Y1 Receptor mRNA Expression Research Articles

Regulation of neuropeptide Y Y1 receptor expression by bone morphogenetic protein 2 in C2C12 myoblasts

The neuropeptide Y (NPY) system is known as one of the major neural signaling pathways. NPY, produced by peripheral tissues including osteoblasts, is known to bind to the Y1 receptor. Recently, osteoblast-specific Y1 receptor knockout mice were developed and were found to have a high bone mass phenotype, indicating a role for the NPY-Y1 receptor axis as a regulator of bone homeostasis. However, regulation of Y1 receptor expression during osteoblastic differentiation remains unexplored. In the present study, we examined the role of bone morphogenetic protein (BMP) 2 signaling in regulating Y1 receptor expression. In C2C12 cells, expression of Y1 receptor mRNA was induced by BMP2. This induction was also observed after co-transfection with Smad1 and Smad4, the intracellular signaling molecules of the BMP2 signaling pathway. In a transfection assay, Smad1/4 up-regulated transcriptional activity through interaction with the Y1 receptor gene promoter. Following transfection of MC3T3-E1 cells with siRNA for the Y1 receptor, the expression of alkaline phosphatase, osteocalcin, Runx2 and osterix were increased. These results show that BMP2 signaling regulates Y1 receptor gene expression, and raises the possibility that NPY acts in osteoblasts via an autocrine mechanism.

Expressions of neuropeptide Y and Y1 receptor in subcutaneous and visceral fat tissues in normal weight and obese humans and their correlations with clinical parameters and peripheral metabolic factors

Recently, neuropeptide Y (NPY) and Y1 receptor (Y1R) were found to be expressed and synthesized in adipose tissue. This study aimed to compare NPY and Y1R mRNA expressions in subcutaneous and visceral fat tissues as well as serum NPY in normal weight and obese humans and their correlations with clinical parameters and peripheral metabolic factors. We demonstrated that NPY mRNA expression was higher in obese than in normal weight humans (p<0.05) in both subcutaneous and visceral adipose tissues and was significantly greater in visceral when compared with subcutaneous fat in overall (p<0.01), obese (p<0.05) and normal weight humans (p<0.05). Y1R mRNA expression was higher in obese than normal weight subjects in visceral (p<0.01) but not in subcutaneous adipose tissue and was statistically greater in subcutaneous when compared to visceral adipose tissue in obese (p<0.05) and overall subjects (p<0.05). Serum NPY was higher in obese than normal weight groups (p<0.05). Obese subjects showed significantly greater levels of systolic blood pressure (SBP) (p<0.01), diastolic blood pressure (DBP) (p<0.05), plasma insulin (p<0.05), and HOMA-IR (p<0.05) when compared with normal weight subjects. Additionally, Y1R mRNA expression in visceral adipose tissue was positively correlated with body weight (R=0.586), BMI (R=0.611), waist (R=0.474) and hip (R=0.483) circumferences, insulin levels (R=0.539), and HOMA-IR (R=0.480). As the result, Y1R expression in visceral adipose tissue might be an indicator of increased risk of metabolic syndrome. Further studies about blocking specific Y1R may propose strategies for risk reduction in metabolic syndrome and prevention or treatment of obesity.

Estrogen Up-regulates Neuropeptide Y Y1 Receptor Expression in a Human Breast Cancer Cell Line

Normal breast tissue mainly expresses the neuropeptide Y (NPY) Y2 receptor whereas primary human breast carcinomas express the Y1 receptor (Y1R) subtype. We hypothesized that activation of estrogen signaling systems plays a role in the induction of Y1R. To investigate this possibility, we used estrogen receptor-positive (ER+) human breast carcinoma cell line, MCF-7, and examined the effect of estrogen on Y1R gene expression and its signaling pathways. Saturation binding studies revealed that MCF-7 cells express high-affinity NPY receptor. NPY inhibited forskolin-stimulated adenosine 3'5'-cyclic monophosphate (cAMP) accumulation and mobilized intracellular Ca(2+) in MCF-7 cells. Chronic estrogen treatment enhanced NPY-mediated inhibition of cAMP accumulation by 4-fold and caused a significant increase in Y1R mRNA expression through ERalpha. Similarly, estrogen increased Y1R mRNA expression in T-47D (ER+) but not in MDA-MB231 or MDA-MB468 (ER-) cell lines. Cycloheximide decreased basal Y1R mRNA expression; however, it did not affect its increase by estrogen. Moreover, estrogen treatment of MCF-7 cells did not increase Y1R mRNA stability. The up-regulation of Y1R expression by estrogen is prevented by hydroxyurea but not by nocodazole or IB-MECA (cell cycle inhibitors). Lastly, NPY inhibited estrogen-induced cell proliferation through Y1R. In conclusion, MCF-7 cells express a functional Y1R coupled to both Ca(2+) and cAMP pathways. Estrogen up-regulates Y1R expression through ERalpha. This effect is independent of increased Y1R mRNA stability or new protein synthesis, and likely occurs during S phase completion of the cell cycle. Estrogen plays an important role in the up-regulation of Y1R, which in turn regulates estrogen-induced cell proliferation in breast cancer cells.

Change in the expression of NPY receptor subtypes Y1 and Y2 in central and peripheral neurons related to the control of blood pressure in rats following experimental hypertension

Neuropeptide Y (NPY) is known to participate in central mechanisms of blood pressure control. However, variations on the expression of its receptors in response to a hypertensive challenge are not well defined, specially when considering that Y1 and Y2 often mediate opposite responses. In this study we have employed in situ hybridization to analyze changes in mRNA expression of NPY receptor subtypes Y1 and Y2 in the nucleus tractus solitarii (NTS), paraventricular nucleus of the hypothalamus (PVN) and petrosal and nodose ganglions 2 h, 3 and 7 days after aortic coarctation induced hypertension. Quantification by image analysis showed significant differences between sham-operated and aortic-coarcted hypertensive rats. Y1 receptor mRNA expression was increased (39%) in petrosal ganglion, 3 days after surgery. Y2 receptor mRNA expression was increased (143%) in the NTS of hypertensive compared with sham rats 2 h after surgery. Y2 receptor mRNA was decreased (62%) in the nodose ganglion of hypertensive compared with sham rats 2 h after surgery. No change was seen in Y1 and Y2 mRNA expression in the PVN in any analyzed period. The data suggest that NPY Y1 and Y2 receptors might participate in the mechanisms involved in the establishment/maintenance of hypertension induced by aortic coarctation. Acute changes seem to be involved with the adaptation to the new hypertensive state.

Estrogen Induces Neuropeptide Y (NPY) Y1 receptor gene expression and responsiveness to NPY in gonadotrope-enriched pituitary cell cultures.

We showed previously that neuropeptide Y1 receptor (Y1R) expression is increased in the hypothalamus on proestrus afternoon and that this up-regulation of Y1R mRNA may permit neuropeptide Y (NPY) to facilitate release of the preovulatory GnRH surge. Because NPY also modulates LH release directly, we examined steroid regulation of Y1R expression in the female rat anterior pituitary. Treatment of female rats with estrogen in vivo decreased the levels of Y1R mRNA in the whole pituitary gland. In lactotrope/somatotrope-enriched pituitary cells separated by unit gravity sedimentation, 17beta-estradiol (E(2)) treatment likewise suppressed Y1R expression. In contrast, E(2) elevated Y1R mRNA in gonadotrope-enriched cell populations, indicating that estrogen regulates Y1R mRNA expression differently in gonadotropes vs. other pituitary cell types. After exposure to E(2), NPY augmented GnRH-induced LH release from gonadotrope-enriched cells in a manner requiring Y1R activation. Without steroid exposure, this augmentation disappeared, and with progesterone alone, NPY reduced GnRH-induced LH release. In addition, NPY inhibited prolactin secretion from primary pituitary cells in a steroid-free environment, but not in the presence of estrogen. These findings demonstrate that E(2) can directly up-regulate gonadotrope responsiveness to NPY and suggest that this action is mediated at least in part by E(2)'s ability to stimulate Y1R gene expression in gonadotropes. Our observations are consistent with the idea that this regulatory mechanism represents a component of E(2)'s positive feedback actions in pituitary gonadotropes. The biological importance of E(2)'s opposite effects on Y1R expression in other pituitary cell types remains to be determined.

Neuropeptide Y Y1 receptor mRNA in rodent brain: Distribution and colocalization with melanocortin-4 receptor

The central neuropeptide Y (NPY) Y1 receptor (Y1-R) system has been implicated in feeding, endocrine, and autonomic regulation. In the present study, we systematically examined the brain distribution of Y1-R mRNA in rodents by using radioisotopic in situ hybridization histochemistry (ISHH) with a novel sensitive cRNA probe. Within the rat hypothalamus, Y1-R-specific hybridization was observed in the anteroventral periventricular, ventromedial preoptic, suprachiasmatic, paraventricular (PVH), dorsomedial, ventromedial, arcuate, and mamillary nuclei. In the rat, Y1-R mRNA expression was also seen in the subfornical organ, anterior hypothalamic area, dorsal hypothalamic area, and in the lateral hypothalamic area. In addition, Y1-R hybridization was evident in several extrahypothalamic forebrain and hindbrain sites involved in feeding and/or autonomic regulation in the rat. A similar distribution pattern of Y1-R mRNA was observed in the mouse brain. Moreover, by using a transgenic mouse line expressing green fluorescent protein under the control of the melanocortin-4 receptor (MC4-R) promoter, we observed Y1-R mRNA expression in MC4-R-positive cells in several brain sites such as the PVH and central nucleus of the amygdala. Additionally, dual-label ISHH demonstrated that hypophysiotropic PVH cells coexpress Y1-R and pro-thyrotropin-releasing hormone mRNAs in the rat. These observations are consistent with the proposed roles of the central NPY/Y1-R system in energy homeostasis.

Demonstration of postsynaptic receptor plasticity in an amphibian neuroendocrine interface.

Pituitary pars intermedia melanotrope cells are often used as a model to study mechanisms of neuroendocrine integration. In the amphibian Xenopus laevis, the synthesis and release of alpha-melanophore-stimulating hormone (alpha-MSH) from these cells is a dynamic process dependent upon the colour of background. In animals on a black background, there is a higher level of synthesis and secretion of alpha-MSH than in animals on a white background, and, consequently, there is skin darkening in animals on a black background. The melanotropes are innervated by hypothalamic neurones that produce neuropeptide Y (NPY), a peptide that inhibits alpha-MSH secretion via the NPY Y1 receptor. The inhibitory neurones have a higher expression of NPY in animals adapted to a white background and both the size and the number of inhibitory synapses on the melanotrope cells are enhanced. The purpose of the present study was to determine if this presynaptic plasticity displayed by the inhibitory neurones is reciprocated by postsynaptic plasticity (i.e. if there is an enhanced expression of the Y1 receptor in melanotropes of animals adapted to a white background). For this purpose quantitative real-time reverse transcriptase-polymerase chain reaction was used to determine the level of Y1 receptor mRNA in melanotropes of animals undergoing the process of background adaptation. The results showed that there is a higher Y1 receptor mRNA expression in melanotropes of white-adapted animals. We conclude that the inhibitory neuroendocrine interface in the Xenopus pars intermedia displays postsynaptic plasticity in response to changes of background colour. To our knowledge, this is the first demonstration of a physiological environmental change leading to changes in postsynaptic receptor expression in a fully identified vertebrate neuroendocrine reflex.

Differential Expression of NPY and Its Receptors in Alcohol‐Preferring AA and Alcohol‐Avoiding ANA Rats

Central neuropeptide Y (NPY) is known to control feeding and stress responses. Recently, it has been suggested that NPY also has a role in regulation of alcohol consumption. NPY and NPY receptor expression in genetically selected alcohol-preferring (AA), alcohol-nonpreferring (ANA), and Wistar rats were investigated. Expression was assessed using in situ hybridization histochemistry with riboprobes specific for preproNPY, Y1, and Y2 receptors. Effects of central NPY administration on ethanol self-administration were also examined in AA, ANA, and Wistar rats by using oral operant self-administration. NPY mRNA expression was higher in ANA than in both AA and Wistar rats in the hippocampal CA region and dentate gyrus, whereas AA and Wistar did not differ from each other. No differences in NPY expression were found in the other regions analyzed: cingulate cortex, medial nucleus of the amygdala, arcuate, and paraventricular nuclei of the hypothalamus. Y1 receptor mRNA expression did not differ between the three lines. Y2 expression was higher in the dentate gyrus of both AA and ANA rats than in Wistar subjects. In the medial amygdala, Y2 mRNA was reduced in the AA line, compared to both ANA and Wistar rats. NPY injected intracerebroventricularly (1.5-3.0 nmol) did not affect operant ethanol self-administration in any of the three lines examined. The NPY system seems to differ in several respects between rat lines with different levels of alcohol preference. Differences observed within the hippocampus could be related to behavioral traits other than alcohol intake but it is also possible that elevated hippocampal expression of NPY in the ANA rats contributes to the low alcohol intake of this line. Aberrant NPY expression and/function within the amygdala complex could contribute to alcohol preference and constitute an anatomic substrate of the effects of NPY expression on alcohol intake observed previously in genetically modified animals.

Vascular neuropeptide Y Y1-receptors in the rat kidney: vasoconstrictor effects and expression of Y1-receptor mRNA

Neuropeptide Y (NPY) -receptor subtypes were studied in the rat kidney in vivo by systemic administration of NPY, the two agonists [Leu 31, Pro 34]NPY (Y1-receptor agonist) and NPY (13–36) (Y2-receptor agonist), or the Y1-receptor antagonist BIBP 3226. Effects on mean arterial blood pressure (MAP) and renal arterial blood flow were recorded. The Y1-receptor agonist evoked a dose-dependent increase in MAP concomitantly with a reduction in renal blood flow. At the largest dose administered (1.42 pmol/g), the Y1-agonist [Leu 31, Pro 34] NPY increased MAP by 20 ± 6 mmHg and reduced the renal vascular conductance by more than 50%. The same dose of the Y2-agonist NPY (13–36) did not evoke any clear-cut effects on the renal blood flow or MAP. Furthermore, administration of the Y1-receptor antagonist BIBP 3226 reduced the NPY-induced renal vasoconstriction, but did not affect the response to angiotensin II or phenylephrine. The effects evoked by 0.71 pmol/g NPY were almost abolished by 3 mg/kg BIBP 3226. In situ hybridization histochemistry was used to study the expression of Y1-receptor mRNA in the developing rat kidney. The levels of Y1-receptor mRNA expression in the vascular smooth muscle of the rat kidney varied at different ages, with low levels at postnatal day 10 and high levels at 20 days and again low levels at 40 days. In summary, the present study show a maturation-specific expression pattern of NPY Y1-receptor mRNA as well as functional effects of vascular NPY receptors of the Y1-subtype in the rat kidney.

Alterations in neuropeptide Y levels and Y1 binding sites in the Flinders Sensitive Line rats, a genetic animal model of depression

Previously, we observed specific alterations of neuropeptide Y (NPY) and Y1 receptor mRNA expression in discrete regions of the Flinders Sensitive Line rats (FSL), an animal model of depression. In order to clarify the correlation between mRNA expression and protein content, radioimmunoassay and receptor autoradiography were currently performed. In the FSL rats, NPY-like immunoreactivity (NPY-LI) was decreased in the hippocampal CA region, while Y1 binding sites were increased; NPY-LI was increased in the arcuate nucleus. Fluoxetine treatment elevated NPY-LI in the arcuate and anterior cingulate cortex and increased Y1 binding sites in the medial amygdala and occipital cortex in both strains. No differences were found regarding the Y2 binding sites. The results demonstrate a good correlation between NPY peptide and mRNA expression, and sustain the possible involvement of NPY and Y1 receptors in depression.

Expression of Neuropeptide Y Receptors mRNA and Protein in Human Brain Vessels and Cerebromicrovascular Cells in Culture

Neuropeptide Y (NPY) has been suggested as an important regulator of CBF. However, except for the presence of Y1 receptors in large cerebral arteries, little is known about its possible sites of action on brain vessels. In this study, we sought to identify the NPY receptors present in the human cerebrovascular bed. Specific Y1 receptor binding sites, localized on the smooth muscle of human pial vessels and potently competed by NPY, polypeptide YY (PYY), and the selective Y1 receptor antagonist BIBP 3226, were identified by quantitative radioautography of the Y1 radioligand [125I]-[Leu31, Pro34]-PYY. In contrast, no specific binding of the Y2-([125I]-PYY3-36) and Y4/Y5-(125I-human pancreatic polypeptide [hPP]) radioligands could be detected. By in situ hybridization, expression of Y1 receptor mRNA was restricted to the smooth muscle layer of pial vessels, whereas no specific signals were detected for either Y2, Y4, or Y5 receptors. Similarly, using reverse transcriptase-polymerase chain reaction (RT-PCR), mRNA for Y1 but not Y2, Y4, or Y5 receptors was consistently detected in isolated human pial vessels, intracortical microvessels, and capillaries. In human brain microvascular cells in culture, PCR products for the Y1 receptors were exclusively found in the smooth muscle cells. In cultures of human brain astrocytes, a cell type that associates intimately with brain microvessels, PCR products for Y1, Y2, and Y4 but not Y5 receptors were identified. Finally, NPY significantly inhibited the forskolin-induced cAMP production in smooth muscle but not in endothelial cell cultures. We conclude that smooth muscle Y1 receptors are the primary if not exclusive NPY receptors associated with human brain extraparenchymal and intraparenchymal blood vessels, where they most likely mediate cerebral vasoconstriction.

Alterations in neuropeptide Y and Y1 receptor mRNA expression in brains from an animal model of depression: region specific adaptation after fluoxetine treatment

To investigate the possible link between neuropeptide Y (NPY) and depression, we analyzed NPY and its receptors in different limbic-related regions in the Flinder sensitive line (FSL), a genetic animal model of depression. In situ hybridization histochemistry was used to measure mRNA expression levels of NPY and NPY receptors, Y1 and Y2, in the FSL as compared to the control Flinder resistant Line rats (FRL). In the FSL rats, NPY mRNA expression levels were significantly decreased in the nucleus accumbens and CA regions, but increased in the arcuate nucleus and anterior cingulate cortex. Y1 receptor mRNA expression was decreased in different cortical regions (retrosplenial, anterior cingulate, and occipital) and in the hippocampal dentate gyrus. Y2 mRNA expression levels did not differ between FSL and FRL animals. The effect of the antidepressant drug fluoxetine (a serotonin reuptake inhibitor) in the two rat strains was also studied. There was an increase of the NPY mRNA hybridization signal in the arcuate nucleus of both strains following the antidepressant treatment (10 μmol/kg; daily for 14 days). However, in other brain regions, fluoxetine administration caused a differential effect on the induction of NPY-related genes in the two rat strains: in the CA region and dentate gyrus NPY mRNA expression was increased in the FSL, but decreased in the FRL. In contrast, Y1 mRNA levels tended to be decreased by fluoxetine in the nucleus accumbens of the FSL rats, but increased in the FRL. These findings suggest an involvement of the Y1, but not the Y2, receptor subtype in depressive disorder. Overall, the results appear to sustain the importance of the FSL rats as an animal model of depression in view of the impairment of NPY genes and the ability of fluoxetine treatment to normalize NPY-related gene expression selectively in this strain.

Suppression of neuropeptide Y1 receptor function in SK-N-MC cells by nitric oxide.

The neuropeptide Y1 receptor (NPY1) predominantly mediates the vasoconstrictor effects of NPY in smooth muscle cells. The present experiments were planned to study the direct influence of the vasodilator nitric oxide (NO) on NPY1-receptor function. SK-N-MC and CHP-234 cells expressing Y1 and Y2 receptor, respectively, were incubated with the NO donors sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1), and S-nitroso-N-acetyl-penicillamine (SNAP). Receptor binding, Y1-receptor mRNA expression by Northern blot, and adenosine 3',5'-cyclic monophosphate (cAMP) and intracellular Ca2+ concentration ([Ca2+]i) responses were studied. SNP, SIN-1, and SNAP decreased normal binding of NPY to the NPY1 receptor in SK-N-MC cells in a concentration-dependent manner. SNP (500 microM), SIN-1 (1,000 microM), and SNAP (500 microM) significantly decreased binding to approximately 50%. The cell viability was not reduced. None of the NO donors affected Y2 receptor binding. Pretreatment with SNP significantly attenuated NPY-induced inhibition of cAMP formation in SK-N-MC cells but had no effect on CHP cells. The NPY-induced [Ca2+]i response was reduced to 50% by SNP pretreatment. NPY1 mRNA expression was reduced to one-third after SNAP treatment of SK-N-MC cells. In vitro, NPY1 receptor function of SK-N-MC cells is inhibited by NO-donor incubation on an mRNA level.

Localization of neuropeptide Y Y1 mRNA in the human brain: abundant expression in cerebral cortex and striatum.

Many neurobiological functions have been ascribed to the NPY Y1 receptor subtype, but autoradiographic analysis has failed to detect Y1 binding sites in most human brain areas, in contrast to the rat. We examined the regional distribution of Y1 mRNA-containing cells in the post-mortem human brain to clarify if there is a major species difference in terms of the existence of Y1 receptors in the human telencephalon, in particular the striatum and cortex. In situ hybridization experiments revealed widespread distribution of Y1 mRNA signals in all layers of most limbic and neocortical regions, predominantly in layer IV (most cortical regions) and layer VI. The striatum showed moderate Y1 receptor mRNA expression levels with intensely expressing cells localized to the nucleus accumbens. The highest Y1 receptor mRNA expression was apparent within the dentate gyrus, and the lowest in the subiculum, parahippocampal gyrus, cerebellum, and thalamus. In vitro autoradiography using [125I]Leu31Pro34-PYY and [125I]PYY with NPY (13-36) or Leu31Pro34-NPY, confirmed the presence of low Y1-like binding in the human brain despite abundant Y1 mRNA expression. However, using a rat model of the human autopsy process, it was apparent that the inability to reveal high Y1- versus Y2-like receptors in the human brain was related in part to marked reductions of Y1-like, but not Y2-like, receptors within a 4 h post-mortem delay. Altogether, the results indicate that the Y1 receptor gene is abundant in the human brain and this receptor may have important roles in cognitive, limbic and motor function.