Hypothalamic glucose-sensing: role of Glia-to-neuron signaling.

The hypothalamus senses hormones and nutrients in order to regulate energy balance. In particular, detection of hypothalamic glucose levels has been shown to regulate both feeding behavior and peripheral glucose homeostasis, and impairment of this regulatory system is believed to be involved in the development of obesity and diabetes. Several data clearly demonstrate that glial cells are key elements in the perception of glucose, constituting with neurons a "glucose-sensing unit". Characterization of this interplay between glia and neurons represents an exciting challenge, and will undoubtedly contribute to identify new candidates for therapeutic intervention. The purpose of this review is to summarize the current data that stress the importance of glia in central glucose-sensing. The nature of the glia-to-neuron signaling is discussed, with a special focus on the endozepine ODN, a potent anorexigenic peptide that is highly expressed in hypothalamic glia.

Secretin as a neurohypophysial factor regulating body water homeostasis.

Hypothalamic magnocellular neurons express either one of the neurohypophysial hormones, vasopressin or oxytocin, along with different neuropeptides or neuromodulators. Axonal terminals of these neurons are generally accepted to release solely the two hormones but not others into the circulation. Here, we show that secretin, originally isolated from upper intestinal mucosal extract, is present throughout the hypothalamo-neurohypophysial axis and that it is released from the posterior pituitary under plasma hyperosmolality conditions. In the hypothalamus, it stimulates vasopressin expression and release. Considering these findings together with our previous findings that show a direct effect of secretin on renal water reabsorption, we propose here that secretin works at multiple levels in the hypothalamus, pituitary, and kidney to regulate water homeostasis. Findings presented here challenge previous understanding regarding the neurohypophysis and could provide new concepts in treating disorders related to osmoregulation.

First evidence for a direct inhibitory effect of kisspeptins on LH expression in the eel, Anguilla anguilla.

The kisspeptin system has emerged as one of the main puberty gatekeepers among vertebrates. The European eel (Anguilla anguilla) is a remarkable model due to its phylogenetical position at the basis of teleosts, and its unique life cycle with a blockade of puberty before reproductive migration. We cloned the full-length coding sequence of a kisspeptin receptor (Kissr) in the eel. Comparison of Kissr sequences assigned the eel Kissr to a basal position in a clade including most of the known teleost Kissr, in agreement with the eel phylogenetical position. Eel Kissr tissue distribution was analyzed by quantitative real-time PCR. Eel Kissr was highly expressed in the brain, especially in the telencephalon and di-/mes-encephalon, while a very low or undetectable expression was observed in various peripheral organs. A high expression of Kissr was also found in the pituitary indicating a possible direct pituitary role of kisspeptin. Primary cultures of eel pituitary cells were performed to investigate the direct effects of kisspeptin on pituitary hormone expression. Human/lamprey kisspeptin exerted a time- and dose-dependent inhibitory effect on LHß expression. All other tested kisspeptins had a similar inhibitory effect on LHß expression. The inhibitory effect of kisspeptins was exerted specifically on LHß as no change was induced on the expression of other glycoprotein hormone subunits (GPα, FSHß and TSHß) nor of growth hormone. These data provide the first evidence for the existence, in the European eel, of a kisspeptin system, which may play a direct inhibitory role on pituitary LHß expression.

Bax siRNA promotes survival of cultured and allografted granule cell precursors through blockade of caspase-3 cleavage.

Transplantation of neuronal precursor cells (NPCs) into the central nervous system could represent a powerful therapeutical tool against neurodegenerative diseases. Unfortunately, numerous NPCs die shortly after transplantation, predominantly due to caspase-dependent apoptosis. Using a culture of cerebellar neuronal precursors, we have previously demonstrated protective effect of the neuropeptide PACAP, which suppresses ceramide-induced apoptosis by blockade of the mitochondrial apoptotic pathway. The main objective of this study was to determine whether Bax repression can promote survival of NPCs allotransplanted into a host animal. In vivo and ex vivo experiments revealed that C2-ceramide increases Bax expression, while PACAP reverses this effect. In vitro tests using cerebellar NPCs demonstrated that the Bax-specific small interfering RNA (siRNA) could reduce their death and caspase-3 cleavage within the first 24 h. BrdU-labelled NPCs were subjected to transfection procedure with or without siRNA introduction before using for in vivo transplantation. Twenty-four hours after, the allografted NPCs containing siRNA showed significantly reduced level of caspase-3 cleavage, and the volume of their implants was almost twofold higher than in the case of empty-transfected precursors. These data evidence an important role of Bax in life/death decision of grafted NPCs and suggest that RNA interference strategy may be applicable for maintaining NPCs survival within the critical first hours after their transplantation.

Interactions of PACAP and ceramides in the control of granule cell apoptosis during cerebellar development.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that belongs to the secretin/glucagon/vasoactive intestinal polypeptide superfamily. The PACAPergic system is actively expressed in the developing cerebellum of mammals. In particular, PACAP receptors are expressed by granule cell precursors suggesting a role of the peptide in neurogenesis of this cell type. Consistent with this hypothesis, several studies reported antiapoptotic effects of PACAP in the developing cerebellum. On the other hand, the sphingomyelin metabolites ceramides are recognized as important signaling molecules that play pivotal roles during neuronal development. Ceramides, which production can be induced by death factors such as FasL or TNFalpha, are involved in the control of cell survival during brain development through activation of caspase-dependent mechanisms. The present review focuses on the interactions between PACAP and ceramides in the control of granule cell survival and on the transduction mechanisms associated with the anti- and proapoptotic effects of PACAP and ceramides, respectively.

Outer-membrane proteomic maps and surface-exposed proteins of Legionella pneumophila using cellular fractionation and fluorescent labelling.

Bacterial surface-associated proteins play crucial roles in host-pathogen interactions and pathogenesis. The identification of these proteins represents an important goal of bacterial proteomics for vaccine development, but also for environmental concerns such as microbial biosensing. Here, we developed such an approach for Legionella pneumophila, a bacterium that causes severe pneumonia. We propose a complementary strategy consisting of (1) a fluorescent labelling of surface-exposed proteins in parallel with (2) a fractionation of the outer-membrane protein extract. These two distinct protein populations were subsequently separated using two-dimensional gel electrophoresis and characterised by mass spectrometry. Within these populations, we found proteins which were expected for the compartments studied, but also a great number of proteins never experimentally described, and also a non-negligible fraction of proteins never described in these fractions. These data provided new routes of inspection for transport and host recognition for Legionella pneumophila. In addition, these results on the membranome and surfaceome show that Legionella in the stationary phase of growth possesses the major determinants to infect host cells.

Rheology of biofilms formed at the surface of NF membranes in a drinking water production unit.

In this study, the mechanical properties of biofilms formed at the surface of nano-filtration (NF) membranes from a drinking water plant were analysed. Confocal laser scanning microscopy observations revealed that the NF biofilms formed a dense and heterogeneous structure at the membrane surface, with a mean thickness of 32.5 +/- 17.7 mum. The biofilms were scraped from the membrane surface and analysed in rotation and oscillation experiments with a RheoStress 150 rotating disk rheometer. During rotation analyses, a viscosity decrease with speed of shearing characteristic of rheofluidification was observed (eta = 300 Pa s for ý = 0.3 s(-1)). In the oscillation analyses with a sweeping of frequency (1-100 Hz), elasticity (G') ranged from 3000 to 3500 Pa and viscosity (G'') from 800 to 1200 Pa. Creep curves obtained with an application of a shear stress of 30 Pa were viscoelastic in nature. The G(0) and eta values were, respectively, 1.4 +/- 0.3 x 10(3) Pa and 3.3 +/- 0.65 x 10(6) Pa s. The relationship between the characteristics of NF biofilms and the flow conditions encountered during NF is discussed.

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.

The pituitary V3 vasopressin receptor and the corticotroph phenotype in ectopic ACTH syndrome.

Ectopic ACTH secretion occurs in highly differentiated and rather indolent tumors like bronchial carcinoids or, in contrast, in various types of aggressive and poorly differentiated neuroendocrine tumors. We explored this phenomenon using the recently cloned human pituitary V3 vasopressin receptor as an alternate molecular marker of the corticotroph phenotype. Expression of V3 receptor, corticotrophin releasing hormone (CRH) receptor, and proopiomelanocortin (POMC) genes was examined in tumors of pituitary and nonpituitary origin. A comparative RT-PCR approach revealed signals for both V3 receptor and CHR receptor mRNAs in 17 of 18 ACTH-secreting pituitary adenomas, and 6 of 6 normal pituitaries; in six growth hormone- or prolactin-secreting adenomas, a very faint V3 receptor signal was observed in three cases, and CRH receptor signal was undetected in all. Six of eight bronchial carcinoids responsible for the ectopic ACTH syndrome had both POMC and V3 receptor signals as high as those in ACTH-secreting pituitary adenomas; in contrast, no POMC signal and only a very faint V3 receptor signal were detected in six of eight nonsecreting bronchial carcinoids. Northern blot analysis showed V3 receptor mRNA of identical size in ACTH-secreting bronchial carcinoids and pituitary tumors. Other types of nonpituitary tumors responsible for ectopic ACTH syndrome presented much lower levels of both POMC and V3 receptor gene expression than those found in ACTH-secreting bronchial carcinoids. In contrast with the V3 receptor, CRH receptor mRNA was detected in the majority of neuroendocrine tumors irrespective of their POMC status. These results show that expression of the V3 receptor gene participates in the corticotroph phenotype. Its striking association with ACTH-secreting bronchial carcinoids defines a subset of nonpituitary tumors in which ectopic POMC gene expression is but one aspect of a wider process of corticotroph cell differentiation, and opens new possibilities of pharmacological investigations and even manipulations of this peculiar ACTH hypersecretory syndrome.

Functional differences between NPFF1 and NPFF2 receptor coupling: high intrinsic activities of RFamide-related peptides on stimulation of [35S]GTPgammaS binding.

By using an optimized [(35)S]GTPgammaS binding assay, the functional activities (potency and efficacy) of peptides belonging to three members of the RFamide family; Neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP) and 26RFamide, were investigated on NPFF(1) and NPFF(2) receptors stably expressed in Chinese Hamster Ovary (CHO) cells. Despite their large differences in affinity and selectivity, all analogues tested behaved as agonists toward NPFF(1) and NPFF(2) receptors. High NaCl concentration in the assay strongly increased the efficacy toward NPFF(2) receptors and augmented differences among agonists. In low sodium conditions, whereas the potencies of agonists correlated with their affinities for NPFF(1) receptors, NPFF(2) receptors exhibited an extraordinary activity since all compounds tested displayed EC(50) values of GTPgammaS binding lower than their K(I) values. Comparisons of functional values between NPFF(1) and NPFF(2) receptors revealed unexpected potent selective NPFF(2) agonists especially for the PLRFamide and the VGRFamide sequences. By using blocker peptides, we also show that Galpha(i3) and Galpha(s) are the main transducers of NPFF(1) receptors while NPFF(2) are probably coupled with Galpha(i2), Galpha(i3), Galpha(o) and Galpha(s) proteins. Our data indicate that NPPF(1) and NPFF(2) receptors are differently coupled to G proteins in CHO cells.

Gastric electrical stimulation modulates hypothalamic corticotropin-releasing factor-producing neurons during post-operative ileus in rat.

High-frequency/low-energy gastric electrical stimulation (GES) is an efficient therapy to treat gastric emptying-related disorders but its mechanism of action remains poorly understood. We aimed to assess the effects of high-frequency/low-energy GES on corticotropin-releasing factor (CRF)-producing neurons in the paraventricular nucleus of the hypothalamus (PVN), which are involved in gastric ileus induced by laparotomy. Two electrodes were implanted in the rat gastric antrum during laparotomy, then stimulation (amplitude: 2 mA; pulse duration 330 micros; frequency: 2 Hz; 1 min ON/2 min OFF) or sham stimulation (control group) were applied. Using immunohistochemistry, the number of c-Fos protein-expressing neurons (c-Fos protein-immunoreactive cells, Fos-IR) was quantified in the PVN after 1 h of stimulation. The number of neurons expressing simultaneously c-Fos protein and CRF mRNA was measured by means of immunocytochemistry combined with in situ hybridization. Finally, c-Fos and CRF mRNA levels in the hypothalamus were determined by in situ hybridization or quantitative reverse transcriptase-polymerase chain reaction. Fos-IR in the PVN was significantly decreased 1 h after GES (P<0.05) but was not affected by sub-diaphragmatic vagotomy. The number of neurons containing c-Fos protein and CRF mRNA was lower in the GES group compared with the control group (P<0.05). In addition, c-Fos and CRF mRNA levels in the PVN were significantly decreased by GES (P

Effect of the diazepam-binding inhibitor-derived peptide, octadecaneuropeptide, on food intake in goldfish.

An endogenous ligand of central-type benzodiazepine receptors (CBR), the endozepine octadecaneuropeptide (ODN), is a very potent inhibitor of food intake in rodents. Although endozepines have been localized and characterized in the trout hypothalamus, so far, the action of these neuropeptides on feeding behavior has never been investigated in fish. In the present study, we have examined the effect of i.c.v. administration of synthetic rat ODN, its C-terminal octapeptide (OP) and the head-to-tail cyclic analog cyclo(1-8)OP (cOP) on feeding behavior in the goldfish model. i.c.v. injection of graded doses of ODN (2.5-10 pmol/g body weight (BW)) induced a dose-dependent inhibition of food intake, a significant decrease in cumulative food intake during the 60-min period after feeding being observed at doses of 5 and 10 pmol/g BW. The inhibitory effect of a 10 pmol/g BW dose of ODN on food consumption (-39%) was mimicked by an equimolar dose of OP (-42%) and cOP (-53%). The food intake-suppressing activity of ODN (10 pmol/g BW) was not affected by pre-injection of the CBR antagonist flumazenil (200 pmol/g BW). In contrast, the anorexigenic effect of ODN (10 pmol/g BW) was totally suppressed by a selective antagonist of metabotropic endozepine receptors, cyclo(1-8)[dLeu(5)]OP. These data indicate that, in goldfish as in rodents, ODN is a potent inhibitor of food consumption, and that the anorexigenic effect of ODN is not mediated through CBR but through the metabotropic endozepine receptor.

The neuropeptide pituitary adenylate cyclase-activating polypeptide exerts anti-apoptotic and differentiating effects during neurogenesis: focus on cerebellar granule neurones and embryonic stem cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally isolated from ovine hypothalamus on the basis of its hypophysiotrophic activity. It has subsequently been shown that PACAP and its receptors are widely distributed in the central nervous system of adult mammals, indicating that PACAP may act as a neurotransmitter and/or neuromodulator. It has also been found that PACAP and its receptors are expressed in germinative neuroepithelia, suggesting that PACAP could be involved in neurogenesis. There is now compelling evidence that PACAP exerts neurotrophic activities in the developing cerebellum and in embryonic stem (ES) cells. In particular, the presence of PACAP receptors has been demonstrated in the granule layer of the immature cerebellar cortex, and PACAP has been shown to promote survival, inhibit migration and activate neurite outgrowth of granule cell precursors. In cerebellar neuroblasts, PACAP is a potent inhibitor of the mitochondrial apoptotic pathway through activation of the MAPkinase extracellular regulated kinase. ES cells and embryoid bodies (EB) also express PACAP receptors and PACAP facilitates neuronal orientation and induces the appearance of an electrophysiological activity. Taken together, the anti-apoptotic and pro-differentiating effects of PACAP characterised in cerebellar neuroblasts as well as ES and EB cells indicate that PACAP acts not only as a neurohormone and a neurotransmitter, but also as a growth factor.

Assessing chemical cleaning of nanofiltration membranes in a drinking water production plant: a combination of chemical composition analysis and fluorescence microscopy.

The efficiency of cleaning procedures to remove the fouling deposit from the surface of NF membranes operating in the drinking water plant of Méry sur Oise (Val d'Oise, France) was assessed by a combination of chemical analysis and fluorescence microscopy. The ATR-FTIR spectra of the fouled membranes revealed the presence of biological matter at the membrane surface, mainly composed of polysaccharides, nucleic acids and proteins. IR bands corresponding to the membrane material were detected for stage 1 but not for stage 3. Confocal laser scanning microscopy (CLSM) observations confirmed the microbial origin of the fouling deposit. After chemical cleaning, the analysis of the inorganic foulants revealed a significant decrease of the inorganic content. Moreover, ATR-FTIR spectra of the fouled membranes were modified, mainly in a broad complex region corresponding to polysaccharides and nucleic acids. The amide bands were also altered for stage 1, and some peaks corresponding to the clean membrane appeared for stage 3 after cleaning. CLSM observations revealed a general decrease of the lectin staining for the two stages with some variations between lectins. A decrease of the DAPI staining indicative of the removal of some microbial cells was also observed for stage 1. In conclusion, cleaning of the NF fouled membranes decreased significantly the inorganic foulants but only partially removed the organic fouling deposit characteristic of a microbial biofilm.

Differential expression and processing of chromogranin A and secretogranin II in relation to the secretory status of endocrine cells.

Chromogranin A (CgA) and secretogranin II (SgII) are neuroendocrine secretory proteins that participate in regulation of the secretory pathway and also serve as precursors of biologically active peptides. To investigate whether there is a relationship between the expression, distribution, and processing of CgA and SgII and the degree of secretory activity, we employed two melanotrope subpopulations of the pituitary intermediate lobe that exhibit opposite secretory phenotypes. Thus, although one of the melanotrope subtypes shows high secretory activity, the other exhibits characteristics of a hormone storage phenotype. Our data show that SgII expression levels were higher in secretory melanotropes, whereas CgA expression showed similar rates in both cell subsets. The use of various antibodies revealed the presence of the unprocessed proteins as well as three CgA-derived peptides (67, 45, and 30 kDa) and six SgII-derived peptides (81, 66, 55, 37, 32, and 30 kDa) in both subpopulations. However, the smallest molecular forms of both granins predominated in secretory melanotropes, whereas the largest SgII- and CgA-immunoreactive peptides were more abundant in storage melanotropes, which is suggestive of a more extensive processing of granins in the secretory subset. Confocal microscopy studies showed that CgA immunoreactivity was higher in storage cells, but SgII immunoreactivity was higher in secretory melanotropes. Taken together, our results indicate that SgII and CgA are differentially regulated in melanotrope subpopulations. Thus, SgII expression is strongly related to the secretory activity of melanotrope cells, whereas CgA expression may not be related to secretory rate, but, rather, to hormone storage in this endocrine cell type.

Pituitary adenylate cyclase-activating polypeptide directly modulates the activity of proopiomelanocortin neurons in the rat arcuate nucleus.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and the proopiomelanocortin (POMC)-derived peptide alpha-melanocyte-stimulating hormone (alpha-MSH) both regulate multiple neuroendocrine functions and feeding behavior. Two subtypes of PACAP receptor mRNAs, pituitary adenylate cyclase-activating polypeptide-specific receptor (PAC1-R) and pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide mutual receptor (VPAC2-R), are actively expressed in the arcuate nucleus of the hypothalamus, where POMC cell bodies are located. This observation led us to investigate the possible regulatory action of PACAP on rat POMC neurons. Double-labeling in situ hybridization histochemistry revealed that approximately 50% of POMC-producing neurons express PAC1-R and/or VPAC2-R mRNAs. The proportion of POMC neurons that also contain PAC1-R mRNA was homogeneous along the rostro-caudal axis of the arcuate nucleus while POMC-positive cell bodies expressing the VPAC2-R subtype were more abundant in the rostral region. Incubation of mediobasal hypothalamic explants with PACAP (10(-7) M; 30 min) increased POMC mRNA expression, and this effect was blocked by PACAP6-38 (10(-6) M). In contrast, incubation with vasoactive intestinal polypeptide (10(-7) M) did not affect POMC mRNA level. Incubation of hypothalamic fragments with PACAP (10(-7) M) caused a significant increase in alpha-MSH content in the tissue and in the incubation medium. Altogether, the present results reveal that exogenous PACAP, acting probably through PAC1-R, regulates the activity of POMC neurons in the rat hypothalamus. These data suggest that the effects of PACAP on the gonadotropin-releasing hormone neuroendocrine axis and the regulation of feeding behavior may be mediated, at least in part, through modulation of POMC neurons.

In vitro effects of crude extracts of Parkia biglobosa (Mimosaceae), Stereospermum kunthianum (Bignoniaceae) and Biophytum petersianum (Oxalidaceae) on corticosteroid secretion in rat.

Previous studies conducted in guinea pig, rat and rabbit have revealed that crude extracts from Parkia biglobosa, Stereospermum kunthianum and Biophytum petersianum exert hypotensive and/or hypoglycemic activities. Since corticosteroids are involved in the control of arterial blood pressure and glycemia, we have investigated the possible effects of these plant extracts on rat adrenal tissue in vitro. Short-term administration of crude semi-ethanolic extracts of P. biglobosa and S. kunthianum to perifused rat adrenal tissue did not induce any significant changes in corticosteroid output. Conversely, the B. petersianum extract caused a dose-dependent increase in corticosterone and aldosterone secretion. Repeated infusions or prolonged administration of B. petersianum extract did not produce any apparent attenuation of the steroid response. Altogether, these data indicate that a semi-ethanolic extract of B. petersianum dose-dependently stimulates corticosterone and aldosterone secretion in rat without any desensitization phenomenon.