This study aimed to examine potential mechanisms for glucocorticoid (GC)-induced decreases in glucose clearance, and to determine if a reduction in oxidative stress load via dietary pre-treatment with an antioxidant-rich diet has a positive net effect on glucose tolerance following a sub-chronic treatment with the GC analogue dexamethasone (DEX). Rats fed a diet supplemented with 700IU of -tocopherol for two weeks had improved glucose clearance after five days of DEX-treatment relative to unsupplemented rats as well as decreased markers of oxidative stress. Following an intraperitoneal bolus of insulin, phosphorylation of AMP activated protein kinase (AMPK) was preserved in the supplemented groups despite no significant differences in upstream insulin signalling cascade intermediates between DEX-treated groups. This was corroborated by a similar increase (p<0.05) in phosphorylation of the downstream AMPK substrate acetyl CoA carboxylase. This study demonstrated that -tocopherol supplementation can attenuate GC-induced decreases in glucose clearance in an AMPK-dependent manner. / Nutrition and Metabolism

Ca2+ is an important second messenger which modulates a variety of signalling pathways in both excitable and non-excitable cells. In the CNS, Ca2+ plays an important role in neurons both physiologically and pathologically. Ca2+ influx following synaptic activity, is important in development, plasticity, redox balance, as well as in neuroprotection, largely through activation of pro-survival pathways downstream of synaptic NMDAR activation, including upregulation of antioxidant defences. However, excessive Ca2+ influx in neurons leads to neuronal damage and excitotoxicity, in which mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniporter (Mcu) resulting in mitochondrial dysfunction is a key player. Excitotoxicity occurs due to glutamate efflux from astrocytes following stroke, traumatic brain injury and in chronic neurodegenerative diseases, leading to excessive neuronal NMDAR activation and triggering of its downstream pro-death pathways. This thesis focuses on understanding the pro-survival and pro-death effects of signalling pathways activated by Ca2+ in neurons, as well as the potential effect of neuronal synaptic activity on influencing neuroprotective gene transcription in astrocytes. I investigated the role of AMPK, a master regulator of metabolism, in NMDA excitotoxicity in cortical neurons as a potential downstream effector of Mcu-dependent excitotoxic death; and found the deletion of AMPKα1/2 to be neuroprotective against NMDA-mediated excitotoxicity, however I found AMPK activation to be independent of Mcu. I also investigated the expression pattern of Mcu and other mitochondrial calcium regulatory genes (MCRGs), and found MCRGs to be differentially expressed in different neural cells (primary neurons vs astrocytes), and neuronal subtypes (CA1 vs CA3 region of the hippocampus), suggesting differing dependence on the various MCRGs in mitochondrial Ca2+ handling in these cell types. A better functional understanding of these genes will allow for investigation of their importance in mitochondrial Ca2+ handling, including their potential role in excitotoxicity. I next investigated the neuroprotective effects of synaptic activity induced Ca2+ influx, focusing on antioxidant target genes of Nrf2, a transcription factor and major regulator of antioxidant genes. I found that unlike astrocytes, neurons express very low levels of Nrf2. However, synaptic activity increased the expression of several Nrf2 target genes in neurons, independently of astrocytes and Nrf2. Additionally, I found no effect of synaptic activity on increasing Nrf2 protein levels, despite previous reports in literature of Nrf2 pathway activation following synaptic activity. Finally, using RNA-seq I identified a list of genes strongly upregulated by a known Nrf2 activator in astrocytes, and found no evidence that neuronal activity triggers expression of these genes independently of neurons, providing further evidence that neuronal activity does not activate the Nrf2 pathway in astrocytes. This suggests that synaptic activity via pathways activated by Ca2+ signalling provides neurons with cell-autonomous antioxidant defences, independently of Nrf2; thus providing a distinct pathway for antioxidant defences in neurons from the Nrf2 pathway, which is activated in astrocytes providing neurons with non-cell autonomous antioxidant support. These results give us further insight into the mechanisms that underlie synaptic and non-synaptic Ca2+ signalling pathways mediating neuronal survival and death, which could help in identifying therapeutic targets to combat excitotoxicity and oxidative stress in various neurological diseases.

This thesis investigates the effects of alanine on rainbow trout’s glucose metabolism at the organismal and molecular levels. Rainbow trout is an important aquaculture species that belongs to the salmonid family. As a carnivorous fish, the requirement of protein/amino acids in trout’s diet is high. In contrast, rainbow trout are poor utilizers of carbohydrates. One prevalent hypothesis suggests that high levels of dietary amino acids could indeed contribute to the poor utilization of carbohydrates in this species. In mammals, there is evidence supporting the importance of alanine as a gluconeogenic precursor. However, a recent study found that alanine stimulates hepatic AMP-activated protein kinase (AMPK) to lower circulating glucose levels in mice. Alanine levels are high in all tissues in rainbow trout. The role of alanine in gluconeogenesis is less clear in trout and there is no evidence, to our knowledge, regarding its effects on glucose kinetics. Therefore, the main goal of the study was to investigate the impact of the continuous infusion of exogenous alanine for 4h on glucose fluxes and to identify potential mechanisms in tissues that could interpret the observed changes in glucose fluxes in vivo. Glucose turnover, appearance and disposal, Rt, Ra and Rd, respectively, were measured to determine the impact of alanine on glucose fluxes. The expression and/or activity of key genes in glucose transport, utilization and gluconeogenesis were assessed in liver and muscle. An additional goal was to assess whether alanine activates AMPK in trout. The levels of phosphorylated AMPK and other signaling proteins known to interact with the latter were quantified. Results show that alanine reduced plasma glucose levels and inhibited Ra and Rd glucose, consistent with previously observed effects of insulin in rainbow trout. The reduction in the expression of a paralogue of glut4, a key gene in glucose transport, and the activity of hexokinase (HK), a key enzyme in glucose utilization, in muscle can partially explain the observed reduction in Rd glucose. Together, these results suggest that glucose was not a preferred substrate under conditions of increased alanine availability and that alanine was probably oxidized to provide energy. Alanine failed to activate AMPK in trout, contrary to mammalian findings. However, it increased AKT (also known as protein kinase B) phosphorylation in muscle, similar to the effect of insulin in trout. In conclusion, my results suggest that alanine mediated at least some of the observed effects by stimulating insulin secretion given the similarities between the effects of exogenous alanine and insulin in rainbow trout as discussed above. Future studies are warranted to investigate the hypothesis that alanine is an insulin secretagogue in rainbow trout.

Immune exhaustion can be a major clinical problem for patients who have cancer or chronic inflammation. Persistent antigen stimulation drives T cells to express multiple surface markers called immune checkpoints. When these markers bind to their corresponding ligands that are expressed by antigen (e.g. tumor cells), T cells become metabolically impaired and lose several important functions; some cell signaling pathways are inhibited, while other intracellular mediators are re-modulated. Eventually, both CD4+ and CD8+ T cells behave dysfunctionally in ways that may facilitate cancer progression. Immune checkpoints are a major hallmark of immune exhaustion. In addition, natural killer (NK) cells, a critical immune cell subset in the peripheral immune system, also express immune checkpoint molecules, and are responsible for detecting and destroying circulating tumor cells. Yet, little research has investigated immune checkpoints on NK cells. Here, we explored the role of Bromodomain and ExtraTerminal domain (BET) proteins (BRD2, BRD3, BRD4), which are important transcriptional co-regulators, and critical for proliferation and metastasis in many cancer types, in the regulation of immune checkpoint molecules in several immune cell subsets, including CD4+ and CD8+ T cells, and NK cells. Through binding to acetylated histone tails of nucleosomal chromatin, BET proteins assist in transcription of multiple genes. Deregulated expression of BET proteins promotes cancer development or tumor cell metastasis, and new data show the BET proteins contribute to immune exhaustion. Furthermore, Type 2 diabetes mellitus (T2DM) is another worrisome problem related to cancer. T2DM patients show increased risk of developing cancer. Patients with both T2DM and any type of cancer show higher risks for metastasis. Significantly, T2DM patients also show immune exhaustion, suggesting a hypothesis that BET proteins may couple immune system dysfunction, abnormal metabolism and cancer incidence or progression. Specifically, T2DM has been defined to be a metabolic and a chronic inflammatory disease. The 5' Adenosine Monophosphate-activated Protein Kinase (AMPK) signaling pathway is a key pivot of cell metabolism and as well a significant target of drugs that normalize blood glucose, such as metformin. Based on published data, we considered that it is important to explore the mechanism of how immune checkpoints are regulated through metabolic pathways, focusing on immune exhaustion in T2DM patients. Moreover, considering that the expression of BET proteins promotes cancer development and progression, and metastasis and immune exhaustion are characteristic of many cancers as well, we suspected a potential relationship among BET proteins, the AMPK metabolic signaling pathway and immune exhaustion is worth exploring. Here, we measure expression of the immune checkpoint molecules TIM-3, TIGIT, PD-1, and CTLA-4 on normal T cells and NK cells by flow cytometry. We demonstrate different degrees of regulation of immune checkpoints by BET proteins on stimulated T cells and NK cells. Comparing stimulated-only cells with stimulated-plus AMPK inhibitor cells, we found that inhibition of the AMPK signaling pathway causes divergent expression patterns for TIM-3 and TIGIT, PD-1 and CTLA-4. Simultaneous inhibition of both BET proteins and the AMPK signaling pathway, shows that BET proteins regulate TIM-3 and TIGIT through an AMPK-independent metabolic pathway and regulate PD-1 and CTLA-4 through an AMPK-dependent pathway. Overall, we show TIM-3 and TIGIT, PD-1 and CTLA-4 display different expression patterns under regulation of the AMPK signaling pathway, and we show that BET proteins regulate TIM-3, TIGIT, PD-1 and CTLA-4 through both AMPK-dependent and -independent pathways. These findings are important because they reveal novel mechanisms of immune checkpoint regulation, which may be valuable for targeting in cancer patients who are being treated with checkpoint inhibitors.

No description available.

The bulk degradative process of macroautophagy requires the dynamic growth of autophagosomes, which carry cellular contents to the lysosome for recycling. Atg9A, a multi-pass transmembrane protein, is an apical regulator of autophagosome growth, yet its regulatory mechanism remains unclear. Our work suggests that hypoxia (low glucose and oxygen) triggers a rearrangement of the small adapter protein 14-3-3ζ interactome. Our data suggest that the localization of mammalian Atg9A to autophagosomes requires phosphorylation on the C terminus of Atg9A at S761, which creates a 14-3-3z docking site. Under basal conditions, this phosphorylation is maintained at a low level and is dependent on both ULK1 and AMPK. However, upon induction of hypoxic stress, activated AMPK bypasses the requirement for ULK1 and mediates S761 phosphorylation directly, resulting in an increase in 14-3-3z interactions, recruitment of Atg9A to LC3-positive autophagosomes, and enhanced autophagosome production. These observations suggested to us that long unstructured C-terminus of Atg9A may be a site of protein docking and regulation. We used BioID, along with conventional interactomics, to map the C- and N-terminal proximity-based interactions of Atg9A. We identified a network of Atg9A C-terminal interactions that include members of the ULK1 complex. Using gel filtration, we find that Atg9A co-immunoprecipitates with the ULK1 complex in high molecular weight fractions. Moreover, phosphorylation of the Atg9A C-terminus at S761 occurs within the ULK1 complex under nutrient-replete conditions, while hypoxia triggers a redistribution of phosphorylated Atg9A to low molecular weight fractions. Probing these relationships further, we find that Atg13, a component of the ULK1 complex, directly interacts with Atg9A and is required for Atg9A C-terminal phosphorylation. Furthermore, a non-phosphorylatable mutant of Atg9A (S761A) accumulates with Atg13 in high molecular weight complexes. Together, these data suggest that Atg13 recruits Atg9A to the ULK1 complex at the phagophore assemble site (PAS) and that S761 phosphorylation triggers Atg9A retrieval from the PAS

Metformin has been used clinically since 1957 for its efficacy and safety as therapy for type 2 diabetes. Besides ameliorating hyperglycemia without risk of hypoglycemia, metformin also lowers plasma triglyceride levels. Furthermore, a wealth of data shows that metformin facilitates weight loss in mice as well as humans. Due to its numerous metabolic benefits, researchers and clinicians are interested in the possibility of using metformin as treatment to combat obesity and other metabolic disorders such as non-alcoholic fatty liver disease (NAFLD). Despite being the most commonly prescribed anti-diabetic, metformin’s complete mechanism(s) for weight loss or for lowering glucose and lipids remains an enigma. Our studies show that metformin-treated mice exhibited decreased caloric intake, providing a viable mechanism for metformin to bring about weight loss. Intriguingly, we found that metformin induces PRDM16 to promote browning of iWAT and increase expression of thermogenic genes such as UCP1 and DIO2. However, metformin did not appear to increase energy expenditure. It’s possible that metformin’s effect on energy expenditure was masked since energy expenditure measurements were taken when metformin-treated mice were still losing weight and were in a state of negative energy balance. Recently, there has been much attention given to AMPK activators as exercise mimetics. Metformin is known to activate AMPK and similarly brings about many beneficial effects as exercise such as alleviation of obesity-induced NAFLD. SIRT1 stimulation by resveratrol and delivery of exogenous FGF21 mimics metformin’s ability to combat obesity and improve NAFLD. Collectively, these results implicate metformin, resveratrol, and exogenous administration of FGF21 as beneficial therapies for weight loss and amelioration of NAFLD.

In 2011, Diabetes and prediabetes affected 9 million Canadians and 366 million people worldwide (Canadian Diabetes Website). The underlying pathophysiology of diabetes is beta cell dysfunction leading to loss of appropriate insulin secretion and resulting in hyperglycemia. I have focused on identifying critical molecular regulators of beta cell function and insulin secretion. The CRTC2-CREB pathway is required for maintaining beta cell mass and insulin secretion. I propose that identifying kinases that regulate CRTC-CREB activity will identify other important regulators of pancreatic beta cell survival and function. First, I have identified several AMP kinases as inhibitors of CRTC2-CREB that are activated by an upstream kinase, LKB1. I then went on to generate mice with a beta cell-specific deletion of LKB1 during adulthood. Loss of LKB1 increased insulin secretion and glucose clearance through enhanced beta cell mass and proliferation. The increased insulin secretion was largely the result of loss of AMPK activity and consequent constitutive mTor activity. AMPK is activated under starvation conditions and as such is thought to be a critical regulator of beta cell function. However, the decrease of AMPK activity in high glucose has been a strong argument against it being a critical effector of insulin secretion. I provide genetic evidence supporting the idea that AMPK activity attenuates insulin secretion. During periods of starvation where AMPK activity is high there is a chronic dampening effect on events that prepare beta cells for the next round of insulin secretion. Surprisingly, another downstream kinase of LKB1, SIK2, has opposing functions in the beta cell. I present evidence that the LKB1-AMPK axis attenuates beta cell functions and that targeting this pathway in beta cells may be of therapeutic benefit for T2D.

Le nombre de lymphocytes T (LT) présents dans l’organisme est contrôlé de manière étroite et maintenu constant. En réponse à une chute de ce nombre, par exemple suite à une infection, un traitement de chimiothérapie ou lors d’une greffe de moelle osseuse, les LT résiduels ou transférés se divisent activement et repeuplent les compartiments lymphoïdes, rétablissant ainsi un nombre «normal» de cellules immunes périphériques. Ce processus, appelé prolifération homéostatique, est soutenu par l’IL-7 ainsi que les signaux TCR et mène à la génération de LT effecteurs/mémoire. Ces cellules effectrices/mémoire peuvent favoriser le développement d’une réponse anti-tumorale mais sont aussi impliquées dans le développement de maladies auto-immunes et inflammatoires.Des changements métaboliques sont étroitement liés à la différenciation et aux fonctions des LT. De plus, un nombre grandissant de données suggère qu’une modulation du métabolisme permet d’influencer le cours d’une réponse immune. L’AMP-activated protein kinase (AMPK) est un senseurmajeur du stress métabolique qui régule l’homéostasie des mitochondries, la glycolyse et l’équilibre rédox. Si l’AMPK est activée lors de la stimulation du TCR, son implication dans la biologie des LT reste confuse.Au cours de cette thèse, nous avons entrepris une série d’expériences afin d’évaluer le rôle de l’AMPK sur la biologie des LT et plus particulièrement au cours de leur prolifération homéostatique. Grâce à différents modèles in vivo et in vitro, nous avons montré que l’AMPK, bien que dispensable pour les étapes précoces de l’activation du TCR, est requise pour soutenir la viabilité et l’expansion des LT au cours de proliférations de longues durées. L’AMPK promeut l’accumulation de LT effecteurs/ mémoire au cours d’une prolifération homéostatique. En accord avec ces données, la transplantation de LT AMPK-KO dans un hôte allogénique conduit au développement d’une réaction du greffon contre l’hôte de moindre gravité.D’un point de vue métabolique, l’AMPK soutient le potentiel de membrane mitochondrial, permet une plus grande flexibilité métabolique, limite la production de dérivés toxiques de l’oxygène (ROS) et protège les LT contre le stress oxydant. En outre, la neutralisation des ROS par un traitement antioxydant restaure partiellement la prolifération des LT AMPK-KO.Nos données suggèrent qu’en limitant l’accumulation de dégâts mitochondriaux et oxydatifs au cours de cycles de divisions prolongées, l’AMPK soutient la viabilité, la prolifération et les fonctions effectrices des LT. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

No description available.