Dicarboxylate Carrier Research Articles

The mitochondrial dicarboxylate carrier mediates in vivo hepatic gluconeogenesis.

Hepatic gluconeogenesis (GNG) is essential for maintaining euglycemia during prolonged fasting. However, GNG becomes pathologically elevated and drives chronic hyperglycemia in type 2 diabetes (T2D). Lactate/pyruvate is a major GNG substrate known to be imported into mitochondria for GNG. Yet, the subsequent mitochondrial carbon export mechanisms required to supply the extra-mitochondrial canonical GNG pathway have not been genetically delineated. Here, we evaluated the role of the mitochondrial dicarboxylate carrier (DiC) in mediating GNG from lactate/pyruvate. We generated liver-specific DiC knockout (DiC LivKO) mice. During lactate/pyruvate tolerance tests, DiC LivKO decreased plasma glucose excursion and 13C-lactate/-pyruvate flux into hepatic and plasma glucose. In a Western diet (WD) feeding model of T2D, acute DiC LivKO after induction of obesity decreased lactate/pyruvate-driven GNG, hyperglycemia, and hyperinsulinemia. Our results show that mitochondrial carbon export through the DiC mediates GNG and that the DiC contributes to impaired glucose homeostasis in a mouse model of T2D.

Human mitochondrial carriers of the SLC25 family function as monomers exchanging substrates with a ping-pong kinetic mechanism

Members of the SLC25 mitochondrial carrier family link cytosolic and mitochondrial metabolism and support cellular maintenance and growth by transporting compounds across the mitochondrial inner membrane. Their monomeric or dimeric state and kinetic mechanism have been a matter of long-standing debate. It is believed by some that they exist as homodimers and transport substrates with a sequential kinetic mechanism, forming a ternary complex where both exchanged substrates are bound simultaneously. Some studies, in contrast, have provided evidence indicating that the mitochondrial ADP/ATP carrier (SLC25A4) functions as a monomer, has a single substrate binding site, and operates with a ping-pong kinetic mechanism, whereby ADP is imported before ATP is exported. Here we reanalyze the oligomeric state and kinetic properties of the human mitochondrial citrate carrier (SLC25A1), dicarboxylate carrier (SLC25A10), oxoglutarate carrier (SLC25A11), and aspartate/glutamate carrier (SLC25A13), all previously reported to be dimers with a sequential kinetic mechanism. We demonstrate that they are monomers, except for dimeric SLC25A13, and operate with a ping-pong kinetic mechanism in which the substrate import and export steps occur consecutively. These observations are consistent with a common transport mechanism, based on a functional monomer, in which a single central substrate-binding site is alternately accessible.

A mitochondrial carrier transports glycolytic intermediates to link cytosolic and mitochondrial glycolysis in the human gut parasite Blastocystis

Stramenopiles form a clade of diverse eukaryotic organisms, including multicellular algae, the fish and plant pathogenic oomycetes, such as the potato blight Phytophthora, and the human intestinal protozoan Blastocystis. In most eukaryotes, glycolysis is a strictly cytosolic metabolic pathway that converts glucose to pyruvate, resulting in the production of NADH and ATP (Adenosine triphosphate). In contrast, stramenopiles have a branched glycolysis in which the enzymes of the pay-off phase are located in both the cytosol and the mitochondrial matrix. Here, we identify a mitochondrial carrier in Blastocystis that can transport glycolytic intermediates, such as dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, across the mitochondrial inner membrane, linking the cytosolic and mitochondrial branches of glycolysis. Comparative analyses with the phylogenetically related human mitochondrial oxoglutarate carrier (SLC25A11) and dicarboxylate carrier (SLC25A10) show that the glycolytic intermediate carrier has lost its ability to transport the canonical substrates malate and oxoglutarate. Blastocystis lacks several key components of oxidative phosphorylation required for the generation of mitochondrial ATP, such as complexes III and IV, ATP synthase, and ADP/ATP carriers. The presence of the glycolytic pay-off phase in the mitochondrial matrix generates ATP, which powers energy-requiring processes, such as macromolecular synthesis, as well as NADH, used by mitochondrial complex I to generate a proton motive force to drive the import of proteins and molecules. Given its unique substrate specificity and central role in carbon and energy metabolism, the carrier for glycolytic intermediates identified here represents a specific drug and pesticide target against stramenopile pathogens, which are of great economic importance.

A mitochondrial carrier transports glycolytic intermediates to link cytosolic and mitochondrial glycolysis in the human gut parasite Blastocystis.

Stramenopiles form a clade of diverse eukaryotic organisms, including multicellular algae, the fish and plant pathogenic oomycetes, such as the potato blight Phytophthora, and the human intestinal protozoan Blastocystis. In most eukaryotes, glycolysis is a strictly cytosolic metabolic pathway that converts glucose to pyruvate, resulting in the production of NADH and ATP (Adenosine triphosphate). In contrast, stramenopiles have a branched glycolysis in which the enzymes of the pay-off phase are located in both the cytosol and the mitochondrial matrix. Here, we identify a mitochondrial carrier in Blastocystis that can transport glycolytic intermediates, such as dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, across the mitochondrial inner membrane, linking the cytosolic and mitochondrial branches of glycolysis. Comparative analyses with the phylogenetically related human mitochondrial oxoglutarate carrier (SLC25A11) and dicarboxylate carrier (SLC25A10) show that the glycolytic intermediate carrier has lost its ability to transport the canonical substrates malate and oxoglutarate. Blastocystis lacks several key components of oxidative phosphorylation required for the generation of mitochondrial ATP, such as complexes III and IV, ATP synthase, and ADP/ATP carriers. The presence of the glycolytic pay-off phase in the mitochondrial matrix generates ATP, which powers energy-requiring processes, such as macromolecular synthesis, as well as NADH, used by mitochondrial complex I to generate a proton motive force to drive the import of proteins and molecules. Given its unique substrate specificity and central role in carbon and energy metabolism, the carrier for glycolytic intermediates identified here represents a specific drug and pesticide target against stramenopile pathogens, which are of great economic importance.

Mitochondrial succinate transport as a novel therapeutic target for cardiac ischemia/reperfusion injury

Abstract Background The accumulation of the mitochondrial metabolite succinate during ischemia and its rapid oxidation by succinate dehydrogenase (SDH) upon reperfusion drives ischemia/reperfusion injury (IRI) in myocardial infarction. Succinate oxidation leads to the production of reactive oxygen species (ROS) by reverse electron transport (RET) at mitochondrial complex I, orchestrating a cascade of events leading to cardiomyocyte death. Modulating succinate metabolism, such as SDH inhibition, is a promising therapeutic strategy. During ischemia, the mitochondria produced succinate is transported into the cytosol, enabling the accumulation of a large succinate pool. On reperfusion, the succinate is transported back into mitochondria and oxidised by SDH, thus we hypothesised that targeting succinate mitochondrial transport could be a novel therapeutic target to prevent cardiac IRI. Purpose To assess the role of succinate mitochondrial transport as a novel therapeutic target to prevent cardiac IRI. Methods Isolated heart mitochondria were used to assess mitochondrial respiration by Oroboros, RET-ROS production by Amplex Red and butylmalonate delivery. Cultured H9C2 myoblasts and isolated primary adult cardiomyocytes from C57BL/6J mice were used to assess butylmalonate uptake and delivery from esters, as well as their impact on succinate accumulation and oxidation in vitro. LAD ligation and reperfusion in C57BL/6J mice was used as an in vivo MI model. Metabolite levels were measured by LC-MS/MS. Results Butylmalonate, an inhibitor of the mitochondrial dicarboxylate carrier (DIC), blocks succinate-dependent respiration and RET-ROS production in isolated mitochondria. However, butylmalonate entry into cells was inefficient and does not impact infarct size after IRI in vivo. We developed novel butylmalonate esters to improve butylmalonate delivery to cardiomyocytes in vivo. We found that diacetoxymethyl butylmalonate (DAB) effectively delivered butylmalonate and could prevent succinate accumulation and oxidation in vitro. DAB could also prevent succinate-mediated respiration and RET-ROS at lower concentrations than butylmalonate alone. Finally, DAB reduced acute infarct size in vivo when administered at reperfusion. Conclusions Blocking succinate transport back into mitochondria at reperfusion prevents the damage in IRI by preventing the oxidation of succinate by SDH and downstream RET-ROS production. Therefore, targeting the mitochondrial transport of succinate may be a promising novel strategy in preventing cardiac IRI.

Kinetic modelling of β-cell metabolism reveals control points in the insulin-regulating pyruvate cycling pathways.

Insulin, a key hormone in the regulation of glucose homoeostasis, is secreted by pancreatic β-cells in response to elevated glucose levels. Insulin is released in a biphasic manner in response to glucose metabolism in β-cells. The first phase of insulin secretion is triggered by an increase in the ATP:ADP ratio; the second phase occurs in response to both a rise in ATP:ADP and other key metabolic signals, including a rise in the NADPH:NADP+ ratio. Experimental evidence indicates that pyruvate-cycling pathways play an important role in the elevation of the NADPH:NADP+ ratio in response to glucose. The authors developed a kinetic model for the tricarboxylic acid cycle and pyruvate cycling pathways. The authors successfully validated the model against experimental observations and performed a sensitivity analysis to identify key regulatory interactions in the system. The model predicts that the dicarboxylate carrier and the pyruvate transporter are the most important regulators of pyruvate cycling and NADPH production. In contrast, the analysis showed that variation in the pyruvate carboxylase flux was compensated by a response in the activity of mitochondrial isocitrate dehydrogenase (ICDm ) resulting in minimal effect on overall pyruvate cycling flux. The model predictions suggest starting points for further experimental investigation, as well as potential drug targets for the treatment of type 2 diabetes.

Mitochondrial ATP Synthase and Mild Uncoupling by Butyl Ester of Rhodamine 19, C4R1.

The homeostasis of the transmembrane potential of hydrogen ions in mitochondria is a prerequisite for the normal mitochondrial functioning. However, in different pathological conditions it is advisable to slightly reduce the membrane potential, while maintaining it at levels sufficient to produce ATP that will ensure the normal functioning of the cell. A number of chemical agents have been found to provide mild uncoupling; however, natural proteins residing in mitochondrial membrane can carry this mission, such as proteins from the UCP family, an adenine nucleotide translocator and a dicarboxylate carrier. In this study, we demonstrated that the butyl ester of rhodamine 19, C4R1, binds to the components of the mitochondrial ATP synthase complex due to electrostatic interaction and has a good uncoupling effect. The more hydrophobic derivative C12R1 binds poorly to mitochondria with less uncoupling activity. Mass spectrometry confirmed that C4R1 binds to the β-subunit of mitochondrial ATP synthase and based on molecular docking, a C4R1 binding model was constructed suggesting the binding site on the interface between the α- and β-subunits, close to the anionic amino acid residues of the β-subunit. The association of the uncoupling effect with binding suggests that the ATP synthase complex can provide induced uncoupling.

Diversity in Acidity between Core and Pulp of Asian Pear Fruit Is Mainly Regulated by the Collaborative Activity of PH8.1 and DIC2 Genes during Fruit Development

The pear (Pyrus pyrifolia) is an important accessory fruit in which the pear core is tarter than the pear pulp. However, the reason for the acidic core and diversity in the taste of the same fruit is not clear. In this study, we observed that the citrate contents were three times higher in the core than in the pulp, while the malate content decreased along with fruit development and was significantly lower in the core than in the pulp at 110 days after flowering. Overall transcript levels for citrate-malate synthesis-related genes increased more in the pear core than the pulp at early fruit development, while degradation-related genes activity was nearly similar or non-significant between the core and pulp during fruit development. The lesser malate accumulation in the pear core compared to the pulp at 110 DAF was possibly due to the reduced activity of tDT2 gene. Regarding citrate accumulation, we identified five important p-type H+-ATPase genes in pear and found that the relative expression level of the PH8.1 gene was four-fold higher in the core than in the pulp during fruit development. Moreover, the expression level of di-carboxylate carrier gene 2 (DIC2) was constantly and significantly higher in the core than in the pulp. In addition, correlation analysis signified that the transcript levels of the two genes PH8.1 and DIC2 positively and significantly correlated with the citrate contents. These results suggested that the increased and collaborative activity of PH8.1 and DIC2 played a key role in the higher citrate accumulation in the core than the pulp, thus, with the help of molecular breeding tools, the citrate contents can be optimized in pear fruit for divers and improved fruit flavoring.

The 2-oxoglutarate/malate carrier enhances the arachidonic acid–mediated proton transport in lipid bilayer membranes

Uncoupling is a process in which the proton gradient at the inner mitochondrial membrane decreases without ATP production. It can be induced by free fatty acids (FA) and involves members of the solute carrier 25 (SLC25) family: uncoupling proteins 1-4, adenine nucleotide translocase 1 (ANT1), dicarboxylate carrier, aspartate/glutamate carrier, and phosphate carrier. Although the 2-oxoglutarate/malate carrier (OGC) was previously considered a negative control for the uncoupling activity, a significant drop in mitochondrial membrane potential following its overexpression in HEK293 cells indicated otherwise1.

Comprehensive In Silico Analysis and Transcriptional Profiles Highlight the Importance of Mitochondrial Dicarboxylate Carriers (DICs) on Hypoxia Response in Both Arabidopsis thaliana and Eucalyptus grandis.

Plant dicarboxylate carriers (DICs) transport a wide range of dicarboxylates across the mitochondrial inner membrane. The Arabidopsis thaliana DIC family is composed of three genes (AtDIC1, 2 and 3), whereas two genes (EgDIC1 and EgDIC2) have been retrieved in Eucalyptus grandis. Here, by combining in silico and in planta analyses, we provide evidence that DICs are partially redundant, important in plant adaptation to environmental stresses and part of a low-oxygen response in both species. AtDIC1 and AtDIC2 are present in most plant species and have very similar gene structure, developmental expression patterns and absolute expression across natural Arabidopsis accessions. In contrast, AtDIC3 seems to be an early genome acquisition found in Brassicaceae and shows relatively low (or no) expression across these accessions. In silico analysis revealed that both AtDICs and EgDICs are highly responsive to stresses, especially to cold and submergence, while their promoters are enriched for stress-responsive transcription factors binding sites. The expression of AtDIC1 and AtDIC2 is highly correlated across natural accessions and in response to stresses, while no correlation was found for AtDIC3. Gene ontology enrichment analysis suggests a role for AtDIC1 and AtDIC2 in response to hypoxia, and for AtDIC3 in phosphate starvation. Accordingly, the investigated genes are induced by submergence stress in A. thaliana and E. grandis while AtDIC2 overexpression improved seedling survival to submergence. Interestingly, the induction of AtDIC1 and AtDIC2 is abrogated in the erfVII mutant that is devoid of plant oxygen sensing, suggesting that these genes are part of a conserved hypoxia response in Arabidopsis.

Fifty Years of Research on Protonophores: Mitochondrial Uncoupling As a Basis for Therapeutic Action.

Protonophores are compounds capable of electrogenic transport of protons across membranes. Protonophores have been intensively studied over the past 50 years owing to their ability to uncouple oxidation and phosphorylation in mitochondria and chloroplasts. The action mechanism of classical uncouplers, such as DNP and CCCP, in mitochondria is believed to be related to their protonophoric activity; i.e., their ability to transfer protons across the lipid part of the mitochondrial membrane. Given the recently revealed deviations in the correlation between the protonophoric activity of some uncouplers and their ability to stimulate mitochondrial respiration, this review addresses the involvement of some proteins of the inner mitochondrial membrane, such as the ATP/ADP antiporter, dicarboxylate carrier, and ATPase, in the uncoupling process. However, these deviations do not contradict the Mitchell theory but point to a more complex nature of the interaction of DNP, CCCP, and other uncouplers with mitochondrial membranes. Therefore, a detailed investigation of the action mechanism of uncouplers is required for a more successful pharmacological use, including their antibacterial, antiviral, anticancer, as well as cardio-, neuro-, and nephroprotective effects.

The versatility of plant organic acid metabolism in leaves is underpinned by mitochondrial malate-citrate exchange.

Malate and citrate underpin the characteristic flexibility of central plant metabolism by linking mitochondrial respiratory metabolism with cytosolic biosynthetic pathways. However, the identity of mitochondrial carrier proteins that influence both processes has remained elusive. Here we show by a systems approach that DICARBOXYLATE CARRIER 2 (DIC2) facilitates mitochondrial malate–citrate exchange in vivo in Arabidopsis thaliana. DIC2 knockout (dic2-1) retards growth of vegetative tissues. In vitro and in organello analyses demonstrate that DIC2 preferentially imports malate against citrate export, which is consistent with altered malate and citrate utilization in response to prolonged darkness of dic2-1 plants or a sudden shift to darkness of dic2-1 leaves. Furthermore, isotopic glucose tracing reveals a reduced flux towards citrate in dic2-1, which results in a metabolic diversion towards amino acid synthesis. These observations reveal the physiological function of DIC2 in mediating the flow of malate and citrate between the mitochondrial matrix and other cell compartments.

Elucidating the contribution of mitochondrial glutathione to ferroptosis in cardiomyocytes

Ferroptosis is a programmed iron-dependent cell death associated with peroxidation of lipids particularly, phospholipids. Several studies suggested a possible contribution of mitochondria to ferroptosis although the mechanisms underlying mitochondria-mediated ferroptotic pathways remain elusive. Reduced glutathione (GSH) is a central player in ferroptosis that is required for glutathione peroxidase 4 to eliminate oxidized phospholipids. Mitochondria do not produce GSH, and although the transport of GSH to mitochondria is not fully understood, two carrier proteins, the dicarboxylate carrier (DIC, SLC25A10) and the oxoglutarate carrier (OGC, SLC25A11) have been suggested to participate in GSH transport. Here, we elucidated the role of DIC and OGC as well as mitochondrial bioenergetics in ferroptosis in H9c2 cardioblasts. Results showed that mitochondria are highly sensitive to ferroptotic stimuli displaying fragmentation, and lipid peroxidation shortly after the onset of ferroptotic stimulus. Inhibition of electron transport chain complexes and oxidative phosphorylation worsened RSL3-induced ferroptosis. LC-MS/MS analysis revealed a dramatic increase in the levels of pro-ferroptotic oxygenated phosphatidylethanolamine species in mitochondria in response to RSL3 (ferroptosis inducer) and cardiac ischemia-reperfusion. Inhibition of DIC and OGC aggravated ferroptosis and increased mitochondrial ROS, membrane depolarization, and GSH depletion. Dihydrolipoic acid, an essential cofactor for several mitochondrial multienzyme complexes, attenuated ferroptosis and induced direct reduction of pro-ferroptotic peroxidized phospholipids to hydroxy-phospholipids in vitro. In conclusion, we suggest that ferroptotic stimuli diminishes mitochondrial bioenergetics and stimulates GSH depletion and glutathione peroxidase 4 inactivation leading to ferroptosis.

Glutathione Metabolism and the Novel Role of Mitochondrial GSH in Retinal Degeneration.

Glutathione (GSH) is present ubiquitously, and its role as a crucial cellular antioxidant in tissues, including the retina, is well established. GSH’s antioxidant function arises from its ability to scavenge reactive oxygen species or to serve as an essential cofactor for GSH S-transferases and peroxidases. This review summarizes the general functions, retinal distribution, disorders linked to GSH deficiency, and the emerging role for mitochondrial GSH (mGSH) in retinal function. Though synthesized only in the cytosol, the presence of GSH in multiple cell organelles suggests the requirement for its active transport across organellar membranes. The localization and distribution of 2-oxoglutarate carrier (OGC) and dicarboxylate carrier (DIC), two recently characterized mitochondrial carrier proteins in RPE and retina, show that these transporters are highly expressed in human retinal pigment epithelium (RPE) cells and retinal layers, and their expression increases with RPE polarity in cultured cells. Depletion of mGSH levels via inhibition of the two transporters resulted in reduced mitochondrial bioenergetic parameters (basal respiration, ATP production, maximal respiration, and spare respiratory capacity) and increased RPE cell death. These results begin to reveal a critical role for mGSH in maintaining RPE bioenergetics and cell health. Thus, augmentation of mGSH pool under GSH-deficient conditions may be a valuable tool in treating retinal disorders, such as age-related macular degeneration and optic neuropathies, whose pathologies have been associated with mitochondrial dysfunction.

The mitochondrial dicarboxylate carrier prevents hepatic lipotoxicity by inhibiting white adipocyte lipolysis

We have previously reported that the mitochondrial dicarboxylate carrier (mDIC [SLC25A10]) is predominantly expressed in the white adipose tissue (WAT) and subject to regulation by metabolic cues. However, the specific physiological functions of mDIC and the reasons for its abundant presence in adipocytes are poorly understood. To systemically investigate the impact of mDIC function in adipocytes invivo, we generated loss- and gain-of-function mouse models, selectively eliminating or overexpressing mDIC in mature adipocytes, respectively. In in vitro differentiated white adipocytes, mDIC is responsible for succinate transport from the mitochondrial matrix to the cytosol, from where succinate can act on the succinate receptor SUCNR1 and inhibit lipolysis by dampening the cAMP- phosphorylated hormone-sensitive lipase (pHSL) pathway. We eliminated mDIC expression in adipocytes in a doxycycline (dox)-inducible manner (mDICiKO) and demonstrated that such a deletion results in enhanced adipocyte lipolysis and promotes high-fat diet (HFD)-induced adipocyte dysfunction, liver lipotoxicity, and systemic insulin resistance. Conversely, in a mouse model with dox-inducible, adipocyte-specific overexpression of mDIC (mDICiOE), we observed suppression of adipocyte lipolysis both in vivo and ex vivo. mDICiOE mice are potently protected from liver lipotoxicity upon HFD feeding. Furthermore, they show resistance to HFD-induced weight gain and adipose tissue expansion with concomitant improvements in glucose tolerance and insulin sensitivity. Beyond our data in rodents, we found that human WAT SLC25A10 mRNA levels are positively correlated with insulin sensitivity and negatively correlated with intrahepatic triglyceride levels, suggesting a critical role of mDIC in regulating overall metabolic homeostasis in humans as well. In summary, we highlight that mDIC plays an essential role in governing adipocyte lipolysis and preventing liver lipotoxicity in response to a HFD. Dysfunctional fat tissue plays an important role in the development of fatty liver disease and liver injury. Our present study identifies a mitochondrial transporter, mDIC, which tightly controls the release of free fatty acids from adipocytes to the liver through the export of succinate from mitochondria. We believe this mDIC-succinate axis could be targeted for the treatment of fatty liver disease.

A transhydrogenase-like mechanism in CHO cells comprising concerted cytosolic reaction and mitochondrial shuttling activities

The proper supply and regeneration of reduction equivalents such as NADH and NADPH is of pivotal importance for living cells. Although both, NADH and NADPH, are electron carriers, they serve for different, complementing needs and require well balanced exchange. In Chinese hamster ovary (CHO) cells, the proton translocator ‘Nicotinamide Nucleotide Transhydrogenase’ (NNT) was annotated recently, but no in vivo operational evidence was given, yet. Here, we present another transhydrogenase-like mechanism building on the adaptive interplay of cytosolic malate dehydrogenase (MDHcyt) and cytosolic malic enzyme (MEcyt) with the mitochondrial shuttles citrate carrier (CIC), dicarboxylate carrier (DIC), and mitochondrial pyruvate carrier (MPC). The system is highly flexible using malate and pyruvate as intermediary electron carriers and quickly adapts to current needs. Indications for the existence of the mechanism are found in different monoclonal antibody producing CHO cell lines and under growing and stationary conditions.

Abstract PO-055: Pan-cancer metabolic profiling of the tumor microenvironment

Abstract Tumors are heterogeneous cellular environments with entwined metabolic dependencies. However, it is challenging to differentially profile metabolism in cancer and infiltrating non-malignant cells inside patient tumors. Here, we used a tumor transcriptome deconvolution approach to profile the metabolic states of cancer and non-cancer (stromal) cells in bulk tumors of 20 solid tumor types. This analysis highlighted metabolic genes and processes recurrently upregulated in cancer cells across tumor types, including the mitochondrial dicarboxylate carrier SLC25A10. Intriguingly, the tryptophan-degrading dioxygenase, IDO1, was highly recurrently overexpressed in stroma compared to cancer cells and normal tissue, suggesting that tryptophan-mediated suppression of antitumor immune response is predominantly constrained by the stroma. Oxidative phosphorylation was unexpectedly the most upregulated metabolic process in cancer cells compared to both stromal cells and a large atlas of cancer cell lines, suggesting that the warburg effect may be less pronounced in cancer cells in vivo. Overall, our analysis highlights fundamental differences in metabolic states of cancer and stromal cells inside tumors and establishes a pan-cancer resource to interrogate tumor metabolism. Citation Format: Neha Rohatgi, Umesh Ghoshdastider, Probhonjon Baruah, Anders Jacobsen Skanderup. Pan-cancer metabolic profiling of the tumor microenvironment [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-055.

Therapeutic potential of tricarboxylic acid cycle metabolite itaconate in cardiovascular diseases.

Therapeutic potential of tricarboxylic acid cycle metabolite itaconate in cardiovascular diseases.

Itaconic acid impairs the mitochondrial function by the inhibition of complexes II and IV and induction of the permeability transition pore opening in rat liver mitochondria

Itaconic acid (methylene-succinic acid, ItA) is an unsaturated dicarboxylic acid that is secreted by mammalian macrophages in response to a pro-inflammatory stimulus and shows an anti-inflammatory/antibacterial effect. Being a mitochondrial metabolite, it exhibits an inhibitory activity on succinate dehydrogenase and subsequently induces mitochondrial dysfunction. The present study has shown that ItA dose-dependently inhibited ADP- and DNP-stimulated (uncoupled) respiration of rat liver mitochondria energized with succinate. This effect of ItA could be related to the suppression of the activity of complex II and the combined activity of complexes II + III of the respiratory chain. At the same time, ItA had no effect on the activity of the dicarboxylate carrier, which catalyzes the transport of succinate across the inner mitochondrial membrane. It was found that 4 mM ItA diminished the rates of ADP- and DNP-stimulated mitochondrial respiration supported by the substrates of complex I glutamate and malate. A study of the effect of ItA on the activity of complexes of the respiratory chain showed that it decreases the activity of complex IV. It was observed that 4 mM ItA inhibited the rate of H2O2 production by mitochondria. At the same time, ItA promoted the opening of the cyclosporin A-sensitive Ca2+-dependent permeability transition pore. The latter was revealed as the decrease in the calcium retention capacity of mitochondria and the stimulation of release of cytochrome c from the organelles. ItA by itself promoted the cytochrome c release from mitochondria. Possible mechanisms of the effect of ItA on mitochondrial function are discussed.

A Fluorescence-Based Method to Measure ADP/ATP Exchange of Recombinant Adenine Nucleotide Translocase in Liposomes.

Several mitochondrial proteins, such as adenine nucleotide translocase (ANT), aspartate/glutamate carrier, dicarboxylate carrier, and uncoupling proteins 2 and 3, are suggested to have dual transport functions. While the transport of charge (protons and anions) is characterized by an alteration in membrane conductance, investigating substrate transport is challenging. Currently, mainly radioactively labeled substrates are used, which are very expensive and require stringent precautions during their preparation and use. We present and evaluate a fluorescence-based method using Magnesium Green (MgGrTM), a Mg2+-sensitive dye suitable for measurement in liposomes. Given the different binding affinities of Mg2+ for ATP and ADP, changes in their concentrations can be detected. We obtained an ADP/ATP exchange rate of 3.49 ± 0.41 mmol/min/g of recombinant ANT1 reconstituted into unilamellar liposomes, which is comparable to values measured in mitochondria and proteoliposomes using a radioactivity assay. ADP/ATP exchange calculated from MgGrTM fluorescence solely depends on the ANT1 content in liposomes and is inhibited by the ANT-specific inhibitors, bongkrekic acid and carboxyatractyloside. The application of MgGrTM to investigate ADP/ATP exchange rates contributes to our understanding of ANT function in mitochondria and paves the way for the design of other substrate transport assays.