The key metabolic regulator, AMP-activated protein kinase (AMPK) is reported to be downregulated in metabolic disorders, but the mechanisms are poorly characterised. Recent studies have identified phosphorylation of the AMPKα1/α2 catalytic subunit isoforms at Ser487/491 respectively as an inhibitory regulation mechanism. Vascular endothelial growth factor (VEGF) stimulates AMPK and protein kinase B (Akt) in cultured human endothelial cells. As Akt has been demonstrated to be an AMPKα1 Ser487 kinase, the effect of VEGF on inhibitory AMPK phosphorylation in cultured primary human endothelial cells was examined. Stimulation of endothelial cells with VEGF rapidly increased AMPKα1 Ser487 phosphorylation in an Akt-independent manner, without altering AMPKα2 Ser491 phosphorylation. In contrast, VEGF-stimulated AMPKα1 Ser487 phosphorylation was sensitive to inhibitors of protein kinase C (PKC) and PKC activation using phorbol esters or overexpression of PKC stimulated AMPKα1 Ser487 phosphorylation. Purified PKC and Akt both phosphorylated AMPKα1 Ser487 in vitro with similar efficiency. PKC activation was associated with reduced AMPK activity, as inhibition of PKC increased AMPK activity and phorbol esters inhibited AMPK, an effect lost in cells expressing mutant AMPKα1 Ser487Ala. Consistent with a pathophysiological role for this modification, AMPKα1 Ser487 phosphorylation was inversely correlated with insulin sensitivity in human muscle. These data indicate a novel regulatory role of PKC to inhibit AMPKα1 in human cells. As PKC activation is associated with insulin resistance and obesity, PKC may underlie the reduced AMPK activity reported in response to overnutrition in insulin-resistant metabolic and vascular tissues.

Perivascular adipose tissue (PVAT) and vascular endothelial cells both have important structural and functional roles in blood vessels and are the focus of this doctoral thesis. Firstly, PVAT has been rediscovered as an endocrine organ, releasing vasorelaxing substances. Secondly, the endothelial monolayer functions as an important barrier, the role of which is to restrict the transfer of molecules or even blood-borne cells between the lumen of the blood vessel and the surrounding tissue. In my main study, the presence of PVAT caused 'anti-contractile' effects, which were reversed by nitric oxide synthase (NOS) inhibition in rat mesenteric arteries and were lost in adiponectin-knockout mice. The β3 adrenoceptor agonist CL-316,243 increased PVAT-dependent anti-contractile effects and caused myocyte hyperpolarisation. Hyperpolarisation to CL-316,243 could be mimicked by the adipokine, adiponectin, and by the 5'AMP kinase (AMPK) activator, A-769662. In addition, the AMPK inhibitor, dorsomorphin, and the selective BKCa channel blocker, iberiotoxin, each blocked hyperpolarisations to CL-316,243, adiponectin and A-769662. The anti-contractile effects of CL-316,243 could also be mimicked by A-769662 but were not blocked by dorsomorphin. Moreover CL-316,243 still had anti-contractile effects in adiponectin-knockout mice. However, inhibiting the production of both NO and hydrogen peroxide reduced anti-contractile effects of CL-316,243. In obese Sprague Dawley rats both the hyperpolarising and the anti-contractile effects to CL-316,243 were impaired, while hyperpolarisation to A-769662 were unchanged. Western blots revealed that NOS, a possible downstream target of AMPK, was phosphorylated in PVAT control samples, a form which was decreased in PVAT from obese rats. These results collectively indicate that the anti-contractile and hyperpolarising effects observed following stimulation with CL-316,243 are due to activation of different PVAT-dependent pathways, both of which probably contribute to vasodilatation in blood vessels. Understanding these pathways is crucial for the development of improved treatments for obesity and hypertension. During my work at Novartis, I found that activation of sphingosine-1-receptors type 1 (S1P1) with the activator FTY720 (Fingolimod, Novartis; used in multiple sclerosis treatment) caused closure of the endothelial barrier in human umbilical vein cells. This effect could be mimicked with a recombinant peptide of nectin, an adherens junction protein. The novel S1P1 antagonists 'A1' and 'A2' (Novartis) inhibited the effect of FTY720, but not those of nectin. The discovery of nectin as a potential barrier closure modulator might contribute to the development of additional treatments for use in multiple sclerosis.

Yes / Recent clinical trials of the hypoglycaemic sodium-glucose co-transporter-2 (SGLT2) inhibitors, which inhibit renal glucose reabsorption, have reported beneficial cardiovascular outcomes. Whether SGLT2 inhibitors directly affect cardiovascular tissues, however, remains unclear. We have previously reported that the SGLT2 inhibitor canagliflozin activates AMP-activated protein kinase (AMPK) in immortalised cell lines and murine hepatocytes. As AMPK has anti-inflammatory actions in vascular cells, we examined whether SGLT2 inhibitors attenuated inflammatory signalling in cultured human endothelial cells. Incubation with clinically-relevant concentrations of canagliflozin, but not empagliflozin or dapagliflozin activated AMPK and inhibited IL-1β-stimulated adhesion of pro-monocytic U937 cells and secretion of IL-6 and monocyte chemoattractant protein-1 (MCP-1). Inhibition of MCP-1 secretion was attenuated by expression of dominant-negative AMPK and was mimicked by the direct AMPK activator, A769662. Stimulation of cells with either canagliflozin or A769662 had no effect on IL-1β-stimulated cell surface levels of adhesion molecules or nuclear factor-κB signalling. Despite these identical effects of canagliflozin and A769662, IL-1β-stimulated IL-6/MCP-1 mRNA was inhibited by canagliflozin, but not A769662, whereas IL-1β-stimulated c-jun N-terminal kinase phosphorylation was inhibited by A769662, but not canagliflozin. These data indicate that clinically-relevant canagliflozin concentrations directly inhibit endothelial pro-inflammatory chemokine/cytokine secretion by AMPK-dependent and -independent mechanisms without affecting early IL-1β signalling. / Project Grant (PG/13/82/30483 to IPS and TMP) and PhD studentships (FS/16/55/32731 and FS/14/61/31284 to DB and AS) from the British Heart Foundation and an equipment grant (BDA11/0004309 to IPS and TMP) from Diabetes UK. OJK was supported by a Scholarship from the Iraqi Ministry of Higher Education and Scientific Research. TAA was supported by a Libyan Ministry of Education PhD Studentship.

Les mutations germinales activatrices de la voie RAS sont responsables de maladies rares regroupées sous le nom de RASopathies : le Syndrome de Noonan, le Syndrome de Noonan avec de Multiples Lentigines, la Neurofibromatose de type 1, le Syndrome de Malformations Capillaires et Malformations Artério-Veinseuses, le Syndrome Cardio-Facio-Cutané, le Syndrome de Legius et le Syndrome de Costello. Cette thèse s’intéresse au syndrome de Costello causé par une mutation hétérozygote de novo du gène HRAS. Ce syndrome est révélé dans les premiers mois de la vie et se caractérise par un retard de croissance postnatal, des traits du visage épais, un déficit intellectuel, des anomalies cutanées, ainsi qu’une prédisposition à développer des tumeurs. De plus, les patients atteints du syndrome de Costello développent une cardiomyopathie hypertrophique, de l’hypertension, une hypotonie et une myopathie d'origine moléculaire inconnue. En lien avec une association de malade et le service de génétique du CHU de Bordeaux, nous avons mené une exploration des anomalies protéomiques dans les tissus d’une souris modèle du syndrome de Costello ainsi que dans des fibroblastes de patients et des cellules modèles exprimant les formes mutées de HRASG12S et HRASG12A. Cette analyse globale et sans a priori a révélé des altérations au niveau du métabolisme énergétique et plus particulièrement de la composition des mitochondries. Le déficit fonctionnel des mitochondries, centrale énergétique du corps humain, a été caractérisé par des approches de biochimie, de bioénergétique et de biologie cellulaire. De plus, l’analyse des données ‘omiques’ a permis de suggérer une nouvelle hypothèse dans la physiopathologie du syndrome de Costello. Cette hypothèse considère l’implication d’un micro-ARN, le miR-221* dans l’inhibition du métabolisme oxydatif. Les analyses génétiques réalisées sur les cellules de patients et les cellules modèles ont démontré l’inhibition de l’expression de la protéine AMPK, un régulateur majeur du métabolisme mitochondrial, par le miR-221* sous le contrôle de HRASG12S et HRASG12A. Ces découvertes ont permis d’élaborer une stratégie thérapeutique visant à réduire la cardiomyopathie dans le syndrome de Costello. Les analyses précliniques effectuées sur les modèles cellulaires et le modèle murin ont permis d’évaluer l’efficacité d’une stimulation pharmacologique du métabolisme mitochondrial. Cette thèse révèle donc l’implication des mitochondries dans le syndrome de Costello et l’analyse moléculaire réalisée propose une série de données ‘Omiques’ qui permettront de progresser dans la compréhension de cette maladie rare. / Germline activating mutations of the RAS pathway are responsible for rare diseases grouped under the name of RASopathies: Noonan Syndrome, Noonan Syndrome with multiple Lentigines, Type 1-neurofibromatosis, Capillaries malformations and arteriovenous malformations syndrome, Cardio-Facio-Cutaneous Syndrome, Legius Syndrome and Costello Syndrome. This Ph.D thesis focuses on Costello syndrome that is caused by a heterozygous de novo mutation of the HRAS gene. This syndrome is revealed in the first months of life and is characterized by postnatal growth retardation, thick facial features, intellectual deficit, skin abnormalities, and a predisposition to developing tumors. In addition, patients with Costello syndrome develop hypertrophic cardiomyopathy, hypertension, hypotonia and myopathy of unknown molecular origin. In connection with a patients association and the genetics department of Bordeaux University Hospital, we conducted an exploration of proteomic abnormalities in the tissues of a mouse model of the Costello syndrome as well as in patients’ fibroblasts and cell models expressing mutated forms of HRASG12S and HRASG12A. This global and unbiased analysis revealed alterations in energy metabolism and more particularly in the composition of mitochondria. The functional deficiency of mitochondria, energy plants of the human body, has been characterized by biochemistry, bioenergetics and cell biology approaches. In addition, the 'omic' analysis of Costello syndrome suggested a new pathophysiology hypothesis that considered the involvement of a microRNA, miR-221* in the alteration of oxidative metabolism. Functional genetic analyzes performed on patient cells and cell models demonstrated the inhibition of the expression of the major mitochondrial metabolism regulator AMPK protein by miR-221* under the control of HRASG12S and HRASG12A. These findings led to the development of a preclinical therapeutic strategy to reduce cardiomyopathy in Costello syndrome. Preclinical investigations performed on the cellular models and the murine model made it possible to evaluate the efficacy of a pharmacological stimulation of mitochondrial metabolism. This thesis thus reveals the involvement of mitochondria in Costello syndrome and the molecular analysis carried out makes available a series of 'Omics' data that will allow progress in the understanding of this rare disease.

O acúmulo de lipídeos associado à obesidade, resistência à insulina (RI) e diabetes mellitus tipo 2 (DM2) pode levar ao desenvolvimento de danos renais, e diversos mecanismos podem estar envolvidos neste processo, dentre os quais: 1) redução na atividade da proteína ativada por AMP (AMPK); 2) hiperativação do sistema renina angiotensina (SRA) e consequente aumento na produção de angiotensina II (Ang II). O treinamento físico aeróbio (TFA) promove melhora metabólica significativa, no entanto, pouco se sabe sobre os mecanismos celulares induzidos pelo TFA contra o desenvolvimento de danos renais associados com doenças metabólicas. Sendo assim, o objetivo deste estudo foi avaliar o potencial do TFA para a prevenção de danos renais induzidos por dieta de cafeteria, e a participação do SRA e da proteína AMPK nessa resposta. Para isso, camundongos machos adultos C57BL6/J foram separados em grupos (n=13/grupo) sedentários (SED) alimentados com dieta normocalórica (NO) ou de cafeteria (CAF) (SED-NO e SED-CAF, respectivamente) e treinados (TF) alimentados com dieta NO ou CAF (TF-NO e TF-CAF, respectivamente). O TFA foi realizado a 60% da capacidade máxima, simultaneamente com as dietas durante 8 semanas. A dieta de cafeteria causou maior adiposidade, intolerância à glicose e RI no grupo SED-CAF, enquanto o TFA preveniu esses prejuízos no grupo TF-CAF. Os animais SED-CAF apresentaram 88% de aumento no ritmo de filtração glomerular (RFG), maior deposição lipídica renal e redução do espaço de Bowman comparado ao SED-NO, as quais foram prevenidas no grupo TF-CAF. Não houve alteração no conteúdo de colágeno IV e fibronectina, entretanto o TNF-alfa aumentou em ambos os grupos alimentados com dieta de cafeteria. Houve aumento de 27% da expressão proteica da p-AMPK no grupo TF-CAF, sem diferenças na expressão de t-ACC, p-ACC, PGC1-alfa e SIRT-1. A expressão gênica do SREBP-1 não diferiu entre os grupos, porém a expressão do SREBP-2 aumentou nos grupos SED-CAF e TF-CAF comparado aos grupos SED-NO e TF-NO. No soro, apenas a atividade da ECA2 aumentou nos grupos TF-NO e TF-CAF comparados aos sedentários. No rim, a atividade da ECA aumentou 46% no grupo SED-CAF comparado ao SED-NO, e o TFA foi capaz de prevenir esse aumento. No entanto, a Ang II renal aumentou nos grupos SED-CAF, TF-NO e TF-CAF comparados ao grupo SED-NO. Não houve diferença nos componentes do SRA ECA2/Ang 1-7/Mas renal. Em conclusão, o TFA preveniu os danos renais causados pela dieta de cafeteria, tais como acúmulo de lipídeos nos rins, aumento do RFG e redução do espaço de Bowman, e essa resposta está associada, pelo menos em parte, com a maior ativação da AMPK independente da contribuição do SRA / Lipid accumulation observed in the obesity, insulin resistance (IR) and Diabetes Mellitus type 2 (DM2) may lead to the development of renal damage, and several mechanisms may be involved in this process, such as: 1) reduction in the AMP-activated protein (AMPK) activity; 2) hyperactivation of the renin angiotensin system (RAS) and consequent increase in the production of Angiotensin II (Ang II). Aerobic exercise training (AET) promotes significant metabolic improvement, however, little is known about the cellular mechanisms induced by AET against the development of kidney damage associated with metabolic diseases. Thus, the present study aimed to evaluate the potential of AET to prevent kidney damage induced by cafeteria diet, and the participation of RAS and AMPK protein in this response. Adult male C57BL6/J mice were separated into sedentary (SED) groups fed a normocaloric (NO) or cafeteria (CAF) (SED-NO and SED-CAF, respectively) and trained (TF) fed a NO or CAF diet (TF-NO and TF-CAF, respectively). The AET was performed at 60% of the maximum capacity simultaneously with the diets during 8 weeks. The cafeteria diet induced adiposity increase, glucose intolerance and IR, while AET prevented these changes. Animals SED-CAF increased 88% of glomerular filtration rate (GFR), increased renal lipid deposition and reduced Bowman\'s space compared to SED-NO, which were prevented by AET in the TF-CAF group. There was no change in the collagen IV and fibronectin, however TNF-alpha increased in both cafeteria diet fed groups. There was a 27% increase in the protein p-AMPK expression in the TF-CAF group, with no changes in t-ACC, p-ACC, PGC1-alpha and SIRT-1 expression. The SREBP-1 gene expression did not change among groups, but SREBP-2 gene expression increased in the SED-CAF and TF-CAF groups compared to the SED-NO and TF-NO groups. In the serum, only the activity of ACE 2 increased in TF-NO and TF-CAF groups compared to sedentary groups. In the kidney, ACE activity increased 46% in the SED-CAF group compared to SED-NO, nevertheless the AET was able to prevent this increase. Renal Ang II concentration increased in SED-CAF, TF-NO and TF-CAF groups compared to the SED-NO. No differences were observed in the components of renal RAS ACE2/Ang 1-7/Mas. In conclusion, AET prevented the renal damage caused by cafeteria diet, such as lipid accumulation, increased GFR and reduced Bowman space, and these responses are associated, at least in part, with greater activation of the AMPK protein independent of the RAS contribution

O acúmulo de lipídeos associado à obesidade, resistência à insulina (RI) e diabetes mellitus tipo 2 (DM2) pode levar ao desenvolvimento de danos renais, e diversos mecanismos podem estar envolvidos neste processo, dentre os quais: 1) redução na atividade da proteína ativada por AMP (AMPK); 2) hiperativação do sistema renina angiotensina (SRA) e consequente aumento na produção de angiotensina II (Ang II). O treinamento físico aeróbio (TFA) promove melhora metabólica significativa, no entanto, pouco se sabe sobre os mecanismos celulares induzidos pelo TFA contra o desenvolvimento de danos renais associados com doenças metabólicas. Sendo assim, o objetivo deste estudo foi avaliar o potencial do TFA para a prevenção de danos renais induzidos por dieta de cafeteria, e a participação do SRA e da proteína AMPK nessa resposta. Para isso, camundongos machos adultos C57BL6/J foram separados em grupos (n=13/grupo) sedentários (SED) alimentados com dieta normocalórica (NO) ou de cafeteria (CAF) (SED-NO e SED-CAF, respectivamente) e treinados (TF) alimentados com dieta NO ou CAF (TF-NO e TF-CAF, respectivamente). O TFA foi realizado a 60% da capacidade máxima, simultaneamente com as dietas durante 8 semanas. A dieta de cafeteria causou maior adiposidade, intolerância à glicose e RI no grupo SED-CAF, enquanto o TFA preveniu esses prejuízos no grupo TF-CAF. Os animais SED-CAF apresentaram 88% de aumento no ritmo de filtração glomerular (RFG), maior deposição lipídica renal e redução do espaço de Bowman comparado ao SED-NO, as quais foram prevenidas no grupo TF-CAF. Não houve alteração no conteúdo de colágeno IV e fibronectina, entretanto o TNF-alfa aumentou em ambos os grupos alimentados com dieta de cafeteria. Houve aumento de 27% da expressão proteica da p-AMPK no grupo TF-CAF, sem diferenças na expressão de t-ACC, p-ACC, PGC1-alfa e SIRT-1. A expressão gênica do SREBP-1 não diferiu entre os grupos, porém a expressão do SREBP-2 aumentou nos grupos SED-CAF e TF-CAF comparado aos grupos SED-NO e TF-NO. No soro, apenas a atividade da ECA2 aumentou nos grupos TF-NO e TF-CAF comparados aos sedentários. No rim, a atividade da ECA aumentou 46% no grupo SED-CAF comparado ao SED-NO, e o TFA foi capaz de prevenir esse aumento. No entanto, a Ang II renal aumentou nos grupos SED-CAF, TF-NO e TF-CAF comparados ao grupo SED-NO. Não houve diferença nos componentes do SRA ECA2/Ang 1-7/Mas renal. Em conclusão, o TFA preveniu os danos renais causados pela dieta de cafeteria, tais como acúmulo de lipídeos nos rins, aumento do RFG e redução do espaço de Bowman, e essa resposta está associada, pelo menos em parte, com a maior ativação da AMPK independente da contribuição do SRA / Lipid accumulation observed in the obesity, insulin resistance (IR) and Diabetes Mellitus type 2 (DM2) may lead to the development of renal damage, and several mechanisms may be involved in this process, such as: 1) reduction in the AMP-activated protein (AMPK) activity; 2) hyperactivation of the renin angiotensin system (RAS) and consequent increase in the production of Angiotensin II (Ang II). Aerobic exercise training (AET) promotes significant metabolic improvement, however, little is known about the cellular mechanisms induced by AET against the development of kidney damage associated with metabolic diseases. Thus, the present study aimed to evaluate the potential of AET to prevent kidney damage induced by cafeteria diet, and the participation of RAS and AMPK protein in this response. Adult male C57BL6/J mice were separated into sedentary (SED) groups fed a normocaloric (NO) or cafeteria (CAF) (SED-NO and SED-CAF, respectively) and trained (TF) fed a NO or CAF diet (TF-NO and TF-CAF, respectively). The AET was performed at 60% of the maximum capacity simultaneously with the diets during 8 weeks. The cafeteria diet induced adiposity increase, glucose intolerance and IR, while AET prevented these changes. Animals SED-CAF increased 88% of glomerular filtration rate (GFR), increased renal lipid deposition and reduced Bowman\'s space compared to SED-NO, which were prevented by AET in the TF-CAF group. There was no change in the collagen IV and fibronectin, however TNF-alpha increased in both cafeteria diet fed groups. There was a 27% increase in the protein p-AMPK expression in the TF-CAF group, with no changes in t-ACC, p-ACC, PGC1-alpha and SIRT-1 expression. The SREBP-1 gene expression did not change among groups, but SREBP-2 gene expression increased in the SED-CAF and TF-CAF groups compared to the SED-NO and TF-NO groups. In the serum, only the activity of ACE 2 increased in TF-NO and TF-CAF groups compared to sedentary groups. In the kidney, ACE activity increased 46% in the SED-CAF group compared to SED-NO, nevertheless the AET was able to prevent this increase. Renal Ang II concentration increased in SED-CAF, TF-NO and TF-CAF groups compared to the SED-NO. No differences were observed in the components of renal RAS ACE2/Ang 1-7/Mas. In conclusion, AET prevented the renal damage caused by cafeteria diet, such as lipid accumulation, increased GFR and reduced Bowman space, and these responses are associated, at least in part, with greater activation of the AMPK protein independent of the RAS contribution

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

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.