Figure 3: S1PR1 requires STAT3 activation to induce anorexia.

(a) Western blottings show leptin-induced STAT3 phosphorylation (upper panel) and S1P-induced STAT3 phosphorylation (lower panel) after ICV stimulation (n=4 in each point). (b) Representation of STAT3 phosphorylation pattern after leptin or S1P ICV injections. (c) Determination of food consumption after ICV injection of saline (2 μl), leptin (10⁻⁶ M) or S1P (50 ng) in control rats (n=10). (d) Western blotting shows the effects of JSI124 (50 μM) on STAT3 phosphorylation in the hypothalamus (n=4). (e) Food intake evaluation after vehicle (2 μl), S1P (50 ng) and JSI124 (50 μM) plus S1P injection. (f) Western blotting shows the effects of JSI124 (50 μM) on S1P-induced STAT3 phosphorylation in the hypothalamus (n=4). (g) Nuclear STAT3 translocation after ICV injection of S1P (50 ng) (n=3, in each point). (h) Correlation matrices (left lower triangle) and pie graphs (right upper triangle) showing correlations among S1pr1, Stat3, Pomc, Mc3r, Mc4r, Npy and Agrp in the hypothalamus of the BXD inbred family (n=33, GSE36674). The depth of the shading at the correlation matrices indicates the magnitude of the correlation. Positive and negative correlations are represented in blue and red, respectively. The pie graphs are filled in proportion to the Pearson’s coefficient values, clockwise for positive correlations (in blue) and anti-clockwise for negative correlations (in red). (i) Interaction network showing correlations of indicated genes. Positive Pearson’s correlation coefficients are represented by blue solid (r=0.5–1.0) and dashed line (r=0.4–0.5). (j) Correlation plots between hypothalamic S1pr1 and each indicated phenotype (n=10 to 29). Pearson’s coefficient values were used to analyse correlations, and Pearson’s r and P-values are indicated. (k) Interaction network showing correlations between hypothalamic S1pr1 and each indicated phenotype. Positive Pearson’s correlation coefficients are represented by blue and red lines (r=0.5–1.0). (l) Food consumption evaluation was performed after vehicle (2 μl), S1P (50 ng) and SHU9119 (1 mmol) plus S1P injection (n=5). Data were expressed by using mean±s.e.m. *P<0.05 versus vehicle, §P<0.05 versus leptin group and #P<0.05 versus vehicle plus S1P. One-way analysis of variance and Bonferroni post test were performed in e,f and l.