Identification of nesfatin-1 as a satiety molecule in the hypothalamus

The brain hypothalamus contains certain secreted molecules that are important in regulating feeding behaviour. Here we show that nesfatin, corresponding to NEFA/nucleobindin2 (NUCB2), a secreted protein of unknown function, is expressed in the appetite-control hypothalamic nuclei in rats. Intracerebroventricular (i.c.v.) injection of NUCB2 reduces feeding. Rat cerebrospinal fluid contains nesfatin-1, an amino-terminal fragment derived from NUCB2, and its expression is decreased in the hypothalamic paraventricular nucleus under starved conditions. I.c.v. injection of nesfatin-1 decreases food intake in a dose-dependent manner, whereas injection of an antibody neutralizing nesfatin-1 stimulates appetite. In contrast, i.c.v. injection of other possible fragments processed from NUCB2 does not promote satiety, and conversion of NUCB2 to nesfatin-1 is necessary to induce feeding suppression. Chronic i.c.v. injection of nesfatin-1 reduces body weight, whereas rats gain body weight after chronic i.c.v. injection of antisense morpholino oligonucleotide against the gene encoding NUCB2. Nesfatin-1-induced anorexia occurs in Zucker rats with a leptin receptor mutation, and an anti-nesfatin-1 antibody does not block leptin-induced anorexia. In contrast, central injection of alpha-melanocyte-stimulating hormone elevates NUCB2 gene expression in the paraventricular nucleus, and satiety by nesfatin-1 is abolished by an antagonist of the melanocortin-3/4 receptor. We identify nesfatin-1 as a satiety molecule that is associated with melanocortin signalling in the hypothalamus

Crosstalk between thyroid hormone receptor and liver X receptor in the regulation of selective Alzheimer's disease indicator-1 gene expression.

Selective Alzheimer's disease (AD) indicator 1 (Seladin-1) has been identified as a gene down-regulated in the degenerated lesions of AD brain. Up-regulation of Seladin-1 reduces the accumulation of β-amyloid and neuronal death. Thyroid hormone (TH) exerts an important effect on the development and maintenance of central nervous systems. In the current study, we demonstrated that Seladin-1 gene and protein expression in the forebrain was increased in thyrotoxic mice compared with that of euthyroid mice. However, unexpectedly, no significant decrease in the gene and protein expression was observed in hypothyroid mice. Interestingly, an agonist of liver X receptor (LXR), TO901317 (TO) administration in vivo increased Seladin-1 gene and protein expression in the mouse forebrain only in a hypothyroid state and in the presence of mutant TR-β, suggesting that LXR-α would compensate for TR-β function to maintain Seladin-1 gene expression in hypothyroidism and resistance to TH. TH activated the mouse Seladin-1 gene promoter (-1936/+21 bp) and site 2 including canonical TH response element (TRE) half-site in the region between -159 and -154 bp is responsible for the positive regulation. RXR-α/TR-β heterodimerization was identified on site 2 by gel-shift assay, and chromatin immunoprecipitation assay revealed the recruitment of TR-β to site 2 and the recruitment was increased upon TH administration. On the other hand, LXR-α utilizes a distinct region from site 2 (-120 to -102 bp) to activate the mouse Seladin-1 gene promoter. Taking these findings together, we concluded that TH up-regulates Seladin-1 gene expression at the transcriptional level and LXR-α maintains the gene expression

Nesfatin-1 induces the phosphorylation levels of cAMP response element-binding protein for intracellular signaling in a neural cell line.

Nesfatin-1 is a novel anorexic peptide that reduces the food intake of rodents when administered either intraventricularly or intraperitoneally. However, the molecular mechanism of intracellular signaling via Nesfatin-1 is yet to be resolved. In the current study, we investigated the ability of different neuronal cell lines to respond to Nesfatin-1 and further elucidated the signal transduction pathway of Nesfatin-1. To achieve this, we transfected several cell lines with various combinations of reporter vectors containing different kinds of response elements and performed reporter assays with Nesfatin-1, its active midsegment encoding 30 amino acid residues (M30) and M30-derived mutants. Notably, we found that both Nesfatin-1 as well as M30, significantly increased cAMP response element (CRE) reporter activity in a mouse neuroblastoma cell line, NB41A3. An antagonist of Melanocortin 3/4 receptor, SHU9119, aborted the promoter activity, and a mutant M30, which exerts no anorexic effect in vivo did not induce the CRE reporter activity in NB41A3 cells. Western blotting analyses revealed that Nesfatin-1 and M30 significantly increased the phosphorylation levels of CRE-binding protein (CREB), without altering the intracellular cAMP levels. Further, our study showed that a mitogen-activated protein kinase (MAPK) kinase inhibitor and an L-type Calcium (Ca(2+)) channel blocker abolished the M30-induced CREB phosphorylation. Furthermore, the radio-receptor assay revealed that (125)I-Nesfatin-1 binds in a saturable fashion to the membrane fractions of the mouse hypothalamus and NB41A3 cells, with Kd values of 0.79 nM and 0.17 nM, respectively. Collectively, our findings indicate the presence of a Nesfatin-1-specific receptor on the cell surface of NB41A3 cells and mouse hypothalamus. Our study highlights that Nesfatin-1, via its receptor, induces the phosphorylation of CREB, thus activating the intracellular signaling cascade in neurons

TR-β is recruited to the site 2 region in the mouse Seladin-1 gene promoter in a T₃-dependent manner.

<p>The location of the PCR primers is indicated in an image above the data as arrows. The closed boxes indicate site 1 and 2 (A). Real-time PCR was performed to determine the relative value of the enrichment of each nuclear receptor. The value is shown as % of input (mean ± S.E., n = 8) (B). TR-β, RXR-α and LXR-α are recruited to the region from −226 bp to +21 bp. The recruitment of TR-β and RXR-α is increased upon T₃ administration. The normal rabbit IgG was used as a negative control. An <i>asterisk</i> indicates that the difference between the denoted pairs is significant at a confidence level of <i>p</i><0.05(*) and <i>p</i><0.0001(****). NS: not significant.</p

Schematic diagram illustrating the hypothetical mechanism of TR and LXR crosstalk on the mouse Seladin-1 gene promoter.

<p>Schematic diagram illustrating the hypothetical mechanism of TR and LXR crosstalk on the mouse Seladin-1 gene promoter.</p

The up-regulation of Seladin-1 gene expression by TO requires LXRs in HTB185 cells.

<p>A) TO failed to induce the Seladin-1 gene expression in LXR-knockdown HTB185 cells. Vehicle for TO was DMSO. Relative values (mean ± S.E., n = 5) normalized by cyclophilin A mRNA levels compared with the expression levels of Seladin-1 mRNA with non-targeting siRNA (sicontrol) are shown as relative expression (fold). B) SiRNAs against LXR-α and LXR-β significantly reduced each gene expression level in HTB185 cells. Relative values (mean ± S.E., n = 6) normalized by cyclophilin A mRNA levels compared with the expression levels of LXR-α mRNA with sicontrol are shown as relative expression (fold). C) TO induced the mouse Seladin-1 gene promoter (−1936/+21 bp) in CV-1 cells in the presence of LXR-α or LXR-β. Relative luciferase activities (mean ± S.E., n = 3) compared to the light units with the −1936/+21 bp construct in the absence of TO (arbitrary light units divided by cellular protein and by β-gal activity) are shown as relative promoter activity (fold). An <i>asterisk</i> indicates that the difference between the denoted pairs is significant at a confidence level of <i>p</i><0.05(*), <i>p</i><0.01(**) and <i>p</i><0.001(***). NS: not significant.</p

RXR-α/TR-β1 binds to site 2.

<p>Oligonucleotide sequences for radiolabelled probes are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054901#pone-0054901-g005" target="_blank">Figure 5A</a>. Flanking regions of two TRE half sites were indicated as site 1 and 2 probes. The consensus TRE half sites were highlighted in italics. Six microliters (indicated as ‘+’) of <i>in vitro</i>-translated human TR-β1 and human RXR-α were incubated with ³²P-radiolabeled DNA probes. For competition experiments, 20- or 40-fold molar excess of cold oligonucleotides was included as indicated. NS: non specific band. α-TR-β: rabbit anti-TR-β antibody. DR-4: Direct repeat-4.</p