Although it has been known for almost 90 years that cancer cells metabolize glucose differently than normal cells, the reason for this difference has been poorly understood and controversial. Unlike healthy cells, which completely oxidize most of their glucose to carbon dioxide, cancer cells metabolize most of their glucose to lactate even in the presence of oxygen. Paradoxically, despite cancer proliferation needing more energy, this metabolism generates less ATP per glucose. This phenomenon, also known as the Warburg Effect, is influenced by the differential isoform expression of pyruvate kinase (PK). PK catalyzes the final step of glycolysis, generating pyruvate and ATP from phosphoenolpyruvate (PEP) and ADP. While many differentiated cells express the M1 isoform (PKM1), cancer cells and other rapidly dividing tissues express the M2 isoform (PKM2). PKM2 has regulatable and overall less activity than PKM1, but it remains unclear how PKM2 benefits rapid proliferation. To study the importance of PKM2 in rapidly dividing cells, we examined the changes to proliferation and metabolism that occur following deletion of PKM2 in primary cells derived from mice harboring a PKM2 conditional allele. We find that acute deletion of PKM2 results in expression of PKM1 and a concomitant arrest in proliferation that is independent of senescence induction and cell death. Co-expression of PKM1 and PKM2 mimics the effect of expressing PKM1 alone, suggesting that gain of PKM1, not loss of PKM2, is responsible for the arrest. Slowed proliferation after exposure of cells to small molecule activators of PKM2 further argues that gain of pyruvate kinase activity is responsible for proliferation arrest. Metabolic flux analysis using U-13C-glucose and mass spectrometry reveals that PKM1 expression reduces flux to specific biosynthetic pathways and increases flux to the TCA cycle. Consistently, U-14C-glucose labeling of CO2 reveals increased glucose oxidation in PKM1-expressing MEFs. Exogenously supplied deoxyribonucleosides were able to rescue the proliferation arrest of PKM1-expressing MEFs. These data suggest that in comparison to PKM1, PKM2 expression promotes anabolism in proliferating cells by favoring metabolic flux that supports nucleotide biosynthesis
