GHRH Expression Research Articles

Hypothalamic GHRH.

Despite initial discovery in pancreatic tumors, GHRH is a 44-amino acid peptide primarily expressed in the hypothalamus. Recent RNA sequencing clarifies GHRH expression: predominantly hypothalamic in humans, with some basal ganglia presence, while extending to additional central nervous system (CNS) regions in other species. GHRH binds to its G-protein coupled receptor (GHRHR) in the arcuate (ARC), ventromedial (VMH), and periventricular (PeN) nuclei of the hypothalamus to exert its effects. Notably, the highest non-brain expression is found in somatotroph cells of the pituitary, directly targeting growth hormone (GH) production. GHRH is the primary regulator of pulsatile GH secretion, counteracted by somatostatin. While early models proposed alternating GHRH/somatostatin bursts, others implicate somatostatin as the primary regulator of GH pulse timing. These models fail to fully explain species and gender differences, particularly regarding nutritional status. The discovery of ghrelin, acting via GHS-R1a on GHRH neurons, significantly advanced understanding of GH regulation. Ghrelin interacts intricately with GHRH, modulating its expression and neuronal activity. Ghrelin also exerts GHRH-independent GH stimulation and synergizes with GHRH. The crucial role of GHRH in GH regulation is demonstrated by its key involvement in the action of other GH regulators, such as leptin, neuropeptide Y (NPY), and orexins. However, these interactions have also revealed that the physiological effects of GHRH extend far beyond its canonical role as a GH secretagogue. In this context, GHRH is thought to be a key regulator of the sleep-wake cycle and may be involved in whole-body energy homeostasis. The objective of this review is to summarize the current knowledge on GHRH and to discuss the potential pleiotropic effect of this hypothalamic neuropeptide, far beyond its classical action as regulator of the somatotroph axis.

Effect of daytime light intensity on daily behaviours and concurrent hypothalamic gene expressions in migratory redheaded bunting.

Animals use photic cues to time their daily and seasonal activity. The role of photoperiod has been much investigated in seasonal responses, but the role of light intensity is less understood in passerine finches. We investigated if and how daytime light intensity influences photoinduced migratory phenologies and hypothalamic mRNA expressions in a Palearctic-Indian migratory finch, redheaded bunting (Emberiza bruniceps). Photoperiodic manipulations were employed to induce winter-nonmigratory (NM), premigratory (PM), and migratory (MIG) states in photosensitive buntings. In each life history state, the birds were further subjected to 0.055 (low), 0.277 (medium), or 1.11W/m2 (high) (N =5 each) light intensity treatment. The low daytime light intensity dampened the locomotor activity rhythm and delayed the onset of Zugunruhe. We found life history-dependant but not light intensity-dependant changes in body mass, fat score, and testis volume. Plasma corticosterone levels were increased under the low-light intensity group in the migratory state. The buntings were foraging throughout the night in the migratory state, aiding body fattening. Front and back sleep were drastically reduced during the migratory phase under all three light intensities. In the migratory state, we found elevated hypothalamic IL1B and IL6 expression in medium and high-light intensity groups, which had significantly reduced sleep duration. In the winter nonmigratory state, the expression of CAMK2 correlated with daytime activity and active wakefulness of buntings. The decreased GHRH expression correlates with the reduction in total sleep in migrating buntings. Overall, daytime light intensity emerges as a key factor that fine-tunes the photoperiodic response and regulates active and sleep behaviour in migratory buntings.

Expression pattern of the fused in sarcoma gene and its contextual influence on the density-specific response of the growth hormone/insulin-like growth factor 1 axis in zig-zag eels (Mastacembelus armatus)

The fused in sarcoma (FUS) protein is a DNA/RNA binding protein from the ten-eleven translocation protein family that is associated with neurodegeneration, and it has been shown to promote cell proliferation through the growth hormone/insulin-like growth factor 1 (Gh/Igf-1) signaling pathway. The zig-zag eel (Mastacembelus armatus) is a newly discovered species exhibiting sexual dimorphism in growth, and the potential role of fus in the growth and development of this species remains largely unknown. Herein, we analyzed the homology, conserved domains, evolutionary characteristics, and conserved syntenies of fus in several teleost species. The expression of fus was predominant in the brain and exhibited sexual dimorphism in the brain, muscle, and liver of zig-zag eels. We found that microRNA (miR)-146-5p, miR-489-3p, and 24 other miRNAs were targeted to the fus 3′ untranslated region, which might affect muscle and bone development in adults. The igf1, insulin-like growth factor 1 receptor a (igf1ra), insulin-like growth factor 2 receptor (igf2r), growth hormone-releasing hormone-like receptor (ghrhrl), growth hormone secretagogue receptor type 1 (ghsr), and glucocorticoid receptor (gr) genes contained a higher abundance of GU-rich fus motifs compared to the other four genes analyzed in zig-zag eels. We also measured the expression of fus mRNA during fish culture at various stocking densities to further elucidate the relationship between fus expression and the Gh/Igf-1 axis. After 100 days of fish cultivation, the expression of fus and ghrhrl decreased and the expression of ghrh and gr increased as the culture density increased (p < 0.05). The expression of fus exhibited a remarkable positive correlation with a specific growth rate. These results indicate that fus mediates growth differences by regulating the expression of several growth-related genes including Gh/Igf-1 axis genes in zig-zag eels.

Experimental Autoimmune Encephalomyelitis Influences GH-Axis in Female Rats.

Inflammation, demyelination, and axonal damage to the central nervous system (CNS) are the hallmarks of multiple sclerosis (MS) and its representative animal model, experimental autoimmune encephalomyelitis (EAE). There is scientific evidence for the involvement of growth hormone (GH) in autoimmune regulation. Previous data on the relationship between the GH/insulin like growth factor-1 (IGF-1) axis and MS/EAE are inconclusive; therefore, the aim of our study was to investigate the changes in the GH axis during acute monophasic EAE. The results show that the gene expression of Ghrh and Sst in the hypothalamus does not change, except for Npy and Agrp, while at the pituitary level the Gh, Ghrhr and Ghr genes are upregulated. Interestingly, the cell volume of somatotropic cells in the pituitary gland remains unchanged at the peak of the disease. We found elevated serum GH levels in association with low IGF-1 concentration and downregulated Ghr and Igf1r expression in the liver, indicating a condition resembling GH resistance. This is likely due to inadequate nutrient intake at the peak of the disease when inflammation in the CNS is greatest. Considering that GH secretion is finely regulated by numerous central and peripheral signals, the involvement of the GH/IGF-1 axis in MS/EAE should be thoroughly investigated for possible future therapeutic strategies, especially with a view to improving EAE disease.

Dynamic Changes and Effects of H2S, IGF-1, and GH in the Traumatic Brain Injury.

The aim of this study was to examine the expression changes of H2S, IGF-1, and GH in traumatic brain injury (TBI) patients and to detect their neuroprotective functions after TBI. In this study, we first collected cerebrospinal fluid (CSF) and plasma from TBI patients at different times after injury and evaluated the concentrations of H2S, IGF-1, and GH. In vitro studies were using the scratch-induced injury model and cell-cell interaction model (HT22 hippocampal neurons co-cultured with LPS-induced BV2 microglia cells). In vivo studies were using the controlled cortical impact (CCI) model in mice. Cell viability was assessed by CCK-8 assay. Pro-inflammatory cytokines expression was determined by qRT-PCR, ELISA, and nitric oxide production. Western blot was performed to assess the expression of CBS, CSE, IGF-1, and GHRH. Moreover, the recovery of TBI mice was evaluated for behavioral function by applying the modified Neurological Severity Score (mNSS), the Rotarod test, and the Morris water maze. We discovered that serum H2S, CSF H2S, and serum IGF-1 concentrations were all adversely associated with the severity of the TBI, while the concentrations of IGF-1 and GH in CSF and GH in the serum were all positively related to TBI severity. Experiments in vitro and in vivo indicated that treatment with NaHS (H2S donor), IGF-1, and MR-409 (GHRH agonist) showed protective effects after TBI. This study gives novel information on the functions of H2S, IGF-1, and GH in TBI.

FRI307 Gigantism Due To Transcriptional Activation Of GHRH: The First Case Of Hormone-encoding Gene Overexpression Underlying A Congenital Disorder

Abstract Disclosure: A. Hattori: None. Y. Katoh-Fukui: None. R. Zhang: None. M. Terao: None. S. Takada: None. K. Nakabayashi: None. K. Hata: None. Y. Yamada: None. N. Matsuura: None. M. Fukami: None. Background: Pituitary gigantism is characterized by tall stature due to GH overproduction. To date, known genetic causes of pituitary gigantism are invariably associated with predispositions to tumors producing GH or GHRH. Clinical and genetic findings of the patient: The patient was a Japanese woman who was born mildly large for gestational age. Her overgrowth started in her first year of life. Despite radiation and pharmacotherapy starting at age three years, she reached an adult height of 197 cm (+7.4 SD). She developed panhypopituitarism associated with empty sella in adulthood and was deceased during sleep at age 53 years. Exome sequencing identified no pathogenic variant causing acromegaly or gigantism. Chromosomal microarray analysis detected a 752-kb deletion upstream of GHRH. This deletion was predicted to create a chimeric gene consisting of the 5´-UTR of TTI1, a ubiquitously expressed gene, and the coding region of GHRH. Reverse transcription PCR (RT-PCR) using mRNA of immortalized lymphoblastoid cells detected the chimeric transcript. Analysis of model mice: We established a mouse model harboring a 676-kb deletion upstream of Ghrh using the CRISPR/Cas9 system. RT-PCR detected a chimeric transcript consisting of the 5´-UTR of Tti1 and the coding region of Ghrh, which mimicked the patient’s chimeric transcript. Mutant mice started to overgrow at two weeks of age and showed high serum GH levels and long tibia at 12-13 weeks of age. Quantitative RT-PCR showed that the median level of Ghrh expression in the hypothalamus of the mutant mice was three-fold higher than that of the wildtype mice. Furthermore, the mutant mice had ectopic Ghrh expression in all analyzed tissues (the pituitary, thymus, heart, liver, and gonad). Discussion: Our findings propose a novel etiology of gigantism. In the present case, the GHRH gene obtained a constitutively active promoter through germline genomic microdeletion. The newly acquired promoter likely led to the constant expression of GHRH in various tissues including the hypothalamus. Possible explanations for GH overproduction in the present case include (1) GHRH overproduction in the hypothalamus, (2) paracrine and autocrine effects of GHRH secreted from the pituitary, and (3) endocrine effects of GHRH produced outside the hypothalamic-pituitary unit. The patient’s clinical course suggests that prenatal GHRH overproduction only mildly affects human growth. More importantly, this is the first case in which transcriptional activation of a hormone-encoding gene caused a human congenital disorder. Presentation: Friday, June 16, 2023

Influence of Leptin on the Secretion of Growth Hormone in Ewes under Different Photoperiodic Conditions.

Leptin is an adipokine with a pleiotropic impact on many physiological processes, including hypothalamic-pituitary-somatotropic (HPS) axis activity, which plays a key role in regulating mammalian metabolism. Leptin insensitivity/resistance is a pathological condition in humans, but in seasonal animals, it is a physiological adaptation. Therefore, these animals represent a promising model for studying this phenomenon. This study aimed to determine the influence of leptin on the activity of the HPS axis. Two in vivo experiments performed during short- and long-day photoperiods were conducted on 12 ewes per experiment, and the ewes were divided randomly into 2 groups. The arcuate nucleus, paraventricular nucleus, anterior pituitary (AP) tissues, and blood were collected. The concentration of growth hormone (GH) was measured in the blood, and the relative expression of GHRH, SST, GHRHR, SSTR1, SSTR2, SSTR3, SSTR5, LEPR, and GH was measured in the collected brain structures. The study showed that the photoperiod, and therefore leptin sensitivity, plays an important role in regulating HPS axis activity in the seasonal ewe. However, leptin influences the release of GH in a season-dependent manner, and its effect seems to be targeted at the posttranscriptional stages of GH secretion.

Chromosomal microdeletion leading to pituitary gigantism through hormone-gene overexpression.

Pituitary gigantism is a rare endocrinopathy characterized by tall stature due to growth hormone (GH) hypersecretion. This condition is generally linked to a genetic predisposition to tumors that produce GH or GH-releasing hormone (GHRH). Here, we report a Japanese woman who exhibited prominent body growth from infancy to reach an adult height of 197.4cm (+7.4 standard deviation). Her blood GH levels were markedly elevated. She carried no pathogenic variants in known growth-controlling genes but had a hitherto unreported 752 kb heterozygous deletion at 20q11.23. The microdeletion was located 8.9 kb upstream of GHRH and encompassed exons 2-9 of a ubiquitously expressed gene TTI1 together with 12 other genes, pseudogenes and non-coding RNAs. Transcript analyses of the patient's leukocytes showed that the microdeletion produced chimeric mRNAs consisting of exon 1 of TTI1 and all coding exons of GHRH. In silico analysis detected promoter-associated genomic features around TTI1 exon 1. Genome-edited mice carrying the same microdeletion recapitulated accelerated body growth from a few weeks after birth. The mutant mice developed pituitary hyperplasia and exhibited ectopic Ghrh expression in all tissues examined. Thus, the extreme phenotype of pituitary gigantism in the patient likely reflects GHRH overexpression driven by an acquired promoter. The results of this study indicate that germline submicroscopic deletions have the potential to cause conspicuous developmental abnormalities due to gene overexpression. Furthermore, this study provides evidence that constitutive expression of a hormone-encoding gene can result in congenital disease.

Enhancing gosling growth and secretion of somatotrophic and thyrotrophic axis hormones through egg turning during incubation

ABSTRACT 1. Growth performance of Yangzhou geese hatched from eggs with turning angles of 50° or 70° was evaluated in association with serum hormones and somatotrophic gene mRNA expression. 2. Egg turning at 70° significantly (P< 0.05) increased hatchability, gosling quality and hatching weight. Gosling post-hatch body weight, leg and breast muscle weight in the 70° turning group was significantly heavier until 50 d of age. 3. Serum concentrations of GH were significantly higher until 30 d of age in the 70° turning group goslings, and those of IGF-I and T3 were higher from hatching to 50 d of age. 4. The mRNA expression of GHRH, pituitary GH, liver and leg muscle IGF-I were all significantly higher at 1 and 30 d of age after hatch, but not at 70 d after hatch, in the 70° turning group. 5. Egg turning at 70° during incubation improves embryo and gosling quality and growth performance through up-regulation of gene expression and secretion of somatotrophic axis hormones, GHRH, GH and IGF-I, as well as T3.

Dietary L-citrulline modulates the growth performance, amino acid profile, and the growth hormone/insulin-like growth factor axis in broilers exposed to high temperature.

Heat stress adversely affects the growth performance, muscle development, and protein metabolism in poultry. l-Citrulline (L-Cit), is a non-essential amino acid that is known to stimulate muscle protein synthesis under stress conditions. This study investigated whether L-Cit could influence the growth performance, amino acid profile, and protein metabolism in broilers exposed to high ambient temperature. In a 2 × 2 factorial arrangement, Arbor acre broilers (288 chickens) were fed with basal diet (CON) or 1% L-Cit supplemented diet and later subjected to either thermoneutral (TNZ: 24°C, 24 h/d) or heat stress (HS: 35°C for 8 h/d) environment for 21 days. The results showed that L-Cit diet promoted the body weight and body weight gain of broilers higher than the CON diet, and it further alleviated HS suppression of body weight and feed intake at certain periods (p < 0.05). Plasma urea, uric acid, glucose, and total cholesterol were elevated during HS, whereas, the triglyceride content was decreased (p < 0.05). Serum amino acids including citrulline, alanine, aspartate, and taurine were decreased by HS. L-Cit supplementation restored the citrulline level and alleviated HS induction of 3-methylhistidine (p < 0.05). L-Cit supplementation increased the plasma growth hormone (GH) and insulin-like growth factor-1 (IGF-1) concentration, as well as the GH concentration in the breast muscle (p < 0.05). The mRNA expression showed that HS elicited tissue-specific responses by upregulating some growth factors in the breast muscle, but downregulated the GH receptor, GH binding protein, and IGF-1 expression in the hypothalamus. L-Cit supplementation upregulated the GHRH and IGFBP2 expression in the hypothalamus. L-Cit also upregulated the expression of IGF-1R and IGFBP2 in the breast muscle of HS broilers. The total mTOR protein level in the breast muscle of HS broilers was also increased by L-Cit diet (p < 0.05). Therefore, this study demonstrated that HS negatively affected the growth performance of broilers and dysregulated the expression of growth factors related to protein metabolism. Contrarily, L-Cit promoted the growth responses of broilers via its stimulation of circulating GH/IGF-1 concentration. To certain extents, L-Cit supplementation elicited protective effects on the growth performance of HS broilers by diminishing protein catabolism.

Reduction of Reactive Oxygen Species by a Growth Hormone‐Releasing Hormone Antagonist in Endothelial Cells

INTRODUCTIONGrowth hormone‐releasing hormone (GHRH) is a hypothalamic hormone regulating the secretion of Growth Hormone (GH) from the anterior pituitary gland. The expression of GHRH and its receptors (full length pituitary‐type and splice variants) in tissues such as breast, prostate and lung, suggested that the activities of that hypothalamic hormone is not limited to the GH ‐ insulin‐like growth factor‐I axis. Reactive Oxygen Species (ROS) are involved in inflammation, disrupt inter‐endothelial junctions, promote cytoskeletal remodeling and induce pro‐inflammatory cytokines (e.g. interleukin [IL]‐1β, IL‐6). Those events have been associated to endothelial hyper‐permeability, the hallmark of Acute Respiratory Distress Syndrome (ARDS). The purpose of the present study is to investigate the effects of GHRH and GHRH antagonists (GHRHAnt) in ROS generation of three different endothelial cell lines.METHODSBovine pulmonary artery endothelial cells (BPAEC), human lung microvascular endothelial cells (HMVEC‐L), human cerebral microvascular endothelial cells (hCMEC/D3) and mouse embryonic fibroblasts (NIH/3T3) were maintained at 37°C in a humidified atmosphere of 5% CO2‐ 95% air per suppliers’ instructions. Cells seeded in 96‐well plates (1×104 cells/well, n=4 wells per group) were treated with GHRH (1µM), or GHRHAnt (1µM), or GHRHAnt (1µM) prior to H2O2 (0.1mM) exposure. To measure ROS levels, the cells were incubated with 25µM of DCFDA for 45 min, and fluorescence intensity was measured by a fluorescence microplate reader using excitation and emission wavelength of 485 nm and 535 nm respectively. Protein expression was measured utilizing Western Blot analysis. The densitometry of immunoblots was accomplished by using Image J software (NIH), and Student's t test was used to determine the statistically significant differences among the groups. A value of p &lt; 0.05 was considered significant.RESULTSThe expression of GHRH receptors in BPAEC, hCMEC/D3 and HMVEC‐L was confirmed by Western Blot. NIH/3T3 cells were used as negative controls, since they do not express receptors responding to GHRH. All cells were exposed to vehicle (0.1% DMSO), GHRH (1µM), or GHRHAnt (1µM) for 8 hours. Our results demonstrate that GHRH significantly increases ROS generation in all three endothelial cell lines, and GHRHAnt reduces ROS production. We also measured the effects of GHRHAnt against H2O2‐induced oxidative stress. Cells were treated with vehicle (0.1% DMSO), or GHRHAnt (1µM) for 8 hours, prior to vehicle (PBS) or H2O2 (0.1mM) exposure (8 hours). H2O2 potentiated ROS generation, and that effect was counteracted by GHRHAnt. Those peptides failed to suppress the H2O2‐induced ROS production in NIH/3T3 cells, since they do not express GHRH receptors.CONCLUSIONSOur results reveal that GHRHAnt suppress ROS production in the endothelium, suggesting that those compounds may be of therapeutic value in diseases related to endothelial barrier dysfunction and oxidative stress (e.g. Acute Respiratory Distress Syndrome and sepsis).

The Potential Prognostic and Therapeutic Implications of Prolactin Receptor and Growth Hormone-releasing Hormone Receptor Expression in Uterine Leiomyosarcomas.

The management of uterine leiomyosarcomas (uLMS) remains challenging. The rate of recurrence and metastasis is high, with 5-yr survival reaching only 40% to 50% in patients with tumor confined to the uterus (FIGO stage I or II). Prolactin receptor (PRLR) and growth hormone-releasing hormone receptor (GHRHR) have been implicated in the carcinogenesis of malignant tumors of the breast, endometrium, ovary, liver, and prostate. GHRHR antagonists inhibit in vitro growth of many human tumors and the expression of PRLR is associated with resistance to chemotherapy. The immunohistochemical expression of PRLR and GHRH in 24 primary and 2 recurrent uLMS was evaluated. Representative sections were stained with PRLR and GHRHR antibodies and immunoreactivity was calculated using H -score. The results were correlated with clinicopathologic data using Kaplan-Meier survival and multivariable Cox proportion hazard regression analyses. All tumors were positive for both markers with predominantly moderate to strong expression of PRLR (89%) and GHRHR (82%). Patients with tumors showing moderate to strong expression of PRLR were significantly less likely to achieve disease-free survival ( P =0.004) and significantly more likely to have a poor overall survival ( P =0.049). No significant difference in mean PRLR expression was found between tumors with higher mitotic counts (>20/10 hpf) and lower mitotic counts (20 or less/10 hpf). Furthermore, in 2 patients where the primary and recurrent tumors were tested, there was stronger expression of PRLR in the recurrence compared with the primary. This correlation was not found with GHRHR. Both PRLR and GHRHR may play a role in carcinogenesis in uLMS, as they do in other malignant neoplasms. To our knowledge, this study is the first evaluating the expression of these receptors in uLMS. Moderate or high expression of PRLR may serve as a prognostic marker associated with recurrences and increased mortality in uLMS patients.

Molecular cloning and functional characterization of growth hormone-releasing hormone in Mastacembelus armatus.

Growth hormone-releasing hormone (GHRH) is a neuropeptide that controls growth hormone (GH) synthesis and release. In this study, the full-length cDNA of Mastacembelus armatus ghrh was obtained by rapid amplification of cDNA ends method. Sequence analysis showed that the cloned sequence is 1090 bp in length, containing an open reading frame (ORF) of 429 bp that encodes a precursor protein of 142 amino acids. Sequence alignment revealed that the 27-amino acid mature peptide of Ghrh in M. armatus is conserved. Real-time PCR showed that ghrh is highly expressed in the brain, with very low or no expression in other tissues. During embryonic and larval development, ghrh expression was low in embryos but increased gradually in the stages of larval development. The biological function of Ghrh peptide was further investigated in vivo. Ghrh injection could significantly upregulate the mRNA expression of growth hormone (gh) and insulin-like growth factor-1/2 (igf-1/2) in M. armatus. Our data indicate that Ghrh is able to activate the GH-IGFs axis in M. armatus.

Growth Hormone-Releasing Hormone Modulation of Neuronal Exosome Biomarkers in Mild Cognitive Impairment.

Age-related changes in cognition are linked to decreased expression of somatotropins, GHRH and IGF-1. Mild cognitive impairment (MCI) and Alzheimer's disease (AD) are heterogeneous conditions. The loss of GHRH signaling in the brain may be mechanistically involved in AD pathogenesis. The consequent need to identify AD at an early and perhaps more treatable stage has fueled research into blood-based, exosome biomarkers. Plasma exosomes from participants enrolled in a randomized, double-blind, placebo-controlled 20-week trial of GHRH administration, were isolated, precipitated, and enriched by immuno-absorption with anti-L1CAM antibody (neural adhesion protein) from adults with MCI and age-matched, cognitively normal controls (CNC). Extracted protein cargo from neuronally-derived exosomes (NDEs) were assessed by ELISAs for protein levels implicated in AD neuropathology and for synaptic proteins altered by AD. Plasma NDE concentrations of Aβ1-42 were significantly increased while plasma NDE concentrations of NRGN, synaptophysin, synaptotagmin, and synaptopodin were significantly decreased in patients with MCI, independent of GHRH treatment. Plasma NDE concentrations of ptau-S396 and GAP43 were not affected by cognitive status (CNC versus MCI) or by GHRH treatment. Aβ1-42, neurogranin (NRGN), synaptophysin, synaptotagmin, and synaptopodin demonstrated the highest diagnostic accuracy for distinguishing between CNC and MCI patients, while synaptophysin and synaptotagmin demonstrated moderate accuracy in distinguishing between placebo-treated and GHRH-treated, MCI patients.

LXR activation increases the expression of GnRH AND αMSH in the rat hypothalamus in vivo.

Liver X receptors (LXR) are important transcription factors involved in the regulation of carbohydrate and lipid metabolism. Recently, we described LXR receptors expression in the hypothalamus but their function in this brain area remains unknown. Here, we evaluated the function of LXR on the expression of factors produced in the hypothalamus in vitro and in vivo by Western blotting and immunocytochemistry. More precisely we studied the expression of GnRH and GHRH, αMSH and NPY in male Sprague-Dawley rats. The effects of two synthetic LXR agonists, T0901317 and GW3965, were first tested in vitro. Hypothalamic explants were treated with either T0901317 or GW3965 (10μM) for 2, 4, 6 and 8h. As a positive control the cholesterol ABCA1 and glucose GLUT2 transporters were used. No changes were observed in the expression of the factors evaluated in vitro. The effects of the LXR agonists were then tested in vivo. Rats were injected ICV into the third ventricle with either T0901317 or GW3965 (2.5μg/5μL ICV) and after 3.5h or 24h the hypothalami were dissected out and rapidly frozen for analysis. αMSH and GnRH expression was significantly increased after 3.5h of T0901317 treatment. Anterior/posterior hypothalamic ratio increases for αMSH expression and decreases for GnRH expression after 24h of LXR activation. Altogether these results show that LXR activation affects the expression of GnRH and αMSH, suggesting that LXR in the hypothalamus is capable of modulating hypothalamic responses related to appetite, sexual behavior and reproductive functions.

Cortistatin Is a Key Factor Regulating the Sex-Dependent Response of the GH and Stress Axes to Fasting in Mice.

Cortistatin (CORT) shares high structural and functional similarities with somatostatin (SST) but displays unique sex-dependent pituitary actions. Indeed, although female CORT-knockout (CORT-KO) mice exhibit enhanced GH expression/secretion, Proopiomelanocortin expression, and circulating ACTH/corticosterone/ghrelin levels, male CORT-KO mice only display increased plasma GH/corticosterone levels. Changes in peripheral ghrelin and SST (rather than hypothalamic levels) seem to regulate GH/ACTH axes in CORT-KOs under fed conditions. Because changes in GH/ACTH axes during fasting provide important adaptive mechanisms, we sought to determine whether CORT absence influences GH/ACTH axes during fasting. Accordingly, fed and fasted male/female CORT-KO were compared with littermate controls. Fasting increased circulating GH levels in male/female controls but not in CORT-KO, suggesting that CORT can be a relevant regulator of GH secretion during fasting. However, GH levels were already higher in CORT-KO than in controls in fed state, which might preclude a further elevation in GH levels. Interestingly, although fasting-induced pituitary GH expression was elevated in both male/female controls, GH expression only increased in fasted female CORT-KOs, likely owing to specific changes observed in key factors controlling somatotrope responsiveness (ie, circulating ghrelin and IGF-1, and pituitary GHRH and ghrelin receptor expression). Fasting increased corticosterone levels in control and, most prominently, in CORT-KO mice, which might be associated with a desensitization to SST signaling and to an augmentation in CRH and ghrelin-signaling regulating corticotrope function. Altogether, these results provide compelling evidence that CORT plays a key, sex-dependent role in the regulation of the GH/ACTH axes in response to fasting.

Melatonin modulates monochromatic light-induced GHRH expression in the hypothalamus and GH secretion in chicks

To study the mechanism by which monochromatic lights affect the growth of broilers, a total of 192 newly hatched broilers, including the intact, sham-operated and pinealectomy groups, were exposed to white light (WL), red light (RL), green light (GL) and blue light (BL) using a light-emitting diode (LED) system for 2 weeks. The results showed that the GHRH-ir neurons were distributed in the infundibular nucleus (IN) of the chick hypothalamus. The mRNA and protein levels of GHRH in the hypothalamus and the plasma GH concentrations in the chicks exposed to GL were increased by 6.83–31.36%, 8.71–34.52% and 6.76–9.19% compared to those in the chicks exposed to WL (P=0.022–0.001), RL (P=0.002–0.000) and BL (P=0.290–0.017) in the intact group, respectively. The plasma melatonin concentrations showed a positive correlation with the expression of GHRH (r=0.960) and the plasma GH concentrations (r=0.993) after the various monochromatic light treatments. After pinealectomy, however, these parameters decreased and there were no significant differences between GL and the other monochromatic light treatments. These findings suggest that melatonin plays a critical role in GL illumination-enhanced GHRH expression in the hypothalamus and plasma GH concentrations in young broilers.

An early reduction in GH peak amplitude in preproghrelin-deficient male mice has a minor impact on linear growth.

Ghrelin is a gut hormone processed from the proghrelin peptide acting as the endogenous ligand of the GH secretagogue receptor 1a. The regulatory role of endogenous ghrelin on pulsatile GH secretion and linear growth had to be established. The aim of the present study was to delineate the endogenous actions of preproghrelin on peripheral and central components of the GH axis. Accordingly, the ultradian pattern of GH secretion was measured in young and old preproghrelin-deficient males. Blood samples were collected by tail bleeding every 10 minutes over a period of 6 hours. Analysis of the GH pulsatile pattern by deconvolution showed that GH was secreted in an ultradian manner in all genotypes, with major secretory peaks occurring at about 3-hour intervals. In older mice, the peak number was reduced and secretion was less irregular compared with younger animals. Remarkably, in young Ghrl(-/-) mice, the amplitude of GH secretory bursts was significantly reduced. In older mice, however, genotype differences were less significant. Changes in GH pulsatility in young Ghrl(-/-) mice were associated with a tendency for reduced GH pituitary contents and plasma IGF-I concentrations, but with only a minor impact on linear growth. In Ghrl(+/-) mice, despite reduced Acyl ghrelin to des-acyl ghrelin ratio, GH secretion was not impaired. Ghrelin deficiency was not associated with a reduction in hypothalamic GHRH content or altered response to GHRH stimulation. Therefore, reduction in GHRH production and/or sensitivity do not primarily account for the altered GH pulsatile secretion of young Ghrl(-/-) mice. Instead, GHRH expression was elevated in young but not old Ghrl(-/-) mice, suggesting that differential compensatory responses resulting from the absence of endogenous ghrelin is occurring according to age. These results show that endogenous ghrelin is a regulator of GH pulse amplitude in growing mice but does not significantly modulate linear growth.

Obestatin Plays an Opposite Role in the Regulation of Pituitary Somatotrope and Corticotrope Function in Female Primates and Male/Female Mice

Obestatin is a 23-amino-acid amidated peptide that is encoded by the ghrelin gene. Previous studies have shown obestatin can modulate the hypothalamic neuronal circuitry that regulates pituitary function, perhaps by modulating the actions of ghrelin. However, the direct actions of obestatin on pituitary function remain controversial. Here, primary pituitary cell cultures from a nonhuman primate (baboon) and mice were used to test the effects of obestatin on pituitary hormone expression and secretion. In pituitary cultures from both species, obestatin had no effect on prolactin, LH, FSH, or TSH expression/release. Conversely, obestatin stimulated proopiomelanocortin expression and ACTH release and inhibited GH expression/release in vitro, actions that were also observed in vivo in mice treated with obestatin. In vitro, obestatin inhibited the stimulatory actions of ghrelin on GH but not ACTH release. The inhibitory effect of obestatin on somatotrope function was associated with an overall reduction in pituitary transcription factor-1 and GHRH receptor mRNA levels in vitro and in vivo as well as a reduction in hypothalamic GHRH and ghrelin expression in vivo. The stimulatory effect of obestatin on ACTH was associated with an increase in pituitary CRF receptors. Obestatin also reduced the expression of pituitary somatostatin receptors (sst1/sst2), which could serve to modify its impact on hormone secretion. The in vitro actions of obestatin on both GH and ACTH release required the adenylyl cyclase and MAPK routes. Taken together, our results provide evidence that obestatin can act directly at the pituitary to control somatotrope and corticotrope function, and these effects are conserved across species.

UII and UT in grouper: cloning and effects on the transcription of hormones related to growth control

Urotensin II (UII) is a cyclic peptide that was originally extracted from the caudal neurosecretory system (CNSS) of fish. UII is well known to exhibit cardiovascular, ventilatory, and motor effects in vertebrates. Studies have reported that UII exerts mitogenic effects and can act as an autocrine/paracrine growth factor in mammals. However, similar information in fish is limited. In this study, the full-length cDNAs of UII and its receptor (UT) were cloned and characterized in the orange-spotted grouper. UII and UT were expressed ubiquitously in various tissues in grouper, and particularly high levels were observed in the CNSS, CNS, and ovary. A functional study showed that UT was coupled with intracellular Ca2+ mobilization in HEK293 cells. Studies carried out using i.p. injections of UII in grouper showed the following: i) in the hypothalamus, UII can significantly stimulate the mRNA expression of ghrh and simultaneously inhibit the mRNA expression of somatostatin 1 (ss1) and ss2 3 h after injection; ii) in the pituitary, UII also significantly induced the mRNA expression of gh 6 and 12 h after injection; and iii) in the liver, the mRNA expression levels of ghr1/ghr2 and igf1/igf2 were markedly increased 12 and 3 h after the i.p. injection of UII respectively. These results collectively indicate that the UII/UT system may play a role in the promotion of the growth of the orange-spotted grouper.