Tesi sul tema "SREBP"

Conselho Nacional de Desenvolvimento Científico e Tecnológico
O estudo teve como objetivo avaliar os efeitos da rosuvastatina (ST) e darosiglitazona sobre a resistência à insulina (RI), morfologia do fígado e do tecido adiposo em camundongos alimentados com dieta hiperlipídica (HF). O tratamento com rosuvastatina resultou em uma acentuada melhoria na sensibilidade à insulina caracterizada pela melhor depuração da glicose durante o teste de tolerância à insulina e uma redução do índice HOMA-IR em 70% (P = 0,0008). O grupo tratado com rosuvastatina apresentou redução no ganho massa corporal (-8%, P <0,01) e menor depósito de gordura visceral (-60%, P <0,01) em comparação com o grupo HF não tratado. Em comparação com camundongos HF, animais do grupo HF+ST reduziram significativamente a massa hepática e a esteatose hepática (-6%; P <0,05% e -21; P <0,01, respectivamente). O grupo HF+ST, reduziu os níveis de triglicerídeos hepáticos em 58% comparado com o grupo HF (P <0,01). Além disso, a expressão de SREBP-1c (proteína 1c ligadora do elemento regulado por esteróis) foi reduzido em 50% no fígado dos animais HF + ST (P <0,01) em comparação com o grupo HF. Os níveis de resistina foram menores no grupo HF + ST comparado com o grupo HF (44% a menos, P <0,01). Em conclusão, demonstramos que camundongos alimentados com dieta HF tratados com rosuvastatina melhoram a sensibilidade à insulina, com redução da esteatose hepática. Além disso, ST reduziu o ganho de massa corporal, melhorou os níveis circulantes de colesterol e triglicerídeo plasmático, com menor conteúdo de hepático de triglicerídeo, que foi concomitante com menor resistina e aumento da adiponectina.
The study aimed to evaluate the effects of rosuvastatin (ST) and rosiglitazone on insulin resistance (IR) and liver and adipose tissue morphologies in mice fed a high-fat (HF) diet. Our data show that treatment with rosuvastatin resulted in a marked improvement in insulin sensitivity characterised by enhanced glucose clearance during insulin tolerance and a decrease in the HOMA-IR index level by 70% (P=0.0008). The group of mice treated with rosuvastatin exhibited reduced body mass gain (-8%; P<0.01) and visceral fat pad thickness (-60%; P<0.01)compared with the untreated HF group. In comparison with HF mice, HF+ST mice showed a significant reduction in hepatomegaly and liver steatosis (-6%; P<0.05 and -21%; P<0.01, respectively). In HF+ST mice, the hepatictriglyceride levels were reduced by 58% compared with the HF group (P <0.01). In addition, the expression of SREBP-1c (sterol regulatory element-binding protein) was decreased by 50% in the livers of HF+ST mice (P<0.01) compared with the HF mice. The levels of resistin were lower in the HF+ST group compared with the HF group (44% less, P< 0.01). In conclusion, we demonstrated that rosuvastatin-treated mice fed HF has been improving in insulin sensitivity, with decreased steatosis found in HF mice. Furthermore, ST reduced body mass gain, improved the circulating levels of plasma cholesterol and triglycerides and reduced hepatic triglycerides, which was concomitant with lower resistin and increased total adiponectin.

High fat feeding increases hepatic fat accumulation and is associated with hepatic insulin resistance. AMP Activated Protein Kinase (AMPK) is thought to inhibit lipid synthesis by the acute inhibition of glycerol-3-phosphate acyltransferase (GPAT) activity and transcriptional regulation via SREBP-1c. The purpose of this study was to determine if chronic activation of AMPK prevented an increase in GPAT1 activity in rats fed a high fat diet. Rats were fed a control (C), or a high fat (HF) diet (60% fat) for 6 weeks and injected with saline or a daily AICAR dose of 0.5 mg/g body weight. Chronic AMPK activation by AICAR injections resulted in a significant reduction in hepatic triglyceride accumulation in both the C and HF fed animals (C, 5.5±0.7; C+AICAR, 2.7 ±0.3; HF, 21.8±3.3; and HF+AICAR, 8.0±1.8 mg/g liver). HF feeding caused an increase in total GPAT and GPAT1 activity which was not affected by chronic AMPK activation (GPAT1 activity vs. C, C+AICAR, 92±19%; HF, 186±43%; HF+AICAR, 234±62%). Markers of oxidative capacity, including citrate synthase activity and cytochrome c abundance, were not affected by chronic AICAR treatment. Interestingly, HF feeding caused a significant increase in LCAD (up 66% from C), a marker of fatty acid oxidation capacity. These results suggest that chronic AMPK activation limits hepatic triglyceride accumulation independent of a reduction in total GPAT1 activity.

Tesis para optar al grado de Doctora en Ciencias con mención en Microbiología
Xanthophyllomyces dendrorhous es una levadura basidiomicete que sintetiza carotenoides, siendo el principal de ellos astaxantina. Esta característica la hace de gran interés comercial y objeto de numerosos estudios; sin embargo, existen muchos aspectos desconocidos relacionados con los mecanismos de regulación transcripcional del proceso de carotenogénesis. Estudios recientes proponen la participación del ergosterol, principal esterol en levaduras, en la regulación de genes carotenogénicos y otros esenciales en la vía del mevalonato. En este aspecto, se ha demostrado en otros hongos que el mecanismo por el que el ergosterol regula la transcripción génica es mediante la vía SREBP (Sterol Regulatory Element Binding Protein). El factor transcripcional Sre1 posee el dominio activador de la transcripción en el amino terminal (Sre1N) y su activación ocurre cuando bajan los niveles de esteroles y/u oxígeno en la célula regulando la transcripción de genes involucrados en la biosíntesis de esteroles y respuesta a hipoxia, entre otros. El objetivo general de este trabajo fue estudiar el mecanismo de regulación de la expresión génica mediada por la vía SREBP dependiente de los niveles de ergosterol y oxígeno en la biosíntesis de carotenoides y esteroles en X. dendrorhous, enfocándose en la función del regulador transcripcional Sre1. En primer lugar, se identificaron y caracterizaron bioinformaticamente posibles genes de la vía SREBP: SRE1, SCP1, STP1 y OFD1 de X. dendrorhous. Para estudiar la funcionalidad del gen SRE1, se construyó mutantes por deleción de este gen: CBS.sre1- y CBS.cyp61-/sre1- que provienen de las cepas parentales CBS 6938 y CBS.cyp61- (esta última no produce ergosterol), respectivamente. Además, se construyó la cepa mutante CBS.gSRE1N, que expresa sólo el dominio activador de la transcripción (Sre1N). Se evaluó el fenotipo de estas cepas en cuanto a la producción de esteroles y carotenoides, crecimiento en presencia de un inhibidor de la síntesis de esteroles (clotrimazol) y la expresión a nivel de transcritos de algunos genes. Como resultado, se observó que la deleción del gen disminuye la producción de ambos tipos de metabolitos y además, SRE1 es esencial para el crecimiento en clotrimazol. Adicionalemente, la cepa CBS.gSRE1N produce doble cantidad de carotenoides y esteroles, respecto a la cepa silvestre. Paralelamente, se realizó un ensayo de complementación heteróloga vía expresión del gen SRE1 de X. dendrorhous en una cepa mutante sre1- de Schizosaccharomyces pombe y se observó que existe una complementación parcial, dado que la cepa complementada presenta un mejor crecimiento en anaerobiosis y cloruro de cobalto, respecto al control sre1-. Por otra parte, se realizaron análisis transcriptómicos de las cepas mutantes en condiciones de normoxia, hipoxia y cloruro de cobalto, dado que este último compuesto se ha descrito como un agente que imita las condiciones de hipoxia. De estos análisis se desprende que Sre1 es necesario para la respuesta a hipoxia y que el cloruro de cobalto imitaría esta respuesta transcripcional en la levadura al menos en algunos genes de la biosíntesis de esteroles. Finalmente, de acuerdo a los resultados obtenidos en este estudio se concluyó que el gen SRE1 identificado en X. dendrorhous es funcional y participa en la regulación de la biosíntesis de esteroles y carotenoides.
Xanthophyllomyces dendrorhous is a basidiomycete yeast that synthesizes carotenoids, mainly astaxanthin, this characteristic is of great commercial interest and subject of numerous studies. However, there are many unknown aspects related to transcriptional regulation mechanisms of carotenogenesis. Recent studies propose that ergosterol, the main sterol in yeast, is involved in the regulation of carotenogenic genes expression and on other genes of the mevalonate pathway. In this aspect, in other fungi it has been demonstrated that the mechanism by which ergosterol regulates gene transcription is through the SREBP (Sterol Regulatory Element Binding Protein) pathway. The transcriptional factor Sre1 contains a transcriptional activation domain at its amino terminal end (Sre1N) that is activated when cellular sterols and/or oxygen levels decrease and by this way, it regulates the transcription of genes involved in the biosynthesis of sterols and response to hypoxia, among others. The general goal of this work was to study if this mechanism, the SREBP pathway, regulates the biosynthesis of carotenoids and sterols in X. dendrorhous, focusing on the function of the transcriptional regulator Sre1. First, X. dendrorhous potential genes of the SREBP pathway, SRE1, SCP1, STP1 and OFD1, were identified and bioinformatically characterized. To study the functionality of the SRE1 gene, deletion mutants of this gene were constructed: CBS.sre1- and CBS.cyp61- / sre1- that derived from the parental strains CBS 6938 and CBS.cyp61- (which does not produce ergosterol), respectively. In addition, the mutant strain CBS.gSRE1N was generated, which expresses only the transcription activating domain (Sre1N). The phenotype of these strains was evaluated in relation to sterol and carotenoids production, growth in the presence of sterol synthesis inhibitor (clotrimazole) and transcript level of some genes. As a result, it was observed that the SRE1 gene deletion, decreases the production of both types of metabolites and also this gene is essential for growth in the presence of clotrimazole. In addition, the production of carotenoids and sterols in the CBS.gSRE1N strain was increased 2-fold compared to the wild-type strain. In parallel, a heterologous complementation assay was performed by expressing the X. dendrorhous SRE1 gene in a Schizosaccharomyces pombe sre1- mutant strain. Partial complementation was observed as the complemented strain displayed better growth in anaerobiosis and in the presence of cobalt chloride (an agent that imitates hypoxia conditions), regarding to the controls strains. On the other hand, transcriptomic analyses of the mutant strains were carried out in conditions of normoxia, hypoxia and cultures in the presence of cobalt chloride. In general, it was observed that Sre1 is necessary for the response to hypoxia conditions and that cobalt chloride would imitate these conditions in the transcriptional response in the yeast, at least in some genes of the sterol biosynthesis. In conclusion, the SRE1 gene identified in X. dendrorhous is functional and it is involved in the regulation of the biosynthesis of sterols and carotenoids in this yeast.
Beca Doctoral y Proyecto FONDECYT 1160202.
20 de febrero 2020.

Les protéines SREBP-1 sont des facteurs de transcription connus pour leur rôle dans la régulation du métabolisme lipidique. Plus récemment des études faites in vitro (myotubes humains en culture primaire) et in vivo (muscle tibial de souris) ont montré que la surexpression de SREBP-1a ou SREBP-1c induit une atrophie musculaire et bloque la différenciation musculaire, en inhibant notamment l'expression des protéines structurales du muscle squelettique et des facteurs de la différenciation musculaire (MRFs). Les travaux de thèse présentés dans ce manuscrit ont eu pour but de décrypter le mécanisme de l'atrophie induite par SREBP-1 et de déterminer comment les protéines SIRT1 pourraient réguler ce facteur de transcription. L'atrophie musculaire résulte d'un déséquilibre entre la quantité de protéines synthétisées et dégradées. Dans nos études, nous montrons que SREBP-1 régule la synthèse protéique et la dégradation protéique, respectivement via le contrôle négatif de l'expression des MRFs et via le contrôle de l'expression des atrogènes, MuRF1 et Atrogin-1. Dans le muscle squelettique, nous démontrons que la désacétylase SIRT1 régule l'activité transcriptionnelle de SREBP-1. Les protéines SREBP-1 et SIRT1 étant toutes deux impliquées dans la régulation du métabolisme lipidique, nous mettons en évidence une nouvelle voie de signalisation reliant le métabolisme énergétique et nutritionnel avec l'activité transcriptionnelle de SREBP-1 dans le muscle. Étant donné le rôle de SIRT1 et SREBP-1 dans le métabolisme lipidique et musculaire, nous nous sommes intéressés au rôle des phospholipides et plus particulièrement des céramides dans la régulation de la masse musculaire.Nos études montrent que la régulation de la quantité de céramides par la cytokine TNFα régule la masse musculaire. Ainsi, nos travaux mettent en évidence de nouveaux liens entre le métabolisme lipidique et la régulation de la masse et du métabolisme musculaire.

La maladie de Parkinson (MP) est caractérisée par une perte progressive des terminaisons présynaptiques dopaminergiques et reste actuellement incurable. Néanmoins, dans les études épidémiologiques, il a été montré que l’utilisation des statines, médicaments hypocholestérolémiants, diminue le risque de développer une MP. Les statines sont également capables d'inhiber les effets neurodégénératifs dans les modèles précliniques in-vitro et in-vivo de la MP. Cependant, les mécanismes moléculaires à l’origine de ces effets neuroprotecteurs ne sont pas encore complétement élucidés. Ainsi, nous avons étudié les effets potentiels des statines sur l'expression des marqueurs synaptiques et sur le transport de la dopamine. Dans nos études, les statines induisent la croissance des neurites dans les cellules dopaminergiques et déclenchent une augmentation de l’expression des protéines synaptiques dopaminergiques telles que le transporteur vésiculaire des monoamines (VMAT2) et le transporteur de la dopamine. Les statines induisent une diminution de la recapture de la dopamine cellulaire et des changements d’affinités aux niveaux des sites de liaison des inhibiteurs sélectifs du VMAT2. L’activation du facteur de transcription nucléaire protéine-1 se liant à l'élément de régulation des stérols (SREBP-1), cholestérol-dépendent, serait l’élément inducteur de la surexpression des marqueurs dopaminergiques présynaptiques induite par les statines. En outre, ces résultats soutiennent un potentiel thérapeutique neuroprotecteur et/ou neurorestaurateur des statines précédemment proposées dans la MP et permettent de mettre en évidence de nouvelles cibles thérapeutiques comme le facteur SREBP
Parkinson disease (PD) is characterized by a progressive loss of dopaminergic presynaptic terminals and remains incurable. However in epidemiological studies, it has been shown that the use of statins, which are hypocholesterolemic drugs, diminishes the risk to develop a PD. Statins are able to inhibit the neurodegenerative effects in in-vitro and in-vivo models of PD. However, the molecular mechanisms driving neuroprotective effects are not yet fully understood. Consequently, we investigated the potential effects of statins on the synaptic expression and dopamine transport function in the dopaminergic system. In our studies, statins enhance the neurite outgrowth in the dopaminergic cells and trigger an increase in the expression levels of presynaptic dopaminergic proteins such as vesicular monoamine transporter 2 (VMAT2) and dopamine transporter. Statins induce a reduction of dopamine cellular uptake and modulate the binding-affinity of the specific inhibitors for VMAT2. The activation of the nuclear transcriptional factor sterol regulatory element-binding protein 1 (SREBP-1), cholesterol-dependent, could be the key element of the overexpression of dopaminergic presynaptic markers induced by the statins. Furthermore, these findings highlight the therapeutic neuroprotective and/or neurorestorative potentials of statins previously proposed in PD and allow to bring out new potential therapeutic targets such as SREBP factor

Ischemic brain injury causes neurodegeneration. In this study, the mechanism of neurodegeneration was investigated by examining the role of sterol regulatory element binding protein-1 (SREBP-1), CCAAT enhancer binding protein&
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(C/EBP&
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), glutathione (GSH), malondialdehyde (MDA), glutathione-S-transferase (GST), and superoxide dismutase (SOD). Carotid artery occlusion (CAO) plus hypotension was produced for 10 minutes. Control groups were sham operated. Animals were sacrificed after 24 hours, 1 week, 2 and 4 weeks of reperfusion periods. The expression of C/EBP&
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and SREBP-1 in rat brain cortex and cerebellum were examined by western blotting. C/EBP&
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expressions significantly increased in both cytosolic (1.19, 1.58 fold) and nuclear (1.73, 1.81 fold) extracts of brain cortex at 24 hours and 1 week CAO groups, respectively. In cerebellum, C/EBP&
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expression significantly increased in 1 week, cytosolic (1.63 fold), and nuclear (1.35 fold) extracts. SREBP-1 expression increased significantly in both cytosolic (2.07 fold) and nuclear (1.41 fold) extracts of brain cortex in 1 week. SREBP-1 expression significantly increased in cytosolic (2.15 fold) and nuclear (1.79 fold) extracts of cerebellum in 1 week. There were no significant alterations in SREBP-1 C/EBP&
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expressions for 2 and 4 weeks in both cytosolic and nuclear extracts of brain cortex and cerebellum. There were insignificant changes in GSH and GST levels in cortex. However, MDA and SOD levels significantly increased by 43.0 % and 47.3 %, respectively, in 24 hours. Our findings indicate that increase in SREBP-1 and C/EBP&
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expressions may be related to oxidative stress during ischemic neurodegenerative processes.

Si l'insulinorésistance joue un rôle central dans la pathogénie des lésions de NASH, celle-ci reste imprécise. Nous avons évalué le rôle des adipokines, des PPAR et de SREBP-1au cours de la stéatose métabolique. Nous avons montré que le rapport adiponectine/leptine sérique permettait de différencier les patients ayant une NASH de ceux ayant une stéatose pure. Nous avons observé une accumulation hépatique de leptine et d'adiponectine en immuno-histochimie, en l'absence de synthèse locale mesurée par PCR en temps réel. Nous avons également mesuré une modulation de l'expression hépatique des PPAR et de SREBP-1 dans différentes populations à risque : patients obèses, patients VIH lipodystrophiques, patients NASH traités par un agoniste de PPAR. Ces résultats suggèrent un rôle de ces molécules dans la pathogénie des lésions de NASH et pourraient ainsi permettre le développement de nouvelles cibles thérapeutiques

Les souris KK-Ay représentent un bon modèle pour étudier les effets d'une alimentation riche en graisse dans le développement des hyperlipidémies de type IIb et IV. Les acides biliaires réduisent l'accumulation des TG dans le foie ainsi que dans le sérum. LXR et LRH-1 induisent l'expression de SREBP-1c, les acides biliaires induisent l'expression de SHP en activant FXR. Ensuite, SHP diminue l'activité transcriptionnelle de LXR et LRH-1 sur le promoteur murin de SREBP-1c. Les acides biliaires réduisent l'accumulation hépatique de TG en réduisant l'expression de SREBP-1c via SHP. La diminution de la sécrétion des VLDL, et donc de TG sérique, est diminuée. Mes résultats suggèrent que des stratégies en vue d'augmenter l'activité de FXR et l'effet répresseur de SHP doivent être explorées pour corriger l'hypertriglycéridémie
KK-Ay mice are useful model to study high fat diet induced type IIb and IV hypertriglyceridemia. Bile acids reduced TG accumulation in liver and TG level in serum. LXR and LRH induce SREBP1c gene expression and bile acids induce mRNA expression of SHP by activating FXR. SHP decreases LXR and LRH transcriptional activity in mouse SREBP1c promoter region. Bile acids reduced liver TG accumulation by suppressing SREBP1c gene expression via SHP. This decreases VLDL secretion and as a result serum TG is reduced. Our results and these data suggest that strategies aimed at increasing FXR activity and the repressive effects of SHP should be explored to correct hypertriglyceridemia

La acuicultura es una importante actividad económica vinculada a la producción de alimentos que se encuentra en etapa de expansión. Sin embargo, la producción de especies carnívoras como la dorada (Sparus aurata) presenta limitaciones, tales como la necesidad de incluir en los piensos altas concentraciones de proteína (incorporada en la forma de harina de pescado). Es por ello que debido a la importancia comercial que representa la dorada en la Unión Europea y particularmente en España, se han hecho grandes esfuerzos en investigación con el fin de entender los mecanismos moleculares encargados de regular el metabolismo intermediario de estos peces. En este sentido, son relevantes y cada vez más abundantes los estudios concernientes a los mecanismos de regulación génica como consecuencia del estado nutricional en peces. Gracias a las tecnologías de secuenciación de nueva generación (que permiten obtener resultados en un período corto de tiempo y a un costo significativamente inferior a los métodos tradicionales), en la última década son diversos los proyectos que han permitido el incremento de la información transcriptómica de dorada. A pesar de ello, ninguno de estos estudios se ha llevado a cabo sobre muestras de hígado, así como tampoco se han estudiado las variaciones consecuencia del estado nutricional y la composición de la dieta. El objetivo principal de esta tesis doctoral fue desarrollar un transcriptoma con muestras de hígado y músculo esquelético de cinco grupos de doradas alimentadas durante un período de 23 días, con piensos de distinta composición de macronutrientes (carbohidratos, proteínas o lípidos), así como de un sexto grupo de peces sometidos a ayuno por ese mismo período de tiempo. Este enfoque metodológico permitió caracterizar el transcriptoma de ambos tejidos, así como la obtención de una base de datos transcriptómicos de amplia cobertura y de interés para el desarrollo de estudios nutricionales. Se ensamblaron 21.093 secuencias únicas a partir de 660.166 y 547.544 lecturas de alta calidad de muestras de hígado y músculo esquelético respectivamente, en un transcriptoma que denominamos híbrido. Estas secuencias fueron utilizadas para el diseño de microarrays de oligonucleótidos que nos permitieron analizar cambios en el patrón de expresión génica como consecuencia del ayuno y la composición de la dieta, con el fin de identificar rutas metabólicas y genes biomarcadores relevantes del estado nutricional y la asimilación de nutrientes por parte de la dorada. Este análisis permitió establecer la importancia de la cadena respiratoria y la fosforilación oxidativa, así como del metabolismo lípidico, como consecuencia del estado nutricional y la composición de la dieta en estos peces. Mediante RT-qPCR se validaron los resultados obtenidos y se identificaron algunos genes biomarcadores, tales como algunos miembros del complejo IV de la cadena respiratoria, que incrementan su expresión en peces sometidos a un ayuno prolongado; o genes relacionados con la síntesis de ácidos grasos y colesterol que varían su expresión en relación con la composición de la dieta. Debido a la importancia que presenta el metabolismo de los lípidos en dorada y a la relación existente del factor de transcripción SREBP-1a tanto con el metabolismo de lípidos como con el metabolismo de carbohidratos, se estudió el efecto metabólico de la sobreexpresión de SREBP-1a en el hígado de doradas alimentadas con dietas de diferente composición. Para ello se administraron nanopartículas de quitosán-tripolifosfato acomplejadas con un plásmido de expresión del fragmento nuclear de SREBP-1a de hámster. El incremento de la expresión hepática de SREBP-1a promovió el aprovechamiento de los carbohidratos de la dieta mediante un incremento en la expresión de genes glucolíticos, así como la de genes relacionados con la síntesis de lípidos. Estos datos sugieren un incremento en la producción de lípidos aparejado a un mayor aprovechamiento de carbohidratos, lo que a su vez permitiría una disminución en la utilización de proteínas de la dieta en la acuicultura.
Aquaculture is an economic activity linked to food production, which is currently an expanding activity. However, the production of carnivorous species such as Gilthead Sea Bream (Sparus aurata) is limited by inclusion of high levels of dietary proteins (fish meal) in feedstuffs. Due to the economic impact of S. aurata farming in the European Union and, in particular, in Spain, remarkable efforts are nowadays devoted to understand the molecular mechanisms that govern the intermediary metabolism of S. aurata. In this regard, studies addressing genetic regulation by the nutritional status in fish are of increasing relevance. The new generation sequencing technologies allow to obtain transcriptomic data in a shorter period of time and at a lower cost than traditional methods. Therefore, during the last decade a number of scientific projects increased transcriptomic data available for S. aurata. In spite of these studies, there are no data concerning the pattern of gene expression in the liver due to changes in the nutritional status and diet composition. The main goal of the present doctoral thesis was to obtain a transcriptome from liver and skeletal muscle samples from five groups of fish fed during 23 days with diets differing in macronutrient composition (carbohydrates, proteins or lipids) and a sixth group of fish subjected to fasting for that same period of time. This approach allowed us to obtain the liver and skeletal muscle transcriptomes of S. aurata and a deep-coverage database of nutritional interest. A total of 21,093 unique sequences were assembled from 660,166 and 547,544 high quality reads of liver and skeletal muscle samples, respectively, to obtain the herein named hybrid transcriptome. Unique sequences were used to design an oligonucleotide microarray that was subsequently used to analyze the pattern of gene expression in fish submitted to starvation and changes in diet composition in order to identify metabolic pathways and biomarker genes relevant for the nutritional status and nutrient utilization in S. aurata. We concluded that nutritional status and diet composition highly affect the respiratory chain, oxidative phosphorylation and lipid metabolism in S. aurata. Microarray data were validated by RT-qPCR, leading to the proposal of several key biomarkers genes, such as some members of complex IV of the respiratory chain, whose gene expression increases in fish subjected to prolonged fasting, and dependence on dietary macronutrient composition of the expression of genes involved in fatty acid and cholesterol biosynthesis. Given the relevance of lipid metabolism in S. aurata and the role exerted by the transcription factor SREBP-1a in the control of both lipid and carbohydrate metabolism, the metabolic effect of SREBP-1a overexpression was studied in the liver of S. aurata fed with different diets. To this end, chitosan-tripolyphosphate nanoparticles complexed with a plasmid expressing the nuclear fragment of hamster SREBP-1a were administered to S. aurata. Overexpression of SREBP-1a stimulated the expression of genes involved in glycolysis and lipid synthesis, suggesting the use of dietary carbohydrates coupled to lipid production, a mechanism that would enable a protein sparing effect in fish farming.

Protein kinase B (PKB/Akt) is a central component of intracellular signaling pathways. It becomes activated downstream of growth factor receptors and has been implicated in cell growth, proliferation and protection from apoptosis. Akt is also a mediator of metabolic insulin action, and stimulates glucose uptake, glycogen synthesis and lipogenesis. Activation of Akt resulted in induction of expression of several enzymes involved in cholesterol and fatty acid biosynthesis. These genes are transcriptional targets of the family of sterol regulatory element-binding proteins (SREBP). Induction of fatty acid synthase and HMG-CoA synthase, two key enzymes of the sterol and fatty acid biosynthesis pathway, by Akt requires SREBP. In addition, activation of Akt results in rapid accumulation of mature SREBP 1 in the nucleus. This process was independent of activation of glycogen synthase kinase 3 (GSK3) but required active complex 1 of the mammalian target of rapamycin (mTOR/TORCl). Analysis of cellular metabolites by NMR revealed that induction of glucose and amino acid uptake, lactate production as well as fatty acid and phosphoglyceride biosynthesis by Akt also requires TORC1 activity. Thus it can be postulated that induction of expression of lipogenic genes through activation of SREBP is part of an anabolic response to activation of the PI3K/Akt/mTOR pathway and may be required for the induction of lipid biosynthesis during cell growth and proliferation. The PI3K/Akt pathway has been implicated in regulation of cell and organ size in Drosophila melanogaster. Several transgenic fly lines carrying an RNAi construct targeting dSREBP expression were generated. Silencing of dSREBP resulted in a significant developmental delay as well as a profound loss of viability. Tissue specific silencing of dSREBP in the wing resulted in a reduction in cell and organ size suggesting that activation of dSREBP by the PI3K/Akt pathway could be involved in cell growth control in flies.

L'athérosclérose, par ses complications telles que les cardiopathies ischémiques et les accidents vasculaires cérébraux, constitue l'une des premières causes de mortalité dans le monde. Ainsi, comprendre les mécanismes mis en jeu dans cette pathologie apparaît comme un enjeu majeur de santé publique. Dans cette optique, notre travail a suivi deux approches. La première, plus appliquée, a cherché à identifier de nouveaux variants génétiques des SREBP (Sterol Regulatory Element Binding Protein) et de la SCAP (SREBP Clivage Activating Protein) puis à étudier leur impact sur les profils lipidique et clinique de sujets hypercholestérolémiques. Nous avons ainsi pu mettre en évidence que certains polymorphismes des gènes étudiés sont associés aux variations de la glycémie à jeun, de l'index de masse corporelle, des paramètres lipidiques et de l'épaisseur intima-média ; ces associations étant observées dans les analyses génotypiques et/ou haplotypiques réalisées pour ce travail. Dans un second temps, par une approche plus fondamentale, nous avons voulu caractériser les mécanismes moléculaires engagés dans le transport intracellulaire du cholestérol et leurs implications dans le maintien de l'homéostasie cellulaire du cholestérol. Nous avons montré l'importance du transport vésiculaire dépendant de la dynamine dans le trafic intracellulaire du cholestérol et, par conséquent, dans la régulation de l'expression des gènes sensibles aux stérols. En conclusion, notre étude a permis de caractériser de nouveaux gènes candidats dans le déterminisme génétique de l'athérosclérose et d'identifier la dynamine comme un acteur de la régulation du transport intracellulaire de cholestérol. Ces résultats permettent d'envisager des perspectives expérimentales mais également diagnostiques (détermination polymorphique des sujets à haut risque cardiovasculaire) et thérapeutiques (identification de nouvelles cibles pharmacologiques pour les traitement de l'athérosclérose)
An estimated 17 million people die of cardiovascular diseases every year in the world, among which approximately 13 million deaths are attributable to atherosclerosis. Thus, understanding mechanisms involved in this pathology represents a major stake in public health. In this context, our work followed two approaches. First, we have characterized the polymorphic structure of SREBPs (Sterol Regulatory Element Binding Proteins) and SCAP (SREBP Cleavage Activating Protein) in order to study the impact of observed genetic variants on biological and clinical profiles of hypercholesterolemic subjects. Our genotypic and haplotypic studies showed that some polymorphisms were associated with lipid parameters, carotid IMT (Intima Media Thickness), fasting glucose, and BMI (Body Mass Index). Second, a more fundamental approach consisted in a better characterization of the molecular mechanisms involved in intracellular cholesterol transport. In this purpose, we examined the role of clathrin- and dynamin-dependent trafficking in the transport of exogenous LDL-derived cholesterol and in cellular cholesterol homeostasis. Thus, we showed a major role for dynamin-dependent vesicular trafficking in cellular cholesterol distribution and homeostasis. In conclusion, our study allowed the characterization of new candidate genes in the genetic determinism of atherosclerosis and the identification of dynamin as a new and crucial actor in the regulation of intracellular cholesterol transport. These results allow to envisage experimental perspectives and will also contribute to the design of new diagnostic and therapeutic approaches

Chez le nématode Caenorhabditis elegans (C. elegans), le cofacteur mdt-15, orthologue à Med15 chez le mammifère, est essentiel à l’homéostasie des lipides. Aussi, l’inhibition pharmacologique de l’interaction entre Med15 et le facteur de transcription SREBP améliore le profil lipidique chez des souris obèses. Le foie, organe important du métabolisme énergétique, peut subir des dérèglements lors du vieillissement et dans l’obésité. La modulation des niveaux hépatiques de Med15 dans ces conditions pathologiques est toutefois inconnue. Cette étude visait donc à évaluer l’expression de Med15 au foie dans le vieillissement et l’obésité. Les modèles de vieillissement étaient des souris C57Black/6J (B6), des rats Sprague-Dawley (SD) et des rats Lou (modèle de vieillissement réussi) de différents groupes d’âges et sous diète faible en gras (LFD). MED15 a également été mesuré dans du foie humain provenant de 3 groupes de patients obèses jeunes, d’âge intermédiaire et vieux. Afin de dissocier les effets du vieillissement de ceux de l’obésité, Med15 a été mesuré dans des souris ob/ob ou db/db sous LFD et des souris B6 sous diète riche en gras (HFD) âgées de 4 mois. L’expression de Med15 était diminuée chez les souris B6 et les rats SD âgés mais demeurait stable chez les rats Lou vieux et les patients âgés. Les niveaux de Med15 étaient diminués chez les souris ob/ob et db/db. En revanche, ses niveaux protéiques hépatiques étaient augmentés chez les souris sous HFD. La conclusion générale, tirée des liens établis entre les résultats présentés ici et la littérature, est que l’expression de Med15 serait bénéfique chez un organisme sain mais que sa diminution permettrait de freiner les troubles métaboliques associés au vieillissement et à l’obésité. Des études in vitro et in vivo sur les impacts des variations observées dans cette étude permettraient de caractériser Med15 comme modulateur métabolique chez le mammifère.
In the nematode Caenorhabditis elegans (C. elegans), the mdt-15 cofactor, orthologous to the mammalian Med15, is essential for lipid homeostasis. Furthermore, pharmacological inhibition of the interaction between Med15 and Sterol Regulatory Element Binding Protein (SREBP) transcription factor improves the lipid profile in obese mice. The liver, important organ of energy metabolism, may undergo disorders during aging and in obesity. Modulation of Med15 hepatic levels under these two conditions is however unknown. This study aimed therefore to evaluate hepatic Med15 expression in several aging and obesity models. The aging models were C57Black/6 Jackson (B6) mice, Sprague-Dawley (SD) rats and Lou rats (a successful aging model) in different age groups and under low-fat diet (LFD). MED15 was also measured in human liver from 3 groups of obese young, middle-aged and old patients. In order to dissociate the effects of aging from those of obesity, Med15 expression was measured in 4 months old ob/ob or db/db mice under LFD and B6 mice under high-fat diet (HFD). Med15 expression was decreased in old B6 mice and SD rats but remained stable in old Lou rats and elderly patients. Med15 levels were diminished in ob/ob and db/db mice. However, Med15 protein levels were increased in mice under HFD. The general conclusion, drawn from links established between the results presented here and the literature, is that Med15 expression would be beneficial in a healthy organism but its decrease would curb the metabolic disorders associated with aging and obesity. In vitro and in vivo studies on the impacts of the variations observed in this study would allow for the Med15 characterization as a key metabolic modulator in mammals.
Résumé en espagnol

Mechanical cues coming from the extracellular matrix (ECM) are key factors in the control of tissue homeostasis in physiology and disease. Cells can sense these physical cues and measure external resisting forces by adjusting their actomyosin cytoskeleton, which in turn regulates intracellular signalling pathways to orchestrate a proper cell response. Thus, ECM stiffness is important for many biological aspects such as proliferation, differentiation and migration. Very little is known instead, about its impact on cellular metabolism, and the molecular players involved in this process are largely unknown. Starting from an unbiased metabolomics approach, we found lipid accumulation as a general response to mechanical signals and to low tension conditions. Mechanicistically, this accumulation is associated with a decreased Lipin1 phosphatidate phosphatase localization at ER/Golgi membranes and decreased Lipin1 activity which ultimately lead to nuclear translocation and activation of SREBP1/2 transcription factors. This occurs independently of YAP/TAZ and mTOR, and in parallel to the feedback control by sterols. Led by our findings, we discovered a coherent regulation of SREBP in stiffened pathological human tissues, and identified SREBP as required for the pro-survival activity of ROCK inhibitors in embryonic stem cells. We thus propose that SREBP is a general mechanism that links the physical cell microenvironment to a key metabolic pathway
Gli stimoli meccanici provenienti dalla matrice extracellulare (ECM) sono fattori chiave nel controllo dell'omeostasi tissutale in condizioni fisiologiche e patologiche. Le cellule possono percepire questi segnali fisici e misurare le forze di resistenza esterne regolando il loro citoscheletro attraverso i filamenti di actomiosina, che a loro volta regolano le vie di segnalazione intracellulare per indurre una risposta cellulare adeguata. Pertanto, la rigidità della matrice extracellulare è importante per molti aspetti biologici come la proliferazione, la differenziazione e la migrazione. Si sa invece molto poco sull’ impatto che essa ha sul metabolismo cellulare, e i fattori molecolari coinvolti in questo processo sono in gran parte sconosciuti. Attraverso un’analisi metabolomica iniziale, abbiamo visto che le cellule tendono ad accumulare lipidi come risposta generale ai segnali meccanici e alle condizioni di bassa tensione. Abbiamo inoltre osservato che questo accumulo è associato ad un cambio di localizzazione della fosfatasi Lipin1, che diminuisce la sua affinità per le membrane del reticolo endoplasmatico e dell’apparato di Golgi e alla ridotta attività di Lipin1 che alla fine porta alla traslocazione nucleare e all'attivazione dei fattori di trascrizione SREBP1 / 2. Ciò si verifica indipendentemente dall’attività di YAP / TAZ e mTOR e in modo parallelo rispetto alla regolazione dei livelli di steroli nella cellula. Cercando una rilevanza biologica per il meccanisco da noi descritto, abbiamo inoltre scoperto che SREBP viene regolato in maniera coerente nei cheloidi, una patologia fibro-proliferativa legata a stress meccanici, e abbiamo identificato SREBP come un fattore importante e richiesto per la sopravvivenza di cellule staminali embrionali mediata dall’inibizione di ROCK. Quindi riassumendo, il nostro modello vede l’inibizione di LIPIN1 e l’attivazione di SREBP come un meccanismo generale che collega le forze fisiche derivanti dal microambiente e il metabolismo cellulare

La stéatose hépatique, caractérisée par l’accumulation de triglycérides dans le foie, est une pathologie associée à l’obésité et l’insulinorésistance. Les mécanismes du développement de la stéatose hépatique ne sont pas complètement caractérisés mais il a été montré que la lipogénèse joue un rôle majeur dans sa pathogénèse. Nous avons cherché à comprendre comment la lipogénèse, physiologiquement contrôlée par l’insuline, est activée dans les foies stéatosés des rongeurs insulinorésistants. Dans un premier temps, nous avons montré que la voie du stress du réticulum endoplasmique (RE) participe directement à l’activation de la lipogénèse dans le foie des souris ob/ob, obèses et insulinorésistantes. Nous avons mis en évidence que l’activation du stress du RE conduit à la maturation du facteur de transcription SREBP-1c, impliqué dans le contrôle de la lipogénèse. Nous montrons que le stress du RE contribue ainsi fortement au développement de la stéatose hépatique et à l’installation de l’insulinorésistance chez la souris ob/ob. Par la suite, nous nous sommes interrogés sur les facteurs déclenchants de l’activation du stress du RE dans le foie des souris ob/ob. Nous avons fait l’hypothèse que l’hyperinsulinémie pourraient être responsable de l’activation du stress du RE dans cette situation. Dans la deuxième partie de ce travail, nous avons montré chez la souris que la réalimentation hyperglucidique conduit rapidement à l’activation du stress du RE dans le foie. Nos travaux ont mis en évidence que l’insuline active partiellement le stress du RE in vitro dans l’hépatocyte suggérant que cette hormone participe à l’induction du stress du RE dans le foie des rongeurs réalimentés

Dans le foie, un certain nombre de genes de la glycolyse et de la lipogenese sont controles par l'insuline seule, comme le gene de la glucokinase (gk) ou par l'insuline et le glucose comme les genes de la synthase des acides gras (fas) et de la proteine s14 (s14). Srebp-1c appartient a une famille de facteurs de transcription decrite initialement comme controlant des genes impliques dans le metabolisme du cholesterol. Dans le foie, srebp-1c est la forme majoritairement exprimee et cette isoforme est specifiquement impliquee dans le metabolisme des acides gras. Dans des hepatocytes de rat en culture primaire, nous avons montre que la transcription du gene srebp-1c est activee par l'insuline et inhibee par le glucagon. Nous avons alors fait l'hypothese que srebp-1c pouvait etre implique dans les effets geniques de l'insuline. Nous avons utilise une forme dominante negative de srebp-1c (srebp-1c dn) inseree dans un vecteur adenoviral pour infecter des hepatocytes en culture primaire. La presence de srebp-1c-dn inhibe les effets inducteurs de l'insuline sur l'expression de la gk. Inversement en absence d'insuline, l'expression du gene gk est induite dans des hepatocytes infectes avec un adenovirus contenant une forme dominante positive de srebp-1c. Pour les genes induits par l'insuline et le glucose (fas, s14), leur expression est inhibee en presence de srebp-1c dn. En l'absence d'insuline et de glucose, le dominant positif srebp-1c est capable d'induire leur expression. Toutefois, cet effet est fortement potentialise par la presence d'une forte concentration en glucose. En conclusion, le facteur srebp-1c est un mediateur essentiel des effets inducteurs de l'insuline sur l'expression des genes hepatiques impliques dans le metabolisme

Les données de la littérature rapportent la supériorité de la chirurgie bariatrique sur le traitement médical optimisé concernant la perte pondérale et l'amélioration du diabète de type 2. Les facteurs prédictifs de bons résultats en terme pondéral et métabolique restent encore méconnus et des échecs sont constatés. Le phénotypage de l'obésité et de son retentissement métabolique semble essentiel afin d'adapter la procédure chirurgicale au cas par cas et améliorer les résultats. Dans ce travail de thèse, par une approche clinique, nous avons cherché à identifier les facteurs prédictifs d'amélioration des paramètres métaboliques et de succès pondéral après chirurgie bariatrique. Nous avons démontré le rôle majeur de la perte de poids après chirurgie dans l'amélioration du métabolisme glucidique et des paramètres métaboliques. Nous avons également montré l'impact positif de la masse musculaire initiale sur la perte pondérale, facteur également déterminant dans le contrôle du métabolisme glucidique. Les marqueurs du dysfonctionnement cellulaire Beta sont également apparus déterminants pour prédire la rémission du diabète de type 2 après chirurgie. Ainsi, l'efficacité de la chirurgie dans le contrôle du syndrome métabolique, au-delà de la technique opératoire, apparaît très dépendante de la perte de poids mais aussi du terrain, confirmant l'importance du phénotypage de l'obésité en préopératoire. Par une approche expérimentale, nous avons cherché à identifier l'impact du tissu adipeux sur les organes sièges de l'insulino-résistance (muscle et foie) impliqués dans le syndrome métabolique. La constitution de la tissuthèque DioMede et l'obtention de milieux conditionnés de tissu adipeux nous ont permis d'étudier l'impact des sécrétions de ce tissu sur les tissus insulino-sensibles en se rapprochant des conditions physiologiques. Nous avons identifié un effet direct du tissu adipeux sur le métabolisme musculaire des acides gras (AG) par la régulation négative du facteur de transcription SREBP-1c. Nos résultats identifient les acides gras insaturés comme les médiateurs de l'inhibition de SREBP-1, conduisant à une diminution de la lipogenèse par l'intermédiaire des gènes cibles de ce facteur de transcription. La composition et les proportions respectives d'AG mono ou poly insaturés et d'AG saturés dans le tissu adipeux, leur niveau de sécrétion, et leur taux circulant apparaissent donc déterminants dans la régulation de la lipogenèse des tissus insulino-sensibles (foie et muscle), et pourraient être un marqueur des obésités avec désordres métaboliques
Literature data reported the superiority of bariatric surgery on optimized medical treatment concerning weight loss outcomes and improvement of type 2 diabetes. Predictive factors of good weight loss results and metabolic control are still unrecognized and failures are recorded. Phenotyping obesity and its metabolic consequences seem essential to tailor the surgical procedure to each patient and to improve the outcomes. In this work, by a clinical approach, we have tried to identify predictive factors of metabolic control and weight loss after bariatric surgery. We have demonstrated the major role of weight loss to achieve glucose homeostasis and metabolic control. We have also reported the positive impact of initial Fat Free Mass on weight loss outcomes and glucose metabolism control. Beta cell dysfunction markers appeared to also have a major impact on Type 2 Diabetes remission after surgery. Thus, the efficacy of surgery on metabolic control, beyond the surgical technique, seems highly related to weight loss and patients history, which underlines the importance of phenotyping obesity before surgery. By an experimental approach, we have tried to identify the impact of adipose tissue on muscle and liver, organs that are involved in the metabolic syndrome. By means of a tissue collection (Diomede) and the use of conditioned media of adipose tissue, we studied the impact of adipose tissue secretions on insulin sensitive tissues, close to physiological conditions. We found a direct effect of adipose tissue on fatty acid metabolism in muscle through SREBP-1c down regulation. Unsaturated Fatty Acids were identified as the mediators of SREBP-1 inhibition, leading to a decrease in lipogenesis through target genes of this transcriptional factor. The composition and the respective proportion of mono or poly unsaturated fatty acids and saturated fatty acids in adipose tissue, their level of secretion, and their circulation rate appear to be determinant in lipogenesis regulation in insuline sensitive tissues (muscle and liver), and could be markers of metabolic disorders in obese patients

Sterol regulatory element-binding proteins (SREBP) are transcription factors that regulate genes involved in lipid metabolism especially in the liver. Therefore, hepatic SREBP is significant regulator of systemic lipid metabolism. Evidence demonstrates that insulin and dietary unsaturated fatty acid (UFA) regulate SREBP1 expression and subsequent SREBP1-mediated gene transcription, events that in many instances result in modulation of systemic fatty acid and triglyceride (TG) homeostasis. A series of investigations was designed to uncover novel regulators of SREBP1. Dietary and exogenous addition of UFA has been shown to regulate SREBP function yet, an endogenous source of UFA capable of modulating SREBP remains elusive. Group VIA calcium-independent phospholipase A2 (iPLA2) releases UFA from the sn-2 position of glycerophospholipids. We hypothesized that iPLA2 provides UFA to suppress SREBP. iPLA2 overexpression and inhibition studies were implemented. iPLA2 inhibition increased SREBP1 expression, SREBP-mediated transcription and the expression of SREBP1 gene targets in vitro. In vivo overexpression of iPLA2 resulted in decreased expression of SREBP1 protein and plasma triglyceride. In contrast, iPLA2 overexpression attenuated SREBP1 expression, SREBP-mediated transcription and expression of SREBP1 targets genes. These data support the hypothesis that iPLA2 generates endogenous UFA that limit SREBP function. Use of a replication-deficient adenovirus 5 (Ad-5) expression vector in the iPLA2 study led to the unexpected observation of hepatic SREBP1 activation following Ad-5 infection. Because of this observation, we tested the hypothesis that replication-deficient Ad-5 might augment lipid synthesis in liver. We demonstrate that first generation Ad-5, a ubiquitous transgene expression vector, induces expression of SREBP1 and its target genes and leads to increases in fatty acid synthesis in vivo and in vitro. The phosphatidylinositol 3-kinase (PI3K) inhibitor, PX-866, suppressed Ad-5-induced SRBEP1 expression and hypertriglyceridemia implicating the PI3K/Akt pathway in Ad-5 activation of SREBP1. Use of PX-866 led to the discovery of a third mechanism of SREBP1 regulation. In vivo studies demonstrate that PX-866 modulates basal lipid metabolism in part through decreasing plasma TG, an increased trend toward decreased SREBP1 expression and a significant increase in plasma cholesterol. These studies characterize three distinct novel regulatory mechanisms of SREBP1.

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FACEPE
O sobrepeso e a obesidade têm sido identificados como os fatores de risco mais importantes para muitas doenças, incluindo doenças cardiovasculares, diabetes tipo 2 e distúrbios lipídicos como a doença do fígado gorduroso não- alcoólica (NAFLD). Atualmente, a NAFLD é considerada como a manifestação hepática da síndrome metabólica, sendo uma das doenças hepáticas mais prevalentes em todo o mundo. Evidências crescentes sugerem que o AMPK e SREBP são reguladores críticos do metabolismo de lipídios no fígado. As tiazolidinadionas (TZDs) são comumente utilizadas para o tratamento de diabetes tipo 2 e outras condições que ofereçam resistência a insulina como a NAFLD. No presente estudo, foi avaliada a atividade biológica do derivado tiazolidínico (LPSF/GQ-02) sobre a via metabólica de lipídios na patogênese da NAFLD. Foram utilizados camundongos deficientes do receptor de LDL (LDLr-/-) dividido em três grupos: 1- Dieta hipercalórica (HFD); 2- HFD + Pioglitazona (20 mg/kg/dia); 3- HFD+LPSF/GQ-02 (30mg/kg/dia). O experimento foi realizado por 12 semanas sendo que nas ultimas 4 semanas as drogas em estudo (PIO e LPSF/GQ-02) foram administradas via gavagem. Os resultados obtidos indicaram que a LPSF/GQ-02 foi eficaz em melhorar a arquitetura hepática diminuindo a acumulação de gordura no fígado, através da inibição da via da lipogênese (LXR/SREBP-1C/ACC/FAS), bem como através da ativação da via lipolítica (AMPK/FoxO1/ATGL). Estes resultados sugerem uma ação direta da LPSF/GQ-02 sobre o metabolismo lipídico e consequentemente na esteatose hepática, devido à diminuição de gordura nos hepatócitos por meio da inativação da via de síntese de lipídios e aumento da β- oxidação dos ácidos graxos e lipólise. Sendo assim, esses dados apoiam os resultados anteriormente publicado pelo Laboratório de Ultraestrutura do Aggeu Magalhães, que mostraram a propriedade hipolipemiante da LPSF/GQ-02, ao reduzir o acúmulo de triglicerídeo no fígado, bem como confirma o potencial desta TZD para o tratamento na NAFLD.
Overweight and obesity have been identified as the more important risk factors for many diseases, including cardiovascular disease, type 2 diabetes and lipid disorders as the disease. Nonalcoholic fatty liver disease (NAFLD). Actually, NAFLD is considered as the hepatic manifestation of metabolic syndrome is one of the most prevalent liver disease worldwide. Growing evidences suggests that AMPK and SREBP are critical regulators of lipid metabolism in the liver. The thiazolidinediones (TZDs) are commonly used for the treatment of type 2 diabetes and other conditions that provide insulin resistance and NAFLD. In the present study, we evaluated the biological activity of LPSF / GQ-02 on the metabolic pathway of lipids in the pathogenesis of NAFLD. We used mice deficient in LDL receptor (LDLr - / -) divided into three groups: 1 hypercaloric diet (HFD); 2- HFD + pioglitazone (20 mg / kg / day); 3- HFD + LPSF / GQ-02 (30mg / kg / day). The experiment was conducted for 12 weeks and in the last four weeks the drugs were administered daily by gavage. The results indicated that LPSF / GQ-02 was effective in improving liver architecture by decreased the accumulation of fat in the liver, by inhibiting the lipogenic via (LXR / SREBP-1C / ACC / FAS), as well as activating the lipolytic pathway (AMPK / FoxO1 / ATGL). These results suggest a direct action of LPSF / GQ-02 on lipid metabolism in hepatic steatosis and, consequently, due to the decrease of fat in hepatocytes through the inactivation of lipid synthesis pathway and increase the β-oxidation of fatty acids and lipolysis. Thus, these data support the results previously published by Ultrastructure Laboratory Aggeu Magalhães, who showed lipid-lowering property of LPSF / QA-02 by reducing triglyceride accumulation in the liver, and confirms the potential of this TZD for treatment in NAFLD.

Sirt1 (Sirtuine 1) est une protéine histone déacétylase dépendante de NAD+ qui stimule la néoglucogénèse et inhibe la glycolyse dans le foie, et qui augmente l'oxydation des acides gras dans le muscle strié squelettique. Le but de ce travail de thèse a été de définir les fonctions métaboliques de Sirt1 dans le muscle strié squelettique. Nous avons tout d'abord montré, à l'aide d'un modèle de souris déficientes pour le gène Sirt1, que Sirt1 régulait l'expression de l'hexokinase II et de SREBP-1c, protéine régulatrice de l'expression de l'hexokinase. De plus, un modèle d'électrotransfert de gènes permettait de mettre en évidence que Sirt1 régulait l'expression de SREBP-1c de façon LXR dépendante. Enfin, l'inhibition de Sirt1 par l'EX527 aboutissait à une diminution de la consommation de glucose chez des myotubes C2C12. Prises ensemble, ces données suggèrent un rôle important de Sirt1 dans la régulation du métabolisme du glucose dans le muscle strié squelettique. Dans un second temps, nous avons déterminé le rôle potentiel de Sirt1 lors d'un jeûne chez des myotubes C2C12. Un jeûne entraînait une augmentation de l'activité cathepsine B + L et une déphosphorylation des protéines AktS473, GSK3S21/S9, p70S6KT412 et S6 S235/S236 qui précédait une amyotrophie des myotubes. La renutrition aboutissait à une rephosphorylation de ces protéines et à un retour à la normale de la taille des myotubes. L'activité cathepsine B + L restait cependant élevée. Enfin, le niveau en ARNm de Sirt1 était augmenté de façon transitoire lors de la renutrition. D'autres mesures de marqueurs des voies protéolytiques et de l'activité de Sirt1 sont à envisager. Nos données ainsi que celles de la littérature suggèrent que Sirt1 pourrait avoir un rôle dans la régulation de l'autophagie lors du jeûne. Pour conclure, ce travail de thèse met en évidence un rôle pour Sirt1 dans la régulation du métabolisme du glucose dans le muscle strié squelettique et apporte de nouvelles perspectives dans l'étude de la régulation de ce métabolisme en conditions pathologiques

Orientador: Ricardo Utsunomia
Resumo: Pesticidas organofosforados são muito utilizados na agricultura, mas são tóxicos para muitos organismos incluindo os seres humanos, podendo atuar como desreguladores endócrinos e como agentes mutagênicos e carcinogênicos. Um dos efeitos desreguladores é a alteração do metabolismo lipídico, que está associado também ao desenvolvimento do câncer. Isso pode ser explicado pela necessidade das células tumorais em utilizar ácidos graxos para compor suas membranas em construção. Dentre os pesticidas organofosforados está o Diclorvós (DDVP), cujo alto consumo e utilização se baseia em sua grande eficácia e baixo custo. Neste sentido, o objetivo do presente estudo foi avaliar a influência do praguicida DDVP sobre os marcadores moleculares do metabolismo lipídico (SREBP, SCAP, LIMP-II e CD36) na próstata de ratos após indução química pelo carcinógeno N-metil-N-nitrosoureia (MNU). Foram utilizados 32 ratos da linhagem Fischer 344, com idade de 90 dias. Os ratos foram separados em quatro grupos experimentais: Controle, DDVP, MNU, MNU+DDVP. Foram feitas análises histopatológicas, imuno-histoquímicas e Western Blotting na próstata ventral dos ratos. Na análise histopatológica, os grupos MNU e MNU+DDVP apresentaram 100% de incidência de hiperplasia. Para a avaliação morfométrico-estereológica, o grupo MNU+DDVP apresentou aumento do volume relativo de epitélio quando comparado com o grupo controle. As proteínas SREBP e CD36 foram encontradas nas células epiteliais luminais na região do Apare... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Organophosphate pesticides are widely used in agriculture, but are toxic to many organisms including humans, and can act as endocrine disrupters and as mutagenic and carcinogenic agents. One of the deregulatory effects is the alteration of lipid metabolism, which is also associated with cancer development. This may be explained by the need for tumor cells to use fatty acids to compose their building membranes. Among the organophosphate pesticides, Dichlorvos (DDVP) is one of the most effective and least expensive. Therefore, the objective of the present study was to evaluate the influence of the DDVP pesticide on the molecular markers of lipid metabolism (SREBP, SCAP, LIMP-II and CD36) in rat prostate after chemical induction by the carcinogen N-methyl-N-nitrosourea (MNU). Thirty two rats from the Fischer 344 lineage aged 90 days were used. The rats were separated into four experimental groups: Control, DDVP, MNU and MNU + DDVP. Histopathological, immunohistochemical and Western Blotting analyzes of the ventral prostate of rats were performed. In the histopathological analysis, the MNU and MNU + DDVP groups had a 100% incidence of hyperplasia. For the morphometric-stereological evaluation, the MNU + DDVP group showed an increase in the relative volume of epithelium when compared with the control group. The SREBP and CD36 proteins were found in luminal epithelial cells in the Golgi Apparatus region, SCAP mainly in the apical region, and LIMP II dispersed in the cytoplasm as cl... (Complete abstract click electronic access below)
Mestre

INTRODUCTION
During the past decade, Nutrition research has been subjected to a shift of focus, from epidemiology and physiology to the comprensión of the molecular basis of nutrients actions.
Thus, the new "-omics" disciplines transcriptomics, proteomics or metabolomics, provide the tools to understand the molecular mechanisms involved in the modulation of gene expression by nutrients. The study of the beneficial properties of wine procyanidins has not avoided this shift of focus. Thus, from the initial studies which defined the "French paradox", to nowadays, a wide array of studies have been focused in defining the properties of the non-alcoholic components of red wine, mainly flavonoids, a family of polyphenolic compounds. The objectives of this thesis have been to define the molecular mechanisms by which grape procyanidins modulate the hepatic metabolism of lipoproteins, reducing the risk of cardiovascular disease and other pathologies which basis is found in the dysregulation of the lipoprotein metabolism.
SUMMARY
In the present thesis, the effect of procyanidins in the hepatic lipoprotein metabolism has been studied. With this objective, HepG2, HeLa and CV-1 cells have been used as invitro models. In vivo studies have been performed in Wistar rats and C57BL6 mice, wild-type and transgenic mice lacking SHP (NR0B2) and FXR (NR5H1).
RESULTS
1. Procyanidins improve plasma lipid profile in the postprandial phase in rats. A single oral dose of procyanidins decreases plasma triglycerides and ApoB levels to 50% of control values. In addition LDL-Cholesterol is significantly reduced, thus improving the atherosclerotic risk index.
2. Procyanidins display a triglyceride-lowering effect both in vivo and in vitro. In rat and mouse, procyanidin treatment triggers a hypotriglyceridemic response. In HepG2 cultures, procyanidins down-regulate the secretion of triglycerides and ApoB, thus showing that these flavonoids act directly on hepatic cells. This fact strongly suggests that, in vivo, a direct action of procyanidins on the liver contributes to their hypotriglyceridemic response.
3. Nuclear receptor Small Heterodimer Partner (SHP) is a target of procyanidins in hepatic cells. Procyanidins modulate the expression of SHP, rapidly increasing its expression in rat liver as well as in HepG2 cultured cells.
4. SHP mediates the triglyceride-lowering activity of procyanidins in vitro and in vivo. When SHP expression is silenced in HepG2 or abolished in SHP-null mice, procyanidins lose their hypotriglyceridemic activity. In contrast, in SHP-silenced HepG2 cells, procyanidins are still able to reduce apoB secretion. Hence, procyanidins reduce triglyceride via a SHP-dependent mechanism, whereas they reduce apoB in a SHPindependent manner.
5. Nuclear receptor Farnesoid X Receptor (FXR) is an essential mediator of the hypotriglyceridemic action of procyanidins upstream SHP. Oral gavage of procyanidins to FXR-null mice have not a hypotriglyceridemic effect. Moreover, luciferase based in vitro assays showed that procyanidins increase the transcriptional activity of FXR. Thus, FXR is an essential component of the signalling pathway used by procyanidins to elicit the triglyceride lowering effect.
6. Key genes of the inflammation process are targets of procyanidins in liver, in the postprandial phase. Oral administration of procyanidins to rats rapidly downregulates the expression, in liver, of transcription factor Egr1, a mediator of the hepatic inflammatory response, and several acute-phase proteins, namely haptoglobin, fibrinogen B and alpha-1 antitrypsin. In addition, expression of DUSP6, a component of the ERK1/2 subfamily of MAPK, is repressed by this treatment. Nfkbia, a repressor of NF-kB activity, is overexpressed upon procyanidin treatment. This expression pattern strongly suggests that procyanidins attenuate the pro-inflammatory state associated to the postprandial phase.
INTRODUCCIÓN
Durante la pasada década, la investigación en nutrición se ha visto sujeta a un cambio en sus objetivos, pasando de los estudios basados en la fisiología y la epidemiología a la comprensión de las bases moleculares implicadas en las acciones biológicas de los nutrientes. Así, las nuevas disciplinas, como la biología molecular o las "-omics", transcriptómica, proteómica o metabolómica, proporcionan las herramientas para el estudio de los mecanismos moleculares implicados en la modulación génica por nutrientes.
El estudio de las propiedades beneficiosas del vino no ha evitado este cambio de foco. Así, desde los primeros estudios que definieron la "paradoja francesa", hasta la actualidad, una ámplia gama de estudios se han dedicado a definir las propiedades de los componentes no alcohólicos del vino, mayoritariamente, los Flavonoides, una familia de compuetos polifenólicos. El objetivo de esta tesis ha sido definir los mecanismos moleculares mediante los cuales las procianidinas de uva modulan el metabolismo de lipoproteínas en el hígado, disminuyendo así el riesgo cardiovascular y diferentes patologías cuya base se encuentra en la desregulación del metabolismo lipoproteico.
MEMORIA
Durante esta tesis se ha estudiado el efecto de las procianidinas sobre el metabolismo lipoproteico en el hígado. Con este objetivo se han usado líneas celulares como modelo in vitro, tanto hepatocitos (HepG2) como líneas accesorias (HeLa y CV-1). Como modelos para el estudio de las procianidinas in vivo se han usado ratas de la cepa Wistar y ratones de la cepa C57BL6, tanto wild-type como dos líneas de transgénicos, Knockout para SHP (NR0B2) y FXR (NR5H1).
RESULTADOS
Se han obtenido los siguientes resultados:
Las procianidinas de uva disminuyen los niveles de lipoproteínas ricas en triglicéridos, así como mejoran los índices de riesgo cardiovascular en ratas.
Estos efectos se deben a la modulación de la expresión génica en el hígado, tejido adiposo y músculo entre otras acciones.
El mecanismo por el cual las procianidinas disminuyen las lipoproteínas ricas en triglicéridos ha sido estudiado in Vitro (HepG2) e in vivo (C57BL6 wild-type y knockout para SHP). Se han definido dos mecanismos principales. El primero implica la señalización de las procianidinas por una vía dependiente de SHP (Small heterodimer partner, NR0B2), un receptor nuclear. El segundo mecanismo es independiente de SHP e inhibe la expresión de MTP (enzima controlador de la síntesis de lipoproteínas) y consecuente secreción de un menor número de lipoproteínas de muy baja densidad (VLDL).
Por encima de SHP, se ha definido FXR (Farnesoid X receptor) como sensor de las procianidinas mediante el uso de ratones C57BL6 KO para FXR y sistemas reporter basados en luciferasa. Estableciendo que el mecanismo de señalización de las procianidinas pasa por FXR, que a su vez induce la expresión de SHP y este inhibe la expresión de SREBP1, factor de transcripción clave para la síntesis de lípidos, disminuyendo así la cantidad de lípidos hepáticos y, consecuentemente, la secreción de lipoproteínas.
DISCUSIÓN
La modulación del metabolismo de lipoproteínas es el principal objetivo para el tratamiento de las diferentes patologías relacionadas con dislipemias. Así, la definición de las procianidinas de uva como agentes hipolipidémicos, las convierte en un componente de la dieta de alta importancia para prevenir y mejorar una ámplia gama de patologías, desde la aterogénesis hasta otros estados metabólicos alterados, causantes de la resistencia a la insulina o el síndrome metabólico.
Por otro lado, el establecimiento de los mecanismos moleculares implicados en los efectos de las procianidinas de uva, aumenta el conocimiento sobre estos compuestos, así como su aplicabilidad en diferentes estados metabólicos alterados. De esta manera, se ha propuesto que la activación de FXR podría usarse como una estrategia en el tratamiento de la hiperlipidemia o la resistencia a la insulina. Así, las procianidinas emergen como un importante agente terapéutico, cuya importancia radica en la amplia presencia de estos compuestos en la dieta.

Introduction. In the late few years reprogramming studies were undergone on somatic cells to get insight into the epigenetic mechanisms of cell differentiation and developmental biology. DNA methylation was identified as having crucial role on the expression of tissue-specific proteins and in gene silencing of embryonic transcriptional factors. On the other hand, demethylation treatments exhibited to be successful in restoring hTERT and OCT4 in animal models and stabilized cell lines. The aim of this study was to investigate the effects of demethylation treatment on somatic cells isolated from human adipose tissue. Materials and methods. In this work, tissue samples from liposuction or abdominoplastic surgery were processed for cell isolation. Optical microscopy and immunofluorescence, together with cytofluorimetric analysis and further western blotting indages were performed to collect informations regarding morphological and hymmunophenotipical features. Preliminary studies were carried out on mitochondrial dehydrogenases activity and proliferative capacity to estimate the cytotoxicity of the demethylation treatment. Results. The out come of cytotoxic analysis was a reduction of mitochondrial activity in treated cells, compared to untreated cells. Furthermore, we obtained indications of working concentration of the demethylation agent. Cell suffering was observed during clonal expansion in populations treated with demethylation agent. Differences on the harvested cell number resulted between treated and control sample, since lower number was registerd for the demethylated population On the other hand, both treated end untreated samples showed comparable proliferative capacity, during and after the incubation time with demethylation agent. Taken together, the last two results give rise the hypothesis that the demethylation treatment may reduce cell adhesion in monolayer culture after clonal expansion, more than affecting the proliferative capacity. No differences between treated and untreated cell samples were observed with phase contrast optical microscopy regarding cell complexity and distribution and with electronic scansion microscopy concenring dimension and cell spreading. Cells isolated from adipose tissue are able of multipotency. Thus, adipogenic and osteogenic differentiation studies were carried out to evaluate wether 5-Azacytidine could affect the differentiation ability of adipose-derived cells, but no differences were observed between treated differentiated and untrated differentiated. The expression of the adipose tissue-specific transcriptional factor SREBP, was evaluated with fluorescence microscopy and western blotting. SREBP signals were registered in treated cells as well as in the papulation of origin. The research of the transcriptional factors NANOG and OCT4, responsible of stem cell self-renewing in pluripotent cells, didn’t get positive results, even in those populations long cultured and expanded after the treatment. Characterization studies with immunofluorescence techniques were undergone on freshly isolated from adipose tissue. Cytometric data revealed the presence of diverse cell linages that were lost during the cell expansion. By contrast an enrichment of the cell component, described elsewhere as bone marrow stem population, CD105+/CD90+/ CD29+, was observed in the III and IV generations. In long-cultured adipose tissue-derived cells was registered lost of adipose tissue-specific marker ADRP as well as the depletion of cytoplasmatic adipose vescicles. The investigation with confocal microscopy and western blotting revealed no modifications in the expression of ADRP and CD105 markers in 5-Azacytidine treated cells, while new signal of STRO1 related to the bone marrow-derived stem cells, was detected in long cultured adipose-derived cells, in treated as well untreated sample. Conclusions. No positive results were obtained through demethylation treatment of human adipose-derived cells with 5-Azacytidine, in contrast to the experiences with animal cell models. Would be extremely interesting to get insight into the methylation level of DNA for defined factors in the long-cultured adipose-derived cells CD105+/ CD90+/STRO1+/ADRP-. Moreover, new approach with association of a variety of demethylation agents in association with stem growth factors may give interesting results.
Introduzione. L’ottenimento di cellule dotate di caratteristiche differenziative di tipo pluripotente a partire da cellule somatiche adulte è reso possibile mediante manipolazione dello status epigenetico. Il processo di metilazione del DNA è responsabile dell’espressione di proteine tessuto-specifiche e del silenziamento genico di fattori di trascrizione tipici della cellula germinale o tumorale. Al contrario, è stato dimostrato che il trattamento con agenti demetilanti è in grado di indurre in cellule animali multipotenti e in cellule stabilizzate l’espressione di proteine e fattori di trascrizione presenti nella cellula germinale (hTERT, OCT4). In questo studio è stato valutato l’effetto di un trattamento demetilante in vitro su cellule umane isolate da tessuto adiposo adulto (PLA cells). Materiali e metodi. Lo studio ha richiesto l’estrazione di cellule da tessuto adiposo omentale ottenuto mediante addominoplastica o liposuzione e l’analisi delle caratteristiche morfologiche e fenotipiche delle PLA cells mediante tecniche di microscopia ottica e a fluorescenza, citofluorimetria e Western blotting a seguito del trattamento con l’agente demetilante 5-Azacitidina. Sono stati condotti studi preliminari sulla citotossicità del trattamento demetilante con saggi sull’attività delle deidrogenasi mitocondriali e sulla capacità proliferativa. Risultati. Negli studi di citotossicità dell’agente demetilante 5-Azacitidina, si è registrato una riduzione dell’attività metabolica mitocondriale nella popolazione trattata rispetto al controllo. Lo studio ha anche fornito le conoscenze per definire la concentrazione di agente demetilante rispetto al range riportato in letteratura. Le popolazioni trattate con l’agente demetilante hanno mostrato sofferenza durante l’espansione generazionale; la popolazione cellulare riseminata dopo distacco enzimatico presenta un numero di cellule significativamente inferiore rispetto al campione di controllo. D’altro lato, la popolazione presenta capacità proliferativa paragonabile a quella dei campioni non trattati, durante e dopo il trattamento demetilante. I due risultati potrebbero indicare che il trattamento demetilante non influisca sulla proprietà replicativa della cellula, ma ne riduca la capacità di adesione nella coltura in monostrato dopo il cambio generazionale. Poiché le PLA cells sono in grado di differenziare in altre tipologie cellulari, sono stati condotti studi di differenziamento in senso adipogenico, osteogenico e miogenico su popolazioni tratte in precedenza con l’agente demetilante. Il confronto con la risposta agli stimoli differenziativi di cellule non trattate ha dimostrato che l’agente demetilante non ha modificato la capacità differenziativa. Le indagini di microscopia a fluorescenza e Western blotting hanno mostrato che il fattore di trascrizione tessuto-specifico SREBP, presente nelle popolazioni di origine, è ugualmente espresso dopo il trattamento con 5-Azacitidina. La ricerca dei fattori di trascrizione NANOG e OCT4, responsabili della capacità di self-renewing nelle cellule pluripotenti non ha dato risultati positivi. Le cellule trattate con 5-Azacitidina sono state mantenute in coltura ed espanse, per valutare un eventuale rimodellamento proteomico nelle cellule replicate, ma non è stata registrata espressione dei due marcatori nucleari. Gli studi di caratterizzazione condotti con immunofluorescenza confocale e analisi citometriche hanno dimostrato che la popolazione cellulare isolata da tessuto adiposo è eterogenea. E’ stato osservato che, l’espansione generazionale determina un arricchimento nella componente cellulare CD90+/CD105+/CD29+, e cioè una popolazione con forti analogie fenotipiche con le cellule staminali stromali del midollo osseo, a scapito delle cellule endoteliali ed ematopoietiche presenti in origine. Inoltre, le cellule mantenute in coltura perdono il segnale dell’ADRP, proteina associata al commitment adipogenico, ed esibiscono una deplezione delle vescicole adipose citoplasmatiche, mentre è stato osservato nelle cellule di terza e quarta generazione la comparsa del marcatore di staminalità mesenchimale STRO1, espresso unicamente nella frazione stromale delle cellule staminali estratte da midollo osseo. Conclusioni. PLA cells di origine umana non si comportano in vitro dopo trattamento demetilante come le corrispondenti cellule di origine animale e cioè non sembrano essere in grado, nelle condizioni sperimentali usate in questo studio, di essere riprogrammate ad un fenotipo diverso da quello di origine. Questo risultato pone il problema dello stato di metilazione del DNA nelle cellule isolate da tessuto adiposo che mantenute a lungo in vitro assumono il fenotipo CD105+/ CD90+/STRO1+/ADRP-, e se un trattamento che preveda l’associazione di diversi agenti demetilanti/deacetilanti in combinazione con fattori di crescita possa realizzare in queste cellule una reale riprogrammazione funzionale in vitro.

Prostate cancer (CaP) is the most commonly diagnosed cancer in males in Australia, North America, and Europe. If found early and locally confined, CaP can be treated with radical prostatectomy or radiation therapy; however, 25-40% patients will relapse and go on to advanced disease. The most common therapy in these cases is androgen deprivation therapy (ADT), which suppresses androgen production from the testis. Lack of the testicular androgen supply causes cells of the prostate to undergo apoptosis. However, in some cases the regression initially seen with ADT eventually gives way to a growth of a population of cancerous cells that no longer require testicular androgens. This phenotype is essentially fatal and is termed castrate resistant prostate cancer (CRPC). In addition to eventual regression, there are many undesirable side effects which accompany ADT, including development of a metabolic syndrome, which is defined by the U.S. National Library of Medicine as “a combination of medical disorders that increase the risk of developing cardiovascular disease and diabetes.” This project will focus on the effect of ADT induced hyperinsulinemia, as mimicked by treating androgen receptor positive CaP cells with insulin in a serum (hormone) deprived environment. While this side effect is not widely explored, in this thesis it is demonstrated for the first time that insulin upregulates pathways important to CaP progression. Our group has previously shown that during CaP progression, the enzymes necessary for de novo steroidogenesis are upregulated in the LNCaP xenograft model, total steroid levels are increased in tumours compared to pre castrate levels, and de novo steroidogenesis from radio-labelled acetate has been demonstrated. Because of the CaP dependence on AR for survival, we and other groups believe that CaP cells carry out de novo steroidogenesis to survive in androgen deprived conditions. Because (a) men on ADT often develop metabolic syndrome, and (b) men with lifestyle-induced obesity and hyperinsulinemia have worse prognosis and faster disease progression, and because (c) insulin causes steroidogenesis in other cell lines, the hypothesis that insulin may contribute to CaP progression through upregulation of steroidogenesis was explored. Insulin upregulates steroidogenesis enzymes at the mRNA level in three AR positive cell lines, as well as upregulating these enzymes at the protein level in two cell lines. It has also been demonstrated that insulin increases mitochondrial (functional) levels of steroid acute regulatory protein (StAR). Furthermore, insulin causes increased levels of total steroids in and induction of de novo steroid synthesis by insulin has been demonstrated at levels induced sufficient to activate AR. The effect of insulin analogs on CaP steroidogenesis in LNCaP and VCaP cells has also been investigated because epidemiological studies suggest that some of the analogs developed may have more cancer stimulatory effects than normal insulin. In this project, despite the signalling differences between glargine, X10, and insulin, these analogs did not appear to induce steroidogenesis any more potently that normal insulin. The effect of insulin of MCF7breast cancer cells was also investigated with results suggesting that breast cancer cells may be capable of de novo steroidogenesis, and that increase in estradiol production may be exacerbated by insulin. Insulin has also been long known to stimulate lipogenesis in the liver and adipocytes, and has been demonstrated to increase lipogenesis in breast cancer cells; therefore, investigation of the effect of insulin on lipogenesis, which is a hallmark of aggressive cancers, was investigated. In CaP progression sterol regulatory element binding protein (SREBP) is dysregulated and upregulates fatty acid synthase (FASN), acetyl CoA-carboxylase, and other lipogenesis genes. SREBP is important for steroidogenesis and in this project has been shown to be upregulated by insulin in CaP cells. Fatty acid synthesis provides building blocks of membrane growth, provides substrates for acid oxidation, the main energy source for CaP cells, provides building blocks for anti-apoptotic and proinflammatory molecules, and provides molecules that stimulate steroidogenesis. In this project it has been shown that insulin upregulates FASN and ACC, which synthesize fatty acids, as well as upregulating hormone sensitive lipase (HSL), diazepam-binding inhibitor (DBI), and long-chain acyl-CoA synthetase 3 (ACSL3), which contribute to lipid activation of steroidogenesis. Insulin also upregulates total lipid levels and de novo lipogenesis, which can be suppressed by inhibition of the insulin receptor (INSR). The fatty acids synthesized after insulin treatment are those that have been associated with CaP; furthermore, microarray data suggests insulin may upregulate fatty acid biosynthesis, metabolism and arachidonic acid metabolism pathways, which have been implicated in CaP growth and survival. Pharmacological agents used to treat patients with hyperinsulinemia/ hyperlipidemia have gained much interest in regards to CaP risk and treatment; however, the scientific rationale behind these clinical applications has not been examined. This thesis explores whether the use of metformin or simvastatin would decrease either lipogenesis or steroidogenesis or both in CaP cells. Simvastatin is a 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) inhibitor, which blocks synthesis of cholesterol, the building block of steroids/ androgens. It has also been postulated to down regulate SREBP in other metabolic disorders. It has been shown in this thesis, in LNCaP cells, that simvastatin inhibited and decreased insulin induced steroidogenesis and lipogenesis, respectively, but increased these pathways in the absence of insulin. Conversely, metformin, which activates AMP-activated protein kinase (AMPK) to shut down lipogenesis, cholesterol synthesis, and protein synthesis, highly suppresses both steroidogenesis and lipogenesis in the presence and absence of insulin. Lastly, because it has been demonstrated to increase steroidogenesis in other cell lines, and because the elucidation of any factors affecting steroidogenesis is important to understanding CaP, the effect of IGF2 on steroidogenesis in CaP cells was investigated. In patient samples, as men progress to CRPC, IGF2 mRNA and the protein levels of the receptors it may signal through are upregulated. It has also been demonstrated that IGF2 upregulates steroidogenic enzymes at both the mRNA and protein levels in LNCaP cells, increases intracellular and secreted steroid/androgen levels in LNCaPs to levels sufficient to stimulate the AR, and upregulated de novo steroidogenesis in LNCaPs and VCaPs. As well, inhibition of INSR and insulin-like growth factor 1 receptor (IGF1R), which IGF2 signals through, suggests that induction of steroidogenesis may be occurring predominantly through IGF1R. In summary, this project has illuminated for the first time that insulin is likely to play a large role in cancer progression, through upregulation of the steroidogenesis and lipogenesis pathways at the mRNA and protein levels, and production levels, and demonstrates a novel role for IGF-II in CaP progression through stimulation of steroidogenesis. It has also been demonstrated that metformin and simvastatin drugs may be useful in suppressing the insulin induction of these pathways. This project affirms the pathways by which ADT- induced metabolic syndrome may exacerbate CaP progression and strongly suggests that the monitoring and modulation of the metabolic state of CaP patients could have a strong impact on their therapeutic outcomes.