S100A8 is a highly-expressed calcium-binding protein in neutrophils and activated macrophages, and has proposed roles in myeloid cell differentiation and host defense. Functions of S100A8 are not fully understood, partly because of difficulties in generating S100A8 knockout mice. Attempts to silence S100A8 gene expression in activated macrophages and fibroblasts using RNA interference (RNAi) technology were unsuccessful. Despite establishing validated small interfering RNA (siRNA) systems, enzymaticallysynthesized siRNA targeted to S100A8 suppressed mRNA levels by only 40% in fibroblasts activated with FGF-2+heparin, whereas chemically-synthesized siRNAs suppressed S100A8 driven by an S100A8-expression vector by ~75% in fibroblasts. Suppression of the gene in activated macrophages/fibroblasts was low, and some enzymatically-synthesized siRNAs to S100A8, and unrelated siRNA to GAPDH, induced/enhanced S100A8 expression in macrophages. This indicated that S100A8 may be upregulated by type-1 interferon (IFN). IFN-β enhanced expression, but did not directly induce S100A8. Poly (I:C), a synthetic dsRNA, directly induced S100A8 through IL-10 and IFN-dependent pathways. Induction by dsRNA was dependent on RNA-dependent protein kinase (PKR), but not cyclooxygenase-2, suggesting divergent pathways in LPS- and dsRNA-induced responses. New mechanisms of S100A8 gene regulation are presented, that suggest functions in anti-viral defense. S100A8 expression was confirmed in lungs from influenza virus-infected mice and from a patient with severe acute respiratory syndrome (SARS). Multiple pathways via mitochondria mediated S100A8 induction in LPS-activated macrophages; Generation of reactive oxygen species via the mitochondrial electron transport chain and de novo synthesis of ATP may be involved. This pathway also regulated IL-10 production, possibly via PKR. Extracellular ATP and its metabolites enhanced S100A8 induction. Results support involvement of cell stress, such as transfection, in S100A8 expression. A breast tumor cell line (MCF-7) in which the S100A8 gene was silenced, was established using micro RNA technology; S100A8 induction by oncostatin M was reduced by >90% in stably-transfected cells. This did not alter MCF-7 growth. The new approach to investigate the role of S100A8 in a human tumor cell line may assist in exploring its functions and lead to new studies concerning its role in cancer.

S100A8 is a highly-expressed calcium-binding protein in neutrophils and activated macrophages, and has proposed roles in myeloid cell differentiation and host defense. Functions of S100A8 are not fully understood, partly because of difficulties in generating S100A8 knockout mice. Attempts to silence S100A8 gene expression in activated macrophages and fibroblasts using RNA interference (RNAi) technology were unsuccessful. Despite establishing validated small interfering RNA (siRNA) systems, enzymaticallysynthesized siRNA targeted to S100A8 suppressed mRNA levels by only 40% in fibroblasts activated with FGF-2+heparin, whereas chemically-synthesized siRNAs suppressed S100A8 driven by an S100A8-expression vector by ~75% in fibroblasts. Suppression of the gene in activated macrophages/fibroblasts was low, and some enzymatically-synthesized siRNAs to S100A8, and unrelated siRNA to GAPDH, induced/enhanced S100A8 expression in macrophages. This indicated that S100A8 may be upregulated by type-1 interferon (IFN). IFN-β enhanced expression, but did not directly induce S100A8. Poly (I:C), a synthetic dsRNA, directly induced S100A8 through IL-10 and IFN-dependent pathways. Induction by dsRNA was dependent on RNA-dependent protein kinase (PKR), but not cyclooxygenase-2, suggesting divergent pathways in LPS- and dsRNA-induced responses. New mechanisms of S100A8 gene regulation are presented, that suggest functions in anti-viral defense. S100A8 expression was confirmed in lungs from influenza virus-infected mice and from a patient with severe acute respiratory syndrome (SARS). Multiple pathways via mitochondria mediated S100A8 induction in LPS-activated macrophages; Generation of reactive oxygen species via the mitochondrial electron transport chain and de novo synthesis of ATP may be involved. This pathway also regulated IL-10 production, possibly via PKR. Extracellular ATP and its metabolites enhanced S100A8 induction. Results support involvement of cell stress, such as transfection, in S100A8 expression. A breast tumor cell line (MCF-7) in which the S100A8 gene was silenced, was established using micro RNA technology; S100A8 induction by oncostatin M was reduced by >90% in stably-transfected cells. This did not alter MCF-7 growth. The new approach to investigate the role of S100A8 in a human tumor cell line may assist in exploring its functions and lead to new studies concerning its role in cancer.

L’hypothalamus participe au contrôle de l’homéostasie énergétique en détectant les signaux circulants tels que le glucose. L’hypothalamus médiobasal (MBH) en particulier, est capable de détecter l’hyperglycémie afin d’initier des réponses physiologiques adaptées, comme par exemple la sécrétion d’insuline via le système nerveux autonome (par un contrôle vagal). Notre équipe a récemment montré que la détection du glucose nécessite la production d’espèces actives de l’oxygène d’origine mitochondriale (mROS), fortement dépendante de la dynamique mitochondriale (fusion et fission). Récemment, l’étude de modèles génétiques ont permis de faire un lien entre ces évènements dynamiques dans le MBH et le développement de pathologies métaboliques. L’objectif de ma thèse a été tout d’abord été de mettre en place un modèle expérimental présentant uniquement une altération de la détection hypothalamique du glucose induite par l’exposition à un régime gras enrichi en saccharose (HFHS) chez le rat. Après avoir caractérisé ce modèle, nos objectifs ont été de déterminer si l’exposition à ce régime hypercalorique avait un impact sur la dynamique mitochondriale ainsi que la signalisation mROS, via la fonction respiratoire de la mitochondrie dans l’hypothalamus. Nous avons finallement réversé quelques acteurs métaboliques dérégulés, potentiellement impliqués dans la dynamique mitochondriale, dans le but de réverser le phénotype observé chez les rats HFHS. Nos résultats montrent qu’après 3 semaines d’exposition au régime HFHS, les rats ont un poids corporel normal malgré l’augmentation de leur masse grasse, comparés aux rats contrôles. Les rats HFHS présentent aussi une intolérance au glucose et une augmentation de la glycémie basale sans modification de leur insulinémie. La sécrétion d’insuline en réponse à la détection hypothalamique du glucose, mesurée après une injection intra-carotidienne de glucose en direction du cerveau qui induit une hyperglycémie uniquement cérébrale, a été fortement diminuée. Cependant, la capacité sécrétoire des îlots pancréatiques est normale chez les rats HFHS. Ces défauts sont associés à une diminution de la production de ROS dans le MBH en réponse au glucose, sans modification du status redox. L’efficacité de la respiration mitochondriale hypothalamique a été mesurée par oxygraphie, et les résultats montrent une déficience de la respiration mitochondriale chez les rats HFHS. La translocation de la protéine de fission DRP1 à la mitochondrie est diminuée en réponse au glucose, suggérant une diminution de la fission mitochondriale. L’augmentation de l’activation de l’AMPK dans l’hypothalamus n’est pas responsable de l’altération de la détection hypothalamique du glucose car sa réversion avec une injection intracérébroventriculaire (ICV) de composé C, n’a pas permis de restaurer la sécrétion d’insuline en réponse à l’hyperglycémie cérébrale. De même, une injection ICV de leptine induisant l’activation de STAT3 n’a pas permis de restaurer la sécrétion d’insuline en réponse à l’hyperglycémie cérébrale. Enfin, la diminution de l’activation d’AKT suggère une résistance centrale à l’insuline. Ces résultats démontrent pour la première fois que l’altération hypothalamique de la signalisation ROS, de la fission et de la respiration mitochondriale, sont présent chez les rats exposés pendant 3 semaines à un régime HFHS. Ces défauts précoces hypothalamiques pourraient ainsi participer à un défaut primaire du contrôle de la sécrétion d’insuline, et finallement, à l’installation d’un phénotype diabétique. / The hypothalamus participates in the control of energy homeostasis by detecting circulating nutrients, such as glucose. The mediobasal hypothalamus (MBH), in particular, senses hyperglycemia and initiates physiological responses, e.g., insulin secretion via the autonomous (vagal) nervous system. We have recently demonstrated that glucose sensing requires mitochondrial reactive oxygen species (mROS) signaling heavily dependant on mitochondrial fusion and fission (dynamics). Recently, genetic models have associated some of these dynamics within the MBH to their obesogenic susceptibility. The aims of my thesis were first to establish a model that only presents a hypothalamic glucose sensing defect induced by a high fat high sucrose (HFHS) feeding in rats. After caracterizing this model, our objectives were to determine whether modulating the diet affects mitochondrial dynamics, and thus, mROS signaling, through the mitochondrial respiratory function in the hypothalamus. We finally reversed some dysregulated metabolic signalings potentially involved in mitochondrial dynamics in order to reverse the phenotype observed in HFHS fed rats. Our results demonstrate that after 3 weeks of HFHS feeding, rats had a normal body weight despite an increase in the fat mass compared to control rats. HFHS fed rats displayed also a glucose intolerance, increased fasting glycemia but no modification of fasting insulinemia. Hypothalamic glucose sensing induced insulin secretion, measured after an intra-carotid glucose injection towards the brain that only increases brain glycemia without alteration in peripheral glycemia, was drastically decreased. However, glucose stimulated insulin secretion in isolated islets was not different compared to controls. These defects correlate with a decrease of MBH ROS production in response to glucose, with no modification in the redox status. Efficiency of hypothalamic mitochondrial respiration was evaluated using oxygraphy, and results showed mitochondrial respiratory deficiencies in HFHS fed rats. The fission protein DRP1 exhibited decreased mitochondrial translocation in the MBH in response to glucose, suggesting decreased mitochondrial fission. The increase of AMPK activation in the hypothalamus was not responsible for the alteration of hypothalamic glucose sensing since its reversal with an intracerebroventricular (ICV) injection of compound C failed to restore brain hyperglycemia induced insulin secretion. Likewise, an ICV injection of leptin that induced STAT3 activation also failed to restore brain hyperglycemia induced insulin secretion. Finally, the decrease in AKT activation suggested a central insulin resistance. These results demonstrate for the first time that hypothalamic alteration of mitochondrial ROS signaling, fission and respiration were present in rats exposed to a 3 weeks HFHS diet. Such hypothalamic glucose sensing defects are early events preceding those in islets. These early but drastic hypothalamic modifications could participate in a primary nervous defect of the control of insulin secretion, and finally, the etablishment of a diabetic phenotype.