Expression of insulin-like growth factor-I during chicken development

We have investigated the expression of insulin-like growth factor-I (IGF-I) during ontogeny in the chick. IGF-I mRNA was first detectable in whole embryos on day 6, while serum IGF-I could be measured on day 9, the earliest time point examined. Serum IGF-I values rose 10-fold from about 3 ng/ml on day 10 to a prehatch peak of 30-35 ng/ml during days 15-17, and then declined to about 10 ng/ml at the time of hatching. On days 17 and 20 of incubation, IGF-I mRNA was detected in eye, skeletal muscle, and brain, but could not be found in liver or heart until after hatching. During the posthatch period, serum IGF-I rose from 10 ng/ml in the first week to 35-40 ng/ml during weeks 3-6, and liver IGF-I mRNA increased nearly 5-fold from weeks 1-7. The increases observed during weeks 1-3 correlated with a posthatch rise in serum GH from 21 to 37 ng/ml, although GH levels declined over the subsequent 4 weeks without an appreciable change in serum IGF-I values. By contrast, before embryonic day 12 no GH could be detected in the circulation, while IGF-I was 19 ng/ml on day 11. These observations suggest that there is both GH-dependent and GH-independent regulation of IGF-I gene expression in the chick, as has been found in mammals, and support the idea that IGF-I plays a role in chicken embryonic development.     

Effects of intravenous insulin-like growth factor-I and insulin administration on insulin-like growth factor-binding proteins in the ovine fetus

The insulin-like growth factors (IGF) are important anabolic hormones in the mammalian fetus; their anabolic actions are potentially modulated by alterations in the IGF-binding proteins (IGFBP). We have previously shown that the nutritional state of the fetus affects both IGF-I and the IGFBP concentrations. The present study was designed to determine the effect of alterations in insulin and IGF-I circulating concentrations on the IGFBPs. Because both insulin and IGF-I elicit decreases in glucose and amino acid concentrations, the concentrations of these substrates were clamped during the hormone infusions. Sixteen ovine fetuses were chronically catheterized at approximately 115 days of gestation, and experimental procedures performed at approximately 130 days of gestation. Insulin, IGF-I or both were infused for an 8-h period. Baseline concentrations of hormones and binding proteins were obtained, and concentrations were also obtained at the end of the infusion. Hepatic IGFBP-1 mRNA expression was also determined. Intravenous infusion of IGF-I significantly increased IGF-I concentrations in plasma in the ovine fetus. Intravenous infusion of insulin inhibited hepatic IGFBP-1 gene expression when amino acids and glucose were clamped. In contrast, intravenous infusion of recombinant human IGF-I (rhIGF-I) enhanced hepatic IGFBP-1 gene expression. Neither insulin nor rhIGF-I treatment had an effect on hepatic IGFBP-3 gene expression. Insulin did not alter plasma IGFBP-1 significantly, but it increased IGFBP-3 in plasma. rhIGF-I increased both IGFBP-1 and IGFBP-3 protein levels in plasma. The responses of IGFBP-1 and IGFBP-3 to increased plasma IGF-I and insulin may serve to protect the fetus from exaggerated anabolic effects and to blunt the hypoglycemic potential of circulating IGFs and insulin.     

Expression of insulin-like growth factor-I and insulin-like growth factor-binding protein-1 in the rat uterus during decidualization.

Decidualization of the uterus involves proliferation and differentiation of uterine cells. The effects of decidualization on uterine expression of insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 (IGFBP-1) have been examined in the hypophysectomized-ovariectomized (hypox-ovx) rat and the pituitary-intact (ovx) rat. Decidualization was induced by uterine stimulation of animals treated with a combination of 17 beta-estradiol and progesterone. The patterns of change in uterine IGF-I mRNA and IGFBP-1 mRNA abundance were similar to hypox-ovx rats, hypox-ovx rats replaced with GH and T4, and ovx rats. The changes in IGF-I mRNA abundance were temporally related to 17 beta-estradiol injections. IGFBP-1 mRNA was undetectable early in the decidualization process and reached maximal levels on day 6. Mechanical separation of the deciduoma tissue from the underlying myometrium revealed that the deciduoma tissue was depleted in IGF-I mRNA, while the majority of the IGFBP-1 was located in the deciduoma tissue. The in situ hybridization technique was used to localize IGF-I and IGFBP-1 mRNA in the decidualized uterus. The majority of the IGF-I expression was localized to the outer stroma and smooth muscle cell layer, whereas IGFBP-1 mRNA was detected in uterine epithelial cells and stromal glands. These experiments demonstrated that uterine IGF-I and IGFBP-1 expression during the process of decidualization are pituitary independent. Furthermore, our observations support the hypothesis that the expression of IGFBP-1, a protein capable of inhibiting the mitogenic activity of IGF-I, in deciduoma tissue may inhibit paracrine IGF-1 actin and allow for the differentiation of stromal tissue.     

Developmental regulation of insulin-like growth factor-I and growth hormone receptor gene expression.

During development, the insulin-like growth factor I (IGF-I) gene is expressed in a tissue specific manner; however, the molecular mechanisms governing its developmental regulation remain poorly defined. To examine the hypothesis that expression of the growth hormone (GH) receptor accounts, in part, for the tissue specific expression of the IGF-I gene during development, the developmental regulation of IGF-I and GH receptor gene expression in rat tissues was examined. The level of IGF-I and GH receptor mRNA was quantified in RNA prepared from rats between day 17 of gestation (E17) and 17 months of age (17M) using an RNase protection assay. Developmental regulation of IGF-I gene expression was tissue specific with four different patterns of expression seen. In liver, IGF-I mRNA levels increased markedly between E17 and postnatal day 45 (P45) and declined thereafter. In contrast, in brain, skeletal muscle and testis, IGF-I mRNA levels decreased between P5 and 4M but were relatively unchanged thereafter. In heart and kidney, a small increase in IGF-I mRNA levels was observed between the early postnatal period and 4 months, whereas in lung, minimal changes were observed during development. The changes in GH receptor mRNA levels were, in general, coordinate with the changes in IGF-I mRNA levels, except in skeletal muscle. Interestingly, quantification of GH receptor levels by Western blot analysis in skeletal muscle demonstrated changes coordinate with IGF-I mRNA levels. The levels of the proteins which mediate GH receptor signaling (STAT1, -3, and -5, and JAK2) were quantified by Western blot analysis. These proteins also are expressed in a tissue specific manner during development. In some cases, the pattern of expression was coordinate with IGF-I gene expression, whereas in others it was discordant. To further define molecular mechanisms for the developmental regulation of IGF-I gene expression, protein binding to IGFI-FP1, a protein binding site that is in the major promoter of the rat IGF-I gene and is important for basal promoter activity in vitro, was examined. Gel shift analyses using a 34-base pair oligonucleotide that contained IGFI-FP1 did not demonstrate changes in protein binding that paralleled those in IGF-I gene expression, suggesting that protein binding to IGFI-FP1 does not contribute to the developmental regulation of IGF-I gene expression, at least in brain and liver. In summary, the present studies demonstrate coordinate expression of the IGF-I gene and GH receptor during development and suggest that GH receptor expression contributes to the tissue specific expression of the IGF-I gene during development.     

Stimulation of hepatic insulin-like growth factor-I gene expression by ovine prolactin: evidence for intrinsic somatogenic activity in the rat

The effect of ovine PRL (oPRL) on hepatic insulin-like growth factor I (IGF-I) gene expression was examined in the hypophysectomized (hypox) rat and compared with that seen in hypox rats treated with ovine GH (oGH) and human GH (hGH). A single injection of oPRL (100 micrograms/100 g BW) resulted in a 15-fold increase in hepatic IGF-I messenger RNA abundance 12 h after administration. Serum IGF-I increased from 82.9 +/- (SE) 9.0 to 137.0 +/- 15.4 ng/ml. In acute studies, where the slopes of 4-point dose-response curves were compared, oPRL was approximately 50% as potent as oGH in stimulating serum IGF-I and hepatic IGF-I messenger RNA accumulation. In chronic experiments, where a single daily injection of oPRL (0, 10, 30, or 90 micrograms/day) was administered for 10 days, a significant increase in body wt was observed at each dose. However, in contrast to hypox rats which received oGH, no dose-response relationship was apparent, and oPRL was less than 10% as potent as oGH in this regard. A small but significant increase in serum IGF-I and hepatic IGF-I expression was observed in hypox rats chronically injected with oPRL when killed 6-7 h after the final injection. However, by extrapolation from the hGH dose-response curves, oPRL was 6.8% and 22.7% as potent as hGH in stimulating serum IGF-I and hepatic IGF-I accumulation respectively. The observations reported here demonstrate that oPRL has significant somatogenic activity in the rat as determined by the three variables measured. Whereas oPRL had significant effects on body weight gain, serum IGF-I and hepatic IGF-I expression, the potency of oPRL by comparison with the two other somatogenic hormones was more marked in acute experiments compared to the effects seen after chronic administration. These results suggest that oPRL, like human GH, has both somatogenic and lactogenic activity in the rat and therefore may be an inappropriate control in studies of the effects of somatogenic hormones in the rat.     

Low prevalence of insulin-like growth factor-I gene mutations in human growth disorders

In an attempt to identify genetic lesions contributing to human growth disorders, we evaluated a prospectively recruited group of children with growth failure for mutations in the insulin-like growth factor-I (IGF-I) gene. Two complementary approaches were used: Southern blot analysis to examine the large scale organization of the gene, and a solution hybridization, nuclease protection assay to identify small alterations, such as point mutations. From a total of 61 subjects studied, 52 had no organic basis for their short stature. Analysis of chromosomal DNA from these individuals failed to reveal any variation in the IGF-I gene except for a HindIII site polymorphism which maps near the 3' end of the last IGF-I exon. No single nucleotide substitutions were found within IGF-I-coding regions. Since the frequency of the length polymorphism was the same for both normal-sized and short individuals, it is unlikely to be associated with growth abnormalities. Our results suggest that there is minimal DNA sequence variability in the human IGF-I gene and that mutations in IGF-I exons are infrequent causes of growth failure.     

Down-regulation of Leydig cell insulin-like growth factor-I gene expression by interleukin-1.

We have reported previously that insulin-like growth factor-I (IGF-I) messenger RNA (mRNA) is expressed in Leydig cells and that IGF-I can enhance androgen production, whereas interleukin-1 (IL-1) is a potent inhibitor of Leydig cell steroidogenesis. Molecular cloning studies have confirmed the existence of at least two species of IL-1: IL-1 alpha and IL-1 beta. Both IL-1 alpha and beta bind to the same receptors and have the same spectrum of biological activities. The purpose of the present study was to elucidate the molecular mechanisms of the interaction between IGF-I and IL-1 both in vivo and in vitro. Adult Sprague-Dawley rats (55-65 days old) were treated with three injections of human recombinant IL-1 beta (1 microgram/rat ip) at 12-h intervals. Rats were killed 2 h after the last injection of IL-1 beta. Purified Leydig cells were isolated and RNA extracted for Northern blot analyses. For in vitro studies, highly purified Leydig cells were cultured in Dulbecco's modified Eagle's medium/F-12 supplemented with 0.1% fetal calf serum with or without IL-1 beta (1-100 ng/ml) for 24 h. RNA was then extracted from these cells. For time course studies, purified Leydig cells were initially cultured for 24 h. Fresh medium was then added with or without IL-1 beta (10 ng/ml), and the cultures were continued for an additional 2, 4, or 6 h. In vivo administration of IL-1 beta inhibited IGF-I mRNA expression in Leydig cells (a 40% reduction, P less than 0.05). IL-1 beta also suppressed IGF-I mRNA expression in Leydig cells in vitro in a time- and dose-dependent fashion. Inhibitory effects of IL-1 beta (10 ng/ml) could be demonstrated as early as 2 h and reached a nadir at 6 h (a 60% reduction, P less than 0.05). IL-1 beta (100 ng/ml) inhibited IGF-I mRNA expression to about 10% of the controls (P less than 0.01). Moreover, the inhibitory effect of IL-1 beta could be reversed by the addition of IL-1 receptor antagonist. In conclusion, IL-1 beta could directly inhibit the mRNA expression in Leydig cells for IGF-I, an important autocrine modulator of Leydig cell function. This suggests that the effect of IL-1 beta on Leydig cell function is, at least in part, achieved by down-regulation of IGF-I mRNA levels.     

Growth hormone and amino acid supply interact synergistically to control insulin-like growth factor-I production and gene expression in cultured ovine hepatocytes.

Many of the anabolic effects of growth hormone (GH) are indirect, occurring through GH-stimulated production of insulin-like growth factor-I (IGF-I) by the liver. As well as being regulated by GH, plasma IGF-I concentrations have been demonstrated to depend upon the level of dietary protein intake, with low protein diets being associated with reduced circulatory IGF-I levels. This inhibitory effect cannot be reversed by GH injection, suggesting that liver sensitivity to GH becomes impaired.To investigate the mechanisms through which protein supply affects GH sensitivity, primary cultures of ovine hepatocytes were grown in defined media, containing various proportions (0.2, 1.0 and 5.0) of jugular amino acid concentrations in fed sheep. Production of IGF-I by these cells was measured after 24 and 48 h in culture by radioimmunoassay. In the first 24-h period basal IGF-I production was the same in all defined media, and GH caused an approximately 2-fold increase in IGF-I release in cells grown in 1.0xor 5.0xamino acid media (P<0.01). Although GH appeared to increase IGF-I release in this period for cells grown in 0.2xamino acid media, this effect was not statistically significant. In the period from 24-48 h in defined media, both basal and GH-stimulated IGF-I production was dependent on amino acid availability (P<0.05 and P<0.001 respectively). Factorial analysis of variance demonstrated a strong positive interaction (P<0.001) between the effects of amino acid availability and GH, such that GH increased IGF-I production by more than 2-fold in cells grown in 5.0xamino acid media (P<0.01) but had no effect on production by cells grown in 1.0xor 0.2xamino acid media.Measurement of steady state concentrations of exon 1-derived IGF-I mRNAs using an RNase protection assay demonstrated that the observed effects on IGF-I peptide secretion were strongly associated with parallel effects at the RNA level.Incorporation of (35)S-methionine into cellular proteins over a 4-h period starting 20 h after transfer to defined culture media was not significantly reduced in 1.0xcompared with 5.0x amino acid media, although rates under both of these conditions were significantly higher than those seen in 0.2xamino acid media (P<0.01). The lack of correspondence between the dose-dependent effects of amino acid supply on cellular protein synthesis and those on basal and GH-stimulated IGF-I production, suggests that amino acid supply modulates IGF-I production through selective mechanisms.Steady state levels of the CCAAT/enhancer-binding protein beta (C/EBPbeta) isoforms, liver-enriched activating protein (LAP) and liver-enriched inhibitory protein (LIP) were determined by Western blotting. When levels of LAP were expressed relative to LIP levels in the same extracts, a significant decrease in the LAP:LIP ratio was observed in response to amino acid limitation (P<0.05).These data strengthen earlier arguments that synergistic interaction between the effects of amino acids and GH on hepatic IGF-I gene expression underlie nutrition-dependent changes in circulating IGF-I titres. The association between these effects and altered levels of C/EBPbeta isoforms suggests that CCAAT/enhancer mediated control of IGF-I gene expression may be involved in this phenomenon.     

Human chorionic gonadotropin up-regulates insulin-like growth factor-I receptor gene expression of Leydig cells.

The effects of hCG, 8-bromo-cAMP, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, and forskolin on insulin-like growth factor-I (IGF-I) receptor gene expression of Leydig cells were studied. The treatment of purified Leydig cells with hCG caused a dose-dependent increase in [125I]IGF-I binding to Leydig cells without changes in binding affinity, indicating that the increased binding was due to increased receptor numbers and not to increased affinity. The minimal time required for hCG to induce IGF-I binding was 6 h, and it had reached a plateau at 16 h. 8-Bromo-cAMP (1 mM) increased IGF-I binding about 2-fold, and forskolin (10 microM) increased binding about 51%. Using the ribonuclease protection assay, we found that hCG and 8-bromo-cAMP could increase IGF-I receptor mRNA expression as early as 2 h before the increase in IGF-I binding. The induction by hCG was over 3.5-fold at 4 h and decreased to about 2-fold at 6 h. 4 beta-Phorbol 12 beta-myristate 13 alpha-acetate had a very small effect on IGF-I receptor mRNA levels (1.5-fold increase at 2 h and no changes at 4 and 6 h). In conclusion, IGF-I receptors can be up-regulated by hCG, 8-bromo-cAMP, and forskolin. The up-regulation of IGF-I receptor number is associated with transient increases in IGF-I receptor mRNA levels. This could be a mechanism by which hCG and IGF-I interact to enhance Leydig cell steroidogenesis.     

Testosterone stimulates insulin-like growth factor-I and insulin-like growth factor-I-receptor gene expression in the mandibular condyle--a model of endochondral ossification

Puberty is associated with an increase in the plasma concentration of sex steroids, GH, and insulin-like growth factor-I (IGF-I). Gonadal steroid hormones are important for the normal pubertal growth spurt and skeletal growth. The mechanism by which gonadal steroids induce skeletal growth is still not fully understood. To better understand the direct effect sex steroids have on bone growth, we studied an isolated organ culture system of the mandibular condyle, derived from 3.5-5.5-week-old male and female mice. We found that testosterone 10(-6) M, but not estradiol, stimulated thymidine incorporation into the DNA of male-derived condyle. Three days of testosterone treatment doubled the condyle size and increased the chondroprogenitor zone, while maintaining the normal gradient of the developing chondrocytes. Immunohistochemistry and in situ hybridization techniques showed that testosterone stimulated IGF-I and IGF-I-R and their messenger RNAs (mRNAs) mainly in the mature chondrocyte layer. Immunoneutralization of IGF-I in the testosterone-treated condyle caused the disappearance of the chondroblast and young chondrocyte layers, though the progenitor cell layer remained almost unaffected. Overtreatment with testosterone (dose or duration) accelerated condylar ossification. In the presence of testosterone 10(-5) M (high dose), calcification "climbs" up to the chondroprogenitor zone, and most of the condylar chondrocytes are replaced by bone tissue. Similar changes occurred after 7 days of testosterone treatment (long duration) with 10(-6) M. In conclusion, testosterone stimulates growth and local production of IGF-I and IGF-I-R in chondrocyte cell layers of an isolated organ culture of mice mandibular condyle. Part of the effect testosterone has on condylar growth is mediated by IGF-I.     

Short- and long-term swimming exercise training increases myocardial insulin-like growth factor-I gene expression.

OBJECTIVES. We investigated the effect of short- and long-term swimming exercise, with or without insulin-like growth factor (IGF)-I administration, on the expression of myocardial IGFs and contractile proteins. METHODS: Sprague-Dawley male rats (n=36) were subjected to swimming exercise for 2 or 6 weeks. IGF-I (0.5mg/rat) was administered continuously for 1 week, using alzet osmotic pumps. Control groups remained sedentary. IGF-I, IGF-I receptor (IGF-IR), IGF-II, skeletal alpha-actin (sk-actin), and beta myosin heavy chain (beta MHC) mRNAs were measured using Northern blot analysis and RT-PCR. RESULTS: A significant 2-fold increase in myocardial IGF-I mRNA was found after 2 and 6 weeks of swimming in both IGF-I treated and untreated rats (p<0.001). IGF-IR mRNA was significantly (p<0.05) increased after 6 weeks of training only in the IGF-I treated animals. IGF-II mRNA remained unchanged at all time points. While beta MHC mRNA was significantly decreased (p=0.003) at 2 and 6 weeks, sk-actin mRNA remained unchanged. CONCLUSIONS: Short- and long-term swimming exercise training increase myocardial expression of IGF-I mRNA. Exogenous administration of IGF-I, during the first week of the exercise session, did not produce any effect on myocardial IGF-I but was associated with increased IGF-IR signal after the long-term exercise training. These data suggest a relationship between IGF-I expression and cardiac adaptation to exercise training.     

Emerging roles of insulin-like growth factor-I in the adult brain.

All tissues in the body are under the influence of insulin-like growth factor-I (IGF-I). Together with insulin, IGF-I is a key regulator of cell metabolism and growth. IGF-I also acts in the central nervous system, where it affects many different cell populations. In this brief review, we discuss the many roles of IGF-I in the adult brain, and present the idea that diseases affecting the brain will perturb IGF-I activity, although more refined studies at the molecular and cellular level are needed before we can firmly established this possibility. We also suggest that under normal physiological conditions IGF-I may play a significant role in higher brain functions underlying cognition, and may serve a homeostatic role during brain aging. Among newly emerging issues, the effects of IGF-I on non-neuronal cells within the nervous system and their impact in brain physiology and pathology are becoming very important in understanding the biology of this peptide in the brain.     

Impaired estrogen-induced uterine insulin-like growth factor-I gene expression in the streptozotocin diabetic rat

Summary Circulating somatomedin-C/insulin-like growth factor-I levels are low in the diabetic rat and unresponsive to exogenous growth hormone. However, the nature of this defect in growth hormone action remains unclear and there is little data on insulin-like growth factor-I gene expression in response to other stimuli and in non-hepatic tissues where insulin-like growth factor-I may have important paracrine and/or autocrine actions. We have previously shown that 17-beta estradiol stimulates uterine insulin-like growth factor-I expression in the ovariectomised rat. In this report uterine and hepatic insulin-like growth factor-I gene expression have been examined in the streptozotocin-diabetic rat. Serum insulin-like growth factor-I concentrations were significantly reduced in diabetic rats compared to normal rats (0.72±0.08 vs 1.23±0.05U/ml, p<0.0005) and hepatic insulin-like growth factor-I mRNA abundance was similarly reduced in diabetic rats to 49±5% of that seen in non-diabetic intact rats (p<0.005). In contrast, uterine insulin-like growth factor-I mRNA abundance was not significantly reduced in diabetic rats compared to control rats (76±12%, p = NS). Although both diabetic and non-diabetic rats demonstrated a significant increase in uterine wet weight following a single injection of 17-beta estradiol the increase in uterine insulinlike growth factor-I expression was significantly less marked in diabetic rats. Acute administration of insulin together with estradiol had no significant effect on serum insulin-like growth factor-I concentrations or hepatic insulin-like growth factor-I mRNA abundance; however, the uterine insulin-like growth factor-I response was significantly (p<0.01) augmented. The observations reported here demonstrate that hepatic insulin-like growth factor-I gene expression is markedly reduced in the diabetic rat and that the estradiol-induced uterine insulin-like growth factor-I response is significantly diminished, consistent with the hypothesis that there is a defect in insulin-like growth factor-I gene activation in the diabetic rat.