Selon l'organisation mondiale de la santé, le diabète de type II (DT2), caractérisé par un défaut de contrôle de la glycémie, est une des causes principales de décès dans le monde. Le GLP-1, sécrété par l'intestin après un repas, contribue au contrôle de la glycémie en stimulant la sécrétion d'insuline par le pancréas et en inhibant la vidange gastrique et la prise alimentaire. Ces actions sont principalement médiées par le nerf vague selon un axe intestin-cerveau-organes périphériques, bien que l'hormone puisse aussi agir de manière endocrine directement sur ses organes cibles via son récepteur (GLP-1r). Des stratégies thérapeutiques basées sur le GLP-1 sont donc utilisées pour traiter les patients diabétiques, mais les réponses sont hétérogènes voire inefficaces pour le contrôle glycémique. Les mécanismes moléculaires responsables sont inconnus mais pourraient être en lien avec la modification du microbiote intestinal, élément déterminant dans le développement des maladies métaboliques. Nous avons d'abord montré que des souris rendues diabétiques (régimes riches en graisse) perdent leur sensibilité aux actions hypoglycémiantes du GLP-1 et présentent une neuropathie entérique, une baisse de l'expression du GLP-1r intestinal et vagal et une altération de l'axe intestin-cerveau. De plus, dans les neurones entériques en culture primaire issus de ces souris diabétiques, la production de NO induite par le GLP-1, est diminuée. Tous ces effets sont retrouvés chez des souris axéniques ou traitées aux antibiotiques sous régime normal démontrant l'implication du microbiote. À l'inverse, des souris sous régime gras traitées aux antibiotiques ont une amélioration de l'action du GLP-1. Cette action hormonale intestinale pourrait aussi dépendre du cycle nycthéméral pour lequel nous avons observé une oscillation de la sécrétion d'insuline, de l'expression du GLP-1r et des bactéries intestinales. De plus, les souris contrôles répondent moins bien à l'hormone au cours du jour que de la nuit et les souris diabétiques, axéniques et antibiotiques - modèles résistants au GLP-1 - ont des variations très marquées et communes de l'expression des "clock genes". L'ensemble de ces résultats montre qu'au cours diabète, l'action du GLP-1 est diminuée. Cette diminution peut s'expliquer par une baisse de l'expression neuronale du GLP-1r et une diminution de la voie de signalisation dépendant du NO capable de réguler la sécrétion d'insuline induite par le GLP-1. Le microbiote et/ou la régulation circadienne semblent déterminants dans la sensibilité au GLP-1. / According to the World Health Organisation, Type II Diabetes, characterized by an alteration of glycemic control, causes numerous death around the world. After a meal, gut secretes Glucagon-Like Peptide one (GLP-1) which regulates glycemia by stimulation of insulin secretion and inhibition of gastric emptying and food intake. Although GLP-1 acts as an endocrine hormone on its target organs through the GLP1 receptor, its action is mainly mediated by nervous pathway involving vagus nerve and gut-brain-periphery axis. Thus, GLP-1 based therapies are used to control glycaemia in type 2 diabetic patients, but, efficiency of the treatment is heterogeneous defining a state of GLP-1 unresponsiveness. Molecular mechanisms involved in this unresponsiveness are not known but could be linked to the changes in gut microbiota (dysbiosis), key element in the development of metabolic diseases. We first found that diabetic mice (high fat diet) are unresponsive to hypoglycemic action of GLP-1 and present enteric neuropathy, impaired gut-brain axis and reduction of GLP-1r and neuronal NO synthase expression in the ileum. In addition, GLP-1-induced nitric oxide production in primary neuron culture is decreased. These effects were also found in germ-free or antibiotic-treated mice under normal chow diet, indicating the involvement of gut microbiota. By contrast, high fat diet mice treated with antibiotics show an improvement of GLP-1 action. This gut incretin action could also depend on the circadian cycle for which we observed a wavering of insulin secretion, GLP-1r expression and gut microbiota. Moreover, the GLP-1 response of control mice is better in the day than in the night and the different mice model resistant to GLP-1 (HFD, axenic or antibiotics) present the same marked variations in the expression of major clock genes. Overall our results show that in type 2 diabetes GLP-1 action is lowered and can be explained by decreased neuronal expression of GLP-1r as well as the NO-dependent signaling pathway regulating insulin secretion induced by GLP-1. Microbiota or the circadian clock seems essential in this GLP-1 sensitivity.

Certains mammifères, dont l’Homme, ont une forte attraction pour les lipides alimentaires. Pendant longtemps, il était admis que ces nutriments étaient détectés uniquement via leurs propriétés olfactives, texturales et post-ingestives. Cependant, l’existence d’une dimension gustative a été suggérée depuis. Dans ce contexte, notre Laboratoire a démontré que la glycoprotéine CD36 exerçait une fonction de lipido-récepteur gustatif impliquée à la fois dans la préférence spontanée pour les lipides alimentaires et la phase céphalique de la digestion induite par la présence de lipides au niveau oral chez la souris. Une autre protéine, GPR120, semble également jouer un rôle dans la détection gustative des lipides alimentaires. Ces travaux de thèse avaient donc pour objectif de comprendre les rôles respectifs de ces deux protéines au sein des bourgeons du goût chez la souris. En utilisant une combinaison d'approches biochimiques, physiologiques et comportementales, nous avons pu montrer que le CD36 lingual était régulé négativement par les lipides alimentaires lors d'une exposition court terme, contrairement au GPR120. Cette régulation pourrait se traduire par une désensibilisation au cours du repas du système de lipido-réception gustatif et donc jouer un rôle dans le rassasiement sensoriel spécifique. Nos résultats suggèrent également que l'activation de GPR120 par les acides gras à longues chaînes peut jouer un rôle dans la modulation de la sensibilité gustative aux saveurs sucrées, voire aux lipides eux-mêmes. Ce phénomène est dépendant de la sécrétion de l'hormone glucagon-like peptide-1 par la papille caliciforme, indépendamment du CD36 lingual. Nous avons également mis en évidence que l'exposition des souris à un régime obésogène chronique perturbe la détection oro-sensorielle des lipides, suggérant une hyposensibilité chez la souris obèse. On peut donc penser qu'une perturbation du système de perception gustative des graisses alimentaires pourrait induire des changements de préférence alimentaire, propice à l’installation d'une obésité. De plus amples investigations sont requises pour explorer le rôle du CD36 et/ou du GPR120 dans ce phénomène. L'aboutissement de telles recherches serait l'émergence de nouvelles voies de traitement de l’obésité et des maladies associées, par des approches pharmacologiques et/ou nutritionnelles ciblées. / Some mammals including humans display a spontaneous attraction for dietary fat. For a long time, it was thought that these nutrients were detected only by olfactory, textural and post-oral cues. However, recent data have indicated that the sense of taste could also contribute to this perception in laboratory rodents. In this context, it was demonstrated by our Laboratory that the glycoprotein CD36 is likely a lingual lipid sensor, involved both in preference for long-chain fatty acids and cephalic phase of digestion secondary to an oral lipid stimulation in mice. Another protein, GPR120, seems also to play a role in the gustatory detection of lipids. The aim of this Thesis was to understand the respective roles of these putative gustatory lipid sensors in mice. Using a combination of biochemical, physiological and behavioral approaches, we showed that the lingual CD36 is negatively regulated by dietary fat during a short term exposure (i.e. during a meal), in contrast to GPR120. This regulation is reminiscent of the receptor desensitization and might play a role in the sensory-specific satiety phenomenon. Our data also suggest that GPR120 activation by long-chain fatty acids plays a role in the modulation of sweet taste sensitivity, and likely of fat taste itself. This phenomenon is dependent from the secretion of the glucagon-like peptide-1 hormone by the circumvallate papillae, independently of lingual CD36. Finally, we showed that chronic exposure of mice to an obesogenic diet disturbs the oro-sensory perception of dietary lipids, suggesting that obese mice become hyposensitive to lipids. This alteration might induce changes in feeding preferences which could lead to obesity. More investigations are required to better understand the role of CD36 and/or GPR120 in this phenomenon. This new field of research might lead to new ways of investigations for the treatment of obesity and related diseases, by using novel pharmacological and/or nutritional approaches.

No description available.

In response to ingestion of nutrients, enteroendocrine L cells secrete the incretin hormone, glucagon-like peptide-1 (GLP-1), to enhance glucose-dependent insulin release. Therapies related to GLP-1 are approved for type 2 diabetes. The GLP-1 receptor (GLP-1R) is expressed in cells of the gastrointestinal tract and elsewhere. In pancreatic beta cells, GLP-1R are coupled to the Gs/cAMP/PKA pathway. The expression and function of GLP-1R in gastrointestinal smooth muscle are not known. Aim. To test the hypothesis that GLP-1 regulates smooth muscle function by acting on GLP-1R expressed on smooth muscle. Methods. Smooth muscle cells (SMC) were isolated and cultured. Expression of GLP-1R mRNA was measured by RT-PCR. Expression of GLP-1R protein was measured by western blot. The effect of GLP-1 (7-36) amide on Gαs activation, cAMP formation, and PKA activity was examined in cultured SMC. The effect of GLP-1 on basal activity and on acetylcholine-induced contraction was measured in intact colon via organ bath. Results. Amplification of GLP-1R mRNA suggested expression of GLP-1R mRNA in mucosal and non-mucosal colon cells, which was confirmed in pure SMC cultures. Similar patterns of protein expression were obtained with western blot. Addition of GLP-1 caused relaxation of phasic activity and agonist-induced tonic contractions in intact colon, suggesting a role of smooth muscle Gs-coupled GLP-1R in mediating relaxation. In SMC, GLP-1 (7-36) amide activated Gas, increased cAMP levels, and stimulated PKA activity. Conclusion. Colonic SMC express GLP-1R, and GLP-1 inhibits both basal and acetylcholine-induced contraction. The GLP1-R is coupled to the heterotrimeric G protein, Gas.

Les cellules β pancréatiques synthétisent et sécrètent l'insuline, seule hormone hypoglycémiante de l'organisme. Dans le cas du diabète de type 2, du diabète de type 1 et suite à une greffe d'îlots de Langherans, on observe une diminution drastique de cette masse fonctionnelle β. L'hyperglycémie chronique et la libération de cytokines proinflammatoires jouent un rôle cytotoxique prépondérant dans ces phénomènes. Dans le but de préserver ou de restaurer cette masse fonctionnelle β chez les patients diabétiques, notre objectif était d'identifier des outils permettant de protéger des effets délétères de l'hyperglycémie chronique et des cytokines proinflammatoires, en s'intéressant à 3 cibles potentielles. Nous montrons tout d'abord que les activités du système ubiquitine protéasome (UPS), impliqué dans la dégradation de protéines, sont altérées en condition d'hyperglycémie chronique. Ces altérations sont corrélées à l'émergence d'un programme apoptotique au sein des cellules β. L'activation du récepteur du GLP-1 (Glucagon-Like Peptide-1), stratégie thérapeutique majeure dans le diabète de type 2, protège l'UPS des effets délétères de l'hyperglycémie chronique. Le facteur de transcription CREB (cAMP Response Element Binding Protein), essentiel pour la survie et la fonction des cellules β, est dégradé par l'hyperglycémie chronique et l'inflammation. Nous montrons que la prévention de sa dégradation prévient les effets de l'hyperglycémie chronique, mais pas de l'inflammation. Ces observations nous ont amenés à étudier la MAP3 kinase Tpl2 (Tumor progression locus 2), impliquée, notamment via l'activation de ERK1/2 (Extra-cellular Regulated Kinases 1/2), dans les processus inflammatoires d'autres types cellulaires. Nous montrons que Tpl2 est exprimé dans la lignée cellulaire β INS-1E, et dans les îlots murins et humains, et qu'elle gouverne spécifiquement l'activation des kinases ERK1/2 induite par les cytokines proinflammatoires IL-1β, TNFα et IFNγ. Cette protéine est surexprimée dans des conditions d'inflammation (in vitro et modèle de diabète murin). L'inhibition de Tpl2 protège contre l'apoptose induite par les cytokines, dans les INS-1E et les îlots de souris et restaure la capacité sécrétrice d'insuline des ilots de souris altérée suite à une exposition aux cytokines. En combinaison avec un analogue du GLP-1, l'inhibition pharmacologique de cette kinase protège totalement contre les effets délétères des cytokines sur la fonction et la survie des îlots humains. Ces données suggèrent que l'inhibition pharmacologique de la kinase Tpl2, seule ou en combinaison avec un analogue du GLP-1, pourrait constituer de nouvelles stratégies thérapeutiques pour protéger contre l'altération de la masse fonctionnelle β pouvant survenir chez des patients diabétiques de type 2 ou après la transplantation d'îlots. / Pancreatic β cells synthesize and secrete insulin, the sole hormone of the organism able to reduce glycemia. In the course of type 2 and type 1 diabetes, and after islet transplantation, there is a drastic loss of function and mass of these cells. Among the common origins of this decrease, chronic hyperglycemia and the release of proinflammatory cytokines play major roles. With the aim to preserve or to restore this functional β cell mass in diabetic patients, our objective was to identify tools able to protect against deleterious effects of these two phenomenons, interesting in three potential targets. We first demonstrated that the ubiquitin-proteasome system (UPS) activities, that degrade proteins, are altered in β cells exposed to chronic hyperglycemia, and correlated with apoptosis. Activation of the GLP-1 (Glucagon-Like Peptide-1) receptor, a key therapeutic strategy in type 2 diabetes, protects UPS from deleterious effects of chronic hyperglycemia. The transcription factor CREB (cAMP Response Element Binding Protein), crucial for β cell survival and function, is involved in deleterious effects of chronic hyperglycemia and inflammation. We demonstrated that prevention of CREB degradation protects β cells from chronic hyperglycemia, but not from the deleterious effects of the proinflammatory cytokines. These observations prompted us to study the MAP3 kinase Tpl2 (Tumor progression locus 2), known to be implicated in inflammatory process in other cell types, through the activation of the kinases ERK1/2 (Extra-cellular Regulated Kinases 1/2). We showed that Tpl2 is expressed in INS-1E clonal β cells and in mouse and human islets, and that it governs specifically the activation of ERK1/2 in response to proinflammatory cytokines IL-1β, TNFα and IFNγ. This protein is overexpressed by inflammatory conditions and in a rat type 2 diabetes model. Inhibition of Tpl2 protects against cytokine-induced apoptosis in INS-1E and in mouse islets. Furthermore, the capacity of mouse islets to secrete insulin in response to glucose, that is altered by a chronic exposure to cytokines, is restored by Tpl2 inhibitor. Finally, we showed that in combination with GLP-1 analog (Exendin-4), Tpl2 inhibitor can entirely restore the survival and function in human islets cultured in pro-inflammatory conditions. These results suggest that pharmacological inhibition of Tpl2, alone or in combination with Exendin-4, may be novel therapeutic strategies to alleviate β-cell failure observed in Type 2 diabetes and islets transplantation.

Made available in DSpace on 2017-07-21T19:59:56Z (GMT). No. of bitstreams: 1 Priscila Costa Estabile.pdf: 1735178 bytes, checksum: b258b31793d5d5954531a50cbe4206cc (MD5) Previous issue date: 2012-02-13 / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná / The Type 2 Diabetes Mellitus, as well as the metabolic syndrome (MS), is a multifactorial and metabolic disorder that now presents itself as a worldwide pandemic with effects on morbidity and mortality, possibly as a result of the mismatch between biological and cultural evolution of man. Was the object of research of this study to analyze the tissue expression of incretin hormones glucagon Pepitide Like-1 (GLP1) and Pepitide YY (PYY3-36), to identify and quantify L cells along the gastrointestinal tract in patients with DM2 subjected to adaptative gastroenteromentectomia with intestinal bipartition (Partial Duodenal Switch - DSP). The study was approved by the Ethics Committee of UEPG and patients informed and educated about the research objectives. The volunteer group consisted of 7 patients aged between 35 and 65 years, body mass index> 25 kg/m2 with T2DM on dietary treatment and medication for a minimum of 2 years and with difficulty on glycemic control and hypertriglyceridemia associated. Samples were obtained from the intestinal mucosa (jejunum and ileum) of DSP in patients undergoing preoperative and postoperative condition of fasting for 12 hours (three and twelve months respectively), through incisional biopsy. These biopsies were designed to test immunohistochemistry, qRT-PCR (Quantitative Real Time PCR) and Western Blott. The results were consistent and indicate a very significant differential expression between the state of pre-and postoperative tests for qRT-PCR (p = 0.1669) and Western Blot (p = 0.1569). Immunohistochemistry also showed low significance (p = 0.0043) of immune marked L cells for the same patients under the same conditions. These data can be interpreted in light of the fast imposed on patients. In addition the results are unprecedented for the immunostaining of L-cells of the human gastrointestinal tract, the data indicate that these cells have basal secretion for GLP-1, even after 12 hours without feed stimulation. In addition, the patients showed normalization of blood glucose levels in the post-surgery, suggesting metabolic improvement. It was also found that the number of L cells marked increases in density along the gastrointestinal tract toward the distal portion of the ileum (p = 0.0409). With these results it was possible to identify, locate and investigate different levels of expression and secretion from intestinal L cells in patients with DM2 and subjected to surgical control. / Diabetes mellitus do tipo 2 (DM2), assim como a Síndrome Metabólica (SM), é uma desordem metabólica e multifatorial que atualmente se apresenta como pandemia mundial com reflexos na morbimortalidade, possivelmente em decorrência do descompasso entre a evolução biológica e cultural do homem. Foi objeto de investigação do presente estudo analisar a expressão tecidual dos hormônios incretínicos Glucagon Like Pepitide-1 (GLP1) e Pepitide YY (PYY3-36), visando identificar e quantificar células L ao longo do trato gastrointestinal, de pacientes portadores de DM2 submetidos a gastroenteromentectomia adaptativa com bipartição intestinal (Duodenal Switch Parcial - DSP). O trabalho foi aprovado pelo Comitê de Ética da UEPG e os pacientes informados e esclarecidos sobre os objetivos da pesquisa. O grupo de voluntários foi composto de 7 pacientes com idade entre 35 e 65 anos, Índice de Massa Corporal > 25 Kg/m2, com DM2 em tratamento dietético e medicamentoso por um período mínimo de 2 anos e com dificuldade de controle glicêmico e hipertrigliceridemia associada. Foram obtidas amostras da mucosa intestinal (jejuno e íleo) dos pacientes submetidos à DSP no pré-operatório e no pós-operatório em condição de jejum de 12 horas (três e doze meses, respectivamente), através de biópsia incisional. Estas biópsias foram destinadas aos ensaios de Imunohistoquímica, qRT-PCR (Quantitative Real Time PCR) e Western Blott. Os resultados obtidos foram congruentes e apontam uma expressão diferencial pouco significativa entre o estado de pré- e pós-operatório para os ensaios de qRT-PCR (p=0,1669) e Western Blott (p=0,1569). A imunohistoquímica mostrou também baixa significância (p=0,0043) de células L imuno marcadas para os mesmos pacientes, nas mesmas condições. Estes dados podem ser interpretados a luz do jejum imposto aos pacientes. Além dos resultados serem inéditos para a imunomarcação de células L do trato gastrointestinal humano, os dados obtidos indicam que estas células apresentam secreção basal para GLP-1, mesmo após 12 horas sem estímulo alimentar. Em adição, os pacientes apresentaram normalização dos níveis de glicemia no estado pós-operado, sugerindo melhora metabólica. Foi ainda constatado que o número de células L marcadas aumenta em densidade ao longo do trato gastrointestinal em direção à porção mais distal do íleo (p=0,0409). Com estes resultados foi possível identificar, localizar e investigar diferentes níveis de expressão e secreção das células L intestinais em pacientes portadores de DM2 e submetidos a controle cirúrgico.

La prévalence de l’obésité et du diabète de type 2 évolue de façon épidémique. Ces pathologies sont caractérisées par un état inflammatoire à bas bruit dont l’origine moléculaire est inconnue. L’une des pistes qui émerge concerne le microbiote intestinal et plus particulièrement des molécules pro-inflammatoires présentes à la surface des bactéries Gram(-) : les lipopolysaccharides (LPS). Nous avons récemment montré que ces LPS augmentent les taux circulants de glucagon-like peptide 1 (GLP-1), une hormone connue pour stimuler la sécrétion d'insuline. Par ailleurs, un lien existerait entre qualité nutritionnelle de l’alimentation et taux de LPS sanguins. Ainsi, alimentation, LPS et GLP-1 pourraient être liés. Ces travaux de thèse portent sur i) les mécanismes moléculaires reliant LPS et GLP-1 et ii) les conséquences physiopathologiques d’une endotoxémie expérimentale lors d’un régime obésogène. Nous montrons, in vitro, ex vivo et in vivo, que les LPS stimulent la sécrétion de GLP-1 par les cellules entéroendocrines via un mécanisme TLR4-dépendant. Les LPS présents dans l’intestin déclenchent cette sécrétion lors de situations pathologiques de dégradation de la muqueuse, faisant du GLP-1 un potentiel marqueur précoce d’altération de la barrière intestinale. Chez des souris sauvages, une endotoxémie expérimentale n’aggrave pas les conséquences métaboliques généralement observées lors d’un régime obésogène, certains paramètres sont même améliorés. Enfin, des souris présentant un défaut de détoxification des LPS nourries avec un régime obésogène prennent plus de masse corporelle que les souris contrôles. Les origines moléculaires de ces différences sont également recherchées. / Obesity and type 2 diabetes are metabolic diseases which have reached epidemic proportions worldwide. These metabolic disorders are related to a low grade inflammation whose molecular origin is still unknown. Previous studies have highlighted the involvement of the gut microbiota and especially components of the cell wall of Gram(-) bacteria: lipopolysaccharides (LPS). We have recently shown that LPS enhance glucagon-like peptide 1 (GLP-1) plasma levels, a hormone which is known to stimulate insulin secretion. Moreover there would be a link between the nutritional qualities of food and LPS plasma levels. Thus diet, LPS and GLP-1 may be closely related. The present work focuses on i) the molecular mechanisms linking LPS to GLP-1 and ii) the pathophysiological consequences of an experimental endotoxemia under obesogenic diet conditions. In vitro, ex vivo and in vivo experiments highlight LPS as potent secretagogues of GLP-1. They are able to induce GLP-1 secretion from enteroendocrine cells through a direct TLR4-dependent mechanism. Luminal LPS trigger GLP-1 secretion only under pathological conditions leading to intestinal mucosal damages. Therefore GLP-1 could be a promising early biomarker for diagnosing gut barrier injuries. Experimentally-induced endotoxemia in wild-type mice does not worsen the usually observed metabolic consequences of an obesogenic diet but rather seems to improve some of them. In addition, under high-fat diet, genetically-engineered mice with a defective LPS detoxification process gain more weight than control mice. The purpose of this thesis is also to disentangle the molecular explanation behind this difference.

Le tractus gastro-intestinal, interface initiale pour la détection, la digestion et l'absorption des nutriments, joue un rôle critique dans la régulation de l'homéostasie énergétique. Les signaux qui proviennent du tractus gastro-intestinal sont nécessaires au contrôle de la fonction intestinale et de la régulation de la prise alimentaire. Les neurones afférents vagaux (NAV) sont une voie importante via laquelle les informations sur les nutriments ingérés atteignent le système nerveux central pour influencer ces deux fonctions. Les NAVs expriment les récepteurs pour la plupart des peptides régulateurs libérés par l'intestin impliqués dans la régulation de la prise alimentaire et du poids corporel. Cette thèse porte sur le rôle de deux peptides de l'intestin, la leptine et le glucagon-like peptide-1 (GLP-1), qui agissent au niveau des NAVs pour inhiber la prise alimentaire. Tout d'abord, nous expliquons le mécanisme d'action du GLP-1 sur les NAVs. La satiété induite par le GLP-1 nécessite un état post-prandial ; les données confirment que le statut nutritionnel régule la localisation du GLP-1R du cytoplasme vers la membrane des cellules neuronales. De plus, la ghréline et son récepteur GHSR1, exprimés par les NAVs, sont impliqués dans la régulation de la translocation du GLP-1R. Deuxièmement, ’utilisation de souris knockout pour le recepteur a la leptine sur les NAVs nous a permis de montrer l’importance de ce recepteur dans la physiopathologie de l’obésité et de l’hyperhagie. En effet, ces souris KO présentent un phénotype obésogène. L'obésité et ses conséquences sur la santé sont des problèmes majeurs de santé dans le monde entier. Les traitements efficaces de prévention ou de l'obésité sont limités. Nos résultats ont apporté des connaissances sur le mécanisme du GLP-1 et sur la signalisation de la leptine au niveau es NAVs. Comprendre la physiologie de la régulation de la prise alimentation est impératif dans le développement des traitements non-invasifs contre l’obésité. / As the initial interface for nutrient sensing, digestion and absorption, the gastrointestinal (GI) tract plays a critical role in the regulation of energy homeostasis. Information that arises from the GI tract is key to normal physiological responses controlling gut function and regulating food intake. Vagal afferent neurons (VAN) are a major pathway by which information about ingested nutrients reaches the central nervous system to influence GI function and food intake behavior. VAN express receptors for many of the regulatory peptides released from the gut that are involved in regulation of food intake and body weight. This dissertation addresses the role of two gut peptides, leptin and glucagon-like peptide-1, acting at the level of VAN, to inhibit food intake. First, the mechanism of action of glucagon-like peptide-1 (GLP-1) on VAN is addressed. GLP-1-induced satiation requires a postprandial state; the data support that feeding changes the localization of GLP-1Rs from the cytoplasm to the neuronal cell membrane. Further, ghrelin and its receptor GHSR1 expressed by VAN is involved in regulating GLP-1 receptor translocation. Second, the importance of leptin receptor expression by VAN in the development of hyperphagia and obesity was demonstrated by selective knockout of the leptin receptor (LepR) in VAN; mice express an obesogenic phenotype. Obesity and its resultant health consequences are a major worldwide health problem. Effective or preventative treatments for obesity are limited. Our findings have filled the gap in our knowledge of the mechanism of GLP-1 and leptin signaling on VAN. Understanding the physiology regulating feeding behavior is imperative in developing non-invasive anti-obesity treatments.

Gut regulatory peptides, such as neuropeptides and incretins, play important roles in hunger, satiety and gastrointestinal motility, and possibly mucosal permeability. Many peptides secreted by myenteric nerves that regulate motor control are also produced in mucosal epithelial cells. Derangements in motility and mucosal permeability occur in many diseases. Current knowledge is fragmentary regarding gut peptide actions and mechanisms in motility and permeability. This thesis aimed to 1) develop probes and methods for gut permeability testing, 2) elucidate the role of neuropeptide S (NPS) in motility and permeability, 3) characterize nitrergic muscle relaxation and 4) characterize mechanisms of glucagon-like peptide 1 (GLP-1) and the drug ROSE-010 (GLP-1 analog) in motility inhibition. A rapid fluorescent permeability test was developed using riboflavin as a transcellular transport probe and the bisboronic acid 4,4'oBBV coupled to the fluorophore HPTS as a sensor for lactulose, a paracellular permeability probe. This yielded a lactulose:riboflavin ratio test. NPS induced muscle relaxation and increased permeability through NO-dependent mechanisms. Organ bath studies revealed that NPS induced NO-dependent muscle relaxation that was tetrodotoxin (TTX) sensitive. In addition to the epithelium, NPS and its receptor NPSR1 localized at myenteric nerves. Circulating NPS was too low to activate NPSR1, indicating NPS uses local autocrine/paracrine mechanisms. Nitrergic signaling inhibition by nitric oxide synthase inhibitor L-NMMA elicited premature duodenojejunal phase III contractions in migrating motility complex (MMC) in humans. L-NMMA shortened MMC cycle length, suppressed phase I and shifted motility towards phase II. Pre-treatment with atropine extended phase II, while ondansetron had no effect. Intestinal contractions were stimulated by L-NMMA, but not TTX. NOS immunoreactivity was detected in the myenteric plexus but not smooth muscle. Food-intake increased motility of human antrum, duodenum and jejunum. GLP-1 and ROSE-010 relaxed bethanechol-induced contractions in muscle strips. Relaxation was blocked by GLP-1 receptor antagonist exendin(9-39) amide, L-NMMA, adenylate cyclase inhibitor 2´5´-dideoxyadenosine or TTX. GLP-1R and GLP-2R were expressed in myenteric neurons, but not muscle. In conclusion, rapid chemistries for permeability were developed while physiological mechanisms of NPS, nitrergic and GLP-1 and ROSE-010 signaling were revealed. In the case of NPS, a tight synchrony between motility and permeability was found.

The syndrome known as Gastric Dilation Air Sacculitis (GDAS) has recently been described by Lumsden et al. (2002) for Chinook salmon (Oncorhynchus tshawytscha, Walbaum), in seawater (SW) culture in New Zealand. The syndrome is characterised by distended abdomens, gastric dilation and air sacculitis, increased feed conversion ratios (FCR) and mortality. Consequently, financial returns on affected stocks are greatly reduced. A study into the epidemiology and physiology of the syndrome was initiated, working with the major aquaculture company, The New Zealand King Salmon Company (NZKS). The study revealed causative factors of GDAS. GDAS was experimentally induced only in saltwater by feeding a commercially manufactured low-cohesion pelleted diet. Control groups were fed a different diet with high physical cohesion. Low-cohesion pellets have previously been associated with a high incidence of GDAS in commercial sea cages. These data implicated osmoregulatory stress and physical properties of the feed in GDAS development. In addition, gastrointestinal (GI) physiology in GDAS -affected and -control fish was characterised. The process of GDAS development in O. tshawytscha is characterised by a loss of smooth muscle tone of the stomach as it distends. Laplace's law (P= 2T/r, where P is the distending pressure, T is the tension in the wall and r is the radius of the cylinder) predicts that unless muscle mass increases, the ability of the stomach wall to contract will be lost and consequently a loss of GI motor function will result. Therefore, GI circular smooth muscle integrity in terms of (1) stimulated and maximal contractility, (2) osmoregulatory ability of the intestine and the (3) control of the GI system was studied in pathologically affected (+ve) and unaffected (-ve) smolt. Affected fish showed changes in GI circular smooth muscle function and osmoregulatory dysfunction. Feeding different diets induced distinct gastric evacuation patterns. The intestinal brake hypothesis is presented and argued to be the probable mechanism for GDAS development. GDAS (+ve) serum showed the presence of factors capable of contracting gut smooth muscle. In addition, potential humoral mediators of the intestinal brake in fish were investigated.