The plant hormone ethylene plays an important role in plant growth, development, and physiology. One of the critical components of the ethylene signal transduction pathway, ctr1 (constitutive triple response), was identified using a particularly useful seedling screen that takes advantage of the profound effects ethylene has on etiolated seedlings, known as triple response. CTR1 is one of six Arabidopsis MAPKKKs that are related to the Raf kinases, and acts as a negative regulator of ethylene response. In this study, isolation and characterization of a family of CTR1-like genes in tomato is reported. Based on amino acid alignments and phylogenetic analysis, the tomato CTR1-like (LeCTR) genes are more similar to Arabidopsis CTR1 (AtCTR1) than any other MAPKKK sequences in the Arabidopsis genome. The capacity of the LeCTR genes to function as negative regulators in ethylene signal transduction was tested through complementation of the Arabidopsis ctr1-8 mutant. Quantitative real-time PCR was carried out to generate an expression profile for the CTR1-like gene family during different stages of development marked by increased ethylene biosynthesis, including fruit ripening. The possibility of a multi-gene family of CTR1-like genes in other species besides tomato was examined through mining of EST and genomic sequence databases. Based on nucleotide and amino acid identity, At4g24480 is most similar to AtCTR1 and could potentially represent a CTR1-like gene in Arabidopsis. Arabidopsis plants carrying a T-DNA insert in the At4g24480 locus were examined for abnormal ethylene response phenotypes including sensitivity to other hormones, signal molecules and abiotic stresses. Two mutant alleles, ctr1-1 and ctr1-8, containing mutations that disrupt kinase activity and receptor association, respectively, were examined for sensitivity to these same treatments in an effort to better characterize ethylene hormone and non-hormone interactions. They also served as controls to determine if At4g24480 indeed possessed CTR1-like function. Arabidopsis and tomato represent species with very distinct fruit ripening/maturation programs. The critical dependence on ethylene for fruit ripening in tomato might have resulted in alteration or modification of the ethylene signal transduction pathway relative to Arabidopsis. Plans to characterize individual functions of the LeCTR genes through over-expression and reduced expression in tomato are outlined.

Interleukin-33 (IL-33) is a multifunctional cytokine that mediates local inflammation upon tissue damage. IL-33 is known to act on multiple cell types including group 2 innate lymphoid cells (ILC2s), Th2 cells, and mast cells to drive production of Th2 cytokines including IL-5 and IL-13. IL-33 signaling activity through transmembrane ST2L can be inhibited by soluble ST2 (sST2), which acts as a decoy receptor. Previous findings suggested that modulation of IL-13 levels in mice lacking decoy IL-13Rα2, or mice lacking IL-13, impacted responsiveness to IL-33. In this study, we used Il13-/- mice to investigate whether IL-13 regulates IL-33 activity by modulating the transmembrane and soluble forms of ST2. In Il13-/- mice, the effects of IL-33 administration were exacerbated relative to wild type (WT). Il13-/- mice administered IL-33 i.p. had heightened splenomegaly, more immune cells in the peritoneum including an expanded ST2L+ ILC2 population, increased eosinophilia in the spleen and peritoneum, and reduced sST2 in the circulation and peritoneum. In the spleen, lung, and liver of mice given IL-33, gene expression of both isoforms of ST2 was increased in Il13-/- mice relative to WT. Because IL-13 and IL-4 signal through a shared receptor complex IL-13Rα1/IL-4Rα, we also studied the combined deficiency of IL-4 and IL-13 using Il4rα-/- mice which are defective in both IL-4 and IL-13 signaling. Responses of Il4rα-/- mice were indistinguishable from those of Il13-/- mice in our model system of IL-33-induced inflammation, suggesting that IL-4 does not play a distinct role separate from IL-13 in regulation of IL-33 activity. Through in vitro experiments, we confirmed fibroblasts to be an IL-13-responsive cell type that can regulate IL-33 activity through production of sST2. This study elucidates the important regulatory activity that IL-13 exerts on IL-33 through induction of IL-33 decoy receptor sST2 and through modulation of ST2L+ ILC2s.

Astrocytic tumors are the most common malignancies of the central nervous system, accounting for more than 60% of all primary brain tumors. The prognosis for high grade astrocytoma patients is dismal and there is no curative treatment, today. A molecular hallmark of astrocytic tumors is dysregulated receptor tyrosine kinase signaling, especially of the epidermal growth factor receptor (EGFR, ErbB1). The aim of the present thesis was to identify endogenous human proteins that downregulate the function of the ErbB1 receptor. We identified a human gene family, the leucine-rich repeats and immunoglobulin-like domains family (LRIG), consisting of LRIG1, LRIG2 and LRIG3, which might fulfill this criterion. Two candidates were identified, LRIG1 and LRIG2, which genes were localized to regions frequently deleted in human cancers, chromosome bands 3p14 and 1p13, respectively. LRIG1 and LRIG2 mRNA were expressed in all tissues analyzed, with high expression in brain and other organs. The LRIG mRNA were predicted to encode integral membrane proteins. Antibodies against LRIG1 and LRIG2 were developed and the protein expression was analyzed. LRIG1 was found to have an apparent molecular weight of 143 kDa and was expressed in most tissues with high expression in glandular tissues of the breast and prostate, and the peptic cells of the stomach. LRIG2 was slightly smaller and had an apparent molecular weight of 132 kDa. The LRIG proteins were localized to the cell surface and to perinuclear structures, where LRIG1 co-localized with the trans-Golgi network and early endosomes. LRIG1 was found to restrict growth factor signaling by enhancing receptor ubiquitylation and degradation. We showed that LRIG1 interacted with all four members of the ErbB family and induced their downregulation. The interaction with ErbB1 involved both the LRR-domains and the Ig-like domains of LRIG1. LRIG1 enhanced receptor degradation by recruiting the E3 ubiquitin ligase c-Cbl to the LRIG1-ErbB1 complex. LRIG1, LRIG2, and LRIG3 were expressed in glioma cell lines and tumor tissues. The LRIG expression was analyzed in 404 astrocytic tumor samples. We found that perinuclear LRIG protein expression correlated with increased survival of patients with astrocytic tumors. Especially perinuclear LRIG3 showed strong correlations with patient survival and tumor cell proliferation index. In summary, this thesis contains the discovery and characterization of human LRIG1 and LRIG2. LRIG1 was found to interact with ErbB receptors and downregulate their function. In a clinical material, expression of LRIG proteins correlated with survival in patients with astrocytic tumors.

Giardia lamblia, a worldwide cause of diarrhoea, must differentiate into environmentally resistant cysts for dissemination and completion of its life cycle. Although G. lamblia is an early diverging eukaryote, encystation involves many complex cellular changes including formation of the cyst wall that contains at least two cyst wall proteins, cyst wall proteins 1 and 2. Cwp genes are transcribed only during encystation. In this study, we examine the regulatory elements for the encystation-specific gene cwp2. The 64 bp immediately upstream of the cwp2 open reading frame (-64 to -1 relative to ATG) was shown to be sufficient for the encystation-specific expression of luciferase. To determine which region(s) within this 64 bp contributed to encystation-specific expression in vivo, a series of deletions were cloned into a Giardia luciferase expression vector and their ability to control encystation-specific expression of luciferase was assessed. Deletion of elements in the -64 to -23 region of the cwp2 promoter significantly increased expression of luciferase in vegetative trophozoites, suggesting that this area contains a negative cis-acting element. Deletions of elements from -23 to -10 led to decreased expression in encysting cells, suggesting that this region may contain positive cis-acting elements. When the A/T-rich initiator was deleted but the cis-acting elements (-64 to -10) were retained, encystation-specific expression of luciferase was maintained but an aberrant transcriptional start site was utilised. These results indicate that Giardia has developed a classic repressor mechanism(s) that allows tight, encystation-specific control by the cwp2 promoter.

No description available.

Η Αυξητική ορμόνη παίζει σημαντικό ρόλο στη μεταγεννητική κατά μήκος αύξηση, στη σκελετική ανάπτυξη, στο μεταβολισμό των πρωτεϊνών, των λιπών και των υδατανθράκων, στην οστική ανακύκλωση και την ανοσιακή λειτουργία. Διαταραχή στην έκκριση ή στη δράση της ορμόνης στα παιδιά προκαλεί, μεταξύ άλλων, ανεπάρκεια αύξησης. Έχουν περιγραφεί αρκετές κλινικές οντότητες ανεπάρκειας αύξησης, που οφείλονται κυρίως σε διαταραχές στην υποφυσιακή έκκριση GH, στην 24ωρη αυθόρμητη έκκριση της GH, στον αριθμό ή τη λειτουργία των υποδοχέων GHR, στη μετά τον υποδοχέα μεταβίβαση του σήματος της GH και στη σύνθεση ή δράση του IGF-I. Η παρούσα μελέτη εξέτασε έναν ασθενή με Διαταραχή στη Μεταγωγή του Σήματος της GH (Growth Hormone Transduction Defect/GHTD). Η οντότητα αυτή χαρακτηρίζεται από σοβαρό κοντό ανάστημα με φυσιολογικές δοκιμασίες φαρμακολογικής πρόκλησης, φυσιολογικές τιμές 24ωρης έκκρισης GH, χαμηλά επίπεδα IGF-I, διαταραχή στη φωσφορυλίωση του μεταγραφικού παράγοντα STAT3 και υπερέκφραση του αναστολέα του κυτταρικού κύκλου p21. Επιπλέον, οι ασθενείς με GHTD παρουσιάζουν σημαντικά αυξημένα επίπεδα IGF-I μετά από επαγωγή με hGH κατά τo IGF-I generation test και σημαντική αναπλήρωση αύξησης μετά από θεραπεία με hGH. Επίσης, χαρακτηρίζονται από απουσία μεταλλάξεων στην πρωτεΐνη STAT3, στον υποδοχέα GHR και στο γονίδιο GH1. Χρησιμοποιήθηκαν πρωτογενείς καλλιέργειες ινοβλαστών από ούλα του προς μελέτη ασθενή κι ενός μάρτυρα. Μελετήθηκαν σηματοδοτικά μόρια του μεταγωγικού μονοπατιού της GH και του μονοπατιού αρνητικής ρύθμισης, και διερευνήθηκε ο ρόλος της πρωτεΐνης CIS στην παθολογική μεταβίβαση του σήματος της GH στον ασθενή, καθώς και η επίδραση της καταστολής του γονιδίου CIS στη σηματοδότηση της GH. Επίσης, διερευνήθηκε η πιθανή διασυνομιλία ανάμεσα στα σηματοδοτικά μονοπάτια της GH και του EGF, καθώς και ο ρόλος της διασυνομιλίας αυτής στην αποκατάσταση της φυσιολογικής σηματοδότησης της GH και, κατ’επέκταση, στην κλινική ανταπόκριση μετά από θεραπεία με εξωγενώς χορηγούμενη ανθρώπινη βιοσυνθετική ορμόνη, παιδιών με GHTD. Η πρωτεϊνική έκφραση των μελετηθέντων πρωτεϊνών μελετήθηκε με ανοσοαποτύπωση κατά Western, η κυτταρική εντόπισή τους με ανοσοφθορισμό και η διαντίδραση ορισμένων από τις πρωτεΐνες με ανοσοσυγκατακρήμνιση. Τα ευρήματα της εργασίας στοιχειοθετούν την αρχική υπόθεση ότι η διαταραγμένη μεταβίβαση του σήματος της GH στα παιδιά με GHTD διαμεσολαβείται μέσω της υπερέκφρασης της ουβικουιτινυλιωμένης μορφής της πρωτεΐνης CIS, η οποίθα προκαλεί ραγδαία και εκσεσημασμένη μεταφορά του GHR στο πρωτεάσωμα για αποδόμηση. Τα αποτελέσματα επίσης έδειξαν ότι η αποκατάσταση της φυσιολογικής σηματοδότησης της GH μετά τη σίγαση του γονιδίου CIS περιλαμβάνει την επαναφορά του GHR στην κυτταροπλασματική μεμβράνη για φυσιολογική ενεργοποίηση από την GH, καθώς και την ενεργοποίηση του σηματοδοτικού μονοπατιού του EGFR. Επιπροσθέτως, υπάρχει έντονη διασυνομιλία μεταξύ των σηματοδοτικών μονοπατιών της GH και του EGF κατά τη χορήγηση εξωγενούς hGH στα παιδιά με GHTD, με αποτέλεσμα την επιτάχυνση της αύξησης που παρατηρείται στα παιδιά αυτά μετά από θεραπεία με hGH. / Growth Hormone (GH) plays an important role in postnatal linear growth, skeletal development, protein, lipid and carbohydrate metabolism, bone turnover and immune function. Defects in the GH secretion and function in children can cause growth retardation. Several clinical entities of growth retardation have been described, including defects in pituitary GH secretion, spontaneous 24h GH secretion, GH receptor number or function, post-receptor signaling and IGF-I synthesis or function. In this study, one patient with Growth Hormone Transduction Defect (GHTD) was studied. GHTD is characterized by severe short stature with normal provoked and spontaneous GH secretion, low IGF-I concentrations, impaired phosphorylation of the transcriptional factor STAT3 and overexpression of the cyclin-dependent kinase inhibitor, p21. Furthermore, GHTD patients have significantly increased IGF-I concentrations after induction with hGH during the IGF-I generation test, and significant ‘catch-up’ growth after hGH therapy. No mutations were found in STAT3, GHR and GH1 gene in the GHTD patients. Primary fibroblast cultures were established from gingival biopsies obtained from the GHTD patient and one control. The GH signaling molecules and the negative regulators of GH were studied, as well as the role of protein CIS in the impaired GH signaling and the effect of CIS silencing on GH signaling. Furthermore, the possible crosstalk between the GH and EGF signaling cascades was examined, as well as its role in the restoration of the impaired GH signaling and the clinical response after therapy with exogenous hGH. The protein expression of the studied molecules was studied by Western Immunoblotting, their cellular localization by Immunofluoresence and the protein-protein interactions by Co-immunoprecipitation. The results of this study support the hypothesis that impaired GH signaling in GHTD children is mediated by the overexpression of ubiquitinated CIS, which causes rapid and excessive translocation of the GHR to the proteasomes for degradation. The results also showed that the restoration of physiological GH signaling after the silencing of CIS involves the restoration of the GHR to the plasma membrane for normal activation by GH, as well as the activation of the EGFR pathway. In addition, there is vigorous crosstalking between the GH and EGF signaling pathways during exogenous hGH treatment in the GHTD children, resulting in the accelerated growth seen in these children after hGH therapy.

IL-1 receptor/Toll-like receptor (IL-1R/TLR) supefamily represents a group of proteins that share highly conserved TIR domain in their cytoplasmic region. Signal transduction mediated by TIR-containing proteins involves the activation of NF-κB transcription factor and thus the members of this superfamily play a key role in many physiological responses related to innate immune defense and inflammation. SIGIRR (single immunoglobulin IL1R-related molecule) is a recently discovered member of the IL-1R family, however it differs from the other group members by its unique structural features. SIGIRRhas been so far considered to be an 'orphan' receptor as no SIGIRR ligand has been identified yet. Moreover, SIGIRR itself is not capable to induce the NF-κB activation. Instead, SIGIRR is supposed to act as a negative regulator for IL- 1Rs/TLRs mediated inflammation. Its inhibitory function has been implemented in several signalling pathways in various cell types and tissues including the kidney, the digestive tract and the lung. Recent reports also suggest that SIGIRR could play a role in early embryonic development. The main aim of this thesis is to characterize the mechanism how SIGIRR negative regulatory function in IL-1R/TLR signalling pathway is delivered. Here we describe the establishment of...

In animals, some of the most critical regulators of gene expression are nuclear hormone receptors (NRs) and their coregulators, specifically the Mediator complex. Of particular interest are the NRs implicated in metabolic and developmental regulation and in carcinogenesis: thyroid hormone receptors (TRs) and retinoid X receptors (RXRs). In this work, I venture to elucidate some aspects of gene expression regulation by these NRs: the degree of evolutionary conservation of signalling based on NRs and their coregulators; the mechanisms of negative regulation by NRs; and possible implications of these findings for clinical medicine. State-of-the-art bioinformatical, genome editing and microscopic techniques are applied at three levels of animal evolution to study NRs and Mediator. Reverse genomics in human patients suffering from the syndrome of resistance to thyroid hormones β are used to infer the structure and function of TRβ subdomains. Alignments, binding studies and in vivo experiments in Trichoplax adhaerens allow identification of a close orthologue of human RXR at the basis of metazoan evolution. Employing database queries, genome editing and microscopy, we describe a correct orthologue of the Mediator subunit 28 in Caenorhabditis elegans, indicating a complete homology of the Mediator complex...

Le diabète de type 2 (DT2) est une maladie métabolique complexe causée par des facteurs génétiques mais aussi environnementaux, tels la sédentarité et le surpoids. La dysfonction de la cellule β pancréatique est maintenant reconnue comme l’élément déterminant dans le développement du DT2. Notre laboratoire s’intéresse à la sécrétion d’insuline par la cellule β en réponse aux nutriments calorigéniques et aux mécanismes qui la contrôle. Alors que la connaissance des mécanismes responsables de l’induction de la sécrétion d’insuline en réponse aux glucose et acides gras est assez avancée, les procédés d’inhibition de la sécrétion dans des contextes normaux ou pathologiques sont moins bien compris. L’objectif de la présente thèse était d’identifier quelques-uns de ces mécanismes de régulation négative de la sécrétion d’insuline dans la cellule β pancréatique, et ce en situation normale ou pathologique en lien avec le DT2. La première hypothèse testée était que l’enzyme mitochondriale hydroxyacyl-CoA déshydrogénase spécifique pour les molécules à chaîne courte (short-chain hydroxyacyl-CoA dehydrogenase, SCHAD) régule la sécrétion d’insuline induite par le glucose (SIIG) par la modulation des concentrations d’acides gras ou leur dérivés tels les acyl-CoA ou acyl-carnitine dans la cellule β. Pour ce faire, nous avons utilisé la technologie des ARN interférants (ARNi) afin de diminuer l’expression de SCHAD dans la lignée cellulaire β pancréatique INS832/13. Nous avons par la suite vérifié chez la souris DIO (diet-induced obesity) si une exposition prolongée à une diète riche en gras activerait certaines voies métaboliques et signalétiques assurant une régulation négative de la sécrétion d’insuline et contribuerait au développement du DT2. Pour ce faire, nous avons mesuré la SIIG, le métabolisme intracellulaire des lipides, la fonction mitochondriale et l’activation de certaines voies signalétiques dans les îlots de Langerhans isolés des souris normales (ND, normal diet) ou nourries à la dière riche en gras (DIO) Nos résultats suggèrent que l’enzyme SCHAD est importante dans l’atténuation de la sécrétion d’insuline induite par le glucose et les acides aminés. En effet, l’oxydation des acides gras par la protéine SCHAD préviendrait l’accumulation d’acyl-CoA ou de leurs dérivés carnitine à chaîne courtes potentialisatrices de la sécrétion d’insuline. De plus, SCHAD régule le métabolisme du glutamate par l’inhibition allostérique de l’enzyme glutamate déshydrogénase (GDH), prévenant ainsi une hyperinsulinémie causée par une sur-activité de GDH. L’étude de la dysfonction de la cellule β dans le modèle de souris DIO a démontré qu’il existe une grande hétérogénéité dans l’obésité et l’hyperglycémie développées suite à la diète riche en gras. L’orginialité de notre étude réside dans la stratification des souris DIO en deux groupes : les faibles et forts répondants à la diète (low diet responders (LDR) et high diet responder (HDR)) sur la base de leur gain de poids corporel. Nous avons mis en lumières divers mécanismes liés au métabolisme des acides gras impliqués dans la diminution de la SIIG. Une diminution du flux à travers le cycle TG/FFA accompagnée d’une augmentation de l’oxydation des acides gras et d’une accumulation intracellulaire de cholestérol contribuent à la diminution de la SIIG chez les souris DIO-HDR. De plus, l’altération de la signalisation par les voies AMPK (AMP-activated protein kinase) et PKC epsilon (protéine kinase C epsilon) pourrait expliquer certaines de ces modifications du métabolisme des îlots DIO et causer le défaut de sécrétion d’insuline. En résumé, nous avons mis en lumière des mécanismes importants pour la régulation négative de la sécrétion d’insuline dans la cellule β pancréatique saine ou en situation pathologique. Ces mécanismes pourraient permettre d’une part de limiter l’amplitude ou la durée de la sécrétion d’insuline suite à un repas chez la cellule saine, et d’autre part de préserver la fonction de la cellule β en retardant l’épuisement de celle-ci en situation pathologique. Certaines de ces voies peuvent expliquer l’altération de la sécrétion d’insuline dans le cadre du DT2 lié à l’obésité. À la lumière de nos recherches, le développement de thérapies ayant pour cible les mécanismes de régulation négative de la sécrétion d’insuline pourrait être bénéfique pour le traitement de patients diabétiques. / Type 2 diabetes (T2D) is a complex metabolic disease caused by genetic as well as environmental factors, such as sedentarity and obesity. Pancreatic β cell dysfunction is now recognized as the key factor in T2D development. Our laboratory is studying the mechanisms of regulation of insulin secretion by the pancreatic β cell in response to nutrients. While the knowledge of the mechanisms responsible for initiation of insulin secretion in response to glucose and fatty acids is quite advanced, the inhibitory processes of insulin secretion in normal or pathological situations are still poorly understood. This doctoral thesis has focused on the identification of some of the mechanisms responsible for negative regulation of insulin secretion in pancreatic β cell. We have addressed this issue under normal situation or pathological conditions related to T2D. We first tested the hypothesis by which a mitochondrial enzyme, short-chain hydroxyacyl-CoA dehydrogenase (SCHAD), negatively regulates glucose-induced insulin secretion (GIIS) by limiting the concentrations of some fatty acids and their derivatives such as acyl-CoA or acyl-carnitine molecules in the β cell. For this purpose, the downregulation of SCHAD by RNA interference (RNAi) was used in the pancreatic β cell line INS832/13. Then, we tested wether a prolonged administration of high-fat diet to mice (diet-induced obesity mouse model, DIO) would modulate intracellular metabolic and molecular pathways responsible for inhibition of insulin secretion. C57BL/6 mice were therefore fed a high-fat diet for 8 weeks followed by insulin secretion, intracellular lipid metabolism, mitochondrial function and intracellular signaling measurements on isolated pancreatic islets of Langerhans of those mice. Our results suggest that SCHAD negatively regulates GIIS and amino acid-induced insulin secretion. We propose that fatty acid oxidation by SCHAD would prevent the accumulation of short-chain acyl-CoAs or acyl-carnitines capable of potentiating insulin secretion. In addition, SCHAD regulates glutamate metabolism by the allosteric inhibition of glutamate dehydrogenase (GDH) preventing the hyperinsulinemia caused by excessive GDH activity. The study of β cell dysfunction in the DIO mouse model stratified LDR and HDR highlighted various fatty acid metabolism pathways involved in the reduction of GIIS. A decrease in the triglycerides/free fatty acid (TG/FFA) cycling associated with an increase in fatty acid oxidation and intracellular accumulation of cholesterol was shown to contribute to the decreased GIIS in DIO-HDR mice. Furthermore, alteration of AMP-activated kinase (AMPK) and protein kinase C epsilon (PKC epsilon) signaling pathways would be responsible for those alterations in metabolic pathways observed in DIO islets and cause decreased insulin secretion. In summary, we have shed light on important pathways negatively regulating insulin secretion in pancreatic β cell. These pathways could either limit the amplitude or duration of insulin secretion after a meal, or help to preserve β-cell function by delaying exhaustion. Some of those signaling pathways could explain the altered insulin secretion observed in T2D obese patients. In light of our research, the development of therapies targeting pathways that negatively regulate insulin secretion may be beneficial for treating diabetic patients.

Le diabète de type 2 (DT2) est une maladie métabolique complexe causée par des facteurs génétiques mais aussi environnementaux, tels la sédentarité et le surpoids. La dysfonction de la cellule β pancréatique est maintenant reconnue comme l’élément déterminant dans le développement du DT2. Notre laboratoire s’intéresse à la sécrétion d’insuline par la cellule β en réponse aux nutriments calorigéniques et aux mécanismes qui la contrôle. Alors que la connaissance des mécanismes responsables de l’induction de la sécrétion d’insuline en réponse aux glucose et acides gras est assez avancée, les procédés d’inhibition de la sécrétion dans des contextes normaux ou pathologiques sont moins bien compris. L’objectif de la présente thèse était d’identifier quelques-uns de ces mécanismes de régulation négative de la sécrétion d’insuline dans la cellule β pancréatique, et ce en situation normale ou pathologique en lien avec le DT2. La première hypothèse testée était que l’enzyme mitochondriale hydroxyacyl-CoA déshydrogénase spécifique pour les molécules à chaîne courte (short-chain hydroxyacyl-CoA dehydrogenase, SCHAD) régule la sécrétion d’insuline induite par le glucose (SIIG) par la modulation des concentrations d’acides gras ou leur dérivés tels les acyl-CoA ou acyl-carnitine dans la cellule β. Pour ce faire, nous avons utilisé la technologie des ARN interférants (ARNi) afin de diminuer l’expression de SCHAD dans la lignée cellulaire β pancréatique INS832/13. Nous avons par la suite vérifié chez la souris DIO (diet-induced obesity) si une exposition prolongée à une diète riche en gras activerait certaines voies métaboliques et signalétiques assurant une régulation négative de la sécrétion d’insuline et contribuerait au développement du DT2. Pour ce faire, nous avons mesuré la SIIG, le métabolisme intracellulaire des lipides, la fonction mitochondriale et l’activation de certaines voies signalétiques dans les îlots de Langerhans isolés des souris normales (ND, normal diet) ou nourries à la dière riche en gras (DIO) Nos résultats suggèrent que l’enzyme SCHAD est importante dans l’atténuation de la sécrétion d’insuline induite par le glucose et les acides aminés. En effet, l’oxydation des acides gras par la protéine SCHAD préviendrait l’accumulation d’acyl-CoA ou de leurs dérivés carnitine à chaîne courtes potentialisatrices de la sécrétion d’insuline. De plus, SCHAD régule le métabolisme du glutamate par l’inhibition allostérique de l’enzyme glutamate déshydrogénase (GDH), prévenant ainsi une hyperinsulinémie causée par une sur-activité de GDH. L’étude de la dysfonction de la cellule β dans le modèle de souris DIO a démontré qu’il existe une grande hétérogénéité dans l’obésité et l’hyperglycémie développées suite à la diète riche en gras. L’orginialité de notre étude réside dans la stratification des souris DIO en deux groupes : les faibles et forts répondants à la diète (low diet responders (LDR) et high diet responder (HDR)) sur la base de leur gain de poids corporel. Nous avons mis en lumières divers mécanismes liés au métabolisme des acides gras impliqués dans la diminution de la SIIG. Une diminution du flux à travers le cycle TG/FFA accompagnée d’une augmentation de l’oxydation des acides gras et d’une accumulation intracellulaire de cholestérol contribuent à la diminution de la SIIG chez les souris DIO-HDR. De plus, l’altération de la signalisation par les voies AMPK (AMP-activated protein kinase) et PKC epsilon (protéine kinase C epsilon) pourrait expliquer certaines de ces modifications du métabolisme des îlots DIO et causer le défaut de sécrétion d’insuline. En résumé, nous avons mis en lumière des mécanismes importants pour la régulation négative de la sécrétion d’insuline dans la cellule β pancréatique saine ou en situation pathologique. Ces mécanismes pourraient permettre d’une part de limiter l’amplitude ou la durée de la sécrétion d’insuline suite à un repas chez la cellule saine, et d’autre part de préserver la fonction de la cellule β en retardant l’épuisement de celle-ci en situation pathologique. Certaines de ces voies peuvent expliquer l’altération de la sécrétion d’insuline dans le cadre du DT2 lié à l’obésité. À la lumière de nos recherches, le développement de thérapies ayant pour cible les mécanismes de régulation négative de la sécrétion d’insuline pourrait être bénéfique pour le traitement de patients diabétiques. / Type 2 diabetes (T2D) is a complex metabolic disease caused by genetic as well as environmental factors, such as sedentarity and obesity. Pancreatic β cell dysfunction is now recognized as the key factor in T2D development. Our laboratory is studying the mechanisms of regulation of insulin secretion by the pancreatic β cell in response to nutrients. While the knowledge of the mechanisms responsible for initiation of insulin secretion in response to glucose and fatty acids is quite advanced, the inhibitory processes of insulin secretion in normal or pathological situations are still poorly understood. This doctoral thesis has focused on the identification of some of the mechanisms responsible for negative regulation of insulin secretion in pancreatic β cell. We have addressed this issue under normal situation or pathological conditions related to T2D. We first tested the hypothesis by which a mitochondrial enzyme, short-chain hydroxyacyl-CoA dehydrogenase (SCHAD), negatively regulates glucose-induced insulin secretion (GIIS) by limiting the concentrations of some fatty acids and their derivatives such as acyl-CoA or acyl-carnitine molecules in the β cell. For this purpose, the downregulation of SCHAD by RNA interference (RNAi) was used in the pancreatic β cell line INS832/13. Then, we tested wether a prolonged administration of high-fat diet to mice (diet-induced obesity mouse model, DIO) would modulate intracellular metabolic and molecular pathways responsible for inhibition of insulin secretion. C57BL/6 mice were therefore fed a high-fat diet for 8 weeks followed by insulin secretion, intracellular lipid metabolism, mitochondrial function and intracellular signaling measurements on isolated pancreatic islets of Langerhans of those mice. Our results suggest that SCHAD negatively regulates GIIS and amino acid-induced insulin secretion. We propose that fatty acid oxidation by SCHAD would prevent the accumulation of short-chain acyl-CoAs or acyl-carnitines capable of potentiating insulin secretion. In addition, SCHAD regulates glutamate metabolism by the allosteric inhibition of glutamate dehydrogenase (GDH) preventing the hyperinsulinemia caused by excessive GDH activity. The study of β cell dysfunction in the DIO mouse model stratified LDR and HDR highlighted various fatty acid metabolism pathways involved in the reduction of GIIS. A decrease in the triglycerides/free fatty acid (TG/FFA) cycling associated with an increase in fatty acid oxidation and intracellular accumulation of cholesterol was shown to contribute to the decreased GIIS in DIO-HDR mice. Furthermore, alteration of AMP-activated kinase (AMPK) and protein kinase C epsilon (PKC epsilon) signaling pathways would be responsible for those alterations in metabolic pathways observed in DIO islets and cause decreased insulin secretion. In summary, we have shed light on important pathways negatively regulating insulin secretion in pancreatic β cell. These pathways could either limit the amplitude or duration of insulin secretion after a meal, or help to preserve β-cell function by delaying exhaustion. Some of those signaling pathways could explain the altered insulin secretion observed in T2D obese patients. In light of our research, the development of therapies targeting pathways that negatively regulate insulin secretion may be beneficial for treating diabetic patients.