Orexigenic action of peripheral ghrelin is mediated by neuropeptide Y and agouti-related protein.

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.

Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass.

Genetic and pharmacological studies have defined a role for the melanocortin-4 receptor (Mc4r) in the regulation of energy homeostasis. The physiological function of Mc3r, a melanocortin receptor expressed at high levels in the hypothalamus, has remained unknown. We evaluated the potential role of Mc3r in energy homeostasis by studying Mc3r-deficient (Mc3r(-/-)) mice and compared the functions of Mc3r and Mc4r in mice deficient for both genes. The 4-6-month Mc3r-/- mice have increased fat mass, reduced lean mass and higher feed efficiency than wild-type littermates, despite being hypophagic and maintaining normal metabolic rates. (Feed efficiency is the ratio of weight gain to food intake.) Consistent with increased fat mass, Mc3r(-/-) mice are hyperleptinaemic and male Mc3r(-/-) mice develop mild hyperinsulinaemia. Mc3r(-/-) mice did not have significantly altered corticosterone or total thyroxine (T4) levels. Mice lacking both Mc3r and Mc4r become significantly heavier than Mc4r(-/-) mice. We conclude that Mc3r and Mc4r serve non-redundant roles in the regulation of energy homeostasis.

Role of the melanocortin-4 receptor in metabolic rate and food intake in mice.

We evaluated the role of the melanocortin-4 receptor (MC-4R) in the control of metabolic rate and food intake in mice. Intraperitoneal administration of the non-selective MC-R agonist melanotan II (MT-II; a cyclic heptapeptide) increases metabolic rate in wildtype mice, while MC-4R knockout mice are insensitive to the effects of MT-II on metabolic rate. MC-4R knockout mice are also insensitive to the effects of MT-II on reducing food intake. We conclude that MC-4R can mediate control of both metabolic rate and food intake in mice. We infer that a role for MC-3R in mediating the acute effects of MT-II on basal metabolic rate and food intake in wildtype mice seems limited.

Increases in circulating insulin-like growth factor I levels by the oral growth hormone secretagogue MK-0677 in the beagle are dependent upon pituitary mediation.

It has been well established that the spiroindoline sulfonamide MK-0677 stimulates GH secretion from the pituitary both in vitro and in vivo. MK-0677 has also been shown to increase serum insulin-like growth factor I (IGF-I) and cortisol levels in vivo; these increases are assumed to be driven by the increased serum GH and ACTH levels, respectively. However, such increases could also be due to a direct stimulatory action of MK-0677 at the level of the liver and adrenal cortex. To address this possibility, we investigated whether MK-0677 increased IGF-I and cortisol levels in hypophysectomized dogs. Baseline GH, IGF-I, and cortisol responses to MK-0677 (1 mg/kg, orally) were initially determined. Hypophysectomy (hypox; n = 7) or sham surgery (sham; n = 5) was then carried out. Six days postsurgery, the GH and cortisol responses to MK-0677 were reevaluated in each dog. In addition, each dog was treated with porcine GH (PST; 0.1 IU/kg, s.c.) to confirm the responsiveness of the GH-IGF-I axis. The mean peak GH increases in response to MK-0677 in the presham dogs (83.7 +/- 19.2 ng/ml), post-sham dogs (108 +/- 26.2 ng/ml), and pre-hypox dogs (121.2 +/- 13.6 ng/ml) were not significantly different. Mean peak GH levels were unchanged after MK-0677 administration in the hypox dogs (2.3 +/- 0.7 ng/ml). Before surgery, serum IGF-I levels increased to 243 +/- 27 and 224 +/- 47 ng/ml in the sham and hypox groups, respectively, after MK-0677 administration. Surgery was associated with a marked (> or =50%) decrease in serum IGF-I levels. MK-0677 administration increased IGF-I levels in the sham dogs from 78 +/- 14 to 187 +/- 31 ng/ml, whereas IGF-I levels remained unchanged (17.7 +/- 2.4 ng/ml) in the-hypox dogs. PST treatment increased IGF-I levels in the sham dogs from 162 +/- 30 to 325 +/- 32 ng/ml. In the hypox dogs PST treatment restored IGF-I to physiological levels (from 17.7 +/- 2.4 to 199 +/- 41 ng/ml). Cortisol was increased after MK-0677 administration 3.7-fold in the pre-sham, 3.6-fold in the post-sham, and 3.6-fold in the pre-hypox dogs, but no increase was seen in the post-hypox dogs. ACTH GEL administration (2.2 U/kg, i.m.) to hypox dogs returned cortisol to normal physiological levels, demonstrating the functional integrity of the adrenal cortex. This study demonstrates that the GH secretagogue MK-0677 does not directly stimulate an increase in serum IGF-I or cortisol levels, but depends upon the presence of an intact pituitary.

Ophiobolin M and analogues, noncompetitive inhibitors of ivermectin binding with nematocidal activity.

A series of ophiobolins were isolated from a fungal extract based on their nematocidal activity. These compounds are non-competitive inhibitors of ivermectin binding to membranes prepared from the free-living nematode, Caenorhabditis elegans, with an inhibition constant of 15 microM. The ophiobolins which were most potent in the biological assays, ophiobolin C and ophiobolin M, were also the most potent compounds when evaluated in a C. elegans motility assay. These data suggest that the nematocidal activity of the ophiobolins is mediated via an interaction with the ivermectin binding site. The isolation, structure and biological activity of ophiobolins have been described.

The mechanism of action of avermectins in Caenorhabditis elegans: correlation between activation of glutamate-sensitive chloride current, membrane binding, and biological activity.

Xenopus laevis oocytes were injected with mRNA isolated from the free-living nematode Caenorhabditis elegans and the activation and potentiation of a glutamate-sensitive chloride current by a series of avermectin analogs and milbemycin D were determined. There was a strong correlation between the EC50 value determined for current activation in oocytes, the LD95 value for nematocidal activity, and also for the Ki value determined in a [3H]ivermectin competition binding assay. Four of the analogs were tested for potentiation of glutamate-sensitive current and the rank order for potentiation correlated with the EC50 for direct activation of current. We conclude that avermectins and milbemycins mediate their nematocidal effects on C. elegans via an interaction with a common receptor molecule, glutamate-gated chloride channels.

Nematocidal activity of MK-801 analogs and related drugs. Structure-activity relationships.

A series of dibenzo[a,d]cycloalkenimines were evaluated for their affinity to the (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) binding site in Caenorhabditis elegans membranes and their nematocidal activity. The (+)-MK-801 enantiomer (1) had a higher affinity (Kd = 240 nM) for its specific binding site and was a more potent nematocidal agent than the (-)-MK-801 enantiomer (-1). Ring expansion to form the dibenzo[a,d]cyclooctenimine analogs generally resulted in more potent compounds. The most potent of this series (23) was approximately 7-fold more potent than (+)-MK-801. A good correlation was established between binding affinities and nematocidal activity for all of the analogs that were tested. However, there was no correlation between binding to C. elegans membranes and affinity for mammalian MK-801 binding sites. Other noncompetitive inhibitors of the mammalian N-methyl-D-aspartate site were examined, and a series of diphenylguanidines were identified as potent competitive inhibitors of MK-801 binding to C. elegans membranes, in addition to displaying potent nematocidal activity. The most potent diphenylguanidine analog (24) was approximately 80-fold more potent than (+)-MK-801 in both its affinity for the MK-801 binding site and as a nematocidal agent. Molecular modeling studies support the hypothesis that the diphenylguanidines and MK-801 are binding to the same site and suggest that more potent compounds may be developed by effective modeling of the existing compounds.

Novel nematocidal agents from Curcuma comosa.

Curcuma comosa is a member of the economically important plant family, Zingiberaceae. A methanolic extract of C. comosa was shown to be nematocidal when tested against the free-living nematode Caenorhabditis elegans. Five diphenylheptanoids [1-5], one new and four known, have been isolated and shown to be responsible for the activity. This is the first report of three of these compounds [1, 2, 4] being isolated from a natural source.

Cochlioquinone A, a nematocidal agent which competes for specific [3H]ivermectin binding sites.

Cochlioquinone A, isolated from the fungus Helminthosporium sativum, was found to have nematocidal activity. Cochlioquinone A is a competitive inhibitor of specific [3H]ivermectin binding suggesting that cochlioquinone A and ivermectin interact with the same membrane receptor.

Lack of cross-resistance between efrotomycin and antibacterial agents used in the therapy of human and animal infections.

Efrotomycin is an N-methylhydroxypyridone glycoside antibiotic with activity primarily against Gram-positive bacteria. It is intended for use as a feed additive for swine. Although efrotomycin is unrelated to any antibacterial drug used in human or veterinary medicine, the possibility of cross-resistance with other antibacterials is of concern. The minimum inhibitory concentrations (MICs) of efrotomycin were determined for a broad panel of bacterial isolates. In addition, the susceptibility of each isolate to 12-15 antibacterials was determined using a standardized disk susceptibility test. No evidence of cross-resistance between efrotomycin and any of the 12-15 antibacterial compounds was observed. When the MIC of efrotomycin for nine selected isolates was increased from 16- to greater than 100-fold by serial passage in subinhibitory concentrations of efrotomycin, no increased resistance to the 15 antibacterials was noted. Subinhibitory concentrations of efrotomycin had no effect on the conjugative transfer of antibacterial-resistance plasmids between K-12 strains of Escherichia coli. The data from this study suggest that if resistance to efrotomycin should occur, it is unlikely to result in the appearance of multiply-resistant bacterial populations.