Introdução: Ao ser usado o retalho de omento dissecado por videolaparoscopia no tratamento de deformidades da mama, foi constatado um significativo aumento do seu volume nos primeiros meses após a sua transposição, em todas as pacientes operadas, o que não é visto com essa magnitude em nenhum outro retalho adiposo. Métodos: Para se estudar o motivo desse aumento de volume, foram realizados estudos histológicos de amostras de omento coletadas no primeiro tempo cirúrgico, logo após sua transposição da cavidade abdominal para a região mamária e, no segundo tempo cirúrgico, durante a complementação de tratamento para a simetrização das mamas de oito pacientes. Foram documentadas as modificações nas medidas morfométricas dos adipócitos (perímetro, diâmetro e área), na densidade microvascular mediante o marcador endotelial CD31 e na expressão imunohistoquímica do fator de crescimento do endotélio vascular (VEGF). Resultados: O aumento do tamanho dos adipócitos e da densidade microvascular foi estatisticamente significativo (P≤0,012). O valor do VEGF foi menor na segunda amostra em relação com a primeira, redução esta que não atingiu significância considerável (P<0,093). Conclusão: Estes resultados sinalizam um aumento no volume celular que se mostrou consistente quando foram utilizados três diferentes processos de medida: perímetro, diâmetro e área dos adipócitos. Além disso, o aumento do número de vasos na segunda amostra sugere que tenha ocorrido neoangiogênese estimulada pelo aumento inicial dos valores do VEGF. Portanto o aumento do volume do retalho se deve a neovascularização e hipertrofia do adipócito. / Introduction: When laparoscopically harvested omentum flap was used to treat breast deformities, a significant volume increase, which had never been noticed in any other adipose flap, was observed in all the patients in the first months following its transposition. Methods: Histological studies of omentum samples were performed to study the reason for this increase. Samples were harvested at the first surgical time, right after the transposition of the omentum from the abdominal cavity to the breast region, and at the second surgical time, during treatment complementation for breast symmetrization of eight patients submitted to the transposition of the omentum flap. Modifications in the morphometric measurements of the adipocytes (perimeter, diameter, and area), in the microvascular density by the CD31 endothelial marker and in the imunohistochemic expression of VEGF were documented. Results: the increase in adipocyte size and microvascular density was statistically significant (P≤0.012). The value of VEGF was lower in the second sample, which was not significant (P<0.093). Conclusion: These results suggest an increase in cellular volume that was consistent when three different measurement procedures were used: adipocyte perimeter, diameter, and area. Moreover, the increase in the number of vessels in the second sample suggests neoangiogenesis stimulated by the initial increase in VEGF values obtained in the first sample. The flap increase was probably caused by adipocyte hypertrophy, resulting from the neoangiogenesis.

Une augmentation de l’hypertrophie et l'hyperplasie des adipocytes est au coeur du développement de l'obésité. Nous avons déjà constaté que 14-3-3zeta (14-3-3ζ), une protéine d’échafaudage moléculaire, a plusieurs rôles essentiels dans l'adipogenèse. Cependant, les contributions de 14-3-3ζ dans la fonction des adipocytes matures ne sont pas connues. Les cellules 3T3-L1 et souris dépourvues de 14-3-3ζ dans les adipocytes (adi14-3-3ζKO) ont été utilisés pour examiner le rôle de 14-3-3ζ dans la lipolyse. L’élimination de 14-3-3ζ dans les cellules 3T3-L1 par l’ARNi a réduit significativement la lipolyse stimulée par l'isoprotérénol (un agoniste bêta adrénergique), la forskoline (un activateur de l’adénylate cyclase) et le dibutyryl AMPc (dbcAMP). Les analyses par qPCR ont démontré des réductions significatives d’adipose triglyceride lipase (Atgl) et lipase hormonsensible (Hsl) au niveau transcriptionnel. De plus, une réduction au niveau des substrats de la PKA phosphorylés et totaux tels que HSL et CREB, a été détectée par Western Blot dans les 3T3-L1 appauvris en 14-3-3ζ. Ces résultats in vitro ont été récapitulés in vivo, car des diminutions des taux phosphorylés et totaux de HSL ont été observés dans le tissu adipeux gonadique des souris adi14-3-3ζKO. Les souris adi14-3-3ζKO et les explants gonadiques ont également montré une lipolyse affaiblie après des injections i.p de l’agoniste bêta 3-adrénergique CL-316,243 et un traitement de l’isoprotérénol respectivement. De manière intéressante, une diminution de l’expression de 14-3-3ζ dans les cellules 3T3-L1 et les souris adi14-3-3ζKO a mené à une diminution des caractéristiques des adipocytes matures telles que les niveaux d’ARNm de Pparg, Lpl et Fabp4, les niveaux de PPARγ, le contenu en triglycérides et l'incorporation de Oil Red-O. Collectivement, ces résultats démontrent que 14-3-3ζ joue un rôle essentiel en facilitant la lipolyse et en déterminant la maturité des adipocytes. / Altered hypertrophy and hyperplasia of adipocytes lie at the core of the development of obesity. We previously demonstrated that the molecular scaffold 14-3-3zeta (14-3-3ζ) had essential roles in adipogenesis. However, the contributions of 14-3-3ζ to mature adipocyte function are not known. 3T3-L1 cells and tamoxifen-inducible adipocyte-specific 14-3-3ζ knockout mice (adi14-3-3ζKO) models were used to examine the roles of 14-3-3ζ in lipolysis. siRNA-mediated knockdown of 14-3-3ζ impaired lipolysis in 3T3-L1 cells stimulated by the beta-adrenergic agonist isoproterenol (Iso), forskolin (an activator of adenylyl cyclase) and dibutyryl cAMP (dbcAMP). qPCR analyses revealed significant reductions in lipase transcript levels (Atgl and Hsl). Furthermore, reductions in the phosphorylated and total levels of PKA substrates such as HSL and CREB were detected in 14-3-3ζ-depleted 3T3-L1 lysates by immunoblotting. These findings were recapitulated in vivo, as reductions in phosphorylated and total HSL levels were detected in the gonadal adipose tissue of adi14-3-3ζKO mice. adi14-3-3ζKO mice and gonadal explants also displayed impaired lipolysis following i.p CL-316,243 (a beta-3 adrenergic agonist) injections and Iso treatment respectively. Interestingly, decreased 14-3-3ζ expression in 3T3-L1 cells and mice revealed reductions in characteristics of a mature adipocyte, such as Pparg, Lpl, and Fabp4 transcript levels, PPARγ levels, triglyceride content, and Oil Red O (ORO) incorporation. Collectively, these results demonstrate that 14-3-3ζ has essential roles in facilitating lipolysis and determining adipocyte maturity.

L'hormone angiotensine II (Ang II) possède deux principaux récepteurs, le récepteur AT1 (R-AT1) et le récepteur AT2 (R-AT2). Le R-AT1 est reconnu pour assurer la majorité des effets de l'Ang II, alors que les rôles et les mécanismes d'action du R-AT2 font l'objet de nombreux débats. Ces controverses peuvent être expliquées, d'une part par les nombreux modèles cellulaires ou animaux utilisés, de par son faible niveau d'expression et d'autre part par l'absence de ligand sélectif non-peptidique ciblant celui-ci. Dans le laboratoire, nous avons caractérisé un nouvel agoniste hautement sélectif pour le R-AT2 et non peptidique appelé le composé 21 (C21) ou M24. Grâce à ce composé, nous avons pu étudier une partie des rôles associés au R-AT2 dans la fonction adipocytaire. Tout d'abord, nous avons utilisé un modèle de cultures primaires de préadipocytes de rats pour étudier la différenciation des préadipocytes provenant de différents dépôts de tissus adipeux. Dans un deuxième temps, nous avons utilisé un modèle animal de résistance à l'insuline qui a été induit par une diète riche en gras et en fructose (HFHF). Les objectifs de mon projet étaient de déterminer 1) le rôle du R-AT2 sur l'adipogenèse et 2) l'effet de l'activation du R-AT2 dans un modèle de rats résistants à l'insuline. Nos résultats montrent que l'expression protéique du R-AT2 est plus importante en début de différenciation. Par ailleurs, l'activation du R-AT2, par le M24 (1 nM), augmente l'expression de PPAR[gamma]. De plus, l'activation du R-AT2 augmente l'accumulation de lipides dans les adipocytes sous-cutanés, mais pas dans les adipocytes rétropéritonéaux, tandis que le R-AT1 diminue l'accumulation de lipides dans les deux types cellulaires. Les résultats obtenus avec un shARN contre le R-AT2, dans ces mêmes cultures, indiquent que l'absence du R-AT2 empêche la différenciation des préadipocytes en adipocytes matures. D'autre part, chez les rats soumis à la diète HFHF, nos résultats montrent que le M24, ainsi que le losartan, préviennent la résistance à l'insuline. Par ailleurs, nous avons observé d'importants changements morphologiques dans les tissus adipeux après traitements au M24. En effet, le M24 diminue le nombre de gros adipocytes dans le tissu adipeux sous-cutané. Sachant que les nouveaux petits adipocytes sous-cutanés corrèlent avec une meilleure sensibilité à l'insuline, nos études suggèrent que le M24 améliore la sensibilité à l'insuline de nos animaux, principalement, en régulant la physiologie du tissu adipeux. Ensemble, mes résultats montrent que le R-AT2 joue un rôle important dans la physiologie des adipocytes et par conséquent du tissu adipeux. De plus, l'agoniste M24 ouvre la porte à de nouvelles études sur le R-AT2 pour étudier son potentiel thérapeutique dans les pathologies métaboliques telles que la résistance à l'insuline, le diabète de type 2 et l'obésité.

Humans are continuously exposed to mixtures of environmental pollutants. Polycyclic aromatic hydrocarbons (PAHs), such as 2-naphthol, and heavy metals, such as lead, are some of these pollutants. Results from epidemiological studies show associations between exposure to 2-naphthol, exposure to lead, and obesity. However, the individual and combined effects of 2-naphthol and lead on fat cell development (adipogenesis) have not been directly characterized in a biological system. In this study, we evaluated the effects of 2-naphthol and/or lead on adipogenesis using mouse 3T3-L1 cells. Cells were exposed to different doses of 2-naphthol and/or lead. Induced terminal differentiation was evaluated by cell morphology, lipid production, and mRNA expression of marker genes characteristic of either early adipocyte differentiation: CCAAT-enhancer-binding protein β (C/EBPβ), insulin receptor substrate 2 (IRS2), and sterol responsive element binding protein 1 c (SREBP1c); or terminal differentiation: C/EBPα, peroxisome proliferator-activated receptor-γ (PPARγ), and fatty acid binding protein 4 (aP2). Production of antimicrobial peptide cathelicidin (Camp), which is produced by differentiating adipocytes and modulates inflammation and immunity, was also evaluated. Cell morphology changes and increased lipid accumulation indicated that, individually, 2-naphthol and lead induced 3T3-L1 differentiation; however, the highest dose of lead (10 μM) showed the lowest induction level. During terminal differentiation, 2-naphthol and low doses of lead increased C/EBPα, PPARγ, and aP2 expression, whereas 10 μM lead suppressed PPARγ and aP2. During early differentiation, 2-naphthol stimulated C/EBPβ, IRS2, and SREBP1c expression, while lead upregulated C/EBPα and aP2. The 2-naphthol/10 μM lead mixture induced a counterbalancing effect on 3T3-L1 adipogenesis, where 10 μM lead suppressed 2-naphthol-induced adipogenesis. Moreover, 2-naphthol elevated Camp expression in a dose-dependent manner, whereas lead slightly increased Camp at lower doses but suppressed it at 10 μM. The 2-naphthol/10 μM lead mixture showed no effect on Camp expression. In conclusion, 2-naphthol and low lead doses accelerate adipocyte differentiation and Camp production in 3T3-L1 cells; however, high doses of lead attenuate the induction. This effect of lead at high dose counterbalances the upregulation of adipocyte differentiation and Camp production by 2-naphthol. Together, these findings indicate that 2-naphthol and lead play potential roles in the development of inflammation and obesity.

The increasing prevalence of obesity and related health complications, such as type 2 diabetes, cardiovascular disease and cancer, demands thorough investigation of the underlying processes. One of the key tissues investigated in this context is adipose tissue. It is becoming increasingly clear that adipose tissue is a very dynamic and heterogenic organ. This thesis provides an overview of various aspects of adipose biology that illustrate its heterogenic nature and describes my own scientific contributions to this field. We typically distinguish between thermogenic, energy-expending brown adipocytes and energy-storing white adipocytes that are located in anatomically distinct adipose depots. In addition, brite (or beige) adipocytes are functionally thermogenic, but are located among white adipocytes. Related to functional variation, adipocytes and adipose tissues display a wide range of morphological appearances. An additional property that illustrates the heterogeneity among adipose cells and depots is the variation of cellular responses to physiological cues, such as changes in diet or environmental temperature. Furthermore, the developmental origins of various adipose types display great heterogeneity, which may explain some of the functional and dynamic differences that are observed. In line with the complexity of developmental origins, molecular markers that were initially proposed to distinguish between brown, brite/beige and white adipose subtypes have added to the notion that the composition of the adipose organ is much more complex than has long been appreciated. My own work has contributed to the enhancement of our understanding of the heterogeneity of adipose subtypes. In particular, my findings related to marker gene expression patterns have led to increased appreciation of the complex nature of adipose gene expression patterns and the complications of translating results obtained in mice to humans. Some of my other contributions have increased the understanding of the differences and similarities in thermogenic adipose tissue functionality and dynamics. With cell culture studies, I have revealed new characteristics of pre-adipose cells from various depots that further add to the appreciation of the adipose heterogeneity. Overall, this thesis provides an overview of important characteristics of the adipose organ, illustrating its heterogenic nature. Realization of this heterogeneity is of importance in order to properly study the adipose organ to ultimately understand how the adipose organ can be therapeutically targeted to effectively treat adipose-related diseases. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 7: Manuscript. Paper 8: Manuscript.</p>

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.

Brown and beige fat are specialized adipose tissues found in almost all mammals that can increase energy expenditure and produce heat. Cold exposure and b3-adrenergic stimulation has been extensively shown to activate brown adipose tissue (BAT) in rodents, which promotes uncoupled respiration of glucose and lipid substrates via uncoupling protein 1 (UCP1). Prolonged stimulation can induce white adipose browning, which leads to the emergence of thermogenic cells within white fat depots, called beige adipocytes. The beige adipocyte possesses a unique molecular signature, yet shares several characteristics of brown adipocytes, including high mitochondrial content. When activated, beige fat can be induced to initiate a thermogenic transcriptional program similar to that of BAT. Recent human studies have identified brown and/or beige fat in the supraclavicular region using various radiation imaging modalities. This remarkable discovery has reinvigorated scientific interest in adipose browning and brown/beige fat activation as possible therapeutic targets for obesity. Like in rodents, several groups have previously tested the potential impact of cold exposure and b3-adrenergic agonism on BAT-mediated thermogenesis in humans. However, even though these approaches were shown to significantly increase energy expenditure and promote weight loss in obese individuals, they are not ideal clinical interventions. Cold exposure is uncomfortable and requires prolonged treatment, while b3-adrenergic agonists may lead to many adverse effects like cardiovascular problems. This thesis will evaluate the therapeutic potential and clinical relevance of alternative anti-obesity approaches that target adipose browning and beige adipose activation.

Les apolipoprotéines L (APOL) forment une famille de protéines conservées chez les mammifères. L’APOL6 murine est principalement exprimée par les adipocytes présents dans le tissu adipeux. Dans un modèle de culture d’adipocytes, l’adipogénèse a causé l’induction de l’expression de l’APOL6. Celle-ci a pu encore être modulée à la hausse par de l’IFNγ, et à la baisse par du TGFβ. Des facteurs élevant la concentration en AMP cyclique ont aussi permis de diminuer l’expression d’APOL6. In vivo, lorsque des souris APOL6 KO ont été nourries par un régime riche en graisses, elles ont pris moins de poids que les souris WT correspondantes. De plus, les adipocytes des souris APOL6 KO obèses étaient plus petits que ceux des contrôles WT. Finalement, la recherche de protéines interagissant avec l’APOL6 par immunoprécipitation a permis de mettre en évidence une majorité de protéines associées au cytosquelette d’actine. En conclusion, l’APOL6 semble être associée au cytosquelette d’actine des adipocytes et permettrait la régulation de la taille de leurs gouttelettes lipidiques. / Apolipoproteins L (APOL) are a family of conserved proteins among mammals. Murine APOL6 is mainly expressed by adipocytes in the adipose tissue. In a model of in vitro adipocyte cell culture, adipogenesis induced the expression of APOL6. This expression increased with IFNγ and decreased with TGFβ. Cyclic-AMP elevating agents also decreased the expression of APOL6. In vivo, APOL6 KO mice that were fed with a high fat diet gained less weight than their wild type (WT) counterparts. Furthermore, adipocytes from obese APOL6 KO mice were smaller than those from WT controls. Finally, immunoprecipitation experiments showed that APOL6 probably interacted with actin cytoskeleton proteins within adipocytes. In conclusion, APOL6 is likely associated with the actin cytoskeleton in adipocytes and could be involved in the regulation of the size of lipid droplets. / Option Biologie moléculaire du Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Adipose tissues have long been considered to derive from a common origin. Even the functionally different brown and white adipose tissues were generalized to share a common origin. Brown adipose tissue is a highly innervated and vascularised tissue containing multilocular and multimitochondrial brown adipocytes. Brown adipose tissue expends energy through sympathetic nervous system-mediated non-shivering thermogenesis, where uncoupling protein 1 (UCP1) is the key player. In contrast, white adipose tissue consists of unilocular white adipocytes with a main role to store energy in the form of the lipid droplet. We know today that this generalisation is exaggerated since adipocytes can derive from more than one origin and not only be brown or white. We and others have demonstrated that the brown adipocyte has a dermomyotomal origin and derives from the adipomyocyte, the precursor cell that can also become a myocyte, whereas white adipocytes are suggested to derive from pericytes, cells that are embedded within the vascular vessel walls. For a long time there has been evidence that energy-expending adipocytes reside within certain white adipose tissues, based on the fact that cold exposure, by switching on the sympathetic nervous system, leads to levels of UCP1 that are not detectable in mice housed at thermoneutrality. We demonstrated that these cells have a molecular signature that is distinct from brown and white adipocytes. Since these energy-expending cells reside within certain white adipose tissues, we chose to name them brite (brown in white) adipocytes. Moreover, we also identified regulatory factors that were specifically expressed in each adipocyte type, thus, facilitating the possibility to identify the three adipocytes: the brown, the white and the brite. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.

Obesity is associated with a state of chronic inflammation, which is believed to contribute to insulin resistance. We previously identified interferon regulatory factor 3 (IRF3) as an anti-adipogenic transcription factor with high expression in adipocytes. Because IRF3 is known to drive expression of pro-inflammatory genes in immune cells, we hypothesized that it may also promote inflammation and insulin resistance in adipocytes. Consistent with our expectations, we found that the expression of inflammatory genes in adipocytes was induced by IRF3 overexpression, while knockdown of IRF3 had the opposite effect. Despite this effect on local adipocyte gene expression, we found that \(Irf3^{-/-}\) mice did not show evidence of altered systemic inflammation. Nonetheless, \(Irf3^{-/-}\) mice did display altered metabolism relative to their wild type (WT) littermates. For example, high fat diet (HFD) fed \(Irf3^{-/-}\) mice exhibited increased lean mass and decreased fat mass compared to WT, accompanied by increased food intake and energy expenditure. Further investigation showed that the white adipose tissue (WAT) of \(Irf3^{-/-}\) mice had increased expression of brown adipocyte selective genes compared to WT, and the inguinal WAT of the \(Irf3^{-/-}\) mouse contain multilocular adipocytes that resemble brown adipocytes. These data suggest that IRF3 affects energy homeostasis by regulating the development of brown adipocyte-like cells in WAT. Additionally, \(Irf3^{-/-}\) mice are significantly more insulin sensitive and glucose tolerant compared to WT when kept on HFD. Consistent with in vivo observations, IRF3 knockdown in 3T3-L1 adipocytes resulted in enhanced insulin-stimulated glucose uptake and lipogenesis, while overexpression of constitutively active IRF3 had the opposite effect. Several IRF3 target genes in adipocytes were identified using transcriptional profiling. Interestingly, the expression level of Slc2a4 (encoding the Glut4 protein) was inversely correlated with that of IRF3 in both WAT and cultured adipocytes. Analysis of the Slc2a4 proximal promoter identified a putative IRF3 binding site upstream of the transcription start site, and luciferase assay in 3T3-L1 adipocytes showed that IRF3 negatively regulates Slc2a4 expression via this site. Taken together, these data indicate that IRF3 plays a role in whole body glucose homeostasis by repressing thermogenic gene expression as well as the expression of adipose Glut4.