Ligands for Melanocortin Receptors: Beyond Melanocyte-Stimulating Hormones and Adrenocorticotropin.

The discovery of melanocortins in 1916 has resulted in more than 100 years of research focused on these peptides. Extensive studies have elucidated well-established functions of melanocortins mediated by cell surface receptors, including MSHR (melanocyte-stimulating hormone receptor) and ACTHR (adrenocorticotropin receptor). Subsequently, three additional melanocortin receptors (MCRs) were identified. Among these five MCRs, MC3R and MC4R are expressed primarily in the central nervous system, and are therefore referred to as the neural MCRs. Since the central melanocortin system plays important roles in regulating energy homeostasis, targeting neural MCRs is emerging as a therapeutic approach for treating metabolic conditions such as obesity and cachexia. Early efforts modifying endogenous ligands resulted in the development of many potent and selective ligands. This review focuses on the ligands for neural MCRs, including classical ligands (MSH and agouti-related peptide), nonclassical ligands (lipocalin 2, ß-defensin, small molecules, and pharmacoperones), and clinically approved ligands (ACTH, setmelanotide, bremelanotide, and several repurposed drugs).

Development of a Therapeutic Peptide for Cachexia Suggests a Platform Approach for Drug-like Peptides.

During the development of a melanocortin (MC) peptide drug to treat the condition of cachexia (a hypermetabolic state producing lean body mass wasting), we were confronted with the need for peptide transport across the blood-brain barrier (BBB): the MC-4 receptors (MC4Rs) for metabolic rate control are located in the hypothalamus, i.e., behind the BBB. Using the term "peptides with BBB transport", we screened the medical literature like a peptide library. This revealed numerous "hits"-peptides with BBB transport and/or oral activity. We noted several features common to most peptides in this class, including a dipeptide sequence of nonpolar residues, primary structure cyclization (whole or partial), and a Pro-aromatic motif usually within the cyclized region. Based on this, we designed an MC4R antagonist peptide, TCMCB07, that successfully treated many forms of cachexia. As part of our pharmacokinetic characterization of TCMCB07, we discovered that hepatobiliary extraction from blood accounted for a majority of the circulating peptide's excretion. Further screening of the literature revealed that TCMCB07 is a member of a long-forgotten peptide class, showing active transport by a multi-specific bile salt carrier. Bile salt transport peptides have predictable pharmacokinetics, including BBB transport, but rapid hepatic clearance inhibited their development as drugs. TCMCB07 shares the general characteristics of the bile salt peptide class but with a much longer half-life of hours, not minutes. A change in its C-terminal amino acid sequence slows hepatic clearance. This modification is transferable to other peptides in this class, suggesting a platform approach for producing drug-like peptides.

Demonstration of a Common DPhe7 to DNal(2')7 Peptide Ligand Antagonist Switch for Melanocortin-3 and Melanocortin-4 Receptors Identifies the Systematic Mischaracterization of the Pharmacological Properties of Melanocortin Peptides.

Melanocortin peptides containing a 3-(2-naphthyl)-d-alanine residue in position 7 (DNal(2')7), reported as melanocortin-3 receptor (MC3R) subtype-specific agonists in two separate publications, were found to lack significant MC3R agonist activity. The cell lines used at the University of Arizona for pharmacological characterization of these peptides, consisting of HEK293 cells stably transfected with human melanocortin receptor subtypes MC1R, MC3R, MC4R, or MC5R, were then obtained and characterized by quantitative polymerase chain reaction (PCR). While the MC1R cell line correctly expressed only hMCR1, the three other cell lines were mischaracterized with regard to receptor subtype expression. The demonstration that a 3-(2-naphthyl)-d-alanine residue in position 7, irrespective of the melanocortin peptide template, results primarily in the antagonism of MC3R and MC4R then allowed us to search the published literature for additional errors. The erroneously characterized DNal(2')7-containing peptides date back to 2003; thus, our analysis suggests that systematic mischaracterization of the pharmacological properties of melanocortin peptides occurred.

Exploring Pleiotropic Functions of Canine ß-Defensin 103: Nasal Cavity Expression, Antimicrobial Activity, and Melanocortin Receptor Activity.

Canine ß-defensin 103 (cBD103) and its common variant cBD103ΔG23 are multitasking polypeptides. As a ß-defensin, cBD103 is one of many antimicrobial agents used by the innate immunity to thwart pathogenic colonization. In this study, we showed that cBD103 was expressed throughout the nasal cavity, with primary expression in the nares as well as respiratory and olfactory epithelia. In the rostral nasal concha, cBD103 was expressed in the epithelium, and to a lesser degree in the lamina propria, but was absent in goblet cells. In the main olfactory epithelium, virtually all cells in the epithelial layer and select cells associated with Bowman's glands expressed cBD103. We also showed that the ΔG23 mutation did not appreciably alter the antimicrobial activity of the peptide against several species of microorganisms tested in nutrient-rich or minimal media or minimal media with salt added. Moreover, we showed antimicrobial activity in minimal media did not necessarily predict the inhibitory action of the peptide in nutrient-rich media. Both forms of cBD103 caused ultrastructural changes (membrane blebbing, condensation of intracellular contents and cell wall lysis) in Escherichia coli and Staphylococcus aureus. As a ligand of the melanocortin receptors, we showed that cBD103ΔG23 increased ERK1/2 activation and cAMP accumulation when bound to the human or canine melanocortin-4 receptor, acting as a weak allosteric agonist.

Alanine Scanning Mutagenesis of the DRYxxI Motif and Intracellular Loop 2 of Human Melanocortin-4 Receptor.

The melanocortin-4 receptor (MC4R) is a member of the G-protein-coupled receptor (GPCR) superfamily, which has been extensively studied in obesity pathogenesis due to its critical role in regulating energy homeostasis. Both the Gs-cAMP and ERK1/2 cascades are known as important intracellular signaling pathways initiated by the MC4R. The DRYxxI motif at the end of transmembrane domain 3 and the intracellular loop 2 (ICL2) are thought to be crucial for receptor function in several GPCRs. To study the functions of this domain in MC4R, we performed alanine-scanning mutagenesis on seventeen residues. We showed that one residue was critical for receptor cell surface expression. Eight residues were important for ligand binding. Mutations of three residues impaired Gs-cAMP signaling without changing the binding properties. Investigation on constitutive activities of all the mutants in the cAMP pathway revealed that six residues were involved in constraining the receptor in inactive states and five residues were important for receptor activation in the absence of an agonist. In addition, mutations of four residues impaired the ligand-stimulated ERK1/2 signaling pathway without affecting the binding properties. We also showed that some mutants were biased to the Gs-cAMP or ERK1/2 signaling pathway. In summary, we demonstrated that the DRYxxI motif and ICL2 were important for MC4R function.

Endothelial Cell Cystathionine γ-Lyase Expression Level Modulates Exercise Capacity, Vascular Function, and Myocardial Ischemia Reperfusion Injury.

Background Hydrogen sulfide (H2S) is an important endogenous physiological signaling molecule and exerts protective properties in the cardiovascular system. Cystathionine γ-lyase (CSE), 1 of 3 H2S producing enzyme, is predominantly localized in the vascular endothelium. However, the regulation of CSE in vascular endothelium remains incompletely understood. Methods and Results We generated inducible endothelial cell-specific CSE overexpressed transgenic mice (EC-CSE Tg) and endothelial cell-specific CSE knockout mice (EC-CSE KO), and investigated vascular function in isolated thoracic aorta, treadmill exercise capacity, and myocardial injury following ischemia-reperfusion in these mice. Overexpression of CSE in endothelial cells resulted in increased circulating and myocardial H2S and NO, augmented endothelial-dependent vasorelaxation response in thoracic aorta, improved exercise capacity, and reduced myocardial-reperfusion injury. In contrast, genetic deletion of CSE in endothelial cells led to decreased circulating H2S and cardiac NO production, impaired endothelial dependent vasorelaxation response and reduced exercise capacity. However, myocardial-reperfusion injury was not affected by genetic deletion of endothelial cell CSE. Conclusions CSE-derived H2S production in endothelial cells is critical in maintaining endothelial function, exercise capacity, and protecting against myocardial ischemia/reperfusion injury. Our data suggest that the endothelial NO synthase-NO pathway is likely involved in the beneficial effects of overexpression of CSE in the endothelium.

Regulation of Melanocortin-4 Receptor Pharmacology by Two Isoforms of Melanocortin Receptor Accessory Protein 2 in Topmouth Culter (Culter alburnus).

Melanocortin-4 receptor (MC4R) plays important roles in regulation of multiple physiological processes, and interaction of MC4R and melanocortin receptor accessory protein 2 (MRAP2) is suggested to play pivotal role in energy balance of vertebrates. Topmouth culter (Culter alburnus) is an economically important freshwater fish in China. Herein we cloned culter mc4r, mrap2a, and mrap2b. Culter mc4r consisted of a 981 bp open reading frame encoding a protein of 326 amino acids. qRT-PCR revealed that mc4r, mrap2a, and mrap2b were primarily expressed in the central nervous system. In the periphery, mc4r and mrap2b were expressed more widely in the male, while mrap2a was expressed more widely in the female. Culter MC4R could bind to four peptide agonists and increase intracellular cAMP production dose dependently. Culter MC4R was constitutively active in both cAMP and ERK1/2 pathways, which was differentially regulated by culter MRAP2a and MRAP2b. Culter MRAP2a significantly increased Bmax and decreased agonist-stimulated cAMP, while MRAP2b increased cell surface and total expression but did not affect Bmax and agonist-stimulated cAMP. These results will aid the investigation of the potential physiological processes that MC4R might be involved in topmouth culter.

Nerve growth factor in metabolic complications and Alzheimer's disease: Physiology and therapeutic potential.

As the population ages, obesity and metabolic complications as well as neurological disorders are becoming more prevalent, with huge economic burdens on both societies and families. New therapeutics are urgently needed. Nerve growth factor (NGF), first discovered in 1950s, is a neurotrophic factor involved in regulating cell proliferation, growth, survival, and apoptosis in both central and peripheral nervous systems. NGF and its precursor, proNGF, bind to TrkA and p75 receptors and initiate protein phosphorylation cascades, resulting in changes of cellular functions, and are associated with obesity, diabetes and its complications, and Alzheimer's disease. In this article, we summarize changes in NGF levels in metabolic and neuronal disorders, the signal transduction initiated by NGF and proNGF, the physiological and pathophysiological relevance, and therapeutic potential in treating chronic metabolic diseases and cognitive decline.

Regulation of melanocortin-1 receptor pharmacology by melanocortin receptor accessory protein 2 in orange-spotted grouper (Epinephelus coioides).

Melanocortin-1 receptor (MC1R) has important roles in regulating pigmentation and inflammation. Melanocortin receptor accessory protein 2 (MRAP2) modulates trafficking, ligand binding, and signaling of mammalian melanocortin receptors. However, the effect of MRAP2 on fish MC1R has not been extensively studied. Herein, we cloned the orange-spotted grouper (Epinephelus coioides) mc1r, which had a 972 bp open reading frame encoding a putative protein of 323 amino acids. Grouper mc1r was mainly expressed in the brain, skin, testis, spleen, head kidney, and kidney. EcoMC1R showed high constitutive activities in both Gs-cAMP and ERK1/2 pathways, which could be differentially modulated by grouper MRAP2 (EcoMRAP2). Three agonists, including α-melanocyte-stimulating hormone (MSH), ß-MSH, and ACTH, could bind to EcoMC1R and dose-dependently increase intracellular cAMP production. EcoMRAP2 had no effect on the IC50 in binding assay or EC50 in cAMP assay; however, it dose-dependently decreased the cell surface expression and maximal response to the three agonists. EcoMRAP2 increased basal ERK1/2 activation but did not alter α-MSH-stimulated ERK1/2 activation. This study extends the knowledge base of fish MC1R pharmacology and its regulation by MRAP2.

Orange-spotted grouper melanocortin-4 receptor: Modulation of signaling by MRAP2.

Melanocortin-4 receptor (MC4R) and melanocortin receptor accessory protein 2 (MRAP2) play important roles in the melanocortin system, and interaction of MC4R and MRAP2 is suggested to play pivotal role in energy balance of vertebrates. Orange-spotted grouper (Epinephelus coioides) is a widely cultured marine fish with high economic value in Asia. To explore potential interaction between grouper MC4R and MRAP2, herein we cloned grouper mc4r and mrap2. Grouper mc4r consisted of a 981 bp ORF encoding a putative protein of 327 amino acids, while the grouper mrap2 consisted of a 696 bp ORF encoding a putative protein of 232 amino acids. Sequence and phylogenetic analysis revealed that the grouper MC4R and MRAP2 were highly homologous at amino acid levels to several teleost MC4Rs and MRAP2s, respectively. qRT-PCR results showed that both mc4r and mrap2 were expressed primarily in the central nervous system. In the periphery, these genes were expressed more widely in male fish. The cloned grouper MC4R was functional, exhibiting high constitutive activity in cAMP pathway, capable of binding to three peptide agonists and increasing intracellular cAMP production dose-dependently. MRAP2 significantly decreased basal and agonist-stimulated cAMP signaling. MRAP2 also increased basal ERK1/2 activation but decreased ligand-induced stimulation when expressed at high levels. These data will facilitate future investigation of these molecules in regulating diverse physiological processes in orange-spotted grouper.

Characterization of channel catfish (Ictalurus punctatus) melanocortin-3 receptor reveals a potential network in regulation of energy homeostasis.

The melanocortin-3 receptor (MC3R) is known to be involved in regulation of energy homeostasis, regulating feed efficiency and nutrient partitioning in mammals. Its physiological roles in non-mammalian vertebrates, especially economically important aquaculture species, are not well understood. Channel catfish (Ictalurus punctatus) is the main freshwater aquaculture species in North America. In this study, we characterized the channel catfish MC3R. The mc3r of channel catfish encoded a putative protein (ipMC3R) of 367 amino acids. We transfected HEK293T cells with ipMC3R plasmid for functional studies. Five agonists, including adrenocorticotropin, α-melanocyte stimulating hormone (α-MSH), ß-MSH, [Nle4, D-Phe7]-α-MSH, and D-Trp8-γ-MSH, were used in the pharmacological studies. Our results showed that ipMC3R bound ß-MSH with higher affinity and D-Trp8-γ-MSH with lower affinity compared with human MC3R. All agonists could stimulate ipMC3R and increase intracellular cAMP production with sub-nanomolar potencies. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation could also be triggered by ipMC3R. The ipMC3R exhibited constitutive activities in both cAMP and ERK1/2 pathways, and Agouti-related protein served as an inverse agonist at ipMC3R, potently inhibiting the high basal cAMP level. Moreover, we showed that melanocortin receptor accessory protein 2 (MRAP2) preferentially modulated ipMC3R in cAMP production rather than ERK1/2 activation. Our study will assist further investigation of the physiological roles of the ipMC3R, especially in energy homeostasis, in channel catfish.

Mutations in GPR101 as a potential cause of X-linked acrogigantism and acromegaly.

X-linked acrogigantism (XLAG) is a recently described early-onset gigantism due to GPR101 duplication that induces growth hormone (GH) oversecretion. GPR101, which belongs to Family A rhodopsin-like family of G protein-coupled receptors, is predominantly expressed in hypothalamus and pituitary, suggesting that GPR101 might be important in regulating diverse functions such as energy balance and reproduction. Most mammalian GPR101s have extremely long third intracellular loops (ICL3); however, zebrafish GPR101 has a much shorter ICL3, but a longer C-terminus. GnRH-(1-5), a GnRH metabolite, can modulate the hypothalamus-pituitary-gonad axis and cancer cell migration via activating GPR101. GPR101 couples to both Gαs and Gαi proteins. GPR101 duplication has a causative role in XLAG, while GPR101 variants, especially c.924G>C (E308D), located at ICL3, are attributed to acromegaly. Some GPR101 mutations that are associated with a small proportion of pituitary tumors without GH oversecretion have also been identified recently. This chapter will summarize studies on GPR101, including its molecular cloning and tissue distribution, physiology, pharmacology, and pathophysiology.

Pharmacology of the giant panda (Ailuropoda melanoleuca) melanocortin-3 receptor.

The melanocortin-3 receptor (MC3R) is a member of the G protein-coupled receptor superfamily that plays a critical role in controlling energy balance and metabolism. Although pharmacological characterization of MC3R has been reported previously in several other species, there is no report on the MC3R from giant panda (Ailuropoda melanoleuca). This ancient species is known as a 'living fossil' and is among the most endangered animals in the world. Giant panda survive on a specialized diet of bamboo despite possessing a typical carnivorous digestive system. We report herein the molecular cloning and pharmacological characterization of amMC3R. Homology and phylogenetic analysis showed that amMC3R was highly homologous (>85%) to several other mammalian MC3Rs. Using human MC3R (hMC3R) as a control, the binding of five agonists, [Nle4, D-Phe7]-α-melanocyte stimulating hormone (NDP-MSH), α-, ß-, γ-, and D-Trp8-γ-MSH, was investigated, as well as Gs-cAMP and pERK1/2 signaling. The results showed that amMC3R bound NDP- and D-Trp8-γ-MSH with the highest affinity, followed by α-, ß-, and γ-MSH, with the same rank order as hMC3R. When stimulated with agonists, amMC3R displayed increased intracellular cAMP and activation of pERK1/2. These data suggest that the cloned amMC3R was a functional receptor. The availability of amMC3R and knowledge of its pharmacological functions will assist further investigation of its role in controlling energy balance and metabolism.

Regulation of prostate cancer by hormone-responsive G protein-coupled receptors.

Regulation of prostate cancer by androgen and androgen receptor (AR), and blockade of AR signaling by AR antagonists and steroidogenic enzyme inhibitors have been extensively studied. G protein-coupled receptors (GPCRs) are a family of membrane receptors that regulate almost all physiological processes. Nearly 40% of FDA-approved drugs in the market target GPCRs. A variety of GPCRs that mediate reproductive function have been demonstrated to be involved in the regulation of prostate cancer. These GPCRs include gonadotropin-releasing hormone receptor, luteinizing hormone receptor, follicle-stimulating hormone receptor, relaxin receptor, ghrelin receptor, and kisspeptin receptor. We highlight here GPCR regulation of prostate cancer by these GPCRs. Further therapeutic approaches targeting these GPCRs for the treatment of prostate cancer are summarized.

Expression of estrogen receptors in female rainbow trout (Oncorhynchus mykiss) during first ovarian development and under dense rearing condition.

To study the expression of four estrogen receptor genes (erα1, erα2, erß1, erß2) of female rainbow trout (Oncorhynchus mykiss) during first ovarian development, trouts were sampled from different ovarian stages. Serum E2 (estradiol) was measured by ELISA and estrogen receptors mRNA expression were examined by qRT-PCR. Our results showed a close association between increased erα1 and vitellogenin mRNA expression during ovarian maturation and increased erα2 mRNA expression in mature ovarian stages. Correlation analysis revealed that a negative relationship between serum E2 and ovarian erß1 (or hepatic erß2), but ovarian erß2 mRNA expression was relatively unchanged during first ovarian development. Trout were also reared in different densities as stocking density 1, 2 and 3 (SD1, 4.6-31.1 kg/m3; SD2, 6.6-40.6 kg/m3; SD3, 8.6-49.3 kg/m3) to elucidate effects of high density on estrogen receptor expression. Histology observation showed ovarian development of trout in higher densities were retard with a relatively early stage and fewer vitellogenin accumulation. Trout in high densities showed significantly decreased serum E2, erα mRNA expression and increasing trends of erß mRNA expression. A noticeable increase of ovarian erß2 mRNA expression was seen in trout when density is approaching to 50 kg/m3. In conclusion, we may hypothesize that increased erß mRNA expression triggered by high density result in decreased erα mRNA expression and vitellogenesis. As a result, ovarian development in higher densities was retard.

Effects of melanocortin-4 receptor agonists and antagonists on expression of genes related to reproduction in spotted scat, Scatophagus argus.

Melanocortin-4 receptor (Mc4r) function related to reproduction in fish has not been extensively investigated. Here, we report on gene expression changes by real-time PCR following treatment with Mc4r agonists and antagonists in the spotted scat (Scatophagus argus). Using in vitro incubated hypothalamus, the Mc4r nonselective agonist NDP-MSH ([Nle4, D-Phe7]-α-melanocyte stimulating hormone; 10-6 M) and selective agonist THIQ (N-[(3R)-1, 2, 3, 4-Tetrahydroisoquinolinium-3-ylcarbonyl]- (1R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl) piperidin-1-yl]-2-oxoethylamine; 10-7 M) significantly increased the expression of gnrh (Gonadotropin releasing hormone), while the Mc4r nonselective antagonist SHU9119 (Ac-Nle-[Asp-His-DPhe/DNal(2')-Arg-Trp-Lys]-NH2; 10-6 M) and selective antagonist Ipsen 5i (compound 5i synthesized in Ipsen Research Laboratories; 10-6 M) significantly inhibited gnrh expression after 3 h of incubation. In incubated pituitary tissue, NDP-MSH and THIQ significantly increased the expression of fshb (Follicle-stimulating hormone beta subunit) and lhb (Luteinizing hormone beta subunit), while SHU9119 and Ipsen 5i significantly decreased fshb and lhb expression after 3 h of incubation. During the in vivo experiment, THIQ (1 mg/kg bw) significantly increased gnrh expression in hypothalamic tissue, as well as the fshb and lhb expression in pituitary tissue 12 h after abdominal injection. Furthermore, Ipsen 5i (1 mg/kg bw) significantly inhibited gnrh expression in hypothalamic tissue, as well as fshb and lhb gene expression in pituitary tissue 12 h after abdominal injection. In summary, Mc4r singling appears to stimulate gnrh expression in the hypothalamus, thereby modulating the synthesis of Fsh and Lh in the pituitary. In addition, Mc4r also appears to directly regulate fshb and lhb levels in the pituitary in spotted scat. Our study suggests that Mc4r, through the hypothalamus and pituitary, participates in reproductive regulation in fish.

Hypothalamus-pituitary-gonad axis of rainbow trout (Oncorhynchus mykiss) during early ovarian development and under dense rearing condition.

The objective of this study was to determine the hypothalamus-pituitary-gonad (HPG) axis of female rainbow trout (Oncorhynchus mykiss) during early ovarian development and under high rearing density. Trouts were sampled from 240 (ovarian stage II) to 540 (ovarian stage IV) days following hatching (DFH) as control group (Ctrl, 4.6-31.1kg/m(3)) to determine HPG axis during early ovarian development. Trouts from the same batch of fertilized eggs were reared in two higher densities during 240-540 DFH as stocking density 1 and 2 (SD1, 6.6-40.6kg/m(3); SD2, 8.6-49.3kg/m(3)) to elucidate effects of high density on reproductive parameters. Dopamine, E2 (estradiol), 17α,20ß-P (17α,20ß-dihydroxy4-pregnen-3-one) and P4 (progesterone) were evaluated by radioimmunoassay or ELISA. mRNA expression of hypothalamic gnrh-1, -2 (gonadotropin-releasing hormone-1, -2), pituitary gonadotropins (fsh/lh, follicle-stimulating hormone/luteinizing hormone) and their cognate receptors (fshr/lhr) in ovaries were examined by qRT-PCR. Our findings demonstrated mRNA expression of hypothalamic sgnrh-1, pituitary fsh and ovarian fshr increased in early ovarian development (360 DFH). Serum 17α,20ß-P and pituitary lh mRNA expression first increased when trouts were in ovarian stage III (420 DFH). Ovaries were at different stages when reared in different densities. Long-term high density treatment (over 31.7kg/m(3)) resulted in decreased hypothalamic sgnrh-1, pituitary fsh, ovarian fshr, serum E2, and increased hypothalamus gnrh-2 and serum dopamine during vitellogenin synthesis, suggesting HPG of rainbow trout might be retarded under dense rearing condition.

Biased signaling initiated by agouti-related peptide through human melanocortin-3 and -4 receptors.

The neural melanocortin receptors (MCRs), melanocortin-3 and -4 receptors (MC3R and MC4R), have been increasingly recognized as important regulators of energy homeostasis. The orexigenic agouti-related peptide (AgRP), initially identified as an endogenous antagonist for both neural MCRs, has been suggested to be a biased agonist of MC4R independent of its antagonizing effects. In the present study, we sought to determine the potential of AgRP to regulate the activation of intracellular kinases, including extracellular signal-regulated kinase 1 and 2 (ERK1/2), AKT and AMP-activated protein kinase (AMPK), through neural MCRs. We showed that AgRP acted as a biased agonist in human MC3R (hMC3R), decreasing cAMP activity of constitutively active mutant (F347A) hMC3R but stimulating ERK1/2 activation in both wide type and F347A hMC3Rs. AgRP-stimulated ERK1/2 phosphorylation through MC3R was abolished by protein kinase A (PKA) inhibitor H-89 but not Rp-cAMPS, whereas AgRP-initiated ERK1/2 activation through MC4R was inhibited by phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. Both NDP-MSH and AgRP treatment induced significant AKT phosphorylation in GT1-7 cells but not in MC3R- or MC4R-transfected HEK293T cells. The phosphorylated AMPK levels in both GT1-7 cells and HERK293T cells transfected with neural MCRs were significantly decreased upon stimulation with NDP-MSH but not with AgRP. In summary, we provided novel data for AgRP-initiated multiple intracellular signaling pathways, demonstrating biased agonism of AgRP in both neural MCRs, leading to a better understanding of neural MCR pharmacology.

Functions of DPLIY motif and helix 8 of human melanocortin-3 receptor.

The melanocortin-3 receptor (MC3R) is a member of the family A G protein-coupled receptors (GPCRs). The MC3R remains the most enigmatic of the melanocortin receptors with regard to its physiological functions, especially its role in energy homeostasis. The N/DPxxY motif and the eighth helix (helix 8) in the carboxyl terminus of GPCRs have been identified to be important for receptor expression, ligand binding, signal transduction and internalization. To gain a better understanding of the structure-function relationship of MC3R, we performed a systematic study of all 20 residues in this domain using alanine-scanning mutagenesis. We showed that although all mutants were expressed normally on the cell surface, eleven residues were important for ligand binding and one was indispensable for downstream cAMP generation. F347A showed constitutive activity in cAMP signaling while all the other mutants had normal basal activities. We studied the signaling capacity of nine mutants in the ERK1/2 signaling pathway. All of these mutants showed normal basal ERK1/2 phosphorylation levels. The pERK1/2 levels of six binding- or signaling-defective mutants were enhanced upon agonist stimulation. The unbalanced cAMP and pERK1/2 signaling pathways suggested the existence of biased signaling in MC3R mutants. In summary, we showed that the DPLIY motif and helix 8 was important for MC3R activation and signal transduction. Our data led to a better understanding of the structure-function relationship of MC3R.

Therapeutic potential of sustained-release sodium nitrite for critical limb ischemia in the setting of metabolic syndrome.

Nitrite is a storage reservoir of nitric oxide that is readily reduced to nitric oxide under pathological conditions. Previous studies have demonstrated that nitrite levels are significantly reduced in cardiovascular disease states, including peripheral vascular disease. We investigated the cytoprotective and proangiogenic actions of a novel, sustained-release formulation of nitrite (SR-nitrite) in a clinically relevant in vivo swine model of critical limb ischemia (CLI) involving central obesity and metabolic syndrome. CLI was induced in obese Ossabaw swine (n = 18) by unilateral external iliac artery deployment of a full cross-sectional vessel occlusion device positioned within an endovascular expanded polytetrafluoroethylene-lined nitinol stent-graft. At post-CLI day 14, pigs were randomized to placebo (n = 9) or SR-nitrite (80 mg, n = 9) twice daily by mouth for 21 days. SR-nitrite therapy increased nitrite, nitrate, and S-nitrosothiol in plasma and ischemic skeletal muscle. Oxidative stress was reduced in ischemic limb tissue of SR-nitrite- compared with placebo-treated pigs. Ischemic limb tissue levels of proangiogenic growth factors were increased following SR-nitrite therapy compared with placebo. Despite the increases in cytoprotective and angiogenic signals with SR-nitrite therapy, new arterial vessel formation and enhancement of blood flow to the ischemic limb were not different from placebo. Our data clearly demonstrate cytoprotective and proangiogenic signaling in ischemic tissues following SR-nitrite therapy in a very severe model of CLI. Further studies evaluating longer-duration nitrite therapy and/or additional nitrite dosing strategies are warranted to more fully evaluate the therapeutic potential of nitrite therapy in peripheral vascular disease.