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Background and aims: The rapid reversibility of recent type 2 diabetes (T2D) by very low-calorie diet in some patients correlates with a marked improvement of glucose-stimulated insulin secretion (GSIS), emphasizing the role of β-cell dysfunction in the early stages of the disease. In search of human-specific mechanisms of β-cell dysfunction, we extensively characterized the glucotoxic alterations of insulin secretion and upstream coupling events in human pancreatic islets cultured for 1 to 3 weeks at ~5, 8, 10 or 20 mmol/l glucose and acutely stimulated by a stepwise increase in glucose concentration. Materials and methods: Islets from 46 non-diabetic (ND) and 6 type 2 diabetic (T2D) donors were obtained from 5 isolation centers over the last 10 years. The islets were precultured 3-7 days in RPMI containing 5 mmol/l glucose and 10% FBS. They were then cultured for 1-3 weeks in the same medium containing 5.5, 8.5, 10.5 or 20.5 mmol/l glucose before measurements of insulin secretion during culture, islet insulin/DNA content ratio, β-cell apoptosis, cytosolic and mitochondrial thiol redox state, and assessment of dynamic insulin secretion and upstream coupling events during stepwise stimulation with glucose (NAD(P)H autofluorescence, ATP/(ATP+ADP) ratio, electrical activity, cytosolic Ca2+ concentration ([Ca2+]c)). Results: Culture of ND-islets for 1 to 3 weeks at ~8, 10 or 20 vs 5 mmol/l glucose did not increase β-cell apoptosis or oxidative stress but concentration-dependently decreased insulin content and increased the β-cell sensitivity to subsequent stimulation with glucose. The islet glucose responsiveness (max amplitude of GSIS per islet) was larger after culture at 8 or 10 vs 5 mmol/l glucose but was markedly reduced after culture at 20 vs 5 mmol/l glucose. In the latter islets, [Ca2+]c and insulin secretion responses to acute stepwise stimulation with glucose were bell-shaped, with a maximal stimulation at 5 to 10 mmol/l glucose followed by a rapid sustained inhibition above that concentration. This glucotoxic alteration was a robust characteristic of human islets. It resulted from long-term stimulation of glucose metabolism and was fully reversible after culture at 5 mmol/l glucose. Finally, acute activation/inhibition of glucokinase during perifusion of islets cultured at 20 mmol/l glucose indicated that the acute reduction of [Ca2+]c and insulin secretion above 10 mmol/l glucose was due to overstimulation rather than inhibition of glucose metabolism. Similar results were obtained in islets from T2D-donors. Conclusion: Long-term exposure of human islets to mildly elevated glucose concentrations markedly increases their glucose sensitivity and reveals a bell-shaped glucose response curve for changes in [Ca2+]c and insulin secretion. This human-specific glucotoxic alteration may contribute to β-cell dysfunction in T2D independently from a detectable increase in β-cell apoptosis and oxidative stress.

Background and aims: The mechanisms responsible for β cell compensation in obesity and for β cell failure in type 2 diabetes (T2D) are poorly defined. Metallothioneins play a role in both Zn2+ homeostasis and the regulation of cellular redox state. The mRNA levels of several metallothionein genes are upregulated in islets from subjects with T2D, but their role in β cells is not clear. Here we examined: 1) the temporal changes of islet Mt1 and Mt2 gene expression in models of β cell compensation and failure, and 2) the role of Mt1 and Mt2 in β cell function and glucose homeostasis. Materials and methods: Mt1 and Mt2 expression was assessed in islets from control lean (chow diet) and diet-induced obese (DIO) mice (8 weeks high fat diet), and pre-diabetic (6-week-old) and diabetic (16-week-old) db/db mice and age-matched db/+ (control) mice. Mt1-Mt2 double knockout (KO) mice, Mt1 overexpressing transgenic mice (Tg-Mt1) and corresponding control mice were studied. Mt1 and Mt2 were inhibited in MIN6 cells by small interfering RNAs. mRNA levels were assessed by real-time RT-PCR, plasma insulin and islet metallothionein levels by ELISA, glucose tolerance by i.p. glucose tolerance tests (ipGTT) and fasting-1h refeeding tests, insulin secretion by RIA, cytosolic free Ca2+ with Fura-2 LR, NAD(P)H by autofluorescence and cytosolic redox state using roGFP1 ratiometric thiol redox probe. Results: Increased plasma insulin levels (β cell compensation) correlated with marked downregulation of Mt1 and Mt2 mRNA levels in islets of DIO mice (Mt1: ~4-fold, p

International audience; Pregnancy and obesity are frequently associated with diminished insulin sensitivity, which is normally compensated for by an expansion of the functional β cell mass that prevents chronic hyperglycemia and development of diabetes mellitus. The molecular basis underlying compensatory β cell mass expansion is largely unknown. We found in rodents that β cell mass expansion during pregnancy and obesity is associated with changes in the expression of several islet microRNAs, including miR-338-3p. In isolated pancreatic islets, we recapitulated the decreased miR-338-3p level observed in gestation and obesity by activating the G protein-coupled estrogen receptor GPR30 and the glucagon-like peptide 1 (GLP1) receptor. Blockade of miR-338-3p in β cells using specific anti-miR molecules mimicked gene expression changes occurring during β cell mass expansion and resulted in increased proliferation and improved survival both in vitro and in vivo. These findings point to a major role for miR-338-3p in compensatory β cell mass expansion occurring under different insulin resistance states.

OBJECTIVE: Sarco-endoplasmic reticulum Ca(2+)-ATPase 2b (SERCA2b) and SERCA3 pump Ca(2+) in the endoplasmic reticulum (ER) of pancreatic β-cells. We studied their role in the control of the free ER Ca(2+) concentration ([Ca(2+)](ER)) and the role of SERCA3 in the control of insulin secretion and ER stress. RESEARCH DESIGN AND METHODS: β-Cell [Ca(2+)](ER) of SERCA3(+/+) and SERCA3(-/-) mice was monitored with an adenovirus encoding the low Ca(2+)-affinity sensor D4 addressed to the ER (D4ER) under the control of the insulin promoter. Free cytosolic Ca(2+) concentration ([Ca(2+)](c)) and [Ca(2+)](ER) were simultaneously recorded. Insulin secretion and mRNA levels of ER stress genes were studied. RESULTS: Glucose elicited synchronized [Ca(2+)](ER) and [Ca(2+)](c) oscillations. [Ca(2+)](ER) oscillations were smaller in SERCA3(-/-) than in SERCA3(+/+) β-cells. Stimulating cell metabolism with various [glucose] in the presence of diazoxide induced a similar dose-dependent [Ca(2+)](ER) rise in SERCA3(+/+) and SERCA3(-/-) β-cells. In a Ca(2+)-free medium, glucose moderately raised [Ca(2+)](ER) from a highly buffered cytosolic Ca(2+) pool. Increasing [Ca(2+)](c) with high [K] elicited a [Ca(2+)](ER) rise that was larger but more transient in SERCA3(+/+) than SERCA3(-/-) β-cells because of the activation of a Ca(2+) release from the ER in SERCA3(+/+) β-cells. Glucose-induced insulin release was larger in SERCA3(-/-) than SERCA3(+/+) islets. SERCA3 ablation did not induce ER stress. CONCLUSIONS: [Ca(2+)](c) and [Ca(2+)](ER) oscillate in phase in response to glucose. Upon [Ca(2+)](c) increase, Ca(2+) is taken up by SERCA2b and SERCA3. Strong Ca(2+) influx triggers a Ca(2+) release from the ER that depends on SERCA3. SERCA3 deficiency neither impairs Ca(2+) uptake by the ER upon cell metabolism acceleration and insulin release nor induces ER stress.

Effects of deglycating enzyme Fructosamine-3-kinase gene knockout on pancreatic beta cell glucotoxicity

Background and Aim: We previously tested the effects of a 18h culture in 2, 5, 10 and 30 mmol/l glucose (G2, G5, G10 and G30) on the transcriptome of cultured rat islets and identified 18 clusters with distinct glucose-dependent mRNA profiles. Of these, genes that were up-regulated between G10 and G30 are particularly interesting, as they may contribute to beta-cell glucotoxicity or protection against it. Besides genes involved in the unfolded protein response, this cluster contains most glycolytic enzymes and other genes typically induced by hypoxia, such as Adrenomedullin (Adm). Hypothesis: High glucose, which increases O2 consumption in β-cells, may induce hypoxia and thereby cause glucotoxicity. In this study, we tested whether overnight culture in high glucose activates the hypoxia-inducible transcription factor HIF in cultured rat beta-cells. Material and Methods: Male Wistar rat islets were precultured for one week in serum-free RPMI medium containing 10 mmol/l glucose (G10) and 5 g/l BSA, during which islets with signs of central necrosis were systematically discarded. INS1-E cells were cultured as monolayers (passage 72 to 80) in the presence of 10% FBS. Rat islets and INS1-E cells (70% confluence) were then cultured 18h in the presence of increasing glucose concentrations (G2, G5, G10 and G30) with various test substances and at different O2 concentrations (1%, 20% or 60% corresponding to a pO2 of 7.6, 152 and 456 mmHg). Results: Compared with G2, culture in G30 significantly increased 4-fold the protein levels of HIF1α and HIF2α, and 2-fold their dimerization partner HIF1β in INS1-E cell nuclear extracts while significantly decreasing HIF1β protein levels by ~50% in cytosolic extracts. High glucose also significantly up-regulated the mRNA levels of several HIF target genes (Adm, Glyceraldehyde phosphate dehydrogenase, Aldolase A, Lactate dehydrogenase A). These glucose effects, which were confirmed in whole rat islets, were mimicked by 6h exposure to a low pO2 (1% O2) or by 18h treatment with 10-30 µmol/l CoCl2, a known activator of HIF. In contrast, they were almost completely inhibited by 60% O2 or by various agents that inhibit Ca2+ influx and insulin secretion (250 µmol/l diazoxide, 1 µmol/l nimodipine and 1 µmol/l clonidine) and thereby likely reduce without suppressing the glucose stimulation of β-cell O2 consumption. In comparison with HIF target genes, Thioredoxin interacting protein (Txnip), one of the most glucose-responsive genes in cultured rat islets, was regulated in a completely different manner. Thus, Txnip mRNA levels were not increased by CoCl2 and 1% O2, and their increase by G30 was not reduced by 60% O2 and was markedly enhanced by diazoxide, nimodipine and clonidine. Conclusion: High glucose induces HIF1α and HIF2α protein stabilization, HIF1/2α-HIF1β dimer translocation to the nucleus, and increased expression of glycolytic enzymes and other HIF target genes in cultured rat beta-cells. These effects likely result from the glucose stimulation of ATP utilization and O2 consumption which may induce beta-cell hypoxia. These effects could contribute to in vitro β-cell glucotoxicity not only in whole islets but also in insulin-secreting cells cultured as monolayers.

The contribution of Ca(2+) release from intracellular stores to the rise in the free cytosolic Ca(2+) concentration ([Ca(2+)](c)) triggered by Ca(2+) influx was investigated in mouse pancreatic beta-cells. Depolarization of beta-cells by 45 mm K(+) (in the presence of 15 mm glucose and 0.1 mm diazoxide) evoked two types of [Ca(2+)](c) responses: a monotonic and sustained elevation; or a sustained elevation superimposed by a transient [Ca(2+)](c) peak (TCP) (40-120 s after the onset of depolarization). Simultaneous measurements of [Ca(2+)](c) and voltage-dependent Ca(2+) current established that the TCP did not result from a larger Ca(2+) current. Abolition of the TCP by thapsigargin and its absence in sarco-endoplasmic reticulum Ca(2+)-ATPase 3 (SERCA3) knockout mice show that it is caused by Ca(2+) mobilization from the endoplasmic reticulum. A TCP could not be evoked by the sole depolarization of beta-cells but required a rise in [Ca(2+)](c) pointing to a Ca(2+)-induced Ca(2+) release (CICR). This CICR did not involve inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) because it was resistant to heparin. Nor did it involve ryanodine receptors (RyRs) because it persisted after blockade of RyRs with ryanodine, and was not mimicked by caffeine, a RyR agonist. Moreover, RyR1 and RyR2 mRNA were not found and RyR3 mRNA was only slightly expressed in purified beta-cells. A CICR could also be detected in a limited number of cells in response to glucose. Our data demonstrate, for the first time in living cells, the existence of an atypical CICR that is independent from the IP(3)R and the RyR. This CICR is prominent in response to a supraphysiological stimulation with high K(+), but plays little role in response to glucose in non-obese mouse pancreatic beta-cells.

BACKGROUND AND AIMS: The low-affinity Ca2+-ATPase SERCA3 is highly expressed in p-cells. It mediates uptake of cytosolic Ca2+ by the endoplasmic reticulum during each period of Ca2+ influx from the extracellular medium, whereas the high-affinity SERCA2b controls basal [Ca'+t For unknown reasons, p-cells within mouse islets display variable patterns of [Ca2 +], oscillations during glucose (G) stimulation. These oscillations can be either regular and fast, or mixed with rapid oscillations superimposed on slow ones. We studied the role of the membrane potential (MP) and the possible intervention of SERCA3 in the generation of both types of [Ca2 +], oscillations. [...]

BACKGROUND AND AIMS: In pancreatic ß-cells, Ca2+ influx through voltagedependent Ca2+ channels plays a prominent role in the depolarizationinduced rise in free cytosolic Ca2+ concentration ([Ca2+]c). The contribution of Ca2+ release from the endoplasmic reticulum (ER) to this [Ca2+]c elevation is largely debated and was investigated here [...]

Chronic hyperglycemia exerts deleterious effects on glucose-induced insulin secretion by pancreatic beta cells. In 90% pancreatectomized rats, chronic hyperglycemia induces beta cell hypertrophy and loss of differentiation associated with increased expression of the early response gene c-Myc. Hyperglycemia also stimulates c-Myc expression in vivo in islets from glucose-infused rats and in vitro in cultured rat islets. This effect requires the elevation of cytosolic Ca2+ concentration produced by glucose. Our results suggest that overstimulation of c-Myc expression by chronic hyperglycemia may be the cause of beta cell hypertrophy and loss of differentiation and function observed in animal models of type 2 diabetes.

A growing body of evidence indicates that mitochondria play a key role in many disorders as well as in cancer progression and response to treatment. Next to being the main cellular energy generator through respiration, mitochondria are also the major producer of superoxide and other downstream reactive oxygen species in the cell. In this thesis, we aimed to develop an integrated electron paramagnetic resonance (EPR) toolbox enabling the assessment of mitochondrial (dys)function by measuring the oxygen consumption rate and superoxide production in a same cellular or mitochondrial preparation. This EPR toolbox was used for the benefit of two different projects: discovering potential radiosensitizers in anticancer therapy (like statins) and assessing the impact of agents such as Boscalid and Bixafen, which are succinate dehydrogenase inhibitors (SDHI), on the mitochondrial function of human cells. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2021

Platelets are central actors in atherosclerosis and atherothrombosis. Stimulation of the AMP-activated protein kinase (AMPK) leads to phosphorylation and inhibition of acetyl-CoA carboxylase (ACC), the first committed enzyme for the de novo fatty acids synthesis in cells, including platelets. Given the impact of atherogenic environment on platelets, we hypothesized that AMPK-ACC signaling is activated in coronary artery disease (CAD) patients. The ACCTHEROMA trial (NCT03034148) reveals platelet ACC phosphorylation (phosphoACC) as a promising marker for risk stratification in suspected CAD patients. It identifies high-risk patients and correlates with severity of coronary artery calcification. The triglycerides/high-density lipoprotein cholesterol ratio is strongly associated with increased phosphoACC, a metabolic signature of the platelet-atherogenic lipid interplay in CAD patients. Phosphorylation and inhibition of ACC impacts platelet lipid content by down-regulating triglyceride lipid species, which in turn may affect platelet functions. (MED - Sciences médicales) -- UCL, 2019

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. Nous avons démontré que l’exposition de ces cellules à des concentrations physiologiques de Palm entraîne une cytotoxicité dose-et temps-dépendante, via l’initiation d’un stress du réticulum endoplasmique (RE) et l’activation des voies ERK et NFκB. En outre, l’AGL saturé induit un état pro-inflammatoire en augmentant l’expression du toll-like receptor 4 et la production des interleukines (IL) 6 et 8. Nous avons montré que les Ob présentent une sensibilité accrue au Palm, associée à une exacerbation du stress du RE et de la réponse pro-inflammatoire. L’Ole n’a pas d’effet délétère sur les MSC et les Ob, et de surcroit, il bloque les différentes voies activées par le Palm, neutralisant ainsi totalement la lipotoxicité induite par l’AGL saturé. Nous avons démontré par ailleurs que l’AGL monoinsaturé favorise l’estérification et le stockage du Palm dans des gouttelettes lipidiques, contrant ainsi ses effets délétères. Nous avons ensuite étudié les effets de ces deux AGL, sur des MSC isolées de patients atteints d’ostéonécrose de la tête fémorale (ON-MSC), comparativement aux HV-MSC. Lors de la procédure d’isolation des MSC, nous avons conservé le surnageant obtenu après la première centrifugation (bone marrow supernatant fluid, BMSF) pour une analyse de sa composition lipidique, celle du sérum étant réalisée en parallèle. Nous avons démontré que l’exposition au Palm favorise la différenciation des MSC vers la lignée adipogénique, au détriment du phénotype ostéoblastique. De plus, nous avons observé que les ON-MSC possèdent une capacité de différenciation adipogénique supérieure à celles des HV-MSC.D’autre part, nos résultats ont montré que les ON-MSC sont plus sensibles à la lipotoxicité que les HV-MSC, cette hypersensibilité étant associée à un dérèglement de plusieurs mécanismes cellulaires impliqués dans la survie ou les processus de protection cellulaire :le niveau d’activation basal de la voie ERK est supérieur à celui des HV-MSC et la régulation de l’expression génique des enzymes stearoyl-CoA desaturase 1 (SCD1) et carnitine palmitoyl transferase 1 (CPT1), favorisant respectivement la désaturation des AGL saturés et leur β-oxydation mitochondriale, est altérée dans les ON-MSC. Par ailleurs, dans ces cellules, la production des cytokines IL-6 et IL-8 est triplée par rapport à celle des HV-MSC.La caractérisation du profil lipidique du sérum n’a pas mis en évidence de différence significative entre les sujets sains et ostéonécrotiques. Cependant, de profondes modifications du contenu en AGL du BMSF ont été observées, montrant un enrichissement important en acide palmitique, palmitoléique, oléique, vaccénique et linoléique chez les sujets ostéonécrotiques. Ces modifications reflètent un changement du microenvironnement osseux chez ces patients qui pourrait être lié à l’activité sécrétrice des adipocytes médullaires et altérer le fonctionnement des cellules voisines.L’ensemble de nos travaux suggère qu’au sein du microenvironnement osseux, l’accumulation d’adipocytes médullaires pourrait avoir un effet délétère sur les cellules responsables de la formation osseuse, notamment via la libération d’AGL. Ces AGL étant cytotoxiques pour les cellules ostéoformatrices mais bénéfiques pour les cellules responsables de la résorption osseuse, ils pourraient favoriser un déséquilibre du remodelage osseux. En lien avec ces observations, nos résultats obtenus avec les ON-MSC suggèrent également que la lipotoxicité pourrait participer aux mécanismes pathogéniques qui initient et/ou entretiennent l’ostéonécrose., Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

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

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

While pioneering studies suggested that enhanced glycolysis, a hallmark of cancer, was caused by an irreversible impairment in mitochondrial respiration, more recent data reported functional mitochondrial activity in many cancer cells. In this thesis, we thus intended to investigate whether metabolic modulations could improve the therapeutic outcome of cancer. We found that dichloroacetate, a new clinically tested compound, induced a switch of glycolytic cancer cells to a more oxidative phenotype, and decreased tumor cell proliferation through a decrease in the activity of the pentose phosphate pathway. In a second part of this work, we investigated whether targeting mitochondrial respiration with H2S, the last gaseous transmitter identified in mammals, improved tumor response to radiotherapy. By rapidly alleviating hypoxia inside solid tumors, the single injection of a H2S donor increased the radioresponse of cancer in mice. Overall, our thesis work emphasizes the ability of cancer cells to remodel their energetic metabolism in response to external stimuli and supports that both glycolysis and oxidative phosphorylation are attractive targets for cancer therapy. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2016

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

Glucose stimulation of insulin secretion by pancreatic β cells is essential to maintain glucose homeostasis. In addition to this short-term effect, glucose is also important to maintain β cell survival and differentiated phenotype. Indeed, both chronic hypo- and hyper-glycemia alter β-cell gene expression, survival and function. Similarly, in vitro, rat β-cell gene expression, function and survival are optimally preserved by culture in the presence of 10 mM glucose (G10) and markedly impaired by culture in either lower (2 or 5 mmol/l glucose, G2 or G5) or higher (30 mmol/l glucose, G30) glucose concentrations. It therefore seems that glucose stimulation exerts beneficial effects on β cell function and survival between G2 and G10 and a deleterious effect on these parameters between G10 and G30. The precise molecular mechanisms behind such phenotypical plasticity are poorly understood, but they could involve an increase in oxidative stress. Indeed, mRNA levels of oxidative stress-response genes like Metallothionein 1a/2a (Mt1a/2a), Heme oxygenase 1 (Hmox1) or c-Mycfollow an asymmetric V-shaped profile similar to that of β cell dysfunction and apoptosis. We therefore hypothesized that extreme glucose concentrations increases oxidative stress in β cells. In a first study, we developed a method to measure the β cell redox status in our experimental conditions. Our results showed that mt-HyPer, besides its expected sensitivity to H2O2, was also highly pH-sensitive. As glucose stimulation increases mitochondrial pH in β cells, that probe could not be used in our experimental model. In contrast, the fluorescence ratio of mt-roGFP1, which measures the thiol/disulfide equilibrium, was only slightly affected by pH. Using that probe, we demonstrated that mitochondrial thiol oxidation in rat β cells reversibly increases when glucose is lowered from 10 to 2 mmol/l. In contrast, acutely increasing glucose concentration from 10 to 30 mmol/l did not increase mt-roGFP1 oxidation in β cell. In a second study, I first demonstrated that 18 to 24h culture of rat islet cell clusters in the présence of G5 or G30 vs. G10 increases mitochondrial glutathione oxidation, thereby confirming our initial hypothesis. I also tested the effect of ZnCl2, a potent inducer of Mt1a, on β cell alterations induced by prolonged exposure to low and high glucose concentrations. My results show that addition of 50μM ZnCl2partially reduced mt-roGFP oxidation after 18-24h culture in G5, and tended to do so after culture in G30.Addition of 100μM ZnCl2also significantly decreased late β-cell apoptosis after prolonged culture in G5 or G30. Theses protective effects of ZnCl2 did not correct β cell dysfunction induced by culture in G5 and G30. In a third study, using islets from Mt1/2 knock-out mice, I wanted to determine the role of MT1/2 on the beneficial effects of Zn2+. I also wanted to test whether the deficiency in Mt1/2 increases β cell apoptosis and mitochondrial glutathione oxidation induced by culture in a low glucose concentration. In contrast with what I observed in rat islets, ZnCl2, despite increasing Mt1expression, did not protect wild-type mouse islets from apoptosis induced by culture in a low glucose concentration, thereby preventing me from studying the role of MT1/2 expression in this effect. Moreover, deficiency in Mt1/2 in mouse islets did not increase apoptosis induced by culture in a low glucose concentration. These results help us to understand the molecular mechanisms underlying the plasticity of the β cell phenotype and may help in the development of new therapeutic strategies for T2D. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2013

Adipose tissue (AT) has been recognized as an active secretory organ, which releases a number of bioactive peptides, collectively named adipokines. They play a central role in energy and vascular homeostasis as well as in immunity. Dysregulation of adipokines triggers the development of a low-grade pro-inflammatory state, which is considered to build the common soil for the development of obesity-linked disorders. Thus, resetting the immunological balance in obese AT is a fundamental therapeutic strategy for the metabolic syndrome. ApN has emerged as a master regulator of immune/inflammatory homeostasis. Yet, the autocrine/paracrine effects of ApN on AT inflammation have been poorly investigated. We have previously generated a transgenic mouse model allowing persistent and moderate overexpression of ApN (ApN-Overex) specifically in white AT. I therefore took advantage of this unique model to unravel the effects of ApN on AT secretory function. I confirmed that ApN regulated in vivo the secretion of downstream adipokines, decreasing those with pro-inflammatory properties, while enhancing those with anti-inflammatory effects. These changes were specific: they occurred before the emergence of any metabolic confounding factors (such as improvement of insulin action or decrease of fat mass). Moreover, a reverse profile of expression was observed for most adipokines in ApN-knockout (ApN-KO) mice. Thus ApN induced in vivo a shift of the immune balance in AT toward a less inflammatory phenotype. Moreover, this shift induced by ApN was associated with reduced activity of NF-κB and Extracellular Signal-Regulated kinases (ERK1/2), as well as increased activity/expression of AMPK in AT. MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression by inducing target mRNA degradation or blocking translation. Deregulation of miRNAs is closely associated with obesity-related metabolic disorders. I therefore used the same ApN-Overex mice to investigate whether miRNAs regulated by ApN contribute to the anti-inflammatory effects of ApN in AT and whether these miRNAs represent novel mechanisms for controlling AT inflammation. Four miRNAs were modified in AT of ApN-Overex mice. Expression of miR532-5p and miR1983 was down-regulated, while that of miR883b-5p and miR1934 was up-regulated. miR883b-5p was identified by computational analysis as being involved in inflammatory pathways. By using gain-of or loss-of function approaches, I further validated that miR883b-5p repressed the lipopolysaccharide (LPS) facilitator, LPS-binding protein (LBP) in 3T3-F442A cells. miR883b-5p blockade also abolished the protective effects of ApN on induction of pro-inflammatory adipokines. These data were recapitulated in de novo AT (formed from engineered 3T3-F442A preadipocytes transplanted in nude mice) where miR883b-5p silencing induced LBP production and tissue inflammation. Eventually, miR883b-5p expression was down-regulated in AT of obese subjects, concomitantly with reduced ApN expression. Altogether, these data suggest that miR883b-5p may be an important mediator of the anti-inflammatory action of ApN in adipocytes. These newly identified downstream adipokines and miRNAs may open new therapeutic perspectives for the management of metabolic syndrome. (SBIM 3) -- UCL, 2012

A promising way to cure diabetes is to implant in vitro produced β-cells, or to induce β-cell regeneration in vivo. Multiple attempts have been made to coax different cell types into β-cells. One of these cell types is the pancreatic duct cell, but its potential as a β-cell precursor remains unclear. In this work we examine the plasticity of duct cells in mice deficient for Hepatocyte Nuclear Factor 6 (HNF6), a transcriptional regulator of pancreatic cell differentiation. In hnf6-/- mice, the number of β-cells is reduced at birth, yet it subsequently undergoes a partial recovery. We show in these mice that a postnatalβ-cell neogenesis occurs. Neogenesis neither results from a higher proliferation rate of mature β-cells nor from delayed differentiation of β-cell precursors. The presence of transition cells that co-express insulin and duct markers, as well as genetic lineage tracing, indicate that duct cells transdifferentiate postnatally to β-cells in hnf6-/- mice. Therefore, our results provide evidence that in the absence of the transcription factor HNF6, duct cells have the potential to generate β-cells. This suggests new strategies for the production of β-cells from duct cells. (SBIM 3) -- UCL, 2010