Insulin-like growth factors: a role in growth hormone negative feedback and body weight regulation via brain.

Intracerebroventricular administration of ILA's, a preparation enriched in insulin-like growth factors, caused a marked decrease in growth hormone secretory episodes and in body weight associated with reduced food intake over 24 hours. Central injection of insulin and bovine serum albumin had no such effects. These findings suggest that insulin-like growth factors play a role in growth hormone negative feedback and body weight regulation at the level of the central nervous system.

Correlation of serum follicular stimulating hormone (FSH) and luteinizing hormone (LH) as measured by radioimmunoassay in disorders of sexual development.

Serum FSH and LH levels in 104 patients with disorders of sexual development were determined by radioimmunoassay and compared with serum FSH and LH levels in 164 normal individuals.32 of 35 gonadal dysgenesis patients (ages 4.8-18.9 yr) had serum FSH levels which were elevated above the range of normal for chronological age, and 19 had serum LH levels similarly elevated. All patients with elevated serum LH levels were 11 yr of age or older. However, 8 of 10 gonadal dysgenesis patients, ages 4.8-10.9 yr, had serum FSH levels elevated above the normal range. In accord with these observations was the finding that in normal girls, serum FSH levels may increase at an earlier age than do serum LH levels (FSH, 5-8 yr of age; LH, 9-10 yr of age). These data indicate that serum FSH determinations may be helpful in diagnosing gonadal dysgenesis during childhood. Serum gonadotropin levels within the range of normal for chronological age were found in 2 of 18 girls with idiopathic isosexual precocity. The other 16 had serum FSH levels elevated above the range of normal for chronological age, and 8 also had serum LH levels similarly elevated. In all instances serum FSH and LH levels were in the range expected for the stage of sexual development. In 35 boys, ages 13.1-17.8 yr, with delayed adolescence, serum gonadotropin levels correlated with stage of sexual development and, therefore, were often less than those expected for age.8 patients with premature pubarche, 5 patients with premature thelarche, and 3 patients with adolescent gynecomastia had serum gonadotropin levels within the range of normal for chronological age.

Effects of hypophysectomy and hormone replacement on the local and metastatic growth of Morris hepatoma 44.

We have demonstrated recently that the local metastatic growth of Morris hepatoma 44 is thyroid dependent ( Mishkin , S., Morris, H. P., Yalovsky , M., and Murthy , P. V. N. Gastroenterology, 77; 547-555, 1979; Mishkin , S. Y., Pollack , R., Morris, H. P., Yalovsky , M., and Mishkin , S. Cancer Res., 41: 3040-3045, 1981) and that exogenous thyroxine (8 micrograms/kg/day) and prolactin (100 micrograms/day) significantly stimulated tumor growth, while growth hormone (100 micrograms/day) failed to do so ( Pollack , R., Mishkin , S. Y., Morris, H. P., and Mishkin , S. Hepatology, 2: 836-842, 1982). In the present study, thyroid ablation (hypothyroidism) and hypophysectomy inhibited tumor growth significantly. These effects were almost totally reversed by administration of exogenous thyroxine to hypothyroid rats. While prolactin or growth hormone or thyroxine alone failed to restore tumor growth in hypophysectomized animals, administration of all three hormones partially but significantly reversed the inhibition of tumor growth. The number and size of pulmonary metastases paralleled local growth in all the above-mentioned conditions. Plasma membrane lactogenic receptors, measured using human growth hormone, were decreased in hypothyroidism and hypophysectomy groups. Binding levels were restored in those groups in which tumor growth was stimulated. In summary, the local and metastatic growth of Morris hepatoma 44 is affected by anterior pituitary hormones. Plasma membrane lactogenic receptors may mediate these effects.

Acute reversal of the enhanced insulin action in trained athletes. Association with insulin receptor changes.

We studied the effect of aerobic training and detraining on insulin-stimulated glucose disposal and on erythrocyte insulin receptor binding. Seven endurance-trained athletes were studied at 12 h, 60 h, and 7 days after cessation of training and compared with three untrained, age- and weight-matched controls. The metabolic clearance rate of glucose as measured by the euglycemic clamp technique was 15.6 +/- 1.8 ml/kg/min (mean +/- SEM) in the trained subjects 12 h after the last bout of exercise compared with 7.8 +/- 1.2 ml/kg/min in the untrained control group. When the trained subjects refrained from physical training, the metabolic clearance rate decreased to 10.1 +/- 1.0 ml/kg/min at 60 h and further to 8.5 +/- 0.5 ml/kg/min after 7 days of detraining. The percentage of specific insulin binding to young erythrocytes (density 1.089-1.092), isolated by density gradient centrifugation, decreased from 10.4 +/- 0.9 at 12 h after the last exercise to 8.1 +/- 0.7%/3 X 10(9) cells after 60 h of detraining (P less than 0.001). The decrease in insulin binding to erythrocytes was almost entirely accounted for by a decrease in the number of insulin receptors. We conclude that the increase in peripheral insulin action seen in trained athletes is rapidly reversed, possibly by a mechanism separate from other phenomena associated with chronic training. The parallel findings of decreased in vivo insulin action and decreased insulin binding in young erythrocytes suggest that modulation of in vivo insulin response by detraining may be at least partially mediated by changes in insulin receptor number.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of Growth Hormone in children with short stature and normal Growth Hormone secretion: a growing problem.

The ethical, economic, psychologic, social, and growth attainment outcome issues related to the use of GH therapy in normal children with short stature are discussed. Although some short children accelerate their growth velocity with GH treatment, the limited available data do not suggest a significant benefit in final height attainment. An international survey of 99 normal short children treated with GH for at least 3 years reported a net improvement in mean height gain of <1 cm/year. Only in one-third was the result considered very good or good; 40% stated that there was no benefit. Thus, it seems unlikely that GH will dramatically increase final height in short children. On this basis, the use of GH for the treatment of the normal child with short stature outside of carefully controlled clinical trials cannot be recommended at present.

Binding and internalization of epidermal growth factor in human term placental cells in culture.

Continuous labeling of primary cultures of human syncytiotrophoblasts with [125I]iodoepidermal growth factor (iodo-EGF) at 37 C revealed a progressive increase in intracellular EGF with 5% of total label observed in intracellular organelles at 2 min and 88% by 60 min. Detailed analysis by electron microscope radioautography showed a progressive transfer of [125I]iodo-EGF from microvillar processes to the bases of microvilli followed by uptake into vesicular and polymorphic endosomes as well as multivesicular bodies. Quantitative analysis revealed an association of radiolabel with the membrane of vesicular endosomes but more generally dispersed over the content of multivesicular bodies. Chloroquine (100 microM), colchicine (100 microM), and bacitracin (1 mg/ml) augmented EGF binding from 45% specific binding in control cultures to 60%, 60%, and 65% specific binding, respectively, with a majority of the ligand (72-83%) observed in vesicular and polymorphic endosomes as well as in multivesicular bodies, i.e. similar to controls. Functional studies indicated a significant enhancement of [14C]-3-O-methylglucose uptake by EGF. Chloroquine-treated cell cultures significantly augmented the stimulation of 3-O-methylglucose uptake by EGF.

Increase in the number of type II insulin-like growth factor receptors during propylthiouracil-induced hyperplasia in the rat thyroid.

We have observed that membranes isolated from rat thyroids contain receptors for the insulin-like growth factors (IGF). As IGFs are known to be important mediators of tissue growth, we conducted this study to determine whether modulation of thyroid IGF receptors might be involved in TSH-stimulated hyperplasia. A substantial increase in both the weight of the thyroid and its DNA content was observed within 2 days of exposing adult male rats to 0.1% propylthiouracil (PTU) in their drinking water. Serum T4 reached unmeasurable levels and serum TSH rose 3-fold over control by the tenth day of treatment. [125I]Iodo-human(h)IGF-II binding to membranes isolated from hyperplastic glands was significantly higher than control beginning at 2 days. A maximum was reached after 5 days (13.3 +/- 0.8%/25 micrograms protein vs. a control level of 6.7 +/- 0.7%, mean +/- SEM). The increase had disappeared by 15 days of PTU exposure, paralleling the drastic fall in the growth rate of the glands. This increase in binding was specific for the thyroid, as it was not seen in other organs. In both treated and control animals, the receptor involved was shown to be type II by preferential binding to IGF-II, lack of interaction with insulin, and molecular sizing. The observed increase in binding could be accounted for by an increase in receptor site number, the affinity remaining essentially the same. We conclude that the TSH-stimulated hyperplasia of the rat thyroid, induced by PTU, is associated with an increase in the binding sites of the type II IGF receptor. This observation raises the possibility that modulation of this receptor may play a role in the mediation of the mitogenic effect of TSH on the thyroid gland.

Effects of mannose-6-phosphate on receptor-mediated endocytosis of insulin-like growth factor-II.

The insulin-like growth factor-II (IGF-II) and lysosomal enzymes containing a mannose-6-phosphate (M6P) recognition site bind to different sites of the same receptor molecule. We have observed that M6P increases the receptor-mediated uptake of IGF-II into IM-9 cells. We now confirm this phenomenon in a different line, the H-35 rat hepatoma cells, and present additional characterization of the underlying mechanisms. When incubated in the presence of radiolabeled IGF-II, H-35 cells accumulated, in a time-dependent fashion, radioactivity that was resistant to removal by trypsin digestion at 15 C, indicating that it was endocytosed. In the presence of 3 mM M6P, endocytosed counts were approximately 2-fold higher after 5 min of incubation, an enhancement that peaked at 10 min, then declined, but was still evident after 40 min (1.5-fold). The rate of release of cell-associated IGF-II, degraded or intact, as measured in a chase experiment, was not affected by M6P. These observations indicate that M6P increased accumulation of IGF-II by accelerating its rate of endocytosis rather than by interfering with IGF-II degradation or with the recycling of intact hormone-receptor complexes to the cell surface. Electrophoresis after affinity cross-linking of labeled cells demonstrated that the enhancement in radioactivity could be located at a molecular size of approximately 250 kDa, corresponding to IGF-II-receptor complexes. Preincubation with M6P did not significantly alter the specific binding of IGF-II to the cell surface of H-35 cells, as measured by a binding assay at 4 C. Finally, pretreatment with cycloheximide for up to 8 h, to remove all newly synthesized lysosomal enzymes bound to the M6P/IGF-II receptor, did not affect IGF-II endocytosis beyond what could be accounted for by some loss of receptor, suggesting that the observed effect of M6P is due to the binding of M6P itself to the receptor and not to displacement of lysosomal enzymes. We conclude that simultaneous occupancy of the M6P/IGF-II receptor by ligands on both binding sites enhances its rate of endocytosis.

The differential regulation by glucagon and growth hormone of insulin-like growth factor (IGF)-I and IGF binding proteins in cultured rat hepatocytes.

The liver is a major site of production of insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGF-BPs). GH decisively influences IGF-I production. To study the role of GH and glucagon in the regulation of IGF-I and IGF-BP production, we examined IGF-I and IGF-BPs secreted by primary rat hepatocytes cultured in a serum-free medium. Glucagon (1 x 10(-8) M) stimulated IGF-I secretion and IGF-BP secretion. Bovine GH (bGH, 300 ng/ml) stimulated IGF-I secretion but suppressed IGF-BP secretion. Combining bGH and glucagon significantly augmented IGF-I secretion above the level seen with each individual agent. The inhibitory effect of bGH on IGF-BP secretion was reversed by glucagon. The major species of IGF-BPs secreted by hepatocytes were found, on Western ligand blotting, to be 24K and 30-34K. All species of secreted IGF-BPs appeared to be comparably affected by glucagon, bGH, and their combination. Northern analysis of IGF-I mRNA revealed three transcripts of 0.7-1.1 kilobases (kb), 1.8 kb, and 7.0 kb. Glucagon stimulated IGF-I mRNA levels 1.8- to 2.0-fold, whereas bGH stimulated IGF-I mRNA levels 2.0- to 2.5-fold. When hepatocytes were incubated with glucagon and bGH for 6 h, IGF-I mRNA levels were augmented 10-fold. Glucagon, in the presence of 50 ng/ml bGH, had a dose-dependent effect on IGF-I mRNA accumulation from a 6-fold level of stimulation at 50 ng/ml of glucagon to a 9-fold level of stimulation at 1000 ng/ml glucagon to a 9-fold level of stimulation at 1000 ng/ml glucagon. This study has demonstrated that glucagon, as well as GH, has significant effects on the production of both IGF-I and IGF-BPs. Of particular interest was the marked augmentation of hepatic IGF-I messenger RNA levels and the reversal of the low levels of IGF-BP production seen on adding glucagon to bGH.

Receptors for insulin-like peptides (ILAs) in rat reticulocytes and erythrocytes.

Rat reticulocytes possess receptors for an insulin-like somatomedin peptide ILAs. At 20 C, pH 7.4 and 10(8) cells per ml, the binding of 125I-ILAs reached a steady-state at 120 min. Binding was partially reversible at 20 C and totally reversible at 37 C. Insulin did not compete with ILAs at concentrations as high as 10 microgram/ml. Insulin-like growth factor II (IGF II) competed with the same potency as ILAs itself, while insulin-like growth factor I (IGF I) was five times less potent. The optimum pH for 125I-ILAs binding was 8.5. Mature erythrocytes possessed seven times less binding sites than reticulocytes. These data suggest that the somatomedins might mediate the effect of growth hormone on erythropoiesis.

Insulin-like growth factor-binding proteins in hypophysectomized rat liver: characterization and subcellular localization.

We have characterized binding proteins for insulin-like growth factors (IGFs) in hepatic subcellular fractions and in the washed supernatants of these fractions in normal and hypophysectomized (hypox) rats. In the course of assessing IGF-II-binding sites on rat liver microsomes, we observed that [125I] IGF-II binding to the microsomal membranes of hypox rats was much lower than that in normal rats. Paradoxically, binding increased in hypox animals at low concentrations (0.5-5 ng/ml) of unlabeled IGF-II. After resuspension and centrifugation (washing) of the microsomes, no difference was found in [125I]IGF-II binding to hypox vs. normal microsomes. However, the binding of [125I]IGF-II to the washing supernatant (SN) from hypox rat microsomes was greater than binding to that from normal animals. Binding to SN was inhibited by unlabeled IGF-II in a dose-dependent manner. Scatchard analyses indicated that the affinity constant for binding by hypox SN was higher than that of normal SN and the microsomal fractions of both hypox and normal rats. After further subfractionation of the liver, no binding activity was found in SN from plasmalemma, whereas it was about 20% of input counts per min of [125I]IGF-II in SN from combined Golgi-endosome fractions of hypox rat liver. We next compared IGF-binding moieties in microsomal SN with those in plasma using cross-linking of [125I]IGF-II followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In normal rat plasma, we observed the presence of 42K, 39K, 31K, and 27K binding complexes. In hypox rat plasma only a 42-39K doublet was found. In the SN of normal rat microsomes, the predominant complex migrated at 39K and was distinguishable only after acidification. In the SN of hypox rat microsomes, the 42K complex was predominant, with a minor 34K complex. These studies have identified IGF-binding moieties in hepatic tissues, particularly in hepatic vesicular elements, which interfere in the binding of IGF-II to membrane receptors. Their presence in these receptor-rich elements may influence IGF binding to intracellular receptors and, hence, the biological activity of the peptide.

Effects of insulin-like growth factors on adult male rat pituitary function in tissue culture.

UNLABELLED: To determine the direct effects of insulin-like growth factors (IGFs) on pituitary secretion of GH, PRL, and ACTH, adult male rat pituitary explant cultures were tested with acute (3-4 h) or chronic (24 h) exposure to a semipurified preparation of IGF peptides, free of immunoreactive insulin, containing IGF-I and IGF-II in a ratio of approximately 1:4. To examine the effect of serum binding proteins on IGF bioactivity, certain experiments were run in parallel using culture medium supplemented with 10% fetal bovine serum or 1% purified BSA. To compare IGF effects with those of known regulators of pituitary function, cultures were also tested with SRIF, TRH, human pancreatic GH-releasing factor, insulin, and human GH (hGH). IGFs, at 10-100 ngeq/ml, were able to inhibit significantly both basal and (1 mM) theophylline-stimulated rat GH (rGH) and rat PRL (rPRL) release during acute (3-4 h) exposure. Only the higher concentration (100 ngeq/ml) was consistently effective in inhibiting rGH and rPRL output after 24 h in culture, due to gradual metabolism of IGF peptides by the cells. Parallel experiments carried out in medium containing 10% fetal bovine serum or 1% BSA gave similar results, demonstrating that IGF serum binding proteins did not interfere with IGF bioactivity in this test system. Chronic 5-day exposure to IGFs, at 100 ngeq/ml, resulted in a significant inhibition of rGH release for the entire 5-day period and rPRL release for the first 3 days. IGFs (10-100 ngeq/ml) had no acute or chronic effect on basal or theophylline-stimulated ACTH release. Purified IGF-I (50 ng/ml) and IGF-II (50 ng/ml) gave approximately equivalent effects on basal rGH and rPRL release during an acute (3 h) exposure suggesting that both IGFs can exert inhibitory influence on pituitary function. Ten thousand nanograms per ml insulin and 10(-9) M SRIF had acute inhibitory effects on rGH and rPRL release similar to what were observed for 100 ngeq/ml semipurified IGFs. hGH (200 and 1000 ng/ml) had no effect on rGH, rPRL, or ACTH release when administered either acutely (3-4 h) or chronically (24 h). CONCLUSIONS: These studies demonstrate that IGFs, administered acutely or chronically, directly inhibit basal as well as theophylline-stimulated rGH and rPRL output by the rat pituitary; ACTH release remains unaltered. Insulin, at high concentrations, can mimic these effects, whereas hGH has no effect either acutely or chronically.

Characterization of insulin-like growth factor receptors in rat anterior pituitary, hypothalamus, and brain.

Studies were undertaken to determine whether the insulin-like growth factors (IGF-I and -II), bind to specific membrane receptors in the pituitary and brain. Anterior pituitary glands, hypothalami, and brains (minus hypothalami) were obtained from adult male Sprague-Dawley rats (225-300 g) and 15,000 X g membranes prepared by differential centrifugation. Binding of 125I-IGF-I and 125I-IGF-II to all three membrane preparations was specific, time and temperature dependent, reversible, and increased in proportion to increasing concentrations of membrane protein or labeled ligand. Neither the pH of the assay buffer (6.5-8.5) nor the presence or absence of 1 mg/ml bacitracin had any significant effect on the levels of specific binding. In all three membrane preparations IGF-II specific binding was 3-5 times higher than that observed for IGF-I, and unlabeled IGF-II displaced either 125I-IGF-I or 125I-IGF-II better than comparable concentrations of IGF-I. All three membrane preparations showed similar low specific binding of 125I-insulin (1.3-2.2%) and negligible specific binding of 125I-rat GH (less than 0.5%). The presence of specific IGF and insulin receptors in rat anterior pituitary, hypothalamic, and brain tissue is additional evidence that IGFs and insulin are involved in modulating brain and pituitary function.

Metabolic effects of growth factors and polycyclic aromatic hydrocarbons on cultured human placental cells of early and late gestation.

The metabolic effects of epidermal growth factor (EGF), insulin, insulin-like growth factor-I (IGF-I), and IGF-II were determined on human placental cells in monolayer culture obtained from early gestation (less than 20 weeks) and late gestation (38-42 weeks). Parameters studied were uptake of aminoisobutyric acid (AIB), uptake of 3-O-methylglucose and [3H]thymidine incorporation into cell protein. Since benzo[alpha]pyrene (BP) inhibits EGF binding and autophosphorylation in cultured human placental cells, particularly in early gestation, we also studied the effect of benzo[alpha]pyrene and other polycyclic aromatic hydrocarbons (PAHs) on EGF-mediated AIB uptake. The metabolic effects of EGF, insulin, and the IGFs in cultured human placental cells varied with gestational age and the growth factor studied. All three classes of growth factors stimulated AIB uptake in both early and late gestation at concentrations from 10-100 micrograms/L, well within a physiological range. However, insulin stimulation of AIB uptake was maximal at a high concentration (200 micrograms/L) in both early and late gestation cells, suggesting an action via type 1 IGF receptors rather than via insulin receptors. EGF stimulated 3-O-methylglucose uptake only in term placental cells. No significant stimulation of [3H]thymidine incorporation by any of the growth factors tested was seen with either early or late gestation cells. The effect of PAHs on AIB uptake by cultured placental cells was variable. BP alone stimulated AIB uptake by both very early and late gestation cells and enhanced EGF-stimulated AIB uptake. alpha-naphthoflavone alone inhibited AIB uptake at all gestational ages and inhibited EGF-stimulated AIB uptake. beta-Naphthoflavone and 3-methylcholanthrene minimally inhibited AIB uptake by early gestation cells and did not modify EGF-stimulated uptake at any gestational period. Our prior results demonstrated that BP more significantly inhibited EGF than IGF or insulin receptor binding as well as autophosphorylation in early gestation placenta, and that BP was the most potent of the PAHs tested. Thus, the direct effect of the PAHs on placental EGF receptors and amino acid transport may indicate altered function of EGF in the regulation of placental growth in smoking mothers that is developmentally regulated.

Characterization and regulation of epidermal growth factor receptors in human placental cell cultures.

We have confirmed that cultured human placental cells rapidly release hCG. Preincubation with epidermal growth factor (EGF) for 24 h significantly increased the amount of hCG released and also increased human placental lactogen release by these cells. To better understand the mechanisms of action of EGF on the feto-placental unit, we studied EGF receptor binding and regulation by examining the characteristics and specificity of EGF receptors in human placental syncytiotrophoblast cultures. Maximal [125I]EGF binding occurred at pH 7.5 and 4 C, and exhibited a high degree of specificity. In the presence or absence of Bacitracin at 4 C, specific binding values were similar, and labeled EGF was physically intact, as assessed by trichloroacetic acid precipitation or rebinding to human placental membranes. The percent specific binding was proportional to cell and ligand concentrations and was significantly increased in term (52.9 +/- 1.2%; n = 11) compared to early gestation placental cells (22.7 +/- 3.4%; n = 7; P less than 0.001). Both term and midterm EGF displacement curves generated curvilinear Scatchard plots, suggesting receptor heterogeneity. Pretreatment of cells with EGF resulted in a dose and time-dependent decrease in specific binding, which was maximal (80%) at 200 ng/ml EGF. This loss of binding was due to decreases in the number of both high and low affinity receptor sites, with no significant change in the apparent affinity. The induction of EGF receptor loss by EGF was a specific effect on the EGF receptor. Preincubation of these same cells with insulin caused a decrease in the number of insulin receptors, while the number of EGF receptors remained unaltered. Conversely, preincubation with EGF, in a dose that down-regulated EGF receptors, did not alter insulin receptor number or affinity. Down-regulation of EGF receptors was reversible, with 50% recovery by 16 h. However, cycloheximide (10 micrograms/ml) blocked EGF-induced down-regulation and receptor recovery. The presence of EGF receptors in human placental cells and the ontogenic changes found suggest that EGF may be involved in the regulation of fetal growth and development. These studies indicate the feasibility of using human placental cells in culture as a model system to probe hormone-cell interaction in the fetoplacental unit.

Receptors for the insulin-like growth factors on human erythrocytes.

We have examined the presence and properties of specific receptors for the insulin-like growth factors (IGFs) on human erythrocytes (RBC). HPLC purified IGF-I and II peptides were used as ligands. RBCs were separated in density fractions representing cell groups of different ages. Binding to all cell fractions was found for both IGF tracers. Porcine insulin displaced this binding only partially, with a potency that was at least 10 times lower. IGF-II was equipotent with IGF-I in displacing [125I] IGF-I binding, but was somewhat more potent than IGF-I in displacing its homologous tracer. In the youngest cell fraction from 7 normal adults, the level of specific binding per 3 X 10(9) cells was 5.2 +/- 0.4% (mean +/- SE) and 6.3 +/- 0.5% of added radioactivity for IGF-I and IGF-II respectively. It declined rapidly as a function of cell age. Binding for unfractionated cell samples was 2.2 +/- 0.1% and 3.1 +/- 0.1% respectively. We conclude that: a) Specific receptors for the IGFs exist on human erythrocytes. b) Binding to these receptors is dependent on cell age. c) The binding in isolated young RBCs is of sufficient magnitude to permit their use for the clinical measurement of these receptors.

Daytime pulsatile growth hormone secretion during childhood and adolescence.

Spontaneous GH secretory patterns were studied in 91 subjects (84 children, 2-18 yr old, at various stages of pubertal development and 7 healthy adults). Plasma GH was determined every 20 min for 6 h (0900-1500 h), and at least 1 spontaneous GH secretory episode (peak, greater than or equal to 5 ng/ml) was evident in 61 children and 5 of 7 adults. There was no significant difference in the mean number of GH secretory episodes or the mean 6-h plasma GH levels in 40 children with short stature compared to those in children of the same sex and pubertal maturation with normal or tall stature. The mean number of GH secretory episodes observed during the sampling period was significantly less in Tanner Stage II males (1.3 +/- 0.15) than in Tanner Stage III males (2.1 +/- 0.20; P less than 0.05). Also, the mean 6-h plasma GH level and the amplitude of the highest GH peak in Tanner Stage III (or greater) boys were greater than those in the prepubertal, early pubertal, or adult male subjects. Among females there was no difference in the number of peaks, mean 6-h plasma GH, or mean peak amplitude in prepubertal, pubertal, or adult subjects. Furthermore, there was no significant difference in overall mean 6-h plasma GH levels between male and female subjects. The frequency of GH secretory bursts was greater between 0830-0930 and 1330-1430 h. The GH secretory profiles were not different in children fed 1 or 2 meals. Children failing to show spontaneous peaks had GH deficiency secondary to central nervous system pathology (n = 10), psychosocial GH deficiency (n = 4), estrogen-dependent GH deficiency (n = 2), and optic nerve hypoplasia (n = 3). There were 2 false negatives and 2 children who were not retested. Pulsatile GH secretion is present during the daytime in children of all ages and stages of puberty. Determination of spontaneous GH secretory bursts is a safe and effective method for assessing GH deficiency.

Free insulin-like growth factor I (IGF-I) and IGF-II in human saliva.

We found that human saliva contains both insulin-like growth factor I (IGF-I) and IGF-II but no significant binding proteins, and that salivary IGF-I levels correlated with plasma GH levels. Mixed saliva had globular proteins precipitated by freezing/thawing. After centrifugation the clear supernatant was used directly in the IGF-I RIA (Van Wyk and Underwood antibody) and in a human placental membrane RRA for IGF-II. The lower limits of detection for IGF-I and IGF-II were 0.7 ng/mL (micrograms/L) and 1.2 ng/mL (micrograms/L), respectively. Iodinated IGF added to saliva was not degraded, as assessed by trichloroacetic acid precipitability and placental membrane binding. In saliva from 14 normal subjects, IGF-I was measurable in all. IGF-II was detectable only in 8 of 14 subjects; the mean value in these 8 subjects was 2.6 +/- 0.6 (+/- SE) ng/mL (micrograms/L). The mol wt of salivary IGF was similar to that of free plasma IGF after acid or neutral pH gel chromatography. Human saliva contained no significant IGF-binding protein. Eluates from neutral gel chromatography of concentrated (20-fold) normal saliva did not inhibit IGF-II binding to placental membrane receptors. Eluted proteins from saliva samples subjected to prior acid gel chromatography failed to bind radiolabeled IGF after neutralization. Saliva samples assayed for binding protein using an amniotic fluid binding protein RIA had values at or below the lower limit of detection [less than 0.06 micrograms eq/mL (mgeq/L)]. Salivary IGF-I concentrations did not change with increasing salivary flow rates above normal, with time of day, or with storage at room temperature for up to 24 h before freezing. The mean IGF-I concentration in mixed saliva from 14 normal young adults (8 men) was 2.3 +/- 0.3 (+/- SE) ng/mL (micrograms/L), and their mean plasma IGF-I level was 315 +/- 27 ng/mL (micrograms/L). Mean salivary IGF-I was significantly lower in 15 patients with GH deficiency [1.3 +/- 0.2 ng/mL (micrograms/L); P less than 0.01] and 8-fold higher in 5 acromegalic patients [17.2 +/- 6.3 ng/mL (micrograms/L); P less 0.01]. Removal of their GH adenomas led to a fall in salivary IGF-I to 5.6 +/- 1.3 ng/mL (micrograms/L); P less than 0.05). In summary, saliva contains free IGFs but no significant quantities of specific binding proteins. Salivary IGF-I levels reflect the GH status of the donor.

Isolation of a somatomedin-binding protein from preterm amniotic fluid. Development of a radioimmunoassay.

Amniotic fluid binding protein (AFBP) is a heat and acid stable somatomedin (Sm)-binding protein with a mol wt of 35-40,000 and an isoelectric point of +/- 4.7. It is reactive in RRAs for Sm and inhibits Sm activity in Sm bioassays. AFBP was purified from midgestational human amniotic fluid (AF) using acid-ethanol extraction, Sephadex G-150 chromatography, high speed gel filtration chromatography, and disc gel-electrophoresis. Specific binding activity (microgram equivalents per mg protein) was quantitated by incubation with 125I-insulin-like growth factor II and dextran-coated charcoal separation. Protein recovery was less than 1%. AFBP antiserum was produced by immunizing rabbits with purified AFBP. The antiserum was cleared of human serum albumin antibodies by affinity chromatography. Immunoelectrophoresis of 20x concentrated preterm AF and fetal serum resulted in one precipitin line. AFBP was labeled by the chloramine-T method. The AFBP antiserum specifically bound +/- 35% of added 125I-AFBP at a final dilution of 1:5000. A double antibody RIA was developed. The AFBP level measured by RIA in midgestation AF (n = 30) was 148 +/- 18 (SEM) and in term AF (n = 12) 72 +/- 36 mu geq/ml. Insulin-like growth factor I/Sm-C values (determined by RIA) in the same samples were uniformly very low (less than 0.10 U/ml). When serum was chromatographed on Sephadex G-200 at pH 2.2, AFBP-RIA activity eluted in one peak corresponding to a mol wt of 35-40,000. Highest activity was found in fetal serum (gestational age +/- 20 weeks) and lowest in serum from adults. The development of the AFBP-RIA may contribute to further elucidation of the physiological importance of Sm and the Sm-binding proteins in pre- and postnatal growth.

Partial purification, characterization, and assay of a slightly acidic insulin-like peptide (ILAs) from human plasma.

An insulin radioreceptor assay (RRA) using human placental microsomal membranes was used to measure insulin-like activity (ILA) extracted from human plasma concentrates (Cohn fraction IV-4) by acid ethanol. The soluble activity (ILAs), chromatographed on Sephadex G-75 in 1 M acetic acid, migrated as a small molecule (fractional elution volume, 0.56) ahead of insulin (fractional elution volume, 0.70), whereas at neutral pH, ILAs migrated as a large molecular weight species. The ILAs peak from acid gel filtration on Sephadex was further purified by chromatography on carboxymethyl cellulose (CMC). The ILAs peak from both Sephadex and CMC diluted parallel to the porcine insulin standard in the insulin RRA and was totally unreactive in an insulin RIA. The CMC-purified material was iodinated and purified by binding to and elution from human placental membranes. The binding of [125I]ILAs to human placental membranes was inhibited only minimally by insulin and proinsulin and not at all by epidermal growth factor, nerve growth factor, glucagon, or lactogenic hormones, including human growth hormone. Multiplication-stimulating activity (MSA) inhibited in a manner parallel to ILAs. A Scatchard plot of the binding data was nonlinear. Sephadex ILAs was subjected to isoelectric focusing. The fractions assayed in both insulin and ILAs RRAs yielded comparable results. Peaks of ILA were observed at pHs 5.3, 6.6, and 8.4. When CMC-ILA was subjected to isoelectric focusing in polyacrylamide, a single peak of activity migrating between pH 6.2-6.8 was seen. [125I]ILAs focused at exactly the same pH. Electrophoresis of CMC-ILAs in acid-urea revealed a sharp peak of activity migrating with one of the five protein bands seen after staining. Again, [125I]ILAs comigrated with unlabeled ILAs. The molecular weight of ILAs, as determined on a calibrated Sephadex G-150 column at neutral pH, was 9,000-10,000 daltons. CMC-ILAs stimulated [14C]glucose incorporation into triglycerides of rat adipose tissue and augmented [3H]thymidine incorporation into human fibroblasts, chicken embryo fibroblasts, and BALB 3T3 cells as well as [35S]sulfate incorporation into macromolecules of rabbit chondrocyte culture medium. In summary, ILAs isolated on the basis of a RRA for insulin is a slightly acidic peptide with some of the biological activities expected of a somatomedin.