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.     

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. This secondary activation of NPY gene expression occurred with a slow, two-fold increase in serum LH at 1500 h, followed by a rapid ascension to peak levels at 1800 h and was associated with an increase at 1400 h of serum progesterone levels which reached their peak at 1800 h. These results demonstrate that a dynamic, biphasic augmentation in hypothalamic NPY gene expression occurs selectively on proestrus, and that the first incremental response is observed some time before the onset of preovulatory LH hypersecretion. Because preproNPY mRNA levels at 1000 h on proestrus were similar to the low levels seen on the preceding diestrous II phase, a neural timing mechanism, and not changes in ovarian hormone levels during this phase may be responsible for the increase in NPY gene expression after 1000 h of proestrus. Because of our previous observations that progesterone can rapidly augment preproNPY mRNA in the MBH and because a rise in serum progesterone occurs hours before the onset of the LH surge, we suggest that the secondary rise in preproNPY mRNA is facilitated by this antecedent increase in serum progesterone. Cumulatively, these results are in accord with the thesis that activation of hypothalamic NPY gene expression is one of the key early neural events initiated by the neural clock that times the preovulatory 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 (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.     

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 d-fenfluramine on neuropeptide Y concentration and release in the paraventricular nucleus of food-deprived rats.

Recent evidence indicates that Neuropeptide Y (NPY) is an important signal in the hypothalamic neural circuitry that stimulates feeding in the rat. Administration of d-fenfluramine (FEN) has been shown to rapidly inhibit feeding in the rat. Because food deprivation increases the levels and release of NPY in the paraventricular nucleus (PVN) of the hypothalamus, the aim of this study was to investigate whether the rapid anorectic effects of FEN in food-deprived (FD) rats are associated with alterations in the hypothalamic NPYergic system. In the first experiment, the effect of FEN (10 mg/kg) on NPY concentrations in nine microdissected hypothalamic sites was assessed by radioimmunoassay (RIA) in rats either food deprived for 3 days or fed ad lib during the experimental period. In response to food deprivation, NPY concentrations increased significantly in the PVN and arcuate nucleus, but NPY levels remained unchanged in the remaining seven hypothalamic sites. In control rats maintained on ad lib food supply, FEN injection produced little effect on NPY concentration in hypothalamic sites. However, FEN suppressed NPY levels selectively in the PVN of FD rats, so that NPY concentrations measured in the nucleus were within the range found in satiated control rats. In the second experiment, the effect of FEN on NPY release in the PVN was examined in FD rats by the push-pull cannula (PPC) technique. NPY levels in the PPC perfusate were unchanged in FD rats during the period 30-120 min after saline or FEN injection. Also, the mean rate of NPY release was similar in vehicle- and FEN-treated FD rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Disruption in neuropeptide Y and leptin signaling in obese ventromedial hypothalamic-lesioned rats

Electrolytic lesions placed in the ventromedial hypothalamus (VMH) of rats induce instant hyperphagia and excessive weight gain. Since neuropeptide Y (NPY) is a potent hypothalamic orexigenic signal, and leptin secreted by adipocytes regulates NPY output, we tested the hypothesis that altered NPYergic-leptin signaling may underlie hyperphagia in VMH-lesioned rats. VMH-lesioned rats exhibiting hyperphagia and excessive weight gain in a time-related fashion were sacrificed on days 2, 7, and 21 post-surgery. Quite unexpectedly, NPY concentrations in the hypothalamic paraventricular nucleus (PVN), a major site of NPY release for stimulation of feeding, and in other sites, such as the dorsomedial nucleus, lateral hypothalamic area and median eminence-arcuate nucleus decreased, with the earliest diminution occurring on day 2 in the PVN only. In vitro basal and K⁺-evoked NPY release from the PVN of VMH-lesioned rats was significantly lower than that of controls. Analysis of hypothalamic NPY gene expression showed that although the daily decrease in NPY mRNA from 0800 to 2200 h occurred as in control rats, NPY mRNA concentrations were markedly reduced at these times in the hypothalami of VMH-lesioned rats. Leptin synthesis in adipocytes as indicated by leptin mRNA levels was also profoundly altered in VMH-lesioned rats. The daily pattern of increase in adipocyte leptin mRNA at 2200 h from 0800 h seen in controls was abolished, higher levels of leptin gene expression at 2200 h were maintained at 0800 h. The pattern of increase in serum leptin and insulin levels diverged in VMH-lesioned rats. Serum insulin concentration increased to maximal on day 2 and remained at that level on day 21-post-lesion; serum leptin levels on the other hand, increased slowly in a time-related fashion during this period. These results demonstrate that hyperphagia and excessive weight gain in VMH-lesioned rats are associated with an overall decrease in hypothalamic NPY and augmented leptin signaling to the hypothalamus. The divergent time course of increases in serum leptin and insulin levels suggest independent mechanisms responsible for their augmented secretion, and neither these hormones nor VMH lesions altered the daily rhythm in NPY gene expression. These observations underscore the existence of an independent mechanism controlling the daily rhythm in hypothalamic NPY gene expression and suggest that leptin feedback action requires an intact VMH.     

Galanin and alcohol dependence: neurobehavioral research

It is known that microinjection of galanin (GAL) intraventricularly or in specific hypothalamic sites increases food consumption and, conversely, the intake of food increases the expression of GAL in hypothalamic sites. Ethanol (EtOH) is a calorie-rich food as well as a drug of abuse. The research reviewed here shows that GAL may play a similar role in alcohol intake. First, experiments in which GAL was microinjected into the third ventricle or the paraventricular nucleus (PVN) showed increases in EtOH consumption. The increase in EtOH consumption occurred during both the light and dark cycles after GAL injection in the third ventricle in rats with limited EtOH access. Injection of GAL did not increase food intake in rats that had been chronically drinking alcohol. GAL receptor blockade reversed these increases. Microinjection of GAL directly into the PVN also increased ad libitum EtOH intake and blockade of these receptors in the PVN inhibited ad libitum EtOH consumption. Secondly, rats administered EtOH showed increases in GAL in the PVN and related hypothalamic sites. EtOH injection and voluntary intake, both ad libitum and limited access, increased GAL gene and peptide expression in the PVN consistently across administration procedures. These experiments show that GAL injection increases alcohol intake and that the intake of alcohol increases GAL, suggesting a positive feedback relationship between alcohol intake and specific hypothalamic GAL systems. Such a relationship may contribute to the motivation to consume excessive alcoholic beverages and the development of alcohol dependence.     

c-fos expression in the rat brain following central administration of neuropeptide Y and effects of food consumption

Administration of neuropeptide Y (NPY) intracerebroventricularly (i.c.v.) results in the release of a number of hypothalamic and pituitary hormones and stimulation of feeding and suppression of sexual behavior. In this study, we sought to identify cellular sites of NPY action by evaluating perikaryal Fos-like immunoreactivity (FLI), a marker of cellular activation, in those hypothalamic and extrahypothalamic sites previously implicated in the control of neuroendocrine function and feeding behavior. Additionally, we compared the topography of FLI in these brain sites when food was either available ad libitum or withheld after NPY injection (1 nmol/3 μl, i.c.v.). The results showed that one hour after NPY injection a large number of cells in the parvocellular region of the paraventricular nucleus (PVN) were FLI-positive in the absence of food consumption. However, in association with food intake, a significant number of cells were intensely stained in the magnocellular region of the PVN. An analogous increase in FLI in association with feeding was apparent in the supraotic nucleus (SON), the dorsomedial nucleus and the bed nucleus of the stria terminalis in the hypothalamus. Anong the extrahypothalamic sites, feeding facilitated FLI in a large number of cells located in the lateral subdivision of the central amygdaloid nucleus and the lateral subdivision of the solitary tract. FLI was observed in a moderate number of cells in the hypothalamic arcuate nucleus (ARC) and ventromedial nucleus, and this response was not changed by feeding. Cumulatively, these results show that neurons in a number of discrete hypothalamic and extrahypothalamic sites, previously implicated in the control of neuroendocrine function and feeding behavior, are activated by NPY and further, a divergent pattern of c-fos expression emerged in some of these sites if feeding occurs after NPY injection. Stimulation of FLI in cells of the PVN, SON and ARC by NPY imply the presence of NPY target cells that play a role in the neuroendocrine control of pituitary function. The finding that NPY induced FLI in cells located in the parvocellular subdivision of the PVN even in the absence of feeding, imply that cells involved in initiation of food intake by NPY may reside in this subdivision of the PVN. On the other hand, the appearance of Fos-cells in the magnocellular subdivision of the PVN in response to feeding, suggests neural mechanisms that operate during the post-ingestion period, including those associated with termination of NPY-induced feeding, may impinge upon this subdivision of the PVN.     

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.     

Effects of food restriction on the hypothalamic prepro-orexin gene expression in genetically obese mice

Orexins, which are identical to hypocretins, are novel hypothalamic orexigenic peptides. We examined the effects of food restriction on the expression of the prepro-orexin gene in control (C57Bl/6J) and genetically obese mice (ob/ob and db/db), using in situ hybridization histochemistry. Dry food was given 3 g/day to each obese mouse for 2 weeks. Food restriction caused a significant increase of the prepro-orexin gene expression in obese mice in comparison with ad libitum fed animals. Although the levels of the expression of the prepro-orexin gene in obese mice were significantly lower than those in C57Bl/6J mice during feeding ad libitum, food restriction caused an increase in the expression of the prepro-orexin gene in the hypothalamus of obese mice. The expression of the neuropeptide Y (NPY) gene was increased significantly in the arcuate nucleus of obese mice compared to that of control mice during feeding ad libitum. Food restriction for 2 weeks also caused a significant increase of the expression in the NPY gene in all groups. These results indicate that the hypothalamic prepro-orexin gene could be upregulated by food restriction without leptin signal in genetically obese mice.     

Gene expression of neuropeptide Y in the nucleus of the solitary tract is activated in rats under restricted daily feeding but not under 48-h food deprivation

The two neuropeptide Y (NPY) systems innervating the hypothalamic paraventrivular nucleus were examined regarding their roles in the prefeeding corticosterone peak developed under restricted daily feeding (RF). Protein and mRNA levels of NPY were measured in the arcuate nucleus (ARC) and the nucleus of the solitary tract (NST) in rats under 48-h food deprivation (48-hFD), RF, and 72-h food deprivation imposed after RF (post-RF 72-hFD) with 7 days of ad libitum feeding in between. NPY protein and mRNA levels in the ARC significantly increased with 48-hFD and decreased with re-feeding, whereas those in the NST were not changed by 48-hFD. When rats had RF imposed with free access to food from 10.00 to 12.00 h (lights on from 06.00 to 18.00 h) for 3 weeks, NPY concentrations in the ARC increased at 10.00 h, just prior to the daily meal, but those in the NST did not change significantly throughout the period examined. On the other hand, NPY mRNA levels in both the ARC and NST increased before the meal supply and remained high for 4 h after feeding. Under post-RF 72-hFD, the prefeeding peak of NPY mRNA was detected in the NST, but NPY mRNA levels in the ARC were continuously high throughout the 24-h period. These findings indicate that the NPY neurons from the NST are specifically activated by RF, whereas those from the ARC are generally stimulated by an increased food demand.     

Reduction of Luteinzing Hormone Secretion Induced by Long-Term Feed Restriction in Male Rats Is Associated with Increased Expression of GABA-Synthesizing Enzymes Without Alterations of GnRH Gene Expression

In rats, fasting or restriction of feed intake impairs the activity of the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator which results in reduced luteinizing hormone (LH) secretion. It is still unknown which neurotransmitters are involved in this phenomenon. However, it is known that increased GABA concentrations in the hypothalamus reduce GnRH biosynthesis and release. Therefore, we examined whether 17 days of feed restriction in male rats affected the hypothalamic gene expression of GnRH and the GABA-synthesizing enzymes glutaminase (GLS) and glutamic acid decarboxylase-which exists in two forms, GAD67 and GAD65-in the mammalian brain. Furthermore, the expression of the GnRH receptor (GnRH-R) and the GABA transporter 1 (GAT-1) were investigated. Feed restriction resulted in a 75% reduction in body weight (b.w.) compared to rats fed ad libitum. Serum concentrations of LH and testosterone in the feed restricted group were significantly reduced to approximately 15% of that of rats fed ad libitum, while the FSH concentration remained unchanged. In the mediobasal hypothalamus (MBH) where GnRH is released into the portal vessels, mRNA levels of GAD67 and GLS were increased twofold compared to rats fed ad libitum while no changes were observed in the preoptic area of the hypothalamus (POA) where GnRH is biosynthesised. Neither the expression of preoptic GnRH mRNA nor the expression of GAD65 and of GnRH-R mRNA in both hypothalamic structures was affected by feed restriction. In the anterior pituitary, a significant reduction of the expression of GnRH-R, LH-beta and the alpha subunit was observed in the feed restricted rats, whereas FSH-beta mRNA levels remained constant. Thus, feed restriction selectively increased the expression of GABA-synthesizing enzymes in the MBH but did not modify GnRH expression in the POA. However, the reduced expression of the LH-beta- and alpha-subunit and of the GnRH-R in the anterior pituitary indicates that pulsatile GnRH release may have been attenuated or even abolished. We suggest, that enhanced expression of GABA-synthesizing enzymes reflects increased GABAergic neurotransmission and thereby reducing GnRH release from the MBH.     

Neuropeptide Y (NPY) May Integrate Responses of Hypothalamic Feeding Systems and the Hypothalamo-Pituitary-Adrenal Axis

Neuropeptide Y (NPY) is a powerful stimulus to food intake in the rat. Exogenous NPY given into the third ventricle or into the paraventricular nucleus (PVN) of the hypothalamus stimulates both food consumption as well as the hypothalamus-pituitary-adrenal (HPA) axis. Presumably NPY activates the adrenocortical system through direct stimulation of CRF containing cells in the PVN. Food intake is also a major regulator of adrenocortical activation. Rhythms in HPA axis activity follow rhythms in food consumption, and rats that have been food deprived overnight have inhibited HPA axis responses to restraint stress and corticosteroid feedback the following morning. To investigate the interaction of NPY with both feeding and HPA axis activation three sets of experiments were performed: Animals fed ad lib were injected icv with NPY (2.5 μg) and allowed access to food or not post injection; animals were fasted overnight prior to NPY injection; finally, dose response experiments were performed to examine the relative sensitivities of feeding and HPA axis activation to exogenous NPY. Ad lib fed animals allowed access to food after NPY injection had slightly greater ACTH responses to NPY while glucocorticoid and insulin responses were not significantly different from ad lib fed animals not allowed access to food post injection. Animals allowed to eat post injection had significantly decreased food consumption the night following injection, however, total 24 h food consumption was not different between these animals and those given food 8 h post NPY injection. In overnight fasted animals NPY injections produced ACTH responses of equal magnitude to those in ad lib fed animals. Insulin responses to NPY were significantly elevated compared to CSF controls in overnight fasted animals. Dose response studies revealed that the adrenocortical system responds to icv NPY with at least as great sensitivity as feeding systems. NPY is discussed as a potential integrator of feeding and responsiveness in the HPA axis.     

Leptin Regulation of Agrp and Npy mRNA in the Rat Hypothalamus

Agouti-related protein (AGRP) is synthesized in the same neurones in the arcuate nucleus as neuropeptide Y (NPY), another potent orexigenic peptide. AGRP antagonizes the action of alpha-melanocyte stimulating hormone, a derivative of pro-opiomelanocortin (POMC) at the hypothalamic MC4 receptor to increase food intake. Although leptin has been shown to regulate Agrp/Npy and Pomc-expressing neurones, there are differences with respect to Agrp regulation in leptin receptor-deficient mice and rats. Unlike the obese leptin receptor-deficient db/db mouse, which exhibits upregulation of Agrp mRNA expression in the medial basal hypothalamus (MBH) compared to lean controls, the obese leptin receptor-deficient (faf; Koletsky) rat does not exhibit upregulation of Agrp expression. To determine whether this represents a general difference between leptin receptor-deficient mice and rats, neuropeptide gene expression was analysed in the MBH of lean and obese rats segregating for a different leptin receptor mutation, Leprfa (Zucker). Fasting in lean rats (+/fa) for 72 h significantly increased Agrp and Npy mRNA expression, and decreased Pomc mRNA expression as detected by a sensitive solution hybridization/S1 nuclease protection assay. Npy mRNA levels were significantly increased in fed obese fa/fa compared to lean rats, and further increased in the obese animals after fasting. In contrast, Agrp mRNA levels did not differ between fed lean and fed obese rats, and fasting did not significantly change Agrp levels in obese rats. To determine whether the change in Agrp expression that occurs with food deprivation in lean rats could be prevented by leptin replacement, Sprague-Dawley rats were fasted and infused via subcutaneous osmotic micropumps for 48 h with either saline or recombinant mouse leptin. Fasting significantly increased Agrp and Npy, and decreased Pomc mRNA levels. Leptin infusion almost completely reversed these changes such that there was no significant difference between the levels in the fasted rats and those that were fed ad libitum. Thus, in fasted lean rats, Agrp and Npy are upregulated in parallel when leptin levels fall and are downregulated by leptin infusion. By contrast, the absence of a functional leptin receptor results in the upregulation of Npy but not Agrp mRNA.     

Effects of repeated administration of mifepristone and 8-OH-DPAT on expression of preproneuropeptide Y mRNA in the arcuate nucleus of obese Zucker rats.

Neuropeptide Y (NPY) is an important hypothalamic regulator of feeding behavior. In this study we have investigated the regulation of the expression of preproNPY mRNA in male obese and lean Zucker rats by in situ hybridization. These animals represent a model of genetic obesity with hyperphagia, hyperinsulinemia and altered endocrine functions. Obese Zucker rats, treated for 12 days with 0.9% saline, had about 210% higher level of basal preproNPY mRNA expression in the arcuate nucleus when compared to their lean littermate controls. Repeated administrations of 8-hydroxy-dipropylaminotetralin (8-OH-DPAT), a serotonergic 5-HT1A agonist, or mifepristone, a glucocorticoid receptor antagonist, did not modify the basal expression of preproNPY mRNA in the Zucker phenotypes. The 8-OH-DPAT treatment significantly reduced hyperinsulinemia in obese Zucker rats without changing plasma glucose levels. The mifepristone treatment significantly increased plasma corticosterone levels in lean animals, but not in obese animals. The present study demonstrates enhanced expression of preproNPY mRNA in the arcuate nucleus in obese Zucker rats suggesting an involvement of NPY in the pathophysiology of the hyperphagic syndrome and genetically determined obesity in Zucker rats. Neither the antagonism of glucocorticoid receptors by mifepristone, nor repeated treatment with 8-OH-DPAT resulting in reduced insulin levels in obese Zucker rats, modified the basal expression of preproNPY mRNA in the arcuate nucleus.     

Diurnal variations in the feeding responses to norepinephrine, neuropeptide Y and galanin in the PVN.

The feeding responses elicited by injection of norepinephrine (NE), neuropeptide Y (NPY) and galanin (GAL) into the paraventricular nucleus (PVN) were studied at two different times of the dark (active) cycle in male Sprague-Dawley rats maintained ad lib on pure nutrient diets. The feeding response elicited by NE in the PVN, characterized by a potent and selective stimulatory effect on ingestion of the carbohydrate diet, was significantly stronger during the early dark period (+11.7 kcal over vehicle baseline) relative to the late dark period (+7.6 kcal). A similar pattern of effects was observed with NPY in the PVN, which also selectively potentiated carbohydrate ingestion. The effects of GAL were different from those observed with NE and NPY. Whereas the total amount of food consumed after PVN GAL injection was similar in the early and late dark periods, the macronutrient selection patterns exhibited at these two times were different. During the early dark period, PVN GAL had a small stimulatory effect on carbohydrate, in addition to a strong enhancement of fat intake; in the late dark period, in contrast, GAL stimulated intake only of the fat diet. These findings may reflect differential functions of these hypothalamic neurotransmitters in controlling nutrient ingestion at different periods of the circadian cycle.     

Increased NPY activity in the PVN contributes to food-restriction induced reductions in blood pressure in aortic coarctation hypertensive rats

We hypothesized that hypothalamic NPYergic mechanisms mediate the blood pressure lowering effect of caloric restriction in hypertensive rats. Aortic coarctation-induced (AC) hypertensive rats (n=25) were assigned to either an ad libitum fed control group (AL) or food restricted group (FR; 60% of AL consumption) for 3 weeks. Rats were instrumented chronically with vascular catheters and bilateral guide cannulae directed at the paraventricular hypothalamic nuclei (PVN). Blood pressure (BP) and heart rate (HR) responses to bilateral PVN microinjection of saline (200 nl) or the putative NPY receptor antagonists [D-Trp32]NPY(1-36) (3.3 micrograms/200 nl) and [D-Tyr27,36 Thr32]NPY(27-36) (D-NPY(27-36); 3.3 micrograms/200 nl) were determined. The FR rats were then refed and cardiovascular responses to PVN injections of NPY receptor antagonists were again determined. FR rats had significantly reduced resting BP (159+/-4 vs. 129+/-4 mmHg) and HR (360+/-11 vs. 326+/-9 bpm) compared to AL controls. Refeeding restored BP and HR of FR rats to levels similar to AL (BP=153+/-4 mmHg, HR=359+/-11 bpm). PVN administration of [D-Trp32]NPY produced foraging behavior and concurrent increases in BP and HR in FR, AL and Re-fed rats. The behavioral activation suggests that [D-Trp32]NPY(1-36) produced activation of NPY receptors. In contrast, D-NPY (27-36) did not produce any behavioral response or affect BP or HR in AL or Re-fed rats. In FR rats, D-NPY (27-36) produced significant increases in BP (peak=15+/-3 mmHg) which partially reversed the effect of FR on BP. Thus, in FR rats with reduced BP, PVN administration of an NPY receptor antagonist increases BP. NPY blockade in the PVN accounted for about 50% of the BP effect of food restriction, thus other mechanisms are likely to be involved. These findings are consistent with the hypothesis that NPYergic mechanisms may contribute to the reduction of BP produced by food restriction.