Context-Dependent Modification of PFKFB3 in Hematopoietic Stem Cells Promotes Anaerobic Glycolysis and Ensures Stress Hematopoiesis
Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in HSCs and elucidate their regulatory mechanisms. Through quantitative13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of adenosine triphosphate (ATP) levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression ofPfkfb3induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss ofPfkfb3suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells. Overall design: 10-week old mice were administered with PBS or 5-FU (150 mg/kg) intraperitoneally 3 times with an interval of 21 days. For 5-FU group, a total of 3000-3500 hematopoietic stem cells (HSCs, CD150+CD48-Lineage-Sca-1+c-Kit+ cells) were isolated 5 days (day 6), and 20 days (day 21) after the first and the third administration of 5-FU. For PBS group, HSCs were isolated 5 days after the first and the third administration of PBS. Untreated fresh HSCs from 2-year-old mice were collected separately. All the samples were subjected to RNA extraction followed by library preparation and RNA-sequencing.