Dissertations / Theses on the topic 'Rat liver mitochondria'

CPT I was localised in the outer mitochondrial membrane and then partially purified. The protein was digested with restriction endonucleases and the fragments N-terminally sequenced and used to generate oligonucleotides for cDNA library screening and polymerase chain reaction (PCR). The partially purified protein was also used to generate polyclonal antibodies, which were used to screen λgt11 cDNA libraries. A positive clone was isolated from the λgt11 cDNA library using affinity purified serum. When sequenced, the clone was identified as long chain fatty acid CoA synthase. CPT II was purified from rat liver mitochondrial inner membranes and n-terminally sequenced. The purified protein was used to generate polyclonal antibodies. Oligonucleotides were generated to the cDNA encoding CPT II reported by Woeltje et al (1990b) and used to PCR up a 300 bp fragment, which when cloned and sequenced was identified as the C-terminus of CPT II. This fragment was then used unsuccessfully to screen λgt10, λgt11 and λgt11-stretch cDNA libraries, in order to find a full length clone. Using the information generated by this thesis along with other recent publications a possible topology of CPT I in the rat liver mitochondrial outer membrane was elucidated.

Le couplage mitochondrial entre oxydation des substrats et phosphorylation est variable et la capacite de dissocier ces deux reactions joue un role primordial dans les phenomenes de regulation du metabolisme cellulaire (production de chaleur, regulation de la production d'especes radicalaires et de la masse corporelle). Nous avons etudie dans ce travail les consequences d'un decouplage de l'oxydation phosphorylante par le 2,4-dinitrophenol sur le metabolisme intermediaire et sur l'etat energetique d'hepatocytes isoles ainsi que la modulation de ce decouplage en fonction des substrats utilises. En presence d'hydrates de carbone (dihydroxyacetone), le decouplage mitochondrial par le 2,4-dinitrophenol, du fait de l'augmentation de la permeabilite de la membrane mitochondrial interne aux protons, conduit a une diminution du potentiel de membrane mitochondrial. Ce qui induit une inhibition de la synthese mitochondriale d'atp qui conduit a l'arret de la neoglucogenese. Ensuite, du fait de l'inhibition des fonctions de la navette malate-aspartate (electrogenique), les potentiels redox cytosolique et mitochondrial se rapprochent de l'equilibre thermodynamique. Il en resulte une oxydation mitochondriale qui induit une inhibition de la respiration. En presence d'octanoate ou de proline, substrats fournissant des equivalents reduits dans la matrice, une augmentation du potentiel de membrane mitochondrial d'hepatocytes decouples est mesuree. Celle ci entraine une augmentation de la synthese mitochondriale d'atp, de la neoglucogenese, de l'activite de la navette malate-aspartate et enfin de la respiration cellulaire. De plus, nous avons observe que le decouplage a des consequences differentes suivant les pompes de la chaine respiratoire etudiees. Les effets sont importants sur le potentiel de membrane et sur la synthese d'atp maintenus par le complexe iii alors que le decouplage est sans effet sur le complexe iv. En conclusion, le decouplage mitochondrial regulateur du metabolisme cellulaire peut etre module par les substrats utilises donc par le regime choisi (hydrates de carbones vs acides gras) et ses consequences sont differents suivant les voies metaboliques etudiees (respiration vs neoglucogenese).

Le travail presenté dans ce mémoire a été entrepris dans le but de purifier et de caractériser le transporteur des dicarboxylates des mitochondries, qui catalyse l'échange un contre un des dicarboxylates entre eux (malate, malonate, succinate) ou contre le phosphate. Cette étude a été effectuée simultanément sur les mitochondries de foie de rat et sur les mitochondries d'une souche de levure: Saccharomyces cerevisiae. Les mitochondries de levure ont été solubilisées par le Triton X-100 et l'extrait a été chromatographié sur hydroxyapatite. Le filtrat, traité par électrophorèse en milieu dénaturant, a révélé par coloration à l'argent la présence d'environ 5 protéines de Mr compris entre 28000 et 35000. Après reconstitution de l'activité de transport dans des liposomes, un accroissement de 10 fois, de l'activité spécifique d'échange a été observé. Le passage des protéines du filtrat d'hydroxyapatite sur une colonne de malate deshydrogénase mitochondriale (EC 1. 1. 1. 37) immobilisée sur Sépharose, a conduit à la purification complète du transporteur. Cette protéine, purifiée à partir des deux types de mitochondries, possède toutes les propriétés caractéristiques du transporteur in situ et présente, en milieu dénaturant, un poids moléculaire de 28000. L'activité du transporteur purifié à partir des mitochondries de levure, reconstituée dans des liposomes, montre un Km pour le succinate de 2 mM et un Vmax de 1,5 lmol. Min-1. Mg-1 protéines tandis que l'activité spécifique augmente de 300 fois par rapport à l'extrait de départ. L'activité d'échange du transporteur reconstituée des deux types de mitochondries est inhibée par un réactif des groupements SH, le p-chloromercuriphénylsulfonate et par un réactif des acides aminés, le phosphate de pyridoxal, suggérant l'implication d'un ou plusieurs groupements SH et d'un ou plusieurs résidus lysine dans le mécanisme catalytique du transporteur. L'analyse de la composition en acides aminés de la protéine de transport des mitochondries de foie de rat indique qu'il s'agit d'une protéine hydrophobe, légèrement acide, avec la partie N-terminale bloquée

In the present study, we have demonstrated that PBR sites are present in the microsomal fraction from rat liver, heart and kidney, which we have named the non-mitochondrial PBR. The non-mitochondrial PBR was pharmacologically characterized by porphyrin competition experiments. The ability of porphyrins to differentially compete for specific (3H) Ro5-4864 or (3H) PK11195 binding demonstrated that the mitochondrial and non-mitochondrial PBR have different affinities for selected porphyrin compounds. These results suggest that the mitochondrial and non-mitochondrial PBR have a different binding pharmacology and support the existence of a non-mitochondrial PBR site. On the other hand, porphyrins show different potencies in competing for specific (3H) Ro5-4864 and (3H) PK11195 binding in the same fraction. In addition, the results of saturation experiments show that there are more PK11195 binding sites than Ro5-4864 binding sites in all fractions examined. This observation suggests that the sites labeled by (3H) Ro5-4864 and (3H) PK11195 are not identical.

INTRODUÇÃO: A lesão de isquemia/reperfusão hepática ocorre durante cirurgias hepáticas de grande porte, transplante de fígado e no trauma abdominal. A lesão de isquemia/reperfusão hepática ocasiona lesões no fígado e pode desencadear uma síndrome inflamatória sistêmica com lesões de órgãos a distância. Estudos anteriores demonstraram que o diazóxido protege outros órgãos (coração, rins, cérebro) da lesão de isquemia/reperfusão destes órgãos. OBJETIVO: Investigar o efeito da administração do diazóxido na lesão de isquemia/reperfusão hepática. MÉTODOS: Ratos Wistar machos foram divididos em 3 grupos. Em 2 grupos, os animais foram submetidos à isquemia hepática parcial realizada por clampeamento do pedículo dos lobos mediano e lateral anterior esquerdo durante uma hora sob ventilação mecânica. Grupo Salina (n=26): ratos receberam solução salina e Grupo Diazóxido (n=26): ratos receberam diazóxido EV ( 3.5mg/kg ) 15 minutos antes da reperfusão hepática. No terceiro grupo, Grupo Controle (n = 22 ), os ratos foram submetidos apenas à anestesia e manipulação cirúrgica. Quatro e 24 horas após os procedimentos, amostras de sangue foram recolhidas para determinações de AST, ALT, TNF-alfa, IL-6, IL-10, de nitrito/nitrato, creatinina. Amostras teciduais do fígado foram analisadas para dosagem do malondialdeído (MDA), para o estudo das funções oxidativas e fosforilativas mitocondriais, e para a análise histológica. Pela coleta de tecido pulomonar, a permeabilidade vascular pulmonar e a atividade da mieloperoxidade (MPO) também foram determinados. RESULTADOS: Quatro horas após, a reperfusão o Grupo Diazóxido apresentou elevações de AST, ALT, TNF-alfa, IL-6, IL-10 e níveis séricos de nitrito/nitrato significativamente menores que o Grupo Controle (p < 0,05). Observou-se uma redução significativa da disfunção mitocondrial hepática no Grupo Diazóxido em comparação com o Grupo Controle (p < 0,05). Não foram observadas diferenças no conteúdo de MDA fígado, na creatinina sérica e na permeabilidade vascular pulmonar, e na atividade da MPO entre os grupos. Vinte e quatro horas após, a reperfusão o Grupo Diazóxido registrou uma redução de AST, ALT quando comparado ao Grupo Controle (p < 0,05). CONCLUSÃO: O Diazóxido mantém a função mitocondrial do fígado, aumenta a tolerância fígado à lesão de Isquemia/Reperfusão e reduz a resposta inflamatória sistêmica. Esses efeitos requerem comprovações adicionais para o uso na prática clínica
INTRODUCTION: Significant liver ischemia/reperfusion injury can occur during hepatic surgeries, liver transplantation and abdominal trauma. Hepatic ischemia/reperfusion can trigger a local and systemic inflammatory syndrome. Previous studies have shown that diazoxide protects other organs (heart, kidneys, brain) from ischemia/reperfusion injury. AIM: To investigate the effect of diazoxide administration on liver ischemic/reperfusion injury. METHODS: Wistar male rats were divided into 3 groups. In two groups the rats underwent partial liver ischemia performed by clamping the pedicle from medium and left anterior lateral segments during an hour under mechanical ventilation. Saline Group (n=26): rats received saline and Diazoxide Group (n=26): rats received IV diazoxide (3.5mg/kg) 15 minutes before liver reperfusion. The third group, the Control Group (n=22) the rats underwent only anesthesia and surgical manipulation. Four and 24 hours after the procedure blood were collected for determinations of AST, ALT, TNF-alfa, IL-6, IL-10, nitrite/nitrate, creatinine. Liver tissues were assembled for mitochondrial oxidation and phosphorylation, malondialdehyde (MDA) content, and histologic analysis. Pulmonary vascular permeability and myeloperoxidade (MPO) were also determined. RESULTS: Four hours after reperfusion Diazoxide Group presented elevation of AST, ALT, TNF-alfa, IL-6, IL-10 and nitrite/nitrate serum levels significantly lower than Control Group (p < 0.05). A significant reduction on liver mitochondrial dysfunction were observed in Diazoxide Group compared to Control Group (p < 0.05). No differences in liver MDA content,serum creatinine and in pulmonary vascular permeability and MPO activity were observed between groups. Twenty four hours after reperfusion Diazoxide Group showed a reduction of AST, ALT and TGF?1 serum levels when compared to Control group (p < 0.05). CONCLUSION: Diazoxide maintains liver mitochondrial function, increases liver tolerance to I/R injury, and reduces systemic inflammatory response. These effects require further evaluations for using in a clinical setting

碩士
台北醫學院
生藥學研究所
88
Liver disease is one of the major causes of human death in Taiwan. There are several complicated factors have been demonstrated in the development of liver diseases such as virus infection, alcoholic, drug abuse, and improper perfusion. Chinese medicinal prescriptions have been used widely in treatment of liver diseases for thousands of years and showed the effective therapeutic effect. However, the scientific evidences for Chinese medicinal prescriptions on therapy of liver diseases are still undefined. In the present study, ten popular Chinese prescriptions including yin-chen-hao tang, xiao chai-hu tang, da chai-hu tang, ba zheng san, zhi-zi bo-pi tang, dang-gui long lui wan, huang-lian jie du tang, jia wei xiao yao san, gui-zhi fu-ling wan and long-dan xie gan tang were used to investigate their antioxidative activities in rat liver mitochondria. To evaluate the antioxidative activities of prescriptions as described above, in vitro lipid peroxide formation induced by several stimulators in rat liver mitochondria was performed and the level of lipid peroxidation was measured by the amount of MDA(TBA)2 complex formation detected by HPLC at wavelength 532 nm. The results revealed that yin-chen-hao tang showed the significant inhibitory effect on t-BOOH, Fe2+, β-NADPH-induced lipid peroxidation, and IC50 values on each stimulator are 1.28, 0.85 and 3.52 (10-2 g/l), respectively. There are three components including alcoholic da-huang (Rhei Rhizoma), yin chen hao (Artemisiae Capillaris Herba), and zhi zi (Gardeniae Fructus) were involved in the prescriptions of yin-chen-hao tang. Therefore, it is interesting to find out which component is major for the antioxidative activity in yin-chen hao tang. As the same part of experiment, the results appeared that alcohoic da-huang is the most potent among these three components, and show the dose-dependent inhibition on t-BOOH, Fe2+, and -NADPH-induced lipid peroxidation. The IC50 values of alcoholic da huang on inhibition of each stimulator induced lipid peroxidation are 1.81, 0.78 and 3.63 (10-2 g/l), respectively. In Chinese medicine, da-huang was cured commonly by different processings such as unhandled, alcoholic, and steamed one. Therefore, evaluation of the anti-lipid peroxidative activities of yin-chen-hao tang with different kinds of da-huang was performed in the following study. The data appeared that alcoholic da-huang used in yin-chen-hao tang showed the most potent anti-lipid peroxidative activities on t-BOOH, Fe2+, and -NADPH induced lipid peroxidation, and IC50 values of each stimulator were 0.86, 0.78, 2.68 (10-2 g/l), respectively. In ancient book “shang han lun”, yin-chen hao tang had been described to exert the potent therapeutic effect on liver diseases. In this study, we provided firstly the scientific evidence to demonstrate the antioxidative activity of yin-chen hao tang in rat liver mitochondria. Based on these data, we proposed that yin-chen hao tang is a Chinese prescription with significant antioxidative activities, and may used as a protectant for liver disease. It should be deserved for further study and for clinical trial in the future.

碩士
國立臺灣大學
毒理學研究所
82
In this thesis, we have studied the effects of silver ion and a stable free radical 1,1-diphenyl-2-picrylhydrazyl(DPPH) and its reduced form 1,1-diphenyl-2-picrylhydrazine(HDPPH) on the respiratory functions of mitochondria isolated from rat liver. We found that silver ion was a powerful uncoupler of oxidative phosphorylation.The minimum concentration to induce uncoupling effect was 5μM and stimulated the rate of state 4 respiration to maximum at 20μM. The silver ion also stimulated state 3 respiration rate, activated ATPase, induced calcium release and a small degree of lipid peroxidation, without any effect on proton conductivity. External addition of the thiol reagent dithiothreitol (1mM) can prevent both the effects on respiratory uncoupling and calcium release. These results are most likely due to the interaction of silver ion with sulfhydryl groups of mitochondrial membrane proteins. 1,1-diphenyl-2-picrylhydrazyl and 1,1-diphenyl-2-picrylhydra- zine were both powerful uncouplers of oxidative phosphorylation. The minimum concentration to induce uncoupling effect was about 10nM and stimulated the rate of state 4 respiration to maximum at 100nM.As silver ion can currently stimulated state 3 respiration rate,activated ATPase, induced mitochondrial calcium release and a small degree of lipid peroxidation, and enhanced proton conduc- tion.Their effects were not prevented by dithiothreitol and Pi/H+ symporter inhibitor N-ethylmaleimide. The present results suggest that 1,1-diphenyl-2-picrylhydrazyl and 1,1-diphenyl-2- picrylhrdr- azine were protonophoric uncouplers which can transport protons across the proton-impermeable membranes to collapse the proton gradient resulted in an uncoupling of oxidative phosphorylation.

Dissertação de mestrado em Bioquímica, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.
Aging is a complex multifactorial process involving alterations at genetic, molecular, cellular, organ, and system levels. The "oxidative stress theory" holds that a progressive and irreversible accumulation of oxidative damage caused by mitochondrial reactive oxygen species impacts on critical aspects of the aging process and contributes to impaired physiological function, increased incidence of disease, and a reduction in life span. Additionally, periods of chronic hypoxia, which can arise from numerous disorders (e.g. chronic vascular diseases) and even aging, potentiate the development of degenerative diseases. In this study we evaluated the effects of age and chronic hypoxia in the oxidative status, mitochondrial enzymatic complexes activity and apoptotic cell death pathway of the liver. For this purpose 3- and 12-month-old male Wistar rats exposed to normoxia (21% O2) or hypoxia (10% O2) during 7 days were used. Several parameters were evaluated: hydrogen peroxide (H2O2) and thiobarbituric acid reactive substances (TBARS) levels, aconitase activity, enzymatic [manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) activities] and non-enzymatic (glutathione and vitamin E levels) antioxidant defenses, the activities of NADH-cytochrome c reductase (mitochondrial complexes I-III), succinate-cytochrome c reductase (mitochondrial complexes II-III), cytochrome c oxidase (mitochondrial complex IV) and ATPase. The activation of caspase-9 and caspase-3 and Bax and Bcl2 protein levels were also analyzed. An age-dependent increase in H2O2 levels and caspases activation and a decrease in aconitase, mitochondrial enzymatic complexes, ATPase and GR activities and glutathione levels were observed. Interestingly, chronic hypoxia in young animals caused a similar pattern of oxidative imbalance and mitochondrial defects compared to that found in 12-month- old animals. In addition, chromic hypoxia potentiated the age-dependent increase in H2O2 levels and decrease in glutathione levels. Curiously, hypoxia decreased caspases activation in 12-month-old animals. Altogether, these results show that age and/or chronic hypoxia enhance liver oxidative imbalance and mitochondrial damage
O envelhecimento é um processo multifactorial complexo que envolve alterações genéticas, moleculares, celulares, nos órgãos e no organismo. A "teoria do stress oxidativo" sustenta que a acumulação progressiva de lesões oxidativas causadas pelas espécies reactivas de oxigénio produzidas pelas mitocôndrias tem um papel chave no processo de envelhecimento contribuindo para uma alteração da função fisiológica, um aumento da incidência de doenças e uma redução no tempo de vida. Além disso, períodos de hipoxia crónica associadas a diversas doenças (ex. doenças vasculares crónicas) e ao processo fisiológico do envelhecimento, potenciam o desenvolvimento de doenças degenerativas. Neste estudo foram avaliados os efeitos da idade e da hipoxia crónica no estado oxidativo, na actividade dos complexos enzimáticos mitocondriais e na via de morte celular por apoptose do fígado. Para este efeito, foram utilizados ratos Wistar de 3 e 12 meses de idade expostos a normoxia (21% O2) ou hipoxia (10% O2) durante 7 dias. Foram avaliados vários parâmetros: os níveis de peróxido de hidrogénio (H2O2) e das substâncias reactivas ao ácido tiobarbitúrico (TBARS), a actividade da aconitase, as defesas antioxidantes enzimáticas [as actividades das enzimas dismutase do superóxido de manganês (MnSOD), catalase (CAT), glutationa peroxidase (GPx) e glutationa redutase (GR)] e não-enzimáticas (níveis de glutationa e vitamina E), as actividades dos complexos enzimáticos mitocondriais NADH- citocromo c redutase (complexos I-III), succinato-citocromo c redutase (complexos II-III), citocromo c oxidase (complexo IV) e ATPase. A activação da caspase-9 e da caspase-3 e os níveis das proteínas Bax e Bcl2 também foram analisados. A idade aumentou os níveis de H2O2 e a activação das caspases e diminui as actividades da aconitase, dos complexos enzimáticos mitocondriais, da ATPase e da GR e os níveis de glutationa. Curiosamente,os animais jovens expostos a hipoxia crónica apresentaram um perfil oxidativo e uma função mitocondrial semelhante aos animais de 12 meses de idade. Além disso, a hipoxia crónica potenciou o aumento nos níveis de H2O2 e glutationa provocados pela idade. Curiosamente, a hipoxia crónica diminui a activação das caspases nos animais de 12 meses de idade. Estes resultados mostram que a idade e/ou a hipoxia crónica potenciam o stress oxidativo e a disfunção mitocondrial.

碩士
國立臺灣大學
生化學研究所
84
The mitochondria palyed a vital role on the bioenergenesis. Many disease free radicals species released from mitochondria. In the present study, we want to test the ability of the anti- oxidative effect of PCA and its oxidative polymer to protect the oxidative damage of mitochondria. Four indices on the oxidative damage of the rat liver mitochondria were used. We directly used ultra-sensitive chemiluminescence technoque to measure the superoxide radical produced from mitochondria; we measured ANS( 1-anilinonphthlen -8-sulfonate) fluorescence intensity to study the integrity of mitochondria; we detect the A520nm for motoring the swelling of mitochondria ; and we measured the MDA amount as the biomarker of lipid peroxidation. Our experimental results showed both protocatechuic acid and its oxidative polymer protected the oxidative damage of mitochondria, but the protective effect of two compounds showed slight differences: the polymer had greater ability on the superoxide anion inhibition on the inhibition of mitochondria swelling, but polymer has the weaker ability on the inhibition of malondialdehyde formation.

碩士
輔仁大學
食品營養學系
92
Previous studies revealed that folate deficiency resulted in large scale deletion of mitochondrial DNA（mtDNA） in rat liver. Effects of folate deficiency on this mtDNA deletion and mitochondrial functions were largely unknown. These questions were studied using animals model as well as primary culture hepatocytes. Liver mitochondria extracted from Wistar rats fed with folate-deficient or folate-supplemented diets were analyzed for common deletion of mtDNA, gene loss and oxidative damage. Primary hepatocytes from those rats were cultured to assess reactive oxygen species（ROS）, mitochondrial membrane poteinal, mitochondrial mass and cytochrome c oxidase activity. Our data showed that folate-deficient rat liver but not folate-supplemented groups exhibited mtDNA common deletion（8103 bp~12936 bp）. Using semiquantitative PCR methods, mitochondrial COXⅢ gene levels in folate-deficient rat liver were significantly decreased to 70% of gene levels in folate-supplemented rat liver. COXⅢ gene encoded on subunit of mitochondrial respiratory complex Ⅳ （cytochrome c oxidase, COX）, which was associated with proton pumping and electron transfer. Primary hepatocytes isolated from folate-deficint rats showed significantly lower levels of enzymatic activity of COX, mitochondrial membrane potenials and mitochondrial mass as compared to those in primary hepatocytes from folate-supplement rats. Supplementation of folate to folate-deficient hepatocyte cultures confer protection against metal ion-induced mitochondrial dysfunction. Mitochondrial dysfunction in folate-deficient hepatocytes were associated with increased intracellular ROS levels, particular for superoxide generation, and oxidative damage to mitochondrial protein . Supplementation of folate to animals or to hepatocytes significantly decreased the severity of oxidative stress（superoxide generation） and prevented from metal ion-induced mitochondrial oxidative damage, particularly for protein oxidative damage. In conclusion, dietary folate deficiency could result in mitochondrial COXⅢ gene loss, mitochondrial dysfunction and increased oxidative damage to mitochondrial protein in rat liver. In vivo and in vitro folate supplementation protected against endogenous or oxidants-induced liver mitochondrial decay and dysfunction.

碩士
高雄醫學大學
醫學研究所
91
Sepsis is regarded as a major initiator of multiple organ dysfunction syndrome that remains a leading cause of mortality in medical critical care unit. Mitochondrial dysfunction plays an important role in the cascade. Our previous studies have shown that the ATP synthesis and the mitochondrial respiratory enzyme activities were all down-regulated in the liver of experimental septic rats. In addition, heat shock response was demonstrated to the protective effect against sepsis in rats while the mechanism is still a mystery. Accordingly, the present study was designed to investigate the proteomic alternation of hepatic mitochondria during sepsis, and explore the molecular protective mechanism of preconditioning heat shock treatment. Spraque-Dawley rats were used as the experimental animal. They were divided into two groups: non-heated and heated. Rats of heated group were whole-bodily heated to 41.5±0.5 °C for 15 min to induce the over-expression of heat shock proteins (Hsps24) hr before sepsis induction. Sepsis was induced by cecal ligation and puncture (CLP). The rats were sacrificed 9h and 18h after CLP that was defined as early and late phase of sepsis, respectively. The mitochondrial proteins of the liver were extracted and separated by two-dimensional electrophoresis with broad immobilized pH gradient strip (PH 3-10) and SDS-PAGE. The protein spots were visualized with silver stain and analyzed by Bio-2D software. Results of Western blot and immunochemical analysis showed that Hsp72 was over-expressed maximally at 24 and sustained till 42 hours after heat shock treatment. Hsp72 was not induced in early and late sepsis, while heated septic rats all showed Hsp72 over-expression. Results of two-dimensional electrophoresis analysis showed that around 120 spots could be separated and visualized distinctly. Among them, 3 spots with same molecular weight (56.4KD) were significantly altered in septic specimens, named MP1 (PI 7.5), MP2 (PI 8.0) and MP3 (PI 8.5). MP1 and MP2 were down-regulated in sepsis while MP3 was up-regulated coincidently. Interestingly, heat shock treatment could reverse the phenomenon. Analyzed by LC/MS/MS, the 3 spots were revealed to be an identical enzyme: aldehyde dehydrogenase 2 (EC 1.2.1.3). However, RT-PCR assay of the enzyme revealed no significance in all specimens. The enzyme activity assay showed that aldehyde dehydrogenase 2 activity was down-regulated in non-heated late septic rats and reversed in heated late septic rats. But the activities between heated and non-heated early septic rats were not different significantly. In conclusion, we suggest that post-translation modification of aldehyde dehydrogenase 2 may play a functional role in the pathogenesis of sepsis and provide a novel protective mechanism of heat shock treatment.

碩士
臺北醫學大學
保健營養學研究所
98
Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease with worldwide prevalence that occurs in subjects who do not abuse alcohol. It is defined as a spectrum of hepatic steatosis, nonalcoholic steatohepatitis (NASH), fibrosis, necrosis and cirrhosis. The prevalence is about 10 to 24% in general population, and the mortality is up to 13%. Simple fatty liver and NASH are the most common characteristics in NAFLD, but the pathogenesis of NASH is not well defined. ”Two-hit hypothesis” have been proposed for the pathophysiology of NAFLD and NASH. Insulin resistance and oxidative stress are the first and second hits, respectively. Mitochondrion is an important organelle confers beta-oxidation of fatty acids and also the major site to produce by-product of oxygen reactive species. Mitochondrial dysfunction might play an important role in both hits. It is reported previously, there are distinct effects on mitochondrial function by treating different kinds of fatty acids. The objects of this study were to investigate whether oils, such as fish oil, olive oil, tea seed oil and lard, could influence mitochondrial function and improve fatty liver disease. Male Sprague Dawley (SD) rats were fed with 71% high fat diet to induce simple fatty liver, afterward, randomly divided into 5 groups: high-lard (HL), low-lard (LL), tea seed oil (TO), olive oil (OO) and fish oil (FO) diets. Liver triglyceride content and HOMA-IR index in LL, TO, OO, FO groups were lower than HL group (P＜0.05), moreover, FO group was significantly reduced in body weight and visceral fat. Respiratory control rate (RCR) in HL group was lower than control group (P＜0.05), and increased in other groups, especially in TO group. The activity of cytochrome c oxidase (CCO) in HL group was lower than control group (P＜0.05), besides, CCO was increased significantly in TO, OO and FO group compared with HL groups (P＜0.05). In conclusion, all of fish oil, olive oil and tea seed oil might provide benefits for fatty liver disease, but they should via diverse mechanisms to affect the progression of NAFLD.

Tese de Doutoramento em Ciências Veterinárias
A cetamina é um anestésico dissociativo usado em anestesiologia humana e veterinária. A sua utilização terapêutica, em doses baixas, para tratar dor crónica e depressão tem vindo a ganhar uma importância crescente. Têm sido relatadas propriedades controversas da cetamina, tanto a nível neuroprotetor/neurotóxico como hepatoprotetor/ hepatotóxico, existindo um interesse renovado no esclarecimento dos seus mecanismos de ação e efeitos adversos. Foi recentemente sugerido que a cetamina pode modificar a funcionalidade mitocondrial promovendo um aumento na produção de radicais livres a nível do encéfalo e fígado. Doses subanestésicas de cetamina induzem efeitos heterogéneos em diversas regiões do encéfalo os quais têm sido associados a défices de memória e a sintomas psicopatológicos. Contudo, existe pouca informação quanto à utilização da cetamina, em doses baixas, na função cerebral e equilíbrio neuroquímico. O objetivo deste estudo é contribuir para o esclarecimento sobre os efeitos da exposição da cetamina no principal órgão alvo - encéfalo, e no órgão de metabolização - fígado. Dois regimes de administração e várias doses de cetamina foram testados no rato adulto. No primeiro estudo, avaliou-se o efeito agudo de uma dose anestésica intraperitoneal de cetamina (50, 100 ou 150 mg/kg) na função mitocondrial e no estado redox cerebral, seis horas após a sua administração. Observou-se que a cetamina aumentou o consumo de oxigénio na respiração mitocondrial no estado 4 e diminuiu a eficiência da fosforilação oxidativa quando se utilizou glutamato-malato. Esta interferência com o complexo I foi confirmada pela diminuição da atividade neste complexo. Simultaneamente, as doses 50 e 100 induziram um aumento na atividade da sintetase óxido nítrico mitocondrial (mtNOS), assim como um aumento global dos níveis de óxido nítrico (NO) plasmático. Além disso, a cetamina induziu um aumento na atividade da superóxido dismutase (SOD). Estes resultados revelam que a administração aguda de cetamina afeta negativamente a função mitocondrial e aumenta a atividade da mtNOS e da SOD, preservando o estado redox geral. No segundo estudo, avaliaram-se os efeitos da administração crónica de cetamina no encéfalo e no fígado. Administrou-se subcutaneamente soro fisiológico, 5 ou 10 mg/kg de cetamina, duas vezes por dia, durante 14 dias consecutivos. A avaliação comportamental iniciou-se 24 horas após a última administração, tendo-se verificado que a cetamina aumentou a ansiedade no teste de exploração do objeto novo. A dose de 5 mg/kg apresentou também uma diminuição no índice de discriminação no teste de reconhecimento do objeto, revelando uma incapacidade para atualizar representações espaciais e uma deficiência no processo de habituação. Os parâmetros neuroquímicos e neuroplásticos foram avaliados, dez dias após a última administração, em regiões do encéfalo sensíveis aos efeitos da cetamina e envolvidas nas alterações comportamentais. Na dose mais baixa, consistentemente com as alterações comportamentais, verificou-se um aumento nos níveis de dopamina na amígdala acompanhados por uma diminuição da funcionalidade na região frontal. Na dose mais elevada verificou-se um aumento dos níveis de serotonina. Os níveis de glutamato diminuíram no córtex pré-frontal (CPF) para a dose mais elevada, enquanto no hipocampo, os níveis de glutamato aumentaram para as duas doses. Os neurónios imunorreativos à parvalbumina diminuíram nas regiões pré-límbica e orbitofrontal do CPF, cíngulo e CA1 do hipocampo. No seu conjunto, estes resultados sugerem um efeito menos deletério da dose mais elevada, consequência duma diminuição da inibição do sistema serotoninérgico (o que pode estar relacionado com o efeito antidepressivo), enquanto a dose mais baixa parece ser prejudicial devido à desregulação dopaminérgica e à diminuição dose-dependente do fluxo glutamatérgico aos neurónios GABAérgicos. Foi também avaliado o efeito da exposição crónica na função mitocondrial encéfálica e hepática. Nas mitocôndrias do encéfalo não foram observadas alterações significativas. No fígado, as duas doses usadas inibiram o complexo I assim como o consumo de oxigénio quando foi utilizado o glutamato-malato como substrato respiratório. A cetamina também diminuiu o conteúdo de glicogénio hepático, o que pode contribuir para a diminuição da resistência hepática às perturbações tóxicas. O tratamento com cetamina induziu uma diminuição no aumento de peso corporal o que sugere alguma precaução na sua utilização. Em geral, este trabalho permite uma melhor compreensão da interferência da cetamina na função mitocondrial, ajudando ao esclarecimento do mecanismo subjacente aos seus efeitos. No uso crónico da cetamina em doses baixas, destaca-se a existência de efeitos dependentes da dose com alterações heterogéneas em diferentes regiões do encéfalo, sendo estas alterações tanto ao nível dos parâmetros neuroquímicos como de neuroplasticidade com consequentes reflexos a nível comportamental.
Ketamine is a dissociative anaesthetic used in Human and Veterinary anaesthesiology. In low doses, ketamine gained an increasing relevance in the therapy of chronic pain and depression. Since ketamine has been reported to have controversial properties such as neuroprotective/hepatoprotective and neurotoxic/hepatotoxic, there is now a renewed interest in clarifying its mechanisms of action and side effects. Recently, it was suggested that ketamine can modify the mitochondrial bioenergetic function, as well as the rate of free radical production, affecting, in particular, the neural and liver tissues. Subanaesthetic ketamine doses induce heterogeneous region effects that were associated with impaired memory function and increased psychopathological symptoms. However, there is a lack of information regarding the safety of chronic low antidepressant or analgesic ketamine doses in the brain function and neurochemical homeostasis. The objective of the present study is to contribute to further characterize the effects of ketamine exposure in its main targets - the brain, and in its metabolisation organ – the liver. Two regimes of ketamine administration and a range of doses were evaluated in an adult rat model. In the first study, the acute effect of a single intraperitoneal anaesthetic ketamine dose (50, 100 or 150 mg/kg) was evaluated six hours later in the brain mitochondrial function and redox state. The ketamine was shown to increase mitochondrial oxygen consumption in the state 4 of respiration and to impair the oxidative phosphorylation efficiency of complex I. This interference with the complex I was confirmed by the observation of its decreased activity. Simultaneously, ketamine lead to increased activity of the mitochondrial nitric oxide synthase (mtNOS) in the lowest doses, and to a global increase of the plasmatic levels of nitric oxide (NO). In addition, ketamine increased the activity of superoxide dismutase (SOD) in all tested doses. These results reveal that acute ketamine administration impairs mitochondrial function and increases the activity of mtNOS. However, ketamine also triggered SOD activity, potentially to preserve the overall redox status. In the second study, the effects of chronic ketamine were evaluated in brain and liver tissues. Rats were administered with saline solution, 5 or 10 mg/kg of ketamine, twice a day, subcutaneously, for 14 consecutive days. Behavioural evaluation started 24 hours after the last administration. Ketamine increased anxiety-like behaviour in the novel object exploration test. The 5 mg/kg group presented also decreased discrimination index in the object recognition task, inability to update spatial representation, and deficient habituation processes. Ten days after the last administration, neurochemical and neuroplastic parameters were evaluated in specific brain regions known to be susceptible to the effects of ketamine, and involved in the reported behavioural changes. Consistently with the behavioural observations for the lower dose, there was an increase in the amygdalar dopamine levels accompanied by frontal hipofunctionality; while a higher dose resulted in increased 5-HT levels. Glutamate levels were decreased in the prefrontal cortex (PFC) for the highest dose; while in the hippocampus, glutamate levels were increased for both doses. Moreover, parvalbumin immunoreactive neurons were reduced in prelimbic and orbitofrontal PFC regions, cingulate cortex and hippocampus CA1 region. Taking all these together, it is propose that a high dose of ketamine may be less deleterious through decreased inhibition of the serotonergic system (a potential antidepressant-like effect), while a lower dose may be detrimental due to altered dopaminergic regulation caused by dose-dependent reduction of the glutamatergic flow to GABAergic neurons. Moreover, the effect of chronic exposure was also assessed in the brain and liver mitochondrial function. While no effects could be observed in brain mitochondria, in the liver both doses inhibited mitochondrial complex I and oxygen consumption when glutamate-malate substrate was used. Ketamine also decreased the hepatic glycogen content, which may contribute to decrease hepatic resistance to toxic insults. Chronic ketamine groups also showed a decreased evolution of body weight gain through the treatment period, clearly suggesting a morbidity induced effect. In general, this work allows a better comprehension of ketamine interference with the mitochondrial function that helps to clarify the mechanism underlying its effects. Moreover, regarding the use of ketamine in chronic low doses, it highlights the existence of dose-dependent effects that lead to heterogeneous changes in different brain regions, either in neurochemical or neuroplastic parameters, with relevant consequences at the behavioural level.

Thesis (M.S.)--University of Wisconsin--Madison, 1990.
Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 111-125).

碩士
臺北醫學大學
保健營養學研究所
102
Non-alcoholic fatty liver disease (NAFLD) is a condition of fat accumulatuin in the liver in the absent of excessive alcohol consumption (less than 20 g per day) and specific causes of hepatic steotosis. An improper dietary habit is one of the factors which induce NAFLD. When consuming too much fat, it will increase triglyceride into liver for metabolism. Meanwhile liver mitochondrial β oxidation increases and produces more reactive oxygen species, which may harm hepatic cells. On the other hand, very low-density lipoprotein can not bring triglyceride out of liver efficiently, and caused NAFLD eventually. n-3 polyunsaturated fatty acid (n-3 PUFA) is a polyunsaturated fatty acid which fist double bond is at third carbon from carbonyl tail. Its physiological functions include cell membrane maintenance, signal transduction, and energy storage. n-3 PUFA’s studies indicated that n-3 PUFA may ameliorate dyslipidemia, insulin resistance, regulate lipid metabolic genes, and reduce inflammatory response. α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are the menbers of n-3 PUFA’s family. In our study, krill oil, fish oil, and linseed oil, which were rich in n-3 PUFA, were used for supplement. Krill oil and fish oil contain EPA and DHA, while linseed oil contains ALA. The purpose of this experiment is to evaluate supplement with oils rich in n-3 PUFA, such as krill oil, fish oil and linseed oil, on plasma and liver lipid profiles, liver fatty acid synthesis enzyme activities, antioxidant enzyme activities, and mitochondrial function in the animal model of NAFLD induced by 45 % high fat diet. 80 Sprague-Dawley (SD) rats were divided into 8 groups, including control group (C), high fat diet group (HF), high fat diet supplied with krill oil, fish oil or linseed oil in one or triple dosage respectively (KO1, KO3, FO1, FO3, LO1, LO3). Except C, rests of the groups were fed with Adjusted Calories Diet (45/Fat) fodder. Except C and HF, rests of the groups were supplied with different oil and different dosage. One and triple dosage were used to control their supplement n-3 PUFA amount, which were 4.2 and 13.0 mg/g fodder/day respectively. After 12 weeks, SD rats were sacrified and analyzed plasma lipid profiles and glucose homeostasis, hepatic lipid profiles, biopsies, fatty acid synthesis enzyme acitivities, antioxidant enzyme activites, mitochondrial respiratory chain complex enzyme activities, and mitochondrial biogenesis. FO1, FO3, LO1, and LO3’s plasma triglyceride were significantly lower than HF. Furthermore, HF, KO3, and FO3’s HOMA-IR increased significantly. KO1, FO1, LO1, and LO3’s hepatic triglyceride content and biopsies were ameliorated significantly. Fatty acid synthesis enzyme activities in KO3 and FO3 were significantly higher than C. Antioxidant enzyme activities, mitochondrial respiratory chain complex enzyme activities, and mitochondrial biogenesis in KO1, FO1, LO1, and LO3 were significantly higher than HF. Supplement with oils rich in n-3 PUFA, such as krill oil 273.4 mg/kg b.w./day, fish oil 149.6 mg/kg b.w./day, and linseed oil 147.0 or 466.5 mg/kg b.w./day may reduce plasma and hepatic lipid profiles, fat accumulation in liver, and fatty acid synthesis; while elevating mitochondrial function, eventually ameliorate NAFLD in animal model induced by 45 % high fat diet.

Acclimation to cold or hardening is known for many decades through its beneficial effects on human health. In contrast, sudden exposure to cold, cold shock, is a great risk of cerebral and cardiac injury, especially in the elderly. There is very little published data on the cellular and molecular mechanisms induced by cold adaptation in heart and brain. The aim of this work was to describe and compare different properties heart, liver, brain and brown adipose tissue mitochondria of rats housed at 25 ± 1 řC and at mild cold (9 ± 1 řC, 5 weeks). The high-resolution oxygraphy, spectrophotometry and Western blotting analyses were used. We found differences in the respiratory control between the heart and liver. Cold acclimation decreased activity of the Krebs cycle enzymes. Fatty acid contribution to the respiration reached the maximum in brown fat and the minimum in the hippocampus. However, further study is necessary.