(A) Organotypic brain slices were incubated with GIP (0.5 μM, 6 h) and then stimulated with leptin (120 nM, 60 min). Images show p-STAT3 immunostaining of fixed slices. Scale bar: 100 μm. (B) GIP inhibited leptin-induced p-STAT3 in a dose- and time-dependent manner (n = 3–14). (C and D) Brain slices were incubated with GIP (0.5 μM), with or without 50 μM ESI-05 (C) or 10 μM PKI1_14–22 (D) for 6 hours and then stimulated with 120 nM leptin for 60 minutes. Representative images and quantification of hypothalamic p-STAT3 (n = 3–5) are shown. Scale bars: 100 μm. (E) Lean mice were i.c.v. administered GIP (3 nmol) for 2 hours. Left: Western blot images of active Rap1, total Rap1, and β-actin (n = 6). Middle: Quantification is shown from 3 independent experiments (n = 17–18). Right: Graph shows Rap1 activity in the brains of lean and obese mice treated with Gipg013 or control IgG (n = 7–10). (F and G) Rap1^ΔCNS or control mice (n = 7–9) were i.c.v. injected with GIP (3 nmol/day) or vehicle and then i.c.v. injected with leptin (5 μg/day) 4 hours later. (F) Body weight change was measured daily. (G) Relative mRNA expression of the indicated genes in the hypothalamus of GIP- or vehicle-treated Rap1^ΔCNS mice. Each data point represents the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA followed by Tukey’s multiple comparisons test (B–E and G); **P < 0.01, by t test (E); and **P < 0.01, by 2-way ANOVA followed by Sidak’s multiple comparisons test (F). ARC, arcuate nucleus; 3V, third ventricle.