Ontogeny of insulin-like growth factors (IGF), IGF binding proteins, IGF receptors, and growth hormone receptor mRNA levels in porcine pancreas

We examined the ontogeny of mRNA levels of IGF-I and -II, IGF type 1 (IGFI-R) and type II receptors (IGFII-R), IGF binding protein-1 and -3 (IGFBP-1 and -3), GH receptor (GHR), and tissue concentrations of IGF and IGFBP in the pancreas of pigs. Tissues were collected from fetuses at 90 and 110 d of gestation and from pigs at 1, 21, 90 and 180 d of age. Northern blots were performed using total RNA hybridized with 32P-labeled cDNA probes (human IGF-I and human IGFI-R) and cRNA probes (rat IGF-II, human IGFII-R, human IGFBP-1, pig IGFBP-3, and pig GHR). There were two accelerated growth stages of the pancreas: the first one at 90 d of fetal life, which is characterized by cell hyperplasia (high ratio of DNA to body weight), and the second one at postnatal 90 d, which is attributed to cell hypertrophy (high ratios of pancreatic weight, RNA, and protein to DNA). The level of IGF-II mRNA and its tissue concentration were predominant during fetal life and low thereafter. The IGF-I mRNA level was high during fetal and early postnatal life and decreased thereafter. Messenger RNA levels of IGFI-R, IGFBP-3, and GHR and concentrations of IGFBP-1 and -2 were abundant during fetal and early postnatal life. In conclusion, IGF may be involved in various physiological periods of pancreatic development in pigs.     

Stimulation of intestinal growth is associated with increased insulin-like growth factor-binding protein 5 mRNA in the jejunal mucosa of insulin-like growth factor-I-treated parenterally fed rats

Surgically stressed rats maintained with total parenteral nutrition (TPN) exhibit jejunal atrophy, which can be attenuated by insulin-like growth factor-I (IGF-I) but not by growth hormone (GH) treatment. In order to understand the basis for the selective action of IGF-I, the levels of mRNAs encoding IGF-I, IGF-binding proteins (IGFBPs), IGF-I receptor, and GH receptor/binding protein (GHR/GHBP) were determined in rats given TPN and treated with GH, IGF-I, or GH + IGF-I. GH treatment significantly stimulated hepatic IGF-I mRNA. IGF-I treatment did not alter liver IGF-I mRNA, nor was there any evidence for interaction between GH and IGF-I. Jejunal mucosa IGF-I mRNA was extremely low and was not altered by TPN or by any of the hormonal treatments. The inability of GH to stimulate jejunal growth was not associated with a deficiency in GHR/GHBP mRNA. In jejunal mucosa, IGF-I and GH treatment independently and synergistically stimulated IGFBP-3 mRNA. IGF-I stimulated jejunal IGFBP-5 mRNA, but GH had no effect on IGFBP-5 mRNA. The levels of IGF-I receptor and IGFBP-1, 2, 4, and 6 mRNAs were extremely low and/or were not altered by any of the treatments. These results suggest that the ability of exogenous IGF-I, but not GH, to induce IGFBP-5 mRNA in jejunal mucosa may lead to the selective growth-promoting effect of IGF-I. Jejunal mucosa IGFBP-3 mRNA levels were not correlated with altered growth. We postulate that IGFBP-5 positively modulates the anabolic effects induced by exogenous IGF-I in the jejunum.     

Expression of insulin-like growth factors (IGFs), their receptors and IGF binding protein-3 in normal, benign and malignant smooth muscle tissues

To assess the role of insulin-like growth factors (IGFs) in growth and transformation of normal (myometrium) and tumorous smooth muscle cell (SMC) tissues, in situ hybridization (ISH) analysis for insulin-like growth factor I and II (IGF-I and IGF-II) mRNAs was combined with detection of IGF peptides, their receptors and IGF binding protein-3 (IGFBP-3). mRNAs for both IGFs were detected in smooth muscle cells in normal, benign and malignant SMC tissues, together with the IGF peptides, both IGF receptors and IGFBP-3. This suggests an autocrine role for both IGFs. Leiomyomas had higher IGF-I peptide levels and higher levels of type I IGF receptors than myometrium, supporting the idea that IGFs play a role in the growth and transformation of these tumours. Low-grade leiomyosarcomas contained more IGF-II mRNAs than myometrium and leiomyoma, fewer type II IGF/mannose 6-phosphate receptors and less IGFBP-3 than myometrium and, in addition, fewer IGF-I mRNAs and type I IGF receptors than leiomyoma. Intermediate- and high-grade leiomyosarcomas had intermediate levels of IGF-II mRNAs and peptide, ranging between those in myometrium and low-grade leiomyosarcomas. Thus, growth and transformation of leiomyosarcomas may be regulated by IGF-II, although more markedly in low-grade than in high-grade leiomyosarcomas. In conclusion, the various categories of SMC tissues are associated with a distinct expression pattern of the IGF system. This suggests that each category of SMC tumours arises as a distinct entity and that there is no progression of transformation in these tissues.     

Developmental pattern of fetal growth hormone, insulin-like growth factor I, growth hormone binding protein and insulin-like growth factor binding protein-3

AIMS: To evaluate the developmental pattern of fetal growth hormone (GH), insulin-like growth factor I (IGF-I), GH binding protein (GHBP) and IGF binding protein-3 (IGF-3); to determine the implications for fetal growth. METHODS: Serum GH, IGF-I, GHBP and IGFBP-3 were measured in 53 fetuses, 41 aged 20-26 weeks (group A) and 12 aged 31-38 weeks (group B). Fetal blood samples were obtained by direct puncture of the umbilical vein in utero. Fetal blood samples were taken to rule out beta thalassaemia, chromosome alterations, mother to fetus transmissible infections, and for maternal rhesus factor. GHBP was determined by gel filtration chromatography of serum incubated overnight with 125I-GH. GH, IGF-I and IGFBP-3 were determined by radioimmunoassay. RESULTS: Fetal serum GH concentrations in group A (median 29 micrograms/l, range 11-92) were significantly higher (P < 0.01) than those of group B (median 16.7 micrograms/l, range 4.5-29). IGF-I in group A (median 20 micrograms/l, range 4.1-53.3) was significantly lower (P < 0.01) than in group B (median 75.2 micrograms/l, range 27.8-122.3). Similarly, IGFBP-3 concentrations in group A (median 950 micrograms/l, range 580-1260) were significantly lower than those of group B (median 1920 micrograms/l, range 1070-1770). There was no significant difference between GHBP values in group A (median 8.6%, range 6.6-12.6) and group B (median 8.3%, range 6-14.3). Gestational age correlated positively with IGF-I concentrations (P < 0.0001) and IGFBP-3 (P < 0.0001) and negatively with GH (P < 0.0001). GHBP values did not correlate with gestational age. Multiple regression analysis showed a negative correlation between GH:IGF-I ratio and fetal growth indices CONCLUSIONS: The simultaneous evaluation of fetal GH, IGF-I, IGFBP-3 and GHBP suggests that the GH-IGF-I axis might already be functional in utero. The progressive improvement in the efficiency of this axis in the last part of gestation does not seem to be due to an increase in GH receptors.     

Long [R3] insulin-like growth factor-I reduces growth, plasma growth hormone, IGF binding protein-3 and endogenous IGF-I concentrations in pigs

Growth hormone (GH) improves growth performance in the pig. Analogues of insulin-like growth factor-I (IGF-I) that bind poorly to IGF binding proteins (IGFBP) stimulate growth in the rat but, in contrast, inhibit growth in the pig. This study was designed to determine the effect of IGF peptides alone or in combination with porcine GH (pGH) on growth characteristics and plasma hormone concentrations in finisher pigs. A four-day infusion of Long [R3] IGF-I (LR3IGF-I; 180 micrograms/kg/day) decreased the average daily gain, food intake, and plasma IGFBP-3, IGF-I and insulin concentrations. The mean plasma GH concentration was decreased by 23% and the area under the GH peaks was reduced by 60%. Co-administration of pGH (30 micrograms/kg/day) with LR3IGF-I had no interactive effect on growth performance, and plasma insulin, IGFBP-3 and IGF-I concentrations remained suppressed. The area under the GH peaks was not restored with this combination treatment although mean plasma GH concentrations were elevated in all animals receiving pGH. Infusion of IGF-I (180 micrograms/kg/day) decreased plasma insulin and mean GH concentrations but had no significant effect on IGFBP-3 concentrations. Average daily gain and feed intake were not changed by IGF-I treatment. A combination of IGF-I and pGH injection (30 micrograms/kg/day) increased plasma IGFBP-3 concentrations but plasma insulin levels remained suppressed. Plasma glucose levels were unaffected by any treatment. The study demonstrates that both IGF-I and LR3IGF-I suppress plasma GH concentrations in finisher pigs. This, in turn, may be responsible for the reduction in the plasma concentration of IGF-I, IGFBP-3 and insulin seen in LR3IGF-I-treated animals. The decrease in these parameters may contribute to the inhibitory effect of LR3IGF-I on growth performance in the pig.     

Insulin-like growth factor binding proteins-2 and -3 stimulate growth hormone receptor binding and mitogenesis in rat osteosarcoma cells

GH exerts its biological actions on osteoblasts through a specific high affinity receptor expressed on these cells. GH receptor binding is positively modulated by a number of factors, including retinoic acid and dexamethasone, whereas fetal calf serum strongly decreases the binding. To identify responsible factors in serum, components of serum, the insulin-like growth factors (IGFs)-I and -II, and IGF binding proteins (IGFBPs)-2 and -3 were tested for a possible negative modulatory role. IGF-I and -II decreased [125I]hGH binding at an optimal concentration of 30 ng/ml for IGF-I and 100 ng/ml IGF-II, reducing the binding to 51% and 55%, respectively, of control values. A stimulation of [125I]hGH binding was observed with IGFBP-2 as well as IGFBP-3, inducing an increase to 148% and 151% of control binding at an optimal concentration of 3000 ng/ml for both peptides. The effects of all peptides were dependent on the incubation time, being significantly increased after 8 h of incubation and reaching the full effect thereafter. The effects were declined at 24 h compared with 16 h for IGFBP-2 and -3 but not for IGF-I and -II. Coincubation of the cells with IGF-I and -II and IGFBP-2 and -3 neutralized the effects of the factors alone. In conclusion, these results show that IGF-I and -II on the one hand and IGFBP-2 and -3 on the other hand exert opposite actions on [125I]hGH binding, IGFBP-2 and -3 exerting probably an IGF-independent effect. Further, IGF-I and -II decreased GH receptor messenger RNA (mRNA) levels, as quantified by a solution hybridization ribonuclease protection assay, from 8.65 +/- 1.78 attomoles (amol)/microgram DNA (control) to 2.4 +/- 0.68 and 2.16 +/- 0.92 amol/microgram DNA, respectively. IGFBP-2 increased GH receptor mRNA levels from 5.26 +/- 1.17 (control) to 13.19 +/- 3.48. Incubation with IGFBP-3 did not result in stimulation of GH receptor mRNA levels (8.59 +/- 2.91 amol/microgram DNA). This shows that the mechanism of regulation of the GH receptor is, except for IGFBP-3, at least in part on the mRNA level. Lastly, IGFBP-2 and IGFBP-3 are mitogenic for UMR-106.01 rat osteosarcoma cells, inducing an increase in cell number to 125% and 142% of control cell counts after 48 h of incubation with 1000 ng/ml IGFBP-2 and -3, whereas IGF-I, IGF-II and Long R3 IGF-I did not stimulate proliferation. IGFBP-2 and -3 potentiate hGH induced mitogenesis at low hGH concentrations of both factors, whereas at higher concentrations no such effect is observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Free and total insulin-like growth factors and insulin-like growth factor binding proteins during 14 days of growth hormone administration in healthy adults

The objective was to investigate the effect of growth hormone (GH) administration on circulating levels of free insulin-like growth factors (IGFs) in healthy adults. Eight healthy male subjects were given placebo and two doses of GH (3 and 6 IU/m2 per day) for 14 days in a double-blind crossover study. Fasting blood samples were obtained every second day. Free IGF-I and IGF-II were determined by ultrafiltration of serum. Total IGF-I and IGF-II were measured after acid-ethanol extraction. In addition, GH, insulin, IGF binding protein 1 (IGFBP-1) and IGFBP-3 were measured. Serum-free and total IGF-I increased in a dose-dependent manner during the 14 days of GH administration. After 14 days, serum-free IGF-I values were 610 +/- 100 ng/l (mean +/- SEM) (placebo), 2760 +/- 190 ng/l (3 IU/ m2) and 3720 +/- 240 ng/l (6 IU/m2) (p = 0.0001 for 3 and 6 IU/m2 vs placebo; p = 0.004 for 3 IU/m2 vs 6 IU/m2). Total IGF-I values were 190 +/- 10 micrograms/l (placebo), 525 +/- 10 (3 IU/m2), and 655 +/- 40 micrograms/l (6 IU/m2) (p < 0.0001 for 3 and 6 IU/m2 vs placebo; p = 0.04 for 3 IU/m2). There were no differences in the levels of free or total IGF-II during the three study periods. Insulin-like growth factor binding protein 1 was decreased during GH administration (p = 0.04 for placebo vs 3 IU/m2; p = 0.006 for placebo vs 6 IU/m2). In conclusion, fasting serum free IGF-I increased dose dependently during GH administration and free IGF-I increased relatively more than total IGF-I. This may partly be due to the decrease in IGFBP-1.     

Use of insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 in the diagnosis of acromegaly and growth hormone deficiency in adults

The goal of our study was to compare the clinical usefulness of plasma insulin-like growth factor-I (IGF-I) (with and without binding protein extraction) and IGF binding protein-3 (IGFBP-3) measurements in the diagnosis of growth hormone (GH) disorders in adults. IGF-I and IGFBP-3 concentrations were measured in 25 acromegalic and 25 GH-deficient adult (GHDA) subjects (20-76 years) by comparison to a control population (n = 81) after age and sex stratification. In untreated acromegaly, IGF-I and IGFBP-3 were clearly increased (10 times the mean of controls for unextracted IGF-I, 4 times for extracted IGF-I and 2 times for IGFBP-3). Using the mean + 2SD of the control population as the cut-off point, the sensitivity of IGF-I for the diagnosis of acromegaly was higher than that of IGFBP-3 (unextracted IGF-I: 96% and extracted IGF-I: 100% vs IGFBP-3: 76%). In GHDAs, IGF-I and IGFBP-3 were decreased (34% of the mean of controls for unextracted IGF-I, 37% for extracted IGF-I and 70% for IGFBP-3). Using the mean - 2SD of the control population as the cut-off point, the sensitivity of IGF-I measurement for the diagnosis of GHDA was relatively low, but better for unextracted (68%) than for extracted IGF-I (52%). The sensitivity of IGFBP-3 was much lower (36%), thus invalidating this parameter for the diagnosis of GHDA. Our observations demonstrate that IGF-I measurement is a more powerful tool than IGFBP-3 measurement for the diagnosis of GH disorders in adults. Both IGF-I and IGFBP-3 are very useful for the diagnosis of acromegaly, but they are less reliable for diagnosing GHDA, as normal IGF-I or IGFBP-3 values do not rule out GH deficiency.     

Feeding colostrum increases circulating insulin-like growth factor I in newborn pigs independent of endogenous growth hormone secretion

Our objective was to examine the influence of feeding and endogenous GH secretion on circulating IGF-I in colostrum-deprived newborn pigs fed colostrum (n = 4), formula (control, n = 4), or water (n = 4). In another four formula-fed pigs, GH was ablated (GRF-A) with two intravenous injections of a GH releasing-factor antagonist (N-Ac-Tyr1,D-Arg2)-GRF(1-29)-NH2. Blood was serially sampled in all pigs to measure plasma IGF-I and GH profiles. Feeding increased plasma IGF-I concentration two- to fourfold and decreased GH secretion. Despite a more than 80% decrease in the plasma GH in GRF-A pigs, the circulating IGF-I concentration was similar to that in control pigs. In colostrum-fed pigs, plasma IGF-I was higher than that in control pigs, despite equal nutrient intake and lower circulating GH. There were no differences in plasma IGF binding protein (IGFBP)-3 levels among the treatment groups. However, the relative abundance of plasma IGFBP-4 was lower, and that of IGFBP-1 higher, in unfed pigs than in any of the three fed groups. The plasma insulin concentration was not different among fed pigs, but it was lower in unfed pigs. Our results indicate that the circulating IGF-I concentration is more dependent on nutrient intake than on GH in newborn pigs, despite relatively high GH concentrations. However, because the nutrient content in the formula was designed to match that of colostrum, a factor other than nutrient intake and GH was responsible for the maximal increase in circulating IGF-I concentration observed in colostrum-fed pigs.     

Short stature associated with high circulating insulin-like growth factor (IGF)-binding protein-1 and low circulating IGF-II: effect of growth hormone therapy

We report a case of short stature associated with high circulating levels of insulin-like growth factor (IGF)-binding protein-1 (IGFBP-10 and low levels of IGF-II responsive to pharmacological treatment with GH. Our patient suffered severe growth failure from birth (2.06 SD below the mean for normal full-term boys, and 5.2 and 7.3 SD below the mean at 5 and 10 months). Studies carried out before referral to our pediatric unit included normal 46,XY karyotype and normal encephalic imaging. Other endocrine and metabolic alterations and other systemic diseases were excluded. At 1.7 yr of age (length, 6.1 SD; weight, 4.6 SD; head circumference, 1.4 SD below the mean, respectively) the patient was referred to our pediatric unit. The baseline GH concentration was 31 microg/L, and the peak after an arginine load was 59.6 microg/L. In the same samples GH bioactivity was nearly superimposable (RIA/Nb2 bioactivity ratio = 0.9). Fasting insulin and glucose concentrations were 7.4 microU/mL and 65 mg/dL, respectively, both normally responsive to an oral glucose load. GH insensitivity was excluded by a basal IGF-I concentration (64 ng/mL) in the normal range for 0- to 5-yr-old boys and its increase after 2 IU/day hGH administration for 4 days. IGFBP-3 (0.5 microg/mL) was slightly reduced, whereas IGFBP-1 (2218 and 1515 ng/mL in two different basal samples) was well above the normal values for age and was suppressible by GH (maximum suppression, -77% at 84 h) and glucose load (maximum suppression, -46% at 150 min). The basal IGF-II concentration was below the normal range (86 ng/mL), whereas IGFBP-2 was normal (258 ng/mL). Analysis of the promoter region of IGFBP-1 and IGF-II failed to find major alterations. Neutral gel filtration of serum showed that almost all IGF-I activity was in the 35- to 45-kDa complex, coincident with IGFBP-1 peak, while the 150-kDa complex was absent, although the acid-labile subunit was normally represented. At 2.86 yr (height, 65.8 cm; height SD score, -7.3; height velocity SD score, -5) the patient underwent treatment with 7 IU/week human GH; after 4 months, the patient's height was 68.5 cm (height SD score, -6.9) corresponding to a growth velocity of 8.3 cm/yr (0.3 height velocity SD score). IGFBP-1 was reduced (216 ng/mL), although still in the high range, whereas IGF-I (71 ng/mL), IGFBP-3 (0.62 microg/mL), and IGF-II (111 ng/mL) were only slightly increased. The IGF-I profile showed activity in the 150-kDa region. In conclusion, we speculate that the increased IGFBP-1 values found in this patient produce 1) inhibition of IGF-I biological activity and, therefore, a resistance to IGF-I not due to a receptor defect for this hormone; 2) inhibition of formation of the circulating 150-kDa ternary complex and, therefore, an accelerated clearance rate of IGF peptides; 3) inhibition of the feedback action on GH, leading to increased GH levels, which could suggest the diagnosis of GH insensitivity syndrome; and 4) inhibition of body growth.     

Growth advantage of human leiomyoma cells compared to normal smooth-muscle cells due to enhanced sensitivity toward insulin-like growth factor I

Human uterine leiomyomas exhibit increased IGF-I binding compared to myometrium, while both tissues show IGF-I gene expression. In this study we have examined the functional importance of these findings by testing the presence of IGF-I in 15 leiomyoma biopsies and in 18 myometrium biopsies and the capacity of smooth-muscle cells cultured from these tissues to react to IGF-I. The mean IGF-I peptide concentration in leiomyomas was 3 times higher than in myometrium. This resulted from increased IGF-I uptake in leiomyomas rather than from increased synthesis, as these tissues contain higher concentrations of type-I IGF receptors, as detected by immunohistochemistry, and equal levels of IGF-I mRNA. Blocking IGF-I transport with cytochalasin-B and with the type-I IGF receptor blocking antibody alpha IR3 in cultured cells induced decreased immunostaining intensity for IGF-I in most myometrium and leiomyoma cultures, indicating that the detected IGF-I is internalized. Depending on the culture conditions, IGF-I administration yielded increased survival or a higher proliferation rate in leiomyoma cultures than in myometrium cultures, indicating the increased importance of exogenous IGF-I for the growth of transformed smooth-muscle cells. We conclude that the increased concentrations of type-I IGF receptors in leiomyoma compared to myometrial smooth-muscle cells are functional with respect to the enhanced internalization of IGF-I and that they provide these tumor cells with a growth advantage compared to their normal counterparts.     

Effect of chronic treatment with octreotide nasal powder on serum levels of growth hormone, insulin-like growth factor I, insulin-like growth factor binding proteins 1 and 3 in acromegalic patients

Octreotide nasal powder is a delivery system of the somatostatin analogue developed to overcome the inconvenience of repeated subcutaneous administrations. Eight patients with clinically active acromegaly were treated for three months with octreotide nasal powder which was administered at the initial dosage of 0.125 mg tid, doubling the dosage up to 2 mg tid in order to obtain a mean GH value below 5 micrograms/l during 8 daytime hours. In 4 of these patients, treatment was prolonged till the sixth month. Blood samples were taken on days 15, 29, 43, 55, 90, 120, 150, 180 for GH, IGF-I, IGFBP-3, IGFBP-1 and insulin measurements. Before treatment, mean daytime GH and morning IGF-I serum levels were both increased but not correlated with each other. Serum IGFBP-3 levels were higher than normal and positively correlated with those of GH, IGF-I and insulin. Insulin levels were elevated and positively correlated with those of GH but not with those of IGF-I and IGFBP-1. Serum IGFBP-1 levels were in the low normal range and not correlated with any of the other parameters. Treatment with octreotide nasal powder induced in all patients a marked decrease of GH which lowered below 5 micrograms/l in 7/8 patients and IGF-I levels, which fell within the normal range in 1 patient. Serum IGFBP-3 and insulin concentrations decreased by 26% and 71%, respectively, and those of IGFBP-1 underwent an only transient increase in 5/8 patients. Opposite changes of insulin and IGFBP-1 levels, with a decrease of the former followed by an increase of the latter were noted during the 8 hours following an octreotide nasal insufflation. During chronic octreotide treatment, positive correlations were found between GH and IGF-I, GH and IGFBP-3, IGF-I and IGFBP-3, insulin and IGFBP-3 and insulin and IGF-I. An improvement of the clinical picture was registered in all patients after a few days of octreotide nasal powder administration. Treatment was well tolerated, with only mild side effects and no significant changes in the nasal mucosa, and the patients' compliance was excellent.     

Growth hormone bioactivity, insulin-like growth factors (IGFs), and IGF binding proteins in obese children

In obese children, both spontaneous and stimulated growth hormone (GH) secretion are impaired but a normal or increased height velocity is usually observed. This study was undertaken to explain the discrepancy between impaired GH secretion and normal height velocity. We evaluated the GH bioactivity (GH-BIO), GH serum level by immunofluorimetric assay (GH-IFMA), insulin-like growth factor-I (IGF-I), IGF-II, and IGF binding protein-1 (IGFBP-1), IGFBP-2, and IGFBP-3 in 21 prepubertal obese children (13 boys and eight girls) aged 5.7 to 9.4 years affected by simple obesity and in 32 (22 boys and 10 girls) age- and sex-matched normal-weight controls. The results were as follows (obese versus [v] controls): GH-IFMA, 4.84 +/- 3.54 v 23.7 +/- 2.04 microg/L (P < .001); GH-BIO, 0.60 +/- 0.45 v 1.84 +/- 0.15 U/mL (P < .001); IGF-I, 173.8 +/- 57.2 v 188.6 +/- 132.6 ng/mL (nonsignificant); IGF-II, 596.1 +/- 139.7 v 439.3 +/- 127.4 ng/mL (P < .001); IGFBP-1, 23.25 +/- 14.25 v 107 +/- 165.7 ng/mL (P < .05); IGFBP-2, 44.37 +/- 62.18 v 385.93 +/- 227.81 ng/mL (P < .001); IGFBP-3, 3.31 +/- 0.82 v 2.6 +/- 0.94 microg/mL (P < .05); and IGFs/IGFBPs, 1.32 +/- 0.32 v 1.07 +/- 0.34 (P < .05). In conclusion, in prepubertal obese children, not only immunoreactive but also bioactive GH concentrations were low. In these subjects, therefore, nutritional factors and insulin may contribute to sustain normal growth also by modulating several components of the IGF-IGFBP system.     

Insulin-like growth factors (IGF) I and II and IGF binding proteins 1, 2 and 3 during low-dose growth hormone (GH) infusion and sequential euglycemic and hypoglycemic glucose clamps: studies in GH-deficient patients

To evaluate the short-term effects of growth hormone (GH), insulin and different levels of glycemia on insulin-like growth factors (IGF) I and II and IGF binding proteins (IGFBP) 1, 2 and 3, we studied six GH-deficient adolescents during a night and the following day in the postabsorptive (basal) state followed by sequential euglycemic (5 mmol/l) and hypoglycemic (3 mmol/l) glucose clamps concomitant with an intravenous infusion (starting at 24.00 h) of GH (35 micrograms/h) or saline. Current GH therapy was withdrawn 24 h prior to each study. Nocturnal levels of IGF-I, IGF-II, IGFBP-2 and IGFBP-3 remained stable during both studies. Nocturnal serum IGFBP-1 increased and correlated inversely with insulin in both studies. Regression analysis revealed a significant inverse correlation between mean nocturnal IGFBP-2 and IGFBP-3 levels. During the daytime, serum IGF-I declined slowly during saline infusion, whereas serum IGF-II remained stable in both studies. Serum IGFBP-1 displayed a gradual significant decline during the basal state and the euglycemic and hypoglycemic clamps seemed to be unaffected by GH levels. By contrast, serum IGFBP-2 remained stable during the same period in both the GH and the saline study. Serum IGFBP-3 declined insignificantly during the daytime in the saline study.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of growth hormone, prolactin, insulin-like growth factors, and gonadotropins on progesterone secretion by porcine luteal cells

Growth hormone (GH) and IGF-I have receptors within the corpus luteum (CL) and stimulate CL function. Our objective was to investigate the effects of GH, prolactin (PRL), IGF-I, IGF-II, LH, and FSH on progesterone secretion by porcine luteal cells during mid-pregnancy. Gilts (crossbred Yorkshire/Landrace) were slaughtered on d 44 of pregnancy and CL were collected. Large and small luteal cells (LLC and SLC, respectively) were obtained from dissociated CL and separated by elutriation. Luteal cells were incubated with 0, 1, 10, or 100 ng/mL of GH, PRL, IGF-I, IGF-II, LH, and FSH or combinations of 10 ng/mL of these reagents for 24 or 48 h. Culture media were harvested and concentrations of progesterone analyzed by radioimmunoassay. Growth hormone, PRL, and IGF-I increased (P < .05; 100 ng/mL dose) concentrations of progesterone in media of LLC. Insulin-like growth factor-II, LH, and FSH had no effect on progesterone in LLC cultures. In SLC cultures, GH, PRL, IGF-I, IGF-II, and FSH failed to stimulate progesterone secretion, whereas LH increased progesterone secretion (linear effect of dose; P < .05). Combinations (10 ng/mL each hormone) of GH and IGF-I or PRL and IGF-I increased progesterone secretion by LLC compared with control, GH, PRL, or IGF-I alone (P < .05). Similar combinations of GH or PRL with IGF-I had no effect on SLC. Conclusions are that GH and PRL are stimulatory to progesterone secretion by LLC (location of GH receptor) and SLC are responsive to LH during mid-pregnancy. Both GH and PRL are synergistic with IGF-I for increased progesterone secretion.     

Biosensor measurement of the binding of insulin-like growth factors I and II and their analogues to the insulin-like growth factor-binding protein-3

Most insulin-like growth factor (IGF) molecules in the circulation are found in a 150-kDa complex containing IGF-binding protein-3 (IGFBP-3) and an acid-labile subunit, which does not itself bind IGF. Affinities (Kd values) between 0.03 and 0.5 nM have been reported for IGF-I/IGFBP-3 binding, but no kinetic data are available. In this study we measured the high affinity binding of unlabeled IGFs and IGF analogues to recombinant unglycosylated IGFBP-3, using a BIAcoretrade mark instrument (Pharmacia Biosensor AB). IGF-I binding showed fast association and slow non-first-order dissociation kinetics, and an equilibrium Kd of 0.23 nM. IGF-II had similar kinetics with slightly higher affinity. Analogues with mutations in the first 3 amino acids of the B-region (des(1-3) IGF-I and long IGF-I) showed 25 and 50 times lower affinity than IGF-I. Replacement of residues 28-37 by Gly-Gly-Gly-Gly or deletion of residues 29-41 in the C-region had little effect on the kinetic parameters, contrasting with the markedly impaired binding of these analogues to the IGF-I receptor. Swapping of the disulfide bridges in IGF-I and the C-region mutants decreased the affinity dramatically for IGFBP-3, primarily by decreasing the association rate. Insulin had approximately 1000 times lower affinity than IGF-I.     

Long-term effects of insulin-like growth factor (IGF)-I on serum IGF-I, IGF-binding protein-3 and acid labile subunit in Laron syndrome patients with normal growth hormone binding protein

A minority of patients with Laron syndrome have normal serum GH binding protein (GHBP), indicating that the defect is elsewhere than in the extracellular domain of the GH receptor. We have evaluated the effect of long-term IGF-I treatment on serum IGF-binding protein (IGFBP)-3 and the acid-labile subunit (ALS) in three sibling with Laron syndrome caused by a GH post-receptor defect and with normal GHBP. The children (a boy aged 3 years, a girl aged 4 years and a boy aged 10 years) were treated by daily s.c. injection of IGF-I in a dose of 150 micrograms/kg. IGFBP-3 was measured by RIA and Western ligand blotting, ALS by RIA. Based values of IGFBP-3 and ALS were low. During IGF-I treatment, the IGFBP-3 concentrations in the girl gradually increased, whereas in the boys there was a 60% decrease during the first week, followed by gradual increase towards baseline. The ALS concentrations followed a similar pattern. We conclude that IGF-I treatment induces and initial suppression and then an increase in the IGFBP-3 and ALS concentrations, confirming data from animal experiments that IGFBP-3 synthesis is not solely under GH control. The differences in responsiveness between the female and male siblings may reflect genetic differences, or lower circulating concentrations of IGF-I in the boys compared with the girl.     

Factors regulating insulin-like growth factor-binding protein-3 binding, processing, and potentiation of insulin-like growth factor action

In this study, we investigated the effects of various biochemical and pharmacological agents on insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) cell binding and action in cultured bovine fibroblasts. When cells were preincubated for 48 h with 50 nM recombinant human (rh) IGFBP-3, IGF-I-stimulated [3H]aminoisobutyric acid ([125H]AIB) uptake was enhanced 2- to 3-fold. The addition of cytoskeletal disrupting agents during the preincubation with rhIGFBP-3 did not affect IGFBP-3 potentiation of IGF-I action, nor did a variety of serine, aspartate, and metalloproteinase inhibitors. On the other hand, ammonium chloride and chloroquine, weak bases that neutralize the pH of acidic cell compartments, blocked IGFBP-3 potentiation of IGF-I-stimulated [3H]AIB uptake. Chloroquine and ammonium chloride had no effect alone and did not inhibit IGF-I receptor binding or action in the absence of rhIGFBP-3. Bafilomycin A, a specific inhibitor of ATP-dependent hydrogen ion pumps, also inhibited IGFBP-3 potentiation of IGF-I-stimulated [3H]AIB uptake. Competitive [125I]IGF-I binding and affinity cross-linking experiments suggested structure/function changes in cell-bound IGFBP-3 that were altered in the presence of chloroquine and bafilomycin. Heparin markedly decreased initial IGFBP-3 cell adherence, but could not promote dissociation of IGFBP-3 from cells after the 48-h preincubation. Moreover, heparin did not inhibit IGFBP-3 potentiation of IGF-I action. In summary, these data indicate that IGFBP-3 undergoes specific pH-dependent structural and/or environmental modifications that mediate the enhancing effect of IGFBP-3 on IGF-I action in bovine fibroblasts. They also suggest that IGFBP-3 binding to heparin-like molecules on the cell surface is not directly involved in this process.