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Vitamin E was first described in 1922 as an unknown factor required for impregnated rats to carry their offspring to term. In fact, when vitamin E was chemically characterized it was given the name "tocopherol" derived from the Greek: tokos = childbirth; phero = to bear; and –ol, indicating an alcohol. Vitamin E is linked to animal health and wellness, maternal fertility and a human neurodegenerative condition, ataxia with vitamin E deficiency However, embryonic vitamin E requirements during development remained unknown. We hypothesized that vitamin E is critical, not only for the mother, but specifically by the embryo for proper development. To separate the embryonic and maternal requirements, we employed an innovative model for the study of vitamin E: the zebrafish. We began by formulating and testing the first fully defined diet sufficient for zebrafish health. We then removed vitamin E from the formula to create our E deficient (E-) diet, which, when fed to adult zebrafish (for >3 months), resulted in E- adults that produced viable, E- gametes. Deficient embryos initially developed normally; however, by 48 hours post fertilization (hpf), E- embryos developed severe malformations leading to significant mortality. Thus, we demonstrated for the first time an embryonic vitamin E requirement. We provided further insight into the embryonic vitamin E requirement by analyzing the transcriptional changes occurring prior to the observed malformations. The transcriptome revealed a putative mechanism of action for vitamin E in development, in which vitamin E deficiency leads to the dysregulation of key metabolic co-activators. Finally, to understand the trafficking of vitamin E, we identified the zebrafish α-tocopherol transfer protein (TTP). We demonstrated that the zebrafish TTP is homologous to its human counterpart, and its expression is both spatially and temporally regulated during embryonic development. Knocking down the expression of TTP, using morpholinos injected at the one-cell stage, resulted in early and severe malformations in the developing head and tail. Consequently we revealed a definitive role for TTP during development. Taken together the work described here presents a new direction for future research into the role of vitamin E and TTP in post-implantation development. / Graduation date: 2012

Vitamin E wird immer noch als das wichtigste lipophile Antioxidanz in biologischen Membranen betrachtet. In den letzten Jahren hat sich jedoch der Schwerpunkt der Vitamin E-Forschung hin zu den nicht-antioxidativen Funktionen verlagert. Besonderes Interesse gilt dabei dem α-Tocopherol, der häufigsten Vitamin E-Form im Gewebe von Säugetieren, und seiner Rolle bei der Regulation der Genexpression. Das Ziel dieser Dissertation war die Untersuchung der genregulatorischen Funktionen von α-Tocoperol und die Identifizierung α-Tocopherol-sensitiver Gene in vivo. Zu diesem Zweck wurden Mäuse mit verschiedenen Mengen α-Tocopherol gefüttert. Die Analyse der hepatischen Genexpression mit Hilfe von DNA-Microarrays identifizierte 387 α-Tocopherol-sensitive Gene. Funktionelle Clusteranalysen der differentiell exprimierten Gene zeigten einen Einfluss von α-Tocooherol auf zelluläre Transportprozesse. Besonders solche Gene, die an vesikulären Transportvorgängen beteiligt sind, wurden größtenteils durch α-Tocopherol hochreguliert. Für Syntaxin 1C, Vesicle-associated membrane protein 1, N-ethylmaleimide-sensitive factor and Syntaxin binding protein 1 konnte eine erhöhte Expression mittels real time PCR bestätigt werden. Ein funktioneller Einfluss von α-Tocopherol auf vesikuläre Transportprozesse konnte mit Hilfe des in vitro β-Hexosaminidase Assays in der sekretorischen Mastzelllinie RBL-2H3 gezeigt werden. Die Inkubation der Zellen mit α-Tocopherol resultierte in einer konzentrationsabhängigen Erhöhung der PMA/Ionomycin-stimulierten Sekretion der β-Hexosaminidase. Eine erhöhte Expression ausgewählter Gene, die an der Degranulation beteiligt sind, konnte nicht beobachtet werden. Damit schien ein direkter genregulatorischer Effekt von α-Tocopherol eher unwahrscheinlich. Da eine erhöhte Sekretion auch mit β-Tocopherol aber nicht mit Trolox, einem hydrophilen Vitamin E-Analogon, gefunden wurde, wurde vermutet, dass α-Tocopherol die Degranulation möglicherweise durch seine membranständige Lokalisation beeinflussen könnte. Die Inkubation der Zellen mit α-Tocopherol resultierte in einer veränderten Verteilung des Gangliosids GM1, einem Lipid raft Marker. Es wird angenommen, dass diese Membranmikrodomänen als Plattformen für Signaltransduktionsvorgänge fungieren. Ein möglicher Einfluss von Vitamin E auf die Rekrutierung/Translokation von Signalproteinen in Membranmikrodomänen könnte die beobachteten Effekte erklären. Eine Rolle von α-Tocopherol im vesikulären Transport könnte nicht nur seine eigene Absorption und seinen Transport beeinflussen, sondern auch eine Erklärung für die bei schwerer Vitamin E-Defizienz auftretenden neuronalen Dysfunktionen bieten. Im zweiten Teil der Arbeit wurde die α-Tocopheroltransferprotein (Ttpa) Knockout-Maus als genetisches Modell für Vitamin E-Defizienz verwendet, um den Effekt von Ttpa auf die Genexpression und die Gewebeverteilung von α-Tocopherol zu analysieren. Ttpa ist ein cytosolisches Protein, das für die selektive Retention von α-Tocopherol in der Leber verantwortlich ist. Die Ttpa-Defizienz resultierte in sehr geringen α-Tocopherol-Konzentrationen im Plasma und den extrahepatischen Geweben. Die Analyse der α-Tocopherol-Gehalte im Gehirn wies auf eine Rolle von Ttpa bei der α-Tocopherol-Aufnahme ins Gehirn hin. / Vitamin E is still considered the most important lipid-soluble antioxidant within biological membranes. However, in the last years the non-antioxidant functions of vitamin E have become the focus of vitamin E research. From the eight members of the vitamin E family, specific emphasis is given to α-tocopherol, the most abundant vitamin E form in mammalian tissues, and its role in the regulation of gene expression. The aim of this thesis was the analysis of the gene regulatory functions of α-tocopherol and the identification of α-tocopherol sensitive genes in vivo. For this purpose mice were fed diets differing in α-tocopherol content. The analysis of hepatic gene expression using DNA microarrays identified 387 α-tocopherol-sensitive genes. Functional cluster analyses of these differentially expressed genes demonstrated an influence of α-tocopherol on cellular transport processes. Especially the expression of genes involved in vesicular trafficking was largely upregulated by α-tocopherol. Upregulation of syntaxin 1C, vesicle-associated membrane protein 1, N-ethylmaleimide-sensitive factor and syntaxin binding protein 1 was verified by real time PCR. A role of α-tocopherol in exocytosis was shown by the in vitro β-hexosaminidase release assay in the secretory mast cell line RBL-2H3. Incubation with α-tocopherol resulted in a concentration dependent increase of PMA/ionomycin-stimulated secretion of β-hexosaminidase. Induction of selected genes involved in degranulation was not observed at any time point. Thus, a direct gene-regulatory effect of α-tocopherol seemed rather unlikely. Since increased secretion was also observed with ß-tocopherol but not with trolox, a water-soluble analog of vitamin E, it was hypothesized that α-tocopherol might affect degranulation through its localization at the plasma membrane. Incubation of cells with α-tocopherol changed the distribution of the gangliosid GM1, a Lipid raft marker. These membrane microdomains are assumed to function as signaling platforms. An possible influence of vitamin E on the recruitment/translocation of signaling proteins into membrane microdomains could explain the observed effects. A role of α-tocopherol in the vesicular transport might not only affect its own absorption and transport but also explain the neural dysfunctions observed in severe α-tocopherol deficiency. In the second part of this dissertation the α-tocopherol transfer protein (Ttpa) knockout-mouse as a model of genetic vitamin E deficiency was used to analyze the effect of Ttpa gene expression and tissue distribution of α-tocopherol. Ttpa is a cytosolic protein, which is responsible for the selective retention of α-tocopherol in the liver. Its deficiency resulted in very low α-tocopherol concentrations in plasma and extrahepatic tissues. Analysis of α-tocopherol contents in brain indicated a role for Ttpa in the uptake of α-tocopherol into the brain.

alpha-tocopherol is the most abundant form of vitamin E in human plasma and tissues. Inter-individual differences in plasma alpha-tocopherol concentration or its response to dietary alpha-tocopherol may be due, in part, to polymorphisms in vitamin E metabolism genes (alpha-tocopherol transfer protein (alpha-TTP), tocopherol associated protein (TAP) and CYP4F2). The thesis objectives were to determine whether common polymorphisms in the alpha-TTP (rs6994076 A>T), TAP (rs2072157 C>T and Arg11Lys) and CYP4F2 (Val433Met) genes influence plasma alpha-tocopherol concentration or modify the association between dietary and plasma alpha-tocopherol. Subjects (n=1248), 20 to 29 years from the Toronto Nutrigenomics and Health study completed a food frequency questionnaire. Fasting blood samples were used for genotyping and to measure plasma alpha-tocopherol concentration. The alpha-TTP and TAP Arg11Lys polymorphisms significantly altered plasma alpha-tocopherol. The alpha-TTP polymorphism only influenced plasma alpha-tocopherol in individuals not using supplements. None of the polymorphisms examined modified the plasma alpha-tocopherol response to dietary alpha-tocopherol.

alpha-tocopherol is the most abundant form of vitamin E in human plasma and tissues. Inter-individual differences in plasma alpha-tocopherol concentration or its response to dietary alpha-tocopherol may be due, in part, to polymorphisms in vitamin E metabolism genes (alpha-tocopherol transfer protein (alpha-TTP), tocopherol associated protein (TAP) and CYP4F2). The thesis objectives were to determine whether common polymorphisms in the alpha-TTP (rs6994076 A>T), TAP (rs2072157 C>T and Arg11Lys) and CYP4F2 (Val433Met) genes influence plasma alpha-tocopherol concentration or modify the association between dietary and plasma alpha-tocopherol. Subjects (n=1248), 20 to 29 years from the Toronto Nutrigenomics and Health study completed a food frequency questionnaire. Fasting blood samples were used for genotyping and to measure plasma alpha-tocopherol concentration. The alpha-TTP and TAP Arg11Lys polymorphisms significantly altered plasma alpha-tocopherol. The alpha-TTP polymorphism only influenced plasma alpha-tocopherol in individuals not using supplements. None of the polymorphisms examined modified the plasma alpha-tocopherol response to dietary alpha-tocopherol.