Oxygen Regulates Human Pluripotent Stem Cell Metabolic Flux

Metabolism has been shown to alter cell fate in human pluripotent stem cells (hPSC). However, current understanding is almost exclusively based on work performed at 20% oxygen (air), with very few studies reporting on hPSC at physiological oxygen (5%). In this study, we integrated metabolic, transcriptomic, and epigenetic data to elucidate the impact of oxygen on hPSC. Using 13C-glucose labeling, we show that 5% oxygen increased the intracellular levels of glycolytic intermediates, glycogen, and the antioxidant response in hPSC. In contrast, 20% oxygen increased metabolite flux through the TCA cycle, activity of mitochondria, and ATP production. Acetylation of H3K9 and H3K27 was elevated at 5% oxygen while H3K27 trimethylation was decreased, conforming to a more open chromatin structure. RNA-seq analysis of 5% oxygen hPSC also indicated increases in glycolysis, lysine demethylases, and glucose-derived carbon metabolism, while increased methyltransferase and cell cycle activity was indicated at 20% oxygen. Our findings show that oxygen drives metabolite flux and specifies carbon fate in hPSC and, although the mechanism remains to be elucidated, oxygen was shown to alter methyltransferase and demethylase activity and the global epigenetic landscape.

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Abstract P443: Loss Of Pkm2 Alters Myocardial Metabolism And Increases Oxidative Stress After Infarction

Circulation Research ◽

10.1161/res.129.suppl_1.p443 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Katie C Lee ◽

Allison L Williams ◽

Ralph V Shohet

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Tca Cycle ◽

Pentose Phosphate ◽

Mitochondrial Enzymes ◽

Rna Seq ◽

Atp Production ◽

The Tca Cycle ◽

Alternatively Spliced ◽

Altered Gene

Introduction: Pyruvate kinase (Pkm1) directs pyruvate to the TCA cycle for oxidative metabolism in the healthy heart. Our lab described a hypoxia-mediated switch to the alternatively spliced isoform Pkm2, enhancing pyruvate to lactate conversion. Recently, we have also found that Pkm2 knockout (KO) mice had profound depletion of basal glucose in the heart compared to control mice. Pkm2 has also been shown to reduce oxidative damage and promote cardiomyocyte cell proliferation after myocardial infarction (MI). We hypothesize that the upregulation of Pkm2 alters metabolic pathways after injury to promote glycolysis and preserve ATP production in hypoxia, which protects the heart from the stresses of hypoxia and injury. Methods: Global Pkm2 KO mice were subjected to permanent ligation of the left anterior descending coronary artery to mimic an MI. RNA-seq analysis of left ventricles from control (n=8) and Pkm2 KO mice (n=8) before and 3 days after sham or MI surgery was performed. Semiquantitative real-time PCR (qPCR) was used to confirm changes in selected genes of interest. Results: Loss of Pkm2 did not alter gene expression substantially at baseline. 68 genes were differentially expressed in Pkm2 KO hearts after MI (q<0.05, FDR<0.05) not observed in control MI hearts. MI in Pkm2 KO hearts resulted in considerable reduction of transcripts of enzymes in the insulin signaling pathway, mitochondrial oxidative phosphorylation, fatty acid metabolism, and increase in transcripts encoding enzymes in the pentose phosphate pathway, response to oxidative stress, and apoptotic signaling. qPCR of selected genes involved in glucose metabolism confirmed RNA-seq results. Conclusions: RNA-seq analysis of Pkm2 KO hearts demonstrated that loss of Pkm2 altered gene expression of metabolic and mitochondrial enzymes. Conversely, Pkm2 KO hearts showed increased abundance of pro-apoptotic markers which may be a result of increased oxidative stress.

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Mitochondrial Mistranslation in Brain Provokes a Metabolic Response Which Mitigates the Age-Associated Decline in Mitochondrial Gene Expression

International Journal of Molecular Sciences ◽

10.3390/ijms22052746 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2746

Author(s):

Dimitri Shcherbakov ◽

Reda Juskeviciene ◽

Adrián Cortés Sanchón ◽

Margarita Brilkova ◽

Hubert Rehrauer ◽

...

Keyword(s):

Gene Expression ◽

Metabolic Response ◽

Mitochondrial Gene ◽

Tca Cycle ◽

Brain Mitochondria ◽

Mitochondrial Gene Expression ◽

Rna Seq ◽

The Tca Cycle ◽

Neurological Phenotype

Mitochondrial misreading, conferred by mutation V338Y in mitoribosomal protein Mrps5, in-vivo is associated with a subtle neurological phenotype. Brain mitochondria of homozygous knock-in mutant Mrps5V338Y/V338Y mice show decreased oxygen consumption and reduced ATP levels. Using a combination of unbiased RNA-Seq with untargeted metabolomics, we here demonstrate a concerted response, which alleviates the impaired functionality of OXPHOS complexes in Mrps5 mutant mice. This concerted response mitigates the age-associated decline in mitochondrial gene expression and compensates for impaired respiration by transcriptional upregulation of OXPHOS components together with anaplerotic replenishment of the TCA cycle (pyruvate, 2-ketoglutarate).

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The transcriptome variations of Panaxnotoginseng roots treated with different forms of nitrogen fertilizers

BMC Genomics ◽

10.1186/s12864-019-6340-7 ◽

2019 ◽

Vol 20 (S9) ◽

Cited By ~ 4

Author(s):

Xiaohong Ou ◽

Shipeng Li ◽

Peiran Liao ◽

Xiuming Cui ◽

Binglian Zheng ◽

...

Keyword(s):

Crop Production ◽

Tca Cycle ◽

Panax Notoginseng ◽

Ammonium Nitrogen ◽

Nitrogen Fertilizers ◽

Rna Seq ◽

Root Yield ◽

Qrt Pcr ◽

The Tca Cycle ◽

Nitrate Fertilizer

Abstract Background The sensitivity of plants to ammonia is a worldwide problem that limits crop production. Excessive use of ammonium as the sole nitrogen source results in morphological and physiological disorders, and retarded plant growth. Results In this study we found that the root growth of Panax notoginseng was inhibited when only adding ammonium nitrogen fertilizer, but the supplement of nitrate fertilizer recovered the integrity, activity and growth of root. Twelve RNA-seq profiles in four sample groups were produced and analyzed to identify deregulated genes in samples with different treatments. In comparisons to NH${~}_{4}^{+}$ 4 + treated samples, ACLA-3 gene is up-regulated in samples treated with NO${~}_{3}^{-}$ 3 − and with both NH$_{4}^{+}$ 4 + and NO${~}_{3}^{-}$ 3 − , which is further validated by qRT-PCR in another set of samples. Subsequently, we show that the some key metabolites in the TCA cycle are also significantly enhanced when introducing NO${~}_{3}^{-}$ 3 − . These potentially enhance the integrity and recover the growth of Panax notoginseng roots. Conclusion These results suggest that the activated TCA cycle, as demonstrated by up-regulation of ACLA-3 and several key metabolites in this cycle, contributes to the increased Panax notoginseng root yield when applying both ammonium and nitrate fertilizer.

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Bovine Oviduct Epithelial Cell-Derived Culture Media and Exosomes Improve Mitochondrial Health by Restoring Metabolic Flux during Pre-Implantation Development

International Journal of Molecular Sciences ◽

10.3390/ijms21207589 ◽

2020 ◽

Vol 21 (20) ◽

pp. 7589

Author(s):

Tabinda Sidrat ◽

Abdul Aziz Khan ◽

Myeon-Don Joo ◽

Yiran Wei ◽

Kyeong-Lim Lee ◽

...

Keyword(s):

Epithelial Cell ◽

Embryonic Development ◽

Metabolic Flux ◽

Culture Media ◽

Tca Cycle ◽

Fine Tuning ◽

The Tca Cycle ◽

Oviduct Epithelial Cell

Oviduct flushing is enriched by a wide variety of nutrients that guide the 3–4 days journey of pre-implantation embryo through the oviduct as it develops into a competent blastocyst (BL). However, little is known about the specific requirement and role of these nutrients that orchestrate the early stages of embryonic development. In this study, we aimed to characterize the effect of in vitro-derived bovine oviduct epithelial cell (BOECs) secretion that mimics the in vivo oviduct micro-fluid like environment, which allows successful embryonic development. In this study, the addition of an in vitro derived BOECs-condition media (CM) and its isolated exosomes (Exo) significantly enhances the quality and development of BL, while the hatching ability of BLs was found to be high (48.8%) in the BOECs-Exo supplemented group. Surprisingly, BOECs-Exo have a dynamic effect on modulating the embryonic metabolism by restoring the pyruvate flux into TCA-cycle. Our analysis reveals that Exo treatment significantly upregulates the pyruvate dehydrogenase (PDH) and glutamate dehydrogenase (GLUD1) expression, required for metabolic fine-tuning of the TCA-cycle in the developing embryos. Exo treatment increases the influx into TCA-cycle by strongly suppressing the PDH and GLUD1 upstream inhibitors, i.e., PDK4 and SIRT4. Improvement of TCA-cycle function was further accompanied by higher metabolic activity of mitochondria in BOECs-CM and Exo in vitro embryos. Our study uncovered, for the first time, the possible mechanism of BOECs-derived secretion in re-establishing the TCA-cycle flux by the utilization of available nutrients and highlighted the importance of pyruvate in supporting bovine in vitro embryonic development.

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IGF-I promotes a shift in metabolic flux in vascular smooth muscle cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00072.2002 ◽

2002 ◽

Vol 283 (3) ◽

pp. E465-E471 ◽

Cited By ~ 16

Author(s):

Jennifer L. Hall ◽

Gary H. Gibbons ◽

John C. Chatham

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Metabolic Flux ◽

Tca Cycle ◽

Muscle Cells ◽

Glucose Flux ◽

The Tca Cycle ◽

Igf I

13C-nuclear magnetic resonance (NMR) spectroscopy was used to test our hypothesis that insulin-like growth factor I (IGF-I) stimulates glucose flux into both nonoxidative and oxidative pathways in vascular smooth muscle cells (VSMC). Rat VSMC were exposed to uniformly labeled [13C]glucose ([U-13C]glucose; 5.5 mM) and [3-13C]pyruvate (1 mM) in the presence and absence of IGF-I (100 ng/ml). IGF-I increased glucose flux through glycolysis and the tricarboxylic acid (TCA) cycle as well as total anaplerotic flux into the TCA cycle. Previous work in our laboratory identified an increase in GLUT1 content and glucose metabolism in neointimal VSMC that was sufficient to promote proliferation and inhibit apoptosis. To test whether IGF-I could potentiate the GLUT1-induced increased flux in the neointima, we utilized VSMC harboring constitutive overexpression of GLUT1. Indeed, IGF-I markedly potentiated the GLUT1-induced increase in glucose flux through glycolysis and the TCA cycle. Taken together, these findings demonstrate that upregulation of glucose transport through either IGF-I or increased GLUT1 content stimulates glucose flux through both nonoxidative and oxidative pathways in VSMC.

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Pyruvate cycle increases aminoglycoside efficacy and provides respiratory energy in bacteria

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1714645115 ◽

2018 ◽

Vol 115 (7) ◽

pp. E1578-E1587 ◽

Cited By ~ 72

Author(s):

Yu-bin Su ◽

Bo Peng ◽

Hui Li ◽

Zhi-xue Cheng ◽

Tian-tuo Zhang ◽

...

Keyword(s):

Metabolic Flux ◽

Carbon Sources ◽

Tca Cycle ◽

Multidrug Resistant ◽

Edwardsiella Tarda ◽

General Mechanism ◽

Resistant Bacteria ◽

Target Drug ◽

The Tca Cycle ◽

P Cycle

The emergence and ongoing spread of multidrug-resistant bacteria puts humans and other species at risk for potentially lethal infections. Thus, novel antibiotics or alternative approaches are needed to target drug-resistant bacteria, and metabolic modulation has been documented to improve antibiotic efficacy, but the relevant metabolic mechanisms require more studies. Here, we show that glutamate potentiates aminoglycoside antibiotics, resulting in improved elimination of antibiotic-resistant pathogens. When exploring the metabolic flux of glutamate, it was found that the enzymes that link the phosphoenolpyruvate (PEP)-pyruvate-AcCoA pathway to the TCA cycle were key players in this increased efficacy. Together, the PEP-pyruvate-AcCoA pathway and TCA cycle can be considered the pyruvate cycle (P cycle). Our results show that inhibition or gene depletion of the enzymes in the P cycle shut down the TCA cycle even in the presence of excess carbon sources, and that the P cycle operates routinely as a general mechanism for energy production and regulation inEscherichia coliandEdwardsiella tarda. These findings address metabolic mechanisms of metabolite-induced potentiation and fundamental questions about bacterial biochemistry and energy metabolism.

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Metabolic flux partitioning between the TCA cycle and glyoxylate shunt combined with a reversible methyl citrate cycle provide nutritional flexibility for Mycobacterium tuberculosis

10.1101/2021.01.29.428863 ◽

2021 ◽

Author(s):

Khushboo Borah ◽

Tom A. Mendum ◽

Nathaniel D. Hawkins ◽

Jane L. Ward ◽

Michael H. Beale ◽

...

Keyword(s):

Metabolic Network ◽

Metabolic Flux ◽

Carbon Sources ◽

Tca Cycle ◽

Flux Analysis ◽

Glyoxylate Shunt ◽

Citrate Cycle ◽

Metabolic Fluxes ◽

The Tca Cycle ◽

Carbon Substrates

AbstractThe utilisation of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection thereby identifying the Mtb specific metabolic pathways and enzymes required for carbon co-metabolism as potential drug targets. Metabolic flux represents the final integrative outcome of many different levels of cellular regulation that contribute to the flow of metabolites through the metabolic network. It is therefore critical that we have an in-depth understanding of the rewiring of metabolic fluxes in different conditions. Here, we employed 13C-metabolic flux analysis using stable isotope tracers (13C and 2H) and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly on physiologically relevant carbon sources in a steady state chemostat. We demonstrate that Mtb is able to efficiently co-metabolise combinations of either cholesterol or glycerol along with C2 generating carbon substrates. The uniform assimilation of the carbon sources by Mtb throughout the network indicated no compartmentalization of metabolism in these conditions however there were substrate specific differences in metabolic fluxes. This work identified that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle as the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide new insights into the metabolic architecture that affords adaptability of Mtb to divergent carbon substrates.ImportanceEach year more than 1 million people die of tuberculosis (TB). Many more are infected but successfully diagnosed and treated with antibiotics, however antibiotic-resistant TB isolates are becoming ever more prevalent and so novel therapies are urgently needed that can effectively kill the causative agent. Mtb specific metabolic pathways have been identified as an important drug target in TB. However the apparent metabolic plasticity of this pathogen presents a major obstacle to efficient targeting of Mtb specific vulnerabilities and therefore it is critical to define the metabolic fluxes that Mtb utilises in different conditions. Here, we used 13C-metabolic flux analysis to measure the metabolic fluxes that Mtb uses whilst growing on potential in vivo nutrients. Our analysis identified selective use of the metabolic network that included the TCA cycle, glyoxylate shunt and methyl citrate cycle. The metabolic flux phenotypes determined in this study improves our understanding about the co-metabolism of multiple carbon substrates by Mtb identifying a reversible methyl citrate cycle and the glyoxylate shunt as the critical metabolic nodes which underlie the nutritional flexibility of Mtb.

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Curcumae Radix Extract Reduces Beta-Amyloid (Αβ) and Tau Protein Through Increased Mitochondrial Respiration

10.21203/rs.3.rs-785607/v1 ◽

2021 ◽

Author(s):

Seong lae Jo ◽

Hyun Yang ◽

Jun H. Heo ◽

Sang R. Lee ◽

Hye Won Lee ◽

...

Keyword(s):

Brain Tumor ◽

Energy Metabolism ◽

Neurodegenerative Diseases ◽

Mitochondrial Respiration ◽

Metabolic Flux ◽

Tca Cycle ◽

Basal Respiration ◽

Atp Production ◽

Inflammatory Conditions ◽

Brain Tumor Cells

Abstract Background: Neurodegenerative diseases are increasingly being studied owing to the increasing proportion of the aging population. Several potential compounds have been studied to prevent neurodegenerative diseases, one of which is Curcumae Radix that is known to be beneficial for inflammatory conditions, metabolic syndrome, and various types of pain. However, it is not well studied and its influence on energy metabolism in neurodegenerative diseases is unclear. We focused on the relationship between neurodegenerative diseases and energy metabolism through Curcumae Radix extract in an animal model. Methods: Mice were treated with Curcumae Radix extract for 5 weeks orally 5 times in a week (50 mg/kg body weight). Murine delayed brain tumor (DBT) cells were supplemented with Curcumae Radix extract. We monitored the neurodegenerative makers and metabolic indicators using Western blotting and qRT-PCR and then assessed the cellular glycolysis and mitochondrial respiration through metabolic flux assay.Results: Low expression levels of Alzheimer’s disease-related markers were observed after treatment with Curcumae Radix extract. It was determined through the pAMPK/AMPK ratio that the ATP state was sufficient in the cerebrum and brain tumor cells. With this, an increase in glycolysis would be expected as glucose is the main energy source of the brain. However, glycolysis-related genes and the extracellular acidification rate showed that glycolysis decreased. Despite this, basal respiration and ATP production through mitochondrial respiration and increased TCA cycle and OXPHOS-related genes were observed in the Curcumae Radix group. Conclusions: In neurodegenerative diseases involving mitochondrial dysfunction, Curcumae Radix may act as a metabolic modulator of brain health to treat and prevent these diseases.

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Quantitative analysis of the physiological contributions of glucose to the TCA cycle

10.1101/840538 ◽

2019 ◽

Cited By ~ 1

Author(s):

Shiyu Liu ◽

Ziwei Dai ◽

Daniel E. Cooper ◽

David G. Kirsch ◽

Jason W. Locasale

Keyword(s):

Quantitative Analysis ◽

Metabolic Flux ◽

Isotope Labeling ◽

Tca Cycle ◽

Central Carbon Metabolism ◽

Flux Analysis ◽

Experimental Conditions ◽

The Tca Cycle ◽

Metabolic Physiology

ABSTRACTThe carbon source for catabolism in vivo is a fundamental question in metabolic physiology. Limited by data and rigorous mathematical analysis, controversy exists over the nutritional sources for carbon in the tricarboxylic acid (TCA) cycle under physiological settings. Using isotope-labeling data in vivo across several experimental conditions, we construct multiple models of central carbon metabolism and develop methods based on metabolic flux analysis (MFA) to solve for the preferences of glucose, lactate, and other nutrients used in the TCA cycle across many tissues. We show that in nearly all circumstances, glucose contributes more than lactate as a nutrient source for the TCA cycle. This conclusion is verified in different animal strains from different studies, different administrations of 13C glucose, and is extended to multiple tissue types. Thus, this quantitative analysis of organismal metabolism defines the relative contributions of nutrient fluxes in physiology, provides a resource for analysis of in vivo isotope tracing data, and concludes that glucose is the major nutrient used for catabolism in mammals.

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N-acetyltaurine and Acetylcarnitine Production for the Mitochondrial Acetyl-CoA Regulation in Skeletal Muscles during Endurance Exercises

Metabolites ◽

10.3390/metabo11080522 ◽

2021 ◽

Vol 11 (8) ◽

pp. 522

Author(s):

Teruo Miyazaki ◽

Yuho Nakamura-Shinya ◽

Kei Ebina ◽

Shoichi Komine ◽

Song-Gyu Ra ◽

...

Keyword(s):

Fatty Acids ◽

Palmitic Acid ◽

Skeletal Muscles ◽

Metabolic Flux ◽

Human Subjects ◽

Tca Cycle ◽

Blood Levels ◽

Acetyl Coa ◽

The Tca Cycle ◽

Human Skeletal Muscle Cells

During endurance exercises, a large amount of mitochondrial acetyl-CoA is produced in skeletal muscles from lipids, and the excess acetyl-CoA suppresses the metabolic flux from glycolysis to the TCA cycle. This study evaluated the hypothesis that taurine and carnitine act as a buffer of the acetyl moiety of mitochondrial acetyl-CoA derived from the short- and long-chain fatty acids of skeletal muscles during endurance exercises. In human subjects, the serum concentrations of acetylated forms of taurine (NAT) and carnitine (ACT), which are the metabolites of acetyl-CoA buffering, significantly increased after a full marathon. In the culture medium of primary human skeletal muscle cells, NAT and ACT concentrations significantly increased when they were cultured with taurine and acetate or with carnitine and palmitic acid, respectively. The increase in the mitochondrial acetyl-CoA/free CoA ratio induced by acetate and palmitic acid was suppressed by taurine and carnitine, respectively. Elevations of NAT and ACT in the blood of humans during endurance exercises might serve the buffering of the acetyl-moiety in mitochondria by taurine and carnitine, respectively. The results suggest that blood levels of NAT and ACT indicate energy production status from fatty acids in the skeletal muscles of humans undergoing endurance exercise.

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