Contrôle transcriptionnel du tissu adipeux par PPARgamma et ses cofacteurs lors du vieillissement

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2010-2011Le vieillissement est un facteur de risque important d'accumulation de masse grasse, particulièrement au niveau viscéral, qui conduit aux maladies métaboliques, telles que l'obésité, le diabète de type 2 et l'athérosclérose. D'autre part, le tissu adipeux semble être lui-même un modulateur de la longévité. Les travaux décrits dans cette thèse montrent des adaptations géniques spécifiques à l'accumulation de masse grasse lors du vieillissement. Dans le tissu adipeux, les mécanismes moléculaires responsables semblent impliquer un changement du milieu transcriptionnel de PPARy, menant ainsi à une capacité transactivatrice plus élevée. Lors du vieillissement, les niveaux d'expression génique et protéique du cofacteur SRC-1 sont diminués chez les souris, les rats et l'humain. De ce fait, l'interaction PPARy/SRC-1 est perdue et la capacité de liaison de PPARy au promoteur de gène aP2 adipogénique est augmentée. Lors du vieillissement, la biologie du tissu adipeux est perturbée, particulièrement avec l'infiltration de lymphocytes T et de macrophages. OCA-B est un cofacteur nucléaire essentiel au développement des lymphocytes B, qui sont recrutés à un stade précoce de ces événements et impliqués dans la prévention de l'infiltration des macrophages. Nous avons tenté de découvrir si le débalancement des niveaux des cofacteurs nucléaires du tissu adipeux allait au-delà de SRC-1. Les résultats de cette étude montrent que l'expression de OCA-B est spécifiquement augmentée dans le dépôt viscéral lors du vieillissement, mais drastiquement diminuée chez les modèles murins obèses. OCA-B est exprimé dans les adipocytes. Il interagit également avec PPARy et se lie aux promoteurs de gènes spécifiques des adipocytes matures, soulignant son implication dans la biologie des adipocytes. Les fibroblastes embryonnaires des souris et les nematodes ne possédant pas OCA-B accumulent plus de lipides. De plus, les vers OCA-B-/- ont par ailleurs une diminution de leur longévité, ce qui suggère qu'OCA-B est un joueur central dans le lien entre masse grasse et espérance de vie. La dernière étude de cette thèse implique le gène klotho qui intervient dans la régulation du vieillissement et dans le contrôle transcriptionnel du métabolisme. Klotho agit comme une hormone sécrétée et favorise la différenciation adipocytaire et la résistance à l'insuline. Nous avons montré chez les souris que, lors du vieillissement, klotho est davantage modulé dans le dépôt sous-cutané, alors que la modulation est beaucoup plus drastique dans le contexte de l'obésité et ce dans le dépôt sous-cutané et viscéral. De plus, selon l'espèce, les résultats divergent puisque chez la femme, klotho est augmenté dans le contexte de l'obésité. Dans les adipocytes 3T3-L1, le traitement aigu avec les TZD augmente l'expression de klotho, et diminue dans les traitements à long terme. La diminution de l'expression de klotho est retrouvée uniquement chez les souris jeunes suite à un traitement d'une semaine avec la pioglitazone. Les résultats de l'ensemble de ces études démontrent que le vieillissement a un impact important sur l'environnement transcriptionnel de PPARy en modifiant notamment l'expression et le recrutement des cofacteurs nucléaires SRC-1 et OCA-B. Ces changements mènent à une empreinte génique spécifique qui stimule l'accumulation de masse grasse et réduit la sensibilité à l'insuline, potentiellement via une altération des molécules sécrétées dont klotho

Crosstalk between alternatively spliced UGT1A isoforms and colon cancer cell metabolism

Alternative splicing at the human glucuronosyltransferase 1 gene locus (UGT1) produces alternate isoforms UGT1A_i2s that control glucuronidation activity through protein-protein interactions. Here, we hypothesized that UGT1A_i2s function into a complex protein network connecting other metabolic pathways with influence on cancer cell metabolism. This is based on a pathway enrichment analysis of proteomic data that identified several high-confidence candidate interaction proteins of UGT1A_i2 proteins in human tissues, namely the rate-limiting enzyme of glycolysis pyruvate kinase (PKM), which plays a critical role in cancer cell metabolism and tumor growth. The partnership of UGT1A_i2 and PKM2 was confirmed by co-immunoprecipitation in the HT115 colon cancer cells and was supported by a partial co-localization of these two proteins. In support of a functional role for this partnership, depletion of UGT1A_i2 proteins in HT115 cells enforced the Warburg effect with higher glycolytic rate at the expense of mitochondrial respiration, and led to lactate accumulation. Untargeted metabolomics further revealed a significantly altered cellular content of 58 metabolites including many intermediates derived from the glycolysis and TCA cycle pathways. These metabolic changes were associated with a greater migration potential. The potential relevance of our observations is supported by the down-regulation of UGT1A_i2s mRNA in colon tumors compared to normal tissues. Alternate UGT1A variants may thus be part of the expanding compendium of metabolic pathways involved in cancer biology directly contributing to the oncogenic phenotype of colon cancer cells. Findings uncover new aspects of UGT functions diverging from their transferase activity

The Retinoblastoma-Histone Deacetylase 3 Complex Inhibits PPARγ and Adipocyte Differentiation

AbstractThe retinoblastoma protein (RB) has previously been shown to facilitate adipocyte differentiation by inducing cell cycle arrest and enhancing the transactivation by the adipogenic CCAAT/enhancer binding proteins (C/EBP). We show here that the peroxisome proliferator-activated receptor γ (PPARγ), a nuclear receptor pivotal for adipogenesis, promotes adipocyte differentiation more efficiently in the absence of RB. PPARγ and RB were shown to coimmunoprecipitate, and this PPARγ-RB complex also contains the histone deacetylase HDAC3, thereby attenuating PPARγ's capacity to drive gene expression and adipocyte differentiation. Dissociation of the PPARγ-RB-HDAC3 complex by RB phosphorylation or by inhibition of HDAC activity stimulates adipocyte differentiation. These observations underscore an important function of both RB and HDAC3 in fine-tuning PPARγ activity and adipocyte differentiation

Sirt1 inhibits resistin expression in aortic stenosis

The development of human calcified aortic stenosis (AS) includes age-dependent processes that have been involved in atherosclerosis, such as infiltration of macrophages in aortic valves, which then promote production of many proinflammatory cytokines, including resistin. However, the molecular mechanisms contributing to these processes are not established. Since Sirt1 has been shown to modulate macrophage biology and inflammation, we examined its levels in human AS and tested its impact on resistin expression. Sirt1 mRNA (p = 0.01) and protein (p,0.05) levels were reduced in explanted valves from AS patients (n = 51) compared to those from control (n = 11) patients. Sirt1 mRNA levels were negatively associated with resistin mRNA levels quantified in AS valves (p = 0.02). Stimulation of Sirt1 by resveratrol or virusdriven overexpression robustly diminished resistin mRNA and protein expression in macrophages, whereas down-regulation of Sirt1 triggered a large increase in resistin expression. These effects were direct, as chromatin immunoprecipitation assays showed that Sirt1 physically interacted with the resistin promoter region at an AP-1 response element. Moreover, Sirt1 blocked c-jun-induced resistin transactivation in gene reporter assays. These findings demonstrate that, in calcified AS, levels of Sirt1 are reduced whereas those of resistin are increased within aortic valve leaflets. Our results also suggest that this loss of Sirt1 expression alleviates its inhibition of resistin transcription in macrophages. Although the overall contribution of this process to the underlying mechanisms for AS disease development remains unresolved, these observations suggest that modification of Sirt1 expression and/or activity could represent a novel approach against inflammation in AS

Methods to extract and study the biological effects of murine gut microbiota using Caenorhabditis elegans as a screening host.

Gut microbiota has been established as a main regulator of health. However, how changes in gut microbiota are directly associated with physiological and cellular alterations has been difficult to tackle on a large-scale basis, notably because of the cost and labor-extensive resources required for rigorous experiments in mammals. In the present study, we used the nematode Caenorhabditis elegans as a model organism to elucidate microbiota-host interactions. We developed a method to extract gut microbiota (MCB) from murine feces, and tested its potential as food source for and its impact on C. elegans biology compared to the standard bacterial diet Escherichia coli OP50. Although less preferred than OP50, MCB was not avoided but had a lower energy density (triglycerides and glucose). Consistently, MCB-fed worms exhibited smaller body length and size, lower fertility, and lower fat content than OP50-fed worms, but had a longer mean lifespan, which resembles the effects of calorie restriction in mammals. However, these outcomes were altered when bacteria were inactivated, suggesting an important role of symbiosis of MCB beyond nutrient source. Taken together, our findings support the effectiveness of gut MCB processing to test its effects in C. elegans. More work comparing MCB of differently treated mice or humans is required to further validate relevance to mammals before large-scale screening assays

Methods to extract and study the biological effects of murine gut microbiota using Caenorhabditis elegans as a screening host

Gut microbiota has been established as a main regulator of health. However, how changes in gut microbiota are directly associated with physiological and cellular alterations has been difficult to tackle on a large-scale basis, notably because of the cost and labor-extensive resources required for rigorous experiments in mammals. In the present study, we used the nematode Caenorhabditis elegans as a model organism to elucidate microbiota-host interactions. We developed a method to extract gut microbiota (MCB) from murine feces, and tested its potential as food source for and its impact on C. elegans biology compared to the standard bacterial diet Escherichia coli OP50. Although less preferred than OP50, MCB was not avoided but had a lower energy density (triglycerides and glucose). Consistently, MCB-fed worms exhibited smaller body length and size, lower fertility, and lower fat content than OP50-fed worms, but had a longer mean lifespan, which resembles the effects of calorie restriction in mammals. However, these outcomes were altered when bacteria were inactivated, suggesting an important role of symbiosis of MCB beyond nutrient source. Taken together, our findings support the effectiveness of gut MCB processing to test its effects in C. elegans. More work comparing MCB of differently treated mice or humans is required to further validate relevance to mammals before large-scale screening assays

PPARgamma controls cell proliferation and apoptosis in an RB-dependent manner

The nuclear receptor PPARgamma is implicated in the control of cell proliferation and apoptosis. However, the molecular mechanisms by which it controls these processes remain largely elusive. We show here that PPARgamma activation in the presence of the retinoblastoma protein (RB) results in the arrest of cells at the G1 phase of the cell cycle, whereas in the absence of RB, cells accumulate in G2/M, endoreduplicate, and undergo apoptosis. Through the use of HDAC inhibitors and coimmunoprecipitations, we furthermore demonstrate that the effects of RB on PPARgamma-mediated control of the cell cycle and apoptosis depend on the recruitment of histone deacetylase 3 (HDAC3) to PPARgamma. In combination, these data hence demonstrate that the effects of PPARgamma on cell proliferation and apoptosis are dependent on the presence of an RB-HDAC3 complex

Cyclin D3 Promotes Adipogenesis through Activation of Peroxisome Proliferator-Activated Receptor γ

In addition to their role in cell cycle progression, new data reveal an emerging role of D-type cyclins in transcriptional regulation and cellular differentiation processes. Using 3T3-L1 cell lines to study adipogenesis, we observed an up-regulation of cyclin D3 expression throughout the differentiation process. Surprisingly, cyclin D3 was only minimally expressed during the initial stages of adipogenesis, when mitotic division is prevalent. This seemingly paradoxical expression led us to investigate a potential cell cycle-independent role for cyclin D3 during adipogenesis. We show here a direct interaction between cyclin D3 and the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ). Our experiments reveal cyclin D3 acts as a ligand-dependent PPARγ coactivator, which, together with its cyclin-dependent kinase partner, phosphorylates the A-B domain of the nuclear receptor. Overexpression and knockdown studies with cyclin D3 had marked effects on PPARγ activity and subsequently on adipogenesis. Chromatin immunoprecipitation assays confirm the participation of cyclin D3 in the regulation of PPARγ target genes. We show that cyclin D3 mutant mice are protected from diet-induced obesity, display smaller adipocytes, have reduced adipogenic gene expression, and are insulin sensitive. Our results indicate that cyclin D3 is an important factor governing adipogenesis and obesity

Impaired pancreatic growth, beta cell mass, and beta cell function in E2F1 (-/-) mice

We evaluated the effects of E2F1 on glucose homeostasis using E2F1(-/-) mice. E2F1(-/-) mice show an overall reduction in pancreatic size as the result of impaired postnatal pancreatic growth. Furthermore, these animals have dysfunctional beta cells, linked to impaired PDX-1 activity. Because of the disproportionate small pancreas and dysfunctional islets, E2F1(-/-) mice secrete insufficient amounts of insulin in response to a glucose load, resulting in glucose intolerance. Despite this glucose intolerance, E2F1(-/-) mice do not develop overt diabetes mellitus because they have insulin hypersensitivity, which is secondary to a diminished adipose tissue mass and altered adipocytokine levels, which compensates for the defect in insulin secretion. These data demonstrate that factors controlling cell proliferation, such as E2F1, determine pancreatic growth and function, subsequently affecting metabolic homeostasis