Nucleobindin-2 mRNA Research Articles

Expression of Kruppel-like factor 6 and nucleobindin-2 genes in gastrointestinal tract of rats with gastric cancer

Objective To discuss the expression of Kruppel-like factor 6 （Klf-6） and nucleo-bindin-2 （NUCB2） genes in gastrointestinal tract of rats with gastric cancer.Methods Ninety-six adult Wister rats were randomly divided into three groups：32 rats each in groups A and B were drenched intraperitoneally with sarcosine ethyl ester hydrochloride to induce gastric cancer for 3 and 6 months respectively,and 32 rats in group C were injected with normal saline.The gastrointestinal tract was examined by electronic scale at 3rd and 6th month after modeling.The Klf-6 and NUCB2 expression in gastrointestinal tract of rats was observed,and the mRNA levels were evaluated by reverse transcription-polymerase chain reaction （RT-PCR） after the rats were decapitated.The statistical analysis was done by using SPSS 14.0 statistical software.Results All the 96 rats were involved in the analysis of results.Positive rate of Klf-6 and NUCB2 in group A was higher than in groups B and C,and that in group B higher than in group C （x2 =3.17,4.95,and 3.79,P ＜0.05）.RT-PCR revealed that the expression levels of Klf-6 and NUCB2 mRNA in group A were higher than in groups B and C,and those in group B higher than in group C （x2 =2.98,4.13 and 3.52,P ＜ 0.05）.Conclusion Combined detection of the expression of Klf-6 and NUCB2 in gastric cancer may give some guideline for the clinical diagnosis,illness evaluation,treatment and prognosis of gastric cancer. Key words: Gastric cancer; Krtippel-like factor 6 ; Nucleobindin-2

Nesfatin-1: An Affair of the Heart

Nesfatin-1 is an 82-amino acid peptide that was identified in 2006 in the rat hypothalamus by Mori and coworkers (1). The polypeptide is proteolytically cleaved from its precursor protein, nucleobindin2 (NUCB2) and named after its initially described function to reduce food intake after third-brain ventricular injection in rats [nucleobindin 2 encoded satiety and fat influencing protein (nesfatin-1)] (1). NUCB2 can be cleaved into three polypeptides: nesfatin-1, nesfatin-2, and nesfatin-3, whereas an anorexigenic effect has so far been described only for nesfatin-1 (1). The brain-inhibitory effect on food intake was confirmed by several independent groups in rodent models (2, 3) as well as nonmammalian vertebrates (4). These initial data fostered research on this peptide, and as observed for many brain peptides before, the role of nesfatin-1 is not restricted to one effect (inhibition of food intake) but also comprises the inhibition of gastric emptying (2) and water intake (5), the modulation of stress-related endocrine and anxiogenic responses (6, 7) and sympathetic outflow (8) and also an involvement in reproduction (9). After the initial reports all focused on NUCB2/nesfatin-1 as a brain peptide, subsequent studies investigated the expression of this peptide in the periphery and detected high levels in the gastric mucosa (10), pancreas (11), adipocytes (12), gonads (13), and blood (14). Interestingly, the levels in the stomach were even higher than those detected in the brain (10), indicating a predominant expression of NUCB2/nesfatin-1 in the periphery. Possible functions attributed to peripheral nesfatin-1 encompassed the modulation of food intake (15), adipogenesis (12), and an involvement in the regulation of glucose homeostasis (16–18). Recently one study reported the expression of NUCB2/ nesfatin-1 in rat cardiac extracts (19). These data were corroborated and extended by a study of Feijoo-Bandin et al published in this issue of Endocrinology (20). The authors present a comprehensive set of experiments using a truly translational approach by means of studying mouse, rat, and human cardiac tissues. First, they show that NUCB2 mRNA is expressed in mouse, human, and rat heart occurring in both atrial and ventricular cardiomyocytes (20). Interestingly, although in mouse and rat, NUCB2 mRNA expression levels are markedly lower in the heart compared with the stomach [the presumed major source of peripheral NUCB2/nesfatin-1 (10)], expression levels are similar in the heart and stomach of humans (20), giving rise to an important role of heart-derived NUCB2/ nesfatin-1 in humans. This expression was also confirmed at the protein level. It is important to note that so far the mature, fully processed 82-amino acid nesfatin-1 has been described only in cerebrospinal fluid of rats (1), rat heart (19), and human plasma using a nesfatin-1 specific sandwich-type ELISA (14). All other studies did not distinguish between full-length NUCB2 and nesfatin-1 or detected the precursor protein, NUCB2, only in tissue extracts (4, 10, 21), whereas exogenous synthetic nesfatin-1 was clearly visible on the Western blot (10). In addition, because NUCB2 is functionally active (1), this led to the assumption that NUCB2 might not be processed at all or might be cleaved more distant from its production site. However, in the present study of Feijoo-Bandin et al (20), mature nesfatin-1 was clearly detectable by Western blot as a 9.5-kDa band in protein samples from mouse, rat, and human cardiomyocytes, indicating the processing of NUCB2 into nesfatin-1 within the heart. This hypothesis was further

High expression of nucleobindin 2 mRNA: an independent prognostic factor for overall survival of patients with prostate cancer

Nucleobindin 2 (NUCB2) has been demonstrated to play critical roles in tumorigenesis and tumor development of breast cancer. The expression change of nucleobindin 2 at mRNA level in prostate cancer (PCa) tissues compared with adjacent benign prostate tissues was detected by using real-time quantitative reverse transcriptase-polymerase chain reaction analysis in our previous study. The data suggests that NUCB2 is a cancer-related gene associated with the aggressive progression and biochemical recurrence-free survival predictor of PCa patients. However, the correlation between the expression of the NUCB2 mRNA and the overall survival of patients with PCa was not analyzed. Thus, the association of NUCB2 mRNA expression with overall survival of PCa patients was analyzed in this study. Kaplan-Meier analysis and Cox proportional hazards regression models were used to investigate the correlation between NUCB2 mRNA expression and prognosis of PCa patients. The Kaplan-Meier survival analysis showed that the high expression of NUCB2 was related to the poor overall survival of patients with PCa. Multivariate Cox analysis showed that NUCB2 mRNA was an independent prognostic factor for overall survival of patients with PCa. In conclusion, we demonstrated that high NUCB2 mRNA expression correlated with poor overall survival in patients with PCa.

Effects of nesfatin-1 on food intake and LH secretion in prepubertal gilts and genomic association of the porcine NUCB2 gene with growth traits

Nesfatin-1, a product of the nucleobindin 2 (NUCB2) gene, purportedly plays important roles in whole-body energy homeostasis. Experiments were conducted to determine how NUCB2 expression in fat depots may be controlled in the pig and to test the hypothesis that nesfatin-1 regulates appetite and LH secretion in the gilt. Prepubertal gilts were used to study expression of NUCB2 in fat and the effects of intracerebroventricular (i.c.v.) injection of nesfatin-1 on food intake and pituitary hormone secretion. Growing pigs (gilts and barrows at 22 wk of age, n = 1,145) or sexually mature gilts (n = 439) were used to test association of SNP in the NUCB2 gene with growth traits. The expression of NUCB2 was similar for subcutaneous fat compared with perirenal fat. An i.c.v. injection of the melanocortin-4 receptor agonist [Nle4, d-Phe7]-α-melanocyte-stimulating hormone did not alter expression of NUCB2 mRNA in the hypothalamus but reduced (P = 0.056) NUCB2 mRNA expression in subcutaneous fat. Short-term (7 d) submaintenance feeding reduced (P < 0.05) BW and did not alter expression of mRNA for NUCB2, visfatin, or leptin but increased (P < 0.05) expression of adiponectin mRNA in fat. Central injection of nesfatin-1 suppressed (P < 0.001) feed intake. Secretion of LH was greater (P < 0.01) after i.c.v. injection of nesfatin-1 than after saline. Single nucleotide polymorphisms in the porcine NUCB2 gene were not associated with adiposity of growing pigs or age at puberty in gilts but were associated (P < 0.05) with BW at puberty. These data indicate that NUCB2 is expressed in fat depots of the pig and that the level of expression is sensitive to stimulation of appetite-regulating pathways in the hypothalamus. It is confirmed herein that nesfatin-1 can regulate appetite in the pig and affect the gonadotropic axis of the prepubertal pig. Association of SNP in the porcine NUCB2 gene with BW at puberty suggests that regulation of appetite by nesfatin-1 in the pig affects growth, which may have important consequences for adult phenotypes.

Increased nucleobindin-2 (NUCB2) transcriptional activity links the regulation of insulin sensitivity in Type 2 diabetes mellitus.

The protein nucleobindin-2 (NUCB2) has been recently identified as a novel satiety regulator. However, its pathophysiological role in humans remains unknown. The aims of the present study are to explore whether plasma NUCB2-1 and NUCB2 transcription activity are increased in newly diagnosed Type 2 diabetes mellitus (nT2DM) and, if so, whether changing NUCB2-1 level is a physiologic response or a compensatory mechanism for impaired insulin action. The nT2DM, impaired glucose tolerance (IGT), and healthy people (NGT, normal glucose tolerance) groups were enrolled in this study. The peripheral and hepatic insulin actions in rats with intracerebroventricular (ICV) NUCB2-1 administration were examined by euglycemic-hyperinsulinemic clamps. Plasma NUCB2-1 levels were elevated in subjects with both nT2DM and IGT compared with normal controls. NUCB2 mRNA and protein contents of muscle and adipose tissues in T2DM patients were also significantly increased compared to controls. ICV NUCB2-1 infusion in rats inhibited hepatic phosphoenolpyruvate carboxykinase (PEPCK) activity, and this was sufficient to induce insulin sensitivity in the liver and peripheral tissues during euglycemic-hyperinsulinemic clamps. In T2DM patients, there were increases in plasma NUCB2-1 levels and increases in NUCB2 mRNA and protein contents in muscle and adipose tissues. These increases are presumably a compensatory response to defective insulin action.

Paraventricular NUCB2/nesfatin-1 rises in synchrony with feeding suppression during early light phase in rats

Obesity often results from hyperphagia and involves rhythm disorder. Circadian feeding pattern is suggested to be implicated in energy homeostasis while its disorder in obesity. However, the mechanism underlying circadian feeding is little known. PVN is considered a regulatory center for feeding and circadian activities of hormone release and autonomic nerve. Nucleobindin2 (NUCB2) and its processing product nesfatin-1 (NUCB2/nesfatin-1) are localized in the hypothalamic paraventricular nucleus (PVN) and implicated in regulation of feeding. This study aimed to clarify whether the PVN NUCB2/nesfatin-1 expression exhibits diurnal rhythm and, if so, whether it is related to circadian feeding. Here we show that NUCB2 mRNA expression in the PVN rises during early light phase (LP) in parallel with suppression of food intake. Immunoneutralization of PVN NUCB2/nesfatin-1 with anti-nesfatin-1 IgG during LP, but not dark phase, increased food intake. PVN-selective shRNA-induced knockdown of NUCB2 mRNA expression elevated food intake. Furthermore, the rise of PVN NUCB2 mRNA during LP was blunted in Zucker-fatty obese rats which exhibited LP-preferential hyperphagia. The increases in food intake during LP and 24h were significantly corrected by intracerebroventricular injection of nesfatin-1 during LP. These results reveal the diurnal rhythm of PVN NUCB2 mRNA expression characterized by early LP rise, which may serve as a factor to limit LP food intake, contributing to circadian feeding. Furthermore, impaired NUCB2/nesfatin-1 rhythm may be related to dysregulated feeding pattern and hyperphagia in Zucker-fatty rats.

Nesfatin-1 as a novel cardiac peptide: identification, functional characterization, and protection against ischemia/reperfusion injury

Nesfatin-1 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, Nesfatin-1 acts as a peripheral cardiac modulator. By western blotting and QT-PCR, we identified the presence of both Nesfatin-1 protein and NUCB2 mRNA in rat cardiac extracts. On isolated and Langendorff-perfused rat heart preparations, we found that exogenous Nesfatin-1 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 kinases1/2 (ERK1/2). Co-immunoprecipitation and bioinformatic analyses supported an interaction between Nesfatin-1 and NPR-A. Lastly, we preliminarily observed, through post-conditioning experiments, that Nesfatin-1 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, ERK1/2, signal transducer and activator of transcription 3, and mitochondrial K(ATP) channels. It also ameliorates contractility recovery. Our data indicate that: (1) the heart expresses Nesfatin-1, (2) Nesfatin-1 directly affects myocardial performance, possibly involving pGC-linked NPR-A, the pGC/PKG pathway, and ERK1/2, (3) the peptide protects the heart against I/R injury. Results pave the way to include Nesfatin-1 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.

Ontogenic pattern of nucleobindin-2/nesfatin-1 expression in the gastroenteropancreatic tissues and serum of Sprague Dawley rats

Nesfatin‐1 is a novel metabolic hormone that has glucose-responsive insulinotropic actions. Islet β-cells and gastrointestinal tissues have been reported as abundant sources of nesfatin-1 and its precursor hormone nucleobindin-2 (NUCB2). While nesfatin-1 is emerging as a multifunctional hormone, there are no reports on the developmental expression of NUCB2/nesfatin-1. The main objective of this study was to examine the ontogenic expression of NUCB2 mRNA, and NUCB2/nesfatin-1 immunoreactivity in the pancreas, stomach and duodenum, and the circulating levels NUCB2/nesfatin-1 in Sprague Dawley rats. In addition, we also determined the co-localization of NUCB2/nesfatin-1 and insulin immunoreactivity during development. NUCB2/nesfatin-1 immunoreactivity was found in the rat stomach from postnatal days 13–27. Furthermore, NUCB2/nesfatin-1 immunoreactivity was also detected in the enteroendocrine cells of the duodenum at postnatal days 13 and 27. Duodenal NUCB2 mRNA expression at postnatal day 27 was highest. Serum NUCB2/nesfatin-1 levels on embryonic day 21 and postnatal day 1 were lower than serum NUCB2/nesfatin-1 levels of adults and neonates at postnatal days 13, 20 and 27, gradually increasing with growth, suggesting an increase in its production and secretion from tissues including the gastrointestinal tract and pancreas. Our findings indicate that NUCB2/nesfatin-1 colocalizes with insulin in the islet β-cells at all developmental stages, but the percentage of colocalization varies in an age-dependent manner. These findings suggest that NUCB2/nesfatin-1 has potential age‐ and tissue‐specific role in the developmental physiology of rats during growth.

Nesfatin‐1 Suppresses Energy Intake, Co‐localises Ghrelin in the Brain and Gut, and Alters Ghrelin, Cholecystokinin and Orexin mRNA Expression in Goldfish

Nesfatin-1 is a novel anorectic peptide encoded in the precursor protein nucleobindin-2 (NUCB2). We recently reported the presence and appetite suppressing effects of nesfatin-1 in goldfish. Nesfatin-1 has been co-localised with ghrelin in the stomach of rats. Whether nesfatin-1 influences other appetite regulatory peptides in goldfish remains unclear. The main objectives of the present study were to investigate whether nesfatin-1 co-localises ghrelin in goldfish, and to test whether exogenous nesfatin-1 influences endogenous ghrelin, cholecystokinin (CCK) and orexin A (OXA). We found co-localisation of nesfatin-1-like and ghrelin-like immunoreactivity in the enteroendocrine cells of the goldfish anterior intestine (J-loop). Furthermore, co-localisation of ghrelin and nesfatin-1 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 nesfatin-1 in goldfish. In support of this, i.c.v. administration of goldfish (gf) nesfatin-1 [25 ng/g body weight (BW)], suppressed food intake and the expression of mRNAs encoding preproghrelin, ghrelin receptor (GHS-R 1a-1), CCK and NUCB2 in the forebrain of fed fish, as well as ghrelin and NUCB2 mRNA in the hypothalamus of unfed fish, both at 1 h post-injection. Nesfatin-1 stimulated hypothalamic CCK mRNA expression at 30 min post-injection in fed fish, and inhibited OXA mRNA in the unfed fish hypothalamus 1 h post-injection. Similarly, i.c.v. injections of gfghrelin (1 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 nesfatin-1 mRNAs encoding these peptides. Our novel results indicate interactions between nesfatin-1 and ghrelin, CCK and orexin, and show that nesfatin-1 acts on other appetite regulatory peptides in a time- and feeding status-dependent, as well as tissue-specific, manner in goldfish.

MTOR-dependent Modulation of Gastric Nesfatin-1/NUCB2

Background: Nesfatin-1, 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 nesfatin-1/NUCB2 is regulated remain unknown. Methods: Nesfatin-1/NUCB2 mRNA and immunoreactivity were examined in gastric tissue and Min-6 cells by RT-PCR and immunofluorescent staining or Western blotting. Results: Nesfatin-1/NUCB2 is co-localized with pS6K1, the downstream target of mammalian target of rapamycin (mTOR), in gastric X/A like cells. A parallel relationship between gastric mTOR signaling and nesfatin-1/NUCB2 was observed during changes in energy status. Both mTOR activity and gastric nesfatin-1/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 nesfatin-1/NUCB2 were increased. Inhibition of the gastric mTOR signaling by rapamycin attenuated the expression of gastric nesfatin-1/NUCB2 mRNA and protein in both lean and obese mice. Attenuation of mTOR activity by rapamycin or over-expression of TSC1 or TSC2 reduced the expression of nesfatin-1/NUCB2 in Min-6 cells, suggesting a direct effect of mTOR signaling. Conclusion: Gastric mTOR is a gastric energy sensor whose activity is linked to the regulation of gastric nesfatin-1/NUCB2.

Possible Involvement of Hypothalamic Nucleobindin-2 in Hyperphagic Feeding in Tsumura Suzuki Obese Diabetes Mice

The aim of this study was to clarify the hypothalamic neuropeptides that are associated with hyperphagic feeding in Tsumura Suzuki Obese Diabetes (TSOD) mice, a model of type 2 diabetes with polygenic abnormalities. TSOD mice showed an increase in body weight and hyperleptinemia from 1 month of age and hyperphagic feeding, hyperglycemia, hyperlipidemia and hyperinsulinemia from 3 to 12 months of age compared with age-matched non-diabetic control Tsumura Suzuki Non Obesity (TSNO) mice. The mRNA level of nucleobindin-2 (NUCB2), the precursor of the anorexigenic neuropeptide nesfatin-1, was significantly decreased in the hypothalamus of TSOD mice compared with that in TSNO mice from 3 to 12 months of age. The protein level of NUCB2 was significantly decreased in the hypothalamus of TSOD mice compared with that in TSNO mice at 3 months of age. The mRNA levels of galanin, melanin-concentrating hormone, neuropeptide Y, and pro-opiomelanocortin were significantly changed in the hypothalamus in TSOD mice at several time points. Another model of type 2 diabetes, db/db mice, which is a mutant mouse that lacks a functional leptin receptor, showed hyperphagic feeding but no change in hypothalamic NUCB2 mRNA compared with non-diabetic control db/+ mice. The results suggest that the disrupted control of hypothalamic NUCB2-mediated signaling may contribute to hyperphagic feeding in TSOD mice. In addition, the mechanism for the development of hyperphagic feeding in TSOD mice is different than that in db/db mice.

Nesfatin-1 stimulates glucagon and insulin secretion and beta cell NUCB2 is reduced in human type 2 diabetic subjects

Nesfatin-1 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, nesfatin-1 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 nesfatin-1 on secretion of insulin and glucagon in vitro and in vivo in mice and in INS-1 (832/13) 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. Nesfatin-1 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 nesfatin-1 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. Nesfatin-1 enhanced glucagon secretion but had no effect on insulin secretion from mouse islets or INS-1 (832/13) cells. On the other hand, nesfatin-1 caused a small increase in insulin secretion and reduced glucose during IVGTT in mice. We conclude that nesfatin-1 is a novel glucagon-stimulatory peptide expressed in the beta cell and that its expression is decreased in T2D islets.

Lipopolysaccharide increases gastric and circulating NUCB2/nesfatin-1 concentrations in rats

Bacterial lipopolysaccharide (LPS) is an established animal model to study the innate immune response to Gram-negative bacteria mimicking symptoms of infection including reduction of food intake. LPS decreases acyl ghrelin associated with decreased concentrations of circulating ghrelin-O-acyltransferase (GOAT) likely contributing to the anorexigenic effect. We also recently described the prominent expression of the novel anorexigenic hormone, nucleobindin2 (NUCB2)/nesfatin-1 in gastric X/A-like cells co-localized with ghrelin in different pools of vesicles. To investigate whether LPS would affect gastric and circulating NUCB2/nesfatin-1 concentration, ad libitum fed rats were equipped with an intravenous (iv) catheter. LPS was injected intraperitoneally (ip, 100μg/kg) and blood was withdrawn before and at 2, 5, 7 and 24h post injection and processed for NUCB2/nesfatin-1 radioimmunoassay. Gastric corpus was collected to measure NUCB2 mRNA expression by RT-qPCR and NUCB2/nesfatin-1 protein concentration by Western blot. Injection of LPS increased plasma NUCB2/nesfatin-1 concentrations by 43%, 78% and 62% compared to vehicle at 2h, 5h and 7h post injection respectively (p<0.05) and returned to baseline at 24h. The plasma NUCB2/nesfatin-1 increase at 2h was associated with increased corpus NUCB2 mRNA expression (p<0.01), whereas NUCB2 mRNA was not detectable in white blood cells. Likewise, gastric NUCB2 protein concentration was increased by 62% after LPS compared to vehicle (p<0.01). These data show that gastric NUCB2 production and release are increased in response to LPS. These changes are opposite to those of ghrelin in response to LPS supporting a differential gastric regulation of NUCB2/nesfatin-1 and ghrelin expression derived from the same cell by immune challenge.

Hypoxia Induces Intestinal Isocitrate Dehydrogenase Expression and Enhances 13C-Octanoate Metabolism in Healthy Mountaineers After Rapid Ascent to 4559 M

Background: Bacterial lipopolysaccharide (LPS) is an established animal model to study the innate immune response to gram-negative bacteria mimicking symptoms of infection including reduction of food intake. We previously reported that LPS decreased acyl ghrelin associated with decreased concentrations of circulating ghrelin-O-acyltransferase (GOAT) likely contributing to the anorexigenic effect. In addition, we recently described the prominent expression of the novel anorexigenic hormone, nucleobindin2 (NUCB2)/nesfatin-1 in gastric X/A-like cells co-localized with ghrelin in different pools of vesicles. Aim: To investigate whether LPS would affect gastric and circulating NUCB2/nesfatin-1 concentration. Methods: Ad libitum fed male Sprague-Dawley rats were equipped with an intravenous (IV) catheter placed in the right jugular vein and allowed to recover for four days. LPS was injected intraperitoneally (IP, 100μg/kg) and blood was withdrawn before and at 2, 5, 7 and 24h post injection and processed for NUCB2/nesfatin-1 measurement by radioimmunoassay (n= 5/group). Gastric corpus was collected at different time points to measure NUCB2 mRNA expression by quantitative RT-PCR and NUCB2/nesfatin-1 protein concentration by Western blot. Results: Injection of LPS significantly increased plasma NUCB2/nesfatin-1 concentrations by 55% compared to vehicle at 2h post injection (248.5 ± 45.5 vs. 161.2 ± 14.3 pg/ ml, P<0.05), whereas at all other time points (5, 7, 24h) no significant alterations were observed. The plasma NUCB2/nesfatin-1 increase at 2h was associated with an increased corpus NUCB2 mRNA expression compared to vehicle (1.22 ± 0.11 vs. 0.58 ± 0.17 pg/ml, P<0.01), whereas NUCB2 mRNA was not detectable in white blood cells. Likewise, gastric NUCB2 protein concentration was increased by 62% after LPS compared to vehicle (concentration normalized to housekeeping protein β-actin: 1.17 ± 0.06 vs. 0.72 ± 0.05, P<0.01). Conclusions: These data show that NUCB2 production and release are increased under conditions of inflammation in response to LPS. The stomach is likely to represent the source of NUCB2/nesfatin-1 as gastric mRNA and protein content was increased at the same time indicating stimulated production, whereas NUCB2 mRNA was not detectable in circulating white blood cells. These changes are in opposition to those observed for ghrelin and support the assumption of a differential regulation of these two food intake regulatory peptides derived from the same cell. The decrease of ghrelin and the increase of nesfatin-1 may act in concert to decrease food intake giving rise to the potential benefits of stimulating ghrelin and reducing nesfatin-1 signaling under conditions of inflammation. Acknowledgement: VA Merit Award (NWGL), Center grant DK-41301 (Animal Core, YT)

Nesfatin-1 Exerts a Direct, Glucose-Dependent Insulinotropic Action on Mouse Islet Beta and MIN6 Cells

Nesfatin-1 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 nesfatin-1 on insulin secretion in vitro using MIN6 cells and islets isolated from C57BL/6 mice. We also examined the expression of the nesfatin-1 precursor protein, nucleobindin 2 (NUCB2) mRNA, and nesfatin-1 immunoreactivity (ir) in the islets of normal mice and in the islets from mice with streptozotocin-induced type 1 diabetes and diet-induced obese (DIO) mice with type 2 diabetes. Nesfatin-1 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 nesfatin-1 in low glucose. In addition, a fourfold increase in nesfatin-1 release from MIN6 cells was observed following incubation in high glucose (16.7 mM) compared to low glucose (2 mM). Furthermore, we observed a significant reduction in both NUCB2 mRNA expression and nesfatin-1-ir in the pancreatic islets of mice with type 1 diabetes, while a significant increase was observed in the islets of DIO mice. Together, our findings indicate that nesfatin-1 is a novel insulinotropic peptide and that the endogenous pancreatic islet NUCB2/nesfatin is altered in diabetes and diet-induced obesity.

Molecular, Cellular and Physiological Evidences for the Anorexigenic Actions of Nesfatin-1 in Goldfish

BackgroundNesfatin-1 is a recently discovered anorexigen encoded in the precursor peptide, nucleobindin-2 (NUCB2) in mammals. To date, nesfatin-1 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 nesfatin-1 in fish.Methodology/Principal FindingsIn the present study, we employed molecular, immunohistochemical, and physiological studies to characterize the structure, distribution, and appetite regulatory effects of nesfatin-1 in a non-mammalian vertebrate. A very high conservation in NUCB2 sequences, especially in the nesfatin-1 region was found in lower vertebrates. Abundant expression of NUCB2 mRNA was detected in several tissues including the brain and liver of goldfish. Nesfatin-1-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 1 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) nesfatin-1 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 gfnesfatin-1 at a dose of 5 ng/g body weight resulted in ∼50% reduction in food intake.Conclusions/SignificanceOur results provide molecular, anatomical and functional evidences to support potential anorectic and metabolic roles for endogenous nesfatin-1 in goldfish. Collectively, we provide novel information on NUCB2 in non-mammals and an anorexigenic role for nesfatin-1 in goldfish.

Stress-related changes in the activity of cocaine- and amphetamine-regulated transcript and nesfatin neurons in the midbrain non-preganglionic Edinger–Westphal nucleus in the rat

Cocaine- and amphetamine-regulated transcript (CART) and nesfatin-1/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 nesfatin 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 nesfatin-like immunoreactivity (LI) as well as mRNA expression were analyzed in the npEW nucleus. Our results depict the following changes: (1) 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 nesfatin-LI, as shown by increased cFos immunoreactivity; (4) Chronic, but not acute stress increased the amount of CART and nesfatin-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 nesfatin are involved in the response of npEW neurons to chronic stress.

Troglitazone, a Ligand of Peroxisome Proliferator-Activated Receptor-γ, Stabilizes NUCB2 (Nesfatin) mRNA by Activating the ERK1/2 Pathway: Isolation and Characterization of the Human NUCB2 Gene

We recently identified a novel satiety peptide, nesfatin-1, containing 82 amino acids derived from the precursor peptide, nucleobindin 2 (NUCB2), from a troglitazone (TZ)-induced cDNA library. We examined the molecular mechanism underlying TZ-induced NUCB2 mRNA expression. Although TZ induced the mRNA expression in HTB185 cells, a nuclear run-on assay revealed no significant change in the transcription of the gene. Surprisingly, HTB185 cells possessed no functional peroxisome proliferator-activated receptor-gamma. We therefore examined the effect of TZ on the mRNA's stability. The half-life of NUCB2 mRNA was approximately 6 h, and incubation with TZ increased this to 27 h. Furthermore, this increase was completely inhibited by an ERK inhibitor, PD98059, and phosphorylated ERK1/2 was significantly increased after 30 min incubation with TZ. In addition, we cloned the entire NUCB2 gene and identified four adenylate/uridylate-rich elements (AREs) in the 3' untranslated region (UTR), to which several proteins of HTB185 extracts treated with TZ bound. The reporter assay fused with 3'UTR showed that the second and third AREs were crucial. Furthermore, the human NUCB2 gene spanned 55 kb and contained 14 exons and 13 introns. The transcriptional start site formed clusters around 246 bp upstream from the translational start site. We confirmed that a construct containing 5889 bp of the promoter region was very active in neuron-derived cell lines but not stimulated by TZ. These findings demonstrated a novel action of derivatives of thiazolidinediones, oral insulin-sensitizing antidiabetic agents, to stabilize the mRNA of NUCB2 through AREs in the 3'UTR by activating the ERK1/2 pathway independently of peroxisome proliferator-activated receptor-gamma.

Identification and Characterization of Nesfatin-1 Immunoreactivity in Endocrine Cell Types of the Rat Gastric Oxyntic Mucosa

Hypothalamic nesfatin-1, derived from the nucleobindin2 (NUCB2) precursor, inhibits nocturnal food intake and body weight gain in rats. Nesfatin-1 is able to cross the blood-brain barrier, suggesting a peripheral source of nesfatin-1. 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 nesfatin-1-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 nesfatin-1 and ghrelin, histidine decarboxylase, or somatostatin revealed two subtypes of nesfatin-1-positive endocrine cells. Cells in the midportion of the glands coexpressed nesfatin-1 and ghrelin, whereas few cells in the glandular base coexpressed nesfatin-1 and somatostatin or histidine decarboxylase. High-resolution three-dimensional volume imaging revealed two separate populations of intracytoplasmic vesicles in these cells, one containing nesfatin-1 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 nesfatin-1 in energy homeostasis.

Distribution and neuropeptide coexistence of nucleobindin-2 mRNA/nesfatin-like immunoreactivity in the rat CNS

The protein fragment nesfatin-1 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 nesfatin-1 antagonism causes increased food intake and overweight. Nesfatin-1 is derived from the precursor nucleobindin-2 (NUCB2). To identify the neurocircuitry underpinning the catabolic effects of NUCB2/nesfatin-1, 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 nesfatin-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 nesfatin 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, nesfatin was extensively colocalized with cocaine- and amphetamine-regulated transcript in almost all NUCB2-expressing brain regions. These data reveal a wider distribution of NUCB2/nesfatin-1 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 nesfatin-like immunoreactivity indicates that this protein may not be processed like a conventional secreted neuromodulator.