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Transfer RNAs (tRNAs) are non-coding RNA molecules essential for protein synthesis. Post-transcriptionally they are heavily modified to improve their function, folding and stability. Intronic polymorphisms in CDKAL1, a tRNA methylthiotransferase, are associated with increased type 2 diabetes risk. Loss-of-function mutations in TRMT10A, a tRNA methyltransferase, are a monogenic cause of early onset diabetes and microcephaly. Here we confirm the role of TRMT10A as a guanosine 9 tRNA methyltransferase, and identify tRNAGln and tRNAiMeth as two of its targets. Using RNA interference and induced pluripotent stem cell-derived pancreatic β-like cells from healthy controls and TRMT10A-deficient patients we demonstrate that TRMT10A deficiency induces oxidative stress and triggers the intrinsic pathway of apoptosis in β-cells. We show that tRNA guanosine 9 hypomethylation leads to tRNAGln fragmentation and that 5'-tRNAGln fragments mediate TRMT10A deficiency-induced β-cell death. This study unmasks tRNA hypomethylation and fragmentation as a hitherto unknown mechanism of pancreatic β-cell demise relevant to monogenic and polygenic forms of diabetes., SCOPUS: ar.j, info:eu-repo/semantics/published

The ability of certain cancer cells to maintain a highly reduced intracellular environment is correlated with aggressiveness and drug resistance. Since the gluthathione (GSH) and thioredoxin (TRX) systems cooperate to a tight regulation of ROS in cell physiology, and to a stimulation of tumor initiation and progression, modulation of the GSH and TRX pathways are emerging as new potential targets in cancer. In vivo methods to assess changes in tumor redox status are critically needed to assess the relevance of redox-targeted agents. The current study assesses in vitro and in vivo biomarkers of tumor redox status in response to treatments targeting the GSH and TRX pathways, by comparing cytosolic and mitochondrial redox nitroxide Electron Paramagnetic Resonance (EPR) probes, and cross-validation with redox dynamic fluorescent measurement. For that purpose, the effect of the GSH modulator buthionine sulfoximine (BSO) and of the TRX reductase inhibitor auranofin were measured in vitro using both cytosolic and mitochondrial EPR and roGFP probes in breast and cervical cancer cells. In vivo, mice bearing breast or cervical cancer xenografts were treated with the GSH or TRX modulators and monitored using the mito-TEMPO spin probe. Our data highlight the importance of using mitochondria targeted spin probes to assess changes in tumor redox status induced by redox modulators. Further in vivo validation of the mito-tempo spin probe with alternative in vivo methods should be considered, yet the spin probe used in vivo in xenografts demonstrated sensitivity to the redox status modulators.

Type 2 diabetes (T2D) arises when the pancreatic beta-cell fails to compensate for increased insulin needs due to insulin resistance. Glucolipotoxicity has been proposed to induce beta-cell dysfunction in T2D by formation of reactive oxygen species (ROS). Here we examined if modelling glucolipotoxic conditions by high glucose-high free fatty acid (FFA) exposure (GLT) regulate beta-cell iron transport, thereby increasing the cytosolic labile iron pool and iron-catalyzed ROS formation. We show that GLT -induced ROS production is regulated by an increased labile iron pool (LIP) associated with elevated expression of genes regulating iron import. Using pharmacological and transgenic approaches, we show that iron reduction and decreased iron import protects from GLT-induced ROS production, prevents impairment of the mitochondrial membrane potential, and inhibits apoptosis. This study identifies a novel pathway underlying GLT-induced apoptosis involving increased iron import, generation of hydroxyl radicals from hydrogen peroxide through the Fenton reaction in the cytosolic compartment associated with dissipation of the mitochondrial membrane potential and beta cell apoptosis.

Ovarian tissue cryopreservation and transplantation is the only way for prepubertal and young female cancer patients to preserve their fertility, but this promising technique (more than 130 births) still has limitations due to follicle loss post-grafting. Indeed, avascular grafts need 5 days to become revascularized, during which time >60% of follicles are lost. In this context, the aim of the present work was to identify a better transplantation technique to limit this follicular loss by stimulating early angiogenesis in ovarian tissue grafts using adipose tissue-derived stem cells (ASCs). This new technique involved a two-step transplantation procedure with (i) preparation of the grafting site and (ii) transplantation of ovarian fragments to the prepared site. A key question that was also addressed in the present work was the mechanism by which ASCs exert their proangiogenic effects in grafted ovarian tissue., La congélation et la transplantation du tissu ovarien est la seule possibilité pour les jeunes patientes prépubères de préserver leur fertilité avant traitement anti-cancéreux. Cette technique prometteuse (plus de 130 naissances) présente néanmoins encore des limitations dans son succès dû à la perte folliculaire post-greffe. En effet, la greffe avasculaire de fragments d’ovaire met 5 jours avant d’être revascularisée, temps pendant lequel >60% des follicules meurent. Dans ce contexte, le présent travail a comme but de trouver une meilleure technique de greffe ovarienne qui a fin de limiter cette perte folliculaire, en stimulant une angiogenèse précoce dans les greffons de tissu ovarien via des cellules souches adipeuses. Cette nouvelle technique consiste en une procédure de transplantation en deux étapes: (i) préparation du site de greffe et (ii) transplantation de tissu ovarien sur le site préparé. Le mécanisme par lequel les cellules souches exercent leurs effets pro-angiogéniques dans le tissu ovarien greffé est aussi analysé., (MED - Sciences médicales) -- UCL, 2018

Type 2 diabetes (T2D) arises when the pancreatic beta-cell fails to compensate for increased insulin needs due to insulin resistance. Glucolipotoxicity (GLT) has been proposed to induce beta-cell dysfunction in T2D by formation of reactive oxygen species (ROS). Here, we examined if modeling glucolipotoxic conditions by high glucose-high free fatty acid (FFA) exposure (GLT) regulates beta-cell iron transport, by increasing the cytosolic labile iron pool (LIP). In isolated mouse islets, the GLT-induced increase in the LIP catalyzed cytosolic ROS formation and induced apoptosis. We show that GLT-induced ROS production is regulated by an increased LIP associated with elevated expression of genes regulating iron import. Using pharmacological and transgenic approaches, we show that iron reduction and decreased iron import protects from GLT-induced ROS production, prevents impairment of the mitochondrial membrane potential (MMP) and inhibits apoptosis. This study identifies a novel pathway underlying GLT-induced apoptosis involving increased iron import, generation of hydroxyl radicals from hydrogen peroxide through the Fenton reaction in the cytosolic compartment associated with dissipation of the MMP and beta-cell apoptosis.

Increasing evidence indicate that lactate, the end-product of a glycolytic metabolism, promotes tumor progression. In addition to being used as an oxidative fuel, lactate indeed stimulates angiogenesis by activating transcription factors HIF-1 in oxidative cancer cells and endothelial cells, and NF-kB in endothelial cells. Our work shows that lactate does not activate NF-kB in oxidative cancer cells, because NADH produced during its oxidation is used in mitochondria rather than fueling NAP(P)H oxidases. Thus, lactate influx inhibition can prevent the oxidative use of lactate in cancer. A main target is inward monocarboxylate transporter 1 (MCT1). We therefore developed a tracer of lactate for positron emission tomography that allows to predict and to document a response to MCT1 inhibitors in vivo. Together, our work contributes to a better understanding and therapeutic exploitation of lactate transport in cancer., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2017

Exercise has anti-inflammatory properties useful to prevent and treat some chronic diseases. The expression of activating transcription factor 3 (ATF3) increases in skeletal muscle after exercise. In many tissues, ATF3 regulates the inflammatory response. Whether ATF3 contributes to the anti-inflammatory properties of exercise in skeletal muscle was unknown. To understand the role of ATF3 in skeletal muscle, we used a mutant mouse model unable to express ATF3 (ATF3-KO). We found that the expression of some inflammation-related genes was higher in ATF3-KO than control mice postexercise. ATF3-KO mice also had an impaired molecular adaptation to training in skeletal muscle. Our data suggest ATF3 is an important regulator of the inflammation postexercise, which could influence training adaptation in skeletal muscle. Since inflammation is involved in the pathogenesis of many diseases, the identification of ATF3 as an exercise-induced anti-inflammatory factor is relevant for future studies., (MOTR - Sciences de la motricité) -- UCL, 2017

Oxygen is essential in wound healing and hypoxia is described as a major cause of wound healing impairment. Tissue oxygenation is an important parameter in the clinical management of chronic wounds such as diabetic ulcers. But until now, no techniques are able to perform absolute, non-invasive and repeated measurements of oxygenation directly in a tissue. Electron Paramagnetic Resonance (EPR) oximetry is a technique that allows this kind of measurements, but was never applied in the context of diabetic wound healing. The aim of the thesis was the implementation of EPR oximetry to follow diabetic wound oxygenation variations. The first part of the thesis consisted in the validation of the technique to measure wound oxygenation. Then, the use of EPR oximetry as a biomarker of treatment response in diabetic wounds was assessed., L’oxygène est essentiel à la cicatrisation des plaies et l’hypoxie est décrite comme une cause majeure de problèmes de cicatrisation. L’oxygénation tissulaire est un paramètre important dans la gestion clinique des plaies chroniques comme les ulcères diabétiques. Mais jusqu’à présent, aucune technique n’est capable de réaliser une mesure absolue, non-invasive et répétée de l’oxygénation directement dans un tissu. L’oxymétrie par Résonance Paramagnétique Electronique (RPE) est une technique qui permet de réaliser ce type de mesures mais n’a jamais été appliquée dans le contexte de cicatrisation de plaies diabétiques. Le but de la thèse était d’implémenter l’oxymétrie RPE pour suivre les variations en oxygénation dans les plaies diabétiques. La première partie de la thèse a consisté en la validation de la technique pour mesurer l’oxygénation dans les plaies. Ensuite, l’utilisation de l’oxymétrie RPE comme biomarqueur de réponse à un traitement dans les plaies diabétiques a été évaluée., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2017

L’os est un tissu dynamique, en remodelage constant grâce à un processus finement régulé impliquant des facteurs locaux et systémiques. Un déséquilibre entre la formation et la résorption osseuses, assurées respectivement par les ostéoblastes et les ostéoclastes, conduit à une altération de la microarchitecture de l’os, une augmentation du risque de fracture, et à l’apparition de pathologies telles que l’ostéonécrose et l’ostéoporose. Ces deux maladies ont pour caractéristiques communes une diminution du nombre de cellules stromales mésenchymateuses (MSC), les progéniteurs des ostéoblastes, et des anomalies fonctionnelles des MSC et des cellules ostéoblastiques (Ob). De surcroit, elles présentent toutes deux une accumulation d’adipocytes au sein de la moelle osseuse. De récentes publications suggèrent que cette accumulation d’adipocytes médullaires pourrait avoir des conséquences délétères sur la physiologie des cellules ostéoformatrices et de leurs progéniteurs, partageant ce même microenvironnement osseux. Une relation inverse entre l’excès d’adiposité médullaire et la masse osseuse est en effet aujourd’hui clairement établie. Par son importante activité sécrétrice de cytokines et d’adipokines ainsi que sa capacité à stocker et libérer des acides gras libres (AGL), l’adipocyte médullaire est capable de modifier la composition du microenvironnement osseux, et ainsi d’influencer le métabolisme et la fonction des cellules avoisinantes, et notamment des cellules osseuses. Afin d’étudier l’influence des AGL sur la survie et la fonction des cellules ostéogéniques, nous avons utilisé un AGL saturé, le palmitate (Palm ;C16 :0) et un AGL monoinsaturé, l’oléate (Ole ;C18 :1), tous deux étant particulièrement abondants dans l’organisme et l’alimentation de l’homme et couramment utilisés dans les études de lipotoxicité. Dans un premier temps, nous avons travaillé sur des MSC isolées de la moelle osseuse de sujets sains (HV-MSC), qui ont éventuellement été différenciées en Ob., Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

info:eu-repo/semantics/published

Cardiac insulin resistance is a common feature of diabetic cardiomyopathy and is characterized by a decreased capacity of insulin to stimulate cardiomyocytic glucose uptake. AMPK is known to restore insulin sensitivity in insulin-resistant cardiomyocytes. In this work, we demonstrate that neither the inhibition of the mTOR/p70S6K pathway, nor the reduction of membrane cholesterol induced by AMPK are sufficient to explain its insulin-sensitizing effect on glucose uptake. On the contrary, we show that an intact actin cytoskeleton, as well as the activation of Rac1, a small GTPase regulating actin rearrangement, are both required for this insulin-sensitizing effect. In conclusion, AMPK and the signaling pathways regulating actin polymerization could be potential targets for the treatment of diabetic cardiomyopathy., L’insulino-résistance cardiaque est un élément notable de la cardiomyopathie diabétique. Elle se caractérise par une diminution de la capacité de l’insuline à stimuler le transport du glucose cardiomyocytaire. L’AMPK est connue pour restaurer la sensibilité à l’insuline dans des cardiomyocytes insulino-résistants. Par ce travail, nous mettons en évidence que ni l’inhibition de la voie mTOR/p70S6K, ni la réduction du cholestérol membranaire induites par l’AMPK ne sont suffisantes pour expliquer son effet insulino-sensibilisateur sur la stimulation du transport du glucose. A l’opposé, nous démontrons que l’intégrité du cytosquelette d’actine ainsi que l’activation de Rac1, une petite GTPase régulant la réorganisation de l’actine, sont toutes deux nécessaires à cet effet insulino-sensibilisateur. En conclusion, l’AMPK et les voies de signalisation régulant la polymérisation de l’actine pourraient être des cibles potentielles pour le traitement de la cardiomyopathie diabétique., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2015

BACKGROUND/AIM: In rat pancreatic islets, beta-cell gene expression, survival and subsequent acute glucose stimulation of insulin secretion (GSIS) are optimally preserved by prolonged culture at 10 mM glucose (G10) and markedly altered by culture at G5 or G30. Here we tested whether pharmacological glucokinase (GK) activation prevents these alterations during culture or improves GSIS after culture. METHODS: Rat pancreatic islets were cultured 1-7 days at G5, G10 or G30 with or without 3 µM of the GK activator Ro 28-0450 (Ro). After culture, beta-cell apoptosis and islet gene mRNA levels were measured, and the acute glucose-induced increase in NAD(P)H autofluorescence, intracellular calcium concentration and insulin secretion were tested in the absence or presence of Ro 28-0450. RESULTS: Prolonged culture of rat islets at G5 or G30 instead of G10 triggered beta-cell apoptosis and reduced their glucose responsiveness. Addition of Ro during culture differently affected beta-cell survival and glucose responsiveness depending on the glucose concentration during culture: it was beneficial to beta-cell survival and function at G5, detrimental at G10, and ineffective at G30. In contrast, acute GK activation with Ro increased the glucose sensitivity of islets cultured at G10, but failed at restoring beta-cell glucose responsiveness after culture at G5 or G30. CONCLUSIONS: Pharmacological GK activation prevents the alteration of beta-cell survival and function by long-term culture at G5, but mimics glucotoxicity when added to G10. The complex effects of glucose on the beta-cell phenotype result from changes in glucose metabolism and not from an effect of glucose per se.

Prostate cancer (PCa) is the most frequent cancer in men above the age of 50 years. Despite radical prostatectomy or radiation therapy as primary treatment, 15-30% of patients will develop an advanced or metastatic cancer requiring systemic therapies. Reference treatment of advanced PCa relies on pharmacological or surgical androgen deprivation therapy. However, despite initial efficacy of androgen deprivation (AD), the tumor inevitably adapts to low testosterone environment and becomes hormone refractory (HRPCa). A better understanding of the mechanisms of androgen independence is necessary to improve the management of HRPCa. Although autophagy confers chemoresistance in some cancers, its role in the development of HRPCa remained unknown. We found that AD or treatment with the anti-androgen bicalutamide promoted autophagy in HRPCa-derived LNCaP cells. This effect was associated with an inhibition of the PI3K/Akt/mTOR pathway and with a disruption of the complex formed by androgen receptor and the regulatory subunit of PI3K p85. Moreover, genetic or pharmacological inhibition of autophagy restored AD-dependent cell death indicating that autophagy is a protective mechanism against AD in HRPCa cells. Besides, in these same cells, we also observed different modifications of Ca2+ homeostasis after androgen withdrawal among which a reduction of the ER Ca2+ content. We discovered that the [Ca2+]ER decrease was due to the presence of IP3R1 made leakier by their phosphorylation by PKA inhibitor H89 or permeant TAT-peptide containing the Ser-1716 consensus phosphorylation sequence sensitized LNCaP cells to AD, suggesting that [Ca2+]ER can control HRPCa cells resistance to AD. Therefore, we identified two distinct mechanisms by which HRPCa-derived cells manage to overcome cell death that they face in the absence of androgenic stimulation. We hope that these findings will help to device new therapeutic strategies to circumvent hormone independence of advanced PCa., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2015

Background and aim: Nicotinamide nucleotide transhydrogenase (NNT) contributes to NADPH production in the mitochondrial matrix of eukaryotic cells. Its inactivation in beta‐cells was previously shown to markedly reduce glucose‐induced rises in ATP, calcium and insulin secretion, but these effects vary between experimental models. We recently found that, in islets from C57BL/6J mice that express a truncated inactive NNT, glucose does not reduce the mitochondrial glutathione redox state measured using GRX1‐roGFP2, in contrast to its effect in islets from C57BL/6N mice that express wild‐type NNT (abstract by Laila RB Santos). Here, we re‐evaluated the role of NNT by simultaneously measuring key stimulus‐secretion coupling events in islets from C57BL/6J and C57BL/6N mice perifused in the presence of increasing glucose concentrations. Results: 1) The glucose‐induced rise in NAD(P)H autofluorescence was significantly larger in N vs. J islets (normalised to the fluorescence in 0.5 mM glucose or after addition of FCCP). 2) The glucose‐induced changes in mitochondrial membrane potential and cytosolic calcium concentration were similar in both types of islets (rhodamine 123 fluorescence normalised to that measured after addition of FCCP). 3) The insulin/DNA content ratio was similar in both types of islets, but the glucose stimulation of insulin secretion was 2 to 2.5 times higher in N vs. J islets (normalized to insulin or DNA content). 4) The stimulation of insulin secretion by high potassium was 2 to 2.5 times higher in N vs. J islets at any glucose concentration, so that the amplifying action of glucose was similar in both types of islets. Conclusion: The lack of functional NNT in J vs. N islets markedly reduced their glucose‐induced rise in NAD(P)H autofluorescence and insulin secretion without affecting mitochondrial membrane hyperpolarisation and rise in cytosolic calcium concentration. These results suggest that the insulin secretion defect of J vs. N islets lies at a

Background and aim: Using GRX1‐roGFP2 as a reporter for changes in glutathione redox state, we recently showed that glucose acutely reduces mitochondrial glutathione in rat islet cell clusters and human islets. Interestingly, this glucose effect was inversely correlated with the increase in NAD(P)H autofluorescence, suggesting a possible link between both events. Among the enzymes involved in mitochondrial NADPH production, nicotinamide nucleotide transhydrogenase (NNT) may play a critical role in beta‐cell function by contributing to mitochondrial ROS detoxification and to the rise in cytosolic NADPH. Here, we tested the impact of NNT inactivation on glucose and H2O2‐mediated changes in cytosolic and mitochondrial glutathione redox state by comparing changes in (mt‐)GRX1‐ roGFP2 fluorescence ratio in islets from C57BL/6J mice with truncated NNT and from C57BL/6N mice with wild‐type NNT (J vs. N islets). Results: 1) The mutation of the NNT gene in J but not N mice was confirmed by PCR on tail DNA. 2) At 10 mM glucose, micromolar amounts of exogenous H2O2 similarly oxidized mitochondrial and cytosolic GRX1‐roGFP2 in J and N islets. 3) At low glucose, mt‐GRX1‐roGFP2 fluorescence ratio was higher in N than J islets. Upon stepwise glucose stimulation, it rapidly decreased in N islets while slightly increasing in J islets. Overexpressing NNT in J islets restored the glucose responsiveness of mt‐GRX1‐roGFP2 fluorescence ratio. 4) In N but not J islets, cytosolic GRX1‐roGFP2 fluorescence ratio slightly increased upon glucose removal and decreased upon stimulation with 2 and 5 mM glucose. 5) Glucose induced a larger increase in NAD(P)H autofluorescence in N vs. J islets. Conclusion: The lack of NNT in islets from C57BL/6J mice is associated with a lower glucose‐induced rise in NAD(P)H autofluorescence and a lack of glucose reduction of the glutathione redox state in the mitochondrial matrix and the cytosol. It does not, however, increase their sensitivity to exogenous H2O2.