京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22304号 / 医博第4545号 / 新制||医||1040(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 坂井 義治, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Finding a source from which high-energy-density biofuels can be derived at an industrial scale has become an urgent challenge for renewable energy production. Some microorganisms can produce free fatty acids (FFA) as precursors towards such high-energy-density biofuels. In particular, photosynthetic cyanobacteria are capable of directly converting carbon dioxide into FFA. However, current engineered strains need several rounds of engineering to reach the level of FFA production for it to be commercially viable. Thus, new chassis strains that require less engineering are needed. Although more than 140 cyanobacterial genomes are sequenced, the natural potential of these strains for FFA production and excretion has not been systematically estimated. In relation to the above-mentioned problems, we developed the first in silico screening method (FFASC) that evaluates the cyanobacterial strains’ potential for FFA production based on the strains’ proteome, which for the first time allows non-experimental selection of the most promising chassis for cyanofactories. The solution is based on the original problem formulation, optimization and ranking. To provide developers and researchers easy means for evaluation and assessment of the cyanobacterial strains potential for production of FFA, we developed the BioPS platform. In addition to being able to compare capacity for FFA production of any novel strain against 140 pre-valuate strains, BioPS can be used to explore characteristics and assessment rules in play for an individual strain. This is the first tool of this type developed. Finally, we developed a novel generic in silico method (PathDES) for ranking and selection of the most suitable pathways / sets of metabolic reactions, which suggests genetic modifications for improved metabolic productivity. The method heavily relies on optimization and integration of disparate information in a novel manner. It has been successfully used in connection with FFASC for design of cyanofactories. In conclusion, this study has contributed novel and unique methods, and tools for the field of bioinformatics, with applications towards the metabolic design of cyanofactories. We believe that these will be of good use to researchers and technology developers in this field.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18327号 / 農博第2052号 / 新制||農||1022(附属図書館) / 学位論文||H26||N4834(農学部図書室) / 31185 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 河田 照雄, 教授 金本 龍平, 教授 入江 一浩 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

No description available.

Student Number : 9404757K - PhD thesis - School of Physiology - Faculty of Science / Natural secretions of oestrogen and progesterone vary according to menstrual phase in eumenorrhoeic women and have an affect on physiological systems that could consequentially influence exercise performance. In a series of menstrual phase comparative investigations (considering mainly the early follicular (EF), late follicular (LF) and mid-luteal (ML) phase), I aimed to elucidate the physiological relation of oestrogen and progesterone to fat metabolism and ventilation during endurance exercise and exercise performance. I measured plasma free fatty acid (FFA) kinetics during prolonged exercise from an intravenous infusion of K+[1-13C]palmitate in eumenorrhoeic women and found the change in palmitate rate of appearance and disappearance between menstrual phases to be related to the oestrogen/progesterone (E/P) ratio. Overall the results from this study suggest that oestrogen promotes increases in FFA availability during exercise, while progesterone mitigates this response. Therefore, a high oestrogen concentration and E/P ratio in the ML phase is required in order to produce a favourable FFA metabolic response for endurance events. In order to estimate plasma FFA oxidation rate using carbon-FFA tracers, the acetate correction factor which accounts for carbon-label retention in secondary metabolic pools, must be applied. When I derived the acetate correction factor by measuring fractional recovery of carbon-13 in expired CO2 from a constant infusion of Na+[1- 13C]acetate during submaximal exercise we found the acetate correction factor to be significantly lower in the ML than EF phase (average change from EF phase -1.8± 0.5%, p<0.05). Failure to account for the lower correction factor in the ML phase will result in a 6% underestimation of plasma FFA oxidation rate. Furthermore, since human serum albumin (HSA) routinely used as a carrier for parenteral delivery of FFA tracers has various disadvantages, I tested the feasibility of using 2-hydroxypropyl-b- cyclodextrin (HP-b-CD) as a possible alternative carrier. A comparison of expired CO2 enrichment following HSA-FFA (7.4±2.0 %o) and HP-b-CD-FFA (8.6±2.1%o) infusion during exercise showed that the HP-b-CD does not compromise natural in vivo behaviour of the FFA tracer (p=0.4). Progesterone-induced hyperventilation is occasionally reported during exercise in the luteal phase. I found that the change in ventilatory parameters (minute ventilation and respiratory rate) during exercise from EF to ML phase is related to both the oestrogen and progesterone concentration in the ML phase. However, the associated increase in respiratory rate throughout prolonged exercise in the ML versus EF phase did not increase metabolic demand and therefore could not be expected to exacerbate fatigue. When exercise performance was evaluated by means of a cycling time trial, I found a trend for best performance in the LF phase versus the EF phase (8 of 11 subjects improved by 5.2±2.9%, p=0.027), while no differences occurred between other menstrual phases. Metabolic and performance benefits of oestrogen may be concealed in the ML phase by the coincident increase in progesterone in this phase and thus a significant effect is often only evident with a high E/P ratio in the ML phase. The transient LF phase, characterised by the pre-ovulatory surge in oestrogen, reveals the maximum benefits of oestrogen on metabolism and performance during submaximal exercise.

Para uma produção eficiente e economicamente viável de ésteres é necessária a utilização de catalisadores durante o processo, uma vez que a transesterificação e a esterificação são reações de equilíbrio, sendo necessário deslocar o equilíbrio de reação na direção desejada. Nesse trabalho, ésteres metílicos e etílicos de ácidos graxos de cadeia longa foram produzidos usando cloretos de cobre como catalisadores. Para definir a melhor condição reacional, foram estudadas três diferentes razões em massa de AGLs de dendê/ metanol (ou etanol) / catalisador. Definida a melhor condição reacional, foram produzidos monoésteres metílicos e etílicos a partir de ácidos graxos livres do destilado da soja assim como das borras do refino correspondente do óleo de soja e de dendê. Os ésteres metílicos e etílicos foram caracterizados através das técnicas de cromatografia gasosa acoplada à espectrometria de massas (CG/MS) e ressonância magnética nuclear de prótons (RMN 1H). Observou-se que as reações empregando cloreto de cobre como catalisador reagem preferencialmente com AGLs em relação aos triésteres. Dentre esses ácidos graxos livres, os de cadeia insaturada, são os que reagem mais facilmente com o catalisador. Em relação ao álcool empregado, observou-se que as reações em etanol são mais rápidas que as realizadas em metanol, devido a facilidade de solubilização do catalisador e matéria prima lipídica no etanol. A reação com metanol, por sua vez ocorre de forma mais lenta, devido a solubilidade parcial entre os reagentes no meio e consequentemente, com separação instantânea de duas fases ao término da agitação, facilitando a extração do produto. A ampliação da estequiometria de reação em 160 e 80 vezes, para o catalisador anidro e hidratado, respectivamente, em relação a massa de AGLs de dendê foi obtida com sucesso em ambos os catalisadores, mostrando a viabilidade do emprego do cloreto de cobre como catalisador em escala industrial e a possibilidade de ser estendido a outras matérias primas. Desse modo, a utilização do cloreto de cobre como catalisador na reação de ácidos graxos livres de cadeia longa ou de óleos vegetais se constitui em uma metodologia simples e prática, sendo uma opção viável para a produção de monoalquil esteres de cadeia longa em condições brandas de reação quando comparada aos métodos já estabelecidos e utilizados inclusive em escala industrial. / For an economically feasible and efficient production of esters is necessary to use catalysts in the esterification and transesterification reactions, which are reversible. It is necessary to shift the reaction equilibrium in the desired direction. In this work we developed a novel reaction to produce methyl and ethyl esters of long chain fatty acids using copper chloride as catalysts. To establish optimal reaction conditions, we studied three different mass ratios of FFAs palm (or soybean) / methanol (or ethanol) / catalyst. Employing the best reaction conditions, methyl and ethyl mono-esters were produced from free fatty acid of the soybean oil deodorization distillate (DDOS) as well as the corresponding refined soybean and palm oil. The methyl and ethyl esters were characterized by gas chromatography coupled to mass spectrometry (GC / MS) and proton nuclear magnetic resonance (1H NMR). It was observed that reactions using copper chloride as catalyst are faster with free fatty acids than with neutrals refined oils. Among these, free fatty acids of unsaturated chain react faster than the saturated ones. Regarding the alcohol employed, it was observed that the reactions in ethanol are faster than those performed in methanol, due to higher of solubility of the catalyst and feedstock lipid in ethanol. The reaction with methanol, in turn occurs more slowly, due to partial solubility of the reactants in the solvent that results in, instantaneous phases separation. The scale up of reaction at 80 and 160 times for both, anhydrous and hydrated catalyst, was successfully performed showing the feasibility of this novel process use in industrial scale. Thus, the use of copper chloride as the catalyst in the reaction of long chain free fatty acids constitutes a simple and practical methodology, being a feasible option for the production of long chain monoalkyl esters under mild conditions compared to methods already established and also used in industrial scale.

O presente estudo teve como objetivo selecionar procedimentos de purificação de óleos vegetais, com enfoque na desacidificação, comparando a eficiência de métodos de refino alternativos (adsorção e extração liquido-liquido) com o convencional (alcalino). Para tanto, foram utilizados óleos com baixo impacto na cadeia alimentícia, como óleo de andíroba e óleo de macaúba, visando contribuir para a ampliação de matérias-primas lipídicas na produção de biodiesel. A triagem do método de purificação mais adequado foi baseada na redução de ácidos graxos livres, na quantidade recuperada de matéria-prima após o refino e na qualidade do biodiesel gerado. Adotando óleos com diferentes teores de ácidos graxos livres, óleo de andíroba (17,7%) e óleo de macaúba (7,9%) a desacidificação por adsorção, empregando diferentes adsorventes foi excluída com base nos baixos valores de desacidificação (<34,1%) e de recuperação em massa de óleo (22,2-43,1%). A acidez mais elevada do óleo de andíroba se mostrou como um fator limitante adicional, portanto, o uso deste óleo foi eliminado para as etapas posteriores de triagem. Em contrapartida, o refino por extração liquido-liquido (ELL) usando etanol como solvente forneceu valores mais elevados de recuperação mássica (>75,9%) e de redução de ácidos graxos livres (>48,8%). Buscou-se por meio da utilização de um método estatístico determinar as condições adequadas de refino por ELL usando etanol hidratado. Esta etapa foi realizada por meio de uma matriz de planejamento fatorial composto de face centrada 22, avaliando simultaneamente a influência da porcentagem mássica de agua no solvente alcoólico (2, 4 e 6%) e da razão mássica solvente/óleo (1:1, 1,5:1, 2:1). De acordo com a análise estatística foi possível estabelecer que a adição de 5,3% de agua no etanol e razão mássica etanol/óleo de 1,79:1 maximizaram a remoção de ácidos graxos livres (64,4%) sem ocasionar elevadas perdas de óleo refinado (93,9%). Adotando as condições otimizadas de ELL, o óleo de macaúba foi refinado em único e duplo estagio. Constatou-se que o teor de ácidos graxos livres em relação ao óleo bruto (7,9%) foi reduzido expressivamente em ambos os casos para valores de 2,9% (refino em único estagio) e de 1,0% (refino em duplo estagio), com a vantagem de não promover oxidação do material lipídico. Entretanto, a recuperação mássica de óleo refinado obtida na ELL em duplo estagio foi menor, da ordem de 78,5%. Para validar a metodologia de purificação, foram realizadas reações de etanólise das amostras de óleo refinado por ELL em único e duplo estagio, óleo bruto e óleo refinado por via alcalina utilizando como catalisador oxido nióbio impregnado com sódio. Enquanto a acidez do óleo bruto inibiu o catalisador, os produtos originados pelas amostras de óleo refinadas por ELL em único e duplo estagio contiveram teores em ésteres etílicos (97,4% e 97,8%, respectivamente) que atenderam as especificações recomendadas pela Agencia Nacional de Petróleo, Gás Natural e Biocombustível para uso como combustível. Esses valores foram similares a conversão alcançada na reação conduzida com o óleo refinado por via alcalina (99,0%), que teve desvantagens como recuperação mássica de 80,1% e aumento do índice de peroxido, indicando que a extração liquido-liquido tem potencial para desenvolvimento e aplicação em processos de refino de óleos vegetais para posterior utilização na síntese de biodiesel. / This study aimed at comparing the purification of non-edible vegetable oils by two different types of refining (adsorption and liquid-liquid extraction) with conventional alkali refining, contributing to the expansion of lipid feedstocks for biodiesel production. The use of non-edible vegetable oils is related to the final cost of biodiesel by increasing product competitiveness. The screening of the purification methods for vegetable oils (andiroba oil and macaw palm oil) was based on the reduction of free fatty acids content, oil mass recovery after the refining and the quality of biodiesel. Adopting oils with different levels of free fatty acids, andiroba oil (17.7%) and macaw palm oil (7.9%), the deacidification by adsorption, employing different adsorbents was excluded on the basis of the low deacidification values (<34.1%) and the oil mass recovered (22.2-43.1%). The higher acidity of andiroba oil was found to be an addition drawback and therefore, this oil was disqualified for further testing. On the other hand, the purification by liquid-liquid extraction (LLE) of macaw palm oil using ethanol as solvent gave higher values for oil mass recovery (>75.9%) and reduction of free fatty acids (>48.8). To determine the suitable conditions for oil refining by LLE using hydrated ethanol as solvent a statistical method was proposed. For this a 22 central composite face-centered experimental design was adopted to simultaneously evaluate the influence of the water percentage in the alcoholic solvent (2, 4, and 6%), and mass ratio of solvent-to-oil (1:1, 1.5:1 and 2:1). According to the statistical analysis it was possible to establish that the addition of 5.3% of water in the ethanol and mass ratio of ethanol to oil of 1.79:1 maximizes the removal of free fatty acids (64.4%) resulting in high refined oil recovered (93.9%). Adopting the optimized LLE conditions, the macaw palm oil was refined in single and double-stage. It was noted that the level of free fatty acids in relation to crude oil (7.9%) was significantly reduced in both cases for values of 2.9% (refining in single stage) and 1.0% (refining in double-stage), with the advantage of not promoting lipid oxidation. However, the percentage of the recovered refined oil obtained in LLE double-stage was slightly lower, in the order of 78.5%. To validate the purification methodology, ethanolysis reactions catalyzed by niobium oxide impregnated with sodium were performed using samples refined by LLE (single and double-stage), oil refined by alkali neutralization and crude oil. The obtained dataset demonstrated that the catalyst was inhibited by the high acidity level of the crude oil, while refined oil by LLE (single-stage and double-stage) gave levels of ethyl esters contents of 97.4% and 97.8%, respectively that meet the criteria established by the Brazilian National Agency of Petroleum, Natural Gas and Biofuels to be used as a fuel. These values were similar to that attained in the reaction using refined oil by alkaline protocol (99.0%), which had disadvantages such as oil mass recovered of 80.1% and oil oxidation (increased peroxide value), indicating that the liquid-liquid extraction has the potential for development and application in vegetable oils refining processes for further using as feedstocks in the biodiesel synthesis.

O presente estudo teve como objetivo selecionar procedimentos de purificação de óleos vegetais, com enfoque na desacidificação, comparando a eficiência de métodos de refino alternativos (adsorção e extração liquido-liquido) com o convencional (alcalino). Para tanto, foram utilizados óleos com baixo impacto na cadeia alimentícia, como óleo de andíroba e óleo de macaúba, visando contribuir para a ampliação de matérias-primas lipídicas na produção de biodiesel. A triagem do método de purificação mais adequado foi baseada na redução de ácidos graxos livres, na quantidade recuperada de matéria-prima após o refino e na qualidade do biodiesel gerado. Adotando óleos com diferentes teores de ácidos graxos livres, óleo de andíroba (17,7%) e óleo de macaúba (7,9%) a desacidificação por adsorção, empregando diferentes adsorventes foi excluída com base nos baixos valores de desacidificação (<34,1%) e de recuperação em massa de óleo (22,2-43,1%). A acidez mais elevada do óleo de andíroba se mostrou como um fator limitante adicional, portanto, o uso deste óleo foi eliminado para as etapas posteriores de triagem. Em contrapartida, o refino por extração liquido-liquido (ELL) usando etanol como solvente forneceu valores mais elevados de recuperação mássica (>75,9%) e de redução de ácidos graxos livres (>48,8%). Buscou-se por meio da utilização de um método estatístico determinar as condições adequadas de refino por ELL usando etanol hidratado. Esta etapa foi realizada por meio de uma matriz de planejamento fatorial composto de face centrada 22, avaliando simultaneamente a influência da porcentagem mássica de agua no solvente alcoólico (2, 4 e 6%) e da razão mássica solvente/óleo (1:1, 1,5:1, 2:1). De acordo com a análise estatística foi possível estabelecer que a adição de 5,3% de agua no etanol e razão mássica etanol/óleo de 1,79:1 maximizaram a remoção de ácidos graxos livres (64,4%) sem ocasionar elevadas perdas de óleo refinado (93,9%). Adotando as condições otimizadas de ELL, o óleo de macaúba foi refinado em único e duplo estagio. Constatou-se que o teor de ácidos graxos livres em relação ao óleo bruto (7,9%) foi reduzido expressivamente em ambos os casos para valores de 2,9% (refino em único estagio) e de 1,0% (refino em duplo estagio), com a vantagem de não promover oxidação do material lipídico. Entretanto, a recuperação mássica de óleo refinado obtida na ELL em duplo estagio foi menor, da ordem de 78,5%. Para validar a metodologia de purificação, foram realizadas reações de etanólise das amostras de óleo refinado por ELL em único e duplo estagio, óleo bruto e óleo refinado por via alcalina utilizando como catalisador oxido nióbio impregnado com sódio. Enquanto a acidez do óleo bruto inibiu o catalisador, os produtos originados pelas amostras de óleo refinadas por ELL em único e duplo estagio contiveram teores em ésteres etílicos (97,4% e 97,8%, respectivamente) que atenderam as especificações recomendadas pela Agencia Nacional de Petróleo, Gás Natural e Biocombustível para uso como combustível. Esses valores foram similares a conversão alcançada na reação conduzida com o óleo refinado por via alcalina (99,0%), que teve desvantagens como recuperação mássica de 80,1% e aumento do índice de peroxido, indicando que a extração liquido-liquido tem potencial para desenvolvimento e aplicação em processos de refino de óleos vegetais para posterior utilização na síntese de biodiesel. / This study aimed at comparing the purification of non-edible vegetable oils by two different types of refining (adsorption and liquid-liquid extraction) with conventional alkali refining, contributing to the expansion of lipid feedstocks for biodiesel production. The use of non-edible vegetable oils is related to the final cost of biodiesel by increasing product competitiveness. The screening of the purification methods for vegetable oils (andiroba oil and macaw palm oil) was based on the reduction of free fatty acids content, oil mass recovery after the refining and the quality of biodiesel. Adopting oils with different levels of free fatty acids, andiroba oil (17.7%) and macaw palm oil (7.9%), the deacidification by adsorption, employing different adsorbents was excluded on the basis of the low deacidification values (<34.1%) and the oil mass recovered (22.2-43.1%). The higher acidity of andiroba oil was found to be an addition drawback and therefore, this oil was disqualified for further testing. On the other hand, the purification by liquid-liquid extraction (LLE) of macaw palm oil using ethanol as solvent gave higher values for oil mass recovery (>75.9%) and reduction of free fatty acids (>48.8). To determine the suitable conditions for oil refining by LLE using hydrated ethanol as solvent a statistical method was proposed. For this a 22 central composite face-centered experimental design was adopted to simultaneously evaluate the influence of the water percentage in the alcoholic solvent (2, 4, and 6%), and mass ratio of solvent-to-oil (1:1, 1.5:1 and 2:1). According to the statistical analysis it was possible to establish that the addition of 5.3% of water in the ethanol and mass ratio of ethanol to oil of 1.79:1 maximizes the removal of free fatty acids (64.4%) resulting in high refined oil recovered (93.9%). Adopting the optimized LLE conditions, the macaw palm oil was refined in single and double-stage. It was noted that the level of free fatty acids in relation to crude oil (7.9%) was significantly reduced in both cases for values of 2.9% (refining in single stage) and 1.0% (refining in double-stage), with the advantage of not promoting lipid oxidation. However, the percentage of the recovered refined oil obtained in LLE double-stage was slightly lower, in the order of 78.5%. To validate the purification methodology, ethanolysis reactions catalyzed by niobium oxide impregnated with sodium were performed using samples refined by LLE (single and double-stage), oil refined by alkali neutralization and crude oil. The obtained dataset demonstrated that the catalyst was inhibited by the high acidity level of the crude oil, while refined oil by LLE (single-stage and double-stage) gave levels of ethyl esters contents of 97.4% and 97.8%, respectively that meet the criteria established by the Brazilian National Agency of Petroleum, Natural Gas and Biofuels to be used as a fuel. These values were similar to that attained in the reaction using refined oil by alkaline protocol (99.0%), which had disadvantages such as oil mass recovered of 80.1% and oil oxidation (increased peroxide value), indicating that the liquid-liquid extraction has the potential for development and application in vegetable oils refining processes for further using as feedstocks in the biodiesel synthesis.

Introduction An increasing proportion of the patients undergoing cardiac surgery have diabetes mellitus, in particular type II diabetes. In spite of this, diabetic patients have received limited attention in this setting. Although diabetes is a metabolic disease cardiac metabolism in association with surgery has previously not been explored in diabetics. This investigation was carried out to describe the metabolic state of the heart in diabetics after cardiac surgery and to study if it is accessible to metabolic intervention with high-dose GIK. Also, the potential hazards associated with such a regime in clinical practice were evaluated. Furthermore, a comparison of the outcome in diabetic and nondiabetic patients after coronary surgery was done. Methods Myocardial metabolism and how it was influenced by high-dose GIK was assessed with coronary sinus catheter technique in a prospective randomized study on 20 type II diabetic patients undergoing CABG (paper I, II). Safety issues concerning high-dose GIK were assessed in two retrospective studies. The potential role of metabolic interventions for neurological injury was assessed in a cohort of 775 consecutive patients undergoing CABG or combined CABG + valve surgery, in whom metabolic interventions gradually replaced traditional treatment for postoperative heart failure (paper III). A detailed analysis of blood glucose and electrolyte control was done in all cases (n=89) receiving high-dose GIK during one year (paper IV). The hemodynamic impact of highdose GIK was assessed with standard postoperative monitoring including Swan-Ganz catheters (paper II, IV). Outcome and prognosis after CABG in diabetic patients (n=540) were compared with nondiabetics (n=2239) with the aid of the institutional database comprising all isolated CABG procedures from 1995-1999 (paper V). Results The metabolism of the diabetic heart after CABG was characterized by predominant uptake of FFA and restricted uptake of carbohydrate substrates. A high extraction rate of beta-hydroxybutyric acid and glutamate was also found. Alanine was released from the heart (paper I). High-dose GIK induced a shift towards uptake of carbohydrates, in particular lactate, at the expense of FFA and betahydroxybutyric acid (paper II). A substantial systemic glucose uptake was found during high-dose GIK treatment but the uptake tended to be lower and blood glucose higher if adrenergic drugs were used or/and if the patient was a diabetic (paper IV). High-dose GIK was associated with beneficial effects on cardiac output both in the prospective and retrospective analyses (paper II, IV). No evidence for untoward neurological effects associated with GIK treatment was found. History of cerebrovascular disease was the most important risk factor for postoperative cerebral complications and in general markers for advanced atherosclerotic disease were found to be of importance (paper III). High-dose GIK in clinical practice was associated with acceptable blood glucose and electrolyte control and no serious adverse events were recorded (paper IV). Patients with diabetes undergoing CABG had an acceptable short-term mortality that did not differ significantly from non-diabetic patients. However, diabetic patients had a higher early postoperative morbidity particularly with regard to stroke, renal- and infectious complications. Also, long-term survival was markedly reduced in diabetic patients, particularly in insulin treated patients (paper V). Comments FFA were the main source of energy for the heart in type II diabetics after CABG whereas the uptake of carbohydrates was restricted. The high extraction rates of beta-hydroxybutyric acid and glutamate may represent an adaptation to the unfavorable metabolic situation of the post-ischemic diabetic heart. High-dose GIK can be used in type II diabetic patients after cardiac surgery to promote carbohydrate uptake at the expense of FFA and beta-hydroxybutyric acid. The magnitude of this shift was sufficient to account for the entire myocardial oxygen consumption assuming that the substrates extracted were oxidized. This could have implications for the treatment of the diabetic heart in association with surgery and ischemia. Provided careful monitoring high-dose GIK can be safely used in clinical practice and this treatment deserves further evaluation in the treatment of postoperative heart failure. High-dose GIK also provides a means for strict blood glucose control and as substantial amounts of glucose can be infused even in critically ill patients, it may prove useful for nutrition in critical care. Several of the risk factors for neurological injury identified constitute markers for advanced atherosclerotic disease, thus, also providing an explanation for the increased risk of neurological injury in diabetics after cardiac surgery. Short-term mortality was acceptable in diabetics after CABG. However, further efforts are warranted to address postoperative morbidity and late outcome. This represents a challenge as diabetic patients are accounting for an increasing proportion of the patients undergoing CABG. / On the day of the public defence the status of article IV was: Submitted and the title of article IV was in the printed version: High-dose GIK in cardiac surgery - clinical safety issues and lessons learned.

Elevated circulating free fatty acids (FFA) cause hepatic insulin resistance; however, the mechanisms for this process are incompletely understood. The objective of the studies in the thesis was to examine whether protein kinase C (PKC)-delta (d), oxidative stress, and the serine kinase IkBa kinase (IKK) B are causally involved in FFA-induced hepatic insulin resistance. To test this, we infused rats with lipid with or without inhibitors of the aforementioned factors for 7h, during the last 2h of which a hyperinsulinemic-euglycemic clamp was performed. In Study 1, inhibition of hepatic PKC-d using antisense oligonucleotide prevented FFA-induced membrane translocation of PKC-d, which is a marker of its activation, in parallel with prevention of lipid-induced hepatic insulin resistance, without affecting lipid-induced peripheral insulin resistance. These results implicate PKC-d as a causal mediator of FFA-induced hepatic insulin resistance. In Study 2, the antioxidant N-acetyl-L-cysteine (NAC) prevented lipid-induced hepatic insulin resistance in conjunction with reversal of lipid-induced increase in markers of IKKB and c-Jun NH2-terminal kinase 1 (JNK1) activation, and of impairment of insulin signaling, without affecting PKC-d membrane translocation and increase in phosphorylated p38 mitogen-activated protein kinase (MAPK) induced by lipid infusion. These findings suggested that oxidative stress is a causal mediator of lipid-induced hepatic insulin resistance upstream of IKKB and JNK1, and potentially downstream of PKC-d and p38 MAPK. In Study 3, sodium salicylate, an IKKB inhibitor, prevented FFA-induced hepatic insulin resistance via restoration of hepatic insulin signaling, thus implicating IKKB as a causal factor in the process. Together, the results from these studies demonstrate that PKC-d, oxidative stress, and IKKB are causally involved in FFA-induced hepatic insulin resistance and suggest that the sequence for the process is: FFA -> PKC-d -> oxidative stress -> IKKB -> impaired hepatic insulin signaling.