Y1 Receptor Research Articles

Neuropeptide Y receptors in GtoPdb v.2023.1

Neuropeptide Y (NPY) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Neuropeptide Y Receptors [158]) are activated by the endogenous peptides neuropeptide Y, neuropeptide Y-(3-36), peptide YY, PYY-(3-36) and pancreatic polypeptide (PP). The receptor originally identified as the Y3 receptor has been identified as the CXCR4 chemokine recepter (originally named LESTR, [139]). The y6 receptor is a functional gene product in mouse, absent in rat, but contains a frame-shift mutation in primates producing a truncated non-functional gene [84]. Three-dimensional structures have been determined for subtype active receptors Y1, Y2 and Y4 [211, 114] and inactive antagonist bound Y1 and Y2 receptors [240, 210]. Many of the agonists exhibit differing degrees of selectivity dependent on the species examined. For example, the potency of PP is greater at the rat Y4 receptor than at the human receptor [62]. In addition, many agonists lack selectivity for individual subtypes, but can exhibit comparable potency against pairs of NPY receptor subtypes, or have not been examined for activity at all subtypes. [125I]-PYY or [125I]-NPY can be used to label Y1, Y2, Y5 and y6 subtypes non-selectively, while [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP may be used to label Y5 receptors preferentially (note that cPP denotes chicken peptide sequence and hPP is the human sequence).

QRFP receptor in GtoPdb v.2023.1

The human gene encoding the QRFP receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on the QRFP receptor [19]; QRFPR, formerly known as the Peptide P518 receptor), previously designated as an orphan GPCR receptor was identified in 2001 by Lee et al. from a hypothalamus cDNA library [17]. However, the reported cDNA (AF411117) is a chimera with bases 1-127 derived from chromosome 1 and bases 155-1368 derived from chromosome 4. When corrected, QRFPR (also referred to as SP9155 or AQ27) encodes a 431 amino acid protein that shares sequence similarities in the transmembrane spanning regions with other peptide receptors. These include neuropeptide FF2 (38%), neuropeptide Y2 (37%) and galanin Gal1 (35%) receptors. QRFP receptor was identified as a Gs-coupled GPCR [6, 14] that's activated by the endogenous peptides QRFP43 (43RFa) and QRFP26 (26RFa) [6, 14, 11]. However, Gq- and Gi/o-mediated signaling was also reported [11, 25]. Two naturally occurring mutations in the human QRFP receptor lead to distinct and opposite 26RFa-evoked signaling bias [20].

Formation of Hemihydrate Crystal form Overcomes Milling Issue Induced by Exposed Functional Groups on Cleavage Plane for a Y5 Receptor Antagonist of Neuropeptide Y

This study aimed to investigate the crystal forms of an originally designed Y5 receptor antagonist of neuropeptide Y. Polymorphic screening was performed via solvent evaporation and slurry conversion using various solvents. The obtained crystal forms α, β, and γ were characterized by X-ray powder diffraction analysis. Thermal analysis determined that forms α, β, and γ were hemihydrate, metastable and stable forms, respectively; the hemihydrate and the stable forms were candidates. To arrange the particle size, forms α and γ were subjected to jet milling. However, form γ could not be milled because of powder stiction to the apparatus, whereas form α could be. To investigate this mechanism, single-crystal X-ray diffraction analysis was performed. The crystal structure of form γ was characterized by two-dimensional hydrogen bonding between neighboring molecules. This revealed that the functional groups forming hydrogen bonds were exposed on the cleavage plane of form γ. The three-dimensional hydrogen-bonding network with water stabilized the hemihydrate form, α. These results indicate that the hydrogen bondable groups exposed on the cleavage plane of form γ should result in stiction of the powder and adherence to the apparatus. It was concluded that crystal conversion is a method to overcome the milling issue.

Characterisation of NPFF-expressing neurons in the superficial dorsal horn of the mouse spinal cord

Excitatory interneurons in the superficial dorsal horn (SDH) are heterogeneous, and include a class known as vertical cells, which convey information to lamina I projection neurons. We recently used pro-NPFF antibody to reveal a discrete population of excitatory interneurons that express neuropeptide FF (NPFF). Here, we generated a new mouse line (NPFFCre) in which Cre is knocked into the Npff locus, and used Cre-dependent viruses and reporter mice to characterise NPFF cell properties. Both viral and reporter strategies labelled many cells in the SDH, and captured most pro-NPFF-immunoreactive neurons (75–80%). However, the majority of labelled cells lacked pro-NPFF, and we found considerable overlap with a population of neurons that express the gastrin-releasing peptide receptor (GRPR). Morphological reconstruction revealed that most pro-NPFF-containing neurons were vertical cells, but these differed from GRPR neurons (which are also vertical cells) in having a far higher dendritic spine density. Electrophysiological recording showed that NPFF cells also differed from GRPR cells in having a higher frequency of miniature EPSCs, being more electrically excitable and responding to a NPY Y1 receptor agonist. Together, these findings indicate that there are at least two distinct classes of vertical cells, which may have differing roles in somatosensory processing.

Combinatorial gene therapy for epilepsy: Gene sequence positioning and AAV serotype influence expression and inhibitory effect on seizures.

Gene therapy with AAV vectors carrying genes for neuropeptide Y and its receptor Y2 has been shown to inhibit seizures in multiple animal models of epilepsy. It is however unknown how the AAV serotype or the sequence order of these two transgenes in the expression cassette affects the actual parenchymal gene expression levels and the seizure-suppressant efficacy. To address these questions, we compared three viral vector serotypes (AAV1, AAV2 and AAV8) and two transgene sequence orders (NPY-IRES-Y2 and Y2-IRES-NPY) in a rat model of acutely induced seizures. Wistar male rats were injected bilaterally with viral vectors and 3 weeks later acute seizures were induced by a subcutaneous injection of kainate. The latency until 1st motor seizure, time spent in motor seizure and latency to status epilepticus were measured to evaluate the seizure-suppressing efficacy of these vectors compared to an empty cassette control vector. Based on the results, the effect of the AAV1-NPY-IRES-Y2 vector was further investigated by in vitro electrophysiology, and its ability to achieve transgene overexpression in resected human hippocampal tissue was evaluated. The AAV1-NPY-IRES-Y2 proved to be better to any other serotype or gene sequence considering both transgene expression and ability to suppress induced seizures in rats. The vector also demonstrated transgene-induced decrease of glutamate release from excitatory neuron terminals and significantly increased both NPY and Y2 expression in resected human hippocampal tissue from patients with drug-resistant temporal lobe epilepsy. These results validate the feasibility of NPY/Y2 receptor gene therapy as a therapeutic opportunity in focal epilepsies.

Abstract 1424: Selective blockade of neuropeptide Y receptor 1 induces cell cycle arrest and promotes apoptosis in colon cancer cells

Abstract Introduction: Colorectal cancer (CRC) is a leading cause of cancer related deaths worldwide. The balance between cancer cell proliferation and apoptosis, which maintains intestinal epithelial cell homeostasis, is disturbed in CRC. Furthermore, deregulation of the apoptotic pathway contributes to therapeutic resistance. Therefore, determination of the mechanisms employed by CRC cells to evade apoptosis is important for development of new therapeutic strategies. Our group has recently reported that neuropeptide Y (NPY), which is a 36-amino acid neuropeptide, is associated with increased angiogenic response in colon cancer. However, the regulatory role of NPY in cellular homeostasis in CRC is largely unknown. NPY is secreted by colon cancer cells that express Y1 receptors (Y1R). The aim of the present study was to investigate the role of NPY in regulation of cell growth and survival in CRC and to determine the apoptosis inducing effects of Y1R antagonist, BIBO 3304 trifluoroacetate (BIBO), in CT26 colon carcinoma cells. Methods: Correlation between NPY expression and cell cycle and apoptosis-related genes were analyzed in cBioPortal for cancer genomics with a sample number of 594 CRC patients from the cancer genome atlas database (TCGA). Based on the TCGA database, 16 apoptosis-related genes (11 proapoptotic and 5 antiapoptotic) were checked. The effect of Y1R blocker (BIBO, Bio-Techne, MN) on CT26 (ATCC) cell proliferation, apoptosis and cell cycle progression was determined by performing in vitro cell proliferation assay and flow cytometry. Results and Conclusion: NPY expression in CRC shows a positive correlation with pro-survival/anti-apoptotic genes (BCL2, BCL2L1, BCL2L2, BCL2A1, MCL1) and negative correlation with key genes associated with the induction of cellular apoptosis (GZMB, TRIB3, TPX2, BAX, BAK1, BAD, BID, BIK, BOK, HRK) in CRC. Decreased cell proliferation and increased apoptosis was observed in CT26 mouse CRC cells upon inhibition of Y1R. An increased number of CT26 cells in early and mid-apoptotic phase was observed upon treatment with Y1R antagonist, BIBO, compared to untreated cells. In addition, cell cycle analysis indicated an increase in cell numbers in the G1/S cell cycle transition phase in the BIBO treated group. Our findings therefore indicate that treatment of CT26 CRC cells with Y1R blocker results in cell cycle arrest, which provides an opportunity for the cells to either undergo repair mechanisms or follow the apoptotic pathway. Blocking the actions of NPY by Y1R blockers thus may be a promising therapeutic approach in CRC patients. Citation Format: Sooraj Kakkat, Steven McClellan, Debanjan Chakroborty, Chandrani Sarkar. Selective blockade of neuropeptide Y receptor 1 induces cell cycle arrest and promotes apoptosis in colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1424.

Abstract 3530: Mechanisms underlying Ewing sarcoma-induced osteolysis

Abstract Ewing Sarcoma (ES) is an aggressive pediatric tumor, which often arises in bones and forms osseous metastases, both of which present as osteolytic lesions. Our previous studies indicated that neuropeptide Y (NPY), which is secreted from ES tumors, stimulates tumor-induced bone degradation via its effect on macrophage recruitment and their subsequent osteoclastic differentiation. This effect was dependent on the autocrine activation of the NPY Y5 receptor (Y5R) expressed by ES cells. However, the exact mechanisms of the osteolytic activity induced by the NPY/Y5R axis remained unknown. To identify the osteolytic factors released from ES cells due to the NPY/Y5R autocrine loop activity, we used shRNA to silence NPY or Y5R in SK-ES-1 ES cells and compared concentrations of known factors involved in the regulation of bone homeostasis in their conditioned media by an antibody array. Four secreted factors were found to be significantly decreased after silencing of NPY signaling: interleukin 8 (IL-8; p<0.005), matrix metalloproteinase 9 (MMP-9; p<0.05), intercellular adhesion molecule 1 (ICAM-1; p<0.005), and monocyte chemoattractant protein 1 (MCP-1; p<0.05). Among these proteins, IL-8 and MMP-9 were selected as the main candidates for mediators of NPY-induced osteolysis. To test their role in ES-mediated stimulation of osteolysis, we co-cultured mouse RAW 264.7 cells, that represent monocyte/macrophage lineage, with SK-ES-1 cells in the presence or absence of the MMP-9 inhibitor (JNJ-0966; 10-7M-10-5M) or anti-IL-8 neutralizing antibody (0.1-0.4µg/ml) and tested their effect on osteoclast differentiation detected by TRAP staining. Inhibiting MMP-9 activity significantly decreased osteoclastogenesis of RAW264.7 cells in dose-dependent manner, while blocking IL-8 had no significant effect on the number of formed osteoclasts. These findings implicate MMP-9 as the mediator of NPY osteolytic activity. Further studies are required to understand the exact mechanisms underlying these effects, as well as potential role of NPY-induced release of MCP-1 in macrophage recruitment to ES tumors. Citation Format: James J. Williams, Joanna B. Kitlinska. Mechanisms underlying Ewing sarcoma-induced osteolysis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3530.

Adipocyte reconstitution of Npy4r gene in Npy4r silenced mice promotes diet-induced obesity.

Neural regulation of adipose tissue is crucial in the homeostasis of energy metabolism. Adipose tissue neuropeptide Y (NPY) and its receptors contribute to the development of diet-induced obesity. NPY1R and NPY2R are major receptors for NPY in peripheral tissues including the adipose tissue. NPY receptor 4 (Npy4r) gene is expressed in adipose tissue. However, it is unknown whether Npy4r is involved in the development of diet-induced obesity. Here, we established an immunofluorescence microscopy technique and generated an adipocyte-reconstituted Npy4r gene knockout mouse. Among six adipose depots, we found that NPY is highly expressed around the vasculature in a dot-like fashion in interscapular brown fat and subcutaneous fat, and NPY receptors are expressed in a depot-specific manner. NPY1R is highly expressed in epidydimal fat, interscapular and peri-aortic brown fat, NPY2R in both interscapular and peri-aortic brown fat, and NPY4R in both brown fat and epidydimal fat. Next, we showed that adipocyte-reconstituted expression of Npy4r promoted diet-induced obesity in mice (P < 0.0001). Overall, this study defines the abundance and distribution of NPY and its receptors 1, 2, and 4 in mouse adipose depots, and demonstrates in an adipocyte-reconstituted gene knockout model that adipocyte Npy4r is sufficient to promote diet-induced obesity.

Towards Probing Conformational States of Y2 Receptor Using Hyperpolarized 129Xe NMR.

G protein-coupled receptors can adopt many different conformational states, each of them exhibiting different restraints towards downstream signaling pathways. One promising strategy to identify and quantify this conformational landscape is to introduce a cysteine at a receptor site sensitive to different states and label this cysteine with a probe for detection. Here, the application of NMR of hyperpolarized 129Xe for the detection of the conformational states of human neuropeptide Y2 receptor is introduced. The xenon trapping cage molecule cryptophane-A attached to a cysteine in extracellular loop 2 of the receptor facilitates chemical exchange saturation transfer experiments without and in the presence of native ligand neuropeptide Y. High-quality spectra indicative of structural states of the receptor-cage conjugate were obtained. Specifically, five signals could be assigned to the conjugate in the apo form. After the addition of NPY, one additional signal and subtle modifications in the persisting signals could be detected. The correlation of the spectroscopic signals and structural states was achieved with molecular dynamics simulations, suggesting frequent contact between the xenon trapping cage and the receptor surface but a preferred interaction with the bound ligand.

Effect of Ganmai Dazao Decoction on ethology of rats with PTSD and its mechanism

This study aimed to explore the effect of Ganmai Dazao Decoction on the ethology of rats with posttraumatic stress disorder(PTSD) and study the related mechanism through the changes in magnetic resonance imaging and protein expression. Sixty rats were randomly divided into 6 groups, namely the normal group, the model group, the low(1 g·kg~(-1)), medium(2 g·kg~(-1)), and high-dose Ganmai Dazao Decoction groups(4 g·kg~(-1)), and the positive control group(intragastric administration with 10.8 mg·kg~(-1) of fluoxetine), with 10 rats in each group. Two weeks after inducing PTSD by single-prolonged stress(SPS) in rats, the positive control group was given fluoxetine hydrochloride capsule by gavage, the low, medium, and high-dose groups were given Ganmai Dazao Decoction by gavage, and both the normal group and the model group were given the same volume of normal saline by gavage, each for 7 days. The open field experiment, elevated cross elevated maze, forced swimming experiment, and new object recognition test were carried out for the behavioral test. Three rats in each group were selected to detect the expression of neuropeptide receptor Y1(NPY1R) protein in the hippocampus by Western blot. Then, the other three rats in each group were selected to use the 9.4T magnetic resonance imaging experiment to observe the overall structural changes in the brain region and the anisotropy fraction of the hippocampus. The results of the open field experiment showed that the total distance and central distance of rats in the model group were significantly lower than those in the normal group, and the total distance and central distance of rats in the middle and high-dose Ganmai Dazao Decoction groups were higher than those in the model group. The results of the elevated cross maze test showed that medium and high-dose Ganmai Dazao Decoction remarkably increased the number of open arm entries and the residence time of open arm of rats with PTSD. The results of the forced swimming experiment showed that the immobility time in the water of the model group rats was significantly higher than that of the normal group, and Ganmai Dazao Decoction hugely reduced the immobility time in the water of rats with PTSD. The results of the new object recognition test showed that Ganmai Dazao Decoction significantly increased the exploration time of new objects and familiar objects in rats with PTSD. The results of Western blot showed that Ganmai Dazao Decoction significantly reduced the expression of NYP1R protein in the hippocampus of rats with PTSD. The 9.4T magnetic resonance examination found that there was no significant difference in the structural image among the groups. In the functional image, the fractional anisotropy(FA value) of the hippocampus in the model group was significantly lower than that in the normal group. The FA value of the hippocampus in the middle and high-dose Ganmai Dazao Decoction groups was higher than that in the model group. Ganmai Dazao Decoction reduces the injury of hippocampal neurons by inhibiting the expression of NYP1R in the hippocampus of rats with PTSD, thereby improving the nerve function injury of rats with PTSD and playing a neuroprotective role.

Abstract PR018: Neuropeptide Y as a metastatic factor

Abstract Although neuropeptide Y (NPY) is known mainly as a sympathetic neurotransmitter, growing evidence indicates its role in tumor biology. Previous studies from our laboratory and other groups suggested associations of high expression of NPY and its Y5 receptor (Y5R) with invasive and metastatic phenotypes of various tumor types; yet the direct evidence for the metastatic activity was lacking. Our recent work began to fill this gap. Using Ewing sarcoma (ES) as a model of NPY-rich tumor, we identified the NPY/Y5R axis as a crucial pathway responsible for hypoxia-induced ES bone metastasis. This osseous dissemination was driven by hypoxia-dependent over-activation of the NPY/Y5R/RhoA pathway, which led to cytokinesis defects and formation of the aneuploid ES cell population with high propensity to bone metastasis. Here, we sought to determine the impact of the Y5R signaling on overall, hypoxia-independent metastatic capabilities of ES cells. To this end, a doxycycline-inducible CRIPSR/Cas9 system was used to knockout Y5R in ES orthotopic xenografts and test its impact on metastasis and cell migration assessed by a transwell assay. The frequency of the NPY5R gene modifications was determined by sequencing and confirmed by immunohistochemistry. FISH was used to identify NPY5R gene gains in ES xenografts. RhoA activity was measured by immunocytochemistry and pull-down assay. As expected, ES/Y5R-sgRNA primary tumors in doxycycline-treated mice consisted of a heterogeneous ES cell population with variable Y5R levels. In contrast, metastases developing from these xenografts were primarily initiated by ES clones with an intact NPY5R gene. The frequency of NPY5R gene modifications in metastases from the doxycycline-treated group was markedly reduced, as compared to the primary tumors. No metastases arising from ES clones lacking intact NPY5R gene were detected. Similarly, metastasis from wild type ES xenografts was associated with a selection of clones with the NPY5R gene gain that was present in a small percent of the parental cells. Subsequent in vitro assays implicated Y5R-dependent ES cell motility driven by the activation of a key cytoskeleton regulator, RhoA, as the mechanism underlying the metastatic effects of NPY. The above results, along with our previously published data, provide direct evidence for the crucial role of the NPY/Y5R axis in metastasis. We have shown that under basal conditions the NPY/Y5R/RhoA axis promotes tumor cell motility and overall cancer cell dissemination, while over-activation of this pathway under hypoxic conditions leads to genomic instability and bone metastasis. While our study focused on ES due to its high endogenous expression of NPY and its receptors, our findings may be relevant to other malignancies rich in NPY and Y5R, such as neuroblastoma, breast and prostate cancer. Given availability of the FDA approved Y5R antagonist, NPY/Y5R pathway may be a promising target for therapies preventing cancer progression. Yet, further research is required to establish effectiveness of such pharmacological interventions. Citation Format: Mina Adnani, Sung-Hyeok Hong, Susana Galli, Akanksha Mahajan, Nouran Abualsaud, Lindsay Caprio, Jason U. Tilan, Olga Rodriguez, Hingkun Wang, Christopher Albanese, Luciane R. Cavalli, Joanna Kitlinska. Neuropeptide Y as a metastatic factor [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR018.

Formation of a Stable Co-Amorphous System for a Brick Dust Molecule by Utilizing Sodium Taurocholate with High Glass Transition Temperature.

Brick dust molecules are usually poorly soluble in water and lipoidal components, making it difficult to formulate them in dosage forms that provide efficient pharmacological effects. A co-amorphous system is an effective strategy to resolve these issues. However, their glass transition temperatures (Tg) are relatively lower than those of polymeric amorphous solid dispersions, suggesting the instability of the co-amorphous system. This study aimed to formulate a stable co-amorphous system for brick dust molecules by utilizing sodium taurocholate (NaTC) with a higher Tg. A novel neuropeptide Y5 receptor antagonist (AntiY5R) and NaTC with Tg of 155 °C were used as the brick dust model and coformer, respectively. Ball milling formed a co-amorphous system for AntiY5R and NaTC (AntiY5R-NaTC) at various molar ratios. Deviation from the theoretical Tg value and peak shifts in Fourier-transform infrared spectroscopy indicated intermolecular interactions between AntiY5R and NaTC. AntiY5R-NaTC at equal molar ratios resulting in an 8.5-fold increase in AntiY5R solubility over its crystalline form. The co-amorphous system remained amorphous for 1 month at 25 °C and 40 °C. These results suggest that the co-amorphous system formed by utilizing NaTC as a coformer could stably maintain the amorphous state and enhance the solubility of brick dust molecules.

Discovery of a potent and long-acting Xenopus GLP-1-based GLP-1/glucagon/Y2 receptor triple agonist

The combination of incretin-based therapies and PYY analogue has shown great potential for the treatment of type 2 diabetes (T2DM) and obesity. In this study we developed the first example of a unimolecular triple agonist peptide to simultaneously target GLP-1, glucagon and Y2 receptors, aiming for superior weight loss and better glycemic control. The strategy for constructing such a unimolecular triple agonist peptide is the conjugation of the GLP-1R/GCGR dual-agonistic moiety and PYY moiety via maleimide-thiol specific reaction. A novel triple agonist peptide, 3b, was identified via stepwise structure optimization, long-acting modification and in vitro receptor screens. Peptide 3b exhibited potent and balanced GCGR and GLP-1R activities as well as potent and highly selective Y2R activity. Peptide 3b potently reduced food intake without triggering nausea associated behavior in kaolin consumption and conditioned taste aversion assays. In diet induced obesity (DIO) mice, a lower dose of 3b achieved significantly better effects on lipid metabolism, body weight, and glycemic control than higher dose of GLP-1R mono-agonist, GLP-1R/GCGR dual agonist and GLP-1R/Y2R dual agonist counterparts. Collectively, these data support the therapeutic potential of our GLP-1R/GCGR/Y2R triple agonist 3b as a novel anti-obesity and anti-diabetic agent. Targeting GLP-1R, GCGR and Y2R with unimolecular triple agonist peptide offers a route to develop new obesity and T2DM treatments.

Structural basis for Y2 receptor-mediated neuropeptide Y and peptide YY signaling

Neuropeptide Y (NPY) and its receptors are expressed in various human tissues including the brain where they regulate appetite and emotion. Upon NPY stimulation, the neuropeptide Y1 and Y2 receptors (Y1R and Y2R, respectively) activate GI signaling, but their physiological responses to food intake are different. In addition, deletion of the two N-terminal amino acids of peptide YY (PYY(3-36)), the endogenous form found in circulation, can stimulate Y2R but not Y1R, suggesting that Y1R and Y2R may have distinct ligand-binding modes. Here, we report the cryo-electron microscopy structures of the PYY(3-36)‒Y2R‒Gi and NPY‒Y2R‒Gi complexes. Using cell-based assays, molecular dynamics simulations, and structural analysis, we revealed the molecular basis of the exclusive binding of PYY(3-36) to Y2R. Furthermore, we demonstrated that Y2R favors G protein signaling over β-arrestin signaling upon activation, whereas Y1R does not show a preference between these two pathways.

A Maternal High-Fat Diet Causes Anxiety-Related Behaviors by Altering Neuropeptide Y1 Receptor and Hippocampal Volumes in Rat Offspring: the Potential Effect of N-Acetylcysteine

The children of obese mothers are known to have a high risk of obesity and metabolic disease and are prone to developing cognitive deficits, although the underlying mechanism is not yet fully understood. This study investigated the relationship between neuropeptide Y1 receptor (NPY1R) and anxiety-like behaviors in the hippocampi of male rat offspring exposed to maternal obesity and the potential neuroprotective effects of N-acetylcysteine (NAC). A maternal obesity model was created using a high-fat (60% k/cal) diet. NAC (150mg/kg) was administered by intragastric gavage for 25days in both the NAC and obesity + NAC (ObNAC) groups. All male rat offspring were subjected to behavioral testing on postnatal day 28, the end of the experiment. Stereological analysis was performed on hippocampal sections, while NPY1R expression was determined using immunohistochemical methods. Stereological data indicated significant decreases in the total volume of the hippocampus and CA1 and dentate gyrus (DG) regions in the obese (Ob) group (p < 0.01). Decreased NPY1R expression was observed in the Ob group hippocampus (p < 0.01). At behavioral assessments, the Ob group rats exhibited increased anxiety and less social interaction, although the ObNAC group rats exhibited stronger responses than the Ob group (p < 0.01). The study results show that NAC attenuated anxiety-like behaviors and NPY1R expression and also protected hippocampal volume against maternal obesity. The findings indicate that a decrease in NPY1R-positive neurons in the hippocampus of male rats due to maternal conditions may be associated with increased levels of anxiety and a lower hippocampal volume. Additionally, although there is no direct evidence, maintenance of NPY1R expression by NAC may be critical for regulating maternal obesity-induced anxiety-related behaviors and hippocampal structure.

TRH and NPY Interact to Regulate Dynamic Changes in Energy Balance in the Male Zebra Finch.

In contrast to mammals, birds have a higher basal metabolic rate and undertake wide range of energy-demanding activities. As a consequence, food deprivation for birds, even for a short period, poses major energy challenge. The energy-regulating hypothalamic homeostatic mechanisms, although extensively studied in mammals, are far from clear in the case of birds. We focus on the interplay between neuropeptide Y (NPY) and thyrotropin-releasing hormone (TRH), 2 of the most important hypothalamic signaling agents, in modulating the energy balance in a bird model, the zebra finch, Taeniopygia guttata. TRH neurons were confined to a few nuclei in the preoptic area and hypothalamus, and fibers widely distributed. The majority of TRH neurons in the hypothalamic paraventricular nucleus (PVN) whose axons terminate in median eminence were contacted by NPY-containing axons. Compared to fed animals, fasting significantly reduced body weight, PVN pro-TRH messenger RNA (mRNA) and TRH immunoreactivity, but increased NPY mRNA and NPY immunoreactivity in the infundibular nucleus (IN, avian homologue of mammalian arcuate nucleus) and PVN. Refeeding for a short duration restored PVN pro-TRH and IN NPY mRNA, and PVN NPY innervation to fed levels. Compared to control tissues, treatment of the hypothalamic superfused slices with NPY or an NPY-Y1 receptor agonist significantly reduced TRH immunoreactivity, a response blocked by treatment with a Y1-receptor antagonist. We describe a detailed neuroanatomical map of TRH-equipped elements, identify new TRH-producing neuronal groups in the avian brain, and demonstrate rapid restoration of the fasting-induced suppression of PVN TRH following refeeding. We further show that NPY via Y1 receptors may regulate PVN TRH neurons to control energy balance in T. guttata.

MTOR-neuropeptide Y signaling sensitizes nociceptors to drive neuropathic pain.

Neuropathic pain is a refractory condition that involves de novo protein synthesis in the nociceptive pathway. The mTOR is a master regulator of protein translation; however, mechanisms underlying its role in neuropathic pain remain elusive. Using the spared nerve injury-induced neuropathic pain model, we found that mTOR was preferentially activated in large-diameter dorsal root ganglion (DRG) neurons and spinal microglia. However, selective ablation of mTOR in DRG neurons, rather than microglia, alleviated acute neuropathic pain in mice. We show that injury-induced mTOR activation promoted the transcriptional induction of neuropeptide Y (Npy), likely via signal transducer and activator of transcription 3 phosphorylation. NPY further acted primarily on Y2 receptors (Y2R) to enhance neuronal excitability. Peripheral replenishment of NPY reversed pain alleviation upon mTOR removal, whereas Y2R antagonists prevented pain restoration. Our findings reveal an unexpected link between mTOR and NPY/Y2R in promoting nociceptor sensitization and neuropathic pain.

The Antagonism of Neuropeptide Y Type I Receptor (Y1R) Reserves the Viability of Bone Marrow Stromal Cells in the Milieu of Osteonecrosis of Femoral Head (ONFH).

Neuropeptide Y (NPY)-Y1 receptor (Y1R) signaling is known to negatively affect bone anabolism. Our study aimed at investigating the impact of NPY-Y1R signaling in the pathogenesis of glucocorticoid-related osteonecrosis of the femoral head (ONFH). Femoral heads were retrieved from 20 patients with and without ONFH, respectively. The bone marrow stromal cells (BMSCs) from ONFH femoral heads were treated with Y1R agonists and antagonists for subsequent analysis. We showed that the local NPY expression level was lower in ONFH heads. The Y1R agonists and antagonists disturb and facilitate the survival of BMSCs. The transcription of stromal derived factor-1 (SDF-1) was enhanced by Y1R antagonists. Our study showed that the local NPY expression level was lower in ONFH heads. Y1R antagonists facilitate the survival of BMSCs and stimulate the transcription of SDF-1 by BMSCs. These findings shed light on the role of NPY-Y1R signaling in the pathogenesis of ONFH.

Involvement of ghrelin in the regulation of swallowing motor activity in an arterially perfused rat preparation

Ghrelin, a peripheral peptide produced in the stomach, is involved in the neural networks that control food intake. Alterations in motor components, such as swallowing, are believed to be significant in the regulation food intake by orexigenic signals. However, there has been no detailed investigation of the relationship between ghrelin and swallowing activities induced in motor nerves innervating the pharyngeal and laryngeal muscles. In this study, we examined the effects of ghrelin administration on swallowing motor activity in arterially perfused rats. Injection of distilled water (0.5 ml) into the oral cavity or electrical stimulation of the superior laryngeal nerve evoked swallowing motor activity in the cervical vagus nerve. Administration of ghrelin (6 nM), but not des-acylated ghrelin (6 nM), into the perfusate increased the peak burst amplitude and burst duration, and shortened the first burst interval of water injection-induced swallowing. These ghrelin-induced changes in swallowing motor activity were blocked by the administration of JMV2959 (6 µM), a growth hormone secretagogue receptor antagonist. In preparations in which the hypothalamus was removed, ghrelin had no effect on swallowing motor activity. Furthermore, ghrelin-induced changes were counteracted by the administration of BIBO3304 (1 µM) or L-152,804 (1 µM), antagonists of neuropeptide Y Y1 and Y5 receptors, respectively, which are essential for ghrelin-induced enhancement of food intake. Ghrelin also increased the peak burst amplitude and burst duration of the swallowing motor activity evoked by electrical stimulation of the superior laryngeal nerve, although the effects of ghrelin on the number of swallowing bursts and burst intervals varied with stimulus intensity. These results suggest that ghrelin enhances the magnitude and frequency of bursts of swallowing motor activity by acting via the hypothalamic neural network, and that neuropeptide Y Y1 and Y5 receptors are involved in this enhancement.

Abstract 11494: Electrophysiological Effects of Sympathoexcitation are Mitigated by Blockade of Neuropeptide Y Y2 Receptors on Cardiac Parasympathetic Nerve Terminals

Introduction: Sympathetic activation releases both norepinephrine and co-transmitter neuropeptide Y (NPY). NPY can predispose to ventricular arrhythmias via receptors on cardiomyocytes. Ex vivo studies suggest that NPY may concomitantly reduce vagal tone via activation of NPY Y2 receptors (Y2R) on parasympathetic nerves, reducing acetylcholine release and further augmenting pro-arrhythmic sympathetic effects. We evaluated if parasympathetic Y2R blockade can prevent NPY-induced vagal inhibition, restore autonomic balance and stabilize cardiac electrophysiology. Methods: Yorkshire pigs (n = 12) underwent bilateral stellate ganglia stimulation (BSGS; 10 Hz, 4 ms) for 45 secs, before and after administration of selective Y2R antagonist BIIE0246 (BIIE; 32 nmol/kg bolus + 2 nmol/kg/min infusion). Hemodynamic and electrophysiological recordings were obtained with a left ventricular pressure (LVP) catheter, surface electrogram and a 56-electrode sock placed over the ventricles to measure activation recovery intervals (ARI), a surrogate of local action potential duration, from unipolar electrograms. Heart rate variability parameters, including low to high frequency ratio (LF/HF), were analyzed pre- and post-BIIE. Results: BSGS significantly increased heart rate (HR) and LVP, and shortened ventricular ARIs. BIIE significantly reduced both the rate of change and the maximal changes in HR and ARIs during BSGS. As anticipated, BSGS significantly increased LF/HF ratio, but this effect was abolished after BIIE. BIIE did not affect BSGS related increases in LVP. Conclusions: Cardiac effects of sympathetic activation are mediated, at least in part, by suppression of vagal tone through binding of NPY to parasympathetic Y2Rs. Blockade of this receptor partially counters pro-arrhythmic electrophysiological effects of BSGS and suggests a possible novel adjuvant role for Y2R blockade as a therapy for reducing consequences of sympatho-excitation.