Nesfatin-1 – More than a food intake regulatory peptide

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Nesfatin-1 – More than a food intake regulatory peptide

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Andreas Stengel ∗ Charité Center for Internal Medicine and Dermatology, Division of General Internal and Psychosomatic Medicine, Charité-Universitätsmedizin Berlin, Campus Mitte, Berlin, Germany

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a r t i c l e

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Article history: Received 16 March 2015 Received in revised form 9 June 2015 Accepted 10 June 2015 Available online xxx

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Keywords: Anxiety Brain-gut Hypothalamus NUCB2 Nucleobindin2 Stomach

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1. Discovery of nesfatin-1

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Nesfatin-1 was discovered a decade ago and despite the fact that it represents just one of a multitude of food intake-inhibiting factors it received increasing attention. This led to a detailed characterization of NUCB2/nesfatin-1’s physiological property to reduce food intake and also gave rise to an involvement in the long term regulation of body weight, especially under conditions of obesity. In addition, studies indicated the involvement of NUCB2/nesfatin-1 in other homeostatic functions as well: glucose homeostasis, water intake, gastrointestinal functions, temperature regulation, cardiovascular functions, puberty onset and sleep. These pleiotropic actions underline the physiological relevance of this peptide. Recently, the involvement of NUCB2/nesfatin-1 in psychiatric disorders such as anxiety has been investigated giving rise to the speculation that NUCB2/nesfatin-1 represents a peptidergic link between eating and anxiety/depression disorders. © 2015 Published by Elsevier Inc.

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The discovery of nesfatin-1 started with the identification of a gene expressed in adipocytes and medulloblastoma cells which was responsive to a ligand for the peroxisome proliferator-activated receptor (PPAR)␥, troglitazone [68]. This gene was subsequently identified as sequence encoding nucleobindin2 [68]. Translation of nucleobindin2 (NUCB2) results in a protein consisting of 396 amino acids (aa) [68] with an aa sequence highly conserved across mammalian and non-mammalian species, a finding indicative of its phylogenetic relevance [62]. NUCB2 is post-translationally cleaved by the enzyme pro-hormone convertase (PC)-1/3 resulting in three different peptide products: N-terminal nesfatin-1 (aa 1–82), nesfatin-2 (aa 85–163) and the C-terminal nesfatin-3 (aa 166–396) [68]. Interestingly, while several biological effects have been described for nesfatin-1 [92], so far no function has been ascribed to nesfatin-2 and nesfatin-3. This review will present the state-of-knowledge on the central and peripheral expression of NUCB2/nesfatin-1 and highlight its implication in the regulation of food intake. However, also the involvement in the modulation of gastrointestinal functions and other behaviors such as drinking and sleep will be discussed.

∗ Correspondence to: Charité Center for Internal Medicine and Dermatology, Division of General Internal and Psychosomatic Medicine, Charitéplatz 1, 10117 Berlin, Germany. Tel.: +49 30 450 553 002; fax: +49 30 450 553 900. E-mail address: [email protected]

Moreover, the involvement in other homeostatic systems such as glucose control, cardiovascular functions as well as reproductive functions will also be discussed. Recent emerging evidence also points toward a role for NUCB2/nesfatin-1 in the mediation of anxiety. Therefore, this interesting topic will be highlighted as well.

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2. Expression of NUCB2/nesfatin-1

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2.1. Brain expression

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In the landmark study of Oh-I and colleagues NUCB2 mRNA and protein expression was detected in the arcuate nucleus (ARC), paraventricular nucleus (PVN) and supraoptic nucleus (SON) as well as the lateral hypothalamic area (LHA) in rats [68], brain nuclei involved in the central regulation of feeding. These expression sites were confirmed by subsequent studies, and in addition, other areas and nuclei expressing NUCB2/nesfatin-1 were identified, including the insular cortex, central amygdaloid nucleus, periventricular nucleus, tuberal hypothalamic area (THA), dorsomedial hypothalamic nucleus, Edinger–Westphal nucleus, the medullary raphe nuclei, ventrolateral medulla (VLM), locus coeruleus (LC), cerebellum, dorsal motor nucleus of the vagus nerve (DMV), nucleus of the solitary tract (NTS) as well as preganglionic sympathetic and parasympathetic neurons of the spinal cord in rats [10,21,23,29,39,40,46] and mice [28]. It is important to note that in mice a novel hypothalamic brain nucleus was identified that prominently expresses NUCB2/nesfatin-1 immunoreactivity and was named based on its localization: intermediate dorsomedial

http://dx.doi.org/10.1016/j.peptides.2015.06.002 0196-9781/© 2015 Published by Elsevier Inc.

Please cite this article in press as: Stengel A. Nesfatin-1 – More than a food intake regulatory peptide. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.06.002

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area of the hypothalamus [28]. The widespread brain distribution of NUCB2/nesfatin-1 already suggested functions beyond the initially described effect on food intake, such as an implication in autonomic functions and a role in the response to stress. In addition to the localization of brain NUCB2/nesfatin-1, several studies investigated the phenotype of the NUCB2/nesfatin1 expressing neurons. NUCB2/nesfatin-1 was colocalized with a multitude of other transmitters involved in the regulation of hunger and satiety but also other homeostatic functions, encompassing urocortin 1 (∼90%), melanin-concentrating hormone (MCH, ∼80%), pro-opiomelanocortin (POMC, ∼60–80%), cocaine-and amphetamine-regulated transcript (CART, ∼70%), ␣-melanocyte-stimulating hormone (␣-MSH, ∼60%), %), vasopressin (∼50%), oxytocin (∼40%), neuropeptide Y (NPY, ∼40%), growth hormone-releasing hormone (GHRH, ∼30%), thyrotropinreleasing hormone (TRH, ∼20%), corticotropin-releasing factor (CRF, ∼20%), somatostatin and neurotensin (∼10%) as well as serotonin [10,21,23,40,46,56,69]. It is important to note that the initial report specifically investigated the expression of NUCB2 as well as processed mature nesfatin-1 [68]. However, subsequent studies did not distinguish between full length NUCB2 and processed nesfatin-1 based on the fact that the antibodies used in these studies were raised against full length nesfatin-1 and consequently also recognize NUCB2 [10,21,28,29,46]. Therefore, the analyte should be described as NUCB2/nesfatin-1 (which has been done in the present review accordingly). Furthermore, it should be noted that mature nesfatin1 (9.7 kDa) has been only described in the initial study of Oh-I et al. with an occurrence in the cerebrospinal fluid and rat hypothalamic extracts [68] as well as in one study investigating human plasma [99]. In contrast, subsequent studies only detected full length NUCB2 in different tissues such as rat hypothalamus, pituitary, adipose tissue, gastric mucosa and pancreas as well as goldfish brain (∼50 kDa) [21,25,31,74,89]. This is likely not due to the detection method since synthetic nesfatin-1 was detectable by Western Blot as indicated by a band at the expected size around 10 kDa [74,89]. Optimized detection methods such as the use of a sandwich-type nesfatin-1 ELISA assay without cross-reaction with full length NUCB2 [99] along with stringent peptidase inhibition to maximize the peptide yield may help to tackle this problem. However, one has to keep in mind that also full length NUCB2 exerts a biological effect on food intake [68] and may therefore be the biologically relevant form. Ten years after the discovery of the ligand and in contrast to the increasing knowledge on the expression of NUCB2/nesfatin-1, the corresponding receptor still remains to be identified. As expected, the receptor is likely to be expressed in the hypothalamus based on data using radiolabeled nesfatin-1 which was shown to bind to membrane preparations of mouse hypothalamus [41]. In addition, first evidence points toward a Gi/o-protein-coupled receptor [10,41] which induces Ca2+ influx via N-type channels in vagal afferent nodose ganglion neurons [42], T-type channels in DMV neurons [104] and L- and P/Q type Ca2+ channels in the hypothalamus [10,56]. Lastly, nesfatin-1 increased the cAMP response element (CRE) reporter activity in a mouse neuroblastoma cell line, NB41A3, an effect abolished by an L-type Ca2+ channel blocker [41]. The identification and subsequent modulation (tissue specific overexpression or knockout) of the receptor will represent a huge leap forward to understand NUCB2/nesfatin-1’s physiology.

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2.2. Peripheral expression

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Besides its expression in the brain, NUCB2/nesfatin-1 is – similarly to a multitude of other peptide hormones – also expressed in peripheral tissues. These encompass anterior pituitary gland, adipose tissue, heart, pancreas, stomach and testis of rat and goldfish

[20,22,31,34,74,89]. Importantly, the expression of NUCB2 mRNA in the stomach by far exceeds the rat brain expression with tentimes higher gastric expression levels [89]. Further purification of the cells expressing NUCB2/nesfatin-1 in the stomach showed a predominant occurrence in gastric small endocrine cells and interestingly, a co-localization with ghrelin in rat gastric X/A-like cells [89]. This finding was also demonstrated in humans with colocalization of ghrelin and NUCB2/nesfatin-1 in human P/D1 cells [90] giving rise to a differential release of these two peptide products and a modulation of hunger and satiety in two directions by this one cell type (stimulation of food intake by ghrelin versus inhibition by NUCB2/nesfatin-1). In the pancreas, NUCB2/nesfatin-1 is co-localized with insulin in ␤-cells of the islets of Langerhans [22]. Taken together, the widespread peripheral distribution of NUCB2/nesfatin-1 points toward an involvement of the peptide in several homeostatic pathways in addition to the modulation of food intake. These effects could be directly mediated in the periphery in an autocrine, paracrine and endocrine manner but also via crossing of the blood-brain barrier which has been shown before [70,73].

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3. Effect of NUCB2/nesfatin-1 on food intake

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Converging evidence describes the anorexigenic effect of centrally injected nesfatin-1. In the initial study nesfatin-1 was injected into the third brain ventricle through a chronically implanted cannula and shown to dose-dependently reduce dark phase food intake in rats fed ad libitum [68]. These findings were confirmed by several independent groups and also extended showing an anorexigenic effect of nesfatin-1 following injection into the lateral, third and fourth brain ventricle, into the cisterna magna or directly into the PVN, LHA or dorsal vagal complex (DVC) in mice [4,30], rats [12,18,48,56,63,88,104,107,108] and goldfish [31,44]. Following up on these initial findings, studies further characterized the kinetics of nesfatin-1’s anorexigenic effects. After injection into the lateral brain ventricle (intracerebroventricular, icv) of rats, nesfatin-1 reduced dark phase food intake with a delayed onset (maximum 87% reduction during the third hour post injection) and a long duration of action (lasting 6–48 h) [48,88]. This finding was reproduced in mice with a delayed (2 h) onset of the anorexigenic effect following icv injection and a duration of 8 h [4]. Interestingly, when injected into the third or fourth brain ventricle or into the cisterna magna, the anorexigenic action of nesfatin-1 was already observed during the first hour post injection in rats [68,88] giving rise to a different downstream mediation of nesfatin-1’s effect in forebrain versus hindbrain. The microstructure underlying these anorexigenic effects was recently characterized using an automated food intake monitoring system. Nesfatin-1 injected acutely icv in mice decreased dark phase food intake via a reduction of meal size (indicating an increase of satiation) as well as meal frequency along with a prolongation of inter-meal intervals (as an indicator of increased satiety) [30]. In addition, the mid fragment nesfatin-130–59 , recently identified as the active core of nesfatin-1 [84], injected icv in rats also reduced food intake via an increase of inter-meal intervals, while meal size was not altered [87]. This difference may be related to species differences in mice versus rats or indicate different receptors/receptor areas responding to full length versus mid fragment nesfatin-1 and/or differential downstream signaling leading to the different microstructures observed. The effect on food intake is likely to be specific as other behaviors such as locomotion [48,68,88] or grooming (including scratching, licking and washing) [88] were not altered (Fig. 1).

Please cite this article in press as: Stengel A. Nesfatin-1 – More than a food intake regulatory peptide. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.06.002

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The anorexigenic action exerted by nesfatin-1 is likely to represent a physiological effect based on several key findings. First, blockade of endogenous brain NUCB2/nesfatin-1 signaling using an anti-nesfatin-1 antibody or an anti-NUCB2 antisense oligonucleotide injected into the third brain ventricle resulted in an increase of food intake in rats [56,68]. Interestingly, hypothalamic knock-down of NUCB2/nesfatin-1 using anti-NUCB2 morpholino oligonucleotides resulted in a more pronounced decrease of hypothalamic NUCB2 content in female compared to male rats, however, daily food intake was not altered [25,106]. Whether sex differences play a role in the nesfatin-1-induced modulation of food intake will have to be further established. Second, hypothalamic NUCB2/nesfatin-1 is regulated dependent on the feeding state as well as according to the light cycle of the animal. NUCB2 mRNA and protein expression in the SON and PVN were reduced under fasting conditions and levels were restored after re-feeding in rats [25,46,68] and goldfish [31]. Moreover, NUCB2 mRNA levels in the PVN were increased during the early light phase, a photoperiod where rats show their minimum food intake, and decreased during the dark phase associated with higher food intake [82]. Lastly, rats with PVN-selective knockdown of NUCB2 mRNA using shRNA or immunoneutralization of PVN NUCB2/nesfatin-1 by an antinesfatin-1-IgG showed an increased food intake during the light but not dark phase [82] giving rise to a physiological inhibitory tone exerted by brain NUCB2/nesfatin-1 on light phase food intake. As observed for several other peptide hormones, NUCB2/nesfatin-1 also interacts with several other transmitters to modulate food intake. Early on, it was demonstrated that nesfatin-1’s anorexigenic effect is independent from leptin signaling since the effect is still observed in Zucker rats with leptin receptor deficiency [56,68] nourishing hope for nesfatin-1 as a promising anti-obesity target since leptin resistance is common under conditions of obesity [78]. Conversely, leptin signaling seems to be dependent on NUCB2/nesfatin-1 as peripheral and central injections of leptin failed to reduce food intake in mice treated with adeno-associated virus vectors encoding shRNA targeting NUCB2 mRNA expression in the PVN [16].

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Brain NUCB2/nesfatin-1 also directly interacts with the wellestablished brain orexigenic peptide neuropeptide Y (NPY). Administration of nesfatin-1 directly onto arcuate neurons in vitro was shown to hyperpolarize NPY positive neurons [72] and downregulated NPY expression in vivo [102], likely to contribute to NUCB2/nesfatin-1’s anorexigenic effect. Conversely, NPY inhibits NUCB2/nesfatin-1 neurons in the PVN in vitro [81]. Early on, an interaction of NUCB2/nesfatin-1 and CRF2 signaling has been described. Activation of the brain CRF2 signaling system is well established to reduce food intake [94,113]. Injection of nesfatin-1 into the third brain ventricle in rats increases the CRF content in the PVN [35]. Moreover, when applied directly onto PVN neurons in vitro, nesfatin-1 increases the excitability of CRF positive neurons [71]. Lastly, when injected icv nesfatin-1 increases plasma levels of ACTH and corticosterone [47] indicating activation of the hypothalamic-pituitary-adrenal axis. The interplay with the CRF signaling system is of functional relevance for icv nesfatin-1’s anorexigenic effect as a blockade of CRF2 receptors by icv injection of the selective peptide CRF2 antagonist, astressin2 -B abolished or third ventricle injection of the CRF1 /CRF2 antagonist, ␣-helical CRF9–41 blunted nesfatin-1’s food intake reducing effect [35,88]. Interestingly, when nesfatin-1 was injected into the cisterna magna, astressin2 -B did not alter the anorexigenic effect [88] giving rise to other downstream mediators involved in nesfatin-1’s anorexigenic effects at the level of the brainstem. Other studies also indicate an involvement of oxytocin and ␣-MSH signaling in the mediation of nesfatin-1’s food intake inhibitory effects. Nesfatin-1 was shown to activate oxytocin containing neurons in the PVN following third ventricular injection [56]. In line with these findings, nesfatin-1 stimulated the release of oxytocin from PVN neurons in vitro [56]. Demonstrating the functional relevance of this interaction, fourth ventricular injection of the oxytocin receptor antagonist, H4928 blocked nesfatin-1’s food intake reducing effect in rats [56,108]. Since oxytocinergic nerve terminals from the PVN that project to the NTS [45] are in close proximity to POMC containing neurons and oxytocin stimulates the release of POMC in the NTS [56], this transmitter was suggested to

Fig. 1. Pleiotropic effects of NUCB2/nesfatin-1. Stimulatory effects are indicated in green, inhibitory in red. ↑ increase, ↓ decrease.

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play a role further downstream to mediate nesfatin-1’s anorexigenic effect. POMC is proteolytically cleaved into several peptides including ␣-MSH, the endogenous ligand of the melanocortin 3/4 receptor [80]. Corroborating the involvement of POMC/␣-MSH signaling in the downstream mediation of nesfatin-1’s food intake suppressive effect, blockade of the ␣-MSH-melanocortin 3/4 receptor with the antagonist SHU9119 injected icv or into the third brain ventricle blocked the anorexigenic effect of nesfatin-1 injected icv or into the third brain ventricle [68,107]. Moreover, ␣-MSH was shown to stimulate NUCB2/nesfatin-1 neurons of the PVN in vitro [81]. Taken together, nesfatin-1 is likely to signal via a hypothalamic-pontine oxytocin-POMC-␣-MSH-melanocortin 3/4 receptor signaling pathway to exert its anorexigenic effect. In addition, limited evidence also indicated an interplay of NUCB2/nesfatin-1 and hypothalamic TRH as the third ventricular injection of a TRH antibody blunted the nesfatin-1 induced anorexigenic effect [35]. Moreover, TRH mRNA expression in the PVN was up-regulated following third ventricular injection of nesfatin1 [35] leading to the speculation that both peptides act together in order to reduce food intake as part of a feed forward mechanism, a hypothesis that warrants further investigation. Pilot evidence also points toward an involvement of NUCB2/nesfatin-1 signaling in the rewarding aspect of food based on the observation that nesfatin-1 hyperpolarized nigral dopaminergic neurons in vitro [51]. This has to be further investigated. Also hypothalamic histamine and serotonin (5-HT) were suggested to contribute to the anorexigenic signaling of NUCB2/nesfatin-1 via an interaction with the H1 and 5-HT2C receptors, respectively [35,66]. Injection of nesfatin-1 into the rat third brain ventricle increased the turnover of hypothalamic histamine, and conversely, histamine increased the expression of NUCB2/nesfatin-1 in the PVN giving rise to a feed forward mechanism [35]. This is likely to be of functional relevance as mice lacking the H1 receptor or rats with inhibited histidine decarboxylase which results in blunted hypothalamic histamine signaling, displayed a reduced anorexigenic effect following third brain ventricular injection of nesfatin-1 [35]. Similarly, also mice lacking the 5-HT2C displayed a reduction of hypothalamic NUCB2 resulting in an increase of food intake [66]. Conversely, intraperitoneal (ip) injection of the 5-HT1B/2C agonist, m-chlorophenylpiperazine (mCPP) up-regulated hypothalamic NUCB2 mRNA expression [66]. Taken together, H1 and 5-HT2C signaling act in concert with NUBC2/nesfatin-1 in order to reduce food intake. In addition to these brain transmitters, also several peripheral peptide hormones were shown to affect NUCB2/nesfatin-1 signaling. Ip injection of desacyl ghrelin, a peptide that was reported to block the orexigenic effect of ghrelin, led to an activation of NUCB2/nesfatin-1 immunoreactive neurons in the ARC [39], a mechanism which may contribute to desacyl ghrelin’s food intake modulatory effects. Moreover, cholecystokinin (CCK), an anorexigenic hormone predominantly produced in endocrine cells of the duodenum [17], injected ip activated NUCB2/nesfatin-1 neurons in the hypothalamus and brainstem as assessed by Fos immunoreactivity [65,88]. Whether NUCB2/nesfatin-1 is involved in the mediation of CCK’s anorexigenic effects warrants further investigation. The characterization of the interactions of NUCB2/nesfatin-1 with other peripheral food intake modulatory transmitters will shed more light on the complex regulation of hunger and satiety.

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Subsequently to the central effects, also peripheral actions of nesfatin-1 on food intake have been investigated. These investigations were driven by the finding of the prominent expression of NUCB2/nesfatin-1 in the stomach [89]. However, one has to

note that the limited amount of peripheral data contrasts with the multitude of studies describing an anorexigenic effect of central NUCB2/nesfatin-1. Early on, it was shown that fasting decreases circulating levels of NUCB2/nesfatin-1 and re-feeding restores these levels, a kinetic of a peptide likely involved in the regulation of food intake [88]. In the stomach the mammalian target of rapamycin (mTOR) was identified to modulate the expression of NUCB2/nesfatin-1 [54] likely underlying the changes observed under conditions of fasting. One study reported a dark phase food intake reducing effect in mice following ip nesfatin-1 injected at a high dose as well as after ip nesfatin-130–59 , the active core of nesfatin-1 [84] (Fig. 1). In line with its central properties, this effect was independent of leptin signaling [84]. Similarly, continuous peripheral infusion of nesfatin-1 reduced cumulative food intake in rats [32]. The gutbrain signaling may involve the vagus nerve as nesfatin-1 activates Ca2+ influx in primary cultured nodose ganglion neurons in vitro [42]. This assumption is further supported by the finding that chemical de-afferentiation using capsaicin prevents the anorexigenic effect of ip nesfatin-130–59 in mice [85]. However, few studies failed to reproduce these data in rats [88] or mice [30], although experimental conditions and doses used were similar. Whether strain differences or other – yet to be better characterized – factors play a role for these inconsistent findings will have to be further established. Lastly, also in goldfish peripheral injection of nesfatin-1 did only subtly alter food intake although used at higher doses compared to the dose used icv which exerted a robust anorexigenic effect [31]. Therefore, the food intake suppressing effect of peripheral NUCB2/nesfatin-1 may not be the only/main effect of this peptide. 4. Other effects of NUCB2/nesfatin-1 that may affect food intake 4.1. Modulation of water intake In line with the well described suppressive effect on food intake, nesfatin-1 injected icv also reduces water intake [106]. This antidipsogenic effect was observed under different conditions such as after water restriction, following injection of angiotensin II or after a hypertonic challenge [106]. Interestingly, the antidipsogenic action of nesfatin-1 seems to be dissociated from the anorexigenic effect as higher doses injected icv are required for suppression of water intake [107]. However, when injected directly into the subfornical organ, a brain structure important in the regulation of fluid homeostasis, low doses similar to those inhibiting food intake also potently stimulated water intake [63]. This response was independent of angiotensinergic pathways and not related to food intake [63]. Further corroborating the importance of these findings, nesfatin-1 injected icv activated neurons in the subfornical organ as assessed by Fos immunohistochemistry [63] and also directly activated these neurons in vitro [49]. Future studies will have to clarify the direction of the alteration of water intake by NUCB2/nesfatin-1 (stimulation versus inhibition) and also establish whether these are physiological effects (Fig. 1). 4.2. Modulation of body temperature Besides the modulation of food intake several peptide hormones also affect energy expenditure [76]. Recently, also nesfatin-1 injected icv was shown to increase body temperature in rats as expressed as dry heat loss [102]. This effect was similar to the thermogenic action of leptin while no additive effects were observed following co-injection of both peptides [102] leading to the suggestion that similar/the same downstream pathways are

Please cite this article in press as: Stengel A. Nesfatin-1 – More than a food intake regulatory peptide. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.06.002

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involved. Interestingly, also the thermogenic effect of nesfatin-1 icv was delayed with an onset during the third hour [102], a kinetic described for food intake before [88]. However, this effect on body temperature seems to be longer lasting since it was observed for up to 48 h and – as described for food intake before – not related to an increase in locomotor activity [48]. The increase in energy expenditure may be part of brain NUCB2/nesfatin-1’s physiological effect to exert a negative energy balance (Fig. 1).

4.3. Modulation of sleep Emerging evidence points toward an association of sleep and food intake and – more importantly – a causal relationship between impaired sleep and the risk for the development of obesity [13,14]. In line with this association, several peptides involved in the regulation of food intake were also shown to affect sleep such as ghrelin [86], leptin [27] and MCH [79]. Since NUCB2/nesfatin-1 is co-localized with MCH in the LHA [21] and THA [43] an involvement in the modulation of sleep has been proposed for NUCB2/nesfatin-1 as well. Inhibition of endogenous NUCB2/nesfatin-1 signaling using icv anti-nesfatin-1 antiserum or anti-NUCB2 antisense reduced paradoxical sleep, while slow wave sleep was not altered [43]. Moreover, nesfatin-1 injected icv reduced rapid eye movement (REM) sleep in rats, while increasing slow wave sleep [100]. Lastly, paradoxical sleep activates NUCB2/nesfatin-1 immunoreactive neurons of the THA as assessed by Fos immunohistochemistry [43], while REM sleep deprivation led to a down-regulation of NUCB2 mRNA and NUCB2/nesfatin-1 peptide levels with a restoration during rebound phases [100]. Taken together, these data point toward an involvement of NUCB2/nesfatin-1 in the modulation of sleep (Fig. 1).

4.4. Modulation of gastrointestinal functions As observed for several food intake regulatory peptides before [91], also NUCB2/nesfatin-1 was implicated in the regulation of gastrointestinal functions. Nesfatin-1 injected into the fourth brain ventricle inhibited the vagally mediated stimulation of gastric acid secretion by 2-deoxy-d-glucose-, whereas the basal or pentagastrin stimulated gastric acid secretion was not altered [104]. In addition, nesfatin-1 was also shown to affect gastrointestinal motility. When injected icv, nesfatin-1 inhibited gastric emptying in rats [88] and mice [28]. In addition, in mice icv nesfatin-1 was also shown to slow down gastroduodenal motility [4]. When injected directly into the ARC [55] or PVN [37], nesfatin-1 also reduced gastric motility in rats (Fig. 1). Conversely, blockade of endogenous NUCB2/nesfatin-1 signaling using an anti-NUCB2/nesfatin-1 antibody injected into the ARC or PVN enhanced gastric motility giving rise to a physiological effect [37,55] which may contribute to the anorexigenic action as gastric distension increases vagal afferent activity [15,95]. In addition, also NUCB2/nesfatin-1 neurons in the NTS were activated by gastric distention [8], which may indicate an additional mechanism to further reduce gastrointestinal motility. Lastly, also peripheral NUCB2/nesfatin-1 may contribute to these alterations as rats with a lesion in the ventromedial hypothalamic nucleus showed increased gastric emptying which was associated with higher expression levels of NUCB2 mRNA and NUCB2/nesfatin-1 peptide in the stomach and duodenum [98]. In line with the assumption of a peripheral action, also intravenous (iv) injection of nesfatin-1 in dogs reduced gastric motility and inhibited cyclical interdigestive migrating contractions under conditions of fasting [101]. These potential peripheral mechanisms will have to be further established.

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5. Implication of NUCB2/nesfatin-1 in homeostatic systems other than food intake 5.1. Implication in glucose control Besides the regulation of food intake, NUCB2/nesfatin-1 was also early on implicated in the regulation of glucose homeostasis. This was suggested by the co-localization of NUCB2/nesfatin-1 and insulin in the rodent and human pancreas [22,33,34,61,77]. Following glucose challenge, NUCB2/nesfatin-1 was shown to be released from these cells [22,32]. However, an oral glucose tolerance test in healthy humans failed to alter circulating NUCB2/nesfatin-1 levels [52,99] pointing toward a local autocrine/paracrine mode of action of NUCB2/nesfatin-1. Giving further rise to a physiological importance of this expression, in vitro studies showed that nesfatin-1 increases the expression of pre-proinsulin mRNA and stimulates the release of insulin induced by glucose via L-type channels [32,33,64]. Interestingly, another study also reported an increase in glucagon release following treatment with nesfatin-1 in isolated mouse islets or INS1 (832/13) cells in vitro [77], a finding to be confirmed in future studies. The effects of nesfatin-1 on insulin were also observed in vivo as an intravenous injection of nesfatin-1 decreased blood glucose levels in hyperglycaemic db/db mice [93], likely due to a stimulated incretin-amplified [75] insulin-induced glucose uptake [32] involving increased GLUT4 membrane translocation in skeletal muscle and adipose tissue [53] (Fig. 1). The implication of NUCB2/nesfatin-1 in glucose control is also supported by alterations observed in the brain where glucose and insulin activated NUCB2/nesfatin-1 containing neurons of the PVN [24], while NUCB2/nesfatin-1 positive neurons of the DMV were activated by hypoglycemia [7], a mechanism that needs to be further studied. The importance of central NUCB2/nesfatin-1 signaling in glucose homeostasis was highlighted in a recent study that showed decreased peripheral glucose uptake following hypothalamic knockdown NUCB2/nesfatin-1 in rats [103]. As expected from a peptide involved in glucose control, the pancreatic expression of NUCB2/nesfatin-1 is altered under conditions of diabetes mellitus. The protein expression of NUCB2/nesfatin-1 is reduced in islets of type 2 diabetic Goto-Kakizaki rats compared to normoglycaemic Wistar rats [22], a finding that held also true in diabetic humans [77]. This altered expression was also associated with changes in the circulating levels of NUCB2/nesfatin-1 with lower levels under conditions of diabetes-associated hyperglycemia in rats [22] and humans [52]. These changes were already visible in subjects with normoglycemic insulin resistance pointing toward an involvement of NUCB2/nesfatin-1 in the early stages of diabetes development [5]. One has to note that one study in Chinese subjects with newly diagnosed type 2 diabetes mellitus or impaired glucose tolerance showed higher circulating NUCB2/nesfatin-1 levels compared to healthy controls [112]. Whether this discrepant result is due to genetic background or other yet to be characterized confounding factors will have to be further established. Taken together, NUCB2/nesfatin-1 is a glucoregulatory insulinotropic peptide that is reduced under conditions of diabetes mellitus, a pathophysiological adaptation that could worsen the condition. This hypothesis is corroborated by the observation that blood glucose and insulin resistance coefficient were reduced following intravenous injection of nesfatin-1 in diabetic mice [19]. 5.2. Implication in cardiovascular functions NUCB2/nesfatin-1 was also implicated in other autonomic regulatory pathways such as the control of blood pressure. Icv injection of nesfatin-1 increased mean arterial pressure in rats [107], an action likely mediated via the NTS based on observations of

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microinjection studies [58] (Fig. 1). The effect was blocked by pretreatment with the melanocortin 3/4 receptor antagonist, SHU9119 [107]. Since also CRF2 as well as oxytocin receptor blockade prevented the hypertensive effect of nesfatin-1, also downstream CRF and oxytocin signaling plays a role in the mediation of the nesfatin-1-induced increase of mean arterial pressure [109]. Since also alpha-adrenergic blockade by intraarterial injection of phentolamine prevented the nesfatin-1-induced rise of mean arterial pressure [107], nesfatin-1 likely exerts its effect via stimulation of sympathetic nerve activity. This hypothesis was corroborated by a subsequent study measuring sympathetic nerve outflow to the kidney in rats [97]. In addition, peripheral nesfatin-1 also inhibited the sodium nitroprusside-induced relaxations of smooth muscle resulting in an increase of blood pressure [105]. Interestingly, microinjection of nesfatin-1 into the nucleus ambiguous induced bradycardia in rats [9] pointing toward a modulation of both sympathetic as well as parasympathetic activity by brain NUCB2/nesfatin-1. 5.3. Implication in reproduction Central NUCB2/nesfatin-1 may play an important role in reproduction as well. During puberty, the hypothalamic expression of NUCB2 mRNA and protein is increased in female rats [25]. This is likely of physiological importance as icv injection of nesfatin-1 stimulated the release of luteinizing hormone and follicle-stimulating hormone in pubertal rats and conversely, the inhibition of NUCB2 signaling using an anti-NUCB2 antisense oligonucleotide injected icv delayed puberty in female rats [25] (Fig. 1). Similar to the effects in female, NUCB2/nesfatin-1 seems to be involved in reproductive functions also in male. NUCB2 mRNA is expressed in interstitial mature Leydig cells of mice, rats and humans and shown to be upregulated by luteinizing hormone [26]. In addition, nesfatin-1 increased the human choriogonadotropinstimulated testosterone secretion in rat testis in vitro [26]. Conversely, also testosterone status in growing male rats affected the expression levels of NUCB2 mRNA in adipose tissue and stomach [83]. Future studies are needed to investigate the physiological relevance of NUCB2/nesfatin-1 in male puberty. 6. Implication of NUCB2/nesfatin-1 in obesity Since nesfatin-1 potently affects food intake and also energy expenditure, several studies investigated NUCB2/nesfatin-1 under conditions of obesity to describe potential associations in the long term regulation of body weight. On a genetic basis, three single nucleotide polymorphisms of the NUCB2 gene were identified to be associated with obesity when investigating a large mixed sex population of 1049 obese and 315 normal weight Caucasian subjects [111]. It is to note that this association – when analyzing the sexes separately – was only observed in male subjects [111], possibly representing a risk factor for male obesity. Moreover, in a population of 471 obese children and adolescents seven sequence variants of the NUCB2 gene were observed [110]. Although these alterations did not affect circulating levels of NUCB2/nesfatin-1 [110], possible changes in NUCB2/nesfatin-1’s brain physiology may increase the susceptibility for the development of obesity. This has to be further investigated. Since congenital prohormone convertase 1 deficiency is characterized by early-onset obesity, this genetic condition may involve altered NUCB2/nesfatin-1 signaling (since prohormone convertase cleaves NUCB2 into nesfatin-1, 2 and 3) [11], a hypothesis that warrants further studies. Lastly, obese Tsumura Suzuki diabetic mice, a polygenic model of obesity, showed reduced NUCB2 mRNA and protein levels in the

hypothalamus which may contribute to the hyperphagia observed in these animals [59]. Taken together, several genetic conditions altering NUCB2/nesfatin-1 signaling may increase the susceptibility for the development of obesity. In humans, the cerebrospinal fluid (CSF)/plasma NUCB2/nesfatin-1 ratio was negatively correlated with body mass index (BMI) and body fat due to an elevation of plasma NUCB2/nesfatin-1 with increasing BMI, while CSF NUCB2/nesfatin-1 levels were only mildly increased [96]. Moreover, NUCB2/nesfatin-1 plasma levels were shown to be lower in female subjects with anorexia nervosa [67], while increased levels have been reported in a mixed sex population of obese [3,74,96] resulting in a positive correlation of NUCB2/nesfatin-1 and BMI [3,67,74]. In line with these findings, gastric NUCB2/nesfatin-1 protein expression was elevated with increasing BMI in a population of male and female obese patients undergoing sleeve gastrectomy bariatric surgery [90]. This increase may represent a compensatory regulatory mechanism to stimulate NUCB2/nesfatin-1’s anorexigenic signaling to prevent further overeating. On the other hand, chronic intake of a high fat obesogenic diet was shown to reduce gastric NUCB2 mRNA expression in mice [60], a regulation that could aggravate the obese situation. Conversely, patients that underwent bariatric surgery displayed decreased NUCB2/nesfatin1 plasma levels at 12 months after surgery, which was related to the decrease in BMI [50]. However, another study failed to show an association between circulating NUCB2/nesfatin-1 levels and BMI in children [2] or even indicated a negative association in a population of non-obese males [99] or normal weight and obese children [1]. It remains to be established whether age (adults versus children), gender differences (females versus males), different assessment methods (ELISA recognizing NUCB2 and nesfatin-1 versus sandwich-type ELISA recognizing only nesfatin-1), the body weight range (normal weight versus underweight and obesity) or yet to be identified confounding factors account(s) for these inconsistent findings.

7. Involvement of NUCB2/nesfatin-1 in anxiety Early on, an involvement of NUCB2/nesfatin-1 in the mediation of anxiety-like behaviors has been suggested. In rats, a dose of 25–80 pmol injected icv induced a heightened startle response and a decrease in the time spent in the open arms of the elevated plus maze in rats [57,107] (Fig. 1). This dose is higher compared to the low dose (5 pmol) necessary to induce the anorexigenic effect following icv injection of nesfatin-1 [68,88]. Therefore, it remains to be established whether this represents a physiological effect or rather a pharmacological property. Several studies in humans support the concept of NUCB2/nesfatin-1 signaling being involved in the modulation of anxiety. In a population of obese patients, the ones with high anxiety assessed by the well-established GAD-7 questionnaire displayed higher circulating levels of NUCB2/nesfatin-1 [38]. This association was also reflected in a positive correlation of plasma NUCB2/nesfatin-1 and anxiety scores [38]. Interestingly, male subjects with generalized anxiety disorder displayed reduced circulating NUCB2/nesfatin-1 levels compared to age matched healthy controls [36], a finding that gives rise to a possible sexspecific regulation of NUCB2/nesfatin-1. The possibility of a sex difference was also supported by a study investigating NUCB2 mRNA expression in the Edinger–Westphal nucleus of subjects who committed suicide because of a major depressive disorder compared to the expression in subjects who died without a psychiatric disorder [6]. While male suicide victims displayed higher NUCB2 mRNA levels in the Edinger–Westphal nucleus, females showed lower expression levels compared to their respective

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non-psychiatric controls [6]. This warrants further investigation. Interestingly, the association between circulating NUCB2/nesfatin1 and anxiety and depression seems to be stronger than the one with BMI, at least in one study investigating obese females over a broad range of BMI and anxiety scores [38]. Whether the observed alterations of NUCB2/nesfatin-1 under conditions of obesity are related to – or even secondary to – differences in anxiety signaling will have to be further characterized in future studies, also investigating NUCB2/nesfatin-1 in a longitudinal manner. 8. Summary and conclusion Since its discovery in 2006, the past 10 years have witnessed a tremendous increase in our knowledge on NUCB2/nesfatin-1 and established this peptide as a physiological regulator of food intake. However, it remains to be established whether this holds true exclusively for brain NUCB2/nesfatin-1 or whether also peripherally expressed NUCB2/nesfatin-1 is involved. If not, the role for peripheral NUCB2/nesfatin-1, especially of gastric origin – since this is by far the major expression site – will have to be established. In line with a role in short term regulation of food intake as well as long term modulation of body weight, several NUCB2 polymorphisms have been identified in obese subjects that may represent a risk factor for the development of obesity. Besides the alteration of food intake, several other pathways are influenced by NUCB2/nesfatin-1 leading to a modulation of gastrointestinal functions, body temperature as well as sleep. Interestingly, increasing evidence points toward an involvement of NUCB2/nesfatin-1 in anxiety as well. Since psychiatric disorders are very common in subjects with obesity, this association warrants further investigation and may indicate NUCB2/nesfatin-1 as a connector between eating and anxiety/depression disorders. Conflicts of interest The author has nothing to disclose. No conflicts of interest exist. Acknowledgements This work was supported by German Research Foundation (STE 1765/3-1) and Charité University Funding (UFF 89-441-176). I would like to thank Dr. Abba Kastin for his enduring support and the always very pleasant and fruitful cooperations. References [1] Abaci A, Catli G, Anik A, Kume T, Bober E. The relation of serum nesfatin-1 level with metabolic and clinical parameters in obese and healthy children. Pediatr Diabetes 2013;14:189–95. [2] Anik A, Catli G, Abaci A, Kume T, Bober E. Fasting and postprandial levels of a novel anorexigenic peptide nesfatin in childhood obesity. J Pediatr Endocrinol Metab 2014;27:623–8. [3] Anwar GM, Yamamah G, Ibrahim A, El-Lebedy D, Farid TM, Mahmoud R. Nesfatin-1 in childhood and adolescent obesity and its association with food intake, body composition and insulin resistance. Regul Pept 2014;188:21–4. [4] Atsuchi K, Asakawa A, Ushikai M, Ataka K, Tsai M, Koyama K, et al. Centrally administered nesfatin-1 inhibits feeding behaviour and gastroduodenal motility in mice. Neuroreport 2010;21:1008–11. [5] Basar O, Akbal E, Koklu S, Kocak E, Tuna Y, Ekiz F, et al. A novel appetite peptide, nesfatin-1 in patients with non-alcoholic fatty liver disease. Scand J Clin Lab Invest 2012;72:479–83. [6] Bloem B, Xu L, Morava E, Faludi G, Palkovits M, Roubos EW, et al. Sex-specific differences in the dynamics of cocaine- and amphetamine-regulated transcript and nesfatin-1 expressions in the midbrain of depressed suicide victims vs. controls. Neuropharmacology 2012;62:297–303. [7] Bonnet MS, Djelloul M, Tillement V, Tardivel C, Mounien L, Trouslard J, et al. Central NUCB2/Nesfatin-1-expressing neurones belong to the hypothalamic-brainstem circuitry activated by hypoglycaemia. J Neuroendocrinol 2013;25:1–13.

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