase is independent from ongoing protein synthesis but dependent on active transcription. Since T(3) and T(2) do not affect IGF-I production, it appears that the iodothyronines affect the hepatic IGF system at the IGFBP level. Our data, demonstrating that T(2) mimics the stimulatory effect of T(3) on IGFBP-4 expression by rat hepatocytes, allow us to include IGFBP-4 gene among the genes regulated by the two iodothyronines.

Induction of insulin-like growth factor binding protein-1 expression in baboon endometrial stromal cells by cells of trophoblast origin.

To determine the influence of the conceptus on the induction of decidualization in endometrial stromal cells from the baboon. For in vivo studies, implantation sites from day 22 of pregnancy in the baboon were analyzed for insulin-like growth factor-II (IGF-II) and insulin-like growth factor binding protein-1 (IGFBP-1) mRNAs using in situ hybridization. For in vitro studies, Jeg-3 cells or primary cytotrophoblasts isolated from term placenta were cocultured with a monolayer of stromal cells from the baboon endometrium. Cytotrophoblasts were placed either directly on top of the stromal cell monolayer or in transwell cell culture inserts and cultured for 7 days. No exogenous hormones were added. Stromal cells were analyzed for IGFBP-1 mRNA and protein by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. Examination of early implantation sites in the baboon revealed a high expression of IGF-II mRNA in the invading cytotrophoblasts. Conversely, the stromal cells of the endometrium directly adjacent to the cytotrophoblasts expressed IGFBP-1 mRNA. Endometrial stromal cells cocultured with Jeg-3 cells or primary cytotrophoblasts for 7 days expressed IGFBP-1 mRNA and protein. This expression occurred in both the direct coculture system and coculture using the cell culture inserts. Cytotrophoblasts also induced the expression of prolactin in stromal fibroblasts following coculture. The conceptus is capable of inducing decidualization of endometrial stromal cells. This is shown in both the in vivo and in vitro systems.

Proteolytic degradation of IGF-binding protein (IGFBP)-2 in equine ovarian follicles: involvement of pregnancy-associated plasma protein-A (PAPP-A) and association with dominant but not subordinated follicles.

In the ovary of mammalian species, terminal follicular growth is accompanied by a decrease in intrafollicular levels of IGF-binding protein-2 (IGFBP-2) and IGFBP-4. The decrease in IGFBP-4 levels is essentially due to an increase in proteolytic cleavage by intrafollicular pregnancy-associated plasma protein-A (PAPP-A) in growing healthy follicles. The decrease in IGFBP-2 levels is partly due to a decrease in mRNA expression by follicular cells. In addition, we have recently shown that IGFBP-2 is also proteolytically cleaved by PAPP-A in bovine and porcine growing follicles. In the present work, we showed that follicular fluid from late dominant equine follicles (35 mm diameter) contains a proteolytic activity against IGFBP-2. First follicular fluid from dominant follicles contained lower levels of native IGFBP-2 than the corresponding serum, as assessed by Western ligand blotting. In contrast, immunoblotting experiments showed much higher levels of a 12 kDa proteolytic fragment in dominant follicular fluid than in the serum. Moreover, equine dominant follicular fluid was able to induce proteolysis of exogenous recombinant bovine (rb)IGFBP-2, this degradation being dose-dependently enhanced by IGFs. The proteolytic activity against IGFBP-2 in equine follicles was partially immunoneutralized by a polyclonal antibody raised against human PAPP-A. Moreover, cleavage of rbIGFBP-2 by equine follicular fluid was dose-dependently inhibited by a peptide derived from the heparin-binding domain of IGFBP-5, as well as by peptides derived from other heparin-binding domain-containing proteins such as connective tissue growth factor, vitronectin and heparin-interacting protein, previously shown to inhibit PAPP-A. Finally, the proteolytic activity was very low in subordinate follicles, was high in both early (25 mm diameter) and late (35 mm diameter) dominant follicles, and was slightly lower in preovulatory follicles recovered 35 h after human chorionic gonadotropin (hCG) treatment.Overall, these data show that in the equine ovary, the selection of dominant follicles is associated with an increase of the proteolytic degradation of IGFBP-2 by PAPP-A, as for IGFBP-4, and potentially other protease(s), probably contributing to the increase in IGF bioavailability. In atretic subordinate follicles, the decrease in the proteolytic degradation of IGFBP-2, probably due in part to a direct inhibition by peptides containing heparin-binding domains, contributes to the increase in IGFBP-2 levels and the decrease in IGF bioavailability. The expression of PAPP-A and IGFBP-2 mRNA during folliculogenesis remain to be investigated in the mare.

Paradoxical Actions of Endogenous and Exogenous Insulin-like Growth Factor-binding Protein-5 Revealed by RNA Interference Analysis

Insulin-like growth factor-binding protein-5 (IGFBP-5) is abundantly expressed in bone cells. To determine the physiological role(s) of endogenous IGFBP-5 in regulating bone cell growth, differentiation, and survival, we used short double-stranded RNA (siRNA) to trigger RNA interference of IGFBP-5 in human osteosarcoma cells. The IGFBP-5 siRNA, targeting against a sequence unique to the IGFBP-5 middle domain, efficiently reduced IGFBP-5 mRNA and protein levels. The IGFBP-5 siRNA did not change the levels of IGFBP-4, a structurally related protein, or glyceraldehyde-3-phosphate dehydrogenase, a housekeeping gene. Knock-down of IGFBP-5 resulted in a significant increase in the number of transferase-mediated dUTP nick end labeling-positive cells and a decrease in a bone differentiation parameter (alkaline phosphatase activity) but had little effect on basal or insulin-like growth factor I-induced proliferation. Overexpression of a siRNA-resistant IGFBP-5 mutant in the IGFBP-5 knock-down cells restored the levels of survival to the control level; overexpression of IGFBP-4 or wild type IGFBP-5 had no such effect. Paradoxically, the addition of exogenous IGFBP-5 not only failed to rescue IGFBP-5 knock-down-induced apoptosis, it caused a further increase in apoptosis. Furthermore, the addition of exogenous IGFBP-5 alone increased apoptosis. This pro-apoptotic action of exogenous IGFBP-5 was abolished when IGF-I was added in excess, suggesting that exogenous IGFBP-5 increases apoptosis by binding to and inhibiting the activities of insulin-like growth factors. These results indicate that endogenous and exogenous IGFBP-5 exhibits opposing biological actions on cell survival and underscore the necessity and utility of studying IGFBP functions through loss-of-function approaches.

Genomic and Proteomic Analyses of Vascular Endothelial Growth Factor and Insulin-Like Growth Factor–Binding Protein 3 in Lung Adenocarcinomas

Vascular endothelial growth factor (VEGF) is regulated by the hypoxia-inducible factor 1 (HIF1) pathway and is implicated in tumor progression and patient survival in many types of cancer. Insulin-like growth factor–binding protein 3 (IGFBP3) is also regulated by HIF1 but works in a completely different manner by modulating the activities of insulin-like growth factors and inducing apoptosis. In this study, 2-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) was used to analyze the protein expression profiles of VEGF and IGFBP3 isoforms in 93 lung adenocarcinomas and 10 uninvolved lung samples. The same samples were examined for messenger RNA (mRNA) expression with use of oligonucleotide arrays. Correlation analysis in the lung adenocarcinomas between mRNA expression levels of VEGF and all 4966 other genes was used to identify other biologic processes that may be associated with increased VEGF expression. Two-dimensional gel separations revealed 7 VEGF protein isoforms and 5 isoforms of IGFBP3. VEGF and IGFBP3 mRNA were found to be overexpressed in bronchial-derived lung adenocarcinomas (P < 0.0001), and expression was decreased in well-differentiated lung adenocarcinomas (P < 0.0002). There was a significant correlation (P < 0.01) between VEGF and IGFBP3 mRNA in lung adenocarcinomas; however, no correlation was detected in uninvolved lung samples. Forty genes were identified as the most significantly associated with VEGF expression (r > 0.38, P < 0.001), 17 of which were also associated with IGFBP3, and 12 were known to be induced through the HIF1 pathway. Among other highly correlated genes, several, including bradykinin receptor B2, suggest additional cellular processes that were not previously known to be associated with VEGF expression in lung adenocarcinoma.

Metalloproteinase pregnancy-associated plasma protein A is a critical growth regulatory factor during fetal development.

Pregnancy-associated plasma protein A (PAPPA) is a metzincin superfamily metalloproteinase in the insulin-like growth factor (IGF) system. PAPPA increases IGF bioavailability and mitogenic effectiveness in vitro through regulated cleavage of IGF-binding protein 4 (IGFBP4). To determine its function in vivo, we generated PAPPA-null mice by gene targeting. Mice homozygous for targeted disruption of the PAPPA gene were viable but 60% the size of wild-type littermates at birth. The impact of the mutation was exerted during the early embryonic period prior to organogenesis, resulting in proportional dwarfism. PAPPA, IGF2 and IGFBP4 transcripts co-localized in wild-type embryos, and expression of IGF2 and IGFBP4 mRNA was not altered in PAPPA-deficient embryos. However, IGFBP4 proteolytic activity was completely lacking in fibroblasts derived from PAPPA-deficient embryos, and IGFBP4 effectively inhibited IGF-stimulated mitogenesis in these cells. These results provide the first direct evidence that PAPPA is an essential growth regulatory factor in vivo, and suggest a novel mechanism for regulated IGF bioavailability during early fetal development.

Growth inhibition of breast epithelial cells by celecoxib is associated with upregulation of insulin-like growth factor binding protein-3 expression

Several experimental and epidemiological studies have suggested a role for the use of cyclooxygenase (COX)-2 inhibitors in the prevention of breast cancer. The relative lack of toxicity associated with these compounds favors their use as chemopreventive agents, but the underlying mechanism of their chemopreventive effect remains unclear. We have observed that the COX-2 inhibitor celecoxib inhibits growth and induces apoptosis in the immortalized breast epithelial cell line 184htert. Microarray gene expression analysis of 184htert cells treated with 50 μM celecoxib for 6 h revealed the modulation of several genes of interest, including a significant induction of expression of the mRNA encoding insulin-like growth factor binding protein-3 (IGFBP-3). IGFBP-3 is a potent pro-apoptotic protein and growth inhibitor of breast cancer cells, which acts mainly by inhibiting the access of the mitogens IGF-I and IGF-II to their cell surface receptor, but also via IGF-independent effects. Quantitative real-time RT PCR demonstrated that 50 μM celecoxib induced a ∼3-fold increase in expression of IGFBP-3 mRNA after 6 h. Furthermore, ligand blot analysis revealed that celecoxib treatment was associated with the upregulation of IGFBP-3 at the protein level. IGFBP-3 (500 ng/ml) treatment of 184htert cells inhibited IGF-I and serum-induced proliferation, but had no effect on cell growth under serum-free conditions, indicating that IGF-independent effects of IGFBP-3 are not observed in this system. Our results suggest that celecoxib may decrease IGF-I-associated breast cancer risk by a mechanism involving induction of expression of IGFBP-3 and subsequent reduced proliferation of at-risk breast epithelial cells.

Insulin-like growth factor-binding protein-3 in porcine ovarian granulosa cells: gene cloning, promoter mapping, and follicle-stimulating hormone regulation.

The role and regulation of IGF-binding protein-3 (IGFBP-3) in the ovary is not fully understood. We cloned and determined the sequence of 12,257 bp of the pig IGFBP-3 gene that includes 4,296 bp of the flanking promoter sequence. The porcine IGFBP-3 promoter sequence shares two highly conserved regions with the human and bovine IGFBP-3 promoters and a mouse DNA clone. The first is a 38 bp region between -1095 and -1058, whereas the second is a 73-bp region between -63 and +10 of the pig sequence. Projected translation of the open reading frame of our sequence gave a peptide sequence identical to that determined by peptide sequencing, but with 27 additional amino acids upstream of this sequence and is highly similar to the human, bovine, rat, and mouse IGFBP-3 peptides. Using RT-PCR we demonstrated that FSH regulates IGFBP-3 mRNA expression in a biphasic manner, with an early induction (maximal at 3 h) and an inhibition at 24 h after FSH treatment. The inhibition at 24 h was not due to changes in IGFBP-3 mRNA stability. A similar pattern of FSH modulation of the IGFBP-3 gene transcription was demonstrated by the reporter activity of granulosa cells transiently transfected with IGFBP-3 promoter constructs. The site for FSH stimulation of the IGFBP-3 gene was localized to the sequence between -61 and -48 relative to the transcription start site. Regulation of IGFBP-3 transcription by FSH suggests a role for IGFBP-3 in follicular development that may be independent of IGF-I.

Insulin-like growth factor binding protein-4 interacts with centrosomes and microtubules in primary astrocytes

Insulin-like growth factor-1 and -2 (IGFs) are important for CNS development and have implications in pathological situations of the brain. Insulin-like growth factor binding protein-4 (IGFBP-4) regulates the biological effects of IGFs. We examined the expression of IGFBP-4 in primary rat and human astrocytes. IGFBP-4 mRNA was detectable by reverse transcription–polymerase chain reaction (RT-PCR) and protein expression was verified by Western blotting of cell lysates as well as conditioned culture medium. When astrocytes were immunostained for IGFBP-4 we detected an intracellular structure that did not derive from organelles involved in cellular trafficking. Ingestion of fluorescein isothiocyanate-labeled transferrin excluded detection of IGFBP-4 within vesicles of endosomal nature, in which the protein might have been incorporated. Double staining with γ-tubulin demonstrated co-localization of IGFBP-4 with centrosomes of these cells. Treatment with nocodazole resulted in absence of IGFBP-4 signal on centrosomes, indicating a dependency on intact microtubules. Immunoelectron microscopy revealed IGFBP-4 localization not only at the centrioles but also a direct interaction with microtubules. There was no binding of IGFBP-4 to centrioles in primary rat oligodendrocytes, microglia or meningeal cells. The association of IGFBP-4 with centrioles and microtubules in astrocytes suggests an involvement of this molecule in microtubule functions of these cells.

Insulin-like growth factor binding protein-3 mediates serum starvation- and doxorubicin-induced apoptosis in H9c2 cardiac cells.

Insulin-like growth factor binding protein 3 (IGFBP-3) modulates the activity of IGF-I, which exerts antiapoptotic action upon the myocardiocyte. IGFBP-3 also exerts IGF-independent actions to inhibit cell growth and induce apoptosis, mediating the effects of several antiproliferative agents. We hypothesized that IGFBP-3 mediates cardiomyocyte apoptosis. IGFBP-3 expression was studied in H9c2 rat cardiac cells cultured in serum-deprived medium in the absence or presence of 1 microM doxorubicin during a 72 h time-span. To a greater degree than serum withdrawal, doxorubicin induced IGFBP-3 up-regulation that was time-dependent. IGFBP-3 mRNA levels positively correlated with the degree of apoptosis. Exogenous IGFBP-3 decreased cell viability and induced apoptosis in serum-starved cells exposed to doxorubicin. IGFBP-3 antisense oligonucleotides markedly decreased apoptosis induced by either serum withdrawal or doxorubicin. Binding studies revealed specific high-affinity sites for IGFBP-3 in H9c2 cardiomyocytes, with binding characteristics typical of receptor-ligand interactions. These findings indicate that IGFBP-3 could play proapoptotic action at the myocardial level and suggest a novel role for this protein in cardiovascular dysfunction.

Urokinase‐Type Plasminogen Activator and Insulin‐Like Growth Factor‐Binding Protein 3 mRNA Expression in Endometriotic Lesions and Eutopic Endometrium

The peritoneal fluid of women with endometriosis contains an increased insulin-like growth factor 1 (IGF-1) bioavailability, which is produced by limited hydrolysis of urokinase-type plasminogen activator (uPA) on IGF-binding protein 3 (IGFBP-3). Recently, IGF-1 was shown to inhibit apoptosis of endometrial-like cells in vitro, suggesting that a microenvironment of increased IGF-1 bioavailability can optimize the survival of endometrial cells grown ectopically. Here the expression of mRNA of IGFBP-3 and uPA in tissue biopsies from eutopic endometrium and endometriotic lesions obtained at laparoscopy from women with endometriosis have been analyzed, and it is documented that both IGFBP-3 and uPA mRNA expression are increased from 3- to 10-fold in endometriotic lesions versus eutopic endometrium. Consequently, the necessary components (uPA and IGFBP-3 expression) of endocrine/autocrine/paracrine enhancement of local IGF bioavailability mediated by uPA hydrolysis of the IGFBP-3 were present in endometriotic lesions. These data possibly explain the origin of the increased content of uPA activity, IGF-1 bioavailability, and NH(2)-truncated forms of IGFBP-3 in the peritoneal fluid of women with endometriosis.

Effect of inducible nitric oxide synthase inhibition by aminoguanidine on insulin-like growth factor binding protein-3 in adjuvant-induced arthritic rats

This study was designed to investigate whether nitric oxide (NO) mediates changes in insulin-like growth factor binding protein-3 (IGFBP-3) levels in rats with adjuvant-induced arthritis. Male Wistar rats were injected with complete Freund's adjuvant, and 20 days afterwards arthritic and control rats were injected daily with an inhibitor of inducible NO synthase (iNOS), aminoguanidine, or vehicle for 8 days. The increase in serum levels of IGFBP-3 induced by arthritis was exacerbated by aminoguanidine treatment. Arthritis increased IGFBP-3 mRNA levels in the kidney but not in the liver. The inhibition of iNOS did not modify IGFBP-3 gene expression in the kidney or in the liver in arthritic rats. However, the inhibitory effect of arthritis on the proteolysis of IGFBP-3 in serum was potentiated by aminoguanidine administration. These results indicate that arthritis increases serum IGFBP-3 by increasing its synthesis in the kidney and decreasing its proteolysis in serum and that these effects are not mediated by NO.

Endotoxin at low doses stimulates pituitary GH whereas it decreases IGF-I and IGF-binding protein-3 in rats.

While it is well known that sepsis inhibits serum IGF-I and its gene expression in the liver, the effect on pituitary GH and IGF-binding protein-3 (IGFBP-3) is poorly understood. The GH-IGF-I-IGFBP-3 response to different doses of lipopolysaccharide (LPS) administration has been investigated in adult male rats. Two experiments were performed, administration of low doses of LPS (5, 10, 50 and 100 microg/kg) and high doses of LPS (100, 250, 500 and 1000 microg/kg). Rats received two i.p. injections of LPS (at 1730 h and 0830 h the following day) and were killed 4 h after the second injection. LPS administration induced a biphasic response in serum concentrations of GH, with an increase at the 10 microg/kg dose, followed by a decrease at higher doses (100 microg/kg on up). Pituitary GH mRNA was also increased by the administration of 10 and 50 microg/kg LPS, whereas at higher doses LPS did not modify pituitary GH mRNA. We also analyzed the GH response to LPS in primary pituitary cell cultures. When exposed to LPS, in the culture medium, there was an increase in GH release at the concentration of 0.1 and 10 ng/ml, whereas more concentrated LPS did not modify GH release. Serum concentrations of IGF-I declined in a dose-dependent fashion after LPS administration in the rats injected with 10 microg/kg LPS on up. This decrease is secondary to modifications in its synthesis in the liver, since endotoxin injection decreased both IGF-I and its mRNA in the liver. The liver GH receptor mRNA was also decreased by LPS administration, but only in the animals injected with high LPS doses. There was a decrease in both the IGFBP-3 serum levels and its gene expression in the liver with all LPS doses studied. These data suggest a biphasic LPS effect on pituitary GH, a stimulatory effect at low doses and an inhibitory effect at higher doses, whereas it has a clear inhibitory effect on IGF-I and IGFBP-3 synthesis in the liver. The decrease in liver IGFBP-3 mRNA and in serum concentrations of IGFBP-3 in the rats injected with LPS may contribute to the decrease in serum concentrations of IGF-I.

Action of mifepristone on the expression of insulin-like growth factor binding protein-1 mRNA and protein during the early luteal phase in the human oviduct

Objective To investigate the effects of the antiprogestin mifepristone on the expression of insulin-like growth factor binding protein-1 (IGFBP-1) mRNA and protein during the early luteal phase in the human oviduct. Design Prospective case-control study. Setting University hospital. Patient(s) Fourteen healthy women with regular menstrual cycles who were admitted to the hospital for voluntary sterilization by the laparoscopic technique. Intervention(s) Treatment with 200 mg of mifepristone was administered on day LH+2. Fallopian tube samples were obtained on days LH+4 to LH+6. Main outcome measure(s) Expression of IGFBP-1 was identified using immunhistochemistry, and mRNA levels were determined with semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Result(s) Immunoreactivity for IGFBP-1 was primarily localized to the cytoplasm of the oviductal epithelial cells. Messenger RNA for IGFBP-1 was identified in total RNA extracted from the same fallopian tube samples. There was a significant increase in the expression of IGFBP-1 immunostaining and mRNA after treatment with mifepristone. Conclusion(s) These data further illustrate the complex actions of mifepristone and support the view that changes in the oviductal environment after treatment with mifepristone may be detrimental to normal gamete transport and function and contribute to the contraceptive action of mifepristone.

Acute in vivo elevation of insulin-like growth factor (IGF) binding protein-1 decreases plasma free IGF-I and muscle protein synthesis.

This study examined whether the acute elevation of IGF-binding protein-1 (IGFBP-1) decreases the plasma free IGF-I concentration and alters in vivo rates of muscle protein synthesis and glucose uptake. The plasma concentration of human IGFBP-1 was increased to approximately 95 ng/ml in conscious catheterized rats infused iv with human IGFBP-1 for 4 h. Infusion of IGFBP-1 also increased the concentration of endogenous (e.g. rat) IGFBP-1 in the blood, and this response was associated with a 2- to 3-fold elevation of IGFBP-1 mRNA in liver and kidney. IGFBP-1 did not significantly alter the plasma concentration of total IGF-I, but decreased circulating free IGF-I levels by about 50%. IGFBP-1 decreased protein synthesis in the predominantly fast-twitch gastrocnemius muscle (20%), and this change resulted from a decreased translational efficiency that was associated with a decreased phosphorylation of S6K1, but not 4E-BP1. Complementary studies demonstrated that IGFBP-1 also decreased the rates of protein synthesis under basal conditions and in response to stimulation by IGF-I when added in vitro to the fast-twitch epitrochlearis muscle. In contrast, IGFBP-1 did not alter in vivo-determined rates of protein synthesis in the slow-twitch soleus muscle, heart, liver, or kidney. The infusion of IGFBP-1 did not significantly alter the plasma glucose or lactate concentration or the whole body rate of glucose production or disposal. The above-mentioned changes were not mediated indirectly by changes in the plasma insulin or corticosterone concentrations, decreased high energy phosphate content in muscle, or hepatoxicity produced by the infused IGFBP-1. These results demonstrate that acute in vivo elevation in IGFBP-1, of the magnitude observed in various catabolic conditions, is capable of selectively decreasing protein synthesis in fast-twitch skeletal muscle and up-regulating the hepatic and renal syntheses of IGFBP-1 per se. Hence, elevations in circulating and tissue levels of IGFBP-1 may be an important mediator for the muscle catabolism observed in various stress conditions.

Expression of insulin-like growth factor-II and insulin-like growth factor binding protein-1 in the placental basal plate from pre-eclamptic pregnancies

Objectives: To determine whether expressions of insulin-like growth factor-II (IGF-II) and insulin-like growth factor binding protein-1 (IGFBP-1) are altered in pre-eclamptic placenta and to elucidate the possible relationship between their expressions and a mechanism for inadequate trophoblast invasion in pre-eclampsia. Methods: Placental tissues were obtained at cesarean delivery from five normotensive, nine mild pre-eclamptic and five severe pre-eclamptic women at 33–39 completed weeks of gestation. After total ribonucleic acid was extracted, reverse transcriptase-polymerase chain reaction was performed to determine IGF-II and IGFBP-1 mRNA expression. Product bands were quantitated by scanning densitometry and results were expressed as ratio of cytokines/β-actin. Western blot analysis was also done to determine IGF-II and IGFBP-1 protein expression. Statistical analysis was determined by Kruskal–Wallis analysis of variance with the Scheffe multiple post-hoc test. Results: The IGF-II mRNA levels of mild and severe pre-eclamptic placenta were significantly lower than that of uncomplicated placenta (P<0.005, P<0.001, respectively), with the level of severe pre-eclamptic placenta being significantly lower than that of mild pre-eclamptic placenta (P<0.05). As for the IGF-II protein expression, a significant decrease was found among the three groups (P<0.001), correlating with the IGF-II mRNA results. However, the mean IGFBP-1 mRNA levels of mild and severe pre-eclamptic placenta were significantly higher than that of uncomplicated placenta (P<0.05, P<0.005, respectively), with the level of severe pre-eclamptic placenta being significantly raised compared with that of mild pre-eclamptic placenta (P<0.05). Finally, a significant increase of IGFBP-1 protein expression was noted among the three groups (P<0.001), correlating with the IGFBP-1 mRNA results. Conclusions: This study suggests that IGF-II and IGFBP-1 might be associated with the impaired trophoblastic invasion that may lead to pathogenesis of pre-eclampsia.