Y1 Receptor mRNA Research Articles

The cardiac sympathetic co-transmitter neuropeptide-Y is pro-arrhythmic in human cardiomyocytes by lengthening activation-recovery interval

Abstract Background Myocardial infarction (MI) is associated with sympathetic overactivity, during which the co-release of neuropeptide-Y (NPY) is prominent. NPY is associated with arrhythmia risk and mortality following ST-elevation MI, although the exact mechanisms for this in human cardiomyocytes remains unclear. Purpose We therefore tested the hypothesis that NPY signalling would alter the electrophysiology of human induced-pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in a way that would promote arrhythmia, and that in patients being treated for MI, high NPY concentrations previously associated with ventricular arrhythmia (>27.3 pg.mL-1) would be associated with comparable ECG changes. Methods Immunohistochemistry and western blot were performed on hiPSC-CMs and ventricular biopsies from human patients undergoing valve surgery. The FRET-based sensor Epac-SH187 was expressed in hiPSC-CMs to monitor the cyclic adenosine 3’,5’-monophosphate (cAMP) response to NPY. A micro-electrode array was used to investigate the effects of NPY on a confluent layer of iPSC-CMs, measuring changes in activation, recovery, conduction velocity and spontaneous automaticity. Additionally, patients presenting with ST-elevation MI had peripheral venous [NPY] measured and compared to 12-lead ECGs recorded following stenting. Results hiPSC-CMs expressed Y1 and Y5 receptor mRNA and protein, which are also expressed in ventricular biopsies from human patients. NPY (1-100 nM) significantly reduced intracellular cAMP levels (n=65), most effectively prevented by Y5 receptor inhibition (1µM CGP71683A, n=40). In a confluent layer of hiPSC-CM, NPY (100 nM) slowed late-repolarisation represented by T-wave peak-end duration (p=0.04), which could be prevented by Y1-receptor inhibition (1 μM BIBO 3304, n=6), and caused prolongation of rate corrected activation-recovery interval (ARIc) (p=0.009, n=6) which could be abolished by Y5-receptor inhibition (n=7). NPY significantly increased automaticity to more than one site of initiation in 4/6 (66.7%) compared to 2/14 (14.3%) control plates (p=0.037), despite no change in conduction velocity. Moreover, in 65 patients being treated for ST elevation MI, those with high peripheral venous NPY concentrations were well matched in terms of cardiovascular risk factors, medications on admission, and pain to balloon times, but had significantly longer corrected QT interval (QTc) on the 12 lead ECG (p=0.04) despite no differences in PR interval, heart rate or electrolyte concentrations. Conclusions NPY slowed late repolarisation and caused ARIc prolongation, which was associated with increased automaticity in human iPSC-CMs. NPY also correlated with QTc-prolongation in patients being treated for MI and may contribute to the increased incidence of ventricular arrhythmia observed in these patients. Antagonists of Y1 and Y5 receptors may therefore offer an adjunct to β-blockade therapy to reduce the risk of ventricular arrhythmia.

Protective effects of silymarin-loaded chitosan nanoparticles in the diet-induced hyperlipidemia rat model.

Obesity is a metabolic syndrome that leads to many chronic diseases worldwide. In this study, we investigate the antihyperlipidemic activities of chitosan nanoparticles (CH NPs) on silymarin (SIL) as a carrier in the drug delivery system that can improve some biochemical parameters and hormones in the model of hyperlipidemic rats receiving a high-fat diet (HFD). Physicochemical characterization of silymarin-loaded chitosannanoparticles (CH-SIL NPs) was done by Fourier-transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), and drug loading efficiency (LE). Diet-induced hyperlipidemic rats were treated with SIL (15 mg/kg/day) and CH-SIL NPs(15 mg/kg/day) for twelve weeks orally daily. The body weight loss (BW), food consumption, serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), levels of fasting blood glucose (FBG) in serum, serum insulin, cortisol, testosterone, and brain neuropeptide Y (NPY), Y1 and Y5 receptor mRNA expression were analyzed. A significant reduction in BW and food consumption from 417 ± 16 g and 33 ± 1.03 in group HFD to 338 ± 10 g and 17.33 ± 1.02 in group CHS+HFD was observed, respectively. This data revealed that CH-SIL NPs improved hyperlipidemia, hyperinsulinemia, and hyperglycemia, reduced serum cortisol, and down-regulated NPY and Y1R with a significant increase in HDL and testosterone hormones compared to the control group. The developed Sil-loaded CH NPs were good agents for improving efficacy. It is the first report of the proposed weight loss mechanism of SIL CH NPs, thereby providing information about the anti-hyperlipidemic and antihyperglycemic effects of silymarin-loaded chitosan nanoparticles, a natural food with proper effects against metabolic disorders in case of hyperlipidemia that may lead to obesity and up-regulation of brain NPY.

Effects of electroacupuncture on the expression of hypothalamic neuropeptide Y and ghrelin in pubertal rats with polycystic ovary syndrome.

BackgroundPolycystic ovary syndrome often starts in puberty, and its pathogenesis is not clear. This study aimed to explore the pathogenesis of pubertal polycystic ovary syndrome (PCOS) and assess the therapeutic effect of electroacupuncture on pubertal PCOS.MethodsDihydrotestosterone (DHT) was used to induce rat models of pubertal PCOS. pubertal rats with PCOS were randomly divided into a model group (M), an electroacupuncture group (EA), and a sham acupuncture group (SA). Age-matched normal rats were regarded as normal controls (N). Rats were treated with EA or SA five times a week for 25 minutes during their 6th–7th week. At the end of the experiment, we observed any changes in ovarian morphology; detected levels of metabolic indices in serum, the hypothalamus and pancreas.ResultsEA significantly improved estrous cycle disorders and the ovarian polycystic morphology in pubertal rats with PCOS, but SA only improved disorders of the estrous cycle. The serum levels of insulin, neuropeptide Y(NPY) and fasting blood glucose(FBG) increased significantly (both p < 0.01), while the serum levels of ghrelin(GHRL) decreased in the model group (p < 0.01). After treatment with EA, the levels of NPY (p < 0.01) and FBG (p < 0.05) went into decrease, whereas the levels of GHRL (p < 0.05) and insulin (p < 0.01) increased. There was few differences in the hypothalamic expression of galanin (GAL), galanin-like peptide (GALP) and ghrelin receptor(GHSR) between the four groups. The upregulation of NPY mRNA and neuropeptide Y2 receptor(NPY2R) mRNA and the downregulation of GHRL protein and mRNA in the hypothalamus, and the increased expression of NPY and NPY2R as well as the decreased expression of GHRL in the arcuate nucleus (ARC) can be rescued by EA. But, surprisingly, SA seem to make no difference to the levels of FBG and insulin, and the protein expression of ghrelin in the hypothalamus and ARC. Co-expression of kisspeptin and GHSR, and co-expression of gonadotrophin releasing hormone(GnRH) and NPY2R were observed in ARC. No differences were found between groups in protein of GAL, GALP and GHRL expression in the pancreas. Neither EA nor SA can attenuate the upregulated kisspeptin protein expression in the pancreas of PCOS model rats.ConclusionsEA and SA improved the symptoms of pubertal PCOS rats, and the mechanism might be associated with regulating hypothalamic NPY and ghrelin levels.

SAT-607 The Vagus Nerve and the Hypothalamus Mediate Different Aspects of the Anorectic Effects of PYY3-36

Background: Drugs that safely promote weight loss are required to treat the obesity crisis. The gut hormone peptide YY 3-36 (PYY3-36) is secreted post-prandially to suppress appetite via the Y2 receptor (Y2R). However, it is unclear whether PYY3-36 acts directly on the Y2R in the hypothalamic arcuate nucleus (ARC) or the afferent vagus nerve to inhibit food intake. Understanding the pathways by which PYY3-36 mediates its anorectic effects may facilitate the therapeutic targeting of this system.Methods: Y2R knockdown in the ARC (ARC-Y2R-KD) was achieved by stereotactic injection of Cre-expressing adeno-associated virus (AAV-Cre) in Y2R-flox C57Bl/6 mice. Y2R KD in the vagus was achieved by bilateral microinjection of AAV-Cre into the nodose ganglia (NG), where the cell bodies of vagal afferents reside. An alternative germline model of sensory nerve Y2R knockdown was generated using Nav1.8-Cre mice crossed with the Y2R-flox strain (Nav1.8-Y2R-KD). Feeding behaviour over 10 days in metabolic cages and the effects of endogenously released (after oral gavage of a nutrient bolus) or exogenously-administered PYY3-36 were investigated.Results: NG-Y2R-KD animals had 60% reduction in NG Y2R mRNA but remained responsive to cholecystokinin, a positive control of vagal functionality. This is the first example of receptor specific adult vagal deafferentation in mice. The Nav1.8-Y2R-KD model achieved 30% receptor KD. Feeding patterns in the ARC-Y2R-KD and NG-Y2R-KD groups were highly different from their controls, with smaller, faster meals in the KD groups. The anorectic effects (at the next meal) of endogenous PYY3-36 were attenuated in NG-Y2R-KD. Low dose exogenous PYY3-36 at 5 µg/kg significantly reduced 2h post injection food intake (FI) in the control groups (n=8; P=0.045) but this was abrogated in the NG-Y2R-KD group. This pattern was mirrored in the Nav1.8-Y2R-KD model: low dose PYY3-36 significantly reduced FI 1h post-IP compared to vehicle in controls (-0.19±0.05 g; P =0.036; n=8) but not in the Nav1.8-Y2R-KD (-0.004±0.111 g; n=3). Peripherally-administered PYY3-36 at a high dose (30 µg/kg) decreased FI in all groups, including ARC-Y2R-KD.Summary: These results suggest that endogenous PYY3-36 modulates meal patterning. The vagus nerve mediates physiological PYY3-36 signalling but alternative pathways, not exclusively via the ARC, may be more important in mediating its pharmacological effects. This is relevant for the design of more effective weight loss agents.

Gene therapy mediated seizure suppression in Genetic Generalised Epilepsy: Neuropeptide Y overexpression in a rat model

Neuropeptide Y (NPY) is an important 36 amino acid peptide that is abundantly expressed in the mammalian CNS and is known to be an endogenous modulator of seizure activity, including in rat models of Genetic Generalised Epilepsy (GGE) with absence seizures. Studies have shown that viral-mediated “gene therapy” with overexpression of NPY in the hippocampus can suppress seizures in acquired epilepsy animal models. This study investigated whether NPY gene delivery to the thalamus or somatosensory cortex, using recombinant adeno-associated viral vector (rAAV), could produce sustained seizure suppression in the GAERS model of GGE with absence seizures. Three cohorts of GAERS were injected bilaterally into the thalamus (short term n = 14 and long term n = 8) or the somatosensory cortex (n = 26) with rAAV-NPY or rAAV-empty. EEG recordings were acquired weekly post-treatment and seizure expression was quantified. Anxiety levels were tested using elevated plus maze and open field test. NPY and NPY receptor mRNA and protein expression were evaluated using quantitative PCR, immunohistochemistry and immunofluorescence. Viral overexpression of human NPY in the thalamus and somatosensory cortex in GAERS significantly reduced the time spent in seizure activity and number of seizures, whereas seizure duration was only reduced after thalamic NPY overexpression. Human and rat NPY and rat Y2 receptor mRNA expression was significantly increased in the somatosensory cortex. NPY overexpression in the thalamus was observed in rAAV-NPY treated rats compared to controls in the long term cohort. No effect was observed on anxiety behaviour. We conclude that virally-mediated human NPY overexpression in the thalamus or somatosensory cortex produces sustained anti-epileptic effects in GAERS. NPY gene therapy may represent a novel approach for the treatment of patients with genetic generalised epilepsies.

Locus coeruleus response to single-prolonged stress and early intervention with intranasal neuropeptide Y.

Dysregulation of the central noradrenergic system is a core feature of post-traumatic stress disorder (PTSD). Here, we examined molecular changes in locus coeruleus (LC) triggered by single-prolonged stress (SPS) PTSD model at a time when behavioral symptoms are manifested, and the effect of early intervention with intranasal neuropeptide Y (NPY). Immediately following SPS stressors, male SD rats were administered intranasal NPY (SPS/NPY) or vehicle (SPS/V). Seven days later, TH protein, but not mRNA, was elevated in LC only of the SPS/V group. Although 90% of TH positive cells expressed GR, its levels were unaltered. Compared to unstressed controls, LC of SPS/V, but not SPS/NPY, expressed less Y2 receptor mRNA with more CRHR1 mRNA in subset of animals, and elevated corticotropin-releasing hormone (CRH) in central nucleus of amygdala. Following testing for anxiety on elevated plus maze (EPM), there were significantly increased TH, DBH and NPY mRNAs in LC of SPS-treated, but not previously unstressed animals. Their levels highly correlated with each other but not with behavioral features on EPM. Thus, SPS triggers long-term noradrenergic activation and higher sensitivity to mild stressors, perhaps mediated by the up-regulation influence of amygdalar CRH input and down-regulation of Y2R presynaptic inhibition in LC. Results also demonstrate the therapeutic potential of early intervention with intranasal NPY for traumatic stress-elicited noradrenergic impairments. Single-prolonged stress (SPS)-triggered long-term changes in the locus coeruleus/norepinephrine (LC/NE) system with increased tyrosine hydroxylase (TH) protein and CRH receptor 1(CRHR1) mRNA and lower neuropeptide Y receptor 2 (Y2R) mRNA levels as well as elevated corticotropin-releasing hormone (CRH) in the central nucleus of amygdala (CeA) that were prevented by early intervention with intranasal neuropeptide Y (NPY). SPS treatment led to increased sensitivity of LC to mild stress of elevated plus maze (EPM), with elevated mRNA for NE biosynthetic enzymes in subset of animals.

Peptide YY signaling in the lateral parabrachial nucleus increases food intake through the Y1 receptor.

Although central PYY delivery potently increases food intake, the sites of action and mechanisms mediating these hyperphagic effects are not fully understood. The present studies investigate the contribution of lateral parabrachial nucleus (lPBN) PYY-Y receptor signaling to food intake control, as lPBN neurons express Y receptors and receive PYY fibers and are known to integrate circulating and visceral sensory signals to regulate energy balance. Immunohistochemical results identified a subpopulation of gigantocellular reticular nucleus PYY-producing neurons that project monosynaptically to the lPBN, providing an endogenous source of PYY to the lPBN. lPBN microinjection of PYY-(1-36) or PYY-(3-36) markedly increased food intake by increasing meal size. To determine which receptors mediate these behavioral results, we first performed quantitative real-time PCR to examine the relative levels of Y receptor expression in lPBN tissue. Gene expression analyses revealed that, while Y1, Y2, and Y5 receptors are each expressed in lPBN tissue, Y1 receptor mRNA is expressed at fivefold higher levels than the others. Furthermore, behavioral/pharmacological results demonstrated that the hyperphagic effects of PYY-(3-36) were eliminated by lPBN pretreatment with a selective Y1 receptor antagonist. Together, these results highlight the lPBN as a novel site of action for the intake-stimulatory effects of central PYY-Y1 receptor signaling.

Cloning, expression, and ligand-binding characterization of two neuropeptide Y receptor subtypes in orange-spotted grouper, Epinephelus coioides.

As one of the most important multifunctional peptides, neuropeptide Y (NPY) performs its physiological functions through different subtype receptors. In this study, full-length cDNAs of two NPY receptors (YRs) in orange-spotted grouper (Epinephelus coioides) were cloned and named npy8br (y8b) and npy2r (y2). Phylogenetic analysis indicated that the Y8b receptor is an ortholog of the teleostean Y8b receptor, which belongs to the Y1 subfamily, and the Y2 receptor is an ortholog of the teleostean Y2 receptor, which belongs to the Y2 subfamily. Both of the YRs have G protein-coupled receptor family profiles. Multiple alignments demonstrate that the extracellular loop regions of YRs have distinctive residues of each species. Expression profile analysis revealed that the grouper Y8b receptor mRNA is primarily expressed in the brain, stomach and intestine, while the grouper Y2 receptor mRNA is primarily expressed in the brain, ovary, liver and heart. Double immunofluorescence analysis determined that the grouper YRs interact with the grouper NPY around the human embryonic kidney 293T cell surface. Furthermore, site-directed mutagenesis in a phage display system revealed that Asp(6.59) might be a common NPY-binding site, while Asp(2.68) of the Y8b receptor and Glu(5.24) of the Y2 receptor could be likely involved in subtype-specific binding. Combining the expression profile and ligand-binding feature, the grouper Y8b receptor could be involved in regulating food intake via the brain-gut axis and the grouper Y2 receptor might play a role in balancing the regulatory activity of the Y8b receptor and participate in metabolism in the liver and ovary.

Neuropeptide Y-stimulated [(35) S]GTPγs functional binding is reduced in the hippocampus after kainate-induced seizures in mice.

Kainate-induced seizures constitute a model of temporal lobe epilepsy where prominent changes are observed in the hippocampal neuropeptide Y (NPY) system. However, little is known about the functional state and signal transduction of the NPY receptor population resulting from kainate exposure. Thus, in this study, we explored functional NPY receptor activity in the mouse hippocampus and neocortex after kainate-induced seizures using NPY-stimulated [(35) S]GTPγS binding. Moreover, we also studied levels of [(125) I]-peptide YY (PYY) binding and NPY, Y1, Y2, and Y5 receptor mRNA in these kainate-treated mice. Functional NPY binding was unchanged up to 12 h post-kainate, but decreased significantly in all hippocampal regions after 24 h and 1 week. Similarly, a decrease in [(125) I]-PYY binding was found in the dentate gyrus (DG) 1 week post-kainate. However, at 2 h, 6 h, and 12 h, [(125) I]-PYY binding was increased in all regions, and in the CA1 also at 24 h post-kainate. NPY mRNA levels were prominently increased in hippocampal regions, reaching maximum at 12 and 24 h. Y1 and Y5 mRNA levels were lowered in the DG at 24 and 2 h, respectively, while Y2 mRNA levels were elevated at 24 h in the DG and CA3. This study confirms rat kainate studies by showing pronounced adaptive changes in the mouse hippocampus both with regard to NPY synthesis and NPY receptor synthesis and binding, which may contribute to regulating neuronal seizure susceptibility after kainate. However, the potential seizure-suppressant effects of increased NPY gene expression at late time points post-kainate could be attenuated by the novel finding of reduced NPY-receptor G-protein activation.

Regulation of neuropeptide Y Y1 receptor expression by bone morphogenetic protein 2 in C2C12 myoblasts

The neuropeptide Y (NPY) system is known as one of the major neural signaling pathways. NPY, produced by peripheral tissues including osteoblasts, is known to bind to the Y1 receptor. Recently, osteoblast-specific Y1 receptor knockout mice were developed and were found to have a high bone mass phenotype, indicating a role for the NPY-Y1 receptor axis as a regulator of bone homeostasis. However, regulation of Y1 receptor expression during osteoblastic differentiation remains unexplored. In the present study, we examined the role of bone morphogenetic protein (BMP) 2 signaling in regulating Y1 receptor expression. In C2C12 cells, expression of Y1 receptor mRNA was induced by BMP2. This induction was also observed after co-transfection with Smad1 and Smad4, the intracellular signaling molecules of the BMP2 signaling pathway. In a transfection assay, Smad1/4 up-regulated transcriptional activity through interaction with the Y1 receptor gene promoter. Following transfection of MC3T3-E1 cells with siRNA for the Y1 receptor, the expression of alkaline phosphatase, osteocalcin, Runx2 and osterix were increased. These results show that BMP2 signaling regulates Y1 receptor gene expression, and raises the possibility that NPY acts in osteoblasts via an autocrine mechanism.

Expressions of neuropeptide Y and Y1 receptor in subcutaneous and visceral fat tissues in normal weight and obese humans and their correlations with clinical parameters and peripheral metabolic factors

Recently, neuropeptide Y (NPY) and Y1 receptor (Y1R) were found to be expressed and synthesized in adipose tissue. This study aimed to compare NPY and Y1R mRNA expressions in subcutaneous and visceral fat tissues as well as serum NPY in normal weight and obese humans and their correlations with clinical parameters and peripheral metabolic factors. We demonstrated that NPY mRNA expression was higher in obese than in normal weight humans (p<0.05) in both subcutaneous and visceral adipose tissues and was significantly greater in visceral when compared with subcutaneous fat in overall (p<0.01), obese (p<0.05) and normal weight humans (p<0.05). Y1R mRNA expression was higher in obese than normal weight subjects in visceral (p<0.01) but not in subcutaneous adipose tissue and was statistically greater in subcutaneous when compared to visceral adipose tissue in obese (p<0.05) and overall subjects (p<0.05). Serum NPY was higher in obese than normal weight groups (p<0.05). Obese subjects showed significantly greater levels of systolic blood pressure (SBP) (p<0.01), diastolic blood pressure (DBP) (p<0.05), plasma insulin (p<0.05), and HOMA-IR (p<0.05) when compared with normal weight subjects. Additionally, Y1R mRNA expression in visceral adipose tissue was positively correlated with body weight (R=0.586), BMI (R=0.611), waist (R=0.474) and hip (R=0.483) circumferences, insulin levels (R=0.539), and HOMA-IR (R=0.480). As the result, Y1R expression in visceral adipose tissue might be an indicator of increased risk of metabolic syndrome. Further studies about blocking specific Y1R may propose strategies for risk reduction in metabolic syndrome and prevention or treatment of obesity.

Maternal low‐protein diet up‐regulates the neuropeptide Y system in visceral fat and leads to abdominal obesity and glucose intolerance in a sex‐ and time‐specific manner

Neuropeptide Y (NPY) mediates stress-induced obesity in adult male mice by activating its Y2 receptor (Y2R) in visceral adipose tissue (VAT). Here, we studied whether the NPY-Y2R system is also activated by maternal low-protein diet (LPD) and linked to obesity in offspring. Prenatal LPD offspring had lower birth weights compared to normal-protein diet (NPD) offspring. Female prenatal and lactation stress (PLS) offspring from mothers fed an LPD developed abdominal adiposity and glucose intolerance associated with a 5-fold up-regulation of NPY mRNA and a 6-fold up-regulation of Y2R mRNA specifically in VAT, in addition to elevated platelet-rich-plasma (PRP) NPY, compared to control females fed a high-fat diet (HFD). Conversely, PLS male offspring showed lower NPY in PRP, a 10-fold decrease of Y2R mRNA in VAT, lower adiposity, and improved glucose tolerance compared to control males. Interestingly, prenatal LPD offspring cross-fostered to control lactating mothers had completely inverse metabolic and NPY phenotypes. Taken together, these findings suggested that maternal LPD activates the VAT NPY-Y2R system and increases abdominal adiposity and glucose intolerance in a sex- and time-specific fashion, suggesting that the peripheral NPY system is a potential mediator of programming for the offspring's vulnerability to obesity and metabolic syndrome.

Neuropeptide Y activates phosphorylation of ERK and STAT3 in stromal vascular cells from brown adipose tissue, but fails to affect thermogenic function of brown adipocytes

The thermogenic function of brown adipose tissue (BAT) is increased by norepinephrine (NE) released from sympathetic nerve endings, but the roles of NPY released along with NE are poorly elucidated. Here, we examined effect of NPY on basal and NE-enhanced thermogenesis in isolated brown adipocytes that express Y1 and Y5 receptor mRNA. Treatment of cells with NPY did not influence the basal and NE-enhanced rates of oxygen consumption and cAMP accumulation. Treatment with NPY also failed to induce ERK (Thr202/Tyr204) phosphorylation in the brown adipocytes. In contrast, treatment with NPY increased ERK phosphorylation in cultured stromal vascular cells from the BAT that express Y1 receptor mRNA. In the latter treatment with NPY also increased STAT3 (Ser727) phosphorylation. These results suggest that NPY mainly acts on stromal vascular cells in BAT and plays roles in the regulation of their gene transcription through ERK and STAT3 pathways, while NPY does not affect the thermogenic function of brown adipocytes.

Y5 neuropeptide Y receptor overexpression in mice neither affects anxiety- and depression-like behaviours nor seizures but confers moderate hyperactivity

Neuropeptide Y (NPY) has been implicated in anxiolytic- and antidepressant-like behaviour as well as seizure-suppressant effects in rodents. Although these effects appear to be predominantly mediated via other NPY receptors (Y1 and/or Y2), several studies have also indicated a role for Y5 receptors. Gene therapy using recombinant viral vectors to induce overexpression of NPY, Y1 or Y2 receptors in the hippocampus or amygdala has previously been shown to modulate emotional behaviour and seizures in rodents. The present study explored the potential effects of gene therapy with the Y5 receptor, by testing effects of recombinant adeno-associated viral vector (rAAV) encoding Y5 (rAAV-Y5) in anxiety- and depression-like behaviour as well as in kainate-induced seizures in adult mice. The rAAV-Y5 vector injected into the hippocampus and amygdala induced a pronounced and sustained increase in Y5 receptor mRNA expression and functional Y5 receptor binding, but no significant effects were found with regard to anxiety- and depression-like behaviours or seizure susceptibility. Instead, rAAV-mediated Y5 receptor transgene overexpression resulted in moderate hyperactivity in the open field test. These results do not support a potential role for single transgene overexpression of Y5 receptors for modulating anxiety-/depression-like behaviours or seizures in adult mice. Whether the induction of hyperactivity by rAAV-Y5 could be relevant for other conditions remains to be studied.

Differential Responses of Orexigenic Neuropeptides to Fasting in Offspring of Obese Mothers

Maternal obesity due to long-term high-fat diet (HFD) consumption leads to faster growth in offspring during suckling, and increased adiposity at 20 days of age. Decreased expression of the orexigenic neuropeptide Y (NPY) and increased anorexigenic proopiomelanocortin (POMC) mRNA expression were observed in the fed state. However, hunger is the major drive to eat and hypothalamic appetite regulators change in response to meals. Therefore, it is important to compare both satiated and fasting states. Female Sprague-Dawley rats (8 weeks old) were fed a cafeteria-style HFD (15.33 kJ/g) or chow for 5 weeks before mating, with the same diet continuing throughout gestation and lactation. At postnatal day 20, male pups were killed either after overnight fasting or in the fed state. Pups from obese dams were hyperphagic during both pre- and postweaning periods. Pups from obese dams had higher hypothalamic mRNA expression of POMC and NPY Y1 receptor, but lower hypothalamic melanocortin-4 receptor (MC4R) and its downstream target single-minded gene 1 (Sim1), in the fed state. Overnight fasting reduced circulating glucose, insulin, and leptin and increased hypothalamic NPY Y1 receptor mRNA in pups from both lean and obese dams. Hypothalamic NPY and agouti-related protein (AgRP) were only increased by fasting in pups from obese dams; reductions in MC4R and Sim1 were only seen in pups from lean dams. At weaning, the suppressed orexigenic signals in offspring from obese dams were normalized after overnight fasting, although anorexigenic signaling appeared impaired in these animals. This may contribute to their hyperphagia and faster growth.

Characterizing the sympathetic neuropeptide Y system in 4T1 murine mammary carcinoma model

Neuropeptide Y (NPY) released from the sympathetic nervous system (SNS) is a potent mitogenic and angiogenic factor. Recent studies have illustrated the prevalence of NPY receptors in many cancer cell lines. Remarkably, little is known of the role that NPY and its Y1‐ and Y2‐receptors play in the progression of breast cancer tumor development. Moreover, no in vivo models have addressed the impact of NPY in breast cancer. In this study we used the murine 4T1 cell line as it mimics growth and metastatic patterns seen in human breast cancer. Cells (103) were injected into the inguinal mammary fat pad of female BALB/c mice (n=10), after 29 days tumors were harvested and prepared for immunofluorescence and molecular analysis (Western blot and qRT‐PCR). 4T1 tumors were characterized by: 1) extensive vascular development that was innervated by sympathetic neurons, and 2) robust expression of NPY, Y1 and Y2 receptors. Additionally, Y1 receptor mRNA was augmented in 4T1 tumors compared to cultured cells (p&lt;0.05), suggesting potential for a Y1‐mediated functional role of NPY in this cancer model. Based on these data we show for the first time that breast cancer tumors raised from the 4T1 cell line have a complete and intact sympathetic NPY system. Thus, we conclude that this is an ideal model to investigate the functional impact of the SNS on breast cancer growth. CIHR

Regulation of Neuropeptide Y Y1 Receptors by Testosterone in Vascular Smooth Muscle Cells in Rat Testis

It is well established that testosterone and neuropeptide Y (NPY), via its Y1 receptor (Y1R), are involved in the central control of the gonadotrope axis in male rats. Here we examined if a similar interaction also occurs in the male peripheral reproductive target organ, the testes. Expression of the Y1R transcript and protein and changes in testicular microcirculation were studied in normal rats and 12 days following hypophysectomy with and without testosterone substitution (1 or 25 mg s.c.). In situ hybridization and immunohistochemistry showed strong expression of, respectively, Y1R messenger RNA (Y1R mRNA) and Y1R-like immunoreactivity (Y1R-LI) in vascular smooth muscles in the testes of control and hypophysectomized rats treated with testosterone, but was not seen without testosterone substitution. In parallel, control animals and hypophysectomized, testosterone-supplemented rats showed a strong (approximately 40%) decrease in testicular blood flow following intratesticular (i.t.) injection of the Y1-R agonists, [Leu³¹, Pro³⁴]NPY, [D-Arg²⁵]NPY or NPY, an effect which was completely blocked by prior intravenous administration of the Y1R antagonist, BIBP3226. No significant change in testicular blood flow following i.t. injection of NPY was seen in hypophysectomized rats without testosterone substitution. These findings suggest that the high levels of Y1R mRNA and Y1R-LI in the testes reflect expression of functional Y1Rs mediating vasoconstriction, and that testosterone regulates expression of functional Y1Rs.

Insulin Secretion Is Increased in Pancreatic Islets of Neuropeptide Y-Deficient Mice

Neuropeptide Y (NPY), whose role in appetite regulation is well known, is also expressed in pancreatic islets. Although previous studies indicated that application of NPY to pancreatic islets inhibits insulin secretion, its physiological role in the regulation of insulin secretion is not fully understood. We hypothesized that NPY in islets tonically suppresses insulin secretion and the reduction of islet NPY increases insulin secretion. To address the hypothesis, islet function of NPY-deficient mice was analyzed. Although there was little change in glucose homeostasis in vivo, pancreatic islets from NPY-deficient mice had higher basal insulin secretion (1.5 times), glucose-stimulated insulin secretion (1.5 times), and islet mass (1.7 times), compared with wild-type mouse. Next we sought to determine whether the expression of NPY and Y(1) receptor in islets was altered in hyperinsulinemia associated with obesity. Islets from C57BL/6J mice on a high-fat diet had 1.9 times higher basal insulin secretion and 2.4 times higher glucose-stimulated insulin secretion than control mice, indicating islet adaptation to obesity. Expression of NPY and Y(1) receptor mRNA levels was decreased by 70 and 64%, respectively, in high-fat diet islets, compared with controls. NPY and Y(1) receptor in islets were also reduced by 91 and 80%, respectively, in leptin-deficient ob/ob mice that showed marked hyperinsulinemia. Together these results suggest that endogenous NPY tonically inhibits insulin secretion from islets and a reduction of islet NPY may serve as one of the mechanisms to increase insulin secretion when islets compensate for insulin resistance associated with obesity.

Neuropeptide Y Y1 and Y5 receptors are co-expressed with GRP mRNA in the rat hypothalamic paraventricular nucleus

A number of hypothalamic neuropeptide systems have been shown to participate in energy homeostasis. We have previously reported that food deprivation time-dependently decreased gastrin-releasing peptide (GRP) gene expression in the medial parvocellular portion of the hypothalamic paraventricular nucleus (PVN) in rats. This region of the hypothalamus receives a dense projection of neuropeptide Y (NPY) neurons from the arcuate nucleus and also expresses NPY receptors. Because this neuropeptide system is engaged during times of negative energy balance, we hypothesized that NPY may directly modulate GRP mRNA expression in the PVN during food deprivation. To address this question we examined whether GRP-containing neurons in the PVN expressed Y1 and Y5 receptors, two NPY receptor subtypes that have been implicated in food intake and energy balance. Double label in situ hybridization was accomplished by hybridization with a combination of radiolabeled probes for either Y1 and Y5 receptors and a digoxigenin-labeled probe for GRP. Emulsion autoradiography was used to detect NPY receptors and Cy3 was used for detection of GRP. Our results indicated that both GRP mRNA and Y1 and Y5 receptor mRNA's were co-localized in this hypothalamic region. This finding supports the possibility that the activity of GRP neurons in the PVN is directly linked to NPY signaling and may contribute to changes in GRP gene expression produced by food deprivation. Supported by NIH DK-046448.

Greater Bone Formation of Y2 Knockout Mice Is Associated with Increased Osteoprogenitor Numbers and Altered Y1 Receptor Expression

Germ line or hypothalamus-specific deletion of Y2 receptors in mice results in a doubling of trabecular bone volume. However, the specific mechanism by which deletion of Y2 receptors increases bone mass has not yet been identified. Here we show that cultured adherent bone marrow stromal cells from Y2(-/-) mice also demonstrate increased mineralization in vitro. Isolation of two populations of progenitor cell types, an immature mesenchymal stem cell population and a more highly differentiated population of progenitor cells, revealed a greater number of the progenitor cells within the bone of Y2(-/-) mice. Analysis of Y receptor transcripts in cultured stromal cells from wild-type mice revealed high levels of Y1 but not Y2, Y4, Y5, or y6 receptor mRNA. Interestingly, germ line Y2 receptor deletion causes Y1 receptor down-regulation in stromal cells and bone tissue possibly due to the lack of feedback inhibition of NPY release and subsequent overstimulation of Y1 receptors. Furthermore, deletion of Y1 receptors resulted in increased bone mineral density in mice. Together, these findings indicate that the greater number of mesenchymal progenitors and the altered Y1 receptor expression within bone cells in the absence of Y2 receptors are a likely mechanism for the greater bone mineralization in vivo and in vitro, opening up potential new treatment avenues for osteoporosis.