Investigating the cellular processes anti-ageing compounds interact with can identify genes and pathways involved in ageing. The macrolide lactone FK506 was identified in a phenotypic screen as extending lifespan in yeast and C. elegans through an unknown mechanism. FK506 also ameliorates neurodegeneration and age-related weight gain in rodents. Here, the mechanism of action of FK506 has been investigated in two experimental systems: C. elegans and 3T3-L1 mouse adipocytes. As the general mechanisms of ageing are well conserved between C. elegans and mammals, C. elegans has been used to understand how FK506 acts at an organismal level. Firstly, the result of the phenotypic screen was confirmed. FK506 treatment induced lifespan extension in C. elegans in the presence of population crowding stress, but not in the absence of crowding. FK506 treatment inhibited neither E. coli OP50 growth nor C. elegans pharyngeal pumping, demonstrating that FK506 did not induce dietary restriction to extend lifespan. FK506 treatment increased C. elegans thrashing and pharynx pumping rates in early adulthood and delayed accumulation of gut bacteria, showing that FK506 extended healthspan. A transcriptome analysis of FK506-treated C. elegans allowed the identification of transcripts whose levels change and potential pathways by which FK506 manifests its effect. To explore this and to identify potential targets of FK506, the cellular functions required for FK506 to extend C. elegans lifespan and healthspan were investigated using RNA-seq, RNAi, genetic mutation and co-treatment with small molecule inhibitors and inducers. Interestingly, FK506 was found to have different mechanisms of action on lifespan and healthspan. The mechanism of FK506 on C. elegans thrashing rate was DAF-16 dependent, did not require population crowding stress, had a partial interaction with FUdR and autophagy, and may involve Ca²⁺ flux. The mechanism of FK506-induced C. elegans lifespan extension overlapped with dietary restriction and was dependent on calcineurin, TOR-independent regulation of autophagy and the presence of population crowding stress. FK506 may modulate body weight by influencing metabolism and/or acting on adipocytes directly. FK506-treated aged 3T3-L1 adipocytes accumulated significantly less lipid, indicating that FK506 acts directly on adipocytes. RNA-seq of FK506-treated adipocytes found that translation-associated RNAs were upregulated whilst RNAs associated with lipid metabolism were downregulated. An ER-localised FK506-binding protein was up regulated in both C. elegans and 3T3-L1 adipocytes, fkb-4 and Fkbp2 respectively. In conclusion, FK506 has been confirmed as a potential anti-ageing treatment, through its ability to extend lifespan and healthspan in C. C. elegans. In addition, FK506 has also been shown to act directly on mouse adipocytes, resulting in a reduction in lipid accumulation. This action could explain how FK506 caused weight loss in obese aged rats, restoring body mass to a healthy adult weight.

Aujourd’hui encore, l’obésité touche de plus en plus d’individus, et sa complexité reflète son aspect multifactoriel. Parmi les facteurs impliqués dans le développement de l'obésité, on compte la génétique qui expliquerait notamment la susceptibilité de certains êtres humains à devenir obèse. D'un point de vue moléculaire, la cascade adipogénique permettant à des préadipocytes de devenir des adipocytes matures est un phénomène dynamique et bien caractérisé. En revanche, les facteurs définissant le potentiel adipogénique des préadipocytes ainsi que le renouvellement des adipocytes demeurent encore inconnus. Notre objectif a été d’identifier et de caractériser de nouveaux gènes définissant l’identité des préadipocytes et pouvant influencer le développement du tissu adipeux. Par dilutions sériées d’une culture de 3T3-L1, nous avons isolé et amplifié 23 nouvelles lignées cellulaires que nous avons classées selon leur potentiel adipogénique. Une puce à ADN nous a permis d’identifier les gènes s’exprimant différemment entre les lignées à fort et à faible potentiel adipogénique. Nous avons identifié un nouveau gène inconnu de la littérature nommé V-set and transmembrane domain containing 2A (Vstm2a). Les travaux réalisés dans le cadre de ma thèse ont été de comprendre l’implication de ce gène dans le développement de l’adipocyte in vitro et dans la formation du tissu adipeux in vivo. Nous avons d’abord identifié Vstm2a comme le gène le plus différentiellement exprimé entre les différentes lignées cellulaires, son expression étant très élevée dans les lignées à fort potentiel adipogénique. Une corrélation positive s’est dessinée entre l’expression de Vstm2a et Peroxisome Proliferator-Activated Receptor γ2 (Pparγ2) dans les préadipocytes. Lors du développement de l’adipocyte in vitro, l’expression de Vstm2a précède toujours l’activation de Pparγ2, un facteur clé régulant l’adipogenèse. Cette expression précoce est retrouvée in vivo, lors de la formation et de l’expansion du tissu adipeux. Les cellules exprimant Vstm2a au stade développemental se retrouvent à proximité de vaisseaux sanguins et de cellules musculaires, une caractéristique de précurseurs adipeux. Chez l’adulte, ces cellules s’apparentent à la population de précurseurs adipeux identifiée à partir des marqueurs de surface LIN⁻CD29⁺CD34⁺SCA1⁺PDGFRα⁺. Nous avons découvert que VSTM2A est une protéine glycosylée et abondamment sécrétée par des cellules ayant un fort potentiel adipogénique. La surexpression de VSTM2A intracellulaire suffit à favoriser la différenciation adipocytaire de cellules fibroblastiques, entre autres par l’induction de Pparγ2. À l’inverse, sa sous-expression dans les préadipocytes entraine un défaut adipogénique, réduisant l’expression de Pparγ2. Nous avons montré que VSTM2A agit en amplifiant la réponse aux Bone Morphogenetic Protein (BMP) favorisant ainsi l’expression de Pparγ2. Nous proposons un modèle dans lequel VSTM2A est essentiel pour maintenir et amplifier le potentiel adipogénique des préadipocytes. Une meilleure compréhension des facteurs régulant les phases précoces de l'adipogenèse pourrait permettre le développement de nouveaux outils régulant la formation du tissu adipeux. / Obesity affects the life of millions of humans worldwide and is the result of complex interactions between genes and environment. Factors involved in the development of obesity include genetics, which may explain why some humans are more susceptible to become obese. From a molecular point of view, the adipogenic cascade allowing preadipocytes to differentiate into mature adipocytes is a dynamic and well characterized phenomenon. However, the factors defining the adipogenic potential of the preadipocytes as well as the renewal of the adipocytes remain unknown. Our objective was to identify and characterize new genes defining the identity of preadipocytes capable of influencing the development of adipose tissue. By performing serial dilutions of a culture of 3T3-L1 preadipocytes, we have isolated and amplified 23 new cell lines that we have classified according to their adipogenic potential. A microarray allowed us to identify the genes differently expressed between high and low adipogenic lines. We have identified a new gene, unknown from the literature, named V-set and transmembrane domain containing 2A (Vstm2a). The objective of my thesis was to understand the implication of this gene in the development of the adipocytes in vitro and in the formation of adipose tissue in vivo. We first identified that Vstm2a was the most differentially expressed gene between the Low and High adipogenic lines, its expression being elevated in lines with a high adipogenic potential. A positive correlation emerged between the expression of Vstm2a and Pparγ2 in the preadipocytes. During adipocyte development in vitro, the expression of Vstm2a always precedes PPARγ2 activation, a key factor regulating adipogenesis. This expression pattern was also found in vivo during adipose tissue formation and expansion. VSTM2A-expressing cells at the developmental stage are found in the vicinity of the vasculature and muscle cells, a characteristic of adipose precursors. In adults, Vstm2a expression is enriched in the adipose precursor population identified with the surface markers LIN⁻CD29⁺CD34⁺SCA1⁺PDGFRα⁺. We found that VSTM2A is a glycosylated protein that is abundantly secreted by cells with a high adipogenic potential. Overexpression of intracellular VSTM2A is sufficient to promote the adipogenic potential of fibroblastic cells, an effect associated with a rise in basal Pparγ2 expression. Conversely, VSTM2A knockdown impairs adipogenesis by reducing the expression of Pparγ2. We found that VSTM2A promotes Pparγ2 expression by amplifying BMP signaling. Here, we propose a model in which VSTM2A is expressed to maintain and amplify the adipogenic potential of adipose precursors. A better understanding of the factors regulating early steps in adipogenesis could allow the development of new tools to regulate adipose tissue formation.

L’obésité, liée à la résistance à l’insuline, au diabète de type 2 et aux maladies cardiovasculaires, est un problème de santé majeur de notre société. Nous avons démontré que la protéine tyrosine phosphatase Shp1, dont l’expression est significativement augmentée dans les tissus cibles de l’insuline chez les souris obèses, est un régulateur de l’homéostasie du glucose dans le foie et le muscle. Shp1 est impliqué dans la modulation de l’expression et de l’activité du récepteur nucléaire PPARγ dans le foie. Nos recherches ont porté sur la caractérisation de Shp1 dans les adipocytes, la signalisation de l’insuline et le transport du glucose soit en sous-exprimant ou en exprimant de façon constitutive Shp1 dans les cellules adipeuses 3T3-L1. L’état physiologique des cellules a été caractérisé par la mesure de la coloration Oil-Red-O, des triglycérides, de l’expression de PPARγ et de ses gènes cibles et de leurs protéines, de la réponse à l’insuline par le transport du glucose ainsi que l’expression et de la phosphorylation des protéines impliquées dans la signalisation de l’insuline. La diminution de l’expression de Shp1 a entraîné un délai lors du début de la différenciation mesurée par l’expression retardée de PPARγ et certains de ses gènes cibles, mais n’a pas beaucoup affecté le phénotype des cellules complètement différenciées. Bien que la réduction de Shp1 ait augmenté la phosphorylation d’Akt stimulée par l’insuline, le transport de glucose n’a pas été modifié dans ces cellules, et l’expression de glut4 a légèrement diminué. L’expression constitutive de Shp1 a entraîné une forte diminution des niveaux de PPARγ, inhibant totalement la différenciation, l’expression de glut4 et le transport du glucose. Nos données suggèrent que Shp1 joue un rôle important dans les adipocytes comme régulateur de l’adipogenèse par la modulation de l’expression et l’activité de PPARγ et par la régulation de la signalisation de l’insuline. / Obesity, which is causally linked to the development of insulin resistance, type 2 diabetes (T2D) and cardiovascular disease (CVD), is a major health issue in our society. We have demonstrated that the protein tyrosine phosphatase Shp1, whose expression is significantly increased in insulin target tissues of obese mice, is a regulator of glucose homeostasis in liver and muscle. Shp1 is implicated in the modulation of expression and activity of the nuclear receptor PPARγ in liver. Here, we describe the characterization of Shp1 in adipocytes by analyzing its role in adipocyte differentiation, insulin signaling and glucose transport by either knocking-down or constitutively expressing Shp1 in 3T3-L1 adipose cells. The physiological state of the cells was characterized by measuring Oil-Red-O staining, triglyceride content, expression of PPARγ and its target genes and their proteins, insulin response by glucose uptake and the expression and phosphorylation of proteins involved in insulin signaling. Knockdown of Shp1 led to a retardation in the onset of differentiation as measured by delayed expression of PPARγ and some of its target genes, but did not much affect the phenotype of fully differentiated cells. Although reducing Shp1 increased insulin-stimulated Akt-phosphorylation, glucose transport was not changed in these cells, and glut4 expression was slightly decreased. Constitutive expression of Shp1, resulted in a strong decrease of PPARγ levels thereby totally inhibiting differentiation, glut4 expression and glucose transport. Our data suggest that Shp1 plays an important role in adipocytes both by acting as a regulator of adipogenesis through modulation of the expression and activity of PPARγ and by regulating the insulin signaling pathway.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 4 uppsatser.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.

No description available.

Thyrotropin (TSH) binds to TSH receptors on thyrocytes to regulate development and growth of the thyroid gland, and to stimulate thyroid hormone production. Thyrotropin has also been shown to act in an extra-thyroidal fashion, and to engage TSH receptors on adipocytes to induce lipolysis, similar to the response seen by stimulation with β-adrenergic receptor agonists (i.e. isoproterenol). In both cell types, cAMP-dependent kinase (PKA) is activated. Recently, PRAJA2, a novel AKAP and E3 ubiquitin ligase that targets the regulatory subunits of PKA was identified. The ubiquitin-dependent proteasomal degradation of the PKA regulatory subunits, due to PKA- phosphorylated PRAJA2, prolongs the catalytic activity of PKA, as shown in neuroblastoma cells by Lignitto et al., 2011. In adipocytes, stimulated PKA activity is required for lipolysis. Additionally, PRAJA2 has been described to have increased expression in TSH-responsive, differentiated thyroid cancer cells when compared to anaplastic thyroid tumor (Cantara et al., 2012) The aim of this study was to characterize PRAJA2 and its potential influence on adipocyte lipolysis. These data confirm that TSH and isoproterenol stimulate lipolysis in primary human differentiated adipocytes. PRAJA2 is expressed at the mRNA and protein level in differentiated adipocytes, with no change following stimulation with TSH or isoproterenol. Stimulation with isoproterenol, but not TSH, increases PKA-dependent phosphorylation of a 122kDa (potentially PRAJA2) and 69kDa protein identified in PRAJA2 immunoprecipitates. These proteins may prove important for lipolytic signaling or other PRAJA2-dependent process in adipocytes. Experimentation was unable to identify interactions between PRAJA2 and PKAR2 in differentiated adipocytes; however further investigations are required before discounting this interaction. An attempt was made to knockdown PRAJA2 in this model, and measure effects on lipolytic response; however, this was unsuccessful. Taken together, PRAJA2 appears to be phosphorylated following β-adrenergic stimulation in human adipocytes; however, further studies are needed to delineate the specific role of PRAJA2 in this human differentiated adipocyte model.

Acute myeloid leukemia (AML) is an aggressive cancer of the blood and bone marrow, affecting 1,100 Canadians annually. Older patients make up 75% of cases yet have the lowest survival rates due to the lack of tolerable treatments. Recently, the combination of Venetoclax and Azacitidine (Ven/Aza) has shown great therapeutic promise, however, chemoresistance has become a growing concern. Current evidence points towards a chemoprotective role from the bone marrow (BM) microenvironment, specifically by BM-derived mesenchymal stromal cells (BMSCs) and adipocytes. AML cells can manipulate BMSCs and adipocytes to create a niche that supports its own growth and evades chemotherapy. However, the role of the microenvironment in Ven/Aza chemoresistance has yet to be studied. Our objective was to study the ability of the microenvironment cells to induce AML chemoresistance to Ven/Aza. We employed a 2-dimensional direct contact co-culture system between MOLM-13 AML cells and BMSCs or adipocytes in both the absence and presence of Ven/Aza to determine the effects on the AML cells. In the absence of Ven/Aza, adipocyte co-cultured AML cells showed a 47% reduction in proliferation, 10% reduction in viability, yet a 1.7-fold increase in Maximal respiration when compared to the monocultured cells. In the presence of Ven/Aza, adipocyte co-cultured AML cells showed a significant increase in both proliferation and viability. Preliminary work investigating the mechanism of action of this support points toward an anti-apoptotic mechanism mediated by the upregulation of MCL-1 upon co-culture with adipocytes. Combination of Venetoclax and Tapotoclax, an MCL-1 inhibitor, abrogated the chemoprotection provided by BMSCs and adipocytes. Overall, our data suggests a dual role of adipocytes, where their inhibition or support of AML is context dependent. Therapeutic targeting of mechanisms for adipocyte chemoprotection such as MCL-1 upregulation may re-sensitize AML cells to Ven/Aza, thereby improving patient outcomes. / Thesis / Master of Science (MSc) / Acute myeloid leukemia (AML) is an aggressive cancer of the blood and bone marrow, affecting 1,100 Canadians annually. Older patients make up 75% of cases yet have the lowest survival rates due to the lack of tolerable treatments. A novel combination of Venetoclax and Azacitidine (Ven/Aza) has shown great therapeutic promise, however, chemoresistance remains an important concern. Previous studies have implicated fat cells, or adipocytes, in AML chemoresistance, however, their role in Ven/Aza treatment has yet to be studied. Here, we show that adipocytes reduce growth of AML cells, yet enhance their metabolism. In the presence of Ven/Aza, adipocytes induce chemoresistance. We show preliminary data that this chemoprotection may be mediated by the upregulation of mitochondrial MCL-1 protein as inhibition of this protein neutralized the protection. By understanding the relationship between adipocytes and AML chemoresistance, we can target this and re-sensitize AML to Ven/Aza, thereby improving older patient outcomes.