Loss of function of the melanocortin 2 receptor accessory protein 2 is associated with mammalian obesity

Abstract

Melanocortin receptor accessory proteins (MRAPs) modulate signaling of melanocortin receptors in vitro. To investigate the physiological role of brain-expressed melanocortin 2 receptor accessory protein 2 (MRAP2), we characterized mice with whole-body and brain-specific targeted deletion of Mrap2, both of which develop severe obesity at a young age. Mrap2 interacts directly with melanocortin 4 receptor (Mc4r), a protein previously implicated in mammalian obesity, and it enhances Mc4r-mediated generation of the second messenger cyclic adenosine monophosphate, suggesting that alterations in Mc4r signaling may be one mechanism underlying the association between Mrap2 disruption and obesity. In a study of humans with severe, early-onset obesity, we found four rare, potentially pathogenic genetic variants in MRAP2, suggesting that the gene may also contribute to body weight regulation in humans.

Fig. 1

Phenotype of Mrap2^−/− mice. (A) Weight curves for Mrap^+/+ vs. Mrap^+/− vs. Mrap2^−/− mice on standard chow (Chow, upper panels: male n= 9 vs. 28 vs. 15, female n=12 vs. 18 vs. 10) or high fat diets (HFD, ages 56–95 days, lower panels, superimposed on standard chow curves: male n= 10 vs. 8 vs. 10, female n=7 vs. 12 vs. 7). For both genders the weight curves of Mrap^+/+ and Mrap^+/− mice on standard chow differ significantly at older (161–175 days) ages, and at younger ages (56–95 days) on a high fat diet. ). *p=.02, **p=.001, ***p=.0003. (B) Fat depots on standard chow diet. Upper panel: White adipose tissue (WAT) weights in Mrap^+/+ vs. Mrap2^−/− (males and females, ages 117–122 days, n=5 vs. 4, respectively). Lower left panel: BAT weight in Mrap^+/+ vs. Mrap2^−/− mice (males and females, ages 117–122 days, n=5 vs. 4). Lower right panel: WAT cell size in in Mrap^+/+ vs. Mrap2^−/− mice (females, 50 cells counted from each mouse). *p=.009, **p=.003, ***p=.0003, ****p<.00001.

Fig. 2

Energy balance in Mrap2^−/− mice. (A) Cumulative food intake (upper panels) and weight (lower panels) in ad libitum fed Mrap^+/+ vs. Mrap2^−/− males (n=10 vs. 11) and females (n=11 vs. 8). (B) Energy expenditure in ad libitum-fed Mrap^+/+ vs. Mrap2^−/− mice. Upper panels, continuous measurement over 3 days, males (n=3 vs. 4), females (n=4 vs. 3), ages 30–34 days. Lower panels: body weight vs. energy expenditure, integrated over 24 h, males (n=18 vs. 14, ages 30–45 days), females (n=16 vs. 11, ages 30–42 days). Analysis by ANCOVA showed no differences between genotypes (males, p=.38, females, p=.67).

Fig. 3

Interaction between Mrap2 and Mc4r. (A) Conditional deletion of Mrap2 in Sim1neurons. Top right, Cre DNA analysis by PCR. HT DNA from Sim1^CreBAC::Mrap2^f/f mice contains Cre (374 bp), but from Mrap2^f/f mice does not. Molecular weight marker (M) on right (bp). Top left, Mrap2 DNA analysis in Sim1^CreBAC::Mrap2^f/f and Mrap2^f/f mice by PCR. Both genotypes contain floxed, intact Mrap2 DNA in CX, HT, and BS (314 bp in upper electropherogram, and 1013 bp in lower electropherogram, molecular weight markers on left). Only Sim1^CreBAC::Mrap2^f/f mice contain Mrap2^Del (400 bp, lower electropherogram), and only in HT and BS, but not in CX, consistent with fluorescent reporter data (Fig. S3A). No PCR products are present without added DNA (H2O). Bottom, Mrap2 mRNA expression in Sim1^CreBAC::Mrap2^f/f and Mrap2^f/f mice by RT-PCR. Both genotypes express floxed, intact Mrap2 mRNA in CX, HT, and BS (247 bp). Only Sim1^CreBAC::Mrap2^f/f mice express Mrap2^Del mRNA (147 bp), and only in HT. Global Mrap2^Del/Del mice express Mrap2^Del mRNA in all 3 sites. (B) Body weights of Mrap2^+/+ (male n=6, female n=11), Mrap2^−/− (male n=11, female n=7), Mrap2^f/f (male n=8, female n=12) and conditional Sim1^CreBAC::Mrap2^f/f (male n=8, female n=7) mice, all age 133 days. *p=.04, **p=.007, ***p=.0002, ****p<.0001. (C) Effect of Mrap2 on Mc4r signaling. Left panel: Level of cAMP reporter activity (CRELuc) in CHO cells alone, or co-transfected with Mc4r, with or without Mrap2 or the Mrap2 knockout construct, Mrap2delE3, 5 h following exposure to 0–10 nM αMSH (n=3/group). Right panel: cAMP activity of these same constructs, expressed as percent induction following 0–10 nM αMSH, relative to 0 nM αMSH. *p<.0001, Mc4r+Mrap2 vs. Mc4r at same [αMSH], by ANOVA. For most data points, error bars are obscured by symbols.