Dans le cadre des recherches menées au sein du laboratoire de Physiologie Animale le gène codant pour la nicotinamide phosphorybosyltransférase (NAmPT) à été identifié et cloné. Au cours de ce travail, nous avons étudié le rôle de cette enzyme du métabolisme du Nicotinamide Adénine Dinucléotide ainsi que celui des enzymes dépendantes du NAD (PARP et sirtuines) dans le contrôle de la réponse inflammatoire.

Non-alcoholic fatty liver disease (NAFLD) is a major chronic liver disease and thus a main reason for liver-related morbidities and mortality. NAFLD covers a wide range of diseases starting with steatosis and frequently progressing to non-alcoholic steatohepatitis (NASH), which is an independent predictor for the development of the hepatocellular carcinoma (HCC). Nicotinamide phosphoribosyltransferase (NAMPT), the key enzyme of the mammalian NAD salvage pathway, recycles nicotinamide to nicotinamide mononucleotide (NMN), which is further converted to nicotinamide adenine dinucleotide (NAD). NAD is not only an important redox partner but also a crucial co-substrate for NAD-dependent enzymes such as sirtuin 1 (SIRT1). Thus, NAD metabolism might be involved in the progression of NAFLD by regulating many cellular processes, such as apoptosis, de novo lipogenesis, glycolysis and gluconeogenesis, in the liver. Interestingly, tumor cells have a high NAD turnover due to their rapid proliferation and high activity of NAD-dependent enzymes. For these reasons, I hypothesized that the NAD salvage pathway is dysregulated during the progression of non-alcoholic fatty liver disease. Therefore, the first study of the present work deals with the role of the NAD salvage pathway in a diet-induced mouse model of hepatic steatosis. In mice fed a high-fat diet for 11 weeks hepatic NAMPT mRNA, protein abundance and activity as well as NAD levels were increased. Additionally, SIRT1 protein abundance was upregulated indicating a higher SIRT1 activity. This could be confirmed by detecting decreased acetylation or transcription of SIRT1 targets. For example, p53 and nuclear factor κB (NF-κB) were less acetylated demonstrating lower activity of key regulators of apoptosis and inflammation, respectively. In the second study of this thesis NAMPT activity was inhibited by applying its specific inhibitor FK866 in hepatocarcinoma cells to investigate whether or not NAMPT inhibition could be a potential novel therapeutic approach in HCC treatment. Hepatocarcinoma cells were more sensitive to NAMPT inhibition by FK866 than primary human hepatocytes, presenting a high number of apoptotic cells after FK866 treatment. FK866 induced NAD and ATP depletion which was associated with activation of the key regulator of energy metabolism 5’-AMP-activated protein kinase (AMPK) and decreased activity of its downstream target mammalian target of rapamycin (mTOR). This thesis shows that the NAD salvage pathway is involved in hepatic steatosis and HCC. During hepatic steatosis NAD metabolism is upregulated to potentially protect against adverse effects of the massive hepatic lipid accumulation. To repress the progression to NASH it might be useful to maintain the hepatic NAD levels during early disease stages by administration of NAD precursors, such as NMN. However, hepatocarcinoma cells have a higher activity of NAMPT and NAD-dependent enzymes. NAMPT inhibition by FK866 could be a potential therapeutic approach in HCC, especially due to the fact that NAD depletion is selectively induced in hepatocarcinoma cells, but not in primary human hepatocytes.

Could NAMPT inhibition become a potential treatment option in hepatocellular carcinoma?

Nicotinamide adenine dinucleotide (NAD) is a key regulator of several metabolic and signaling pathways that are relevant in cancer cell survival. Cancer cells have an increased energy demand associated with an increased NAD turnover. Nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme of the NAD salvage pathway, plays a crucial role in maintaining the intracellular NAD levels and in regulating the activity of NAD-dependent enzymes, such as sirtuins (SIRTs). The inhibition of NAMPT activity and the use of phytochemicals, such as resveratrol, represent novel therapeutic approaches in cancer therapy. Based on these facts, this thesis aimed to investigate (1) the chemotherapeutic potential and molecular mechanisms of FK866, a specific NAMPT inhibitor, and resveratrol on hepatocarcinoma cells and to find out whether there are differences compared to primary human hepatocytes; (2) to address the impact of NAMPT inhibition on the energy metabolism in cancer cells; and (3) to investigate the roles of NAMPT and SIRT1 in resveratrol´s mode of action and chemotherapeutic effects. This work demonstrates that FK866 and resveratrol possess potent chemotherapeutic effects in hepatocarcinoma cells which were absent in human hepatocytes. Hepatocarcinoma cells display a dysregulation in the AMP-activated kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling as well as in the NAMPT-mediated NAD salvage pathway compared to human hepatocytes. FK866-induced NAMPT inhibition induces ATP depletion associated with AMPK activation and mTOR inhibition whereas resveratrol induces caspase3-mediated apoptosis that is not dependent on NAMPT and SIRT1 function. NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and human hepatocytes. This work also reveals that resveratrol activates p53-induced cell cycle arrest in hepatocarcinoma cells which is partly mediated by SIRT1 inhibition. In summary, this thesis provides new insight into the role of the NAMPT-mediated NAD salvage pathway in energy metabolism and characterized FK866 and resveratrol as promising potential chemotherapeutic agents for treatment of hepatocellular carcinoma.

Non-alcoholic fatty liver disease (NAFLD) is a major chronic liver disease and thus a main reason for liver-related morbidities and mortality. NAFLD covers a wide range of diseases starting with steatosis and frequently progressing to non-alcoholic steatohepatitis (NASH), which is an independent predictor for the development of the hepatocellular carcinoma (HCC). Nicotinamide phosphoribosyltransferase (NAMPT), the key enzyme of the mammalian NAD salvage pathway, recycles nicotinamide to nicotinamide mononucleotide (NMN), which is further converted to nicotinamide adenine dinucleotide (NAD). NAD is not only an important redox partner but also a crucial co-substrate for NAD-dependent enzymes such as sirtuin 1 (SIRT1). Thus, NAD metabolism might be involved in the progression of NAFLD by regulating many cellular processes, such as apoptosis, de novo lipogenesis, glycolysis and gluconeogenesis, in the liver. Interestingly, tumor cells have a high NAD turnover due to their rapid proliferation and high activity of NAD-dependent enzymes. For these reasons, I hypothesized that the NAD salvage pathway is dysregulated during the progression of non-alcoholic fatty liver disease. Therefore, the first study of the present work deals with the role of the NAD salvage pathway in a diet-induced mouse model of hepatic steatosis. In mice fed a high-fat diet for 11 weeks hepatic NAMPT mRNA, protein abundance and activity as well as NAD levels were increased. Additionally, SIRT1 protein abundance was upregulated indicating a higher SIRT1 activity. This could be confirmed by detecting decreased acetylation or transcription of SIRT1 targets. For example, p53 and nuclear factor κB (NF-κB) were less acetylated demonstrating lower activity of key regulators of apoptosis and inflammation, respectively. In the second study of this thesis NAMPT activity was inhibited by applying its specific inhibitor FK866 in hepatocarcinoma cells to investigate whether or not NAMPT inhibition could be a potential novel therapeutic approach in HCC treatment. Hepatocarcinoma cells were more sensitive to NAMPT inhibition by FK866 than primary human hepatocytes, presenting a high number of apoptotic cells after FK866 treatment. FK866 induced NAD and ATP depletion which was associated with activation of the key regulator of energy metabolism 5’-AMP-activated protein kinase (AMPK) and decreased activity of its downstream target mammalian target of rapamycin (mTOR). This thesis shows that the NAD salvage pathway is involved in hepatic steatosis and HCC. During hepatic steatosis NAD metabolism is upregulated to potentially protect against adverse effects of the massive hepatic lipid accumulation. To repress the progression to NASH it might be useful to maintain the hepatic NAD levels during early disease stages by administration of NAD precursors, such as NMN. However, hepatocarcinoma cells have a higher activity of NAMPT and NAD-dependent enzymes. NAMPT inhibition by FK866 could be a potential therapeutic approach in HCC, especially due to the fact that NAD depletion is selectively induced in hepatocarcinoma cells, but not in primary human hepatocytes.

Le développement d’agents anticancéreux s’étend de la découverte d’une cible thérapeutique à la commercialisation du médicament. La chimie médicinale intervient dans la conception et la synthèse de molécules. Les essais réalisés in vitro puis in vivo sont ensuite déterminants pour évaluer le mode d’action, l’efficacité et la sélectivité du principe actif. À cette étape, si une molécule n’est pas sélectionnée, son étude s’arrête. Afin de tirer profit du temps et de l’argent investis dans son développement, des stratégies s’inspirant de ces composés peuvent être utilisées, en modifiant leur structure afin d’améliorer leur efficacité. La première méthode exploitée ici consiste à synthétiser des molécules bifonctionnelles, incorporant le pharmacophore du principe actif d’intérêt, ainsi qu’un ligand capable d’induire la dégradation de sa cible. Ces molécules sont appelées AUTAC ou PROTAC, selon le mode de dégradation induit, soit l’autophagie ou la protéolyse. La deuxième méthode repose sur le développement de molécules hybrides, capables d’interagir avec différentes cibles. Deux principes actifs sont alors liés de manière covalente afin de synthétiser une nouvelle molécule dont les propriétés pharmacocinétiques diffèrent. L’hybride pourrait être ainsi plus efficace, cumulant les activités de ses deux ligands de base. Pour viser les cellules cancéreuses, une stratégie attrayante est le ciblage de certaines voies métaboliques. L’ATP étant une source d’énergie essentielle pour l’intense prolifération des cellules cancéreuses, réduire sa production en ciblant la phosphorylation oxydative dans la mitochondrie et la glycolyse peut être un traitement efficace. Les dérivés de biguanides, comme la metformine, sont connus pour inhiber la phosphorylation oxydative. Par ailleurs, le NAD+ étant un cofacteur nécessaire à de nombreux processus biologiques essentiels, notamment la glycolyse, diminuer sa production peut alors induire des effets anticancéreux. De nouveaux dérivés de biguanides et d’inhibiteurs de la NAMPT sont donc synthétisés afin de développer des molécules bifonctionnelles ciblant le métabolisme des cellules cancéreuses. / The development of new anticancer agents covers all the steps from the discovery of a target to the commercialisation of a drug. Medicinal chemists are responsible for the design and synthesis of molecules but the future of the drug relies on their activity in vitro and in vivo. However, compounds that failed because of their lack of efficiency or selectivity can be reused to take advantage of the time and money invested on their development. To develop more potent drugs, these compounds can be modified using different strategies. One of them consists in synthesising bifunctional molecules, also called chimeras, incorporating a pharmacophoric moiety from the drug and a ligand inducing its target’s degradation. These molecular degraders are named according to the mode of degradation involved: AUTAC for Autophagy Targeting Chimera or PROTAC for Proteolysis Targeting Chimera. Another strategy is to design a dual targeting agent based on two different drugs bound covalently. The resulting hybrid is then able to interact with each ligand’s target but exhibits a new pharmacokinetic profile that might increase its efficiency. Targeting cancer cell metabolism is an attractive strategy that can be applied to many different types of cancer. Since ATP is the main energy source of the cells, its production is necessary to insure the intense proliferation rate of cancer cells. Targeting the oxidative phosphorylation in mitochondria and the glycolysis, the two main ways to produce ATP, can be effective to inhibit the proliferation of cancer cells. Biguanides derivatives such as metformin are known to inhibit OXPHOS. Furthermore, NAD+ being an essential cofactor involved in many biological processes including glycolysis, targeting its production by the NAD salvage pathway allows the discovery of effective anticancer agents. New biguanides and NAMPT inhibitors derivatives are thus synthesized for the development of bifunctional molecules targeting the energy metabolism of cancer cells.

Alors que les phases de résorption et de couplage du cycle de remodelage de l’os sont de plus en plus connues et ont permis le développement d’agents thérapeutiques, la phase d’activation reste peu étudiée. L’objectif global de ce travail est d’analyser les évènements cellulaires mis en jeu au cours de la phase d’activation du remodelage de l’os. Les objectifs spécifiques ont été 1- de caractériser le trafic cellulaire dans le périoste au cours de la phase d’activation du remodelage et 2- d’étudier le rôle d’une enzyme, la nicotinamide phosphorybosyl transférase (Nampt) dans ces évènements. Dans notre premier travail, nous montrons dans un modèle de remodelage synchronisé de l’os alvéolaire, une expression précoce de ICAM-1 par les vaisseaux qui serait impliquée dans la diapédèse observée de monocyte-macrophages CD68+. Ces cellules migreraient à travers le compartiment non ostéogénique puis ostéogénique, guidées par des cellules de type fibroblastes puis des OB exprimant VCAM-1. Le nombre des cellules RANKL+ dans le compartiment ostéogénique augmente graduellement lors de la phase d’activation. En parallèle, l’expression de la sémaphorine 3a, qui inhibe l’ostéoclastogénèse, diminue chez les OB et les ostéocytes superficiels. Dans notre second travail, nous trouvons que l’expression basale de la Nampt est accrue dans les cellules de la couche ostéogénique au cours de la phase d’activation du remodelage. Inhiber son activité via le FK866 permet de diminuer l’ostéoclastogenèse indiquant que la Nampt serait impliquée dans le recrutement et l’activité des OC. En culture primaire d’ostéoblastes murins, nous montrons que son expression augmente au cours de la différentiation et qu’elle régule l’expression de marqueurs tardifs de différentiation. L’ensemble de ces données montre une série d’évènements coordonnés qui servent au recrutement des précurseurs ostéoclastiques et à leur migration vers la surface osseuse à résorber. La Nampt semble jouer un rôle dans l’acquisition des ostéoblastes d’un phénotype favorable à ces évènements. / Resorption and inversion phases of bone remodeling are well understood, which have permitted the development of therapeutic agents. At the opposite, activation phase remains poorly characterized. This work aims to analyze cellular events involved in the activation phase of bone remodeling. Specific goals were: 1- To characterize cellular traffic in the periosteum during the activation phase of bone remodeling. 2- To study the role of NicotinAMide Phosphorybosyl Transférase (NAMPT) enzyme during activation. In the first study, we show an early expression of ICAM-1 by vessels in a synchronized alveolar-bone-remodeling model. The ICAM-1 expression may be involved in the observed diapedesis of monocytes – macrophages CD68+. These cells migrate through non osteogenic and osteogenic layers, steered by fibroblast-like cells and then by VCAM+ osteoblasts (OB). The number of RANKL+ cells in osteogenic layer gradually increases during the activation phase. Simultaneously, the expression of semaphorine 3a inhibiting osteoclastogenesis, decreases in osteoblasts and superficial osteocytes. In the second study, we show that basal expression of NAMPT increases in osteogenic-layer cells during the activation phase of bone remodeling. Inhibiting its activity with FK866 enhables to decrease osteoclastogenesis, suggesting an involvement of NAMPT in osteoclast recruitment and activity. In primary culture of murine OB, we show that NAMPT expression increases during differentiation. It also regulates OB late-differentiation markers expression. All these data show a series of coordinated events which serve in osteoclasts precursors’ recruitment and migration towards bone surface. NAMPT seems to contribute to acquiring an OB phenotype more favorable to OC recruitment.

Nicotinamide adenine dinucleotide (NAD) is a key regulator of several metabolic and signaling pathways that are relevant in cancer cell survival. Cancer cells have an increased energy demand associated with an increased NAD turnover. Nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme of the NAD salvage pathway, plays a crucial role in maintaining the intracellular NAD levels and in regulating the activity of NAD-dependent enzymes, such as sirtuins (SIRTs). The inhibition of NAMPT activity and the use of phytochemicals, such as resveratrol, represent novel therapeutic approaches in cancer therapy. Based on these facts, this thesis aimed to investigate (1) the chemotherapeutic potential and molecular mechanisms of FK866, a specific NAMPT inhibitor, and resveratrol on hepatocarcinoma cells and to find out whether there are differences compared to primary human hepatocytes; (2) to address the impact of NAMPT inhibition on the energy metabolism in cancer cells; and (3) to investigate the roles of NAMPT and SIRT1 in resveratrol´s mode of action and chemotherapeutic effects. This work demonstrates that FK866 and resveratrol possess potent chemotherapeutic effects in hepatocarcinoma cells which were absent in human hepatocytes. Hepatocarcinoma cells display a dysregulation in the AMP-activated kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling as well as in the NAMPT-mediated NAD salvage pathway compared to human hepatocytes. FK866-induced NAMPT inhibition induces ATP depletion associated with AMPK activation and mTOR inhibition whereas resveratrol induces caspase3-mediated apoptosis that is not dependent on NAMPT and SIRT1 function. NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and human hepatocytes. This work also reveals that resveratrol activates p53-induced cell cycle arrest in hepatocarcinoma cells which is partly mediated by SIRT1 inhibition. In summary, this thesis provides new insight into the role of the NAMPT-mediated NAD salvage pathway in energy metabolism and characterized FK866 and resveratrol as promising potential chemotherapeutic agents for treatment of hepatocellular carcinoma.

Adipose tissue secreted proteins (adipokines) have been proposed to form a link between obesity and type 2 diabetes (T2D). Resistin and visfatin are two adipokines which have been previously suggested as having roles in the pancreatic islet. The aim of this study was therefore to investigate the regulatory role of the adipokines resistin and visfatin in the pancreatic beta-cell. In order to do this, pancreatic β-cell lines from rat (BRIN-BD11) and mouse (βTC-6) were used to study the effect of exogenous incubation with physiological and pathological concentrations of resistin and visfatin on diverse elements of beta-cell biology including cell viability, gene expression and insulin secretion. In addition to this the expression levels of these two adipokines was also measured in the beta-cell. PCR array analysis showed that resistin and visfatin treatment resulted in significant changes in the expression of key beta-cell specific genes. Interestingly, both resistin and visfatin are highly expressed in the beta-cells. This suggests that the roles of these adipokines are not confined to adipose tissue but also in other endocrine organs. Resistin treatment significantly increased viability of the beta-cells at physiological concentrations however there was no increase with the elevated pathological concentrations. Resistin at elevated concentrations decreased insulin receptor expression in the beta-cells however there was no significant effect at lower concentrations. Both physiological and elevated resistin concentrations did not have any effect on glucose stimulated insulin secretion. Incubation of visfatin induced phosphorylation of insulin receptor and the intracellular signalling MAPK, ERK1/2. Visfatin treatment at 200ng/ml also significantly increased insulin secretion. These effects were replicated by incubation of beta-cells with the product of visfatin’s enzymatic action, nicotinamide mononucleotide and were reversed by visfatin inhibitor FK866. Visfatin treatment at low concentrations did not have any effect on cell viability however the elevated concentrations resulted in a decline. These data indicate that both resistin and visfatin potentially play important roles in beta-cell function and viability and that they form a significant link between adipose tissue and the pancreatic islet in type 2 diabetes.

Pre-B Cell Colony-Enhancing Factor (PBEF) is a cytokine-like molecule that functions as a nicotinamide phosphoribosyl transferase (Nampt) in a salvage pathway of NAD biosynthesis. PBEF has well-characterized activity as an extracellular inflammatory mediator and has been proposed to signal through the insulin receptor (IR). As neutrophils are key effectors of the innate immune response to infection and injury, we hypothesized that PBEF promotes pro-inflammatory function in neutrophils and that these pro-inflammatory effects may occur through interactions with the neutrophil IR or through PBEF���s enzymatic Nampt activity. Our studies focused on two important facets of neutrophil inflammatory function: their ability to generate reactive oxygen species (ROS) and undergo constitutive apoptosis. We found that, although PBEF does not activate oxidative burst on its own, it primes for ROS generation through the NADPH oxidase. PBEF promotes membrane translocation of cytosolic NADPH oxidase subunits p40phox and p47phox, but not p67phox, induces p40phox phosphorylation and activates Rac. Priming, translocation and phosphorylation are dependent on activation of p38 and ERK mitogen activated protein kinases. PBEF priming of neutrophils occurs independent of its Nampt capacity or of interactions with IR. We next investigated the effects of PBEF on neutrophil constitutive apoptosis. Our lab previously established that extracellular PBEF delays neutrophil apoptosis. Accordingly, we next investigated the mechanism through which this delay was occurring. PBEF-induced delayed apoptosis was enhanced in the presence of Nampt substrates, and NAD alone could delay apoptosis to an extent comparable to PBEF. Delayed apoptosis was blocked by a Nampt inhibitor and was lacking when a mutated PBEF deficient in Nampt activity was utilized. The cell-surface NAD glycohydrolase, CD38, can convert NAD to cyclic ADP-ribose (cADPR). Blocking CD38 activity with a blocking antibody partially reversed the delay of apoptosis induced by PBEF in conjunction with its substrates, and delayed apoptosis could be achieved by addition of the CD38 product cADPR. Finally, we found that delayed apoptosis induced by PBEF did not involve IR. These results indicate that PBEF can prime for enhanced oxidative burst and delay apoptosis in neutrophils, and that these phenomena occur through distinct mechanisms.