ATP/ADP ratio, the missed connection between mitochondria and the Warburg effect

Abstract

Non-proliferating cells generate the bulk of cellular ATP by fully oxidizing respiratory substrates in mitochondria. Respiratory substrates cross the mitochondrial outer membrane through only one channel, the voltage dependent anion channel (VDAC). Once in the matrix, respiratory substrates are oxidized in the tricarboxylic acid cycle to generate mostly NADH that is further oxidized in the respiratory chain to generate a proton motive force comprised mainly of membrane potential (ΔΨ) to synthesize ATP. Mitochondrial ΔΨ then drives the release of ATP(4-) from the matrix in exchange for ADP(3-) in the cytosol via the adenine nucleotide translocator (ANT) located in the mitochondrial inner membrane. Thus, mitochondrial function in non-proliferating cells drives a high cytosolic ATP/ADP ratio, essential to inhibit glycolysis. By contrast, the bioenergetics of the Warburg phenotype of proliferating cells is characterized by enhanced aerobic glycolysis and the suppression of mitochondrial metabolism. Suppressed mitochondrial function leads to lower production of mitochondrial ATP and hence lower cytosolic ATP/ADP ratios that favor enhanced glycolysis. Thus, the cytosolic ATP/ADP ratio is a key feature that determines if cell metabolism is predominantly oxidative or glycolytic. Here, we describe two novel mechanisms to explain the suppression of mitochondrial metabolism in cancer cells: the relative closure of VDAC by free tubulin and the inactivation of ANT. Both mechanisms contribute to low ATP/ADP ratios that activate glycolysis.

Keywords: ANT; ATP/ADP ratio; Glycolysis; Oxidative phosphorylation; VDAC; Warburg.

Fig. 1. VDAC closure and inactivation of ANT suppress mitochondrial metabolism and activate glycolysis in the Warburg phenomenon

Respiratory substrates, ADP and Pi first cross mitochondrial outer membranes via VDAC and then mitochondrial inner membranes via individual transporters, including the ANT. Respiratory substrates generate mostly NADH, which feeds into the respiratory chain (Complexes I-IV). Electron transfer leads to proton translocation from the matrix into the intermembrane space, generating Δp as oxygen is reduced to water. Protons return into the matrix through the F_1-F₀-ATP synthase (Complex V) driving synthesis of ATP from ADP and Pi. In aerobic metabolism by non-proliferating differentiated cells (top scheme), newly synthesized ATP exchanges for ADP via ANT and subsequently moves into the cytosol through VDAC. A strongly negative mitochondrial ΔΨ drives ANT-mediated outward electrogenic exchange of ATP⁻⁴ for inwardly directed ADP⁻³, which increases cytosolic relative to mitochondrial ATP/ADP ratios by ~100-fold. In proliferating cells (bottom scheme), high free tubulin causes a relative blockade of VDAC conductance. In addition, eletrogenic ATP/ADP exchange by ANT becomes inactivated and is replaced by electroneutral ATP/ADP exchange likely mediated by the ATP-Mg/Pi carrier. Relative VDAC closure and loss of ANT function together produce global suppression of mitochondrial metabolism and decrease cytosolic ATP/ADP ratios that promote Warburg-type aerobic glycolysis.

Fig 2. VDAC3 knockdown decreases mitochondrial membrane potential, NADH and ATP in HepG2 cells

In A, HepG2 hepatoma cells were transfected with non-target siRNA and siRNA against VDAC3. After 48 h, ΔΨ-indicating TMRM and mitochondrial NAD(P)H-indicating blue autofluorescence were imaged by confocal and multiphoton microscopy, respectively. Note the decrease of TMRM fluorescence and autofluorescence after VDAC3 knockdown, which is quantified in the right panels. Arrows identify 4-μm fiduciary fluorescent beads. In B, total cellular ATP is shown under the same conditions. *, p<0.05. Adapted from (Maldonado, et al., 2013).

Fig. 3. ANT-independent ATP hydrolysis sustains mitochondrial membrane potential in HepG2 hepatoma cells after respiratory inhibition

HepG2 cells were loaded with TMRM, as described in Fig. 2. Note a small of decrease of TMRM fluorescence after myxothiazol (Myx, 10 μM). Persisting mitochondrial TMRM uptake indicates that ATP hydrolysis supports ΔΨ formation during respiratory inhibition (compare right and left upper panels). Subsequent bongkrekic acid (BA, 5 μM), an ANT inhibitor, does not further decrease ΔΨ, indicating that ATP supply to mitochondria is independent of ANT (bottom left panel). Subsequent oligomycin (Oligo, 10 μg/ml) collapses ΔΨ, confirming that ANT-independent ATP entry into mitochondria maintains ΔΨ after respiratory inhibition (right bottom panel). Additions are 30 min apart. Adapted from (Maldonado, et al., 2013).

Fig. 4. Glycolytic ATP supports mitochondrial membrane potential after respiratory inhibition in A549 cells

A549 lung cancer cells were loaded with TMRM, as described in Fig. 2. Note that myxothiazol (Myx) slightly decreases TMRM fluorescence similarly to HepG2 cells (compare left and center panel). Subsequent 2-deoxyglucose (2-DG, 50 mM), a glycolytic inhibitor, collapses ΔΨ virtually completely, indicating that mitochondrial hydrolysis of glycolytic ATP supports mitochondrial ΔΨ formation after respiratory inhibition.