%0 PDF %T Multi-modal Analysis of Metabolic Enzyme Disruption in Adipocytes. %A Sims, James. %8 2017-04-20 %R http://localhost/files/5x21tt22h %X Abstract: Obesity results from a chronic imbalance in caloric intake and expenditure. The excess calories are stored mainly as lipids (triglycerides, TG), leading to an expansion of body fat or adipose tissue through increases in fat cell (adipocyte, AD) size and number. One approach to controlling body fat could be to intervene in the metabolic processes of the adipose tissue that directly contribute to lipid accumulation and cell growth. The goal of this study is to investigate targets for reducing AD size and TG accumulation via RNAi-mediated gene silencing (i.e. siRNA knockdown) of metabolic proteins. Targets for siRNA knockdown were selected based on our knowledge of adipocyte metabolism, and were identified from each stage of lipid accumulation: early synthesis (breakdown and transformation of glycolysis intermediates into precursors of fatty acid synthesis), late synthesis (formation of triglyceride from synthesized precursors), and droplet stability (protection from intracellular lipases and formation of larger lipid droplets). Pyruvate carboxylase (PCX) and isocitrate dehydrogenase (IDH) were identified as targets for early synthesis, diglyceride acyltransferase (DGAT) for late synthesis, and fat-specific protein 27 (FSP) and perilipin (PLIN) for droplet stability. In addition to monitoring TG accumulation via conventional, destructive biochemical assays, we developed an image processing method, which identifies LDs in microscopy images, and quantifies their number, size distribution, and total lipid content. This method could prospectively be used as a high-throughput screening method for a variety of applications. We also conducted a metabolomic analysis and isotopic labeling experiments to understand how disrupting each enzyme affected metabolite concentrations and reaction fluxes across the entire metabolic network. We have shown successful knockdown of several metabolic proteins, which led to reduction in accumulation of triglycerides; however, inhibition of PCX and DGAT had the greatest individual effects. Interestingly, DGAT and FSP had the greatest combined effect on and this combination was selected for further analysis. Prospectively, results from this study could be used to identify potential therapeutic targets for treating obesity, guided by an improved understanding of how the enzymes and LD proteins regulate the balance of TG in adipocytes.; Thesis (Ph.D.)--Tufts University, 2015.; Submitted to the Dept. of Chemical and Biological Engineering.; Advisor: Kyongbum Lee.; Committee: Yaguang Si, Catherine Kuo, and Qiaobing Xu.; Keyword: Chemical engineering. %[ 2022-10-11 %9 Text %~ Tufts Digital Library %W Institution