Direct Measurements of Oscillatory Glycolysis in Pancreatic Islet β-Cells Using Novel Fluorescence Resonance Energy Transfer (FRET) Biosensors for Pyruvate Kinase M2 Activity
Pulses of insulin released from pancreatic β-cells maintain blood glucose in a narrow range, although the source of these pulses is unclear. We and others have proposed that positive feedback mediated by the glycolytic enzyme phosphofructokinase-1 (PFK1) enables β-cells to generate metabolic oscillations via autocatalytic activation by its product fructose 1,6-bisphosphate (FBP). Although much indirect evidence has accumulated in favor of this hypothesis, a direct measurement of oscillating glycolytic intermediates has been lacking. To probe glycolysis directly, we engineered a family of inter- and intramolecular FRET biosensors based on the glycolytic enzyme pyruvate kinase M2 (PKAR; pyruvate kinase activity reporter), which multimerizes and is activated upon binding FBP. When introduced into Min6 β-cells, PKAR FRET efficiency increased rapidly in response to glucose. Importantly, however, metabolites entering downstream of PFK1 (glyceraldehyde, pyruvate, and ketoisocaproate) failed to activate PKAR, consistent with sensor activation by FBP; the dependence of PKAR on FBP was further confirmed using purified sensor in vitro. Using a novel imaging modality for monitoring mitochondrial flavin fluorescence in mouse islets, we show that slow oscillations in mitochondrial redox potential stimulated by 10 mm glucose are in phase with glycolytic efflux through PKM2, measured simultaneously from neighboring islet β-cells expressing PKAR. These results indicate that PKM2 activity in β-cells is oscillatory and are consistent with pulsatile PFK1 being the mediator of slow glycolytic oscillations.
Journal of Biological Chemistry
Merrins, Matthew J.; Van Dyke, Aaron R.; Mapp, Anna K.; Rizzo, Mark A.; and Satin, Leslie S., "Direct Measurements of Oscillatory Glycolysis in Pancreatic Islet β-Cells Using Novel Fluorescence Resonance Energy Transfer (FRET) Biosensors for Pyruvate Kinase M2 Activity" (2013). Chemistry Faculty Publications. 20.
Merrins, Matthew J., Aaron R. Van Dyke, Anna K. Mapp, Mark A. Rizzo, and Leslie S. Satin. "Direct Measurements of Oscillatory Glycolysis in Pancreatic Islet β-Cells Using Novel Fluorescence Resonance Energy Transfer (FRET) Biosensors for Pyruvate Kinase M2 Activity." Journal of Biological Chemistry 288, no. 46 (2013): 33312-33322.