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.     

Attenuation of hypercholesterolemia and hyperglycemia in ob/ob mice by NPY Y2 receptor ablation

Neuropeptide Y (NPY) is a 36 amino acid peptide well known for its role in regulating food intake and energy homeostasis. It has previously been shown that the NPY Y2 receptor is required for a full biological response to leptin in the central nervous system. We have examined the impact of this receptor on plasma levels of lipid and cholesterol in wild type and obese (ob/ob) mice. The results show that an absence of Y2 in female mice has no effect on cholesterol level in normal lean mice but profoundly decreases serum cholesterol and glucose levels in ob/ob mice. We conclude that NPY, interacting with the Y2 receptor, participates in cholesterol and glucose homeostasis of obese mice.     

Reduced anxiety-like and depression-related behavior in neuropeptide Y Y4 receptor knockout mice

Neuropeptide Y (NPY) acting through Y1 receptors reduces anxiety- and depression-like behavior in rodents, whereas Y2 receptor stimulation has the opposite effect. This study addressed the implication of Y4 receptors in emotional behavior by comparing female germ line Y4 knockout (Y4−/−) mice with control and germ line Y2−/− animals. Anxiety- and depression-like behavior was assessed with the open field (OF), elevated plus maze (EPM), stress-induced hyperthermia (SIH) and tail suspension tests (TST), respectively. Learning and memory were evaluated with the object recognition test (ORT). In the OF and EPM, both Y4−/− and Y2−/− mice exhibited reduced anxiety-related behavior and enhanced locomotor activity relative to control animals. Locomotor activity in a familiar environment was unchanged in Y4−/− but reduced in Y2−/− mice. The basal rectal temperature exhibited diurnal and genotype-related alterations. Control mice had temperature minima at noon and midnight, whereas Y4−/− and Y2−/− mice displayed only one temperature minimum at noon. The magnitude of SIH was related to time of the day and genotype in a complex manner. In the TST, the duration of immobility was significantly shorter in Y4−/− and Y2−/− mice than in controls. Object memory 6 h after initial exposure to the ORT was impaired in Y2−/− but not in Y4−/− mice, relative to control mice. These results show that genetic deletion of Y4 receptors, like that of Y2 receptors, reduces anxiety-like and depression-related behavior. Unlike Y2 receptor knockout, Y4 receptor knockout does not impair object memory. We propose that Y4 receptors play an important role in the regulation of behavioral homeostasis.     

Increased brain neuropeptide Y1 and Y2 receptor binding in NPY knock out mice does not result in increased receptor function

The brain neuropeptide Neuropeptide Y (NPY) is an important modulator of a number of centrally mediated processes including feeding, anxiety-like behaviors, blood pressure and others. NPY produces its effects through at least four functional G-protein coupled receptors termed Y1, Y2, Y4 and Y5. In the brain, the Y1 and Y2 receptor subtypes are the predominant receptor population. To better understand the roles of NPY, genetically modified mice lacking NPY were produced but lacked the expected phenotypes. These mice have previously been reported to have a marked increase in Y2 receptor binding. In the present study, we found an upregulation of both Y1 and Y2 receptor binding and extended these findings to the female. These increases were as large as 10-fold or greater in many brain regions. To assess functional coupling of the receptors, we performed agonist-induced [(35)S]GTPgammaS autoradiography. In the mouse brain, the Y1/Y4/Y5 agonist Leu(31),Pro(34)-NPY increased [(35)S]GTPgammaS binding with a regional distribution consistent with that produced when labeling adjacent sections with [(125)I]-Leu(31),Pro(34)-PYY. In a few brain regions, minor increases were noted in the agonist-induced binding when comparing knock out mice to wild type. The Y2 agonist C2-NPY stimulated [(35)S]GTPgammaS binding in numerous brain areas with a regional distribution similar to the binding observed with [(125)I]-PYY3-36. Again, no major increases were noted in the functional activation of Y2 receptors between knock out and wild type mice. Therefore, the increased Y1 and Y2 binding observed in the NPY knock out mice does not represent an increase in NPY receptor mediated signaling and is likely due to an increase in spare (uncoupled) receptors.     

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.     

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.     

Neuropeptide Y (NPY) functional group mimetics: design, synthesis, and characterization as NPY receptor antagonists

N,N′-bis-[2-N-(O-2,6-dichlorobenzyl-L-tyrosyl)aminoethylguanyl]cystamine 3 and N,N′-bis-[2-N-(O-2,6-dichlorobenzyl-L-tyrosyl)aminoethyl]-1,6-hexanediguanidine 4 have been designed as neuropeptide Y (NPY) functional group mimetics. Both 3 and 4 displace N-[propionyl-³H]-NPY from rat brain binding sites, and are NPY receptor antagonists in rat femoral artery ring segments.     

Characterization of NPY receptor subtypes Y2 and Y7 in rainbow trout Oncorhynchus mykiss

We report the cloning and pharmacological characterization of two neuropeptide Y (NPY) receptor subtypes, Y2 and Y7, in rainbow trout (Oncorhynchus mykiss). These subtypes are approximately 50% identical to each other and belong to the Y2 subfamily of NPY receptors. The binding properties of the receptors were investigated after expression in human HEK-293 EBNA cells. Both receptors bound the three zebrafish peptides NPY, PYYa, and PYYb, as well as porcine NPY and PYY, with affinities in the nanomolar range that are similar to mammalian Y2. The affinity of the truncated porcine NPY fragments, NPY 13-36 and NPY 18-36 was markedly lower compared to mammalian and chicken Y2. This suggests that mammalian and chicken Y2 are unique among NPY receptors in their ability to bind truncated peptide fragments. The antagonist BIIE0246, developed for mammalian Y2, did not bind either of the two rainbow trout receptors. Our results support the proposed expansion of this gene family by duplications before the gnathostome radiation. They also reveal appreciable differences in the repertoire and characteristics of NPY receptors between fish and tetrapods stressing the importance of lineage-specific gene loss as well as sequence divergence after duplication.     

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.     

Neuropeptide Y (NPY) Y2 receptor-selective agonist inhibits food intake and promotes fat metabolism in mice: combined anorectic effects of Y2 and Y4 receptor-selective agonists

Peripheral administration of the endogenous Y(2) and Y(4) receptor selective agonists, PYY(3-36) and PP, have been shown to inhibit food intake and body weight gain in rodents, and to reduce appetite and caloric intake in humans. We have previously developed a long-acting, potent and highly selective Y(2) receptor selective agonist, N-alpha-Ac-[Nle(24,28), Trp(30), Nva(31), Psi(35-36)]PYY(22-36)-NH(2) (BT-48). BT-48 (ip) dose-dependently inhibited ad lib food intake and also decreased the respiratory quotient in mice during both the light and dark periods. The latter observation is indicative of enhanced fat metabolism. Moreover, BT-48 also inhibited food intake in fasted mice. Combined ip administration of BT-48 (50nmol/mouse) with a highly potent and selective Y(4) anorectic peptide, BVD-74D (50nmol/mouse), resulted in a powerful and long lasting inhibitory effect on food intake. As expected, this inhibitory effect on food intake was nearly double that exhibited by either peptide (50nmol/mouse) alone. In summary, BT-48, unlike PYY(3-36), exhibits little or no affinity to other "Y" receptors, and may therefore have a better clinical potential than PYY(3-36) for control of food intake. Moreover, it appears that treatment with a combination of Y(2) and Y(4) receptor selective agonists may constitute a more powerful approach to control food intake than treatment with either of these agonists alone.     

Increased ethanol resistance and consumption in Eps8 knockout mice correlates with altered actin dynamics

Dynamic modulation of the actin cytoskeleton is critical for synaptic plasticity, abnormalities of which are thought to contribute to mental illness and addiction. Here we report that mice lacking Eps8, a regulator of actin dynamics, are resistant to some acute intoxicating effects of ethanol and show increased ethanol consumption. In the brain, the N-methyl-D-aspartate (NMDA) receptor is a major target of ethanol. We show that Eps8 is localized to postsynaptic structures and is part of the NMDA receptor complex. Moreover, in Eps8 null mice, NMDA receptor currents and their sensitivity to inhibition by ethanol are abnormal. In addition, Eps8 null neurons are resistant to the actin-remodeling activities of NMDA and ethanol. We propose that proper regulation of the actin cytoskeleton is a key determinant of cellular and behavioral responses to ethanol.     

Molecular dynamics of NPY Y1 receptor activation

A three-dimensional model of the human neuropeptide Y(NPY)Y₁ receptor (hY₁) was constructed, energy refined and used to simulate molecular receptor interactions of the peptide ligands NPY, [L31, P34]NPY, peptide YY (PYY) and pancreatic polypeptide (PP), and of the nonpeptide antagonist R-N²-(diphenylacetyl)-N-(4-hydroxyphenyl)methyl-argininamide (BIBP3226) and its S-enantiomer BIBP3435. The best complementarity in charges between the receptor and the peptides, and the best structural accordance with experimental studies, was obtained with amino acid 1–4 of the peptides interacting with Asp194, Asp200, Gln201, Phe202 and Trp288 in the receptor. Arg33 and Arg35 of the peptides formed salt bridges with Asp104 and Asp287, respectively, while Tyr36 interacted in a binding pocket formed by Phe41, Thr42, Tyr100, Asn297, His298 and Phe302. Calculated electrostatic potentials around NPY and hY₁ molecules indicated that ligand binding is initiated by electrostatic interactions between a highly positive region in the N- and C-terminal parts of the peptides, and a negative region in the extracellular receptor domains. Molecular dynamics simulations of NPY and BIBP3226 interactions with the receptor indicated rigid body motions of TMH5 and TMH6 upon NPY binding as mechanisms of receptor activation, and that BIBP3226 may act as an antagonist by constraining these motions.     

Generation and characterization of Sca2 (ataxin-2) knockout mice

Ataxin-2, the gene product of the Spinocerebellar Ataxia Type 2 (SCA2) gene, is a protein of unknown function with abundant expression in embryonic and adult tissues. Its interaction with A2BP1/Fox-1, a protein with an RNA recognition motif, suggests involvement of ataxin-2 in mRNA translation or transport. To study the effects of in vivo ataxin-2 function, we generated an ataxin-2 deficient mouse strain. Ataxin-2 deficient mice were viable. Genotypic analysis of litters from mating of heterozygous mice showed segregation distortion with a significant reduction in the birth of Sca-/- females. Detailed macroscopic and microscopic analysis of surviving nullizygous Sca2 knockout mice showed no major histological abnormalities. On a fat-enriched diet, ataxin-2 deficient animals had increased weight gain. Our results demonstrate that ataxin-2, although widely expressed, is not essential in development or during adult survival in the mouse, but leads to adult-onset obesity.     

Endocrine function of neuropeptide Y knockout mice

Among its many proposed functions, neuropeptide Y (NPY) is thought to modulate the hypothalamic-pituitary axis. Specifically, increased hypothalamic NPY signaling may be critical in mediating the neuroendocrine response to fasting. To determine the consequences of NPY deficiency on endocrine physiology, multiple hormones were quantitated in wildtype and NPY-knockout mice under fed and fasted conditions. Serum concentrations of leptin, corticosterone, thyroxine, and testosterone were normal in NPY-knockout males fed ad libitum. A 48-hour fast resulted in a 50% reduction in leptin, a 60% reduction in thyroxine, a 75% reduction in testosterone, and a 12-fold increase in corticosterone in both wildtype and NPY-knockout mice. Fasting also increased the estrous cycle length by 3 days in both wildtype and NPY-deficient female mice. We conclude that NPY is not essential for appropriate function of the gonadotropic, thyrotropic, or corticotropic axes under ad lib fed conditions or in response to acute fasting.     

Enhanced proliferation of astrocytes from beta(2)-adrenergic receptor knockout mice is influenced by the IGF system

In the present study, we investigated the IGF system in neonatal astrocytes derived from mice with a targeted disruption of the beta-2 adrenergic receptor (β₂AR). β₂AR knockout astrocytes demonstrated higher proliferation rates and increased expression of the astrogliotic marker GFAP, as compared with wild-type cells. β₂AR deletion also regulated molecules of the IGF system. Although IGF-1 levels remained unaltered, IGF-2 and type 1 IGF receptor expression was increased in β₂AR knockout cells. Furthermore, conditioned medium from knockout astrocytes contained lower levels of IGF binding protein-2 and -4. Our data suggest a deficit of β₂AR on astrocytes, as previously reported in multiple sclerosis, may have implications on proliferative status of astrocytes, a feature that might be attributed to regulation of IGF mitogenic actions.     

Modulation of neuropeptide Y and Y1 receptor expression in the amygdala by fluctuations in the brain content of neuroactive steroids during ethanol drinking discontinuation in Y1R/LacZ transgenic mice

Previous studies have shown that GABAergic neuroactive steroids increase Y₁ receptor (Y₁R) gene expression in the amygdala of Y ₁ R / LacZ transgenic mice, harbouring the murine Y₁R gene promoter linked to a LacZ reporter gene. As ethanol is known to increase GABAergic neuroactive steroids, we investigated the relationship between fluctuations in the brain content of neuroactive steroids induced by chronic voluntary ethanol consumption or ethanol discontinuation and both the level of neuropeptide Y (NPY) immunoreactivity and Y₁R gene expression in the amygdala of Y ₁ R / LacZ transgenic mice. Ethanol discontinuation (48 h) after voluntary consumption of consecutive solutions of 3%, 6%, 10% and 20% (v/v) ethanol over 4 weeks produced an anxiety-like behaviour as measured by elevated plus maze. Voluntary ethanol intake increased the cerebrocortical concentration of the progesterone metabolite 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG) that returned to control level 48 h after discontinuation of ethanol intake. Ethanol discontinuation significantly decreased NPY immunoreactivity and concomitantly increased Y ₁ R / LacZ transgene expression in the amygdala, whereas chronic ethanol intake failed to affect these parameters. The 5α-reductase inhibitor finasteride prevented both the increase in the cerebrocortical concentration of 3α,5α-TH PROG apparent after 4 weeks of ethanol intake and the changes in NPY immunoreactivity and transgene expression induced by ethanol discontinuation. Data suggest that 3α,5α-TH PROG plays an important role in the changes in NPY–Y₁R signalling in the amygdala during ethanol discontinuation.