Metabolic flexibility secures skeletal progenitor function upon reduced glycolysis driven by PFKFB3 loss.

Glycolysis is widely considered as a major metabolic pathway in several bone-residing cell types, including skeletal stem and progenitor cells (SSPCs) that are essential for bone development, maintenance, and regeneration. However, the contribution of glycolysis to the in vivo function of SSPCs remains unknown. To examine how reduced glycolysis affects SSPC biology, we conditionally deleted phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3), a key regulator of glycolytic flux. PFKFB3 deletion decreased glycolytic flux by at least 30%, but also reduced glucose‑carbon incorporation into glycolysis-branching pathways and tricarboxylic acid (TCA) cycle intermediates. Despite this overall attenuation of glucose metabolism, PFKFB3-deficient SSPCs maintained metabolic homeostasis and their functional properties. Bone mass was also preserved in mutant mice, even under anabolic conditions that are associated with increased glycolytic demand. Mechanistically, metabolic profiling revealed that PFKFB3 knockout SSPCs compensated for reduced glucose utilization by increasing the uptake of amino acids and pyruvate, with pyruvate‑carbon contributing to TCA cycle anaplerosis and amino acid synthesis. Together, these findings demonstrate that SSPCs possess substantial metabolic flexibility, allowing them to adapt to reductions in glucose metabolism by rerouting alternative nutrients to biosynthetic and bioenergetic pathways. This metabolic reprogramming likely represents an adaptive mechanism that helps preserve bone formation under metabolic stress.