Intracellular metabolic fluxes provide important information about the internal operation of mammalian cells when exposed to different external conditions. In this work, we have quantified detailed metabolic flux profiles for proliferating, differentiating, and hypoxic mammalian cells. To accomplish this goal, we performed comprehensive stable-isotopic tracer experiments coupled with mass spectrometry and 13C metabolic flux analysis (13C-MFA). Specifically, we performed four parallel labeling experiments under each metabolic condition using the following optimal tracers: [1 -13C]glucose + [4,5,6 - 13C]glucose, [2 -13C]glucose + [4,5,6 - 13C]glucose, [3 -13C]glucose + [4,5,6 - 13C]glucose, and [U -13C]glutamine; subsequently, we collected intracellular mass isotopomer distributions as well as external growth and nutrient uptake and secretion rates and analyzed the data. With this information, we then applied 13C-MFA using the software METEAN to generate detailed metabolic flux maps of intracellular metabolism. Through these comprehensive investigations we identified how metabolic pathways were metabolically rewired in response to different growth phases and oxygenation conditions, including dramatic changes in glycolysis, TCA cycle, oxidative pentose phosphate pathway, and lipogenesis. For example, comparing proliferating to differentiating cellular metabolism, we observed reversal of glutamine flux (i.e. from net consumption to net production), more than 2-fold upregulation of glycolysis flux, more than 4-fold reduction in lactate secretion, and significant upregulation of lipogenesis. Comparing normoxic to hypoxic metabolism, we observed more than 4-fold increase in lactate production and more than 4-fold reduction in citric acid cycle activity. These results provide important new insights into the regulation of mammalian metabolism.

13C metabolic flux analysis quantifies changes in metabolism during proliferation, differentiation, and under low oxygen conditions

ACS Fall 2020 Virtual Meeting

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