Usefulness of different biochemical markers of the insulin-like growth factor (IGF) family in diagnosing growth hormone excess and deficiency in adults.

The diagnostic approach to acromegaly and GH deficiency frequently includes measurement of several components of the insulin-like growth factor (IGF) system. IGF-I levels are reported to be good predictors of active and cured acromegaly, but are commonly found within the normal age-adjusted range in adult GH-deficient (GHD) patients. Circulating concentrations of IGF-binding protein-3 (IGFBP-3), acid-labile subunit (ALS), and free IGF-I reflect the GH secretory status, but their diagnostic accuracy is still debated. In this study serum levels of total and free IGF-I, IGFBP-3, ALS, and IGFBP-3-IGF-I and IGFBP-3-ALS complexes were determined in patients previously diagnosed with active (n = 67) or inactive (n = 16) acromegaly and adult GHD (n = 34) and compared with results obtained in 58 healthy controls. In healthy subjects, IGF-I, IGFBP-3, ALS, and both IGFBP-3 complexes declined with age; a correlation was found between IGF-I and IGFBP-3 (r = 0.59; P < 0.001), ALS (r = 0.67; P < 0.001), and free IGF-I (r = 0.40; P < 0.05). Active acromegalic patients showed a significant increase in all parameters tested. IGF-I concentrations were above +2 SD in 100% of patients, whereas slightly lower sensitivities were shown for IGFBP-3 (85%), ALS (88%), and free IGF-I (94%). In this group, IGF-I exhibited a slightly higher correlation with IGFBP-3 (r = 0.83; P < 0.001) than with ALS levels (r = 0.78; P < 0.001). In cured acromegalic patients, we observed the normalization of all parameters but free IGF-I levels. Adult GHD patients showed a significant reduction of all hormones. Unlike active acromegalic patients, all parameters had only a modest sensitivity in GHD; suppression below -2 SD was observed in 41% of GHD patients for IGF-I, 47% for IGFBP-3, 32% for ALS, and 35% for free IGF-I measurements. Previous radiotherapy and GH peak response below 3 microg/L were associated with significantly lower IGF-I, IGFBP-3, and ALS levels. IGF-I levels were significantly correlated to ALS (r = 0.68; P < 0.001) and IGFBP-3 (r = 0.64; P < 0.001) as well as with free IGF-I (r = 0.67; P < 0.001) levels. By multiple regression analysis, the number of anterior pituitary hormones impaired was the most predictive indicator of IGF-I, IGFBP-3, and free IGF-I levels in GHD patients; conversely, the GH peak response better anticipated ALS concentrations. The pattern of IGFBP-3 complexes paralleled previous hormonal findings. In active acromegalic patients, IGFBP-3-IGF-I levels were 5.4-fold higher than in controls and were above +2 SD in 95% of patients, whereas IGFBP-3-ALS levels were elevated in 15% of cases. On the other hand, both IGFBP-3 complexes were able to predict GHD in only a minority of cases. Taken together, these data support the diagnostic role of IGF-I in acromegaly and suggest that free IGF-I and the IGFBP-3-IGF-I complex can assist diagnostic strategies in this condition. All markers are of limited predictive value in adult GHD, as hormonal values are commonly found within the normal limits. In these patients, low IGFBP-3 and IGF-I concentrations can add further clinical information on the residual GH activity.     

Serum IGF-I and IGFBP-3 levels for the assessment of disease activity of acromegaly

To assess the disease activity of acromegaly in patients, we measured the changes in serum growth hormone (GH) levels during oral glucose tolerance test and the basal serum levels of insulin-like growth factor I (IGF-I) and insulin-like growth factor-binding protein 3 (IGFBP-3) in 29 acromegalic patients and 30 health persons served as normal controls. Based on the clinical and laboratory criteria, acromegaly was in an active state of disease in 18 patients and was inactive in the other 11 patients. Basal serum IGF-I levels were 177+/-116 ng/ml (mean+/-SD), 250+/-135 ng/ml and 810+/-297 ng/ml in the normal subjects, the inactive and active acromegalic patients, respectively. Basal serum IGFBP-3 levels were 1.71+/-1.29 microg/ml, 2.98+/-0.96 microg/ml and 6.82+/-1.31 microg/ml in the normal controls, the inactive and active acromegalic patients, respectively. Serum levels of IGF-I and IGFBP-3 significantly correlated with each other in the normal subjects as well as the patients. Both IGF-I and IGFBP-3 levels were significantly higher in the group of patients with active acromegaly than inactive acromegalic patients and the normal subjects but there was not statistically difference between the normal controls and the inactive acromegalics. While serum IGF-I levels presented considerable overlapping instances among the three groups, the serum IGFBP-3 of inactive patients and the normal controls could rarely reach 4.44 ng/ml, the lowest value of the active acromegalics. The serum IGF-I and IGFBP-3 levels declined with increased age in normal controls, but not in the patients with acromegaly. There was no sex predilection of serum IGF-I and IGFBP-3 found in our study. The results of this study indicated that the serum IGFBP-3 level is an important laboratory parameter for assessing growth hormone function in humans, and might be a more reliable discrimination for the disease activity of acromegaly than the serum IGF-I is.     

Depolarization induces insulin-like growth factor binding protein-2 expression in vivo via NMDA receptor stimulation

The effect of depolarization and N-methyl-D-aspartate (NMDA) receptor blockade on insulin-like growth factor-I (IGF-I), IGF binding protein-2 (IGFBP-2) and IGFBP-4 expression was analysed in vivo. Depolarization was induced in adult rat brains by applying 3 M KCl to the exposed cortex for 10 min. A subgroup of animals also received daily injections of MK-801. Four days after KCl exposure, the brains were analysed by in situ hybridization, immunohistochemistry and TUNEL. A significant upregulation of IGFBP-2 mRNA and protein was detected in astrocytes after KCl exposure This upregulation was reduced by MK-801 treatment. No alterations in IGF-I or IGFBP-4 mRNA levels were noted. We did not detect TUNEL positive cells, morphological signs of necrosis or apoptosis, or neuronal loss in the depolarized zone. Taken together, these findings indicate that upregulation of IGFBP-2 by depolarization is mediated by NMDA receptors, and, as no neuronal damage was detected, astrocytic NMDA receptors may be responsible for this upregulation.     

Diagnostic value of the acid-labile subunit in acromegaly: evaluation in comparison with insulin-like growth factor (IGF) I, and IGF-binding protein-1, -2, and -3

In normal subjects the main form of circulating insulin-like growth factor (IGF) is the 150-kDa complex. This complex is formed by the IGF peptide, the acid-stable IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS). Experimental and clinical data have demonstrated that ALS is primarily under the control of GH and plays a critical role in maintaining constant levels of circulating IGF-I. In this study we evaluated ALS, IGF-I, and IGFBP-1, -2, and -3 in 45 acromegalic patients in basal conditions and, in 37 of these, twice after surgical therapy compared with 100 age- and sex-matched control subjects to estimate their value as parameter of GH secretory state. The results demonstrated that in acromegaly before treatment all parameters (ALS, 523 +/- 26; IGF-I, 129 +/- 6; IGFBP-1, 0.7 +/- 0.1; IGFBP-3, 234 +/- 21; nmol/L; mean +/- SEM) but IGFBP-2 were significantly different (P<0.0001) from those in healthy subjects (ALS, 281 +/- 4; IGF-I, 22 +/- 1; IGFBP-1, 1.6 +/- 0.1; IGFBP-3, 91 +/- 3). IGF-I was more sensitive (100%) than ALS (89%), and both were more predictive of disease status than IGFBP-3, in that 27% of the patients had IGFBP-3 levels within the normal range. Considering the ALS/IGFBP-3 molar ratio, almost 55% of ALS circulated in a free form in active acromegaly. Before treatment, the IGF-I/IGFBPs (-1 + -2 + -3) molar ratio, which can be regarded as free, biologically active, IGF-I, was greatly increased (0.77 +/- 0.06; P<0.0001) compared with that in control subjects (0.23 +/- 0.01). After surgery, all 10 patients with controlled disease showed normalization of ALS (100% sensitivity), whereas 9 of them had normal IGFBP-3; reevaluation after varying lengths of time showed all these parameters within the normal range. In the 27 patients with active disease, IGF-I and ALS were more predictive of disease status (91% and 83% negative predictive values, respectively) than IGFBP-3 (53%). The basal ALS concentration correlated only with IGFBP-3 (r = 0.70; P<0.001). In postsurgery samples (first control) a statistically significant (P<0.001) correlation was found between mean GH values as well as minimum GH after oral glucose tolerance test and ALS (r = 0.72 and 0.83, respectively), IGF-I (r = 0.69 and 0.77), IGFBP-3 (r = 0.50 and 0.72), and IGFBP-2 (r = -0.36 and -0.63). Similarly, IGF-I, IGFBP-3, and ALS were positively correlated among themselves and negatively correlated with IGFBP-2 (P<0.001). In conclusion, in the diagnosis of acromegaly, the measurement of total IGF-I appears to be the most sensitive parameter among the subunits of the 150K complex, and IGFBP-3 the least sensitive. For ALS, this subunit is quite sensitive and appears to be a useful parameter in reassessment after surgical treatment.     

The role of IGF binding protein-3 as a parameter of activity in acromegalic patients.

OBJECTIVE: The production of insulin-like growth factor binding protein-3 (IGFBP-3), the main IGF-I binding protein, is regulated by GH, and its serum levels are increased in acromegaly. We investigated its potential value as a parameter of acromegaly activity or remission in comparison with IGF-I, taking GH suppression below 2 microg/l after glucose load as the normal standard. METHODS: Data from 40 acromegalic patients (12 males and 28 females, aged 28 to 79 years) were obtained retrospectively from stored samples. From these, 145 pairs of IGF-I/IGFBP-3 values were collected; in 67 of them, simultaneous measurement of GH after glucose loading allowed their classification as active or inactive acromegaly. Relationships between IGF-I, IGFBP-3 and GH after glucose load were assessed, as well as differences between IGF-I and IGFBP-3 levels in active and inactive acromegaly. RESULTS: Significant positive correlation between IGF-I and IGFBP-3 in 145 samples was observed (r=0.49, P<0. 0001). As for the 67 samples in which activity or remission could be defined in terms of GH after glucose load, 50 were active and 17 inactive. Both IGF-I and IGFBP-3 significantly correlated with minimum GH (r=0.53, P<0.0001 and r=0.41, P<0.001 respectively). For both parameters, significant differences of means between active and inactive cases were observed (623+/-296 vs 300+/-108 ng/ml, P<0.0001 for IGF-I, and 4.1+/-1.3 vs 3.2+/-0.9 microg/ml, P<0.006 for IGFBP-3). Yet, when comparing in individual cases their classification as active or inactive with the finding of normal or increased IGF-I and IGFBP-3, among active cases 16% appeared as normal according to IGF-I, and 50% appeared as normal in terms of IGFBP-3. Among inactive cases, 23.5% appeared as active according to IGF-I, while 17.5% appeared as active in terms of IGFBP-3. CONCLUSION: Even though IGFBP-3 reflects GH secretion, it offers no advantage over IGF-I in the assessment of acromegaly, and it may underestimate disease activity in acromegalic patients.     

Monitoring of treatment success in patients with acromegaly: the value of serum insulin-like growth factor binding protein-3 and serum leptin measurements in comparison to plasma insulin-like growth factor I determination

The aim of the present study was to determine the value of serum insulin-like growth factor binding protein-3 (IGFBP-3) and serum leptin measurements in comparison with plasma insulin-like growth factor I (IGF-I) measurements as indicators of treatment success in patients with acromegaly. Thirty-five acromegaly patients, 25 female and 10 male, divided into groups of patients with postadenomectomy "active" acromegaly (n = 20) and patients with postadenomectomy "controlled" acromegaly (n = 15), and 44 healthy volunteers sex- and age-matched with the acromegaly patients were included in the present study. We comparatively analyzed plasma IGF-I, serum IGFBP-3, and serum leptin levels in the aforementioned groups. Because serum leptin has sex dimorphism, the groups were divided into sexes when leptin was evaluated. As expected, the patients with active acromegaly had significantly higher mean values of plasma IGF-I and serum IGFBP-3 and lower mean values of serum leptin (only in women) than the control group. However, individual evaluation showed that 1 of 20, 9 of 20, and many patients with postadenomectomy active acromegaly patients had values that overlapped values of control subjects for plasma standard deviation score (SDS)-IGF-I, serum SDS-IGFBP-3, and sex-adjusted serum leptin, respectively. Application of the receiver operating characteristic (ROC) curves method shows that plasma IGF-I measurement has the best discriminatory power to differentiate patients with postsurgical active acromegaly from healthy people. Its area under the curve (AUC) was 0.95, with a sensitivity and specificity of 86% and 94%, respectively. Its positive and negative likelihood ratios were 14 and 0.15. Serum IGFBP-3 has certain discriminatory power, its AUC being 0.89, with a sensitivity and specificity of 83% and 77%. Its positive and negative likelihood ratios were 3.6 and 0.22. Serum leptin, both in women and in men, has a poor performance with sensitivity and specificity of 53% and 50% for women and 55% and 56% for men and positive and negative likelihood ratios of 1.06 and 0.94 for women and 1.26 and 0.8 for men. Application of the ROC curves method and the determination of positive and negative likelihood ratios in comparative evaluation of serum IGFBP-3 and serum leptin with plasma IGF-I as indicators of treatment success in acromegalic patients showed that neither serum IGFBP-3 nor serum leptin determinations have accuracy better than or similar to that of plasma IGF-I for monitoring treatment success in acromegaly patients. Serum IGFBP-3 is accurate but does not increase accuracy for age-adjusted plasma IGF-I, whereas determination of serum leptin level has no value in monitoring these patients.     

Inhibition of human osteoblast marker gene expression by retinoids is mediated in part by insulin-like growth factor binding protein-6

All-trans -retinoic acid (atRA) inhibits osteoblast marker gene expression and markedly increases expression of insulin-like growth factor binding protein-6 (IGFBP-6) in human osteoblasts. The possibility that IGFBP-6 inhibits the osteoblast phenotype and also mediates the inhibitory effect of atRA on osteoblast marker gene expression was explored using an antisense approach. Stable human osteoblast-like osteosarcoma SaOS-2 cells were prepared that expressed antisense IGFBP-6 RNA under basal and atRA-stimulated conditions. The functional expression of IGFBP-6 antisense RNA was confirmed by measuring IGFBP-6 mRNA by Northern analysis or by measuring IGFBP-6 protein in the conditioned media (CM) by radioimmunoassay. Antisense clones produced less mRNA and had less IGFBP-6 protein in the CM than controls. IGFBP-6 protein levels in the CM were inversely correlated with alkaline phosphatase (ALP) activity, whereas IGFBP-3 and IGFBP-4 protein levels were not. We reasoned that atRA would have little or no effect on ALP activity in IGFBP-6 antisense clones if atRA mediated its inhibitory effects by recruiting IGFBP-6. In the majority of IGFBP-6 antisense clones with the lowest IGFBP-6 mRNA and CM protein levels and only modest changes in other IGF system components, atRA did not significantly decrease ALP activity. These findings provide evidence that atRA recruits IGFBP-6 to inhibit the human osteoblast phenotype.     

Acid-labile subunit in growth hormone excess and deficiency in adults: evaluation of its diagnostic value in comparison with insulin-like growth factor (IGF)-I and IGF-binding protein-3

In serum, insulin-like growth factors (IGFs) are primarily present as a approximately 150 kDa ternary protein complex, which consists of IGFs, IGF binding protein-3 (IGFBP-3), and acid-labile subunit (ALS). Like IGF-I and IGFBP-3, serum levels of ALS depend on growth hormone (GH). To date, the diagnostic relevance of ALS in adult GH deficiency (GHD) has remained uncertain. To clarify the clinical utility of ALS measurement in adults, we measured serum ALS levels in patients with adult GHD or acromegaly. We also measured the levels of serum IGF-I and IGFBP-3 in these patients to compare the utility of ALS with IGF-I and IGFBP-3 as a marker of GH secretion. Serum ALS was measured by radioimmunoassay (RIA) kit, and serum IGF-I and IGFBP-3 were measured by immunoradiometric assay (IRMA) kits in 56 patients with adult GHD (adult-onset (AO)/child-onset (CO), 13/43) and 43 patients with acromegaly. Serum ALS levels were less than 5th percentile in 40 of 56 (71%) patients with adult GHD (32/43 (74%) for CO and 8/13 (62%) for AO), and more than 95th percentile in 38 of 43 (88%) patients with acromegaly, respectively. Serum IGF-I levels were less than -1.96 SD in 43 of 56 (77%) patients with adult GHD (35/43 (81%) for CO and 8/13 (62%) for AO) and more than +1.96 SD in 42 of 43 (98%) patients with acromegaly, respectively. Serum IGFBP-3 levels were less than -1.96 SD in 51 of 56 (91%) patients with adult GHD (42/43 (98%) for CO and 9/13 (69%) for AO) and more than +1.96 SD in 31 of 43 (72%) patients with acromegaly, respectively. These data suggested that measurement of ALS offers no advantage over measurements of serum IGF-I and IGFBP-3. Furthermore, our results indicate that serum IGFBP-3 is the most suitable marker of GH secretion for adult GHD, especially CO, while IGF-I may be the most useful in acromegaly.     

Effect of growth hormone (GH) and insulin-like growth factor I on prostate diseases: an ultrasonographic and endocrine study in acromegaly, GH deficiency, and healthy subjects.

The role of insulin-like growth factor I (IGF-I) in prostate development is currently under thorough investigation because it has been claimed that IGF-I is a positive predictor of prostate cancer. To assess the effect of GH and IGF-I levels on prostate pathophysiology, 46 acromegalic (30 in active disease, 10 cured from acromegaly, and 6 affected from GH deficiency) and 30 age-matched male controls, free from previous or concomitant prostate disorders, underwent pituitary, androgen, and prostate hormonal assessments and transrectal ultrasonography. Compared to control values, GH (P < 0.0001), IGF-I (P < 0.0001), and IGFBP-3 (P < 0.001) levels were increased, whereas testosterone (P < 0.0001) and dihydrotestosterone levels (P < 0.0001) were reduced in active acromegalic patients. Hypogonadism was present in 28 of the 46 acromegalic patients (60.8%). The anteroposterior (P < 0.05), and transverse (P < 0.0001) prostate diameters and the transitional zone volume (P < 0.05) were increased in acromegalic patients compared to those in controls. Prostate volume (PV) was significantly higher in untreated acromegalic patients than in controls (41.7 +/- 3.2 vs. 21.9 +/- 1.4 mL; P < 0.0001), cured patients (23.6 +/- 1.6 mL; P < 0.0001), and GH-deficient patients (17.5 +/- 1.1 mL; P < 0.0001). In the patients, PV was correlated with disease duration (r = 0.606; P < 0.0001) and age (r = 0.496; P < 0.0001), whereas in controls it was correlated with age (r = 0.476; P < 0.01) and IGF-I levels (r = -0.448; P < 0.05). Benign prostate hyperplasia (PV > or = 30 mL) was found in 58% of the acromegalics and 26.6% of the controls. When grouped by age (<40, 40-60, and >60 yr), PV was increased in elderly patients compared to younger patients (P < 0.05) and to controls (P < 0.01). The prevalence of structural abnormalities, including calcifications, nodules, cysts, and vesicle inflammation, was significantly increased in patients compared to controls (78.2% vs. 23.3%; chi2 = 5.856; P < 0.05). No clinical, transrectal ultrasonography, or cytological evidence of prostate cancer was detected in acromegalic or control subjects. In conclusion, chronic excess of GH and IGF-I cause prostate overgrowth and further phenomena of rearrangement, but not prostate cancer.     

Effect of two years of growth hormone and insulin-like growth factor-I suppression on prostate diseases in acromegalic patients

The insulin-like growth factors (IGFs) have mitogenic effects on normal and tumoral prostate epithelial cells and have been suggested to be involved in prostate cancer. Moreover, chronic GH and IGF-I excess causes prostate overgrowth in patients with acromegaly. This study was designed to investigate whether the suppression of GH and IGF-I levels by surgery or pharmacotherapy could induce the regression of prostatic hyperplasia in acromegalic patients. To this end, prostate volume (PV) as well as the occurrence of prostatic diseases were studied by transrectal ultrasonography in 23 untreated acromegalic patients (with elevated GH and IGF levels). None of the patients reported symptoms due to prostatic disorders or obstruction. At study entry, prostate hyperplasia was found in half patients. After 2 yr, GH, IGF-I, and IGFBP-3 levels were decreased, whereas prostate-specific antigen levels did not change. PV was decreased in the 16 patients who were well controlled. Among the 6 patients with prostate hyperplasia at study entry who achieved disease control, 4 regained a normal PV at the end of the 2 yr of treatment, whereas none of the 5 patients with prostate hyperplasia at study entry and not achieving disease control normalized their PV. When patients were divided according to age, prostate volume decreased after 2 yr only in the 8 controlled patients aged below 50 yr, but not in those controlled and with age above 50 yr despite similar decrease in GH, IGF-I, and IGFBP3 levels. No clinical, transrectal ultrasonography, or cytological evidence of prostate cancer was detected during the study period. These data suggest that hyperplasia, but not cancer, is frequent in acromegalic men, and that the GH-IGF axis and age are independently associated with the development of this process.     

The relationship between serum levels of insulin-like growth factor-I and its binding proteins and microvascular function in acromegalic patients.

The system of insulin-like growth factor-I (IGF-I) and its binding proteins is thought to be involved in the pathogenesis of vascular damage under different pathological circumstances. The results of various studies are rather controversial. This study considers the relationship between the activity of this system and the function of microcirculation in acromegalic patients. Thirteen patients with hormonally active acromegaly and 15 healthy controls were included in the study. The growth hormone, free IGF-I, IGF-I, IGF binding protein (IGFBP) -1, -2, -3 and -6 serum levels and parameters of lipid metabolism were determined. The function of microcirculation was determined by laser Doppler fluxmetry and the intima media thickness of the common carotid artery was measured by ultrasound. We noted significant reduction in postocclusive reactive hyperaemia (PORH(max)) (P < 0.01), in thermal hyperaemia (TH(max)) (P < 0.05) and in the velocity of reaction in both tests in the group of acromegalic patients. A significant negative correlation between free IGF-I serum levels and maximal perfusion during thermal hyperaemia TH(max) (P < 0.02) was found in the control group. Statistically significant positive correlation between free IGF-I serum levels and the time to maximal perfusion in postocclusive reactive hyperaemia PORH(max) (P < 0.05) was found in the group with hormonally active acromegaly. Moreover, a positive relationship between IGFBP-1 serum levels and serum levels of total (P < 0.01) and low density lipoprotein (LDL) (P < 0.05) cholesterol was found in the group of patients with acromegaly. We conclude that the function of microcirculation is impaired in patients with acromegaly and that free IGF-I serum levels may affect the microvascular function as measured by laser Doppler fluxmetry. In addition, we found a significant relationship between the serum levels of IGFBP-1 and those of total and LDL cholesterol in the group of patients with hormonally active acromegaly.     

Reduced serum insulin-like growth factor-1 (IGF-1) and IGF-binding protein-3 levels in adults with inflammatory bowel disease

In the present study, the changes in circulating IGF-1 and its binding protein IGFBP-3 were determined in adult patients with active inflammatory bowel disease (IBD) in order to assess the effect of this inflammatory condition on the IGF system. IGF-1 and IGFBP-3, as well as interleukin-6 (IL-6) were measured in serum obtained from 22 consecutive newly diagnosed patients (mean age 41.3 years) with active IBD, including 10 patients with Crohn's disease (CD), and 12 with ulcerative colitis (UC). For comparison the same parameters were determined in 30 healthy volunteers matched for age, sex and Body Mass Index (BMI). Serum IGF-1 and IGFBP-3 levels were similar in the two subgroups of patients and the values from all patients were combined for comparison with those from the control group. The mean (+/- SD) serum IGF-1 concentration (178 +/- 91 ng/ml) in the patients with IBD was lower compared with that in the controls (227 +/- 79 ng/ml, P<0.035). Similarly, the mean IGFBP-3 concentration in the patients was lower than in the controls (1.6 +/- 0.6 ng/ml vs 3.2 +/- 0.7 ng/ml respectively, P<0.001), Serum IL-6 levels were higher in the patients compared with the controls (5.5 +/- 4.2 vs 0.65 +/- 0.11 pg/ml, P<0.0001). The reduced IGF-1 and IGFBP-3 levels in patients with active IBD suggest that this systemic inflammatory condition is associated with a degree of acquired GH resistance, possibly induced by inflammatory cytokines.     

The changes of IGF binding proteins after rhGH administration to patients totally dependent on parenteral nutrition

The objective was to study the effect of recombinant human growth hormone (rhGH) administration to patients with chronic malnutrition maintained on total parenteral nutrition (TPN) on the levels of insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) during a double-blind trial. After 1 week of TPN the patients were randomized into group I (placebo) or group II (rhGH). Samples were collected on the first day (start of the TPN) to measure basal values, the seventh day to study the effect of TPN and the 10th, 14th and 21st days to evaluate the rhGH effect. Basal laboratory evaluation, nutritional status and glucose tolerance were assessed using standard laboratory techniques. Radioimmunoassays were used to analyse IGF-I, free IGF-I (fIGF-I) and IGFBP1-3. Electrophoresis with Western ligand blotting and Western immunoblotting was applied to find the pattern of IGFBPs. TPN had no effect on the circulating IGF-I concentration and the pattern of IGFBPs present in the studied groups of patients. The rhGH administration led to significant increases of IGF-I, total IGFBP-3, glycosylated IGFBP-3 (39, 42 kDa) and the 29 kDa fragment of IGFBP-3 and the decrease of IGFBP-2 during the trial (P<0.05). The mean levels of IGFBP-1, fIGF-I and the parameters of nutritional status in group II during the trial were not significantly influenced by rhGH. However, it has been found that IGFBP-1 and fIGF-I levels were correlated with the levels of the weekly nitrogen balance of each patient in group II at the end of the trial. In spite of the significant changes of IGF-I, IGFBP-2, total IGFBP-3 and IGFBP-3 (29 kDa proteolytic fragment) after rhGH administration to patients with malnutrition, maintained on parenteral nutrition, the increase of nitrogen balance was seen only in patients who decreased their IGFBP-1 and increased bioavailable IGF-I as reflected by measurement of fIGF-I. The levels of IGFBP-1 may provide a useful marker of IGF-I bioavailability in monitoring the efficiency of the rhGH therapy in malnourished patients.     

Roles of insulin-like growth factor-I (IGF-I) and IGF-I binding protein-2 (IGFBP2) and -5 (IGFBP5) in developing chick limbs

Insulin-like growth factor-I (IGF-I) and the IGF-I binding proteins (IGFBPs) which modulate IGF-I action have been implicated in the development of the vertebrate limbs and skeleton. We have examined the distribution of IGF-I, IGFBP2 and IGFBP5 in developing chick limb buds and have investigated their functional roles and relationships during chick limb development. IGF-I and IGFBP2 are co-expressed throughout the lateral plate from which limbs form, although IGFBP2, unlike IGF-I, does not promote formation of rudimentary limb buds from non-limb-forming flank regions in vitro. During limb outgrowth, IGF-I is present in non-AER limb ectoderm, but little IGF-I is present in the AER itself, suggesting that restriction of endogenous IGF-I activity may be required for proper AER function. Consistent with this possibility, the ectoderm of mutant limbless and wingless wing buds, which fail to form an AER, continues to express IGF-I. We also found that the AER contains abundant IGFBP2 but that IGFBP2 is not present in limb subridge mesoderm. In contrast, IGFBP2 is present in the distal mesoderm of mutant limbless or wingless limb buds, which fail to grow out. This suggests that attenuation of IGFBP2 expression is controlled by the AER and that cessation of IGFBP2 expression may be necessary for the proliferation and suppression of differentiation of subridge mesoderm that is required for limb outgrowth to occur. Consistent with this possibility, we found that exogenous IGFBP2 inhibits the anti-differentiative activity of the AER in vitro. We also found that regions of cell death in the limb contain abundant IGF-I-immunoreactive cells, consistent with a role for IGF-I in apoptosis. During skeletogenesis, IGF-I and IGFBP2 are co-localized to the condensing central core of the limb, implicating these factors as potential regulators of the onset of chondrogenic differentiation. Intriguingly, we found that IGF-I and IGFBP2 have opposing effects on chondrogenesis, as IGF-I stimulates but IGFBP2 inhibits accumulation of cartilage matrix by micromass cultures in vitro. Long [R(3)] IGF-I, an analog of IGF-I that cannot bind IGFBPs, is more effective than IGF-I in stimulating matrix accumulation, consistent with a negative role for IGFBP2 in chondrogenesis. As the chondrocytes of the limb mature, IGF-I is present only in terminal hypertrophic chondrocytes, which undergo programmed cell death, while IGFBP2 becomes localized to prehypertrophic and hypertrophic chondrocytes, suggesting involvement in chondrocyte maturation. Consistent with this possibility, we found that exogenous IGFBP2 induces precocious expression of Indian hedgehog, a marker of prehypertrophy, in maturing chondrocytes in vitro. IGF-I and IGFBP2 are also present in the osteoblasts, clasts and nascent matrix of the long bones, consistent with roles in endochondral bone formation. Unlike in rodent limbs, IGFBP5 is not expressed by chick limb ectoderm or AER. IGFBP5 expression is highly localized to developing limb musculature and, later, to the developing skeletal elements where it is expressed by osteoblast precursers and osteoblasts. The results of this study suggest potential novel roles for IGF-I and IGFBP2 in several aspects of limb development including limb outgrowth and AER activity, programmed cell death, chondrogenesis and chondrocyte maturation.     

The ratio between serum levels of insulin-like growth factor (IGF)-I and the IGF binding proteins (IGFBP-1, 2 and 3) decreases with age in healthy adults and is increased in acromegalic patients

OBJECTIVE Several in-vitro studies have suggested that the biological actions of IGF-I can be modified by the presence of specific IGF binding proteins. In man, the 24-hour serum levels of IGF-I and IGFBP-3 remain constant, but short-term changes in the IGF-l/IGFBP-3 ratio have been described following GH administration. Serum levels of IGF-I and IGFBP-3 decrease with age in normal adults and are elevated In active acromegaly due to excessive GH secretion. However, the Individual ratios between serum levels of IGF-I and IGFBP-3 in acromegalic and healthy adults have not been described previously. METHODS AND MATERIALS We studied this ratio In 198 healthy adults and In 56 acromegalic patients, grouped according to their serum GH levels (group I GH < 2mLU/l II GH 2–10mLU/l; III GH > 10mLU/l). In all subjects a single blood sample was drawn for IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3 and GH measurements by specific RIAs. In 38 of the patients a 24-hour urinary collection was performed for GH determination. RESULTS In healthy adults serum levels of IGF-I and IGFBP-3 decreased with Increasing age (r =?0.52 and r=?0.34, respectively, P< 0.0001). In addition, the molar IGF-l/IGFBP-3 ratio declined with increasing age (r =?0.44, P – 0.0001). In patients with acromegaly and high serum GH levels (group III), circulating IGF-I was increased 7–97 standard deviations (SDS) and IGFBP-3 was increased 4.20 SOS (P < 0.0001). Serum levels of IGF-II were normal in all three groups (588 ± 240μ/l) whereas IGFBP-1 and IGFBP-2 levels were low and IGFBP-2 levels decreased significantly with increasing serum GH levels (P < 0.0001). The molar IGF-l/IGFBP-3 ratio in the acromegalic patients was significantly higher than in the controls (P < 0.0001) and correlated significantly with urinary GH excretion (r = 0.67, P < 0.0001) as well as with serum GH levels (r = 0.73, P < 0.0001). CONCLUSION We demonstrated a decreasing molar IGF-l/IGFBP-3 ratio with increasing age in healthy adults and an increased ratio between serum IGF-I and IGFBP-3 levels in acromegalic patients. As IGF-II is normal and IGFBP-1 and IGFBP-2 are inversely correlated to the serum GH levels In the acromegalic patients, we speculate that the molar ratio between IGF-I and IGFBP-3 reflects free (biologically active) IGF-I and Is dependent on GH levels.     

The insulin-like growth factor binding protein 3 ternary complex is reduced in cirrhosis

BACKGROUND/AIMS: In healthy adults, serum insulin-like growth factor I (IGF-I), IGF binding protein 3 (IGFBP-3) and acid labile subunit (ALS) form a 150-kDa ternary complex under the control of growth hormone (GH). Approximately 80-90% of circulating IGF-I is bound to the ternary complex. In cirrhosis the GH/IGF axis is severely disturbed and the individual components of the ternary complex are reduced. However, the degree of ternary complex formation in cirrhosis has not previously been described. METHODS: Serum IGF-I, IGFBP-3, ALS, the 150-kDa ternary complex and IGFBP-3 proteolysis were all measured in six compensated and six decompensated cirrhotic patients and compared to six healthy controls. RESULTS: Patients with compensated cirrhosis had decreased levels of IGF-I (55%), IGFBP-3 (64%) and ALS (53%), and in the decompensated patients these levels were decreased even further: IGF-I (32%), IGFBP-3 (37%) and ALS (27%) compared to healthy controls. The levels of the ternary complex followed this pattern, with low levels seen in the compensated patients (66%) and a further reduction in the decompensated patients (27%). Ternary complex levels correlated negatively with the Child-Pugh score. No increase in IGFBP-3 proteolysis was found in cirrhotic patients compared to healthy controls. CONCLUSION: Cirrhosis is associated with reduced levels of the 150-kDa ternary IGFBP-3 complex correlating with the degree of liver disease.     

The high molecular weight insulin-like growth factor-binding protein complex: epitope mapping, immunoassay, and preliminary clinical evaluation.

Measurements of insulin-like growth factor I(IGF-I), IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS) are important in assessing the GH-IGF axis. As nearly all IGF-I, IGFBP-3, and ALS circulate in a GH-dependent ternary protein complex, direct determination of the complex may be of significant analytical and clinical importance. We evaluated a panel of monoclonal antibodies (mAb) to human IGFBP-3 and classified them into four groups (G-1 to G-4). G-1 antibodies recognized epitopes that mapped at or near IGFBP-3 ligand (IGF)-binding site. This region overlapped with the G-2 defined region, which, in turn, overlapped with G-3 epitopes defined by one antibody (mAb 3). Only G-1 and G-3 antibodies paired without interference. mAb 9 recognized a conformational epitope (G-4), and mAb 10 was nonreactive. In pairwise mixed antibody evaluation, mAbs in G-2 and G-3 showed simultaneous binding to serum IGFBP-3 complexes in combination with an anti-IGF-I or an anti-ALS antibody. On this basis, two novel enzyme-linked immunosorbent assays (ELISAs) involving IGFBP-3/IGF-I (ELISA-1) and IGFBP-3/ALS (ELISA-2) recognition partners were developed, both demonstrating acceptable analytical performance characteristics. IGFBP-3 complexes measured by ELISA-1 and -2 in samples from normal individuals and subjects with GH deficiency, acromegaly, and GH receptor deficiency more tightly correlated with IGF-I, IGFBP-3, and ALS than IGF-II. ELISA-1 determinations were comparatively more age dependent and, in comparison to ELISA-2, showed better discriminations among the various sample groups, particularly among GH receptor deficiency, normal, and GH deficiency subjects. The development of IGFBP-3 complex ELISAs may simplify diagnostic applications and facilitate investigations of the physiological relevance of the ternary complex formation.     

A central domain binding site in insulin-like growth factor binding protein-5 for the acid-labile subunit

Like insulin-like growth factor binding protein-3 (IGFBP-3), IGFBP-5 forms a ternary complex with insulin-like growth factor (IGF)-I or IGF-II, and the acid-labile subunit (ALS). The study of IGFBP-5/IGFBP-6 chimeric proteins with amino-terminal and middle domain swaps, has revealed the existence of a site in the middle domain of IGFBP-5, that binds to ALS in the absence of the IGFBP-5 carboxy-terminal domain. An IGFBP-6 chimeric protein containing the central domain of IGFBP-5 complexed efficiently with ALS, and a carboxy-terminally truncated IGFBP-5 mutant, IGFBP-5'(1-169), also bound to ALS in the presence of IGFs, although with much less potency than full length rhIGFBP-5. In contrast to the latter, IGFBP-5(1-169) preferentially formed ternary complexes with IGF-II rather than IGF-I. These results indicate that a site which binds ALS exists in IGFBP-5 mutants which lack the IGFBP-5 carboxy-terminal domain.     

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.     

Estrogens exert route- and dose-dependent effects on insulin-like growth factor (IGF)-binding protein-3 and the acid-labile subunit of the IGF ternary complex

We have previously shown that exogenous estrogens exert route-dependent effects on serum GH and insulin-like growth factor I (IGF-I) levels. IGF-I circulates as a ternary complex with IGF-binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS). It is not known whether IGFBP-3 and ALS in blood are regulated by estrogen and, if so, whether this is also route dependent. In the present study we investigate the effects on IGFBP-3 and ALS of oral and transdermal estrogens (study 1), of different oral estrogen formulations (ethinyl estradiol, conjugated estrogen, and estradiol valerate; study 2), of different estrogen dosages (study 3) in normal postmenopausal women, and of oral estrogen in hypogonadal GH-deficient women (study 4). Administration of oral, but not transdermal, estrogen in normal postmenopausal women significantly decreased serum levels of IGFBP-3 and ALS (P < or = 0.005). The suppressive effects were similar with different oral estrogen formulations, and the degree of suppression increased with estrogen dosage. In hypogonadal GH-deficient women, oral estrogen treatment also significantly reduced IGFBP-3 and ALS (P = 0.02). The changes in IGF-I in each of the four studies paralleled the changes in both IGFBP-3 and ALS. In conclusion, exogenous estrogens suppress serum IGFBP-3 and ALS in a route- and dose-dependent manner, which are in parallel with the effects on serum IGF-I. These actions of oral estrogen are independent of endogenous GH status.