Neuropeptide Y Receptor Research Articles

Galanin and neuropeptide Y interactions elicit antidepressant activity linked to neuronal precursor cells of the dentate gyrus in the ventral hippocampus.

A need for new antidepressants is necessary since traditional antidepressants have several flaws. Neuropeptide Y(NPY) Y1 receptor (NPYY1R) and galanin (GAL) receptor 2 (GALR2) interact in several regions of the limbic system, including the hippocampus. The current study assesses the antidepressant effects induced by GALR2 and NPYY1R coactivation, together with the evaluation of cell proliferation through 5-Bromo-2'-deoxyuridine expression within the dentate gyrus of the ventral hippocampus (vDG). We employed in situ proximity ligation assay to manifest GALR2/NPYY1R heteroreceptor complexes. Additionally, the expression pattern of GALR2 and the activation of the extracellular-regulated kinases (ERK) pathway after GALR2 and NPYY1R costimulation in cell cultures were examined. GALR2 and NPYY1R coactivation resulted in sustained antidepressant behaviors in the FST after 24 h, linked to increased cell proliferation in the vDG. Moreover, an increased density of GALR2/NPYY1R heteroreceptor complexes was observed in vDG, on doublecortin-expressing neuroblasts. Recruitment of the GALR2 expression to the plasma membrane was observed upon the coactivation of GALR2 and NPYY1R in cell cultures, presumably associated to the enhanced effects on the activation of ERK pathway. GALR2 may promote the GALR2/NPYY1R heteroreceptor complexes formation in the ventral hippocampus. It may induce a transformation of cell proliferation toward a neuronal lineage by enhancement of ERK pathway. Thus, it may give the mechanism for the antidepressant behavior observed. These results may provide the basis for the development of heterobivalent agonist pharmacophores, targeting GALR2/NPYY1R heteromers, especially in the neuronal precursor cells of the dentate gyrus in the ventral hippocampus for the novel treatment of depression.

Neuropeptide Y Is an Immunomodulatory Factor: Direct and Indirect.

Neuropeptide Y (NPY), which is widely distributed in the nervous system, is involved in regulating a variety of biological processes, including food intake, energy metabolism, and emotional expression. However, emerging evidence points to NPY also as a critical transmitter between the nervous system and immune system, as well as a mediator produced and released by immune cells. In vivo and in vitro studies based on gene-editing techniques and specific NPY receptor agonists and antagonists have demonstrated that NPY is responsible for multifarious direct modulations on immune cells by acting on NPY receptors. Moreover, via the central or peripheral nervous system, NPY is closely connected to body temperature regulation, obesity development, glucose metabolism, and emotional expression, which are all immunomodulatory factors for the immune system. In this review, we focus on the direct role of NPY in immune cells and particularly discuss its indirect impact on the immune response.

Protective effect of neuropeptide Y2 receptor activation against methamphetamine-induced brain endothelial cell alterations

Methamphetamine (METH) consumption is a health problem that leads to neurological and psychiatric disturbances. The cellular alterations behind these conditions have been extensively investigated and it is now well-established that METH causes cerebrovascular alterations being a key feature in drug-induced neuropathology.Although promising advances in understanding the blood-brain barrier (BBB) alterations induced by METH, there is still no available approach to counteract or diminish such effects. Interestingly, several studies show that neuropeptide Y (NPY) has an important protective role against METH-induced neuronal and glial toxicity, as well as behavioral deficits. Despite these beneficial effects of the NPY system, nothing is known about its role in brain endothelial cells under conditions of METH exposure.Thus, our aim was to unravel the effect of NPY and its receptors against METH-induced endothelial cell dysfunction. For that, we used a human brain microvascular endothelial cell line (hCMEC/D3) and our results demonstrate that endothelial cells express both NPY Y1 (Y1R) and Y2 (Y2R) receptors, but only Y2R is upregulated after METH exposure. Moreover, this drug of abuse induced endothelial cell death and elicited the production of reactive oxygen species (ROS) by these cells, which were prevented by the activation of Y2R. Additional, cell death and oxidative stress triggered by METH were dependent on the concentration of the drug. In sum, with the present study we identified for the first time the NPY system, and particularly the Y2R subtype, as a promising target to protect against METH-induced neurovascular dysfunction.

Probing the Y2 Receptor on Transmembrane, Intra- and Extra-Cellular Sites for EPR Measurements

The function of G protein-coupled receptors is intrinsically linked to their conformational dynamics. In conjugation with site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy provides powerful tools to study the highly dynamic conformational states of these proteins. Here, we explored positions for nitroxide spin labeling coupled to single cysteines, introduced at transmembrane, intra- and extra-cellular sites of the human neuropeptide Y2 receptor. Receptor mutants were functionally analyzed in cell culture system, expressed in Escherichia coli fermentation with yields of up to 10 mg of purified protein per liter expression medium and functionally reconstituted into a lipid bicelle environment. Successful spin labeling was confirmed by a fluorescence assay and continuous wave EPR measurements. EPR spectra revealed mobile and immobile populations, indicating multiple dynamic conformational states of the receptor. We found that the singly mutated positions by MTSL ((1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate) have a water exposed immobilized conformation as their main conformation, while in case of the IDSL (bis(1-oxyl-2,2,5,5-tetramethyl-3-imidazolin-4-yl) disulfide) labeled positions, the main conformation are mainly of hydrophobic nature. Further, double cysteine mutants were generated and examined for potential applications of distance measurements by double electron–electron resonance (DEER) pulsed EPR technique on the receptor.

Mechanisms of action of neuropeptide Y on stem cells and its potential applications in orthopaedic disorders.

Musculoskeletal disorders are the leading causes of disability and result in reduced quality of life. The neuro-osteogenic network is one of the most promising fields in orthopaedic research. Neuropeptide Y (NPY) system has been reported to be involved in the regulations of bone metabolism and homeostasis, which also provide feedback to the central NPY system via NPY receptors. Currently, potential roles of peripheral NPY in bone metabolism remain unclear. Growing evidence suggests that NPY can regulate biological actions of bone marrow mesenchymal stem cells, hematopoietic stem cells, endothelial cells, and chondrocytes via a local autocrine or paracrine manner by different NPY receptors. The regulative activities of NPY may be achieved through the plasticity of NPY receptors, and interactions among the targeted cells as well. In general, NPY can influence proliferation, apoptosis, differentiation, migration, mobilization, and cytokine secretion of different types of cells, and play crucial roles in the development of bone delayed/non-union, osteoporosis, and osteoarthritis. Further basic research should clarify detailed mechanisms of action of NPY on stem cells, and clinical investigations are also necessary to comprehensively evaluate potential applications of NPY and its receptor-targeted drugs in management of musculoskeletal disorders.

Shuttling of Peptide‐Drug Conjugates by G Protein‐Coupled Receptors Is Significantly Improved by Pulsed Application

G protein‐coupled receptors (GPCRs) can be used to shuttle peptide‐drug conjugates into cells. But, for efficient therapy, a high concentration of cargo needs to be delivered. To explore this, we studied the pharmacologically interesting neuropeptide Y1 receptor (Y1R) in one recombinant and three oncogenic cell systems that endogenously express the receptor. We demonstrate that recycled receptors behave identically to newly synthesized receptors with respect to ligand binding and internalization pathways. Depending on the cell system, biosynthesis, recycling efficiency, and peptide uptake differ partially, but shuttling was efficient in all systems. However, by comparing continuous application of the ligand for four hours to four cycles of internalization and recycling in between, a significantly higher amount of peptide uptake was achieved in the pulsed application (150–250 % to 300–400 %). Accordingly, in this well‐suited drug shuttle system pulsed application is superior under all investigated conditions and should be considered for innovative, targeted drug delivery in general.

Sex Differences in the Neuropeptide Y System and Implications for Stress Related Disorders.

The neuropeptide Y (NPY) system is emerging as a promising therapeutic target for neuropsychiatric disorders by intranasal delivery to the brain. However, the vast majority of underlying research has been performed with males despite females being twice as susceptible to many stress-triggered disorders such as posttraumatic stress disorder, depression, anorexia nervosa, and anxiety disorders. Here, we review sex differences in the NPY system in basal and stressed conditions and how it relates to varied susceptibility to stress-related disorders. The majority of studies demonstrate that NPY expression in many brain areas under basal, unstressed conditions is lower in females than in males. This could put them at a disadvantage in dealing with stress. Knock out animals and Flinders genetic models show that NPY is important for attenuating depression in both sexes, while its effects on anxiety appear more pronounced in males. In females, NPY expression after exposure to stress may depend on age, timing, and nature and duration of the stressors and may be especially pronounced in the catecholaminergic systems. Furthermore, alterations in NPY receptor expression and affinity may contribute to the sex differences in the NPY system. Overall, the review highlights the important role of NPY and sex differences in manifestation of neuropsychiatric disorders.

Abstract 5168: The dual regulatory role of Neuropeptide Y in colon cancer

Abstract Introduction: Colon cancer is the third most common cancer and affects approximately one million people every year. Patients with inflammatory bowel diseases (IBDs) have a higher risk of developing colon cancer. The neuropeptides, particularly neuropeptide Y (NPY) which is a 36-amino acid neuropeptide found in the brain, the autonomic nervous system and different peripheral tissues including the colon is associated with the development and progression of IBD. Objective: The objective of this study was to determine the role of NPY in colon cancer and establish NPY and its receptors as new targets for colon cancer. Methods: Immunohistochemical analysis was performed to detect the expression of NPY in colon cancer. Colon cancer (CT26) bearing mice were treated with both NPY Y1 receptor (Y1R) (BIBO 3304) or Y2 receptor (Y2R) (BIIE0246) specific antagonists and the effect on tumor growth and progression was studied. In vitro tumor and endothelial cell proliferation assays and colonic endothelial cell (CEC) migration and tubule forming assays were also conducted. Results and Conclusion: Our results show that there is significant upregulation of NPY in colon cancer and NPY is profusely secreted by colonic epithelial cells. Treatment of CT26 tumor bearing mice with both Y1R and Y2R antagonists resulted in significant reduction of tumor growth. Treatment with Y1R antagonist was associated with reduced number of proliferative cells in tumor tissues, while Y2R antagonist treated tumor tissues showed significant reduction in angiogenesis (micro vessel density). However, tumor cell apoptosis was significantly increased in both the treated groups compared to vehicle treated control group. Our in vitro results showed that treatment with NPY (100nM) significantly increased CT26 tumor cell as well as CEC proliferation compared to respective untreated control cells. However, when CT26 tumor cells were previously treated with Y1R antagonist (1μM) the effect of NPY was abrogated. Y2R antagonist (1μM) prior treated tumor cells didn't show any significant reduction in NPY induced tumor cell proliferation. On the contrary, when CEC were prior treated with Y2R antagonist, NPY mediated CEC proliferation was abrogated. Prior treatment with Y1R antagonist had no effect on this function of NPY. NPY also promoted migration and tube formation capabilities of CEC. Prior treatment with Y2R antagonist also significantly decreased these NPY mediated functions of CEC. Y1 antagonist had no effect on CEC. Our results thus show that NPY by acting through Y1 receptors, which are predominantly expressed by tumor cells, controls the proliferation and growth of the cancer cells. Also, NPY by acting through Y2 receptors that are mostly expressed by the endothelial cells controls the functions of these cells present in the tumor microenvironment. This to our knowledge is the first report about the dual regulatory role of NPY in colon cancer by which it controls both the tumor cells as well as the endothelial cells in the surrounding microenvironment. Citation Format: Sandeep Goswami, Debanjan Chakroborty, Sujit Basu, Wendy Frankel, Chandrani Sarkar. The dual regulatory role of Neuropeptide Y in colon cancer [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 5168.

Abstract 6157: Hypoxia increases motility of neuroblastoma cells induced by the neuropeptide Y/Y5 receptor pathway and exacerbates their metastatic potential

Abstract Neuroblastoma (NB) is a pediatric malignancy that originates from precursors of sympathetic neurons and expresses neuronal proteins, such as neuropeptide Y (NPY) and its receptors. NB cells constitutively express NPY Y2 receptor (Y2R), which maintains their proliferation and tumor vascularization. However, pro-apoptotic conditions additionally induce expression of Y5 receptor (Y5R), which acts as a survival factor for tumor cells. High Y5R expression was also observed in angioinvasive NB cells surrounding blood vessels, suggesting a role for this receptor in NB dissemination. Indeed, in NB patients, high serum levels of NPY associated with the presence of metastases. Hence, the goal of this study was to identify mechanisms that may underlie potential pro-metastatic effects of NPY. We have found that NPY, acting via Y5R, stimulates NB cell motility and acts as a chemotactic factor for these cells in a concentration-dependent manner. Hypoxia, a known condition promoting metastasis, increased expression of Y5R in SK-N-BE(2) and SK-N-AS NB cell lines. Consequently, hypoxic NB cells had increased motility and a chemotactic response to NPY. This increase in NPY-driven migration was fully blocked by Y5R antagonist. Pre-incubation of NB cells in hypoxia before orthotopic injections into adrenal glands of SCID/bg mice exacerbated metastasis in xenografts from both NB cell lines. In mice bearing non-MYCN amplified SK-N-AS tumors, the hypoxia-driven metastases were located in soft tissues, while MYCN-amplified SK-N-BE(2) xenografts derived from hypoxic cells exhibited enhanced dissemination to lungs. The latter pattern of metastases is also observed in NB patients with MYCN amplification. Further studies are required to determine if the hypoxia-driven increase in NB metastasis is Y5R-dependent. Altogether, our data support the role of tumor hypoxia and NPY/Y5R axis in NB dissemination. Since this pathway is also activated during chemotherapy and promotes NB cells resistance to treatment, NPY/Y5R may also be involved in the secondary dissemination of the recurrent tumors. If the role of Y5R in NB metastasis is validated in preclinical models, this NPY receptor may become a potential therapeutic target preventing the disease dissemination. Citation Format: Nouran Abualsaud, Lindsay Caprio, Sung Hyeok Hong, Susana Galli, Joanna Kitlinska. Hypoxia increases motility of neuroblastoma cells induced by the neuropeptide Y/Y5 receptor pathway and exacerbates their metastatic potential [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 6157.

Generation and characterisation of C-terminally stabilised PYY molecules with potential in vivo NPYR2 activity

BackgroundActivation of neuropeptide Y2 receptors (NPYR2) by the N-terminally truncated, dipeptidyl peptidase-4 (DPP-4) generated, Peptide YY (PYY) metabolite, namely PYY(3–36), results in satiating actions. However, PYY(3–36) is also subject to C-terminal enzymatic cleavage, which annuls anorectic effects. MethodsSubstitution of l-Arg35 with d-Arg35 in the DPP-4 stable sea lamprey PYY(1–36) peptide imparts full C-terminal stability. In the current study, we have taken this molecule and introduced DPP-4 susceptibility by Iso3 substitution. ResultsAs expected, [Iso3]sea lamprey PYY(1–36) and [Iso3](d-Arg35)sea lamprey PYY(1–36) were N-terminally degraded to respective PYY(3–36) metabolites in plasma. Only [Iso3](d-Arg35)sea lamprey PYY(1–36) was C-terminally stable. Both peptides possessed similar insulinostatic and anti-apoptotic biological actions to native PYY(1–36) in beta-cells. Unlike native PYY(1–36) and [Iso3](d-Arg35)sea lamprey PYY(1–36), [Iso3]sea lamprey PYY(1–36) displayed some proliferative actions in Npyr1 knockout beta-cells. In addition, [Iso3]sea lamprey PYY(1–36) induced more rapid NPYR2-dependent appetite suppressive effects in mice than its C-terminally stable counterpart. Twice daily administration of either peptide to high fat fed (HFF) mice resulted in significant body weight reduction and improvements in circulating triglyceride levels. [Iso3]sea lamprey PYY(1–36) treatment also prevented elevations in glucagon. Both peptides, and especially [Iso3]sea lamprey PYY(1–36), improved glucose tolerance. The treatment interventions also partially reversed the deleterious effects of sustained high fat feeding on pancreatic islet morphology. ConclusionThe present study confirms that sustained NPYR2 receptor activation by [Iso3](d-Arg35)sea lamprey induced significant weight lowering actions. However, identifiable benefits of this peptide over [Iso3]sea lamprey PYY(1–36), which was not protected against C-terminal degradation, were not pronounced.

Targeting spinal neuropeptide Y1 receptor-expressing interneurons to alleviate chronic pain and itch.

An accelerating basic science literature is providing key insights into the mechanisms by which spinal neuropeptide Y (NPY) inhibits chronic pain. A key target of pain inhibition is the Gi-coupled neuropeptide Y1 receptor (Y1). Y1 is located in key sites of pain transmission, including the peptidergic subpopulation of primary afferent neurons and a dense subpopulation of small, excitatory, glutamatergic/somatostatinergic interneurons (Y1-INs) that are densely expressed in the dorsal horn, particularly in superficial lamina I-II. Selective ablation of spinal Y1-INs with an NPY-conjugated saporin neurotoxin attenuates the development of peripheral nerve injury-induced mechanical and cold hypersensitivity. Conversely, conditional knockdown of NPY expression or intrathecal administration of Y1 antagonists reinstates hypersensitivity in models of chronic latent pain sensitization. These and other results indicate that spinal NPY release and the consequent inhibition of pain facilitatory Y1-INs represent an important mechanism of endogenous analgesia. This mechanism can be mimicked with exogenous pharmacological approaches (e.g. intrathecal administration of Y1 agonists) to inhibit mechanical and thermal hypersensitivity and spinal neuron activity in rodent models of neuropathic, inflammatory, and postoperative pain. Pharmacological activation of Y1 also inhibits mechanical- and histamine-induced itch. These immunohistochemical, pharmacological, and cell type-directed lesioning data, in combination with recent transcriptomic findings, point to Y1-INs as a promising therapeutic target for the development of spinally directed NPY-Y1 agonists to treat both chronic pain and itch.

The Effects of NPY1 Receptor Antagonism on Intervertebral Disc and Bone Changes in Ovariectomized Rats.

Study Design:Basic science.Objective:To compare the effects of a neuropeptide Y1 receptor antagonist (NPY-1RA) to estrogen on maintaining vertebral bone microarchitecture and disc height in a rat model of menopause.Methods:This study was an institutional animal care approved randomized control study with 104 ovariectomized rats and 32 intact control animals. Comparison of disc height, trabecular bone, body weights, circulating levels of NPY and estrogen, and distribution of Y1 receptors in the intervertebral disc in an established rodent osteoporotic model were made at baseline and after 2, 4, and 8 weeks after receiving either an implant containing estrogen or an antagonist to the neuropeptide Y1 receptor. Data was compared statistically using One-way analysis of variance.Results:Circulating levels of estrogen increased and NPY decreased following estrogen replacement, with values comparable to ovary-intact animals. NPY-1RA-treated animals had low estrogen and high NPY circulating levels and were similar to ovariectomized control rats. Both NPY-1RA and estrogen administration were able reduce, menopause associated weight gain. NPY-1RA appeared to restore bone formation and maintain disc height, while estrogen replacement prevented further bone loss.Conclusion:NPY-1RA in osteoporotic rats activates osteoblast production of bone and decreased marrow and body fat more effectively than estrogen replacement when delivered in similar concentrations. Annulus cells had NPY receptors, which may play a role in disc nutrition, extracellular matrix production, and pain signaling cascades.

The anorexigenic effect of beta-melanocyte-stimulating hormone involves corticotrophin-releasing factor and mesotocin in birds

Beta-melanocyte-stimulating hormone (β-MSH), when centrally injected, induces anorexigenic effects in rodents and chickens but its mechanism remains unclear. Thus, the primary goal of this research was to elucidate the hypothalamic mechanism using chickens. Intracerebroventricular injection of 0.3, 1.0 and 3.0 nmol of β-MSH decreased food intake for 540 min. Expression of hypothalamic mRNAs were affected by β-MSH injection, including corticotrophin-releasing factor (CRF) and its receptor subtype 1 (CRFR1), mesotocin (MT) and its receptor (MTR), pro-opiomelanocortin, cocaine- and amphetamine-regulated transcript (CART), growth hormone secretagogue receptor (GHSR) and neuropeptide Y (NPY) receptor subtype 5 (NPYR5). Within the arcuate nucleus, expressions of NPY, agouti-related peptide, MT and MTR were increased by β-MSH injection. β-MSH-treated chicks had more CRF, CRFR1, CRF receptor subtype 2, GHSR, NPY receptor subtype 1 (NPYR1) and NPYR5 mRNA but lower levels of CART and ghrelin, in the paraventricular nucleus. Greater amounts of mRNA for MTR, GHSR, NPYR1 and NPYR5 and less CRF expression were observed in the ventromedial hypothalamus. In conclusion, central injection of β-MSH potently reduced food intake and was associated with changes in mRNA expression of some anorexigenic factors in a hypothalamic nucleus-specific manner.

Determination of neuropeptide Y Y1 receptor antagonist BIBP 3226 and evaluation of receptor expression based on liquid chromatography coupled with tandem mass spectrometry.

Neuropeptide Y (NPY) is a peptide widely distributed throughout the body that is involved in various physiological processes, including the regulation of feeding behavior and energy homeostasis. 5-Carbamimidamido-2-(2,2-diphenylacetamido)-N-[(4-hydroxyphenyl)methyl]pentanamide (BIBP 3226) is a selective NPY Y1 receptor antagonist with recognized application in bone regeneration studies, requiring quantification at picogram levels. Hence, BIBP 3226 determination is proposed here by a validated HPLC-MS/MS method, based on a reversed-phase Kinetex® core-shell C8 column (2.6μm, 150 × 2.1mm) at 30°C, elution in isocratic mode using a mixture of acetonitrile and water (30:70, v/v), containing 0.1% (v/v) formic acid, at 0.25mLmin-1, detection in positive ionization mode, and data acquisition in selected reaction monitoring mode. Calibration curves were linear for concentrations ranging from 0.25 to 30ngmL-1 with LOD and LOQ values as low as 0.1 and 0.3pg in cell extracts and 16 and 48pg in supernatant culture media, respectively. BIBP 3226 was successfully determined in cell extracts and supernatants obtained from internalization assays. Using similar exposure conditions, the amount of BIBP 3226 found in breast cancer cells (MCF7) was 72 to 657 times higher than that found in bone marrow cells (Wt C57BL/6 mice), providing an indirect indicator of NPY Y1 receptor expression.

Effects of Y1 receptor agonist on the pancreatic islet of diet-induced obese and diabetic mice

AimsAgonists of the NPY receptor might be potential in protecting pancreatic islets from injury. We aimed to characterize the role of [Leu31, Pro34]-PYY, an NPYR1 agonist, in pancreatic islets of a diet-induced obesity and insulin resistance model. MethodsWe studied long-term high-fat diet intake as a model and selective agonist of the Y1 receptor to explore the pancreatic islet architecture and stereology, and insulin secretion in isolated islets and a whole animal model. Gene and protein expressions were assessed in isolated islets investigating the signaling cascades involved in inflammation, insulin signaling, and secretion. Also, the insulin release potential was studied in vitro. ResultsOur data reveal that an infusion of NPYR1 for 14 days did not change the body mass of mice and eating behavior. NPYR1 did not modify the islet and beta-cell mass but positively impacted the inflammatory process by lowering the expressions of Tnf alpha and If gamma. Besides, NPYR1 restored the insulin signaling and the exocytose pattern by activating the PDX1/STAT3 pathway and improving the leptin signaling cascade. ConclusionsThe findings are compellingly indicating the potential effect of the NPYR1 as a target for improving the insulin resistance condition. As such, the infusion of the NPYR1 agonist would help to enhance insulin secretion by the beta-cell from the PDX1/STAT3 pathway and the improvement of the inflammatory process.

Type A Trichothecene Diacetoxyscirpenol-Induced Emesis Corresponds to Secretion of Peptide YY and Serotonin in Mink

The trichothecene mycotoxins contaminate cereal grains and have been related to alimentary toxicosis resulted in emetic response. This family of mycotoxins comprises type A to D groups of toxic sesquiterpene chemicals. Diacetoxyscirpenol (DAS), one of the most toxic type A trichothecenes, is considered to be a potential risk for human and animal health by the European Food Safety Authority. Other type A trichothecenes, T-2 toxin and HT-2 toxin, as well as type B trichothecene deoxynivalenol (DON), have been previously demonstrated to induce emetic response in the mink, and this response has been associated with the plasma elevation of neurotransmitters peptide YY (PYY) and serotonin (5-hydroxytryptamine, 5-HT). However, it is found that not all the type A and type B trichothecenes have the capacity to induce PYY and 5-HT. It is necessary to identify the roles of these two emetogenic mediators on DAS-induced emesis. The goal of this study was to determine the emetic effect of DAS and relate this effect to PYY and 5-HT, using a mink bioassay. Briefly, minks were fasted one day before experiment and given DAS by intraperitoneally and orally dosing on the experiment day. Then, emetic episodes were calculated and blood collection was employed for PYY and 5-HT test. DAS elicited robust emetic responses that corresponded to upraised PYY and 5-HT. Blocking the neuropeptide Y2 receptor (NPY2R) diminished emesis induction by PYY and DAS. The serotonin 3 receptor (5-HT3R) inhibitor granisetron totally restrained the induction of emesis by serotonin and DAS. In conclusion, our findings demonstrate that PYY and 5-HT have critical roles in DAS-induced emetic response.

Cardiac sympathetic activation circumvents high-dose beta blocker therapy in part through release of neuropeptide Y

The sympathetic nervous system plays an important role in the occurrence of ventricular tachycardia (VT). Many patients, however, experience VT despite maximal doses of beta blocker therapy, possibly due to the effects of sympathetic cotransmitters such as neuropeptide Y (NPY). The purpose of this study was to determine, in a porcine model, whether propranolol at doses higher than clinically recommended could block ventricular electrophysiological effects of sympathoexcitation via stellate ganglia stimulation, and if any residual effects are mediated by NPY. Greater release of cardiac NPY was observed at higher sympathetic stimulation frequencies (10 and 20 vs. 4 Hz). Despite treatment with even higher doses of propranolol (1.0 mg/kg), electrophysiological effects of sympathetic stimulation remained, with residual shortening of activation recovery interval (ARI), a surrogate of action potential duration (APD). Adjuvant treatment with the NPY Y1 receptor antagonist BIBO 3304, however, reduced these electrophysiological effects while augmenting inotropy. These data demonstrate that high-dose beta blocker therapy is insufficient to block electrophysiological effects of sympathoexcitation, and a portion of these electrical effects in vivo are mediated by NPY. Y1 receptor blockade may represent a promising adjuvant therapy to beta-adrenergic receptor blockade.

SUN-660 Appetite Suppressing Effects of Glucoregulatory Chimeric Peptides Devoid of Nausea

Current treatments for type 2 diabetes (T2D) and obesity do not reliably achieve long-term weight-loss and up to 50% of patients experience nausea and vomiting. Thus, there is a critical need for obesity medications that provide glycemic control with enhanced hypophagic response without nausea. Based on rational design and in silico modelling of the glucagon-like peptide 1-receptor agonist (GLP1RA) exendin-4 (Ex4) and the neuropeptide Y2 receptor (Y2R) agonist peptide YY (PYY) 3-36, our group has developed and tested several new monomeric chimeric peptides that display dual agonism of the anorectic Y2R and the glucoregulatory GLP1R. In addition, we explored a third agonistic behavior at the neuropeptide Y1 receptor (Y1R), which potentially relates to pancreatic beta-cell protection. Our current lead, GEP44, binds and robustly activates the Y2R, Y1R, and GLP1R. In our experiments, half maximal effective concentration (EC50) values for GEP44 were 10 nM at Y2R, 24 nM at Y1R, and 330 pM at GLP1R for dose responses of cell-based fluorescence resonance energy transfer assays. For the control substrates PYY3-36, PYY1-36, and Ex4, EC50 values were 16 nM, 12 nM, and 16 pM at the Y2R, Y1R, and GLP1R, respectively. We tested effects of daily injections of these chimeric peptides in adult Sprague-Dawley rats on food intake (FI), body weight (BW) changes, blood glucose levels and an indicator of nausea (kaolin intake). GEP44 produced profound reduction in FI (2-d average GEP44 at 20 nmol/kg FI -71%; Ex-4 20 nmol/kg FI -40%). Anorectic doses of GEP44 did not trigger a pica response assessed by kaolin consumption in treated rats, while in Ex-4 treated rats, kaolin consumption accounted for 28% of total daily solid intake, indicating a clear nausea response. During 11 d of treatment with GEP44, FI was consistently reduced resulting in a significantly stronger reduction of BW compared to Ex4 at the end of treatment (GEP44 -7.6%, Ex4 -3.7%, p<0.05). Furthermore, 5-day injections of GEP44 showed greater reduction of glucose levels than for Ex4 in diet induced obese rats receiving intraperitoneal glucose. These results were supported by results generated using a customized perifusion system that demonstrated acute stimulation of insulin secretion by exposure of isolated rodent and human islets to GEP44 and Ex4 (both p<0.01 vs. vehicle). In conclusion, simultaneously targeting serial anorectic pathways with single molecule chimeric peptides developed by our group can address multiple coexisting conditions, namely obesity and T2D, to more efficaciously reduce FI, BW and blood glucose levels thereby reducing off target effects.

Neuropeptide Y1 receptor antagonist promotes osteoporosis and microdamage repair and enhances osteogenic differentiation of bone marrow stem cells via cAMP/PKA/CREB pathway

Osteoporosis is a common metabolic bone disorder in the elderly population. The accumulation of bone microdamage is a critical factor of osteoporotic fracture. Neuropeptide Y (NPY) has been reported to regulated bone metabolism through Y1 receptor (Y1R). In this study the effects and mechanisms of Y1R antagonist on prevention for osteoporosis were characterized. In the clinical experiment, compared with osteoarthritis (OA), osteoporosis (OP) showed significant osteoporotic bone microstructure and accumulation of bone microdamage. NPY and Y1R immunoreactivity in bone were stronger in OP group, and were both correlated with bone volume fraction (BV/TV). In vivo experiment, Y1R antagonist significantly improved osteoporotic microstructure in the ovariectomized (OVX) rats. And Y1R antagonist promoted RUNX2, OPG and inhibit RANKL, MMP9 in bone marrow. In vitro cell culture experiment, NPY inhibited osteogenesis, elevated RANKL/OPG ratio and downregulated the expression of cAMP, p-PKAs and p-CREB in BMSCs, treated with Y1R antagonist or 8-Bromo-cAMP could inhibit the effects of NPY. Together, Y1R antagonist improved the bone microstructure and reduced bone microdamage in OVX rats. NPY-Y1R could inhibit osteoblast differentiation of BMSCs via cAMP/PKA/CREB pathway. Our findings highlight the regulation of NPY-Y1R in bone metabolism as a potential therapy strategy for the prevention of osteoporosis and osteoporotic fracture.

Neuropeptide Y Expression Defines a Novel Class of GABAergic Projection Neuron in the Inferior Colliculus.

Located in the midbrain, the inferior colliculus (IC) integrates information from numerous auditory nuclei and is an important hub for sound processing. Despite its importance, little is known about the molecular identity and functional roles of defined neuron types in the IC. Using a multifaceted approach in mice of both sexes, we found that neuropeptide Y (NPY) expression identifies a major class of inhibitory neurons, accounting for approximately one-third of GABAergic neurons in the IC. Retrograde tracing showed that NPY neurons are principal neurons that can project to the medial geniculate nucleus. In brain slice recordings, many NPY neurons fired spontaneously, suggesting that NPY neurons may drive tonic inhibition onto postsynaptic targets. Morphologic reconstructions showed that NPY neurons are stellate cells, and the dendrites of NPY neurons in the tonotopically organized central nucleus of the IC cross isofrequency laminae. Immunostaining confirmed that NPY neurons express NPY, and we therefore hypothesized that NPY signaling regulates activity in the IC. In crosses between Npy1rcre and Ai14 Cre-reporter mice, we found that NPY Y1 receptor (Y1R)-expressing neurons are glutamatergic and were broadly distributed throughout the rostrocaudal extent of the IC. In whole-cell recordings, application of a high-affinity Y1R agonist led to hyperpolarization in most Y1R-expressing IC neurons. Thus, NPY neurons represent a novel class of inhibitory principal neurons that are well poised to use GABAergic and NPY signaling to regulate the excitability of circuits in the IC and auditory thalamus.SIGNIFICANCE STATEMENT The identification of neuron types is a fundamental question in neuroscience. In the inferior colliculus (IC), the hub of the central auditory pathway, molecular markers for distinct classes of inhibitory neurons have remained unknown. We found that neuropeptide Y (NPY) expression identifies a class of GABAergic principal neurons that constitute one-third of the inhibitory neurons in the IC. NPY neurons fire spontaneously, have a stellate morphology, and project to the auditory thalamus. Additionally, we found that NPY signaling hyperpolarized the membrane potential of a subset of excitatory IC neurons that express the NPY Y1 receptor. Thus, NPY neurons are a novel class of inhibitory neurons that use GABA and NPY signaling to regulate activity in the IC and auditory thalamus.