Structural basis for activation of the growth hormone-releasing hormone receptor.

Growth hormone-releasing hormone (GHRH) regulates the secretion of growth hormone that virtually controls metabolism and growth of every tissue through its binding to the cognate receptor (GHRHR). Malfunction in GHRHR signaling is associated with abnormal growth, making GHRHR an attractive therapeutic target against dwarfism (e.g., isolated growth hormone deficiency, IGHD), gigantism, lipodystrophy and certain cancers. Here, we report the cryo-electron microscopy (cryo-EM) structure of the human GHRHR bound to its endogenous ligand and the stimulatory G protein at 2.6 Å. This high-resolution structure reveals a characteristic hormone recognition pattern of GHRH by GHRHR, where the α-helical GHRH forms an extensive and continuous network of interactions involving all the extracellular loops (ECLs), all the transmembrane (TM) helices except TM4, and the extracellular domain (ECD) of GHRHR, especially the N-terminus of GHRH that engages a broad set of specific interactions with the receptor. Mutagenesis and molecular dynamics (MD) simulations uncover detailed mechanisms by which IGHD-causing mutations lead to the impairment of GHRHR function. Our findings provide insights into the molecular basis of peptide recognition and receptor activation, thereby facilitating the development of structure-based drug discovery and precision medicine.

Growth hormone-releasing hormone (GHRH) deficiency promotes inflammation-associated carcinogenesis.

The somatotropic axis, in addition to its well-known metabolic and endocrine effects, plays a pivotal role in modulation of inflammation. Moreover, growth hormone (GH)-releasing hormone (GHRH) has been involved in the development of various human tumors. In this work we aimed to investigate the consequences of GHRH deficiency on the development of inflammation-associated colon carcinogenesis in a mouse model of isolated GH deficiency due to generalized ablation of the GHRH gene [GHRH knock out (GHRHKO)]. Homozygous GHRHKO (-/-) male mice and wild type (C57/BL6, +/+) male mice as control group, were used. After azoxymetane (AOM)/dextran sodium sulfate (DSS) treatment -/- mice displayed higher Disease Activity Index (DAI) score, and more marked weight loss compared to +/+ animals. Additionally, -/- mice showed a significant increase in total tumors, in particular of large size predominantly localized in distal colon. In colonic tissue of AOM/DSS-treated -/- mice we found the presence of invasive adenocarcinomas, dysplasia and colitis with mucosal ulceration. Conversely, AOM/DSS-treated +/+ mice showed only presence of adenomas, without invasion of sub-mucosa. Treatment with AOM/DSS significantly increased prostaglandin (PG)E2 and 8-iso-PGF2α levels along with cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF)-α, nuclear factor kappa B (NF-kB) and inducible nitric oxide synthase (iNOS) gene expression, in colon specimens. The degree of increase of all these parameters was more markedly in -/- than +/+ mice. In conclusion, generalized GHRH ablation increases colon carcinogenesis responsiveness in male mice. Whether this results from lack of GH or GHRH remains to be established.

Increased pain and inflammatory sensitivity in growth hormone-releasing hormone (GHRH) knockout mice.

Growth hormone (GH) and GH-releasing hormone (GHRH), in addition to metabolic and endocrine effects, play a role in the modulation of pain and inflammation. We aimed to elucidate the consequences of GHRH deficiency on acute nociceptive stimulation and on both acute and chronic inflammatory stimuli in a mouse model of GH deficiency. Mice with generalized ablation of the GHRH gene (GHRH knock out, GHRHKO, -/-) were compared to wild type (GHRH +/+) mice. Responsiveness to acute nociceptive stimulation and to acute inflammatory stimulation was evaluated by conventional hot plate apparatus and formalin test, respectively. We also evaluated responsiveness to colonic inflammation induced both in vivo, after dextran sodium sulfate (DSS) treatment, or ex vivo, by incubating colon segments with bacterial lipopolysaccaride (LPS). Macroscopical and histological examinations were performed, prostaglandin (PG) E2 and 8-iso-PGF2α levels and cyclooxigenase (COX)-2 and tumor necrosis factor (TNF)-α gene expression were measured. Compared to controls, -/- mice showed decreased response latency during the hot plate test, and increased licking/biting time in formalin test, particularly in the second phase of inflammation. DSS treated -/- mice showed a significant increase of colonic inflammation compared to controls. Moreover DSS treatment increased PGE2 and 8-iso-PGF2α levels, along with COX-2 and TNF-α gene expression more markedly in colon specimens of -/- mice compared to controls. LPS-induced PGE2 and 8-iso-PGF2α production from colonic segments incubated ex vivo was also increased in -/- mice. Generalized GHRH gene ablation increases sensitivity to thermal pain and both acute and persistent inflammatory stimuli in male mice.

Alterations in oxidative, inflammatory and apoptotic events in short-lived and long-lived mice testes.

Aged testes undergo profound histological and morphological alterations leading to a reduced functionality. Here, we investigated whether variations in longevity affect the development of local inflammatory processes, the oxidative state and the occurrence of apoptotic events in the testis. To this aim, well-established mouse models with delayed (growth hormone releasing hormone-knockout and Ames dwarf mice) or accelerated (growth hormone-transgenic mice) aging were used. We hereby show that the testes of short-lived mice show a significant increase in cyclooxygenase 2 expression, PGD2 production, lipid peroxidation, antioxidant enzymes expression, local macrophages and TUNEL-positive germ cells numbers, and the levels of both pro-caspase-3 and cleaved caspase-3. In contrast, although the expression of antioxidant enzymes remained unchanged in testes of long-lived mice, the remainder of the parameters assessed showed a significant reduction. This study provides novel evidence that longevity confers anti-inflammatory, anti-oxidant and anti-apoptotic capacities to the adult testis. Oppositely, short-lived mice suffer testicular inflammatory, oxidative and apoptotic processes.

Testing for growth hormone deficiency in adults: doing without growth hormone-releasing hormone.

PURPOSE OF REVIEW: This article summarizes recent advances in testing for growth hormone deficiency (GHD) in adults, focusing on critical appraisal of existing growth hormone (GH) provocative tests as well as newer tests in development. RECENT FINDINGS: The diagnosis of GHD can be challenging and often requires the use of GH provocative testing. The most widely validated of these is insulin-induced hypoglycemia (ITT), which requires close supervision and has significant contraindications and side-effects. The arginine-growth hormone-releasing hormone (GHRH) test had become widely used as a safe and accurate alternative to the ITT, but GHRH is currently unavailable for clinical use in the USA. On the basis of review of recent literature we recommend that in the absence of GHRH, glucagon stimulation testing should be the preferred alternative to ITT. Several synthetic GH secretagogues that mimic the gastric peptide ghrelin are currently in development and may become available for use in the diagnosis of GHD in the near future. Other GH provocative tests suitable for use in children lack adequate specificity for the diagnosis of GHD in adults. SUMMARY: Due to the current unavailability of the arginine-GHRH test in the USA, when ITT is contraindicated or impractical we recommend the glucagon stimulation testing as the GH provocative test of choice. There remains a need for a simple, safe and accurate test for the diagnosis of GHD.

Influenza virus-induced glucocorticoid and hypothalamic and lung cytokine mRNA responses in dwarf lit/lit mice.

Influenza virus infection up-regulates cytokines such as interleukin-1beta (IL-1beta) and activates the somatotropic axis and the hypothalamic-pituitary axis. Mice with deficits in growth hormone releasing hormone (GHRH) signaling (lit/lit mice) respond to influenza virus challenge with a progressive decrease in sleep and lower survival rates. Current experiments characterize plasma glucocorticoid responses and hypothalamic and lung mRNA expression of sleep-related genes in lit/lit mice and their heterozygous controls after influenza virus challenge. lit/lit mice had higher basal and post-infection plasma corticosterone levels compared to controls. In contrast, the heterozygous mice increased hypothalamic GHRH-receptor, CRH-type 2 receptor, IL-1beta, and tumor necrosis factor-alpha (TNF-alpha) mRNAs after virus treatment while the lit/lit mice failed to up-regulate these substances. In contrast, lung levels of IL-1beta and TNF-alpha mRNAs were greater in the lit/lit mice. These data are consistent with the hypothesis that the sleep response to influenza infection is mediated, in part, by an up-regulation of hypothalamic sleep-related transcripts and they also show that a primary deficit in GHRH signaling is associated with enhanced corticosterone secretion and attenuated hypothalamic cytokine response to infection.

Hypothalamic growth hormone-releasing hormone (GHRH) deficiency: targeted ablation of GHRH neurons in mice using a viral ion channel transgene.

Animal and clinical models of GHRH excess suggest that GHRH provides an important trophic drive to pituitary somatotrophs. We have adopted a novel approach to silence or ablate GHRH neurons, using a modified H37A variant of the influenza virus M2 protein ((H37A)M2). In mammalian cells, (H37A)M2 forms a high conductance monovalent cation channel that can be blocked by the antiviral drug rimantadine. Transgenic mice with (H37A)M2 expression targeted to GHRH neurons developed postweaning dwarfism with hypothalamic GHRH transcripts detectable by RT-PCR but not by in situ hybridization and immunocytochemistry, suggesting that expression of (H37A)M2 had silenced or ablated virtually all the GHRH cells. GHRH-M2 mice showed marked anterior pituitary hypoplasia with GH deficiency, although GH cells were still present. GHRH-M2 mice were also deficient in prolactin but not TSH. Acute iv injections of GHRH in GHRH-M2 mice elicited a significant GH response, whereas injections of GHRP-6 did not. Twice daily injections of GHRH (100 microg/d) for 7 d in GHRH-M2 mice doubled their pituitary GH but not PRL contents. Rimantadine treatment failed to restore growth or pituitary GH contents. Our results show the importance of GHRH neurons for GH and prolactin production and normal growth.

Ghrelin stimulates GH but not food intake in arcuate nucleus ablated rats.

Ghrelin, an endogenous ligand for the GH secretagogue receptor 1a (GHS-R(1a)), was originally purified from the rat stomach. Ghrelin mRNA and peptide have also been detected in the hypothalamus and pituitary. Ghrelin is a novel acylated peptide that regulates GH release and energy metabolism. GHS-R(1a) mRNA is expressed in the pituitary gland as well as in several areas of the brain including the hypothalamus. In this study, we examined whether ghrelin could stimulate GH secretion and feeding in chronic GHRH, neuropeptide Y, and agouti-related protein deficient rats that were neonatally treated with monosodium glutamate (MSG), which destroys the neurons in the hypothalamic arcuate nucleus (ARC). Intravenous (iv) administration of rat ghrelin (10 micro g/kg body weight) increased plasma GH levels significantly in the normal adult male rats during a GH trough period of pulsatile GH secretion, while iv injection of ghrelin in MSG-treated rats resulted in a markedly attenuated GH response. When rat ghrelin (10 micro g/rat) was administered intracerebroventricular (icv), plasma GH levels were increased comparably in normal control and MSG-treated rats. However, the GH release after icv injection of ghrelin was markedly diminished compared with that after iv administration of a small amount of ghrelin in normal control rats (icv: 10 micro g/rat, iv: approximately 4.0 micro g/rat), indicating that the GH-releasing activity of exogenous ghrelin is route dependent and at least in part via hypothalamic ARC. The icv administration of 1 micro g of ghrelin increased significantly 4-h food intake in normal control, whereas the peptide did not increase food intake in MSG-treated rats, indicating that the feeding response to ghrelin requires intact ARC. Taken together, the primary action of ghrelin on appetite control and GH releasing activity is via the ARC even though it might act on another type of GHS-R besides GHS-R(1a).

Reproducibility of the growth hormone response to stimulation with growth hormone-releasing hormone plus arginine during lifespan.

The reliability and reproducibility of provocative stimuli of growth hormone (GH) secretion in the diagnosis of GH deficiency are still controversial both in childhood and in adulthood. The combined administration of GH-releasing hormone (GHRH) and arginine (ARG), which likely acts via inhibition of hypothalamic somatostatin release, is one of the most potent stimuli known so far and has been proposed recently as the best test to explore the maximal somatotrope capacity of somatotrope cells. However, it is well known that, usually, provocative stimuli of GH secretion suffer from poor reproducibility and that of the GHRH + ARG test has still to be verified. We aimed to verify the between- and within-subject variability of the GH response to the GHRH + ARG test in normal subjects during their lifespan as well as in hypopituitaric patients with GH deficiency (GHD). In 10 normal children (C: six male and four female, age 12.3 +/- 0.9 years, body mass index (BMI) = 16.6 +/- 0.7 kg/m2, pubertal stages I-III), 18 normal young adults (Y: ten male and eight female, age 31.1 +/- 1.3 years, BMI = 21.4 +/- 0.4 kg/m2), 12 normal elderly subjects (E: two male and ten female, age 74.4 +/- 1.8 years, BMI= 22.6 +/- 0.6 kg/m2) and 15 panhypopituitaric GH-deficient patients (GHD: nine male and six female, age 40.9 +/- 4.1 years, BMI= 22.7 +/- 1.0 kg/m2), we studied the inter- and intra-individual variability of the GH response to GHRH (1 microg/kg i.v.) + ARG (0.5 g/kg i.v.) in two different sessions at least 3 days apart. The GH responses to GHRH + ARG in C (1st vs 2nd session: 61.6 +/- 8.1 vs 66.5 +/- 9.4 microg/l), Y (70.4 +/- 10.1 vs 76.2 10.7 microg/l) and E (57.9 14.8 vs 52.1 +/- 8.0 microg/l) were similar and reproducible in all groups. The somatotrope responsiveness to GHRH + ARG also showed a limited within-subject variability (r = 0.71, 0.90 and 0.89 and p < 0.02, 0.0005 and 0.0005 for C, Y and E, respectively). Similarly in GHD, the GH response to the GHRH + ARG test showed a good inter- (1st vs 2nd session: 2.3 +/- 0.5 vs 2.2 +/- 0.6 microg/l) and intra-individual reproducibility (r = 0.70, p < 0.005). The GHRH + ARG-induced GH responses in GHD were markedly lower (p < 0.0005) than those in age-matched controls and no overlap was found between GH peak responses in GHD and normal subjects. In normal subjects, the GH response to GHRH + ARG is very marked, independent of age and shows limited inter- and intra-individual variability. The GH response to the GHRH + ARG test is strikingly reduced in panhypopituitaric patients with GHD, in whom the low somatotrope responsiveness is reproducible. Thus, these findings strengthen the hypothesis that GHRH + ARG should be considered the most reliable test to evaluate the maximal secretory capacity of somatotrope cells and to distinguish normal subjects from GHD patients in adulthood.

Alterations of adenylyl cyclase-coupled growth hormone-releasing hormone (GHRH) pituitary receptors in different conditions of GHRH deprivation.

Previous studies have clearly shown that the progressive decrease of growth hormone (GH) secretion occurring during ageing is coupled with a reduced responsiveness of pituitary GHRH receptors both in terms of GH secretion and activation of the adenylyl cyclase (AC), in the presence of increased basal values of the enzyme. The mechanism(s) subserving the age-associated alterations of GHRH-sensitive AC is likely related to the progressive decrease of hypothalamic GHRH function occurring with ageing. In this context, in old male rats, short-term administration of GHRH decreased the high basal AC activity and enhanced the GHRH-stimulated AC activity. Along this line, we decided to investigate whether experimentally induced abrogation of GHRH function in adult rats would induce the same alterations of AC-coupled GHRH receptors present in aged rats. Passive immunization of male young-adult rats with supra-maximal doses of GHRH antiserum (Ab-GHRH) significantly reduced the AC responsiveness to GHRH, an effect already evident 5 days post-injection and still present at 10 days. At this time interval, the treatment also evoked a significant increase of basal AC levels and of Gs alpha protein in the pituitary and completely blocked the GH-releasing effect of a bolus injection of GHRH. Furthermore, mechanical disruption of brain-pituitary links by complete stereotaxical ablation of the mediobasal hypothalamus induced a significant increase of basal AC levels and Gs alpha protein in the pituitary and a strikingly lower AC responsiveness to GHRH.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth hormone-releasing activity of growth hormone-releasing peptide-1 (a synthetic heptapeptide) in children and adolescents.

The heptapeptide growth hormone-releasing peptide-1 (GHRP-1), one of a series of recently synthesized small growth hormone (GH)-releasing peptides, was administered as an iv bolus (1 microgram/kg) to 15 (six prepubertal, nine pubertal) short but healthy children and adolescents and to eight juvenile patients with pituitary insufficiency (four with isolated growth hormone deficiency, two with multiple pituitary hormone deficiencies, one with partial GH deficiency and one with GH-releasing hormone (GHRH) deficiency). Eleven out of 23 subjects also underwent an in GHRH (1-29) test (1 microgram/kg). All the healthy children responded with a progressive rise in plasma human GH (hGH) peaking at 15-30 min, with a significantly higher rise (p < 0.05) in the pubertal than prepubertal group. The hGH response to GHRH (1-29) in these children was similar or slightly higher. Six hypopituitary patients had no response to either GHRP-1 or GHRH; the patient with partial GH deficiency had a hGH peak of 6.5 micrograms/l (at 5 min) to GHRP-1 and 9.2 micrograms/l (at 15 min) to GHRH. One patient had no response of hGH to hypoglycemia, clonidine and GHRP-1, but the plasma hGH rose to 10 micrograms/l after GHRH. Following the GHRP-1 bolus there was a significant (p < 0.01) rise in plasma free thyroxine and a decrease of thyrotropin (p < 0.01), both in the limits of normal values. There was also a transitory rise of plasma cortisol (p < 0.05). Plasma prolactin, luteinizing hormone and follicle-stimulating hormone did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

Efecto de la administración aguda de rilmenidina sobre la secreción de la hormona de crecimiento: estudio preliminar / Effect of rilmenidine of growth hormone secretion: preliminary study

En el presente estudio preliminar se analiza el efecto de la rilmenidina como estimulante de la secreción de hormona del crecimiento en un grupo de 8 niños pre-puberales con talla por debajo del promedio, pero dentro del rango normal (entre percentil 3 y el 25). Se encuentra que este agente en general es tan eficiente como la clonidina en este aspecto. De acuerdo con estos resultados se considera que la rilmenidina podría ser utilizada como prueba funcional en niños prepuberales con retraso del cresimiento y se plantea la posibilidad de utilizarla en el tratamiento de estos pacientes, en forma similar a la que se ha hecho con la clonidina

Growth induced by pulsatile infusion of an amidated fragment of human growth hormone releasing factor in normal and GHRF-deficient rats.

The discovery of human pancreatic growth hormone releasing factors (GHRFs) and subsequent characterization of human hypothalamic GHRF has led to studies on the role of these peptides in stimulating growth hormone (GH) release, and attempts to use GHRF peptides to increase growth rates in short children are already underway. However, there is no experimental evidence in animals that exogenous GHRF promotes growth in vivo. Although anaesthetized rats release GH reproducibly in response to GHRF injections, the responses in conscious male rats are much more variable, perhaps because of their highly episodic endogenous GH secretory pattern. In contrast, female rats secrete GH in a more continuous pattern and respond reproducibly to repeated injections of GHRF. We report here that it is possible to establish a 'male' type of GH secretory pattern in normal female rats by long-term pulsatile intravenous (i.v.) infusions of the active human GHRF fragment GHRF (1-29)NH2. We found that this treatment accelerates growth and increases pituitary GH content, whereas continuous infusions of this GHRF fragment at the same daily dose are ineffective. Pulsatile, but not continuous GHRF also stimulates growth in animals made GHRF-deficient by neonatal monosodium glutamate treatment. Thus exogenous GHRF will stimulate growth in both GHRF-deficient and normal animals provided it is administered in an appropriate pattern.

Hypothalamic growth hormone releasing factor deficiency following cranial irradiation.

The effect of synthetic human pancreatic tumour GH releasing factor (hp GRF1-44) on GH release has been studied in 10 patients with radiation-induced GH deficiency and four normal subjects. All 10 patients showed subnormal GH responses to both an ITT (median peak GH 3.2 mU/l) and to arginine stimulation (median peak GH 2.9 mU/l), although the remainder of pituitary function was intact. Following an acute intravenous bolus (100 micrograms) of hp GRF1-44, there was no GH response in two patients and a subnormal but definite GH response in a further four. The remaining four patients showed a significant GH response (median peak GH level 29 mU/l; range 22-57 mU/l) to hp GRF1-44, similar in magnitude and timing to that seen in the four normals. This strongly suggests that in these four subjects, the discrepancy in GH responses to hp GRF1-44, ITT and to arginine was a result of radiation-induced hypothalamic damage leading to a deficiency of endogenous GRF. The availability of synthetic hp GRF capable of stimulating GH secretion means that the distinction between hypothalamic and pituitary causes of GH deficiency will be of considerable therapeutic importance in the future.