A Novel FRET Biosensor For Measuring Glycolytic Activity: A Study of Pancreatic Beta-Cells

Abstract

Pulses of insulin from pancreatic beta-cells help maintain blood glucose in a narrow range, although the source of these pulses is unclear. We propose that a positive feedback circuit exists within the glycolytic pathway employing the allosteric enzyme phosphofructokinase-1 (PFK1), which endows beta-cells with the ability to generate oscillations in metabolism via autocatalytic activation by its product Fructose-1,6-bisphosphate (Fru1,6-BP). To test this hypothesis, we have engineered a family of inter- and intramolecular Cerulean/Citrine FRET biosensors based on the glycolytic enzyme pyruvate kinase M2 (PK), which is allosterically activated and reported to multimerize upon binding Fru1,6-BP. When introduced into Min6 beta-cells, intramolecular PK biosensor apparent FRET efficiency increased dose-dependently in response to glucose (0, 2.5, 11, and 25 mM). This change was rapid (within seconds) and reversible. When Min6 cells were stimulated with 25 mM glucose/TEA, oscillations in PK biosensor activity were evident as ratiometric FRET changes, and exhibited a similar period to slow oscillations in intracellular calcium or NAD(P)H (3-4 min). Our results suggest that glycolysis in beta-cells is oscillatory and that PFK1 is indeed an attractive candidate for the oscillatory generator. More broadly, this family of PK biosensor constructs could be useful for exploring the magnitude and kinetics of glycolytic activity in living cells. Supported by F32DK085960 (M.J.M.) and R01DK46409 (L.S.).