Pertussis toxin inhibits neuropeptide Y-induced feeding in rats

Neuropeptide Y (NPY) is the most powerful peptide drug stimulating feeding in rats. Rats with paraventricular hypothalamic (PVH) cannulae were used to investigate the mechanisms involved in NPY-induced feeding. Consistent with previous reports, injection of 2 μg of NPY into the PVH significantly increased the cumulative food intake over 1-, 2- and 4-hr periods. Ad lib feeding decreased significantly two days after pertussis toxin (PT) administration, but recovered to nearly normal levels on the fourth day. PT had no immediate effect on NPY-induced feeding; however, four days after PT was injected NPY (2 μg) did not increase the food intake compared to control. In vitro investigations showed that isoproterenol-stimulated adenylate cyclase activity in the hypothalamus of control rats was inhibited by NPY. In PT-treated rats, however, no inhibition of cAMP production was observed. These results suggest that cAMP may mediate NPY-induced feeding and that a PT-sensitive G protein may be involved in this signal transduction.     

Peripheral ghrelin participates in the glucostatic signaling mediated by the ventromedial and lateral hypothalamus neurons

Employing immunohistochemistry techniques, we examined the c-fos expression in different hypothalamic areas, when plasma glucose levels were modified by the administration of insulin and 2-deoxyglucose (2-DG) respectively. Subsequently, the hypoglycemia produced by an injection of insulin significantly increased feeding concomitant to higher c-fos expression in the arcuate nucleus (ARC), paraventricular nucleus (PVN), dorsomedial hypothalamus (DMH) and lateral hypothalamus (LH), while no statistical changes in the ventromedial hypothalamus (VMH) were found. Also, the glucopenia induced by 2-DG administration produced similar stimulatory effects on appetite and the neuronal activity affecting all the hypothalamic areas studied, including the VMH. The peripheral blockade of the orexigenic hormone ghrelin with a specific antibody (AGA) significantly decreased food intake as induced from acute hypoglycemia and glucopenia. Curiously, the conjoint AGA and insulin or 2-DG administration produced a differential effect on the hypothalamic neurons analyzed, by increasing the number of c-fos positive neurons in the ARC, PVN and DMH, but not in the VMH and LH. This outcome suggests an interactive effect of the glucostatic pathways involving these two areas with the ghrelin signaling.     

A role for NPY overexpression in the dorsomedial hypothalamus in hyperphagia and obesity of OLETF rats

Otsuka Long-Evans Tokushima Fatty (OLETF) rats lacking CCK-A receptors are hyperphagic, obese, and diabetic. We have previously demonstrated that these rats have a peripheral satiety deficit resulting in increased meal size. To examine the potential role of hypothalamic pathways in the hyperphagia and obesity of OLETF rats, we compared patterns of hypothalamic neuropeptide Y (NPY), proopiomelanocortin (POMC), and leptin receptor mRNA expression in ad libitum-fed Long-Evans Tokushima (LETO) and OLETF rats and food-restricted OLETF rats that were pair-fed to the intake of LETO controls. Pair feeding OLETF rats prevented their increased body weight and elevated levels of plasma insulin and leptin and normalized their elevated POMC and decreased NPY mRNA expression in the arcuate nucleus. In contrast, NPY expression was upregulated in the dorsomedial hypothalamus (DMH) in pair-fed OLETF rats. A similar DMH NPY overexpression was evident in 5-wk-old preobese OLETF rats. These findings suggest a role for DMH NPY upregulation in the etiology of OLETF hyperphagia and obesity.     

Peptides that regulate food intake: glucagon-like peptide 1-(7-36) amide acts at lateral and medial hypothalamic sites to suppress feeding in rats

Glucagon-like peptide 1-(7-36) amide (GLP-1) potently inhibits rat feeding behavior after central administration. Because third ventricular injection of GLP-1 appeared to be less effective than lateral ventricular injection, we have reexamined this issue. In addition, we attempted to identify brain regions other than the paraventricular nucleus of the hypothalamus that are sensitive toward GLP-1-induced feeding suppression. Finally, we examined the local role of endogenous GLP-1 by specific GLP-1 receptor blockade. After lateral ventricular injection, GLP-1 significantly inhibited food intake of 24-h-fasted rats in a dose-dependent fashion with a minimal effective dose of 1 microg. After third ventricular injection, GLP-1 (1 microg) was similarly effective in suppressing food intake, which extends previous findings. Intracerebral microinjections of GLP-1 significantly suppressed food intake in the lateral (LH), dorsomedial (DMH), and ventromedial hypothalamus (VMH), but not in the medial nucleus of the amygdala. The minimal effective dose of GLP-1 was 0.3 microg at LH sites and 1 microg at DMH or VMH sites. LH microinjections of exendin-(9-39) amide, a GLP-1 receptor antagonist, at 1 or 2.5 microg did not alter feeding behavior in 24-h-fasted rats. In satiated animals, however, a single LH injection of 1 microg exendin-(9-39) amide significantly augmented food intake, but only during the first 20 min (0.6 vs. 0.1 g). With three repeated injections of 2.5 microg exendin-(9-39) amide every 20 min, 1-h food intake was significantly increased by 300%. These data strongly support and extend the concept of GLP-1 as a physiological regulator of food intake in the hypothalamus.     

The mPVN mediates blockade of NPY-induced feeding by a Y5 receptor antagonist: a c-FOS analysis

To identify the site(s) of NPY Y5 receptor (Y5R) mediation of NPY-induced feeding, we employed c-Fos immunostaining and a selective Y5R antagonist (Y5R-A), CGP71683A, in adult male rats. Intracerebroventricular (icv) administration of NPY stimulated feeding and c-Fos-like immunoreactivity (FLI) in the dorsomedial hypothalamus, supraoptic nucleus and the two subdivision of the hypothalamic paraventricular nucleus (pPVN), the parvocellular (pPVN), and magnocellular (mPVN). Y5R-A on its own, injected either intraperitoneally or icv, neither affected feeding nor FLI in hypothalamic sites. However, Y5R-A pretreatment suppressed NPY-induced food intake and FLI selectively in the mPVN. Taken together with our previous similar finding of Y1R involvement, these results suggest that NPY receptor sites concerned with feeding behavior reside selectively in the mPVN and Y1 and Y5 receptors are either coexpressed or expressed separately in those target neurons that promote appetitive drive.     

A Very-Low-Calorie Conventional Japanese Diet: Its Implications for Prevention of Obesity

A very-low-calorie conventional Japanese diet of 370 kcal/day has been shown to be useful for weight reduction and its long-term maintenance. Sources of the diet were mainly from chicken fillet, egg white, fish white-meat, mushroom, seaweed and low-or non-calorie fiber-rich vegetable, and contained 4.4g fat, 38.1g protein, 45.2g carbohydrate and essential minerals and vitamins. However, metabolic and neural implications of a commercial very-low-calorie liquid formula diet have rarely been investigated from the view point of food intake and appetite regulation. Animal studies have demonstrated the rationale for efficacy of the very-low calorie conventional Japanese diet as follows: (1)Increased hypothalamic histamine suppressed food intake through H₁-receptors in the ventromedial hypothalamus (VMH) and paraventricular nucleus, the satiety centers. (2) Low energy intake enhanced satiety and decreased food intake through histaminergic activation of VMH neurons. (3) Mastication activated afferent signal transmission from proprioceptors in the oral cavity to the mesencephalic trigeminal nucleus(Me5). Histaminergic systems in the hypothalamus were activated by mastication and low energy supply, which was accompanied by satiation through the action of histamine in the VMH. Usefulness of the very-low-calorie conventional Japanese diet derives from utilization of conventional Japanese food stuffs as a fiber rich, low energy food source, and from enhancement of satiation by increased mastication required of the diet. The properties of the diet seemed to effect a closed positive feedback loop between histaminergic activation in hypothalamic satiety centers and behavioral changes to enhance satiation and cause feeding suppression.     

Participation of cAMP in a signal-transduction pathway relating erythrocyte deformation to ATP release

Previously, we reportedthat red blood cells (RBCs) of rabbits and humans release ATP inresponse to mechanical deformation and that this release of ATPrequires the activity of the cystic fibrosis transmembrane conductanceregulator (CFTR). It was reported that cAMP, acting through acAMP-dependent protein kinase, PKA, is an activator of CFTR. Here weinvestigate the hypothesis that cAMP stimulates ATP release from RBCs.Incubation of human and rabbit RBCs with the direct activator ofadenylyl cyclase, forskolin (10 or 100 µM), with IBMX (100 µM),resulted in ATP release and increases in intracellular cAMP. Inaddition, epinephrine (1 µM), a receptor-mediated activator ofadenylyl cyclase, stimulated ATP release from rabbit RBCs. Moreover,incubation of human and rabbit RBCs with an active cAMP analog[adenosine 3'5'-cyclic monophosphorothioate Sp-isomer (Sp-cAMP, 100 µM)] resulted in ATP release. In contrast, forskolin and Sp-cAMPwere without effect on dog RBCs, cells known not to release ATP inresponse to deformation. When rabbit RBCs were incubated with theinactive cAMP analog and inhibitor of PKA activity, adenosine3',5'-cyclic monophosphorothioate Rp-isomer (100 µM),deformation-induced ATP release was attenuated. These results areconsistent with the hypothesis that adenylyl cyclase and cAMP arecomponents of a signal-transduction pathway relating RBC deformation toATP release from human and rabbit RBCs.

     

Deoxyglucose-autoradiography of the hypothalamus after food deprivation in the mouse

[14C] deoxyglucose-autoradiography of mouse brain after food deprivation showed no specific changes in the ratio of autoradiographic optical density of the lateral nucleus of the hypothalamus (LH) versus ventromedial nucleus of the hypothalamus (VMH). The results show that even if there were differences in functional activity between these hypothalamic nuclei during conditions of hunger and satiety, they are not reflected as autoradiographically detectable in relative deoxyglucose uptake.     

Brain-derived neurotrophic factor in the ventromedial nucleus of the hypothalamus reduces energy intake

Recent studies show that brain-derived neurotrophic factor (BDNF) decreases feeding and body weight after peripheral and ventricular administration. BDNF mRNA and protein, and its receptor TrkB, are widely distributed in the hypothalamus and other brain regions. However, there are few reports on specific brain sites of actions for BDNF. We evaluated the effect of BDNF, given into the ventromedial nucleus of the hypothalamus (VMH), on normal and deprivation- and neuropeptide Y (NPY)-induced feeding behavior and body weight. BDNF injected unilaterally or bilaterally into the VMH of food-deprived and nondeprived rats significantly decreased feeding and body weight gain within the 0- to 24-h and the 24- to 48-h postinjection intervals. Doses effectively producing inhibition of feeding behavior did not establish a conditioned taste aversion. BDNF-induced feeding inhibition was attenuated by pretreatment of the TrkB-Fc fusion protein that blocks binding between BDNF and its receptor TrkB. VMH-injected BDNF significantly decreased VMH NPY-induced feeding at 1, 2, and 4 h after injection. In summary, BDNF in the VMH significantly decreases food intake and body weight gain, by TrkB receptor-mediated actions. Furthermore, the anorectic effects of BDNF in this site appear to be mediated by NPY. These data suggest that the VMH is an important site of action for BDNF in its effects on energy metabolism.     

Differential body weight and feeding responses to high-fat diets in rats and mice lacking cholecystokinin 1 receptors

Prior data demonstrated differential roles for cholecystokinin (CCK)1 receptors in maintaining energy balance in rats and mice. CCK1 receptor deficiency results in hyperphagia and obesity of Otsuka Long-Evans Tokushima Fatty (OLETF) rats but not in mice. To ascertain the role of CCK1 receptors in high-fat-diet (HFD)-induced obesity, we compared alterations in food intake, body weight, fat mass, plasma glucose, and leptin levels, and patterns of hypothalamic gene expression in OLETF rats and mice lacking CCK1 receptors in response to a 10-wk exposure to HFD. Compared with Long-Evans Tokushima Otsuka (LETO) control rats, OLETF rats on HFD had sustained overconsumption over the 10-wk period. High fat feeding resulted in greater increases in body weight and plasma leptin levels in OLETF than in LETO rats. In situ hybridization determinations revealed that, while HFD reduced neuropeptide Y (NPY) mRNA expression in both the arcuate nucleus (Arc) and the dorsomedial hypothalamus (DMH) of LETO rats, HFD resulted in decreased NPY expression in the Arc but not in the DMH of OLETF rats. In contrast to these results in OLETF rats, HFD increased food intake and induced obesity to an equal degree in both wild-type and CCK1 receptor(-/-) mice. NPY gene expression was decreased in the Arc in response to HFD, but was not detectable in the DMH in both wild-type and CCK1 receptor(-/-) mice. Together, these data provide further evidence for differential roles of CCK1 receptors in the controls of food intake and body weight in rats and mice.     

Peripheral and central satiety factors in neuropeptide Y-induced feeding in rats

In the first study, injection of NPY into the hypothalamic paraventricular nucleus (PVN) caused a robust feeding effect that was attenuated by intravenous infusions of glucose, but not fructose. It is suggested that increased blood glucose has a direct central effect on NPY feeding mechanisms. In a second study, and contrary to a previous report, peripheral administration of CCK-8 had a marked satiety effect in NPY-treated rats. Thus, NPY-induced feeding is, at least for a short time, possibly subject to the satiating action of prandially-released CCK. In a final study, NPY was shown to be without effect on gastric emptying of a solid test meal.     

Acute effects of PYY3-36 on food intake and hypothalamic neuropeptide expression in the mouse

It has recently been suggested that gut-derived PYY(3-36) may be involved in the central mediation of post-prandial satiety signals. We have examined the acute effects of peripherally administered PYY(3-36) on food intake and hypothalamic gene expression of neuropeptides in mice. A single intraperitoneal injection of PYY(3-36) to mice that had been fasted for 24h resulted in a highly significant reduction in food intake at 6 and 24h post-injection but not at 48h. However, in freely fed mice, food intake was unaltered by PYY(3-36) administration. In the arcuate nucleus POMC mRNA expression was significantly elevated at 6h and remained elevated at 24h following PYY(3-36) injection. By contrast NPY mRNA expression in the arcuate nucleus was suppressed at 6h but not at 24h post-injection. In the lateral hypothalamus there were no differences in MCH mRNA expression at either time point. In conclusion, peripherally administered PYY(3-36) has a suppressive effect on food intake that is more prominent in recently fasted mice and lasts up to 24 h. This is associated with a short-lived suppression of NPY mRNA, a longer lasting increase in POMC mRNA but no change in MCH mRNA expression.     

Hypothalamic neuropeptide Y receptor characteristics and NPY-induced feeding responses in lean and obese Zucker rats.

Increased hypothalamic neuropeptide Y levels have previously been demonstrated in several hypothalamic nuclei of the (fa/fa) Zucker rat. This study set out to characterise hypothalamic NPY receptors in both genotypres and to study the effect of exogenous NPY on feeding behavior in these rats. Spontaneous daytime food intake was raised in the obese rat (p less than 0.05). Total hypothalamic receptor density (Bmax) was reduced in the obese rat compared with the lean rat (by 56%, p less than 0.005), but affinity remained unaltered. The lowest dose of NPY tested (23.5 pmol) stimulated daytime feeding in lean rats after 1, 2 and 3 hours but was inaffective in the obese rat (p less than 0.05). At two higher doses (235 pmol and 2.35 nmol), NPY was equipotent in both genotypes over 1 and 2 hours but NPY-induced feeding was attenuated over 3 hours in the obese rat. These results suggest an overactive endogenous NPYergic system in the obese (fa/fa) rat which might contribute to hyperphagia and obesity in this strain.     

Inhibitory effect of ghrelin on food intake is mediated by the corticotropin-releasing factor system in neonatal chicks

It is known that, in rats, central and peripheral ghrelin increases food intake mainly through activation of neuropeptide Y (NPY) neurons. In contrast, intracerebroventricular (ICV) injection of ghrelin inhibits food intake in neonatal chicks. We examined the mechanism governing this inhibitory effect in chicks. The ICV injection of ghrelin or corticotropin-releasing factor (CRF), which also inhibits feeding and causes hyperactivity in chicks. Thus, we examined the interaction of ghrelin with CRF and the hypothalamo-pituitary-adrenal (HPA) axis. The ICV injection of ghrelin increased plasma corticosterone levels in a dose-dependent or a time-dependent manner. Co-injection of a CRF receptor antagonist, astressin, attenuated ghrelin-induced plasma corticosterone increase and anorexia. In addition, we also investigated the effect of ghrelin on NPY-induced food intake and on expression of hypothalamic NPY mRNA. Co-injection of ghrelin with NPY inhibited NPY-induced increase in food intake, and the ICV injection of ghrelin did not change NPY mRNA expression. These results indicate that central ghrelin does not interact with NPY as seen in rodents, but instead inhibits food intake by interacting with the endogenous CRF and its receptor.     

Decreased orexigenic response to neuropeptide Y in rats with obstructive cholestasis

Neuropeptide Y (NPY) is a key factor in the neurochemical control of food intake, and obstructive cholestasis can be associated with disturbances in food intake. Our aim in this study was to determine whether obstructive cholestasis in the rat is associated with defective central responsiveness to NPY. Cholestasis was induced in rats by surgical bile duct resection. Rats with obstructive cholestasis exhibited a 20% reduction in food intake 2 days after laparotomy (compared with sham-resected controls) that had resolved by 4 days after surgery. Responsiveness to the orexigenic action of NPY was tested by measuring food intake after intracerebroventricular injection of NPY. In sham-resected rats, NPY infusion strikingly increased food intake, whereas bile duct-resected (BDR) rats showed a consistent significantly impaired feeding response to NPY at postlaparotomy days 2, 4, and 7. Separate experiments measured specific binding of [(3)H]NPY to hypothalamic receptors. Fos protein expression was measured in the hypothalamic paraventricular nucleus (PVN) as a marker of NPY-induced neuronal activation. The decreased orexigenic responsiveness to NPY was not caused by altered NPY binding at hypothalamic receptors or its ability to activate neurons in the PVN. Therefore, cholestatic rats demonstrate an attenuated NPY-induced orexigenic drive that occurs early after biliary obstruction, when cholestatic rats exhibit reduced food intake, and persists despite the return of food intake to normal levels and the presence of intact central NPY-related neuronal pathways.     

Endocannabinoids gate state-dependent plasticity of synaptic inhibition in feeding circuits

Changes in food availability alter the output of hypothalamic nuclei that underlie energy homeostasis. Here, we asked whether food deprivation impacts the ability of GABA synapses in the dorsomedial hypothalamus (DMH), an important integrator of satiety signals, to undergo activity-dependent changes. GABA synapses in DMH slices from satiated rats exhibit endocannabinoid-mediated long-term depression (LTD(GABA)) in response to high-frequency stimulation of afferents. When CB1Rs are blocked, however, the same stimulation elicits long-term potentiation (LTP(GABA)), which manifests presynaptically and requires heterosynaptic recruitment of NMDARs and nitric oxide (NO). Interestingly, NO signaling is required for eCB-mediated LTD(GABA). Twenty-four hour food deprivation results in a CORT-mediated loss of CB1R signaling and, consequently, GABA synapses only exhibit LTP(GABA). These observations indicate that CB1R signaling promotes LTD(GABA) and gates LTP(GABA). Furthermore, the satiety state of an animal, through regulation of eCB signaling, determines the polarity of activity-dependent plasticity at GABA synapses in the DMH.     

Ghrelin microinjection into forebrain sites induces wakefulness and feeding in rats

Ghrelin, a gut-brain peptide, is best known for its role in the stimulation of feeding and growth hormone release. In the brain, orexin, neuropeptide Y (NPY), and ghrelin are parts of a food intake regulatory circuit. Orexin and NPY are also implicated in maintaining wakefulness. Previous experiments in our laboratory revealed that intracerebroventricular injections of ghrelin induce wakefulness in rats. To further elucidate the possible role of ghrelin in the regulation of arousal, we studied the effects of microinjections of ghrelin into hypothalamic sites, which are implicated in the regulation of feeding and sleep, such as the lateral hypothalamus (LH), medial preoptic area (MPA), and paraventricular nucleus (PVN) on sleep in rats. Sleep responses, motor activity, and food intake after central administration of 0.04, 0.2, or 1 mug (12, 60, or 300 pmol) ghrelin were recorded. Microinjections of ghrelin into the LH had strong wakefulness-promoting effects lasting for 2 h. Wakefulness was also stimulated by ghrelin injection into the MPA and PVN; the effects were confined to the first hour after the injection. Ghrelin's non-rapid-eye-movement sleep-suppressive effect was accompanied by attenuation in the electroencephalographic (EEG) slow-wave activity and changes in the EEG power spectrum. Food consumption was significantly stimulated after microinjections of ghrelin into each hypothalamic site. Together, these results are consistent with the hypothesis that forebrain ghrelinergic mechanisms play a role in the regulation of vigilance, possibly through activating the components of the food intake- and arousal-promoting network formed by orexin and NPY.     

Reduced feeding response to muscimol and neuropeptide Y in senescent F344 rats

Many mammals experience spontaneous declines in their food intake and body weight near the end of life, a stage we refer to as senescence. We have previously demonstrated that senescent rats have blunted food intake responses to intracerebroventricular injections of neuropeptide Y (NPY). In the present study, we tested the hypothesis that responsiveness to GABA, a putative potentiator of NPY's effect, is also diminished. Young and old male F344 rats received injections of NPY, muscimol, (MUS, a GABA-A receptor agonist), combinations of these two agents, and vehicle [artificial cerebrospinal fluid (aCSF)] into the hypothalamic paraventricular nucleus (PVN). Both young and old presenescent rats increased their food intake in response to NPY, MUS, and the combination of the two (in comparison to injections of aCSF). The combination treatment was generally more effective than either NPY or MUS alone. These data are consistent with suggestions that both NPY and GABA play a role in the regulation of feeding behavior. Senescent rats exhibited an attenuated NPY-induced food intake, no increase in response to MUS, and a response to NPY + MUS that was no larger than that of NPY alone. We conclude that PVN injections of GABA, as well as NPY, are less effective in stimulating feeding in senescent rats and suggest that alterations in their signaling pathways play a role in the involuntary feeding decrease seen near the end of life.     

Acute food deprivation and chronic food restriction differentially affect hypothalamic NPY mRNA expression

Although acute food deprivation and chronic food restriction both result in body weight loss, they produce different metabolic states. To evaluate how these two treatments affect hypothalamic peptide systems involved in energy homeostasis, we compared patterns of hypothalamic neuropeptide Y (NPY), agouti-related protein (AgRP), proopiomelanocotin (POMC), and leptin receptor gene expression in acutely food-deprived and chronically food-restricted rats. Both acute food deprivation and chronic food restriction reduced body weight and circulating leptin levels and resulted in increased arcuate NPY and decreased arcuate POMC gene expression. Arcuate AgRP mRNA levels were only elevated in acutely deprived rats. NPY gene expression was increased in the compact subregion of the dorsomedial hypothalamus (DMH) in response to chronic food restriction, but not in response to acute food deprivation. Leptin receptor expression was not affected by either treatment. Double in situ hybridization histochemistry revealed that, in contrast to the situation in the arcuate nucleus, NPY and leptin receptor mRNA-expressing neurons were not colocalized in the DMH. Together, these data suggest that arcuate and DMH NPY gene expression are differentially regulated. DMH NPY-expressing neurons do not appear to be under the direct control of leptin signaling.