Ghrelin, a stomach-derived orexigenic hormone, has stimulated great interest as a potential target for obesity control. Pharmacological evidence indicates that ghrelin’s effects on food intake are ...mediated by neuropeptide Y (NPY) and agouti-related protein (AgRP) in the central nervous system. These include intracerebroventricular application of antibodies to neutralize NPY and AgRP, and the application of an NPY Y1 receptor antagonist, which blocks some of the orexigenic effects of ghrelin. Here we describe treatment of Agrp−/−;Npy−/− and Mc3r−/−;Mc4r−/− double knockout mice as well as Npy−/− and Agrp−/− single knockout mice with either ghrelin or an orally active nonpeptide ghrelin agonist. The data demonstrate that NPY and AgRP are required for the orexigenic effects of ghrelin, as well as the involvement of the melanocortin pathway in ghrelin signaling. Our results outline a functional interaction between the NPY and AgRP pathways. Although deletion of either NPY or AgRP caused only a modest or nondetectable effect, ablation of both ligands completely abolished the orexigenic action of ghrelin. Our results establish an in vivo orexigenic function for NPY and AgRP, mediating the effect of ghrelin.
The recently discovered growth hormone secretagogue, ghrelin, is a potent agonist at the human growth hormone secretagogue receptor 1a (hGHSR1a). To elucidate structural features of this peptide ...necessary for efficient binding to and activation of the receptor, several analogues of ghrelin with various aliphatic or aromatic groups in the side chain of residue 3, and several short peptides derived from ghrelin, were prepared and tested in a binding assay and in an assay measuring intracellular calcium elevation in HEK-293 cells expressing hGHSR1a. Bulky hydrophobic groups in the side chain of residue 3 turned out to be essential for maximum agonist activity. Also, short peptides encompassing the first 4 or 5 residues of ghrelin were found to functionally activate hGHSR1a about as efficiently as the full-length ghrelin. Thus the entire sequence of ghrelin is not necessary for activity: the Gly-Ser-Ser(n-octanoyl)-Phe segment appears to constitute the “active core” required for agonist potency at hGHSR1a.
Small synthetic molecules termed growth hormone secretagogues (GHSs) act on the pituitary gland and the hypothalamus to stimulate and amplify pulsatile growth hormone (GH) release. A heterotrimeric ...GTP-binding protein (G protein)-coupled receptor (GPC-R) of the pituitary and arcuate ventro-medial and infundibular hypothalamus of swine and humans was cloned and was shown to be the target of the GHSs. On the basis of its pharmacological and molecular characterization, this GPC-R defines a neuroendocrine pathway for the control of pulsatile GH release and supports the notion that the GHSs mimic an undiscovered hormone.
The superfamily of seven-transmembrane-domain G-protein-coupled receptors (GPCRs) is the largest and most diverse group of transmembrane proteins involved in signal transduction. Each of the ∼1000 ...family members found in vertebrates responds to stimuli as diverse as hormones, neurotransmitters, odorants and light, which selectively activate intracellular signaling events mediated by heterotrimeric G proteins. Because GPCRs are centrally positioned in the plasma membrane to initiate a cascade of cellular responses by diverse extracellular mediators, it is not surprising that modulation of GPCR function has been successful in the development of many marketed therapeutic agents. It has become clear that GPCRs for which a natural activating ligand has not yet been identified (orphan GPCRs) might provide a path to discovering new cellular substances that are important in human physiology. The process of ‘de-orphanizing’ these novel proteins has accelerated significantly and opened up new avenues for research in human physiology and pharmacology.
Neuromedin U (NMU) is a neuropeptide with potent activity on smooth muscle
which was isolated first from porcine spinal cord and later from other species. It is widely distributed in the gut and ...central
nervous system. Peripheral activities of NMU include stimulation
of smooth muscle, increase of blood pressure,
alteration of ion transport in the gut, control of local blood
flow and regulation of adrenocortical function.
An NMU receptor has not been molecularly identified. Here we show that the
previously described orphan G-protein-coupled receptor FM-3 (ref. 15) and a newly discovered one (FM-4) are cognate receptors
for NMU. FM-3, designated NMU1R, is abundantly expressed in peripheral tissues
whereas FM-4, designated NMU2R, is expressed in specific regions of the brain.
NMU is expressed in the ventromedial hypothalamus in the rat brain, and its
level is significantly reduced following fasting. Intracerebroventricular
administration of NMU markedly suppresses food intake in rats. These findings
provide a molecular basis for the biochemical activities of NMU and may indicate
that NMU is involved in the central control of feeding.
Complete 18S ribosomal RNA gene sequences were determined for 8 Eimeria species of chickens and for Eimeria bovis of cattle. Sequences were aligned with each other and with sequences from 2 ...Sarcocystis spp., Toxoplasma gondii, Neospora caninum, and 4 Cryptosporidium spp. Aligned sequences were analyzed by maximum parsimony to infer evolutionary relationships among the avian Eimeria species. Eimeria bovis was found to be the sister taxon to the 8 Eimeria species infecting chickens. Within the avian Eimeria species, E. necatrix and E. tenella were sister taxa; this clade attached basally to the other chicken coccidia. The remaining Eimeria spp. formed 3 clades that correlated with similarities based on oocyst size and shape. Eimeria mitis and Eimeria mivati (small, near spherical oocysts) formed the next most basal clade followed by a clade comprising Eimeria praecox, Eimeria maxima, and Eimeria brunetti (large, oval oocysts), which was the sister group to Eimeria acervulina (small, oval oocysts). The 4 clades of avian Eimeria species were strongly supported in a bootstrap analysis. Basal rooting of E. necatrix and E. tenella between E. bovis and the remaining Eimeria species and the apparent absence of coccidia that infect the ceca of jungle fowl all suggest that E. necatrix and E. tenella may have arisen from a host switch, perhaps from the North American turkey, Meleagris gallopavo.
Central administration of the neuropeptide melanin-concentrating hormone (MCH) stimulates feeding in rodents. We studied the effects of intracerebroventricular (i.c.v.) administration of an MCH-1 ...receptor agonist (Compound A) and an MCH-1 receptor antagonist (Compound B) on feeding in satiated rats. Compound B (10 μg, i.c.v.) blocked the acute orexigenic effect of Compound A (5 μg, i.c.v.). In an experiment designed to either stimulate or inhibit MCH-1 receptor signaling over an extended period, rats received continuous i.c.v. infusions of vehicle (saline), Compound A (30 μg/day), Compound B (30 or 48 μg/day) or neuropeptide Y (24 μg/day, as positive control) via implantable infusion pumps. Continuous MCH-1 receptor activation recapitulated the obese phenotype of MCH-over-expressor mice, manifest as enhanced feeding (+23%,
P<0.001), caloric efficiency and body weight gain (+38%,
P<0.005) over the 14-day period relative to controls. Chronic MCH-1 receptor activation also elevated plasma insulin and leptin levels significantly. Conversely, continuous MCH-1 receptor antagonism led to sustained reductions in food intake (−16%,
P<0.001), body weight gain (−35%,
P<0.01), and body fat gain relative to controls, without an effect on lean mass. Antagonism of the MCH-1 receptor may be an effective approach for the treatment of obesity.
Melanin-concentrating hormone (MCH) is a 19-aa cyclic neuropeptide originally isolated from chum salmon pituitaries. Besides its effects on the aggregation of melanophores in fish several lines of ...evidence suggest that in mammals MCH functions as a regulator of energy homeostasis. Recently, several groups reported the identification of an orphan G protein-coupled receptor as a receptor for MCH (MCH-1R). We hereby report the identification of a second human MCH receptor termed MCH-2R, which shares about 38% amino acid identity with MCH-1R. MCH-2R displayed high-affinity MCH binding, resulting in inositol phosphate turnover and release of intracellular calcium in mammalian cells. In contrast to MCH-1R, MCH-2R signaling is not sensitive to pertussis toxin and MCH-2R cannot reduce forskolin-stimulated cAMP production, suggesting an exclusive Gα qcoupling of the MCH-2R in cell-based systems. Northern blot and in situ hybridization analysis of human and monkey tissue shows that expression of MCH-2R mRNA is restricted to several regions of the brain, including the arcuate nucleus and the ventral medial hypothalamus, areas implicated in regulation of body weight. In addition, the human MCH-2R gene was mapped to the long arm of chromosome 6 at band 6q16.2-16.3, a region reported to be associated with cytogenetic abnormalities of obese patients. The characterization of a second mammalian G protein-coupled receptor for MCH potentially indicates that the control of energy homeostasis in mammals by the MCH neuropeptide system may be more complex than initially anticipated.
I. Introduction
II. Identification of Peptidomimetic GH Secretagogues
A. Mechanism of action of GHRH, GHRP-6, and somatostatin
B. In vitro assays
III. Molecular Design by Medicinal Chemistry
A. ...Benzolactams and L-692,429
B. Spiroindanes and MK-0677
C. Isonipecotic acid peptidomimetics
IV. Characterization of the MK-0677 Receptor
A. Pituitary gland
B. Hypothalamus
V. Signal Transduction Pathway
VI. Cloning the GH Secretagogue Receptor
A. Chromosomal localization
VII. Action of the Peptidomimetic GH Secretagogues in the Central
Nervous System
VIII. Peptidomimetic GH Secretagogues in Vivo
A. Animal models
B. Clinical studies in humans
IX. Regulation of Pulsatile GH Release
A. GHRH and somatostatin
B. The role of GHS-R
X. Concluding Comments
The recent cloning of a growth hormone secretagogue receptor (GHS-R) from human pituitary gland and brain identified a third G protein-coupled receptor (GPC-R) involved in the control of growth ...hormone release. The nucleotide sequence of the GHS-R is most closely related to the neurotensin receptor-1 (NT-R1) (35% overall protein identity). Two human GPC-Rs related to both the type 1a GHS-R and NT-Rs were cloned and characterized. Hybridization at low posthybridizational stringency with restriction enzyme-digested human genomic DNA resulted in the identification of a genomic clone encoding a first GHS-R/NT-R family member (GPR38). A cDNA clone was identified encoding a second GHS-R-related gene (GPR39). GPR38 and GPR39 share significant amino acid sequence identity with the GHS-R and NT-Rs 1 and 2. An acidic residue (E124) in TM-3, essential for the binding and activation of the GHS-R by structurally dissimilar GHSs, was conserved in GPR38 and GPR39. GPR38 is encoded by a single gene expressed in thyroid gland, stomach, and bone marrow. GPR39 is encoded by a highly conserved single-copy gene, expressed in brain and other peripheral tissues. Fluorescencein situhybridization localized the genes for GPR38 and GPR39 to separate chromosomes, distinct from the gene encoding the GHS-R and NT-R type 1. The ligand-binding and functional properties of GPR38 and GPR39 remain to be determined.
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