Neuropeptide Y Gene Transcription Research Articles

Lesion of area postrema attenuated hyperphagic responses to glucoprivation, but not transcriptional activation of the neuropeptide Y gene in rats

The area postrema (AP) is a circumventricular organ that lacks a blood-brain barrier. Previous studies have shown that the lesion of AP (APX) attenuated hyperphagic responses to glucoprivation. As the orexigenic neuropeptide Y (NPY) neurons have been implicated in the regulation of food intake, we examined whether the activation of NPY neurons by glucoprivation is mediated through the AP as well. In agreement with previous studies, hyperphagic responses to an injection of 2-deoxy-D-glucose that blocks glucose utilization were significantly attenuated in the APX group compared with the sham-operated (Sham) group. However, the expression levels of NPY heteronuclear RNA, a sensitive indicator for the gene transcription, were significantly increased in the arcuate nucleus by a 2-deoxy-D-glucose injection in both the APX and the Sham groups, and there were no significant differences in the values between groups. These data suggest that the hyperphagic response to glucoprivation, but not the activation of NPY gene transcription in the arcuate nucleus, was mediated through the AP in the hindbrain.

Effects of orchidectomy on adaptation of arcuate neuropeptide Y, proopiomelanocortin, and cocaine- and amphetamine-related transcript gene profiles to recurring insulin-induced hypoglycemia in the male rat

Our studies show that recurring insulin-induced hypoglycemia (RIIH) diminishes neuronal activation in several key components of the central metabolic regulatory circuitry, including the hypothalamic arcuate nucleus (ARH), and that orchidectomy (ORDX) modifies this impact of RIIH on Fos protein expression in this and other select neural structures. The testicular hormone, testosterone, regulates the expression of ARH neuropeptide genes of characterized metabolic relevance, including neuropeptide Y (NPY) and proopiomelanocortin (POMC). We investigated the hypothesis that acute hypoglycemia-associated patterns of ARH NPY, POMC, and cocaine and amphetamine-related transcript (CART) gene transcription, and potential RIIH-associated adaptive modifications in these expression profiles are regulated by testes-dependent mechanisms. ARH tissue was micropunched from serial frozen brains sections obtained from sham-operated (SHAM) or bilaterally ORDX male rats after sc injection of one or four doses of neutral protamine Hagedorn insulin, over as many days, or vehicle alone, and analyzed by quantitative real-time RT-PCR. In SHAM rats, acute hypoglycemia increased ARH NPY mRNA; precedent hypoglycemia elevated baseline gene expression in this group, but suppressed transcription during RIIH. In ORDX rats, ARH NPY mRNA was decreased during acute hypoglycemia and after multiple exposures; however, gene expression was not further suppressed by RIIH. ARH POMC gene transcription was not modified by acute or recurring hypoglycemia in the SHAM group. ORDX caused a reduction in both basal and acute hypoglycemic patterns of POMC transcription, relative to the SHAM controls, but enhanced baseline and RIIH-associated patterns of gene expression. ARH CART transcripts were not altered by acute or recurring hypoglycemia in SHAM rats, whereas ORDX animals exhibited elevated CART gene expression during RIIH. These data show that acute and recurring hypoglycemia exert opposite effects on ARH NPY gene expression in testes-intact male rats, and that these transcriptional responses are abolished by ORDX. Hypoglycemia had no impact on POMC nor CART mRNA profiles in the SHAM group, but both genes were upregulated during RIIH in ORDX rats. Collectively, these results demonstrate that in the male rat, testes-dependent mechanisms underlie patterns of acclimated or unvarying reactivity to RIIH of the specific ARH metabolic neuropeptide genes evaluated here.

Insulin is not a prerequisite for rapid regulation of neuropeptide Y gene transcription in the arcuate nucleus in food-restricted rats

Neuropeptide Y (NPY), synthesized in the arcuate nucleus of the hypothalamus, is one of the most potent orexigenic neuropeptides in the brain. The NPY neurons project to other hypothalamic nuclei, such as paraventricular nucleus (PVN), and it is reported that NPY contents in the PVN, but not NPY mRNA levels in the arcuate nucleus, decreased rapidly after food consumption. While many signals reflecting energy balance in the periphery are integrated at the NPY neurons, insulin has been implicated as one of the key regulators for NPY neurons. In the present study, we first examined whether there exist dynamic changes in NPY gene transcription in the arcuate nucleus in association with food intake in rats which had access to food only 4h a day. To detect possible changes in NPY gene transcription, we measured the expression levels of NPY heteronuclear (hn) RNA, a sensitive indicator of gene transcription, with intronic in situ hybridization. Our data showed that NPY hnRNA levels in the arcuate nucleus decreased rapidly after food consumption. We next examined whether postprandial increases in insulin release might contribute to the rapid downregulation of NPY gene transcription. To do so, insulin-deficient rats by streptozotocin injection were subjected to the same paradigm. Our data showed that NPY hnRNA levels also decreased rapidly after food consumption, suggesting that the postprandial increase in insulin release is not a prerequisite for the rapid downregulation of NPY gene transcription in the arcuate nucleus.

Ghrelin Increases Neuropeptide Y and Agouti-Related Peptide Gene Expression in the Arcuate Nucleus in Rat Hypothalamic Organotypic Cultures

Ghrelin, which was identified from the rat stomach, is a potent stimulant for food intake. Several lines of evidence suggest that the orexigenic action of ghrelin is mediated via the neuropeptide Y (NPY) neurons in the arcuate nucleus, although the detailed mechanisms by which ghrelin stimulates NPY neurons are not clear. In this study, we examined the gene regulation of NPY and agouti-related peptide (AGRP), another orexigenic peptide synthesized in the NPY neurons, in the arcuate nucleus by ghrelin in hypothalamic organotypic cultures. Incubation of the hypothalamic explants with ghrelin significantly increased NPY and AGRP mRNA expression in the presence, but not absence, of dexamethasone. Glucocorticoids were also necessary for ghrelin action in vivo because an intracerebroventricular injection of ghrelin significantly increased NPY and AGRP mRNA expression in the arcuate nucleus only in sham-operated, but not in adrenalectomized rats. The stimulatory effects of ghrelin on gene expression were not blocked by a sodium channel blocker tetrodotoxin in the organotypic cultures. Ghrelin also increased NPY heteronuclear (hn) RNA expression, the first transcript that has been used as an indicator for gene transcription. The stimulatory effects of ghrelin on NPY gene expression were abolished in the presence of cycloheximide, which blocks translation, suggesting that de novo protein synthesis is required for ghrelin action. These data suggest that ghrelin stimulates NPY and AGRP gene expression independently of action potentials only in the presence of glucocorticoids. Furthermore, our data demonstrate stimulatory action of ghrelin on NPY gene transcription, which requires de novo protein synthesis.

Effect of fasting and leptin deficiency on hypothalamic neuropeptide Y gene transcription in vivo revealed by expression of a lacZ reporter gene.

Neuropeptide Y (NPY), a peptide synthesized in the hypothalamic arcuate nucleus, is implicated in the physiologic control of food intake and body weight. Because both genetic (e.g. in obese ob/ob mice) and acquired leptin deficiency (e.g. fasting in normal mice) increase hypothalamic NPY accumulation, and as leptin administration reverses this effect, we hypothesized that leptin inhibits transcription of the NPY gene by arcuate nucleus neurons. To test this hypothesis, we studied mice with a targeted mutation of the NPY gene (NPY knockout mice), in which the lacZ reporter gene was inserted into the first exon of the NPY gene. As a result, these mice express beta-galactosidase (beta gal; the enzyme encoded by lacZ) in neurons that normally express the NPY gene. To determine whether beta gal staining provides a valid measure of lacZ expression, we used a histochemical method to count the number of beta gal+ neurons in coronal sections of brain tissue from mice bearing either one (NPY+/-) or two (NPY-/-) mutant alleles. In both the arcuate nucleus and the thalamic reticular nucleus, beta gal+ cell number was 260% higher in NPY-/- than in NPY+/- mice (P < 0.05). Fasting for 48 h also increased the mean beta gal+ cell number in the arcuate nucleus of NPY+/- mice by 260% (P < 0.001), but had no effect in the thalamic reticular nucleus. Similarly, obese leptin-deficient ob/ob, NPY+/- mice had a 67.3% increase in arcuate nucleus beta gal+ cell number compared with lean ob/+, NPY+/- littermates (P < 0.05), and this effect was attenuated 36.6% (P < 0.05) by leptin administration (70 microg/day, i.p., for 4 days). Based on the results of this novel method for measuring NPY gene transcription in vivo, we conclude that both fasting and genetic leptin deficiency increase NPY gene transcription in the arcuate nucleus and that this transcriptional response is attenuated by leptin administration in ob/ob, NPY+/- mice.

Ca2+/calmodulin-dependent transcriptional activation of neuropeptide Y gene induced by membrane depolarization: determination of Ca(2+)- and cyclic AMP/phorbol 12-myristate 13-acetate-responsive elements.

Membrane depolarization stimuli (high potassium concentration and veratridine) increased neuropeptide Y (NPY) mRNA abundance time-dependently, without a change in beta-actin mRNA level, in NG108-15 and PC12 cells. Although the induction by veratridine was blocked completely by tetrodotoxin, the induction by potassium was suppressed minimally. Voltage-dependent Ca channel blockers and calmodulin antagonists inhibited the increases by both depolarization stimuli completely, suggesting involvement of Ca2+/calmodulin-dependent kinases (CaM kinases). Transient assay using chloramphenicol acetyltransferase reporter genes containing the rat NPY gene promoter indicated that membrane depolarization and Ca entry stimulate transcription of the NPY gene. The depolarization-induced transactivation was also blocked by CaM kinase inhibitors. The 200-bp 5'-upstream region (-344/-145) was localized as a Ca2+/ calmodulin-responsive element (CaMRE), which confers depolarization-induced transactivation. It is interesting that this CaMRE did not contain the canonical Ca-responsive elements such as CRE, SRE, NF-AT, or the C/EBP beta-binding site and was separated from a 64-bp cyclic AMP/ phorbol 12-myristate 13-acetate-responsive element (-144/-81). These findings suggested that membrane depolarization regulates the NPY gene transcription positively through the unique CaMRE by activation of CaM kinases following Ca entry through L-type Ca channels.

Differential stimulation of somatostatin but not neuropeptide Y gene expression by quinolinic acid in cultured cortical neurons.

Somatostatin (SS) and neuropeptide Y (NPY) are coproduced in a subpopulation of neurons that are selectively resistant to NMDA neurotoxicity. We have previously reported that quinolinic acid (QUIN), an NMDA receptor agonist, augments SS mRNA in cultured fetal rat cortical neurons. This study examines coregulation of SS and NPY by QUIN and NMDA in cultured cortical neurons and compares the effects of these agents with those of forskolin and phorbol 12-myristate 13-acetate (PMA), known to activate SS and NPY gene transcription by protein kinase A- and protein kinase C-dependent mechanisms. In addition, transcriptional regulation of the SS gene was investigated by acute transfection of cortical cultures with an SS promoter-chloramphenicol acetyltransferase (CAT) construct. QUIN and NMDA displayed dose-dependent fourfold augmentation of levels of mRNA for SS but not for NPY. In contrast, forskolin and PMA increased both SS and NPY mRNA levels. QUIN- and NMDA-mediated induction of SS mRNA was blocked by the NMDA receptor antagonist (-)-2-amino-5-phosphonovaleric acid and displayed regional brain specificity because it was not observed in fetal hypothalamic cell cultures. In time course studies, the effects of QUIN/NMDA on SS mRNA occurred after a latency of 8 h, indicating a delayed effect. Cortical cells transfected with pSS-750 CAT showed three- to fourfold stimulation of CAT activity with forskolin but not by QUIN or NMDA. These data reveal a dose-dependent, tissue-specific, NMDA receptor-mediated stimulation of SS but not NPY mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of direct action of D-fenfluramine on arcuate preproNPY mRNA levels in the rat.

Hypothalamic neuropeptide Y (NPY) and 5-hydroxytryptamine (5-HT) may interact to regulate food intake. This study investigated the effects of the 5-HT reuptake blocker and secretagogue D-fenfluramine (D-FEN) on preproNPY mRNA abundance in the hypothalamic arcuate nucleus (ARC) of free-feeding and food-deprived (FD) rats in two treatment regimens. ARC preproNPY mRNA levels were significantly increased in FD rats, to the same extent as in combined FD/D-FEN animals. D-FEN treatments producing different-sized reductions in food intake (and body weight) produced corresponding changes in ARC preproNPY mRNA abundance. These findings suggest that ARC preproNPY mRNA levels are not directly altered by D-FEN, and that D-FEN-induced anorexia does not involve effects on the hypothalamic NPY system at the level of NPY gene transcription.

Neuropeptide Y mRNA and immunoreactivity in hypothalamic neuroendocrine neurons: effects of adrenalectomy and chronic osmotic stimulation

Neuropeptide Y (NPY) coexists with vasopressin or oxytocin in magnocellular neurons of the hypothalamo-neurohypophysial tract. Using quantitative in situ hybridization histochemistry and immunohistochemistry, we have studied the effects of adrenalectomy and chronic osmotic stimulation, either alone or in combination, on NPY mRNA expression and NPY immunoreactivity in magnocellular neurons of the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, and arcuate nucleus (Arc). Adrenalectomy and chronic osmotic stimulation each increased NPY mRNA levels in magnocellular neurons of the PVN and SON, while the combination of both treatments had an additive effect. In the Arc, only the combination of adrenalectomy and chronic osmotic stimulation increased NPY mRNA levels. Chronic osmotic stimulation also resulted in a marked increase of NPY-immunoreactive magnocellular perikarya in the PVN and SON. In contrast, adrenalectomy had only minor effects on the number of NPY-immunoreactive magnocellular PVN/SON perikarya. Neither chronic osmotic stimulation nor adrenalectomy affected the number of NPY-immunoreactive Arc perikarya. However, adrenalectomy decreased the number of NPY-immunoreactive nerve terminals in the external zone of the median eminence, while chronic osmotic stimulation increased the number of immunoreactive nerve fibers in the internal zone of the median eminence. The present study provides evidence that adrenalectomy and chronic osmotic stimulation can separately influence NPY gene transcription in magnocellular hypothalamo-neurohypophysial neurons, while only the combined effect of adrenalectomy and chronic osmotic stimulation increases NPY mRNA expression in neurons of the Arc.

CENTRAL INSULIN ADMINISTRATION REDUCES NEUROPEPTIDE Y mRNA EXPRESSION IN THE ARCUATE NUCLEUS OF FOOD-DEPRIVED LEAN (Fa/Fa) BUT NOT OBESE (fa/fa) ZUCKER RATS

By acting in the brain, insulin suppresses food intake, whereas neuropeptide Y (NPY) has the opposite effect. Since fasting increases NPY gene expression in the hypothalamic arcuate nucleus (ARC) and also lowers circulating insulin levels, we hypothesized that the anorexiant effect of insulin could result from insulin inhibition of NPY gene transcription in the ARC. Therefore, we determined whether the administration of insulin (200 mU per 12 hrs) into the 3rd cerebral ventricle of lean (Fa/Fa) female Zucker rats (n = 5) during 48 hrs of food deprivation reduces the expression of preproNPY mRNA in the ARC compared to vehicle-treated controls (n = 5). Coronal sections of rat brain were hybridized with an oligonucleotide probe complementary to preproNPY mRNA and apposed to x-ray film. Hybridization was quantified in both the ARC and the hippocampal dentate gyrus by computerized image analysis of the resulting autoradiographs. Central insulin significantly reduced the area of hybridization in the ARC (0.235 +/- 0.017 mm2; mean +/- SE) compared to vehicle-treated controls (0.331 +/- 0.037 mm2; p less than 0.05), but was without effect in the hippocampus. Thus, insulin reduced the expression of mRNA for NPY specifically in the ARC. Since the genetically obese (fa/fa) Zucker rat is insensitive to the anorexiant effect of insulin and over-expresses NPY in the ARC, we next tested the hypothesis that insulin does not suppress NPY mRNA expression in the ARC of these rats. Consistent with this hypothesis, central insulin administration to obese Zucker rats during 48 hrs of food deprivation (n = 6) did not lower hybridization area in the ARC compared to vehicle alone (n = 4) (0.286 +/- 0.036 vs. 0.248 +/- 0.019 mm2; p greater than 0.05). We conclude that insulin suppresses the expression of mRNA for NPY in the ARC of fasted lean but not obese Zucker rats. Regulation of hypothalamic NPY gene expression by insulin may account for its anorexiant effect, and a defect in this action may contribute to certain forms of obesity.

Rapid decrease in neuropeptide Y gene expression in rat adrenal gland induced by the α2‐adrenoceptor agonist, clonidine

1 The mechanism of regulation of the neuropeptide Y (NPY) gene by pharmacological treatment with the alpha 2-adrenenoceptor agonist, clonidine, was investigated by quantitative Northern blot analysis of the effects of this drug on the NPY mRNA levels in rat adrenal gland and medulla oblongata/pons. 2 In the adrenal gland, clonidine-treatment (50 microgram kg-1, s.c., once daily) resulted in decrease in the amount of NPY mRNA to 44 +/- 10% of the control level in 24 h and then its increase to 162 +/- 16% of the control level after 5 days. Concomitant changes in putative NPY pre-mRNA species (7.0 and 3.3 kb) were observed, probably due to changes at the level of NPY gene transcription. 3 The short-term (24 h) effect of clonidine was blocked by yohimbine (5 mg kg-1, i.p., once daily). Yohimbine alone tended to increase the NPY mRNA level after 24h. 4 The recovery/increase in the NPY mRNA level in the adrenal gland after 5 days treatment with clonidine was similar to its increase after treatment with reserpine (0.5 mg kg-1, i.p., once daily). 5 NPY gene expression in the medulla oblongata/pons was not changed by short- or long-term treatment with clonidine. 6 These results suggest that clonidine suppresses NPY gene expression in the adrenal gland, probably at the level of transcription, by activation of the alpha 2-adrenoceptor.

Expression of the human neuropeptide Y gene.

Cis-acting sequences involved in the expression of the human neuropeptide Y (NPY) gene were identified by transient expression and in vitro transcription assays using the Lan-5 cell system. Sequences between -63 and -51 were necessary for NPY gene expression. Gel retardation analyses utilizing this region indicate that a sequence-specific DNA-binding protein interacts with the sequence CCCCTCC. An in vitro nuclear transcription system was prepared from Lan-5 cell nuclei in order to determine a functional correlation between the CCCCTCC-binding protein and promoter activity. The NPY promoter was accurately transcribed in this system; furthermore, the data obtained from the in vitro transcription experiments were similar to those obtained from the in vivo transfection studies. Competition experiments in which an oligonucleotide containing either CCCCTCC or Sp1 (GGCGGG) sequences was added in excess to the transcription extracts demonstrated the functional importance of an Sp1-like protein in NPY gene transcription.

Techniques for the simultaneous demonstrations of neuropeptide y gene expression and peptide storage in single neurons of the human brain.

The distribution of mRNA encoding neuropeptide Y (NPY) in neurons of the normal human cerebral cortex in surgical biopsy specimens and postmortem brain was studied by in situ hybridization techniques. A 32P-labeled complementary RNA (cRNA) probe was used on cryostat sections of formaldehyde-fixed cortical biopsy specimens. Hybridization to NPY mRNA was found in all samples: after autoradiography, discrete deposits of silver granules were observed on neuronal cell bodies abundantly distributed in the deep layers of the cortex, particularly laminae IV and VI, and on smaller cell bodies in the white matter. The localization of the neurons hybridized for NPY mRNA was comparable to that of NPY-immunoreactive cells as shown in sections from the same tissue blocks immunostained by using NPY antibodies. The specificity of the in situ hybridization technique was confirmed by hybridization analysis of electrophoretically fractionated RNA. This study clearly demonstrated the consistent localization of NPY gene transcription and expression in normal mature human cortical neurons. A combination of the methods of in situ hybridization for mRNA encoding Neuropeptide Y (NPY) using a 32P-labeled complementary RNA probe and immunocytochemistry using polyclonal antibodies against NPY were applied on human cortical brain samples. The manipulation of these two techniques allowed successful simultaneous demonstration of NPY gene expression and peptide storage in single neurons of the human brain.

Localization of neuropeptide Y mRNA in neurons of human cerebral cortex by means of in situ hybridization with a complementary RNA probe.

The distribution of mRNA encoding neuropeptide Y (NPY) in neurons of the normal human cerebral cortex in surgical biopsy specimens and postmortem brain was studied by in situ hybridization techniques. A 32P-labeled complementary RNA (cRNA) probe was used on cryostat sections of 13 formaldehyde-fixed cortical biopsy specimens. Hybridization to NPY mRNA was found in all samples: after autoradiography, discrete deposits of silver granules were observed on neuronal cell bodies abundantly distributed in the deep layers of the cortex, particularly laminae IV and VI, and on smaller cell bodies in the white matter. The localization of the neurons hybridized for NPY mRNA was comparable to that of NPY-immunoreactive cells as shown in sections from the same tissue blocks immunostained by using NPY antibodies. The specificity of the in situ hybridization technique was confirmed by blot hybridization analysis of electrophoretically fractionated RNA. This study clearly demonstrated the consistent localization of NPY gene transcription and expression in normal mature human cortical neurons.