GHRH-stimulated GH Secretion Research Articles

Paltusotine, a novel oral once-daily nonpeptide SST2 receptor agonist, suppresses GH and IGF-1 in healthy volunteers

PurposeEvaluate the pharmacodynamics, pharmacokinetics, and safety of paltusotine, an orally bioavailable, nonpeptide, somatostatin receptor subtype 2 (SST2) agonist being developed for the treatment of acromegaly and neuroendocrine tumors.MethodsA randomized, double-blind, placebo-controlled, single center, single and multiple ascending dose phase 1 study was conducted in healthy male volunteers who received (i) single-dose of oral paltusotine 1.25, 2.5, 5, 10, and 20 mg (solution); and 40 and 60 mg (capsules) or (ii) multiple-dose oral paltusotine capsules once daily 5 mg (× 7 days), 10, 20, and 30 mg (× 10 days). Main outcome measures were pharmacodynamics (changes in growth hormone-releasing hormone [GHRH] stimulated growth hormone [GH] and insulin-like growth factor 1 [IGF-1]), pharmacokinetics, safety, and tolerability.ResultsSingle-dose cohorts: n = 41 active, n = 14 placebo. Multiple-dose cohorts: n = 24 active, n = 12 placebo. Paltusotine was well tolerated, orally bioavailable, associated with increased plasma concentrations to doses up to 40 mg, and was eliminated with a half-life of approximately 30 h. Single-dose paltusotine 1.25 to 20 mg suppressed GHRH-stimulated GH secretion by 44% to 93% compared to 15% with placebo. Multiple-dose paltusotine 5 to 30 mg administered once daily for 10 days suppressed IGF-1 by 19% to 37% compared to an increase of 2.4% with placebo.ConclusionsPaltusotine suppresses GH and IGF-1 in a dose-dependent fashion, with a safety profile similar to currently approved SST2 receptor ligands. Paltusotine is a promising once-daily oral nonpeptide SST2 agonist candidate for managing acromegaly and neuroendocrine tumors.Trial registrationNCT03276858, registered September 8, 2017, retrospectively registered.

Octreotide administration, under particular temporal conditions, enhances the responses of growth hormone to growth hormone-releasing hormone in normal subjects

Somatostatin not only inhibits basal and GHRH-stimulated GH secretion but might also enhance pituitary GH responsivity to GHRH under different temporal conditions. We investigated whether octreotide, a long-acting somatostatin analogue, has any positive actions on GHRH-induced GH release in normal human subjects. The study consisted of three protocols. At 0800 hours, after fasting overnight, all subjects received 1 microgram/kg GHRH i.v. bolus at 0 minutes. In each protocol, either octreotide (200 micrograms s.c.) or placebo were given respectively 8, 12, or 16 hours prior to GHRH challenge. Three groups of eight normal volunteers (four female and four male in each group), aged 18-35 years, were randomly assigned to each protocol. Growth hormone was measured by IRMA. Samples for GH assay were taken at -30 and 0 minutes and then at 15-minute intervals up to 120 minutes. When placebo or octreotide were administered 8 hours before GHRH, peak GH levels were respectively (mean +/- SE, mU/l) 56.2 +/- 16.6 and 60.8 +/- 11.4 (NS). Also, when placebo or the somatostatin analogue were administered 16 hours prior to GHRH, peak GH levels were comparable (61.0 +/- 7.4 vs 58.8 +/- 7.4, NS). However, in the group receiving placebo or octreotide 12 hours prior to GHRH, the GH responses to GHRH were clearly enhanced by octreotide administration (peak GH levels, mU/l, 55.6 +/- 21.6 vs 104.0 +/- 17.4, P < 0.02). This enhancement of GH responses was observed in all subjects. Octreotide administration did not affect GH responses to GHRH when given either 8 or 16 hours prior to GHRH. However, octreotide enhanced GHRH-induced GH release when administered 12 hours prior to GHRH. It thus appears that, under particular temporal conditions, octreotide may act positively on GH secretion in man.

Beta‐adrenergic agonism does not impair the GH response to acylated ghrelin in humans

Acylated ghrelin (AG) is a physiological GH secretion amplifier, in part stimulating GHRH neurones and antagonizing somatostatin activity. In humans, AG is one of the most potent pharmacological stimuli of GH secretion and, unlike GHRH, is refractory to the inhibitory effect of glucose, free fatty acids (FFA) and somatostatin. Somatotroph secretion is also profoundly modulated by the adrenergic system. Indeed, beta-adrenergic agonists abolish spontaneous and GHRH-stimulated GH secretion. Based on these data, the aim of the present study was to investigate the effects of beta adrenergic agonism on the GH response to AG. Six young healthy male volunteers underwent: (a) acute AG intravenous (iv) administration (1.0 microg/kg); (b) salbutamol infusion (SLB; 0.06 microg/kg/min iv); (c) AG + SLB; and (d) saline infusion. In all sessions GH levels were assayed every 15 min from time -30 to +210 min. SLB induced a significant (P < 0.05) inhibition of spontaneous GH secretion that persisted up to 75 min after SLB withdrawal. AG induced a marked increase (P < 0.01) in GH that was not modified by SLB. The GH-releasing effect of AG is refractory to the inhibitory effect of SLB-induced beta-adrenergic receptor activation. Although further studies are needed to confirm these results during the lifespan and particularly during prolonged exposure to beta agonists, the present data clearly suggest that, among GH stimulatory tests, AG administration might be the most suitable in clinical conditions of chronic treatment with beta-2 agonists, such as in asthmatic disease.

Age and Secretagogue Type Jointly Determine Dynamic Growth Hormone Responses to Exogenous Insulin-Like Growth Factor-Negative Feedback in Healthy Men

The primary cause of waning GH and IGF-I concentrations in healthy aging adults is not established. To test the postulate that age influences negative feedback by IGF-I in a secretagogue-specific fashion, 17 normal men (nine young and eight older) each completed eight randomly ordered injections of placebo or recombinant human (rh) IGF-I (20 microg/kg sc), followed by saline/rest, aerobic exercise, GHRH (1 microg/kg iv bolus), or GH-releasing peptide-2 (1 microg/kg iv bolus) stimulation. GH secretion was monitored by sampling blood every 10 min for 7 h, high-sensitivity immunochemiluminometric assay, and deconvolution analysis conditioned on prior pulse-onset times and biexponential kinetics. Analysis of covariance showed that age (P = 0.028), secretagogue (P < 0.001), and rhIGF-I (P < 0.005) individually determine pulsatile GH secretion and exhibit a strong 3-fold interaction (P < 10(-5)). Post hoc comparisons revealed that elderly subjects manifest less IGF-I inhibition of a maximal GHRH stimulus (P = 0.013 vs. young), blunted initial IGF-I suppression of fasting GH release (P = 0.038), and impaired IGF-I feedback on the regularity of GH secretion (P = 0.023). Age stratum did not influence peak IGF-I and nadir GH concentrations or rhIGF-I-induced inhibition of GH secretion stimulated by exercise or GH-releasing peptide-2. In summary, experimental elevation of IGF-I concentrations unmasks reduced rhIGF-I-dependent feedback inhibition of fasting and GHRH-stimulated GH secretion in healthy older men, indicating that aging selectively modulates the autoinhibition process.

The role of the GH/IGF system in pituitary tumorigenesis.

Pituitary adenomas are mostly benign tumours that originate from differentiated anterior pituitary cells. Altered expression of growth factors or their receptors could enhance clonal expansion of pituitary adenoma cells. GHRH overstimulation or an activating point mutation in the Gs a-subunit leads to increased GH secretion and tumour formation. In contrast, IGF-I suppresses basal and GHRH-stimulated GH secretion in pituitary adenoma cells, whereas prolactin secretion is unaffected. Somatostatin analogues and pegvisomant, a novel growth hormone-receptor antagonist, results in a reduction of serum IGF-I levels and clinical improvement in patients suffering from pituitary adenoma. Thus, this review focuses on the role of the growth hormone/insulin-like growth factor system in pituitary tumorigenesis with particular focus on the genetic alterations described in pituitary adenomas up to now.

Effects of leptin on basal and GHRH-stimulated GH secretion from the bovine adenohypophysis are dependent upon nutritional status.

We have shown recently that leptin modulates at least two aspects of anterior pituitary LH release in ruminants: basal and GnRH-mediated release. To test the hypothesis that leptin directly affects basal and GHRH-mediated GH secretion from the adenohypophysis, we examined the effects of various doses of recombinant ovine leptin (oleptin) on perifused adenohypophyseal (AP) explants and compared responses of tIssues from control and fasted cows. Ten mature, ovariectomized and estradiol-implanted cows were assigned to one of two dietary groups: (1) normal-fed (n=5) and (2) fasted for 72 h (n=5). At the end of the fasting period, cows were euthanized and pituitaries were collected. Adenohypophyseal explants were perifused for a total of 6.5 h, including a 2-h treatment at 2.5 h with Krebs-Ringer bicarbonate buffer containing 0, 5, 10, 50, or 100 ng/ml oleptin, and a challenge with GHRH at 4.5 h. All doses of oleptin greater than 5 ng/ml decreased (P<0.01) basal GH secretion compared with controls in tIssues collected from normal-fed cows. In contrast, GH release from AP explants from fasted cows treated with the lowest dose of oleptin was 28% (P<0.002) higher than control explants, but larger doses had no effect. Leptin caused an inversely related, dose-dependent increase in GHRH-mediated GH release in tIssues from normal-fed cows. Marked increases (P<0.01-P<0.001) in GH release were observed for the 5 and 10 ng/ml oleptin, with lesser (P<0.08) and no effects observed at the 50 and 100 ng/ml doses respectively. In fasted cows, oleptin had no stimulatory effect on GHRH-induced GH release. Results show that leptin can act directly at the anterior pituitary level to modulate GH release, and this effect is dependent upon nutritional history.

The in vitro regulation of growth hormone secretion by orexins.

Orexins, orexigenic neuropeptides, have recently been discovered in lateral hypothalamus and play an important role in the regulation of pituitary hormone secretion. Two subtypes of orexin receptors (orexin-1 and orexin-2) have been demonstrated in pituitaries. In this experiment, the effects of orexins on voltage-gated Ca2+ currents and the GH release in primary cultured ovine somatotropes were examined. Voltage-gated Ca2+ currents were isolated in ovine somatotropes as L, T, and N currents using whole-cell patch-clamp techniques and specific Ca2+ channel blocker and toxin. Application of orexin-A or orexin-B (100 nM) significantly, dose-dependently, and reversibly increased only nifedipine-sensitive L-type Ca2+ current. Inhibitors of PKC (calphostin C, PKC inhibitory peptide) but not inhibitors of PKA (H89, PKA inhibitory peptide) cancelled the increase in the L current by orexins. Co-administration of orexin-A and GHRH (10 nM) showed an additive effect on the L current. Specific intracellular Ca2+-store-depleting reagent, thapsigargin (1 microM), did not affect the orexin-induced increase in the L current. Orexin-B alone slightly increased GH release and co-administration of orexin-A and GHRH synergistically stimulated GH secretion in vitro. It is therefore suggested that orexins may play an important role in regulating GHRH-stimulated GH secretion through an increase in the L-type Ca2+ current and the PKC-mediated signaling pathways in ovine somatotropes.

In vitro effect of human recombinant leptin and expression of leptin receptors on growth hormone-secreting human pituitary adenomas.

Leptin is a circulating hormone secreted by adipose tissue and a few other tissues. It has recently been demonstrated that leptin and leptin receptors are expressed in normal and adenomatous pituitary cells. The aim of this study was to investigate the effect of recombinant human leptin on GH release from adenomatous GH-secreting cells in culture. Specimens were obtained from 10 patients with acromegaly who had undergone selective transsphenoidal adenomectomy. Cells (2 x 10(5)/well) preincubated for 24 h with leptin (10(-10)-10(-8) m) or control medium were exposed to GHRH for 2 h. The GH released into the medium was measured before and after GHRH incubation. The expression of leptin receptor isoforms was evaluated by reverse-transcriptase polymerase chain reaction (RT-PCR) in cells obtained from five adenomas. After the first incubation, there was a slight dose-dependent leptin-induced decrease in GH released into the medium. A significant increase in GH release after GHRH was noted from cells previously exposed to leptin in comparison with cells incubated with medium alone. Expression of leptin receptors was found in two out of five GH-secreting adenomas evaluated. Our data confirm that some, but not all, GH-secreting adenomas express leptin receptors. Leptin seems to exert both a slight inhibitory effect on spontaneous GH secretion and a stimulatory effect on GHRH-stimulated GH secretion from GH-secreting adenomatous tissue.

Accelerated Escape from GH Autonegative Feedback in Midpuberty in Males: Evidence for Time-Delimited GH-Induced Somatostatinergic Outflow in Adolescent Boys

A single injected pulse of GH inhibits the time-delayed secretion of GH in the adult by way of central mechanisms that drive somatostatin and repress GHRH outflow. The marked amplification of spontaneous GH pulse amplitude in puberty poses an autoregulatory paradox. We postulated that this disparity might reflect unique relief of GH-induced autonegative feedback during this window of development. The present study contrasts GH autonegative feedback in: 1) normal prepubertal boys (PP) (n = 6; Tanner genital stage I, chronologically aged 8 yr, 9 months to 10 yr, 1 month; median bone age 8.5 yr); 2) longitudinally identified midpubertal boys (MP) (n = 6; Tanner genital stages III/IV, aged 12 yr, 6 months to 15 yr, 6 months; median bone age 15 yr); and 3) healthy young men (YM) (n = 6, aged 18–24 yr; bone age >18 yr). Subjects each underwent four randomly ordered tandem peptide infusions on separate mornings while fasting: i.e. 1) saline/saline infused iv bolus at 0830 h and 1030 h; 2) saline/GHRH (0.3 μg/kg iv bolus) at the foregoing times; 3) recombinant human (rh) GH (3 μg/kg as a 6-min square-wave iv pulse)/saline; and 4) rhGH and GHRH. To monitor GH autofeedback effects, blood samples were obtained every 10 min for 5.5 h beginning at 0800 h (30 min before GH or saline infusion). Serum GH concentrations were quantitated by ultrasensitive chemiluminometry (threshold 0.005 μg/liter). On the day of successive saline/saline infusion, MP boys maintained higher serum concentrations of: 1) GH (μg/liter), 2.2 ± 0.25, compared with PP (0.61 ± 0.10) or YM (0.88 ± 0.36) (P = 0.011); 2) IGF-I (μg/liter), 493 ± 49 vs. PP (134 ± 16) and YM (242 ± 22) (P < 0.001); 3) T (ng/dl), 524 ± 58 vs. PP (<20) (P < 0.001); and 4) E2 (pg/ml),19 ± 3 vs. PP (< 10) (P = 0.030) (mean ± sem). Consecutive saline/GHRH infusion elicited comparable peak (absolute maximal) serum GH concentrations (micrograms per liter) in the three study groups, i.e. 18 ± 5.0 (PP), 9.6 ± 1.7 (MP), and 14 ± 5.3 (YM) (each P < 0.01 vs. saline; P = NS cohort effect). Injection of rhGH attenuated subsequent GHRH-stimulated peak serum GH concentrations (micrograms per liter) to 7.8 ± 1.9 (PP), 5.8 ± 1.2 (MP), and 4.8 ± 1.1 (YM) (each P < 0.01 vs. saline; P = NS pubertal effect). GH autofeedback reduced non-GHRH-stimulated (basal) serum GH concentrations by 0.74 ± 0.28 (PP), 5.7 ± 1.7 (MP) and 1.4 ± 0.27 (YM) fold, compared with saline (P = 0.016 for MP vs. PP or YM). In addition to greater fractional autoinhibition, MP boys exhibited markedly accentuated postnadir escape (4.6-fold steeper slope) of suppressed GH concentrations (P < 0.001 vs. PP or YM). Linear regression analysis of data from all 18 subjects revealed that the fasting IGF-I concentration negatively predicted fold-autoinhibition of GHRH-stimulated peak GH release (r = −0.847, P = 0.006) and positively forecast fold-autoinhibition of basal GH release (r = +0.869, P < 0.001). In contrast, the kinetics of rhGH did not differ among the three study cohorts. In summary, boys in midpuberty manifest equivalent responsiveness to exogenous GHRH-stimulated GH secretion; heightened susceptibility to rhGH-induced fractional inhibition of endogenous secretagogue-driven GH release, compared with the prepubertal or adult male; and accelerated recovery of GH output after acute autonegative feedback. This novel tripartite mechanism could engender recurrent high-amplitude GH secretory bursts that mark sex hormone-dependent activation of the human somatotropic axis.

Is GHRH receptor essential to GHRP-2-induced GH secretion in primary cultured rat pituitary cells?

It is still controversial in rat whether the stimulation of GH secretion by GH-releasing peptides (GHRP) requires both GHRP receptor (GHRP-R) and GH-releasing hormone receptor (GHRH-R). To clarify this issue, we have postulated that inhibition of GHS-R or GHRH-R gene transcription should block GHRP-2-induced GH secretion. Rat pituitary cells were incubated for 3 days in the presence or absence of antisense 18-mer phosphorothiate oligonucleotides (ONs) complementary to the codon region of GHS-R or GHRH-R mRNAs. A significant decrease in GHRH-R and GHS-R mRNA levels was found in corresponding antisense-treated cells compared with the control cells treated with sense ON. Treatment with antisense GHS-R ON reduced (but not abolished) GHRP-2-induced GH secretion although GHRH-induced GH secretion was not altered. GHRH-stimulated GH secretion was totally abolished by the treatment with antisense GHRH-R ON, whereas GHRP-2 induced GH secretion was not affected. Treatment of cells with both GHS-R and GHRH-2 ONs however completely inhibited GHRH and GHRP-2-stimulated GH secretion. These results suggest that GHRH-R is vital for GHRH-induced GH secretion but only partially involved in GHRP-2-stimulated GH secretion under the condition of down-regulation of GHS-R gene transcription.

Administration of recombinant bovine tumor necrosis factor-alpha affects intermediary metabolism and insulin and growth hormone secretion in dairy heifers.

Four experiments were conducted to clarify the effect of intravenous (i.v.) administration of recombinant bovine tumor necrosis factor alpha (rbTNF) on selected metabolites and on hormone secretion in Holstein heifers (n = 6; 347.0 kg average BW). In Exp. 1, rbTNF was injected at three dosage levels in a Latin square; 0 (CONT), 2.5 (TNF2.5), or 5.0 (TNF5) microg/kg BW. Plasma glucose and triglyceride concentrations were at first elevated (P < .05) by rbTNF treatment and then were decreased (P < .05) by TNF2.5 and TNF5. Plasma NEFA concentrations were increased (P < .05) in rbTNF-treated groups. The injection of rbTNF resulted in an increase in plasma insulin levels (P < .05 with TNF5) during the period between 2 and 24 h, except for the period between 6 and 8 h, after the treatment. In Exp. 2, 3, and 4, each heifer received i.v. injections of glucose (.625 mM/kg BW) + rbTNF (5 microg/kg) or glucose + saline (10 mL) (Exp. 2), insulin (0.2 U/kg) + rbTNF or insulin + saline (Exp. 3), and GHRH (0.25 microg/kg) + rbTNF or GHRH + saline (Exp. 4) at 1-wk intervals. In Exp. 2, rbTNF inhibited (P < .05) glucose-stimulated insulin secretion during the initial phase. Thereafter, plasma insulin was higher (P < .01) with the glucose + rbTNF treatment than with the glucose + saline treatment. Treatment with rbTNF inhibited the insulin-stimulated glucose utilization (Exp. 3) and GHRH-stimulated GH secretion (Exp. 4) during the initial phase. These results suggest that rbTNF directly and(or) indirectly affects the intermediary metabolism and hormone secretion in Holstein heifers.

In vivo semiquantification of hypothalamic growth hormone-releasing hormone (GHRH) output in humans: evidence for relative GHRH deficiency in aging.

GH secretion declines with aging. The neuroendocrine mechanisms of somatopause are uncertain. To semiquantify endogenous hypothalamic GHRH output, we measured the suppressibility of spontaneous and GHRH-stimulated GH secretion by graded doses of a specific competitive GHRH receptor antagonist (N-Ac-Tyr1,D-Arg2)GHRH-(1-29) in healthy young and elderly men. Nocturnal GH was about 30% lower in the elderly than in the young. Graded boluses of GHRH elicited dose-dependent GH responses, with no difference between the two age groups. Graded infusions of GHRH antagonist suppressed GH responses to GHRH in a dose-dependent manner, but with similar potency in both groups. The degree of inhibition depended on the magnitude of GHRH bolus: the dose-inhibition curves for the low GHRH boluses were shifted to the left compared to those with the high GHRH bolus (P = 0.01). Similarly, the dose-inhibition curve for spontaneous GH secretion was shifted to the left for the elderly compared to the young men (P = 0.01). Thus, the model of graded infusions of GHRH antagonist differentiates between different amounts of GHRH presented to the pituitary, permitting semiquantification of the endogenous hypothalamic GHRH output in vivo. Our data suggest that there is an age-dependent decrease in the endogenous hypothalamic GHRH output contributing to the age-associated GH decline.

LH and FSH secretory responses to GnRH in normal individuals: a non-parametric deconvolution approach.

To reconstruct the instantaneous secretion rate (ISR) of LH and FSH after GnRH administration in normal volunteers using non-parametric deconvolution, and to derive a direct integration formula to evaluate the amount of LH and FSH secreted during the first 60 min after the stimulus. First, the deconvolution method was validated in vivo by reconstructing doses ranging from 7.5 IU to 75 IU injected in three healthy adult volunteers whose endogenous LH had previously been downregulated by pretreating them, 3-4 weeks earlier, with 3.75 mg GnRH agonist i.m. Then, 40 healthy adult male volunteers were tested with a single 100 microg GnRH bolus, administered at 0 min. LH and FSH concentrations were determined at -30, 0, 15, 30, 45, 60, 90, and 120 min. The validation study, conducted over a 10-fold range of doses, demonstrated that non-parametric deconvolution provided a reasonably accurate estimate of the amount of hormone entering the circulation. Applying deconvolution to the LH and FSH responses to GnRH, the ISRs of both hormones were shown to have a similar pattern, with a clearly delimited pulse after the GnRH bolus. In conjunction with earlier analyses of estimates of GHRH-stimulated GH secretion, we conclude that secretagogues evoke discrete LH, FSH, and GH secretory bursts of about 60 min total duration, despite markedly unequal (glyco-)protein hormone half-lives (18-500 min). With respect to the assessment of total hormone release during the first 60 min after the stimulus, the integration formula provided a reliable approximation of the result obtained by deconvolution, and had a negligible dependence on the samples at times 90 and 120 min.

The in vitro Effect of Leptin onBasal and Growth Hormone-Releasing Hormone-StimulatedGrowth Hormone Secretion from theOvine Pituitary Gland

We have studied the direct effect of leptin on the secretion of GH from the anterior pituitary gland of the sheep. Using primary cultures of ovine pituitary cells, leptin (10^–9–10^–7 M) treatment for 30 min did not affect basal or GHRH (10^–7 M)-stimulated GH secretion. Following treatment for 24 h, a dose of 10^–7 M leptin stimulated basal GH secretion. In contrast, doses of 10^–7 and 10^–8 M leptin inhibited GHRH-stimulated GH secretion after 24 h of treatment. These results suggest that leptin can have long-term effects on the somatotropes, but no acute effect. Furthermore, leptin appears to have opposite effects on basal and GHRH-stimulated GH secretion.

Responsiveness of chicken embryonic somatotropes to somatostatin (SRIF) and IGF-I

We have previously demonstrated that chicken embryonic somatotropes are responsive to GHRH shortly after they differentiate. In contrast, relatively little is known about the regulation of GH secretion by somatostatin (SRIF) and IGF-I during chicken embryonic development. In the present study, anterior pituitary cells were isolated from day. 16, 18 and 20 embryos and subjected to reverse hemolytic plaque assays (RHPAs) for chicken GH to assess the effect of SRIF and IGF-I on basal GH release and SRIF on GHRH-stimulated GH secretion. We found that all three ages responded to SRIF, under both basal and stimulated conditions. SRIF inhibition of basal GH release was evident for day 18 and 20 cells by 9 h, while 18 h were required for day 16 cells. After 18 h, 10(-11) M SRIF depressed basal GH secretion by day 16 and 18 cells, while 10(-9) M SRIF was required to depress GH plaque percentages by day 20 cells. GHRH stimulated GH release from all ages tested. After 2 h, SRIF partially suppressed GHRH-stimulated GH release by day 20 cells. After 6 h, day 18 cells responded to SRIF, reducing the percentage of plaque-forming cells under GHRH stimulated conditions. After 18 h, the percentage of day 16 cells forming GH plaques was reduced for cells treated with GHRH and SRIF, compared with cells treated with GHRH alone. All ages examined also responded to IGF-I. After 36 h, GH release by day 16 and 18 cells was decreased when exposed to IGF-I. While IGF-I decreased the relative amount of GH secreted per somatotrope on day 20, a paradoxical increase in the percentage of cells secreting GH was noted. These results indicate that anterior pituitary somatotropes are responsive to SRIF and IGF-I during late embryonic development of chickens.

Involvement of nitric oxide in the interferon- γ-induced inhibition of growth hormone and prolactin secretion in anterior pituitary cell cultures

In previous work it was shown that the immune cytokine interferon- γ (IFN- γ) inhibits hormone secretion in anterior pituitary (AP) cell cultures, an action most likely mediated by folliculostellate (FS) cells. In the present study, we wanted to investigate whether nitric oxide (NO) is involved in this inhibitory action of IFN- γ. NO synthase (NOS) inhibitors with affinity for the inducible (iNOS) and the constitutive (cNOS) isoform such as N G-monomethyl- l-arginine ( l-NMMA) and S-methyl- l-thiocitrulline (SMLT) dose-dependently blocked the inhibitory action of IFN- γ on GHRH-stimulated GH secretion, and partially reversed the inhibitory effect on basal prolactin (PRL) release. In the absence of IFN- γ these inhibitors significantly augmented basal PRL release and slightly enhanced GHRH-stimulated GH release. l- N 6-(1-iminoethyl)lysine ( l-NIL), a NOS inhibitor with preferential affinity for iNOS, abrogated the IFN- γ effect on GHRH-stimulated GH secretion and partially reversed IFN- γ inhibition of PRL release. However, l-NIL did not exert a stimulatory effect on basal PRL and GHRH-stimulated GH release by its own. 2,4-diamino-6-hydroxypyrimidine (DAHP), a NOS inhibitor by interfering with tetrahydrobiopterin (BH4) cofactor availability, showed the same activity profile as l-NIL. NOS inhibitors blocked or reduced the production of NO as detected by measuring nitrite (NO − 2) levels in AP cell cultures and cGMP levels in the NO-reporter cell line RFL-6. The NOS inhibiting action of l-NMMA was confirmed by competition experiments with the natural NOS substrate l-arginine. Thus, in culture medium with lower amounts of l-arginine, l-NMMA blocked the IFN- γ-induced inhibition of GHRH-stimulated GH release at a lower dose. The inhibition of PRL and GH release by IFN- γ was markedly reduced in l-arginine-depleted medium. The NO donor sodium nitroprusside (SNP) mimicked the inhibitory action of IFN- γ on GHRH-stimulated GH and basal PRL release. Similarly to IFN- γ, SNP did not affect basal GH release. As previously reported, inhibition by IFN- γ occurred only in AP cell populations containing a minimal proportion of FS cells. As studied in different cell populations obtained by unit gravity sedimentation in a serum albumin gradient, l-NMMA reversed the IFN- γ effect in the same populations enriched in FS cells. Interestingly, in the absence of IFN- γ l-NMMA strongly stimulated basal PRL release in the population most enriched in FS cells. It is concluded that IFN- γ through activation of the iNOS pathway probably in FS cells enhances the production of NO and that this effect is responsible for the inhibitory action of IFN- γ on GHRH-stimulated GH release and partially for the IFN- γ-induced decrease in basal PRL release. On the other hand, NO, likely produced by cNOS, appears to exert a tonic inhibitory effect on GHRH-stimulated GH and basal PRL release. It seems therefore that low amounts of NO produced constitutively may take charge of subtle physiological adaptations, and higher levels of NO produced by iNOS under the influence of IFN- γ may attenuate PRL and GH release during emergency conditions of immune and inflammatory reactions.

Activity of GH/IGF-I axis in catabolic patients with sepsis or trauma

Increased GH together with decreased IGF-I levels suggesting a peripheral GH insensitivity in critically ill patients have been reported by some but not by other authors. To clarify the activity of GH-IGF-I axis in catabolic states, basal and GHRH-stimulated GH secretion and IGF-I levels have been evaluated in patients with sepsis [SEP, n = 11; age (mean ± SEM) 56.1 ± 2.7 years] or trauma (TRA, n = 13; age 42.4 ± 4.1 years) on 1st, 3rd, 5th and 7th day after ICU admission, during artificial nutrition. SAPS II and MOF scores overlapped in both groups. Basal GH and IGF-I levels were also assayed in 24 normal subjects (NS, age 48.1 ± 4.3 years), 54 adult hypopituitaric patients with severe GH deficiency (GHD, age 44.8 ± 2.3 years), 19 patients with anorexia nervosa (AN, age 18.6 ± 0.6 years) and 12 patients with liver cirrhosis (LC, age 50.4 ± 2.8 years). In GHD, basal GH levels were lower than in NS (0.3 ± 0.1 versus 1.4 ± 0.2 μg/l, P < 0.01); similarly, IGF-I levels were markedly reduced and lower than in NS (68.6 ± 6.1 versus 213.5 ± 15.4 μg/l, P < 0.01). AN and LC basal GH levels were similar (10.0 ± 2.8 and 7.7 ± 2.1 μg/l) and higher than in NS (P < 0.01). AN and LC IGF-I levels were similar (70.4 ± 9.1 and 52.4 ± 10.5 μg/l), lower than in NS (P < 0.01) while overlapping with those in GHD. On the 1st day of ICU admission basal GH levels in SEP (0.7 ± 0.3 μg/l) and TRA (1.8 ± 0.6 μg/l) were similar to those in NS. IGF-I levels in SEP were lower than in TRA (82.9 ± 13.0 versus 127.0 ± 16.4 μg/l, P < 0.05) and both lower than in NS (P < 0.01). IGF-I levels in SEP were similar to those in GHD, AN and LC which were lower than in TRA (P < 0.01). GH levels in SEP and TRA remained similar up to the 7th day. In both groups the GH response to GHRH on the 3rd day was nearly abolished, clearly lower than in age-matched NS (GH peak, 2.5 ± 1.2 versus 10.5 ± 1.9 μg/l, P < 0.01). During parenteral nutrition, IGF-I levels increased both in SEP and TRA (P < 0.05 and P < 0.01, respectively). However, on the 7th day, IGF-I levels in SEP were clearly lower than in TRA (110.3 ± 9.5 versus 211.4 ± 25.5 μg/l, P < 0.01), overlapping the latter with those in NS. In conclusion, in severe catabolic conditions due to trauma and furthermore to sepsis, IGF-I levels are markedly low and similar to those in GHD. Somatotrope secretion is also impaired in both these catabolic conditions. Therefore, in patients with severe catabolism due to trauma or, particularly to sepsis, peripheral GH insensitivity and somatotrope insufficiency could both severely impair the activity of GH-IGF-I axis. Finally, artificial nutrition clearly increases IGF-I levels in post traumatic but not in septic catabolic states.

Involvement of endogneous nitric oxide in the control of pituitary responsiveness to different elicitors of growth hormone release in prepubertal rats.

Recently, nitric oxide (NO) has emerged as an important modulator of several neuroendocrine events. Present studies were designed in order to evaluate the involvement of endogenous NO in the control of GH secretion. In a first set of experiments, in vivo pituitary responsiveness to the elicitors of GH release, GH-releasing hormone (GHRH), GH-releasing hexapeptide (GHRP-6) and the agonist of excitatory amino acid receptors, N-methyl-D-aspartate (NMDA) was tested in prepubertal male rats after blockade of endogenous NO by pretreatment (45 min before the tests) with the competitive NO synthase (NOS) inhibitors NW-nitro-L-arginine methyl ester (NAME) and NW-nitro-L-arginine (NA). In addition, simultaneous administration of NAME and GHRH to prepubertal males was performed and the effects of NO deprivation on GHRH- and NMDA-stimulated GH release in prepubertal female rats were studied. In a second set of experiments, the involvement of NO in the modulation of GH secretion was analyzed in vitro. GH release by dispersed adenohypophysial cells was evaluated after challenge with the NO donors, sodium nitroprusside (SNP) and S-nitrosoacetylpenicillamine (SNAP), and cGMP, the classical second messenger for NO actions. In addition, the ability of NAME, SNP and cGMP to affect the GHRH-induced GH release in vitro was also tested. Our results showed that endogenous NO deprivation significantly attenuated in vivo GHRH-, GHRP-6- and NMDA-induced GH secretion in prepubertal male rats. However, the releasing ability of GHRH was not abolished by simultaneous administration of NAME. In addition, pretreatment with NAME blunted GHRH- and NMDA-stimulated GH release in prepubertal females. In vitro, neither SNP, SNAP, NAME nor cGMP modified GH secretion by pituitary cells in culture but NO deprivation by NAME completely blocked GHRH-induced GH release. In addition, cGMP but not SNP slightly enhanced GHRH-stimulated GH secretion. Taken together, these data strongly suggest that endogenous NO plays a permissive role in the control of stimulated GH release and that endogenous NO is an important factor eliciting GH secretion in prepubertal rats.

Short-term fasting in obesity fails to restore the blunted GH responsiveness to GH-releasing hormone alone or combined with arginine.

Fasting is known to clearly increase both spontaneous and GHRH-stimulated GH secretion in normal subjects and this effect is likely to be due to hypothalamic mechanism(s). Our aim was to clarify the effect of a 3 or 4-day fast, on the GH response to GHRH alone or combined with arginine, an amino acid probably acting via inhibition of hypothalamic somatostatin release. Two tests with GHRH (1 microgram/kg i.v.), administered either alone or in combination with arginine (ARG, 0.5 g/kg i.v.) were performed, in a randomized order at least 3 days apart. In obese women the two tests were repeated after a 3 or 4-day fast. Seven obese women (OB, aged 17-54 years, BMI 42.4 +/- 3.6 kg/m2, waist-hip ratio (WHR) 0.85 +/- 0.01) and ten healthy women, as control subjects (CS, aged 20-44 years, BMI 23.1 +/- 1.1 kg/m2, WHR 0.79 +/- 0.01) were studied. Serum GH and IGF-I levels were measured by radioimmunoassay. The GH secretory responses were expressed either as absolute values (mU/l) or as areas under the curve (AUC, mU/l/h) calculated by trapezoidal integration. IGF-I concentrations were expressed as absolute values (microgram/l) with reference to a pure recombinant IGF-I preparation. Results are expressed as mean +/- SEM. Basal GH and IGF-I levels in OB were lower than in CS (0.8 +/- 0.2 vs 4.8 +/- 1.0 mU/l, P < 0.0001 and 120.1 +/- 21.4 vs 188.7 +/- 13.1 micrograms/l, P < 0.02, respectively). The GHRH-induced GH rise in OB was lower (P < 0.00001) than in CS (AUC 340.2 +/- 81.0 vs 2125.0 +/- 199.6 mU/l/h). ARG increased the GHRH-induced GH rise in both groups, but in OB the GH response to ARG+GHRH (1458.4 +/- 439.0 mU/l/h, P < 0.03 vs GHRH alone) remained lower (P < 0.0001) than in CS (6396.2 +/- 772.2 mU/l/h, P < 0.01 vs GHRH alone). In spite of a reduction in body weight and IGF-I, insulin and glucose levels, in OB fasting failed to modify both the basal GH levels and the somatotroph responsiveness to GHRH when administered either alone or combined with ARG. An increase in free fatty acids (FFA) was also found after fasting. The results of this study demonstrate that in obesity the somatotroph hyporesponsiveness to GHRH, either alone or combined with arginine, is not improved by short-term fasting. As fasting is considered a CNS mediated stimulus to GH secretion, its ineffectiveness in obesity does not support a hypothalamic pathogenesis and suggests that long standing metabolic alterations, such as hyperinsulinaemia and/or elevated free fatty acids, could play a major role in causing GH insufficiency in obese patients.

Long-term changes of somatotrophic function induced by deprivation of growth hormone-releasing hormone during the fetal life of the rat

We have studied the effects of intra-amniotic administration of an anti-GH-releasing hormone serum (GHRH-Ab) on day 16 of fetal life in the rat, when the ontogenetic development of the GHRH neuronal system occurs. Control animals received normal rabbit serum. Following delivery, body weight was monitored for the next 30 days as an index of somatic growth, and the following indices of somatotrophic function were determined: plasma and pituitary GH, pituitary GH mRNA, hypothalamic GHRH and somatostatin mRNA, and the in vivo GH responsiveness to GHRH. At birth, GHRH-Ab-treated rats had a body weight that was equivalent to that of control rats but, starting from postnatal day 6 up to day 30, they had a significantly reduced body weight. Pituitary weight, the absolute pituitary GH content and GH mRNA levels were lower in experimental compared with control rats, while pituitary GH concentrations were similar in the two groups, thus implying that there was a defect, not only in GH synthesis, but also in GH release. In agreement with this theory, basal GH levels and GHRH-stimulated GH secretion were reduced in GHRH-Ab-treated rats but, in contrast, hypothalamic regulation of GH secretion appeared to be working in these rats as they were still able to respond to the low plasma GH by increasing GHRH and decreasing somatostatin mRNA levels. These findings indicate that deprivation of GHRH during fetal life induces long-lasting changes of growth rate and somatotrophic function.(ABSTRACT TRUNCATED AT 250 WORDS)