Comparison of physiological functions between neuromedin U-related peptide and neuromedin S-related peptide in the rat central nervous system.

Two novel peptides, neuromedin U precursor-related peptide (NURP) and neuromedin S precursor-related peptide (NSRP), are produced from neuromedin U (NMU) and neuromedin S (NMS) precursors, respectively, as these precursors have multiple consensus sequences for proteolytic processing. Our group has shown previously that one of these two novel peptides, NURP, stimulates body temperature and locomotor activity, but not food intake. However, the physiological function of the other peptide, NSRP, has remained unclear. Therefore, the aim of this study was to characterize differences in the regions of the rat brain targeted by the NMU/NMS peptide family, including NURP and NSRP, and their physiological functions. First, we explored the regions of c-Fos expression after intracerebroventricular (i.c.v.) injection of NURP and NSRP and found that these were fewer than after i.c.v. injection of NMU and NMS in the hypothalamus, possibly because NURP and NSRP cannot activate NMU/NMS receptors. In the ventral subiculum, which is one region of the hippocampus, c-Fos expression was evident only after i.c.v. injection of NURP. We also examined the effects of NSRP on food intake, body temperature and locomotor activity. Like NURP, NSRP increased both body temperature and locomotor activity, but not food intake, indicating that NSRP is also a functional peptide. However, these effects of NSRP were distinctly weaker than those of NURP. These findings suggest differences in the affinity of NURP and/or NSRP for specific receptors, or in their respective biological activities.

Neuromedin U suppresses prolactin secretion via dopamine neurons of the arcuate nucleus.

Neuromedin U (NMU) has a precursor that contains one additional peptide consisting of 33 or 36 amino acid residues. Recently, we identified this second peptide from rat brain and designated it neuromedin U precursor-related peptide (NURP), showing it to stimulate prolactin release from the pituitary when injected via the intracerebroventricular (icv) route. Here, we examined whether NMU, like NURP, also stimulates prolactin release. Unlike NURP, icv injection of NMU significantly decreased the secretion of prolactin from the pituitary. This suppression of prolactin release by NMU was observed in hyper-prolactin states such as lactation, stress, pseudopregnancy, domperidone (dopamine antagonist) administration, and icv injection of NURP. Immunohistochemical analysis revealed that icv injection of NMU induced cFos expression in dopaminergic neurons of the arcuate nucleus, but not the substantia nigra. Mice with double knockout of NMU and neuromedin S (NMS), the latter also binding to NMU receptors, showed a significant increase of the plasma prolactin level after domperidone treatment relative to wild-type mice. These results suggest that NMU and NURP may play important reciprocal roles in physiological prolactin secretion.

Purification and identification of native forms of goldfish neuromedin U from brain and gut.

Neuromedin U (NMU) plays important roles in energy homeostasis in rodents and birds. Previously, our group has isolated four cDNAs encoding precursor proteins of NMU from the goldfish brain and gut, and it was assumed that these transcripts are produced by alternative splicing. We have also demonstrated that intracerebroventricular (ICV) injection of putative goldfish NMU inhibits food intake. However, as native goldfish NMU has not yet been identified, we attempted to purify it from goldfish brain and gut extracts. To assess NMU activity in fractions at each purification step, we measured changes in the intracellular concentrations of Ca2+ using HEK293 cells expressing goldfish NMU-R1 or -R2. We isolated a 25-amino-acid peptide (NMU-25) from the brain and gut and found that its primary structure is similar to that of mammalian NMU. Another 21-amino-acid peptide (NMU-21) was purified from the brain, but not from the gut. Furthermore, a 9-amino-acid peptide (NMU-9) identical to the C-terminus of NMU-21 and -25 was also isolated from the brain and gut. Treatment with synthetic NMU-9, -21 and -25 dose-dependently increased the intracellular Ca2+ concentration in mammalian cells expressing goldfish NMU-R1 and -R2. We also examined the effect of ICV-administered synthetic goldfish NMUs on goldfish food intake. NMU-25 inhibited food intake to the same degree as NMU-21. However, the inhibitory effect of NMU-9 was slightly weaker than those of NMU-21 and -25. These results indicate that several molecular forms of NMU exist in the goldfish brain and gut, and that all of them play physiological roles via NMU-R1 and NMU-R2.

Neuromedin U precursor-related peptide (NURP) exerts neuromedin U-like sympathetic nerve action in the rat.

It has been suggested that novel peptide that is produced from the neuromedin U (NMU) precursor may exist, as this precursor contains multiple consensus sequences for proteolytic processing. Recently, we identified two mature novel peptides comprising 33 and 36 residues in the rat brain, which were designated neuromedin U precursor-related peptide (NURP) 33 and 36. In the present study, we compared the roles of NURP33 and 36 with that of NMU, as neither activates the NMU receptors. Immunoreactivity for NMU and NURPs was widely present in the central nervous system and showed a similar distribution. Intracerebroventricular (icv) injection of NURP33 in rats increased locomotor activity, energy expenditure, heart rate and back surface temperature (BS-T), similarly to NMU or NURP36. NMU treatment reduced food intake, but NURP33 did not. Pretreatment with the ß3 blocker, SR59230A, and the cyclooxygenase blocker, indomethacin, inhibited the NURP33- or NMU-induced increase of BS-T. In addition, icv injection of NURP33 or NMU increased the expression of mRNA for cyclooxygenase 2 in the hypothalamus and for uncoupling protein 1 in the brown adipose tissue. These results suggest that although NURP33 and 36 do not activate the NMU receptors, they might exert NMU-like sympathetic nerve action in the brain.

Involvement of endogenous neuromedin U and neuromedin S in thermoregulation.

We investigated the possible involvement of neuromedin U (NMU) and neuromedin S (NMS) in thermoregulation in rats. Intracerebroventricular (icv) injection of NMU or NMS increased the back surface temperature (BS-T) in a dose-dependent manner during both the light and dark periods. Pre-treatment with the ß3 blocker SR59230A, and the cyclooxygenase blocker indomethacin, inhibited the increase in BS-T induced by NMS. Icv injection of NMS and NMU increased the expression of mRNAs for prostaglandin E synthase and cyclooxygenase 2 (COX2) in the hypothalamus, and that of mRNA for uncoupling protein 1 (UCP1) in the brown adipose tissue. Comparison of thermogenesis in terms of body temperature under normal and cold conditions revealed that NMS-KO and double-KO mice had a significantly low BS-T during the active phase, whereas NMU-KO mice did not. Exposure to low temperature decreased the BS temperature in all KO mice, but BS-T was lower in NMS-KO and double-KO mouse than in NMU-KO mice. Calorie and oxygen consumption was also significantly lower in all KO mice than in wild-type mice during the dark period. These results suggest that NMU and NMS are involved in thermoregulation via the prostaglandin E2 and ß3 adrenergic receptors, but that endogenous NMS might play a more predominant role than NMU.

Periostin promotes the generation of fibrous membranes in proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is a severe, vision-threatening disorder characterized by the fibrous membrane formation that leads to tractional retinal detachment. There has been no effective therapeutic approach other than vitreoretinal surgery. In this study, DNA microarray analysis of the fibrous membranes revealed significant up-regulation of periostin. We also found increased periostin expression in the vitreous and retinal pigment epithelial (RPE) cells from fibrous membranes of PVR patients. In vitro, periostin increased proliferation, adhesion, migration, and collagen production in RPE cells through integrin αV-mediated FAK and AKT phosphorylation. Periostin blockade suppressed migration and adhesion induced by TGFß2 and PVR vitreous. In vivo, periostin inhibition had the inhibitory effect on progression of experimental PVR in rabbit eyes without affecting the viability of retinal cells. These results identified periostin as a pivotal molecule for fibrous membrane formation as well as a promising therapeutic target for PVR.

Mice heterozygous for the xanthine oxidoreductase gene facilitate lipid accumulation in adipocytes.

OBJECTIVE: Xanthine oxidoreductase (XOR) catalyzes the production of uric acid with concomitant generation of reactive oxygen species. XOR has been shown to regulate adipogenesis through the control of peroxisome proliferator-activated receptor γ, but its role in adipose tissue remains unclear. The aim of this study was to examine the role of XOR in adipose tissue using XOR genetically modified mice. APPROACH AND RESULTS: Experiments were performed using 2-, 4-, and 18-month-old XOR heterozygous mice (XOR(+/-)) and their wild-type littermates to evaluate the physiological role of XOR as the mice aged. Stromal vascular fraction cells were prepared from epididymal white adipose tissue in 2-month-old XOR mice to assess adipogenesis. At 18 months, XOR(+/)- mice had significantly higher body weight, higher systolic blood pressure, and higher incidence of insulin resistance compared with wild-type mice. At 4 months, blood glucose and the expressions of CCAAT enhancer-binding protein ß, peroxisome proliferator-activated receptor γ, monocyte chemoattractant protein-1, and tumor necrosis factor α mRNA in epididymal white adipose tissue were significantly higher in XOR(+/-) than in wild-type mice. Furthermore, histological analysis of epididymal white adipose tissue in XOR(+/-) mice revealed that adipocyte size and the F4/80-positive macrophage count were increased. Experiments with a high-fat diet exhibited that body weight gain was also significantly higher in XOR(+/-) than in wild-type mice. In stromal vascular fraction cells derived from XOR(+/-) mice, the levels of peroxisome proliferator-activated receptor γ, fatty acid-binding protein 4, and CCAAT enhancer-binding protein α mRNA were upregulated, and oxidative stress levels were elevated during differentiation into adipocytes. CONCLUSIONS: These results suggest that the reduction in XOR gene expression in mice augments lipid accumulation in adipocytes, accompanied by an increase in oxidative stress, and induces obesity with insulin resistance in older age.

Epigenetic switching by the metabolism-sensing factors in the generation of orexin neurons from mouse embryonic stem cells.

The orexin system plays a central role in the integration of sleep/wake and feeding behaviors in a broad spectrum of neural-metabolic physiology. Orexin-A and orexin-B are produced by the cleavage of prepro-orexin, which is encoded on the Hcrt gene. To date, methods for generating other peptide neurons could not induce orexin neurons from pluripotent stem cells. Considering that the metabolic status affects orexin expression, we supplemented the culture medium with a nutrient factor, ManNAc, and succeeded in generating functional orexin neurons from mouse ES cells. Because DNA methylation inhibitors and histone deacetylase inhibitors could induce Hcrt expression in mouse ES cells, the epigenetic mechanism may be involved in this orexin neurogenesis. DNA methylation analysis showed the presence of a tissue-dependent differentially methylated region (T-DMR) around the transcription start site of the Hcrt gene. In the orexin neurons induced by supplementation of ManNAc, the T-DMR of the Hcrt gene was hypomethylated in association with higher H3/H4 acetylation. Concomitantly, the histone acetyltransferases p300, CREB-binding protein (CBP), and Mgea5 (also called O-GlcNAcase) were localized to the T-DMR in the orexin neurons. In non-orexin-expressing cells, H3/H4 hypoacetylation and hyper-O-GlcNAc modification were observed at the T-DMRs occupied by O-GlcNAc transferase and Sirt1. Therefore, the results of the present study suggest that the glucose metabolite, ManNAc, induces switching from the inactive state by Ogt-Sirt1 to the active state by Mgea5, p300, and CBP at the Hcrt gene locus.

Characterization of a novel genetically obese mouse model demonstrating early onset hyperphagia and hyperleptinemia.

Obesity is a critical risk factor for the development of metabolic syndrome, and many obese animal models are used to investigate the mechanisms responsible for the appearance of symptoms. To establish a new obese mouse model, we screened ∼13,000 ICR mice and discovered a mouse demonstrating spontaneous obesity. We named this mouse "Daruma" after a traditional Japanese ornament. Following the fixation of the genotype, these animals exhibited obese phenotypes according to Mendel's law of inheritance. In the Daruma mouse, the leptin receptor gene sequence carried two base mutations that are good candidates for the variation(s) responsible for the obese phenotype. The Daruma mice developed characteristic visceral fat accumulation at 4 wk of age, and the white adipose and liver tissues exhibited increases in cell size and lipid droplets, respectively. No histological abnormalities were observed in other tissues of the Daruma mice, even after the mice reached 25 wk of age. Moreover, the onset of impaired leptin signaling was early and manifested as hyperleptinemia and hyperinsulinemia. Pair feeding completely inhibited obesity, although these mice rapidly developed hyperphagia and obesity followed by hyperleptinemia when pair feeding ceased and free-access feeding was permitted. Therefore, the Daruma mice exhibited unique characteristics and may be a good model for studying human metabolic syndrome.

Central and peripheral des-acyl ghrelin regulates body temperature in rats.

In the present study using rats, we demonstrated that central and peripheral administration of des-acyl ghrelin induced a decrease in the surface temperature of the back, and an increase in the surface temperature of the tail, although the effect of peripheral administration was less marked than that of central administration. Furthermore, these effects of centrally administered des-acyl ghrelin could not be prevented by pretreatment with [D-Lys3]-GHRP-6 GH secretagogue receptor 1a (GHS-R1a) antagonists. Moreover, these actions of des-acyl ghrelin on body temperature were inhibited by the parasympathetic nerve blocker methylscopolamine but not by the sympathetic nerve blocker timolol. Using immunohistochemistry, we confirmed that des-acyl ghrelin induced an increase of cFos expression in the median preoptic nucleus (MnPO). Additionally, we found that des-acyl ghrelin dilated the aorta and tail artery in vitro. These results indicate that centrally administered des-acyl ghrelin regulates body temperature via the parasympathetic nervous system by activating neurons in the MnPO through interactions with a specific receptor distinct from the GHS-R1a, and that peripherally administered des-acyl ghrelin acts on the central nervous system by passing through the blood-brain barrier, whereas it exerts a direct action on the peripheral vascular system.

Assessment of pulmonary arterial pressure by velocity-encoded cine magnetic resonance imaging in children with congenital heart disease.

BACKGROUND: Velocity-encoded cine magnetic resonance imaging (VEC-MRI) has recently been reported as effective for assessing not only pulmonary blood flow (Qp) but also pulmonary arterial pressure (PAP) in adults. However, there have been few reports on the usefulness of VEC-MRI for assessing PAP in children with congenital heart disease (CHD). METHODS AND RESULTS: We evaluated 34 children with CHD. Qp and systemic blood flows (Qs) were determined by cardiac catheterization and VEC-MRI. The right-to-left Qp ratio (R/L) was measured by pulmonary perfusion scintigraphy and VEC-MRI. The pulmonary-to-systemic blood pressure ratio (Pp/Ps) was determined by cardiac catheterization. The acceleration time (AcT), ejection time (ET), peak velocity (PV), acceleration volume (AcV), and maximal change in flow rate during ejection (MCFR) in the pulmonary arteries, which were standardized by body surface area, were determined by VEC-MRI. The children were divided into 2 groups according to Pp/Ps. The Qs, R/L ratio and Qp/Qs obtained by VEC-MRI strongly correlated with those obtained by catheterization and scintigraphy. No significant differences in AcT, ET, AcT/ET, PV, or AcV were observed between the 2 groups. However, a significant difference was observed in MCFR. Furthermore, a significant correlation was observed between the MCFR and Pp/Ps. CONCLUSIONS: This study clearly demonstrated that VEC-MRI is useful for assessing not only blood flow, but also PAP, by referring to MCFR in children.

Effects of the superoxide dismutase mimetic tempol on impaired endothelium-dependent and endothelium-independent relaxations in type II diabetic rats.

Endothelium-derived hyperpolarizing factor (EDHF)-mediated hyperpolarization and relaxation, and endothelium-independent relaxations to the nitric oxide donor sodium nitroprusside and the adenosine 5'-triphosphate (ATP)-sensitive K(+)-channel opener levcromakalim were both impaired in mesenteric arteries of type II diabetic Goto-Kakizaki rats. The treatment with the superoxide dismutase mimetic tempol or its combination with the angiotensin II type 1 receptor blocker candesartan failed to improve EDHF-mediated responses, although both treatments partially improved endothelium-independent relaxations. These findings suggest that increased oxidative stress may in part account for the impaired endothelium-independent relaxations in diabetes, while it does not play a major role in the impaired EDHF-mediated responses.

Somatostatin is involved in anorexia in mice fed a valine-deficient diet.

The ingestion of a valine (Val)-deficient diet results in a significant reduction of food intake and body weight within 24 h, and this phenomenon continues throughout the period over which such a diet is supplied. Both microarray and real-time PCR analyses revealed that the expression of somatostatin mRNA was increased in the hypothalamus in anorectic mice that received a Val-deficient diet. On the other hand, when somatostatin was administered intracerebroventricularly to intact animals that were fed a control diet, their 24-h food intake decreased significantly. In addition, Val-deficient but not pair-fed mice or those fasted for 24 h showed a less than 0.5-fold decrease in the hypothalamic mRNA expression levels of Crym, Foxg1, Itpka and two unknown EST clone genes and a more than twofold increase in those of Slc6a3, Bdh1, Ptgr2 and one unknown EST clone gene. These results suggest that hypothalamic somatostatin and genes responsive to Val deficiency may be involved in the central mechanism of anorexia induced by a Val-deficient diet.

Peripheral ghrelin transmits orexigenic signals through the noradrenergic pathway from the hindbrain to the hypothalamus.

Ghrelin, a gastrointestinal peptide, stimulates feeding when administered peripherally. Blockade of the vagal afferent pathway abolishes ghrelin-induced feeding, indicating that the vagal afferent pathway may be a route conveying orexigenic ghrelin signals to the brain. Here, we demonstrate that peripheral ghrelin signaling, which travels to the nucleus tractus solitarius (NTS) at least in part via the vagus nerve, increases noradrenaline (NA) in the arcuate nucleus of the hypothalamus, thereby stimulating feeding at least partially through alpha-1 and beta-2 noradrenergic receptors. In addition, bilateral midbrain transections rostral to the NTS, or toxin-induced loss of neurons in the hindbrain that express dopamine beta hydroxylase (an NA synthetic enzyme), abolished ghrelin-induced feeding. These findings provide new evidence that the noradrenergic system is necessary in the central control of feeding behavior by peripherally administered ghrelin.

Identification of the endogenous cysteine-rich peptide trissin, a ligand for an orphan G protein-coupled receptor in Drosophila.

There are many orphan G protein-coupled receptors (GPCRs), for which ligands have not yet been identified, in both vertebrates and invertebrates, such as Drosophila melanogaster. Identification of their cognate ligands is critical for understanding the function and regulation of such GPCRs. Indeed, the discovery of bioactive peptides that bind GPCRs has enhanced our understanding of mechanisms underlying many physiological processes. Here, we identified an endogenous ligand of the Drosophila orphan GPCR, CG34381. The purified ligand is a peptide comprised of 28 amino acids with three intrachain disulfide bonds. The preprotein is coded for by gene CG14871. We designated the cysteine-rich peptide "trissin" (it means for triple S-S bonds) and characterized the structure of intrachain disulfide bonds formation in a synthetic trissin peptide. Because the expression of trissin and its receptor is reported to predominantly localize to the brain and thoracicoabdominal ganglion, trissin is expected to behave as a neuropeptide. The discovery of trissin provides an important lead to aid our understanding of cysteine-rich peptides and their functional interaction with GPCRs.

Identification of the novel bioactive peptides dRYamide-1 and dRYamide-2, ligands for a neuropeptide Y-like receptor in Drosophila.

A number of bioactive peptides are involved in regulating a wide range of animal behaviors, including food consumption. Vertebrate neuropeptide Y (NPY) is a potent stimulator of appetitive behavior. Recently, Drosophila neuropeptide F (dNPF) and short NPF (sNPF), the Drosophila homologs of the vertebrate NPY, were identified to characterize the functions of NPFs in the feeding behaviors of this insect. Dm-NPFR1 and NPFR76F are the receptors for dNPF and sNPF, respectively; both receptors are G protein-coupled receptors (GPCRs). Another GPCR (CG5811; NepYR) was indentified in Drosophila as a neuropeptide Y-like receptor. Here, we identified 2 ligands of CG5811, dRYamide-1 and dRYamide-2. Both peptides are derived from the same precursor (CG40733) and have no significant structural similarities to known bioactive peptides. The C-terminal sequence RYamide of dRYamides is identical to that of NPY family peptides; on the other hand, dNPF and sNPF have C-terminal RFamide. When administered to blowflies, dRYamide-1 suppressed feeding motivation. We propose that dRYamides are related to the NPY family in vertebrates, similar to dNPF and sNPF.

Neuromedin U has a novel anorexigenic effect independent of the leptin signaling pathway.

Neuromedin U (NMU) is a hypothalamic neuropeptide that regulates body weight and composition. Here we show that mice lacking the gene encoding NMU (Nmu(-/-) mice) develop obesity. Nmu(-/-) mice showed increased body weight and adiposity, hyperphagia, and decreased locomotor activity and energy expenditure. Obese Nmu(-/-) mice developed hyperleptinemia, hyperinsulinemia, late-onset hyperglycemia and hyperlipidemia. Notably, however, treatment with exogenous leptin was effective in reducing body weight in obese Nmu(-/-) mice. In addition, central leptin administration did not affect NMU gene expression in the hypothalamus of rats. These results indicate that NMU plays an important role in the regulation of feeding behavior and energy metabolism independent of the leptin signaling pathway. These characteristic functions of NMU may provide new insight for understanding the pathophysiological basis of obesity.

Transitional change in rat fetal cell proliferation in response to ghrelin and des-acyl ghrelin during the last stage of pregnancy.

Expression of mRNA for the ghrelin receptor, GHS-R1a, was detected in various peripheral and central tissues of fetal rats, including skin, bone, heart, liver, gut, brain and spinal cord, on embryonic day (ED)15 and ED17. However, its expression in skin, bone, heart and liver, but not in gut, brain and spinal cord, became relatively weak on ED19 and disappeared after birth (ND2). Ghrelin and des-acyl ghrelin facilitated the proliferation of cultured fetal (ED17, 19), but not neonatal (ND2), skin cells. On the other hand, with regard to cells from the spinal cord and hypothalamus, the proliferative effect of ghrelin continued after birth, whereas the effect of des-acyl ghrelin on neurogenesis in these tissues was lost at the ED19 fetal and ND2 neonatal stages. Immunohistochemistry revealed that the cells in the hypothalamus induced to proliferate by ghrelin at the ND2 stage were positive for nestin and glial fibrillary acidic protein. These results suggest that in the period immediately prior to, and after birth, rat fetal cells showing proliferation in response to ghrelin and des-acyl ghrelin are at a transitional stage characterized by alteration of the expression of GHS-R1a and an undefined des-acyl ghrelin receptor, their responsiveness varying among different tissues.

Variation in fecal testosterone hormone concentration with season and harem size in Misaki feral horses.

On Misaki peninsula, Japan, fecal samples were collected from 14 Misaki stallions at monthly intervals for 12 consecutive months. The fecal testosterone concentration was measured by radioimmunoassay. We examined monthly fecal testosterone hormone patterns and the relationship between fecal testosterone concentration and breeding season and later harem size. Marked monthly variations in fecal testosterone concentration were observed. The fecal testosterone concentration began rising in March; the highest mean monthly concentration, 2.87 +/- 0.18 ng/g, was found in April, and the level remained elevated until the end of August and thereafter decreased. A significant correlation was found between the fecal testosterone concentrations and harem size in both the breeding and non-breeding season among the 14 stallions. It is therefore possible that the testosterone levels in feces, instead of blood, correlate very well with harem size in Misaki stallions. Our findings emphasized that the fecal testosterone concentration can be a powerful indicator for monitoring of endocrine status in wild stallions.

Comparison of glucose tolerance between wild-type mice and mice with double knockout of neuromedin U and neuromedin S.

Recently, it has been proposed that neuromedin U (NMU) is "decretin", which suppresses insulin secretion from the pancreas in vitro. Here we examined the possible involvement of NMU in insulin secretion in vivo by comparing the plasma glucose and insulin levels of wild-type mice with those of double knockout (D-KO) of the NMU and neuromedin S (NMS) genes, as NMS binds to the neuromedin U receptor. If NMU is, in fact, "decretin", which inhibits insulin secretion from the pancreas, then NMU-deficient mice might result in higher plasma insulin levels than is the case in wild-type mice, or injection of NMU lead to suppression of plasma insulin level. In this study, we found that the fasting plasma level of insulin was not increased in D-KO mice. Glucose tolerance tests revealed no significant difference in plasma insulin levels between wild-type mice and D-KO mice under non-fasting conditions. After peripheral injection of NMU, plasma glucose and insulin levels did not show any significant changes in either wild-type or D-KO mice. Glucose tolerance testing after 3 weeks of high fat feeding revealed no significant difference in plasma insulin levels during 60 min after glucose injection between wild-type and D-KO mice. These results suggest that even if NMU is a decretin candidate, its physiological involvement in suppression of insulin secretion may be very minor in vivo.