Splicing machinery is profoundly altered in systemic lupus erythematosus and antiphospholipid syndrome and directly linked to key clinical features.

OBJECTIVES: To characterize the splicing machinery (SM) of leukocytes from primary antiphospholipid syndrome (APS), systemic lupus erythematosus (SLE) and antiphospholipid syndrome with lupus (APS + SLE) patients, and to assess its clinical involvement. METHODS: Monocytes, lymphocytes and neutrophils from 80 patients (22 APS, 35 SLE and 23 APS + SLE) and 50 HD were purified, and 45 selected SM components were evaluated by qPCR-microfluidic array. Relationship with clinical features and underlying regulatory mechanisms were assessed. RESULTS: APS, SLE and APS + SLE leukocytes displayed significant and specific alterations in SM-components (SMC), associated with clinical features [autoimmune profiles, disease activity, lupus nephritis (LN), and CV-risk markers]. A remarkable relationship among dysregulated SMC in monocytes and the presence of LN in SLE was highlighted, revealing a novel pathological mechanism, which was further explored. Immunohistology analysis of renal biopsies highlighted the pathological role of the myeloid compartment in LN. Transcriptomic analysis of monocytes from SLE-LN(+) vs SLE-LN(-) identified 271 genes differentially expressed, mainly involved in inflammation and IFN-signaling. Levels of IFN-related genes correlated with those of SMC in SLE-LN(+). These results were validated in two external SLE-LN(+) datasets of whole-blood and kidney biopsies. In vitro, SLE-LN(+)-serum promoted a concomitant dysregulation of both, the IFN signature and several SMC, further reversed by JAKinibs treatment. Interestingly, IFNs, key inflammatory cytokines in SLE pathology, also altered SMC. Lastly, the over/down-expression of selected SMC in SLE-monocytes reduced the release of inflammatory cytokines and their adhesion capacity. CONCLUSION: Overall, we have identified, for the first time, a specific alteration of SMC in leukocytes from APS, SLE and APS + SLE patients that would be responsible for the development of distinctive clinical profiles.

Metabolic Fingerprint of Acromegaly and its Potential Usefulness in Clinical Practice.

Insulin-like growth factor-1 (IGF-1) and growth hormone (GH) levels are the main targets for monitoring acromegaly activity, but they are not in close relationship with the clinical course of the disease and the associated comorbidities. The present study was aimed at identifying metabolites that could be used as biomarkers for a better disease phenotyping. For this purpose, metabolic fingerprint using an untargeted metabolomic approach was examined in serum from 30 patients with acromegaly and 30 age-matched controls. Patients with acromegaly presented fewer branched-chain amino acids (BCAAs) compared to the control group (valine: 4.75 ± 0.87 vs. 5.20 ± 1.06 arbitrary units (AUs), p < 0.05; isoleucine: 2.54 ± 0.41 vs. 2.80 ± 0.51 AUs; p < 0.05). BCAAs were also lower in patients with active disease compared to patients with normal levels of IGF-1 with or without medical treatment. GH, but not IGF-1, serum levels were inversely correlated with both valine and isoleucine. These findings indicate that low levels of BCAAs represent the main metabolic fingerprint of acromegaly and that GH, rather than IGF-1, might be the primary mediator. In addition, our results suggest that the assessment of BCAAs could help to identify active disease and to monitor the response to therapeutic strategies.

Multiple signaling pathways convey central and peripheral signals to regulate pituitary function: Lessons from human and non-human primate models.

The anterior pituitary gland is a key organ involved in the control of multiple physiological functions including growth, reproduction, metabolism and stress. These functions are controlled by five distinct hormone-producing pituitary cell types that produce growth hormone (somatotropes), prolactin (lactotropes), adrenocorticotropin (corticotropes), thyrotropin (thyrotropes) and follicle stimulating hormone/luteinizing hormone (gonadotropes). Classically, the synthesis and release of pituitary hormones was thought to be primarily regulated by central (neuroendocrine) signals. However, it is now becoming apparent that factors produced by pituitary hormone targets (endocrine and non-endocrine organs) can feedback directly to the pituitary to adjust pituitary hormone synthesis and release. Therefore, pituitary cells serve as sensors to integrate central and peripheral signals in order to fine-tune whole-body homeostasis, although it is clear that pituitary cell regulation is species-, age- and sex-dependent. The purpose of this review is to provide a comprehensive, general overview of our current knowledge of both central and peripheral regulators of pituitary cell function and associated intracellular mechanisms, focusing on human and non-human primates.

Translational research in neuroendocrine tumors: pitfalls and opportunities.

Interest in research on neuroendocrine tumors (NETs) has grown in the past 10 years, coinciding with improvements in our understanding of the molecular pathogenesis of NETs. In addition, NETs have become one of the most exciting settings for drug development. Two targeted agents for the management of advanced pancreatic NETs have been approved, but the development of targeted agents for NETs is limited by problems with both patient selection and demonstration of activity. In this review, we analyze these limitations and discuss ways to increase the predictive value of preclinical models for target discovery and drug development. The role of translational research and 'omics' methodologies is emphasized, with the final aim of developing personalized medicine. Because NETs usually grow slowly and metastatic tumors are found at easily accessible locations, and owing to improvements in techniques for liquid biopsies, NETs provide a unique opportunity to obtain tumor samples at all stages of the evolution of the disease and to adapt treatment to changes in tumor biology. Combining clinical and translational research is essential to achieve progress in the NET field. Slow growth and genetic stability limit and challenge both the availability and further development of preclinical models of NETs, one of the most crucial unmet research needs in the field. Finally, we suggest some useful approaches for improving clinical drug development for NETs: moving from classical RECIST-based response end points to survival parameters; searching for different criteria to define response rates (for example, antiangiogenic effects and metabolic responses); implementing randomized phase II studies to avoid single-arm phase II studies that produce limited data on drug efficacy; and using predictive biomarkers for patient selection.

Not So Giants: Mice Lacking Both Somatostatin and Cortistatin Have High GH Levels but Show No Changes in Growth Rate or IGF-1 Levels.

Somatostatin (SST) and cortistatin (CORT) are two highly related neuropeptides involved in the regulation of various endocrine secretions. In particular, SST and CORT are two primary negative regulators of GH secretion. Consequently, single SST or CORT knockout mice exhibit elevated GH levels; however, this does not lead to increased IGF-1 levels or somatic growth. This apparent lack of correspondence has been suggested to result from compensatory mechanisms between both peptides. To test this hypothesis, in this study we explored, for the first time, the consequences of simultaneously deleting endogenous SST and CORT by generating a double SST/CORT knockout mouse model and exploring its endocrine and metabolic phenotype. Our results demonstrate that simultaneous deletion of SST and CORT induced a drastic elevation of endogenous GH levels, which, surprisingly, did not lead to changes in growth rate or IGF-1 levels, suggesting the existence of additional factors/systems that, in the absence of endogenous SST and CORT, could counteract GH actions. Notably, elevation in circulating GH levels were not accompanied by changes in pituitary GH expression or by alterations in the expression of its main regulators (GHRH and ghrelin) or their receptors (GHRH receptor, GHS receptor, or SST/CORT receptors) at the hypothalamic or pituitary level. However, although double-SST/CORT knockout male mice exhibited normal glucose and insulin levels, they had improved insulin sensitivity compared with the control mice. Therefore, these results suggest the existence of an intricate interplay among the known (SST/CORT), and likely unknown, inhibitory components of the GH/IGF-1 axis to regulate somatic growth and glucose/insulin homeostasis.

Gastrointestinal neuroendocrine tumors (NETs): new diagnostic and therapeutic challenges.

This paper summarizes the current understanding of the biology of somatostatin receptor (sst), role of immunotherapy in neuroendocrine tumor (NET), new agents for PPRT, and methods to assess response and clinical benefit in NET. One of the most interesting aspects of sst biology is the recent discovery of truncated variants of the sst5 receptor subtype with unique tissue distribution and response to somatostatin (SST). These truncated receptors are associated with bad patient prognosis, decreased response to SST analogs, and may be new targets for diagnoses and treatment. IFN remains a cost-effective agent, particularly in classic mid gut carcinoids, and there is interest to continue examining immunotherapy's in this disease. PRRT remains a key strategy for treatment and imaging. In addition to the classic agents, there are a series of new agents targeting other receptors such as the incretin receptors (GLP-1R; GIPR) and other G-protein coupled receptors with great potential. With regards to therapy monitoring, the most commonly used criteria are Response Criteria Evaluation in Solid Tumors (RECIST). However, for different reasons, these criteria are not very useful in NET. Incorporation of other criteria such as Choi as well as functional imaging assessment with PET would be of great interest in this area.

A cellular and molecular basis for the selective desmopressin-induced ACTH release in Cushing disease patients: key role of AVPR1b receptor and potential therapeutic implications.

CONTEXT: Desmopressin is a synthetic agonist of vasopressin receptors (AVPRs). The desmopressin stimulation test is used in the diagnosis and postsurgery prognosis of Cushing disease (CD). However, the cellular and molecular mechanisms underlying the desmopressin-induced ACTH increase in patients with CD are poorly understood. OBJECTIVE: The objectives of this study were to determine, for the first time, whether desmopressin acts directly and exclusively on pituitary corticotropinoma cells to stimulate ACTH expression/release and to elucidate the cellular and molecular mechanisms involved in desmopressin-induced ACTH increase in CD. DESIGN: A total of 8 normal pituitaries (NPs), 23 corticotropinomas, 14 nonfunctioning pituitary adenomas, 17 somatotropinomas, and 3 prolactinomas were analyzed for AVPR expression by quantitative real-time RT-PCR. Primary cultures derived from corticotropinomas, nonfunctioning pituitary adenomas, somatotropinomas, prolactinomas, and NPs were treated with desmopressin, and ACTH secretion/expression, [Ca(2+)]i kinetics, and AVPR expression and/or proliferative response were evaluated. The relationship between AVPR expression and plasma adrenocorticotropin/cortisol levels obtained from desmopressin tests was assessed. RESULTS: Desmopressin affects all functional parameters evaluated in corticotropinoma cells but not in NPs or other pituitary adenomas cells. These effects might be due to the dramatic elevation of AVPR1b expression levels found in corticotropinomas. In line with this notion, the use of an AVPR1b antagonist completely blocked desmopressin stimulatory effects. Remarkably, only AVPR1b expression was positively correlated with elevated plasma adrenocorticotropin levels in corticotropinomas. CONCLUSIONS: The present results provide a cellular and molecular basis to support the desmopressin stimulation test as a reliable, specific test for the diagnosis and postsurgery prognosis of CD. Furthermore, our data indicate that AVPR1b is responsible for the direct/exclusive desmopressin stimulatory pituitary effects observed in CD, thus opening the possibility of exploring AVPR1b antagonists as potential therapeutic tools for CD treatment.

The new truncated somatostatin receptor variant sst5TMD4 is associated to poor prognosis in breast cancer and increases malignancy in MCF-7 cells.

Somatostatin receptors (sst1-5) are present in different types of tumors, where they inhibit key cellular processes such as proliferation and invasion. Although ssts are densely expressed in breast cancer, especially sst2, their role and therapeutic potential remain uncertain. Recently, we identified a new truncated sst5 variant, sst5TMD4, which is related to the abnormal response of certain pituitary tumors to treatment with somatostatin analogs. Here, we investigated the possible role of sst5TMD4 in breast cancer. This study revealed that sst5TMD4 is absent in normal mammary gland, but is abundant in a subset of poorly differentiated human breast tumors, where its expression correlated to that of sst2. Moreover, in the MCF-7 breast cancer model cell, sst5TMD4 expression increased malignancy features such as invasion and proliferation abilities (both in cell cultures and nude mice). This was likely mediated by sst5TMD4-induced increase in phosphorylated extracellular signal-regulated kinases 1 and 2 and p-Akt levels, and cyclin D3 and Arp2/3 complex expression, which also led to mesenchymal-like phenotype. Interestingly, sst5TMD4 interacts physically with sst2 and thereby alters its signaling, enabling disruption of sst2 inhibitory feedback and providing a plausible basis for our findings. These results suggest that sst5TMD4 could be involved in the pathophysiology of certain types of breast tumors.

Somatostatin dramatically stimulates growth hormone release from primate somatotrophs acting at low doses via somatostatin receptor 5 and cyclic AMP.

Somatostatin and cortistatin have been shown to act directly on pituitary somatotrophs to inhibit growth hormone (GH) release. However, previous results from nonprimate species indicate that these peptides can also directly stimulate GH secretion, at low concentrations. The relevance of this phenomenon in a nonhuman primate model was investigated in the present study by testing the impact of somatostatin/cortistatin on GH release in primary pituitary cell cultures from baboons. High doses (> 10(-10) m) of somatostatin/cortistatin did not alter basal GH secretion but blocked GH-releasing hormone (GHRH)- and ghrelin-induced GH release. However, at low concentrations (10(-17)-10(-13) m), somatostatin/cortistatin dramatically stimulated GH release to levels comparable to those evoked by GHRH or ghrelin. Use of somatostatin receptor (sst) specific agonists/antagonists, and signal transduction blockers indicated that sst2 and sst1 activation via intact adenylate cylcase and mitogen-activated protein kinase systems mediated the inhibitory actions of high-concentration somatostatin. By contrast, the stimulatory actions of low-dose somatostatin on GH release were mediated by sst5 signalling through adenylate cylcase/cAMP/protein kinase A and intracellular Ca(2+) pathways, and were additive with ghrelin (not GHRH). Notably, low-concentrations of somatostatin, similar to sst5-agonists, inhibited prolactin release. These results clearly demonstrate that the ultimate impact of somatostatin/cortistatin on hormone release is dose-dependent, cell type-selective and receptor-specific, where the stimulatory effects of low-concentration somatostatin/cortistatin on GH release extend to primates, thereby supporting the notion that this action is relevant in regulating GH secretion in humans.

Self-limited acute hepatotoxicity caused by pegvisomant.

We present a case of acute severe hepatitis in a patient with acromegaly receiving combination therapy with somatostatin analogs and pegvisomant. Hepatitis resolved completely 18 weeks after diagnosis of hypertransaminasemia without discontinuation of therapy and with a close clinical and biochemical follow-up. In this case, despite the severity of the hepatitis, therapy could be continued as hypertransaminasemia was gradually decreasing after the maximum peak. We also review the literature on toxic hepatitis associated to pegvisomant therapy analyzing the etiology, clinical predisposing factors and natural evolution.

Somatotroph tumor progression during pegvisomant therapy: a clinical and molecular study.

CONTEXT: There is concern that pegvisomant could be associated with a higher risk of tumor growth. The rate and possible determinants of this tumor growth are unknown. OBJECTIVE: The objective of the study was to investigate the clinical, immunohistological, and molecular factors conditioning tumor growth in patients taking pegvisomant. DESIGN AND SETTING: This was a cross-sectional study performed from 2004 to 2010 in four university hospitals in Spain. PATIENTS: Seventy-five acromegalic patients with active disease resistant to somatostatin analogs treated with pegvisomant were followed up for a mean of 29 ± 20 months. MAIN OUTCOME MEASURES: Magnetic resonance images before initiation of pegvisomant, at 6 months, and then yearly were examined in all patients. Immunohistological and molecular studies were performed in tumors that grew. RESULTS: A significant increase in tumor size was observed in five patients (6.7%). Absence of previous irradiation (P = 0.014) and shorter duration of prepegvisomant somatostatin analog therapy (P < 0.001) were associated with an increased risk of tumor growth. A stepwise multivariate linear regression analysis (R(2) = 0.334, P < 0.001) identified the duration of somatostatin analog therapy prior to pegvisomant (beta = -4.509, P = 0.014) as the only significant predictor of tumor growth. In those tumors that grew, GH expression and insulin receptor expression were higher (P = 0.033 in both cases) than in the control group. CONCLUSIONS: No previous radiotherapy, shorter duration of prepegvisomant somatostatin analog therapy, and higher tumor expression of GH and insulin receptor could be risk factors for tumor growth during pegvisomant therapy.

Identification of novel genes involved in the plasticity of pituitary melanotropes in amphibians.

Melanotrope cells from the amphibian intermediate lobe are composed of two subpopulations that exhibit opposite secretory behavior: hypersecretory and hormone-storage hyposecretory melanotropes. Isolation of these subpopulations allowed a comparison of their gene expression profiles by differential display, leading to the identification of a number of genes differentially expressed in hypersecretory or hyposecretory melanotropes. Among them, we chose two (preferentially expressed in hyposecretory cells) of unknown function but structurally related to proteins involved in the secretory process: Rab18 and KIAA0555. We demonstrate that, upon activation of the regulated secretory pathway, Rab18 associates with secretory granules, inhibits their mobilization, and, consequently, reduces the secretory capacity of neuroendocrine cells. The other gene, KIAA0555, was predicted by in silico analysis to encode a protein with a long coiled-coil domain, a structural feature also shared by different proteins related to intracellular membrane traffic (i.e., golgins), and a hydrophobic C-terminal domain that could function as a transmembrane domain. A database search unveiled the existence of a KIAA0555 paralogue, KIAA4091, displaying a long coiled-coil region highly similar to that of KIAA0555 and an identical C-terminal transmembrane domain. Both KIAA0555 and KIAA4091 were found to be predominantly expressed in tissues containing cells with regulated secretory pathway, that is, endocrine and neural tissues. Moreover, when exogenously expressed in HEK293 cells, both proteins showed a yuxtanuclear distribution, which partially overlaps with that of a Golgi complex marker, thus suggesting a possible role of these two proteins in the control of the secretory process.

Novel expression and direct effects of adiponectin in the rat testis.

Adiponectin is an adipocyte hormone, with relevant roles in lipid metabolism and glucose homeostasis, recently involved in the control of different endocrine organs, such as the placenta, pituitary and, likely, the ovary. However, whether as described previously for other adipokines, such as leptin and resistin, adiponectin is expressed and/or conducts biological actions in the male gonad remains unexplored. In this study, we provide compelling evidence for the expression, putative hormonal regulation, and direct effects of adiponectin in the rat testis. Testicular expression of adiponectin was demonstrated along postnatal development, with a distinctive pattern of RNA transcripts and discernible protein levels that appeared mostly located at interstitial Leydig cells. Testicular levels of adiponectin mRNA were marginally regulated by pituitary gonadotropins but overtly modulated by metabolic signals, such as glucocorticoids, thyroxine, and peroxisome proliferator-activated receptor-gamma, whose effects were partially different from those on circulating levels of adiponectin. In addition, expression of the genes encoding adiponectin receptor (AdipoR)-1 and AdipoR2 was detected in the rat testis, with developmental changes and gonadotropin regulation for AdipoR2 mRNA, and prominent levels of AdipoR1 in seminiferous tubules. Moreover, recombinant adiponectin significantly inhibited basal and human choriogonadotropin-stimulated testosterone secretion ex vivo, whereas it failed to change relative levels of several Sertoli cell-expressed mRNAs, such as stem cell factor and anti-Müllerian hormone. In summary, our data are the first to document the expression, regulation and functional role of adiponectin in the rat testis. Taken together with its recently reported expression in the ovary and its effects on LH secretion and ovarian steroidogenesis, these results further substantiate a multifaceted role of adiponectin in the control of the reproductive axis, which might operate as endocrine integrator linking metabolism and gonadal function.

Ghrelin induces growth hormone secretion via a nitric oxide/cGMP signalling pathway.

The presence of ghrelin and its receptor, growth hormone (GH) secretagogue receptor, in the hypothalamus and pituitary, and its ability to stimulate GH release in vivo and in vitro, strongly support a significant role for this peptide in the control of somatotroph function. We previously demonstrated that ghrelin elicits GH secretion directly in somatotrophs by activating two major signalling cascades, which involve inositol phosphate and cAMP. In as much as nitric oxide (NO) and its mediator cGMP have been recently shown to contribute substantially to the response of somatotrophs to key regulatory hormones, including GH-releasing hormone, somatostatin and leptin, we investigated the possible role of this signalling pathway in ghrelin-induced GH release in vitro. Accordingly, cultures of pituitary cells from prepuberal female pigs were challenged with ghrelin (10(-8) m, 30 min) in the absence or presence of activators or blockers of key steps of the NO synthase (NOS)/NO/guanylate cyclase (GC)/cGMP route and GH secretion was measured. Two distinct activators of the NO route, S-nitroso-N-acetylpenicillamine (SNAP) (5 x 10(-4) m) and L-arginine methyl ester hydrochloride (L-AME) (10(-3) m), comparably stimulated GH secretion when applied alone. The presence of L-AME enhanced ghrelin-stimulated GH secretion, whereas SNAP did not alter its effect. Conversely, two different NOS/NO pathway inhibitors, N(w)-nitro-L-arginine methyl ester hydrochloride (10(-5) m) or haemoglobin (20 microg/ml), similarly blocked ghrelin-induced (but not basal) GH release, thus indicating that NO contributes critically to ghrelin action in somatotrophs. Moreover, incubation with a permeable cGMP analogue, 8-Br-cGMP (10(-8) m) stimulated GH secretion, but did not modify the stimulatory action of ghrelin, suggesting that cGMP could mediate the action of NO. Indeed, inhibition of GC by 10 microm LY-53,583 did not alter basal GH secretion but abolished the GH-releasing action of ghrelin. Taken together, our results provide novel evidence indicating that ghrelin requires activation of the NOS/NO route, and its subsequent GC/cGMP signal transduction pathway, as necessary steps to induce GH secretion from somatotrophs.

Identification and characterization of two novel (neuro)endocrine long coiled-coil proteins.

We have identified a novel vertebrate-specific gene by applying a Differential Display method on two distinct subtypes of pituitary melanotropes showing divergent secretory phenotypes of hypo- and hypersecretion. A paralogue of this gene was also identified. The existence of a long coiled-coil domain and a C-terminal transmembrane domain in the sequences, together with the Golgi distribution of the proteins in transfected cells, suggest that they can be considered as new members of the golgin family of proteins. Both genes were primarily expressed in (neuro)endocrine tissues in vertebrates thus supporting a role for these proteins in the regulated secretory pathway.

Direct pituitary effects of kisspeptin: activation of gonadotrophs and somatotrophs and stimulation of luteinising hormone and growth hormone secretion.

Recent, compelling evidence indicates that kisspeptins, the products of KiSS-1 gene, and their receptor GPR54, represent key elements in the neuroendocrine control of reproduction, and that they act primarily by regulating gonadotrophin-releasing hormone (GnRH) secretion at the hypothalamus. Conversely, and despite earlier reports showing GPR54 expression in the pituitary, the potential physiological roles of kisspeptins at this gland have remained elusive. To clarify this issue, cultures of rat pituitary cells were used to evaluate expression of KiSS-1 and GPR54, and to monitor the ability of kisspeptin-10 to stimulate Ca(2+) responses in gonadotrophs and to elicit luteinising hormone (LH) secretion in vitro. The results obtained show that both GPR54 and KiSS-1 are expressed in the pituitary of peripubertal male and female rats. Moreover, kisspeptin-10 induced a rise in free cytosolic Ca(2+) concentration ([Ca(2+)](i)) in approximately 10% of male rat pituitary cells. Intriguingly, kisspeptin-responsive cells included not only gonadotrophs, in which a 62.8 +/- 16.0%[Ca(2+)](i) rise was observed, but also somatotrophs, wherein kisspeptin induced a 60.3 +/- 5.5%[Ca(2+)](i) increase. Accordingly, challenge of dispersed pituitary cells with increasing kisspeptin-10 concentrations induced dose-related LH and growth hormone (GH) secretory responses, which were nevertheless of lower magnitude than those evoked by the primary regulators GnRH and GH-releasing hormone, respectively. In particular, 10(-8) M kisspeptin caused maximal increases in LH release (218.7 +/- 23.6% and 180.4 +/- 7.2% in male and female rat pituitary cells, respectively), and also stimulated maximally GH secretion (181.9 +/- 14.9% and 260.2 +/- 15.9% in male and female rat pituitary cells, respectively). Additionally, moderate summation of kisspeptin- and GnRH-induced LH responses was observed after short-term incubation of male rat pituitary cells. In conclusion, our results provide unequivocal evidence that kisspeptins exert direct pituitary effects in peripubertal male and female rats and suggest a possible autocrine/paracrine mode of action. The precise relevance and underlying mechanisms of this potential new actions of kisspeptins (i.e. the direct modulation of gonadotrophic and somatotrophic axis at the pituitary) deserve further analysis.

Ontogeny and mechanisms of action for the stimulatory effect of kisspeptin on gonadotropin-releasing hormone system of the rat.

Kisspeptins have recently emerged as essential regulators of gonadotropin secretion and puberty onset. These functions are primarily conducted by stimulation of hypothalamic gonadotropin-releasing hormone (GnRH) secretion. However, relevant aspects of KiSS-1 physiology, including the ontogeny and major signaling systems of its stimulatory action, remain to be fully elucidated. To cover these issues, the effects of kisspeptin-10 on GnRH and LH secretion were monitored at early stages of postnatal maturation, and potential changes in the sensitivity to kisspeptin were assessed along the pubertal transition in the rat. In addition, the signaling cascades involved in kisspeptin-induced GnRH secretion were explored by means of pharmacological blockade using rat hypothalamic explants. Despite sexual immaturity, kisspeptin-10 potently elicited GnRH release ex vivo and LH secretion in vivo at early stages (neonatal to juvenile) of postnatal development. Yet, LH responsiveness to low doses of kisspeptin was enhanced in peri-pubertal animals. Concerning GnRH secretion, the stimulatory action of kisspeptin-10 required activation of phospholipase-C, mobilization of intracellular Ca2+ and recruitment of ERK1/2 and p38 kinases, but was preserved after blockade of type 2 cyclo-oxygenase and prostaglandin synthesis. In summary, our present data document the ontogeny, sensitivity and intracellular signals for the stimulatory action of kisspeptin on the GnRH/LH axis in the rat. Although LH responses to low doses of kisspeptin appeared to be enhanced at puberty, kisspeptin was able to readily activate the GnRH system at early stages of postnatal maturation. These observations further stress the essential role of kisspeptin in normal, and eventually pathological, timing of puberty.

Cortistatin mimics somatostatin by inducing a dual, dose-dependent stimulatory and inhibitory effect on growth hormone secretion in somatotropes.

Cortistatin is a recently discovered neuropeptide that is structurally related to somatostatin, the classic inhibitor of growth hormone (GH) release. Cortistatin binds with high affinity to all five somatostatin receptors (sst1-5), and, like somatostatin, cortistatin inhibits in vivo GH release in man and rats. In this report, we compared the in vitro actions of cortistatin and somatostatin using primary pig pituitary cell cultures. In this species, we have previously reported that somatostatin not only inhibits GH-releasing hormone (GHRH)-stimulated GH release at high doses, but also stimulates basal GH release at low (pM) doses, a dual response that is markedly dependent on the subpopulation of pituitary somatotropes examined. Results reported herein demonstrate that cortistatin closely mimics the dose-dependent inhibitory and stimulatory effects of somatostatin on GH secretion. As cortistatin, unlike somatostatin, binds to the human receptor for ghrelin/GH secretagogs (GHS-R), we also investigated whether cortistatin stimulates GH release through this receptor by using a synthetic, short form of cortistatin, cortistatin-8 (CST8), which lacks the sst-binding capacity of full-length cortistatin but retains its GHS-R-binding capacity. Interestingly, CST8 stimulated GH release only at low doses (10(-15) M), and did not reduce GH secretion stimulated by GHRH, ghrelin, or low-dose, full-length cortistatin, yet it counteracted that induced by a nonpeptidyl GHS, L-163 255. Taken together, our results indicate that the dual, inhibitory and stimulatory effects of cortistatin on GH release closely parallel those of somatostatin and are probably mediated by the same receptor(s) and signaling pathway(s) for both peptides. Furthermore, they suggest that the pathway(s) activated by cortistatin (and somatostatin) to stimulate GH release are not initiated by GHS-R activation.

Increased expression of alpha- and beta-globin mRNAs at the pituitary following exposure to estrogen during the critical period of neonatal sex differentiation in the rat.

Deterioration of reproductive health in human and wildlife species during the past decades has drawn considerable attention to the potential adverse effects of exposure to xenosteroids during sensitive periods of sex development. The hypothalamic-pituitary (HP) unit is a key element in the neuroendocrine system controlling development and function of the reproductive axis; the HP unit being highly sensitive to the organizing effects of endogenous and exogenous sex steroids. To gain knowledge on the molecular mode of action and potential biomarkers of exposure to estrogenic compounds at the HP unit, we screened for differentially expressed genes at the pituitary and hypothalamus of rats after neonatal exposure to estradiol benzoate. Our analyses identified persistent up-regulation of alpha- and beta-globin mRNAs at the pituitary following neonatal estrogenization. This finding was confirmed by combination of RT-PCR analyses and in situ hybridization. Induction of alpha- and beta-globin mRNA expression at the pituitary by neonatal exposure to estrogen was demonstrated as dose-dependent and it was persistently detected up to puberty. In contrast, durable up-regulation of alpha- and beta-globin genes was not detected at the hypothalamus, cortex, cerebellum, liver and testis. Finally, enhanced levels of alpha- and beta-globin mRNAs at the pituitary were also demonstrated after neonatal administration of the anti-androgen flutamide. In summary, alpha- and beta-globin genes may prove as sensitive, pituitary-specific biomarkers of exposure to estrogenic (and/or anti-androgenic) compounds at critical periods of sex development, whose potential in the assessment of endocrine disrupting events at the HP unit merits further investigation.