Loss of Function of the Melanocortin 2 Receptor Accessory Protein 2 Is Associated with Mammalian Obesity
July 2013
Abstract
Melanocortin receptor accessory proteins (MRAPs) modulate signaling of melanocortin receptors. 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 AMP, 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 and potentially pathogenic genetic variants in MRAP2. Results suggest the gene may also contribute to body weight regulation.
Membrane-expressed G protein-coupled receptors (GPCRs) modulate cellular responses to numerous physiological stimuli. The melanocortin receptors (MCRs) are a subfamily of GPCRs that mediate signaling in response to the pro-opiomelanocortin-derived peptides, adrenocorticotropic hormone (ACTH) and ?-melanocyte–stimulating hormone (?-MSH) and their competitive antagonists, agouti and agouti-related protein. The MCRs mediate a diverse range of physiological functions]MC1R[/b] is involved in skin pigmentation, MC2R plays a critical role in the hypothalamic-pituitary-adrenal axis, MC3R and MC4R are involved in energy homeostasis and MC5R is implicated in exocrine function.
There is increasing recognition that accessory proteins can modulate GPCR trafficking, as well as ligand binding and signaling. An accessory protein for MC2R, MC2R accessory protein (MRAP), is required for the trafficking of MC2R to the surface of adrenal cells and for signaling in response to ACTH Loss of either MC2R or MRAP causes severe resistance to ACTH, with resulting glucocorticoid deficiency.
All mammals have a paralogous gene, MRAP2, which, like MC3R and MC4R, is predominantly expressed in the brain, most prominently in the pons and cerebellum, but including in regions involved in energy homeostasis such as the hypothalamus and brain-stem. Within the paraventricular nucleus of the hypothalamus (PVN), Mrap2 and Mc4r mRNAs are co-expressed in many cells. We hypothesized that Mrap2 might modulate signaling through a melanocortin receptor and potentially affect energy homeostasis. We therefore performed targeted deletion of Mrap2, using Cre-lox-mediated excision of the 100 bp exon 3 (which encodes the highly conserved transmembrane domain) to create test subjects with normal levels of mRNA predicted to encode a truncated protein that includes the first 55 amino acids of Mrap2, with the transmembrane domain replaced by 11 aberrant amino acids specified by the out-of-frame exon 4, followed by a stop codon. Normal levels of the mutant mRNA indicate preservation of Mrap2-containing neurons in null...although these neurons likely do not express the predicted mutant protein, since mutant Mrap2 mRNA, but not protein, is present in cells transfected with the same Mrap2 mutant construct used to create the null lab guinea pig.
Mrap2 null rodents appeared normal at birth, with normal weight gain and post-weaning food intake during early life (0–32 days and 23–32 days, respectively), although young Mrap2?/? male mice trended toward greater weight and food intake with advancing age. However, null mice of both genders gradually became extremely obese on a diet of regular chow ad libitum. Heterozygous mice were significantly heavier than wild-type animals on standard chow (160–175 days; males, Mrap2+/+ 26.0±0.4 g, Mrap2+/? 29.9±0.9 g; females Mrap2+/+ 24.5±0.9 g, Mrap2+/? 28.1±0.7 g), and at younger ages (56–95 days) on a high fat diet. In addition, Mrap2?/? mice had increased length and per cent of weight due to fat, and decreased per cent of weight due to lean mass. Both genders of Mrap2?/? mice had increased visceral adiposity, over twice the normal white adipose tissue cell size, enlarged brown adipose tissue depots, normal liver histology on a regular chow diet, but much greater hepatic adipose degeneration compared with wild-type mice on a high fat diet. Adult Mrap2 null mice had, as expected, elevated leptin concentrations corresponding to their increased fat mass, which normalized with diet-induced weight normalization. Obese adult mice had normal fasting insulin and normal tolerance to intraperitoneal glucose injection.
Mrap2 has been postulated to play a role in the adrenal response to ACTH. We therefore measured diurnal rhythmicity and stress responsiveness of the adrenal axis in Mrap2 null mice, which were normal. Thyroid hormone levels were also normal. Epinephrine and norepinephrine excretion were reduced in male Mrap2?/? mice only, but Ucp1 mRNA concentrations increased appropriately in both genders of null mice following exposure to 4°C for 18h. Hypothalamic Agrp mRNA concentration was reduced in the Mrap2 null group, whereas Pomc mRNA was normal.
In summary, we have found that global/ brain-specific inactivation of Mrap2 causes obesity in the rodent and that rare heterozygous variants in MRAP2 are associated with early-onset, severe obesity in humans. The mechanism(s) by which Mrap2 exerts effect on body weight regulation remain to be firmly established but likely involve altered signaling through Mc4r and perhaps other melanocortins. This evolutionary conservation, plus the extreme disease phenotype caused by loss of Mrap2 function, supports the importance of Mrap2 in biology today.
Melanotan has been known to be of help in obesity and body weight problems. But these findings about the melanocortin accessory proteins also have significant value since they are directly involved with obesity. MRAP 2 inactivation causes obesity. So it is important to use products that activate that pathway in order to control obesity. If there are researches about genetic problems with MRAP 2 that interfere with its activation, then this can surely make these findings more accurate. For now, this is such a great study on such a relevant topic.
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