The effect of adrenalectomy on ghrelin secretion and orexigenic action

Ghrelin is an orexigenic peptide made both in the periphery and in the central nervous system. Relatively little is known about the factors that regulate ghrelin secretion. Because both ghrelin and glucocorticoids are increased during fasting, we hypothesised that ghrelin secretion from the stomach is stimulated by glucocorticoids. Plasma ghrelin concentrations were determined by radioimmunoassay in fed and fasted adrenalectomised (ADX) and sham-operated rats. Fasting plasma ghrelin concentrations were significantly increased in ADX relative to sham rats and were normalised by glucocorticoid replacement. Several lines of evidence suggest that the orexigenic action of ghrelin is mediated through neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurones. Because ADX reduces the orexigenic actions of NPY and AgRP, we hypothesised that ADX would also reduce the orexigenic action of ghrelin. Food intake was assessed in ADX and sham rats following an intra-third-ventricular injection of either saline or ghrelin (1, 5 or 10 microg in 2 microl). ADX rats were equally sensitive to the orexigenic action of ghrelin compared to sham rats. Given that ghrelin has been shown to stimulate glucocorticoid secretion, the current data imply the existence of a regulatory feedback loop whereby glucocorticoids inhibit further ghrelin secretion. The results also suggest that, unlike the orexigenic effects of NPY and AgRP, the ability of ghrelin to stimulate food intake is maintained in ADX rats.     

Middle-aged female rats retain sensitivity to the anorexigenic effect of exogenous estradiol

It is well established that estradiol (E2) decreases food intake and body weight in young female rats. However, it is not clear if female rats retain responsiveness to the anorexigenic effect of E2 during middle age. Because middle-aged females exhibit reduced responsiveness to E2, manifesting as a delayed and attenuated luteinizing hormone surge, it is plausible that middle-aged rats are less responsive to the anorexigenic effect of E2. To test this we monitored food intake in ovariohysterectomized young and middle-aged rats following E2 treatment. E2 decreased food intake and body weight to a similar degree in both young and middle-aged rats. Next, we investigated whether genes that mediate the estrogenic inhibition of food intake are similarly responsive to E2 by measuring gene expression of the anorexigenic genes corticotropin-releasing hormone (CRH), proopiomelanocortin (POMC), the long form of the leptin receptor (Lepr) and serotonin 2C receptors (5HT2CR) and the orexigenic genes agouti-related peptide (AgRP), neuropeptide Y (NPY), prepromelanin-concentrating hormone (pMCH) and orexin in the hypothalamus of young and middle-aged OVX rats treated with E2. As expected, E2 increased expression of all anorexigenic genes while decreasing expression of all orexigenic genes in young rats. Although CRH, 5HT2CR, Lepr, AgRP, NPY and orexin were also sensitive to E2 treatment in middle-aged rats, POMC and pMCH expression were not influenced by E2 in middle-aged rats. These data demonstrate that young and middle-aged rats are similarly sensitive to the anorexigenic effect of E2 and that most, but not all feeding-related genes retain sensitivity to E2.     

Regulation of NPY and α-MSH expression by estradiol in the arcuate nucleus of Wistar female rats: a stereological study

Objectives: Feeding behavior in both animals and humans is modulated by estrogens, as shown by the increased adiposity observed in women and rats upon the drop of estradiol levels at menopause. Estradiol action on food intake is mediated through its cognate receptors within several hypothalamic nuclei, namely the arcuate nucleus (ARN). The ARN contains two neuronal populations expressing peptides that exert opposing effects on the central control of feeding: the orexigenic neuropeptide Y (NPY) and the anorexigenic α-melanocyte-stimulating hormone (α-MSH).

Methods: To understand the role played by estradiol in the modulation of food intake, we have used an animal model of cyclic 17β-estradiol benzoate (EB) administration and stereological methods to estimate the total number of neurons immunoreactive for NPY and α-MSH in the ARN of ovariectomized rats.

Results: Present results show that the experimentally induced EB cyclicity prompted a decrease in food consumption and in body weight. Data also show that ovariectomy induced an increase in NPY expression and a decrease in α-MSH expression in the ARN that were reverted by EB administration. Conversely, EB blocked the expression of NPY and increased the synthesis of α-MSH in ARN neurons, without affecting the overall sum of NPY and α-MSH neurons.

Discussion: These results suggest that estradiol affects food intake and, consequently, body weight gain, through an overriding mechanism superimposed in the physiological balance between both peptides in the ARN of female rats.     

Anorexia is Associated with Stress‐Dependent Orexigenic Responses to Exogenous Neuropeptide Y

Chicken lines that have been divergently selected for either low (LWS) or high (HWS) body weight at 56 days of age for more than 57 generations have different feeding behaviours in response to a range of i.c.v. injected neurotransmitters. The LWS have different severities of anorexia, whereas the HWS become obese. Previously, we demonstrated that LWS chicks did not respond, whereas HWS chicks increased food intake, after central injection of neuropeptide Y (NPY). The present study aimed to determine the molecular mechanisms underlying the loss of orexigenic function of NPY in LWS. Chicks were divided into four groups: stressed LWS and HWS on day of hatch, and control LWS and HWS. The stressor was a combination of food deprivation and cold exposure. On day 5 post‐hatch, each chick received an i.c.v. injection of vehicle or 0.2 nmol of NPY. Only the LWS stressed group did not increase food intake in response to i.c.v. NPY. Hypothalamic mRNA abundance of appetite‐associated factors was measured at 1 h post‐injection. Interactions of genetic line, stress and NPY treatment were observed for the mRNA abundance of agouti‐related peptide (AgRP) and synaptotagmin 1 (SYT1). Intracerebroventricular injection of NPY decreased and increased AgRP and SYT1 mRNA, respectively, in the stressed LWS and increased AgRP mRNA in stressed HWS chicks. Stress was associated with increased NPY, orexin receptor 2, corticotrophin‐releasing factor receptor 1, melanocortin receptor 3 (MC3R) and growth hormone secretagogue receptor expression. In conclusion, the loss of responsiveness to exogenous NPY in stressed LWS chicks may be a result of the decreased and increased hypothalamic expression of AgRP and MC3R, respectively. This may induce an intensification of anorexigenic melanocortin signalling pathways in LWS chicks that block the orexigenic effect of exogenous NPY. These results provide insights onto the anorexic condition across species, and especially for forms of inducible anorexia such as human anorexia nervosa.     

Involvement of specific orexigenic neuropeptides in sweetener-induced overconsumption in rats

Palatability is one of the factors that regulates food and fluid intake and contributes to overconsumption in turn contributing to obesity. To elucidate the brain mechanisms of the palatability-induced ingestion, we explored the roles of six hypothalamic orexigenic neuropeptides, orexin, melanin-concentrating hormone (MCH), neuropeptide Y (NPY), agouti-related protein (AgRP), ghrelin and dynorphin, in the intake of a palatable solution, saccharin. Of the six peptides, intracerebroventricular (i.c.v.) administrations of orexin, MCH and NPY increased the intake of saccharin. Drinking of saccharin in turn elevated the mRNA levels of orexin and NPY, but not MCH. Pre-treatments of naloxone, an opioid antagonist, blocked the orexigenic effects of orexin and NPY. Specific gastric motor responses induced by central orexin-A and NPY are well known, however, MCH did not induce such responses. The i.c.v. administration of orexin-A facilitated gastric emptying. These results suggest that the overconsumption promoted by sweet and palatable tastes is attributed to the activation of orexigenic neuropeptides, such as orexin and NPY, and a downstream opioid system together with enhanced digestive functions.     

Cyclic estradiol replacement attenuates stress-induced c-Fos expression in the PVN of ovariectomized rats

Estradiol modulates stress reactions in female rats. Several studies showed anxiolytic effects of estradiol in behavioral tests, but the underlying mechanisms are still unclear. The aim of the current study was to explore how estradiol-treated rats respond to acute and chronic stress compared to ovariectomized rats. Ovariectomized rats received vehicle or 17beta-estradiol injections (10 microg/250 g) once every 4 days, which induced alternating high and low plasma 17beta-estradiol levels. Stress was presented by daily exposure to an adverse environment in which the animals received five footshocks for either 3 or 22 days. Under control conditions no differences were observed, but as soon as stress was applied, reactions of ovariectomized and estradiol-treated rats diverged. Both acute and chronic stress increased the c-Fos protein expression in the paraventricular nucleus (PVN) of the hypothalamus. Cyclic estradiol treatment reduced this stress-induced activation of the PVN, an effect that seems to be dependent on the plasma estradiol levels. No differences in stress-induced corticosterone responses were revealed between the treatment groups. An increase in the number of ERbeta-expressing cells in the PVN of ovariectomized and estradiol-treated rats during chronic stress implied increased ERbeta-mediated mechanisms during these conditions. The dampening effect of estradiol on the excessive stress-induced activity in the PVN may be beneficial for the animal in its response to chronic recurrent stress by reducing the output of the PVN.     

Neuropeptide Y facilitates activity-based-anorexia

The hypothesis that treatment with neuropeptide Y (NPY) can increase running activity and decrease food intake and body weight was tested. Female rats with a running wheel lost more weight than sedentary rats and ran progressively more as the availability of food was gradually reduced. When food was available for only 1h/day, the rats lost control over body weight. Correlatively, the level of NPY mRNA was increased in the hypothalamic arcuate nucleus. This phenomenon, activity-based-anorexia, was enhanced by intracerebroventricular infusion of NPY in rats which had food available during 2h/day. By contrast, NPY stimulated food intake but not wheel running in rats which had food available continuously. These findings are inconsistent with the prevailing theory of the role of the hypothalamus in the regulation of body weight according to which food intake is a homeostatic process controlled by "orexigenic" and "anorexigenic" neural networks. However, the finding that treatment with NPY, generally considered an "orexigen", can increase physical activity and decrease food intake and cause a loss of body weight is in line with the clinical observation that patients with anorexia nervosa are physically hyperactive and eat only little food despite having depleted body fat and up-regulated hypothalamic "orexigenic" peptides.     

Dipeptidyl peptidase IV (DPP4)-deficiency attenuates diet-induced obesity in rats: possible implications for the hypothalamic neuropeptidergic system

The underlying mechanisms controlling food intake and satiety are thoroughly controlled, but seem to be insufficient under conditions of almost unlimited food supply. Hence, overweight and obesity are serious problems especially in industrialized countries. To assess the possible influence of CD26, exerting a dipeptidyl peptidase activity (DPP4) cleaving several energy homeostasis-relevant peptides, we investigated wild type and DPP4-deficient dark agouti rats in a model of diet-induced obesity and found a reduced weight gain in DPP4-deficient rats. When investigating the specific increase of whole body fat volume by MRI to assess the distribution pattern (subcutaneous vs. intraabdominal), there was an altered ratio under dietary conditions only in DPP4-deficient rats, which was due to lower intraabdominal fat amounts. Furthermore, we investigated the number of cells immunopositive for the leptin receptor (OB-R), the orexigenic leptin antagonist neuropeptide Y (NPY), as well as of the NPY receptors Y1, Y2, and Y5 within hypothalamic nuclei. Independent from the body weight, higher levels of NPY and all receptors were expressed in DPP4-deficent rats. Under obese conditions, hypothalamic Y2-levels were reduced in both strains. Concerning NPY and Y1, there were partly oppositional effects, with reduced hypothalamic Y1 levels only in wild types, and increased NPY levels only in DPP4-deficient rats. These effects might be responsible for unaltered food intake in DPP4-deficent rats compared to wild types, despite reduced weight gain. However, since the food intake remained unaffected, these effects suggest that DPP4 exerts its effects on intraabdominal fat also via peripheral actions.     

Neuropeptide-Y: a possible mediator of prolactin-induced feeding and regulator of energy balance in the ring dove (Streptopelia risoria)

Although neuropeptide-Y (NPY) has been widely reported to be a potent stimulator of feeding activity and regulator of energy homeostasis, most of the supportive evidence for such effects has been gathered in mammalian species. This study characterized the orexigenic potency of NPY in an avian species, the ring dove, and measured changes in hypothalamic NPY-immunoreactive (NPY-ir) cell numbers in response to energy state fluctuations or intracranial administration of the potent orexigenic hormone prolactin. Food intake was significantly elevated in male doves at 1 h after intracerebroventricular (i.c.v.) injection of 0.25 and 0.5 microg NPY but not after injection of a higher dose (1.0 microg). In time course studies, food intake was increased at 1 h after i.c.v. injection of 0.5 microg NPY but was not elevated at 2, 3, or 4 h. The number of NPY-ir cell bodies in the infundibular region of the dove hypothalamus increased two to four-fold following acute food deprivation, chronic food restriction, or repeated i.c.v. injections of prolactin. No additive effects were observed when food restriction and prolactin treatment were combined. These findings suggest that NPY is involved in energy homeostasis in doves and are consistent with the hypothesis that prolactin-induced hyperphagia is mediated in part by NPY.     

Early leptin intervention reverses perturbed energy balance regulating hypothalamic neuropeptides in the pre‐ and postnatal calorie‐restricted female rat offspring

Pre‐ and postnatal calorie restriction is associated with postnatal growth restriction, reduced circulating leptin concentrations, and perturbed energy balance. Hypothalamic regulation of energy balance demonstrates enhanced orexigenic (NPY, AgRP) and diminished anorexigenic (POMC, CART) neuropeptide expression (PN21), setting the stage for subsequent development of obesity in female Sprague‐Dawley rats. Leptin replenishment during the early postnatal period (PN2‐PN8) led to reversal of the hypothalamic orexigenic:anorexigenic neuropeptide ratio at PN21 by reducing only the orexigenic (NPY, AgRP), without affecting the anorexigenic (POMC, CART) neuropeptide expression. This hypothalamic effect was mediated via enhanced leptin receptor (ObRb) signaling that involved increased pSTAT3/STAT3 but reduced PTP1B. This was further confirmed by an increase in body weight at PN21 in response to intracerebroventricular administration of antisense ObRb oligonucleotides (PN2‐PN8). The change in the hypothalamic neuropeptide balance in response to leptin administration was associated with increased oxygen consumption, carbon dioxide production, and physical activity, which resulted in increased milk intake (PN14) with no change in body weight. This is in contrast to the reduction in milk intake with no effect on energy expenditure and physical activity observed in controls. We conclude that pre‐ and postnatal calorie restriction perturbs hypothalamic neuropeptide regulation of energy balance, setting the stage for hyperphagia and reduced energy expenditure, hallmarks of obesity. Leptin in turn reverses this phenotype by increasing hypothalamic ObRb signaling (sensitivity) and affecting only the orexigenic arm of the neuropeptide balance. © 2015 Wiley Periodicals, Inc.     

Effects of neuropeptide Y and corticotropin-releasing factor on ethanol intake in Wistar rats: interaction with chronic ethanol exposure

Neuropeptide Y (NPY) and corticotropin-releasing factor (CRF) have opposing effects on stress-associated and consummatory behaviors in rodents. Recent studies also suggest that both peptides influence ethanol intake. In the present study, the effects of administration of CRF and NPY into the lateral ventricle on ethanol intake in naive and ethanol-vapor-exposed Wistar rats were examined. A limited access paradigm was used to measure intake of a 10% (v/v) ethanol solution in Wistar rats trained to drink using a sucrose fading procedure. Ethanol vapor exposure for 8 weeks significantly elevated ethanol intake in this limited access paradigm relative to pre-exposure levels. The effects of icv administration of CRF (1 microg), NPY (10 microg) or NPY/CRF combined (10 and 1 microg, respectively) on ethanol intake were then assessed. In non-vapor-exposed subjects, icv infusion of NPY had no effect on ethanol intake, while a significant suppression of drinking was seen following icv administration of CRF. Administration of NPY in combination with CRF had no effect on ethanol intake in non-ethanol-vapor-exposed rats. In vapor-exposed subjects, both NPY and CRF reduced ethanol intake, but when given in combination, no difference from vehicle was detected. Locomotor activity was measured during drinking sessions and was unaffected by peptide administration. These studies underscore the importance of a history of exposure to chronic ethanol vapor in the regulation of ethanol intake by NPY. Furthermore, the results presented here suggest that a balance between the stress-related peptides NPY and CRF may be involved in the regulation of ethanol intake.     

Adiponectin at physiological level glucose-independently enhances inhibitory postsynaptic current onto NPY neurons in the hypothalamic arcuate nucleus

Adiponectin regulates glucose and lipid metabolism, acting against atherosclerosis and metabolic syndrome. Accumulating evidences suggest that adiponectin acts on the brain including the arcuate nucleus of hypothalamus (ARC). The ARC contains orexigenic neuropeptide Y (NPY)/agouti related peptide (AgRP) neurons and anorexigenic proopiomelanocortin (POMC) neurons, the first order neurons for feeding regulation. We recently reported that intracerebroventricular injection of adiponectin at low glucose level suppressed food intake, while at elevated glucose level it promoted food intake, exhibiting glucose-dependent reciprocal effects. As an underlying neuronal mechanism, physiological level of adiponectin at low glucose activated ARC POMC neurons and at high glucose inactivated them. Now, whether physiological level of adiponectin also affects NPY/AgRP neurons is essential for fully understanding the adiponectin action, but it remains to be clarified. We here report that a physiological dose of adiponectin, in both high and low glucose conditions, attenuated action potential firing without altering resting membrane potential in ARC NPY neurons. This adiponectin effect was abolished by GABA_A receptor blockade. Adiponectin enhanced amplitude but not frequency of inhibitory postsynaptic current (IPSC) onto NPY neurons. These results demonstrate that adiponectin enhances IPSC onto NPY neurons to attenuate action potential firing in NPY neurons in a glucose-independent manner, being contrasted to its glucose-dependent effect on POMC neurons.     

Role of leptin in the control of feeding of goldfish Carassius auratus: interactions with cholecystokinin,neuropeptide Y and orexin A,and modulation by fasting

To assess the role of leptin on food intake regulation in goldfish, we examined the effects of central (intracerebroventricular, ICV) and peripheral (intraperitoneal, IP) injections of recombinant murine leptin on feeding behavior. Centrally (100 ng/g) and peripherally (300 ng/g) injected leptin both caused a significant decrease in food intake, compared to the saline-treated controls. To test the hypothesis that leptin influenced orexigenic neuropeptide systems in goldfish, fish were co-injected with neuropeptide Y (NPY) or orexin A and leptin. Both NPY (5 ng/g) and orexin A (10 ng/g) significantly increased food intake. Fish co-injected ICV with NPY (5 ng/g) or orexin A (10 ng/g) and leptin (1 or 10 ng/g) had a food intake lower than that of fish treated with NPY or orexin A alone. NPY mRNA expression in goldfish brain was reduced 2 and 6 h following central injection of leptin. To test the hypothesis that the cholecystokinin (CCK) mediates the effects of leptin in goldfish, fish were simultaneously injected ICV with an ineffective dose of leptin (10 ng/g) and either ICV or IP with an ineffective doses of CCK (1 ng/g ICV or 25 ng/g IP). These fish had a food intake lower than vehicle-treated fish, suggesting that leptin potentiates the satiety actions of CCK. CCK hypothalamic mRNA expression was increased 2 h following central treatment with leptin. The CCK receptor antagonist proglumide blocked both central and peripheral CCK satiety effects. Blockade of CCK brain receptors by proglumide resulted in an inhibition of the leptin-induced decrease in food intake and an attenuation of the inhibiting action of leptin on both NPY- and orexin A-induced feeding. These data suggests that CCK has a role in mediating the effects of leptin on food intake. Fasting potentiated the actions of leptin and attenuated the effects of CCK. Whereas fasting had no effects on the brain mRNA expression of CCK, it increased the brain mRNA expression of NPY and decreased the expression of CART. These changes in neuropeptide expression were partially reversed when fish were treated ICV with leptin. These results provide strong evidence that, in goldfish, leptin influences food intake, in part by modulating the orexigenic effects of NPY and orexin and that its actions are mediated, at least in part, by CCK.     

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.     

Intake inhibition by NPY and CCK-8: A challenge of the notion of NPY as an "Orexigen"

We tested the hypothesis that neuropeptide Y (NPY) interacts with cholecystokinin octapeptide (CCK-8) in inhibition of intake of an intraorally infused solution of sucrose, a test of consummatory ingestive behavior. Both intracerebroventricular infusion of NPY (10 microg) and intraperitoneal injection of CCK-8 (0.5 micro/kg) reduced the intake of a 1M solution of sucrose infused intraorally at a rate of 0.5 ml/min in ovariectomized female rats, but the two peptides did not interact in inhibiting intraoral intake. By contrast, NPY increased intake if the sucrose solution was ingested from a bottle, a test demanding both appetitive and consummatory ingestive responses. CCK-8 inhibited intake in this test and its inhibitory effect was increased by simultaneous treatment with NPY. The activity in the nucleus of the solitary tract (NTS), a brainstem relay mediating inhibition of intake, judged by the expression of c-fos-like immunoreactivity, was significantly increased after treatment with CCK-8 or NPY to approximately the same extent. Combined treatment with NPY and CCK-8 did not increase the c-fos-like immunoreactivity in the NTS above treatment with NPY or CCK-8 alone. These results strengthen the hypothesis that NPY, like CCK-8, is an inhibitor of consummatory ingestive behavior and suggest that this inhibition is mediated via the NTS.     

Glucokinase inhibitor glucosamine stimulates feeding and activates hypothalamic neuropeptide Y and orexin neurons

Maintaining glucose levels within the appropriate physiological range is necessary for survival. The identification of specific neuronal populations, within discreet brain regions, sensitive to changes in glucose concentration has led to the hypothesis of a central glucose-sensing system capable of directly modulating feeding behaviour. Glucokinase (GK) has been identified as a glucose-sensor responsible for detecting such changes both within the brain and the periphery. We previously reported that antagonism of centrally expressed GK by administration of glucosamine (GSN) was sufficient to induce protective glucoprivic feeding in rats. Here we examine a neurochemical mechanism underlying this effect and report that GSN stimulated food intake is highly correlated with the induction of the neuronal activation marker cFOS within two nuclei with a demonstrated role in central glucose sensing and appetite, the arcuate nucleus of the hypothalamus (ARC) and lateral hypothalamic area (LHA). Furthermore, GSN stimulated cFOS within the ARC was observed in orexigenic neurons expressing the endogenous melanocortin receptor antagonist agouti-related peptide (AgRP) and neuropeptide Y (NPY), but not those expressing the anorectic endogenous melanocortin receptor agonist alpha-melanocyte stimulating hormone (α-MSH). In the LHA, GSN stimulated cFOS was found within arousal and feeding associated orexin/hypocretin (ORX), but not orexigenic melanin-concentrating hormone (MCH) expressing neurons. Our data suggest that GK within these specific feeding and arousal related populations of AgRP/NPY and ORX neurons may play a modulatory role in the sensing of and appetitive response to hypoglycaemia.