Nigel Levens

Acute and chronic administration of melanin-concentrating hormone enhances food intake and body weight in Wistar and Sprague-Dawley rats

AIM: Although melanin-concentrating hormone (MCH) is believed to be an important regulator of feeding behavior, both its acute and chronic effects on food intake as well as its interaction with other brain peptides involved in the control of appetite remain unclear. Therefore, the acute effects of MCH on food intake and the chronic effect of MCH on food intake and the gene expression of various hypothalamic peptides involved in the control of appetite were studied in rats.METHODS AND RESULTS: Either the acute or the continuous intraventricular infusion of MCH for 12 days stimulated feeding in both Wistar or Sprague–Dawley rats. Removal of the hypothalamus at the end of the chronic infusion studies allowed measurement of the expression of mRNAs encoding for MCH, neuropeptide Y (NPY), orexin, agouti gene-related peptide, cocaine and amphetamine-related transcript and neurotensin–neuropeptides involved in the control of appetite. Chronic intraventricular infusion of MCH activated only NPY mRNA synthesis in Sprague–Dawley rats. The increase in food intake in response to MCH in Sprague–Dawley rats did not appear to be due to the release of NPY since combination studies demonstrated consistently additive effects of the two peptides on food intake at maximum or near maximum doses.CONCLUSIONS: These results strongly suggest that MCH is an orexigenic peptide involved in the control of both short- and long term food intake in satiated rats and further indicate that the MCH pathway is a possible target for the control of food intake and obesity.

Structure-activity relationship studies of melanin-concentrating hormone (MCH)-related peptide ligands at SLC-1, the human MCH receptor.

Melanin-concentrating hormone (MCH) is a cyclic nonadecapeptide involved in the regulation of feeding behavior, which acts through a G protein-coupled receptor (SLC-1) inhibiting adenylcyclase activity. In this study, 57 analogues of MCH were investigated on the recently cloned human MCH receptor stably expressed in HEK293 cells, on both the inhibition of forskolin-stimulated cAMP production and guanosine-5′-O-(3-[35S]thiotriphosphate ([35S]- GTPγS) binding. The dodecapeptide MCH-(6–17) (MCH ring between Cys7 and Cys16, with a single extra amino acid at the N terminus (Arg6) and at the C terminus (Trp17)) was found to be the minimal sequence required for a full and potent agonistic response on cAMP formation and [35S]- GTPγS binding. We Ala-scanned this dodecapeptide and found that only 3 of 8 amino acids of the ring, namely Met8, Arg11, and Tyr13, were essential to elicit full and potent responses in both tests. Deletions inside the ring led either to inactivity or to poor antagonists with potencies in the micromolar range. Cys7 and Cys16 were substituted by Asp and Lys or one of their analogues, in an attempt to replace the disulfide bridge by an amide bond. However, those modifications were deleterious for agonistic activity. In [35S]- GTPγS binding, these compounds behaved as weak antagonists (K B 1–4 μm). Finally, substitution in MCH-(6–17) of 6 out of 12 amino acids by non-natural residues and concomitant replacement of the disulfide bond by an amide bond led to three compounds with potent antagonistic properties (K B = 0.1–0.2 μm). Exploitation of these structure-activity relationships should open the way to the design of short and stable MCH peptide antagonists.

Reduced food intake in response to CGP 71683A may be due to mechanisms other than NPY Y5 receptor blockade

INTRODUCTION: The purpose of this study was to test the continuing validity of the hypothesis that neuropeptide Y (NPY) produced in the brain controls food intake through an interaction with the NPY Y5 receptor subtype.METHODS: The hypothesis was tested using CGP 71683A a potent and highly selective non-peptide antagonist of the NPY Y5 receptor which was administered into the right lateral ventricle of obese Zucker fa/fa rats.RESULTS: Intraventricular injection of 3.4 nmol/kg NPY increased food intake during a 2 h test period. Doses of CGP 71683A in excess of 15 nmol/kg (i.cv.) resulted in blockade of the increase in food intake produced by NPY. Repeated daily injection of CGP 71683A (30–300 nmol/kg, i.cv.) immediately before the dark phase produced a dose-dependent and slowly developing decrease in food intake. CGP 71683A has a low affinity for NPY Y1, Y2 and Y4 receptors but a very high affinity for the NPY Y5 receptor (Ki, 1.4 nM). Surprisingly, CGP 71683A had similarly high affinity for muscarinic receptors (Ki, 2.7 nM) and for the serotonin uptake recognition site (Ki, 6.2 nM) in rat brain. Anatomic analysis of the brain after treatment with CGP 71683A demonstrated an inflammatory response associated with the fall in food intake.CONCLUSIONS: While the fall in food intake in response to CGP 71683A may have a Y5 component, interactions with other receptors or inflammatory mediators may also play a role. It is concluded that CGP 71683A is an imprecise tool for investigating the role of the NPY Y5 receptor in the control of physiological processes including food intake.

Appetite suppression based on selective inhibition of NPY receptors.

AIM: The aim of this review is to critically assess available evidence that blockade of the actions of NPY at one of the five NPY receptor subtypes represents an attractive new drug discovery target for the development of an appetite suppressant drug.RESULTS: Blockade of the central actions of NPY using anti-NPY antibodies, antisense oligodeoxynucleotides against NPY and NPY receptor antagonists results in a decrease in food intake in energy-deprived animals. These results appear to show that endogenous NPY plays a role in the control of appetite. The fact that NPY receptors exist as at least five different subtypes raises the possibility that the actions of endogenous NPY on food intake can be adequately dissociated from other effects of the peptide. Current drug discovery has produced a number of highly selective NPY receptor antagonists which have been used to establish the NPY Y1 receptor subtype as the most critical in regulating short-term food intake. However, additional studies are now needed to more clearly define the relative contribution of NPY acting through the NPY Y2 and NPY Y5 receptors in the complex sequence of physiological and behavioral events that underlie the long-term control of appetite.CONCLUSIONS: Blockade of the NPY receptor may produce appetite-suppressing drugs. However, it is too early to state with certainty whether a single subtype selective drug used alone or a combination of NPY receptor selective antagonists used in combination will be necessary to adequately influence appetite regulation.

NPY Receptors as Drug Targets for the Central Regulation of Body Weight

Neuropeptide Y (NPY) is present in the hypothalamus, where it is believed to play a key role in the control of food intake. Evidence for this assertion has come from studies demonstrating that acute administration of NPY into the hypothalamus or into the brain ventricles leads to increased food intake. In the case of chronic administration, the hyperphagic effects of NPY are prolonged leading to the development of an obese state. NPY levels in the hypothalamus are temporally correlated with food intake and are markedly elevated in response to energy depletion. However, attempts to demonstrate an important role for NPY in the control of food intake using NPY knockout mice, NPY antisense oligodeoxynucleotides and anti-NPY antibodies has produced equivocal results. Despite this many pharmaceutical companies have moved ahead with the search for agonists and antagonists of NPY receptor subtypes as anti-obesity agents. Antagonists of the NPY Y(1) and NPY Y(5) receptor subtype initially looked promising since analogs of NPY with high selectivity for these receptors strongly stimulated food intake. However, attempts to inhibit the signaling of NPY through the NPY Y(1) and NPY Y(5) receptors has produced equivocal effects on food intake. Recent observations that the gut derived peptide PYY(3-36) suppresses appetite by stimulating both peripherally and centrally located NPY Y(2) receptors remain controversial in animals but the effects look promising in human studies. Whether this will be the long awaited therapy based on manipulation of NPY receptors will await further studies of long term efficacy and more importantly a favorable side effect profile.

Endothelin‐1 inhibits TNFα‐induced iNOS expression in 3T3‐F442A adipocytes

Endothelin‐1 (ET‐1) and tumor necrosis factor α (TNFα) by their action on adipocytes have been independently linked to the pathogenesis of insulino‐resistance. In isolated adipocytes, TNFα induces the expression of the inducible nitric oxide synthase (iNOS). The purpose of the present work was, in the 3T3‐F442A adipocyte cell line, to characterise TNFα‐induced iNOS expression and to determine whether or not ET‐1 could influence TNFα‐induced iNOS expression and NO production. In differentiated 3T3‐F442A, treatment with TNFα (20 ng ml−1) induced the expression of a functional iNOS as demonstrated by nitrite assay, Western blot, reverse transcription–polymerase chain reaction and Northern blot analysis. TNFα‐induced iNOS expression requires nuclear factor κB activation, but does not necessitate the activation of the PI‐3 kinase/Akt and P38–MAP kinase pathways. ET‐1, but not ET‐3, inhibited the TNFα‐induced expression of iNOS protein and mRNA as well as nitrite production. The effects of ET‐1 were blocked by a specific ETA (BQ123, pA2 7.4) but not by a specific ETB receptor antagonist (BQ788). 3T3‐F442A adipocytes express the mRNAs for prepro‐ET‐1 and the ET‐A receptor subtype, but not for the ET‐B subtype. The inhibitory effect of ET‐1 was not affected by bisindolylmaleimide, SB 203580 or indomethacin, inhibitors of protein kinase C, p38‐MAP kinase and cyclooxygenase, respectively, and was not associated with cAMP production. However, the effect of ET‐1 was partially reversed by wortmannin, suggesting the involvement of PI3 kinase in the transduction signal of ET‐1. Differentiated 3T3‐F442A adipocytes did not release ET‐1 with or without exposure to TNFα, although the mRNA for preproET‐1 was detected in both pre‐ and differentiated adipocytes. Thus, these results confirm that adipocytes are a target for circulating ET‐1 and demonstrate that the activation of the ETA receptor subtype can prevent TNFα‐induced iNOS expression.