Ontogeny of a new enteric peptide, neuropeptide W (NPW), in the developing rat stomach

Neuropeptide W (NPW), a novel endogenous peptide for G protein-coupled receptors GPR7 and GPR8, is expressed in the gastric antral mucosa of rat, mouse, and human stomachs. Here, we studied the ontogeny of NPW in the developing rat stomach. Real-time RT-PCR showed that NPW gene expression was initially detectable in embryonic day 14 (E14) stomach and gradually increased during the progress of age until birth, postnatal day 1 (P1). NPW mRNA level in the stomach increased again from the weaning period (P21) until reaching adulthood. Immunohistochemistry using polyclonal antibodies raised against rat NPW revealed that NPW-positive cells were detected in the P1 antral stomach and gradually increased during the development of age. Furthermore, double immunohistochemistry demonstrated that NPW colocalized with gastrin in P1 rat stomach. These data will provide clues to physiological functions of NPW in the development of rat stomach.     

Neuropeptide W is expressed in the noradrenalin-containing cells in the rat adrenal medulla

Neuropeptide W (NPW) is an endogenous ligand for GPR7, a member of the G-protein-coupled receptor family. NPW plays an important role in the regulation of both feeding and energy metabolism, and is also implicated in modulating responses to an acute inflammatory pain through activation of the hypothalamus-pituitary-adrenal axis. GPR7 mRNA has been shown to be expressed in the hypothalamus, pituitary gland and adrenal cortex. Similarly, NPW expression has been demonstrated in the brain and pituitary gland. However, the precise distribution of NPW-producing cells in the adrenal gland remains unknown. The aim of this study was to explore the distribution and localization of NPW immunoreactivity in the rat adrenal gland. Total RNA was prepared from the hypothalamus, pituitary gland and adrenal gland. RT-PCR revealed the expression of NPW mRNA in these tissues, while in situ hybridization demonstrated the presence of NPW mRNA in the adrenal medulla. When immunohistochemistry was performed on sections of adrenal gland, NPW-like immunoreactivity (NPW-LI) was observed in the medulla but not in the cortex. Moreover, NPW-LI was found to be co-localized in cells which expressed dopamine beta hydroxylase but not phenylethanolamine-N-methyltransferase. The finding that NPW is expressed in noradrenalin-containing cells in the adrenal medulla suggests that it may play an important role in endocrine function in the adrenal gland.     

Quantitative Analysis of Long-Form Aromatase mRNA in the Male and Female Rat Brain

In vitro studies show that estrogens acutely modulate synaptic function in both sexes. These acute effects may be mediated in vivo by estrogens synthesized within the brain, which could fluctuate more rapidly than circulating estrogens. For this to be the case, brain regions that respond acutely to estrogens should be capable of synthesizing them. To investigate this question, we used quantitative real-time PCR to measure expression of mRNA for the estrogen-synthesizing enzyme, aromatase, in different brain regions of male and female rats. Importantly, because brain aromatase exists in two forms, a long form with aromatase activity and a short form with unknown function, we targeted a sequence found exclusively in long-form aromatase. With this approach, we found highest expression of aromatase mRNA in the amygdala followed closely by the bed nucleus of the stria terminalis (BNST) and preoptic area (POA); we found moderate levels of aromatase mRNA in the dorsal hippocampus and cingulate cortex; and aromatase mRNA was detectable in brainstem and cerebellum, but levels were very low. In the amygdala, gonadal/hormonal status regulated aromatase expression in both sexes; in the BNST and POA, castration of males down-regulated aromatase, whereas there was no effect of estradiol in ovariectomized females. In the dorsal hippocampus and cingulate cortex, there were no differences in aromatase levels between males and females or effects of gonadal/hormonal status. These findings demonstrate that long-form aromatase is expressed in brain regions that respond acutely to estrogens, such as the dorsal hippocampus, and that gonadal/hormonal regulation of aromatase differs among different brain regions.     

A chronic high fat diet alters the homologous and heterologous control of appetite regulating peptide receptor expression

Leptin, ghrelin and neuropeptide W (NPW) modulate vagal afferent activity, which may underlie their appetite regulatory actions. High fat diet (HFD)-induced obesity induces changes in the plasma levels of these peptides and alters the expression of receptors on vagal afferents. We investigated homologous and heterologous receptor regulation by leptin, ghrelin and NPW. Mice were fed (12 weeks) a standard laboratory diet (SLD) or HFD. Nodose ganglia were cultured overnight in the presence or absence of each peptide. Leptin (LepR), ghrelin (GHS-R), NPW (GPR7) and cholecystokinin type-1 (CCK1R) receptor mRNA, and the plasma leptin, ghrelin and NPW levels were measured. SLD: leptin reduced LepR, GPR7, increased GHS-R and CCK1R mRNA; ghrelin increased LepR, GPR7, CCK1R, and decreased GHS-R. HFD: leptin decreased GHS-R and GPR7, ghrelin increased GHS-R and GPR7. NPW decreased all receptors except GPR7 which increased with HFD. Plasma leptin was higher and NPW lower in HFD. Thus, HFD-induced obesity disrupts inter-regulation of appetite regulatory receptors in vagal afferents.     

Neuronal interactions between neuropeptide W- and orexin- or melanin-concentrating hormone-containing neurons in the rat hypothalamus

Neuropeptide W (NPW) was recently discovered as the endogenous ligand for GPR7 and GPR8, which are orphan G protein-coupled receptors isolated from the porcine brain. These receptors are assumed to be involved in feeding regulation and/or energy homeostasis. Recent anatomical studies have revealed that high levels of GPR7 mRNA are distributed in the brain, including the hypothalamus and amygdala. However immunohistochemical studies on the distribution and localization of NPW have revealed differing results concerning whether or not NPW-containing cell bodies and their processes are present in the hypothalamus. Only a few immunohistochemical reports have been published concerning the presence of NPW-containing neurons in the brains of rodents, while there have been no anatomical studies of the co-localization of this neuropeptide with other transmitters. On this basis, we used a specific antiserum against NPW to determine immunohistochemically the presence of NPW-containing neurons in the rat hypothalamus. Many NPW-like immunoreactive cell bodies and their processes could be detected in the caudal region of the lateral hypothalamus but not in its anterior or middle regions. Given this positive identification of NPW-containing neurons in the lateral hypothalamus, we further studied the nature of interaction between NPW-containing neurons and neurons containing feeding regulating peptides such as orexin- and melanin-concentrating hormone (MCH). Very close interactions between NPW-containing nerve processes and orexin- and MCH-containing neuronal cell bodies and processes could be observed. These morphological findings strongly suggest that NPW is involved in the regulation of feeding and/or sleep/arousal behavior through orexin- and/or MCH-mediated neuronal pathways.     

Neuropeptide B and W regulate leptin and resistin secretion, and stimulate lipolysis in isolated rat adipocytes

Neuropeptide B (NPB) and W (NPW) regulate food intake and energy homeostasis in humans via two G-protein-coupled receptor subtypes, termed as GPR7 and GPR8. Rodents express GPR7 only. In animals, NPW decreases insulin and leptin levels, whereas the deletion of either NPB or GPR7 leads to obesity and hyperphagia. Metabolic and endocrine in vitro activities of NPW/NPB in adipocytes are unknown. We therefore characterize the effects of NPB and NPW on the secretion and expression of leptin and resistin, and on lipolysis, using rat adipocytes. Isolated rat adipocytes express GPR7 mRNA. NPB and NPW are expressed in macrophages and preadipocytes but are absent in mature adipocytes. Both, NPB and NPW reduce the secretion and expression of leptin from isolated rat adipocytes. NPB stimulates the secretion and expression of resistin, whereas both, NPB and NPW increase lipolysis. Our study demonstrates for the first time that NPB and NPW regulate the expression and secretion of leptin and resistin, and increase lipolysis in isolated rat adipocytes. These effects are presumably mediated via GPR7. The increase of resistin secretion, stimulation of lipolysis and the decrease of leptin secretion may represent mechanisms, through which NPB and NPW can affect glucose and lipid homeostasis, and food intake in rodents.     

Regulation of Resistin by Gonadal,Thyroid Hormone,and Nutritional Status

Objective: Resistin was recently identified as a hormone secreted by adipocytes that is under hormonal and nutritional control. This hormone has been suggested to be the link between obesity and type 2 diabetes. The aim of this study was to assess the influence of gender, gonadal status, thyroid hormones, pregnancy, and food restriction on resistin mRNA levels in adipose tissue of rats. Research Methods and Procedures: We have determined resistin mRNA expression by Northern blot analysis in all experimental sets. Results: Resistin mRNA expression is influenced by age, with the highest hormone levels existing at 45 days after birth and decreasing thereafter. Resistin mRNA expression is higher in men than in women. Moreover, we studied the effect of orchidectomy and ovariectomy in rats of different ages and showed that gonadal hormones increase adipose tissue resistin mRNA expression in male rats. Resistin is also regulated by thyroid hormones; it is severely decreased in hyperthyroid rats. Our results clearly show that chronic food restriction (30% of ad libitum food intake) led to a decrease in adipose tissue mRNA levels in normal cycling female rats and pregnant rats. In pregnancy, resistin mRNA levels were enhanced particularly at midgestation. Discussion: Our observations indicate that resistin is influenced by gender, gonadal status, thyroid hormones, and pregnancy. These findings suggest that resistin could explain the decreased insulin sensitivity during puberty and could be the link between sex steroids and insulin sensitivity. Moreover, resistin could mediate the effect of thyroid hormones on insulin resistance and the state of insulin resistance present during pregnancy.     

Neuropeptide W in the rat pancreas: potentiation of glucose-induced insulin release and Ca2+ influx through L-type Ca2+ channels in beta-cells and localization in islets

Neuropeptide W (NPW) is a regulatory peptide that acts via two subtypes of G protein-coupled receptors, GPR7 and GPR8. Evidence has been provided that NPW is involved in the central regulation of energy homeostasis and feeding behavior. In this study, we examined the effects of NPW on insulin release and localization of NPW in the rat pancreas. NPW (10-100 nM) significantly increased insulin release in the presence of 8.3 mM, but not 2.8 mM, glucose in the isolated rat islets. By fura-2 microfluorometry, NPW (1-100 nM) concentration-dependently increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) at 8.3 mM glucose in rat single beta-cells. The NPW-induced [Ca(2+)](i) increase was abolished under external Ca(2+)-free conditions and by an L-type Ca(2+) channel blocker nifedipine (10 microM). RT-PCR analysis revealed that mRNA for NPW was expressed in the rat pancreas and hypothalamus. Double immunohistochemical analysis showed that NPW-immunoreactivity was found in islets and co-localized with insulin-containing beta-cells, but not glucagon-containing alpha-cells and somatostatin-containing delta-cells. These results suggest that NPW could serve as a local modulator of glucose-induced insulin release in rat islets. NPW directly activates beta-cells to enhance Ca(2+) influx through voltage-dependent L-type Ca(2+) channels and potentiates glucose-induced insulin release.     

Neuropeptide W regulates proliferation and differentiation of ATDC5: Possible involvement of GPR7 activation,PKA and PKC‐dependent signalling cascades

Various neuropeptides related to the energy equilibrium affect bone growth in humans and animals. Neuropeptides W (NPW) are identical in the internal ligands of the two G‐protein receptors (GPRs) included in subtypes 7 and 8. Neuropeptides W inhibits proliferation in the cultivated rat calvarial osteoblast‐like (ROB) cells. This study examines the expression of NPW and GPR7 in murine chondrocyte and their function. An immunohistochemical analysis showed that NPW and GPR7 were expressed in the proliferative chondrocytes of the growth plates in the hind limbs of mice. The NPW mRNA quickly elevated in the early differentiation (7‐14 days) of ATDC5 cells, while NPW and GPR7 mRNA were reduced during the late stage (14‐21 days) of differentiation. Neuropeptide W‐23 (NPW‐23) promoted the proliferation of ATDC5 cells, which was attenuated by inhibiting the GPR7, protein kinase A (PKA), protein kinase C (PKC) and ERK1/2 pathways. Neuropeptide W‐23 enhanced the early cell differentiation, as evaluated by collagen type II and the aggrecan gene expression, which was unaffected by inhibiting the ERK1/2 pathway, but significantly decreased by inhibiting the PKA, PKC and p38 MAPK pathways. In contrast, NPW‐23 was not involved in the terminal differentiation of the chondrocytes, as evaluated by the mineralization of the chondrocytes and the activity of the alkaline phosphatase. Neuropeptides W stimulated the PKA, PKC, p38 MAPK and ERK1/2 activities in a dose‐ and time‐dependent manner in the ATDC5 cells. These results show that NPW promotes the proliferation and early differentiation of murine chondrocyte via GPR7 activation, as well as PKA and PKC‐dependent signalling cascades, which may be involved in endochondral bone formation.     

Injection of neuropeptide W into paraventricular nucleus of hypothalamus increases food intake

Neuropeptide W (NPW) is an endogenous ligand for G protein-coupled receptor 7 (GPR7). There are two forms of the peptide, designated as neuropeptide W-23 (NPW23) and neuropeptide W-30 (NPW30). In the current study we found that intracerebroventricular administration of NPW23 increased c-Fos immunoreactivity (IR) in a variety of brain sites, many of which are involved in the regulation of feeding. In particular, we noted that c-Fos IR levels were increased in hypocretin-expressing neurons in the perifornical region of the lateral hypothalamus (LH). We then studied whether injection of NPW23 into the paraventricular nucleus of the hypothalamus (PVN) and the LH increased food intake over a 24-h time period. Intra-PVN injection of NPW23 at doses ranging from 0.1 to 3 nmol increased feeding for up to 4 h, and doses ranging from 0.3 to 3 nmol increased feeding for up to 24 h. In contrast, only the 3-nmol dose of NPW23 increased feeding after administration into the LH. Together, these data suggest a modulatory role for NPW in the control of food intake.     

Sex-role reversal is reflected in the brain of African black coucals (Centropus grillii)

In most bird species males compete over access to females and have elevated circulating androgen levels when they establish and defend a breeding territory or guard a mate. Testosterone is involved in the regulation of territorial aggression and sexual display in males. In few bird species the traditional sex-roles are reversed and females are highly aggressive and compete over access to males. Such species represent excellent models to study the hormonal modulation of aggressive behavior in females. Plasma sex steroid concentrations in sex-role reversed species follow the patterns of birds with "traditional" sex-roles. The neural mechanisms modulating endocrine secretion and hormone-behavior interactions in sex-role reversed birds are currently unknown. We investigated the sex differences in the mRNA expression of androgen receptors, estrogen receptor alpha, and aromatase in two brain nuclei involved in reproductive and aggressive behavior in the black coucal, the nucleus taeniae and the bed nucleus of the stria terminalis. In the bed nucleus there were no sex differences in the receptor or aromatase expression. In the nucleus taeniae, however, we show for the first time, that females have a higher mRNA expression of androgen receptors than males. These results suggest that the expression of agonistic and courtship behavior in females does not depend on elevated blood hormone levels, but may be regulated via increased steroid hormone sensitivity in particular target areas in the brain. Hence, aggression in females and males may indeed be modulated by the same hormones, but regulated at different levels of the neuroendocrine cascade.     

Expression of juvenile hormone acid O‐methyltransferase and juvenile hormone synthesis in Blattella germanica

Juvenile hormone (JH), a sesquiterpenoid synthetized by the insect corpora allata (CA), plays critical roles in metamorphosis and reproduction. Penultimate or last step of JH synthesis is catalyzed by juvenile hormone acid O‐methyltransferase (JHAMT). Here we report the cloning and expression analysis of the JHAMT orthologue in the cockroach, Blattella germanica (L.) (BgJHAMT). BgJHAMT is mainly expressed in CA, with only expression traces in ovary. Three different isoforms, differing in the 3′‐UTR sequence, were identified. Isoform A shows between 35 and 65 times higher expression than B and C in CA from penultimate nymphal instar and adult females. RNAi‐triggered knock down of BgJHAMT produces a dramatic reduction of JH synthesis, concomitant with a decrease of fat body vitellogenin expression and basal follicle length. BgJHAMT mRNA levels in CA of females along the gonadotrophic cycle parallel, with a slight advancement, JH synthesis profile. BgJHAMT mRNA levels were reduced in starved females and in females in which we reduced nutritional signaling by knocking down insulin receptor and target of rapamycin (TOR). Results show that conditions that modify JH synthesis in adult B. germanica females show parallel changes of BgJHAMT mRNA levels and that the JH‐specific branch of the JH synthesis pathway is regulated in the same way as the mevalonate branch. Furthermore, we demonstrate that nutrition and its signaling through the insulin receptor and TOR pathways are essential for activating BgJHAMT expression, which suggests that this enzyme can be a checkpoint for the regulation of JH production in relation to nutritional status.     

Dietary and hormonal regulation of L-type pyruvate kinase gene expression in rat small intestine

L-type pyruvate kinase is an enzyme of the glycolytic pathway whose activity and mRNA levels fluctuate in the small intestine according to dietary status. Both the enzyme activity and mRNA concentration decline during fasting and increase upon refeeding either a glucose-rich or a fructose-rich diet. Using a single-strand M 13 phage complementary to L-type pyruvate kinase mRNA as probe, we determined the level of the mRNA in the small intestine of normal, adrenalectomized, thyroidectomized, diabetic and glucagon-treated or cAMP-treated animals refed either a glucose-rich or a fructose-rich diet. The specific mRNA is present in the small intestine of normal fasted rats and increases twofold and threefold on refeeding glucose and fructose respectively. However, the hormonal control of the gene expression differs according to the dietary carbohydrate. The L-type pyruvate kinase mRNA increase, induced by glucose feeding, is hormone-dependent and requires the presence of thyroid hormones and insulin. In fructose-fed rats a certain level of mRNA increase occurs regardless of the hormonal status of the animals, but the full induction of the mRNA by fructose requires the presence of glucocorticoids, thyroid hormones and insulin. Thus, the hormonal regulation of L-type pyruvate kinase gene expression in the small intestine is largely similar to that described in normal rat liver but the basal mRNA level and the stimulation of the mRNA increase by fructose are higher in the small intestine.     

Identification of neuropeptide W as the endogenous ligand for orphan G-protein-coupled receptors GPR7 and GPR8

The structurally related orphan G-protein-coupled receptors GPR7 and GPR8 are expressed in the central nervous system, and their ligands have not been identified. Here, we report the identification of the endogenous ligand for both of these receptors. We purified the peptide ligand from porcine hypothalamus using stable Chinese hamster ovary cell lines expressing human GPR8 and cloned the cDNA encoding its precursor protein. The cDNA encodes two forms of the peptide ligand with lengths of 23 and 30 amino acid residues as mature peptides. We designated the two ligands neuropeptide W-23 (NPW23) and neuropeptide W-30 (NPW30). The amino acid sequence of NPW23 is completely identical to that of the N-terminal 23 residues of NPW30. Synthetic NPW23 and NPW30 activated and bound to both GPR7 and GPR8 at similar effective doses. Intracerebroventricular administration of NPW23 in rats increased food intake and stimulated prolactin release. These findings indicate that neuropeptide W is the endogenous ligand for both GPR7 and GPR8 and acts as a mediator of the central control of feeding and the neuroendocrine system.     

Obestatin induction of early-response gene expression in gastrointestinal and adipose tissues and the mediatory role of G protein-coupled receptor, GPR39

Obestatin was identified as a brain/gut peptide hormone encoded by the ghrelin gene and found to interact with the G protein-coupled receptor, GPR39. We investigated target cells for obestatin based on induction of an early-response gene c-fos in different tissues. After ip injection of obestatin, c-fos staining was found in the nuclei of gastric mucosa, intestinal villi, white adipose tissues, hepatic cords, and kidney tubules. Immunohistochemical analyses using GPR39 antibodies further revealed cytoplasmic staining in these tissues. In cultured 3T3-L1 cells, treatment with obestatin, but not motilin, induced c-fos expression. In these preadipocytes, treatment with obestatin also stimulated ERK1/2 phosphorylation. Because phenotypes of GPR39 null mice are partially consistent with a role of GPR39 in mediating obestatin actions, we hypothesized that inconsistencies on the binding of iodinated obestatin to GPR39 are due to variations in the bioactivity of iodinated obestatin. We obtained monoiodoobestatin after HPLC purification and demonstrated its binding to jejunum, stomach, ileum, pituitary, and white adipose tissue. Furthermore, human embryonic kidney 293T cells transfected with plasmids encoding human or mouse GPR39 or a human GPR39 isoform, but not the ghrelin receptor, exhibited high-affinity binding to monoiodoobestatin. Binding studies using jejunum homogenates and recombinant GPR39 revealed obestatin-specific displacement curves. Furthermore, treatment with obestatin induced c-fos expression in gastric mucosa of wild-type, but not GPR39 null, mice, underscoring a mediating role of this receptor in obestatin actions. The present findings indicate that obestatin is a metabolic hormone capable of binding to GPR39 to regulate the functions of diverse gastrointestinal and adipose tissues.     

Molecular dynamics simulation of neuropeptide B and neuropeptide W in the dipalmitoylphosphatidylcholine membrane bilayer

GPR7 and GPR8 are recently deorphanized G-protein-coupled receptors that are implicated in the regulation of neuroendocrine function, feeding behavior, and energy homeostasis. Neuropeptide B (NPB) and neuropeptide W (NPW) are two membrane-bound hypothalamic peptides, which specifically antagonize GPR7 and GPR8. Despite years of research, an accurate estimation of structure and molecular recognition of these neuropeptide systems still remains elusive. Herein, we investigated the structure, orientation, and interaction of NPB and NPW in a dipalmitoylphosphatidylcholine bilayer using long-range molecular dynamics (MD) simulation. During 30-ns simulation, membrane-embedded helical axes of NPB and NPW tilted 30 and 15°, respectively, from the membrane normal in order to overcome possible hydrophobic mismatch with the lipid bilayer. The calculation of various structural parameters indicated that NPW is more rigid and compact as compared to NPB. Qualitatively, the peptides exhibited flexible N-terminal (residues 1–12) and rigid C-terminal α-helical parts (residues 13–21), confirming previous NMR data. A strong electrostatic attraction between C-termini and headgroup atoms caused translocation of the peptides towards lower leaflet of the bilayer. The stabilizing hydrogen bonds (H-bonds) between phosphate groups and Trp1, Lys3, and Arg15 of the peptides played important roles for membrane anchoring. MD simulations of Alanine (Ala) mutants revealed that WYK->Ala variant of NPB/NPW lacked crucial H-bond interactions with phospholipid headgroups and also caused severe misfolding in NPB. Altogether, the knowledge of preferred structural fold and interaction of neuropeptides within the membrane bilayer will be useful to develop synthetic agonist or antagonist peptides for GPR7 and GPR8.     

The Orphan G Protein-Coupled Receptor Gene GPR178 Is Evolutionary Conserved and Altered in Response to Acute Changes in Food Intake

G protein-coupled receptors (GPCRs) are a class of integral membrane proteins mediating physiological functions fundamental for survival, including energy homeostasis. A few years ago, an amino acid sequence of a novel GPCR gene was identified and named GPR178. In this study, we provide new insights regarding the biological significance of Gpr178 protein, investigating its evolutionary history and tissue distribution as well as examining the relationship between its expression level and feeding status. Our phylogenetic analysis indicated that GPR178 is highly conserved among all animal species investigated, and that GPR178 is not a member of a protein family. Real-time PCR and in situ hybridization revealed wide expression of Gpr178 mRNA in both the brain and periphery, with high expression density in the hypothalamus and brainstem, areas involved in the regulation of food intake. Hence, changes in receptor expression were assessed following several feeding paradigms including starvation and overfeeding. Short-term starvation (12–48h) or food restriction resulted in upregulation of Gpr178 mRNA expression in the brainstem, hypothalamus and prefrontal cortex. Conversely, short-term (48h) exposure to sucrose or Intralipid solutions downregulated Gpr178 mRNA in the brainstem; long-term exposure (10 days) to a palatable high-fat and high-sugar diet resulted in a downregulation of Gpr178 in the amygdala but not in the hypothalamus. Our results indicate that hypothalamic Gpr178 gene expression is altered during acute exposure to starvation or acute exposure to palatable food. Changes in gene expression following palatable diet consumption suggest a possible involvement of Gpr178 in the complex mechanisms of feeding reward.