Neurons have an extremely high rate of energy consumption and use mitochondrial-derived ATP as the primary energy source to drive biochemical processes involved in various functions. Consequently, neurons produce reactive oxygen species (ROS) as 'by-products' of oxidative phosphorylation. Excessive levels of ROS are highly detrimental to neurons as ROS can directly oxidize and induce damage to cellular macromolecules including lipids, DNA and proteins. Hence, the high-energy demands of neurons, together with their high levels of ROS production, place them at risk during conditions of stress, which occur during aging and in neurodegenerative disorders including Alzheimer's and Huntington's disease. Uncoupling proteins (UCPs) belong to a family of inner mitochondrial membrane proteins initially identified as regulators of thermogenesis in fat cells wherein they uncouple energy-substrate oxidation from mitochondrial ATP production, resulting in the production of heat. UCPs also regulate ROS production from mitochondria by physiologically lowering the mitochondrial membrane potential below the critical level for ROS production. Because of their important role in co-regulating energy metabolism and ROS production, there has been considerable interest in the functions of UCPs. Neurons express at least three UCPs including the widely expressed UCP2 and the brain- specific UCP4 and UCP5. Despite a great deal of interest, to date neither the molecular mechanism nor the biochemical and physiological functions of brain UCPs are well understood. Our previous studies showed that UCP4 is highly expressed in subpopulations of neurons with high energy demands. Knockdown ofUCP4 expression in cultured primary neurons markedly enhances neuronal death suggesting that endogenous UCP4 is critical for neuronal survival. Expression of UCP4 shifts cellular ATP synthesis from oxidative phosphorylation to anaerobic glycolysis, which might be beneficial to cell survival. In this study, we investigated the underlying mechanism of UCP4-mediated metabolic adaptation in response to mitochondrial inhibition. We found that UCP4 enhances glucose uptake and glycolysis which may compensate for the reduced supply of ATP from compromised mitochondria. In addition, the activation of mitogen activated protein kinases (MAPKs) and several transcription factors play a role in augmenting nonoxidative synthesis of ATP in response to metabolic stress possibly by acting downstream of UCP4. Elucidating the underlying mechanism(s) whereby this brain UCP mediates metabolic adaptation in response to mitochondrial inhibition will likely lead to the development of novel preventative and therapeutic strategies for neurodegenerative disorders.

Uncoupling protein, a mitochondrial inner membrane protein found in mammalian brown adipose tissue, functions as an uncoupler of oxidative phosphorylation by serving as a proton carrier when activated, resulting in heat production, the function of the tissue. Unlike most nuclear-encoded mitochondrial proteins, uncoupling protein is not made with a cleavable presequence. With the availability of an uncoupling protein cDNA clone, the region responsible for targeting uncoupling protein to mitochondria was examined using in vitro transcription and translation and import into isolated mitochondria. In order to localize the targeting sequence of uncoupling protein, fusion proteins containing portions of uncoupling protein and uncoupling protein modified by site-directed mutagenesis were constructed and analysed for their ability to be imported. Previously it has been shown that there was a targeting signal within uncoupling protein amino acids 13 to 105 (Liu et al., 1988). However, amino acids 13 to 51 did not target a passenger protein to mitochondria (Liu et al., 1988). Here the role of amino acids 53 to 105 of uncoupling protein in targeting was examined with two new constructs, uncoupling protein amino acids 53 to 105 joined to rat ornithine carbamoyltransferase amino acids 147 to 354 and to mouse dihydrofolate reductase. These two constructs along with uncoupling protein with amino acids 2 to 51 deleted were imported into mitochondria consistent with uncoupling protein amino acids 53 to 105 having a potential targeting role in uncoupling protein. Further, these three constructs were processed upon import. The major processed forms of all three constructs are approximately 20 amino acids smaller than the initial translation product. Both fusion constructs also have an intermediate-sized processed form approximately 14 amino acids smaller than the initial translation product. Processing suggests that at least the amino terminus of these proteins has reached the mitochondrial matrix. The location of the proteins was examined using Na2CO3 extraction. Uncoupling protein and U13-105-OCT (uncoupling protein amino acids 13 to 105 joined to ornithine carbamoyltransferase amino acids 147 to 354) were found in the membrane fraction while the processed forms of Ud2-51 (uncoupling protein with amino acids 2 to 52 deleted) and U53-105-DHFR (uncoupling protein amino acids 53 to 105 joined to dihydrofolate reductase) were found in the aqueous fraction suggesting that uncoupling protein amino acids 2 to 52/53 are involved in membrane localization. Analysis of Ud2-35 (uncoupling protein with amino acids 2 to 35 deleted) revealed that it was associated with both the membrane and aqueous fractions. Analysis of uncoupling protein amino acids 53 to 105 revealed the potential existence of two positively charged amphipathic a-helices. Based on the sizes of processed forms and on the helical wheel projection for the first possible sequence, arginine54 , lysine56 and lysine67 were changed to glutamines, individually and in various combinations using oligonucleotide site-directed mutagenesis. All mutant proteins were imported into mitochondria even when all three basic amino acids were replaced. The results suggest that this portion of uncoupling protein, amino acids 54 to 67, is not a targeting signal in the protein. / Thesis / Master of Science (MS)

High circulating glucose and non-esterified free fatty acid (NEFA) levels can cause pancreatic β-cell failure. The molecular mechanisms of this β-cell glucolipotoxicity are yet to be established conclusively. In this thesis by exploring mitochondrial energy metabolism in INS-1E insulinoma cells and isolated pancreatic islets, a role of mitochondria in pancreatic β-cell glucolipotoxicity is uncovered. It is reported that prolonged palmitate exposure at high glucose attenuates glucose-stimulated mitochondrial respiration which is coupled to ADP phosphorylation. These mitochondrial defects coincide with an increased level of mitochondrial reactive oxygen species (ROS), impaired glucose-stimulated insulin secretion (GSIS) and decreased cell viability. Palmitoleate, on the other hand, does not affect mitochondrial ROS levels or cell viability and protects against the adverse effects of palmitate on these phenotypes. Interestingly, palmitoleate does not significantly protect against mitochondrial respiratory or insulin secretion defects and in pancreatic islets tends to limit these functions on its own. Furthermore, strong evidence suggests that glucolipotoxic-induced ROS are of a mitochondrial origin and these ROS are somehow linked with NEFA-induced loss in cell viability. To explore the mechanism of glucolipotxic-induced mitochondrial ROS and associated cell loss, uncoupling protein-2 (UCP2) protein levels and activity were probed in NEFA exposed INS-1E cells. It is concluded that UCP2 neither mediates palmitate-induced mitochondrial ROS production and the related cell loss, nor protects against these deleterious effects. Instead, UCP2 dampens palmitoleate protection against palmitate toxicity. Collectively, these data shed important new light on the area of glucolipotoxicity in pancreatic β-cells and provide novel insights into the pathogenesis of Type 2 diabetes.

The prototype uncoupling protein 1 (UCP1) mediates proton leak-dependent thermogenesis in mammals, but the physiological functions of the novel UCP2-5 are unclear. Nematodes only express one uncoupling protein that is most similar to UCP4 in the human brain, which is believed to be the most evolutionarily conserved of the uncoupling proteins. Consistent with reported UCP functions in mammals, we observed that ceUCP4-null nematodes had decreased metabolic rates and increased adiposity compared to wild type. Surprisingly, these phenotypes corresponded to decreased succinate-mediated mitochondrial respiration without apparent changes in mitochondrial uncoupling. ceUCP4-null mitochondria exhibited normal electron transport chain functions, but had a decreased capacity for succinate import. Supporting the functional importance of ceUCP4-dependent complex II regulation in vivo, ceUCP4 deficiency was demonstrated to result in a selectively lethal response to genetic and pharmacological inhibition of Complex I. Similarly, ceUCP4-deficiency significantly prolonged lifespan in the short-lived mev-1 mutant that generates deleterious complex II-derived reactive oxidants. These results define a new physiological function for the ancestral ceUCP4 in the regulation of complex II-mediated oxidative phosphorylation through an unexpected effect on mitochondrial succinate transport. The data described in this dissertation also describe a novel mechanism by which uncoupling proteins mediate mitochondrial bioenergetics. / text

Traumatic brain injury (TBI) is becoming a national epidemic, as it accounts for 1.5 million cases each year. This disorder affects primarily the young population and elderly. Currently, there is no treatment for TBI, which means that ~2% of the U.S. population is currently living with prolonged neurological damage and dysfunction. Recently, there have been many studies showing that TBI negatively impacts mitochondrial function. It has been proposed that in order to save the cell from destruction mitochondrial function must be preserved. The ketogenic diet, originally designed to mimic fasting physiology, is effective in treating epilepsy. Therefore, we have used fasting as a post injury treatment and attempted to elucidate its underlying mechanism. 24 hours of fasting after a moderate TBI increased tissue sparing, cognitive recovery, improved mitochondrial function, and decreased mitochondrial biomarkers of injury. Fasting results in hypoglycemia, the production of ketones, and the upregulation of free fatty acids (FFA). As such, we investigated the neuroprotective effect of hypoglycemia in the absence of fasting through insulin administration. Insulin administration was not neuroprotective and increased mortality in some treatment groups. However, ketone administration resulted in increased tissue sparing. Also, reduced reactive oxygen species (ROS) production, increased the efficiency of NADH utilization, and increased respiratory function. FFAs and uncoupling proteins (UCP) have been implicated in an endogenously regulated anti-ROS mechanism. FFAs of various chain lengths and saturation were screened for their ability to activate UCP mediated mitochondrial respiration and attenuate ROS production. We also measured FFA levels in serum, brain, and CSF after a 24 hour fast. We also used UCP2 transgenic overexpressing and knockout mice in our CCI injury model, which showed UCP2 overexpression increased tissue sparing, however UCP2 deficient mice did not show a decrease in tissue sparing, compared with their wild type littermates. Together our results indicate that post injury initiated fasting is neuroprotective and that this treatment is able to preserve mitochondrial function. Our work also indicates ketones and UCPs may be working together to preserve mitochondrial and cellular function in a concerted mechanism, and that this cooperative system is the underlying mechanism of fasting induced neuroprotection.

Myocardial and skeletal muscle high energy phosphate metabolism is abnormal in heart failure, but the pathophysiology is not understood. Plasma non-esterified fatty acids (NEFA) increase in heart failure due to increased sympathetic drive, and regulate the transcription of mitochondrial uncoupling protein-3 (UCP3), through peroxisome proliferator-activated receptor-α. The aim of the work in this thesis was to determine whether cardiac PCr/ATP ratios and skeletal muscle PCr kinetics during exercise were related to cardiac and skeletal muscle UCP3 levels respectively, thus providing a mechanism for the apparent mitochondrial dysfunction observed in heart failure. Patients having cardiac surgery underwent pre-operative testing, including cardiac and gastrocnemius 31P magnetic resonance spectroscopy. Intra-operatively, ventricular, atrial and skeletal muscle biopsies were taken for measurement of mitochondrial protein levels by immunoblotting, along with mitochondrial function by tissue respiration rates. Fasting plasma NEFA concentrations increased in patients with ventricular dysfunction and with New York Heart Association (NYHA) class. Ventricular UCP3 levels increased and cardiac PCr/ATP decreased with NYHA class, however, demonstrated no relationship to each other. In skeletal muscle, maximal rates of oxidative ATP synthesis (Qmax) related to functional capacity. Skeletal muscle UCP3 levels increased with NYHA class but were unrelated to skeletal muscle Qmax. Tissue respiration experiments revealed no relationship between ventricular function and indices of mitochondrial coupling, furthermore, indices of mitochondrial coupling were unrelated to tissue UCP3 levels. No evidence was found to support mitochondrial uncoupling, mediated through UCP3, as a cause of the abnormalities in cardiac and skeletal muscle high energy phosphate metabolism.

A obesidade é atualmente um dos problemas públicos de saúde mais visível e negligenciado. Ainda, essa condição pode causar sérios problemas de saúde como doenças cardiovasculares, diabetes, hipertensão arterial e diversos problemas fisiológicos. Dietas alimentares e/ou o uso de fármacos tem demonstrado pouca eficiência (e grandes efeitos colaterais) em reduzir a incidência de sobrepeso e obesidade no mundo. Dessa forma outras estratégias são requeridas para auxiliar no controle desta epidemia global. Nesse contexto, alguns ácidos graxos específicos podem desempenhar um importante papel na regulação da expressão de genes que possuem a habilidade de modular metabolismo. O ácido linoléico conjugado (CLA, 18:2) e o ácido oléico (18:1) tem sido descritos com propriedades anti-obesidade: a respeito de CLA, são bem conhecidos os seus efeitos adipotróficos; quanto ao oléico, são bem conhecidos os seus efeitos anti-diabetes; outros efeitos desses ácidos graxos sobre o metabolismo corporal são desconhecidos ou controversos. Dessa forma, o objetivo deste trabalho foi avaliar os efeitos da suplementação da dieta de camundongos (C57bl6) com esses dois ácidos graxos, individualmente ou em conjunto, na modulação do metabolismo corporal e mitocondrial como uma possível estratégia de combate à obesidade. Foram analisados parâmetros bioquímicos, moleculares, fisiológicos, morfológicos e funcionais. Nossos resultados demonstram que o ácido graxo indutor de aumento do gasto energético corporal e mitocondrial hepático é o CLA e não o oléico; quando suplementados em conjunto, os efeitos metabólicos do CLA se sobrepõem aos do oléico, mantendo esses metabolismos elevados. O aumento de metabolismo mitocondrial está relacionado ao aumento de expressão/atividade de proteínas desacopladoras, as quais parecem ser controladas por espécies reativas de O2 mitocondriais. Demonstramos também que no tecido adiposo branco o ácido oléico não exerce efeitos atróficos como o CLA, os quais estão relacionados a inibição da expressão de PPAR1. Por outro lado, verificamos que a suplementação conjunta com ácido oléico previne efeitos adversos da suplementação da dieta com CLA, como a hipertrofia hepática e resistência a insulina. Concluímos dessa forma que a suplementação da dieta com CLA aumenta o metabolismo corporal e mitocondrial; a suplementação com ácido oléico em conjunto não potencializa os efeitos do CLA, porém previne seus efeitos adversos como a hipertrofia hepática e resistência a insulina. Em conjunto esses ácidos graxos constituem uma boa estratégia alimentar contra o ganho de peso corporal. / Obesity is one of the major public health problems. This condition can leads to the development of other complications such as cardiovascular disease, diabetes and hypertension. Diet and/or drug treatments have shown low efficiency (and major side effects) in reducing the incidence of overweight and obesity in worldwide. On the other hand, some specific fatty acids may play an important role regulating the expression of genes that modulate the metabolism. Conjugated linoleic acid (CLA, 18:2) and oleic acid (18:1) have been known for their anti-obesity properties: CLA is well described inducer of atrophy in adipose tissue, while oleic acid is known by its anti-diabetic effects; other effects of these fatty acids on body metabolism are unknown or controversial. Therefore, the aim of this study was to evaluate the effects of a murine diet supplemented with CLA and oleic acid, individually or together, in the modulation of body and mitochondrial metabolism. Biochemical, molecular, physiological, morphological and functional parameters were analyzed in mice. Our results show that CLA, and not oleic acid, is the fatty acid inductor of increase in body metabolism and liver mitochondrial energy expenditure; when supplemented together, the metabolic effects of CLA overlap the oleic effects, maintaining the high metabolism. Increased mitochondrial metabolism is associated with an increased expression/activity of uncoupling proteins, which appear to be controlled by mitochondrial oxygen reactive species. It was also demonstrated that, in white adipose tissue, oleic acid exerts no atrophic effects as compared to CLA, which are related to inhibition of PPAR1 expression. On the other hand, we demonstrated that the supplementation with oleic acid prevents adverse effects of dietary supplementation with CLA, such as liver hypertrophy and insulin resistance in mice. In conclusion, supplementing the murine diet with CLA increases body and mitochondrial metabolism and reduces the adiposity; in addition, supplementation with oleic acid does not potentialize the metabolic effects of CLA, but prevents its adverse effects. Together CLA and oleic in conjunction appears as a good dietary strategy against the excessive weight gain.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / Diversos estudos tÃm evidenciado que a principal funÃÃo da proteÃna desacopladora mitocondrial de plantas (pUCP) està relacionada a regulaÃÃo de espÃcies reativas de oxigÃnio (EROs). AnÃlises in silico sugerem a existÃncia de famÃlias multigÃnicas para a codificaÃÃo de pUCPs, porÃm novos estudos ainda sÃo necessÃrios para estabelecer o perfil de expressÃo gÃnica das pUCPs, assim como a quantidade de genes em cada espÃcie e suas relaÃÃes filogenÃticas. O presente trabalho teve como objetivo caracterizar, analisar filogeneticamente e avaliar o perfil de expressÃo da famÃlia multigÃnica da pUCP em diferentes tecidos durante o desenvolvimento da soja [Glycine max (L.) MERR.] e em condiÃÃes de estresse. Foi realizada uma anÃlise in silico no genoma da soja e de outras leguminosas disponÃveis no banco de dados WGS, revelando uma famÃlia multigÃnica codificadora da pUCP, UCP1 e 2 com nove Ãxons, UCP 3 com 2 Ãxons, e UCP 4 e 5 com apenas um Ãxon. Dentre as leguminosas analisadas a soja se destacou com o maior nÃmero de genes, 10 genes no total, sendo quatro genes GmUCP1, uma GmUCP2, uma GmUCP3, dois GmUCP4 e dois GmUCP5, alÃm da presenÃa de um splicing alternativo no gene GmUCP1b1. Primers especÃficos foram desenhados para cada membro da GmUCP a fim de analisar os perfis de expressÃo em diferentes tecidos (semente seca e embebida, flores, vagens, cotilÃdones, folhas unifolioladas e trifolioladas, raÃzes, hipocÃtilos e epicÃtilos) durante o desenvolvimento da soja. Para os ensaios em condiÃÃes de estresse foram utilizadas folhas e raÃzes de soja com treze dias apÃs a semeadura (DAS) que foram submetidas a estresse osmÃtico promovido pela aplicaÃÃo de polietileno glicol (PEG) e estresse biÃtico atravÃs de Ãcido salicÃlico (AS). O RNA total de cada amostra foi extraÃdo para a realizaÃÃo de RT-qPCR. Os valores de ct foram obtidos pelo programa realplex e analisados pelo programa GeNorm. O perfil de expressÃo gÃnica mostrou que todos os genes GmUCP foram expressos em todos os tecidos/ÃrgÃos analisados durante o desenvolvimento da soja, com exceÃÃo de alguns genes em semente seca e epicÃtilo. Os diferentes perfis de expressÃo de cada gene durante o desenvolvimento de cada tecido/ÃrgÃo sugerem que ocorra uma regulaÃÃo gÃnica espacial/temporal entre os membros da GmUCP. Os perfis de expressÃo dos genes GmUCP em soja durante as condiÃÃes de estresses foi diversificado, visto que 2 genes apresentaram expressÃo estÃvel em ambos tecidos/estresse, 7 genes apresentaram queda do perfil de expressÃo, enquanto apenas 4 genes apresentaram aumento dos nÃveis de transcritos. / Several studies have evidenced that the main function of the mitochondrial uncoupling protein in plants (pUCP) is related to reactive oxygen species (ROS) regulation. In silico analysis suggests the existence of multigenic families to pUCPs codification, however further studies are yet needed to establish the pUCPs genetic expression profile, just like the gene amount in each species and their phylogenetic relations. The current work had as objective to characterize, analyze phylogeneticly and evaluate the expression profile of the pUCP multigenic family in different tissues during the soybean development [Glycine max (L.) MERR.] and in stress conditions. It has been performed an in silico analysis on the soybean genome and on other legumes available in the database WGS, revealing a codifier multigenic family for pUCP, UCP1 and 2 with 9 exons, UCP3 with 2 exons, and UCP4 and 5 with only 1 exon. Amongst the legumes analyzed, the soybean stood out with the greater number of genes, 10 genes in total, giving four GmUCP1 genes, one GmUCP2, one GmUCP3, two GmUCP4 and two GmUCP5, along with the presence of an alternative splicing on GmUCP1b1 gene. Specific primers have been designed for each GmUCP member in order to analize the expression profiles in different tissues (dry and doused seed, flowers, pods, cotyledons, unifoliate and trifoliate leaves, roots, hypocotyl and epicotyl) during the soybean development. For the assays in stress conditions have been used soybean leaves and roots with thirteen days after sowing (DAS) which have been subjected to osmotic stress caused by the application of polyethylene glycol (PEG) and biotic stress caused by salicylic acid (SA). The total RNA from each sample has been extracted in order to perform the RT-qPCR. The ct values have been obtained through the realplex program and analyzed through the GeNorm program. The genetic expression profile has shown that all genes were expressed in every tissue/organ analyzed during the soybean development, with the exception on some genes in dry seeds and epicotyl. The different expression profiles of each gene during the development of each tissue/organ suggest that occurs a spatial/temporal gene regulation among the GmUCP members. The expression profiles of the GmUCP genes in soybean during the stress conditions have varied, once 2 genes have shown steady expression in both tissues/ stress, 7 genes have shown a drop in the expression profile, while only 4 genes have shown an increase of the transcript levels.

Uncoupling protein-2 (UCP2) may be linked to the attenuation of reactive oxygen species (ROS), but it is unclear whether this phenomenon pertains to the pancreatic beta cell. In this study, a UCP2-deficient mouse model was used to assess the importance of UCP2 to beta cell viability. We investigated the effect of UCP2 absence in response to a beta cell cytotoxic model of diabetes induction. In vivo treatment by the cytotoxic agent streptozotocin led to overall beta cell loss, but severity was not exacerbated by UCP2 deficiency. We also examined ROS production and cell viability in islet cells exposed to various stressors associated with oxidative stress. In vitro measurements of ROS and cell death in islet cells demonstrated that the response was not influenced by UCP2 expression. In contrast with UCP2 overexpression studies showing cytoprotection, this study reveals that beta cell survival is not compromised by the absence of UCP2.

Uncoupling protein-2 (UCP2) may be linked to the attenuation of reactive oxygen species (ROS), but it is unclear whether this phenomenon pertains to the pancreatic beta cell. In this study, a UCP2-deficient mouse model was used to assess the importance of UCP2 to beta cell viability. We investigated the effect of UCP2 absence in response to a beta cell cytotoxic model of diabetes induction. In vivo treatment by the cytotoxic agent streptozotocin led to overall beta cell loss, but severity was not exacerbated by UCP2 deficiency. We also examined ROS production and cell viability in islet cells exposed to various stressors associated with oxidative stress. In vitro measurements of ROS and cell death in islet cells demonstrated that the response was not influenced by UCP2 expression. In contrast with UCP2 overexpression studies showing cytoprotection, this study reveals that beta cell survival is not compromised by the absence of UCP2.