preproNPY mRNA Levels Research Articles

Intake of High Fat Diet leads to Over‐expression of CD36, QRFP and QRFPr in the Gustatory Neural Circuit

Taste is a primary sensory modality that guides food preferences. Orosensory fat perception, which is mediated by lingual CD36, contributes to the intake and preference of dietary fat. Our previous studies indicate that feeding patterns differentially affect lingual CD36 mRNA expression and expression of homeostatic and hedonic markers in the brain. QRFP, an orexigenic peptide expressed primarily in the mediobasal hypothalamus (MBH), selectively increases the intake of high fat diet (HFD). The relationship between lingual CD36 and hypothalamic QRFP has yet to be investigated. Therefore, the goal of the current study was to conduct a time course analysis of continuous access to HFD on neural markers of food intake, including QRFP in the MBH and its receptor, QRFPr, in the nucleus accumbens (NAc). Male Sprague‐Dawley rats were fed a low fat diet (LFD, 10% kcal from fat) or HFD (55% kcal from fat) for 1 day, 3 days or 14 days. Body weight, food intake, and body adiposity were assessed. Real time PCR was used to determine the expression of CD36 mRNA on the circumvallate papillae of the tongue, expression of prepro‐QRFP, prepro‐NPY, and AgRP mRNA in the MBH, and dopamine receptor 1 (D1), 2 (D2) and QRFPr mRNA levels in the NAc. Rats fed HFD consumed more kilocalories than rats consuming the LFD, and gained more body adiposity, but not body weight, after 14 days of HFD intake. Lingual expression of CD36 mRNA was increased after 3 and 14 days of HFD intake. Intake of HFD increased hypothalamic expression of prepro‐QRFP mRNA but did not alter AgRP or prepro‐NPY mRNA levels. QRFPr expression in the NAc was significantly elevated by HFD intake, while D1 and D2 mRNA levels were differentially altered across days of HFD intake, with D1 levels decreasing after 14 days and D2 levels decreasing following 3 days access to HFD. Lingual CD36 mRNA was positively correlated with body weight gain and body adiposity and negatively correlated with AgRP mRNA levels. Hypothalamic prepro‐QRFP levels were positively correlated with kilocalorie intake. QRFPr mRNA levels in the NAc were positively correlated with body adiposity and negatively correlated with D1 and D2 mRNA levels. Prepro‐NPY and AgRP mRNA levels were negatively correlated with weight gain, body adiposity and QRFPr expression and AgRP levels were positively correlated with prepro‐NPY and D2 expression. Hypothalamic neuropeptides regulate food intake, particularly homeostatic eating. In this study, a time course analysis of HFD‐induced changes in gene expression suggest that while prepro‐NPY and AgRP levels were not altered, prepro‐QRFP mRNA levels were elevated. In the NAc, a brain region regulating hedonic eating, expression of D1 and D2, regulators of dopamine signaling, decreased, while QRFPr expression increased with HFD intake. These data, combined with the positive relationship between QRFP and kilocalories consumed and negative relationships between prepro‐NPY and AgRP and weight gain, suggest that CD36, QRFP and its receptor, QRFPr, may serve as positive regulators of caloric intake and weight gain by mediating the continued overconsumption of dietary fat.

Olfactory bulbectomy increases met-enkephalin- and neuropeptide-Y-like immunoreactivity in rat limbic structures

Bilateral olfactory bulbectomy (OBX) in rats produces a well-characterized syndrome of behavioral, physiological, and neurochemical changes identical to those seen in depression. Previous experiments using in situ hybridization histochemistry have demonstrated that OBX increases prepro-neuropeptide-Y (NPY) and prepro-enkephalin (ENK) mRNA levels in limbic structures. The present experiments determined whether increases in peptide immunoreactivity occur in conjunction with increases in mRNA levels following OBX. In situ hybridization analyses in olfactory bulbectomized and sham-operated rats revealed increased prepro-ENK mRNA in the piriform cortex (PIR) and olfactory tubercles (OTs) of bulbectomized rats. Prepro-NPY mRNA levels were significantly increased in the PIR of bulbectomized rats as comapred to controls. Radioimmunoassays (RIAs) revealed significant elevations in ENK-like immunoreactivity in the OTs following OBX. NPY-like immunoreactivity was significantly elevated in the PIR following OBX. These data reveal that OBX-induced increases in ENK-like immunoreactivity occur concomitantly with increases in prepro-ENK mRNA, and NPY-like immunoreactivity occur concomitantly with increases in prepro-NPY mRNA.

Involvement of hippocampal neuropeptide Y in mediating the chronic actions of lithium, electroconvulsive stimulation and citalopram

Neuropeptide Y (NPY) has been considered to be involved in the pathogenesis of affective disorders, and chronic treatment with lithium or electroconvulsive stimuli (ECS) has been shown to increase mRNA and peptide levels of NPY in rat brain tissue. Consequently, parameters reflective of NPYergic neurotransmission were studied in the hippocampus of rats following chronic treatment with lithium, ECS or the selective serotonin re-uptake inhibitor (SSRI), citalopram. Lithium (28 days, diet) and ECS (10 days, once daily) treatments caused a marked increase in levels of preproNPY mRNA in the CA1 area and dentate gyrus (DG). This increase was accompanied by an increase in extracellular levels of NPY in the dorsal hippocampus of freely moving rats as determined by microdialysis, suggesting that lithium and ECS treatments lead to an increased biosynthesis and release of NPY in this area. 125I-peptide YY (PYY) binding was reduced by 40 and 60% respectively in the DG following the same treatments, showing that the increased release is accompanied by a down-regulation of corresponding binding sites. In contrast, citalopram (10 mg/kg i.p., twice daily for 28 days) caused a 100% increase in 125I-PYY binding in CA, CA3 and DG while levels of preproNPY mRNA and extracellular NPY in the hippocampus were unaffected. The results indicate that various agents and stimuli exerting antidepressant effects in humans, such as chronic lithium, ECS and citalopram all increase NPYergic neurotransmission in the hippocampus by distinct modes of action. Moreover, NPY (6 μg) given intracerebroventricularly (i.c.v.) induced an antidepressant-like effect in the forced swim test. It is hypothesised that the increase in NPYergic neurotransmission may be associated with the mechanism of action of various antidepressant treatments in the alleviation of depression.

Absence of increased neuropeptide Y neuronal activity before and during the luteinizing hormone (LH) surge may underlie the attenuated preovulatory LH surge in middle-aged rats.

A large body of evidence suggests that the neuroendocrine axis plays a major role in the reproductive aging of female rats. Since increased hypothalamic neuropeptide Y (NPY) neurosecretion is crucial in the preovulatory LH discharge in young rats, we tested the hypothesis that diminution in the preovulatory LH surge in middle-aged (MA) rats may be due to altered neurosecretory activity in NPYergic neurons. In Exp 1, we examined NPY levels in six microdissected hypothalamic nuclei, including median eminence (ME), arcuate nucleus (ARC), and medial preoptic area (MPOA), at 1000, 1200, 1400, 1600, 1800, 2000, or 2200 h on the day of proestrus in young (2.5- to 3-month old) and MA (7- to 9-month old) regularly cycling rats. At 1000 h, ME NPY levels in young rats were significantly lower than those in MA rats. In young rats, the ME NPY levels were significantly increased at 1400 h before the LH surge in the afternoon and thereafter decreased progressively during the interval of the LH surge. In MA rats, however, ME NPY levels decreased in the afternoon in association with an attenuated LH surge. In addition, in the ARC and MPOA, the other hypothalamic sites associated with induction of LH surge, NPY levels increased before and during the LH surge in young rats, no change in NPY levels in these nuclei was observed in association with the attenuated LH surge in MA rats. Also, NPY levels in the ARC and MPOA during the afternoon were significantly lower in MA compared with those in young animals. These results demonstrated the absence of an antecedent increase in NPY levels, specifically in the ME and ARC, during the afternoon of proestrus in MA animals. In a second experiment, we evaluated whether the absence of dynamic changes in NPY levels in the ME and ARC in MA rats was due to altered hypothalamic NPY gene expression. Regularly cycling young (2.5- to 3-month-old) and MA (8- to 10-month-old) rats were killed at 1000, 1200, 1400, 1600, 1800, 2000, or 2200 h on the day of proestrus. The medial basal hypothalamus was processed for prepro-NPY messenger RNA (mRNA) measurement by ribonuclease protection assay. In young rats, prepro-NPY mRNA levels were significantly increased at 1200 h and remained elevated throughout the afternoon. In contrast, in MA rats prepro-NPY mRNA levels remained unchanged before and during the attenuated LH surge. These results clearly indicate that the augmentation in NPY neuronal activity before and during the LH surge seen in young rats fails to manifest itself in middle-aged rats. As hypothalamic NPY participates in the induction of LHRH surge, our results suggest that reduced LHRH and LH surges in MA rats may be due to diminution in NPY secretion in these animals.

Leptin and ciliary neurotropic factor (CNTF) inhibit fasting-induced suppression of luteinizing hormone release in rats: role of neuropeptide Y

Periods of chronic undernutrition and short periods of fasting suppress pituitary luteinizing hormone (LH) secretion and upregulate hypothalamic neuropeptide Y (NPY), the orexigenic peptide. The effect of suppression of NPY upregulation with ciliary neurotropic factor (CNTF), a cytokine, and leptin, an adipocyte hormone, on pituitary LH secretion was evaluated in fasted rats. In the first experiment, daily injection of CNTF (0.2 nmol) intracerebroventricularly (i.c.v.) for 4 days drastically reduced food intake and body weight gain similar to the weight loss seen in pair-fed rats. Food deprivation (FD) also decreased body weight. Despite drastic loss in body weight, plasma LH was reduced in FD and pair-fed rats, but not in CNTF-treated rats. In the second experiment, FD rats received either control vehicle, CNTF (0.2 nmol) or leptin (0.2 nmol) daily for 4 days. FD increased steady state levels of preproNPY mRNA in the hypothalamus over the control freely-fed rats. However, both CNTF and leptin suppressed hypothalamic gene expression and significantly attenuated LH suppression in response to FD. Taken together, these results support the hypothesis that the upregulation of hypothalamic NPY system may underlie diminution in pituitary gonadotropin secretion and that the NPYergic pathway may serve as a communication bridge between the neural processes that regulate reproduction and those that maintain energy balance.

Neuropeptide Y-Induced Feeding and Its Control

Publisher Summary This chapter critically reviews evidence of physiologically relevant appetite stimulation by neuropeptide Y (NPY) and to forecast how this knowledge might be applied in the context of eating disorders. Recently, a novel receptor for NPY, termed “Y5”, has been cloned from rat hypothalamus and appears to meet all the criteria necessary to be designated the “feeding” receptor distinct from previously cloned Y-type receptors. Striking appetite stimulation by NPY receptor agonists under pharmacological conditions can only elucidate the true physiological role of this neuropeptide if NPY receptor antagonists are found to be equally effective in blunting feeding produced under naturalistic conditions. Evidence for insulin actions on NPY gene expression was provided by studies that demonstrated that central infusion of insulin could depress elevated preproNPY mRNA levels induced by food deprivation of lean Zucker rats but was without effect on preproNPY mRNA content in fed or fasted obese Zucker rats. The extrapolation of the NPY-induced overeating and overweight syndrome in rodents to human obesity must await knowledge about the role of NPY in the human brain.

Increased receptor sensitivity to neuropeptide Y in the hypothalamus may underlie transient hyperphagia and body weight gain

Disruption of neural signaling by microinjection of a neurotoxin, colchicine (COL), in the ventromedial hypothalamus (VMH) of rats results in rapid and transient hyperphagia and body weight gain. Since neuropeptide Y (NPY) is a potent hypothalamic orexigenic signal and continuous NPY receptor activation by intracerebroventricular (icv) NPY infusion results in hyperphagia and obesity, we tested the hypothesis that altered NPYergic signaling may underlie the transient hyperphagia in COL-injected rats. Immediately following COL (4 μg) microinjections in the ventromedial nucleus (VMN) rats displayed hyperphagia both during the lights-on and lights-off periods. Concomitant with hyperphagia, preproNPY mRNA levels in the arcuate nucleus and NPY levels in the paraventricular nucleus decreased in a time-dependent manner. However, food intake in response to intracerebroventricular injections of NPY (29, 117 and 470 pmole) was significantly higher in COL-injected rats and the latency to initiation of feeding was markedly reduced as compared to controls. The smallest dose of NPY which was virtually ineffective in control rats, evoked near maximal intake in COL-injected rats. This enhanced response lasted for only 4 days paralleling the transient hyperphagia. The NPY Y1 receptor antagonist 1229U91 (5 or 30 μg/rat, icv) significantly suppressed feeding in COL-treated rats thereby indicating that hyperphagia in these rats was dependent upon endogenous NPY. Overall, these studies demonstrate that not only high levels, but low levels of NPY may also result in hyperphagia and increased body weight and this hyperphagia may be attributed to the rapid development of NPY Y1 receptor hypersensitivity.

Disinhibition from opioid influence augments hypothalamic neuropeptide Y (NPY) gene expression and pituitary luteinizing hormone release: effects of NPY messenger ribonucleic acid antisense oligodeoxynucleotides.

It is well known that hypothalamic endogenous opioid peptides (EOP) exert an inhibitory influence on LH release, and that a restraint on this inhibitory tone triggers preovulatory LHRH and LH hypersecretion. Recent evidence suggests that hypothalamic neuropeptide Y (NPY) is an important component of the neural circuitry that participates in induction of the LH surge. We have reported previously that blockade of EOP influence by the opioid receptor antagonist, naloxone (NAL), stimulated NPY accumulation in the median eminence (ME) in association with increased LH release in estradiol-17 beta-primed ovariectomized rats. To evaluate whether a restraint on the EOP system will result in an increase in NPY synthesis, the effects of NAL infusion on preproNPY messenger RNA (mRNA) levels in the medial basal hypothalamus were studied by solution hybridization/RNase protection assay and by in situ hybridization. NAL (2 mg/0.6 ml.h) or saline (SAL, 0.6 ml/h) was infused for 3 h (1100-1400 h) via an intrajugular cannula in estradiol-17 beta-primed ovariectomized rats. In accord with previous studies, NAL infusion significantly increased plasma LH levels at 1400 h. concomitant with this activation of LH release, NPY gene expression was also augmented. As compared with initial control levels at 1100 h, preproNPY mRNA levels in the medial basal hypothalamus increased at 1400 h in SAL (106%)- and NAL (202%)-infused rats, and at this time, preproNPY mRNA levels were significantly higher in NAL-infused rats than in SAL-infused rats. In situ hybridization studies showed that NAL infusion significantly increased the preproNPY mRNA signal at 1400 h mainly in the rostral and middle regions of the arcuate nucleus (ARC) as compared with that seen at 1100 and 1400 h in SAL-infused rats. To examine further the relationship between the NAL-induced increase in LH release and increase in NPY gene expression, the effects of NPY mRNA antisense oligonucleotides (oligos) on NPY levels in the ME-ARC and on plasma LH levels were studied in NAL-infused rats. In NAL-infused rats, intracerebroventricular administration of a NPY antisense oligo (25 micrograms/rat) at 1000, 1200, and 1300 h decreased NPY levels in the ME-ARC and blocked the increase in plasma LH levels at 1500 h, whereas control missense oligos had no effect. Collectively, these results show that a decrease in opioid influence rapidly augments NPY gene expression in a subpopulation of neurons in the ARC, and support the hypothesis that a disinhibition from opioid influence acutely promotes NPY synthesis and release, which is necessary for phasic LH discharge in rats.

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.

Evidence that hypothalamic neuropeptide Y gene expression increases before the onset of the preovulatory LH surge.

Neuropeptide Y (NPY), a 36 amino acid residue peptide, is involved in stimulation of LHRH and LH surges on proestrus and those induced by ovarian steroids in ovariectomized (ovx) rats. Recently, we observed that NPY gene expression in the medial basal hypothalamus (MBH) was increased before the onset of the LH surge in the ovarian steroid-primed ovx rats. Since the ovarian steroidal milieu during the estrous cycle is markedly different from that prevailing after ovarian steroid injections in ovx rats, we evaluated in cycling rats the temporal relationship between MBH preproNPY mRNA levels and the preovulatory LH surge on the day of proestrus and compared that with diestrus II, concomitant with basal LH levels. PreproNPY mRNA levels in the MBH were measured by solution hybridization/RNAse protection assay, using a cRNA probe. On the day of diestrus II, preproNPY mRNA levels changed little between 1000 and 1800 h. Quite unexpectedly, preproNPY mRNA levels at 1000 h on proestrus were similar to diestrus II levels, despite additional exposure to ovarian steroids during this interval. However, from these low levels at 1000 h, the preproNPY mRNA profile displayed a biphasic rise. During the first phase, preproNPY mRNA rose significantly at 1200 h and remained elevated at 1300 and 1400 h concomitant with basal serum LH levels. Thereafter, a second rise in preproNPY mRNA began at 1500 h, peaked rapidly at 1600 h and declined significantly at 1800 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Stress elicits trans-synaptic activation of adrenal neuropeptide Y gene expression

Stress triggers responses important to maintain internal homeostasis, and yet when prolonged can cause medical consequences that are most likely mediated by changes in gene expression. In this study we examine the alterations in gene expression of neuropeptide Y (NPY), a potent vasoconstrictor, with stress. Stressors, such as immobilization and cold, were found to increase adrenal NPY gene expression in rats. Adrenal prepro-NPY mRNA levels were elevated by a relatively short period of stress. A single immobilization was sufficient for an increase in prepro-NPY mRNA, that remained elevated for as long as one day later. This rise in adrenal NPY mRNA was abolished by the transcriptional inhibitor actinomycin D. Repeated (2 and 7) daily immobilizations led to a further rise and sustained elevations of prepro-NPY mRNA levels. This increase persisted 2–3 days after the cessation of repeated stress. The stress-elicited increase in NPY gene expression is transsynaptic requiring splanchnic innervation and mediated by nicotinic receptors. Hypophysectomy did not prevent the stress elicited rise in adrenal prepro-NPY mRNA levels. These results suggest that long lasting changes in NPY gene expression might be an important component in the homeostatic mechanisms triggered by chronic stress.

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.

Age-related decrease in neuropeptide-Y gene expression in the arcuate nucleus of the male rat brain is independent of testicular feedback.

Neuropeptide-Y (NPY) is thought to modulate reproductive function and food intake. NPY neuronal activity is modulated by sex steroids, and NPY secretion declines with aging. We hypothesized that reduced NPY secretion with aging is due to decreased NPY gene expression, and that this decrease is independent of testicular feedback. To test this hypothesis, arcuate nucleus (ARC) prepro-NPY (ppNPY) mRNA levels, determined by in situ hybridization, and serum testosterone levels, determined by RIA, were compared in sham-operated and orchidectomized young (3 months old), middle-aged (13 months old), and old (23 months old) male Brown Norway (BN) rats. Hybridization area and average optical density (OD) were used as indices of ARC ppNPY mRNA content. In sham-operated rats, both ppNPY mRNA hybridization area and OD decreased progressively with aging, whereas serum testosterone levels were decreased only in old, not in middle-aged or young, rats. In orchidectomized rats, ppNPY mRNA hybridization area also decreased significantly with aging, although OD did not change significantly. The ppNPY mRNA hybridization area was lower in orchidectomized than in intact young and middle-aged rats, whereas OD was unchanged by orchidectomy. Furthermore, the effects of aging and orchidectomy on ppNPY mRNA levels were not localized to a specific region of the ARC. We conclude that in the male BN rat, ARC NPY gene expression is decreased with aging independently of the effects of testicular feedback. This reduction in NPY synthetic capacity may contribute to altered reproductive function and food intake with aging.

Electroconvulsive shocks increase the expression of neuropeptide Y (NPY) mRNA in the piriform cortex and the dentate gyrus

Repeated electroconvulsive stimulations represent one treatment modality for depressive disorders, but the mechanism leading to its effect is largely unknown. Studies of humans and rats have indicated that neuropeptide Y (NPY) is involved in major depression and anxiety. The purpose of the present investigation was to detect changes in the expression of preproNPY mRNA in the limbic cortex of rats exposed to electroconvulsive shocks (ECS) daily for 14 days. Twenty-four hours after the last ECS, the animals were sacrificed, brain sections were hybridized with a synthetic oligonucleotide probe complimentary to rat preproNPY mRNA. Semi-quantitative in situ hybridization histochemistry revealed an about ten-fold increase of preproNPY mRNA levels over the dentate gyrus and the piriform cortex in animals exposed to ECS compared to sham-treated controls. In the dentate gyrus dipped sections showed that the increase of gene expression took place in individual neurons in the polymorph layer. In the piriform cortex a moderate increase in the number of grains was observed over many individual cells in the pyramidal layer. These data show that the expression of preproNPY mRNA is markedly increased in specific brains regions after ECS, but whether this increase is a result of the ECS-induced seizures per se, or rather should be regarded as a protective adaptation to changes in neuronal activity pattern remains to be established.

Chronic Administration of Glucocorticoids Directly Upregulates Prepro‐Neuropeptide Y and Y1‐Receptor mRNA Levels in the Arcuate Nucleus of the Rat

The complete sequence of the cDNA encoding the neuropeptide Y (NPY) Y1-receptor has recently been deduced from a rat brain library, and the presence of messenger ribonucleic acid (mRNA) encoding Y1-receptor protein has been demonstrated within the brain. Using quantitative in situ hybridization histochemistry, the content and distribution of Y1-receptor and preproNPY mRNAs have been investigated in the hypothalamic arcuate nucleus of adrenalectomized rats receiving glucocorticoid replacement therapy for 12 days by means of either high doses of dexamethasone in their drinking water or by subcutaneous corticosterone pellets. Basal metabolic parameters such as weight gain or loss, blood glucose and plasma insulin were monitored: Dexamethasone treatment induced weight loss and a state of hyperinsulinemia with normoglycemia, while corticosterone treated animals displayed metabolic parameters identical to sham ADX animals. Within the arcuate nucleus of glucocorticoid treated animals, levels of Y1-receptor and preproNPY mRNAs were increased. In contrast, adrenalectomy itself had no effect upon Y1-receptor mRNA levels or preproNPY mRNA levels in the arcuate nucleus. These studies demonstrate that glucocorticoids exert a stimulatory action on levels of Y1-receptor mRNA and preproNPY mRNA levels in the hypothalamic arcuate nucleus. This is the first evidence to suggest that the expression of a neuropeptide-receptor gene in the central nervous system may be directly sensitive to peripheral hormonal signals.

Hypothalamic neuropeptide-Y gene expression increases before the onset of the ovarian steroid-induced luteinizing hormone surge.

Neuropeptide-Y (NPY), a hypothalamic peptide, is involved in stimulation of LHRH and LH surges during proestrus and those induced by ovarian steroids in ovariectomized (ovx) rats. The NPY neurons that reside in the arcuate nucleus of the medial basal hypothalamus (MBH) and accumulate 17 beta-estradiol participate in the initiation of LHRH and LH surges. To determine whether NPY synthesis is altered in conjunction with the LH surge, we studied the dynamic changes in prepro-NPY mRNA levels in the MBH in association with the LH surge elicited by estradiol benzoate (EB) alone or by progesterone (P) in EB-primed ovx rats. Five days after ovariectomy, rats received oil or EB (30 micrograms/rat) at 1000 h on day 0. On day 2, these rats were injected with either oil or P (2 mg/rat) at 1000 h. Rats were killed before (1000 h) and at 2-h intervals after oil or P injection. The MBHs were dissected out and processed for determination of prepro-NPY mRNA levels by solution hybridization/RNase protection assay using a cRNA probe. Although in control ovx rats, prepro-NPY mRNA levels remained unchanged between 1000-1600 h, prepro-NPY mRNA levels showed dynamic changes in steroid-primed rats. In the EB-primed rats, prepro-NPY mRNA levels rose significantly (100%) at 1200 and 1400 h before the LH rise at 1600 h, and the levels remained elevated up to 1800 h. After P injection to the EB-primed rats, this response was further augmented, with a slightly different temporal pattern. Prepro-NPY mRNA levels rose at 1400 h (600%) before the onset of the LH rise at 1600 h and declined steadily to significantly lower values at 1800 h, coincident with the highest rate of LH secretion. These studies demonstrate dynamic shifts in hypothalamic NPY gene expression in association with the LH (LHRH) surge, and that maximal increases occur before the onset of the LH rise, but thereafter, NPY gene expression diverged in the two ovarian-steroid treatment models. These findings along with previous evidence of similar antecedent increases in NPY content in the median eminence, followed by release, suggest that augmented NPY synthesis and release are two temporally dissociable neural events for the LHRH and LH surges.

Hypothalamic neuropeptide-Y gene expression increases before the onset of the ovarian steroid-induced luteinizing hormone surge

Neuropeptide-Y (NPY), a hypothalamic peptide, is involved in stimulation of LHRH and LH surges during proestrus and those induced by ovarian steroids in ovariectomized (ovx) rats. The NPY neurons that reside in the arcuate nucleus of the medial basal hypothalamus (MBH) and accumulate 17 beta-estradiol participate in the initiation of LHRH and LH surges. To determine whether NPY synthesis is altered in conjunction with the LH surge, we studied the dynamic changes in prepro-NPY mRNA levels in the MBH in association with the LH surge elicited by estradiol benzoate (EB) alone or by progesterone (P) in EB-primed ovx rats. Five days after ovariectomy, rats received oil or EB (30 micrograms/rat) at 1000 h on day 0. On day 2, these rats were injected with either oil or P (2 mg/rat) at 1000 h. Rats were killed before (1000 h) and at 2-h intervals after oil or P injection. The MBHs were dissected out and processed for determination of prepro-NPY mRNA levels by solution hybridization/RNase protection assay using a cRNA probe. Although in control ovx rats, prepro-NPY mRNA levels remained unchanged between 1000-1600 h, prepro-NPY mRNA levels showed dynamic changes in steroid-primed rats. In the EB-primed rats, prepro-NPY mRNA levels rose significantly (100%) at 1200 and 1400 h before the LH rise at 1600 h, and the levels remained elevated up to 1800 h. After P injection to the EB-primed rats, this response was further augmented, with a slightly different temporal pattern. Prepro-NPY mRNA levels rose at 1400 h (600%) before the onset of the LH rise at 1600 h and declined steadily to significantly lower values at 1800 h, coincident with the highest rate of LH secretion. These studies demonstrate dynamic shifts in hypothalamic NPY gene expression in association with the LH (LHRH) surge, and that maximal increases occur before the onset of the LH rise, but thereafter, NPY gene expression diverged in the two ovarian-steroid treatment models. These findings along with previous evidence of similar antecedent increases in NPY content in the median eminence, followed by release, suggest that augmented NPY synthesis and release are two temporally dissociable neural events for the LHRH and LH surges.

Differential effect of fasting on hypothalamic expression of genes encoding neuropeptide Y, galanin, and glutamic acid decarboxylase

The effect of caloric deprivation to stimulate hypothalamic neuropeptide Y (NPY) gene expression is hypothesized to represent a physiologically important adaptation in body weight homeostasis. To evaluate the specificity of this response, we used in situ hybridization histochemistry to measure hypothalamic expression of mRNA encoding NPY, galanin, and the two isoforms of glutamic acid decarboxylase (GAD 67 and GAD 65) in male Wistar rats either fed ad lib or deprived of food for 24 or 48 h. As expected, food deprivation for 24 and 48 h increased preproNPY mRNA levels in the arcuate nucleus by 43 ± 13% ( p = NS) and 127 ± 29% ( p < 0.05 vs. both fed and 24-h fasted groups) when compared to ad lib-fed controls, and hypothalamic preproNPY mRNA levels were significantly correlated to the percent change in body weight over the three groups of rats ( r = −0.72; p < 0.05). In contrast, no significant effects of either 24 or 48 h of fasting were observed on hypothalamic levels of preprogalanin, GAD 67, or GAD 65 mRNA, and no relationship between percent change in body weight and expression of any of these mRNA species could be demonstrated. In conclusion, fasting increases preproNPY mRNA levels in the arcuate nucleus but does not alter expression of other hypothalamic mRNA species pertinent to feeding behavior. This supports the hypothesis that stimulation of NPY gene expression represents an important component of the hypothalamic response to caloric deprivation.

Expression of preproNPY and precursor VIP mRNAs in rats under hypo- or hyperthyroid conditions

Both neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) are present in thyroid nerves and have been shown to alter thyroid activity. The present study was conducted to determine whether hypo- or hyperthyroidism is associated with changes in the expression of the mRNAs for these neuropeptides in the major ganglia which supply nerves to the thyroid or within the thyroid gland itself. Hypo- or hyperthyroid conditions were induced by the administration of propylthiouracil (PTU) or thyroxine (T 4), respectively, for 6 days. Control rats received vehicle injections. Total RNA from superior cervical ganglia (SCG), local thyroid ganglia, thyroid gland, and selected other tissues was extracted and mRNA levels were analyzed using Northern blot procedures. No significant changes in preproNPY or precursor VIP mRNA levels were detected in the SCG or the local thyroid ganglia in response to PTU or T 4 treatment. However, PTU treatment was associated with an increase in preproNPY mRNA levels in the thyroid gland itself. These results indicate that changes within the thyroid axis in response to these hypo- and hyperthyroid conditions do not include alterations in steady-state preproNPY or precursor VIP mRNA concentrations in the major ganglia which supply nerves to the thyroid gland. However, intrathyroidal preproNPY mRNA levels are increased as a consequence of the thyroidal adaptation to a PTU challenge.

Glucocorticoids are Required for Food Deprivation‐Induced Increases in Hypothalamic Neuropeptide Y Expression

Neuropeptide Y (NPY), a 36 amino-acid peptide found within the hypothalamus, is thought to be an important regulator of food intake. Hypothalamic NPY gene expression, synthesis and secretion are all known to be increased in models of increased metabolic demand in which serum glucocorticoids are also elevated. The present studies were designed to test the hypothesis that glucocorticoids are required for increased hypothalamic preproNPY mRNA levels induced by food deprivation (FD). First, animals underwent bilateral sham-adrenalectomy (sham) or not (control), and were subjected to 72 h FD, or not. Total RNA was isolated from hypothalamic tissue blocks and the content of preproNPY mRNA was measured by solution hybridization/RNase protection analysis. This study revealed that there was no significant difference in hypothalamic preproNPY mRNA content between shamfed and control-fed groups, or between sham-FD and control-FD groups. In the second experiment, animals underwent bilateral adrenalectomy (ADX), were allowed to feed ad libitum and were sacrificed 1 day, 4 days and 7 days after ADX. Nuclease protection analysis revealed no significant effect of ADX on hypothalamic preproNPY mRNA levels over this time-course. Finally, we examined the role of glucocorticoids in regulating NPY gene expression following FD. Animals underwent bilateral ADX, or not. At the time of surgery, ADX animals received placebo, or corticosterone (B) replacement in the form of constant release pellets, at one of two doses. Food was removed from half of the animals in each group 24 h after surgery; all animals were sacrificed 72 h thereafter. There was no difference in preproNPY mRNA content between the ADX-FD and ADX-fed groups, relative to the fed controls. Replacement with corticosterone [ADX(B)] did not alter preproNPY mRNA content in fed animals, however preproNPY mRNA content in FD animals was increased 2.5-fold. These studies demonstrate that glucocorticoids are necessary and serve a stimulatory role in the increase in hypothalamic preproNPY mRNA levels observed under conditions of FD, and suggest that hypothalamic NPY gene expression may be directly responsive to peripheral metabolic and hormonal signals.