A seven transmembrane G-protein coupled receptor has been cloned from Drosophila melanogaster. This receptor shows structural similarities to vertebrate Neuropeptide Y2 receptors and is activated by endogenous Drosophila peptides, recently designated as short neuropeptide Fs (sNPFs). sNPFs have so far been found in neuroendocrine tissues of four other insect species and of the horseshoe crab. In locusts, they accelerate ovarian maturation, and in mosquitoes, they inhibit host-seeking behavior. Expression analysis by RT-PCR shows that the sNPF receptor (Drm-sNPF-R) is present in several tissues (brain, gut, Malpighian tubules and fat body) from Drosophila larvae as well as in ovaries of adult females. All 4 Drosophila sNPFs clearly elicited a calcium response in receptor expressing mammalian Chinese hamster ovary cells. The response is dose-dependent and appeared to be very specific. The short NPF receptor was not activated by any of the other tested arthropod peptides, not even by FMRFamide-related peptides (also ending in RFamide), indicating that the Arg residue at position 4 from the amidated C-terminus appears to be crucial for the response elicited by the sNPFs (Mertens, 2002).
Since the Drosophila genome encodes at least 4 receptors belonging to the NPY subgroup of receptors, the Drosphila EST database was searched for the presence of EST clones, encoding one of the NPY-type receptors. PCR amplification of the EST clone (GH23382) with oligonucleotide primers specific for the predicted ORF of CG7395 (Neuropeptide F-like Receptor 76F) produced a single product of approximately 1800 bp. Sequence determination of the TA-cloned PCR product revealed a DNA insert of 1803 bp, corresponding to the sequence and size of the predicted receptor in the cDNA database of BDGP. The deduced protein encoded by the ORF of Drm-sNPF-R is 600 amino acids long. Analysis by the TMHMM program revealed that this protein is predicted to have seven transmembrane domains along with the intracellular and extracellular loops, consistent with the known G-protein coupled receptors. The N-terminal extracellular region exhibits no O-glycosylation, 2 N-glycosylation sites, along with 7 Ser/Thr phosphorylation sites (Mertens, 2002).
A phylogenetic tree based on Clustal W alignment of Drm-sNPF-R and various known NPY receptors indicates that Drm-sNPF-R is most closely related to the vertebrate neuropeptide Y2 receptors, i.e., of the domestic guinea pig (33% identity and 49% homology), humans (33% identity and 49% homology), the domestic pig (33% identity and 48% homology), and the rat (33% identity and 48% homology). An NPY-like orphan GPCR of C. elegans (C53C7.1) displays 33% identity and 47% homology. Sequence conservation among Drm-sNPF-R, the human Y2 receptor, and the C. elegans orphan receptor is depicted by similarities shown in their alignment by the AlignX program (Mertens, 2002).
Short NPFs and 'head' peptides display substantial sequence similarities and appear to belong to the same family. All NPFs have a typical R(K)–X1–R–X2amide motif, where the first amino acid of this motif is always a basic amino acid residue such as Arg or Lys. X1 can be L, T or P and X2 is always an aromatic amino acid residue such as Phe or Trp. It is proposed to (re)name all peptides with the R(K)–X1–R–X2amide C-terminal motif, as short NPFs; these peptides do not only occur in the head or central nervous system, but instead reach 10 times higher amounts in the abdomen and the midgut. In addition, their precursor in the Drosophila genome is annotated as the short NPF precursor. The present identification of a specific short NPF receptor in Drosophila is in favor of the presence of functional short NPFs. Several reports indicate that short NPFs have a hormonal function in insects, associated with reproduction and digestion. The expression of the short NPF receptor not only in the nervous system, but also in peripheral targets (ovaries, gut) is in agreement with a hormonal function of short NPFs. Hemolymph from sugar-fed mosquito females contains 414 fmol/μl immunoreactive short NPF. Short NPFs are also abundantly present in endocrine cells of the midgut, suggesting that they might have a function in digestion. The demonstration of the presence of the short NPF receptor transcript in the midgut favors this hypothesis (Mertens, 2002).
A cDNA clone encoding a seven-transmembrane domain, G-protein-coupled receptor (Neuropeptide F-like Receptor 76F, NPFR76F, or GPCR60), has been isolated from Drosophila melanogaster. Deletion mapping showed that the gene encoding this receptor is located on the left arm of the third chromosome at position 76F. Northern blotting and whole mount in situ hybridization have shown that this receptor is expressed in a limited number of neurons in the central and peripheral nervous systems of embryos and adults. Analysis of the deduced amino acid sequence suggests that this receptor is related to vertebrate neuropeptide Y receptors. This Drosophila receptor shows 62%-66% similarity and 32%-34% identity to type 2 neuropeptide Y receptors cloned from a variety of vertebrate sources. Coexpression in Xenopus oocytes of NPFR76F with the promiscuous G-protein Galpha16 showed that this receptor is activated by the vertebrate neuropeptide Y family to produce inward currents due to the activation of an endogenous oocyte calcium-dependent chloride current. Maximum receptor activation was achieved with short, putative Drosophila neuropeptide F peptides (Drm-sNPF-1, 2 and 2s). Neuropeptide F-like peptides in Drosophila have been implicated in a signalling system that modulates food response and social behaviour. The identification of this neuropeptide F-like receptor and its endogenous ligand by reverse pharmacology will facilitate genetic and behavioural studies of neuropeptide functions in Drosophila (Feng, 2003).
Since the NPFR76F receptor was maximally activated by a short insect NPF-like sequence from Leptinotarsa, genome mining was used to search for Drosophila peptides which might be functionally equivalent to (or better than) the Leptinotarsa I peptide at activating NPFR76F. Initially, one precursor sequence, a gene (npf) encoding Drosophila NPF was identified at chromosome location 89D3. This gene was found by blasting an incomplete version of the Drosophila genome (October 1999) with the amino acid sequence of Aplysia NPY. This revealed a precursor molecule encoding a 36 amino acid peptide with sequence similarity to NPF. The completed Drosophila genome sequence has now been searched and no other potential NPF precursors were identified. Since this peptide (NPF-A1) contained a potential dibasic amino acid cleavage site within its sequence, both the shorter 28 amino acid form (NPF-A2) and the full-length peptide (NPF-A1) were sequenced for testing (Feng, 2003).
A second open reading frame in the Drosophila genome encodes a precursor peptide for two short NPF-like peptides (Drm-sNPF-1, AQRSPSLRLRFamide and Drm-sNPF-2, WFGDVNQKPIRSPSLRLRFamide). The precursor for these peptides is encoded by the short NPF precursor (sNPF) gene (CG13968) and maps to position 38A7 on the left arm of Drosophila chromosome 2. The WFGDVNQKPIRSPSLRLRFamide peptide (Drm-sNPF-2) contains a potential single basic amino acid-processing site. This peptide (Drm-sNPF-2), its shorter form (peptide Drm-sNPF-2 s, PIRSPSLRLRFamide) and the Drm-sNPF-1 peptide (all encoded by the sNPF gene) were synthesized. The same precursor was predicted to include the sequences for two other short peptides, PQRLRWamide and PMRLRWamide, which have been designated Drm-sNPF-3 and Drm-sNPF-4, respectively. These peptides were synthesized and tested (Feng, 2003).
When tested at 1 µm, the shorter NPF-like peptides derived from the sNPF gene (peptides Drm-sNPF-1 and Drm-sNPF-2) were more effective than the original Leptinotarsa I NPF-like sequence at inducing inward currents in Xenopus oocytes expressing NPFR76F and Galpha16. Shortening of peptide Drm-sNPF-2 to the Drm-sNPF-2s form may slightly increase its effectiveness. The longer Drosophila peptides derived from the precursor gene npf at 89D3 were much less effective than the original Leptinotarsa I NPF-like sequence at inducing inward currents. In addition, the PQRLRWamide (Drm-sNPF-3) and PMRLRWamide (Drm-sNPF4) peptides were much less effective than the original Leptinotarsa I sequence at inducing inward currents (Feng, 2003).
Dose–response curves for the shorter endogenous Drosophila NPF-like peptides encoded by the sNPF gene at 38A7 reveal that AQRSPSLRLRFamide (peptide Drm-sNPF-1) is the most potent peptide tested (pEC50 = −8.84). It showed a threshold for the generation of inward currents between 100 pm and 1 nm and a maximal effect at 100 nm. The second putative endogenous Drosophila NPF-like peptide encoded by the sNPF gene, PIRSPSLRFamide (peptide Drm-sNPF-2 s) (pEC50 = −7.62), and the original Leptinotarsa I NPF-like sequence, ARGPQLRLRFamide (pEC50 = −7.83), were an order of magnitude less potent than AQRSPSLRLRFamide. These results justify the classification of NPFR76F as a NPF-like receptor and suggest that the short peptide AQRSPSLRLRFamide (sNPF-1) may be the endogenous agonist for this receptor. The other two short peptides encoded by the sNPF gene, PQRLRWamide (pEC50 = −6.1) and PMRLRWamide (pEC50 = −7.30), were 2.7 and 1.5 orders of magnitude, respectively, less potent than the AQRSPSLRLRFamide sequence, leading to questioning of their designation as true short NPF-like peptides (Feng, 2003).
Expression of NPFR76F transcripts was assessed by Northern blot analysis of poly(A)+ RNA prepared from adult body parts. A single transcript of 6.5 kb was detected in both heads and appendages (legs and antennae), suggesting that NPFR76F is expressed in both the central and peripheral nervous systems. In addition to finding transcript in heads and appendages, trace amounts were also seen in bodies. When compared with the amount of RNA loaded from each body part (as indicated by the ubiquitous rp49 loading control), the relative abundance of transcript in bodies is very low. This distribution of the NPFR76F transcript is consistent with a role for this NPF-like receptor in the Drosophila nervous system (Feng, 2003).
To further refine the NPFR76F receptor transcript expression, in situ hybridization with a digoxigenin-labelled antisense RNA probe to whole mounts of the mature embryos was used. The central nervous system in embryos is composed of two dorsal brain hemispheres and a fused ventral ganglion. The NPFR76F receptor is expressed both in the dorsal brain and in the ventral ganglion. In the brain the receptor is strongly expressed in the specific cells in the dorsal posterior region. It is estimated that there are 22–24 cells in each brain lobe expressing NPFR76F, including the strongly expressing cells. In the ventral ganglion, pairs of cells along the ventral midline, as well as cells found in a bilaterally symmetric pattern in a more lateral position from the midline, also express receptor mRNA. In each full segment of the ventral ganglion, the receptor is strongly expressed in eight to 12 cells, including a pair of cells at the midline in each segment (Feng, 2003).
In the peripheral nervous system the receptor is expressed in a subset of sensilla and in the anterior sensory complex. It is estimated that there are 10–14 cells in the anterior sensory complex, including the antennomaxillary complex, the labral sensory complex and the labial sensory complex, that express NPFR76F. Finally, in the posterior sensilla, there are eight cells that express NPFR76F. The expression pattern of the NPFR76F receptor in many specific cells in the dorsal brain, the ventral ganglion, lateral sensilla, the anterior sensory complex and the posterior sensilla suggests that this receptor is involved in a widespread modulation of neuronal activity (Feng, 2003).
A Drosophila melanogaster G-protein-coupled receptor (NPFR76F) that is activated by neuropeptide F-like peptides has been expressed in Xenopus oocytes to determine its ability to regulate heterologously expressed G-protein-coupled inwardly rectifying potassium channels. The activated receptor produced inwardly rectifying potassium currents by a pertussis toxin-sensitive G-protein-mediated pathway and the effects were reduced in the presence of proteins, such as the betaARK 1 carboxy-tail fragment and alpha-transducin, which bind G-protein betagamma-subunits. Short Drosophila NPF-like peptides are more potent than long NPF-like peptides at coupling the receptor to the activation of inwardly rectifying potassium channels. The putative endogenous short Drosophila NPF-like peptides showed agonist-specific coupling depending on whether their actions were assessed as the activation of the inwardly rectifying potassium channels or as the activation of endogenous inward chloride channels through a co-expressed promiscuous G-protein, Galpha16. As inwardly rectifying potassium channels are known to be encoded in the Drosophila genome and the NPFR76F receptor is widely expressed in the Drosophila nervous system, the receptor could function to control neuronal excitability or slow wave potential generation in the Drosophila nervous system (Reale, 2004).
Neuropeptides regulate a wide range of animal behavior including food consumption, circadian rhythms, and anxiety. Recently, Drosophila neuropeptide F, which is the homolog of the vertebrate neuropeptide Y, was cloned, and the function of Drosophila neuropeptide F in feeding behaviors was well characterized. However, the function of the structurally related short neuropeptide F (sNPF) was unknown. This study reports the cloning, RNA, and peptide localizations, and functional characterizations of the Drosophila sNPF gene. The sNPF gene encodes the preprotein containing putative RLRF amide peptides and was expressed in the nervous system of late stage embryos and larvae. The embryonic and larval localization of the sNPF peptide in the nervous systems revealed the larval central nervous system neural circuit from the neurons in the brain to thoracic axons and to connective axons in the ventral ganglion. In the adult brain, the sNPF peptide was localized in the medulla and the mushroom body. However, the sNPF peptide was not detected in the gut. The sNPF mRNA and the peptide were expressed during all developmental stages from embryo to adult. From the feeding assay, the gain-of-function sNPF mutants expressed in nervous systems promoted food intake, whereas the loss-of-function mutants suppressed food intake. Also, sNPF overexpression in nervous systems produced bigger and heavier flies. These findings indicate that the sNPF is expressed in the nervous systems to control food intake and regulate body size in Drosophila melanogaster (Lee, 2004).
Various evidence suggests that sNPF and dNPF peptides have different functions. The sNPF peptide is found only in nervous systems, whereas the neuropeptide F (dNPF) is found as the Drosophila brain-gut peptide. The expression patterns of sNPF and dNPF differ in the larval brain. For example, the sNPF expression is found in the anterior dorsal neurons of the brain, whereas the dNPF expression is detected in the four neurons of larval brain. In the feeding behavior analysis, overexpression of sNPF in wandering larvae did not extend the feeding period, contrary to the extension of the feeding period in the wandering larval stage by overexpression of dNPF. At the receptor level, each peptide works in different receptors; for example, the NPFR76F receptor is for sNPF peptides, and the DmNPFR1 receptor is for the dNPF. These differences indicate that the dNPF and sNPF peptides function in different neurons and may regulate different aspects of feeding behaviors in Drosophila (Lee, 2004).
Like other neuropeptides, the sNPF peptide may be involved in regulating various physiological processes other than regulating food intake because the sNPF peptide and transcript were expressed during all developmental stages, and the sNPF is localized in the mushroom body calyx and medulla of the adult brain. The mushroom body is involved in learning and memory. These unknown multi-functions of the sNPF peptide in various biological processes are the subjects of future studies (Lee, 2004).
Among insects, short neuropeptide Fs (sNPF) have been implicated in regulation of reproduction and feeding behavior. For Drosophila melanogaster, the nucleotide sequence for the sNPF precursor protein encodes four distinctive candidate sNPFs. In the present study, all four peptides were identified by mass spectrometry in body extracts of D. melanogaster; some also were identified in hemolymph, suggesting potential neuroendocrine roles. Actions of sNPFs in D. melanogaster are mediated by the G protein-coupled receptor Drm-NPFR76F. Mammalian CHO-K1 cells were stably transfected with the Drm-NPFR76F receptor for membrane-based radioreceptor studies. Binding assays revealed that longer sNPF peptides comprised of nine or more amino acids are clearly more potent than shorter ones of eight or fewer amino acids. These findings extend understanding of the relationship between structure and function of sNPFs (Garczynski, 2006).
The sNPFs of D. melanogaster differ in their interactions with the sNPF receptor Drm-NPFR76F, as analyzed directly by radioreceptor assay. A wide variety of D. melanogaster sNPFs were assayed for their ability to inhibit the binding of 125I-[D-Y1]-Drm-sNPF1 to membranes prepared from cells stably transfected with Drm-NPFR76F. Two distinctive classes of activity of sNPFs were readily apparent. The sNPF peptides containing nine or more amino acids typically exhibited high affinity, as judged by an IC50 < 1 nM, whereas peptides containing eight for fewer amino acids exhibited IC50 values of >5 nM. Those exhibiting lower affinity included sNPF3 and sNPF4, peptides with the C-terminal RLRWa sequence. The minimum length of the highly active group was represented by sNPF211–19. The sequence of this sNPF211–19 (RSPSLRLRFa) differs from sNPF14–11 (SPSLRLRFa) only by a single arginine residue, which appears to confer a substantial increase in binding affinity. To test this hypothesis, an alanine substituted analog, Drm-sNPF211–19R11A, was assayed and found to exhibit a substantial drop in activity, with an IC50 of only 12.5 nM, indicating the crucial role of this arginine residue (Garczynski, 2006).
For further tests of these apparent structure-function relations, putative sNPFs identified in the genomes of A. gambiae and A. aegypti also were examined in the Drm-NPFR76F radioreceptor assay. Each of these mosquito sNPF peptides conformed to the distinctive pattern of length-associated activity established previously for those of D. melanogaster. In contrast, the A. aegypti head peptides which partly resemble sNPFs were either weakly active, Aea-HP-I, or inactive, Aea-HP-III, despite being of sufficient length and having the requisite arginine. Accordingly, additional structural features in the C-terminus common to sNPF peptides appear important for high affinity binding (Garczynski, 2006).
The genome of Anopheles gambiae contains sequences encoding a neuropeptide F (Ang-NPF) and NPF receptor (Ang-NPFR) related to the neuropeptide Y signaling family. cDNAs for each were cloned and sequenced. Ang-NPFR was stably expressed for radioligand binding analysis. Ang-NPF exhibited high affinity (IC50 approximately 3 nM) membrane binding; NPFs from Aedes aegypti (Aea-NPF) and Drosophila melanogaster (Drm-NPF) were less potent, with the rank order: Ang-NPF>Aea-NPF>Drm-NPF>Drm-NPF8-36. RT-PCR analysis revealed Ang-NPF and Ang-NPFR transcripts in all life stages. Ang-NPF and Ang-NPFR may be strategically positioned for signaling in relation to nutritional status in the African malaria mosquito (Garczynski, 2005).
A neuropeptide F (NPF) was isolated from an extract of adult Aedes aegypti mosquitoes based on its immunoreactivity in a radioimmunoassay for Drosophila NPF. After sequencing the peptide, cDNAs encoding the NPF were identified from head and midgut. These cDNAs encode a prepropeptide containing a 36 amino acid peptide with an amidated carboxyl terminus, and its sequence shows it to be a member of the neuropeptide F/Y superfamily. Immunocytochemistry and Northern blots confirmed that both the brain and midgut of females are likely sources of NPF, found at its highest hemolymph titer before and 24 h after a blood meal (Stanek, 2002).
Natural isolates of C. elegans exhibit either solitary or social feeding behavior. Solitary foragers move slowly on a bacterial lawn and disperse across it, while social foragers move rapidly on bacteria and aggregate together. A loss-of-function mutation in the npr-1 gene, which encodes a predicted G protein-coupled receptor similar to neuropeptide Y receptors, causes a solitary strain to take on social behavior. Two isoforms of NPR-1 that differ at a single residue occur in the wild. One isoform, NPR-1 215F, is found exclusively in social strains, while the other isoform, NPR-1 215V, is found exclusively in solitary strains. An NPR-1 215V transgene can induce solitary feeding behavior in a wild social strain. Thus, isoforms of a putative neuropeptide receptor generate natural variation in C. elegans feeding behavior (de Bono, 1998).
Wild isolates of Caenorhabditis elegans can feed either alone or in groups. This natural variation in behaviour is associated with a single residue difference in NPR-1, a predicted G-protein-coupled neuropeptide receptor related to Neuropeptide Y receptors. The NPR-1 isoform associated with solitary feeding acts in neurons exposed to the body fluid to inhibit social feeding. Furthermore, suppressing the activity of these neurons, called AQR, PQR and URX, using an activated K(+) channel, inhibits social feeding. NPR-1 activity in AQR, PQR and URX neurons seems to suppress social feeding by antagonizing signalling through a cyclic GMP-gated ion channel encoded by tax-2 and tax-4. Mutations in tax-2 or tax-4 disrupt social feeding, and tax-4 is required in several neurons for social feeding, including one or more of AQR, PQR and URX. The AQR, PQR and URX neurons are unusual in C. elegans because they are directly exposed to the pseudocoelomic body fluid. The data suggest a model in which these neurons integrate antagonistic signals to control the choice between social and solitary feeding behaviour (Coates, 2002).
Variation in the acute response to ethanol between individuals has a significant impact on determining susceptibility to alcoholism. The degree to which genetics contributes to this variation is of great interest. Allelic variation that alters the functional level of NPR-1, a neuropeptide Y (NPY) receptor-like protein, can account for natural variation in the acute response to ethanol in wild strains of C. elegans. NPR-1 negatively regulates the development of acute tolerance to ethanol, a neuroadaptive process that compensates for effects of ethanol. Furthermore, dynamic changes in the NPR-1 pathway provide a mechanism for ethanol tolerance in C. elegans. This suggests an explanation for the conserved function of NPY-related pathways in ethanol responses across diverse species. Moreover, these data indicate that genetic variation in the level of NPR-1 function determines much of the phenotypic variation in adaptive behavioral responses to ethanol that are observed in natural populations (Davies, 2003).
Neuropeptide Y (NPY), a 36-amino-acid transmitter distributed throughout the nervous system, is thought to function as a central stimulator of feeding behaviour. NPY has also been implicated in the modulation of mood, cerebrocortical excitability, hypothalamic-pituitary signalling, cardiovascular physiology and sympathetic function. However, the biological significance of NPY has been difficult to establish owing to a lack of pharmacological antagonists. Mice deficient for NPY have normal food intake and body weight, and become hyperphagic following food deprivation. Mutant mice decrease their food intake and lose weight, initially to a greater extent than controls, when treated with recombinant leptin. Occasional, mild seizures occur in NPY-deficient mice and mutants are more susceptible to seizures induced by a GABA (gamma-aminobutyric acid) antagonist. These results indicate that NPY is not essential for certain feeding responses or leptin actions but is an important modulator of excitability in the central nervous system (Erickson, 1996a).
The obesity syndrome of ob/ob mice results from lack of leptin, a hormone released by fat cells that acts in the brain to suppress feeding and stimulate metabolism. Neuropeptide Y (NPY) is a neuromodulator implicated in the control of energy balance and is overproduced in the hypothalamus of ob/ob mice. To determine the role of NPY in the response to leptin deficiency, ob/ob mice deficient for NPY were generated. In the absence of NPY, ob/ob mice are less obese because of reduced food intake and increased energy expenditure, and are less severely affected by diabetes, sterility, and somatotropic defects. These results suggest that NPY is a central effector of leptin deficiency (Erickson, 1996b).
An extensive behavioral characterization was conducted with mice lacking the gene for neuropeptide Y (NPY) including response to 24 and 48 h fast and challenge with small molecule antagonists of NPY receptors implicated in mediating the feeding effects of NPY (i.e., Y1 and Y5). In addition, wildtype (WT) and NPY knockout (KO) mice were tested in locomotor monitors, elevated plus maze, inhibitory avoidance, acoustic startle, prepulse inhibition, and hot plate assays. One of the major findings was that the NPY KO mice have a reduced food intake relative to WT controls in response to fasting. Also, based on data from the behavioral models, the NPY KO mice may have an anxiogenic-like phenotype, and appear to be hypoalgesic in the hot plate paradigm. The data from these studies provide further evidence of involvement of NPY in energy balance, anxiety, and possibly nociception (Bannon, 2000).
Agouti-related protein (AgRP), a neuropeptide abundantly expressed in the arcuate nucleus of the hypothalamus, potently stimulates feeding and body weight gain in rodents. AgRP is believed to exert its effects through the blockade of signaling by alpha-melanocyte-stimulating hormone at central nervous system (CNS) melanocortin-3 receptor (Mc3r) and Mc4r. AgRP-deficient (Agrp-/-) mice were generated to examine the physiological role of AgRP. Agrp-/- mice are viable and exhibit normal locomotor activity, growth rates, body composition, and food intake. Additionally, Agrp-/- mice display normal responses to starvation, diet-induced obesity, and the administration of exogenous leptin or neuropeptide Y (NPY). In situ hybridization failed to detect altered CNS expression levels for proopiomelanocortin, Mc3r, Mc4r, or NPY mRNAs in Agrp-/- mice. Since AgRP and the orexigenic peptide NPY are coexpressed in neurons of the arcuate nucleus, AgRP and NPY double-knockout (Agrp-/-;Npy-/-) mice were generated to determine whether NPY or AgRP plays a compensatory role in Agrp-/- or NPY-deficient (Npy-/-) mice, respectively. Similar to mice deficient in either AgRP or NPY, Agrp-/-;Npy-/- mice suffer no obvious feeding or body weight deficits and maintain a normal response to starvation. These results demonstrate that neither AgRP nor NPY is a critically required orexigenic factor, suggesting that other pathways capable of regulating energy homeostasis can compensate for the loss of both AgRP and NPY (Qian, 2003).
Neuropeptide Y (NPY) is an orexigenic (appetite-stimulating) peptide that plays an important role in regulating energy balance. When administered directly into the central nervous system, animals exhibit an immediate increase in feeding behavior, and repetitive injections or chronic infusions lead to obesity. Surprisingly, initial studies of Npy-/- mice on a mixed genetic background did not reveal deficits in energy balance, with the exception of an attenuation in obesity seen in ob/ob mice in which the NPY gene was also deleted. On a C57BL/6 background, NPY ablation is associated with an increase in body weight and adiposity and a significant defect in refeeding after a fast. This impaired refeeding response in Npy-/- mice resulted in a deficit in weight gain in these animals after 24 h of refeeding. These data indicate that genetic background must be taken into account when the biological role of NPY is evaluated. When examined on a C57BL/6 background, NPY is important for the normal refeeding response after starvation, and its absence promotes mild obesity (Segal-Lieberman, 2003).
Neuropeptide Y (NPY) is a potent orexigenic peptide that is implicated in the feeding response to a variety of stimuli. The current studies employed mice lacking NPY (Npy-/-) and their wild-type (Npy+/+) littermates to investigate the role of this peptide in the feeding response to circadian and palatability cues. To investigate the response to a circadian stimulus, food intake was assessed during the 4-h period following dark onset, a time of day characterized by maximal rates of food consumption. Compared to Npy+/+ controls, intake of Npy-/- mice was reduced by 33% during this period. In contrast, intake did not differ between genotypes when measured over a 24-h period. Furthermore, reduced dark cycle 4h food intake in Npy-/- mice was not evident after a 24-h fast, despite a pronounced delay in the initiation of feeding. To investigate the role of NPY in the feeding response to palatability cues, mice were presented with a highly palatable diet (HP) for 1h each day (in addition to having ad libitum access to chow) for 18 days. Npy+/+ mice rapidly increased daily HP intake such that by the end of the first week, they derived a substantial fraction of daily energy from this source. By comparison, HP intake was markedly reduced in Npy-/- mice during the first week, although it eventually increased (by Day 9) to values comparable to those of Npy+/+ controls. These experiments suggest that NPY contributes to the mechanism whereby food intake increases in response to circadian and palatability cues and that mechanisms driving food intake in response to these stimuli differ from those activated by energy restriction (Sindelar, 2005).
Exogenous neuropeptide Y (NPY) reduces experimental anxiety in a wide range of animal models. The generation of an NPY-transgenic rat has provided a unique model to examine the role of endogenous NPY in control of stress and anxiety-related behaviors using paradigms previously used by pharmacological studies. Locomotor activity and baseline behavior on the elevated plus maze were normal in transgenic subjects. Two robust phenotypic traits were observed. (1) Transgenic subjects showed a markedly attenuated sensitivity to behavioral consequences of stress, in that they were insensitive to the normal anxiogenic-like effect of restraint stress on the elevated plus maze and displayed absent fear suppression of behavior in a punished drinking test. (2) A selective impairment of spatial memory acquisition was found in the Morris water maze. Control experiments suggest these traits to be independent. These phenotypic traits were accompanied by an overexpression of prepro-NPY mRNA and NPY peptide and decreased NPY-Y1 binding within the hippocampus, a brain structure implicated both in memory processing and stress responses. Data obtained using this unique model support and extend a previously postulated anti-stress action of NPY and provide novel evidence for a role of NPY in learning and memory (Thorsell, 2000).
Neuropeptide Y (NPY), one of the most abundant peptide transmitters in the mammalian brain, is assumed to play an important role in feeding and body weight regulation. However, there is little genetic evidence that overexpression or knockout of the NPY gene leads to altered body weight regulation. NPY-overexpressing mice have been developed by using the Thy-1 promoter, which restricts NPY expression strictly within neurons in the central nervous system, but the obese phenotype was not observed in the heterozygote. In the homozygous mice, overexpression of NPY leads to an obese phenotype, but only after appropriate dietary exposure. NPY-overexpressing mice exhibit significantly increased body weight gain with transiently increased food intake after 50% sucrose-loaded diet, and later they developed hyperglycemia and hyperinsulinemia without altered glucose excursion during 1 year of the observation period (Kaga, 2001).
Despite numerous experiments showing that administration of neuropeptide Y (NPY) to rodents stimulates feeding and obesity, whereas acute interference with NPY signaling disrupts feeding and promotes weight loss, NPY-null mice have essentially normal body weight regulation. These conflicting observations suggest that chronic lack of NPY during development may lead to compensatory changes that normalize regulation of food intake and energy expenditure in the absence of NPY. To test this idea, gene targeting was used to introduce a doxycycline (Dox)-regulated cassette into the Npy locus, such that NPY would be expressed until the mice were given Dox, which blocks transcription. Compared with wild-type mice, adult mice bearing this construct expressed approximately 4-fold more Npy mRNA, which fell to approximately 20% of control values within 3 days after treatment with Dox. NPY protein also fell approximately 20-fold, but the half-life of approximately 5 days was surprisingly long. The biological effectiveness of these manipulations was demonstrated by showing that overexpression of NPY protected against kainate-induced seizures. Mice chronically overexpressing NPY had normal body weight, and administration of Dox to these mice did not suppress feeding. Furthermore, the refeeding response of these mice after a fast was normal. It is concluded that, if there is compensation for changes in NPY levels, then it occurs within the time it takes for Dox treatment to deplete NPY levels. These observations suggest that pharmacological inhibition of NPY signaling is unlikely to have long-lasting effects on body weight (Ste Marie, 2005).
Central neuropeptide Y (NPY) injection has been reported to cause hyperphagia and in some cases also hypometabolism or hypothermia. Chronic central administration induced a moderate rise of short duration in body weight, without consistent metabolic/thermal changes. In the present studies the acute and subsequent subacute ingestive and metabolic/thermal changes were studied following intracerebroventricular (i.c.v.) injections of NPY in cold-adapted and non-adapted rats, or the corresponding chronic changes following i.c.v. NPY infusion. Besides confirming basic earlier data, this study demonstrated novel findings: a temporal relationship for the orexigenic and metabolic/thermal effects, and differences of coordination in acute/subacute/chronic phases or states. The acute phase (30-60 min after injection) was anabolic: coordinated hyperphagia and hypometabolism/hypothermia. NPY evoked a hypothermia by suppressing any (hyper)metabolism in excess of basal metabolic rate, without enhancing heat loss. Thus, acute hypothermia was observed in sub-thermoneutral but not thermoneutral environments. The subsequent subacute catabolic phase exhibited opposite effects: slight increase in metabolic rate, rise in body temperature, reaching a plateau within 3-4 h after injection -- this was maintained for at least 24 h; meanwhile the food intake decreased and the normal daily weight gain stopped. This rebound is only indirectly related to NPY. Chronic (7-day long) i.c.v. NPY infusion induced an anabolic phase for 2-3 days, followed by a catabolic phase and fever, despite continued infusion. In cold-adaptation environment the primary metabolic effect of the infusion induced a moderate hypothermia with lower daytime nadirs and nocturnal peaks of the circadian temperature rhythm, while at near-thermoneutral environments in non-adapted rats the infusion attenuated only the nocturnal temperature rise by suppressing night-time hypermetabolism. Further finding is that in cold-adapted animals, the early feeding effect of NPY-infusion was enhanced, whereas the early hypothermic effect in cold was limited by interference with competing thermoregulatory mechanisms (Szekely, 2005)
Neuropeptide Y (NPY) is thought to have a major role in the physiological control of energy homeostasis. Among five NPY receptors described, the NPY Y5 receptor (Y5R) is a prime candidate to mediate some of the effects of NPY on energy homeostasis, although its role in physiologically relevant rodent obesity models remains poorly defined. The effect was examined of a potent and highly selective Y5R antagonist in rodent obesity and dietary models. The Y5R antagonist selectively ameliorates diet-induced obesity (DIO) in rodents by suppressing body weight gain and adiposity while improving the DIO-associated hyperinsulinemia. The compound does not affect the body weight of lean mice fed a regular diet or genetically obese leptin receptor-deficient mice or rats, despite similarly high brain Y5R receptor occupancy. The Y5R antagonist acts in a mechanism-based manner, since the compound does not affect DIO of Y5R-deficient mice. These results indicate that Y5R is involved in the regulation and development of DIO and suggest utility for Y5R antagonists in the treatment of obesity (Ishihara, 2006).
Neuropeptide Y (NPY) is a 36-amino-acid neurotransmitter that is widely distributed throughout the central and peripheral nervous system. NPY involvement has been suggested in various physiological responses including cardiovascular homeostasis and the hypothalamic control of food intake. At least six subtypes of NPY receptors have been described. Because of the lack of selective antagonists, the specific role of each receptor subtype has been difficult to establish. This study describes mice deficient for the expression of the Y1 receptor subtype. Homozygous mutant mice demonstrate a complete absence of blood pressure response to NPY, whereas they retain normal response to other vasoconstrictors. Daily food intake, as well as NPY-stimulated feeding, are only slightly diminished, whereas fast-induced refeeding is markedly reduced. Adult mice lacking the NPY Y1 receptor are characterized by increased body fat with no change in protein content. The higher energetic efficiency of mutant mice might result, in part, from the lower metabolic rate measured during the active period, associated with reduced locomotor activity. These results demonstrate the importance of NPY Y1 receptors in NPY-mediated cardiovascular response and in the regulation of body weight through central control of energy expenditure. In addition, these data are also indicative of a role for the Y1 receptor in the control of food intake (Pedrazzini, 1998).
Neuropeptide Y (NPY) and Agouti-related peptide (AgRP) stimulate feeding, whereas NPY also facilitates the estrogen-mediated preovulatory GnRH surge. In addition to regulating reproductive function, estrogen also acts as an anorexigenic hormone, although it is not yet known which hypothalamic neurons are involved in this process. It is hypothesized that estrogen may directly control hypothalamic NPY and/or AgRP synthesis to influence energy homeostasis. Using two clonal, murine hypothalamic neuronal cell models, N-38 and N-42, it has been demonstrated that 17beta-estradiol differentially regulates estrogen receptor (ER)alpha and ERbeta levels, as well as NPY and AgRP gene expression in a manner that is temporally coordinated with the changes in ER abundance. The estrogen-mediated repression of NPY and AgRP mRNA levels in N-38 and N-42 neurons require either ERalpha and ERbeta or ERalpha alone, respectively, whereas the induction of NPY and AgRP in N-38 neurons is strictly ERbeta-dependent, as assessed by ER-specific agonists and siRNA knockdown of ERalpha or ERbeta. Through transient transfection analysis in N-38 neurons, the estrogen-mediated repression of NPY was mapped to within -1078 of the 5' regulatory region of the NPY gene. These results provide the first evidence that NPY and AgRP gene expression is directly regulated by estrogen in specific hypothalamic neurons, and that this regulation is dependent upon the ratio of ERbeta to ERalpha. The biphasic control of neuronal NPY/AgRP transcription may be a mechanism by which estrogen has distinct effects on both energy homeostasis and reproduction (Titolo, 2006).
Hypothalamic neurons that express neuropeptide Y (NPY) and agouti-related protein (AgRP) are thought to be critical regulators of feeding behavior and body weight. To determine whether NPY/AgRP neurons are essential in mice, the human diphtheria toxin receptor was targeted to the Agrp locus, which allows temporally controlled ablation of NPY/AgRP neurons to occur after an injection of diphtheria toxin. Neonatal ablation of NPY/AgRP neurons has minimal effects on feeding, whereas their ablation in adults causes rapid starvation. These results suggest that network-based compensatory mechanisms can develop after the ablation of NPY/AgRP neurons in neonates but do not readily occur when these neurons become essential in adults (Luquiet, 2005).
Genetic linkage analysis of rats that were selectively bred for alcohol preference identified a chromosomal region that includes the neuropeptide Y (NPY) gene. Alcohol-preferring rats have lower levels of NPY in several brain regions compared with alcohol-non-preferring rats. Alcohol consumption by mice that completely lack NPY as a result of targeted gene disruption was studied. NPY-deficient mice show increased consumption, compared with wild-type mice, of solutions containing 6%, 10% and 20% (v/v) ethanol. NPY-deficient mice are also less sensitive to the sedative/hypnotic effects of ethanol, as shown by more rapid recovery from ethanol-induced sleep, even though plasma ethanol concentrations do not differ significantly from those of controls. In contrast, transgenic mice that overexpress a marked NPY gene in neurons that usually express it have a lower preference for ethanol and are more sensitive to the sedative/hypnotic effects of this drug than controls. These data are direct evidence that alcohol consumption and resistance are inversely related to NPY levels in the brain (Thiele, 1998).
Voluntary ethanol consumption and resistance to ethanol-induced sedation are inversely related to neuropeptide Y (NPY) levels in NPY-knock-out (NPY-/-) and NPY-overexpressing mice. Knock-out mice completely lacking the NPY Y1 receptor (Y1-/-) were studied to further characterize the role of the NPY system in ethanol consumption and neurobiological responses to this drug. Male Y1-/- mice show increased consumption of solutions containing 3, 6, and 10% (v/v) ethanol when compared with wild-type control mice. Female Y1-/- mice show increased consumption of a 10% ethanol solution. In contrast, Y1-/- mice show normal consumption of solutions containing either sucrose or quinine. Relative to Y1(+/+) mice, male Y1-/- mice were found to be less sensitive to the sedative effects of 3.5 and 4.0 gm/kg ethanol as measured by more rapid recovery from ethanol-induced sleep, although plasma ethanol levels did not differ significantly between the genotypes. Finally, male Y1-/- mice showed normal ethanol-induced ataxia on the rotarod test after administration of a 2.5 gm/kg dose. These data suggest that the NPY Y1 receptor regulates voluntary ethanol consumption and some of the intoxicating effects caused by administration of ethanol (Thiele, 2002).
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