Melanocortin Receptor Research Articles

Additive and Dominance Effects of MC4R and PIT1 Polymorphisms on Production and Carcass Traits in Duroc Pigs

This study aimed to estimate the allelic and genotypic frequencies of single nucleotide polymorphisms (SNPs) of MC4R (melanocortin-4-receptor gene) and PIT1 (pituitary specific transcription factor-1) genes, and their additive dominance genetic effects on production and carcass traits in Duroc pigs. A total of 2170 tail tissue samples (1728 gilts and 442 male pigs) were collected from Dabaco Nucleus Breeding Pig Company, Bac Ninh province, Vietnam from January 2017 to June 2020. Polymorphisms in the MC4R and PIT1 genes were detected using the PCR-RFLP method. For MC4R, the frequencies of alleles A and G were 0.430 and 0.570, respectively. For PIT1, the frequencies of alleles A and B were 0.460 and 0.540, respectively. The genotype frequency distribution of the MC4R gene was in Hardy-Weinberg equilibrium (P = 0.375), but the genotype frequency distribution of PIT1 (P <0.001) was not. The MC4R and PIT1 polymorphisms were significantly associated with final body weight and average daily gain (P <0.001). Significant additive effects were also found (P <0.05) for the two traits. The selection of individuals with the MC4R genotype AA and PIT1 genotype AA could improve final body weight and average daily gain in Duroc pigs.

Feeding and meat qualities of young pigs of different genotypes according to melanocortin 4 receptor (Mc4r) gene and interbreed differentiation according to the coefficient of decrease in growth intensity in early ontogenesis

The paper presents the results of studies of fattening and meat qualities of young large white pigs of different genotypes for the melanocortin receptor 4 (Mc4r) gene and the decline in growth intensity in early ontogeny. The research was carried out in the agricultural formations of the Dnipropetrovsk region, the Jazz meat processing plant, the laboratory of the genetics of the Institute of Pig Breeding and APV of the National Academy of Sciences, and the laboratory of animal husbandry of the State Institution “Institute of Grain Crops of the National Academy of Sciences”. The work was carried out following the scientific research program of the National Academy of Sciences No. 30, “Innovative technologies of breeding, industrial and organic production of pig farming products” (“Pig farming”). Assessment of animals for fattening and meat quality was carried out taking into account the following characteristics: average daily gain of live weight during the period of control fattening, g; the age of reaching 100 kg live weight, days, length of the chilled carcass, cm; length of the bacon half of the cooled carcass, cm; thickness of lard at the level of 6–7 thoracic vertebrae, mm. The coefficient of decline in growth intensity was calculated according to the method of Yu. K. Sviechin. Biometric research results were processed using generally accepted methods. It was established that according to live weight at 4 and 6 months of age, fattening and meat qualities (age of reaching a live weight of 100 kg, days; lard thickness at the level of 6–7 thoracic vertebrae, mm; length of the chilled carcass, cm) young pigs the controlled population belongs to the I class and the elite class. The coefficient of growth decline in animals of the controlled population ranges from 108.57 to 142.51 points. The data analysis shows that according to the live weight at 4 and 6 months of age, the age of reaching the live weight of 100 kg, the fat thickness at the level of 6–7 thoracic vertebrae, and the length of the chilled carcass, the young pigs of the controlled population belong to the I class and the elite class. Animals of the Mc4r АГ genotype prevail over peers of the Mc4r AA genotype in terms of fattening and meat qualities by an average of 5.90 %. The interbreed differentiation of young pigs by the coefficient of the intensity of growth decline (∆K) shows that the difference between the animals of the experimental groups in terms of the average daily gain in live weight is 23.3 g (td = 2.62), the age of reaching 100 kg live weight is 2.7 days (td = 1.59), the length of the cooled carcass is 1.4 mm (td = 2.12). The number of reliable correlations between fattening and meat qualities, coefficient of the intensity of growth decline (∆K), and Tyler B. index is 75.0 %, which indicates the possibility of their use in selection and breeding work. The use of young pigs of the Mc4r АG genotype and animals of the I group, in which the coefficient of the intensity of growth decline (∆K) ranges from 115.61 to 123.27 points, provides additional production at the level of +3.68 – +1.75 % respectively.

Interaction between ultra-processed food intake and genetic risk score on mental health and sleep quality.

Mental health and sleep quality are associated with genetics and nutrient and energy intake. The present study examined the association between ultra-processed food (UPF) intake and genetic risk score (GRS) and their interactions on mental health and sleep quality in Iranian women. A cross-sectional study was conducted on 278 overweight and obese females aged between 18 and 56years. According to the NOVA classification system, 37 food groups and beverages were collected using a 147-item semi-quantitative food frequency questionnaire (FFQ). The blood parameters of all participants were assessed. Mini-column kit (type G; Genall; Exgene) and the PCR-RFLP method were used to extract DNA and determine gene polymorphism, respectively. Three single nucleotide polymorphisms (SNPs), including Caveolin_1 (Cav_1), Melanocortin4 receptor (MC4R), and cryptochrome circadian regulator 1 (CRY1), were used to calculate GRS. The individual risk allele (0, 1, 2) for each SNP was calculated using the incremental genetic model. After controlling for confounders, a significant interaction was found for depression (β = 0.026, 95% CI 0.003, 0.049, P = 0.028) and depression anxiety stress scales (DASS) score (β = 0.059, 95% CI 0.001, 0.117, P = 0.046) on the NOVA classification system and GRS. The findings of this study showed a significant interaction between GRS and the NOVA classification system on mental disorders, including depression, DASS score and stress. There was also a significant relationship between the NOVA classification system and anxiety, DASS score, sleep quality and depression. Furthermore, a partially significant association was observed between GRS and stress. Level V, cross-sectional descriptive study.

Molecular cloning and functional characterization of melanocortin-3 receptor in grass carp (Ctenopharyngodon idella).

The melanocortin-3-receptor (MC3R) plays an important role in mammals' food intake and energy homeostasis. However, its physiological role in bony fishes, such as grass carp, has not been well understood. This study reports the molecular cloning, tissue distribution, and pharmacological characterization of grass carp melanocortin-3-receptor (ciMC3R). Phylogenetic and chromosomal synteny analyses indicated that ciMC3R was closest to cyprinid fishes in evolution. Quantitative PCR experiments revealed that the mRNA of ciMC3R was highly expressed in the brain of grass carp. The cytological function of ciMC3R was investigated by the co-transfection of pcDNA3.1-ciMC3R and the signal-pathway-specific luciferase into the HEK293T cells. Results revealed that the four agonists, α-MSH, β-MSH, ACTH, and NDP-MSH, potentiate the activation of ciMC3R and further increase the production of cAMP and upregulate the MAPK/ERK signaling, respectively. Our study will provide basic data for exploring the physiological functions of grass carp MC3R, especially in energy homeostasis and food intake.

Activation of amygdala prokineticin receptor 2 neurons drives the anorexigenic activity of the neuropeptide PK2

Energy homeostasis is a complex system involving multiple hormones, neuropeptides, and receptors. Prokineticins (PK1 and PK2) are agonists to two G protein-coupled receptors, prokineticin receptor 1 and 2 (PKR1 and PKR2), which decrease food intake when injected in rodents. The relative contribution of PKR1 and PKR2 to the anorexigenic effect of PK2 and their site of action in the brain have not yet been elucidated. While PKR1 and PKR2 are both expressed in the hypothalamus, a central region involved in the control of energy homeostasis, PKR2 is also present in the amygdala, which has recently been shown to regulate food intake in response to several anorexigenic signals. PKR trafficking and signaling are inhibited by the melanocortin receptor accessory protein 2 (MRAP2), thus suggesting that MRAP2 has the potential to alter the anorexigenic activity of PK2 invivo. In this study, we investigated the importance of PKR1 and PKR2 for PK2-mediated inhibition of food intake, the brain region involved in this function, and the effect of MRAP2 on PK2 action invivo. Using targeted silencing of PKR2 and chemogenetic manipulation of PKR2 neurons, we show that the anorexigenic effect of PK2 is mediated by PKR2 in the amygdala and that altering MRAP2 expression in PKR2 neurons modulates the activity of PK2. Collectively, our results provide evidence that inhibition of food intake by PKs is not mediated through activation of hypothalamic neurons but rather amygdala PKR2 neurons and further establishes the importance of MRAP2 in the regulation of energy homeostasis.

The interaction of MC3R and MC4R with MRAP2a in rainbow trout (Oncorhynchus mykiss).

Melanocortin 3 and 4 receptors are two important neural G protein-coupled receptors that regulate energy homeostasis in vertebrates. Melanocortin receptor accessory protein 2 (MRAP2) is also involved in the regulation of food intake and body weight as a variable regulator of melanocortin receptors. Rainbow trout (Oncorhynchus mykiss) is a valuable cold-water fish cultured worldwide. In the rainbow trout model, we cloned and identified mrap2a, a paralog of mrap2. Rainbow trout mrap2a consisted of a 690bp ORF and was expected to encode a putative protein of 229 amino acids. The qPCR results showed that rainbow trout mrap2a was expressed at high levels in brain tissue similar to mc3r and mc4r. In addition, co-immunoprecipitation verified that MRAP2a interacts with MC3R and MC4R in vitro and that MRAP2a is involved in and regulates the constitutive activity and signaling of MC3R and MC4R. MRAP2a reduced constitutive and agonist-stimulated cAMP levels of MC3R; furthermore, MRAP2a increased constitutive ERK1/2 activation but reduced ligand-induced stimulation at high levels of expression. For MC4R, MRAP2a showed decreased cAMP basal activity but increased agonist-stimulated cAMP signaling and increased ACTH ligand sensitivity. However, MRAP2a failed to affect MC4R constitutive activity and agonist-induced ERK1/2 signaling. Undoubtedly, our study will have great significance for revealing the conserved role of MC4R and MC3R signaling in teleost fish, especially in cold-water fish growth and energy homeostasis.

A thalamic circuit facilitates stress susceptibility via melanocortin 4 receptor‐mediated activation of nucleus accumbens shell

Central melanocortin 4 receptor (MC4R) has been reported to induce anhedonia via eliciting dysfunction of excitatory synapses. It is evident that metabolic signals are closely related to chronic stress-induced depression. Here, we investigated that a neural circuit is involved in melanocortin signaling contributing to susceptibility to stress. Chronic social defeat stress (CSDS) was used to develop depressive-like behavior. Electrophysiologic and chemogenetic approaches were performed to evaluate the role of paraventricular thalamus (PVT) glutamatergic to nucleus accumbens shell (NAcsh) circuit in stress susceptibility. Pharmacological and genetic manipulations were applied to investigate the molecular mechanisms of melanocortin signaling in the circuit. CSDS increases the excitatory neurotransmission in NAcsh through MC4R signaling. The enhanced excitatory synaptic input in NAcsh is projected from PVT glutamatergic neurons. Moreover, chemogenetic manipulation of PVTGlu -NAcsh projection mediates the susceptibility to stress, which is dependent on MC4R signaling. Overall, these results reveal that the strengthened excitatory neurotransmission in NAcsh originates from PVT glutamatergic neurons, facilitating the susceptibility to stress through melanocortin signaling. Our results make a strong case for harnessing a thalamic circuit to reorganize excitatory synaptic transmission in relieving stress susceptibility and provide insights gained on metabolic underpinnings of protection against stress-induced depressive-like behavior.

Production of immunoregulatory molecules by induced erythroblasts at various stages of cell differentiation

Introduction. Bone marrow erythroblasts produce a wide range of cytokines with opposite biological effects. This may be due to a change in the spectrum of production of immunoregulatory mediators during differentiation and small qualitative and quantitative differences in the spectrum of cytokines produced at each stage of differentiation, which may be important for the regulation of hemo- and immunopoiesis. The aim. To study the spectrum of production of mediators by erythroblasts at different stages of differentiation. Methods. Erythroblasts were obtained from CD34+ bone marrow cells of healthy donors in the presence of recombinant cytokines. Phenotype assessment was performed using flow cytometry for erythroid (CD45, CD71, CD235a, CD44) and lymphoid markers (CD3, CD4, CD8, CD16, CD19). Blocking of erythroblast differentiation at different stages was carried out using specific blocking monoclonal antibodies to melanocortin receptors (MCR) of types 1, 2 and 5. Cytokine analysis in conditioned erythroblast media was performed using the Bio-Plex Pro Human Cytokine 48-Plex Screening Panel (Bio-Rad Laboratories, USA). Cytokine production was analyzed using the CytokineExplore online tool. Results. The resulting erythroblasts are divided into positive and negative populations according to the CD45 marker, carry markers of erythroid cells CD71, CD235a and do not express linear markers of lymphoid cells. In type 1 MCR blockage, polychromatophilic erythroblasts predominate, in type 2 MCR blockage, basophilic erythroblasts predominate, and in type 5 MCR blockage, orthochromatophilic erythroblasts accumulate. According to the production of cytokines, it was shown that when using any of the blocking antibodies, we obtain cells that differ qualitatively and quantitatively in a number of mediators from the initial population of induced erythroblasts. Conclusion. Thus, we have shown qualitative and quantitative differences in the production of mediators by erythroblasts depending on the stage of differentiation, which can lead to different regulatory effects.

Genetic loci, rs17817449 and rs6567160, known for obesity and the risk of stroke events among middle-aged and older Chinese people.

Fat Mass and Obesity-Associated (FTO) and the Melanocortin-4 Receptor (MC4R) genes are strongly associated with obesity, an established risk factor for stroke. We aimed to assess the associations between rs17817449 at the FTO and rs6567160 at the MC4R and the risk of stroke events in middle-aged and older Chinese people. Study data were obtained from the Guangzhou Biobank Cohort Study; a total of 148 participants with a self-reported history of stroke and an equal volume of age- and sex-matched participants were selected as the cases and the controls in a case-control study; a total of 13,967 participants at the first follow-up and all participants with fatal stroke (up to April 2021) were included in a retrospective cohort study. Conditional logistic regression and the Cox proportional hazards regression analyses were used to assess the associations of the two genetic loci with the risk of stroke events. After adjusting for age, sex, education, job, smoking, alcohol consumption, body mass index, physical activity, hypertension, diabetes, and dyslipidemia, rs17817449 and rs6567160 shared minor alleles G and C, respectively, in the case-control analyses. The genotypes GG+GT of rs17817449 at the FTO were significantly associated with a decreased risk of fatal stroke occurrence, with fatal all strokes having an adjusted hazard ratio (aHR) of 0.71 (95% confidence intervals (CI) 0.52-0.97, P = 0.04) and fatal ischemic stroke having an aHR of 0.64 (95% CI 0.41-1.00, P = 0.05), when the genotype TT was taken as a reference and a series of multiplicities were adjusted; the risk of fatal all strokes was lowered by dyslipidemia (aHR = 0.63, 95% CI 0.39-1.00, P = 0.05) and non-diabetes (aHR = 0.68, 95% CI 0.46-0.99, P = 0.049) in the retrospective cohort analyses. Significances were observed neither in the associations between rs6567160 and the risk of stroke events nor in an interaction between rs17817449 and rs6567160 in the two-stage analyses. The G allele of rs17817449 at the FTO, not rs6567160 at the MC4R, was associated with a decreased risk of fatal stroke occurrence; its functional role in stroke should be explored in relatively healthy middle-aged to older Chinese people.

SIGNS OF POSTEMBRYONAL DEVELOPMENT YOUNG PIGS OF DIFFERENT GENOTYPES BY MELANOCORTIN 4 (MC4R) RECEPTOR GENE AND THEIR PRODUCTIVITY

The results of studies of indicators of individual development, fattening, and meat qualities of young pigs of the large white breed of different genotypes according to the melanocortin receptor 4 (Mc4r) gene are presented, and the level of correlations between traits is calculated, as well as the economic efficiency of the use of animals in experimental groups. The research was carried out in agricultural formations of the Dnipropetrovsk region (Ltd. «Druzhba-Kaznacheivka»), the meat processing plant «Jazz», the laboratory of the genetics of the Institute of Pig Breeding and APP of the National Academy of Sciences, and the laboratory of animal husbandry of the State Institution «Institute of Grain Crops of the National Academy of Sciences». The work was carried out following the scientific research program of the National Academy of Sciences No. 30, «Innovative technologies of breeding, industrial and organic production of pig farming products» («Pig farming»). Assessment of animals according to growth indicators in early ontogenesis, fattening, and meat qualities were carried out taking into account the following characteristics: live weight at the time of birth, at the age of 3 and 6 months (kg), average daily increase in live weight during the period of control fattening, g; the age of reaching 100 kg live weight, days, length of a chilled carcass, cm; length of the bacon half of the cooled carcass, cm; thickness of lard at the level of 6-7 thoracic vertebrae, mm. The «intensity of formation» index (Δt; age periods 0-3-6) was calculated according to the method of Svichin (1985). Biometric processing of research results and calculation of the economic efficiency of research results was carried out according to generally accepted methods. It was established that according to live weight at 3 and 6 months of age, fattening and meat qualities (age of reaching a live weight of 100 kg, days; lard thickness at the level of 6-7 thoracic vertebrae, mm; length of the chilled carcass, cm) young pigs the population under control belongs to the I class and the elite class. Young pigs of the Mc4r AG genotype outperform peers of the Mc4r AA genotype in terms of average daily live weight gain, age at which live weight is 100 kg, lard thickness at the level of 6-7 thoracic vertebrae, and chilled carcass length by an average of 4.50 %. According to the Tyler B. index, the difference between the groups is 11.82 points (td=3.70; P<0.01). The intra-breed differentiation of young pigs according to the «formation intensity» index (Δt; age periods 0-3-6) shows that the difference between the animals of the III and I experimental groups in terms of the average daily increase in live weight is 6.65 %, the age of reaching the live weight of 100 kg - 3.77 %, the thickness of lard at the level of 6-7 thoracic vertebrae - 0.98% and the length of the chilled carcass - 1.13 %. The number of reliable correlations between fattening and meat qualities, the «formation intensity» index (Δt; age periods 0-3-6), and Tyler B. index is 75.0%, which indicates the possibility of their use in breeding tribal work. The use of young pigs of the genotype Mc4r AG and animals of the III group, in which the index «intensity of formation» (Δt; age periods 0-3-6) ranges from 0.715 to 0.947 provides additional production at the level of +2.71 - + 4.77 %, respectively.

Identification of a novel specific small-molecule melanocortin-2-receptor antagonist.

The overproduction of adrenocorticotropic hormone (ACTH), in conditions such as Cushing's disease and congenital adrenal hyperplasia (CAH), leads to significant morbidity. Current treatment with glucocorticoids does not adequately suppress plasma ACTH, resulting in excess adrenal androgen production. At present, there is no effective medical treatment in clinical use that would directly block the action of ACTH. Such a therapy would be of great clinical value. ACTH acts via a highly selective receptor, the melanocortin-2 receptor (MC2R) associated with its accessory protein MRAP. ACTH is the only known naturally occurring agonist for this receptor. This lack of redundancy and the high degree of ligand specificity suggest that antagonism of this receptor could provide a useful therapeutic strategy in the treatment of conditions of ACTH excess. To this end, we screened an extensive library of low-molecular-weight drug-like compounds for MC2R antagonist activity using a high-throughput homogeneous time-resolved fluorescence cAMP assay in Chinese hamster ovary cells stably co-expressing human MC2R and MRAP. Hits that demonstrated MC2R antagonist properties were counter-screened against the β2 adrenergic receptor and dose-response analysis undertaken. This led to the identification of a highly specific MC2R antagonist capable of antagonising ACTH-induced progesterone release in murine Y-1 adrenal cells and having selectivity for MC2R amongst the human melanocortin receptors. This work provides a foundation for the clinical investigation of small-molecule ACTH antagonists as therapeutic agents and proof of concept for the screening and discovery of such compounds.

Effect of the Melanocortin 4-Receptor Ile269Asn Mutation on Weight Loss Response to Dietary, Phentermine and Bariatric Surgery Interventions.

The loss of function melanocortin 4-receptor (MC4R) Ile269Asn mutation has been proposed as one of the most important genetic contributors to obesity in the Mexican population. However, whether patients bearing this mutation respond differently to weight loss treatments is unknown. We tested the association of this mutation with obesity in 1683 Mexican adults, and compared the response of mutation carriers and non-carriers to three different weight loss interventions: dietary restriction intervention, phentermine 30 mg/day treatment, and Roux-en-Y gastric bypass (RYGB) surgery. The Ile269Asn mutation was associated with obesity [OR = 3.8, 95% CI (1.5-9.7), p = 0.005]. Regarding interventions, in the dietary restriction group only two patients were MC4R Ile269Asn mutation carriers. After 1 month of treatment, both mutation carriers lost weight: -4.0 kg (-2.9%) in patient 1, and -1.8 kg (-1.5%) in patient 2; similar to the mean weight loss observed in six non-carrier subjects (-2.9 kg; -2.8%). Phentermine treatment produced similar weight loss in six carriers (-12.7 kg; 15.5%) and 18 non-carriers (-11.3 kg; 13.6%) after 6 months of pharmacological treatment. RYGB also caused similar weight loss in seven carriers (29.9%) and 24 non-carriers (27.8%), 6 months after surgery. Our findings suggest that while the presence of a single MC4R loss of function Ile269Asn allele significantly increases obesity risk, the presence of at least one functional MC4R allele seems sufficient to allow short-term weight loss in response to dietary restriction, phentermine and RYGB. Thus, these three different interventions may be useful for the short-term treatment of obesity in MC4R Ile269Asn mutation carriers.

Role of astroglial ACBP in energy metabolism flexibility and feeding responses to metabolic challenges in male mice.

Acyl-CoA binding protein (ACBP), also known as diazepam binding inhibitor (DBI), has recently emerged as a hypothalamic and brainstem gliopeptide regulating energy balance. Previous work has shown that the ACBP-derived octadecaneuropeptide exerts strong anorectic action via proopiomelanocortin (POMC) neuron activation and the melanocortin-4 receptor. Importantly, targeted ACBP loss-of-function in astrocytes promotes hyperphagia and diet-induced obesity while its overexpression in arcuate astrocytes reduces feeding and body weight. Despite this knowledge, the role of astroglial ACBP in adaptive feeding and metabolic responses to acute metabolic challenges has not been investigated. Using different paradigms, we found that ACBP deletion in glial fibrillary acidic protein (GFAP)-positive astrocytes does not affect weight loss when obese male mice are transitioned from a high fat diet to a chow diet, nor metabolic parameters in mice fed with a normal chow diet (e.g., energy expenditure, body temperature) during fasting, cold exposure and at thermoneutrality. In contrast, astroglial ACBP deletion impairs meal pattern and feeding responses during refeeding after a fast and during cold exposure, thereby showing that ACBP is required to stimulate feeding in states of increased energy demand. These findings challenge the general view that astroglial ACBP exerts anorectic effects and suggest that regulation of feeding by ACBP is dependent on metabolic status.

Human loss-of-function variants in the serotonin 2C receptor associated with obesity and maladaptive behavior

Serotonin reuptake inhibitors and receptor agonists are used to treat obesity, anxiety and depression. Here we studied the role of the serotonin 2C receptor (5-HT2CR) in weight regulation and behavior. Using exome sequencing of 2,548 people with severe obesity and 1,117 control individuals without obesity, we identified 13 rare variants in the gene encoding 5-HT2CR (HTR2C) in 19 unrelated people (3 males and 16 females). Eleven variants caused a loss of function in HEK293 cells. All people who carried variants had hyperphagia and some degree of maladaptive behavior. Knock-in male mice harboring a human loss-of-function HTR2C variant developed obesity and reduced social exploratory behavior; female mice heterozygous for the same variant showed similar deficits with reduced severity. Using the 5-HT2CR agonist lorcaserin, we found that depolarization of appetite-suppressing proopiomelanocortin neurons was impaired in knock-in mice. In conclusion, we demonstrate that 5-HT2CR is involved in the regulation of human appetite, weight and behavior. Our findings suggest that melanocortin receptor agonists might be effective in treating severe obesity in individuals carrying HTR2C variants. We suggest that HTR2C should be included in diagnostic gene panels for severe childhood-onset obesity.

Aberrant expression of agouti signaling protein (ASIP) as a cause of monogenic severe childhood obesity

Here we report a heterozygous tandem duplication at the ASIP (agouti signaling protein) gene locus causing ubiquitous, ectopic ASIP expression in a female patient with extreme childhood obesity. The mutation places ASIP under control of the ubiquitously active itchy E3 ubiquitin protein ligase promoter, driving the generation of ASIP in patient-derived native and induced pluripotent stem cells for all germ layers and hypothalamic-like neurons. The patient’s phenotype of early-onset obesity, overgrowth, red hair and hyperinsulinemia is concordant with that of mutant mice ubiquitously expressing the homolog nonagouti. ASIP represses melanocyte-stimulating hormone-mediated activation as a melanocortin receptor antagonist, which might affect eating behavior, energy expenditure, adipocyte differentiation and pigmentation, as observed in the index patient. As the type of mutation escapes standard genetic screening algorithms, we rescreened the Leipzig Childhood Obesity cohort of 1,745 patients and identified four additional patients with the identical mutation, ectopic ASIP expression and a similar phenotype. Taken together, our data indicate that ubiquitous ectopic ASIP expression is likely a monogenic cause of human obesity.

A New Organotypic 3D Slice Culture of Mouse Meibomian Glands Reveals Impact of Melanocortins

The meibomian glands (MGs) within the eyelids produce a lipid-rich secretion that forms the superficial layer of the tear film. Meibomian gland dysfunction (MGD) results in excessive evaporation of the tear film, which is the leading cause of dry eye disease (DED). To develop a research model similar to the physiological situation of MGs, we established a new 3D organotypic slice culture (OSC) of mouse MGs (mMGs) and investigated the effects of melanocortins on exocrine secretion. Tissue viability, lipid production and morphological changes were analyzed during a 21-day cultivation period. Subsequently, the effects on lipid production and gene expression were examined after stimulation with a melanocortin receptor (MCR) agonist, α-melanocyte-stimulating hormone (α-MSH), and/or an MCR antagonist, JNJ-10229570. The cultivation of mMGs OSCs was possible without impairment for at least seven days. Stimulation with the MCR agonists induced lipid production in a dose-dependent manner, whereas this effect was tapered with the simultaneous incubation of the MCR antagonist. The new 3D OSC model is a promising approach to study the (patho-) physiological properties of MG/MGD while reducing animal studies. Therefore, it may accelerate the search for new treatments for MGD/DED and lead to new insights, such as that melanocortins likely stimulate meibum production.

Genes Predisposing to Monogenic, Polygenic, and Syndromic Obesity: A Review of Current Trends and Prospects for Standard Obesity Genetic Testing

Objective: The burden of obesity is currently enormous, necessitating a novel strategy to complement the existing ones. Accordingly, genetic predisposition is suspected in many cases of the disease, which can potentially be used as therapeutic targets. However, there are differing viewpoints on the suspect genes, prompting the current review to articulate the genes and their mechanisms. Eight (16%) of the genes singularly predispose humans to obesity (called monogenic obesity), 22 (43%) interact with other genes and the environment to predispose humans to obesity (called polygenic obesity), and 21 (41%) cause syndromic obesity. Monogenic obesity is often caused by three genes [the leptin (LEP), the leptin receptor (LEPR), and the melanocortin 4 receptor (MC4R) genes], polygenic obesity [fat mass and obesity-associated (FTO) gene], and syndromic obesity (Prader-Willi Syndrome). These genes control food intake and energy expenditure, and so mutations in them cause overeating, adiposity, and hyperphagia. Based on these findings, two geneticallybased drugs, named recombinant human leptin and setmelanotide, have been formulated and shown to significantly reduce food intake, body weight, and fat mass. This suggests that when the genetic etiology of obesity is fully understood, the disease’s treatment and prevention will improve. Healthcare providers are urged to develop genetically-based personalized treatments for obese patients.

Pharmacology of orange-spotted grouper (Epinephelus coioides) melanocortin-5 receptor and its modulation by Mrap2

The mammalian melanocortin-5 receptors (MC5Rs) are involved in various functions, including exocrine gland secretion, glucose uptake, adipocyte lipolysis, and immunity. However, the physiological role of fish Mc5r is rarely studied. Melanocortin-2 receptor accessory protein 2 (MRAP2) modulates pharmacological properties of melanocortin receptors. Herein, to lay the foundation for future physiological studies, we cloned the orange-spotted grouper (Epinephelus coioides) mc5r, with a 1008 bp open reading frame and a predicted protein of 334 amino acids. Grouper mc5r had abundant expression in the brain, skin, and kidney. Four ligands could bind to grouper Mc5r and dose-dependently increase intracellular cAMP levels. Grouper Mrap2 did not affect binding affinity or potency of Mc5r; however, grouper Mrap2 decreased cell surface expression and maximal binding of Mc5r. Mrap2 also significantly decreased the maximal response to a superpotent agonist but not the endogenous agonist. This study provided new data on fish Mc5r pharmacology and its regulation by Mrap2.

Pro-resolving and anti-arthritic properties of the MC1 selective agonist PL8177.

Melanocortins are peptides endowed with anti-inflammatory and pro-resolving activities. Many of these effects are mediated by the Melanocortin receptor 1 (MC1) as reported in several experimental settings. As such, MC1 can be a viable target for the development of new therapies that mimic endogenous pro-resolving mediators. The aim of this study was to assess the immunopharmacology of a selective MC1 agonist (PL8177) in vitro and in a mouse model of inflammatory arthritis. PL8177 and the natural agonist αMSH were tested for activation of mouse and human Melanocortin receptors (MC1,3,4,5), monitoring cAMP accumulation and ERK1/2 phosphorylation, using transiently transfected HEK293A cells. The anti-inflammatory and pro-resolving effects of PL8177 and αMSH were evaluated using mouse peritoneal Macrophages. Finally, a model of K/BxN serum transfer induced arthritis was used to determine the in vivo potential of PL8177. PL8177 activates mouse and human MC1 with apparent EC50 values of 0.01 and 1.49 nM, respectively, using the cAMP accumulation assay. Similar profiles were observed for the induction of ERK phosphorylation (EC50: 0.05 and 1.39 nM). PL8177 displays pro-resolving activity (enhanced Macrophage efferocytosis) and counteracts the inflammatory profile of zymosan-stimulated macrophages, reducing the release of IL-1β, IL-6, TNF-α and CCL-2. In the context of joint inflammation, PL8177 (3mg/kg i.p.) reduces clinical score, paw swelling and incidence of severe disease as well as the recruitment of immune cells into the arthritic joint. These results demonstrate that the MC1 agonism with PL8177 affords therapeutic effects in inflammatory conditions including arthritis. Drugs targeting the Melanocortin system have emerged as promising therapeutics for several conditions including inflammation or obesity. Multiple candidates are under clinical development, and some have already reached approval. Here we present the characterization of a novel drug candidate, PL8177, selective for the Melanocortin 1 receptor (MC1), demonstrating its selectivity profile on cAMP and ERK1/2 phosphorylation signaling pathways, of relevance as selective drugs will translate into lesser off-target effect. PL8177 also demonstrated, not only anti-inflammatory activity, but pro-resolving actions due to its ability to enhance efferocytosis (i.e. the phagocytosis of apoptotic cells), endowing this molecule with therapeutic advantages compared to classical anti-inflammatory drugs. Using a mouse model of inflammatory arthritis, the compound demonstrated in vivo efficacy by reducing clinical score, paw swelling and overall disease severity. Taken together, these results present Melanocortin-based therapies, and specifically targeting MC1 receptor, as a promising strategy to manage chronic inflammatory diseases.

Effect of the renin-angiotensin system on the exacerbation of adrenal glucocorticoid steroidogenesis in diabetic mice: Role of angiotensin-II type 2 receptor.

Prior investigation shows an increase in the activity of both hypothalamus-pituitary-adrenal (HPA) axis and the renin-angiotensin system (RAS) in diabetic patients. Moreover, activation of angiotensin-II type 1 receptor (AT1) has been associated with adrenal steroidogenesis. This study investigates the role of RAS on the overproduction of corticosterone in diabetic mice. Diabetes was induced by intravenous injection of alloxan into fasted Swiss-webster mice. Captopril (angiotensin-converting enzyme inhibitor), Olmesartan (AT1 receptor antagonist), CGP42112A (AT2 receptor agonist) or PD123319 (AT2 receptor antagonist) were administered daily for 14 consecutive days, starting 7 days post-alloxan. Plasma corticosterone was evaluated by ELISA, while adrenal gland expressions of AT1 receptor, AT2 receptor, adrenocorticotropic hormone receptor MC2R, pro-steroidogenic enzymes steroidogenic acute regulatory protein (StAR), and 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) were assessed using immunohistochemistry or western blot. Diabetic mice showed adrenal gland overexpression of AT1 receptor, MC2R, StAR, and 11βHSD1 without altering AT2 receptor levels, all of which were sensitive to Captopril or Olmesartan treatment. In addition, PD123319 blocked the ability of Olmesartan to reduce plasma corticosterone levels in diabetic mice. Furthermore, CGP42112A significantly decreased circulating corticosterone levels in diabetic mice, without altering the overexpression of MC2R and StAR in the adrenal glands. Our findings revealed that inhibition of both angiotensin synthesis and AT1 receptor activity reduced the high production of corticosterone in diabetic mice via the reduction of MC2R signaling expression in the adrenal gland. Furthermore, the protective effect of Olmesartan on the overproduction of corticosterone by adrenals in diabetic mice depends on both AT1 receptor blockade and AT2 receptor activation.