Lipoprotein lipase (LPL) is the main enzyme hydrolyzing triglyceride-rich lipoproteins in plasma. Proteoglycan-bound LPL on the vascular endothelium represent the functional pool of active enzyme. LPL is regulated in a tissue specific manner according to metabolic demands. Rapid regulation of LPL activity is necessary to provide free fatty acids for storage or energy production. This regulatory mechanism appears to be post-translational and requires synthesis of other protein/proteins. Recently it was demonstrated that angiopoietin-like protein 4 (ANGPTL4) is involved in the metabolism of plasma triglycerides and that it is able to inhibit LPL activity in vitro. These properties were linked to the N-terminal coiled-coil domain of ANGPTL4 (ccd-ANGPTL4), but the mechanism for the inhibition was not known. The aim of this thesis was to investigate the molecular mechanism for inhibition of LPL by ccd-ANGPTL4, to characterize regions in ccd-ANGPTL4 that are important for inactivation of LPL and to study the role of ANGPTL4 for regulation of LPL in vivo. Binding of ccd-ANGPTL4 to LPL was demonstrated by several methods, including surface plasmon resonance. The interaction was transient and resulted in conversion of the enzyme from catalytically active dimers to inactive monomers with decreased affinity for heparin. We have shown that ANGPTL4 mRNA in rat adipose tissue turns over rapidly and that changes in the ANGPTL4 mRNA abundance were inversely correlated to LPL activity, both during the fed to fasted and the fasted to fed transitions. We conclude that ANGPTL4 is a fasting-induced controller of LPL in adipose tissue, acting extracellularly on the native conformation of LPL in an unusual fashion, like an unfolding molecular chaperone. Site directed mutagenesis was used to explore regions in ccd-ANGPTL4 important for inactivation of LPL, and for binding of ANGPTL4 to heparin. Others had shown that ccd-ANGPTL4 forms higher oligomers. Structure prediction analyses demonstrated that the coiled-coil domain of ccd-ANGPTL4 probably forms three consecutive α-helices with strong hydrophobic faces, and that there are clusters of positively charged residues both on the helices and in intervening sequences. We made replacements of hydrophobic residues, positively charged residues, cysteine residues and negatively charged residues in ccd-ANGPTL4. In addition, helix-breaking proline residues were introduced in all three helices. We found that hydrophobic residues are important for oligomer formation. The higher oligomers appeared to be stabilized by disulfide bonds, but cysteines are not crucial for oligomerization. Introduction of Pro-residues in the first and second helix prevented formation of higher oligomers and reduced the ability of ccd-ANGPTL4 to inactivate LPL. We found that negatively charged residues in ccd-ANGPTL4 are important for inactivation of LPL. A heparin binding site was localized in the C-terminal end of ccd-ANGPTL4 (amino acid residues 114-140). To investigate whether LPL is differently processed in different depots of adipose tissue we measured the levels of LPL mRNA, protein and activity in omental and subcutaneous adipose tissue in human subjects undergoing elective surgery. Our results show that, although the expression level of LPL was higher in subcutaneous adipose tissue, the specific LPL activity (ratio of activity over the LPL protein mass) was higher in omental adipose tissue. Interestingly, the levels of ANGPTL4 mRNA were lower in omental compared to subcutaneous adipose tissue in most of the studied subjects. This difference can possibly explain the higher specific activity of LPL in omental adipose tissue and indicated that ANGPTL4 is involved in regulation of LPL activity also in humans. LPL produced by macrophages in the artery wall promotes local accumulation of lipids in these cells, and thereby plays an important role in development of atherosclerosis. The known association between type 2 diabetes and atherosclerosis forwarded us to study production of LPL by THP-1 macrophages under hyperglycemic conditions and under treatment with a peroxisome proliferator-activated receptor delta (PPARδ) agonist (GW501516). We found that LPL activity (but not LPL mass) produced by macrophages was decreased by GW501516. The loss of LPL activity coincided with increased level of ANGPTL4 mRNA, indicating that the agonist regulates LPL activity through expression of ANGPTL4. This effect was even more pronounced in cells grown under hyperglycemic conditions. Our data suggest that a suitable PPARδ agonist, like GW501516, may have protective effects against development of atherosclerosis in subjects with diabetes type 2.

The thesis is aimed at understanding of molecular mechanisms of autophagic degradation of peroxisomes (pexophagy) in the yeast Yarrowia lipolytica. This microorganism has been extensively used to explore peroxisome biogenesis (Titorenko and Rachubinski, 2000). Gunkel et al. (1999) intoduced Y. lypolitica into pexophagy studies. However, the field of pexophagic research on this yeast remains quite unexplored. This work involved following tasks: (1) the development and optimization of Y. lipolytica as a model system to study peroxisome degradation; (2) Y. lipolytica genes and proteins implicated in pexophagy should be found and characterized; (3) a proper easy-to-handle selection procedure to isolate novel peroxisome degradation-deficient(pdd) mutants of Y. lipolytica should be devised.

Peroxisome biogenesis is complex, involving a diverse array of intracellular pathways and mechanisms that mediate their biogenesis and cellular functions. Relevant to our understanding of peroxisome biogenesis is the utilization of peroxisomal membranes for viral genome replication as observed in plant cells infected by several members of the Tombusviridae family of positive-strand RNA viruses. Tomato Bushy Stunt Virus (TBSV), for instance, usurps an array of host factors that facilitate the transformation of peroxisomes into peroxisomal multivesicular bodies (pMVB) the sites of viral RNA replication. In this study, pMVB topology and biogenesis was investigated using transmission electron and epifluorescence microscopy of tobacco and wildtype or mutant budding yeast that were transformed with TBSV replicase proteins and a defective interfering viral RNA. Overall, the results suggest that host-virus interactions specifically associated with Endosomal Sorting Complex Required for Transport (ESCRT) and lipid metabolism are involved in TBSV replication and pMVB biogenesis.

[Truncated abstract] Glucocorticoids are critical for the maturation of the fetus late in pregnancy. Indeed, clinical administration of glucocorticoids is used to accelerate fetal lung maturation in mothers at risk of pre-term delivery. Increased glucocorticoid exposure, however, can have detrimental effects on fetal and placental growth and increase the risk of disease in later life. Many studies have focused on the effect of an increase in the transplacental passage of glucocorticoids on both fetal growth and subsequent postnatal development. But there is a growing body of evidence to suggest that the impact of glucocorticoids on fetal growth is mediated, in part, via their direct effects on the placenta . . . Overall, these studies quantify the labyrinth zone-specific increases in placental expression of PPARG and VEGF in association with a marked increase in vascularisation observed near term. Furthermore, this study demonstrates for the first time that these increases in gene expression are prevented by maternal dexamethasone treatment which also inhibits growth of the fetal capillary network. Elevated expression of SFRP4 in the regressing basal zone late in gestation and in both placental zones after dexamethasone-induced placental growth restriction is consistent with a role for SFRP4 in glucocorticoid-mediated inhibition of wnt signalling. Collectively, the data presented in this thesis show that glucocorticoid inhibition of fetal growth is mediated in large part via effects on the placenta, specifically through inhibition of signals that promote proliferation and vascularisation.

Os receptores ativadores da proliferação de peroxissomos (PPARs) pertencem à superfamília de receptores nucleares que funcionam como fatores transcricionais. Eles exercem um papel fundamental em processos que envolvem, principalmente, o metabolismo lipídico, em resposta à ativação por ligantes naturais e sintéticos como os ácidos graxos e os fibratos, respectivamente. A crescente descoberta de importantes funções fisiológicas, coordenadas pelos PPARs, e a necessidade de se conhecer como os agonistas, atualmente disponíveis, atuam nesses receptores, têm incitado pesquisas que vislumbram sua melhor exploração nos tratamentos de doenças metabólicas e inflamatórias, minimizando os efeitos adversos de ativações suprafisiológicas. Nesse cenário, o presente trabalho buscou compreender melhor as bases estruturais envolvidas nas funções atribuídas aos PPARs e explicar como as interações com seus ligantes ocorrem. Para isso, foram realizadas a subclonagem do domínio de ligação ao ligante do PPARα, sua expressão e purificação, seguidas de ensaios cristalográficos e biofísicos, além da abordagem de testes funcionais. Uma vez que a formação de oligômeros está relacionada à funcionalidade desses receptores, foram abordados estudos de oligomerização dos PPARs α e γ, compreendendo tanto o processo de homo- quanto o de heterodimerização. Os ensaios de cristalização do hPPARα LBD complexado a ligantes naturais e sintéticos, resultaram em estruturas cristalográficas que permitiram a identificação dos resíduos envolvidos no reconhecimento dos ligantes e a caracterização de sítios de ligação nunca antes descritos. A presença de ligantes nessas regiões afeta a conformação da proteína e, consequentemente, a modulação de sua função e o recrutamento da maquinaria transcricional. Adicionalmente, as estruturas cristalográficas da proteína complexada a ácidos graxos auxiliaram na compreensão de como essa importante classe de ligantes naturais possui efeitos farmacológicos similares aos de ligantes sintéticos. Esses resultados têm imediato impacto na procura racional de agonistas para esses receptores e se inserem em uma perspectiva de promoção do desenvolvimento científico-tecnológico na área de endocrinologia molecular. / The peroxisome proliferation-activated receptors (PPARs) belong to the nuclear receptors superfamily, acting as transcriptional factors. They play a key role in processes involving essentially lipid metabolism in response to activation by natural and synthetic ligands such as fatty acids and fibrates, respectively. The rising discovery of important physiological functions coordinated by PPARs and the necessity to know how the currently available agonists act on these receptors, have encouraged researches envisioning a better receptor exploration in the treatment of metabolic and inflammatory diseases, minimizing the adverse effects of supraphysiological activations. In this scenario, the present study aimed to better understand the structural basis involved in PPARs functions and elucidates how the interactions with their ligands takes place. For this, the ligand-binding domain of PPARα was subjected to subcloning, expression and purification steps, followed by crystallographical and biophysical assays, in addition to functional testing approaches. Since the degree of oligomerization is related to the functionality of these receptors, oligomeric studies of PPARs α and γ oligomerization were also achieved, comprising both homo- and hetero-dimerization. The co-crystallization assays of hPPARα LBD complexed with natural and synthetic ligands resulted in crystallographic structures that allowed the identification of residues involved in ligand recognition and the characterization of novel binding sites. The presence of ligands in these regions affects the conformation of the protein and thereby modulates their function and transcriptional machinery recruitment. Additionally, the crystallographic structures of the protein complexed to fatty acids were valuable for the understanding of how this important class of natural ligands has similar pharmacological effects to those of synthetic ligands. These results have direct impact on rational agonists design to these receptors and are inserted in a perspective of scientifical promotion and technological development in the field of molecular endocrinology.

Defeitos peroxissomais estão associados a diversas doenças complexas. O peroxissomo é responsável pela beta-oxidação de ácidos graxos, quando é gerado peróxido de hidrogênio. A catalase A, de ocorrência peroxissomal, é frequentemente considerada a única defesa antioxidante dessa organela, porém, em diversos organismos, a ausência dessa enzima não acarreta uma alteração fenotípica clara. Em Saccharomyces cerevisiae, linhagens mutantes deficientes em catalase A (Δcta1) apresentam viabilidade muito similar à linhagem selvagem correspondente. Trabalhamos com a hipótese de que peroxidases baseadas em cisteína compensam a ausência de catalase A, contribuindo para a detoxificação de peróxidos provenientes do peroxissomo. De fato, linhagens com os genes para as peroxirredoxinas Ahp1 e Tsa1 nocauteados mostraram-se mais sensíveis a hidroperóxido de terc-butila (tBHP) em comparação a linhagem selvagem. A linhagem de levedura deficiente nas cinco peroxirredoxinas (prxΔ) mostrou-se ainda mais sensível a tBHP. Em relação ao estresse induzido por peróxido de hidrogênio, a prxΔ apresentou maior sensibilidade do que as linhagens selvagem e mutantes com deleções simples, apesar da presença de catalases (peroxissomal e citossólica). Esses dados estão de acordo com resultados obtidos no nosso grupo demonstrando um aumento da expressão de genes referentes às peroxirredoxinas Ahp1, Prx1 e Tsa2 em células Δ cta1 crescidas em condições de alta atividade peroxissomal (oleato), indicando uma cooperação entre catalase e peroxirredoxinas na proteção antioxidante. A peroxirredoxina Ahp1 pode apresentar localização organelar (possivelmente mitocondrial ou peroxissomal), o que sugere que Ahp1 pode ser um componente relevante da defesa contra oxidantes provenientes do peroxissomo. No entanto, a linhagem Δ ahp1, normalmente sensível a peróxido orgânico, apresentou ganho de resistência na ausência de atividade de catalase (com a adição de ATZ e na linhagem duplo-mutante Δcta1/ahp1), indicando a existência de uma via antioxidante compensatória induzida pela ausência de catalase A. A construção das linhagens duplo-mutantes Δcta1/ahp1, Δcta1/tsa1, Δcta1/tsa2, Δ cta1/prx1 e Δcta1/dot5 foi realizada com o objetivo de investigar mecanismos compensatórios entre enzimas que podem proteger a levedura contra os oxidantes provenientes do peroxissomo. Para tanto, foram realizados ensaios de viabilidade comparativa em condições de alta atividade peroxissomal. Além disso, os níveis comparativos de proteínas carboniladas foram analisados nessas linhagens. Os resultados indicaram maior sensibilidade a peróxido e maiores níveis de danos oxidativos na linhagem Δcta1/tsa2, apontando a peroxirredoxina Tsa2 como candidata a importante componente da via antioxidante de compensação à ausência de catalase A. Nesses ensaios, também foram utilizadas a linhagem quíntupla mutante (prxΔ) e uma linhagem deficiente nas cinco peroxirredoxinas e três glutationa peroxidases - deficiente em oito tiól-peroxidases baseadas em cisteína (Δ8). A comparação das linhagens prxΔ e Δ8 com as linhagens selvagem, simples-mutantes e duplo-mutantes evidenciou a importância das peroxirredoxinas na defesa antioxidante da célula e o fato das tiól-peroxidases serem imprescindíveis em condições de estresse oxidativo. Ao examinar a expressão gênica de TSA2 em células crescidas em oleato, foi verificada a indução do gene na ausência de catalase A, em condição basal. Os resultados obtidos indicam a existência de uma eficiente via de defesa antioxidante, na qual estão envolvidas tiól-peroxidases, que compensa a ausência de catalase A na célula e que protege leveduras contra estresse induzido tanto por peróxido de hidrogênio como peróxido orgânico. A peroxirredoxina Tsa2 parece estar envolvida na via compensatória à ausência de catalase peroxissomal através de um mecanismo ainda não esclarecido / Defects in peroxisomes are associated with several complex diseases. Beta-oxidation of fatty acids takes place in these organelles, with the concomitant generation of hydrogen peroxide. Generally, it is assumed that peroxisomal catalase is the enzyme responsible for degradation of hydrogen peroxide, but in several organisms, deletion of its gene results in no clear phenotype. In Saccharomyces cerevisiae, catalase A- null (Δcta1) mutant strains exhibit very similar viability levels when compared with the corresponding wild-type strain. We hypothesized here that Cys-based peroxidases compensate the absence of catalase A, contributing to the detoxification of peroxides derived from the peroxisome. Indeed, null mutante strains for the peroxiredoxins Ahp1 and Tsa1 displayed increased sensitivity for tert-butylhydroperoxide (tBHP) in comparison to the wild type strain. Furthermore, a mutant strain whose five genes for peroxiredoxins were interrupted (prxΔ) was even more sensitive to tBHP. In regards to hydrogen peroxide insult, the prxΔ strain was more susceptible to oxidative stress than the single mutant and wild-type strains, despite the activity of catalases. These data are in agreement with previous results from our group demonstrating increased expression of genes encoding the three peroxiredoxin enzymes: Ahp1, Prx1 and Tsa2 in Δcta1 cells at high peroxisomal activity (media containing oleate). Indeed, a yeast strain deleted of all five peroxiredoxin genes is more sensitive to peroxides than the corresponding wild type cells. These results indicated that catalase and peroxiredoxins cooperate to protect yeast in conditions of high fatty acid intake. There are evidences of an organellar location of Ahp1 (possible peroxisomal or mitochondrial), suggesting it could be a relevant component of antioxidant defense relative to the insult derived from the peroxisome. Nonetheless, the ahp1-null strain (Δahp1), which is usually sensitive to organic peroxide, displayed a gain of resistance in the absence of catalase activity (in the presence of ATZ and in the double-mutant strain Δcta1/ahp1), indicating the existance of a compensatory antioxidant pathway induced in the absence of catalase A. The double-mutant strains Δcta1/ahp1, Δcta1/tsa1, Δcta1/tsa2, Δcta1/prx1 and Δcta1/dot5 were developed in order to elucidate the identity of the enzymes that cooperate to protect yeast against oxidative insult derived from the peroxisome. To this end, comparative viability assays in conditions of high peroxisomal activity were realised, as well as assays in comparative total protein carbonyl levels. Among the double-mutant strains, Δcta1/tsa2 displayed higher sensibility to peroxide and higher levels of oxidative damage, suggesting that the peroxiredoxin Tsa2 may be an important component in the antioxidant pathway that compensates the lack of catalase A. In addition, a quintuple mutant strain, lacking all peroxiredoxins, and a mutant strain lacking all eight Cys-based, thiol peroxidases were used in these assays. The comparison of these strains with the wild-type, single-mutant and double-mutant strains demonstrated the importance of peroxiredoxins in the cellular antioxidant defence and that thiol-peroxidases are vital in conditions of oxidative stress. The expression of the TSA2 was induced in the absence of catalase A in cells grown in oleate and with no exogenous oxidants. The results suggest the existence of an efficient pathway of antioxidant defense, involving thiol-peroxidases, which compensates the absence of catalase A in the cell and protects yeast against oxidative stress induced by both hydrogen peroxide and organic peroxide. The peroxiredoxin Tsa2 may be involved in the antioxidant pathway that compensates the absence of peroxisomal catalase through an unknown mechanism.

Marine environments are under constant pressure from anthropogenic pollution. Chemical pollutants are introduced into the aquatic environment through waste disposal, sewage, land runoff and environmental exploitation (harbours, fisheries, tourism) leading to disastrous effects on the marine wildlife. Developmental malformations, reproduction failure including sex changes and high death rates are commonly observed in aquatic animal populations around the world. Unfortunately, the underlying molecular mechanisms of these pollution effects, in particular for marine invertebrate species, are often unknown. One proposed mechanism through which environmental pollution affects wildlife, is the disruption of nuclear receptors (NRs), ligand-binding transcription factors in animals. Environmental pollutants can directly interact with nuclear receptors, inducing incorrect signals for gene expression and subsequently disrupt developmental and physiological processes. Elucidation of the exact mechanism in invertebrates, however, is sparse due to limited understanding of invertebrate endocrinology and molecular regulatory mechanisms. Here, I have investigated the presence, expression and function of NRs in the Pacific oyster, Crassostrea gigas, and explored their interrelation with known environmental pollutants. Using a suite of molecular techniques and bioinformatics tools I demonstrate that the Pacific oyster possesses a large variety of NR homologs (43 NRs), which display individual expression profiles during embryo/larval development and supposedly fulfil distinct functions in developmental and physiological processes. Functional studies on a small subset of oyster NRs provided evidence for their ability to regulate gene expression, including interactions with DNA, other NRs or small molecules (ligand-binding). Oyster receptors also show a high likeliness to be disrupted by environmental pollutants. Computational docking showed that the retinoid X receptor ortholog, CgRXR, is able to bind and be activated by 9-cis retinoic acid and by the well-known environmental contaminant tributyltin. A potential interaction between tributyltin and the peroxisome proliferator-activated receptor ortholog CgPPAR has also been found. In addition, exposure of oyster embryos to retinoic acids and tributyltin resulted in shell deformations and developmental failure. In contrast, computer modelling of another putative target for pollutants, the retinoic acid receptor ortholog CgRAR, did not indicate interactions with common retinoic acids, supporting a recently developed theory of loss of retinoid binding in molluscan RARs. Sequence analyses revealed six residues in the receptor sequence, which prevent the successful interaction with retinoid ligands. In conclusion, this investigative work aids the understanding of fundamental processes in invertebrates, such as gene expression and endocrinology, as well as further understanding and prediction of effects of environmental pollutants on marine invertebrates.

La stéatohépatite non alcoolique (NASH) est une maladie du foie à évolution clinique grave, dont la prévalence est en constante progression. La stéatohépatite non alcoolique est caractérisée par un dépôt excessif de lipides dans les hépatocytes (stéatose) associé à une inflammation chronique, au contraire de la stéatose hépatique (NAFLD), manifestation initiale mais bénigne d'un dérèglement métabolique. Le NASH augmente le risque de progression vers la fibrose, la cirrhose et le carcinome hépatocellulaire et ne peut être soigné que par une greffe hépatique. Le risque de développer un diabète de type est aussi significativement augmenté chez les patients atteints de NASH. PPAR⍺ est un récepteur nucléaire connu pour réguler l'utilisation des acides gras dans le foie et réprimer les voies de signalisation pro-inflammatoires. [...]Nous avons conçu mutant de PPAR⍺ dont l'activité de liaison à l'ADN est abolie. La comparaison de ses activités transcriptionnelles in vitro avec le PPAR⍺ non muté démontre que les activités de contrôle du métabolisme sont abolies pour ce mutant, alors que les activités anti-inflammatoires restent intactes. [...] Dans cette étude, nous montrons donc pour la première fois que PPAR⍺ inhibe la progression de la stéatose vers le NASH et la fibrose par un mécanisme anti-inflammatoire direct, indépendant de son effet sur le métabolisme lipidique hépatique. / Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent liver condition characterized by excessive lipid deposition in the hepatocytes steatohepatitis (NASH) is hallamarked by chronic inflammation. NASH markedly increases the risk of progression towards liver fibrosis, cirrhosis ans hepatocellular carcinoma. The nuclear peroxisome proliferator-activated receptor alpha (PPAR⍺) regulates hepatic fatty acid utilization and represses pro-inflammatory signaling pathways. [...]Liver-specific expression of wild type or DNA binding-deficient PPAR⍺ in acute and chronic models of inflammation demonstrated that PPAR's anti-inflammatory, but not metabolic activities, result from DNA binding-independent mechanisms in vivo. We futher show that PPAR⍺ inhits the transition from steatosis toward NASH and fibrosis through a direct, anti-inflammatory mechanism independent of its effetc on hepatic lipid metabolism.

L’Adrénoleucodystrophie liée à l’X est une maladie neurodégénérative rare due à des mutations dans le gène ABCD1. Ce gène code un demi-transporteur ABC peroxysomal, impliqué dans l’importation d’acides gras à très longue chaîne. Deux autres demi-transporteurs sont localisés dans la membrane peroxysomale : ABCD2 et ABCD3. La surexpression d’ABCD2 permet de compenser la déficience en ABCD1, ouvrant ainsi des perspectives thérapeutiques. Dans cette optique, l’objectif principal de ma thèse était d’étudier la fonction et la structure d’ABCD2, et plus largement des transporteurs ABC peroxysomaux.Les demi-transporteurs doivent au minimum se dimériser pour constituer un transporteur fonctionnel. Leur dimérisation alternative pourrait moduler leur spécificité de substrat. Afin de tester cette hypothèse, nous avons réalisé des constructions plasmidiques codant différents dimères chimériques, dont la fonctionnalité a été vérifiée par transfection transitoire dans deux modèles cellulaires (fibroblastes humains et levures). D’après nos résultats, ABCD1 et ABCD2 seraient fonctionnels quel que soit leur agencement dimérique. De plus, comme d’autres transporteurs ABC, les transporteurs ABC peroxysomaux pourraient s’oligomériser. En utilisant différentes techniques biochimiques (co-immunoprécipitation, sédimentation sur gradient de sucrose et électrophorèse en conditions natives), sur un modèle cellulaire surexprimant ABCD2-EGFP, nous démontrons qu’ABCD2-EGFP interagit avec ABCD1 et ABCD3, et que les transporteurs ABC peroxysomaux sont capables de s’oligomériser. Il reste désormais à déterminer les facteurs qui contrôlent cette oligomérisation et comprendre la valeur fonctionnelle de ces interactions. / X-linked Adrenoleukodystrophy (X-ALD) is a rare neurodegenerative disease caused by deficiency of the peroxisomal half-transporter ABCD1, implicated in very long chain fatty acids import. Two additional half-transporters are located in the peroxisomal membrane: ABCD2 and ABCD3. Over-expression of ABCD2 is known to compensate for ABCD1 deficiency, making ABCD2 a therapeutic target for X-ALD treatment. In this context, the main objective of my thesis was to investigate the function and the structure of ABCD2, and more broadly, of peroxisomal ABC transporters.Half-transporters must at least dimerize to form a functional transporter. Alternative dimerization could modulate substrate specificity. In order to test this hypothesis, we engineered plasmidic constructs encoding chimeric ABCD dimers, whose functionality has been evaluated by transient transfection in two cell models (human fibroblasts and yeasts). Our results show that, ABCD1 and ABCD2 are functional whatever their dimeric organization. Besides, like other ABC transporters, peroxisomal ABC transporters could oligomerize. By using a multi-technical approach (co-immunoprecipitation, velocity sucrose gradient and native polyacrylamide gel electrophoresis experiments) on stably transfected hepatoma cells expressing ABCD2-EGFP, we demonstrate that ABCD2-EGFP interacts with ABCD1 and ABCD3, and that peroxisomal ABC transporters oligomerize. The perspectives will consist in determining which factors control the oligomerization process and understanding the functional value of these interactions.

Although once considered a simple energy storage depot, the adipose tissue is now known to be a powerful regulator of whole body insulin sensitivity and energy metabolism. This metabolically dynamic organ functions to safely store excess fatty acid as triglyceride, thereby preventing lipotoxicity in peripheral tissues and the development of insulin resistance. In addition, the adipose tissue acts as an endocrine organ and secretes factors, called adipokines, which influence whole body insulin sensitivity and glucose homeostasis. Therefore, understanding adipose tissue development and biology is essential to understanding whole body energy metabolism. A master regulator of adipose tissue development and whole body insulin sensitivity is the nuclear receptor, PPARγ. Due to the importance of this nuclear receptor in maintaining adipocyte function, disruptions in PPARγ activity result in severe metabolic abnormalities, such as insulin resistance and type 2 diabetes. Conversely, PPARγ activation by synthetic agonists ameliorates these conditions, demonstrating the potent control this nuclear receptor has on whole body metabolism. Therefore, understanding how PPARγ expression and activity are regulated, particularly in the adipose tissue, is paramount to understanding the pathogenesis of type 2 diabetes. While there are several synthetic PPARγ agonists available, identifying the endogenous ligand or ligands is still an area of intense investigation. Since fatty acids can induce PPARγ activation, in the first part of this thesis, I screened several fatty acid metabolizing enzymes present in the adipocyte to identify novel modulators of PPARγ activity. These studies revealed that the fatty acid Δ9 desaturase, Stearoyl CoA Desaturase 2 (SCD2), is absolutely required for 3T3-L1 adipogenesis and to maintain adipocyte-specific gene expression in fully differentiated cells. Although SCD2 does not appear to regulate PPARγ ligand production, it does potently regulate PPARγ activity by maintaining the synthesis of PPARγ protein. Surprisingly, this effect was found only with SCD2 and not with the highly homologous protein, SCD1. Therefore, these findings identify separate cellular functions for these SCD isoforms and reveal a novel and essential role for fatty acid desaturation in the adipocyte. Equally important to understanding PPARγ regulation is identifying the downstream mechanisms by which PPARγ activation improves insulin sensitivity. Evidence suggests that the PPARγ target gene, Cidea, is involved in mediating insulin sensitivity by binding to lipid droplets and promoting lipid storage in the adipocyte. Therefore, the second part of thesis provides mechanistic detail into Cidea function by showing that the carboxy terminal 104 amino acids is necessary and sufficient for lipid droplet targeting and the stimulation of triglyceride storage. However, these studies also identified a novel function for Cidea, which requires both the carboxy and amino termini: to induce larger and fewer droplets from smaller dispersed droplets, indicating the possible fusion of droplets. Perhaps this striking change in lipid droplet morphology allows tighter packing and more efficient storage of triglyceride and identifies a novel role for Cidea in lipid metabolism. The results presented in this thesis elucidate key aspects of lipid metabolism that maintain adipocyte function: SCD2 is required to maintain PPARγ protein expression in the mouse; Cidea is a downstream effector of PPARγ activity by promoting efficient triglyceride storage. Therefore, these findings enhance our understanding of adipocyte biology.