Neuropeptide Y (NPY) is an endogenous neuropeptide that is abundantly expressed in the central nervous system. NPY is involved in various neurological processes and neuropsychiatric disorders, including fear learning and anxiety disorders. Reduced levels of NPY are reported in Post-Traumatic Stress Disorder (PTSD) patients, and NPY has been proposed as a potential therapeutic target for PTSD. It is therefore important to understand the effects of chronic enhancement of NPY on anxiety and fear learning. Previous studies have shown that acute elevation of NPY reduces anxiety, fear learning and locomotor activity. Models of chronic NPY overexpression have produced mixed results, possibly caused by ectopic NPY expression. NPY is expressed primarily by a subset of GABAergic interneurons, providing specific spatiotemporal release patterns. Administration of exogenous NPY throughout the brain, or overexpression in cells that do not normally release NPY, can have detrimental side effects, including memory impairment. In order to determine the effects of boosting NPY only in the cells that normally release it, we utilized a transgenic mouse line that overexpresses NPY only in NPY+ cells. We tested for effects on anxiety related behaviors in adolescent mice, an age with high incidence of anxiety disorders in humans. Surprisingly, we did not observe the expected reduction in anxiety-like behavior in NPY overexpression mice. There was no change in fear learning behavior, although there was a deficit in nest building. The effect of exogenous NPY on synaptic transmission in acute hippocampal slices was also diminished, indicating that the function of NPY receptors is impaired. Reduced NPY receptor function could contribute to the unexpected behavioral outcomes. We conclude that overexpression of NPY, even in cells that normally express it, can lead to reduced responsiveness of NPY receptors, potentially affecting the ability of NPY to function as a long-term therapeutic.

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.

An extensive behavioral characterization was conducted with mice lacking the gene for neuropeptide Y (NPY) including response to 24 and 48 h fast and challenge with small molecule antagonists of NPY receptors implicated in mediating the feeding effects of NPY (i.e., Y1 and Y5). In addition, wildtype (WT) and NPY knockout (KO) mice were tested in locomotor monitors, elevated plus maze, inhibitory avoidance, acoustic startle, prepulse inhibition, and hot plate assays. One of the major findings was that the NPY KO mice have a reduced food intake relative to WT controls in response to fasting. Also, based on data from the behavioral models, the NPY KO mice may have an anxiogenic-like phenotype, and appear to be hypoalgesic in the hot plate paradigm. The data from these studies provide further evidence of involvement of NPY in energy balance, anxiety, and possibly nociception.

The neuropeptide Y (NPY) system is a multireceptor/multiligand system consisting of four receptors in humans (hY(1), hY(2), hY(4), hY(5)) and three agonists (NPY, PYY, PP) that activate these receptors with different potency. The relevance of this system in diseases like obesity or cancer, and the different role that each receptor plays influencing different biological processes makes this system suitable for the design of subtype selectivity studies. In this review we focus on the latest findings within the NPY system, we summarize recent mutagenesis studies, structure activity relationship studies, receptor chimera, and selective ligands focusing also on the binding mode of the native agonists.

Neuropeptide Y (NPY) is involved in multiple processes such as behavior, energy and bone metabolism. Previous studies have relied on global NPY depletion to examine its effects on bone. However, this approach is unable to distinguish the central or local source of NPY influencing bone. Our aim was to identify which cells within the skeleton express Npy and establish a model that will enable us to differentiate effects of NPY derived from different cell types. We have generated the NPY floxed (NPYflox) mice using CRISPR technology. By crossing the NPYflox mice with Hypoxanthine Phosphoribosyltransferase 1 (Hprt)-cre to generate a global knockout, we were able to validate and confirm loss of Npy transcript and protein in our global NPYKO. Global deletion of NPY results in a smaller femoral cortical cross-sectional area (-12%) and reduced bone strength (-18%) in male mice. In vitro, NPY-deficient bone marrow stromal cells (BMSCs) showed increase in osteogenic differentiation detected by increases in alkaline phosphatase staining and bone sialoprotein and osteocalcin expression. Despite both sexes presenting with increased adiposity, female mice had no alterations in bone mass, suggesting that NPY may have sex-specific effects on bone. In this study we identified Npy expression in the skeleton and examined the effect of global NPY depletion to bone mass. The differential impact of NPY deletion in cortical and cancellous compartments along with differences in phenotypes between in vitro and in vivo, highlights the complex nature of NPY signaling, indicative of distinct sources that can be dissected in the future using this NPYflox model.

Sleep is an essential and evolutionarily conserved behavioral state whose regulation remains poorly understood. To identify genes that regulate vertebrate sleep, we recently performed a genetic screen in zebrafish, and here we report the identification of neuropeptide Y (NPY) as both necessary for normal daytime sleep duration and sufficient to promote sleep. We show that overexpression of NPY increases sleep, whereas mutation of npy or ablation of npy-expressing neurons decreases sleep. By analyzing sleep architecture, we show that NPY regulates sleep primarily by modulating the length of wake bouts. To determine how NPY regulates sleep, we tested for interactions with several systems known to regulate sleep, and provide anatomical, molecular, genetic, and pharmacological evidence that NPY promotes sleep by inhibiting noradrenergic signaling. These data establish NPY as an important vertebrate sleep/wake regulator and link NPY signaling to an established arousal-promoting system.

Neuropeptide Y (NPY) is a multifunctional neurotransmitter acting via G protein-coupled receptors - Y1R, Y2R and Y5R. NPY activities, such as its proliferative effects, are mediated by multiple receptors, which have the ability to dimerize. However, the role of this receptor interplay in NPY functions remains unclear. The goal of the current study was to identify NPY receptor interactions, focusing on the ligand-binding fraction, and determine their impact on the mitogenic activity of the peptide. Y1R, Y2R and Y5R expressed in CHO-K1 cells formed homodimers detectable on the cell surface by cross-linking. Moreover, Y1R and Y5R heterodimerized, while no Y2R/Y5R heterodimers were detected. Nevertheless, Y5R failed to block internalization of its cognate receptor in both Y1R/Y5R and Y2R/Y5R transfectants, indicating Y5R transactivation upon stimulation of the co-expressed receptor. These receptor interactions correlated with an augmented mitogenic response to NPY. In Y1R/Y5R and Y2R/Y5R transfectants, the proliferative response started at picomolar NPY concentrations, while nanomolar concentrations were needed to trigger proliferation in cells transfected with single receptors. Thus, our data identify direct and indirect heterotypic NPY receptor interactions as the mechanism amplifying its activity. Understanding these processes is crucial for the design of treatments targeting the NPY system.

Postmenopausal osteoporosis (PMO) is often accompanied by neuroendocrine changes in the hypothalamus, which closely associates with the microbial diversity, community composition, and intestinal metabolites of gut microbiota (GM). With the emerging role of GM in bone metabolism, a potential neuroendocrine signal neuropeptide Y (NPY) mediated brain-gut-bone axis has come to light. Herein, it is reported that exogenous overexpression of NPY reduced bone formation, damaged bone microstructure, and up-regulated the expressions of pyroptosis-related proteins in subchondral cancellous bone in ovariectomized (OVX) rats, but Y1 receptor antagonist (Y1Ra) reversed these changes. In addition, it is found that exogenous overexpression of NPY aggravated colonic inflammation, impaired intestinal barrier integrity, enhanced intestinal permeability, and increased serum lipopolysaccharide (LPS) in OVX rats, and Y1Ra also reversed these changes. Most importantly, NPY and Y1Ra modulated the microbial diversity and changed the community composition of GM in OVX rats, and thereby affecting the metabolites of GM (e.g., LPS) entering the blood circulation. Moreover, fecal microbiota transplantation further testified the effect of NPY-mediated GM changes on bone. In vitro, LPS induced pyroptosis, reduced viability, and inhibited differentiation of osteoblasts. The study demonstrated the existence of NPY-mediated brain-gut-bone axis and it might be a novel emerging target to treat PMO.

Previous work has demonstrated that neuropeptide tyrosine (NPY), Y(1) receptor and Y(2) receptor are critical in modulation of pain after nerve injury. We hypothesized that NPY was important for nociception after surgical incision. As a model of postoperative pain, rats underwent a plantar incision in one hindpaw. Western blots were used to quantify changes in protein expression of NPY, Y(1) receptor and Y(2) receptor after incision in skin, muscle, and dorsal root ganglion (DRG). Pain-related behaviors were tested after incision in rats treated with intrathecal NPY, Y(1) receptor antagonist (BIBO3304--Chemical Name: N-[(1R)-1-[[[[4-[[(Aminocarbonyl)amino]methyl]phenyl]methyl]amino]carbonyl]-4-[(aminoiminomethyl)amino]butyl]-α-phenyl-benzeneacetamide ditrifluoroacetate), Y(2) receptor antagonist (BIIE0246--Chemical Name: N-[(1S)-4-[(Aminoiminomethyl)amino]-1-[[[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]amino]carbonyl]butyl]-1-[2-[4-(6,11-dihydro-6-oxo-5H-dibenz[b,e]azepin-11-yl)-1-piperazinyl]-2-oxoethyl]-cyclopentaneacetamide), combined NPY+antagonists, morphine, or vehicle. Pain behaviors were tested after incision in rats treated with locally applied intraplantar injections of NPY, Y(1) receptor and Y(2) receptor antagonists or vehicle. NPY protein expression was significantly downregulated in muscle for two days after incision. In contrast, Y(1) receptor and Y(2) receptor protein expression was upregulated in both skin and muscle. A single intrathecal injection of NPY reduced cumulative guarding pain scores, as did morphine. The intrathecal administration of Y(2) receptor antagonist also reduced pain scores; findings that were not observed when drugs were administered locally. Intrathecal Y(2) receptor antagonists and NPY improved mechanical threshold and heat withdrawal latency 2h after incision. Intrathecal administration of NPY and/or central blockade of Y(2) receptor attenuated pain behaviors early after incision (postoperative day (POD) 1-2). Y(1) receptor antagonist administration blocked the anti-hyperalgesic effect of NPY. Together these data suggest a role for spinal NPY in postoperative pain.

Neurogenic inflammation plays a major role in the pathogenesis of inflammatory bowel disease (IBD). We examined the role of neuropeptide Y (NPY) and neuronal nitric oxide synthase (nNOS) in modulating colitis.

Neuropeptide Y (NPY) is highly abundant in the brain and involved in various physiological processes related to food intake and anxiety, as well as human diseases such as obesity and cancer. However, the molecular details of the interactions between NPY and its receptors are poorly understood. Here, we report a cryo-electron microscopy structure of the NPY-bound neuropeptide Y1 receptor (Y1R) in complex with Gi1 protein. The NPY C-terminal segment forming the extended conformation binds deep into the Y1R transmembrane core, where the amidated C-terminal residue Y36 of NPY is located at the base of the ligand-binding pocket. Furthermore, the helical region and two N-terminal residues of NPY interact with Y1R extracellular loops, contributing to the high affinity of NPY for Y1R. The structural analysis of NPY-bound Y1R and mutagenesis studies provide molecular insights into the activation mechanism of Y1R upon NPY binding.

Peptide YY (PYY) and neuropeptide Y (NPY) activate Y receptors, targets under consideration as treatments for diarrhoea and other intestinal disorders. We investigated the gastrointestinal consequences of selective PYY or NPY ablation on mucosal ion transport, smooth muscle activity and transit using wild-type, single and double peptide knockout mice, comparing mucosal responses with those from human colon.

Neuropeptide Y (NPY) plays a trophic role in the nervous and vascular systems and in cardiac hypertrophy. However, there is no report concerning the expression of NPY and its receptors in the heart during postnatal development. In the current study, immunohistochemistry and Western blot analysis was used to label NPY, and Y1R, Y2R, and Y5R receptors in the heart tissue and intramural cardiac ganglia from rats of different ages (newborn, 10 days old, 20 days old, 30 days old, 60 days old, 1 year old, and 2 years old).The obtained data suggest age-dependent changes of NPY-mediated heart innervation. The density of NPY-immunoreactive (IR) fibers was the least in newborn animals and increased in the first 20 days of life. In the atria of newborn and 10-day-old rats, NPY-IR fibers were more abundant compared with the ventricles. The vast majority of NPY-IR fibers also contained tyrosine hydroxylase, a key enzyme in catecholamine synthesis.The expression of Y1R increased between 10 and 20 days of life. Faint Y2R immunoreactivity was observed in the atria and ventricles of 20-day-old and older rats. In contrast, the highest level of the expression of Y5R was found in newborn pups comparing with more adult rats. All intramural ganglionic neurons were also Y1R-IR and Y5R-IR and Y2R-negative in all studied animals.Thus, the increasing of density of NPY-containing nerve fibers accompanies changes in relation of different subtypes of NPY receptors in the heart during development.

Machado-Joseph disease (MJD) is the most common dominantly-inherited ataxia worldwide with no effective treatment to prevent, stop or alleviate its progression. Neuropeptide Y (NPY) is a neuroprotective agent widely expressed in the mammalian brain. Our previous work showed that NPY overexpression mediated by stereotaxically-injected viral vectors mitigates motor deficits and neuropathology in MJD mouse models. To pursue a less invasive translational approach, we investigated whether intranasal administration of NPY would alleviate cerebellar neuropathology and motor and balance impairments in a severe MJD transgenic mouse model. For that, a NPY solution was administered into mice nostrils 5 days a week. Upon 8 weeks of treatment, we observed a mitigation of motor and balance impairments through the analysis of mice behavioral tests (rotarod, beam walking, pole and swimming tests). This was in line with a reduction of cerebellar pathology, evidenced by a preservation of cerebellar granular layer and of Purkinje cells and reduction of mutant ataxin-3 aggregate numbers. Furthermore, intranasal administration of NPY did not alter body weight gain, food intake, amount of body fat nor cholesterol or triglycerides levels. Our findings support the translational potential of intranasal infusion of NPY as a pharmacological intervention in MJD.

Although the olfactory bulb contains one of the highest concentrations of neuropeptide Y in the CNS, its function in the bulb remains unclear. In this study, we used whole-cell electrophysiological, molecular, and primary culture techniques to investigate neuropeptide Y gene expression and neuromodulatory actions of neuropeptide Y on rat olfactory bulb neurons. Northern analysis showed that neuropeptide Y mRNA increases with animal age or time in culture, in a parallel manner. In electrophysiology experiments, agonists that activate neuropeptide Y receptors (whole neuropeptide Y) and the Y2 receptor subtype (neuropeptide Y 13-36) reduced spontaneous excitatory activity in bulb interneurons. In investigating potential presynaptic effects, both agonists reduced the amplitude of calcium channel currents in the presynaptic (mitral/tufted) cell. Also consistent with a presynaptic effect, both agonists reduced the frequency but not the amplitude of miniature excitatory postsynaptic currents (or "minis") in interneurons. In examining potential postsynaptic effects, both agonists slightly increased membrane resistance but had no effect on currents evoked by glutamate. Together, these data suggest that neuropeptide Y inhibits excitatory neurotransmission between olfactory bulb neurons via a presynaptic effect on transmitter (glutamate) release.

Exogenous neuropeptide Y has antiepileptic effects; however, the underlying mechanism and optimal administration method for neuropeptide Y are still unresolved. Previous studies have used intracerebroventricular injection of neuropeptide Y into animal models of epilepsy. In this study, a recombinant adeno-associated virus expression vector carrying the neuropeptide Y gene was injected into the lateral ventricle of rats, while the ipsilateral hippocampus was injected with kainic acid to establish the epileptic model. After transfection of neuropeptide Y gene, mossy fiber sprouting in the hippocampal CA3 region of epileptic rats was significantly suppressed, hippocampal synaptophysin (p38) mRNA and protein expression were inhibited, and epileptic seizures were reduced. These experimental findings indicate that a recombinant adeno-associated virus expression vector carrying the neuropeptide Y gene reduces mossy fiber sprouting and inhibits abnormal synaptophysin expression, thereby suppressing post-epileptic synaptic reconstruction.

Neuropeptides play key roles in shaping the organization and function of neuronal circuits. In the inferior colliculus (IC), which is located in the auditory midbrain, Neuropeptide Y (NPY) is expressed by a large class of GABAergic neurons that project locally as well as outside the IC. The IC integrates information from numerous auditory nuclei making the IC an important hub for sound processing. Most neurons in the IC have local axon collaterals, however the organization and function of local circuits in the IC remains largely unknown. We previously found that neurons in the IC can express the NPY Y1 receptor (Y 1 R + ) and application of the Y 1 R agonist, [Leu 31 , Pro 34 ]-NPY (LP-NPY), decreases the excitability of Y 1 R + neurons. To investigate how Y 1 R + neurons and NPY signaling contribute to local IC networks, we used optogenetics to activate Y 1 R + neurons while recording from other neurons in the ipsilateral IC. Here, we show that 78.4% of glutamatergic neurons in the IC express the Y1 receptor, providing extensive opportunities for NPY signaling to regulate excitation in local IC circuits. Additionally, Y 1 R + neuron synapses exhibit modest short-term synaptic plasticity, suggesting that local excitatory circuits maintain their influence over computations during sustained stimuli. We further found that application of LP-NPY decreases recurrent excitation in the IC, suggesting that NPY signaling strongly regulates local circuit function in the auditory midbrain. Together, our data show that excitatory neurons are highly interconnected in the local IC and their influence over local circuits is tightly regulated by NPY signaling.

Pancreatic β-cells regulate glucose homeostasis by secreting insulin in response to glucose elevation and G protein-coupled receptor (GPCR) activation. Neuropeptide Y (NPY) and somatostatin (SST) attenuate insulin secretion through G(i) activation of Y(1) and SSTR(1&5) receptors, respectively. The downstream pathways altered by NPY and SST are poorly understood. Thus, we investigated these underlying mechanisms. NPY and SST increase cellular redox potential, suggesting that their inhibitory effect may not be mediated through metabolic inhibition. NPY does not affect intracellular calcium ([Ca(2+)](i)) activity upon glucose stimulation, whereas SST alters this response. G(βγ)-subunit inhibition by gallein attenuates insulin secretion but does not alter metabolism or [Ca(2+)](i). mSIRK-induced G(βγ) activation does not modulate glucose metabolism but increases [Ca(2+)](i) activity and potentiates insulin release. Cotreatment with gallein and NPY or SST reduces insulin secretion to levels similar to that of gallein alone. mSIRK and NPY cotreatment potentiates insulin secretion similarly to mSIRK alone, whereas mSIRK and SST treatment decreases insulin release. The data support a model where SST attenuates secretion through G(βγ) inhibition of Ca(2+) activity, while NPY activates a Ca(2+)-independent pathway mediated by G(α). GPCR ligands signal through multiple pathways to inhibit insulin secretion, and determining these mechanisms could lead to novel diabetic therapies.

Elevated triglyceride (TG) is the major plasma lipid abnormality in obese and diabetic patients and contributes to cardiovascular morbidity in these disorders. We sought to identify novel mechanisms leading to hypertriglyceridemia. Resistance to negative feedback signals from adipose tissue in key central nervous system (CNS) energy homeostatic circuits contributes to the development of obesity. Because triglycerides both represent the largest energy depot in the body and are elevated in both the plasma and adipose in obesity and diabetes, we hypothesized that the same neural circuits that regulate energy balance also regulate the secretion of TGs into plasma.

Neuroblastoma (NB) is a pediatric malignant neoplasm of sympathoadrenal origin. Challenges in its management include stratification of this heterogeneous disease and a lack of both adequate treatments for high-risk patients and noninvasive biomarkers of disease progression. Our previous studies have identified neuropeptide Y (NPY), a sympathetic neurotransmitter expressed in NB, as a potential therapeutic target for these tumors by virtue of its Y5 receptor (Y5R)-mediated chemoresistance and Y2 receptor (Y2R)-mediated proliferative and angiogenic activities. The goal of this study was to determine the clinical relevance and utility of these findings. Expression of NPY and its receptors was evaluated in corresponding samples of tumor RNA, tissues, and sera from 87 patients with neuroblastic tumors and in tumor tissues from the TH-MYCN NB mouse model. Elevated serum NPY levels correlated with an adverse clinical presentation, poor survival, metastasis, and relapse, whereas strong Y5R immunoreactivity was a marker of angioinvasive tumor cells. In NB tissues from TH-MYCN mice, high immunoreactivity of both NPY and Y5R marked angioinvasive NB cells. Y2R was uniformly expressed in undifferentiated tumor cells, which supports its previously reported role in NB cell proliferation. Our findings validate NPY as a therapeutic target for advanced NB and implicate the NPY/Y5R axis in disease dissemination. The correlation between elevated systemic NPY and NB progression identifies serum NPY as a novel NB biomarker.