Differential regulation of the insulin-like growth factors (IGF-I and -II) and IGF binding proteins during malnutrition in the neonatal rat.

Insulin-like growth factor (IGF)-I and -II are known to play a major role in fetal and early postnatal growth. The IGF binding proteins (IGFBPs) are thought to be important in modulating the actions of the IGFs. In this paper, the effect of malnutrition in the neonatal rat on serum IGFs and IGFBPs and hepatic IGFBP messenger (m) RNA was examined. Control (C) dams (n = 9) were allowed ad libitum intake, whereas restricted (R) dams (n = 9) were limited to 50% of ad libitum intake throughout lactation, which results in decreased milk production and malnutrition of pups suckling on restricted dams. A subset of pups were cross-fostered from the R-dams to the C-dams from days 15-19 postpartum (PP) to investigate the effect of nutritional repletion (refed). Pups were killed on days 8, 12, 15, and 19 PP and liver and blood collected. Serum IGF-I and -II concentrations were measured by RIA after acid-chromatography to remove IGFBPs. Serum IGFBPs were characterized by Western ligand blot. Hepatic mRNA for IGFBP-1, -2, and -3 were determined by northern analysis. Body weight (BW) of R-pups was significantly less than C-pups by day 10 PP (P less than or equal to 0.05), and mean BW at day 19 was 56% of the C-pups. Refeeding from days 15-19 resulted in a significantly greater rate of growth vs. R-pups (3.2 vs. 0.9 g/day), and mean BW of refed pups at day 19 PP was 75% of C-pups. Malnutrition caused a significant reduction in both serum IGF-I and -II after day 12 PP, while causing an elevation in serum IGFBP-2. IGFBP-1 and IGFBP-2 mRNA expression were not significantly affected at days 8 and 12, but were elevated in livers of day 15 and 19 pups. Malnutrition caused a delay in the development shift from IGFBP-2 to IGFBP-3, which normally occurs between day 15 and 19 in the rat. Refeeding raised serum IGF-I and -II levels to those found in the C-pups and a trend toward normalization of IGFBP profiles. In conclusion, IGFs and IGFBPs are differentially regulated during neonatal malnutrition. The decrease in IGF peptide and induction of IGFBP-1 and -2 may provide protective mechanisms by inhibiting growth during malnutrition.     

Contributions of the N- and C-terminal domains of IGF binding protein-6 to IGF binding

Insulin-like growth factors IGF-I and IGF -II are important mediators of growth. A family of six high affinity IGF binding proteins (IGFBPs) modulate IGF action. IGFBPs have three domains, of which the N- and C-domains are involved in high affinity IGF binding. IGFBP-6 is unique in its 20-100-fold IGF-II binding specificity over IGF-I. The aim of this study was to determine the contributions of the N- and C-domains of IGFBP-6 to its IGF binding properties. We confirmed that differential dissociation kinetics are responsible for the IGF-II binding preference of IGFBP-6. The N-domain has rapid association kinetics, similar to full-length IGFBP-6, but both IGF-I and -II dissociate rapidly from this domain, thereby reducing its binding affinity for IGF-II approximately 50-fold. However, the N-domain binds IGF-I and -II with similar affinities and it has a similar IGF-I binding affinity to full-length IGFBP-6. This suggests that the C-domain confers the IGF-II binding preference of IGFBP-6; indeed, IGF-I bound inconsistently with very low affinity to the C-domain. Coincubation studies showed that isolated N- and C-domains of IGFBP-6 do not strongly cooperate to enhance IGF binding. The results of the binding studies are supported by the effects of the IGFBP-6 domains on IGF-induced colon cancer cell proliferation; the N-domain inhibited IGF-II induced proliferation with approximately 20-fold lower potency than IGFBP-6 and it was equipotent in inhibiting IGF-I- and IGF-II-induced proliferation. Coincubation of C-domain had no additional effect on N-domain-induced inhibition of proliferation. In conclusion, both the N- and C-domains of IGFBP-6 are involved in IGF binding, the C-domain is responsible for the IGF-II binding preference of IGFBP-6 and intact IGFBP-6 is necessary for high affinity IGF binding.     

Insulin-like growth factor I (IGF-I) and long R(3)IGF-I differently affect development and messenger ribonucleic acid abundance for IGF-binding proteins and type I IGF receptors in in vitro produced bovine embryos

The insulin-like growth factor (IGF) system is a complex network, including ligands (IGF-I and -II), binding proteins (IGFBP-1 to -6), and receptors, of which the type I IGF receptor (IGF-I-R) is important for transmission of most biological effects of IGFs. As IGFs are secreted in large amounts by the female reproductive tract, it has been hypothesized that maternal IGFs may affect embryonic growth and differentiation in a fine-tuned manner, involving modulation of IGF effects by embryonic IGFBP and IGF-I-R expression. To address this point, we cultured in vitro produced bovine embryos in a chemically defined culture system in the presence (100 ng/ml) of recombinant human IGF-I, long R(3)IGF-I (LR(3)), or without IGF supplementation (control). The affinity of LR(3) to IGFBPs measured by competition assays and Western ligand blots is at least 3 orders of magnitude lower than that of IGF-I. LR(3) was most efficient in stimulating early embryonic cleavage, whereas further development was most potently supported by IGF-I. Total cell numbers of blastocysts were highest in the presence of LR(3) (105 +/- 4), followed by IGF-I (96 +/- 5), and the control group (91 +/- 3; P < 0.05). Differential cell staining of blastocysts revealed that these differences were mainly represented by trophectoderm cell numbers. Analysis of messenger RNA (mRNA) expression for IGFBPs and IGF-I-R was performed by RT-real-time PCR, using expression of the nonregulated housekeeping gene glyceraldehyde-3-phosphate dehydrogenase for normalization. Embryonic IGFBP-2 mRNA levels in the LR(3) treatment group were 1.7-fold (P < 0.001) and 2.8-fold (P < 0.001) higher than those in the IGF-I and control groups, respectively. IGFBP-5 mRNA levels were about 2-fold (P < 0.001) elevated in both IGF treatment groups, with slightly (P < 0.05) higher levels in IGF-I- than in LR(3)-treated embryos. Similarly, IGFBP-3 mRNA abundance was increased (P < 0.05) in embryos from the IGF-I vs. the LR(3) culture system. IGF-I-R mRNA levels were reduced by IGF-I (80% of control; P < 0.01), but increased by LR(3) (1.3-fold vs. control; P < 0.001). These data show that the affinity for IGFBPs of IGF peptides is relevant for their effects on preimplantation embryos and affects different parameters, i.e. development, cell numbers, and mRNA expression for components of the IGF system, in different directions.     

Insulin and insulin-like growth factors (IGFs) stimulate production of IGF-binding proteins by ovarian granulosa cells.

Ligand blot analysis of granulosa cell (GC)-conditioned culture medium revealed several easily measurable insulin-like growth factor (IGF)-binding proteins (IGFBPs), including IGFBP-3 [40-44 kilodaltons (kDa)] and IGFBP-2 (34 kDa). In the present study, IGF-I, in a dose-dependent manner, significantly stimulated the production of these IGFBPs. Insulin, but not IGF-II, mimicked IGF-I's action on IGFBP-3 and -2 production, but was less potent. The synthetic IGF, long R3-IGF-I, which has very low affinity for IGFBPs and only slightly reduced affinity for the IGF-I (type I) receptor, had significantly greater potency in stimulating IGFBP-3 and -2 production compared to IGF-I. Des-(1-3)-IGF-I had similar effects. IGF-I, IGF-II, and the IGF-I analogs, but not insulin, also induced production of an unidentified 30-kDa IGFBP not normally detectable in these cultures. However, in the presence of epidermal growth factor (which was without independent effect on the 30-kDa IGFBP), insulin also induced this 30-kDa IGFBP. By Northern analysis the expression of IGFBP-3 mRNA was found to be significantly stimulated by IGF-I. In summary, insulin stimulated IGFBP-3 and -2 production in a manner that mimics that of IGF-I and the more potent long R3-IGF-I. However, its low potency suggested that IGFBP production is regulated via the IGF-I (type I) receptor. The much higher potency of long R3-IGF-I compared to that of IGF-I suggests that the IGFBPs themselves modulate the action of IGFs by sequestering exogenous IGFs. Thus, one cellular response to IGF stimulation is the production of IGFBPs, which, in turn, reduce or negate the biological activity of the IGFs. The effects of insulin-like peptides are exerted at least in part by increasing levels of mRNA for specific BPs.     

Regulation of insulin-like growth factor binding protein synthesis and secretion in a bovine epithelial cell line.

The Madin-Darby bovine kidney cell line was used to examine regulation of insulin-like growth factor binding protein (IGFBP) synthesis by epithelial cells. Ligand and immunoblot analysis of conditioned media indicated that IGFBP-2 was the predominant IGFBP secreted by untreated cells. Treatment with forskolin decreased secretion of IGFBP-2 by 75 +/- 3% and induced the appearance of IGFBP-3 and 24,000 Mr IGFBP. Although insulin alone did not induce the appearance of either band, in the presence of forskolin it increased the IGFBP-3 and 24,000 Mr bands 4.2 +/- 1.1 and 7.3 +/- 0.9-fold, respectively, above the values for forskolin treatment alone. Exposure to forskolin resulted in a 3-fold decrease in the abundance of IGFBP-2 messenger RNA (mRNA), and a 30-fold increase in IGFBP-3 mRNA. An additional 2- to 3-fold increase in IGFBP-3 mRNA was observed when cells were treated with insulin plus forskolin. Treatment with insulin plus forskolin increased cell number 2-fold, compared to small increases (26%) observed with forskolin treatment alone. Since treatment with IGF-I or -II did not result in similar responses to those of insulin, IGF analogs with differing affinities for IGFBP and IGF type I receptor were tested. B-chain IGF-I (decreased affinity for IGFBP) increased cell number and enhanced forskolin's effects on IGFBP-3 secretion and mRNA abundance to the same extent as insulin, whereas [Leu24,1-62]IGF-I (decreased affinity for the type I IGF receptor) did not. Therefore, activation of the type I IGF receptor was required to elicit increases in cell number and IGFBP synthesis and secretion, and the actions of IGF-I and II were likely blocked by binding to the large amounts of IGFBP-2 that were secreted. These results are in direct contrast to studies with human fibroblasts in which IGF-I and [Leu24,1-62]IGF-I stimulate IGFBP-3 secretion, whereas B-chain IGF-I has only a minimal effect. The ability to differentially regulate secretion of different forms of IGFBPs by epithelial cells and the finding that regulation is distinct from that of fibroblasts may have important implications for understanding mechanisms by which IGFs and IGFBPs interact to regulate epithelial cell growth.     

Identification and characterization of insulin-like growth factor (IGF)-binding protein expression and secretion by adult human adrenocortical cells: differential regulation by IGFs and adrenocorticotropin

In previous studies we have shown that IGF-II stimulates basal as well as ACTH-induced cortisol secretion from adult human adrenocortical cells more potently than IGF-I, and that both IGFs predominantly stimulate androgen biosynthesis. The steroidogenic effect of IGF-I and IGF-II is mediated through interaction with the IGF-I receptor, and modified by locally produced IGF-binding proteins (IGFBPs). In the present study, we identified and characterized IGFBP synthesis in normal adult human adrenocortical cells in primary culture, and investigated the effect of ACTH and recombinant human IGF-I and -II on the regulation of IGFBP expression and secretion. Using RT-PCR, we identified the mRNA of all six high-affinity IGFBPs, in both adrenocortical tissue and monolayer cell cultures of adrenocortical cells. Using Western ligand and immunoblotting and two-dimensional Western ligand blotting we confirmed the secretion of IGFBP-1, -2, -3, -4 and -5 by adrenocortical cells in primary culture. The quantification of IGFBPs indicated that IGFBP-3 accounts for almost half the binding activity in conditioned medium of unstimulated cells (47%), followed by IGFBP-4 (20%), IGFBP-5 (15%), IGFBP-2 (12%) and IGFBP-1 (6%). After treatment with ACTH, the abundance of IGFBP-1 was upregulated significantly 2.6-fold, while IGFBP-3 was induced only slightly (1.3-fold). IGFBP-2, -4 and -5 remained unchanged. In contrast, IGF-I and -II (6.5 nM) predominantly induced the abundance of IGFBP-5 (2- and 1.6-fold respectively) and IGFBP-3 (2- and 1.7-fold respectively), while IGFBP-1, -2 and -4 were unaltered. The induction of IGFBP-1 and -5 by ACTH and IGFs, respectively, was paralleled by an increase in the amount of IGFBP-1 and -5 mRNA in these cells. In conclusion, all six high-affinity IGFBPs are expressed in the adult human adrenal gland, and the presence of at least five high-affinity IGFBPs has been demonstrated in conditioned medium of adult human adrenocortical cells. Furthermore, the expression and secretion of IGFBP-1 is upregulated by ACTH, whereas IGFBP-5 is induced by IGF-I and -II. Together with earlier findings, these results suggest that IGFBPs play an important modulatory role in the regulation of the differentiated adrenocortical function.     

Role of insulin-like growth factor binding proteins in controlling IGF actions

The insulin-like growth factors (IGF) stimulate growth in multiple connective tissue cell types. The capacity of IGF-I and -II to access cell surface receptors is controlled by insulin-like growth factor binding proteins (IGFBPs). Connective tissue cells synthesize four of the IGFBPs (IGFBP-2 through -5). Synthesis is controlled by growth hormone and several other growth factors. In addition to regulating synthesis, other variables regulate the abundance of the IGFBPs including specific serine proteases that are produced for each form of IGFBP. Following cleavage, the IGFBPs have reduced affinity for IGF-I and -II, thus allowing release to receptors. Variables that regulate the amount of proteolysis have been shown to regulate IGF action. In addition to being proteolytically cleaved, three forms of IGFBPs (IGFBP-2, -3 and -5) can associate with extracellular matrix (ECM). In the case of IGFBP-5 binding to ECM, its affinity is lowered substantially allowing IGF to better equilibrate with the receptors. This event results in a potentiation of IGF-I action on fibroblasts and smooth muscle cells (SMC). In summary, IGFBPs are important molecules for regulating the bioavailability of IGF-I and -II to receptors. Understanding the variables that regulate their abundance may lead to a better understanding of the factors that regulate IGF action in skeletal tissues.     

Differential effects of IGF-binding proteins, IGFBP-3 and IGFBP-5, on IGF-I action and binding to cell membranes of immortalized human chondrocytes

Insulin-like growth factor-I (IGF-I) is an important anabolic factor for cartilage tissue and its action is, in part, regulated by IGF-binding proteins (IGFBPs). The object of this study was to investigate the effects of IGFBPs on IGF-I action and on binding of IGF-I to cells using a reproducible immortalized human chondrocyte culture model. Treatment of the C-28/I2 cells with IGF-I or des(1-3)IGF-I in serum-free medium stimulated cell proliferation in a dose-dependent manner. However, the effect of des(1-3)IGF-I was more potent, thereby suggesting that endogenously produced IGFBPs inhibited IGF action. The stimulatory effect of IGF-I was inhibited significantly by addition of IGFBP-3 but enhanced slightly by IGFBP-5. However, neither IGFBP-3 nor IGFBP-5 had an effect on basal cell growth. Binding of (125)I-labeled IGF-I to the cells was displaced by both IGFBP-3 and IGFBP-5, although higher concentrations of unlabeled IGFBP-5 were required to displace IGF-I to the same extent as IGFBP-3. Treatment of the cells with IGF-I increased the levels of IGFBP-5 protein measured by Western ligand blotting, and stimulated a corresponding increase in IGFBP-5 mRNA while increasing type II collagen mRNA. Our findings indicate that the balance between IGFBP-3 and IGFBP-5 influences IGF receptor binding and its action on chondrocyte proliferation, and may thereby modulate cartilage metabolism.     

The expression of the IGF system in the bovine uterus throughout the oestrous cycle and early pregnancy

The IGF system is expressed in the uterus during the oestrous cycle and early pregnancy and is likely to play an important role in regulating the development of the embryo and uterus. The IGF peptides (IGF-I and -II) mediate their effects through the type 1 IGF receptor (IGF-1R), while the IGF-binding proteins (IGFBP-1 to -6) modulate their interaction with the receptor. In this study, the expression of the IGF system in the bovine uterus was determined throughout the oestrous cycle and on day 16 of pregnancy. Endometrial biopsy samples were collected from four cows over three cycles such that there were samples for every 2 days from day 0 (oestrus) to day 14 and then every day until day 21. To assess the effect of pregnancy, uterine horn cross-sections were collected on day 16 from 15 pregnant (PREG), five inseminated non-pregnant (INP) and nine uninseminated cyclic controls (CONT). The expression of mRNA for the IGFs, IGF-1R and IGFBP-1 to -5 was determined by in situ hybridisation and the results were quantified by measuring the optical density units from autoradiographs. The main region of IGF-I mRNA expression was the sub-epithelial stroma underlying the luminal epithelium. The expression of IGF-I mRNA was highest at oestrus and lowest during the early and late luteal phases. On day 16, IGF-I mRNA levels were low in all groups, with pregnancy having no effect on the IGF-I mRNA concentrations. The strongest expression of IGF-II mRNA was in the caruncular stroma, with pregnancy having no significant effect in this region. IGF-1R mRNA was also present in the caruncles and was strongly expressed in all epithelial cells both throughout the oestrous cycle and during early pregnancy. The expression of IGFBP-1 mRNA was confined to the luminal epithelium, with the strongest expression seen on day 14 of the cycle. On day 16 the expression of IGFBP-1 mRNA was higher in the PREG group compared with the CONT group. The expression of IGFBP-2 mRNA was localised to the sub-epithelial stroma with more INP than PREG cows showing detectable levels of IGFBP-2. The strongest expression of IGFBP-3 mRNA was in the caruncular stroma; expression in the endometrial stroma was similarly decreased during early pregnancy. IGFBP-5 mRNA was mainly expressed in the inner ring of myometrium and was not affected by pregnancy on day 16. In conclusion, these results show that many components of the uterine IGF system are differentially regulated during the oestrous cycle and early pregnancy and suggest that modulation of the IGF system may influence uterine activity during this period.     

Changes of serum growth factors (IGF-I,-II and IGFBP-2,-3) prior to and after stem cell transplantation in children with acute leukemia

Summary:Insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) may play an important role in tumor proliferation. This study aimed to investigate the IGF system in children with acute leukemia prior to and after hematological stem cell transplantation (HSCT). In 51 patients (AML n=27; ALL n=24; mean age 11.2+/-4.8 years), serum parameters (IGF-I,-II, IGFBP-2,-3) were investigated up to 18 months after HSCT by RIA. Patients with AML showed a significant increase of IGFBP-2 up to 100 days after HSCT (mean +/-s.d. prior to HSCT: 3.2+/-3.6 SDS vs 100 days after HSCT: 5.3 degrees +/-3.4 SDS, P=0.005). Furthermore, IGF-I and IGFBP-3 were significantly decreased (IGF-I: -0.3+/-1.5 vs -0.7 +/-1.2 SDS, P=0.001; IGFBP-3: -0.3+/-1.1 vs -1.0+/-1.1 SDS, P=0.02). Children with AML showed significantly higher IGFBP-2 (P=0.04) and significantly lower IGF-I (P=0.03) and IGFBP-3 (P=0.05) levels than children with ALL at day 100 after HSCT. We conclude that children with acute leukemia show important changes in the IGF system after HSCT. In particular, IGFBP-2 was significantly elevated at day 100 after HSCT. Increased IGFBP-2 and decreased IGF-I and IGFBP-3 may be associated with the increased proliferation rate of transplanted bone marrow.     

Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta 1 release IGF-binding protein-3 from human fibroblasts by different mechanisms.

Human neonatal fibroblasts in monolayer culture secrete insulin-like growth factor-binding proteins (IGFBPs), which may modulate IGF action. To examine whether an increase in extracellular concentrations of IGFBPs in response to IGF-I is due to the release of cell-associated IGFBPs, we measured secreted and cell-associated IGFBP-3 immunologically in fibroblast monolayers treated with IGF-I and IGF analogs with altered affinities for the IGF receptors and IGFBPs. IGFBP-3 in medium conditioned by fibroblasts treated with IGF-I was significantly increased (P < 0.05) compared with that in medium from untreated cultures; concomitantly, cell-associated IGFBP-3 was significantly decreased (P < 0.05). [Ser24]IGF-I (reduced affinity for IGF receptors) also increased secreted IGFBP-3 and decreased cell-associated IGFBP-3. In contrast, IGFBP-3 concentrations in medium conditioned by fibroblasts treated with B-chain IGF-I (reduced affinity for IGFBPs) were not significantly increased, and cell-associated IGFBP-3 was unchanged. Heparin, which releases proteins attached to cell surface proteoglycans, increased medium concentrations of IGFBP-3 and decreased IGFBP-3 binding to fibroblasts. An IGFBP of 29-31 kilodaltons (kDa) showed a pattern of regulation similar to that of IGFBP-3, while a third IGFBP, of 24 kDa, was decreased in IGF-I- and [Ser24]IGF-I-conditioned medium and unchanged by B-chain IGF-I and heparin. Preincubation with transforming growth factor-beta 1 (TGF beta 1), which stimulates fibroblast IGFBP-3 production, or human serum-derived IGFBP-3 did not increase cell-associated IGFBP-3. Analysis of total RNA isolated from fibroblasts revealed that IGFBP-3 mRNA was increased by TGF beta 1, but not by IGF-I. These data suggest that IGFs and TGF beta 1 release fibroblast IGFBPs by distinct mechanisms: IGFs by binding and subsequent release of cell-associated IGFBP-3 and 29- to 31-kDa IGFBP, and TGF beta 1 by increased de novo synthesis of IGFBP-3.     

Endocrine actions of myostatin: systemic regulation of the IGF and IGF binding protein axis

Myostatin's inhibitory actions on striated muscle growth are believed to be directly mediated by locally produced myostatin and possibly by IGF binding proteins (IGFBPs). We therefore measured skeletal muscle, heart, and liver expression, in neonates and adults, and circulating levels of various IGF axis components (IGF-I, IGFBP-1 to IGFBP-3, and acid labile subunit) in wild-type and mstn-/- mice. Compared with wild type, differences in muscle expression were tissue specific, although IGF-I receptor expression was higher in all mstn-/- neonatal tissues and in adult gastrocnemius. Liver expression of several components also differed between genotype as IGF-I receptor, IGFBP-3 and IGFBP-5 expression was higher in mstn-/- neonates and IGF-I and IGFBP-3 in adults. Circulating IGF-I levels were also higher in mstn-/- adults, whereas IGFBP-1 and IGFBP-2 levels were lower. Comparing IGF-I:IGFBP molar ratios suggested that the relative IGF-binding capacity was potentially lower in mstn-/- mice, and thus, total and "free" IGF-I levels may be elevated. This in turn may increase negative feedback control on GH, because mstn-/- liver weights were lower. Bone growth was similar in both genotypes, suggesting that changes in circulating IGF-I may be more important to muscle, whose mass is enhanced in mstn-/- mice, than to bone. Myostatin receptors, but not myostatin itself, are expressed in the liver. Changes in hepatic production of circulating IGF axis components could therefore result from the loss of endocrine myostatin. Thus, myostatin may inhibit striated muscle growth directly at the cellular level and indirectly through systemic effects on the IGF axis.     

The relationship between the circulating IGF system and the presence of retinopathy in Type 1 diabetic patients.

BACKGROUND: Patients with proliferative diabetic retinopathy (PDR) have increased vitreous levels of insulin-like growth factor (IGF)-I, IGF-II and IGF binding proteins (IGFBPs). This accumulation is probably caused by increased leakiness of the blood-retina barrier and influx of circulating IGFs and IGFBPs. To date, interest has focused on the role of circulating total IGF-I in the development of PDR, and there are only sparse data on circulating levels of free IGF-I and IGFBPs. METHODS: We compared fasting serum samples from matched groups of Type 1 diabetic patients with no retinopathy (n = 29), non-PDR (n = 13) and PDR (n = 16). We also included matched controls (n = 26). Serum was analysed for free and total IGF-I and -II, free plus dissociable IGF-I, IGFBP-1, -2 and -3, IGFBP-1-bound IGF-I as well as IGFBP-3 proteolysis. RESULTS: When compared with controls, diabetic patients (n = 58) showed reduced (P < 0.0005) levels of free and total IGFs, free plus dissociable IGF-I and IGFBP-3, whereas levels of IGFBP-1, IGFBP-1-bound IGF-I and IGFBP-2 were elevated. IGFBP-3 proteolysis remained unaltered. When comparing diabetic patients with different degrees of retinopathy, IGFBP-2 and IGFBP-1-bound IGF-I were the only parameters that differed significantly. Patients with retinopathy (non-PDR as well as PDR) had elevated IGFBP-2 (P < 0.03) and reduced IGFBP-1-bound IGF-I (P < 0.03), when compared with patients without retinopathy. Noteworthy, both parameters correlated (0.11 < r2 < 0.14, P < 0.02) with the severity of retinopathy. CONCLUSION: Our study gives no evidence of a direct role of circulating free IGF-I in the development of PDR, whereas IGFBP-2 and IGFBP-1-bound IGF-I showed a relationship with the degree of retinopathy. However, further investigations are needed in order to clarify the basis and clinical relevance of this finding.     

Using green fluorescent protein to study intracellular signalling

Insulin-like growth factors (IGFs) stimulate growth rate in a number of animal species and are likely to contribute to genetic variations of growth potential. The present study was designed to link levels of IGF-I and IGF-II mRNA and peptides with growth rate in divergently selected genotypes of chickens with high (HG) or low (LG) growth rates. Circulating IGF-I and -II and hepatic mRNA levels were measured under ad libitum feeding conditions from 1 to 12 weeks of age, and at 6 weeks of age under three different nutritional conditions (fed, fasted for 16 or 48 h, re-fed for 4 or 24 h after a 48-h fast). IGF binding proteins (IGFBPs) were also measured. Circulating IGFs increased with age and were higher in HG chickens from 1 to 6 weeks. They decreased with fasting and only IGF-II was fully restored after 24 h of re-feeding, while IGF-I remained low. A significant decrease in steady state IGF-I mRNA levels was also observed with fasting. Across the nutritional study, hepatic IGF-I mRNAs were significantly higher in HG chickens. Variations of IGF-II mRNA levels with nutritional state or genotype exhibited a similar trend. IGFBP (28, 34 and 40 kDa) levels increased with age, while only faint differences were observed between genotypes. IGFBP-28 transiently increased with fasting and was inversely related to blood glucose and insulin levels, suggesting that it is equivalent to mammalian IGFBP-1. In HG chickens, IGFBP-28 and IGFBP-34 levels decreased markedly following re-feeding. Therefore, high and low growth rates were respectively associated with high and low IGF-I and -II levels, supporting the hypothesis of a stimulatory role for both IGFs during post-hatching growth of chickens.     

Effects of androgens on the insulin-like growth factor system in an androgen-responsive human osteoblastic cell line

Although androgens have significant effects on bone metabolism, the mediators of their effects are still unclear. As the insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) have important effects on osteoblast proliferation and differentiation, we examined androgen effects on the IGF system in a conditionally immortalized human fetal osteoblastic cell line, hFOB/AR-6, which displays a mature osteoblastic phenotype and physiological levels of functional androgen receptors. The nonaromatizable androgen, 5alpha-dihydrotestosterone (5alphaDHT), and testosterone, but not dehydroepiandrosterone, increased IGF-I messenger RNA (mRNA) levels up to 4-fold in a dose (10(-12)-10(-6) M)- and time (2-72 h)-dependent fashion. These changes were prevented by the specific androgen receptor antagonist, hydroxyflutamide. In addition, 5alpha-DHT decreased IGFBP-4 mRNA and protein levels by 2- and 4-fold, respectively, and increased IGFBP-2 and -3 mRNA and protein levels by 6- and 7-fold (for mRNA) and 3- and 5-fold (for protein), respectively. hFOB/AR-6 cells expressed the type-I IGF receptor, but this was not regulated by 5alphaDHT. 5alphaDHT and IGFBP-3 specifically increased hFOB/AR-6 cell proliferation, and a monoclonal antibody specific for IGF-I blocked this effect. Thus, androgens increase the expression of IGF-I, IGFBP-2, and IGFBP-3, but decrease levels of the inhibitory IGFBP-4 in an androgen-responsive human osteoblastic cell line. Our data are consistent with the hypothesis that the effects of androgen on bone cells may be mediated at least in part by increases in IGF-I production and by differential regulation of IGFBPs.     

Spatial and temporal regulation of GH-IGF-related gene expression in growth plate cartilage

Previous studies of the GH-IGF system gene expression in growth plate using immunohistochemistry and in situ hybridization have yielded conflicting results. We therefore studied the spatial and temporal patterns of mRNA expression of the GH-IGF system in the rat proximal tibial growth plate quantitatively. Growth plates were microdissected into individual zones. RNA was extracted, reverse transcribed and analyzed by real-time PCR. In 1-week-old animals, IGF-I mRNA expression was minimal in growth plate compared with perichondrium, metaphyseal bone, muscle, and liver (70-, 130-, 215-, and 400-fold less). In contrast, IGF-II mRNA was expressed at higher levels than in bone and liver (65- and 2-fold). IGF-II expression was higher in the proliferative and resting zones compared with the hypertrophic zone (P < 0.001). GH receptor and type 1 and 2 IGF receptors were expressed throughout the growth plate. Expression of IGF-binding proteins (IGFBPs)-1 through -6 mRNA was low throughout the growth plate compared with perichondrium and bone. With increasing age (3-, 6-, 9-, and 12-week castrated rats), IGF-I mRNA levels increased in the proliferative zone (PZ) but remained at least tenfold lower than levels in perichondrium and bone. IGF-II mRNA decreased dramatically in PZ (780-fold; P < 0.001) whereas, type 2 IGF receptor and IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4 increased significantly with age in growth plate and/or surrounding perichondrium and bone. These data suggest that IGF-I protein in the growth plate is not produced primarily by the chondrocytes themselves. Instead, it derives from surrounding perichondrium and bone. In addition, the decrease in growth velocity that occurs with age may be caused, in part, by decreasing expression of IGF-II and increasing expression of type 2 IGF receptor and multiple IGFBPs.     

Regulation of insulin-like growth factor (IGF)-binding protein expression by growth factors and cytokines alters IGF-mediated proliferation of postnatal lung fibroblasts

Postnatal day 5 is the beginning of septation and the peak of postnatal fibroblast proliferation. The author and colleagues studied fibroblasts from this developmental time period to determine factors that regulate cell proliferation. Exposure of cells to insulin-like growth factor (IGF)-I for 48 hours increased cell number whereas exposure to epithelial growth factor (EGF), platelet-derived growth factor (PDGF)-BB, fibroblast growth factor (FGF)-7, FGF-2, tumor necrosis factor-alpha (TNF-alpha), or interleukin (L)-1beta did not alter cell number. Long[R3]IGF-I (a synthetic IGF analog with reduced affinity for IGF-binding proteins [IGFBPs]) was more potent than IGF-I, with half-maximal stimulation at a dose of 0.6 nM for long[R3]IGF-I compared to 1.5 nM for IGF-I, suggesting that IGFBPs in the conditioned medium (CM) inhibit IGF activity. Addition of exogenous IGFBP-3 inhibited the IGF-stimulated increase in cell number. Addition of IGFBP-4 did not alter IGF activity because IGF-I stimulated proteolysis of IGFBP-4. The expression of mRNA for PAPP-A (a known IGFBP-4 protease) suggests that the clearance of IGFBP-4 is mediated by pregnancy-associated plasma protein (PAPP)-A. Exposure of cells to TNF-alpha or IL-1beta increased IGFBP-3 mRNA abundance and IGFBP-3 protein in CM. PDGF-BB and IL-1beta increased IGFBP-4 protein abundance and PDGF-BB and dibutyryl cAMP increased IGFBP-4 mRNA. The increase in CM IGFBP-3 following TNF-alpha exposure blocked IGF-mediated cell proliferation, suggesting that the growth factor- and cytokine-mediated changes in IGFBP abundance regulate postnatal fibroblast cell proliferation.