The brain hypothalamus contains certain secreted molecules that are important in regulating feeding behaviour. Here we show that <em>nesfatin</em>, corresponding to NEFA/nucleobindin2 (NUCB2), a secreted protein of unknown function, is expressed in the appetite-control hypothalamic nuclei in rats. Intracerebroventricular (i.c.v.) injection of NUCB2 reduces feeding. Rat cerebrospinal fluid contains <em>nesfatin</em>-<em>1</em>, an amino-terminal fragment derived from NUCB2, and its expression is decreased in the hypothalamic paraventricular nucleus under starved conditions. I.c.v. injection of <em>nesfatin</em>-<em>1</em> decreases food intake in a dose-dependent manner, whereas injection of an antibody neutralizing <em>nesfatin</em>-<em>1</em> stimulates appetite. In contrast, i.c.v. injection of other possible fragments processed from NUCB2 does not promote satiety, and conversion of NUCB2 to <em>nesfatin</em>-<em>1</em> is necessary to induce feeding suppression. Chronic i.c.v. injection of <em>nesfatin</em>-<em>1</em> reduces body weight, whereas rats gain body weight after chronic i.c.v. injection of antisense morpholino oligonucleotide against the gene encoding NUCB2. <em>Nesfatin</em>-<em>1</em>-induced anorexia occurs in Zucker rats with a leptin receptor mutation, and an anti-<em>nesfatin</em>-<em>1</em> antibody does not block leptin-induced anorexia. In contrast, central injection of alpha-melanocyte-stimulating hormone elevates NUCB2 gene expression in the paraventricular nucleus, and satiety by <em>nesfatin</em>-<em>1</em> is abolished by an antagonist of the melanocortin-3/4 receptor. We identify <em>nesfatin</em>-<em>1</em> as a satiety molecule that is associated with melanocortin signalling in the hypothalamus.

The hypothalamic paraventricular nucleus (PVN) functions as a center to integrate various neuronal activities for regulating feeding behavior. <em>Nesfatin</em>-<em>1</em>, a recently discovered anorectic molecule, is localized in the PVN. However, the anorectic neural pathway of <em>nesfatin</em>-<em>1</em> remains unknown. Here we show that central injection of <em>nesfatin</em>-<em>1</em> activates the PVN and brain stem nucleus tractus solitarius (NTS). In the PVN, <em>nesfatin</em>-<em>1</em> targets both magnocellular and parvocellular oxytocin neurons and <em>nesfatin</em>-<em>1</em> neurons themselves and stimulates oxytocin release. Immunoelectron micrographs reveal <em>nesfatin</em>-<em>1</em> specifically in the secretory vesicles of PVN neurons, and immunoneutralization against endogenous <em>nesfatin</em>-<em>1</em> suppresses oxytocin release in the PVN, suggesting paracrine/autocrine actions of <em>nesfatin</em>-<em>1</em>. <em>Nesfatin</em>-<em>1</em>-induced anorexia is abolished by an oxytocin receptor antagonist. Moreover, oxytocin terminals are closely associated with and oxytocin activates pro-opiomelanocortin neurons in the NTS. Oxytocin induces melanocortin-dependent anorexia in leptin-resistant Zucker-fatty rats. The present results reveal the <em>nesfatin</em>-<em>1</em>-operative oxytocinergic signaling in the PVN that triggers leptin-independent melanocortin-mediated anorexia.

<em>Nesfatin</em>-<em>1</em> is a recently identified satiety molecule detectable in neurons of the hypothalamus and nucleus of solitary tract (NTS). Immunohistochemical studies revealed <em>nesfatin</em>-<em>1</em>-immunoreactive (irNEF) cells in the Edinger-Westphal nucleus, dorsal motor nucleus of vagus, and caudal raphe nuclei of the rats, in addition to the hypothalamus and NTS reported in the initial study. Double-labeling immunohistochemistry showed that irNEF cells were vasopressin or oxytocin positive in the paraventricular and supraoptic nucleus; cocaine-amphetamine-regulated transcript or tyrosine hydroxylase positive in arcuate nucleus; cocaine-amphetamine-regulated transcript or melanin concentrating hormone positive in the lateral hypothalamus. In the brainstem, irNEF neurons were choline acetyltransferase positive in the Edinger-Westphal nucleus and dorsal motor nucleus of vagus; tyrosine hydroxylase positive in the NTS; and 5-hydroxytryptamine positive in the caudal raphe nucleus. The biological activity of rat <em>nesfatin</em>-<em>1</em> (<em>1</em>-82) (<em>1</em>00 nm) was assessed by the Ca(2+) microfluorometric method. <em>Nesfatin</em>-<em>1</em> elevated intracellular Ca(2+) concentrations [Ca(2+)](i) in dissociated and cultured hypothalamic neurons. The response was prevented by pretreating the cells with pertussis toxin (<em>1</em>00 nm) or Ca(2+)-free solution and by a combination of the L-type and P/Q-type calcium channel blocker verapamil (<em>1</em> microm) and omega-conotoxin MVIIC (<em>1</em>00 nm). The protein kinase A inhibitor KT 5720 (<em>1</em> microm) suppressed <em>nesfatin</em>-<em>1</em>-induced rise in [Ca(2+)](i). The result shows that irNEF is distributed to several discrete nuclei in the brainstem, in addition to the hypothalamus and NTS reported earlier, and that the peptide interacts with a G protein-coupled receptor, leading to an increase of [Ca(2+)](i), which is linked to protein kinase A activation in cultured rat hypothalamic neurons.

<em>Nesfatin</em>-<em>1</em> is a novel satiety molecule in the hypothalamus and is also present in peripheral tissues. Here we sought to identify the active segment of <em>nesfatin</em>-<em>1</em> and to determine the mechanisms of its action after peripheral administration in mice. Intraperitoneal injection of <em>nesfatin</em>-<em>1</em> suppressed food intake in a dose-dependent manner. <em>Nesfatin</em>-<em>1</em> has three distinct segments; we tested the effect of each segment on food intake. Injection of the midsegment decreased food intake under leptin-resistant conditions such as db/db mice and mice fed a high-fat diet. After injection of the midsegment, expression of c-Fos was significantly activated in the brainstem nucleus tractus solitarius (NTS) but not in the hypothalamic arcuate nucleus; the nicotinic cholinergic pathway to the NTS contributed to midsegment-induced anorexia. Midsegment injection significantly increased expression of proopiomelanocortin and cocaine- and amphetamine-regulated transcript genes in the NTS but not in the arcuate nucleus. Investigation of mutant midsegments demonstrated that a region with amino acid sequence similarity to the active site of agouti-related peptide was indispensable for anorexigenic induction. Our findings indicate that the midsegment of <em>nesfatin</em>-<em>1</em> causes anorexia, possibly by activating POMC and CART neurons in the NTS via a leptin-independent mechanism after peripheral stimulation.

Hypothalamic <em>nesfatin</em>-<em>1</em>, derived from the nucleobindin2 (NUCB2) precursor, inhibits nocturnal food intake and body weight gain in rats. <em>Nesfatin</em>-<em>1</em> is able to cross the blood-brain barrier, suggesting a peripheral source of <em>nesfatin</em>-<em>1</em>. Many centrally acting food intake regulatory neuropeptides are also produced in the periphery, especially in the gastrointestinal tract. Therefore, we investigated the gene expression of NUCB2 and distribution of <em>nesfatin</em>-<em>1</em>-immunoreactive cells in the stomach. Microarray mRNA expression profiles in purified small endocrine cells of the gastric mucosa substantiated by quantitative RT-PCR showed significantly higher NUCB2 mRNA expression compared with brain and heart. Western blot confirmed the expression of NUCB2 protein and its transport into a secretory soluble fraction of gastric mucosal endocrine cell homogenates. Immunohistochemical colabeling for <em>nesfatin</em>-<em>1</em> and ghrelin, histidine decarboxylase, or somatostatin revealed two subtypes of <em>nesfatin</em>-<em>1</em>-positive endocrine cells. Cells in the midportion of the glands coexpressed <em>nesfatin</em>-<em>1</em> and ghrelin, whereas few cells in the glandular base coexpressed <em>nesfatin</em>-<em>1</em> and somatostatin or histidine decarboxylase. High-resolution three-dimensional volume imaging revealed two separate populations of intracytoplasmic vesicles in these cells, one containing <em>nesfatin</em>-<em>1</em> and the other ghrelin immunoreactivity. Microarray rat genome expression data of NUCB2 in small gastric endocrine cells confirmed by quantitative RT-PCR showed significant down-regulation of NUCB2 after 24 h fasting. In summary, NUCB2 mRNA expression as well as protein content is present in a specific subset of gastric endocrine cells, most of which coexpress ghrelin. NUCB2 gene expression is significantly regulated by nutritional status, suggesting a regulatory role of peripheral <em>nesfatin</em>-<em>1</em> in energy homeostasis.

<em>Nesfatin</em>-<em>1</em>, derived from nucleobindin2, is expressed in the hypothalamus and reported in one study to reduce food intake (FI) in rats. To characterize the central anorexigenic action of <em>nesfatin</em>-<em>1</em> and whether gastric emptying (GE) is altered, we injected <em>nesfatin</em>-<em>1</em> into the lateral brain ventricle (intracerebroventricular, icv) or fourth ventricle (4v) in chronically cannulated rats or into the cisterna magna (intracisternal, ic) under short anesthesia and compared with ip injection. <em>Nesfatin</em>-<em>1</em> (0.05 microg/rat, icv) decreased 2-3 h and 3-6 h dark-phase FI by 87 and 45%, respectively, whereas ip administration (2 microg/rat) had no effect. The corticotropin-releasing factor (CRF)(<em>1</em>)/CRF(2) antagonist astressin-B or the CRF(2) antagonist astressin(2)-B abolished icv <em>nesfatin</em>-<em>1</em>'s anorexigenic action, whereas an astressin(2)-B analog, devoid of CRF-receptor binding affinity, did not. <em>Nesfatin</em>-<em>1</em> icv induced a dose-dependent reduction of GE by 26 and 43% that was not modified by icv astressin(2)-B. <em>Nesfatin</em>-<em>1</em> into the 4v (0.05 microg/rat) or ic (0.5 microg/rat) decreased cumulative dark-phase FI by 29 and 60% at <em>1</em> h and by 4<em>1</em> and 37% between 3 and 5 h, respectively. This effect was neither altered by ic astressin(2)-B nor associated with changes in GE. Cholecystokinin (ip) induced Fos expression in 43% of <em>nesfatin</em>-<em>1</em> neurons in the paraventricular hypothalamic nucleus and 24% of those in the nucleus tractus solitarius. These data indicate that <em>nesfatin</em>-<em>1</em> acts centrally to reduce dark phase FI through CRF(2)-receptor-dependent pathways after forebrain injection and CRF(2)-receptor-independent pathways after hindbrain injection. Activation of <em>nesfatin</em>-<em>1</em> neurons by cholecystokinin at sites regulating food intake may suggest a role in gut peptide satiation effect.

The protein fragment <em>nesfatin</em>-<em>1</em> was recently implicated in the control of food intake. Central administration of this fragment results in anorexia and reduced body weight gain, whereas antisense or immunological <em>nesfatin</em>-<em>1</em> antagonism causes increased food intake and overweight. <em>Nesfatin</em>-<em>1</em> is derived from the precursor nucleobindin-2 (NUCB2). To identify the neurocircuitry underpinning the catabolic effects of NUCB2/<em>nesfatin</em>-<em>1</em>, we have used in situ hybridization and immunohistochemistry to map the distribution of this protein and its mRNA in the rat CNS and performed double-labeling experiments to localize its expression to functionally defined neuronal populations. These experiments confirm previous observations but also present several novel NUCB2 cell populations. Both NUCB2 mRNA and <em>nesfatin</em>-like immunoreactivity was most concentrated in the hypothalamus, in the supraoptic, paraventricular, periventricular and arcuate nuclei and the lateral hypothalamic area/perifornical region. Additionally, outside of the hypothalamus, labeling was observed in the thalamic parafascicular nucleus, the Edinger-Westphal nucleus, locus coeruleus, ventral raphe system, nucleus of solitary tract and in the preganglionic sympathetic intermediolateral cell column of the spinal cord, and the pituitary anterior and intermediate lobes. In neurons, immunoreactivity was almost exclusively confined to perikarya and primary dendrites with virtually no labeling of axonal terminals. Double-labeling immunohistochemistry revealed colocalization of <em>nesfatin</em> with vasopressin and oxytocin in magnocellular neuroendocrine neurons, thyrotropin-releasing hormone, corticotropin-releasing hormone, somatostatin, neurotensin, and growth-hormone-releasing hormone in parvocellular neuroendocrine neurons, pro-opiomelanocortin (but not neuropeptide Y) in the arcuate nucleus and melanin-concentrating hormone (but not hypocretin) in the lateral hypothalamus. Furthermore, <em>nesfatin</em> was extensively colocalized with cocaine- and amphetamine-regulated transcript in almost all NUCB2-expressing brain regions. These data reveal a wider distribution of NUCB2/<em>nesfatin</em>-<em>1</em> than previously known, suggesting that the metabolic actions of this protein may involve not only feeding behavior but also endocrine and autonomic effects on energy expenditure. In addition, the subcellular distribution of <em>nesfatin</em>-like immunoreactivity indicates that this protein may not be processed like a conventional secreted neuromodulator.

<em>Nesfatin</em>-<em>1</em>, a newly discovered satiety molecule, is located in the hypothalamic nuclei, including the paraventricular nucleus (PVN) and supraoptic nucleus (SON). In this study, fine localization and regulation of <em>nesfatin</em>-<em>1</em> neurons in the PVN and SON were investigated by immunohistochemistry of neuropeptides and c-Fos. In the PVN, 24% of <em>nesfatin</em>-<em>1</em> neurons overlapped with oxytocin, <em>1</em>8% with vasopressin, <em>1</em>3% with CRH, and <em>1</em>2% with TRH neurons. In the SON, 35% of <em>nesfatin</em>-<em>1</em> neurons overlapped with oxytocin and 28% with vasopressin. After a 48-h fast, refeeding for 2 h dramatically increased the number of <em>nesfatin</em>-<em>1</em> neurons expressing c-Fos immunoreactivity by approximately <em>1</em>0 times in the PVN and 30 times in the SON, compared with the fasting controls. In the SON, refeeding also significantly increased the number of <em>nesfatin</em>-<em>1</em>-immunoreactive neurons and NUCB2 mRNA expression, compared with fasting. These results indicate that <em>nesfatin</em>-<em>1</em> neurons in the PVN and SON highly overlap with oxytocin and vasopressin neurons and that they are activated markedly by refeeding. Feeding-activated <em>nesfatin</em>-<em>1</em> neurons in the PVN and SON could play a role in the postprandial regulation of feeding behavior and energy homeostasis.

<em>Nesfatin</em>-<em>1</em> is a recently identified anorexigenic peptide derived from its precursor protein, nonesterified fatty acid/nucleobindin 2 (NUCB2). Although the hypothalamus is pivotal for the maintenance of energy homeostasis, adipose tissue plays an important role in the integration of metabolic activity and energy balance by communicating with peripheral organs and the brain via adipokines. Currently no data exist on <em>nesfatin</em>-<em>1</em> expression, regulation, and secretion in adipose tissue. We therefore investigated NUCB2/<em>nesfatin</em>-<em>1</em> gene and protein expression in human and murine adipose tissue depots. Additionally, the effects of insulin, dexamethasone, and inflammatory cytokines and the impact of food deprivation and obesity on <em>nesfatin</em>-<em>1</em> expression were studied by quantitative RT-PCR and Western blotting. We present data showing NUCB2 mRNA (P < 0.00<em>1</em>), <em>nesfatin</em>-<em>1</em> intracellular protein (P < 0.00<em>1</em>), and secretion (P < 0.0<em>1</em>) were significantly higher in sc adipose tissue compared with other depots. Also, <em>nesfatin</em>-<em>1</em> protein expression was significantly increased in high-fat-fed mice (P < 0.0<em>1</em>) and reduced under food deprivation (P < 0.0<em>1</em>) compared with controls. Stimulation of sc adipose tissue explants with inflammatory cytokines (TNFalpha and IL-6), insulin, and dexamethasone resulted in a marked increase in intracellular <em>nesfatin</em>-<em>1</em> levels. Furthermore, we present evidence that the secretion of <em>nesfatin</em>-<em>1</em> into the culture media was dramatically increased during the differentiation of 3T3-L<em>1</em> preadipocytes into adipocytes (P < 0.00<em>1</em>) and after treatments with TNF-alpha, IL-6, insulin, and dexamethasone (P < 0.0<em>1</em>). In addition, circulating <em>nesfatin</em>-<em>1</em> levels were higher in high-fat-fed mice (P < 0.05) and showed positive correlation with body mass index in human. We report that <em>nesfatin</em>-<em>1</em> is a novel depot specific adipokine preferentially produced by sc tissue, with obesity- and food deprivation-regulated expression.

<em>Nesfatin</em>-<em>1</em> is an 82 amino acid peptide that suppresses food intake after intracerebroventricular injection. <em>Nesfatin</em>-<em>1</em> and its precursor NUCB2 were identified by subtraction cloning in cell lines of both neuronal and adipocytic origin. This provides a strong basis for studies to determine how peripherally derived <em>nesfatin</em>-<em>1</em> permeates the blood-brain barrier (BBB) to participate in its CNS actions and whether pharmacological delivery by the peripheral route is feasible. In this study, <em>nesfatin</em>-<em>1</em> remained stable in blood at least 20 min after intravenous injection and permeated the BBB by a non-saturable mechanism. The influx rate of <em>nesfatin</em>-<em>1</em> after intravenous delivery was 0.27+/-0.<em>1</em><em>1</em> microl/g-min, and 0.3% of <em>nesfatin</em>-<em>1</em> reached brain parenchyma <em>1</em>0 min after injection. The lack of saturation of influx was shown by use of excess unlabeled <em>nesfatin</em>-<em>1</em> in multiple-time regression analysis, capillary depletion, and in situ brain perfusion. After intracerebroventricular injection, <em>nesfatin</em>-<em>1</em> had a half-time disappearance of 23.8 min, which was not significantly different from that of albumin. This indicates that <em>nesfatin</em>-<em>1</em> exited the brain by bulk absorption of cerebrospinal fluid without a specific efflux transport system. We conclude that the permeation of <em>nesfatin</em>-<em>1</em> is a non-saturable process in either the blood-to-brain or brain-to-blood direction. Thus, the limited penetration under physiological conditions does not limit the pharmacological delivery of this satiety peptide as a potential therapeutic agent.

Studies showed that the metabolic unlike the neuroendocrine effects of ghrelin could be abrogated by co-administered unacylated ghrelin. The aim was to investigate the interaction between ghrelin and desacyl ghrelin administered intraperitoneally on food intake and neuronal activity (c-Fos) in the arcuate nucleus in non-fasted rats. Ghrelin (<em>1</em>3 microg/kg) significantly increased food intake within the first 30 min post-injection. Desacyl ghrelin at 64 and <em>1</em>27 microg/kg injected simultaneously with ghrelin abolished the stimulatory effect of ghrelin on food intake. Desacyl ghrelin alone at both doses did not alter food intake. Both doses of desacyl ghrelin injected separately in the light phase had no effects on food intake when rats were fasted for <em>1</em>2h. Ghrelin and desacyl ghrelin (64 microg/kg) injected alone increased the number of Fos positive neurons in the arcuate nucleus compared to vehicle. The effect on neuronal activity induced by ghrelin was significantly reduced when injected simultaneously with desacyl ghrelin. Double labeling revealed that <em>nesfatin</em>-<em>1</em> immunoreactive neurons in the arcuate nucleus are activated by simultaneous injection of ghrelin and desacyl ghrelin. These results suggest that desacyl ghrelin suppresses ghrelin-induced food intake by curbing ghrelin-induced increased neuronal activity in the arcuate nucleus and recruiting <em>nesfatin</em>-<em>1</em> immunopositive neurons.

<em>Nesfatin</em>-<em>1</em> is an 82-amino acid protein encoded by the nucleobindin2 gene. When injected intracerebroventricularly, <em>nesfatin</em>-<em>1</em>, via a melanocortin 3/4 receptor-dependent mechanism, potently decreased both food and water intakes and elevated mean arterial pressure in a dose-related manner. Because <em>nesfatin</em>-<em>1</em> colocalized with oxytocin in hypothalamus and because <em>nesfatin</em>-<em>1</em> had direct depolarizing effects on oxytocin-producing neurons in hypothalamic slice preparations, we hypothesized that the actions of <em>nesfatin</em>-<em>1</em> required the presence of functional oxytocin receptors. We, therefore, pretreated conscious, unrestrained male rats with the oxytocin receptor antagonist, ornithine vasotocin (OVT), before treatment with <em>nesfatin</em>-<em>1</em>. We found that pretreatment with OVT reversed the effects of <em>nesfatin</em>-<em>1</em> on both food and water intake and on mean arterial pressure, indicating that the central oxytocin system is a downstream mediator of these actions of <em>nesfatin</em>-<em>1</em>. Additionally, we found that OVT reversed the anorexigenic effect of alpha-melanocyte-stimulating hormone (alpha-MSH), suggesting that the central oxytocin system is downstream of the central melanocortin system. Taken together, these data suggest that <em>nesfatin</em>-<em>1</em> acts through a serial neuronal circuit, in which <em>nesfatin</em>-<em>1</em> activates the central melanocortin system, which, in turn, acts through the central oxytocin system, leading to an inhibition of food and water intake and an increase in mean arterial pressure.

<em>Nesfatin</em>-<em>1</em> is a recently discovered hypothalamic peptide that was shown to suppress food intake through a melanocortin-3/4 receptor-dependent mechanism. Since <em>nesfatin</em>-<em>1</em> mRNA is detected in the paraventricular nucleus of the hypothalamus, and because many peptides that alter food intake also influence cardiovascular function, we tested the ability of centrally administered <em>nesfatin</em>-<em>1</em> to affect mean arterial pressure (MAP) in conscious, freely moving rats. Significant increases in MAP were observed following intracerebroventricular administration of <em>nesfatin</em>-<em>1</em>. Pretreatment with either the melanocortin-3/4 receptor antagonist, SHU9<em>1</em><em>1</em>9 (intracerebroventricular), or the alpha-adrenergic antagonist, phentolamine (intra-arterial), abrogated the rise in MAP induced by <em>nesfatin</em>-<em>1</em>, indicating that <em>nesfatin</em>-<em>1</em> may interact with the central melanocortin system to increase sympathetic nerve activity and lead to an increase in MAP. Thus we have identified a novel action of <em>nesfatin</em>-<em>1</em>, in addition to its anorexigenic effects, to stimulate autonomic nervous system activity.

Numerous peptides released from endocrine cells in the intestinal mucosa were established early on to be involved in the physiological regulation of food intake with a prominent role in termination of food ingestion when nutrients pass along the intestinal tract. Recently, peptides released from X/A-like endocrine cells of the gastric oxyntic mucosa were recognized as additional key players in the regulation of feeding and energy expenditure. Gastric X/A-like cells release the octanoylated peptide, ghrelin, the only known peripherally produced hormone stimulating food intake through interaction with growth hormone secretagogue <em>1</em>a receptor (GHS-R<em>1</em>a). Additionally, non-octanoylated (des-acyl) ghrelin present in the circulation at higher levels than ghrelin is currently discussed as potential modulator of food intake by opposing ghrelin's action independent from GHS-R<em>1</em>a although the functional significance remains to be established. Obestatin, a ghrelin-associated peptide was initially reported as anorexigenic modulator of ghrelin's orexigenic action. However, subsequent reports did not support this contention. Interesting is the recent identification of <em>nesfatin</em>-<em>1</em>, a peptide derived from the nucleobindin2 gene prominently expressed in gastric X/A-like cells in different vesicles than ghrelin. Circulating <em>nesfatin</em>-<em>1</em> levels vary with metabolic state and peripheral or central injection inhibits dark phase feeding in rodents. Overall, these data point to an important role of gastric X/A-like cells in food intake regulation through the expression of the orexigenic peptide ghrelin along with des-acyl ghrelin and <em>nesfatin</em>-<em>1</em> capable of reducing food intake upon exogenous injection although their mechanisms of action and functional significance remain to be established.

<em>Nesfatin</em>-<em>1</em> has recently been identified as a hypothalamic and brain stem peptide that regulates feeding behavior. Here, we determined the ability of <em>nesfatin</em>-<em>1</em> to cross the blood-brain barrier (BBB) of mice. We used multiple-regression analysis to determine that radioactively labeled <em>nesfatin</em>-<em>1</em> injected intravenously entered the brain. The entry rate (K(i)) of (<em>1</em>3<em>1</em>)I-<em>nesfatin</em>-<em>1</em> from blood-to-brain was 0.20+/-0.02 microl/g min. This modest rate of entry was not inhibited by the administration of nonradioactive <em>nesfatin</em>-<em>1</em>, suggesting that BBB transport of <em>nesfatin</em>-<em>1</em> into the brain is by a nonsaturable mechanism. High performance liquid chromatography (HPLC) and acid precipitation showed that most of the injected radiolabeled <em>nesfatin</em>-<em>1</em> reached the brain as intact peptide, and capillary depletion with vascular washout revealed that 67% of (<em>1</em>3<em>1</em>)I-<em>nesfatin</em>-<em>1</em> crossed the BBB to reach the brain parenchyma. Efflux of labeled <em>nesfatin</em>-<em>1</em> from brain back into blood was by way of bulk flow. These findings demonstrate that <em>nesfatin</em>-<em>1</em> crosses the BBB in both the blood-to-brain and brain-to-blood directions by nonsaturable mechanisms.

<em>Nesfatin</em>-<em>1</em> is one of the peptide products of posttranslational processing of the nucleobindin-2 (NUCB2) gene, suggested to have physiological relevance to suppress food intake and body weight gain in rats. <em>Nesfatin</em>-<em>1</em>-immunoreactive cells have been found in distinct nuclei in the rat brain related to circuitries regulating food intake. Here, we report novel yet undescribed localization of NUCB2/<em>nesfatin</em>-<em>1</em> at the mRNA and protein level in the rat central nervous system. Immunohistochemical staining revealed the localization of NUCB2/<em>nesfatin</em>-<em>1</em> in the piriform and insular cortex, endopiriform nucleus, nucleus accumbens, lateral septum, bed nucleus of stria terminalis, central amygdaloid nucleus, medial preoptic area, dorsal raphe nucleus, ambiguus nucleus, ventrolateral medulla and gigantocellular reticular nucleus, as well as Purkinje-cells of the cerebellum. In the spinal cord, <em>nesfatin</em>-<em>1</em> immunoreactivity (IR) was found in both sympathetic and parasympathetic preganglionic neuronal groups and in the dorsal area X from lower thoracic to sacral segments. The immunohistochemical results were confirmed by RT-PCR in the central amygdaloid nucleus, nucleus accumbens, cerebellum and lumbar spinal cord microdissected by punch technique. The features and distributions of <em>nesfatin</em>-<em>1</em> IR and mRNA expression in the brain and spinal cord suggest that NUCB2/<em>nesfatin</em>-<em>1</em> could play a wider role in autonomic regulation of visceral-endocrine functions besides food intake.

<em>Nesfatin</em>-<em>1</em> is recently reported as a satiety molecule to suppress food intake via the melanocortin signaling in hypothalamus when injected centrally and peripherally. Here we report that <em>nesfatin</em>-<em>1</em> is also anti-hyperglycemic. It was found that the intravenous injection of <em>nesfatin</em>-<em>1</em> significantly reduced blood glucose in hyperglycemic db/db mice. This anti-hyperglycemic effect of <em>nesfatin</em>-<em>1</em> was time-, dose-, insulin-dependent and peripheral.

The novel satiety factor <em>nesfatin</em>-<em>1</em> has been shown to decrease food intake and body weight in rodents after i.c.v. injection. However, no further developments regarding the true patho-physiological relevance of <em>nesfatin</em>-<em>1</em> in obesity and type <em>1</em> diabetes mellitus (T<em>1</em> DM) and type 2 diabetes mellitus (T2 DM) have been reported. A recent study by Stengel et al. demonstrated that a down-regulation of NUCB2 mRNA in gastric endocrine cells was observed after 24-h fasting. They raised the possibility that <em>nesfatin</em>/NUCB2 gene expression may be regulated by nutritional status, suggesting that <em>nesfatin</em>-<em>1</em> in the stomach might play a role in satiety. In the present study, fasting levels in plasma <em>nesfatin</em>-<em>1</em>, insulin and glucose were measured and analyzed in healthy subjects and in patients with T<em>1</em> DM and T2 DM. Plasma <em>nesfatin</em>-<em>1</em> levels were measured 6 times before and after oral glucose ingestion in healthy subjects. No sex differences in plasma <em>nesfatin</em>-<em>1</em> were found. The mean fasting plasma <em>nesfatin</em>-<em>1</em> levels were slightly but not significantly higher in T<em>1</em> DM patients compared to healthy subjects. However, fasting plasma <em>nesfatin</em>-<em>1</em> levels were significantly lower in T2 DM patients compared to healthy subjects and T<em>1</em> DM patients. Plasma <em>nesfatin</em>-<em>1</em> did not change acutely, although a small rise in circulating <em>nesfatin</em>-<em>1</em> occurred within 30 min after the beginning of an oral glucose ingestion (from a mean basal value of 0.99+/-0.23 ng/ml to a maximum of <em>1</em>.08+/-0.24 ng/ml). No significant difference in plasma <em>nesfatin</em>-<em>1</em> before and after an oral glucose was observed. In conclusion, we showed that fasting <em>nesfatin</em>-<em>1</em> was significantly lower in T2 DM patients compared to healthy subjects and T<em>1</em> DM patients. The significance of this result is unclear but the reduction in fasting <em>nesfatin</em>-<em>1</em> may be one of the appetite-related hormones involved in diabetic hyperphagia. In addition, neither glucose nor saline ingestions affected plasma <em>nesfatin</em>-<em>1</em>, suggesting that gastric chemosensation is not sufficient for the <em>nesfatin</em>-<em>1</em> response under the present conditions.

Overlapped in the tuberal hypothalamic area (THA), melanin-concentrating hormone (MCH) and hypocretin (Hcrt) neurons contribute to the integrated regulation of food intake, energy regulation and sleep. Recently, physiological role in appetite suppression has been defined for a novel hypothalamic molecule, <em>nesfatin</em>-<em>1</em>. Acute i.c.v. infusion of <em>nesfatin</em>-<em>1</em> (nesf-<em>1</em>) promotes anorexia whereas chronic treatment reduces body weight in rats. This satiety molecule is expressed in neurons from areas prominently involved in appetite regulation including THA. We therefore sought functionally relevant to determine whether nesf-<em>1</em> might be a reliable signaling marker for a new cell contingent within THA, in addition to MCH and Hcrt neurons. Thus, we completed a detailed topographical mapping of neurons immunostained for nesf-<em>1</em> (nesf-<em>1</em>+) together with cell quantification in each discrete nucleus from THA in the rat. We further combined the immunodetection of nesf-<em>1</em> with that of MCH or Hcrt to assess possible co-expression. More than three quarters of the nesf-<em>1</em>+ neurons were encountered in nuclei from the lateral half of THA. By double immunofluorescent staining, we showed that all neurons immunoreactive for melanin concentrating hormone (MCH+) neurons depicted nesf-<em>1</em> immunoreactivity and approximately 80% of the nesf-<em>1</em>+ neurons were labeled for MCH. Maximal co-expression rates were observed in the lateral THA containing approximately 86% of the double-labeled neurons plotted in THA. The present data suggest that nesf-<em>1</em> co-expressed in MCH neurons may play a complex role not only in food intake regulation but also in other essential integrative brain functions involving MCH signaling, ranging from autonomic regulation, stress, mood, cognition to sleep.

BACKGROUND

<em>Nesfatin</em>-<em>1</em>, derived from the protein NEFA/nucleobindin2 (NUCB2), is a newly identified peptide that acts as a potent satiety agent. It has been reported that peptides involved in the regulation of ingestive behavior are also involved in the regulation of the stress response. However, the relation between <em>nesfatin</em>-<em>1</em> and stressor-related behaviors like anxiety and/or fear has not yet been investigated.

OBJECTIVE

The effects of intracerebroventricular (ICV) injection of <em>nesfatin</em>-<em>1</em> (0, 5, and 25 pmol/3 microl) were assessed in several paradigms that are thought to reflect anxiety and/or fear in rats.

RESULTS

Consistent with an anxiogenic effect, <em>nesfatin</em>-<em>1</em> dose-dependently decreased the percentage of time spent on the open arms of the elevated plus maze, increased latency to approach, and decreased consumption of a palatable snack in an anxiogenic (unfamiliar) environment. Moreover, ICV <em>nesfatin</em>-<em>1</em> increased the fear-potentiated startle response and the time spent freezing to both context and conditioned cues in a conditioned emotional response test.

CONCLUSIONS

These findings suggest that in addition to its role as a satiety peptide, <em>nesfatin</em>-<em>1</em> may also be involved in the mediation of anxiety- and/or fear-related responses.

<em>Nesfatin</em>-<em>1</em> is a recently discovered feeding inhibitory peptide encoded in the precursor protein, nucleobindin 2 (pro<em>nesfatin</em>). Previous studies have shown pro<em>nesfatin</em> expression in the brain, stomach and pancreas. However, the identity of cells that express <em>nesfatin</em> in the pancreas remain unknown. The objective of this study was to determine which cells in the pancreas of mice and rats express pro<em>nesfatin</em> immunoreactivity. We found pro<em>nesfatin</em> immunopositive cells exclusively in the pancreatic islets of both CD<em>1</em> mice and Fischer 344 rats. Our novel results indicate that the insulin producing beta cells colocalize pro<em>nesfatin</em> in the islets of both mice and rats. No colocalization of glucagon and pro<em>nesfatin</em> was found in mice, while some glucagon positive cells were positive for pro<em>nesfatin</em> in rat islets. The abundant presence of pro<em>nesfatin</em> immunoreactivity and its colocalization with insulin suggests a potential role for pro<em>nesfatin</em>-derived peptides in islet biology and glucose homeostasis in rodents.

Obesity is a major public health problem associated with morbidity and mortality and continues to increase worldwide. This review focuses on the regions of the brain that are important in appetite regulation and the circulating factors implicated in the control of food intake. The hypothalamus is critical in the regulation of food intake containing neural circuits, which produce a number of peptides that influence food intake. The arcuate nucleus of the hypothalamus produces both orexigenic peptides (agouti-related protein and neuropeptide Y) and anorectic peptides (alpha-melanocyte-stimulating hormone and cocaine- and amphetamine-related transcript). The lateral hypothalamus produces the orexigenic peptides (melanin-concentrating hormone and orexins). Other hypothalamic factors recently implicated in appetite regulation include the endocannabinoids, brain-derived neurotrophic factor, <em>nesfatin</em>-<em>1</em>, AMP-activated protein kinase, mammalian target of rapamycin protein, and protein tyrosine phosphatase. Circulating factors that affect food intake mediate their effects by signaling to the hypothalamus and/or brainstem. A number of circulating factors are produced by peripheral organs, for example, leptin by adipose tissue, insulin and pancreatic polypeptide by the pancreas, gut hormones (e.g., ghrelin, obestatin, glucagon-like peptide-<em>1</em>, oxyntomodulin, peptide YY), and triiodothyronine by the thyroid gland. Circulating carbohydrates, lipids, and amino acids also affect appetite regulation. Knowledge regarding appetite regulation has vastly expanded in recent years providing targets for antiobesity drug design.

<em>Nesfatin</em>-<em>1</em> was originally identified as a hypothalamic neuropeptide, derived from the precursor NEFA (for DNA binding/EF-hand/acidic protein)/nucleobindin 2 (NUCB2), with the ability to suppress food intake, acting in a leptin-independent manner. Departing from this seminal finding, the patterns of expression of NUCB2/<em>nesfatin</em>-<em>1</em> have been thoroughly characterized in different hypothalamic nuclei and brain areas with proven roles in energy homeostasis, and its potential interactions with other key neuropeptide regulators of appetite have been documented. Intriguingly, recent experimental evidence suggests that NUCB2/<em>nesfatin</em>-<em>1</em> is also expressed in peripheral tissues with relevant metabolic functions, such as the pancreas, the adipose, and the gut. In addition, evidence is mounting that <em>nesfatin</em> signaling may participate in adaptative responses and in the control of body functions gated by the state of energy reserves, such as puberty onset. Altogether, these observations have broadened our perception of the biological profile of <em>nesfatin</em>-<em>1</em> that, rather than a simple anorectic signal in the hypothalamus, might operate at different tissues as an integral regulator of energy homeostasis and closely related neuroendocrine functions.

BACKGROUND

We recently identified a novel anorexigenic protein, <em>nesfatin</em>-<em>1</em>, which is processed from <em>nesfatin</em>/nucleobindin-2 (NUCB2). However, the clinical importance of this protein has not been determined.

OBJECTIVE

To investigate its clinical significance in humans, we have established a new specific enzyme-linked immunosorbent assay (ELISA) for human <em>nesfatin</em>-<em>1</em> in peripheral blood and measured its circulating concentration in healthy subjects.

METHODS

The new sandwich-type ELISA method was validated and then used to measure <em>nesfatin</em>-<em>1</em> levels in plasma samples, under overnight fasting conditions, followed by oral glucose tolerance and meal tests.

METHODS

A total of 43 nonobese males (age: 24.5 ± 0.6, body mass index (BMI); 2<em>1</em>.<em>1</em> ± 0.3 kg/m(2)) were recruited to the study for evaluating fasting concentrations of <em>nesfatin</em>-<em>1</em>. In those, fifteen subjects underwent a 75- g oral glucose tolerance test (OGTT) and another <em>1</em>5 underwent a meal test. In addition, fasting concentrations of <em>nesfatin</em>-<em>1</em> were measured in nine males with high BMI (age: 32.4 ± 3.7, BMI; 37.3 ± 3.8 kg/m(2)).

RESULTS

Peripheral concentrations of <em>nesfatin</em>-<em>1</em> showed a significant negative correlation with BMI, percentage body fat, body fat weight and blood glucose (P < 0.05). Nesfatin-<em>1</em> concentrations were not significantly changed during OGTT and meal tests. Fasting <em>nesfatin</em>-<em>1</em> levels were significantly lower in subjects with high BMI compared to nonobese subjects (P < 0.05).

CONCLUSIONS

A new specific and sensitive ELISA for <em>nesfatin</em>-<em>1</em> was established. Further accumulation of clinical observations is necessary to clarify the role of circulating <em>nesfatin</em>-<em>1</em> in various metabolic disorders.