Yes / Chemerin is an adipokine involved in inflammation, adipogenesis, angiogenesis and energy metabolism, and has been hypothesized as a link between obesity and type II diabetes. In humans affected by obesity, chemerin gene expression in peripheral tissues and circulating levels are elevated. In mice, plasma levels of chemerin are upregulated by high-fat feeding and gain and loss of function studies show an association of chemerin with body weight, food intake and glucose homeostasis. Therefore, chemerin is an important blood-borne mediator that, amongst its other functions, controls appetite and body weight. Almost all studies of chemerin to date have focused on its release from adipose tissue and its effects on peripheral tissues with the central effects largely overlooked. To demonstrate a central role of chemerin, we manipulated chemerin signaling in the hypothalamus, a brain region associated with appetite regulation, using pharmacological and genetic manipulation approaches. Firstly, the selective chemerin receptor CMKLR1 antagonist α-NETA was administered i.c.v. to rats to test for an acute physiological effect. Secondly, we designed a short-hairpin-RNA (shRNA) lentivirus construct targeting expression of CMKLR1. This shRNA construct, or a control construct was injected bilaterally into the arcuate nucleus of male Sprague Dawley rats on high-fat diet (45%). After surgery, rats were maintained on high-fat diet for 2 weeks and then switched to chow diet for a further 2 weeks. We found a significant weight loss acutely and inhibition of weight gain chronically. This difference became apparent after diet switch in arcuate nucleus-CMKLR1 knockdown rats. This was not accompanied by a difference in blood glucose levels. Interestingly, appetite-regulating neuropeptides remained unaltered, however, we found a significant reduction of the inflammatory marker TNF-α suggesting reduced expression of CMKLR1 protects from high-fat diet induced neuroinflammation. In white and brown adipose tissue, mRNA expression of chemerin, its receptors and markers of adipogenesis, lipogenesis and brown adipocyte activation remained unchanged confirming that the effects are driven by the brain. Our behavioral analyses suggest that knockdown of CMKLR1 had an impact on object recognition. Our data demonstrate that CMKLR1 is functionally important for the central effects of chemerin on body weight regulation and neuroinflammation. / This work was funded in part by the Academy of Medical Sciences, the Wellcome Trust, the Government of Business, Energy and Industrial Strategy and the British Heart Foundation and Diabetes United Kingdom [SBF004/1063] (GH), the Society for Endocrinology Equipment Grant (GH, RD), the University of Bradford (GH, KP, SK) and Nottingham Trent University (RD).

Yes / Wnt signalling and its downstream effectors are well known for their roles in embryogenesis and tumourigenesis, including the regulation of cell proliferation, survival and differentiation. In the nervous system, Wnt signalling has been described mainly during embryonic development, although accumulating evidence suggests that it also plays a major role in adult brain morphogenesis and function. Studies have predominantly concentrated on memory formation in the hippocampus, although recent data indicate that Wnt signalling is also critical for neuroendocrine control of the developed hypothalamus, a brain centre that is key in energy balance regulation and whose dysfunction is implicated in metabolic disorders such as type 2 diabetes and obesity. Based on scattered findings that report the presence of Wnt molecules in the tanycytes and ependymal cells lining the third ventricle and arcuate nucleus neurones of the hypothalamus, their potential importance in key regions of food intake and body weight regulation has been investigated in recent studies. The present review brings together current knowledge on Wnt signalling in the hypothalamus of adult animals and discusses the evidence suggesting a key role for members of the Wnt signalling family in glucose and energy balance regulation in the hypothalamus in diet-induced and genetically obese (leptin deficient) mice. Aspects of Wnt signalling in seasonal (photoperiod sensitive) rodents are also highlighted, given the recent evidence indicating that the Wnt pathway in the hypothalamus is not only regulated by diet and leptin, but also by photoperiod in seasonal animals, which is connected to natural adaptive changes in food intake and body weight. Thus, Wnt signalling appears to be critical as a modulator for normal functioning of the physiological state in the healthy adult brain, and is also crucial for normal glucose and energy homeostasis where its dysregulation can lead to a range of metabolic disorders.

The subfornical organ (SFO) is a sensory circumventricular organ, lacking a proper blood-brain barrier. Neurons of the SFO are exposed directly to the ionic environment and circulating signaling molecules in the plasma, providing a unique window for communication of physiological status from the periphery to the central nervous system (CNS). The SFO is recognized as a key site for hydromineral balance, cardiovascular regulation and energy homeostasis. Neuropeptide Y (NPY) is a potent stimulator of food intake when released centrally, and has well-documented pressor effects when released peripherally. It has been demonstrated that the SFO expresses NPY receptors, however the effects of NPY on SFO neurons has never been investigated. The aim of this study was to determine the effects of NPY on the electrophysiological properties of SFO neurons dissociated from Sprague Dawley rats. Using whole cell patch clamp techniques in the current-clamp configuration, we report that 300 nM NPY caused 16% of SFO neurons to depolarize and 26% to hyperpolarize. The remaining neurons were insensitive to NPY. These effects were dose-dependent with a combined EC50 of 3.7 nM. Specific NPY receptor antagonists were applied, suggesting that the Y5 receptor predominately elicited a hyperpolarizing effect, while the Y1 receptor had a mixed response that was predominately hyperpolarizing, and the Y2 receptor had a mixed response that was predominately depolarizing. Using the voltage-clamp configuration, it was also observed that NPY caused an increase in the voltage-gated K+ current density as well as a shift in membrane activation of the persistent Na+ current, mediating the hyperpolarizing and depolarizing effects, respectively. These findings indicate that NPY elicits electrophysiological changes on SFO neurons, suggesting that the SFO is a key site of action for NPY in mediating energy regulation and/or cardiovascular output. / February 2017

Anthropogenic activities are fundamentally altering the chemistry of the world’s oceans. Many of these modifications could have a significant impact on the health of marine organisms. Yet, despite being proposed as one of the most significant threats that marine ecosystems face, to date very little is known about the impact of anthropogenic climate change, and ocean acidification in particular, on host defence. The aims of this thesis are to investigate the impact of environmental stressors on the invertebrate immune response, providing empirical data on how anthropogenically induced stressors will impact the invertebrate immune system and how this will impact organism condition and subsequent physiological trade-offs. Exposure to reduced seawater pH and increased temperature significantly reduced the immune response in the blue mussel, Mytilus edulis. This reduction in immune response could indicate stress-induced immune dysfunction. However, the immune system protects an organism from infectious disease, ensuring survival, and should therefore be evaluated functionally rather than immunologically. By subsequently exposing mussels to a bacterial challenge this study demonstrated that an earlier study which measured a reduction in host defence represented a trade-off of immune system maintenance costs, with mussels maintaining a capacity to up-regulate immune defence when required. However, whilst this immune plasticity ensures mussels are able to survive a pathogen exposure, such a strategy appears to be physiologically costly. This cost is seen as a reduction in reproductive investment, an altered energy metabolism and an altered fatty acid composition in organisms exposed to low pH. Therefore the overarching picture that emerges is, without measuring physiological processes functionally, and in neglecting any physiological trade-offs, it is possible that many studies may misinterpret the complex physiological responses of marine organisms to ocean acidification.

Ghrelin is a unique hunger-inducing stomach-borne hormone. It activates orexigenic circuits in the central nervous system (CNS) when acylated with a fatty acid residue by the Ghrelin O-acyltransferase (GOAT). Soon after the discovery of ghrelin a theoretical model emerged which suggests that the gastric peptide ghrelin is the first “meal initiation molecule”. Ghrelin is also termed “hunger hormone” with a potentially important role as an endogenous regulator of energy balance. However, genetic deletion of ghrelin or its receptor, the growth hormone secretagogue receptor (GHSR), has only limited effects on appetite and obesity. Here we introduce novel mouse models of altered ghrelin, GHSR and GOAT function to reevaluate the role of the ghrelin system in regulating energy homeostasis. Simultaneous loss of ghrelin and GHSR function leads to decreased body weight and body fat, likely caused by increased energy expenditure and locomotor activity. Similarly, GOAT deficient mice are lighter and leaner than the wild-type controls. Mice overexpressing ghrelin and GOAT have increased body weight and fat mass along with decreased energy expenditure. Wild-type mouse studies show that fasting induces downregulation of the GOAT gene Mboat4 and decreases acyl ghrelin concentration in blood. We therefore hypothesized that GOAT rather depends on dietary than endogenous derived lipids for ghrelin acylation. Feeding studies show that GOAT uses the unnatural fatty acid heptanoate (C7) to acylate ghrelin, which clearly supports our theory. Further, acylation of overproduced ghrelin in our transgenic mouse model requires dietary supplementation of medium-chain-triglycerides, the preferred GOAT substrate. Our genetic models suggest that the ghrelin system plays an important physiological role in the control of energy metabolism. Thus, GOAT offers a novel peripheral drug target for the treatment of metabolic diseases. Moreover, our results suggest that ghrelin signaling may not be a result of absent nutrient intake, but indicate the availability of dietary lipids. We therefore propose that the ghrelin system functions as a novel lipid sensor, linking specific dietary lipids with the central-nervous control of energy metabolism. / Ghrelin ist ein einzigartiges im Magen produziertes Hormon, da es von dem Enzym Ghrelin O-acyltransferase (GOAT) mit einer mittelkettigen Fettsäure acyliert werden muss, um biologische Aktivität zu erlangen. Kurz nach seiner Entdeckung entstand die Hypothese, dass Ghrelin das „Hungerhormon“ sei und eine wichtige Rolle in der Regulation des Energiehaushalts spiele. Die genetische Manipulation von Ghrelin und seinem Rezeptor, dem GHSR, hat jedoch nur geringe Auswirkung auf Appetit und Körpergewicht. In der hier vorliegenden Studie stellen wir neuartige Mausmodelle mit abgewandelter Ghrelin-, GHSR- und GOATfunktion vor, um den Einfluss des Ghrelinsystems auf die Regulation der Energiehomöostase zu reevaluieren. Weiterhin wird die endogene Regulation von GOAT erstmalig beschrieben. Double-knockout Mäuse, die gleichzeitig defizitär für Ghrelin und GHSR sind, haben ein geringeres Körpergewicht, weniger Fettmasse und einen niedrigeren Energieverbrauch als Kontrolltiere. Knockout Mäuse für das GOAT Gen Mboat4 sind leichter und schlanker als Kontrolltiere. Dementsprechend haben transgene Mäuse, die Ghrelin und GOAT überproduzieren, eine erhöhte Fettmasse und einen verminderten Energieverbrauch. Weiterhin können wir zeigen, dass GOAT, anders als auf Grund der allgemein bekannten Ghrelinfunktion angenommen, nicht durch Hungern aktiviert wird. Bei Mäusen, die gefastet haben, ist die Genexpression von Mboat4 deutlich herunterreguliert, woraus ein geringer Blutspiegel von Acyl-Ghrelin resultiert. Daraus haben wir geschlossen, dass GOAT eventuell Nahrungsfette und nicht die durch Hungern freigesetzten endogen Fettsäuren zur Ghrelinacylierung benutzt. Fütterungsversuche bestätigen diese Hypothese, da GOAT die unnatürliche Fettsäure Heptan Säure (C7), die der Tiernahrung beigefügt wurde, zur Ghrelinacylierung verwendet. Ein weiteres Indiz für die Notwendigkeit von Nahrungsfetten für die Ghrelinacylierung ist, dass die transgenen Ghrelin/GOAT Mäuse nur massiv Acyl-Ghrelin produzieren, wenn sie mit einer Diät gefüttert werden, die mit mittelkettigen Fettsäuren angereichert ist. Zusammenfassend zeigt die Studie, dass das Ghrelinsystem maßgeblich an der Regulation der Energiehomöostase beteiligt ist und dass die Ghrelinaktivierung direkt von Nahrungsfetten beeinflusst wird. Daraus könnte geschlossen werden, dass Ghrelin wohlmöglich nicht das Hungerhormon ist, wie bisher generell angenommen wurde. Ghrelin könnte vielmehr ein potentieller “Fettsensor” sein, der dem Gehirn die Verfügbarkeit von fettreicher Nahrung signalisiert und somit den Metabolismus zur optimalen Verwertung und Speicherung der aufgenommenen Energie beeinflusst.

Hypothalamic lactate metabolism regulates hepatic glucose and lipid homeostasis, however it remains unclear whether hypothalamic lactate also controls energy homeostasis. Furthermore, the precise downstream molecular and signaling pathway(s) involved in hypothalamic lactate-sensing is yet to be fully elucidated. To specifically address these two questions, we tested the hypothesis that hypothalamic lactate metabolism regulates energy homeostasis (Study 1) and assessed whether the activation of N-methyl-D-aspartate (NMDA) receptors in the nucleus of the solitary tract (NTS) of the brainstem is required for hypothalamic lactate, and sufficient per se, to regulate glucose homeostasis (Study 2). In an in vivo rat model, we reported in Study 1 that central lactate lowers food intake and body weight through its metabolism into pyruvate. In Study 2, we identified that hypothalamic lactate metabolism requires the activation of NMDA receptors in the NTS to lower hepatic glucose production. Moreover, we showed that the activation of NTS NMDA receptors per se lowers hepatic glucose production. In summary, these findings advance the understanding of central nutrient-sensing in the regulation of energy and glucose homeostasis, which is critical in bridging the therapeutic gap of obesity and type 2 diabetes.

Hypothalamic lactate metabolism regulates hepatic glucose and lipid homeostasis, however it remains unclear whether hypothalamic lactate also controls energy homeostasis. Furthermore, the precise downstream molecular and signaling pathway(s) involved in hypothalamic lactate-sensing is yet to be fully elucidated. To specifically address these two questions, we tested the hypothesis that hypothalamic lactate metabolism regulates energy homeostasis (Study 1) and assessed whether the activation of N-methyl-D-aspartate (NMDA) receptors in the nucleus of the solitary tract (NTS) of the brainstem is required for hypothalamic lactate, and sufficient per se, to regulate glucose homeostasis (Study 2). In an in vivo rat model, we reported in Study 1 that central lactate lowers food intake and body weight through its metabolism into pyruvate. In Study 2, we identified that hypothalamic lactate metabolism requires the activation of NMDA receptors in the NTS to lower hepatic glucose production. Moreover, we showed that the activation of NTS NMDA receptors per se lowers hepatic glucose production. In summary, these findings advance the understanding of central nutrient-sensing in the regulation of energy and glucose homeostasis, which is critical in bridging the therapeutic gap of obesity and type 2 diabetes.

L’hypothalamus joue un rôle clef dans la régulation de l’homéostasie énergétique grâce à la présence de circuits neuronaux contrôlant la prise alimentaire. Ces circuits peuvent être remodelés dans le cerveau. Nous avons émis l’hypothèse que la plasticité de ces circuits intervient en conditions physiologiques. Nous avons démontré que les synapses des neurones à pro-opiomélanocortine sont modifiées en fonction de l’alimentation. Ce processus de plasticité est indispensable pour ajuster la prise alimentaire et nécessite la présence d’un polymère glucidique appelé PSA (acide polysialique), se fixant sur les protéines d’adhésion cellulaire NCAM et limitant les contacts synaptiques. Nous avons évalué le lien entre la plasticité cérébrale et la vulnérabilité à développer une obésité chez des souris placées en régime gras pendant 3 mois. La réponse comportementale au régime gras était variable, et prédictive de la prise de poids à terme, et était liée aux taux de PSA hypothalamique. La déplétion chronique de PSA dans l’hypothalamus a accéléré la prise de poids et l’adiposité des animaux. Ces résultats suggèrent qu’une capacité réduite de plasticité synaptique est un facteur de risque de l’obésité. En parallèle, nous nous sommes intéressés à l’homéostasie du cholestérol circulant contrôlée par le système à mélanocortine. Il existe un mécanisme de régulation du cholestérol circulant dépendant de PSA dans l’hypothalamus. Une dérégulation de ce mécanisme a provoqué l’accumulation de dépôts graisseux dans les vaisseaux sanguins. L’ensemble de ces travaux a permis de mettre en évidence le rôle de la plasticité synaptique hypothalamique dans la régulation de l’homéostasie énergétique. / Hypothalamus plays a major role in the regulation of energy homeostasis by the presence of neural circuits controlling food intake. These circuits are plastic and can be rewired during adulthood. We hypothesized that synaptic plasticity can occur during physiological conditions. We have shown that synaptic contact on hypothalamic anorexigen POMC neurons are rewired in mouse upon high fat diet (HFD). This synaptic process is mandatory to adjust energy intake and requires the glycan PSA (polysialic acid). PSA promotes synaptic plasticity in the brain by the weakening of cell-to-cell interaction by addition on NCAM (neural cell adhesion molecule). We hypothesized that a defect in brain synaptic plasticity capacity could be a risk factor in the etiology of metabolic diseases. We show that homeostatic feeding response to HFD ingestion was predictive to weight gain observed three months after HFD introduction. The feeding response to HFD was correlated with the hypothalamus PSA level. We show that chronic depletion of hypothalamic PSA accelerate the onset of diet induced obesity. These results indicate that a low hypothalamic PSA level prone to diet induced obesity. In parallel, we focus on the hypothalamic regulation of circulating cholesterol. Melanocortin system control level of circulating cholesterol. Using our model of diet induced synaptic plasticity; we show that there is a link between hypothalamic PSA and circulating cholesterol. A long term reduction of hypothalamic PSA level, lead to an accumulation of fat deposit in blood vessels. This whole work allows us to underscore the role of diet induced synaptic plasticity in the regulation of energy homeostasis.

Abstract Orexins, or hypocretins, are peptides originally found in the hypothalamus, and have been shown to be involved in the stabilization and maintenance of sleep and wakefulness. In addition, these peptides are known for their actions on energy homeostasis by increased heat production or physical activity. Previous results suggest them to be also involved in peripheral actions on the regulation of intestinal secretion, depending on the subject’s nutritional status (fasted-fed). Orexin-A and Orexin-B peptides, are derived from the prepro-orexin precursor protein. These ligands bind to two G-protein-coupled receptors, orexin-1 and -2 -receptors. Despite intensive research, the role of orexins has not yet been clarified. The aim of the present study was to investigate the role of orexins and their receptors on sleep and wake patterns, energy homeostasis and intestinal secretion. The effects of orexins on sleep and wakefulness, and energy homeostasis were studied in a novel transgenic mouse line, overexpressing the human prepro-orexin gene. The overexpression of prepro-orexin and orexin-A was confirmed in the hypothalami of transgenic mice. The transgenic mice showed a significant reduction in their REM sleep during day and night time, and differences in their vigilance states in the light/dark transition periods. In addition, the mice demonstrated a significantly elevated day time food intake at room temperature, and an increased metabolic heat production independent of uncoupling protein 1 mediated thermogenesis in brown adipose tissue. Instead, transgenic mice showed increased levels of uncoupling protein 2 in white adipose tissue. Furthermore, transgenic mice significantly decreased their total locomotor activity during the first two nights in response to cold exposure (+4°C). The effect of orexins and their receptors on duodenal HCO3– secretion were studied after an overnight (16 h) food deprivation in an in situ perfused organ. Fasting decreased the expression of orexin receptors in rat duodenal mucosa and in acutely isolated duodenal enterocytes. Furthermore, food deprivation abolished OXA induced duodenal mucosal HCO3– secretion in rats, and intracellular calcium signalling in isolated rat and human duodenal enterocytes. In conclusion, the present thesis demonstrates that orexins inhibit REM sleep. In addition, peptides affect increasingly on metabolic heat production, independent of uncoupling protein 1 mediated thermogenesis. Furthermore, the orexin system has a significant role in duodenal bicarbonate secretion, which is regulated by the presence of food in the intestine.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13168号 / 論医博第2155号 / 新制||医||1029(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 川口 義弥, 教授 近藤 玄 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM