Regulation of the growth hormone-independent growth factor-binding protein in children

Regulation of the diurnal variation of the GH-independent insulin-like growth factor-binding protein (BP-28) was studied in 53 children who underwent various investigations for possible endocrine abnormalities. The plasma BP-28 levels increased 12-fold from 8 +/- 2 (+/-SE) micrograms/L at 2100 h to a peak level of 109 +/- 15 micrograms/L between 0600 and 0800 h. This rise was inversely related to plasma insulin levels and was unrelated to plasma cortisol levels. The overnight rise of plasma BP-28 was significantly altered in children who ate a light meal at 0130 h; in them BP-28 levels started to fall after 0300 h, reached nadir levels at 0400 h, began to rise again by 0700 h, and returned to control levels by 0800 h. Such changes did not occur in children given water alone. From the peak early morning level, plasma BP-28 fell to basal levels in children given oral glucose at 0800 h; the t1/2 of the fall was 55 +/- 9 (+/-SE) min. In children who continued to fast, plasma BP-28 did not fall but, rather, increased from 144 +/- 12 micrograms/L at 0800 h after 10 h of fasting to 239 +/- 30 micrograms/L by 1600 h. The induction of hypoglycemia by insulin given at 0945 h after an overnight fast caused a similar but more rapid rise in plasma BP-28 to 668 +/- 317 micrograms/L (range, 208-1763 micrograms/L) by 1230 h. These results suggest that the diurnal variation of plasma BP-28 concentrations in children is not due to an intrinsic rhythm, but is regulated by the metabolic status of the child.     

Endogenous and stimulated GH secretion,urinary GH excretion,and plasma IGF-I and IGF-II levels in prepubertal children with short stature after intrauterine growth retardation*

OBJECTIVE The pathophysiological mechanisms underlying the failure of catch up-growth in children with short stature after Intrauterine growth retardation (IUGR) remain obscure. Since GH secretion disturbances might play a role in the growth retardation of these children we have Investigated various aspects of the GH/IGF axis. DESIGN Cross-sectional study in one group of patients. PATIENTS Forty prepubertal children (15 glrls/25 boys; mean age (range) 7·5 years (3·4-10·8)) with short stature (height below the third centile) after IUGR, defined as a birth length below the third centile for gestational age, were studied. MEASUREMENTS GH secretion was determined by a 24-hour plasma GH profile (sampling every 20 minutes) and, on a separate occasion, by a standard arginine provocation test (ATT). Plasma IGF-I and IGF-II levels were measured at the start of the OH profile. Urine was collected to measure urinary OH levels. Plasma and urinary OH were determined by double antibody RIA. IGF-I and IGF-II were determined by specific RIA after acid chromatography. The 24-hour OH profiles were analysed using Pulsar. RESULTS Endogenous GH secretion was similar for boys and girls. Boys had significantly lower mean GH levels compared to healthy controls. Forty per cent of the children met our criterla for a normal 24-hour GH profile (group A; n = 16) and 60% (n = 24) did not. We subdivided these 24 children into two groups: group B (n = 14) (children with either mean GH levels less than controls but with at least one spontaneous GH peak above 20 μ/I and children with normal mean GH levels but with no OH peak above 20 mull (subnormal 24-hour GH profile)) and group C (n = 10) (children with mean GH levels less than controls and no OH peak above 20 μ/l (low 24-hour GH profile)). The GH secretory abnormalities were due to a decrease in pulse amplitude, not in pulse frequency. Mean (SD) maximal GH response during ATT was 22·3 (12·1) μ/I. Nineteen children (47·5%) had a maximal OH value <20mU/I. Moderate, but significant, correlations were found between several 24-hour GH profile characteristics and the maximal OH response during All (r = 0·31-0·35; P<0·05). Mean (SD) overnight urinary GH excretion was 3·8 (2·1) and 4·4 (3·5) μU/night for boys and girls, respectively. Compared to healthy schoolchildren, overnight urinary GH was lower in boys, but not in girls. Mean (SD) IGF-I and IGF-II SDS levels for chronological age were ?0·88 (1·40) and ?0·64 (1·48), respectively. Plasma IGF-I and IGF-II levels were significantly reduced compared to controls. Height SDS_CA or height velocity SDS_CA did not correlate with either spontaneous or stimulated OH secretion, urinary OH excretion or plasma IGF-I or IGF-II levels. CONCLUSIONS Our study indicates that 50-60% of children with short stature after Intrauterine growth retardation have 24-hour GH profile abnormalities and/or subnormal responses to arginine provocation, while mean plasma IGF-I and IGF-II levels are significantly reduced, indicating GH Insufficiency. Urinary GH excretion is lower in boys, but not in girls. The precise mechanism of the failure to catch up growth needs further elucidation. It seems justified to start clinical trials in order to investigate whether treatment with exogenous GH might be beneficial for these Children.     

Growth hormone increases insulin-like growth factor-I (IGF-I) and decreases IGF-II in plasma of growing pigs

Insulin-like growth factor-I (IGF-I) and IGF-II have been measured in plasma obtained from male and female pigs of two strains during daily administration of pituitary-derived porcine GH (pGH; 100 micrograms/kg) from 60 to 90 kg body weight. Each plasma sample was first chromatographed to separate the IGF from binding proteins in order to obtain reliable measurements. IGF-I concentrations showed no differences between strains, but were higher in untreated males (497 +/- 43 (S.E.M.) micrograms/l) than females (299 +/- 15 micrograms/l). GH-treated animals had two-fold higher concentrations of IGF-I. IGF-II concentrations were not significantly different between sexes or strains, but were decreased in pigs treated with pGH (299 +/- 28 micrograms/1) compared with controls (431 +/- 32 micrograms/l). Binding protein concentrations, measured as interference in the IGF-I and IGF-II assays, were not different between sexes or strains, but were increased in pGH-treated animals. Taken together, these results indicate that in addition to the expected increase in IGF-I concentrations, exogenous administration of pGH to pigs leads to an increase in IGF-binding protein and a depression in IGF-II concentrations.     

Two immunoreactive binding proteins for insulin-like growth factors in human amniotic fluid: relationship to fetal maturity

A binding protein for insulin-like growth factors (IGFs) has been purified from human amniotic fluid by IGF-I affinity chromatography and high performance reverse phase chromatography. This protein, with an apparent molecular mass of 28K nonreduced and 34K reduced, had an identical amino-terminus to a previously purified binding protein from amniotic fluid and to placental protein 12. The purified preparation (BP-28) bound both IGFs with high affinity [Ka, 6.55 +/- 2.24 (+/- SD) L/nmol for IGF-I and 3.23 +/- 1.05 L/nmol for IGF-II], with approximately 0.5 mol binding sites/mol BP-28 for either ligand. A 53K IGF-binding protein purified from human plasma (BP-53) did not cross-react in a RIA for BP-28, and BP-28 had less than 0.1% molar cross-reactivity in a RIA for BP-53. Human amniotic fluid reacted strongly in both assays. Fractionation of amniotic fluid samples by reverse phase chromatography showed that BP-28 and BP-53 immunoreactivities were present on separate proteins. In 40 third trimester amniotic fluid samples selected to cover a wide range of lecithin to sphingomyelin ratios, the mean concentrations of BP-28 and BP-53 were 37.6 +/- 17.6 (+/- SD) and 4.6 +/- 1.6 mg/L, respectively. Significant negative correlations were found between the levels of both BP-28 and BP-53 and the lecithin to sphingomyelin ratio, suggesting an association between the levels of both proteins and the degree of fetal maturity. A significant positive association was also found between the levels of BP-28 and BP-53. We conclude that the 28K IGF-binding protein from amniotic fluid, like the previously purified 53K binding protein, has high affinity for both IGF-I and IGF-II, that it coexists in amniotic fluid with BP-53 or a related protein, and that the levels of both proteins decline with increasing fetal maturity.     

Changes in insulin-like growth factors I and II and their binding protein after a single intramuscular injection of growth hormone

The concentrations of insulin-like growth factors I and II (IGF-I and IGF-II) and their binding proteins in serum were measured in 10 GH-deficient patients before and after a single 6-IU injection of GH. Serum IGF-I concentrations were initially low, increased significantly by 8 and 24 h, and decreased to pretreatment levels 48 and 72 h after GH administration. Serum IGF-II concentrations also were low initially and did not increase by 8 and 24 h, but were, however, significantly higher 48 and 72 h after GH administration. In GH-deficient patients before GH administration, binding of IGF-I or IGF-II to serum proteins was restricted primarily to proteins of 50K mol wt. Little or no binding to proteins of 150,000 mol wt was found. By 8 and 24 h after GH injection, IGF-I, but not IGF-II, bound primarily to a protein(s) of 150K mol wt, as in normal subjects. IGF-II remained bound to a 50K mol wt protein. By 48 and 72 h after administering GH, however, the binding pattern was reversed, and IGF-II, but not IGF-I, bound predominantly to a protein(s) of 150K mol wt. Our data demonstrate both a temporal dissociation in the responses of IGF-I and IGF-II to GH and a similar temporal dissociation in the binding of IGF-I and IGF-II to the large mol wt (150K) binding protein. This dissociation, particularly the latter, may provide a means for better characterization of protein fractions in binding IGF, particularly in terms of specificity.     

Measurement of developmental changes in plasma insulin-like growth factor-I levels of broiler chickens by radioreceptor assay and radioimmunoassay

The main purpose of this study was to examine the relationship between insulin-like growth factor-I (IGF-I) and growth hormone (GH) during embryonic and posthatching development of broiler chickens. Two heterologous assays were validated for measurement of IGF-I in chicken and turkey plasma. A radioreceptor assay (RRA), utilizing microsomal membranes prepared from human placenta, was modified and validated for measurement of IGF peptide (mainly IGF-I). A double-antibody radioimmunoassay (RIA) was validated for measurement of immunoreactive IGF-I levels in chicken and turkey plasma. In both assay systems, recombinant-derived human IGF-I was used for standards and trace hormone. Hypophysectomy in turkey poults reduced plasma levels of IGF (RRA) by 35% and IGF-I (RIA) by 59% as compared to intact control turkeys. In Experiment 1, 14 chicken embryos were bled at 15, 17, 19, and 21 days of incubation and at 1 week of age to determine plasma levels of IGF-I and GH. Plasma IGF levels (RRA) remained constant during late incubation, but increased significantly (P less than 0.05) at 1 week of age. Plasma IGF-I levels (RIA) declined 2 days before hatching; however, plasma levels of IGF-I were sharply elevated (P less than 0.05) at 1 week of age. Plasma GH concentrations were low in embryos and were greatly elevated (P less than 0.05) at hatching (21 days of incubation) and at 1 week of age. In Experiment 2, 12 different broiler cockerels were weighed and then bled by cardiac puncture each week from hatching (1 day of age) to 7 weeks of age. The plasma profiles of IGF, IGF-I, GH, triiodothyronine (T3), and thyroxine (T4) were each compared to relative growth rate by analysis of covariance. Plasma IGF and IGF-I levels increased progressively from 0 to 3 weeks of age and were maintained in a plateau from 3 to 7 weeks of age. Plasma GH levels reached a peak at 4 weeks of age, but declined sharply thereafter, while IGF and IGF-I levels remained elevated. Plasma T3 concentrations were progressively increased and reached peak concentrations at 3 weeks of age, while plasma T4 levels increased only at 6 and 7 weeks of age. There was a high correlation (P less than 0.01) between relative growth rate and age-related changes in plasma levels of IGF (r = 0.96), IGF-I (r = 0.97), and T3 (r = 0.94); however, there was no correlation between relative growth rate and changes in plasma GH or T4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synergistic interaction between insulin-like growth factors-I and -II in central regulation of pulsatile growth hormone secretion.

Insulin-like growth factor (IGF)-I and -II peptides, receptors, mRNAs, and binding proteins are widely distributed in the central nervous system (CNS), yet their physiological role in the brain remains largely unknown. While earlier in vivo studies in the rat suggested that IGF-I may participate in feedback regulation of GH secretion at a CNS level, the preparations used were only partially pure. The recent availability of purified recombinant IGF-I and -II peptides prompted us to reexamine the involvement of the IGFs in vivo in central regulation of pulsatile GH secretion. Five groups of free-moving adult male rats bearing chronic intracerebroventricular (icv) and intracardiac venous cannulae were icv administered IGF-I (in doses of 0.5, 2, 3, and 10 micrograms) or the acid-saline vehicle; an additional group received 1 microgram of the potent IGF-I analog, long R3 IGF-I. Spontaneous 6-h plasma GH secretory profiles were obtained from all groups. Vehicle-injected control animals exhibited the typical pulsatile pattern of GH secretion, with most peak GH values above 150 ng/ml and trough levels below 1.2 ng/ml. Central administration of IGF-I alone or long R3 IGF-I at all doses tested failed to alter the pulsatile pattern of GH release; there were no significant differences in GH peak amplitude, GH trough level, GH interpeak interval, or mean 6-h plasma GH level compared to those in vehicle-injected controls. In a second study, designed to determine the effects of central administration of IGF-I and IGF-II, in combination, icv injection of 1 microgram IGF-I and 1 microgram IGF-II resulted in a marked suppression in the amplitude of spontaneous GH secretory bursts approximately 3 h after injection; both GH pulse amplitude (43.5 +/- 5.6 vs. 130.6 +/- 14.6 ng/ml; P less than 0.001) and mean plasma GH level (16.3 +/- 1.9 vs. 35.2 +/- 1.8 ng/ml; P less than 0.001) were severely reduced 3-6 h after injection compared to those in vehicle-injected controls. These results demonstrate that IGF-I alone does not play a physiologically important role in feedback regulation of GH secretion at the level of the CNS. Our findings suggest a synergistic interaction between IGF-I and -II in the brain for central control of pulsatile GH secretion.     

Insulin-like growth factor-binding proteins (IGF-BPs) produced by human skin fibroblasts: immunological relationship to other human IGF-BPs

We have characterized the insulin-like growth factor-binding protein (IGF-BP) produced by neonatal human skin fibroblasts in monolayer culture using antibodies specific for the acid-stable subunit of the 150K GH-dependent IGF-BP complex, BP-53, and the amniotic fluid IGF-BP, BP-28. Fibroblasts produced 65.3 +/- 10.4 ng/ml.72 h (SE; n = 6) immunoreactive BP-53 in serum-free medium; this was stimulated by increasing fetal bovine serum in the medium up to 385.3 +/- 49.0 ng/ml.72 h at 10% serum. Epidermal growth factor (EGF) also caused dose-dependent stimulation of BP-53 production, with a maximal effect (3-fold increase) at 30 ng/ml EGF. No immunoreactive BP-28 production was detectable in the presence or absence of serum or EGF. Neutral gel chromatography of serum-free medium revealed a peak of immunoreactive BP-53 at about 50K, with a smaller species at 20-30 K. Serum- and EGF-stimulated cells produced higher levels of about 50K BP-53, and an additional peak of immunoreactivity at 150K was present in serum-stimulated, but not EGF-stimulated, samples. Comparison of IGF-I and IGF-II binding by fibroblast BP-53 revealed slightly higher IGF-II than IGF-I binding, and association constants of 3-4 x 10(10) liter/mol for both IGFs, similar to BP-53 from human plasma. Affinity labeling of acid-stripped medium followed by nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed specifically cross-linked IGF-binding species of 60K (identical to labeled plasma BP-53), 42K, and 37K. Only the 60K and 42K complexes were precipitable by antiserum to plasma BP-53, and none was precipitable by anti-BP-28 serum, suggesting that the 37K band might represent a third class of IGF-BP. We conclude that neonatal skin fibroblasts produce no BP-28, but do produce two IGF-BPs immunologically homologous to human plasma BP-53, one of which shows size and IGF-binding characteristics identical to the plasma protein.     

Circulating levels and molecular distribution of the acid-labile (alpha) subunit of the high molecular weight insulin-like growth factor-binding protein complex.

A RIA is described for the acid-labile (alpha) subunit of the high mol wt insulin-like growth factor (IGF)-binding protein complex, a glycoprotein of approximately 85,000 daltons (approximately 85K) which combines with the GH-dependent binding protein (BP-53 or IGFBP-3) and IGF-I or IGF-II to form the complex. The assay shows relative specificity for higher primate species. Whereas amniotic fluid, cerebrospinal fluid, and seminal plasma contain virtually no immunoreactive alpha-subunit, the protein is easily detectable in 0.5-microL serum samples. Serum alpha-subunit levels are markedly age dependent, rising over 5-fold from birth to puberty, then remaining relatively constant throughout adulthood. In 170 children, there was a strong association between alpha-subunit and IGFBP-3 levels. Mean alpha-subunit levels (+/- SD) in adults were 24.2 +/- 4.7 mg/L in 93 normal subjects, 54.1 +/- 15.5 mg/L in 12 acromegalics, 6.5 +/- 4.8 mg/L in 10 GH-deficient subjects, and 31.5 +/- 5.7 mg/L in 18 third term pregnant women. In serum fractionated by gel chromatography, alpha-subunit appeared as a broad 100-150K peak. After depleting serum samples of IGFBP-3 by immunoaffinity chromatography, approximately one third of alpha-subunit remained, in a peak of about 100K, suggesting that two thirds of the total alpha-subunit in serum is present in the approximately 150K complex, and one third is uncomplexed. Development of this RIA for alpha-subunit will allow further study of regulation of the GH-dependent complex.     

Serum concentrations of insulin-like growth factor II are not changed by short-term fasting and refeeding

To determine the factors that regulate insulin-like growth factor II (IGF-II), we raised polyclonal antibodies to this peptide and developed a RIA that measures IGF-II in serum or plasma samples after extraction of IGF-binding proteins by C18 cartridge chromatography. The IGF-II antiserum was highly specific, exhibiting no cross-reactivity with IGF-I or insulin at the highest concentrations tested (10(-6) mol/L). As little as 0.43 micrograms/L IGF-II was detectable, and 50% displacement of tracer occurred at 1.7 microgram/L. The serum IGF-II concentrations of normal adults [mean, 634 +/- 170 (+/- SD) micrograms/L], patients with acromegaly (570 +/- 146 micrograms/L), and patients with hypopituitarism (156 +/- 58 micrograms/L) were similar to those reported by others. In eight obese subjects injected with GH (0.1 mg/kg ideal BW, im, every 48 h for 16 days), serum IGF-II concentrations did not rise significantly, whereas IGF-I concentrations increased 67%. Sixteen normal subjects, within 15% of ideal body weight, were fasted for 5 days on two to four occasions and refed diets of differing protein and calorie contents. Their mean serum IGF-II concentration before fasting (691 +/- 26 micrograms/L) was not significantly different from that after fasting (674 +/- 21 micrograms/L) or after refeeding (641 +/- 20 micrograms/L). In contrast, their mean IGF-I concentration decreased 42% with fasting and rose with refeeding. Unlike IGF-I, serum IGF-II concentrations do not appear to be regulated by short term changes in nutritional status. It is clear from this study and others that IGF-II and IGF-I are regulated differently despite their structural homology and the similarity of their actions in vitro.     

Increased insulin-like growth factor II production and consequent suppression of growth hormone secretion: a dual mechanism for tumor-induced hypoglycemia

We investigated the pathophysiology of fasting hypoglycemia associated with large tumors of mesenchymal origin. We studied two patients with symptomatic fasting hypoglycemia (plasma glucose, 1.92 and 2.03 mmol/L) and a large mesenchymal neoplasm. Before therapy, the plasma insulin-like growth factor II (IGF-II) level measured by RIA was elevated (1879 and 1084 micrograms/L; normal range, 358-854 micrograms/L), the serum GH response to hypoglycemia was impaired, and the plasma IGF-I level was low in both patients. After treatment of the tumor, all of these abnormalities resolved in both patients. Northern blot analysis of tumor RNA revealed extremely high levels of IGF-II mRNA (greater than 100-fold higher than those in normal adult liver). Tumor fragments released IGF-II into tissue culture medium (2.1 and 7.2 micrograms IGF-II/g tissue.24 h). These findings indicate that secretion of IGF-II into the circulation by the tumor was the likely source of the elevated plasma IGF-II levels. We suggest that the primary event in tumor-induced hypoglycemia is overproduction of IGF-II by the tumor, which gives rise to hypoglycemia by a dual mechanism: increased glucose utilization mediated by the insulin-like actions of IGF-II and inhibition of GH secretion.     

Cultured human fibroblasts secrete insulin-like growth factor IA prohormone

Nucleotide sequencing of the two known cDNAs encoding human insulin-like growth factor I (IGF-I) predicts the existence of two different prohormone forms of IGF-I. The E peptide regions extend the carboxy-terminus of IGF-I by either an additional 35 (IGF-IA) or 77 (IGF-IB) amino acids. With a specific and sensitive RIA employing an antiserum directed against a synthetic peptide that is unique to the E peptide region of IGF-IA prohormone (EIA), we have identified EIA-immunoreactive material in the conditioned medium of fetal and postnatal human fibroblasts in culture. Incubation of postnatal human fibroblasts with GH increased specific immunoreactive EIA secretion 2- to 3-fold. There was no immunologically detectable 7.5K IGF-I or IGF-II peptide in acid-chromatographed human fibroblast-conditioned medium under either basal or GH-stimulated conditions. Acid chromatography of human fibroblast-conditioned medium on Sephadex G-75 revealed a single elution peak of EIA immunoreactivity corresponding to a mol wt of 9-17 K. With neutral chromatography, EIA immunoreactivity eluted at 25-38K mol wt. These data suggest that the E peptide region of IGF-IA is translated and released as part of the prohormone form in cultured human fibroblasts, and that the levels of this prohormone are regulated by GH.     

Metabolic regulation of the growth hormone independent insulin-like growth factor binding protein in human plasma

This study examines the regulation of circulating GH-independent insulin-like growth factor binding protein, BP-28. Commencing at 22.00 h, BP-28 in 5 normal adults rose 11-fold to peak values of 120 +/- 12 micrograms/l, remained elevated between 01.00 and 08.00 h, then fell rapidly following a meal. If meals were omitted, BP-28 remained at peak levels throughout the day. The fasting BP-28 level was higher in women (141 +/- 22 micrograms/l, N = 5) than men (59 +/- 14 micrograms/l, N = 7), and pregnancy caused a further 2-fold elevation. Oral glucose rapidly lowered BP-28 in diabetic and nondiabetic pregnant women, nonpregnant women, and men. In a heterogeneous group of 18 subjects, insulin (0.1 U/kg iv), with or without simultaneous administration of GnRH and TRH, elicited a 3- to 4-fold rise in BP-28, commencing 60 min after the nadir of plasma glucose, and independent of the response in GH, PRL, TSH, LH or cortisol. We conclude that BP-28 levels in adults are metabolically regulated, and postulate a role for this protein in the maintenance of glucose homeostasis.     

Twenty-four-hour plasma growth hormone (GH) profiles, urinary GH excretion, and plasma insulin-like growth factor-I and -II levels in prepubertal children with chronic renal insufficiency and severe growth retardation

We studied 24-h plasma GH profiles, maximal GH responses to arginine provocation and insulin-like growth factor-I (IGF-I) and IGF-II levels in plasma in 22 euthyroid prepubertal children (mean age, 9.5 yr) with chronic renal insufficiency (glomerular filtration rate, less than 20 mL/min.1.73 m2) and severe growth retardation [mean (+/- SD) height SD score (SDS), -2.8 (1.1)]. The 24-h GH profiles were analyzed using the Pulsar program. Girls had significantly higher 24-h GH secretion than boys (P less than 0.004). Children with end-stage nephrotic syndrome had higher baseline GH levels and total area under the curve (AUCo) than patients with dysplastic kidneys (P less than 0.05), while the area under the curve above baseline (AUCb) was similar in all types of renal diseases. The type of treatment (conservative, peritoneal, hemodialysis) did not significantly influence the 24-h GH secretion. No correlation was found between 24-h GH profiles and age, height SDS for chronological age, height velocity SDS for bone age, and weight for height. Fourteen children showed a normal 24-h GH profile, defined as a GH profile with well defined, regular GH peaks returning to baseline GH levels and a distinct day and night pattern (AUCb, 90-300 micrograms/L.24 h). Four children had low profiles, with GH peaks below 10 micrograms/L, returning to baseline GH levels and occurring almost exclusively during the night (AUCb, less than 90 micrograms/L.24 h). The remaining four children had elevated 24-h GH profiles, with GH peaks on top of elevated baseline GH levels of more than 3 micrograms/L (AUCb, 35-205 micrograms/L.24 h; AUCo greater than 300 micrograms/L.24 h). In all patients 24-h urinary GH and beta 2-globulin excretion was 100-1000 times higher than that in controls. The urinary GH excretion correlated significantly with all characteristics of the 24-h GH profiles (r = 0.57-0.59; P less than 0.05). The maximal GH response during the arginine tolerance test was normal in 66% of the children. The mean (+/- SD) SDS for bone age for the IGF-I plasma levels was +1.1 (1.9), and that for IGF-II was +3.6 (3.4). IGF-I levels correlated significantly with the AUCb, maximum GH, and GH peak characteristics of the 24-h GH profiles (r = 0.05-0.73; P less than 0.02-0.001). IGF-II levels did not show any correlation with the characteristics of the endogenous GH secretion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Coordinated regulation of the GH/IGF system genes during refeeding in rainbow trout (Oncorhynchus mykiss)

The GH/IGF system is a complex regulation network strongly dependent on nutrient availability. While the effect of starvation on the GH/IGF system has been extensively studied, the time course of events leading to the restoration of GH/IGF system activity after starvation is largely unknown. We, therefore, measured the plasma levels of GH, IGF-I and IGF-II and the expression of the GH/IGF system in liver and muscle. Starvation increased the plasma GH level and 1 day of refeeding completely restored it (1.10 +/- 0.27 vs 1.12 +/- 0.28 ng/ml). Thereafter, plasma GH continued to decrease until day 7 and returned to control values from day 15. Starvation decreased plasma IGF-I and IGF-II and refeeding raised plasma IGF-I only from day 4. In contrast, the plasma IGF-II level doubled after 1 day's refeeding (26.5 +/- 1.9 vs 44.0 +/- 3.4 ng/ml; P < 0.01). Starved fish exhibited higher GH receptor (GHR)1 mRNA abundance in liver and muscle than in controls, whereas GHR2 mRNA abundance was increased only in muscle. In liver, 1 day of refeeding, decreased GHR1 (twofold), but increased GHR2 mRNA abundance (twofold). Thereafter, a progressive return to normal values was observed. Liver IGFBP-4 mRNA abundance was lowered in starved fish followed by a progressive restoration during refeeding. Starvation had no effect on liver IGFBP-2 and IGFBP-6 mRNA abundance, whereas refeeding provoked a peak of IGFBP-2 and IGFBP-6 expression at day 7. In muscle, starvation led to a decrease of the IGFBP-2 mRNA level, which was restored only from day 7. IGFBP-4 mRNA abundance in starved fish was lower than in the controls and refeeding led to a transient upregulation (sevenfold) of IGFBP-4 gene at day 1. IGF-I, IGFBP-5, and IGFBP-related protein 1 (rP1) expression profiles were similar, showing a decrease of expression after starvation, a first peak of expression at day 2, a second peak at day 7, and a return to normal value from day 15. Moreover, IGF-I, IGFBP-5, and IGFBP-rP1 mRNA abundance were positively correlated (r = 0.6-0.8; P < 0.0001). In conclusion, plasma IGF-I was restored later than plasma GH level, which suggests that plasma IGF-I levels cannot account for plasma GH changes. The coordinated regulation of IGF-I, IGFBP-5, and IGFBP-rP1 expression would be a signature for the resumption of myogenic activity.     

Spontaneous pulsatile growth hormone release in male and female premature infants.

Although premature infants have high umbilical cord GH levels, little is known about spontaneous GH release in these individuals. The purpose of our study was to investigate spontaneous 12-h GH release in appropriate for gestational age male and female premature infants. We studied 22 premature infants (15 males and 7 females) of appropriate length and weight for age. Gestational ages, birth weights, birth lengths, and ages at the time of study were similar in male or female infants. All infants were biochemically euthyroid. Blood was taken every 30 min over a 12-h period from an indwelling umbilical catheter; no stress occurred during the blood withdrawal. GH was determined by a double antibody RIA, using 0.01 mL plasma. GH pulse detection was undertaken using Cluster, a computerized pulse detection algorithm. Total insulin-like growth factor-I and -II (IGF-I and -II) levels were measured after separation of the IGFs from the serum binding proteins. Spontaneous pulsatile GH release was observed in all infants studied. No differences were found between males and females in the pulse characteristics of frequency, maximal amplitude, incremental amplitude, width, or area. The GH pulse frequency per 12 h was 3.2 +/- 0.3 (mean +/- SE) in males and 3.0 +/- 0.7 in females. The maximal pulse amplitude was 50.7 +/- 6.2 micrograms/L in males and 44.7 +/- 9.0 micrograms/L in females. The incremental pulse amplitude was 24.3 +/- 3.2 micrograms/L in males and 20.2 +/- 3.9 micrograms/L in females. The mean 12-h GH level was 37.1 +/- 4.8 micrograms/L in males and 35.8 +/- 8.5 micrograms/L in females; the average GH nadir was 26.1 +/- 4.0 micrograms/L in males and 24.4 +/- 8.3 micrograms/L in females. Both IGF-I and IGF-II concentrations were similar in males and females. The mean IGF-I levels were 10.7 +/- 1.5 ng/mL in males and 7.5 +/- 1.1 ng/mL in females; IGF-II levels were 96.0 +/- 12.0 ng/mL in males and 115.4 +/- 17.1 ng/mL in females. These results demonstrate similar pulsatile GH release in male and female premature infants at a mean age of 32-33 weeks. Compared with previously reported values for mean GH concentration and average GH nadir in older children, the values in these premature infants were much higher. We speculate that the higher GH levels in premature infants may result from decreased negative feedback associated with low IGF-I levels. The premature infant's somatotrophs may also not fully respond to the GH inhibitory action of somatostatin.     

Characterization of insulin-like growth factor binding proteins in ovine tissue fluids

Competitive tracer binding studies using radioiodinated insulin-like growth factor-I and -II (125I-labelled IGF-I and 125I-labelled IGF-II) together with size exclusion chromatography and IGF-I affinity chromatography have been used to characterize IGF binding protein activity in ovine tissue fluids. Binding proteins of greater than 200, 150 and 40-50 kDa were revealed in these studies and shown to be widely distributed in body fluids. Thus, the greater than 200 kDa binding protein, which is IGF-II specific, is present in plasma from mature sheep, colostrum and follicular fluid as well as fetal sheep plasma. This may be the ovine equivalent of the soluble type-2 IGF receptor recently identified in rat serum. The presence of a 150 kDa binding protein, of mixed specificity for IGF-I and IGF-II, in fetal and mature sheep plasma was confirmed in these studies. This protein, previously believed to be restricted to vascular fluids, was also identified in mammary lymph, follicular fluid and as a minor component in vitreous humor. Binding proteins of 40-50 kDa were revealed in every fluid tested and multiple variants with distinct specificities were also suggested. This was investigated by IGF-I affinity chromatography using mature sheep plasma. Following passage through the affinity adsorbent, binding of 125I-labelled IGF-I to proteins in the 40-50 kDa region was abolished but when 125I-labelled IGF-II was used as tracer, a binding protein of 40-50 kDa was still observed. Thus sheep plasma contains at least two 40-50 kDa binding proteins. The competitive tracer binding studies indicated that one such protein demonstrates mixed specificity for IGF-I and -II while the other strongly favours IGF-II.     

Circadian variation in serum free and total insulin-like growth factor (IGF)-I and IGF-II in untreated and treated acromegaly and growth hormone deficiency

OBJECTIVE: It is generally accepted that there is no clinically significant circadian variation in total insulin-like growth factor (IGF)-I or total IGF-II in healthy subjects. In contrast there is a significant nocturnal decrease in free IGF-I in healthy subjects, corresponding to the nocturnal increase in IGF binding protein-1. In this study we have investigated the circadian variation in circulating free IGF-I and IGF-II in patients with acromegaly and patients with adult onset growth hormone deficiency. PATIENTS: Seven acromegalic patients were studied with and without treatment with a slow-release formulation of octreotide. Seven GH-deficient patients were studied without GH replacement. In addition 5 of the GH-deficient patients were studied during GH replacement. DESIGN: Serum samples were obtained every hour for 24 h. Free IGF-I and IGF-II were measured every 2nd hour. Total IGF-I and IGF-II were measured every 2nd hour (acromegalic patients) or every 4th hour (GH deficient patients). IGF binding protein (IGFBP)-1 was measured every 2nd hour (acromegalic patients) or every hour (GH deficient patients). RESULTS: In the untreated acromegalic patients there was a significant nocturnal decrease in free IGF-I, but not free IGF-II, before treatment. During treatment there was a significant nocturnal decrease in both free IGF-I and free IGF-II. Peak values of free IGF-I were 112% and 75% above trough (treatment and withdrawal, respectively). In the GH-deficient patients there were no significant circadian variations in free IGF-I or free IGF-II in either of the two occasions. In contrast, there was a significant circadian variation of total IGF-I after adjustment for changes in plasma volume in both treated and untreated acromegaly and GH deficiency in all cases with a peak between 0300 h and 0400 h. The nocturnal increase in total IGF-I ranged from 20% to 35%. CONCLUSIONS: A significant circadian variation in free IGF-I and IGF-II was demonstrated in acromegalic patients. In contrast no significant circadian variation in free IGF-I and IGF-II was found in GH-deficient patients. Part of the variations may be due to poorly understood variations in IGF-I release. It is not clear whether and to what extent the observed circadian changes in free and total IGF-I are involved in circadian changes in IGF-I bioactivity.     

Hypoglycemia due to paraneoplastic secretion of insulin-like growth factor-I in a patient with metastasizing large-cell carcinoma of the lung

CONTEXT: Nonpancreatic tumors may cause recurrent hypoglycemia known as nonislet cell tumor hypoglycemia. It is due to overproduction and secretion by the tumor of incompletely processed IGF-II, termed big IGF-II. We recently identified a patient with recurrent hypoglycemia and low insulin, but without elevated big IGF-II. Multiple small lung nodules were detected by computed tomography scan. An undifferentiated large-cell carcinoma was diagnosed from an axillary lymph node metastasis. OBJECTIVE: The objective was to investigate whether the patient's hypoglycemia was due to excessive IGF-I production by the tumor. METHODS: Serum IGF- I and IGF-II, insulin, and GH were measured by RIA; the distribution of IGFs between IGF binding protein complexes in serum was analyzed after neutral gel filtration. Tissue IGF-I was identified by immunohistochemistry and in situ hybridization, and by RT-PCR after RNA extraction. RESULTS: Total and free serum IGF-I, but not total, free, and big IGF-II, was increased, and the IGF-I content of the two IGF binding protein complexes was elevated. Immunohistochemistry demonstrated IGF-I peptide in situ hybridization IGF-I mRNA in the lymph node metastasis. Combined GH/glucocorticoid treatment prevented hypoglycemia, but did not lower IGF-I. After chemotherapy with carboplatinum/etoposide, the lung nodules largely regressed, and serum IGF-I and the IGF-I content of the two binding protein complexes became normal. Hypoglycemia did not recur despite discontinuation of GH/glucocorticoid treatment. CONCLUSION: Our findings are compatible with a new form of tumor hypoglycemia caused by circulating tumor-derived IGF-I.     

Wasting in the acquired immune deficiency syndrome is associated with multiple defects in the serum insulin-like growth factor system

OBJECTIVE The aim of this investigation was to characterize the GH–IGF axis of patients with AIDS associated wasting. A special emphasis was placed on determining whether IGF binding proteins (IGFBPs) of patients who have lost more than 10% of their ideal body mass are structurally different from the IGFBPs of patients with no weight loss. DESIGN AND PATIENTS A cross-sectional study of 11 AIDS patients was performed to determine whether the IGF system is abnormal in AIDS patients with wasting. Seven additional AIDS patients were followed longitudinally to determine whether AIDS patients experience long-term changes to their IGF system. MEASUREMENTS Serum levels of GH and IGF-I were measured by radioimmunoassay, IGF-II was measured by radioreceptor assay, and IGFBP-1 was measured by an enzyme linked immunoassay. IGFBP-3 and IGFBP-3 protease activity were measured by ligand blotting and a BP-3 protease assay, respectively. IGFBP-3 ternary complex formation and IGFBP-1 phosphovariants were analysed by non-denaturing PAGE. RESULTS AIDS patients who had lost more than 10% of their ideal body mass demonstrated a 55% reduction in serum IGF-I (81 vs 179 μg/l) and a 70% reduction in IGF-II (226 vs 776 μg/l), compared to healthy HIV negative subjects. IGF-I levels were depressed, in some patients, despite high serum levels of GH. AIDS patients who had lost more than 10% of their ideal body mass had low levels of IGFBP-3 and a reduced ability to form the IGFBP-3 ternary complex. The IGFBP-3 ternary complex could be restored only upon addition of pure IGFBP-3 and acid labile subunit to serum. Serum IGFBP-1 was increased more than threefold compared to control subjects (90 vs 24 μg/l). IGFBP-1 was present as a free phosphoprotein in AIDS patients with low levels of IGF-I and in a bound form when serum IGF-I levels were normal. Changes in the GH–IGF axis were sustained for up to 25 months in AIDS patients with wasting. CONCLUSIONS AIDS wasting is associated with a GH resistant state that results in low levels of serum IGF-I, IGF-II and IGFBP-3, elevated levels of phosphorylated IGFBP-1, and a reduced ability to form the IGFBP-3 ternary complex.