Mitoenergetic Dysfunction Triggers a Rapid Compensatory Increase in Steady-State Glucose Flux

Abstract

ATP can be produced in the cytosol by glycolytic conversion of glucose (GLC) into pyruvate. The latter can be metabolized into lactate, which is released by the cell, or taken up by mitochondria to fuel ATP production by the tricarboxylic acid cycle and oxidative phosphorylation (OXPHOS) system. Altering the balance between glycolytic and mitochondrial ATP generation is crucial for cell survival during mitoenergetic dysfunction, which is observed in a large variety of human disorders including cancer. To gain insight into the kinetic properties of this adaptive mechanism we determined here how acute (30 min) inhibition of OXPHOS affected cytosolic GLC homeostasis. GLC dynamics were analyzed in single living C2C12 myoblasts expressing the fluorescent biosensor FLII(12)Pglu-700μδ6 (FLII). Following in situ FLII calibration, the kinetic properties of GLC uptake (V1) and GLC consumption (V2) were determined independently and used to construct a minimal mathematical model of cytosolic GLC dynamics. After validating the model, it was applied to quantitatively predict V1 and V2 at steady-state (i.e., when V1 = V2 = Vsteady-state) in the absence and presence of OXPHOS inhibitors. Integrating model predictions with experimental data on lactate production, cell volume, and O2 consumption revealed that glycolysis and mitochondria equally contribute to cellular ATP production in control myoblasts. Inhibition of OXPHOS induced a twofold increase in Vsteady-state and glycolytic ATP production flux. Both in the absence and presence of OXPHOS inhibitors, GLC was consumed at near maximal rates, meaning that GLC consumption is rate-limiting under steady-state conditions. Taken together, we demonstrate here that OXPHOS inhibition increases steady-state GLC uptake and consumption in C2C12 myoblasts. This activation fully compensates for the reduction in mitochondrial ATP production, thereby maintaining the balance between cellular ATP supply and demand.

Acute stimulation of glucose influx upon mitoenergetic dysfunction requires LKB1, AMPK, Sirt2 and mTOR–RAPTOR

“…During the last 15 min of this incubation, glucose consumption was blocked by adding the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) inhibitor iodoacetic acid (IAA; 500 µM). Importantly, under these conditions glucose consumption was completely blocked (Liemburg-Apers et al, 2015a). This allowed quantification of the maximum rate of [GLC] c increase ( Fig.…”

“…Glucose binding and unbinding induces conformational changes that affect the efficiency of fluorescence resonance energy transfer (FRET) from CFP to citrine. Previously, we have calibrated the citrine FRET :CFP emission ratio signal in C2C12 cells, allowing quantification of the free cytosolic glucose concentration ([GLC] c ) (Liemburg-Apers et al, 2015a). To study the effects of mitochondrial dysfunction, we used our recently described incubation protocol (Liemburg-Apers et al, 2015a).…”

“…Previously, we have calibrated the citrine FRET :CFP emission ratio signal in C2C12 cells, allowing quantification of the free cytosolic glucose concentration ([GLC] c ) (Liemburg-Apers et al, 2015a). To study the effects of mitochondrial dysfunction, we used our recently described incubation protocol (Liemburg-Apers et al, 2015a). First, cells were pretreated (30 min) with vehicle (0.01% ethanol), the complex-I inhibitor piericidin A (100 nM), or the complex-III inhibitor antimycin A (20 nM).…”

“…This allowed quantification of the maximum rate of [GLC] c increase ( Fig. 1A; lines), which is a measure of the maximal rate of glucose uptake, upon extracellular addition of 2 mM of glucose (Liemburg-Apers et al, 2015a).…”

“…Mechanistically, Glut1 transport capacity can be stimulated by (i) increasing the translocation of Glut1-containing vesicles from the cytosol to the plasma membrane (Cura and Carruthers, 2012;Jing et al, 2008;Wheeler, 1988) or (ii) stimulating the activity of Glut1 transporters that are already present at the plasma membrane (Abbud et al, 2000;Barnes et al, 2002;Hamrahian et al, 1999;Loaiza et al, 2003;Mercado et al, 1989;Shetty et al, 1993;Shi et al, 1995). In a recent study (Liemburg-Apers et al, 2015a), we have demonstrated that acute (30 min) inhibition of complex I (with piericidin A) or of complex III (with antimycin A) increases glucose uptake and consumption in C2C12 myoblasts. This suggests that mitoenergetic dysfunction evokes a rapid compensatory increase in steady-state glucose uptake and consumption to prevent energy crisis in cells that are compromised in mitochondrial ATP generation.…”