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

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

IGF-I/IGF-binding protein-3 combination improves insulin resistance by GH-dependent and independent mechanisms

IGF-I has been shown to enhance insulin sensitivity in patients with type I and type II diabetes. IGF-I suppresses GH, and this raises the question of whether its ability to enhance insulin sensitivity is mediated solely through a reduction in GH's antiinsulin actions. This study was conducted to determine whether administration of a GH receptor antagonist to patients with acromegaly and insulin resistance would result in improvement in insulin sensitivity and whether IGF-I had any additional insulin-sensitizing effects over and above those induced by its ability to suppress GH secretion. Five patients with active acromegaly were treated for 2 wk with a GH receptor antagonist. The GH receptor antagonist was effective, as IGF-I fell 65%, and mean GH values rose 42%. Mean fasting insulin fell from 39 +/- 6 to 30 +/- 7 micro U/ml, and this was accompanied by a 9% decrease in fasting glucose. After treatment the insulin sensitivity index was 2.7 +/- 1.0 x 10(-4)/min. micro U/ml compared with a baseline value of 1.65 +/- 0.8 x 10(-4)/min. micro U/ml (P < 0.015). Subsequently, the subjects were treated with the receptor antagonist plus IGF-I/IGF-binding protein-3 given by sc injection (1 mg/kg daily). After 2 wk of the combined treatment, fasting insulin fell from 49 +/- 9 to 29 +/- 7 micro U/ml, and fasting glucose fell by 14%. The insulin sensitivity index improved to 4.34 +/- 1.3 x 10(-4)/min. micro U/ml, which was significantly greater than the value obtained after treatment with the GH antagonist alone. Although only a limited number of subjects were studied, the results strongly suggest that IGF-I has insulin-sensitizing actions that are independent of its ability to suppress GH secretion. These findings necessitate further studies into the non-GH-related mechanism by which IGF-I enhances insulin sensitivity.     

Insulin-like growth factor I (IGF-I) and IGF-binding protein-3 in benign prostatic hyperplasia and prostate cancer

In view of evidence indicating significant involvement of the insulin-like growth factor (IGF) system in the pathogenesis of prostate cancer, we measured serum IGF-I and IGF-binding protein-3 (IGFBP-3) in men with benign prostatic hyperplasia (BPH; n = 75) or prostatic carcinoma (CaP; n = 84). The age-matched patient populations were selected to have circulating prostate-specific antigen (PSA), the most reliable predictor of CaP, in the overlapping diagnostic gray zone range of approximately 4--10 microg/L. Of particular interest was investigation of intact, fragment, and total IGFBP-3 levels in relation to PSA, which is also a well established IGFBP-3 protease. Among the key findings were significantly higher IGF-I and intact IGFBP-3 levels in CaP vs. BPH (P < 0.001), whereas changes in fragment and total IGFBP-3 were statistically insignificant. As expected, total PSA levels were similar in the two groups of patients (P = 0.173), whereas free PSA levels were significantly lower in those with CaP (P < 0.001). IGF-I and IGFBP-3 (intact and total) correlated significantly (P = 0.024 to <0.001) and inversely (r = -0.26 to -0.35) with free PSA in BPH, but not in CaP, and no correlations were found in comparisons involving total PSA. Statistical analysis of the various markers and their combinations indicated enhanced performance of IGF-I/free PSA [receiver operating characteristics area under the curve (AUC) = 0.728] and intact IGFBP-3/free PSA (AUC = 0.737) ratios in discriminating between BPH and CaP compared with the currently used free/total PSA ratio (AUC = 0.689). Multivariate logistic regression models confirmed the observed relationships and identified IGF-I/free PSA and intact IGFBP-3/free PSA as independent factors in predicting the presence of CaP. We conclude that increases in IGF-I and intact IGFBP-3 levels are positively associated with the presence of CaP in this group of patients with low to moderately elevated PSA, and that their measurements in relation to PSA may help improve diagnostic discrimination between BPH and prostate cancer.     

Augmented serum levels of the IGF-I/IGF-binding protein-3 ratio in pre-menopausal patients with type I breast cysts

OBJECTIVE: Gross cystic disease (GCD) is the most common benign breast pathology. Although breast cysts are not considered pre-malignant lesions, an increased risk of breast cancer has been reported for patients with type I cysts (Na(+)/K(+)<3). Furthermore, an augmented IGF-I/IGF-binding protein-3 (IGFBP-3) ratio has been described in breast cancer patients. The objective was to evaluate serum IGF-I and binding protein concentrations of type I and type II cyst patients as compared with healthy women. METHODS: Twenty-four patients with type I cysts, 17 with type II cysts and 25 healthy women were evaluated. Serum IGF-I, IGFBP-3 and IGFBP-1 concentrations were measured by IRMA. RESULTS: IGF-I concentrations were significantly higher in sera from patients with type I cysts than in patients with type II cysts. A highly significant decrease of IGFBP-3, the major IGFBP, was found in patients with type I cysts with respect to healthy women, whereas no significant difference was evident between the different cyst types. The IGF-I/IGFBP-3 ratio, an estimate of biologically active IGF-I, was very significantly higher in patients with type I cysts than in both type II patients and healthy women. IGFBP-1 levels were significantly lower in patients with type I than in controls and type II cysts. The IGF-I/IGFBP-1 ratio was significantly higher in patients with type I cysts than in type II bearers and healthy women. Estrogen levels correlated with IGF-I in patients and controls. CONCLUSIONS: The enhanced levels of IGF-I/IGFBP-3 found in patients with type I cysts could eventually be associated with the increased risk of breast cancer described for this group.     

Effect of recombinant porcine IGF-binding protein-3 on proliferation of embryonic porcine myogenic cell cultures in the presence and absence of IGF-I

IGF-binding protein (IGFBP)-3 is produced by cultured porcine embryonic myogenic cell (PEMC) cultures and is secreted into the medium. Levels of secreted IGFBP-3 and IGFBP-3 mRNA are significantly reduced during differentiation and increase after differentiation is complete, suggesting that IGFBP-3 may play some role in myogenesis and/or in changes in myogenic cell proliferation that accompany differentiation. IGFBP-3 reportedly may either suppress or stimulate proliferation of cultured cells depending on cell type. Additionally, IGFBP-3 has been shown to affect proliferation via both IGF-dependent and IGF-independent mechanisms in some cell types but not all. Currently, the effect, if any, of IGFBP-3 on myogenic cell proliferation is not known. Consequently, the goal of this study was to assess the IGF-I-dependent and IGF-I-independent actions of recombinant porcine IGFBP-3 on proliferation of cultured porcine myogenic cells. To facilitate these investigations, we have expressed porcine IGFBP-3 in the baculovirus system, purified and characterized the expressed recombinant porcine IGFBP-3 (rpIGFBP-3), and produced and characterized an anti-porcine IGFBP-3 antibody that neutralizes the biological activity of porcine IGFBP-3. rpIGFBP-3 suppressed IGF-I-stimulated proliferation of PEMCs in a concentration-dependent manner with equimolar concentrations of IGF-I and rpIGFBP-3, resulting in complete suppression of IGF-I-stimulated proliferation. rpIGFBP-3 also suppressed Long-R3-IGF-I-stimulated proliferation of PEMC, indicating that rpIGFBP-3 possesses IGF-independent activity in this cell system. These data have established that IGFBP-3 has the potential to affect proliferation of PEMCs during critical periods of muscle development that may impact ultimate muscle mass achievable postnatally.     

Diagnostic efficiency of serum IGF-I, IGF-binding protein-3 (IGFBP-3), IGF-I/IGFBP-3 molar ratio and urinary GH measurements in the diagnosis of adult GH deficiency: importance of an appropriate reference population

OBJECTIVE: To analyse the diagnostic role of serum IGF-I, IGF-binding protein-3 (IGFBP-3), IGF-I/IGFBP-3 molar ratio and urinary GH (uGH) excretion in adult GH deficiency (GHD). DESIGN: Twenty-seven adults (age range: 18-71 years) with severe GHD, defined by a peak GH response to an insulin tolerance test below 3microg/l in patients with at least one additional pituitary hypofunction. Reference values were established from a selected age- and body mass index-matched population (154 healthy adults grouped in four age groups). METHODS: IGF-I and IGFBP-3 were measured by RIA (Nichols) and results expressed as standard deviation (s.d.) scores from our reference population and assay normative data (s.d. score Nichols). uGH was measured by IRMA. RESULTS: Within the control group, IGF-I, IGFBP-3, IGF-I/IGFBP-3 ratio standardisation regarding our control population and IGF-I with respect to the assay normative data resulted in disappearance of age-related differences. However, IGFBP-3 s.d. score Nichols resulted in mean values between +1.4 and +2.5 s.d. score. Greatest diagnostic efficiency was for IGF-I standardised with respect to our controls (97.2%), followed by s.d. score IGFBP-3 (92.9%). s.d. score IGF/IGFBP-3 ratio and uGH showed poor diagnostic efficiency. Any combination of at least two abnormal parameters raised specificity to 100%. IGF-I standardised with respect to assay reference (s.d. score Nichols) showed similar diagnostic value (95.0%) whereas IGFBP-3 showed low sensitivity (33. 3%). Within the GHD patients, those with three or more additional deficiencies had lower s.d. score IGF-I than those with only two or one. CONCLUSION: We underline the importance of an appropriate reference population for correct interpretation of GH secretion markers. Considering our results, specificity obtained with two simultaneous abnormal parameters when referred to an adequate reference population may add valuable information to alternative GH stimulation tests to confirm adult GHD.     

Production of recombinant porcine IGF-binding protein-5 and its effect on proliferation of porcine embryonic myoblast cultures in the presence and absence of IGF-I and Long-R3-IGF-I

IGF-binding protein-5 (IGFBP-5) is produced by porcine embryonic myogenic cell (PEMC) cultures and is secreted into the medium. IGFBP-5 may play some role in myogenesis and/or in changes in myogenic cell proliferation that accompany differentiation. IGFBP-5 reportedly may either suppress or stimulate proliferation or differentiation of cultured cells depending on cell type and culture conditions. Additionally, IGFBP-5 has been shown to possess both IGF-dependent and IGF-independent actions in some cell types. The goal of this study was to produce recombinant porcine IGFBP-5 (rpIGFBP-5) and assess its IGF-I-dependent and IGF-I-independent actions on the proliferation of PEMCs. To accomplish this, we have expressed porcine IGFBP-5 in the baculovirus system, purified and characterized the expressed rpIGFBP-5 and produced an anti-porcine IGFBP-5 antibody that neutralizes the biological activity of porcine IGFBP-5. rpIGFBP-5, purified to 98% homogeneity using nickel affinity chromatography and IGF-I affinity chromatography, suppressed IGF-I-stimulated proliferation of PEMCs in a concentration-dependent manner (P>0.05). rpIGFBP-5 also suppressed Long-R3-IGF-I-stimulated proliferation of PEMCs (P>0.05), even in the presence of significant molar excess of Long-R3-IGF-I compared with rpIGFBP-5, demonstrating the IGF-independent activity that rpIGFBP-5 possesses in PEMCs, since Long-R3-IGF-I is an IGF analog that has very low affinity for the IGFBPs but retains its ability to bind to the type I IGF receptor and thereby can stimulate proliferation. The anti-rpIGFBP-5 IgY produced against rpIGFBP-5 specifically recognized native porcine IGFBP-5 in PEMC media that also contained porcine IGFBP-2, -3, and -4. This antibody is capable of neutralizing the effects of both rpIGFBP-5 and endogenously produced porcine IGFBP-5 on PEMCs as well as detecting IGFBP-5 in Western blots. The production of rpIGFBP-5 and a neutralizing antibody to porcine IGFBP-5 provides a powerful tool to investigate the role of IGFBP-5 in porcine myogenic cell proliferation and differentiation. The data provided here demonstrated that IGFBP-5 has the potential to affect proliferation of PEMCs during critical periods of in vitro muscle cell development and therefore may impact the capacity for ultimate postnatal muscle mass development in vivo.     

Smallness for gestational age is associated with persistent change in insulin-like growth factor I (IGF-I) and the ratio of IGF-I/IGF-binding protein-3 in adulthood

CONTEXT: Implication of the IGF-IGF-binding protein (IGFBP) axis in the development of metabolic and cardiovascular diseases has been well documented. It has also been shown that an adverse intrauterine environment alters the IGF-IGFBP axis during childhood. OBJECTIVE: The objective of this study was to investigate whether these alterations persist into adulthood. DESIGN AND METHODS: Fasting serum IGF-I, IGFBP-3, and insulin concentrations were measured, and their determinants were analyzed in a cohort of young adult subjects (22 yr of age) born either small (SGA; n = 461) or appropriate (AGA; n = 568) for gestational age. RESULTS: In adulthood, subjects born SGA had significantly lower mean serum IGF-I (320 +/- 137 vs. 348 +/- 143 microg/liter; P = 0.0015), IGFBP-3 (4700 +/- 700 vs. 4800 +/- 800 microg/liter; P = 0.04), and IGF-I/IGFBP-3 ratio (0.067 +/- 0.026 vs. 0.072 +/- 0.025; P = 0.01) than those born AGA. The fasting IGF-I concentration and the IGF-I/IGFBP-3 ratio were significantly inversely associated with age, body mass index, smoking, and oral contraception and were positively related to birth weight and fasting insulin levels. The IGFBP-3 concentration was significantly negatively correlated to age and smoking and was positively related to insulin concentration and oral contraception. After adjustment for age, height, body mass index, gender, smoking, and oral contraception, the mean IGF-I concentration and the mean IGF-I/IGFBP-3 ratio remained significantly lower in the SGA compared with the AGA group (P = 0.003 and P = 0.01, respectively). CONCLUSIONS: Serum IGF-I concentrations and the IGF-I/IGFBP-3 ratio are lower in adult subjects born SGA. Although the origin of this persisting alteration of the IGF-IGFBP axis in adulthood needs to be elucidated, its potential contribution to the long-term metabolic and cardiovascular complications associated with fetal growth restriction is important to consider in the future.     

Body mass index, circulating levels of sex-steroid hormones, IGF-I and IGF-binding protein-3: a cross-sectional study in healthy women

OBJECTIVE: Excess weight has been associated with increased risk of cancer at several organ sites. In part, this effect may be modulated through alterations in the metabolism of sex steroids and IGF-I related peptides. The objectives of the study were to examine the association of body mass index (BMI) with circulating androgens (testosterone, androstenedione and dehydroepiandrosterone sulfate (DHEAS)), estrogens (estrone and estradiol), sex hormone-binding globulin (SHBG), IGF-I and IGF-binding protein (IGFBP)-3, and the relationship between sex steroids, IGF-I and IGFBP-3. DESIGN AND METHODS: A cross-sectional analysis was performed using hormonal and questionnaire data of 620 healthy women (177 pre- and 443 post-menopausal). The laboratory measurements of the hormones of interest were available from two previous case-control studies on endogenous hormones and cancer risk. RESULTS: In the pre-menopausal group, BMI was not related to androgens and IGF-I. In the post-menopausal group, estrogens, testosterone and androstenedione increased with increasing BMI. The association with IGF-I was non-linear, with the highest mean concentrations observed in women with BMI between 24 and 25. In both pre- and post-menopausal subjects, IGFBP-3 did not vary across BMI categories and SHBG decreased with increasing BMI. As for the correlations between peptide and steroid hormones, in the post-menopausal group, IGF-I was positively related to androgens, inversely correlated with SHBG, and not correlated with estrogens. In the pre-menopausal group, similar but weaker correlations between IGF-I and androgens were observed. CONCLUSIONS: These observations offer evidence that obesity may influence the levels of endogenous sex-steroid and IGF-related hormones in the circulation, especially after menopause. Circulating IGF-I, androgens and SHBG appear to be related to each other in post-menopausal women.     

IGF-I and IGF-binding protein-3 measurements on filter paper blood spots in children and adolescents on GH treatment: use in monitoring and as markers of growth performance

BACKGROUND/AIM: In childhood an appropriate response to GH treatment is achieved by titration of growth response against dose administered, with careful observation for side-effects. In order to evaluate the potential use of IGF monitoring in children treated with GH, a cross-sectional study has been carried in 215 children and adolescents (134 with GH deficiency (GHD), 54 with Turner syndrome (TS) and 27 with non-GHD growth disorders) treated with GH for 0.2-13.7 years. METHODS: IGF-I and IGF-binding protein-3 (IGFBP-3) were measured in ELISAs, using dried capillary blood collected onto filter papers. Results were expressed as the mean S.D. range (SDS). Values of either analyte < -2 or > +2 SDS were considered abnormal. RESULTS: IGF-I and IGFBP-3 SDS were higher in the TS and non-GHD groups (mean +0.01 and +0.1 respectively) than in those with GHD (mean value -0.6). Nineteen per cent of the IGF-I values (13% low, 6% high) and 12% of IGFBP-3 values were abnormal (10% low, 2% high). Abnormalities, either low or high, were most common in the GHD group. There was a weak but significant relationship between change in height SDS over the Year up to the time of sampling in the whole group and IGF-I SDS. Satisfactory growth performance (+0.5>change in height SDS> -0.5) was found in those with high (7.2%), normal (60%) and low (9.3%) IGF-I levels. Overall, it was estimated that 26% of the tests would indicate that an adjustment to GH dose (up in 18% and down in 8%) could be considered. CONCLUSIONS: From this cross-sectional study of IGF monitoring across a broad range of diagnoses and ages, it can be concluded that the majority of children on GH have normal levels of IGF-I and IGFBP-3, but 26% of tests could suggest that a change of GH dose should be considered. Regular monitoring of IGF-I and IGFBP-3 should be considered in any child on GH treatment.     

Changes in bioactive IGF-I and IGF-binding protein-1 during an oral glucose tolerance test in patients with liver cirrhosis

OBJECTIVE: Liver cirrhosis is characterized by reduced circulating IGF-I and this has been linked to an adverse clinical outcome. Therefore, we investigated the dynamic changes in circulating total, free, and bioactive IGF-I, IGF-binding protein (IGFBP)-1, IGFBP-2, and IGFBP-1-bound IGF-I (binary complex) during an oral glucose tolerance test (OGTT) in patients with liver cirrhosis. METHODS: Seven Caucasian males with liver cirrhosis and seven healthy males matched for age (54.4+/-3.2 vs 54.6+/-4.4 years) and body mass index (25.3+/-1.2 vs 25.9+/-1.3 kg/m2) were studied. Blood samples were drawn at 0, 30, 60, 90, 120, 150, and 180 min for determination of serum total and free IGF-I, IGFBP-1, IGFBP-2, and binary complex, while bioactive IGF-I was measured at 0, 30, 60, 120, and 180 min. RESULTS: In comparison with healthy subjects, baseline levels of total (47%), free (36%), and bioactive IGF-I (51%) were lower, while IGFBP-1 (268%) was higher (P<0.05), IGFBP-2 (172%) tended to be higher (P>0.05), and the binary complex unchanged (approximately 100%) in cirrhotic patients. Serum total and free IGF-I, and IGFBP-2 remained unchanged in both study groups during the OGTT. Bioactive IGF-I decreased by 29% from baseline to 60 min in cirrhotic patients and remained low at the end of the OGTT (P<0.05). A similar tendency was observed in healthy controls (P=0.052). Concomitantly, IGFBP-1, binary complex, and IGFBP-1 saturation index decreased significantly in both groups. The disappearance of the binary complex was about twofold faster than that of IGFBP-1 (P < 0.05). CONCLUSION: Despite unchanged concentrations of total and free IGF-I, bioactive IGF-I declined significantly after an oral glucose load in patients with liver cirrhosis and the same tendency was observed in healthy subjects. We speculate that the reduction in bioactive IGF-I may be related to the higher levels of free IGFBP-1 and the faster disappearance of IGFBP-1-bound IGF-I.     

Regulation of insulin-like growth factor (IGF)-I action by matrix metalloproteinase-3 involves selective disruption of IGF-I/IGF-binding protein-3 complexes

IGF-I and IGF-II play important roles in growth and development via interactions with cell-surface receptors; however, in nature, IGFs are sequestered by at least six soluble, high-affinity IGF-binding proteins (IGFBPs), namely IGFBPs 1-6. Herein, we demonstrate that the stromal cell-derived extracellular matrix-degrading metalloproteinase stromelysin 1 (matrix metalloproteinase 3) disrupts IGF/IGFBP-3 complexes and liberates free, intact IGFs, leading to phosphorylation of cell surface type 1 IGF receptors and cellular proliferation. Tissue inhibitor of metalloproteinases (TIMP-1) or an antibody to the type 1 IGF receptor mitigates IGF-mediated cellular proliferation. Thus, these studies suggest that matrix metalloproteinases, beyond their effects on extracellular matrix turnover, regulate cellular proliferation by modulating the bioavailability of IGFs, an event critical for such diverse phenomena as embryo development, morphogenesis, angiogenesis, and tumorigenesis.     

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.     

Arthritis-induced increase in serum levels of IGF-binding protein-3 in rats is secondary to the decrease in its proteolytic activity

Adjuvant-induced arthritis is a chronic inflammatory illness that induces a catabolic state, with a decrease in pituitary GH and hepatic IGF-I synthesis. We have previously observed an increase in serum IGF-binding protein-3 (IGFBP-3) in arthritic rats, and found that GH administration prevents the increase in circulating IGFBP-3 in arthritic rats. The aim of this work was therefore to study IGFBP-3 synthesis in the liver as well as its proteolysis in serum as the two possible causes of the increased circulating IGFBP-3 in arthritic rats. The effect of recombinant human GH (rhGH) administration was also analysed. Adult male Wistar rats were injected with complete Freund's adjuvant or vehicle, and 14 days later they were injected s.c. daily until day 22 after adjuvant injection with rhGH (3 IU/kg) or saline. Three hours after the last GH injection, all rats were killed by decapitation. Arthritis increased serum IGFBP-3 levels (P<0.01). The increase in serum IGFBP-3 levels in arthritic rats seems to be due to decreased proteolysis (P<0.01) rather than to an increased synthesis, since liver IGFBP-3 mRNA content was not modified by arthritis. GH administration to control rats resulted in an increase in both hepatic IGFBP-3 mRNA content and in serum IGFBP-3 levels in spite of the increase in IGFBP-3 proteolysis in serum. In arthritic rats, GH treatment did not modify liver IGFBP-3 synthesis, but it increased serum proteolysis of IGFBP-3, leading to a serum concentration of IGFBP-3 similar to that of control rats. Furthermore, there was a negative correlation between circulating IGFBP-3 and its proteolytic activity in the serum of adjuvant-induced arthritic rats. These data suggest that in chronic arthritis the increase in IGFBP-3 serum concentration is secondary to a decrease in proteolytic activity, rather than to an increase in hepatic IGFBP-3 gene expression.     

Development and clinical evaluation of a novel immunoassay for the binary complex of IGF-I and IGF-binding protein-1 in human serum.

Correlation studies have suggested that IGF-binding protein (IGFBP)-1 is a dynamic regulator of free IGF-I. To further study this, we developed a monoclonal immunofluorometric assay specific for the binary complex of IGF-I and IGFBP-1 in human serum. An IGFBP-1 antibody, which recognizes all phospho-forms of IGFBP-1, was used for coating. An europium-labeled IGF-I antibody served as tracer. Assay incubation was performed at conditions approaching those in vivo (i.e. pH 7.4, 37 C). The assay was highly specific: no signal was obtained unless both IGF-I and IGFBP-1 were present and neither IGFBP-2, -3, -4, nor IGF-II caused any cross-reaction. The linear standard curve covered 3 orders of magnitude, and within and in-between assay coefficients of variation were less than 5 and 15%, respectively. To study the dynamic relationship between free IGF-I and binary complex formation, seven healthy subjects were fasted for 72 h. Samples were collected every 3 h. During fasting, free IGF-I was reduced by two thirds (P < 0.0001). IGFBP-1 and the binary complex increased in parallel (P < 0.0001), and levels correlated positively in all subjects (0.89 < or = r < or = 0.98; P < 0.0001). Free IGF-I correlated inversely with IGFBP-1 (-0.81 < or = r < or = -0.48; 0.0001 < or = P < or = 0.05) and the binary complex (-0.79 < or = r < or = -0.41; 0.0001 < or = P < or = 0.05). To study overnight fasting levels, we compared healthy controls and patients with type 1 diabetes and chronic renal failure (n = 10), because these patients show profound alterations in their IGF-system. In both groups, the binary complex was increased about 2.5-fold (P < 0.0001), whereas IGFBP-1 was increased by 5- to 6-fold (P < 0.0001). Accordingly, free IGF-I was severely reduced (P < 0.0001). In conclusion, the assay enables us to study the role of IGFBP-1 as a dynamic regulator of free IGF-I. Our results clearly show that IGFBP-1 and free IGF-I are tightly associated peptides. Furthermore, it has now become possible to compare levels of IGF-I carried within the binary complex IGFBP-1:IGF-I in different (patho-) physiological conditions.     

Inactivation of Kupffer cells by gadolinium administration prevents lipopolysaccharide-induced decrease in liver insulin-like growth factor-I and IGF-binding protein-3 gene expression

Gram-negative bacterial infection or treatment of animals with bacterial lipopolysaccharide (LPS) induces a catabolic state with proteolysis, liver injury and an inhibition of the insulin-like growth factor-I (IGF-I) system. The purpose of this work was to elucidate the role of Kupffer cells in LPS-induced inhibition of the IGF-I/IGF-binding protein-3 (IGFBP-3) system. Adult male Wistar rats were either pretreated with the Kupffer cell inhibitor gadolinium chloride (10 mg/kg, i.v., 24 h prior to LPS exposure) or saline vehicle. Rats received two i.p. injections of 1 mg/kg LPS (at 17:30 and 08:30 h the following day) and were killed 4 h after the second injection. LPS administration induced a significant decrease in body weight and in serum concentrations of IGF-I and IGFBP-3 (P < 0.01), as well as in their gene expression in the liver. LPS-injected rats had increased serum concentrations of ACTH, corticosterone (P < 0.05), tumour necrosis factor-alpha (TNF-alpha) and nitrites (P < 0.01). Pretreatment of the animals with gadolinium chloride blocked the inhibitory effect of LPS on body weight, and on serum concentrations of IGF-I, IGFBP-3 and nitrites, as well as growth hormone receptor (GHR), IGF-I and IGFBP-3 gene expression in the liver. In contrast, gadolinium chloride administration did not modify the stimulatory effect of LPS on serum concentrations of ACTH, corticosterone and TNF-alpha. These results suggest that Kupffer cells are important mediators in the inhibitory effect of LPS on GHR, IGF-I and IGFBP-3 gene expression in the liver, leading to a decrease in serum concentrations of IGF-I and IGFBP-3.     

Glucocorticoids are not necessary for the inhibitory effect of endotoxic shock on serum IGF-I and hepatic IGF-I mRNA

The aim of this work was to elucidate the possible role of glucocorticoids in the bacterial lipopolysaccharide (LPS)-induced decrease in hepatic IGF-I synthesis. For this purpose, we studied the effect of LPS on IGF-I in two rat strains, Wistar and Lewis, which have different adrenal responses to inflammation. Compared with Wistar rats, Lewis rats have a reduced hypothalamic-pituitary-adrenal response to inflammatory stimuli. Rats received two i.p. injections of 1 mg/kg LPS and were killed 4 h after the second injection. LPS induced an increase in serum concentrations of both ACTH and corticosterone, the increase being more pronounced in Wistar than in Lewis rats. LPS decreased hepatic GH receptor (GHR) and IGF-I mRNA only in Wistar rats. However, serum concentrations of IGF-I were significantly decreased (P<0.01) in both Wistar and Lewis rats. These data indicate that the adrenal axis may mediate the inhibitory effect of LPS on GHR and IGF-I synthesis in the liver. In a second experiment, adrenalectomized or sham-operated Wistar rats were injected with LPS. Two LPS injections (0.1 mg/kg) decreased serum concentrations of IGF-I in both type of rat; however, the inhibitory effect of LPS on liver GHR and IGF-I mRNA was observed in adrenalectomized rats, but not in intact rats. All these data suggest that some component of the adrenal axis, other than glucocorticoids, mediates the inhibitory effect of LPS on liver GHR and IGF-I.     

Differential expression of IGF-binding protein-3 in normal and malignant colon and its influence on apoptosis

IGF-binding protein-3 (IGFBP-3) has been reported to exert a protective influence on the pathogenesis of colorectal cancer. This may reflect its modulation of IGF-I bioactivity as well as IGF-I-independent effects on cell proliferation and apoptosis. Although local expression of IGF-I in the colon is increasingly recognised as having important regulatory consequences, the role of locally expressed IGFBP-3 remains unknown. The aims of the present study were: (i) to quantify and localise the expression of IGFBP-3 in human normal and malignant colon; (ii) to relate this expression to that of other components of the IGF-I axis; and (iii) to investigate the effects of IGFBP-3 on colonic epithelial cell proliferation and apoptosis. RNA was extracted from 46 paired samples of normal and malignant colonic tissue. IGFBP-3, IGF-I, IGF-I receptor and GH receptor mRNA levels were quantified using real-time RT-PCR. Laser-capture microdissection of the same samples was used to isolate mRNA from epithelium and stromal components and localise mRNA expression. Expression was confirmed at a protein level by immunohistochemistry. Human colorectal cancer HT-29 and CaCo-2 cells were cultured with IGFBP-3 (200 ng/ml), +/- IGF-I (20 ng/ml), +/- sodium butyrate (5 mM). Cell number was assessed by an MTS assay (a modification of the MTT assay), and apoptosis assessed by cell morphology and FACS analysis using both annexin and propidium iodide staining. UO146, a MAP kinase inhibitor, and wortmannin, an inhibitor of the phosphatidylinositol 3-kinase (PI-3K) pathway, were used to determine the contribution of these signalling pathways on the effects of IGFBP-3. IGFBP-3 mRNA was detected in all samples (mean copy number/mug total RNA in normal colon, 2.6 x 10(6) compared with 1.3 x 10(7) in the cancers; P < 0.0001). Immunohistochemistry confirmed the expression and showed it to be equally distributed between epithelial and stromal components in normal tissue, but to be mainly restricted to the stromal component of malignant tissue. This differential expression was confirmed by RT-PCR of RNA from laser-capture microdissected samples. IGF-I mRNA was detected in 31 samples of normal colon; mean IGFBP-3 copy number was higher in the IGF-I-positive samples compared with IGF-I-negative samples. IGFBP-3 on its own induced apoptosis in HT-29 cells (P < 0.001). Co-incubation of 200 ng/ml IGFBP-3 with butyrate (5 mM) resulted in the potentiation of its apoptosis (P < 0.0001), which was not rescued by co-incubation with IGF-I (P < 0.0001). The addition of UO126 caused a decrease in cell number and increased the effects of IGFBP-3. IGFBP-3 is differentially expressed between stromal and epithelial components of normal and malignant colon, which may reflect its pro-apoptotic, IGF-I-independent effect on colonic epithelial cells. These effects are mediated in part by the PI-3K pathway in contrast to the MAP kinase pathway used by IGF-I.