Neuropeptide Y Peptide Levels Research Articles

Sibutramine reduces feeding, body fat and improves insulin resistance in dietary-obese male Wistar rats independently of hypothalamic neuropeptide Y.

We studied the effects of the novel noradrenaline and serotonin (5-HT) reuptake inhibitor sibutramine on feeding and body weight in a rat model of dietary obesity, and whether it interacts with hypothalamic neuropeptide Y (NPY) neurones. Chow-fed and dietary-obese (DIO) male Wistar rats were given sibutramine (3 mg kg(-1) day(-1) p.o.) or deionized water for 21 days. Sibutramine decreased food intake throughout the treatment period in both dietary-obese rats (P<0.0001) and lean rats (P<0.0001). Weight gain was reduced so that final body weight was 10% lower in dietary-obese (P<0.005) and 8% lower in lean (P<0.05) rats versus their untreated controls. Plasma leptin concentration was lower in sibutramine-treated dietary-obese rats (P<0.05), and in treated lean rats (P<0.05). Using the homeostasis model assessment (HOMA) as a measure of insulin resistance, untreated DIO rats were significantly more insulin resistant than controls (P<0.005), and this was corrected by sibutramine treatment (P<0.05). Neither hypothalamic NPY mRNA nor NPY peptide levels in a number of hypothalamic nuclei were significantly altered by sibutramine compared to untreated controls. The hypophagic and anti-obesity effects of sibutramine in dietary-obese Wistar rats appear not to be mediated by inhibition of ARC NPY neurones.

Zinc Deficiency Increases Hypothalamic Neuropeptide Y and Neuropeptide Y mRNA Levels and Does Not Block Neuropeptide Y–Induced Feeding in Rats

Zinc deficiency reduces intake and produces an unusual ∼3.5-d cycle of intake in rats. The mechanism underlying the anorexia and cycling has not yet been defined; current hypotheses suggest that alterations in amino acid metabolism and neurotransmitter concentrations may be a part of this anorexia. Recent reports indicate that appetite-stimulating neuropeptide Y (NPY) may be elevated during zinc deficiency. This suggests that a resistance to NPY may exist during zinc deficiency because NPY levels are high, yet appetite is low. The purpose of this study was to measure NPY peptide and mRNA concentrations during zinc deficiency in specific nuclei of the hypothalamus in which peptide and mRNA for NPY are known to be associated with appetite, and also to determine whether zinc-deficient rats are responsive to central infusions of NPY. Both NPY peptide levels in the paraventricular nucleus and NPY mRNA levels in the arcuate nucleus were higher (P < 0.05) in zinc-deficient rats than in zinc-adequate rats. When rats were administered exogenous NPY to the paraventricular nucleus, both zinc-deficient and zinc-adequate rats responded similarly by increasing food intake. These results suggest that NPY is elevated during zinc deficiency in an attempt to restore normal food intake levels, rather than being reduced and thereby contributing to the anorexia associated with zinc deficiency. During zinc deficiency, NPY receptors are able to bind NPY and initiate an orexigenic response.

Neuropeptide Y gene expression in immortalized rat hippocampal and pheochromocytoma-12 cell lines

Employing clonal cell lines derived from rat embryonic hippocampal cells, we detected neuropeptide Y (NPY) mRNA in three progenitor subcloned cell lines. These cell lines upon differentiation express markers indicative of commitment to either neuronal (H19-7; NF+, GFAP−), glial (H19-5; GFAP+, NF−), or bipotential (H583-5; NF+, GFAP+) lineages. Induction of differentiation was associated with the persistence of the NPY mRNA, however, in the differentiated H19-7 cells a 20-fold increase in NPY mRNA levels was observed ( P<0.05). NPY immunoreactivity was observed only in cells with a differentiated neuronal phenotype. The cellular radioimmunoassayable NPY peptide levels increased twelve-fold without a change in extracellular NPY peptide levels by multi-factorially induced neuronal or glial cell differentiation. The differentiated H19-5 cells expressed lower levels of NPY that could not be immunocytochemically detected. The peripheral sympathetic PC-12 neuronal cells examined in the undifferentiated and nerve growth factor-driven differentiated states expressed NPY only upon differentiation. We conclude that NPY is expressed by the cultured undifferentiated and differentiated rat hippocampal clonal cell lines, while the peripheral sympathetic PC-12 neuronal cell line only expresses the NPY gene upon differentiation. These immortalized embryonic neural cell line(s) will provide a hippocampal cell line(s) to conduct future in-vitro investigations targeted at determining the cellular and molecular mechanisms governing NPY gene expression.

Maternal diabetes-induced hyperglycemia and acute intracerebral hyperinsulinism suppress fetal brain neuropeptide Y concentrations.

We examined the effect of streptozotocin-induced maternal diabetes of 6-day duration and 4- to 24-h intracerebroventricular and systemic hyperinsulinism on fetal brain neuropeptide Y (NPY) synthesis and concentrations. Maternal diabetes (n = 6) leading to fetal hyperglycemia (5-fold increase; P < 0.05) and normoinsulinemia caused a 40% decline (P < 0.05) in fetal brain NPY messenger RNA (mRNA) and a 50% decline (P < 0.05) in NPY radioimmunoassayable levels compared to levels in streptozotocin-treated nondiabetic (n = 7) and vehicle-treated control (n = 8) animals. In contrast, systemic hyperinsulinemia (n = 7) of 5- to 100-fold increase (P < 0.05) over the respective control (n = 7) with normoglycemia caused an insignificant (20-30%) decrease in fetal brain NPY mRNA and protein concentrations. However, fetal intracerebroventricular hyperinsulinism (n = 7) with no change in fetal glucose concentrations caused a 50-60% decline (P < 0.05) in only the NPY peptide levels, with no change in the corresponding mRNA amounts. We conclude that fetal hyperglycemia of 6-day duration and intracerebroventricular hyperinsulinism of 4-24 h suppress fetal brain NPY concentrations, the former by a pretranslational and the latter by either a translational/posttranslational mechanism or depletion of intracellular secretory stores. We speculate that fetal hyperglycemia and intracerebroventricular hyperinsulinism additively can inhibit various intrauterine and immediate postnatal NPY-mediated biological functions.

IMMORTALIZED FETAL RAT NEURONAL CELLS SYNTHESIZE NEUROPEPTIDE Y.† 384

Neuropeptide Y (NPY), an orexigenic peptide synthesized in-vivo within the arcuate nucleus and released in the paraventricular nucleus, causes hyperphagia and anxiolysis. To determine the presence of NPY synthesis and secretion by fetal rat neural cells that were immortalized by the temperature sensitive large T antigen, we undertook the present study. Using undifferentiated (33°C, serum-containing medium) and differentiated(39°C, serum-free defined medium) neural cells predominantly expressing neuronal (neurofilament [NF] positive, glial fibrillary acidic protein [GFAP] negative), and glial (NF -ve, GFAP +ve) phenotypes, we characterized the presence of NPY mRNA by Northern blot analysis (n=2), cellular peptide by immunohistochemical analysis (n=4), and extracellular secretion of the NPY peptide over 20 min by radioimmunoassay of Kreb's buffer conditioned medium(n=10). Differentiation of neuronal and glial enriched cell cultures led to a 24-fold and 13-fold increase in NPY mRNA (0.8 kb) concentrations respectively(p <0.01). A similar change was noted in the number of cells expressing NPY immunoreactivity, this immunoreactivity being noted only in well differentiated neuronal cells and their processes. The increase in NPY immunoreactive cell number with differentiation was not paralleled by an increase in the extracellularly secreted NPY peptide levels. Employing 10μm of antisense oligonucleotide complementary to 20 bp downstream starting from the ATG-translation start site codon of the rat preproNPY gene, the number of NPY immunoreactive neuronal cells diminished 6-fold when compared to corresponding sense and missense oligonucleotide treated cells (n=3 each; p.<0.05). We conclude that 1] immortalized fetal neuronal cells synthesize and actively secrete NPY, the rate of synthesis being higher in differentiated cells, and 2] appropriately synthesized antisense oligonucleotides suppress neuronal NPY peptide production. These in-vitro studies will set the stage for undertaking in-vivo selective neuronal knock out of the hypothalamic NPY, thereby allowing phenotypic characterization of postnatal animals in the absence of hypothalamic NPY production.

Central administration of antisense oligodeoxynucleotides to neuropeptide Y (NPY) mRNA reveals the critical role of newly synthesized NPY in regulation of LHRH release

Compelling evidence shows that the episodic and cyclic secretion of hypothalamic luteinizing hormone releasing hormone (LHRH), the primary stimulator of pituitary LH release, is subject to regulation by neuropeptide Y (NPY). We have reported earlier that sequential treatment of ovariectomized (ovx) rats with estrogen and progesterone to stimulate a preovulatory-type LH surge elevated the levels of both NPY and preproNPY mRNA levels in the hypothalamus concomitant with dynamic changes in LHRH activity. The present study was designed to determine whether these elevations in NPY content and gene expression represent new synthesis of NPY that is crucial to elicit LHRH discharge. Ovx, steroid-primed rats received intracerebroventricular injections of an unmodified 20-mer oligodeoxynucleotide (oligo) complementary to the NPY mRNA sequence. Control rats were injected similarly with either saline or the sense or missense oligos. Results showed that control rats displayed a characteristic surge-type elevation in plasma LH levels that was not affected by the administration of missense or sense oligos. However, in rats injected with the antisense oligo, the steroid-induced LH surge was completely blocked. In an additional experiment, NPY peptide levels were measured in microdissected hypothalamic sites following the injection of antisense or missense oligos. NPY antisense oligo administration blocked the significant increases in NPY levels in the median eminence-arcuate area, the medial preoptic area and lateral preoptic area seen in control rats. These results suggest that sequential ovarian steroid treatment augments NPY synthesis in the hypothalamus and this newly synthesized NPY is critical for induction of the LHRH and LH surge.

Nicotine administration reduces neuropeptide Y and neuropeptide Y mRNA concentrations in the rat hypothalamus: NPY may mediate nicotine's effects on energy balance

Neuropeptide Y (NPY) is synthesized in arcuate (ARC) neurons which project principally to the paraventricular nucleus (PVN). NPY injected into the PVN causes hyperphagia, reduced energy expenditure and eventually obesity, effects which are opposed by nicotine. We aimed to investigate whether nicotine's effects on energy balance might be mediated by inhibition of hypothalamic NPYergic neurons. Nicotine or saline was given for 1 or 12 days using osmotic minipumps, and additional groups of rats were food-restricted to the intake of the nicotine-treated groups to allow for the effects of hypophagia on hypothalamic NPY. One day's nicotine treatment (12 mg/kg/day) reduced food intake by 30% ( P < 0.001) and body weight by 2% ( P < 0.01 vs. controls). NPY mRNA levels were significantly reduced by 40% ( P < 0.05) and NPY concentrations fell significantly by 33% in the ARC and PVN (both P < 0.01). Matched food restriction also reduced NPY levels significantly in the ARC and PVN ( P < 0.02 vs. controls) but had no effect on NPY mRNA. 12 days' nicotine treatment (12 mg/kg/day) lowered cumulative food intake by 8% ( P = 0.02) and body weight by 10% ( P < 0.05). NPY mRNA levels rose by 40% ( P < 0.05), while NPY levels again fell in the ARC and PVN (both P < 0.05). Food restriction, which induced weight loss comparable with that during nicotine treatment, increased NPY mRNA to levels that were 100% above controls ( P < 0.01) and also significantly higher than in the nicotine-treated group ( P < 0.05). Food restriction also reduced NPY peptide levels in the PVN ( P < 0.02), but did not affect those in the ARC. In addition, 12 days' nicotine treatment significantly reduced plasma insulin levels compared with controls ( P < 0.05). We suggest that nicotine may inhibit NPY synthesis in the hypothalamus, independently of any effects due to altered energy balance. Reduced activity of NPYergic neurons in the ARC-PVN projection may mediate the effects of nicotine on energy balance.

Hypothalamic Neuropeptide Y and Its Gene Expression: Relation to Light/Dark Cycle and Circulating Corticosterone

The hypothalamic neuropeptide Y (NPY) system, along with levels of circulating corticosterone (CORT), were examined in rats at different times across the light/dark cycle. Tissue samples were taken from the mediobasal hypothalamus (MBH), which contains the primary hypothalamic NPY cell group of the arcuate nucleus (ARC), and the mediodorsal (MDH) hypothalamus, which contains the paraventricular and dorsomedial nuclei that receive a dense NPY innervation from the ARC. In these dissections, measurements of NPY mRNA and peptide levels were taken using a solution hybridization/nuclease protection assay procedure and radioimmunoassay. The results demonstrate that (i) NPY mRNA levels in the MBH, but not MDH, vary significantly in relation to the light/dark cycle, showing a sharp rise 4-6 h before dark onset, sustained high levels over the next 3-4 h and then, a sharp decline 1 h before dark onset; (ii) this rise in NPY mRNA in the MBH before dark onset, while associated with stable levels of MBH NPY during this time, is followed 2-4 h later, around dark onset, by a rise in NPY peptide levels of the MDH simultaneous to a decrease in NPY levels of the MBH; (iii) levels of circulating CORT shift dramatically across the light-dark cycle, exhibiting an increase from basal levels (<0.3 μg/dl) to 5 μg/dl approximately 4 h before dark onset, a further rise that peaks at 26 μg/dl around dark onset, and then a significant decline to 16 μg/dl at 2 h after dark onset; and (iv) there exists a positive relationship between CORT and NPY mRNA or peptide levels in the MBH during the 4-6 h before dark onset, while in the MDH, a positive relationship between this steroid and NPY peptide levels is obtained at dark onset. It is proposed that these rhythms, involving a predark rise in CORT and NPY gene expression leading to a peak in CORT and peptide levels at dark onset, are active in stimulating feeding behavior, particularly carbohydrate ingestion, which predominates at that time.