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The mitochondrial pyruvate carrier (MPC) resides in the mitochondrial inner membrane, where it links cytosolic and mitochondrial metabolism by transporting pyruvate produced in glycolysis into the mitochondrial matrix. Due to its central metabolic role, it has been proposed as a potential drug target for diabetes, non-alcoholic fatty liver disease, neurodegeneration, and cancers relying on mitochondrial metabolism. Little is known about the structure and mechanism of MPC, as the proteins involved were only identified a decade ago and technical difficulties concerning their purification and stability have hindered progress in functional and structural analyses. The functional unit of MPC is a hetero-dimer comprising two small homologous membrane proteins, MPC1/MPC2 in humans, with the alternative complex MPC1L/MPC2 forming in the testis, but MPC proteins are found throughout the tree of life. The predicted topology of each protomer consists of an amphipathic helix followed by three transmembrane helices. An increasing number of inhibitors are being identified, expanding MPC pharmacology and providing insights into the inhibitory mechanism. Here, we provide critical insights on the composition, structure, and function of the complex and we summarize the different classes of small molecule inhibitors and their potential in therapeutics.

Pharmacological AMPK activation represents an attractive approach for the treatment of type 2 diabetes (T2D). AMPK activation increases skeletal muscle glucose uptake, but there is controversy as to whether AMPK activation also inhibits hepatic glucose production (HGP) and pharmacological AMPK activators can have off-target effects that contribute to their anti-diabetic properties. The main aim was to investigate the effects of 991 and other direct AMPK activators on HGP and determine whether the observed effects were AMPK-dependent. In incubated hepatocytes, 991 substantially decreased gluconeogenesis from lactate, pyruvate and glycerol, but not from other substrates. Hepatocytes from AMPKβ1−/− mice had substantially reduced liver AMPK activity, yet the inhibition of glucose production by 991 persisted. Also, the glucose-lowering effect of 991 was still seen in AMPKβ1−/− mice subjected to an intraperitoneal pyruvate tolerance test. The AMPK-independent mechanism by which 991 treatment decreased gluconeogenesis could be explained by inhibition of mitochondrial pyruvate uptake and inhibition of mitochondrial sn-glycerol-3-phosphate dehydrogenase-2. However, 991 and new-generation direct small-molecule AMPK activators antagonized glucagon-induced gluconeogenesis in an AMPK-dependent manner. Our studies support the notion that direct pharmacological activation of hepatic AMPK as well as inhibition of pyruvate uptake could be an option for the treatment of T2D-linked hyperglycemia.

Malate oxidation by plant mitochondria enables the generation of both oxaloacetate and pyruvate for tricarboxylic acid (TCA) cycle function, potentially eliminating the need for pyruvate transport into mitochondria in plants. Here, we show that the absence of the mitochondrial pyruvate carrier 1 (MPC1) causes the co-commitment loss of its putative orthologs, MPC3/MPC4, and eliminates pyruvate transport into Arabidopsis thaliana mitochondria, proving it is essential for MPC complex function. While the loss of either MPC or mitochondrial pyruvate-generating NAD-malic enzyme (NAD-ME) did not cause vegetative phenotypes, the lack of both reduced plant growth and caused an increase in cellular pyruvate levels, indicating a block in respiratory metabolism, and elevated the levels of branched-chain amino acids at night, a sign of alterative substrate provision for respiration. 13C-pyruvate feeding of leaves lacking MPC showed metabolic homeostasis was largely maintained except for alanine and glutamate, indicating that transamination contributes to the restoration of the metabolic network to an operating equilibrium by delivering pyruvate independently of MPC into the matrix. Inhibition of alanine aminotransferases when MPC1 is absent resulted in extremely retarded phenotypes in Arabidopsis, suggesting all pyruvate-supplying enzymes work synergistically to support the TCA cycle for sustained plant growth.

Mitochondrial pyruvate carrier 1 (MPC1) is a key metabolic protein that regulates the transport of pyruvate into the mitochondrial inner membrane. MPC1 deficiency may cause metabolic reprogramming. However, whether and how MPC1 controls mitochondrial oxidative capacity in cancer are still relatively unknown. MPC1 deficiency was recently found to be strongly associated with various diseases and cancer hallmarks. We utilized online databases and uncovered that MPC1 expression is lower in many cancer tissues than in adjacent normal tissues. In addition, MPC1 expression was found to be substantially altered in five cancer types: breast-invasive carcinoma (BRCA), kidney renal clear cell carcinoma (KIRC), lung adenocarcinoma (LUAD), pancreatic adenocarcinoma (PAAD), and prostate adenocarcinoma (PRAD). However, in KIRC, LUAD, PAAD, and PRAD, high MPC1 expression is closely associated with favourable prognosis. Low MPC1 expression in BRCA is significantly associated with shorter overall survival time. MPC1 expression shows strong positive and negative correlations with immune cell infiltration in thymoma (THYM) and thyroid carcinoma (THCA). Furthermore, we have comprehensively summarized the current literature regarding the metabolic reprogramming effects of MPC1 in various cancers. As shown in the literature, MPC1 expression is significantly decreased in cancer tissue and associated with poor prognosis. We discuss the potential metabolism-altering effects of MPC1 in cancer, including decreased pyruvate transport ability; impaired pyruvate-driven oxidative phosphorylation (OXPHOS); and increased lactate production, glucose consumption, and glycolytic capacity, and the underlying mechanisms. These activities facilitate tumour progression, migration, and invasion. MPC1 is a novel cancer biomarker and potentially powerful therapeutic target for cancer diagnosis and treatment. Further studies aimed at slowing cancer progression are in progress.

Glyoxylate is an important chemical and is also an intermediate involved in metabolic pathways of living microorganisms. However, it cannot be rapidly utilized by many microbes. We observed a very long lag phase (up to 120 h) when E. coli is growing in a mineral medium supplemented with 50 mM glyoxylate. To better understand this strange growth pattern on glyoxylate and accelerate glyoxylate utilization, a random genomic library of E. coli was transformed into E. coli BW25113, and mutants that showed significantly shortened lag phase on glyoxylate were obtained. Interestingly, mutations in BtsT/BtsS, a two component system that is involved in pyruvate transport, were found to be a common feature in all mutants retrieved. We further demonstrated, through reverse engineering, that the mutations in BtsT/BtsS can indeed increase glyoxylate uptake. Growth experiments with different concentration of glyoxylate also showed the higher the concentration of glyoxylate, the shorter the lag phase. These new findings thus increased our understanding on microbial utilization of glyoxylate.

Lactate/pyruvate transport between glial cells and neurons is thought to play an important role in how brain cells sustain the high-energy demand that neuronal activity requires. However, the in vivo mechanisms and characteristics that underlie the transport of monocarboxylates are poorly described. Here, we use Drosophila expressing genetically encoded FRET sensors to provide an ex vivo characterization of the transport of monocarboxylates in motor neurons and glial cells from the larval ventral nerve cord. We show that lactate/pyruvate transport in glial cells is coupled to protons and is more efficient than in neurons. Glial cells maintain higher levels of intracellular lactate generating a positive gradient toward neurons. Interestingly, during high neuronal activity, raised lactate in motor neurons is dependent on transfer from glial cells mediated in part by the previously described monocarboxylate transporter Chaski, providing support for in vivo glia-to-neuron lactate shuttling during neuronal activity.

Proton-linked monocarboxylate transporters (MCTs) must transport monocarboxylate efficiently to facilitate monocarboxylate efflux in glycolytically active cells, and transport monocarboxylate slowly or even shut down to maintain a physiological monocarboxylate concentration in glycolytically inactive cells. To discover how MCTs solve this fundamental aspect of intracellular monocarboxylate homeostasis in the context of multicellular organisms, we analyzed pyruvate transport activity of human monocarboxylate transporter 2 (MCT2). Here we show that MCT2 transport activity exhibits steep dependence on substrate concentration. This property allows MCTs to turn on almost like a switch, which is physiologically crucial to the operation of MCTs in the cellular context. We further determined the cryo-electron microscopy structure of the human MCT2, demonstrating that the concentration sensitivity of MCT2 arises from the strong inter-subunit cooperativity of the MCT2 dimer during transport. These data establish definitively a clear example of evolutionary optimization of protein function., Proton-linked monocarboxylate transporters (MCTs) facilitate monocarboxylate efflux in glycolytically active cells and regulate transport down in glycolytically inactive cells. Here authors show a steep dependence of human MCT2 activity on substrate concentration and show the structural basis of cooperative transport.

The majority of the pyruvate inside plant mitochondria is either transported into the matrix from the cytosol via the mitochondria pyruvate carrier (MPC) or synthesised in the matrix by alanine aminotransferase (AlaAT) or NAD-malic enzyme (NAD-ME). Pyruvate from these origins could mix into a single pool in the matrix and contribute indistinguishably to respiration, or they could maintain a degree of independence in metabolic regulation. Here, we demonstrated that feeding isolated mitochondria with U-13C-pyruvate and unlabelled malate enables the assessment of pyruvate contribution from different sources to TCA cycle intermediate production. Imported pyruvate is the preferred source for citrate production even when the synthesis of NAD-ME-derived pyruvate was optimised. Genetic or pharmacological elimination of MPC activity removed this preference and allowed an equivalent amount of citrate to be generated from the pyruvate produced by NAD-ME. Increasing mitochondrial pyruvate pool size by exogenous addition only affected metabolites from pyruvate transported by MPC whereas depleting pyruvate pool size by transamination to alanine only affected metabolic products derived from NAD-ME. Together, these data reveal respiratory substrate supply in plants involves distinct pyruvate pools inside the matrix that can be flexibly mixed based on the rate of pyruvate transport from the cytosol. These pools are independently regulated and contribute differentially to organic acids export from plant mitochondria.Significance statementPyruvate is the primary respiratory substrate for energy production to support plant growth and development. However, it is also the starting material of many other pathways. Prioritisation of respiratory use over other competing pathways would enable a level of control when pyruvate is delivered to mitochondria via the mitochondrial pyruvate transporter. We demonstrated the existence of two distinct pyruvate pools in plant mitochondria suggesting inner mitochondrial organisation allows metabolic heterogeneity, hence metabolic specialisation. This explains why NAD-ME flux into plant respiration is low and confirms the prominent link between imported pyruvate and energy production. This compartmentation also reveals how NAD-ME supplies substrate to the mitochondrial pyruvate exporter in plants, especially during C4 metabolism.

Monocarboxylates, such as lactate and pyruvate, are precursors for biosynthetic pathways, including those for glucose, lipids, and amino acids via the tricarboxylic acid (TCA) cycle and adjacent metabolic networks. The transportation of monocarboxylates across the cellular membrane is performed primarily by monocarboxylate transporters (MCTs), the membrane localization and stabilization of which are facilitated by the transmembrane protein basigin (BSG). Here, we demonstrate that the MCT/BSG axis sits at a crucial intersection of cellular metabolism. Abolishment of MCT1 in the plasma membrane was achieved by Bsg depletion, which led to gluconeogenesis impairment via preventing the influx of lactate and pyruvate into the cell, consequently suppressing the TCA cycle. This net anaplerosis suppression was compensated in part by the increased utilization of glycogenic amino acids (e.g., alanine and glutamine) into the TCA cycle and by activated ketogenesis through fatty acid β-oxidation. Complementary to these observations, hyperglycemia and hepatic steatosis induced by a high-fat diet were ameliorated in Bsg-deficient mice. Furthermore, Bsg deficiency significantly improved insulin resistance induced by a high-fat diet. Taken together, the plasma membrane-selective modulation of lactate and pyruvate transport through BSG inhibition could potentiate metabolic flexibility to treat metabolic diseases.

Pyruvate metabolism requires the mitochondrial pyruvate carrier (MPC) proteins to transport pyruvate from the intermembrane space through the inner mitochondrial membrane to the mitochondrial matrix. The lack of the atomic structures of MPC hampers the understanding of the functional states of MPC and molecular interactions with the substrate or inhibitor. Here, we develop the de novo models of human MPC complexes and characterize the conformational dynamics of the MPC heterodimer formed by MPC1 and MPC2 (MPC1/2) by computational simulations. Our results reveal that functional MPC1/2 prefers to adopt an inward-open conformation, with the carrier open to the matrix side, whereas the outward-open states are less populated. The energy barrier for pyruvate transport in MPC1/2 is low enough, and the inhibitor UK5099 blocks the pyruvate transport by stably binding to MPC1/2. Notably, consistent with experimental results, the MPC1 L79H mutation significantly alters the conformations of MPC1/2 and thus fails for substrate transport. However, the MPC1 R97W mutation seems to retain the transport activity. The present de novo models of MPC complexes provide structural insights into the conformational states of MPC complexes and mechanistic understanding of interactions between the substrate/inhibitor and MPC proteins.

Pyruvate is a key metabolite in living cells and has been shown to play a crucial role in the virulence of several bacterial pathogens. The bioluminescent Vibrio campbellii, a severe infectious burden for marine aquaculture, excretes extraordinarily large amounts of pyruvate during growth and rapidly retrieves it by an as-yet unknown mechanism. We have now identified the responsible pyruvate transporter, here named BtsU, and our results show that it is the only pyruvate transporter in V. campbellii. Expression of btsU is tightly regulated by the membrane-integrated LytS-type histidine kinase BtsS, a sensor for extracellular pyruvate, and the LytTR-type response regulator BtsR. Cells lacking either the pyruvate transporter or sensing system show no chemotactic response towards pyruvate, indicating that intracellular pyruvate is required to activate the chemotaxis system. Moreover, pyruvate sensing and uptake were found to be important for the resuscitation of V. campbellii from the viable but nonculturable (VBNC) state and the bacterium's virulence against brine shrimp larvae. IMPORTANCE Bacterial infections are a serious threat to marine aquaculture, one of the fastest growing food sectors on earth. Therefore, it is extremely important to learn more about the pathogens responsible, one of which is Vibrio campbellii. This study sheds light on the importance of pyruvate sensing and uptake for V. campbellii, and reveals that the bacterium possesses only one pyruvate transporter, which is activated by a pyruvate-responsive histidine kinase/response regulator system. Without the ability to sense or take up pyruvate, the virulence of V. campbellii towards gnotobiotic brine shrimp larvae is strongly reduced.

Over the past years, systemic derived cues that regulate cellular metabolism have been implicated in the regulation of immune responses. Ghrelin is an orexigenic hormone produced by enteroendocrine cells in the gastric mucosa with known immunoregulatory roles. The mechanism behind the function of ghrelin in immune cells, such as macrophages, is still poorly understood. Here, we explored the hypothesis that ghrelin leads to alterations in macrophage metabolism thus modulating macrophage function. We demonstrated that ghrelin exerts an immunomodulatory effect over LPS-activated peritoneal macrophages, as evidenced by inhibition of TNF-α and IL-1β secretion and increased IL-12 production. Concomitantly, ghrelin increased mitochondrial membrane potential and increased respiratory rate. In agreement, ghrelin prevented LPS-induced ultrastructural damage in the mitochondria. Ghrelin also blunted LPS-induced glycolysis. In LPS-activated macrophages, glucose deprivation did not affect ghrelin-induced IL-12 secretion, whereas the inhibition of pyruvate transport and mitochondria-derived ATP abolished ghrelin-induced IL-12 secretion, indicating a dependence on mitochondrial function. Ghrelin pre-treatment of metabolic activated macrophages inhibited the secretion of TNF-α and enhanced IL-12 levels. Moreover, ghrelin effects on IL-12, and not on TNF-α, are dependent on mitochondria elongation, since ghrelin did not enhance IL-12 secretion in metabolic activated mitofusin-2 deficient macrophages. Thus, ghrelin affects macrophage mitochondrial metabolism and the subsequent macrophage function.

Malate oxidation by plant mitochondria enables the generation of both oxaloacetate (OAA) and pyruvate for tricarboxylic acid (TCA) cycle function, potentially eliminating the need for pyruvate transport into mitochondria in plants. Here we show that the absence of the mitochondrial pyruvate carrier 1 (MPC1) causes the co-commitment loss of its orthologs, MPC3/MPC4, and eliminates pyruvate transport into Arabidopsis mitochondria, proving it is essential for MPC complex function. While the loss of either MPC or mitochondrial pyruvate-generating NAD-malic enzyme (NAD-ME) did not cause vegetative phenotypes, the lack of both reduced plant growth and caused an increase in cellular pyruvate levels, indicating a block in respiratory metabolism, and elevated the levels of branched-chain amino acids at night, a sign of alterative substrate provision for respiration.13C-pyruvate feeding of leaves lacking MPC showed metabolic homeostasis were largely maintained except for alanine and glutamate, indicating that transamination contributes to restoration of the metabolic network to an operating equilibrium by delivering pyruvate independently of MPC into the matrix. Inhibition of alanine aminotransferases (AlaAT) when MPC1 is absent resulted in extremely retarded phenotypes in Arabidopsis, suggesting all pyruvate-supplying enzymes work synergistically to support the TCA cycle for sustained plant growth.

Glucose is an essential nutrient for every cell but its metabolic fate depends on cellular phenotype. Normally, the product of cytosolic glycolysis, pyruvate, is transported into mitochondria and irreversibly converted to acetyl coenzyme A by pyruvate dehydrogenase complex (PDC). In some pathological cells, however, pyruvate transport into the mitochondria is blocked due to the inhibition of PDC by pyruvate dehydrogenase kinase. This altered metabolism is referred to as aerobic glycolysis (Warburg effect) and is common in solid tumors and in other pathological cells. Switching from mitochondrial oxidative phosphorylation to aerobic glycolysis provides diseased cells with advantages because of the rapid production of ATP and the activation of pentose phosphate pathway (PPP) which provides nucleotides required for elevated cellular metabolism. Molecules, called glycolytics, inhibit aerobic glycolysis and convert cells to a healthier phenotype. Glycolytics often function by inhibiting hypoxia-inducible factor-1α leading to PDC disinhibition allowing for intramitochondrial conversion of pyruvate into acetyl coenzyme A. Melatonin is a glycolytic which converts diseased cells to the healthier phenotype. Herein we propose that melatonin’s function as a glycolytic explains its actions in inhibiting a variety of diseases. Thus, the common denominator is melatonin’s action in switching the metabolic phenotype of cells.

In insect, pyruvate is generally the predominant oxidative substrate for mitochondria. This metabolite is transported inside mitochondria via the mitochondrial pyruvate carrier (MPC), but whether and how this transporter controls mitochondrial oxidative capacities in insects is still relatively unknown. Here, we characterize the importance of pyruvate transport as a metabolic control point for mitochondrial substrate oxidation in two genotypes of an insect model, Drosophila melanogaster, differently expressing MPC1, an essential protein for the MPC function. We evaluated the kinetics of pyruvate oxidation, mitochondrial oxygen consumption, metabolic profile, activities of metabolic enzymes, and climbing abilities of wild-type (WT) flies and flies harboring a deficiency in MPC1 (MPC1def). We hypothesized that MPC1 deficiency would cause a metabolic reprogramming that would favor the oxidation of alternative substrates. Our results show that the MPC1def flies display significantly reduced climbing capacity, pyruvate-induced oxygen consumption, and enzymatic activities of pyruvate kinase, alanine aminotransferase, and citrate synthase. Moreover, increased proline oxidation capacity was detected in MPC1def flies, which was associated with generally lower levels of several metabolites, and particularly those involved in amino acid catabolism such as ornithine, citrulline, and arginosuccinate. This study therefore reveals the flexibility of mitochondrial substrate oxidation allowing Drosophila to maintain cellular homeostasis.

International audience; Synthetic Biology (SB) aims at the rational design and engineering of novel biological functions and systems. By facilitating the engineering of living organisms, SB promises to facilitate the development of many new applications for health, biomanufacturing, and the environment. Over the last decade, SB promoted the construction of libraries of components enabling the fine-tuning of genetic circuits expression and the development of novel genome engineering methodologies for many organisms of interest. SB thus opened new perspectives in the field of metabolic engineering, which was until then mainly limited to (over) producing naturally synthesized metabolic compounds. To engineer efficient cell factories, it is key to precisely reroute cellular resources from the central carbon metabolism (CCM) to the synthetic circuitry. This task is however difficult as there is still significant lack of knowledge regarding both the function of several metabolic components and the regulation of the CCM fluxes for many industrially important bacteria. Pyruvate is a pivotal metabolite at the heart of the CCM and a key precursor for the synthesis of several commodity compounds and fine chemicals. Numerous bacterial species can also use it as a carbon source when present in the environment but bacterial, pyruvate-specific uptake systems were to be discovered. This is an issue for metabolic engineering as one can imagine to make use of pyruvate transport systems to replenish synthetic metabolic pathways towards the synthesis of chemicals of interest. Here we describe a recent study (MBio 8(5): e00976-17), which identified and characterized a pyruvate transport system in the Gram-positive (G(+ve)) bacterium Bacillus subtilis, a well-established biotechnological workhorse for the production of enzymes, fine chemicals and antibiotics. This study also revealed that the activity of the two-component system (TCS) responsible for its induction is retro-inhibited by the level of pyruvate influx. Following up on the open question which is whether this retro-inhibition is a generic mechanism for TCSs, we will discuss the implications in metabolic engineering.

Alterations in mitochondrial function contribute to diabetic cardiomyopathy. We have previously shown that heart mitochondrial proteins are hyperacetylated in OVE26 mice, a transgenic model of type 1 diabetes. However, the universality of this modification and its functional consequences are not well established. In this study, we demonstrate that Akita type 1 diabetic mice exhibit hyperacetylation. Functionally, isolated Akita heart mitochondria have significantly impaired maximal (state 3) respiration with physiological pyruvate (0.1 mm) but not with 1.0 mm pyruvate. In contrast, pyruvate dehydrogenase activity is significantly decreased regardless of the pyruvate concentration. We found that there is a 70% decrease in the rate of pyruvate transport in Akita heart mitochondria but no decrease in the mitochondrial pyruvate carriers 1 and 2 (MPC1 and MPC2). The potential role of hyperacetylation in mediating this impaired pyruvate uptake was examined. The treatment of control mitochondria with the acetylating agent acetic anhydride inhibits pyruvate uptake and pyruvate-supported respiration in a similar manner to the pyruvate transport inhibitor α-cyano-4-hydroxycinnamate. A mass spectrometry selective reactive monitoring assay was developed and used to determine that acetylation of lysines 19 and 26 of MPC2 is enhanced in Akita heart mitochondria. Expression of a double acetylation mimic of MPC2 (K19Q/K26Q) in H9c2 cells was sufficient to decrease the maximal cellular oxygen consumption rate. This study supports the conclusion that deficient pyruvate transport activity, mediated in part by acetylation of MPC2, is a contributor to metabolic inflexibility in the diabetic heart.

Androgen receptor (AR) signaling plays a central role in metabolic reprogramming for prostate cancer (PCa) growth and progression. Mitochondria are metabolic powerhouses of the cell and support several hallmarks of cancer. However, the molecular links between AR signaling and the mitochondria that support the metabolic demands of PCa cells are poorly understood. Here, we demonstrate increased levels of dynamin-related protein 1 (DRP1), a mitochondrial fission mediator, in androgen-sensitive and castration-resistant AR-driven PCa. AR signaling upregulates DRP1 to form the VDAC-MPC2 complex, increases pyruvate transport into mitochondria, and supports mitochondrial metabolism, including oxidative phosphorylation and lipogenesis. DRP1 inhibition activates the cellular metabolic stress response, which involves AMPK phosphorylation, induction of autophagy, and the ER unfolded protein response, and attenuates androgen-induced proliferation. Additionally, DRP1 expression facilitates PCa cell survival under diverse metabolic stress conditions, including hypoxia and oxidative stress. Moreover, we found that increased DRP1 expression was indicative of poor prognosis in patients with castration-resistant PCa. Collectively, our findings link androgen signaling-mediated mitochondrial dynamics to metabolic reprogramming; moreover, they have important implications for understanding PCa progression.

The mitochondrial pyruvate carriers mediate pyruvate import into the mitochondria, which is key to the sustenance of the tricarboxylic cycle and oxidative phosphorylation. However, inhibition of mitochondria pyruvate carrier-mediated pyruvate transport was recently shown to be beneficial in experimental models of neurotoxicity pertaining to the context of Parkinson’s disease, and is also protective against excitotoxic neuronal death. These findings attested to the metabolic adaptability of neurons resulting from MPC inhibition, a phenomenon that has also been shown in other tissue types. In this short review, I discuss the mechanism and potential feasibility of mitochondrial pyruvate carrier inhibition as a neuroprotective strategy in neuronal injury and neurodegenerative diseases.

Aims Podocytes play an important role in the development of diabetic kidney disease (DKD). Mitochondria are the source of energy for cell survival, and mitochondrial abnormalities have been shown to contribute to podocyte injury in DKD. In high glucose (HG)-treated podocytes, mitochondrial function and dynamics are abnormal, and intracellular metabolism is often disrupted. However, the molecular mechanism is still unclear. Mitochondrial pyruvate carrier 2 (MPC2) mediates pyruvate transport from the cytoplasm to the mitochondrial matrix, which determines the cellular energy supply and cell survival. Here, we hypothesize that MPC2 damages mitochondria and induces apoptosis in HG-treated podocytes. Main methods We used Western blotting, immunofluorescence and immunoprecipitation to detect the expression of MPC2 in HG-treated podocytes. Pyruvate levels were measured to evaluate metabolic station. Mitochondrial membrane potential (MMP) was measured by inverted fluorescence microscopy and flow cytometry. Mitochondrial morphology was assayed by MitoTracker Red staining, and cellular apoptosis was examined by flow cytometry. Furthermore, we treated podocytes with UK5099 and MPC2 siRNA to assess the outcomes of UK5099 treatment and MPC2 knockdown. Key findings Intracellular pyruvate accumulated, the mitochondria were damaged and cellular apoptosis increased in podocytes cultured with HG compared to that in control podocytes. MPC2 acetylation was significantly increased in HG-treated podocytes. Furthermore, the mitochondrial morphology changed, the MMP decreased, and cellular apoptosis increased. Inhibition of MPC2 function by UK5099 or MPC2 knockdown by siRNA produced the same abnormal effects observed following treatment with HG. Significance MPC2 may mediate mitochondrial dysfunction in HG-treated podocytes, ultimately leading to cell apoptosis.

The Mitochondrial Pyruvate Carrier (MPC) occupies a central metabolic node by transporting cytosolic pyruvate into the mitochondrial matrix and linking glycolysis with mitochondrial metabolism. Two reported human MPC1 mutations cause developmental abnormalities, neurological problems, metabolic deficits, and for one patient, early death. We aimed to understand biochemical mechanisms by which the human patient C289T and T236A MPC1 alleles disrupt MPC function. MPC1 C289T encodes two protein variants, a mis-spliced, truncation mutant (A58G) and a full length point mutant (R97W). MPC1 T236A encodes a full length point mutant (L79H). Using human patient fibroblasts and complementation of CRISPR-deleted, MPC1 null mouse C2C12 cells, we investigated how MPC1 mutations cause MPC deficiency. Truncated MPC1 A58G protein was intrinsically unstable and failed to form MPC complexes. The MPC1 R97W protein was less stable but when overexpressed formed complexes with MPC2 that retained pyruvate transport activity. Conversely, MPC1 L79H protein formed stable complexes with MPC2, but these complexes failed to transport pyruvate. These findings inform MPC structure-function relationships and delineate three distinct biochemical pathologies resulting from two human patient MPC1 mutations. They also illustrate an efficient gene pass-through system for mechanistically investigating human inborn errors in pyruvate metabolism.

Objective The objectives of this study were to assess the role of mitochondrial pyruvate carriers (MPCs) in adipocyte development in vitro and determine whether MPCs are regulated in vivo by high-fat feeding in male and female C57BL/6J mice. Methods This study utilized small interfering RNA-mediated knockdown to assess the requirement of MPC1 for adipogenesis in the 3T3-L1 model system. Treatment with UK-5099, a potent pharmacological MPC inhibitor, was also used to assess the loss of MPC activity. Western blot analysis was performed on adipose tissue samples from mice on a low-fat diet or a high-fat diet (HFD) for 12 weeks. Results The loss of MPC expression via small interfering RNA-mediated knockdown or pharmacological inhibition did not affect adipogenesis of 3T3-L1 cells. In vivo studies indicated that expression of MPCs was significantly decreased in brown adipose tissue of male mice, but not female, on an HFD. Conclusions Although MPCs are essential for pyruvate transport, MPCs are not required for adipogenesis in vitro, suggesting that other substrates can be used for energy production when the MPC complex is not functional. Also, a significant decrease in MPC1 and 2 expression occurred in brown fat, but not white fat, of male mice fed an HFD.

The mitochondrial pyruvate carrier (MPC) occupies a carbohydrate metabolism nexus and is a potential target of thiazolidinediones (TZDs) prescribed to treat the metabolic syndrome. MSDC-0602, a TZD-like, PPARγ sparing molecule, is currently in phase 2B clinical for NASH treatment following positive results in animal studies. Previous research demonstrates that MSDC-0602 inhibits mitochondrial pyruvate oxidation and physically interacts with the MPC. However, the extent to which MSDC-0602 directly inhibits mitochondrial pyruvate uptake has not been fully resolved. Here, we aimed to comprehensively test the action of MSDC-0206 as a specific MPC inhibitor. We examined MSDC-0602 treatment effects on pyruvate oxidation and pyruvate transport by isolated liver mitochondria from normal chow (NCD)- and high fat diet (HFD)-fed mice. MSDC-0602 inhibited pyruvate-driven State III and State IV+Uncoupled respiration in a dose dependent manner. MSDC-0602 reduced pyruvate-driven respiration to levels observed for liver mitochondria isolated from MPC liver knockout (MPC LivKO) mice. NCD and HFD mice showed similar response to MSDC-0602. These data confirm that MSDC-0602 inhibits pyruvate-driven respiration. However, pyruvate oxidation is an indirect measure of MPC activity because it depends upon pyruvate-oxidizing enzymes and electron transport. To directly test the action of MSDC-0602 on mitochondrial pyruvate transport, we performed isolated mitochondria 14C-pyruvate uptake assays. Pyruvate uptake was significantly decreased by MSDC-0602 to rates observed for MPC LivKO mouse liver mitochondria. Finally, we examined effects of MSDC-0602 metabolites and found them to also inhibit isolated liver mitochondria pyruvate oxidation. Together, these data demonstrate that MSDC-0602 directly and specifically inhibits MPC activity as a mechanism for impairing downstream pyruvate utilization. The therapeutic effects of MSDC-0602 may be conferred by MPC inhibition and resulting metabolic adaptations. Disclosure A.J. Rauckhorst: None. L. Oonthonpan: None. E.B. Taylor: Research Support; Self; Cirius Therapeutics. Funding American Diabetes Association (1-18-PDF-060 to A.J.R.); National Institutes of Health (DK104998); Cirius Therapeutics

The transport of lactate and pyruvate between glial cells and neurons plays an important role in the nervous system metabolic coupling. However, the mechanisms and characteristics that underlie the transport of monocarboxylates (MC-T)in vivoare poorly described. Here we useDrosophilaexpressing genetically-encoded FRET sensors to provide anex vivocharacterization of the MC-T in motor neurons and glial cells from the ventral nerve cord. We show that lactate/pyruvate transport on glial cells is coupled to protons and is more efficient than in neurons. Glial cells maintain higher levels of intracellular lactate generating a positive gradient towards neurons. Moreover, our results show that under increased activity lactate and pyruvate rise on motor neurons and suggest that this depends on the transfer of lactate from glial cells mediated in part by the previously described MC transporter Chaski, giving support to thein vivoglia to neurons lactate shuttling during activity.

Appropriate substrate selection between fats and glucose is associated with the success of interventions that maintain health such as exercise or caloric restriction, or with the severity of diseases such as diabetes or other metabolic disorders. Although the interaction and mutual inhibition between glucose and fatty-acids (FAs) catabolism has been studied for decades, a quantitative and integrated understanding of the control and regulation of substrate selection through central catabolic pathways is lacking. We addressed this gap here using a computational model representing cardiomyocyte catabolism encompassing glucose (Glc) utilization, pyruvate transport into mitochondria and oxidation in the tricarboxylic acid (TCA) cycle, -oxidation of palmitate (Palm), oxidative phosphorylation, ion transport, pH regulation, and ROS generation and scavenging in cytoplasmic and mitochondrial compartments. The model is described by 82 differential equations and 119 enzymatic, electron transport and substrate transport reactions accounting for regulatory mechanisms and key players, namely pyruvate dehydrogenase (PDH) and its modulation by multiple effectors. We applied metabolic control analysis to the network operating with various Glc to Palm ratios. The flux and metabolites’ concentration control were visualized through heat maps providing major insights into main control and regulatory nodes throughout the catabolic network. Metabolic pathways located in different compartments were found to reciprocally control each other. For example, glucose uptake and the ATP demand exert control on most processes in catabolism while TCA cycle activities and membrane-associated energy transduction reactions exerted control on mitochondrial processes namely beta-oxidation. PFK and PDH, two highly regulated enzymes, exhibit opposite behavior from a control perspective. While PFK activity was a main rate-controlling step affecting the whole network, PDH played the role of a major regulator showing high sensitivity (elasticity) to substrate availability and key activators/inhibitors, a trait expected from a flexible substrate selector strategically located in the metabolic network. PDH regulated the rate of Glc and Palm consumption, consistent with its high sensitivity towards AcCoA, CoA and NADH. Overall, these results indicate that the control of catabolism is highly distributed across the metabolic network suggesting that fuel selection between FAs and Glc goes well beyond the mechanisms traditionally postulated to explain the glucose-fatty-acid cycle

Summary The reactions catalyzed by the delta-5 and delta-6 desaturases (D5D/D6D), key enzymes responsible for highly unsaturated fatty acid (HUFA) synthesis, regenerate NAD+ from NADH. Here, we show that D5D/D6D provide a mechanism for glycolytic NAD+ recycling that permits ongoing glycolysis and cell viability when the cytosolic NAD+/NADH ratio is reduced, analogous to lactate fermentation. Although lesser in magnitude than lactate production, this desaturase-mediated NAD+ recycling is acutely adaptive when aerobic respiration is impaired in vivo. Notably, inhibition of either HUFA synthesis or lactate fermentation increases the other, underscoring their interdependence. Consistent with this, a type 2 diabetes risk haplotype in SLC16A11 that reduces pyruvate transport (thus limiting lactate production) increases D5D/D6D activity in vitro and in humans, demonstrating a chronic effect of desaturase-mediated NAD+ recycling. These findings highlight key biologic roles for D5D/D6D activity independent of their HUFA end products and expand the current paradigm of glycolytic NAD+ regeneration.

The discovery that the MPC1 and MPC2 genes encode the protein components of the mitochondrial pyruvate carrier (MPC) has invigorated studies of mitochondrial pyruvate transport and its regulation in normal and disease states. Indeed, recent reports have demonstrated MPC involvement in the control of cell fate in cancer and gluconeogenesis in models of type 2 diabetes. Biochemical measurements of MPC activity are foundational for understanding the role of pyruvate transport in health and disease. We developed a 96-well scaled method of [14C]pyruvate uptake that markedly decreases sample requirements and increases throughput relative to previous techniques. This method was applied to determine the mouse liver MPC Km (28.0 ± 3.9 μm) and Vmax (1.08 ± 0.05 nmol/min/mg), which have not previously been reported. Km and Vmax of the rat liver MPC were found to be 71.2 ± 17 μm and 1.42 ± 0.14 nmol/min/mg, respectively. Additionally, we performed parallel pyruvate uptake and oxidation experiments with the same biological samples and show differential results in response to fasting, demonstrating the continued importance of a direct MPC activity assay. We expect this method will be of value for understanding the contribution of the MPC activity to health and disease states where pyruvate metabolism is expected to play a prominent role.

Pyruvate is a key product of glycolysis that regulates the energy metabolism of cells. In Trypanosoma brucei, the causative agent of sleeping sickness, the fate of pyruvate varies dramatically during the parasite life cycle. In bloodstream forms, pyruvate is mainly excreted, whereas in tsetse fly forms, pyruvate is metabolized in mitochondria yielding additional ATP molecules. The character of the molecular machinery that mediates pyruvate transport across mitochondrial membrane was elusive until the recent discovery of mitochondrial pyruvate carrier (MPC) in yeast and mammals. Here, we characterized pyruvate import into mitochondrion of T. brucei. We identified mpc1 and mpc2 homologs in the T. brucei genome with attributes of MPC protein family and we demonstrated that both proteins are present in the mitochondrial membrane of the parasite. Investigations of mpc1 or mpc2 gene knock-out cells proved that T. brucei MPC1/2 proteins facilitate mitochondrial pyruvate transport. Interestingly, MPC is expressed not only in procyclic trypanosomes with fully activated mitochondria but also in bloodstream trypanosomes in which most of pyruvate is excreted. Moreover, MPC appears to be essential for bloodstream forms, supporting the recently emerging picture that the functions of mitochondria in bloodstream forms are more diverse than it was originally thought.

Summary Chronic exposure of pancreatic β-cells to excess glucose can lead to metabolic acceleration and loss of stimulus-secretion coupling. Here, we examined how exposure to excess glucose (defined here as concentrations above 5 mM) affects mTORC1 signaling and the metabolism of β-cells. Acute exposure to excess glucose stimulated glycolysis-dependent mTORC1 signaling, without changes in the PI3K or AMPK pathways. Prolonged exposure to excess glucose led to hyperactivation of mTORC1 and metabolic acceleration, characterized by higher basal respiration and maximal respiratory capacity, increased energy demand, and enhanced flux through mitochondrial pyruvate metabolism. Inhibition of pyruvate transport to the mitochondria decelerated the metabolism of β-cells chronically exposed to excess glucose and re-established glucose-dependent mTORC1 signaling, disrupting a positive feedback loop for mTORC1 hyperactivation. mTOR inhibition had positive and negative impacts on various metabolic pathways and insulin secretion, demonstrating a role for mTOR signaling in the long-term metabolic adaptation of β-cells to excess glucose., Graphical Abstract, Highlights • Acute glucose stimulates mTORC1 in β-cells through a glycolysis-dependent mechanism • Chronic excess glucose promotes mTOR-dependent metabolic acceleration of β-cells • Metabolic acceleration activates a positive feedback loop for mTORC1 hyperactivation • mTOR hyperactivation disturbs the metabolism and insulin secretion patterns of β-cells, Molecular Mechanism of Behavior; Endocrinology; Diabetology; Specialized Functions of Cells

Mitochondrial pyruvate carrier (MPC), which transports pyruvate into mitochondria, is a key regulatory element in the material metabolism and energy metabolism. Since MPC was firstly identified in yeast in 2012, many groups have investigated the function of MPC. As MPC is a classic material transporter, the focus of previous studies has been placed on its role in pyruvate transport. In this study, we discovered a novel Cd resistant gene, stress-seventy subfamily A 4 (SSA4), which can recover the Cd sensitive phenotype in the yeast MPC1 mutant strain. It is suggested that, except for adjusting metabolism, MPC can regulate stress tolerance by regulating downstream genes in yeast. Previously, we discovered a Cd related gene, AGP30, which is associated with MPC1 in Arabidopsis. These results indicate that MPC can regulate Cd tolerance through downstream genes in both Arabidopsis and yeast. This study will pave the way for further exploring the bypass pathways of MPC at the molecular level, and the interaction between MPC and the downstream genes in biology.

Pyruvate is an important intermediate of central carbon metabolism and connects a variety of metabolic pathways in Escherichia coli . Although the intracellular pyruvate concentration is dynamically altered and tightly balanced during cell growth, the pyruvate transport system remains unclear. Here, we identified a pyruvate transporter in E. coli using high-throughput transposon sequencing. The transposon mutant library (a total of 5 × 10 5 mutants) was serially grown with a toxic pyruvate analog (3-fluoropyruvate [3FP]) to enrich for transposon mutants lacking pyruvate transport function. A total of 52 candidates were selected on the basis of a stringent enrichment level of transposon insertion frequency in response to 3FP treatment. Subsequently, their pyruvate transporter function was examined by conventional functional assays, such as those measuring growth inhibition by the toxic pyruvate analog and pyruvate uptake activity. The pyruvate transporter system comprises CstA and YbdD, which are known as a peptide transporter and a conserved protein, respectively, whose functions are associated with carbon starvation conditions. In addition to the presence of more than one endogenous pyruvate importer, it has been suggested that the E. coli genome encodes constitutive and inducible pyruvate transporters. Our results demonstrated that CstA and YbdD comprise the constitutive pyruvate transporter system in E. coli , which is consistent with the tentative genomic locus previously suggested and the functional relationship with the extracellular pyruvate sensing system. The identification of this pyruvate transporter system provides valuable genetic information for understanding the complex process of pyruvate metabolism in E. coli . IMPORTANCE Pyruvate is an important metabolite as a central node in bacterial metabolism, and its intracellular levels are tightly regulated to maintain its functional roles in highly interconnected metabolic pathways. However, an understanding of the mechanism of how bacterial cells excrete and transport pyruvate remains elusive. Using high-throughput transposon sequencing followed by pyruvate uptake activity testing of the selected candidate genes, we found that a pyruvate transporter system comprising CstA and YbdD, currently annotated as a peptide transporter and a conserved protein, respectively, constitutively transports pyruvate. The identification of the physiological role of the pyruvate transporter system provides valuable genetic information for understanding the complex pyruvate metabolism in Escherichia coli .

The active transport of glycolytic pyruvate across the inner mitochondrial membrane is thought to involve two mitochondrial pyruvate carrier subunits, MPC1 and MPC2, assembled as a 150 kDa heterotypic oligomer. Here, the recombinant production of human MPC through a co-expression strategy is first described; however, substantial complex formation was not observed, and predominantly individual subunits were purified. In contrast to MPC1, which co-purifies with a host chaperone, we demonstrated that MPC2 homo-oligomers promote efficient pyruvate transport into proteoliposomes. The derived functional requirements and kinetic features of MPC2 resemble those previously demonstrated for MPC in the literature. Distinctly, chemical inhibition of transport is observed only for a thiazolidinedione derivative. The autonomous transport role for MPC2 is validated in cells when the ectopic expression of human MPC2 in yeast lacking endogenous MPC stimulated growth and increased oxygen consumption. Multiple oligomeric species of MPC2 across mitochondrial isolates, purified protein and artificial lipid bilayers suggest functional high-order complexes. Significant changes in the secondary structure content of MPC2, as probed by synchrotron radiation circular dichroism, further supports the interaction between the protein and ligands. Our results provide the initial framework for the independent role of MPC2 in homeostasis and diseases related to dysregulated pyruvate metabolism.

Metabolism provides substrates for reactive oxygen species (ROS) and nitric oxide (NO) generation, which are a part of the macrophage (mφ) anti-microbial response. Mφs infected with Trypanosoma cruzi (Tc) produce insufficient levels of oxidative species and lower levels of glycolysis compared to classically-activated mφs. How mφs fail to elicit a potent ROS/NO response during infection and its link to glycolysis is unknown. Herein, we evaluated for ROS, NO, and cytokine production in the presence of metabolic modulators of glycolysis and the Krebs cycle. Metabolic status was analyzed by Seahorse Flux Analyzer and mass spectrometry, and validated by RNAi. Tc infection of RAW264.7 or bone marrow-derived mφs elicited a substantial increase in peroxisome proliferator-activated receptor (PPAR)-α expression and pro-inflammatory cytokine release, and moderate levels of ROS/NO. Interferon (IFN)-γ addition enhanced the Tc-induced ROS/NO release and shut down mitochondrial respiration to the levels noted in classically activated mφs. Inhibition of PPAR-α attenuated the ROS/NO response and was insufficient for complete metabolic shift. Deprivation of glucose and inhibition of pyruvate transport showed that Krebs cycle and glycolysis support ROS/NO generation in Tc+IFN-γ-stimulated mφs. Metabolic profiling and RNAi studies showed that glycolysis-pentose phosphate pathway (PPP) at 6-phosphogluconate dehydrogenase was essential for ROS/NO response and control of parasite replication in mφ. We conclude that IFN-γ, but not inhibition of PPAR-α, supports metabolic upregulation of glycolytic-PPP for eliciting potent ROS/NO response in Tc-infected mφs. Chemical analogs enhancing the glucose-PPP will be beneficial in controlling Tc replication and dissemination by mφs.

Some of the most commonly occurring but difficult to isolate halophilic prokaryotes, Archaea as well as Bacteria, require or prefer pyruvate as carbon and energy source. The most efficient media for the enumeration and isolation of heterotrophic prokaryotes from natural environments, from freshwater to hypersaline, including the widely used R2A agar medium, contain pyruvate as a key ingredient. Examples of pyruvate-loving halophiles are the square, extremely halophilic archaeon Haloquadratum walsbyi and the halophilic gammaproteobacterium Spiribacter salinus. However, surprisingly little is known about the availability of pyruvate in natural environments and about the way it enters the cell. Some halophilic Archaea (Halorubrum saccharovorum, Haloarcula spp.) partially convert sugars and glycerol to pyruvate and other acids (acetate, lactate) which are excreted to the medium. Pyruvate formation from glycerol was also shown during a bloom of halophilic Archaea in the Dead Sea. However, no pyruvate transporters were yet identified in the genomes of halophilic Archaea, and altogether, our understanding of pyruvate transport in the prokaryote world is very limited. Therefore, the preference for pyruvate by fastidious and often elusive halophiles and the empirically proven enhanced colony recovery on agar media containing pyruvate are still poorly understood.

Pyruvate is the end-product of glycolysis, a major substrate for oxidative metabolism, and a branching point for glucose, lactate, fatty acid and amino acid synthesis. The mitochondrial enzymes that metabolize pyruvate are physically separated from cytosolic pyruvate pools and rely on a membrane transport system to shuttle pyruvate across the impermeable inner mitochondrial membrane (IMM). Despite long-standing acceptance that transport of pyruvate into the mitochondrial matrix by a carrier-mediated process is required for the bulk of its metabolism, it has taken almost 40 years to determine the molecular identity of an IMM pyruvate carrier. Our current understanding is that two proteins, mitochondrial pyruvate carriers MPC1 and MPC2, form a hetero-oligomeric complex in the IMM to facilitate pyruvate transport. This step is required for mitochondrial pyruvate oxidation and carboxylation–critical reactions in intermediary metabolism that are dysregulated in several common diseases. The identification of these transporter constituents opens the door to the identification of novel compounds that modulate MPC activity, with potential utility for treating diabetes, cardiovascular disease, cancer, neurodegenerative diseases, and other common causes of morbidity and mortality. The purpose of the present review is to detail the historical, current and future research investigations concerning mitochondrial pyruvate transport, and discuss the possible consequences of altered pyruvate transport in various metabolic tissues.

At the heart of central carbon metabolism, pyruvate is a pivotal metabolite in all living cells. Bacillus subtilis is able to excrete pyruvate as well as to use it as the sole carbon source. We herein reveal that ysbAB (renamed pftAB), the only operon specifically induced in pyruvate-grown B. subtilis cells, encodes a hetero-oligomeric membrane complex which operates as a facilitated transport system specific for pyruvate, thereby defining a novel class of transporter. We demonstrate that the LytST two-component system is responsible for the induction of pftAB in the presence of pyruvate by binding of the LytT response regulator to a palindromic region upstream of pftAB. We show that both glucose and malate, the preferred carbon sources for B. subtilis, trigger the binding of CcpA upstream of pftAB, which results in its catabolite repression. However, an additional CcpA-independent mechanism represses pftAB in the presence of malate. Screening a genome-wide transposon mutant library, we find that an active malic enzyme replenishing the pyruvate pool is required for this repression. We next reveal that the higher the influx of pyruvate, the stronger the CcpA-independent repression of pftAB, which suggests that intracellular pyruvate retroinhibits pftAB induction via LytST. Such a retroinhibition challenges the rational design of novel nature-inspired sensors and synthetic switches but undoubtedly offers new possibilities for the development of integrated sensor/controller circuitry. Overall, we provide evidence for a complete system of sensors, feed-forward and feedback controllers that play a major role in environmental growth of B. subtilis., IMPORTANCE Pyruvate is a small-molecule metabolite ubiquitous in living cells. Several species also use it as a carbon source as well as excrete it into the environment. The bacterial systems for pyruvate import/export have yet to be discovered. Here, we identified in the model bacterium Bacillus subtilis the first import/export system specific for pyruvate, PftAB, which defines a novel class of transporter. In this bacterium, extracellular pyruvate acts as the signal molecule for the LytST two-component system (TCS), which in turn induces expression of PftAB. However, when the pyruvate influx is high, LytST activity is drastically retroinhibited. Such a retroinhibition challenges the rational design of novel nature-inspired sensors and synthetic switches but undoubtedly offers new possibilities for the development of integrated sensor/controller circuitry.

The liver toxicity of valproic acid (VPA) is an established side effect of this widely used antiepileptic drug, which is extremely problematic for patients with metabolic epilepsy and particularly epilepsy due to mitochondrial dysfunction. In the present report, we investigated the reason for liver mitochondrial toxicity of VPA and several acid and amide VPA analogues. While the pyruvate and 2-oxoglutarate oxidation rates of rat brain mitochondria were nearly unaffected by VPA, rat liver mitochondrial pyruvate and 2-oxoglutarate oxidation was severely impaired by VPA concentrations above 100 µM. Among the reactions involved in pyruvate oxidation, pyruvate transport and dehydrogenation steps were not affected by VPA, while α-lipoamide dehydrogenase was strongly inhibited. Strong inhibition of α-lipoamide dehydrogenase was also noted for the VPA one-carbon homolog sec -butylpropylacetic acid (SPA) and to a lesser extent for the VPA constitutional isomer valnoctic acid (VCA), while the corresponding amides of the above three acids valpromide (VPD), sec -butylpropylacetamide (SPD) and valnoctamide (VCD) showed only small effects. We conclude that the active inhibitors of pyruvate and 2-oxoglutarate oxidation are the CoA conjugates of VPA and its acid analogues affecting selectively α-lipoamide dehydrogenase in liver. Amide analogues of VPA, like VCD, show low inhibitory effects on mitochondrial oxidative phosphorylation in the liver, which might be relevant for treatment of patients with mitochondrial epilepsy.

Pyruvate is an important organic acid that plays a key role in the central metabolic pathway. Manipulating transporters is an efficient strategy to enhance production of target organic acids and a means to understand the effects of altered intracellular pyruvate content on global metabolic networks. Efforts have been made to manipulate mitochondrial pyruvate carrier (MPC) to transport pyruvate into different subcellular compartments in Candida glabrata to demonstrate the effects of the subcellular distribution of pyruvate on central carbon metabolism. By increasing the mitochondrial pyruvate content through enhancing the rate of pyruvate transport into mitochondria, a high central carbon metabolism rate, specific growth rate and specific pyruvate production rate were obtained. Comparing the intracellular pyruvate content of engineered and control strains showed that higher intracellular pyruvate levels were not conducive to improving pyruvate productivity or central carbon metabolism. Plasma membrane expression of MPCs significantly increased the expression levels of key rate-limiting glycolytic enzymes. Moreover, pyruvate production of CGΔura3-Sp-MPC1, CGΔura3-Sp-MPC2, and CGΔura3-Sp-MPC1-Sp-MPC2 increased 134.4%, 120.3%, and 30.0%, respectively. In conclusion, lower intracellular pyruvate content enhanced central carbon metabolism and provided useful clues for improving the production of other organic acids in microorganisms.

The MTT assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium in the cell cytoplasm to a strongly light absorbing formazan is among the most commonly used methods for determination of cell viability and activity of NAD-dependent oxidoreductases. In the present study, the effects of MTT (0.1 mg/ml) on mitochondrial potential (ΔΨm), intracellular NADH, and respiration of cultured rat cerebellum neurons and isolated rat liver mitochondria were investigated. MTT caused rapid quenching of NADH autofluorescence, fluorescence of MitoTracker Green (MTG) and ΔΨm-sensitive probes Rh123 (rhodamine 123) and TMRM (tetramethylrhodamine methyl ester). The Rh123 signal, unlike that of NADH, MTG, and TMRM, increased in the nucleoplasm after 5-10 min, and this was accompanied by the formation of opaque aggregates of formazan in the cytoplasm and neurites. Increase in the Rh123 signal indicated diffusion of the probe from mitochondria to cytosol and nucleus due to ΔΨm decrease. Inhibition of complex I of the respiratory chain decreased the rate of formazan formation, while inhibition of complex IV increased it. Inhibition of complex III and ATP-synthase affected only insignificantly the rate of formazan formation. Inhibition of glycolysis by 2-deoxy-D-glucose blocked the MTT reduction, whereas pyruvate increased the rate of formazan formation in a concentration-dependent manner. MTT reduced the rate of oxygen consumption by cultured neurons to the value observed when respiratory chain complexes I and III were simultaneously blocked, and it suppressed respiration of isolated mitochondria if substrates oxidized by NAD-dependent dehydrogenases were used. These results demonstrate that formazan formation in cultured rat cerebellum neurons occurs primarily in mitochondria. The initial rate of formazan formation may serve as an indicator of complex I activity and pyruvate transport rate.

SummaryCarrier-facilitated pyruvate transport across the inner mitochondrial membrane plays an essential role in anabolic and catabolic intermediary metabolism. Mitochondrial pyruvate carrier 2 (Mpc2) is believed to be a component of the complex that facilitates mitochondrial pyruvate import. Complete MPC2 deficiency resulted in embryonic lethality in mice. However, a second mouse line expressing an N-terminal truncated MPC2 protein (Mpc2Δ16) was viable but exhibited a reduced capacity for mitochondrial pyruvate oxidation. Metabolic studies demonstrated exaggerated blood lactate concentrations after pyruvate, glucose, or insulin challenge in Mpc2Δ16 mice. Additionally, compared with wild-type controls, Mpc2Δ16 mice exhibited normal insulin sensitivity but elevated blood glucose after bolus pyruvate or glucose injection. This was attributable to reduced glucose-stimulated insulin secretion and was corrected by sulfonylurea KATP channel inhibitor administration. Collectively, these data are consistent with a role for MPC2 in mitochondrial pyruvate import and suggest that Mpc2 deficiency results in defective pancreatic β cell glucose sensing.

NADPH is the primary source of reducing equivalents in the cytosol. Its major source is considered to be the pentose phosphate pathway, but cytosolic NADP(+)-dependent dehydrogenases using intermediates of mitochondrial pathways for substrates have been known to contribute. Photoreceptors, a nonproliferating cell type, provide a unique model for measuring the functional utilization of NADPH at the single cell level. In these cells, NADPH availability can be monitored from the reduction of the all-trans-retinal generated by light to all-trans-retinol using single cell fluorescence imaging. We have used mouse rod photoreceptors to investigate the generation of NADPH by different metabolic pathways. In the absence of extracellular metabolic substrates, NADPH generation was severely compromised. Extracellular glutamine supported NADPH generation to levels comparable to those of glucose, but pyruvate and lactate were relatively ineffective. At low extracellular substrate concentrations, partial inhibition of ATP synthesis lowered, whereas suppression of ATP consumption augmented NADPH availability. Blocking pyruvate transport into mitochondria decreased NADPH availability, and addition of glutamine restored it. Our findings demonstrate that in a nonproliferating cell type, mitochondria-linked pathways can generate substantial amounts of NADPH and do so even when the pentose phosphate pathway is operational. Competing demands for ATP and NADPH at low metabolic substrate concentrations indicate a vulnerability to nutrient shortages. By supporting substantial NADPH generation, mitochondria provide alternative metabolic pathways that may support cell function and maintain viability under transient nutrient shortages. Such pathways may play an important role in protecting against retinal degeneration.

Physiological role of mitochondrial uncoupling proteins UCP2 and UCP3, homologous to UCP1 from brown adipose tissue, is unclear. It was proposed recently that UCP2 and UCP3 are metabolic triggers that switch oxidation of glucose to oxidation of fatty acids, exporting pyruvate from mitochondria. In the present study we tried to verify this hypothesis using ground squirrels (Spermophilus undulatus), since expression of all UCPs in different tissues increases during winter season, and UCP1 is abundant in brown fat. We confirmed the possibility of nonspecific transport of pyruvate through UCP1 in brown fat mitochondria and tried to identify similar transport in liver and skeletal muscle mitochondria where UCP2 and UCP3 are expressed. Transport of pyruvate mediated by UCP1 in mitochondria of brown fat was observed using valinomycin-induced swelling of non-respiring mitochondria in 55 mM potassium pyruvate and was inhibited by GDP. In contrast, mitochondria of liver and skeletal muscles in similar conditions did not exhibit electrogenic transport of pyruvate anions that could be related to functioning of UCP2 and UCP3. At the same time, functioning of pyruvate carrier was detected in these mitochondria by nigericin-induced passive swelling or valinomycin-induced active swelling in potassium pyruvate that was inhibited by α-CHC, a specific inhibitor of the pyruvate carrier. Thus, our results suggest that in contrast to UCP1 of brown fat, UCP2 and UCP3 from intact liver and skeletal muscle mitochondria of winter active ground squirrels are unable to carry out pyruvate transport.

Metabolic remodeling is a major determinant for many cell fate decisions, and a switch from respiration to aerobic glycolysis is generally considered as a hallmark of cancer cell transformation. Pyruvate is a key metabolite at the major junction of carbohydrate metabolism between cytosolic glycolysis and the mitochondrial Krebs cycle. In this issue of The EMBO Journal, Bender et al show that yeast cells regulate pyruvate uptake into mitochondria, and thus its metabolic fate, by expressing alternative pyruvate carrier complexes with different activities.

Facilitated pyruvate transport across the mitochondrial inner membrane is a critical step in carbohydrate, amino acid, and lipid metabolism. We report that clinically relevant concentrations of thiazolidinediones (TZDs), a widely used class of insulin sensitizers, acutely and specifically inhibit mitochondrial pyruvate carrier (MPC) activity in a variety of cell types. Respiratory inhibition was overcome with methyl pyruvate, localizing the effect to facilitated pyruvate transport, and knockdown of either paralog, MPC1 or MPC2, decreased the EC 50 for respiratory inhibition by TZDs. Acute MPC inhibition significantly enhanced glucose uptake in human skeletal muscle myocytes after 2 h. These data ( i ) report that clinically used TZDs inhibit the MPC, ( ii ) validate that MPC1 and MPC2 are obligatory components of facilitated pyruvate transport in mammalian cells, ( iii ) indicate that the acute effect of TZDs may be related to insulin sensitization, and ( iv ) establish mitochondrial pyruvate uptake as a potential therapeutic target for diseases rooted in metabolic dysfunction.

Pyruvate is a central metabolite that must be imported into organelles for specific metabolic processes, including C4 photosynthesis. Plastidial and mitochondrial pyruvate transporter molecules were recently identified: the former was found based on C4 photosynthesis transcriptome analysis and the latter using a bioinformatics approach in yeast. The transport activities of these molecules were recently investigated in heterologous expression systems: Escherichia coli and Lactococcus lactis, respectively. These studies demonstrated the important roles of the NHD1/Bass2-protein coupling function and the mitochondria pyruvate carrier protein complex in pyruvate uptake. Here, I summarize the approaches used to isolate these proteins and the issues that remain to be investigated.

Alterations in cell metabolism are increasingly recognized as a hallmark of cancer and are being exploited for the development of diagnostic tools and targeted therapeutics. Recently, 13C magnetic resonance spectroscopy (MRS)-detectable hyperpolarized pyruvate to lactate conversion was validated in models as a noninvasive imaging method for the detection of tumors and treatment response, and successfully passed phase I clinical trials. To date, response to treatment was associated with a drop in hyperpolarized lactate production. Here, we monitored the effect of treatment with the MEK inhibitor U0126 in prostate and breast cancer cells. Following treatment we observed a 31% drop in flux of hyperpolarized 13C label in treated MCF-7 breast cancer cells compared to controls. In contrast and unexpectedly, flux increased to 167% in treated PC3 prostate cancer cells. To mechanistically explain these observations we investigated treatment-induced changes in the different factors known to affect pyruvate to lactate conversion. NADH levels remained unchanged whereas lactate dehydrogenase expression and activity as well as intracellular lactate increased in both cell lines, providing an explanation for the elevated hyperpolarized lactate observed in PC3 cells. The expression of MCT1, which mediates pyruvate transport, dropped in treated MCF-7 but not in PC3 cells. This identifies pyruvate transport as rate limiting in U0126-treated MCF-7 cells and explains the drop in hyperpolarized lactate observed in those cells following treatment. Our findings highlight the complexity of interactions between MEK and metabolism, and the need for mechanistic validation before hyperpolarized 13C MRS can be used for monitoring treatment-induced molecular responses.

Fisetin is a flavonoid dietary ingredient found in the smoke tree (Cotinus coggyria) and in several fruits and vegetables. The effects of fisetin on glucose metabolism in the isolated perfused rat liver and some glucose-regulating enzymatic activities were investigated. Fisetin inhibited glucose, lactate, and pyruvate release from endogenous glycogen. Maximal inhibitions of glycogenolysis (49%) and glycolysis (59%) were obtained with the concentration of 200 microM. The glycogenolytic effects of glucagon and dinitrophenol were suppressed by fisetin 300 microM. No significant changes in the cellular contents of AMP, ADP, and ATP were found. Fisetin increased the cellular content of glucose 6-phosphate and inhibited the glucose 6-phosphatase activity. Gluconeogenesis from lactate and pyruvate or fructose was inhibited by fisetin 300 microM. Pyruvate carboxylation in isolated intact mitochondria was inhibited (IC(50) = 163.10 +/- 12.28 microM); no such effect was observed in freeze-thawing disrupted mitochondria. It was concluded that fisetin inhibits glucose release from the livers in both fed and fasted conditions. The inhibition of pyruvate transport into the mitochondria and the reduction of the cytosolic NADH-NAD(+) potential redox could be the causes of the gluconeogenesis inhibition. Fisetin could also prevent hyperglycemia by decreasing glycogen breakdown or blocking the glycogenolytic action of hormones.

Metabolic fluxes can serve as specific biomarkers for detecting malignant transformations, tumor progression, and response to microenvironmental changes and treatment procedures. We present noninvasive hyperpolarized 13 C NMR investigations on the metabolic flux of pyruvate to lactate, in a well-controlled injection/perfusion system using T47D human breast cancer cells. Initial rates of pyruvate-to-lactate conversion were obtained by fitting the hyperpolarized 13 C and ancillary 31 P NMR data to a model, yielding both kinetic parameters and mechanistic insight into this conversion. Transport was found to be the rate-limiting process for the conversion of extracellular pyruvate to lactate with K m = 2.14 ± 0.03 mM, typical of the monocarboxylate transporter 1 (MCT1), and a V max = 27.6 ± 1.1 fmol·min −1 ·cell −1 , in agreement with the high expression level of this transporter. Modulation of the environment to hypoxic conditions as well as suppression of cells' perfusion enhanced the rate of pyruvate-to-lactate conversion, presumably by up-regulation of the MCT1. Conversely, the addition of quercetin, a flavonoidal MCT1 inhibitor, markedly reduces the apparent rate of pyruvate-to-lactate conversion. These results suggest that hyperpolarized 13 C 1 -pyruvate may be a useful magnetic resonance biomarker of MCT regulation and malignant transformations in breast cancer.

The action of gatifloxacin, the broad-spectrum fluoroquinolone antibiotic commonly used in the therapy of various bacterial infections, was investigated in isolated rabbit hepatocytes and kidney-cortex tubules by measuring the activity of gluconeogenesis, a process that maintains whole body glucose homeostasis. The data show that in kidney-cortex tubules, application of gatifloxacin at up to 100 μM was followed by a marked accumulation of the drug in the intracellular milieu and a decrease in the rate of glucose formation from pyruvate by 20–50%. Gatifloxacin did not affect the rate of gluconeogenesis from either alanine + glycerol + octanoate or aspartate + glycerol + octanoate. At concentrations between 25 and 200 μM the drug decreased mitochondrial oxygen consumption by 20–45% with pyruvate + malate and ADP. As in the case of alpha-cyano-4-hydroxycinnamate, a well-established inhibitor of the mitochondrial pyruvate transporter, it diminished pyruvate uptake by both renal and hepatic mitochondria. The inhibitory action of gatifloxacin was less pronounced in hepatocytes where reduction in pyruvate-dependent glucose formation and mitochondrial respiration was by no more than 25%. The antibiotic did not influence mitochondrial oxygen consumption with glutamate + malate in either kidney-cortex or liver mitochondria. A differential substrate dependence of gatifloxacin action on gluconeogenesis and mitochondrial respiration combined with a decrease in pyruvate uptake by mitochondria suggest that the inhibitory action of this drug on gluconeogenesis might result from its impairment of pyruvate transport into mitochondria.

Cardiac mitochondria were isolated from Bufo marinus and Rana catesbeiana, two species of amphibian whose cardiovascular systems are adapted to either predominantly aerobic or glycolytic modes of locomotion. Mitochondrial oxidative capacity was compared using VO2 max and respiratory control ratios in the presence of a variety of substrates including pyruvate, lactate, oxaloacetate, β-hydroxybutyrate, and octanoyl-carnitine. B. marinus cardiac mitochondria exhibited VO2 max values twice that of R. catesbeiana cardiac mitochondria when oxidizing carbohydrate substrates. Pyruvate transport was measured via a radiolabeled-tracer assay in isolated B. marinus and R. catesbeiana cardiac mitochondria. Time-course experiments described both α-cyano-4-hydroxycinnamate-sensitive (MCT-like) and phenylsuccinate-sensitive pyruvate uptake mechanisms in both species. Pyruvate uptake by the MCT-like transporter was enhanced in the presence of a pH gradient, whereas the phenylsuccinate-sensitive transporter was inhibited. Notably, anuran cardiac mitochondria exhibited activities of lactate dehydrogenase and pyruvate carboxylase. The presence of both transporters on the inner mitochondrial membrane affords the net uptake of monocarboxylates including pyruvate, β-hydroxybutyrate, and lactate; the latter potentially indicating the presence of a lactate/pyruvate shuttle allowing oxidation of extramitochondrial NADH. Intramitochondrial lactate dehydrogenase and pyruvate carboxylase enables lactate to be oxidized to pyruvate or converted to anaplerotic oxaloacetate. Kinetics of the MCT-like transporter differed significantly between the two species, suggesting differences in aerobic scope may be in part attributable to differences in mitochondrial carbohydrate utilization. J. Exp. Zool. 307A:425–438, 2007. © 2007 Wiley-Liss, Inc.