Differential modulation of gonadotropin responses to kisspeptin by aminoacidergic, peptidergic, and nitric oxide neurotransmission.

Kisspeptins (Kp), products of the Kiss1 gene, have emerged as essential elements in the control of GnRH neurons and gonadotropic secretion. However, despite considerable progress in the field, to date limited attention has been paid to elucidate the potential interactions of Kp with other neurotransmitters known to centrally regulate the gonadotropic axis. We characterize herein the impact of manipulations of key aminoacidergic (glutamate and GABA), peptidergic (NKB, Dyn, and MCH), and gaseous [nitric oxide (NO)] neurotransmission on gonadotropin responses to Kp-10 in male rats. Blockade of ionotropic glutamate receptors (of the NMDA and non-NMDA type) variably decreased LH responses to Kp-10, whereas activation of both ionotropic and metabotropic receptors, which enhanced LH and FSH release per se, failed to further increase gonadotropin responses to Kp-10. In fact, coactivation of metabotropic receptors attenuated LH and FSH responses to Kp-10. Selective activation of GABA(A) receptors decreased Kp-induced gonadotropin secretion, whereas their blockade elicited robust LH and FSH bursts and protracted responses to Kp-10 when combined with GABA(B) receptor inhibition. Blockade of Dyn signaling (at κ-opioid receptors) enhanced LH responses to Kp-10, whereas activation of Dyn and NKB signaling modestly reduced Kp-induced LH and FSH release. Finally, MCH decreased basal LH secretion and modestly reduced FSH responses to Kp-10, whereas LH responses to Kp-10 were protracted after inhibition of NO synthesis. In summary, we present herein evidence for the putative roles of glutamate, GABA, Dyn, NKB, MCH, and NO in modulating gonadotropic responses to Kp in male rats. Our pharmacological data will help to characterize the central interactions and putative hierarchy of key neuroendocrine pathways involved in the control of the gonadotropic axis.

Effects of intracerebroventricular and intra-accumbens melanin-concentrating hormone agonism on food intake and energy expenditure.

The brain melanin-concentrating hormone (MCH) system represents an anabolic system involved in energy balance regulation through influences exerted on the homeostatic and nonhomeostatic controls of food intake and energy expenditure. The present study was designed to further delineate the effect of the MCH system on energy balance regulation by assessing the actions of the MCH receptor 1 (MCHR1) agonism on both food intake and energy expenditure after intracerebroventricular (third ventricle) and intra-nucleus-accumbens-shell (intraNAcSH) injections of a MCHR1 agonist. Total energy expenditure and substrate oxidation were assessed following injections in male Wistar rats using indirect calorimetry. Food intake was also measured. Pair-fed groups were added to evaluate changes in thermogenesis that would occur regardless of the meal size and its thermogenic response. Using such experimental conditions, we were able to demonstrate that acute MCH agonism in the brain, besides its orexigenic effect, induced a noticeable change in the utilization of the main metabolic fuels. In pair-fed animals, MCH significantly reduced lipid oxidation when it was injected in the third ventricle. Such an effect was not observed following the injection of MCH in the NAcSH, where MCH nonetheless strongly stimulated appetite. The present results further delineate the influence of MCH on energy expenditure and substrate oxidation while confirming the key role of the NAcSH in the effects of the MCH system on food intake.

Cannabinoids excite hypothalamic melanin-concentrating hormone but inhibit hypocretin/orexin neurons: implications for cannabinoid actions on food intake and cognitive arousal.

Cannabinoids modulate energy homeostasis and decrease cognitive arousal, possibly by acting on hypothalamic neurons including those that synthesize melanin-concentrating hormone (MCH) or hypocretin/orexin. Using patch-clamp recordings, we compared the actions of cannabinoid agonists and antagonists on identified MCH or hypocretin neurons in green fluorescent protein-expressing transgenic mice. The cannabinoid type-1 receptor (CB1R) agonist R-(+)-[2,3-dihydro-5-methyl-3-(4-morpho linylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN55,212,2) depolarized MCH cells and increased spike frequency; in contrast, WIN55,212,2 hyperpolarized and reduced spontaneous firing of the neighboring hypocretin cells, both results consistent with reduced activity seen with intracerebral cannabinoid infusions. These effects were prevented by AM251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide], a CB1R antagonist, and by tetrodotoxin, suggesting no postsynaptic effect on either neuron type. In MCH cells, depolarizing WIN55,212,2 actions were abolished by the GABA(A) receptor antagonist bicuculline, suggesting that the CB1R-mediated depolarization was attributable to reduced synaptic GABA release. WIN55,212,2 decreased spontaneous IPSCs, reduced the frequency but not amplitude of miniature IPSCs, and reduced electrically evoked synaptic currents in MCH cells. Glutamate microdrop experiments suggest that WIN55,212,2 acted on axons arising from lateral hypothalamus local inhibitory cells that innervate MCH neurons. In hypocretin neurons, the reduced spike frequency induced by WIN55,212,2 was attributable to presynaptic attenuation of glutamate release; CB1R agonists depressed spontaneous and evoked glutamatergic currents and reduced the frequency of miniature EPSCs. Cannabinoid actions on hypocretin neurons were abolished by ionotropic glutamate receptor antagonists. Together, these results show that cannabinoids have opposite effects on MCH and hypocretin neurons. These opposing actions could help explain the increase in feeding and reduction in arousal induced by cannabinoids.

Synthesis and biological evaluation in vitro of a selective, high potency peptide agonist of human melanin-concentrating hormone action at human melanin-concentrating hormone receptor 1.

Human melanin-concentrating hormone (hMCH) is a nonselective natural ligand for the human melanin-concentrating hormone receptors: hMCH-1R and hMCH-2R. Similarly, the smaller peptide encompassing the disulfide ring and Arg(6) of hMCH, Ac-Arg(6)-cyclo(S-S)(Cys(7)-Met(8)-Leu(9)-Gly(10)-Arg(11)-Val(12)-Tyr(13)-Arg(14)-Pro(15)-Cys(16))-NH(2), Ac-hMCH(6-16)-NH(2), binds to and activates equally well both human MCH receptors present in the brain. To separate the physiological functions of hMCH-1R from those of hMCH-2R, new potent and hMCH-1R selective agonists are necessary. In the present study, analogs of Ac-hMCH(6-16)-NH(2) were prepared and tested in binding and functional assays on cells expressing the MCH receptors. In these peptides, Arg in position 6 was replaced with various d-amino acids and/or Gly in position 10 was substituted with various L-amino acids. Several of the new compounds turned out to be potent agonists at hMCH-1R with improved selectivity over hMCH-2R. For example, peptide 26 with d-Arg in place of L-Arg in position 6 and Asn in place of Gly in position 10, Ac-dArg(6)-cyclo(S-S)(Cys(7)-Met(8)-Leu(9)-Asn(10)-Arg(11)-Val(12)-Tyr(13)-Arg(14)-Pro(15)-Cys(16))-NH(2), was a potent hMCH-1R agonist (IC(50) = 0.5 nm, EC(50) = 47 nm) with more than 200-fold selectivity with respect to hMCH-2R. Apparently, these structural changes in positions 6 and 10 results in peptide conformations that allow for efficient interactions with hMCH-1R but are unfavorable for molecular recognition at hMCH-2R.

Visualizing differences in ligand-induced beta-arrestin-GFP interactions and trafficking between three recently characterized G protein-coupled receptors.

beta-Arrestin 1-GFP or beta-arrestin 2-GFP were coexpressed transiently with G protein-coupled receptor kinase 2 within cells stably expressing the orexin-1, apelin or melanin-concentrating hormone (MCH), receptors. In response to agonist ligands both the orexin-1 and apelin receptors were able to rapidly translocate both beta-arrestin 1-GFP and beta-arrestin 2-GFP from cytoplasm to the plasma membrane. For the MCH receptor this was only observed for beta-arrestin 2-GFP. beta-Arrestin 1-GFP translocated by the apelin receptor remained at the plasma membrane during prolonged exposure to ligand even though the receptor became internalized. By contrast, for the orexin-1 receptor, internalization of beta-arrestin 1-GFP within punctate vesicles could be observed for over 60 min in the continued presence of agonist. Co-internalization of the orexin-1 receptor was observed by monitoring the binding and trafficking of TAMRA-(5- and 6-carboxytetramethylrhodamine) labelled orexin-A. Subsequent addition of an orexin-1 receptor antagonist resulted in cessation of incorporation of beta-arrestin 1-GFP into vesicles at the plasma membrane and a gradual clearance of beta-arrestin 1-GFP from intracellular vesicles. For the melanin-concentrating hormone receptor the bulk of translocated beta-arrestin 2-GFP was maintained at concentrated foci close to, or at, the plasma membrane. These results demonstrate very distinct features of beta-arrestin-GFP interactions and trafficking for three G protein-coupled receptors for which the natural ligands have only recently been identified and which were thus previously considered as orphan receptors.