Within the cytosol of the cell, glucose is converted into pyruvate that is used as a fuel for mitochondria. The latter supply the cell with energy in the form of ATP (adenosine triphosphate) and also play an important role in many other cellular functions like calcium homeostasis. Glucose-to-pyruvate conversion is mediated by the glycolysis pathway, which also generates ATP. Interestingly, the balance between glycolytic and mitochondrial ATP production differs between cell types and metabolic conditions. Moreover, this balance is often altered during pathological conditions. We studied the effect of acute (30 min) mitochondrial dysfunction, induced by mitochondrial inhibitors, on the balance between glycolytic and mitochondrial ATP production. To this end we developed a strategy to analyze the uptake and consumption rate of glucose in single living muscle cells (C2C12 cells). Cytosolic glucose concentration was measured using the FRET-based glucose nanosensor FLII (FLII12Pglu-700μδ6), which was developed by the Frommer group (Takanaga et al., Biochim. Biophys. Acta 1778:1091-1099, 2008). This sensor consists of a glucose binding domain sandwiched between two fluorescent proteins (CFP and Citrine).
As exemplified by the cover illustration, glucose binding to FLII triggers a conformational change that increases the energy transfer from CFP to Citrine. This is reflected by an increase in Citrine fluorescence intensity (CitrineFRET; upper panel), paralleled by a decrease in CFP fluorescence intensity (middle panel). As a consequence, the ratio between these signals (CitrineFRET/CFP; lower panel) increases and can be used as a readout of free cytosolic glucose concentration. Calibration of the ratio signal and the use of specific inhibitors of glucose uptake/consumption allowed construction of a quantitative mathematical model to predict the steady-state glucose flux (in mM/min). We demonstrated that this flux was rapidly increased upon mitochondrial inhibition, and that this increase fully compensated for the loss in mitochondrial ATP production. The latter suggests that cells can alter the balance between glycolytic and mitochondrial ATP production on demand, to prevent energy crisis and maintain their viability. Our strategy can also be applied to study glucose uptake and consumption in other pathophysiological models such as cancer cells. Since individual cells are analyzed, it is possible to perform inter-cell variability analysis and correlative studies with other readouts. We are currently applying FLII to investigate glucose uptake and consumption in cells with inherited metabolic dysfunction. More information about our research is provided here.