Thesis advisor: Thomas N. Seyfried / Two major metabolic phenomena observed in cancer cells include the Warburg effect and Crabtree effect. The Crabtree effect is the in vitro inhibition of respiration by glucose. The influence of glucose on the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of tumorigenic RAW264.7 and VM-M3 macrophage cells, as well as non-tumorigenic BV-2 microglia cells, was studied using the Seahorse XF96 extracellular flux analyzer. RAW264.7, VM-M3, and BV-2 cells incubated in glucose medium displayed a significantly lower OCR and higher ECAR compared to cells incubated in no glucose medium. Furthermore, when glucose medium was added to the RAW264.7 and BV-2 cells in real-time using the Seahorse XF96 injection ports, a rapid decrease in OCR and increase and ECAR was observed. Therefore, RAW264.7, VM-M3, and BV-2 cells display a robust Crabtree effect in vitro, as assessed by OCR and ECAR. Additionally, it is important to consider the Crabtree effect when studying in vitro energy metabolism of all cell and tissue types. It was also found that the elimination of the Crabtree effect through glucose deprivation resulted in dynamic cardiolipin (CL) fatty acid changes in VM-M3 cells. VM-M3 cells incubated in 10 mM glucose medium for four hours displayed a short-chain, saturated (immature) CL fatty acid composition, while VM-M3 cells incubated in no glucose media for four hours displayed long-chain, unsaturated (mature) CL fatty acid composition. Cardiolipin (CL) is a phospholipid highly enriched in the inner mitochondrial membrane. Mature, long-chain, unsaturated CL molecular species are involved in maintaining mitochondrial function and membrane integrity. Overall, these data suggest that CL fatty acid composition may function as a structural component of the Crabtree effect in vitro. The Warburg effect, or aerobic glycolysis, is the observation that tumor cells consume less oxygen and more glucose than normal, untransformed cells in the presence of oxygen. It has been shown that immune cells display a Warburg effect upon activation by changing their core metabolism from oxidative phosphorylation to glycolysis. In this study, it was observed that both RAW264.7 macrophage cells and BV-2 microglia cells display a significantly lower OCR and higher ECAR following LPS-activation. However, this observation is dependent on the concentration of LPS. Therefore, these data suggest that both RAW264.7 and BV-2 cells display a LPS concentration-dependent change in metabolism from oxidative phosphorylation to glycolysis upon LPS-activation in vitro. The in vitro lipid profiles that resulted from the Crabtree effect and the LPS-activated Warburg effect were also studied in the RAW264.7 cell line. The lipids phosphatidylserine (PS) and cardiolipin (CL) displayed the most robust changes in the RAW264.7 cells. Both PS and CL have been shown to be associated with cellular respiration. / Thesis (MS) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.
| Identifer | oai:union.ndltd.org:BOSTON/oai:dlib.bc.edu:bc-ir_106811 |
| Date | January 2016 |
| Creators | Brown, Ashley Kaye |
| Publisher | Boston College |
| Source Sets | Boston College |
| Language | English |
| Detected Language | English |
| Type | Text, thesis |
| Format | electronic, application/pdf |
| Rights | Copyright is held by the author, with all rights reserved, unless otherwise noted. |
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