Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Des études épidémiologiques ont montré que l’obésité est associée à une diminution du taux plasmatique en rétinol et en 25 hydroxyvitamine D. Il a été également montré qu’il existe une corrélation entre carences en vitamines A et D et de nombreuses pathologies notamment l’insulino-résistance et l’inflammation. Notre hypothèse était de savoir s’il existe un lien entre vitamines A/D et inflammation au niveau du tissu adipeux (TA). Quelques études avaient montré que les formes actives de ces vitamines limitaient l’inflammation au niveau du TA en s’intéressant uniquement à quelques marqueurs inflammatoires. Nous avons alors voulu étudier l’impact global de ces deux vitamines sur le transcriptome d’adipocytes soumis à un stress inflammatoire en ayant recours à des approches haut débit. Nous avons montré que les vitamines A et D limitent l’inflammation au niveau des adipocytes humains et murins, in vitro, en diminuant l’expression de cytokines et de chimiokines ce qui induit une diminution de la migration macrophagique. Nous avons confirmé l’effet anti-inflammatoire de ces deux vitamines in vivo en ayant recours à des modèles murins d’inflammation chronique et aigüe. Nous nous sommes ensuite intéressés aux microARN dérégulés dans les adipocytes en condition inflammatoire. Nous avons mis en évidence le rôle clé du miR-155 dans l’amplification du signal inflammatoire. Nous avons montré que les vitamines A et D réduisent l’expression du miR-155 mais également celle des miR-146a et miR-150 au niveau des adipocytes murins et humains. Enfin, nous avons montré que les effets anti-inflammatoires des vitamines A et D sont médiés par une inhibition de la voie de signalisation NF-κB. / Epidemiological studies have shown that obesity is associated with a decrease in plasma retinol and 25-hydroxyvitamin D. It was also shown a correlation between insufficiencies of vitamins A and D and many diseases including insulin resistance and inflammation. Our working hypothesis was to test whether there is a link between vitamins A/D and inflammation in adipose tissue. Some studies have shown that the active forms of vitamins A and D limited inflammation in adipose tissue by looking only at few inflammatory markers. On the basis of these promising preliminary results, we wanted to examine the overall impact of these two vitamins on the transcriptome of adipocytes submitted to an inflammatory stress by using high-throughput methodologies. We have shown that vitamins A and D are able to limit inflammation in human and murine adipocytes, in vitro, by decreasing the expression of cytokines and chemokines which induces a decrease of macrophage migration. We have confirmed the anti-inflammatory effect of these two vitamins in vivo by using of chronic and acute inflammation mouse models. We then focused on microRNA which were deregulated in adipocytes in inflammatory conditions. We highlighted the key role of miR-155 in the amplification of the inflammatory signal. We have shown that vitamins A and D reduce the expression of miR-155 but also that of miR-146a and miR-150 in murine and human adipocytes. Finally, we showed that the anti-inflammatory effects of vitamins A and D are mediated by an inhibition of the signaling pathway NF-κB.

Longtemps décrit comme un simple tissu de réserve énergétique, le tissu adipeux blanc est, depuis l’identification de la leptine en 1994, considéré comme un véritable organe endocrine. En effet, ce tissu secrète de nombreuses hormones et cytokines agissant de manière paracrine et endocrine pour contrôler le métabolisme énergétique. Par ailleurs, en plus des préadipocytes et des adipocytes, le tissu adipeux blanc contient également des cellules immunes innées et adaptatives ; lui conférant ainsi un rôle important dans le développement et le contrôle de l’immunité. Cependant, le rôle joué par le tissu adipeux blanc dans les infections - notamment pulmonaires - reste encore peu étudié. C’est dans ce cadre général que s’est inscrit ce travail de Thèse. La susceptibilité accrue des individus obèses (expansion du tissu adipeux blanc) à l’infection par le virus de la grippe (influenza) est largement étayée dans la littérature. Nous avons évalué l’impact de l’infection par le virus influenza sur le tissu adipeux blanc, chez des souris minces et des souris obèses. Nos résultats montrent que, de manière inattendue, le virus est détecté dans les tissus adipeux, sous-cutané (inguinal) et viscéral (périgonadique), de souris infectées par voie intra-nasale (détection du génome viral par RT-qPCR). La présence de virus dans le tissu adipeux est associée à l’augmentation de la sécrétion de cytokines pro- et anti-inflammatoires, à la diminution de l’expression de gènes impliqués dans la lipolyse et la lipogénèse, et à l’augmentation de l’expression des gènes impliqués dans l’induction d’une réponse immune anti-virale. De manière intéressante, l’infection par le virus influenza est associée au brunissement du tissu adipeux sous-cutané chez les souris minces. Chez les souris obèses, l’infection par le virus de la grippe n’induit pas l’effet dépôt spécifique observé chez la souris mince et ne montre pas de brûnissement au niveau du tissu adipeux sous-cutané 7 jours p.i. In vitro, nous montrons que le virus influenza peut infecter les préadipocytes et les adipocytes (lignée murine et cellules primaires humaines). Cependant, alors que le virus effectue la totalité de son cycle dans l’adipocyte, le préadipocyte libère très peu, voire pas, de nouveaux virions infectieux (PCR, transcriptomique, technique de plages de lyse, microscopie confocale et électronique). Ainsi nos résultats, très originaux, identifient le tissu adipeux blanc comme un nouveau tissu cible de l’infection par le virus de la grippe, in vivo. Au sein de ce tissu, les préadipocytes et les adipocytes sont potentiellement infectés par le virus, comme le montrent nos données in vitro, les adipocytes seuls permettant la production de nouvelles particules infectieuses.Contrairement à l’infection grippale, les données épidémiologiques et/ou expérimentales concernant la susceptibilité des obèses à l’infection par la bactérie Streptococcus pneumoniae sont contradictoires, du fait de l’utilisation de différents modèles d’obésité d’origine génétique et de sérotypes de pneumocoques. Dans ce projet, nous avons utilisé un modèle d’obésité d’origine nutritionnelle ; le modèle de souris nourries par un régime enrichi en lipides. Nous montrons que les souris obèses infectées (sérotype Sp1) développent un syndrome de type méningite, mortel, tandis que les souris minces contrôlent l’infection. Si les réponses pulmonaires à l’infection sont comparables entre les souris minces et obèses (dénombrement des colonies bactériennes, histologie, PCR, ELISA, cytométrie en flux), le nombre de bactéries dans le cerveau est significativement plus élevé chez les souris obèses, associé à une altération de la perméabilité de la barrière hématoencéphalique [...] / Since the identification of leptin in 1994, the white adipose tissue (WAT) is no longer considered to solely be an inert tissue storing fat. As an endocrine organ, the adipose tissue synthesizes and secretes several hormones and cytokines involved in the control of whole-body metabolism. In addition, besides preadipocytes and adipocytes, WAT also contains innate and adaptive immune cells; thereby contributing to the development and control of immunity. However, the role played by the adipose tissue during infections - notably during pulmonary infections, such as those resulting from influenza virus or Streptococcus pneumoniae infections - has barely been investigated. This defines the general scope of this work. Epidemiological and experimental data convincingly report that obese individuals are more susceptible to influenza infection. During this project, we therefore questioned whether influenza infection may impact on adipose tissues, both subcutaneous (inguinal, SCAT) and visceral (perigonadal, EWAT) depots, in lean and high-fat diet-induced obese mice. We unexpectedly showed that influenza virus is detected in SCAT and EWAT (quantification of the viral genome by RtqPCR) and that this was associated with drastic changes in the tissue such as: increased secretion of pro- and anti-inflammatory cytokines, decreased expression of genes involved in lipogenesis and lipolysis, as well as increased expression of genes related to the induction of anti-viral immunity. Interestingly enough, influenza infection was associated with the development of brown-like adipocytes in the SCAT, only in lean animals. Moreover, we demonstrated in vitro that preadipocytes and adipocytes (murine cell-line and human primary cells) are permissive to infection, yet with different outcomes. Indeed, only adipocytes allowed the release of new infectious particles (RtqPCR, transcriptomics, quantification of infectious particules on MDCK cells, confocal and transmission electron microscopy). Altogether, our findings revealed, for the first time, that the white adipose tissue, an organ at the crossroads of metabolism and immunity, is deeply affected by influenza infection and might thus be undervalued in influenza pathophysiology.In opposite to influenza infection, the impact of obesity on the outcome of Streptococcus pneumoniae (S.p.) infection remains uncertain, due to the different models (genetically-based obesity, and bacterial strains) used. During this work, we investigated S. pneumoniae (Sp1 strain, sublethal dose) infection in lean and high-fat diet-induced obese mice. We showed that obese mice died from sublethal S. pneumoniae infection, compared to lean animals. The increased mortality induced by infection did not result from impaired pulmonary response but rather from the development of a meningitis-like syndrom likely resulting from an increased bacterial dissemination through the bloodbrain barrier into the brain. We propose that the model of dietary obesity induced by consumption of fat-enriched diet, may be envisaged as a novel and valuable experimental model of memingitis to study Streptococcus pneumoniae travel through the blood-brain barrier and the subsequent immune consequences.

Parmi les cellules stromales présentes dans le microenvironnement du cancer du sein, les adipocytes représentent des acteurs émergents dans la progression tumorale. Etudier le dialogue croisé entre adipocytes et cellules tumorales est d'importance en médecine, car l'obésité est un facteur reconnu de mauvais pronostic dans de nombreux cancers, notamment le cancer du sein. Nos résultats montrent que lorsque les adipocytes sont maintenus en présence des cellules tumorales pendant un temps prolongé, ils se réorientent progressivement en cellules fibroblastiques, présentant des caractéristiques de fibroblastes-Associés au Cancer. Nous avons nommé ces cellules, ADFs pour " Adipocytes Derived Fibroblasts " et montré que ces derniers, présents dans des tumeurs mammaires humaines, favorisent de manière importante l'invasion des cellules tumorales. D'autre part, nous avons démontré que les adipocytes favorisent une résistance pléiotropique aux cellules tumorales via un mécanisme d'efflux original et que cette chimiorésistance médiée par les adipocytes est amplifiée en conditions d'obésité. Ces travaux pourraient expliquer, en moins en partie, le mauvais pronostic des cancers du sein chez les patientes obèses. / Among stromal cells present in breast cancer, adipocytes represent emerging players in tumor progression. Studying the role of adipocytes in cancer is a major interest since epidemiological studies have convincingly established that obesity is associated with a poor outcome for several cancers, especially breast cancer. We demonstrated that some CAFs (Cancer Associated Fibroblasts) present in the breast tumor stroma arise from the "dedifferenciation" of adipocytes upon prolonged stimulation by tumor cells. This population was named ADFs (Adipocytes-Derived Fibroblasts) and was found in clinical samples of breast cancer. We further demonstrated that ADFs stimulate the invasive capacities of tumor cells. Moreover, our results suggest that adipocytes promote multidrug resistance in breast cancer cell lines mediated by an original efflux mechanism. Finally, we demonstrated that adipocyte-induced chemoresistance is amplified by obesity. This work may explain, at least in part, the poor prognosis of breast cancer in obese patients.

Les omégas trois ont un bénéfice prouvé dans la prévention de maladie cardiovasculaire et l’inflammation. Un apport optimal peut être réalisé avec des modifications diététiques simples permettant d’avoir un enrichissement des membranes cellulaires et un effet métabolique. Le tissu adipeux de part son rôle important dans la genèse du syndrome métabolique semble être une cible importante du traitement par oméga trois. Les patients avec une maladie rénale chronique (MRC) ont un risque cardiovasculaire accru et cumulent les perturbations métaboliques comme le syndrome métabolique et un état micro inflammatoire. Des doses supra physiologiques d’oméga trois ont été utilisés dans le passé dans des études de prévention rénale ou traitement de dyslipidémie. Or l’effet métabolique en fonction de la dose d’oméga 3 n’est pas connu. En accord, avec les études chez le sujet sain, en fonction de la dose administrée, les omégas 3 ont un impact différent métabolique et sur l’expression génique. Des études complémentaires sont nécessaires pour vérifier la faisabilité et l’impact métabolique d’une modification de régime afin de diminuer le rapport n-6/n-3, ainsi que l’effet à long terme des omégas trois chez ces patients. Par ailleurs, les mécanismes impliqués dans les différences de dose réponse devront être caractérisés sur un modèle animal / Omega 3 fatty acids play an important modulatory role in metabolic and inflammatory responses, the progression of atherosclerosis and gene expression. Recent studies suggest their beneficial impact on adipocyte morphology and function. Chronic kidney disease (CKD) patients have an increased cardiovascular morbi-mortality and suffer from a cluster of metabolic disorders. On the basis of previous studies there are reasons to suggest that omega 3 supplementation may offer a host of benefits to CKD patients. Unfortunatly, published studies on the effect of such supplementation are characterized by supra physiological omega 3 doses, that may be difficult to implement for extended periods in one hand and in the other hand the metabolic effect of different doses of omega 3 hasn’t been studied in detail. Simple dietary modifications can help achieve the recommended n-6/n-3 ratio in healthy subjects. In CKD patients supplementation with n-3 shows a differential dose response effect. Further studies are required to test the faisability and metabolic impact of dietary modifications in order to decrease n-6/n-3 ratio and to assess the long term effect of omega supplementation in CKD patients. Finally the molecular pathways implicated in this differential dose response should be assessed in animal models

Chronic exposure of humans or rodents to high calorie diets leads to hypertriglyceridemia and ectopic lipid deposition throughout the body, resulting in metabolic disease. Cellular lipids are stored in organelles termed lipid droplets (LDs) that are regulated by tissue-specific LD proteins. These proteins are critical for lipid homeostasis, as humans with LD protein mutations manifest metabolic dysfunction. Identification of novel components of the LD machinery could shed light on human disease mechanisms and suggest potential therapeutics for Type 2 Diabetes. Microarray analyses pinpointed the largely unstudied Hypoxia-Inducible Gene 2 (Hig2) as a gene that was highly expressed in obese human adipocytes. Imaging studies demonstrated that Hig2 localized to LDs in mouse hepatocytes and the human SGBS adipocyte cell line. Thus, this work examined the role of Hig2 as a LD protein in liver and adipose tissue. Hig2 deficiency reduced triglyceride deposition in hepatocytes; conversely, ectopic Hig2 expression promoted lipid deposition. Furthermore, liver-specific Hig2-deficient mice displayed improved glucose tolerance and reduced liver triglyceride content. Hig2 deficiency increased lipolysis and -oxidation, accounting for the reduced triglyceride accumulation. Similarly, adipocyte-specific Hig2-deficient mice displayed improved glucose tolerance, reduced adipose tissue weight and brown adipose tissue that was largely cleared of lipids. These improvements were abrogated when the animals were placed in thermoneutral housing and brown adipocyte-specific Hig2-deficient mice also displayed improved glucose tolerance, suggesting that active brown fat largely mediates the metabolic phenotype of Hig2 deletion. Thus, this work demonstrates that Hig2 localizes to LDs in liver and adipose tissue and promotes glucose intolerance.

Reduced neo-adipogenesis and dysfunctional lipid-overloaded adipocytes are hallmarks of hypertrophic obesity linked to insulin resistance. Identifying molecular features of hypertrophic adipocytes requires appropriate in vitro models. We describe the generation of a model of human hypertrophic-like adipocytes directly comparable to normal adipose cells and the pathologic evolution toward hypertrophic state. We generate in vitro hypertrophic cells from mature adipocytes, differentiated from human mesenchymal stem cells. Combining optical, confocal, and transmission electron microscopy with mRNA/protein quantification, we characterize this cellular model, confirming specific alterations also in subcutaneous adipose tissue. Specifically, we report the generation and morphological/molecular characterization of human normal and hypertrophic-like adipocytes. The latter displays altered morphology and unbalance between canonical and dominant negative (PPARGΔ5) transcripts of PPARG, paralleled by reduced expression of PPARγ targets, including GLUT4. Furthermore, the unbalance of PPARγ isoforms associates with GLUT4 down-regulation in subcutaneous adipose tissue of individuals with overweight/obesity or impaired glucose tolerance/type 2 diabetes, but not with normal weight or glucose tolerance. In conclusion, the hypertrophic-like cells described herein are an innovative tool for studying molecular dysfunctions in hypertrophic obesity and the unbalance between PPARγ isoforms associates with down-regulation of GLUT4 and other PPARγ targets, representing a new hallmark of hypertrophic adipocytes.

Numerous epidemiology studies suggest a correlation between exposures to polychlorinated biphenyls (PCBs) and the development and severity of type 2 diabetes (T2D); however, mechanisms remain largely unknown. Previous studies demonstrated that PCBs that are ligands of the aryl hydrocarbon receptor (AhR) promote the expression of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), that are linked to insulin resistance in adipocytes. To explore potential mechanisms linking PCB exposures to diabetes, we developed a mouse model of glucose and insulin intolerance induced by acute and chronic exposures to PCB-77. We hypothesized that PCB ligands of AhR result in adipocyte-specific elevations in TNF-α and dysregulated glucose homeostasis. Results demonstrated that PCB77 resulted in rapid and sustained glucose and insulin intolerance in low fat (LF)-fed mice, and that these effects were associated with adipose-specific elevations in TNF-α. When mice were made obese from consumption of a high fat (HF) diet, effects of PCB77 were lost presumably due to concentration of the toxin in adipose lipids. However, upon weight loss, mice exposed to PCB77 exhibit impaired glucose homeostasis. These results suggest that lipophilic PCBs redistribute from adipose lipids with weight loss and mitigate beneficial effects to improve glucose homeostasis. To define the role of adipocyte AhR in PCB-induced diabetes, we created a mouse 3 model of adipocyte AhR deficiency using the Cre/LoxP system. Adipocyte-AhR deficiency conferred protection from the development of PCB-77-induced impairments in glucose and insulin tolerance in obese mice undergoing weight loss. Unexpectedly, adipocyte-AhR deficient mice fed the HF diet exhibited adipocyte hypertrophy, increased adipose mass and elevated body weight. These results suggest that (1) adipocyte AhRs are responsible for effects of PCB77 to impair glucose homeostasis during weight loss and (2) adipocyte AhRs respond to the HF diet to regulate adipose mass and body weight. We used resveratrol as a putative AhR antagonist to determine if the polyphenol confers protection against PCB-77-induced diabetes. Resveratrol abolished acute effects of PCB77 to impair glucose and insulin tolerance in LF-fed mice. Notably, PCB77 administration abolished insulin-induced phosphorylation of Akt in adipose tissue and these effects were abolished by resveratrol. Resveratrol also abolished marked suppressions in glucose uptake in adipocytes exposed to PCB77. These studies suggest the adipocyte AhR plays a potentially significant role in the development of diabetes and obesity, and that resveratrol may represent a novel therapeutic for PCB exposed populations.

The present study was undertaken to investigate the effects of the T-type calcium channel blocker, Mibefradil, on white adipocytes. Unexpected for a T-type channel blocker, Mibefradil was found to increase intracellular calcium levels, cause lipid droplet fusion, and result in cell death. Calcium imaging of white adipocytes showed an increase of calcium concentration by Mibefradil at concentrations ranging from10-50 µM. The elevation in calcium by Mibefradil was significantly reduced by pretreatment of cells with Thapsigargin, an endoplasmic reticulum (ER) specific Ca ATPase inhibitor. Additionally, lipid droplet fusion and cell death were also attenuated by Thapsigargin pretreatment in white adipocytes. We conclude that Mibefradil elevated intracellular calcium levels, induced lipid droplet fusion and cell death in white adipocytes via mobilizing intracellular calcium stores from the ER. These results describe novel effects of Mibefradil on white adipocytes and may provide new insight into how this drug might be repurposed in obesity research.

Background: Genome-wide association studies have identified variants within the FTO (fat mass and obesity associated) locus as the strongest predictors of obesity amongst all obesity-associated gene loci. Recent evidence suggests that variants in FTO directly affect human adipocyte function through targeting IRX3 and IRX5 and thermogenesis regulation. Aim: We addressed the relevance of this proposed FTO-IRX pathway in adipose tissue (AT) of children. Results: Expression of IRX3 was higher in adipocytes compared to SVF. We found increased adipocyte-specific expression of IRX3 and IRX5 with the presence of the FTO risk haplotype in lean children, whereas it was unaffected by risk variants in obese peers. We further show that IRX3 expression was elevated in isolated adipocytes and AT of lean compared to obese children, particularly in UCP1-negative adipocytes, and inversely correlated with BMI SDS. Independent of BMI, IRX3 expression in adipocytes was significantly related to adipocyte hypertrophy, and subsequent associations with AT inflammation and HOMA-IR in the children. Conclusion: One interpretation of our observation of FTO risk variants linked to IRX3 expression and adipocyte size restricted to lean children, along with the decreased IRX3 expression in obese compared to lean peers, may reflect a defense mechanism for protecting body-weight, which is pertinent for lean children.