1734-P: ADP Privation of Human Beta-Cell Mitochondria by Pyruvate Kinase

Introduction & Objective: A revised mechanism for glucose stimulated insulin secretion (GSIS) in beta-cells endorses that pyruvate kinase (PK) outcompetes oxidative phosphorylation (OxPhos) for ADP and generate the ATP/ADP ratio to close K_ATP channels. Here we investigate how PK deprives mitochondria of ADP.

Methods: Competition between OxPhos vs. PK for ADP was assessed by monitoring OxPhos-dependent oxygen consumption rate (OCR) in intact and permeabilized human islets using allosteric, nutrient modulation of enzyme and mitochondrial function including substrate transport.

Results: Up and down titration of mitochondrial coupling ruled out significant substrate-driven OxPhos. Phosphoenolpyruvate (PEP) decreased OxPhos-dependent OCR in a dose-dependent manner when ADP was limiting (33±0.6, 40±1.3 and 59±0.7% reduction by PEP at 156, 313 and 625µM, respectively; p<0.0001). At submaximal PEP, the PK activator PKa1 further reduced respiration 10µM (55±2.3% reduction respect to PEP; p<0.01). At maximal PEP, 4mM of the PK inhibitor L-phenylalanine partially improved OxPhos by 65±1.8% (p<0.0001). Likewise, 10mM creatine rescued OCR by 35% (p<0.0001) OxPhos by regenerating intramitochondrial ADP. PEP inhibition of OxPhos required 1) PEP hydrolysis to pyruvate by PK, 2) ADP/ATP exchange, and 3) ADP privation but 4) did not inhibit leak pathways 5) regardless of whether electrons entered at Complex I, II, III, or IV.

Conclusion: PEP metabolism by PK starves OxPhos by ristricting mitochondrial ADP availability in human beta-cells. These data are consistent with the newly proposed role for PEP and PK regulating the ATP/ADP mediated closure of K_ATP channels during GSIS.