Relationship between serum growth hormone binding protein levels and height in young men.

The biochemical mediators responsible for variations in stature among normal subjects are largely unknown. To obtain some initial information about potential endocrine factors, we measured the serum concentrations of GH, IGF-1, IGFBP-3 and GHBP in healthy young men shorter than 159 cm and taller than 187 cm. We studied 14 volleyball and basketball players (tall group), and 14 jockey students from a horse racetrack (short group). A careful medical history was taken, including dietary intake, and physical examination with special attention to the possible presence of genetic stigmata was performed. Serum prealbumin was determined as an index of nutritional status. A buccal smear was performed to exclude Klinefelter's syndrome. The BMI and serum prealbumin levels were comparable in both groups of individuals. The nutritional survey, however, revealed that the tall subjects had a higher intake of calories (42.2+/-11.2 vs. 30.1+/-15.15 kcal/kg, p<0.05), and protein (1.5+/-0.6 vs. 0.8+/-0.4 mg/kg, p<0.01). Serum concentrations of GHBP did not differ in the two groups (0.95+/-0.37 nmol/l in the tall, and 0.95+/-0.53 nmol/l in the short group), and did not correlate with height, serum IGF-I levels, or BMI. We observed a significant difference in the serum concentrations of IGF-I in the two groups of individuals (42.02+/-9.37 nmol/l in the tall and 31.79+/-3.18 nmol/l in the short group, p<0.05), and this growth factor showed a positive correlation with height (r = 0.5, p<0.01). These preliminary findings suggest that final height differences in young men do not appear to be mediated by variations in GHBP concentrations.

Long-acting peptidomimergic control of gigantism caused by pituitary acidophilic stem cell adenoma.

Gigantism is caused by GH hypersecretion occurring before epiphyseal long bone closure and usually is associated with pituitary adenoma. A 15-yr-old female patient presented with accelerated growth due to a large pituitary tumor that was surgically resected to relieve pressure effects. Second surgery to remove residual tumor tissue was followed by administration of octreotide LAR, a long-acting depot somatostatin analog, together with long-acting cabergoline. Height was over the 95th percentile, with evidence of a recent growth spurt. Serum GH levels were more than 60 ng/mL (normal, <10 ng/mL) with no suppression to 75 g oral glucose, and serum PRL (>8,000 ng/mL; normal, <23 ng/mL) and insulin-like growth factor I levels (845 ng/mL; age-matched normal, 242-660 ng/mL) were elevated. Histology, immunostaining, and electron microscopy demonstrated a pituitary acidophil stem cell adenoma. Tumor tissue expressed both somatostatin receptor type 2 and dopamine receptor type 2. The Gs alpha subunit, GHRH receptor, and MEN1 genes were intact, and tumor tissue abundantly expressed pituitary tumor transforming gene (PTTG). Serum GH and PRL levels were controlled after two surgeries, and with continued cabergoline and octreotide LAR GH, PRL, and insulin-like growth factor I levels were normalized. In conclusion, administration of long-acting somatostatin analog every 4 weeks in combination with a long-acting dopamine agonist biweekly controlled biochemical parameters and accelerated growth in a patient with gigantism caused by a rare pituitary acidophil stem cell adenoma.

Pituitary hypoplasia in patients with a mutation in the growth hormone-releasing hormone receptor gene.

BACKGROUND AND PURPOSE: Several anatomic abnormalities of the pituitary gland have been described as occurring in association with congenital growth hormone deficiency, including hypoplasia of the adenohypophysis, truncation of the pituitary stalk, and ectopia of the neurohypophysis. Their pathogenesis, however, is obscure. Normal pituitary development is dependent on the sequential expression of a series of ontogenetic factors. Growth hormone-releasing hormone (GHRH) is known to stimulate somatotroph proliferation, and a dwarf mouse model with a mutant GHRH receptor, the "little mouse," has a small anterior pituitary due to hypoplasia of the somatotrophs. We recently described the human homolog of the little mouse (dwarfism of Sindh), caused by a homozygous nonsense mutation in the GHRH receptor gene in a Pakistani kindred. We investigated MR imaging characteristics to gain information regarding the potential role of GHRH in human pituitary organogenesis. METHODS: MR images of the head were obtained of four affected male patients (age range, 22-29 years). Maximal anterior pituitary dimensions were determined from sagittal and coronal images, and pituitary volumes were estimated from cubic and ellipsoid formulae. The measurements were compared with normative values matched for age and sex. RESULTS: The adenohypophysis was small in each of the four patients. The maximal height for the anterior pituitary was 3 mm in three patients and 2 mm in one (mean +/- SD, 2.75 +/- 0.5 mm), which is significantly (P < .001) less than the expected height of 5.6 +/- 1.0 mm for men in this age group. Estimates of anterior pituitary volume in the patients ranged from 75 to 124 mm3 (104 +/- 21 mm3), which corresponds to 35% to 52% of the normal mean volume corrected for small head size (P < .005). No other cranial abnormalities were identified. CONCLUSION: We describe significant hypoplasia of the adenohypophysis occurring in four dwarfs with a nonsense mutation in the GHRH receptor. In addition to isolated growth hormone deficiency and severe dwarfism, affected patients have anterior pituitary hypoplasia, presumably due to somatotroph maldevelopment. Resistance to GHRH explains the hypoplasia of the adenohypophysis--a feature that contributes to growth hormone deficiency in this syndrome. This is one of the few instances in which the molecular basis of pituitary dysmorphogenesis has been identified.

Familial dwarfism due to a novel mutation of the growth hormone-releasing hormone receptor gene.

Isolated growth hormone (GH) deficiency (IGHD) is a rare cause of short stature. The same mutation of the gene encoding the growth hormone-releasing hormone receptor (GHRHR) has been identified as the basis for IGHD in three families from the Indian subcontinent. The prevalence and heterogeneity of defects in the GHRHR gene are not known. Twenty-two dwarf members of a large, extended kindred containing at least 105 affected members with autosomal recessive short stature underwent extensive endocrine evaluation, which confirmed markedly reduced or undetectable serum concentrations of GH that did not increase in response to different stimuli. DNA sequences of the 13 exons and intron-exon boundaries of the GHRHR gene were determined in an index patient. A novel homozygous 5' splice site mutation (G-->A at position +1) in IVS1 was found. Thirty of the affected subjects tested were homozygous for this mutation, and 64 clinically unaffected patients were either heterozygous for the mutation (n = 41, including 9 obligate carriers) or homozygous for the wild-type sequence (n = 23). We describe a novel mutation in the GHRHR gene as cause of dwarfism in the largest kindred with familial IGHD described to date.

Selective lack of growth hormone (GH) response to the GH-releasing peptide hexarelin in patients with GH-releasing hormone receptor deficiency.

The mechanism of the synergistic relationship between GH-releasing peptide (GHRP) and GHRH with respect to GH secretion is poorly understood. We report the response to hexarelin, a potent GHRP, in patients affected with a homozygous mutation in the GHRH receptor gene, with consequent GHRH resistance and GH-deficient dwarfism. This newly described syndrome is the human homolog of the little (lit/lit) mouse. Intravenous administration of hexarelin (2 microg/kg) to four male adult patients (dwarfs of Sindh) resulted in a complete lack of elevation in plasma GH levels (< 1 ng/mL), an at least 50- to 100-fold deviation from the normal response. In contrast, plasma PRL, ACTH, and cortisol levels rose in a normal manner in response to hexarelin. We conclude that an intact GHRH signaling system is critical for GHRPs to exert their effect on GH release, but that the GHRH system is not necessary for the effect of GHRP on PRL and ACTH secretion. Hexarelin (and probably other GHRPs) are not effective agents for the treatment of patients with GHRH resistance due to GHRH receptor deficiency.

Phenotype and genetic analysis of a syndrome caused by an inactivating mutation in the growth hormone-releasing hormone receptor: Dwarfism of Sindh.

We report, in detail, a new form of familial dwarfism, including its phenotypic features, hormonal profile, and molecular basis. Following a newspaper report of severe dwarfism in two villages in the province of Sindh, Pakistan, we organized an expedition to study its clinical, genetic, and molecular characteristics. We identified 18 dwarfs (15 male, 3 female), all members of a consanguineous kindred, ranging in age from newborn to 28 yr. Mean height was 7.2 SD below the norm, with mean adult heights of 130 cm for males and 113.5 cm for females. Body proportions and habitus were normal; but head circumference was 4.1 SD, and blood pressure approximately 3 SD below the norm. There was no dysmorphism, no microphallus, and no history of hypoglycemia. Serum GH did not respond to provocative stimuli (GHRH, L-dopa, or clonidine). Insulin-like growth factor I (IGF-I) and IGF-binding protein 3 were low (5.2 +/- 2.0 ng/mL and 0.42 +/- 0.13 microg/mL, respectively; mean +/- SD) but rose normally with GH treatment. One affected, dwarfed couple had a son, demonstrating fertility in both sexes. Clinical and endocrinological evidence suggested isolated GH deficiency with a recessive inheritance pattern. The GH-N gene was found to be intact. Linkage analysis of microsatellite chromosomal markers near other candidate genes yielded a high LOD score (6.26) for the GHRH receptor (GHRH-R) locus. DNA sequencing revealed a nonsense mutation (Glu50-->Stop) in the extracellular domain of the GHRH-R. This mutation predicts a severely truncated GHRH-R; it is identical to that recently reported in four patients from two other families. Inheritance is autosomal recessive (chromosome 7p) with a high degree of penetrance. Relatives heterozygous for the mutation had moderately decreased IGF-I levels and slightly blunted GH responses to GHRH and L-dopa, but they showed only minimal or no height deficit. This syndrome represents the human homologue of the little (lit/lit) mouse and closely resembles its phenotype. It demonstrates the absolute requirement of GHRH signaling for pituitary GH secretion and postnatal growth in humans, and its relatively minor (but discernible) biological importance in extrapituitary sites. The syndrome is distinct from other forms of GH deficiency with respect to microcephaly, asymptomatic hypotension, and absence of features such as facial dysplasia, significant truncal obesity, microphallus, or hypoglycemia. Its discovery raises the possibility of milder mutations in the GHRH-R gene as potential causes for partial GH insufficiency and idiopathic short stature.

Blockade of growth hormone receptor shedding by a metalloprotease inhibitor.

GH, an important growth-promoting and metabolic hormone, exerts its biological effects by interacting with cell surface GH receptors (GHRs). The GHR is a single membrane-spanning protein that binds GH via its extracellular domain. The high affinity GH-binding protein (GHBP), which corresponds to a soluble form of the GHR extracellular domain, carries a substantial fraction of the GH in the circulation of various species and probably has a role in modulation of the hormone's bioavailability. Although in rodents, it is believed that the GHBP is largely derived by translation of an alternatively spliced GHR messenger RNA, in humans and rabbits, proteolytic cleavage of the membrane-anchored receptor releases the GHR extracellular domain, which is believed to thereby become the GHBP. In this study, we used human IM-9 lymphocytes and GHR antibodies to study this proteolytic shedding of the GHBP. As determined by immunoblotting with anti-GHR cytoplasmic domain serum, addition of phorbol 12-myristate 13-acetate (PMA; 1 microg/ml) to serum-starved cells led to rapid loss (roughly 60% decline after 1 h; t(1/2) = approximately 5 min) of mature GHRs (115-140 kDa) from either total cell or detergent-soluble extracts. Loss of full-length GHRs was accompanied by accumulation of four proteins (65-68 kDa), each reactive with the cytoplasmically directed antiserum. The pattern of appearance of these GHR ctyoplasmic domain proteins, the electrophoretic and immunological characteristics of which are similar to those of a recombinant rabbit GHR mutant that lacks the extracellular domain, was such that progressively faster migrating forms were evident between 5-60 min of PMA exposure. Treatment with N-ethylmaleimide (NEM; 5 mM), an agent known to cause GHBP shedding from IM-9 cells, promoted a similar rapid loss of full-length GHRs and an accumulation of GHR cytoplasmic domain remnant proteins. PMA-induced, but not NEM-induced, GHR proteolysis was blocked by the protein kinase C inhibitor, GF109203X. Both PMA- and NEM-induced receptor proteolysis were, however, inhibited by the metalloprotease inhibitor, Immunex Compound 3 (minimum effective concentration, 10 microM). Notably, PMA and NEM also promoted shedding of GHBP into the conditioned medium of the cells, as determined by a chromatographic [125I]human GH binding assay; this GHBP shedding was also inhibited by Immunex Compound 3. These results strongly implicate a member(s) of the metalloprotease family as a potential GHBP-generating enzyme.

A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse).

Laron syndrome [growth hormone (GH) insensitivity syndrome] is a hereditary dwarfism resulting from defects in the GH receptor (GHR) gene. GHR deficiency has not been reported in mammals other than humans. Many aspects of GHR dysfunction remain unknown because of ethical and practical limitations in studying humans. To create a mammalian model for this disease, we generated mice bearing a disrupted GHR/binding protein (GHR/BP) gene through a homologous gene targeting approach. Homozygous GHR/BP knockout mice showed severe postnatal growth retardation, proportionate dwarfism, absence of the GHR and GH binding protein, greatly decreased serum insulin-like growth factor I and elevated serum GH concentrations. These characteristics represent the phenotype typical of individuals with Laron syndrome. Animals heterozygous for the GHR/BP defect show only minimal growth impairment but have an intermediate biochemical phenotype, with decreased GHR and GH binding protein expression and slightly diminished insulin-like growth factor I levels. These findings indicate that the GHR/BP-deficient mouse (Laron mouse) is a suitable model for human Laron syndrome that will prove useful for the elucidation of many aspects of GHR/BP function that cannot be obtained in humans.

Alteration in IGF-I binding in the cerebral cortex and cerebellum of neonatal rats during protein-calorie malnutrition.

Neonatal brain development in the rat is adversely affected by malnutrition. Alterations in tissue binding of IGF-I in the malnourished brain were tested in rat pups from mothers who were fed a 20% protein diet (C) or a 4% protein diet (M) starting from day 21 of gestation and continued throughout suckling. IGF-I binding in both cortex and cerebellum decreased progressively in C and M groups from day 6 to day 13. At day 9, 11, and 13, the binding was significantly greater (p < 0.02) in M compared to C groups. To investigate whether these changes might be related to the alteration in receptor activity, membranes were incubated with 125I-IGF in the presence of excess insulin with or without unlabeled IGF-I. In the absence of insulin, specific IGF-I binding in the M group was increased by 41.8 +/- 13.8% (mean +/- SEM p < 0.05) relative to C group. Insulin produced a consistent but incomplete inhibition of binding in both C and M, of 75% and 67% respectively. In addition, the specific IGF-I binding in the presence of insulin was increased in M group by 70.2 +/- 9.4% relative to C, p < 0.05. To characterize the nature of this binding, cerebral cortical membranes, from both groups, incubated with 125I-IGF-I were cross-linked, and electrophoresed on 6% and 10% SDS-PAGE gels under reducing conditions. Autoradiography of the 6% gel showed two specific bands at 115 kD and 240 kD, consistent with monomeric and dimeric forms of the IGF-I receptor, which were inhibited by excess insulin. In contrast, a 10% gel showed an additional band at 35 kD (IGF-binding protein) that was not inhibited by insulin. In both gels, membrane preparations from the M group showed a heightened intensity of the bands relative to C. The increase in binding protein relative to the receptor suggests a disequilibrium that may limit the availability of exogenous IGF-I to the tissues.

The evolution of changes in immunoreactive serum insulin-like growth factors (IGFs), IGF-binding proteins, circulating growth hormone (GH) and GH-binding protein as a result of short-term dexamethasone treatment.

Inhibition of growth in man and laboratory animals by glucocorticoid treatment is well recognized, yet we have previously shown that glucocorticoids may paradoxically enhance GH secretion and increase serum insulin-like growth factor (IGF) levels. IGFs circulate bound to high-affinity binding proteins (IGFBPs) which modulate their actions, and circulating GH may be associated with two binding proteins (GHBPs) of which the high-affinity GHBP has been characterized and is structurally identical to the extracellular domain of the GH receptor. We have investigated the time-course of changes in GH, IGFs and their binding proteins induced by glucocorticoid treatment in normal male volunteers (n = 12, age range 22-31 years) sampled at 0800 h daily before and during treatment with dexamethasone (2 mg twice daily) for 5 days. In addition, subjects were sampled at 30-min intervals over 7-h periods (0730-1430 h) during the day prior to dexamethasone (day 0), on day 1 following the first dose of dexamethasone and on day 5 following the last dose of dexamethasone. Mean serum IGF-I rose over the initial 72 h and remained elevated at 96 h (297 +/- 11.5 compared with basal levels of 215.5 +/- 9.3 micrograms/l, P < 0.001) whereas IGF-II levels did not change (472.6 +/- 20.5 vs 450.3 +/- 21.7 micrograms/l, P = 0.97). There was a concomitant rise in serum IGFBP-3 from basal levels of 3.69 +/- 0.23 mg/l to a peak at 5 days of 4.16 +/- 0.21 (P = 0.003 vs day 1).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of hypothyroidism and hyperthyroidism on insulin-like growth factors (IGFs) and growth hormone- and IGF-binding proteins.

Normal thyroid status is a prerequisite for the normal growth and development of many tissues. The interrelationships between the thyroid and pituitary-GH-insulin-like growth factor (IGF) axes are complex and not yet fully understood. We have studied the effects of hypothyroidism (n = 22) and hyperthyroidism (n = 17) on levels of serum immunoreactive IGF-I and II, IGF-binding proteins (IGFBP-1 and -3), and IGF bioactivity before and during treatment. We have also assessed changes in GH-binding activity (GHBP). Mean immunoreactive (IR) IGF-I levels in the hypothyroid group rose from 106.6 +/- 10.6 micrograms/L at diagnosis to 139.9 +/- 12.7 micrograms/L (P = 0.009) on normalization of thyroid function. In hyperthyroidism, mean IGF-I levels (258.9 +/- 33.9 micrograms/L) were high initially and fell to 188.7 +/- 14.8 micrograms/L (P = 0.04) after treatment. IR IGF-I levels correlated positively with free T3 and free T4 and negatively with TSH levels. Mean serum IGF-II levels were low in hypothyroid patients (375.2 +/- 37.3) and rose during treatment (516.9 +/- 59.4 micrograms/L; P = 0.04). In the hyperthyroid subjects, however, there was no significant change during therapy (625.0 +/- 66.9 vs. 621.9 +/- 120.8 micrograms/L; P = 0.98). IGF bioactivity potency ratios were low in the hypothyroid group (0.26 +/- 0.03 U/mL) and rose to 0.71 +/- 0.10 U/mL (P = 0.01) during treatment. IGF bioactivity in the hyperthyroid group was also low (0.38 +/- 0.05 U/mL) and rose significantly during treatment (0.81 +/- 0.06 U/mL; P = 0.003). Mean IGFBP-1 levels (29.8 +/- 5.7 micrograms/L) were unaltered by treatment of hypothyroid subjects (28.4 +/- 4.8 micrograms/L). In contrast, IGFBP-1 levels in the hyperthyroid subjects were high at diagnosis (134.6 +/- 26.6 micrograms/L) and fell significantly (71.3 +/- 14.3 micrograms/L; P = 0.04) during treatment. In the hypothyroid group, IGFBP-3 levels rose from an initial mean of 1.98 +/- 0.17 to 2.67 +/- 0.27 mg/L (P = 0.04) during treatment. The higher mean pretreatment levels in the thyrotoxic group (3.46 +/- 0.32 mg/L) were unaltered by treatment (3.20 +/- 0.51 mg/L; P = 0.71). GHBP was low in the hypothyroid group at diagnosis (28.5 +/- 2.5%) and rose during treatment to 40.6 +/- 3.9% (P = 0.02). We have confirmed that IR IGF-I levels are low in hypothyroidism and have demonstrated a reduction in IGF bioactivity and IGF-II and IGFBP-3 levels, and low GH-binding activity, which may reflect a reduction in the processing of GH receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Growth hormone binding protein in patients with renal failure.

To investigate changes in the growth hormone binding protein (GH-BP) in renal disease, gel chromatography was used to separate free and bound hormone after incubation with 125I-GH, the results being expressed as a percentage of radioactive GH eluting in a high molecular weight (70-80 kD) peak. In 26 normal individuals, binding was 39.3 +/- 8.0%, while in 11 patients with renal disease who were off dialysis binding was reduced to 16.8 +/- 5.6%. Similarly, in 9 patients undergoing hemodialysis binding was reduced to 24.6 +/- 6.8%, in 8 patients undergoing chronic ambulatory peritoneal dialysis binding was reduced to 25.7 +/- 7.6%, and in 9 patients within three months of a renal transplant binding was reduced to 25.1 +/- 8.6%. Scatchard analysis showed that these changes were not a result of decreased affinity of GH-BP for GH, and receptor binding studies showed that uremic serum was not inhibiting binding. The decreased concentration of GH-BP may indicate decreased expression of the GH receptor in target tissues, and hence diminished responsiveness to GH in renal failure.

Laron-type dwarfism with apparently normal high affinity serum growth hormone-binding protein.

OBJECTIVE: Normal serum contains a high affinity GH-binding protein, which appears to be identical with the extracellular domain of the GH receptor. It is normally absent from the serum of patients with Laron-type dwarfism. We wished in this study to define the serum GH-binding protein status of a family with Laron-type dwarfism. DESIGN AND PATIENTS: We performed an open case study of an Asian family in which three sisters (aged 3 to 15 years) had the phenotype of Laron-type dwarfism. Sera from a fourth, unrelated girl with Laron-type dwarfism and subjects without endocrine disorders were used as control samples. MEASUREMENTS: Laron-type dwarfism was confirmed by demonstration of elevated serum GH levels and low serum IGF-I levels on immunoassay, with serum IGF-I levels failing to rise during treatment with GH. Serum GH-binding proteins were characterized using gel chromatography on Sephacryl S-100HR following incubation of serum with 125I-GH, Scatchard analysis of ligand binding, and by polyacrylamide gel electrophoresis after covalent cross-linking to 125I-GH. RESULTS: All members of the family had high affinity serum GH-binding protein activity similar in size, circulating levels and apparent affinity for GH to that of normal subjects. This contrasted with the very low serum GH-binding protein activity in the unrelated child with Laron-type dwarfism and previous reports of serum GH-binding protein levels in this disorder. CONCLUSIONS: The affected patients may possess a novel biochemical defect which results in GH-resistance and reduced production of IGF-I in the presence of normal serum GH-binding protein levels.