GLUCOSE-1,6-BISPHOSPHATE: A BRAIN-ENRICHED REGULATORY METABOLITE MODULATING MITOCHONDRIAL PYRUVATE UPTAKE

Brain activity and energy metabolism are intimately linked through a precisely regulated, compartmentalized network of biochemical reactions. However, whether and how brain cells control mitochondrial fuel selection remains unclear, representing a significant gap in our understanding of brain metabolic plasticity. Here, we identify glucose-1,6-bisphosphate (G-1,6-BP), a brain-enriched glycolytic byproduct synthesized by phosphoglucomutase 2-like 1 (PGM2L1), as a critical regulator of mitochondrial pyruvate uptake. G-1,6-BP levels are highly sensitive to glycolytic flux and cellular energy status, as it is degraded by inosine monophosphate–activated phosphomannomutase 1. Using siRNA-mediated PGM2L1 knockdown, ¹³C-glucose isotope tracing, BRET-based mitochondrial pyruvate carrier (MPC) sensors, Drug Affinity Responsive Target Stability (DARTS) assays, and isolated mitochondria, we show that G-1,6-BP directly interacts with MPC subunits and dose-dependently enhances pyruvate uptake and mitochondrial respiration. We further demonstrate that PGM2L1 is essential for post-ischemic neuronal survival and that its activity is regulated by upstream kinases. To contextualize these findings, we examined PGM2L1 expression across different brain cell types, and investigated its role in modulating respiration driven by alternative substrates. Collectively, these results reveal a novel protein–metabolite–dependent mechanism by which brain cells regulate mitochondrial pyruvate uptake, a process responsive to glycolytic flux, cellular energy status, and kinase signaling, providing multiple layers of control to fine-tune this critical metabolic step.