Peripheral activation of corticotropin-releasing factor receptor 2 inhibits food intake and alters meal structures in mice.

The orexigenic effect of urocortins (Ucns), namely Ucn 1, Ucn 2 and Ucn 3 through activation of corticotropin-releasing factor (CRF) receptors, has been well characterized after injection into the brain but not in the periphery. We examined the role of CRF receptor subtype 2 (CRF(2)) in the regulation of food intake using intraperitoneal (ip) injection of Ucns and the selective CRF(2) antagonist, astressin(2)-B, and CRF(2) knockout (-/-) mice. Meal structures were monitored using an automated episodic solid food intake monitoring system. Ucn 2 (3, 10 or 30 µg/kg, ip) induced a rapid in onset, long lasting and dose-dependent decrease (38%, 66% and 86%, respectively at 4h) of cumulative food intake after an overnight fast in mice. Ucn 3 anorexic effect was 10-times less potent. Astressin(2)-B (30 or 100 µg/kg) injected ip, but not intracerebroventricularly, blocked the inhibitory effect of ip Ucn 1 and Ucn 2 (10 µg/kg). Fasted CRF(2-/-) mice did not respond to ip Ucn 1 (10 µg/kg). Meal microstructure analysis of the 4-h re-feeding response to an overnight fast showed that Ucn 2 (10 µg/kg, ip) decreased meal size and duration, but increased meal frequency. In mice fed ad libitum, Ucn 2 (30 µg/kg) injected ip before the dark phase decreased the 4-h nocturnal meal size and duration without influencing meal frequency while the 10 µg/kg dose had no effect. These data indicate that Ucns, through peripheral CRF(2) receptor-mediated induction of satiation, inhibit the eating response to a fast more potently than the physiological nocturnal feeding in mice.

Lipopolysaccharide increases plasma levels of corticotropin-releasing hormone in rats.

BACKGROUND: Corticotropin-releasing hormone (CRH) is expressed in the brain, immune cells and the gut, where gene expression is upregulated by lipopolysaccharide (LPS) 6 h after injection. Whether these changes are reflected by increased circulating levels of CRH and adrenocorticotropic hormone (ACTH) is unknown. METHODS: LPS (100 µg/kg) was injected intraperitoneally in conscious rats, and blood processed for CRH using the new RAPID (reduced temperatures, acidification, protease inhibition, isotopic exogenous controls and dilution) method compared with EDTA blood with or without plasma methanol extraction. Hormone levels were measured by commercial radioimmunoassay. RESULTS: The RAPID method improved blood recovery of ¹²5I-CRH in vitro compared to EDTA only added to the blood without or with methanol extraction (90.8 ± 2.0 vs. 66.9 ± 2.6 and 47.5 ± 2.0%, respectively; p < 0.001 vs. RAPID). Basal CRH levels from blood processed by the RAPID method were 28.9 ± 2.8 pg/ml, and by other methods below the radioimmunoassay detection limit (<10 pg/ml). At 6 h after LPS, CRH plasma levels increased significantly by 2.9 times, and in the proximal colon tended to decrease (-27.6 ± 5.7%; p > 0.05), while circulating levels were unchanged at 3 or 4 h. ACTH levels rose compared to control rats (135.3 ± 13.8 vs. 101.4 ± 6.0 pg/ml; p < 0.05) 30 min after the increase in CRH, while at 3 or 6 h after LPS, the levels were not changed. CONCLUSION: Intraperitoneal LPS induces a delayed rise in plasma CRH levels associated with an elevation in ACTH plasma levels 30 min later, suggesting that under conditions of immune challenge, CRH of peripheral origin may also contribute to pituitary activation, as detected using the RAPID method of blood processing, which improves CRH recovery.

Central nesfatin-1 reduces the nocturnal food intake in mice by reducing meal size and increasing inter-meal intervals.

Nesfatin-1 is well established to reduce food intake upon brain injection in rats, while in mice its anorexigenic action and brain expression are largely unexplored. We characterized the influence of intracerebroventricular (icv) and peripheral (intraperitoneal, ip, subcutaneous, sc) injection of nesfatin-1 on dark phase ingestive behavior using an automated feeding monitoring system and co-localized NUCB2/nesfatin-1 immunoreactivity in the associated brain areas. Nesfatin-1 (0.3, 1 or 3 µg/mouse, icv) caused a dose-related reduction of 4-h dark phase food intake by 13%, 27%, and 46% respectively. Nesfatin-1 (3 µg/mouse, icv) action had a 2-h delayed onset, 82% peak inhibition occurring at 3-4h post-injection and was long lasting (30% reduction for 12h period post-injection). Nesfatin-1 (3 µg/mouse, icv)-treated mice had a 46% lower meal frequency associated with 2-times longer inter-meal intervals and a 35% reduction in meal size compared to vehicle during the 1-4h post-injection (p<0.05). NUCB2/nesfatin-1-immunopositive neurons were found in hypothalamic (supraoptic, paraventricular, arcuate, dorsomedial, lateral) and brainstem (dorsal vagal complex) feeding regulatory nuclei. When injected peripherally, neither food intake nor feeding microstructure parameters were altered. These results demonstrate that NUCB2/nesfatin-1 is prominently expressed in mouse hypothalamus and medulla and acts in the brain to curtail the dark phase feeding by inducing satiation and satiety indicated by reduced meal size and prolonged inter-meal intervals respectively. The lack of nesfatin-1 effect when injected peripherally at a 23-times higher dose indicates a primarily central site of the anorexigenic action for nesfatin-1 in mice.

Sulfated cholecystokinin-8 activates phospho-mTOR immunoreactive neurons of the paraventricular nucleus in rats.

The serin/threonin-kinase, mammalian target of rapamycin (mTOR) was detected in the arcuate nucleus (ARC) and paraventricular nucleus of the hypothalamus (PVN) and suggested to play a role in the integration of satiety signals. Since cholecystokinin (CCK) plays a role in the short-term inhibition of food intake and induces c-Fos in PVN neurons, the aim was to determine whether intraperitoneally injected CCK-8S affects the neuronal activity in cells immunoreactive for phospho-mTOR in the PVN. Ad libitum fed male Sprague-Dawley rats received 6 or 10 µg/kg CCK-8S or 0.15M NaCl ip (n=4/group). The number of c-Fos-immunoreactive (ir) neurons was assessed in the PVN, ARC and in the nucleus of the solitary tract (NTS). CCK-8S increased the number of c-Fos-ir neurons in the PVN (6 µg: 103 ± 13 vs. 10 µg: 165 ± 14 neurons/section; p<0.05) compared to vehicle treated rats (4 ± 1, p<0.05), but not in the ARC. CCK-8S also dose-dependently increased the number of c-Fos neurons in the NTS. Staining for phospho-mTOR and c-Fos in the PVN showed a dose-dependent increase of activated phospho-mTOR neurons (17 ± 3 vs. 38 ± 2 neurons/section; p<0.05), while no activated phospho-mTOR neurons were observed in the vehicle group. Triple staining in the PVN showed activation of phospho-mTOR neurons co-localized with oxytocin, corresponding to 9.8 ± 3.6% and 19.5 ± 3.3% of oxytocin neurons respectively. Our observations indicate that peripheral CCK-8S activates phospho-mTOR neurons in the PVN and suggest that phospho-mTOR plays a role in the mediation of CCK-8S's anorexigenic effects.

Cold ambient temperature reverses abdominal surgery-induced delayed gastric emptying and decreased plasma ghrelin levels in rats.

We investigated whether acute cold-induced vagal activation through brainstem thyrotropin-releasing hormone (TRH) signaling influences abdominal surgery-induced delayed gastric emptying (GE) in fasted rats. Laparotomy and cecal palpation or sham (short anesthesia alone) was performed 10 min before or 30 min after cold exposure (4-6°C) lasting 90 min. Non-nutrient GE was assessed during 70-90 min of cold exposure. Control groups remained at room temperature (RT). The stable TRH analog, RX-77368 (50 ng/rat) was injected intracisternally immediately before surgery and GE monitored 30-50 min postsurgery in rats maintained at RT. Plasma acyl (AG) and total ghrelin levels were assessed using the new RAPID blood processing method and radioimmunoassays. Desacyl ghrelin (DAG) was derived from total minus AG. In rats maintained at RT, abdominal surgery decreased GE by 60% compared to sham. Cold before or after surgery or RX-77368 normalized the delayed GE. In non-fasted rats, cold exposure increased plasma AG and DAG levels at 2 h (2.4- and 2.7-times, respectively) and 4 h (2.2- and 2.0-times, respectively) compared to values in rats maintained at RT. In fasted rats, abdominal surgery decreased AG and DAG levels by 2.4- and 2.1-times, respectively, at 90 min. Cold for 90 min after surgery normalized AG and DAG levels to those observed in sham-treated animals kept at RT. These data indicate that endogenous (cold exposure) and exogenous (TRH analog) activation of medullary TRH vagal signaling prevent abdominal surgery-induced delayed GE. The restoration of circulating AG levels inhibited by abdominal surgery may contribute to alleviate postoperative gastric ileus.

Activation of brain somatostatin 2 receptors stimulates feeding in mice: analysis of food intake microstructure.

We recently reported that the oligosomatostatin receptor agonist, ODT8-SST increases food intake in rats via the somatostatin 2 receptor (sst(2)). We characterized ingestive behavior following intracerebroventricular (icv) injection of a selective sst(2) agonist in freely fed mice during the light phase. The sst(2) agonist (0.01, 0.03, 0.1, 0.3 or 1µg/mouse) injected icv under short inhalation anesthesia dose-dependently increased cumulative light phase food intake over 4h compared to vehicle with a 3.1-times increase at 1µg/mouse (p<0.05). Likewise, the sst(2,3,5) agonist octreotide (0.3 or 1µg/mouse) dose-dependently increased 4-h food intake, whereas selective sst(1) or sst(4) agonists at 1µg/mouse did not. In vehicle-treated mice, high fat diet increased caloric intake/4h by 2.8-times compared to regular diet (p<0.05) and values were further increased 1.4-times/4h by the sst(2) agonist. Automated continuous assessment of food intake established a 6.6-times higher food intake during the dark phase due to increased number of meals, meal size, meal duration and rate of ingestion compared to non-treated mice during the light phase. During the first 4h post icv sst(2) agonist injection, mice had a 57% increase in number of meals with a 60% higher rate of ingestion, and a 61% reduction in inter-meal intervals, whereas meal sizes were not altered compared to vehicle. These data indicate that the activation of brain sst(2) receptors potently stimulates the light phase ingestive behavior under basal or high fat diet-stimulated conditions in mice. The shortened inter-meal interval suggests an inhibitory effect of the sst(2) agonist on "satiety", whereas "satiation" is not altered as indicated by normal meal size.

Lipopolysaccharide differentially decreases plasma acyl and desacyl ghrelin levels in rats: potential role of the circulating ghrelin-acylating enzyme GOAT.

Bacterial lipopolysaccharide (LPS) in rodents is an established model for studying innate immune responses to gram-negative bacteria and mimicking symptoms of infections including reduced food intake associated with decreased circulating total ghrelin levels. The ghrelin-acylating enzyme, ghrelin-O-acyltransferase (GOAT) involved in the formation of acyl ghrelin (AG) was recently identified. We investigated changes in circulating AG, desacyl ghrelin (DG) and GOAT induced by intraperitoneal LPS (100 microg/kg) and associated changes in food intake. Plasma AG and total ghrelin were assessed by radioimmunoassay, GOAT protein by Western blot and mRNA by RT-qPCR. DG was derived from total minus AG. Plasma AG and DG were decreased at 2, 5 and 7 h (p<0.01) post-injection compared to vehicle and recovered at 24 h. At 2 h there was a significantly greater decrease of AG (-53%) than DG (-28%) resulting in a decreased AG/DG ratio (1:5, p<0.01), which thereafter returned to pre-injection values (1:3). This altered ratio was associated with a 38% decrease in plasma GOAT protein compared to vehicle (p<0.001), whereas gastric GOAT protein was slightly increased by 10% (p<0.05). GOAT mRNA expression was unchanged. Food intake was reduced by 58% measured during the 1.5-2 h period post-LPS injection. Decreased plasma AG and DG preceded the rise in rectal temperature and blood glucose that peaked at 7 h. These data indicate that LPS induces a long-lasting reduction of AG and DG levels that may have a bearing with the decrease in food intake. The faster drop in AG than DG within 2 h is associated with reduced circulating GOAT.

Pattern of Fos expression in the brain induced by selective activation of somatostatin receptor 2 in rats.

Central activation of somatostatin (sst) receptors by oligosomatostatin analogs inhibits growth hormone and stress-related rise in catecholamine plasma levels while stimulating grooming, feeding behaviors, gastric transit and acid secretion, which can be mimicked by selective sst(2) receptor agonist. To evaluate the pattern of neuronal activation induced by peptide sst receptor agonists, we assessed Fos-expression in rat brain after intracerebroventricular (i.c.v.) injection of a newly developed selective sst(2) agonist compared to the oligosomatostatin ODT8-SST, a pan-sst(1-5) agonist. Ninety min after injection of vehicle (10 microl) or previously established maximal orexigenic dose of peptides (1 microg=1 nmol/rat), brains were assessed for Fos-immunohistochemistry and doublelabeling. Food and water were removed after injection. The sst(2) agonist and ODT8-SST induced a similar Fos distribution pattern except in the arcuate nucleus where only the sst(2) agonist increased Fos. Compared to ODT8-SST, the sst(2) agonist induced higher Fos-expression by 3.7-times in the basolateral amygdaloid nucleus, 1.2-times in the supraoptic nucleus (SON), 1.6-times in the magnocellular paraventricular hypothalamic nucleus (mPVN), 4.1-times in the external lateral parabrachial nucleus, and 2.6-times in both the inferior olivary nucleus and superficial layer of the caudal spinal trigeminal nucleus. Doublelabeling in the hypothalamus showed that ODT8-SST activates 36% of oxytocin, 63% of vasopressin and 79% of sst(2) immunoreactive neurons in the mPVN and 28%, 55% and 25% in the SON, respectively. Selective activation of sst(2) receptor results in a more robust neuronal activation than the pan-sst(1-5) agonist in various brain regions that may have relevance in sst(2) mediated alterations of behavioral, autonomic and endocrine functions.

Central injection of the stable somatostatin analog ODT8-SST induces a somatostatin2 receptor-mediated orexigenic effect: role of neuropeptide Y and opioid signaling pathways in rats.

Somatostatin and octreotide injected into the brain have been reported to modulate food intake. However, little is known regarding the underlying mechanisms. The stable oligosomatostatin analog, des-AA(1,2,4,5,12,13)-[DTrp(8)]-somatostatin (ODT8-SST), like somatostatin, binds to all five somatostatin receptors (sst(1-5)). We characterized the effects of ODT8-SST injected intracerebroventricularly (i.c.v.) on food consumption and related mechanisms of action in freely fed rats. ODT8-SST (0.3 and 1 microg per rat, i.c.v.) injected during the light or dark phase induced an early onset (within 1 h) and long-lasting (4 h) increase in food intake in nonfasted rats. By contrast, i.p. injection (0.3-3 mg/kg) or i.c.v. injection of selective sst(1) or sst(4) agonists (1 microg per rat) had no effect. The 2 h food intake response during the light phase was blocked by i.c.v. injection of a sst(2) antagonist, the neuropeptide Y (NPY) Y(1) receptor antagonist, BIBP-3226, and ip injection of the mu-opioid receptor antagonist, naloxone, and not associated with changes in plasma ghrelin levels. ODT8-SST (1 microg per rat, i.c.v.) stimulated gastric emptying of a solid meal which was also blocked by naloxone. The increased food intake was accompanied by a sustained increase in respiratory quotient, energy expenditure, and drinking as well as mu-opioid receptor-independent grooming behavior and hyperthermia, while ambulatory movements were not altered after ODT8-SST (1 microg per rat, i.c.v.). These data show that ODT8-SST acts primarily through brain sst(2) receptors to induce a long-lasting orexigenic effect that involves the activation of Y(1) and opiate-receptors, accompanied by enhanced gastric transit and energy expenditure suggesting a modulation of NPYergic and opioidergic orexigenic systems by brain sst(2) receptors.

Peripherally injected CCK-8S activates CART positive neurons of the paraventricular nucleus in rats.

Cholecystokinin (CCK) plays a role in the short-term inhibition of food intake. Cocaine- and amphetamine-regulated transcript (CART) peptide has been observed in neurons of the paraventricular nucleus (PVN). It has been reported that intracerebroventricular injection of CART peptide inhibits food intake in rodents. The aim of the study was to determine whether intraperitoneally (ip) injected CCK-8S affects neuronal activity of PVN-CART neurons. Ad libitum fed male Sprague-Dawley rats received 6 or 10 microg/kg CCK-8S or 0.15M NaCl ip (n=4/group). The number of c-Fos-immunoreactive neurons was determined in the PVN, arcuate nucleus (ARC), and the nucleus of the solitary tract (NTS). CCK-8S dose-dependently increased the number of c-Fos-immunoreactive neurons in the PVN (mean+/-SEM: 102+/-6 vs. 150+/-5 neurons/section, p<0.05) and compared to vehicle treated rats (18+/-7, p<0.05 vs. 6 and 10 microg/kg CCK-8S). CCK-8S at both doses induced an increase in the number of c-Fos-immunoreactive neurons in the NTS (65+/-13, p<0.05, and 182+/-16, p<0.05). No effect on the number of c-Fos neurons was observed in the ARC. Immunostaining for CART and c-Fos revealed a dose-dependent increase of activated CART neurons (19+/-3 vs. 29+/-7; p<0.05), only few activated CART neuron were observed in the vehicle group (1+/-0). The present observation shows that CCK-8S injected ip induces an increase in neuronal activity in PVN-CART neurons and suggests that CART neurons in the PVN may play a role in the mediation of peripheral CCK-8S's anorexigenic effects.

Mice overexpressing corticotropin-releasing factor show brain atrophy and motor dysfunctions.

Chronic stress and persistently high glucocorticoid levels can induce brain atrophy. Corticotropin-releasing factor (CRF)-overexpressing (OE) mice are a genetic model of chronic stress with elevated brain CRF and plasma corticosterone levels and Cushing's syndrome. The brain structural alterations in the CRF-OE mice, however, are not well known. We found that adult male and female CRF-OE mice had significantly lower whole brain and cerebellum weights than their wild type (WT) littermates (347.7+/-3.6mg vs. 460.1+/-4.3mg and 36.3+/-0.8mg vs. 50.0+/-1.3mg, respectively) without sex-related difference. The epididymal/parametrial fat mass was significantly higher in CRF-OE mice. The brain weight was inversely correlated to epididymal/parametrial fat weight, but not to body weight. Computerized image analysis system in Nissl-stained brain sections of female mice showed that the anterior cingulate and sensorimotor cortexes of CRF-OE mice were significantly thinner, and the volumes of the hippocampus, hypothalamic paraventricular nucleus and amygdala were significantly reduced compared to WT, while the locus coeruleus showed a non-significant increase. Motor functions determined by beam crossing and gait analysis showed that CRF-OE mice took longer time and more steps to traverse a beam with more errors, and displayed reduced stride length compared to their WT littermates. These data show that CRF-OE mice display brain size reduction associated with alterations of motor coordination and an increase in visceral fat mass providing a novel animal model to study mechanisms involved in brain atrophy under conditions of sustained elevation of brain CRF and circulating glucocorticoid levels.

Differential distribution of ghrelin-O-acyltransferase (GOAT) immunoreactive cells in the mouse and rat gastric oxyntic mucosa.

The enzyme that acylates ghrelin was recently identified in mice as the fourth member of the membrane-bound O-acyltransferases superfamily (MBOAT4) and named ghrelin-O-acyltransferase (GOAT). Only one report showed GOAT mRNA expression in ghrelin-expressing cells of the mouse stomach. We investigated the distribution of GOAT protein in peripheral tissues and co-expression with endocrine markers in the gastric mucosa using a custom-made anti-GOAT antibody. Tissues were collected from male Sprague-Dawley rats and C57BL/6 mice. Western blot revealed two immunoreactive bands in rat and mouse gastric corpus mucosal proteins, a 50 kDa band corresponding to the GOAT protein and a 100 kDa band likely corresponding to a dimer. Western blot also detected GOAT in the plasma and levels were strongly increased after 24-h fasting in mice and slightly in rats. GOAT-immunoreactive cells were located in the gastric corpus mucosa and the anterior pituitary gland, whereas other peripheral tissues of rats and mice examined were negative. In mice, GOAT-immunoreactive cells were mainly distributed throughout the middle portion of the oxyntic glands, whereas in rats they were localized mainly in the lower portion of the glands. Double labeling showed that 95+/-1% of GOAT-immunoreactive cells in mice co-labeled with ghrelin, whereas in rats only 56+/-4% of GOAT-positive cells showed co-expression of ghrelin. The remainder of the GOAT-immunopositive cells in rats co-expressed histidine decarboxylase (44+/-3%). No co-localization was observed with somatostatin in rats or mice. These data suggest species differences between rats and mice in gastric GOAT expression perhaps resulting in a different role of the MBOAT4 enzyme in the rat stomach. Detection of GOAT in the plasma raises the possibility that ghrelin octanoylation may occur in the circulation and the fasting-induced increase in GOAT may contribute to the increase of acylated ghrelin after fasting.

Ghrelin, des-acyl ghrelin and nesfatin-1 in gastric X/A-like cells: role as regulators of food intake and body weight.

Numerous peptides released from endocrine cells in the intestinal mucosa were established early on to be involved in the physiological regulation of food intake with a prominent role in termination of food ingestion when nutrients pass along the intestinal tract. Recently, peptides released from X/A-like endocrine cells of the gastric oxyntic mucosa were recognized as additional key players in the regulation of feeding and energy expenditure. Gastric X/A-like cells release the octanoylated peptide, ghrelin, the only known peripherally produced hormone stimulating food intake through interaction with growth hormone secretagogue 1a receptor (GHS-R1a). Additionally, non-octanoylated (des-acyl) ghrelin present in the circulation at higher levels than ghrelin is currently discussed as potential modulator of food intake by opposing ghrelin's action independent from GHS-R1a although the functional significance remains to be established. Obestatin, a ghrelin-associated peptide was initially reported as anorexigenic modulator of ghrelin's orexigenic action. However, subsequent reports did not support this contention. Interesting is the recent identification of nesfatin-1, a peptide derived from the nucleobindin2 gene prominently expressed in gastric X/A-like cells in different vesicles than ghrelin. Circulating nesfatin-1 levels vary with metabolic state and peripheral or central injection inhibits dark phase feeding in rodents. Overall, these data point to an important role of gastric X/A-like cells in food intake regulation through the expression of the orexigenic peptide ghrelin along with des-acyl ghrelin and nesfatin-1 capable of reducing food intake upon exogenous injection although their mechanisms of action and functional significance remain to be established.

Abdominal surgery activates nesfatin-1 immunoreactive brain nuclei in rats.

Abdominal surgery-induced postoperative gastric ileus is well established to induce Fos expression in specific brain nuclei in rats within 2-h after surgery. However, the phenotype of activated neurons has not been thoroughly characterized. Nesfatin-1 was recently discovered in the rat hypothalamus as a new anorexigenic peptide that also inhibits gastric emptying and is widely distributed in rat brain autonomic nuclei suggesting an involvement in stress responses. Therefore, we investigated whether abdominal surgery activates nesfatin-1-immunoreactive (ir) neurons in the rat brain. Two hours after abdominal surgery with cecal palpation under short isoflurane anesthesia or anesthesia alone, rats were transcardially perfused and brains processed for double immunohistochemical labeling of Fos and nesfatin-1. Abdominal surgery, compared to anesthesia alone, induced Fos expression in neurons of the supraoptic nucleus (SON), paraventricular nucleus (PVN), locus coeruleus (LC), Edinger-Westphal nucleus (EW), rostral raphe pallidus (rRPa), nucleus of the solitary tract (NTS) and ventrolateral medulla (VLM). Double Fos/nesfatin-1 labeling showed that of the activated cells, 99% were nesfatin-1-immunoreactive in the SON, 91% in the LC, 82% in the rRPa, 74% in the EW and VLM, 71% in the anterior parvicellular PVN, 47% in the lateral magnocellular PVN, 41% in the medial magnocellular PVN, 14% in the NTS and 9% in the medial parvicellular PVN. These data established nesfatin-1 immunoreactive neurons in specific nuclei of the hypothalamus and brainstem as part of the neuronal response to abdominal surgery and suggest a possible implication of nesfatin-1 in the alterations of food intake and gastric transit associated with such a stressor.

Novel insight in distribution of nesfatin-1 and phospho-mTOR in the arcuate nucleus of the hypothalamus of rats.

Recently, two proteins have been localized in the arcuate nucleus (ARC) and implicated in the regulation of food intake: the serine-threonine-kinase mammalian target of rapamycin (mTOR) as part of the TOR signaling complex 1 (TORC1), and nesfatin-1 derived from the precursor protein nucleobindin2. However, the exact cell types are not well described. Therefore, we performed double-labeling studies for NPY, CART, nesfatin-1 and pmTOR in the ARC. In this study, we showed that nesfatin-1 is not only intracellularly co-localized with cocaine- and amphetamine-regulated transcript (CART) peptide as reported before, but also with phospho-mTOR (pmTOR) and neuropeptide Y (NPY) in ARC neurons. Quantification revealed that 59+/-5% of the pmTOR-immunoreactive (ir) neurons were immunoreactive for nesfatin-1. Moreover, double labeling for nesfatin-1 and NPY exhibited that 19+/-5% of the NPY positive cells were also immunoreactive for nesfatin-1. Furthermore, we could also confirm results from previous studies, showing that the majority of nesfatin-1 neurons are also positive for CART peptide, whereas most of the pmTOR is co-localized with NPY and only to a lesser extent with CART.

Central nesfatin-1 reduces dark-phase food intake and gastric emptying in rats: differential role of corticotropin-releasing factor2 receptor.

Nesfatin-1, derived from nucleobindin2, is expressed in the hypothalamus and reported in one study to reduce food intake (FI) in rats. To characterize the central anorexigenic action of nesfatin-1 and whether gastric emptying (GE) is altered, we injected nesfatin-1 into the lateral brain ventricle (intracerebroventricular, icv) or fourth ventricle (4v) in chronically cannulated rats or into the cisterna magna (intracisternal, ic) under short anesthesia and compared with ip injection. Nesfatin-1 (0.05 microg/rat, icv) decreased 2-3 h and 3-6 h dark-phase FI by 87 and 45%, respectively, whereas ip administration (2 microg/rat) had no effect. The corticotropin-releasing factor (CRF)(1)/CRF(2) antagonist astressin-B or the CRF(2) antagonist astressin(2)-B abolished icv nesfatin-1's anorexigenic action, whereas an astressin(2)-B analog, devoid of CRF-receptor binding affinity, did not. Nesfatin-1 icv induced a dose-dependent reduction of GE by 26 and 43% that was not modified by icv astressin(2)-B. Nesfatin-1 into the 4v (0.05 microg/rat) or ic (0.5 microg/rat) decreased cumulative dark-phase FI by 29 and 60% at 1 h and by 41 and 37% between 3 and 5 h, respectively. This effect was neither altered by ic astressin(2)-B nor associated with changes in GE. Cholecystokinin (ip) induced Fos expression in 43% of nesfatin-1 neurons in the paraventricular hypothalamic nucleus and 24% of those in the nucleus tractus solitarius. These data indicate that nesfatin-1 acts centrally to reduce dark phase FI through CRF(2)-receptor-dependent pathways after forebrain injection and CRF(2)-receptor-independent pathways after hindbrain injection. Activation of nesfatin-1 neurons by cholecystokinin at sites regulating food intake may suggest a role in gut peptide satiation effect.

The RAPID method for blood processing yields new insight in plasma concentrations and molecular forms of circulating gut peptides.

The correct identification of circulating molecular forms and measurement of peptide levels in blood entails that the endocrine peptide being studied is stable and recovered in good yields during blood processing. However, it is not clear whether this is achieved in studies using standard blood processing. Therefore, we compared peptide concentration and form of 12 (125)I-labeled peptides using the standard procedure (EDTA-blood on ice) and a new method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls, and Dilution (RAPID). During standard processing there was at least 80% loss for calcitonin-gene-related peptide and cholecystokinin-58 (CCK-58) and more than 35% loss for amylin, insulin, peptide YY forms (PYY((1-36)) and PYY((3-36))), and somatostatin-28. In contrast, the RAPID method significantly improved the recovery for 11 of 12 peptides (P < 0.05) and eliminated the breakdown of endocrine peptides occurring after standard processing as reflected in radically changed molecular forms for CCK-58, gastrin-releasing peptide, somatostatin-28, and ghrelin. For endogenous ghrelin, this led to an acyl/total ghrelin ratio of 1:5 instead of 1:19 by the standard method. These results show that the RAPID method enables accurate assessment of circulating gut peptide concentrations and forms such as CCK-58, acylated ghrelin, and somatostatin-28. Therefore, the RAPID method represents an efficacious means to detect circulating variations in peptide concentrations and form relevant to the understanding of physiological function of endocrine peptides.

Restraint stress activates nesfatin-1-immunoreactive brain nuclei in rats.

Nesfatin-1 is a newly discovered peptide that was reported to reduce food intake when injected centrally. We recently described its wide distribution in rat brain autonomic nuclei which implies potential recruitment of nesfatin-1 by stress. We investigated whether restraint, a mixed psychological and physical stressor, activates nesfatin-1-immunoreactive (ir) neurons in the rat brain. Male Sprague-Dawley rats were either subjected to 30 min restraint or left undisturbed and 90 min later brains were processed for double immunohistochemical labeling of Fos and nesfatin-1. Restraint induced significant Fos expression in neurons of the supraoptic nucleus (SON), paraventricular nucleus (PVN), locus coeruleus (LC), rostral raphe pallidus (rRPa), nucleus of the solitary tract (NTS), and ventrolateral medulla (VLM). Double Fos/nesfatin-1 labeling revealed that Fos-ir neurons comprised 95% of nesfatin-1-ir cells in the SON, 90% in the VLM, 80% in the LC, 48% in the caudal NTS, 57% in the rRPa, 48% in the anterior parvicellular PVN, 27% in the medial magnocellular PVN, 18% in the lateral magnocellular PVN and 10% in the medial parvicellular PVN. These data demonstrate that nesfatin-1 neurons are part of the hypothalamic and hindbrain neuronal cell groups activated by restraint suggesting a possible role of nesfatin-1 in the response to stress.

CCK-8S activates c-Fos in a dose-dependent manner in nesfatin-1 immunoreactive neurons in the paraventricular nucleus of the hypothalamus and in the nucleus of the solitary tract of the brainstem.

Recently, a new neuropeptide, named nesfatin-1, was discovered. It has been reported that nesfatin-1 inhibits food intake after injection into the third ventricle as well as intraperitoneal (ip) injection. Cholecystokinin (CCK) is well established to play a role in the regulation of food intake. The aim of the study was to examine whether CCK-8S injected ip modulates neuronal activity in nesfatin-1 immunoreactive (ir) neurons localized in the PVN and in the nucleus of the solitary tract (NTS). Additionally, tyrosine hydroxylase-immunoreactivity (TH-ir) in the PVN was determined to assess the distribution of TH-ir fibers in relation to nesfatin-1-ir. Non-fasted male Sprague-Dawley rats received 6 or 10 microg CCK-8S/kg or vehicle solution (0.15M NaCl; n=4 all groups) ip. The number of c-Fos-ir neurons was determined in the PVN, arcuate nucleus (ARC), and NTS. Double staining procedure for nesfatin-1 and c-Fos revealed that CCK-8S increased significantly and in a dose-dependent manner the number of c-Fos positive nesfatin-1-ir neurons in the PVN ( approximately 4-fold and approximately 7-fold) and NTS ( approximately 9-fold and approximately 26-fold). Triple staining in the PVN showed a dose-dependent neuronal activation of nesfatin-1 neurons that were colocalized with CRF and oxytocin. Double labeling against nesfatin-1 and TH revealed that nefatin-1-ir neurons were encircled in a network of TH-ir fibers in the PVN. No effect on the number of c-Fos-ir neurons was observed in the ARC. These results suggest that the effects of CCK on the HPA axis and on food intake may, at least in part, be mediated by nesfatin-1-ir neurons in the PVN.

Nesfatin-1 immunoreactivity in rat brain and spinal cord autonomic nuclei.

Nesfatin-1 is one of the peptide products of posttranslational processing of the nucleobindin-2 (NUCB2) gene, suggested to have physiological relevance to suppress food intake and body weight gain in rats. Nesfatin-1-immunoreactive cells have been found in distinct nuclei in the rat brain related to circuitries regulating food intake. Here, we report novel yet undescribed localization of NUCB2/nesfatin-1 at the mRNA and protein level in the rat central nervous system. Immunohistochemical staining revealed the localization of NUCB2/nesfatin-1 in the piriform and insular cortex, endopiriform nucleus, nucleus accumbens, lateral septum, bed nucleus of stria terminalis, central amygdaloid nucleus, medial preoptic area, dorsal raphe nucleus, ambiguus nucleus, ventrolateral medulla and gigantocellular reticular nucleus, as well as Purkinje-cells of the cerebellum. In the spinal cord, nesfatin-1 immunoreactivity (IR) was found in both sympathetic and parasympathetic preganglionic neuronal groups and in the dorsal area X from lower thoracic to sacral segments. The immunohistochemical results were confirmed by RT-PCR in the central amygdaloid nucleus, nucleus accumbens, cerebellum and lumbar spinal cord microdissected by punch technique. The features and distributions of nesfatin-1 IR and mRNA expression in the brain and spinal cord suggest that NUCB2/nesfatin-1 could play a wider role in autonomic regulation of visceral-endocrine functions besides food intake.