Melanocortin Receptor Research Articles

Melanocortin-4 receptor and proopiomelanocortin: Candidate genes for obesity in domestic shorthair cats.

Obesity is an escalating global health problem affecting both humans and companion animals. In cats it is associated with increased mortality and multiple diseases, including diabetes mellitus. Two genes coding for proteins known to play a critical role in energy homeostasis across species are the proopiomelanocortin (POMC) gene and the melanocortin-4 receptor (MC4R) gene. A missense variant in the coding sequence of the feline MC4R (MC4R:c.92C>T) has been reported to be associated with diabetes and overweight in domestic shorthair cats, and while variants in the POMC gene are known to cause obesity in humans and dogs, variants in POMC and their association with feline obesity and diabetes mellitus have not been investigated to date. The current study aimed to assess the association between the previously described MC4R variant and body condition score (BCS), as well as body fat content (%BF) in 89 non-diabetic domestic shorthair cats. Furthermore, we investigated the feline POMC gene as a potential candidate gene for obesity. Our results indicate that the MC4R:c.92C>T polymorphism is not associated with BCS or %BF in non-diabetic domestic shorthair cats. The mutation analysis of all POMC exons identified two missense variants, with a variant in exon 1 (c.28G>C; p.G10R) predicted to be damaging. The variant was subsequently assessed in all 89 cats, and cats heterozygous for the variant had a significantly increased body condition score (p = 0.03) compared with cats homozygous for the wild-type allele. Results from our study provide additional evidence that the previously described variant in MC4R is not associated with obesity in domestic shorthair cats. More importantly, we have identified a novel variant in the POMC gene, which might play a role in increased body condition score and body fat content in domestic shorthair cats.

Targeting the central melanocortin system for the treatment of metabolic disorders.

A large body of preclinical and clinical data shows that the central melanocortin system is a promising therapeutic target for treating various metabolic disorders such as obesity and cachexia, as well as anorexia nervosa. Setmelanotide, which functions by engaging the central melanocortin circuitry, was approved by the FDA in 2020 for use in certain forms of syndromic obesity. Furthermore, the FDA approvals in 2019 of two peptide drugs targeting melanocortin receptors for the treatment of generalized hypoactive sexual desire disorder (bremelanotide) and erythropoietic protoporphyria-associated phototoxicity (afamelanotide) demonstrate the safety of this class of peptides. These approvals have also renewed excitement in the development of therapeutics targeting the melanocortin system. Here, we review the anatomy and function of the melanocortin system, discuss progress and challenges in developing melanocortin receptor-based therapeutics, and outline potential metabolic and behavioural disorders that could be addressed using pharmacological agents targeting these receptors.

1608-P: Phenotype of Liver-Specific MC3R Knockout Mice

The melanocortin 3 receptor (MC3R) is a key regulator of energy homeostasis and feeding behavior. Global Mc3r knockout mice exhibit greater fat mass and reduced lean mass. Our previous research has found that Mc3r transcripts can be detected in liver and the MC3R deficiency phenotype is partially reversed in liver-specific MC3R recovery mice on a chow diet, in both male and female mice, and high fat diet, in females only. To further examine the role of hepatic MC3R, we produced a liver-specific Mc3r knockout model by crossing mice expressing albumin-Cre recombinase (Alb-Cre+/+) to mice with a floxed Mc3r gene (LoxP+/+), and placed them on a 45 kcal% energy from fat diet for 12 weeks. Female liver-specific Mc3r-deficient mice with diet-induced obesity displayed nonsignificant trends towards worsened glucose tolerance during a glucose tolerance test (glucose AUC Lox+/+, Alb-Cre+/+: n=10, 24036±1644; Alb-Cre+/+: n=10, 19745±1364; BL/6: n=11, 21072±1152 min*mg/dL, p=0.059) and insulin resistance during a glucose tolerance test (glucose AUC LoxP+/+, Alb-Cre+/+: n=10, 14938±812.2;Alb-Cre+/+: n=14, 13062±850; BL/6: n=11, 11725±1024 min*mg/dL, p=0.12), but no differences in morning serum C-peptide (LoxP+/+, Alb-Cre+/+: n=8, 0.74±0.07; Alb-Cre+/+: n=4, 0.55±0.15; BL/6: n=8, 0.29±0.05 ng/mL, p=0.32). Similar patterns were observed for total food consumption (LoxP+/+, Alb-Cre+/+: n=10, 37.2±1.2; Alb-Cre+/+: n=15, 34.5±0.7; BL/6: n=10, 35.7±1.4 g/15 days, p=0.082), with no differences in feeding efficiency (LoxP+/+, Alb-Cre+/+: n=10, 0.027±0.008; Alb-Cre+/+: n=15, 0.009±0.011; BL/6: n=10, 0.017±0.025 g/kcal, p=0.19). No notable trends were observed in male mice. These data suggest that hepatic Mc3r deficiency alone is at most a minor contributor to the obesity phenotypes associated with HFD-treated Mc3r-deficient mice. Additional analysis of tissue samples from these mice and future studies examining Mc3r function in the liver will further elucidate the role of hepatic MC3R. Disclosure J.Chen: None. J.K.Elmquist: Research Support; Regeneron Pharmaceuticals Inc. J.A.Yanovski: Research Support; Hikma Pharmaceuticals, Rhythm Pharmaceuticals, Inc., Soleno Therapeutics, Inc., Versanis Bio Inc. T.P.Patel: None. A.Jain: None. D.Elizondo: None. E.K.Altman: None. N.Tugarinov: None. M.Hollis: None. R.Roberson: None. A.Caron: None.

Prevalence of Deleterious Variants in MC3R in Patients With Constitutional Delay of Growth and Puberty.

The melanocortin 3 receptor (MC3R) has recently emerged as a critical regulator of pubertal timing, linear growth, and the acquisition of lean mass in humans and mice. In population-based studies, heterozygous carriers of deleterious variants in MC3R report a later onset of puberty than noncarriers. However, the frequency of such variants in patients who present with clinical disorders of pubertal development is currently unknown. This work aimed to determine whether deleterious MC3R variants are more frequently found in patients clinically presenting with constitutional delay of growth and puberty (CDGP) or normosmic idiopathic hypogonadotropic hypogonadism (nIHH). We examined the sequence of MC3R in 362 adolescents with a clinical diagnosis of CDGP and 657 patients with nIHH, experimentally characterized the signaling properties of all nonsynonymous variants found and compared their frequency to that in 5774 controls from a population-based cohort. Additionally, we established the relative frequency of predicted deleterious variants in individuals with self-reported delayed vs normally timed menarche/voice-breaking in the UK Biobank cohort. MC3R loss-of-function variants were infrequent but overrepresented in patients with CDGP (8/362 [2.2%]; OR = 4.17; P = .001). There was no strong evidence of overrepresentation in patients with nIHH (4/657 [0.6%]; OR = 1.15; P = .779). In 246 328 women from the UK Biobank, predicted deleterious variants were more frequently found in those self-reporting delayed (aged ≥16 years) vs normal age at menarche (OR = 1.66; P = 3.90E-07). We have found evidence that functionally damaging variants in MC3R are overrepresented in individuals with CDGP but are not a common cause of this phenotype.

352-OR: Regulation of Melanocortin Receptor 4 (MC4R) Ciliary Trafficking

The primary cilium, a hair-like projection at the surface of nearly every cell, functions as a signaling antenna in processes as diverse as vision and body weight homeostasis. Obesity is a cardinal symptom of many ciliopathies including Bardet-Biedl Syndrome (BBS). While we are beginning to understand how cilia participate in body weight homeostasis, the molecular pathway that leads to obesity in BBS patients remains unclear. We now know that the melanocortin receptor 4, a GPCR that regulates feeding behavior, localizes to cilia in hypothalamic neurons. Meanwhile, most BBS proteins assemble or regulate the BBSome, a protein complex that removes activated signaling receptors from cilia. To characterize the stimuli and mechanisms that regulate MC4R trafficking in and out of cilia, we recapitulated MC4R trafficking in ciliated cell lines amenable to rapid manipulation. Surprisingly, levels of MC4R in cilia remain under the detection limit in most cells unless treated with an inverse agonist of MC4R. In addition, interference with BBSome function also increases ciliary MC4R levels in all cells. Thus, the tonic activity of MC4R promotes the constitutive clearance of MC4R from cilia via BBSome-mediated exit. These results suggest that artificially increasing the ciliary levels of MC4R may augment its signaling activity. To selectively recognize activated GPCRs, the BBSome relies on specific ubiquitin chains linked at lysine 63 (K63Ub chains) that become attached onto activated GPCRs via β-arrestin-associated ubiquitin ligases. Depletion of β-arrestin blocked the constitutive exit of MC4R from cilia and mutation of ubiquitin acceptor lysines from MC4R and MRAP2 results in a measurable increase in the ciliary levels of MC4R. In addition, targeting a protease that specifically cleaves K63Ub chains to cilia completely blocks the exit of MC4R from cilia. We conclude that the tonically active MC4R is ubiquitinated in cilia and actively removed by the BBSome. Disclosure M.Nachury: None. Funding American Diabetes Association (1-20-VSN-03)

260-LB: The Effect of LXR Activation on Liver Metabolism and Diet-Induced Steatohepatitis in Mice

While nonalcoholic fatty liver disease (NAFLD) affects approx. 30% of the US population, there is currently no approved pharmaceutical treatment to inhibit the progression of NAFLD to its more severe stage of nonalcoholic steatohepatitis (NASH). The liver X receptors (LXRs) are key regulators of cholesterol and fatty acid homeostasis in liver. However, the effects of LXR activation on liver metabolism in NASH pathogenesis are not well understood. We hypothesize that LXR activation might reduce NAFLD progression by augmenting lipid composition and metabolism in hepatocytes. To test this hypothesis, 8-week-old male wild type (WT) or obese, melanocortin-4 receptor knockout (Mc4r-/-) mice were placed on either chow or Western diet (WD) for 8 weeks. Half of the WD-fed mice were given WD supplemented with GW3965 (100mg/kg), a selective, orally active agonist for the LXRs. Animals fed GW3965-supplemented diet had increased gene expression of Abca1 and Lpcat3, both transcriptional targets of LXR action. LXR activation significantly decreased body mass of both WT and Mc4r-/- mice, which was correlated with reduction of subcutaneous fat. GW3965 supplementation greatly improved glucose homeostasis of Mc4r-/-. LXR activation significantly decreased hepatic triglycerides, cholesterol esters and ceramides content which was accompanied by decreased expression of lipogenic factors (Fasn, Dgat1 and 2, Fatp5, Elovl1 and 6) in Mc4r-/-. Contrary expression of genes involved in fatty acid β-oxidation together with pyruvate cycling fluxes (VPC, VPK+ME, VPEPCK) were elevated in Mc4r-/- fed diet supplemented with GW3965. These metabolic changes were related to decrease in hepatic fibrosis, expression of NF-κB pathway genes as well as inflammatory (Itgam, Cd274), oxidative (Sod2), and endoplasmic reticulum stress markers (Chop, Atf4 and 6). Taken together, these data suggest that LXR activation improves phenotype of genetically obese WD-fed animals and inhibits progression from NAFLD to NASH. Disclosure T. Bednarski: None. M. Rahim: None. N. Whittenbarger: None. A. J. Olichwier: None. J. Young: Consultant; Pfizer Inc. Stock/Shareholder; Metalytics, Inc. Consultant; Metalytics, Inc. Research Support; Merck Sharp & Dohme Corp. Funding National Institutes of Health (R01DK106348); Vanderbilt University; University of Nebraska-Lincoln Nutrition and Health Sciences Fund

Understanding the Genetics of Early-Onset Obesity in a Cohort of Children From Qatar.

Monogenic obesity is a rare form of obesity due to pathogenic variants in genes implicated in the leptin-melanocortin signaling pathway and accounts for around 5% of severe early-onset obesity. Mutations in the genes encoding the MC4R, leptin, and leptin receptor are commonly reported in various populations to cause monogenic obesity. Determining the genetic cause has important clinical benefits as novel therapeutic interventions are now available for some forms of monogenic obesity. To unravel the genetic causes of early-onset obesity in the population of Qatar. In total, 243 patients with early-onset obesity (above the 95% percentile) and age of onset below 10 years were screened for monogenic obesity variants using a targeted gene panel, consisting of 52 obesity-related genes. Thirty rare variants potentially associated with obesity were identified in 36 of 243 (14.8%) probands in 15 candidate genes (LEP, LEPR, POMC, MC3R, MC4R, MRAP2, SH2B1, BDNF, NTRK2, DYRK1B, SIM1, GNAS, ADCY3, RAI1, and BBS2). Twenty-three of the variants identified were novel to this study and the rest, 7 variants, were previously reported in literature. Variants in MC4R were the most common cause of obesity in our cohort (19%) and the c.485C>T p.T162I variant was the most frequent MC4R variant seen in 5 patients. We identified likely pathogenic/pathogenic variants that seem to explain the phenotype of around 14.8% of our cases. Variants in the MC4R gene are the commonest cause of early-onset obesity in our population. Our study represents the largest monogenic obesity cohort in the Middle East and revealed novel obesity variants in this understudied population. Functional studies will be required to elucidate the molecular mechanism of their pathogenicity.

A genome-wide association study for loin depth and muscle pH in pigs from intensely selected purebred lines

BackgroundGenome-wide association studies (GWAS) aim at identifying genomic regions involved in phenotype expression, but identifying causative variants is difficult. Pig Combined Annotation Dependent Depletion (pCADD) scores provide a measure of the predicted consequences of genetic variants. Incorporating pCADD into the GWAS pipeline may help their identification. Our objective was to identify genomic regions associated with loin depth and muscle pH, and identify regions of interest for fine-mapping and further experimental work. Genotypes for ~ 40,000 single nucleotide morphisms (SNPs) were used to perform GWAS for these two traits, using de-regressed breeding values (dEBV) for 329,964 pigs from four commercial lines. Imputed sequence data was used to identify SNPs in strong (ge 0.80) linkage disequilibrium with lead GWAS SNPs with the highest pCADD scores.ResultsFifteen distinct regions were associated with loin depth and one with loin pH at genome-wide significance. Regions on chromosomes 1, 2, 5, 7, and 16, explained between 0.06 and 3.55% of the additive genetic variance and were strongly associated with loin depth. Only a small part of the additive genetic variance in muscle pH was attributed to SNPs. The results of our pCADD analysis suggests that high-scoring pCADD variants are enriched for missense mutations. Two close but distinct regions on SSC1 were associated with loin depth, and pCADD identified the previously identified missense variant within the MC4R gene for one of the lines. For loin pH, pCADD identified a synonymous variant in the RNF25 gene (SSC15) as the most likely candidate for the muscle pH association. The missense mutation in the PRKAG3 gene known to affect glycogen content was not prioritised by pCADD for loin pH.ConclusionsFor loin depth, we identified several strong candidate regions for further statistical fine-mapping that are supported in the literature, and two novel regions. For loin muscle pH, we identified one previously identified associated region. We found mixed evidence for the utility of pCADD as an extension of heuristic fine-mapping. The next step is to perform more sophisticated fine-mapping and expression quantitative trait loci (eQTL) analysis, and then interrogate candidate variants in vitro by perturbation-CRISPR assays.

The Parallel Structure-Activity Relationship Screening of Three Compounds Identifies the Common Agonist Pharmacophore of Pyrrolidine Bis-Cyclic Guanidine Melanocortin-3 Receptor (MC3R) Small-Molecule Ligands.

The melanocortin receptors are involved in numerous physiological pathways, including appetite, skin and hair pigmentation, and steroidogenesis. In particular, the melanocortin-3 receptor (MC3R) is involved in fat storage, food intake, and energy homeostasis. Small-molecule ligands developed for the MC3R may serve as therapeutic lead compounds for treating disease states of energy disequilibrium. Herein, three previously reported pyrrolidine bis-cyclic guanidine compounds with five sites for molecular diversity (R1-R5) were subjected to parallel structure-activity relationship studies to identify the common pharmacophore of this scaffold series required for full agonism at the MC3R. The R2, R3, and R5 positions were required for full MC3R efficacy, while truncation of either the R1 or R4 positions in all three compounds resulted in full MC3R agonists. Two additional fragments, featuring molecular weights below 300 Da, were also identified that possessed full agonist efficacy and micromolar potencies at the mMC5R. These SAR experiments may be useful in generating new small-molecule ligands and chemical probes for the melanocortin receptors to help elucidate their roles in vivo and as therapeutic lead compounds.

Single transmembrane GPCR modulating proteins: neither single nor simple.

The discovery of G-protein coupled receptor (GPCR) accessory proteins has fundamentally redefined the pharmacological concept of GPCR signaling, demonstrating a more complex molecular basis for receptor specificity on the plasma membrane and impressionable downstream intracellular cascades. GPCR accessory proteins not only contribute to the proper folding and trafficking of receptors, but also exhibit selectable receptor preferences. The melanocortin receptor accessory proteins (MRAP1 and MRAP2) as well as receptor activity-modifying proteins (RAMPs) are two well-known single transmembrane partners for the regulation of the melanocortin receptors (MC1R-MC5R) and the glucagon receptor (GCGR), respectively. Especially, MRAP family participates in the pathological control of multiple endocrine disorders and RAMPs contribute to the endogenous regulation of glucose homeostasis. However, the precise mechanisms by which the MRAP and RAMP proteins regulate receptor signaling at the atomic resolution remain unknown. Recent progress made in the determination of RAMP2-bound GCGR complexes published on Cell (Krishna Kumar et al., 2023) unraveled the importance of RAMP2 for the promotion of extracellular receptor dynamics leading to cytoplasmic surface inactivation. Moreover, the new findings on Cell Research (Luo et al., 2023) of the adrenocorticotropic hormone (ACTH)-bound MC2R-Gs-MRAP1 complex disclosed the essential role of MRAP1 for MC2R activation and specificity of ligand recognition. In this article, we reviewed a series of key findings of MRAP proteins in the last decade, and the recent structural investigation of MRAP-MC2R and RAMP-GCGR functional complex and the expanded identification of new GPCR partners of MRAP proteins. In-depth understanding of GPCR modulation by single transmembrane accessory proteins will provide valuable insights for the therapeutic drug development to treat multiple GPCR associated human disorders.

Discovery of a Pan-Melanocortin Receptor Antagonist [Ac-DPhe(pI)-Arg-Nal(2')-Orn-NH2] at the MC1R, MC3R, MC4R, and MC5R that Mediates an Increased Feeding Response in Mice and a 40-Fold Selective MC1R Antagonist [Ac-DPhe(pI)-DArg-Nal(2')-Arg-NH2

Discovery of pan-antagonist ligands for the melanocortin receptors will help identify the physiological activities controlled by these receptors. The previously reported MC3R/MC4R antagonist Ac-DPhe(pI)-Arg-Nal(2')-Arg-NH2 was identified herein, for the first time, to possess MC1R and MC5R antagonist activity. Further structure-activity relationship studies probing the second and fourth positions were performed toward the goal of identifying potent melanocortin antagonists. Of the 21 tetrapeptides synthesized, 13 possessed MC1R, MC3R, MC4R, and MC5R antagonist activity. Three tetrapeptides were more than 10-fold selective for the mMC1R, including 8 (LTT1-44, Ac-DPhe(pI)-DArg-Nal(2')-Arg-NH2) that possessed 80 nM mMC1R antagonist potency and was at least 40-fold selective over the mMC3R, mMC4R, and mMC5R. Nine tetrapeptides were selective for the mMC4R, including 14 [SSM1-8, Ac-DPhe(pI)-Arg-Nal(2')-Orn-NH2] with an mMC4R antagonist potency of 1.6 nM. This compound was administered IT into mice, resulting in a dose-dependent increase in the food intake and demonstrating the in vivo utility of this compound series.

A Review of Genes Associated with Obesity Susceptibility: Findings from Association Studies

Obesity is described as the accumulation of excess body fat. Several health issues are caused by excess fat, including cancer, type 2 diabetes, and cardiovascular disease. Additionally, obesity rates among schoolchildren and young adults are rising globally, putting young people at risk of chronic diseases. Genetics, epigenetic modification, epigenomics, and environmental factors influence inheritance patterns significantly. This systematic study aimed to classify and investigate the polymorphisms of novel candidate obesity genes. Several genes have been suggested, includingat mass and obesity-associated gene (FTO), leptin gene (LEP), leptin receptor gene (LEPR), peroxisome proliferatoractivated receptor gamma gene (PPARG), melanocortin 4 receptor (MC4R), insulin-induced gene 2 (INSIG2), proprotein convertase subtilisin/kexin type 1 (PCSK1), adrenoceptor beta 2 (ADRB2), and uncoupling protein 2 (UCP2). The study’s literature review identified genes in scientific papers published in databases such as Web of Science, PubMed, Google Scholar, Embase, and others over the past three decades. There is evidence that genetic variations contribute to childhood obesity, adolescent obesity, and young adult obesity. Identifying functional differences and further defining the implicated molecularly and physiologically involved genes andpathways in efficient therapeutic approaches in fighting. Technological advances have recently demonstrated that genetic changes and mutations can be used as biological markers, risk indicators, and therapeutic targets.

Hypophagia induced by intracerebroventricular injection of apelin-13 is mediated via CRF1/CRF2 and MC3/MC4 receptors in neonatal broiler chicken

Previous studies have shown the role of apelin and its receptors in the regulation of food intake. In the present study, we investigate the mediating role of melanocortin, corticotropin, and neuropeptide Y systems in apelin-13- induced food intake in broilers. Eight trials were run in the current investigation to ascertain the relationships between the aforementioned systems and apelin-13 on food intake and behavioral changes after apelin-13 administration. In experiment 1, hens were given an intracerebroventricular administration of a solution for control in addition to apelin-13 (0.25, 0.5, and 1 µg). Astressin-B (a CRF1/CRF2 receptor antagonist, 30 µg), apelin-13 (1 µg), and administration of astressin-B and apelin-13 concurrently, were all injected into the birds in experiment 2. Experiments 3 through 8 were quite similar to experiment 2, with the exception of astressin2-B (CRF2 receptor antagonist, 30 µg), SHU9119 (MC3/MC4 receptor antagonist, 0.5 nmol), MCL0020 (MC4 receptor antagonist, 0.5 nmol), BIBP-3226 (NPY1 receptor antagonist, 1.25 nmol), BIIE 0246 (NPY2 receptor antagonist, 1.25 nmol), and CGP71683A (NPY5 receptor antagonist, 1.25 nmol) were injected instead of astressin-B. After then, total food consumption was monitored for 6 h. Apelin-13 injections of 0.5 and 1 µg decreased feeding (P < 0.05). The hypophagic effects of apelin were attenuated following the simultaneous administration of Astressin-B and Astressin2-B with apelin-13 (P > 0.05). Co-infusion of SHU9119 and apelin-13 reduced the appetite-decreasing effects of apelin-13 (P > 0.05). When MCL0020 and apelin-13 were injected at the same time, the hypophagia that apelin-13 induced was eliminated (P > 0.05). BIBP-3226, BIIE 0246, and CGP71683A had no effect on the hypophagia brought on by apelin-13 (P > 0.05). Also, apelin-13 significantly increased number of steps, jumps, exploratory food, pecks and standing time while decreased siting time (P < 0.05). These findings suggest that apelin-13-induced hypophagia in hens may involve the CRF1/CRF2 and MC3/MC4 receptors.

Quercetin Exerts a Protective Effect on Ischemic Stroke-induced Memory Deficits in Mice

Objectives Brain injury resulting from an ischemic stroke affects cognitive performance by disrupting the hippocampus. Several processes are involved in brain injury progression, including inflammation, glutamate excitotoxicity, and modulated brain peptide systems such as the melanocortin system. Reports show that quercetin exerts neuroprotective activity. This study investigates quercetin’s role in the cognitive function of ischemic stroke-induced mice and the possible mechanisms involved. Materials and Methods ICR mice were used. The left unilateral common carotid artery occlusion was conducted for 4 h to induce an ischemic stroke in the mice. Quercetin 50, 100, and 200 mg/kg were administered to separate groups intraperitoneally for 7 days. Cognitive function was examined using the T-maze test. The hippocampal mRNA expressions of NR2A, NR2B, melanocortin 4 receptor (MC4R), pro-opiomelanocortin precursors (POMC), and nuclear factor 2 (Nrf2) were examined using reverse transcription-polymerase chain reaction. Results It was found that stroke disrupted cognitive function. Quercetin administration ameliorated cognitive impairment. Quercetin attenuated the stroke-induced decrease in MC4R mRNA expression. Moreover, quercetin suppressed the stroke-induced increase in the hippocampal mRNA expression of NR2A. Conclusion Quercetin ameliorates cognitive deficits and normalizes impaired hippocampal melanocortin and glutamatergic signaling in ischemic stroke-induced mice.

Effect of melanocortin-4 receptor antagonist TCMCB07 on chemotherapy-induced anorexia and weight loss in rats.

e15196 Background: We previously reported that peripheral administration of TCMCB07, a synthetic antagonist of the type 4 melanocortin receptor (MC4R), ameliorates cancer- and chronic kidney disease-associated cachexia in rats. In this study, we used a rat model of cisplatin and 5-FU to evaluate the efficacy of TCMCB07 in alleviating chemotherapy-induced anorexia, weight loss and muscle wasting. Methods: Two separate experiments were performed in a total of 58 rats. 1) Cisplatin (Cis)/TCMCB07 experiment with 3 groups: Cis/saline (n = 11), Cis/TCMCB07 (n = 11), Saline/saline (n = 6). 2) 5-FU/TCMCB07 experiment with 3 groups: 5-FU/saline (n = 12), 5-FU/TCMCB07 (n = 12), Saline/saline (n = 6). Rats were treated with Cis (2.5mg/kg), 5-FU (70 mg/kg), or saline via IP injection once weekly for 3 weeks. Rats were also treated with TCMCB07 (3 mg/kg/day) or saline via daily SC injection for 21 days. Food intake and body weight were measured daily. Body composition was analyzed pre- and post-treatment by MRI. Heart and gastrocnemius were dissected and weighed after euthanasia. Results: Total food intake over 21 days was significantly increased in Cis/TCMCB07 vs Cis/saline group (p = 0.0006) and 5-FU/TCMCB07 vs 5-FU/saline group (p = 0.0134), and decreased in Cis/saline vs Saline/saline group (p = 0.001), but was no significant change in Cis/TCMCB07 vs Saline/saline group and 5-FU/saline or 5-FU/TCMCB07 vs Saline/saline group. Body weight was robustly increased in Cis/TCMCB07 vs Cis/saline group (p = 0.0007) and 5-FU/TCMCB07 vs 5-FU/saline group (p = 0.0013), and decreased in Cis/saline vs Saline/saline group (p = 0.0001) and 5-FU/saline vs Saline/saline group (p = 0.0041), but was no significant change in Cis/TCMCB07 vs Saline/saline group and 5-FU/TCMCB07 vs Saline/saline group. Cardiac and skeletal muscle loss was markedly attenuated in Cis/TCMCB07 vs Cis/saline (cardiac: 3.12% vs -11.95%, p &lt; 0.0001; skeletal: -3.88% vs -9.02%, p = 0.0259). Cardiac muscle loss was significantly attenuated in 5-FU/TCMCB07 vs 5-FU/saline group (1.40% vs -6.09%, p = 0.0131) and skeletal muscle was modestly changed (-4.17% vs -7.24%, p = 0.1782). While Cis/saline and 5-FU/saline groups lost 91.33% and 52.61% of fat mass respectively compared to Saline/saline group, Cis/TCMCB07 and 5-FU/TCMCB07 groups gained 37.68% (p = 0.0002) and 70.19% (p &lt; 0.0001). Conclusions: This preclinical trial provides evidence supporting peripheral TCMCB07 antagonism of central melanocortin signaling, increasing food intake and anabolism, ultimately alleviating anorexia, weight loss and muscle wasting over multiple cycles of cisplatin or 5-FU chemotherapy.

RAMP and MRAP accessory proteins have selective effects on expression and signalling of the CB1, CB2, GPR18 and GPR55 cannabinoid receptors.

Receptor activity-modifying proteins (RAMPs) and melanocortin receptor accessory proteins (MRAPs) modulate expression and signalling of calcitonin and melanocortin GPCRs. Interactions with other GPCRs have also been reported. The cannabinoid receptors, CB1 and CB2, and two putative cannabinoid receptors, GPR18 and GPR55, exhibit substantial intracellular expression and there are discrepancies in ligand responsiveness between studies. We investigated whether interactions with RAMPs or MRAPs could explain these phenomena. Receptors and accessory proteins were co-expressed in HEK-293 cells. Selected receptors were studied at basal expression levels and also with enhanced expression produced by incorporation of a preprolactin signal sequence/peptide (pplss). Cell surface and total expression of receptors and accessory proteins were quantified using immunocytochemistry. Signalling was measured using cAMP (CAMYEL) and G protein dissociation (TRUPATH Gα13) biosensors. MRAP2 enhanced surface and total expression of GPR18. Pplss-GPR18 increased detection of cell surface MRAP2. MRAP1α and MRAP2 reduced GPR55 surface and total expression, correlating with reduced constitutive, but not agonist-induced, signalling. GPR55, pplss-CB1 and CB2 reduced detection of MRAP1α at the cell surface. Pplss-CB1 agonist potency was reduced by MRAP2 in Gα13 but not cAMP assays, consistent with MRAP2 reducing pplss-CB1 expression. Some cannabinoid receptors increased RAMP2 or RAMP3 total expression without influencing surface expression. Mutual influences on expression and/or function for specific accessory protein-receptor pairings raises the strong potential for physiological and disease-relevant consequences. Sequestration and/or hetero-oligomerisation of cannabinoid receptors with accessory proteins is a possible novel mechanism for receptor crosstalk. This article is part of a themed issue Therapeutic Targeting of G Protein-Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc.

Hranidbena i mesna svojstva mladih svinja različitog intenziteta formiranja u ranoj ontogenezi i unutarpasminske diferencijacije prema genu za melanokortin 4 (Mc4r) receptor

The aim of this study was to investigate young pig fattening and meat quality due to the varying formation intensities in early on- togenesis and two genotypes based on the mel- anocortin receptor 4 (Mc4r) gene. To calculate the correlations between traits and the econom- ic efficiency of using animals of experimental groups in industrial complex conditions, the assessment of animals for fattening and meat quality examined the following characteris- tics: average daily live weight gain during the control fattening period in grams; age of reaching 100 kg live weight in days, chilled car- cass length in centimetres; length of the bacon half of the cooled carcass in centimetres; lard thickness at the thoracic vertebrae T6-T7 in millimetres. Further, the biometric processing of research results and calculation of the eco- nomic efficiency was performed. Young pigs of the controlled population met the requirements of the 1st class and the elite class for fattening and meat qualities. In examining the interbreed differentiation of large white breed animals, the melanocortin 4 receptor (Mc4r) gene determined that the Mc4rAG genotype outperformed the Mc4r AA genotype for average daily live weight gain, age at live weight of 100 kg, lard thickness at T6-T7, and chilled carcass length by an average of 4.50%. The difference between groups for the Tyler B's index was 11.82 points, and according to the average daily increase in live weight, the disparity between young pigs of interbreed differentiation according to the formation intensity index was 4.69%, the age of reaching 100 kg live weight was 3.10%, and the chilled carcass length was 1.23%. The num- ber of reliable connections between fattening and meat qualities, and the formation inten- sity and Tyler B indices was 75.0%, indicating their promise for use in selection and breeding work. The maximum increase in additional production was obtained from young pigs of the Mc4r AG genotype (+2.71%) and of group I, in which the formation intensity index ranged from 0.935 to 1.087 points (+2.65%).

(033) Examining the Neuroanatomical Basis of Bremelanotide, a Drug for Low Sexual Desire in Women

Abstract Introduction Low sexual desire is a commonly reported and often distressing form of sexual dysfunction in women. Characterized by diminished interest in sex, disinclination to initiate sex, and a loss of pleasure during sex, disorders of sexual desire among women are not only poorly understood from a psychological perspective, but also in terms of their underlying neurobiology. The drug bremelanotide, trade name Vyleesi, was approved by the FDA in 2019 to treat hypoactive sexual desire disorder in women. The drug is a synthetic analogue of the melanocortin receptor (MCR) agonist alpha-MSH and thus thought to bind to the melanocortin-4 receptor (MC4R) in the brain. However, despite its FDA approval, few preclinical studies or clinical trials have been performed on the drug. Objective We attempted to elucidate bremelanotide’s central nervous system effects in an established rodent model of female sexual behavior. Methods Female Syrian hamsters underwent 0, 2 or 5 weeks of sexual conditioning in the conditioned place preference apparatus, a validated model that is designed to test the rewarding properties of sexual behavior. Some hamsters that were conditioned for 2 weeks were also given a subcutaneous injection of bremelanotide. After the sexual conditioning experiment, brains were collected for assessment of MC4R expression in the striatum. mRNA expression of dopamine-1 and 2 receptors and MC4R were carried out using Syrian hamster customized RNAscope probes. Results Analysis of behavior data indicated the longer (5-week) sexual conditioning period was most effective for increasing responses indicative of reward, though the shorter (2-week) period also increased reward. However, neither the low dose nor high dose of bremelanotide increased reward responding as compared to control hamsters. Further, no brain changes in MC4R expression in the striatum were detected as a function of either sexual experience or bremelanotide treatment. MC4R was not co-expressed with dopamine-1 or dopamine-2 receptors in the nucleus accumbens or dorsal striatum. Conclusions Bremelanotide did not increase responding indicative of reward derived from sex in the female Syrian hamster model. Given that MC4R expression was unaffected by sexual experience and/or bremelanotide, our findings did not provide evidence that the drug acts via an MC4R-related mechanism in the central nervous system. MC4R was also not co-expressed with dopamine-1 or 2 receptors, suggesting the MC4R may instead be expressed by interneurons in the striatum. Despite our lack of evidence to support bremelanotide’s efficacy in animals, limitations of the rodent model of female sex behavior include the obvious psychosocial aspects of human sexuality that cannot be replicated. Further research is warranted to clarify bremelanotide’s mechanism of action. Disclosure No

(112) Treatment Options for Female Sexual Dysfunction in Patients with Epilepsy: A Systematic Review

Abstract Introduction Epilepsy is one of the most common neurologic conditions worldwide, affecting approximately 5 million people globally every year. Many with this diagnosis rely on lifelong medications for seizure prophylaxis, which involve complex mechanisms of action with various hormones. The myriad of therapeutic side effects and the debilitating nature of this disease process make sexual dysfunction a common occurrence, with low libido, arousal, and orgasm being the primary complaints for 30-67% of female patients with epilepsy. Prioritizing quality of life from a biopsychosocial perspective is imperative – the need for investigating diverse treatment options for sexual dysfunction in these female patients becomes increasingly evident. Objective This study aims to discuss the available treatment options for sexual dysfunction in females with diagnosed epilepsy. Methods A systematic review in the PubMed database that spanned between January 2015 and November 2022 was executed. Six authors independently and thoroughly screened titles, abstracts, and full-text articles. They assessed the relevant medical literature that acknowledged the presence of female sexual dysfunction and explicitly stated treatment options for patients who have epilepsy. MeSH advanced key terms used to refine the search include: [“epilepsy”], [“female sexual dysfunction”], AND [“treatment”]. Results The published articles (n = 26) that fit the inclusion criteria were reviewed. The pathophysiology behind sexual dysfunction in patients with epilepsy is multifactorial. Seizure activity preferentially occurring in the temporal and frontal lobes alters several fundamental functions of the brain and nervous system, including the HPA axis and the release of gonadal hormones. Furthermore, anti-epileptic drugs target complex substrates that influence the limbic system, hypothalamus, pituitary, endocrine glands, as well as steroid and protein synthesis and metabolism in the liver and adipose tissue. Specifically, drugs documented to correlate with sexual dysfunction negatively are valproate, topiramate, and gabapentin. Agents such as lamotrigine, oxcarbazepine, and levetiracetam are reported to result in fewer sexual side effects and may even have a protective role against sexual dysfunction. Non-pharmacological approaches such as treating underlying comorbidities, addressing psychosocial variables, and mindfulness-based cognitive therapy have also proven significantly beneficial in treating and preventing adverse sexual outcomes. Conclusions Sexual dysfunction, secondary to epilepsy-induced seizures or anti-epileptic drug management, is not adequately explored and treated. The body of literature supports treating sexual dysfunction in females with epilepsy from a multidimensional standpoint; however, there remains a lack of thorough discussion on treatment options tailored to female pelvic anatomy. Going forward, continued research on how the pathologic consequences of epilepsy affect sexual function, unrelated to the use of anti-epileptic drugs, could enhance the prevention and comprehensive management of female sexual dysfunction in this population. Additionally, evaluating the effectiveness of topical estrogens, 5-HT1a agonists/5HT2a antagonist like Flibanserin, and melanocortin receptor agonists like Bremelanotide in female patients with epilepsy and other neurologic disorders may aid in the understanding of this relationship and spark further discussion. Sexual dysfunction and related symptoms have a devastating impact, indicating the need for this research, standardized guidelines, and a wide array of biopsychosocial treatment options. Disclosure No

Structural Prediction of Melanocortin Receptor (MC4R) of Carassius Auratus: An In-silico Approach

Carassius auratus commonly known as goldfish is one of most attractive fish used as ornamental fishes throughout the world. From the ancient times, the melanocortin system is considered to be conserved ranging from teleosts to mammals. Various researches in several fish species presented melanocortin receptors 4 (MC4R) is involved in different functions in fish body. Therefore, in-silico analysis of MC4R of Carassius auratus has been carried out in the study to know the physicochemical properties and 3D structural confirmation of the protein. Sequence containing accession number XM_026207258.1 was taken from National Center for Biotechnology Information (NCBI) and then processed to Expasy’s protparam for the physicochemical characterization, SOPMA for secondary structure prediction. Template search was done by SWISSMODEL/Workspace and Swiss-Pdb-Viewer was used for prediction of 3D structure of the concerned protein. The results suggested MC4R is a stable, hydrophobic and slightly basic nature of protein. The secondary structure of the analysed MC4R protein suggested presence of 50.77% alpha helix, 19.69% extended strands and 3.38% beta turns along with 26.15% random coils. Moreover, it might be resolved from the ramachandran plot that the structural prediction of MC4R is correct in prediction. Further, predicted 3D structure of MC4R protein can also be utilized for docking and simulation studies in future for finding out its relative binding affinity and functional action mechanism aspects. Moreover, the study is an approach for reducing the sequence data and solved structures gap by X-ray crystallography and NMR spectroscopy, which are also tedious and expensive laboratory techniques in application as well. Keywords: Melanocortin receptors 4 (MC4R); In silico analysis; 3D structural prediction; Carassius auratus.