Orexin-A-induced feeding is dependent on nitric oxide

Abstract

Orexin-A is a peptide produced in the lateral hypothalamus/perifornical area, which stimulates feeding. The production of orexin-A is determined by the metabolic state of the animal. We have previously shown that nitric oxide (NO) plays an important role as a mediator of feeding induced by a variety of neuropeptides. This raises the question of whether orexin-A's effects are NO dependent. Here, we first determined that intracerebroventricular administration of 25 ng of orexin-A significantly increased food intake in satiated mice. We next examined the effects of N_ω-nitro-l-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, on orexin-A-induced increase in food intake. L-NAME (50 mg/kg; SC) significantly blocked the orexin-A-induced increase in food intake. Orexin-A administration increased the levels of nitric oxide synthase in the hypothalamus. To further verify the importance of NO in the orexin-A-induced increase in food intake, we compared the ability of orexin-A to increase food intake in neuronal nitric oxide synthase knockout (NOS-KO) mice and their wild-type controls. Orexin-A failed to increase food intake in the NOS-KO mice, whereas it did increase food intake in the wild-type controls. This supports the hypothesis that nitric oxide is a central regulator of food consumption.

Introduction

The orexins, also known as hypocretins, are a family of hormones discovered in the late 1990's [10], [29]. The orexin's are processed from the same prohormone and are produced in the lateral hypothalamus/perifornical area, a region known to be important in feeding behavior [29]. Orexin-A (hypocretin-1) is a 33 amino acid peptide with two internal disulfide linkages, a pyroglutamyl residue in the N-terminus, and an amidated C-terminus. Orexin B (hypocretin-2) is 28 amino acids in length, lacks internal disulfide bonds, but similarly has an amidated C-terminus. Two orexin receptors, OX₁R and OX₂R were also identified at the time of the identification as the peptides [29]. Both are G-protein coupled receptors and OX₁R appears to couple via the G_q/11 subclass of heteromeric receptors, whereas OX₂R may couple through either the G_q/11 or the G_i/o subclasses [29], [34]. It is clear that OX₁R prefers orexin-A as a ligand, whereas OX₂R binds orexin-A or orexin-B with similar affinities.

Exogenously applied orexin-A can stimulate feeding and orexin-A production is determined by the metabolic state of the animal [18]. Convincing evidence for a physiologically relevant role for the endogenous orexin-A in the CNS control of feeding comes from passive immunoneutralization studies in which compromise of the bioactivity of endogenous orexin [27], [38] with neutralizing antibodies reduced feeding in response to an overnight fast. Administration of the relatively selective, non-peptide OX₁R antagonist, SB-334867, decreased feeding in response to exogenously applied orexin or post-fasting feeding [28]. Orexin knockout mice (preprohormone gene deletion) eat less than wild-type littermates [3] again suggesting a physiologically relevant role for endogenous orexin in the regulation of appetite. However, these mice grew normally, thus some other determinant of growth or body weight must compensate for the decreased feeding.

Orexin-A is part of a complex pathway involved in energy intake and expenditure [11]. This pathway within the brain includes agouti-related protein (AgRP), neuropeptide Y (NPY), proopiomelanocortin (PMOC) and cocaine- and amphetamine-related transcript (CART) in the arcuate nucleus, melanocortin 4 receptor (MC4R) in the paraventricular and ventromedial hypothalamus (PVH/VMH) and orexin and melanin-concentrating hormone (MCH) in the lateral hypothalamus. Substances from the periphery that regulate the processes in these regions include leptin produced from fat, insulin produced in the pancreas and protein YY (PYY), glucagons-like peptide-1 (GLP-1), (cholecystokinin) CCK and ghrelin produced in the stomach. Previous experiments have shown NPY and ghrelin produced in the periphery promote food consumption through a nitric oxide-dependent (NO) pathway, whereas leptin decreased nitric oxide synthase levels [13], [26], [33]. NO is synthesized by nitric oxide synthase (NOS) from arginine. The NOS enzyme belongs to the cytochrome P450 protein family and is inhibited by several arginine analogs including N_ω-nitro-l-arginine methyl ester (L-NAME) [6], [7], [8], [9], [19], [24], [37]. Nitric oxide functions as a gaseous hormone that is involved in a myriad of systems throughout the body. Included are the nervous, cardiovascular, renal, pulmonary, endocrine, and immune systems, where NO acts as a second messenger [6]. Evidence is emerging that NO is an important regulator of food intake [7], [8], [9], [19], [21], [22], [23], [24], [25], [36], [37].

Evidence suggests that substances, which stimulate feeding, such as NPY and ghrelin, stimulate food intake through a NO pathway and evidence that neuronal NOS and orexin are co-expressed in the dorsomedial perifornical arcuate and ventromedial hypthothalamic neurons [5]. We examined the effects of the NOS inhibitor (L-NAME) on orexin-A-induced food consumption in mice. A dose–response curve was constructed for the orexin-A to determine the optimal dose for testing L-NAME. To further investigate this mechanism, we tested the ability of orexin-A to increase food intake in NOS knockout (NOS-KO) and their wild-type controls. NOS levels in the hypothalamus were measured following ICV injection of orexin-A and in the NOS-KO's and their controls.