Figure 5: Hypothalamic RIIβ–PKA is regulated by feeding states and leptin.

(a) Representative images of multiple sections from three mice showing co-immunofluorescent staining of RIIβ and pSTAT3 in the hypothalamus of WT mice treated with leptin (1 h after leptin i.p. 2.0 mg kg⁻¹). RIIβ is localized in soma while pSTAT3 is localized in cell nucleus. Scales bars, 200 μm (upper panel) and 50 μm (lower panel). (b) Representative western blots (experiment repeated 3 times) of whole hypothalamic pRIIβ (S114), pCREB (S133), RIIβ and β-actin from fed, 24-h fast, 24-h fast+2-h re-feeding and 24-h fast+2-h leptin treatment (3 mg kg⁻¹ BW, i.p.) of WT mice. (c) Representative images of pRIIβ immunofluorescnet staining in hypothalamic regions of 24-h fasted WT mice (n=3) with or without 2-h re-feeding. ARC, arcuate nucleus; VMH, ventromedial hypothalamic nucleus; DMH, dorsomedial hypothalamic nucleus. pRIIβ immunostaining in the cortex (CTX) was shown as internal control. Insets show the co-staining of neurons for pRIIβ and a marker for nuclear DNA (ToPro3) as indicated by arrowheads. Scale bars, 100 μm. (d) Representative western blots and the quantification of hypothalamic pCREB from fed, 24 h fasted, and 2 h re-fed WT and RIIβ KO mice. Total CREB, RIIβ and β-actin were blotted as internal control (n=3 for each condition and genotype). Data are presented as mean±s.e.m. and analysed by two-tailed Student’s t-test (*P<0.05, ***P<0.001 ). (e) Representative immunofluorescent staining of pCREB (S133) in the hypothalamus of 24-h fasted WT and RIIβ KO mice with or without 2-h re-feeding (three animals for each condition and genotype). Scale bars, 200 μm. Full blots are shown in Supplementary Fig. 3.