Thermogenic brown adipose tissue generates heat via mitochondrial uncoupling protein-1 (UCP-1), increases whole-body energy expenditure and may protects against obesity and metabolic disorders. White adipocytes store excess energy in the form of triglycerides. UCP-1 positive adipocytes develop within white adipose tissue (beige or brite adipocytes) in response to cold exposure or β3 adrenergic agonists. It was known that beige adipocytes arise from a distinct lineage compared with brown adipocytes, but the developmental origin of the beige adipocytes is still unclear. Signaling pathways that control beige adipocyte determination and formation are essentially unknown. Here, we identified a novel signaling pathway that regulates the lineage specification of beige adipocytes. Bone morphogenetic protein 7 (BMP7), a known brown adipogenesis inducer, suppresses Rho-GTPase kinase (ROCK) and depolymerizes F-actin (filamentous actin) into G-actin (globular actin) in mesenchymal stem cells. G-actin regulates myocardin-related transcription factor A (MRTFA) that co-transactivates serum response factor (SRF) and promotes smooth muscle cell differentiation in various organs. Subcutaneous white adipose tissue from MRTFA-/- mice had enhanced accumulation of UCP-1+ adipocytes and elevated levels of brown-selective proteins. Compared with wild type (WT) controls, MRTFA-/- mice exhibited improved metabolic profiles and were protected from diet-induced obesity and insulin resistance, suggesting that the beige adipocytes are physiologically functional. Compared to WT mice, stromal vascular cells from MRTFA-/- mice expressed higher levels of distinct beige progenitor markers and reduced levels of smooth muscle markers. Our studies demonstrate a novel ROCK-actin-MRTFA/SRF pathway that contributes to the development of beige adipocytes.

A atividade do complexo 1 da proteína alvo mecanístico da rapamicina (mTORC1), importante regulador da adiposidade e do metabolismo de lipídeos, está aumentada no tecido adiposo de camundongos obesos. A inibição completa de mTORC1 reduz a adiposidade, enquanto que sua inibição parcial potencializa a obesidade induzida por dieta. Assim, hipotetizamos que um nível ótimo de ativação de mTORC1 é necessário para promover aumento da adiposidade, de forma que sua superativação é tão inibitória para a deposição de gordura quanto sua inibição completa. Para testar esta hipótese, investigamos os efeitos da ativação constitutiva de mTORC1, induzida pela deleção de Tsc1, especificamente em adipócitos na adiposidade in vivo. A deleção de Tsc1 reduziu a massa do tecido adiposo visceral, mas não do subcutâneo, que foi associado ao aumento da lipólise e browning. Além disso, aumentou em ambos tecidos adiposos a massa e atividade oxidativa mitocondrial. Esses dados apoiam nossa hipótese de que é necessário um nível ótimo de ativação de mTORC1 para promover aumento da adiposidade. / The activity of mechanistic target of rapamycin complex 1 (mTORC1), an important regulator of adiposity and lipid metabolism, is increased in adipose tissue of obese mice. Complete mTORC1 inhibition reduces adiposity, whereas partial mTORC1 inhibition enhances diet-induced obesity. Therefore, we hypothesized that an optimal level of mTORC1 activity is required to increase adiposity, in such a manner that mTORC1 overactivation is as inhibitory to fat deposition as its complete inhibition. To test this hypothesis, we investigated the effects of constitutive mTORC1 activation, induced by Tsc1 deletion, specifically in adipocytes on adiposity in vivo. Tsc1 deletion reduced visceral, but not subcutaneous, fat mass, which was associated with increased lipolysis and browning. Moreover, it enhanced mitochondrial mass and oxidative activity in both visceral and subcutaneous fat. These data support our hypothesis that an optimal level of mTORC1 activation is necessary to increase adiposity.

A atividade do complexo 1 da proteína alvo mecanístico da rapamicina (mTORC1), importante regulador da adiposidade e do metabolismo de lipídeos, está aumentada no tecido adiposo de camundongos obesos. A inibição completa de mTORC1 reduz a adiposidade, enquanto que sua inibição parcial potencializa a obesidade induzida por dieta. Assim, hipotetizamos que um nível ótimo de ativação de mTORC1 é necessário para promover aumento da adiposidade, de forma que sua superativação é tão inibitória para a deposição de gordura quanto sua inibição completa. Para testar esta hipótese, investigamos os efeitos da ativação constitutiva de mTORC1, induzida pela deleção de Tsc1, especificamente em adipócitos na adiposidade in vivo. A deleção de Tsc1 reduziu a massa do tecido adiposo visceral, mas não do subcutâneo, que foi associado ao aumento da lipólise e browning. Além disso, aumentou em ambos tecidos adiposos a massa e atividade oxidativa mitocondrial. Esses dados apoiam nossa hipótese de que é necessário um nível ótimo de ativação de mTORC1 para promover aumento da adiposidade. / The activity of mechanistic target of rapamycin complex 1 (mTORC1), an important regulator of adiposity and lipid metabolism, is increased in adipose tissue of obese mice. Complete mTORC1 inhibition reduces adiposity, whereas partial mTORC1 inhibition enhances diet-induced obesity. Therefore, we hypothesized that an optimal level of mTORC1 activity is required to increase adiposity, in such a manner that mTORC1 overactivation is as inhibitory to fat deposition as its complete inhibition. To test this hypothesis, we investigated the effects of constitutive mTORC1 activation, induced by Tsc1 deletion, specifically in adipocytes on adiposity in vivo. Tsc1 deletion reduced visceral, but not subcutaneous, fat mass, which was associated with increased lipolysis and browning. Moreover, it enhanced mitochondrial mass and oxidative activity in both visceral and subcutaneous fat. These data support our hypothesis that an optimal level of mTORC1 activation is necessary to increase adiposity.

Les sécrétines de l’hormone de croissance (GHRPs) sont de petits peptides synthétiques capables de stimuler la sécrétion de l’hormone de croissance à partir de l’hypophyse via leur liaison au récepteur de la ghréline GHS-R1a. Le GHRP hexaréline a été utilisé afin d’étudier la distribution tissulaire de GHS-R1a et son effet GH-indépendant. Ainsi, par cette approche, il a été déterminé que l’hexaréline était capable de se lier à un deuxième récepteur identifié comme étant le récepteur scavenger CD36. Ce récepteur possède une multitude de ligands dont les particules oxLDL et les acides gras à longue chaîne. CD36 est généralement reconnu pour son rôle dans l’athérogénèse et sa contribution à la formation de cellules spumeuses suite à l’internalisation des oxLDL dans les macrophages/monocytes. Auparavant, nous avions démontré que le traitement des macrophages avec l’hexaréline menait à l’activation de PPARƔ via sa liaison à GHS-R1a, mais aussi à CD36. De plus, une cascade d’activation impliquant LXRα et les transporteurs ABC provoquait également une augmentation de l’efflux du cholestérol. Une stimulation de la voie du transport inverse du cholestérol vers les particules HDL entraînait donc une diminution de l’engorgement des macrophages de lipides et la formation de cellules spumeuses. Puisque CD36 est exprimé dans de multiples tissus et qu’il est également responsable du captage des acides gras à longue chaîne, nous avons voulu étudier l’impact de l’hexaréline uniquement à travers sa liaison à CD36. Dans le but d’approfondir nos connaissances sur la régulation du métabolisme des lipides par CD36, nous avons choisi des types cellulaires jouant un rôle important dans l’homéostasie lipidique n’exprimant pas GHS-R1a, soient les adipocytes et les hépatocytes. L’ensemble de mes travaux démontre qu’en réponse à son interaction avec l’hexaréline, CD36 a le potentiel de réduire le contenu lipidique des adipocytes et des hépatocytes. Dans les cellules adipeuses, l'hexaréline augmente l’expression de plusieurs gènes impliqués dans la mobilisation et l’oxydation des acides gras, et induit également l’expression des marqueurs thermogéniques PGC-1α et UCP-1. De même, hexaréline augmente l’expression des gènes impliqués dans la biogenèse mitochondriale, un effet accompagné de changements morphologiques des mitochondries; des caractéristiques observées dans les types cellulaires ayant une grande capacité oxydative. Ces résultats démontrent que les adipocytes blancs traités avec hexaréline ont la capacité de se transformer en un phénotype similaire aux adipocytes bruns ayant l’habileté de brûler les acides gras plutôt que de les emmagasiner. Cet effet est également observé dans les tissus adipeux de souris et est dépendant de la présence de CD36. Dans les hépatocytes, nous avons démontré le potentiel de CD36 à moduler le métabolisme du cholestérol. En réponse au traitement des cellules avec hexaréline, une phosphorylation rapide de LKB1 et de l’AMPK est suivie d’une phosphorylation inhibitrice de l’HMG-CoA réductase (HMGR), l’enzyme clé dans la synthèse du cholestérol. De plus, la liaison d'hexaréline à CD36 provoque le recrutement d’insig-2 à HMGR, l’étape d’engagement dans sa dégradation. La dégradation de HMGR par hexaréline semble être dépendante de l’activité de PPARƔ et de l’AMPK. Dans le but d’élucider le mécanisme d’activation par hexaréline, nous avons démontré d’une part que sa liaison à CD36 provoque une déphosphorylation de Erk soulevant ainsi l’inhibition que celui-ci exerce sur PPARƔ et d’autre part, un recrutement de l’AMPK à PGC-1α expliquant ainsi une partie du mécanisme d’activation de PPARƔ par hexaréline. Les résultats générés dans cette thèse ont permis d’élucider de nouveaux mécanismes d’action de CD36 et d'approfondir nos connaissances de son influence dans la régulation du métabolisme des lipides. / Growth hormone releasing peptides (GHRPs) are small synthetic peptides aimed at stimulating GH release from the pituitary through their binding to ghrelin receptor known as growth hormone secretagogue receptor 1a (GHS-R1a). Using the GHRP, hexarelin to study tissue distribution of GHS-R1a and its GH-independent effect, it was observed that hexarelin was capable of binding to a second receptor identified as scavenger receptor CD36. While having multiple ligands, CD36 is mainly known for binding and internalizing oxLDL and long chain fatty acids. CD36 is thought to play a detrimental role in macrophage derived foam cell formation and development of atherosclerosis. Previously, we have shown that in macrophages, expressing both GHS-R1a and CD36, hexarelin promoted an activation of PPARƔ via GHS-R1a but also through its binding to CD36. This activation led to the induction of the LXRα-ABC transporters pathway and an increase in cholesterol efflux, reducing lipid-laden macrophage content. This positive effect on macrophages was reproduced in apolipoprotein E-null mice on a high fat diet treated with hexarelin. A significant reduction in the size of atherosclerotic lesions was observed while similar increases in the expression of PPARƔ, LXRα and ABC transporters occurred in isolated peritoneal macrophages. CD36 also plays a role in fatty acid uptake, and to further investigate the impact of the interaction of hexarelin with CD36, we aimed at evaluating the role of CD36 in regulating lipid metabolism in cells devoid of GHS-R1a such as adipocytes and hepatocytes. In the present thesis, we demonstrated through its interaction with hexarelin, the ability of CD36 to decrease intracellular lipid content in both adipocytes and hepatocytes. In adipocytes, hexarelin was able to increase the expression of several genes involved in fatty acid mobilization, fatty acid oxidation but also to induce the expression of the thermogenic markers, PGC-1α and UCP-1. In addition, hexarelin increased the expression of genes involved in mitochondrial biogenesis which was accompanied by mitochondrial morphological changes in agreement with what is usually seen in highly oxidative cells. In support of these findings, we also observed an increase in the activity of cytochrome c oxidase (a component of the respiratory chain) which could reflect an increase in oxidative phosphorylation. The results generated with cultured white adipocytes suggest the ability of hexarelin to promote changes toward a brown fat-like phenotype which also occurred in vivo and was dependent on the presence of CD36. In hepatocytes, CD36 was capable of regulating cholesterol metabolism by rapidly phosphorylating LKB1 and AMPK which subsequently resulted in the inactivating phosphorylation of HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. Hexarelin via CD36 also induced the recruitment of insig-2 to HMGR, the committed step in HMGR degradation while lifting the exerted inhibitory effect of Erk on nuclear receptor PPARƔ activity, and promoting the recruitment of AMPK to PPARƔ coactivator PGC-1α, suggesting an enhanced transcriptional potential of PPARƔ. The results generated during my graduate studies represent unique and novel mechanisms by which CD36 is capable of regulating lipid metabolism.

Les sécrétines de l’hormone de croissance (GHRPs) sont de petits peptides synthétiques capables de stimuler la sécrétion de l’hormone de croissance à partir de l’hypophyse via leur liaison au récepteur de la ghréline GHS-R1a. Le GHRP hexaréline a été utilisé afin d’étudier la distribution tissulaire de GHS-R1a et son effet GH-indépendant. Ainsi, par cette approche, il a été déterminé que l’hexaréline était capable de se lier à un deuxième récepteur identifié comme étant le récepteur scavenger CD36. Ce récepteur possède une multitude de ligands dont les particules oxLDL et les acides gras à longue chaîne. CD36 est généralement reconnu pour son rôle dans l’athérogénèse et sa contribution à la formation de cellules spumeuses suite à l’internalisation des oxLDL dans les macrophages/monocytes. Auparavant, nous avions démontré que le traitement des macrophages avec l’hexaréline menait à l’activation de PPARƔ via sa liaison à GHS-R1a, mais aussi à CD36. De plus, une cascade d’activation impliquant LXRα et les transporteurs ABC provoquait également une augmentation de l’efflux du cholestérol. Une stimulation de la voie du transport inverse du cholestérol vers les particules HDL entraînait donc une diminution de l’engorgement des macrophages de lipides et la formation de cellules spumeuses. Puisque CD36 est exprimé dans de multiples tissus et qu’il est également responsable du captage des acides gras à longue chaîne, nous avons voulu étudier l’impact de l’hexaréline uniquement à travers sa liaison à CD36. Dans le but d’approfondir nos connaissances sur la régulation du métabolisme des lipides par CD36, nous avons choisi des types cellulaires jouant un rôle important dans l’homéostasie lipidique n’exprimant pas GHS-R1a, soient les adipocytes et les hépatocytes. L’ensemble de mes travaux démontre qu’en réponse à son interaction avec l’hexaréline, CD36 a le potentiel de réduire le contenu lipidique des adipocytes et des hépatocytes. Dans les cellules adipeuses, l'hexaréline augmente l’expression de plusieurs gènes impliqués dans la mobilisation et l’oxydation des acides gras, et induit également l’expression des marqueurs thermogéniques PGC-1α et UCP-1. De même, hexaréline augmente l’expression des gènes impliqués dans la biogenèse mitochondriale, un effet accompagné de changements morphologiques des mitochondries; des caractéristiques observées dans les types cellulaires ayant une grande capacité oxydative. Ces résultats démontrent que les adipocytes blancs traités avec hexaréline ont la capacité de se transformer en un phénotype similaire aux adipocytes bruns ayant l’habileté de brûler les acides gras plutôt que de les emmagasiner. Cet effet est également observé dans les tissus adipeux de souris et est dépendant de la présence de CD36. Dans les hépatocytes, nous avons démontré le potentiel de CD36 à moduler le métabolisme du cholestérol. En réponse au traitement des cellules avec hexaréline, une phosphorylation rapide de LKB1 et de l’AMPK est suivie d’une phosphorylation inhibitrice de l’HMG-CoA réductase (HMGR), l’enzyme clé dans la synthèse du cholestérol. De plus, la liaison d'hexaréline à CD36 provoque le recrutement d’insig-2 à HMGR, l’étape d’engagement dans sa dégradation. La dégradation de HMGR par hexaréline semble être dépendante de l’activité de PPARƔ et de l’AMPK. Dans le but d’élucider le mécanisme d’activation par hexaréline, nous avons démontré d’une part que sa liaison à CD36 provoque une déphosphorylation de Erk soulevant ainsi l’inhibition que celui-ci exerce sur PPARƔ et d’autre part, un recrutement de l’AMPK à PGC-1α expliquant ainsi une partie du mécanisme d’activation de PPARƔ par hexaréline. Les résultats générés dans cette thèse ont permis d’élucider de nouveaux mécanismes d’action de CD36 et d'approfondir nos connaissances de son influence dans la régulation du métabolisme des lipides. / Growth hormone releasing peptides (GHRPs) are small synthetic peptides aimed at stimulating GH release from the pituitary through their binding to ghrelin receptor known as growth hormone secretagogue receptor 1a (GHS-R1a). Using the GHRP, hexarelin to study tissue distribution of GHS-R1a and its GH-independent effect, it was observed that hexarelin was capable of binding to a second receptor identified as scavenger receptor CD36. While having multiple ligands, CD36 is mainly known for binding and internalizing oxLDL and long chain fatty acids. CD36 is thought to play a detrimental role in macrophage derived foam cell formation and development of atherosclerosis. Previously, we have shown that in macrophages, expressing both GHS-R1a and CD36, hexarelin promoted an activation of PPARƔ via GHS-R1a but also through its binding to CD36. This activation led to the induction of the LXRα-ABC transporters pathway and an increase in cholesterol efflux, reducing lipid-laden macrophage content. This positive effect on macrophages was reproduced in apolipoprotein E-null mice on a high fat diet treated with hexarelin. A significant reduction in the size of atherosclerotic lesions was observed while similar increases in the expression of PPARƔ, LXRα and ABC transporters occurred in isolated peritoneal macrophages. CD36 also plays a role in fatty acid uptake, and to further investigate the impact of the interaction of hexarelin with CD36, we aimed at evaluating the role of CD36 in regulating lipid metabolism in cells devoid of GHS-R1a such as adipocytes and hepatocytes. In the present thesis, we demonstrated through its interaction with hexarelin, the ability of CD36 to decrease intracellular lipid content in both adipocytes and hepatocytes. In adipocytes, hexarelin was able to increase the expression of several genes involved in fatty acid mobilization, fatty acid oxidation but also to induce the expression of the thermogenic markers, PGC-1α and UCP-1. In addition, hexarelin increased the expression of genes involved in mitochondrial biogenesis which was accompanied by mitochondrial morphological changes in agreement with what is usually seen in highly oxidative cells. In support of these findings, we also observed an increase in the activity of cytochrome c oxidase (a component of the respiratory chain) which could reflect an increase in oxidative phosphorylation. The results generated with cultured white adipocytes suggest the ability of hexarelin to promote changes toward a brown fat-like phenotype which also occurred in vivo and was dependent on the presence of CD36. In hepatocytes, CD36 was capable of regulating cholesterol metabolism by rapidly phosphorylating LKB1 and AMPK which subsequently resulted in the inactivating phosphorylation of HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. Hexarelin via CD36 also induced the recruitment of insig-2 to HMGR, the committed step in HMGR degradation while lifting the exerted inhibitory effect of Erk on nuclear receptor PPARƔ activity, and promoting the recruitment of AMPK to PPARƔ coactivator PGC-1α, suggesting an enhanced transcriptional potential of PPARƔ. The results generated during my graduate studies represent unique and novel mechanisms by which CD36 is capable of regulating lipid metabolism.