Palmitate-mediated induction of neuropeptide Y expression occurs through intracellular metabolites and not direct exposure to proinflammatory cytokines.

A contributing factor to the development of obesity is the consumption of a diet high in saturated fatty acids, such as palmitate. These fats induce hypothalamic neuroinflammation, which dysregulates neuronal function and induces orexigenic neuropeptide Y (Npy) to promote food intake. An inflammatory cytokine array identified multiple candidates that could mediate palmitate-induced up-regulation of Npy mRNA levels. Of these, visfatin or nicotinamide phosphoribosyltransferase (NAMPT), macrophage migratory inhibitory factor (MIF), and IL-17F were chosen for further study. Direct treatment of the neuropeptide Y/agouti-related peptide (NPY/AgRP)-expressing mHypoE-46 neuronal cell line with the aforementioned cytokines demonstrated that visfatin could directly induce Npy mRNA expression. Preventing the intracellular metabolism of palmitate through long-chain acyl-CoA synthetase (ACSL) inhibition was sufficient to block the palmitate-mediated increase in Npy gene expression. Furthermore, thin-layer chromatography revealed that in neurons, palmitate is readily incorporated into ceramides and defined species of phospholipids. Exogenous C16 ceramide, dipalmitoyl-phosphatidylcholine, and dipalmitoyl-phosphatidylethanolamine were sufficient to significantly induce Npy expression. This study suggests that the intracellular metabolism of palmitate and elevation of metabolites, including ceramide and phospholipids, are responsible for the palmitate-mediated induction of the potent orexigen Npy. Furthermore, this suggests that the regulation of Npy expression is less reliant on inflammatory cytokines per se than palmitate metabolites in a model of NPY/AgRP neurons. These lipid species likely induce detrimental downstream cellular signaling events ultimately causing an increase in feeding, resulting in an overweight phenotype and/or obesity.

Sensitivity of olfactory sensory neurons to food cues is tuned to nutritional states by Neuropeptide Y signaling.

Modulation of sensory perception by homeostatic feedback from physiological states is central to innate purposive behaviors. Olfaction is an important predictive modality for feeding-related behaviors and its modulation has been associated with hunger-satiety states. However, the mechanisms mapping internal states to chemosensory processing in order to modify behavior are poorly understood. In the zebrafish olfactory epithelium, a subset of olfactory sensory neurons (OSNs) and the terminal nerve projections express neuropeptide Y (NPY). Using a combination of neuronal activity and behavioral evaluation, we find that NPY signaling in the peripheral olfactory system of zebrafish is correlated with its nutritional state and is both necessary and sufficient for the olfactory perception of food-related odorants. NPY activity dynamically modulates the microvillar OSN activation thresholds and acts cooperatively with amino acid signaling resulting in a switch-like increase in OSN sensitivity in starved animals. We suggest that cooperative activation of phospholipase C by convergent signaling from NPY and amino acid receptors is central to this heightened sensitivity. This study provides ethologically relevant, physiological evidence for NPY signaling in the modulation of OSN sensitivity to food-associated amino acid cues. We demonstrate sensory gating directly at the level of OSNs and identify a novel mechanistic framework for tuning olfactory sensitivity to prevailing energy states. Cover Image for this issue: https://doi.org/10.1111/jnc.15091.

Chronic oral nicotine administration and withdrawal regulate the expression of neuropeptide Y and its receptors in the mesocorticolimbic system.

Neuropeptide Y (NPY) and its receptors are involved in the regulation of mood, stress, and anxiety. In parallel, NPY signaling may play a vital role in the negative affective state induced by drug withdrawal. This study examined the changes in the transcript levels of NPY, Y1, Y2, and Y5 receptors in the mesocorticolimbic system during chronic nicotine exposure and withdrawal. Rats were administered with nicotine (initial dose: 25 µg/ml, maintenance dose: 50 µg/ml, free base) in drinking water for 12 weeks. Control group received only tap water. In the final week of the study, some of the nicotine-treated animals continued to receive nicotine (0-W), whereas some were withdrawn for either 24 (24-W) or 48 (48-W) h. All animals were decapitated after the evaluation of somatic signs (frequency of gasps, eye blinks, ptosis, shakes, teeth chatter) and the duration of locomotor activity and immobility. mRNA levels of NPY, Y1, Y2, and Y5 receptors in the mesocorticolimbic system were measured by quantitative real-time PCR (qRT-PCR). Results showed that nicotine withdrawal increased overall somatic signs. Moreover, chronic nicotine treatment increased the duration of locomotor activity, whereas withdrawal increased the duration of immobility. qRT-PCR analysis revealed that chronic nicotine treatment increased NPY mRNA levels in the hippocampus. On the other hand, 24- and 48-h withdrawals increased NPY mRNA levels in the amygdala and medial prefrontal cortex (mPFC), Y1 and Y2 mRNA levels in the nucleus accumbens and mPFC, and Y5 mRNA levels in the mPFC. These findings suggest that nicotine withdrawal enhances NPY signaling in the mesocorticolimbic system, which could be an important mechanism involved in regulating the negative affective state triggered during nicotine withdrawal.

Adequate expression of neuropeptide Y is essential for the recovery of zebrafish motor function following spinal cord injury.

In strong contrast to limited repair within the mammalian central nervous system, the spinal cord of adult zebrafish is capable of almost complete recovery following injury. Understanding the mechanism underlying neural repair and functional recovery in zebrafish may lead to innovative therapies for human spinal cord injury (SCI). Since neuropeptide Y (NPY) plays a protective role in the pathogenesis of several neurological diseases, in the present study, we evaluated the effects of NPY on neuronal repair and subsequent recovery of motor function in adult zebrafish following SCI. Real-time quantitative PCR (qRT-PCR), in situ hybridization and immunostaining for NPY revealed decreased NPY expression at 12 hours (h), 6 and 21 days (d) after SCI. Double-immunostaining for NPY and islet-1, a motoneuron marker, showed that NPY was expressed in spinal cord motoneurons. Morpholino (MO) treatment for suppressing the expression of NPY inhibited supraspinal axon regrowth and locomotor recovery, in which double-staining for proliferating cell nuclear antigen (PCNA) and islet-1 showed a reduction in motoneuron proliferation. Similarly, a downregulated mRNA level of Y1 receptor of NPY (NPY1R) was also detected at 12 h, 6 and 21 d after injury. Immunostaining for NPY and in situ hybridization for NPY1R revealed that NPY1R was co-localized with NPY. Collectively, the results suggest that NPY expression in motoneurons promotes descending axon regeneration and locomotor recovery in adult zebrafish after SCI, possibly by regulating motoneuron proliferation through activation of NPY1R.

Double immunofluorescent evidence that oxidative stress-associated activation of JNK/AP-1 signaling participates in neuropeptide-mediated appetite control.

Amphetamine (AMPH), an appetite suppressant, alters expression levels of neuropeptide Y (NPY) and cocaine- and amphetamine-regulated transcript (CART) in the hypothalamus. This study explored the potential role of cJun-N-terminal kinases (JNK) in appetite control, mediated by reactive oxygen species (ROS) and activator protein-1 (AP-1) in AMPH-treated rats. Rats were given AMPH daily for 4 days. Changes in feeding behavior and expression levels of hypothalamic NPY, CART, cFos, cJun, phosphorylated JNK (pJNK), as well as those of anti-oxidative enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GP) and glutathione S-transferase (GST), were examined and compared. Following AMPH treatment, food intake and NPY expression decreased, whereas the other proteins expression and AP-1/DNA binding activity increased. Both cerebral cJun inhibition and ROS inhibition attenuated AMPH anorexia and modified detected protein, revealing a crucial role for AP-1 and ROS in regulating AMPH-induced appetite control. Moreover, both pJNK/CART and SOD/CART activities detected by double immunofluorescent staining increased in hypothalamic arcuate nucleus in AMPH-treated rats. The results suggested that pJNK/AP-1 signaling and endogenous anti-oxidants participated in regulating NPY/CART-mediated appetite control in rats treated with AMPH. These findings advance understanding of the molecular mechanism underlying the role of pJNK/AP-1 and oxidative stress in NPY/CART-mediated appetite suppression in AMPH-treated rats.

Attenuation of stress-induced weight loss with a ketogenic diet.

Ketogenic diets (KDs) are high-fat, low-carbohydrate diets that have been used therapeutically for decades, most notably for the treatment of epilepsy and diabetes. Recent data, however, suggest that KD may impart protective effects on mood disorders. The current experiments test the hypothesis that KDs can protect from stress-induced symptoms of mood disorders. To test this, we assessed behavioral and neuroendocrine effects of KD in male and female Long Evans rats. Animals experienced three weeks of chronic mild stress (CMS) while consuming KD or control chow (CH). Body weight and food intake data were recorded daily and behaviors were assayed after three weeks. Plasma beta-hydroxybutyrate (ßHB), corticosterone (CORT) and interleukin-1 beta (IL-1ß) were measured after behavioral testing, along with hypothalamic corticotropin-releasing hormone (CRH) and neuropeptide Y (NPY) mRNA expression. CMS induced weight loss in the CH groups, however the KD-fed rats were resistant to CMS-induced weight loss. Female rats fed KD were protected from CMS-induced reductions in plasma CORT and hypothalamic NPY expression. Collectively, these data suggest protective potential of KDs against chronic stress, particularly in females.

Potential role of sympathetic activity on the pathogenesis of massive pulmonary embolism with circulatory shock in rabbits.

BACKGROUND: We recently showed that intravenous sodium nitroprusside treatment (SNP) could relieve the pulmonary vasospasm of pulmonary embolism (PE) and non-pulmonary embolism (non-PE) regions in a rabbit massive pulmonary embolism (MPE) model associated with shock. The present study explored the potential role of cardiopulmonary sympathetic activity on the pathogenesis and the impact of vasodilators on cardiopulmonary sympathetic activity in this model. METHODS: Rabbits were randomly divided into sham operation group (S group, n = 8), model group (M, equal volume of saline intravenously, n = 11), SNP group (3.5 µg/kg/min intravenously, n = 10) and diltiazem group (DLZ, 6.0 µg/kg/min intravenously, n = 10). RESULTS: MPE resulted in reduced mean arterial pressure and increased mean pulmonary arterial pressure as well as reduced PaO2 in the M, SNP and DLZ groups. Tyrosine hydroxylase (TH), neuropeptide Y (NPY) and endothelin-1 (ET-1) expression levels were significantly increased, while nitric oxide (NO) levels were reduced in both PE and non-PE regions in the M group. Both SNP and DLZ decreased mean pulmonary arterial pressure, reversed shock status, downregulated the expression of TH, NPY and ET-1, and increased NO levels in PE and non-PE regions. CONCLUSION: Present results indicate that upregulation of the sympathetic medium transmitters TH and NPY in whole lung tissues serves one of the pathological features of MPE. The vasodilators SNP and DLZ could relieve pulmonary vasospasm in both embolization and non-embolization regions and reverse circulatory shock, thereby indirectly downregulating the sympathetic activation of the whole lung tissues and breaking a vicious cycle related to sympathetic activation in this model.

Effect of newer anti-epileptic drugs (AEDs) on the cognitive status in pentylenetetrazol induced seizures in a zebrafish model.

Epilepsy is marked by seizures that are a manifestation of excessive brain activity and is symptomatically treatable by anti-epileptic drugs (AEDs). Unfortunately, the older AEDs have many side effects, with cognitive impairment being a major side effect that affects the daily lives of people with epilepsy. Thus, this study aimed to determine if newer AEDs (Zonisamide, Levetiracetam, Perampanel, Lamotrigine and Valproic Acid) also cause cognitive impairment, using a zebrafish model. Acute seizures were induced in zebrafish using pentylenetetrazol (PTZ) and cognitive function was assessed using the T-maze test of learning and memory. Neurotransmitter and gene expression levels related to epilepsy as well as learning and memory were also studied to provide a better understanding of the underlying processes. Ultimately, impaired cognitive function was seen in AED treated zebrafish, regardless of whether seizures were induced. A highly significant decrease in γ-Aminobutyric Acid (GABA) and glutamate levels was also discovered, although acetylcholine levels were more variable. The gene expression levels of Brain-Derived Neurotrophic Factor (BDNF), Neuropeptide Y (NPY) and Cyclic Adenosine Monophosphate (CAMP) Responsive Element Binding Protein 1 (CREB-1) were not found to be significantly different in AED treated zebrafish. Based on the experimental results, a decrease in brain glutamate levels due to AED treatment appears to be at least one of the major factors behind the observed cognitive impairment in the treated zebrafish.

Single prolonged stress PTSD model triggers progressive severity of anxiety, altered gene expression in locus coeruleus and hypothalamus and effected sensitivity to NPY.

PTSD is heterogeneous disorder that can be long lasting and often has delayed onset following exposure to a traumatic event. Therefore, it is important to take a staging approach to evaluate progression of biological mechanisms of the disease. Here, we begin to evaluate the temporal trajectory of changes following exposure to traumatic stressors in the SPS rat PTSD model. The percent of animals displaying severe anxiety on EPM increased from 17.5% at one week to 57.1% two weeks after SPS stressors, indicating delayed onset or progressive worsening of the symptoms. The LC displayed prolonged activation, and dysbalance of the CRH/NPY systems, with enhanced CRHR1 gene expression, coupled with reduced mRNAs for NPY and Y2R. In the mediobasal hypothalamus, increased CRH mRNA levels were sustained, but there was a flip in alterations of HPA regulatory molecules, GR and FKBP5 and Y5 receptor at two weeks compared to one week. Two weeks after SPS, intranasal NPY at 300 µg/rat, but not 150 µg which was effective after one week, reversed SPS triggered elevated anxiety. It also reversed SPS elicited depressive/despair symptoms and hyperarousal. Overall, the results reveal time-dependent progression in development of anxiety symptoms and molecular impairments in gene expression for CRH and NPY systems in LC and mediobasal hypothalamus by SPS. With longer time afterwards only a higher dose of NPY was effective in reversing behavioral impairments triggered by SPS, indicating that therapeutic approaches should be adjusted according to the degree of biological progression of the disorder.

Effect of the algal alkaloid caulerpin on neuropeptide Y (NPY) expression in the central nervous system (CNS) of Diplodus sargus.

Recent studies have suggested that Mediterranean indigenous fish species are affected by bioactive metabolites coming from marine invasive species via food web interactions. In particular, both physiological and behavioural changes in the white sea bream Diplodus sargus were related to caulerpin (CAU), a bisindolic alkaloid particularly abundant in the invasive alga Caulerpa cylindracea, on which the fish actively feed. Dietary administration of CAU decreased aggressiveness in D. sargus, suggesting an anxiolytic-like effect of CAU possibly mediated by endogenous anxiolytic agents. This hypothesis is supported here by the finding of a significant increase of NPY transcriptional expression in the brain of fish fed with CAU enriched food, shedding more light on the neural mechanisms behind the altered behaviour of D. sargus.

Age-related central regulation of orexin and NPY in the short-lived African killifish Nothobranchius furzeri.

Orexin A (OXA) and neuropeptide Y (NPY) are two hypothalamic neuropeptides involved in the regulation of feeding behavior and food intake in all vertebrates. Accumulating evidences document that they undergo age-related modifications, with consequences on metabolism, sleep/wake disorders and progression of neurodegenerations. The present study addressed the age related changes in expression and distribution of orexin A (its precursor is also known as hypocretin-HCRT) and NPY, and their regulation by food intake in the short-lived vertebrate model Nothobranchius furzeri. Our experiments, conducted on male specimens, show that: (a) HCRT and OXA and NPY mRNA and protein are localized in neurons of diencephalon and optic tectum, as well as in numerous fibers projecting through the entire neuroaxis, and are colocalized in specific nuclei; (b) in course of aging, HCRT and NPY expressing neurons are localized also in telencephalon and rhombencephalon; (c) HCRT expressing neurons increased slightly in the diencephalic area of old animals and in fasted animals, whereas NPY increased sharply; (d) central HCRT levels are not regulated neither in course of aging nor by food intake; and (e) central NPY levels are augmented in course of aging, and regulated by food intake only in young. These findings represent a great novelty in the study of central orexinergic and NPY-ergic systems in vertebrates', demonstrating an uncommon and unprecedented described regulation of these two orexigenic neuropeptides.

Central Dicer-miR-103/107 controls developmental switch of POMC progenitors into NPY neurons and impacts glucose homeostasis.

Proopiomelanocortin (POMC) neurons are major negative regulators of energy balance. A distinct developmental property of POMC neurons is that they can adopt an orexigenic neuropeptide Y (NPY) phenotype. However, the mechanisms underlying the differentiation of Pomc progenitors remain unknown. Here, we show that the loss of the microRNA (miRNA)-processing enzyme Dicer in POMC neurons causes metabolic defects, an age-dependent decline in the number of PomcmRNA-expressing cells, and an increased proportion of Pomc progenitors acquiring a NPY phenotype. miRNome microarray screening further identified miR-103/107 as candidates that may be involved in the maturation of Pomc progenitors. In vitro inhibition of miR-103/107 causes a reduction in the number of Pomc-expressing cells and increases the proportion of Pomc progenitors differentiating into NPY neurons. Moreover, in utero silencing of miR-103/107 causes perturbations in glucose homeostasis. Together, these data suggest a role for prenatal miR-103/107 in the maturation of Pomc progenitors and glucose homeostasis.

Uneven balance of power between hypothalamic peptidergic neurons in the control of feeding.

Two classes of peptide-producing neurons in the arcuate nucleus (Arc) of the hypothalamus are known to exert opposing actions on feeding: the anorexigenic neurons that express proopiomelanocortin (POMC) and the orexigenic neurons that express agouti-related protein (AgRP) and neuropeptide Y (NPY). These neurons are thought to arise from a common embryonic progenitor, but our anatomical and functional understanding of the interplay of these two peptidergic systems that contribute to the control of feeding remains incomplete. The present study uses a combination of optogenetic stimulation with viral and transgenic approaches, coupled with neural activity mapping and brain transparency visualization to demonstrate the following: (i) selective activation of Arc POMC neurons inhibits food consumption rapidly in unsated animals; (ii) activation of Arc neurons arising from POMC-expressing progenitors, including POMC and a subset of AgRP neurons, triggers robust feeding behavior, even in the face of satiety signals from POMC neurons; (iii) the opposing effects on food intake are associated with distinct neuronal projection and activation patterns of adult hypothalamic POMC neurons versus Arc neurons derived from POMC-expressing lineages; and (iv) the increased food intake following the activation of orexigenic neurons derived from POMC-expressing progenitors engages an extensive neural network that involves the endogenous opioid system. Together, these findings shed further light on the dynamic balance between two peptidergic systems in the moment-to-moment regulation of feeding behavior.

Hippocampal neuropeptide Y protein expression following controlled cortical impact and posttraumatic epilepsy.

This study assessed neuropeptide Y (NPY) expression in the hippocampus after long-term survival following traumatic brain injury (TBI) induced by controlled cortical impact (CCI) with or without the development of posttraumatic epilepsy (PTE). We hypothesized that following long-term survival after CCI, the severity of tissue injury and the development of PTE would correlate with the degree of hippocampal neurodegeneration as reflected by NPY+ and neuronal nuclear antigen (NeuN)+ cell loss. Adult Sprague-Dawley rats of 2-3 months of age were lesioned in the right parietal cortex and monitored for seizure activity by video and/or video-EEG. Neuropeptide Y and NeuN immunoreactivities (IRs) were quantified by light microscopy and semiautomatic image analysis approaches for unbiased quantification. Severely injured animals, marked by extensive tissue loss in the ipsilateral neocortex and adjacent hippocampus, resulted in significantly lower NeuN+ hilar cell density and NPY+ cell loss in the contralateral Cornu Ammonis (CA)-3 and dentate hilus (DH). The degree of NPY+ cell loss was more severe in CCI-injured animals with PTE than those animals that did not develop PTE. Mildly injured animals demonstrated no significant change of NPY expression compared with control animals. Our findings of long-term alterations of NPY expression in the hippocampus of severely brain-injured animals can provide important insights into the cellular and molecular consequences of severe TBI and posttraumatic epileptogenesis.

Two types of somatostatin-expressing GABAergic interneurons in the superficial layers of the mouse cingulate cortex.

Somatostatin-expressing (SOM+), inhibitory interneurons represent a heterogeneous group of cells and given their remarkable diversity, classification of SOM+ interneurons remains a challenging task. Electrophysiological, morphological and neurochemical classes of SOM+ interneurons have been proposed in the past but it remains unclear as to what extent these classes are congruent. We performed whole-cell patch-clamp recordings from 127 GFP-labeled SOM+ interneurons ('GIN') of the superficial cingulate cortex with subsequent biocytin-filling and immunocytochemical labeling. Principal component analysis followed by k-means clustering predicted two putative subtypes of SOM+ interneurons, which we designated as group I and group II GIN. A key finding of our study is the fact that these electrophysiologically and morphologically distinct groups of SOM+ interneurons can be correlated with two neurochemical subtypes of SOM+ interneurons described recently in our laboratory. In particular, all SOM+ interneurons expressing calbindin but no calretinin could be classified as group I GIN, whereas all but one neuropeptide Y- and calretinin-positive interneurons were found in group II.

Anesthetic drugs modulate feeding behavior and hypothalamic expression of the POMC polypeptide precursor and the NPY neuropeptide.

BACKGROUND: Several hypnotic drugs have been previously identified as modulators of food intake, but exact mechanisms remain unknown. Feeding behavior implicates several neuronal populations in the hypothalamic arcuate nucleus including orexigenic neuropeptide Y and anorexigenic pro-opiomelanocortin producing neurons. The aim of this study was to investigate in mice the impact of different hypnotic drugs on food consumption and neuropeptide Y or pro-opiomelanocortine mRNA expression level in the hypothalamic arcuate nucleus. METHODS: Saline control, isoflurane, thiopental, midazolam or propofol were administered to C57Bl/6 mice. Feeding behavior was evaluated during 6 h. In situ hybridization of neuropeptide Y and pro-opiomelanocortine mRNAs in the hypothalamus brain region was also performed. Data were analyzed by Kruskal Wallis test and analysis of variance (p < 0.05). RESULTS: Midazolam, thiopental and propofol induced feeding behavior. Midazolam and thiopental increased neuropeptide Y mRNA level (respectively by 106 and 125%, p < 0.001) compared with control. Propofol and midazolam decreased pro-opiomelanocortine mRNA level by 31% (p < 0,01) compared with control. Isoflurane increased pro-opiomelanocortine mRNA level by 40% compared with control. CONCLUSION: In our murine model, most hypnotics induced food consumption. The hypnotic-induced regulation of neuropeptide Y and pro-opiomelanocortine hypothalamic peptides is associated with this finding. Our data suggest that administration of some hypnotic drugs may affect hypothalamic peptide precursor and neuropeptide expression and concomittantly modulate food intake. Thus, this questions the choice of anesthetics for better care management of patients undergoing major surgery or at risk of undernutrition.

Neuropeptide Y as a possible homeostatic element for changes in cortical excitability induced by repetitive transcranial magnetic stimulation.

BACKGROUND: Repetitive transcranial magnetic stimulation (rTMS) is able to modify cortical excitability. Rat rTMS studies revealed a modulation of inhibitory systems, in particular that of the parvalbumin-expressing (PV+) interneurons, when using intermittent theta-burst stimulation (iTBS). OBJECTIVE: The potential disinhibitory action of iTBS raises the questions of how neocortical circuits stabilize excitatory-inhibitory balance within a physiological range. Neuropeptide Y (NPY) appears to be one candidate. METHODS: Analysis of cortical expression of PV, NPY and vesicular glutamate transporter type 1 (vGluT1) by immunohistochemical means at the level of cell counts, mean neuropil expression and single cell pre-/postsynaptic expression, with and without intraventricular NPY-injection. RESULTS: Our results show that iTBS not only reduced the number of neurons with high-PV expression in a dose-dependent fashion, but also increased the cortical expression of NPY, discussed to reduce glutamatergic transmission, and this was further associated with a reduced vGluT1 expression, an indicator of glutamateric presynaptic activity. Interneurons showing a low-PV expression exhibit less presynaptic vGluT1 expression compared to those with a high-PV expression. Intraventricular application of NPY prior to iTBS prevented the iTBS-induced reduction in the number of high-PV neurons, the reduction in tissue vGluT1 level and that presynaptic to high-PV cells. CONCLUSIONS: We conclude that NPY, possibly via a global but also slow homeostatic control of glutamatergic transmission, modulates the strength and direction of the iTBS effects, likely preventing pathological imbalance of excitatory and inhibitory cortical activity but still allowing enough disinhibition beneficial for plastic changes as during learning.

Impact of type 1 diabetes mellitus and sitagliptin treatment on the neuropeptide Y system of rat retina.

BACKGROUND: Neuropeptide Y (NPY) is a neuromodulator that is expressed in the retina. Increasing evidence suggests that NPY has pronounced anti-inflammatory effects, which might depend on the inhibition of dipeptidyl-peptidase-IV (DPP-IV). The aim of this study was to investigate the impact of type 1 diabetes mellitus (DM) and sitagliptin, a DPP-IV inhibitor, on the NPY system in the retina using an animal model. METHODS: Type 1 DM was induced in male Wistar rats by an intraperitoneal injection of streptozotocin. Starting 2 weeks after DM onset, animals were treated orally with sitagliptin (5 mg/kg.day) for 2 weeks. The expression of NPY and NPY receptors (Y1 , Y2 and Y5 receptors) was measured by quantitative polymerase chain reaction, Western blot and/or enzyme-linked immunosorbent assay. The immunoreactivity of NPY and NPY receptors was evaluated by immunohistochemistry, and the [35 S]GTPγS binding assay was used to assess the functional binding of NPY receptors. RESULTS: DM decreased the mRNA levels of NPY in the retina, as well as the protein levels of NPY and Y5 receptor. No changes were detected in the localization of NPY and NPY receptors in the retina and in the functional binding of NPY to all receptors. Sitagliptin alone reduced retinal NPY mRNA levels. The effects of DM on the NPY system were not affected by sitagliptin. CONCLUSION: DM modestly affects the NPY system in the retina and these effects are not prevented by sitagliptin treatment. These observations suggest that DPP-IV enzyme is not underlying the NPY changes detected in the retina induced by type 1 DM.

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

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

AgRP knockdown blocks long-term appetitive, but not consummatory, feeding behaviors in Siberian hamsters.

Arcuate hypothalamus-derived agouti-related protein (AgRP) and neuropeptide Y (NPY) are critical for maintaining energy homeostasis. Fasting markedly upregulates AgRP/NPY expression and circulating ghrelin, and exogenous ghrelin treatment robustly increases acute food foraging and food intake, and chronic food hoarding behaviors in Siberian hamsters. We previously demonstrated that 3rd ventricular AgRP injection robustly stimulates acute and chronic food hoarding, largely independent of food foraging and intake. By contrast, 3rd ventricular NPY injection increases food foraging, food intake, and food hoarding, but this effect is transient and gone by 24h post-injection. Because of this discrepancy in AgRP/NPY-induced ingestive behaviors, we tested whether selective knockdown of AgRP blocks fasting and ghrelin-induced increases in food hoarding. AgRP gene knockdown by a novel DICER small interfering RNA (AgRP-DsiRNA) blocked food-deprivation induced increases in AgRP expression, but had no effect on NPY expression. AgRP-DsiRNA attenuated acute (1day), and significantly decreased chronic (4-6days), food deprivation-induced increases in food hoarding. In addition, AgRP-DsiRNA treatment blocked exogenous ghrelin-induced increases in food hoarding through day 3, but had no effect on basal food foraging, food intake, or food hoarding prior to ghrelin treatment. Lastly, chronic AgRP knockdown had no effect on body mass, fat mass, or lean mass in either food deprived or ad libitum fed hamsters. These data collectively suggest that the prolonged increase in food hoarding behavior following energetic challenges, and food deprivation especially, is primarily regulated by downstream AgRP signaling.