Einleitung: Die Transitphase ist bei Milchkühen mit gravierenden Veränderungen im Metabolismus verbunden. Darüber hinaus ist sie mit Umstellungen im Haltungsumfeld verbunden, welche die Adaptationsmechanismen der Kühe beanspruchen. Aufgrund dieser Herausforderungen erhöht sich das Risiko für produktionsbedingte Erkran¬kungen. Die Leber übernimmt aufgrund ihrer vielfältigen Funktionen, u.a. als Hauptorgan des Stoffwechsels, eine besondere Rolle. Nach wie vor sind die zu Grunde liegenden pathophysiologischen Abläufe nicht vollständig ergründet. Neue Analysemethoden, wie die Metabolomics Technologie, ermöglichen tiefere Einsichten in Stoffwechselvorgänge und Ursachen für individuelle Unterschiede der Erkran¬kungswahrscheinlichkeit sowie die Wirkweise von metaphylaktischen bzw. therapeu¬tischen Maßnahmen. Die positiven Wirkungen von Butafosfan und Cyanocobalamin (BCC) hinsichtlich Ketoseprävention und Leistung wurden bereits aufgezeigt. Die zugrundeliegenden Wirkmechanismen sind jedoch unzureichend erforscht. Zur Erlan¬gung evidenzbasierter Erkenntnisse sind auch in der Buiatrik aktuell noch prospektive, randomisierte, verblindete Tierversuche nötig. Sie sind mit Risiken für die Gesundheit und Produktivitätsverlusten bei den Versuchstieren verbunden. Genaue Angaben zu Qualität und Quantität derartiger Ausfälle für Feldversuche gibt es allerdings kaum. Ziele der Untersuchung: Die Dissertationsschrift hatte das Ziel, das Metabolom im Lebergewebe in Bezug zu klinischen und laborchemischen Merkmalen sowie zu Gesundheits- und Produktionsdaten von Milchkühen während der Transitphase zu charakterisieren. Weiterhin sollte der Effekt der metaphylaktischen Behandlung mit BCC auf das Leber-Metabolom evaluiert sowie die Auswirkungen eines intensiven Versuchsprotokolls auf die Gesundheit und Leistungskennzahlen erfasst werden. Tiere, Material und Methoden: Aus einer Herde mit 660 Deutsch-Holstein-Kühen wurden im ersten Teil der Studie 87 gesunde Kühe einbezogen (Publikation 1). Diese wurden ab Tag 14 ante partum (Tag –14) bis Tag 49 post partum (Tag 49) täglich klinisch überwacht. An den Tagen –7 bis –5 und 1 bis 3 wurden die Kühe intravenös mit BCC in zwei Dosierungen (5 / 10 ml Catosal® pro 100 kg Körpermasse; 0,05 mg / ml Cyanocobalamin, 100 mg / ml Butafosfan; Bayer Animal Health GmbH, Leverkusen, Deutschland) bzw. Natriumchlorid-haltigem Placebo behandelt. Die statistische Analyse wurde mit multivariaten [partielle Diskriminanzanalyse der kleinsten Quadrate (PLS-DA)] und univariaten Methoden (lineares gemischtes Modell) durchgeführt. Den Kühen wurden mehrfach Blut- (n = 8), Lebergewebe- (n = 4) und Harnproben (n = 13) entnommen. Zu sieben Zeitpunkten wurde die Leber sonographisch untersucht bzw. die Rückenfettdicke vermessen. Sämtliche Gesundheits-, Leistungs- und Reproduk¬tionsmerkmale wurden digital im Programm Herde® (dsp-Agrosoft GmbH, Ketzin / Havel, Deutschland) dokumentiert. Im zweiten Teil der Studie wurden gesundheitliche Schäden, die potenziell mit den Versuchsmaßnahmen im Zusammenhang standen, erfasst und ausgewertet. Dazu wurden 206 Kühe als nicht manipulierte Vergleichstiere für die Gesundheits- und Produktionsmerkmale hinzugezogen (Publikation 2). Die Milchleistung Ergebnisse: Insgesamt bestätigen die Ergebnisse stoffwechselassoziierte Veränder¬ungen im Metabolom während der Transitphase. Die Kühe wurden anhand derartiger Unterschiede des Leber-Metaboloms in drei Metabotypen (A – C) eingeteilt. Die Be¬handlung mit BCC wirkte sich unterschiedlich auf diese Stoffwechselprofile aus. Die Veränderungen im Leber-Metabolom deuten auf die Steigerung der Beta-Oxidation und den effizienteren Triacylglycerol-Export in der Leber hin. Dieser Effekt war auf phänotypische Merkmale beim Einsatz der höheren Dosis im Vergleich zur niedrigeren Dosis signifikant. Der deutlichste Behandlungseffekt wurde beim Metabotyp B am Tag 7 beobachtet. Für den zweiten Teil der Dissertation wurden die Folgen des durch¬geführten Tierversuches näher untersucht. Die meisten Schäden waren mit der Entnahme der Leberbiopsien assoziiert (Biopsiestelle: diffuse Entzündung der Bauch¬wand, erhöhte Echogenität im Lebergewebe). Die Milchleistung der Versuchskühe lag unter und die somatische Zellzahl über der der nicht-manipulierten Vergleichskühe (statistisch signifikant, P=0.001). Bei den Versuchskühen wurde eine geringere Totgeburtenrate erreicht. Der Versuch hatte keinen Einfluss auf die Überlebensdauer der Versuchstiere. Schlussfolgerungen: Es konnten Kühe identifiziert werden, die aufgrund ihres Metabotyps während der Transitphase einem besonderen Risiko ausgesetzt waren (Metabotyp B). Für diese Risikotiere konnte eine dosisabhängige, positive Wirkung der Behandlung mit BCC auf den Gesundheitsstatus erzielt werden. Weiterhin zeigte sich, dass ein intensives Studienprotokoll bei Milchkühen im Herkunftsbetrieb unterschiedliche Auswirkungen auf Gesundheits- und Produktionskennzahlen haben kann. Diese Analyse trägt zur Abschätzung der Risiken derartiger Verfahren bei und kann somit hilfreich bei der Planung und Durchführung weiterer Studien sein.:1. EINLEITUNG 1 2. LITERATURÜBERSICHT 4 2.1 Die Transitphase bei Milchkühen 4 2.1.1 Physiologie der Adaptation 4 2.1.2 Das Lipomobilisationssyndrom 7 2.2 Untersuchungen des Metaboloms zur Ergründung pathophysiologischer Reaktionsnetzwerke in der Transitphase 10 2.2.1 „Omics“-Methoden 10 2.2.2 Metabolomics 11 2.2.3 Technische Durchführung 11 2.2.4 Anwendung in der Veterinärmedizin 12 2.3 Prinzipien pro- und metaphylaktischer Maßnahmen in der Transitphase 14 2.3.1 Zufuhr von Energieträgern 19 2.3.2 Beeinflussung des Pansenmikrobioms 19 2.3.3 Beeinflussung des Leberstoffwechsels 220 2.4 Auswirkungen von Studienprotokollen auf den Organismus von Versuchstieren 23 2.4.1 Evidenzbasierte Medizin 23 2.4.2 Evidenzbasierte Veterinärmedizin: Versuche am Tier 24 2.4.3 Belastungen durch Studienprotokolle 25 2.4.4 Beurteilung studienassoziierter Belastungen 26 2.5 Schlussfolgerungen aus der Literaturrecherche und Arbeitshypothesen 27 3. MATERIAL UND METHODEN 29 3.1 Studiendesign 29 3.1.1 Tierversuchsantrag und Präambel 29 3.1.2 Patientenmaterial 29 3.2 Spezielle Untersuchungsgänge der Kühe der Versuchsgruppe 30 3.2.1 Untersuchungen 30 3.2.2 Probenentnahme 30 3.2.3 Probenhandling und Analyse von Blut-, Leber- und Harnproben 32 3.2.4 Sonographische Untersuchung 321 4. PUBLIKATION 1 332 5. PUBLIKATION 2 565 6. DISKUSSION 743 6.1 Präambel 743 6.2 Diskussion der Eignung des Versuchsprotokolls zur Beantwortung der Zielstellung 74 6.3 Angewandtes Therapieschema und Effekte der Butafosfan- und Cyanocobalamintherapie 75 6.3.1 Dosierung, Applikationsart und Behandlungshäufigkeit 754 6.3.2 Diskussion des Therapieschemas im Kontext der neuen EU-Tierarznei¬mittel-Verordnung 78 6.3.3 Mögliche Effekte bei erkrankten Tieren, Ausblick 79 6.4 Diskussion der Auswirkungen des intensiven Versuchsprotokolls auf die Gesundheit und Leistungskennzahlen der Milchkühe während des Versuchs 80 6.4.1. Direkte Schäden an den Tieren durch studienbedingte Manipulationen 80 6.4.2. Auswirkung auf die Gesamtgesundheit der Tiere 821 6.4.3. Einfluss auf das Abgangsgeschehen bei den Tieren 843 6.5 Bedeutung der Befunddokumentation in Protokollen und Verwendung von Scoring-Sheets zur Datenerhebung sowie der Wert einer Überwachung der Versuchstiere durch erfahrene Tierärzte 84 7 ZUSAMMENFASSUNG 87 8 SUMMARY 89 9 LITERATURVERZEICHNIS 91 10 ANHANG 118 11 DANKSAGUNG 151 / Introduction: The transition period in dairy cows is characterised by significant metabolic changes. Management changes commonly occur at the same time, causing stress and impairing the adaptation mechanisms of the cows, which increases the risk of production diseases. The liver is the main metabolic organ and therefore plays a crucial role in the adaptive changes that occur in the transition period. However, several basic pathophysiological mechanisms that occur in the transition period are not completely understood. Novel methods of analysis such as metabolomics technology improve the interpretation of metabolic processes and the individual differences in the probability of illness among cows. Furthermore, these techniques allow a better understanding of the modes of action of metaphylactic and therapeutic measures. The beneficial effects of Butafosfan and cyanocobalamin (BCC) with respect to ketosis prevention and milk yield have been established but the underlying mechanisms of action are not completely understood. Prospective, randomised and blinded animal testing remains a cornerstone of the search for evidence-based knowledge in many medical fields including buiatrics. However, animal testing is associated with an increased risk of health problems and loss of production, and the magnitude and types of risks are not clearly defined. Objectives: The primary goal of this dissertation was to characterise the metabolome of liver tissue in relation to clinical and laboratory variables as well as health and production parameters in transitional dairy cows. A secondary goal was to investigate the effect of metaphylactic treatment with BCC on the metabolome of the liver and the effect of an intensive experimental protocol on the health and production parameters in dairy cows. Animals, Materials and Methods: In the first part of the study (publication 1), 87 healthy cows from a herd of 660 German Holstein cows were used. The cows were monitored clinically every day from day 14 antepartum (day –14) until day 49 postpartum (day 49). On days –7 to –5 and 1 to 3, the cows received either 5 or 10 ml BCC (Catosal® per 100 kg body weight; 0.05 mg/ml cyanocobalamin, 100 mg/ml Buta¬fosfan; Bayer Animal Health GmbH, Leverkusen, Germany) or a placebo of coloured isotonic saline solution administered intravenously. Statistical analysis was performed using multivariate [partial least squares discriminant analysis (PLS-DA)] and univariate methods (linear mixed model). The cows underwent serial blood (n = 8), liver biopsy sample (n = 4) and urine (n = 13) collection and sonographic examination of the liver (n = 7) and measurement of the back fat (n = 7). The herd management software Herde® (dsp-Agrosoft GmbH, Ketzin/ Havel, Germany) was used to document health, fertility and production parameters. The second part of the study (publication 2) dealt with adverse health effects that were potentially associated with the experimental protocol. For this purpose, 206 control cows were used for the comparison of health and production data. Results: Overall, the findings of this study confirm the occurrence of metabolomic changes related to metabolic processes during the transition period. The cows were divided into 3 metabolomic profiles (A to C) based on the changes in the liver metabolome. The effect of BCC on these profiles differed. The changes in the liver metabolome suggested an increase in β-oxidation and more efficient triacylglycerol export in the liver of cows treated with the higher dose of BCC. A comparison of the 2 doses of BCC showed that the higher dose had a significant effect on phenotypic traits. The largest treatment effect was seen in metabolomic profile B on day 7. Most study protocol-associated lesions were related to the liver biopsy procedure and included diffuse inflammation of the abdominal wall at the biopsy site and increased echogenicity of the hepatic tissue. In the experimental cows, the milk yield was lower, the somatic cell count was higher and the stillbirth rate was lower compared with the control cows (statistically significant, P=0.001). The survival rate did not differ between the experimental and control cows. Conclusions: This study allowed the identification of cows with an increased risk of metabolic disease based on their metabolic profile (profile B) during the transition period. A positive and dose-dependent effect of BCC on the health status was seen in those cows. An intensive experimental protocol can have different effects on health and production parameters in dairy cows. This study improves risk assessment for intensive experimental protocols and facilitates the planning and execution of future studies that involve animal testing.:1. EINLEITUNG 1 2. LITERATURÜBERSICHT 4 2.1 Die Transitphase bei Milchkühen 4 2.1.1 Physiologie der Adaptation 4 2.1.2 Das Lipomobilisationssyndrom 7 2.2 Untersuchungen des Metaboloms zur Ergründung pathophysiologischer Reaktionsnetzwerke in der Transitphase 10 2.2.1 „Omics“-Methoden 10 2.2.2 Metabolomics 11 2.2.3 Technische Durchführung 11 2.2.4 Anwendung in der Veterinärmedizin 12 2.3 Prinzipien pro- und metaphylaktischer Maßnahmen in der Transitphase 14 2.3.1 Zufuhr von Energieträgern 19 2.3.2 Beeinflussung des Pansenmikrobioms 19 2.3.3 Beeinflussung des Leberstoffwechsels 220 2.4 Auswirkungen von Studienprotokollen auf den Organismus von Versuchstieren 23 2.4.1 Evidenzbasierte Medizin 23 2.4.2 Evidenzbasierte Veterinärmedizin: Versuche am Tier 24 2.4.3 Belastungen durch Studienprotokolle 25 2.4.4 Beurteilung studienassoziierter Belastungen 26 2.5 Schlussfolgerungen aus der Literaturrecherche und Arbeitshypothesen 27 3. MATERIAL UND METHODEN 29 3.1 Studiendesign 29 3.1.1 Tierversuchsantrag und Präambel 29 3.1.2 Patientenmaterial 29 3.2 Spezielle Untersuchungsgänge der Kühe der Versuchsgruppe 30 3.2.1 Untersuchungen 30 3.2.2 Probenentnahme 30 3.2.3 Probenhandling und Analyse von Blut-, Leber- und Harnproben 32 3.2.4 Sonographische Untersuchung 321 4. PUBLIKATION 1 332 5. PUBLIKATION 2 565 6. DISKUSSION 743 6.1 Präambel 743 6.2 Diskussion der Eignung des Versuchsprotokolls zur Beantwortung der Zielstellung 74 6.3 Angewandtes Therapieschema und Effekte der Butafosfan- und Cyanocobalamintherapie 75 6.3.1 Dosierung, Applikationsart und Behandlungshäufigkeit 754 6.3.2 Diskussion des Therapieschemas im Kontext der neuen EU-Tierarznei¬mittel-Verordnung 78 6.3.3 Mögliche Effekte bei erkrankten Tieren, Ausblick 79 6.4 Diskussion der Auswirkungen des intensiven Versuchsprotokolls auf die Gesundheit und Leistungskennzahlen der Milchkühe während des Versuchs 80 6.4.1. Direkte Schäden an den Tieren durch studienbedingte Manipulationen 80 6.4.2. Auswirkung auf die Gesamtgesundheit der Tiere 821 6.4.3. Einfluss auf das Abgangsgeschehen bei den Tieren 843 6.5 Bedeutung der Befunddokumentation in Protokollen und Verwendung von Scoring-Sheets zur Datenerhebung sowie der Wert einer Überwachung der Versuchstiere durch erfahrene Tierärzte 84 7 ZUSAMMENFASSUNG 87 8 SUMMARY 89 9 LITERATURVERZEICHNIS 91 10 ANHANG 118 11 DANKSAGUNG 151

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Abstract Metabolic syndrome is a constellation of metabolic abnormalities predisposing to cardiovascular diseases (CVD), type 2 diabetes, and increased mortality. Due to the high prevalence and severe co-morbidities, metabolic syndrome constitutes a major burden for both public health and the global economy. Improved understanding of the detailed molecular mechanisms could provide novel strategies for the treatment and preferably prevention of the metabolic syndrome-related health issues. Recent advancements in ‘omics’ technologies have facilitated the development of novel tools to examine the links between genetic variation and human health. The new techniques allow determination of millions of genotypes or quantification of hundreds of metabolic measures from a single blood sample. In this thesis, genomics and metabolomics approaches are coupled to improve our understanding of the metabolic syndrome-related health issues. More precisely, my projects evaluate the metabolic effects of two lipid-lowering therapies and non-alcoholic fatty liver, as well as assess genetic determinants of chronic inflammation. The present results indicate generally consistent metabolic effects of statins and proprotein convertase subtilisin/kexin type 9 (PCSK9) genetic inhibition. The subtle discrepancies observed could potentially contribute to differences in the efficacy to lower CVD risk between statins and PCSK9 inhibitors. The dissimilar metabolic effects of the four genetic variants that increase the risk of non-alcoholic fatty liver disease (NAFLD) highlight the heterogeneity of the molecular mechanisms involved in NAFLD pathogenesis. The results further suggest that fatty liver by itself might not promote unfavourable metabolic aberrations associated with fatty liver on a population level. The newly identified loci associating with inflammatory phenotypes elucidate the genetic mechanisms contributing to the inflammatory load. In particular, the present results suggest the important role of the locus determining the ABO blood types in the regulation of the soluble adhesion molecule levels. To conclude, this thesis successfully complements the knowledge of the molecular mechanisms involved in metabolic syndrome-related traits and provides examples of how to couple omics technologies in the study of complex traits or in the evaluation of drug effects. / Tiivistelmä Metabolinen oireyhtymä on tila, jossa useiden aineenvaihdunnallisten riskitekijöiden kasautuminen suurentaa riskiä sairastua tyypin 2 diabetekseen ja sydän- ja verisuonitauteihin sekä lisää kokonaiskuolleisuutta. Vakavista liitännäissairauksista ja suuresta esiintyvyydestä johtuen metabolinen oireyhtymä kuormittaa merkittävästi sekä terveydenhuoltoa että kansantaloutta. Jotta metabolisen oireyhtymän hoitoon ja ennaltaehkäisyyn voitaisiin kehittää uusia keinoja, on tärkeää ymmärtää paremmin oireyhtymän syntyyn vaikuttavat täsmälliset molekyylimekanismit. Niin sanottujen ’omiikka-tekniikoiden’ viimeaikainen kehitys tarjoaa uusia mahdollisuuksia tutkia geenimuutosten vaikutuksia terveyteen. Uusien tekniikoiden avulla voidaan määrittää miljoonia genotyyppejä tai satoja aineenvaihdunnan merkkiaineita yhdestä verinäytteestä. Tässä väitöskirjatyössä yhdistetään genomiikan ja metabolomiikan menetelmiä metaboliseen oireyhtymään liittyvien terveysongelmien tutkimiseksi. Väitöskirjani osatöissä arvioin kahden lipidilääkkeen sekä ei-alkoholiperäisen rasvamaksan aineenvaihdunnallisia vaikutuksia sekä pyrin tunnistamaan krooniseen tulehdukseen vaikuttavia geneettisiä tekijöitä. Tulosten mukaan statiinien ja PCSK9:n (engl. proprotein convertase subtilisin/kexin type 9) geneettisen eston aineenvaihduntavaikutukset ovat hyvin samankaltaiset. Kuitenkin havaitut pienet poikkeavuudet tietyissä merkkiaineissa voivat vaikuttaa eroavaisuuksiin siinä, kuinka tehokkaasti lääkeaineet alentavat sydäntautiriskiä. Suuret erot rasvamaksan riskiä lisäävien geenimuutosten vaikutuksissa aineenvaihduntaan korostavat rasvamaksaan liittyvien molekyylimekanismien monimuotoisuutta. Tulosten perusteella vaikuttaa siltä, että rasvan kertyminen maksaan ei luultavasti itsessään aiheuta suuria muutoksia verenkierron aineenvaihduntatuotteiden pitoisuuksiin. Tulehdusmerkkiaineisiin assosioituvat uudet geenialueet täydentävät tulehduksen molekyylimekanismeihin liittyvää tietoa. Tulokset korostavat ABO-veriryhmän määräävän geenin vaikutusta liukoisten adheesiomolekyylien pitoisuuksiin. Kaiken kaikkiaan väitöskirjan osatyöt tuovat uutta tietoa metaboliseen oireyhtymään liittyvien terveysongelmien molekyylimekanismeihin. Projektit havainnollistavat, miten omiikka-tekniikoita voidaan hyödyntää monitekijäisten fenotyyppien tutkimuksessa sekä lääkeaineiden aineenvaihduntavaikutusten arvioinnissa.

Introdução: A quantidade e o tipo de gordura alimentar exercem importante influência no desenvolvimento de doença cardiovascular (DCV) e podem contribuir para o desenvolvimento de esteatose hepática. Os ácidos graxos saturados e trans são consensualmente apontados como aterogênicos; já os poli-insaturados parecem exercer ação antiaterogênica. Com relação a esteatose hepática, sabe-se que os ácidos graxos saturados estão associados com o seu desenvolvimento; porém, a ação dos ácidos graxos trans na gênese e no desenvolvimento de esteatose hepática não está totalmente elucidada. Neste estudo, avaliou-se o efeito do consumo de dietas enriquecidas com ácidos graxos saturados (SAT), poli-insaturados (POLI) ou trans (TRANS) sobre componentes envolvidos na indução e na progressão da placa aterosclerótica, bem como sobre o desenvolvimento da doença hepática gordurosa não alcoólica. Métodos: Camundongos com ablação gênica para o receptor de LDL (LDLr-KO) foram alimentados com dietas hiperlipídicas (40% do valor calórico total sob a forma de gordura), enriquecidas com ácidos graxos SAT, POLI ou TRANS por 16 semanas e ao final submetidos a: 1) análises plasmáticas: colesterol total (CT), triglicérides (TG), insulina, glicose, aspartato aminotransferase (AST) e alanina aminotransferase (ALT), interleucina-6 (IL-6), fator de necrose tumoral-? (TNF-alfa) e perfil de lipoproteínas; 2) determinação da lesão aterosclerótica: área de lesão (Oil Red-O), conteúdo de ATP-binding cassette transporter A1 (ABCA1) e infiltrado de macrófagos (imuno-histoquímica), colocalização de ABCA1 e macrófagos (microscopia confocal) e conteúdo de colágeno (Picrosirius-Red) na raiz aórtica; 3) conteúdo de CT, colesterol éster (CE) e colesterol livre (CL) na aorta total; 4) macrófagos peritoneais foram tratados com lipopolissacarídeo (LPS), e IL-6, TNF-alfa e interleucina-10 (IL-10) medidas no meio de cultura; 5) no fígado: grau da doença hepática gordurosa não alcoólica, concentração de CT e TG e expressão de RNA mensageiro (mRNA) de PPAR-gama, PPAR-gama, SREBP-1c, MTP, CPT-1 e ABCA1 por RT-qPCR; 6) determinação do conteúdo de tecido adiposo visceral e subcutâneo na carcaça. Resultados: O consumo de dieta não diferiu entre os grupos; comparado à dieta POLI, TRANS induziu menor ganho de peso refletido por menor conteúdo de tecido adiposo. TRANS induziu hepatomegalia, desenvolvimento de esteato-hepatite não alcoólica (NASH) e piora da sensibilidade insulínica (evidenciada pelo índice HOMAIR). As concentrações de AST e ALT não diferiram entre os grupos. A dieta TRANS elevou a expressão de mRNA de genes relacionados à lipogênese hepática (PPAR-gama e SREBP-1c) comparada à SAT e POLI e reduziu a expressão de MTP comparada à dieta POLI. Não houve diferença entre os grupos com relação à expressão de genes envolvidos na oxidação hepática de lípides (PPAR-gama e CPT-1). As concentrações plasmáticas de CT e TG foram maiores no grupo TRANS comparado a SAT e POLI. POLI apresentou menor área de lesão, infiltrado de macrófagos e conteúdo de ABCA1 comparados a SAT e TRANS. Macrófagos e ABCA1 não se colocalizaram na área de lesão. O conteúdo de CT na parede arterial foi menor no grupo POLI comparado a TRANS; CL foi menor no grupo POLI comparado a SAT e TRANS; CE não diferiu entre os grupos. Comparado a POLI, SAT e TRANS apresentaram maior conteúdo de colágeno e núcleos necróticos na placa aterosclerótica. A concentração plasmática de IL-6 não diferiu entre os grupos; já a concentração de TNF-alfa foi maior nos grupos POLI e TRANS em comparação a SAT. Em relação à resposta inflamatória de macrófagos ao LPS, POLI e TRANS apresentaram maiores concentrações de IL-6 e TNF-alfa comparadas a SAT. POLI apresentou menores concentrações de IL-10 em comparação aos demais grupos. A expressão hepática de ABCA1 não diferiu entre os grupos. Conclusão: O consumo de dieta TRANS induziu perfil lipídico proaterogênico, hipercolesterolemia, hipertrigliceridemia, hiperglicemia e severo desenvolvimento de aterosclerose, além de hepatomegalia, maior acúmulo hepático de lípides e desenvolvimento de NASH. Por outro lado, POLI preveniu o desenvolvimento de aterosclerose, independentemente de sua ação inflamatória. / Introduction: The amount and type of dietary fat play important roles on the development of cardiovascular disease (CVD) and on the development of hepatic steatosis. Saturated (SAT) and trans (TRANS) fatty acids are known as pro-atherogenic, while the polyunsaturated (POLY) fats seem to exert an antiatherogenic action. Regarding hepatic steatosis, it is known that SAT are associated with its development, however, the role of TRANS in the genesis and development of hepatic steatosis is not fully undestood. This study evaluated the effect of the intake of diets enriched with SAT, POLY or TRANS on the parameters involved in the progression of the atherosclerotic plaque and also on the development of the nonalcoholic fatty liver disease (NAFLD). Methods: LDL receptor knock-out (LDLR-KO) male mice were fed for 16 weeks a high fat diet (40% of calories as fat) enriched with SAT, POLY or TRANS, for 16 weeks. The following parameters were mesured: 1) plasma: total cholesterol (TC), triglyceride (TG), insulin, glucose, aspartate aminotransferase (AST) and alanine amino transferase (ALT), interleukin-6 (IL-6), tumor necrosis factor- ? (TNF-?) and lipoprotein profile; 2) atherosclerotic lesion - lesion area (Oil Red-O), ATP-binding cassette transporter A1 (ABCA1) content and macrophage infiltration (immunohistochemistry), co-localization of ABCA1 and macrophages (confocal microscopy) and collagen content (Picrosirius-Red); 3) TC, cholesteryl ester (CE) and free cholesterol (FC) content of the total aorta; 4) interleukin-6 and 10 (IL-6 and IL-10) and TNF-alfa in the culture medium of peritoneal macrophages after treatment with lipopolysaccharide (LPS); 5) liver: degree of fat liver disease, concentration of TC and TG and mRNA expression (RT-qPCR) of PPAR-gama, PPAR-gama, SREBP-1c, MTP, ABCA1 and CPT-1; 6) visceral and subcutaneous adipose tissue contents in the carcass of the animals. Results: Food intake did not differ amongst the groups, however, compared to POLY, TRANS induced less weight gain, due to lower adipose tissue content. TRANS induced hepatomegaly, nonalcoholic steatohepatitis (NASH) and worsening of insulin sensitivity, as evidenced by the index HOMAIR. The concentrations of ALT and AST did not differ among groups. TRANS increased the mRNA expression of the hepatic lipogenic genes (PPAR-gama and SREBP-1c) compared to the SAT and POLY and reduced the mRNA expression of MTP compared to POLI. There was no difference among the groups regarding the mRNA expression of genes involved in hepatic lipid oxidation (PPAR-gama and CPT-1). Plasma concentrations of TC and TG were higher in TRANS compared to SAT and POLY. POLY showed lower arterial lesion area, macrophage infiltration and ABCA1 content compared to SAT and TRANS. ABCA1 and macrophages did not colocalize in the lesion area. The TC content in the arterial wall was lower on POLY compared to TRANS; FC was lower on POLY compared to SAT and TRANS; CE did not differ among groups. Compared to POLY, SAT and TRANS showed higher collagen content and necrotic core in atherosclerotic plaques. The plasma concentration of IL-6 did not differ among groups, however, TNF-alfa plasma concentration was higher in POLY and TRANS compared to SAT. Regarding the macrophage inflammatory response to LPS, POLY and TRANS showed higher concentrations of IL-6 and TNF-alfa compared to SAT. Moreover, POLY had the lowest concentration of the anti-inflammatory cytokine IL-10. The hepatic expression of ABCA1 did not differ amongst the groups. Conclusion: TRANS induced pro-atherogenic lipid profile, hypercholesterolemia, hypertriglyceridemia, hyperglycemia, and severe atherosclerosis, and in addition, elicted hepatomegaly, increased hepatic lipid accumulation and NASH. On the other hand, POLY prevented the development of atherosclerosis, independently of their pro-inflammatory activity.

Este estudo teve, por finalidade, estabelecer o valor da ressonância magnética em 30 doadores potenciais do lobo direito do fígado, na determinação dos seguintes fatores: esteatose hepática; anatomia biliar; anatomias arterial hepática, venosas portal e hepática, e volume hepático lobar, comparando-os, respectivamente, com os achados anatomopatológicos da biópsia hepática, da colangiografia intraoperatória, da angiografia digital e/ou com os achados cirúrgicos, e com o peso real do enxerto. A RM subestimou a infiltração gordurosa hepática; permitiu identificar a anatomia biliar, com concordância em 83% dos casos; apresentou 100% de concordância na avaliação das anatomias arterial e venosas portal e hepática, e superestimou, em pequeno grau, o volume hepático lobar / The purpose of this study was to establish the value of the magnetic resonance in 30 potential donors for right lobe living liver transplantation. The main goal was to determine the following factors: steatosis; biliar anatomy; hepatic arterial anatomy; portal and hepatic venous anatomy, and lobar liver volume, comparing them to liver biopsy results, to intraoperative colangiography, to digital angiography and/or surgical findings, and to the real graft weight, respectively. The MR has underestimated liver steatosis; it has identified biliar anatomy with 83% of agreement; it has had 100% of agreement in the evaluation of arterial and portal and hepatic venous anatomy, and it has overestimated with small degree the lobar liver volume

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O objetivo do presente estudo foi avaliar se o Bezafibrato, um agonista PAN-PPAR, é capaz de aliviar a doença não alcoólica do fígado gorduroso (NAFLD) na prole de machos de mães C57BL/6 obesas. Fêmeas virgens foram alimentadas com uma dieta HL (hiperlipídica, 49% de lipídios) ou uma dieta C (controle, 10% de lipídios) por oito semanas antes do acasalamento e durante os períodos de gestação e lactação. A prole de machos foi subdividida em quatro grupos: C (dieta controle para as mães e filhotes); C/BZ (dieta controle para as mães e filhotes com tratamento com Bezafibrato[100mg/Kg]); HL (dieta HL para as mães e dieta controle para os filhotes); e HL/BZ (dieta HL para as mães e dieta controle para os filhotes com tratamento com Bezafibrato [100mg/Kg]). O tratamento com Bezafibrato começou na 12 semana e se manteve por três semanas. Análise do metabolismo, bioquímica, estereológica e por western-blotting foram realizadas. A dieta HL causou um fenótipo de sobrepeso nas mães e acarretou em uma intolerância oral à glicose com aumento da glicemia de jejum. A prole HL apresentou hiperfagia, ganho de massa corporal, altos níveis de triglicerídeo hepático e plasmático, esteatose hepática e aumento da expressão de proteínas lipogênicas concomitante com diminuição do receptor ativador de proliferação peroxissomal alfa (PPARα), que é responsável pela β-oxidação e aumento do receptor ativador de proliferação peroxissomal gama (PPARγ) e do elemento regulador de esterol ligante da proteína 1 (SREBP-1c) proteínas envolvidas na lipogênese hepática. Por outro lado, o tratamento com o Bezafibrato reverteu o quadro da programação metabólica no fígado, com uma melhora dos parâmetros morfológicos, bioquímicos e moleculares do fígado dos animais, com um aumento da ativação de PPARα em associação a uma diminuição do PPARγ e não alterando a expressão de SREBP-1c. Em conclusão, nós demonstramos que o tratamento com Bezafibrato melhora a NAFLD causada pela obesidade materna. / The aim of the present study was to evaluate whether Bezafibrate , a PAN-PPAR agonist, could attenuate non-alcoholic fatty liver disease (NAFLD) of male offspring from obese C57BL/6 dams. Dams were fed on a HF (high-fat, 49% lipids) diet or SC (standard chow; 10% lipids) diet for 8 weeks before mating and during gestation and lactation periods. Male offspring were subdivided into 4 groups: SC (standard-chow for dams and offspring); SC/BZ [standard-chow for dams and offspring with treatment with BZ (100mg/Kg)]; HF (high-fat diet for dams and standard-chow for offspring); HF/BZ [high-fat diet for dams and standard-show for offspring with treatment with Bezafibrate (100mg/Kg)]. Treatment with Bezafibrate started at 12th week and was maintained for 3 weeks. Metabolic measurements, biochemical analysis, stereological tools and western-blotting were performed. The HF diet yielded an overweight phenotype and an increase in oral glucose tolerance and fasting glucose of dams. The HF offspring presented hyperphagia, body mass gain, high levels of plasmatic and hepatic triglycerides, impairment of glucose metabolism, hepatic steatosis and high expression of lipogenic proteins concomitant to decreased expression of PPARα, which is responsible for β-oxidation. On the other hand, treatment with Bezafibrate reverted hepatic outcomes of metabolic programming, with an improvement of morphological, biochemical and molecular parameters of animals livers, with an increase of PPARα activation in association with a decrease of PPARγ expression and no changes in SREBP-1c expression. In conclusion, we demonstrated that treatment with Bezafibrate improved NAFLD caused by maternal obesity.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O objetivo do presente estudo foi avaliar se o Bezafibrato, um agonista PAN-PPAR, é capaz de aliviar a doença não alcoólica do fígado gorduroso (NAFLD) na prole de machos de mães C57BL/6 obesas. Fêmeas virgens foram alimentadas com uma dieta HL (hiperlipídica, 49% de lipídios) ou uma dieta C (controle, 10% de lipídios) por oito semanas antes do acasalamento e durante os períodos de gestação e lactação. A prole de machos foi subdividida em quatro grupos: C (dieta controle para as mães e filhotes); C/BZ (dieta controle para as mães e filhotes com tratamento com Bezafibrato[100mg/Kg]); HL (dieta HL para as mães e dieta controle para os filhotes); e HL/BZ (dieta HL para as mães e dieta controle para os filhotes com tratamento com Bezafibrato [100mg/Kg]). O tratamento com Bezafibrato começou na 12 semana e se manteve por três semanas. Análise do metabolismo, bioquímica, estereológica e por western-blotting foram realizadas. A dieta HL causou um fenótipo de sobrepeso nas mães e acarretou em uma intolerância oral à glicose com aumento da glicemia de jejum. A prole HL apresentou hiperfagia, ganho de massa corporal, altos níveis de triglicerídeo hepático e plasmático, esteatose hepática e aumento da expressão de proteínas lipogênicas concomitante com diminuição do receptor ativador de proliferação peroxissomal alfa (PPARα), que é responsável pela β-oxidação e aumento do receptor ativador de proliferação peroxissomal gama (PPARγ) e do elemento regulador de esterol ligante da proteína 1 (SREBP-1c) proteínas envolvidas na lipogênese hepática. Por outro lado, o tratamento com o Bezafibrato reverteu o quadro da programação metabólica no fígado, com uma melhora dos parâmetros morfológicos, bioquímicos e moleculares do fígado dos animais, com um aumento da ativação de PPARα em associação a uma diminuição do PPARγ e não alterando a expressão de SREBP-1c. Em conclusão, nós demonstramos que o tratamento com Bezafibrato melhora a NAFLD causada pela obesidade materna. / The aim of the present study was to evaluate whether Bezafibrate , a PAN-PPAR agonist, could attenuate non-alcoholic fatty liver disease (NAFLD) of male offspring from obese C57BL/6 dams. Dams were fed on a HF (high-fat, 49% lipids) diet or SC (standard chow; 10% lipids) diet for 8 weeks before mating and during gestation and lactation periods. Male offspring were subdivided into 4 groups: SC (standard-chow for dams and offspring); SC/BZ [standard-chow for dams and offspring with treatment with BZ (100mg/Kg)]; HF (high-fat diet for dams and standard-chow for offspring); HF/BZ [high-fat diet for dams and standard-show for offspring with treatment with Bezafibrate (100mg/Kg)]. Treatment with Bezafibrate started at 12th week and was maintained for 3 weeks. Metabolic measurements, biochemical analysis, stereological tools and western-blotting were performed. The HF diet yielded an overweight phenotype and an increase in oral glucose tolerance and fasting glucose of dams. The HF offspring presented hyperphagia, body mass gain, high levels of plasmatic and hepatic triglycerides, impairment of glucose metabolism, hepatic steatosis and high expression of lipogenic proteins concomitant to decreased expression of PPARα, which is responsible for β-oxidation. On the other hand, treatment with Bezafibrate reverted hepatic outcomes of metabolic programming, with an improvement of morphological, biochemical and molecular parameters of animals livers, with an increase of PPARα activation in association with a decrease of PPARγ expression and no changes in SREBP-1c expression. In conclusion, we demonstrated that treatment with Bezafibrate improved NAFLD caused by maternal obesity.

This thesis focuses on the effects of n-3 polyunsaturated fatty acids (n-3 PUFA) on development of non-alcoholic fatty liver disease (NAFLD) in experiment, on prevalence of this condition in patients with type 2 diabetes mellitus and metabolic syndrome and also on non-invasive diagnostics. The aim was to study the effect of n-3 PUFA on NAFLD development in an experimental model and based on analysis of a group of patients with type 2 diabetes and metabolic syndrome to assess the prevalence of this condition. Lastly we aimed to evaluate non-invasive diagnostic methods of liver fibrosis and NASH. We demonstrated beneficial effects of n-3 PUFA administration on NAFLD development in a C57/Bl6 mice high fat methionin-cholin defficient dietary model of NAFLD. n-3 PUFA administration led to biochemical improvement, decrease of lipid accumulation in the liver as well as improvement of histology. These effects are determined by complex modulation of lipid metabolism, mainly due to decrease in availability of fatty acids for triglyceride synthesis in the liver, changes of adipokine levels and amelioration of proinflammatory status in the liver. In a group of type 2 diabetics we found NAFLD prevalence of almost 80%, 14% of these patients had also signs of liver fibrosis or cirrhosis. Non-invasive methods...

Este estudo teve, por finalidade, estabelecer o valor da ressonância magnética em 30 doadores potenciais do lobo direito do fígado, na determinação dos seguintes fatores: esteatose hepática; anatomia biliar; anatomias arterial hepática, venosas portal e hepática, e volume hepático lobar, comparando-os, respectivamente, com os achados anatomopatológicos da biópsia hepática, da colangiografia intraoperatória, da angiografia digital e/ou com os achados cirúrgicos, e com o peso real do enxerto. A RM subestimou a infiltração gordurosa hepática; permitiu identificar a anatomia biliar, com concordância em 83% dos casos; apresentou 100% de concordância na avaliação das anatomias arterial e venosas portal e hepática, e superestimou, em pequeno grau, o volume hepático lobar / The purpose of this study was to establish the value of the magnetic resonance in 30 potential donors for right lobe living liver transplantation. The main goal was to determine the following factors: steatosis; biliar anatomy; hepatic arterial anatomy; portal and hepatic venous anatomy, and lobar liver volume, comparing them to liver biopsy results, to intraoperative colangiography, to digital angiography and/or surgical findings, and to the real graft weight, respectively. The MR has underestimated liver steatosis; it has identified biliar anatomy with 83% of agreement; it has had 100% of agreement in the evaluation of arterial and portal and hepatic venous anatomy, and it has overestimated with small degree the lobar liver volume

Introdução: A quantidade e o tipo de gordura alimentar exercem importante influência no desenvolvimento de doença cardiovascular (DCV) e podem contribuir para o desenvolvimento de esteatose hepática. Os ácidos graxos saturados e trans são consensualmente apontados como aterogênicos; já os poli-insaturados parecem exercer ação antiaterogênica. Com relação a esteatose hepática, sabe-se que os ácidos graxos saturados estão associados com o seu desenvolvimento; porém, a ação dos ácidos graxos trans na gênese e no desenvolvimento de esteatose hepática não está totalmente elucidada. Neste estudo, avaliou-se o efeito do consumo de dietas enriquecidas com ácidos graxos saturados (SAT), poli-insaturados (POLI) ou trans (TRANS) sobre componentes envolvidos na indução e na progressão da placa aterosclerótica, bem como sobre o desenvolvimento da doença hepática gordurosa não alcoólica. Métodos: Camundongos com ablação gênica para o receptor de LDL (LDLr-KO) foram alimentados com dietas hiperlipídicas (40% do valor calórico total sob a forma de gordura), enriquecidas com ácidos graxos SAT, POLI ou TRANS por 16 semanas e ao final submetidos a: 1) análises plasmáticas: colesterol total (CT), triglicérides (TG), insulina, glicose, aspartato aminotransferase (AST) e alanina aminotransferase (ALT), interleucina-6 (IL-6), fator de necrose tumoral-? (TNF-alfa) e perfil de lipoproteínas; 2) determinação da lesão aterosclerótica: área de lesão (Oil Red-O), conteúdo de ATP-binding cassette transporter A1 (ABCA1) e infiltrado de macrófagos (imuno-histoquímica), colocalização de ABCA1 e macrófagos (microscopia confocal) e conteúdo de colágeno (Picrosirius-Red) na raiz aórtica; 3) conteúdo de CT, colesterol éster (CE) e colesterol livre (CL) na aorta total; 4) macrófagos peritoneais foram tratados com lipopolissacarídeo (LPS), e IL-6, TNF-alfa e interleucina-10 (IL-10) medidas no meio de cultura; 5) no fígado: grau da doença hepática gordurosa não alcoólica, concentração de CT e TG e expressão de RNA mensageiro (mRNA) de PPAR-gama, PPAR-gama, SREBP-1c, MTP, CPT-1 e ABCA1 por RT-qPCR; 6) determinação do conteúdo de tecido adiposo visceral e subcutâneo na carcaça. Resultados: O consumo de dieta não diferiu entre os grupos; comparado à dieta POLI, TRANS induziu menor ganho de peso refletido por menor conteúdo de tecido adiposo. TRANS induziu hepatomegalia, desenvolvimento de esteato-hepatite não alcoólica (NASH) e piora da sensibilidade insulínica (evidenciada pelo índice HOMAIR). As concentrações de AST e ALT não diferiram entre os grupos. A dieta TRANS elevou a expressão de mRNA de genes relacionados à lipogênese hepática (PPAR-gama e SREBP-1c) comparada à SAT e POLI e reduziu a expressão de MTP comparada à dieta POLI. Não houve diferença entre os grupos com relação à expressão de genes envolvidos na oxidação hepática de lípides (PPAR-gama e CPT-1). As concentrações plasmáticas de CT e TG foram maiores no grupo TRANS comparado a SAT e POLI. POLI apresentou menor área de lesão, infiltrado de macrófagos e conteúdo de ABCA1 comparados a SAT e TRANS. Macrófagos e ABCA1 não se colocalizaram na área de lesão. O conteúdo de CT na parede arterial foi menor no grupo POLI comparado a TRANS; CL foi menor no grupo POLI comparado a SAT e TRANS; CE não diferiu entre os grupos. Comparado a POLI, SAT e TRANS apresentaram maior conteúdo de colágeno e núcleos necróticos na placa aterosclerótica. A concentração plasmática de IL-6 não diferiu entre os grupos; já a concentração de TNF-alfa foi maior nos grupos POLI e TRANS em comparação a SAT. Em relação à resposta inflamatória de macrófagos ao LPS, POLI e TRANS apresentaram maiores concentrações de IL-6 e TNF-alfa comparadas a SAT. POLI apresentou menores concentrações de IL-10 em comparação aos demais grupos. A expressão hepática de ABCA1 não diferiu entre os grupos. Conclusão: O consumo de dieta TRANS induziu perfil lipídico proaterogênico, hipercolesterolemia, hipertrigliceridemia, hiperglicemia e severo desenvolvimento de aterosclerose, além de hepatomegalia, maior acúmulo hepático de lípides e desenvolvimento de NASH. Por outro lado, POLI preveniu o desenvolvimento de aterosclerose, independentemente de sua ação inflamatória. / Introduction: The amount and type of dietary fat play important roles on the development of cardiovascular disease (CVD) and on the development of hepatic steatosis. Saturated (SAT) and trans (TRANS) fatty acids are known as pro-atherogenic, while the polyunsaturated (POLY) fats seem to exert an antiatherogenic action. Regarding hepatic steatosis, it is known that SAT are associated with its development, however, the role of TRANS in the genesis and development of hepatic steatosis is not fully undestood. This study evaluated the effect of the intake of diets enriched with SAT, POLY or TRANS on the parameters involved in the progression of the atherosclerotic plaque and also on the development of the nonalcoholic fatty liver disease (NAFLD). Methods: LDL receptor knock-out (LDLR-KO) male mice were fed for 16 weeks a high fat diet (40% of calories as fat) enriched with SAT, POLY or TRANS, for 16 weeks. The following parameters were mesured: 1) plasma: total cholesterol (TC), triglyceride (TG), insulin, glucose, aspartate aminotransferase (AST) and alanine amino transferase (ALT), interleukin-6 (IL-6), tumor necrosis factor- ? (TNF-?) and lipoprotein profile; 2) atherosclerotic lesion - lesion area (Oil Red-O), ATP-binding cassette transporter A1 (ABCA1) content and macrophage infiltration (immunohistochemistry), co-localization of ABCA1 and macrophages (confocal microscopy) and collagen content (Picrosirius-Red); 3) TC, cholesteryl ester (CE) and free cholesterol (FC) content of the total aorta; 4) interleukin-6 and 10 (IL-6 and IL-10) and TNF-alfa in the culture medium of peritoneal macrophages after treatment with lipopolysaccharide (LPS); 5) liver: degree of fat liver disease, concentration of TC and TG and mRNA expression (RT-qPCR) of PPAR-gama, PPAR-gama, SREBP-1c, MTP, ABCA1 and CPT-1; 6) visceral and subcutaneous adipose tissue contents in the carcass of the animals. Results: Food intake did not differ amongst the groups, however, compared to POLY, TRANS induced less weight gain, due to lower adipose tissue content. TRANS induced hepatomegaly, nonalcoholic steatohepatitis (NASH) and worsening of insulin sensitivity, as evidenced by the index HOMAIR. The concentrations of ALT and AST did not differ among groups. TRANS increased the mRNA expression of the hepatic lipogenic genes (PPAR-gama and SREBP-1c) compared to the SAT and POLY and reduced the mRNA expression of MTP compared to POLI. There was no difference among the groups regarding the mRNA expression of genes involved in hepatic lipid oxidation (PPAR-gama and CPT-1). Plasma concentrations of TC and TG were higher in TRANS compared to SAT and POLY. POLY showed lower arterial lesion area, macrophage infiltration and ABCA1 content compared to SAT and TRANS. ABCA1 and macrophages did not colocalize in the lesion area. The TC content in the arterial wall was lower on POLY compared to TRANS; FC was lower on POLY compared to SAT and TRANS; CE did not differ among groups. Compared to POLY, SAT and TRANS showed higher collagen content and necrotic core in atherosclerotic plaques. The plasma concentration of IL-6 did not differ among groups, however, TNF-alfa plasma concentration was higher in POLY and TRANS compared to SAT. Regarding the macrophage inflammatory response to LPS, POLY and TRANS showed higher concentrations of IL-6 and TNF-alfa compared to SAT. Moreover, POLY had the lowest concentration of the anti-inflammatory cytokine IL-10. The hepatic expression of ABCA1 did not differ amongst the groups. Conclusion: TRANS induced pro-atherogenic lipid profile, hypercholesterolemia, hypertriglyceridemia, hyperglycemia, and severe atherosclerosis, and in addition, elicted hepatomegaly, increased hepatic lipid accumulation and NASH. On the other hand, POLY prevented the development of atherosclerosis, independently of their pro-inflammatory activity.