Aging is a major risk factor for many chronic diseases and a complex biological phenomenon. The most well studied and characterized pathways involved in metabolism and known to regulate longevity include sirtuins, AMP-activated protein kinase, insulin-like growth factor (IGF) and the mechanistic target of rapamycin (mTOR).1 These signaling pathways and related transcriptional factors are evolutionarily conserved from yeast to primates. Evidence suggests adipose tissue plays an important role in the regulation of lifespan particularly through energy homeostasis during times of scarcity and excess. Our laboratory has shown adipose triglyceride lipase (ATGL), the rate-limiting enzyme within the lipolytic pathway, is the target of dietary restriction and insulin/IGF-1 signaling pathways, both of which regulate lifespan.22 Given the convergence and necessity of ATGL-1 in the longevity response of dietary restriction and reduced insulin/IGF1 signaling pathways and the uncertainty of the downstream effects TOR has on longevity, we hypothesize that ATGL-1 plays an important role in CeTOR regulated longevity in C. elegans. This investigation was carried out by (a) determining whether levels of ATGL-1 are influenced by TOR inhibition via rapamycin and TOR specific RNA interference (RNAi) and (b) examining the role of ATGL-1 in CeTOR regulated longevity in C. elegans. We have found that rapamycin treatment does not increase expression of ATGL-1::GFP in C. elegans, however, continued research with CeTOR inhibition using rapamycin and RNAi treatment is necessary. The RNAi and longevity experiments need to be conducted. Tissue specific regulation of ATGL expression has been shown to be implicated in chronic disease and in longevity. However, there are still many insights to be discovered and understood about its role in longevity pathways, including feedback mechanisms and second messengers lipolytic products play. Elucidating the downstream effects of ATGL within model organisms will impact future chronic disease research and longevity studies. Given that these pathways are widely evolutionarily conserved, future findings will aid in understanding longevity regulatory mechanisms in humans.

Os hormônios tireoidianos (HT) executam um papel lipolítico importante no Tecido Adiposo Branco (TAB), sendo este efeito mediado por meio do aumento da expressão de receptores beta adrenérgicos na membrana do adipócito, o que aumenta a sensibilidade deste tecido as catecolaminas. Sabe-se que os principais efetores da ação lipolítica nesse tecido são a lipase hormônio sensível (LHS) e a lipase dos triglicerídeos dos adipócitos (ATGL), as quais hidrolisam os triglicerídeos em ácidos graxos e glicerol. Além disso, outros componentes estão envolvidos na atividade lipolítica, como as perilipinas, proteínas estas que envolvem a gota de gordura, formando uma barreira contra a ação da LHS e ATGL, de modo que precisam ser fosforiladas para que a LHS e ATGL possam exercer seu efeito lipolítico. Considerando: (a) a importância do tecido adiposo na homeostase energética e como fonte de citocinas, as quais estão relacionadas com a sensibilidade tecidual à insulina; (b) que a função e o metabolismo do tecido adiposo variam com a sua distribuição regional, e (c) que as ações lipolíticas dos HT, importantes reguladores da homeostase energética, têm sido muito pouco exploradas, pretendemos investigar, em ratos, (i) se os HT interferem na expressão da LHS, ATGL, perilipina A e dos receptores beta3 adrenérgicos no tecido adiposo branco, e (ii) se essas ações diferem nos distintos depósitos de gordura, o que poderia ampliar o campo de conhecimento sobre os efeitos lipolíticos destes hormônios e a nossa compreensão sobre a contribuição deles nas complicações associadas à obesidade e suas co-morbidades. / Thyroid hormones (TH) play an important lipolytic role in white adipose tissue (WAT). This effect is mediated by increased expression of beta-adrenergic receptors on adipocytes membrane, which increases the sensitivity of that tissue to catecholamines. It is known that the main effectors of the lipolytic action in WAT enzymatic activity, especially: hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), which hydrolyze triglycerides into fatty acids and glycerol. In addition, other components are involved in the lipolytic activity, such as perilipin. These proteins support the fat droplet, forming a protective barrier against HSL and ATGL action. Considering: (a) the importance of adipose tissue in energy homeostasis and as a source of cytokines which are related to insulin tissue sensitivity; (b) function and metabolism of adipose tissue vary with their regional distribution; and (c) lipolytic actions of HT, important regulators of energy homeostasis, have been little explored, we investigated in rats with hypothyroidism and submitted to T3 treatment: (i) TH effects on the expression of hormone sensitive lipase (HSL), adipose triglyceride lipase (ATGL),perilipin A and beta-3 adrenergic receptors in WAT, and (ii) if this action are different on subcutaneous and visceral fat depot. This study has increased our understanding about the contribution of these hormones on WAT metabolism and metabolic disease as obesity.

Os hormônios tireoidianos (HT) executam um papel lipolítico importante no Tecido Adiposo Branco (TAB), sendo este efeito mediado por meio do aumento da expressão de receptores beta adrenérgicos na membrana do adipócito, o que aumenta a sensibilidade deste tecido as catecolaminas. Sabe-se que os principais efetores da ação lipolítica nesse tecido são a lipase hormônio sensível (LHS) e a lipase dos triglicerídeos dos adipócitos (ATGL), as quais hidrolisam os triglicerídeos em ácidos graxos e glicerol. Além disso, outros componentes estão envolvidos na atividade lipolítica, como as perilipinas, proteínas estas que envolvem a gota de gordura, formando uma barreira contra a ação da LHS e ATGL, de modo que precisam ser fosforiladas para que a LHS e ATGL possam exercer seu efeito lipolítico. Considerando: (a) a importância do tecido adiposo na homeostase energética e como fonte de citocinas, as quais estão relacionadas com a sensibilidade tecidual à insulina; (b) que a função e o metabolismo do tecido adiposo variam com a sua distribuição regional, e (c) que as ações lipolíticas dos HT, importantes reguladores da homeostase energética, têm sido muito pouco exploradas, pretendemos investigar, em ratos, (i) se os HT interferem na expressão da LHS, ATGL, perilipina A e dos receptores beta3 adrenérgicos no tecido adiposo branco, e (ii) se essas ações diferem nos distintos depósitos de gordura, o que poderia ampliar o campo de conhecimento sobre os efeitos lipolíticos destes hormônios e a nossa compreensão sobre a contribuição deles nas complicações associadas à obesidade e suas co-morbidades. / Thyroid hormones (TH) play an important lipolytic role in white adipose tissue (WAT). This effect is mediated by increased expression of beta-adrenergic receptors on adipocytes membrane, which increases the sensitivity of that tissue to catecholamines. It is known that the main effectors of the lipolytic action in WAT enzymatic activity, especially: hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), which hydrolyze triglycerides into fatty acids and glycerol. In addition, other components are involved in the lipolytic activity, such as perilipin. These proteins support the fat droplet, forming a protective barrier against HSL and ATGL action. Considering: (a) the importance of adipose tissue in energy homeostasis and as a source of cytokines which are related to insulin tissue sensitivity; (b) function and metabolism of adipose tissue vary with their regional distribution; and (c) lipolytic actions of HT, important regulators of energy homeostasis, have been little explored, we investigated in rats with hypothyroidism and submitted to T3 treatment: (i) TH effects on the expression of hormone sensitive lipase (HSL), adipose triglyceride lipase (ATGL),perilipin A and beta-3 adrenergic receptors in WAT, and (ii) if this action are different on subcutaneous and visceral fat depot. This study has increased our understanding about the contribution of these hormones on WAT metabolism and metabolic disease as obesity.

L’hydrolyse des triglycérides (TG) du tissu adipeux est initialisée par l’action de l’adipocyte TG lipase (ATGL), activée par son cofacteur la protéine CGI-58. Des mutations du gène codant les protéines CGI-58 ou ATGL sont respectivement à l’origine du syndrome de Chanarin-Dorfmann et de maladies de stockagede lipides neutres. La protéine CGI-58 appartient à la famille des a/ß-hydrolases, elle en possède la triade catalytique Ser-Asp/Glu-His caractéristique des carboxylester hydrolases, ainsi que le motif HX4D, caractéristique d’une activité acyltransférase. A ce jour, le rôle de la protéine CGI-58, chez les mammifères ou chez les plantes, n’est pas totalement éclairci, de même que son activité enzymatique. Dans le but de mieux comprendre le rôle de cette protéine CGI-58 dans le métabolisme lipidique, nous avons développé une nouvelle stratégie, en générant notamment de nouveaux plasmides qui nous ont permis d’exprimer et de purifier la protéine CGI-58 de souris et de plantes, ainsi que l’ATGL murine, dans différents souches d’E. coli, pour tester l’activité in vitro de ces protéines. De plus, nous avons mis en place, en utilisant ces plasmides générés et différentes souches E. coli, un système qui nous a permis de tester in vivo, dans E. coli, l’activité acyltransférase (LPAAT et/ou LPGAT) de la protéine CGI-58. En utilisant ces différentes techniques, nous avons pu montrer, aussi bien in vivo qu’in vitro, que la protéine CGI-58 de plante, ainsique celle de mammifère, ne possède ni activité LPAAT ni activité LPGAT, et que la protéine CGI-58 de plante est dépourvue d’activité TG lipase ou phospholipase. Cependant, nous avons montré, en analysant, par chromatographie sur couche mince et par spectrométrie de masse, des extraits lipidiques des différentes souches d’E. coli exprimant la protéine CGI-58, que l’expression de la protéine de plante, et non celle de mammifère, aboutit à une diminution du taux de phosphatidylglycérol (PG) dans les différentes souchestestées, et a contrario nous avons montré que la mutation de la sérine, ainsi que la mutation de l’histidine de la triade catalytique potentielle, restaure le phénotype sauvage. Ces résultats nous ont permis de proposer que la protéine CGI-58 de plante est impliquée dans le métabolisme du PG / Triglycerides (TG) hydrolysis in adipose tissue is initialized by the action of the adipose TG lipase (ATGL) and its cofactor, the CGI-58 protein. Mutations in the gene coding the CGI-58 or ATGL proteins are the cause of the syndrome of Chanarin-Dorfman and of neutral lipids storage disease, respectively. CGI-58 protein belongs to the a/ß-hydrolase family, harboring the catalytic triad Ser-Asp/Glu-His, characteristic of carboxylester hydrolases, as well as the HX4D motif, characteristic of acyltransferase activity. Nowadays, the role and the enzymatic activity of the CGI-58 protein, in mammalians and plants, are not quite clear. In order to better understand the function of CGI-58 protein in lipid metabolism, we developed a new strategy using a new set of plasmids that enabled us, with the use of pET28b(+) plasmid, to express and purify the CGI-58 protein of mice and plants as well as the murine ATGL. These proteins were expressed in different E. coli strains, to test in vitro their activity. In addition, we have set up, using the generated plasmids and different E. coli strains, a system that allowed us to test the in vivo acyltransferase activity (LPAAT and/or LPGAT) of the CGI-58 proteins expressed in E. coli. Using these techniques, we demonstrated in vivo and in vitro that both mice and plant CGI-58 proteins, are neither able to catalyze a LPAAT or a LPGAT reaction, and that the plant CGI-58 protein is devoid of TG lipase or phospholipase activity. However we have shown, by analyzing lipid extracts, by thin layer chromatography and by mass spectrometry of different E. coli strains expressing the CGI-58 protein, that the expression of the plant CGI-58 protein, but not the mice one, results in a decrease of phosphatidylglycerol (PG) content in the different strains tested. However the mutation of the serine or histidine residue of the putative catalytic triad restores the wild-typephenotype. These results allowed us to propose that plant CGI-58 protein is involved in the metabolism of PG

No description available.

Mon travail de thèse a été axé sur l'étude du rôle des lipides musculaires dans la régulation du métabolisme énergétique et la sensibilité à l'insuline. Les lipides sont présents sous deux formes au sein du muscle squelettique : soit sous forme d'adipocytes insérés entre les fibres/faisceaux musculaires, soit sous forme de gouttelettes lipidiques à l'intérieur des fibres musculaires (i.e. triglycérides intramyocellulaires ou IMTG). Ces deux dépôts de lipides, lorsqu'ils sont présents en excès, sont associés à la mise en place de l'insulino-résistance musculaire chez l'homme, via l'accumulation intracellulaire d'espèces lipidiques lipotoxiques altérant la signalisation insulinique pour les IMTG, et par un mécanisme inconnu pour les adipocytes. Dans un premier temps, nous avons isolé et mieux caractérisé, à partir de biopsies musculaires humaines, deux populations de cellules progénitrices. La première population présente un potentiel de différenciation myogénique en culture, il s'agit des cellules satellites (cellules progénitrices musculaires). La deuxième population est composée de cellules capables d'acquérir les propriétés phénotypiques et métaboliques d'adipocytes blancs matures, il s'agit des progéniteurs fibro/adipocytaires (FAPs). Grace à ces modèles d'étude, nous avons mis en évidence que les sécrétions des adipocytes dérivés des FAPs sont capables d'altérer la voie de signalisation et les effets de l'insuline sur des fibres musculaires humaines in vitro. Cet effet paracrine pourrait en partie expliquer la corrélation négative observée entre le contenu en adipocytes intramusculaires et la sensibilité à l'insuline chez l'homme. Dans un second temps, nous avons étudié le rôle de deux protéines, G0/G1 Switch Gene 2 (G0S2) et la périlipine 5 (PLIN5), dans la dynamique des gouttelettes lipidiques ainsi que leur impact sur le métabolisme des lipides et la sensibilité à l'insuline. Nous avons montré in vitro que ces deux protéines jouent un rôle clé dans le contrôle de la lipolyse musculaire (i.e. hydrolyse des IMTG) via l'adipose triglyceride lipase (ATGL, enzyme limitante de la lipolyse musculaire), et que G0S2 et PLIN5 inhibent l'activité de l'ATGL par des mécanismes directs et indirects, respectivement. Par ailleurs, nos données ont montré que l'invalidation de G0S2 et PLIN5 dans le muscle squelettique active la lipolyse, augmente la lipotoxicité et diminue la sensibilité à l'insuline in vivo chez la souris. Nous avons également démontré un rôle important de PLIN5 dans la régulation de l'oxydation des acides gras en ajustant finement leur disponibilité aux besoins énergétiques des cellules. En résumé, ces travaux démontrent d'une part qu'une communication entre adipocytes et fibres au sein du muscle peut entraîner une altération de la sensibilité à l'insuline musculaire chez l'homme, et d'autre part que G0S2 et PLIN5, deux protéines de la gouttelette lipidique, sont au centre du contrôle de l'homéostasie lipidique et du maintien de l'insulino-sensibilité musculaire. Ces données permettent ainsi d'élargir les connaissances existantes sur le lien entre les lipides musculaires et la sensibilité à l'insuline chez l'homme. / My PhD research work was focused on the role of muscle lipids in the regulation of energy metabolism and insulin sensitivity. Lipids can be found under two different forms in skeletal muscle: adipocytes located between muscle fibers/bundles and lipid droplets inside muscle fibers (i.e. intramyocellular triacylglycerols or IMTG). These depots, when present in excess, have both been associated with insulin-resistance in humans, mainly because of intracellular lipotoxic lipid accumulation known to impair insulin signaling for IMTG, and through a yet unknown mechanism for adipocytes. First, we isolated and characterized two distinct populations of progenitor cells from human muscle biopsies. The first population is composed of satellite cells (muscle progenitor cells) and display a myogenic differentiation potential in vitro. The second population is composed of cells that acquire the phenotypic and metabolic properties of functional white adipocytes, called fibro/adipogenic progenitors (FAPs). By using these cell models, we showed that FAPs-derived adipocytes secretions are able to impair insulin signaling and action in human skeletal muscle fibers in vitro. This paracrine effect could explain, at least partly, the inverse relationship observed between intramuscular adipocyte content and insulin sensitivity in humans. Secondly, we studied the role of two proteins, G0/G1 Switch Gene 2 (G0S2) and perilipin 5 (PLIN5), in lipid droplets dynamics as well as their impact on lipid metabolism and insulin sensitivity. We showed in vitro that these two proteins play a key role in the control of muscle lipolysis (i.e. IMTG hydrolysis) via the adipose triglyceride lipase (ATGL, catalyzing the limiting step of muscle lipolysis), and that G0S2 and PLIN5 inhibit ATGL activity through direct and indirect mechanisms, respectively. Furthermore, our data showed that G0S2 and PLIN5 invalidation in vivo in mouse skeletal muscle activates lipolysis, increases lipotoxicity and impairs insulin sensitivity. We have also highlighted an important role for PLIN5 in the regulation of fatty acids oxidation, by finely adjusting their availability to energy demand. Overall, these results clearly show on one hand that a crosstalk between adipocytes and fibers within skeletal muscle can lead to an alteration of insulin sensitivity in humans, and on the other hand that G0S2 and PLIN5, two lipid droplet proteins, play a central role in the control of muscle lipid homeostasis and insulin sensitivity. These data help to develop our current understanding of the link between muscle lipids and insulin sensitivity in humans.

No description available.