Mitochondrial Pyruvate Transport Research Articles

Metabolic Engineering of Saccharomyces cerevisiae for Ethyl Acetate Biosynthesis

Ethyl acetate can be synthesized from acetyl-CoA and ethanol via a reaction by alcohol acetyltransferases (AATase) in yeast. In order to increase the yield of acetyl-CoA, different terminators were used to optimize the expressions of acetyl-CoA synthetase (ACS1/2) and aldehyde dehydrogenase (ALD6) to increase the contents of acetyl-CoA in Saccharomyces cerevisiae. ATF1 coding AATase was coexpressed in expression cassettes of ACS1/ACS2 and ALD6 to promote the carbon flux toward ethyl acetate from acetyl-CoA. Further to improve ethyl acetate production, four heterologous AATase including HuvEAT1 (Hanseniaspora uvarum), KamEAT1 (Kluyveromyces marxianus), VAAT (wild strawberry), and AeAT9 (kiwifruit) were introduced. Subsequently mitochondrial transport and utilization of pyruvate and acetyl-CoA were impeded to increase the ethyl acetate accumulation in cytoplasm. Under the optimal fermentation conditions, the engineered strain of PGAeΔPOR2 produced 1.69 g/L ethyl acetate, which was the highest value reported to date by metabolic engineering methods.

TAMI-38. CYSTEINE-PROMOTING COMPOUNDS INDUCE MITOCHONDRIAL TOXICITY IN GLIOBLASTOMA THROUGH ALTERED PYRUVATE AND SERINE METABOLISM

Abstract Glioblastoma (GBM) remains a poorly treatable disease with high mortality. Tumor metabolism in GBM is a critical mechanism responsible for accelerated growth because of upregulation of glucose, amino acid, and fatty acid utilization. However, little is known about the metabolic alterations that are specific to GBM and that are targetable with FDA-approved compounds. To investigate tumor metabolism signatures unique to GBM, we interrogated the TCGA and a cancer metabolite database for alterations in glucose and amino acid signatures in GBM relative to other human cancers and relative to low-grade glioma. From these analyses, we found that GBM exhibits the highest levels of cysteine and methionine pathway gene expression of 32 human cancers and that GBM exhibits high levels of cysteine-related metabolites compared to low-grade gliomas. To study the role of cysteine in GBM pathogenesis, we treated patient-derived GBM cells with a variety of FDA-approved cyst(e)ine-promoting compounds in vitro, including N-acetylcysteine (NAC) and the cephalosporin antibiotic, Ceftriaxone (CTX), which induces cystine import through System Xc transporter upregulation. Cysteine-promoting compounds, including NAC and CTX, inhibit growth of GBM cells, which is exacerbated by glucose deprivation. This growth inhibition is associated with reduced mitochondrial metabolism, manifest by reduction in ATP, NADPH/NADP+ ratio, mitochondrial membrane potential, and oxygen consumption rate. Metabolic tracing experiments with 13C6-glucose demonstrate that L-serine is rapidly depleted in GBM cells upon treatment with NAC and CTX, and exogenous serine rescues NAC- and CTX-mediated cell growth inhibition. In addition, these compounds reduce GBM mitochondrial pyruvate transport. We show that cysteine-promoting compounds reduce cell growth and induce mitochondrial toxicity in GBM, which may be due to rapid serine depletion and reduced mitochondrial pyruvate transport. This metabolic phenotype is exacerbated by glucose deprivation. This pathway is targetable with FDA-approved cysteine-promoting compounds and could synergize with glucose-lowering treatments, including the ketogenic diet, for GBM.

Enhanced Production of Ethyl Lactate in Saccharomyces cerevisiae by Genetic Modification.

Ethyl lactate is an important flavor substance in baijiu, and it is also one of the common raw materials in the production of flavors and spices. In this study, we first established the ethyl lactate biosynthesis pathway in Saccharomyces cerevisiae α(L) by introducing propionyl coenzyme A transferase (Pct) and alcohol acyltransferase (AAT), and the results showed that strain α(L)-CP-Ae produced the most ethyl lactate 239.53 ± 5.45 mg/L. Subsequently, the copy number of the Pctcp gene and AeAT9 gene was increased, and the modified strain α(L)-tCP-tAe produced 346.39 ± 3.99 mg/L ethyl lactate. Finally, the porin gene (por2) and the mitochondrial pyruvate carrier gene (MPC2) were knocked to impede mitochondrial transport of pyruvate, and the final modified strain α(L)-tCP-tAeΔpor2 produced ethyl lactate 420.48 ± 6.03 mg/L.

Ketone bodies for the starving heart.

The heart is an organ with high energy demands and metabolic flexibility, thus allowing for various energy substrates for ATP production under different physiological conditions. Zhang et al., Fernandez-Caggiano et al. and McCommis et al. converge on the mitochondrial pyruvate transporter as a key metabolic hub for the maintenance of cardiac metabolism and a critical determinant of cardiac metabolic plasticity during heart failure.

Augmented mitochondrial energy metabolism is an early response to chronic glucose stress in human pancreatic beta cells

Aims/hypothesisIn islets from individuals with type 2 diabetes and in islets exposed to chronic elevated glucose, mitochondrial energy metabolism is impaired. Here, we studied early metabolic changes and mitochondrial adaptations in human beta cells during chronic glucose stress.MethodsRespiration and cytosolic ATP changes were measured in human islet cell clusters after culture for 4 days in 11.1 mmol/l glucose. Metabolomics was applied to analyse intracellular metabolite changes as a result of glucose stress conditions. Alterations in beta cell function were followed using insulin secretion assays or cytosolic calcium signalling after expression of the calcium probe YC3.6 specifically in beta cells of islet clusters.ResultsAt early stages of glucose stress, mitochondrial energy metabolism was augmented in contrast to the previously described mitochondrial dysfunction in beta cells from islets of diabetic donors. Following chronic glucose stress, mitochondrial respiration increased (by 52.4%, p < 0.001) and, as a consequence, the cytosolic ATP/ADP ratio in resting human pancreatic islet cells was elevated (by 27.8%, p < 0.05). Because of mitochondrial overactivation in the resting state, nutrient-induced beta cell activation was reduced. In addition, chronic glucose stress caused metabolic adaptations that resulted in the accumulation of intermediates of the glycolytic pathway, the pentose phosphate pathway and the TCA cycle; the most strongly augmented metabolite was glycerol 3-phosphate. The changes in metabolites observed are likely to be due to the inability of mitochondria to cope with continuous nutrient oversupply. To protect beta cells from chronic glucose stress, we inhibited mitochondrial pyruvate transport. Metabolite concentrations were partially normalised and the mitochondrial respiratory response to nutrients was markedly improved. Furthermore, stimulus–secretion coupling as assessed by cytosolic calcium signalling, was restored.Conclusion/interpretationWe propose that metabolic changes and associated mitochondrial overactivation are early adaptations to glucose stress, and may reflect what happens as a result of poor blood glucose control. Inhibition of mitochondrial pyruvate transport reduces mitochondrial nutrient overload and allows beta cells to recover from chronic glucose stress.Graphical abstract

FXR Isoforms Control Different Metabolic Functions in Liver Cells via Binding to Specific DNA Motifs

The nuclear receptor subfamily 1 group H member 4 (NR1H4, also called FXR) is a ligand-activated transcription factor that, upon binding of bile acids, regulates the expression of genes involved in bile acid, fat, sugar, and amino acid metabolism. Transcript variants encode the FXR isoforms alpha 1, alpha 2, alpha 3, and alpha 4, which activate different genes that regulate metabolism. Little is known about the mechanisms by which the different isoforms regulate specific genes or how the expression of these genes affects the outcomes of patients given drugs that target FXR. We determined genome-wide binding of FXR isoforms in mouse liver organoids that express individual FXR isoforms using chromatin immunoprecipitation, followed by sequencing analysis and DNA motif discovery. We validated regulatory DNA sequences by mobility shift assays and with luciferase reporters using mouse and human FXR isoforms. We analyzed mouse liver organoids and HepG2 cells that expressed the FXR isoforms using chromatin immunoprecipitation, quantitative polymerase chain reaction, and immunoblot assays. Organoids were analyzed for mitochondrial respiration, lipid droplet content, and triglyceride excretion. We used the FXR ligand obeticholic acid to induce FXR activity in organoids, cell lines, and mice. We collected data on the binding of FXR in mouse liver and the expression levels of FXR isoforms and gene targets in human liver tissue and primary human hepatocytes from the Gene Expression Omnibus. In mouse liver cells, 89% of sites that bound FXR were bound by only FXRα2 or FXRα4, via direct interactions with the DNA sequence motif ER-2. Via DNA binding, these isoforms regulated metabolic functions in liver cells, including carbon metabolism and lipogenesis. Incubation with obeticholic acid increased mitochondrial pyruvate transport and reduced insulin-induced lipogenesis in organoids that expressed FXRα2 but not FXRα1. In human liver tissues, levels of FXRα2 varied significantly and correlated with expression of genes predicted to be regulated via an ER-2 motif. Most metabolic effects regulated by FXR in mouse and human liver cells are regulated by the FXRα2 isoform via specific binding to ER-2 motifs. The expression level of FXRα2 in liver might be used to predict responses of patients to treatment with FXR agonists.

Mitochondrial pyruvate and fatty acid flux modulate MICU1-dependent control of MCU activity.

The tricarboxylic acid (TCA) cycle converts the end products of glycolysis and fatty acid β-oxidation into the reducing equivalents NADH and FADH2 Although mitochondrial matrix uptake of Ca2+ enhances ATP production, it remains unclear whether deprivation of mitochondrial TCA substrates alters mitochondrial Ca2+ flux. We investigated the effect of TCA cycle substrates on MCU-mediated mitochondrial matrix uptake of Ca2+, mitochondrial bioenergetics, and autophagic flux. Inhibition of glycolysis, mitochondrial pyruvate transport, or mitochondrial fatty acid transport triggered expression of the MCU gatekeeper MICU1 but not the MCU core subunit. Knockdown of mitochondrial pyruvate carrier (MPC) isoforms or expression of the dominant negative mutant MPC1R97W resulted in increased MICU1 protein abundance and inhibition of MCU-mediated mitochondrial matrix uptake of Ca2+ We also found that genetic ablation of MPC1 in hepatocytes and mouse embryonic fibroblasts resulted in reduced resting matrix Ca2+, likely because of increased MICU1 expression, but resulted in changes in mitochondrial morphology. TCA cycle substrate-dependent MICU1 expression was mediated by the transcription factor early growth response 1 (EGR1). Blocking mitochondrial pyruvate or fatty acid flux was linked to increased autophagy marker abundance. These studies reveal a mechanism that controls the MCU-mediated Ca2+ flux machinery and that depends on TCA cycle substrate availability. This mechanism generates a metabolic homeostatic circuit that protects cells from bioenergetic crisis and mitochondrial Ca2+ overload during periods of nutrient stress.

A highly responsive pyruvate sensor reveals pathway-regulatory role of the mitochondrial pyruvate carrier MPC.

Mitochondria generate ATP and building blocks for cell growth and regeneration, using pyruvate as the main substrate. Here we introduce PyronicSF, a user-friendly GFP-based sensor of improved dynamic range that enables real-time subcellular quantitation of mitochondrial pyruvate transport, concentration and flux. We report that cultured mouse astrocytes maintain mitochondrial pyruvate in the low micromolar range, below cytosolic pyruvate, which means that the mitochondrial pyruvate carrier MPC is poised to exert ultrasensitive control on the balance between respiration and anaplerosis/gluconeogenesis. The functionality of the sensor in living tissue is demonstrated in the brain of Drosophila melanogaster larvae. Mitochondrial subpopulations are known to coexist within a given cell, which differ in their morphology, mobility, membrane potential, and vicinity to other organelles. The present tool can be used to investigate how mitochondrial diversity relates to metabolism, to study the role of MPC in disease, and to screen for small-molecule MPC modulators.

Exposure of Pancreatic β-Cells to Excess Glucose Results in Bimodal Activation of mTORC1 and mTOR-Dependent Metabolic Acceleration.

SummaryChronic 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.

Necessary and Sufficient? Solving the Mystery of the Mitochondrial Pyruvate Transporter

While there are several available lessons for teaching introductory biology students about diffusion, facilitated diffusion, and active transport, fewer materials exist to support upper-division students&#39; understanding of the proteins that mediate these forms of transport. In the 1970s, mitochondrial pyruvate carrier (MPC) proteins were predicted to import pyruvate from the cytoplasm into mitochondria for cellular respiration. Yet it was not until 2012 that the identity of the proteins responsible for this transport was confirmed in two seminal publications. In this Lesson, students will use their background knowledge of transport mechanisms to analyze data from those papers to determine which of the predicted MPC proteins are actually part of the mitochondrial pyruvate transporter. Student will also learn how scientists test whether a protein is necessary and sufficient. The Lesson is written in the style of process-oriented guided inquiry learning (POGIL). POGIL is a teaching approach that requires students to work collaboratively in small groups to answer a set of questions based on scientific data. Questions in the POGIL activity, called the problem set, are structured so that each question leads to the next, helping to guide students to a deeper understanding of the content. During this Lesson, the instructor acts as a facilitator to guide student learning. Several forms of assessment are included within the Lesson, allowing instructors to assess learning gains. This Lesson has been used multiple times by over 10 faculty in an upper-division Cell Biology course and can also be used in other upper-division biology courses.

Activated but Not Quiescent Hematopoietic Stem Cells Rely Readily on Glycolysis As Their Main Source of Energy

Elucidating mechanisms that regulate hematopoietic stem cells (HSCs) quiescence is critical for improving bone marrow transplantation. It is postulated that quiescent HSCs rely mostly on glycolysis rather than mitochondrial oxidative phosphorylation (OXPHOS) as their energy source. We have identified a population of HSCs with low mitochondrial activity (LSKCD150+CD48- within the 25% lowest mitochondrial membrane potential; MMP-low) that we show are highly enriched in quiescent HSCs (~95% ±2.65 in G0) as measured by pyronin Y/Hoechst staining in contrast to LSKCD150+CD48- within 25% highest MMP (MMP-high) that are in majority in G1/S/G2/M phase of cell cycle (55.4%±16; p&amp;lt;0.05; n=3). MMP-low HSCs exhibit greater in vivo competitive repopulation ability (3.7 fold; p=0.021; n=10 mice) at 16 weeks as compared to MMP-high fractions, show higher self renewal ability and are enriched in label-retaining H2B-GFP+ cells (~3 fold; p&amp;lt;0.01; n=3). Conversely, label-retaining GFP+HSCs maintain lower MMP than non-label-retaining cells. Altogether these results reinforce the notion that MMP-low HSCs are quiescent whereas MMP-high HSCs are primed/activated. Using single cell RNA-sequencing (scRNA-seq) analysis to interrogate the transcriptome by a Fluidigm C1 platform within MMP-high versus MMP-low HSCs we found major metabolic pathways including OXPHOS, exhibit significantly greater expression within the MMP-high than in the MMP-low HSC fraction. Seahorse analysis confirmed that MMP-high but not -low hematopoietic stem and progenitor cells (HSPCs) use OXPHOS as their source of energy (3.9 fold; n=16 mice). Unexpectedly, glycolytic gene expression was also enriched in the primed MMP-high HSCs and relatively low in quiescent MMP-low HSCs. qRT-PCR analysis further confirmed that the expression of glycolytic enzymes and other genes including glucose transporter 1 (Glut1, slc2a1) that is the main glucose transporter on HSCs is greater in MMP-high relative to -low HSCs. These unforeseen findings raised the potential that despite the current consensus in HSC biology, glycolysis may more readily support activated rather than quiescent HSCs. We thus measured the glucose uptake in MMP-low vs -high HSCs under metabolic (pyruvate, glucose and glutamine)-restricted conditions. Using 2NBDG, a fluorescently tagged glucose analog, we found that MMP-high HSCs uptake 3.3-fold more glucose and contained three times more 2NBDG+ cells as compared to MMP-low HSCs (p&amp;lt;0.001 for each; n=3). Pharmacological inhibition of Glut1 reduced glucose uptake in MMP-high but not -low HSCs suggesting the specific sensitivity of MMP-high HSCs to the glucose inhibition. In addition, activation of tricarboxylic acid cycle TCA cycle with combined methyl-pyruvate and dimethyl-alpha-ketoglutarate led to a greater glucose uptake (~3.5 fold) in MMP-high as compared to MMP low HSCs (~2.2 fold; p&amp;lt;0.0001). To address the degree to which glycolysis is necessary, FACS-purified MMP-low and -high HSCs were incubated with 2-Deoxy D-Glucose (2DG) - a glucose analog - that inhibits glycolysis via its action on hexokinase. While glucose deprivation with 50 mM dose of 2DG within 12 hours did not have much effect on MMP-low HSCs, over 60% of MMP-high HSCs died (p&amp;lt;0.001; n=3), suggesting that MMP-high but not -low HSCs rely readily on glycolysis for their survival. Also, the inhibition of mitochondrial transport of pyruvate that is the end product of glycolysis (α-cyano-4-hydroxycinnamate (CHC)), decreased survival in MMP-high HSCs by 80% with negligible effect on MMP-low HSCs (p&amp;lt;0.01; n=3). Finally 50 FACS-purified HSCs (LSKCD150+CD48-MMP-low or -high) were transplanted in lethally irradiated mice along with 200,000 unfractionated bone marrow cells in a competitive repopulation assay and mice were subsequently treated with 2DG or control vehicle every other day for two weeks. Two months later, 2DG treatment led to significantly enhanced reconstitution levels in MMP-high HSCs with no or little effect on MMP low reconstitution levels (ongoing). These combined results suggest that primed MMP-high rather than quiescent MMP-low HSCs rely on glycolysis as their main source of energy. These findings are consistent with the concept that glycolysis is key in sustaining rapidly dividing cells such as embryonic stem cells and cancer cells. Disclosures Ghaffari: Rubius Therapeutics: Consultancy.

Repression of mitochondrial metabolism for cytosolic pyruvate-derived chemical production in Saccharomyces cerevisiae

BackgroundSaccharomyces cerevisiae is a suitable host for the industrial production of pyruvate-derived chemicals such as ethanol and 2,3-butanediol (23BD). For the improvement of the productivity of these chemicals, it is essential to suppress the unnecessary pyruvate consumption in S. cerevisiae to redirect the metabolic flux toward the target chemical production. In this study, mitochondrial pyruvate transporter gene (MPC1) or the essential gene for mitophagy (ATG32) was knocked-out to repress the mitochondrial metabolism and improve the production of pyruvate-derived chemical in S. cerevisiae.ResultsThe growth rates of both aforementioned strains were 1.6-fold higher than that of the control strain. 13C-metabolic flux analysis revealed that both strains presented similar flux distributions and successfully decreased the tricarboxylic acid cycle fluxes by 50% compared to the control strain. Nevertheless, the intracellular metabolite pool sizes were completely different, suggesting distinct metabolic effects of gene knockouts in both strains. This difference was also observed in the test-tube culture for 23BD production. Knockout of ATG32 revealed a 23.6-fold increase in 23BD titer (557.0 ± 20.6 mg/L) compared to the control strain (23.5 ± 12.8 mg/L), whereas the knockout of MPC1 revealed only 14.3-fold increase (336.4 ± 113.5 mg/L). Further investigation using the anaerobic high-density fermentation test revealed that the MPC1 knockout was more effective for ethanol production than the 23BD production.ConclusionThese results suggest that the engineering of the mitochondrial transporters and membrane dynamics were effective in controlling the mitochondrial metabolism to improve the productivities of chemicals in yeast cytosol.

Elevated Exogenous Pyruvate Potentiates Mesodermal Differentiation through Metabolic Modulation and AMPK/mTOR Pathway in Human Embryonic Stem Cells.

SummaryPyruvate is a key metabolite in glycolysis and the tricarboxylic acid (TCA) cycle. Exogenous pyruvate modulates metabolism, provides cellular protection, and is essential for the maintenance of human preimplantation embryos and human embryonic stem cells (hESCs). However, little is known about how pyruvate contributes to cell-fate determination during epiblast stage. In this study, we used hESCs as a model to demonstrate that elevated exogenous pyruvate shifts metabolic balance toward oxidative phosphorylation in both maintenance and differentiation conditions. During differentiation, pyruvate potentiates mesoderm and endoderm lineage specification. Pyruvate production and its mitochondrial metabolism are required in BMP4-induced mesoderm differentiation. However, the TCA-cycle metabolites do not have the same effect as pyruvate on differentiation. Further study shows that pyruvate increases AMP/ATP ratio, activates AMPK, and modulates the mTOR pathway to enhance mesoderm differentiation. This study reveals that exogenous pyruvate not only controls metabolism but also modulates signaling pathways in hESC differentiation.

1178-P: MSDC-0602 Directly Inhibits Mitochondrial Pyruvate Transport

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

PF781 FATTY ACID OXIDATION AND MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION REGULATE T‐CELL EFFECTOR FUNCTION DURING GRAFT‐VERSUS‐HOST DISEASE

Background:Upon activation, effector T‐cells preferably utilize glucose for rapid and extensive proliferation, and cytokine production. Until recently, it has been thought that extracellular fatty acid (FA) uptake and oxidation are severely reduced in alloreactive T‐cells; however, recent studies have suggested that lipid metabolism is rather increased in alloreactive T‐cells, and that metabolic pathways of FA can be a promising therapeutic target for GVHD.Aims:To determine the role of lipid metabolism in human alloreactive T‐cells, we investigated the metabolic changes in human T‐cells in vivo using xenogeneic GVHD models.Methods:NOG mice received 250cGy of total body irradiation (TBI) and were subsequently injected intravenously with human pan T‐cells. Under this condition, all mice developed severe GVHD and died within 2 weeks. Human T‐cells were harvested from GVHD target organs of mice at day 9 after transplantation. For the measurement of glucose and FA uptake by flow cytometry, cells were stained with fluorescent‐labeled deoxyglucose analogue (2‐NBDG) and long‐chain FA analogue (BODIPY 500/510 C12), respectively. PCR array and extracellular flux analysis were performed according to the manufacturer's instructions.Results:Glucose uptake was significantly increased in human T‐cells obtained from GVHD mice as expected. Extracellular FA uptake was also increased in human T‐cells of GVHD mice, and was associated with cell proliferation rate. Effector memory T‐cells followed by central memory T‐cells showed a higher FA uptake than did naive T‐cells. These findings were similarly observed in both CD4+ and CD8+ T‐cells. Extracellular uptake of FA as well as glucose in T‐cells was significantly higher in MHC+/+ GVHD mice compared with MHC−/− GVHD mice, suggesting that the recognition of host MHC molecules is the initial trigger for metabolic shift in alloreactive T‐cells. Quantitative real‐time PCR analysis demonstrated up‐regulation of mRNAs encoding the enzymes involved in FA transport including carnitine palmitoyltransferase (CPT1B), FA binding protein (FABP1–4, FABP6, and FABP7), and FA oxidation (FAO) pathway including acyl‐CoA synthase (ACSBG2) and acyl‐CoA dehydrogenase (ACAD9–11, ACADS, and ACADL) in human T‐cells of GVHD mice. Similarly, the expression of genes encoding the enzymes in triacylglycerol metabolism such as glycerol kinase (GK, GK2) and lipoprotein lipase (LPL) was up‐regulated in GVHD mice. Furthermore, the expression of genes associated with mevalonate pathways such as HMG‐CoA synthase (HMGCS1, HMGCS2), was also upregulated. These observations suggest that alloreactive T‐cells increase lipid hydrolysis, mitochondrial FA transport, and FAO, resulting in greater utilization of intra‐ and extracellular FA. Extracellular flux analysis demonstrated that mitochondrial FA transport blockade by specific CPT1 inhibitor, etomoxir, significantly decreased the maximum respiration and spare respiratory capacity in human T‐cells in MHC+/+ GVHD mice but not in MHC−/− GVHD mice. Even after glucose deprivation or inhibition of mitochondrial pyruvate transport by UK5099, the oxygen consumption rate was precipitously reduced by inhibition of CPT1, suggesting that increasing FAO supported oxidative phosphorylation (OXPHOS) in alloreactive T‐cells of GVHD mice.Summary/Conclusion:Our results clearly indicate that alloreactive T‐cells increase FA uptake, intra‐ and extracellular lipid metabolism, and OXPHOS, and suggest that lipid metabolic processes in T‐cells have potential as a therapeutic target for GVHD.image

Does shuttling of glycogen-derived lactate from astrocytes to neurons take place during neurotransmission and memory consolidation?

Glycogen levels in resting brain and its utilization rates during brain activation are high, but the functions fulfilled by glycogenolysis in living brain are poorly understood. Studies in cultured astrocytes have identified glycogen as the preferred fuel to provide ATP for Na+ ,K+ -ATPase for the uptake of extracellular K+ and for Ca2+ -ATPase to pump Ca2+ into the endoplasmic reticulum. Studies in astrocyte-neuron co-cultures led to the suggestion that glycogen-derived lactate is shuttled to neurons as oxidative fuel to support glutamatergic neurotransmission. Furthermore, both knockout of brain glycogen synthase and inhibition of glycogenolysis prior to a memory-evoking event impair memory consolidation, and shuttling of glycogen-derived lactate as neuronal fuel was postulated to be required for memory. However, lactate shuttling has not been measured in any of these studies, and procedures to inhibit glycogenolysis and neuronal lactate uptake are not specific. Testable alternative mechanisms to explain the observed findings are proposed: (i) disruption of K+ and Ca2+ homeostasis, (ii) release of gliotransmitters, (iii) imposition of an energy crisis on astrocytes and neurons by inhibition of mitochondrial pyruvate transport by compounds used to block neuronal monocarboxylic acid transporters, and (iv) inhibition of astrocytic filopodial movements that secondarily interfere with glutamate and K+ uptake from the synaptic cleft. Evidence that most pyruvate/lactate derived from glycogen is not oxidized and does not accumulate suggests predominant glycolytic metabolism of glycogen to support astrocytic energy demands. Sparing of blood-borne glucose for use by neurons is a reasonable explanation for the requirement for glycogenolysis in neurotransmission and memory processing.

Enhanced mitochondrial pyruvate transport elicits a robust ROS production to sensitize the antitumor efficacy of interferon-γ in colon cancer

Metabolic reprogramming is a feature of cancer cells and crucial for tumor growth and metastasis. Interferon-γ (IFNγ) is a cytokine that plays a pivotal role in host antitumor immunity. However, little is known about the roles of metabolic reprogramming in immune responses. Here, we show that colon cancer cells reprogram metabolism to coordinate proper cellular responses to IFNγ by downregulating mitochondrial pyruvate carrier (MPC)1 and 2 via STAT3 signaling. Forced overexpression of MPC promote the production of reactive oxygen species and enhance the apoptosis induced by IFNγ in colon cancer cells. Moreover, inhibiting STAT3 sensitize the antitumor efficacy of IFN-γ against colon cancer cells. Our findings present a previously unrecognized mechanism that colon cancer manipulate to resist IFNγ mediated antitumor immunity that have implications for targeting a unique aspect of this disease.

Exploration of the Regulatory Mechanism of Secondary Metabolism by Comparative Transcriptomics in Aspergillus flavus.

Mycotoxins cause a huge threaten to agriculture, food safety, and human and animal life. Among them, aflatoxins (AFs) have always been considered the most potent carcinogens, and filamentous fungi from Aspergillus genus are their major producers, especially A. flavus. Although the biosynthesis path of these chemicals had been well-identified, the regulatory mechanisms controlling expression of AF gene cluster were poorly understood. In this report, genome-wide transcriptome profiles of A. flavus from AF conducing [yeast sucrose media (YES)] and non-conducing [yeast peptone media (YEP)] conditions were compared by using deep RNA sequencing (RNA-seq), and the results revealed that AF biosynthesis pathway and biosynthesis of amino acids were significantly upregulated in YES vs. YEP. Further, a novel LaeA-like methyltransferase AFLA_121330 (Lael1) was identified for the first time, to play a specific role in the regulation of AF biosynthesis. Contrary to LaeA, which gene deletion reduced the level, lael1 deletion resulted in a significant increase in AF production. Further, co-expression network analysis revealed that mitochondrial pyruvate transport and signal peptide processing were potentially involved in AF synthesis for the first time, as well as biological processes of ribosome, branched-chain amino acid biosynthetic process and translation were co-regulated by AfRafA and AfStuA. To sum up, our analyses could provide novel insights into the molecular mechanism for controlling the AF and other secondary metabolite synthesis, adding novel targets for plant breeding and making fungicides.

Abstract 3527: Inhibition of mitochondrial pyruvate transport selectively sensitizes cancer cells to metabolic oxidative stress

Abstract To determine if mitochondrial pyruvate transport could represent a therapeutic target for sensitizing cancer cells to oxidative stress, lung and breast cancer cells were treated with 5 µM UK5099 to inhibit the mitochondrial pyruvate carrier (MPC). Treatment with UK5099 selectively sensitized lung and breast cancer cells to clonogenic cell killing when combined with depletion of glutathione using 1 mM buthionine sulfoximine (BSO; a glutathione synthesis inhibitor) for 48 h and 72 h, relative to normal lung and breast epithelial cells. Furthermore, cancer cell killing mediated by UK5099 combined with BSO was inhibited by the thiol antioxidant, N-acetylcysteine (NAC; 20 mM), independent of GSH levels, indicating a mechanism of toxicity involving reduced thiols and metabolic oxidative stress. In addition, treatment with UK5099 alone for 48 h also decreased levels of total glutathione in cancer cells that could be reversed by NAC. Using oxidation-sensitive fluorescent dyes (CDCFH2 and MitoSOX), treatment of lung and breast cancer cells for 24 h and 48 h with UK5099 induced increases in steady state levels of pro-oxidants (presumably hydroperoxides and mitochondrial superoxide), which were further increased with BSO. Finally, treatment of breast cancer cells with UK5099 for 24 and 48 hours significantly sensitized breast cancer cells to clonogenic cell killing mediated by paclitaxel. These data support the hypothesis that inhibition of the MPC selectively causes an impairment of antioxidant capability in cancer cells that is enhanced by depletion of glutathione. Furthermore, these results also support the hypothesis that inhibition of the MPC represents a significant target for sensitizing human breast cancer cells to chemotherapy agents thought to induce oxidative stress. Supported by R01CA182804. Citation Format: Shane R. Solst, Samuel N. Rodman, Melissa A. Fath, Eric B. Taylor, Douglas R. Spitz. Inhibition of mitochondrial pyruvate transport selectively sensitizes cancer cells to metabolic oxidative stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3527.

Interruption of lactate uptake by inhibiting mitochondrial pyruvate transport unravels direct antitumor and radiosensitizing effects

Lactate exchange between glycolytic and oxidative cancer cells is proposed to optimize tumor growth. Blocking lactate uptake through monocarboxylate transporter 1 (MCT1) represents an attractive therapeutic strategy but may stimulate glucose consumption by oxidative cancer cells. We report here that inhibition of mitochondrial pyruvate carrier (MPC) activity fulfils the tasks of blocking lactate use while preventing glucose oxidative metabolism. Using in vitro 13C-glucose and in vivo hyperpolarized 13C-pyruvate, we identify 7ACC2 as a potent inhibitor of mitochondrial pyruvate transport which consecutively blocks extracellular lactate uptake by promoting intracellular pyruvate accumulation. Also, while in spheroids MCT1 inhibition leads to cytostatic effects, MPC activity inhibition induces cytotoxic effects together with glycolysis stimulation and uncompensated inhibition of mitochondrial respiration. Hypoxia reduction obtained with 7ACC2 is further shown to sensitize tumor xenografts to radiotherapy. This study positions MPC as a control point for lactate metabolism and expands on the anticancer potential of MPC inhibition.