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Moderate long-term modulation of neuropeptide Y in hypothalamic arcuate nucleus induces energy balance alterations in adult rats.

Sousa-Ferreira L, Garrido M, Nascimento-Ferreira I, Nobrega C, Santos-Carvalho A, Alvaro AR, Rosmaninho-Salgado J, Kaster M, Kügler S, de Almeida LP, Cavadas C - PLoS ONE (2011)

Bottom Line: Neuropeptide Y (NPY) produced by arcuate nucleus (ARC) neurons has a strong orexigenic effect on target neurons.We achieved a physiological overexpression (3.6-fold increase) and down-regulation (0.5-fold decrease) of NPY in the rat ARC by injection of AAV vectors expressing NPY and synthetic microRNA that target the NPY, respectively.In addition, moderate down-regulation of NPY did not affect basal feeding or normal body weight gain but the response to food deprivation was compromised since fasting-induced hyperphagia was inhibited and fasting-induced decrease in locomotor activity was absent.These results highlight the importance of the physiological ARC NPY levels oscillations on feeding regulation, fasting response and body weight preservation, and are important for the design of therapeutic interventions for obesity that include the NPY.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.

ABSTRACT
Neuropeptide Y (NPY) produced by arcuate nucleus (ARC) neurons has a strong orexigenic effect on target neurons. Hypothalamic NPY levels undergo wide-ranging oscillations during the circadian cycle and in response to fasting and peripheral hormones (from 0.25 to 10-fold change). The aim of the present study was to evaluate the impact of a moderate long-term modulation of NPY within the ARC neurons on food consumption, body weight gain and hypothalamic neuropeptides. We achieved a physiological overexpression (3.6-fold increase) and down-regulation (0.5-fold decrease) of NPY in the rat ARC by injection of AAV vectors expressing NPY and synthetic microRNA that target the NPY, respectively. Our work shows that a moderate overexpression of NPY was sufficient to induce diurnal over-feeding, sustained body weight gain and severe obesity in adult rats. Additionally, the circulating levels of leptin were elevated but the immunoreactivity (ir) of ARC neuropeptides was not in accordance (POMC-ir was unchanged and AGRP-ir increased), suggesting a disruption in the ability of ARC neurons to response to peripheral metabolic alterations. Furthermore, a dysfunction in adipocytes phenotype was observed in these obese rats. In addition, moderate down-regulation of NPY did not affect basal feeding or normal body weight gain but the response to food deprivation was compromised since fasting-induced hyperphagia was inhibited and fasting-induced decrease in locomotor activity was absent.These results highlight the importance of the physiological ARC NPY levels oscillations on feeding regulation, fasting response and body weight preservation, and are important for the design of therapeutic interventions for obesity that include the NPY.

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In vitro validation of NPY vector and synthetic microRNA for down regulation of NPY expression.(A) Schematic representation of the AAV vectors. The NPY expression vector contains one cassette to express NPY under a neuronal specific promoter. The microRNA expression vector contains one cassette with co-cistronic expression of EGFP and miR under the neuronal specific promoter. ITR, inverted terminal repeats; hSyn, human Synapsin promoter; WPRE, woodchuck hepatitis virus posttranslational control element; PolyA, polyadenosine sequence. Small human synapsin 1 gene promoter has shown to drive long-term neuron specific transgene expression from AAV-2 vectors [88]. (B) Three synthetic microRNA directed to different regions of NPY mRNA and one control microRNA (random sequence) were cloned. (C) Screen of microRNA targeting NPY. Representative image of NPY expression and densitometry band evaluation normalized to α-tubulin and shown as percentage of miR-ctr. Cells co-transfected with the NPY vector and one of the miR-NPY vectors, in a 1∶1 ratio. NPY expression is significantly reduced with the miR-NPY-3 vector. n = 3 independent cell transfection; * p<0.05 compared to miR-ctr control. (D) Transduction of NPY positive neurons (red) by AAV-hSyn-miR cassette (EGFP-green) in primary neuronal culture. Scale bar, 20 µm.
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pone-0022333-g001: In vitro validation of NPY vector and synthetic microRNA for down regulation of NPY expression.(A) Schematic representation of the AAV vectors. The NPY expression vector contains one cassette to express NPY under a neuronal specific promoter. The microRNA expression vector contains one cassette with co-cistronic expression of EGFP and miR under the neuronal specific promoter. ITR, inverted terminal repeats; hSyn, human Synapsin promoter; WPRE, woodchuck hepatitis virus posttranslational control element; PolyA, polyadenosine sequence. Small human synapsin 1 gene promoter has shown to drive long-term neuron specific transgene expression from AAV-2 vectors [88]. (B) Three synthetic microRNA directed to different regions of NPY mRNA and one control microRNA (random sequence) were cloned. (C) Screen of microRNA targeting NPY. Representative image of NPY expression and densitometry band evaluation normalized to α-tubulin and shown as percentage of miR-ctr. Cells co-transfected with the NPY vector and one of the miR-NPY vectors, in a 1∶1 ratio. NPY expression is significantly reduced with the miR-NPY-3 vector. n = 3 independent cell transfection; * p<0.05 compared to miR-ctr control. (D) Transduction of NPY positive neurons (red) by AAV-hSyn-miR cassette (EGFP-green) in primary neuronal culture. Scale bar, 20 µm.

Mentions: To over-express NPY in the ARC neurons we cloned the Npy cDNA into the AAV back-bone (Fig. 1A). To down-regulate the NPY expression we cloned three synthetic microRNAs (miR) targeting different sites of the NPY mRNA (Fig. 1B) into the AAV backbone (Fig. 1A). Additionally, we cloned one microRNA with a random sequence into the AAV backbone to be used as control (miR-ctr).


Moderate long-term modulation of neuropeptide Y in hypothalamic arcuate nucleus induces energy balance alterations in adult rats.

Sousa-Ferreira L, Garrido M, Nascimento-Ferreira I, Nobrega C, Santos-Carvalho A, Alvaro AR, Rosmaninho-Salgado J, Kaster M, Kügler S, de Almeida LP, Cavadas C - PLoS ONE (2011)

In vitro validation of NPY vector and synthetic microRNA for down regulation of NPY expression.(A) Schematic representation of the AAV vectors. The NPY expression vector contains one cassette to express NPY under a neuronal specific promoter. The microRNA expression vector contains one cassette with co-cistronic expression of EGFP and miR under the neuronal specific promoter. ITR, inverted terminal repeats; hSyn, human Synapsin promoter; WPRE, woodchuck hepatitis virus posttranslational control element; PolyA, polyadenosine sequence. Small human synapsin 1 gene promoter has shown to drive long-term neuron specific transgene expression from AAV-2 vectors [88]. (B) Three synthetic microRNA directed to different regions of NPY mRNA and one control microRNA (random sequence) were cloned. (C) Screen of microRNA targeting NPY. Representative image of NPY expression and densitometry band evaluation normalized to α-tubulin and shown as percentage of miR-ctr. Cells co-transfected with the NPY vector and one of the miR-NPY vectors, in a 1∶1 ratio. NPY expression is significantly reduced with the miR-NPY-3 vector. n = 3 independent cell transfection; * p<0.05 compared to miR-ctr control. (D) Transduction of NPY positive neurons (red) by AAV-hSyn-miR cassette (EGFP-green) in primary neuronal culture. Scale bar, 20 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3142146&req=5

pone-0022333-g001: In vitro validation of NPY vector and synthetic microRNA for down regulation of NPY expression.(A) Schematic representation of the AAV vectors. The NPY expression vector contains one cassette to express NPY under a neuronal specific promoter. The microRNA expression vector contains one cassette with co-cistronic expression of EGFP and miR under the neuronal specific promoter. ITR, inverted terminal repeats; hSyn, human Synapsin promoter; WPRE, woodchuck hepatitis virus posttranslational control element; PolyA, polyadenosine sequence. Small human synapsin 1 gene promoter has shown to drive long-term neuron specific transgene expression from AAV-2 vectors [88]. (B) Three synthetic microRNA directed to different regions of NPY mRNA and one control microRNA (random sequence) were cloned. (C) Screen of microRNA targeting NPY. Representative image of NPY expression and densitometry band evaluation normalized to α-tubulin and shown as percentage of miR-ctr. Cells co-transfected with the NPY vector and one of the miR-NPY vectors, in a 1∶1 ratio. NPY expression is significantly reduced with the miR-NPY-3 vector. n = 3 independent cell transfection; * p<0.05 compared to miR-ctr control. (D) Transduction of NPY positive neurons (red) by AAV-hSyn-miR cassette (EGFP-green) in primary neuronal culture. Scale bar, 20 µm.
Mentions: To over-express NPY in the ARC neurons we cloned the Npy cDNA into the AAV back-bone (Fig. 1A). To down-regulate the NPY expression we cloned three synthetic microRNAs (miR) targeting different sites of the NPY mRNA (Fig. 1B) into the AAV backbone (Fig. 1A). Additionally, we cloned one microRNA with a random sequence into the AAV backbone to be used as control (miR-ctr).

Bottom Line: Neuropeptide Y (NPY) produced by arcuate nucleus (ARC) neurons has a strong orexigenic effect on target neurons.We achieved a physiological overexpression (3.6-fold increase) and down-regulation (0.5-fold decrease) of NPY in the rat ARC by injection of AAV vectors expressing NPY and synthetic microRNA that target the NPY, respectively.In addition, moderate down-regulation of NPY did not affect basal feeding or normal body weight gain but the response to food deprivation was compromised since fasting-induced hyperphagia was inhibited and fasting-induced decrease in locomotor activity was absent.These results highlight the importance of the physiological ARC NPY levels oscillations on feeding regulation, fasting response and body weight preservation, and are important for the design of therapeutic interventions for obesity that include the NPY.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.

ABSTRACT
Neuropeptide Y (NPY) produced by arcuate nucleus (ARC) neurons has a strong orexigenic effect on target neurons. Hypothalamic NPY levels undergo wide-ranging oscillations during the circadian cycle and in response to fasting and peripheral hormones (from 0.25 to 10-fold change). The aim of the present study was to evaluate the impact of a moderate long-term modulation of NPY within the ARC neurons on food consumption, body weight gain and hypothalamic neuropeptides. We achieved a physiological overexpression (3.6-fold increase) and down-regulation (0.5-fold decrease) of NPY in the rat ARC by injection of AAV vectors expressing NPY and synthetic microRNA that target the NPY, respectively. Our work shows that a moderate overexpression of NPY was sufficient to induce diurnal over-feeding, sustained body weight gain and severe obesity in adult rats. Additionally, the circulating levels of leptin were elevated but the immunoreactivity (ir) of ARC neuropeptides was not in accordance (POMC-ir was unchanged and AGRP-ir increased), suggesting a disruption in the ability of ARC neurons to response to peripheral metabolic alterations. Furthermore, a dysfunction in adipocytes phenotype was observed in these obese rats. In addition, moderate down-regulation of NPY did not affect basal feeding or normal body weight gain but the response to food deprivation was compromised since fasting-induced hyperphagia was inhibited and fasting-induced decrease in locomotor activity was absent.These results highlight the importance of the physiological ARC NPY levels oscillations on feeding regulation, fasting response and body weight preservation, and are important for the design of therapeutic interventions for obesity that include the NPY.

Show MeSH
Related in: MedlinePlus