Macrophage-conditioned medium (MacCM) inhibits the differentiation of rodent and human preadipocytes. Previous studies report that murine J774A.1-MacCM inhibits clonal expansion (early required phase of adipogenesis), including Rb phosphorylation. I hypothesized that MacCM induced alterations in cyclins and/or cyclin-dependent kinases (CDKs) were responsible for impairing Rb phosphorylation. My first objective was to assess the effect of J774A.1-MacCM on CDK4, CDK2, and their regulatory cyclins. Murine 3T3-L1 preadipocytes were differentiated with control medium or J774A.1-MacCM. Expression of cyclin D and A was inhibited by J774A.1-MacCM. Inhibition of cyclin A expression was associated with reduced differentiation-induced CDK2 activity. My second objective was to assess the expression patterns of cell cycle proteins in differentiating human abdominal subcutaneous preadipocytes, which do not undergo clonal expansion in culture. Cyclin E expression increased with differentiation. THP-1-MacCM (a human macrophage cell line) further enhanced this increase. My studies suggest MacCM leads to alterations in cyclin/CDK regulation during adipogenesis in murine and human preadipocyte models.

Elevated Thyroid-Stimulating Hormone (TSH) is associated with an increased risk of cardiovascular disease (CVD). We hypothesized that TSH-stimulated FA release from adipocytes contributes to macrophage inflammation. 3T3-L1 and human subcutaneous differentiated adipocytes were treated with TSH for 4 hours under various conditions and lipolysis assessed via glycerol secretion. Optimal conditions were determined and protein expression of ATGL, HSL and perilipin remained stable. TSH-stimulated 3T3-L1 or human adipocyte-conditioned medium (T-ACM) was placed on murine J774 or human THP-1 macrophages, respectively, and macrophage cytokine mRNA levels (IL-1β, IL-6, MCP-1, and TNFα) were measured by real-time RT-PCR. T-ACM did not change cytokine mRNA expression in J774 macrophages or THP-1 macrophages when compared to ACM. Absence of BSA in the medium may have hindered release of FA from differentiated adipocytes into the medium, BSA may be required to permit adequate FA accumulation in the medium to then evaluate the effect of T-ACM on macrophages. Further investigation is required to determine the effect of FA on J774 and THP-1 inflammatory response.

Le cil primaire (CP) est une organelle présente chez l’Homme dans la grande majorité des cellules. Lors du développement le CP est d’une importance capitale, puisqu’il contrôle les voies de signalisation comme Hedgehog ou Wnt. Certaines pathologies génétiques affectant spécifiquement le CP, engendrent une obésité. Au cours de ma thèse je me suis intéressé à l’évolution du CP au cours de l’adipogenèse des cellules souches mésenchymateuses humaines. Les résultats que nous avons obtenus indiquent que le cil est présent dans les cellules indifférenciées, qu’il subit une élongation importante suite à l’induction de la différenciation, suivi d’une diminution de sa taille et fini par disparaitre dans les adipocytes. L’élongation de la taille du cil ne semble pas affecter la localisation des protéines qui lui sont associées comme Kif3-A ou Smoothened, une protéine importante de la voie Hedgehog. Néanmoins, il apparait que la voie de signalisation Hedgehog est inhibée après trois jours de différenciation et que les cellules ont développé une résistance à Sonic Hedgehog. La déacétylase de la tubuline acétylée HDAC6 est apparue comme étant une bonne cible puisque son expression augmente au cours de la différenciation et qu’elle est décrite pour être responsable de la perte du cil pendant la mitose. Les données que nous avons obtenues ont permis de montrer que l’inhibition, ou la surexpression d’HDAC6 au cours de l’adipogenèse engendrent une inhibition de l’élongation du cil associée à une forte inhibition de la différenciation adipocytaire. Ces résultats permettront, à terme de mieux comprendre les liens entre le cil primaire et la différenciation adipocytaire. / The primary cilium (PC) is an organelle present in almost all cell types of the organism. During development, the PC plays an important function by driving signaling pathways such as Hedgehog or Wnt. Some genetic syndromes affecting specifically the PC are associated with obesity. My project has consisted to analyze the evolution of the PC during adipocyte differentiation of human mesenchymal stems cells. Our results indicate that the PC is present in undifferentiated cells, then it undergoes a strong elongation at the beginning of the differentiation followed by a decreased of its size, and disappears in differentiated cells. This increase in the cilium size does not affect the localization of its associated proteins such as KIF3-A and Smoothened an important protein of the Hedgehog signaling pathway. However, this pathway is inhibited after three days of differentiation and cells have developed a Sonic Hedgehog resistance. The tubulin deacetylase HDAC6 appeared as a good target because its expression increases during differentiation and it is known to be responsible for the loss of the cilium during mitosis. Our data show that an inhibition or an overexpression of HDAC6 lead to a decrease in the cilium elongation associated with an inhibition of adipocyte differentiation. These results will ultimately lead to a better understanding of the connections between the PC and adipocyte differentiation.

Diploma thesis deals with developing and testing of revitalization program which has positive effect on Cellulite-dermopanniculosis deformans. The research presents and evaluates results of revitalization program which took 6 months. Analysis of technical terms, that are closely related to cellulite, is presented in the theoretical part. There are presented views of Czech and foreign experts who deal with issues of cellulit. At the end of theoretical part we focused on available revitalizing methods that are aiming to elliminate cellulit. Experimental investigation was carried out on 60 women who were divided into experimental and control groups. Results are statisticaly processed in the form of graphs and tables and acompanied with discussion.

Chronic low-grade endotoxemia is an important player in obesity and insulin resistance associated to a high-fat diet (HFD). On the other hand, although it is known that intense endotoxemia and infection reduce appetite and induce intense catabolism, leading to weight loss during the acute inflammatory phase, the late effects of an intense endotoxemia were previously unexplored. Here we report that, besides the concurrent effects, multiple and intense endotoxemia causes long lasting biochemical alterations in the adipose tissue that intensify the harmful effects of a HFD. Mice submitted to multiple and severe endotoxemia had increased the adipose tissue expression of TLR-4, CD14 and SAA3, remaining altered after one week in recovery. When associated to a HFD, mice previously submitted to acute endotoxemia showed a more severe weight gain and impaired insulin sensitivity. Adopting the HFD as an obesogenic stimulus, we evaluated the participation of the protein serum amyloid A (SAA) in obesity development. Using a SAA-targeted antisense oligonucleotide, we observed that the depletion of SAA prevented metabolic alterations, endotoxin elevation, weight gain and insulin resistance in a diet-induced obesity protocol. Inadequate sleep is another important factor to be considered in the obesity epidemic. We found that sleep restriction (SR) causes biochemical and morphological alterations in mice adipose tissue. The levels of serum resistin and the adipose tissue mRNA expression of resistin, TNF-α and IL-6 were increased after SR. When associated to a HFD, mice previously submitted to SR gained more weight with increased macrophage infiltration in the epididymal adipose tissue, and insulin resistance. SAA is also part of the initial biochemical alterations caused by SR. It was observed that the expression of SAA in liver and adipose tissue is upregulated, with return to baseline when sleep is restored. Furthermore, 48 hours of total sleep restriction in healthy human volunteers also caused a serum elevation in SAA concentrations. Considering that SAA induces cell proliferation, we suggest that situations with an increase in SAA production and the consecutive preadipocyte proliferation would prime the adipose tissue to further adipocyte differentiation and hypertrophy. Furthermore, we suggest that SAA alter LPS signaling, possibly inhibiting its clearance. The mechanism associating inflammation and obesity is complex and encompass a diversity of factors; the inflammatory protein SAA may be one of them. In conclusion, our data describes the relationship between SAA, acute inflammation, sleep restriction and obesity. / Endotoxemia crônica de baixo grau tem um importante papel na obesidade e resistência à insulina associada a uma ração hiperlipídica. Por outro lado, embora se saiba que a endotoxemia intensa e infecção reduzam o apetite e induzam a um intenso catabolismo, conduzindo a perda de peso durante a fase aguda da inflamação, os efeitos tardios da endotoxemia intensa nunca foram explorados. Aqui mostramos que, além dos efeitos correntes, a endotoxemia aguda provoca alterações bioquímicas prolongadas no tecido adiposo que intensificam os efeitos deletérios de uma ração hiperlipídica. Camundongos submetidos à endotoxemia aguda apresentaram aumento na expressão de TLR-4, CD14 e SAA3 no tecido adiposo, permanecendo alteradas após uma semana em recuperação. Quando associado a uma ração hiperlipídica, os camundongos previamente submetidos à endotoxemia aguda mostraram um ganho de peso mais pronunciado e uma maior resistência à insulina. Adotando a ração hiperlipídica como um estímulo obesogênico, foi avaliada a participação da proteína amilóide sérica A (SAA) no desenvolvimento da obesidade. Usando um oligonucleotídeo antisense anti-SAA, observamos que a depleção da SAA previne as alterações metabólicas, elevação de endotoxina, ganho de peso e resistência à insulina associadas a ração rica em gordura. O sono inadequado é outro fator importante a ser considerado na epidemia de obesidade. Descobrimos que a restrição do sono (SR) provoca alterações bioquímicas e morfológicas no tecido adiposo de camundongos. A concentração de resistina no soro e a expressão de mRNA no tecido adiposo de resistina, TNF-α e IL- 6 foram aumentadas após SR. Quando associado a uma ração hiperlipídica, os camundongos submetidos previamente à SR ganharam mais massa com aumento da infiltração de macrófagos no tecido adiposo epididimal, e resistência à insulina. SAA também faz parte das alterações bioquímicas iniciais provocadas pelo SR. Observou-se que a expressão de SAA no fígado e tecido adiposo é regulada positivamente, com retorno ao basal quando o sono é restaurado. Além disso, 48 horas de restrição de sono total em voluntários humanos saudáveis também causou uma elevação nas concentrações séricas de SAA. Considerando que SAA induz proliferação, sugerimos que situações onde ocorra aumento na produção de SAA e a consecutiva proliferação celular, o tecido adiposo se tornaria predisposto a futura diferenciação e hipertrofia. Além disso, sugerimos que SAA altera a sinalização de LPS, possivelmente inibindo sua depuração. O mecanismo de associação entre a inflamação e a obesidade é complexo e inclui uma diversidade de fatores; a proteína inflamatória SAA pode ser um deles. Em conclusão, nossos dados descrevem a relação entre SAA, inflamação aguda, restrição do sono e obesidade.

A ausência de nutrientes durante o jejum leva a intensa mobilização de ácidos graxos (AG) do adipócito. A intensidade deste fenômeno deve ser controlada, pois o excesso de AG está associado a condições patológicas. Nestas condições, a lipogênese torna-se útil e a Gliceroneogênese indispensável. Nesta via, o lactato seria um substrato fisiológico e provável. Metodologia. Ratos machos Wistar foram divididos em grupos, Alimentado (Al) e Jejum (J) e os coxins subcutâneo (SC) e visceral retroperitoneal (RP) submetidos aos testes biológicos e moleculares. No Teste de Incorporação de [14C]-Acido Lático em Glicerol e no teste de captação de [14C]-Acido Lático o grupo Al mostrou maior capacidade (Al > J; *p<0.05; [N=8]). Nestes testes, a glicose (1 ou 4 mM) foi fundamental e a presença de insulina (10 nM) ampliou estes resultados em ambos os tecidos. Na expressão do transportador de monocarboxilatos 1 (MCT1) e da enzima fosfoenol piruvato carboxiquinase (PEPCK), não houve diferenças entre Al e J. Concluímos que a alimentação promove aumento da Glicroneogênese a partir do ácido lático e a expressão da PEPCK não exerceu influencia neste processo. No entanto, a glicose e a insulina, mostraram-se como potencializadores da Gliceroneogênese. / The absence of nutrients during fasting leads to intensive mobilization of adipocyte fatty acids (FA). The intensity of this phenomenon should be controlled because excess of the AG is associated with pathological conditions. Under these conditions, the lipogenesis and gliceroneogenesis are useful and indispensable. In this pathway, the lactate and likely would be a physiological substrate. Methodology. Male Wistar rats were divided into groups: (Al) and Fasting (J) and fat pad subcutaneous (SC) and visceral retroperitoneal (RP) subject to biological and molecular assays. The Test of Incorporation of [14C]-Latic Acid into Glycerol and test of uptake lactic-acid, Al group showed greater capacity (Al> J; * p <0.05; [N = 8]). In these tests, glucose (1 or 4 mM) was fundamental and the presence of insulin (10 nM) extended these findings in both tissues. In monocarboxylate transporter 1 expression (MCT1) and the enzyme phosphoenol pyruvate carboxykinase (PEPCK), there was no difference between Al and J. We concluded that feeding promotes increased Glicroneogênese from lactic acid and expression of PEPCK did not exercised influence in this process. However, glucose and insulin appeared as potentiators of Gliceroneogênese.

The small GTP-binding protein Rad (RRAD, Ras associated with diabetes) is the founding member of the RGK (Rad, Rem, Rem2, and Gem/Kir) family that regulates voltage-dependent calcium channel function. Given its expression in both excitable and non-excitable cell types, the control mechanisms for Rad regulation and the potential for novel functions for Rad beyond calcium channel modulation are open questions. Here we report a novel interaction between Rad and Enigma, a scaffolding protein that also binds to the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1). Overexpression of Smurf1, but not of a catalytically inactive mutant enzyme, results in ubiquitination of Rad and down regulation of Rad protein levels. The Smurf1-mediated decrease in Rad levels is sensitive to proteasome inhibition and requires the ubiquitination site Lys204, suggesting that Smurf1 targets Rad for degradation. Rad protein levels, but notably not mRNA levels, are increased in the hearts of Enigma-/- mice, leading to the hypothesis that Enigma may function as a scaffold to enhance Smurf1 regulation of Rad. In addition to ubiquitination, phosphorylation of RGK proteins represents another potential means of regulation. Indeed, Rem phosphorylation has been shown to abolish calcium channel inhibition. We demonstrate that b-adrenergic signaling promotes Rad phosphorylation at Ser39. Rad Ser39 phosphorylation is correlated with a decrease in the interaction between Rad and the CaVb subunit of the calcium channel and an increase in Rad binding to 14-3-3. Interestingly, Enigma overexpression promotes an increase in Rad Ser39 phosphorylation as well. Despite an interaction between Enigma and the CaV1.2 calcium channel subunit, overexpression of Enigma had no effect on Rad-mediated channel inhibition. Thus, Rad Ser39 phosphorylation alters its association with the calcium channel, but its impact on calcium channel regulation has yet to be determined. Finally, we report a novel function for Rad in the regulation of bone homeostasis. Rad deletion in mice results in a significant decrease in bone mass. Dynamic histomorphometry in vivo and primary calvarial osteoblast assays in vitro demonstrate that bone formation and osteoblast mineralization rates are depressed in the absence of Rad. Microarray analysis revealed that canonical osteogenic gene expression is not altered in Rad-/- osteoblasts; instead robust up-regulation of matrix Gla protein (MGP, +11-fold), an inhibitor of mineralization and a protein secreted during adipocyte differentiation, was observed. Strikingly, Rad deficiency also resulted in significantly higher bone marrow adipose tissue (BMAT) levels in vivo and promoted spontaneous in vitro adipogenesis of primary calvarial osteoblasts. Adipogenic differentiation of WT osteoblasts resulted in the loss of endogenous Rad protein, further supporting a role for Rad in the control of BMAT levels. These findings reveal a novel in vivo function for Rad signaling in the complex physiological control of skeletal homeostasis and bone marrow adiposity. In summary, this dissertation expands our understanding of Rad regulation through identification of a novel binding partner and characterization of post-translational regulatory mechanisms for Rad function. This work also defines a new role for Rad that may not depend upon its calcium channel regulatory properties: regulation of the bone-fat balance. These findings suggest that the regulation of Rad GTPase is likely more complex than guanine nucleotide cycling and that functions of Rad in non-excitable tissues warrant further study.

Le tissu adipeux est un organe complexe qui centralise de nombreuses fonctions métaboliques. La principale est la régulation de la balance énergétique. Depuis quelques années, il est reconnu pour être le siège d’une activité sécrétoire importante dont les produits sont les adipokines. Au cours du développement de l’obésité, il concentre de nombreux dysfonctionnements cellulaires qui vont avoir des répercutions fonctionnelles importantes, sur lui-même, mais également sur d’autres tissus. Ces dysfonctionnements vont êtres à l’origine de nombreuses complications liées à l’obésité dont l’insulino-résistance, le diabète de type II ou les maladies cardiovasculaires. Dans ce cadre, des études épidémiologiques ont montré que la consommation de fruits et légumes avait un impact bénéfique, attribué en partie à certains micronutriments dont les caroténoïdes, sur ces pathologies et sur certains types de cancer. Parmi ces caroténoïdes, le lycopène et le ß-carotène occupent une place importante. Ils représentent les deux principaux caroténoïdes de notre alimentation et sont stockés physiologiquement au niveau du tissu adipeux. Des études ont suggéré qu’ils étaient spécifiquement et individuellement liés à une diminution des pathologies cardiovasculaires et de ses complications. De plus, compte tenu du lien très fort existant entre tissu adipeux, obésité et les pathologies associées, des études ont suggéré qu’il existait une relation entre ces deux caroténoïdes, le tissu adipeux et ces pathologies. Cependant, à ce jour, il n’existe quasiment pas d’explications sur les liens entre ces trois facteurs. Le but de cette thèse est d’apporter les premières pistes explicatives. Nous avons ainsi évalué les effets du lycopène et du ß-carotène sur certains aspects de la biologie du tissu adipeux et des fonctions adipocytaires. Nous montrons que les deux isomères principaux du lycopène (all-trans et 5-cis), son métabolite et le ß-carotène influencent fortement le transcriptome et le microtranscriptome de l’adipocyte. De plus, le lycopène diminue la réponse inflammatoire en réponse à un régime riche en gras via une diminution de l’activité NF-?B, et que un de ses métabolites, l’acide apo-10’-lycopénoïque transactive RAR in vivo dans le tissu adipeux. Il possède également la même capacité antiinflammatoire. Le ß-carotène quant à lui diminue l’adiposité chez des souris. Ce mécanisme est BCMO1 et PPAR? dépendant. Enfin, L’ensemble de nos résultats apportent de nouvelles perspectives et élargissent les connaissances sur ces deux caroténoïdes. Ils sont capables d’influencer de façon importante de nombreuses fonctions adipocytaires en lien avec l’obésité et ses complications dont les maladies cardiovasculaires selon différents mécanismes. / Adipose tissue is a complex organ who exerts several metabolic functions. It is involved in the regulation of the energy balance and since a decade is well known as an exocrine organ who secrets several proteins collectively called adipokines. It is one of the main organs involved in obesity where it concentrates several cellular dysfunctions which will have important physiological consequences such as development of insulin resistance, type II diabetes and cardiovascular diseases. Epidemiological studies have reported that high consumption of fruits and vegetables is link to a decrease of pathologies such as cardiovascular diseases, type II diabetes and cancer; due to the presence of micronutrients notably carotenoids. Among these carotenoids, lycopene and ß-carotene play an important role. They are the main carotenoids in our diet, and are physiologically store in adipose tissue. Furthermore, others studies point that the high consumption or concentration of these compounds in adipose tissue is associated to a decrease of the risk to develop cardiovascular diseases. However, the link between carotenoids, adipose tissue, obesity-associated pathologies is unclear. The aim of this thesis was to bring some explanatory way to this association. An analyze of the transcriptome and microtranscriptome of adipocytes in response to lycopene (all-trans and 5-cis, the two main isomers and the metabolite) and ß-carotene also reveal that these compounds influence a large amount of genes and miRNAs. These effects can also explain a part of the positives effects attributed to these carotenoids. We found that lycopene decrease the inflammatory state of adipose tissue in response to a high fat diet. This phenomenon is explained by a decrease of the NF-?B activity. We also found that a metabolite of lycopene, the apo-10’-lycopenoic acid is able to transactivate RAR in vivo in different tissues included adipose tissue and possess the same anti inflammatory property than all-trans lycopene. In another article, we show that, a diet rich in ß-carotene in mouse lead to a decrease of adiposity. This effect is BCMO1 and PPAR? dependant. This enzyme, BCMO1, seems to play a key role in adipose tissue. Taking all these results together, we open new ways related to the effect of lycopene and ß- carotene on adipose tissue. These results can explain at least in part the benefic effects observed in several studies.

Macrophage-conditioned medium (MacCM) inhibits the differentiation of rodent and human preadipocytes. Previous studies report that murine J774A.1-MacCM inhibits clonal expansion (early required phase of adipogenesis), including Rb phosphorylation. I hypothesized that MacCM induced alterations in cyclins and/or cyclin-dependent kinases (CDKs) were responsible for impairing Rb phosphorylation. My first objective was to assess the effect of J774A.1-MacCM on CDK4, CDK2, and their regulatory cyclins. Murine 3T3-L1 preadipocytes were differentiated with control medium or J774A.1-MacCM. Expression of cyclin D and A was inhibited by J774A.1-MacCM. Inhibition of cyclin A expression was associated with reduced differentiation-induced CDK2 activity. My second objective was to assess the expression patterns of cell cycle proteins in differentiating human abdominal subcutaneous preadipocytes, which do not undergo clonal expansion in culture. Cyclin E expression increased with differentiation. THP-1-MacCM (a human macrophage cell line) further enhanced this increase. My studies suggest MacCM leads to alterations in cyclin/CDK regulation during adipogenesis in murine and human preadipocyte models.

Elevated Thyroid-Stimulating Hormone (TSH) is associated with an increased risk of cardiovascular disease (CVD). We hypothesized that TSH-stimulated FA release from adipocytes contributes to macrophage inflammation. 3T3-L1 and human subcutaneous differentiated adipocytes were treated with TSH for 4 hours under various conditions and lipolysis assessed via glycerol secretion. Optimal conditions were determined and protein expression of ATGL, HSL and perilipin remained stable. TSH-stimulated 3T3-L1 or human adipocyte-conditioned medium (T-ACM) was placed on murine J774 or human THP-1 macrophages, respectively, and macrophage cytokine mRNA levels (IL-1β, IL-6, MCP-1, and TNFα) were measured by real-time RT-PCR. T-ACM did not change cytokine mRNA expression in J774 macrophages or THP-1 macrophages when compared to ACM. Absence of BSA in the medium may have hindered release of FA from differentiated adipocytes into the medium, BSA may be required to permit adequate FA accumulation in the medium to then evaluate the effect of T-ACM on macrophages. Further investigation is required to determine the effect of FA on J774 and THP-1 inflammatory response.