Cell turnover is a common feature of many organs in all animals and is required to maintain organ structure and function. It is achieved by a tightly regulated balance between cell death and cell division, which can be re-adjusted in response to injury and nutrient availability. How the balance between dying and dividing cells is coordinated has however remained unclear. Planarians represent an important model for studying cell turnover in adult animals, because all tissues undergo continuous cell turnover and a single stem cell type – the neoblast – is the exclusive source of all new cells. Moreover, planarians change their body size proportionally and reversibly depending on the nutritional status: feeding induces rapid and transient neoblast proliferation that results in animal growth, while starvation increases the rate of cell death, leading to de-growth. Importantly, also during starvation neoblasts keep proliferating at a basal-level. The hypothesis I addressed with my thesis research is that planarian energy metabolism might be a central mediator of cell turnover, particularly proliferation control and growth. I approached this hypothesis at several levels, including the characterization of the planarian energy metabolism and energy stores, the dependency of proliferation on the diet, and genetic requirements of proliferation control during starvation and feeding. I found that planarians have orthologs of key enzymes of most animal metabolic pathways, but, surprisingly, seem to lack fatty acid synthase. This suggests that planarians are likely not only auxotrophic for cholesterol, but also for fatty acids. I described that planarians store energy as triacylglycerols (TAGs, stored in lipid droplets) and glycogen, with the intestine as the main storage organ. Interestingly, the amount of TAGs and glycogen changes with size and is higher for larger animals, suggesting a regulatory interplay with the known size-dependency of growth/degrowth rates. Further, we demonstrated that the energy stores are the physiological basis of Kleiber’s law that describes the near-universal scaling between metabolic rate and body mass. I further showed that proliferation occurs in three different modes, one during starvation when proliferation is maintained at basal levels and two after feeding, an initial proliferation mode (at three hours after feeding), which is diet independent and a later proliferation (at 24 hours after feeding), which is diet dependent. The two feeding-induced proliferation modes differ not only in their diet-dependencies, but also in their gene expression profiles, as assessed by RNA-sequencing. To identify genes involved in proliferation regulation, I assessed the requirements of different candidate genes in all three proliferation modes in a small-scale RNA interference screen. This screen revealed that insulin signaling, TORC1 and FGFR are involved in regulating basal proliferation during starvation and – most interestingly –that AMP-activated protein kinase (AMPK)-depleted animals showed increased proliferation during starvation at levels characteristic of recently fed animals. This result uncovered AMPK as a modulator that adjusts the neoblast proliferative activity to the nutritional state, potentially independently of TOR. In sum, my work shows how energy metabolism and storage are coordinated with proliferation and growth in planarians and identified AMPK as a central modulator that adjust proliferation to cellular energy states. I discuss potential mechanisms by which AMPK modulates proliferation and putative links between AMPK and cell death, the second process of cell turnover. The energy state as the central mediator of cell turnover and the key players and mechanisms that my work revealed in planarians might also apply across different species:Chapter 1 1. Introduction 1 1.1 Cell turnover is a crucial process for tissue homeostasis 1 1.2 Cell division 2 1.2.1 Control mechanisms of cell division 2 1.2.1.1 Cell cycle machinery 2 1.2.1.2 Organization of the cell cycle control system – cell-cycle intrinsic regulation by Cdk-cyclin complexes 3 1.2.1.3 External control of cell cycle progression 4 1.2.1.4 Metabolic control of cell cycle progression 6 1.2.2 Metabolic requirements of proliferating cells 10 1.2.2.1 The energy stores 11 1.3 Cell death 13 1.4 Suggested mechanisms that coordinate cell death and division and their caveats 14 1.5 Planarians as a model to study cell turnover 16 1.6 Planarian body anatomy 18 1.7 Planarian stem cell system 19 1.7.1 Neoblasts form a heterogeneous population 19 1.7.2 Neoblast proliferative activity 21 1.7.3 Neoblast cell cycle machinery 22 1.7.4 Regulation of neoblast proliferative activity 22 1.8 Cell death in planarians 23 1.9 Mechanisms that coordinate the rate of dividing and dying cells in planarians still remain elusive 24 1.10 Scope of the thesis 24 Chapter 2 2. Planarian energy metabolism and the regulation of planarian growth dynamics 26 2.1 Introduction 26 2.2 Part 1: Planarian energy metabolism 27 2.2.1 The metabolic machinery of S. mediterranea 27 2.2.2 Planarian energy stores 30 2.2.2.1 Visualization of lipid and glycogen storage compartments in planarians 30 2.2.2.2 Investigation of feeding-dependent changes in lipid and glycogen stores 31 2.3 Part 2: Role of planarian organismal energy stores in regulating their growth and degrowth dynamics 36 2.3.1 Background information about known aspects of growth and degrowth dynamics in planarians 36 2.3.1.1 Growth and degrowth arise mainly from changes in cell number 36 2.3.1.2 Growth and degrowth rates are size dependent 37 2.3.2 Energy stores increase disproportionately with size and strongly contribute to the size-dependent dry mass increase 38 2.3.3 Metabolic rate and energy intake are unlikely causes of the size-dependency of the energy stores 41 2.4 Summary and Discussion 43 2.4.1 Part 1: First insights into planarian energy metabolism 43 2.4.1.1 Core planarian metabolic pathways 43 2.4.1.2 Characterization of planarian energy stores 44 2.4.2 Part 2: Implications of size-dependent behavior of planarian energy stores 44 2.4.2.1 Role of energy stores as the physiological origin of Kleiber’s law in planarians 44 2.5 Outlook 46 Chapter 3 3. Towards understanding a systems-level regulation of neoblast proliferative activity 48 3.1 Introduction 48 3.2 Assay development for quantitative determination of proliferating cells 50 3.3 Food quantity and quality affect the later proliferation phase, but not the initial response to feeding 53 3.4 Deep sequencing time course provides insights into gene-expression changes in response to feeding 56 3.5 Discussion 59 3.5.1 Evidence for feeding-induced neoblast regulation at the G0/G1-to-S transition 59 3.5.2 Three distinct modes of neoblast proliferation 59 3.5.3 Early and late proliferation modes show distinct transcriptional profiles 59 3.5.4 Implications from feeding and gene expression profiling experiments 60 3.5.4.1 Potential explanations for diet dependence of the late proliferation mode 60 3.5.4.2 Potential mechanisms of diet-independent early proliferation response 61 3.5.5 Summary and Outlook 61 Chapter 4 4. Towards identifying the mechanisms underlying the regulation of neoblast proliferation 63 4.1 Introduction 63 4.1.1 Chosen gene candidates and their known role in proliferation 64 4.2 RNAi-mediated depletion of candidate genes to test their regulatory role in proliferation 67 4.2.1 Assay design and optimization for the functional RNAi screen 67 4.2.2 Results of small-scale RNAi screen 69 4.3 AMPK - a potential integrator of neoblast proliferation to the nutritional state of the animal 73 4.3.1 AMPK and LKB1 knockdown increases proliferation during starvation 73 4.3.2 AMPK depletion-phenotype of increased proliferation during starvation seems to be TOR independent 73 4.4 Discussion 76 4.4.1 Evidence for a mechanism that regulates basal proliferation during starvation 76 4.4.2 AMPK integrates neoblast activity in response to feeding 77 4.4.2.1 Implications of my observations 77 4.4.2.2 Possible experiments to test the role of AMPK during the regulation of proliferation 78 4.4.3 AMPK potentially regulates proliferation independently of TOR 79 4.4.4 An evolutionarily conserved stem cell switch? 80 4.4.5 Summary and Outlook 80 Chapter 5 5. Discussion and Outlook 81 5.1 Cell-autonomous roles of AMPK in proliferation regulation 83 5.1.1 Independent regulation of ribosomal translation elongation as a potential modulator of neoblast proliferation 83 5.1.2 AMPK might regulate cell cycle progression directly 85 5.1.3 AMPK might regulate symmetric versus asymmetric cell division 85 5.2 Cell non-autonomous roles of AMPK in proliferation regulation 86 5.2.1 AMPK might modulate the release of lipid stores 86 5.3 Possible role of AMPK in regulation of autophagic cell death 87 5.4 AMPK as a potential modulator of cell turnover that couples cell proliferation and cell death to the animal’s energy state 88 5.5 Summary and Outlook 89 Materials and Methods 91 List of Figures 106 List of Tables 107 Acknowledgments 108 References 110

Le suppresseur de tumeur et sérine/thréonine kinase LKB1 est un régulateur clé de la polarité cellulaire et du métabolisme énergétique en partie grâce à l'activation de sa kinase substrat AMPK. Cette protéine est un senseur métabolique pour adapter les apports énergétiques aux besoins nutritionnels des cellules confrontées à un stress. Pour cela, AMPK phosphoryle divers substrats qui activent les réactions cataboliques et inhibent les processus anaboliques dont la kinase mTOR.Au cours de ma thèse, via l’utilisation de modèles murins d’inactivation conditionnelle, j'ai découvert que Lkb1 est crucial pour la formation des cellules de crête neurale (CCN). Ces cellules multipotentes, originaires du tube neural, donnent naissance à divers dérivés, comme les cellules des os et cartilage de la face, les cellules pigmentées de la peau et les cellules gliales et neurales des nerfs périphériques et du système nerveux entérique. J'ai démontré que Lkb1 est essentiel pour la formation de la tête des vertébrés et pour la différenciation et le maintien des dérivés des CCN dans le système nerveux périphérique. J'ai également mis en évidence l’acétylation de LKB1 sur la lysine 48 par l'acétyltransférase GCN5 et son rôle dans l'ontogenèse des CCN céphaliques et la formation de la tête. De plus, j'ai découvert que Lkb1 contrôle la différenciation des cellules gliales en réprimant un programme de biosynthèse d’acides aminés couplé à la transamination du pyruvate en alanine, en amont de la voie de signalisation mTOR.Les phénotypes dus à la perte de Lkb1 dans les CCN récapitulent les caractéristiques cliniques de maladies humaines appelées neurocristopathies. L’activation anormale du suppresseur de tumeur p53 est également associée à certaines neurocristopathies et l’ablation de p53 sauve le phénotype pathologique. Ainsi, j'ai montré que Lkb1 dans les cellules gliales contrôle p53 en limitant les dommages à l’ADN. Lkb1 est aussi essentiel pour maintenir l’homéostasie lysosomale et le recyclage des protéines et ainsi empêcher la formation de granules nommés lipofuscine, chargés en protéines et lipides oxydés. De façon intéressante, les voies mTOR et LKB1/AMPK sont activées à la surface des lysosomes de façon dépendante des niveaux d’acides aminés. Des données récentes de la littérature suggèrent que les lysosomes constitueraient une plateforme de signalisation pour contrôler la protéolyse et le devenir cellulaire. Ainsi, nos données proposent que les signalisations Lkb1 et p53 pourraient réguler l'homéostasie lysosomale et en conséquence le vieillissement cellulaire.De façon intéressante, les cellules de Sertoli, des cellules somatiques épithéliales, localisées dans les tubes séminifères des testicules, et qui régissent la maturation des cellules germinales et l'homéostasie testiculaire, partagent des fonctions métaboliques similaires avec les cellules gliales. En effet, ces cellules sécrètent le lactate et l'alanine qui alimentent les mitochondries des cellules voisines (cellules germinales ou neurones respectivement) contrôlant ainsi leur survie et leur maturation. Au cours de ma thèse, nous avons observé que Lkb1 est requis pour l'homéostasie testiculaire et la spermatogenèse en régulant la polarité des cellules de Sertoli et leur métabolisme énergétique par le cycle pyruvate-alanine. Ces résultats suggèrent une conservation des régulations métaboliques par Lkb1 dans divers tissus.Dans leur ensemble, mes travaux de thèse ont apporté une meilleure connaissance des mécanismes sous-jacents des régulations métaboliques lors du devenir cellulaire. Ces résultats fournissent de nouvelles perspectives sur le développement des CCN et élargissent notre compréhension du contrôle métabolique exercé par LKB1. Enfin, mes projets de doctorat ont mis en évidence l'existence d'une communication entre les voie de signalisation Lkb1 et p53 et suggèrent l’importance de cette communication dans les pathologies humaines dues à des défauts des CCN. / The tumor suppressor LKB1 codes for a serine/threonine kinase. It acts as a key regulator of cell polarity and energy metabolism partly through the activation of the AMP-activated protein kinase (AMPK), a sensor that adapts energy supply to the nutrient demands of cells facing situations of metabolic stress. To achieve metabolic adaptations, AMPK phosphorylates numerous substrates which inhibit anabolic processes while activating catabolic reactions. In particular, AMPK inhibits the mammalian target of rapamycin (mTOR).During my PhD, based on genetically engineered mouse models, I uncovered that Lkb1 signaling is essential for neural crest cells (NCC) formation. NCC are multipotent cells that originate from the neural tube and give rise to various derivatives including bones and cartilage of the face, pigmented cells in the skin and glial and neural cells in peripheral nerves and the enteric nervous system. I demonstrated that Lkb1 is essential for vertebrate head formation and for the differentiation and maintenance of NCC-derivatives in the peripheral nervous system. I also emphasized that LKB1 is acetylated on lysine 48 by the acetyltransferase GCN5 and that this acetylation could regulates cranial NCC ontogeny and head formation. Furthermore, I discovered that Lkb1 controls NCC-derived glial differentiation through metabolic regulations involving amino acid biosynthesis coupled to pyruvate-alanine cycling upstream of mTOR signaling.Phenotypes due to Lkb1 loss in NCC recapitulate clinical features of human disorders called neurocristopathies and therefore suggest that aberrant Lkb1 metabolic signaling underlies the etiology of these pathologies. Abnormal activation of the tumor suppressor p53 has been described in some NCC disorders and p53 inactivation in neurocristopathy mouse models rescues the pathological phenotype. By using a NCC line that can be cultivated as progenitors or differentiated in glial cells in vitro, I demonstrated that Lkb1 expression in NCC-derivatives controls p53 activation by limiting oxidative DNA damage and prevents the formation of lysosomes filled with oxidized proteins and lipids called lipofuscin granules. Interestingly, activation of mTOR and LKB1/AMPK pathways is governed by amino acid sensors and takes place at the lysosome surface. Lysosomes have been proposed as a signaling hub controlling proteolysis and aging. Thus Lkb1 and p53 signaling could converge especially through lysosome homeostasis thereby potentially impacting cellular aging.Strikingly, Sertoli cells, that are epithelial somatic cells, located in seminiferous tubules in testes, and which govern germ cells maturation and whole testis homeostasis, share similar metabolic functions with glial cells. For example, they secrete lactate and alanine to fuel mitochondria of neighboring cells (germ cells or neurons respectively) to control their survival and maturation. During my PhD, we highlighted that Lkb1 is essential for testis homeostasis and spermatogenesis by regulating Sertoli cell polarity and, as observed in glial cells, energy metabolism through pyruvate-alanine cycling. These data suggest that this particular Lkb1 metabolic regulation is conserved in tissues with similar function.Taken together, these studies reveal the underlying molecular mechanisms that coordinately regulate energy metabolism and cell fate. They provide new insights into NCC development and expand our understanding of the role of LKB1 as an energy metabolic regulator. Finally, my PhD projects uncover the existence of a crosstalk between Lkb1 and p53 and underline its importance in NCC disorders.

Invadolysin (inv) is a member of the M8 class of zinc-metalloproteases and is conserved throughout metazoans. It is essential for development and invadolysin homozygous Drosophila mutants are third instar larval lethal. These larvae exhibit a reduced larval brain size and an absence of imaginal discs. Detailed analysis showed that inv mutants exhibit pleiotropic effects, including defects with chromosome architecture, cell cycle progression, spindle assembly, nuclear envelope dynamics, protein turnover and problems with germ cell migration. These findings indicated that Invadolysin must have a critical role in Drosophila. In order to better understand these roles, I set out to identify genetic interactors of invadolysin. I performed a genetic screen scoring for enhancer/suppressor modification of a ‘rough eye’ phenotype induced by invadolysin overexpression. Screening against the Drosdel ‘deficiency kit’ identified numerous genetic interactors including genes linked to energy regulation, glucose and fatty acid pathways. Immunofluorescence experiments in cultured cells showed that H. sapiens Invadolysin localises to the surface of lipid droplets (LD), and subcellular fractionation confirmed its enrichment to these structures. Lipid droplets are highly dynamic organelles involved not only in energy storage but also in protein sequestration, protein and membrane trafficking, and cell signaling. Drosophila fat bodies are enriched in LDs and therefore important energy stores. In addition, they are nutritional sensors and regulators, which are proposed to be the ortholog of vertebrate liver and adipose tissue. Mutant inv fat bodies appeared smaller and thinner than wild type fat body, and accumulated lower levels of triacylgylcerides. This indicated that the loss of invadolysin might be affecting lipid metabolism and storage, confirming the genetic data. However, it was not clear whether these effects were due to the direct action of Invadolysin. Hence, transgenic fly lines expressing either HA, RFP or FLAG tagged forms of Invadolysin were generated to identify physical interactors of Invadolysin. Subsequent mass spectrometry analysis detected ATP synthase-α, -β and -d as interactors. This result suggested that Invadolysin might play a role in regulating mitochondrial function, which might then be manifest in the fat body as the defects previously observed. Energy levels are known to affect the cell cycle, cell growth, lipid metabolism and inevitably development. Further in vivo and in vitro experiments confirmed this hypothesis. Genetic crosses confirmed the interaction of invadolysin with ATP-synthase subunit-α, whilst staining of mitochondria in mutant third instar larval fat bodies suggested decreased mitochondrial activity. Mutants also showed lower ATP levels and an accumulation of reactive oxygen species, hence indicating the possibility of a dysfunctional electron transport chain. Lipid droplets are known to interact with mitochondria, whilst ATP synthase has been found on lipid droplets by proteomic studies in Drosophila. Therefore, based on these data, we propose that Invadolysin is found, with ATP synthase, on lipid droplets, where Invadolysin (likely acting as a protease) could be aiding the normal processing or assembly of ATP synthase. This interaction is vital for the proper functioning of ATP synthase, and hence mitochondria. In this scenario, cellular ATP needs are not met, energy levels drop which results in an inhibition of fatty acid synthesis, cell and organismal growth defects.

<p>Calcium (Ca2+) is a known important second messenger. Calcium/Calmodulin (CaM) dependent protein kinase kinase 2 (CaMKK2) is a crucial kinase in the calcium signaling cascade. Activated by Ca2+/CaM, CaMKK2 can phosphorylate other CaM kinases and AMP-activated protein kinase (AMPK) to regulate cell differentiation, energy balance, metabolism and inflammation. Outside of the brain, CaMKK2 can only be detected in hematopoietic stem cells and progenitors, and in the subsets of mature myeloid cells. CaMKK2 has been noted to facilitate tumor cell proliferation in prostate cancer, breast cancer, and hepatic cancer. However, whethter CaMKK2 impacts the tumor microenvironment especially in hematopoietic malignancies remains unknown. Due to the relevance of myeloid cells in tumor growth, we hypothesized that CaMKK2 has a critical role in the tumor microenvironment, and tested this hyopothesis in murine models of hematological and solid cancer malignancies. </p><p>We found that CaMKK2 ablation in the host suppressed the growth of E.G7 murine lymphoma, Vk*Myc myeloma and E0771 mammary cancer. The selective ablation of CaMKK2 in myeloid cells was sufficient to restrain tumor growth, of which could be reversed by CD8 cell depletion. In the lymphoma microenvironment, ablating CaMKK2 generated less myeloid-derived suppressor cells (MDSCs) in vitro and in vivo. Mechanistically, CaMKK2 deficient dendritic cells showed higher Major Histocompatibility Class II (MHC II) and costimulatory factor expression, higher chemokine and IL-12 secretion when stimulated by LPS, and have higher potent in stimulating T-cell activation. AMPK, an anti-inflammatory kinase, was found as the relevant downstream target of CaMKK2 in dendritic cells. Treatment with CaMKK2 selective inhibitor STO-609 efficiently suppressed E.G7 and E0771 tumor growth, and reshaped the tumor microenvironment by attracting more immunogenic myeloid cells and infiltrated T cells.</p><p>In conclusion, we demonstrate that CaMKK2 expressed in myeloid cells is an important checkpoint in tumor microenvironment. Ablating CaMKK2 suppresses lymphoma growth by promoting myeloid cells development thereby decreasing MDSCs while enhancing the anti-tumor immune response. CaMKK2 inhibition is an innovative strategy for cancer therapy through reprogramming the tumor microenvironment.</p> / Dissertation

Since its discovery, it was appreciated that the antifolate pemetrexed had multiple targets within folate metabolism. This laboratory was instrumental in showing that pemetrexed elicited its primary action as a thymidylate synthase inhibitor. Unusual for an antifolate, pemetrexed showed significant clinical activity against malignant pleural mesothelioma and non-small cell lung cancer. Accordingly, the FDA recently issued first-line approvals for pemetrexed in these diseases, leading us to question whether the effects of pemetrexed on other folate-dependent targets could explain this atypical clinical activity of the drug. Studies in this dissertation showed that in addition to thymidylate synthase inhibition, pemetrexed was also an inhibitor of aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART), the second folate- dependent enzyme of de novo purine synthesis. Consequent of AICART inhibition, pemetrexed caused robust activation of a key energy-sensing regulatory enzyme of the PI3K-AKT signal transduction pathway, AMP-activated protein kinase (AMPK). AMPK activation resulted from xx accumulation of the AMP-mimetic, ZMP, behind the AICART block. Constituents of the PI3K- AKT cascade are frequently deregulated in human carcinomas, uncoupling nutrient supply from proliferative capacity. Therefore, interventions that reinstate control over aberrant signaling along this axis, such as AMPK activation, are of significant cancer therapeutic interest. The cellular consequences of AMPK activation in response to pemetrexed were assessed. In particular, effects on the downstream target of PI3K-AKT signaling, the mammalian target of rapamycin complex 1 (mTORC1), were studied. Unlike targeted mTORC1 inhibitors, such as rapamycin and its analogs, pemetrexed-mediated activation of AMPK also signaled to mTOR- independent controlling elements of protein and lipid synthesis, highlighting additional benefits of AMPK activating agents that extend beyond effects on mTOR signaling. We therefore propose that the unusual activity of pemetrexed in mesothelioma and non-small cell lung cancer is due in part to effects on signaling processes downstream of AMPK activation. These findings present a novel approach to AMPK activation secondary to an AICART block, define pemetrexed as a molecularly targeted agent, and ultimately extend the utility of antifolates beyond their traditional function.

Pemetrexed (PTX), an antifolate drug, has been approved by the US FDA for first line therapy of mesothelioma and non-small cell lung cancer. In addition to its primary site of action on thymidylate synthase (TS), PTX also inhibits the second folate-dependent enzyme of purine biosynthesis aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART). The accumulation of the substrate for AICART, ZMP, in PTX-inhibited cancer cells leads to activation of AMP-activated protein kinase (AMPK) with subsequent inhibition of mammalian target of rapamycin (mTOR) and hypophosphorylation of its downstream targets responsible for protein synthesis and cell proliferation. Inhibitors of mTORC1 like Rapamycin and its analogs (rapalogs) have only partial effects on tumor cells as they do not inhibit mTORC2, which phosphorylates Akt subsequently relieving the inhibition of mTORC1, thus leading to poor cytotoxicity by rapalogs. AMPK exerts control on mTORC1 kinase activity and PTX mediated activation of AMPK leads to its subsequent downregulation and hence, would be expected to have a therapeutic interaction with direct mTOR inhibitors. AZD8055, an ATP-competitive inhibitor of mTOR kinase, potently inhibits both mTORC1 and mTORC2 and therefore, can overcome the feedback mechanism(s) limiting the action of rapalogs to cytostatic effects. To study the effects of AMPK activation and mTOR inhibition pharmacologically, we performed growth suppression assays using pemetrexed, AICAR, RAD001, and AZD8055. The effect of inhibition of mTOR with these drugs was assessed by examining the dephosphorylation of mTORC1 substrates S6K1 and 4E-BP1, as single agents and in combination, at their 50% inhibitory concentrations (IC50) by western blotting. Our data suggested that AMPK activation via PTX mediated AICART inhibition in combination with direct mTOR inhibition by AZD8055 has a synergistic interaction on the proliferation of NSCLC cells in culture. Inhibition of mTOR endogenously by pemetrexed, along with direct pharmacological inhibition of mTOR prevents the feedback circuit which may compromise the therapeutic efficacy of rapamycin analogs. Pemetrexed and AZD8055, as single agents, demonstrated inhibitory activity on phosphorylation events of mTORC1 substrates. This activity was markedly increased by combining both the drugs. Our findings suggest that direct inhibitors of mTOR enhance the effects of activators of AMPK. These effects appear to be mediated via combined effects on mTORC1. Taken together, the combination of catalytic site mTOR inhibitors and pemetrexed is a promising therapeutic strategy and calls for further preclinical and clinical investigations.

The tumor suppressor TP53 is the most frequently altered gene in human cancers. The growth-promoting complex, mTORC1 plays a part of the oncogenic profile caused by dysfunctional p53. mTORC1 sits downstream of AMPK and other crucial tumor suppressors/oncogenes, PTEN, LKB1, and Akt. The antifolate pemetrexed was found by this laboratory to activate AMPK via the inhibition of the enzyme AICART in de novo purine synthesis. This work presents a mechanism of mTORC1 activation with p53 loss, as well as of mTORC1 inhibition by pemetrexed-induced AMPK. We have found that mTORC1 activity was substantially upregulated by the loss or mutation of p53. This activation involves the loss of TSC2 from lysosomal membranes, the site of mTORC1 activation by Rheb. We demonstrate that loss of lysosomal TSC2 increased the levels of lysosomal Rheb. Control of mTORC1 was restored by overexpression of TSC2, which correlated with decreased lysosomal Rheb. Surprisingly, pemetrexed-activated AMPK did not phosphorylate TSC2 because of an accumulation of nonfunctional p53, and a subsequent decrease in TSC2 mRNA. Accordingly, lysosomal TSC2 decreased, however, the levels of lysosomal Rheb decreased. Future studies will question whether the robust Raptor phosphorylation by pemetrexed is involved in this decrease in lysosomal Rheb. AMPK activation by pemetrexed also significantly increased the translocation of AMPK to the nucleus, and we will explore the function of this nuclear AMPK. Overall, these findings present a mechanism involved in the oncogenic signaling of mTORC1 with loss of p53 and offer insight into how pemetrexed reinstates control.

GLUT4 trafficking in muscle cells has been studied to determine how distinct signalling pathways induce GLUT4 translocation. Two different cell models were adopted for these investigations; cardiomyocytes isolated from a transgenic mouse line expressing HA-GLUT4-GFP in muscle and L6 myotubes retrovirally expressing HA-GLUT4. The GLUT4 constructs were largely excluded from the external membrane under basal conditions in both cell models. GLUT4 was trafficked to the external membrane in to response all stimuli studied in cardiomyocytes (insulin, contraction and hypoxia) and L6 myotubes (insulin, AICAR and A-769662). By comparing the anti-HA and GFP signals at the sarcolemma and transverse tubules in cardiomyocytes, it has also be possible to observe an enhancement of GSV fusion with the sarcolemma following stimulation with insulin and contraction. This effect was specific to these stimuli and to the sarcolemma. Insulin-stimulation of GLUT4 exocytosis was not detected under steady-state conditions in L6 myotubes. Here, the major effect of insulin-stimulation and AMPK-activation was on GLUT4 internalisation. The rate constant for GLUT4 internalisation was very rapid in basal cells and was decreased during the steady-state responses to insulin and the AMPK-activators AICAR and A-769662. In cardiomyocytes, internalising GLUT4 colocalised with clathrin at puncta at the sarcolemma. This indicates that GLUT4 is internalised via a clathrin-mediated route. Investigations into the amount of GLUT4 recycling in L6 cells under steady-state conditions revealed that a large proportion of cellular GLUT4 recycles with the cell surface under basal conditions. Insulin-stimulation and AMPK-activation additively mobilised GLUT4 in L6 cells. This implies a non-convergent mobilisation of GLUT4 in response to activation of the PKB/Akt and AMPK signalling pathways. Data obtained from an in vitro kinase assay confirmed that serine 237 of TBC1D1 is a bone fide AMPK phosphorylation site. Furthermore, phosphorylation of this site in L6 myotubes incubated with AMPK activators has been confirmed using a novel antibody specific to TBC1D1 phosphorylated at serine 237. This thesis discusses the consequences and importance of multiple controls impinging on GLUT4 traffic and highlights the advantages and limitations of kinetic studies of these processes.

No description available.

abstract: Objectives: To investigate the potential of vinegar supplementation as a means for reducing visceral fat in healthy overweight and obese adults, and to evaluate its effects on fasting blood glucose and fasting insulin. Subjects and Methods: Forty-five sedentary overweight and obese adult participants with a waist circumference greater than 32 inches for women and 37 inches for men were randomly assigned to one of two groups, the vinegar group (VIN, n=21) or the control group (CON, n=24), and instructed to consume either two tablespoons of liquid red wine vinegar (3.6g acetic acid) or a control pill (0.0225g acetic acid) twice daily at the beginning of a meal for 8 weeks. Participants were also instructed to maintain normal diet and physical activity levels. Anthropometric measures, dual-energy x-ray absorptiometry (DXA) scans, blood samples, and 24-hour dietary recalls were collected at baseline and at end of trial. A compliance calendar was provided for daily tracking of vinegar supplementation. Results: Compliance to vinegar supplementation averaged 92.7 ±13.3% among the VIN group and 89.1 ±18.9% among the CON group. There were no statistically significant differences in anthropometric measurements between baseline and week 8: weight (P=0.694), BMI (P=0.879), and waist circumference (P=0.871). Similarly, DXA scan data did not show significant changes in visceral fat (P=0.339) or total fat (P=0.294) between baseline and week 8. The VIN group had significant reductions in fasting glucose (P=0.003), fasting insulin (P <0.001), and homeostatic model assessment of insulin resistance scores (P <0.001) after treatment. Conclusions: These data do not support the findings from previous studies that indicated a link between vinegar supplementation and increased fat metabolism, specifically visceral fat reduction. / Dissertation/Thesis / Masters Thesis Nutrition 2019