Although the novel satiety factor <em>nesfatin</em>-<em>1</em> has been shown to influence feeding behavior through effects on melanocortin signaling, the specific hypothalamic neuronal substrates through which such effects are mediated have yet to be elucidated. To identify neuronal cell types potentially important in mediating <em>nesfatin</em>-<em>1</em>'s effects, whole cell current clamp recordings were made from hypothalamic arcuate nucleus neurons and the effects of bath applied <em>nesfatin</em>-<em>1</em> on membrane excitability determined. Neurons then underwent phenotypic identification post hoc using single cell RT-PCR. <em>Nesfatin</em>-<em>1</em> (<em>1</em>0 nM) had effects on the majority of arcuate nucleus neurons tested, with most responsive cells hyperpolarizing following exposure. Furthermore, 9 of <em>1</em><em>1</em> identified NPY neurons hyperpolarized in response to <em>nesfatin</em>-<em>1</em> exposure. Pharmacological experiments revealed that glibenclamide (500 nM), an ATP-sensitive potassium conductance antagonist, prevented <em>nesfatin</em>-<em>1</em>-induced hyperpolarization. Therefore, <em>nesfatin</em>-<em>1</em>-induced inhibition of feeding may be mediated through the inhibition of orexigenic NPY neurons.

<em>Nesfatin</em>/nucleobindin 2 (NUCB2) is expressed in the appetite-control hypothalamic nuclei and brainstem nuclei. <em>Nesfatin</em>/NUCB2 expression in the paraventricular nucleus of the hypothalamus was modulated by starvation and refeeding. Intracerebroventricular administration of <em>nesfatin</em>-<em>1</em> dose-dependently inhibited food intake for 6 hours in male Wistar and leptin resistant, Zucker fatty rats. Intraperitoneal administration of <em>nesfatin</em>-<em>1</em> and its mid-segment (M30) dosedependentlyinhibited food intake for 3 hours in male ICR mice. Intraperitoneal administration of M30 also decreased foodintake in leptin-resistant, genetically obese (ob/ob), diabetic (db/db) mice and mice fed a 45% high fat diet for 28 days. Intraperitoneal administration of M30 increased proopiomelanocortin and cocaine- and amphetamine- related peptide mRNA expression in the nucleus of the solitary tract of mice. In addition, intranasal administration of <em>nesfatin</em>-<em>1</em> significantly inhibited food intake for 6 hours in male Wistar rats. We summarize recent observations about <em>nesfatin</em>-<em>1</em>, and attempt to present future direction of <em>nesfatin</em>-<em>1</em> research for developing a new anti-obesity treatment.

<em>Nesfatin</em>-<em>1</em>, derived from nucleobindin 2, was recently identified as an anorexigenic signal peptide. However, its neural role in glucose homeostasis and insulin sensitivity is unknown. To evaluate the metabolic impact and underlying mechanisms of central <em>nesfatin</em>-<em>1</em> signaling, we infused <em>nesfatin</em>-<em>1</em> in the third cerebral ventricle of high-fat diet (HFD)-fed rats. The effects of central <em>nesfatin</em>-<em>1</em> on glucose metabolism and changes in transcription factors and signaling pathways were assessed during euglycemic-hyperinsulinemic clamping. The infusion of <em>nesfatin</em>-<em>1</em> into the third cerebral ventricle markedly inhibited hepatic glucose production (HGP), promoted muscle glucose uptake, and was accompanied by decreases in hepatic mRNA and protein expression and enzymatic activity of PEPCK in both standard diet- and HFD-fed rats. In addition, central <em>nesfatin</em>-<em>1</em> increased insulin receptor (InsR)/insulin receptor substrate-<em>1</em> (IRS-<em>1</em>)/AMP-dependent protein kinase (AMPK)/Akt kinase (Akt)/target of rapamycin complex (TORC) 2 phosphorylation and resulted in an increase in Fos immunoreactivity in the hypothalamic nuclei that mediate glucose homeostasis. Taken together, these results reveal what we believe to be a novel site of action of <em>nesfatin</em>-<em>1</em> on HGP and the PEPCK/InsR/IRS-<em>1</em>/AMPK/Akt/TORC2 pathway and suggest that hypothalamic <em>nesfatin</em>-<em>1</em> action through a neural-mediated pathway can contribute to increased peripheral and hepatic insulin sensitivity by decreasing gluconeogenesis and promoting peripheral glucose uptake in vivo.

An anorexigenic peptide, <em>nesfatin</em>-<em>1</em> was found in rat hypothalamus, and its expression in the paraventricular nucleus of the hypothalamus was reduced by starvation. Intracerebroventricular administration dose-dependently inhibited food intake for 6 h in male Wistar and leptin resistant, Zucker fatty rats. There may be a crosstalk between <em>nesfatin</em>-<em>1</em> pathway and melanocortin pathway in the brain. <em>Nesfatin</em>-<em>1</em> neurons co-express with oxytocin, vasopressin and melanin concentrating hormone in the hypothalamus. Intraperitoneal administration of <em>nesfatin</em>-<em>1</em> and its mid-segment dose-dependently inhibited food intake for 3 h. Mid-segment of <em>nesfatin</em>-<em>1</em> decreased food intake under leptin-resistant animal models of obesity. Intraperitoneal administration of the mid-segment of <em>nesfatin</em>-<em>1</em> increased proopiomelanocortin and cocain- and amphetamine-related peptide mRNA expression in the nucleus of the solitary tract, but not in arcuate nucleus of the hypothalamus. In this review, we summarized recent progress in the research about the possible mechanism of <em>nesfatin</em>-<em>1</em>-induced anorexia.

Nucleobindin-2 (NUCB2)-derived <em>nesfatin</em>-<em>1</em> located in the brain has been implicated in the satiety and control of energy metabolism. <em>Nesfatin</em>-<em>1</em> is also produced in the periphery and present in the plasma. It has recently been reported that NUCB2/<em>nesfatin</em>-<em>1</em> is localized in pancreatic islet β-cells in mice and rats and released from islets. However, its function in islets remains largely unknown. This study examined direct effects of <em>nesfatin</em>-<em>1</em> on insulin release from pancreatic islets and on cytosolic Ca(2+) concentration ([Ca(2+)](i)) in single β-cells from ICR mice. In the presence of 8.3 mmol/L glucose, <em>nesfatin</em>-<em>1</em> at <em>1</em>0(-<em>1</em>0)-<em>1</em>0(-9) mol/L tended to increase and at <em>1</em>0(-8) mol/L increased insulin release from isolated islets, while at 2.8 mmol/L glucose <em>nesfatin</em>-<em>1</em> had no effect. Furthermore, <em>nesfatin</em>-<em>1</em> at <em>1</em>0(-<em>1</em>0)-<em>1</em>0(-8) mol/L increased [Ca(2+)](i) in single β-cells in the presence of 8.3 but not 2.8 mmol/L glucose. The <em>nesfatin</em>-<em>1</em>-induced [Ca(2+)](i) increase and insulin release were inhibited by removal of extracellular Ca(2+) and by addition of nitrendipine, a blocker of voltage-dependent L-type Ca(2+) channels. Unexpectedly, the [Ca(2+)](i) responses to <em>nesfatin</em>-<em>1</em> were unaltered by inhibitors of protein kinase A (PKA) and phospholipase A(2) (PLA(2)). These results indicate that nesfain-<em>1</em> potentiates glucose-induced insulin secretion by promoting Ca(2+) influx through L-type Ca(2+) channels independently of PKA and PLA(2) in mouse islet β-cells.

The hypothalamic peptide, <em>nesfatin</em>-<em>1</em>, derived from the precursor NEFA/nucleobindin 2 (NUCB2), was recently identified as anorexigenic signal, acting in a leptin-independent manner. Yet its participation in the regulation of other biological functions gated by body energy status remains unexplored. We show herein that NUCB2/<em>nesfatin</em>-<em>1</em> is involved in the control of female puberty. NUCB2/<em>nesfatin</em> mRNA and protein were detected at the hypothalamus of pubertal female rats, with prominent signals at lateral hypothalamus (LHA), paraventricular (PVN), and supraoptic (SON) nuclei. Hypothalamic NUCB2 expression raised along pubertal transition, with detectable elevations of its mRNA levels at LHA, PVN, and SON, and threefold increase of its total protein content between late-infantile and peripubertal periods. Conditions of negative energy balance, such as 48 h fasting or sustained subnutrition, decreased hypothalamic NUCB2 mRNA and/or protein levels in pubertal females. At this age, central administration of <em>nesfatin</em>-<em>1</em> induced modest but significant elevations of circulating gonadotropins, whose magnitude was notably augmented in conditions of food deprivation. Continuous intracerebroventricular infusion of antisense morpholino oligonucleotides (as-MONs) against NUCB2 along pubertal maturation, which markedly reduced hypothalamic NUCB2 protein content, delayed vaginal opening and decreased ovarian weights and serum luteinizing hormone (LH) levels. In contrast, in adult female rats, intracerebroventricular injection of <em>nesfatin</em> did not stimulate LH or follicle-stimulating hormone secretion; neither did central as-MON infusion alter preovulatory gonadotropin surges, despite suppression of hypothalamic NUCB2. In sum, our data are the first to disclose the indispensable role of NUCB2/<em>nesfatin</em>-<em>1</em> in the central networks driving puberty onset, a function that may contribute to its functional coupling to energy homeostasis.

<em>Nesfatin</em>-<em>1</em> is well established to reduce food intake upon brain injection in rats, while in mice its anorexigenic action and brain expression are largely unexplored. We characterized the influence of intracerebroventricular (icv) and peripheral (intraperitoneal, ip, subcutaneous, sc) injection of <em>nesfatin</em>-<em>1</em> on dark phase ingestive behavior using an automated feeding monitoring system and co-localized NUCB2/<em>nesfatin</em>-<em>1</em> immunoreactivity in the associated brain areas. <em>Nesfatin</em>-<em>1</em> (0.3, <em>1</em> or 3 μg/mouse, icv) caused a dose-related reduction of 4-h dark phase food intake by <em>1</em>3%, 27%, and 46% respectively. <em>Nesfatin</em>-<em>1</em> (3 μg/mouse, icv) action had a 2-h delayed onset, 82% peak inhibition occurring at 3-4h post-injection and was long lasting (30% reduction for <em>1</em>2h period post-injection). <em>Nesfatin</em>-<em>1</em> (3 μg/mouse, icv)-treated mice had a 46% lower meal frequency associated with 2-times longer inter-meal intervals and a 35% reduction in meal size compared to vehicle during the <em>1</em>-4h post-injection (p<0.05). NUCB2/<em>nesfatin</em>-<em>1</em>-immunopositive neurons were found in hypothalamic (supraoptic, paraventricular, arcuate, dorsomedial, lateral) and brainstem (dorsal vagal complex) feeding regulatory nuclei. When injected peripherally, neither food intake nor feeding microstructure parameters were altered. These results demonstrate that NUCB2/<em>nesfatin</em>-<em>1</em> is prominently expressed in mouse hypothalamus and medulla and acts in the brain to curtail the dark phase feeding by inducing satiation and satiety indicated by reduced meal size and prolonged inter-meal intervals respectively. The lack of <em>nesfatin</em>-<em>1</em> effect when injected peripherally at a 23-times higher dose indicates a primarily central site of the anorexigenic action for <em>nesfatin</em>-<em>1</em> in mice.

The central melanocortin system regulates body energy homeostasis including the melanocortin-4 receptor (MC4R). The lateral hypothalamic area (LHA) receives dense melanocortinergic inputs from the arcuate nucleus of the hypothalamus and regulates multiple processes including food intake, reward behaviors, and autonomic function. By using a mouse line in which green fluorescent protein (GFP) is expressed under control of the MC4R gene promoter, we systemically investigated MC4R signaling in the LHA by combining double immunohistochemistry, electrophysiology, and retrograde tracing techniques. We found that LHA MC4R-GFP neurons coexpress neurotensin as well as the leptin receptor but do not coexpress other peptide neurotransmitters found in the LHA including orexin, melanin-concentrating hormone, and <em>nesfatin</em>-<em>1</em>. Furthermore, electrophysiological recording demonstrated that leptin, but not the MC4R agonist melanotan II, hyperpolarizes the majority of LHA MC4R-GFP neurons in an ATP- sensitive potassium channel-dependent manner. Retrograde tracing revealed that LHA MC4R-GFP neurons do not project to the ventral tegmental area, dorsal raphe nucleus, nucleus accumbens, and spinal cord, and only limited number of neurons project to the nucleus of the solitary tract and parabrachial nucleus. Our findings provide new insights into MC4R signaling in the LHA and its potential implications in homeostatic regulation of body energy balance.

A recently discovered satiety molecule, <em>nesfatin</em>-<em>1</em>, is localized in neurons of the hypothalamus and brain stem and colocalized with stress-related substances, corticotropin-releasing hormone (CRH), oxytocin, proopiomelanocortin, noradrenaline (NA) and 5-hydroxytryptamine (5-HT). Intracerebroventricular (icv) administration of <em>nesfatin</em>-<em>1</em> produces fear-related behaviors and potentiates stressor-induced increases in plasma adrenocorticotropic hormone (ACTH) and corticosterone levels in rats. These findings suggest a link between <em>nesfatin</em>-<em>1</em> and stress. In the present study, we aimed to further clarify the neuronal network by which <em>nesfatin</em>-<em>1</em> could induce stress responses in rats. Restraint stress induced c-Fos expressions in <em>nesfatin</em>-<em>1</em>-immunoreactive neurons in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus, and in the nucleus of solitary tract (NTS), locus coeruleus (LC) and dorsal raphe nucleus (DR) in the brain stem, without altering plasma <em>nesfatin</em>-<em>1</em> levels. Icv <em>nesfatin</em>-<em>1</em> induced c-Fos expressions in the PVN, SON, NTS, LC, DR and median raphe nucleus, including PVN-CRH, NTS-NA, LC-NA and DR-5-HT neurons. <em>Nesfatin</em>-<em>1</em> increased cytosolic Ca2+ concentration in the CRH-immunoreactive neurons isolated from PVN. Icv <em>nesfatin</em>-<em>1</em> increased plasma ACTH and corticosterone levels. These results indicate that the central <em>nesfatin</em>-<em>1</em> system is stimulated by stress and activates CRH, NA and 5-HT neurons and hypothalamic-pituitary-adrenal axis, evoking both central and peripheral stress responses.

Numerous bioactive peptides (such as ghrelins) have been identified in breast milk but there is no information concerning apelin and <em>nesfatin</em>-<em>1</em>. Therefore, present study was designated to explore whether breast milk contains apelin and <em>nesfatin</em>-<em>1</em>, to determine the concentrations and to compare these with serum levels. In addition, the concentrations of these peptides were compared in patients with gestational diabetes and normal lactating samples. Furthermore, this study explored the effectivity of various commercial diagnostic kits for determining ghrelin concentrations in breast milk. Ten gestational diabetic lactating women (29.<em>1</em>±2.2 years old and BMI: 33.2±4.8) and <em>1</em>0 control lactating women (28.2±<em>1</em>.8 years old and BMI: 39.48±<em>1</em>.7) were enrolled in the study. An ELISA was used to determine concentrations of apelin-36 and -<em>1</em>2, <em>nesfatin</em>-<em>1</em>, and acylated and desacylated ghrelin in serum, colostrum and mature milk. Serum apelin-36 and -<em>1</em>2 concentrations were correlated with colostrum and mature milk, and the same trends were observed for <em>nesfatin</em>-<em>1</em>. Apelins and <em>nesfatin</em>-<em>1</em> concentrations were higher in mature milk than in colostrum (P<0.05). The concentration of apelins, ghrelins and <em>nesfatin</em>-<em>1</em> in serum and milk in gestational diabetic lactating women was lower than in control samples. The majority of ghrelin circulating and in milk was the free form (desacylated) in both groups of women. This is the first report to describe the presence of apelins and <em>nesfatin</em>-<em>1</em> in breast milk. It is suggested that the source of ghrelins, apelins and <em>nesfatin</em>-<em>1</em> in breast milk is likely to be breast tissue (autonomous production). These bioactive peptides found in breast milk could be important for growth, energy regulation and maturation of the gastrointestinal system in neonates.

<em>Nesfatin</em>-<em>1</em> is a newly-discovered satiety peptide found in several nuclei of the hypothalamus, including the paraventricular nucleus. To begin to understand the physiological mechanisms underlying these satiety-inducing actions, we examined the effects of <em>nesfatin</em>-<em>1</em> on the excitability of neurones in the paraventricular nucleus. Whole-cell current-clamp recordings from rat paraventricular nucleus neurones showed <em>nesfatin</em>-<em>1</em> to have either hyperpolarizing or depolarising effects on the majority of neurones tested. Both types of response were observed in neurones irrespective of classification based on electrophysiological fingerprint (magnocellular, neuroendocrine or pre-autonomic) or molecular phenotype (vasopressin, oxytocin, corticotrophin-releasing hormone, thyrotrophin-releasing hormone or vesicular glutamate transporter), determined using single cell reverse transcription-polymerase chain reaction. Consequently, we provide the first evidence that this peptide, which is produced in the paraventricular nucleus, has effects on the membrane potential of a large proportion of different subpopulations of neurones located in this nucleus, and therefore identify <em>nesfatin</em>-<em>1</em> as a potentially important regulator of paraventricular nucleus output.

Adipose tissue is now regarded as an active endocrine organ which can secrete various cytokines. Adipocyte-derived cytokines are termed adipocytokines (adipocytes+cytokine). Adipocytokines can affect vascular systems to prevent or exacerbate obesity-related vascular complications, including diabetes-related vascular dysfunction, hypertension, and atherosclerosis. However, their basic vascular functions remain to be fully determined. In this manuscript, I summarize our recent findings on the vascular effects of 5 newly identified adipocytokines (omentin, visfatin, <em>nesfatin</em>, vaspin, and chemerin), with a special focus on <em>1</em>) vascular contractile reactivity, and 2) vascular inflammatory response/injury. These novel adipocytokines may be important future targets for the development of drugs and therapy for treating metabolic vascular disorders.

<em>Nesfatin</em>-<em>1</em> reduces food intake when injected centrally in rodents. We recently described wide distribution of nucleobindin2 (NUCB2)/<em>nesfatin</em>-<em>1</em> immunoreactivity in rat brain autonomic nuclei activated by various stressors. We used C57BL/6 mice to localize brain NUCB2/<em>nesfatin</em>-<em>1</em> immunoreactivity and assessed activation of NUCB2/<em>nesfatin</em> <em>1</em> neurons after water avoidance stress (WAS). Gastric emptying of a non-nutrient liquid was also determined. NUCB2/<em>nesfatin</em>-<em>1</em> immunoreactivity was detected in cortical areas including piriform, insular, cingulate and somatomotor cortices, the limbic system including amygdaloid nuclei, hippocampus and septum, the basal ganglia, bed nucleus of the stria terminalis, the thalamus including paraventricular and parafascicular nuclei, the hypothalamus including supraoptic, periventricular, paraventricular (PVN), arcuate nuclei and ventromedial and lateral hypothalamic areas. Intensely labeled NUCB2/<em>nesfatin</em>-<em>1</em> neurons were detected in a previously undefined region which we named intermediate dorsomedial hypothalamus. In the brainstem, NUCB2/<em>nesfatin</em>-<em>1</em> immunoreactivity was detected in the raphe nuclei, Edinger-Westphal nucleus, locus coeruleus (LC), lateral parabrachial nucleus, ventrolateral medulla (VLM) and dorsal vagal complex. WAS induced Fos expression in 35% of NUCB2/<em>nesfatin</em>-<em>1</em>-immunoreactive neurons in the PVN, 50% in the LC, 54% in the rostral raphe pallidus, 58% in the VLM, 39% in the middle part of the nucleus of the solitary tract (NTS) and 33% in the caudal NTS. <em>Nesfatin</em>-<em>1</em> injected intracerebroventricularly significantly decreased gastric emptying. These data showed that NUCB2/<em>nesfatin</em>-<em>1</em> immunoreactivity is distributed in mouse brain areas involved in the regulation of stress response and visceral functions activated by an acute psychological stressor suggesting that <em>nesfatin</em>-<em>1</em> might play a role in the efferent component of the stress response.

<em>Nesfatin</em>-<em>1</em> was recently identified as a peptide with anorexigenic effects that is localized in the hypothalamus and adipocytes. Not much is known about the effect of <em>nesfatin</em>-<em>1</em> on gut motility. Food intake was measured after intracerebroventricular administration of <em>nesfatin</em>-<em>1</em> in food-deprived mice. Antral and duodenal motility was assessed by using a manometric method in conscious fed mice. We found that centrally administered <em>nesfatin</em>-<em>1</em> decreased food intake and inhibited gastroduodenal motility in mice. These results suggest that <em>nesfatin</em>-<em>1</em> influences gut motility and feeding behaviour.

<em>Nesfatin</em>-<em>1</em>, product of the precursor NEFA/nucleobindin2 (NUCB2), was initially identified as anorectic hypothalamic neuropeptide, acting in a leptin-independent manner. In addition to its central role in the control of energy homeostasis, evidence has mounted recently that <em>nesfatin</em>-<em>1</em> is also produced in peripheral metabolic tissues, such as pancreas, adipose, and gut. Moreover, <em>nesfatin</em>-<em>1</em> has been shown to participate in the control of body functions gated by whole-body energy homeostasis, including puberty onset. Yet, whether, as is the case for other metabolic neuropeptides, NUCB2/<em>nesfatin</em>-<em>1</em> participates in the direct control of gonadal function remains unexplored. We document here for the first time the expression of NUCB2 mRNA in rat, mouse, and human testes, where NUCB2/<em>nesfatin</em>-<em>1</em> protein was identified in interstitial mature Leydig cells. Yet in rats, NUCB2/<em>nesfatin</em>-<em>1</em> became expressed in Sertoli cells upon Leydig cell elimination and was also detected in Leydig cell progenitors. Although NUCB2 mRNA levels did not overtly change in rat testis during pubertal maturation and after short-term fasting, NUCB2/<em>nesfatin</em>-<em>1</em> content significantly increased along the puberty-to-adult transition and was markedly suppressed after fasting. In addition, testicular NUCB2/<em>nesfatin</em>-<em>1</em> expression was up-regulated by pituitary LH, because hypophysectomy decreased, whereas human choriogonadotropin (super-agonist of LH receptors) replacement enhanced, NUCB2/<em>nesfatin</em>-<em>1</em> mRNA and peptide levels. Finally, <em>nesfatin</em>-<em>1</em> increased human choriogonadotropin-stimulated testosterone secretion by rat testicular explants ex vivo. Our data are the first to disclose the presence and functional role of NUCB2/<em>nesfatin</em>-<em>1</em> in the testis, where its expression is regulated by developmental, metabolic, and hormonal cues as well as by Leydig cell-derived factors. Our observations expand the reproductive dimension of <em>nesfatin</em>-<em>1</em>, which may operate directly at the testicular level to link energy homeostasis, puberty onset, and gonadal function.

Epilepsy is a common neurological disorder characterized by recurrent seizures. These seizures are due to abnormal excessive and synchronous neuronal activity in the brain caused by a disruption of the delicate balance between excitation and inhibition. Neuropeptides can contribute to such misbalance by modulating the effect of classical excitatory and inhibitory neurotransmitters. In this review, we discuss 2<em>1</em> different neuropeptides that have been linked to seizure disorders. These neuropeptides show an aberrant expression and/or release in animal seizure models and/or epilepsy patients. Many of these endogenous peptides, like adrenocorticotropic hormone, angiotensin, cholecystokinin, cortistatin, dynorphin, galanin, ghrelin, neuropeptide Y, neurotensin, somatostatin, and thyrotropin-releasing hormone, are able to suppress seizures in the brain. Other neuropeptides, such as arginine-vasopressine peptide, corticotropin-releasing hormone, enkephalin, β-endorphin, pituitary adenylate cyclase-activating polypeptide, and tachykinins have proconvulsive properties. For oxytocin and melanin-concentrating hormone both pro- and anticonvulsive effects have been reported, and this seems to be dose or time dependent. All these neuropeptides and their receptors are interesting targets for the development of new antiepileptic drugs. Other neuropeptides such as <em>nesfatin</em>-<em>1</em> and vasoactive intestinal peptide have been less studied in this field; however, as <em>nesfatin</em>-<em>1</em> levels change over the course of epilepsy, this can be considered as an interesting marker to diagnose patients who have suffered a recent epileptic seizure.

<em>Nesfatin</em>-<em>1</em> has been demonstrated to possess anti-inflammatory and anti-apoptotic effects in the rat brain with subarachnoid hemorrhage. The study was designed to investigate the influence of <em>nesfatin</em>-<em>1</em> on inflammatory responses and neuronal cell apoptosis after traumatic brain injury. Wistar rats were subjected to 5, <em>1</em>0 or 20 μg/kg of <em>nesfatin</em>-<em>1</em> at designed time points (0.5, 2, 4 or 8h after head trauma) intraperitoneally. Rats were sacrificed at hours 2, 6 and <em>1</em>2, as well as day <em>1</em>, 2, 3 and 5 after head trauma. The administration of <em>1</em>0 or 20 μg/kg of <em>nesfatin</em>-<em>1</em> at hour 0.5 after head trauma could significantly suppress gene expressions of nuclear factor kappa-B, lessen concentrations of tumor necrosis factor-alpha, interleukin-<em>1</em>beta and interleukin-6, diminish caspase-3 activity as well as reduce number of apoptotic neuronal cells in traumatic rat brain tissues (P<0.05), but the administration of 5 μg/kg of <em>nesfatin</em>-<em>1</em> not (P>0.05). Moreover, 20 μg/kg <em>nesfatin</em>-<em>1</em> also significantly suppressed the inflammation and neuronal cell apoptosis when applied 2, 4 or 8h after head trauma. However, a clear concentration-response or time-response relationship was not found. These findings suggest that <em>nesfatin</em>-<em>1</em> may inhibit nuclear factor kappa-B-dependent inflammatory responses, and lessen caspase-3-mediated neuronal cell apoptosis after traumatic brain injury in rats.

OBJECTIVE

To determine whether the anorexigenic peptide, <em>nesfatin</em>-<em>1</em> affects energy expenditure, and to follow the time course of its effects.

METHODS

Food intake duration, core body temperature, locomotor activity and heart rate of rats were measured by telemetry for 48 h after a single intracerebroventricular injection of 25 or <em>1</em>00 pmol <em>nesfatin</em>-<em>1</em> applied in the dark or the light phase of the day. Body weight, food and water intake changes were measured daily. Furthermore, cold-responsive <em>nesfatin</em>-<em>1</em>/NUCB2 neurons were mapped in the brain.

RESULTS

Nesfatin-<em>1</em> reduced duration of nocturnal food intake for 2 days independently of circadian time injected, and raised body temperature immediately, or with little delay depending on the dose and circadian time applied. The body temperature remained higher during the next light phases of the 48 h observation period, and the circadian curve of temperature flattened. After light phase application, the heart rate was elevated transiently. Locomotion did not change. Daily food and water intake, as well as body weight measurements point to a potential decrease in all parameters on the first day and some degree of compensation on the second day. Cold-activated (Fos positive) <em>nesfatin</em>-<em>1</em>/NUCB2 neurones have been revealed in several brain nuclei involved in cold adaptation. Nesfatin-<em>1</em> co-localised with prepro-thyrotropin-releasing hormone in cold responsive neurones of the hypothalamic paraventricular nucleus, and in neurones of the nucleus raphe pallidus and obscurus that are premotor neurones regulating brown adipose tissue thermogenesis and skin blood flow.

CONCLUSIONS

Nesfatin-<em>1</em> has a remarkably prolonged effect on food intake and body temperature. Time course of <em>nesfatin</em>-<em>1</em>'s effects may be varied depending on the time applied. Many of the <em>nesfatin</em>-<em>1</em>/NUCB2 neurones are cold sensitive, and are positioned in key centres of thermoregulation. Nesfatin-<em>1</em> regulates energy expenditure a far more potent way than it was recognised before making it a preferable candidate anti-obesity drug.

<em>Nesfatin</em>-<em>1</em> is a recently discovered multifunctional metabolic hormone abundantly expressed in the pancreatic islets. The main objective of this study is to characterize the direct effects of <em>nesfatin</em>-<em>1</em> on insulin secretion in vitro using MIN6 cells and islets isolated from C57BL/6 mice. We also examined the expression of the <em>nesfatin</em>-<em>1</em> precursor protein, nucleobindin 2 (NUCB2) mRNA, and <em>nesfatin</em>-<em>1</em> immunoreactivity (ir) in the islets of normal mice and in the islets from mice with streptozotocin-induced type <em>1</em> diabetes and diet-induced obese (DIO) mice with type 2 diabetes. <em>Nesfatin</em>-<em>1</em> stimulated glucose-induced insulin release in vitro from mouse islets and MIN6 cells in a dose-dependent manner. No such stimulation in insulin secretion was found when MIN6 cells/islets were incubated with <em>nesfatin</em>-<em>1</em> in low glucose. In addition, a fourfold increase in <em>nesfatin</em>-<em>1</em> release from MIN6 cells was observed following incubation in high glucose (<em>1</em>6.7 mM) compared to low glucose (2 mM). Furthermore, we observed a significant reduction in both NUCB2 mRNA expression and <em>nesfatin</em>-<em>1</em>-ir in the pancreatic islets of mice with type <em>1</em> diabetes, while a significant increase was observed in the islets of DIO mice. Together, our findings indicate that <em>nesfatin</em>-<em>1</em> is a novel insulinotropic peptide and that the endogenous pancreatic islet NUCB2/<em>nesfatin</em> is altered in diabetes and diet-induced obesity.

Abdominal surgery-induced postoperative gastric ileus is well established to induce Fos expression in specific brain nuclei in rats within 2-h after surgery. However, the phenotype of activated neurons has not been thoroughly characterized. <em>Nesfatin</em>-<em>1</em> was recently discovered in the rat hypothalamus as a new anorexigenic peptide that also inhibits gastric emptying and is widely distributed in rat brain autonomic nuclei suggesting an involvement in stress responses. Therefore, we investigated whether abdominal surgery activates <em>nesfatin</em>-<em>1</em>-immunoreactive (ir) neurons in the rat brain. Two hours after abdominal surgery with cecal palpation under short isoflurane anesthesia or anesthesia alone, rats were transcardially perfused and brains processed for double immunohistochemical labeling of Fos and <em>nesfatin</em>-<em>1</em>. Abdominal surgery, compared to anesthesia alone, induced Fos expression in neurons of the supraoptic nucleus (SON), paraventricular nucleus (PVN), locus coeruleus (LC), Edinger-Westphal nucleus (EW), rostral raphe pallidus (rRPa), nucleus of the solitary tract (NTS) and ventrolateral medulla (VLM). Double Fos/<em>nesfatin</em>-<em>1</em> labeling showed that of the activated cells, 99% were <em>nesfatin</em>-<em>1</em>-immunoreactive in the SON, 9<em>1</em>% in the LC, 82% in the rRPa, 74% in the EW and VLM, 7<em>1</em>% in the anterior parvicellular PVN, 47% in the lateral magnocellular PVN, 4<em>1</em>% in the medial magnocellular PVN, <em>1</em>4% in the NTS and 9% in the medial parvicellular PVN. These data established <em>nesfatin</em>-<em>1</em> immunoreactive neurons in specific nuclei of the hypothalamus and brainstem as part of the neuronal response to abdominal surgery and suggest a possible implication of <em>nesfatin</em>-<em>1</em> in the alterations of food intake and gastric transit associated with such a stressor.

The protein nucleobindin 2 (NUCB2) or NEFA (DNA binding/EF-hand/acidic amino acid rich region) was identified over a decade ago and implicated in intracellular processes. New developments came with the report that post-translational processing of hypothalamic NUCB2 may result in <em>nesfatin</em>-<em>1</em>, <em>nesfatin</em>-2 and <em>nesfatin</em>-3 and convergent studies showing that <em>nesfatin</em>-<em>1</em> and full length NUCB2 injected in the brain potently inhibit the dark phase food intake in rodents including leptin receptor deficient Zucker rats. <em>Nesfatin</em>-<em>1</em> also reduces body weight gain, suggesting a role as a new anorexigenic factor and modulator of energy balance. In light of the obesity epidemic and its associated diseases, underlying new mechanisms regulating food intake may be promising targets in the drug treatment of obese patients particularly as the vast majority of them display reduced leptin sensitivity or leptin resistance while <em>nesfatin</em>-<em>1</em>'s mechanism of action is leptin independent. Although much progress on the localization of NUCB2/<em>nesfatin</em>-<em>1</em> in the brain and periphery as well as on the understanding of <em>nesfatin</em>-<em>1</em>'s anorexic effect have been achieved during the past three years, several important mechanisms have yet to be unraveled such as the identification of the <em>nesfatin</em>-<em>1</em> receptor and the regulation of NUCB2 processing and <em>nesfatin</em>-<em>1</em> release.

<em>Nesfatin</em>-<em>1</em>, processed from nucleobindin 2, is an anorexigenic peptide expressed in the brain and several peripheral tissues including the stomach and pancreas. Peripheral, as well as intracerebroventricular, administration of <em>nesfatin</em>-<em>1</em> suppresses feeding behavior, though underlying mechanisms are unknown. In this study, we examined effects of <em>nesfatin</em>-<em>1</em> on cytosolic Ca(2+) concentration ([Ca(2+)](i)) in the neurons isolated from the vagal afferent nodose ganglion of mice. <em>Nesfatin</em>-<em>1</em> at <em>1</em>0(-<em>1</em>0)-<em>1</em>0(-8)M increased [Ca(2+)](i) in the isolated neurons in a concentration-dependent manner, and at <em>1</em>0(-8)M it increased [Ca(2+)](i) in 33 out of 263 (<em>1</em>2.5%) neurons. These responses were inhibited under Ca(2+)-free conditions and by N-type Ca(2+) channel blocker, omega-conotoxin GVIA. All the <em>nesfatin</em>-<em>1</em>-responsive neurons also exhibited [Ca(2+)](i) responses to capsaicin and cholecystokinin-8. These results provide direct evidence that <em>nesfatin</em>-<em>1</em> activates vagal afferent neurons by stimulating Ca(2+) influx through N-type channels, demonstrating the machinery through which peripheral <em>nesfatin</em>-<em>1</em> can convey signals to the brain.

Leptin is critical for normal food intake and energy metabolism. While leptin receptor (ObR) function has been well studied in hypothalamic feeding circuitries, the functional relevance of ObR in extrahypothalamic areas is largely unknown. Central regulatory pathways involved in food intake utilize various neuropeptides, such as urocortin <em>1</em> (Ucn<em>1</em>), cocaine- and amphetamine-regulated transcript peptide (CART) and <em>nesfatin</em>-<em>1</em>. Ucn<em>1</em> is most abundantly expressed in the non-preganglionic Edinger-Westphal nucleus (npEW). In addition to Ucn<em>1</em>, other satiety signals, such as CART and <em>nesfatin</em>-<em>1</em>, are highly expressed in neurons of the npEW. Using immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR), we here show the presence of short and long forms of ObR in the rat npEW. Then, we tested our hypothesis that a change in plasma leptin will modulate the activity of npEW neurons containing Ucn<em>1</em>, CART and <em>nesfatin</em>-<em>1</em>. First, by double-labeling immunocytochemistry, we observed that almost all npEW neurons colocalizing Ucn<em>1</em>, CART and <em>nesfatin</em>-<em>1</em> also contain ObR. Fasting rats for two days caused a marked body weight loss and reduced leptin plasma level in both genders. Ucn<em>1</em> mRNA and CART mRNA were upregulated after fasting in males (3.3 and 2.4 times, respectively; P<0.05) but not in females. However, their peptide levels were not significantly changed. The peptide level and mRNA of <em>nesfatin</em>-<em>1</em> were unaffected by fasting. We conclude that npEW-neurons containing Ucn<em>1</em>, CART and <em>nesfatin</em>-<em>1</em> co-express ObR, and may be involved in leptin-mediated feeding control in male rats only.

BACKGROUND

<em>Nesfatin</em>-<em>1</em> is a recently discovered anorexigen encoded in the precursor peptide, nucleobindin-2 (NUCB2) in mammals. To date, <em>nesfatin</em>-<em>1</em> has not been described in any non-mammalian species, although some information is available in the sequenced genomes of several species. Our objective was to characterize <em>nesfatin</em>-<em>1</em> in fish.

RESULTS

In the present study, we employed molecular, immunohistochemical, and physiological studies to characterize the structure, distribution, and appetite regulatory effects of <em>nesfatin</em>-<em>1</em> in a non-mammalian vertebrate. A very high conservation in NUCB2 sequences, especially in the <em>nesfatin</em>-<em>1</em> region was found in lower vertebrates. Abundant expression of NUCB2 mRNA was detected in several tissues including the brain and liver of goldfish. <em>Nesfatin</em>-<em>1</em>-like immunoreactive cells are present in the feeding regulatory nucleus of the hypothalamus and in the gastrointestinal tract of goldfish. Approximately 6-fold increase in NUCB2 mRNA levels was found in the liver after 7-day food-deprivation, and a similar increase was also found after short-term fasting. This points toward a possible liver specific role for NUCB2 in the control of metabolism during food-deprivation. Meanwhile, ∼2-fold increase at <em>1</em> and 3 h post-feeding and an ∼3-fold reduction after a 7-day food-deprivation was observed in NUCB2 mRNA in the goldfish hypothalamus. In vivo, a single intraperitoneal injection of the full-length native (goldfish; gf) <em>nesfatin</em>-<em>1</em> at a dose of 50 ng/g body weight induced a 23% reduction of food intake one hour post-injection in goldfish. Furthermore, intracerebroventricular injection of gf<em>nesfatin</em>-<em>1</em> at a dose of 5 ng/g body weight resulted in ∼50% reduction in food intake.

CONCLUSIONS

Our results provide molecular, anatomical and functional evidences to support potential anorectic and metabolic roles for endogenous <em>nesfatin</em>-<em>1</em> in goldfish. Collectively, we provide novel information on NUCB2 in non-mammals and an anorexigenic role for <em>nesfatin</em>-<em>1</em> in goldfish.