Estudamos a homeostase energética no cérebro (córtex cerebral, estriado e hipocampo) e tecidos periféricos (coração e músculo esquelético) de camundongos selvagens (WT) e nocaute para a enzima glutaril-CoA desidrogenase (Gcdh-/-), modelo animal genético para estudo da acidemia glutárica tipo I (AG I), com 15 e 30 dias de vida. Esses animais também foram submetidos a uma sobrecarga de lisina através de uma injeção intraperitoneal (8 μmol/g) desse aminoácido ou de uma dieta rica em lisina (4,7 %) por 60 horas. Os parâmetros da homeostase energética analisados foram as atividades dos complexos I-III, II, II-III e IV da cadeia respiratória, das enzimas do ciclo do ácido cítrico (CAC) citrato sintase (CS), aconitase, isocitrato desidrogenase (IDH), α- cetoglutarato desidrogenase, sucinato desidrogenase e malato desidrogenase, da creatina quinase (CK) e da Na+, K+ - ATPase, bem como a liberação de lactato, os parâmetros respiratórios mitocondriais estados 3 e 4, razão de controle respiratório e o estado desacoplado, além do potencial de membrana mitocondrial na presença ou ausência de Ca2+. Estudos histológicos também foram conduzidos no córtex cerebral e estriado dos camundongos WT e Gcdh-/- de 30, 60 e 90 dias de vida submetidos por um pequeno (60 horas) ou longo (30 dias) período com dieta com alta concentração de lisina (4,7 %). Verificamos leves alterações nas atividades dos complexos da cadeia respiratória no cérebro, coração e músculo esquelético dos animais Gcdh-/- quando comparados aos WT com 15 e 30 dias de vida. Além disso, demonstramos uma diminuição significativa das atividades da CS e IDH em preparações mitocondriais de estriado de camundongos Gcdh-/- submetidos a uma sobrecarga de lisina associada a um pequeno aumento na liberação de lactato. No entanto, não encontramos alterações nos parâmetros respiratórios e no potencial de membrana em mitocôndrias de estriado dos camundongos Gcdh-/-quando comparados aos WT. Por outro lado, as atividades da Na+, K+-ATPase (cérebro) e CK (cérebro e músculo esquelético) foram significativamente menores em camundongos Gcdh-/- com 15 dias de vida quando submetidos a uma injeção intraperitoneal de lisina. Além disso, encontramos uma redução na atividade da Na+, K+-ATPase associada com uma diminuição da sua expressão em córtex cerebral, mas não em estriado e hipocampo, de camundongos Gcdh-/- com 30 dias de vida submetidos ou não a uma dieta rica em lisina. Finalmente, a análise histológica revelou a presença de vacúolos no córtex cerebral dos camundongos Gcdh-/- com 60 e 90 dias de vida, bem como no estriado dos animais Gcdh-/- com 90 dias de vida que foram alimentados com uma dieta rica em lisina por 30 dias. Concluindo, presumimos que uma redução das atividades da Na+, K+-ATPase e CK possa contribuir para o dano neurológico encontrado nos camundongos Gcdh-/- e possivelmente nos pacientes com AG I. / We studied energy homeostasis in the brain (cerebral cortex, striatum and hippocampus) and peripheral tissues (heart and skeletal muscle) from 15 and 30- day-old wild type (WT) and glutaryl-CoA dehydrogenase deficient (Gcdh-/-) mice, which is a genetic animal model to study glutaric academia type I (GA I). These animals were also submitted to lysine overload through an intraperitoneal injection (8 μmol/g) of this amino acid or supplementing the mice with a high lysine (4.7 %) diet for 60 hours. The energy homeostasis parameters evaluated were the activities of the respiratory chain complexes I-III, II, II-III and IV, of the citric acid cycle (CAC) enzymes citrate synthase (CS), aconitase, isocitrate dehydrogenase (IDH), α-ketoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase, creatine kinase (CK) and Na+, K+ - ATPase, as well as the lactate release, the mitochondrial respiratory parameters states 3 and 4, respiratory control ratio and uncoupled state, besides the mitochondrial membrane potential in the presence or absence of Ca2+. Histological studies were also conducted in the cerebral cortex and striatum from 30, 60 and 90-day-old WT and Gcdh-/- mice submitted for a short (60 hours) or long (30 days) period to a high lysine (4.7 %) diet. We verified mild alterations in the respiratory chain activity in the brain, heart and skeletal muscle from Gcdh-/- animals when compared to 15 and 30-day-old WT mice. Furthermore, we demonstrated a reduction in the activities of CS and IDH in striatum mitochondrial preparations from Gcdh-/- mice submitted to a lysine overload associated with a mild increase of lactate release. However, we did not find alterations in the respiratory parameters and membrane potential in striatum mitochondria from Gcdh-/- mice when compared to WT. On the other hand, the activities of Na+, K+-ATPase (brain) and CK (brain and skeletal muscle) were significantly reduced in 15-day-old Gcdh-/- mice when received an intraperitoneal injection of lysine. Moreover, a reduction in the Na+, K+-ATPase activity associated with a diminution of its expression was observed in the cerebral cortex, but not in striatum and hippocampus, from 30-day-old Gcdh-/- mice submitted or not to a high lysine diet. Finally, the histological analyses revealed the presence of vacuoles in the cerebral cortex from 60 and 90-day-old Gcdh-/- mice, as well as in the striatum from 90-day-old Gcdh-/- animals that were fed a high Lys chow for 30 days. In conclusion, we presume that a reduction in the activities of Na+, K+- ATPase and CK may contribute to the brain damage found in Gcdh-/- mice and possibly in GA I patients.

Orientador: Otavio Rizzi Coelho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T04:45:29Z (GMT). No. of bitstreams: 1 Almeida_RaitanyCostade_M.pdf: 2666422 bytes, checksum: 10b733ae4ca57255b813baf100fd8a15 (MD5) Previous issue date: 2009 / Resumo: Entre as operações não cardíacas, as vasculares apresentam os índices de complicações cardiovasculares mais elevados. A doença arterial coronariana (D/KC) é a principal causa de morte após operação vascular. Apesar dos efeitos benéficos comprovados das estatinas em termos de prevenção primária e secundária de eventos cardiovasculares, não existem estudos analisando o efeito de diferentes doses de estatinas na morbimortalidade cardiovascular em uma população de pacientes submetidos a operações não cardíacas. Este estudo clínico, prospectivo, aleatorizado, controlado e duplo-cego, conduzido pela Disciplina de Cardiologia no Hospital de Clínicas da Universidade Estadual de Campinas, foi desenvolvido para avaliar se atorvastatina 80 mg reduz mortalidade e morbídade cardiovascular periope rato ria nos 30 dias após cirurgia vascular em comparação com a dose atorvastatina 20 mg. Foram definidos como eventos cardiovasculares: morte de causa cardíaca; infarto agudo do miocárdio não fatal e acidente vascular cerebral. O objetivo secundário do estudo foi avaliar o efeito das diferentes doses de atorvastatina nos níveis séricos de LDL-C, HDL-C, Colesterol total, triglicérides e proteína C-reativa. Segurança foi avaliada através de monitoramento de níveis de creatinoquinase e aminotransferases hepáticas. Os pacientes incluídos foram aleatorizados para receber, diariamente, por um período de 60 dias, 80 mg de atorvastatina ou 20 mg de atorvastatina. A operação vascular ocorreu durante o período de administração do medicamento do estudo. Acompanhamos 106 pacientes, sendo que, 53 em cada grupo. No total 10 pacientes (9,43%) apresentaram algum dos eventos pesquisados.Três pacientes pertenciam ao grupo atorvastatina 80 mg e 7 pacientes pertenciam ao grupo atorvastatina 20 mg. Observou-se incidência duas vezes maior no grupo atorvastatina 20 mg (13,20%) do que no grupo atorvastatina 80 mg (5,66%), sendo esta diferença não estatisticamente significativa (p= 0,319). A análise da probabilidade de sobrevida dos pacientes em 30 dias evidenciou uma diferença não estatisticamente significativa entre as curvas de sobrevida dos dois grupos (p= 0.179), sendo que a probabilidade livre de eventos cardiovasculares caiu mais rapidamente para o grupo atorvastatina 20mg, sugerindo que dose alta de atorvastatina é um fator de proteção para esses pacientes. Ocorreu uma redução, significativamente, maior no níveis de Colesterol total (36% x 28% p=0.013 ), LDL-C (47% X 35% - p= 0.011) e Proteína C reativa (25% x 20% p=0.022) no grupo atorvastatina 80 mg. Não houve diferença significativa entre os grupos quanto a elevação de creatinofosfoquinase e transaminases hepáticas. Com estes dados concluímos que o uso de atorvastatina 80 mg tende a reduzir mais eventos cardiovasculares adversos do que 20mg de atorvastatina no perioperatório de cirurgia vascular / Abstract: Among noncardiac operations, the vascular surgery has been associated with highest incidence of cardiovascular complications. Coronary artery disease (CAD) has been the most common cause of death after major vascular surgery. Despite its beneficial effects on primary and secondary prevention of cardiovascular events, there is no clinical trial analyzing the effect of different doses statins on perioperative cardiovascular mortality and morbidity of patients undergoing -noncardiac surgery. This study is a clinical, prospective, randomized, controlled and double-blind trial, developed by Cardiology Department of Clinics Hospital -Campinas University to analyze the effects of atorvastatin 80 mg compared to atorvastatin 20 mg on the occurrence of a 30 days composite of perioperative cardiac mortality and cardiovascular events (non-fatal myocardial infarction and stroke) after noncardiac vascular surgery. Secondary endpoint was the analysis of the effect on Low Density Lipoprotein-Cholesterol, High Density Lipoprotein-Cholesterol, Total Cholesterol, Triglycerides and C-reactive protein levels. Safety was assessed by monitoring levels of liver enzymes and creatine-kinase.Subjects who satisfied the inclusion criteria were randomly assigned to receive once a day, for 60 days, 80 mg of atorvastatin or 20 mg atorvastatin. We prospectively studied 106 patients during 30 days after vascular surgery, 53 patients in each group. Ten patients (9,43%) developed primary endpoint, of which 3 belonged to atorvastatin 80 mg group and 7 to atorvastatin 20 mg group. An incidence of cardiac events two times higher at atorvastatin 20 mg group (13,20%) than at atorvastatin 80 mg group (5,66% ), but there is no statistically differences (p= 0,319 ). The analysis of survival-probability of patients at 30 days showed a difference not statistically significant between the survival curves of the two groups (p = 0,179), and the probability free of cardiovascular events fell faster for atorvastatin 20 mg group, suggesting that high dose of atorvastatin is a protective factor for these patients. There was a reduction, significantly higher in levels of total cholesterol (36% vs. 28% p = 0.013), LDL-C (47% vs. 35% - p = 0,011) and C-reactive protein (25% vs. 20% p = 0.022) in the atorvastatin 80 mg group. There was no significant difference between groups in the elevation of creatine kinase and liver transaminases. With-these data we conclude that the use of atorvastatin 80 mg tends to reduce adverse cardiovascular events more than 20 mg of atorvastatin in the perioperative of vascular surgery / Mestrado / Clinica Medica / Mestre em Clinica Medica

Abstract In the cells, the last step of the beta-oxidation cycle, aiming at the degradation of fatty acids, is catalyzed by the enzyme named thiolase. It shortens the acyl chain of the acyl-CoA by two carbons. The reaction is reversible, it can proceed for both directions. Thiolases are divided into two categories, synthetic and degradative ones. These two classes of thiolases differ not only by their biological function, but also by their substrate specificity. Degradative thiolases accept substrates with various lengths but synthetic thiolases only accept short chain-acyl-CoAs as a substrate. In humans, at least six isozymes of thiolases are found. The mitochondrial biosynthetic thiolase, T2, differs from other thiolases by getting activated by potassium. In addition, it accepts branched acyl-CoA, namely 2-methyl-acetoacetyl-CoA, as a substrate. This molecule is an important reaction intermediate in the degradation of the amino acid isoleucine. Many human patients have been diagnosed to have a mutation in the gene of T2, and they are treated with a special diet. The results of this theses show that potassium ion rigidifies the groups of the T2 protein involved in the substrate binding. The presence of potassium increases the reaction rate and it also raises the affinity towards some of the substrates. The enzyme mechanistic studies with bacterial thiolase revealed that the oxyanion hole 1, formed by a water molecule and histidine side chain, is important for the synthetic reaction, not so much for the degradative direction. Binding studies showed that both the terminal sulfur of the substrate and the sulfur of the catalytic cysteine are important for the right positioning of the substrate. The electrostatics of the active site also have a significant role in the catalysis. These studies give a good basis for future studies aiming at drug development against this enzyme in pathogenic species.

Abstract Fatty acids are one of the most essential categories of biological lipids and their synthesis and degradation are vital for all organisms. Severely compromised phenotypes of yeast mutants and human patients, which have defective components in their degradative or synthetic processes for fatty acid metabolism, have highlighted the importance of these processes for overall metabolism. Most fatty acids are degraded by β-oxidation, which occurs in mitochondria and peroxisomes in mammals, whereas synthesis is catalyzed by cytosolic multifunctional peptides, although a synthesis system involving individual enzymes in mitochondria has been also proposed. In this study a novel mitochondrial 2-enoyl thioester reductase Etr1p from the yeast Candida tropicalis, its homolog Mrf1p from Saccharomyces cerevisiae, and their mammalian ortholog were identified and characterized. Observations indicating that mitochondrial localization as well as enzymatic activity is needed to complement the respiratory-deficient phenotype of the mrf1Δ strain from S. cerevisiae suggests that Etr1p and Mrf1p might act as a part of the mitochondrial fatty acid synthesis machinery, the proper function of which is essential for respiration and the maintenance of mitochondrial morphology in yeast. The mammalian enzyme, denoted Nrbf-1p, showed similar localization, enzymatic activity, and ability to rescue the growth of the mrf1Δ strain suggesting that mammals are also likely to possess the ability and required machinery for mitochondrial fatty acid synthesis. This study further included the characterization of another mitochondrial thioester reductase, 2,4-dienoyl-CoA reductase, which acts as an auxiliary enzyme in the β-oxidation of unsaturated fatty acids. The function of this gene was analyzed by creating a knock-out mouse model. While unstressed mice deficient in 2,4-dienoyl-CoA reductase were asymptomatic, metabolically challenged mice showed symptoms including hypoglycemia, hepatic steatosis, accumulation of acylcarnitines, and severe intolerance to acute cold exposure. Although the oxidation of saturated fatty acids proceeds normally, the phenotype was in many ways similar to mouse models of the disrupted classical β-oxidation pathway, except that an altered ketogenic response was not observed. This mouse model shows that a proper oxidative metabolism for unsaturated fatty acids is important for balanced fatty acid and energy metabolism.

Abstract α-Methylacyl-CoA racemase (Amacr) is an enzyme at the merging point of two important pathways of lipid metabolism: elimination of methyl-branched fatty acids and synthesis of bile acids. Amacr is regarded as obligatory for these processes. Patients with Amacr-deficiency suffer from adult onset sensory motor neuropathy and/or severe neonatal cholestasis with coagulopathy and fat-soluble vitamin malabsorption. Amacr is also linked to cancer and so far has been proposed as a new marker for diagnosis of at least prostate and colon cancers. Common sources of phytol derived branched-chain fatty acids for man are ruminant fats, meat and dairy products. The bile acid synthesis is the main pathway for cholesterol catabolism. Amacr is considered to be a member of family III of the CoA transferases (L-carnitine dehydratase - bile acid inducible protein F (CaiB-BaiF) family) and localized to two subcellular compartments, mitochondria and peroxisomes. In this work the mouse gene encoding Amacr was characterized, the gene was inactivated and mutational and structural studies were used to determine the loop and the active site structure of the enzyme. It was shown that mouse Amacr which locates both to mitochondria and peroxisomes, is an identical product of a single gene, which is located at chromosome 15, region 15B1. Neither alternative replication, splicing, or any post-translational modifications of the enzyme occur. The mouse model for Amacr-deficiency indicated a role of Amacr in detoxification of methyl-branched fatty acids, and suggested that a diet free from these phytol metabolites may function as a treatment for the deficiency. Furthermore, major changes were observed in the bile acid pool of the knock-out mice compared to wild type mice. However, the study suggests that there is an Amacr-independent pathway for synthesis of bile acids albeit of low capacity, which provides a way for Amacr-deficient individuals to survive. The mutational and structural studies confirmed Amacr as a member of family III of the CoA transferases. Furthermore, according to comparisons of the structural data of Amacr and other members of the family (FRC, YfdW), the superfamily can be divided into two subgroups, racemases and transferases. Proteins in the subfamilies share the CoA-binding mode, but the substrate specificities as well as the catalysed reaction differ greatly.

No description available.

In this study, we examined the effects of thyroid hormone on regulatory processes of cholesterol metabolism. Specifically, the pathways of cholesterol synthesis and cholesterol efflux were investigated. Hepatic HMG-CoA reductase (HMGR) is the rate-limiting enzyme in cholesterol synthesis. Hypothyroid rats exhibit decreased expression of this gene, which can be induced by subsequent treatment with thyroid hormone. The mechanism of this activation was previously unknown. Utilizing in vivo electroporation, we identified HMGR promoter elements necessary for the induction of HMGR by thyroid hormone. The -316/-321 element, the sterol response element, and nuclear factor-y site were all found to be necessary to induce HMGR promoter activity by thyroid hormone. We used electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) studies to demonstrate increased binding of upstream transcription factor-2 (USF-2) to the -316/-321 element in the HMGR promoter in response to thyroid hormone. Finally, co-electroporation of the wild-type HMGR plasmid with siRNA to USF-2, SREBP-2, or NF-Y nearly abolished the T3 induction as measured by promoter activity. Microarray and real-time PCR analysis demonstrated an induction of the apolipoproteins ApoA-I and ApoA-IV mRNA by T3. Serum levels of ApoA-I and ApoA-IV proteins were induced by T3. We collected serum from rats treated with or without T3 and used these sera in an in vitro macrophage efflux model. We found that T3 promoted cholesterol efflux via the ABCA1 cholesterol transporter and not via the ABCG1 transporter. We propose that the induction of serum ApoA-I and ApoA-IV by thyroid hormone promotes cholesterol efflux via the ABCA1 cholesterol transporter. Hepatic ABCG5 and ABCG8 are cholesterol transporters that promote biliary secretion of cholesterol. We utilized EMSAs to scan the shared ABCG5/G8 rat promoter for a thyroid hormone response element (TRE). We identified a TRß binding site at -392/-376 of the ABCG8 promoter. Collectively, these observations provide new insight into the cholesterol-lowering function of thyroid hormone.

No description available.

Acetyl-CoA is a metabolite at the crossroads of central metabolism and the substrate of histone acetyltransferases regulating gene expression. In many tissues fasting or lifespan extending calorie restriction (CR) decreases glucose-derived metabolic flux through ATP-citrate lyase (ACLY) to reduce cytoplasmic acetyl-CoA levels to decrease activity of the p300 histone acetyltransferase (HAT) stimulating pro-longevity autophagy. Because of this, compounds that decrease cytoplasmic acetyl-CoA have been described as CR mimetics. But few authors have highlighted the potential longevity promoting roles of nuclear acetyl-CoA. For example, increasing nuclear acetyl-CoA levels increases histone acetylation and administration of class I histone deacetylase (HDAC) inhibitors increases longevity through increased histone acetylation. Therefore, increased nuclear acetyl-CoA likely plays an important role in promoting longevity. Although cytoplasmic acetyl-CoA synthetase 2 (ACSS2) promotes aging by decreasing autophagy in some peripheral tissues, increased glial AMPK activity or neuronal differentiation can stimulate ACSS2 nuclear translocation and chromatin association. ACSS2 nuclear translocation can result in increased activity of CREB binding protein (CBP), p300/CBP-associated factor (PCAF), and other HATs to increase histone acetylation on the promoter of neuroprotective genes including transcription factor EB (TFEB) target genes resulting in increased lysosomal biogenesis and autophagy. Much of what is known regarding acetyl-CoA metabolism and aging has come from pioneering studies with yeast, fruit flies, and nematodes. These studies have identified evolutionary conserved roles for histone acetylation in promoting longevity. Future studies should focus on the role of nuclear acetyl-CoA and histone acetylation in the control of hypothalamic inflammation, an important driver of organismal aging.

No description available.