INTRODUCTION: Retinal Pigment Epithelia (RPE) degeneration is a key event in the development of age-related macular degeneration (AMD). RPE dysfunction in AMD is thought to occur through the accumulation of reactive oxygen species (ROS) and oxidative damage. The transcriptional co-activators, PGC-1α and PGC-1β, are important regulators of mitochondrial biogenesis and anti-oxidant capacity. Our group has previously shown that the PGC-1α protein promotes RPE oxidative metabolism and that overexpression of the PGC-1α gene protects cells from AMD-associated pro-oxidants. On the other hand, PGC-1β gene expression has been found to be upregulated in patients with neovascular AMD, and in-vitro overexpression of PGC-1β damages cells and induces pro-oxidant conditions. OBJECTIVE: Given the divergence of PGC-1α and PGC-1β functions in RPE and their clinical relevance in AMD pathogenesis, this project will seek to investigate the impact of the upregulation of PGC-1α and PGC-1β in RPE metabolism. PGC-1α will be upregulated through treatment with compound ZLN005. A new methodology for PGC-1β expression will be developed to closely modulate in-vitro PGC-1β induction. METHODS: In-vitro experiments were performed on the ARPE-19 cell line. Cells were treated with 10µM of ZLN005 for 24 hours. Oxidative stress was induced by exposure to H2O2 and NaIO3 under serum-free conditions. Lactate dehydrogenase (LDH) levels were used to quantify cell death. Quantitative PCR (qPCR) and Western Blot were performed to measure changes in gene and protein expression respectively. Superoxide production by the mitochondria was measured to evaluate ROS levels within the cell. Intravitreal injections of 20µM ZLN005 were performed on eight-week old male C57BL/6J mice. After 24 and 72 hours of treatment, the mice were euthanized and the enucleated eyes were dissected to obtain the RPE and neural retina layers. Total RNA was extracted from these layers and qPCR was performed to measure gene expression. A tetracycline-inducible PGC-1β plasmid was designed and transfected into ARPE-19 cells. The cells were exposed to 0.01-100µg/ml doxycycline for 48-hours and qPCR was used to measure gene expression. Transfected cells were treated with ZLN005 and cell death upon exposure to oxidative stress was quantified. RESULTS: Gene expression analysis on ARPE-19 cells treated with ZLN005 showed robust upregulation of PGC-1α, PGC-1β and their associated transcription factors and enzymes. Induction of PGC-1α at the protein level was also confirmed. ZLN005 efficiently protected ARPE-19 cells from H2O2 and NaIO3 cytotoxicity and its protection was negated in PGC-1α-silenced cells. Treatment with ZLN005 also decreased mitochondrial superoxide production. ZLN005 intravitreal injections were safely administered to the animals and did not cause cataracts or other damage to the ocular tissues. While statistical significance in gene expression changes was limited due to the small sample size, anti-oxidants GPX1 and TXN2, and electron transport chain gene, ATP50, were found to be potentially induced in the neuro-retina, while FOXO3 was found to be downregulated. Evaluation of our novel tetracycline-inducible PGC-1β adenoviral vector showed that upregulation of PGC-1β was efficiently controlled by the addition of doxycycline to transfected cells. Upon exposure to H2O2, transfected cells treated with doxycycline experienced greater cell death than transfected cells not exposed to doxycycline. ZLN005 treatment was able to decrease cell death in both conditions. CONCLUSION: The present study shows that ZLN005 efficiently protects RPE cells from oxidative damage through selective induction of PGC-1α. While still preliminary, the in-vivo study indicates that ZLN005 is safe to be injected into the eye and may be able to increase the expression of mito-protective and anti-oxidant genes in the neuronal retina. In addition, our design of the tetracycline inducible PGC-1β plasmid allows for tight control over PGC-1β expression through doxycycline addition. Upregulation of PGC-1β at levels similar to those observed in clinical conditions caused increased pro-oxidant induced cell death and treatment with ZLN005 was able to protect against cell death. / 2021-06-30T00:00:00Z

Age-Related Macular Degeneration (AMD) is a retinal eye disease that is the leading cause of blindness in those over 50 years of age throughout the developed world. Oxidative and metabolic dysfunction of the retinal pigment epithelium (RPE) has been shown to play an important role in AMD. However, the mechanism of dysfunction in the RPE is poorly understood. The peroxisome proliferator-activated receptor-gamma coactivator 1α and β (PGC1A and PGC1B) are coactivators that interact with transcription factors to regulate mitochondria metabolism. In a previous study, it was demonstrated that one of the isoforms, PGC1A, protects RPE cells from oxidative stress through the upregulation of transcription factors that regulate important antioxidant enzymes. There is experimental and clinical evidence that demonstrates that PGC1B may play a deleterious role in the RPE cell. The objective of this study is to characterize the pathological effect of PGC1B on the RPE cell. PGC1B was overexpressed in the human retinal pigment epithelium cell line (ARPE-19) and expression of the PGC1 isoforms and their main gene targets was evaluated using quantitative polymerase chain reaction (qPCR). Cell death was evaluated under basal and pro-oxidant conditions by quantification of lactate dehydrogenase (LDH) release from the RPE cell. The effect of PGC1B gain of function on the RPE pro-angiogenic function was evaluated using the choroid explant sprouting assay and by testing the proliferative, migratory, and tube formation potential of RPE-derived conditioned media on the rhesus monkey chorioretinal cell line (RF/6A). Quantitative PCR analysis showed that overexpression of PGC1B in ARPE-19 cells leads to increased mitochondrial metabolism and decreased antioxidant enzyme expression, causing oxidative stress. After treatment with H2O2, PGC1B overexpression caused ARPE-19 cells to become more susceptible to cytotoxicity. The ex vivo choroid sprouting assay demonstrated that PGC1B overexpression in RPE is pro-angiogenic. However, cell proliferation as measured by MTT and the cell migration assay provided conflicting results on the pro-angiogenic effect of PGC1B. Previous research has demonstrated that oxidative stress in the RPE cell plays a role in AMD progression. It has been demonstrated in this study that PGC1B expression leads to increased mitochondrial metabolism and repression of antioxidant enzymes needed to prevent oxidative stress and dysfunction in the RPE cell. While experiments to test the effect of PGC1B on angiogenesis provided conflicting results, a different endothelial cell model may be better suited in demonstrating the pro-angiogenic effect of PGC1B. The hope is that the information provided from this study may be used to further our understanding of AMD and lead to the development of therapeutic targets to combat the effects of AMD.

Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants that are known to elicit adverse health effects including skin toxicity and cancer to animals and humans. 4-Monochlorobiphenyl (PCB3), a low-chlorinated airborne PCB conger is present in human blood and the environment. 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ), a quinone metabolite of PCB3, has been shown to induce oxidative stress and toxicity in human mammary and prostate epithelial cells. These studies were designed to investigate and characterize the cellular responses to 4-ClBQ in HaCaT human skin keratinocytes. We found that 4-ClBQ treatment increased cellular reactive oxygen species (ROS) production, inhibited cell proliferation, and induced toxicity in HaCaT cells. Results from a Human Antioxidant Mechanism PCR array and quantitative RT-PCR assay showed that the mRNA levels of antioxidant gene selenoprotein P (sepp1) and catalase were significantly downregulated by the treatment, which correlated with evident decreases in their protein levels and catalase enzymatic activity. Pharmacological (sodium selenite supplementation) and molecular (sepp1overexpression) manipulation of SEPP1 expression significantly suppressed 4-ClBQ induced oxidative stress and toxicity. Additional results demonstrated that decreased catalase expression was associated with an inhibition in transcriptional coactivator peroxisome proliferator activated receptor Γ coactivator 1α (PGC-1α) expression. Overexpression of pgc-1α restored catalase expression and activity and consequently protected HaCaT cells from 4-ClBQ induced oxidative stress and toxicity. Furthermore, results from metabolic flux analysis using Seahorse XF96 Analyzer showed that 4-ClBQ treatment increased extracellular acidification rate, proton production rate, and oxygen consumption rate, which were associated with increases in glucose uptake and in the expression of glucose metabolism regulatory gene hexokinase 2, pyruvate kinase M2, and glucose-6-phosphate dehydrogenase (G6PD). G6PD is the rate-limiting enzyme of the pentose phosphate pathway. The enhanced expression of G6PD correlated with an increase in cellular glutathione content; and inhibition of G6PD activity sensitized HaCaT cells to 4-ClBQ induced toxicity, suggesting that the protective function of the pentose phosphate pathway is active in 4-ClBQ treated cells. Interestingly, we also found that 4-ClBQ selectively and significantly decreased mitochondrial complex II subunits C (sdhc) and D (sdhd) mRNA expression and subsequently reduced complex II activity leading to metabolic oxidative stress and toxicity, which were significantly suppressed by overexpressing sdhc and sdhd in HaCaT cells. Taken together, findings from this project demonstrate that 4-ClBQ treatment increases ROS production through perturbing cellular metabolism and mitochondrial function and decreases antioxidant capacity by inhibiting SEPP1 and catalase expression in HaCaT cells. This imbalance due to increased mitochondrial prooxidant production and decreased antioxidant capacity leads to oxidative stress and toxicity. Importantly, antioxidant supplementation could abrogate 4-ClBQ induced toxicity, suggesting that antioxidants, especially nutrient-based manipulation of selenoproteins could be promising countermeasures for PCB induced adverse health effects in humans.

L'énergie indispensable au fonctionnement de la cellule est produite principalement par la mitochondrie grâce au mécanisme de phosphorylation oxydative impliquant des protéines codées par le génome nucléaire et celui de la mitochondrie. La coordination transcriptionnelle de ces génomes est nécessaire à la biogenèse de mitochondries fonctionnelles, et est assurée par divers facteurs de transcription, tels que les NRFs (Nuclear Respiratory Factors) et les ERRs (Estrogen-Related Receptors). Leur efficacité transcriptionnelle est contrôlée par les coactivateurs de la famille PGC-1 (Peroxisome proliferator-activated receptor γ Coactivator-1) – PGC-1α, PGC-1β et PRC (PGC-1-Related Coactivator) – dont l'expression dépend de signaux endogènes ou environnementaux. Afin de préciser le rôle de PRC dans le dialogue nucléo-mitochondrial, nous avons utilisé plusieurs modèles cellulaires de carcinomes folliculaires thyroïdiens humains (RO82 W-1, FTC-133 et XTC.UC1) présentant une richesse en mitochondries, une orientation métabolique et des niveaux d'expression de PRC et de PGC-1α différents. Ce travail a mis en évidence le rôle clef du complexe ERRα–PRC dans la biogenèse de mitochondries fonctionnelles. PRC semble par ailleurs coordonner les phases du cycle cellulaire selon l'efficacité du métabolisme énergétique mitochondrial et le statut redox de la cellule. Dans ces modèles, notre travail a mis en évidence un rôle du monoxyde d'azote et du calcium comme régulateurs de la biogenèse et de la fonction mitochondriales PRC-dépendantes. L'ensemble de ces données fait du coactivateur PRC et des voies qui régulent sa fonction des cibles thérapeutiques potentielles dans les tumeurs.

La Diabetis Mellitus tipus 2 (DM2) és una malaltia metabòlica complexa que afecta entre un 4 i un 5% de la població en les societats industrialitzades. Aquesta patologia es caracteritza per la presència, en la seva fase inicial, de resistència a la insulina (RI). Freqüentment, una de les primeres alteracions que s´observen en els individus amb predisposició a patir RI/DM2 és l´acumulació de grassa intraabdominal. De fet, la relació epidemiològica entre l´obesitat i la RI és molt sòlida. A més, en l´última dècada nombroses evidències han posat de manifest l´existència d´una estreta relació entre un estat d´inflamació crònic de baixa intensitat i la presència d´obesitat-RI-DM2. De tota manera, tot i que el vincle entre l´increment d´àcids grassos lliures en plasma i la diabetis està ben acceptat, els mecanismes implicats en l´aparició de RI i DM2 induïdes per aquests àcids grassos no són ben coneguts. Per aquest motiu, l´objectiu d´aquesta tesi doctoral ha estat aprofundir en els mecanismes implicats en l´aparició de RI induïda per l´àcid gras saturat palmitat i estudiar la funció de l´enzim COX-2 en aquestes condicions, així com també determinar la capacitat de l´àcid gras monoinsaturat oleat i de l´agonista PPARdelta GW501516 per a prevenir la inflamació i la RI induïdes pel palmitat en miotubs de ratolí C2C12.Els estudis realitzats indiquen que la presència elevada de l´àcid gras saturat palmitat provoca una disminució de PGC-1alfa(coactivador que controla l´expressió de gens mitocondrials) a través de l´activació de la via ERK-MAPK-NF-kB, així com també l´acumulació de diacilglicerol intramiocel·lular, fent que apareguin estats d´inflamació i RI. A més, s´ha observat que un augment agut de determinats marcadors inflamatoris, com la COX-2, contribueixen a resoldre aquest procés inflamatori generat per l´acumulació de lípids, tot i que la seva presència de manera crònica accentua l´estat inflamatori. Segons hem pogut constatar amb el nostre model in vitro de RI, l´àcid gras monoinsaturat oleat i el GW501516 podrien ser dues noves possibilitats terapèutiques per evitar la inflamació induïda per àcids grassos i millorar la sensibilitat a la insulina, ja que tenen la capacitat d´incrementar la beta-oxidació mitocondrial impedint així que s´acumulin metabolits lipotòxics com el diacilglicerol. / Insulin resistance (IR) is a major characteristic of type 2 diabetes mellitus and is also associated with obesity. Impairment of glucose utilization and insulin sensitivity has been related to the presence of high free fatty acids in plasma and a low-grade chronic systemic inflammation. However, the mechanisms by which free fatty results in inflammation and IR are not well understood. After exposing C2C12 cells (mouse myotubes) to the saturated fatty acid palmitate, we observed, on the one hand, a reduction in PGC-1-alpha gene expression through the activation of ERK-MAPK-NF-kB pathway, and on the other hand, an increase in diacylglycerol accumulation. Moreover, we observed that the presence of palmitate increased COX-2 expression, which seems to contribute to resolve the acute, but not chronic, inflammation. Our in vitro model of IR also showed that the monounsaturated fatty acid oleate and the PPAR-delta agonist GW501516, could avoid the development of inflammation and IR induced by fatty acids through an increase in mitochondrial beta-oxidation, thus preventing the accumulation of lipotoxic metabolites, such as dicylglycerol.

<p>Sepsis is a major cause of morbidity and mortality in the United States, and Staphylococcus aureus (S. aureus) is the bacteria most commonly cultured from septic patients. In severe sepsis, the relationship between the systemic inflammatory response and the resulting mitochondrial and metabolic dysfunction is not fully understood, especially with respect to the mechanisms of mitochondrial damage resolution. The process of mitochondrial biogenesis, which leads to the restoration of metabolic and anti-oxidative functions in damaged or stressed cells and tissues, is pro-survival and is a critical protective response in sepsis. Mitochondrial biogenesis requires the coordinated expression of multiple regulatory proteins, including the PPARgamma-coactivator (PGC) family of proteins. Previous work in sepsis has focused on mitochondrial biogenesis in response to late signals of mitochondrial damage; however, for acute sepsis, we have hypothesized a direct and early link between the innate immune response and the transcriptional activation of mitochondrial biogenesis. Since the Toll-like receptors (TLRs) are a major part of the innate immune response, we hypothesized that they could activate mitochondrial biogenesis in bacterial sepsis. Earlier work showed that TLR4 (which responds to components of Gram-negative bacteria) was necessary for mitochondrial biogenesis induction in response to heat-killed E. coli challenge. For this work, the objective was to investigate whether signaling by TLR2 (which responds to components of Gram-positive bacteria) would activate mitochondrial biogenesis in response to S. aureus sepsis in mice. The sepsis model was initially characterized in wild-type (WT) mice by PCR analysis of hepatic RNA, in which the up-regulation of several regulatory proteins for mitochondrial biogenesis, including all three PGC family members, was observed. In contrast, in both TLR2-/- and TLR4-/- mice, the mitochondrial biogenesis response was deficient and delayed. In addition, PGC-1alpha and PGC-1beta were differentially regulated in WT, TLR2-/-, and TLR4-/- mice. To identify the mechanisms involved in this induction pattern, the known TLR signaling pathways were systematically probed for activation using several strains of genetic knockout mice. These data demonstrated that the differential regulation of the PGC family is independent of the MyD88 adapter protein and is caused in part by IRF7 signaling. IRF7 is a pro-inflammatory transcription factor that is normally involved in the interferon response; in this case, IRF7 was found to be necessary but not sufficient for PGC-1alpha/beta induction. In addition, a second level of regulation was identified in the microRNA mmu-mir-202-3p, which is inversely correlated with the expression of PGC-1alpha and PGC-1beta mRNA in WT, TLR2-/-, and TLR4-/- mice and was shown to functionally decrease PGC-1alpha mRNA. If these observations are confirmed in humans, IRF7 and mir-202-3p may be potential therapeutic targets for the up-regulation of PGC-1alpha/beta levels in the clinical setting of sepsis and impaired mitochondrial biogenesis.</p> / Dissertation

H οικογένεια των γουανυλικών κυκλασών περιλαμβάνει δύο μέλη, μία διαλυτή μορφή (sGC) και μια μορφή που είναι συνδεδεμένη στην κυτταρική μεμβράνη (pGC). Αυτές ενεργοποιούνται από τα διαφορετικούς προσδέτες. Η μεν sGC από το μονοξείδιο του αζώτου (NO), η δε pGC από τα νατριουρητικά πεπτίδια και με την ενεργοποίησή τους παράγουν την κυκλική GMP (cGMP). Σε πολλές περιπτώσεις, η ξεχωριστή αυτή παραγωγή του cGMP από sGC έναντι αυτής που προέρχεται από την pGC οδηγεί σε διαφορετικές βιολογικές δράσεις. Προηγούμενη εργασία στο εργαστήριό μας έχει δώσει έμφαση στη σημασία της από sGC παραγόμενης cGMP στην αγγειογένεση. Ωστόσο, περιορισμένες πληροφορίες είναι διαθέσιμες όσον αφορά στις αγγειογενετικές δράσεις της pGC στο ενδοθήλιο. Επομένως επιδιώξαμε να καθορίσουμε τα αποτελέσματα της ενεργοποίησης pGC στις ιδιότητες των ενδοθηλιακών κυττάρων που σχετίζονται με την αγγειογένεση. Αρχικά, ερευνήσαμε την επίδραση του κολπικού νατριουρητικού πεπτιδίου (ANP) στο σχηματισμό αγγείων αίματος in vivo, χρησιμοποιώντας ως πρότυπο μοντέλο τη χοριοαλλαντοϊκή μεμβράνη εμβρύου όρνιθας (CAM). Το ANP (0.1-10 μmole) ενίσχυσε τη νεοαγγειογένεση με δοσοεξαρτώμενο τρόπο, όπως διαπιστώθηκε από την αύξηση στα σημείων διακλάδωσης και το μήκος αγγείων. In vitro, το ANP ενίσχυσε τον πολλαπλασιασμό (0.01-1 μΜ) και τη μετανάστευση (10 μΜ) ενδοθηλιακών κυττάρων ανθρώπινου ομφάλιου λώρου (HUVEC) και υποκίνησε το σχηματισμό δομών αγγειακού τύπου από τα HUVEC 10 μΜ) σε υπόστρωμα Matrigel. Προκειμένου να μελετηθούν οι μηχανισμοί που ενέχονται στην επαγόμενη από ANP αγγειογένεση, εξετάσαμε τη φωσφορυλίωση δύο κινασών (MAPK), των ERK1/2 και p38, ως πιθανά μόρια-στόχουςστο μονοπάτι της σηματοδότησης του ANP. Το ANP ενεργοποίησε τόσο την ERK1/2, όσο και την p38 MAPKs κατά χρονοεξαρτώμενο τρόπο. Για να εξασφαλίσουμε στοιχεία για τη λειτουργική σημασία p38 χρησιμοποιήσαμε ένα φαρμακολογικό της αναστολέα, τον παράγοντα SB203580. Προεπώαση των κυττάρων με SB203580 ανέστειλε εν μέρει τη μετανάστευση των HUVEC. Εν περιλήψει, τα στοιχεία μας δείχνουν ότι το ANP προάγει την αγγειογένεση in vivo και ενισχύει τις σχετιζόμενες με αγγειογένεση, ιδιότητες των ενδοθηλιακών κυττάρω in vitro με τη ρύθμιση της φωσφορυλίωσης των MAPK. Η ενεργοποίηση λοιπόν της pGC να είναι ευεργετική όταν απαιτείται ο σχηματισμός νέων αγγείων αίματος. / The guanylyl cyclase (GC) family comprises of two members, a soluble one (sGC) and a membrane-bound GC, the particulate GC (pGC). These are activated by different ligands; sGC by nitric oxide (NO) and pGC by natriuretic peptides and upon activation produce cyclic GMP (cGMP). In many instances compartmentalized production of cGMP by sGC vs pGC results in different biological responses. Previous work in our laboratory has highlighted the importance of sGC-derived cGMP in angiogenesis. However, limited information is available with regard to the angiogenic actions of pGC in the endothelium. We therefore sought to determine the effects of pGC activation on angiogenesis-related properties of EC. Initially, we investigated the effects of atrial natriuretic peptide (ANP) on blood vessel formation in vivo, using as a model the chick embryo chorioallantoic membrane (CAM). ANP (0.1-10 μmole) enhanced neovascularization in a dose-dependent manner, as shown by the increase in branching points and vessel length. In vitro, ANP increased human umbilical vein endothelial cell (HUVEC) growth (0.01-1 μΜ) and migration (10 μΜ) and stimulated the assembly of HUVEC into tube-like networks (10 μΜ) on Matrigel. In order to study the mechanisms implicated in ANP-induced angiogenesis, we examined the phosphorylation of two MAP Kinases (MAPK), ERK1/2 and p38, as possible downstream targets of ANP signaling. ANP activated both ERK1/2 and p38 MAPKs in a time-dependent manner. To provide evidence for the functional significance of p38 we used the pharmacological inhibitor SB203580. Pretreatment of cells with SB203580 inhibited ANF-stimulated migration of HUVEC. In summary, our data show that ANP promotes angiogenesis in vivo and enhances angiogenesis-related properties of endothelial cells in vitro by modulating phosphorylation of MAPK. pGC activation might be beneficial when new blood vessel formation is desired.

Abstract Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a master regulator of energy metabolism and mitochondrial biology in high-energy cell types and tissues. The regulation of PGC-1α is versatile, and both transcriptional and post-transcriptional mechanisms play major roles. External stimuli affect PGC-1α-regulation which in turn adapts cellular signals to meet them. For example, conditions like fasting and diabetes mellitus (DM) are known to activate PGC-1α expression in the liver, resulting in enhanced de novo glucose production, gluconeogenesis. In the present study, the mechanisms of hepatic PGC-1α regulation and PGC-1α-regulated functions were elucidated. We found that PGC-1α was induced by oral type 2 diabetes therapeutic metformin, via AMPK and SIRT1, regulating the mitochondrial gene response, against previous assumptions. Simultaneously, gluconeogenesis was repressed by other means. Furthermore, PGC-1α upregulated the anti-inflammatory interleukin 1 receptor antagonist (IL1Rn). PGC-1α also diminished interleukin 1β-mediated inflammatory response in hepatocytes. Novel, xenobiotic and endobiotic metabolizing Cytochrome P450 enzymes regulated by PGC-1α were also identified in this thesis. CYP2A5 was induced by PGC-1α through hepatocyte nuclear factor 4α (HNF-4α) coactivation. Also, vitamin D metabolizing CYP2R1 and CYP24A1 were identified as novel genes regulated by PGC-1α, suggesting a role for PGC-1α in the regulation of active vitamin D levels. The findings presented in this thesis provide insight into the pathology of glucose perturbations such as type 2 diabetes, and stimulate discovery of therapeutic agents to treat this disease. Furthermore, the findings suggest that vitamin D metabolism and energy metabolism are tightly linked, with PGC-1α emerging as a novel mediator. / Tiivistelmä Peroksisomiproliferaattori-aktivoituvan reseptori γ:n koaktivaattori 1α (PGC-1α) on merkittävä glukoosiaineenvaihdunnan ja mitokondrioiden toiminnan säätelijä korkeaenergisissä soluissa ja kudoksissa. PGC-1α:a säädellään monin tavoin: sekä transkriptionaalisella säätelyllä että transkription jälkeisellä muokkauksella on merkittävä rooli. Monet ulkoiset tekijät säätelevät PGC-1α:n aktiivisuutta, joka puolestaan säätelee solunsisäisiä signaalireittejä vastaamaan tähän signaaliin. Esimerkiksi paasto ja diabetes mellitus (DM) ovat fysiologisia tiloja, jotka lisäävät voimakkaasti PGC-1α:n ilmentymistä maksassa, jolloin glukoosin uudistuotanto eli glukoneogeneesi kiihtyy. Tässä väitöskirjassa tutkittiin PGC-1α:n säätelyä sekä PGC-1α -säädeltyjä signaalireittejä maksassa. Osoitimme, että tyypin 2 diabeteslääke metformiini indusoi PGC-1α:n ilmentymistä maksassa, vastoin aikaisempia käsityksiä. PGC-1α indusoitui AMPK:n ja SIRT1:n välityksellä, säädelleen edelleen mitokondriaalisten geenien aktiivisuutta. Samalla glukoneogeneesi kuitenkin repressoitui muilla mekanismeilla. Lisäksi osoitimme, että PGC-1α indusoi tulehdusreaktiota vaimentavaa interleukiini 1 reseptorin antagonistia (IL1Rn). PGC-1α esti interleukiini 1β:n aiheuttamaa tulehdusvastetta hepatosyyteissä. Lisäksi väitöskirjassa tunnistettiin uusia, PGC-1α -säädeltyjä lääkeaineita ja elimistön sisäisiä yhdisteitä metaboloivia sytokromi P450 -entsyymejä (CYP). Hiiren CYP2A5:n ilmentymisen osoitettiin olevan PGC-1α- ja HNF4α-välitteistä. Lisäksi osoitettiin, että D-vitamiinia metaboloivat CYP2R1 ja CYP24A1 ovat uusia PGC-1α -säädeltyjä geenejä. Tämä löydös viittaa siihen, että PGC-1α:lla on rooli aktiivisen D-vitamiinin säätelyssä. Tämän väitöskirjan löydökset lisäävät tietoa glukoosiaineenvaihdunnan häiriöiden kuten tyypin 2 diabeteksen molekulaarisista mekanismeista, joita voidaan hyödyntää mahdollisten uusien lääkeaineiden kehittämisessä. Lisäksi väitöskirjassa osoitettiin, että D-vitamiinimetabolia on kytköksissä energia-aineenvaihduntaan ja että PGC-1α:lla on tässä rooli, jota ei aiemmin ole tunnettu.

Les glucocorticoïdes (GCs) ont des effets diabétogènes avérés. Précédemment, notre équipe a également pu montrer que les GCs, en association avec le corégulateur transcriptionnel PGC-1α, sont impliqués dans la programmation fœtale du diabète de type 2 (DT2). Le DT2 est une maladie métabolique, conséquence à la fois de l’insulinorésistance et d’un défaut de sécrétion d’insuline en partie dû à la diminution de la masse des cellules β. Au laboratoire nous nous intéressons donc d’une part aux mécanismes sous-jacents des effets diabétogènes des GCs et d’autre part, aux mécanismes permettant d’améliorer la sécrétion d’insuline en restaurant une masse fonctionnelle de cellules β. Dans la première partie de cette thèse, nous avons montré que PGC-1α, dont l’expression est stimulée par les GCs dans les cellules β, induit un double stress énergétique et oxydatif impliqué dans l’altération de la sécrétion d’insuline. Dans la deuxième partie, nous avons montré grâce à un model murin d’insulinorésistance sévère par surexposition aux GCs, que l’adaptation compensatrice de la masse fonctionnelle des cellules β se fait par un processus de néogenèse, impliquant la réexpression du facteur Ngn3. Ce processus, indépendant de l’effet des GCs sur le pancréas, alimente l’hypothèse d’un facteur circulant libéré par les organes insulinorésistants pour instruire le pancréas endocrine et initier la néogenèse des cellules β. En conclusion, nos travaux associent indirectement les GCs : 1/ à un effet délétère sur la sécrétion et impliquant PGC-1α et 2/ à un effet bénéfique sur la masse β et impliquant Ngn3. Ces deux voies constituent des perspectives thérapeutiques intéressantes du DT2. / Glucocorticoids (GCs) are hormones secreted in response to stress and that display diabetogenic effects. Previously, our team was able to demonstrate that GCs, in combination with the transcriptional co-regulator PGC-1α, are involved in fetal programming of type 2 diabetes (T2D). T2D is a metabolic disease characterized by fasting hyperglycemia, consequence of both insulin resistance and an insulin secretory defect, partly due to the decrease of the mass of β cells. In the laboratory we are therefore interested in understanding the mechanisms underlying diabetogenic effects of GCs, and mechanisms that improve insulin secretion and functional β-cell mass. In the first part of this thesis, we have shown that PGC-1α, whose expression is strongly stimulated by GCs in β cells, induces both energy and oxidative stress involved in impaired insulin secretion. In the second part of this thesis, we demonstrated through a murine model of massive GCs overexposure – which induces severe insulin resistance – that the adaptation of the functional β-cell mass in order to counteract insulin resistance occurs through a neogenesis process, involving the re-expression of Ngn3 factor. This process is independent of the effect of GCs on the pancreas. We hypothesize that a circulating factor released by insulin-resistant organs will instruct the endocrine pancreas to initiate β-cells neogenesis. In conclusion, our work indirectly associate GCs: 1/ to a deleterious effect on the secretion involving PGC-1α and 2/ to a beneficial effect on the β-cell mass and involving Ngn3. These two pathways are interesting therapeutic perspectives for curing T2D.

Die Herzinsuffizienz ist eine der häufigsten chronischen Erkrankungen mit progressivem Krankheitsverlauf. Dieser ist verschiedenen Kompensationsmechanismen geschuldet, die zunächst zur Verbesserung, über einen längeren Zeitraum hinweg jedoch zur Verschärfung der Symptomatik führen. Durch erhöhte Konzentrationen inflammatorischer Zytokine im Rahmen dieser Krankheit kommt es unter anderem zu pathologischen Veränderungen in Muskel- als auch Fettgewebe. Das kürzlich in der Literatur als PGC-1α reguliert beschriebene Molekül FNDC5 (Spaltprodukt Irisin) wird als Myokin bezeichnet, welches zwischen Muskel- und Fettgewebe vermittelt. Bis dato ist jedoch ungeklärt, ob und inwiefern es einen Zusammenhang zwischen einer Herzinsuffizienz, erhöhten Konzentrationen proinflammatorischer Zytokine und dem Molekül FNDC5 in der Skelettmuskulatur gibt. Um diesen Fragestellungen nachzugehen, wurde in der vorliegenden Arbeit zum einen durch LAD-Ligatur in Ratten eine Herzinsuffizienz induziert, um die systemischen Verhältnisse dieser Erkrankung im Tiermodell bestmöglich nachzuempfinden. Zum anderen wurden Mäuse mit TNF-α oder Ang II behandelt und C2C12-Myotuben mit TNF-α, Ang II sowie einer Zytokinkombination inkubiert, um den Einfluss der Zytokine bzw. des Hormons auf die FNDC5-Expression zu untersuchen. Anschließend wurden die Expression von FNDC5 auf mRNA- und Proteinebene sowie die Expression der PGC-1α-mRNA in Skelettmuskelbiopsien bestimmt. Es konnte nachgewiesen werden, dass eine Herzinsuffizienz sowie der Einfluss inflammatorischer Zytokine zur signifikanten Expressionsverminderung von FNDC5 führt. Ang II hingegen bewirkte nicht in allen Versuchen eine deutliche Reduktion der FNDC5-Expression. Außerdem wurde gezeigt, dass die Signaltransduktionswege über p38 und p42/44 MAPK nicht für die Wirkung der Zytokinkombination auf die FNDC5-Expression verantwortlich sind.