ATP/ADP Turnover and Import of Glycolytic ATP into Mitochondria in Cancer Cells Is Independent of the Adenine Nucleotide Translocator

Eduardo N Maldonado,David N Dehart,Jyoti Patnaik,Sandra C Klatt,Monika Beck Gooz,John J Lemasters

doi:10.1074/jbc.m116.734814

Eduardo N Maldonado, David N Dehart + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m116.734814

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Abstract

Non-proliferating cells oxidize respiratory substrates in mitochondria to generate a protonmotive force (Δp) that drives ATP synthesis. The mitochondrial membrane potential (ΔΨ), a component of Δp, drives release of mitochondrial ATP(4-) in exchange for cytosolic ADP(3-) via the electrogenic adenine nucleotide translocator (ANT) located in the mitochondrial inner membrane, which leads to a high cytosolic ATP/ADP ratio up to >100-fold greater than matrix ATP/ADP. In rat hepatocytes, ANT inhibitors, bongkrekic acid (BA), and carboxyatractyloside (CAT), and the F1FO-ATP synthase inhibitor, oligomycin (OLIG), inhibited ureagenesis-induced respiration. However, in several cancer cell lines, OLIG but not BA and CAT inhibited respiration. In hepatocytes, respiratory inhibition did not collapse ΔΨ until OLIG, BA, or CAT was added. Similarly, in cancer cells OLIG and 2-deoxyglucose, a glycolytic inhibitor, depolarized mitochondria after respiratory inhibition, which showed that mitochondrial hydrolysis of glycolytic ATP maintained ΔΨ in the absence of respiration in all cell types studied. However in cancer cells, BA, CAT, and knockdown of the major ANT isoforms, ANT2 and ANT3, did not collapse ΔΨ after respiratory inhibition. These findings indicated that ANT was not mediating mitochondrial ATP/ADP exchange in cancer cells [corrected]. We propose that suppression of ANT contributes to low cytosolic ATP/ADP, activation of glycolysis, and a Warburg metabolic phenotype in proliferating cells.

Highlights

Cellular bioenergetics in cancer and other proliferating cells is characterized by enhanced aerobic glycolysis and suppression of mitochondrial metabolism despite adequate oxygenation [1, 2]
bongkrekic acid (BA), CAT, and OLIG each decreased respiration to at or below basal noninduced levels, indicating that ureagenic respiration depends on ATP synthesis driven by the ATP synthase and that ATP turnover depends on adenine nucleotide translocator (ANT) function (Fig. 1A)
Respiration drives the formation of ⌬⌿ across the mitochondrial inner membrane, which in turns drives ATP formation by the F1FO-ATP synthase [12]

Summary

Results

Bongkrekic Acid and Carboxyatractyloside Decrease Respiration in Rat Hepatocytes but Not in Cancer Cells—Respiration by cultured rat hepatocytes was measured in a Seahorse XF24 extracellular flux analyzer under basal conditions and after the addition of ureagenic substrates (3 mM NH4Cl, 5 mM L-ornithine, and 5 mM lactate) [26]. Subsequent addition of MYX after OLIG, BA, or CAT collapsed ⌬⌿ of hepatocytes These results showed that hydrolysis of cytosolic ATP entering mitochondria via ANT by the ATP synthase, working in reverse, maintained mitochondrial ⌬⌿ in hepatocytes after respiratory inhibition. To show that mitochondrial ATP hydrolysis was still ongoing after ANT inhibitors, OLIG was added after MYX plus BA and MYX plus CAT, which decreased TMRM uptake by 89 and 85%, respectively (Fig. 5, B–D). Subsequent inhibition of respiration with MYX (10 ␮M) only slightly decreased ⌬⌿, indicating that ⌬⌿ was still maintained by hydrolysis of ATP Further addition of both BA (10 ␮M) and CAT (20 ␮M) after MYX failed to collapse ⌬⌿, which indicated that mitochondrial ATP/ADP exchange continued even after exposure to ANT inhibitors. The addition of 2DG after MYX caused a similar large release of mitochondrial TMRM in all single and double knockdown cells compared with wild type cells (Fig. 10, A–D)

Discussion

Experimental Procedures

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