Nesfatin-1 Receptor: Distribution, Signaling and Increasing Evidence for a G Protein-Coupled Receptor – A Systematic Review

Background Nesfatin-1 is an 82-amino acid polypeptide, cleaved from the 396-amino acid precursor protein nucleobindin-2 (NUCB2) and discovered in 2006 in the rat hypothalamus. In contrast to the growing body of evidence for the pleiotropic effects of the peptide, the receptor mediating these effects and the exact signaling cascades remain still unknown. Methods This systematic review was conducted using a search in the Embase, PubMed, and Web of Science databases. The keywords “nesfatin-1” combined with “receptor”, “signaling”, “distribution”, “pathway”, g- protein coupled receptor”, and “binding” were used to identify all relevant articles reporting about potential nesfatin-1 signaling and the assumed mediation via a Gi protein-coupled receptor. Results Finally, 1,147 articles were found, of which 1,077 were excluded in several steps of screening, 70 articles were included in this systematic review. Inclusion criteria were studies investigating nesfatin-1’s putative receptor or signaling cascade, observational preclinical and clinical studies, experimental studies, registry-based studies, cohort studies, population-based studies, and studies in English language. After screening for eligibility, the studies were assigned to the following subtopics and discussed regarding intracellular signaling of nesfatin-1 including the potential receptor mediating these effects and downstream signaling of the peptide. Conclusion The present review sheds light on the various effects of nesfatin-1 by influencing several intracellular signaling pathways and downstream cascades, including the peptide’s influence on various hormones and their receptors. These data point towards mediation via a Gi protein-coupled receptor. Nonetheless, the identification of the nesfatin-1 receptor will enable us to better investigate the exact mediating mechanisms underlying the different effects of the peptide along with the development of agonists and antagonists.


INTRODUCTION
Since the discovery of nesfatin-1 in 2006 in the rat hypothalamus (1), there has been a vast amount of research to further explore the pleiotropic physiological functions of this peptide. Nesfatin-1 is an 82-amino acid polypeptide generated via post-translational processing of hypothalamic nucleobindin-2 (NUCB2), a protein of 396 amino acids, whose sequence is highly conserved from fish to mammals (2) pointing towards its physiological relevance. The peptide is predominantly localized in food intake-regulatory nuclei e.g., the arcuate nucleus (Arc), the paraventricular nucleus (PVN) and the nucleus of the solitary tract (3,4). Accordingly, the peptide became first known for its anorexigenic effects; however, subsequent studies unraveled its many other properties, such as cardiovascular effects, lipid metabolism, reproduction functions, and emotion-related functions (5)(6)(7). Subsequently, the peptides' central part, nesfatin-1  , which has been identified as the active core of the peptide, was reported to reduce food intake after intracerebroventricular (icv) injection in mice (8,9) and in rats (10).
However, multiple studies have indicated that nesfatin-1 is also secreted by peripheral tissues such as adipose tissue, gastric mucosa, pancreatic endocrine beta cells and testes and its expression level was found to be 20 times higher in endocrine cells of the oxyntic gastric mucosa than in the brain (11)(12)(13).
Numerous studies have reported multifunctional effects of nesfatin-1: peripheral nesfatin-1 affects glucose homeostasis (14) and shows anti-apoptotic and anti-inflammatory effects (15). In addition, peripherally administered nesfatin-1 induces vasoconstriction and elevates blood pressure (16), and moreover decreases antral and duodenal motility (17) and reduces gastric emptying (18). Taken together, the role of nesfatin-1 goes far beyond its initially observed function as a negative modulator of food intake.
In contrast to the growing body of evidence of the pleiotropic peptide's effects, the receptor mediating these effects is still largely unknown. Although several studies suggest that the peptide activates extracellular and intracellular regulatory pathways involving multiple putative binding sites (19), a specific nesfatin-1 receptor has not yet been identified. Therefore, the present paper was designed to systematically review the findings about nesfatin-1 signaling, especially by focusing on its potential receptor. This systematic review will discuss the state of knowledge on nesfatin-1 signaling, the putative mediation via a G i protein-coupled receptor and highlight respective direct and indirect evidence. Lastly, gaps in knowledge will be highlighted to encourage further research hopefully leading to the identification of the nesfatin-1 receptor.

METHODS
We followed the preferred reporting items for systematic reviews and meta-analyses (PRISMA) (20) to report the results of this review.

Search Strategy
This article is a systematic review; articles related to the topic, were searched, collected, and screened. Articles released from the earliest day of publication to the day the search was performed were included. The search was conducted on November 22 nd in 2020. We searched Embase, PubMed and Web of Science using the following keywords: "nesfatin-1" combined with "receptor", "signaling", "distribution", "pathway", G-protein coupled receptor", and "binding" ( Table 1). All studies that contained material applicable to the topic were considered. We investigated the reference lists of the included studies to find other potential articles for inclusion. Local experts in the field were consulted for additional studies. Retrieved manuscripts were extracted using a standardized collection tool. Gray literature was not considered due to lack of essential information usually affecting this type of research. There was no date (all studies until November 2020) or species restriction in the search, but the search was limited to texts in English.

Inclusion and Exclusion Criteria
The inclusion criteria were as follows: (1) observational preclinical and clinical studies, experimental studies, registrybased studies, cohort studies, population-based studies; (2) Studies that were written in English (3) Studies investigating nesfatin-1 putative receptor or signaling cascade.
The exclusion criteria were as follows: (1) Review articles, surveys, case reports, comments, letters, conference abstracts or posters, and economic evaluation; (2) Studies for which abstracts or full-text articles were not available; (3) Studies that were not available in English; (4) Studies with absence of outcome data.

Study Selection and Data Extraction
One reviewer screened all titles and abstracts retrieved from the electronic searches to identify potentially eligible articles. Full texts of the potentially eligible articles were retrieved. Two reviewers (S.R. and E.W.) screened all full text articles and potentially eligible or unclear full-text articles, determined whether they were eligible or not eligible and recorded the reason for exclusion. Any disagreements between both reviewers were resolved through discussion.
The quality of the studies included in this review was assessed with respect to risk of bias within individual and across studies by thoroughly evaluating the study designs, selection of population/species, methodological procedures applied as well as presentation of the results. Following a full-text review of the eligible studies, one reviewer (E.W.) extracted the relevant data. From each included study the following information was extracted: first author of the publication, year of publication, title, population/species, size of the respective population/ species, study type, research question/purpose, and key findings ( Table 2).

RESULTS
1,147 articles were identified after searching the databases using the keywords mentioned above. Reviews, surveys, case reports, comments, letters, conference abstracts or posters, as well as economic evaluation and non-English language studies or studies with no full text available, and duplicates were excluded. This deceased the number to 425 articles. Next, title and abstract were screened and studies which deviated from the main topic were excluded; thus, 128 studies required full-text screening. Here, studies not related to the review topic were excluded. Ultimately, 70 articles were selected for this systematic review. The PRISMA flow diagram schematically depicts the article selection process ( Figure 1). Table 2 shows the main results of these articles.

Quality Assessment
The current review consists of observational preclinical and clinical studies or experimental studies, assuming a risk for bias. There are several issues that may contribute to limited comparability between studies and thus limited transferability of study results in general. First, the studies included different species or population, which may lead to limited comparability. Second, the respective study populations differed in sample size, while the study methods, treatments and observation periods used were also very heterogeneous. Furthermore, we only reviewed the abstracts and full texts published in English. Studies in other languages have not been included. Additionally, we searched only three databases for potentially eligible studies. Taken together, bias cannot be ruled out for the studies included in this systematic review; thus, these limitations should be kept in mind when interpreting the results discussed here.

DISCUSSION
The aim of this systematic review was to identify and summarize the state of knowledge on signaling cascades of nesfatin-1 and to shed light on the peptide's potential receptor. Nesfatin-1 affects multiple sites in the organism and elicits a variety of intracellular effects, which accounts for the pleiotropic nature of this peptide. The multiple intracellular signaling cascades triggered by nesfatin-1 ( Figure 2) only reinforce the multifaceted nature of this peptide and highlight that it may be of great importance to understand the exact cascades by further investigating the particular type and localization of the potential receptor in the future.

Intracellular Signaling and Potential Receptor
By using autoradiography binding of radiolabeled nesfatin-1 in the gastric mucosa of corpus and antrum, in duodenum, jejunum and ileum and centrally in the cortex, paraventricular nucleus of the hypothalamus, area postrema, dorsal motor nucleus of the vagus nerve and cerebellum was shown, giving rise to respective expression sited of the receptor (62). Since nesfatin-1 is known to be able to cross the blood-brain barrier, peripheral nesfatin-1 might access and then bind to these central receptors (87). In addition, NUCB2/Nesfatin-1 was found to be widely distributed in the central nervous system of mice (88) and rats (3, 89).

AMPK Signaling Pathway
Converging evidence points towards a mediation of nesfatin-1's effects via a G-protein coupled receptor: Previous results suggest the presence of a nesfatin-1 receptor in hepatocytes whose activation stimulates the phosphorylation of AMPK, reducing hepatic lipid accumulation. These alterations were associated with a significant attenuation of peroxisome proliferator-activated receptor g (PPARg) and sterol-regulatory element-binding protein-1 (SREBP1), which are known lipogenesis-related transcription factors (79). Furthermore, in diabetic mice low-dose injection of nesfatin-1 also regulated fatty acid metabolism via activation of an AMPK-ACC pathway (30). The assumption of an influence of nesfatin-1 on AMPK signaling cascades is consistent with recent findings, highlighting that nesfatin-1/NUCB2 enhances invasion, migration, and mesenchymal phenotype in colon cancer via liver kinase B1 (LKB1)/AMPK/target of rapamycin kinase complex I (TORC1)/ ZEB1 signaling pathways (44). In line with this, it was further shown that nesfatin-1 suppresses peripheral artery remodeling likely by increasing NO production and LKB1-mediated activation of AMPK in vascular endothelial cells in mice (54). However, in the testis of mice nesfatin-1-induces an increase in G protein-coupled receptor 54 (GPR-54) accompanied by increases in PCNA, Bcl2, androgen receptor (AR), GLUT8, insulin receptor and gonadotropin-releasing hormone receptor (GnRH-R) (64,65). In addition, a potential role of protein kinase B (AKT) as a signaling mechanism for nesfatin-1induced glucose uptake and activation of the AMP-activated protein kinase (AMPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) system was found through which nesfatin-1 enhances insulin sensitivity in the testis of mice (64,66). Supporting these findings, icv injection of nesfatin-1 increased peripheral and hepatic insulin sensitivity by decreasing gluconeogenesis and promoting peripheral glucose uptake through the AMPK/AKT/TORC2 pathway (78).

MAPK Signaling Pathway
Furthermore, previous results assumed the presence of a nesfatin-1-specific-receptor, likely a Gi/o protein-coupled receptor, on the cell surface of a murine neuroblastoma cell line (NB41A3 cells) and mouse hypothalamus. Here, binding of nesfatin-1 activates the cAMP-response reporter (CRE) and further phosphorylates CREB in these cells by utilizing Ca 2+ influx and/or the MAPK signaling pathway (40). Other studies Nesfatin-1 elevated mean arterial pressure and modulates heart rate in rats via the central cholinergic system.

Selection bias
Barutcigil A 2018 Male Wistar rats Not indicated Experimental intervention study Nesfatin-1 dose-dependently induced a relaxation on the endothelium-intact thoracic aorta of rats and produced positive inotropic and chronotropic effects on atria. These effects might be beta-1 receptor independent, while involving the NO-cGMP cascade.

Selection bias
Brailoiu GC Nesfatin-1 treatment increased dry heat loss, iBAT and tail temperature through activation of the melanocortin system.

Experimental intervention study
Nesfatin-1 had neuroprotective effects in neuronal cells and neuroinflammatory processes caused by brain ischemia by decreasing activation of caspase-3.

Experimental intervention study
Nesfatin-1 required GHSR for mediating its effects on food intake and glucose metabolism.    Nesfatin-1 signaling in the lateral hypothalamic area modulated the activation of GD-responsive neurons, gastric motility and gastric secretion also involving melanin-concentrating hormone signaling.

Experimental intervention study
Nesfatin-1 administration into the BMA increased firing rate of GD-excitatory neurons, while decreasing firing rates of GDinhibitory neurons. Nesfatin-1 in the BMA is involved in decreasing gastric motility and the Arc may also play a role in this regulating process.

CRF Signaling Pathway
Regarding nesfatin-1's influence on CRF signaling pathways, gastric distension (GD)-responsive neurons were found in ventromedial hypothalamic nucleus (VMH), whose firing rate was altered by nesfatin-1, most likely through interactions with CRF signaling pathways (35). This hypothesis was strengthened by the observed association of nesfatin-1 and irritable bowel syndrome-like visceral hypersensitivity that is also mediated, at least in part, by CRF/CRF 1 signaling pathways in the brain (42).
In consistence with these findings, it was reported that nesfatin-1 reduced dark-phase food intake via CRF pathways (18). Furthermore, in neonatal chicks nesfatin-1 used CRF 1 /CRF 2 as well as histamine (H) 1 and H 3 receptors to mediate its anorexigenic effect. Interestingly, co-injection of nesfatin-1 and chlorpheniramine (H 1 antagonist) blunted nesfatin-1-induced hypophagia, while co-administration with thioperamide (H 3 antagonist) reinforced the hypophagic effect of nesfatin-1. This divergence in the effects of H 1 and H 3 receptors can be explained by the fact that H 1 is a postsynaptic receptor, while H 3 is a presynaptic autoreceptor, leading to the hypothesis that H 1 and H 3 play a different regulatory role in the feeding behavior influenced by nesfatin-1 (39).

Melanocortin Signaling Pathway
Using the melanocortin signaling pathway, nesfatin-1 was found to alter firing rates of GD-responsive neurons in the VMH (36), baso-medial amygdala (BMA) (77), arcuate nucleus (49) and central nucleus of the amygdala (73). Also, by involving the melanocortin system nesfatin-1 alters the excitability of glucose sensitive neurons in the lateral parabrachial nucleus (LPBN) and increases the level of uncoupling protein (UCP) expression in brown adipose tissue (BAT) (82). In accordance, it was assumed that nesfatin-1-induced satiety is associated with leptinindependent melanocortin signaling in the hypothalamus (1). This hypothesis was strengthened by a study showing that hypothalamic nesfatin-1 regulates feeding behavior through the MC 3/4 -ERK signaling pathway. Interestingly, in this study, hypothalamic nesfatin-1 mediates its effects via MC 3/4 , while not altering expression of MC 3/4 (84). Furthermore, through activation of the melanocortin system icv nesfatin-1 treatment increases dry heat loss, interscapular (i) BAT and tail temperature. Moreover, nesfatin-1 administration upregulated the expression of POMC and MC 3 mRNA in the hypothalamus along with an elevation of iodothyronine deiodinase 2 (Dio2), UCP1 and PPARg-1 alpha mRNA in the iBAT (31). Furthermore, the nesfatin-1-induced elevation in the mRNA expression of the cAMP-responsive gene Dio2 strengthens the hypothesis of an activation of the b-adrenergic/cAMP signaling pathway, which is in line with other findings emphasizing the established role of nesfatin-1 in sympathetic nerve activity by activating the central melanocortin system (70). On the other hand, only the midsegment of nesfatin-1 proves effective and leads to a higher activation of c-Fos in the brainstem nucleus of the solitary tract (NTS). Furthermore, administration of the middle segment of nesfatin-1 results in an increase in the expression of proopiomelanocortin (POMC), cocaine-and amphetamine-regulated transcript (CART) mRNA in the NTS (9). In adult ventricular myocytes nesfatin-1 targets MC 4 , sequentially coupling to the G bg subunits of G i/o , leading to the subsequent activation of the novel protein kinase C (PKC) q isoform, subsequently resulting in an inhibition of L-Type Ca 2+ channels associated with a hyperpolarizing shift in the voltagedependence of inactivation. Notable the nesfatin-1 mediated inhibition of calcium-influx was not affected by Kt-5720, a PKA antagonist indicating that the cAMP/PKA pathway is not involved in the nesfatin-1-induced L-Type Ca 2+ channel response in ventricular myocytes (80). In contrast, it was observed that both nesfatin-1 and nesfatin-1-like peptide directly affect somatotrophs via binding to a GPCR containing a G-a-i subunit and utilizing the adenylyl cyclase (AC)/protein kinase A (PKA)/CREB signaling pathway subunit, thereby downregulating the synthesis of ghrelin hormone (72).

Ion Currents
However, in cardiac vagal neurons nesfatin-1 may increase cytosolic Ca 2+ levels via a G i/o -coupled mechanism by involving P/Q-type voltage-activated Ca 2+ channels (25), while in hypothalamic neurons the peptide might mediate Ca 2+ influx also via a GPCR, most likely by affecting both Land P/Q-Ca 2+ channels. Surprisingly, Ca 2+ influx was significantly reduced by a PKA blocker, indicating an involvement of PKA in hypothalamic neurons (26). The effects of nesfatin-1 in sensory neurons may be mediated through the participation of a G i/o protein coupled receptor as it is speculated that the nesfatin-1-induced Ca 2+ increase may result from a Ca 2+ influx from both extracellular and intracellular sources (58). Furthermore, several other studies proposed an influence of nesfatin-1 on calcium levels in various tissues: Exposure of the isolated and perfused working heart to nesfatin-1 results in positive inotropism mediated through cAMP, PKA, L-type Ca 2+ channels, sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA2a) pumps, ERK1/2 and phospholamban (PLN) (53). On the other hand, it was found that nesfatin-1 leads to increased expression of Atp2a2a mRNA encoding SERCA2a, while there were no changes observed in the expression of ryanodine receptor 1b (RyR1b) encoding mRNA (55). Furthermore, a different study demonstrated that nesfatin-1 induces heart failure during clinical treatments by increasing expression of the cardiac L-type Ca 2+ channel a1c subunit (22). Likewise, it was shown that in mice, nesfatin-1 dose-dependently stimulates intracellular Ca 2+ levels via L-type Ca 2+ channels independently of PKA and phospholipase A2 (PLA2) in beta-cells under elevated plasma glucose concentration (56). One mechanism of the elevated Ca 2+ influx could be an increase in membrane Na + permeability, which depolarizes the membrane to open voltage-gated calcium channels (VDCC) (90), a mechanism used by glucagon-like peptide 1 (GLP-1) (91). Another potential explanation is an involvement of protein kinase C, which has been reported to increase glucose-stimulated Ca 2+ influx through VDCC and insulin secretion (92). In contrast, another study reported that peripheral nesfatin-1 stimulates Ca 2+ influx through voltage-gated N-type channels, thereby directly activating afferent vagal neurons (41). On the other hand, nesfatin-1 showed an effect on vagal neurons by mediating Ca 2+ signaling through T-type channels, which are low-voltage activated channels localized in different areas of the central nervous system (75,93,94). The ultimate role of T-type Ca 2+ channel activation in the response of vagal neurons to nesfatin-1 and the downstream target/effect of this signaling pathway remain unknown. However, nesfatin-1 not only regulates the calcium influx via various mechanisms, but also influences the levels of other ions: More precisely, nesfatin-1 directly induces an inhibition of the voltage-dependent potassium (Kv) current by directly binding to Kv2.1 channels to exert its effect on pancreatic beta cells, as these Kv channels have been identified as the major contributors to Kv currents in these cells. Since nesfatin-1 is localized in beta cells, it has been hypothesized that nesfatin-1 may affect beta cell function in an autocrine/paracrine manner (51). Another finding assumes that nesfatin-1 activates K ATP channels, thereby inhibiting orexigenic neuropeptide Y (NPY) neurons of the arcuate nucleus and consequently leading to satiety (61).

NO-cGMP System
Interestingly, nesfatin-1 also targets the NO-cGMP system; again, the particular receptor(s) is/are still unknown and require further research: In atrial tissue nesfatin-1 might increase the sensitivity of smooth muscle to cGMP-mediated relaxing mechanisms rather than increasing the NO levels. Interestingly, these positive chronotropic effects in the atrial tissue were independent of the b1-adrenergic receptor (24). On the other hand, nesfatin-1 also impairs the SNP-induced cGMP production, thereby inhibiting the NO donor-induced smooth muscle relaxations. Since these effects were persistent in the presence of IBMX, a phosphodiesterase (PDE) inhibitor, it can be suggested that these effects may be mediated via inhibition of soluble guanylate cyclase activity rather than activation of PDEs (16). However, other findings indicated that by recruiting guanylyl cyclase-linked receptors, namely natriuretic peptide receptor type A (NPR-A), and thereby involving the cGMP/ protein kinase G pathway and ERK1/2, nesfatin-1 mediates negative inotropic and lusitropic effects in rats. Since the use of pertussistoxin did not inhibit the effects of nesfatin-1, the hypothesis that nesfatin-1 binds to a G i/o cannot be confirmed by these observations (21).

Colocalization With Other Peptides
Furthermore, PVN neurons in rats are directly depolarized by nesfatin-1, suggesting a mediation of these effects through the activation of a GPCR. Since PVN neurons respond to nesfatin-1 and further produce nesfatin-1, there might be potential interactions between various subgroups of PVN neurons engaged in the control of several autonomic outputs. The specificity of the effects may also arise from the co-expression of other receptors on nesfatin-1-sensitive neurons. An alternative explanation is that multiple nesfatin-1 receptors exist that are separately responsible for hyperpolarizing or depolarizing responses (60). It was shown that in the PVN, nesfatin-1 targets magnocellular and parvocellular Oxt neurons as well as nesfatin-1 neurons themselves, further stimulating Oxt release. Furthermore, the presence of nesfatin-1 specifically in the secretory vesicles of PVN neurons was found, indicating paracrine/autocrine actions of nesfatin-1 (52). On the other hand, it was suggested that the hypertensive effect of nesfatin-1 may require both activation of Oxt neurons and recruitment of CRF neurons (81). Moreover, by targeting ghrelin and Oxt receptors nesfatin-1 suppresses neutrophil infiltration and improves glutathione levels in acute pancreatitis (27) and colitis (59). Interestingly, the effects of nesfatin-1 on myeloperoxidase activity, lipid peroxidation and glutathione level were abrogated by application of melanocortin receptor antagonist. Despite previously demonstrating that nesfatin-1 utilizes the melanocortin signaling pathway for its effects on food intake, these findings indicate that nesfatin-1 may also utilize the receptors mediating its anorexigenic effect for antiinflammatory effects (59). Since the effects of nesfatin-1 on GDexcitatory neurons and GD-inhibitory neurons in the PVN were attenuated in the presence of H4928, an Oxt receptor antagonist, it can be assumed that nesfatin-1involves Oxt receptors to modulate gastric function (38). Moreover, in goldfish nesfatin-1 inhibits ghrelin mRNA expression in the brain and vice versa, leading to the assumption of a negative feedback inhibition of endogenous ghrelin and nesfatin-1 (45). Another study indicates that peripheral nesfatin-1 most likely exerts part of its effect directly via ghrelin receptor (GHSR) signaling. Furthermore, the results here show that the effects of nesfatin-1 in mice fed a normal chow diet, such as improvement of glucose tolerance, upregulation and phosphorylation of AKT-kinase mRNA and GLUT4 membrane translocation, were dependent on the presence of GHSR. In high-fat diet (HFD) fed mice, nesfatin-1 additionally led to a raise of AKT levels in liver tissues, which is also a GHSR-dependent mechanism (33). Therefore, it is possible that nesfatin-1 acts as an endogenous inverse agonist of the GHSR and influences the structure or activity of the GHSR. Furthermore, it was found that nesfatin-1 inhibits the excitability of dopaminergic neurons in the ventral tegmental area (VTA) (28) and the substantia nigra (47), thereby decreasing dopamine release (28). In addition, nesfatin-1 signaling in the lateral hypothalamic area (LHA) or electric stimulation of the Arc modulate the activation of GD-responsive neurons, gastric motility and gastric secretion involving melanin-concentrating hormone signaling indicating the potential presence of nesfatin-1 specific receptors in these neurons (76). Recent findings also provide evidence that nesfatin-1 administered into the amygdala targets glucocorticoid and mineral corticoid receptor pathways, thereby being involved in the pathophysiology of irritable bowel syndrome (IBS)-like visceral hypersensitivity (86). Lastly, predominantly via the CCK-CCK1R signaling pathway nesfatin-1 reduces food intake in Siberia sturgeon (85).
Involving cholinergic pathways, central administration of nesfatin-1 elevates mean arterial pressure and modulates heart rate in rats. These cardiovascular effects were mediated by both nicotinic and muscarinic receptors. Considering that recent studies have shown that both muscarinic and nicotinic acetylcholine receptors interact with G-coupled proteins (95), it may be that nesfatin-1 mediates its cardiovascular effects via central muscarinic and nicotinic receptors because of its affinity to GPCRs (23).
Furthermore, it was found that central nesfatin-1 can increase peripheral sympathetic outflow and thus b-adrenergic activation, resulting in iBAT thermogenesis and body weight loss. It was demonstrated that the thermogenetic effect of nesfatin-1 mainly depends on b3-adrenergic stimulation. In addition, levels of Dio2 and cell death inducing DFFA like effector A (CIDEA) mRNA were increased in brown adipose tissue after nesfatin-1 administration, which is plausible as both are involved in regulating the thermogenic program (46). Since the direct effects of peripherally injected nesfatin-1 on vascular smooth muscle were attenuated by pretreatment with propranolol, an involvement of the b-adrenergic system can also be suspected here (57).

Implications for Future Research
The described various distribution of the nesfatin-1 receptor further supports the assumption that nesfatin-1 may be involved in the regulation of various homeostatic functions, and the various signaling pathways underlying the actions of nesfatin-1 emphasize its multiple effects. The recent discoveries about the diverse effects of nesfatin-1 indicate the importance of evaluating and determining the potential use of nesfatin-1 in a therapeutic context in the future. For this reason, further research is needed to fully understand the exact cascades of the peptide and its complex interplay with other hormones.

CONCLUSION
This present review highlights nesfatin-1 as a pleotropic peptide that acts at multiple levels in the organism, thereby eliciting a wide variety of effects. As diverse as the effects of the peptide, so are the intracellular signaling pathways and downstream effects summarized here, including the influence of nesfatin-1 on various hormones and their receptors. The identification of the so far unknown nesfatin-1 receptor will represent a major leap forward in our understanding of the physiology of nesfatin-1 and will allow us to better investigate the precise mechanisms underlying the many different effects of the peptide. Further research is needed so we might be able to consider a therapeutic use of nesfatin-1 in the future.