Inhibition of the melanocortin-3 receptor (MC3R) causes generalized sensitization to anorectic agents

Abstract

The melanocortin-3 receptor (MC3R) acts presynaptically to regulate GABA release from agouti-related protein (AgRP) nerve terminals and thus may be a negative regulator of multiple circuits involved in feeding behavior and energy homeostasis. Here, we examined the role of MC3R in regulating the response to various anorexigenic agents. Our findings reveal that genetic deletion or pharmacological inhibition of MC3R improves 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 other agents, including the acute satiety factors peptide YY (PYY_3-36) and cholecystokinin (CCK) and the long-term adipostatic factor, leptin, demonstrated that increased sensitivity to anorectic agents is a generalized result of MC3R antagonism. Enhanced neuronal activation in multiple nuclei, including ARH, VMH, and DMH, was observed using Fos immunohistochemistry following low-dose liraglutide in MC3R knockout mice (Mc3r^-/-), supporting the hypothesis that the MC3R is a negative regulator of circuits regulating 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 GLP1R analogs, suggesting that MC3R antagonists may have value in enhancing the dose-response range of obesity therapeutics.

Figure 1.. Loss of MC3R increases responsiveness to GLP1 drugs.

Liraglutide (0.05mg/kg – 0.4mg/kg) administration results in more significant inhibition of (A) food intake and (B) weight loss in Mc3r ^−/− male mice compared to Mc3r ^+/+ mice in a dose-dependent manner after 24-hours (N = 7–8/group). (C) Liraglutide-induced feeding (D) and body weight changes of Mc3r ^+/+ and Mc3r ^−/− female mice (n = 7–8/group; 0.05mg/kg – 0.4mg/kg). Tirzepatide (TZ, 1nmol/kg – 4nmol/kg) and coadministration of tirzepatide and C11- induced (E) feeding responses and (F) body weight changes of wildtype male mice (vehicle, n = 10, all other groups n = 8) at 24-hours post-injection. (G) 24-hour feeding and (H) body weight change after chronic injections of tirzepatide (2nmol/kg), C11 (0.5nmol), tirzepatide and C11, and vehicle. Data is expressed as mean ± SEM, and statistical tests were two-way ANOVA with Tukey’s test for post hoc analysis for (G–J). For all the dose-response curve data, repeated measures of two-way ANOVA were corrected for multiple comparisons using the Tukey–Kramer method for each time point, and data were fitted with four parameters: nonlinear fit, *p < 0.05, *p <0.01, ***p <0.001.

Figure 2.. MC3R deletion has no effect on the incretin activity or malaise associated with liraglutide.

Glucose levels and area under the curve before and after oral administration of glucose at 0.5 g/kg (A), 1.0 g/kg (B), 2.0 g/kg (C) after liraglutide (0.2 mg/kg), and vehicle treatment in Mc3r^+/+ and Mc3r^−/− male mice (n = 7/group). (D) Glucose levels before and after insulin injection (0.75 IU insulin/kg) in Mc3r^+/+ and Mc3r^−/− male mice treated with liraglutide and vehicle (n = 8/group). CTA test days 1 (E) and 2 (F) after liraglutide administration in Mc3r^+/+ and Mc3r^−/− male mice. (G) Representative images from the AP showing Fos IHC after saline or liraglutide injection were obtained from Mc3r^+/+ and Mc3r^−/− male mice. (H) quantified cells expressing Fos after saline or liraglutide injection in Mc3r^+/+ and Mc3r^−/− male mice. (I) Representative images from the hypothalamus showing Fos IHC after liraglutide treatment in Mc3r^+/+ and Mc3r^−/− mice. (J) quantified cells expressing Fos in ARH, VMH, and DMH after liraglutide treatment.

Figure 3.. Deletion of MC3R results in generalized enhanced sensitivity to anorectic hormones.

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

Figure 4:. MC3R deletion enhances the ability of an MC4R agonist to increase sensitivity to liraglutide.

(A, C) 24-hr food intake and (B, D) body weight changes in response to CTX-1211 (2mg/kg, i.p., n =8/group), liraglutide (0.05 mg/kg – 0.4 mg/kg, sc, n = 8) in male and female mice. (E) 24-hr food intake and (F) body weight changes in response to setmelanotide (1.5 mg/kg, i.p., n = 9), liraglutide (0.2mg/kg, sc, n= 9), liraglutide and setmelanotide (n = 9), and vehicle (n= 9) in Mc3r^+/+ and Mc3r^−/− mice. Means ± SEM are shown. Statistical analysis was done with 2-way ANOVA with repeated measures, *p < 0.05, *p <0.01, ****p <0.001.

Figure 5.. Specific deletion of MC3R in AgRP neurons results in increased responsiveness to liraglutide and leptin.

(A) Time course feeding in Agrp-iCre and (B) Agrp:iCre;Mc3r^fl/fl in response to 1mg/kg leptin (ip) (n= 8/group). 24-hr food intake and change in body weight in (C, D) males and (E, F) in females in response to liraglutide (0.1 mg/kg, sc) (n =8/group/males, n =4–6/group/females). Means ± SEM are shown. Statistical analysis was done with 2-way ANOVA with repeated measures (A, B) with Tukey’s post hoc, student t-test (C-F), *p < 0.05, **p <0.01, ****p <0.001.

Figure 6.. Genetic deletion of the neuronal POMC gene abolishes the response to liraglutide in males.

(A, C) 24-hour food intake and percent body weight change 24-h post-injection of liraglutide in (A, B) males (0.2mg/kg, sc, n= 7/group) and (C, D) females (0.2mg/kg, sc, n =4/group). Data is expressed as mean ± SEM. Statistical analysis was done with repeated 2-way ANOVA with Tukey post hoc analysis. *p < 0.05, **p <0.01, ***p <0.001.