Antisense and sense RNA probe hybridization to immobilized crude cellular lysates: a tool to screen growth hormone antagonists.

The growth-promoting effect of growth hormone (GH) is primarily mediated by insulin-like growth factor-1 (IGF-1). The liver is the main source of circulating IGF-I. The authors have used rodent primary hepatocytes for studies on pharmacological intervention of IGF-I mRNA expression. A 96-well nonradioactive IGF-1 mRNA quantification assay was developed, based on the hybridization of sense and antisense RNA probes, to replicate membranes with crude hepatocyte lysates. The sense hybridization was used as an internal standard. The antagonistic properties of a set of GH-receptor binding compounds were evaluated. Two compounds were found to down-regulate IGF-I mRNA. Effects due to metabolic inhibition or toxicity were excluded using a cell proliferation assay. To investigate potential unspecific transcriptional effects, the mRNA levels of the housekeeping genes, beta-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were determined. Two other GH-regulated genes, cytochrome P450 2C12 (CYP2C12) and a rat homologue to the human alpha1B-glycoprotein (A1BG), were quantified by RNase protection assays and found to be down-regulated, confirming the antagonistic property of 1 compound. In conclusion, a direct filter hybridization assay of hepatocyte lysates using nonradioactive sense and antisense probes can be used for quantitative mRNA measurements and could constitute a valuable tool in screening for pharmacologically active compounds.

Differential inhibition of postnatal brain, spinal cord and body growth by a growth hormone antagonist.

BACKGROUND: Growth hormone (GH) plays an incompletely understood role in the development of the central nervous system (CNS). In this study, we use transgenic mice expressing a growth hormone antagonist (GHA) to explore the role of GH in regulating postnatal brain, spinal cord and body growth into adulthood. The GHA transgene encodes a protein that inhibits the binding of GH to its receptor, specifically antagonizing the trophic effects of endogenous GH. RESULTS: Before 50 days of postnatal age, GHA reduces spinal cord weight more than brain weight, but less than body weight. Thereafter, GHA ceases to inhibit the increase in body weight, which approaches control levels by day 150. In contrast, GHA continues to act on the CNS after day 50, reducing spinal cord growth to a greater extent and for a longer duration than brain growth. CONCLUSIONS: Judging from its inhibition by GHA, GH differentially affects the magnitude, velocity and duration of postnatal growth of the brain, spinal cord and body. GH promotes body enlargement more than CNS growth early in postnatal life. Later, its CNS effects are most obvious in the spinal cord, which continues to exhibit GH dependence well into adulthood. As normal CNS growth slows, so does its inhibition by GHA, suggesting that reduced trophic effects of GH contribute to the postnatal slowing of CNS growth. GHA is a highly useful tool for studying the role of endogenous GH on organ-specific growth during aging.

Photic stimulation inhibits growth hormone secretion in rats: a hypothalamic mechanism for transient entrainment.

It is well established that photic cues are used by the suprachiasmatic nucleus (SCN) to entrain circadian rhythms to the light/dark cycle, but the role of photic stimuli in the regulation of ultradian neuroendocrine rhythms is ill defined. In relation to the rhythms of GH secretion, recent studies have shown that nocturnal photic stimulation induces gene expression not only in the SCN but also in periventricular (PeN) somatostatin (SRIF) neurons. We have, therefore, investigated the effect of nocturnal photic stimulation on spontaneous and induced GH secretion in conscious rats. Nocturnal photic stimulation (lights on at 2400 h for 1 h) suppressed spontaneous GH secretion in male and female rats and reduced the GH response to SRIF withdrawal and iv injection of GH-releasing factor. A similar trough in GH secretion was also observed during the first hour of the normal light phase (0600 h). Using immunohistochemical analysis, we have also shown that expression of the transcription factor, Egr-1, is induced at the commencement of the light phase in the SCN, PeN, and medial preoptic nucleus. This effect is abolished by maintaining rats in the dark during this period. These data, together with our previous demonstration that 50% of SRIF-positive neurons in the PeN coexpress Egr-1 after photic stimulation, suggest that activation of SRIF neurons in the PeN may entrain the episodes of GH secretion to the dark/light interface. However, the absence of synchrony in GH pulses between animals by the second half of the light period suggests that this entrainment is transient.

Pattern-dependent suppression of growth hormone (GH) pulsatility by ghrelin and GH-releasing peptide-6 in moderately GH-deficient rats.

The peptide hormone ghrelin binds to the GH secretagogue receptor (GHS-R), stimulates GH secretion, and promotes adipogenesis. However, continuous GHS infusion does not stimulate skeletal growth and is associated with desensitization to further GH secretagogue treatment. In this study, 7-d intermittent (i.e. every 3 h) infusion of ghrelin, or the GH secretagogue, GH-releasing peptide-6, in the moderately GH- deficient transgenic growth-retarded rat, augmented GH secretion, leading to a sustained acceleration in skeletal growth. In contrast, continuous infusion of ghrelin, or GH-releasing peptide-6, suppressed the amplitude of spontaneous GH secretory episodes and produced only a transient increase in body weight gain. The reduction in GH secretion seen with continuous GHS-R activation was not associated with a desensitization of the pituitary to GH-releasing factor or to down-regulation of hypothalamic GHS-R mRNA expression. Continuous ghrelin treatment elicited an increase in somatostatin mRNA expression in the periventricular nuclei. Thus, exposure to continuously elevated circulating ghrelin may be responsible for the suppression of GH secretion reported in rats after prolonged starvation.

Insulin-like growth factor I (IGF-I) replacement during growth hormone receptor antagonism normalizes serum IGF-binding protein-3 and markers of bone formation in ovariectomized rhesus monkeys.

Previous work from this laboratory has shown that the constant sc infusion of insulin-like growth factor I (IGF-I) to normal pituitary monkeys results in a sustained elevation in circulating concentrations of IGF-binding protein-3 (IGFBP-3), whereas the acute administration of IGF-I to monkeys pretreated with a GH receptor antagonist produces a brief, but significant, elevation in serum IGFBP-3. The present study tested the hypothesis that the constant infusion of IGF-I would normalize serum concentrations of IGFBP-3 in females treated with the GH receptor antagonist. To assess the biological significance of these effects, serum levels of the acid-labile subunit (ALS) and biomarkers for bone formation, osteocalcin, and collagen type I C-terminal propeptide, were also examined. Five female rhesus monkeys were studied over 21 consecutive days involving 7 days of baseline, 7 days of treatment with the GH receptor antagonist (1.0 mg/kg-week, sc), and 7 days of treatment with the GH receptor antagonist supplemented with IGF-I (120 microg/kg x day, sc infusion with osmotic minipump). Within 48 h of the initiation of treatment with the GH receptor antagonist, serum IGF-I and IGFBP-3 were decreased by 40% and 18% from baseline, respectively, and levels continued to decline through the remainder of treatment. However, within 48 h of the initiation of IGF-I administration during GH receptor antagonist treatment, both serum IGF-I and IGFBP-3 were elevated and normalized to baseline values. Serum concentrations of ALS were also decreased by GH antagonism, but levels increased in some (n = 2), but not all, subjects upon administration of IGF-I. Size exclusion ultrafiltration indicated that the amount of IGF-I found in the high molecular mass complex (>100 kDa) decreased significantly during GH antagonism, but was similar during the baseline and IGF-I infusion phases. Finally, treatment with the GH receptor antagonist also significantly reduced serum levels of osteocalcin and collagen type I C-terminal propeptide, an effect reversed by the addition of IGF-I. These data support the hypothesis that IGF-I increases serum concentrations of IGFBP-3 when endogenous GH action is compromised and that such treatment produces biologically active IGF-I, as evidenced by normalization of biomarkers for bone formation. These results indicate that IGF-I administration during GH receptor antagonism restores circulating levels of IGFBP-3 and the amount of IGF-I found in the high molecular mass complex to levels observed during baseline conditions. It remains to be determined whether IGF-I directly affects hepatic synthesis and secretion of IGFBP-3 and what role IGF-I has in the direct regulation of ALS in the monkey.

Inhibition of GH in maternal separation may be mediated through altered serotonergic activity at 5-HT2A and 5-HT2C receptors.

The hyposecretion of growth hormone (GH) in maternal separation (MS) of rat pups is remarkably similar to the specific suppression of GH secretion to evocative tests in infants diagnosed with Reactive Attachment Disorder of Infancy (RADI). Growth hormone-releasing factor (GRF) and somatostatin (SS) provide opposing regulation of GH secretion, and both are modified by noradrenergic and serotonergic stimuli in neonatal and adult rats. In this study, GRF administration reversed MS-induced suppression of GH secretion in 10-day-old pups, but this action of GRF was prevented by pretreatment with cyproheptadine (Cypro), a serotonergic antagonist. The normalization of GH secretion after return to the dam was not altered by pretreatment with SS. Indirect 5-HT agonists, fluoxetine (FLX) and 5-HTP, both stimulated GH secretion in 10-day-old pups. All mixed serotonin- and 5-HT1A-receptor agonists suppressed GH secretion in 10-day-old pups. Antagonists Cypro and ketanserin (Ket) suppressed FLX-induced GH secretion. In contrast, only Cypro suppressed 5-HTP-induced GH secretion. Maternal separation inhibited GH secretion stimulated by 5-HTP, but not by FLX. The serotonergic pathway acting on 5-HT2A receptors may be obligatory for GRF-mediated stimulation and is sensitive to inhibition by Cypro. In addition, a Ket-sensitive serotonergic parallel pathway acting on 5-HT2C receptors may also stimulate GH secretion by acting on GRF or SS. However, only the obligate 5-HT2A pathway appears to be suppressed in MS. These data and observations by others indicate that specific suppression of GH secretion in MS may derive from a reduction in GRF release through noradrenergic neurons, possibly impinging upon serotonergic terminals in the hypothalamus. This study may also provide insight into mechanisms by which GH secretion is suppressed in humans with RADI.

Terminal differentiation of mouse preadipocyte cells: the mitogenic-adipogenic role of growth hormone is mediated by the protein kinase C signalling pathway.

The role of growth hormone (GH) in the differentiation process of Ob1771 mouse preadipocyte cells has been studied under culture conditions that were serum-free and hormone-supplemented and which were previously shown to lead to terminal differentiation. In the absence of GH, a dramatic decrease in the adipogenic activity of the culture medium could be observed, as indicated 12 days after confluence by the low levels of glycerol-3-phosphate dehydrogenase activity and the sharp reduction of the number of triacylglycerol-containing cells. This decrease in adipogenic activity was accompanied by a parallel loss of the mitogenic potency of the culture medium. Determination of the half-maximal and maximal concentrations of GH required for the restoration of growth and differentiation were identical, 0.5 and 2 nM, respectively. Despite the presence of insulin-like growth factor-I (IGF-I) to substitute for supraphysiological concentrations of insulin and to saturate IGF-I receptor, GH was still required to induce terminal differentiation of a maximal number of cells. However, protein kinase C activators such as prostaglandin F2 alpha, phorbol esters and diacylglycerol were able to mimic GH in promoting a maximal mitogenic-adipogenic response, indicating that the ability of GH to induce diacylglycerol production (Doglio et al., 1989; Catalioto et al., 1990) plays a prominent role in this process. Furthermore, in agreement with the fact that the mitoses which precede terminal differentiation of Ob1771 preadipocytes are strictly controlled by cAMP and only modulated by protein kinase C, terminal differentiation of Ob1771 preadipocytes occurred in the absence of GH upon supplementation with high concentrations of carbaprostacyclin, added as a cAMP-elevating agent or with 8-Br-cAMP, added as a cAMP analogue. It is concluded that the control exerted by GH on terminal differentiation of mouse preadipocytes corresponds to a modulating mitogenic effect mediated through protein kinase C activation and leading to a potentiation of the cAMP and IGF-I mitogenic signalling pathways.

The glucocorticoid hormone dexamethasone reverses the growth hormone-releasing properties of the cholinomimetic carbachol.

We recently reported that dexamethasone (DEX) enhances acetylcholine (ACh) release from pituitary cell aggregates. In the present study, the effect of DEX on the GH-releasing properties of the cholinergic agonist carbachol (CCh) was investigated. Perifusion of hemipituitaries from 14-day-old rats with CCh stimulated basal GH release. CCh also increased basal GH release from organ-cultured pituitaries and from pituitary cells cultured as reaggregates, but only when the thyroid hormone T3 was supplemented to the culture medium. Pretreatment of the animals in vivo with DEX abolished the CCh-induced increase in basal GH release from hemipituitaries tested in vitro. Treatment of pituitary organ cultures and reaggregate cell cultures with DEX reversed the stimulation of basal GH release by CCh into an inhibition. CCh also inhibited isoproterenol- and GRF-stimulated GH release from DEX-treated pituitary cell reaggregates. In contrast, the responsiveness of tumoral GH3 cell aggregates to CCh was not dependent on T3 or DEX during culture. The half-maximal concentration of CCh for inhibition was significantly lower than that for stimulation (1 and 10 microM, respectively). Perifusion with CCh of DEX-treated cell reaggregates consisting of a highly enriched somatotroph population (greater than 90% GH immunoreactive cells), obtained by sequential velocity and buoyant density sedimentation of dispersed cells, also inhibited basal GH release. Pretreatment of pituitary cell reaggregates cultured in DEX-supplemented medium with pertussis toxin completely abolished the inhibition by CCh. The inhibition of GH release by CCh was not affected by the Na+ conductance blocker tetrodotoxin, the Cl- channel blocker picrotoxin, or the K+ channel blocker caesium, but was abolished by the Ca2+ channel blockers cadmium and verapamil. In conclusion, CCh is capable of both stimulating and inhibiting GH release in different pituitary in vitro assay systems; the inhibition is dependent on glucocorticoids and the stimulation on the thyroid hormone T3. The mechanism of action of the inhibition seems to involve a GTP-binding protein and most probably a decrease in calcium conductance in the somatotroph.

Activation of cholinergic neurotransmission by pyridostigmine reverses the inhibitory effect of hyperglycemia on growth hormone (GH) releasing hormone-induced GH secretion in man: does acute hyperglycemia act through hypothalamic release of somatostatin?

Acute hyperglycemia blocks growth hormone (GH) secretion in response to provocative stimuli including growth hormone releasing hormone (GHRH) administration. However, the precise mechanism of glucose action is unknown. To determine if enhanced somatostatinergic stimulation accounts for the decreased GH secretion, we studied the effect of enhanced cholinergic tone by pyridostigmine on the hyperglycemia blockade of GH release in 7 normal subjects. Pyridostigmine, an acetylcholinesterase inhibitor, has been postulated as an inhibitor of somatostatin release. Each subject underwent 4 tests with GHRH injection (100 micrograms i.v. at 0 min). In the first (control) test, placebo was administered before GHRH. In the second test, 100 g of glucose was administered p.o. 45 min before GHRH. In the third test, pyridostigmine, 120 mg p.o., was administered 60 min before GHRH, and in the fourth test, pyridostigmine, glucose and GHRH were administered at -60, -45 and 0 min, respectively. GHRH-induced GH secretion of 25.8 +/- 4.5 ng/ml was significantly reduced by previous glucose administration (12.1 +/- 4.5 ng/ml) and significantly potentiated by previous pyridostigmine pretreatment (56.5 +/- 16.8 ng/ml). In the fourth test (pyridostigmine plus glucose plus GHRH) the GH peak of 42.4 +/- 9.2 ng/ml was significantly higher than after GHRH alone and not different to the pyridostigmine-GHRH test. In conclusion, central cholinergic activation by pyridostigmine reversed the hyperglycemic blockade of GHRH-induced GH secretion. In addition, hyperglycemia was unable to reduce the potentiating effect of pyridostigmine on GH secretion elicited by GHRH. Based on the reported actions of pyridostigmine, acute hyperglycemia might act over GH release by inducing hypothalamic somatostatin release.

Mechanisms of calcitonin-induced growth hormone (GH) suppression: roles of somatostatin and GH-releasing factor.

Calcitonin (CT) binds to specific receptors in the hypothalamus and has been localized in the pituitary, suggesting a potential neuroendocrine role for this peptide. We and others have previously shown that CT given centrally markedly suppresses pulsatile GH secretion. However, the mechanism mediating this response remains to be elucidated. In the present study, we assessed the involvement of the two hypothalamic GH-regulatory peptides, somatostatin (SRIF) and GH-releasing factor (GRF), using a combination of in vivo and in vitro techniques. Six-hour GH secretory profiles were obtained from eight groups of freely moving rats bearing chronic intracerebroventricular (icv) and intraatrial cannulae. In four groups, salmon (s) CT (250 ng/10 microliters) was administered icv, whereas the remaining four groups received either normal saline (NS) icv or sCT iv. Central injection of sCT caused a severe suppression in amplitude of spontaneous GH pulses compared to NS icv-treated control rats, whereas the same dose of sCT iv had no significant effect. Passive immunization of sCT icv-injected rats with a specific antiserum to SRIF failed to restore the amplitude of GH pulses to normal values. In addition, in vitro basal and 50 mM K+-stimulated SRIF release from incubated hypothalamic fragments was not altered by sCT in doses ranging from 10(-10) to 10(-6) M. The iv administration of a bolus of rat GRF (1-29)NH2 (1 microgram) 1 h after sCT icv injection also failed to augment plasma GH levels compared to sCT iv-treated rats (16.6 +/- 10.0 vs. 326.6 +/- 63.6 ng/ml; P less than 0.001) and NS icv controls (407.2 +/- 145.4 ng/ml; P less than 0.01). Blood calcium levels decreased similarly 1 h after iv and icv sCT administration. These results demonstrate that: sCT inhibits pulsatile GH secretion via a central nervous system site of action, GH suppression induced by sCT is apparently not due solely to increased hypothalamic SRIF release, and centrally administered sCT produces an acute loss of responsiveness of somatotrophs to GRF, which can be dissociated from peripheral blood calcium levels.

Growth hormone pretreatment in man blocks the response to growth hormone-releasing hormone; evidence for a direct effect of growth hormone.

The effect of pretreatment with biosynthetic methionyl human GH (hGH) on the GH response to GHRH has been studied in normal subjects. Eight volunteers were given either 4 IU hGH or placebo s.c. 12-hourly for 72 h before a GHRH test, or a single s.c. dose of 4 IU hGH 12 h before a GHRH test. Somatomedin-C (Sm-C) levels at the time of the GHRH tests were significantly elevated after treatment with hGH compared to placebo, and the GH response to GHRH was significantly attenuated. A further six subjects were given 2 IU hGH or placebo i.v., and i.v. GHRH 3 h later; there was no rise in Sm-C for the 5 h of the study after either treatment; nevertheless, the response to GHRH was completely abolished by pretreatment with hGH. These results demonstrate that GH can regulate its own secretion independently of changes in Sm-C levels, through a mechanism other than the inhibition of GHRH release. The attenuated response to GHRH in the presence of elevated Sm-C levels may be related to Sm-C, or be a more direct effect of the recently elevated GH levels.

Growth hormone inhibition causes increased selenium levels in Duchenne muscular dystrophy: a possible new approach to therapy.

Nine children with Duchenne muscular dystrophy were given Sanorex (mazindol), a growth hormone inhibitor, daily for 6 months. There was no significant change in their muscle function, but there was a significant reduction in weight gain and in levels of growth hormone, somatomedin C, hair zinc, serum zinc, and serum LDH. Selenium and glutathione peroxidase in the serum increased significantly. Thirteen other children with growth hormone deficiency had a significant reduction in hair selenium following growth hormone administration. These results show a significant relationship between growth hormone and selenium nutritional status and confirm our previous reports indicating an effect of growth hormone on zinc nutritional status. It is possible that prolonged therapy with a growth hormone inhibitor would attenuate the course and improve the longevity of patients with muscular dystrophy.

Somatostatin-induced changes in insulin and glucagon secretion in normal and diabetic dogs.

In conscious dogs intravenously infused somatostatin (3.3 mug per min for 1 h) caused prompt and sustained declines in mean plasma insulin and glucagon, even during alanine infusion and intraduodenal casein hydrolysate feeding; plasma glucose declined, but not significantly. 6.7 mug per min of somatostatin significantly lowered pancreatoduodenal vein glucagon and insulin within 2.5 min and profoundly suppressed their secretion throughout the infusion. Consistent bihormonal suppression occurred at rates as low as 24 ng per kg per min, but was variable at 12 and 2.4 ng per kg per min. When somatostatin-induced (3.3 mug per min) hypoglucagonemia was corrected by exogenous glucagon, hyperglycemia occurred. In dogs with long-standing insulin-requiring alloxan diabetes 3.3 mug per min of somatostatin suppressed glucagon to 55 pg per ml throughout the 30-min infusion and lowered glucose by 36.4+/-6.1 mg per dl, about 1 mg per dl per min. Glucagon suppression was maintained despite alanine infusion, and glucose, which rose 29 mg per dl during alanine infusion without somatostatin, declined 58 mg per dl in the somatostatin-treated diabetic dogs despite alanine. Continuous infusion of somatostatin for 24 h in five insulin-requiring alloxan-diabetic dogs suppressed glucagon and lowered glucose significantly, usually to below normal. It is concluded that in normal dogs pharmacologic doses of somatostatin virtually abolish insulin and glucagon secretion in the basal state and during hyperaminoacidemia. Hyperglycemia occurs during somatostatin-induced insulin lack only if hypoglucagonemia is corrected. Somatostatin suppresses glucagon in diabetic dogs and lowers their plasma glucose approximately 1 mg per dl per min, even when the gluconeogenic substrate alanine is abundant. Glucagon suppression can be maintained for several hours in such dogs and hyperglycemia is thereby reduced.