Insulin-like Growth Factor-1 mRNA Expression Research Articles

Effects of ovariectomy and anabolic steroid implantation on the somatotrophic axis in feedlot heifers

A 2 x 2 factorial experimental design was used to evaluate effects of ovariectomy and implantation (200 mg trenbolone acetate and 28 mg estradiol benzoate; Synovex-Plus) on the performance, serum urea nitrogen, serum insulin like growth factor -1 (IGF-1), and mRNA expression of hepatic IGF-1, growth hormone (GH) receptor and estrogen receptor-α as well as pituitary GH, estrogen receptor-α and GH releasing hormone receptor in feedlot heifers. Thirty-two British x Continental heifers were randomly assigned to either an ovariectomized (OVX) or intact group and then to either a control group or a Synovex-Plus implant group. Heifers were fed a 900 g/kg concentrate steam-flaked maize based diet for 42 d. Liver biopsies were taken before OVX for baseline expression of IGF-1, GH receptor and hepatic estrogen receptor-α, which did not differ between treatment groups. Blood and body weight (BW) were taken on d 0, 28, and 42 and one heifer/pen was slaughtered on d 42 for liver and pituitary tissue samples. Initial and final BW did not differ due to OVX or implant. No gender x treatment interaction was observed for average daily gain (ADG) or final BW. Neither OVX nor implant affected ADG for the final 14 d of the feeding period, but 28 and 42 d ADG were greater in implanted than in control heifers. No 2- or 3-way interactions with day were observed for serum urea nitrogen. Serum urea nitrogen was greater in control heifers than in implanted heifers, but gender had no effect. Both implant and OVX increased serum IGF-1 and a gender x treatment interaction was observed. Neither gender nor implant treatment affected hepatic estrogen receptor-α, hepatic IGF-1, hepatic GH receptor, GH releasing hormone receptor, GH, or pituitary estrogen receptor-α. Data indicate that implanting heifers with Synovex-plus increases serum IGF-1, but the reason for this increase cannot be explained by mRNA expression of key somatotropic genes in the present study.A 2 x 2 factorial experimental design was used to evaluate effects of ovariectomy and implantation (200 mg trenbolone acetate and 28 mg estradiol benzoate; Synovex-Plus) on the performance, serum urea nitrogen, serum insulin like growth factor -1 (IGF-1), and mRNA expression of hepatic IGF-1, growth hormone (GH) receptor and estrogen receptor-α as well as pituitary GH, estrogen receptor-α and GH releasing hormone receptor in feedlot heifers. Thirty-two British x Continental heifers were randomly assigned to either an ovariectomized (OVX) or intact group and then to either a control group or a Synovex-Plus implant group. Heifers were fed a 900 g/kg concentrate steam-flaked maize based diet for 42 d. Liver biopsies were taken before OVX for baseline expression of IGF-1, GH receptor and hepatic estrogen receptor-α, which did not differ between treatment groups. Blood and body weight (BW) were taken on d 0, 28, and 42 and one heifer/pen was slaughtered on d 42 for liver and pituitary tissue samples. Initial and final BW did not differ due to OVX or implant. No gender x treatment interaction was observed for average daily gain (ADG) or final BW. Neither OVX nor implant affected ADG for the final 14 d of the feeding period, but 28 and 42 d ADG were greater in implanted than in control heifers. No 2- or 3-way interactions with day were observed for serum urea nitrogen. Serum urea nitrogen was greater in control heifers than in implanted heifers, but gender had no effect. Both implant and OVX increased serum IGF-1 and a gender x treatment interaction was observed. Neither gender nor implant treatment affected hepatic estrogen receptor-α, hepatic IGF-1, hepatic GH receptor, GH releasing hormone receptor, GH, or pituitary estrogen receptor-α. Data indicate that implanting heifers with Synovex-plus increases serum IGF-1, but the reason for this increase cannot be explained by mRNA expression of key somatotropic genes in the present study. Keywords : Synovex-plus, mRNA, gender South African Journal of Animal Science Vol. 38 (3) 2008: pp. 207-216

Th2 Cytokine-Induced Alterations in Intestinal Smooth Muscle Function Depend on Alternatively Activated Macrophages

Enteric nematode infection induces a strong type 2 T helper cell (Th2) cytokine response characterized by increased infiltration of various immune cells, including macrophages. The role of these immune cells in host defense against nematode infection remains poorly defined. The present study investigated the role of macrophages and the arginase pathway in nematode-induced changes in intestinal smooth muscle function and worm expulsion. Mice were infected with Nippostrongylus brasiliensis and treated with clodronate-containing liposome to deplete macrophages or given S-(2-boronoethyl)-I-cysteine in drinking water to inhibit arginase activity. Segments of intestinal smooth muscle were suspended in organ baths to determine responses to acetylcholine, 5-hydroxytryptamine, or nerve stimulation. The phenotype of macrophages was monitored by measuring mRNA expression of the specific molecular markers by real-time polymerase chain reaction or viewed by immunofluorescence staining. Infection increased the infiltration of macrophages and up-regulation alternatively activated macrophage markers by a mechanism dependent on interleukin-4 (IL-4) or interleukin-13 (IL-13) activation of signal transducer and activator of transcription 6. Elimination of alternatively activated macrophages blocked smooth muscle hypercontractility and the increased smooth muscle thickness, and impaired worm expulsion. In addition, specific inhibition of arginase activity interfered with smooth muscle contractility, but only partially affected the protective immunity of the host. These data show that the phenotype of macrophages is determined by the local immune environment and that alternatively activated macrophages play a major role in the effects of Th2 cytokines, IL-4 and IL-13, on intestinal smooth muscle function.

Estrogen Effects on Skeletal Muscle Insulin-Like Growth Factor–1 and Myostatin in Ovariectomized Rats

Previous work showed that estrogen replacement attenuates muscle growth in immature rats. The present study examined muscle insulin-like growth factor-1 (IGF-1) and myostatin expression to determine whether these growth regulators might be involved in mediating estrogen's effects on muscle growth. IGF-1 and myostatin message and protein expression in selected skeletal muscles from 7-week-old sham-ovariectomized (SHAM) and ovariectomized rats that received continuous estrogen (OVX/E2) or solvent vehicle (OVX/CO) from an implant for 1 week or 5 weeks was measured. In the 1-week study, ovariectomy increased IGF-1 mRNA expression in fast extensor digitorum longus and gastrocnemius muscles; the increase was reversed by estrogen replacement. A similar trend was observed in the slow soleus muscle, although the change was not statistically significant. In contrast to mRNA, muscle IGF-1 protein expression was not different between SHAM and OVX/ CO animals in the 1-week study. One week of estrogen replacement significantly decreased IGF-1 protein level in all muscles examined. Myostatin mRNA expression was not different among the 1-week treatment groups. One week of estrogen replacement significantly increased myostatin protein in the slow soleus muscle but not the fast extensor digitorum longus and gastrocnemius muscles. There was no treatment effect on IGF-1 and myostatin expression in the 5-week study; this finding suggested a transient estrogen effect or upregulation of a compensatory mechanism to counteract the estrogen effect observed at the earlier time point. This investigation is the first to explore ovariectomy and estrogen effects on skeletal muscle IGF-1 and myostatin expression. Results suggest that reduced levels of muscle IGF-1 protein may mediate estrogen's effect on growth in immature, ovariectomized rats. Increased levels of muscle myostatin protein may also have a role in mediating estrogen's effects on growth in slow but not fast skeletal muscle.

Effects of Bushen Tiaochong Recipe (补肾调冲方) containing serum on ovarian granulosa cell proliferation, steroidogenesis and associated gene expression in rats

To observe the effect of bushen tiaochong recipe (BSTCR) on rats' ovarian granulosa cell (GC) proliferation, steroidogenesis and follicle-stimulating hormone receptor (FSHR), and insulin-like growth factor-1 (IGF-1) mRNA expression using serum pharmacological method. Rats' GCs were incubated with 10% blank serum (as negative control group), follicle-stimulating hormone (FSH)-containing serum (S-FSH, as positive control group), or BSTCR (in different dosages) containing serum (S-BSTCR, as the BSTCR groups) for 48 h. 3H-TdR incorporation was then performed; DNA was measured to analyze the distribution of GCs in the cell cycle and their proliferation index (PI) using a flow cytometer; estradiol (E2) and progesterone (P) content in the culture fluid were examined by radioimmunoassay; and levels of FSHR and IGF-1 mRNA expression in GCs were measured by real-time RT-PCR. A dose-dependent increase of 3H-TdR incorporation in GC was shown in the BSTCR groups. Cells in G0/G1 phase had markedly less, while those in S phase had a significantly higher increase in the BSTCR groups compared with the negative control group. A high value of PI was also shown in the BSTCR groups, especially in the high dose group where the influence of cell proliferation was stronger than that in the positive control group. The levels of E2 and P in the BSTCR groups of all dosages were significantly higher than those in the negative control group, and did not show any significant difference compared with those in the positive control group. Levels of FSHR and IGF-1 mRNA expression in the BSTCR groups increased in a dose-dependent manner at levels higher than those in the negative control group. S-BSTCR can obviously stimulate the proliferation and steroidogenesis of ovarian GCs. It is speculated that BSTCR could play a regulatory action on ovarian function through two different pathways of endocrine and autocrine by promoting FSHR and IGF-1 mRNA expression.

Correlation between growth hormone/insulin-like growth factor-1 resistance and insulin resistance in non catch-up growth rats born small for gestational age

To investigate the post-receptor signaling mechanism responsible for insulin resistance-induced growth hormone (GH) resistance in non-catch-up (NCU) growth rats born small for gestational age (SGA). Twenty pregnant female SD rats were fed with restricted food (40% of normal intake, 9 g/d) throughout the pregnancy so as to develop NCU-SGA rats. The rats with their length and body weight < or = -2SD were out into the NCU-SGA group, and those with their length and body weight > -2SD were out into the catch-up (CU) growth group. Rats born to normally-fed pregnant rats were set as normal control (control Group, C Group, n = 17). The body weight and length were measured every 2 weeks. At the age of 4 weeks, 24 h urine was collected to measure the urine GH (U-GH). Then blood samples were collected to measure the serum insulin-like growth factor-1 (IGF-1), fasting insulin (FINS), and glucose levels, and the liver was taken out to detect the expression of STAT5 signal. Twelve 3-week NCU-SGA rats were divided into 2 equal groups: P13K blocking group, undergoing intraperitoneal injection of LY294002, blocker of P13K twice every 3 days, and solvent control group, undergoing intraperitoneal injection of DMSO. At the age of 4 weeks, blood samples were collected and then the liver was taken out to detect the IGF-1 mRNA and STAT5 signal. (1) The body weight and length at birth of the NCU-SGA group were (4.4 +/- 0.5) g and (4.5 +/- 0.2) cm, both significantly lower than those of Group C [(6.8 +/- 0.6) g and (5.3 +/- 0.2) cm respectively], and the body weight and length at 4 weeks of age of the NCU-SGA group were (63 +/- 12) g and (13.2 +/- 1.0) cm respectively, both significantly lower than those of the C group [(88 +/- 12) g and (15.3 +/- 0.5) cm respectively, all P < 0.01]. The serum IGF-1 level, IGF-1 mRNA expression, and total and phosphate STAT5 level in liver of the NCU-SGA group were (248 +/- 58) ng/ml, (6.1 +/- 0.3) copies, and (61 +/- 22)% respectively, all significantly lower than those of the C group [(383 +/- 62) ng/ml, (6.6 +/- 0.4) copies, and (91 +/- 29)%, all P < 0.01]. There was no statistic difference in 24 h U-GH between the NCU-SGA and C groups (P > 0.05). The FINS and glucose level of the NCU-SGA group were (24.7 +/- 9.6) mU/ml and (5.4 +/- 0.3) mmol/L respectively, both significantly higher than those of the C group [(9.8 +/- 2.8) mU/ml and (4.5 +/- 1.7) mmol/L respectively, both P < 0.05]. The level of 24 h U-GH was positively correlated with FINS (r = 0.680, P = 0.000). No correlation was found between IGF-1 and fasting insulin level. (2) After the PI3K pathway was chronically blocked, the NCU-SGA rats lost weight and developed a more severe insulin resistance, decreased serum IGF-1 level and the IGF-1 mRNA expression level of the PI3K inhibitor group were (218 +/- 60) ng/ml and (6.1 +/- 0.3) copies respectively, both significantly lower than those of the solvent control group [(286 +/- 45) ng/ml and (6.3 +/- 0.3) copies, both P < 0.05]. No statistically significant difference in total and phosphate STAT5 levels in liver between the P13K blocker and solvent groups. GH resistance is closely associated with insulin resistance in the NCU-SGA rats. GH resistance-induced failure of catch-up growth is related to the impairment of JAK2-STAT5 pathway. Insulin resistance exacerbates growth axis resistance and growth retardation in NCU-SGA rats via a non-STAT5 dependent pathway.

INTRAUTERINE GROWTH RESTRICTION AFFECTS INSULIN GROWTH FACTOR BINDING PROTEIN 3 BUT NOT INSULIN-LIKE GROWTH FACTOR 1 RECEPTOR EXPRESSION IN THE DEVELOPING RAT LUNG.

Background Intrauterine growth restriction (IUGR) predisposes neonates toward chronic lung changes characterized by mesenchymal thickening. Insulin-like growth factor 1 (IGF-1) plays a crucial role in normal lung growth and development. Key modulators of IGF-1 activity include IGFBP-3 and IGF-1r. Previous studies have shown that IGFBP-3 binds IGF-1 and decreases its activity. In addition, evidence suggests that IGFBP-3 may exhibit growth inhibitory effects independent of its ability to down-regulate IGF-1 activity. Specifically, IGFBP-3 has been shown to inhibit the growth of fibroblasts derived from IGF-1r knockout mice. IGF-1r is a tyrosine kinase receptor that mediates the effects of IGF-1. Objective We hypothesized that IUGR would decrease IGFBP-3 expression in the developing rat lung and thereby contribute to increased IGF-1 activity and mesenchymal thickening. Furthermore, we hypothesized that these changes would be independent of IGF-1r expression. Design/Methods To prove these hypotheses, we used a rat model of IUGR induced through uteroplacental insufficiency. Lungs of control and IUGR rats ( n = 6-8 litters per group) at day 0 and day 21 were harvested and flash frozen. We measured relative mRNA levels of IGFBP-3 and IGF-1r with RT-PCR at day 0 and day 21. We also measured IGFBP-3 protein levels in day 0 rats. Results Significant results are expressed as percent of control ± SEM. IGF-1r mRNA levels were not different between IUGR and control rat lungs at either day 0 ( p = .66) or day 21 ( p = .32). While IGFBP-3 mRNA and protein levels did not differ at day 0 ( p = .81 and .90, respectively), mRNA levels differed significantly by day 21 (40.1% ± 7.0%, p = .05). Conclusions We conclude that IUGR decreases IGFBP-3 mRNA levels in the postnatal rat lung. IGF-1r expression is unaffected by IUGR. These results are particularly intriguing because although IUGR leads to mesenchymal thickening in the day 21 rat lung, we have previously demonstrated that IGF-1 mRNA expression in the postnatal IUGR rat lung is unchanged. We speculate that decreased IGFBP-3 during alveolarization results in increased IGF-1 bioavailability, which contributes to the mesenchymal thickening seen in the postnatal IUGR rat lung. This may be secondary to IUGR-induced imprinting on the IGFBP-3 gene that subsequently decreases its expression. Supported by the CHRC.

INTRAUTERINE GROWTH RETARDATION ALTERS EXPRESSION OF INSULIN-LIKE GROWTH FACTOR 1 IN FEMALE RAT THYMUS.

Background Intrauterine growth retardation (IUGR) predisposes human infants and children to infectious diseases. We have previously shown that IUGR decreases the overall number of lymphocytes and alters T-cell phenotype. IUGR has also been shown to affect males to a greater degree than females. The exact mechanisms underlying these effects are unknown; however, the growth factor insulin-like growth factor 1 (IGF-1) is known to play a significant role in thymic development and lymphocyte proliferation. Additionally, IGF-1 levels in other organ systems are altered by IUGR. Furthermore, IGF-1 has several splice variants, which are preferentially expressed due to epigenetic mechanisms known to be active in IUGR. Objective We therefore hypothesized that IUGR would decrease thymic size and alter IGF-1 mRNA expression and its splice variants in male and female rat thymus. Methods Bilateral uterine artery ligation was performed on pregnant Sprague-Dawley rats to induce IUGR. Thymus tissue was harvested from IUGR and sham controls and weighed at d0 and d21. mRNA levels of the IGF-1 transcription start sites P1 and P2, as well as splice variants IGF-1A and IGF-1B, were quantified with real-time RT-PCR. Results At d0, male IUGR rat thymus had an average weight of only 0.0073 g ± 0.001 compared with sham at 0.0138 g ± 0.002 (p Conclusion The overall expression of IGF-1 was increased in IUGR female rat pups at d0 and the splice variant IGF-1A at d21. There were no significant changes in IGF-1 expression in male IUGR rat pups at d0 or d21. We know that IGF-1 is involved in the proliferation of lymphocytes and is increased in female IUGR rats and female lymphocyte phenotype and number are less affected by IUGR. We speculate that the increased IGF-1 levels and splice variants in females may explain why their immune systems are less severely affected by IUGR.

Effect of construct properties on encapsulated chondrocyte expression of insulin-like growth factor-1

Hydrogels are a promising type of biomaterial for articular cartilage constructs since they have been shown to enable encapsulated chondrocytes to express their predominant phenotypic marker, type II collagen. Endogenously expressed signaling molecules, such as insulin-like growth factor-1 (IGF-1), are also known to facilitate the retention of this chondrocytic phenotype. Recent investigations have attempted to enhance the ability of encapsulated chondrocytes to regenerate cartilage through delivery of exogenous signaling molecules. However, we hypothesize that by altering construct properties, such as cell density and polymer concentration, we can augment the expression of endogenous IGF-1 in chondrocytes. To this end, bovine articular chondrocytes were encapsulated within alginate hydrogels at two different cell densities (25,000 and 100,000 cells/bead) and various alginate concentrations (0.8%, 1.2%, and 2.0% w/v). These parameters were chosen to simultaneously investigate cell-to-cell distance on paracrine signaling and water content on IGF-1 diffusion by chondrocytes. At 1, 4, and 8d, chondrocytes were analyzed for protein and mRNA expression of IGF-1 as well as type II collagen. Results suggest that cell density and alginate concentration at high cell density can significantly affect the endogenous IGF-1 expression by chondrocytes. Therefore, these results indicate that construct properties can impact chondrocyte gene expression and should be considered in order to create a proper engineered articular cartilage construct.

Upregulation of Transforming Growth Factor‐β1 and Vascular Endothelial Growth Factor Gene and Protein Expression in Cyclosporin‐Induced Overgrown Edentulous Gingiva in Rats

To examine the effects of cyclosporin A (CsA) on the expression of growth factors in induced gingival overgrowth with limited contributing factors arising from local inflammation caused by bacterial plaque, this study of gingival overgrowth was designed on the edentulous ridge of rats. After a 3-week healing period following maxillary molar extractions, 16 five-week-old male Sprague-Dawley rats were assigned to CsA and control groups. Animals in the CsA group were fed 30 mg/kg CsA daily, whereas the control rats received a mineral oil vehicle instead. After 4 weeks, all animals were sacrificed, and the morphology of edentulous ridges was recorded by dental impression. The gingivae on the left-hand side were dissected and stored for mRNA analysis, whereas the gingivae on the right-hand side were fixed in 4% paraformaldehyde for immunohistochemistry (IHC) analysis of transforming growth factor-beta1 (TGF-beta1), platelet-derived growth factor beta (PDGF-beta), insulin-like growth factor-1 (IGF-1), and vascular endothelial growth factor (VEGF). The edentulous gingivae were enlarged and the body weights were reduced in the CsA-treated animals compared to controls. The mRNA expressions of TGF-beta1, IGF-1, and VEGF were higher in the gingivae of the CsA group than in the control group. In addition, a greater mRNA expression (7.21-fold) of VEGF was demonstrated in the CsA group than in the control group by real-time polymerase chain reaction (PCR). The percentages of cells staining positive for TGF-beta1 and VEGF were significantly greater in the CsA rats than in the control rats. Greater mRNA expression and positive staining for TGF-beta1 and VEGF were observed in the edentulous gingivae of rats that received CsA. Therefore, CsA may upregulate TGF-beta1 and VEGF gene expression and protein secretion in CsA-induced gingival overgrowth.

Modulation of insulin-like growth factor 1 levels in human osteoarthritic subchondral bone osteoblasts

Human osteoarthritis (OA) is characterized by cartilage loss, bone sclerosis, osteophyte formation and inflammation of the synovial membrane. We previously reported that OA osteoblasts (Ob) show abnormal phenotypic characteristics possibly responsible for bone sclerosis and that two subgroups of OA patients can be identified by low or high endogenous production of prostaglandin E 2 (PGE 2) by OA Ob. Here, we determined that the elevated PGE 2 levels in the high OA subgroup were linked with enhanced cyclooxygenase-2 (COX-2) protein levels compared to normal and low OA Ob. A linear relationship was observed between endogenous PGE 2 levels and insulin-like growth factor 1 (IGF-1) levels in OA Ob. As parathyroid hormone (PTH) and PGE 2 are known stimulators of IGF-1 production in Ob, we next evaluated their effect in OA Ob. Both subgroups increased their IGF-1 production similarly in response to PGE 2, while the high OA subgroup showed a blunted response to PTH compared to the low OA group. Conversely, only the high OA group showed a significant inhibition of IGF-1 production when PGE 2 synthesis was reduced with Naproxen, a non-steroidal antiinflammatory drug (NSAID) that inhibits cyclooxygenases (COX). The PGE 2-dependent stimulation of IGF-1 synthesis was due in part to the cAMP/protein kinase A pathway since both the direct inhibition of this pathway with H-89 and the inhibition of EP2 or EP4 receptors, linked to cAMP production, reduced IGF-1 synthesis. The production of the most abundant IGF-1 binding proteins (IGFBPs) in bone tissue, IGFBP-3, -4, and -5, was lower in OA compared to normal Ob independently of the OA group. Under basal condition, OA Ob expressed similar IGF-1 mRNA to normal Ob; however, PGE 2 stimulated IGF-1 mRNA expression more in OA than normal Ob. These data suggest that increased IGF-1 levels correlate with elevated endogenous PGE 2 levels in OA Ob and that higher IGF-1 levels in OA Ob could be important for bone sclerosis in OA.

Bone Microenvironment-related Growth Factors, Zoledronic Acid and Dexamethasone Differentially Modulate PTHrP Expression in PC-3 Prostate Cancer Cells

Bone metastasis microenvironment-related growth factors such as insulin-like growth factor 1 (IGF-1), transforming growth factor beta 1 (TGF-beta1), basic fibroblast growth factor (bFGF) and interleukin 6 (IL-6) show survival factor activity, thereby inhibiting chemotherapy-induced apoptosis of PC-3 prostate cancer cells in vitro. Recently, zoledronic acid has been shown to induce apoptosis in PC-3 prostate cancer cells while overexpression of parathyroid hormone-related protein (PTHrP) inhibits serum deprivation-induced apoptosis in PC-3 cells. Consequently, we have investigated whether IGF-1, TGF-beta1, bFGF, IL-6, zoledronic acid and/or dexamethasone affect the expression of the PTHrP and type I PTH/PTHrP receptor (PTH.1R) in PC-3 prostate cancer cells using relative quantitative PCR and real-time PCR (expression at mRNA level) and immunocytochemical and immunofluorescence analysis (expression at protein level). Our data show that IGF-1, TGF-beta1, bFGF and IL-6 increase PTHrP mRNA expression and its perinuclear localization, while zoledronic acid (50 muM, 100 muM for 24 h and 48 h) and dexamethasone suppress PTHrP expression in PC-3 cells. We did not detect any appreciable change of the PTH.1R expression due to IGF-1, TGF- beta1, bFGF, IL-6, zoledronic acid or dexamethasone in PC-3 cells. Therefore, it is conceivable that bone metastasis microenvironment-related survival factor/anti-apoptotic activity and zoledronic acid anticancer action/pro-apoptotic activity on PC-3 cells is mediated, at least in part, by differential modulation of PTHrP expression.

The upregulation of insulin-like growth factor-1 in oral submucous fibrosis

Insulin-like growth factor-1 (IGF-1) is a member of a family of two interacting polypeptide hormone ligands with close homology to proinsulin. IGF-1 can influence mesenchymal cell migration, proliferation, and extracellular matrix deposition, thus implicating it in the progression of fibrotic disorders. Currently, there is limited information about the regulation of IGF-1 expression in areca quid-associated oral submucous fibrosis (OSF). The aim of this study was to compare IGF-1 expression in normal human buccal mucosa and OSF specimens and further explore the potential mechanism that may lead to induce IGF-1 expression. Twenty OSF specimens and 10 normal buccal mucosa were examined by immunohistochemistry. The activity of IGF-1 from cells cultured from OSF and normal buccal mucosa were by using reverse-transcriptase polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Furthermore, the effect of arecoline, the major areca nut alkaloid, was added to explore the potential mechanism that may lead to induce IGF-1 expression. IGF-1 expression was significantly higher in OSF specimens (p<0.05) and expressed mainly by fibroblasts, endothelial cells, and inflammatory cells. OSF demonstrated significantly higher IGF-1 protein expression than normal buccal mucosa fibroblast (BMF) both in mRNA and protein levels (p<0.05). In addition, arecoline was also found to elevate IGF-1 mRNA and protein expression in a dose-dependent manner (p<0.05). Taken together, the data presented here demonstrated that IGF-1 expression is significantly upregulated in OSF from areca quid chewers and arecoline may be responsible for the enhanced IGF-1 expression in vivo.

Insulin-like growth factor-1 expression in reflux nephropathy.

Reflux nephropathy (RN) is recognised as a major cause of end-stage renal failure in children and young adults. Insulin-like growth factor-1 (IGF-1), a peptide growth factor produced by collecting ducts, and its receptor, insulin-like growth factor-1 receptor (IGF-1R), are present in the glomeruli and basolateral membrane of renal proximal tubular cells. Exogenous IGF-1 has been shown to enhance proliferation and reduce apoptosis of tubular cells following renal injury. We designed this study to investigate the expression of IGF-1 in RN. The kidney specimens from 15 children with RN were obtained at the time of nephrectomy. Control material included normal kidney specimens obtained from adult patients during partial nephrectomy for an incidentaloma. Single-label immunofluorescence histochemistry was carried out using polyclonal antibodies to IGF-1 and IGF-1R employing laser scanning confocal microscopy. Double-label immunofluorescence histochemistry was carried out using monoclonal antibodies to vimentin and clusterin to assess tubulointerstitial fibrosis. IGF-1 and IGF-1R gene expression were evaluated by in situ hybridisation (ISH). The TUNEL method was utilised to assess tubular apoptosis. In the normal kidney there was strong IGF-1 and IGF-1R immunoreactivity in the proximal tubules, whereas IGF-1 and IGF-1R immunoreactivity was markedly reduced in RN specimens. Strong IGF-1 and IGF-1R mRNA expression was observed in the proximal tubules in normal kidneys, whereas IGF-1 and IGF-1R mRNA expression was undetectable in RN. Renal tubulointerstitial expression of vimentin and clusterin was markedly increased in RN kidneys. Decreased IGF-1 and IGF-1R expression in RN strongly correlated with severity of tubular apoptosis in RN compared with controls. These data suggest that the downregulation of IGF-1 and IGF-1R may play an important role in the pathogenesis of RN, at least in part by increasing interstitial collagen deposition and tubular apoptosis.

MODULATION OF THE GENE EXPRESSION IN THE PROTECTIVE EFFECTS OF ELECTROACUPUNCTURE AGAINST CEREBRAL ISCHEMIA: A cDNA MICROARRAY STUDY

In the present study, a cDNA microarray technology was used to analyze the gene expression profile in ischemia and in electroacupuncture (EA)-treated ischemia. A focal cerebral ischemia/reperfusion model on Macaca mulatta was performed with a modified middle cerebral artery occlusion method. Among the nearly 8000 genes, approximately 8% of the total number of genes examined were affected after ischemia/reperfusion injury. Major altered genes were downregulated. In EA-treated monkeys, approximately 10% of the total number of genes examined were affected. Major altered genes were upregulated, including signal transduction-, cell-cycle-, metabolism-, stress response-, DNA repair-related genes. One of the representative upregulated genes encodes insulin-like growth factor-1 (IGF-1) was confirmed using in situ hybridization. Results showed that after ischemia/reperfusion injury, IGF-1 mRNA expression decreased in ipsilateral striatum, whereas increased in ipsilateral hippocampus. No expression changes were observed in cortex. EA treatment could obviously upregulate the IGF-1 mRNA expression in striatum, and further enhance its expression in hippocampus. Therefore, the data presented suggest a possible mediator underlying the mechanisms of anti-ischemic effect of acupuncture. In conclusion, the protective mechanisms of EA against stroke include several related pathways and gene expressions. Microarray analysis may provide a framework for understanding these complicated mechanisms and yield valuable, clinically relevant insights and potentially therapeutic targets of stroke.

Effects of Selective Nitric Oxide Synthase Inhibition on IGF-1, Caspases and Cytokines in a Newborn Piglet Model of Perinatal Hypoxia-Ischaemia

Selective inhibition of neuronal and inducible nitric oxide synthase (NOS) with 2-iminobiotin previously showed a reduction in brain cell injury. In the present study, we investigated the effects of 2-iminobiotin treatment on insulin-like growth factor-1 (IGF-1) expression, caspase activity and cytokine expression in a newborn piglet model of perinatal hypoxia-ischaemia. Newborn piglets were subjected to 1 h of hypoxia-ischaemia and were treated intravenously with vehicle or 2-iminobiotin. Vehicle-treated piglets showed reduced IGF-1 mRNA expression and increased caspase-3 activity and DNA fragmentation. 2-Iminobiotin treatment, administered immediately upon reperfusion, prevented these observations. No differences in caspase-8 and -9 activity and cytokine [interleukin (IL)-1α/β, IL-6, tumour necrosis factor (TNF)-α, transforming growth factor (TGF)-β] mRNA expression were demonstrated between vehicle- and 2-iminobiotin-treated piglets at 24 h following hypoxia-ischaemia. IGF-1 mRNA correlated inversely with caspase-3 and transferase-mediated dUTP-biotin in situ nick end labelling score in the cortex, but positively with caspase-8. Cytokine mRNA did not correlate with IGF-1 mRNA, caspase-3 activity or DNA fragmentation. The present results indicate that the previously demonstrated neuroprotective effect of 2-iminobiotin treatment after perinatal hypoxia-ischaemia coincided with a preservation of the endogenous IGF-1 production and reduced caspase-3 activity, but not with a significant decrease in cytokine production.

Estrogen induces insulin-like growth factor-1 mRNA expression in the immortalized hippocampal cell: determination by quantitative real-time polymerase chain reaction.

The basic mechanisms of nerve protection by estrogen remain to be clarified. This study was undertaken to confirm estrogen-induced insulin-like growth factor 1 (IGF-1) mRNA expression in the immortalized rat hippocampal cell H19-7 using a real-time quantitative polymerase chain reaction (PCR) assay, which has considerably increased accuracy and rapidity over other current methods. Upon stimulation by estradiol, the copy number of ERalpha mRNA showed a 1.4-fold increase, and that of IGF-1 mRNA showed a 38.5-fold increase when compared with the control value. ICI182,780 inhibited the estradiol-induced upregulation of ERalpha mRNA completely, while it inhibited estradiol-stimulated IGF-1 mRNA expression partially. The increase of the copy number of IGF-1 mRNA was accomanied by enhancement of IGF-1 protein as observed by Western blot analysis.

Effect of epristeride on the expression of IGF-1 and TGF-beta receptors in androgen-induced castrated rat prostate.

The development of benign prostatic hyperplasia (BPH) is an androgen-dependent process that may be mediated by a number of locally produced growth factors. Among them, insulin-like growth factor 1 (IGF-1) and transforming growth factor beta (TGF beta) are thought important in regulating prostate growth and homeostasis, and their expression undergoes changes in proliferative prostatic disease. Epristeride, a 5 alpha-reductase inhibitor, is an effective drug in the treatment of BPH, inducing regressive changes in the prostate. This study was designed to assess the effects of epristeride on expression of these two factors at mRNA and protein levels in castrated rats maintained with exogenous testosterone. Epristeride treatment caused significant reduction in ventral prostate weight in a dose-dependent manner. There was a positive correlation between IGF-1 mRNA expression and ventral prostate weight and an inverse correlation between TGF-beta 1 mRNA expression and ventral prostate weight. Immunohistochemistry showed strong IGF-1 receptor immunoreactivity in the prostatic epithelial cells of untreated animals. In situ hybridization demonstrated high levels of IGF-1 mRNA expression both in the prostatic stromal and epithelial cells of untreated rats. In treated rats, both IGF-1 receptor protein and IGF-1 mRNA levels decreased significantly, and IGF-1 mRNA was mainly expressed in prostatic stromal cells. Weak expression of TGF beta receptors at the protein level and TGF beta at the mRNA level were found in the prostatic hyperplastic epithelial cells of untreated rats. In treated animals, intense T beta RII immunoreactivity was observed in epithelial cells, and a higher level of TGF beta mRNA was observed in both epithelial cells and stromal cells compared with control animals. In our opinion, the effect of epristeride on rat prostatic atrophy might be mediated via local growth factor(s).

Beneficial effects of growth hormone on bacterial translocation during the course of acute necrotizing pancreatitis in rats.

Because bacterial translocation from the gut is one of the important sources of bacterial infection in acute necrotizing pancreatitis (ANP) and growth hormone (GH) has the ability to promote the intestinal epithelial proliferation, we investigated the effects of GH on bacterial translocation in a rat ANP model. ANP was induced in rats by injection of 5% sodium taurocholate into the biliopancreatic duct. The rats with ANP were treated with either human recombinant GH or placebo. Laparotomized animals without induction of ANP (sham operation [SO]) served as controls. At 24 hours after operation, blood was drawn for bacterial culture and determination of amylase, lipase, and endotoxin. Peritoneal fluid and specimens of mesenteric lymph nodes (MLN), liver, pancreas, and spleen were taken for bacterial culture by standard techniques. Intestinal mucosal permeability was assessed by measuring the movement of 125I-labeled albumin from blood to intestinal lumen. Insulin-like growth factor-1 (IGF-1) mRNA was detected in the liver and ileum by reverse transcriptase-polymerase chain reaction (RT-PCR). Morphologic changes of pancreas and ileum were also analyzed. Administration of GH significantly decreased the serum amylase, lipase activities, plasma endotoxin level, and incidence of bacterial translocation. Moreover, the survival rate of ANP rats was improved. The severity of inflammation in pancreas and ileum was alleviated by GH treatment. Ileal mucosal thickness, villus height, and crypt depth in GH treatment rats were obviously increased compared with those of ANP rats. The intestinal permeability was markedly improved in the GH group versus the ANP group. GH treatment resulted in up-regulation of IGF-1 mRNA expression in ileum, but not in liver. These results suggested that exogenous GH had beneficial effects in maintaining the integrity of intestinal mucosal barrier and reducing the incidence of bacterial translocation in rats with ANP. One of the mechanisms might be the up-regulation of IGF-1 mRNA in intestine by GH treatment.

Growth hormone therapy during neonatal hypoxia in rats: body composition, bone mineral density, and insulin-like growth factor-1 expression.

Hypoxia from birth results in a decrease in body weight gain, body size, and bone mineral density (BMD). The purpose of the present study was to determine whether short-term administration of growth hormone (GH) (rat GH; 100 microg/d) could attenuate some of these effects of neonatal hypoxia. Rat pups (with their lactating dams) were exposed to hypoxia (vs normoxic control) from birth. Hypoxia was continued until 14 d of age, with rat GH (vs vehicle control) administered daily. Hypoxia significantly inhibited body weight gain; GH therapy did not reverse this effect. GH therapy did reverse the inhibitory effect of hypoxia on tail length but not on body length. Hypoxia decreased BMD analyzed by dual X-ray absorptiometry (DXA); this effect was not reversed by GH therapy. Both GH therapy and hypoxia decreased the percentage of body fat analyzed by DXA, the effects of which were additive when combined. There were minimal effects of hypoxia and GH therapy on plasma insulin-like growth factor-1 (IGF-1), IGF-binding protein-3, and hepatic IGF-1 mRNA expression. We conclude that some of the effects of hypoxia on body habitus are reversed by GH therapy, but that short-term GH therapy did not prevent a loss of BMD. GH therapy for more than 14 days may be necessary to appreciate fully its potential in the treatment of the sequelae of neonatal hypoxia.

Postnatal function of the somatotrophic axis in pigs born naturally or by caesarian section

This study was designed to determine what effects the birth process would have on development of the somatotrophic axis in neonatal pigs. Eight crossbred sows were selected ( n = 4 natural birth and n = 4 Caesarian section) for the present study. Blood and tissue samples from 38 pigs were collected at birth. Twenty pigs were maintained with natural birth sows until sacrificed for blood and tissue collection at 2 wk of age. Gestational age at birth did not differ (P > 0.16) between natural birth and C-section pigs. Average daily gain (ADG) from birth until 2 wk of age was reduced (P < 0.0001) by 39.3% in the C-section pigs as compared to the natural birth pigs. Serum growth hormone (GH) did not differ (P > 0.86) at birth, but was greater (P < 0.024) at 2 wk in C-section pigs. Serum insulin-like growth factor 1 (IGF-1) was greater at birth (P < 0.0025) and at 2 wk of age (P < 0.035) in the natural birth pigs. Serum concentration of IGF-2 did not differ at birth (P > 0.8) but was greater (P < 0.043) in natural birth pigs at 2 wk of age. Pituitary content of GH mRNA and GH-releasing hormone receptor mRNA did not differ (P > 0.90) between groups regardless of age; however, expression of both mRNAs declined (P < 0.0003) from birth until 2 wk of age. There tended to be a birth type X age interaction (P < 0.082) for liver IGF-1 mRNA such that C-section pigs had a greater expression at 2 wk of age. Liver IGF-1 mRNA expression increased (P < 0.0001) in both groups from birth to 2 wk of age. Liver expression of GH receptor mRNA was greater in C-section pigs at birth (P < 0.04) and 2 wk of age (P < 0.03). These data provide evidence that the natural birth process affects postnatal development and/or function of the somatotrophic axis in the neonatal pig.