Figure 2: Enhanced leptin-induced STAT3 phosphorylation and POMC expression in the hypothalamus of RIIβ KO mice.

(a) Representative images of pSTAT3 immunofluorescent staining in hypothalamic regions of fasted (24 h) WT and RIIβ KO mice (n=3 for each group) with vehicle or leptin i.c.v injections at different time points. pSTAT3, tyrosine⁷⁰⁵-phosphorylated STAT3. ARC, arcuate nucleus; VMH, ventromedial hypothalamic nucleus; DMH, dorsomedial hypothalamic nucleus. Scales bars, 200 μm. (b) Representative immunoblots of pSTAT3 (from five mice per group in three independent experiments) with hypothalamic lysates from WT and RIIβ KO mice at different time points after 100 ng leptin (1, 6 and 12 h) or vehicle i.c.v. injection (0 h). Each lane represents one mouse. (c) STAT3 phosphorylation normalized to total STAT3 levels quantified by densitometry. For each treatment group, n=5. Inset shows the initial difference in STAT3 phosphorylation of fasted mice. Prior to leptin treatment animals were fasted for 24 h. (d) Hypothalamic Pomc mRNA from WT and RIIβ KO mice treated with vehicle (0 h) or leptin (100 ng, i.c.v.). Animals (males) were fasted for 24 h prior to treatment. N values for WT:KO were 0 h (6:6), 1 h (4:4), 6 h (7:8) and 12 h (8:9). (e) Hypothalamic Socs3 mRNA for WT and RIIβ KO mice that were fasted for 24 h and then either re-fed for 6 h or treated with leptin as indicated. N values for WT:KO were fasted (12:12), fed (7:9) and leptin 2 h (8:8). (f) Representative images of FoxO1 immunostaining in the ARC of vehicle or leptin-treated (100 ng leptin i.c.v., 3 h) WT and RIIβ KO mice (n=3 per genotype for each treatment). RIIβ KO mice showed enhanced leptin-induced FoxO1 exclusion from the nucleus and degradation. ToPro3 was used to visualize the cell nucleus. Scale bars, 20 μm. Male mice were used in these studies. Data are presented as mean±s.e.m. and two-way analysis of variance was followed by Bonferroni’s post hoc test to determine significance (*P<0.05, **P<0.01.) Full blots are shown in Supplementary Fig. 3.