Tissue-specific expression of insulin-like growth factor binding protein-3 protease activity during rat pregnancy.

The abundances of insulin-like growth factor binding proteins (IGFBPs) in sera and tissue homogenates of rats at days 12, 15, and 18 of pregnancy were determined by ligand- and immunoblotting. As in serum, IGFBP-3 was abundant in day 12 uterus, placenta, and fetuses, and decreased by day 15. On day 18 of pregnancy, IGFBP-2 was predominant and IGFBP-3 was less than 25% of day 12 values in fetus and placenta, and was undetectable in uterus and decidua. In contrast, IGFBPs in the nonreproductive tissues did not change significantly. IGFBP-3 was more abundant in muscle than heart and liver, and was not detected in lung, kidney, or brain. The decrease in IGFBP-3 in serum and reproductive tissues between days 12 and 15 of pregnancy was temporally related to the appearance of IGFBP-3 protease activity. Proteolytic activity was detectable only at low levels in brain, liver, and spleen, and was undetectable in lung, heart, muscle, and kidney. The specific protease inhibitors that blocked IGFBP-3 proteolytic activities in pregnant rat serum and decidua were virtually identical and suggested inhibition of a divalent cation-dependent tryptic-like serine protease. Furthermore, exposure of bovine IGFBP-3 (in nonpregnant bovine serum) or radiolabeled recombinant human IGFBP-3 to day 18 pregnant rat serum, decidua or uterus resulted in the generation of IGFBP-3 fragments with the same apparent Mrs (29-31 K and 18-23 K). We postulate that tissue-specific degradation may be as important as synthesis in determining IGFBP-3 abundance, and that the dramatic changes in IGFBPs in reproductive and fetal tissues may cause changes in IGF availability which are necessary for rapid tissue growth and differentiation.     

Lack of insulin-like growth factor binding protein-3 protease activation by venous cannulation.

Disruption of the endothelium activates thrombogenic and fibrinolytic enzymes that cleave insulin-like growth factor binding protein-3 (IGFBP-3) in vitro. The aim of the present human study was to determine whether blood sampling, i.e., venous stasis and cannulation increase IGFBP-3 proteolysis before and/or after surgery by activating these enzymes. Serum samples obtained immediately after cannulation were compared with samples obtained from a previously inserted venous catheter. Cannulation did not increase serum IGFBP-3 proteolytic activity pre- and post-operatively, as determined by in vitro degradation of 125I-IGFBP-3. Furthermore, there was no effect on in vivo IGFBP-3 fragmentation assessed by western immunoblot. In addition, a standardized venous stasis did not affect IGFBP-3 proteolytic activity or fragmentation. Comparison of IGFBP-3 proteolytic activity before and after surgery demonstrated a significant post-operative increase. However, this could not be demonstrated immediately after the initial cannulation, due to a large individual variation at this time-point before surgery.     

Structural and biological characterization of bovine insulin-like growth factor binding protein-3.

Insulin-like growth factor binding protein-3 (IGFBP-3) purified from bovine serum shares 19 of 25 amino-terminal amino acid residues with IGFBP-3 purified from human, rat, and porcine sources. A newly characterized bovine fibroblast model was used to investigate the biological effects of purified bovine IGFBP-3 (bIGFBP-3). Coincubation of insulin-like growth factor I (IGF-I) with increasing concentrations of bIGFBP-3 produced a dose-dependent inhibition of IGF-I-stimulated [3H]aminoisobutyric acid (AIB) uptake in cultured bovine fibroblasts. Inhibition was complete at equimolar concentrations of IGF-I and bIGFBP-3. Inhibition of IGF-I-stimulated [3H]AIB uptake paralleled the ability of bIGFBP-3 to prevent [125I]IGF-I cell surface binding. In contrast, preincubation with bIGFBP-3 resulted in a dose-dependent enhancement of IGF-I-stimulated [3H]AIB uptake; a 32-86% increase in IGF-I bioactivity was seen after a 24 h preexposure to 10 nM bIGFBP-3, and a 2- to 6-fold potentiation was seen after a 72 h preincubation. Preincubation with bIGFBP-3 increased both the sensitivity and maximal responsiveness of the cells to IGF-I. The potentiating effects of bIGFBP-3 were associated with increased [125I]IGF-I binding to cultured bovine fibroblasts. Affinity cross-linking experiments indicated that the increase in IGF-I binding was due to increased membrane-associated bIGFBP-3 rather than to a bIGFBP-3-induced increase in type I IGF receptors. bIGFBP-3 had no effect on insulin stimulation of [3H]AIB uptake under either experimental condition. These data suggest that soluble bIGFBP-3 inhibits IGF-I action by sequestering and preventing IGF-I receptor binding, whereas surface-associated bIGFBP-3 enhances the growth-promoting effects of IGF-I in bovine fibroblasts. We propose that IGFBP-3 serves a dual function in modulating IGF action in vivo.     

Characterisation of recombinant glycosylation variants of insulin-like growth factor binding protein-3

There are three potential N-glycosylation sites in the non-conserved central region of the insulin-like growth factor binding protein-3 (IGFBP-3) sequence (N89AS, N109AS, N172FS). IGFBP-3 exists as two glycoforms which reduce to a single form on enzymatic deglycosylation. To determine the functional significance of the carbohydrate chains, the N-glycosylation sites were mutated singly and in combinations by substituting Asn residues with Ala. Each recombinant glycoform was detected by radioimmunoassay, indicating that glycosylation is not essential for secretion in Chinese hamster ovary cells. Ligand blotting of the conditioned media using [125I]IGF-I indicated that all seven mutants are active. On the basis of the number and molecular masses of the bands detected for each glycoform, there is approximately 4, 4.5 and 5 kDa of carbohydrate on Asn89, Asn109 and Asn172 respectively, with variable occupancy of Asn172. Ternary complex formation by the glycovariants in the presence of ALS and excess IGF-I was not significantly different from that of fully glycosylated recombinant human (rh)IGFBP-3 [Ka (fully glycosylated)=12.5+/-4.1 l/nmol; mean Ka (all mutants)=22.1+/-3.0 l/nmol]. In contrast, Asn to Asp substitutions decreased acid-labile subunit (ALS) binding activity. Cell-surface association experiments indicate that glycosylation may influence the partitioning of IGFBP-3 between the extracellular milieu and the cell surface. Therefore, while the carbohydrate units appear to be non-essential to ALS or IGF binding, they may modulate other biological activities of IGFBP-3.     

Time dependent impact of perinatal hypoxia on growth hormone,insulin-like growth factor 1 and insulin-like growth factor binding protein-3

Hypoxic-ischemia (HI) is a widely used animal model to mimic the preterm or perinatal sublethal hypoxia, including hypoxic-ischemic encephalopathy. It causes diffuse neurodegeneration in the brain and results in mental retardation, hyperactivity, cerebral palsy, epilepsy and neuroendocrine disturbances. Herein, we examined acute and subacute correlations between neuronal degeneration and serum growth factor changes, including growth hormone (GH), insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein-3 (IGFBP-3) after hypoxic-ischemia (HI) in neonatal rats. In the acute phase of hypoxia, brain volume was increased significantly as compared with control animals, which was associated with reduced GH and IGF-1 secretions. Reduced neuronal survival and increased DNA fragmentation were also noticed in these animals. However, in the subacute phase of hypoxia, neuronal survival and brain volume were significantly decreased, accompanied by increased apoptotic cell death in the hippocampus and cortex. Serum GH, IGF-1, and IGFBP-3 levels were significantly reduced in the subacute phase of HI. Significant retardation in the brain and body development were noted in the subacute phase of hypoxia. Here, we provide evidence that serum levels of growth-hormone and factors were decreased in the acute and subacute phase of hypoxia, which was associated with increased DNA fragmentation and decreased neuronal survival.     

Post-transcriptional and post-translational regulation of insulin-like growth factor binding protein-3 and -4 by insulin-like growth factor-I in uterine myometrial cells.

Involution of the uterus induced by oestrogen depletion is associated with a decrease in uterine insulin-like growth factor (IGF)-I and an increase in IGF binding protein (IGFBP) gene expression. We examined the effects of IGF-I on primary uterine myometrial cell proliferation, and on IGFBP-3 and IGFBP-4 gene expression. IGF-I enhanced DNA synthesis in these cells. In conditioned media, IGF-I increased IGFBP-3 accumulation by release of cell associated IGFBP-3. A low dose of IGF-I increased IGFBP-4 accumulation, and a high dose caused IGFBP-4 to disappear. In cell-free conditioned media IGF-I protected IGFBP-3 and enhanced IGFBP-4 proteolysis. Co-incubation of [(125)I]-IGFBP-4 with cell-free conditioned media cleaved IGFBP-4 into 18 and 12 kDa fragments. Northern blot analysis indicated that IGF-I increased IGFBP-4 mRNA accumulation by stabilizing the mRNA while IGFBP-3 gene expression was slightly decreased. The results demonstrate that IGF-I regulates IGFBP-4 post-trancriptionally and post-translationally, whereas IGFBP-3 is only affected post-translationally. By enhancing IGFBP-4 proteolysis, increasing cell-associated IGFBP-3 and stabilizing IGFBP-3, IGF-I may initiate a mitogenic response.     

Serum concentrations of insulin-like growth factor-I and insulin-like growth factor binding protein-3 in patients with chronic hepatitis

BACKGROUND: Insulin-like growth factor-I is a liver-derived humoral factor, which has important anabolic and metabolic actions and is predominantly bound by insulin-like growth factor binding protein-3. Low serum concentrations of both insulin-like growth factor-I and insulin-like growth factor binding protein-3 have been reported in patients with chronic liver disease, especially cirrhosis, but their conditions in chronic hepatitis are uncertain. The aim of this study was to evaluate the effect of chronic hepatitis on serum concentrations of insulin-like growth factor-I and insulin-like growth factor binding protein-3 and their association with hepatic inflammation activity and fibrosis. METHODS: Serum insulin-like growth factor-I and insulin-like growth factor binding protein-3 were measured by RIA (ng/ml) in 17 patients with mild to severe chronic viral hepatitis (12 chronic hepatitis C, 5 chronic hepatitis B) and 16 healthy subjects. The hepatic inflammation activity and the severity of fibrosis were evaluated using Desmet classification. RESULTS: Both insulin-like growth factor-I and insulin-like growth factor binding protein-3 levels did not correlate with inflammation activity, fibrosis or transaminase levels. In the chronic hepatitis group, insulin-like growth factor-I levels were significantly higher than the control group (mean, 263.8 +/- 27.33 versus 127.14 +/- 10.83 ng/ml, P < 0.001, respectively), whereas insulin-like growth factor binding protein-3 levels were significantly lower when compared with the controls (1643.47 +/- 60.68 versus 2728.87 +/- 284.61 ng/ml, P < 0.05, respectively). CONCLUSIONS: These results suggest that the concomitant states of serum insulin-like growth factor-I and insulin-like growth factor binding protein-3 levels in patients with chronic hepatitis may be different from cirrhotic patients and high serum IGF-I levels may be a specific finding of the stage of chronic hepatitis before developing cirrhosis.     

The insulin-like growth factor binding protein-1 in low and high insulin responders before and during dexamethasone treatment.

To investigate the influence of normal insulin levels on levels of the insulin-like growth factor binding protein-1 (IGFBP-1) we measured this peptide postabsorptively and during hyperglycemic clamp in 17 healthy subjects, nine with low insulin response (LIR) and eight with high insulin response (HIR). The study was performed before and after 60 hours of treatment with dexamethasone 6 mg/d. The fasting levels of IGFBP-1 were significantly higher in LIR, 36 +/- 2.5 micrograms/L, than in HIR, 22 +/- 2.6 micrograms/L (P less than .01), while no differences in glucose, insulin, and C-peptide concentrations were found. Dexamethasone induced an increase in basal concentrations of insulin, while IGFBP-1 levels decreased to 18.8 +/- 2 micrograms/L in LIR (P less than .01) and to 14.0 +/- 0.9 micrograms/L in HIR (P less than .05). There was no correlation between the individual basal IGFBP-1 concentrations and basal insulin levels. In contrast, basal levels of IGFBP-1 were inversely correlated to the integrated insulin or C-peptide concentrations during the hyperglycemic clamp both before (r = -.67, P less than .01) and during dexamethasone (r = -.79, P less than .001). Dexamethasone, which increased the insulin resistance, did not change the relationship between basal IGFBP-1 and the glucose-induced insulin release. In conclusion, the morning levels of IGFBP-1 in healthy subjects reflect the acute beta-cell responsiveness to glucose, which may correspond to integrated diurnal insulin levels. The inhibitory effects of dexamethasone on the morning levels of IGFBP-1 can be explained by attendant hyperinsulinemia.     

Impaired adipogenesis in insulin-like growth factor binding protein-1 transgenic mice.

Differentiation of precursor cells into mature fat cells is accompanied by enhanced expression of insulin-like growth factor (IGF)-I and is stimulated by multiple hormones including growth hormone, glucocorticoids, IGF-I and insulin. We used transgenic mice that overexpress insulin-like growth factor binding protein-1 to investigate the role of IGF-I in the accumulation of fat tissue. In response to a sucrose-enriched diet, transgenic mice gained significantly less body weight and the epididymal fat mass was significantly reduced compared with wild-type mice. The increase in adipocyte size was also significantly reduced in transgenic mice compared with wild-type mice. Fewer colonies were generated from adipose tissue from transgenic mice and the mitogenic response of these cells to IGF-I was significantly reduced compared with those from wild-type mice. Induction of glycerol-3-phosphate dehydrogenase, a measure of adipocyte differentiation, by IGF-I but not insulin, was reduced in preadipocytes from transgenic mice. These data indicate that IGF-I has a critical role in the proliferation of adipocyte precursors, the differentiation of preadipocytes and the development of obesity in response to calorie excess.     

Regulation of proliferation of prostate epithelial cells by 1,25-dihydroxyvitamin D3 is accompanied by an increase in insulin-like growth factor binding protein-3

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to regulate the proliferation of human prostate epithelial cell lines. Since the insulin-like growth factor (IGF) system is involved in the transformation process of epithelial cells, the following study was undertaken to determine if the IGF system, in particular IGF binding protein-3 (IGFBP-3), is altered by 1,25-(OH)2D3 in normal prostate epithelial cells as part of a mechanism for inhibition of transformation. Two cell systems were used in this study: (1) primary cultures of benign human prostate epithelial cells (PECs) and (2) an SV40-T immortalized prostate epithelial cell line (P153) that is non-tumorigenic. 1,25-(OH)2D3 was added to parallel sets of PECs and P153 cells in addition to the presence or absence of IGF-I or des(1-3)IGF-I. Treatment with 1,25-(OH)2D3 resulted in significant growth inhibition of both PECs and P153 cells. Furthermore, 1,25-(OH)2D3 inhibited IGF-induced proliferation, but this was partially reversed by high concentrations of IGF-I. Western ligand blots of condition media demonstrated a significant increase in IGFBP-3; likewise Northern blots demonstrated an increase in mRNA for IGFBP-3. Proliferation assays using an antibody designed to block the IGF-independent effects of IGFBP-3 failed to reverse the inhibitory effect of 1,25-(OH)2D3. Thus, IGFBP-3 acts in an IGF-dependent manner to inhibit cell growth of benign prostate epithelial cells.