Dissertations / Theses on the topic 'Metabolom'

From its first use in the field of biochemistry, instrumental analysis offered a variety of invaluable tools for the comprehensive description of biological systems. Multi-selective methods that aim to cover as many endogenous compounds as possible in biological samples use different analytical platforms and include methods like gene expression profile and metabolite profile analysis. The enormous amount of data generated in application of profiling methods needs to be evaluated in a manner appropriate to the question under investigation. The new field of system biology rises to the challenge to develop strategies for collecting, processing, interpreting, and archiving this vast amount of data; to make those data available in form of databases, tools, models, and networks to the scientific community.

On the background of this development a multi-selective method for the determination of phytohormones was developed and optimised, complementing the profile analyses which are already in use (Chapter I). The general feasibility of a simultaneous analysis of plant metabolites and phytohormones in one sample set-up was tested by studies on the analytical robustness of the metabolite profiling protocol. The recovery of plant metabolites proved to be satisfactory robust against variations in the extraction protocol by using common extraction procedures for phytohormones; a joint extraction of metabolites and hormones from plant tissue seems practicable (Chapter II).

Quantification of compounds within the context of profiling methods requires particular scrutiny (Chapter II). In Chapter III, the potential of stable-isotope in vivo labelling as normalisation strategy for profiling data acquired with mass spectrometry is discussed. First promising results were obtained for a reproducible quantification by stable-isotope in vivo labelling, which was applied in metabolomic studies.

In-parallel application of metabolite and phytohormone analysis to seedlings of the model plant Arabidopsis thaliana exposed to sulfate limitation was used to investigate the relationship between the endogenous concentration of signal elements and the ‘metabolic phenotype’ of a plant. An automated evaluation strategy was developed to process data of compounds with diverse physiological nature, such as signal elements, genes and metabolites – all which act in vivo in a conditional, time-resolved manner (Chapter IV). Final data analysis focussed on conditionality of signal-metabolome interactions.
Die instrumentelle Analytik stellt mit ihrem unschätzbaren Methodenreichtum Analysenwerkzeuge zur Verfügung, die seit ihrem Einzug in die Biologie die Aufzeichnung immer komplexerer ‚Momentaufnahmen’ von biologischen Systemen ermöglichen. Konkret hervorzuheben sind dabei vor allem die sogenannten ‚Profilmethoden’. Die Anwendung von Profilmethoden zielt darauf ab, aus einer bestimmten Stoffklasse so viele zugehörige Komponenten wie nur möglich gleichzeitig zu erfassen.

Für die Auswertung derart komplexer Daten müssen nun auch entsprechende Auswertungsmethoden bereit gestellt werden. Das neu entstandene Fachgebiet der Systembiologie erarbeitet deshalb Strategien zum Sammeln, Auswerten und Archivieren komplexer Daten, um dieses gesammelte Wissen in Form von Datenbanken, Modellen und Netzwerken der allgemeinen Nutzung zugänglich zu machen.

Vor diesem Hintergrund wurde den vorhandenen Profilanalysen eine Methode zur Erfassung von Pflanzenhormonen hinzugefügt. Verschiedene Experimente bestätigten die Möglichkeit zur Kopplung von Pflanzenhormon- und Pflanzeninhaltsstoff(=metabolit)-Profilanalyse. In weiteren Untersuchungen wurde das Potential einer innovativen Standardisierungstechnologie für die mengenmässige Erfassung von Pflanzeninhaltsstoffen in biologischen Proben betrachtet (in vivo labelling mit stabilen Isotopen).

Hormon- und Metabolitprofilanalyse wurden dann parallel angewandt, um Wechselwirkungen zwischen der Konzentration von Signalkomponenten und der Ausprägung des Stoffwechsels in Keimlingen der Modellpflanze Arabidopsis thaliana zu untersuchen. Es wurde eine Prozessierungsmethode entwickelt, die es auf einfache Art und Weise erlaubt, Daten oder Komponenten verschiedenen Ursprungs wie Signalelemente, Gene und Metabolite, die in biologischen Systemen zeitlich versetzt aktiv oder verändert erscheinen, im Zusammenhang zu betrachten. Die abschließende Analyse aller Daten richtet sich auf die Abschätzung der Bedingtheit von Signal-Metabolismus Interaktionen.

Introdução - A metabolômica permite determinar padrões de variação dos metabólitos entre indivíduos doentes e não doentes, com ou sem ingestão de um determinado alimento ou dieta. Compreender a relação entre metabólitos e doenças metabólicas pode ajudar a combater obesidade e doenças crônicas. Objetivo - Investigar a associação entre o perfil metabolômico de aminoácidos, a ingestão dietética e o estado nutricional em adultos participantes do Inquérito de Saúde no município de São Paulo, Brasil. Métodos - Foram avaliados dados de 168 indivíduos. A análise metabolômica para identificação de 21 aminoácidos foi realizada nas amostras de plasma, utilizando kit AbsoluteIDQTMp180 da Biocrates Life Science AG (Innsbruck, Austria). O consumo alimentar foi estimado por meio da aplicação do questionário de frequência alimentar. Os grupos de aminoácidos e de alimentos foram submetidos à análise fatorial por componente principal. A variável Metabolicamente Saudável foi construída considerando-se as Diretrizes da Sociedade Brasileira de Cardiologia para o diagnóstico e tratamento da síndrome metabólica. A regressão linear múltipla foi aplicada para avaliar as associações, tendo como variáveis de ajustes: etnia, idade, renda familiar, sexo, dieta, atividade física. A comparação entre grupos foi feita por MANOVA e a confirmação da diferença significativa pelo teste de Bonferroni. Resultados - A porcentagem de indivíduos com obesidade foi de 24%, sendo que a média de IMC correspondeu a 26 kg/m2. A população estudada apresentou média de idade de 50 anos, sendo a maioria de etnia branca (56%) e do sexo masculino (52%), com pouca adesão à atividade física (21%). Os parâmetros bioquímicos estavam, em média, abaixo das concentrações estabelecidas como normais, exceto a insulina, cuja média foi de 20,8 ?UI/mL. Todavia, ao estratificar pelo estado nutricional e metabolicamente saudável, os parâmetros bioquímicos se mostraram diferentes, tais como a glicemia, triglicerídeos e colesterol total. A ingestão dietética foi igual entre os grupos estudados. O perfil de aminoácido revelou potencial de diferenciar os estados nutricional e metabólico, destacando os aminoácidos de cadeia ramificada (Leu, Ile, Val). Foram identificados dois padrões de aminoácidos relacionados a beta oxidação e aos aminoácidos glicogênicos. O primeiro teve relação positiva para os indivíduos com obesidade e metabolicamente não saudável. O segundo, apresentou relação inversa para ambos os estados. O consumo de manganês pela população foi de 2,5 mg/dia, sendo infusão de mate a principal fonte (28mg Mn/porção). Os indivíduos obesos ingeriram menor quantidade de manganês, que apresentou relação inversa ao estado nutricional e ao padrão de aminoácidos relacionado ao metabolismo não saudável. Identificaram-se três padrões alimentares: perfil saudável, tradicional e moderno. A associação com o estado nutricional e metabolicamente saudável foi positiva para o padrão saudável. Conclusão - Os aminoácidos de cadeia ramificada se revelaram biomarcadores para identificar o estado nutricional e metabólico de indivíduos adultos. Os indivíduos que tiveram baixo consumo de manganês apresentaram maior adesão ao perfil metabolômico de aminoácidos relacionados com beta oxidação.
Introduction - The metabolomics allows to determine patterns of variation of the metabolites between people with or without illness, considering or not the food consumption. Understanding the relationship between metabolites and metabolic disorders, it is possible to deal with obesity and chronic diseases. Objective - To investigate the association between the metabolic profile of amino acids and dietary intake and nutritional status in adults from the household survey conducted in the city of São Paulo, Brazil. Method - The 21 amino acids were identified by metabolomic analysis, using Absolute IDQTMp 180 kit from Biocrates Life Science AG (Innsbruck, Austria). Food intake was estimated using the food frequency questionnaire. The amino acid and food groups were submitted to factorial analysis by main component. The Metabolically Healthy variable was constructed considering the Guidelines of the Brazilian Society of Cardiology for the diagnosis and treatment of the metabolic syndrome. Multiple linear regression was applied to evaluate the associations, adjusted by the variables: ethnicity, age, family income, sex, diet, physical activity. The comparison between groups was made by MANOVA, and the confirmation of the significant difference, by the Bonferroni test. Results - The percentage of people with obesity was 24%, although the mean BMI corresponded to 26 kg/m2. The population studied presented a mean age of 50 years, most of them white (56%) and male (52%), with little adherence to physical activity (21%). The biochemical parameters were, on average, below the established normal concentrations, except for insulin (20,8 ?UI/mL). However, when stratified by nutritional status and metabolically healthy, the biochemical parameters were statistically different. The dietary intake was the same among the groups studied. The amino acid profile revealed potential to differentiate the nutritional and metabolic states, highlighting the branched chain amino acids. Two amino acid patterns related to beta-oxidation and glycogenic amino acids had been identified. The former had a positive relationship for obese and metabolically unhealthy people. The second presented an inverse relation for both states. The manganese consumption by the population was 2.5 mg / day, and the mate infusion was the main source (28mg Mn/serving). Obese subjects consumed less manganese, which had an inverse relationship to nutritional status and to the amino acid pattern related to unhealthy metabolism. Three dietary patterns were identified: healthy, traditional and modern profiles. The association with nutritional and metabolically healthy status was positive for the healthy pattern. Conclusion - The branched-chain amino acids had been revealed to be biomarkers to identify the nutritional and metabolic status of adult individuals. The individuals that had low manganese consumption showed greater adhesion to the metabolic profile of amino acids related to beta-oxidation, and could be used as biomarker for the ingestion of this micronutrient.

When microalgae are exposed to changing environmental conditions, e.g., light-dark cycles or oscillations in nutrient availability (CO2, nitrogen, phosphate or silicate) they respond with metabolic changes in the carbon allocation pattern. Short time regulations in the time range of few seconds to minutes can be mirrored best by mass spectroscopy based metabolomics. However, these snap shots do not reflect the alterations in the carbon flow to the cellular macromolecules like protein, carbohydrate or lipid. In this review it is shown how the combination of FTIR spectroscopy and Chla-in-vivo-fluorescence based electron transport rates can reveal changes in the metabolic flux rates of carbon during a shift of the environmental conditions. The review will demonstrate in which time range FTIR spectroscopy can deliver significant information and how FTIR spectroscopy data can synergistically support metabolome analysis by mass-spectroscopy.

Background: The metabolic syndrome is associated with an increased risk of diabetes and vascular disease. In order to understand the pathophysiological processes underlying such risk, it is necessary to develop a better understanding of normal fat metabolism and abnormalities associated with the syndrome. The hypothesis tested in this thesis is that specific abnormalities in adipose tissue and muscle fat metabolism characterise the metabolic syndrome. Methods: Fasting biochemical parameters were measured in a cohort of overweight men with and without the metabolic syndrome. Stable-isotope labeling and arterio-venous difference measurements were conducted in 18 men to elucidate pathways of exogenous and endogenous fat metabolism under fasting and postprandial conditions in adipose tissue and skeletal muscle. In addition, a pilot study of the effects of heat and electrical stimulation on adipose tissue metabolism was undertaken. Results: Cohort study - The prevalence of the metabolic syndrome depended on the definition used. Total cholesterol and apoB were greater in those with the metabolic syndrome than in those without. There was no difference in fasting NEFAs. Metabolic investigation - There was significant postprandial uptake of NEFA from the circulating NEFA pool by adipose tissue. Chylomicrons were confirmed as the preferred substrate of LPL. There was preferential uptake of FAs derived from chylomicron hydrolysis. There was release of NEFA across muscle. In the metabolic syndrome, adipose tissue NEFA output is lower during fasting and falls less following a meal than in the healthy obese. Clearance of dietary-derived TG is lower across both adipose tissue and muscle in the metabolic syndrome. Pilot study – Heat increased measures of lipolysis whereas electrical stimulation had no effect. Conclusions: Fat metabolism in individuals with the metabolic syndrome is characterised by metabolic inflexibility but not insulin resistance.

Background Prostate cancer (PCa) is the most frequently diagnosed malignant disease among adult males in the USA and the second leading cause of cancer deaths in men. Due to the lack of diagnostic tools that are able to differentiate highly malignant and aggressive cases from indolent tumors, overtreatment has become very common in the era of prostate specific antigen (PSA) screening. New diagnostic methods to determine biological status, malignancy, aggressiveness and extent of PCa are urgently needed. 1H High Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy (1H HRMAS MRS) can be used to establish PCa metabolomic profiles while preserving tissue architecture for subsequent histopathological analysis. Immunohistochemistry (IHC), as opposed to conventional histopathology methods, has the potential to provide objective, more accurate and quantitative knowledge of tissue pathology. This diagnostic- accuracy study sought to evaluate a novel approach to quantitatively identify metabolomic markers of PCa by exploring the potential of PCa immunomarkers to quantify metabolomic profiles established by 1H HRMAS MRS. Material and Methods 1H HRMAS MRS was performed on tissue samples of 51 prostate cancer patients using a 14.1 Tesla NMR spectrometer (BRUKER Biospin, Billerica, MA) with a rotor synchronized CPMG pulse sequence. Spectral intensities of 36 regions of interest were measured as integrals of curve fittings with Lorentzian-Gaussian line shapes. Immunohistochemistry (IHC) was carried out following the spectroscopy scan, using three prostate immunomarkers to identify cancerous and benign glands: P504S (Alpha-methylacyl-CoA-racemace), CK903 (high-molecular weight cytokeratin) and p63. The immunostaining quality following 1H HRMAS MRS was evaluated and compared to unscanned sections of the same sample, to verify the stability and accessibility of the proposed immunomarkers. IHC images were automatically and quantitatively evaluated, using a quantitative image analysis program (QIAP), to determine the percentage of cancerous and benign epithelia in the tissue cross- sections. The results of the program were validated by a correlation with the results of a quantitative IHC review and quantitative conventional histopathology analysis performed by an experienced pathologist. Ultimately, spectral intensities and the cancer epithelium percentage, obtained from quantitative immunohistochemistry, were correlated in order to validate PCa metabolomic markers identified by 1H HRMAS MRS. Patient outcomes and incidence of recurrence were determined by retrospective review of medical records five years after initial surgery. Categories of recurrence were correlated to spectral intensities to explore potential metabolomic markers of recurrence in the cohort. Results Immunostainings with P504S and CK903 showed excellent staining quality and accessibility following 1H HRMAS MRS, suggesting these markers to be suitable for the presented quantitative approach to determine metabolomics profiles of PCa. In contrast, the quality of p63 IHC was impaired after previously performed spectroscopy. IHC using the immunomarkers P504S and CK903 on adjacent slides was found to present a feasible quantitative diagnostic method to distinguish between benign and cancerous conditions in prostate tissue. The cancer epithelium percentage as determined by QIAP showed a significant correlation to the results of quantitative IHC analysis performed by a pathologist (p < 0.001), as well as to a quantitative conventional histopathology review (p = 0.001). The same was true for the benign epithelium percentage (p < 0.001 and p = 0.0183), validating the presented approach. Two metabolomic regions showed a significant correlation between relative spectral intensities and the cancer epithelium percentage as determined by QIAP: 3.22 ppm (p = 0.015) and 2.68 ppm (p = 0.0144). The metabolites corresponding to these regions, phosphocholine and citrate, could be identified as metabolomic markers of PCa in the present cohort. 45 patients were followed for more than 12 months. Of these, 97.8% were still alive five years after initial surgery. 11 patients (24.4%) experienced a recurrence during the follow- up time. The categories of recurrence showed a correlation to the spectral intensities of two regions, 2.33 – 2.3 ppm (p = 0.0403) and 1.28 ppm (p = 0.0144), corresponding to the metabolites phosphocreatine and lipids. Conclusion This study introduces a method that allows an observer-independent, quantitative analysis of IHC to help establish metabolomic profiles and identify metabolomic markers of PCa from spectral intensities obtained with 1H HRMAS NMR Spectroscopy. The immunomarkers P504S and CK903 have been found suitable IHC analysis following 1H HRMAS MRS. A prospective in vivo application of PCa metabolite profiles and metabolomic markers determined by the presented method could serve as highly sensitive, non- invasive diagnostic tool. This observer- independent, computer- automated, quantitative analysis could help to distinguish highly aggressive tumors from low-malignant conditions, avoid overtreatment and reduce risks and complications for cancer patients in the future. Further studies are needed to verify the identified PCa metabolomic markers and to establish clinical applicability
Einführung Prostatakrebs ist eine häufigsten Krebserkrankungen in den USA und die zweithäufigste malignom- assoziierte Todesursache männlicher Patienten weltweit. Seit der Einführung des Prostata- spezifischen Antigen (PSA)- Screeningtests wird diese Krebsart in früheren Stadien diagnostiziert und therapiert, wodurch die Mortalitätsrate in den letzten Jahren deutlich reduziert werden konnte. Da moderne diagnostische Methoden bislang jedoch nicht ausreichend in der Lage sind, suffizient zwischen hochmalignen und weniger aggressiven Varianten dieses bösartigen Krebsleidens zu unterscheiden, werden häufig auch Patienten aggressiv therapiert, deren niedriggradiges Prostatakarzinom keine klinische Relevanz gehabt hätte. Es besteht daher ein großes wissenschaftliches Interesse an der Entwicklung neuer diagnostischer Methoden zur akkuraten Bestimmung von biologischem Status, Malignität, Aggressivität und Ausmaß einer Prostatakrebserkrankung. \\\\\\\"1H High Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy\\\\\\\" (1H HRMAS MRS) ist eine vielversprechende diagnostische Methode, welche es ermöglicht, metabolomische Profile von Prostatakrebs zu erstellen, ohne die Gewebsstruktur der analysierten Proben zu zerstören. Durch anschließende histopathologische Begutachtung lassen sich die erstellten Metabolitprofile validieren und evaluieren. Im Gegensatz zu konventionellen histopathologischen Methoden können durch immunhistochemische Verfahren dabei objektivere, akkuratere und quantifizierbare histopathologische Erkenntnisse gewonnen werden. Die vorliegende Studie präsentiert einen neuentwickelten diagnostischen Ansatz zur quantitativen Bestimmung von metabolomischen Markern von Prostatakrebs, basierend auf der Durchführung von 1H HRMAS NMR Spektroskopie und quantitativer Immunhistochemie. Material und Methoden Einundfünfzig Gewebsproben von Prostatakrebspatienten wurden mittels 1H HRMAS MRS an einem 14.1 T BRUKER NMR Spektrometer unter Einsatz einer CPMG-Pulssequenz untersucht. Spektrale Intensitäten in 36 Metabolitregionen wurden gemessen. Anschließend wurden die analysierten Gewebeproben mit drei Immunfärbemarkern für sowohl malignes (P504S, Alpha-methylacyl-CoA-racemase) als auch benignes (CK903, High-molecular weight cytokeratin, und p63) Prostatagewebe angefärbt und quantitativ mit Hilfe eines Bildanalyseprogramms (QIAP) ausgewertet. Die Anwendbarkeit und Auswertbarkeit der genannten Immunomarker nach Spektroskopie wurde evaluiert und mit der Färbungsqualität von nicht- gescannten Schnitten verglichen. Die Resultate der automatischen Auswertung durch QIAP konnten durch einen erfahrenen Pathologen in einer quantitativen Analyse der Immunfärbungen sowie konventioneller histologischer Färbungen derselben Gewebsproben validiert werden. Die spektralen Intensitäten aus den Messungen mit 1H HRMAS MRS wurden mit den korrespondierenden Ergebnissen der quantitativen Auswertung der Immunfärbungen korreliert, um metabolomische Marker von Prostatakrebs zu identifizieren. Der klinische Verlauf und die Rezidivrate der Patienten wurden 5 Jahre nach der initialen Prostatektomie retrospektiv bestimmt. Rezidivkategorien wurden erstellt und mit den bestimmten spektralen Intensitäten korreliert, um metabolomische Marker für das Auftreten von Prostatakrebsrezidiven zu identifizieren. Ergebnisse Die Immunfärbungen mit P504S und CK903 zeigten exzellente Qualität und Auswertbarkeit nach vorheriger 1H HRMAS MRS. Beide Marker eigneten sich zur Durchführung von quantitativer Immunhistochemie an spektroskopierten Gewebeproben. Im Gegensatz dazu war die Qualität der Immunfärbungen mit p63 nach Spektroskopie vermindert. Quantitative Immunfärbungen unter Einsatz der Immunmarker P504S und CK903 stellten eine praktikable diagnostische Methode dar, um zwischen malignen und benignem Prostatagewebe zu unterscheiden. Der Anteil von bösartig verändertem Prostatagewebe, bestimmt durch QIAP, korrelierte signifikant mit den Ergebnissen der quantitativen Analyse der Immunfärbungen durch den Pathologen (p < 0.001), sowie mit der quantitativen Auswertung der konventionellen histopathologischen Färbung (p = 0.001). Ebenso ließ sich die Bestimmung des Anteils von benignem Gewebe mit QIAP zu den Ergebnissen der pathologischen Analyse korrelieren (p < 0.001 und p = 0.0183). Für zwei metabolomische Regionen konnte ein signifikante Korrelation zwischen relativen spektralen Intensitäten, bestimmt mit 1H HRMAS NMR Spektroskopie, und dem Anteil von malignem Epithelium in derselben Gewebeprobe, ermittelt durch QIAP, festgestellt werden: 3.22 ppm (p = 0.015) und 2.68 ppm (p = 0.0144). Die zu diesen Regionen korrespondierenden Metaboliten, Phosphocholin und Zitrat, konnten als potentielle metabolomische Marker für Prostatakrebs identifiziert werden. Die retrospektiven Analyse der klinischen Daten der Patienten fünf Jahre nach Prostatektomie ergab eine Überlebensrate von 97.8%. Elf dieser Patienten (24.4%) erlitten ein Rezidiv ihrer Erkrankung. Die bestimmten Rezidivkategorien korrelierten signifikant mit zwei metabolomischen Regionen (2.33 – 2.3 ppm, p = 0.0403 und 1.28 ppm, p = 0.0144), welche zu den Metaboliten Phosphokreatin und Lipiden korrespondierten. Schlussfolgerung Die vorliegende Studie präsentiert einen diagnostischen Ansatz zur objektiven und quantitativen Bestimmung metabolomischer Marker von Prostatakrebs unter Verwendung von 1H HRMAS MRS und Immunhistochemie. P504S und CK903 eignen sich als Immunmarker für quantitative Immunfärbungen nach vorheriger Durchführung von 1H HRMAS MRS. Die Metaboliten Phosphocholin und Zitrat konnten in der vorliegenden Patientenkohorte als potentielle metabolomische Marker für Prostatakrebs identifiziert werden. Eine mögliche in vivo Anwendung der gefundenen metabolomischen Marker könnte als hochsensitives, objektives und nicht- invasives diagnostisches Werkzeug der Prostatakrebsdiagnostik dienen. Der vorliegende untersucherunabhängige, automatisierte und quantitative diagnostischer Ansatz hat das Potential, zwischen hochmalignen und weniger aggressiven Krebsfällen zu unterscheiden und somit unnötige Risiken und Komplikationen für Prostatakrebspatienten zu reduzieren. Weitere Untersuchungen sind notwendig, um die identifizierten metabolomischen Marker zu verifizieren und eine klinische Anwendung zu etablieren

Chronic renal failure (CRF) is associated with loss of lean body mass, a high incidence of malnutrition, and with insulin resistance. CRF is often complicated by metabolic acidosis. Metabolic acidosis is known to alter both protein and carbohydrate metabolism. A series of studies have been undertaken to investigate the effect of metabolic acidosis on protein metabolism in both normal and CRF human subjects, and to study whether metabolic acidosis in CRF affects insulin sensitivity. Protein turnover was studied using the technique of primed constant infusions of L-[1-¹³C]leucine. Normal subjects were studied before and after ammonium chloride induced metabolic acidosis. Acidosis was associated with increased protein turnover and amino acid oxidation. In CRF subjects, correction of acidosis with sodium bicarbonate decreased protein turnover and amino acid oxidation. The effect of acidosis in CRF on insulin mediated carbohydrate metabolism was studied using the technique of the hyperinsulinaemic euglycaemic clamp. Insulin sensitivity increased with correction of acidosis. By combining L-[1-¹³C]leucine infusions with hyperinsulinaemic euglycaemic clamps, the response of protein metabolism to hyperinsulinaemia was measured before and after correction of acidosis. The presence of acidosis did not impair the ability of insulin to modulate protein metabolism. There is therefore, dissociation between the effects of acidosis in CRF on insulin mediated carbohydrate metabolism and insulin mediated protein metabolism. In summary, metabolic acidosis increases protein catabolism in both normal and CRF man and may contribute to the loss of lean body mass characteristic of CRF. Insulin resistance in CRF improves with correction of acidosis. However the effects of acidosis on protein metabolism are not mediated via alterations in insulin sensitivity.

There is increasing evidence demonstrating a determinant role of gut microbiota in host health, and one underlying mechanism is via gut-microbial metabolites-host interaction in modulating host's cellular functions. SCFAs, main fermentation products of dietary fibres by gut microbiota in GI tract, are considered to be generally beneficial to the host. It has been shown that SCFAs act as substrates for energy metabolism, receptor agonists, and as histone deacetylase (HDAC) inhibitors. However, there are still gaps in the knowledge of their biological effects. It has been hypothesised that SCFAs may act as substrate for energy metabolism as well as ligand for several GPCRs, thus play roles in many cellular functions. Combining 1H nuclear magnetic resonance (NMR) spectroscopy with multivariate statistical analysis of the effect of SCFAs on the hepatic cancer metabolic network identified a metabolic signature associated dose- and time- related SCFA exposure. TCA cycle intermediate, α-keto-β-methylvalerate was strongly correlated with the treatment of SCFAs suggesting the preferential of the cells to SCFAs for energy production. This signature further confirms that SCFAs play direct role in host energy metabolism. Having implemented a harmonised pharmacological assessment of a comprehensive SCFA panel on FFAR2, FFAR3, and GPR109A, this study is the first to reveal that isobutyrate and isovalerate are novel partial agonists for GPR109A. Furthermore, niacin, a classical agonist for GPR109A also has been shown to activate FFAR2 and FFAR3 but with much lower affinity. In another study, the effect of SCFAs on 3T3-L1 adipogenesis and adipocytes lipolysis has been characterised. Dosing the 3T3-L1 cells with isobutyrate, valerate, and isovalerate significantly induce the adipogenesis of the cells. In contrast, other SCFAs have no effect of the 3T3-L1 cell differentiation. Subsequent study focused on the effect of SCFAs on basal adipocyte lipolysis. Mature adipocytes were treated with 100 μM of SCFAs for 3hr and glycerol release was measured. This study revealed the anti-lipolytic property of propionate, butyrate, and valerate by significantly inhibits lipolysis in mature 3T3-L1 adipocytes. Thus, the role of SCFAs in regulating adipocytes functions may be particular important and beneficial in regulating plasma lipid profile and possibly aspects of metabolic syndrome. Together, these data enhance our understanding on the role of SCFAs on important metabolic tissues, which are hepatocyte and adipocyte.

Specialized metabolism (secondary metabolism) in glandular trichomes of sweet basil (Ocimum basilicum L.) and accumulation of specialized metabolites (secondary metabolites) in rhizomes of turmeric (Curcuma longa L.) was investigated using proteomic and metabolomic approaches, respectively. In an effort to further clarify the regulation of metabolism in the glandular trichomes of sweet basil, we utilized a proteomics-based approach that applied MudPIT (multidimensional protein identification technology) and GeLC-MS/MS (gel enhanced LC-MS/MS) to protein samples from isolated trichomes of four different basil lines: MC, SW, SD, and EMX-1. Phosphorylation, ubiquitination and methylation of proteins in these samples were detected using X!tandem. Significant differences in distribution of the 755 non-redundant protein entries demonstrated that the proteomes of the glandular trichomes of the four basil lines were quite distinct. Correspondence between proteomic, EST, and metabolic profiling data demonstrated that both transcriptional regulation and post-transcriptional regulation contribute to the chemical diversity. One very interesting finding was that precursors for different classes of terpenoids, including mono- and sesquiterpenoids, appear to be almost exclusively supplied by the MEP (2-C-methyl-D-erythritol 4- phosphate) pathway, but not the mevolonate pathway, in basil glandular trichomes. Our results suggest that carbon flow can be readily redirected between the phenylpropanoid and terpenoid pathways in this specific cell type. To investigate the impact of genetic, developmental and environmental factors on the accumulation of phytochemicals in rhizomes of turmeric, we performed metabolomic analysis in a 2x2x4 full factorial design experiment using GC-MS, LC-MS, and LC-PDA. Our results showed that growth stage had the largest effect on levels of the three major curcuminoids. Co-regulated metabolite modules were detected, which provided valuable information for identification of phytochemicals and investigation of their biosynthesis. Based on LC-MS/MS data, 4 new diarylheptanoids were tentatively identified in turmeric rhizomes using Tandem-MSASC, a home-made software tool that automatically recognizes spectra of unknown compounds using three approaches. Based on our metabolomic results, we proposed two new strategies, “metabolomics-guided discovery” and “correlation bioassay”, to identify bioactive constituents from plant extracts based on information provided by metabolomic investigation.

[Truncated abstract] Arachidonic acid is oxidised in vivo by non-enzymatic (free radical) or enzymatic pathways (cyclooxygenase, lipoxygenase, and cytochrome P450) to form a range of biologically active eicosanoids. Specifically, arachidonic acid is metabolised by cytochrome P450 -hydroxylase to produce vasoactive 20-hydroxyeicosatetraenoic acid (20-HETE), and by 5-lipoxygenase to produce proinflammatory leukotriene B4 (LTB4), which can further be metabolised by -hydroxylase to from 20-OH-LTB4 and 20-COOH-LTB4. F2-Isoprostanes (F2-IsoPs) are produced through free radical attack on arachidonic acid and have been recognised as the most reliable markers of lipid peroxidation in vivo. The metabolic syndrome (MetS) is characterised by abdominal obesity, hypertension, insulin resistance, glucose intolerance, and dyslipidemia. It is associated with low-grade inflammation and oxidative stress and an increased risk of developing cardiovascular diseases. Dietary weight loss is strongly recommended for the management of the MetS and can potentially minimise the risk of cardiovascular diseases and diabetes in individuals with the MetS. Little is known regarding the role of these arachidonic acid metabolites in the MetS and the effect of weight loss on their metabolism. Chapter three comprised of three in vitro studies aimed to examine 20-HETE synthesis in human blood cells. 20-HETE acts as a second messenger for vasoconstrictor actions of angiotensin II (Ang II) and endothelin-1 (ET-1) in renal and mesenteric beds. Human neutrophils and platelets are integral to the inflammatory process. ... Production of LTB4 and 20-OH-LTB4 was significantly lower compared with controls (P<0.005) and remained so after adjustment for neutrophil count (P<0.05).The weight loss intervention resulted in a 4.6kg reduction in body weight and a 6.6cm decrease in waist circumference and a significant increase in LTB4 and 20-OH- LTB4 in the weight loss group. Chapter Five continued to investigate the role of other arachidonic acid metabolites, 20-HETE and F2-IsoPs in the MetS and the effect of weight loss. In the case-control study (Human study 1), plasma and urinary 20-HETE and F2-IsoPs were significantly elevated in the MetS group, but no significant difference was found in stimulated-neutrophil 20-HETE. A significant gender x group interaction was observed in that women with the MetS had higher urinary 20-HETE and F2-IsoPs compared to controls (P<0.0001). In a randomised controlled trial (Human study 2), relative to the weight- maintenance group, a 4.6 kg loss in weight resulted in a 2 mmHg fall in blood pressure but did not alter the production of 20-HETE or F2-IsoPs. No significant differences were shown in 20-HETE released from stimulated-neutrophils before and after weight loss. 20-HETE and oxidative stress may be important mediators of cardiovascular disease risk in the MetS. Although a 4% reduction in body weight reduced BP, there were no changes in plasma or urinary 20-HETE or F2-IsoPs. In summary, in vitro studies show that human neutrophils and platelets can produce 20-HETE in response to Ang II and ET-1, and human studies demonstrate that the presence of MetS has a significant impact on arachidonic acid metabolism and effective weight loss can restore leukocyte synthesis of LTB4.

Biological variation is evident in all aspects of nutrition, particularly total daily energy expenditure (TDEE) and basal metabolic rate (BMR). The impact of ethnicity on biological variation on certain aspects of energy metabolism has been investigated. Predictive equations to estimate BMR were published by the FAOIWHOIUNU (1985) in a report entitled EllerK}' and Protein Requirements. Since its publication, serious concern has been expressed on the validity of these equations, largely because the analysis appears to be based on a biased and incomplete analysis of the world literature on BMR. Using an expanded database (n=10,004) and stringent selection criteria the global BMR data has been re-analysed and new predictive equations presented. The robustness of the new equations were tested by comparing their predictive accuracy with those of existing ones. The BMR of 77 women aged 18-30 years were found to be best described by the newly developed Oxford Brookes equations and the Henry & Rees (1991) equations for tropical peoples (P < 0.05 and NS respectively). Furthermore, the new expanded data base was used to plot nonnal curves for BMR for individuals which compared ethnic differences. It was observed that when BMR is expressed per kilogram of body mass, ethnic differences are diminished. The necessity to estimate TDEE in various ethnic groups prompted the evaluation of a non-invasive easy to use technique. Heart rate monitoring as a means of estimating TDEE is reviewed and compares favourably to the use of activity diaries. Whilst highly variable, heart rate monitoring is acknowledged as a valuable tool in the estimation of energy expenditure and activity in individuals and populations Ethnic differences in energy intake in a migrating student population were assessed. Malaysian students recently arrived in the UK were requested to keep food diaries in order to detect changes in their diets. Particular emphasis was placed on changes in the energy densities of food consumed and their effect on body weight and energy balance. After 6 months, it was observed that energy balance was maintained despite the energy density of food consumed in the UK being significantly (P < 0.05) more energy dense than food consumed in their native Malaysia. This illustrates the precise way in which the human body can maintain energy balance. Anthropometric differences were compared in 553 individuals from 4 ethnic groups in a study investigating the ann-span and height relationship. Differences were found between the ethnic groups (P < O.OI). The use of ann-span as a proxy for height should be used with caution. It is imperative that more global data collected under strict control is required before human biological variation can be attributed to true ethnic differences and not merely individual variation.

The metabolic and biochemical factors involved in the regulation of fuel and pathway selection, at appropriate rates and times, during elevated metabolic demands remains to be resolved. Therefore, the purpose of this investigation was to examine the metabolic interrelationships involved in energy provision and its regulation during exercise at different intensities. Specifically, fuel selection and its control by adenine nucleotides and cytosolic redox and were investigated in red and white muscle of rainbow trout during various progressive intensities of exercise. The control of glycolysis was examined with respect to glycolytic enzyme disequilibrium. The effects of substrate limitations and end product accumulations were examined with regard to fatigue. Finally, the buffering capacity and various potential buffering constituents were investigated. It would appear that the energy turnovers required to perform the varied exercise intensities in this study were achieved by selecting the fiber type, fuel and pathway for optimizing ATP production rate versus substrate and proton accumulation. The purine nucleotide cycle was found to be operational within both fiber types. Fuel selection appeared to be intimately related to myofibrillar ATPase activation with free ADP acting as the metabolic signal to coordinate the phasing in of appropriate fuels/pathways at appropriate rates. HK, phos, PFK and PK were identified as regulatory enzymes in both fiber types. As well, the GPDH.PEK complex also appeared to exhibit regulation when glycogen was limiting and this regulation appeared to have been induced by a decreased ATP/ADP₊ ratio. The redox state of the NAD couple became more oxidized in both tissues when muscle glycogen was low. This finding was attributed to an induced shift in the equilibrium of LDH in the direction of NAD and lactate. The simultaneous ATP/ADP₊ induced disequilibrium of the PGK reaction would inhibit flux through the GPDH.PGK complex. Skeletal muscle buffering was found to be dominated by protein, inorganic phosphate and histidine related compounds. Thus, these metabolic and biochemical adjustments, allowed a coordinated integration of fiber type, fuel and pathway selection, to achieve the appropriate coupling of myofibrillar ATPase activity to ATP turnover, while minimizing substrate depletion and proton accumulations.
Science, Faculty of
Zoology, Department of
Graduate

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009.
Cataloged from PDF version of thesis.
Includes bibliographical references.
The integrated network of biochemical reactions known collectively as metabolism is essential for life, and dysfunction in parts of this network causes human disease - both rare, inherited disorders and common diseases such as diabetes mellitus. The study of metabolic disease depends upon quantitative methods which are traditionally custom-tailored to a given compound. Recent advances in technologies such as mass spectrometry now enable the simultaneous measurement of a diverse metabolite collection spanning multiple biological pathways, an approach known as metabolic profiling or metabolomics. This dissertation describes the development of one such metabolic profiling system and its application to the study of two major topics in human energy metabolism: the fasting:feeding transition and mitochondrial disease. In the first study, we profile human plasma in response to glucose ingestion, detecting dozens of metabolite changes and identifying several distinct effects of insulin. Based on these observations, we propose a multivariate view of insulin sensitivity, and show that individuals at risk for developing diabetes mellitus can differ in their insulin response profile, a concept of potential value for estimating disease risk and progression. In the second study, we elucidate a metabolic signature of human mitochondrial disease that reflects substrate oxidation, biosynthesis and energy charge.
(cont.) We demonstrate that the culture media profile of a cellular disease model of mitochondrial dysfunction reflects the plasma profile of human patients, an approach that could be applicable to other diseases as well. In addition, we show that a combination of metabolites distinguishes individuals with mitochondrial disease from healthy individuals better than the currently used diagnostic markers. Our findings provide insight into human disorders of energy metabolism, and demonstrate the utility of a profiling approach for the understanding of metabolic disease.
by Oded Shaham.
Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, September, 2020
Cataloged from the official PDF of thesis. "July 2020."
Includes bibliographical references.
Cancer is the second leading cause of death following heart diseases in the U.S. During the past two decades, cancer metabolism has emerged as an indispensable part of contemporary cancer research. Various types of metabolic alterations in cancer cells have been documented, prompting extensive investigation of the link between reprogrammed cell signaling pathways and rewired cellular metabolism. In addition, drug targeting of rewired metabolic pathways has been demonstrated to be a promising cancer treatment strategy. Despite this progress, a fundamental question remains unanswered: whether there is a difference in the metabolism of cancerous, fast growing cells and normal proliferative cells. This deficiency has hindered our understanding of cancer metabolism and the efforts to develop effective cancer therapies targeting metabolism with reduced side effects.
In this thesis, we used ¹³C-isotope tracing and metabolic flux analysis (MFA) to study cancer metabolism and identify metabolic pathways differentially activated in cancer cells. To support efforts to design effective therapeutic therapies, we sought to distinguish metabolic behavior in cancer versus normal cells growing at the same speed, and obtain a systematic understanding of cancer metabolism. To this end, we dissected bioreaction networks in human mammary epithelial cells (HMECs) that have been genetically modified to exhibit different levels of tumorigenicity. We discovered distinct substrate utilization pattern in the tricarboxylic acid (TCA) cycle and de novo lipogenesis. Specifically, we found that glucose was catabolized in the TCA cycle up to the formation of citrate, which was then used primarily for lipogenesis. The majority of the TCA cycle flux, however, was maintained by glutamine anaplerosis.
¹³CMFA further revealed that some metabolic reactions were more activated in tumorigenic HMECs. By introducing a new quantity termed metabolic flux intensity, defined as pathway flux divided by the specific growth rate, we identified three most enhanced reactions - oxidative pentose phosphate pathway (oxPPP), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in the most tumorigenic HMEC. Targeting of these three pathways with small molecule inhibitors selectively reduced growth in the cancerous HMEC line. In addition, our study provides direct evidence that metabolism may be dually controlled by proliferation and oncogenotypes.
by Wentao Dong.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Chemical Engineering

Dissertation presented to obtain the Ph.D degree in Biochemistry, Neuroscience
Brain energy metabolism results from a complex group of pathways and trafficking mechanisms between all cellular components in the brain, and importantly provides the energy for sustaining most brain functions. In recent decades, 13C nuclear magnetic resonance (NMR) spectroscopy and metabolic modelling tools allowed quantifying the main cerebral metabolic fluxes in vitro and in vivo. These investigations contributed significantly to elucidate neuro-glial metabolic interactions, cerebral metabolic compartmentation and the individual contribution of neurons and astrocytes to brain energetics. However, many issues in this field remain unclear and/or under debate.
To the financial support provided by Fundação para a Ciência a Tecnologia (SFRH/BD/29666/2006; PTDC/BIO/69407/2006) and to the Clinigene – NoE (LSHBCT2006- 010933). I further acknowledge the Norwegian Research Council for a fellowship that allowed me to perform part of my PhD work at NTNU, Norway.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2006.
Title from electronic title page (viewed Oct. 10, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biomedical Engineering." Discipline: Biomedical Engineering; Department/School: Medicine.

It is clear that sub-clinical inflammation is a key factor that triggers type 2 diabetes mellitus (T2DM) and is directly influenced by weight gain. Current studies highlight that obesity, particularly central obesity, heightens the pathogenesis of T2DM. Additionally, factors produced by adipose tissue (AT), referred to as adipocytokines, can influence the degree of insulin resistance as well as inflammation, due to their duality of function. A recently implicated adipocytokine, visfatin, has been identified as a potential insulin mimetic, an enzyme associated with mitochondrial biogenesis and an inflammatory factor. However, current studies lack a clear understanding as to the role and influence of visfatin in human AT and insulin resistant states. Therefore, this thesis examined visfatin expression within specific human AT depots and the influence of adiposity - with specific consideration given to insulin resistant states including T2DM, non-alcoholic fatty liver disease (NAFLD) and human immunodeficiency virus (HIV). Further, serum studies addressed how different insulin resistant states influenced circulating visfatin levels, whilst mechanistic studies explored how the role of visfatin was altered by insulin, an insulin sensitiser, inflammation and/or disease. This current thesis identified that visfatin was abundant in both abdominal depots, with highest expression in the omental (Om) AT, and isolated adipocytes, with an apparent relationship with insulin resistance. Subsequent in vivo and in vitro analysis further identified that, whilst insulin appeared to increase visfatin protein expression in isolated abdominal subcutaneous (Abd Sc) adipocytes, the use of an insulin sensitiser - either used in cultured Abd Sc adipocytes or as part of an oral therapeutic treatment in subjects with T2DM - decreased circulating visfatin levels. In addition, intracellular signalling studies highlighted that visfatin regulation within AT appeared to be dependent upon both nuclear factor (NF)-B and c-Jun N-terminal kinase (JNK) activation, influencing interleukin (IL)-6 as part of a visfatin regulatory feedback mechanism. Following the potential influence of visfatin in T2DM, this thesis explored its’ potential role in disease associated with other insulin resistant phenotypes, such as liver disease and HIV. From serum assessment of visfatin in subjects with NAFLD, it was identified that progression of liver disease was accompanied by a reduction in circulating visfatin levels – a finding that occurred independently of diabetic status. However, circulating visfatin still remained significantly higher in NAFLD with T2DM than those without. Finally, this thesis examined a potentially severe insulin resistant phenotype noted in HIV patients, due to an apparent lipodystrophy - a condition which alters fat oxidation and mitochondrial activation or regulation. In such a condition, circulating visfatin levels and visfatin mRNA AT expression remained unaltered by HIV status or drug therapy. Due to the capacity of visfatin to act as an enzyme involved in nicotinamide adenine dinucleotide (NAD) biosynthesis, essential for mitochondrial function and oxidative phosphorylation (OXPHOS), its role in AT remained unchanged by disease status, whilst other mitochondrial and fat metabolism factors were altered by both disease state and drug treatment. Taken together, these current data suggest a duality of function of which visfatin appears to be regulated by insulin, in addition to inflammation, in different disease states and therefore expands our current understanding of this multi-functional adipocytokine.

Aerobic fermentations of Escherichia coli grown with glucose accumulate organic acids in the culture medium. These by-products of glucose metabolism are synthesised and excreted when the glucose uptake rate is greater than its conversion to biomass and carbon dioxide. Acetate is the most abundant overflow metabolite produced in aerobic cultures of E. coli. Its production is strain and media specific, being greatest in dense nutrient-rich cultures. In industrial fermentations, E. coli is widely employed for production of recombinant proteins. However, acetate excretion reduces process efficiency by lowering cell growth rate, and decreasing the amount of substrate carbon converted to recombinant protein product. This thesis describes the construction of three plasmid vectors designed to express antisense RNA targeted against phosphotransacetylase and acetate kinase, which convert acetyl CoA to acetate. Antisense RNA was used as a metabolic engineering tool in this study, to enable examination of the central carbon flux distribution before, during and after enzyme downregulation. The aim was to decrease expression of these enzymes in E. coli, and thus decrease acetate production. E. coli MG1655 was transformed with a) a plasmid construct encoding an antisense RNA fragment, b) two compatible plasmids encoding different antisense RNA fragments. The resulting strains were cultivated in a 2L bioreactor, and the effect of antisense RNA expression evaluated. Assays to monitor the enzyme activities of phosphotransacetylase and acetate kinase were conducted, along with metabolite analysis to determine organic acid excretion profiles. Flux balance analysis, which is a method of modelling metabolism, was applied to the central carbon pathways in E. coli to provide insight into the partitioning of internal carbon fluxes. Information about the mechanisms used by E. coli to cope with partial shutdown of the acetate synthesis pathway was gained by comparing the flux distribution of a control strain with an antisense RNA-expressing strain.

Hyperhomocysteinemia, an elevation of blood homocysteine levels, is a metabolic disorder associated with dysfunction of multiple organs. Previous studies have shown that hyperhomocysteinemia is related to fatty liver. However, the underlying mechanism remains speculative. The objective of the present study is to investigate the regulatory mechanism of hepatic inflammatory response and cholesterol metabolism during metabolic disorders. In the present study, hyperhomocysteinemia was induced in Sprague-Dawley rats by feeding a high-methionine diet. The mRNA and protein expression of cyclooxygenase-2 (COX-2), a pro-inflammatory factor, were significantly elevated in the liver of hyperhomocysteinemic rats. An activation of NF-B and a stimulation of oxidative stress were observed in the same liver tissue in which COX-2 was induced. Inhibition of NF-B or oxidative stress effectively abolished hepatic COX-2 expression, inhibited the formation of inflammatory foci, and improved liver function. Activity of HMG-CoA reductase, the rate-limiting enzyme of cholesterol biosynthesis, was markedly elevated in the liver of hyperhomocysteinemic rats, which may contribute to the hepatic lipid accumulation induced by hyperhomocysteinemia. Administration of Berberine (5mg/ kg body weight/ day for 5 days) inhibited HMG-CoA reductase activity via upregulating AMP-activated protein kinase (AMPK)-mediated phosphorylation of HMG-CoA reductase. Berberine treatment reduced hepatic cholesterol content and ameliorated liver function. In addition, the regulatory mechanism of HMG-CoA reductase activation was investigated in C57BL/6 mice fed a high-fat diet. There was a significant increase in hepatic HMG-CoA reductase mRNA and protein expression as well as enzyme activity. The DNA binding activity of sterol regulatory element binding protein (SREBP)-2 (a transcription factor of HMG-CoA reductase) and Sp1 (a transcription factor of SREBP-2) were both increased in the liver of mice fed a high-fat diet. The in vitro study in palmitic acid-treated HepG2 cells further confirmed that inhibition of Sp1 by siRNA transfection abolished palmitic acid-induced SREBP-2 and HMG-CoA reductase mRNA expression. In conclusion, the present study have demonstrated that (1) Hepatic COX-2 expression is induced via oxidative stress mediated NF-B activation during hyperhomocysteinemia; (2) Dietary berberine reduces cholesterol biosynthesis by elevating AMPK-mediated HMG-CoA reductase phosphorylation; (3) HMG-CoA reductase is upregulated by Sp1-mediated SREBP-2 activation in the liver during high-fat diet feeding.

Recently, persistent organic pollutants such as polychlorinated biphenyls (PCBs) were classified as “metabolic disruptors” for their suspected roles is altering metabolic and energy homeostasis through bioaccumulation in liver and adipose tissues. Among PCBs, a specific congener, 3,3',4,4',5-pentachlorobiphenyl (PCB126), is a potent arylhydrocarbon receptor (AhR) agonist and elicits toxicity similar to the classic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). PCB126 levels found in human blood are particularly associated with diabetes and nonalcoholic fatty liver disease (NAFLD) in humans, however the mechanisms are unclear. We hypothesized that the accumulation of PCB126 disrupts carbohydrate and lipid metabolism by altering the functions of liver and adipose tissues. Hence, our objective was to characterize PCB126 induced-metabolic disruption and the underlying molecular mechanisms that cause toxicity. Separate animal studies were performed using a rat model to understand the time- and dose-dependent effects after PCB126 administration. The chronology of PCB126 toxicity showed early decreases in serum glucose level at 9 h, worsened in a time-dependent way until the end of the study at 12 d. Lipid accumulation and the liver pathology also worsened over time between 3 d and 12 d post administration. These observed effects in the liver were also found to be dose-dependent. The decrease in serum glucose was a result of a decrease in the transcript levels of gluconeogenic and glycogenolytic enzymes, necessary for hepatic glucose production and hence the maintenance of steady glucose levels in the blood. Phosphoenolpyruvate carboxykinase (PEPCK-C), the rate limiting enzyme of gluconeogenesis, was found to be significantly decreased upon exposure to PCB126. The expression levels of peroxisome proliferator-activated receptor alpha (Pparα) and some of its targets involved in fatty acid oxidation were also found to be time and dose-dependently decreased upon exposure to PCB126. In an attempt to understand the molecular targets that may cause these dual effects on both gluconeogenic and fatty acid oxidation, we found that PCB126 significantly decreases phosphorylation of the cAMP response element-binding protein (CREB). CREB is a nuclear transcription factor that is activated in the liver through phosphorylation; to switch-on the transcription of enzymes that catalyze gluconeogenesis and fatty acid oxidation, in order to meet energy demands, especially during fasting. Further, to understand the toxicity of PCB126 on adipose tissue, a human pre-adipocyte model that can be differentiated into mature adipocytes was used. In these studies, we found that exposure of preadipocytes to PCB126 resulted in a significant reduction in their ability to differentiate into adipocytes. This results in decreased lipid accumulation in the adipocyte. Reduction in the differentiation by PCB126 was associated with down regulation in transcript levels of a key adipocyte transcription factor, PPARγ and its transcriptional targets necessary for adipogenesis and adipocyte function. These inhibitory effects of PCB126 on the regulation of PPARγ and the initiation of adipogenesis were mediated through activation of AhR. Overall, this work shows that PCB126 disrupts nutrient homeostasis through its effects on the function of target tissues; liver and adipose. PCB126 significantly alters the nutrient homeostasis through its effects on gluconeogenesis and fatty-acid oxidation necessary for glucose and energy regulation during fasting. In addition, PCB126 interrupts the storage functions of adipose tissue by inhibiting adipogenesis and thus disrupts lipid storage and distribution

Trypanosoma brucei is a parasitic protozoan that can cause human African trypanosomiasis (HAT) and Nagana in cattle. Human African trypanosomiasis is deadly when left untreated, and thus there is an urgent need to develop new drugs against this disease. As trypanosomes are early diverged eukaryotes, it is anticipated that studying their metabolism can identify novel drug targets. The main drug currently in use against the late encephalitic stage, Eflornithine, was shown to inhibit an essential pathway in trypanosomes (Yarlett and Bacchi, 1989). In this Thesis three approaches were used to apply metabolomic and proteomic techniques for protein function identification and to investigate metabolic pathways. The genome of T. brucei has been published (Berriman et al., 2005) and data is available via databases, such as TriTrypDB, a database dedicated to the trypanosomatids (Aslett et al., 2009). An estimated 40% of the identified genes in this organism are annotated with an unknown or putative function. In 2006, Saito et al. developed a systematic method to ascertain enzyme function based on an in vitro assay, in combination with metabolite profiling. This approach was successfully applied in several other studies. Here, I investigate the use of this method for its application in a high throughput approach for unknown enzyme identification in trypanosomes. Seven putative identified enzymes were randomly selected from TriTrypDB, cloned and expressed in E. coli and a function could be attributed to at least one of the enzymes. Furthermore, the amino acid metabolism in trypanosomes was investigated; using stable isotope labelling combined with metabolomics. The flux of labelled compounds could be traced through the organism showing the active metabolic pathways of L-methionine, L-proline and L-arginine in T. brucei. Two T. b. brucei strains used in this study, GVR35 and 427, cause different forms of infections in their mammalian host. GVR35 causes a chronic infection and invades the central nervous system (CNS) with varying parasitemia in mice, whereas infection with strain 427 presents an acute form with high parasitaemia, causing high mortality, without invading the CNS. What causes this difference in the progression of infection? Secreted or excreted proteins from the parasites, referred to as the secretome, have been described as being important factor for virulence and avoiding the host immune response (Geiger et al., 2010) and Garzon et al. (2006) showed that excreted/secreted proteins can inhibit the maturation of dentritic cells and stop them from inducing a lymphocytic allogenic response. Significant differences in proteins secreted from these two strains are discussed; although the results are preliminary.

Dissertation presented to obtain a Ph. D. degree in Biochemistry by Universidade Nova de Lisboa, Instituto de Tecnologia Química e Biológica.
The human brain is the product of 600 million years of evolution and it is by far the most complex structure in the known universe. The vertebrates’ brain is composed of several different cell types, which perform the different functions required. The two most abundant cell types are neurons, which are the brain function unit, and glial cells, which are responsible for a myriad of housekeeping, homeostatic and structural functions. Glial metabolism is so far interconnected with the neuronal metabolism, that it is difficult to state where one finishes and the other starts. Trafficking between the two compartments include metabolites of almost all metabolic pathways (glycolysis, TCA, amino acids, ketone bodies, etc) and also ions.(...)
Fundação para a Ciência e a Tecnologia – PRAXIS XXI, for the financial support that made this doctoral work possible (PRAXIS XXI/BD/21532/99).

A demanda comercial de produtos derivados da cana-de-açúcar é a grande motivadora do aprimoramento genético com a finalidade de proporcionar a planta aumento no acúmulo de sucrose e/ou de resistência a ataques de herbívoros. Assim sendo, este trabalho abrange o estudo metabolomico de duas variedades transgênicas da cana-de-açúcar (Bowman-Birk-SBBI e Kunitz- SKTI) e seus respectivos controles através de duas metodologias distintas: a análise quimiométrica do perfil cromatográfico dos polifenóis existentes nas variedades transgênicas e o estudo metabolomico por ressonância magnética nuclear a fim de identificar possíveis diferenças entre as plantas transgênicas em relação aos seus respectivos controles. Nas amostras de folhas foram encontradas nove regiões cromatográficas representativas para a discriminação das variedades SBBI e SKTI. Análises de HPLC-MS/MS foram empregadas para a identificação parcial dos biomarcadores selecionados pelo método OPS, dentre eles: ácido cafeoílaquínico, ácido feruloílaquínico, shaftosídeo ou isoshafitosídeo, além de quatro substâncias parcialmente identificadas: um derivado de apigenina, um glicosídeo da tricina--O-(metoxicinamato), um derivado de flavonóide metoxilado e um derivado de catequina. Através do estudo metabolomico obteve-se uma visão geral sobre os metabólitos produzidos pelas variedades transgênicas e controles e através das análises dos espectros de RMN 1H, J-resolved, COSY 1H-1H, HMBC 13C-1H foi possível identificar a presença de ácidos orgânicos, amino-ácidos, açúcares, flavonóides e fenilpropanóides. Adicionalmente, a análise quimiométrica dos espectros de RMN 1H mostrou não haver diferença significativa entre folhas SBBI e controles e SKTI e controles. Como conclusão pôde-se sugerir que a inserção dos genes inibidores de protease na cana-de-açúcar não afeta as rotas biosintéticas da planta, mas apenas confere maior resistência contra a broca da cana-de-açúcar (Diatrea saccharilis). Entretanto diferenças significativas entre as variedades transgênicas foram encontradas: a variedade SBBI apresenta teor de açucares e fenilpropanóides mais elevados que a variedade SKTI. Conclui-se também que as alterações metabólicas encontradas neste trabalho não são provenientes dos genes inseridos na planta, mas por outro tipo de efeito sofrido nos controles das duas variedades, como por exemplo, variação somaclonal que antecedem a modificação genética.
Comercial demanding of industrial products from sugarcane plants are reponsable for researchs on genetic improvements with the aim to increase sucrose plant acumulation and/or provide resistence against herbivors. Thus, the present work covers metabolomic study of two transgenics varieties of sugarcane (Bowman-Birk-SBBI e Kunitz- SKTI) and their respectives wild (control) plants through two distinct methodologies: chemometrics analysis of polyphenols chromatography profile found into the transgenics varieties and the metabolomic study by nuclear magnetic resonance for identification of possible metabolic alterations comparing transgenics plants and their respective controls. Nine discriminants chromatography regions were detected on SBBI and SKTI leaves samples. HPLC-MS/MS analyses were performed for biomarkers identification selected by OPS method, among then: caffeoylquinic acid, feruloyl quinic acid, shaftoside or isoshaftoside, apigenin derivatives, tricin-O-(methoxycinamate)-glicosilated, methoxylated flavone and catechine derivative. A general vision about metabolites produced for transgenics and control plants could be achived through metabolomic study. Identification of organic acids, amino acids, sugars, phenylpropanoids and flavonoids were possible through 1H NMR, J-resolved, COSY 1H-1H, HMBC 13C-1H. Moreover, chemometrics analysis of the 1H NMR spectra had shown no significant differences between SBBI with wild plant and SKTI with control plants. These results suggest that the biosyntheses pathways are not affected by protease inhibitors genes introduced on sugarcane, but these genes just provides resistence against Diatrea saccharilis. However, metabolic alterations were found between the transgenic plants (SBBI and SKTI). Specially, the variety SBBI presents high levels of sugars and phenylpropanoids compared with SKTI variety. In conclusion, the metabolic variation found in the present are not due to genes introduction, but are originated by other type of effect on wild plants, such as, somaclonal variation before genetical modification.

A alimentação humana requer o suprimento de nutrientes de uma forma balanceada. A carne vermelha é uma perfeita fonte de proteína de alta qualidade por suprimir a necessidade diária de ferro, zinco e de várias vitaminas. Porém, a carne vermelha aumenta o risco de induzir estresse oxidativo no organismo através da formação de radicais e espécies reativas catalisadas pelo ferro, elevando a incidência de câncer colorretal e do sistema gastrointestinal. Lipídeos e proteínas são os principais alvos de tais processos oxidativos, levando à formação de produtos potencialmente mutagênicos, pró-oxidantes e substâncias de aroma indesejado. Com o aumento da população mundial, há uma projeção de acentuado aumento do consumo de carne no mundo demanda por produtos de maior qualidade e estabilidade de prateleira para suprir uma dieta mais saudável. A produção de carne e derivados de superior qualidade sensorial e nutricional é mandatária, além de configurar um desafio científico e tecnológico suprir essa demanda e fornecer uma ingestão saudável de carne. Estudos recentes revelaram que as folhas da erva-mate (Ilex paraguariensis) possuem propriedades antioxidantes e anti-coccídeas. A utilização de metodologias modernas analíticas, de acordo com abordagens \"ômicas\", permite a caracterização de matrizes complexas por determinação do \"perfil metabólito ou metabólico\". Sendo assim, o presente trabalho se propôs a investigar o padrão metabólico e metabonômico da carne proveniente de animal suplementado com extrato de erva-mate a fim de verificar seu impacto na estabilidade redox do produto. Não foi observada uma mudança qualitativa no perfil metabólico dos animais, apenas uma mudança nos teores dos metabólitos investigados. As maiores concentrações de metabólitos, na maioria das amostras, foram encontradas em animais suplementados com teores de 1,0% e 1,5% de extrato de erva-mate na dieta, apontando uma influência desta no metabolismo animal. Em relação à influência da dieta dos animais no status redox de carne bovina, amostras provenientes de animais alimentados sem suplementação e com 1,5 % de extrato de erva-mate apresentaram, respectivamente, as taxas de formação de radicais mais altas e mais baixas. De fato, houve uma tendência de gerar uma menor concentração de radicais a uma menor taxa em conformidade com o aumento do nível de extrato de erva-mate na dieta.
The human nutrition demands the supply of nutrients in a balanced way. The red meat is a perfect source of protein with a high quality, once it supplies the daily needs of iron, zinc and many vitamins. Although, the red meat increases the risk of inducing the oxidative stress in the organism through the formation of radicals and reactive species catalyzed by iron, increasing the incidence of colorectal and gastro-intestinal system cancer. Lipids and proteins are the main targets of such oxidative process, leading to the formation of products which are potentially mutagenic, prooxidant and substances of an unpleasant smell. Considering the increase of the world population, there is a projection of a sharp increase of meat consumption which is demanded by products of higher quality and stability of shelves to supply a healthier diet. The production of meat and its derivatives of a higher sensorial and nutritional quality is mandatory, besides of characterizing a scientific and technological challenge to supply this demand and offer a healthier ingestion of meat. Recent studies pointed out that the leaves of yerba mate (Ilex paraguariensis) present antioxidant and anticcocidial properties. The usage of modern analytical methodologies, according to omic approaches, allows the characterization of complex matrices by the determination of the \"metabolite or metabolic profile\". On this way, the present study aims at investigating the metabolic and metabonomic pattern and of meat which came from the animal supplemented with the extract of yerba mate in order to analyzing its impact on the redox stability of the product. It was not observed a qualitative change on the metabolic profile of animals, just a change on the levels of the investigated metabolites. The highest concentrations of metabolites, on the major part of samples, were found on animals supplemented with levels of 1.0% and 1.5% of yerba mate extract on the diet, pointing out an influence of it on the metabolism of the animal. Regarding to the influence of the diet on the redox status of the bovine meat, samples from the animals fed without supplementation and with 1.5% of yerba mate extract presented, respectively, the formation rates of higher and shorter radicals. Indeed, there was a tendency to create a lower concentration of radicals and a lower rate in accordance with the increase of the level of the yerba mate extract on the diet.

La biologia molecular ha avançat considerablement gràcies a importants progressos com la seqüenciació del ADN o la seva modificació per CRISPR. Tot i això, per entendre el metabolisme requerim estudiar els perfils metabòlics i les seves reaccions metabòliques. L™objectiu d™aquesta tesi és contribuir en aquest estudi del metabolism, el qual unifica dels camps de la proteòmica i la metabolòmica. Tradicionalment, l™anàlisi de dades òmiques es basa en el tractament independent de les diferents variables encara que està profundament establert que els mecanismes moleculars són controlats per la interacció de diferents molècules, i per tant seria més correcte tractar les dades de la mateixa manera. Avui dia, s™han descrit una gran quantitat de vies metabòliques, incluint els enzims responsables de les transformacions dels metabòlits que les formen, aquesta informació s™ha recopilat en bases de dades, que a la vegada poden ser utilitzades per a construir xarxes metabòliques. En aquesta tesi, s™han utilitzat xarxes metabòliques per a desenvolupar un algoritme que prediu metabòlits desregulats basant-se en el perfil d™expressió d™enzims gràcies a proteòmica quantitativa. Per a validar tals prediccions, és possible mesurar l™abundància d™aquests metabòlits, o el seu flux, o sigui la velocitat a la que s™han transformat, utilitzant experiments de marcatge amb isòtops estables, mesures completades mitjançant metabolòmica. Aqui, mostrem els productes del desenvolupament de dos mètodes per a l™anàlisi de dades de metabolòmica per a experiments amb isòtops estables: el primer per a la quantificació dirigida del flux en metabòlits del metabolisme central; i un segon, per la detecció no-dirigida de metabòlits marcats amb isòtops en altres vies metabòliques. Aquests mètodes han sigut provats en diferents estudis on han aportat resultats remarcables, revelant nous mecanismes moleculars en una complicació de la diabetes o en relació al metabolisme del càncer.
La biología molecular ha avanzado considerablemente gracias a progresos como la secuenciación de ADN o su modificación por CRISPR. Sin embargo, para entender el metabolismo es indispensable estudiar los perfiles metabólicos y sus reacciones metabólicas. El objetivo de esta tesis es contribuir en el estudio del metabolismo, el cual implica los campos de la proteómica y la metabolómica. Tradicionalmente, el análisis de datos ómicas se basa en el tratamiento independiente de las diferentes variables aunque está profundamente aceptado que los mecanismos moleculares son controlados por la interacción de diferentes moléculas, y por lo tanto sería más correcto tratar los datos de esa manera. Hoy día, se han descrito una gran cantidad de vías metabólicas, incluyendo las enzimas responsables de las transformaciones de los metabolitos que las forman, esta información se ha recopilado en bases de datos, que a su vez pueden ser utilizadas para construir redes metabólicas . En esta tesis, se han utilizado redes metabólicas para desarrollar un algoritmo que predice metabolitos desregulados basándose en el perfil de expresión de enzimas por proteómica cuantitativa. Para validar tales predicciones, es posible medir la abundancia de estos metabolitos, o su flujo, o sea la velocidad a la que se han transformado, utilizando experimentos de marcado con isótopos estables, estas medidas se obtienen por metabolómica. Aquí, mostramos los productos del desarrollo de dos métodos para el análisis de datos de metabolómica para experimentos con isótopos estables: el primero para la cuantificación dirigida del flujo en metabolitos del metabolismo central; y un segundo, para la detección no-dirigida de metabolitos marcados con isótopos en otras vías metabólicas. Estos métodos han sido probados en diferentes estudios donde han aportado resultados interesantes, revelando nuevos mecanismos moleculares en una complicación de la diabetes o en relación al metabolismo del cáncer.
Understanding the molecular basis of life has been in the spotlight of biochemistry research for more than a century already. Molecular biology has taken medicine forward thanks to technological breakthroughs like DNA sequencing and CRISPR editing. However, in order to understand metabolism we must rely on the study of metabolite profiles and metabolic reactions. The purpose of this thesis to contribute to this area, which unites the fields of proteomics and metabolomics. Traditionally, omics data analysis treats variables independently even if it is strongly settled that molecular mechanisms involve the interaction of diverse pathways, therefore data should be analyzed correspondingly. A vast amount of metabolic pathways have been described, together with enzymes that are responsible for metabolite transformations, this information has been assembled in databases that, in turn, can be used to build metabolic networks. In here, we use metabolic networks to predict metabolite dysregulation based on quantitative proteomics profiles. To validate the predictions, it is possible to measure the abundance of metabolites or their flux, namely the rate at which they are transformed, using stable isotope labelling experiments, both measurements can be performed by metabolomics. In this thesis, two different metabolomics-based stable isotope labelling approaches have been developed, one for the study of central carbon metabolites and one for the unbiased detection of deregulated fluxes in other metabolic pathways. These approaches have been tested on different datasets and have proven valuable to obtain remarkable results, unraveling molecular mechanisms in diabetes complications or novel metabolic hallmarks of cancer.

1n NMR spectroscopy and gas chromatography (GC) based metabolimics approaches were used to characterize the metabolic profiles of a selection of mouse models of atherosclerosis, including the wildtype C57BL/6 mice, the low-density lipoprotein receptor null (LDLR^-/-) mice, and the Apolipoprotein E null (ApoE^-/-) mice. The LDLR^-/- mice demonstrated a profound perturbation in lipid and choline metabolism, notably the choline oxidation pathway, which results in reduced concentrations of choline derivatives in urine. A consistent change in this pathway was also observed in the ApoE^-/- mice. The ApoE^-/- mice exhibited significant changes in energy metabolism, favouring the utilisation of fatty acids for energy production. These findings were also compared with the ApoE^-/- mice that are haplodeficient for the Ataxia telangiectasia mutated (ATM) gene. The ATM^+/-/ApoE^-/- mice demonstrated accelerated atherosclerosis and more severe abnormalities in energy metabolism, as compared with the ATM^+/+/ApoE^-/- mice. Finally, a metabolomic study on an inducible Akt1 transgenic mouse model was conducted. These mice exhibit inducible muscle hypertrophy, as well as a significant reduction in fat mass. The muscle-specific Akt1 activation caused increased glycolysis in gastronemius muscle, as well as increased gluconeogenesis, glycogenolysis and ketogenesis in the liver of the mice. These data demonstrate how hypertrophic muscle affects systemic metabolism, and influences distant organs to feed its active cell growth. The studies demonstrated the impact of a muscle-specific Akt1 activation on systemic metabolism, which leads to a reduction in risks associated with cardiovascular disease.

Advances in bioinformatics and computational biology have enabled integration of an enormous amount of known biological interactions. This has enabled researchers to use models and data to design experiments and guide new discovery as well as test for consistency. One such computational method is constraint-based metabolic flux modeling. This is performed using genome-scale metabolic models (GEMs) that are a collection of biochemical reactions, derived from a genome's annotation. This type of flux modeling enables prediction of net metabolite conversion rates (metabolic fluxes) to help understand metabolic activities under specific environmental conditions. It can also be used to derive metabolic engineering strategies that involve genetic manipulations. Over the past decade, GEMs have been constructed for several different microbes, plants, and animal species. Researchers have also developed advanced algorithms to use GEMs to predict genetic modifications for the overproduction of biofuel and valuable commodity chemicals. Many of the predictive algorithms for microbes were validated with experimental results and some have been applied industrially. However, there is much room for improvement. For example, many algorithms lack straight-forward predictions that truly help non-computationally oriented researchers understand the predicted necessary metabolic modifications. Other algorithms are limited to simple genetic manipulations due to computational demands. Utilization of GEMs and flux-based modeling to predict in vivo characteristics of multicellular organisms has also proven to be challenging. Many researchers have created unique frameworks to use plant GEMs to hypothesize complex cellular interactions, such as metabolic adjustments in rice under variable light intensity and in developing tomato fruit. However, few quantitative predictions have been validated experimentally in plants. This research demonstrates the utility of GEMs and flux-based modeling in both metabolic engineering and analysis by tackling the challenges addressed previously with alternative approaches. Here, a novel predictive algorithm, Node-Reward Optimization (NR-Opt) toolbox, was developed. It delivers concise and accurate metabolic engineering designs (i.e. genetic modifications) that can truly improve the efficiency of strain development. As a proof-of-concept, the algorithm was deployed on GEMs of E. coli and Arabidopsis thaliana, and the predicted metabolic engineering strategies were compared with results of well-accepted algorithms and validated with published experimental data. To demonstrate the utility of GEMs and flux-based modeling in analyzing plant metabolism, specifically its response to changes in the signaling pathway, a novel modeling framework and analytical pipeline were developed to simulate changes of growth and starch metabolism in Arabidopsis over multiple stages of development. This novel framework was validated through simulation of growth and starch metabolism of Arabidopsis plants overexpressing sucrose non-fermenting related kinase 1.1 (SnRK1.1). Previous studies suggest that SnRK1.1 may play a critical signaling role in plant development and starch level (a critical carbon source for plant night growth). It has been shown that overexpressing of SnRK1.1 in Arabidopsis can delay vegetative-to-reproductive transition. Many studies on plant development have correlated the delay in developmental transition to reduction in starch turnover at night. To determine whether starch played a role in the delayed developmental transition in SnRK1.1 overexpressor plants, starch turnover was simulated at multiple developmental stages. Simulations predicted no reduction in starch turnover prior to developmental transition. Predicted results were experimentally validated, and the predictions were in close agreement with experimental data. This result further supports previous data that SnRK1.1 may regulate developmental transition in Arabidopsis. This study further validates the utility of GEMs and flux-based modeling in guiding future metabolic research.
Ph. D.

The worldwide prevalence of obesity and metabolic disease is increasing at an exponential rate and current projections provide no indication of relief. This growing burden of obesity-related metabolic disorders, including type 2 diabetes mellitus (T2DM), highlights the importance of identifying how lifestyle choices, genetics and physiology play a role in metabolic disease and place obese individuals at a greater risk for obesity-related complications including insulin resistance (IR). This increased risk of IR, which is characterized by a decreased response to insulin in peripheral tissues including adipose tissue (AT) and liver, is associated with a chronic, low grade inflammatory state; however, the causative connections between obesity and inflammation remains in question. Experimental evidence suggests that adipocytes and macrophages can profoundly influence obesity-induced IR because adipocyte dysfunction leads to ectopic lipid deposition in peripheral insulin sensitive tissues, and obese AT is characterized by increased local inflammation and macrophage and other immune cell populations. Attempts to delineate the individual roles of macrophage-derived pro-inflammatory cytokines, like tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β), have demonstrated causative roles in impaired systemic insulin sensitivity, adipocyte function and hepatic glucose and lipid metabolism in obese animal models. Thus, the attenuation of macrophage-derived inflammation is an evolving area of interest to provide insight into the underlying mechanism(s) leading to obesity-induced IR. Thus, in the first chapter of this thesis, I describe experiments to refine the current paradigm of obesity-induced AT inflammation by combining gene expression profiling with computational analysis of two anatomically distinct AT depots, visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) to address whether the inflammatory signature of AT is influenced by diet-induced obesity (DIO). Microarray and qRT-PCR analysis data revealed that DIO mouse SAT is resistant to high fat diet (HFD)-induced inflammation and macrophage infiltration, and our data support the current model of obesity-induced visceral adipose tissue macrophage (VATM) enrichment. Our data demonstrated robust increases in VAT pro-inflammatory cytokine expression, which are consistent with the significant increases in macrophage-specific gene expression and consistent with previous reports in which VAT inflammation is enhanced and attributed to classically activated (M1) macrophage infiltration. However, these data are only observed relative to the expression of invariant housekeeping gene expression. When M1-specific genes are expressed relative to macrophage-specific standards like F4/80 expression, these inflammatory makers are unchanged. These data indicate that the changes in the overall inflammatory profile of DIO mouse VAT is because of quantitative changes in adipose tissue macrophage (ATM) number and not qualitative changes in activation state. These observations are consistent with the idea that infiltrating ATMs may have roles other than the previously described role in mediating inflammation in obese adipose tissue. Hepatic IR occurs partly as a consequence of adipocyte dysfunction because the liver becomes a reservoir for AT-derived fatty acids (FAs), which leads to obesity-related non-alcoholic fatty liver disease (NAFLD). In the second part of my thesis, I used clodronate liposome-mediated macrophage depletion to define the role of macrophages in hepatic lipid metabolism regulation. We discovered that i.p. administration of clodronate liposomes depletes Kupffer cells (KCs) in ob/ob mice without affecting VATM content, whereas clodronate liposomes depletes both KCs and VATMs in DIO mice. To this end, we established that clodronate liposome-mediated KC depletion, regardless of VATM content in obese mice, abrogated hepatic steatosis by reducing hepatic de novo lipogenic gene expression. The observed reductions in hepatic inflammation in macrophage-depleted obese mice led to the hypothesis that IL-1β may be responsible for obesity-induced increased hepatic triglyceride (TG) accumulation. We determined that IL-1β treatment increases fatty acid synthase (Fas) protein expression and TG accumulation in primary mouse hepatocytes. Pharmacological inhibition of interleukin-1 (IL-1) signaling by interleukin-1 receptor antagonist (IL-1Ra) administration recapitulated these results by reducing hepatic TG accumulation and lipogenic gene expression in DIO mice. Thus, these data highlight the importance of the inflammatory cytokine IL-1β in obesity-driven hepatic steatosis and suggests that liver inflammation controls hepatic lipogenesis in obesity. To this end, the studies described herein provide new insight and appreciation to the multi-functional nature of macrophages and clinical implications for anti-inflammatory therapy in obesity and NAFLD treatment. We demonstrate the complexities of macrophage-mediated functions in insulin sensitive tissues and a role for obesity-induced inflammatory cytokine IL-1β in hepatic lipid metabolism modulation, which is reversed via IL-1Ra intervention. The use of anti-inflammatory therapy to ameliorate obesity-associated NAFLD was perhaps the most important contribution to this body of work and is full of promise for future clinical application. It is likely that the future of therapeutics will be multi-faceted and combine therapeutic approaches to enhance glucose tolerance and overall health in obese, IR and T2DM patients.

The worldwide prevalence of obesity and metabolic disease is increasing at an exponential rate and current projections provide no indication of relief. This growing burden of obesity-related metabolic disorders, including type 2 diabetes mellitus (T2DM), highlights the importance of identifying how lifestyle choices, genetics and physiology play a role in metabolic disease and place obese individuals at a greater risk for obesity-related complications including insulin resistance (IR). This increased risk of IR, which is characterized by a decreased response to insulin in peripheral tissues including adipose tissue (AT) and liver, is associated with a chronic, low grade inflammatory state; however, the causative connections between obesity and inflammation remains in question. Experimental evidence suggests that adipocytes and macrophages can profoundly influence obesity-induced IR because adipocyte dysfunction leads to ectopic lipid deposition in peripheral insulin sensitive tissues, and obese AT is characterized by increased local inflammation and macrophage and other immune cell populations. Attempts to delineate the individual roles of macrophage-derived pro-inflammatory cytokines, like tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β), have demonstrated causative roles in impaired systemic insulin sensitivity, adipocyte function and hepatic glucose and lipid metabolism in obese animal models. Thus, the attenuation of macrophage-derived inflammation is an evolving area of interest to provide insight into the underlying mechanism(s) leading to obesity-induced IR. Thus, in the first chapter of this thesis, I describe experiments to refine the current paradigm of obesity-induced AT inflammation by combining gene expression profiling with computational analysis of two anatomically distinct AT depots, visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) to address whether the inflammatory signature of AT is influenced by diet-induced obesity (DIO). Microarray and qRT-PCR analysis data revealed that DIO mouse SAT is resistant to high fat diet (HFD)-induced inflammation and macrophage infiltration, and our data support the current model of obesity-induced visceral adipose tissue macrophage (VATM) enrichment. Our data demonstrated robust increases in VAT pro-inflammatory cytokine expression, which are consistent with the significant increases in macrophage-specific gene expression and consistent with previous reports in which VAT inflammation is enhanced and attributed to classically activated (M1) macrophage infiltration. However, these data are only observed relative to the expression of invariant housekeeping gene expression. When M1-specific genes are expressed relative to macrophage-specific standards like F4/80 expression, these inflammatory makers are unchanged. These data indicate that the changes in the overall inflammatory profile of DIO mouse VAT is because of quantitative changes in adipose tissue macrophage (ATM) number and not qualitative changes in activation state. These observations are consistent with the idea that infiltrating ATMs may have roles other than the previously described role in mediating inflammation in obese adipose tissue. Hepatic IR occurs partly as a consequence of adipocyte dysfunction because the liver becomes a reservoir for AT-derived fatty acids (FAs), which leads to obesity-related non-alcoholic fatty liver disease (NAFLD). In the second part of my thesis, I used clodronate liposome-mediated macrophage depletion to define the role of macrophages in hepatic lipid metabolism regulation. We discovered that i.p. administration of clodronate liposomes depletes Kupffer cells (KCs) in ob/ob mice without affecting VATM content, whereas clodronate liposomes depletes both KCs and VATMs in DIO mice. To this end, we established that clodronate liposome-mediated KC depletion, regardless of VATM content in obese mice, abrogated hepatic steatosis by reducing hepatic de novo lipogenic gene expression. The observed reductions in hepatic inflammation in macrophage-depleted obese mice led to the hypothesis that IL-1β may be responsible for obesity-induced increased hepatic triglyceride (TG) accumulation. We determined that IL-1β treatment increases fatty acid synthase (Fas) protein expression and TG accumulation in primary mouse hepatocytes. Pharmacological inhibition of interleukin-1 (IL-1) signaling by interleukin-1 receptor antagonist (IL-1Ra) administration recapitulated these results by reducing hepatic TG accumulation and lipogenic gene expression in DIO mice. Thus, these data highlight the importance of the inflammatory cytokine IL-1β in obesity-driven hepatic steatosis and suggests that liver inflammation controls hepatic lipogenesis in obesity. To this end, the studies described herein provide new insight and appreciation to the multi-functional nature of macrophages and clinical implications for anti-inflammatory therapy in obesity and NAFLD treatment. We demonstrate the complexities of macrophage-mediated functions in insulin sensitive tissues and a role for obesity-induced inflammatory cytokine IL-1β in hepatic lipid metabolism modulation, which is reversed via IL-1Ra intervention. The use of anti-inflammatory therapy to ameliorate obesity-associated NAFLD was perhaps the most important contribution to this body of work and is full of promise for future clinical application. It is likely that the future of therapeutics will be multi-faceted and combine therapeutic approaches to enhance glucose tolerance and overall health in obese, IR and T2DM patients.

This project aimed to investigate the role of the γ-lactone of 3,4-dihydroxybutanoic acid (3,4DB) which was claimed by Japanese investigators to be one of several endogenous γ-lactones involved in the control of food intake. Tissue, plasma and urine organic acid profiles were screened for the γ-lactone of 3,4DB using both GC and GCMS. Careful mass spectral analysis and in vitro acid-γ-lactone exchange analyses with structural validation studies showed that the γ-lactone of 3,4DB did not occur in vivo but that the free acid of 3,4DB did. This rejects previous claims to the contrary. Fasting increased rat plasma 3,4DB with urinary output significantly elevated for the first 24h of fasting. A metabolic route from glutamate to acetate is proposed with 3,4DB as an intermediate. Peripheral administration of glutamate, γ-aminobutyric acid (GABA) and 4-hydroxybutyric acid (4HB) to rats reduced food intake with increasing effects at each stage of the pathway. The γ-lactone 2-buten-4-olide is toxic and other γ-lactones had more potent effects than their free acids on rats. The intragastric administration of GABA stimulated 4HB production in vivo and GABA or 4HB increased plasma 3,4DB levels, thus implying that 3,4DB occurs at an intermediate in our proposed pathway. A likely endogenous source of 3,4DB is as a minor GABA metabolite. The response of the free acid of 3,4DB to fasting followed a similar trend as that reported for its γ-lactone and related γ-lactones are unlikely to occur in vivo. Given the similarities of 4HB (a precursor of 3,4DB) and 3,4DB in structure to the ketone body 3-hydroxybutyrate (3HB) and the similarity of 3,4DB in fasting response to 3HB, both 4HB and 3,4DB may initiate anorexia in fasting.