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Title:Natural versus synthetic alpha-tocopherol using cell and animal models
Author(s):Ranard, Katherine Marie
Director of Research:Erdman, John W
Doctoral Committee Chair(s):Jeffery, Elizabeth H
Doctoral Committee Member(s):Juraska, Janice M; Nakamura, Manabu T
Department / Program:Nutritional Sciences
Discipline:Nutritional Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):vitamin E
natural alpha-tocopherol
synthetic alpha-tocopherol
aortic smooth muscle cells
aortic endothelial cells
Ttpa-null mouse model
Abstract:Vitamin E (α-tocopherol, α-T) is one of nature’s famous lipid-soluble chain-breaking antioxidants. Although originally discovered in the 1920s as an essential rodent fertility factor, decades of research also demonstrate α-T’s essentiality in the lipid-rich, oxidative-stress-prone environment of the central nervous system. Indeed, severe α-T deficiency is characterized by neurologic symptoms, such as impaired motor coordination and peripheral neuropathy. α-T’s known anti-inflammatory properties may also benefit other areas of health, especially cardiovascular disease and immune response. Unlike wild animals that obtain their α-T exclusively from nature, humans consume naturally-occurring α-T from plant components, as well as a synthetic form from α-T-fortified food products and supplements. These two α-T sources differ in their stereochemistry: Synthetic α-T is an equal mixture of the molecule’s eight possible stereoisomers, while natural α-T includes only one of these eight stereoisomers. Due principally to these stereochemical differences, natural α-T is more biologically potent than synthetic α-T. However, the exact ratio of biopotency between natural and synthetic α-T is debated. Rat fetal-resorption bioassays suggest a 1.36:1 ratio, but human bioaccumulation data and the current dietary intake recommendations support a 2:1 ratio. Importantly, these data are not sufficient to determine the ratio of biopotency; fetal-resorption is not a translatable functional endpoint for humans, and bioaccumulation data do not necessarily reflect biological activity in tissues. Our studies are linked by a common objective: To directly compare dietary natural vs. synthetic α-T using human-relevant functional outcomes. These are the first steps towards assigning the ratio of biopotency and setting appropriate α-T intake recommendations. To study α-T in the context of cardiovascular diseases, we treated human aortic vascular cell lines with either natural or synthetic α-T (Chapter 2). We then stimulated cells with a pro-inflammatory cytokine (tumor necrosis factor-α, TNF-α), and measured the treatments’ effects on genes related to inflammation and cardiovascular disease. Pre-treatment with either α-T source prevented the TNF-α-induced increase of TNF. However, pre-treatment with a different vitamin E-like antioxidant (γ-tocopherol) did not prevent this response. The differing gene expression profiles between α-T and γ-tocopherol support two important notions: 1) α-T has distinct anti-inflammatory properties, and 2) α-T may function through non-antioxidant mechanisms to modulate gene expression, perhaps through indirect cell signaling pathways and/or direct transcriptional regulation. α-T has an unequivocal role in neurological health. We, therefore, compared the effects of α-T sources in the mouse central nervous system and did so during two vulnerable stages of life: adolescence, when the brain is still developing, and adulthood, when the brain is more susceptible to oxidative-stress and functional decline. Brain α-T levels are not easily depleted in wild-type mice through dietary α-T restriction alone, so we used the transgenic α-T transfer protein-null (Ttpa-/-) mouse model. These mice develop ataxia and other neurological α-T deficiency symptoms similar to humans. Ttpa+/- dams were used to generate Ttpa-/- and Ttpa+/+ (wild-type) littermates for our studies. The α-T-content in Ttpa+/- dams’ diet must be sufficient for maintaining fertility, while also minimizing the transfer of α-T to brains of the offspring to be used for studies. Therefore, we developed two diet strategies to optimize these parameters. The first strategy included α-T-dosing cycles and was implemented in the adolescent mouse study. The second strategy provided dams with a consistent low dietary α-T concentration throughout breeding and was implemented in the adult mouse study. As a secondary analysis, we assessed and compared the effectiveness and feasibility of these two breeder diet strategies (Chapter 3). The optimal diet strategy highly depends on the study design and objectives. Our findings will help standardize the breeding methodology used to generate Ttpa-/- mice for future neurological α-T studies. In the adolescent mouse study, we evaluated the effects of α-T dose and source on brain α-T accumulation and cerebellar gene expression (Chapter 4). Three-week old male Ttpa-/- weanlings were fed 1 of 4 AIN-93G-based diets until 7 weeks of age: vitamin E deficient (VED); natural α-T, 600 mg/kg diet (NAT); synthetic α-T, 816 mg/kg diet (SYN), i.e. 1.36-times the mass of NAT; or high synthetic α-T, 1200 mg/kg diet (HSYN), i.e. 2-times the mass of NAT. Male Ttpa+/+ littermates fed AIN-93G served as controls (CON). Total brain α-T concentrations were increased in NAT, SYN, and HSYN groups compared to the VED group, but α-T levels were still significantly lower than CON. Brain α-T stereoisomer compositions were substantially different between natural and synthetic α-T groups. The naturally-occurring stereoisomer was predominant in brains of mice fed the NAT diet, while the majority of α-T in SYN and HSYN brains consisted of synthetic stereoisomers. Very few of the 16,774 cerebellar genes measured via RNA-sequencing were differentially expressed. However, compared with the NAT diet, HSYN significantly downregulated genes related to myelin, a neuron insulator that increases signal transmission efficiency. The modulated genes included two key transcription factors: SRY-box transcription factor 10 (Sox10) and myelin regulatory factor (Myrf), as well as several downstream target genes. These α-T source-dependent gene expression changes in the adolescent mouse cerebellum could lead to morphological and functional abnormalities later in life. Our adult Ttpa-/- mouse study used the same α-T diets as the adolescent mouse study to maximize comparisons between life stages (Chapter 5). For the adult study, weanling mice were fed their respective diets until 10 months of age. We conducted analyses on both the spinal cord and cerebellum, as α-T-deficiency pathologies have been detected in these two tissues. Spinal cords of VED-fed mice had increased expression of neuroinflammatory genes, such as Tnf and Ccl2. Both α-T sources normalized this outcome in the spinal cord. Cerebellar pathology was not evident in our 10 month old Ttpa-/- mice; these histological aberrations may only occur during late adulthood. The spinal cord appeared to be particularly sensitive to α-T status, and these molecular findings aligned with the known morphological and neurobehavioral deficits that accompany α-T deficiency. We studied the biological effects of dietary natural vs. synthetic α-T using both cell and animal models. Overall, our contributions confirm the anti-inflammatory properties of α-T in the contexts of both cardiovascular and neurological health. We also explored multiple human-relevant outcomes, which are foundational for future studies comparing the two α-T sources. Looking to the future of α-T research, there are many potential areas of focus. The relative biopotency between α-T sources remains an open question; the α-T dose comparisons in our studies were limited. Continuing to test how α-T status affects gene expression could hint at α-T’s mechanism of action in tissues, whether it be through antioxidant or non-antioxidant pathways. More targeted research using a variety of models will help establish the role of α-T source in specific health outcomes across the lifespan, and ultimately inform α-T intake recommendations.
Issue Date:2020-11-20
Type:Thesis
URI:http://hdl.handle.net/2142/109496
Rights Information:Copyright 2020 Katherine Marie Ranard
Date Available in IDEALS:2021-03-05
Date Deposited:2020-12


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