Rapid communication R7 Journal of Endocrinology (1999) 160, R7–R12 Accepted 15 January 1999 0022-0795/99/0160-00R7 © 1999 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology.org The effect of the orexins on food intake: comparison with neuropeptide Y, melanin-concentrating hormone and galanin C M B Edwards, S Abusnana, D Sunter, K G Murphy , M A Ghatei and S R Bloom ICSM Endocrine Unit, Hammersmith Hospital, London W12 0NN, UK (Requests for offprints should be addressed to S R Bloom) Abstract Orexin-A and orexin-B (the hypocretins) are recently described neuropeptides suggested to have a physiological role in the regulation of food intake in the rat. We compared the orexigenic effect of the orexins administered intra- cerebroventricular (ICV) with other known stimulants of food intake, one strong, neuropeptide Y (NPY), and two weaker, melanin-concentrating hormone (MCH) and galanin. Orexin-A consistently stimulated food intake, but orexin-B only on occasions. Both peptides stimulated food intake significantly less than NPY, but to a similar extent to MCH (2 h food intake: NPY 3 nmol, 7.2±0.9 g vs saline, 1.5±0.2 g, P<0.001, MCH 3 nmol, 3.2±0.8 g vs saline, P<0.01, orexin-B 30 nmol, 2.6±0.5 g vs saline, P=0.11) and to galanin (1 h food intake: galanin 3 nmol, 2.0±0.4 g vs saline, 0.8±0.2 g, P<0.05, orexin-A 3 nmol, 2.2±0.4 g vs saline, P=0.01; 2 hour food intake: orexin- B 3 nmol, 2.4±0.3 g vs saline, 1.3±0.2 g, P<0.05). Following ICV orexin-A, hypothalamic c-fos, a marker of neuronal activation, was highly expressed in the paraventricular nucleus (PVN), and the arcuate nucleus (P<0.005 for both). IntraPVN injection of orexin-A stimulated 2 h food intake by one gram (orexin-A 0.03 nmol, 1.6±0.3 g vs saline, 0.5±0.3 g, P<0.005). These findings support the suggestion that the orexins stimulate food intake. However, this effect is weak and may cast doubt upon their physiological importance in appetite regulation in the rat. Introduction Originally reported as the hypocretins, due to their hypothalamic location and sequence homology to secretin, two novel peptides have been demonstrated to be present in neuronal cell bodies of the dorsal and lateral hypothalamus, with fibres projecting to multiple targets including the brain stem (De Lecea et al. 1998). Later identified and named as orexin-A and orexin-B, these same peptides were reported to powerfully stimulate feeding (Sakurai et al. 1998), 3 nmol orexin-B stimulating 2 h food intake 5-fold and the same dose of orexin-A stimulating 6-fold over saline control. The genetic precursor to both peptides, prepro-orexin, was shown to be up- regulated 2.4-fold after a 48 h fast, greater than the up- regulation of NPY mRNA under the same experimental conditions. Two receptors for the peptides, OX 1 and OX 2 were expressed exclusively in the brain, though to date the distribution of the receptors has not been reported. Such evidence led the authors to suggest the orexins as mediators in the central feedback mechanisms that regulate feeding behaviour (Sakurai et al. 1998). We have used orexin-A, and orexin-B from two separate sources, to compare their effects with more established orexigenic agents. We also assessed the effect of orexin-A on the induction of the immediate early gene c-fos, a marker of neuronal activation (Sagar et al. 1988). Finally, we investigated whether the orexins act through the paraventricular nucleus (PVN), by injecting directly into the nucleus. Materials and Methods Peptides Orexin-A and orexin-B were purchased from Pheonix Pharm. Inc., Mountain View, CA, USA. NPY, MCH, galanin and orexin-B were synthesised using fmoc chemistry on an Advanced ChemTech 396MPS peptide synthesiser. The products comprised one major peak which was purified to homogeneity by reversed phase HPLC on a C8 column (Phenomenex, Macclesfield, UK) using a gradient of acetonitrile in 0.1% trifluoroacetic acid. Electrospray and MALDI-TOF mass spectrometry was used to confirm the peptides identity. Animals Adult male Wistar rats (250-300 g) were maintained under controlled conditions of temperature (21-23 ° C) and light (12 h light, 12 h dark) with ad libitum access to food (RM1 diet, SDS UK Ltd) and water. All animals were handled daily from recovery after surgery to the time of study. In the case of the c- fos studies, the animals were each handled for ten minutes daily for ten days to minimise stress, which readily activates c- fos (Arnold et al. 1992).