Résumé: La subérine, un polymère lipidique et complexe des plantes est retrouvé dans divers tissus dont le périderme de la pomme de terre. Le processus biologique de sa dégradation reste encore peu connu et est attribué aux champignons. Des échantillons de sol provenant d'un champ de pommes de terre ont été inoculés dans un milieu de culture contenant de la subérine comme source de carbone. Une approche métaprotéomique a été utilisée pour identifier les populations bactériennes qui se développent en présence de la subérine sur une période d'incubation de 60 jours. Le nombre de spectres normalisé (NSpC) des protéines extracellulaires produites par la communauté bactérienne du sol ont considérablement diminué du jour 5 au jour 20, puis ont augmenté lentement, révélant une succession de bactéries, où la population des bactéries du genre Pseudomonas à croissance rapide a diminué et a été remplacée par d’autres espèces bactériennes qui pouvaient se développer en présence de la subérine. La récalcitrance de la subérine a été démontrée par l'émergence de bactéries auxotrophes telles qu’Oscillatoria dans les derniers jours de la culture bactérienne. Néanmoins, l'identification de deux lipases dans le surnageant de la culture suggère qu'au moins certaines espèces bactériennes peuvent dégrader la subérine. Une des lipases (I4WGM2) a été associée à Rhodanobacter thiooxydans. Lorsque cultivée dans un milieu contenant de la subérine, la souche de R. thiooxidans LCS2 a produit trois lipases, dont I4WGM2. R. thiooxidans LCS2 a également produit d'autres protéines liées au métabolisme des lipides, des transporteurs de chaines d’acide gras et les enzymes de la [béta]-oxydation. Ceci suggère que R. thiooxydans pourrait participer à la dégradation de la subérine. / Abstract: Suberin is a complex lipidic plant polymer found in various tissues including potato periderm. The biological degradation process of suberin is poorly characterized and is attributed to fungi. Soil samples from a potato field were used to inoculate a culture medium containing suberin as carbon source and a metaproteomics approach was used to identify bacterial populations that develop in the presence of suberin, over a 60-day incubation period. The normalized spectral counts of predicted extracellular proteins produced by the soil bacterial community drastically decreased from day 5 to day 20 and then slowly increased, revealing a succession of bacteria. The population of fast-growing pseudomonads declined and was replaced by species that could develop in the presence of suberin. The recalcitrance of suberin was demonstrated by the emergence of auxotrophic bacteria such as Oscillatoria in the last days of the assay. Nevertheless, the identification of two putative lipases in the culture supernatants suggests that at least some bacterial species could degrade suberin. One of the lipases (I4WGM2) was associated with Rhodanobacter thiooxydans. When grown in a suberin-containing medium, R. thiooxydans strain LCS2 produced three lipases, including I4WGM2. This strain also produced other proteins linked to lipid metabolism, including fatty acid and lipid transporters and [beta]-oxidation enzymes, suggesting that R. thiooxydans could participate in suberin degradation.

Abstract Fatty acid degradation in living organisms occurs mainly via the β-oxidation pathway. When this work was started, it was known that the hydration and dehydrogenation reactions in mammalian peroxisomal β-oxidation were catalyzed by only multifunctional enzyme type 1 (MFE-1; Δ2-Δ3-enoyl-CoA isomerase/2-enoyl-CoA hydratase 1/(3S)-hydroxyacyl-CoA dehydrogenase) via the S-specific pathway, whereas in the yeast peroxisomes via the R-specific pathway by multifunctional enzyme type 2 (MFE-2; 2-enoyl-CoA hydratase 2/(3R)-hydroxyacyl-CoA dehydrogenase). The work started with the molecular cloning of the rat 2-enoy-CoA hydratase 2 (hydratase 2). The isolated cDNA (2205 bp) encodes a polypeptide with a predicted molecular mass of 79.3 kDa, which contains a potential peroxisomal targeting signal (AKL) in the carboxyl terminus. The hydratase 2 is an integral part of the cloned polypeptide, which is assigned to be a novel mammalian peroxisomal MFE-2. The physiological role of the mammalian hydratase 2 was investigated with the recombinant hydratase 2 domain derived from rat MFE-2. The protein hydrates a physiological intermediate (24E)-3α, 7α, 12α-trihydroxy-5β-cholest-24-enoyl-CoA to (24R, 25R)-3α, 7α, 12α, 24-tetrahydroxy-5β-cholestanoyl-CoA in bile acid synthesis. The sequence alignment of human MFE-2 with MFE-2(s) of different species reveals 12 conserved protic amino acid residues, which are potential candidates for catalysis of the hydratase 2. Each of these residues was replaced by alanine. Complementation of Saccharomyces cerevisiae fox-2 (devoid of endogenous MFE-2) with human MFE-2 provided a model system for examing the in vivo function of the variants. Two protic residues, Glu366 and Asp510, of the hydratase 2 domain of human MFE-2 have been identified and are proposed to act as a base and an acid in catalysis. Mammalian MFE-2 has a (3R)-hydroxyacyl-CoA dehydrogenase domain, whereas the yeast MFE-2 has two dehydrogenase domains, A and B. The present work, applying site-directed mutagenesis to dissect the two domains, shows that the growth rates of fox-2 cells expressing a single functional domain are lower than those of cells expressing S. cerevisiae MFE-2. Kinetic experiments with the purified proteins demonstrate that domain A is more active than domain B in catalysis of medium- and long-chain (3R)-hydroxyacyl-CoA, whereas domain B is solely responsible for metabolism of short-chain substrates. Both domains are required when yeast cells utilize fatty acids as the carbon source.

The current dietary recommendations for optimal health are designed to increase our intake of two bioactive omega-3 (n-3) fatty acids, eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), abundant naturally in fatty fish such as salmon. Health authorities recommend that the general population consume two to three fatty fish meals per week (1) for optimal health and for the prevention of cardiovascular disease. However, some modern Western societies consume only modest amounts of fish and seafood (2;3). Land based vegetable oils may provide an alternative to meet these needs. Linseed and canola oils are rich in alpha-linolenic acid (ALA, 18:3n-3) (4). ALA can be converted endogenously to EPA and DHA and suggests that increasing the dietary intake of ALA may increase the conversion and accumulation of DHA in tissues and plasma. However, elevated dietary intakes of ALA in animals and humans results in an increased level of EPA in tissues yet there is little or no change in the level of DHA (5-7). The current consensus is that the synthesis of DHA from ALA in humans is limited yet the mechanisms involved in regulating the accumulation of DHA in tissues are poorly understood. The reputed rate-limiting enzyme in the conversion of fatty acids is delta 6 desaturase (D6D). ALA is a substrate for D6D and undergoes a series of desaturation and elongation reactions to yield n-3 long chain polyunsaturated fatty acids (LCPUFA). The final step in the synthesis of DHA from ALA involves translocation of its immediate fatty acid precursor, 24:6n-3 from the endoplasmic reticulum to the peroxisome to be partially beta-oxidised to yield DHA. The involvement of multiple enzymes in the desaturation-elongation pathway, and the integration of other pathways, such as phospholipid biosynthesis, suggests there are various steps that may regulate the accumulation of DHA in cell membranes. This thesis aimed to examine the possible regulatory steps in the conversion of fatty acids to LCPUFA, particularly in the synthesis of DHA from n-3 fatty acid precursors. The human hepatoma cell line, HepG2, was used as an in vitro cell system to examine the accumulation of individual fatty acids and their metabolites in isolation from other competing fatty acid substrates. The accumulation of linoleic acid (LA, 18:2n-6) and ALA in HepG2 cell phospholipids following supplementation with increasing concentrations of each respective fatty acid correlated with that described in vivo, as was the accumulation of their conversion products. The accumulation of DHA in cells supplemented with ALA reached a plateau at concentrations above 5 micro g/ml and paralleled the accumulation of 24:6n-3 in cell phospholipids, suggesting that the delta 6 desaturation of 24:6n-3 was prevented by increasing concentrations of ALA, thereby limiting the accumulation of DHA. The accumulation of DHA in cells supplemented with eicosapentaenoic acid (EPA, 20:5n-3) or docosapentaenoic acid (DPA, 22:5n-3) was significantly greater than the level of DHA that accumulated in cells supplemented with ALA. However, regardless of substrate, the level of DHA in cell membranes reached a plateau at substrate concentrations above 5 micro g/ml. This thesis further aimed to examine the effect of fatty acid supplementation on the mRNA expression of D6D in HepG2 cells. The expression and activity of D6D mRNA is subject to nutritional and hormonal regulation. The mRNA expression of D6D in HepG2 cells following supplementation with oleic acid (OA, 18:1n-9), LA, ALA, arachidonic acid (AA, 20:4n-6) or EPA was examined by real time RT PCR. The expression of D6D mRNA was reduced by up to 50% in cells supplemented with OA, LA, ALA , AA or EPA compared with control cells and suggests that fatty acids modulate the expression of the key enzyme involved in the conversion of fatty acids. The effect of fatty acid co-supplementation on the fatty acid composition of HepG2 cell phospholipids was also examined in an attempt to gain insights into the role of D6D and the enzymes involved in peroxisomal beta-oxidation on the accumulation of DHA from n-3 fatty acid precursors. The reduction in the accumulation of DHA in cells co-supplemented with DPA and docosatetraenoic acid (DTA, 22:4n-6) was greater than in cells co-supplemented with DPA and LA, suggesting that peroxisomal beta-oxidation may have a greater role in determining the accumulation of DHA from DPA than the activity of D6D. Further investigation should be directed towards understanding the role that peroxisomal beta-oxidation may play in the synthesis of DHA from precursor fatty acids. The fatty acid composition of cell membranes in vivo is a result of several physiological processes including dietary intake, phospholipids biosynthesis and fatty acid conversion as well as catabolic processes. This thesis demonstrates that a greater understanding of the regulation of the conversion of fatty acids will help to define dietary approaches that enhance the synthesis of n-3 LCPUFA from n-3 fatty acid precursors to lead to improved outcomes for health.

Vitamin E ist der Überbegriff für 4 Tocopherole (α, β, γ und δ) sowie 4 Tocotrienole (α, β, γ und δ), die als gemeinsames Merkmal ein Chromanolringsystem sowie eine gesättigte (Tocopherole) bzw. ungesättigte (Tocotrienole) Seitenkette aufweisen. Neben ihrer antioxidativen Wirkung (Schutz von Membranen vor Lipidperoxidaton) konnten für einige Vitamin E - Formen auch eine Reihe von hochspezifischen, nicht-antioxidativen Wirkungen in vitro nachgewiesen werden. Meist bleibt jedoch unklar, ob ein solcher Effekt auch in vivo, also im Tiermodel oder direkt im Menschen, gefunden werden kann. In erster Linie müsste hierbei geklärt werden, ob die jeweilige Vitamin E - Form auch bioverfügbar, also in für eine Wirkung ausreichender Konzentration im Organismus vorhanden ist, oder aber vorher eliminiert und ausgeschieden wird. In dieser Doktorarbeit wurden deshalb wichtige Grundlagen zum Abbau der Tocopherole und Tocotrienole erarbeitet. • In HepG2-Zellen konnte der Abbau der Tocotrienole mit Hilfe flüssig- sowie gaschromatographischer Analysemethoden vollständig aufgeklärt werden. Wie sich hierbei ergab, verläuft der Abbau weitgehend in Analogie zum Abbau der Tocopherole über eine durch Cytochrom P450 katalysierte initiale ω-Hydroxylierung mit 5 nachfolgenden β-Oxidationsschritten. • In vitro konnten in HepG2 – Zellen die Abbauraten der verschiedenen Vitamin E - Formen bestimmt werden. Dies nahmen in folgender Reihenfolge zu: α-Tocopherol < γ-Tocopherol < α-Tocotrienol < γ-Tocotrienol. • Wie sich mit Hilfe eines mit Cytochrom P450 hochangereicherten Homogenats aus Rattenlebern ergab, stellt die initiale ω-Hydroxylierung einen geschwindigkeitsbestimmenden Schritt des Abbaus dar: α-Tocopherol wurde weit langsamer hydroxyliert als alle anderen Vitamin E – Formen. • Der unterschiedliche Abbau von α-Tocopherol und γ-Tocotrienol konnte auch im Mäuseversuch in vivo bestätigt werden. Nach Fütterung von Mäusen mit α-Tocopherol wurden nur geringe Mengen von α-Tocopherolmetaboliten im Urin der Mäuse gefunden, während nach Applikation von γ-Tocotrienol hohe Konzentrationen der γ-Tocotrienolmetabolite nachgewiesen wurden. In Plasma und Leber wiederum wurden (dem Futtergehalt entsprechende) hohe α-Tocopherolkonzentrationen entdeckt, während γ-Tocotrienol selbst nach hoher Gabe nicht oder nur in Spuren nachweisbar war. In HepG2 – Zellen konnte gezeigt werden, dass γ-Tocotrienol eine cytotoxische Wirkung auf die Hepatocarcinoma-Zelllinie HepG2 entfalten kann, indem durch die Aktivierung der proteolytischen Caspase 3 die Induktion des programmierten Zelltodes (Apoptose) ausgelöst wird. Abschliessend lässt sich festhalten, dass der Körper lediglich das natürliche α-Tocopherol vor dem Abbau bewahrt, die anderen Vitamin E – Formen jedoch als Fremdstoffe behandelt und rapide ausscheidet. Als doppelter Schutz vor Verlust des “wertvollen” α-Tocopherol dienen hierbei das α-Tocopherol Transfer Protein sowie die in dieser Arbeit gefundenen Unterschiede im ersten Schritt des Abbaus, der Cytochrom P450 - katalysierten ω-Hydroxylierung. Beides erklärt die bevorzugte Retention von α-Tocopherol im Organsimus und seine hohe Bioaktivität. Will man deshalb in vitro Ergebnisse anderer Vitamin E – Formen auf die in vivo Situation übertragen, muss man die geringe Bioverfügbarkeit dieser Substanzen berücksichtigen. / The vitamin E family is comprised of 4 different tocopherols (Toc: α, β, γ, δ) and 4 different tocotrienols (T3: α, β, χ, δ). All share a hydroxychromanol ring and a saturated (Toc) or unsaturated (T3) side chain. Apart from their role as anti-oxidants (protection of membranes from lipid peroxidation), recent attention has focused on novel molecular, non-antioxidative functions. Numerous specific effects of tocopherols and tocotrienols were uncovered by a large variety of in vitro studies, in vivo - based evidence, however, is scarce. Moreover, little information exists on the bioavailabilty of the different vitamin E - forms. To better understand the biological role of the different tocopherols and tocotrienols, this thesis therefore aimed to address the basic but important aspect of tocopherol and tocotrienol metabolism. • In HepG2 cells, the metabolic pathway of α- and γ-T3 could be elucidated by the identification of all intermediary degradation products by using high performance liquid- as well as gas-chromatography. Thus, tocotrienols are degraded in analogy to tocopherols with an initial ω-hydroxylation and 5 subsequent β-oxidation steps. • In vitro (HepG2 cells), tocotrienols were degraded to a larger extent than tocopherols, and γ-Toc to a larger extent than α-Toc. Differences reached two orders of magnitude with α-Toc < γ-Toc < α-T3 < γ-T3. • By using rat liver microsomes that were highly enriched with cytochrome P450 enzymes, the initial ω-hydroxylation was shown to be a rate limiting step in the degradation of vitamin E: α-Toc is hydrolysed to a much smaller extent than all other vitamin E forms. • The differences in vitamin E metabolism were confirmed in vivo using male mice. After supplementation with α-Toc, only little amounts of α-Toc metabolites were found in urine, while oral administration of γ-T3 led to the rapid excretion of large amounts of γ-T3 metabolites. Correspondingly, in plasma and liver α-Toc levels were high but γ-T3 could hardly be detected. • γ-T3 but no other vitamin E – form was shown to be highly cytotoxic for HepG2 cells. Immunohistochemistry stainings revealed that γ-T3 induced apoptosis by activation of the proteolytic caspase 3. To summarize, α-Toc is metabolized to a much smaller extent than all other vitamin E - forms. Both the α-tocopherol transfer protein as well as the here described differences in the ω-hydroxylation rates provide a double protection for the “valuable” α-Toc from degradation. Both phenomena explain the high retention of α-Toc in the organism and its higher bioactivity, compared to other Vitamin E forms. The differences in the metabolism of vitamin E might therefore lead to an inequivalence of biological activities found in vitro vs. in vivo.

Alcohol use disorder (AUD) is the fourth leading cause of preventable death in the United States, and the fifth leading risk factor for premature death and disability, globally. There are currently very few treatment options for AUD and there is a need for effective preventive and treatment strategies for this condition. AUD risk has a significant hereditary component, with the contribution of genetic factors being estimated to be about 50%. The Davies-Bettinger laboratory uses C. elegans as a model organism to study the contribution of genetic factors in modulating neuronal responses to ethanol. In this project, we examined the role of mitochondrial beta-oxidation of fatty acids (FA) in altering ethanol responses using loss-of-function (lf) mutants and RNAi-mediated knockdown of specific genes in this pathway. We tested a total of 34 genes and found that lf in 13 genes significantly affected ethanol response phenotypes. We conclude that mitochondrial beta-oxidation of FA is essential for ethanol response behavior in C. elegans. Further experiments need to be conducted to dissect the specific contribution of various components of mitochondrial beta-oxidation in modifying the neuronal responses to ethanol.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that function as ligand activated transcription factors. There are three identified isotypes: PPAR alpha, PPAR gamma and PPAR delta, together controlling the expression of genes involved in inflammation, cell differentiation, proliferation, lipid and carbohydrate metabolism and energy homeostasis. The PPARs are potential targets for the treatment of dyslipidaemia, type II diabetes mellitus and the metabolic syndrome. This thesis uses a multi-platform metabolomics approach, 13C-isotope substrate flux analysis, respirometry and transcriptomics to determine the role PPAR delta and PPAR gamma play in metabolic control both in adipose tissue and systemically. To achieve this, the metabolic phenotype of the 3T3-L1 adipocyte cell line was defined to generate a metabolically phenotyped in vitro model of adipose tissue. The importance of fatty acid alpha-oxidation in the differentiation of adipocytes was emphasised The effects of PPAR delta and PPAR gamma activation in white adipose tissue from the ob/ob mouse model of insulin resistance, and in the phenotyped 3T3-L1 adipocyte model, were investigated. PPAR delta activation was distinguished by oxidative catabolism of fatty acids and citric acid cycle intermediates. Conversely, PPAR gamma activation was identified by the sequestration of lipids into adipose tissue. Moreover, to address the systemic influence of PPAR activation, with a focus on the Cori cycle and the interactions of the liver and skeletal muscle, the metabolic changes that occur in these tissues following PPAR delta and PPAR gamma activation in the ob/ob mouse were examined. PPAR delta activation was characterised by the mobilisation and release of triacylglycerols (TAGs) into circulation as an energy source for peripheral tissues whereas PPAR gamma activation was defined by a reduction and sequestration of circulating TAGs. This thesis has better characterised the role of the PPARs as master regulators of metabolism and emphasised their potential as therapeutic targets for metabolic diseases of global importance.

No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24287号 / 医博第4903号 / 新制||医||1061(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 寺田 智祐, 教授 松田 道行, 教授 YOUSSEFIAN Shohab / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Cardiac energy metabolism is the one of the most complex system in the body. To sustain life, but also to respond quickly to any sudden changes (e.g. running, emotional stress), the heart has developed a unique ability and has become a metabolic "omnivore". At physiological conditions, long chain fatty acids (LCFAs) present the major energetic source for the adult myocardium. However, the cardiac energy metabolism may be compromised during pathophysiological states. One of the most dangerous is, undoubtedly, ischaemia-reperfusion injury with its acute form, myocardial infarction. However, the adaptation to chronic hypoxia has been known for decades for its cardioprotective effect against I/R. Changes of cardiac energy metabolism induced by the adaptation have not been fully explored and the system conceals still too many secrets. This thesis has aimed to determine how adaptation to chronic hypoxia affects the cardiac metabolism of the rat LVs in the following set-ups: 1. The effect of chronic normobaric hypoxia (CNH; 3 weeks, 5500m) during a brief I/R protocol in vitro on the protein kinase B/hexokinase (Akt/HK) pathway, including the expression and phosphorylation of Akt, the expression and localization of HK, the expression of mitochondrial creatine kinase (mtCKS), and the level of Bcl-2 family...

Chronic hypoxia is a state of oxygen limitation that is common in many aquatic and terrestrial environments. Metabolic suppression is an essential strategy that is used by hypoxia-tolerant champions such as goldfish and naked mole-rats to cope with prolonged low oxygen. This thesis examines the physiological processes used by goldfish and naked mole-rats to survive in low oxygen environments. It proposes a novel mechanism - the remodeling of membrane lipids - to reduce ATP use and production. Temperature (homeoviscous adaptation), diet (natural doping in migrant birds) and body mass (membrane pacemaker of metabolism) have an impact on the lipid composition of membranes that, in turn, modulates metabolism. In chapters 2 and 3 of this thesis, I demonstrate that vertebrate champions of hypoxia tolerance undergo extensive changes in membrane lipid composition upon in vivo exposure to low oxygen. These changes and those observed in hibernating mammals can promote the downregulation of Na⁺/K⁺-ATPase (major ATP consumers), mitochondrial respiration capacity [OXPHOS (phosphorylating conditions), proton leak (non-phosphorylating conditions), cytochrome c oxidase], and energy metabolism (β-oxidation and glycolysis) as discussed in chapters 3 and 4. A common membrane signal regulating the joint inhibition of ion pumps and channels could be an exquisite way to preserve the balance between ATP supply and demand in hypometabolic states. In chapter 5, I show that the reduction in ATP turnover is also orchestrated by mechanisms that involve post-translational and post-transcriptional modifications and epigenetic changes. Membrane remodeling, together with these more traditional molecular mechanisms, could work in concert to cause metabolic suppression.