Dissertations / Theses on the topic 'Leptin signaling'

Le traitement de l’obésité est devenu un problème majeur de santé publique aussi bien dans les pays industrialisés que dans les pays en voie de développement. La leptine est une hormone clé dans le contrôle de l’homéostasie énergétique et glucidique en agissant au niveau du noyau arqué de l’hypothalamus (ARC). La résistance à la leptine au niveau de ce noyau hypothalamique contribue au développement de l’obésité. Par conséquent, prévenir son installation et lever cette résistance constitue un enjeu thérapeutique majeur. Notre laboratoire à récemment mis en évidence que la protéine Endospanine 1 est un régulateur négatif des fonctions du récepteur de la leptine (OB-R). En effet, cette protéine, exprimée à partir du même gène qu’OB-R, est capable d’interagir avec ce récepteur et de le retenir dans le compartiment intracellulaire. L’extinction d’Endospanine 1 spécifiquement au niveau de l’ARC prévient l’installation d’une obésité induite par un régime gras. Ces données démontrent donc, in vivo, l’importance de cette protéine dans la régulation des fonctions d’OB-R. Suite a ces résultats prometteurs, l’objectif premier de ma thèse a été d’approfondir nos connaissances sur le rôle joué, in vitro et in vivo, par Endospanine 1, ainsi que par son homologue Endospanine 2, dans la régulation des fonctions d’OB-R. Le second objectif de ce travail de thèse a été d’identifier de nouveaux composés capables de sensibiliser la réponse à la leptine chez les individus obèses. Ce travail a permis de montré que des souris rendues obèses par un régime gras présentaient une surexpression d’Endospanine 1 dans l’ARC suggérant ainsi une potentielle implication de cette protéine dans le développement de la résistance à la leptine. D’autre part, nous avons démontré que l’extinction d’Endospanine 1, dans l’ARC, permet de prévenir l’installation d’une obésité et de la corriger chez des souris obèses et conduit à une altération de la sécrétion d’insuline par le pancréas. Cet effet double sur le poids corporel et sur la glycémie pourrait être expliqué par l’effet différentiel de l’extinction d’Endospanine 1 sur l’activation des voies STAT3 et PI3K/AKT. En effet, en absence d’Endospanine 1 la voie STAT3 est suractivée tandis que la voie PI3K est inhibée. De façon surprenante, l’extinction d’Endospanine 2, second membre de la famille des Endospanines, inhibe de façon drastique l’activation de la voie STAT3 suggérant que ces protéines pourraient jouer des rôles différents dans la régulation des fonctions d’OB-R. Ce travail a également permis de décrire, pour la première fois, les conséquences d’une déficience en Endospanine 1 chez l’Homme. Nos données suggèrent qu’Endospanine 1 ne régule pas les fonctions d’autres protéines, définissant ainsi Endospanine 1 comme une cible thérapeutique hautement spécifique dans la correction de la signalisation leptine.La dernière partie de cette étude a consisté en l’identification de nouveaux composés capables d’activer OB-R ou d’augmenter son expression de surface. Deux tests de criblages ont permis d’identifier de telles molécules. Après validation et caractérisation, de tels composés pourraient être utilisés comme outils thérapeutique afin de restaurer la sensibilité à la leptine perdue chez les individus obèses
Obesity is one of the greatest current public health challenges, not only in industrialized countries but also in developing countries. The hypothalamic arcuate nucleus (ARC) is a major integration centre for energy and glucose homeostasis that responds to peripheral hormones such as leptin. Resistance to leptin in the ARC is an important component of obesity development and its prevention or reversal represents a major therapeutic goal. Our laboratory recently described endospanin 1 as a negative regulator of the leptin receptor (OB-R) that by interacting with OB-R retains the receptor inside the cell. Interestingly, both proteins are expressed from the same promoter. Silencing of endospanin 1 in the ARC prevented the development of diet-induced obesity demonstrating the importance of this protein on OB-R in vivo function.Based on these encouraging findings the first aim of this thesis was to extend our understanding of the in vitro and in vivo role of endospanin 1 and its homologue, endospanin 2, on OB-R function. The second aim consisted in the identification of chemical compounds able to sensitize the leptin response in obese patients. We show here that endospanin 1 is up-regulated in the ARC of obese mice suggesting a potential contribution of this protein to the development of leptin resistance. Its silencing in the ARC of naïve and obese mice reverses obesity development and impairs pancreatic insulin secretion. This dual effect correlates with the differential effect of endospanin 1 on OB-R signaling, inhibition of the STAT3 pathway and activation of the AKT pathway. Intriguingly, endospanin 2, the second member of the endospanin family, promotes efficient STAT3 activation suggesting differential roles of both endospanins on OB-R function.We characterized an obese patient carrying a homozygous deletion in the chromosomal 1p31.3 region coding for endospanin 1. This is the first report defining the consequences of endospanin 1-deficiency in humans. Our data suggest that endospanin 1 has no major OB-R-independent functions thus defining endospanin 1 as an attractive and highly specific therapeutic target for the improvement of impaired leptin signaling.In the last part of the thesis two screening assays were developed to identify compounds that either activate OB-R or promote its cell surface expression. Primary screens were successfully performed for both assays and positive hits identified. Validated hits might be useful to resensitize the impaired leptin response in obese patients

Extreme deviations from what is considered normal body weight, from anorexia to obesity, have been linked to reduced reproductive function in females. Discovered in 1994, leptin is a signaling hormone released from adipose tissue to mediate satiety effects in the hypothalamus. Leptin secretion is directly proportional to amount of body fat and evidence has accumulated that leptin and its receptor (Lepr) are found in a variety of tissues including granulosa cells (GCs) of the ovary. Thus, leptin through its receptor may play a role in reproductive function in females. Many studies have examined the effects of Lepr in GCs and the ovary, however the results are contradictory and all have been in vitro. Here we present the first in vivo study to examine the role of Lepr in GCs during follicular development and ovulation. Immature superovulated mice were used in all studies and GCs collected by follicle puncture. We first determined the expression profiles of Lepr isoforms (LeprA, LeprB) during follicular and luteal development along with leptin-related signaling molecules and targets. We also analyzed transcription factors potentially regulating Lepr expression in GCs. To examine the response of GCs to leptin in vivo, leptin hormone was administered at various times of follicular and luteal development. Lastly, we blocked Lepr action using a Lepr antagonist (SMLA) and determined its effects on ovulation. LeprA and LeprB were upregulated at 4h post- human chorionic gonadotropin (hCG) with LeprA being the most abundant isoform showing a 23-fold increase from 0 to 4h post-hCG. Leptin was upregulated at the same time and Lepr signaling molecules: signal transducer and activator of transcription 3 (Stat3), and suppressor of cytokine signaling 3 (Socs3), were upregulated just after Lepr induction at 7 and 12h post-hCG, respectively. CCAAT/enhancer-binding protein beta (Cebpb), which was induced at 1h post-hCG, was shown to associate with the Lepr promoter and thus regulate Lepr expression. Early growth response protein 1 (Egr1) protein and mRNA data revealed it to be another potential regulatory transcription factor with upregulation at 1h post-hCG, just prior to Lepr upregulation. Thus, the mRNA profiles of genes examined provide evidence of a role for Lepr during the periovulatory period. This was further confirmed as the in vivo response of GCs to a physiological dose of leptin was enhanced at 6h post-hCG evidenced by phosphorylation of mitogen-activated protein kinase (Mapk) and Stat3 proteins; however showed no change during the early follicular or luteal periods. Leptin treatment also increased expression of ovulation genes: a disintegrin and metalloproteinase with thrombospondin motifs 1 (Adamts1), programmed cell death 1 (Pdcd1), and Egr1. Antagonizing Lepr action reduced ovulation rate by 60% in SMLA-treated animals. This reduction appeared, at least in part, to be due to deregulated gene expression of Adamts10, Adamts19, Hyaluronan synthase 2 (Has2), amphiregulin (Areg), Pentraxin-related protein (Ptx3), and Forkhead box protein O1 (Foxo1). Overall, the results of this study provide molecular mechanisms for Lepr induction and signaling in GCs. In addition, it provides evidence that leptin and Lepr play a positive role during ovulation and are thus essential for optimal female fertility.
Les écarts extrêmes de poids par rapport à ce qui est considéré comme un poids normal, telles que l'anorexie et l'obésité, sont liées à des problèmes de la fonction reproductrice chez la femme. Découverte en 1994, la leptine est une hormone sécrétée par le tissu adipeux dans le but d'informer l'hypothalamus sur l'état de satiété de l'organisme. La libération de leptine est directement proportionnelle à la quantité de tissu adipeux et la présence de l'hormone et de son récepteur (Lepr) a été montrée dans différents tissus incluant les cellules de la granulosa des ovaires. Par conséquent, la leptine, via son récepteur, joue un rôle dans la fonction reproductrice de la femme. De nombreuses études ont étudié les effets de Lepr dans les cellules de la granulosa et dans l'ovaire, mais elles ont toutes été réalisées in vitro et les résultats sont contradictoires. Nous présentons ici la première étude in vivo dans le but d'examiner le rôle de Lepr dans les cellules de la granulosa pendant le développement folliculaire et l'ovulation. Des souris immature produisant un grand nombre d'ovocytes ont été utilisées dans toutes nos expériences et les cellules de la granulosa ont été collectées par ponction folliculaire. Les profils d'expression des isoformes de Lepr (LeprA et LeprB) durant les développements folliculaire et lutéal ont été d'abord déterminés, ainsi que ceux des molécules de la voie de signalisation de la leptine et leurs cibles. Les facteurs de transcription régulant potentiellement l'expression de Lepr dans les cellules de la granulosa ont aussi été analysés. Pour évaluer la réponse des cellules de la granulosa à la leptine in vivo, l'hormone leptine a été administrée à différents moments des développements folliculaire et lutéal. Enfin, le récepteur Lepr a été bloqué grâce à l'utilisation d'un antagoniste de Lepr (SMLA) et les effets de ce blocage sur l'ovulation ont été analysés. L'expression de LeprA et LeprB ont augmenté 4h après administration d'hCG, LeprA étant l'isoforme la plus abondante et présentant une expression 23 fois plus importante de 0 à 4h post-hCG. L'expression de la leptine a augmenté durant le même temps ainsi que celle des molécules de la voie de signalisation de Lepr, Stat3 et Socs3, juste après l'induction de Lepr, respectivement 7h et 12h post-hCG. Cebpb, qui a été induit 1h post-hCG, a été identifié comme étant associé au promoteur de Lepr et donc comme étant un régulateur de l'expression de Lepr. Les données concernant la protéine Egr1 et ses ARNm suggèrent qu'il peut s'agir d'un autre potentiel facteur de transcription régulant l'expression de Lepr, notamment en raison d'une augmentation de son expression 1h post-hCG, juste avant l'augmentation de l'expression de Lepr. Les profils d'ARNm des gènes examinés ont donc fourni la preuve du rôle de Lepr durant la période périovulatoire. Ceci a été ensuite confirmé par l'augmentation de la réponse des cellules de la granulosa in vivo suite à une dose physiologique de leptine 6h post-hCG , mise en évidence par la phosphorylation des protéines Mapk et Stat3. Le traitement avec la leptine a aussi accru l'expression des gènes impliqués dans l'ovulation Adamts1, Pdcd1 et Egr1. Le blocage de l'action de Lepr a réduit le taux d'ovulation de 60% chez les animaux traités avec SMLA. Cette réduction apparaît être due, au moins en partie, à la dérégulation de l'expression des gènes de Adamts10, Adamts19, Has2, Areg, Ptx3, et Foxo1. En conclusion, les résultats de cette étude éclairent les mécanismes moléculaires de l'induction du récepteur Lepr ainsi que de la voie de signalisation qui lui est associée dans les cellules de la granulosa. Pour finir, cette étude fournit des preuves concernant le rôle positif joué par la leptine et Lepr durant l'ovulation, ce qui est essentiel pour optimiser la fertilité de la femme.

Obesity over the last several has become a major health concern in our country as well as the world. Obesity is also one of the risk factors which lead to several inflammatory complications such as diabetes, artherosclerosis, etc. Two leading factors involved in the causes of inflammatory complications include leptin and low dose endotoxin lipopolysaccharide (LPS). However, the mechanism underlying the involvement of these two mediators is not clearly understood. The purpose of this study is to understand the mechanism underlying inflammatory complications caused by leptin and low dose endotoxin most recently coined metabolic endotoxemia. Interleukin-Receptor Associated Kinase 1 (IRAK-1) is an intracellular signaling component shown to activate NFκB which leads to the induction of proinflammatory mediators. Deletion of IRAK-1 in mice has beneficial effects in alleviating inflammatory complications and human variations in IRAK-1 gene are correlated with higher risks for inflammatory diseases. Therefore, we hypothesized that IRAK-1 is critically involved for the induction of proinflammatory mediators induced by leptin and low dose LPS. IL-6 mRNA levels were measured in THP-1 (human monocytic cells) and wild type and IRAK-deficient bone marrow derived macrophages (BMDM) challenged with different combinations of leptin and LPS. Data shows that leptin alone will not induce inflammatory mediators. However, increased induction of IL-6 was observed in a synergistic manner involving both LPS and leptin in an IRAK-1 dependent manner causing a robust inflammatory response. With regard to the effect of low dose LPS, we observed that human monocytic cells treated with low concentrations of LPS showed a mild yet sustained induction of proinflammatory cytokines, which is contrast to the robust and transient induction of cytokines by a high dose LPS. To further determine the molecular mechanisms, we measured several key signaling molecules that include IRAK-1, IKKepsilon, and C/EBPdelta. Our study revealed a novel mechanism that appears to be distinct from the traditional NFï «B pathway responsible for the effect of low dose LPS.
Ph. D.

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

A restrição de sal na dieta está associada com resistência à ação da insulina e obesidade. O mecanismo molecular pelo qual a dieta hipossódica (HO) pode induzir resistência à insulina e obesidade não está totalmente compreendido. O objetivo do presente estudo foi avaliar a influência da ingestão crônica de sal sobre o peso corporal (PC), sinalização da insulina no fígado, músculo e tecido adiposo branco (TAB) e sua associação com adiposidade e resistência à insulina. Com esta finalidade, ratos Wistar foram alimentados com dieta HO, normossódica (NR) ou hipersódica (HR) desde o desmame. O PC foi avaliado desde o desmame. Ao completarem 12 semanas de vida, foram avaliados pressão arterial, balanço energético, consumo de ração, glicemia, angiotensina II (ANGIO II) plasmática e perfil hormonal. A atividade motora espontânea foi estudada em ratos com 8 e 12 semanas. A sensibilidade à insulina foi analisada pelo índice de HOMA. A expressão da proteína desacopladora mitocondrial 1 (UPC-1) foi quantificada no tecido adiposo marrom (TAM) e o conteúdo de ANGIO II no TAM, TAB e hipotálamo. As etapas iniciais da sinalização da insulina foram avaliadas por imunoprecipitação e immunoblotting das proteínas envolvidas como o receptor da insulina (IR), substrato 1 e 2 do IR (IRS-1 e IRS-2), enzima fosfatidilinositol 3 – quinase (PI-3q), proteína quinase B (Akt/PKB), ativação da proteína c-jun NH2-terminal quinase (JNK) e fosforilação em serina 307 do IRS-1. O PC no desmame foi semelhante entre os grupos de dieta. No entanto, na idade adulta os ratos em dieta HO apresentaram maior PC, adiposidade visceral, glicemia e insulinemia de jejum, concentração de ANGIO II plasmática e aumento do conteúdo de ANGIO II no TAM. Por outro lado, nestes mesmos animais a dieta HO diminuiu o consumo de ração, o gasto energético, a expressão da proteína UCP-1, adiponectina plasmática e o conteúdo de ANGIO II no TAB. A atividade motora não foi diferente entre os grupos estudados. A dieta HO diminuiu a via IR/PI-3q/Akt/Foxo1 de sinalização da insulina no fígado e músculo. Por outro lado, parte desta via (IRS-2/Akt/Foxo1) mostrou-se aumentada no TAB. No fígado e músculo houve um aumento da fosforilação da proteína JNK associada com maior fosforilação do IRS-1ser307 no grupo HO. Em conclusão, a restrição ou sobrecarga crônica de sal altera a evolução ponderal associada com modificações no balanço energético e no perfil hormonal na idade adulta. A resistência à insulina induzida pela dieta HO é tecido-específico e foi acompanhada por uma ativação da proteína JNK e um aumento da fosforilação dos resíduos de serina 307 do IRS-1.
Restriction of sodium chloride intake has been associated with insulin resistance (INS-R) and obesity. The molecular mechanisms by which the low salt diet (LSD) can induce INS-R and obesity have not yet been established.The aim of the present study was to evaluate the influences of salt intake on body weight (BW) and on insulin signaling in liver, muscle and white adipose tissue (WAT). Wistar rats were fed a LSD, normal (NSD), or high (HSD) salt diet since weaning. At 12 weeks of age, BW, blood pressure(BP),energy balance, food intake, plasma glucose and angiotesin II (ANGIO II), and hormonal profile were evaluated. Afterward, motor activity, HOMA index, uncoupling protein 1 expression (UCP-1) and tissue adipose ANGIO II content was determined. The early steps of insulin signaling (IR: insulin receptor, IRS-1 and IRS-2: IR substrate 1 and 2, PI-3K: phosphatidylinositol 3-kinase), Akt (protein kinase B) phosphorylation, JNK (c-jun NH2-terminal kinase) activation and IRS-1ser307 (serine 307 of IRS-1) phosphorylation were evaluated by immunoprecipitation and immunoblotting. LSD increased BW, visceral adiposity, blood glucose, insulin, leptin, plasma ANGIO II and its content in BAT. Otherwise, LSD decreased food intake, energy expenditure, UCP-1 expression, adiponectin and ANGIO II content in WAT. Motor activity was not influenced by the dietary salt content. In LSD, a decreasing in IR/PI-3K/Akt/Foxo1 was observed in liver and muscle and an increase in this pathway was showed in adipose tissue. JNK activity and IRS-1ser307 phosphorylation were higher in liver and muscle. In conclusion, LSD induced obesity and insulin resistance due to changes in energy expenditure, SRA and insulin signaling. The INS-R is tissuespecific and is accompanied by JNK activation and IRS-1ser307 phosphorylation.

L’insuline et la sérotonine (5-HT) sont deux acteurs majeurs du maintien de l’homéostasie énergétique, fonction placée sous le contrôle de l’hypothalamus. En ciblant cette région, l’insuline remplit de nombreuses fonctions métaboliques via l’activation de la voie PI3K/Akt. La 5-HT exercent des effets biologiques similaires mais les voies de signalisation impliquées dans ces processus étaient jusqu’alors mal connues. De plus, il avait été démontré que la 5-HT est capable d’activer la voie PI3K/Akt/GSK3β dans l’hippocampe, mécanisme sous-tendant potentiellement les effets antidépresseurs du neurotransmetteur. Les principaux objectifs de cette thèse étaient d’étudier 1/ l’activation de la voie PI3K/Akt par la 5-HT dans l’hypothalamus de rats diabétiques (modèle Goto-Kakizaki) et chercher un potentiel dialogue avec l’insuline and 2/ les mécanismes sous-tendant l’induction de la dépression par une alimentation hyperlipidique, par l’analyse de la phosphorylation d’Akt et GSK3β sous l’action de l’insuline, de la leptine et de la 5-HT dans l’hippocampe de rat.Ici on montre que 1/ la 5-HT stimule la voie PI3K/Akt dans l’hypothalamus et que la phosphorylation d’Akt induite par la 5-HT est atténuée dans des conditions d’insulino-résistance, suggérant l’existence d’un dialogue entre les voies de signalisation de l’insuline et de la 5-HT. Par ailleurs, nos résultats indiquent qu’une alimentation hyperlipidique induit un comportement dépressif réversible chez le rat, qui pourrait impliquer la voie PI3K/Akt/GSK3β dans les neurones subgranulaires du gyrus denté. La mise en évidence d’un dialogue entre les voies de signalisation de la 5-HT, de la leptine et de l’insuline au niveau central enrichit nos connaissances sur le rôle de ces facteurs dans la régulation de l’homéostasie énergétique et de l’humeur, et propose un lien moléculaire entre diabète de type 2, obésité et dépression
Insulin and serotonin (5-HT) are two key players in the maintenance of energy homeostasis which is controlled by the hypothalamus. In this brain region, insulin mediates numerous metabolic effects via the activation of the PI3K/Akt signaling pathway. 5-HT exerts similar biological properties by acting in the hypothalamus but the signaling pathways accountable for these effects are still unclear. Moreover, it has been reported that 5-HT induces the activation of the PI3K/Akt pathway in the hippocampus and the inhibition of GSK3β, suggesting this action as a potential mechanism for the antidepressant effects of this neurotransmitter.The main objectives of this thesis were to study 1/ the serotonin-induced activation of the PI3K/Akt in the hypothalamus of wild type and diabetic rats (Goto-Kakizaki model) and search a potential cross-talk with insulin and, 2/ the mechanisms underlying the high-fat diet induced depression by investigating the role of the phosphorylation of Akt and GSK3β by 5-HT, insulin and leptin in the hippocampus of rats.Here, we show that 5-HT triggers the PI3K/Akt signaling pathway in the rat hypothalamus, and that this activation is attenuated in insulin-resistant conditions, suggesting a cross-talk between insulin and 5-HT. Moreover, we reported that high-fat diet feeding induces a reversible depressive-like behavior, which may involve the PI3K/Akt/GSK3β pathway in subgranular neurons of the dentate gyrus. In conclusion, the activation of the PI3K/Akt pathway and its target GSK3β by 5-HT in the hypothalamus and in the dentate gyrus, respectively, can be impaired in insulin-/leptin-resistant states, which may underlie a link between metabolic diseases and depression

L’obesitat és una patologia àmpliament estesa per tot el món i un dels principals factors relacionats amb altres malalties cròniques. Les teràpies convencionals utilitzades per prevenir o pal·liar l’obesitat, basades en l’exercici i en la reducció del consum d’aliments molt energètics, són inefectives. En aquest sentit, l’ús de compostos bioactius com són els polifenols, metabòlits secundaris de les plantes amb un ampli espectre d’efectes beneficiosos per la salut, es presenta com a una estratègia innovadora per combatre l’obesitat. La leptina és una hormona que es secreta proporcionalment a la quantitat d’adipòcits i que actua principalment en el sistema nerviós central controlant el balanç energètic. En aquest procés, el transport de la leptina a través de la barrera hematoencefàlica és especialment important. També està implicada en la regulació de l’homeòstasi perifèrica, principalment modulant el metabolisme lipídic i dels carbohidrats. L’obesitat està relacionada amb un deteriorament de l’acció de la leptina, la resistència a la leptina, causant hiperleptinèmia i un increment en la ingesta energètica. En aquest context, l’objectiu de la tesi és identificar compostos fenòlics amb la capacitat de restaurar la condició obesitat-resistència a la leptina causada en teixits perifèrics (fetge, múscul esquelètic i teixit adipós blanc de l’epidídim) i que puguin incrementar el transport de la leptina a través de la barrera hematoencefàlica. Els nostres resultats demostren els efectes del resveratrol i els seus metabòlits actuant en la via perifèrica de senyalització de la leptina, reduint l’acumulació de greix corporal en un model de rata obesa. A més a més, el resveratrol restaura la sensibilitat a la leptina en un model esteatòtic de cèl·lules cancerígenes hepàtiques humanes, incrementant el contingut del receptor de la leptina. Finalment, s’ha descrit la capacitat de diferents compostos fenòlics en incrementar el contingut del receptor de la leptina i en protegir contra un dany induït per citocines pro-inflamatòries en cèl·lules endotelials de cervell de rata. Aquesta investigació proporciona una informació innovadora que pot ser útil per a la indústria d’aliments funcionals, identificant compostos bioactius que poden ser utilitzats per a tractar potencialment l’obesitat i les seves patologies associades.
La obesidad es una patología extendida por todo el mundo y uno de los principales factores relacionados con otras enfermedades crónicas. Las terapias convencionales utilizadas para prevenir o paliar la obesidad, ejercicio y reducción del consumo de alimentos muy energéticos, son inefectivas. En este sentido, el uso de compuestos bioactivos como son los polifenoles, metabolitos secundarios de las plantas con un amplio espectro de beneficios para la salud, se presenta como una estrategia innovadora para combatir la obesidad. La leptina es una hormona secretada proporcionalmente a la cantidad de adipocitos y que actúa principalmente en el sistema nervioso central controlando el balance energético. En este proceso, el transporte de la leptina a través de la barrera hematoencefálica es importante. También está implicada en la regulación de la homeóstasis periférica, modulando el metabolismo lipídico y de los carbohidratos. La obesidad está relacionada con un deterioro de la acción de la leptina, la resistencia a la leptina, causando hiperleptinemia y un incremento en la ingesta energética. En este contexto, el objetivo de la tesis es identificar compuestos fenólicos con la capacidad de restaurar la condición de obesidad-resistencia a la leptina causada en tejidos periféricos (hígado, músculo esquelético y tejido adiposo epididimal) y que puedan incrementar el transporte de la leptina a través de la barrera hematoencefálica. Nuestros resultados demuestran los efectos del resveratrol y sus metabolitos actuando en la vía periférica de señalización de la leptina, disminuyendo la grasa corporal en un modelo de rata obesa. Además, el resveratrol restaura la sensibilidad a la leptina en un modelo esteatótico de células cancerígenas hepáticas humanas, incrementando el contenido del receptor de la leptina. Finalmente, se ha descrito la capacidad de diferentes compuestos fenólicos para incrementar el contenido del receptor de la leptina y la capacidad de éstos en proteger contra el daño inducido por citoquinas pro-inflamatorias en células endoteliales de cerebro de rata. Esta investigación proporciona una información innovadora que puede ser útil para la industria de alimentos funcionales, identificando compuestos bioactivos que pueden ser usados para tratar potencialmente la obesidad y sus patologías asociadas.
Obesity is a current and worldwide extended problem and one of the main factors related with other chronic pathologies. Conventional therapies, normally based on increasing the exercise and reducing the consumption of energy-dense food used to prevent or palliate obesity, are ineffective. In this sense, the use of bioactive compounds as polyphenols, a group of plant secondary metabolites with a wide range of beneficial healthy effects, arises as a novel strategy to combat obesity and its related pathologies. Leptin is a key hormone secreted proportionally by the amount of adipocytes that acts primarily in the central nervous system controlling the energy balance. In this process, leptin transport across the blood-brain barrier is especially important. In addition, leptin is implicated in the regulation of peripheral homeostasis, mainly modulating the lipid and carbohydrate metabolism, in organs such as liver, muscle and white adipose tissue. However, obesity is related with an impaired action of leptin, namely leptin resistance, causing hyperleptinemia and an increase in the energy intake. In this context, the aim of this thesis is to identify phenolic compounds with the capacity to restore the obesogenic-leptin resistance condition caused in peripheral tissues (liver, skeletal muscle and epididymal white adipose tissue) and to increase the leptin transport across the blood-brain barrier. Our results demonstrate the effects of resveratrol and its metabolites acting in the peripheral leptin signalling pathway on reducing body fat accumulation in an obesogenic rat model. Moreover, resveratrol restores the leptin sensitivity in a palmitate-induced model of steatotic human hepatocellular carcinoma cell line by increasing the leptin receptor content. Finally, the capacity of different phenolic compounds to increase the leptin receptor content and to protect against pro-inflammatory cytokine-induced damage in rat brain endothelial cells is described. This research provides novel information that can be useful for the functional food industry identifying bioactive compounds that can be used to potentially treat obesity and its related pathologies.

Oscillations in cytoplasmic Ca2+ concentration ([Ca2+]i) is the key signal in glucose-stimulated β-cells governing pulsatile insulin release. The glucose response of mouse β-cells is often manifested as slow oscillations and rapid transients of [Ca2+] i. In the present study, microfluorometric technique was used to evaluate the role of amino acids, glucagon, ryanodine and caffeine on the generation and maintenance of [Ca2+] i oscillations and transients in individual murine β-cells and isolated mouse pancreatic islets. The amino acids glycine, alanine and arginine, at around their physiological concentrations, transformed the glucose-induced slow oscillations of [Ca2+] i in isolated mouse β-cells into sustained elevation. Increased Ca2+ entry promoted the reappearance of the slow [Ca2+] i oscillations. The [Ca2+] i oscillations were more resistant to amino acid transformation in intact islets, supporting the idea that cellular interactions are important for maintaining the oscillatory activity. Individual rat β-cells responded to glucose stimulation with slow [Ca2+] i oscillations due to periodic entry of Ca2+ as well as with transients evoked by mobilization of intracellular stores. The [Ca2+] i oscillations in rat β-cells had a slightly lower frequency than those in mouse β-cells and were more easily transformed into sustained elevation in the presence of glucagon or caffeine. The transients of [Ca2+] i were more common in rat than in mouse β-cells and often appeared in synchrony also in cells lacking physical contact. Depolarization enhanced the generation of [Ca2+] i transients. In accordance with the idea that β-cells have functionally active ryanodine receptors, it was found that ryanodine sometimes restored oscillatory activity abolished by caffeine. However, the IP3 receptors are the major Ca2+ release channels both in β-cells from rats and mice. Single β-cells from ob/ob mice did not differ from those of lean controls with regard to frequency, amplitudes and half-widths of the slow [Ca2+] i oscillations. Nevertheless, there was an excessive firing of [Ca2+] i transients in the β-cells from the ob/ob mice, which was suppressed by leptin at close to physiological concentrations. The enhanced firing of [Ca2+] i transients in ob/ob mouse β-cells may be due to the absence of leptin and mediated by activation of the phospholipase C signaling pathway.

Oscillations in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) is the key signal in glucose-stimulated β-cells governing pulsatile insulin release. The glucose response of mouse β-cells is often manifested as slow oscillations and rapid transients of [Ca²⁺] _i. In the present study, microfluorometric technique was used to evaluate the role of amino acids, glucagon, ryanodine and caffeine on the generation and maintenance of [Ca²⁺] _i oscillations and transients in individual murine β-cells and isolated mouse pancreatic islets. The amino acids glycine, alanine and arginine, at around their physiological concentrations, transformed the glucose-induced slow oscillations of [Ca²⁺] _i in isolated mouse β-cells into sustained elevation. Increased Ca²⁺ entry promoted the reappearance of the slow [Ca²⁺] _i oscillations. The [Ca²⁺] _i oscillations were more resistant to amino acid transformation in intact islets, supporting the idea that cellular interactions are important for maintaining the oscillatory activity. Individual rat β-cells responded to glucose stimulation with slow [Ca²⁺] _i oscillations due to periodic entry of Ca²⁺ as well as with transients evoked by mobilization of intracellular stores. The [Ca²⁺] _i oscillations in rat β-cells had a slightly lower frequency than those in mouse β-cells and were more easily transformed into sustained elevation in the presence of glucagon or caffeine. The transients of [Ca²⁺] _i were more common in rat than in mouse β-cells and often appeared in synchrony also in cells lacking physical contact. Depolarization enhanced the generation of [Ca²⁺] _i transients. In accordance with the idea that β-cells have functionally active ryanodine receptors, it was found that ryanodine sometimes restored oscillatory activity abolished by caffeine. However, the IP3 receptors are the major Ca²⁺ release channels both in β-cells from rats and mice. Single β-cells from ob/ob mice did not differ from those of lean controls with regard to frequency, amplitudes and half-widths of the slow [Ca²⁺] _i oscillations. Nevertheless, there was an excessive firing of [Ca²⁺] _i transients in the β-cells from the ob/ob mice, which was suppressed by leptin at close to physiological concentrations. The enhanced firing of [Ca²⁺] _i transients in ob/ob mouse β-cells may be due to the absence of leptin and mediated by activation of the phospholipase C signaling pathway.

Incidences of obesity and type 2 diabetes have risen worldwide at alarming rates. While there is an undeniable correlation between obesity and type 2 diabetes, a clear mechanistic link between these two conditions has not been fully elucidated. The adipocyte-derived hormone leptin may play a role linking obesity and type 2 diabetes. Leptin can regulate body weight through its effects on the brain to decrease food intake and increase energy expenditure, but these effects are disrupted in obesity. Interestingly, leptin also has effects on glucose and lipid metabolism independent of its effects on body weight, so it is possible that disrupted leptin signalling during obesity can also perturb glucose and lipid metabolism, leading to symptoms associated with type 2 diabetes. Since the liver plays a critical role in integrating and controlling glucose and lipid metabolism, it was hypothesized that leptin resistance in the liver could play a role in the development of diabetic symptoms. To investigate this hypothesis, three complementary mouse models were used to help identify the role of leptin signalling specifically in the liver. It was found that in lean mice, hepatic leptin resistance results in increased insulin sensitivity in the liver, leading to reduced hepatic glucose output but also increased lipid accumulation and secretion of larger, more triglyceride-rich very low density lipoprotein (VLDL) particles without an increase in total plasma triglycerides. In obese, hyperinsulinemic mice lacking hepatic leptin signalling, the effects of lost leptin signalling on triglyceride metabolism were exacerbated, resulting in decreased triglyceride clearance and elevated plasma triglycerides compared to controls. These effects on plasma triglycerides were reversed when hepatic leptin signalling was restored in a mouse model of total leptin resistance. Collectively, these data reveal a possible role for hepatic leptin resistance in the development of diabetic symptoms during obesity.

There is a wealth of evidence to support the anti-inflammatory properties of the prototypical second messenger cyclic-AMP (cAMP), notably with regard to endothelial function. Many studies have shown that cAMP can limit vascular permeability by enhancing barrier function and reducing pro-inflammatory effects of cytokines. Although the protective effects of cAMP elevation on limiting endothelial dysfunction have been well documented, the exact molecular mechanisms remain unclear. Using two endothelial cell types, namely human umbilical vein endothelial cells (HUVECs) and a novel human endothelial angiosarcoma-derived cell line (AS-M), this study has further characterised the cAMP-mediated inhibitory mechanism on the signalling pathways of two cytokines; interleukin-6 (IL-6) and leptin. Both cytokines have been implicated in the regulation of the immune response and both have been shown to play important pathological roles in various inflammatory diseases. In preliminary studies, cAMP elevation was shown to induce suppressor of cytokine signalling 3 (SOCS3) in HUVECs. Further investigation of this SOCS protein in the context of IL-6 and leptin signalling in endothelial cells would be of interest in terms of possibly elucidating the molecular mechanisms underlying the protective effects of cAMP. Results from this study demonstrated a cAMP-mediated inhibition of soluble IL-6Rα (sIL-6R)/IL-6-stimulated extracellular regulated mitogen-activated protein kinase 1, 2 (ERK1,2) and signal transducer and activator of transcription 3 (STAT3) activation in HUVECs, which was independent of cAMP-dependent protein kinase A (PKA). Instead, results demonstrated the involvement of the other major cAMP sensor; exchange protein activated by cAMP 1 (Epac1). Moreover, this inhibition was shown to be SOCS3-dependent. There also appeared to be a requirement for ERK1,2 activation in the cAMP-mediated inhibition of sIL-6R/IL-6-stimulated STAT3 activation in HUVECs. In contrast to these findings, cAMP-mediated inhibition of leptin-stimulated STAT3 activation in HUVECs was shown to occur via a SOCS3-independent mechanism. The responses to cAMP elevation on sIL-6R/IL-6- and leptin-stimulated ERK1,2 activation in AS-Ms were variable, since basal levels of ERK1,2 activation were high. Furthermore, the responses to cAMP elevation on sIL-6R/IL-6- and leptin-stimulated STAT3 activation in AS-Ms were either very modest or showed no effect, respectively. SOCS3 was not shown to be involved in the cAMP-mediated inhibition of sIL-6R/IL-6-stimulated ERK1,2 and STAT3 activation in AS-Ms. In conclusion, this study further characterised the cAMP-mediated inhibitory mechanism in HUVECs and AS-Ms, with a particular focus on the ERK1,2 signalling pathway of IL-6 and leptin. Despite varying results between both cell types, this study also identified AS-Ms as a useful and tractable cell model to study in the context of endothelial biology. Thus, a potentially new pathway has been identified which inhibits cytokine receptor activation of ERK1,2 and STAT3 in endothelial cells. A better understanding of this mechanism could contribute towards new therapeutics in the area of chronic inflammatory diseases, such as atheroscleriosis.

La leptine circule en proportion de la masse graisseuse du corps et la transduction de son signal à travers la forme longue de son récepteur via un certain nombre de voies neurales , y compris MAPK, PI3-K ,AMPK et JAK2 - STAT3 . Il faut noter que STAT3 constitue une voie clée au récepteur de la leptine par laquelle la leptine module l'expression des gènes impliqués dans la régulation du bilan énergétique. La plupart des recherches ont porté sur la fonction du récepteur de la leptine au sein de l' hypothalamus, en particulier la fonction du récepteur de la leptine dans le noyau arqué. Toutefois, les récepteurs de la leptine sont également exprimés sur les neurones dopaminergiques de l'aire tégmentale ventrale et la leptine agit sur cette région du cerveau pour influencer la prise alimentaire, la motivation, la locomotion, l'anxiété et la transmission de la dopamine. De plus, la leptine active la STAT3 dans les dopaminergiques et GABAergiques populations neuronales. Bien que ces résultats contribuent à notre compréhension des multiples actions de la leptine dans le système nerveux central, il reste à résoudre les cellules et la signalisation du récepteur de la leptine qui sont responsables des effets neurocomportementaux de la leptine dans le mésencéphale. Visant à déterminer la contribution de la voie de signalisation STAT3 dans les neurones dopaminergiques du mésencéphale, nous avons généré une lignée de souris knockout conditionnel dans lequel l'activation du gène de STAT3 sur son résidu tyrosine 705 ( Tyr 705 ) est absent spécifiquement dans les neurones dopaminergiques. Avec l'utilisation de ce modèle de souris génétique, nous avons évalué l'impact de l'ablation de la signalisation STAT3 dans les neurones dopaminergiques sur un certain nombre de fonctions liées à la dopamine, y compris l'alimentation, la locomotion, les comportements liés à la récompense, l'émotion et la libération de dopamine dans le noyau accumbens. Fait intéressant, nous avons observé un dimorphisme sexuel dans le phénotype des souris STAT3DAT-KO. L'activation de la voie de signalisation STAT3 dans les neurones dopaminergiques est responsable de l'action de la leptine dans la réduction de la locomotion, récompense liée à l'activité physique, et de l'augmentation de la libération et de la disponibilité de la dopamine chez les souris mâles. Cependant, il ne module pas le comportement émotionnel. D'autre part, les souris femelles STAT3DAT-KO augmentent les niveaux d'anxiété et les niveaux plasmatiques de corticostérone, sans provoquer de changements de la dépression. Cependant, la perte d'activation de STAT3 dans les neurones dopaminergiques ne module pas le comportement locomoteur chez les souris femelles. Notamment, les actions de la leptine dans le mésencéphale pour influencer le comportement alimentaire ne sont pas médiées par l'activation de STAT3 dans les neurones dopaminergiques, considérant que les souris mâles et femelles ont un comportement alimentaire normal. Nos résultats démontrent que la voie de signalisation STAT3 dans les neurones dopaminergiques est responsable des effets anxiolytiques de la leptine, et soutient l'hypothèse que la leptine communique l'état d'énergie du corps (i.e. la relation entre la dépense et les apports énergétiques) pour les régions mésolimbiques pour atténuer les effets de motivation et de récompense de plusieurs comportements qui servent à réhabiliter ou à épuiser les réserves d'énergie. En outre, ce travail souligne l'importance d'étudier la modulation de la signalisation de la leptine dans différente types de cellules, afin d'identifier les voies de signalisation et les mécanismes cellulaires impliqués dans les différentes fonctions neuro-comportementales de la leptine.
The adipocyte-derived hormone leptin circulates in proportion to the body fat content and transduces its signal through the long form of its receptor via a number of neural pathways, including MAPK, PI3-K, AMPK and JAK2-STAT3. Of note, STAT3 constitutes a key pathway downstream to the leptin receptor by which leptin modulates the expression of genes involved in energy balance. Most research has focused on leptin receptor function within the hypothalamus, in particular leptin receptor function within the arcuate nucleus. However, leptin receptors are also expressed on dopaminergic neurons of the ventral tegmental area, and leptin has been shown to target this brain region to influence feeding, motivation, locomotion, anxiety and dopamine tone. Moreover, leptin activates STAT3 in both dopaminergic and GABAergic neuronal populations. Although these findings contribute to our understanding of the multiple actions of leptin in the central nervous system, it remains to be resolved which cells and leptin receptor signaling pathway mediates the neurobehavioral effects of leptin in the midbrain. Aiming at determining the contribution of STAT3 signaling in midbrain DA neurons, we generated a line of conditional knockout mice in which the main activation site of STAT3 gene (tyr 705) is absent specifically in dopaminergic neurons (STAT3DAT-KO mice). Using this genetic mouse model, we assessed the impact of ablation of STAT3 signaling in dopaminergic neurons on a number of dopamine-related functions, including feeding, locomotion, reward-related behaviors, emotion and nucleus accumbens dopamine release. Interestingly, we observed a sexual dimorphism in the phenotype of STAT3DAT-KO mice. STAT3 signaling in DA neurons mediates the actions of leptin in the midbrain to decrease locomotion and running reward, and to increase dopamine release and availability in male mice. However, it does not modulate emotional behavior. On the other hand, STAT3DAT-KO female mice exhibited increased anxiety-like behavior accompanied by increased plasma corticosterone levels, without changes in behavioral despair relative to littermate controls. However, loss of STAT3 activation in dopaminergic neurons does not modulate locomotor behavior in female mice. Notably, the actions of leptin in the midbrain to influence feeding behavior are not mediated by STAT3 signaling in dopaminergic neurons, as both male and female STAT3DAT-KO mice have normal feeding behavior as compared to littermate controls. Our results demonstrate that STAT3 signaling in dopaminergic neurons mediates the anxiolytic actions of leptin, and support the hypothesis that leptin communicates body energy status (defined as a relationship between energy intake and energy expenditure) to mesolimbic regions to adjust the motivational and rewarding effects of multiple behaviors that serve to either restore or deplete energy stores. In addition, this work highlight the importance of studying cell-type specific modulation of leptin signaling molecules to tease apart pathways and the mechanisms involved in the different neurobehavioral functions of this adipocyte-derived hormone.

碩士
國立臺灣大學
獸醫學研究所
93
Leptin, mainly secreted from adipose tissues surrounding adrenal glands, is proposed to locally control the biosynthesis of adrenal steroid hormones. The human adrenocortical NCI-H295 cells were treated with or without leptin, adrenocorticotropic reagent, or both. Two major adrenal steroid products, progesterone and cortisol secreted in their cultured media were measured by ELISA. Cholera toxin, an activator of cAMP-protein kinase A pathway, mimicked ACTH effect to stimulate the secretion of progesterone and cortisol in time- and dose-dependent fashions. Leptin did not affect basal secretion of both steroid hormones; however, it effectively inhibited ACTH- or cholera toxin-induced secretion of progesterone and cortisol. Furthermore, the induction of cholera toxin on the protein amounts of P450scc, the first and rate-limiting steroidogenic enzyme, 3b-HSD, the essential enzyme for synthesis of bioactive steroids, and P450c21, the critical enzyme in secreting corticoids, were reduced by leptin. Similar inhibition of leptin was observed at the mRNA levels of P450scc and 3b-HSD. The involvement of leptin in regulating CYP11A1 promoter activity was evaluated by 5’-serial deletion. The deletion clones containing CYP11A1 promoter over 1.7 kb were responsive, whereas the shorter clone with 1.5-kb CYP11A1 promoter was silent to cAMP stimulation. The cAMP-inducible promoter activity was decreased by leptin. The inhibition of leptin on cAMP-regulated steroidogenesis and CYP11A1 promoter activity was prevented by the JAK1/2 specific inhibitor AG490 and PI3 kinase specific inhibitor Wortmannin as well as a general PDE inhibitor IBMX and a PDE3 selective inhibitor SKF94836; moreover, leptin failed to interfere the induction of N6-MB-cAMP, a PDE3B resistant cAMP analogue. Collectively, this study demonstrated leptin reduced adrenocorticotropic reagent-induced steroidogenesis possibly through a hypothesized JAK1/2-PI3 kinase-PDE3B-cAMP pathway.

碩士
國立陽明大學
神經科學研究所
104
Alzheimer’s disease (AD) is an age-dependent neurodegenerative disease with unknown etiology. The hallmarks of AD include β-amyloid (Aβ) containing senile plaques and neurofibrillary tangles in the brain. Neuroinflammation, neuronal insulin resistance and impaired energy homeostasis have been reported to modulate AD pathogenesis. The food intake and energy expenditure majorly modulated by the hypothalamus are impaired in AD patients, but underlying mechanisms remain unclear. Western diet contains high sucrose and high fat. High fat diet (HFD) induces obesity and accelerates the AD pathogenesis, but less is known about the impact of high sucrose diet (HSD) and HFD on central leptin signaling and AD pathology. This study is aimed to investigate individual impacts of HSD and HFD on the pathology and leptin signaling of APP/PS1 transgenic mice (AD mice). Wild type (WT) and AD mice fed on HSD, HFD or normal chow diet (NCD) were applied in this study. Compared with NCD, my data showed that HSD and HFD both increased the level of serum Aβ and induced neuroinflammation. HSD elevated the level of cortical Aβ and HFD induced hyperleptinemia and increased soluble leptin receptor. However, no hyperleptinemia and obesity was observed in HSD WT and AD mice, in spite of severe leptin resistance of HSD AD mice was observed in our laboratory. My data suggested that HSD increased soluble leptin receptor but not leptin of both HSD WT and AD mice. Furthermore, the level of soluble leptin receptor of HSD WT was significantly higher than that of HSD AD mice along aging. Nevertheless, both leptin and soluble leptin receptor of HSD WT and AD mice responded comparably in the fasting-refeeding experiment. In conclusion, metabolic stresses induced by HFD and HSD accelerate AD central pathology and modulate on leptin signaling which can be important risk factors of metabolic syndrome and AD.

The central nervous system (CNS) melanocortin signaling pathway plays a critical role in the regulation of metabolism. However, the regulatory effects of CNS melanocortin signaling on hepatic lipid metabolism and fatty liver disease have not been well established. Although the activity of the CNS melanocortin system is regulated by metabolic signals, the mechanism for this regulation is not fully understood. Variants of the FTO (fat mass and obesity-associated) gene are associated with obesity and FTO is expressed in the hypothalamic neurons including proopiomelanocortin (POMC) neurons. Therefore, it is hypothesized that hypothalamic FTO plays a role in the regulation of metabolism by mediating the effect of metabolic signals on hypothalamic melanocortinergic neurons, and that impairments in this regulation may cause metabolic impairments including obesity and fatty liver disease. Intracerebroventricular (i.c.v.) treatment with SHU9119, a melanocortin antagonist, increased hepatic lipid accumulation and the expression of genes encoding lipogenic enzymes in lean mice. Conversely, i.c.v. treatment with MTII, a melanocortin agonist, reduced the expression of hepatic lipogenic genes in association with reduction in body weight in ob/ob mice, a mouse model of fatty liver disease. Immunohistochemical analysis demonstrated that Fto is co-expressed in both POMC and agouti-related protein (AgRP) neurons in the mouse hypothalamus. Fto mRNA and protein expression was reduced by fasting and increased by glucose treatment in nutritionally important hypothalamic nuclei. Fasting-induced reduction in hypothalamic Fto expression was observed in both lean wild-type and obese ob/ob mice, while the stimulatory effect of glucose on hypothalamic Fto expression was absent in ob/ob mice. These findings support the hypothesis that central melanocortin signaling regulates hepatic lipid metabolism in part by regulating de novo lipogenesis. Impairments in the central melanocortin signaling lead to the development of hepatic steatosis, while enhanced melanocortin signaling may be beneficial in reversing abnormal hepatic lipid metabolism in fatty liver disease (Poritsanos et al., 2008). These findings also support the hypothesis that Fto is expressed in the hypothalamic melanocortinergic neurons and is regulated by metabolic signals involving changes in CNS glucose availability and/or glucose action. Impairments in this regulation may cause metabolic impairments including obesity and fatty liver disease.

Background: The stomach has the ability to respond to chemical and mechanical stimuli to mediate satiety through vagal pathways. Within the stomach specialised endocrine and epithelial cells synthesise and secrete leptin and ghrelin, which influence food intake through vagal afferent pathways. However, it remains to be determined if mechanosensitive gastric vagal afferent signalling is disrupted in obesity and whether this may play a role in the overconsumption of energy required for the maintenance of diet induced obesity. Furthermore, whether leptin can modulate mechanically sensitive gastric vagal afferents and whether any ability of leptin and ghrelin to modulate mechanically sensitive endings is altered in obesity has not been conclusively determined. Aims: To determine in lean mice and in high fat diet induced obese mice: 1) The effect of gastric peptides ghrelin and leptin on gastric vagal afferent mechanosensitivity. 2) The effect of gastric peptides on the expression of their own and other peptide receptors. 3) The reversibility of diet-induced obesity. Methods: Lean and diet-induced obese mice were created by feeding 8 week old female C57BL/6 mice a standard chow diet (N=4-20; 7% energy from fat) or a high-fat diet (N=4-20; 60% of energy from fat) respectively. An in vitro gastro-oesophageal vagal flat sheet preparation was utilised to determine the mechanosensitivity of vagal afferent endings and the effect of leptin, ghrelin and diet-induced obesity on this mechanosensitivity. Messenger RNA (mRNA) content in nodose ganglia was measured by QRT-PCR. Specific gastric vagal afferent cell bodies were identified by retrograde labelling and this technique was combined with QRT-PCR to determine mRNA content in specific gastric cell bodies. Anterograde tracing by injection of tracer into the nodose ganglia allowed visualisation of the distribution of gastric vagal afferents in relation to leptin and ghrelin positive cells. Nodose ganglia were cultured overnight in medium containing leptin, ghrelin or neuropeptide W (NPW) followed by QRTPCR to determine any homologous or heterologous receptor expression regulation. Results: Diet-induced obesity caused a reduction in the mechanosensitivity of gastric tension receptors. Furthermore, it increased the inhibitory effect of ghrelin on gastric vagal afferent mechanosensitivity and resulted in a switch in the effect of leptin from potentiating to inhibitory. The gut peptides leptin, ghrelin and NPW modified the mRNA content of their own and each other‘s receptors in a manner that was dependent on dietary group. Placing obese mice back on a chow diet resulted in an initial weight loss but subsequent increased food consumption and weight gain. The decrease in mechanosensitivity caused by the high fat diet was not reversible by placing diet-induced obese mice back on a chow diet and the effects of leptin were only partially reversed. Conclusions: Vagal afferent function is altered in diet-induced obesity to the extent that both the baseline response and the effects of leptin and ghrelin may act to facilitate increased food intake. Given the lack of reversibility of changes observed in diet-induced obesity this suggests that gastric vagal afferents may play a role in the maintenance of obesity and may act to oppose weight loss.
Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2013

JAK/STAT signaling pathway is one of the most studied intracellular cascades transmitting signals from the extracellular environment to the cell nucleus in order to affect expression of target genes. Circadian clocks localized in the suprachiasmatic nuclei (SCN) of the hypothalamus are sensitive especially to light but they can respond to non-photic stimuli such as growth factors, opioids, leptin and cytokines that have been demonstrated to perform its function via the JAK/STAT signaling pathway. The recent findings of our laboratory demonstrated that STAT3 protein is highly produced by SCN of rat. Primary aim of our experiments was to test the circadian regulation of STAT3 production in SCN and describe the effect of exogenously administered leptin on STAT3 phosphorylation in the SCN, pineal gland and hypothalamic structures responsible for regulated feeding behavior and energy metabolism. Because activation of leptin receptors may stimulate a number of other signaling cascades, we chose phosphorylated forms of kinase ERK1/2 and GSK-3β as other markers of intracellular changes after administration of leptin in the studied structures. Our results proved rhythmic production of STAT3 protein in SCN of rat and indicated circadian regulation of sensitivity to leptin in hypothalamic structures. The data...