Characterization,tissue distribution and regulation of neuropeptideY in Schizothorax prenanti

In this study, the full‐length neuropeptide Y (npy) complementary (c)DNA was cloned in ya fish Schizothorax prenanti. npy cDNA was composed of 789 nucleotides with a 288 nucleotide open reading frame encoding a protein of 96 amino acids. The deduced amino acid sequences contained a 28 amino acids signal peptide followed by a 36 amino acids mature neuropeptide Y (NPY). The npy mRNA was expressed mainly in the brain and eye as detected by real‐time quantitative polymerase chain reaction RT‐PCR (rt‐qPCR). The S. prenanti NPY was detectable from blastulation to hatch, suggesting that npy might be involved in the late embryonic development of S. prenanti. An experiment was conducted to determine the expression profile of npy during feeding of a single meal and during long‐term fasting. The expression level of npy in fed fish was significantly decreased at 0·5, 1·5, 3 and 9 h post‐feeding (hpf) than in fasting fish. Fasting for 14 days induced an increase in npy messenger (m)RNA expression in the brain. Overall, the results suggest that NPY is a conserved peptide that might be involved in the regulation of feeding and other physiological function in S. prenanti.     

Energy Metabolic Profile of Mice after Chronic Activation of Central NPY Y1, Y2, or Y5 Receptors**

Objective: Neuropeptide Y (NPY), a 36‐amino acid peptide with orexigenic properties, is expressed abundantly in the central nervous system and binds to several NPY receptor subtypes. This study examines the roles of the NPY Y1, Y2, and Y5 receptor(s) in energy homeostasis. Research Methods and Procedures: We administered intracerebroventricular NPY (3 μg/d) or selective peptide agonists for the Y1, Y2, and Y5 receptor subtypes to C57Bl/6 mice for 6 days by mini‐osmotic pumps to assess the role of each receptor subtype in NPY‐induced obesity. Energy expenditure (EE) and respiratory quotient (RQ) were studied using indirect calorimetry. Adiposity was measured by DXA scanning and fat pad dissection. Insulin sensitivity was tested by whole‐blood glucose measurement after an insulin challenge. Results: Central administration of the selective Y1 agonist, Y5 agonist, or NPY for 6 days in mice significantly increased body weight, adiposity, and RQ, with significant hyperphagia in the Y5 agonist‐ and NPY‐treated groups but not in the Y1 agonist‐treated group. The NPY, Y1, or Y5 agonist‐treated mice had little change in total EE during ad libitum and pair‐feeding conditions. Conversely, selective activation of the Y2 receptor reduced feeding and resulted in a significant, but transient, weight loss. Discussion: Central activation of both Y1 and Y5 receptors increases RQ and adiposity, whereas only Y5 receptor activation reduces energy expended per energy ingested. Selective activation of Y2 autoreceptors leads to hypophagia and transient weight loss, with little effect on total EE. Our study indicates that all three NPY receptor subtypes may play a role in regulating energy homeostasis in mice.     

Altered Hypothalamic Signaling and Responses to Food Deprivation in Rats Fed a Low‐Carbohydrate Diet

Objective: To model how consuming a low‐carbohydrate (LC) diet influences food intake and body weight. Research Methods and Procedures: Food intake and body weight were monitored in rats with access to chow (CH), LC‐high‐fat (HF), or HF diets. After 8 weeks, rats received intracerebroventricular injections of a melanocortin agonist (melanotan‐II) and antagonist (SHU9119), and feeding responses were measured. At sacrifice, plasma hormones and hypothalamic expression of mRNA for proopiomelanocortin (POMC), melanocortin‐4 receptor, neuropeptide Y (NPY), and agouti related protein (AgRP) were assessed. A second set of rats had access to diet (chow or LC‐HF) for 4 weeks followed by 24 h food deprivation on two occasions, after which food intake and hypothalamic POMC, NPY, and AgRP mRNA expression were measured. Results: HF rats consumed more food and gained more weight than rats on CH or LC‐HF diets. Despite similar intakes and weight gains, LC‐HF rats had increased adiposity relative to CH rats. LC‐HF rats were more sensitive to melanotan‐II and less sensitive to SHU9119. LC‐HF rats had increased plasma leptin and ghrelin levels and decreased insulin levels, and patterns of NPY and POMC mRNA expression were consistent with those of food‐deprived rats. LC‐HF rats did not show rebound hyperphagia after food deprivation, and levels NPY, POMC, and AgRP mRNA expression were not affected by deprivation. Discussion: Our results demonstrate that an LC diet influences multiple systems involved in the controls of food intake and body weight. These data also suggest that maintenance on an LC‐HF diet affects food intake by reducing compensatory responses to food deprivation.     

Viral mediated neuropeptide Y expression in the rat paraventricular nucleus results in obesity

Objective: Chronic central administration of neuropeptide Y (NPY) has dramatic effects on energy balance; however, the exact role of the hypothalamic paraventricular nucleus (PVN) in this is unknown. The aim of this study was to further unravel the contribution of NPY signaling in the PVN to energy balance. Research Methods and Procedures: Recombinant adeno‐associated viral particles containing NPY (rAAV‐NPY) were injected in the rat brain with coordinates targeted at the PVN. For three weeks, body weight, food intake, endocrine parameters, body temperature, and locomotor activity were measured. Furthermore, effects on insulin sensitivity and expression of NPY, agouti‐related protein (AgRP), and pro‐opiomelanocortin in the arcuate nucleus were studied. Results: Food intake was increased specifically in the light period, and dark phase body temperature and locomotor activity were reduced. This resulted in obesity characterized by increased fat mass; elevated plasma insulin, leptin, and adiponectin; decreased AgRP expression in the arcuate nucleus; and decreased insulin sensitivity; whereas plasma corticosterone was unaffected. Discussion: These data suggest that increased NPY expression targeted at the PVN is sufficient to induce obesity. Interestingly, plasma concentrations of leptin and insulin were elevated before a rise in food intake, which suggests that NPY in the PVN influences leptin and insulin secretion independently from food intake. This strengthens the role of the PVN in regulation of energy balance by NPY.     

Wolbachia infection and an all-female trait in Ostrinia orientalis and Ostrinia zaguliaevi

Sex ratio distortion toward females (SR trait), induced by a Wolbachia bacterium, has been reported in two species of the Ostrinia furnacalis group, viz., O. furnacalis, and O. scapulalis (Lepidoptera: Crambidae). In addition, an SR trait caused by abacterial, unidentified agent(s) is known in O. scapulalis. Here we examined the SR trait in four other species of the furnacalis group, viz., O. orientalis, O. zaguliaevi, and O. zealis from Japan, and O. nubilalis from central and eastern Europe. Wolbachia infection was detected in three O. orientalis females and in one O. zaguliaevi female, and the infection was always associated with the SR trait. In terms of wsp and ftsZ gene sequences, Wolbachia strains in O. orientalis and O. zaguliaevi were indistinguishable from each other, or from those in O. furnacalis and O. scapulalis. These findings suggest that Wolbachia strains in the four Ostrinia species are probably identical. In addition, one O. zealis female, which was negative in diagnostic PCRs for Wolbachia and general bacteria, produced an all‐female brood. This trait was very similar to the SR trait caused by abacterial agent(s) in O. scapulalis.     

A reverse temperature‐size rule associated with a negative relationship between larval development time and pupal weight in a tropical population of Ostrinia furnacalis

Empirical studies demonstrate a negative relationship between body size and temperature [i.e. the temperature‐size rule (TSR)] and a positive relationship between body size and development time (a trade‐off). However, many exceptions are also reported. The present study tests the two relationships in a tropical population of the Asian corn borer Ostrinia furnacalis under a wide range of temperatures from 18 to 31 °C. The results show that larval time is decreased significantly and pupal weight is increased significantly with an increasing rearing temperature, exhibiting the reverse‐TSR. Growth rate is positively correlated with temperature. The relationships between body size and development time among individuals show a negative slope (i.e. pupal weight tends to decrease with increases in development time). Females are significantly larger than males at all temperatures, showing a female‐biased sex size dimorphism. It is also found that development time and pupal weight vary greatly among individuals, particularly at lower temperatures. The results of the present study demonstrate a reverse‐TSR associated with a negative relationship between larval development time and pupal weight in a tropical population of O. furnacalis.     

Effects of azadirachtin on six inorganic cation distributions in Ostrinia furnacalis (G.)

Azadirachtin (Az), as a botanical insecticide, is relatively safe and biodegradable. It affects a wide vaariety of biological processes, including the reduction of feeding, suspension of molting, death of larvae and pupae, and sterility of emerged adults in a dose-dependent manner. However, the mode of action of this toxin remains obscure. By using ion chromatography, we analyzed changes in six inorganic cation (Li⁺, Na⁺, NH₄ ⁺, K⁺, Mg²⁺, and Ca²⁺) distributions of the whole body and hemolymph in Ostrinia furnacalis (G.) after exposure to sublethal doses of Az. The results showed that Az dramatically interfered with Na⁺, NH₄ ⁺, K⁺, Mg²⁺, and Ca²⁺ distributions in hemolymph of O. furnacalis (G.) and concentrations of these five cations dramatically increased. However, in the whole body, the levels of K⁺, Mg²⁺, and Ca²⁺ significantly, decreased after exposure to Az, except that Na⁺ and NH₄ ⁺ remained constant. Li⁺ was undetected in both the control and treated groups in the whole body and hemolymph. It is suggested that Az exerts its insecticidal effects on O. furnacalis (G.) by interfering with the inorganic cation distributions related to ion channels.     

Unique gene alterations are induced in FACS‐purified Fos‐positive neurons activated during cue‐induced relapse to heroin seeking

Cue‐induced heroin seeking after prolonged withdrawal is associated with neuronal activation and altered gene expression in prefrontal cortex (PFC). However, these previous studies assessed gene expression in all neurons regardless of their activity state during heroin seeking. Using Fos as a marker of neural activity, we describe distinct molecular alterations induced in activated versus non‐activated neurons during cue‐induced heroin seeking after prolonged withdrawal. We trained rats to self‐administer heroin for 10 days (6 h/day) and assessed cue‐induced heroin seeking in extinction tests after 14 or 30 days. We used fluorescent‐activated cell sorting (FACS) to purify Fos‐positive and Fos‐negative neurons from PFC 90 min after extinction testing. Flow cytometry showed that Fos‐immunoreactivity was increased in less than 10% of sparsely distributed PFC neurons. mRNA levels of the immediate early genes fosB, arc, egr1, and egr2, as well as npy and map2k6, were increased in Fos‐positive, but not Fos‐negative, neurons. In support of these findings, double‐label immunohistochemistry indicated substantial coexpression of neuropeptide Y (NPY)‐ and Arc‐immunoreactivity in Fos‐positive neurons. Our data indicate that cue‐induced relapse to heroin seeking after prolonged withdrawal induces unique molecular alterations within activated PFC neurons that are distinct from those observed in the surrounding majority of non‐activated neurons.     

Importance of Orexigenic Counter‐Regulation for Multiple Targeted Feeding Inhibition

Objective: Central feeding regulation involves both anorectic and orexigenic pathways. This study examined whether targeting both systems could enhance feeding inhibition induced by anorectic neuropeptides. Research Methods and Procedures: Experiments were carried out in 24‐hour fasted rats. Intracerebroventricular (ICV) injections were accomplished through stereotaxically implanted cannulae aimed at the lateral cerebral ventricle. Food intake of standard rat chow pellets was subsequently recorded for 2 hours. Results: Blockade of orexigenic central opioids and neuropeptide Y (NPY) by ICV naloxone (25 μg) or the NPY receptor antagonist [d‐Trp³²]NPY (NPY‐Ant; 10 μg) powerfully augmented the feeding suppression induced by ICV glucagon‐like peptide 1 (7‐36)‐amide (GLP‐1; 10 μg) or xenin‐25 (xenin; 15 μg) in 24‐hour fasted rats. Most importantly, in combination with naloxone or NPY‐Ant, even a low and ineffective dose of GLP‐1 (5 μg) caused a 40% reduction of food intake, which was augmented further when both antagonists were given in combination with GLP‐1. The combination of GLP‐1 (5 μg) and xenin (10 μg) at individually ineffective doses caused a 46% reduction of food intake, which was abolished at a 10‐fold lower dose. This ineffective dose, however, reduced food intake by 72% when administered in combination with naloxone and NPY‐Ant. Discussion: Targeting up to four pathways of feeding regulation in the central nervous system by blockade of endogenous feeding stimuli and simultaneous administration of anorectic neuropeptides potentiated reduction of food intake. This raises a promising perspective for treatment of obesity.     

Susceptibility of Nontarget Hosts to Nosema furnacalis (Microsporida: Nosematidae), a Potential Biological Control Agent of the European Corn Borer, Ostrinia nubilalis (Lepidoptera: Pyralidae)

Nosema furnacalis Wenn whose normal host is the Asian corn borer, Ostrinia furnacalis (Guenee), is also pathogenic to the European corn borer, Ostrinia nubilaus (Hübner), making N. furnacalis a candidate biological control agent for O. nubilalis. Before N. furnacalis can be used in classical biological control, its effect on natural enemies of O. nubilalis needs to be assessed. We fed nine predator species and one parasitoid of O. nubilalis either purified N. furnacalis spores or N. furnacalis-infected larvae. Using microscopic examination and a species-specific serological assay within these natural enemies, we determined whether N. furnacalis spores were present and whether the spores had germinated. Of the 424 adult predators fed N. furnacalis spores or N. furnacalis-infected corn horers, only 2 tested weakly positive with the enzyme-linked immunosorbent assay (ELISA), indicating that ingested spores did not germinate in the adult predators. Fifteen of 130 immature predators tested weakly positive with the ELISA, but because relatively few spores were found upon microscopic examination, we concluded there was no evidence of an active N. furnacalis infection in any of the nine predator species tested. In contrast, the parasitoid Macrocentrus grandii Goidanich always tested strongly positive in the ELISA (mean O.D._{405 nm} = 1.36), indicating that sporoplasm antigens were present. Microscopic examination of this parasitoid showed that a high number of spores were present (mean spore number/larvae 1.4 × 10⁷). From these data, M. grandii might become infected with N. furnacalis.     

Sustained NPY Overexpression in the PVN Results in Obesity via Temporarily Increasing Food Intake

Increasing neuropeptide Y (NPY) signaling in the paraventricular nucleus (PVN) by recombinant adeno‐associated virus (rAAV)‐mediated overexpression of NPY in rats, results in hyperphagia and obesity in rats. To determine the importance of hyperphagia in the observed obesity phenotype, we pair‐fed a group of AAV‐NPY‐injected rats to AAV‐control‐injected rats and compared parameters of energy balance to ad libitum fed AAV‐NPY‐injected rats. For 3 weeks, AAV‐NPY‐injected rats, received the same amount of food as ad libitum‐fed rats injected with control rAAV They did not gain more body weight than these controls. When allowed access to food ad libitum, these AAV‐NPY‐injected rats increased food intake, which subsequently decreased when rats reached the same body weight as AAV‐NPY‐injected rats that were fed ad libitum for the entire study. These data indicate that overexpression of NPY in the PVN results in obesity by increasing food intake until a certain body weight is achieved.     

CONTROL OF SEX PHEROMONE BIOSYNTHETIC PATHWAY BY PBAN IN ASIAN CORN BORER OSTRINIA FURNACALlS*

Abstract Sex pheromone titer in Ostrinia furnacalis was significantly decreased to a very low level by decapitation, but it could be restored by injection of head extract prepared from both male and female moths or synthetic pheromone biosynthesis activating neuropepide (PBAN). This fact indicates that pheromone production is under the control of a PBAN-like factor. The sex pheromone biosynthetic pathway of O. furnacalis originates with the biosynthesis of palmitic acid and followed by A14 desaturation, chain shortening, reduction and acetylation to form the pheromone components, (Z) and (E)-12-tetradecenyl acetate. In order to determine which step in the pathway is controlled by PBAN, the incorporation of different labeled precursors into the pheromone and its intermediate were studied. Our results suggest that PBAN controls pheromone biosynthesis in O. furnacalis by mainly regulating an early step from acetate to palmitic acid.     

Looking for Food: Molecular Neuroethology of Invertebrate Feeding Behavior

The assignment of complex behavior of animals to the function of specific genes has seen significant advances in the past decade. The advent of modern tools of genetics and genomics permitted analyses that revealed a good number of neural system enriched genes whose products modulate, and whose polymorphism qualitatively or quantitatively influenced invertebrate feeding behavior. The most prominent of these genes are orthologues of foraging (for) and the neuropeptide Y (NPY)/NPY receptor. The former encodes a cyclic‐GMP‐dependent protein kinase, which functional genetics have been characterized in Drosophila melanogaster, Apis mellifera and Caenorhabditis elegans. Allelic variations and changes in the expression of the above genes could influence the initiation of particular feeding behaviors or related social phenotype. These genes have provided the first molecular insights towards feeding behavior in invertebrates. Besides detailed investigations into the neural pathways involved and mechanisms of function of the gene products, parallel studies in other animal models is imperative to understand ecological drivers of animal feeding behavior.     

Mapping of neuropeptide Y expression in Octopus brains

Neuropeptide Y (NPY) is an evolutionarily conserved neurosecretory molecule implicated in a diverse complement of functions across taxa and in regulating feeding behavior and reproductive maturation in Octopus. However, little is known about the precise molecular circuitry of NPY-mediated behaviors and physiological processes, which likely involve a complex interaction of multiple signal molecules in specific brain regions. Here, we examined the expression of NPY throughout the Octopus central nervous system. The sequence analysis of Octopus NPY precursor confirmed the presence of both, signal peptide and putative active peptides, which are highly conserved across bilaterians. In situ hybridization revealed distinct expression of NPY in specialized compartments, including potential “integration centers,” where visual, tactile, and other behavioral circuitries converge. These centers integrating separate circuits may maintain and modulate learning and memory or other behaviors not yet attributed to NPY-dependent modulation in Octopus. Extrasomatic localization of NPY mRNA in the neurites of specific neuron populations in the brain suggests a potential demand for immediate translation at synapses and a crucial temporal role for NPY in these cell populations. We also documented the presence of NPY mRNA in a small cell population in the olfactory lobe, which is a component of the Octopus feeding and reproductive control centers. However, the molecular mapping of NPY expression only partially overlapped with that produced by immunohistochemistry in previous studies. Our study provides a precise molecular map of NPY mRNA expression that can be used to design and test future hypotheses about molecular signaling in various Octopus behaviors.     

Novel Cell Line Selectively Expressing Neuropeptide Y‐Y2 Receptors

Abstract

Neuropeptide Y (NPY) recognition by the human neuroblastoma cell lines SiMa, Kelly, SH‐SY5Y, CHP‐234, and MHH‐NB‐11 was analyzed in radioactive binding assays using tritiated NPY. For the cell lines CHP‐234 and MHH‐NB‐11 binding of [³H]propionyl‐NPY was observed with K_d‐values of 0.64 ± 0.07 nM and 0.53 ± 0.12 nM, respectively, determined by saturation analysis with non‐linear regression. The receptor subtype was determined by competition analysis using the subtype selective NPY analogues [Leu³¹, Pro³⁴]‐NPY (NPY‐Y₁, NPY‐Y₅), [Ahx^5‐24]‐NPY (NPY‐Y₂), [Ala³¹, Aib³²]‐NPY (NPY‐Y₅), NPY [3‐36] (NPY‐Y₂, NPY‐Y₅), and NPY [13‐36] (NPY‐Y₂). Both cell lines, CHP‐234 and MHH‐NB‐11, the latter one being characterized for NPY receptors for the first time, showed exclusive expression of NPY‐Y₂ receptors. In both cell lines binding of NPY induced signal transduction, which was monitored as reduction of forskolin‐induced cAMP production in an ELISA.