Duplication of neuropeptide Y and peptide YY in Nile tilapia Oreochromis niloticus and their roles in food intake regulation

In vertebrates, the neuropeptide Y (NPY) family peptides have been recognized as key players in food intake regulation. NPY centrally promotes feeding, while peptide YY (PYY) and pancreatic polypeptide (PP) mediate satiety. The teleost tetraploidization is well-known to generate duplicates of both NPY and PYY; however, the functional diversification between the duplicate genes, especially in the regulation of food intake, remains unknown. In this study, we identified the two duplicates of NPY and PYY in Nile tilapia (Oreochromis niloticus). Both NPYa and NPYb were primarily expressed in the central nervous system (CNS), but the mRNA levels of NPYb were markedly lower than those of NPYa. Hypothalamic mRNA expression of NPYa, but not NPYb, decreased after feeding and increased after 7-days of fasting. However, both NPYa and NPYb caused a significant increase in food intake after an intracranial injection of 50 ng/g body weight dose. PYYb, one of the duplicates of PYY, had an extremely high expression in the foregut and midgut, whereas another form of duplicate PYYa showed only moderate expression in the CNS. Both hypothalamic PYYa and foregut PYYb mRNA expression increased after feeding and decreased after 7-days of fasting. Furthermore, the intracranial injection of PYYb decreased food intake, but PYYa had no significant effect. Our results suggested that although the mature peptides of NPYa and NPYb can both stimulate food intake, NPYa is the main endogenous functional NPY for feeding regulation. A functional division has been identified in the duplicates of PYY, which deems PYYb as a gut-derived anorexigenic peptide and PYYa as a CNS-specific PYY in Nile tilapia.     

Increased food intake by neuropeptide Y is due to an increased motivation to eat

Neuropeptide Y (NPY), administered intracerebroventricularly, is a potent orexigenic agent. To determine if NPY-induced eating represented an increase in motivation to eat (e.g., hunger) rather than pathological or stimulus-bound eating, we determined its effect on eating in three paradigms, including lever press, appetitive passive avoidance and quinine-adulterated milk. NPY-injected mice consumed more milk when required to work for it in a lever press apparatus and tolerated shock to the tongue for drinking milk. Increasing the dose of NPY also allowed mice to overcome a taste aversion for quinine-adulterated milk. Overall, these studies support the hypothesis that NPY causes a specific increase in the motivation to eat, rather than nonspecific or stimulus-bound behavior.     

Peripheral insulin administration attenuates the increase in neuropeptide Y concentrations in the hypothalamic arcuate nucleus of fasted rats

Fasting increases neuropeptide Y (NPY) concentrations in the arcuate nucleus (ARC), its site of synthesis, and in other regions of the rat hypothalamus. Neuropeptide Y is a potent central orexigenic agent and may therefore stimulate appetite during fasting. We tested the hypothesis that low plasma insulin levels stimulate ARC levels of NPY in fasted rats. Compared with freely fed controls (n = 8), rats fasted for 72 h (n = 8) showed significantly lower plasma insulin levels (28.9 ± 1.6 vs. 52.6 ± 5.7 pmol/l; p < 0.001) and higher ARC NPY concentrations (14.2 ± 1.8 vs. 8.4 ± 2.2 fmol/μg protein; p < 0.001). Fasted rats treated with subcutaneous insulin (5 U/kg/day; n = 10), which nearly normalized plasma insulin (46.6 ± 2.8 pmol/l), showed intermediate ARC NPY levels (11.2 ± 1.4 fmol/μg protein; p < 0.01 vs. controls and untreated fasted rats). Insulin administered peripherally, therefore, attenuates fasting-induced NPY increases in the ARC, supporting the hypothesis that hypoinsulinemia stimulates hypothalamic NPY.     

Reserpine-induced immunocytochemical change of neuropeptide Y in the hypothalamic arcuate nucleus

The effect of reserpine on neuropeptide Y immunoreactive (NPY-IR) neurons in the rat hypothalamic arcuate nucleus was examined by immunocytochemical techniques. Although only NPY-IR fibers and terminals were distributed in this nucleus in untreated and saline treated rats, single treatment of reserpine (10 mg/kg, i.p.) visualized abundant NPY-IR neuronal cell bodies: the increase began at 12 h of postinjection, reached its maximal level at 48 h, and returned to its normal level at 96 h. Pretreatment of nialamide, a monoamine oxidase inhibitor, prevented these acute reserpine-induced changes, suggesting reserpine acts on NPY neurons through monoaminergic mechanism. Chronic treatment of haloperidol (5 mg/kg, once daily for 5 days) a dopamine receptor antagonist, could induce the similar increase of NPY immunoreactivity. However, interruption of adrenergic and serotonergic neurotransmissions by chronic treatment of propranorol and methysergide, or chemical lesions of ascending noradrenergic and serotononergic pathways by 6-hydroxydopamine and 5,6-dihydroxytryptamine, could not induce any immunoreactive increase of NPY in arcuate neurons. These findings strongly suggest that reserpine-induced NPY increase occurs through dopaminergic afferents in hypothalamic arcuate neurons. Special issue dedicated to Dr. Kinya Kuriyama.     

Decreased circulating leptin and increased neuropeptide Y gene expression are implicated in food deprivation-induced hyperactivity in striped hamsters, Cricetulus barabensis

Physiological and behavioral adjustments of small mammals are important strategies in response to variations in food availability. Although numerous of studies have been carried out in rodents, behavioral patterns in response to food deprivation and re-feeding (FD–RF) are still inconsistent. Here we examined effects of a 24 h FD followed by RF on general activity, serum leptin concentrations and gene expression of orexigenic and anorexigenic hypothalamic neuropeptides in striped hamsters (Cricetulus barabensis) with/without leptin supplements. The time spent on activity was increased by 2.5 fold in FD hamsters compared with controls fed ad libitum (P < 0.01). Body mass, fat mass as well as serum leptin concentrations were significantly decreased in FD hamsters in comparison with ad libitum controls, which were in parallel with hyperactivity. During re-feeding, leptin concentrations increased rapidly to pre-deprivation levels by 12 h, but locomotor activity decreased gradually and did not return to pre-deprivation levels until 5 days after re-feeding. Leptin administration to FD hamsters significantly attenuated the increased activity. Gene expression of hypothalamic neuropeptide Y (NPY) was upregulated in FD hamsters and fell down to control levels when hamsters were re-fed ad libitum, similar to that observed in activity behavior. Leptin supplement induced increases in serum leptin concentrations (184.1%, P < 0.05) in FD hamsters and simultaneously attenuated the increase in activity (45.8%, P < 0.05) and NPY gene expression (35%, P < 0.05). This may allow us to draw a more generalized conclusion that decreased leptin concentrations function as a starvation signal in animals under food shortage; to induce an increase in activity levels, leading animals to forage and/or migrate, and consequently increasing the chance of survival. Decreased concentrations of serum leptin in animals subjected to food shortage may induce an upregulation of gene expression of hypothalamus NPY, consequently driving a significant increase in foraging behavior.     

Viral mediated neuropeptide Y expression in the rat paraventricular nucleus results in obesity

Objective: Chronic central administration of neuropeptide Y (NPY) has dramatic effects on energy balance; however, the exact role of the hypothalamic paraventricular nucleus (PVN) in this is unknown. The aim of this study was to further unravel the contribution of NPY signaling in the PVN to energy balance. Research Methods and Procedures: Recombinant adeno‐associated viral particles containing NPY (rAAV‐NPY) were injected in the rat brain with coordinates targeted at the PVN. For three weeks, body weight, food intake, endocrine parameters, body temperature, and locomotor activity were measured. Furthermore, effects on insulin sensitivity and expression of NPY, agouti‐related protein (AgRP), and pro‐opiomelanocortin in the arcuate nucleus were studied. Results: Food intake was increased specifically in the light period, and dark phase body temperature and locomotor activity were reduced. This resulted in obesity characterized by increased fat mass; elevated plasma insulin, leptin, and adiponectin; decreased AgRP expression in the arcuate nucleus; and decreased insulin sensitivity; whereas plasma corticosterone was unaffected. Discussion: These data suggest that increased NPY expression targeted at the PVN is sufficient to induce obesity. Interestingly, plasma concentrations of leptin and insulin were elevated before a rise in food intake, which suggests that NPY in the PVN influences leptin and insulin secretion independently from food intake. This strengthens the role of the PVN in regulation of energy balance by NPY.     

Bilateral neural transections at the level of mesencephalon increase food intake and reduce latency to onset of feeding in response to neuropeptide Y

Administration of neuropeptide Y (NPY) into the IIIrd ventricle of the rat brain induces robust ingestive behavior with a latency to onset of feeding (LOF) ranging from 12 to 20 min. Since substantial amounts of NPY found in hypothalamic sites that mediate the control of feeding behavior originate from the brain stem, we studied the effects of NPY on LOF and food intake in male and female rats after bilateral severing of brain stem NPY input to the hypothalamus at the level of the mesencephalon. NPY in doses of 117 pmol significantly increased food intake and decreased LOF in both male and female transected rats. Higher doses of 470 pmol NPY decreased only the LOF in transected rats as compared to sham control rats. Additionally, 117 pmol NPY in transected rats elicited food consumption equivalent to that produced by 470 pmol NPY in control rats. These studies show that decreases in NPY levels found in the paraventricular nucleus and neighboring hypothalamic sites as a result of these neural transections may render rats hyperresponsive to NPY, presumably due to denervation-induced hypersensitivity in these sites.     

Effect of chronic infusion of olanzapine and clozapine on food intake and body weight gain in male and female rats

Many antipsychotics cause weight gain in humans, but usually not in rats, when injected once or twice daily. Since blood antipsychotic half-lives are short in rats, compared to humans, chronic administration by constant infusion may be necessary to see consistent weight gain in rats. Male and female rats were implanted with mini-pumps for constant infusion of olanzapine (5 mg/kg/day), clozapine (10 mg/kg/day) or vehicle for 11 days. Food intake and body weight were measured; blood drug levels were measured by HPLC. Olanzapine increased food intake and body weight in female, but not male rats. Serum olanzapine concentrations were 30-35 ng/ml. Clozapine had no effect on food intake or body weight in female or male rats. Serum clozapine concentrations were about 75 ng/ml. Single-dose pharmacokinetic analysis revealed a serum terminal half-life of 1.2-1.5 h for each drug, with no sex differences. Despite the fact that olanzapine and clozapine promote weight gain in humans, these drugs appear to have minimal effects on body weight and food intake in rats, except for a modest effect of olanzapine in female rats, even though therapeutic levels of olanzapine are achieved in serum during chronic infusion. Hence, the rapid clearance of drug following single administration in previous studies cannot explain the weak or absent effects of antipsychotics on weight gain in this species. The rat thus appears to be an inadequate model of weight gain produced by some antipsychotics in humans.     

Synaptic interactions between ghrelin- and neuropeptide Y-containing neurons in the rat arcuate nucleus

Morphological relationships between neuropeptide Y- (NPY) like and ghrelin-like immunoreactive neurons in the arcuate nucleus (ARC) were examined using light and electron microscopy techniques. At the light microscope level, both neuron types were found distributed in the ARC and could be observed making contact with each other. Using a preembedding double immunostaining technique, some NPY-immunoreactive axon terminals were observed at the electron microscope level to make synapses on ghrelin-immunoreactive cell bodies and dendrites. While the axo-somatic synapses were mostly symmetric in nature, the axo-dendritic synapses were both symmetric and asymmetric. In contrast, ghrelin-like immunoreactive (ghrelin-LI) axon terminals were found to make synapses on NPY-like immunoreactive (NPY-LI) dendrites although no NPY-like immunoreactive perikarya were identified receiving synapses from ghrelin-LI axon terminals. NPY-like axon terminals were also found making synapses on NPY-like neurons. Axo-axonic synapses were also identified between NPY- and ghrelin-like axon terminals. The present study shows that NPY- and ghrelin-LI neurons could influence each other by synaptic transmission through axo-somatic, axo-dendritic and even axo-axonic synapses, and suggests that they participate in a common effort to regulate the food-intake behavior through complex synaptic relationships.     

Pituitary adenylate cyclase activating polypeptide (PACAP) reduces food intake in mice

Pituitary adenylate cyclase activating peptide (PACAP) is a peptide that is present in the hypothalamus and other areas of the rat brain. This study demonstrates that PACAP reduces food intake after intracerebroventricular injection in food-deprived mice. Behavioral analysis suggests that this decrease in food intake is, in part, compensated for by an increase in other behaviors. Pituitary adenylate cyclase activating peptide also was demonstrated to antagonize increased food intake resulting from administration of neuropeptide Y. Thus, PACAP joins a growing list of neuropeptides involved in the central regulation of food intake.     

Cardiovascular effects of neuropeptide Y in the nucleus tractus solitarius of rats: relationship with noradrenaline and vasopressin

The central haemodynamic effects of neuropeptide Y (NPY), both alone and together with either noradrenaline (NA) or vasopressin (AVP), have been investigated by microinjecting synthetic peptide into the nucleus tractus solitarius (NTS) of anaesthetized rats. NPY alone elicited dose-dependent changes in blood pressure (BP) and heart rate (HR); 470 fmol inducing a pressor response, and 4.7 pmol a fall in BP. The hypotensive response to 20 nmol NA was significantly modified by both simultaneous and prior injection of an ineffective dose (47 fmol) of NPY. Prior injection of a similar dose of NPY also modified the NTS pressor effect of 10 ng AVP. A relationship between the action of AVP and NPY in the NTS was further indicated by the finding that prior injection of an ineffective dose of AVP (1 ng) reduced the hypotensive response to 4.7 pmol NPY, and by the demonstration of contrasting effects of 4.7 pmol NPY in AVP-deficient Brattleboro rats compared to parent strain LE rats. These results, taken together with the recent localization of NPY-like immunoreactivity in the NTS, suggest a role for NPY in central cardiovascular control. In addition, NPY has been shown to exhibit functional interactions with both an amine neurotransmitter and a neuropeptide present in the NTS of rats.     

The arcuate nucleus and neuropeptide Y contribute to the antitumorigenic effect of calorie restriction

Calorie restriction (CR) is known to have profound effects on tumor incidence. A typical consequence of CR is hunger, and we hypothesized that the neuroendocrine response to CR might in part mediate CR's antitumor effects. We tested CR under appetite suppression using two models: neuropeptide Y (NPY) knockout mice and monosodium glutamate-injected mice. While CR was protective in control mice challenged with a two-stage skin carcinogenesis model, papilloma development was neither delayed nor reduced by CR in the monosodium glutamate-treated and NPY knockout mice. Adiponectin levels were also not increased by CR in the appetite-suppressed mice. We propose that some of CR's beneficial effects cannot be separated from those imposed on appetite, and that NPY neurons in the arcuate nucleus of the hypothalamus are involved in the translation of reduced intake to downstream physiological and functional benefits.     

Differential expression and localization of neuropeptide Y peptide in pancreatic islet of diabetic and high fat fed rats

Neuropeptide Y (NPY) inhibits insulin secretion. Increased numbers of pancreatic islet cells expressing NPY have been observed in type 1 diabetic rats. To understand the functional significance of NPY expression in islet cells, we investigated the effects of high fat feeding and diabetic conditions on the expression and location of NPY expressing cells in normal and diabetic rats. Twenty rats were maintained on either normal chow (ND) or a high fat dietary regimen (HFD) for 4 weeks. In half of each group, type 1 or type 2 diabetes (groups T1DM and T2DM, respectively) was induced by injection of streptozotocin. At 8 weeks rats were euthanized and the pancreases were processed for immunofluorescence labeling (NPY/insulin, NPY/glucagon, NPY/somatostatin, and NPY/pancreatic polypeptide). Compared with the ND group, HFD rats had significantly fewer alpha cells, but beta cells were similar, while T1DM and T2DM rats showed significant increases in the proportions of alpha, delta, and PP cells. Robust increases in NPY-positive islet cells were found in the HFD, T1DM, and T2DM rats compared with ND controls. In ND rats, 99.7% of the NPY-positive cells were PP cells. However, high fat feeding and diabetes resulted in significant increases in NPY-positive delta cells, with concomitant decreases in NPY-positive PP cells. In summary, high-fat feeding and diabetes resulted in changes in the hormonal composition of pancreatic islet and increased number of NPY-expressing islet cells. Under diabetic conditions NPY expression switched from predominantly a characteristic of PP cells to predominantly that of delta cells. This may be a factor in reduced pancreatic hormone secretion during diabetes.     

Possible involvement of neuropeptide Y Y1 receptors in antidepressant like effect of agmatine in rats

Agmatine and neuropeptide Y (NPY) are widely distributed in central nervous system and critically involved in modulation of depressive behavior in experimental animals. However their mutual interaction, if any, in regulation of depression remain largely unexplored. In the present study we explored the possible interaction between agmatine and neuropeptide Y in regulation of depression like behavior in forced swim test. We found that acute intracerebroventricular (i.c.v.) administration of agmatine (20–40 μg/rat), NPY (5 and 10 μg/rat) and NPY Y1 receptor agonist, [Leu³¹, Pro³⁴]-NPY (0.4 and 0.8 ng/rat) dose dependently decreased immobility time in forced swim test indicating their antidepressant like effects. In combination studies, the antidepressant like effect of agmatine (10 μg/rat) was significantly potentiated by NPY (1 and 5 μg/rat, icv) or [Leu³¹, Pro³⁴]-NPY (0.2 and 0.4 ng/rat, icv) pretreatment. Conversely, pretreatment of animals with NPY Y1 receptor antagonist, BIBP3226 (0.1 ng/rat, i.c.v.) completely blocked the antidepressant like effect of agmatine (20–40 μg/rat) and its synergistic effect with NPY (1 μg/rat, icv) or [Leu³¹, Pro³⁴]-NPY (0.2 ng/rat, icv). The results of the present study showed that, agmatine exerts antidepressant like effects via NPYergic system possibly mediated by the NPY Y1 receptor subtypes and suggest that interaction between agmatine and neuropeptide Y may be relevant to generate the therapeutic strategies for the treatment of depression.     

PYY(3-36) into the arcuate nucleus inhibits food deprivation-induced increases in food hoarding and intake

Central administration of neuropeptide Y (NPY) increases food intake in laboratory rats and mice, as well as food foraging and hoarding in Siberian hamsters. The NPY-Y1 and Y5 receptors (Rs) within the hypothalamus appear sufficient to account for these increases in ingestive behaviors. Stimulation of NPY-Y2Rs in the Arcuate nucleus (Arc) has an anorexigenic effect as shown by central or peripheral administration of its natural ligand peptide YY (3-36) and pharmacological NPY-Y2R antagonism by BIIE0246 increases food intake. Both effects on food intake by NPY-Y2R agonism and antagonism are relatively short-lived lasting ∼4 h. The role of NPY-Y2Rs in appetitive ingestive behaviors (food foraging/hoarding) is untested, however. Therefore, Siberians hamsters, a natural food hoarder, were housed in a semi-natural burrow/foraging system that had (a) foraging requirement (10 revolutions/pellet), no free food (true foraging group), (b) no running wheel access, free food (general malaise control) or (c) running wheel access, free food (exercise control). We microinjected BIIE0246 (antagonist) and PYY_(3-36) (agonist) into the Arc to test the role of NPY-Y2Rs there on ingestive behaviors. Food foraging, hoarding, and intake were not affected by Arc BIIE0246 microinjection in fed hamsters 1, 2, 4, and 24 h post injection. Stimulation of NPY-Y2Rs by PYY_(3-36) inhibited food intake at 0–1 and 1–2 h and food hoarding at 1–2 h without causing general malaise or affecting foraging. Collectively, these results implicate a sufficiency, but not necessity, of the Arc NPY-Y2R in the inhibition of food intake and food hoarding by Siberian hamsters.     

Experimental dissociation of food intake and plasma beta-endorphin following 2-deoxy-D-glucose in rats

The present studies were undertaken to further assess the role of plasma beta-endorphin (beta-EP) in the hyperphagia induced by the glucose antimetabolite, 2-deoxy-D-glucose (2-DG). Plasma concentrations of immunoreactive beta-EP (ir-beta-EP) were measured at the end of the first hour of feeding in all animals treated with 400 mg/kg 2-DG. Previous studies had shown a consistent, positive association between 2-DG hyperphagia and plasma ir-beta-EP concentrations, but the present data revealed dissociations between hyperphagia and plasma ir-beta-EP. Dexamethasone administration blocked the 2-DG-induced rise in plasma ir-beta-EP, but had no effect on the 2-DG hyperphagia measured at 1 hour. Forced drinking of a 2% NaCl solution decreased 2-DG hyperphagia, but not the 2-DG induced rise in plasma ir-beta-EP. Thus, elevations in plasma ir-beta-EP are not necessary for the full expression of 2-DG-induced hyperphagia in dexamethasone-treated rats. Furthermore, decreased feeding responses to 2-DG could coexist with increased levels of plasma ir-beta-EP in NaCl-treated normal rats. Elevations in plasma ir-beta-EP do not appear to be the critical opiate link in 2-DG induced hyperphagia.     

The effects of pancreatic polypeptides and neuropeptide Y on the rat vas deferens

In order to study the physiological significance of the coexistence of pancreatic polypeptide and norepinephrine (NE) in peripheral noradrenergic nerves, the effects of pancreatic polypeptides of several species were tested on the isolated rat vas deferens. Neuropeptide Y (NPY) was also studied because of its sequence homology to the pancreatic polypeptides. The contractile responses, which were mediated predominantly by activation of noradrenergic nerves following electrical stimulation, were inhibited by bovine pancreatic polypeptide (BPP), human pancreatic polypeptide (HPP), avian pancreatic polypeptide (APP) and NPY in a dose-dependent manner using a constant flow bath. The decreasing order of the inhibitory responses was as follows: BPP = HPP greater than NPY greater than APP. The inhibitory responses produced by BPP and HPP lasted more than 1 hr and displayed a marked tachyphylaxis. In contrast, the inhibitory effects induced by NPY and APP usually returned to the control level after 20-30 min and had minimal tachyphylaxis. The inhibitory action of NPY was still present during alpha-adrenergic blockade. Contractions produced by a single submaximal dose of exogenous NE or serotonin (5-HT) in unstimulated preparations were not affected by pretreatment with NPY. The amplitude of contractions was partially reduced 1 min after pretreatment with BPP or HPP; recovery occurred about 15 min after peptide pretreatment in a constant flow bath. These results suggest that an NPY receptor exists presynaptically in the rat vas deferens and that stimulation of the receptor by NPY inhibits the release of NE from noradrenergic nerves.(ABSTRACT TRUNCATED AT 250 WORDS)