Dimers of the neuropeptide Y (NPY) Y2 receptor show asymmetry in agonist affinity and association with G proteins

In conditions precluding activation of G proteins, the binding of agonists to dimers of the neuropeptide Y (NPY) Y2 receptor shows two components of similar size, but differing in affinity. The dimers of all NPY receptors are solubilized as approximately 180-kDa complexes containing one G protein alpha beta gamma trimer. These heteropentamers are stable to excess agonists, chelators, and alkylators. However, dispersion in the weak surfactant cholate releases approximately 300-kDa complexes. These findings indicate that both protomers in the Y2 dimer are associated with G protein heterotrimers, but the extent of interaction depends on affinity for the agonist peptide. The G protein in contact with the first-liganded, higher-affinity protomer should have a stronger interaction with the receptor and a larger probability of activation.     

Impaired angiogenesis in neuropeptide Y (NPY)-Y2 receptor knockout mice

Which of Y1-Y5 receptors (Rs) mediate NPY's angiogenic activity was studied using Y2R-null mice and R-specific antagonists. In Y2R-null mice, NPY-induced aortic sprouting and in vivo Matrigel capillary formation were decreased by 50%; Y1R-antagonist blocked the remaining response. NPY-induced sprouting was equally inhibited by Y2R- (and Y5R- but less by Y1R-) antagonists in wild type mice. Spontaneous and NPY-induced revascularization of ischemic gastrocnemius muscles were similarly reduced in Y2R-null mice. Thus, NPY-induced angiogenesis, spontaneous and ischemic, is primarily mediated by Y2Rs. However, Y5Rs and, to a lesser degree Y1Rs, also may play a role in NPY-mediated angiogenesis.     

Genome wide association (GWA) study for early onset extreme obesity supports the role of fat mass and obesity associated gene (FTO) variants

Background

Obesity is a major health problem. Although heritability is substantial, genetic mechanisms predisposing to obesity are not very well understood. We have performed a genome wide association study (GWA) for early onset (extreme) obesity.

Methodology/Principal Findings

a) GWA (Genome-Wide Human SNP Array 5.0 comprising 440,794 single nucleotide polymorphisms) for early onset extreme obesity based on 487 extremely obese young German individuals and 442 healthy lean German controls; b) confirmatory analyses on 644 independent families with at least one obese offspring and both parents. We aimed to identify and subsequently confirm the 15 SNPs (minor allele frequency ≥10%) with the lowest p-values of the GWA by four genetic models: additive, recessive, dominant and allelic. Six single nucleotide polymorphisms (SNPs) in FTO (fat mass and obesity associated gene) within one linkage disequilibrium (LD) block including the GWA SNP rendering the lowest p-value (rs1121980; log-additive model: nominal p = 1.13×10⁻⁷, corrected p = 0.0494; odds ratio (OR)_CT 1.67, 95% confidence interval (CI) 1.22–2.27; OR_TT 2.76, 95% CI 1.88–4.03) belonged to the 15 SNPs showing the strongest evidence for association with obesity. For confirmation we genotyped 11 of these in the 644 independent families (of the six FTO SNPs we chose only two representing the LD bock). For both FTO SNPs the initial association was confirmed (both Bonferroni corrected p<0.01). However, none of the nine non-FTO SNPs revealed significant transmission disequilibrium.

Conclusions/Significance

Our GWA for extreme early onset obesity substantiates that variation in FTO strongly contributes to early onset obesity. This is a further proof of concept for GWA to detect genes relevant for highly complex phenotypes. We concurrently show that nine additional SNPs with initially low p-values in the GWA were not confirmed in our family study, thus suggesting that of the best 15 SNPs in the GWA only the FTO SNPs represent true positive findings.     

Synthesis of porcine neuropeptide Y(NPY) in solution

The porcine neuropeptide Y (NPY), a 36-residue peptide amide, was synthesized by assembling six peptide fragments followed by thioanisole-mediated deprotection with trifluoromethanesulfonic acid in trifluoroacetic acid. beta-Cycloheptyl aspartate, Asp(OChp), was employed to suppress base-catalyzed succinimide formation. When administered to dogs, the purified peptide (10 micrograms/kg) caused prolonged increase of systemic arterial blood pressure and decreased pancreatic blood flow.     

Cloned neuropeptide Y (NPY) Y1 and pancreatic polypeptide Y4 receptors expressed in Chinese hamster ovary cells show considerable agonist-driven internalization, in contrast to the NPY Y2 receptor.

Guinea-pig neuropeptide Y1 and rat pancreatic polypeptide Y4 receptors expressed in Chinese hamster ovary cells were internalized rapidly upon attachment of selective peptide agonists. The Y1 and Y2, but not the Y4, receptor also internalized the nonselective neuropeptide Y receptor agonist, human/rat neuropeptide Y. The internalization of guinea-pig neuropeptide Y2 receptor expressed in Chinese hamster ovary cells was small at 37 degrees C, and essentially absent at or below 15 degrees C, possibly in connection to the large molecular size of the receptor-ligand complexes (up to 400 kDa for the internalized fraction). The rate of intake was strongly temperature dependent, with essentially no internalization at 6 degrees C for any receptor. Internalized receptors were largely associated with light, endosome-like particulates. Sucrose dose-dependently decreased the internalization rate for all receptors, while affecting ligand attachment to cell membrane sites much less. Internalization of the Y1 and the Y4 receptors could be blocked, and that of the Y2 receptor significantly inhibited, by phenylarsine oxide, which also unmasked spare cell-surface receptors especially abundant for the Y2 subtype. The restoration of Y1 and Y4 receptors after agonist peptide pretreatment was decreased significantly by cycloheximide and monensin. Thus, in Chinese hamster ovary cells the Y1 and Y4 receptors have much larger subcellular dynamics than the Y2 receptor. This differential could also hold in organismic systems, and is comparable with the known differences in internalization of angiotensin, bradykinin, somatostatin and opioid receptor subtypes.     

神经肽Y(NPY)的生理功能研究进展

神经肽Y(NPY)是机体内的一种重要且保守的神经递质,一般以前体形式存在,释放的有活性的NPY主要通过与其受体结合发挥作用。NPY受体包含了亚型Y1、Y2、Y3、Y4、Y5、Y6、Y7、Y8。Y1和Y2是NPY发挥收缩血管作用的关键受体;Y1、Y2和Y5是NPY调节动物摄食行为的关键受体;Y1、Y2和Y4是NPY调控动物焦虑、沮丧行为的必要受体。着重对NPY与其各种受体结合后如何行使动物的相关生理功能的情况进行了阐述。     

Immunocytochemical localization of neuropeptide Y (NPY) in the human hypothalamus

Summary In order to study the distribution of neuropeptide Y-like immunoreactivity in the human hypothalamus, an immunocytochemical localization of this peptide was performed. Using antibodies developed against synthetic porcine neuropeptide Y (NPY), we have been able to localize immunoreactivity in neuronal cell bodies located exclusively in the infundibular nucleus. Immunostained fibers were found in several regions in the hypothalamus with a high concentration in the periventricular areas. Fibers were also found in the neurovascular zone of the median eminence, the pituitary stalk and the posterior pituitary. These results suggest that immunoreactive material related to porcine NPY is present in the human hypothalamus, with a distribution similar to that observed in the rat.     

Targeted deletion of neuropeptide Y (NPY) modulates experimental colitis

Background

Neurogenic inflammation plays a major role in the pathogenesis of inflammatory bowel disease (IBD). We examined the role of neuropeptide Y (NPY) and neuronal nitric oxide synthase (nNOS) in modulating colitis.

Methods

Colitis was induced by administration of dextran sodium sulphate (3% DSS) or streptomycin pre-treated Salmonella typhimurium (S.T.) in wild type (WT) and NPY (NPY^−/−) knockout mice. Colitis was assessed by clinical score, histological score and myeloperoxidase activity. NPY and nNOS expression was assessed by immunostaining. Oxidative stress was assessed by measuring catalase activity, glutathione and nitrite levels. Colonic motility was assessed by isometric muscle recording in WT and DSS-treated mice.

Results

DSS/S.T. induced an increase in enteric neuronal NPY and nNOS expression in WT mice. WT mice were more susceptible to inflammation compared to NPY^−/− as indicated by higher clinical & histological scores, and myeloperoxidase (MPO) activity (p<0.01). DSS-WT mice had increased nitrite, decreased glutathione (GSH) levels and increased catalase activity indicating more oxidative stress. The lower histological scores, MPO and chemokine KC in S.T.-treated nNOS^−/− and NPY^−/−/nNOS^−/− mice supported the finding that loss of NPY-induced nNOS attenuated inflammation. The inflammation resulted in chronic impairment of colonic motility in DSS-WT mice. NPY –treated rat enteric neurons in vitro exhibited increased nitrite and TNF-α production.

Conclusions

NPY mediated increase in nNOS is a determinant of oxidative stress and subsequent inflammation. Our study highlights the role of neuronal NPY and nNOS as mediators of inflammatory processes in IBD.     

Functional reconstitution of human neuropeptide Y (NPY) Y(2) and Y(4) receptors in Sf9 insect cells

The four functionally expressed human neuropeptide Y receptor subtypes (hY(1)R, hY(2)R, hY(4)R, hY(5)R) belong to class A of the G-protein-coupled receptors (GPCRs) and interact with pertussis toxin-sensitive G(i/o)-proteins. The number of small molecules described as ligands for hY(1)R and hY(5)R exceeds by far those for hY(2)R. Potent non-peptidergic ligands for the hY(4)R are not available so far. Here, we report on the functional reconstitution of the hY(2)R and the hY(4)R in Sf9 insect cells using the baculovirus system. Sf9 cells were genetically engineered by infection with up to four different baculoviruses, combining the receptors with G-proteins of the G(i/o) family and regulators of G-protein signaling (RGS) proteins to improve signal-to-noise ratio. In steady-state GTPase assays, using pNPY (Y(2)) and hPP (Y(4)), the GPCRs coupled to various G(i)/G(o)-proteins and both, RGS4 and GAIP, enhanced the signals. Co-expression systems hY(2)R + G?(i2) and hY(4)R + G?(i2)/G?(o) + RGS4, combined with G?(1)?(2), yielded best signal-to-noise ratios. hY(2)R function was validated using both agonistic peptides (NPY, PYY, NPY(13?36)) and selective non-peptidergic antagonists (BIIE0246 and derivatives), whereas the hY(4)R model was characterized with peptidergic agonists (PP, NPY, GW1229, and BW1911U90). Tunicamycin inhibited receptor N-glycosylation diminished NPY signals at hY(2)R and abolished hY(4)R function. Investigations with monovalent salts showed sensitivity of hY(4)R toward Na(+), revealing moderate constitutive activity. After validation, an acylguanidine (UR-PI284) was identified as a weak non-peptide Y(4)R antagonist. In summary, the established steady-state GTPase assays provide sensitive test systems for the characterization of Y(2) and Y(4) receptor ligands.     

Influence of diet composition on food intake and neuropeptide Y (NPY) gene expression in goldfish brain.

In this study, goldfish demonstrate preference for high carbohydrate and high fat diets, with no preference shown for high protein diets. Fish fed high (45% and 55%) carbohydrate (CHO) diets for 1 and 4 weeks exhibited decreased NPY gene expression in telencephalon-preoptic area (TEL-POA) and optic tectum-thalamus (OT-THAL) compared to fish fed low CHO (35% and 40%) diets. In hypothalamus (HYP), NPY gene expression was significantly increased after 1 week in fish fed both low and high CHO diets compared to control diet (40% CHO); after 4 weeks, the pattern in HYP was reversed. Fish fed a high fat (9%) diet had low NPY gene expression in TEL-POA after 1 and 4 weeks; however, HYP NPY expression was increased in fish fed a low (3%) fat diet after 1 week, and 2% and 3% fat diets after 4 weeks. In OT-THAL, NPY gene expression was decreased in fish fed a 2% fat diet for 1 week, and increased after 4 weeks. Feeding diets with different protein contents for 1 or 4 weeks did not influence NPY gene expression in goldfish brain. The results demonstrate, for the first time in a lower vertebrate, that NPY gene expression in goldfish brain is influenced by macronutrient intake.     

Pancreatic hormone response to neuropeptide Y (NPY) perifusion in vitro

Available data on the effect of neuropeptide Y (NPY) on insulin release are conflicting and little data exist regarding the effect of NPY on glucagon secretion. The purpose of the present study, therefore, was to characterize the direct effect of NPY on the release of these pancreatic hormones and to examine the role of glucose on these interactions. Using a perifused mouse islet system, we found that NPY suppressed both basal and glucose-stimulated insulin secretion. Thus, basal insulin release assessed as mean integrated area under the curve/20 min (AUC/20 min) decreased from 1446 +/- 143 pg to 651 +/- 112 pg (P less than 0.05) with the addition of 2 x 10(-8) M NPY and the AUC/20 min for glucose stimulated insulin output decreased from 1973 +/- 248 pg to 1426 +/- 199 pg (P less than 0.05). In both cases, this inhibitory effect was followed after removing NPY by a stimulation of insulin secretion which was typical of a 'rebound off-response'. In contrast, NPY exerted a stimulatory effect on basal glucagon release and significantly reversed the suppressive effect of high glucose on glucagon output. The basal glucagon AUC/20 min increased from 212 +/- 103 pg to 579 +/- 316 pg (P less than 0.05), while glucagon secretion in the presence of 27.7 mM glucose increased from 75 +/- 26 pg to 255 +/- 28 pg (P less than 0.01). In conclusion, we have shown that the direct effect of NPY on the endocrine pancreas is to suppress insulin but stimulate glucagon secretion. These data are compatible with a role for NPY in the regulation of pancreatic hormone output.     

Ligands of the neuropeptide Y Y2 receptor

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian brain and exerts a variety of physiological processes in humans via four different receptor subtypes Y1, Y2, Y4 and Y5. Y2 receptor is the most abundant Y subtype receptor in the central nervous system and implicated with food intake, bone formation, affective disorders, alcohol and drugs of abuse, epilepsy, pain, and cancer. The lack of small molecule non-peptidic Y2 receptor modulators suitable as in vivo pharmacological tools hampered the progress to uncover the precise pharmacological role of Y2. Only in recent years, several potent, selective and non-peptidic Y2 antagonists have been discovered providing the tools to validate Y2 receptor as a therapeutic target. This Letter reviews Y2 receptor modulators mainly non-peptidic antagonists and their structure–activity relationships.     

Innervation of the mink pineal gland with neuropeptide Y (NPY)-containing nerve fibers

Summary An immunohistochemical investigation of the mink pineal gland was performed by use of antibodies raised in rabbits against neuropeptide Y (NPY) and Cys-NPY (32–36)-amide recognizing neuropeptide Y with an amidation at position 36 (NPYamide). NPY-immunoreactive nerve fibers were located predominantly in the rostral part of the pineal gland and in the pineal stalk. Immunoreactive nerve fibers were found throughout the pineal gland, but the number of fibers in the caudal part of the gland was low. The fibers were present both in the perivascular spaces and between the pinealocytes. Many NPY-immunoreactive fibers were also located in the posterior and habenular commissures; some of these fibers were connected with the fibers in the rostral part of the mink pineal gland, indicating that at least some of the NPY-immunoreactive nerve fibers are of central origin. The nerve fibers immunoreactive to amidated NPY were distributed in a similar manner. However, the number of fibers immunoreactive to NPYamide was lower than the number of fibers immunoreactive to NPY itself. After removal of the superior cervical ganglia bilaterally 22 days or 12 months before sacrifice, NPY-immunoreactive nerve fibers remained in the gland. This immunohistochemical study of the mink pineal gland therefore shows that the NPY/NPYamide-immunoreactive nerve fibers innervating the pineal gland in this spegcies are a component of the central innervation or originnate from extracerebral parasympathetic ganglia.     

Mapping of the transcription start site (TSS) and identification of SNPs in the bovine neuropeptide Y (NPY) gene

Background

The domestic goat is one of the important livestock species of India. In the present study we assess genetic diversity of Indian goats using 17 microsatellite markers. Breeds were sampled from their natural habitat, covering different agroclimatic zones.

Results

The mean number of alleles per locus (NA) ranged from 8.1 in Barbari to 9.7 in Jakhrana goats. The mean expected heterozygosity (He) ranged from 0.739 in Barbari to 0.783 in Jakhrana goats. Deviations from Hardy-Weinberg Equilibrium (HWE) were statistically significant (P < 0.05) for 5 loci breed combinations. The D_A measure of genetic distance between pairs of breeds indicated that the lowest distance was between Marwari and Sirohi (0.135). The highest distance was between Pashmina and Black Bengal. An analysis of molecular variance indicated that 6.59% of variance exists among the Indian goat breeds. Both a phylogenetic tree and Principal Component Analysis showed the distribution of breeds in two major clusters with respect to their geographic distribution.

Conclusion

Our study concludes that Indian goat populations can be classified into distinct genetic groups or breeds based on the microsatellites as well as mtDNA information.     

Decreased neuropeptide Y (NPY) expression in the infundibular nucleus of patients with nonthyroidal illness

In patients with a variety of illnesses, serum concentrations of T3 decrease without giving rise to elevated serum levels of TSH, a phenomenon known as the sick euthyroid syndrome or nonthyroidal illness (NTI). Our previous studies in postmortem brain material showed decreased thyrotropin-releasing hormone (TRH) messenger RNA (mRNA) in the paraventricular nucleus (PVN) of patients with NTI, suggesting a role for TRH cells in the persistence of low TSH levels in NTI.In the present study, we hypothesized that changes in neuropeptide Y (NPY) input from the infundibular nucleus (IFN) to TRH cells in the PVN might be a determinant of decreased TRH expression in NTI. We investigated the hypothalamus of nine patients whose endocrine status had been assessed in a serum sample taken less than 24h before death and we examined NPY expression in the IFN by means of immunocytochemistry and mRNA in situ hybridization using an image analysis system. There was a negative correlation (r = -0.88; p = 0.01) between serum leptin concentrations and total NPY mRNA in the IFN. The total amount of NPY immunoreactivity in the IFN correlated with total NPY mRNA (r = 0.69; p = 0.04). In contrast to the situation in food-deprived rodents, total NPY immunoreactivity in the IFN showed a positive correlation with total TRH mRNA in the PVN (r = 0.77; p = 0.02). The results suggest a role for decreased NPY input from the IFN in the resetting of thyroid hormone feedback on hypothalamic TRH cells in NTI.     

The neuropeptide Y (NPY) Y2 receptors are largely dimeric in the kidney, but monomeric in the forebrain

The neuropeptide Y(NPY) Y2 receptors are detected largely as dimers in the clonal expressions in CHO cells and in particulates from rabbit kidney cortex. However, in two areas of the forebrain (rat or rabbit piriform cortex and hypothalamus), these receptors are found mainly as monomers. Evidence is presented that this difference relates to large levels of G proteins containing the Gi alpha -subunit in the forebrain areas. The predominant monomeric status of these Y2 receptors should also be physiologically linked to large synaptic inputs of the agonist NPY. The rabbit kidney and the human CHO cell-expressed Y2 dimers are converted by agonists to monomers in vitro at a similar rate in the presence of divalent cations.     

Oligomerization of neuropeptide Y (NPY) Y2 receptors in CHO cells depends on functional pertussis toxin-sensitive G-proteins

Human neuropeptide Y Y2 receptors expressed in CHO cells are largely oligomeric, and upon solubilization are recovered by density gradient centrifugation as approximately 180 kDa complexes of receptor dimers and G-protein heterotrimers. A large fraction of the receptors is inactivated in the presence of pertussis toxin, in parallel with inactivation of Gi alpha subunits (with half-periods of about 4 h for both). This is accompanied by a very long-lasting loss of receptor dimers and of masked surface Y2 sites (an apparent receptor reserve pre-coupled mainly to Gi alpha subunit-containing G-proteins). However, surface Y2 receptors accessible to large peptide agonists are much less sensitive to the toxin. All surface Y2 receptors are rapidly blocked by Y2 antagonist BIIE0246, with a significant loss of the dimers, but with little change of basal Gi activity. However, both dimers and Y2 receptor compartmentalization are restored within 24 h after removal of the antagonist. In CHO cells, the maintenance and organization of Y2 receptors appear to critically depend on functional pertussis toxin-sensitive G-proteins.     

Agonist internalization by cloned Y1 neuropeptide Y (NPY) receptor in Chinese hamster ovary cells shows strong preference for NPY,endosome-linked entry and fast receptor recycling

In Chinese hamster ovary (CHO) cells expressing the cloned guinea-pig Y1 receptor, the saturable, receptor-linked internalization of NPY (NPY)-related peptides showed the rank order of human/rat neuropeptide Y (hNPY)>pig/rat peptide YY (pPYY)>=(Pro(34))human PYY>(Leu(31),Pro(34))hNPY>(Leu(31),Pro(34))hPYY>BVD-11 (a selective Y1 antagonist). All agonists accessed similar numbers of Y1 sites in particulates from disrupted cells, with relatively small affinity variation. The rate of internalization could significantly depend on the overall interactivity of the agonist peptide (reflected in sensitivity to chaotropic agents, as well as in the level of non-saturable binding and internalization). Concentration-dependent inhibition of the agonist-driven CHO-Y1 internalization was found with filipin III (a cholesterol-complexing macrolide), and confirmed with inhibitors of clathrin lattice formation, phenylarsine oxide (PAO) and sucrose. In the concentration range affecting Y1 internalization, none of the above treatments or agents significantly alter agonist affinity for Y1 cell surface or particulate receptors. Largely similar responses to the above inhibitors were observed in CHO-Y1 cells for internalization of human transferrin. Internalization of CHO-Y1 receptor apparently is driven by NPY in strong preference to other naturally encountered agonists. At 37 degrees C, most of the internalized receptor is rapidly recycled through endosome-like membrane elements, detectable in Percoll gradients.