Reserpine-induced immunocytochemical change of neuropeptide Y in the hypothalamic arcuate nucleus

The effect of reserpine on neuropeptide Y immunoreactive (NPY-IR) neurons in the rat hypothalamic arcuate nucleus was examined by immunocytochemical techniques. Although only NPY-IR fibers and terminals were distributed in this nucleus in untreated and saline treated rats, single treatment of reserpine (10 mg/kg, i.p.) visualized abundant NPY-IR neuronal cell bodies: the increase began at 12 h of postinjection, reached its maximal level at 48 h, and returned to its normal level at 96 h. Pretreatment of nialamide, a monoamine oxidase inhibitor, prevented these acute reserpine-induced changes, suggesting reserpine acts on NPY neurons through monoaminergic mechanism. Chronic treatment of haloperidol (5 mg/kg, once daily for 5 days) a dopamine receptor antagonist, could induce the similar increase of NPY immunoreactivity. However, interruption of adrenergic and serotonergic neurotransmissions by chronic treatment of propranorol and methysergide, or chemical lesions of ascending noradrenergic and serotononergic pathways by 6-hydroxydopamine and 5,6-dihydroxytryptamine, could not induce any immunoreactive increase of NPY in arcuate neurons. These findings strongly suggest that reserpine-induced NPY increase occurs through dopaminergic afferents in hypothalamic arcuate neurons. Special issue dedicated to Dr. Kinya Kuriyama.     

Peripheral insulin administration attenuates the increase in neuropeptide Y concentrations in the hypothalamic arcuate nucleus of fasted rats

Fasting increases neuropeptide Y (NPY) concentrations in the arcuate nucleus (ARC), its site of synthesis, and in other regions of the rat hypothalamus. Neuropeptide Y is a potent central orexigenic agent and may therefore stimulate appetite during fasting. We tested the hypothesis that low plasma insulin levels stimulate ARC levels of NPY in fasted rats. Compared with freely fed controls (n = 8), rats fasted for 72 h (n = 8) showed significantly lower plasma insulin levels (28.9 ± 1.6 vs. 52.6 ± 5.7 pmol/l; p < 0.001) and higher ARC NPY concentrations (14.2 ± 1.8 vs. 8.4 ± 2.2 fmol/μg protein; p < 0.001). Fasted rats treated with subcutaneous insulin (5 U/kg/day; n = 10), which nearly normalized plasma insulin (46.6 ± 2.8 pmol/l), showed intermediate ARC NPY levels (11.2 ± 1.4 fmol/μg protein; p < 0.01 vs. controls and untreated fasted rats). Insulin administered peripherally, therefore, attenuates fasting-induced NPY increases in the ARC, supporting the hypothesis that hypoinsulinemia stimulates hypothalamic NPY.     

Moderate long-term modulation of neuropeptide Y in hypothalamic arcuate nucleus induces energy balance alterations in adult rats

Neuropeptide Y (NPY) produced by arcuate nucleus (ARC) neurons has a strong orexigenic effect on target neurons. Hypothalamic NPY levels undergo wide-ranging oscillations during the circadian cycle and in response to fasting and peripheral hormones (from 0.25 to 10-fold change). The aim of the present study was to evaluate the impact of a moderate long-term modulation of NPY within the ARC neurons on food consumption, body weight gain and hypothalamic neuropeptides. We achieved a physiological overexpression (3.6-fold increase) and down-regulation (0.5-fold decrease) of NPY in the rat ARC by injection of AAV vectors expressing NPY and synthetic microRNA that target the NPY, respectively. Our work shows that a moderate overexpression of NPY was sufficient to induce diurnal over-feeding, sustained body weight gain and severe obesity in adult rats. Additionally, the circulating levels of leptin were elevated but the immunoreactivity (ir) of ARC neuropeptides was not in accordance (POMC-ir was unchanged and AGRP-ir increased), suggesting a disruption in the ability of ARC neurons to response to peripheral metabolic alterations. Furthermore, a dysfunction in adipocytes phenotype was observed in these obese rats. In addition, moderate down-regulation of NPY did not affect basal feeding or normal body weight gain but the response to food deprivation was compromised since fasting-induced hyperphagia was inhibited and fasting-induced decrease in locomotor activity was absent.These results highlight the importance of the physiological ARC NPY levels oscillations on feeding regulation, fasting response and body weight preservation, and are important for the design of therapeutic interventions for obesity that include the NPY.     

Peripheral ghrelin deepens torpor bouts in mice through the arcuate nucleus neuropeptide Y signaling pathway

Many small mammals have the ability to enter torpor, characterized by a controlled drop in body temperature (Tb). We hypothesized that ghrelin would modulate torpor bouts, because torpor is induced by fasting in mice coincident with elevated circulating ghrelin. Female National Institutes of Health (NIH) Swiss mice were implanted with a Tb telemeter and housed at an ambient temperature (Ta) of 18 degrees C. On fasting, all mice entered a bout of torpor (minimum Tb: 23.8+/-2.0 degrees C). Peripheral ghrelin administration (100 microg) during fasting significantly deepened the bout of torpor (Tb minimum: 19.4+/-0.5 degrees C). When the arcuate nucleus (ARC) of the hypothalamus, a ghrelin receptor-rich region of the brain, was chemically ablated with monosodium glutamate (MSG), fasted mice failed to enter torpor (minimum Tb=31.6+/-0.6 degrees C). Furthermore, ghrelin administration had no effect on the Tb minimum of ARC-ablated mice (31.8+/-0.8 degrees C). Two major pathways that regulate food intake reside in the ARC, the anorexigenic alpha-melanocyte stimulating hormone (alpha-MSH) pathway and the orexigenic neuropeptide Y (NPY) signaling pathway. Both Ay mice, which have the alpha-MSH pathway blocked, and Npy-/-mice exhibited shallow, aborted torpor bouts in response to fasting (Tb minimum: 29.1+/-0.6 degrees C and 29.9+/-1.2 degrees C, respectively). Ghrelin deepened torpor in Ay mice (Tb minimum: 22.8+/-1.3 degrees C), but had no effect in Npy-/-mice (Tb minimum: 29.5+/-0.8 degrees C). Collectively, these data suggest that ghrelin's actions on torpor are mediated via NPY neurons within the ARC.     

Effects of pinealectomy and exogenous melatonin on ghrelin and peptide YY in gastrointestinal system and neuropeptide Y in hypothalamic arcuate nucleus: immunohistochemical studies in male rats

It is reported that the pineal gland and its main hormone melatonin may have a role in the regulation of ghrelin synthesis in the brain. Stomach is the place where ghrelin is predominantly expressed and secreted. One aim of this study was to investigate possible effects of pinealectomy and melatonin treatment on gastric ghrelin amount. The studies on the effects of the pineal gland on leptin and ghrelin arises the question whether the pineal gland has also effects on the other energy-regulatory peptides such as peptide YY (PYY) and neuropeptide Y (NPY). Therefore, we also aimed to investigate the changes in the immunohistochemical staining of intestinal PYY and hypothalamic NPY following pinealectomy and melatonin treatment. Serum PYY levels were also investigated. Sprague-Dawley rats were divided into four groups as sham-operated (SHAM), sham-operated with melatonin treatment (SHAM-MT), pinealectomised (PNX) and melatonin-treated PNX (PNX-MT) groups. The cells immunostained for ghrelin were abundant throughout the gastric mucosa in all the groups. Neither pinealectomy nor exogenous melatonin affected significantly immunohistochemical staining of ghrelin in stomach. Pinealectomy resulted in a significant increase in immunohistochemical staining of PYY in ileum. The results of serum PYY measurement corresponded closely to the data obtained by immunohistochemical analysis of PYY in ileum, being significantly lower and higher in SHAM and PNX groups, respectively. Pinealectomy caused a decrease in NPY synthesis in ARC as understood from low immunohistochemical staining of NPY. Melatonin treatment increased NPY synthesis in SHAM rats and restored reduction in NPY synthesis caused by pinealectomy. In conclusion, the pineal gland and its main hormone melatonin can be suggested to have a role in the regulation of NPY synthesis in ARC and PYY in gastrointestinal system.     

The arcuate nucleus and neuropeptide Y contribute to the antitumorigenic effect of calorie restriction

Calorie restriction (CR) is known to have profound effects on tumor incidence. A typical consequence of CR is hunger, and we hypothesized that the neuroendocrine response to CR might in part mediate CR's antitumor effects. We tested CR under appetite suppression using two models: neuropeptide Y (NPY) knockout mice and monosodium glutamate-injected mice. While CR was protective in control mice challenged with a two-stage skin carcinogenesis model, papilloma development was neither delayed nor reduced by CR in the monosodium glutamate-treated and NPY knockout mice. Adiponectin levels were also not increased by CR in the appetite-suppressed mice. We propose that some of CR's beneficial effects cannot be separated from those imposed on appetite, and that NPY neurons in the arcuate nucleus of the hypothalamus are involved in the translation of reduced intake to downstream physiological and functional benefits.     

Decreased sensory responses in osteocalcin null mutant mice imply neuropeptide function

Osteocalcin, the most abundant member of the family of extracellular mineral binding gamma-carboxyglutamic acid proteins is synthesized primarily by osteoblasts. Its affinity for calcium ions is believed to limit bone mineralization. Several of the numerous hormones that regulate synthesis of osteocalcin, including glucocorticoids and parathyroid hormone, are also affected by stressful stimuli that require energy for an appropriate response. Based on our observations of OC responding to stressful sensory stimuli, the expression of OC in mouse and rat sensory ganglia was confirmed. It was thus hypothesized that the behavioral responses of the OC knockout mouse to stressful sensory stimuli would be abnormal. To test this hypothesis, behaviors related to sensory aspects of the stress response were quantified in nine groups of mice, aged 4-14 months, comparing knockout with their wild-type counterparts in six distinctly different behavioral tests. Resulting data indicated the following statistically significant differences: open field grooming frequency following saline injection, wild-type > knockout; paw stimulation with Von Frey fibers, knockout < wild-type; balance beam, knockout mobility < WT; thermal sensitivity to heat (tail flick), knockout < wild-type; and cold, knockout < wild-type. Insignificant differences in hanging wire test indicate that these responses are unrelated to reduced muscle strength. Each of these disparate environmental stimuli provided data indicating alterations of responses in knockout mice that suggest participation of osteocalcin in transmission of information about those sensory stimuli.     

Neither agouti-related protein nor neuropeptide Y is critically required for the regulation of energy homeostasis in mice

Agouti-related protein (AgRP), a neuropeptide abundantly expressed in the arcuate nucleus of the hypothalamus, potently stimulates feeding and body weight gain in rodents. AgRP is believed to exert its effects through the blockade of signaling by alpha-melanocyte-stimulating hormone at central nervous system (CNS) melanocortin-3 receptor (Mc3r) and Mc4r. We generated AgRP-deficient (Agrp(-/-)) mice to examine the physiological role of AgRP. Agrp(-/-) mice are viable and exhibit normal locomotor activity, growth rates, body composition, and food intake. Additionally, Agrp(-/-) mice display normal responses to starvation, diet-induced obesity, and the administration of exogenous leptin or neuropeptide Y (NPY). In situ hybridization failed to detect altered CNS expression levels for proopiomelanocortin, Mc3r, Mc4r, or NPY mRNAs in Agrp(-/-) mice. As AgRP and the orexigenic peptide NPY are coexpressed in neurons of the arcuate nucleus, we generated AgRP and NPY double-knockout (Agrp(-/-);Npy(-/-)) mice to determine whether NPY or AgRP plays a compensatory role in Agrp(-/-) or NPY-deficient (Npy(-/-)) mice, respectively. Similarly to mice deficient in either AgRP or NPY, Agrp(-/-);Npy(-/-) mice suffer no obvious feeding or body weight deficits and maintain a normal response to starvation. Our results demonstrate that neither AgRP nor NPY is a critically required orexigenic factor, suggesting that other pathways capable of regulating energy homeostasis can compensate for the loss of both AgRP and NPY.     

Paraventricular nucleus injections of peptide YY and neuropeptide Y preferentially enhance carbohydrate ingestion

Neuropeptide Y (NPY) injected into the paraventricular nucleus (PVN) is known to elicit a powerful feeding response in satiated, brain-cannulated rats [41, 42, 43]. The present experiment investigates the effect of peptide YY (PYY), a structurally-related peptide, on feeding behavior and, in addition, the effects of both PYY and NPY on the pattern of macronutrient selection. Injection of PYY directly into the PVN, in doses ranging from 7.8 to 235 pmol/0.3 μl, caused a strong, dose-dependent stimulation of feeding behavior, as well as a small stimulation of drinking behavior, in satiated rats. The mean latency to eat was 9.3 min, with substantial feeding occurring within 30 min of the injection. At low doses, the increase in feeding was seen predominantly during the first hr. At the highest dose, in contrast, food intake continued to increase progressively over the next few hr, such that by 4 hr postinjection food intake was more than 20 g over vehicle baseline. In 1 hr tests with 3 pure macronutrient (protein, fat and carbohydrate) diets simulataneously available, PYY and NPY (78 pmol/0.3 μl) both elicited a strong and selective increase in carbohydrate consumption, with little or no effect on protein or fat consumption. These results suggest that hypothalamic receptors sensitive to PYY and NPY may participate in the control of carbohydrate consumption.     

Opposite regulation of hypothalamic orexin and neuropeptide Y receptors and peptide expressions in obese Zucker rats

Many hyothalamic neuropeptides are involved in the regulation of food intake and body weight. The orexins (OX) which are synthesized in the lateral hypothalamus are among the most recently characterized whereas neuropeptide Y (NPY) belongs to a group of "older" peptides extensively studied for their effects on feeding behavior. Both stimulate food ingestion in rodents. In this experiment, we measured the expressions of these peptides as well as of their receptors (OX1-R and OX2-R, Y1 and Y5) in the hypothalamus of obese hyperphagic and lean Zucker rats by real-time RT-PCR using the TaqMan apparatus. NPY mRNA expression in the obese rats was significantly increased by a factor of 10 (P < 0.002) whereas expressions of the Y1 and Y5 receptors were decreased by 25% (P < 0.01) and 50% (P < 0.002), respectively. Their prepro-orexin mRNA expression was more than twofold decreased (P < 0.01) and expressions of their OX receptors 1 and 2 mRNA were five- and fourfold increased (P < 0.05), respectively. An inverse phenomenon was therefore noted between the two peptides: for NPY, increased levels and downregulation of receptors; and for OX, diminished levels with upregulation of receptors. The reasons for these changes might be linked to the absence of leptin signaling as similar profiles are found in the ob/ob mice. For orexins at least, other factors such as hyperglycemia might be involved. Based on anatomical considerations, a direct effect of NPY or of other brain peptides such as CRH cannot be excluded. We conclude that the diminution in the OX tone might participate in a counterregulatory system necessary to limit the noxious effects of NPY on food intake and body weight.     

Synaptic interactions between ghrelin- and neuropeptide Y-containing neurons in the rat arcuate nucleus

Morphological relationships between neuropeptide Y- (NPY) like and ghrelin-like immunoreactive neurons in the arcuate nucleus (ARC) were examined using light and electron microscopy techniques. At the light microscope level, both neuron types were found distributed in the ARC and could be observed making contact with each other. Using a preembedding double immunostaining technique, some NPY-immunoreactive axon terminals were observed at the electron microscope level to make synapses on ghrelin-immunoreactive cell bodies and dendrites. While the axo-somatic synapses were mostly symmetric in nature, the axo-dendritic synapses were both symmetric and asymmetric. In contrast, ghrelin-like immunoreactive (ghrelin-LI) axon terminals were found to make synapses on NPY-like immunoreactive (NPY-LI) dendrites although no NPY-like immunoreactive perikarya were identified receiving synapses from ghrelin-LI axon terminals. NPY-like axon terminals were also found making synapses on NPY-like neurons. Axo-axonic synapses were also identified between NPY- and ghrelin-like axon terminals. The present study shows that NPY- and ghrelin-LI neurons could influence each other by synaptic transmission through axo-somatic, axo-dendritic and even axo-axonic synapses, and suggests that they participate in a common effort to regulate the food-intake behavior through complex synaptic relationships.     

Role of dorsomedial hypothalamic neuropeptide Y in energy homeostasis

Hypothalamic neuropeptide Y (NPY) is primarily expressed in the arcuate nucleus (Arc) and the dorsomedial hypothalamus (DMH). Although Arc NPY gene expression is responsive to circulating leptin, the regulation of DMH NPY expression is leptin-independent. DMH NPY expression is increased in response to chronic food restriction, but not acute food deprivation. DMH NPY expression is elevated in pair-fed OLETF rats lacking cholecystokinin (CCK)-1 receptors. A role for CCK in controlling DMH NPY expression is demonstrated by the down-regulation of DMH NPY by parenchymal DMH CCK administration in intact rats. Moreover, access to running wheels normalizes body weight and prevents altered DMH NPY expression of OLETF rats. Together, these data suggest that DMH NPY plays an important role in feeding and body weight control.     

AAV mediated expression of anti-sense neuropeptide Y cRNA in the arcuate nucleus of rats results in decreased weight gain and food intake

Neuropeptide Y (NPY) is the most potent stimulant of feeding when administered by intracerebroventricular injection. Despite this, there is conflicting evidence as to its importance in the regulation of daily food intake and energy balance. It has been suggested that whilst it is important in the response to starvation it has little role in the regulation of daily food intake. To investigate the role of NPY in the regulation of food intake, anti-sense cRNA to NPY was expressed in the arcuate nucleus of adult male rats. The anti-sense NPY (AS-NPY) construct was initially tested in vitro and there was a decrease of approximately 50% in NPY release from anti-sense treated cells compared to controls (16.3 +/- 2.0 fmol/L [AS-NPY] vs 37.3 +/- 7.7 fmol/L [control], mean +/- SEM p < 0.05). NPY release from hypothalamic explants from anti-sense injected animals was decreased by over 50% compared to those from controls at both 15 and 20 days after AAV injection (15 days 42% +/- 6.5% [AS-NPY] vs 100% +/- 36% [control], 20 days 41% +/- 6% [AS-NPY] vs 100% +/- 27% [control] mean+/-SEM, p < 0.05). In a study lasting for 50 days, weight gain was significantly lower in anti-sense injected animals from day 16 (day 16: 6.25 +/- 1.10 g [AS-NPY] vs 9.42 +/- 0.65 g [control] mean +/- SEM, p < 0.05) and remained so until the end of the study when they had gained approximately 40% less weight than controls (day 50: 52.0 +/- 9.6 g [AS-NPY] vs 82.0 +/- 6.3 g [control] mean +/- SEM, p < 0.01). Cumulative food intake was significantly lower in the anti-sense injected animals from day 23 (day 23: 225.8 +/- 1.9 g [AS-NPY] vs 250.6 +/- 8.7 g [control], mean +/- SEM, p < 0.05) and remained so until the end of the study (day 50: 834.5 +/- 14.8 g [AS-NPY] vs 926.0 +/- 31.7 g [control], mean +/- SEM, p < 0.05). Similarly mean daily food intake was also reduced in the anti-sense injected animals (days 7-14: 24.9 +/- 0.4 g/day [AS-NPY] vs 27.2 +/- 0.4 g/day [control], mean +/- SEM, p < 0.01). These data are supportive of a role for NPY in the regulation of daily food intake as well as in response to starvation.     

Expression of NPY Y1 and Y5 receptors in the hypothalamic paraventricular nucleus of aged Fischer 344 rats

Many mammals, nearing the end of life, spontaneously decrease their food intake and body weight, a stage we refer to as senescence. The spontaneous decrease in food intake and body weight is associated with attenuated responses to intracerebroventricular injections of neuropeptide Y (NPY) compared with old presenescent or with young adult rats. In the present study, we tested the hypothesis that this blunted responsiveness involves the number and expression of hypothalamic paraventricular nucleus (PVN) Y(1) and/or Y(5) NPY receptors, both of which are thought to mediate NPY-induced food intake. We found no significant difference in mRNA levels, via quantitative PCR, for Y(1) and Y(5) receptors in the PVN of senescent vs. presenescent rats. In contrast, immunohistochemistry indicated that the number of PVN neurons staining for Y(1) receptor protein was greater in presenescent compared with senescent rats. We conclude that a decreased expression and number of Y(1) or Y(5) receptors in the PVN cannot explain the attenuated responsiveness of the senescent rats to exogenous NPY.     

Neuronal interactions between neuropeptide Y (NPY) and catecholaminergic systems in the rat arcuate nucleus as shown by dual immunocytochemistry

Several recent studies have suggested interactions between catecholamine (CA) and neuropeptide Y (NPY) neuronal systems in the rat brain. In order to obtain morphological evidence for such CA/NPY interactions in the arcuate nucleus, we have used a double immunostaining procedure using an anti-tyrosine hydroxylase (TH) antiserum as a marker for catecholamine neurons and an anti-NPY antiserum. This double staining, where the first staining is silver-gold intensified, was detectable at both light and electron microscopic levels. In semi-thin sections, a substantial overlap and close proximity of TH-immunopositive neurons and NPY neuronal elements could be seen within the arcuate nucleus. At the electron microscopic level, direct appositions between TH- and NPY-immunoreactive structures could be detected. These appositions were of axosomatic, axodendritic or axoaxonic types without any synaptic membrane differentiation. Moreover, direct appositions between NPY-immunoreactive structures have also been observed. This morphological study showing appositions between TH and NPY neuronal systems suggest direct interactions between these two systems in the arcuate nucleus.