NPY-mediated synaptic plasticity in the extended amygdala prioritizes feeding during starvation.

Efficient control of feeding behavior requires the coordinated adjustment of complex motivational and affective neurocircuits. Neuropeptides from energy-sensing hypothalamic neurons are potent feeding modulators, but how these endogenous signals shape relevant circuits remains unclear. Here, we examine how the orexigenic neuropeptide Y (NPY) adapts GABAergic inputs to the bed nucleus of the stria terminalis (BNST). We find that fasting increases synaptic connectivity between agouti-related peptide (AgRP)-expressing 'hunger' and BNST neurons, a circuit that promotes feeding. In contrast, GABAergic input from the central amygdala (CeA), an extended amygdala circuit that decreases feeding, is reduced. Activating NPY-expressing AgRP neurons evokes these synaptic adaptations, which are absent in NPY-deficient mice. Moreover, fasting diminishes the ability of CeA projections in the BNST to suppress food intake, and NPY-deficient mice fail to decrease anxiety in order to promote feeding. Thus, AgRP neurons drive input-specific synaptic plasticity, enabling a selective shift in hunger and anxiety signaling during starvation through NPY.

High-Caloric Diets in Adolescence Impair Specific GABAergic Subpopulations, Neurogenesis, and Alter Astrocyte Morphology.

We compared the effects of two different high-caloric diets administered to 4-week-old rats for 12 weeks: a diet rich in sugar (30% sucrose) and a cafeteria diet rich in sugar and high-fat foods. We focused on the hippocampus, particularly on the gamma-aminobutyric acid (GABA)ergic system, including the Ca2+-binding proteins parvalbumin (PV), calretinin (CR), calbindin (CB), and the neuropeptides somatostatin (SST) and neuropeptide Y (NPY). We also analyzed the density of cholinergic varicosities, brain-derived neurotrophic factor (BDNF), reelin (RELN), and cyclin-dependent kinase-5 (CDK-5) mRNA levels, and glial fibrillary acidic protein (GFAP) expression. The cafeteria diet reduced PV-positive neurons in the granular layer, hilus, and CA1, as well as NPY-positive neurons in the hilus, without altering other GABAergic populations or overall GABA levels. The high-sugar diet induced a decrease in the number of PV-positive cells in CA3 and an increase in CB-positive cells in the hilus and CA1. No alterations were observed in the cholinergic varicosities. The cafeteria diet also reduced the relative mRNA expression of RELN without significant changes in BDNF and CDK5 levels. The cafeteria diet increased the number but reduced the length of the astrocyte processes. These data highlight the significance of determining the mechanisms mediating the observed effects of these diets and imply that the cognitive impairments previously found might be related to both the neuroinflammation process and the reduction in PV, NPY, and RELN expression in the hippocampal formation.

High-throughput assay for regulated secretion of neuropeptides in mouse and human neurons.

Neuropeptides are the largest group of chemical signals in the brain. More than 100 different neuropeptides modulate various brain functions and their dysregulation has been associated with neurological disorders. Neuropeptides are packed into dense core vesicles (DCVs), which fuse with the plasma membrane in a calcium-dependent manner. Here, we describe a novel high-throughput assay for DCV exocytosis using a chimera of Nanoluc luciferase and the DCV-cargo neuropeptide Y (NPY). The NPY-Nanoluc reporter colocalized with endogenous DCV markers in all neurons with little mislocalization to other cellular compartments. NPY-Nanoluc reported DCV exocytosis in both rodent and induced pluripotent stem cell-derived human neurons, with similar depolarization, Ca2+, RAB3, and STXBP1/MUNC18 dependence as low-throughput assays. Moreover, NPY-Nanoluc accurately reported modulation of DCV exocytosis by known modulators diacylglycerol analog and Ca2+ channel blocker and showed a higher assay sensitivity than a widely used single-cell low-throughput assay. Lastly, we showed that Nanoluc coupled to other secretory markers reports on constitutive secretion. In conclusion, the NPY-Nanoluc is a sensitive reporter of DCV exocytosis in mammalian neurons, suitable for pharmacological and genomic screening for DCV exocytosis genes and for mechanism-based treatments for central nervous system disorders.

Pain-related single nucleotide polymorphisms: association with lumbar spinal stenosis patient experience and non-surgical treatment outcomes.

PURPOSE: Lumbar spinal stenosis (LSS) is common in our aging population resulting in pain and functional impairment. Recent advances in pain research have identified several single nucleotide polymorphisms (SNP) associated with inter-individual symptom and treatment response. The goal of the current study was to investigate the association of SNPs in Neuropeptide Y (NPY) and Catechol-O-methyltransferase (COMT) with pain, function, and treatment outcomes in Lumbar spinal stenosis (LSS) patients receiving non-surgical treatments. METHODS: An exploratory observational biomarker study was performed ancillary to a previously published clinical trial evaluating three different non-surgical treatments for LSS. Saliva samples were obtained for single nucleotide polymorphism genotyping and blood samples were collected for NPY protein. Data on pain and function collected as part of the clinical trial at baseline, 2 and 6 months were examined for association with known polymorphisms in NPY and COMT. RESULTS: Subjects with the NPY rs16147 TT genotype exhibited higher baseline symptom severity but also a higher likelihood of responding to non-surgical treatments. Subjects with the COMT rs4680 GG genotype also exhibited higher baseline symptom severity but did not demonstrate greater response to treatment. CONCLUSIONS: NPY rs16147 and COMT rs4680 are important potential biomarkers associated with pain and function. NPY genotype may be useful in predicting response to non-surgical treatments in older adults with LSS.

Topical Neuropeptide Y for Ischemic Skin Wounds.

Our objective was to investigate the effects of topically applied neuropeptide Y (NPY) on ischemic wounds. Initially, the animal model for ischemic wound healing was validated using 16 male Sprague Dawley albino rats. In the intervention study, an additional 28 rats were divided into three groups: NPY (0.025%), the positive control insulin-like growth factor-I (IGF-I, 0.0025%), and the hydrogel carrier alone (control). The hydrogel was selected due to its capacity to prolong NPY release (p < 0.001), as demonstrated in a Franz diffusion cell. In the animals, an 8 mm full-thickness wound was made in a pedunculated dorsal ischemic skin flap. Wounds were then treated and assessed for 14 days and collected at the end of the experiment for in situ hybridization analysis (RNAscope®) targeting NPY receptor Y2R and for meticulous histologic examination. Wound healing rates, specifically the percentage changes in wound area, did not show an increase with NPY (p = 0.907), but there was an increase with rhIGF-I (p = 0.039) compared to the control. Y2R mRNA was not detected in the wounds or adjacent skin but was identified in the rat brain (used as a positive control). Light microscopic examination revealed trends of increased angiogenesis and enhanced inflammatory cell infiltration with NPY compared to control. An interesting secondary discovery was the presence of melanophages in the wounds. Our findings suggest the potential of NPY to enhance neovascularization under ischemic wound healing conditions, but further optimization of the carrier and dosage is necessary. The mechanism remains elusive but likely involves NPY receptor subtypes other than Y2R.

Npy transcription is regulated by noncanonical STAT3 signaling in hypothalamic neurons: Implication with lipotoxicity and obesity.

Neuropeptide Y (Npy) is an abundant neuropeptide expressed in the central and peripheral nervous systems. NPY-secreting neurons in the hypothalamic arcuate nucleus regulate energy homeostasis, and Npy mRNA expression is regulated by peripheral nutrient and hormonal signals like leptin, interleukin-6 (IL-6), and fatty acids. This study demonstrates that IL-6, which phosphorylates tyrosine 705 (Y705) of STAT3, decreased Npy mRNA in arcuate immortalized hypothalamic neurons. In parallel, inhibitors of STAT3-Y705 phosphorylation, stattic and cucurbitacin I, robustly upregulated Npy mRNA. Chromatin-immunoprecipitation showed high baseline total STAT3 binding to multiple regulatory regions of the Npy gene, which are decreased by IL-6 exposure. The STAT3-Npy interaction was further examined in obesity-related pathologies. Notably, in four different hypothalamic neuronal models where palmitate potently stimulated Npy mRNA, Socs3, a specific STAT3 activity marker, was downregulated and was negatively correlated with Npy mRNA levels (R2 = 0.40, p < 0.001), suggesting that disrupted STAT3 signaling is involved in lipotoxicity-mediated dysregulation of Npy. Finally, human NPY SNPs that map to human obesity or body mass index were investigated for potential STAT3 binding sites. Although none of the SNPs were linked to direct STAT3 binding, analysis show that rs17149106 (-602 G > T) is located on an upstream enhancer element of NPY, where the variant is predicted to disrupt validated binding of KLF4, a known inhibitory cofactor of STAT3 and downstream effector of leptin signaling. Collectively, this study demonstrates that STAT3 signaling negatively regulates Npy transcription, and that disruption of this interaction may contribute to metabolic disorders.

Early starvation in European seabass (Dicentrarchus labrax) larvae has no drastic effect on hepatic intermediary metabolism in juveniles.

The present study aims to investigate nutritional programming through early starvation in the European seabass (Dicentrarchus labrax). European seabass larvae were fasted at three different developmental periods for three durations from 60 to 65 dph (F1), 81 to 87 dph (F2), and 123 to 133 dph (F3). Immediate effects were investigated by studying gene expression of npy (neuropeptide Y) and avt (Arginine vasotocin) in the head, while potential long-term effects (i.e., programming) were evaluated on intermediary metabolism later in life (in juveniles). Our findings indicate a direct effect regarding gene expression in the head only for F1, with higher avt mRNA level in fasted larved compared to controls. The early starvation periods had no long-term effect on growth performance (body weight and body length). Regarding intermediary metabolism, we analyzed related key plasma metabolites which reflect the intermediary metabolism: no differences for glucose, triglycerides, and free fatty acids in the plasma were observed in juveniles irrespective of the three early starvation stimuli. As programming is mainly linked to molecular mechanisms, we then studied hepatic mRNA levels for 23 key actors of glucose, lipid, amino acid, and energy metabolism. For many of the metabolic genes, there was no impact of early starvation in juveniles, except for three genes involved in glucose metabolism (glut2-glucose transporter and pk-pyruvate kinase) and lipid metabolism (acly-ATP citrate lyase) which were higher in F2 compared to control. Together, these results highlight that starvation between 81 to 87 dph may have more long-term impact, suggesting the existence of a developmental window for programming by starvation. In conclusion, European seabass appeared to be resilient to early starvation during larvae stages without drastic impacts on intermediary metabolism later in life.

Regulation of colonic neuropeptide Y expression by the gut microbiome in patients with ulcerative colitis and its association with anxiety- and depression-like behavior in mice.

Patients with inflammatory bowel disease (IBD), including ulcerative colitis (UC), show an increased incidence of anxiety and depression; however, the association between UC-associated psychiatric disorders and the gut microbiota is unclear. This study aimed to examine whether gut microbiota from patients with UC can alter colonic gene expression, leading to anxiety- and depression-like behavior in mice receiving fecal microbiota transplantation (FMT). RNA sequencing transcriptome analyses revealed a difference in colonic gene expression between mice receiving FMT from patients with UC (UC-FMT mice) and those receiving FMT from healthy controls (HC-FMT mice). Gene ontology analysis revealed the downregulation of neuropeptide signaling pathways, including neuropeptide Y (NPY) expression, in the colons of UC-FMT mice. The protein levels of NPY also decreased in the colon and plasma of UC-FMT mice compared to those in HC-FMT mice. The oral administration of Enterococcus mundtii (EM), a bacterium isolated from the feces of patients with UC, reduced NPY expression in the colons of mice and induced intestinal inflammation, anxiety, and depression-like behavior. Reduced NPY protein levels were also observed in the plasma and hippocampus of EM-treated mice. Intraperitoneal administration of NPY significantly alleviated anxiety- and depressive-like behaviors induced by EM in mice. Capsular polysaccharide in EM was associated with EM-induced NPY downregulation in the colon. Analysis of Gene Expression Omnibus datasets showed markedly reduced NPY expression in the inflamed colons of patients with UC compared with that in the colons of healthy controls. In summary, EM-induced reduction in the colonic expression of NPY may be associated with a decrease in hippocampal NPY and anxiety- and depression-like behavior in mice.

Neuropeptide Y gene variants and Agreeableness: interaction effect with the birth cohort and the serotonin transporter promoter polymorphism.

OBJECTIVE: Neuropeptide Y (NPY) is a powerful regulator of anxious states, including social anxiety, but evidence from human genetic studies is limited. Associations of common gene variants with behaviour have been described as subject to birth cohort effects, especially if the behaviour is socially motivated. This study aimed to examine the association of NPY rs16147 and rs5574 with personality traits in highly representative samples of two birth cohorts of young adults, the samples having been formed during a period of rapid societal transition. METHODS: Both birth cohorts (original n = 1238) of the Estonian Children Personality Behaviour and Health Study (ECPBHS) self-reported personality traits of the five-factor model at 25 years of age. RESULTS: A significant interaction effect of the NPY rs16147 and rs5574 and birth cohort on Agreeableness was found. The T/T genotype of NPY rs16147 resulted in low Agreeableness in the older cohort (born 1983) and in high Agreeableness in the younger cohort (born 1989). The C/C genotype of NPY rs5574 was associated with higher Agreeableness in the younger but not in the older cohort. In the NPY rs16147 T/T homozygotes, the deviations from average in Agreeableness within the birth cohort were dependent on the serotonin transporter promoter polymorphism. CONCLUSIONS: The association between the NPY gene variants and a personality domain reflecting social desirability is subject to change qualitatively in times of rapid societal changes, serving as an example of the relationship between the plasticity genes and environment. The underlying mechanism may involve the development of the serotonergic system.

Neuropeptide Y gene polymorphisms and chronic kidney disease progression.

BACKGROUND: Neuropeptide Y (NPY) is a neurotransmitter expressed in both the central and peripheral nervous systems, which is involved in regulating a multitude of physiological processes ranging from arterial pressure, energy balance, the immune response and inflammation and renal electrolyte transport. In a cohort of chronic kidney disease (CKD) patients, we recently showed that high plasma NPY levels predict renal disease progression independently of hypertension and other risk factors but the causal nature of this association remains unproven. METHODS: In the same cohort of the previous study, we tested the relationship of NPY gene variability, as assessed by five single nucleotide polymorphisms (SNPs) that explained the whole gene variability, with the incidence rate of a predefined combined renal endpoint (dialysis/transplantation/estimated glomerular filtration rate reduction >30%) over a median follow up of 36 months (inter-quartile range 35-37 months) in 735 ethnically homogeneous patients with stage 2-5 CKD. RESULTS: Two variants [rs16131 (recessive model for the T risk allele: TT, n  = 563; CT + CC, n  = 172) and rs16140 (dominant model for the G risk allele: GG + CG, n  = 413; CC, n  = 322)] were coherently associated with the incidence rate of renal events [hazard ratio (HR) ranging from 1.39 to 1.57, P  ≤ 0.015] and this was also true when the two SNPs were jointly introduced into the same Cox model ( P  ≤ 0.043). The analysis of the biological interaction showed a significant synergism between the NPY rs16131 and rs16140 variants. Indeed, patients harboring NPY rs16131 TT and NPY rs16140 GG + CG risk genotypes had a much higher HR of renal events [HR: 1.80, 95% confidence interval (CI):1.16-2.79, P  = 0.009] than that expected in the absence of biological interaction under both the additive and multiplicative models and the attributable proportion due to interaction (AP) was 25% and 38% on crude and adjusted analyses, respectively. CONCLUSION: This study, based on the Mendelian randomization approach and using NPY gene variants as instrumental variables to test the link between NPY and CKD progression, is in line with findings indicating that high plasma NPY levels predict an increased risk for renal events and lend support to the hypothesis that NPY is causally involved in renal disease progression.

Neuropeptide Y, a potential marker for lupus, promotes lupus development.

OBJECTIVE: Relationship between neuropeptide Y (NPY) serum levels, NPY genetic mutation with systemic lupus erythematosus (SLE) pathogenesis is yet to be clarified, and role of NPY in development of SLE needs elucidation. METHOD: This study included 460 SLE patients, 472 non-SLE cases, 500 healthy volunteers. Serum NPY, matrix metalloproteinase-1 (MMP-1) and MMP-8 levels were tested by ELISA. Genotyping 7 NPY single nucleotides polymorphisms (SNPs) (rs5573, rs5574, rs16129, rs16138, rs16140, rs16147, rs16478) was obtained by Kompetitive Allele-Specific PCR (KASP) method. Pristane-induced lupus mice were treated with NPY-Y1 receptor antagonist, and histological analysis, serological changes of the mice were evaluated. RESULTS: NPY serum concentrations were significantly increased in SLE patients when compared to that in healthy volunteers, non-SLE cases. Rs5573 G allele, rs16129 T allele, rs16147 G allele frequencies were significantly different between SLE cases and healthy controls. Rs5574 TT + TC genotypes were related to levels of IgG, C3, C4 and erythrocyte sedimentation rate, and rs16138 GG + GC genotypes correlated with SLE cases with anti-double-stranded deoxyribonucleic acid antibody (anti-dsDNA) (+). Serum MMP-1, MMP-8 concentrations were higher in SLE patients, and NPY levels were significantly related to MMP-1, MMP-8 levels. After treatment of lupus mice with NPY-Y1 receptor antagonist, damage of liver, spleen and kidney was alleviated, production of autoantibodies (anti-nuclear antibody (ANA), total IgG, anti-dsDNA) and MMP-1, MMP-8 was down-regulated, and differentiation of CD3+, CD8+ T cells, B cells, monocytes, macrophages, T helper 1 (Th1), Th2, Th17 cells was reversed. CONCLUSION: NPY may be a biomarker for lupus, which may promote occurrence and development of lupus.

Possible role of neuropeptide Y on zymosan- and lipopolysaccharide-induced change in gastrointestinal feed passage via the medulla oblongata in chicks.

Zymosan is a fungi-derived pathogen-associated molecular pattern. It activates the immune system and induces the reduction of feed passage rate in the gastrointestinal tract of vertebrates including birds. However, the mechanism mediating the zymosan-induced inhibition of feed passage in the gastrointestinal tract remains unknown. Since the medulla oblongata regulates the digestive function, it is plausible that the medulla oblongata is involved in the zymosan-induced inhibition of feed passage. The present study was performed to identify the genes that were affected by zymosan within the medulla oblongata of chicks (Gallus gallus) using an RNA sequencing approach. We found that mRNAs of several bioactive molecules including neuropeptide Y (NPY) were increased with an intraperitoneal (IP) injection of zymosan. The increase of mRNA expression of NPY in the medulla oblongata was also observed after the IP injection of lipopolysaccharide, derived from gram-negative bacteria. These results suggest that medullary NPY is associated with physiological changes during fungal and bacterial infection. Furthermore, we found that intracerebroventricular injection of NPY and its receptor agonists reduced the feed passage from the crop. Additionally, the injection of NPY reduced the feed passage from the proventriculus to lower digestive tract. NPY also suppressed the activity of duodenal activities of amylase and trypsin. The present study suggests that fungi- and bacteria-induced activation of the immune system may activate the NPY neurons in the medulla oblongata and thereby reduce the digestive function in chicks.

Cotadutide (GLP-1/Glucagon dual receptor agonist) modulates hypothalamic orexigenic and anorexigenic neuropeptides in obese mice.

The hypothalamic neuropeptides linked to appetite and satiety were investigated in obese mice treated with cotadutide (a dual receptor agonist of glucagon-like peptide 1 (GLP-1R)/Glucagon (GCGR)). Twelve-week-old male C57BL/6 mice were fed a control diet (C group, n = 20) or a high-fat diet (HF group, n = 20) for ten weeks. Each group was further divided, adding cotadutide treatment and forming groups C, CC, HF, and HFC for four additional weeks. The hypothalamic arcuate neurons were labeled by immunofluorescence, and protein expressions (Western blotting) for neuropeptide Y (NPY), proopiomelanocortin (POMC), agouti-related protein (AgRP), and cocaine- and amphetamine-regulated transcript (CART). Cotadutide enhanced POMC and CART neuropeptides and depressed NPY and AGRP neuropeptides. In addition, gene expressions (RT-qPCR) determined that Lepr (leptin receptor) and Calcr (calcitonin receptor) were diminished in HF compared to C but enhanced in CC compared to C and HFC compared to HF. Besides, Socs3 (suppressor of cytokine signaling 3) was decreased in HFC compared to HF, while Sst (somatostatin) was higher in HFC compared to HF; Tac1 (tachykinin 1) and Mc4r (melanocortin-4-receptor) were lower in HF compared to C but increased in HFC compared to HF. Also, Glp1r and Gcgr were higher in HFC compared to HF. In conclusion, the findings are compelling, demonstrating the effects of cotadutide on hypothalamic neuropeptides and hormone receptors of obese mice. Cotadutide modulates energy balance through the gut-brain axis and its associated signaling pathways. The study provides insights into the mechanisms underlying cotadutide's anti-obesity effects and its possible implications for obesity treatment.

Alleviation of neuropathic pain with neuropeptide Y requires spinal Npy1r interneurons that coexpress Grp.

Neuropeptide Y targets the Y1 receptor (Y1) in the spinal dorsal horn (DH) to produce endogenous and exogenous analgesia. DH interneurons that express Y1 (Y1-INs; encoded by Npy1r) are necessary and sufficient for neuropathic hypersensitivity after peripheral nerve injury. However, as Y1-INs are heterogenous in composition in terms of morphology, neurophysiological characteristics, and gene expression, we hypothesized that a more precisely defined subpopulation mediates neuropathic hypersensitivity. Using fluorescence in situ hybridization, we found that Y1-INs segregate into 3 largely nonoverlapping subpopulations defined by the coexpression of Npy1r with gastrin-releasing peptide (Grp/Npy1r), neuropeptide FF (Npff/Npy1r), and cholecystokinin (Cck/Npy1r) in the superficial DH of mice, nonhuman primates, and humans. Next, we analyzed the functional significance of Grp/Npy1r, Npff/Npy1r, and Cck/Npy1r INs to neuropathic pain using a mouse model of peripheral nerve injury. We found that chemogenetic inhibition of Npff/Npy1r-INs did not change the behavioral signs of neuropathic pain. Further, inhibition of Y1-INs with an intrathecal Y1 agonist, [Leu31, Pro34]-NPY, reduced neuropathic hypersensitivity in mice with conditional deletion of Npy1r from CCK-INs and NPFF-INs but not from GRP-INs. We conclude that Grp/Npy1r-INs are conserved in higher order mammalian species and represent a promising and precise pharmacotherapeutic target for the treatment of neuropathic pain.

Dietary zinc levels affect growth, appetite, and lipid metabolism of Chinese perch (Siniperca chuatsi).

An 84-day feeding experiment was conducted to investigate the effects of dietary Zn (zinc) on growth performance, food intake, and lipid metabolism of Chinese perch (Siniperca chuatsi). Five isonitrogenous and isolipidic diets with differential Zn contents (67, 100, 149, 230, and 410 mg/kg) were fed to 270 fish (35.47 ± 0.49 g). Results showed that fish growth and food intake increased markedly with the dietary 149 mg/kg Zn levels. Meanwhile, the food intake of 149 mg/kg group was significantly higher than that of other treatment groups after feeding for 8 weeks (P < 0.05). The qRT-PCR results showed that the expression of center appetite regulation factors in the hypothalamus was significantly regulated, and 149 mg/kg significantly increased mRNA expression of npy (neuropeptide Y) and decreased pomc (anorexigenic proopiomelanocortin) and cart (cocaine- and amphetamine-regulated transcript) gene expression. Meanwhile, the expressions of the main genes (such as leptin A and ghrelin) involved in peripheral appetite regulation factors were significantly up-regulated firstly and then reduced with the dietary Zn level increased, whereas the expression of cck (cholecystokinin) was significantly up-regulated. Serum AST (aspartate transaminase) and ALT (alanine transaminase) activities in fish fed the diets containing 230 and 410 mg/kg were significantly higher than that in other groups (P < 0.05). The lipid content of liver in 67 and 100 mg/kg groups was significantly higher than other groups (P < 0.05). Furthermore, dietary Zn significantly elevated the serum TG (triglyceride) and TCHO (total cholesterol) content levels (P < 0.05). Fish fed a high Zn diet (149, 230, and 410 mg/kg) dramatically down-regulated expression of srebp1 (sterol regulatory element binding proteins1c) and fas (fatty acid synthetase), but up-regulated expression of pparα (peroxisome proliferators-activated receptor-α) and cpt1 (carnitine palmitoyl transferase I) in the liver. The optimal dietary Zn inclusion level ranged from 146.69 to 152.86 mg/kg diet, based on two-slope broken-line regression analysis of WGR (weight gain rate) and FCR (feed conversion rate) for Chinese perch.

Neuropeptide Y directly reduced apoptosis of granulosa cells, and the expression of NPY and its receptors in PCOS subjects.

BACKGROUND: Most women with anovulatory infertility show polycystic ovarian syndrome (PCOS), and androgen excess is known as a key factor involved in pathogenicity of PCOS. However, the mechanism of follicular developmental arrest in PCOS is not completely understood. The reproductive function of Neuropeptide Y (NPY) in the ovary during folliculogenesis was previously reported; NPY function in apoptosis and proliferation of granulosa cells (GCs) is follicular-stage dependent. The objective of this study was to investigate the role of NPY in ovarian follicular development and the pathogenesis of PCOS. METHODS: To simulate the PCOS phenotype using a rat model, 21-day old Sprague Dawley rats were implanted with dihydrotestosterone (DHT) capsule (83 µg/day) and euthanized after 28 days. mRNA and protein content of NPY and its receptors were assessed in GCs from DHT treated rats using RT-qPCR and Western blot, respectively. Proliferation and apoptosis of GCs was assessed using Ki67- and TUNEL assays. Finally, NPY levels were measured in human follicular fluid (FF) from matched PCOS and non-PCOS patients using ELISA. RESULTS: GCs from DHT treated rats (PCOS-GCs) contained significantly less NPY protein and Npy mRNA by 0.16- and 0.56-fold, respectively, and more NPY receptor type 2 and 5 protein by 2.21- and 3.17-fold, respectively, when compared to sham control. Addition of recombinant NPY to PCOS-GCs culture did not alter Ki67-positive but significantly decreased TUNEL-positive cells by 0.65-fold, but not to baseline levels. There was no significant difference in NPY levels in FF between PCOS and non-PCOS subjects. CONCLUSIONS: These results indicate that DHT modulates expression of NPY and its receptors, NPY decreases DHT-induced GCs apoptosis. That alterations in NPY's function might be involved in follicular developmental failure of PCOS.

Neuropeptide Y-expressing dorsal horn inhibitory interneurons gate spinal pain and itch signalling.

Somatosensory information is processed by a complex network of interneurons in the spinal dorsal horn. It has been reported that inhibitory interneurons that express neuropeptide Y (NPY), either permanently or during development, suppress mechanical itch, with no effect on pain. Here, we investigate the role of interneurons that continue to express NPY (NPY-INs) in the adult mouse spinal cord. We find that chemogenetic activation of NPY-INs reduces behaviours associated with acute pain and pruritogen-evoked itch, whereas silencing them causes exaggerated itch responses that depend on cells expressing the gastrin-releasing peptide receptor. As predicted by our previous studies, silencing of another population of inhibitory interneurons (those expressing dynorphin) also increases itch, but to a lesser extent. Importantly, NPY-IN activation also reduces behavioural signs of inflammatory and neuropathic pain. These results demonstrate that NPY-INs gate pain and itch transmission at the spinal level, and therefore represent a potential treatment target for pathological pain and itch.

Pharmacological inhibition of neuropeptide Y receptors Y1 and Y5 reduces hypoxic breast cancer migration, proliferation, and signaling.

BACKGROUND: Neuropeptide Y (NPY) is an abundant neurohormone in human breast carcinomas that acts on a class of G-protein coupled receptors, of which NPY1R and NPY5R are the most highly expressed. This abundance is exploited for cancer imaging, but there is interest in pharmacological inhibition of the NPYRs to interrogate their functional relevance in breast cancer. We previously reported that NPY1R and NPY5R mRNA abundance is increased by hypoxia inducible factors, which sensitizes these receptors to NPY stimulation leading to enhanced migration and proliferation. METHODS/RESULTS: Here, we measured the effects of NPY1R and NPY5R antagonists in normoxia and hypoxia on migration, proliferation, invasion, and signaling in 2D and 3D models of breast cancer cell lines MDA-MB-231 and MCF7. Antagonizing NPY1R and/or NPY5R in hypoxia compared to normoxia more greatly reduced MAPK signaling, cell proliferation, cell migration and invasion, and spheroid growth and invasion. The estrogen receptor positive MCF7 cells were significantly less invasive in 3D spheres when NPY5R was specifically inhibited. There were some discrepancies in the responses of each cell line to the isoform-specific antagonists and oxygen availability, therefore further investigations are required to dissect the intricacies of NPYR signaling dynamics. In human breast tumor tissue, we show via immunofluorescence that NPY5R protein levels and colocalization with hypoxia correlate with advanced cancer, and NPY1R protein correlates with adverse outcomes. CONCLUSIONS: Antagonizing the NPYRs has been implicated as a treatment for a wide variety of diseases. Therefore, these antagonists may aid in the development of novel cancer therapeutics and patient-based treatment plans.

CaMK1D signalling in AgRP neurons promotes ghrelin-mediated food intake.

Hypothalamic AgRP/NPY neurons are key players in the control of feeding behaviour. Ghrelin, a major orexigenic hormone, activates AgRP/NPY neurons to stimulate food intake and adiposity. However, cell-autonomous ghrelin-dependent signalling mechanisms in AgRP/NPY neurons remain poorly defined. Here we show that calcium/calmodulin-dependent protein kinase ID (CaMK1D), a genetic hot spot in type 2 diabetes, is activated upon ghrelin stimulation and acts in AgRP/NPY neurons to mediate ghrelin-dependent food intake. Global Camk1d-knockout male mice are resistant to ghrelin, gain less body weight and are protected against high-fat-diet-induced obesity. Deletion of Camk1d in AgRP/NPY, but not in POMC, neurons is sufficient to recapitulate above phenotypes. In response to ghrelin, lack of CaMK1D attenuates phosphorylation of CREB and CREB-dependent expression of the orexigenic neuropeptides AgRP/NPY in fibre projections to the paraventricular nucleus (PVN). Hence, CaMK1D links ghrelin action to transcriptional control of orexigenic neuropeptide availability in AgRP neurons.

Neuropeptide Y regulates osteocyte phenotype and function through AHNAK-Smad signalling.

Neuropeptide Y (NPY) is a widespread hormone in the central and peripheral nervous systems that maintains body homeostasis. Central actions of hypothalamic NPY have been identified in bone metabolism. Osteocytes are the main source of NPY in bone tissue, indicating that osteocytic NPY could be a local alternative pathway for hypothalamic mediated regulation of bone and bone cells. Here, we show that osteocytic NPY induces cell viability and proliferation. Osteocyte-derived factors are also closely associated with changes in cellular NPY mRNA levels. Furthermore, osteoblast mineralization was significantly induced by conditioned medium collected from NPY-overexpressing osteocytes (P < 0.05). Importantly, the NPY-AHNAK interaction was identified for the first time by co-immunoprecipitation, and significant inactivation of p-Smad1/5/9 was found in osteocytes with NPY or AHNAK insufficiency (P < 0.05). The activation of p-Smad1/5/9 reversed NPY insufficiency-caused decreases in the expression of osteocytic proliferating cell nuclear antigen and osteoblast markers including osteocalcin and Runx2 (P < 0.05); these findings showed an additional molecular mechanism by which NPY acts on cells through AHNAK-mediated Smad1/5/9 signalling. Collectively, our findings provide novel insights into the function of NPY in regulating osteocyte phenotype and function and provide new insights for further investigation into osteocytic NPY-mediated therapy.