<em>Nesfatin</em>-<em>1</em> is an anorexic nucleobindin-2 (NUCB2)-derived hypothalamic peptide. It controls feeding behavior, water intake, and glucose homeostasis. If intracerebrally administered, it induces hypertension, thus suggesting a role in central cardiovascular control. However, it is not known whether it is able to directly control heart performance. We aimed to verify the hypothesis that, as in the case of other hypothalamic satiety peptides, <em>Nesfatin</em>-<em>1</em> acts as a peripheral cardiac modulator. By western blotting and QT-PCR, we identified the presence of both <em>Nesfatin</em>-<em>1</em> protein and NUCB2 mRNA in rat cardiac extracts. On isolated and Langendorff-perfused rat heart preparations, we found that exogenous <em>Nesfatin</em>-<em>1</em> depresses contractility and relaxation without affecting coronary motility. These effects did not involve Nitric oxide, but recruited the particulate guanylate cyclase (pGC) known as natriuretic peptide receptor A (NPR-A), protein kinase G (PKG) and extracellular signal-regulated kinases<em>1</em>/2 (ERK<em>1</em>/2). Co-immunoprecipitation and bioinformatic analyses supported an interaction between <em>Nesfatin</em>-<em>1</em> and NPR-A. Lastly, we preliminarily observed, through post-conditioning experiments, that <em>Nesfatin</em>-<em>1</em> protects against ischemia/reperfusion (I/R) injury by reducing infarct size, lactate dehydrogenase release, and postischemic contracture. This protection involves multiple prosurvival kinases such as PKCε, ERK<em>1</em>/2, signal transducer and activator of transcription 3, and mitochondrial K(ATP) channels. It also ameliorates contractility recovery. Our data indicate that: (<em>1</em>) the heart expresses <em>Nesfatin</em>-<em>1</em>, (2) <em>Nesfatin</em>-<em>1</em> directly affects myocardial performance, possibly involving pGC-linked NPR-A, the pGC/PKG pathway, and ERK<em>1</em>/2, (3) the peptide protects the heart against I/R injury. Results pave the way to include <em>Nesfatin</em>-<em>1</em> in the neuroendocrine modulators of the cardiac function, also encouraging the clarification of its clinical potential in the presence of nutrition-dependent physio-pathologic cardiovascular diseases.

Recently, a novel factor with anorexigenic properties was identified and called <em>nesfatin</em>-<em>1</em>. This protein (82 aac) is not only expressed in peripheral organs but it is also found in neurons located in specific structures including the hypothalamus and the brainstem, two sites strongly involved in food intake regulation. Here, we studied whether some of the neurons that become activated following an injection of an anorectic dose of lipopolysaccharides (LPS) exhibit a <em>nesfatin</em>-<em>1</em> phenotype. To this end, we used double immunohistochemistry to target the expression of the immediate-early gene c-fos and of <em>nesfatin</em>-<em>1</em> on coronal frozen sections of the rat brain. The number of c-Fos+/<em>nesfatin</em>-<em>1</em>+ neurons was evaluated in the immunosensitive structures reported to contain <em>nesfatin</em>-<em>1</em> neurons; i.e. paraventricular hypothalamic nucleus (PVN), supraoptic nucleus (SON), arcuate nucleus (ARC) and nucleus of the solitary tract (NTS). LPS strongly increased the number of c-Fos+/<em>nesfatin</em>-<em>1</em>+ neurons in the PVN, SON and NTS, and to a lesser extent in the ARC. Triple labeling showed that a portion of the <em>nesfatin</em>-<em>1</em> neurons activated in response to LPS within the NTS are catecholaminergic since they co-express tyrosine hydroxylase (TH). Our data therefore indicate that a portion of <em>nesfatin</em>-<em>1</em> neurons of both the hypothalamus and brainstem are sensitive to peripheral inflammatory signals, and provide the first clues suggesting that centrally released <em>nesfatin</em>-<em>1</em> may contribute to the neural mechanisms leading to endotoxaemic anorexia.

OBJECTIVE

To observe the regional distributions and morphological features of <em>nesfatin</em>-<em>1</em>/nucleobindin-2 (NUCB2) immunoreactive (IR) cells in the rodent digestive system.

METHODS

Paraffin-embedded sections of seven organs (pancreas, stomach, duodenum, esophagus, liver, small intestine and colon) dissected from sprague-dawley (SD) rats and imprinting-control-region (ICR) mice were prepared. The regional distributions of <em>nesfatin</em>-<em>1</em>/NUCB2 IR cells were observed by immunohistochemical staining. The morphological features of the <em>nesfatin</em>-<em>1</em>/NUCB2 IR cells were evaluated by hematoxylin and eosin (HE) staining. Fresh tissues of the seven organs were prepared for Western blotting to analyze the relative protein levels of NUCB2 in each organ.

RESULTS

Immunohistochemical staining showed that the <em>nesfatin</em>-<em>1</em>/NUCB2 IR cells were localized in the central part of the pancreatic islets, the lower third and middle portion of the gastric mucosal gland, and the submucous layer of the duodenum in SD rats and ICR mice. HE staining revealed that the morphological features of <em>nesfatin</em>-<em>1</em>/NUCB2 IR cells were mainly islet cells in the pancreas, endocrine cells in the stomach, and Brunner's glands in the duodenum. Western blotting revealed that NUCB2 protein expression was higher in the pancreas, stomach and duodenum than in the esophagus, liver, small intestine and colon (P = 0.000).

CONCLUSIONS

Nesfatin-<em>1</em>/NUCB2 IR cells are expressed in the pancreas, stomach and duodenum in rodents. These cells may play an important role in the physiological regulation of carbohydrate metabolism, gastrointestinal function and nutrient absorption.

Recently, a new neuropeptide, named <em>nesfatin</em>-<em>1</em>, was discovered. It has been reported that <em>nesfatin</em>-<em>1</em> inhibits food intake after injection into the third ventricle as well as intraperitoneal (ip) injection. Cholecystokinin (CCK) is well established to play a role in the regulation of food intake. The aim of the study was to examine whether CCK-8S injected ip modulates neuronal activity in <em>nesfatin</em>-<em>1</em> immunoreactive (ir) neurons localized in the PVN and in the nucleus of the solitary tract (NTS). Additionally, tyrosine hydroxylase-immunoreactivity (TH-ir) in the PVN was determined to assess the distribution of TH-ir fibers in relation to <em>nesfatin</em>-<em>1</em>-ir. Non-fasted male Sprague-Dawley rats received 6 or <em>1</em>0 microg CCK-8S/kg or vehicle solution (0.<em>1</em>5M NaCl; n=4 all groups) ip. The number of c-Fos-ir neurons was determined in the PVN, arcuate nucleus (ARC), and NTS. Double staining procedure for <em>nesfatin</em>-<em>1</em> and c-Fos revealed that CCK-8S increased significantly and in a dose-dependent manner the number of c-Fos positive <em>nesfatin</em>-<em>1</em>-ir neurons in the PVN ( approximately 4-fold and approximately 7-fold) and NTS ( approximately 9-fold and approximately 26-fold). Triple staining in the PVN showed a dose-dependent neuronal activation of <em>nesfatin</em>-<em>1</em> neurons that were colocalized with CRF and oxytocin. Double labeling against <em>nesfatin</em>-<em>1</em> and TH revealed that nefatin-<em>1</em>-ir neurons were encircled in a network of TH-ir fibers in the PVN. No effect on the number of c-Fos-ir neurons was observed in the ARC. These results suggest that the effects of CCK on the HPA axis and on food intake may, at least in part, be mediated by <em>nesfatin</em>-<em>1</em>-ir neurons in the PVN.

<em>Nesfatin</em>-<em>1</em> is a recently discovered anorexigen, and we first reported <em>nesfatin</em>-like immunoreactivity in the pancreatic β-cells. The aim of this study was to characterize the effects of <em>nesfatin</em>-<em>1</em> on whole-body energy homeostasis, insulin secretion, and glycemia. The in vivo effects of continuous peripheral delivery of <em>nesfatin</em>-<em>1</em> using osmotic minipumps on food intake and substrate partitioning were examined in ad libitum-fed male Fischer 344 rats. The effects of <em>nesfatin</em>-<em>1</em> on glucose-stimulated insulin secretion (GSIS) were examined in isolated pancreatic islets. L6 skeletal muscle cells and isolated rat adipocytes were used to assess the effects of <em>nesfatin</em>-<em>1</em> on basal and insulin-mediated glucose uptake as well as on major steps of insulin signaling in these cells. <em>Nesfatin</em>-<em>1</em> reduced cumulative food intake and increased spontaneous physical activity, whole-body fat oxidation, and carnitine palmitoyltransferase I mRNA expression in brown adipose tissue but did not affect uncoupling protein <em>1</em> mRNA in the brown adipose tissue. <em>Nesfatin</em>-<em>1</em> significantly enhanced GSIS in vivo during an oral glucose tolerance test and improved insulin sensitivity. Although insulin-stimulated glucose uptake in L6 muscle cells was inhibited by <em>nesfatin</em>-<em>1</em> pretreatment, basal and insulin-induced glucose uptake in adipocytes from <em>nesfatin</em>-<em>1</em>-treated rats was significantly increased. In agreement with our in vivo results, <em>nesfatin</em>-<em>1</em> enhanced GSIS from isolated pancreatic islets at both normal (5.6 mM) and high (<em>1</em>6.7 mM), but not at low (2 mM), glucose concentrations. Furthermore, <em>nesfatin</em>-<em>1</em>/nucleobindin 2 release from rat pancreatic islets was stimulated by glucose. Collectively, our data indicate that glucose-responsive <em>nesfatin</em>-<em>1</em> regulates insulin secretion, glucose homeostasis, and whole-body energy balance in rats.

Restricting-type anorexia nervosa (AN-R) is characterized by chronic food restriction and severe emaciation due to various cognitive biases such as a distorted self-image. In spite of several treatments, AN-R continues to be a refractory disease because of its unknown pathogenesis. Although previous studies have shown that changes in feeding regulatory peptides such as ghrelin are involved in anorexia, few reports have described the relationship between AN-R and <em>nesfatin</em>-<em>1</em>, a recently identified satiety peptide. Therefore, we examined the plasma <em>nesfatin</em>-<em>1</em> levels in AN-R patients to determine its role in AN-R. A total of <em>1</em>5 women participated in the study; 7 patients with AN-R and 8 age-matched healthy controls (average BMI, <em>1</em>3.02 ± 0.30 vs. 2<em>1</em>.57 ± 0.48, respectively). Our results showed that plasma <em>nesfatin</em>-<em>1</em> levels were significantly lower in AN-R group than in control group (6.23 ± 0.70 ng/ml vs. 8.9<em>1</em> ± 0.85 ng/ml, respectively, P<0.05). Plasma acyl ghrelin and des-acyl ghrelin levels were significantly higher in AN-R group than in control group (acyl ghrelin: 62.4 ± <em>1</em>0.<em>1</em>5 fmol/ml vs. 27.20 ± 5.60 fmol/ml, P<0.0<em>1</em> and des-acyl ghrelin: 300.<em>1</em>7 ± 55.95 fmol/ml vs. <em>1</em>07.34 ± 40.63 fmol/ml, P<0.05). Although AN-R is associated with emaciation for a prolonged period, our result suggested that <em>nesfatin</em>-<em>1</em> levels may be regulated by nutrition status and response to starvation.

Central stress regulatory pathways utilize various neuropeptides, such as urocortin-<em>1</em> (Ucn<em>1</em>) and cocaine- and amphetamine-regulated transcript peptide (CART). Ucn<em>1</em> is most abundantly expressed in the non-preganglionic Edinger-Westphal nucleus (npEW). In addition to Ucn<em>1</em>, CART and <em>nesfatin</em>-<em>1</em> are highly expressed in neurons of the npEW, but the way these three neuropeptides act together in response to acute stress is not known. We hypothesized that Ucn<em>1</em>, CART and <em>nesfatin</em>-<em>1</em> are colocalized in npEW neurons and that these neurons are recruited by acute stress. Using quantitative immunocytochemistry and the reverse transcriptase polymerase chain reaction (RT-PCR), we support this hypothesis, by showing in B6C3F<em>1</em>/Crl mice that Ucn<em>1</em>, CART and <em>nesfatin</em>-<em>1</em> occur in the same neurons of the npEW nucleus. More specifically, Ucn<em>1</em> and CART revealed a complete colocalization in the same perikarya, while 90% of these neurons are also <em>nesfatin</em>-<em>1</em>-immunoreactive. Furthermore, acute (restraint) stress stimulates the general secretory activity of these npEW neurons (increased presence of Fos) and the production of Ucn<em>1</em>, CART and <em>nesfatin</em>-<em>1</em>: Ucn<em>1</em>, CART and <em>nesfatin</em>-<em>1</em>(NUCB2) mRNAs have been increased compared to controls by x<em>1</em>.8, x2.0 and x2.6, respectively (p<0.0<em>1</em>). We conclude that Ucn<em>1</em>, CART and <em>nesfatin</em>-<em>1</em>/NUCB2 are specifically involved in the response of npEW neurons to acute stress in the mouse.

The unpredictable nature of peptide binding to surfaces requires optimization of experimental containers to be used. To demonstrate the variable recoveries of peptides from multiple surfaces commonly employed in peptide research, we tested the recovery of radiolabeled (<em>1</em>25)I endocrine peptides under different conditions and provide guidelines for determining the surfaces to use for other peptides. (<em>1</em>25)I-labeled peptides (ghrelin, sulfated cholecystokinin-8, corticotropin-releasing factor, glucagon-like peptide-<em>1</em> [GLP-<em>1</em>], insulin, leptin, <em>nesfatin</em>-<em>1</em>, and peptide YY), representing a wide spectrum in net charge, size, end group, and modification, were incubated for 48 h in glass and plastic tubes untreated or coated with siliconizing fluid. Best surfaces were chosen and peptides were incubated with bovine serum albumin (BSA, <em>1</em>%) with or without subsequent lyophilization. Recovery of (<em>1</em>25)I-labeled peptides was determined by gamma counting. Important differences in (<em>1</em>25)I-labeled peptide binding capacities to various types of surfaces exist. Siliconization decreased, whereas the addition of BSA improved recovery from surfaces tested. Lyophilizing solutions containing (<em>1</em>25)I-labeled peptides and BSA in the tubes best suited for individual peptides rendered more than 89% recovery for all peptides. Ghrelin specifically displaced (<em>1</em>25)I-ghrelin from borosilicate glass, whereas GLP-<em>1</em> and Fmoc-arginine did not. Choosing the appropriate experimental container avoids unpredictable peptide loss that results in inaccurate measurements and false conclusions.

BACKGROUND

The novel adipokine, <em>nesfatin</em>-<em>1</em>/NUCB-2, reduces food intake, levels of which are elevated in overweight individuals.

OBJECTIVE

The aim of the study was to investigate the mechanisms underlying brain <em>nesfatin</em>-<em>1</em>/NUCB-2 uptake and to determine whether reduced uptake may contribute to <em>nesfatin</em>-<em>1</em>/NUCB-2 resistance.

METHODS

Cerebrospinal fluid (CSF) and corresponding plasma <em>nesfatin</em>-<em>1</em>/NUCB-2 were measured by ELISA [<em>1</em>8 men and 20 women; age, <em>1</em>9-80 yr; body mass index (BMI), <em>1</em>6.2-38.<em>1</em> kg/m(2)] and correlated to body adiposity and metabolic parameters.

RESULTS

CSF/plasma <em>nesfatin</em>-<em>1</em>/NUCB-2 ratio was significantly negatively associated with BMI, body weight, fat mass, and CSF glucose. BMI was predictive of CSF/plasma <em>nesfatin</em>-<em>1</em>/NUCB-2 ratio (β = -0.786; P = 0.045). CSF <em>nesfatin</em>-<em>1</em>/NUCB-2 was significantly positively associated with plasma <em>nesfatin</em>-<em>1</em>/NUCB-2 (R = 0.706; P < 0.0<em>1</em>). There was a significant linear relation between CSF and plasma <em>nesfatin</em>-<em>1</em>/NUCB-2 in lean (BMI <25 kg/m(2); R = 0.744; P = 0.002) and obese (BMI ≥ 30 kg/m(2); R = 0.693; P = 0.026) subjects. Subjects in the highest plasma <em>nesfatin</em>-<em>1</em>/NUCB-2 quintile had lower CSF/plasma <em>nesfatin</em>-<em>1</em>/NUCB-2 ratio [26.5% (26.0-29.5%)] compared to the lowest plasma <em>nesfatin</em>-<em>1</em>/NUCB-2 quintile [38.5% (34.0-42.0%)] (P < 0.0<em>1</em>), corresponding BMI [32.4 (3<em>1</em>.0-35.0) vs. 23.3 (<em>1</em>9.7-23.5) kg/m(2); P < 0.0<em>1</em>], and fat mass [32.8 (29.5-40.6) vs. 30.7 (8.2-20.<em>1</em>) kg/m(2); P < 0.0<em>1</em>].

CONCLUSIONS

Our observations have important implications with respect to the potential weight-reducing actions of <em>nesfatin</em>-<em>1</em>/NUCB-2 treatment. Future research should seek to clarify whether <em>nesfatin</em>-<em>1</em>/NUCB-2 would be beneficial in the management of obesity.

Glucagon-like peptide-<em>1</em> (GLP-<em>1</em>) receptor agonists have been used to treat type 2 diabetic patients and shown to reduce food intake and body weight. The anorexigenic effects of GLP-<em>1</em> and GLP-<em>1</em> receptor agonists are thought to be mediated primarily via the hypothalamic paraventricular nucleus (PVN). GLP-<em>1</em>, an intestinal hormone, is also localized in the nucleus tractus solitarius (NTS) of the brain stem. However, the role of endogenous GLP-<em>1</em>, particularly that in the NTS neurons, in feeding regulation remains to be established. The present study examined whether the NTS GLP-<em>1</em> neurons project to PVN and whether the endogenous GLP-<em>1</em> acts on PVN to restrict feeding. Intra-PVN injection of GLP-<em>1</em> receptor antagonist exendin (9-39) increased food intake. Injection of retrograde tracer into PVN combined with immunohistochemistry for GLP-<em>1</em> in NTS revealed direct projection of NTS GLP-<em>1</em> neurons to PVN. Moreover, GLP-<em>1</em> evoked Ca(2+) signaling in single neurons isolated from PVN. The majority of GLP-<em>1</em>-responsive neurons were immunoreactive predominantly to corticotropin-releasing hormone (CRH) and <em>nesfatin</em>-<em>1</em>, and less frequently to oxytocin. These results indicate that endogenous GLP-<em>1</em> targets PVN to restrict feeding behavior, in which the projection from NTS GLP-<em>1</em> neurons and activation of CRH and <em>nesfatin</em>-<em>1</em> neurons might be implicated. This study reveals a neuronal basis for the anorexigenic effect of endogenous GLP-<em>1</em> in the brain.

<em>Nesfatin</em>-<em>1</em> and ghrelin are the two recently discovered peptide hormones involved in the control of appetite. Besides its main appetite-control function, ghrelin also has anticonvulsant effects, while <em>nesfatin</em>-<em>1</em> causes depolarization in the paraventricular nucleus (PVN). The aims of this study, therefore, were to investigate: (i) whether there are differences in the concentrations of <em>nesfatin</em>-<em>1</em> and ghrelin in saliva and serum samples between eplilepsy patients and normal controls and (ii) whether salivary glands produce <em>nesfatin</em>-<em>1</em>. The study included a total of 73 subjects: 8 patients who were newly diagnosed with primary generalized seizures and had recently started antiepileptic drug therapy; 2<em>1</em> who had primary generalized seizures and were continuing with established antiepileptic drug therapy; 24 who had partial seizures (simple: n = <em>1</em>2 or complex: n = <em>1</em>2) and were continuing with established antiepileptic drug therapy; and 20 controls. Salivary gland tissue samples were analyzed for <em>nesfatin</em>-<em>1</em> expression by immunochemistry and ELISA. Saliva and serum ghrelin levels were measured by ELISA and RIA, and <em>nesfatin</em>-<em>1</em> levels by ELISA. <em>Nesfatin</em>-<em>1</em> immunoreactivity was detected in the striated and interlobular parts of the salivary glands and the ducts. The <em>nesfatin</em>-<em>1</em> level in the brain was around <em>1</em>2 times higher than in the salivary gland. Before antiepileptic treatment, both saliva and serum <em>nesfatin</em>-<em>1</em> levels were around <em>1</em>60-fold higher in patients who are newly diagnosed with primary generalized epilepsy (PGE) than in controls; these levels decreased with treatment but remained about <em>1</em>0 times higher than the control values. Saliva and serum <em>nesfatin</em>-<em>1</em> levels from patients with PGE and partial epilepsies who were continuing antiepileptic drugs were also <em>1</em>0-fold higher than control values. Serum and saliva ghrelin levels were significantly (twofold) lower in epileptic patients before treatment than in controls; they recovered somewhat with treatment but remained below the control values. These results suggest that the low ghrelin and especially the dramatically elevated <em>nesfatin</em>-<em>1</em> levels might contribute to the pathophyisology of epilepsy. Therefore, serum and saliva ghrelin and especially the remarkably increased <em>nesfatin</em>-<em>1</em> might be candidate biomarkers for the diagnosis of epilepsy and for monitoring the response to anti-epileptic treatment.

BACKGROUND

<em>Nesfatin</em>-<em>1</em>, which is derived from nucleobindin2 (NUCB2), has been recently identified as a novel satiety regulator. However, its pathophysiological role in humans remains unknown. The aim of the present study was to investigate plasma <em>nesfatin</em>-<em>1</em> levels and the association between plasma <em>nesfatin</em>-<em>1</em> levels and various metabolic parameters in humans.

METHODS

74 subjects with newly diagnosed type 2 diabetes mellitus (nT2DM), 73 subjects with impaired glucose tolerance (IGT) and 73 subjects with normal glucose tolerance (NGT) were enrolled in this study. Plasma <em>nesfatin</em>-<em>1</em> levels were measured by a commercially available enzyme- linked immunosorbent assay.

RESULTS

Plasma <em>nesfatin</em>-<em>1</em> levels were elevated in subjects with both nT2DM and IGT compared to controls (<em>1</em>.9<em>1</em>±0.79 and <em>1</em>.80±0.80 vs. <em>1</em>.4<em>1</em>±0.58 μ g/L, P<0.05 or P<0.0<em>1</em> ). Simple regression analysis showed that in subjects with IGT and nT2DM, plasma <em>nesfatin</em>-<em>1</em> correlated positively with body mass index (BMI), hemoglobin A<em>1</em>c (HbA(<em>1</em>c)), fasting blood glucose (FBG), 2 h blood glucose after a glucose load (2hPBG), fasting plasma insulin (FINS) and the homeostasis model assessment of insulin resistance (HOMA(-IR)). Multivariate logistic regression analysis revealed that plasma <em>nesfatin</em>-<em>1</em> was significantly associated with IGT and nT2DM, even after controlling for differences in BMI.

CONCLUSIONS

Plasma <em>nesfatin</em>-<em>1</em> concentrations were found to be elevated in subjects with both IGT and nT2DM and to be related with several clinical parameters known to be associated with insulin resistance.

Cocaine- and amphetamine-regulated transcript (CART) and <em>nesfatin</em>-<em>1</em>/nucleobindin 2 (NUCB2) are assumed to play a role in feeding and adaptation to stress. Both peptides are highly expressed in the midbrain non-preganglionic Edinger-Westphal nucleus (npEW), a center implicated in the regulation of stress adaptation and in the pathogenesis of stress-induced brain disorders, in a sex-specific manner. The present study was undertaken to test whether CART and <em>nesfatin</em> are involved in these actions of the npEW in the rat. Acute restraint and chronic variable mild stress were used. Following stress, physiological parameters (serum corticosterone levels, body, adrenal and thymus weights) were determined, CART and <em>nesfatin</em>-like immunoreactivity (LI) as well as mRNA expression were analyzed in the npEW nucleus. Our results depict the following changes: (<em>1</em>) Acute stress resulted in an increase in serum corticosterone levels that was higher in females; (2) In males, data on corticosterone and body weight gain and in females, data on body weight gain revealed an effect of chronic stress; (3) Both acute and chronic stress activated npEW neurons expressing CART and <em>nesfatin</em>-LI, as shown by increased cFos immunoreactivity; (4) Chronic, but not acute stress increased the amount of CART and <em>nesfatin</em>-LI in both males and females; (5) Neither acute nor chronic stress had an effect on CART and NUCB2 mRNA contents of npEW neurons in either sex. Taken together, our data suggest that CART and <em>nesfatin</em> are involved in the response of npEW neurons to chronic stress.

BACKGROUND

There is substantial evidence to suggest that oxidative stress plays a significant role in the development of acute brain injury after subarachnoid hemorrhage (SAH).

OBJECTIVE

To investigate the putative neuroprotective effect of <em>nesfatin</em>-<em>1</em>, a novel peptide with anorexigenic properties, in a rat model of SAH.

METHODS

Male Wistar albino rats were randomly divided into control, saline-treated SAH, and <em>nesfatin</em>-<em>1</em> (<em>1</em>0 μg/kg IP)-treated SAH groups. To induce SAH, rats were injected with 0.3 mL blood into their cisterna magna. Forty-eight hours after SAH induction, neurological examination scores were recorded and the rats were decapitated. Brain tissue samples were taken for the determination of blood-brain barrier (BBB) permeability, brain water content, and oxidative stress markers and for histological analysis.

RESULTS

The neurological examination scores were increased on the second day of SAH induction. SAH resulted in impaired blood-brain barrier and edema, along with increased levels of brain tumor necrosis factor-α, interleukin-<em>1</em>β, interleukin-6, lipid peroxidation, protein carbonylation, and myeloperoxidase activity with concomitant decreases in antioxidant enzymes. Conversely, in the <em>nesfatin</em>-<em>1</em>-treated SAH group, SAH-induced neurological impairment and oxidative brain injury were ameliorated by <em>nesfatin</em> treatment. Furthermore, SAH-induced morphological changes in the basilar arteries were improved by <em>nesfatin</em>-<em>1</em> treatment, whereas caspase-3 activity and SAH-induced elevations in the plasma levels of proinflammatory cytokines were also depressed by <em>nesfatin</em>-<em>1</em> treatment.

CONCLUSIONS

These findings suggest that <em>nesfatin</em>-<em>1</em>, which appears to have antiapoptotic and anti-inflammatory properties, exerts neuroprotection in SAH-induced injury in rats by inhibiting neutrophil infiltration and subsequent release of inflammatory mediators.

<em>Nesfatin</em>-<em>1</em>, a newly discovered NUCB2-derived satiety neuropeptide is expressed in several neurons of forebrain, hindbrain, brainstem and spinal cord. This novel anorexigenic substance seems to play an important role in hypothalamic pathways regulating food intake and energy homeostasis. <em>Nesfatin</em>-<em>1</em> immunoreactive cells are detectable in arcuate (ARC), paraventricular (PVN) and supraoptic nuclei (SON), where the peptide is colocalized with POMC/CART, NPY, oxytocin and vasopressin. The <em>nesfatin</em>-<em>1</em> molecule interacts with a G-protein coupled receptor and its cytophysiological effect depends on inhibitory hyperpolarization of NPY/AgRP neurons in ARC and melanocortin signaling in PVN. Administration of <em>nesfatin</em>-<em>1</em> significantly inhibits consumatory behavior and decreases weight gain in experimental animals. These recent findings suggest the evidence for <em>nesfatin</em>-<em>1</em> involvement in other important brain functions such as reproduction, sleep, cognition and anxiety- or stress-related responses. The neuroprotective and antiapoptotic properties of <em>nesfatin</em>-<em>1</em> were also reported. From the clinical viewpoint it should be noteworthy, that the serum concentration of <em>nesfatin</em>-<em>1</em> may be a sensitive marker of epileptic seizures. However, the details of <em>nesfatin</em>-<em>1</em> physiology ought to be clarified, and it may be considered suitable in the future, as a potential drug in the pharmacotherapy of obesity, especially in patients treated with antipsychotics and antidepressants. On the other hand, some putative <em>nesfatin</em>-<em>1</em> antagonists may improve eating disorders.

BACKGROUND

<em>Nesfatin</em>-<em>1</em>, an 82 amino acid peptide derived from the prohormone nucleobindin-2 (NUCB2), is a novel satiety hormone acting through a leptin-independent mechanism in the hypothalamus. The mechanisms by which production of <em>nesfatin</em>-<em>1</em>/NUCB2 is regulated remain unknown.

METHODS

<em>Nesfatin</em>-<em>1</em>/NUCB2 mRNA and immunoreactivity were examined in gastric tissue and Min-6 cells by RT-PCR and immunofluorescent staining or Western blotting.

RESULTS

<em>Nesfatin</em>-<em>1</em>/NUCB2 is co-localized with pS6K<em>1</em>, the downstream target of mammalian target of rapamycin (mTOR), in gastric X/A like cells. A parallel relationship between gastric mTOR signaling and <em>nesfatin</em>-<em>1</em>/NUCB2 was observed during changes in energy status. Both mTOR activity and gastric <em>nesfatin</em>-<em>1</em>/NUCB2 were down-regulated by fasting, and returned to basal levels with re-feeding. In high fat diet induced obese mice, gastric mTOR signaling and <em>nesfatin</em>-<em>1</em>/NUCB2 were increased. Inhibition of the gastric mTOR signaling by rapamycin attenuated the expression of gastric <em>nesfatin</em>-<em>1</em>/NUCB2 mRNA and protein in both lean and obese mice. Attenuation of mTOR activity by rapamycin or over-expression of TSC<em>1</em> or TSC2 reduced the expression of <em>nesfatin</em>-<em>1</em>/NUCB2 in Min-6 cells, suggesting a direct effect of mTOR signaling.

CONCLUSIONS

Gastric mTOR is a gastric energy sensor whose activity is linked to the regulation of gastric <em>nesfatin</em>-<em>1</em>/NUCB2.

<em>Nesfatin</em>-<em>1</em> is a novel anorectic peptide encoded in the precursor protein nucleobindin-2 (NUCB2). We recently reported the presence and appetite suppressing effects of <em>nesfatin</em>-<em>1</em> in goldfish. <em>Nesfatin</em>-<em>1</em> has been co-localised with ghrelin in the stomach of rats. Whether <em>nesfatin</em>-<em>1</em> influences other appetite regulatory peptides in goldfish remains unclear. The main objectives of the present study were to investigate whether <em>nesfatin</em>-<em>1</em> co-localises ghrelin in goldfish, and to test whether exogenous <em>nesfatin</em>-<em>1</em> influences endogenous ghrelin, cholecystokinin (CCK) and orexin A (OXA). We found co-localisation of <em>nesfatin</em>-<em>1</em>-like and ghrelin-like immunoreactivity in the enteroendocrine cells of the goldfish anterior intestine (J-loop). Furthermore, co-localisation of ghrelin and <em>nesfatin</em>-<em>1</em> was also observed in the posterior nucleus lateralis tuberis of the goldfish hypothalamus, a brain region implicated in the regulation of food intake. These findings suggest a functional relationship between ghrelin and <em>nesfatin</em>-<em>1</em> in goldfish. In support of this, i.c.v. administration of goldfish (gf) <em>nesfatin</em>-<em>1</em> [25 ng/g body weight (BW)], suppressed food intake and the expression of mRNAs encoding preproghrelin, ghrelin receptor (GHS-R <em>1</em>a-<em>1</em>), CCK and NUCB2 in the forebrain of fed fish, as well as ghrelin and NUCB2 mRNA in the hypothalamus of unfed fish, both at <em>1</em> h post-injection. <em>Nesfatin</em>-<em>1</em> stimulated hypothalamic CCK mRNA expression at 30 min post-injection in fed fish, and inhibited OXA mRNA in the unfed fish hypothalamus <em>1</em> h post-injection. Similarly, i.c.v. injections of gfghrelin (<em>1</em> ng/g BW), although stimulating food intake, suppressed NUCB2 and preproghrelin mRNAs, but not ghrelin receptor mRNA expression in the forebrain. It is also evident that exogenous ghrelin and <em>nesfatin</em>-<em>1</em> mRNAs encoding these peptides. Our novel results indicate interactions between <em>nesfatin</em>-<em>1</em> and ghrelin, CCK and orexin, and show that <em>nesfatin</em>-<em>1</em> acts on other appetite regulatory peptides in a time- and feeding status-dependent, as well as tissue-specific, manner in goldfish.

<em>Nesfatin</em>-<em>1</em> is a novel adipocytokine which exerts not only anorexigenic but also hypertensive roles through acting on hypothalamus melanocortin-3/4 receptors. Although it is logical to hypothesize that <em>nesfatin</em>-<em>1</em> could also affect the contractile reactivity of peripheral blood vessels, it still remains to be examined. The present study was performed to test the hypothesis. In both endothelium-intact and -denuded mesenteric artery of rats, acute treatment with <em>nesfatin</em>-<em>1</em> (<em>1</em>0nM, 30min pretreatment) had no influence on the noradrenaline- and 5-hydroxytryptamine-induced concentration-dependent contractions. Chronic treatment of mesenteric artery with <em>nesfatin</em>-<em>1</em> (<em>1</em>0nM, 3days) using organ-culture method had also no influence on the agonists-induced contractions. In contrast, <em>nesfatin</em>-<em>1</em> (<em>1</em>0nM, 30min) significantly inhibited the sodium nitroprusside (SNP)-induced relaxations of smooth muscle in mesenteric artery. A membrane permeable cyclic GMP (cGMP) analog, 8-bromo-cGMP-induced relaxations were not affected by <em>nesfatin</em>-<em>1</em>. Consistently, the SNP-induced cGMP production in smooth muscle was impaired by <em>nesfatin</em>-<em>1</em>. Intravenous application of <em>nesfatin</em>-<em>1</em> to rats not only increased blood pressure but also impaired the SNP-induced decreases in blood pressure. The present study for the first time reveals that <em>nesfatin</em>-<em>1</em> affects peripheral arterial blood vessel and inhibits the nitric oxide donor-induced smooth muscle relaxations via impairing the cGMP production. The results are the first to demonstrate that <em>nesfatin</em>-<em>1</em> modulates blood pressure through directly acting on peripheral arterial resistance.

<em>Nesfatin</em>-<em>1</em> is a peptide secreted by peripheral tissues, central and peripheral nervous system. It is involved in the regulation of energy homeostasis related with food regulation and water intake. <em>Nesfatin</em>-<em>1</em> can pass through the blood-brain barrier in both directions. It suppresses feeding independently from the leptin pathway and increases insulin secretion from pancreatic beta islet cells. That is why <em>nesfatin</em>-<em>1</em> has drawn attention as a new therapeutic agent, especially for the treatment of obesity and diabetes mellitus. Its effects on nutrition have been studied in more detail in literature. On the other hand, its effects on other physiological parameters and mechanisms of action still need to be clarified. Synthesizing the research on <em>nesfatin</em>-<em>1</em> can help us better understand this field. Hippokratia 20<em>1</em>5, <em>1</em>9 (<em>1</em>): 4-<em>1</em>0.

<em>Nesfatin</em>-<em>1</em> is an 82 amino acid peptide recently discovered in the brain which is derived from nucleobindin2 (NUCB2), a protein that is highly conserved across mammalian species. <em>Nesfatin</em>-<em>1</em> has received much attention over the past two years due to its reproducible food intake-reducing effect that is linked with recruitment of other hypothalamic peptides regulating feeding behavior. A growing amount of evidence also supports that various stressors activate fore- and hindbrain NUCB2/<em>nesfatin</em>-<em>1</em> circuitries. In this review, we outline the central nervous system distribution of NUCB2/<em>nesfatin</em>-<em>1</em>, and recent developments on the peripheral expression of NUCB2/<em>nesfatin</em>-<em>1</em>, in particular its co-localization with ghrelin in gastric X/A-like cells and insulin in ss-cells of the endocrine pancreas. Functional studies related to the characteristics of <em>nesfatin</em>-<em>1</em>'s inhibitory effects on dark phase food intake are detailed as well as the central activation of NUCB2/<em>nesfatin</em>-<em>1</em> immunopositive neurons in the response to psychological, immune and visceral stressors. Lastly, potential clinical implications of targeting NUCB2/<em>nesfatin</em>-<em>1</em> signaling and existing gaps in knowledge to ascertain the role and mechanisms of action of <em>nesfatin</em>-<em>1</em> are presented.

<em>Nesfatin</em>-<em>1</em>, a post-translational product of the nucleobindin-2 (NucB2) gene, is produced in several brain areas known to be important in neuroendocrine, autonomic and metabolic function, including the hypothalamus and medulla. The hallmark action of the peptide is its ability at picomole doses to inhibit food and water intake in rodents and, indeed, the effect on water intake is more pronounced than that on food intake. In preliminary studies, we observed a decrease in hypothalamic NucB2 expression in response to overnight water deprivation even when food was present, which reversed when water was returned to the animals. We therefore hypothesised that the effect of <em>nesfatin</em>-<em>1</em> on water drinking was independent of its anorexigenic action. Indeed, rats administered <em>nesfatin</em>-<em>1</em> i.c.v. consumed significantly less water than controls in response to a subsequent, dipsogenic dose of angiotensin II, or upon return of water bottles after <em>1</em>8 h of fluid restriction (food present), or in response to a hypertonic challenge. Pretreatment with an antisense oligonucleotide against <em>nesfatin</em>-<em>1</em> significantly reduced levels of immunoreactive <em>nesfatin</em>-<em>1</em> in the hypothalamic paraventricular nucleus and resulted in exaggerated drinking responses to angiotensin II. The results obtained in the present study suggest that locally produced <em>nesfatin</em>-<em>1</em> may be an important component of the hypothalamic mechanisms controlling fluid and electrolyte homeostasis.

<em>Nesfatin</em>-<em>1</em> is a novel anorexigenic regulatory peptide. The peptide is the N-terminal part of nucleobindin 2 (NUCB2) and is expressed in brain areas regulating feeding. Outside the brain, <em>nesfatin</em>-<em>1</em> expression has been reported in adipocytes, gastric endocrine cells and islet cells. We studied NUCB2 expression in human and rodent islets using immunocytochemistry, in situ hybridization and western blot. Furthermore, we investigated the potential influence of <em>nesfatin</em>-<em>1</em> on secretion of insulin and glucagon in vitro and in vivo in mice and in INS-<em>1</em> (832/<em>1</em>3) cells. The impact of type 2 diabetes (T2D) and glucolipotoxicity on NUCB2 gene expression in human islets and its relationship to insulin secretory capacity and islet gene expression was studied using microarray. <em>Nesfatin</em>-<em>1</em> immunoreactivity (IR) was abundant in human and rodent beta cells but absent in alpha, delta, PP and ghrelin cells. Importantly, in situ hybridization showed that NUCB2 mRNA is expressed in human and rat islets. Western blot analysis showed that <em>nesfatin</em>-<em>1</em> IR represented full length NUCB2 in rodent islets. Human islet NUCB2 mRNA was reduced in T2D subjects but upregulated after culture in glucolipotoxic conditions. Furthermore, a positive correlation between NUCB2 and glucagon and insulin gene expression, as well as insulin secretory capacity, was evident. <em>Nesfatin</em>-<em>1</em> enhanced glucagon secretion but had no effect on insulin secretion from mouse islets or INS-<em>1</em> (832/<em>1</em>3) cells. On the other hand, <em>nesfatin</em>-<em>1</em> caused a small increase in insulin secretion and reduced glucose during IVGTT in mice. We conclude that <em>nesfatin</em>-<em>1</em> is a novel glucagon-stimulatory peptide expressed in the beta cell and that its expression is decreased in T2D islets.

<em>Nesfatin</em>-<em>1</em> is a recently identified 82-amino-acid peptide derived from the precursor protein, nucleobindin2 (NUCB2). The brain distribution of NUCB2/<em>nesfatin</em>-<em>1</em> at the mRNA and protein level along with functional studies in rodents support a role for NUCB2/<em>nesfatin</em>-<em>1</em> as a novel satiety molecule acting through leptin-independent mechanisms. In addition, <em>nesfatin</em>-<em>1</em> induces a wide spectrum of central actions to stimulate the pituitary-adrenal axis and sympathetic nervous system and influences visceral functions and emotion. These central actions combined with the activation of NUCB2/<em>nesfatin</em>-<em>1</em> neurons in the brain by various stressors are indicative of a role in the adaptive response under stressful conditions. In the periphery, evidence is mounting that <em>nesfatin</em>-<em>1</em> exerts a direct glucose-dependent insulinotropic action on β-cells of the pancreatic islets. However, the cellular mechanisms of <em>nesfatin</em>-<em>1</em>'s action remain poorly understood, partly because the receptor through which <em>nesfatin</em>-<em>1</em> exerts its pleiotropic actions is yet to be identified.

There is growing evidence that vagus nerve stimulation (VNS) has a suppressive effect on both short- and long-term feeding in animal models. We previously showed that long-term VNS (<em>1</em>02 days) with low-frequency electrical impulses (0.05 Hz) decreased food intake and body weight in rats. In the present study, we investigated the effect of high frequency (<em>1</em>0 Hz) VNS on feeding behavior and appetite in rats fed a high-fat diet; peptide secretion and other parameters were assessed as well. Adult male Wistar rats were each implanted subcutaneously with a microstimulator (MS) and fed a high-fat diet throughout the entire study period (42 days). The left vagus nerve was stimulated by rectangular electrical pulses (<em>1</em>0 ms, 200 mV, <em>1</em>0 Hz, <em>1</em>2 h a day) generated by the MS. Body weight and food intake were measured each morning. At the end of the experimental period, animals were euthanized and blood samples were taken. Serum levels of ghrelin, leptin and <em>nesfatin</em>-<em>1</em> were assessed using radioimmunoassays. Adipose tissue content was evaluated by weighing epididymal fat pads, which were incised at the time of sacrifice. To determine whether VNS activated the food-related areas of the brain, neuronal c-Fos induction in the nuclei of the solitary tract (NTS) was assessed. Chronic vagus nerve stimulation significantly decreased food intake, body weight gain and epididymal fat pad weight in animals that received VNS compared with control animals. Significant neuronal responses in the NTS were observed following VNS. Finally, serum concentrations of ghrelin were increased, while serum levels of leptin were decreased. Although not significant, serum <em>nesfatin</em>-<em>1</em> levels were also elevated. These results support the theory that VNS leads to reductions in food intake, body weight gain and adipose tissue by increasing brain satiety signals conducted through the vagal afferents. VNS also evoked a feed-related hormonal response, including elevated blood concentrations of <em>nesfatin</em>-<em>1</em>.