Glycolysis Blockade in a Cell Line Lacking Mitochondrial DNA
A ρ0 cell line (no mitochondrial DNA, no electron transport chain) is grown in medium with 25 mM glucose and 2 mM glutamine. Investigators add 2-deoxyglucose (2-DG) at 10 mM, which is phosphorylated by hexokinase but not metabolized further, trapping phosphate and inhibiting hexokinase. Within 30 minutes ATP falls to 15% of baseline, and the cells accumulate fructose-2,6-bisphosphate. Separately, a control ρ+ line under the same 2-DG exposure maintains ATP at 80% of baseline. The ρ0 cells show 8-fold higher hexokinase activity and 5-fold higher lactate secretion than ρ+ cells. Glutamine is present but the cells lack a functional malate-aspartate shuttle.
1. Explain why the ρ0 cells are far more sensitive to 2-DG than the ρ+ cells, using the bioenergetic logic of ATP yield.
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ρ0 cells have no oxidative phosphorylation, so ATP comes almost entirely from glycolysis: 2 ATP per glucose (net) plus lactate dehydrogenase recycling NAD+. Blocking hexokinase with 2-DG cuts the only ATP source, so ATP collapses (to 15%). ρ+ cells can oxidize pyruvate and glutamine in the TCA cycle and use the ETC, yielding far more ATP per carbon (~30–32 ATP per glucose), so a glycolytic block is partially buffered (ATP at 80%). The 8-fold hexokinase and 5-fold lactate data confirm the ρ0 cells are glycolytic-adapted (Warburg-like). Judgment: the sensitivity difference reflects pathway redundancy and ATP yield per substrate, not a difference in 2-DG uptake.
2. Would supplementing the medium with 10 mM pyruvate rescue the 2-DG-treated ρ0 cells? Explain.
Read the worked answer
No. Pyruvate can be converted to lactate by LDH, regenerating NAD+ and allowing glycolysis to continue-but glycolysis is blocked at hexokinase, so there is no flux to sustain. Pyruvate cannot enter the TCA cycle productively because the ETC is absent (NADH cannot be reoxidized by oxidative phosphorylation), and the malate-aspartate shuttle is nonfunctional, so reducing equivalents cannot be moved to the matrix. Pyruvate could serve as an anaplerotic carbon source only if a carboxylation route to malate/oxaloacetate existed and downstream NADH oxidation were possible-neither holds. Some ATP could come from substrate-level phosphorylation in the TCA cycle if acetyl-CoA were formed, but without NAD+ regeneration the cycle stalls. Expect little to no rescue; the lesion is upstream and the NAD+ recycling capacity is insufficient.