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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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ARC NPY overexpression increases adipocytes size and decreases PPAR-gamma-2 adipogenic marker in white adipose tissue.Evaluation of adipocytes size and differentiation markers (Peroxisome Proliferator Activated Receptor type 2, PPAR-gamma-2), eight weeks after AAV injection. (A) Representative images of adipocytes auto-fluorescence from ARC-miR-ctr, ARC-miR-NPY and ARC-NPY groups. Scale bar, 100 µm. (B) Adipocytes size distribution represented as percentage of total number of adipocytes. n = 6 rats per group; ns, non-significant, ** p<0.01; *** p<0.001 compared to ARC-miR-ctr group size range. (C) Evaluation of PPAR-gamma isoforms in epididymal adipose tissue extracts by immunobloting. Representative images of protein level. The expression of PPAR-gamma-2 isoform is lower on ARC-NPY obese rats, compared to ARC-miR-ctr rats. (D) Densitometry band evaluation normalized to β-actin and shown as percentage of ARC-miR-ctr. n = 3/4 rats per group; ns p>0.5; * p<0.5, compared to ARC-miR-ctr group. ir, immunoreactivity.
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pone-0022333-g004: ARC NPY overexpression increases adipocytes size and decreases PPAR-gamma-2 adipogenic marker in white adipose tissue.Evaluation of adipocytes size and differentiation markers (Peroxisome Proliferator Activated Receptor type 2, PPAR-gamma-2), eight weeks after AAV injection. (A) Representative images of adipocytes auto-fluorescence from ARC-miR-ctr, ARC-miR-NPY and ARC-NPY groups. Scale bar, 100 µm. (B) Adipocytes size distribution represented as percentage of total number of adipocytes. n = 6 rats per group; ns, non-significant, ** p<0.01; *** p<0.001 compared to ARC-miR-ctr group size range. (C) Evaluation of PPAR-gamma isoforms in epididymal adipose tissue extracts by immunobloting. Representative images of protein level. The expression of PPAR-gamma-2 isoform is lower on ARC-NPY obese rats, compared to ARC-miR-ctr rats. (D) Densitometry band evaluation normalized to β-actin and shown as percentage of ARC-miR-ctr. n = 3/4 rats per group; ns p>0.5; * p<0.5, compared to ARC-miR-ctr group. ir, immunoreactivity.

Mentions: We assessed possible phenotype alterations on the white adipose tissue (WAT) upon modulation of ARC NPY by evaluation of adipocytes size and differentiation marker Peroxisome Proliferator Activated Receptor type 2 (PPAR-gamma-2), eight weeks after AAV injection. Sections from epididymal fat pad were analyzed by fluorescence microscopy (Fig. 4A) and adipocytes average diameter was measured and grouped by size range (Fig. 4B). The distribution of adipocytes size was not different between the ARC-miR-ctr and ARC-miR-NPY group. However, the obese ARC-NPY group showed a higher percentage of large adipocytes and a reduced percentage of small adipocytes, compared to lean ARC-miR-ctr group (large adipocytes: range of 140–180 µm 34.6±6.3% and 0.8±0.6%, respectively and range of 180–220 µm 19.7±7.5% and 0.0%, respectively; small adipocytes: range of 60–100 µm 11.9±3.8% and 57.2±14.6%, respectively).


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)

ARC NPY overexpression increases adipocytes size and decreases PPAR-gamma-2 adipogenic marker in white adipose tissue.Evaluation of adipocytes size and differentiation markers (Peroxisome Proliferator Activated Receptor type 2, PPAR-gamma-2), eight weeks after AAV injection. (A) Representative images of adipocytes auto-fluorescence from ARC-miR-ctr, ARC-miR-NPY and ARC-NPY groups. Scale bar, 100 µm. (B) Adipocytes size distribution represented as percentage of total number of adipocytes. n = 6 rats per group; ns, non-significant, ** p<0.01; *** p<0.001 compared to ARC-miR-ctr group size range. (C) Evaluation of PPAR-gamma isoforms in epididymal adipose tissue extracts by immunobloting. Representative images of protein level. The expression of PPAR-gamma-2 isoform is lower on ARC-NPY obese rats, compared to ARC-miR-ctr rats. (D) Densitometry band evaluation normalized to β-actin and shown as percentage of ARC-miR-ctr. n = 3/4 rats per group; ns p>0.5; * p<0.5, compared to ARC-miR-ctr group. ir, immunoreactivity.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3142146&req=5

pone-0022333-g004: ARC NPY overexpression increases adipocytes size and decreases PPAR-gamma-2 adipogenic marker in white adipose tissue.Evaluation of adipocytes size and differentiation markers (Peroxisome Proliferator Activated Receptor type 2, PPAR-gamma-2), eight weeks after AAV injection. (A) Representative images of adipocytes auto-fluorescence from ARC-miR-ctr, ARC-miR-NPY and ARC-NPY groups. Scale bar, 100 µm. (B) Adipocytes size distribution represented as percentage of total number of adipocytes. n = 6 rats per group; ns, non-significant, ** p<0.01; *** p<0.001 compared to ARC-miR-ctr group size range. (C) Evaluation of PPAR-gamma isoforms in epididymal adipose tissue extracts by immunobloting. Representative images of protein level. The expression of PPAR-gamma-2 isoform is lower on ARC-NPY obese rats, compared to ARC-miR-ctr rats. (D) Densitometry band evaluation normalized to β-actin and shown as percentage of ARC-miR-ctr. n = 3/4 rats per group; ns p>0.5; * p<0.5, compared to ARC-miR-ctr group. ir, immunoreactivity.
Mentions: We assessed possible phenotype alterations on the white adipose tissue (WAT) upon modulation of ARC NPY by evaluation of adipocytes size and differentiation marker Peroxisome Proliferator Activated Receptor type 2 (PPAR-gamma-2), eight weeks after AAV injection. Sections from epididymal fat pad were analyzed by fluorescence microscopy (Fig. 4A) and adipocytes average diameter was measured and grouped by size range (Fig. 4B). The distribution of adipocytes size was not different between the ARC-miR-ctr and ARC-miR-NPY group. However, the obese ARC-NPY group showed a higher percentage of large adipocytes and a reduced percentage of small adipocytes, compared to lean ARC-miR-ctr group (large adipocytes: range of 140–180 µm 34.6±6.3% and 0.8±0.6%, respectively and range of 180–220 µm 19.7±7.5% and 0.0%, respectively; small adipocytes: range of 60–100 µm 11.9±3.8% and 57.2±14.6%, respectively).

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