beta-cell deletion of the PKm1 and PKm2 isoforms of pyruvate kinase in mice reveals their essential role as nutrient sensors for the K-ATP channel

Pyruvate kinase (PK) and the phosphoenolpyruvate (PEP) cycle play key roles in nutrient-stimulated K-ATP channel closure and insulin secretion. To identify the PK isoforms involved, we generated mice lacking beta-cell PKm1, PKm2, and mitochondrial PEP carboxykinase (PCK2) that generates mitochondrial PEP. Glucose metabolism was found to generate both glycolytic and mitochondrially derived PEP, which triggers K-ATP closure through local PKm1 and PKm2 signaling at the plasma membrane. Amino acids, which generate mitochondrial PEP without producing glycolytic fructose 1,6-bisphosphate to allosterically activate PKm2, signal through PKm1 to raise ATP/ADP, close K-ATP channels, and stimulate insulin secretion. Raising cytosolic ATP/ADP with amino acids is insufficient to close K-ATP channels in the absence of PK activity or PCK2, indicating that K-ATP channels are primarily regulated by PEP that provides ATP via plasma membrane-associated PK, rather than mitochondrially derived ATP. Following membrane depolarization, the PEP cycle is involved in an 'off-switch' that facilitates K-ATP channel reopening and Ca2+ extrusion, as shown by PK activation experiments and beta-cell PCK2 deletion, which prolongs Ca2+ oscillations and increases insulin secretion. In conclusion, the differential response of PKm1 and PKm2 to the glycolytic and mitochondrial sources of PEP influences the beta-cell nutrient response, and controls the oscillatory cycle regulating insulin secretion.