Subthreshold activation of the melanocortin system causes generalized sensitization to anorectic agents in mice

Abstract

The melanocortin-3 receptor (MC3R) regulates GABA release from agouti-related protein (AgRP) nerve terminals and thus tonically suppresses multiple circuits involved in feeding behavior and energy homeostasis. Here, we examined the role of the MC3R and the melanocortin system in regulating the response to various anorexigenic agents. The genetic deletion or pharmacological inhibition of the MC3R, or subthreshold doses of an MC4R agonist, improved the dose responsiveness to glucagon-like peptide 1 (GLP1) agonists, as assayed by inhibition of food intake and weight loss. An enhanced anorectic response to the acute satiety factors peptide YY (PYY3-36) and cholecystokinin (CCK) and the long-term adipostatic factor leptin demonstrated that increased sensitivity to anorectic agents was a generalized result of MC3R antagonism. We observed enhanced neuronal activation in multiple hypothalamic nuclei using Fos IHC following low-dose liraglutide in MC3R-KO mice (Mc3r-/-), supporting the hypothesis that the MC3R is a negative regulator of circuits that control multiple aspects of feeding behavior. The enhanced anorectic response in Mc3r-/- mice after administration of GLP1 analogs was also independent of the incretin effects and malaise induced by GLP1 receptor (GLP1R) analogs, suggesting that MC3R antagonists or MC4R agonists may have value in enhancing the dose-response range of obesity therapeutics.

Keywords: Metabolism; Obesity.

Figure 1. MC3R loss increases responsiveness to GLP1 drugs.

Liraglutide (0.05–0.4 mg/kg) administration resulted in more significant inhibition of (A) food intake and (B) weight loss in Mc3r^–/– male mice compared with Mc3r^+/+ mice in a dose-dependent manner after 24 hours (n = 7–8/group). (C) Liraglutide-induced feeding and (D) body weight changes of Mc3r^+/+ and Mc3r^–/– female mice (n = 7–8/group; 0.05–0.4 mg/kg). Tirzepatide (TZ) (1–4 nmol/kg) and coadministration of tirzepatide and C11-induced (E) feeding responses and (F) body weight changes of WT male mice (vehicle, n = 10; all other groups, n = 8) at 24 hours after injection. (G) Twenty-four-hour feeding and (H) body weight changes after chronic injections of tirzepatide (2 nmol/kg), C11 (0.5 nmol), tirzepatide plus C11, and vehicle. Data are expressed as the mean ± SEM, and statistical tests were performed by 2-way ANOVA with Tukey’s test for post hoc analysis (G–H). For all the dose-response curve data, a repeated-measures 2-way ANOVA was corrected for multiple comparisons using the Tukey-Kramer method for each time point, and data were fitted with 4 parameters: nonlinear fit, **P < 0.01.

Figure 2. Mc3r deletion has no effect on the incretin activity or malaise associated with liraglutide.

(A–F) Glucose levels and AUC before and after oral administration of glucose (1 g/kg) after liraglutide (lira) (0.01–0.2 mg/kg) or vehicle (veh) treatment in Mc3r^+/+ and Mc3r^–/– male mice (n = 6–7/group). CTA test day 1 (G) and test day 2 (H) after liraglutide administration in Mc3r^+/+ and Mc3r^–/– male mice. (I) Representative images from the AP showing Fos IHC after saline or liraglutide injection from Mc3r^+/+ and Mc3r^–/– male mice. CC, central canal. Scale bars: 100 μm. (J) Quantification of cells expressing Fos after saline or liraglutide injection in Mc3r^+/+ and Mc3r^–/– male mice. (K) Representative images from the hypothalamus showing Fos IHC after liraglutide treatment in Mc3r^+/+ and Mc3r^–/– mice. Scale bars: 100 μm. (L) Quantification of cells expressing Fos in the ARH, VMH, and DMH after liraglutide treatment. Statistical analysis was done by 2-tailed Student’s t test, *P < 0.05, and ****P < 0.0001 (J, G, H, and L).

Figure 3. Deletion of Mc3r results in generalized enhanced sensitivity to anorectic hormones.

Nocturnal feeding in response to leptin (0.1–1 mg/kg) 12 hours after injection (vehicle, n = 11, n = 6/group) in Mc3r^+/+ and Mc3r^–/– (A) male and (D) female mice. Acute dark-phase feeding after administration of PYY_3–36 (n = 7/group) in Mc3r^+/+ and Mc3r^–/– (B ) male and (E) female mice. Acute dark-phase feeding after administration of CCK (n = 7–8/group) in Mc3r^+/+ and Mc3r^–/– (C ) male and (F) female mice. Data represent the mean ± SEM. Statistical analysis was done by 2-way, repeated-measures ANOVA with Tukey’s post hoc test (A–F).

Figure 4. Mc3r deletion enhances the ability of an MC4R agonist to increase sensitivity to liraglutide.

(A, C, E, and G) Twenty-four-hour food intake and (B, D, F, and H) body weight changes in response to CTX 1211 or setmelanotide (2 mg/kg, i.p., n = 8/group) or to liraglutide alone (0.05–0.4 mg/kg, s.c., n = 8), as indicated in male and female mice. (I and K) Twenty-four-hour food intake and (J and L) body weight changes in response to CTX 1211 (1.5 mg/kg, i.p., n = 5–6) or setmelanotide (1.5 mg/kg, i.p., n = 9) alone, liraglutide alone (0.2 mg/kg, s.c.), liraglutide plus CTX 21 or setmelanotide, and vehicle in Mc3r^+/+ and Mc3r^–/– mice. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-way, repeated-measures ANOVA.

Figure 5. MC4R agonist synergistically increases the sensitivity of liraglutide.

(A and C). Twenty-four-hour food intake and (B and D) body weight changes in response to vehicle (n = 10/group), liraglutide (0.1 mg/kg, n = 10/group), CTX 1211 (1 mg/kg, n = 10/group), and the combination of liraglutide plus CTX 1211 in male (A and B) and female(C and D) mice. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-way ANOVA.

Figure 6. Specific deletion of Mc3r in AgRP neurons increases the responsiveness to liraglutide and leptin.

(A) Time-course feeding for Agrp-Cre and (B) Agrp-Cre Mc3r^fl/fl mice in response to leptin (1 mg/kg, i.p., n = 8/group). Twenty-four-hour food intake and change in body weight in (C and D) male and (E and F) female mice in response to liraglutide (0.1 mg/kg, s.c., n = 8/group/males, n = 4–6/group/females). Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-way, repeated-measures ANOVA with Tukey’s post hoc test (A and B) and 2-tailed Student’s t test (C–F).

Figure 7. The reduction in food intake and weight loss induced by liraglutide persists with DCZ-mediated activation of hM3Dq-DREADD in AgRP neurons.

(A) Experimental timeline for DREADD experiments. (B) AAV-mediated functional expression of hM3Dq-mCherry in AgRP neurons after DCZ (0.3 mg/kg) or saline treatment. Representative confocal images are shown after staining for c-Fos. Scale bars: 50 μm. (C and E) Twenty-four-hour food intake and (D and F) changes in body weight after liraglutide (0.1 mg/kg, s.c.) or vehicle treatment in the presence of DCZ (0.3 mg/kg, i.p.) or saline in Agrp-Cre and Agrp-Cre Mc3r^fl/fl mice. Data are from both male and female mice and represent the mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001, by 2-way, repeated-measures ANOVA with Tukey’s post hoc test (A and B) and 2-tailed Student’s t test (C–F).

Figure 8. Genetic deletion of the neuronal Pomc gene abolishes the response to liraglutide and leptin in male mice.

(A) Schematic of the FED3 feeding device in free-feeding mode. Twenty-four-hour food intake and changes in body weight 24 hours after injection of liraglutide in (B and C) male mice (0.2 mg/kg, s.c., n = 7/group) and (D and E) female mice (0.2 mg/kg, s.c., n = 4/group). (F and G) Twenty-four-hour food intake and body weight changes in response to leptin (3 mg/kg, i.p.) in male mice (n = 5/group). Data are expressed as the mean ± SEM. **P < 0.01, ***P < 0.001, and ****P < 0.0001, by repeated-measures 2-way ANOVA with Tukey’s post hoc analysis.