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Title Oxidized soybean oil alters the expression of PPAR gamma and target genes in 3T3-L1 cells
Publication Date
Degree MS
Discipline/Department Nutrition
Degree Level thesis
University/Publisher Georgia State University
Abstract <strong>Background: </strong> The typical western diet contains foods with modest amounts of lipid oxidation products. Previous work by us and others have demonstrated that mildly oxidized lipids promote a gain in fat mass while highly oxidized lipids decrease fat mass in rodents and triglyceride (TAG) accumulation in 3T3-L1 cells. Adipocyte differentiation is regulated by a key nuclear transcription factor known as PPARγ. <strong>Objective:</strong> To investigate if the alterations in triglyceride accumulation in 3T3-L1 cells pretreated with oxidized soy oil are due to 1) a change in PPARg DNA interactions 2) changes in the expression of SREBP-1c, PPARg, and/or its target genes. <strong>Main Methods: </strong> Confluent 3T3-L1 cells were pretreated for 24hours with 0.01% soy oil (SO) which was either unheated (unheated SO) or heated for 3, (3h-SO), 6 (6h-SO), or 9hours (9h-SO). The effect of 24hour soy oil exposure was assessed at several time points throughout the differentiation process. Alterations in PPARg DNA interaction was assessed using a PPARγ transcription factor assay kit while alterations in the expression of genes upstream and downstream of PPARγ was determined by RT-PCR. Primary and secondary products of oxidation within the SO were determined by spectrophotometry. <strong>Results:</strong> The 6hr-SO contained the greatest concentration of peroxides whereas both the 6hr-SO and 9hr-SO contained a significantly higher concentration of conjugated dienes and aldehydes.Nuclear extracts from 3T3-L1 cells pretreated with 6h-SO demonstrated the greatest reduction in PPARγ DNA binding. Compared to the unheated SO and mildly oxidized 3h-SO, cells treated with the 6h-SO had a significant reduction in SREBP-1c, PPARg, LPL, and GLUT4 expression occurring early in the differentiation process. Variations in the gene expression of 6hr-SO pretreated cells persisted within partially differentiated and mature adipocytes. <strong>Conclusions: </strong>Pre-treatment of preadipocytes with soy oil heated for ³ 6h greatly decreases the activity of PPARγ in the nucleus and adipogenic gene expression . These changes seen in early differentiation seem to correlate the best with the phenotype of reduced triglyceride accumulation seen in mature adipocytes.
Subjects/Keywords PPAR gamma; oxidized lipids; gene expression; adipocyte differentiation; triglyceride accumulation; 3T3-L1 cells
Contributors Meera Penumetcha; Nalini Santanam; Barbara Hopkins
Country of Publication us
Record ID oai:scholarworks.gsu.edu:nutrition_theses-1039
Repository georgia-state
Date Retrieved
Date Indexed 2020-09-25
Created Date 2012-11-15 08:00:00

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…confirmed by the presence of solid, intact bands using gel electrophoresis 25 6 Influence of unheated SO and oxidized SO on adipogenic gene expression in 3T3-L1 cells 26 7 PPARγ protein levels determined by Western blot 27 v ABBREVIATIONS CD…

…x29; in partially differentiated cells and Day 15 (B) in Mature, fully differentiated cells. PPARγ DNA activity is expressed as percent change relative to the unheated SO. Gene Expression in 3T3-L1 cells after a one time treatment with…

…oxidized soy oil: RNA quality was confirmed by gel electrophoresis within 100% confluent cells, control cells, unheated SO, 3hr-SO, 6hr-SO, and 9hr-SO treated cells (Figure 5). The gene expression of SREBP-1c, PPARγ, LPL, and GLUT-4 within 3T3-L1

…adipogenic gene expression in 3T3-L1 cells Figure 6. SREBP1c (6A), PPARγ (6B), LPL (6C) and GLUT-4 (6D) mRNA expression within 3T3-L1 pretreated for 24 hours before the induction of differentiation with unheated SO…

…TABLES Table Page Number 1. PCR Primer Sequence 19 2. Effect of heat duration on soy oil oxidation 22 iv FIGURES Figure Page Number 1 PPARγ pathway of gene transcription, regulation, and adipogenesis 6 2 Timeline of Soy oil experiment…

…16 3 Differentiation of untreated 3T3-L1 cells 23 4 PPARγ transcription factor binding in nuclear extracts from 3T3-L1 after a one time treatment with 0.01% unheated or heated soybean oil 24 5 The presence and quality of isolated RNA were…

…responds by promoting the transcription/repression of specific target genes6. Without ligand binding, PPARγ is inactive; nuclear corepressors bind to the heterodimer and downregulate gene transcription by histone deactylase recruitment8. PPARs are unique…

…considerable variability exists in the ability of endogenous ligands to activate PPARγ and influence gene transcription 10. Of the natural ligands, PUFAs and their derivatives, fatty acid metabolites, hormones, and certain prostanoids, have all demonstrated a…