scholarly journals Colorectal cancers utilize glutamine as an anaplerotic substrate of the TCA cycle in vivo

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Yiqing Zhao ◽  
Xuan Zhao ◽  
Vanessa Chen ◽  
Ying Feng ◽  
Lan Wang ◽  
...  
Keyword(s):  
Tca Cycle ◽  
Genetically Engineered ◽  
Glutamine Metabolism ◽  
Colorectal Cancers ◽  
Wild Type ◽  
Colon Tumors ◽  
The Tca Cycle ◽  
Genetically Engineered Mice ◽  
Tca Cycle Intermediates

AbstractCancer cells in culture rely on glutamine as an anaplerotic substrate to replenish tricarboxylic acid (TCA) cycle intermediates that have been consumed. but it is uncertain whether cancers in vivo depend on glutamine for anaplerosis. Here, following in vivo infusions of [13C5]-glutamine in mice bearing subcutaneous colon cancer xenografts, we showed substantial amounts of infused [13C5]-glutamine enters the TCA cycle in the tumors. Consistent with our prior observation that colorectal cancers (CRCs) with oncogenic mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic (PIK3CA) subunit are more dependent on glutamine than CRCs with wild type PIK3CA, labeling from glutamine to most TCA cycle intermediates was higher in PIK3CA-mutant subcutaneous xenograft tumors than in wild type PIK3CA tumors. Moreover, using orthotopic mouse colon tumors estalished from human CRC cells or patient-derived xenografts, we demonstrated substantial amounts of infused [13C5]-glutamine enters the TCA cycle in the tumors and tumors utilize anaplerotic glutamine to a greater extent than adjacent normal colon tissues. Similar results were seen in spontaneous colon tumors arising in genetically engineered mice. Our studies provide compelling evidence CRCs utilizes glutamine to replenish the TCA cycle in vivo, suggesting that targeting glutamine metabolism could be a therapeutic approach for CRCs, especially for PIK3CA-mutant CRCs.

scholarly journals Evidence for reverse flux through pyruvate kinase in skeletal muscle

2009 ◽  
Vol 296 (4) ◽  
pp. E748-E757 ◽  
Author(s):  
Eunsook S. Jin ◽  
A. Dean Sherry ◽  
Craig R. Malloy
Keyword(s):  
Skeletal Muscle ◽  
Glucose Production ◽  
Tca Cycle ◽  
Hepatic Glycogen ◽  
Direct Transfer ◽  
The Tca Cycle ◽  
Tca Cycle Intermediates ◽  
Minced Muscle ◽  
Labeling Pattern

Conversion of lactate to glucose was examined in myotubes, minced muscle tissue, and rats exposed to 2H2O or 13C-enriched substrates. Myotubes or minced skeletal muscle incubated with [U-13C3]lactate released small amounts of [1,2,3-13C3]- or [4,5,6-13C3]glucose. This labeling pattern is consistent with direct transfer from lactate to glucose without randomization in the tricarboxylic acid (TCA) cycle. After exposure of incubated muscle to 2H2O, [U-13C3]lactate, glucose, and glutamine, there was minimal release of synthesized glucose to the medium based on a low level of 2H enrichment in medium glucose but 50- to 100-fold greater 2H enrichment in glucosyl units from glycogen. The 13C enrichment pattern in glycogen from incubated skeletal muscle was consistent only with direct transfer of lactate to glucose without exchange in TCA cycle intermediates. 13C nuclear magnetic resonance (NMR) spectra of glutamate from the same tissue showed flux from lactate through pyruvate dehydrogenase but not flux through pyruvate carboxylase into the TCA cycle. Carbon from an alternative substrate for glucose production that requires metabolism through the TCA cycle, propionate, did not enter glycogen, suggesting that TCA cycle intermediates do not exchange with phospho enolpyruvate. In vivo, the 13C labeling patterns in hepatic glycogen and plasma glucose after administration of [U-13C3]lactate did not differ significantly. However, skeletal muscle glycogen was substantially enriched in [1,2,3-13C3]- and [4,5,6-13C3]glucose units that could only occur through skeletal muscle glyconeogenesis rather than glycogenesis. Lactate serves as a substrate for glyconeogenesis in vivo without exchange into symmetric intermediates of the TCA cycle.

Formation of Platelet Strings and Microthrombi in the Presence of ADAMTS13 Inhibitor Does Not Require P-Selectin or β3 Integrin.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1782-1782
Author(s):  
Anil K. Chauhan ◽  
Denisa D. Wagner
Keyword(s):  
Calcium Ionophore ◽  
Genetically Engineered ◽  
Ionophore A23187 ◽  
Type I ◽  
Wild Type ◽  
Endothelial Surface ◽  
Genetically Engineered Mice ◽  
Β3 Integrin ◽  
Mean Time

Abstract VWF is synthesized in megakaryocytes and endothelial cells and stored in a-granules and Weibel-Palade bodies respectively. VWF released from the storage granules upon cellular activation or stimulation with secretagogues are extremely large and known as Ultra-Large von Willebrand factor (UL-VWF). Such filaments were first shown by Dong and colleagues (Dong et. al., 2002) and platelets were seen to align as string of beads on these UL-VWF multimers. These platelet strings are then rapidly cleaved to smaller size by the metalloprotease ADAMTS-13 (A Disintegrin-like And Metalloprotease with Thrombospondin type I repeats-13). Recently, we have similarly shown that in Adamts13−/− mice platelet strings form and remain on stimulated endothelium in veins, where they wave in the blood stream for many minutes (Motto et. al., 2005). We have also shown that antibody to ADAMTS13 induces platelet strings in activated venules and transient thrombi formation in activated microvenules of wild type similar to Adamts13−/− mice (Chauhan et. al., 2006). The obvious question is what anchors the UL-VWF mulimers to the endothelial surface? In a flow chamber model, using polyclonal antibodies to P-selectin or soluble P-selectin, it was proposed that P-selectin anchors the UL-VWF multimers to the endothelial surface. The present study was designed to address the role of P-selectin and that of the platelet and endothelial integrin β3 in platelet string formation in vivo using intravital microscopy and genetically engineered mice. We demonstrate that in histamine activated venules, causing Weibel-Palade bodies secretion, P-selectin is not required for formation or retention of VWF platelet strings on endothelium in vivo. The mean time the platelet strings (~30–100 μm long) anchored onto the endothelium was 45 s. This duration was significantly longer than in P-selectin−/− mice infused with control Ig (P<0.001) and similar to WT mice treated with the inhibitory antibody. Similar to wild type, 45 s after topical superfusion of calcium ionophore A23187, platelet strings forming transient thrombi formation was seen in microvenules (25–30 μm in diameter) of P-selectin−/− mice when the ADAMTS13 activity was inhibited. Platelet strings were either not seen or were very short lived in the β3−/− mice infused with control Ig, whereas in the mice infused with anti- ADAMTS13 antibody platelet strings could be seen that anchored to the endothelium with a mean time of 50 s (P<0.0001). These observations show that platelet strings are not held by β3 integrin and platelets lacking β3 integrin can adhere firmly to the VWF strings in the histamine activated venules. VWF containing platelet strings could also entangle and form emboli in the absence of β3 integrin.

scholarly journals TAMI-42. H3K27M MUTANT GLIOMAS HIJACK A CONSERVED AND CRITICAL METABOLIC PATHWAY USED BY IDH1 MUTANT GLIOMAS TO MAINTAIN THEIR PREFERRED EPIGENETIC STATE

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii222-ii222
Author(s):  
Chan Chung ◽  
Stefan Sweha ◽  
Drew Pratt ◽  
Benita Tamrazi ◽  
Pooja Panwalkar ◽  
...  
Keyword(s):  
Glutamate Dehydrogenase ◽  
Metabolic Pathway ◽  
Tca Cycle ◽  
Chromatin Accessibility ◽  
Metabolic Reprogramming ◽  
Glutamine Metabolism ◽  
Wild Type ◽  
Hexokinase 2

Abstract H3K27M-midline gliomas are fatal tumors that mainly harbor H3.3K27M mutations resulting in global H3K27me3 reduction that impacts neuroglial-differentiation. However, the exact mechanisms by which H3.3K27M mutations promote cancer are poorly understood. Metabolic reprogramming is a hallmark of cancer and we hypothesized that H3.3K27M mutations can reprogram metabolism to support uncontrolled growth. We demonstrate that H3.3K27M-mutant cells show elevated levels of critical enzymes related to glycolysis and TCA cycle metabolism including hexokinase-2, isocitrate dehydrogenase (IDH)-1 and glutamate dehydrogenase. H3.3K27M cells also demonstrated enhanced glycolysis, glutamine and TCA-cycle metabolism accompanied by increased alpha-ketoglutarate (α-KG) production. Mutant IDH (mIDH)1/2 converts α-KG to D-2-hydroxyglutarate (D-2HG). D-2HG increases H3K27me3 by inhibiting α-KG’s function to drive H3K27-demethylases. We discovered that H3.3K27M cells use α-KG in an opposing manner to maintain low H3K27me3. Inhibiting enzymes related to α-KG generation including hexokinase-2, glutamate-dehydrogenase and wild type-IDH1 increased global H3K27me3, altered chromatin accessibility at neuroglial-differentiation factors and lowered tumor cell proliferation. In vivo inhibition of glutamine metabolism and/ or wild type-IDH1 using blood-brain barrier penetrant small molecule inhibitors increased overall survival in vivo in two independent H3.3K27M animal models (p< 0.0001). H3K27M and mIDH1 were mutually exclusive in patient tumor samples and D-2HG treatment or forced-mIDH1 expression in H3.3K27M cells increased global H3K27me3 and cell death. Finally H3.3K27M and mIDH1 were synthetic lethal in vitro. Our data suggest that H3.3K27M and mIDH1 hijack a critical and conserved metabolic pathway in opposing manners to regulate global H3K27me3. These results have implications for understanding the pathogenesis of fatal H3K27M-gliomas and for developing therapeutic strategies by disruption of an integrated metabolic/epigenetic-axis.

Growth of Paracoccus denitrificans on [2, 3- 13 C]succinate and [1, 4- 13 C]succinate. I. The flux of carbon in energy metabolism and the operation of the TCA cycle

1987 ◽  
Vol 231 (1264) ◽  
pp. 339-347 ◽  
Keyword(s):  
Mass Spectrometry ◽  
Energy Metabolism ◽  
Malic Enzyme ◽  
Paracoccus Denitrificans ◽  
Tca Cycle ◽  
Labelling Pattern ◽  
Carboxyl Groups ◽  
The Tca Cycle ◽  
Tca Cycle Intermediates

The metabolism of Paracoccus denitrificans , grown on either [2, 3- 13 C]- succinate or [1, 4- 13 C]succinate, was investigated by using gas chromato­graphy-mass spectrometry. The distribution of label in a group of metabolites closely related to the TCA-cycle intermediates showed that the flux of carbon from succinate in energy metabolism in vivo was via pyruvate (malic enzyme) and acetyl CoA. The labelling pattern of the carboxyl groups showed that one fifth of the succinate pool was formed by the regeneration of succinate via the TCA cycle, and four fifths was supplied externally as substrate from the medium.

scholarly journals The mitochondrial HSP90 paralog TRAP1 forms an OXPHOS-regulated tetramer and is involved in maintaining mitochondrial metabolic homeostasis

2019 ◽  
Author(s):  
Abhinav Joshi ◽  
Joyce Dai ◽  
Jungsoon Lee ◽  
Nastaran Mohammadi Ghahhari ◽  
Gregory Segala ◽  
...  
Keyword(s):  
Tca Cycle ◽  
Mitochondrial Metabolism ◽  
Glutamine Metabolism ◽  
Mitochondrial Proteins ◽  
Mitochondrial Oxidative Phosphorylation ◽  
Wild Type ◽  
Cellular Energy ◽  
Cellular Energy Metabolism ◽  
Tca Cycle Intermediates ◽  
Mitochondrial Chaperones

AbstractBackgroundThe molecular chaperone TRAP1, the mitochondrial isoform of cytosolic HSP90, remains poorly understood with respect to its pivotal role in the regulation of mitochondrial metabolism. Most studies have found it to be an inhibitor of mitochondrial oxidative phosphorylation (OXPHOS) and an inducer of the Warburg phenotype of cancer cells. However, others have reported the opposite and there is no consensus on the relevant TRAP1 interactors. This calls for a more comprehensive analysis of the TRAP1 interactome and of how TRAP1 and mitochondrial metabolism mutually affect each other.ResultsWe show that the disruption of the gene for TRAP1 in a panel of cell lines dysregulates OXPHOS by a metabolic rewiring that induces the anaplerotic utilization of glutamine metabolism to replenish TCA cycle intermediates. Restoration of wild-type levels of OXPHOS requires full-length TRAP1. Whereas the TRAP1 ATPase activity is dispensable for this function, it modulates the interactions of TRAP1 with various mitochondrial proteins. Quantitatively by far the major interactors of TRAP1 are the mitochondrial chaperones mtHSP70 and HSP60. However, we find that the most stable stoichiometric TRAP1 complex is a TRAP1 tetramer, whose levels change in response to both a decline or an increase in OXPHOS.ConclusionsOur work provides a roadmap for further investigations of how TRAP1 and its interactors such as the ATP synthase regulate cellular energy metabolism. Our results highlight that TRAP1 function in metabolism and cancer cannot be understood without a focus on TRAP1 tetramers as potentially the most relevant functional entity.

scholarly journals NMR Determination of the TCA Cycle Rate and α-Ketoglutarate/Glutamate Exchange Rate in Rat Brain

1992 ◽  
Vol 12 (3) ◽  
pp. 434-447 ◽  
Author(s):  
Graeme F. Mason ◽  
Douglas L. Rothman ◽  
Kevin L. Behar ◽  
Robert G. Shulman
Keyword(s):  
Exchange Rate ◽  
Rat Brain ◽  
Tca Cycle ◽  
Isotopic Labeling ◽  
Cycle Rate ◽  
The Tca Cycle ◽  
The Difference ◽  
Tca Cycle Intermediates

A mathematical model of cerebral glucose metabolism was developed to analyze the isotopic labeling of carbon atoms C4 and C3 of glutamate following an intravenous infusion of [1-13C]glucose. The model consists of a series of coupled metabolic pools representing glucose, glycolytic intermediates, tricarboxylic acid (TCA) cycle intermediates, glutamate, aspartate, and glutamine. Based on the rate of 13C isotopic labeling of glutamate C4 measured in a previous study, the TCA cycle rate in rat brain was determined to be 1.58 ± 0.41 μmol min−1 g−1 (mean ± SD, n = 5). Analysis of the difference between the rates of isotopic enrichment of glutamate C4 and C3 permitted the rate of exchange between α-ketoglutarate (α-KG) and glutamate to be assessed in vivo. In rat brain, the exchange rate between α-KG and glutamate is between 89 ± 35 and 126 ± 22 times faster than the TCA cycle rate (mean ± SD, n = 4). The sensitivity of the calculated value of the TCA cycle rate to other metabolic fluxes and to concentrations of glycolytic and TCA cycle intermediates was tested and found to be small.

scholarly journals Propionate metabolism in the rat heart by 13C n.m.r. spectroscopy

1988 ◽  
Vol 254 (2) ◽  
pp. 593-598 ◽  
Author(s):  
A D Sherry ◽  
C R Malloy ◽  
R E Roby ◽  
A Rajagopal ◽  
F M Jeffrey
Keyword(s):  
Rat Heart ◽  
Tca Cycle ◽  
Methylmalonic Aciduria ◽  
Non Invasive ◽  
Propionate Metabolism ◽  
The Tca Cycle ◽  
Exogenous Substrate ◽  
Oxidative Pathway ◽  
Tca Cycle Intermediates

High-resolution 13C n.m.r. spectroscopy has been used to examine propionate metabolism in the perfused rat heart. A number of tricarboxylic acid (TCA) cycle intermediates are observable by 13C n.m.r. in hearts perfused with mixtures of pyruvate and propionate. When the enriched 13C-labelled nucleus originates with pyruvate, the resonances of the intermediates appear as multiplets due to formation of multiply-enriched 13C-labelled isotopomers, whereas when the 13C-labelled nucleus originates with propionate, these same intermediates appear as singlets in the 13C spectrum since entry of propionate into the TCA cycle occurs via succinyl-CoA. An analysis of the isotopomer populations in hearts perfused with [3-13C]pyruvate plus unlabelled propionate indicates that about 27% of the total pyruvate pool available to the heart is derived directly from unlabelled propionate. This was substantiated by perfusing a heart for 2 h with [3-13C]propionate as the only available exogenous substrate. Under these conditions, all of the propionate consumed by the heart, as measured by conventional chemical analysis, ultimately entered the oxidative pathway as [2-13C] or [3-13C]pyruvate. This is consistent with entry of propionate into the TCA cycle intermediate pools as succinyl-CoA and concomitant disposal of malate to pyruvate via the malic enzyme. 13C resonances arising from enriched methylmalonate and propionylcarnitine are also detected in hearts perfused with [3-13C] or [1-13C]propionate which suggests that 13C n.m.r. may be useful as a non-invasive probe in vivo of metabolic abnormalities involving the propionate pathway, such as methylmalonic aciduria or propionic acidaemia.

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

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).

scholarly journals Cytochrome c Deficiency Differentially Affects the In Vivo Mitochondrial Electron Partitioning and Primary Metabolism Depending on the Photoperiod

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 444
Author(s):  
Igor Florez-Sarasa ◽  
Elina Welchen ◽  
Sofia Racca ◽  
Daniel H. Gonzalez ◽  
José G. Vallarino ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Tca Cycle ◽  
Day Length ◽  
Primary Metabolism ◽  
Stress Signaling ◽  
Alternative Pathways ◽  
Tca Cycle Intermediates ◽  
Growth Adaptation ◽  
Plant Respiration

Plant respiration provides metabolic flexibility under changing environmental conditions by modulating the activity of the nonphosphorylating alternative pathways from the mitochondrial electron transport chain, which bypass the main energy-producing components of the cytochrome oxidase pathway (COP). While adjustments in leaf primary metabolism induced by changes in day length are well studied, possible differences in the in vivo contribution of the COP and the alternative oxidase pathway (AOP) between different photoperiods remain unknown. In our study, in vivo electron partitioning between AOP and COP and expression analysis of respiratory components, photosynthesis, and the levels of primary metabolites were studied in leaves of wild-type (WT) plants and cytochrome c (CYTc) mutants, with reduced levels of COP components, under short- and long-day photoperiods. Our results clearly show that differences in AOP and COP in vivo activities between WT and cytc mutants depend on the photoperiod likely due to energy and stress signaling constraints. Parallel responses observed between in vivo respiratory activities, TCA cycle intermediates, amino acids, and stress signaling metabolites indicate the coordination of different pathways of primary metabolism to support growth adaptation under different photoperiods.

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