Multi-modal Analysis of Metabolic Enzyme Disruption in Adipocytes.
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 c... read moreontribute 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
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.read less