Formation and utilization of acetoin, an unusual product of pyruvate metabolism by Ehrlich and AS30-D tumor mitochondria

[14C]Pyruvate was rapidly non-oxidatively decarboxylated by Ehrlich tumor mitochondria at a rate of 40 nmol/min/mg of protein in the presence or absence of ADP. A search for decarboxylation products led to significant amounts of acetoin formed when Ehrlich tumor mitochondria were incubated with 1 mM [14C] pyruvate in the presence of ATP. Added acetoin to aerobic tumor mitochondria was rapidly utilized in the presence of ATP at a rate of 65 nmol/min/mg of protein. Citrate has been found as a product of acetoin utilization and was exported from the tumor mitochondria. Acetoin has been found in the ascitic liquid of Ehrlich and AS30-D tumor-bearing animals. These unusual reactions were not observed in control rat liver mitochondria.     

Oxidation of reduced nicotinamide-adenine dinucleotide by the malate—Aspartate shuttle in Ehrlich ascites tumour cells

The capability of ascites tumour mitochondria to oxidize externally formed NADH has been investigated in intact cells. Lactate has been used as the source of reducing equivalents and the oxidation of this substrate to pyruvate has been estimated. Ascites cells, under conditions of endogenous metabolism, are able to produce pyruvate upon addition of lactate. This effect is prevented by aminooxyacetate, an inhibitor of glutamate—oxalacetate transaminase (EC 2.6.1.1). Half-maximal inhibition by aminooxyacetate is attained at a concentration of approx. 30 μM. Oxidation of lactate is also sensitive to inhibitors of mitochondrial electron and energy transfer and it is enhanced by -oxoglutarate plus aspartate. These data demonstrate that reducing equivalents can be transported across the mitochondrial membrane of intact Ehrlich ascites tumour cells by the malate—aspartate shuttle.     

The proton stoichiometry of electron transport in Ehrlich ascites tumor mitochondria.

Initial rate measurements of the stoichiometric relationships between H+ ejection, K+ and Ca2+ uptake, and electron transport were carried out on mitochondria from Ehrlich ascites tumor cells grown in mice. With succinate as substrate and N-ethylmaleimide to prevent interfering H+ reuptake via the phosphate carrier, close to 8 H+ were ejected per oxygen atom reduced (H+/O ejection ratio = 8.0); with the NAD-linked substrates pyruvate or pyruvate + malate, the H+/O ejection ratio was close to 12. The average H+/site ratio (H+ ejected/2e-/energy-conserving site) was thus close to 4. The simultaneous uptake of charge-compensating cations, either K+ (in the presence of valinomycin) or Ca2+, was also measured, yielding average K+/site uptake ratios of very close to 4 and Ca2+/site ratios close to 2. It was also demonstrated that each calcium ion enters the respiring tumor mitochondria carrying two positive electric charges. These stoichiometric data observed in mitochondria from Ehrlich ascites tumor cells thus are in complete agreement with similar data on normal rat liver and rat heart mitochondria and suggest that the H+/site ratio of mitochondrial electron transport may be 4 generally. It was also observed that the rate of deltaH+ back-decay in anaerobic tumor mitochondria following oxygen pulses is some 6- to 8-fold greater than in rat liver mitochondria tested at equal amounts of mitochondrial protein.     

Enhanced rate of citrate export from cholesterol-rich hepatoma mitochondria. The truncated Krebs cycle and other metabolic ramifications of mitochondrial membrane cholesterol

Mitochondria isolated from rapidly growing, poorly differentiated Morris hepatoma 3924A have been found to export the citrate they generate from pyruvate, at a rate greater than four times that of control liver preparations. These 3924A mitochondria fail to exhibit state 3 respiration when either pyruvate or citrate are supplied as respiratory fuels. Nevertheless, substrates that join the Krebs cycle beyond citrate (viz. isocitrate, glutamate, alpha-ketoglutarate, and succinate) are readily oxidized by tumor 3924A mitochondria. Blocking the tricarboxylate anion exchange carrier with the citrate transport inhibitor 1,2,3-benzenetricarboxylate restores the ability of tumor 3924A mitochondria to respire with pyruvate or citrate. Slowly growing, minimally deviated Morris hepatoma 16 possesses mitochondria that do not display discernably altered respiratory patterns with pyruvate or citrate, but they do exhibit a 30% increase in the rate of citrate export relative to control liver preparations. Paralleling the preferential citrate export from tumor mitochondria is a dramatic enrichment of the tumor mitochondrial membranes with cholesterol. Hepatoma 3924A mitochondria possess a more than 5-fold enrichment in cholesterol, and those from tumor 16 display a 2-fold enrichment. When normal mitochondria, isolated from ACI strain rat liver, were enriched with cholesterol in vitro via a solid-state molecule transfer method employing Sephadex G-10 beads coated with cholesterol, they exhibited altered patterns of Krebs cycle metabolism that were qualitatively identical to those obtained with isolated Morris hepatoma mitochondria (which become enriched in membrane cholesterol endogenously during tumorigenesis). The enrichment of mitochondrial membranes with cholesterol, either by experimental manipulation in vitro or during the proliferation of the tumor in the host animal, promotes these metabolic changes directly, apparently by effecting a functional alteration in the operation of the tricarboxylate (citrate) exchange carrier of the inner mitochondrial membrane. These results highlight two related but incompletely understood phenomena as follows: 1) a functionally truncated Krebs cycle in cholesterol-rich tumor mitochondria, and 2) a mechanism for providing higher cytoplasmic levels of precursor metabolite intermediates which help sustain deregulated cholesterogenesis in hepatomas and other malignant neoplasms.     

Mitochondrial dysfunction and cancer metastasis

Mitochondria have an essential role in powering cells by generating ATP following the metabolism of pyruvate derived from glycolysis. They are also the major source of generating reactive oxygen species (ROS), which have regulatory roles in cell death and proliferation. Mutations in mitochondrial DNA (mtDNA) and dysregulation of mitochondrial metabolism have been frequently described in human tumors. Although the role of oxidative stress as the consequence of mtDNA mutations and/or altered mitochondrial functions has been demonstrated in carciongenesis, a causative role of mitochondria in tumor progression has only been demonstrated recently. Specifically, the subject of this mini-review focuses on the role of mitochondria in promoting cancer metastasis. Cancer relapse and the subsequent spreading of cancer cells to distal sites are leading causes of morbidity and mortality in cancer patients. Despite its clinical importance, the underlying mechanisms of metastasis remain to be elucidated. Recently, it was demonstrated that mitochondrial oxidative stress could actively promote tumor progression and increase the metastatic potential of cancer cells. The purpose of this mini-review is to summarize current investigations of the roles of mitochondria in cancer metastasis. Future development of diagnostic and therapeutic strategies for patients with advanced cancer will benefit from the new knowledge of mitochondrial metabolism in epithelial cancer cells and the tumor stroma.     

l-Lactate metabolism in HEP G2 cell mitochondria due to the l-lactate dehydrogenase determines the occurrence of the lactate/pyruvate shuttle and the appearance of oxaloacetate, malate and citrate outside mitochondria

As part of an ongoing study of l-lactate metabolism both in normal and in cancer cells, we investigated whether and how l-lactate metabolism occurs in mitochondria of human hepatocellular carcinoma (Hep G2) cells. We found that Hep G2 cell mitochondria (Hep G2-M) possess an l-lactate dehydrogenase (ml-LDH) restricted to the inner mitochondrial compartments as shown by immunological analysis, confocal microscopy and by assaying ml-LDH activity in solubilized mitochondria. Cytosolic and mitochondrial l-LDHs were found to differ from one another in their saturation kinetics. Having shown that l-lactate itself can enter Hep G2 cells, we found that Hep G2-M swell in ammonium l-lactate, but not in ammonium pyruvate solutions, in a manner inhibited by mersalyl, this showing the occurrence of a carrier-mediated l-lactate transport in these mitochondria. Occurrence of the l-lactate/pyruvate shuttle and the appearance outside mitochondria of oxaloacetate, malate and citrate arising from l-lactate uptake and metabolism together with the low oxygen consumption and membrane potential generation are in favor of an anaplerotic role for l-LAC in Hep G2-M.     

Synergistic stimulation of pregnenolone synthesis in rat adrenal mitochondria by n-hexane and cardiolipin

n-Hexane and cardiolipin each stimulate pregnenolone production by isolated rat adrenal mitochondria. Following corticotropin (ACTH) stimulation, mitochondrial cholesterol metabolism exhibits a fast phase lasting 2 min, followed by a 10-fold slower metabolism. ACTH suppression by dexamethazone or cycloheximide (CX) treatment removes this fast phase. n-Hexane, at concentrations approaching 80% of the aqueous solubility limit (approximately 0.08 mM), selectively stimulates the slow phase of metabolism, while cardiolipin (100 microM) stimulates only the fast phase. Other alkanes and ethers are effective. The effect of n-hexane is dependent on mitochondrial integrity, as evidenced by decreased effects in hypoosmotically shocked mitochondria (outer membrane disrupted) and ineffectiveness in sonicated mitochondria (both membranes disrupted). n-Hexane apparently enhances the transfer of outer membrane cholesterol to inner membrane P-450scc. Stimulation by cardiolipin is retained by disrupted mitochondria and may involve enhanced availability of P-450scc to inner membrane cholesterol. When added together, these agents produce more than additive effects on cholesterol metabolism. Preincubation with n-hexane did not increase reactive cholesterol, suggesting that enhanced cholesterol transport occurs only in concert with metabolism of inner membrane cholesterol. Uptake of alkanes into mitochondrial membranes may effect structural changes that facilitate outer to inner membrane cholesterol transfer, but major changes are excluded by the effectiveness of isocitrate as a reductant for P-450scc. In combination, n-hexane and cardiolipin reproduce the effect of the ACTH-sensitive sterol regulatory peptide on mitochondria [R. C. Pedersen and A. C. Brownie (1983) Proc. Natl. Acad. Sci. USA 80, 1882-1886], suggesting that peptide action on adrenal mitochondria may resolve into two analogous components.     

Stimulation of tumor-cell respiration by inhibitors of pyruvate kinase.

In a model system consisting of highly coupled rat liver mitochondria respiring in the presence of substrate, pyruvate kinase, phosphoenolpyruvate, ATP, hexokinase and glucose, the increase in the mitochondrial concentration results in a progressive decrease in the activity of pyruvate kinase. These results are in accord with a role of pyruvate kinase as a determinant of glycolytic activity by competing with mitochondrial oxidative phosphorylation for the available ADP. The addition of adequate amounts of the amino acids, cysteine, alanine and phenylalanine, known as inhibitors of pyruvate kinase, to living Ehrlich ascites tumor cell suspensions results in a stimulation of the respiratory rate and in a decrease of the glycolytic rate of the cells. Concomitant with these changes, there is an accumulation of intracellular phosphoenolpyruvate and ADP, and a decrease in pyruvate and ATP. These results provide additional evidence for paying attention to pyruvate kinase as another key enzyme whose properties and activities may be major determinants for the control of glycolysis and the Crabtree and Pasteur effects of tumor cells.     

The voltage dependent anion channel affects mitochondrial cholesterol distribution and function

We have observed abnormally high membrane cholesterol levels and a subsequent deficiency of oxidative energy production in mitochondria from cultured Morris hepatoma cells (MH7777). Using cholesterol affinity chromatography and MALDI-TOF Mass Spectrometry, we have identified the voltage dependent anion channel (VDAC) as a necessary component of a protein complex involved in mitochondrial membrane cholesterol distribution. VDAC is known to associate strongly with hexokinase, particularly in glycolytic cancers. By constructing an E72Q mutant form of VDAC that inhibits its binding of hexokinase, we report an increase in oxidative phosphorylation activity of MH7777 cells, as well as reduced membrane cholesterol ratios to levels near that of normal liver mitochondria. This paper demonstrates that the ability of VDAC to influence mitochondrial membrane cholesterol distribution may have implications on mitochondrial characteristics such as oxidative phosphorylation and induction of apoptosis, as well as the propensity of cancer cells to exhibit a glycolytic phenotype.     

Mitochondrial membrane cholesterol,the voltage dependent anion channel (VDAC), and the Warburg effect

Normal cells of aerobic organisms synthesize the energy they require in the form of ATP via the process of oxidative phosphorylation. This complex system resides in the mitochondria of cells and utilizes oxygen to produce a majority of cellular ATP. However, in most tumors, especially those with elevated cholesterogenesis, there is an increased reliance on glycolysis for energy, even in conditions where oxygen is available. This aerobic glycolysis (the Warburg effect) has far reaching ramifications on the tumor itself and the cells that surround it. In this brief review, we will discuss how abnormally high membrane cholesterol levels can result in a subsequent deficiency of oxidative energy production in mitochondria from cultured Morris hepatoma cells (MH-7777). We have identified the voltage dependent anion channel (VDAC) as a necessary component of a protein complex involved in mitochondrial membrane cholesterol distribution and transport. Work in our laboratory demonstrates that the ability of VDAC to influence mitochondrial membrane cholesterol distribution may have implications on mitochondrial characteristics such as oxidative phosphorylation and induction of apoptosis, as well as the propensity of cancer cells to exhibit a glycolytic phenotype.     

Isolated tumoral pyruvate dehydrogenase can synthesize acetoin which inhibits pyruvate oxidation as well as other aldehydes

Oxidation of 1 mM pyruvate by Ehrlich and AS30-D tumor mitochondria is inhibited by acetoin, an unusual and important metabolite of pyruvate utilization by cancer cells, by acetaldehyde, methylglyoxal and excess pyruvate. The respiratory inhibition is reversed by other substrates added to pyruvate and also by 0.5 mM ATP. Kinetic properties of pyruvate dehydrogenase complex isolated from these tumor mitochondria have been studied. This complex appears to be able to synthesize acetoin from acetaldehyde plus pyruvate and is competitively inhibited by acetoin. The role of a new regulatory pattern for tumoral pyruvate dehydrogenase is presented.     

A cholesterol-binding and transporting protein from rat liver mitochondria

In this communication, we present results indicating a protein isolated from rat liver mitochondrial intermembrane space that is capable of binding cholesterol and transporting it between the inner and outer mitochondrial membranes. This protein has a molecular weight of 57.5 kDa by SDS-PAGE; however, under native conditions, there is cholesterol-binding capability only as a 115 kDa dimer. Our data show that this dimeric protein may play a role in the regulation of mitochondrial membrane cholesterol levels, a prerequisite for the optimal activity of inner mitochondrial membrane-associated enzyme complexes. In addition, it appears that this protein is largely responsible for the differences in membrane cholesterol levels observed in normal and hepatoma mitochondria, a discrepancy which may help to explain the lack of energy production via oxidative phosphorylation in malignant tumor mitochondria.     

A re-evaluation of the role of mitochondrial pyruvate transport in the hormonal control of rat liver mitochondrial pyruvate metabolism.

The inhibitor of mitochondrial pyruvate transport alpha-cyano-beta-(1-phenylindol-3-yl)-acrylate was used to inhibit progressively pyruvate carboxylation by liver mitochondria from control and glucagon-treated rats. The data showed that, contrary to our previous conclusions [Halestrap (1978) Biochem. J. 172, 389-398], pyruvate transport could not regulate metabolism under these conditions. This was confirmed by measuring the intramitochondrial pyruvate concentration, which almost equilibrated with the extramitochondrial pyruvate concentration in control mitochondria, but was significantly decreased in mitochondria from glucagon-treated rats, where rates of pyruvate metabolism were elevated. Computer-simulation studies explain how this is compatible with linear Dixon plots of the inhibition of pyruvate metabolism by alpha-cyano-4-hydroxycinnamate. Parallel measurements of the mitochondrial membrane potential by using [3H]triphenylmethylphosphonium ions showed that it was elevated by about 3 mV after pretreatment of rats with both glucagon and phenylephrine. There was no significant change in the transmembrane pH gradient. It is shown that the increase in pyruvate metabolism can be explained by a stimulation of the respiratory chain, producing an elevation in the protonmotive force and a consequent rise in the intramitochondrial ATP/ADP ratio, which in turn increases pyruvate carboxylase activity. Mild inhibition of the respiratory chain with Amytal reversed the effects of hormone treatment on mitochondrial pyruvate metabolism and ATP concentrations, but not on citrulline synthesis. The significance of these observations for the hormonal regulation of gluconeogenesis from L-lactate in vivo is discussed.     

Inhibition of Mitochondrial Pyruvate Transport by Zaprinast Causes Massive Accumulation of Aspartate at the Expense of Glutamate in the Retina

Transport of pyruvate into mitochondria by the mitochondrial pyruvate carrier is crucial for complete oxidation of glucose and for biosynthesis of amino acids and lipids. Zaprinast is a well known phosphodiesterase inhibitor and lead compound for sildenafil. We found Zaprinast alters the metabolomic profile of mitochondrial intermediates and amino acids in retina and brain. This metabolic effect of Zaprinast does not depend on inhibition of phosphodiesterase activity. By providing ¹³C-labeled glucose and glutamine as fuels, we found that the metabolic profile of the Zaprinast effect is nearly identical to that of inhibitors of the mitochondrial pyruvate carrier. Both stimulate oxidation of glutamate and massive accumulation of aspartate. Moreover, Zaprinast inhibits pyruvate-driven O₂ consumption in brain mitochondria and blocks mitochondrial pyruvate carrier in liver mitochondria. Inactivation of the aspartate glutamate carrier in retina does not attenuate the metabolic effect of Zaprinast. Our results show that Zaprinast is a potent inhibitor of mitochondrial pyruvate carrier activity, and this action causes aspartate to accumulate at the expense of glutamate. Our findings show that Zaprinast is a specific mitochondrial pyruvate carrier (MPC) inhibitor and may help to elucidate the roles of MPC in amino acid metabolism and hypoglycemia.     

Bioflavonoid regulation of ATPase and hexokinase activity in Ehrlich ascites cell mitochondria.

(1) The mitochondrial ATPase (EC 3.6.1.3) Ehrlich ascites cell mitochondria, was inhibited by D-glucose under physiological concentrations of ATP. The generation of ADP by the mitochondrial bound hexokinase, seems to be the reason for the D-glucose inhibitory effect. Reversal of the inhibitory effect of ADP on Ehrlich ascites cell mitochondria ATPase by an ATP-regenerating system was achieved. (2) Dissociation of mitochondrial bound hexokinase from the mitochondria eliminated the inhibitory effect of D-glucose. Rebinding of the hexokinase to the mitochondria regenerated the D-glucose inhibitory effect on Ehrlich ascites cell mitochondria ATPase. (3) Bioflavonoids such as quercetin inhibit the mitochondrial hexokinase activity, but do not change the mitochondrial ATPase activity of isolated Ehrlich ascites tumor cell mitochondria. (4) The inhibitory effect of bioflavonoids on mitochondrial bound hexokinase activity is shown to be dissociable from the ascites tumor cell mitochondria and seems to be associated with regulatory rather than catalitic sites of the enzyme.     

Characterization of aconitate hydratase from mitochondria and cytoplasm of ascites tumor cells

This paper describes the characterization of aconitate hydratase (EC 4.2.1.3) in cytoplasmic and mitochondrial extracts from Ehrlich ascites tumor cells carried by BALB/C mice. The results show a similar distribution of aconitate hydratase in both extracts, with specific activities much lower than those found in pig and mouse tissues. Mitochondrial aconitate hydratase shows a substrate inhibition by citrate with a Km similar to that found in cytoplasm (Km = 1.0 mM and 0.9 mM, respectively). Oxalacetate produces a mixed type of inhibition in both cytoplasmic and mitochondrial aconitate hydratases with different inhibition constants (Ki = 0.3 mM and 1.0 mM, respectively). Moreover, the specific activities of aconitate hydratase in both cytoplasm and mitochondria decrease when the tumor progresses in the peritoneum of BALB/C mice, as well as the percentage of aconitate hydratase activity in the presence of oxalacetate as the inhibitor. These results indicate that the activity and kinetics of aconitate hydratase are markedly altered by neoplastic transformation as occurs in Ehrlich ascites tumor cells. Since aconitate hydratase is not a key enzyme, these unexpected data are of interest in the study of cancer biochemistry.     

Glutamine effect on cultured granule neuron death induced by glucose deprivation and chemical hypoxia

Using a specific fluorescent probe of mitochondrial membrane potential (tetramethylrhodamine ethyl ester), we have shown that glucose deprivation (GD) of cultured cerebellar granule neurons (CGN) for 3 h lowers mitochondrial membrane potential in these cells. Longer glucose starvation (24 h) causes CGN death that is not prevented by blockers of ionotropic glutamate receptors (MK-801 (10 μM) and NBQX (10 μM)). Glutamine or pyruvate (2 mM) maintain membrane potential of mitochondria and decrease CGN death under GD conditions. In the presence of glucose the mitochondrial respiratory chain blocker rotenone induces neuron death potentiated by glutamine. The potentiation effect is completely prevented by blockers of ionotropic glutamate receptors. These results show that glutamine under conditions of GD can be utilized by mitochondria as substrate, but at the same time, in the case of mitochondrial function deterioration, metabolism of this amino acid results in glutamate accumulation to toxic level.     

Altered cholesterol metabolism in Niemann-Pick type C1 mouse brains affects mitochondrial function

Niemann-Pick type C1 (NPC1) disease is a fatal hereditary disorder characterized by a defect in cholesterol trafficking and progressive neurodegeneration. Although the NPC1 gene has been identified, the molecular mechanism responsible for neuronal dysfunction in brains of patients with NPC1 disease remains unknown. This study demonstrates that the amount of cholesterol within mitochondria membranes is significantly elevated in NPC1 mouse brains and neural cells. In addition, the mitochondrial membrane potential, the activity of ATP synthase, and henceforth the level of ATP are markedly decreased in NPC1 mouse brains and neurons. Importantly, reducing the level of cholesterol within mitochondrial membranes using methyl-beta-cyclodextrin can restore the activity of ATP synthase. Finally, NPC1 neurons show an impaired neurite outgrowth, which can be rescued by exogenous ATP. These results suggest that mitochondrial dysfunctions and subsequent ATP deficiency, which are induced by altered cholesterol metabolism in mitochondria, may be responsible for neuronal impairment in NPC1 disease.     

The specificity and metabolic implications of the inhibition of pyruvate transport in isolated mitochondria and intact tissue preparations by alpha-Cyano-4-hydroxycinnamate and related compounds.

1. Effects of alpha-cyano-4-hydroxycinnamate and alpha-cyanocinnamate on a number of enzymes involved in pyruvate metabolism have been investigated. Little or no inhibition was observed of any enzyme at concentrations that inhibit completely mitochondrial pyruvate transport. At much higher concentrations (1 mM) some inhibition of pyruvate carboxylase was apparent. 2. Alpha-Cyano-4-hydroxycinnamate (1-100 muM) specifically inhibited pyruvate oxidation by mitochondria isolated from rat heart, brain, kidney and from blowfly flight muscle; oxidation of other substrates in the presence or absence of ADP was not affected. Similar concentrations of the compound also inhibited the carboxylation of pyruvate by rat liver mitochondria and the activation by pyruvate of pyruvate dehydrogenase in fat-cell mitochondria. These findings imply that pyruvate dehydrogenase, pyruvate dehydrogenase kinase and pyruvate carboxylase are exposed to mitochondrial matrix concentrations of pyruvate rather than to cytoplasmic concentrations. 3. Studies with whole-cell preparations incubated in vitro indicate that alpha-cyano-4-hydroxycinnamate or alpha-cyanocinnamate (at concentrations below 200 muM) can be used to specifically inhibit mitochondrial pyruvate transport within cells and thus alter the metabolic emphasis of the preparation. In epididymal fat-pads, fatty acid synthesis from glucose and fructose, but not from acetate, was markedly inhibited. No changes in tissue ATP concentrations were observed. The effects on fatty acid synthesis were reversible. In kidney-cortex slices, gluconeogenesis from pyruvate and lactate but not from succinate was inhibited. In the rat heart perfused with medium containing glucose and insulin, addition of alpha-cyanocinnamate (200 muM) greatly increased the output and tissue concentrations of lactate plus pyruvate but decreased the lactate/pyruvate ratio. 4. The inhibition by cyanocinnamate derivatives of pyruvate transport across the cell membrane of human erythrocytes requires much higher concentrations of the derivatives than the inhibition of transport across the mitochondrial membrane. Alpha-Cyano-4-hydroxycinnamate appears to enter erythrocytes on the cell-membrane pyruvate carrier. Entry is not observed in the presence of albumin, which may explain the small effects when these compounds are injected into whole animals.     

Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism

Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of the matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth.