Gut-derived GIP activates central Rap1 to impair neural leptin sensitivity during overnutrition

Abstract

Nutrient excess, a major driver of obesity, diminishes hypothalamic responses to exogenously administered leptin, a critical hormone of energy balance. Here, we aimed to identify a physiological signal that arises from excess caloric intake and negatively controls hypothalamic leptin action. We found that deficiency of the gastric inhibitory polypeptide receptor (Gipr) for the gut-derived incretin hormone GIP protected against diet-induced neural leptin resistance. Furthermore, a centrally administered antibody that neutralizes GIPR had remarkable antiobesity effects in diet-induced obese mice, including reduced body weight and adiposity, and a decreased hypothalamic level of SOCS3, an inhibitor of leptin actions. In contrast, centrally administered GIP diminished hypothalamic sensitivity to leptin and increased hypothalamic levels of Socs3. Finally, we show that GIP increased the active form of the small GTPase Rap1 in the brain and that its activation was required for the central actions of GIP. Altogether, our results identify GIPR/Rap1 signaling in the brain as a molecular pathway linking overnutrition to the control of neural leptin actions.

Keywords: G-protein coupled receptors; Leptin; Metabolism; Neuroscience; Obesity.

Figure 1. Brain GIPR controls body weight and adiposity in obese mice.

The GIPR monoclonal antibody Gipg013 was centrally infused (1 μg, every other day) into HFD-induced obese mice (A–C, 20 weeks of HFD feeding, n = 11–13), normal chow–fed (lean) mice (D–F, n = 6–7), and ob/ob mice (G–I, n = 8–9). Body weight (A, D, and G) and food intake (B, E, and H) were measured daily. Body composition (C, F, and I) was measured on day 14 of Gipg013 treatment. (J) Relative mRNA expression of the indicated genes in the hypothalamus after 15 days of Gipg013 injection. (K) Western blot quantification of SOCS-3 protein in the hypothalamus of Gipg013-treated mice (n = 7–13). β-Actin was used as a loading control. (L–N) HFD-induced obese mice fed for 49 weeks were i.c.v. infused with Gipg013 or control IgG (1 μg every 4 days, arrows) and in combination with an i.p. injection of liraglutide or saline (0.3 mg/kg once a day) (n = 9–11). (L) Body weight, (M) body weight change, and (N) food intake were measured during the treatment. Each data point represents the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-way ANOVA followed by Sidak’s multiple comparisons tests (A–I and L–N); 1-way ANOVA followed by Tukey’s multiple comparisons test (K); and t test (J).

Figure 2. GIP negatively regulates neural leptin actions.

(A and B) Leptin or vehicle was i.c.v. infused into WT and Gipr-KO mice after 4 weeks of a normal chow diet (A) or a HFD (B) (n = 7–11). Body weight and food intake were measured daily. (C) Normal chow–fed mice (n = 11–12, 16 weeks of age) were i.c.v. administered GIP (30 pmol/day) or vehicle. Leptin (5 μg/day) or vehicle was i.c.v. administered. Body weight and food intake were measured. (D) Mice (n = 3) were i.c.v. administered GIP or vehicle followed by leptin (5 μg) 3 hours later. p-STAT3 immunohistochemistry and quantification. Scale bar: 100 μm. (E) Electrophysiological recordings demonstrated that GIP pretreatment (6 h) occluded the leptin-induced depolarization of POMC neurons. The inhibitory effect of GIP on leptin-induced activation of POMC neurons is summarized in the histogram (n = 8–9). (F) GIP (administered i.c.v.) increased hypothalamic mRNA expression of Socs-3, Ptp1b, and Tcptp. Data are from 3 different experiments (n = 17–18). (G) Mice received once-daily i.p. injections of GIP for 3 days and then i.c.v. injections of leptin (5 μg) 2 hours after the last GIP injection. Body weight and food intake were measured 24 hours after leptin injection. n = 11 for groups without GIP treatment, n = 9 for GIP (30 pmol) treatment, and n = 4 for GIP (300 pmol) treatment. Each data point represents the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with control mice, by 2-way ANOVA followed by Sidak’s multiple comparisons test (A–D and G); ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, and ^####P < 0.0001, compared with control mice on day 6 (A and B) and on day 3 (C), by 1-way ANOVA followed by Tukey’s multiple comparisons test; and *P < 0.05 and ***P < 0.001 compared with control, by t test (E and F). Data represent the mean ± SEM of 2 different experiments.

Figure 3. Rap1 mediates the effects of centrally administered GIP.

(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.