En cas de lymphopénie -diminution du nombre de lymphocytes T (LT) présents en périphérie-, les LT restants prolifèrent. Ce processus, dit de prolifération homéostatique, est régulé par la disponibilité de la cytokine IL-7 ainsi que par des interactions TcR/MHC et mène à la différenciation de LT effecteurs/mémoires. La prolifération homéostatique peut augmenter l’efficacité des immunothérapies anti-cancéreuses ou avoir des conséquences délétères pour l’organisme (réaction du greffon contre l’hôte, auto-immunité). Les LT naïfs, effecteurs et de mémoire produisent leur énergie via des mécanismes différents et la capacité des lymphocytes à enclencher le programme métabolique adéquat au bon moment joue un rôle essentiel dans leur différenciation. En outre, l’inhibition de la glycolyse ou de la respiration mitochondriale altère la prolifération homéostatique, suggérant que ces deux voies métaboliques sont importantes dans ce processus. Les mécanismes par lesquels les LT accroissent leur métabolisme énergétique lors de la prolifération homéostatique ne sont, à ce jour, pas encore élucidés. L’AMPK est un régulateur essentiel du métabolisme cellulaire et est la cible de différents composés déjà utilisés chez l’homme. L’objectif de notre travail a été de déterminer les conséquences de l’absence d’AMPK sur la capacité des LT à proliférer de manière homéostatique et à adapter leur métabolisme en réponse à l’IL-7. Notre hypothèse a été que l’AMPK, en permettant de réorienter le métabolisme des LT, jouerait un rôle important dans le processus de prolifération homéostatique. Au cours de ce travail, nous avons démontré que l’AMPK favorise la prolifération homéostatique des LT ainsi que l’acquisition des fonctions effectrices de type Th1. Nos résultats mettent également en évidence un rôle de l’AMPK dans le processus de graft-versus-host disease (GVHD). En effet, les LT déficients pour l’AMPK induisent une GVHD de moindre gravité. D’un point de vue métabolique, nous avons montré que les LT déficients pour l’AMPK présentent un potentiel de membrane mitochondriale réduit ainsi qu’une sensibilité accrue aux dérivés réactifs de l’oxygène (ROS). Les LT déficients pour l’AMPK ont, de plus, un défaut de switch glycolytique lors de stress mitochondriaux ainsi que lors d’une stimulation secondaire en présence d’anticorps anti-CD3/anti-CD28. Une réduction de la toxicité des ROS, associée à une plus grande flexibilité énergétique, pourrait conférer un avantage prolifératif aux LT soumis à des stimuli antigéniques de faibles affinités, tels que rencontrés lors de proliférations induites par la lymphopénie. Nos résultats suggèrent également qu’une régulation fine du métabolisme pourrait réduire la sévérité de la GVHD. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Orientador: Antonio Carlos Boschero / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T15:59:13Z (GMT). No. of bitstreams: 1 Santos_GustavoJorge_M.pdf: 2550633 bytes, checksum: 914361a57f5efd5794bcee99ff8ce2a4 (MD5) Previous issue date: 2011 / Resumo: Diabetes Mellitus (DM) é uma síndrome metabólica, de etiologia múltipla, caracterizada por hiperglicemia crônica, decorrente da falta de insulina, às vezes, associado à resistência dos tecidos periféricos a esse hormônio. O DM1 é caracterizado pela infiltração de macrófagos e linfócitos do tipo T-CD4+ e T-CD8+ no pâncreas, devido a uma falha do reconhecimento no sistema autoimune, que desencadeiam processos inflamatórios com liberação de óxido nítrico (NO), de radicais livres e de citocinas tais como: interleucina-1? (IL-1 ?) e Interferon- ? (IFN- ?). Essas citocinas pró-inflamatórias ativam mecanismos que levam a morte celular por apoptose, com perda da massa funcional das células beta. Esses mecanismos podem ser reproduzidos in-vitro pela exposição de células beta a essas citocinas ou à Aloxana. O CNTF é uma citocina de sobrevivência neuronal, e em células beta age sobre o controle glicêmico inibindo secreção de insulina e promovendo sobrevivência de ilhotas. A AMPK é uma proteína quinase que em células beta atua como um sensor do estado energético celular e, quando as concentrações intracelulares de ATP diminuem, a AMPK é ativada estimulando geração de ATP e inibindo o consumo desse nucleotídeo. Em ilhotas, a AMPK desempenha função importante na regulação da secreção de insulina sendo que a inibição de AMPK protege as células beta da apoptose mediada por citosinas (IL-1? e IFN-?) ou induzidas por células T do tipo CD8+ e CD4+. Diante do exposto, conclui-se que CNTF e AMPK desempenham funções importantes e correlatas nas células beta pancreáticas e, podem ser considerados alvos terapêuticos para o tratamento do DM tipo I. Contudo, a interação entre esses dois fatores (CNTF e AMPK) em células secretoras de insulina permanece desconhecida. Assim, o objetivo deste trabalho foi investigar a possível papel da interação entre CNTF e AMPK na morte celular induzida pelos agentes diabetogênicos IL-1? e Aloxana. Nossos resultados indicaram que: A Aloxana e a IL-1? dependem da via da AMPK para induzir apoptose em células MIN6; O CNTF modula a via da AMPK em células MIN6 e ilhotas de camundongos Swiss neonato. O CNTF foi capaz de impedir a morte celular induzida por Aloxana e por IL-1? através da downregulation da via da AMPK em células MIN6. / Abstract: Diabetes Mellitus (DM) is a metabolic syndrome of multiple etiologies, resulting from the lack of insulin sometime associated with an increase in the resistance to the hormone by insulin-target tissues. DM1 is characterized by the infiltration of macrophages and T-type CD4 + and T-CD8 + cells in the pancreas, due to a failure of the autoimmune system, causing inflammation and leading to the release of nitric oxide (NO), free radicals, and cytokines such as interleukin-1? (IL-1?) and interferon-? (IFN-?). These pro-inflammatory cytokines activate pro-apoptotic mechanisms, triggering beta cell death apoptosis, leading to a loss in functional beta cell mass. These mechanisms may be reproduced in vitro with exposure of beta cells to pro-inflammatory cytokines such as IL-1? or to Alloxan. The cytokine CNTF is a neuronal survival factor. Besides, CNTF modulates glycemia, inhibiting insulin secretion and promoting islet cells survival. AMPK is a protein kinase that acts on pancreatic beta cells as a sensor of the cellular energy state, and is activated when the cellular ATP concentrations decrease, stimulating ATP generation and inhibiting ATP consumption. In islets, AMPK plays an important role in regulating insulin secretion and inhibition of AMPK protects beta cells from apoptosis mediated by either cytokines (IL-1? and IFN-?) and/or induced T cell CD8 + and CD4 +. AIMS: Given that, both CNTF and AMPK play important role in beta cells and may be used as therapeutic targets for the treatment of DM1. However, the interaction between these two factors (CNTF and AMPK) in pancreatic beta cells remains unknown. Thus, the objective of this work was to investigate the relationship between interaction of AMPK and CNTF on pancreatic beta cell death, induced by Alloxan or IL-1?. Our results indicated that both Alloxan and IL-1? are dependent of AMPK pathway to induce apoptosis in MIN6 cells; CNTF inhibits AMPK pathway in MIN6 cells as well as in islets of newborn Swiss mice; CNTF prevents beta cell death, induced by Alloxan and IL-1?, through downregulation of AMPK pathway in MIN6 cells. / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Orientador: Helena Coutinho Franco de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-19T14:08:09Z (GMT). No. of bitstreams: 1 Kato_LarissaSayuri_M.pdf: 3085711 bytes, checksum: 2c9b592f72e52ceb332be3af215a87fe (MD5) Previous issue date: 2011 / Resumo: O estudo das vias de sinalização envolvidas no metabolismo energético é de grande relevância fisiológica, uma vez que um desequilíbrio da homeostase energética pode resultar em obesidade e/ou síndrome metabólica e aumento da mortalidade por doença cardiovascular. Estudos recentes de nosso grupo em três modelos experimentais que exibem distintos tipos de dislipidemias revelaram alterações significativas da composição corporal, gasto energético e padrão de ingestão alimentar. Neste trabalho estudamos a homeostase energética desses modelos dislipidêmicos avaliando: (1) a expressão e fosforilação da proteína quinase dependente de AMP (AMPK), um importante regulador do metabolismo energético, bem como de seu alvo, a enzima acetil-CoA carboxilase (ACC), em fígado e músculo esquelético de camundongos hipoalfalipoproteinêmicos e hipercolesterolêmicos e (2) o efeito da alimentação pareada em animais hipertrigliceridêmicos que apresentam alterações de comportamento alimentar, metabolismo corporal e maior atividade do canal mitocondrial de potássio sensível ao ATP (mitoKATP). Considerando os animais hipoalfalipoproteinêmicos (transgênicos para CETP), os quais apresentam aumento de gasto energético global, verificamos que estes apresentam redução da massa relativa dos depósitos adiposos quando comparados os controles wild type (WT). O estudo da ativação da AMPK e da ACC mostra que o estado energético dos tecidos muscular e hepático parece não diferir nos animais CETP e WT. Tanto no fígado como no músculo dos animais CETP não houve alteração da massa e do estado de ativação da AMPK e da ACC. Estes resultados sugerem que não ocorrem variações significativas na síntese, armazenamento e "exportação" de lípides no fígado destes animais. Em relação ao músculo sóleo, pode-se concluir que não há alteração de síntese e catabolismo lipídico nos animais CETP. De modo geral, podemos dizer que a sinalização da AMPK não está ativada nestes tecidos e, portanto, que o maior metabolismo corporal observado nestes animais deve estar sendo causado por outros tecidos do organismo, por exemplo, o próprio tecido adiposo. Em animais machos e fêmeas hipercolesterolêmicas (LDLR0) observamos redução da massa corporal, porém sem alteração significativa da massa relativa dos depósitos adiposos quando comparados aos animais controles wild type. Os resultados sobre ativação da AMPK e da ACC mostram que o estado energético em tecidos periféricos é diferente nos animais LDLR0 e controles (WT). No fígado das fêmeas hipercolesterolêmicas observamos aumento da ativação da AMPK sem alteração significativa da fosforilação da ACC. Isso significa que não houve inibição da lipogênese ou ativação da beta-oxidação no fígado dos animais hipercolesterolêmicos, embora possa ter havido aumento de catabolismo de outros nutrientes. No músculo sóleo das fêmeas e dos machos não houve alteração de fosforilação de ambas AMPK e ACC. Pode-se então concluir que não há deprivação energética no músculo destes animais. Considerando o estudo em animais hipertrigliceridêmicos (HTG), quando submetidos ao regime de paired feeding (PF) observamos uma redução de 17% no consumo alimentar nas fêmeas e nos machos HTG quando comparados aos HTG alimentados ad libitum (ad lib). Isso levou a uma redução significativa do ganho de peso dos HTG-PF comparados aos WT-ad lib, em ambos os sexos. Os animais HTG-PF mantiveram a massa dos depósitos adiposos da carcaça semelhantes aos WT-ad lib e HTG-ad lib. No entanto, o depósito adiposo visceral das fêmeas HTG-ad lib é menor que dos WT-ad lib, enquanto nos machos, os HGT-PF apresentaram maior adiposo visceral que os HTG-ad lib. Quando comparados aos WT-ad lib, verificamos que as fêmeas HTG-PF mantiveram aumento significativo da atividade (abertura) do canal de potássio mitocondrial sensível ao ATP (mitoKATP) e da produção corporal de CO2. No entanto, nos machos HTG-PF houve fechamento dos mitoKATP, redução da produção de CO2 e manutenção da massa corporal. Assim, pode-se inferir que o metabolismo corporal (produção de CO2) reflete o aumento do metabolismo celular causado por aumento da atividade do mitoKATP que desacopla levemente as mitocôndrias e que estas adaptações são revertidas pela restrição alimentar nos machos, mas não nas fêmeas HTG / Abstract: An imbalance of energy homeostasis can result in obesity and/or metabolic syndrome and increased mortality from cardiovascular disease. Recent studies by our group in three experimental models that exhibit different types of dyslipidemia have shown significant changes in body composition, energy expenditure and food intake. In this work we studied the energy homeostasis in these models through: (1) quantifying the expression and phosphorylation of AMP-dependent protein kinase (AMPK), a key regulator of energy metabolism, as well as its target, the enzyme acetyl-CoA carboxylase (ACC) in liver and skeletal muscle in hypoalphalipoproteinemic and hypercholesterolemic mice and (2) the effect of paired feeding regimen on hypertriglyceridemic mice that present increased food intake, body CO2 production and increased activity of the mitochondrial potassium channel sensitive to ATP (mitoKATP). Considering the hypoalphalipoproteinemic mice (transgenic for CETP), which show an increased overall energy expenditure, we found that these mice have reduced relative fat depot mass when compared to wild type controls (WT). Western blot analyses showed that, in both tissues, liver and muscle, there were no changes in mass and state of activation of AMPK and ACC in CETP compared to WT mice. These results suggest that no significant variations in the synthesis, storage and secretion of lipids in the liver of these mice. Regarding the soleus muscle, these results suggest that there is no change in lipid synthesis and catabolism in CETP mice. Overall we may say that AMPK signaling is not activated in liver and muscle tissues and, therefore, that the increased body metabolism observed in these CETP mice must be caused by other body tissues, for example, the adipose tissue itself. In hypercholesterolemic male and female mice (LDLR0) we observed a reduction in body mass, but no significant change in the relative mass of fat depots when compared to WT. The results on activation of AMPK and ACC show that the liver of LDLR0 females had increased activation of AMPK without significant change in the phosphorylation of ACC. This means that there was no inhibition of lipogenesis and activation of ?-oxidation in the liver of hypercholesterolemic mice, although there may have been increased catabolism of other nutrients. In the soleus muscle of females and males there were no changes in the phosphorylation state of both AMPK and ACC. Then, we can conclude that there is no energy deprivation in the muscle of these LDLR0 mice. Considering the study with hypertriglyceridemic (HTG) mice, when subjected to the paired feeding (PF) we observed a 17% reduction in food intake of females and males when compared to HTG mice fed ad libitum (ad lib). This led to a significant reduction in HTG-PF weight gain compared to WT-ad lib in both sexes. HTG-PF mice retained the mass of carcass fat deposits similar to WT and HTG ad lib. Compared to WT-ad lib, HTG-PF mice maintained significant increased activity (opening) of the mitoKATP and body CO2 production. These data showed that the regimen of paired feeding in which HTG mice were submitted did not change the high rate body metabolism and mitochondrial resting respiration observed in HTG-ad lib mice. These results suggest that the metabolic adaptation of HTG (higher activity of mitoKATP) is not sensitive to changes in food restriction and compromises the rate of body growth / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Rapidly growing tumor cells outgrow their blood supply resulting in a microenvironment with reduced oxygen and nutrients. Using an in vitro transformation model we found that cancer cells expressing the SV40 ST antigen (+ST cells) are more resistant to glucose deprivation-induced cell death than cells lacking the SV40 ST antigen (−ST cells). Mechanistically, we found that the ST antigen mediates this effect by activating a nutrient-sensing kinase, AMP-activated protein kinase (AMPK). We further show that AMPK mediates its effects, at least in part, by inhibiting mTOR (mammalian target of rapamycin), thereby shutting down protein translation, and by inducing autophagy as an alternate energy source. Resistance to anoikis upon anchorage-deprivation is yet another form of stress tolerated by both normal stem/progenitor cells of various tissues in our body and by cancer cells. Using mammospheres as a model to enrich for stem/progenitor cells we found that mammosphere formation is accompanied with increased activation of AMPK. Concomitant with AMPK activation, we detected increased phosphorylation of the anti-apoptotic protein PED/PEA15. We further demonstrate that AMPK directly interacts with and phosphorylates PEA15 at Ser116, thus establishing PEA15 as a new AMPK target. Thus, our study has identified AMPK-PEA15 signaling as a key component of sphere formation by both normal and cancerous breast tissues. During metastasis, epithelial cells lose attachments to their neighbors, acquire a mesenchymal-like morphology, a process termed as epithelial-mesenchymal transition (EMT) and become motile. Our results indicate that AMPK regulates EMT by both transcriptional and post-translational modification of EMT-inducing transcription factor, Twist. Thus, our study has identified a role for AMPK in nutrient deprivation, anchorage-independent growth, and epithelial-mesenchymal transition involved in metastasis. In addition, we have identified two novel substrates of AMPK, PEA15 and Twist, that may play key roles in cancer progression. Thus, our study suggests that targeting AMPK, or its newly identified substrates, can be explored as possible anti-cancer mechanisms.

AMP-activated protein kinase (AMPK) is an important metabolic sensor in eukaryotic organisms and it plays an important role in regulating energy homeostasis, at both the cells and the whole organism. AMPK controls glucose and lipid metabolism by direct stimulation of enzymes or by long term stimulation of the gene expression of energy metabolism. Skeletal muscles significantly contribute to the total body weight and metabolic rate and to the maintenance of glucose homeostasis. Due to the ability of the muscle to increase energy expenditure to 95% of whole-body energy expenditure, could be the proper development and programming of metabolism in the early postnatal period crucial for the further development of the organism in adulthood. Early postnatal development leads to substantial changes in energy requirements of the body and this suggests the significant involvement of AMPK in this period. The aim of this thesis was to study the activity and expression of isoforms of the catalytic subunit of AMPK in skeletal muscle during early postnatal development of both mouse strains A/J and C57BL/6 that differ in the development of diet-induced obesity. The next task was to analyze the expression of selected genes involved in energy metabolism - GLUT4, PGC-1α and UCP3 that AMPK regulates. It was found that the...

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

The adrenal cortex is a crucial endocrine gland in the mammalian stress response. In chronic inflammatory stress, cortisol is elevated whereas adrenal androgens are decreased. Furthermore, ACTH levels have poor correlation with the plasma cortisol in these conditions, thus suggesting that other factors are driving the stress response during chronic inflammatory stress. Interleukin-6 (IL-6), a cytokine which is released during chronic inflammatory stress, is assumed to be one such factor. Thus the biochemical pathways by which IL-6 increases cortisol release from the zona fasciculata (ZF), and decreases adrenal androgen release from the zona reticularis (ZR) were investigated. Since IL-6 activates AMP-activated kinase (AMPK) in skeletal muscle, AMPK was investigated for IL-6- induced effects in ZF and ZR tissue. The effects of AMPK activation and IL-6 exposure on the expression of the steroidogenic proteins, steroidogenic acute regulatory protein (StAR) and cholesterol side chain cleavage enzyme (P450scc), and on the steroidogenic nuclear factors steroidogenic factor-1 (SF-1) and adrenal hypoplasia congenita, critical region on the X chromosome, gene-1 (DAX-1) were investigated. AMPK activation and IL-6 exposure increased the expression of StAR, P450scc, and SF-1, and decreased DAX-1 in the ZF. Meanwhile, AMPK activation and IL-6 exposure decreased the expression of StAR, P450scc, and SF-1, and increased DAX-1 in the ZR. AMPK inhibition blocked the effects of AMPK activation and IL-6 on the ZF and ZR. Activator Protein-1 (AP-1) was the second biochemical intermediate studied since in other tissues AMPK activation increases the expression and phosphorylation of AP-1 subunits. IL-6 stimulation and AMPK activation increased the expression of the AP-1 subunits cFOS, cJUN, JUN B, and JUN D, while increasing the phosphorylation of cJUN in both the ZF and the ZR. These effects were blocked by AMPK inhibition. Inhibition of AP-1 leads to decreased StAR, P450scc, and SF-1, and increased DAX-1 in the ZF. Meanwhile, AP-1 inhibition leads to increased StAR, P450scc, SF-1, and decreased DAX-1 in the ZR. Therefore the AP-1 complex functions as a biochemical intermediate in the IL-6 and AMPK regulation of steroidogenic enzymes in the ZF and ZR. Overall, the results suggest that IL-6 activates AMPK, which increases the expression and phosphorylation of AP-1 subunits in the ZF and the ZR. However, increased AP-1 activation leads to increased StAR and P450scc in the ZF, but decreased StAR and P450scc in the ZR.

Yes / AMP-activated protein kinase (AMPK) is a pivotal regulator of metabolism at the cellular and organismal levels. AMPK also suppresses inflammation. We found that pharmacological activation of AMPK rapidly inhibited the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway in various cells. In vitro kinase assays revealed that AMPK directly phosphorylated two residues (Ser515 and Ser518) within the SH2 domain of JAK1. Activation of AMPK enhanced the interaction between JAK1 and 14-3-3 proteins in cultured vascular endothelial cells and fibroblasts, an effect which required the presence of Ser515 and Ser518 and was abolished in cells lacking AMPK catalytic subunits. Mutation of Ser515 and Ser518 abolished AMPKmediated inhibition of JAK-STAT signaling stimulated either by the sIL-6Rα/IL-6 complex or by expression of a constitutively active V658F-mutant JAK1 in human fibrosarcoma cells. Clinically used AMPK activators metformin and salicylate enhanced the inhibitory phosphorylation of endogenous JAK1 and inhibited STAT3 phosphorylation in primary vascular endothelial cells. Therefore our findings reveal a mechanism by which JAK1 function and inflammatory signaling may be suppressed in response to metabolic stress and provide a mechanistic rationale for the investigation of AMPK activators in a range of diseases associated with enhanced activation of the JAK-STAT pathway.

Insulin resistance is an early marker of Type II diabetes. Excessive lipid accumulation in muscle and liver leads to insulin resistance, and lowering tissue lipids causes an enhancement of insulin action. The enzyme AMP-activated protein kinase (AMPK) is activated when cellular energy levels are compromised, such as during exercise; this enhances fuel oxidation and inhibits energy consuming processes. The hypothesis in this thesis was that activating AMPK in a lipid-induced insulin resistant state leads to tissue lipid reduction and improved insulin sensitivity. Insulin resistant high-fat fed (HF-) rats were administered 5-aminoimidazole-4-carboxamide-1-??-D-ribofuranoside (AICAR), a specific AMPK activator. During an euglycaemic hyperinsulinaemic clamp performed 24h later, HF-rats showed increased whole body, muscle and liver insulin action, independent of changes in PKB-phosphorylation. The liver had reduced triglycerides, malonyl-CoA and increased IkB-a content. A lowering of muscle malonyl-CoA was consistent with conditions favouring increased lipid utilisation. Normal, chow-fed rats also showed improved insulin action post-AICAR. Further studies showed that basal glucose uptake was not increased 24h after AICAR, suggesting that AMPK activation had caused an increase in insulin sensitivity. Diacylglycerols and triglycerides, but not ceramides, were reduced in the liver of AICAR treated HF-rats, suggesting lipid reduction as a likely mediator of enhanced liver insulin action. These lipid species were not reduced in muscle. AICAR administration to HF-rats lowered plasma glucose and fatty acids (FA) acutely, probably due to increased muscle glucose uptake and FA oxidation. Glycogen was reduced in liver and increased in muscle, suggesting glucose mobilisation from liver to muscle. Adrenergic blockade excluded the sympathetic nervous system in the acute AICAR effects. AMPK was activated in white muscle and liver of HF-rats immediately after AICAR, the same tissues that exhibited later improved insulin sensitivity. Tracer technologies used to investigate glucose and lipid fluxes showed that AMPK activation in white muscle simultaneously increased both glucose and FA uptake and their metabolism, with glucose also being stored as glycogen. The liver showed lower lipid synthesis, consistent with reduced liver lipid accumulation observed 24h post-AICAR. In conclusion, these results suggest that activation of AMPK leads to selective tissue lipid reduction and improved insulin action, and is a potential target for the treatment of insulin resistance and type II diabetes.

La protéine kinase activée par AMP (AMPK) est un senseur et régulateur central de l'état énergétique cellulaire, mais ces voies de signalisation ne sont pour le moment que partiellement comprises. Deux criblages non-biaisés pour la recherche de partenaires d'interaction et de substrats d'AMPK ont précédemment été réalisés dans le laboratoire. Ces derniers ont permis l'identification de plusieurs candidats (protéines), mais leur rôle fonctionnel et physiologique n'était pas encore établi. Ici nous avons caractérisé la fonction de la relation entre AMPK et quatre partenaires d'interaction : gluthation S-transferases (GSTP1 and GSTM1), fumarate hydratase (FH), l'E3 ubiquitine-ligase (NRDP1), et les protéines associées à la membrane (VAMP2 and VAMP3). Chacune de ces interactions parait avoir un rôle différent dans la signalisation AMPK, agissant en amont ou en aval de la protéine AMPK. GSTP1 et GSTM1 contribueraient à l'activation d'AMPK en facilitant la S-glutathionylation d'AMPK en conditions oxydatives moyennes. Cette régulation non-canonique suggère que l'AMPK peut être un senseur de l'état redox cellulaire. FH mitochondrial est l'unique substrat AMPK clairement identifié. Etonnamment le site de phosphorylation se trouve dans le peptide signal mitochondrial, ce qui pourrait affecter l'import mitochondrial. NRDP1, protéine pour laquelle nous avons pour la première fois développé un protocole de production de la protéine soluble, est faiblement phosphorylée par l'AMPK. L'interaction ne sert pas à l'ubiquitination d'AMPK, mais affecte le renouvellement de NRDP1. Finalement, l'interaction de VAMP2/3 avec AMPK n'implique pas d'évènement de phosphorylation ou d'activation d'un des partenaires. Nous proposons un mécanisme de recrutement d'AMPK par VAMP2/3 (" scaffold ") au niveau des vésicules en exocytose. Ce recrutement favoriserait la phosphorylation de substrats de l'AMPK à la surface des vésicules en exocytoses. Une fois mis en commun, nos résultats enrichissent les connaissances sur les voies de signalisation AMPK, et suggèrent une grande complexité de ces dernières. Plus que les kinases en amont et des substrats en aval, la régulation de la signalisation d'AMPK se fait via des modifications secondaires autres que la phosphorylation, via des effets sur le renouvellement de protéines, et probablement via un recrutement spécifique de l'AMPK dans certains compartiments cellulaires.