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Inhoff T.,Charite - Medical University of Berlin | Stengel A.,Digestive Diseases Research Center | Peter L.,Charite - Medical University of Berlin | Goebel M.,Digestive Diseases Research Center | And 6 more authors.
Peptides | Year: 2010

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. © 2009 Elsevier Inc. All rights reserved. Source

Peter L.,Charite - Medical University of Berlin | Stengel A.,Digestive Diseases Research Center | Noetzel S.,Charite - Medical University of Berlin | Inhoff T.,Charite - Medical University of Berlin | And 9 more authors.
Peptides | Year: 2010

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 μg/kg CCK-8S or 0.15 M 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 μg/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. © 2010 Elsevier Inc. Source

Stengel A.,University of California at Los Angeles | Goebel M.,University of California at Los Angeles | Wang L.,University of California at Los Angeles | Rivier J.,La Jolla Salk Institute | And 3 more authors.
Physiology and Behavior | Year: 2010

We recently reported that the oligosomatostatin receptor agonist, ODT8-SST increases food intake in rats via the somatostatin 2 receptor (sst2). We characterized ingestive behavior following intracerebroventricular (icv) injection of a selective sst2 agonist in freely fed mice during the light phase. The sst2 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 sst2,3,5 agonist octreotide (0.3 or 1μg/mouse) dose-dependently increased 4-h food intake, whereas selective sst1 or sst4 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 sst2 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 sst2 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 sst2 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 sst2 agonist on "satiety", whereas "satiation" is not altered as indicated by normal meal size. © 2010. Source

Goebel-Stengel M.,Institute of Neurogastroenterology | Goebel-Stengel M.,Digestive Diseases Research Center | Goebel-Stengel M.,University of California at Los Angeles | Stengel A.,Digestive Diseases Research Center | And 6 more authors.
Analytical Biochemistry | Year: 2011

The unpredictable nature of peptide binding to surfaces requires optimization of experimental containers to be used. To demonstrate the variable recoveries of peptides from multiple surfaces commonly employed in peptide research, we tested the recovery of radiolabeled 125I endocrine peptides under different conditions and provide guidelines for determining the surfaces to use for other peptides. 125I-labeled peptides (ghrelin, sulfated cholecystokinin-8, corticotropin-releasing factor, glucagon-like peptide-1 [GLP-1], insulin, leptin, nesfatin-1, and peptide YY), representing a wide spectrum in net charge, size, end group, and modification, were incubated for 48 h in glass and plastic tubes untreated or coated with siliconizing fluid. Best surfaces were chosen and peptides were incubated with bovine serum albumin (BSA, 1%) with or without subsequent lyophilization. Recovery of 125I-labeled peptides was determined by gamma counting. Important differences in 125I-labeled peptide binding capacities to various types of surfaces exist. Siliconization decreased, whereas the addition of BSA improved recovery from surfaces tested. Lyophilizing solutions containing 125I-labeled peptides and BSA in the tubes best suited for individual peptides rendered more than 89% recovery for all peptides. Ghrelin specifically displaced 125I-ghrelin from borosilicate glass, whereas GLP-1 and Fmoc-arginine did not. Choosing the appropriate experimental container avoids unpredictable peptide loss that results in inaccurate measurements and false conclusions. © 2011 Elsevier Inc. All rights reserved. Source

Teuffel P.,Universitaetsmedizin Berlin | Wang L.,University of California at Los Angeles | Prinz P.,Universitaetsmedizin Berlin | Goebel-Stengel M.,Institute of Neurogastroenterology | And 7 more authors.
Journal of Physiology and Pharmacology | Year: 2015

The ghrelin acylating enzyme ghrelin-O-acyltransferase (GOAT) was recently identified and implicated in several biological functions. However, the effects on food intake warrant further investigation. While several genetic GOAT mouse models showed normal food intake, acute blockade using a GOAT inhibitor resulted in reduced food intake. The underlying food intake microstructure remains to be established. In the present study we used an automated feeding monitoring system to assess food intake and the food intake microstructure. First, we validated the basal food intake and feeding behavior in rats using the automated monitoring system. Afterwards, we assessed the food intake microstructure following intraperitoneal injection of the GOAT inhibitor, GO-CoA-Tat (32, 96 and 288 μg/kg) in freely fed male Sprague-Dawley rats. Rats showed a rapid habituation to the automated food intake monitoring system and food intake levels were similar compared to manual monitoring (P = 0.43). Rats housed under these conditions showed a physiological behavioral satiety sequence. Injection of the GOAT inhibitor resulted in a dose-dependent reduction of food intake with a maximum effect observed after 96 μg/kg (-27%, P = 0.03) compared to vehicle. This effect was delayed in onset as the first meal was not altered and lasted for a period of 2 h. Analysis of the food intake microstructure showed that the anorexigenic effect was due to a reduction of meal frequency (-15%, P = 0.04), whereas meal size (P = 0.29) was not altered compared to vehicle. In summary, pharmacological blockade of GOAT reduces dark phase food intake by an increase of satiety while satiation is not affected. © 2015 Polish Physiological Society. All rights reserved. Source

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