GH Release Research Articles

Differential regulation of gonadotropins and glycoprotein hormone α-subunit by IGF-I in anterior pituitary cells from male rats

IGF-I has been demonstrated to stimulate basal and GnRH-induced gonadotropin release. IGF-I also elicites alpha-subunit secretion in human pituitary tumor cells. The aims of this study were to evaluate both the effect of IGF-I on gonadotropin LH-beta and FSH-beta mRNA levels and glycoprotein alpha-subunit gene expression in cultured rat anterior pituitary cells. The exposure of pituitary cells to recombinant human IGF-I (rhlGF-I; 2 microg/ml) for 72 h markedly stimulated basal LH and FSH release whereas their mRNA levels remained unmodified. IGF-I elicited a-subunit release from pituitary cells (p < 0.01) and augmented its mRNA levels. Exposure to IGF-I consistently reduced GH release from pituitary cells. This study shows that the gonadotropin-releasing effects of IGF-I are not paralleled by changes in their mRNAs whereas IGF-I stimulates not only alpha-subunit release but also its mRNA levels. This study provides the first observation of alpha-subunit regulation by IGF-I in normal pituitary cells, where a differential regulation between release and synthesis for gonadotropin a-and 1-subunits is also shown.

Suppressing effects of bisphenol A on the secretory function of ovine anterior pituitary cells.

We investigated the action of bisphenol A (BPA) on cellular GH release and content, cell number, GHmRNA expression, and concentrations of cellular cyclic AMP ([cAMP]c) and calcium ion ([Ca2+]c) in primary cultured ovine anterior pituitary cells. The following results were found: (1) BPA as well as nonylphenol (NP) at 10(-6) to 10(-3) M significantly and concentration-dependently suppressed basal and GHRH-stimulated GH release, and the cellular GH content, (2) BPA suppressed the cell number in a time- and concentration-dependent manner, (3) 10(-4)M BPA suppressed GHmRNA expression to 68% of control (BPA-free), and abolished GHRH (10(-8) M)-induced increases in [cAMP]c and [Ca2+]c. From these findings we conclude that BPA possesses a suppressing action on GH synthesis and release, and this suppressing action is probably related to impairment of cellular signal transduction systems in ovine anterior pituitary cells.

The novel somatostatin ligand (SOM230) regulates human and rat anterior pituitary hormone secretion.

Currently available somatostatin analogs predominantly bind to the somatostatin receptor subtype (SSTR)2 subtype, and control GH and IGF-I secretion in approximately 65% of patients with acromegaly, their efficacy relating to receptor density and subtype expression. SOM230 is a somatostatin ligand with high affinity to four SSTR subtypes. In primary cultures of rat pituicytes, SOM230 dose-dependently inhibited GH release (P = 0.002) with an IC50 of 1.2 nM. Ten nanomoles SOM230 inhibited GH and TSH release by 40 +/- 7% (P < 0.001) and 47 +/- 21% (P = 0.09), respectively. No effect of SOM230 was observed on prolactin (PRL) or LH release. In cultures of human fetal pituitary cells, SOM230 inhibited GH secretion by 42 +/- 9% (P = 0.002) but had no effect on TSH release. SOM230 inhibited GH release from GH-secreting adenoma cultures by 34 +/- 8% (P = 0.002), PRL by 35 +/- 4% from PRL-secreting adenomas (P = 0.01), and alpha-subunit secretion from nonfunctioning pituitary adenomas by 46 +/- 18% (P = 0.34). In contrast, octreotide inhibited GH, PRL, and alpha-subunit from the respective adenoma by 18 +/- 12 (P = 0.39), 22 +/- 4 (P = 0.04), and 20 +/- 10% (P = 0.34). In all culture systems, no significant difference in the inhibitory action of SOM230, octreotide, and somatostatin 14 on hormone release was observed. SOM230, similar to somatostatin, has high-affinity binding to SSTR1, 2, 3, and 5 and, in keeping with this, has an equivalent inhibitory effect on pituitary hormone secretion. As a consequence of its broader binding profile, SOM230 is likely to find clinical utility in treating tumors resistant to SSTR-2-preferential analogs.

Pituitary Mapping

1662 PURPOSE: The purpose of this study was to examine the responses and adaptations of pituitary gland to produce immunoreactive growth hormone (iGH) in response to an acute resistance exercise stress before and after training in rats. METHODS: A 4-group block design: pre-training (i.e., pre-exercise group and post-exercise group) and post-training (pre-exercise group and post-exercise groups) was used. Twelvemonth old male Sprage-Dawley rats were randomly assigned to one of the 4 groups (n = 9/group). The acute exercise stress and the resistance training protocol consisted of climbing a 1-meter long ladder set at an 85o angle with a weight attached to a tail sleeve. The exercise session consisted of 10 individual repetitions of climbing with each repetition separated by a 2 min rest period. The training program was performed 3 days per week for 7 weeks. The mass lifted was gradually increased from 50 g to 535 g by the conclusion of the training program. Rats were kept on a 12: 12 h light dark cycle at 21o C and provided standard laboratory animal chow ad libitum. The Institutional Animal Care and Use Committee approved all procedures. Rats were euthanized by decapitation and immediately following death the pituitary was harvested and subsequently analyzed dividing the gland into eight distinct anatomical sections. Basal GH release was measured from sections placed in gyrotory incubation for three consecutive periods (20 min each). Tissues and cells were analyzed by light microscopy and flow cytometry. Immunoassay was used to detect iGH signals in both the peripheral blood and pituitary media with immunocytohistochemistry performed on pituitary slices to determine GH activity. RESULTS: Exercise significantly (P<0.05) increased GH concentrations and number of GH producing cells in the pituitary after exercise. With training the number of GH producing cells after exercise increased as well as [GH] in the blood and media when compared to pre-training values. No significant changes for iGH were observed pre-exercise. Surprisingly, no significant differences were observed among the different quadrants of the pituitary for the iGH signals. CONCLUSIONS: These data indicate that post-exercise increases in the concentrations of iGH with resistance exercise and training are due to an increased number of GH producing cells throughout the pituitary gland. A lack of changes at rest for iGH supports human observations of little or no changes in iGH with resistance training. How other molecular variants of the GH molecule or aggregates respond to resistance exercise and training and are anatomically located in the pituitary remains to be further characterized.

Homologous and heterologous in vitro regulation of pig pituitary somatostatin receptor subtypes, sst1, sst2 and sst5 mRNA.

Somatostatin (SRIF) is commonly regarded as an inhibitor of GH release in rodents and humans. However, in pigs, SRIF can stimulate the release of GH at low (picomolar) doses, while inhibiting GHRH-stimulated GH release at high (nanomolar) doses in primary pituitary cell cultures. One possible mechanism by which pig cells respond differently to the actions of SRIF is by differential expression and regulation of SRIF receptor subtypes. As no information is available on the homologous regulation of SRIF receptors in pigs, we examined the acute (4 h) in vitro effects of SRIF on mRNA levels of SRIF receptors sst1, sst2 and sst5 by multiplex RT-PCR. These particular sst subtypes were selected because all three have been implicated in the inhibitory effects of SRIF on GH release in both rodents and humans. At a high dose (10(-7) M), SRIF stimulated the expression of sst1, sst2 and sst5 in pig pituitary cell cultures. At a low dose (10(-13) M), SRIF markedly increased sst1, without affecting sst2 or sst5. Given that our laboratory has shown SRIF at high and low doses stimulates cAMP production in a subpopulation of pig somatotropes, we sought to determine if this signaling pathway may be responsible for the stimulatory effect of SRIF on its own receptor expression. The receptor-independent cAMP activator forskolin elevated sst1 and sst2 mRNA levels but did not affect sst5 expression, suggesting the stimulatory actions of high- and low-dose SRIF on sst1 and high-dose SRIF on sst2 mRNA levels can be mediated by activation of cAMP, whereas the stimulatory effect of high-dose SRIF on sst5 mRNA is elicited by a cAMP-independent pathway. Interestingly, both GHRH (10(-8) M) and ghrelin (10(-6) M), which release GH in pig pituitary cell cultures via cAMP-dependent mechanisms, decreased sst5 without altering sst1 or sst2 mRNA levels. Since the actions of GHRH and ghrelin on sst expression markedly contrasted with that observed for SRIF and forskolin these results clearly indicate GHRH and ghrelin are regulating sst5 mRNA levels by a cAMP-independent signaling pathway. Taken together, our results demonstrate that expression of pig SRIF receptors is under a complex, receptor subtype-selective regulation, wherein the concerted actions of key regulators of somatotrope function would play divergent and dose-dependent effects.

Ghrelin stimulation of growth hormone release and appetite is mediated through the growth hormone secretagogue receptor.

Synthetic agonists of the growth hormone secretagogue receptor (GHSR) rejuvenate the pulsatile pattern of GH-release in the elderly, and increase lean but not fat mass in obese subjects. Screening of tissue extracts in a cell line engineered to overexpress the GHSR led to the identification of a natural agonist called ghrelin. Paradoxically, this hormone was linked to obesity. However, it had not been directly shown that the GHSR is a physiologically relevant ghrelin receptor. Furthermore, ghrelin's structure is significantly different from the synthetic agonist (MK-0677) used to expression-clone the GHSR. To address whether the GHSR mediates ghrelin's stimulatory effects on GH release and appetite, we generated Ghsr-null mice. In contrast to wild-type mice, acute treatment of Ghsr-null mice with ghrelin stimulated neither GH release nor food intake, showing that the GHSR is a biologically relevant ghrelin receptor. Nevertheless, Ghsr-null mice are not dwarfs; their appetite and body composition are comparable to that of wild-type littermates. Furthermore, in contrast to suggestions that ghrelin regulates leptin and insulin secretion, fasting-induced changes in serum levels of leptin and insulin are identical in wild-type and null mice. Serum insulin-like growth factor 1 levels and body weights of mature Ghsr-null mice are modestly reduced compared to wild-type littermates, which is consistent with ghrelin's property as an amplifier of GH pulsatility and its speculated role in establishing an insulin-like growth factor 1 set-point for maintaining anabolic metabolism. Our results suggest that chronic treatment with ghrelin antagonists will have little effect on growth or appetite.

The cholinergic system influences both ghrelin concentrations and ghrelin induced GH release

The stomach-derived endogenous ligand of the GHS-receptor ghrelin acts as GH releaser on the pituitary gland, but also – and probably more importantly – on the level of the hypothalamus on both GHRH release and appetite regulation. The central cholinergic system is apparently involved in mediating several ghrelin actions – among them gastric acid secretion, GH release, and appetite stimulation. The cholinergic system has been shown to influence GH release mainly via somatostatin. The aim of our study was to determine the influence of cholinergic manipulation by the cholinergic antagonist atropine (A) and the cholinergic agonist pyridostigmine (PD) on ghrelin plasma levels and ghrelin induced GH release at different ghrelin dosages in 12 young, healthy volunteers in a randomized, double-blind, placebo-controlled, crossover study design. After administration of A alone ghrelin levels were significantly reduced, whereas under PD alone ghrelin levels were unaltered. Ghrelin induced GH peak levels and AUCs were significantly lower after A than after PD treatment for both the lower (0.25µg/kg) and the higher (1µg/kg) ghrelin dosage. Ghrelin in both dosages did also lead to a significant decrease in insulin levels and increase in blood glucose levels, which were not different between A and PD treatment. It is concluded that the cholinergic system influences both ghrelin concentrations and ghrelin ability to induce GH release.

Fasting-induced changes in the hypothalamic-pituitary-GH axis in the absence of GH expression: lessons from the spontaneous dwarf rat.

Fasting results in a reciprocal shift in hypothalamic neuropeptide Y (NPY) and GH-releasing hormone (GHRH) expression in the adult male rat. It is hypothesized that the fasting-induced rise in NPY is responsible for the GHRH decline and subsequent attenuation of pulsatile GH release. Fasting also leads to a decrease in circulating IGF-I, attributed to both reduced GH release and peripheral GH resistance. Although pituitary GH output is suppressed in the fasted rat, we report herein that pituitary GHRH receptor (GHRH-R) and GH secretagogue receptor (GHS-R) mRNA levels are increased, while pituitary expression of the somatostatin receptor subtype 2 (sst2) and 5 (sst5) is decreased, as determined by real-time reverse transcription (RT)-PCR. A shift in the expression of pituitary receptor subtypes to favor GH synthesis and release may be due, at least in part, to a decline in GH/IGF-I negative feedback. In order to test this hypothesis, we compared hypothalamic and pituitary response to fasting (72 h) in normal male rats and rats with isolated GH deficiency (spontaneous dwarf rats (SDR)). Circulating GH levels were undetectable in SDR, and IGF-I levels were less than 10% of normal controls. Fasting stimulated NPY mRNA levels in SDR; however, the rise in NPY mRNA levels was not accompanied by a fall in GHRH mRNA, as observed in fasted normal rats. In fact, GHRH mRNA levels paradoxically rose in the fasted SDR to 135% of fed controls. At the pituitary level, fasting did not alter sst2 and sst5 mRNA levels in SDR but did stimulate the expression of GHRH-R and GHS-R to 165% and 149% of fed controls, respectively. These results demonstrate that the fasting-induced changes in pituitary expression of sst2 and sst5, but not GHRH-R and GHS-R, are GH/IGF-I dependent. In addition, these results argue against the theory that the negative association of NPY and GHRH expression observed following fasting represents a simple cause-and-effect relationship and suggest that GH, either directly or indirectly, mediates the effects of fasting on hypothalamic GHRH expression.

Somatostatin analogs: a new tool for the management of Graves' ophthalmopathy.

Somatostatin (SM), a peptide inhibiting the release of GH, is present and plays an inhibitory role in the regulation of several organ systems in men and other species. Various SM analogs (SM-As) have been developed and used in clinical practice because the short half-life of SM makes it unsuitable for routine treatment. Recently it has been shown that SM-As might be of therapeutic value in the treatment of active thyroid ophthalmopathy. So far, 61 patients have been treated with octreotide and the results have been published in the literature. It was found that in 41 patients the drug had a beneficial effect. Ten patients were given lanreotide and 8 of those had a positive response, while 23 patients were treated with long-acting release octreotide and 16 improved. The exact mechanism of action of SM-As has not yet been fully clarified. One possible mechanism could be a direct inhibition of insulin-like growth factor (IGF)-I mediated effects. A second mechanism could be an inhibition of the release of lymphokines and inflammatory mediators from T-lympocytes. Finally, SM-As may act directly on target cells through specific cell surface receptors. With the introduction from Novartis of SOM-230, a compound which binds with high affinity to SM-receptors (SM-Rs) subtypes 1, 2, 3 and 5, with lower affinity to SM-Rs 4, and which has a very favorable T1/2 of nearly 24 h, a much better therapeutic outcome is expected. Thus, SM-As may provide a well-tolerated therapeutic alternative to corticosteroids. However, prospective placebo-controlled studies with large numbers of patients are needed before their role in the treatment of Graves' opthalmopathy is definitely established.

Ceramide inhibits L-type calcium channel currents in GH 3 cells

In this study, we investigated the effect of ceramide on the L-type Ca 2+ channel (L-channel) in GH 3 cells. We found that C6-ceramide, but not C6-dihydroceramide, the inactive analogue, had an inhibitory effect on BayK 8644-stimulated GH release. Using patch clamp analysis, C6- and C2-ceramide, but not C6-dihydroceramide, were found to inhibit the L-channel current. Increasing intracellular ceramide level with sphingomyelinase also inhibited the L-channel current. The inhibitory effect of ceramide on the L-channel current was attenuated by calphostin C, a myristolated pseudosubstrate protein kinase C (PKC) inhibitor, and lavendustin A, a tyrosine kinase inhibitor. Combined treatment with lavendustin A and the myristolated PKC inhibitor blocked the effect of ceramide on the L-channel current. These results indicate that ceramide, a lipid messenger of the sphingomyelin pathway, is an important regulator of the L-channel in GH 3 cells and both tyrosine kinase and PKC are involved in this effect of ceramide.

Increased activity of antagonists of growth hormone-releasing hormone substituted at positions 8, 9, and 10.

Antagonists of human growth hormone-releasing hormone (hGHRH) with increased potency and improved enzymatic and chemical stability are needed for potential clinical applications. We synthesized 21 antagonistic analogs of hGHRH(1-29)NH(2), substituted at positions 8, 9, and 10 of the common core sequence [phenylacetyl-Tyr(1), d-Arg(2,28), para-chloro-phenylalanine 6, Arg(9)/homoarginine 9, Tyr(10)/O-methyltyrosine 10, alpha-aminobutyric acid 15, norleucine 27, Har(29)] hGHRH(1-29)NH(2). Inhibitory effects on hGHRH-induced GH release were evaluated in vitro in a superfused rat pituitary system, as well as in vivo after i.v. injection into rats. The binding affinities of the peptides to pituitary GHRH receptors were also determined. Introduction of para-amidinophenylalanine 10 yielded antagonists JV-1-62 and -63 with the highest activities in vitro and lowest receptor dissociation constants (K(i) = 0.057-0.062 nM). Antagonists JV-1-62 and -63 also exhibited the strongest effect in vivo, significantly (P < 0.05-0.001) inhibiting hGHRH-induced GH release for at least 1 h. Para-aminophenylalanine 10 and O-ethyltyrosine 10 substitutions yielded antagonists potent in vitro, but His(10), 3,3'-diphenylalanine 10, 2-naphthylalanine 10, and cyclohexylalanine 10 modifications were detrimental. Antagonists containing citrulline 9 (in MZ-J-7-72), amidinophenylalanine 9 (in JV-1-65), His(9), d-Arg(9), citrulline 8, Ala(8), d-Ala(8), or alpha-aminobutyric acid 8 substituents also had high activity and receptor affinity in vitro. However, in vitro potencies of analogs with substitution in position 9 correlated poorly with acute endocrine effects in vivo, as exemplified by the weak and/or short inhibitory actions of antagonists JV-1-65 and MZ-J-7-72 on GH release in vivo. Nevertheless, antagonist JV-1-65 was more potent than JV-1-63 in tests on inhibition of the growth of human prostatic and lung cancer lines xenografted into nude mice. This indicates that oncological activity may be based on several mechanisms. hGHRH antagonists with improved efficacy could be useful for treatment of cancers that depend on insulin-like growth factors or GHRH.

Production and enhanced biological activity of a novel GHRH analog, hGHRH with an N-terminal Pro–Pro extension

Growth Hormone Releasing Hormone (GHRH) is one of the most important hormones in life. Because of its potential clinical importance, its short half-life, and its expensive chemical synthesis, an analog of hGHRH with a prolonged half-life and better activity has been studied for clinical application, especially for the treatment of muscle wasting, type II diabetes, or sleep disorders. The Pro–Pro–hGHRH(1–44) peptide has better activity. The fusion partner gene with 127 amino acid residues of the C-terminus from l-asparaginase was recombined with asp-pro–pro–hGHRH(1–44) gene synthesized by PCR method to form a fusion protein with the unique acid labile linker Asp–Pro. The recombinant protein was expressed to high levels in Escherichia coli BL21 (DE3). The Pro–Pro–hGHRH(1–44) peptide was purified to homogeneity by means of cell disruption, washing, ethanol precipitation, acid hydrolysis, and SP-Sephadex C-25, and Sephadex G-25 column chromatography. The fold of the purification was about 88 times and the yield was 1.1% of the total protein weight of the inclusion body. The peptide molecular mass of 5235.25 Da was determined by ESI mass spectroscopy. Its purity was determined by SDS–PAGE. In the study of the activity, we measured GH release of rat pituitary by using the antiserum kit against human GH. The peptide doses of 0.01, 0.1, 1.0, 7.72, and 20.9 μg/ml used, respectively, released the GH values of 0.1 ± 0.1, 12.5 ± 7.3, 16.6 ± 5.8, 49.8 ± 7.6, and 79.5 ± 5.7 ng/ml whereas their blank controls, respectively, were 0.5 ± 0.8, 4.1 ± 2.6, 3.1 ± 3.1, 4.7 ± 1.8, and 1.2 ± 0.3 ng/ml. The activity results of all dose groups except 0.01 μg/ml Pro–Pro–hGHRH(1–44) group and hGHRH(1–40) group showed that there were significant differences between GH released by the peptide and that by its blank control. With the increase of dosage, the differences were more significant. hGHRH(1–40) showed no measured GH release when the dose was up to 2 μg/ml. The activity results show that the Pro–Pro–hGHRH(1–44) peptide is a potential GH releasing analog.

Effects of long-term GH-releasing factor administration on patterns of GH and LH secretion in growing female buffaloes (Bubalus bubalis).

To investigate the effects of long-term GH-releasing factor (GRF) administration on the patterns of GH and LH secretion in growing female Murrah buffalo (Bubalus bubalis) calves, 12 buffaloes of 6-8 months of age were divided into two groups (treatment and control groups) of six each in such a way that average body weight between the groups did not differ significantly (P > 0.05). Both the groups were administered i.v. with either synthetic bovine GRF (bGRF(1-44)-NH(2)) at 10 microg/100 kg body weight (treatment group) or an equal volume of normal saline (control group) at intervals of 15 days until 18 injections had been completed (9 months). Blood samples collected prior to and after the first and last injection of GRF at -60, -45, -30, -15, -10, -5 min and +5, +10, +15, +30 min, and thereafter at intervals of 15 min up to 8 h post-injection, were assayed for plasma GH and LH. Plasma progesterone was also estimated in twice-a-week samples to assess whether either group had begun ovarian cyclicity. The body weight of all animals was recorded twice a week. In all animals, a peak of GH was recorded within 5-20 min and 5-30 min after the first and last GRF injections and post-injection mean values for plasma GH were significantly (P < 0.01) higher compared with the control group of animals. Although peak GH values after the first and last GRF injection did not differ (P > 0.05), GH levels were maintained at a higher level for a longer time after the last GRF injection compared with the first (240 vs 150 min). The area under the GH response curve after the last GRF injection was found to be significantly (P < 0.01) higher than after the first injection (9344 +/- 99.7 vs 7763 +/- 112.4 ng/ml x min). The mean post-injection plasma LH levels of the treatment group were significantly (P < 0.01) higher after both the first and last GRF injections than in the control group of animals. Interestingly, compared with the first GRF injection, the pre-injection plasma LH level was found to be significantly higher (P < 0.01) at the last injection. The plasma LH concentrations around the last injection of GRF were significantly higher (P < 0.01) than those recorded at the time of the first injection in treated buffaloes. Correspondingly, the plasma LH concentrations in controls were also higher (P < 0.01) around the last injection of GRF vis-à-vis the first injection. The hormone concentration exhibited a higher pulsatility with greater amplitude after the last injection as compared with that recorded after the first injection. Although pulses of LH were also recorded in controls following the last injection, these were fewer and of lower magnitude than those seen in treated animals. No animal from either group reached puberty. GRF-treated buffaloes attained higher (P < 0.001) body weight than the controls. In conclusion, long-term administration of GRF induces and even enhances GH release without any sign of refractoriness, and significantly increases plasma LH also. Hence, long-term treatment with GRF may be used to maintain a sustained increased level of plasma GH in buffaloes and it may assist the animals of this species to grow faster.

A new plasmid-mediated approach to supplement somatotropin production in pigs.

Tremendous progress has been made in the identification of the stimulatory molecules that regulate growth, the mechanisms of action, and the potential application of these molecules for livestock production. A parallel and significant effort is now focused on the discovery and development of economically feasible gene delivery technologies. Plasmid-mediated GHRH gene transfer has emerged as an excellent candidate for agricultural applications to optimize production and animal welfare. We have engineered a GHRH-expressing plasmid that is efficiently expressed in skeletal muscle following intramuscular injection enhanced by electroporation. The GHRH is synthesized in the injected muscle, from which it is secreted to circulate and stimulate normal pituitary GH production and release. Young pigs directly injected with as little as 0.1 mg of a GHRH-expressing plasmid had greater (P < 0.01) weight gain than controls, and a increase (P < 0.05) in fat-free mass. We also have demonstrated that the offspring of gilts injected intramuscularly at d 85 of gestation with a GHRH-expressing plasmid have optimized growth characteristics due to both improved intrauterine weight gain and enhanced maternal lactation performance. Thus, the piglets from treated gilts were larger at birth and weaning compared to controls and reached market weight earlier (P < 0.001). Additionally, pituitaries collected from this group contained an increased number of somatotrophs and lactotrophs (P < 0.001) at birth and at 100 kg. An additional advantage of administering the GHRH plasmid to the gilt compared with the administration of growth-promoting agents to the individual adult animal is a substantial decrease in offspring morbidity and mortality (P < 0.01), which has always represented a major economic loss for the swine industry.

Physiological Studies of Transgenic Mice Overexpressing Growth Hormone (GH) Secretagogue Receptor 1A in GH-Releasing Hormone Neurons

The type 1A GH secretagogue (GHS) receptor (GHSR) has been proposed to mediate the effects of ghrelin on GH release, food intake, and body composition. We have overexpressed GHSR in GH-producing GC cells and GHRH neurons in an attempt to enhance signaling via this pathway selectively, in the GH axis. Constitutive overexpression of human GHSR in rat GC cell lines resulted in increased basal phosphoinositol turnover and rendered them responsive to GHS ligands. We then generated transgenic mice overexpressing human GHSR in GHRH neurons using a 38-kb rat GHRH cosmid promoter. GHRH-GHSR transgenic mice showed increased hypothalamic GHRH expression, pituitary GH contents, and postweaning growth rates. Body weights of the transgenic mice became similar in adulthood, whereas adipose mass was reduced, particularly so in female GHRH-GHSR mice. Organ and muscle weights of transgenic mice were increased despite chronic exposure to a high fat diet. These results suggest that constitutive overexpression of GHSR in GHRH neurons up-regulates basal activity in the GHRH-GH axis. However, GHRH-GHSR mice showed no evidence of increased sensitivity to acute or chronic treatment with exogenous GHS ligands. Food intake and adipose tissue responses to chronic high fat feeding and treatment with GHS ligands were unaffected, as were locomotor and anxiety behaviors, although GHRH-GHSR mice remained significantly leaner than wild-type littermates. Thus, constitutive overexpression of GHSR can up-regulate basal signaling activity in the GHRH/GH axis and reduce adiposity without affecting other GHSR-mediated signals.

The relationship of ghrelin to biochemical and anthropometric markers of adult growth hormone deficiency.

Ghrelin is the natural ligand of the growth hormone secretagogue receptor (GHS-R) and potently stimulates GH release in humans. Ghrelin is found in the hypothalamus, but most circulating ghrelin is derived from the stomach. Ghrelin stimulates food intake but circulating levels are low in obesity. We hypothesized that GH deficiency (GHD) might be associated with increased circulating ghrelin concentrations as a result of low GH levels. We therefore measured circulating ghrelin concentrations, leptin and body composition in subjects with GHD and healthy controls. Subjects with GHD (n = 18) were compared to healthy control subjects (n = 18), matched for body mass index (BMI). They underwent assessment of body composition [waist circumference, BMI and percentage body fat (using bioimpedance)]. Plasma ghrelin, leptin, insulin, GH and IGF-1 were measured in the fasting state. Plasma ghrelin was measured using a specific radioimmunassay, and the other hormones using commercially available assays. The groups were well-matched for BMI (GHD vs. control; 32.9 +/- 10.8 vs. 31.3 +/- 11.7, P = ns) and waist circumference (GHD vs. control; 102.9 +/- 20.0 vs. 99.8 +/- 25.2, P = ns), but percentage body fat (GHD vs. control; 37.0 +/- 9.1 vs. 29.4 +/- 13.0, P = 0.06) tended to be higher in the GHD group. As expected, IGF-1 was lower in GHD (GHD vs. control; 12.5 +/- 6.8 vs. 19.2 +/- 5.8 nmol/l, P = 0.003). Ghrelin [GHD vs. controls; geometric mean (95% CI); 828.8 (95% CI 639.9-1074.2) vs. 487.9 (95% CI 297.2-800.2) pmol/l] and leptin [GHD vs. controls; 13.2 (95% CI 6.6-26.5) vs. 7.9 (95% CI 3.7-16.9) ng/ml] were similar in the two groups. Plasma ghrelin correlated inversely with waist circumference and waist hip ratio in GHD subjects (r = -0.6, P = 0.02) but not with IGF-1 or GH concentrations. There was no significant correlation in the control subjects. Circulating ghrelin concentrations are influenced by body fat distribution, but not by levels of either GH or IGF-1. However, given that obesity is associated with reduced ghrelin concentrations and that GHD is commonly associated with increased body fat, it is possible that these two opposing influences on circulating ghrelin levels result in normal concentrations in subjects with GHD.

Transgenic mice over-expressing galanin exhibit pituitary adenomas and increased secretion of galanin, prolactin and growth hormone.

Galanin is a biologically active 29 amino acid peptide, widely distributed in the central and peripheral nervous system, and most abundantly in the hypothalamus where it may serve in the regulation of anterior pituitary hormones. We herein report that mice carrying the rat preprogalanin cDNA specifically targeted to the somatomammotroph cell lineage, under the control of the rat GH promoter, over-express and over-secrete galanin. Galanin peptide is localised within the GH and prolactin secretory granules. GH and prolactin release is increased as well, predominantly in males, while older transgenic animals develop pituitary hyperplasia and adenoma. In both male and female transgenic mice there is a significant increase in serum galanin (P<0.00003 and P<0.001 respectively) and prolactin (P<0.002 and P<0.05 respectively) levels, while only in male transgenic mice is there a significant increase in the serum levels of GH. Furthermore, in male transgenic mice serum prolactin levels are significantly correlated with the serum galanin levels (P<0.03). We conclude that galanin plays a key role in the process of pituitary hyperplasia, acting as a growth factor to promote pituitary cell proliferation, and participates in pituitary adenoma formation not necessarily dependent on oestrogens. Targeted over-expression and over-secretion of galanin in the somatomammotroph cell lineage stimulates predominantly hyperprolactinaemia in an oestrogen-independent manner.

Ghrelin concentrations in healthy children and adolescents.

In addition to its regulation by GH releasing hormone (GHRH) and somatostatin, release of GH from the pituitary is modulated by a third factor, ghrelin, which is expressed in high concentration in the stomach and is present in the circulation. Ghrelin has also been shown to cause weight gain by increasing food intake and decreasing fat utilization. Ghrelin is a potential candidate hormone to influence nutrient intake and growth. Its role through normal childhood and adolescence has not been fully defined. Cross-sectional study in 121 healthy children (65 male, 56 female) aged 5-18 years, in whom height, weight, body mass index (BMI), pubertal status and measurements of IGF-I, IGFBP-3, IGFBP-1 and leptin were available. Serum ghrelin concentrations have been measured in radioimmunoassay (RIA; Phoenix, AZ, USA) that detects active and inactive human ghrelin. Relationships between ghrelin and anthropometric data and growth factors were assessed by correlation and regression analyses. Ghrelin was detected in all samples, with a median concentration of 162 pg/ml, range 60-493 pg/ml. Prepubertal children had higher ghrelin concentrations than those in puberty [218 pg/ml (n = 42) and 157 pg/ml (n = 79), P < 0.001], with significant negative correlations between ghrelin and age (rs = -0.39, P < 0.001) and pubertal stage (rs = -0.42, P < 0.001). The decrease in ghrelin with advancing pubertal stage/age was more marked in boys than girls. In the whole group, ghrelin was negatively correlated to BMI SD (rs = -0.24, P = 0.006) and to weight SD (rs = -0.24, P = 0.008) but not height sds. Ghrelin was also negatively correlated to IGF-I (rs = -0.48, P < 0.001), IGFBP-3 (rs = -0.32, P < 0.001) and leptin (rs = -0.22, P = 0.02) but not IGF-II. It was positively related to IGFBP-1 (rs = +0.46, P < 0.001). In stepwise multiple regression, 30% of the variability in ghrelin through childhood could be accounted for by log IGF-I (24%) and log IGFBP-1 (6%). The fall in ghrelin over childhood and with puberty does not suggest that it is a direct growth-promoting hormone. However in view of the negative relationship with IGF-I and the positive relationship with IGFBP-1, this fall in ghrelin could facilitate growth acceleration over puberty.

Ghrelin-induced GH secretion in domestic fowl in vivo and in vitro

Although avian and mammalian species differ significantly in their regulation of GH secretion, preliminary studies have demonstrated in vivo GH responses to ghrelin in chickens, as in mammals. However, the relative potency of ghrelin as a GH-releasing hormone (GHRH) in birds is uncertain, as is its site of action. The intravenous administration of human ghrelin to immature chickens promptly increased the circulating GH concentration (within 10 min), although this was transitory and was only maintained for 20 min. This GH response was dose-related with an EC50 of approximately 3.0 microg/kg, comparable with the reported potency of human GHRH in chickens. When incubated with dispersed pituitary cells, human ghrelin induced dose-dependent GH release over a range of 10(-6) to 10(-9) M, with an EC50 of 7.0 x 10(-8) M, comparable with that induced by human GHRH (EC50 6.0 x 10(-8) M), although it was less effective at doses of 10(-6) to 10(-8) M. This was due to direct effects on pituitary somatotrophs, since human ghrelin increased GH release (determined by the reverse hemolytic plaque assay) from individual pituitary cells. The incubation of these cells with human ghrelin induced a dose-dependent increase in the numbers of somatotrophs secreting GH and in the amount of GH released by each cell. In summary, these results demonstrated that ghrelin is a dose-related GH-releasing factor in chickens with a potency comparable with that induced by human GHRH. The GH-releasing action of ghrelin is due, at least in part, to stimulatory actions on the numbers of somatotrophs induced to release GH and upon the amount of GH released from individual somatotrophs.

Endotoxin at low doses stimulates pituitary GH whereas it decreases IGF-I and IGF-binding protein-3 in rats.

While it is well known that sepsis inhibits serum IGF-I and its gene expression in the liver, the effect on pituitary GH and IGF-binding protein-3 (IGFBP-3) is poorly understood. The GH-IGF-I-IGFBP-3 response to different doses of lipopolysaccharide (LPS) administration has been investigated in adult male rats. Two experiments were performed, administration of low doses of LPS (5, 10, 50 and 100 microg/kg) and high doses of LPS (100, 250, 500 and 1000 microg/kg). Rats received two i.p. injections of LPS (at 1730 h and 0830 h the following day) and were killed 4 h after the second injection. LPS administration induced a biphasic response in serum concentrations of GH, with an increase at the 10 microg/kg dose, followed by a decrease at higher doses (100 microg/kg on up). Pituitary GH mRNA was also increased by the administration of 10 and 50 microg/kg LPS, whereas at higher doses LPS did not modify pituitary GH mRNA. We also analyzed the GH response to LPS in primary pituitary cell cultures. When exposed to LPS, in the culture medium, there was an increase in GH release at the concentration of 0.1 and 10 ng/ml, whereas more concentrated LPS did not modify GH release. Serum concentrations of IGF-I declined in a dose-dependent fashion after LPS administration in the rats injected with 10 microg/kg LPS on up. This decrease is secondary to modifications in its synthesis in the liver, since endotoxin injection decreased both IGF-I and its mRNA in the liver. The liver GH receptor mRNA was also decreased by LPS administration, but only in the animals injected with high LPS doses. There was a decrease in both the IGFBP-3 serum levels and its gene expression in the liver with all LPS doses studied. These data suggest a biphasic LPS effect on pituitary GH, a stimulatory effect at low doses and an inhibitory effect at higher doses, whereas it has a clear inhibitory effect on IGF-I and IGFBP-3 synthesis in the liver. The decrease in liver IGFBP-3 mRNA and in serum concentrations of IGFBP-3 in the rats injected with LPS may contribute to the decrease in serum concentrations of IGF-I.