Melanocortin-4 Receptor Signaling Research Articles

Activation of the Melanocortin-4 receptor signaling by \u03b1-MSH stimulates nerve-dependent mouse digit regeneration

BackgroundExpression of Mc4r in peripheral organs indicates it has broader roles in organ homeostasis and regeneration. However, the expression and function of Mc4r in the mouse limb and digit has not been fully investigated. Our previous work showed that Mc4r−/− mice fail to regenerate the digit, but whether activation of MC4R signaling could rescue digit regeneration, or stimulate proximal digit regeneration is not clear.ResultsWe analyzed the expression dynamics of Mc4r in the embryonic and postnatal mouse limb and digit using the Mc4r-gfp mice. We found that Mc4r-GFP is mainly expressed in the limb nerves, and in the limb muscles that are undergoing secondary myogenesis. Expression of Mc4r-GFP in the adult mouse digit is restricted to the nail matrix. We also examined the effect of α-MSH on mouse digit regeneration. We found that administration of α-MSH in the Mc4r+/− mice rescue the delayed regeneration of distal digit tip. α-MSH could rescue distal digit regeneration in denervated hindlimbs. In addition, α-MSH could stimulate regeneration of the proximally amputated digit, which is non-regenerative.ConclusionsMc4r expression in the mouse limb and digit is closely related to nerve tissues, and α-MSH/MC4R signaling has a neurotrophic role in mouse digit tip regeneration.

Paraventricular Calcitonin Receptor-Expressing Neurons Modulate Energy Homeostasis in Male Mice.

The paraventricular nucleus of the hypothalamus (PVH) is a heterogeneous collection of neurons that play important roles in modulating feeding and energy expenditure. Abnormal development or ablation of the PVH results in hyperphagic obesity and defects in energy expenditure whereas selective activation of defined PVH neuronal populations can suppress feeding and may promote energy expenditure. Here, we characterize the contribution of calcitonin receptor-expressing PVH neurons (CalcRPVH) to energy balance control. We used Cre-dependent viral tools delivered stereotaxically to the PVH of CalcR2Acre mice to activate, silence, and trace CalcRPVH neurons and determine their contribution to body weight regulation. Immunohistochemistry of fluorescently-labeled CalcRPVH neurons demonstrates that CalcRPVH neurons are largely distinct from several PVH neuronal populations involved in energy homeostasis; these neurons project to regions of the hindbrain that are implicated in energy balance control, including the nucleus of the solitary tract and the parabrachial nucleus. Acute activation of CalcRPVH neurons suppresses feeding without appreciably augmenting energy expenditure, whereas their silencing leads to obesity that may be due in part due to loss of PVH melanocortin-4 receptor signaling. These data show that CalcRPVH neurons are an essential component of energy balance neurocircuitry and their function is important for body weight maintenance. A thorough understanding of the mechanisms by which CalcRPVH neurons modulate energy balance might identify novel therapeutic targets for the treatment and prevention of obesity.

Quantitative high-throughput assay to measure MC4R-induced intracellular calcium.

The melanocortin-4 receptor (MC4R), a critical G-protein-coupled receptor (GPCR) regulating energy homeostasis, activates multiple signalling pathways, including mobilisation of intracellular calcium ([Ca2+]i). However, very little is known about the physiological significance of MC4R-induced [Ca2+]i since few studies measure MC4R-induced [Ca2+]i. High-throughput, read-out assays for [Ca2+]i have proven unreliable for overexpressed GPCRs like MC4R, which exhibit low sensitivity mobilising [Ca2+]i. Therefore, we developed, optimised, and validated a robust quantitative high-throughput assay using Fura-2 ratio-metric calcium dye and HEK293 cells stably transfected with MC4R. The quantitation enables direct comparisons between assays and even between different research laboratories. Assay conditions were optimised step-by-step to eliminate interference from stretch-activated receptor increases in [Ca2+]i and to maximise ligand-activated MC4R-induced [Ca2+]i. Calcium imaging was performed using a PheraStar FS multi-well plate reader. Probenecid, included in the buffers to prevent extrusion of Fura-2 dye from cells, was found to interfere with the EGTA-chelation of calcium, required to determine Rmin for quantitation of [Ca2+]i. Therefore, we developed a method to determine Rmin in specific wells without probenecid, which was run in parallel with each assay. The validation of the assay was shown by reproducible α-melanocyte-stimulating hormone (α-MSH) concentration-dependent activation of the stably expressed human MC4R (hMC4R) and mouse MC4R (mMC4R), inducing increases in [Ca2+]i, for three independent experiments. This robust, reproducible, high-throughput assay that quantitatively measures MC4R-induced mobilisation of [Ca2+]i in vitro has potential to advance the development of therapeutic drugs and understanding of MC4R signalling associated with human obesity.

Melanocortin receptor activation alleviates amyloid pathology and glial reactivity in an Alzheimer\u2019s disease transgenic mouse model

Alzheimer’s disease (AD) is a devastating neurodegenerative disorder with no disease-modifying treatment. AD progression is characterized by cognitive decline, neuroinflammation, and accumulation of amyloid-beta (Aβ) and neurofibrillary tangles in the brain, leading to neuronal and glial dysfunctions. Neuropeptides govern diverse pathophysiological processes and represent key players in AD pathogenesis, regulating synaptic plasticity, glial cell functions and amyloid pathology. Activation of the pro-opiomelanocortin (POMC)-derived neuropeptide and its receptor from the melanocortin receptor (MCR) family have previously been shown to rescue the impairment in hippocampus-dependent synaptic plasticity in the APP/PS1 mouse model of AD. However, the functional roles of MCR signaling in AD conditions, particularly in glial functions, are largely unknown. In this study, we investigated the potential benefits of MCR activation in AD. In APP/PS1 transgenic mice, we demonstrate that MCR activation mediated by the central administration of its agonist D-Tyr MTII substantially reduces Aβ accumulation, while alleviating global inflammation and astrocytic activation, particularly in the hippocampus. MCR activation prominently reduces the A1 subtype of reactive astrocytes, which is considered a key source of astrocytic neurotoxicity in AD. Concordantly, MCR activation suppresses microglial activation, while enhancing their association with amyloid plaques. The blunted activation of microglia may contribute to the reduction in the neurotoxic phenotypes of astrocytes. Importantly, transcriptome analysis reveals that MCR activation restores the impaired homeostatic processes and microglial reactivity in the hippocampus in APP/PS1 mice. Collectively, our findings demonstrate the potential of MCR signaling as therapeutic target for AD.

Peripheral actions and direct central–local communications of melanocortin 4 receptor signaling

Melanocortin 4 receptor (MC4R), the most important monogenetic cause of human metabolic disorders, has been of great interest to many researchers in the field of energy homeostasis and public health. Because MC4R is a vital pharmaceutical target for maintaining controllable appetite and body weight for professional athletes, previous studies have mainly focused on the central, rather than the peripheral, roles of MC4R. Thus, the local expression of MC4R and its behavioral regulation remain unclear. In an attempt to shed light on different directions for future studies of MC4R signaling, we review a series of recent and important studies exploring the peripheral functions of MC4R and the direct physiological interaction between peripheral organs and central MC4R neurons in this article.

Pharmacological evaluation of MRAP proteins on Xenopus neural melanocortin signaling.

Melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R), two neural G protein-coupled receptors are known to be functionally critical for energy balance in vertebrates. As allosteric regulators of melanocortin receptors, melanocortin accessory proteins (MRAPs) are also involved in energy homeostasis. The interaction of MRAPs and melanocortin signaling was previously shown in mammals and zebrafish, but nothing had been reported in amphibians. As the basal class of tetrapods, amphibians occupy a phylogenetic transition between teleosts and terrestrial animals. Here we examined the evolutionary conservation of MC3R, MC4R, and MRAPs between diploid Xenopus tropicalis (xt-) and other chordates and investigated the pharmacological regulatory properties of MRAPs on the neural MC3R and MC4R signaling. Our results showed that xtMRAP and xtMRAP2 both exerted robust potentiation effect on agonist (α-MSH and adrenocorticotropin [ACTH]) induced activation and modulated the basal activity and cell surface translocation of xtMC3R and xtMC4R. In addition, the presence of two accessory proteins could convert xtMC3R and xtMC4R into ACTH-preferred receptors. These findings suggest that the presence of MRAPs exhibits fine control over the pharmacological activities of the neuronal MC3R and MC4R signaling in the Xenopus tropicalis, which is physiologically relevant with the complicated transition of feeding behaviors during their life history.

Pharmacological modulation of dual melanocortin-4 receptor signaling by melanocortin receptor accessory proteins in the Xenopus laevis.

Physiological modulation of melanocortin-4 receptor (MC4R) signaling by MRAP2 proteins plays an indispensable role in appetite control and energy homeostasis in the central nervous system. Great interspecies differences of the interaction and regulation of melanocortin receptors by MRAP protein family have been reported in several diploid vertebrates but never been investigated in the tetrapod amphibian Xenopus laevis. Here, we performed phylogenetic and synteny-based analyses to explore the evolutionary aspects of dual copies of X. laevis MC4R (xlMC4R) and MRAP2 (xlMRAP2) proteins. Our data showed that xlMRAPs directly interacted with xlMC4Rs on the cell surface as a functional antiparallel dimeric topology and pharmacological studies suggested a homology specific regulatory pattern of the melanocortin system in X. laevis.

Bariatric Surgery Improves Glycemia But Not Obesity in a Family with Inactive Melanocortin 4 Receptor Signaling

We conducted an observational long-term study with a median follow-up of 102 months on the outcomes of bariatric surgery in a family that carries a deleterious mutation in the melanocortin 4 receptor (MC4R) gene. In homozygous patients, bariatric surgery led to transient weight-loss and improvements in associated comorbidities, but in the long-term did not resolve morbid obesity: median presurgical/final body mass index 71.2/64.3 kg/m2. Glycemia however was improved despite regaining: median presurgical/final fasting blood glucose 218/101 mg/dL. Heterozygous patients responded normally to bariatric surgery. This unique case series, the largest and longest of its kind, demonstrates that bariatric surgery is dependent on MC4R signaling for sustained weight-loss, but its anti-diabetic effect is also independent of weight-loss. Bariatric surgery provides temporary relief in comorbidities for super-obese patients, may improve long-term glycemic control, and should be considered as a metabolic treatment for such genetic cases.

Structural Complexity and Plasticity of Signaling Regulation at the Melanocortin-4 Receptor.

The melanocortin-4 receptor (MC4R) is a class A G protein-coupled receptor (GPCR), essential for regulation of appetite and metabolism. Pathogenic inactivating MC4R mutations are the most frequent cause of monogenic obesity, a growing medical and socioeconomic problem worldwide. The MC4R mediates either ligand-independent or ligand-dependent signaling. Agonists such as α-melanocyte-stimulating hormone (α-MSH) induce anorexigenic effects, in contrast to the endogenous inverse agonist agouti-related peptide (AgRP), which causes orexigenic effects by suppressing high basal signaling activity. Agonist action triggers the binding of different subtypes of G proteins and arrestins, leading to concomitant induction of diverse intracellular signaling cascades. An increasing number of experimental studies have unraveled molecular properties and mechanisms of MC4R signal transduction related to physiological and pathophysiological aspects. In addition, the MC4R crystal structure was recently determined at 2.75 Å resolution in an inactive state bound with a peptide antagonist. Underpinned by structural homology models of MC4R complexes simulating a presumably active-state conformation compared to the structure of the inactive state, we here briefly summarize the current understanding and key players involved in the MC4R switching process between different activity states. Finally, these perspectives highlight the complexity and plasticity in MC4R signaling regulation and identify gaps in our current knowledge.

High-fat food biases hypothalamic and mesolimbic expression of consummatory drives.

SummaryMaintaining healthy body weight is increasingly difficult in our obesogenic environment. Dieting efforts are often overpowered by the internal drive to consume energy-dense foods. While the selection of calorically-rich substrates over healthier options is identifiable across species, the mechanisms behind this choice remain poorly understood. Using a passive devaluation paradigm, we found that exposure to high-fat diet (HFD) suppresses the intake of nutritionally-balanced standard chow diet (SD) irrespective of age, sex, body mass accrual and functional leptin or melanocortin-4 receptor signaling. Longitudinal recordings revealed this SD devaluation and subsequent shift toward HFD consumption is encoded at the level of hypothalamic Agouti-related peptide (AgRP) neurons and mesolimbic dopamine signaling. Prior HFD consumption vastly diminished the capacity of SD to alleviate the negative valence associated with hunger and the rewarding properties of food discovery even after periods of HFD abstinence. These data reveal a neural basis behind the hardships of dieting.

372-OR: Ablation of the Hepatokine TSK Protects Mice against Obesity and Metabolic Disorders Caused by MC4R Deficiency

Endocrine hormones released by peripheral tissues, such as adipose tissue, skeletal muscle and the liver, play an important role in metabolic crosstalk and homeostasis and contribute to disease pathogenesis. We recently identified Tsukushi (TSK) as a liver-enriched secreted factor that is highly inducible in response to increased energy expenditure. TSK null mice were resistant to diet-induced obesity, insulin resistance, and diet-induced NASH as a result of enhanced sympathetic activation and brown fat thermogenesis. However, the mechanisms underlying TSK action remain incompletely elucidated. Melanocortin 4 receptor (MC4R) is a key regulator of energy balance and its mutations represent the most common monogenic cause of obesity in human. In this study, we generated TSK/MC4R double knockout mice to explore potential crosstalk between the TSK and MC4R signaling pathways. While MC4R-null mice developed severe obesity when fed standard rodent chow, TSK inactivation nearly completely abolished the defects of energy balance caused by MC4R deficiency. Remarkably, ablation of TSK normalized food intake and corrected hepatic steatosis, adipose tissue inflammation, and insulin resistance in MC4R null mice. TSK deficiency also rescued the impairments of thermogenic gene expression caused by MC4R deficiency. Those results suggest that TSK likely acts downstream or in parallel to MC4R signaling to regulate feeding behavior and energy expenditure. Our work supports a rationale for therapeutic blockade of TSK for the treatment of obesity and metabolic disease caused by MC4R mutations. Disclosure Q. Wang: None. J. Lin: None. Funding National Institutes of Health (DK102456, AG055379, DK114220)

OR26-01 Epinephrine Is Essential for Normal Renal Glucose Reabsorption via the Glucose Transporter GLUT2

Humans and mice with Melanocortin 4 receptor (MC4R) deficiency remain protected from hyperglycemia despite chronic obesity and insulin resistance. We have observed that elevated glycosuria in MC4R deficient mice protects them from hyperglycemia. Moreover, our results indicate that circulating epinephrine may couple MC4R signaling with kidney glucose reabsorption. However, the direct role of epinephrine in regulating kidney glucose reabsorption remains unclear. We hypothesize that epinephrine is essential for maintaining glucose homeostasis via kidney glucose reabsorption. To test this hypothesis, we performed oral glucose tolerance tests (OGTTs) and intraperitoneal insulin tolerance tests (ITTs) in phenylethanolamine-N-methyltransferase (Pnmt) knockout (KO) mice that specifically lack epinephrine but have normal norepinephrine levels. Pnmt KO mice exhibited reduced insulin sensitivity compared to their Wild-Type (WT) littermates (Area under the curve for ITT: 9,700±256 vs. 8,482±417 mg/dL.min, p<0.05). Paradoxically, we observed improved rather than impaired glucose tolerance in Pnmt KO mice compared to their WT controls (Area under the curve for OGTT: 32,546±1,592 vs. 40,058±1,918 mg/dL.min, p<0.05). To ascertain if Pnmt KO mice, like MC4R deficient mice, show elevated glycosuria, we quantified their 24 urine glucose levels after oral glucose (250 mg) challenge. Indeed, Pnmt KO mice demonstrated elevated glycosuria compared to their WT littermates (Urine glucose: Baseline, 24.63±2.2 vs. 11.14±0.82 mg/dl; post glucose challenge: 67.83±5 vs. 16.09±1.13 mg/dl, p<0.001), again validating the phenotype similar to that of MC4R deficient mice. To determine the glucose transporters involved in mediating elevated glycosuria in the Pnmt KO mice, we measured the levels of different renal glucose transporters using western blot. We found that GLUT2 was decreased by ~26% in Pnmt KO mice compared to their WT littermates. Levels of other glucose transporters were not changed, indicating that suppression of renal GLUT2 mediates elevated glycosuria in the epinephrine deficient mice. We validated the direct effect of epinephrine on GLUT2 levels in vitro using mouse primary renal proximal tubule epithelial cells. Indeed, epinephrine selectively increased GLUT2, but did not affect other glucose transporters in the mouse kidney primary cells. Our findings establish the essential role of epinephrine in glucose reabsorption via the renal glucose transporter GLUT2. Therefore, modulating the renal adrenergic system, or, kidney-specific GLUT2 may afford alternative strategies to regulate glycosuria and ultimately mitigate diabetes.

The Role of PVN MC4R Neural Pathway in Autonomic Control of Metabolic Homeostasis and Cardiovascular Function

Central melanocortin pathways, mainly via acting on melanocortin‐4 receptor (MC4R), play an important role in both energy balance and sympathetic traffics affecting cardiovascular function. MC4R deficiency in both humans and rodents causes obesity, insulin resistance and diabetes while maintaining low to normal sympathetic tone and blood pressure, suggesting a critical role of MC4R signaling in metabolic homeostasis and obesity‐associated sympathetic overdrive and hypertension. MC4R is widely expressed in the brain including hypothalamic paraventricular nucleus (PVN) which functions as an integrative center for energy metabolism and autonomic regulation. However, whether and how PVNMC4R pathways contribute to the sympathetic control of cardiovascular function and metabolic homeostasis remain incompletely understood. In present study, we first comprehensively mapped afferent projections of PVNMC4R neurons by stereotaxically delivering Cre‐dependent AAV driving expression of eYFP into the PVN of MC4R‐Cre knock‐in mice, which allow targeted anterograde tract‐tracing of PVNMC4R neurons throughout the brain. In addition to known brain region for feeding behavior (i.e. parabrachial nucleus), we found broad innervations of PVNMC4R neurons to various brain regions important for autonomic‐cardiovascular control and energy metabolism, including but not limited to nucleus of solitary tractus , dorsal motor nucleus of vagus, rostral ventrolateral medulla (RVLM) and spinal cord. Using a combination of sophisticated viral tools, we further reveal that thoracic spinal cord‐projecting PVNMC4R neurons are exclusively located in the posterolateral PVN and send collateral projections to RVLM. Functionally, microinjection of synthetic MC4R agonist (MTII) into the PVN evokes renal sympathetic nerve activity in anesthetized mice and chemogenetic activation of PVNMC4R neurons elevates blood pressure and decreases food intake in freely moving animals. Additionally, genetic ablation of PVNMC4R neurons in adult mice using Cre‐dependent AAV expressing Casp3 causes hyperphagia and massive weight gain (over 80% increase within 6 weeks). These results provide an important insight into the role of PVNMCR neural circuits in regulating metabolic homeostasis and cardiovascular function. Optogenetic and chemogenetic studies are ongoing to functionally dissect the neural circuits downstream of PVNMC4R neurons.Support or Funding InformationNational Institute of Health (R01), AHA Postdoctoral Grant Award (19POST34450083)

Differential Signaling Profiles of MC4R Mutations with Three Different Ligands.

The melanocortin 4 receptor (MC4R) is a key player in hypothalamic weight regulation and energy expenditure as part of the leptin–melanocortin pathway. Mutations in this G protein coupled receptor (GPCR) are the most common cause for monogenetic obesity, which appears to be mediated by changes in the anorectic action of MC4R via GS-dependent cyclic adenosine-monophosphate (cAMP) signaling as well as other signaling pathways. To study potential bias in the effects of MC4R mutations between the different signaling pathways, we investigated three major MC4R mutations: a GS loss-of-function (S127L) and a GS gain-of-function mutant (H158R), as well as the most common European single nucleotide polymorphism (V103I). We tested signaling of all four major G protein families plus extracellular regulated kinase (ERK) phosphorylation and β-arrestin2 recruitment, using the two endogenous agonists, α- and β-melanocyte stimulating hormone (MSH), along with a synthetic peptide agonist (NDP-α-MSH). The S127L mutation led to a full loss-of-function in all investigated pathways, whereas V103I and H158R were clearly biased towards the Gq/11 pathway when challenged with the endogenous ligands. These results show that MC4R mutations can cause vastly different changes in the various MC4R signaling pathways and highlight the importance of a comprehensive characterization of receptor mutations.

Activation of Melanocortin-4 Receptor Inhibits Both Neuroinflammation Induced by Early Exposure to Ethanol and Subsequent Voluntary Alcohol Intake in Adulthood in Animal Models: Is BDNF the Key Mediator?

The concept that neuroinflammation induced by excessive alcohol intake in adolescence triggers brain mechanisms that perpetuate consumption has strengthened in recent years. The melanocortin system, composed of the melanocortin 4 receptor (MC4R) and its ligand α-melanocyte-stimulating hormone (α-MSH), has been implicated both in modulation of alcohol consumption and in ethanol-induced neuroinflammation decrease. Chronic alcohol consumption in adolescent rats causes a decrease in an α-MSH release by the hypothalamus, while the administration of synthetic agonists of MC4R causes a decrease in neuroinflammation and a decrease in voluntary alcohol consumption. However, the mechanism that connects the activation of MC4R with the decrease of both neuroinflammation and voluntary alcohol consumption has not been elucidated. Brain-derived neurotrophic factor (BDNF) has been implicated in alcohol drinking motivation, dependence and withdrawal, and its levels are reduced in alcoholics. Deficiencies in BDNF levels increased ethanol self-administration in rats. Further, BDNF triggers important anti-inflammatory effects in the brain, and this could be one of the mechanisms by which BDNF reduces chronic alcohol intake. Interestingly, MC4R signaling induces BDNF expression through the activation of the cAMP-responsive element-binding protein (CREB). We hypothesize that ethanol exposure during adolescence decreases the expression of α-MSH and hence MC4R signaling in the hippocampus, leading to a lower BDNF activity that causes dramatic changes in the brain (e.g., neuroinflammation and decreased neurogenesis) that predispose to maintain alcohol abuse until adulthood. The activation of MC4R either by α-MSH or by synthetic agonist peptides can induce the expression of BDNF, which would trigger several processes that lead to lower alcohol consumption.

Bromocriptine improves glucose tolerance independent of circadian timing, prolactin, or the melanocortin-4 receptor.

Bromocriptine, a dopamine D2 receptor agonist originally used for the treatment of hyperprolactinemia, is largely successful in reducing hyperglycemia and improving glucose tolerance in type 2 diabetics. However, the mechanism behind bromocriptine's effect on glucose intolerance is unclear. Here, we tested three hypotheses, that bromocriptine may exert its effects on glucose metabolism by 1) decreasing prolactin secretion, 2) indirectly increasing activity of key melanocortin receptors in the central nervous system, or 3) improving/restoring circadian rhythms. Using a diet-induced obese (DIO) mouse model, we established that a 2-wk treatment of bromocriptine is robustly effective at improving glucose tolerance. We then demonstrated that bromocriptine is effective at improving the glucose tolerance of both DIO prolactin-deficient and melanocortin-4 receptor (MC4R)-deficient mice, pointing to bromocriptine's ability to affect glucose tolerance independently of prolactin or MC4R signaling. Finally, we tested bromocriptine's dependence on the circadian system by testing its effectiveness in environmental (e.g., repeated shifts to the light-dark cycle) and genetic (e.g., the Clock mutant mouse) models of circadian disruption. In both models of circadian disruption, bromocriptine was effective at improving glucose tolerance, indicating that a functional or well-aligned endogenous clock is not necessary for bromocriptine's effects on glucose metabolism. Taken together, these results do not support the role of prolactin, MC4R, or the circadian clock as integral to bromocriptine's underlying mechanism. Instead, we find that bromocriptine is a robust diabetic treatment and resilient to genetically induced obesity, diabetes, and circadian disruption.

Pharmacological modulation of melanocortin-4 receptor by melanocortin receptor accessory protein 2 in Nile tilapia

The melanocortin-4 receptor (MC4R) acts as a member of G-protein coupled receptors and participate in food intake and energy expenditure. Melanocortin 2 receptor accessory protein 2 (MRAP2) plays a critical role in regulating MC4R signaling in mammals and zebrafish. However, evidence on their interaction in other teleost species remains elusive. Here, we cloned and assessed the evolutionary aspect and pharmacological modulation of MRAP2 on MC4R signaling in Nile tilapia (Oreochromis niloticus). Tissue distribution analysis of tmc4r and tmrap2 confirmed their co-expression in the brain region. tMRAP2 protein could form antiparallel homo-dimer and directly interacted with tMC4R in vitro and presence of tMRAP2 led to the reduction of agonist response and surface expression of tMC4R. Overall, our findings provide a comparative overview on the evolutionary conservation, genomic distribution, tissue-specific expression and pharmacological profile of the MC4R and MRAP2 in another non-mammalian teleost.

Hypertension Editors' Picks Preeclampsia.

Hypertension Editors' Picks Preeclampsia.

2009-P: Molecular Mechanisms of Obesity-Associated Vascular Vulnerability via Signals of Appetite Regulation

Background: Obesity is a risk factor of hypertension and abdominal aortic aneurysm (AAA). Melanocortin 4 receptor (MC4R) is activated by α-melanocyte-stimulating hormone (α-MSH) to mediate most of the anorectic effects of leptin. In humans, deficiency or mutation of MC4R is the most common monogenic form of obesity. However, it remains unknown whether MC4R signaling affects the development of obesity-associated hypertension, vascular vulnerability and aortic aneurysm. Methods and Results: MC4R-knockout (KO) mice fed a high-fat/high-sucrose diet (HF/HS) for 20 weeks displayed obesity associated with hyperleptinemia compared to HF/HS-fed wild type (WT) mice. MC4R-KO mice exhibited hypertension, increase of medial thickness and elastic lamina destruction, and elevation of inflammatory and fibrosis-related genes including TGF-β and osteopontin (OPN) in aortae. MC4R-KO mice showed higher incidence of angiotensin II (Ang II, 500 ng/kg/min)-induced AAA (73%) than WT mice (8%). MC4R-KO mice with ob/ob background exhibited lower incidence of Ang II-induced AAA (50%) than MC4R-KO mice, but higher than ob/ob mice (12.5%). Treatment of OPN-blocking antibody decreased Ang II-induced AAA incidence in HF/HS-fed MC4R-KO mice. In vascular smooth muscle cells (VSMCs), leptin (100 ng/ml) significantly upregulated OPN gene expression. α-MSH suppressed AngII-induced TGF-β gene expression in VSMCs, and LPS/IFN-γ-induced iNOS gene expression in peritoneal macrophages. However, these effects of α-MSH were abolished in primary macrophages from MC4R-KO mice and RAW264.7 cells pretreated with MC4R antagonist HS024. Conclusion: Our observation indicates that deficiency of MC4R promotes the vascular vulnerability in mice. It suggests that, not only hyperleptinemia, but also impairment of α-MSH-MC4R signaling in VSMCs and macrophages, may also promote the vascular vulnerability. Disclosure K. Mori: None. K. Tsuchiya: None. S. Nakamura: None. K. Shiba: None. Y. Miyachi: None. Y. Ogawa: None. K. Kitamura: None.

Current Mechanistic and Pharmacodynamic Understanding of Melanocortin-4 Receptor Activation.

In this work we summarize our understanding of melanocortin 4 receptor (MC4R) pathway activation, aiming to define a safe and effective therapeutic targeting strategy for the MC4R. Delineation of cellular MC4R pathways has provided evidence for distinct MC4R signaling events characterized by unique receptor activation kinetics. While these studies remain narrow in scope, and have largely been explored with peptidic agonists, the results provide a possible correlation between distinct ligand groups and differential MC4R activation kinetics. In addition, when a set of small-molecule and peptide MC4R agonists are compared, evidence of biased signaling has been reported. The results of such mechanistic studies are discussed.