Effects Of NPY Research Articles

The Role of Neuropeptide Y in the Pathogenesis of Alzheimer’s Disease: Diagnostic Significance and Neuroprotective Functions

Background. Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases. It has been suggested that the factors that cause pathologic changes and lead to the development of AD may also include changes in certain neuropeptides. The implication of the neuropeptide (NPY) in the pathogenesis of AD and its potential therapeutic role is possible due to the following properties: involvement in adult neurogenesis, regulatory effects on the immune system, the inhibition of potential-dependent Ca2+ channels, and the reduction in glutamate excitotoxicity. The aim of our review was to summarize recent data on the role of NPY in AD development and to explore its potential as a biomarker and a possible therapeutic target. Materials and methods. We performed a systematic review of studies, for which we search using the keywords “Alzheimer’s disease and neuropeptide Y”, “Alzheimer’s disease and NPY”, “AD and NPY”, “Neuropeptide Y and Neurodegenerative disease”. Nineteen articles were included in the review. Results. The NPY levels in cerebrospinal fluid and plasma have been found to be reduced or unchanged in AD patients; however, these findings need to be confirmed in more recent studies. Data obtained in transgenic animal models support the role of NPY in AD pathogenesis. The neuroprotective effects of NPY have been demonstrated in vitro and in vivo in AD models. Conclusion. The findings may open new possibilities for using NPY as a diagnostic marker to detect AD at earlier stages of the disease or as a potential therapeutic target due to its neuroprotective properties.

Ghrelin Induces the Production of Hypothalamic NPY Through the AMPK-mTOR Pathway to Alleviate Cancer-induced Bone Pain.

Cancer-induced bone pain (CIBP) is one of the most common symptoms of bone metastasis of tumor cells. The hypothalamus may play a pivotal role in the regulation of CIBP. However, little is known about the exact mechanisms. First, we established a CIBP model to explore the relationship among hypothalamic ghrelin, NPY and CIBP. Then, we exogenously administered NPY and NPY receptor antagonists to investigate whether hypothalamic NPY exerted an antinociceptive effect through binding to NPY receptors. Finally, we exogenously administered ghrelin to investigate whether ghrelin alleviated CIBP by inducing the production of hypothalamic NPY through the AMPK-mTOR pathway. Body weight, food intake and behavioral indicators of CIBP were measured every 3 days. Hypothalamic ghrelin, NPY and the AMPK-mTOR pathway were also measured. The expression of hypothalamic ghrelin and NPY was simultaneously decreased in cancer-bearing rats, which was accompanied by CIBP. Intracerebroventricular (i.c.v.) administration of NPY significantly alleviated CIBP in the short term. The antinociceptive effect of NPY was reversed with the i.c.v. administration of the Y1R and Y2R antagonists. The administration of ghrelin activated the AMPK-mTOR pathway and induced hypothalamic NPY production to alleviate CIBP. This effect of ghrelin on NPY and antinociception was reversed with the administration of a GHS-R1α antagonist. Ghrelin could induce the production of hypothalamic NPY through the AMPK-mTOR pathway to alleviate CIBP, which can provide a novel therapeutic mechanism for CIBP.

Effect of Adrenoreceptor Stimulation on Peptidergic Regulation of Cardiac Activity in Newborn Rats.

We studied the effect of adrenoreceptor stimulation on the frequency of spontaneous activity and amplitude-time parameters of isometric contraction of the atrial myocardial strips from newborn rats, as well as the effect of Y receptor stimulation against the background of adrenoreceptor activation. After addition of Y1,5 receptor agonist [Leu31, Pro34] NPY (10-7 M), a tendency to a decrease in the effect of β1,2-adrenoreceptor agonist isoproterenol (10-5 M) on the frequency of spontaneous activity and atrial myocardial contractility was observed. The age-related features of the effect of NPY on the frequency of spontaneous activity and contractility of myocardial strips from newborn and adult rats were revealed.

SAT592 Sexually Dimorphic Modulation Of Arcuate Kisspeptin Neurons Activity In Response To NPY

Abstract Disclosure: N.D. Mansano: None. R. Frazao: None. Kisspeptin neurons are part of an intricate brain circuit that receives neuronal inputs related tonutritional status from at least three neuronal populations, including NPY/AgRP and POMC neurons of the arcuate nucleus of the hypothalamus (ARH) and cells in the ventral premammillary nucleus. Its well-known action on the HPG axis, hypothalamic kisspeptin neurons also modulate the activity of specific neurons of the paraventricular and the dorsomedial nuclei of the hypothalamus, regions involved in the regulation of appetite and energy expenditure. However, it is not completely elucidated if NPY can influence the HPG axis through the modulation of kisspeptin neuron activity. To determine the NPY effect in the hypothalamic kisspeptin neurons we performed whole-cell patch-clamp recordings. The hypothalamic slices were obtained from the adult Kiss1/hrGFP female (diestrus-stage) or male mice. Kisspeptin cells located in the arcuate nucleus of the hypothalamus (ARHkisspeptin) and at the anteroventral periventricular and rostral periventricular nuclei (AVPV/PeNkisspeptin) were recorded. On female mice, ARHkisspeptin neurons exhibited a rest membrane potential (RMP) average of -52.5±2.5 mV (-40 to -62 mV) and 1.3±0.2 GΩ input resistance (IR). NPY induced the hyperpolarization of 45% of the ARHkisspeptin neurons (n= 9 of 20 cells, out of 9 mice). ARHkisspeptin neuron’s RMP hyperpolarization was followed by a significant decrease in IR and frequency of action potentials (fAPS). In the AVPV/PeNkisspeptin neurons showed RMP average of -69.6±1.9 mV (-51 to - 88 mV) and IR of 0.8±0.09 GΩ (n=18 cells out of 9 mice). No significant NPY-induced effect was noted when recordings were performed on AVPV/PeNkisspeptin cells from female mice. To elucidate if the ARHkisspeptin neuron’s hyperpolarization depends on action potentials (APs) mediated by synaptic transmission we further evaluated NPY effects in the presence of TTX and amino acid receptor antagonists (6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10μM), DL-2-amino-5-phosphonopentanoic acid (AP5, 50μM), and picrotoxin (50μM). NPY induced no effect on ARHkisspeptin neuron’s RMP, and IR in the presence of TTX and amino acid receptor antagonists. Suggesting that NPY-induced hyperpolarization of ARHkisspeptin neurons is an indirect effect. To further verify whether sex must be considered as a biological variable to study the NPY effect, we recorded ARHkisspeptin cells from male mice. ARHkisspeptin neurons recorded from male mice exhibited RPM average of -55.6±1.5 (-45 to -66 mV) and IR of 1.5±0.15 GΩ (n= 13 cells out of 10 mice). Interestingly, NPY induced no effect on RMP, IR, or fAPs in male animals. In conclusion, our results suggest that NPY-induced hyperpolarization of ARHkisspeptin cells depends on AP’s mediate synaptic transmission, as a sex-specific effect. Presentation: Saturday, June 17, 2023

Fetal origins of obesity: a novel pathway of regulating appetite neurons in the hypothalamus of growth-restricted rat offspring

PurposeFetal growth restriction causes a series of sequelae, some of which, such as hyperphagia, reduced satiety and postnatal obesity, are believed to be associated with embryonic hypothalamic neurons impairment. The mechanisms underlying the linkage of fetal brain injuries to break the energy homeostasis have not been elucidated completely. Here, we aim to investigate the effect of intrauterine energy restriction on remodeling appetite neurons in the hypothalamus of fetal and postnatal infant rats.MethodsLow-protein (8%) diet combined with 75% energy restriction was used to establish an animal model. Rats offspring brain tissues, harvested from embryo day 18 and postnatal infant day 1, were sampled for dependent regulator analyses and master neuron assessment.ResultsGrowth-restricted rats showed the increased expression of Bsx and NPY in the hypothalamus as well as remodeling hypothalamic neurons differentiation compared to controls. Intriguingly, in cells cultured in vitro test, we found that activated effects of Bsx and NPY could be exacerbated by DNMT1 inhibitor.ConclusionsIn embryonic and early postnatal stage of FGR rats, we detected high concentrations of orexigenic neurons in the hypothalamus. DNMT1 activity is correlated with early embryonic neurogenesis by mediating the expression of Bsx and NPY. It may be one of the reasons for the abnormal development of the appetite regulation pathway and higher susceptibility to obesity in FGR offspring.

Transcranial direct current stimulation alters anxious-like behavior and neural parameters in rats with chronic pain exposed to alcohol

The aim of this study was to evaluate the effects of transcranial direct current stimulation (tDCS) on anxiety-like behavior and neural parameters in rats with chronic pain exposed to alcohol. Thirty-six adult male Wistar rats were randomly assigned to control (CT), neuropathic pain (NP), NPtDCS, NP + alcohol (NPAL), or NPALtDCS groups, subjected to sciatic nerve chronic constriction injury (CCI) and exposed to alcohol (20% v/v solution, 4 g/kg) or vehicle by gavage for 15 days. Afterward, rats were treated using bimodal tDCS (0.5 mA/20 min/8 days) and tested in the open field. Rats were killed 24 h after the last behavioral assessment, and brain and spinal cord tissue samples were collected and processed for NPY immunohistochemistry, expression of Il1a and Il1b in the spinal cord, cerebellum, and hippocampus, and levels of IL-1α and IL-1β in the same brain structures and the striatum. tDCS reverted the anxiety-like behavior induced by CCI and alcohol, and the increased expression of Il1a in the spinal cord induced by alcohol, which increased the expression of Il1b in the cerebellum. In addition, tDCS modulated the hypothalamic NPY-immunoreactivity, increased the levels of IL-1α in the hippocampus (like NP and AL), and increased the expression of Il1b in the spinal cord (like AL). Thus, this study shows that tDCS changes NP and alcohol-induced anxiety-like behavior, possibly through its central modulatory effect of NPY and spinal cord expression of Il1a and Il1b, being considered a treatment option for alcohol and NP-induced anxiety symptoms.

Abstract 6159: Blocking neuropeptide Y Y5 receptor prevents hypoxia-induced Ewing sarcoma metastasis

Abstract Ewing sarcoma (ES) is the second most prevalent malignant bone tumor after osteosarcoma in children and adolescents. While survival of patients with localized ES has improved, patients with metastatic ES still lack adequate therapies, which results in poor survival rates. Thus, there is an important need for new therapies targeting ES metastases and preventing its dissemnation. Previously, we have shown that ES cells constitutively express high levels of NPY and its receptors - Y1R and Y5R. However, tumor hypoxia changes expression pattern and functions of NPY system, leading to the activation of its Y2R and Y5R, which stimulate ES cell motility and invasiveness under these conditions. Hence, the goal of this study was to determine the role of NPY in hypoxia-induced ES metastases. Here, we focused on Y5R, since our data from other malignancies implicated this receptor as the major mediator of NPY's pro-metastatic effects. To this end, we used doxycycline (DOX)-inducible CRIPSR/Cas9 system to create an SK-ES1/Y5R knockout (KO) cell line. The Y5R KO significantly decreased motility of SK-ES1 cells. To determine the impact of this phenomenon on ES metastases, SK-ES1/Cas9/Y5RsgRNA cells were injected into gastrocnemius muscles of SCID/bg mice and the animals were fed with DOX+ or control diet. The Y5R KO did not affect primary tumor growth. Once the xenografts reached a volume of 0.25cm3, a femoral artery ligation (FAL) was performed in the tumor-bearing limbs to induce hypoxic conditions. Both primary tumors and their corresponding metastases were sequenced to confirm the efficiency of gene editing. Notably, majority of metastases arising in mice bearing SKES-1/Y5RsgRNA+DOX xenografts were initiated by the clones without Y5R gene modification, confirming the role of Y5R in ES dissemination. The frequency of metastases with efficient Y5R KO originating from hypoxic tumors was markedly decreased, as compared to the control group (-DOX). Moreover, Y5R KO prevented hypoxia-induced increase in osseous metastases previously reported by our group. Collectively, these data validate NPY/Y5R axis as a crucial mediator of the hypoxia-induced ES metastasis and implicate Y5R receptor as a novel target for therapies preventing ES dissemination. Importantly, the Y5R antagonist is already available and used in clinical trials. Citation Format: Mina T. Adnani, Susana Galli, Sung Hyeok Hong, Jason U. Tilan, Joanna Kitlinska. Blocking neuropeptide Y Y5 receptor prevents hypoxia-induced Ewing sarcoma metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6159.

Direct inhibition of arcuate kisspeptin neurones by neuropeptide Y in the male and female mouse.

Adverse energy states exert a potent suppressive influence on the reproductive axis by inhibiting the pulsatile release of gonadotrophin-releasing hormone and luteinising hormone. One potential mechanism underlying this involves the metabolic-sensing pro-opiomelanocortin and agouti-related peptide/neuropeptide Y (AgRP/NPY) neuronal populations directly controlling the activity of the arcuate nucleus kisspeptin neurones comprising the gonadotrophin-releasing hormone pulse generator. Using acute brain slice electrophysiology and calcium imaging approaches in Kiss1-GFP and Kiss1-GCaMP6 mice, we investigated whether NPY and α-melanocyte-stimulating hormone provide a direct modulatory influence on the activity of arcuate kisspeptin neurones in the adult mouse. NPY was found to exert a potent suppressive influence upon the neurokinin B-evoked firing of approximately one-half of arcuate kisspeptin neurones in both sexes. This effect was blocked partially by the NPY1R antagonist BIBO 3304, whereas the NPY5R antagonist L152,804 was ineffective. NPY also suppressed the neurokinin B-evoked increase in intracellular calcium levels in the presence of tetrodotoxin and amino acid receptor antagonists, indicating that the inhibitory effects of NPY are direct on kisspeptin neurones. By contrast, no effects of α-melanocyte-stimulating hormone were found on the excitability of arcuate kisspeptin neurones. These studies provide further evidence supporting the hypothesis that AgRP/NPY neurones link energy status and luteinising hormone pulsatility by demonstrating that NPY has a direct suppressive influence upon the activity of a subpopulation of arcuate kisspeptin neurones.

Hypothalamic mechanisms associated with corticotropin-releasing factor-induced anorexia in chicks

Central administration of corticotropin-releasing factor (CRF), a 41-amino acid peptide, is associated with potent anorexigenic effects in rodents and chickens. However, the mechanism underlying this effect remains unclear. Hence, the objective of the current study was to elucidate the hypothalamic mechanisms that mediate CRF-induced anorexia in 4 day-old Cobb-500 chicks. After intracerebroventricular (ICV) injection of 0.02 nmol of CRF, CRF-injected chicks ate less than vehicle chicks while no effect on water intake was observed at 30 min post-injection. In subsequent experiments, the hypothalamus samples were processed at 60 min post-injection. The CRF-injected chicks had more c-Fos immunoreactive cells in the arcuate nucleus (ARC), dorsomedial nucleus (DMN), ventromedial hypothalamus (VMH), and paraventricular nucleus (PVN) of the hypothalamus than vehicle-treated chicks. CRF injection was associated with decreased whole hypothalamic mRNA abundance of neuropeptide Y receptor sub-type 1 (NPYR1). In the ARC, CRF-injected chicks expressed more CRF and CRF receptor sub-type 2 (CRFR2) mRNA but less agouti-related peptide (AgRP), NPY, and NPYR1 mRNA than vehicle-injected chicks. CRF-treated chicks expressed greater amounts of CRFR2 and mesotocin mRNA than vehicle chicks in the PVN and VMH, respectively. In the DMN, CRF injection was associated with reduced NPYR1 mRNA. In conclusion, the results provide insights into understanding CRF-induced hypothalamic actions and suggest that the anorexigenic effect of CRF involves increased CRFR2-mediated signaling in the ARC and PVN that overrides the effects of NPY and other orexigenic factors.

Endocannabinoid and nitric oxide systems of the hypothalamic paraventricular nucleus mediate effects of NPY on energy expenditure

ObjectiveNeuropeptide Y (NPY) is one of the most potent orexigenic peptides. The hypothalamic paraventricular nucleus (PVN) is a major locus where NPY exerts its effects on energy homeostasis. We investigated how NPY exerts its effect within the PVN. MethodsPatch clamp electrophysiology and Ca2+ imaging were used to understand the involvement of Ca2+ signaling and retrograde transmitter systems in the mediation of NPY induced effects in the PVN. Immuno-electron microscopy were performed to elucidate the subcellular localization of the elements of nitric oxide (NO) system in the parvocellular PVN. In vivo metabolic profiling was performed to understand the role of the endocannabinoid and NO systems of the PVN in the mediation of NPY induced changes of energy homeostasis. ResultsWe demonstrated that NPY inhibits synaptic inputs of parvocellular neurons in the PVN by activating endocannabinoid and NO retrograde transmitter systems via mobilization of Ca2+ from the endoplasmic reticulum, suggesting that NPY gates the synaptic inputs of parvocellular neurons in the PVN to prevent the influence of non-feeding-related inputs. While intraPVN administered NPY regulates food intake and locomotor activity via NO signaling, the endocannabinoid system of the PVN selectively mediates NPY-induced decrease in energy expenditure. ConclusionThus, within the PVN, NPY stimulates the release of endocannabinoids and NO via Ca2+-influx from the endoplasmic reticulum. Both transmitter systems appear to have unique roles in the mediation of the NPY-induced regulation of energy homeostasis, suggesting that NPY regulates food intake, energy expenditure, and locomotor activity through different neuronal networks of this nucleus.

Hypothalamic AgRP neurons and the regulation of appetite and body weight: A review

The maintenance of energy balance is essential for the survival of animals. The hypothalamus in the brain plays a central role in this process through the integration of neural, nutritional and hormonal signals. Multiple and distributed neuronal populations in the hypothalamus form complex circuits to regulate appetite and body weight. Located in the arcuate nucleus of hypothalamus, agouti-related peptide (AgRP)-expressing cells are known as the hunger-promoting neurons. Over the past three decades, extensive studies have been performed to understand the role of AgRP neurons in energy balance control. AgRP neurons are both necessary and sufficient to orchestrate feeding behavior. The AgRP neuron activity adapts to and thereby is regulated by the nutritional status in the body. Furthermore, we now know that this alternation is driven by various circulating hormones, such as Leptin and Ghrelin, as well as synaptic inputs from upstream neurons. Neurotransmitters (e.g. glutamate and GABA) play a vital role in the regulation of AgRP neuron activity. The excitatory glutamatergic input is required in fasting-related activation of AgRP neurons, whereas the inhibitory GABAergic inputs is crucial in the rapid reduction of AgRP neuron activity in response to food-related cues, and thus, to terminate a meal. In addition to these molecules, signaling molecules and pathways in AgRP neurons, such as receptors, ion channels, kinases (e.g. IKKβ), endoplasmic reticulum stress (ER stress) and mitochondrial dynamic, are critically involved in the control of its activity. Moreover, AgRP neurons release neuropeptides AgRP and NPY, as well as neurotransmitter GABA to regulate neuronal activity. Both AgRP and NPY potently promote appetite. Although NPY is widely expressed in the central nervous system, it is mainly co-expressed with AgRP in the arcuate nucleus of hypothalamus. The appetite-promoting effect of NPY is mediated by Y1 and Y5 receptors, while AgRP exert its orexigenic effect mainly by antagonizing the central melanocortin system. Surprisingly, ablation of AgRP neurons in neonates has little effect on body weight, whereas these neurons are essential for the maintenance of appetite in adult mice. Moreover, accumulating evidences indicate that GABA released by AgRP neurons is required to retain animals appetite. At the circuit level, AgRP neurons project to both intra- and extra-hypothalamic regions to regulate energy balance. For example, in arcuate nucleus, AgRP neurons innervate and inhibit POMC neurons via the inhibitory GABAergic synapses. Conversely, POMC neurons release β-endorphin, a μ-opioid receptor agonist, to inhibit the activity of AgRP neurons. In summary, AgRP neurons play a crucial role in the regulation of appetite and energy balance. Targeting hypothalamic AgRP neurons might be a relevant approach to treat common obesity.

Abstract 1926: Npy1r as a prognostic marker and a novel drug target in estrogen receptor-positive breast cancer

Abstract Despite the crucial role of some G protein-coupled receptors (GPCRs) in cancer progression and metastases reported in recent studies, the function of majority of GPCRs, as a class, remains largely unexplored in breast cancer. In this study, we evaluated transcriptome, proteome and phosphoproteome data for 398 non-sensory GPCRs (IUPHAR v1.0) using the landmark TCGA breast cancer proteogenomics dataset and Illumina HiSeq dataset. Neuropeptide Y Receptor Y1 (NPY1R) was the top most hypo-phosphorylated (at S368) GPCR in 92 out of 99 evaluable samples. Furthermore, NPY1R gene expression was found to be significantly lower in tumor samples than tumor-matched normal (n=112). Among tumor samples, the LumA subtype had significantly higher NPY1R expression than LumB, Basal and Her2 subtype (p<0.05, One-way ANOVA, Tukey test). Interestingly, the trend of NPY1R gene expression, protein expression and phosphosite expression was decreasing in the order of LumA, LumB, Basal and Her2 subtype (p<0.01, Jonckheere trend test). Using publicly available dataset of 22,277 genes on survival in 2,422 patients, we found that higher NPY1R expression predicted better overall survival and recurrence-free survival in estrogen receptor-positive (ER+) breast cancer patients. In light of this, we interrogated NPY1R gene expression in endocrine sensitive and resistant cell line models and the impact of NPY1R antagonist in ER+ breast cancer cell lines. NPY1R expression was increased in response to estradiol in various ER+ breast cancer cell line models (MCF7, T47D, and BT474) in publicly available datasets. Conversely, we found that tamoxifen or estrogen deprivation (ED) treatment reduced NPY1R expression in MCF7 and T47D cells. In our endocrine-resistant (tamoxifen-resistant, estrogen deprivation-resistant, and fulvestrant-resistant) cell line models, NPY1R expression remained significantly lower compared to the parental cells in vitro and in vivo (p<0.05, One-way ANOVA, Tukey test). Treatment of MCF7 parental cells with 100 nM NPY significantly reduced estrogen-stimulated cell growth (p<0.05, One-way ANOVA, Tukey test). Chronic treatment of cells with 1 μM BIBP3226, an NPY1R antagonist, for 2 weeks reversed the effect of NPY on the estradiol-stimulated cell growth. Estradiol-stimulated cell growth was modest (~40%) in ED-resistant MCF7 cells compared to that (~250%) in parental MCF7 cells. While 100 nM NPY treatment did not affect the estrogen-stimulated growth in ED-resistant MCF7 cells, which can be explained by the undetectable expression of NPY1R, it caused significant inhibition of the estrogen-stimulated growth in the same cells treated chronically with BIBP3226. Molecular mechanisms to explain these effects of chronic NPY1R antagonism are underway. Our ongoing studies will further elucidate the role of NPY1R as a novel drug target in ER+ breast cancer. Citation Format: Raksha Bhat, Suhas Vasaikar, Leon Bae, De Angelis Carmine, Maria Letizia Cataldo, Sarmistha Nanda, Bing Zhang, Rachel Schiff, Meghana V. Trivedi. Npy1r as a prognostic marker and a novel drug target in estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1926.

26. Investigating the Role of Neuropeptide Y Infusions to the Ventral Hippocampus in Mediating Defensive Burying Behavior in Rats in the Shock-Probe Test

The current study will investigate the role of NPY in the ventral hippocampus in anxiety. NPY is a neuropeptide found in many structures in the brain, including the hippocampus, and is implicated in regulation of anxiety related behaviors. The hippocampus has also been found to play a role in anxiety and defensive behaviors – specifically, the ventral hippocampus regulates innate defensive behaviors. The effect of NPY in the ventral hippocampus will be investigated by infusing either NPY (n=12) or physiological saline (n=12) into the rat ventral hippocampus followed by behavioural testing in an animal model of anxiety; i.e., the shock-probe burying task. I expect to find a selective reduction in burying duration in NPY-infused rats.

Dietary macronutrient composition and central neuropeptide Y injection affect dietary preference and hypothalamic gene expression in chicks

Objective: The objective of this study was to determine the influence of dietary macronutrient composition on central NPY's orexigenic effect in chicks.Methods: Day-of-hatch chicks were fed one of three diets (3000 kcal ME/kg) ad libitum from hatch: high carbohydrate (HC), high fat (HF; 30% ME derived from soybean oil), and high protein (HP; 25 vs. 22% CP). In Experiment 1, chicks received intracerebroventricular injections of 0 (vehicle), 0.2, or 2.0 nmol NPY on day 4 and food intake was recorded for 6 hours. In Experiment 2, chicks were given all three diets before and after injection. In Experiment 3, hypothalamus was collected at 1-hour post-injection for gene expression analysis.Results: The HC diet-fed chicks responded with a greater increase, while the chicks fed the HF diet had a lower threshold response in food intake to NPY. Neuropeptide Y dose-dependently increased food intake in chicks fed the HC and HP diets. Chicks administered 0.2 nmol NPY preferred the HC and HP diets over the HF diet. Relative quantities of hypothalamic NPYR1 and MC4R mRNA were reduced by NPY in chicks that consumed the HP and HC diets, respectively.Discussion: Consumption of the HC diet was associated with the most robust NPY-induced increase in food intake. Injection of NPY accentuated differences among dietary groups in hypothalamic gene expression of several appetite-associated factors, results suggesting that the NPY/agouti-related peptide and melanocortin pathways are associated with some of the diet- and NPY-induced differences observed in this study.

NPY and CGRP Inhibitor Influence on ERK Pathway and Macrophage Aggregation during Fracture Healing

Aim: The aims of this study are to investigate the effects of neurotransmitters NPY and CGRP on ERK signaling in fracture healing, and to identify the correlation between macrophage aggregation and fracture healing. Methods: Male Sprague-Dawley rats were used to build a fracture model. The neurotransmitter receptor inhibitors were injected intraperitoneally into the rats. Immunofluorescence staining and ELISA were employed to determine the expression of NPY and CGRP in fracture area and the peripheral blood, respectively. Micro-CT together with histological staining were utilized to assess the fracture healing conditions. Relative protein expression was determined using western blot. Immunofluorescence staining was used to detect the aggregation of macrophages in the injury area. Results: During fracture healing, the serum NPY and CGRP significantly increased. The levels of NPY and CGRP reached a peak in the 8^th week and reduced significantly thereafter. NPY and CGRP inhibitors could inhibit fracture healing and down-regulate the phosphorylated ERK. Macrophages (NPY+ and CGRP+) aggregated in the injury area. Conclusion: NPY and CGRP participated in fracture healing, in which they were also shown to influence phosphorylated ERK expression. In addition, macrophages are involved in the fracture healing process.

Neuroprotecitve effect of NPY in in vitro models of Alzheimer's disease, focus on neurotrophins

neurodegenerative disorder associated with an expanded polyglutamine tract within the ataxin-3 protein, and characterized by progressive impairment of motor coordination, associated to neurodegeneration of specific brain regions including cerebellum and striatum. The currently available therapies do not allow modification of disease progression. NPY has been shown to exert potent neuroprotective effects by multiple pathways associated to the MJD mechanisms of disease. Thus, we evaluated NPY levels in MJD and investigated whether raising NPY by gene transfer would alleviate neuropathological and behavioural deficits in cerebellar and striatal mouse models of the disease. For that, a cerebellar transgenic and a striatal lentiviral-based models of MJD were used. NPY overexpression in the affected brain regions in these two mouse models was obtained by stereotaxic injection of adeno-associated viral vectors encoding NPY; 8 weeks after viral injection, balance and motor coordination behaviour and neuropathology were analysed. NPY levels were decreased in two MJD patients' cerebella and in striata and cerebella of disease mouse models. Furthermore, NPY overexpression alleviated motor coordination impairments and attenuated related neuropathological cerebellar parameters and decreasing mutant ataxin-3 aggregation. Additionally, NPY mediated increase of brain-derived neurotrophic factor levels and decreased neuroinflammation markers. Our data suggest that NPY is a potential therapeutic strategy for MJD. This work was supported by The Richard Chin and Lily Lock Research Fund; E-Rare4/0003/2012; FCT SFRH/BD/74993/2010, SFRH/BD/87404/ 2012, SRFH/BPD/87341/2012; QREN – Projeto Mais Centro – New Strategies to Manage Brain Diseases CENTRO-07-ST24-FEDER-002002, FEDER and COMPETE – UID/NEU/04539/2013.

NPY/Y2 gene therapeutic overexpression in hippocampus of experimental Beagle dogs

people worldwide. Current treatment is only symptomatic and fails to control seizures in about 40% of patients. Gene therapy with NPY overexpression has emerged as an alternative treatment strategy for epilepsy, showing inhibitory effect on seizures in animal models. This effect seems to be mediated mostly via Y2 receptors, while Y1 receptor activation has an opposite outcome. To enhance NPY gene transfer effect on seizures, we additionally overexposed Y2 receipts using AAV vectors in the hippocampus of epileptic rats. We demonstrate that transgene Y2 receptors are functional and even potentiate transgene NPY effect on seizures. These data suggest that combinatorial NPY/Y2 gene therapy could be an alternative strategy to single NPY gene overexertion.

Hypoxic Remodeling of Fetal Cerebral Arteries Involves The NPY/Y1 Pathway

The sympathetic perivascular innervation has long been suggested to exert trophic influences on cerebral arteries, particularly during hypoxic adaptation. Although most of this effect has been attributed to the actions of norepinephrine, sympathetic nerves also co‐release NPY together with norepinephrine. This study tests the hypothesis that NPY released from sympathetic nerves also contributes to hypoxic remodeling of fetal lamb cerebral arteries. Hypoxic acclimatization for 110 days increased NPY levels by 230% in nerve intact arteries. Whereas sympathectomy (SANX) had no significant effect in normoxic arteries, it decreased NPY levels in hypoxic arteries (25%). Hypoxia also increased Y1 receptor levels (70%) but only in nerve intact arteries. SANX decreased Y1 levels (30%) in hypoxic arteries. Confocal microscopy revealed that organ culture with 100 nM NPY potently decreased the colocalization of Non‐Muscle Myosin with Smooth Muscle Alpha‐Actin, indicating increased contractile differentiation. In nerve intact arteries, NPY decreased myogenic tone whereas in SANX arteries NPY increased myogenic tone. Co‐culture with the Y1 antagonist BIBP‐3226 (5 µM) reversed these effects of NPY. Taken together, these results suggest that NPY released from sympathetic nerves contributes to remodeling of the fetal cerebral vasculature during hypoxic acclimatization.

Effects of Intranasal Neuropeptide Y and HS014 in Preventing Anxiety and Depressive‐like Behavior Elicited by Single Prolonged Stress Animal Model of PTSD: Comparative Study

Stress triggered neuropsychiatric disorders are a serious societal problem. Prophylactic treatment or early intervention has great potential in increasing resilience to traumatic stress and reducing its harmful impact. Previously, we demonstrated proof of concept that intranasal administration of NPY or the MC4 receptor antagonist, HS014, prior to single prolonged stress (SPS) rodent PTSD model, can prevent or attenuate many PTSD associated impairments. Here, we compare effects of NPY and HS014 given 30 min before or immediately after SPS stressors on development of anxiety, depressive‐like behavior and associated biochemical abnormalities. SPS triggered anxiety on EPM was reduced by intranasal pre‐administration of 100 µg NPY and to even greater extent HS014 (3.5 ng or 100 µg). The SPS‐elicited depressive‐like behavior on FST was prevented with 100 µg NPY or the high dose HS014. Combined administration of low HS014 and NPY, ineffective by themselves, prevented development of depressive‐like behavior. Reductions in stress triggered activation of LC/noradrenergic system and HPA axis were observed with both HS014 and NPY. In contrast to NPY, early intervention with HS014 immediately after SPS did not prevent the development of anxiogenic behavior on EPM. However, HS014 given after SPS stressors, even at very low dose, prevented development of depressive‐like behavior. Thus, both MC4R antagonist and NPY, alone or combined, have potential for prophylactic treatment against traumatic stress triggered anxiety or depressive‐like behaviors, while NPY has more widespread potential for early intervention. (Supported by US Amy grant DM102281)

Neuropeptide Y promotes adipogenesis in chicken adipose cells in vitro

Neuropeptide Y is an evolutionarily conserved neurotransmitter that stimulates food intake in higher vertebrate species and promotes adipogenesis in mammals. The objective of this study was to determine if NPY also enhances adipogenesis in birds, using chickens as a model. The stromal–vascular fraction of cells was isolated from the abdominal fat of 14day-old broiler chicks and effects of exogenous chicken NPY on proliferation and differentiation determined. Based on a thymidine analog incorporation assay and gene expression analysis, there was no effect of NPY on proliferation during the first 12hours post-treatment in cells that were induced to proliferate. However, there were effects of NPY treatment on proliferation and lipid accumulation during the first 6days post-induction of differentiation. Neuropeptide Y supplementation during induction of differentiation was associated with greater glycerol-3-phosphate dehydrogenase activity and staining for neutral lipids, indicative of augmented lipid accumulation. This was also accompanied by increased proliferation during differentiation, which was characterized by up-regulation of proliferation and preadipocyte marker mRNA, and a greater number of proliferating cells in groups that were treated with NPY. Additionally, NPY treatment was associated with increased expression of fatty acid binding protein 4 and lipoprotein lipase during differentiation. In conclusion, these results suggest that NPY plays a role in promoting adipogenesis in chickens and that the mechanisms involve an increase in the synthesis of new preadipocytes and increased lipid synthesis and storage.