**1. Introduction**

Nucleobindin (NUCB) belongs to the family of calcium and DNA binding proteins and comprises two members, NUCB1 and NUCB2. In mammals, NUCB1 is expressed within the pituitary, liver, and kidney, where it regulates calcium homeostasis and G protein signaling [1,2], while NUCB2 is expressed in the appetite-controlling hypothalamic nuclei, such as the lateral area of the hypothalamus (LHA), paraventricular nucleus (PV), arcuate nucleus (ARC), supraoptic nucleus (SON), tractus

solitarius nucleus (NTS), and dorsal nucleus of the vagus [3]. NUCB2 is post-translationally cleaved by prohormone convertases into one N-terminal fragment, Nesfatin-1 (residues 1–82), and two C-terminal peptides, Nesfatin-2 (residues 85–163) and Nesfatin-3 (residues 166–396) [4]. Only the mid-segment of Nesfatin-1 (Nesf-1) is considered the bioactive core, exercising anorexigenic effects [3–5]. In fact, Kohno and colleagues [6] showed that in PV and SON nuclei, which are immunoreactive (ir) to oxytocin and vasopressin, Nesf-1 positive neurons play a key role in the post-prandial regulation of food intake and peripheral metabolism. Hypothalamic neurons which co-expressed Nesf-1, antidiuretic hormone (ADH), corticotropin (CRH), and thyrotropin-releasing hormone (TRH) constitute an important network which regulates food intake. This network acts through the anorexigenic system of the melanocortin above all. It has been shown that the intracerebro-ventricular administration of both NUCB2 and Nesf-1 reduces food intake and body weight as well as increases sympathetic nerve activity and blood pressure in rats [3]. However, the use of anti-Nesf-1 antibodies is not enough to inhibit the sense of satiety induced by leptin: therefore, Nesf-1 is an anorexigenic molecule with a leptin-independent action in mammals [7]. As with many other central appetite regulators, NUCB2/Nesf-1 was also detected in peripheral tissues in different vertebrate models that had an important role in energy homeostasis, such as adipose tissue [8], pancreas [9,10], and in middle and lower segments of gastric mucosal glands [11], as well as in the submucosal layer of the duodenum [9]. Stengel and colleagues described NUCB2/Nesf-1 in the gastric X/A-like endocrine cells of the stomach and showed co-localization with the orexigenic hormone ghrelin in mammals. Moreover, expression levels of NUCB2/Nesf-1 in purified small endocrine cells of gastric mucosa have been reported to be 10-fold higher compared to brain levels [11]. Based on these indications, the stomach is considered one of the main sources of circulating NUCB2/Nesf-1 [12], supporting the hypothesis that NUCB2 is cleaved and Nesf-1 is produced at the gastric level [13].

In the last few years, several studies have described the distribution pattern of NUCB2/Nesf-1, also reporting the appetite regulatory effect in non-mammalian vertebrates. Particularly, in teleost fish, NUCB2 mRNA is widely expressed in central and peripheral tissues, mostly in the brain and gut. Similarly to mammals, the highest NUCB2/Nesf-1 mRNA expression was found in the gastrointestinal tract [14–16]. Nesf-1 ir-cells were found in the feeding regulatory nuclei of the hypothalamus and anterior intestine of the goldfish [14] and in the mucosal cellular layer of the anterior gastrointestinal tract in zebrafish [16]. NUCB2/Nesf-1 and ghrelin co-localize in the enteroendocrine cells of the anterior intestine [17] and in the hepatopancreas of goldfish [18], in the gut mucosal cells of zebrafish [16], and in intestinal enteroendocrine cells of pejerry [19]. Furthermore, the anorexigenic role of Nesf-1 seems to also be conserved in goldfish and Ya-fish, where central or peripheral administration of Nesf-1 induces feeding behavior suppression [14,15,17] as well as in Siberian sturgeon, where it acts through the cholecystokinin signal pathway [20,21].

Despite the fact that tissue distribution patterns are well described in vertebrates, few studies are dedicated to the determination of the expression of NUCB2 mRNA and Nesf-1 immunoreactivity during ontogenesis and postnatal development. Mohan and Unniappan [22] demonstrated that NUCB2/Nesf-1 immunoreactivity in the pancreas and gastrointestinal tract of rats increased from embryonic day 21 through to postnatal day 27, which is likely related to weaning. Senin and colleagues [23] confirmed and expanded previous data on gastroenteropancreatic tissues of rats by analyzing NUCB2/Nesf-1 expression from 2 until 8 weeks of age, corresponding to adulthood. However, there are no reports regarding age-related changes of NUCB2/Nesf-1 in the brain and stomach of any vertebrate. Here, we investigate the expression of Nesf-1 in the African turquoise killifish *Nothobranchius furzeri,* a well consolidated model organism for aging research. *N. furzeri* is the shortest-lived vertebrate that can be kept in captivity, with a lifespan of certain strains of 4 to 6 months in optimal laboratory conditions (6–10 times shorter than the lifespan of mice and zebrafish, respectively) [24]. In fact, *N. furzeri* is characterized by rapid growth, early sexual maturation [25,26], and the development of several biomarkers of aging during the short lifespan [27].

Thus, the aim of the present study is to investigate for the first time the age-related central and peripheral expression of NUCB2/Nesf-1 in *N. furzeri,* achieving deeper knowledge in food intake regulation during aging. Additionally, this study contributes to widely characterize the food intake regulation of the African turquoise killifish [28] and enrich data on neuropeptides that regulate food intake in fish [29–32].
