MC4R Localizes at Excitatory Postsynaptic and Peri-Postsynaptic Sites of Hypothalamic Neurons in Primary Culture.

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that is expressed in several brain locations encompassing the hypothalamus and the brainstem, where the receptor controls several body functions, including metabolism. In a well-defined pathway to decrease appetite, hypothalamic proopiomelanocortin (POMC) neurons localized in the arcuate nucleus (Arc) project to MC4R neurons in the paraventricular nuclei (PVN) to release the natural MC4R agonist α-melanocyte-stimulating hormone (α-MSH). Arc neurons also project excitatory glutamatergic fibers to the MC4R neurons in the PVN for a fast synaptic transmission to regulate a satiety pathway potentiated by α-MSH. By using super-resolution microscopy, we found that in hypothalamic neurons in a primary culture, postsynaptic density protein 95 (PSD95) colocalizes with GluN1, a subunit of the ionotropic N-methyl-D-aspartate receptor (NMDAR). Thus, hypothalamic neurons form excitatory postsynaptic specializations. To study the MC4R distribution at these sites, tagged HA-MC4R under the synapsin promoter was expressed in neurons by adeno-associated virus (AAV) gene transduction. HA-MC4R immunofluorescence peaked at the center and in proximity to the PSD95- and NMDAR-expressing sites. These data provide morphological evidence that MC4R localizes together with glutamate receptors at postsynaptic and peri-postsynaptic sites.

Melanocortin-4 Receptor PLC Activation Is Modulated by an Interaction with the Monocarboxylate Transporter 8.

The melanocortin-4 receptor (MC4R) is a key player in the hypothalamic leptin-melanocortin pathway that regulates satiety and hunger. MC4R belongs to the G protein-coupled receptors (GPCRs), which are known to form heterodimers with other membrane proteins, potentially modulating receptor function or characteristics. Like MC4R, thyroid hormones (TH) are also essential for energy homeostasis control. TH transport across membranes is facilitated by the monocarboxylate transporter 8 (MCT8), which is also known to form heterodimers with GPCRs. Based on the finding in single-cell RNA-sequencing data that both proteins are simultaneously expressed in hypothalamic neurons, we investigated a putative interplay between MC4R and MCT8. We developed a novel staining protocol utilizing a fluorophore-labeled MC4R ligand and demonstrated a co-localization of MC4R and MCT8 in human brain tissue. Using in vitro assays such as BRET, IP1, and cAMP determination, we found that MCT8 modulates MC4R-mediated phospholipase C activation but not cAMP formation via a direct interaction, an effect that does not require a functional MCT8 as it was not altered by a specific MCT8 inhibitor. This suggests an extended functional spectrum of MCT8 as a GPCR signaling modulator and argues for the investigation of further GPCR-protein interactions with hitherto underrepresented physiological functions.

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

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.

Melanocortin-receptor 4 activation modulates proliferation and differentiation of rat postnatal hippocampal neural precursor cells.

Postnatal hippocampal neurogenesis is essential for learning and memory. Hippocampal neural precursor cells (NPCs) can be induced to proliferate and differentiate into either glial cells or dentate granule cells. Notably, hippocampal neurogenesis decreases dramatically with age, partly due to a reduction in the NPC pool and a decrease in their proliferative activity. Alpha-melanocyte-stimulating hormone (α-MSH) improves learning, memory, neuronal survival and plasticity. Here, we used postnatally-isolated hippocampal NPCs from Wistar rat pups (male and female combined) to determine the role of the melanocortin analog [Nle4, D-Phe7]-α-MSH (NDP-MSH) in proliferation and fate acquisition of NPCs. Incubation of growth-factor deprived NPCs with 10 nM NDP-MSH for 6 days increased the proportion of Ki-67- and 5-bromo-2'-deoxyuridine (BrdU)-positive cells, compared to the control group, and these effects were blocked by the MC4R antagonist JKC-363. NDP-MSH also increased the proportion of glial fibrillar acidic protein (GFAP)/Ki-67, GFAP/sex-determining region Y-box2 (SOX2) and neuroepithelial stem cell protein (NESTIN)/Ki-67-double positive cells (type-1 and type-2 precursors). Finally, NDP-MSH induced peroxisome proliferator-activated receptor (PPAR)-γ protein expression, and co-incubation with the PPAR-γ inhibitor GW9662 prevented the effect of NDP-MSH on NPC proliferation and differentiation. Our results indicate that in vitro activation of MC4R in growth-factor-deprived postnatal hippocampal NPCs induces proliferation and promotes the relative expansion of the type-1 and type-2 NPC pool through a PPAR-γ-dependent mechanism. These results shed new light on the mechanisms underlying the beneficial effects of melanocortins in hippocampal plasticity and provide evidence linking the MC4R and PPAR-γ pathways in modulation of hippocampal NPC proliferation and differentiation.

Automated Patch Clamp Recordings of GPCR-Gated Ion Channels: Targeting the MC4-R/Kir7.1 Potassium Channel Complex.

Automated patch clamp recording is a valuable technique in drug discovery and the study of ion channels. It allows for the precise measurement and manipulation of channel currents, providing insights into their function and modulation by drugs or other compounds. The melanocortin 4 receptor (MC4-R) is a G protein-coupled receptor (GPCR) crucial to appetite regulation, energy balance, and body weight. MC4-R signaling is complex and involves interactions with other receptors and neuropeptides in the appetite-regulating circuitry. MC4-Rs, like other GPCRs, are known to modulate ion channels such as Kir7.1, an inward rectifier potassium channel, in response to ligand binding. This modulation is critical for controlling ion flow across the cell membrane, which can influence membrane potential, excitability, and neurotransmission. The MC4-R is the target for the anti-obesity drug Imcivree. However, this drug is known to lack optimal potency and also has side effects. Using high-throughput techniques for studying the MC4-R/Kir7.1 complex allows researchers to rapidly screen many compounds or conditions, aiding the development of drugs that target this system. Additionally, automated patch clamp recording of this receptor-channel complex and its ligands can provide valuable functional and pharmacological insights supporting the development of novel therapeutic strategies. This approach can be generalized to other GPCR-gated ion channel functional complexes, potentially accelerating the pace of research in different fields with the promise to uncover previously unknown aspects of receptor-ion channel interactions.

Therapeutic Strategies Against Metabolic Imbalance in a Male Mouse Model With 5-HT2CR Loss-of-Function.

The serotonin 2C receptor (5-HT2CR)-melanocortin pathway plays well-established roles in the regulation of feeding behavior and body weight homeostasis. Dysfunctions in this system, such as loss-of-function mutations in the Htr2c gene, can lead to hyperphagia and obesity. In this study, we aimed to investigate the potential therapeutic strategies for ameliorating hyperphagia, hyperglycemia, and obesity associated with a loss-of-function mutation in the Htr2c gene (Htr2cF327L/Y). We demonstrated that reexpressing functional 5-HT2CR solely in hypothalamic pro-opiomelanocortin (POMC) neurons is sufficient to reduce food intake and body weight in Htr2cF327L/Y mice subjected to a high-fat diet (HFD). In addition, 5-HT2CR expression restores the responsiveness of POMC neurons to lorcaserin, a selective agonist for 5-HT2CR. Similarly, administration of melanotan II, an agonist of the melanocortin receptor 4 (MC4R), effectively suppresses feeding and weight gain in Htr2cF327L/Y mice. Strikingly, promoting wheel-running activity in Htr2cF327L/Y mice results in a decrease in HFD consumption and improved glucose homeostasis. Together, our findings underscore the crucial role of the melanocortin system in alleviating hyperphagia and obesity related to dysfunctions of the 5-HT2CR, and further suggest that MC4R agonists and lifestyle interventions might hold promise in counteracting hyperphagia, hyperglycemia, and obesity in individuals carrying rare variants of the Htr2c gene.

A molecularly defined orbitofrontal cortical neuron population controls compulsive-like behavior, but not inflexible choice or habit.

Habits are familiar behaviors triggered by cues, not outcome predictability, and are insensitive to changes in the environment. They are adaptive under many circumstances but can be considered antecedent to compulsions and intrusive thoughts that drive persistent, potentially maladaptive behavior. Whether compulsive-like and habit-like behaviors share neural substrates is still being determined. Here, we investigated mice bred to display inflexible reward-seeking behaviors that are insensitive to action consequences. We found that these mice demonstrate habitual response biases and compulsive-like grooming behavior that was reversible by fluoxetine and ketamine. They also suffer dendritic spine attrition on excitatory neurons in the orbitofrontal cortex (OFC). Nevertheless, synaptic melanocortin 4 receptor (MC4R), a factor implicated in compulsive behavior, is preserved, leading to the hypothesis that Mc4r+ OFC neurons may drive aberrant behaviors. Repeated chemogenetic stimulation of Mc4r+ OFC neurons triggered compulsive and not inflexible or habitual response biases in otherwise typical mice. Thus, Mc4r+ neurons within the OFC appear to drive compulsive-like behavior that is dissociable from habitual behavior. Understanding which neuron populations trigger distinct behaviors may advance efforts to mitigate harmful compulsions.

Control of goal-directed and inflexible actions by dorsal striatal melanocortin systems, in coordination with the central nucleus of the amygdala.

The dorsomedial striatum (DMS) is associated with flexible goal seeking, as opposed to routinized habits. Whether local mechanisms brake this function, for instance when habits may be adaptive, is incompletely understood. We find that a sub-population of dopamine D1 receptor-containing striatal neurons express the melanocortin-4 receptor (MC4R) for α-melanocyte stimulating hormone. These neurons within the DMS are necessary and sufficient for controlling the capacity of mice to flexibly adjust actions based on the likelihood that they will be rewarded. In investigating MC4R function, we found that it suppresses immediate-early gene levels in the DMS and concurrently, flexible goal seeking. MC4R+ neurons receive input from the central nucleus of the amygdala, and behavioral experiments indicate that they are functionally integrated into an amygdalo-striatal circuit that suppresses action flexibility in favor of routine. Publicly available spatial transcriptomics datasets were analyzed for gene transcript correlates of Mc4r expression across the striatal subregions, revealing considerable co-variation in dorsal structures. This insight led to the discovery that the function of MC4R in the dorsolateral striatum complements that in the DMS, in this case suppressing habit-like behavior. Altogether, our findings suggest that striatal MC4R controls the capacity for goal-directed and inflexible actions alike.

IMPROVE 2022 International Meeting on Pathway-Related Obesity: Vision of Excellence.

Nearly 90 clinicians and researchers from around the world attended the first IMPROVE 2022 International Meeting on Pathway-Related Obesity. Delegates attended in person or online from across Europe, Argentina and Israel to hear the latest scientific and clinical developments in hyperphagia and severe, early-onset obesity, and set out a vision of excellence for the future for improving the diagnosis, treatment, and care of patients with melanocortin-4 receptor (MC4R) pathway-related obesity. The meeting co-chair Peter Kühnen, Charité Universitätsmedizin Berlin, Germany, indicated that change was needed with the rapidly increasing prevalence of obesity and the associated complications to improve the understanding of the underlying mechanisms and acknowledge that monogenic forms of obesity can play an important role, providing insights that can be applied to a wider group of patients with obesity. World-leading experts presented the latest research and led discussions on the underlying science of obesity, diagnosis (including clinical and genetic approaches such as the role of defective MC4R signalling), and emerging clinical data and research with targeted pharmacological approaches. The aim of the meeting was to agree on the questions that needed to be addressed in future research and to ensure that optimised diagnostic work-up was used with new genetic testing tools becoming available. This should aid the planning of new evidence-based treatment strategies for the future, as explained by co-chair Martin Wabitsch, Ulm University Medical Center, Germany.

Age-related ciliopathy: Obesogenic shortening of melanocortin-4 receptor-bearing neuronal primary cilia.

Obesity is often associated with aging. However, the mechanism of age-related obesity is unknown. The melanocortin-4 receptor (MC4R) mediates leptin-melanocortin anti-obesity signaling in the hypothalamus. Here, we discovered that MC4R-bearing primary cilia of hypothalamic neurons progressively shorten with age in rats, correlating with age-dependent metabolic decline and increased adiposity. This "age-related ciliopathy" is promoted by overnutrition-induced upregulation of leptin-melanocortin signaling and inhibited or reversed by dietary restriction or the knockdown of ciliogenesis-associated kinase 1 (CILK1). Forced shortening of MC4R-bearing cilia in hypothalamic neurons by genetic approaches impaired neuronal sensitivity to melanocortin and resulted in decreased brown fat thermogenesis and energy expenditure and increased appetite, finally developing obesity and leptin resistance. Therefore, despite its acute anti-obesity effect, chronic leptin-melanocortin signaling increases susceptibility to obesity by promoting the age-related shortening of MC4R-bearing cilia. This study provides a crucial mechanism for age-related obesity, which increases the risk of metabolic syndrome.

The melanocortin-4 receptor pathway and the emergence of precision medicine in obesity management.

Over the past few decades, there has been a global surge in the prevalence of obesity, rendering it a globally recognized epidemic. Contrary to simply being a medical condition, obesity is an intricate disease with a multifactorial aetiology. Understanding the precise cause of obesity remains a challenge; nevertheless, there seems to be a complex interplay among biological, psychosocial and behavioural factors. Studies on the genetic factors of obesity have revealed several pathways in the brain that play a crucial role in food intake regulation. The best characterized pathway, thus far, is the leptin-melanocortin pathway, from which disruptions are responsible for the majority of monogenic obesity disorders. The effectiveness of conservative lifestyle interventions in addressing monogenic obesity has been limited. Therefore, it is crucial to complement the management strategy with pharmacological and surgical options. Emphasis has been placed on developing drugs aimed at replacing the absent signals, with the goal of restoring the pathway. In both monogenic and polygenic forms of obesity, outcomes differ across various interventions, likely due to the multifaceted nature of the disease. This underscores the need to explore alternative therapeutic strategies that can mitigate this heterogeneity. Precision medicine can be regarded as a powerful tool that can address this concern, as it values the understanding of the underlying abnormality triggering the disease and provides a tailored treatment accordingly. This would assist in optimizing outcomes of the current therapeutic approaches and even aid in the development of novel treatments capable of more effectively managing the global obesity epidemic.

Protection against overfeeding-induced weight gain is preserved in obesity but does not require FGF21 or MC4R.

Overfeeding triggers homeostatic compensatory mechanisms that counteract weight gain. Here, we show that both lean and diet-induced obese (DIO) male mice exhibit a potent and prolonged inhibition of voluntary food intake following overfeeding-induced weight gain. We reveal that FGF21 is dispensable for this defense against weight gain. Targeted proteomics unveiled novel circulating factors linked to overfeeding, including the protease  legumain (LGMN). Administration of recombinant LGMN lowers body weight and food intake in DIO mice. The protection against weight gain is also associated with reduced vascularization in the hypothalamus and sustained reductions in the expression of the orexigenic neuropeptide genes, Npy and Agrp, suggesting a role for hypothalamic signaling in this homeostatic recovery from overfeeding. Overfeeding of melanocortin 4 receptor (MC4R) KO mice shows that these mice can suppress voluntary food intake and counteract the enforced weight gain, although their rate of weight recovery is impaired. Collectively, these findings demonstrate that the defense against overfeeding-induced weight gain remains intact in obesity and involves mechanisms independent of both FGF21 and MC4R.

Stress integration by an ascending adrenergic-melanocortin circuit.

Stress is thought to be an important contributing factor for eating disorders; however, neural substrates underlying the complex relationship between stress and appetite are not fully understood. Using in vivo recordings from awake behaving mice, we show that various acute stressors activate catecholaminergic nucleus tractus solitarius (NTSTH) projections in the paraventricular hypothalamus (PVH). Remarkably, the resulting adrenergic tone inhibits MC4R-expressing neurons (PVHMC4R), which are known for their role in feeding suppression. We found that PVHMC4R silencing encodes negative valence in sated mice and is required for avoidance induced by visceral malaise. Collectively, these findings establish PVHMC4R neurons as an effector of stress-activated brainstem adrenergic input in addition to the well-established hypothalamic-pituitary-adrenal axis. Convergent modulation of stress and feeding by PVHMC4R neurons implicates NTSTH → PVHMC4R input in stress-associated appetite disorders.

Activating MC4R Promotes Functional Recovery by Repressing Oxidative Stress-Mediated AIM2 Activation Post-spinal Cord Injury.

Spinal cord injury (SCI) is a destructive neurological trauma that induces permanent sensory and motor impairment as well as a deficit in autonomic physiological function. Melanocortin receptor 4 (MC4R) is a G protein-linked receptor that is extensively expressed in the neural system and contributes to inhibiting inflammation, regulating mitochondrial function, and inducing programmed cell death. However, the effect of MC4R in the modulation of oxidative stress and whether this mechanism is related to the role of absent in melanoma 2 (AIM2) in SCI are not confirmed yet. In the current study, we demonstrated that MC4R is significantly increased in the neurons of spinal cords after trauma and oxidative stimulation of cells. Further, activation of MC4R by RO27-3225 effectively improved functional recovery, inhibited AIM2 activation, maintained mitochondrial homeostasis, repressed oxidative stress, and prevented Drp1 translocation to the mitochondria. Meanwhile, treating Drp1 inhibitors would be beneficial in reducing AIM2 activation, and activating AIM2 could abolish the protective effect of MC4R on neuron homeostasis. In conclusion, we demonstrated that MC4R protects against neural injury through a novel process by inhibiting mitochondrial dysfunction, oxidative stress, as well as AIM2 activation, which may serve as an available candidate for SCI therapy.

A human obesity-associated MC4R mutation with defective Gq/11α signaling leads to hyperphagia in mice.

Melanocortin 4 receptor (MC4R) mutations are the most common cause of human monogenic obesity and are associated with hyperphagia and increased linear growth. While MC4R is known to activate Gsα/cAMP signaling, a substantial proportion of obesity-associated MC4R mutations do not affect MC4R/Gsα signaling. To further explore the role of specific MC4R signaling pathways in the regulation of energy balance, we examined the signaling properties of one such mutant, MC4R (F51L), as well as the metabolic consequences of MC4RF51L mutation in mice. The MC4RF51L mutation produced a specific defect in MC4R/Gq/11α signaling and led to obesity, hyperphagia, and increased linear growth in mice. The ability of a melanocortin agonist to acutely inhibit food intake when delivered to the paraventricular nucleus (PVN) was lost in MC4RF51L mice, as well as in WT mice in which a specific Gq/11α inhibitor was delivered to the PVN; this provided evidence that a Gsα-independent signaling pathway, namely Gq/11α, significantly contributes to the actions of MC4R on food intake and linear growth. These results suggest that a biased MC4R agonist that primarily activates Gq/11α may be a potential agent to treat obesity with limited untoward cardiovascular and other side effects.

Novel Melanocortin-3 and -4 Receptor Functional Variants in Asian Children With Severe Obesity.

CONTEXT: Genetic variants in melanocortin 3 receptor (MC3R) and melanocortin 4 receptor (MC4R) genes are strongly associated with childhood obesity. OBJECTIVE: This study aims to identify and functionally characterize MC3R and MC4R variants in an Asian cohort of children with severe early-onset obesity. METHODS: Whole-exome sequencing was performed to screen for MC3R and MC4R coding variants in 488 Asian children with severe early-onset obesity (body mass index for age ≥97th percentile). Functionality of the identified variants were determined via measurement of intracellular cyclic adenosine monophosphate (cAMP) concentrations and luciferase activity. RESULTS: Four MC3R and 2 MC4R heterozygous nonsynonymous rare variants were detected. There were 3 novel variants: MC3R c.151G > C (p.Val51Leu), MC4R c.127C > A (p.Gln43Lys), and MC4R c.272T > G (p.Met91Arg), and 3 previously reported variants: MC3R c.127G > A (p.Glu43Lys), MC3R c.97G > A (p.Ala33Thr), and MC3R c.437T > A (p.Ile146Asn). Both MC3R c.127G > A (p.Glu43Lys) and MC4R c.272T > G (p.Met91Arg) variants demonstrated defective downstream cAMP signaling activity. The MC4R c.127C > A (p.Gln43Lys) variant showed reduced cAMP signaling activity at low substrate concentration but the signaling activity was restored at high substrate concentration. The MC3R c.151G > C (p.Val51Leu) variant did not show a significant reduction in cAMP signaling activity compared to wild-type (WT) MC3R. Coexpression studies of the WT and variant MC3R/MC4R showed that the heterozygous variants did not exhibit dominant negative effect. CONCLUSION: Our functional assays demonstrated that MC3R c.127G > A (p.Glu43Lys) and MC4R c.272T > G (p.Met91Arg) variants might predispose individuals to early-onset obesity, and further studies are needed to establish the causative effect of these variants in the pathogenesis of obesity.

Predisposition of the Common MC4R rs17782313 Female Carriers to Elevated Obesity and Interaction with Eating Habits.

The global rise in obesity is attributed to genetic predisposition interaction with an obesogenic environment. Melanocortin 4 receptor (MC4R) rs17782313 polymorphism has been linked to common obesity with varying influence across different populations. MC4R is a crucial player in the leptin proopiomelanocortin pathway that regulates weight hemostasis. We aimed to study MC4R rs17782313 and its interaction with eating behaviors on obesity predisposition in the Israeli population. Adults' (n = 5785, >18 y) genotype and anthropometric and demographic data were analyzed using logistic regression models adjusting for age, sex, T1DM, and T2DM. MC4R rs17782313 significantly predisposes to elevated obesity risk under the recessive and additive models (OR = 1.38, 95% CI: 1.1-1.72, p = 0.005 and OR = 1.1, 95% CI: 1.01-1.2, p = 0.03, respectively) adjusted for confounders (age, sex, T1DM, and T2DM). Stratification by sex demonstrated that carrying the common MC4R rs17782313 is significantly associated with an elevated predisposition to obesity under the recessive model among females only (OR = 1.41, 95% CI: 1.09-1.82, p = 0.01), with an average of 0.85 BMI increment compared with wild type and one risk allele carriers. MC4R rs17782313 significantly interacted with several eating behaviors to enhance the risk of obesity. Our findings demonstrate that MC4R rs17782313 homozygous female carriers are significantly predisposed to obesity amplified by eating behaviors.

Functional Characterization of Novel MC4R Variants Identified in Two Unrelated Patients with Morbid Obesity in Qatar.

The leptin-melanocortin pathway is pivotal in appetite and energy homeostasis. Pathogenic variants in genes involved in this pathway lead to severe early-onset monogenic obesity (MO). The MC4R gene plays a central role in leptin-melanocortin signaling, and heterozygous variants in this gene are the most common cause of MO. A targeted gene panel consisting of 52 obesity-related genes was used to screen for variants associated with obesity. Variants were analyzed and filtered to identify potential disease-causing activity and validated using Sanger sequencing. We identified two novel heterozygous variants, c.253A>G p.Ser85Gly and c.802T>C p.Tyr268His, in the MC4R gene in two unrelated patients with morbid obesity and evaluated the functional impact of these variants. The impact of the variants on the MC4R gene was assessed using in silico prediction tools and molecular dynamics simulation. To further study the pathogenicity of the identified variants, GT1-7 cells were transfected with plasmid DNA encoding either wild-type or mutant MC4R variants. The effects of allelic variations in the MC4R gene on cAMP synthesis, MC4R protein level, and activation of PKA, ERB, and CREB signaling pathways in both stimulated and unstimulated ɑ-MSH paradigms were determined for their functional implications. In silico analysis suggested that the variants destabilized the MC4R structure and affected the overall dynamics of the MC4R protein, possibly leading to intracellular receptor retention. In vitro analysis of the functional impact of these variants showed a significant reduction in cell surface receptor expression and impaired extracellular ligand binding activity, leading to reduced cAMP production. Our analysis shows that the variants do not affect total protein expression; however, they are predicted to affect the post-translational localization of the MC4R protein to the cell surface and impair downstream signaling cascades such as PKA, ERK, and CREB signaling pathways. This finding might help our patients to benefit from the novel therapeutic advances for monogenic forms of obesity.

Melanocortin-4 receptor in macrophages attenuated angiotensin II-induced abdominal aortic aneurysm in mice.

Obesity is recognized as an independent risk factor for abdominal aortic aneurysm (AAA). While mutations in the melanocortin-4 receptor (MC4R) gene is the most common cause of obesity caused by mutations in a single gene, the link between MC4R function and vascular disease has still remained unclear. Here, by using melanocortin-4 receptor (MC4R) deficient mice, we confirmed MC4R deficiency promotes AAA and atherosclerosis. We demonstrated the contribution of two novel factors towards vascular vulnerability in this model: leptin signaling in vascular smooth muscle cells (VSMCs) and loss of MC4R signaling in macrophages. Leptin was shown to promote vascular vulnerability via PI3K-dependent upregulation of Spp1 expression in VSMC. Additionally, Ang II-induced AAA incidence was significantly reduced when MC4R gene expression was myeloid cell-specifically rescued in MC4R deficient (MC4RTB/TB) mice. Ex vivo analysis showed a suppression in NF-κB activity in bone marrow-derived macrophages from LysM(+);MC4RTB/TB mice compared to LysM(-);MC4RTB/TB mice, which exaggerates with endogenous MC4R ligand treatment; α-MSH. These results suggest that MC4R signaling in macrophages attenuates AAA by inhibiting NF-κB activity and subsequent vascular inflammation.

Computational analysis of missense variants of human MC4R and childhood obesity.

Industrialized and developing nations face severe public health problems related to childhood obesity. Previous studies revealed that the melanocortin-4 receptor gene (MC4R) is the most prevalent monogenic cause of severe early obesity. Due to its influence on food intake and energy expenditure via neuronal melanocortin-4 receptor pathways, MC4R is recognized as a regulator of energy homeostasis. This study used a variety of computational systems to analyze 273 missense variations of MC4R in silico. Several tools, including PolyPhen, PROVEAN, SIFT, SNAP2, MutPred2, PROVEAN, SNP&GO and Mu-Pro, I-Mutant, PhD-SNP, SAAFEC-SEQ I-Mutant, and ConSurf, were used to make predictions of 13 extremely confident nsSNPs that are harmful and disease-causing (E308k, P299L, D298H, C271F, C271R, P260L, T246N, G243R, C196Y, W174C, Y157S, D126Y, and D90G). The results of our study suggest that these MC4R nsSNPs may disrupt normal protein function, leading to an increased risk of childhood obesity. These results highlight the potential use of these nsSNPs as biomarkers to predict susceptibility to obesity and as targets for personalized interventions.