Altered CELF4 splicing factor enhances pancreatic neuroendocrine tumors aggressiveness influencing mTOR and everolimus response.

Pancreatic neuroendocrine tumors (PanNETs) comprise a heterogeneous group of tumors with growing incidence. Recent molecular analyses provided a precise picture of their genomic and epigenomic landscape. Splicing dysregulation is increasingly regarded as a novel cancer hallmark influencing key tumor features. We have previously demonstrated that splicing machinery is markedly dysregulated in PanNETs. Here, we aimed to elucidate the molecular and functional implications of CUGBP ELAV-like family member 4 (CELF4), one of the most altered splicing factors in PanNETs. CELF4 expression was determined in 20 PanNETs, comparing tumor and non-tumoral adjacent tissue. An RNA sequencing (RNA-seq) dataset was analyzed to explore CELF4-linked interrelations among clinical features, gene expression, and splicing events. Two PanNET cell lines were employed to assess CELF4 function in vitro and in vivo. PanNETs display markedly upregulated CELF4 expression, which is closely associated with malignancy features, altered expression of key tumor players, and distinct splicing event profiles. Modulation of CELF4 influenced proliferation in vitro and reduced in vivo xenograft tumor growth. Interestingly, functional assays and RNA-seq analysis revealed that CELF4 silencing altered mTOR signaling pathway, enhancing the effect of everolimus. We demonstrate that CELF4 is dysregulated in PanNETs, where it influences tumor development and aggressiveness, likely by modulating the mTOR pathway, suggesting its potential as therapeutic target.

The uprise of RNA biology in neuroendocrine neoplasms: altered splicing and RNA species unveil translational opportunities.

Neuroendocrine neoplasms (NENs) comprise a highly heterogeneous group of tumors arising from the diffuse neuroendocrine system. NENs mainly originate in gastrointestinal, pancreatic, and pulmonary tissues, and despite being rare, show rising incidence. The molecular mechanisms underlying NEN development are still poorly understood, although recent studies are unveiling their genomic, epigenomic and transcriptomic landscapes. RNA was originally considered as an intermediary between DNA and protein. Today, compelling evidence underscores the regulatory relevance of RNA processing, while new RNA molecules emerge with key functional roles in core cell processes. Indeed, correct functioning of the interrelated complementary processes comprising RNA biology, its processing, transport, and surveillance, is essential to ensure adequate cell homeostasis, and its misfunction is related to cancer at multiple levels. This review is focused on the dysregulation of RNA biology in NENs. In particular, we survey alterations in the splicing process and available information implicating the main RNA species and processes in NENs pathology, including their role as biomarkers, and their functionality and targetability. Understanding how NENs precisely (mis)behave requires a profound knowledge at every layer of their heterogeneity, to help improve NEN management. RNA biology provides a wide spectrum of previously unexplored processes and molecules that open new avenues for NEN detection, classification and treatment. The current molecular biology era is rapidly evolving to facilitate a detailed comprehension of cancer biology and is enabling the arrival of personalized, predictive and precision medicine to rare tumors like NENs.

The oncogenic role of the spliced somatostatin receptor sst5TMD4 variant in prostate cancer.

sst5TMD4, a splice variant of the sst5 gene, is overexpressed and associated with aggressiveness in various endocrine-related tumors, but its presence, functional role, and mechanisms of actions in prostate cancer (PCa)-the most common cancer type in males-is completely unexplored. In this study, formalin-fixed, paraffin-embedded prostate pieces from patients with localized PCa, which included tumoral and nontumoral adjacent regions (n = 45), fresh biopsies from patients with high-risk PCa (n = 52), and healthy fresh prostates from cystoprostatectomies (n = 14) were examined. In addition, PCa cell lines and xenograft models were used to determine the presence and functional role of sst5TMD4. Results demonstrated that sst5TMD4 is overexpressed (mRNA/protein) in PCa samples, and this is especially drastic in metastatic and/or high Gleason score tumor samples. Remarkably, sst5TMD4 expression was associated with an altered frequency of 2 single-nucleotide polymorphisms: rs197055 and rs12599155. In addition, PCa cell lines and xenograft models were used to demonstrate that sst5TMD4 overexpression increases cell proliferation and migration in PCa cells and induces larger tumors in nude mice, whereas its silencing decreased proliferation and migration. Remarkably, sst5TMD4 overexpression activated multiple intracellular pathways (ERK/JNK, MYC/MAX, WNT, retinoblastoma), altered oncogenes and tumor suppressor gene expression, and disrupted the normal response to somatostatin analogs in PCa cells. Altogether, we demonstrate that sst5TMD4 is overexpressed in PCa, especially in those patients with a worse prognosis, and plays an important pathophysiologic role in PCa, which suggesting its potential as a biomarker and/or therapeutic target.-Hormaechea-Agulla, D., Jiménez-Vacas, J. M., Gómez-Gómez, E., L.-López, F., Carrasco-Valiente, J., Valero-Rosa, J., Moreno, M. M., Sánchez-Sánchez, R., Ortega-Salas, R., Gracia-Navarro, F., Culler, M. D., Ibáñez-Costa, A., Gahete, M. D., Requena, M. J., Castaño, J. P., Luque, R. M. The oncogenic role of the spliced somatostatin receptor sst5TMD4 variant in prostate cancer.

The truncated somatostatin receptor sst5TMD4 stimulates the angiogenic process and is associated to lymphatic metastasis and disease-free survival in breast cancer patients.

The truncated somatostatin receptor sst5TMD4 is associated with poor prognosis in breast cancer and increases breast cancer cell malignancy. Here, we examined the cellular/molecular mechanisms underlying this association, aiming to identify new molecular tools to improve diagnosis, prognosis or therapy. A gene expression array comparing sst5TMD4 stably-transfected MCF-7 cells and their controls (empty-plasmid) revealed the existence of profound alterations in the expression of genes involved in key tumoral processes, such as cell survival or angiogenesis. Moreover, sst5TMD4-overexpressing MCF-7 and MDA-MB-231 cells demonstrated increased expression/production of pro-angiogenic factors and enhanced capacity to form mammospheres. Consistently, sst5TMD4-expressing MCF-7 cells induced xenografted tumors with higher VEGF levels and elevated number of blood vessels. Importantly, sst5TMD4 was expressed in a subset of breast cancers, where it correlated with angiogenic markers, lymphatic metastasis, and reduced disease-free survival. These results, coupled to our previous data, support a relevant role of sst5TMD4 in the angiogenic process and reinforce the role of sst5TMD4 in breast cancer malignancy and metastatic potential, supporting its possible utility to develop new molecular biomarkers and drug therapies for these tumors.

Dietary fat alters the expression of cortistatin and ghrelin systems in the PBMCs of elderly subjects: putative implications in the postprandial inflammatory response.

SCOPE: Dietary fat influences systemic inflammatory status, which determines the progression of age-associated diseases. Since somatostatin (SST), cortistatin (CORT), and ghrelin systems modulate inflammatory response, we aim to comprehensively characterize the presence and regulation of the components of these systems in the peripheral blood mononuclear cells (PMBCs), a subset of white blood cells placed at the crossroad between diet and inflammation, in response to diets with different fat composition, and during the postprandial phase in elderly subjects. METHODS AND RESULTS: The applied nutrigenomic, inflammation-related PBMC-based approach revealed that the majority of components of SST/CORT and ghrelin systems are present in the human PBMCs. Particularly, CORT, SST/CORT receptors (sst2, sst3, sst5, and sst5TMD4), ghrelin, its acylating enzyme (GOAT), In1-ghrelin variant, and GHSR1b were detected in PBMCs. Their expression was altered in the long-term by diet composition, and in the short-term, during the postprandial phase. Of particular relevance is the postprandial elevation of CORT, sst2, and sst5 expression in PBMCs of subjects under n-3 PUFAs-enriched diet. CONCLUSION: Our results suggest a potential relevant role of CORT/ssts and ghrelin systems in regulating PBMCs response to nutrient intake, which could help to explain the positive effects of n-3 PUFAs-enriched diets in reducing the inflammatory response.

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.

Porcine sst1 can physically interact with other somatostatin receptors, and its expression is regulated by metabolic/inflammatory sensors.

The majority of the biological actions attributed to somatostatin (SST) are thought to be mediated by SST receptor 2 (sst2), the most ubiquitous sst, and, to a lesser extent, by sst5. However, a growing body of evidence suggests a relevant role of sst1 in mediating SST actions in (patho)physiological situations (i.e., endometriosis, type 2 diabetes). Moreover, sst1 together with sst2 and sst5 is involved in the well-known actions of SST on pituitary somatotropes in pig and primates. Here, we cloned the porcine sst1 (psst1) and performed a structural and functional characterization using both primary and heterologous models. The psst1 sequence presents the majority of signature motifs shared among G protein-coupled receptors and, specifically, among ssts and exhibits a high homology with other mammalian sst1, with only minor differences in the amino-terminal domain, reinforcing the idea of an early evolutive divergence between mammalian and nonmammalian sst1s. psst1 is functional in terms of decreasing cAMP levels in response to SST when transfected in heterologous models. The psst1 receptor is expressed in several tissues, and analyses of gene cis elements predict regulation by multiple transcription factors and metabolic stimuli. Finally, psst1 is coexpressed with other sst subtypes in various tissues, and in vitro data demonstrate that psst1 can interact with itself forming homodimers and with other ssts forming heterodimers. These data highlight the functional importance of sst1 on the SST-mediated effects and its functional interaction with different ssts, which point out the necessity of exploring the consequences of such interactions.

Ghrelin gene products, receptors, and GOAT enzyme: biological and pathophysiological insight.

Ghrelin is a 28-amino acid acylated hormone, highly expressed in the stomach, which binds to its cognate receptor (GHSR1a) to regulate a plethora of relevant biological processes, including food intake, energy balance, hormonal secretions, learning, inflammation, etc. However, ghrelin is, in fact, the most notorious component of a complex, intricate regulatory system comprised of a growing number of alternative peptides (e.g. obestatin, unacylated ghrelin, and In1-ghrelin, etc.), known (GHSRs) and, necessarily unknown receptors, as well as modifying enzymes (e.g. ghrelin-O-acyl-transferase), which interact among them as well as with other regulatory systems in order to tightly modulate key (patho)-physiological processes. This multiplicity of functions and versatility of the ghrelin system arise from a dual, genetic and functional, complexity. Importantly, a growing body of evidence suggests that dysregulation in some of the components of the ghrelin system can lead to or influence the development and/or progression of highly concerning pathologies such as endocrine-related tumors, inflammatory/cardiovascular diseases, and neurodegeneration, wherein these altered components could be used as diagnostic, prognostic, or therapeutic targets. In this context, the aim of this review is to integrate and comprehensively analyze the multiple components and functions of the ghrelin system described to date in order to define and understand its biological and (patho)-physiological significance.

The long coiled-coil protein NECC2 is associated to caveolae and modulates NGF/TrkA signaling in PC12 cells [corrected].

TrkA-mediated NGF signaling in PC12 cells has been shown to be compartimentalized in specialized microdomains of the plasma membrane, the caveolae, which are organized by scaffold proteins including the member of the caveolin family of proteins, caveolin-1. Here, we characterize the intracellular distribution as well as the biochemical and functional properties of the neuroendocrine long coiled-coil protein 2 (NECC2), a novel long coiled-coil protein selectively expressed in neuroendocrine tissues that contains a predicted caveolin-binding domain and displays structural characteristics of a scaffolding factor. NECC2 distributes in caveolae, wherein it colocalizes with the TrkA receptor, and behaves as a caveolae-associated protein in neuroendocrine PC12 cells. In addition, stimulation of PC12 cells with nerve growth factor (NGF) increased the expression and regulated the distribution of NECC2. Interestingly, knockdown as well as overexpression of NECC2 resulted in a reduction of NGF-induced phosphorylation of the TrkA downstream effector extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2) but not of Akt. Altogether, our results identify NECC2 as a novel component of caveolae in PC12 cells and support the contribution of this protein in the maintenance of TrkA-mediated NGF signaling.

Role of endogenous cortistatin in the regulation of ghrelin system expression at pancreatic level under normal and obese conditions.

Ghrelin-system components [native ghrelin, In1-ghrelin, Ghrelin-O-acyltransferase enzyme (GOAT) and receptors (GHS-Rs)] are expressed in a wide variety of tissues, including the pancreas, where they exert different biological actions including regulation of neuroendocrine secretions, food intake and pancreatic function. The expression of ghrelin system is regulated by metabolic conditions (fasting/obesity) and is associated with the progression of obesity and insulin resistance. Cortistatin (CORT), a neuropeptide able to activate GHS-R, has emerged as an additional link in gut-brain interplay. Indeed, we recently reported that male CORT deficient mice (cort-/-) are insulin-resistant and present a clear dysregulation in the stomach ghrelin-system. The present work was focused at analyzing the expression pattern of ghrelin-system components at pancreas level in cort-/- mice and their control littermates (cort +/+) under low- or high-fat diet. Our data reveal that all the ghrelin-system components are expressed at the mouse pancreatic level, where, interestingly, In1-ghrelin was expressed at higher levels than native-ghrelin. Thus, GOAT mRNA levels were significantly lower in cort-/- mice compared with controls while native ghrelin, In1-ghrelin and GHS-R transcript levels remained unaltered under normal metabolic conditions. Moreover, under obese condition, a significant increase in pancreatic expression of native-ghrelin, In1-ghrelin and GHS-R was observed in obese cort+/+ but not in cort-/- mice. Interestingly, insulin expression and release was elevated in obese cort+/+, while these changes were not observed in obese cort-/- mice. Altogether, our results indicate that the ghrelin-system expression is clearly regulated in the pancreas of cort+/+ and cort -/- under normal and/or obesity conditions suggesting that this system may play relevant roles in the endocrine pancreas. Most importantly, our data demonstrate, for the first time, that endogenous CORT is essential for the obesity-induced changes in insulin expression/secretion observed in mice, suggesting that CORT is a key regulatory component of the pancreatic function.

Role of SST, CORT and ghrelin and its receptors at the endocrine pancreas.

Somatostatin (SST), cortistatin (CORT), and its receptors (sst1-5), and ghrelin and its receptors (GHS-R) are two highly interrelated neuropeptide systems with a broad range of overlapping biological actions at central, cardiovascular, and immune levels among others. Besides their potent regulatory role on GH release, its endocrine actions are highlighted by SST/CORT and ghrelin influence on insulin secretion, glucose homeostasis, and insulin resistance. Interestingly, most components of these systems are expressed at the endocrine pancreas and are actively involved in the modulation of pancreatic islet function and, consequently influence glucose homeostasis. In addition, some of them also participate in islet survival and regeneration. Furthermore, under severe metabolic condition as well as in endocrine pathologies, their expression profile is severely deregulated. These findings suggest that SST/CORT and ghrelin systems could play a relevant role in pancreatic function under metabolic and endocrine pathologies. Accordingly, these systems have been therapeutically targeted for the prevention or amelioration of certain metabolic conditions (obesity) as well as for tumor growth inhibition and/or hormonal regulation in endocrine pathologies (neuroendocrine tumors). This review focuses on the interrelationship between SST/CORT and ghrelin systems and their role in severe metabolic conditions and some endocrine disorders.

Truncated variants of pig somatostatin receptor subtype 5 (sst5) act as dominant-negative modulators for sst2-mediated signaling.

Somatostatin (SST) and its related peptide cortistatin (CORT) exert their multiple actions through binding to the SST receptor (sst) family, generally considered to comprise five G protein-coupled receptors with seven transmembrane domains (TMD), named sst1-sst5, plus a splice sst2B variant. However, we recently discovered that human and rodent sst5 gene expression also generates, through noncanonical alternative splicing, novel truncated albeit functional sst5 variants with less than seven TMD. Here, we cloned and characterized for the first time the porcine wild-type sst5 (psst5, full-length) and identified two novel truncated psst5 variants with six and three TMD, thus termed psst5TMD6 and psst5TMD3, respectively. In line with that observed in human and rodent truncated sst5 variants, psst5TMD6 and psst5TMD3 are functional (e.g., activate calcium signaling), selectively respond to SST and CORT, respectively, and exhibit specific tissue expression profiles that differ from full-length psst5 and often overlaps with psst2 expression. Moreover, fluorescence resonance energy transfer analysis shows that psst5 truncated variants physically interact with psst2, thereby altering their localization at the plasma membrane and specifically disrupting the cellular response to SST and/or CORT. These results represent the first characterization of a key porcine SST receptor, psst5, and, together with our previous results, provide strong evidence that alternative splicing-derived, truncated sst5 variants with distinct functional capacities exist in the mammalian lineage, where they can act as dominant-negative receptors, by interacting directly with long, seven TMD variants, potentially contributing to modulate normal and pathological SST and CORT signaling.

The Golgi-associated long coiled-coil protein NECC1 participates in the control of the regulated secretory pathway in PC12 cells.

Golgi-associated long coiled-coil proteins, often referred to as golgins, are involved in the maintenance of the structural organization of the Golgi apparatus and the regulation of membrane traffic events occurring in this organelle. Little information is available on the contribution of golgins to Golgi function in cells specialized in secretion such as endocrine cells or neurons. In the present study, we characterize the intracellular distribution as well as the biochemical and functional properties of a novel long coiled-coil protein present in neuroendocrine tissues, NECC1 (neuroendocrine long coiled-coil protein 1). The present study shows that NECC1 is a peripheral membrane protein displaying high stability to detergent extraction, which distributes across the Golgi apparatus in neuroendocrine cells. In addition, NECC1 partially localizes to post-Golgi carriers containing secretory cargo in PC12 cells. Overexpression of NECC1 resulted in the formation of juxtanuclear aggregates together with a slight fragmentation of the Golgi and a decrease in K+-stimulated hormone release. In contrast, NECC1 silencing did not alter Golgi architecture, but enhanced K+-stimulated hormone secretion in PC12 cells. In all, the results of the present study identify NECC1 as a novel component of the Golgi matrix and support a role for this protein as a negative modulator of the regulated trafficking of secretory cargo in neuroendocrine cells.

Revisiting the regulated secretory pathway: from frogs to human.

The regulated secretory pathway is a hallmark of endocrine and neuroendocrine cells. This process comprises different sequential steps, including ER-associated protein synthesis, ER-to-Golgi protein transport, Golgi-associated posttranslational modification, sorting and packing of secretory proteins into carrier granules, cytoskeleton-based granule transport towards the plasma membrane and tethering, docking and fusion of granules with specialized releasing zones in the plasma membrane. Each one of these steps is tightly regulated by a large number of factors that function in a spatially and temporarily coordinated fashion. During the past three decades, much effort has been devoted to characterize the precise role of the yet-known proteins participating in the different steps of this process and to identify new regulatory factors in order to obtain a unifying picture of the secretory pathway. In spite of this and given the enormous complexity of the process, certain steps are not fully understood yet and many players remain to be identified. In this review, we offer a summary of the current knowledge on the main molecular mechanisms that govern and ensure the correct release of secretory proteins. In addition, we have integrated the advance on the field made possible by studies carried out in non-mammalian vertebrates, which, although not very numerous, have substantially contributed to acquire a mechanistic understanding of the regulated secretory pathway.

Cortistatin is not a somatostatin analogue but stimulates prolactin release and inhibits GH and ACTH in a gender-dependent fashion: potential role of ghrelin.

Cortistatin (CST) and somatostatin (SST) evolve from a common ancestral gene and share remarkable structural, pharmacological, and functional homologies. Although CST has been considered as a natural SST-analogue acting through their shared receptors (SST receptors 1-5), emerging evidence indicates that these peptides might in fact exert unique roles via selective receptors [e.g. CST, not SST, binds ghrelin receptor growth hormone secretagogue receptor type 1a (GHS-R1a)]. To determine whether the role of endogenous CST is different from SST, we characterized the endocrine-metabolic phenotype of male/female CST null mice (cort-/-) at hypothalamic-pituitary-systemic (pancreas-stomach-adrenal-liver) levels. Also, CST effects on hormone expression/secretion were evaluated in primary pituitary cell cultures from male/female mice and female primates (baboons). Specifically, CST exerted an unexpected stimulatory role on prolactin (PRL) secretion, because both male/female cort-/- mice had reduced PRL levels, and CST treatment (in vivo and in vitro) increased PRL secretion, which could be blocked by a GHS-R1a antagonist in vitro and likely relates to the decreased success of female cort-/- in first-litter pup care at weaning. In contrast, CST inhibited GH and adrenocorticotropin-hormone axes in a gender-dependent fashion. In addition, a rise in acylated ghrelin levels was observed in female cort-/- mice, which were associated with an increase in stomach ghrelin/ghrelin O-acyl transferase expression. Finally, CST deficit uncovered a gender-dependent role of this peptide in the regulation of glucose-insulin homeostasis, because male, but not female, cort-/- mice developed insulin resistance. The fact that these actions are not mimicked by SST and are strongly gender dependent offers new grounds to investigate the hitherto underestimated physiological relevance of CST in the regulation of physiological/metabolic processes.

Rab18 dynamics in adipocytes in relation to lipogenesis, lipolysis and obesity.

Lipid droplets (LDs) are organelles that coordinate lipid storage and mobilization, both processes being especially important in cells specialized in managing fat, the adipocytes. Proteomic analyses of LDs have consistently identified the small GTPase Rab18 as a component of the LD coat. However, the specific contribution of Rab18 to adipocyte function remains to be elucidated. Herein, we have analyzed Rab18 expression, intracellular localization and function in relation to the metabolic status of adipocytes. We show that Rab18 production increases during adipogenic differentiation of 3T3-L1 cells. In addition, our data show that insulin induces, via phosphatidylinositol 3-kinase (PI3K), the recruitment of Rab18 to the surface of LDs. Furthermore, Rab18 overexpression increased basal lipogenesis and Rab18 silencing impaired the lipogenic response to insulin, thereby suggesting that this GTPase promotes fat accumulation in adipocytes. On the other hand, studies of the ß-adrenergic receptor agonist isoproterenol confirmed and extended previous evidence for the participation of Rab18 in lipolysis. Together, our data support the view that Rab18 is a common mediator of lipolysis and lipogenesis and suggests that the endoplasmic reticulum (ER) is the link that enables Rab18 action on these two processes. Finally, we describe, for the first time, the presence of Rab18 in human adipose tissue, wherein the expression of this GTPase exhibits sex- and depot-specific differences and is correlated to obesity. Taken together, these findings indicate that Rab18 is involved in insulin-mediated lipogenesis, as well as in ß-adrenergic-induced lipolysis, likely facilitating interaction of LDs with ER membranes and the exchange of lipids between these compartments. A role for Rab18 in the regulation of adipocyte biology under both normal and pathological conditions is proposed.

Expression of the ghrelin and neurotensin systems is altered in the temporal lobe of Alzheimer's disease patients.

Ghrelin and neurotensin (NTS) are neuroendocrine peptides that exert opposite effects on food intake and energy homeostasis, but share comparable actions in improving memory and learning. Ghrelin and NTS mediate their effects via receptors with high evolutionary identity: two ghrelin G-protein coupled receptors (GPCRs; GHS-R1a/1b) and three NTS-receptors, two GPCRs (NTSR1/2) and one non-GPCR (NTSR3). Because ghrelin and NTS systems are tightly linked to energy balance regulation and cognitive processes, they have been proposed to be altered in Alzheimer's disease (AD), a dementia syndrome markedly influenced by the metabolic status. Although it has been demonstrated that ghrelin and NTS can attenuate AD-related cognitive impairment, a comprehensive analysis of these systems in AD has not been conducted. Here, we used quantitative real time-RT-PCR to analyze expression of the ghrelin/NTS axis in one of the cortical regions most affected in AD, the temporal gyrus. Results unveiled a striking reduction of mRNA levels for ghrelin, and its newly discovered In2-ghrelin variant, as well as for the enzyme responsible for ghrelin acylation, ghrelin-O-acyltransferase and GHS-R1a, while expression of GHS-R1b was markedly increased. In addition, expression levels of NTSR1 and NTSR2 were profoundly decreased in AD, whereas mRNA levels of NTS only declined slightly, and those of NTSR3 (which is involved in neuronal apoptosis) did not vary. Taken together, our results provide the first quantitative evidence showing that ghrelin/NTS systems are markedly altered in the brain of AD patients, thereby suggesting that these systems may contribute to the severe cognitive deficit observed in this pathology.

Somatostatin and its receptors from fish to mammals.

Somatostatin (SST) and its receptors (sst) make up a molecular family with unique functional complexity and versatility. Widespread distribution and frequent coexpression of sst subtypes underlies the multiplicity of (patho)physiological processes controlled by SST (central nervous system functions, endocrine and exocrine secretion, cell proliferation). This complexity is clearly reflected in the intricate evolutionary development of this molecular family. Recent studies postulate the existence of an ancestral somatostatin/urotensin II (SST/UII) gene, which originated two ancestral, SST and UII, genes by local duplication. Subsequently, segment duplication would have originated two diverging SST genes in both fish (SS1/SS2) and tetrapods [(SST/cortistatin(CST))]. SST/CST actions are mediated by a family of GPCRs (sst1-5) encoded by five different genes. sst1-4 sequences are highly conserved compared with sst5, suggesting unique evolutionary and functional relevance for the latter. Indeed, we recently identified novel truncated but functional sst5 variants in several species, which may help to explain part of the complexity of the SST/CST/sst family. Comparative and phylogenetic analysis of this molecular family would enhance our understanding of its paradigmatic evolutionary complexity and functional versatility.

A potential inhibitory role for the new truncated variant of somatostatin receptor 5, sst5TMD4, in pituitary adenomas poorly responsive to somatostatin analogs.

CONTEXT: Somatostatin (SST) receptors, specially sst2 and sst5, provide a valuable target to inhibit excessive hormone release and cell growth in pituitary tumors by using SST analogs (SSAs). Unfortunately, an appreciable proportion of tumors fail to respond to SSA despite expressing high levels of one or more ssts. Recently we identified two novel truncated sst5 variants, sst5TMD5, and sst5TMD4, absent in normal pituitary but expressed in pituitary tumors. OBJECTIVE AND DESIGN: We aimed at exploring the potential role of sst5TMD5 and sst5TMD4 in the poor response of some tumors to SSA in vivo and in vitro. Specifically, 25 somatotropinomas showing different responses to octreotide in vivo and sst2 (BIM-23197)- and sst5(BIM-23268)-selective compounds in vitro were screened for sst5TMD5/sst5TMD4 expression by real-time PCR. Relationships between ssts expression and in vivo and in vitro secretory response of the corresponding pituitary samples were assessed. RESULTS: sst5TMD5 was absent in all samples analyzed. sst5TMD4 was found in 85% of tumors, and its expression was positively correlated to that of sst5 (R(2) = 0.79, P < 0.001). Expression of sst5TMD4 was negatively correlated with the ability of octreotide to reduce GH levels in vivo and partially negatively correlated with inhibition of GH secretion by an sst5 selective agonist (BIM-23268) in vitro. CONCLUSIONS: These results indicate that sst5TMD4 is related to the reduced ability of octreotide at normalizing hormone secretion in poorly responsive tumors in vivo. Further studies will help to evaluate the potential use of sst5TMD4 expression in surgically removed pituitary adenomas as a predictor of the subsequent response of different pituitary tumors to SSA therapy.

Resistin regulates pituitary somatotrope cell function through the activation of multiple signaling pathways.

The adipokine resistin is an insulin-antagonizing factor that also plays a regulatory role in inflammation, immunity, food intake, and gonadal function. Although adipose tissue is the primary source of resistin, it is also expressed in other tissues and organs, including the pituitary. However, there is no information on whether resistin, as described previously for other adipokines such as leptin and adiponectin, could regulate this gland. Likewise, the molecular basis of resistin actions remains largely unexplored. Here we show that administration of resistin to dispersed rat anterior pituitary cells increased GH release in both the short (4 h) and long (24 h) term, decreased mRNA levels of the receptor of the somatotrope regulator ghrelin, and increased free cytosolic Ca(2+) concentration in single somatotropes. By means of a pharmacological approach, we found that the stimulatory action of resistin occurs through a Gs protein-dependent mechanism and that the adenylate cyclase/cAMP/protein kinase A pathway, the phosphatidylinositol 3-kinase/Akt pathway, protein kinase C, and extracellular Ca(2+) entry through L-type voltage-sensitive Ca(2+) channels are essential players in mediating the effects of resistin on somatotropes. Taken together, our results demonstrate for the first time a regulatory role for resistin on somatotrope function and provide novel insights on the intracellular mechanisms activated by this protein.