Regulation of mitochondrial pyruvate uptake by alternative pyruvate carrier complexes

At the pyruvate branch point, the fermentative and oxidative metabolic routes diverge. Pyruvate can be transformed either into lactate in mammalian cells or into ethanol in yeast, or transported into mitochondria to fuel ATP production by oxidative phosphorylation. The recently discovered mitochondrial pyruvate carrier (MPC), encoded by MPC1, MPC2, and MPC3 in yeast, is required for uptake of pyruvate into the organelle. Here, we show that while expression of Mpc1 is not dependent on the carbon source, expression of Mpc2 and Mpc3 is specific to fermentative or respiratory conditions, respectively. This gives rise to two alternative carrier complexes that we have termed MPC_FERM and MPC_OX. By constitutively expressing the two alternative complexes in yeast deleted for all three endogenous genes, we show that MPC_OX has a higher transport activity than MPC_FERM, which is dependent on the C‐terminus of Mpc3. We propose that the alternative MPC subunit expression in yeast provides a way of adapting cellular metabolism to the nutrient availability.     

Regulation of cellular energy metabolism. The Crabtree effect

The Crabtree effect (inhibition of respiration by glycolysis) is observed in cells with approximately equal glycolytic and respiratory capacities for ATP synthesis. Addition of glucose to aerobic suspensions of glucose-starved cells (Sarcoma 180 ascites tumor cells) causes a burst of respiration and lactate production due to ATP utilization for glucose phosphorylation by hexokinase and phosphofructokinase. This burst of activity is followed by inhibition of both respiration and glycolysis, the former to below the value before glucose addition (Crabtree effect). Both the respiratory rate and the glycolytic flux appear to be regulated by the cytosolic $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ albeit by completely different mechanisms. Respiration is regulated by the free energy of hydrolysis of ATP, such that the rate increases as the $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ decreases and decreases as the $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ increases. The regulatory enzymes of glycolysis are activated by ADP (AMP) and P_i and inhibited by ATP. Thus both respiration and glycolysis increase or decrease as the $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ decreases or increases. The parallel regulation of both ATP-producing pathways by this common metabolite ratio is consistent with the cytoplasmic $\text{[}\text{ATP}\text{]}\text{[}\text{ADP}\text{][}\text{P}{}_{\mathrm{i}}\text{]}$ being an important determinant of homeostatic regulation of cellular energy metabolism.     

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.     

Hispidulin: antioxidant properties and effect on mitochondrial energy metabolism

Hispidulin (6-methoxy-5,7,4'-trihydroxyflavone) and eupafolin (6-methoxy-5,7,3',4'-tetrahydroxyflavone), are flavonoids found in the leaves of Eupatorium litoralle. They have recognized antioxidant and antineoplastic properties, although their action mechanisms have not been previously described. We now report the effects of hispidulin on the oxidative metabolism of isolated rat liver mitochondria (Mit) and have also investigated the prooxidant and antioxidant capacity of both flavonoids. Hispidulin (0.05-0.2 mM) decreased the respiratory rate in state III and stimulated it in state IV, when glutamate or succinate was used as oxidizable substrate. Hispidulin inhibited enzymatic activities between complexes I and III of the respiratory chain. In broken Mit hispidulin (0.2 mM) slightly inhibited ATPase activity (25%). However, when intact Mit were used, the flavonoid stimulated this activity by 100%. Substrate energized mitochondrial swelling was markedly inhibited by hispidulin. Both hispidulin and eupafolin were able to promote iron release from ferritin, this effect being more accentuated with eupafolin with the suggestion of a possible involvement of H₂O₂ in the process. Hispidulin was incapable of donating electrons to the stable free radical DPPH, while eupafolin reacted with it in a similar way to ascorbic acid. The results indicate that hispidulin as an uncoupler of oxidative phosphorylation, is able to release iron from ferritin, but has distinct prooxidant and antioxidant properties when compared to eupafolin.     

Defects in the mitochondrial energy metabolism outside the respiratory chain and the pyruvate dehydrogenase complex

Disturbances in substrate oxidations in muscle mitochondria from patients with a suspicion of a mitochondrial myopathy may arise from a deficiency of one or more of the complexes of the respiratory chain or of the pyruvate dehydrogenase complex. However, we found no clear-cut defect in a substantial part of such patients. In this report we discuss some of the other possibilities which could account for the disturbed substrate oxidation rates. Special attention will be paid to defects which are localized outside the respiratory chain, such as defects in post-respiratory chain enzymes, defects in transport mechanisms of the mitochondrial inner or outer membrane, deficiency of cofactors and deficiency of heat-shock protein. (Mol Cell Biochem 174: 243–247, 1997)     

Regulation of muscle pyruvate metabolism during exercise

Pyruvate dehydrogenase and phosphoenolpyruvate carboxykinase are important enzymes in the regulation of muscle pyruvate metabolism and their in vitro measured activities have been studied in muscle from rested and exercised rats. In addition, the muscle concentration of metabolic intermediates associated with pyruvate metabolism has been measured after exercise. Phosphoenolpyruvate concentration was decreased to less than half the value found in rested muscle but pyruvate concentration did not change. This suggests an increase in the in vivo rate of conversion of phosphoenolpyruvate to pyruvate. Concentrations of malate and aspartate increased two- to threefold which suggests that oxaloacetate concentration was also increased. An increase in oxaloacetate availability would increase acetyl CoA metabolism and therefore would increase pyruvate dehydrogenase activity in vivo. The basal activity of pyruvate dehydrogenase measured in vitro increased approximately twofold after 2 hr of exercise and returned to control values 5 min after the cessation of exercise. Total pyruvate dehydrogenase activity (activated to the maximal extent) was not changed by exercise. Muscle PEPCK activity was also increased during exercise suggesting an increased rate of conversion of oxaloacetate to pyruvate to provide net oxidation of oxaloacetate and other citric acid cycle intermediates. Results of this study demonstrate that the rates of formation and metabolism of pyruvate are increased during exercise.     

Regulation of pyruvate metabolism via pyruvate carboxylase in rat brain mitochondria

1. The fixation of CO(2) by pyruvate carboxylase in isolated rat brain mitochondria was investigated. 2. In the presence of pyruvate, ATP, inorganic phosphate and magnesium, rat brain mitochondria fixed H(14)CO(3) (-) into tricarboxylic acid-cycle intermediates at a rate of about 250nmol/30min per mg of protein. 3. Citrate and malate were the main radioactive products with citrate containing most of the radioactivity fixed. The observed rates of H(14)CO(3) (-) fixation and citrate formation correlated with the measured activities of pyruvate carboxylase and citrate synthase in the mitochondria. 4. The carboxylation of pyruvate by the mitochondria had an apparent K(m) for pyruvate of about 0.5mm. 5. Pyruvate carboxylation was inhibited by ADP and dinitrophenol. 6. Malate, succinate, fumarate and oxaloacetate inhibited the carboxylation of pyruvate whereas glutamate stimulated it. 7. The results suggest that the metabolism of pyruvate via pyruvate carboxylase in brain mitochondria is regulated, in part, by the intramitochondrial concentrations of pyruvate, oxaloacetate and the ATP:ADP ratio.     

Regulation of gluconeogenesis and lipogenesis. The regulation of mitochondrial pyruvate metabolism in guinea-pig liver synthesizing precursors for gluconeogenesis

1. The carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase in guinea-pig liver mitochondria was determined by measuring the amount of (14)C from H(14)CO(3) (-) fixed into organic acids in the presence of pyruvate, ATP, Mg(2+) and P(i). The main products of pyruvate carboxylation were malate, fumarate and citrate. Pyruvate utilization, metabolite formation and incorporation of (14)C from H(14)CO(3) (-) into these metabolites in the presence and the absence of ATP were examined. The synthesis of phosphoenolpyruvate from pyruvate and bicarbonate is minimal during continued oxidation of pyruvate. Larger amounts of phosphoenolpyruvate are formed from alpha-oxoglutarate than from pyruvate. Addition of glutamate, alpha-oxoglutarate or fumarate did not appreciably increase formation of phosphoenolpyruvate when pyruvate was used as substrate. With alpha-oxoglutarate as substrate addition of fumarate resulted in increased formation of phosphoenolpyruvate, whereas addition of succinate inhibited phosphoenolpyruvate formation. In the presence of added oxaloacetate guinea-pig liver mitochondria synthesized phosphoenolpyruvate in amount sufficiently high to play an appreciable role in gluconeogenesis. 2. Addition of fatty acids of increasing carbon chain length caused a strong inhibition of pyruvate oxidation and phosphoenolpyruvate formation, and greatly promoted carbon dioxide fixation and malate, citrate and acetoacetate accumulation. The incorporation of (14)C from H(14)CO(3) (-), [1-(14)C]pyruvate and [2-(14)C]pyruvate into organic acids formed was examined. 3. It is concluded that guinea-pig liver pyruvate carboxylase contributes significantly to gluconeogenesis and that fatty acids and metabolites play an important role in its regulation.     

The role of mitochondrial transport in energy metabolism

Since mitochondria are closed spaces in the cell, metabolite traffic across the mitochondrial membrane is needed to accomplish energy metabolism. The mitochondrial carriers play this function by uniport, symport and antiport processes. We give here a survey of about 50 transport processes catalysed by more than 30 carriers with a survey of the methods used to investigate metabolite transport in isolated mammalian mitochondria. The role of mitochondria in metabolic pathways including ammoniogenesis, amino acid metabolism, mitochondrial shuttles etc. is also reported in more detail, mainly in the light of the existence of new transport processes.     

Regulation of mitochondrial function by voltage dependent anion channels in ethanol metabolism and the Warburg effect

Voltage dependent anion channels (VDAC) are highly conserved proteins that are responsible for permeability of the mitochondrial outer membrane to hydrophilic metabolites like ATP, ADP and respiratory substrates. Although previously assumed to remain open, VDAC closure is emerging as an important mechanism for regulation of global mitochondrial metabolism in apoptotic cells and also in cells that are not dying. During hepatic ethanol oxidation to acetaldehyde, VDAC closure suppresses exchange of mitochondrial metabolites, resulting in inhibition of ureagenesis. In vivo, VDAC closure after ethanol occurs coordinately with mitochondrial uncoupling. Since acetaldehyde passes through membranes independently of channels and transporters, VDAC closure and uncoupling together foster selective and more rapid oxidative metabolism of toxic acetaldehyde to nontoxic acetate by mitochondrial aldehyde dehydrogenase. In single reconstituted VDAC, tubulin decreases VDAC conductance, and in HepG2 hepatoma cells, free tubulin negatively modulates mitochondrial membrane potential, an effect enhanced by protein kinase A. Tubulin-dependent closure of VDAC in cancer cells contributes to suppression of mitochondrial metabolism and may underlie the Warburg phenomenon of aerobic glycolysis. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

Regulation of mitochondrial pyruvate carboxylation in isolated hepatocytes by acute insulin treatment.

The effect of acute insulin treatment of hepatocytes on pyruvate carboxylation in both isolated mitochondria and cells rendered permeable by filipin was examined. Challenging the cells with insulin alone had no effect on either the basal rate of pyruvate carboxylation or gluconeogenesis, although it did suppress the responses to both glucagon and catecholamines. Insulin treatment was unable to antagonize the enhanced rate of pyruvate carboxylation caused by stimulation of the cells with either angiotensin or vasopressin. Neither insulin nor the gluconeogenic hormones altered the total extractable pyruvate carboxylase activity in the isolated mitochondria, suggesting that the effect of hormones at the level of the isolated intact organelle was mediated via alterations in the intramitochondrial concentrations of effector molecules, notably ATP and the [ATP]/[ADP] ratio and substrate availability. The alterations in pyruvate carboxylation correlate well with glucose synthesis in terms of sensitivity to effector molecules, putative second messengers and time of onset of the response, indicating that alterations in the flux through this enzyme are compatible with it being an important site in the control of gluconeogenesis from C3 precursors.     

Regulation of cyclosporin A sensitive mitochondrial permeability transition by the redox state of pyridine nucleotides

The mechanisms involved in the induction of cyclosporine A sensitive mitochondrial swelling by oxidative stress were investigated in isolated guinea pig liver mitochondria. The aim of our study was to investigate, if swelling is inevitably associated with the oxidation of pyridine nucleotides, and if the oxidized pyridine nucleotides have to be hydrolysed for the induction of mitochondrial swelling. Quantitative measurement of oxidized pyridine nucleotides was performed with HPLC. Mitochondrial swelling was recorded by monitoring the decrease in light scattering of the mitochondrial suspension. Reduction and oxidation of pyridine nucleotides were followed by monitoring the changes of the autofluorescence signal of reduced pyridine nucleotides. Qualitative measurement of mitochondrial membrane potential was performed with the fluorescence indicator rhodamine 123. Neither t-butyl hydroperoxide nor the dissipation of the mitochondrial inner membrane potential with FCCP (carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone) induced the opening of the membrane permeability transition pore, unless an extensive oxidation of mitochondrial pyridine nucleotides took place. Mitochondrial swelling induced by our experimental conditions was always sensitive to cyclosporine A and accompanied by a cyclosporine A sensitive release of inner mitochondrial pyridine nucleotides without pyridine nucleotide hydrolysis. Not the cycling of calcium across the mitochondrial inner membrane but the accumulation of calcium inside the mitochondria was a prerequisite for mitochondrial swelling. The mitochondrial membrane permeability transition is neither caused nor accompanied by the hydrolysis of mitochondrial pyridine nucleotides.