Ghrelin receptor inverse agonists as a novel therapeutic approach against obesity-related metabolic disease.

AIMS: Ghrelin is implicated in the control of energy balance and glucose homeostasis. The ghrelin receptor exhibits ligand-independent constitutive activity, which can be pharmacologically exploited to induce inverse ghrelin actions. Because ghrelin receptor inverse agonists (GHSR-IA) might be effective for the treatment of obesity-related metabolic disease, we tested 2 novel synthetic compounds GHSR-IA1 and GHSR-IA2. MATERIALS AND METHODS: In functional cell assays, electrophysiogical and immunohistochemical experiments, we demonstrated inverse agonist activity for GHSR-IA1 and GHSR-IA2. We used healthy mice, Zucker diabetic fatty (ZDF) rats and diet-induced obese (DIO) mice to explore effects on food intake (FI), body weight (BW), conditioned taste aversion (CTA), oral glucose tolerance (OGT), pancreatic islet morphology, hepatic steatosis (HS), and blood lipids. RESULTS: Both compounds acutely reduced FI in mice without inducing CTA. Chronic GHSR-IA1 increased metabolic rate in chow-fed mice, suppressed FI, and improved OGT in ZDF rats. Moreover, the progression of islet hyperplasia to fibrosis in ZDF rats slowed down. GHSR-IA2 reduced FI and BW in DIO mice, and reduced fasting and stimulated glucose levels compared with pair-fed and vehicle-treated mice. GHSR-IA2-treated DIO mice showed decreased blood lipids. GHSR-IA1 treatment markedly decreased HS in DIO mice. CONCLUSIONS: Our study demonstrates therapeutic actions of novel ghrelin receptor inverse agonists, suggesting a potential to treat obesity-related metabolic disorders including diabetes mellitus.

Oral Treatment with the Ghrelin Receptor Agonist HM01 Attenuates Cachexia in Mice Bearing Colon-26 (C26) Tumors.

The gastrointestinal hormone ghrelin reduces energy expenditure and stimulates food intake. Ghrelin analogs are a possible treatment against cancer anorexia-cachexia syndrome (CACS). This study aimed to investigate whether oral treatment with the non-peptidergic ghrelin receptor agonist HM01 counteracts CACS in colon-26 (C26) tumor-bearing mice. The C26 tumor model is characterized by pronounced body weight (BW) loss and muscle wasting in the absence of severe anorexia. We analyzed the time course of BW loss, body composition, muscle mass, bone mineral density, and the cytokines interleukin-6 (IL-6) and macrophage-inhibitory cytokine-1 (MIC-1). Moreover, we measured the expression of the muscle degradation markers muscle RING-finger-protein-1 (MuRF-1) and muscle atrophy F-box (MAFbx). After tumor inoculation, MIC-1 levels increased earlier than IL-6 and both cytokines were elevated before MuRF-1/MAFbx expression increased. Oral HM01 treatment increased BW, fat mass, and neuronal hypothalamic activity in healthy mice. In tumor-bearing mice, HM01 increased food intake, BW, fat mass, muscle mass, and bone mineral density while it decreased energy expenditure. These effects appeared to be independent of IL-6, MIC-1, MuRF-1 or MAFbx, which were not affected by HM01. Therefore, HM01 counteracts cachectic body weight loss under inflammatory conditions and is a promising compound for the treatment of cancer cachexia in the absence of severe anorexia.

The ghrelin receptor agonist HM01 mimics the neuronal effects of ghrelin in the arcuate nucleus and attenuates anorexia-cachexia syndrome in tumor-bearing rats.

The gastric hormone ghrelin positively affects energy balance by increasing food intake and reducing energy expenditure. Ghrelin mimetics are a possible treatment against cancer anorexia-cachexia syndrome (CACS). This study aimed to characterize the action of the nonpeptidergic ghrelin receptor agonist HM01 on neuronal function, energy homeostasis and muscle mass in healthy rats and to evaluate its possible usefulness for the treatment of CACS in a rat tumor model. Using extracellular single-unit recordings, we tested whether HM01 mimics the effects of ghrelin on neuronal activity in the arcuate nucleus (Arc). Furthermore, we assessed the effect of chronic HM01 treatment on food intake (FI), body weight (BW), lean and fat volumes, and muscle mass in healthy rats. Using a hepatoma model, we investigated the possible beneficial effects of HM01 on tumor-induced anorexia, BW loss, muscle wasting, and metabolic rate. HM01 (10(-7)-10(-6) M) mimicked the effect of ghrelin (10(-8) M) by increasing the firing rate in 76% of Arc neurons. HM01 delivered chronically for 12 days via osmotic minipumps (50 µg/h) increased FI in healthy rats by 24%, paralleled by increased BW, higher fat and lean volumes, and higher muscle mass. Tumor-bearing rats treated with HM01 had 30% higher FI than tumor-bearing controls and were protected against BW loss. HM01 treatment resulted in higher muscle mass and fat mass. Moreover, tumor-bearing rats reduced their metabolic rate following HM01 treatment. Our studies substantiate the possible therapeutic usefulness of ghrelin receptor agonists like HM01 for the treatment of CACS and possibly other forms of disease-related anorexia and cachexia.

Involvement of the extracellular signal-regulated kinase 1/2 signaling pathway in amylin's eating inhibitory effect.

Peripheral amylin inhibits eating via the area postrema (AP). Because amylin activates the extracellular-signal regulated kinase 1/2 (ERK) pathway in some tissues, and because ERK1/2 phosphorylation (pERK) leads to acute neuronal responses, we postulated that it may be involved in amylin's eating inhibitory effect. Amylin-induced ERK phosphorylation (pERK) was investigated by immunohistochemistry in brain sections containing the AP. pERK-positive AP neurons were double-stained for the calcitonin 1a/b receptor, which is part of the functional amylin-receptor. AP sections were also phenotyped using dopamine-ß-hydroxylase (DBH) as a marker of noradrenergic neurons. The effect of fourth ventricular administration of the ERK cascade blocker U0126 on amylin's eating inhibitory action was tested in feeding trials. The number of pERK-positive neurons in the AP was highest ∼10-15 min after amylin treatment; the effect appeared to be dose-dependent (5-20 µg/kg amylin). A portion of pERK-positive neurons in the AP carried the amylin-receptor and 22% of the pERK-positive neurons were noradrenergic. Pretreatment of rats with U0126 decreased the number of pERK-positive neurons in the AP after amylin injection. U0126 also attenuated the ability of amylin to reduce eating, at least when the animals had been fasted 24 h prior to the feeding trial. Overall, our results suggest that amylin directly stimulates pERK in AP neurons in a time- and dose-dependent manner. Part of the AP neurons displaying pERK were noradrenergic. At least under fasting conditions, pERK was shown to be a necessary part in the signaling cascade mediating amylin's anorectic effect.

Lipopolysaccharide inhibits ghrelin-excited neurons of the arcuate nucleus and reduces food intake via central nitric oxide signaling.

Lipopolysaccharide (LPS) induces anorexia and expression of inducible nitric oxide synthase (iNOS) in the hypothalamic arcuate nucleus (Arc). Peripheral administration of the iNOS inhibitor 1400 W counteracts the anorectic effects of LPS. Here we investigated the role of central NO signaling in LPS anorexia. In electrophysiological studies we tested whether 1400 W counteracts the iNOS-dependent inhibition of Arc neurons triggered by in vivo or in vitro stimulation with LPS. We used the hormone ghrelin as a functional reference stimulus because ghrelin is known to activate orexigenic Arc neurons. Further, we investigated whether in vitro LPS stimulation induces an iNOS-mediated formation of the second messenger cGMP. Since the STAT1 pathway contributes to the regulation of iNOS expression we investigated whether LPS treatment induces STAT1 phosphorylation in the Arc. Finally we tested the effect of intracerebroventricular injection of 1400 W on LPS-induced anorexia. Superfusion with 1400 W (10(-4) M) increased neuronal activity in 37% of neurons in Arc slices from LPS treated (100 µg/kg ip) but not from saline treated rats. Similarly, 1400 W excited 45% of Arc neurons after in vitro stimulation with LPS (100 ng/ml). In both approaches, a considerable percentage of 1400 W sensitive neurons were excited by ghrelin (10(-8)M; 50% and 75%, respectively). In vitro stimulation with LPS induced cGMP formation in the Arc, which was blocked by co-incubation with 1400 W. LPS treatment elicited a pSTAT1 response in the Arc of mice. Central 1400 W injection (4 µg/rat) attenuated LPS-induced anorexia and counteracted the LPS-dependent decrease in respiratory quotient and energy expenditure. In conclusion, the current findings substantiate a role of central iNOS dependent NO formation in LPS-induced effects on eating and energy homeostasis. A pharmacological blockade of NO formation might be a therapeutic approach to ameliorate disease-related anorexia.

Reduced fasting-induced activation of hypothalamic arcuate neurons is associated with hyperleptinemia and increased leptin sensitivity in obese mice.

Fasting increases c-Fos expression in neuropeptide Y (NPY) neurons of the hypothalamic arcuate nucleus (ARC) in lean, but not in hyperleptinemic mice with late-onset obesity (LOO). Although obesity is associated with leptin resistance, we hypothesized that under fasting conditions, leptin sensitivity might be restored and that hyperleptinemia may counteract the neuronal response to fasting. We investigated whether the reduced fasting response of ARC neurons in LOO is paralleled by an increase in leptin sensitivity, as measured by leptin-induced STAT-3 phosphorylation. To assess leptin's role in the modulation of the fasting-induced ARC activation, we investigated c-Fos responses and hormone and metabolite levels in hyperleptinemic diet-induced obese (DIO) and in leptin-deficient ob/ob mice. Leptin induced a stronger STAT-3 phosphorylation in fasted LOO and lean mice than in ad libitum-fed animals. Similar to LOO, hyperleptinemic DIO mice showed no c-Fos response after fasting, while ob/ob mice showed a stronger response than lean control mice. Mimicking hyperleptinemia by repeated leptin injections in lean mice during fasting attenuated the fasting-induced c-Fos expression. Our findings indicate that high leptin levels prevent the fasting-induced activation of ARC neurons in mice. Moreover, leptin sensitivity is dynamic in obese subjects and depends on the feeding status. During short-term increases in leptin sensitivity, e.g., during fasting, leptin signaling appears to be effective, even in hyperleptinemic obesity. As reflected by the blockade of the fasting-induced ARC activation, fasting seems to interfere with the responsiveness of the ARC to signals related to the status of energy intake.

Effects of glucagon-like peptide 1 and oxyntomodulin on neuronal activity of ghrelin-sensitive neurons in the hypothalamic arcuate nucleus.

Glucagon-like peptide 1 (GLP-1) and oxyntomodulin (OXM) are structurally related gastrointestinal hormones that are secreted in response to food intake. They reduce food intake and body weight and exert partly overlapping actions on glucose homeostasis and gastrointestinal function. The hypothalamic arcuate (ARC) nucleus is among the central structures expressing a high density of GLP-1 receptors (GLP-1R), which are known to be activated by both peptides. It was the aim of our electrophysiological studies to characterize the effects of GLP-1 and OXM on functionally defined ghrelin-sensitive ARC neurons. GLP-1 and OXM (10(-7) M) exerted excitatory effects in about two-thirds of ghrelin-inhibited neurons and in approximately one-third of ghrelin-excited cells. In addition, a minor fraction of the ghrelin-excited cells was inhibited by both peptides. There was a high degree of cosensitivity to GLP-1 and OXM, and the effects of both hormones were blocked by the GLP-1R antagonist exendin(9-39). The GLP-1R-mediated excitations and inhibitions persisted under synaptic blockade, indicating a direct postsynaptic mode of action. Our results demonstrate that GLP-1 and OXM directly and similarly alter neuronal activity in the ARC, probably via a common GLP-1R-mediated mechanism. Ghrelin-antagonistic effects on neuronal activity, which might be implicated in ghrelin-antagonistic in vivo actions, resulting from GLP-1R stimulation (e.g., GLP-1R dependent supression of food intake), predominated in ghrelin-inhibited ARC neurons. However, a subset of ghrelin-excited ARC neurons showed responses to OXM or GLP-1, suggesting the existence of a common mode of action for these hormones; the functional relevance of this effect remains to be elucidated.

Noradrenergic neurons of the area postrema mediate amylin's hypophagic action.

Circulating amylin inhibits food intake via activation of the area postrema (AP). The aim of this study was to identify the neurochemical phenotype of the neurons mediating amylin's hypophagic action by immunohistochemical and feeding studies in rats. Expression of c-Fos protein was used as a marker for neuronal activation and dopamine-beta-hydroxylase (DBH), the enzyme-catalyzing noradrenaline synthesis, as a marker for noradrenergic neurons. We found that approximately 50% of amylin-activated AP neurons are noradrenergic. To clarify the functional role of these neurons in amylin's effect on eating, noradrenaline-containing neurons in the AP were lesioned using a saporin conjugated to an antibody against DBH. Amylin (5 or 20 microg/kg s.c.)-induced anorexia was observed in sham-lesioned rats with both amylin doses. Rats with a lesion of > 50% of the noradrenaline neurons were unresponsive to the low dose of amylin (5 microg/kg) and only displayed a reduction in food intake 60 min after injection of the high amylin dose (20 microg/kg). In a terminal experiment, the same rats received amylin (20 microg/kg) or saline. The AP and nucleus of the solitary tract (NTS) were stained for DBH to assess noradrenaline lesion success and for c-Fos expression to evaluate amylin-induced neuronal activation. In contrast to sham-lesioned animals, noradrenaline-lesioned rats did not show a significant increase in amylin-induced c-Fos expression in the AP and NTS. We conclude that the noradrenergic neurons in the AP mediate at least part of amylin's hypophagic effect.

Brainstem prolactin-releasing peptide contributes to cancer anorexia-cachexia syndrome in rats.

Up to 80% of cancer patients are affected by the cancer anorexia-cachexia syndrome (CACS), which leads to excessive body weight loss, reduced treatment success and increased lethality. The area postrema/nucleus of the solitary tract (AP/NTS) region emerged as a central nervous key structure in this multi-factorial process. Neurons in this area are targeted by cytokines and signal to downstream sites involved in energy homeostasis. NTS neurons expressing prolactin-releasing peptide (PrRP) are implicated in the control of energy intake and hypothalamus-pituitary-adrenal (HPA) axis activation, which contributes to muscle wasting. To explore if brainstem PrRP neurons contribute to CACS, we selectively knocked down PrRP expression in the NTS of hepatoma tumor-bearing rats by an AAV/shRNA gene silencing approach. PrRP knockdown reduced body weight loss and anorexia compared to tumor-bearing controls treated with a non-silencing AAV. Gastrocnemius and total hind limb muscle weight was higher in PrPR knockdown rats. Corticosterone levels were increased in the early phase after tumor induction at day 6 in both groups but returned to baseline levels at day 21 in the PrRP knockdown group. While we did not detect significant changes in gene expression of markers for muscle protein metabolism (MuRF-1, myostatin, mTOR and REDD1), mTOR and REDD1 tended to be lower after disruption PrRP signalling. In conclusion, we identified brainstem PrRP as a possible neuropeptide mediator of CACS in hepatoma tumor-bearing rats. The central and peripheral downstream mechanisms require further investigation and might involve HPA axis activation.

Diet-derived nutrients mediate the inhibition of hypothalamic NPY neurons in the arcuate nucleus of mice during refeeding.

Fasting activates orexigenic neuropeptide Y neurons in the hypothalamic arcuate nucleus (ARC) of mice, which is reversed by 2 h refeeding with standard chow. Here, we investigated the contribution of diet-derived macronutrients and anorectic hormones to the reversal of the fasting-induced ARC activation during 2 h refeeding. Refeeding of 12-h-fasted mice with a cellulose-based, noncaloric mash induced only a small reduction in c-Fos expression. Refeeding with diets, containing carbohydrates, protein, or fat alone reversed it similar to chow; however, this effect depended on the amount of intake. The fasting-induced ARC activation was unchanged by subcutaneously injected amylin, CCK (both 20 microg/kg), insulin (0.2 U/kg and 0.05 U/kg) or leptin (2.6 mg/kg). Insulin and leptin had no effect on c-Fos expression in neuropeptide Y or proopiomelanocortin-containing ARC neurons. Interestingly, CCK but not amylin reduced the ghrelin-induced c-Fos expression in the ARC in ad libitum-fed mice, suggesting that CCK may inhibit orexigenic ARC neurons when acting together with other feeding-related signals. We conclude that all three macronutrients and also non-nutritive, ingestion-dependent signals contribute to an inhibition of orexigenic ARC neurons after refeeding. Similar to the previously demonstrated inhibitory in vivo action of peptide YY, CCK may be a postprandial mediator of ARC inhibition.

Blunted fasting-induced hypothalamic activation and refeeding hyperphagia in late-onset obesity.

Hormonal and metabolic factors signal the status of energy balance to hypothalamic nuclei. Obesity is characterized by neuronal, metabolic and hormonal alterations. We therefore hypothesized that hypothalamic responses to challenges of energy balance may differ between lean and obese animals. To test this, we compared c-Fos expression in the hypothalamic arcuate (ARC) and paraventricular nuclei (PVN) and the lateral hypothalamic area (LHA) of mice (1-year-old) with late-onset obesity (LOO) and of lean controls under different feeding conditions. Fourteen hours of fasting induced high c-Fos expression in neuropeptide-Y-positive ARC neurons, in the PVN and in the rostral LHA in lean but not in LOO mice. c-Fos expression in melanin-concentrating hormone (MCH) and orexin-containing neurons in the caudal LHA was not affected by fasting. LOO mice showed fasting hyperinsulinemia, hyperleptinemia, elevated fasting blood glucose and an attenuated hyperphagic response during refeeding. Moreover, the anorectic response to leptin and hypoglycemic response to insulin were reduced in LOO mice. We conclude that adiposity blunts the neuronal responses to metabolic challenges in hypothalamic centers which control feeding behavior and energy balance. Elevated blood glucose may be one factor that suppresses hypothalamic responsiveness in obese mice. A similar impact of hyperinsulinemia and hyperleptinemia in LOO mice is also likely although under the current experimental conditions responsiveness to some effects of these hormones appeared to be reduced.

Inhibitory effects of lipopolysaccharide on hypothalamic nuclei implicated in the control of food intake.

The arcuate nucleus (Arc) and the lateral hypothalamic area (LHA), two key hypothalamic nuclei regulating feeding behavior, express c-Fos, a marker of neuronal activation in fasted animals. This is reversed by refeeding. In the present study we tested whether an anorectic dose of lipopolysaccharide (LPS), the cell wall component of Gram-negative bacteria, also inhibits fasting-induced c-Fos expression in these hypothalamic nuclei. This would suggest that they are involved in anorexia during bacterial infections as well. We also studied whether LPS modulates the activity of orexin-A positive (OX+) LHA neurons. Food deprived BALB/c mice were injected with LPS or saline and were sacrificed 4 or 6h later. Four hours after injection, LPS reduced the number of c-Fos positive cells in the Arc and in the LHA, but had no effect on c-Fos in OX+ neurons. Six hours after injection, LPS reduced c-Fos expression in the LHA, both in the OX- and OX+ neurons, but not in the Arc. These results show that LPS modulates neuronal activity in the Arc and LHA similar to feeding-related stimuli, suggesting that the observed effects might contribute to the anorectic effect of LPS. Thus, physiological satiety signals released during refeeding and anorexia during bacterial infection seem to engage similar neuronal substrates.

Glucose reverses fasting-induced activation in the arcuate nucleus of mice.

The hypothalamic arcuate nucleus is an important target for metabolic and hormonal signals controlling food intake. As demonstrated by c-Fos studies, arcuate neurons are activated in food-deprived mice, whereas refeeding reverses the fasting-induced activation. To evaluate whether an increase in blood glucose has an inhibitory effect on these neurons, we analyzed the c-Fos response to an intraperitoneal glucose injection in fasted mice. This treatment increased blood glucose levels twofold and reduced 2-h food intake. Similar to refeeding, it also reversed the fasting-induced activation in the arcuate nucleus. Therefore, an increase in blood glucose might be an important feeding-related signal acting via the arcuate nucleus to oppose orexigenic stimuli.

Brainstem GLP-1 signalling contributes to cancer anorexia-cachexia syndrome in the rat.

The cancer anorexia-cachexia syndrome (CACS) is a frequent and severe condition in cancer patients. Currently, no pharmacological treatment is approved for the therapy of CACS. Centrally, glucagon-like peptide-1 (GLP-1) is expressed in the nucleus tractus solitarii (NTS) and is implicated in malaise, nausea and food aversion. The NTS is reciprocally connected to brain sites implicated in the control of energy balance including the area postrema (AP), which mediates CACS in certain tumour models. Given the role of GLP-1 as a mediator of anorexia under acute sickness conditions, we hypothesized that brainstem GLP-1 signalling might play a role in the mediation of CACS. Using hepatoma tumour-bearing (TB) rats, we first tested whether the chronic delivery of the GLP-1R antagonist exendin-9 (Ex-9) into the fourth ventricle attenuates CACS. Second, we investigated whether a genetic knockdown of GLP-1 expression in the NTS ameliorates CACS. Ex-9 attenuated anorexia, body weight loss, muscle and fat depletion compared to TB controls. Similarly, TB animals with a knockdown of GLP-1 expression in the NTS had higher food intake, reduced body weight loss, and higher lean and fat mass compared to TB controls. Our study identifies brainstem GLP-1 as crucial mediator of CACS in hepatoma TB rats. The GLP-1R represents a promising target against CACS and possibly other forms of disease-related anorexia/cachexia.

In-vitro and in-vivo antagonistic action of an anti-amylin Spiegelmer.

The anorectic and dipsogenic effects of the pancreatic hormone amylin are mediated by the area postrema and the subfornical organ. We tested the effectiveness of a new amylin antagonist, a so-called RNA Spiegelmer, by electrophysiological in-vitro recordings from the rat subfornical organ and by immunohistological c-Fos studies in the area postrema. Amylin's excitatory effect on subfornical organ neurons was blocked by the anti-amylin Spiegelmer. Peripheral administration 5 h prior to amylin also suppressed the amylin-induced activation (c-Fos expression) in the area postrema. The biostable anti-amylin Spiegelmer may be therapeutically beneficial in conditions associated with high plasma amylin levels, such as cancer anorexia occurring during certain pancreatic tumors.

Diet-derived nutrients modulate the effects of amylin on c-Fos expression in the area postrema and on food intake.

The pancreatic hormone amylin decreases food intake via activation of area postrema (AP) neurons. We investigated whether amylin's potency to reduce food intake and to induce c-Fos expression in the AP/nucleus of the solitary tract region is affected by the feeding conditions and specifically by the macronutrient composition of the diet. Whereas a low dose of amylin (5 microg/kg s.c.) induced very little c-Fos expression in ad libitum chow fed rats, it caused a strong c-Fos expression in 24-hour food-deprived rats and in rats that received a nutrient-deficient non-caloric mash (NCM; vanilla-flavoured cellulose) 24 h before injection. To reveal the contribution of single nutrients to the low c-Fos expression after chow feeding, amylin-induced c-Fos was analyzed after feeding NCM that was selectively supplemented with glucose, fat (lard), or protein (casein), matching the intake of these nutrients of chow-fed rats. While the rats fed NCM supplemented with glucose or fat displayed an equally strong amylin-induced activation as fasted rats or rats fed plain NCM, a significantly lower c-Fos expression was observed in rats fed a protein-supplemented NCM or a NCM containing all three nutrients. In line with this lower activation, the same dose of amylin failed to reduce food intake in NCM/protein-fed rats, while amylin caused a reduction in feeding when animals received NCM, NCM/glucose, or NCM/fat. Interestingly, amylin effectively reduced food intake in ad libitum chow fed rats despite the low level of amylin-induced c-Fos expression in the AP under these conditions. We conclude that the anorectic potential of amylin may be attenuated by diet-derived proteins, whereas this effect appears to be overridden when the amount of carbohydrates/fat is high relative to the protein content, such as, e.g., in standard chow.

Lesion of the lateral parabrachial nucleus attenuates the anorectic effect of peripheral amylin and CCK.

Amylin and CCK activate the area postrema (AP)/nucleus of the solitary tract (NTS) - lateral parabrachial nucleus (LPBN) - central amygdala (CeA) pathway. However, except for the brainstem structures the role of these nuclei for the anorectic effect of these peptides is not yet well characterized. The current study investigated the role of the LPBN in mediating the inhibitory effect of peripheral amylin and CCK on feeding behavior. Rats with electrolytic lesions in the LPBN (LPBN-X) were used in behavioral as well as in immunohistological c-Fos studies. LPBN-X significantly reduced the anorectic effect of amylin (5 microg/kg, i.p.). The effect of a higher amylin dose (10 microg/kg, i.p.) was only slightly attenuated in the LPBN-X rats. In agreement with previous studies, LPBN lesions also reduced the inhibitory effect of CCK on food intake. In the immunohistological experiments, amylin and CCK induced c-Fos expression in the AP, NTS, LPBN and CeA in the SHAM rats. Both the amylin- and CCK-induced activation of the CeA was completely abolished in the animals with a LPBN lesion. These results clearly suggest that the signal transduction pathway between the AP/NTS and CeA has been disrupted by the LPBN ablation. We conclude that the LPBN is a crucial brain site mediating the anorectic effect of amylin and CCK. Furthermore, an intact LPBN seems to be essential for the c-Fos response in the CeA induced by these peptides.

Involvement of Amylin and Leptin in the Development of Projections from the Area Postrema to the Nucleus of the Solitary Tract.

The area postrema (AP) and the nucleus of the solitary tract (NTS) are important hindbrain centers involved in the control of energy homeostasis. The AP mediates the anorectic action and the inhibitory effect on gastric emptying induced by the pancreatic hormone amylin. Amylin's target cells in the AP project to the NTS, an integrative relay center for enteroceptive signals. Perinatal hormonal and metabolic factors influence brain development. A postnatal surge of the adipocyte-derived hormone leptin represents a developmental signal for the maturation of projections between hypothalamic nuclei controlling energy balance. Amylin appears to promote neurogenesis in the AP in adult rats. Here, we examined whether amylin and leptin are required for the development of projections from the AP to the NTS in postnatal and adult mice by conducting neuronal tracing studies with DiI in amylin- (IAPP-/-) and leptin-deficient (ob/ob) mice. Compared to wild-type littermates, postnatal (P10) and adult (P60) IAPP-/- mice showed a significantly reduced density of AP-NTS projections. While AP projections were also reduced in postnatal (P14) ob/ob mice, AP-NTS fiber density did not differ between adult ob/ob and wild-type animals. Our findings suggest a crucial function of amylin for the maturation of neuronal brainstem pathways controlling energy balance and gastrointestinal function. The impaired postnatal development of neuronal AP-NTS projections in ob/ob mice appears to be compensated in this experimental model during later brain maturation. It remains to be elucidated whether an amylin- and leptin-dependent modulation in neuronal development translates into altered AP/NTS-mediated functions.

Anorexia-cachexia syndrome in hepatoma tumour-bearing rats requires the area postrema but not vagal afferents and is paralleled by increased MIC-1/GDF15.

BACKGROUND: The cancer-anorexia-cachexia syndrome (CACS) negatively affects survival and therapy success in cancer patients. Inflammatory mediators and tumour-derived factors are thought to play an important role in the aetiology of CACS. However, the central and peripheral mechanisms contributing to CACS are insufficiently understood. The area postrema (AP) and the nucleus tractus solitarii are two important brainstem centres for the control of eating during acute sickness conditions. Recently, the tumour-derived macrophage inhibitory cytokine-1 (MIC-1) emerged as a possible mediator of cancer anorexia because lesions of these brainstem areas attenuated the anorectic effect of exogenous MIC-1 in mice. METHODS: Using a rat hepatoma tumour model, we examined the roles of the AP and of vagal afferents in the mediation of CACS. Specifically, we investigated whether a lesion of the AP (APX) or subdiaphragmatic vagal deafferentation (SDA) attenuate anorexia, body weight, muscle, and fat loss. Moreover, we analysed MIC-1 levels in this tumour model and their correlation with tumour size and the severity of the anorectic response. RESULTS: In tumour-bearing sham-operated animals mean daily food intake significantly decreased. The anorectic response was paralleled by a significant loss of body weight and muscle mass. APX rats were protected against anorexia, body weight loss, and muscle atrophy after tumour induction. In contrast, subdiaphragmatic vagal deafferentation did not attenuate cancer-induced anorexia or body weight loss. Tumour-bearing rats had substantially increased MIC-1 levels, which positively correlated with tumour size and cancer progression and negatively correlated with food intake. CONCLUSIONS: These findings demonstrate the importance of the AP in the mediation of cancer-dependent anorexia and body weight loss and support a pathological role of MIC-1 as a tumour-derived factor mediating CACS, possibly via an AP-dependent action.

Nitric oxide directly inhibits ghrelin-activated neurons of the arcuate nucleus.

The hypothalamic arcuate nucleus (Arc) is a target site for signals regulating energy homeostasis. The orexigenic hormone ghrelin directly activates neurons of the medial arcuate nucleus (ArcM) in rats. Nitric oxide (NO) is a neuromodulator implicated in the control of food intake and body weight. NO is produced by nitric oxide synthase (NOS) and induces the formation of cyclic guanosine monophosphate (cGMP) via a stimulation of soluble guanylate cyclase (sGC). Both enzymes NOS and sGC have been identified in the Arc. Using extracellular recordings we characterized the effects of NO signaling on ArcM neurons and their co-sensitivity to ghrelin. The artificial NO donor sodium nitroprusside (10(-4) M) reversibly inhibited 91% of all ArcM neurons by a direct postsynaptic mechanism. 52% of ArcM neurons were excited by ghrelin. In all but one of these neurons SNP caused inhibitory responses. The SNP-induced inhibitions were mediated by cGMP since they were blocked by the specific sGC inhibitor ODQ (1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one, 10(-4) M). Furthermore, the membrane permeating cGMP analogue 8-Br-cGMP (10(-4) M) mimicked the inhibitory responses of SNP. In immunohistological in vitro studies SNP induced a cGMP formation, which could also be blocked by ODQ. The current studies demonstrate that NO/cGMP signaling inhibits a large population of ArcM neurons including ghrelin-excited cells. Since an activation of the latter neurons is regarded as a correlate of negative energy balance, NO may represent an anorectic neuromodulator in the Arc and/or restrain the action of signals promoting energy intake. NO signaling in the Arc is also induced following inflammation suggesting a possible role of Arc-intrinsic NO in disease-related anorexia.