Changes in intracellular redox homeostasis are implicated in both normal cell signaling and as pathophysiological mechanisms contributing to a variety of age-related diseases, including diabetes, atherosclerosis, neurodegenerative conditions, and cancer. Though a variety of well described mechanisms exist to counterbalance the overproduction of cellular oxidants and maintain optimal intracellular redox poise, the understanding of the mechanism(s) through which cellular redox homeostasis regulates cell signaling functions is less well understood. Here, we demonstrate that signaling by the immediate early gene / orphan nuclear hormone receptor NGFI-B (Nur77, TR3), which functions pleiotropically in the regulation of cell growth, metabolism, differentiation and death in diverse tissues, is redox-regulated at both the level of induction and NGFI-B-dependent gene transcription. Using co-immunoprecipitation experiments in cells, we also identified a novel interaction between NGFI-B and the cytoplasmic thiol-reducing catalyst thioredoxin1 (Trx1), that, similar to DTT, blocks NGFI-B-dependent gene expression in a manner that depends on the Trx1 active site cysteines. Together these observations add NGFI-B-dependent gene expression to a growing portfolio of transcription factor pathways that are redox-regulated. NGFI-B, in addition, appears to regulate the mitochondrial membrane potential in L6 skeletal myoblasts. NGFI-B is indispensible for T-cell receptor-mediated apoptosis and induces cell death in a variety of cell types in response to diverse pro-apoptotic stimuli. Like p53, translocation of NGFI-B from the nucleus to the mitochondria may be a critical aspect of its pro-apoptotic function. Interestingly, we found that enforced NGFI-B expression in L6 skeletal muscle myoblasts led to a significant decrease of MMP that peaked 48hr after transfection and did not require a cell death-inducing stimulus. Moreover, NGFI-B transfected cells had no increase in mitochondrial cytochrome C release despite loss of MMP at 48 hr. Combined, these data suggest that loss of MMP in muscle cells may be an early event in the apoptotic process regulated by NGFI-B. This, along with the redox regulation of NGFI-B, provides unique evidence of a relationship between the mitochondria, mitochondrial by-products, ROS, and the regulation of and by the transcription factor NGFI-B. / text

This thesis is a series of investigations into the regulation of skeletal muscle mitochondrial metabolism. Novel regulatory mechanisms regarding mitochondrial fatty acid oxidation are continually being identified and alterations in skeletal muscle mitochondrial metabolism have been implicated in the pathogenesis of type II diabetes (T2DM). Therefore, advancing our basic understanding of mitochondrial regulatory processes is required to provide insight into the progression of T2DM. In study one, the utilization of knockout mice for the putative mitochondrial fatty acid transport protein FAT/CD36, showed that mitochondrial FAT/CD36 plays a functional role in mitochondrial long chain fatty acid (LCFA) oxidation. Specifically, FAT/CD36 was found to be located on the outer mitochondrial membrane (OMM) upstream of acyl-CoA synthetase. In study two, it was observed that in rat muscle, malonyl-CoA (M-CoA) inhibition kinetics of carnitine palmitoyltransferase I (CPT-I) display a more physiological IC50 in permeabilzed muscle fibre bundles (PmFB) compared to isolated mitochondria. These data suggest that the cytoskeleton may have a role in regulating M-CoA inhibition. Additionally, a significant effect of LCFA-CoA on M-CoA inhibition kinetics was observed. These data indicate that M-CoA content does not need to decrease to promote an increase in CPT-I flux. Finally, in a model of T2DM (ZDF rat), submaximal ADP-stimulated respiration rates and the content of adenine nucleotide translocase 2 (ANT2) content were depressed compared to lean control animals. Resveratrol treatment in ZDF rats recovered these declines concomitantly with improving insulin-stimulated skeletal muscle glucose uptake and the cellular redox state. A number of novel findings are presented, specifically, 1) a functional role for mitochondrial FAT/CD36 in mitochondrial LCFA oxidation was confirmed and the topology of this protein along the OMM is expanded upon, 2) M-CoA inhibition kinetics of CPT-I were re-evaluated in PmFB and a regulatory role of LCFA-CoA on M-CoA inhibition kinetics is established, and 3) submaximal ADP-stimulated respiration rates and ANT2 content are depressed in the ZDF rat and resveratrol supplementation prevents these decrements.

The estuarine sheepshead minnow (Cyprinodon variegatus) is the most eurythermic fish species, with a thermal tolerance window between 0.6°C and 45.1°C. However, little is known about the physiological mechanisms that allow this species to survive this temperature range. In order to understand how sheepshead minnow physiology is affected by temperature acclimation and acute changes in temperature, I conducted research on this species using a multi-level approach. I began at the organismal level, and examined the effects of these temperature changes on the sheepshead minnow's metabolic rate and swimming performance. The next chapter investigated the effects of changing temperatures on cardiac function (i.e., tissue/organ specific effects). In the final chapter, I conducted research at the sub-cellular level, and determined how mitochondrial bioenergetics / function is impacted by changing temperatures. This research shows that while sheepshead minnows are able to sustain heart function and mitochondrial respiration over a broad range of temperatures; they also display a plastic temperature response which is associated with the downregulation of standard metabolic rate and cardiac remodeling to maintain force generation. Collectively, these physiological responses may contribute to the sheepshead minnow's ability to maintain physiological and organismal function across a large temperature range.

Gap junctions are highly-conserved communicating junctions composed of the connexin family of proteins. In addition to this channel function, gap junctions mediate adhesive contacts at extracellular domains, and are host to a variety of signalling metabolites at intracellular surfaces. In this thesis, I explore the emerging theme of the connexin interactome. Starting with a non-biased proteomic approach, I identified endogenous protein interactions with the predominant liver and oligodendrocyte connexin, connexin32 (Cx32). Here, I identified novel mitochondrial protein interactions suggesting that Cx32 might localize to mitochondrial membranes, as has been reported for cardiac Cx43. Following proteomic quantitation of WT and Cx32 KO membranes, I determined that loss of Cx32 specifically induces mitochondrial protein expression. Bioenergetic analysis of isolated mitochondria then confirmed that oxygen consumption and rates of reactive oxygen species (ROS) generation were elevated in Cx32 KO mitochondria. In addition to novel intracellular connexin protein interactions, we hypothesized that connexin-mediated glial cell:cell interactions were responsible for mediating fate decisions in the complex hippocampal neurogenic niche environment. We identified that Cx32-mediated glial cell:cell interactions exert significant proliferative and fate specifying pressures on hippocampal progenitor cell types, wherein the loss of Cx32 enables improved histological and functional regeneration following excitotoxic injury. Together, this thesis identifies novel connexin-mediated signalling pathways that provide mechanistic insight into both intracellular and extracellular interactomedependent functions for Cx32, and outlines a potentially transformative avenue for brain repair.

Background: Adipose-derived stem/stromal cells (ASCs) isolated from the stromal vascular fraction are a source of mesenchymal stem cells that have been shown to be beneficial in many regenerative medicine applications. ASCs are an attractive source of stem cells in particular, due to their lack of immunogenicity. This study examines differences between mitochondrial bioenergetic profiles of ASCs isolated from adipose tissue of five peri-organ regions: pericardial, thymic, knee, shoulder, and abdomen. Results: Flow cytometry showed that the majority of each ASC population isolated from the adipose tissue of 12 donors, with an n = 3 for each tissue type, were positive for MSC markers CD90, CD73, and CD105, and negative for hematopoietic markers CD34, CD11B, CD19, and CD45. Bioenergetic profiles were obtained for ASCs with an n = 4 for each tissue type and graphed together for comparison. Mitochondrial stress tests provided the following measurements: basal respiration rate (measured as oxygen consumption rate [pmol O-2/min], ATP production, proton leak, maximal respiration, respiratory control ratio, coupling efficiency, and non-mitochondrial respiration. Glycolytic stress tests provided the following measurements: basal glycolysis rate (measured as extracellular acidification rate [mpH/min]), glycolytic capacity, glycolytic reserve, and non-glycolytic acidification. Conclusions: The main goal of this manuscript was to provide baseline reference values for future experiments and to compare bioenergetic potentials of ASCs isolated from adipose tissue harvested from different anatomical locations. Through an investigation of mitochondrial respiration and glycolysis, it was demonstrated that bioenergetic profiles do not significantly differ by region due to depot-dependent and donor-dependent variability. Thus, although the physiological function, microenvironment and anatomical harvest site may directly affect the characteristics of ASCs isolated from different organ regions, the ultimate utility of ASCs remains independent of the anatomical harvest site.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A obesidade, doença resultante do acúmulo excessivo de gordura corporal, é importante fator de risco para diabetes mellitus tipo 2, dislipidemias e doenças cardiovasculares, doenças de alta prevalência em todo o mundo. O processo de transição nutricional decorrente da globalização contribuiu para o crescente número de indivíduos com obesidade, principalmente pela modificação nos hábitos alimentares da população, com ampla inclusão de produtos industrializados ricos em gordura saturada, sal e açúcar, denominada dieta ocidental. Os mecanismos pelos quais a obesidade induzida por dieta leva ao desenvolvimento de doenças cardiovasculares ainda não estão completamente esclarecidos na literatura, porém sabe-se que a obesidade leva ao comprometimento da função cardíaca e do metabolismo energético, aumentando a morbidade e mortalidade. Em grande parte dos estudos relacionados à obesidade, o metabolismo energético celular comprometido associa-se à disfunção mitocondrial. Neste contexto, torna-se importante avaliar a função mitocondrial na obesidade, visto que as mitocôndrias são organelas com funções-chave no metabolismo energético. No presente estudo, avaliamos inicialmente o efeito obesogênico da dieta ocidental em camundongos Swiss por 16 semanas a partir do desmame. Para tal, analisamos a ingestão alimentar, evolução da massa corporal, Índice de Lee, peso das gorduras epididimal e retroperitoneal, peso e morfologia do fígado, relação entre o peso do fígado/massa corporal, peso do ventrículo esquerdo (VE)/massa corporal, glicemia de jejum e teste intraperitoneal de tolerância à glicose. Avaliamos também o consumo de oxigênio das fibras cardíacas através da respirometria de alta resolução. Além disso, o conteúdo das proteínas envolvidas no metabolismo energético: Carnitina Palmitoil Transferase 1 (CPT1), proteína desacopladora 2 (UCP2), Transportadores de glicose 1 e 4 (GLUT1 e GLUT4), proteína quinase ativada por AMP (AMPK), proteína quinase ativada por AMP fosforilada (pAMPK), receptor de insulina &#946; (IR&#946;) e substrato do receptor de insulina 1 (IRS-1) foi determinado por western blotting. Nossos resultados confirmaram o caráter obesogênico da dieta ocidental, visto que os camundongos submetidos a esta dieta (GO), apresentaram-se hiperfágicos (P<0,001) e obesos (72,031,82, P<0,001), com aumento progressivo no ganho de massa corporal. Além do aumento significativo dos parâmetros: Índice de Lee (362,902,44, P<0,001), gorduras epididimal e retroperitonial (3,310,15 e 1,610,11, P<0,001), relação entre o peso do fígado/massa corporal (0,060,003, P<0,001) e peso de ventrículo esquerdo (VE)/massa corporal (0,080,002, P<0,01), hiperglicemia de jejum (192,1014,75, P<0,01), intolerância à glicose (P<0,05, P<0,01) e deposição ectópica de gordura no fígado. A respirometria de alta resolução evidenciou disfunção mitocondrial cardíaca no grupo GO, com reduzida capacidade de oxidação de carboidratos e ácidos graxos (P<0,001) e aumento do desacoplamento entre a fosforilação oxidativa e a síntese de ATP (P<0,001). Os resultados de western blotting evidenciaram aumento nos conteúdos de CPT1 (1,160,08, P<0,05) e UCP2 (1,080,06, P<0,05) e redução no conteúdo de IRS-1 (0,600,08, P<0,05). Não houve diferença significativa nos conteúdos de GLUT1, GLUT4, AMPK, pAMPK, pAMPK/AMPK e IR&#946;. Em conclusão, o consumo da dieta ocidental resultou no desenvolvimento de obesidade com disfunção mitocondrial associada a alterações no metabolismo energético. / Obesity, a disease resulting from excessive accumulation of body fat is a risk factor for type 2 diabetes, dyslipidemia and cardiovascular diseases, which are of high prevalence worldwide. Nutritional transition, a process associated with globalization, has contributed to growing obesity, mainly by changing eating habits of the population, with broad inclusion of industrial products high in saturated fat, salt and sugar, the called Western diet. The mechanisms by which diet-induced obesity leads to cardiovascular disease are not completely understood, but it is known that obesity leads to impairment of cardiac function and energy metabolism, increasing morbidity and mortality. In most obesity studies, the related cellular energy metabolism is compromised associated with mitochondrial dysfunction. In this context, it becomes important to asses mitochondrial function in obesity, since mitochondria are organelles with key roles in energy metabolism. In the present study, we evaluated the effect of the Western diet in Swiss mice for 16 weeks from weaning. We analyzed the food intake, changes in body weight, Lee index, weight of epididymal and retroperitoneal fat, weight and morphology of the liver, the ratio of liver weight /body weight, weight of the left ventricle (LV)/body weight , fasting plasma glucose and intraperitoneal glucose tolerance test. We also evaluated the oxygen consumption of cardiac fibers by high-resolution respirometry. Furthermore, proteins content involved in energy metabolism: carnitine palmitoyl transferase 1 (CPT1), uncoupling protein 2 (UCP2), glucose transporters 1 and 4 (GLUT1 and GLUT4), AMP-activated protein kinase (AMPK), AMP-activated protein kinase phosphorylated (pAMPK), insulin receptor &#946; (IR&#946;) and insulin receptor substrate 1 (IRS-1) was determined by western blotting. Our results confirmed the obesogenic role of the Western diet. Thus, mice subjected to Western diet (WG), presented hyperphagia (P<0.001) and obesity (72.031.82, P<0.001), with a progressive increase in body mass gain. Also, the WG group compared to control had statistically significant increase of the all parameters studied: Lee index (362.902.44, P<0.001), epididymal and retroperitoneal fat (3.310.15 and 1.610.11, P<0.001), ratio of liver weight/body weight (0.060.003, P<0.001) and weight of the left ventricle (LV)/body weight (0.080.002, P<0.01), fasting hyperglycemia (192.1014.75, P<0.01), glucose intolerance (P<0.05, P<0.01) and ectopic fat deposition in liver. High-resolution respirometry showed cardiac mitochondrial dysfunction in the WG group, with reduced capacity of oxidation of carbohydrates and fatty acids and increased uncoupling between oxidative phosphorylation and ATP synthesis. Western blotting results revealed an increase in CPT1 (1.160.08, P<0.05) and UCP2 (1.080.06, P<0.05) content and reduction in IRS-1 content (0.600.08, P<0.05). There was no statistically significant difference in the GLUT1, GLUT4, AMPK, pAMPK, pAMPK/AMPK and IR&#946; content. In conclusion, the Western diet consumption resulted in the development of obesity with mitochondrial dysfunction associated to alterations in energy metabolism.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / A obesidade, doença resultante do acúmulo excessivo de gordura corporal, é importante fator de risco para diabetes mellitus tipo 2, dislipidemias e doenças cardiovasculares, doenças de alta prevalência em todo o mundo. O processo de transição nutricional decorrente da globalização contribuiu para o crescente número de indivíduos com obesidade, principalmente pela modificação nos hábitos alimentares da população, com ampla inclusão de produtos industrializados ricos em gordura saturada, sal e açúcar, denominada dieta ocidental. Os mecanismos pelos quais a obesidade induzida por dieta leva ao desenvolvimento de doenças cardiovasculares ainda não estão completamente esclarecidos na literatura, porém sabe-se que a obesidade leva ao comprometimento da função cardíaca e do metabolismo energético, aumentando a morbidade e mortalidade. Em grande parte dos estudos relacionados à obesidade, o metabolismo energético celular comprometido associa-se à disfunção mitocondrial. Neste contexto, torna-se importante avaliar a função mitocondrial na obesidade, visto que as mitocôndrias são organelas com funções-chave no metabolismo energético. No presente estudo, avaliamos inicialmente o efeito obesogênico da dieta ocidental em camundongos Swiss por 16 semanas a partir do desmame. Para tal, analisamos a ingestão alimentar, evolução da massa corporal, Índice de Lee, peso das gorduras epididimal e retroperitoneal, peso e morfologia do fígado, relação entre o peso do fígado/massa corporal, peso do ventrículo esquerdo (VE)/massa corporal, glicemia de jejum e teste intraperitoneal de tolerância à glicose. Avaliamos também o consumo de oxigênio das fibras cardíacas através da respirometria de alta resolução. Além disso, o conteúdo das proteínas envolvidas no metabolismo energético: Carnitina Palmitoil Transferase 1 (CPT1), proteína desacopladora 2 (UCP2), Transportadores de glicose 1 e 4 (GLUT1 e GLUT4), proteína quinase ativada por AMP (AMPK), proteína quinase ativada por AMP fosforilada (pAMPK), receptor de insulina &#946; (IR&#946;) e substrato do receptor de insulina 1 (IRS-1) foi determinado por western blotting. Nossos resultados confirmaram o caráter obesogênico da dieta ocidental, visto que os camundongos submetidos a esta dieta (GO), apresentaram-se hiperfágicos (P<0,001) e obesos (72,031,82, P<0,001), com aumento progressivo no ganho de massa corporal. Além do aumento significativo dos parâmetros: Índice de Lee (362,902,44, P<0,001), gorduras epididimal e retroperitonial (3,310,15 e 1,610,11, P<0,001), relação entre o peso do fígado/massa corporal (0,060,003, P<0,001) e peso de ventrículo esquerdo (VE)/massa corporal (0,080,002, P<0,01), hiperglicemia de jejum (192,1014,75, P<0,01), intolerância à glicose (P<0,05, P<0,01) e deposição ectópica de gordura no fígado. A respirometria de alta resolução evidenciou disfunção mitocondrial cardíaca no grupo GO, com reduzida capacidade de oxidação de carboidratos e ácidos graxos (P<0,001) e aumento do desacoplamento entre a fosforilação oxidativa e a síntese de ATP (P<0,001). Os resultados de western blotting evidenciaram aumento nos conteúdos de CPT1 (1,160,08, P<0,05) e UCP2 (1,080,06, P<0,05) e redução no conteúdo de IRS-1 (0,600,08, P<0,05). Não houve diferença significativa nos conteúdos de GLUT1, GLUT4, AMPK, pAMPK, pAMPK/AMPK e IR&#946;. Em conclusão, o consumo da dieta ocidental resultou no desenvolvimento de obesidade com disfunção mitocondrial associada a alterações no metabolismo energético. / Obesity, a disease resulting from excessive accumulation of body fat is a risk factor for type 2 diabetes, dyslipidemia and cardiovascular diseases, which are of high prevalence worldwide. Nutritional transition, a process associated with globalization, has contributed to growing obesity, mainly by changing eating habits of the population, with broad inclusion of industrial products high in saturated fat, salt and sugar, the called Western diet. The mechanisms by which diet-induced obesity leads to cardiovascular disease are not completely understood, but it is known that obesity leads to impairment of cardiac function and energy metabolism, increasing morbidity and mortality. In most obesity studies, the related cellular energy metabolism is compromised associated with mitochondrial dysfunction. In this context, it becomes important to asses mitochondrial function in obesity, since mitochondria are organelles with key roles in energy metabolism. In the present study, we evaluated the effect of the Western diet in Swiss mice for 16 weeks from weaning. We analyzed the food intake, changes in body weight, Lee index, weight of epididymal and retroperitoneal fat, weight and morphology of the liver, the ratio of liver weight /body weight, weight of the left ventricle (LV)/body weight , fasting plasma glucose and intraperitoneal glucose tolerance test. We also evaluated the oxygen consumption of cardiac fibers by high-resolution respirometry. Furthermore, proteins content involved in energy metabolism: carnitine palmitoyl transferase 1 (CPT1), uncoupling protein 2 (UCP2), glucose transporters 1 and 4 (GLUT1 and GLUT4), AMP-activated protein kinase (AMPK), AMP-activated protein kinase phosphorylated (pAMPK), insulin receptor &#946; (IR&#946;) and insulin receptor substrate 1 (IRS-1) was determined by western blotting. Our results confirmed the obesogenic role of the Western diet. Thus, mice subjected to Western diet (WG), presented hyperphagia (P<0.001) and obesity (72.031.82, P<0.001), with a progressive increase in body mass gain. Also, the WG group compared to control had statistically significant increase of the all parameters studied: Lee index (362.902.44, P<0.001), epididymal and retroperitoneal fat (3.310.15 and 1.610.11, P<0.001), ratio of liver weight/body weight (0.060.003, P<0.001) and weight of the left ventricle (LV)/body weight (0.080.002, P<0.01), fasting hyperglycemia (192.1014.75, P<0.01), glucose intolerance (P<0.05, P<0.01) and ectopic fat deposition in liver. High-resolution respirometry showed cardiac mitochondrial dysfunction in the WG group, with reduced capacity of oxidation of carbohydrates and fatty acids and increased uncoupling between oxidative phosphorylation and ATP synthesis. Western blotting results revealed an increase in CPT1 (1.160.08, P<0.05) and UCP2 (1.080.06, P<0.05) content and reduction in IRS-1 content (0.600.08, P<0.05). There was no statistically significant difference in the GLUT1, GLUT4, AMPK, pAMPK, pAMPK/AMPK and IR&#946; content. In conclusion, the Western diet consumption resulted in the development of obesity with mitochondrial dysfunction associated to alterations in energy metabolism.

The overall objective of this thesis was to examine mitochondrial bioenergetics in muscle biopsies from humans with type 1 diabetes (T1D) to gain a deeper understanding of the cellular mechanism(s) underlying changes to skeletal muscle health reported in T1D, a phenotype we have referred to as ‘diabetic myopathy’. It was hypothesized that humans with T1D, compared to their matched counterparts without diabetes (control), would demonstrate significant deficiencies in muscle mitochondrial function and ultrastructure/content as determined by the gold-standard in vitro methodology: high-resolution respirometry and transmission electron microscopy, respectively. It was further hypothesized that sex differences would not exist in mitochondrial function with T1D, and mitochondrial deficiencies would be more dramatic at an earlier age with T1D. Adults with uncomplicated T1D and strictly matched controls (age, sex, BMI, self-reported physical activity levels) were recruited from surrounding university-dwelling communities. Site-specific deficiencies in mitochondrial respiration, H2O2 emission, and calcium retention capacity were found in young, physically active adults with T1D despite normal mitochondrial content. Further experiments revealed that muscle mitochondrial respiration in women and men differentially adapt to the T1D environment where men with T1D have lower complex II but higher complex I respiration compared to women with T1D, while women (irrespective of T1D) have lower ADP sensitivity. Women with T1D also demonstrated lower H2O2 emission compared to men with T1D. In contrast, despite a lower mitochondrial content in middle- to older-aged adults with T1D, mitochondrial respiration (normalized to content) was either normal or increased in adults with T1D compared to control, with observable differences between sexes. Overall, this research has demonstrated that despite being recreationally to physically active, adults with uncomplicated T1D with moderately well-managed glycemia demonstrate alterations in skeletal muscle mitochondrial function and ultrastructure, including differences between sexes. / Dissertation / Doctor of Science (PhD) / Type 1 diabetes (T1D) is a complex disease that still has no known cure. Current treatment focuses on managing blood sugar levels with exogenous insulin injections and frequent blood sugar checks. However, over time, people with T1D still develop serious complications that inevitably impact their quality of life and lifespan. A potential adjuvant therapy to prevent complications in T1D is improving the health of skeletal muscle through exercise given its role in stabilizing blood sugar/lipid levels and whole-body insulin sensitivity. However, this area continues to be severely understudied in the T1D population. Thus, this thesis examined skeletal muscle metabolic ‘health’ from adults with T1D who do not have major diabetes complications and manage their blood glucose moderately-well. Through a series of novel experiments, we found that young and middle- to older-aged adults with T1D have alterations in the metabolic engines of their muscles, and depending on biological sex, the alterations manifest as either heightened or degraded cellular function. These findings are the first to provide a comprehensive cellular investigation of the impact of T1D on the metabolic health of skeletal muscle in people with T1D and provide the foundation for future research examining skeletal muscle as an essential and early adjuvant therapy in this population.