Interactions of neuropeptide Y,hypocretin-I (orexin A) and melanin-concentrating hormone on feeding in rats

Amongst various neuropeptidergic systems, neuropeptide Y (NPY), hypocretin-1 and melanin-concentrating hormone (MCH) producing neurons have been shown to play an important role in the regulation of food intake and body weight. All of these neuropeptides are orexigenic signals and recent evidence suggests the existence of morphological connections between these neuronal systems in the hypothalamus. However, the functional interactions between these neuronal systems are not clearly understood. Therefore, in the present study, we examined whether there is a cooperative action on food intake between these neuropeptides after third intracerebroventricular (icv) injection in the rat. The icv administration of NPY (0.118, 0.588, 1.176 nmol), hypocretin-1 (1, 3 nmol) and MCH (0.42, 1.048, 2.096 nmol) stimulated food intake in a dose dependent manner. Coinjection with 0.118 nmol of NPY and hypocretin-1 (1, 3 nmol) or MCH (0.42, 1.048, 2.096 nmol) had no additive effect on food intake as compared to that of NPY alone. However, coinjection with lower dose of NPY (0.023 nmol) and hypocretin-1 (0.25 nmol), that did not have any effect alone, significantly induced food intake. In contrast, combination of a lower dose of NPY (0.023 nmol) or hypocretin-1 (0.25 nmol) with lower stimulatory dose (0.21, 0.42 nmol) of MCH did not result in further increase in food intake as compared to that of MCH alone. Also, combination of 0.25 nmol hypocretin-1 and a non-stimulatory dose of MCH (0.105 nmol) was ineffective in stimulating food intake. Finally, coinjection with of 0.023 nmol NPY and 0.105 nmol MCH significantly induced food intake as compared to saline control group but not as compared to NPY or MCH treated groups. In total, these results show (1) a synergistic action between NPY and hypocretin-1, (2) no interaction between hypocretin-1 and MCH and (3) very little interaction, if any, between NPY and MCH in inducing food intake. In conclusion, these results provide a physiological concomitant to the previous demonstration of morphological contacts between NPY and hypocretin producing neurons by suggesting an interaction between these two orexigenic signals in control of food intake, and further suggest that MCH's action on feeding may be independent of NPY and hypocretin-1 action.     

Effects of (+)‐pentazocine on the antinociceptive effects of (–)‐pentazocine in mice

Previous studies have shown that sigma‐1 receptor chaperone (Sig‐1R) ligands can regulate pain‐related behaviors, and Sig‐1R itself can regulate μ‐opioid receptor functions as well as signal transduction. Even though (±)‐pentazocine has been used clinically for the treatment of pain through opioid receptors, (+)‐pentazocine is known to be a selective Sig‐1R agonist. To the best of our knowledge, there is no information available regarding the involvement of Sig‐1R agonistic action in the antinociceptive effects of (±)‐pentazocine. Therefore, the present study was designed to investigate the effects of (+)‐pentazocine on the antinociceptive effects of (–)‐pentazocine in mice. Both and (–)‐pentazocine induced biphasic antinociceptive effects as measured by the warm‐plate test. The early phase, but not the delayed phase, of the antinociceptive effects induced by (–)‐pentazocine, which are mediated by the activation of μ‐opioid receptors, were suppressed by pretreatment with (+)‐pentazocine. These results suggest that the innate antinociceptive action of (±)‐pentazocine could be marginally reduced by the effects of (+)‐pentazocine, but (+)‐pentazocine can suppress the antinociceptive effects of (–)‐pentazocine at certain time points. Synapse 69:166–171, 2015. © 2015 Wiley Periodicals, Inc.     

[Leu,Pro]Neuropeptide Y (NPY), but not NPY 20–36, produces discriminative stimulus effects similar to NPY and induces food intake

Rats were trained to discriminate between an intracerebroventricular injection of 1.15 nmol of Neuropeptide Y (NPY) and a sham injection. Rats rapidly learned to press the appropriate lever during training. NPY's discriminative stimulus effects were compared to those of saline, and 1.15–3.45 nmol [Leu³¹,Pro³⁴]NPY, a Y₁ receptor agonist and NPY 20–36, Y₂ receptor agonist. [Leu³¹,Pro³⁴]NPY resulted in NPY-appropriate responding, whereas saline and NPY 20–36 did not. [Leu³¹,Pro³⁴]NPY also increased food intake, but NPY 20–36 did not. This suggests that NPY's discriminative stimulus and orexigenic effects involve the Y₁, but not the Y₂, receptor.     

Microinjection of galanin-like peptide into the medial preoptic area stimulates food intake in adult male rats

Galanin-like peptide (GALP) is a neuropeptide implicated in the regulation of feeding behaviour, metabolism and reproduction. GALP is an endogenous ligand of the galanin receptors, which are widely expressed in the hypothalamus. GALP is predominantly expressed in arcuate nucleus (ARC) neurones, which project to the paraventricular nucleus (PVN) and medial preoptic area (mPOA). Intracerebroventricular or intraparaventricular (iPVN) injection of GALP acutely increases food intake in rats. The effect of GALP injection into the mPOA on feeding behaviour has not previously been studied. In the present study, intra-mPOA (imPOA) injection of GALP potently increased 0-1-h food intake in rats. The dose-response effect of imPOA GALP administration on food intake was similar to that previously observed following iPVN administration. The effects of GALP (1 nmol) or galanin (1 nmol) on food intake were then compared following injection into the PVN, mPOA, ARC, dorsal medial nucleus (DMN), lateral hypothalamus and rostral preoptic area (rPOA). GALP (1 nmol) increased food intake to a similar degree when injected into the imPOA or iPVN, but produced no significant effect when injected into the ARC, DMN, lateral hypothalamus or rPOA. Similarly, galanin (1 nmol) significantly increased food intake following injection imPOA and iPVN. However, the effect was significantly smaller than that following administration of GALP (1 nmol). Galanin also had no significant effect on food intake when administered into the ARC, DMN, lateral hypothalamus and rPOA. These data suggest that the mPOA and the PVN may have specific roles in mediating the orexigenic effect of GALP and galanin.     

Anthocyanins attenuate body weight gain via modulating neuropeptide Y and GABAB1 receptor in rats hypothalamus

Anthocyanins in a variety of plant species have been identified and are known for its hypolipidemic and anti-obesity effects. The effect of anthocyanins extracted from black soybean on body weight and daily food intake in adult rats raised on normal diet were studied. Male Sprague–Dawley rats were daily intra-gastric administered water or anthocyanins 6 mg/kg and 24 mg/kg for 40 days. During this period daily food intake and body weight were measured prior to anthocyanins treatment. These findings showed that anthocyanins treatment resulted in significantly lowered body weight and food intake compared with water treated rats. In addition, anthocyanins dose dependently reduced the adipose tissue size compared with control group. Western blot analysis showed that high dose of anthocyanins treatment significantly reduced the expression of neuropeptide Y (NPY) and increased γ-amino butyric acid receptor (GABA_B1R) in hypothalamus. Furthermore, these events were followed by a decreased in expression of GABA_B1R downstream signaling molecules protein kinase A-α (PKA) and phosphorylated cAMP-response element binding protein (p-CREB) in hypothalamus. These data support the concept that anthocyanins even in normal circumstances have the capability to reduce body weight and food intake through its modulatory effect on NPY and GABA_B1R in hypothalamus. These results suggest that anthocyanins from black soybean seed coat might have a novel role in preventing obesity in rats on normal diet.     

Neuropeptide Y suppresses absence seizures in a genetic rat model primarily through effects on Y receptors

Neuropeptide Y (NPY) potently suppresses absence seizures in a model of genetic generalized epilepsy, genetic absence epilepsy rats of Strasbourg (GAERS). Here we investigated the Y-receptor subtype(s) on which NPY exerts this anti-absence effect. A dual in vivo approach was used: the cumulative duration of seizures was quantified in adult male GAERS in 90-min electroencephalogram recordings following intracerebroventricular (i.c.v.) injection of: (i) subtype-selective agonists of Y1 ([Leu31Pro34]NPY, 2.5 nmol), Y2 (Ac[Leu(28,31)]NPY24-36, 3 nmol), Y5 receptors [hPP1(-17),Ala31,Aib32]NPY, 4 nmol), NPY (3 nmol) or vehicle; and following (ii) i.c.v. injection of antagonists of Y1 (BIBP3226, 20 nmol), Y2 (BIIE0246, 20 nmol) and Y5 (NPY5RA972, 20 nmol) receptors or vehicle, followed by NPY (3 nmol). Injection of the Y1- and Y5-selective agonists resulted in significantly less mean seizure suppression (37.4% and 53.9%, respectively) than NPY (83.2%; P < 0.05), while the Y2 agonist had similar effects to NPY (62.3% suppression, P = 0.57). Food intake was not increased following injection of the Y2 agonist, while significant increases in food intake were seen following NPY and the other Y-subtype agonists. Compared with vehicle, NPY injection suppressed seizures following the Y1 and Y5 antagonists (45.3% and 80.1%, respectively, P < 0.05), but not following the Y2 antagonist (5.1% suppression, P = 0.46). We conclude that NPY Y2 receptors are more important than Y1 and Y5 receptors in mediating the effect of NPY to suppress absence seizures in a genetic rat model. Y2 receptor agonists may represent targets for novel drugs against genetic generalized epilepsies without resulting in appetite stimulation.     

Evidence that hypothalamic neuropeptide Y gene expression and NPY levels in the paraventricular nucleus increase before the onset of hyperphagia in experimental diabetes

Neuropeptide Y (NPY) is the most potent endogenous orexigenic signal. Several lines of evidence indicate that the site of NPY action in transducing feeding signal may reside in the paraventricular nucleus (PVN) and neighboring sites in the hypothalamus. To test the hypothesis that an increase in NPY activity in the ARC-PVN pathway precedes the onset of diabetic hyperphagia, we evaluated NPY levels in seven hypothalamic nuclei and NPY gene expression in the hypothalamus at 48, 72 or 96 h after streptozotocin (STZ) treatment in rat. In STZ-treated diabetic rats, NPY gene expression in the hypothalamus and NPY levels only in the PVN significantly elevated at 48 h, while hyperphagia occurred sometimes after 48 h post-injection. These results show that augmentation in NPY neuronal activity in the ARC-PVN axis precedes the onset of increased food intake produced by STZ-induced insulinopenia. These findings affirm the hypothesis that increased NPY neurosecretion in the PVN may underlie the diabetes-induced hyperphagia.     

5-HT(2A/2C) receptor antagonists in the paraventricular nucleus attenuate the action of DOI on NPY-stimulated eating.

Hypothalamic neuropeptide Y (NPY) and serotonin (5-HT)-containing neurons are believed to exert an interactive effect on ingestive behavior. The present study examined the ability of two serotonergic antagonists, spiperone (SPIP), a 5-HT2A antagonist, and mianserin (MIAN), a 5-HT(2A/2C) antagonist, to block the inhibitory action of the 5-HT(2A/2C) receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) on NPY-stimulated eating. Drugs were injected directly into the paraventricular nucleus (PVN), the perifornical (PFH) or the ventromedial hypothalamus (VMH) at the onset of the dark cycle. PVN, PFH and VMH injections of NPY potentiated food intake although only PVN pretreatment with DOI (5-20 nmol) suppressed NPY-induced eating. SPIP or MIAN, injected immediately prior to PVN DOI, reversed the suppressive effect of DOI on NPY feeding. These findings are consistent with other recent data showing that 5-HT2A receptors within the PVN modulate NPY's effect on food intake at the start of the nocturnal period.     

The kappa-opioid antagonist GNTI reduces U50,488-, DAMGO-, and deprivation-induced feeding, but not butorphanol- and neuropeptide Y-induced feeding in rats

Antagonists selective for either kappa- [e.g. nor-binaltorphimine (nor-BNI)] and mu- (e.g. beta-funaltrexamine) opioid receptors have previously been shown to reduce both kappa- and mu-opioid-induced feeding. In the present studies, the anorectic effects of GNTI, a newly synthesized antagonist selective for kappa-opioid receptors, were studied in rats. GNTI (0.032-0.32 nmol; i.c.v.), administered 15 min prior to food access, reduced feeding induced by the kappa-opioid agonist U50,488 (producing a 70% maximal decrease), the mu-opioid agonist DAMGO (90% maximal decrease), and 24 h acute food deprivation (60% maximal decrease). GNTI did not reduce the orexigenic effects of butorphanol, an agonist that binds to both kappa- and mu-opioid receptors, and neuropeptide Y (NPY). Taken together, these results suggest that GNTI is a potent anorectic agent and opioid antagonist in rats. Like nor-BNI, GNTI reduced feeding induced by both kappa- and mu-opioid agonists. However, unlike nor-BNI, GNTI did not alter the orexigenic effects of butorphanol or NPY. Given the selectivity of GNTI and its effectiveness in several of the present experiments, its potency, and its short duration of action compared to nor-BNI, GNTI may serve to be a useful tool to study behavioral effects mediated by kappa-opioid receptors.     

Neuropeptide Y facilitates activity-based-anorexia

The hypothesis that treatment with neuropeptide Y (NPY) can increase running activity and decrease food intake and body weight was tested. Female rats with a running wheel lost more weight than sedentary rats and ran progressively more as the availability of food was gradually reduced. When food was available for only 1h/day, the rats lost control over body weight. Correlatively, the level of NPY mRNA was increased in the hypothalamic arcuate nucleus. This phenomenon, activity-based-anorexia, was enhanced by intracerebroventricular infusion of NPY in rats which had food available during 2h/day. By contrast, NPY stimulated food intake but not wheel running in rats which had food available continuously. These findings are inconsistent with the prevailing theory of the role of the hypothalamus in the regulation of body weight according to which food intake is a homeostatic process controlled by "orexigenic" and "anorexigenic" neural networks. However, the finding that treatment with NPY, generally considered an "orexigen", can increase physical activity and decrease food intake and cause a loss of body weight is in line with the clinical observation that patients with anorexia nervosa are physically hyperactive and eat only little food despite having depleted body fat and up-regulated hypothalamic "orexigenic" peptides.     

Lack of effects by sigma ligands on neuropeptide Y-induced G-protein activation in rat hippocampus and cerebellum

It has been suggested that neuropeptide Y (NPY) and sigma (sigma) receptor ligands may share a putative NPY/sigma receptor in rat brain. To study whether NPY and sigma receptor ligands have an inverse agonism at this putative NPY/sigma receptor, we measured their effects on G-protein activity in rat brain. Using [35S]GTPgammaS autoradiography, we found that NPY-induced G-protein activation exhibited a discrete distribution pattern in rat brain. G-protein activation in superficial cortical layers and hippocampal CA1-3 region was mainly attributed to Y1 and Y2 receptors, respectively. In the presence of 10 microM sigma-receptor agonist BD737 or 10 microM sigma-receptor antagonist haloperidol, the distribution and density of [35S]GTPgammaS binding stimulated by 10 nM NPY was not significantly altered. In rat cerebellar membranes, NPY stimulated high-affinity GTPase activity in a dose-related manner, with maximal effects of 29% increase over basal level seen at 500 nM. This NPY-elicited GTPase activity was not significantly affected by micromolar concentrations of the sigma-receptor antagonists Dup734 or haloperidol. Since no significant effects by sigma-receptor ligands on NPY-induced G-protein activation were observed, we did not see an inverse agonism of NPY and sigma-receptor ligands at the putative NPY/sigma receptor measured at the level of G-protein activation, suggesting that sigma receptors and NPY receptors do not represent a common population in rat hippocampus and cerebellum. It is also suggested that G-protein activation is not a convergent point for the signal transduction mechanisms of NPY receptors and sigma receptors.     

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.     

Gonadotropin-inhibitory hormone-stimulation of food intake is mediated by hypothalamic effects in chicks

Gonadotropin-inhibitory hormone (GnIH), a 12 amino acid peptide, is expressed in the avian brain and inhibits luteinizing hormone secretion. Additionally, exogenous injection of GnIH causes increased food intake of chicks although the central mechanism mediating this response is poorly understood. Hence, the purpose of our study was to elucidate the central mechanism of the GnIH orexigenic response using 12 day post hatch layer-type chicks as models. Firstly, via mass spectrometry we deduced the chicken GnIH amino acid sequence: SIRPSAYLPLRFamide. Following this we used chicken GnIH to demonstrate that intracerebroventricular (ICV) injection of 2.6 and 7.8 nmol causes increased food intake up to 150 min following injection with no effect on water intake. The number of c-Fos immunoreactive cells was quantified in appetite-associated hypothalamic nuclei following ICV GnIH and only the lateral hypothalamic area (LHA) had an increase of c-Fos positive neurons. From whole hypothalamus samples following ICV GnIH injection abundance of several appetite-associated mRNA was quantified which demonstrated that mRNA for neuropeptide Y (NPY) was increased while mRNA for proopiomelanocortin (POMC) was decreased. This was not the case for mRNA abundance in isolated LHA where NPY and POMC were not affected but melanin-concentrating hormone (MCH) mRNA was increased. A comprehensive behavior analysis was conducted after ICV GnIH injection which demonstrated a variety of behaviors unrelated to appetite were affected. In sum, these results implicate activation of the LHA in the GnIH orexigenic response and NPY, POMC and MCH are likely also involved.     

Neuronal relationship between orexin-A- and neuropeptide Y-induced orexigenic actions in goldfish

Orexin-induced orexigenic action is mediated by neuropeptide Y (NPY) in goldfish and rodents. A previous study indicated that NPY-induced orexigenic action may also be mediated by orexin-A in goldfish. However, there is little information about the mutual actions of orexin-A and NPY in the goldfish. Therefore, using their specific receptor antagonists, we examined whether the orexigenic actions of orexin-A and NPY mutually interact in the goldfish. The stimulatory effect of intracerebroventricular injection of NPY at 1 pmol/g body weight (BW) on food intake was abolished by treatment with the orexin receptor-1 antagonist, SB334867, at 10 pmol/g BW whereas the NPY Y1-receptor antagonist, BIBP3226, at 100 pmol/g BW attenuated orexin-A (at 2.8 pmol/g BW)-stimulated feeding. This led us, using a double-immunostaining method and confocal laser scanning microscopy, to investigate whether orexin-A- and NPY-containing neurons in the goldfish brain have direct mutual inputs. Orexin-A- and NPY-like immunoreactivities were distributed throughout the brain, especially in the diencephalon. Orexin-A- and NPY-containing neurons were located in a region of the hypothalamus, the nucleus posterioris periventricularis (NPPv), in close proximity to each other: NPY-containing nerve fibers or endings lay in close apposition to orexin-A-containing neurons in the NPPv, and orexin-A-containing nerve fibers or endings also lay in close apposition to NPY-containing neurons in the same region. These results indicate that, in goldfish, orexin-A- and NPY-induced orexigenic actions are mediated by mutual signaling pathways.     

Effects of three neurochemical stimuli on delayed feeding and energy metabolism

Infusions of norepinephrine (NE), the gamma-aminobutyric acid agonist, muscimol (MUS), or neuropeptide Y (NPY) into the paraventricular nucleus (PVN) of the hypothalamus all increase food intake. Such feeding may be due to direct activation of behavioral processes driving ingestion and/or to alterations in nutrient metabolism that feeding serves to normalize. To examine these possibilities, male Sprague-Dawley rats received PVN infusions of vehicle, 20 nmol NE, 1 nmol MUS or 100 pmol NPY at dark onset, then food intake was measured under three feeding conditions: (1) 1 and 2 h immediately after injections, (2) 1 h after a 1 h delay between injections and access to food, and (3) 1 h after a 1 h feeding delay, but with injections occurring just before presenting food. Measures of energy expenditure (EE) and respiratory quotients (RQs) in the absence of food were made over 2 h in parallel experiments. Results confirmed that NE, MUS and NPY all increased dark-onset feeding, but only NPY increased intake above control levels after a 1 h feeding delay. No neurochemically-induced changes in EE were observed, nor were there changes in RQs after NE or MUS. However, NPY reliably enhanced RQs from 30 to 120 min of testing. Our findings imply that NE and MUS initiate relatively immediate, short-term feeding that is not associated with changes in nutrient metabolism and does not summate with cues stimulated by delayed access to food. NPY initiates more protracted feeding temporally linked to enhanced carbohydrate metabolism. This may indicate that part of NPY's feeding stimulatory effects are secondary to physiological processes driving ingestion.     

Microinjection of the α1-agonist methoxamine into the paraventricular hypothalamus induces anorexia in rats

Adrenergic receptors within the paraventricular hypothalamus (PVN) play a prominent role in the control of food intake: stimulation of α2-adrenoceptors induces food intake whereas stimulation of α1-adrenoceptors suppresses food intake. This study further examines the role of PVN α1-adrenoceptors hy examining the effects on food and water intake of the α1-adrenergic agonist methoxamine (100, 200, 400 nMol) microinjected into the rat paraventricular hypothalamus. Methoxamine suppressed food intake but not water intake. Doses of 100, 200, and 400 nMol methoxamine suppressed food intake by 47%. 64%, and 96%, respectively. These results further confirm the hypothesis that administration of α1-agonists into the PVN acts to significantly suppress food intake; an action that is in opposition to the facilitory effects of α2-adrenergic agonists on food intake.     

Estradiol decreases the orexigenic effect of neuropeptide Y, but not agouti-related protein, in ovariectomized rats

Available data suggest that estradiol exerts an inhibitory effect on food intake by modulating the actions of multiple gut- and brain-derived peptides implicated in the control of food intake. For example, recent studies have shown that estradiol decreases the orexigenic effects of ghrelin and melanin-concentrating hormone. In the present study, we examined estradiol's ability to decrease the actions of two additional orexigenic peptides, neuropeptide Y (NPY) and agouti-related protein (AgRP). Food intake was monitored following lateral ventricular infusions of 5 microg NPY, 10 microg AgRP, or saline vehicle in ovariectomized rats treated with either 1 microg estradiol or sesame oil vehicle. NPY increased food intake for 2h in both oil- and estradiol-treated ovariectomized rats. During this interval, the orexigenic effect of NPY was significantly greater in oil-treated rats, relative to estradiol-treated rats. In contrast to the short-term action of NPY, a single injection of AgRP increased food intake for 3 days in oil- and estradiol-treated rats. Meal pattern analysis revealed that the orexigenic effect of AgRP is mediated by an increase in meal size, not meal number. Unlike that observed following NPY treatment, estradiol failed to modulate the magnitude by which AgRP increased food intake and meal size. We conclude that a physiological regimen of estradiol treatment decreases the orexigenic effect of NPY, but not AgRP, in ovariectomized rats.