Astressin B, a nonselective corticotropin-releasing hormone receptor antagonist, prevents the inhibitory effect of ghrelin on luteinizing hormone pulse frequency in the ovariectomized rhesus monkey.

Administration of ghrelin, a key peptide in the regulation of energy homeostasis, has been shown to decrease LH pulse frequency while concomitantly elevating cortisol levels. Because increased endogenous CRH release in stress is associated with an inhibition of reproductive function, we have tested here whether the pulsatile LH decrease after ghrelin may reflect an activated hypothalamic-pituitary-adrenal axis and be prevented by a CRH antagonist. After a 3-h baseline LH pulse frequency monitoring, five adult ovariectomized rhesus monkeys received a 5-h saline (protocol 1) or ghrelin (100-microg bolus followed by 100 microg/h, protocol 2) infusion. In protocols 3 and 4, animals were given astressin B, a nonspecific CRH receptor antagonist (0.45 mg/kg im) 90 min before ghrelin or saline infusion. Blood samples were taken every 15 min for LH measurements, whereas cortisol and GH were measured every 45 min. Mean LH pulse frequency during the 5-h ghrelin infusion was significantly lower than in all other treatments (P < 0.05) and when compared with the baseline period (P < 0.05). Pretreatment with astressin B prevented the decrease. Ghrelin stimulated cortisol and GH secretion, whereas astressin B pretreatment prevented the cortisol, but not the GH, release. Our data indicate that CRH release mediates the inhibitory effect of ghrelin on LH pulse frequency and suggest that the inhibitory impact of an insufficient energy balance on reproductive function may in part be mediated by the hypothalamic-pituitary-adrenal axis.

Complete hypogonadotropic hypogonadism associated with a novel inactivating mutation of the gonadotropin-releasing hormone receptor.

In this study, we describe a patient with a phenotype of complete hypogonadotropic hypogonadism who presented primary failure of pulsatile GnRH therapy, but responded to exogenous gonadotropin administration. This patient bore a novel point mutation (T for A) at codon 168 of the gene encoding the GnRH receptor (GnRH-R), resulting in a serine to arginine change in the fourth transmembrane domain of the receptor. This novel mutation was present in the homozygous state in the patient, whereas it was in the heterozygous state in both phenotypically normal parents. When introduced into the complementary DNA coding for the GnRH-R, this mutation resulted in the complete loss of the receptor-mediated signaling response to GnRH. In conclusion, we report the first mutation of the GnRH-R gene that can induce a total loss of function of this receptor and is associated with a phenotype of complete hypogonadotropic hypogonadism.

Gonadotropin-releasing hormone secretion from hypothalamic neurons: stimulation by insulin and potentiation by leptin.

Insulin and leptin are peripheral metabolic factors signaling the body needs in energy to the central nervous system. Because energy homeostasis and reproductive function are closely related phenomena, we investigated the respective roles played by insulin and leptin in the hypothalamic control of GnRH secretion. We observed that increasing circulating insulin levels, by performing hyperinsulinemic clamp studies in male mice, was associated with a significant rise in LH secretion. This effect of insulin is likely mediated at the hypothalamic level, because it was also found to stimulate the secretion and the expression of GnRH by hypothalamic neurons in culture. Leptin was found to potentiate the effect of insulin on GnRH secretion in vitro but was devoid of any effect on its own. These data represent the first evidence of direct insulin sensing by hypothalamic neurons involved in activating the neuroendocrine gonadotrope axis. They also demonstrate that these neurons can integrate different hormonal signals to modulate net hypothalamic GnRH output. We propose that such integration is an essential mechanism for the adaptation of reproductive function to changes in the metabolic status of an individual.

Mechanisms of the pressor response to intravenous thyrotropin-releasing hormone in the rat.

1. The purpose of this study was to examine the contribution of the sympatho-adrenomedullary system to the blood pressure response to an intravenous bolus of thyrotropin-releasing hormone (TRH) in conscious medullectomized and sham-operated rats. 2. The peak pressor effect of 0.5 mg TRH was significantly increased in rats having no adrenal medulla (+24.2 +/- 1.6 mmHg, mean +/- s.e.m., P < 0.01) as compared to sham-operated animals (+12.2 +/- 3.0 mmHg). 3. Blockade of alpha-adrenergic receptors with phentolamine abolished the pressor effect of TRH in control rats (+2.1 +/- 1.9 mmHg) but did not attenuate the blood pressure response of medullectomized rats (+21.5 +/- 4.7 mmHg). In contrast, beta-blockade with propranolol blunted the blood pressure responsiveness of rats subjected to adrenal medullectomy (+12.4 +/- 2.6 mmHg) but did not modify the effect of TRH in sham-operated controls (+10.9 +/- 2.9 mmHg). 4. The direct in vitro effect of TRH on isolated mesenteric rat arteries was also evaluated. TRH did not induce contractions of isolated arteries. 5. These results suggest that in rats with intact adrenals, the pressor effect of intravenous TRH is mediated primarily by a stimulation of alpha-adrenergic receptors. Adrenal medullectomy appears to enhance the blood pressure response to intravenous TRH. Activation of cardiac beta-adrenoceptors seems to contribute to the blood pressure increasing effect of intravenous TRH in medullectomized animals.

Thyrotropin-releasing hormone-stimulated thyrotropin expression involves islet-brain-1/c-Jun N-terminal kinase interacting protein-1.

Islet-brain-1 (IB1)/c-Jun N-terminal kinase interacting protein 1 (JIP-1) is a scaffold protein that is expressed at high levels in neurons and the endocrine pancreas. IB1/JIP-1 interacts with the c-Jun N-terminal kinase and mediates the specific physiological stimuli (such as cytokines). However, the potential role of the protein in the pituitary has not been evaluated. Herein, we examined expression of the gene encoding IB1/JIP-1 and its translated product in the anterior pituitary gland and a pituitary cell line, GH3. We then examined the potential role of IB1/JIP-1 in controlling TSH-beta gene expression. Exposure of GH3 cells to TRH stimulated the expression of IB1/JIP-1 protein levels, mRNA, and transcription of the promoter. The increase of IB1/JIP-1 content by transient transfection study of a vector encoding IB1/JIP-1 or by the stimulation of TRH stimulates TSH-beta promoter activity. This effect is not found in the presence of a mutated nonfunctional (IB1S59N) IB1/JIP-1 protein. Together, these facts point to a central role of the IB1/JIP-1 protein in the control of TRH-mediated TSH-beta stimulation.

Quetiapine affects neuropeptide Y and corticotropin-releasing hormone in cerebrospinal fluid from schizophrenia patients: relationship to depression and anxiety symptoms and to treatment response.

Cumulative evidence indicates that neuropeptides play a role in the pathophysiology of schizophrenia. Early data showed increased neuropeptide Y (NPY) in cerebrospinal fluid (CSF) from schizophrenia patients and data from rodents show that antipsychotic drugs modulate NPY levels in and release from selected rat brain regions. In view of these findings we investigated whether the atypical antipsychotic quetiapine, originally used as an antipsychotic but subsequently shown to be efficient also in major depressive disorder and in both poles of bipolar disorder, would affect NPY-like immunoreactivity (-LI), and corticotropin-releasing hormone (CRH)-LI levels in CSF of schizophrenia patients. NPY-LI and CRH-LI in CSF were determined in 22 patients with schizophrenia. Lumbar puncture was performed at baseline and again after 4 wk of quetiapine treatment (600 mg/d). Patients were assessed with the Positive and Negative Syndrome Scale (PANSS) at baseline and at weekly intervals. Quetiapine treatment was associated with a significant increase in NPY-LI (p<0.001) and decrease in CRH-LI (p<0.01). Stepwise multiple regression analysis revealed that ΔNPY-LI and ΔCRH-LI levels predicted 63% (p<0.001) of the variability of the ΔPANSS total score, ΔNPY-LI 42% (p<0.05) of the ΔPANSS anxiety items (G2) and ΔCRH-LI 40% (p=0.05) of the ΔPANSS depression items (G6). These results suggest that while quetiapine's effects on monoamines are probably related to its antipsychotic properties, the modulation of NPY and CRH accounts for its antidepressant and anxiolytic effects and can be markers of response.

The fetal hypothalamus has the potential to generate cells with a gonadotropin releasing hormone (GnRH) phenotype.

BACKGROUND: Neurospheres (NS) are colonies of neural stem and precursor cells capable of differentiating into the central nervous system (CNS) cell lineages upon appropriate culture conditions: neurons, and glial cells. NS were originally derived from the embryonic and adult mouse striatum subventricular zone. More recently, experimental evidence substantiated the isolation of NS from almost any region of the CNS, including the hypothalamus. METHODOLOGY/FINDINGS: Here we report a protocol that enables to generate large quantities of NS from both fetal and adult rat hypothalami. We found that either FGF-2 or EGF were capable of inducing NS formation from fetal hypothalamic cultures, but that only FGF-2 is effective in the adult cultures. The hypothalamic-derived NS are capable of differentiating into neurons and glial cells and most notably, as demonstrated by immunocytochemical detection with a specific anti-GnRH antibody, the fetal cultures contain cells that exhibit a GnRH phenotype upon differentiation. CONCLUSIONS/SIGNIFICANCE: This in vitro model should be useful to study the molecular mechanisms involved in GnRH neuronal differentiation.

Neuropeptide Y and corticotropin-releasing hormone in CSF mark response to antidepressive treatment with citalopram.

Neuropeptides appear to play a role in the pathophysiology of depression and electroconvulsive treatment and lithium affect these compounds in human cerebrospinal fluid (CSF) and rodent brain. Consequently, we investigated whether long-term treatment with the selective serotonin reuptake inhibitor (SSRI) citalopram (Cit) would also affect neuropeptides in CSF of depressed patients. Changes in CSF monoamine metabolites were also explored. CSF concentrations of corticotropin-releasing hormone (CRH)-like immunoreactivity (-LI), neuropeptide Y (NPY)-LI, and Cit were determined in 21 patients with major depression. Lumbar puncture was performed in the morning at baseline and was repeated after at least 4 wk of Cit treatment (40 mg/d). The severity of depression was assessed by the Hamilton Rating Scale for Depression (HAMD). Cit treatment was associated with a significant increase in NPY-LI and decrease in CRH-LI. An evaluation of the relationship between changes in concentrations of NPY-LI, CRH-LI, and the clinical response showed significant correlations between these parameters. Significant NPY and CRH changes in CSF following treatment as well as correlations to changes in HAMD support the hypothesis that these two peptides play a role in affective disorders and are markers of therapeutic response.

Dysfunctional SEMA3E signaling underlies gonadotropin-releasing hormone neuron deficiency in Kallmann syndrome.

Individuals with an inherited deficiency in gonadotropin-releasing hormone (GnRH) have impaired sexual reproduction. Previous genetic linkage studies and sequencing of plausible gene candidates have identified mutations associated with inherited GnRH deficiency, but the small number of affected families and limited success in validating candidates have impeded genetic diagnoses for most patients. Using a combination of exome sequencing and computational modeling, we have identified a shared point mutation in semaphorin 3E (SEMA3E) in 2 brothers with Kallmann syndrome (KS), which causes inherited GnRH deficiency. Recombinant wild-type SEMA3E protected maturing GnRH neurons from cell death by triggering a plexin D1-dependent (PLXND1-dependent) activation of PI3K-mediated survival signaling. In contrast, recombinant SEMA3E carrying the KS-associated mutation did not protect GnRH neurons from death. In murine models, lack of either SEMA3E or PLXND1 increased apoptosis of GnRH neurons in the developing brain, reducing innervation of the adult median eminence by GnRH-positive neurites. GnRH neuron deficiency in male mice was accompanied by impaired testes growth, a characteristic feature of KS. Together, these results identify SEMA3E as an essential gene for GnRH neuron development, uncover a neurotrophic function for SEMA3E in the developing brain, and elucidate SEMA3E/PLXND1/PI3K signaling as a mechanism that prevents GnRH neuron deficiency.

A growth hormone-releasing peptide that binds scavenger receptor CD36 and ghrelin receptor up-regulates sterol transporters and cholesterol efflux in macrophages through a peroxisome proliferator-activated receptor gamma-dependent pathway.

Macrophages play a central role in the pathogenesis of atherosclerosis by accumulating cholesterol through increased uptake of oxidized low-density lipoproteins by scavenger receptor CD36, leading to foam cell formation. Here we demonstrate the ability of hexarelin, a GH-releasing peptide, to enhance the expression of ATP-binding cassette A1 and G1 transporters and cholesterol efflux in macrophages. These effects were associated with a transcriptional activation of nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma in response to binding of hexarelin to CD36 and GH secretagogue-receptor 1a, the receptor for ghrelin. The hormone binding domain was not required to mediate PPARgamma activation by hexarelin, and phosphorylation of PPARgamma was increased in THP-1 macrophages treated with hexarelin, suggesting that the response to hexarelin may involve PPARgamma activation function-1 activity. However, the activation of PPARgamma by hexarelin did not lead to an increase in CD36 expression, as opposed to liver X receptor (LXR)alpha, suggesting a differential regulation of PPARgamma-targeted genes in response to hexarelin. Chromatin immunoprecipitation assays showed that, in contrast to a PPARgamma agonist, the occupancy of the CD36 promoter by PPARgamma was not increased in THP-1 macrophages treated with hexarelin, whereas the LXRalpha promoter was strongly occupied by PPARgamma in the same conditions. Treatment of apolipoprotein E-null mice maintained on a lipid-rich diet with hexarelin resulted in a significant reduction in atherosclerotic lesions, concomitant with an enhanced expression of PPARgamma and LXRalpha target genes in peritoneal macrophages. The response was strongly impaired in PPARgamma(+/-) macrophages, indicating that PPARgamma was required to mediate the effect of hexarelin. These findings provide a novel mechanism by which the beneficial regulation of PPARgamma and cholesterol metabolism in macrophages could be regulated by CD36 and ghrelin receptor downstream effects.

Feedback on hypothalamic TRH transcription is dependent on thyroid hormone receptor N terminus.

The beta thyroid hormone receptor (TRbeta), but not TRalpha1, plays a specific role in mediating T(3)-dependent repression of hypothalamic TRH transcription. To investigate the structural basis of isoform specificity, we compared the transcriptional regulation and DNA binding obtained with chimeric and N-terminally deleted TRs. Using in vivo transfection assays to follow hypothalamic TRH transcription in the mouse brain, we found that TRbeta1 and chimeras with the TRbeta1 N terminus did not affect either transcriptional activation or repression from the rat TRH promoter, whereas N-terminally deleted TRbeta1 impaired T(3)-dependent repression. TRalpha1 or chimeras with the TRalpha1 N terminus reduced T(3)-independent transcriptional activation and blocked T(3)-dependent repression of transcription. Full deletion of the TRalpha1 N terminus restored ligand-independent activation of transcription. No TR isoform specificity was seen after transcription from a positive thyroid hormone response element. Gel mobility assays showed that all TRs tested bound specifically to the main negative thyroid hormone response element in the TRH promoter (site 4). Addition of neither steroid receptor coactivator 1 nor nuclear extracts from the hypothalamic paraventricular nuclei revealed any TR isoform specificity in binding to site 4. Thus N-terminal sequences specify TR T(3)-dependent repression of TRH transcription but not DNA recognition, emphasizing as yet unknown neuron-specific contributions to protein-promoter interactions in vivo.

Transcriptional activation of the macrophage migration-inhibitory factor gene by the corticotropin-releasing factor is mediated by the cyclic adenosine 3',5'- monophosphate responsive element-binding protein CREB in pituitary cells.

Macrophage migration-inhibitory factor (MIF) has recently been identified as a pituitary hormone that functions as a counterregulatory modulator of glucocorticoid action within the immune system. In the anterior pituitary gland, MIF is expressed in TSH- and ACTH-producing cells, and its secretion is induced by CRF. To investigate MIF function and regulation within pituitary cells, we initiated the characterization of the MIF 5'-regulatory region of the gene. The -1033 to +63 bp of the murine MIF promoter was cloned 5' to a luciferase reporter gene and transiently transfected into freshly isolated rat anterior pituitary cells. This construct drove high basal transcriptional activity that was further enhanced after stimulation with CRF or with an activator of adenylate cyclase. These transcriptional effects were associated with a concomitant rise in ACTH secretion in the transfected cells and by an increase in MIF gene expression as assessed by Northern blot analysis. A cAMP-responsive element (CRE) was identified within the MIF promoter region which, once mutated, abolished the cAMP responsiveness of the gene. Using this newly identified CRE, DNA-binding activity was detected by gel retardation assay in nuclear extracts prepared from isolated anterior pituitary cells and AtT-20 corticotrope tumor cells. Supershift experiments using antibodies against the CRE-binding protein CREB, together with competition assays and the use of recombinant CREB, allowed the detection of CREB-binding activity with the identified MIF CRE. These data demonstrate that CREB is the mediator of the CRF-induced MIF gene transcription in pituitary cells through an identified CRE in the proximal region of the MIF promoter.