Preproneuropeptide Y Messenger Ribonucleic Acid in the Hypothalamic Arcuate Nucleus of the Rat is Increased by Food Deprivation or Dehydration

The distribution of messenger ribonucleic acid (mRNA) encoding preproneuropeptide Y (prepro-NPY) in the hypothalamus of rats subjected to food deprivation or dehydration has been investigated by quantitative in situ hybridization. Levels of prepro-NPY mRNA in the arcuate nucleus (ARC) were selectively increased by both treatments. The very high concentration of prepro-NPY mRNA seen following 96 h of food deprivation had returned towards control levels after 24 h of refeeding. Levels of preprogalanin (prepro-GAL) mRNA throughout the hypothalamus were essentially unaffected by both regimes. These results demonstrate that hypothalamic NPY gene expression is regulated by peripheral metabolic status (and osmolality), and confirm the key physiological role of NPY in controlling ingestive behaviour.     

Metabolic fuels, neuropeptide Y, and estrous behavior in Syrian hamsters

Of the various environmental factors influencing reproduction, food availability plays a particularly significant role, and an insufficient supply of oxidizable metabolic fuels inhibits reproduction in female mammals. When ovariectomized, steroid-primed hamsters are food deprived for 48 h, estrous behavior is suppressed. However, the specific neuroendocrine alterations that mediate the suppression of estrous behavior are unknown. Several conditions that inhibit female sexual behavior are thought to be associated with altered neuropeptide Y (NPY) activity in the brain. Intracerebroventricular (ICV) infusion of NPY inhibits estrous behavior in ovariectomized steroid-primed rats and hamsters. Furthermore, food-deprived rats have an increase in NPY mRNA in the arcuate nucleus (ARC) of the hypothalamus. Unlike rats, studies in Syrian hamsters have failed to detect any alterations in ARC NPY mRNA following food deprivation. Here we show that ARC NPY immunoreactivity and mRNA is increased in food-deprived hamsters but not in hamsters given other metabolic challenges that inhibit estrous behavior. These findings support the hypothesis that NPY contribute to, but not be critical for, the nutritional inhibition of sexual receptivity.     

Neuropeptide Y mRNA and serotonin innervation in the arcuate nucleus of anorexia mutant mice

The anorexia (anx) mutation causes reduced food intake in preweanling mice, resulting in death from starvation within 3–4 weeks. In wild-type rodents, starvation induces increased neuropeptide Y (NPY) mRNA levels in the arcuate nucleus that promotes compensatory hyperphagia. Despite severely decreased body weight and food intake at 3-weeks age, anx/anx mice do not show elevated NPY mRNA levels in the hypothalamic arcuate nucleus compared to wild-type/heterozygous littermates. The NPY mRNA levels can be upregulated in normal mice at this chronological age, because 24-h food deprivation increased arcuate NPY mRNA in wild-type littermates. The unresponsiveness of NPY expression in the arcuate of anx/anx mice was paralleled by serotonergic hyperinnervation of the arcuate nucleus, comparable to the serotonergic hyperinnervation previously reported in the rest of the anx/anx brain. This result is consistent with the hypothesis that wasting disorders are accompanied by disregulation of NPY mRNA expression in the arcuate nucleus, and suggests that reduced food intake, the primary behavioral phenotype of the anx/anx mouse, may be the result of altered hypothalamic mechanisms that normally regulate feeding.     

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 (GAD₆₇ and GAD₆₅) 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, GAD₆₇, or GAD₆₅ 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.     

Anorectic Effect of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) in Rats: Lack of Evidence for Involvement of Hypothalamic Neuropeptide Gene Expression

We investigated the effect of centrally administered pituitary adenylate cyclase activating polypeptide (PACAP) on feeding in rats, and the involvement of hypothalamic neuropeptide gene expression using in situ hybridization. lntracerebroventricular injection of PACAP (1000  pmol/rat) significantly decreased food intake in a dose-dependent manner. In PACAP-treated rats, neuropeptide Y (NPY) mRNA levels in the arcuate nucleus and galanin mRNA levels in the paraventricular nucleus increased, and corticotropin-releasing hormone (CRH) mRNA levels in the paraventricular nucleus decreased. In rats fasted for 72  h, NPY mRNA levels increased, and CRH mRNA levels decreased, but galanin mRNA levels were unchanged. These results indicate that the anorectic function of PACAP is not mediated by NPY or CRH, and that PACAP increases galanin synthesis.     

Glucocorticoids are required for meal-induced changes in the expression of hypothalamic neuropeptides

Glucocorticoid deficiency is associated with a decrease of food intake. Orexigenic peptides, neuropeptide Y (NPY) and agouti related protein (AgRP), and the anorexigenic peptide proopiomelanocortin (POMC), expressed in the arcuate nucleus of the hypothalamus (ARC), are regulated by meal-induced signals. Orexigenic neuropeptides, melanin-concentrating hormone (MCH) and orexin, expressed in the lateral hypothalamic area (LHA), also control food intake. Thus, the present study was designed to test the hypothesis that glucocorticoids are required for changes in the expression of hypothalamic neuropeptides induced by feeding. Male Wistar rats (230-280 g) were subjected to ADX or sham surgery. ADX animals received 0.9% NaCl in the drinking water, and half of them received corticosterone in the drinking water (B: 25 mg/L, ADX+B). Six days after surgery, animals were fasted for 16 h and they were decapitated before or 2 h after refeeding for brain tissue and blood collections. Adrenalectomy decreased NPY/AgRP and POMC expression in the ARC in fasted and refed animals, respectively. Refeeding decreased NPY/AgRP and increased POMC mRNA expression in the ARC of sham and ADX+B groups, with no effects in ADX animals. The expression of MCH and orexin mRNA expression in the LHA was increased in ADX and ADX+B groups in fasted condition, however there was no effect of refeeding on the expression of MCH and orexin in the LHA in the three experimental groups. Refeeding increased plasma leptin and insulin levels in sham and ADX+B animals, with no changes in leptin concentrations in ADX group, and insulin response to feeding was lower in this group. Taken together, these data demonstrated that circulating glucocorticoids are required for meal-induced changes in NPY, AgRP and POMC mRNA expression in the ARC. The lower leptin and insulin responses to feeding may contribute to the altered hypothalamic neuropeptide expression after adrenalectomy.     

Evidence that hypothalamic neuropeptide Y gene expression and NPY levels in the paraventricular nucleus increase before the onset of hyperphagia in experimental diabetes

Neuropeptide Y (NPY) is the most potent endogenous orexigenic signal. Several lines of evidence indicate that the site of NPY action in transducing feeding signal may reside in the paraventricular nucleus (PVN) and neighboring sites in the hypothalamus. To test the hypothesis that an increase in NPY activity in the ARC-PVN pathway precedes the onset of diabetic hyperphagia, we evaluated NPY levels in seven hypothalamic nuclei and NPY gene expression in the hypothalamus at 48, 72 or 96 h after streptozotocin (STZ) treatment in rat. In STZ-treated diabetic rats, NPY gene expression in the hypothalamus and NPY levels only in the PVN significantly elevated at 48 h, while hyperphagia occurred sometimes after 48 h post-injection. These results show that augmentation in NPY neuronal activity in the ARC-PVN axis precedes the onset of increased food intake produced by STZ-induced insulinopenia. These findings affirm the hypothesis that increased NPY neurosecretion in the PVN may underlie the diabetes-induced hyperphagia.     

Localization of neuropeptide Y mRNA and peptide in the chicken hypothalamus and their alterations after food deprivation, dehydration, and castration.

Localization of neuropeptide Y (NPY) mRNA in the hypothalamus of chickens was studied by in situ hybridization with digoxigenin-labeled chicken NPY cRNA probe. The largest number of perikarya-expressing NPY mRNA was found within the mediobasal hypothalamus, including the infundibular nucleus, inferior hypothalamic nucleus, and median eminence. Many NPY perikarya were noted to surround the nucleus rotundus and to be present in the supraoptic nucleus. Moreover, some perikarya were detected in the nucleus of basal optic root, bed nucleus pallial commissure, and nucleus striae terminalis close to the lateral forebrain bundle. NPY-immunoreactive nerve fibers were densely distributed in these regions containing the NPY mRNA-expressing perikarya. Following food deprivation for four days, perikarya-expressing NPY mRNA and peptide were markedly increased in the mediobasal hypothalamus and particularly so in the infundibular nucleus. No changes, however, were detected in other regions containing NPY-positive perikarya. Water deprivation induced less increase in NPY-positive perikarya in the mediobasal hypothalamus compared to food deprivation. After gonadectomy, the number of NPY-positive perikarya in the mediobasal hypothalamus was unaltered. Northern blot analysis with (32)P-labeled chicken NPY cDNA probe demonstrated that a 2.7-fold increase of NPY mRNA was induced by starvation and a 1.5-fold increase was induced by dehydration, whereas the NPY mRNA band remained unchanged after gonadectomy. Thus, it seems that NPY neurons located in the mediobasal hypothalamus are involved in feeding behavior but not reproductive activity.     

Rapid and localized alterations of neuropeptide Y in discrete hypothalamic nuclei with feeding status

Neuropeptide Y (NPY) is believed to regulate the normal eating behavior and body weight in rats via central mechanisms. We have investigated whether NPY, which stimulates food intake, may in turn be modified by the nutritional state of the animals. Thus the impact of food deprivation (FD) (48 h) and subsequent refeeding on the levels of NPY in discrete hypothalamic areas was examined in this study. The results showed site specific change in only 3 of 7 hypothalamic sites. A 5-fold increment in NPY was reported in the paraventricular nucleus (PVN) and a 10-fold increase was observed in the arcuate nucleus-median eminence (ARC-ME). While subsequent refeeding for 6 h reversed the effect of FD in the ARC-ME, the levels of NPY in the PVN remained high in the refed rats. The perifornical lateral hypothalamus displayed a different pattern, namely, a significant increase in NPY content in refed as compared to satiated and deprived rats. The NPY levels in 4 other hypothalamic sites, namely, the dorsomedian, ventromedian, supraoptic and suprachiasmatic nuclei, and two extrahypothalamic sites, namely caudate nucleus and nucleus accumbens, showed total resistance to any change following deprivation and refeeding. These data emphasize the important and specific role of the paraventricular and arcuate nuclei in NPY's regulation of food intake and provide support for the idea that the variations of hypothalamic NPY after food deprivation reflect a specific physiological response of feeding regulatory system to alterations in the animal nutritional state and body weight.     

Rapid changes in hypothalamic neuropeptide Y produced by carbohydrate-rich meals that enhance corticosterone and glucose levels

Prior studies have demonstrated that chronic consumption over several weeks of a high-carbohydrate (65%) diet, compared to a moderate-carbohydrate (45%) or low-carbohydrate (15%) diet, potentiates the expression, synthesis and release of hypothalamic NPY. This effect occurs specifically in neurons of the arcuate nucleus (ARC) which project to the paraventricular nucleus (PVN). In the present experiments, tests involving acute manipulations were conducted to determine whether such diet-induced changes in NPY can occur rapidly, perhaps within 1-2 h, and whether these effects can be linked to specific changes in circulating glucoregulatory hormones or glucose itself., In adult, albino rats maintained on lab chow, the acute manipulations included the presentation of either a high-carbohydrate, moderate-carbohydrate or high-fat diet for 90 min at the onset of the natural feeding cycle. They also involved manipulations of glucose itself, either through the ingestion of a glucose (20%) solution in a drinking tube or intraperitoneal injection of a glucose solution (10%). After a high-carbohydrate meal compared to a moderate-carbohydrate or high-fat meal, NPY gene expression examined via in situ hybridization is found to be significantly enhanced in the ARC. The high-carbohydrate meal also potentiates NPY immunoreactivity in the ARC and PVN but has little effect on NPY in other hypothalamic areas examined and actually causes a reduction in the feeding-stimulatory peptide, galanin, specifically in the PVN. The meal-induced increase in NPY is associated with specific endocrine patterns, as revealed by measurements in serum collected from trunk blood or from rats implanted with a chronic jugular catheter. After a high-carbohydrate meal, levels of glucose, together with corticosterone and insulin, are significantly elevated, while non-esterified fatty acids are reduced. A possible effect of circulating glucose on hypothalamic NPY is further suggested by the finding that the consumption or a single injection of a glucose solution at the onset of the feeding cycle similarly elevates NPY mRNA and peptide immunoreactivity in the ARC and PVN. These results demonstrate that hypothalamic NPY can change rapidly in response to dietary carbohydrate. They also suggest that this effect may be related to changes in circulating CORT as well as to the availability or utilization of glucose.     

Fasting activates neuropeptide Y neurons in the arcuate nucleus and the paraventricular nucleus in the rhesus macaque

It is well accepted that neuropeptide Y (NPY) plays a pivotal role in the regulation of food intake and energy homeostasis in the rodent, with NPY neurons in the arcuate nucleus (ARH) being thought of as the major contributor to the complex central feeding circuitry. Recent data from our group also indicate that NPY is important in the regulation of energy homeostasis in the nonhuman primate (NHP); exogenous NPY administration into the 3rd ventricle is a potent stimulator of food intake in the male rhesus macaque. The purpose of this study was to determine if NPY neurons in the rhesus macaque respond to a metabolic challenge, induced by 48 h of fasting, in a manner similar to that seen in the rodent. NPY mRNA was detected in hypothalamic sections from 48-h fasted or fed rhesus monkeys by in situ hybridization, using a [35S]UTP-labeled riboprobe specific for human NPY. Not surprisingly, NPY mRNA was abundant in the ARH of the NHP; however, of great interest was the expression of NPY mRNA in neurons within the paraventricular nucleus of the hypothalamus (PVH) and the supraoptic nucleus (SON). This raised the question as to whether all of these populations of NPY neurons are sensitive to changes in energy availability. Indeed, NPY expression in the ARH and PVH was significantly elevated in response to fasting; however, no significant change was detected in the SON. These data indicate that the NPY neurocircuitry involved in the regulation of food intake is more complex in the NHP than in rodents.     

Increased feeding and neuropeptide Y (NPY) but not NPY mRNA levels in the hypothalamus of the rat following central administration of the serotonin synthesis inhibitorp-chlorophenylalanine

Neurons containing serotonin (5-HT), a potent anorexic agent, come into contact with neuropeptide Y-ergic neurons, that project from the arcuate nucleus (ARC) to the paraventricular nucleus (PVN). NPY powerfully stimulates feeding and induces obesity when injected repeatedly into the PVN. We hypothesize that 5-HT tonically inhibits the ARC-PVN neurons and that balance between the two systems determines feeding and energy homeostasis. This study aimed to determine whether central injection of the 5-HT synthesis inhibitor p-chlorophenylalanine (pCPA), which increases feeding, increased hypothalamic NPY and NPY mRNA levels. pCPA (10 mg/kg in 3 μl) was administered into the third ventricle either as a single injection (n = 8) or daily for 7 days (n = 8). Control rats received a similar injection of saline. pCPA significantly increased food intake compared with controls after both single and repeated injections (P < 0.05). NPY levels were measured by radioimmunoassay in microdissected hypothalamic extracts. NPY levels in the acutely treated group were significantly increased in the paraventricular nucleus (PVN; by 41%,P = 0.01), anterior hypothalamic area (AHA; by 34%,P < 0.01) and lateral hypothalamic area (LHA; by 41%,P < 0.02). In the 7-day-treated group, NPY levels were also increased in the same areas, i.e. PVN (by 24%,P < 0.01), AHA (by 30%,P < 0.01) and LHA (by 38%,P = 0.01). There were no significant changes in the ARC or any other region or in hypothalamic NPY mRNA levels. pCPA administration increased NPY levels in several regions notably the PVN. This is a major site of NPY release, where NPY injection induces feeding. We suggest that the hyperphagia induced by pCPA is mediated by increased NPY levels and secretion in the PVN. This is further evidence for interactions between NPY and 5-HT in the control of energy homeostasis.     

Increased neuropeptide Y secretion in the hypothalamic paraventricular nucleus of obese (fa/fa) Zucker rats

NPY is synthesized in the hypothalamic arcuate nucleus (ARC), and NPY injected into the paraventricular nucleus (PVN), the main site of NPY release, induces hyperphagia and reduces energy expenditure. Hypothalamic NPY and mRNA and NPY levels are increased in fatty Zucker rats, consistent with increased NPY release. This could explain the hyperphagia and reduced energy expenditure, which lead to obesity in the fatty Zucker rat. We have therefore compared NPY secretion in the PVN of conscious fatty and lean Zucker rats using push-pull sampling. The NPY secretory profile was consistently higher in fatty Zucker rats than in lean rats throughout the 3-h study period (P < 0.01), and mean NPY secretion over the whole 3 h was increased 2-fold in the fatty rats (P < 0.001). We conclude that fatty Zucker rats have increased NPY release in the PVN. This observation further supports the hypothesis that increased activity of the NPYergic ARC-PVN pathway may contribute to obesity in the fatty Zucker syndrome.     

Oestrogenic regulation of pro-opiomelanocortin, neuropeptide Y and corticotrophin-releasing hormone mRNAs in mouse hypothalamus

It is well documented that oestrogen suppresses food intake by an action at the hypothalamic level. Using in situ hybridisation, we studied the effect of castration (CX) and short-term administration of oestradiol (E2) in CX female mice for three neuropeptides involved in feeding behaviour: two anorexigenic peptides, (i) the pro-opiomelanocortin (POMC)-derived peptide alpha-melanocyte-stimulating hormone and (ii) corticotrophin-releasing hormone (CRH), and the orexigenic peptide, (iii) neuropeptide Y (NPY). POMC-expressing neurones were mostly laterally located in the arcuate nucleus. POMC mRNA expression was decreased following CX and a single injection of E2 induced an increase in mRNA levels at 12- and 24-h time intervals. In the parvocellular area of the paraventricular nucleus, CRH mRNA levels were similarly decreased after CX and completely restored to normal levels at 12 and 24 h following E2 injection. On the other hand, the levels of NPY mRNA expressed in neurones located in the inner zone of the arcuate nucleus were increased by CX and decreased to the levels observed in intact animals by E2 injection (3-24 h). The present data suggest that oestrogen might exert an anorexigenic action by stimulating POMC and CRH mRNA expression and decreasing NPY mRNA expression in the hypothalamus.     

Effect of peripheral 2-DG on opioid and neuropeptide Y gene expression

It is well known that 2-Deoxy-d-glucose (2-DG) blocks intracellular utilization of glucose and increases food intake. The aim of the present study was to determine whether administration of 2-DG alters gene expression of the orexigenic peptides, neuropeptide Y (NPY) and endogenous opioids, in the arcuate nucleus of the hypothalamus (ARC). Male Sprague–Dawley rats were injected peripherally (i.p.) with 2-DG (200 or 400 mg/kg body weight) and were sacrificed at 2 or 6 h post injection. Half of the animals were given ad libitum access to food whereas the other half of the animals were food-deprived. 2-DG increased food intake fourfold compared to saline injected animals, but did not affect NPY mRNA levels after 2 h. Messenger RNA levels of ProDynorphin (proDYN), but not pro-opiomelanocortin (POMC) nor proEnkephalin (proENK) were significantly decreased 2 h after 2-DG injection. Administration of 400 mg/kg of 2-DG increased mRNA levels of NPY in the arcuate nucleus after six h, but only in those animals not receiving food.     

Effects of palatability-induced hyperphagia and food restriction on mRNA levels of neuropeptide-Y in the arcuate nucleus

This study examined the effect of feeding either a bland cornstarch-based diet (BCD) or a highly palatable, high fat diet containing sucrose (HPD) on hypothalamic arcuate nucleus (ARC) gene expression for neuropeptide-Y (NPY). Male Sprague–Dawley rats received either BCD ad libitum, HPD ad libitum, HPD pair-fed to the caloric intake of the BCD, or the HPD at 60% of ad libitum HPD intake for 7 days. Animals receiving the HPD ad libitum consumed more calories and gained more weight than animals receiving the BCD (P<0.001). The HPD did not affect ARC NPY mRNA levels, whether the subjects were allowed to overeat or pair-fed to the BCD (P>0.05). However, feeding the HPD at 60% of ad libitum intake of the HPD, increased NPY mRNA levels in the ARC relative to the other treatments (P<0.01). The present data are consistent with the view that NPY in ARC responds to energy deficits rather than to hyperphagia stimuli related to palatability.     

Effect of food deprivation and leptin repletion on the plasma levels of estrogen (E2) and NADPH-d reactivity in the ventromedial and arcuate nuclei of the hypothalamus in the female rats

The exact role of leptin in fasting has not been completely elucidated. To determine whether leptin can act in fasting to influence plasma estrogen levels and nitric oxide synthase reactivity in food regulating centers of the brain, we fasted female rats for 4 days and treated them i.p. with vehicle or 100 microg of recombinant mouse leptin as 1 ml on the 3rd and 4th day twice daily (10.00 and 17.00 h). Proestrus blood was collected at 10.00, 14.00, 18.00 and at 22.00 h, plasma obtained and assayed for estrogen (E2) and leptin levels. Verification of ovulation occurrence was by examining the oviduct for extruded ovum. The rat brains were removed and processed for nitric oxide synthase reactivity in the ventromedial hypothalamus (VMH) and arcuate nucleus (ARC) using NADPH-diaphorase histochemistry, a marker for neurons expressing NOS enzyme. Leptin effect on dependable variables such as food intake, water intake and body weight gain was also investigated. Four days fasting significantly decreased body weight, estrogen and postfast leptin levels, nitric oxide reactivity in the VMH and ARC nucleus and stopped ovulation in many (4 out of 5) rats fasted and given vehicle. Leptin treatment significantly increased plasma estrogen and postfast leptin levels, restored ovulation in many (4 out of 5) rats and increased nitric oxide reactivity in the VMH and ARC. Leptin significantly inhibited food intake, water intake and gain in body weight during recommenced feeding. These observations suggest that leptin could act in the pituitary-ovarian axis during fasting to improve reproductive function by partly stimulating estrogen secretion.