d that the metabolism of glutamine through reductive carboxylation is more active in PC-3M cells than in PC-3S cells. These results are consistent with a higher ability of PC-3M cells for using alternative metabolic pathways to sustain energy production and to rapidly adapt to changes in nutrient availability. PDH is a key enzyme that A-83-01 site mediates the entry of pyruvate to mitochondria whose activity is regulated by its phosphorylation status through the activity of pyruvate dehydrogenase kinase . Treatment of cells with the PDHK inhibitor dichloroacetic acid or the LDH inhibitor oxamate resulted in a more pronounced increase in mitochondrial respiration in PC-3M cells than in PC-3S cells, particularly in response to DCA. This suggests that PDH has a lower baseline activity in PC-3M cells than in PC-3S cells. Indeed, PDH was markedly more phosphorylated in PC-3M cells than in PC-3S cells and transcript levels for PDHK1 were higher and the PDH phosphatase PDP2 lower in PC-3M cells than in 
PC-3S cells. We next used Isodyn to integrate 13C-based isotopologue distributions and biochemical data to estimate comparative metabolic flux distributions relative to glucose uptake in central carbon metabolic networks in both cell types. The resulting metabolic flux simulations involving TCA cycle-related reactions lend further support to the occurrence of an enhanced entry of pyruvate via PDH into the TCA cycle, increased TCA cycle fluxes and a higher flux associated with mitochondrial respiration in PC-3S cells than in PC-3M cells, consistent with a higher diversion of glucose into mitochondrial metabolism. Glutaminolysis protects cells from acidity generated from a marked Warburg effect: an Achilles heel of e-CSCs To explore the role of an enhanced glutamine metabolism in PC-3M cells, we first determined the expression levels of glutaminase, a direct MYC target frequently expressed at high levels in rapidly proliferating cells and CSCs. GLS1 was expressed at significantly higher levels in PC-3M cells than PC-3S cells, in particular the glutaminase C isoform. The higher expression levels of GAC vs. the kidney-type glutaminase in PC-3M cells were partly dependent on the maintenance of an epithelial gene program, as shown by a diminished GAC/KGA ratio in PC-3M/Snai1 cells as compared to control PC-3M cells. Exposure to the glutaminase inhibitor BPTES inhibited the proliferation of PC-3M cells more significantly than that of PC-3S cells, associated with a decreased proportion of cells in the S phase of the cell cycle. The sensitivity of PC-3M cells to growth inhibition by BPTES was attenuated by overexpression of Snai1, illustrating that the strong dependence on glutaminolysis of PC-3M cells relies on the maintenance of an epithelial gene program. Both PC-3M and PC-3S cells increased lactate production upon treatment with BPTES, suggesting the participation of metabolic compensatory mechanisms, and thus we tested the effects on viability of a combined inhibition of glutaminolysis and glycolysis. We observed a marked additive effect on cell death by combining BPTES and 2-DG treatments Stem Cells. Author manuscript; available in PMC 2017 May 01. Author Manuscript Author Manuscript Author PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19858123 Manuscript Author Manuscript Aguilar et al. Page 9 in PC-3M cells but not in PC-3S cells, indicating that glycolysis and glutaminolysis are essential for the optimal growth and survival of PC-3M cells. Glutamine metabolism has been described to be important for several cance
