Various mouse models have allowed for identification of several transcription factors that are necessary for pituitary development. Lesions in the transcription factor genes result in pituitary hormone deficiency. Hormone deficiencies occur in approximately one in 4000 live births. Pituitary hormone deficiency may occur due to loss of a single hormone causing isolated hormone deficiency or several hormones leading to combined pituitary hormone deficiency (CPHD). Defects in genes such as LHX3, LHX4, RPX, PROP1, and PIT1 are known to contribute to CPHD in humans. FOXO1 is a member of a large family of forkhead transcription factors. FOXO1 is expressed in various tissues where it functions to regulate metabolism, maintenance of cell differentiation, vascular development, cell cycle progression and apoptosis. Previous studies in our laboratory found that FOXO1 is expressed in different subsets of pituitary cells during embryonic pituitary development, with almost 50% of GH positive somatotropes also immunopositive for FOXO1. However, the roles of FOXO1 during pituitary development have not been extensively explored. Therefore, this research focuses on the contributions of FOXO1 to pituitary development and exploration of FOXO1 as a candidate gene for CPHD. In this study, a mouse model (Foxo1 cKO) is used wherein the Foxo1 gene has been deleted in the pituitary gland by Cre-LoxP recombination system. First, expression of several genes was examined that might be associated with loss of FOXO1 in the pituitary with an aim to place FOXO1 within the hierarchy of transcription factors critical for pituitary development. The early pituitary organizers, PITX2, PITX3, LHX3, LHX4, are not affected due to deletion of Foxo1 suggesting that FOXO1 is not critical for the initial induction of oral ectoderm to form Rathke’s pouch during the early stages of pituitary development. PIT1 marks the progenitors committed to becoming somatotropes, thyrotropes or lactotropes. No apparent difference in Pit1 mRNA level as well as PIT1 immunostaining between cKO and wildtype embryos suggests that FOXO1 does not affect the commitment of progenitors to cells of the PIT1 lineage. The most significant effects of Foxo1 deletion in the pituitary gland was observed in somatotrope differentiation. There was a drastically decreased mRNA level of Ghrhr, a marker of terminally differentiated somatotropes as well as reduced expression of Neurod4 in Foxo1 cKO embryos compared to wildtype littermates. NEUROD4 is downstream of FOXO1. Another study suggests that Neurod4 deletion in the pituitary gland affects maturation of somatotrope while preserving other cell types of anterior pituitary. NEUROD4 is essential for expression of Ghrhr during embryonic development as Neurod4 deletion results in fewer somatotropes and a complete lack of Ghrhr. Therefore, it can be implied that NEUROD4 may act as an intermediate in FOXO1 mediated terminal somatotrope differentiation and loss of FOXO1 in pituitary tissue is impeding somatotrope differentiation. We also assessed the functional consequences of loss of FOXO1 in postnatal mice. The delay in differentiation of somatotropes that was evident during embryonic development seems to have recovered by P10. Therefore, we suggest that FOXO1 is important for somatotrope differentiation embryonically. FOXO1 is important for somatotrope function postnatally also. Gh1 expression, GH pituitary content and serum IGF1 levels are significantly reduced at P21. However, the cKO mice do not exhibit any growth deficit indicating that FOXO1 is dispensable for postnatal somatotrope expansion and growth. Our results show that the embryonic somatotrope phenotype associated with deletion of Foxo1 does not result in any morphological changes in postnatal cKO mice. A gene expression profiling study was done to ascertain the changes in transcriptome of the embryonic pituitary lacking FOXO1. We identified Slc25a33 and Deptor as differentially expressed genes. SLC25A33 is involved with transport of pyrimidine nucleotides across mitochondrial membrane and such transport is essential for mitochondrial DNA and RNA metabolism. Studies involving overexpression of Slc25a33 in human cells have shown it enhances cell size and mitochondrial thymidine triphosphate level but decreases ROS. Its knockdown causes depletion of mitochondrial DNA, reduced oxidative phosphorylation, cell size, and mitochondrial UTP levels, and increased ROS levels. SLC25A33 essentially maintains mitochondrial function as it regulates mitochondrial DNA replication and the ratio of transcription of mitochondrial genes relative to nuclear genes. DEPTOR acts as an intermediate in BAF60c-induced AKT activation that results in a metabolic switch from oxidative phosphorylation to glycolysis in fast-twitch myofiber. Such a switch is considered to protect mice from diet induced insulin resistance and glucose intolerance in diabetic state. In differentiating cells, significant oxidative damage occurs, which can be attributed to higher mitochondrial activity. Embryonic stem cells undergoing differentiation exhibit increased mitochondrial activity associated with mitochondrial DNA replication to encode mitochondrial electron transport chain components. During osteogenic differentiation, there is a significant increase in expression of Slc25a33, with concomitant increase in mitochondrial oxygen consumption. Similar changes have been reported during spontaneous embryonic stem cell differentiation with increase in ROS production associated with differentiation. Identification of Slc25a33 and Deptor brings to our attention, a possible mechanism which might explain the delayed somatotrope differentiation phenotype in Foxo1 cKO pituitary. We hypothesize that loss of FOXO1 and resulting suppression of Slc25a33 and Deptor results in perturbation in mitochondrial replication and imbalances ROS homeostasis, which thereby affects mitochondrial function. This hinders the somatotrope’s ability to switch to more energetically demanding metabolic pathways that are essential during terminal differentiation. A metabolic switch may be the key to the terminal differentiation of mature somatotropes from their committed progenitor cells. Our findings thus provide new insights and opens avenues for future research to investigate mechanisms of somatotrope development.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18420号 / 生博第300号 / 新制||生||39(附属図書館) / 31278 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 西田 栄介, 教授 上村 匡, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

No description available.

Les lymphocytes T (LT) perçoivent et intègrent en permanence des signaux solubles et cellulaires, conditionnant leur comportement et leur devenir. A l’état de repos, les propriétés des LT s’appuient sur un réseau moléculaire caractéristique, au sein duquel les facteurs de transcription FoxOs jouent un rôle majeur. En effet, ces derniers sont impliqués dans le maintien de la quiescence et de la capacité circulatoire des LT, de par le profil transcriptionnel qu’ils induisent. Nous avons identifié Fam65b comme une nouvelle cible transcriptionnelle de FOXO1. D’un point de vue fonctionnel, nous avons démontré que Fam65b régule négativement le seuil de prolifération des LT en réponse à une stimulation du récepteur à l’antigène (TCR) ou du récepteur aux chimiokines CCR7. In vivo, dans un modèle de souris transgénique pour le TCR, ces caractéristiques fonctionnelles se traduisent par une réponse secondaire plus efficace en absence de Fam65b. Physiologiquement, la moindre expression de Fam65b que nous avons observé dans les LT mémoires par comparaison aux LT naïfs corrèle avec leur plus grande réactivité. L’ensemble de ces résultats suggère que Fam65b pourrait être un marqueur fonctionnel des LT mémoires. Enfin, nous avons pu démontrer que les effets fonctionnels de Fam65b résultent d’une inhibition de l’activité de RhoA.Fam65b est donc un régulateur de la quiescence et de la migration des LT. De par son rôle de régulateur de l’activité de RhoA, Fam65b constitue un nouveau lien fonctionnel entre deux familles majeures, contrôlant la physiologie des LT : les Rho-GTPases et les FoxOs. / T cells continually sense numerous soluble and cellular signals, which determine their behavior and differentiation pattern. At the steady state, lymphocytes properties rely on a specific network, in which FoxOs transcription factors play a central role. Indeed, FoxOs-induced transcriptome is involved in the maintenance of T cell quiescence and circulation.We identified a new transcriptional target of FoxO1 named Fam65b. We show that Fam65b acts as a brake on T lymphocyte activation downstream of the T cell receptor. Functionally, we demonstrate that Fam65b negatively regulates the threshold of T cell activation downstream of TCR or CCR7 stimulation. These characteristics allow a more efficient secondary response as Fam65b expression is inhibited. Physiologically, the lower expression of Fam65b in memory T cells compared to naïve T cells takes part in their enhanced reactivity. This suggests that Fam65b could be a functional marker of memory T cells. Finally, we demonstrated that the role of Fam65b is mediated by an inhibition of RhoA activity. Therefore, Fam65b is a regulator of T cell quiescence and migration. Because he regulates the activity of RhoA, Fam65b constitutes a functional link between two major families of proteins which control T cell physiology: Rho GTPases and FoxOs.

<p>Rhabdomyosarcoma is the most common pediatric soft tissue sarcoma and demonstrates features of skeletal muscle. Of the two predominant (pediatric) subtypes, embryonal (eRMS) and alveolar (aRMS), aRMS has the poorer prognosis, with a 5-year survival rate of <50%. The majority of aRMS tumors express the fusion protein PAX3/7-FOXO1. As PAX3/7-FOXO1 is not currently druggable, we aimed to identify proteins that are downstream from or cooperate with PAX3-FOXO1 (PF) to enable tumorigenesis with the hope that these proteins may be more amenable to pharmacological inhibition.</p><p>First, in a microarray analysis of the transcriptomes of human skeletal muscle myoblasts expressing PF, we observed alterations of several Wnt pathway genes, including the Wnt inhibitor Secreted Frizzled Related Protein 3 (SFRP3). Loss-of-function studies interrogated the role of SFRP3 in human aRMS cell lines using shRNAs. Suppression of SFRP3 inhibited aRMS cell growth, reduced proliferation accompanied by a G1 arrest and induction of p21, and induced apoptosis. SFRP3 suppression modestly increased Wnt signaling; however, activation of the Wnt pathway in human aRMS cells in vitro and in a xenograft murine model of aRMS in vivo only partially recapitulated the phenotype observed with SFRP3 suppression. To identify other signaling pathways downstream of SFRP3 signaling, we conducted an oncogenic signaling pathways screen and a microarray. In the former, we identified Notch signaling as conferring resistance to SFRP3 suppression-mediated decreased cell growth and confirmed Notch crosstalk with Wnt signaling and SFRP3 in aRMS cells. In the latter, SFRP3 suppression increased genes associated with skeletal muscle differentiation and decreased those associated with cell cycle progression. </p><p>Second, we established a role for SFRP3 in a conditional xenograft murine model of aRMS. Doxycycline-inducible suppression of SFRP3 reduced aRMS tumor growth and weight by more than three-fold. Analysis of the tumors by qPCR and IHC revealed an increase in myogenic differentiation and β-catenin signaling. The combination of SFRP3 suppression and vincristine was more effective at reducing aRMS cell growth in vitro than either treatment alone, and ablated tumorigenesis in vivo. In conclusion, SFRP3 is necessary for the growth of human aRMS cells both in vitro and in vivo and is a promising new target for investigation in aRMS.</p> / Dissertation

The present study revealed that FOXO1 is present in the nuclei of non-dividing pituitary cells and in a subset of differentiated cells with highest level of expression in somatotrophs, followed by corticotrophs, thyrotrophs and gonadotrophs throughout development and in adulthood stage. A significant difference in Foxo1 transcript between age-matched males and females at 8-9 weeks of age was demonstrated in the anterior pituitary for the first time. IHC data demonstrating (i) FOXO1 co-localization with p27kip1 (ii) an increase in FOXO1 immunopositive cells within anterior pituitary in p27KO embryos compared to WT (iii) absence of FOXO1 in the nucleus of BrdU positive cells suggested that in absence of p27Kip1 FOXO1 might be important for preventing unbridled cell proliferation. Data suggested that FOXO1 might not be important for initiating pituitary cell differentiation but might be involved with p27kip1 in maintaining pituitary cell quiescence. Increase in nuclear localization of FOXO1 in the pituitary of Foxp3 mutant (lacking insulin signaling) suggested that it might be a down-stream target of insulin/PI3K/PKB pathway in the pituitary as it is in several other tissues.

The ability to adapt fuel usage to nutrient availability is termed metabolic flexibility, and is influenced by activity of the pyruvate dehydrogenase complex (PDC). The Virginia lines of chickens are a unique model of anorexia and obesity that have resulted from 56 generations of artificial selection for high (HWS) or low (LWS) juvenile body weight. We hypothesized that hyperphagia and obesity in juvenile HWS chickens are associated with altered fatty acid oxidation efficiency and metabolic flexibility in tissues associated with energy sensing and storage, and relative cellular hypertrophy in white adipose tissue. Hypothalamus, liver, Pectoralis major, gastrocnemius, abdominal fat, clavicular fat and subcutaneous fat were collected from juvenile (56-65 day-old) HWS and LWS chickens for metabolic, gene expression and histological assays. The HWS chickens had reduced fatty acid oxidation efficiency in abdominal fat (P < 0.0001) and reduced rates of oxidation in abdominal fat and gastrocnemius (P < 0.0001) as compared to LWS. There was reduced citrate synthase activity in white adipose tissue (P < 0.0001) and greater metabolic inflexibility in skeletal muscle (P = 0.006) of HWS compared to LWS. Greater pyruvate dehydrogenase kinase 4 (PDK4) and forkhead box O1 (FoxO1) mRNA were found in skeletal muscle and white adipose tissue of 56-day-old HWS than LWS. Expression of peroxisome proliferator-activated receptor γ (PPARγ) in all adipose tissue depots was greater (P < 0.05) in LWS than in HWS chickens. The HWS chickens had larger (P < 0.0001) and fewer (P < 0.0001) adipocytes per unit area than LWS. These results suggest that the HWS chickens have impaired metabolic flexibility and fatty acid oxidation efficiency due to an up-regulation of pyruvate dehydrogenase activity to accommodate the influx of acetyl CoA from fatty acid oxidation in skeletal muscle and white adipose tissue. These metabolic adaptations can be linked to differences in gene expression regulation and body composition between the lines. Adipocyte cellularity data are consistent with greater oxidative efficiency in the adipose tissue of LWS, because of the greater number of unfilled cells in all depots that were sampled. Results can be extrapolated to agricultural production in the understanding of factors regulating the amount of lipid deposition in chicken carcass fat. Results may also provide insight into eating disorders and the development of obesity. / Master of Science

No description available.

No description available.

Type 2 diabetes (T2D) is a health problem throughout the world. In 2010, there were nearly 230 million individuals with diabetes worldwide and it is estimated that in the economically advanced countries the cases will increase about 50% in the next twenty years. Insulin resistance is one of major features in T2D, which is also a risk factor for metabolic and cardiovascular complications. Epidemiological and animal studies have shown that the consumption of vegetables and fruits can delay or prevent the development of the disease, although the underlying mechanisms of these effects are still unclear. Brassica species such as broccoli (Brassica oleracea var. italica) and nasturtium (Tropaeolum majus) possess high content of bioactive phytochemicals, e.g. nitrogen sulfur compounds (glucosinolates and isothiocyanates) and polyphenols largely associated with the prevention of cancer. Isothiocyanates (ITCs) display their anti-carcinogenic potential by inducing detoxicating phase II enzymes and increasing glutathione (GSH) levels in tissues. In T2D diabetes an increase in gluconeogenesis and triglyceride synthesis, and a reduction in fatty acid oxidation accompanied by the presence of reactive oxygen species (ROS) are observed; altogether is the result of an inappropriate response to insulin. Forkhead box O (FOXO) transcription factors play a crucial role in the regulation of insulin effects on gene expression and metabolism, and alterations in FOXO function could contribute to metabolic disorders in diabetes. In this study using stably transfected human osteosarcoma cells (U-2 OS) with constitutive expression of FOXO1 protein labeled with GFP (green fluorescent protein) and human hepatoma cells HepG2 cell cultures, the ability of benzylisothiocyanate (BITC) deriving from benzylglucosinolate, extracted from nasturtium to modulate, i) the insulin-signaling pathway, ii) the intracellular localization of FOXO1 and iii) the expression of proteins involved in glucose metabolism, ROS detoxification, cell cycle arrest and DNA repair was evaluated. BITC promoted oxidative stress and in response to that induced FOXO1 translocation from cytoplasm into the nucleus antagonizing the insulin effect. BITC stimulus was able to down-regulate gluconeogenic enzymes, which can be considered as an anti-diabetic effect; to promote antioxidant resistance expressed by the up-regulation in manganese superoxide dismutase (MnSOD) and detoxification enzymes; to modulate autophagy by induction of BECLIN1 and down-regulation of the mammalian target of rapamycin complex 1 (mTORC1) pathway; and to promote cell cycle arrest and DNA damage repair by up-regulation of the cyclin-dependent kinase inhibitor (p21CIP) and Growth Arrest / DNA Damage Repair (GADD45). Except for the nuclear factor (erythroid derived)-like2 (NRF2) and its influence in the detoxification enzymes gene expression, all the observed effects were independent from FOXO1, protein kinase B (AKT/PKB) and NAD-dependent deacetylase sirtuin-1 (SIRT1). The current study provides evidence that besides of the anticarcinogenic potential, isothiocyanates might have a role in T2D prevention. BITC stimulus mimics the fasting state, in which insulin signaling is not triggered and FOXO proteins remain in the nucleus modulating gene expression of their target genes, with the advantage of a down-regulation of gluconeogenesis instead of its increase. These effects suggest that BITC might be considered as a promising substance in the prevention or treatment of T2D, therefore the factors behind of its modulatory effects need further investigation. / Diabetes mellitus Typ 2 stellt auf der ganzen Welt ein Gesundheitsproblem dar. Im Jahr 2010 waren annähernd 230 Millionen Personen weltweit an Diabetes erkrankt und innerhalb der nächsten 20 Jahre wird in industrialisierten Ländern eine Steigerung der Fälle um 50% erwartet. Eines der Hauptmerkmale des Typ 2 Diabetes ist die Insulinresistenz, die auch als Risikofaktor für metabolische und kardio-vaskuläre Komplikationen gilt. Epidemiologische Studien und Tierversuche haben ergeben, dass durch Verzehr von Gemüse und Obst eine Prävention oder Verzögerung der Entwicklung dieser Krankheit erreicht werden kann, jedoch sind die zugrunde liegenden Mechanismen dieser Effekte noch nicht aufgeklärt. Brassica Spezies wie Broccoli (Brassica oleracea var. italica) und Nasturtium (Tropaeolum majus) enthalten einen hohen Anteil an bioaktiven Pflanzen-inhaltsstoffen, wie z. B. stickstoff- und schwefelhaltige Verbindungen (Glukosinolate und Isothiocyanate) und Polyphenole, die bisher hauptsächlich mit der Prävention von Krebs assoziiert wurden. Isothiocyanate (ITCs) erreichen ihr antikanzerogenes Potential durch die Induktion von entgiftenden Phase II Enzymen und eine Anhebung der Glutathion (GSH)-Spiegel im Gewebe. Diabetes Typ2 geht einher mit einem Anstieg der Glukoneogenese und Triglycerid-Synthese, sowie einer Reduktion der Fettsäure-Oxidation in Verbindung mit erhöhten Spiegeln an reaktiven Sauerstoffspezies (ROS) insgesamt als Resultat einer unangemessenen Insulinantwort. Forkhead box O (FOXO) Transkriptionsfaktoren spielen eine wesentliche Rolle in der Regulation der Insulineffekte in Bezug auf die vermittelte Genexpression und den Metabolismus, wobei Veränderungen in der Funktion von FOXO zu metabolischen Entgleisungen im Diabetes beitragen können. In dieser Studie wurde unter Verwendung von stabil transfizierten humanen Osteosarkoma-Zellen (U-2 OS) mit konstitutiver Expression von GFP (grün fluoreszierendes Protein)-markiertem FOXO1 und humanen Hepatoma-Zellen (HepG2) die Wirkung von Benzylisothiocyanat (BITC), dessen Vorstufe Benzylglukosinolat aus Nasturtium isoliert wurde, in Zellkulturen evaluiert wie Modulationen der i) Insulin-Signal-Kaskade, ii) intrazellulären Lokalisation von FOXO1 und iii) Expression beteiligter Proteine am Glucose Metabolismus, der ROS Detoxifikation, Zellzyklus-Fixierung und DNA-Reparatur. BITC erzeugte oxidativen Stress und induzierte als Antwort darauf eine Translokation von FOXO1 aus dem Zytoplasma in den Zellkern antagonisierend zum Insulin-Effekt. Eine Stimultion mit BITC war in der Lage, die Expression von Enzymen der Gluconeogenese herunter zu regulieren, was als antidiabetogener Effekt betrachtet werden kann, eine antioxidative Resistenz durch Induktion der Mangan-Superoxid-Dismutase (MnSOD) und entgiftender Enzyme zu erzeugen, Autophagie zu modulieren durch Induktion von BECLIN1 und Herunterregulation des „mammalian target of rapamycin complex1 (mTORC1)-Stoffwechselwegs, den Zellzyklus zu fixieren und DNA-Reparatur zu induzieren durch Hochregulation des Cyclin- abhängigen Kinase- Inhibitors p21CIP und GADD45 (growth arrest and DNA damage repair). Mit Ausnahme des nuklearen Faktors (erythroid derived)-like2 (NRF2) und dessen Einfluss auf die Genexpression von Entgiftungsenzymen waren alle beobachteten Effekte unabhängig von FOXO1, Proteinkinase B (PKB/AKT) und der NAD-abhängigen Deacetylase Sirtuin-1 (SIRT1). Die gegenwärtige Studie liefert Anhaltspunkte dafür, dass Isothiocayanate neben dem antikanzerogenen Potential eine Rolle bei der Prävention von Typ 2 Diabetes spielen könnten. BITC-Stimulationen ahmen einen Fastenzustand nach, in dem kein Insulin-Signal ausgelöst wird, FOXO Proteine im Zellkern verbleiben und die Expression von Target-Genen modulieren, mit dem Vorteil einer Herunterregulation der Glukoneogenese anstelle seiner Zunahme. Diese Effekte legen nahe, dass BITC als vielversprechende Substanz zur Prävention und Behandlung von Typ 2 Diabetes angesehen werden könnte. Deshalb benötigen die Faktoren, die dessen modulatorische Effekte hervorrufen, weitere Untersuchungen.