Motivation to obtain preferred foods is enhanced by ghrelin in the ventral tegmental area

Ghrelin is an orexigenic peptide that acts within the central nervous system to stimulate appetite and food intake via the growth hormone secretagogue receptor (GHS-R). It has been hypothesized that ghrelin modulates food intake in part by stimulating reward pathways in the brain and potentially stimulating the intake of palatable foods. Here we examined the effects of chronic ghrelin administration in the ventral tegmental area (VTA) via osmotic minipumps on 1) ad libitum food intake and bodyweight; 2) macronutrient preference; and 3) motivation to obtain chocolate pellets. In the first study rats receiving ghrelin into the VTA showed a dose-dependent increase in the intake of regular chow, also resulting in increased body weight gain. A second study revealed that intra-VTA delivery of the ghrelin receptor antagonist [Lys-3]-GHRP-6 selectively reduced caloric intake of high-fat chow and reduced body weight gain relative to control and ghrelin treated rats. The third study demonstrated that food restricted rats worked harder for food pellets when infused with ghrelin than when infused with vehicle or ghrelin receptor antagonist treated rats. Finally, rats trained on an FR1 schedule but returned to ad libitum during ghrelin infusion, responded at 86% of baseline levels when they were not hungry, whereas saline infused rats responded at 36% of baseline. Together, these results suggest that ghrelin acts directly on the VTA to increase preference for and motivation to obtain highly-palatable food.     

Motivation to obtain preferred foods is enhanced by ghrelin in the ventral tegmental area

Ghrelin is an orexigenic peptide that acts within the central nervous system to stimulate appetite and food intake via the growth hormone secretagogue receptor (GHS-R). It has been hypothesized that ghrelin modulates food intake in part by stimulating reward pathways in the brain and potentially stimulating the intake of palatable foods. Here we examined the effects of chronic ghrelin administration in the ventral tegmental area (VTA) via osmotic minipumps on 1) ad libitum food intake and bodyweight; 2) macronutrient preference; and 3) motivation to obtain chocolate pellets. In the first study rats receiving ghrelin into the VTA showed a dose-dependent increase in the intake of regular chow, also resulting in increased body weight gain. A second study revealed that intra-VTA delivery of the ghrelin receptor antagonist [Lys-3]-GHRP-6 selectively reduced caloric intake of high-fat chow and reduced body weight gain relative to control and ghrelin treated rats. The third study demonstrated that food restricted rats worked harder for food pellets when infused with ghrelin than when infused with vehicle or ghrelin receptor antagonist treated rats. Finally, rats trained on an FR1 schedule but returned to ad libitum during ghrelin infusion, responded at 86% of baseline levels when they were not hungry, whereas saline infused rats responded at 36% of baseline. Together, these results suggest that ghrelin acts directly on the VTA to increase preference for and motivation to obtain highly-palatable food.     

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.     

The Amygdala as a Neurobiological Target for Ghrelin in Rats: Neuroanatomical,Electrophysiological and Behavioral Evidence

Here, we sought to demonstrate that the orexigenic circulating hormone, ghrelin, is able to exert neurobiological effects (including those linked to feeding control) at the level of the amygdala, involving neuroanatomical, electrophysiological and behavioural studies. We found that ghrelin receptors (GHS-R) are densely expressed in several subnuclei of the amygdala, notably in ventrolateral (LaVL) and ventromedial (LaVM) parts of the lateral amygdaloid nucleus. Using whole-cell patch clamp electrophysiology to record from cells in the lateral amygdaloid nucleus, we found that ghrelin reduced the frequency of mEPSCs recorded from large pyramidal-like neurons, an effect that could be blocked by co-application of a ghrelin receptor antagonist. In ad libitum fed rats, intra-amygdala administration of ghrelin produced a large orexigenic response that lasted throughout the 4 hr of testing. Conversely, in hungry, fasted rats ghrelin receptor blockade in the amygdala significantly reduced food intake. Finally, we investigated a possible interaction between ghrelin''s effects on feeding control and emotional reactivity exerted at the level of the amygdala. In rats allowed to feed during a 1-hour period between ghrelin injection and anxiety testing (elevated plus maze and open field), intra-amygdala ghrelin had no effect on anxiety-like behavior. By contrast, if the rats were not given access to food during this 1-hour period, a decrease in anxiety-like behavior was observed in both tests. Collectively, these data indicate that the amygdala is a valid target brain area for ghrelin where its neurobiological effects are important for food intake and for the suppression of emotional (anxiety-like) behaviors if food is not available.     

Nitric oxide is a central component in neuropeptide regulation of appetite

In recent years, there have been a large number of neuropeptides discovered that regulate food intake. Many of these peptides regulate food intake by increasing or decreasing nitric oxide (NO). In the current study, we compared the effect of the food modulators ghrelin, NPY and CCK in NOS KO mice. Satiated homozygous and heterozygous NOS KO mice and their wild type controls were administered ghrelin ICV. Food intake was measured for 2 h post injection. Ghrelin did not increase food intake in the homozygous NOS KO mice compared to vehicle treated NOS KO mice, whereas food intake was increased in the wild type controls compared to vehicle treated wild type controls. NPY was administered ICV and food intake measured for 2 h. Homozygous NOS KO mice showed no increase in food intake after NPY administration, whereas the wild type controls did. In our final study, we administered CCK intraperitoneally to homozygous and heterozygous NOS KO mice and their wild type controls after overnight food deprivation. Food intake was measured for 1 h after injection. CCK inhibited food intake in wild type mice after overnight food deprivation, however, CCK failed to inhibit food intake in the NOS KO mice. The heterozygous mice showed partial food inhibition after the CCK. The current results add further support to the theory that NO is a central mediator in food intake.     

Ghrelin mediates anticipation to a palatable meal in rats

Food anticipatory activity (FAA) is displayed in rats when access to food is restricted to a specific time frame of their circadian phase, a behavior thought to reflect both hunger and the motivation to eat. Rats also display FAA in a feeding schedule with ad libitum access to normal chow, but limited availability of a palatable meal, which is thought to involve mainly motivational aspects. The orexigenic hormone ghrelin has been implicated in FAA in rodents with restricted access to chow. Because ghrelin plays an important role not only in the control of food intake, but also in reward, we sought to determine the role of ghrelin in anticipation to a palatable meal. Plasma ghrelin levels of non-restricted rats that anticipated chocolate correlated positively with FAA and were increased compared with chow-fed control rats. Furthermore, centrally injected ghrelin increased, whereas an antagonist of the ghrelin receptor decreased, the anticipation to chocolate. Therefore, we hypothesize that central ghrelin signaling is able to mediate the motivational drive to eat.     

Changes in glucose tolerance and leptin responsiveness of rats offered a choice of lard, sucrose, and chow

Rats offered chow, lard, and 30% sucrose solution (choice) rapidly become obese. We tested metabolic disturbances in rats offered choice, chow+lard, or chow+30% sucrose solution [chow+liquid sucrose (LS)] and compared them with rats fed a composite 60% kcal fat, 7% sucrose diet [high-fat diet (HFD)], or a 10% kcal fat, 35% sucrose diet [low-fat diet (LFD)]. Choice rats had the highest energy intake, but HFD rats gained the most weight. After 23 days carcass fat was the same for choice, HFD, chow+lard, and chow+LS groups. Glucose clearance was the same for all groups during an intraperitoneal glucose tolerance test (GTT) on day 12, but fasting insulin was increased in choice, LFD fed, and chow+LS rats. By contrast, only choice and chow+LS rats were resistant to an intraperitoneal injection of 2 mg leptin/kg on day 17. In experiment 2 choice rats were insulin insensitive during an intraperitoneal GTT, but this was corrected in an oral GTT due to GLP-1 release. UCP-1 protein was increased in brown fat and inguinal white fat in choice rats, and this was associated with a significant increase in energy expenditure of choice rats during the dark period whether expenditure was expressed on a per animal or a metabolic body size basis. The increase in expenditure obviously was not great enough to prevent development of obesity. Further studies are required to determine the mechanistic basis of the rapid onset of leptin resistance in choice rats and how consumption of sucrose solution drives this process.     

Obestatin对血浆ghrelin和nesfatin-1水平及胃排空的影响

目的:探讨侧脑室注射obestatin对大鼠血浆酰基化ghrelin、去酰基化ghrelin、nesfatin-1水平的影响以及对胃排空的调控。方法:侧脑室注射obestatin,采用酶免疫测定(EIA)法检测血浆酰基化ghrelin、去酰基化ghrelin、nesfatin-1水平以及胃排空率的变化。结果:侧脑室分别注射0.1、0.3或1.0 nmol obestatin,大鼠血浆酰基化ghrelin、去酰基化ghrelin以及nesfatin-1水平无显著改变(P0.05),且酰基化ghrelin与去酰基化ghrelin比率无显著改变(P0.05);侧脑室注射obestatin,大鼠摄食量无显著改变,但胃排空率明显增加(P0.05);胃排空率明显延迟(P0.05)。与侧脑室注射1.0 nmol Obestatin组相比,注射1.0 nmol Obestatin+CRF,大鼠摄食量无显著改变,胃排空率明显延迟(P0.05)。各组摄食量及进入十二指肠内食物量无明显差异(P0.05)。结论:中枢obestatin促进大鼠的胃排空,可能与h/r CRF通路有关。     

Stimulatory effect of n-octanoylated ghrelin on locomotor activity in the goldfish, Carassius auratus

Ghrelin is implicated in growth and feeding regulation in fish. The influence of ghrelin on behavior has not been well studied and the physiological role of des-fatty acid modification of this peptide is unclear. Therefore, the effects of intracerebroventricular (ICV) and intraperitoneal (IP) administration of synthetic n-octanoylated (acyl) goldfish ghrelin and des-n-octanoylated (des-acyl) ghrelin on locomotor and orexigenic activity in the goldfish were examined. ICV administration of acyl ghrelin at doses of 1 and 2 pmol/g body weight (BW) and IP administration at 16 pmol/g BW both induced significant increases in locomotor activity during for 45-60 min after treatment. Cumulative food intake was significantly increased by ICV injection of acyl ghrelin at doses of 1 and 2 pmol/g BW and IP injection at 8 and 16 pmol/g BW during the 60-min post-treatment observation period. In contrast, ICV and IP administration of des-acyl ghrelin produced no changes in locomotor and orexigenic activity. We also analyzed fasting-induced changes in the expression of ghrelin mRNA in the brain and intestine using a real-time PCR method. The level of ghrelin mRNA in the intestine, but not in the brain, obtained from fish fasted for 7 days was significantly higher than that in fish that had been fed normally. These results suggest that, in the goldfish, acyl ghrelin, but not des-acyl ghrelin, stimulates locomotor activity and enhances food intake via central and peripheral pathways.     

Effects of systemic or intracerebroventricular naloxone injection on basal and 2-deoxy-D-glucose-induced ingestive behavior

In order to examine the role and site of action of opiates on both hunger and thirst and food and water intake in rats after short term (3 hr.) food deprivation alone or in combination with 2DG-induced glucoprivic stress, naloxone was given to rats in either the jugular vein or the lateral ventricle. Both basal and 2DG-induced food and water intake were reduced by naloxone injected either peripherally or centrally. Latencies to eat and drink were used as measures of hunger and thirst respectively. Only central injection of naloxone significantly reduced 2DG-induced but not basal hunger. These results suggest a central site of action of naloxone on both food and water intake even if some peripheral effects cannot be totally ruled out. Our findings indicate central nervous system opiate receptor involvement in the hunger response to 2DG-induced glucoprivation. In all other treatment conditions, decreases in food intake cannot be related to reduction of hunger but may be due to potentiation of satiation during opiate receptor blockade.     

Peptide S is a novel potent inhibitor of voluntary and fast-induced food intake in rats

Peptide S (NPS or PEPS) and its cognate receptor have been recently identified both in the central nervous system and in the periphery. NPS/PEPS promotes arousal and has potent anxiolytic-like effects when it is injected centrally in mice. In the present experiment, we tested by different approaches its central effects on feeding behaviour in Long-Evans rats. PEPS at doses of 1 and 10 microg injected in the lateral brain ventricle strongly inhibited by more than 50% chow intake in overnight fasted rats with effects of longer duration with the highest dose (P<0.0001). A similar decrease was observed for the spontaneous intake of a high-energy palatable diet (-48%; P<0.0001). This anorexigenic effect was comparable to that induced by corticotropin-releasing hormone in fasted rats at equimolar doses. However, peptide S did not modify food intake stimulated by neuropeptide Y (NPY) at equimolar doses. It also did not affect the fasting concentrations of important modulators of food intake like leptin, ghrelin, and insulin in circulation. This study therefore showed that peptide S is a new potent anorexigenic agent when centrally injected. Its inhibitory action appears to be independent of the NPY, ghrelin, and leptin pathways. Development of peptide S agonists could constitute a new approach for the treatment of obesity.     

Differential Modulation of Arcuate Nucleus and Mesolimbic Gene Expression Levels by Central Leptin in Rats on Short-Term High-Fat High-Sugar Diet

Objective

Leptin resistance is a common hallmark of obesity. Rats on a free-choice high-fat high-sugar (fcHFHS) diet are resistant to peripherally administered leptin. The aim of this study was to investigate feeding responses to central leptin as well as the associated changes in mRNA levels in hypothalamic and mesolimbic brain areas.

Design and Methods

Rats on a CHOW or fcHFHS diet for 8 days received leptin or vehicle intracerebro(lateral)ventricularly (ICV) and food intake was measured 5 h and 24 h later. Four days later, rats were sacrificed after ICV leptin or vehicle and mRNA levels were quantified for hypothalamic pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) and for preproenkephalin (ppENK) in nucleus accumbens and tyrosine hydroxylase (TH) in ventral tegmental area (VTA).

Results

ICV leptin decreased caloric intake both in CHOW and fcHFHS rats. In fcHFHS, leptin preferentially decreased chow and fat intake. Leptin increased POMC and decreased NPY mRNA in CHOW, but not in fcHFHS rats. In CHOW rats, leptin had no effect on ppENK mRNA and decreased TH mRNA. In fcHFHS, leptin decreased ppENK mRNA and increased TH mRNA.

Conclusion

Despite peripheral and arcuate leptin resistance, central leptin suppresses feeding in fcHFHS rats. As the VTA and nucleus accumbens are still responsive to leptin, these brain areas may therefore, at least partly, account for the leptin-induced feeding suppression in rats on a fcHFHS diet.     

Regulation of food intake in the goldfish by interaction between ghrelin and orexin

Intracerebroventricular (ICV) administration of ghrelin, orexin and neuropeptide Y (NPY) stimulates food intake in goldfish. Orexin and NPY interact with each other in the regulation of feeding, while ghrelin-induced feeding has also shown to be mediated by NPY in the goldfish model. To investigate the interaction between ghrelin and orexin, we examined the effects of a selective orexin receptor-1 antagonist, SB334867, and a growth hormone secretagogue-receptor antagonist, [D-Lys(3)]-GHRP-6, on ghrelin- and orexin-A-induced feeding. Ghrelin-induced food intake was completely inhibited for 1h following ICV preinjection of SB334867, while [D-Lys(3)]-GHRP-6 attenuated orexin-A stimulated feeding. Furthermore, ICV administration of ghrelin or orexin-A at a dose sufficient to stimulate food intake increased the expression of each other's mRNA in the diencephalon. These results indicate that, in goldfish, ghrelin and orexin-A have interacting orexigenic effects in the central nervous system. This is the first report that orexin-A-induced feeding is mediated by the ghrelin signaling in any animal model.     

Purification and identification of neuromedin U as an endogenous ligand for an orphan receptor GPR66 (FM3)

GPR66 is an orphan G-protein-coupled receptor (GPCR) whose structure is similar to the ghrelin and motilin receptors. We have tried to purify a natural ligand for GPR66 in rat tissues and identified a 23-amino-acid peptide as the endogenous ligand. Sequence analysis revealed the peptide as neuromedin U (NMU), a smooth-muscle-contracting peptide that was first purified from porcine spinal cord by our group. NMU binds to GPR66-expressing cells with high specificity to induce intracellular calcium mobilization. When NMU was injected intracerebroventricularly (ICV) into rats, it potently suppressed food intake. In contrast, ICV injection of NMU-antibody increased food intake. These results suggest that NMU is a potent endogenous anorexic peptide.     

Food intake, gastric secretion, and motility as affected by avian pancreatic polypeptide administered centrally in chickens

The effects of intracerebroventricular (ICV) injections of avian pancreatic polypeptide (APP) on food intake, gizzard motility, gastric secretion volume, pH, and pepsin concentration was investigated using 16-20-week-old Single-Comb White Leghorn hens. Birds were stereotaxically cannulated in the right lateral ventricle. In addition, a strain gauge was attached to the gizzard to measure motility and a polyethylene cannula was implanted into the caudoventral margin of the proventriculus to collect glandular secretions. All birds were fasted for 18 hr prior to the injection of APP. In Experiment 1 food was made available immediately following the injection of APP while in Experiment 2 food was withheld for an additional one hr post-injection. The ICV injection of APP significantly increased food intake but had no significant effect on gizzard motility, gastric secretion volume, pH, or pepsin concentration in birds given access to food immediately after injection. In birds which remained fasted after injection, pepsin concentration was decreased by APP injection, but gizzard motility, gastric secretion volume, and pH were not affected. Because ICV injections of APP significantly increased food intake and, in fasted birds, decreased pepsin concentration, it appears that APP is involved in the central nervous system control of food intake and pepsin secretion in the domestic fowl.     

Ghrelin enhances cue-induced bar pressing for high fat food

Ghrelin is an orexigenic hormone produced by the stomach that acts on growth hormone secretagogue receptors (GHSRs) both peripherally and centrally. The presence of GHSRs in the ventral tegmental area (VTA) suggests that ghrelin signaling at this level may increase the incentive value of palatable foods as well as other natural and artificial rewards. The present investigation sought to determine if ghrelin plays a role in relapse to such foods following a period of abstinence. To achieve this, thirty-six male Long Evans rats were trained to press a lever to obtain a high fat chocolate food reward on a fixed ratio schedule of 1. Following an extinction period during which lever presses were not reinforced, rats were implanted with a cannula connected to a minipump that continuously delivered ghrelin, a GHSR antagonist ([d-Lys-3]-GHRP-6), or saline in the VTA for 14 days. One week later, food reward-associated cues, food reward priming, and an overnight fast were used to induce reinstatement of the lever pressing response. Our results indicate that intra-VTA ghrelin enhances cue-induced reinstatement of responses for palatable food pellets. To the extent that the reinstatement paradigm is considered a valid model of relapse in humans, this suggests that ghrelin signaling facilitates relapse to preferred foods in response to food cues through GHSR signaling in the VTA.     

Decreased behavioral effects of daily intracerebroventricular bombesin

Intracerebroventricular (ICV) bombesin increases grooming and decreases food intake in rats. We examined tolerance to these effects by administering a daily injection of either saline or 25 ng bombesin to rats for 8 days via lateral ventricular cannulas. Food intake and grooming were monitored. After 8 days bombesin no longer increased grooming or decreased food intake in bombesin-treated rats, but did increase grooming and decrease food intake in saline-treated rats. This development of behavioral tolerance conflicts with previous reports using larger doses and demonstrates that repeated small doses of ICV bombesin produce different effects from larger doses.     

Memantine reduces consumption of highly palatable food in a rat model of binge eating

Excessive consumption of highly palatable food has been linked to the development of eating disorders and obesity, and can be modeled in non-food-deprived rats by offering them a limited (2-h daily) access to an optional dietary fat. Since the glutamatergic system has recently emerged as a viable target for binge-eating medication development, we compared the effects of subchronic treatment with glutamatergic receptor antagonists to the effects of a reference appetite-suppressing agent sibutramine on highly palatable food (lard) and normal chow intake. In three separate experiments, the consumption of a standard laboratory chow and lard were measured during 12 days of medication treatment and for 6 days afterwards. Generalized estimating equations analysis demonstrated that sibutramine (7.5 mg/kg, PO) significantly decreased lard consumption, with a concurrent increase in chow consumption. Sibutramine effects disappeared after treatment discontinuation. The NMDA receptor antagonist memantine (5 mg/kg, IP) significantly decreased lard consumption and increased chow consumption, comparable to effects of sibutramine; however, memantine’s effects persisted after treatment discontinuation. The effects of the mGluR5 antagonist MTEP (7.5 mg/kg, IP) on food consumption were in the same direction as seen with memantine, but the observed differences were not significant. In an additional control experiment, sibutramine and memantine reduced unlimited (24 h) chow intake during the treatment phase. Present results provide evidence that glutamatergic neurotransmission might be involved in the regulation of excessive consumption of highly palatable foods, and suggest that NMDA receptor may be an attractive target for developing obesity and disordered eating pharmacotherapies.     

Overeating of palatable food is associated with blunted leptin and ghrelin responses

Palatable food is rich in fat and/or sucrose. In this study we examined the long-term effects of such diets on food intake, body weight, adiposity and circulating levels of the satiety peptide leptin and the hunger peptide ghrelin. The experiments involved rats and mice and lasted 5 weeks. In rats, we examined the effect of diets rich in fat and/or sucrose and in mice the effect of a high fat diet with or without sucrose in the drinking water. Animals fed with the palatable diets had a larger intake of calories, gained more weight and became more adipose than animals fed standard rat chow. Fasted animals are known to have low serum leptin and high serum ghrelin and to display elevated serum leptin and lowered serum ghrelin postprandially. With time, a sucrose-rich diet was found to raise the fasting level of leptin and to lower the fasting level of ghrelin in rats. A fat-rich diet suppressed serum ghrelin without affecting serum leptin; high sucrose and high fat in combination greatly reduced serum ghrelin and raised serum leptin in the fasted state. The mRNA expression of leptin in the rat stomach was up-regulated by sucrose-rich (but not by fat-rich) diets, whereas the expression of ghrelin seemed not to be affected by the palatable diets. Mice responded to sucrose in the drinking water with elevated serum leptin (fasted state) and to all palatable diets with low serum ghrelin. The expression of both leptin and ghrelin mRNA in the stomach was suppressed in fasted mice that had received a high fat diet for 5 weeks. We conclude that the expression of leptin mRNA in stomach and the concentration of leptin in serum were elevated in response to sucrose-rich rather than fat-rich diets, linking leptin with sucrose metabolism. In contrast, the expression of ghrelin and the serum ghrelin concentration were suppressed by all palatable diets, sucrose and fat alike. In view of the increased body weight and adiposity neither elevated leptin nor suppressed ghrelin were able to control/restrain the overeating that is associated with palatable diets.     

Ghrelin acts in the central nervous system to stimulate gastric acid secretion

Ghrelin is a novel acylated peptide that functions in the regulation of growth hormone release and energy metabolism. It was isolated from rat stomach as an endogenous ligand for growth hormone secretagogue receptor. Ghrelin is also localized in the arcuate nucleus of rat hypothalamus. Intracerebroventricular (ICV) administration increases food intake and body weight. We examined the effect of ghrelin on gastric acid secretion in urethane-anesthetized rats and found that ICV administration of ghrelin increased gastric acid output in a dose-dependent manner. Vagotomy and administration of atropine abolished the gastric acid secretion induced by ghrelin. ICV administration of ghrelin also induced c-fos expression in the neurons of the nucleus of the solitary tract and the dorsomotor nucleus of the vagus, which are key sites in the central nervous system for regulation of gastric acid secretion. Our results suggest that ghrelin participates in the central regulation of gastric acid secretion by activating the vagus system.