Nuclear Hormone Receptors (NRs) are hormone dependent DNA binding proteins that translate physiological signals into gene expression. Gene products have been identified that belong to the NR superfamily on the basis of homology. However, the endogenous and /or synthetic ligands that regulate their activity remain unknown, consequently, this subgroup of proteins are designated as orphans). Retinoic acid receptor related orphan receptors alpha and gamma(RORα and γ) are orphan NRs, and are preferentially expressed in skeletal muscle a major metabolic tissue and other tissues including pancreas, thymus, prostate, liver, adipose and testis. Surprisingly, the specific roles of ROR α and γ in skeletal muscle, a peripheral tissue, have not been examined. Muscle is one of the most energy demanding tissues which accounts for ~40% of the total body mass and energy expenditure, ~75% of glucose disposal and relies heavily on β-oxidation of fatty acids. We hypothesize that ROR α and γ regulates metabolism in this major mass lean tissue. Initially, this hypothesis was examined by “gain and loss” of function studies in an in-vitro mouse skeletal muscle cell culture model. Previous in vitro studies analyzed the role of RORα in the regulation of lipid homeostasis in skeletal muscle cells. We similarly conducted in vitro RORγ gain and loss of function studies in skeletal muscle cells to understand the role of this isoform in metabolism. We utilized stable ectopic over-expression of VP16-RORγ (gain of function), native RORγ and RORγΔH12 (loss of function) vectors to modulate RORγ mRNA expression and function. Candidate driven expression profiling of lines that ectopically express the native and variant forms of RORγ suggested that this orphan NR has a function in regulating the expression of genes that control lipid homeostasis (fatty acid-binding protein 4), CD36 (fatty acid translocase), lipoprotein lipase and uncoupling protein 3), carbohydrate metabolism (GLUT5 (fructose transporter), adiponectin receptor 2 and interleukin 15 (IL-15)) and muscle mass (including myostatin and IL-15). Interestingly, our study revealed a function for RORγ in the pathway that regulates production of reactive oxygen species which was also correlated with increased expression of UCP3 mRNA. Subsequently, we conducted in vivo studies with mouse models displaying global and muscle specific perturbation in RORα expression and function to elucidate the physiological role of this orphan NR in the context of metabolism.Along these lines, we characterized homozygous staggerer mice (sg/sg) in the context of lipid, carbohydrate and energy homeostasis. Staggerer mice were characterized by decreased and dysfunctional retinoic acid receptor-related orphan receptor alpha (RORα) expression. We observed decreases in serum (and liver) triglycerides and total and high density lipoprotein serum cholesterol in staggerer mice. Moreover, the staggerer mice were associated with reduced adiposity, decreased fat pad mass and adipocyte size. Candidate-based expression profiling demonstrated that the dyslipidemia in staggerer mice was associated with decreased hepatic expression of SREBP-1c, and the reverse cholesterol transporters, ABCA1 and ABCG1. This was consistent with the reduced serum lipids. Furthermore, the lean phenotype in staggerer mice was also characterized by significantly increased expression of PGC-1α, PGC-1β, and lipin1mRNAin liver and white and brown adipose tissue from staggerer mice. In addition, we observed a significant 4-fold increase in β2-adrenergic receptor mRNA in brown adipose tissue. Finally, dysfunctional RORα expression protects against diet-induced obesity. Following a 10-week high fat diet, wild-type (but not sg/sg) mice exhibited a ~20% weight gain, increased hepatic triglycerides, and notable white and brown adipose tissue accumulation. In summary, these changes in gene expression (that modulate lipid homeostasis) in metabolic tissues were involved in decreased adiposity and resistance to diet induced obesity in the sg/sg mice, despite hyperphagia. Finally, we specifically modulated RORα signaling in skeletal muscle by the targeted over-expression of truncated RORαΔDE (lacking the ligand binding domain) driven by a myogenic specific promoter, to investigate the contribution of this peripheral tissue to the RORα phenotype. Interestingly, transgenic heterozygous animals exhibit increased fasting blood glucose levels and mild glucose intolerance. Expression profiling (and western analysis) identified perturbations in the insulin signaling cascade. For example, we observed attenuation of p85alpha (PI3K) and Akt2 (mRNA and protein) expression; and insulin dependent induction of phospho-Akt2. In concordance, significantly increased levels of active phospho-AMPK were detected in the muscle of transgenic mice (relative to wt littermates). The increase in phospho-AMPK correlated with: (i) the suppression of lipogenic gene expression; and (ii) increased phospho-ACC and activation of genes involved in fatty acid oxidation in the skeletal muscle of transgenic animals. In conclusion, we suggest these orphan nuclear receptors (RORα and γ) are key modulators of fat and carbohydrate homeostasis in skeletal muscle tissue. Specifically, we propose that, RORα plays vital role in fat accumulation in adipose tissue and insulin mediated glucose homeostasis in skeletal muscle. Therefore we suggest that selective muscle specific RORα modulators may have utility in the treatment of type2 diabetes and obesity.

Thermogenic brown and beige adipocytes convert chemical energy to heat by metabolizing glucose and lipids via uncoupling protein 1 (Ucp1), a process known as non-shivering thermogenesis. Serotonin (5-HT) neurons in the ventral medulla are known to regulate sympathetic efferent neurons in the intermediolateral nucleus (IML) necessary to maintain brown adipose tissue (BAT) activity. Previous studies show that mice lacking central 5-HT neurons are incapable of maintaining body temperature in cold, ambient conditions. Due to this direct linkage between 5-HT and thermoregulation, we hypothesized that central 5-HT neurons may be vital to the regulation of brown and beige adipocyte activity. Given that BAT consumes large amounts of substrate when active, we also hypothesized that inactivation of BAT due to deletion of the regulatory neural circuitry (5-HT neurons) would cause metabolic dysregulation. To test this, we generated mice in which the human diphtheria toxin (DT) receptor was selectively expressed in central 5-HT neurons under control of a Pet-1 promoter. Pet-1 is a transcription factor selectively located in mature, central 5-HT neurons. Coincidentally, some cells within pancreatic islets also express Pet-1, and contain adequate machinery to produce, release, and uptake 5-HT. Systemic treatment with DT eliminated 5-HT neurons and caused loss of thermoregulation, BAT steatosis, and a >50% decrease in Ucp1 expression in BAT and beige fat, indicative of reduced thermal production. In parallel, blood glucose increased 3.5-fold, free fatty acids 13.4-fold and triglycerides 6.5-fold. Intracerebroventricular (ICV) treatment with 1/30th the systemic dose of DT induced an even greater thermoregulatory impairment. The metabolic deficits following systemic DT treatment indicate that central 5-HT neurons are essential for proper metabolic regulation. However, such high levels of glucose and lipids also indicate failure of the pancreatic endocrine program following systemic treatment, likely due to moderate destruction of β-cells expressing Pet-1 and the DT receptor. Because ICV treatment caused even greater thermoregulatory and metabolic deficits, where little, if any, of the toxin would spread systemically, central 5-HT neurons are clearly essential for normal central regulation of metabolism. Interestingly, similar BAT and beige fat defects occurred in Lmx1bf/f/p mice, in which 5-HT neurons fail to develop in utero. Assessment of systemically treated animals using a euglycemic/hyperinsulinemic clamp showed extensive fasting hyperglycemia and systemic insulin resistance, coinciding with reduced glucose uptake in skeletal muscle and BAT. The hyperinsulinemic clamp failed to suppress hepatic glucose and fatty acid production, leading to the conclusion that loss of central 5-HT neurons disrupts central hepatic regulation. In attempts to induce BAT thermogenesis and metabolism, we optogenetically stimulated 5-HT neurons in the rostral raphe pallidus and measured BAT and body temperature along with blood glucose. Unfortunately, these stimulations were incapable of increasing BAT temperature and lowering blood glucose, perhaps limiting therapeutic potential of these 5-HT neurons. We conclude that 5-HT neurons are major players in central regulation of metabolic homeostasis, in part through recruitment and activation of brown and beige adipocytes and hepatic substrate production. Data also suggest that 5-HT neurons regulate glucose homeostasis via undefined neural mechanisms independently of BAT activity and pancreatic insulin secretion. Cumulative data on central 5-HT neurons indicate they are master regulators of whole-body metabolism.

L’accumulation de cellules spumeuses dans l’intima des artères est le point critique de l’initiation de l’athérosclérose. L’accumulation de cholestérol et l’activation des voies pro-inflammatoires sont responsables de l’acquisition par les macrophages de ce phénotype délétère. Les dérivés oxydés du cholestérol accumulés vont stimuler les récepteurs nucléaires LXR et incidemment l’efflux de cholestérol athéroprotecteur, tandis que la phagocytose de cholestérol cristallisé dans la lésion causera une perturbation du trafic vésiculaire qui activera l’inflammasome NLRP3, verrou de l’inflammation IL-1. Cette étude a pour but de décrypter ces voies dans le macrophage humain. La stimulation de l’efflux de cholestérol par un agoniste LXR dans le macrophage humain m’a est médiée par l’activation transcriptionnelle LXRα-dépendante d’ARL7 - qui permet le transport du cholestérol libre de la membrane des endosomes et lysosomes aux radeaux lipidiques – et du transporteur ABCA1 – l’exportant vers les HDL, lipoprotéines athéroprotectrices. L’activation du complexe multi-protéique NLRP3 dans le macrophage, et la sécrétion des cytokines délétères IL-1β et IL-18, est également réprimée par l’agoniste LXR. L’activation des cathepsines B et L par l’enzyme AEP est aussi nécessaire pour l’activation de NLRP3 par des cristaux. Inactiver l’AEP protège ainsi contre le développement de l’athérosclérose. Ces travaux ont démontré la nécessité des études chez l’humain pour la mise en évidence des mécanismes moléculaires et la nécessité d’intégrer les signalisations lipidiques et inflammatoires étroitement liées dans la cellule spumeuse. / Foam cell accumulation in arterial walls is the critical initiating event of atheroma plaque development. Macrophages acquire this phenotype by cholesterol ester accumulation and pro-inflammatory signaling pathways activation. Oxydized form of cholesterol activates LXR and subsequently atheroprotective cellular cholesterol efflux. In parallel, crystallized cholesterol phagocytosis will impair vesicular traffic, activating NLRP3 and deleterious IL-1 cytokines release. Here we describe further those pathways in human macrophages and to evidence new key factors in atherosclerosis development. First, stimulation of cellular cholesterol efflux to ApoA-I and HDL from human macrophage by LXR agonist is LXRα-dependent. Transcriptional activation of ARL7 increases cholesterol transport from endolysosomal membrane to efflux-prone plasmic membrane pools, lipid rafts. From there, cholesterol will be exported on ApoA-I containing lipoproteins by ABCA1 transporter, whose expression is stimulated by LXRα agonists as well. Cholesterol crystals phagocytose will lead to inflammasome NLRP3 activation, leading to pro-atherogenous IL-1β and IL-18 secretion. We first showed LXR agonist GW3965 role in repressing transcription of NLRP3 partners. Also, we evidenced the importance of cathepsin B and L maturation by asparagin endopeptidase (AEP) in human macrophages, and atheroprotective properties of AEP silencing. Overall, this work demonstrated the necessity of using human models to confirm murine data about molecular mechanisms. Also, it is important to integrate lipid homeostasis and inflammation signaling in foam cells, for there is a strong molecular link between both.

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Le MGDG (monogalactosyldiacylglycerol) et le DGDG (digalactosyldiacylglycerol) sont les lipides les plus abondants des membranes du chloroplaste. Ils sont synthétisés exclusivement dans l'enveloppe plastidiale par l'action des MGDG synthases (MGD1, MGD2 et MGD3) et des DGDG synthases (DGD1 et DGD2). Les galactolipides sont essentiels pour la structuration des photosystèmes et la biogenèse des thylacoïdes. En carence de phosphate, les galactolipides deviennent une source de lipides pour composer certaines membranes en dehors du chloroplaste. Suivant une stratégie de criblage pharmacologique à haut débit, une nouvelle molécule appelée galvestine-1 a pu être identifiée et caractérisée comme un inhibiteur des MGDG synthases. La galvestine-1 agit par compétition avec le diacylglycérol. Cet outil moléculaire permet donc de perturber le système complet constitué par l'ensemble des réactions de synthèses, de conversions et de trafics lipidiques, aboutissant à cet état stable que nous appelons homéostasie des lipides. Le but de cette thèse est de mettre en évidence, à l'aide de la galvestine-1, de nouveaux acteurs ou nouvelles voies permettant l'établissement de l'homéostasie lipidique à l'échelle de la cellule végétale. Pour cela, j'ai réalisé un criblage de mutants EMS (ethyl methanesulfonate) dans le but d'isoler des mutants résistants à la galvestine-1 et d'identifier les gènes mutés conférant cette résistance. Des données transcriptomiques (Affymetrix genome array genechip, ATH1) d'Arabidopsis thaliana traité en présence de galvestine-1 ont par ailleurs été obtenues avant le début des travaux de thèse. Ces données ont permis de cibler des gènes dont l'expression variait et possiblement impliqués dans l'homéostasie lipidique. En parallèle de l'approche sans a priori, j'ai donc réalisé une étude suivant une stratégie de gènes candidats sur ALA10, un gène codant pour une flippase putative, sur-exprimé après traitement à la galvestine-1 et en carence de phosphate. Le second volet de cette thèse vise donc à comprendre la relation entre l'expression d'ALA10 et les gènes impliqués dans la synthèse des galactolipides chez la plante. / MGDG (monogalactosyldiacylglycerol) and DGDG (digalactosyldiacylglycerol) are the most abundant membrane lipids of the chloroplast. They are synthesized exclusively in the chloroplast envelope by the action of MGDG synthases (MGD1, MGD2 and MGD3) and DGDG synthases (DGD1 and DGD2). Galactolipids are known to be essential for the structure (and function) of the photosystems and for the biogenesis of thylakoids. In phosphate deprivation, galactolipids become a source of lipid for other cell membranes, outside the chloroplast. Based on a high throughput chemical screen, a new molecule called galvestine-1 has been identified and characterized as an inhibitor of MGDG synthases. Galvestine-1 competes with the binding of the diacylglycerol substrate to MGDs. This molecular tool can be used to disturb the system comprising all lipid biosynthesis reactions, conversions, and lipid trafficking, responsible for the membrane lipid steady state observed at the whole cell level, or membrane lipid homeostasis. Perturbation of the system occurs at the level of MGDG synthases. The aim of this thesis is to use the effect of galvestine-1 to identify new actors or new pathways involved in the control of lipid homeostasis in plant cells. To this purpose, I designed and performed a screening of a collection of EMS (ethyl methanesulfonate) mutants, in order to isolate galvestine-1-resistant mutants and to identify mutated genes conferring this resistance. Transcriptomic data (Affymetrix genome array genechip, ATH1) of Arabidopsis thaliana treated in the presence of galvestine-1 had been obtained prior to the PhD project. These data were used to identify genes whose expression varied and possibly involved in lipid homeostasis. Based on a complementary candidate gene approach, I focused on Ala10, a putative flippase, which gene is over-expressed after treatment with galvestine-1 and following phosphate deprivation. The purpose of this second part of this thesis is to understand the relationship between the expression of ALA10 and genes involved in galactolipid synthesis in plants.

The mammalian brain is the most cholesterol-rich organ of the body, requiring in situ de novo cholesterol synthesis to maintain its cholesterol requirement. Defects in brain cholesterol homeostasis are implicated in cognitive deficits related to aging and in neurodegenerative diseases such as Alzheimer's Disease (AD). Oxysterol-binding protein (OSBP) - related proteins are highly conserved cytosolic proteins that coordinate lipid homeostasis by regulating cell signaling, inter-organelle membrane contact sites and non-vesicular transport of cholesterol. Previously, ORP6, a poorly characterized member of this family, was found to be part of complex transcriptional cascade coordinated by SBREP2 and emerged as a novel regulator of intracellular cholesterol trafficking in hepatocytes and macrophages. Yet how ORP6 regulates these pathways and its function in the brain where it is most highly expressed is unknown. Here, we show that ORP6 is highly expressed in the brain, where it exhibits spatial and cell-type specific expression. ORP6 expression is enriched in the hippocampus and caudal-putamen brain regions, specifically within neurons and astrocytes. ORP6 knockdown in astrocytes altered the expression of cholesterol biosynthesis, cholesterol efflux and cholesterol esterification genes, resulting in the accumulation of esterified cholesterol within cytoplasmic lipid droplets and reduced cholesterol efflux highlighting a role for ORP6 in astrocytic cholesterol metabolism. We also present in this thesis, the newly generated second viable ORP family member knockout mouse. ORP6 ablation in mice results in the dysregulation of brain and whole-body lipid homeostasis, increased Aβ deposition in the brain and neuroanatomical alterations. Together, our findings highlight a critical role for cholesterol trafficking proteins in brain cholesterol homeostasis and identify ORP6 as a potential novel target for AD.

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