Cross-species studies implicate the melanocortin 3 receptor more strongly in the control of pubertal development than energy balance

Abstract

Hypothalamic neurons expressing either POMC or AGRP sense nutritional state directly and indirectly and transmit these neuropeptide signals to other brain centres through the melanocortin 3 and 4 receptors. MC4R is primarily concerned with the control of appetite and energy expenditure while MC3R is more closely related to the control of linear growth and the timing of puberty. The role of MC3R in the long-term control of energy balance and body composition is less clear, particularly in humans. We have undertaken studies in humans, domestic dogs and mice with the goal of clarifying the relative impact of MC3R deficiency on energy balance, growth and sexual development. By studying three large consanguineously enriched cohorts, totalling approximately 300K people, we identified nine individuals who are homozygous for functionally null MC3R variants. The body mass index (BMI) of the homozygous MC3R variant carriers was not significantly different from that of age, sex and demographically matched controls, with six of the nine homozygotes having a BMI <30 kg/m². We detected a canine MC3R missense variant (p.M320I) which is common in labrador retrievers and showed that this significantly impairs receptor signalling. Dogs homozygous for p.M320I were lighter and showed delayed pubertal development but were not significantly more obese than wild-type or heterozygous dogs. We also established that the lack of Mc3r delayed pubertal development in both male and female mice. Finally, we studied growth and pubertal trajectories of individuals carrying rare loss-of-function MC3R variants and found that male carriers had delayed peak weight velocity and genital development but had no evidence for excess body fat compared to non-carriers. Our results support MC3R having a conserved role across mammals in controlling growth and pubertal timing. While MC3R deficiency may influence linear growth and body composition, complete loss of MC3R does not result in a penetrant human obesity syndrome.

Keywords: ALSPAC; MC3R; Melanocortins; Obesity; Puberty.

Graphical abstract

Figure 1

Characterisation of MC3R LoF variants and the weight, height and BMI of homozygous carriers. (A) In vitro measurement of cAMP response of overexpressed MC3R WT and homozygous mutations, upon stimulation with NDP-MSH ligand, reported as percentage of Emax of WT. Error bars = SEM. N = 3–6 biological replicates. (B–D) Z-scored plots of weight, height and BMI for the cohorts B) BELIEVE (two p.V63M homozygotes, in blue), C) PGR (two p.V63M and one p.D84N homozygotes, in green), and D) MCPS (four p.Y143∗ homozygotes, in red). Homozygotes for CLoF MC3R mutations are Z-scored against an age, sex and ethnicity-matched population, then Z-scores combined for plotting. Each homozygote is represented as a coloured dot. Boxplot represents median with 25th and 75th percentiles, and whiskers extend to 1.5 times the interquartile range. Outliers are plotted as individual greyscale dots.

Figure 2

Loss of function MC3R variants in ALSPAC and carrier associations with pubertal milestones. (A–B) cAMP response of MC3R WT and heterozygous variants upon stimulation with NDP-MSH ligand, reported as percentage of Emax of WT, where (A) are partial LoF variants, and (B) are complete LoF variants. Error bars = SEM. N = 2–7 biological replicates. (C) For carriers of mutations in (A) or (B), results of linear models for 9 derived puberty phenotypes for females and males. Filled in circles represent unadjusted p < 0.05. Hollow circles represent unadjusted p ≥ 0.05. Lines represent lower and upper 95% confidence intervals. Numbers for carriers and non-carriers included in the analysis can be found in Supplementary Table 4.

Figure 3

Loss of MC3R function is associated with delayed onset of puberty in dogs and reduced body condition score and weight. (A) cAMP generation and (B) β-arrestin recruitment measured in vitro for overexpressed MC3R construct, either WT or carrying the p.M320I mutation, upon stimulation with ⍺-MSH. Error bars represent SEM. P-value reported for WT vs p.M320I Emax using unpaired two-tailed t-test. N = 3–4 biological replicates. (C–E) Partial regression violin plots for C) weight, D) food motivation and E) body condition score (BCS) from Labrador retrievers, per each allele dose of p.M320I. Test statistic and effect estimate obtained by ANOVA with allele effect modelled as additive. Beta-adjusted phenotypes calculated using residuals from linear regression coefficients for the respective phenotype (see methods for details). (F) Violin plots for 80 female, non-neutered Labrador retrievers and their age of first season. Overlayed boxplots show the median with 25th and 75th percentiles, and whiskers extend to 1.5 times the interquartile range.

Figure 4

Mc3r deficient mice exhibit delayed pubertal onset (A) Age of vaginal opening in female mice and (B) age of preputial separation in male mice, in WT or Mc3r^−/− mice. Error bars represent mean and SEM. P-value calculated by unpaired, two-tailed t-test between genotypes.