Palmitoylethanolamide dampens neuroinflammation and anxiety-like behavior in obese mice.

High-fat diet (HFD) consumption leads to obesity and a chronic state of low-grade inflammation, named metainflammation. Notably, metainflammation contributes to neuroinflammation due to the increased levels of circulating free fatty acids and cytokines. It indicates a strict interplay between peripheral and central counterparts in the pathogenic mechanisms of obesity-related mood disorders. In this context, the impairment of internal hypothalamic circuitry runs in tandem with the alteration of other brain areas associated with emotional processing (i.e., hippocampus and amygdala). Palmitoylethanolamide (PEA), an endogenous lipid mediator belonging to the N-acylethanolamines family, has been extensively studied for its pleiotropic effects both at central and peripheral level. Our study aimed to elucidate PEA capability in limiting obesity-induced anxiety-like behavior and neuroinflammation-related features in an experimental model of HFD-fed obese mice. PEA treatment promoted an improvement in anxiety-like behavior of obese mice and the systemic inflammation, reducing serum pro-inflammatory mediators (i.e., TNF-α, IL-1ß, MCP-1, LPS). In the amygdala, PEA increased dopamine turnover, as well as GABA levels. PEA also counteracted the overactivation of HPA axis, reducing the expression of hypothalamic corticotropin-releasing hormone and its type 1 receptor. Moreover, PEA attenuated the immunoreactivity of Iba-1 and GFAP and reduced pro-inflammatory pathways and cytokine production in both the hypothalamus and hippocampus. This finding, together with the reduced transcription of mast cell markers (chymase 1 and tryptase ß2) in the hippocampus, indicated the weakening of immune cell activation underlying the neuroprotective effect of PEA. Obesity-driven neuroinflammation was also associated with the disruption of blood-brain barrier (BBB) in the hippocampus. PEA limited the albumin extravasation and restored tight junction transcription modified by HFD. To gain mechanistic insight, we designed an in vitro model of metabolic injury using human neuroblastoma SH-SY5Y cells insulted by a mix of glucosamine and glucose. Here, PEA directly counteracted inflammation and mitochondrial dysfunction in a PPAR-α-dependent manner since the pharmacological blockade of the receptor reverted its effects. Our results strengthen the therapeutic potential of PEA in obesity-related neuropsychiatric comorbidities, controlling neuroinflammation, BBB disruption, and neurotransmitter imbalance involved in behavioral dysfunctions.

Overexpression of melanocortin 2 receptor accessory protein 2 (MRAP2) in adult paraventricular MC4R neurons regulates energy intake and expenditure.

OBJECTIVE: Melanocortin 2 receptor accessory protein 2 (MRAP2) has a critical role in energy homeostasis. Although MRAP2 has been shown to regulates a number of GPCRs involved in metabolism, the key neurons responsible for the phenotype of gross obesity in MRAP2 deficient animals are unclear. Furthermore, to date, all the murine MRAP2 models involve the prenatal deletion of MRAP2. METHODS: To target Melanocortin 4 receptor (MC4R)-expressing neurons in the hypothalamic paraventricular nucleus (PVN), we performed stereotaxic surgery using AAV to selectively overexpress MRAP2 postnatally in adult Mc4r-cre mice. We assessed energy homeostasis, glucose metabolism, core body temperature, and response to MC3R/MC4R agonist MTII. RESULTS: Mc4r-crePVN-MRAP2 female mice on a standard chow diet had less age-related weight gain and improved glucose/insulin profile compared to control Mc4r-crePVN-GFP mice. These changes were associated with a reduction in food intake and increased energy expenditure. In contrast, Mc4r-crePVN-MRAP2 male mice showed no improvement on a chow diet, but improvement of energy and glucose metabolism was observed following high fat diet (HFD) feeding. In addition, an increase in core body temperature was found in both females fed on standard chow diet and males fed on HFD. Mc4r-crePVN-MRAP2 female and male mice showed increased neuronal activation in the PVN compared to controls, with further increase in neuronal activation post MTII treatment in females. CONCLUSIONS: Our data indicate a site-specific role for MRAP2 in PVN MC4R-expressing neurons in potentiating MC4R neuronal activation at baseline conditions in the regulation of food intake and energy expenditure.

Aromatase and estrogen receptor immunoreactivity in the coronary arteries of monkeys and human subjects.

OBJECTIVE: The objective of this study was to determine whether estrogen could be formed locally in the coronary arteries. DESIGN: Coronary arteries were examined from monkeys (Macaca fascicularis, one male and one female) and human subjects (one premenopausal woman, one postmenopausal woman, and one man) by immunocytochemistry, using purified antisera against human placental estrogen synthetase (aromatase) and ER α. The arteries were graded for the amount of atherosclerosis. RESULTS: There was clear immunopositivity for both aromatase and estrogen receptors in all arteries studied. Although all endothelial cells (CD31 positive) stained for both antigens, the staining in macrophages, fibroblasts, and smooth muscle cells was irregular. CONCLUSION: The present results provide the first evidence for the local formation of estrogen in the coronary arteries. In addition to complementing the evidence of a cardioprotective effect of estrogen on the coronary circulation, our results highlight the potential importance of local regulation of estrogen formation and the role of available precursor androgens in maintaining the cardiovascular system.

Hypothalamic Ventromedial Lin28a Enhances Glucose Metabolism in Diet-Induced Obesity.

The Lin28a/Let-7 axis has been studied in peripheral tissues for its role in metabolism regulation. However, its central function remains unclear. Here we found that Lin28a is highly expressed in the hypothalamus compared with peripheral tissues. Its expression is positively correlated with positive energy balance, suggesting a potential central role for Lin28a in metabolism regulation. Thus, we targeted the hypothalamic ventromedial nucleus (VMH) to selectively overexpress (Lin28aKIVMH ) or downregulate (Lin28aKDVMH ) Lin28a expression in mice. With mice on a standard chow diet, body weight and glucose homeostasis were not affected in Lin28aKIVMH or Lin28aKDVMH mice. On a high-fat diet, although no differences in body weight and composition were observed, Lin28aKIVMH mice showed improved glucose tolerance and insulin sensitivity compared with controls. Conversely, Lin28aKDVMH mice displayed glucose intolerance and insulin resistance. Changes in VMH AKT activation of diet-induced obese Lin28aKIVMH or Lin28aKDVMH mice were not associated with alterations in Let-7 levels or insulin receptor activation. Rather, we observed altered expression of TANK-binding kinase-1 (TBK-1), which was found to be a direct Lin28a target mRNA. VMH-specific inhibition of TBK-1 in mice with diet-induced obesity impaired glucose metabolism and AKT activation. Altogether, our data show a TBK-1-dependent role for central Lin28a in glucose homeostasis.

PPARγ ablation sensitizes proopiomelanocortin neurons to leptin during high-fat feeding.

Activation of central PPARγ promotes food intake and body weight gain; however, the identity of the neurons that express PPARγ and mediate the effect of this nuclear receptor on energy homeostasis is unknown. Here, we determined that selective ablation of PPARγ in murine proopiomelanocortin (POMC) neurons decreases peroxisome density, elevates reactive oxygen species, and induces leptin sensitivity in these neurons. Furthermore, ablation of PPARγ in POMC neurons preserved the interaction between mitochondria and the endoplasmic reticulum, which is dysregulated by HFD. Compared with control animals, mice lacking PPARγ in POMC neurons had increased energy expenditure and locomotor activity; reduced body weight, fat mass, and food intake; and improved glucose metabolism when exposed to high-fat diet (HFD). Finally, peripheral administration of either a PPARγ activator or inhibitor failed to affect food intake of mice with POMC-specific PPARγ ablation. Taken together, our data indicate that PPARγ mediates cellular, biological, and functional adaptations of POMC neurons to HFD, thereby regulating whole-body energy balance.

Hypothalamic prolyl endopeptidase (PREP) regulates pancreatic insulin and glucagon secretion in mice.

Prolyl endopeptidase (PREP) has been implicated in neuronal functions. Here we report that hypothalamic PREP is predominantly expressed in the ventromedial nucleus (VMH), where it regulates glucose-induced neuronal activation. PREP knockdown mice (Prep(gt/gt)) exhibited glucose intolerance, decreased fasting insulin, increased fasting glucagon levels, and reduced glucose-induced insulin secretion compared with wild-type controls. Consistent with this, central infusion of a specific PREP inhibitor, S17092, impaired glucose tolerance and decreased insulin levels in wild-type mice. Arguing further for a central mode of action of PREP, isolated pancreatic islets showed no difference in glucose-induced insulin release between Prep(gt/gt) and wild-type mice. Furthermore, hyperinsulinemic euglycemic clamp studies showed no difference between Prep(gt/gt) and wild-type control mice. Central PREP regulation of insulin and glucagon secretion appears to be mediated by the autonomic nervous system because Prep(gt/gt) mice have elevated sympathetic outflow and norepinephrine levels in the pancreas, and propranolol treatment reversed glucose intolerance in these mice. Finally, re-expression of PREP by bilateral VMH injection of adeno-associated virus-PREP reversed the glucose-intolerant phenotype of the Prep(gt/gt) mice. Taken together, our results unmask a previously unknown player in central regulation of glucose metabolism and pancreatic function.

Mitochondrial uncoupling protein 2 (UCP2) in glucose and lipid metabolism.

Nutrient availability is critical for the physiological functions of all tissues. By contrast, an excess of nutrients such as carbohydrate and fats impair health and shorten life due by stimulating chronic diseases, including diabetes, cancer and neurodegeneration. The control of circulating glucose and lipid levels involve mitochondria in both central and peripheral mechanisms of metabolism regulation. Mitochondrial uncoupling protein 2 (UCP2) has been implicated in physiological and pathological processes related to glucose and lipid metabolism, and in this review we discuss the latest data on the relationships between UCP2 and glucose and lipid sensing from the perspective of specific hypothalamic neuronal circuits and peripheral tissue functions. The goal is to provide a framework for discussion of future therapeutic strategies for metabolism-related chronic diseases.

Nicotine decreases food intake through activation of POMC neurons.

Smoking decreases appetite, and smokers often report that they smoke to control their weight. Understanding the neurobiological mechanisms underlying the anorexic effects of smoking would facilitate the development of novel treatments to help with smoking cessation and to prevent or treat obesity. By using a combination of pharmacological, molecular genetic, electrophysiological, and feeding studies, we found that activation of hypothalamic α3ß4 nicotinic acetylcholine receptors leads to activation of pro-opiomelanocortin (POMC) neurons. POMC neurons and subsequent activation of melanocortin 4 receptors were critical for nicotinic-induced decreases in food intake in mice. This study demonstrates that nicotine decreases food intake and body weight by influencing the hypothalamic melanocortin system and identifies critical molecular and synaptic mechanisms involved in nicotine-induced decreases in appetite.

Alpha-melanocyte stimulating hormone: production and degradation.

Proopiomelanocortin (POMC) is a polypeptide hormone precursor that is expressed in the brain and in peripheral tissues such as in the pituitary gland, immune system, and skin. In the brain, POMC is processed to form several peptides including alpha-melanocyte stimulating hormone (α-MSH). alpha-MSH is expressed in the hypothalamic arcuate nucleus and in the nucleus tractus solitarius of the brainstem where it has a crucial role in the regulation of metabolic functions. Specifically, α-MSH is an anorexigenic peptide. Its production and maturation processes have been shown to be regulated according to the metabolic condition of the organism. This review summarizes our current knowledge on α-MSH processing including its maturation and degradation processes and pharmacological aspects of its manipulation.

NTPDases in the neuroendocrine hypothalamus: possible energy regulators of the positive gonadotrophin feedback.

BACKGROUND: Brain-derived ectonucleoside triphosphate diphosphohydrolases (NTPDases) have been known as plasma membrane-incorporated enzymes with their ATP-hydrolyzing domain outside of the cell. As such, these enzymes are thought to regulate purinergic intercellular signaling by hydrolyzing ATP to ADP-AMP, thus regulating the availability of specific ligands for various P2X and P2Y purinergic receptors. The role of NTPDases in the central nervous system is little understood. The two major reasons are the insufficient knowledge of the precise localization of these enzymes in neural structures, and the lack of specific inhibitors for the various NTPDases. To fill these gaps, we recently studied the presence of neuron-specific NTPDase3 in the mitochondria of hypothalamic excitatory neurons by morphological and functional methods. Results from those studies suggested that intramitochondrial regulation of ATP levels may play a permissive role in the neural regulation of physiological functions by tuning the level of ATP-carried energy that is needed for neuronal functions, such as neurotransmission and/or intracellular signaling. PRESENTATION OF THE HYPOTHESIS: In the lack of highly specific inhibitors, the determination of the precise function and role of NTPDases is hardly feasable. Yet, here we attempt to find an approach to investigate a possible role for hypothalamic NTPDase3 in the initiation of the midcycle luteinizing hormone (LH) surge, as such a biological role was implied by our recent findings. Here we hypothesize that NTPDase-activity in neurons of the AN may play a permissive role in the regulation of the estrogen-induced pituitary LH-surge. TESTING THE HYPOTHESIS: We propose to test our hypothesis on ovariectomized rats, by stereotaxically injecting 17beta-estradiol and/or an NTPDase-inhibitor into the arcuate nucleus and determine the consequential levels of blood LH, mitochondrial respiration rates from arcuate nucleus synaptosomal preparations, NTPDase3-expression from arcuate nucleus tissue samples, all compared to sham and intact controls. IMPLICATIONS OF THE HYPOTHESIS: Results from these studies may lead to the conclusion that estrogen may modulate the activity of mitochondrial, synapse-linked NTPDase3, and may show a correlation between mitochondrial NTPDase3-activity and the regulation of LH-release by estrogen.

Ecto-nucleoside triphosphate diphosphohydrolase 3 in the ventral and lateral hypothalamic area of female rats: morphological characterization and functional implications.

BACKGROUND: Based on its distribution in the brain, ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) may play a role in the hypothalamic regulation of homeostatic systems, including feeding, sleep-wake behavior and reproduction. To further characterize the morphological attributes of NTPDase3-immunoreactive (IR) hypothalamic structures in the rat brain, here we investigated: 1.) The cellular and subcellular localization of NTPDase3; 2.) The effects of 17beta-estradiol on the expression level of hypothalamic NTPDase3; and 3.) The effects of NTPDase inhibition in hypothalamic synaptosomal preparations. METHODS: Combined light- and electron microscopic analyses were carried out to characterize the cellular and subcellular localization of NTPDase3-immunoreactivity. The effects of estrogen on hypothalamic NTPDase3 expression was studied by western blot technique. Finally, the effects of NTPDase inhibition on mitochondrial respiration were investigated using a Clark-type oxygen electrode. RESULTS: Combined light- and electron microscopic analysis of immunostained hypothalamic slices revealed that NTPDase3-IR is linked to ribosomes and mitochondria, is predominantly present in excitatory axon terminals and in distinct segments of the perikaryal plasma membrane. Immunohistochemical labeling of NTPDase3 and glutamic acid decarboxylase (GAD) indicated that gamma-amino-butyric-acid- (GABA) ergic hypothalamic neurons do not express NTPDase3, further suggesting that in the hypothalamus, NTPDase3 is predominantly present in excitatory neurons. We also investigated whether estrogen influences the expression level of NTPDase3 in the ventrobasal and lateral hypothalamus. A single subcutaneous injection of estrogen differentially increased NTPDase3 expression in the medial and lateral parts of the hypothalamus, indicating that this enzyme likely plays region-specific roles in estrogen-dependent hypothalamic regulatory mechanisms. Determination of mitochondrial respiration rates with and without the inhibition of NTPDases confirmed the presence of NTPDases, including NTPDase3 in neuronal mitochondria and showed that blockade of mitochondrial NTPDase functions decreases state 3 mitochondrial respiration rate and total mitochondrial respiratory capacity. CONCLUSION: Altogether, these results suggest the possibility that NTPDases, among them NTPDase3, may play an estrogen-dependent modulatory role in the regulation of intracellular availability of ATP needed for excitatory neuronal functions including neurotransmission.

Hormonal regulation of the arcuate nucleus melanocortin system.

Over the past century, the hypothalamus has emerged as one of the critical sites involved in energy homeostasis. Degeneration studies in rats performed some six decades ago, first led to identifying hypothalamic subregions controlling food intake and body weight. The idea that the central nervous system (CNS), and the hypothalamus in particular, are key in metabolism regulation was reinforced by the discovery of leptin in 1994. Since the identification of leptin, enormous progress has been made in the understanding of the regulation of hypothalamic and extrahypothalamic brain regions that control food intake and energy expenditure by peripheral signals such as hormones. An important challenge is to decipher these complicated interactions between peripheral signals and neuronal circuits to better understand the etiology of metabolic disorders and to identify opportunities to intervene with pharmacological treatment. In this review, we focus on the hormonal regulation of the neuronal circuits of the arcuate nucleus of the hypothalamus: the melanocortin system.

Anorectic estrogen mimics leptin's effect on the rewiring of melanocortin cells and Stat3 signaling in obese animals.

Metabolic hormones, such as leptin, alter the input organization of hypothalamic circuits, resulting in increased pro-opiomelanocortin (POMC) tone, followed by decreased food intake and adiposity. The gonadal steroid estradiol can also reduce appetite and adiposity, and it influences synaptic plasticity. Here we report that estradiol (E2) triggers a robust increase in the number of excitatory inputs to POMC neurons in the arcuate nucleus of wild-type rats and mice. This rearrangement of synapses in the arcuate nucleus is leptin independent because it also occurred in leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) mice, and was paralleled by decreased food intake and body weight gain as well as increased energy expenditure. However, estrogen-induced decrease in body weight was dependent on Stat3 activation in the brain. These observations support the notion that synaptic plasticity of arcuate nucleus feeding circuits is an inherent element in body weight regulation and offer alternative approaches to reducing adiposity under conditions of failed leptin receptor signaling.

A central thermogenic-like mechanism in feeding regulation: an interplay between arcuate nucleus T3 and UCP2.

The active thyroid hormone, triiodothyronine (T3), regulates mitochondrial uncoupling protein activity and related thermogenesis in peripheral tissues. Type 2 deiodinase (DII), an enzyme that catalyzes active thyroid hormone production, and mitochondrial uncoupling protein 2 (UCP2) are also present in the hypothalamic arcuate nucleus, where their interaction and physiological significance have not been explored. Here, we report that DII-producing glial cells are in direct apposition to neurons coexpressing neuropeptide Y (NPY), agouti-related protein (AgRP), and UCP2. Fasting increased DII activity and local thyroid hormone production in the arcuate nucleus in parallel with increased GDP-regulated UCP2-dependent mitochondrial uncoupling. Fasting-induced T3-mediated UCP2 activation resulted in mitochondrial proliferation in NPY/AgRP neurons, an event that was critical for increased excitability of these orexigenic neurons and consequent rebound feeding following food deprivation. These results reveal a physiological role for a thyroid-hormone-regulated mitochondrial uncoupling in hypothalamic neuronal networks.

Ghrelin controls hippocampal spine synapse density and memory performance.

The gut hormone and neuropeptide ghrelin affects energy balance and growth hormone release through hypothalamic action that involves synaptic plasticity in the melanocortin system. Ghrelin binding is also present in other brain areas, including the telencephalon, where its function remains elusive. Here we report that circulating ghrelin enters the hippocampus and binds to neurons of the hippocampal formation, where it promotes dendritic spine synapse formation and generation of long-term potentiation. These ghrelin-induced synaptic changes are paralleled by enhanced spatial learning and memory. Targeted disruption of the gene that encodes ghrelin resulted in decreased numbers of spine synapses in the CA1 region and impaired performance of mice in behavioral memory testing, both of which were rapidly reversed by ghrelin administration. Our observations reveal an endogenous function of ghrelin that links metabolic control with higher brain functions and suggest novel therapeutic strategies to enhance learning and memory processes.

Leptin induces nitric oxide synthase type II in C6 glioma cells. Role for nuclear factor-kappaB in hormone effect.

Astrocytes in the CNS produce inflammatory mediators in response to several stimuli and cytokines. Here we investigated the in vitro effect of leptin on inducible nitric oxide synthase (iNOS) expression in a glioma cell line (C6). After hormone stimulation, culture media were analysed for accumulated stable oxidation products of NO (NO2(-) and NO3(-), designated as NO(x)), cellular RNA was extracted to determine iNOS mRNA level by RT-PCR and cellular lysates were prepared for protein expression. Leptin induced a concentration-dependent increase of NO release, related to iNOS induction. This effect was potentiated by IFN-gamma, or TNF-alpha, or IFN-gamma plus IL-1beta. Pyrrolidine dithiocarbamate (PDTC) and N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), two inhibitors of NF-kappaB activation, as well as the specific proteasome inhibitor MG132, blocked leptin-induced iNOS. The role of NF-kappaB was also confirmed by time course studies on degradation of IkappaB-alpha, which began to degrade 5 min after treatment with leptin and returned to basal level after 30-60 min. Pre-incubation of cells with MG132 inhibited leptin-induced IkappaB-alpha degradation. These results confirm the pro-inflammatory role of leptin and identify it as a potential up-regulator of cytokine-induced inflammatory response in the CNS.

A novel growth hormone secretagogue-1a receptor antagonist that blocks ghrelin-induced growth hormone secretion but induces increased body weight gain.

Ghrelin, the natural ligand for the growth hormone secretagogue-1a (GHS-1a) receptor, has received a great deal of attention due to its ability to stimulate weight gain and the hope that an antagonist of the GHS-1a receptor could be a treatment for obesity. We have discovered an analog of full-length human ghrelin, BIM-28163, which fully antagonizes GHS-1a by binding to but not activating the receptor. We further demonstrate that BIM-28163 blocks ghrelin activation of the GHS-1a receptor, and inhibits ghrelin-induced GH secretion in vivo. Unexpectedly, however, BIM-28163 acts as an agonist with regard to stimulating weight gain. These results may suggest the presence of an unknown ghrelin receptor that modulates ghrelin actions on weight gain. In keeping with our results on growth hormone (GH) secretion, BIM-28163 acts as an antagonist of ghrelin-induced Fos protein immunoreactivity (Fos-IR) in the medial arcuate nucleus, an area involved in the ghrelin modulation of GH secretion. However, in the dorsal medial hypothalamus (DMH), a region associated with regulation of food intake, both ghrelin and BIM-28163 act as agonists to upregulate Fos-IR. The observation that ghrelin and BIM-28163 have different efficacies in inducing Fos-IR in the DMH, and that concomitant administration of ghrelin and an excess of BIM-28163 results in the same level of Fos-IR as BIM-28163 administered alone may demonstrate that in the DMH both ghrelin and BIM-28163 act via the same receptor. If so, it is unlikely that this receptor is GHS-1a. Collectively, our findings suggest that the action of ghrelin to stimulate increased weight gain may be mediated by a novel receptor other than GHS-1a, and further imply that GHS-1a may not be the appropriate target for anti-obesity strategies.

Obesity and the neuroendocrine control of energy homeostasis: the role of spontaneous locomotor activity.

Obesity represents one of the most urgent global health threats as well as one of the leading causes of death throughout industrialized nations. Efficacious and safe therapies remain at large. Attempts to decrease fat mass via pharmacological reduction of energy intake have had limited potency or intolerable side effects. Increasingly widespread sedentary lifestyle is often cited as a major contributor to the increasing prevalence of obesity. Moreover, low levels of spontaneous physical activity (SPA) are a major predictor of fat mass accumulation during overfeeding in humans, pointing to a substantial role for SPA in the control of energy balance. Despite this, very little is known about the molecular mechanisms by which SPA is regulated. The overview will attempt to summarize available information on neuroendocrine factors regulating SPA.

Novel analogs of ghrelin: physiological and clinical implications.

Ghrelin, the 28 amino acid peptide recently identified as the natural ligand for the growth hormone (GH) secretagogue (GHS) receptor, has multiple activities in addition to stimulation of GH secretion, including stimulation of feeding and weight gain. To utilize these actions for potential therapeutic benefit, we have produced analogs of human ghrelin with enhanced metabolic stability, affinity for the GHS receptor, and efficacy in stimulating weight gain. We have also discovered an analog of ghrelin, BIM-28163, that is an antagonist at the GHS receptor and that fully inhibits GHS receptor activation induced by native ghrelin. In vivo, BIM-28163 does not increase GH secretion but fully blocks ghrelin-induced GH secretion. In contrast, BIM-28163 acts as a full agonist with regard to the ghrelin actions of stimulating weight gain and food intake. These results suggest that a receptor other than the GHS receptor mediates the actions of ghrelin on feeding and weight gain. This concept is strengthened by our observation that at certain hypothalamic sites, BIM-28163 acts as an antagonist of ghrelin-induced neuronal activation, while at other sites, both ghrelin and BIM-28163 induce neuronal activation via the same receptor. Collectively, these results indicate the existence of a novel ghrelin receptor that may regulate the feeding activity of ghrelin. Using BIM-28163 as a tool to define the endogenous role of ghrelin in normal GH secretion, we have demonstrated that antagonism of the GHS receptor in normal rats does not impair the pulsatility of GH secretion but lowers the pulse amplitude and mean GH level. These results demonstrate that endogenous ghrelin acts to amplify the basic pattern of GH secretion established by the interplay of hypothalamic GH-releasing hormone and somatostatin. These studies demonstrate the feasibility of creating ghrelin analogs that are selective for specific activities, as well as their utility in dissecting the role of ghrelin in both normal physiology and specific pathologies.