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.     

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.     

The circulating IGF system and its relationship with 24-h glucose regulation and insulin sensitivity in healthy subjects

OBJECTIVE AND DESIGN: It has been suggested that circulating free IGF-I participates in glucose homeostasis and that IGFBP-1 reflects changes in insulin sensitivity. To study this further, we examined 10 healthy, nonobese subjects under standardized conditions for 24 h with and without an intravenous infusion of glucose, the latter in order to augment insulin sensitivity. Serum was collected every 2 h for analysis of free and total IGFs, IGFBP-1, - 2 and - 3 and the acid labile subunit (ALS). Insulin sensitivity was estimated at the end of each 24-h study period by use of the hyperinsulinaemic euglycaemic clamp technique. RESULTS: Glucose infusion resulted in mild hyperglycaemia (P < 0.0001), a reduction in IGFBP-1 by approximately 40% (P < 0.0003), and increased insulin and C-peptide levels (P < 0.0001). Glucose infusion also increased insulin sensitivity (P < 0.003). However, despite the reduction in IGFBP-1, glucose infusion did not increase free IGF-I over the control level, and free IGF-II was slightly reduced (P < 0.02). Irrespective of glucose infusion, free IGF-I and -II remained stable during daytime (i.e. they were unresponsive to meal-related changes in plasma glucose), but both free fractions decreased during the night, reaching nadir at 04.00 h. None of the other members of the IGF system showed any relationship with plasma glucose levels. Finally, we failed to observe any relationship between changes in insulin sensitivity and the circulating IGF system. CONCLUSION: We found no evidence that the circulating IGF system is involved in meal-related blood glucose regulation or that it reflects short-term changes in insulin sensitivity in healthy, nonobese subjects. However, we cannot preclude that the observed changes in circulating IGFBP-1 may affect the glucose-lowering effect of IGF-I and -II at the local tissue level.     

Paradoxical elevations in serum IGF-II and IGF binding protein-2 in acromegaly: insights into the regulation of these peptides

OBJECTIVE: Circulating insulin-like growth factor (IGF)-II and IGF binding protein-2 (IGFBP-2) are frequently altered, often in parallel, in numerous pathologies including neoplastic disease but little is known about their normal regulation. This study compared serum IGF-II and IGFBP-2 distributions between acromegalics and a large normal adult population to explore possible determinants. PATIENTS: Sixty acromegalic patients undergoing screening colonoscopy (age range 25-81 years); normative data from 306 healthy adults (age range 20-89 years). MEASUREMENTS: Serum IGF-I, IGF-II, IGFBP-2 and IGFBP-3 were measured in healthy adults and acromegalics. Mean growth hormone (GH) levels were obtained for acromegalic patients. Differences were compared using t-tests (unadjusted) and multiple regression models (adjusted for age and gender). Correlations were expressed as Pearson's coefficient (r). RESULTS: For acromegalic patients, GH was significantly correlated with IGF-I (r = 0.50; P < 0.001) and IGFBP-3 (r = 0.29; P = 0.03) but not IGF-II or IGFBP-2. Contrary to expectations, mean IGF-II and IGFBP-2 levels were significantly raised in the acromegalics compared with normals [adjusted mean difference (95% CI) = 226 (181, 271) microg/l and 305 (200, 410) microg/l, respectively]. Ten acromegalic patients had colorectal neoplasia but their presence did not contribute to the elevations in serum IGF-II and IGFBP-2. The (IGF-I + IGF-II)/IGFBP-3 molar ratios were remarkably constant in both healthy adults and acromegalics, but the relationships of the ligands individually with IGFBP-3 were not linear: as IGFBP-3 increased, IGF-I also increased whereas IGF-II initially increased but then decreased. IGFBP-2 did not correlate with IGF-II, but molar concentration significantly correlated with the IGF-II/IGFBP-3 molar ratio (r = 0.40; P = 0.001). CONCLUSIONS: Serum IGF-II and IGFBP-2 levels were paradoxically elevated in acromegalics, independent of the presence of colorectal neoplasia. The (IGF-I + IGF-II)/IGFBP-3 molar ratio appears to be pivotal in determining IGF-II values, which, in turn, expressed as a ratio of IGFBP-3, is related to IGFBP-2. These observations offer new insights into the regulation of these peptides.     

Insulin-like growth factor (IGF) I, -II, and IGF binding protein-3 and risk of ischemic stroke

BACKGROUND: Low IGF-I levels may be associated with the development of stroke; however, prospective data appear to be unavailable. METHODS: This was a nested case-control study within a Danish follow-up study, including 57,053 men and women. Baseline data included circulating IGF-I, IGF-II, and IGF binding protein (IGFBP)-3 concentrations as well as lifestyle factors and medical history. We identified 254 cases with incident ischemic stroke and 254 gender- and age-matched controls. RESULTS: Participants in the bottom quartiles of IGF-I and IGFBP-3 levels (median concentrations, 72 and 2937 ng/ml, respectively) were at increased risk of ischemic stroke, e.g. adjusted odds ratios (ORs) of 2.06 [95% confidence interval (CI), 1.05-4.03] and 2.29 (95% CI, 1.17-4.49), respectively, when compared with participants in the top quartiles (median concentrations, 125 and 4835 ng/ml, respectively). A negative, although weaker, association was also found for IGF-II (adjusted OR 1.44, 95% CI 0.79-2.64) when comparing the bottom quartile with the top quartile. No substantial associations were seen for IGF-I and IGF-II when also adjusting for IGFBP-3; adjusting IGFBP-3 for IGF-I and -II had only a minor impact on the risk estimates. CONCLUSION: These findings give some support to the hypothesis that the IGF axis is involved in the pathogenesis of ischemic stroke.     

Size at birth and cord blood levels of insulin, insulin-like growth factor I (IGF-I), IGF-II, IGF-binding protein-1 (IGFBP-1), IGFBP-3, and the soluble IGF-II/mannose-6-phosphate receptor in term human infants. The ALSPAC Study Team. Avon Longitudinal Study of Pregnancy and Childhood

Experimental rodent studies demonstrate that insulin-like growth factor II (IGF-II) promotes fetal growth, whereas the nonsignaling IGF-II receptor (IGF2R) is inhibitory; in humans their influence is as yet unclear. A soluble, circulating form of IGF2R inhibits IGF-II mediated DNA synthesis and may therefore restrain fetal growth. We measured cord blood levels of IGF-II, soluble IGF2R, insulin, IGF-I, IGF-binding protein-1 (IGFBP-1), and IGFBP-3 and examined their relationships to weight, length, head circumference, ponderal index, and placental weight at birth in 199 normal term singletons. IGF-II levels correlated with levels of IGF-I (r = 0.29; P < 0.0005), IGFBP-3 (r = 0.45; P < 0.0005), and soluble IGF2R (r = 0.20; P < 0.005). Insulin and IGF-I were positively related to all parameters of size at birth. IGF-II was weakly related to ponderal index (r = 0.18; P < 0.05) and placental weight (r = 0.18; P < 0.05), and the molar ratio of IGF-II to IGF2R was also related to birth weight (r = 0.15; P < 0.05). Correlations between the IGFs and size at birth were stronger in nonprimiparous pregnancies; in these, IGF-I (r = 0.52; P < 0.0005), IGFBP-3 (r = 0.41; P < 0.0005), and the IGF-II to IGF2R ratio (r = 0.40; P < 0.0005) were most closely related to placental weight, together accounting for 39% of its variance. We demonstrate for the first time relationships between circulating IGF-II and soluble IGF2R levels and size at birth, supporting their putative opposing roles in human fetal growth.     

Free and total insulin-like growth factor (IGF)-I, -II,and IGF binding protein-1, -2, and -3 serum levels in patients with active thyroid eye disease

To determine whether serum levels of total (T) and free (F) IGF-I and -II and IGF binding protein (IGFBP),-1, -2, and -3 are normal in euthyroid patients with Graves' disease and active thyroid ophthalmopathy, we investigated the above-mentioned parameters in 21 patients (11 male, 10 female) aged 50.8 +/- 11.8 yr (range 35-70) and 19 healthy individuals matched for age, gender, and body mass index. All patients had active thyroid eye disease (TED) with clinical activity scores > or = 4 and positive orbital octreoscan in both eyes. Serum T and F IGF-I and IGF-II were determined using noncompetitive time-resolved monoclonal immunofluorometric assays, IGFBP-1 was determined by an in-house RIA, IGFBP-2 by a novel in-house time-resolved immunofluorometric assay, whereas IGFBP-3 by an immunoradiometric assay. All data are expressed as mean +/- SD. Our results show that T and F IGF-I, -II, and IGFBP-1, -2, and -3 levels in patients were similar to controls and did not show any significant difference. Specifically, mean T IGF-I for patients group was 131 (61), F IGF-I was 0.47 (0.16), T IGF-II was 1056 (300), F IGF-II was 1.45 (0.54), IGFBP-1 was 33 (14), IGFBP-2 was 848 (377), and finally IGFBP-3 was 3953 (1422). For controls, mean T IGF-I was 146 (51), F IGF-I was 0.85 (0.43), T IGF-II was 939 (197), F IGF-II was 1.53 (0.53), IGFBP-1 was 44 (24), IGFBP-2 was 764 (316) and finally IGFBP-3 was 3721 (1017). Furthermore, no statistically differences emerged in the ratio between molar weights of T IGF-I/IGFBP-3 and T IGF-II/IGFBP-3, as well as to the F/T IGF-I and F/T IGF-II. Finally, no relationship was found between the levels of the above-mentioned parameters and clinical activity scores, octreoscan scores, and thyroid hormones. Our data demonstrate for the first time that serum levels of IGFs (including free fractions) and IGFBPs are not increased in euthyroid Graves' patients with active TED. The increased IGF levels in retrobulbar tissues previously described, appear to be independent of serum IGFs concentration and probably represent autocrine and/or paracrine activity.     

Interaction of insulin-like growth factor (IGF)-I and -II with IGF binding protein-2: mapping the binding surfaces by nuclear magnetic resonance

The interaction of IGF binding protein-2 (IGFBP-2) with IGF-I and -II has been investigated in solution using nuclear magnetic resonance (NMR) spectroscopy. Chemical shift perturbations in 15N- and 2H/15N-labelled IGF-I or -II upon binding to unlabelled thioredoxin-tagged bovine IGFBP-2 (Trx(1-279)IGFBP-2) have been monitored to identify residues involved directly in the binding interaction as well as any affected by conformational changes associated with the interaction. A key step in obtaining high-quality spectra of the complexes was the use of transverse relaxation optimised spectroscopy (TROSY) methods with partially deuterated ligands. Indeed, because the effects of conformational averaging and aggregation are eliminated in IGF-I and -II bound to IGFBP-2, the spectra of the complexes are actually superior to those of the free ligands. Comparison of our results with the crystal structure of the complex between IGF-I and an N-terminal fragment of IGFBP-5 allowed identification of those residues perturbed by the C-domain of IGFBP-2. Other perturbations, such as those of Gly 19 and Asp 20 of IGF-I (and the corresponding residues in IGF-II) - which are located in a reverse turn linking N-domain and C-domain interactive surfaces - are due to local conformational changes in the IGF-I and -II. Our results confirm that the C-domain of IGFBP-2 plays a key role in binding regions of IGF-I and -II that are also involved in binding to the type-1 IGF receptor and thereby blocking ligand binding to this receptor.     

The relationship among circulating insulin-like growth factor components, biochemical markers of bone turnover and bone mineral density in postmenopausal women under the age of 60

OBJECTIVES: The changes in circulating IGF components after the menopause and the potential role of new markers of bone turnover and circulating IGF components in predicting bone mass in postmenopausal women are still controversial and the relationship between these two systems has not been investigated. The aims of this study were to investigate the changes in circulating IGF components after the menopause, to evaluate whether new markers of bone turnover and circulating IGF components reflect bone mass in postmenopausal women under the age of 60 and to study the relationship between these two systems. DESIGN, PATIENTS AND MEASUREMENTS: Serum IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3, osteocalcin (OST), bone specific alkaline phosphatase (BAP), urinary deoxypyridinoline (DPYD) and N-telopeptide of type I collagen (NTX) were measured in 31 premenopausal women aged 31-43 and 65 postmenopausal women aged 47-60: this latter group comprised 30 normal healthy women and 35 osteoporotic women. RESULTS: Compared with premenopausal women or normal postmenopausal women, serum IGF-1 and IGFBP-3 levels were significantly lower in osteoporotic postmenopausal women while no significant differences in serum levels of IGF-II, IGFBP-1 and IGFBP-2 were observed. The correlations between bone turnover markers and circulating IGF components (except between serum BAP and IGF-II), and between bone turnover markers and bone mineral density (BMD) in postmenopausal women were not significant. However, serum IGF-I and IGFBP-3 correlated positively with BMD of the lumbar spine and/or Ward's triangle even if age, BMI and menopause duration were taken into account in a multiple regression analysis model. CONCLUSIONS: Circulating IGF-I and IGFBP-3 may be involved in the mechanism of bone loss in postmenopausal women under the age of 60. They may also provide indirect information on the current bone microenvironment different from that provided by new markers of bone turnover.     

The divergent effect of insulin-like growth factor binding protein (IGFBP) - 1 on IGF-induced Steroidogenesis in bovine adrenocortical cells is not due to its phosphorylation status.

In previous studies we have shown that insulin-like growth factor (IGF)-II stimulates basal as well as ACTH-induced cortisol secretion from bovine adrenocortical cells more potently than IGF-I. The steroidogenic effect of both, IGF-I and IGF-II, is mediated through the IGF-I receptor and modified by locally produced IGF binding proteins. Further studies demonstrate that bovine adrenocortical cells do synthesize IGFBP-1 to -4 and that their secretion is regulated differentially by IGFs and ACTH. Since IGFBP-1 selectively is induced 3- to 4- fold by ACTH, the aim of the following studies was to evaluate the effect of IGFBP-1 on IGF-induced cortisol secretion from bovine adrenocortical cells in primary culture. Coincubation of bovine adrenocortical cells with purified IGFBP-1 (30 nM) induced a time--and dose-dependent potentiating effect (38+/-2%) on IGF-I-stimulated (6.5 nM) cortisol-secretion, wheras the IGF-II induced steroidogenic effect was inhibited (40+/-3%) by IGFBP-1. Similar results were found when bovine adrenocortical cells were preincubated with IGFBP-1 before stimulation experiments with IGFs were performed. In order to evaluate the influence of different phosphorylation states of IGFBP-1 on the steroidogenic effect of IGF-I and IGF-II and on the affinity to IGFs, a highly phosphorylated (pIGFBP-1) and a dephosphorylated isoform (dIGFBP-1) of IGFBP-1 have been separated by anion exchange chromatography for further incubation experiments and binding studies. No different effects on IGF-I or IGF-II-induced steroidogenesis were observed when a highly phosphorylated IGFBP-1 isoform was used, compared to the dephosphorylated IGFBP-1 isoforms. In binding studies IGFBP-1 did show high affinity binding for IGF-I with a calculated association constant (K (a)) of 2,17 x 10 (9) M (-1). Similar association constants were calculated for dIGFBP-1 and pIGFBP-1 (1,93 x 10 (9) M (-1) and 2,67 x 10 (9) M (-1) respectively) and no difference in binding capacity was observed when IGF-II was used as ligand. IN CONCLUSION, these results demonstrate that in bovine adrenocortical cells IGFBP-1 time- and dose-dependently inhibits the steroidogenic effect of IGF-II and stimulates the effect of IGF-I. The observed modulatory effect of IGFBP-1 is independent of the mode of incubation and its phosphorylation status. The previously observed stronger steroidogenic potency of IGF-II in bovine adrenocortical cells therefore can not be explained by an interaction with IGFBP-1. The mechanisms by which IGFBP-1 divergently regulates the steroidogenic effect of IGF-I and IGF-II remain unclear at present and further investigation is necessary to elucidate the mechanisms by which IGFBP-1 modulates IGF action in the adult adrenal gland.     

Association between insulin-like growth factor status and physical activity levels in rheumatoid arthritis

OBJECTIVE: To determine if the altered insulin-like growth factor (IGF) status in rheumatoid arthritis (RA) is due to inflammation, altered body composition, or lack of exercise. METHODS: Subjects included 73 patients with RA, 54 patients with other rheumatic diseases, both inflammatory and noninflammatory, and 28 healthy, physically active controls. Serum levels of IGF-I, IGF-II, and IGF binding protein-3 (IGFBP-3) were measured by radioimmunoassay. Body composition was estimated by bioelectrical impedance analysis, and habitual exercise level approximated by questionnaire. Statistical analysis was performed by 2 and 3 way ANOVA and moderated hierarchical regression. RESULTS: Serum IGF-I (p < 0.001), IGFBP-3 (p < 0.001), and the BP-3:total IGF molar ratio (p < 0.001) were depressed in both patient groups relative to controls. In contrast, IGF-II levels were depressed only in patients with RA (p < 0.01). Differences in the IGF proteins between patients and controls could not be attributed to inflammation. Habitual exercise level, but not body composition, was shown to be a significant predictor for IGF-I, IGFBP-3, and BP-3:total IGF molar ratio (p < 0.001). CONCLUSION: Our results indicate that the reduction in circulating IGF proteins observed in our patients is more related to their sedentary lifestyle than to the inflammatory process. This conclusion is in agreement with reports that show that highly active individuals typically exhibit higher levels of systemic IGF proteins than age matched sedentary controls.     

Insulin-like growth factor (IGF)-binding protein-6 inhibits IGF-II-induced but not basal proliferation and adhesion of LIM 1215 colon cancer cells

IGF-II is an autocrine growth factor for many colon cancer cells. This study aimed to determine the role of IGF-II in proliferation and adhesion of LIM 1215 colon cancer cells. RT-PCR demonstrated expression of IGF-I and IGF-II mRNA. Addition of IGF-I or -II increased monolayer proliferation in a dose-dependent manner. Although addition of IGFBP-6 had no effect on basal proliferation, coincubation of IGFBP-6 decreased IGF-II but not IGF-I-induced proliferation. Colony formation in agar was increased by IGF-II, an effect inhibited by coincubation with IGFBP-6. IGFBP-6 alone significantly decreased colony formation. Preincubation of cells with IGF-II increased adhesion to type IV collagen, fibronectin and laminin. IGFBP-6 had no effect on basal cell adhesion but completely inhibited the effects of IGF-II. LIM 1215 colon cancer cells are therefore IGF-responsive but IGF-II is not a major autocrine factor for these cells in monolayer, suggesting heterogeneity between colon carcinoma cell lines with respect to the role of the IGF system.     

Associations of childhood and adulthood height and the components of height with insulin-like growth factor levels in adulthood: a 65-year follow-up of the Boyd Orr cohort

CONTEXT: Taller individuals with longer legs have a higher risk of cancer but a lower risk of coronary heart disease. OBJECTIVE: We investigated whether childhood height and its components are associated with the IGF system in adulthood. DESIGN AND PARTICIPANTS: We analyzed data from 429 participants of the Boyd Orr cohort, for whom height measured in childhood (mean age, 7.4 yr) in 1937-1939 could be related to levels of IGF-I, IGF-II, IGF binding protein (IGFBP)-2, and IGFBP-3 in adulthood (mean age, 71.1 yr). In 385 participants, measured height in adulthood could be related to IGF levels. RESULTS: In fully adjusted models (controlling for age, sex, socioeconomic factors, lifestyle, and body mass index), childhood height and its components were not associated with adult circulating IGF-I, IGF-II, or IGFBP-2 levels. IGFBP-3 was 85.5 ng/ml higher (95% confidence interval, -11.6 to 182.5; P = 0.08) per sd increase in childhood trunk length and 83.6 ng/ml lower (95% confidence interval, -10.3 to 177.5; P = 0.08) per sd increase in childhood leg/trunk ratio. Height in adulthood was not associated with IGF-I, IGF-II, or IGFBP-3 and was inversely associated with IGFBP-2 (P = 0.05) after additionally controlling for childhood height. CONCLUSION: There was no evidence that associations of childhood height with cancer and coronary heart disease risk are mediated by IGF-I in adulthood. The anthropometric associations with IGFBP-2 and IGFBP-3 could be chance findings but warrant additional investigation. IGF levels in childhood may be more important determinants of long-term disease risk than adult levels.     

Clinical significance of serum IGF-Ⅰ, IGF-Ⅱ and IGFBP-3 in liver cirrhosis

AIM: To investigate the relationship between insulin-like growth factor-Ⅰ, -Ⅱ (IGF-Ⅰ and IGF-Ⅱ), IGF-binding protein 3(IGFBP-3) and Child-Pugh score in patients with liver cirrhosis,and to search for potential clinical markers of liver function.METHODS: Forty-four patients with advanced liver cirrhosis of viral origin were divided into 3 groups according to severity of cirrhosis (Child-Pugh score) and 38 healthy subjects served as controls. Serum levels of IGF-Ⅰ, IGF-Ⅱ and IGFBP-3 were measured by immunoradiometric assay.RESULTS: Serum IGF-Ⅰ, IGF-Ⅱ and IGFBP-3 levels were significantly lower in patients with cirrhosis than in controls,and serum concentrations of IGF-Ⅰ, IGF-Ⅱ and IGFBP-3 were associated with the severity of liver dysfunction, and dropped sharply during the progression of liver failure. Among these 3 parameters, serum IGF-Ⅱ was the most sensitive and effective indicator for liver dysfunction. Concentrations of IGF-Ⅰ &lt;30 ng/mL, IGF-Ⅱ &lt;200 ng/mL and IGFBP-3 &lt;6 ng/mL implied a negative prognosis for patients with liver cirrhosis.CONCLUSION: Serum IGF-Ⅰ, IGF-Ⅱ and IGFBP-3 may provide a new dimension in the assessment of liver dysfunction.Combined detection of serum IGF-Ⅰ, IGF-Ⅱ and IGFBP-3 with Child-Pugh score is more effective in predicting prognosis than Child-Pugh score alone.     

Inaccuracy of insulin-like growth factor (IGF) binding protein (IGFBP)-3 assessment in the diagnosis of growth hormone (GH) deficiency from childhood to young adulthood: association to low GH dependency of IGF-II and presence of circulating IGFBP-3 18-kilodalton fragment

CONTEXT: Poor sensitivity of IGF binding protein (IGFBP)-3 assessment in the work-up of GH deficiency (GHD) has been ascribed to the equal affinity of IGFBP-3 for IGF-I and IGF-II and to IGFBP-3 proteolysis. OBJECTIVE: The objective of this study was to determine the IGF-II GH dependency and IGFBP-3 proteolysis in patients with GHD from childhood to young adulthood. DESIGN: This study was cross-sectional. SETTING: This was a national multicenter study performed in university hospitals. PATIENTS: One hundred thirty-one subjects (chronological age, 1.3-25 yr), 72 patients with GHD and 59 subjects with idiopathic short stature, were studied. INTERVENTIONS: IGF-I, IGF-II, and IGFBP-3 serum concentrations were measured by immunoradiometric assay. IGFBP-3 circulating forms were assessed by Western immunoblot (WIB) analysis. MAIN OUTCOME MEASURES: Main outcome measures were sensitivity and specificity of IGF-I, IGF-II, and IGFBP-3 measurements. RESULTS: Sensitivity and specificity of IGFBP-3 measurement were 27 and 100%, respectively. IGFBP-3 sensitivity was 46% in young adulthood. Sensitivity and specificity of IGF-I were 69 and 81%, respectively. Sensitivity and specificity of IGF-II assessment were 23 and 97%, respectively. IGFBP-3 WIB revealed the presence of the intact form and the major 29-kDa fragment in both GHD and subjects with idiopathic short stature. In patients with GHD, WIB showed the presence of an additional smaller IGFBP-3 fragment migrating at approximately 18 kDa. CONCLUSIONS: Our results suggest that in children and young adults with GHD, the low GH dependency of IGF-II together with IGFBP-3 proteolytic activity yielding the 18-kDa fragment concur to reduce the sensitivity of IGFBP-3 assessment, ultimately making it too inaccurate as a screening test in the work-up of GHD.     

An acid-stable insulin-like growth factor (IGF)-binding protein from pig serum inhibits binding of IGF-I and IGF-II to vascular endothelial cells

The effects of a porcine insulin-like growth factor (IGF)-binding protein on binding of IGF-I and IGF-II to porcine aortic endothelial cells (PAEC) were determined. Binding of 125I-labelled IGF-I and -II to IGF receptors was inhibited by IGF-binding protein. IGF-binding protein inhibited binding of IGF-I and -II in a dose-dependent manner with half-maximal inhibition occurring at 5.43 and 108 micrograms/l respectively. A 125I-labelled IGF-I--IGF-binding protein complex, formed by incubating 125I-labelled IGF-I with IGF-binding protein overnight at 4 degrees C, did not effectively bind to endothelial IGF receptors. Addition of IGF-binding protein to PAEC previously incubated with IGF-I caused a marked dissociation of bound IGF-I (47% dissociation within 12h). These results indicate that the acid-stable IGF-binding protein which appears to be a part of the 150 kDa GH-dependent binding protein, blocks binding of IGF-I and -II by the IGF receptors and appears to exhibit a higher affinity for IGF-I than the endothelial type-I IGF receptor. The ramifications of this latter point with respect to transfer of circulating IGFs (bound to their IGF-binding proteins) across the vascular endothelium are not clear.     

Inflammation is a modulator of the insulin-like growth factor (IGF)/IGF-binding protein system inducing reduced bioactivity of IGFs in cystic fibrosis

OBJECTIVE: In inflammatory bowel diseases, increased serum interleukin (IL)-6 levels are associated with high serum insulin-like growth factor-binding protein 2 (IGFBP-2) levels, and cytokines modify the insulin-like growth factor (IGF)/IGFBP system in models in vitro. In cystic fibrosis (CF) the IGF/IGFBP system has not been extensively studied, and relationships with proinflammatory cytokines have not been explored. The aim of this study was to investigate the IGF/IGFBP system and verify changes dependent on IL-1beta, IL-6, tumour necrosis factor alpha (TNFalpha), and insulin. METHODS: Eighteen subjects with CF (mean age 26.6 +/- 1.1 years) and 18 controls, comparable for age, sex, and body mass index, were enrolled. Serum IGF-I, IGF-II, IGFBP-2, IGFBP-3, IL-1beta, IL-6, TNFalpha, insulin and C-peptide were measured. Different molecular forms of IGFBP-2 and IGFBP-3 were investigated by Western immunoblotting. The patients were analysed as a whole and as two subgroups depending on established clinical criteria (Swachman-Kulczycki score). RESULTS: Patients had higher serum concentrations of IL-1beta, IL-6, TNFalpha and IGFBP-2 than controls. Serum concentrations of IGF-I and IGF-II were significantly lower and insulin and C-peptide levels significantly increased in CF compared with healthy controls whereas IGFBP-3 serum concentrations were similar, with comparable IGF-I/IGFBP-3 and decreased IGF-I/IGFBP-2 and IGF-II/IGFBP-2 molar ratios. From correlation analysis we detected a significant positive correlation between IGFBP-2 and IL-6 and a negative correlation between IGFBP-2 and IGFBP-3. CONCLUSIONS: Our findings suggest that inflammation is an important modulator of the IGF/IGFBP system with an overall reduction in IGF bioactivity in CF.     

A quantitative PCR measurement of messenger RNA expression of IGF-I, IGF-II and IGFBP-5 in human skeletal muscle.

Insulin-like growth factor-I and -II (IGF-I and IGF-II) and their binding proteins are important components in growth promotion and tissue maintenance. We determined the presence of IGF-I, -II, and binding protein 5 (IGFBP-5) gene expression in human skeletal muscle and that mRNA abundance is not altered by nutrients and insulin. In the first protocol, (control) subjects were given water. In the second protocol, half of these subjects drank Polycose (carbohydrate) and the remaining subjects drank equal calories as a mixed meal. Quadriceps muscle biopsies were taken at 10 h. A semi-quantitative polymerase chain reaction was designed to measure gene expression. IGF-I, IGF-II and IGFBP-5 mRNA are present in adult human skeletal muscle, but no significant changes between meal groups were observed for IGF-I, IGF-II or IGFBP-5 mRNA levels, indicating that the expression of these genes are not altered acutely by nutrients and insulin.     

Primary acid-labile subunit deficiency due to recessive IGFALS mutations results in postnatal growth deficit associated with low circulating insulin growth factor (IGF)-I, IGF binding protein-3 levels, and hyperinsulinemia

CONTEXT: Up to 90% of circulating IGF-I and IGF-II are carried bound to either IGF binding protein (IGFBP)-3 or IGFBP-5 and the acid-labile subunit (ALS) in the form of tertiary complexes that extend their circulating half-life. Three cases of complete ALS deficiency have been recently reported in short-stature patients with very low circulating IGF-I and IGFBP-3 levels who presented with homozygous or compound heterozygous mutations in the ALS encoding gene (IGFALS; 16p13.3), thus supporting a role for ALS in the regulation of the bioavailability of IGFs during postnatal growth. OBJECTIVE: We present the molecular and clinical characterization of two novel IGFALS mutations that caused complete ALS deficiency in three unrelated patients with postnatal growth deficit, low IGF-I and IGFBP-3 levels, and no GH deficiency. RESULTS: IGFALS mutation screening identified a novel homozygous IGFALS missense mutation, which altered a conserved residue, N276S, in two of the probands. The third proband presented a novel homozygous nonsense mutation, Q320X, that is predicted to generate a severely truncated ALS protein. The affected probands presented a similar phenotype characterized by a moderate postnatal growth deficit associated with undetectable ALS, low IGF-I, IGF-II, and IGFBP-3, and hyperinsulinemia, and, in two cases, delayed puberty. CONCLUSIONS: Primary ALS deficiency due to IGFALS mutations should be considered as a possible cause of postnatal growth deficit in IGF-I-deficient patients in the absence of GH deficiency or insensitivity. Determination of serum ALS levels and basal insulinemia can be helpful in the differential diagnosis of patients with idiopathic IGF-I deficiency.