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This project investigated the effects of exercise and epinephrine on the mRNA expression of peroxisome proliferator activated receptor gamma coactivator-1 alpha (PGC-1 alpha), a master regulator of mitochondrial biogenesis, in rat adipose tissue. Rats that swam 2 hours daily for 4 weeks had increased mitochondrial marker proteins and PGC-1 alpha mRNA expression in epididymal and retroperitoneal adipose tissue (p<0.05). Adipose tissue organ culture treatment with epinephrine increased (p<0.05) PGC-1 alpha mRNA expression in both depots, but only epididymal adipose responded to a supra-physiological dose. Beta blockade attenuated the effects of an acute bout of exercise on PGC-1 alpha mRNA expression in epididymal, but not in retroperitoneal adipose tissue. This is the first study to demonstrate that rat white adipose tissue PGC-1 alpha mRNA expression is increased by acute and chronic exercise and epinephrine. Increases in circulating catecholamine levels might be one potential mechanism mediating exercise induced increases in PGC-1 alpha mRNA expression in rat abdominal adipose tissue. / Nutrition and Metabolism

Primordial gonocytes (PGCs) in some animals, including fish arise after fertilization in extragonadal region from maternally inherited germline cytoplasm, and migrate to the future gonads region during embryogenesis, where differentiate into gametes. PGCs formation and migration patterns have been studied in several species models, and it is known that these patterns differ from each other. Sturgeons belong to class ray-finned fishes(Actinoptergii), in which the sturgeon phylogenetic position is an out-group to teleost fishes, the sturgeon development pattern is more similar to amphibians than teleost fishes. In this study, we demonstrate an injection technique for sturgeon PGCs visualization by GFP nos1 3'UTR mRNA. We found that the Sterlet(A. ruthenus) PGCs are specified in the vegetative pole of the embryo. Subsequently, we reported the PGCs migration route. The arisen PGCs actively migrated on the yolky cell mass, yolky extension, and after that passively moved to gonadal ridge. This study provides evidence that the PGCsare specified by maternally inherited germplasm, located in the vegetative part of the embryo. Sturgeon PGCs specification was similar to that of anuras, but the migration pattern resembled that of teleost. Furthermore, we successfully isolated PGCs to next needed studies.

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is known as the master regulator of mitochondrial biogenesis. PGC-1α holds this role by acting as a transcriptional coactivator for an array of transcription factors and nuclear hormone receptors, such as NRF-1/2 and ERRα/γ, whose downstream targets function in mitochondrial biogenesis and oxidative phosphorylation. PGC-1α is regulated both at the transcriptional and post-translational level in several signaling pathways, including p38 MAPK and AMPK. This regulation affects which transcription factor binding events can occur in a given tissue, and thus affects regulation of PGC-1α target genes. PGC-1α is downregulated in many neurodegenerative disorders as well as in muscular dystrophies, diabetes, and aging. Therefore, PGC-1α is prized as a potential therapeutic target to create novel treatments for these various diseases.However, details governing the spatio-temporal regulation of PGC-1α are not completely understood, and overexpression of PGC-1α throughout the body or even in certain tissues or subsets of cells have had detrimental effects in animal and cell models. Therefore, it is necessary to gain knowledge of how to modulate PGC-1α in a tissue-specific manner utilizing these different levels of regulation in order to develop novel therapies. In order to further understand all the functions that have been attributed to PGC-1α, the PGC-1α isoforms need to be accounted for and understood in human tissues. Several murine isoforms have been published, as well as several human brain and muscle isoforms. However, most of these isoforms have only been validated as mature transcripts, and it is not known whether they produce functional protein. Our lab has identified the isoform b transcript in human brain tissue via 5’ RACE and have developed an isoform b specific antibody. This project aimed to characterize the isoform b transcripts and also to validate and optimize this antibody for immunoblotting conditions for detection of further PGC-1α-b isoform protein variants in human tissues. Preliminary studies in our lab have shown that in postmortem frontal cortex from age-matched PD and healthy patients, isoform a transcript levels were 10-15 times more abundant than that of isoform b. These differences in regulation could be partially attributed to the isoform b promoter region being heavily methylated, as shown in this thesis through bisulphite cloning and sequencing as well as 454 bisulphite sequence analysis. The high degree of methylation, correlated with the low level of isoform b transcript in brain and it is not known whether this transcript would be translated into protein in this tissue. In order to probe for isoform b protein expression using human cell lines and tissues, however, it was necessary to create a recombinant protein in order to have a positive control with which to optimize our novel antibody. In our previous 5’ RACE studies, an alternatively spliced PGC-1α-b transcript was found which coded for an early stop codon. This truncated isoform was called PGC-1α-b-3T1, and mature transcript was found in both human skeletal muscle and brain. For this project, PGC-1a-b-3T1 was cloned from human skeletal muscle into a bacterial expression vector to create a recombinant GST fusion protein. This protein was used to validate and optimize our PGC-1α-b specific antibody as well as to determine sensitivity and specificity. The purified recombinant protein contained 3 bands of lower molecular weight that were detected via western blot with both GST and the PGC-1α-b specific antibody. These bands were trypsin cleaved and subjected to mass spectrometry analysis, which verified that all bands detected by the PGC-1a-b specific antibody contained the epitope sequence, and thus binding was specific. This protein was then used to determine western blotting conditions and sensitivity, which is 10 ng using a 1:100 dilution of the antibody. This antibody was then used to probe SH-SY5Y WCL, a human neuroblastoma cell line. Peptide competition assay confirmed 5 PGC-1α-b specific proteins in these lysates. The sizes of these proteins matched to several murine PGC-1α-b isoforms as well as putative PGC-1α-b versions of PGC-1a-a isoforms. These findings provided the putative identities of several endogenous functional human PGC-1α-b isoforms. Mammalian overexpression vectors of these isoforms are still in development. By using this antibody and these expression vectors to further characterize these isoforms, including determining tissue specificity, more knowledge of PGC-1α will be gained. This information could then be used to develop novel, tissue specific treatments for pharmacological intervention of diseases characterized by PGC-1α misregulation.

The transcriptional coactivator PPARgamma coactivator 1alpha (PGC-1α) is a regulator of mitochondrial biogenesis and function and is decreased in the striatum of patients with Huntington’s Disease (HD). HD is an autosomal dominant neurological disorder caused by a polyglutamine repeat in the huntingtin protein which leads to degeneration of striatal and cortical tissues. PGC-1α undergoes targeted downregulation by mutant huntingtin protein (mtHtt) and PGC-1α knockout mice have striatal lesions similar to HD transgenic mice. Exogenous PGC-1α partially reverses the toxic effects of mutant huntingtin in cultured striatal neurons while in vivo administration of PGC-1α to the striatum in a mouse model of HD reduces neuronal volume loss. Synaptic N-methyl-D-aspartate receptor (NMDAR)- activity can drive the expression of PGC-1α which is neuroprotective against oxidative and excitotoxic stress in vitro whereas extrasynaptic NMDAR expression is increased in HD. Excessive NMDAR activity, specifically through extrasynaptic rather than synaptic NMDARs, leads to excitotoxic death in neurons and its regulation has been targeted in the search for therapeutic interventions for multiple neurological disorders. The data presented in this thesis show that the repression of PGC-1α by mtHtt may be significant in the dysregulation of NMDARs in HD. Both PGC-1α knockdown and mutant huntingtin are found to increase extrasynaptic NMDAR activity and excitotoxicity in a non-additive way, suggesting common regulatory mechanisms. Furthermore exogenous PGC- 1α expression is sufficient to reverse this increase in extrasynaptic NMDAR currents and excitotoxicity by mtHtt. This thesis adds mechanistic insight into previous understanding of the synergistic roles of mtHtt, NMDAR activity and PGC-1α in HD. Finally, we show that chronic knockout of PGC-1α in the PGC-1α(-/-) mouse causes distinct alterations in glutamatergic signaling that do not mimic the observation of acute knockdown of PGC-1α. We propose that the loss of PGC-1α in a number of neurological disorders contributes to concurrent increases in aberrant glutamate signaling and excitotoxicity in these diseases.

Obesity is associated with greater rates of cardiovascular disease, dyslipidemia and dysfunctional lipid metabolism. Exercise may provide an effective therapeutic tool to ameliorate dyslipidemia. However, how exercise attenuates dyslipidemia with obesity is not fully understood. Additionally, whether acute exercise or exercise training is the primary driver of such changes in this population is unknown. Furthermore, mechanisms mediating these exercise responses are not elucidated. The peroxisome proliferator-activated receptors (PPARs) provide a likely mechanism through enhanced expression of oxidative metabolism and cholesterol transport proteins augmenting fatty acid oxidation and cholesterol transport. Study one describes blood lipid and lipoprotein responses to acute aerobic exercise and exercise training in obese men and women. The primary measured effects include: increased HDL-C in men following 12 wks exercise training, and a shift from HDL3-C to HDL2-C, with concomitantly reduced HDL-C mean density and LDL3-C in women. Acute exercise of 400 kcal duration performed before and after training, yielded a decreased TC: HDL-C ratio in men, which was unaffected by training. Thus, the primary exercise-based treatment for dyslipidemia with obesity appears to be exercise training. In study two, PPARδ and PGC-1α content were significantly enhanced after acute exercise, whereas PPARα and AMPKα content were augmented only after training. These effects were seen with concomitantly increased content of target proteins involved in oxidative and lipoprotein metabolism including lipoprotein lipase, CPT-I, COX-IV, and FAT/CD36. PPARδ expression was correlated with total and LDL-cholesterol concentrations. AMPKα expression was correlated with the concentration of HDL-C and its subfractions, suggesting regulation of blood cholesterols by PPARδ and AMPKα. Study three demonstrates comparative responses to high volume resistance exercise (RE) in lean and obese Zucker rats. RE enhanced PPARδ expression regardless of phenotype, but PGC-1α in obese only. Mitochondrial biogenesis was enhanced in lean animals only, indicating PPARδ and PGC-1α content is disconnected from mitochondrial biogenesis with obesity. These studies enhance our understanding of exercise as a therapeutic tool in treating dyslipidemia and dysregulated lipid metabolism often associated with obesity. They further demonstrate the necessity for exercise training to attenuate dyslipidemia, while illustrating PPAR-mediated augmentations in oxidative and lipoprotein metabolism following exercise with obesity.

Mitochondrial content, an important determinant of muscle metabolic capacity, changes in individuals during development, and in response to physiological and environmental challenges. This phenotypic plasticity is attributed to the coactivator PPARγ coactivator-1α (PGC-1α) but it remains unclear if this transcriptional regulator accounts for evolutionary variation in mitochondrial content. In an attempt to explain why some species have higher muscle mitochondrial enzyme levels than other species, I examined if the transcriptional mechanisms that control mitochondrial content of a tissue in an individual are also responsible for differences between species. If PGC-1α creates differences between the mitochondrial content of species based on variation in promoter binding motifs, then cis-factor evolution may be the guiding force in scaling trends. In this thesis I explored the basis of size-dependent patterns by looking at layers of regulation, from catalytic activities to promoter evolution and regulation. A representative family, Rodentia, was used to collect muscle samples from a size range of approximately 20g up to 17 kg. As expected, in rodent lower limb muscles, mitochondrial and glycolytic enzyme activity exhibited reciprocal scaling patterns, though the scope differed between muscles. Very little of the variation was accounted for when the activity was related to DNA content. However, when COX activities were expressed relative to DNA, the scaling patterns were similar among the 3 muscles. To determine if interspecies differences were linked to transcriptional regulation, ~800bp of the PGC-1α promoter from 56 terrestrial mammals (5g-5000kg) was examined. The basal placental mammalian promoter possesses putative elements for Sp1, HNF3, myogenic factors and metabolic effectors, which have been retained in mammals with little change in order or spacing. To investigate the ability of these promoters to control PGC-1α expression, rodent promoters were cloned into luciferase reporter gene constructs and transfected into a common mouse myoblast background (Sol8 cells). Unlike mitochondrial content, promoter activity did not vary with body size across the rodent family. Likewise, PGC-1α transcript levels did not vary in rodent muscles in a way that would explain differences in COX activity. This suggests that though PGC-1α may be crucial for within species variation, transcriptional regulation of PGC-1α is not responsible for interspecies variation in mitochondrial content. / Thesis (Master, Biology) -- Queen's University, 2010-06-09 10:50:02.133

Le récepteur relié au récepteur à l'estrogène alpha (ERR?) est un récepteur orphelin de la superfamille des récepteurs nucléaires impliqué dans la régulation du métabolisme énergétique. II cause l'expression de gènes impliqués dans les différentes voies métaboliques et peut ainsi augmenter le potentiel de production d'énergie des cellules. Fidèle à son état d'orphelin, l'activité de ERR? ne semble pas être régulée via la liaison d'un ligand, mais plutôt via la présence des corégulateurs. Les corégulateurs sont des protéines ayant pour fonction d'assister ou de nuire à l'action des récepteurs nucléaires et autres facteurs de transcription dans l'expression de leurs gènes cibles. L'étude de l'interaction entre ERR? et ses corégulateurs s'avère donc être incontournable pour la compréhension de son rôle dans les divers processus où il est impliqué et dans les pathologies auxquelles il est associé comme le cancer qui est un dérèglement cellulaire causé par des altérations génétiques, et est caractérisé par une prolifération incontrôlée de cellules au sein d'un organisme. Pour qu'une cellule devienne cancéreuse, elle doit acquérir des mutations précises au niveau du génome causant des modifications dans la régulation de différents processus. Un de ces processus est le métabolisme énergétique. Le but de ma maîtrise était d'identifier et d'étudier les corégulateurs de ERR? (PGC-1?, PGC-1? et PRC) et leur modulation de gènes cibles de ERR? dans le contexte du métabolisme énergétique du cancer. L'expression de ERR? et des trois membres de la famille PGC-1 a été mesurée par qPCR sur des tissus colorectaux sains et cancéreux appropriés. L'expression de ERR? ne semble pas significativement différente suivant l'apparition de la pathologie, alors que l'expression des coactivateurs PGC1-? et PGC-1? s'avère moins élevée dans les tissus cancéreux par rapport aux tissus sains. L'expression de PRC est plus grande dans le cancer colorectal. Ces résultats suggèrent que PRC pourrait posséder un rôle, exclusif à sa famille, dans le carcinome colorectal. Afin de trouver de nouveaux corégulateurs de ERR?, nous avons procédé à une expérience de double hybride sur levure qui a permis d'identifier 6 nouveaux partenaires d'interactions du récepteur : La leucine aminopeptidase 3 (LAP3), le synaptic nuclear envelope protein 1 (SYNE1 /nesprinl ), le proteasome macropain 26S subunit ATPase (PSMC5), la DEAD box polypeptide 1 (DDXI), le ring finger protein 2 (RNF2/Ring2) et l'interferon-related developmental regulator IFRD1. Leurs fonctions connues peuvent toutes être associées à l'action de ERR?, soit en régulant sa dégradation ou son activité. En particulier, nos résultats pour 1FRD1 montrent que son expression est supérieure dans la presque totalité des cancers colorectaux étudiés par rapport aux tissus sains adjacents. On peut ainsi supposer que cette protéine possède une fonction encourageant la carcinogenèse. De plus, des essais luciférases ont montré qu'IFRD1 augmente l'activité du récepteur nucléaire ERR?. Le partenariat entre ERR? et IFRD I pourrait donc être pertinent lors de la carcinogenèse colorectale. ERR?, PGC-1, PRC, IFRD1.

PGC-1 beta é um co-ativador de transcrição gênica responsável pela regulação do metabolismo celular, principalmente na biogênese e função mitocondrial, disponibilidade de substrato e síntese de lipídios. Nos últimos anos, outras isoformas de PGC-1 têm sido descritos como participantes na gênese e manutenção de tumores. Portanto, nosso objetivo foi determinar se o PGC-1beta está relacionado ao aumento da proliferação celular de células de melanoma. Inicialmente, foi demonstrado que os níveis de RNAm e proteína de PGC-1beta são muito mais elevados em linhagens de células de melanoma (Tm1 e Tm5) do que na linhagem parental de melanócitos não tumorais (Melan-a) como detectado por PCR quantitativa e western blotting. A fim de descobrir uma relação causal entre a expressão de PGC-1? e crescimento celular da linhagem Tm5, células de tal linhagem foram transfectadas com um oligonucleotídeo antisense (ASO) contra PGC-1beta. As células tratados com ASO apresentaram níveis mais baixos de RNAm e proteína PGC-1beta, além de redução em sua atividade avaliada pela expressão de genes PGC-1beta dependentes. Além disso, as células transfectadas apresentaram uma taxa de proliferação inferior em comparação com células de controle Tm5. Este fenômeno também foi observado in vivo. Quando injetadas em camundongos, as células Tm5 desenvolvem-se em um tumor que atinge 1,34 ± 0,20 cm3 após nove dias. Tumores tratados com ASO após o mesmo tempo apresentaram volume tumoral de 0,75 ± 0,05 cm3. Este crescimento não estava relacionada à necrose tumoral, mas sim com a proliferação reduzida de células. Finalmente, verificamos se o mesmo fenômeno seria observado em humanos. A expressão PGC-1beta foi muito maior em amostras de melanoma do que em nevos, alterações não-malignas da pele com alto conteúdo de melanina. Por conseguinte, conclui-se que a expressão PGC-1? está aumentada no melanoma, tanto murino e humano, e que o bloqueio da sua atividade leva à diminuição da proliferação celular e crescimento tumoral / PGC-1beta is a co-activator of gene transcription primarily responsible for the regulation of cellular metabolism, mainly in mitochondrial biogenesis and function and also substrate and lipid synthesis. In recent years, other isoforms of PGC-1 have been described as participating in the genesis and maintenance of tumors. Therefore, our objective was to determine whether PGC-1beta is related to increased proliferation of melanoma cells. Initially, it was demonstrated that mRNA and protein levels of PGC-1beta are much higher in melanoma cell lines (Tm1 and TM5) than in the non-tumoral parental lineage melanocytes (melan-a) as detected by quantitative PCR and Western blotting. In order to find a causal relationship between the expression of PGC-1beta and cell growth, Tm5 lineage cells were transfected with an antisense oligonucleotide (ASO) against PGC-1beta. The cells treated with ASO had lower levels of PGC-1beta mRNA and protein, as well as reduction in its activity detected by quantitation of PGC-1beta dependent genes expression. Furthermore, transfected cells showed a lower rate of proliferation compared to Tm5control cells. This phenomenon was also observed in vivo. When injected into mice, Tm5 cells develop a tumor which reaches 1.34 ± 0.20 cm3 after nine days. Tumors treated with ASO, after the same time, presented tumor volume of 0.75 ± 0.05 cm 3. This growth was not related to tumor necrosis, but with reduced cell proliferation. Finally, we checked whether the same phenomenon would be observed in humans. The PGC-1beta expression was much higher in melanoma samples than in nevi, a non-malignant skin alteration filled with melanin. Therefore, we concluded that PGC-1beta expression in melanoma is increased, both in murine and human, and that blocking its activity leads to decreased cell proliferation and tumor growth

A capacidade de sincronizar vias metabólicas a estímulos ambientais é um aspecto central da homeostase em mamíferos. Dentro desse contexto, o controle molecular da função mitocondrial representa um aspecto fundamental e defeitos na integridade desse sistema podem levar a severas perturbações à homeostase celular levando a um amplo espectro de doenças como a obesidade e o diabetes tipo 2. O controle transcricional do metabolismo energético é um processo dinâmico que depende da ação coordenada de fatores de transcrição, enzimas modificadoras de cromatina e coreguladores transcricionais. Co-reguladores podem agir como interruptores transcricionais ativando ou reprimindo a atividade de receptores nucleares. Neste estudo, demonstramos que o coativador PGC-1? e o co-repressor NCoR1 são importantes mediadores do metabolismo energético e da homeostase redox mitocondrial em células musculares. Nossos resultados sugerem que os efeitos desses co-reguladores são mediados pela transativação do elemento responsivo de PPAR (PPRE) em promotores de seletos grupos de genes. Ainda, a indução da capacidade oxidativa e da defesa antioxidante pelo silenciamento de NCoR1 ou pela expressão de PGC-1? atenua a produção de espécies reativas de oxigênio e a morte celular induzida por estresse metabólico. Essas evidências sugerem que o equilíbrio entre a ativação e a repressão transcricional em promotores contendo PPREs exerce um papel central na função mitocondrial em células musculares esqueléticas. Coletivamente, os resultados deste estudo indicam que o antagonismo entre os coreguladores PGC-1? e NCoR1 é um componente central no controle da função mitocondrial representando uma interface promissora para o desenvolvimento de novas abordagens terapêuticas para o tratamento e prevenção da disfunção metabólica. / The ability to synchronize metabolic pathways to environmental stimuli is a central aspect of mammalian homeostasis. Within this context, the molecular control of mitochondrial function represents a fundamental aspect and defects in the integrity of this system can lead to severe disturbances to cellular homeostasis causing a wide spectrum of pathologies such as obesity and type 2 diabetes. Transcriptional control of energy metabolism is a dynamic process that depends on the coordinated action of transcription factors, chromatin modifying enzymes, and transcriptional co-regulators. Co-regulators can act as transcriptional switches activating or repressing the activity of nuclear receptors. In this study, we demonstrated that the co-activator PGC-1? and NCoR1 co-repressor are essential mediators of energy metabolism and mitochondrial redox homeostasis in muscle cells. Our results suggest that the effects of these co-regulators are mediated by the transactivation of the PPAR responsive elements (PPREs) in promoters of selected gene groups. Furthermore, the oxidative capacity and antioxidant defense induction by either NCoR1 knockdown or PGC-1? overexpression attenuates the production of reactive oxygen species and cell death induced by metabolic stress. These evidence suggest that the balance between activation and transcriptional repression in promoters containing PPREs exert a central role in mitochondrial function in skeletal muscle cells. Collectively, the results of this study indicate that the antagonism between the co-regulators PGC-1? and NCoR1 is a central component of mitochondrial function representing a promising interface for the development of novel therapeutic approaches for the treatment and prevention of metabolic dysfunction.