At this point, many important questions regarding the properties of the pyruvate transporter remained problematic. Various studies were providing contradictory conclusions regarding metabolite effects on pyruvate transport [ 25 , 28 , 29 , 32 ].
Providing definitive answers surrounding the phenomenon of mitochondrial pyruvate transport would require the identity of the genes and proteins involved to allow biochemical purification and reconstitution in an isolated system.
Studying transport properties in the context of the mitochondrial membrane is extremely difficult due to the presence of other transporters and ongoing metabolism. The purification, identification and reconstitution of the transporter would solve many of these issues. As a result, great effort was expended to accomplish these goals. Purification of the transporter and reconstitution into liposomes would provide the single best system for characterizing transport properties as well as sharpening the focus onto this one activity.
Such an isolated system is perhaps the highest threshold to be achieved in biochemical studies and is one of the most difficult enterprises to undertake, made even more difficult by the lack of identity of the proteins. Without knowing their identity, overexpressing and tagging of the proteins for purification would not be possible. In spite of the challenges, the purification and reconstitution of pyruvate transport activity from mitochondria was demonstrated in [ 33 , 34 ].
Specifically, pyruvate exchange activity across proteoliposomal membranes was studied and shown to be sensitive to 2-cyanohydroxycinnamate. While this represented a critical step in the studies of mitochondrial pyruvate transport, the MPC was not purified. Attempts to reconstitute a purified preparation of the MPC were first described in castor bean mitochondria [ 34 ]. These data led the authors to conclude that one or more of the proteins was the pyruvate transporter, but complete purification remained elusive.
New strategies would likely be required to achieve this goal. Studies using a 14 C labeled inhibitor provided information on binding and dissociation beyond what could be learned from activity measurements. Using this system, the authors drew several parallels between inhibitor binding and inhibition of pyruvate transport, thus providing support for a direct relationship between the inhibitor, the MPC and pyruvate transport [ 35 ].
The data supporting a stable inhibitor-bound complex led to the idea of using an immobilized inhibitor as a means of purifying the MPC. The Azzi group that had previously reconstituted pyruvate transport activity using hydoxyapatite chromatography refined their methods in an attempt to identify the specific proteins involved.
For these studies they covalently immobilized the inhibitor 2-cyanohydroxycinnamate on sepharose. Following one-step hydroxyapatite purification, the mitochondrial fraction was passed through the cinnamate column. We currently have no way to explain the molecular weights of the proteins found in this study in light of the smaller size of the newly discovered MPC proteins, but it is possible that the observed proteins were monocarboxylate MCT transporters, which are predominantly at the plasma membrane, but have been proposed to exist in mitochondria [ 38 ].
The authors did find that higher concentrations of inhibitor were required to block transport activity compared to isolated intact mitochondria and Halestrap previously showed that this is the case for the MCT pyruvate transporter compared to the mitochondrial carrier. Now with the identification of the MPC it will be possible to go back and re-evaluate these studies. The MCTs may also represent another mode of pyruvate transport with low sensitivity that is active at higher concentrations although there is no definitive data on this.
In addition to the size discrepancy, it was found that their activities were different, with the yeast carrier being more active but less abundant than what was purified and reconstituted from bovine and rat tissue [ 40 ].
Even with these purification procedures in hand, the identity of the transporter remained a mystery. From what had been uncovered regarding mitochondrial transport of other metabolites, it seemed likely that pyruvate transport would be facilitated by a member of the mitochondrial carrier family MCF [ 42 ]. In , a report was published claiming to have finally identified the mitochondrial pyruvate carrier [ 2 ]. Using S. In total, 16 had been previously characterized and had known substrates.
Of the remaining proteins, 1 was essential for growth under respiratory conditions, which left 18 to characterize. A systematic analysis of strains individually lacking each of the 18 MCF proteins produced 1 candidate, the This mutant displayed the lowest level of pyruvate uptake and weakest response to UK, but had limited conservation from yeast to Drosophila , mouse, and man.
Indeed, phylogenetic analysis showed a clustering of this MCF protein with other nucleotide transporters. The approach employed by Hilyard et al. It cannot, however, be understated how difficult these transporter studies were, especially with unidentified proteins.
Characterizing basic biochemical attributes of membrane proteins is difficult and tedious work and achieving reproducible results requires zealous oversight. The work done over several decades by Halestrap, Palmieri, Papa, Azzi and their colleagues and many others represents an incredible body of work that facilitated the identification of the MPC.
Following these two papers, one claiming to have identified the mitochondrial pyruvate carrier and the second calling this into question, studies on the carrier slowed.
Those that were carried out became increasingly focused: pyruvate transport in frog mitochondria and cinnamate-resistant sake yeast [ 44 , 45 ]. The eventual identification of the MPC came from two groups, neither one of which had set out to identify the carrier. Bricker et al. Genetic ablation of the MPC genes in yeast and flies led to an increase in glycolytic intermediates and pyruvate with reductions in acetyl-CoA and TCA cycle metabolites. Herzig et al.
These two different approaches and very different experimental strategies led to the same conclusion: the proteins under investigation assemble to form the elusive MPC. The proteins found to be required for pyruvate transport are quite small.
The MPC inhibitor UK was used differently by the two groups, but the data generated was complementary in establishing the necessity and sufficiency of the MPC proteins for mitochondrial pyruvate uptake. They expressed murine MPC1 and MPC2 in Lactococcus lactis and showed that the two genes could confer pyruvate uptake, but neither gene alone had any effect. Sequencing revealed two mutations in highly conserved regions of MPC1 and metabolic studies showed that the defect in pyruvate metabolism could be rescued by expression of wild-type MPC1.
Despite the hidden identity of the mitochondrial pyruvate carrier, many studies have provided important information about the physiological significance and regulation of this process. These studies touch on the relationship with gluconeogenesis, MPC control by hormones and drugs and include relevance to pathological conditions including hyperthyroidism, aging, and diabetes [ 47 - 55 ]. The clinical relationship has also been investigated with respect to inborn errors of metabolism.
As expected, a defect in mitochondrial pyruvate transport causes a phenotype that is similar to a mutation in one of the components of the pyruvate dehydrogenase complex [ 4 , 5 ]. Now that the MPC genes have been identified, we are now in a position to determine whether patients that appear to have a PDH mutation but retain normal enzymatic activity have a mutation in one of these genes. As the epidemiology of PDH mutations remains unknown it is difficult to estimate how many potential idiopathic pyruvate metabolism defects with normal PDH activity, of which there are many, may be due to mutations of MPC [ 56 ].
Apart from basic metabolic disorders, the MPC may also exert a significant pathophysiological effect on the metabolic alterations found in cancer. At the most simplistic level, the Warburg effect could be described as a loss or decrease of MPC function. Many other well-written reviews have discussed cancer metabolism in depth so we will attempt to focus on aspects with relevant to mitochondrial pyruvate transport [ 57 , 58 ].
The study of metabolism in cancer has expanded significantly with many studies examining the role of glycolysis, oxidative phosphorylation, fatty acid oxidation, the TCA cycle, and hypoxia but previous studies have not been able to provide models that integrate mitochondrial pyruvate transport [ 59 - 61 ].
The limited work on mitochondrial pyruvate transport in cancer has supported the expectation that changes in the MPC may promote the glycolytic metabolic profile. One early study found that the activity of the mitochondrial pyruvate transporter was an order of magnitude lower in Ehrlich tumor cells compared to normal liver cells implicating the MPC in the metabolic phenotype of these cells [ 62 ].
A follow-up study comparing Ehrlich ascites, Morris hepatoma 44, and Morris hepatoma A cells with normal rat liver cells found that the V max of the transporter was decreased and pyruvate supported respiration was similarly reduced in each of the cancer cell preparations. There were no significant changes in transmembrane pH gradient, which may have explained reduced transport activity. The authors concluded the defect was due to lower transporter activity due to either a reduction in the abundance of the carrier or due to alterations in the cellular environment that may affect the transporter [ 63 ].
In both the Morris hepatoma 44 and Morris hepatoma A cells, the K M for pyruvate transport was increased, implying that the MPC might actually be different in those cells and exhibit a lower affinity for pyruvate.
Despite the limited data available on the connection between mitochondrial pyruvate transport and cancer metabolism, the possible relationships and their implications are exciting.
The proximity of the MPC to metabolic enzymes with a validated role in cancer metabolism draws immediate attention to the MPC and its modulation in cancer.
Lactate dehydrogenase, the M2 isoform of pyruvate kinase, and pyruvate dehydrogenase act directly on pyruvate metabolism and could indirectly affect its movement into mitochondria [ 64 - 66 ]. Each of these enzymes is altered in cancers, thereby perturbing pyruvate metabolism. We therefore consider it reasonable to anticipate a role for the MPC in cancer-relevant control of pyruvate metabolism. Many outstanding issues regarding how the MPC may contribute to the Warburg effect remain to be addressed, including how these various enzymes and their regulation interact in the context of cancer metabolism.
For example, reduced MPC expression or activity in the face of altered cytosolic pyruvate metabolism, namely through increased pyruvate kinase isozyme M2 PKM2 and lactate dehydrogenase LDH , might provide only a marginal exacerbation of the Warburg effect.
In contrast, reducing MPC in cancers with preserved cytosolic pyruvate metabolism might profoundly increase lactate production and the manifestation of the Warburg effect. There are many avenues of investigation available now with the identification of proteins necessary for the mitochondrial import of pyruvate. Understanding how the MPC fits into the complex environment of cancer metabolism sits at the forefront.
The metabolic profile of cancer has recently come back into vogue with people espousing the century-old contributions of biochemists, most notably Otto Warburg. Many sophisticated studies in the past few years have placed cancer metabolism in a deserved position of prominence in the field of oncology. The initial observation that tumor cells produce high levels of lactate despite adequate oxygenation was essential for future investigations of the mechanisms and physiological importance of the Warburg effect.
Key tumor suppressors have been shown to regulate metabolism and by doing so alter the fate of the cell. By controlling the mitochondrial flow of pyruvate, a cancer cell can tune its biology to meet the demands of rapid growth. Now with the identification of the mitochondrial pyruvate carrier we are poised to add yet another integral piece to this story and in doing so hopefully gain a better understanding that will ultimately translate into therapy. JCS and JR wrote and edited the manuscript.
Both authors read and approved the final manuscript. National Center for Biotechnology Information , U. Journal List Cancer Metab v. Cancer Metab. Published online Jan John C Schell 1 and Jared Rutter 1. Author information Article notes Copyright and License information Disclaimer. Corresponding author. John C Schell: ude. Received Jul 30; Accepted Sep 4. This article has been cited by other articles in PMC. Abstract The extraction of energy and biosynthetic building blocks from fuel metabolism is a fundamental requisite for life.
Review An extensive body of work has accumulated over decades on the subject of mitochondrial pyruvate transport. Membrane transport Membranes provide the cell with the essential ability to delineate the unregulated external environment from the specific and homeostatically controlled internal milieu.
Pyruvate metabolism Pyruvate is a key node in the branching pathways of glucose, fatty acid and amino acid metabolism. Open in a separate window. Figure 1. Upon entering the mitochondrial matrix, a multi-enzyme complex converts pyruvate into acetyl CoA. In the process, carbon dioxide is released and one molecule of NADH is formed.
Step 1. A carboxyl group is removed from pyruvate, releasing a molecule of carbon dioxide into the surrounding medium. The result of this step is a two-carbon hydroxyethyl group bound to the enzyme pyruvate dehydrogenase.
This is the first of the six carbons from the original glucose molecule to be removed. This step proceeds twice remember: there are two pyruvate molecules produced at the end of glycolysis for every molecule of glucose metabolized; thus, two of the six carbons will have been removed at the end of both steps.
Step 2. Step 3. An acetyl group is transferred to conenzyme A, resulting in acetyl CoA. The enzyme-bound acetyl group is transferred to CoA, producing a molecule of acetyl CoA. Note that during the second stage of glucose metabolism, whenever a carbon atom is removed, it is bound to two oxygen atoms, producing carbon dioxide, one of the major end products of cellular respiration. Therefore, the present study sought to examine the role of pyruvate in methylmercury toxicity, using budding yeast as a model organism.
Yeast has been established as a model organism in which powerful genetic approaches can be used to elucidate fundamental but complex eukaryotic processes Recently, yeast has been used as a model system to study the mechanisms of human neurodegenerative disorders 12 , 13 , Moreover, the similarities between yeast and human mitochondria facilitate the study of mitochondrial functions Examinations were also performed using IMR cells, which are human-derived neuroblastoma cells.
In the glycolytic system, glucose is converted to glycerate 2-phosphate in several stages and then to phosphoenolpyruvate; this conversion is mediated by Eno1 and Eno2. Phosphoenolpyruvate undergoes subsequent conversion to pyruvate, in a process mediated by Cdc19 Because Cdc19 acts downstream of Eno2, we examined how the overexpression of Cdc19 could potentially influence the sensitivity of yeast to methylmercury.
In this approach, yeast cells that overexpressed Cdc19 demonstrated a high sensitivity to methylmercury Fig. When non-toxic concentrations of pyruvate were added to the culture media, yeast growth was more intensively inhibited by methylmercury and was dependent on the concentration of pyruvate Fig. These results suggest that an increase in pyruvate levels in yeast is involved in intensifying the toxicity of methylmercury.
Pyruvate is produced in the cytoplasm and is then transported into the mitochondria, where it is converted to acetyl-CoA; acetyl-CoA subsequently reacts with oxaloacetate to form citrate, thus entering the tricarboxylic acid TCA cycle. Following the suggestion that pyruvate is involved in methylmercury toxicity, we examined the influence of methylmercury on the level and distribution of pyruvate in yeast.
Because spheroplasting during cell fractionation may alter the metabolism or distribution of pyruvate, in this study we spheroplasted yeast cells prior to treatment with methylmercury. Moreover, we used a large quantity of spheroplasts to measure the pyruvate levels because endogenous pyruvate levels can be measured by using a large quantity of spheroplasts. The accuracy of cell fractionation was confirmed via Western blotting, using antibodies against cytochrome c oxidase subunit III, a mitochondrial protein marker, or 3-phosphoglycerate kinase, a cytoplasmic protein marker Supplementary Figure 1.
In this study, pyruvate levels in the cytoplasmic fraction obtained by removing nuclei from the homogenate tended to increase after exposure to methylmercury, although the change was not statistically significant Fig. In addition, pyruvate levels in the mitochondria exhibited a significant increase depending on the concentration of added methylmercury and pyruvate levels in the post-mitochondrial fraction obtained by removing mitochondria from the cytoplasm in contrast decreased, depending on the concentration of methylmercury Fig.
Measurement of the transport of radioactive pyruvate into isolated mitochondria showed increased transportation of pyruvate into the mitochondria and was dependent on the concentration of methylmercury used during the treatment Fig.
Based on these observations, we hypothesize that methylmercury promotes the transport of pyruvate into mitochondria. Pyruvate synthesized in the cytoplasm of yeast is transported into the mitochondrial matrix, a process mediated by the Yilw transporter that is present in the inner mitochondrial membrane We therefore examined the sensitivity of Yilw-deleted yeast to methylmercury to determine the relationship between methylmercury toxicity and pyruvate transport into mitochondria.
In addition, in Yilw-deleted yeast the toxicity of methylmercury was only minimally intensified by pyruvate Fig. Based on these observations, we hypothesize that methylmercury toxicity is intensified by promoting the transport of pyruvate into mitochondria, a process mediated by Yilw. Pyruvate dehydrogenase is an enzyme that converts pyruvate to acetyl-CoA and is composed of several proteins: Pda1, Pdb1, Lat1, Lpd1 and Pdx1 To determine the relationship between methylmercury toxicity and the conversion of pyruvate to acetyl-CoA, we examined the influence of the removal of pyruvate dehydrogenase components Pda1, Pdb1, Lat1 and Lpd1.
In this approach, yeast with deletions of the respective components, compared to wild-type yeast, consistently exhibited a higher sensitivity to methylmercury data not shown. We subsequently examined how the addition of pyruvate to the culture media influenced methylmercury toxicity using yeast cells with Lat1 deleted dihydrolipoyl transacetylase, the active centre of pyruvate dehydrogenase 19 , Based on these results, we hypothesize that pyruvate that is transported into mitochondria is involved in intensifying methylmercury toxicity without being converted to acetyl-CoA.
Our observations in yeast suggested that methylmercury produces cytotoxicity by increasing the pyruvate levels in mitochondria. We next investigated the influence of pyruvate on methylmercury toxicity; we used human neuroblastoma cells IMR because methylmercury is a neurotoxic substance. In this approach, using an Alamar blue assay Fig. We next examined the influence of methylmercury on pyruvate levels in the mitochondria of IMR cells.
However, it is very difficult to measure the levels of endogenous pyruvate in human cultured cells. In this approach, a brief treatment with methylmercury significantly increased the level of radioactive pyruvate in the mitochondria Fig.
In addition, the effectiveness of cell fractionation was confirmed via western blotting, using antibodies against cytochrome c oxidase subunit IV, a mitochondrial marker protein, or glyceraldehyde 3-phosphate dehydrogenase, a cytoplasmic protein marker Supplementary Figure 3.
These results suggested that methylmercury causes cellular disorders by promoting the transport of pyruvate into mitochondria in human-derived IMR cells as well as in yeast.
Mitochondria are organelles that produce energy by promoting membrane electrogenesis, due to a pH or potential difference between the outer side and matrix side of the inner membrane.
If the membrane potential decreases, low-molecular-weight substances including protons flow into the mitochondria, causing mitochondrial dysfunction 21 , The results shown in Fig. There is a possibility that pyruvate transported into mitochondria influences the mitochondrial membrane potential as an organic acid. We therefore investigated the relationship between the mitochondrial membrane potential and the intensification of methylmercury toxicity mediated by pyruvate.
The membrane potential was measured using rhodamine , a cationic fluorescent substance that accumulates in response to an increase of anions on the matrix side of the mitochondria This result suggests that mitochondrial dysfunction is involved in the intensification of methylmercury toxicity mediated by pyruvate.
In addition, methylmercury is known to promote the production of reactive oxygen species in mitochondria 24 , 25 , 26 , 27 , We therefore investigated the relationship between reactive oxygen species and the intensification of methylmercury toxicity mediated by pyruvate. However, treatment with pyruvate alone did not influence the intracellular levels of reactive oxygen species. These results suggest that the production of reactive oxygen species via the reduction of the mitochondrial membrane potential may be involved in the intensification of methylmercury toxicity by pyruvate.
Next, we examined the influence of N -acetylcysteine NAC , an antioxidant, on the intensification of methylmercury toxicity mediated by pyruvate. NAC is a cysteine derivative that may bind to methylmercury in culture media. In this approach, treatment with NAC significantly alleviated methylmercury toxicity and significantly inhibited the intensification of methylmercury toxicity mediated by pyruvate Fig.
These results strongly suggest that reactive oxygen species are involved in the intensification of methylmercury toxicity mediated by pyruvate.
Pyruvate produced from glycolysis is involved in the production of ATP and in homeostasis of carbohydrates, fats and amino acids 29 , It has also been reported that some cells release pyruvate into blood plasma and serum and the released pyruvate reacts with extracellular H 2 O 2 independently of enzymes to produce an antioxidative action 31 , Interestingly, our study found that excessive pyruvate transported into mitochondria intensified the toxicity of methylmercury without being metabolized to acetyl-CoA.
Excessive pyruvate added to culture media did not change pyruvate levels in mitochondria and did not lead to cytotoxicity data not shown , indicating that the normal level of pyruvate in mitochondria is under strict control. However, in the presence of methylmercury, we observed enhanced transport of pyruvate into mitochondria. Furthermore, the deletion of the Yilw transporter which is involved in transporting pyruvate into mitochondria did not result in any intensification of pyruvate-mediated methylmercury toxicity.
Based on these observations, we can hypothesize that the toxicity of methylmercury is intensified by increasing the pyruvate levels in mitochondria. Normally, pyruvate is involved in the production of ATP via the TCA cycle, following its transportation into mitochondria. However, deletion of pyruvate dehydrogenase, which catalyses the metabolism of pyruvate, resulted in a further intensification of pyruvate-mediated methylmercury toxicity, suggesting that pyruvate intensifies the toxicity of methylmercury without being metabolized in the TCA cycle.
However, it cannot be excluded that pyruvate dehydrogenase is somehow involved in the pyruvate-mediated intensification of methylmercury toxicity. Pyruvate dehydrogenase, which converts pyruvate to acetyl-CoA, is a complex composed of three enzymes and the primary active site is comprised of lipoic acid bound to a Lys residue of dihydrolipoyl transacetylase Lat1 This lipoic acid has sulfhydryl groups that form a disulphide bond in a reversible manner and the resulting redox reaction decarboxylates pyruvate and transfers an acetyl group to the decarboxylated pyruvate.
Methylmercury is known to bind strongly to sulfhydryl groups and block the activity of sulfhydryl enzymes This leaves the possibility that methylmercury transported into mitochondria binds to the sulfhydryl groups of Lat1 and thus blocks the activity of pyruvate dehydrogenase, resulting in accumulation of pyruvate within the mitochondria. Therefore, we examined the influence of methylmercury on the activity of pyruvate dehydrogenase. We can thus hypothesize that blockage of pyruvate dehydrogenase activity by methylmercury is essentially not involved when methylmercury increases the pyruvate levels in mitochondria.
Some authors, in examining mitochondria isolated from rat liver cells, have suggested that methylmercury opens a mitochondrial membrane permeability transition pore and promotes influx of anions such as nitrate, thus inducing mitochondrial swelling and leading to cell death Previous studies have indicated that pyruvate strengthens the dihydrolipoate-induced mitochondrial permeability transition and mitochondrial swelling in isolated rat liver mitochondria The increase in mitochondrial permeability has been reported to be involved in neuronal injury 37 and mitochondrial swelling has been found to be induced in the cortices in an Alzheimer disease mouse model Therefore, the disruption of the mitochondrial permeability transition and of mitochondrial swelling may be involved in the mechanism of pyruvate-mediated methylmercury toxicity.
It is possible that methylmercury has some influence on transporters that are present in mitochondria, such as Yilw and hence promotes the transport of pyruvate into mitochondria. The results in Fig.
However, transport mechanisms other than Yilw do not appear to be involved when methylmercury promotes the transport of pyruvate into mitochondria, given that methylmercury did not increase pyruvate concentrations in mitochondria following the deletion of Yilw. One study has reported that methylmercury exhibits cytotoxicity by increasing mitochondrial membrane permeability, thus increasing the release of calcium from mitochondria to the cytoplasm 39 , Other reports have raised the possibility that methylmercury promotes the production of reactive oxygen species by inhibiting the activity of complex III of the mitochondrial electron transport chain 41 and that methylmercury blocks production of ATP by inhibiting the function of complex IV Thus, mitochondria can be regarded as intracellular targets for methylmercury.
The addition of pyruvate minimally influenced the cytotoxicity of cadmium, arsenic trioxide and other substances data not shown , which suggests that the intensification of toxicity mediated by pyruvate may be specific to methylmercury toxicity. Our present findings suggest the existence of a new mechanism for the toxicity of methylmercury that targets mitochondria, in addition to the above-mentioned known toxic mechanism of methylmercury acting through mitochondria.
The mechanisms related to methylmercury toxicity may be clarified by studying in detail the relationship between the level of pyruvate in mitochondria and the known toxic mechanism of methylmercury acting via mitochondria. Plasmid DNA was introduced into BY cells using the high-efficiency lithium acetate transformation method Yeast cells 6. Spheroplasts and mitochondria were prepared according to previously published methods When the yeast cells appeared to be collapsed due to the osmotic pressure, they were considered to be completely spheroplasted.
Methylmercury-treated spheroplasts were washed twice with ice-cold spheroplasting buffer, suspended in 3 mL of mitochondrial isolation buffer MIB 0. The homogenate was divided into mitochondrial, post-mitochondrial and post-nuclear fractions. The supernatant was combined with the previously prepared supernatant. This supernatant was used as the post-nuclear fraction. Crude mitochondria were isolated according to previously published methods After the incubation, the cell suspension was homogenized using 30 strokes with a Dounce homogenizer with a tight-fitting pestle on ice.
The pellet was washed twice with mitochondrial isolation buffer then suspended in PBS and used as the crude mitochondrial fraction.
The cells were treated with 0. After incubation, the mitochondria were isolated from the cells. The amount of [2- 14 C] pyruvate incorporated into the mitochondria was measured using a liquid scintillation counter and was normalized to the amount of mitochondrial protein How to cite this article : Lee, J. Transport of pyruvate into mitochondria is involved in methylmercury toxicity. Castoldi, A. Neurotoxic and molecular effects of methylmercury in humans.
Health 18, 19—31
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