Ghrelin-induced Increases Research Articles

Ghrelin Action in the PVH of Male Mice: Accessibility, Neuronal Targets, and CRH Neurons Activation.

The hormone ghrelin displays several well-characterized functions, including some with pharmaceutical interest. The receptor for ghrelin, the growth hormone secretagogue receptor (GHSR), is expressed in the hypothalamic paraventricular nucleus (PVH), a critical hub for the integration of metabolic, neuroendocrine, autonomic, and behavioral functions. Here, we performed a neuroanatomical and functional characterization of the neuronal types mediating ghrelin actions in the PVH of male mice. We found that fluorescent ghrelin mainly labels PVH neurons immunoreactive for nitric oxide synthase 1 (NOS1), which catalyze the production of nitric oxide [NO]). Centrally injected ghrelin increases c-Fos in NOS1 PVH neurons and NOS1 phosphorylation in the PVH. We also found that a high dose of systemically injected ghrelin increases the ghrelin level in the cerebrospinal fluid and in the periventricular PVH, and induces c-Fos in NOS1 PVH neurons. Such a high dose of systemically injected ghrelin activates a subset of NOS1 PVH neurons, which do not express oxytocin, via an arcuate nucleus-independent mechanism. Finally, we found that pharmacological inhibition of NO production fully abrogates ghrelin-induced increase of calcium concentration in corticotropin-releasing hormone neurons of the PVH whereas it partially impairs ghrelin-induced increase of plasma glucocorticoid levels. Thus, plasma ghrelin can directly target a subset of NO-producing neurons of the PVH that is involved in ghrelin-induced activation of the hypothalamic-pituitary-adrenal neuroendocrine axis.

Ghrelin-induced Food Intake, but not GH Secretion, Requires the Expression of the GH Receptor in the Brain of Male Mice.

Ghrelin stimulates both GH secretion and food intake. The orexigenic action of ghrelin is mainly mediated by neurons that coexpress agouti-related protein (AgRP) and neuropeptide Y (NPY) in the arcuate nucleus of the hypothalamus (ARH). GH also stimulates food intake and, importantly, ARHAgRP/NPY neurons express GH receptor (GHR). Thus, ghrelin-induced GH secretion may contribute to the orexigenic effect of ghrelin. Here, we investigated the response to ghrelin in male mice carrying GHR ablation specifically in neurons (brain GHR knockout [KO] mice) or exclusively in ARHAgRP/NPY neurons (AgRP GHR KO mice). Although brain GHR KO mice showed normal ghrelin-induced increase in plasma GH levels, these mutants lacked the expected orexigenic response to ghrelin. Additionally, brain GHR KO mice displayed reduced hypothalamic levels of Npy and Ghsr mRNA and did not elicit ghrelin-induced c-Fos expression in the ARH. Furthermore, brain GHR KO mice exhibited a prominent reduction in AgRP fiber density in the ARH and paraventricular nucleus of the hypothalamus (PVH). In contrast, AgRP GHR KO mice showed no changes in the hypothalamic Npy and Ghsr mRNAs and conserved ghrelin-induced food intake and c-Fos expression in the ARH. AgRP GHR KO mice displayed a reduced AgRP fiber density (~16%) in the PVH, but this reduction was less than that observed in brain GHR KO mice (~61%). Our findings indicate that GHR signaling in the brain is required for the orexigenic effect of ghrelin, independently of GH action on ARHAgRP/NPY neurons.

Ghrelin Signaling Affects Feeding Behavior, Metabolism, and Memory through the Vagus Nerve

Vagal afferent neuron (VAN) signaling sends information from the gut to the brain and is fundamental in the control of feeding behavior and metabolism [1]. Recent findings reveal that VAN signaling also plays a critical role in cognitive processes, including affective motivational behaviors and hippocampus (HPC)-dependent memory [2-5]. VANs, located in nodose ganglia, express receptors for various gut-derived peptide signals; however, the function of these receptors with regard to feeding behavior, metabolism, and memory control is poorly understood. We hypothesized that VAN-mediated processes are influenced by ghrelin, a stomach-derived orexigenic hormone, via communication to its receptor (GHSR) expressed on gut-innervating VANs. To examine this hypothesis, rats received nodose ganglia injections of an adeno-associated virus (AAV) expressing short hairpin RNAs targeting GHSR (or a control AAV) for RNAi-mediated VAN-specific GHSR knockdown. Results reveal that VAN GHSR knockdown induced various feeding and metabolic disturbances, including increased meal frequency, impaired glucose tolerance, delayed gastric emptying, and increased body weight compared to controls. Additionally, VAN-specific GHSR knockdown impaired HPC-dependent contextual episodic memory and reduced HPC brain-derived neurotrophic factor expression, but did not affect anxiety-like behavior or general activity levels. A functional role for endogenous VAN GHSR signaling was further confirmed by results revealing that VAN signaling is required for the hyperphagic effects of ghrelin administered at dark onset, and that gut-restricted ghrelin-induced increases in VAN firing rate require intact VAN GHSR expression. Collective results reveal that VAN GHSR signaling is required for both normal feeding and metabolic function as well as HPC-dependent memory.

Fasting- and ghrelin-induced food intake is regulated by NAMPT in the hypothalamus.

Neurons in the arcuate nucleus of the hypothalamus are involved in regulation of food intake and energy expenditure, and dysregulation of signalling in these neurons promotes development of obesity. The role of the rate-limiting enzyme in the NAD+ salvage pathway, nicotinamide phosphoribosyltransferase (NAMPT), for regulation energy homeostasis by the hypothalamus has not been extensively studied. We determined whether Nampt mRNA or protein levels in the hypothalamus of mice were affected by diet-induced obesity, by fasting and re-feeding, and by leptin and ghrelin treatment. Primary hypothalamic neurons were treated with FK866, a selective inhibitor of NAMPT, or rAAV carrying shRNA directed against Nampt, and levels of reactive oxygen species (ROS) and mitochondrial respiration were assessed. Fasting and ghrelin-induced food intake was measured in mice in metabolic cages after intracerebroventricular (ICV)-mediated FK866 administration. NAMPT levels in the hypothalamus were elevated by administration of ghrelin and leptin. In diet-induced obese mice, both protein and mRNA levels of NAMPT decreased in the hypothalamus. NAMPT inhibition in primary hypothalamic neurons significantly reduced levels of NAD+ , increased levels of ROS, and affected the expression of Agrp, Pomc and genes related to mitochondrial function. Finally, ICV-induced NAMPT inhibition by FK866 did not cause malaise or anhedonia, but completely ablated fasting- and ghrelin-induced increases in food intake. Our findings indicate that regulation of NAMPT levels in hypothalamic neurons is important for the control of fasting- and ghrelin-induced food intake.

Gut ghrelin regulates hepatic glucose production and insulin signaling via a gut-brain-liver pathway

BackgroundGhrelin modulates many physiological processes. However, the effects of intestinal ghrelin on hepatic glucose production (HGP) are still unclear. The current study was to explore the roles of intestinal ghrelin on glucose homeostasis and insulin signaling in the liver.MethodsThe system of intraduodenal infusion and intracerebral microinfusion into the nucleus of the solitary tract (NTS) in the normal chow-diet rats and pancreatic-euglycemic clamp procedure (PEC) combined with [3-3H] glucose as a tracer were used to analyze the effect of intestinal ghrelin. Intraduodenal co-infusion of ghrelin, tetracaine and Activated Protein Kinase (AMPK) activator (AICAR), or pharmacologic and molecular inhibitor of N-methyl-D-aspartate receptors within the dorsal vagal complex, or hepatic vagotomy in rats were used to explore the possible mechanism of the effect of intestinal ghrelin on HGP.ResultsOur results demonstrated that gut infusion of ghrelin inhibited duodenal AMP-dependent protein kinase (AMPK) signal pathways, increased HGP and expression of gluconeogenic enzymes, and decreased insulin signaling in the liver of the rat. Intraduodenal co-infusion of ghrelin receptor antagonist [D-Lys3]-GHRP-6 and AMPK agonist with ghrelin diminished gut ghrelin-induced increase in HGP and decrease in glucose infusion rate (GIR) and hepatic insulin signaling. The effects of gut ghrelin were also negated by co-infusion with tetracaine, or MK801, an N-methyl-D-aspartate (NMDA) receptor inhibitor, and adenovirus expressing the shRNA of NR1 subunit of NMDA receptors (Ad-shNR1) within the dorsal vagal complex, and hepatic vagotomy in rats. When ghrelin and lipids were co-infused into the duodenum, the roles of gut lipids in increasing the rate of glucose infusion (GIR) and lowering HGP were reversed.ConclusionsThe current study provided evidence that intestinal ghrelin has an effect on HGP and identified a neural glucoregulatory function of gut ghrelin signaling.

Capsaicin-Sensitive Sensory Nerves Are Necessary for the Protective Effect of Ghrelin in Cerulein-Induced Acute Pancreatitis in Rats.

Ghrelin was shown to exhibit protective and therapeutic effect in the gut. Aim of the study was to investigate the role of sensory nerves (SN) in the protective effect of ghrelin in acute pancreatitis (AP). Studies were performed on male Wistar rats or isolated pancreatic acinar cells. After capsaicin deactivation of sensory nerves (CDSN) or treatment with saline, rats were pretreated intraperitoneally with ghrelin or saline. In those rats, AP was induced by cerulein or pancreases were used for isolation of pancreatic acinar cells. Pancreatic acinar cells were incubated in cerulein-free or cerulein containing solution. In rats with intact SN, pretreatment with ghrelin led to a reversal of the cerulein-induced increase in pancreatic weight, plasma activity of lipase and plasma concentration of tumor necrosis factor-α (TNF-α). These effects were associated with an increase in plasma interleukin-4 concentration and reduction in histological signs of pancreatic damage. CDSN tended to increase the severity of AP and abolished the protective effect of ghrelin. Exposure of pancreatic acinar cells to cerulein led to increase in cellular expression of mRNA for TNF-α and cellular synthesis of this cytokine. Pretreatment with ghrelin reduced this alteration, but this effect was only observed in acinar cells obtained from rats with intact SN. Moreover, CDSN inhibited the cerulein- and ghrelin-induced increase in gene expression and synthesis of heat shock protein 70 (HSP70) in those cells. Ghrelin exhibits the protective effect in cerulein-induced AP on the organ and pancreatic acinar cell level. Sensory nerves ablation abolishes this effect.

Ghrelin receptors mediate ghrelin-induced excitation of agouti-related protein/neuropeptide Y but not pro-opiomelanocortin neurons.

Ghrelin increases food intake and body weight by stimulating orexigenic agouti-related protein (AgRP)/neuropeptide Y (NPY) neurons and inhibiting anorexic pro-opiomelanocortin (POMC) neurons in the hypothalamus. Growth hormone secretagogue receptor (Ghsr) mediates the effect of ghrelin on feeding behavior and energy homeostasis. However, the role of Ghsr in the ghrelin effect on these two populations of neurons is unclear. We hypothesized that Ghsr mediates the effect of ghrelin on AgRP and POMC neurons. In this study, we determined whether Ghsr similarly mediates the effects of ghrelin on AgRP/NPY and POMC neurons using cell type-specific Ghsr-knockout mice. Perforated whole-cell recordings were performed on green fluorescent protein-tagged AgRP/NPY and POMC neurons in the arcuate nucleus in hypothalamic slices. In Ghsr+/+ mice, ghrelin (100nM) significantly increased the firing activity of AgRP/NPY neurons but inhibited the firing activity of POMC neurons. In Ghsr-/- mice, the excitatory effect of ghrelin on AgRP/NPY neurons was abolished. Ablation of Ghsr also eliminated ghrelin-induced increases in the frequency of GABAergic inhibitory postsynaptic currents of POMC neurons. Strikingly, ablation of Ghsr converted the ghrelin effect on POMC neurons from inhibition to excitation. Des-acylated ghrelin had no such effect on POMC neurons in Ghsr-/- mice. In both Ghsr+/+ and Ghsr-/- mice, blocking GABAA receptors with gabazine increased the basal firing activity of POMC neurons, and ghrelin further increased the firing activity of POMC neurons in the presence of gabazine. Our findings provide unequivocal evidence that Ghsr is essential for ghrelin-induced excitation of AgRP/NPY neurons. However, ghrelin excites POMC neurons through an unidentified mechanism that is distinct from conventional Ghsr.

Ghrelin enhances food intake and carbohydrate oxidation in a nitric oxide dependent manner

In the present study we sought to investigate interactions between hypothalamic nitric oxide (NO) and ghrelin signaling on food intake and energy substrate utilization as measured by the respiratory exchange ratio (RER). Guide cannulae were unilaterally implanted in either the arcuate (ArcN) or paraventricular (PVN) nuclei of male Sprague-Dawley rats. Animals were pretreated with subcutaneous (2.5–10mg/kg/ml) or central (0–100pmol) N-nitro-l-Arginine methyl ester (l-NAME) followed by 50pmol of ghrelin administered into either the ArcN or PVN. Both l-NAME and ghrelin were microinjected at the onset of the active cycle and food intake and RER were assessed 2h postinjection. RER was measured as the ratio of the volume of carbon dioxide expelled relative to the volume of oxygen consumed (VCO2/VO2) using an open-circuit indirect calorimeter. Our results demonstrated that peripheral and central l-NAME pretreatment dose-dependently attenuated ghrelin induced increases in food intake and RER in either the ArcN or PVN. In fact the 100pmol dose largely reversed the metabolic effects of ghrelin in both anatomical regions. These findings suggest that ghrelin enhancement of food intake and carbohydrate oxidation in the rat ArcN and PVN is NO-dependent.

Intracerebroventricular injection of ghrelin decreases wheel running activity in rats

There is an increasing interest in elucidating the molecular mechanisms by which voluntary exercise is regulated. In this study, we examined how the central nervous system regulates exercise. We used SPORTS rats, which were established in our laboratory as a highly voluntary murine exercise model. SPORTS rats showed lower levels of serum ghrelin compared with those of the parental line of Wistar rats. Intracerebroventricular and intraperitoneal injection of ghrelin decreased wheel-running activity in SPORTS rats. In addition, daily injection of the ghrelin inhibitor JMV3002 into the lateral ventricles of Wistar rats increased wheel-running activity. Co-administration of obestatin inhibited ghrelin-induced increases in food intake but did not inhibit ghrelin-induced suppression of voluntary exercise in rats. Growth hormone secretagogue receptor (GHSR) in the hypothalamus and hippocampus of SPORTS rats was not difference that in control rats. We created an arcuate nucleus destruction model by administering monosodium glutamate (MSG) to neonatal SPORTS rats. Injection of ghrelin into MSG-treated rats decreased voluntary exercise but did not increase food intake, suggesting that wheel-running activity is not controlled by the arcuate nucleus neurons that regulate feeding. These results provide new insights into the mechanism by which ghrelin regulates voluntary activity independent of arcuate nucleus neurons.

Central ghrelin increases food foraging/hoarding that is blocked by GHSR antagonism and attenuates hypothalamic paraventricular nucleus neuronal activation.

The stomach-derived "hunger hormone" ghrelin increases in the circulation in direct response to time since the last meal, increasing preprandially and falling immediately following food consumption. We found previously that peripheral injection of ghrelin potently stimulates food foraging (FF), food hoarding (FH), and food intake (FI) in Siberian hamsters. It remains, however, largely unknown if central ghrelin stimulation is necessary/sufficient to increase these behaviors regardless of peripheral stimulation of the ghrelin receptor [growth hormone secretagogue receptor (GHSR)]. We injected three doses (0.01, 0.1, and 1.0 μg) of ghrelin into the third ventricle (3V) of Siberian hamsters and measured changes in FF, FH, and FI. To test the effects of 3V ghrelin receptor blockade, we used the potent GHSR antagonist JMV2959 to block these behaviors in response to food deprivation or a peripheral ghrelin challenge. Finally, we examined neuronal activation in the arcuate nucleus and paraventricular hypothalamic nucleus in response to peripheral ghrelin administration and 3V GHSR antagonism. Third ventricular ghrelin injection significantly increased FI through 24 h and FH through day 4. Pretreatment with 3V JMV2959 successfully blocked peripheral ghrelin-induced increases in FF, FH, and FI at all time points and food deprivation-induced increases in FF, FH, and FI up to 4 h. c-Fos immunoreactivity was significantly reduced in the paraventricular hypothalamic nucleus, but not in the arcuate nucleus, following pretreatment with intraperitoneal JMV2959 and ghrelin. Collectively, these data suggest that central GHSR activation is both necessary and sufficient to increase appetitive and consummatory behaviors in Siberian hamsters.

Anti-ghrelin Spiegelmer inhibits exogenous ghrelin-induced increases in food intake, hoarding, and neural activation, but not food deprivation-induced increases

Circulating concentrations of the stomach-derived "hunger-peptide" ghrelin increase in direct proportion to the time since the last meal. Exogenous ghrelin also increases food intake in rodents and humans, suggesting ghrelin may increase post-fast ingestive behaviors. Food intake after food deprivation is increased by laboratory rats and mice, but not by humans (despite dogma to the contrary) or by Siberian hamsters; instead, humans and Siberian hamsters increase food hoarding, suggesting the latter as a model of fasting-induced changes in human ingestive behavior. Exogenous ghrelin markedly increases food hoarding by ad libitum-fed Siberian hamsters similarly to that after food deprivation, indicating sufficiency. Here, we tested the necessity of ghrelin to increase food foraging, food hoarding, and food intake, and neural activation [c-Fos immunoreactivity (c-Fos-ir)] using anti-ghrelin Spiegelmer NOX-B11-2 (SPM), an l-oligonucleotide that specifically binds active ghrelin, inhibiting peptide-receptor interaction. SPM blocked exogenous ghrelin-induced increases in food hoarding the first 2 days after injection, and foraging and food intake at 1-2 h and 2-4 h, respectively, and inhibited hypothalamic c-Fos-ir. SPM given every 24 h across 48-h food deprivation inconsistently inhibited food hoarding after refeeding and c-Fos-ir, similarly to inabilities to do so in laboratory rats and mice. These results suggest that ghrelin may not be necessary for food deprivation-induced foraging and hoarding and neural activation. A possible compensatory response, however, may underlie these findings because SPM treatment led to marked increases in circulating ghrelin concentrations. Collectively, these results show that SPM can block exogenous ghrelin-induced ingestive behaviors, but the necessity of ghrelin for food deprivation-induced ingestive behaviors remains unclear.

Sa1725 A Novel Rat Cachexia Model With Possible Ghrelin Resistance Made by Inoculation of a Gastric 85as2 Cancer Cell Line: A Traditional Japanese Medicine Rikkunshito Ameliorates Cachexia Symptoms by Potentiation of Ghrelin Receptor-Mediated Signaling

Aims: Gastric cancer patients have high frequency of weight loss and anorexia, causing highest tendency of cachexia. Cancer cachexia (CC) is a multifactorial syndrome characterized by anorexia, low fat-free mass (FFM) and weight loss. We established a novel rat CC model by inoculation of a gastric cancer 85AS2 cell line. To improve gastrointestinal-related anorexia and weight loss, treatment with an orexygenic peptide ghrelin has been tested in clinical trials. However, plasma ghrelin levels in CC patients are relatively high possibly due to compensatory mechanisms. We evaluated the effects of ghrelin or a traditional Japanese medicine rikkunshito (RKT), effective for the treatment of gastrointestinal disorders, on the CC symptoms. We postulate that RKT has other mechanisms in addition to elevate ghrelin concentrations (Takeda et al., Gastroenterology (2008)). Accordingly we further investigated the effects of RKT on the ghrelin-mediated cellular signaling. Methods: To establish CC models, 85As2 cell lines derived from gastric MKN-45 cancer cells was inoculated subcutaneously into nude rats. We evaluated the effects of single intraperitoneal ghrelin or oral RKT administration for 7 days after the onset of CC rats. Further, the effects of RKT on ghrelin receptor activity was investigated with cells stably expressing ghrelin receptors; ghrelininduced increases in intracellular Ca2+ concentrations ([Ca2+]i) was measured with Ca2+ imaging asssay. Ghrelin receptor binding assay was also performed with radiolabelled ghrelin in the ghrelin receptor-expressing cells. Results: 85As2-inoculated rats showed significant weight loss, anorexia, low FFM (including loss of skeletal muscle) and fat mass were observed with 100% efficacy. Increased acute phase proteins and decreased albumin levels were observed in the plasma of all CC rats. Ghrelin administration significantly increased food intake in non-tumor-bearing rats but not in 85As2-induced CC rats; higher plasma ghrelin levels were detected in all CC rats. Although ghrelin failed to improve the CC symptoms, RKT significantly ameliorated anorexia, weight loss and decreased FFM. In ghrelin receptorexpressed cells, RKT enhanced ghrelin-induced [Ca2+]i in a concentration-dependent manner. We screened 43 compounds contained in RKT, and 2 (atractylodin and atractylodinol) showed marked increases in ghrelin receptor binding capacity. We finally found atractylodin potentiated the ghrelin-induced increase in [Ca2+]i as well as RKT. Conclusions: We established the novel CC model by using 85As2 cells and this model showed ghrelin resistance. The ameliorative effects of CC symptoms by RKT may be due to enhanced ghrelin receptor activity. RKT as well as atractylodin may contribute to the development of novel therapeutics for CC patients even ghrelin concentrations in plasma were higher.

Ghrelin Increases GABAergic Transmission and Interacts with Ethanol Actions in the Rat Central Nucleus of the Amygdala

The neural circuitry that processes natural rewards converges with that engaged by addictive drugs. Because of this common neurocircuitry, drugs of abuse have been able to engage the hedonic mechanisms normally associated with the processing of natural rewards. Ghrelin is an orexigenic peptide that stimulates food intake by activating GHS-R1A receptors in the hypothalamus. However, ghrelin also activates GHS-R1A receptors on extrahypothalamic targets that mediate alcohol reward. The central nucleus of the amygdala (CeA) has a critical role in regulating ethanol consumption and the response to ethanol withdrawal. We previously demonstrated that rat CeA GABAergic transmission is enhanced by acute and chronic ethanol treatment. Here, we used quantitative RT-PCR (qRT-PCR) to detect Ghsr mRNA in the CeA and performed electrophysiological recordings to measure ghrelin effects on GABA transmission in this brain region. Furthermore, we examined whether acute or chronic ethanol treatment would alter these electrophysiological effects. Our qRT-PCR studies show the presence of Ghsr mRNA in the CeA. In naive animals, superfusion of ghrelin increased the amplitude of evoked inhibitory postsynaptic potentials (IPSPs) and the frequency of miniature inhibitory postsynaptic currents (mIPSCs). Coapplication of ethanol further increased the ghrelin-induced enhancement of IPSP amplitude, but to a lesser extent than ethanol alone. When applied alone, ethanol significantly increased IPSP amplitude, but this effect was attenuated by the application of ghrelin. In neurons from chronic ethanol-treated (CET) animals, the magnitude of ghrelin-induced increases in IPSP amplitude was not significantly different from that in naive animals, but the ethanol-induced increase in amplitude was abolished. Superfusion of the GHS-R1A antagonists D-Lys3-GHRP-6 and JMV 3002 decreased evoked IPSP and mIPSC frequency, revealing tonic ghrelin activity in the CeA. D-Lys3-GHRP-6 and JMV 3002 also blocked ghrelin-induced increases in GABAergic responses. Furthermore, D-Lys3-GHRP-6 did not affect ethanol-induced increases in IPSP amplitude. These studies implicate a potential role for the ghrelin system in regulating GABAergic transmission and a complex interaction with ethanol at CeA GABAergic synapses.

Abstract 344: Intrarenal Ghrelin Receptors Mediate Sodium Reabsorption via an ENaC-Dependent Pathway

Intrarenal ghrelin infusion stimulates distal nephron-dependent sodium (Na+) reabsorption in normal rats, but the mechanism is unknown. The main Na+ transporters of the distal nephron segment are the Na+-Cl- co-transporter (NCC) and the epithelial Na+ channel (ENaC). To determine which of these transporters is involved in the antinatriuretic actions of intrarenal ghrelin receptors, uninephrectomized Sprague-Dawley rats received 3 cumulative 1h renal interstitial (RI) infusions of ghrelin (0.3-3 μg/min, N=8) or vehicle (5% dextrose in water, D5W, N=8), prior to harvesting the infused kidney for determination of NCC and ENaC protein expression. Ghrelin-infused rats demonstrated significantly reduced Na+ excretion rates (UNaV) compared to D5W-infused rats (66.7±7.1% of baseline, P<0.01) and significantly increased cortical collecting duct ENaC expression (2.46±0.17 and 1.56±0.19 densitometric units respectively, P<0.01). Renal NCC expression did not change in response to either ghrelin or D5W infusion (4.21±0.58 and 4.13±0.44 densitometric units respectively, P=NS). To test whether the ghrelin-induced increase in collecting duct ENaC expression was responsible for the antinatriuretic actions of ghrelin, we infused ghrelin into the kidney in the presence of amiloride, a selective inhibitor of ENaC activity. Following uninephrectomy, rats were implanted with either a subcutaneous osmotic minipump which systemically delivered amiloride (1.4 ng/min) for 72h to block ENaC activity (N=6), or a minipump that was filled with D5W (N=6). On the day of the study, RI ghrelin (0.3-3 μg/min) or D5W infusion was initiated. RI ghrelin infusion (in the absence of amiloride) significantly reduced UNaV to 58.9±7.2% of baseline, P<0.01; however, in the presence of amiloride, RI ghrelin failed to reduce UNaV, demonstrating values identical to rats that did not receive RI ghrelin. In contrast, studies carried out in the presence of chlorothiazide pumps (to block NCC activity, 1.1 μg/min, N=6), continued to demonstrate ghrelin-induced antinatriuresis (UNaV decreased to 65.3±8.8% of baseline, P<0.01). Mean arterial pressures did not change during any of the acute RI infusions. Thus, intact ENaC function is necessary for ghrelin-induced Na+ reabsorption.

In vitro selection of a peptide antagonist of growth hormone secretagogue receptor using cDNA display

G protein-coupled receptors (GPCRs) are major drug targets, and their ligands are currently being explored and developed by many pharmaceutical companies and independent researchers. Class A (rhodopsin-like) GPCRs compose a predominant GPCR family; therefore, class A GPCR ligands are in demand. Growth hormone secretagogue receptor (GHS-R) is a class A GPCR that stimulates food intake by binding to its peptide ligand, ghrelin. Therefore, antagonists of GHS-R are expected to exert antiobesity function. In this article, we describe the use of cDNA display to screen for successfully and identify an antagonistic peptide of GHS-R. The antagonistic peptide inhibited the ghrelin-induced increase in intracellular Ca(2+) in vitro (IC(50)=approximately10μM) and repressed the contraction of isolated animal stomach in response to ghrelin. Furthermore, peripheral administration of the peptide inhibited the food intake of mice. This work provides new insight into the development of antiobesity drugs and describes a method for the discovery of unique peptide ligands for class A GPCRs.

Ghrelin increases the motivation to eat, but does not alter food palatability.

Homeostatic eating cannot explain overconsumption of food and pathological weight gain. A more likely factor promoting excessive eating is food reward and its representation in the central nervous system (CNS). The anorectic hormones leptin and insulin reduce food reward and inhibit related CNS reward pathways. Conversely, the orexigenic gastrointestinal hormone ghrelin activates both homeostatic and reward-related neurocircuits. The current studies were conducted to identify in rats the effects of intracerebroventricular ghrelin infusions on two distinct aspects of food reward: hedonic valuation (i.e., "liking") and the motivation to self-administer (i.e., "wanting") food. To assess hedonic valuation of liquid food, lick motor patterns were recorded using lickometry. Although ghrelin administration increased energy intake, it did not alter the avidity of licking (initial lick rates or lick-cluster size). Several positive-control conditions ruled out lick-rate ceiling effects. Similarly, when the liquid diet was hedonically devalued with quinine supplementation, ghrelin failed to reverse the quinine-associated reduction of energy intake and avidity of licking. The effects of ghrelin on rats' motivation to eat were assessed using lever pressing to self-administer food in a progressive-ratio paradigm. Ghrelin markedly increased motivation to eat, to levels comparable to or greater than those seen following 24 h of food deprivation. Pretreatment with the dopamine D1 receptor antagonist SCH-23390 eliminated ghrelin-induced increases in lever pressing, without compromising generalized licking motor control, indicating a role for D1 signaling in ghrelin's motivational feeding effects. These results indicate that ghrelin increases the motivation to eat via D1 receptor-dependent mechanisms, without affecting perceived food palatability.

Ghrelin Interacts with Neuropeptide Y Y1 and Opioid Receptors to Increase Food Reward

Ghrelin, a stomach-derived hormone, is an orexigenic peptide that was recently shown to potently increase food reward behavior. The neurochemical circuitry that links ghrelin to the mesolimbic system and food reward behavior remains unclear. Here we examined the contribution of neuropeptide Y (NPY) and opioids to ghrelin's effects on food motivation and intake. Both systems have well-established links to the mesolimbic ventral tegmental area (VTA) and reward/motivation control. NPY mediates the effect of ghrelin on food intake via activation of NPY-Y1 receptor (NPY-Y1R); their connection with respect to motivated behavior is unexplored. The role of opioids in any aspect of ghrelin's action on food-oriented behaviors is unknown. Rats were trained in a progressive ratio sucrose-induced operant schedule to measure food reward/motivation behavior. Chow intake was measured immediately after the operant test. In separate experiments, we explored the suppressive effects of a selective NPY-Y1R antagonist or opioid receptor antagonist naltrexone, injected either intracerebroventricularly or intra-VTA, on ghrelin-induced food reward behavior. The ventricular ghrelin-induced increase in sucrose-motivated behavior and chow intake were completely blocked by intracerebroventricular pretreatment with either an NPY-Y1R antagonist or naltrexone. The intra-VTA ghrelin-induced sucrose-motivated behavior was blocked only by intra-VTA naltrexone. In contrast, the intra-VTA ghrelin-stimulated chow intake was attenuated only by intra-VTA NPY-Y1 blockade. Finally, ghrelin infusion was associated with an elevated VTA μ-opioid receptor expression. Thus, we identify central NPY and opioid signaling as the necessary mediators of food intake and reward effects of ghrelin and localize these interactions to the mesolimbic VTA.

Ghrelin and obestatin modulate growth hormone-releasing hormone release and synaptic inputs onto growth hormone-releasing hormone neurons

Ghrelin, a natural ligand of the growth hormone secretagogue receptor (GHS-R), is synthesized in the stomach but may also be expressed in lesser quantity in the hypothalamus where the GHS-R is located on growth hormone-releasing hormone (GHRH) neurons. Obestatin, a peptide derived from the same precursor as ghrelin, is able to antagonize the ghrelin-induced increase of growth hormone (GH) secretion in vivo but not from pituitary explants in vitro. Thus, the blockade of ghrelin-induced GH release by obestatin could be mediated at the hypothalamic level by the neuronal network that controls pituitary GH secretion. Ghrelin increased GHRH and decreased somatostatin (somatotropin-releasing inhibitory factor) release from hypothalamic explants, whereas obestatin only reduced the ghrelin-induced increase of GHRH release, thus indicating that the effect of ghrelin and obestatin is targeted to GHRH neurons. Patch-clamp recordings on mouse GHRH-enhanced green fluorescent protein neurons indicated that ghrelin and obestatin had no significant effects on glutamatergic synaptic transmission. Ghrelin decreased GABAergic synaptic transmission in 44% of the recorded neurons, an effect blocked in the presence of the GHS-R antagonist BIM28163, and stimulated the firing rate of 78% of GHRH neurons. Obestatin blocked the effects of ghrelin by acting on a receptor different from the GHS-R. These data suggest that: (i) ghrelin increases GHRH neuron excitability by increasing their action potential firing rate and decreasing the strength of GABA inhibitory inputs, thereby leading to an enhanced GHRH release; and (ii) obestatin counteracts ghrelin actions. Such interactions on GHRH neurons probably participate in the control of GH secretion.

Ghrelin reduces rat myocardial calcification induced by nicotine and vitamin D3 in vivo

Ghrelin, a newly discovered bioactive peptide, initially was identified as a strong stimulant for the release of growth hormone (GH) and that has improved cardiac function in patients suffering from end-stage chronic heart failure. Increasing evidence has demonstrated that ghrelin may have myocardial protective effects. However, the role of ghrelin in the pathogenesis of cardiovascular diseases remains unclear. In this study, an in vivo model of rat myocardial calcification induced by vitamin D3 and nicotine was used to study the possible mechanism in the regulatory action of ghrelin on the calcified myocardium. Calcification increased total Ca2+ content and 45Ca2+ deposition in the myocardium and alkaline phosphatase (ALP) activation in the plasma. Compared with the control group, ghrelin mRNA expression was up-regulated and the myocardium calcium content was significantly increased in vitamin D3 and nicotine-treated rats. Rats were subcutaneously injected with 1 or 10 nmol/kg ghrelin. Rats treated with both low- and high-dose ghrelin decreased total Ca2+ content and 45Ca2+ deposition in cardiac muscle and inhibited ALP activation in the myocardium and plasma, in a concentration-dependent manner. In addition, osteopontin (OPN) mRNA expression significantly decreased and that of endothelin (ET-1) significantly increased with myocardial calcification. Ghrelin treatment increased OPN expression at the mRNA level and reduced ET-1 mRNA expression in a dose-dependent manner. These results indicate that exogenous administration with ghrelin attenuates myocardial calcification induced by nicotine and vitamin D3, and that the possible mechanism is via the ghrelin-induced increase in the OPN mRNA levels and decrease in the ET-1 mRNA expression in the myocardium.

Ghrelin stimulates corticotropin-releasing factor and vasopressin gene expression in rat hypothalamic 4B cells

Corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) play a central role in regulating the stress response. In response to stress, CRF and AVP neurons in the hypothalamic paraventricular nucleus secrete the peptides to stimulate the release of adrenocorticotropic hormone from the anterior pituitary. Ghrelin, an endogenous ligand of the growth hormone-releasing peptide receptors (GHSR), has been shown to stimulate the release of CRF and AVP by rat hypothalamic explants. However, little is known about the ability of the ghrelin signaling pathways to activate the CRF and AVP genes in the hypothalamus. In the present study, we examined the direct effect of ghrelin on CRF and AVP gene expression in hypothalamic 4B cells, which show the characteristics of the hypothalamic parvocellular paraventricular nucleus neurons. Cells were transfected with CRF or AVP promoter to examine the activity of each promoter. Ghrelin stimulated the promoter activities and mRNA levels for both CRF and AVP. The involvement of a protein kinase pathway was examined using inhibitors. Protein kinase A and phospholipase C pathways were shown to be involved in ghrelin-induced increases in both CRF and AVP promoter activities. GHSR type 1a (GHSR1a) mRNA levels were also increased by ghrelin, and these ghrelin-induced levels were suppressed by a GHSR1a antagonist. Thus, ghrelin-dependent pathways are involved in the regulation of CRF and AVP gene expression in the hypothalamus: ghrelin, an orexigenic hormone, stimulates CRF, an anorexigenic/anxiogenic factor in the hypothalamus, resulting in hypothalamic–pituitary–adrenal axis activation to stimulate the release of glucocorticoids.