Prevalence of obesity and associated diseases like type 2 diabetes has increased rapidly during last years. These diseases closely relate to each other. Obesity leads to insulin resistence, which directly precedes type 2 diabetes. Metformin is the most prescribed medicament for type 2 diabetic patients and insulin resistant people. It improves glucose tolerance and insulin resistance. Enzyme AMP-activated protein kinase (AMPK) is strogly involved in metformin action. The latest studies using transgenic models lacking AMPK suggest, that notable part of mechanisms involved in metformin action is independent on AMPK. n-3 polyunsaturated fatty acids (n-3 PUFA), namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are abundant in sea fish, have beneficial effects on metabolism. These fatty acids lower plasma lipids and exert cardioprotective effects. n-3 PUFA also prevent development of insulin resistence and type 2 diabetes in rodents. The aim of this thesis was to characterise acute effects of metformin on glucose homeostasis, impact of short term diet intervention with diet rich in n-3 PUFA on metformin action and the role of insulin stimulated signalling pathways and AMPK. Results suggest that early effect of metformin is dose dependent and that single dose of metformin...

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No / Nutritionally important PUFAs (polyunsaturated fatty acids) mediate some of their bioactivities through formation of oxygenated metabolites. These bioactive lipids are formed by COX (cyclo-oxygenase), LOX (lipoxygenase) and cytochrome-P450-catalysed reactions, as well as non-enzymatic lipid peroxidation. These reactions produce numerous species, some of which can be formed through more than one pathway. MS-based lipidomics offers the selectivity and sensitivity required for qualitative and quantitative analysis of multiple lipid species, in a variety of biological systems, and can facilitate the study of these mediators.

No / Lipid mediators are produced from the oxidation of polyunsaturated fatty acids through enzymatic and free radical-mediated reactions. When subject to oxygenation via cyclooxygenases, lipoxygenases, and cytochrome P450 monooxygenases, polyunsaturated fatty acids give rise to an array of metabolites including eicosanoids, docosanoids, and octadecanoids. These potent bioactive lipids are involved in many biochemical and signaling pathways, with inflammation being of particular importance. Moreover, because they are produced by more than one pathway and substrate, and are present in a variety of biological milieus, their analysis is not always possible with conventional assays. Liquid chromatography coupled to electrospray mass spectrometry offers a versatile and sensitive approach for the analysis of bioactive lipids, allowing specific and accurate quantitation of multiple species present in the same sample. Here we explain the principles of this approach to mediator lipidomics and present detailed protocols for the assay of enzymatically produced oxygenated metabolites of polyunsaturated fatty acids that can be tailored to answer biological questions or facilitate assessment of nutritional and pharmacological interventions.

The main source of omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA) in our diet is supplied by fish, and an ever-increasing proportion of these are being produced by aquaculture. The drive for the growing market demand and production from sustainable sources has led to the use of high-energy (fat) diets and, recently, to the replacement of fishmeal and fish oil with non-marine components, such as plant meals and vegetable oils that are devoid of n-3 LC-PUFA. Both changes impact greatly on lipid and fatty acid metabolism in fish, with health implications for the fish and the human consumer. This impact highlights the need to investigate the basic molecular mechanisms underlying the regulation of lipid and fatty acid metabolism in fish, specifically focussing on the pathways of lipid homeostasis and LC-PUFA synthesis. The aim of this study was to develop and utilise Chinook salmon embryo (CHSE-214) cell line as a model for Atlantic salmon, Salmo salar L., to enable an integrated approach to study the biochemical and molecular regulation of lipid metabolism in fish. In particular, α-linolenic acid (LNA, 18:3n-3) and linoleic acid (LOA, 18:2n-6), which are essential fatty acids abundantly found in vegetable oils, and are precursors of LC-PUFA, were supplemented in combination with other fatty acids, to explore the effect of these on total lipid content, lipid class, FA composition and gene expression of CHSE-214 cell line. Total lipid content was extracted, followed by determination of lipid class and fatty acid analyses. Gene expression analyses of transcription/nuclear factors and various target genes in Atlantic salmon, including those involved in pathways of LC-PUFA synthesis and fatty acid oxidation, were carried out. The results demonstrated that CHSE-214 cell line, under experimental conditions, is able to convert LNA to eicosapentaenoic acid (EPA, 20:5n-3), and LOA to arachidonic acid (ARA, 20:4n-6), but not LNA and/or EPA to docosahexaenoic acid (DHA, 22:6n-3), highlighting the activity of elongase and desaturase enzymes during the conversion process. Changes occurring on the fatty acid profile and also at molecular level were observed. Understanding the role that transcription factors play in the regulation of lipid biosynthesis in fish will allow endogenous LC-PUFA synthesis to be optimised. The results from this study could be used to improve the efficiency of alternative, sustainable diets in aquaculture, while maintaining the nutritional quality of farmed fish for the final consumer. CHSE-214 cell line can therefore be used as a model to study the molecular mechanisms involved in the LC-PUFA biosynthesis, particularly in the conversion of LNA to EPA, which can then be reproduced in vivo, saving time and money.

Der globale Prävalenzanstieg allergischer Erkrankungen wird mit der westlichen Ernährung und einem sich ändernden Fettsäurespektrum assoziiert. Die omega-3 Fettsäure Docosahexaensäure (DHA) wurde bereits bei verschiedenen chronisch-entzündlichen Erkrankungen erfolgreich therapeutisch eingesetzt. Die dabei zugrunde liegenden Wirkmechanismen sind jedoch nicht vollständig geklärt. Deshalb wurde hier der molekulare Mechanismus der DHA-vermittelten Hemmung der IgE-Produktion in humanen B-Zellen sowie der verminderten Differenzierung IgE-produzierender Plasmazellen in vitro untersucht. Analysen der beteiligten Signaltransduktionswege offenbarten eine Reduktion der IL-4-abhängigen STAT6-Phosphorylierung und der CD40-vermittelten NFkappaB-Translokation, was zu einer Inhibition des IgE-Klassenwechsels auf dem Niveau des epsilon-Keimbahntranskriptes sowie der Aktivierungsinduzierten Desaminase führte. Weiterhin wurde in einer randomisierten, kontrollierten Doppelblindstudie die Supplementierung mit hochdosierter DHA bei Patienten mit atopischem Ekzem hinsichtlich klinischer und immunologischer Parameter geprüft. Dabei führte DHA zu einer Reduktion des Schweregrades der Erkrankung und zu einer verminderten IgE-Produktion anti-CD40/IL 4-stimulierter Blutzellen ohne Beeinflussung des Serum-IgE-Spiegels. Schließlich wurden die lokalen Prozesse nach DHA-Verabreichung in einem Mausmodell für proteininduzierte Dermatitis analysiert. Dabei war die Reduktion der klinischen Ekzemausprägung mit der verminderten Zahl dermaler CD8+ T-Zellen verbunden. Andere histologische Parameter und das Serum-IgE blieben jedoch unbeeinflusst. Durch die Fähigkeit von DHA, in den IgE-Klassenwechsel in B-Zellen einzugreifen, stellt die Supplementierung mit DHA somit eine mögliche präventive Maßnahme gegen allergische Erkrankungen dar. Ebenso ist DHA in der Lage, den Schweregrad des atopischen Ekzems durch die positive Beeinflussung lokaler inflammatorischer Prozesse signifikant zu verbessern. / The prevalence of allergic diseases has increased worldwide. Westernised diet with its changing polyunsaturated fatty acid (PUFA) proportions is considered to contribute to this development. The omega-3 PUFA Docosahexaenoic acid (DHA) has been reported to be antiinflammatory, but its way of action is not completely understood. Initially, the molecular mechanisms of DHA impact on IgE production in human B cells were examined in vitro. Thereby, DHA inhibited IgE production and the differentiation of IgE secreting cells. This was mediated through direct inhibition of the immunoglobulin isotype switching process by decreasing epsilon germline transcript and activation induced desaminase transcription. Analysis of involved signalling pathways revealed an inhibition of IL-4 driven STAT6 phosphorylation and a reduced NFkappaB translocation into nucleus upon CD40 ligation. Next, in a randomised, double bind, controlled clinical study the efficacy of high-dose DHA supplementation in atopic eczema was determined by investigating the impact on clinical and immunological parameters. In the DHA, but not in control group a clinical improvement of atopic eczema and a reduction of CD40/IL-4 mediated IgE synthesis of peripheral blood cells were observed whereas serum IgE levels remained unchanged. Finally, in a mouse model the impact of oral DHA application on allergen induced dermatitis as well as the underlying local mechanisms were investigated. Thereby, the DHA mediated reduction of clinical skin score was associated with decreased dermal CD8+ T cell numbers, whereas other histological parameters or serum IgE values were not affected. In summary the results indicate that dietary DHA may be effective in prevention of allergic diseases by interference with the IgE switching process and improve the clinical outcome of atopic eczema by its positive impact on local inflammatory processes.

La qualité des lipides des préparations pour nourrissons est primordiale, notamment en termes d’acides gras polyinsaturés (AGPI) comme l’acide arachidonique (ARA) et docosahexaénoïque (DHA). Ces derniers pourraient favoriser le développement neurosensoriel de l’enfant. Nous avons ainsi évalué 4 standards commerciaux contenant des lipides végétaux ou laitiers et supplémentés ou non en ARA/DHA, sur le développement neurosensoriel au travers d’un modèle d’allaitement artificiel « pups in the cups ». En TEP-cs, nous observons que la supplémentation en ARA/DHA permet de normaliser le fonctionnement cérébral.L’exploration des lipides tissulaires indique des différences en DHA particulièrement bas avec l’allaitement en lipides végétaux purs. Nous proposons un algorithme de prédiction du DHA cérébrale et oculaire via les profils en acides gras érythrocytaires. Dans ces tissus un tiers des espèces à DHA sont affectées et corrélées à l’activité cérébrale. Les neuromédiateurs issus de l’AL, ARA, DHA par la voie LOX sont impactés ainsi que la distribution spatiale en DHA en IMS. Les autres données omiques soulignaient l’impact des interactions fond lipidique x ajout DHA/ARA (transcriptomique) ou fond lipidique (métabolomique) sur la régulation du métabolisme cérébral impactant le métabolisme neuronal et le métabolisme cérébral du microbiote probablement via l’axe de signalisation intestin-cerveau. Nous identifions alors un métagénome sensible à l’ajout DHA/ARA corrélé à la fonction cérébrale. Enfin, des modifications épigénétiques (méthylation du génome et miARN) touchant le groupe FC suggèrent potentiellement un impact à long terme. / The quality of lipids in infant formula is essential, especially in terms of polyunsaturated fatty acids (PUFAs) such as arachidonic acid (ARA) and docosahexaenoic acid (DHA). These could promote the neurosensory development of the child. We thus evaluated 4 commercial standards containing plant or dairy lipids and supplemented or not with ARA / DHA, on the neurosensory development through an artificially feeding model "pups in the cups". In PET-cs, we observe that the supplementation in ARA / DHA makes it possible to normalize the cerebral functioning. The exploration of tissue lipids indicates differences in DHA which are particularly low with pure plant lipids intake. We propose an algorithm for predicting cerebral and ocular DHA via erythrocyte fatty acids profiles. In these tissues one-third of the DHA species are affected and correlated with brain activity. The neuromediators resulting from AL, ARA, DHA by the LOX pathway are impacted as well as the spatial distribution of DHA in IMS imaging. Other omics data underlined the impact of lipid background x combination DHA / ARA (transcriptomics) or lipid background (metabolomics) on the regulation of cerebral metabolism impacting neuronal metabolism and brain metabolism of the microbiota probably through the signalling of gut-brain axis. We then identify a metagenome sensitive to the addition of DHA / ARA correlated to brain function. Finally, epigenetic modifications (methylation of the genome and miRNA) affecting the FC group potentially suggest a long-term impact.

A inflamação sistêmica é uma das características que marcam o diagnóstico da caquexia associada ao câncer. Entre as interações tumor-hospedeiro, o tecido adiposo branco contribui à inflamação, uma vez que ele sofre uma reorganização morfológica e lipólise, liberando ácidos graxos livres (AGLs), mediadores lipídicos (LMs) e citocinas pró-inflamatórias, que acentuam a ativação de vias de sinalização pró-inflamatória e o recrutamento de células do sistema imunológico para o tecido. O objetivo deste projeto foi investigar quais fatores inflamatórios sistêmicos estão envolvidos na inflamação do tecido adiposo e qual é a influência desses fatores sobre as enzimas envolvidas no metabolismo dos AGs ou LMs em indivíduos saudáveis (Controle), pacientes com câncer gastrointestinal com peso estável (WSC) e pacientes com câncer e caquexia (CC). Os resultados demonstraram que a resposta inflamatória sistêmica é diferente da resposta encontrada no tecido adiposo. A inflamação sistêmica dos pacientes com câncer e caquexia (CC) foi caracterizada por níveis circulantes mais elevados de ácidos graxos saturados (SFAs), tumor-necrosis-factor-&#945 (TNF-&#945), Interleukin IL-6, IL-8 e proteina Creativa (PCR), enquanto os níveis de ácidos graxos poliinsaturados (PUFAs), especialmente n3-PUFAs, foram menores em CC que nos demais grupos. In vitro e em explantes de tecido adiposo, citocinas pró-inflamatórias e SFAs aumentaram a expressão das quimiocinas IL-8 e CXCL10. E também observamos um aumento na expressão destas quimiocinas na inflamação do tecido adiposo no CC, que era mais profundo no tecido adiposo visceral (VAT) quando comparado ao tecido adiposo subcutâneo (SAT). A inflamação sistêmica foi negativamente associada com a expressão de enzimas sintetizadoras dos PUFAs, embora a expressão gênica e protéica mostraram somente pequenas diferencias entre os grupos. Os efeitos dos fatores inflamatórios sobre as enzimas no tecido adiposo podem ter sido mascarados pela modulação diferenciada dos diversos tipos celulares constituintes desse tecido. Experimentos in vitro mostraram que a expressão de enzimas que modificam os AGs, tais como as dessaturases e elongases em adipócitos e macrófagos, foram reguladas em direções opostas por TNF-&#945, IL-6, LPS e palmitato. Mesmo os pacientes CC demonstrando uma maior concentração plasmática da Resolvina D1, que é um mediador lipídico de resolução da inflamação, ainda assim, a inflamação sistêmica é maior nesses pacientes, e os resultados indicam que as citoquinas inflamatórias interferem com as vias de síntese das LMs da resolução. Concluímos que, os dados revelaram um crosstalk inter-tecidual e intercelular complexo mediado por citocinas pró-inflamatórias e compostos lipídicos que aumentam a inflamação na caquexia associada ao câncer por mecanismos autoregulação. / Systemic inflammation is a hallmark of cancer cachexia. Among tumor-host interactions, the white adipose tissue (WAT) is an important contributor to inflammation as it suffers morphological reorganization and lipolysis, releasing free fatty acids (FA), bioactive lipid mediators (LM) and pro-inflammatory cytokines, which accentuate the activation of proinflammatory signaling pathways and the recruitment of immune cells to the tissue. This project aimed to investigate which inflammatory factors are involved in the local adipose tissue inflammation and what is the influence of such factors upon enzymes involved in FA or LM metabolism in healthy individuals (Control), weight stable gastro-intestinal cancer patients (WSC) and cachectic cancer patients (CC). The results demonstrated that the inflammatory signature of systemic inflammation is different from local adipose tissue inflammation. The systemic inflammation of the cachectic cancer patients was characterized by higher levels of circulating saturated fatty acids (SFA), tumor-necrosisfactor- &#945 (TNF- &#945), interleukins IL-6, IL-8 and CRP while levels of polyunsaturated fatty acids (PUFAs), especially n3-PUFAs, were lower in CC than in the other groups. In vitro and in adipose tissue explants, pro-inflammatory cytokines and SFAs were shown to increase the chemokines IL-8 and CXCL10 that were found to be augmented in adipose tissue inflammation in CC which was more profound in the visceral adipose tissue (VAT) than in subcutaneous adipose tissue (SAT). Systemic inflammation was negatively associated with the expression of PUFA synthesizing enzymes, though gene and protein expression did hardly differ between groups. The effects of inflammatory factors on enzymes in the whole tissue could have been masked by differentiated modulation of the diverse cell types in the same tissue. In vitro experiments showed that the expression of FA-modifying enzymes such as desaturases and elongases in adipocytes and macrophages was regulated into opposing directions by TNF- &#945, IL-6, LPS or palmitate. The higher plasma concentration of the pro-resolving LM resolvin D1 in CC cannot compensate the overall inflammatory status and the results indicate that inflammatory cytokines interfere with synthesis pathways of pro-resolving LM. In summary, the data revealed a complex inter-tissue and inter-cellular crosstalk mediated by pro-inflammatory cytokines and lipid compounds enhancing inflammation in cancer cachexia by feedforward mechanisms.

A few abnormal cells found in a small piece of prostate tissue are most consequential for a man’s future. The prevalence of prostate cancer (PCa) is increasing globally. The main instigating factor for this cancer is not yet known, but it appears to be the consequence of many variables such as an increasingly older population, more frequent PSA-testing, and factors involving lifestyle. Prostate cancer screening, as an equivalent for breast cancer screening, has been suggested but unfortunately there are no accurate diagnostic tools available for this type of screening. The reason for this is simply that the prostate is one of the most difficult organs to diagnose and, consequently, PCa screening would generate far too many false-positive and false-negative results.  The prostate is not easily accessible as it is deeply-seated in the male pelvic area, wrapped around the urethra and surrounded by sensitive vital organs.  Furthermore, PCa is frequently multi-focal, and the cancer cells have a tendency of assimilating among normal cells and, thus, do not always form solid lumps.  Therefore, prostate tumors are often not felt by digital rectal examination (DRE) or identified by imaging.  The PSA-test is not reliable as it is more prostate-specific than cancer-specific.  Due to increasing prostate awareness, more early-stage and locally confined PCa are being detected. This is saving lives, although there is a high risk of over treatment and unnecessary side-effects.  The increased detection of PCa requires sophisticated diagnostic methods and highly skilled clinicians who can discern between indolent and aggressive cancers.  The current “gold-standard” for PCa diagnosis is biopsy grading by pathologists using the Gleason score system, which is a difficult task.  Therefore, innovative methods to improve the precision of prostate diagnosis, by increased biopsy sensitivity and tumor localization, are of essence. In light of these difficulties, the metabolomic approach using 1D and 2D high-resolution magic angle spinning (HRMAS) NMR spectroscopy combined with histopathology on intact prostatectomy specimens was evaluated in this research project.  The non-destructive nature of HRMAS NMR enables spectroscopic analysis of intact tissue samples with consecutive histological examinations under light microscope. Metabolomics aids in the unraveling and the discovery of organ-specific endogenous metabolites that have the potential to be reliable indicators of organ function and viability, extrinsic and intrinsic perturbations, as well as valuable markers for treatment response. The results may, therefore, be applied clinically to characterize an organ by utilizing biomarkers that have the capacity to distinguish between disease and health. The aim was to characterize the human and the rat prostate in terms of its intermediary metabolism, which I show here to differ between species and anatomical regions.  Furthermore, the aim is to seek the verification of HRMAS NMR derived metabolites which are known to be a part of the prostate metabolome such as, citrate, choline, and the polyamines which were performed, but also the identification of metabolites not previously identified as part of the local prostate metabolism, such as Omega-6, which was detected in tumors.  The extended aim was to elucidate novel bio-markers with clinical potential. In this study, the common phyto-nutrient, inositol, which appears to possess protective properties, was identified as being a potentially important PCa bio-marker for the distinction between the more indolent Gleason score 6 and the more aggressive Gleason score 7 in non-malignant prostate tissues with tumors elsewhere in the organ. Further studies in this area of PCa research are therefore warranted.

Malgré les avancées thérapeutiques récentes, un nombre significatif de patients décèdent de leur cancer suite au développement de métastases. Les molécules conventionnelles de chimiothérapie ont un rôle pivot à ce stade, mais leur efficacité qui est dépendante de la dose, est limitée par leur toxicité aux tissus non tumoraux, par manque de spécificité. L’enjeu est de développer des approches spécifiques qui augmentent la toxicité de ces molécules pour les tumeurs sans affecter les autres tissus. L’acide docosahexaènoïque (DHA) est capable d’augmenter la sensibilité des tumeurs à la chimiothérapie de façon spécifique sans sensibiliser les tissus non tumoraux. Ce travail de thèse présente 1) une synthèse des études existantes supportant cette hypothèse, 2) l’évaluation de la faisabilité d’une supplémentation orale en DHA au cours de la chimiothérapie chez des patientes présentant un cancer du sein métastasé, 3) l’exploration des mécanismes impliqués dans la sensibilisation spécifique des tumeurs, 4) l’effet du DHA sur la perte de poids en cours de traitement, et 5) le profil d’incorporation du DHA au niveau des tumeurs et la relation avec son taux plasmatique. / Despite great therapeutic improvements, a significant proportion of patients still die from cancer, mainly because of the development of metastases. At this stage, treatments rely heavily on conventional chemotherapy, but their efficacy, which is dose-dependent, is limited by its toxicity to non-tumor tissues, as a result of their poor selectivity. The challenge is to develop approaches aimed at increasing chemotherapy cytotoxicity to tumor tissue while not affecting non-tumor tissues. Docosahexaenoic acid (DHA), a lipid of marine origin, has the potential to selectively sensitize tumor tissue to anticancer drugs without sensitizing nontumor tissues. This manuscript reports 1) a review of existing studies supporting this hypothesis, 2) an assessement of the feasiblility of supplementing breast cancer patients with DHA during an anthracycline-based chemotherapy for metastases, 3) an exploration of the mechanisms involved in the selective sensitization of tumors by DHA, 4) the effect of DHA on weight loss related to chemotherapy, and 5) the profile of DHA incorporation into tumor tissue and the relation with its level in plasma.