CHARACTERIZATION OF NESFATIN-1 IN THE BRAIN AND STOMACH OF THE TELEOSTEAN MODEL FOR AGING RESEARCH NOTHOBRANCHIUS FURZERI

The anorexigenic peptide Nesfatin-1 (Nesf-1) and its precursor NEFA/nucleobindin 2 (NUCB2) showed a highly conserved structure between mammalian and non-mammalian species [1]. In Mammals, NUCB2/Nesf-1 were detected both in appetite-control hypothalamic nuclei [1] and in peripheral tissues involved in energy metabolism. In particular, NUCB2/Nesf-1 were described in gastric X/A-like endocrine cells and the highest mRNA expression allow to considered stomach as the main sources of circulating Nesf-1 [2]. In Teleosts, similarly to Mammals, NUCB2/Nesf-1 were mostly detected in feeding regulatory hypothalamic nucleus and in gut, with higher expression in the anterior gastrointestinal tract respect to the brain [3-4]. Although tissue distribution of NUCB2/Nesf-1 is well known in Vertebrates, there are few reports on their expression during ontogenesis. This study aimed to examine for the first time the age-related central and peripheral expression of NUCB2/Nesf-1 in the teleost fish Nothobranchius furzeri, a well consolidated model organism for aging research. Experiments were performed on brain and stomach of N. furzeri belonging to MZM 04/10 strain, at the following time points: 5 weeks post hatching (wph) (young-adult, age of sexual maturity) and 27wph (old). Phylogenetic analysis displayed that gene structure was well conserved in N. furzeri, showing 78% of similarity with medaka, the closest related species. Real-Time PCR revealed increased expression levels in old fishes compared to young both in brain and stomach. Then we carried out in situ hybridization against NUCB2 and immunohistochemistry against Nesf-1. Western blot on homogenates of brain confirmed the specificity of Nesf-1 antibody, revealing a band of about 40 kDa. Either in young and aged brains both NUCB2 mRNA transcript and Nesf-1 immunoreactivity cells were detected in the hypothalamic area. Interestingly mRNA expression and protein distribution were observed in non diencephalic regions, specifically telencephalon, optic tectum and semicircular tori at all age stages analyzed. Telencephalon, diencephalon and optic tectum are brain regions involved in appetite control of Teleosts [5]. Either in young and aged stomachs NUCB2 mRNA transcript was detected mainly in the lining epithelium while Nesf-1 immunoreactive cells were distributed in the submucosae. This study represents a step for understanding the regulation of NUCB2/Nesf-1 during vertebrate aging processes