scholarly journals The SDHB Arg230His mutation causing familial paraganglioma alters glycolysis in a new Caenorhabditis elegans model

2020 ◽  
Vol 13 (10) ◽  
pp. dmm044925 ◽  
Author(s):  
Éva Saskői ◽  
Zoltán Hujber ◽  
Gábor Nyírő ◽  
István Likó ◽  
Barbara Mátyási ◽  
...  
Keyword(s):  
Tricarboxylic Acid Cycle ◽  
Familial Cancer ◽  
Tca Cycle ◽  
Metabolic Reprogramming ◽  
Sdhb Mutation ◽  
Mutation Site ◽  
Atp Production ◽  
B Subunit ◽  
Mitochondrial Number

ABSTRACTThe conserved B-subunit of succinate dehydrogenase (SDH) participates in the tricarboxylic acid cycle (TCA) cycle and mitochondrial electron transport. The Arg230His mutation in SDHB causes heritable pheochromocytoma/paraganglioma (PPGL). In Caenorhabditiselegans, we generated an in vivo PPGL model (SDHB-1 Arg244His; equivalent to human Arg230His), which manifests delayed development, shortened lifespan, attenuated ATP production and reduced mitochondrial number. Although succinate is elevated in both missense and null sdhb-1(gk165) mutants, transcriptomic comparison suggests very different causal mechanisms that are supported by metabolic analysis, whereby only Arg244His (not null) worms demonstrate elevated lactate/pyruvate levels, pointing to a missense-induced, Warburg-like aberrant glycolysis. In silico predictions of the SDHA-B dimer structure demonstrate that Arg230His modifies the catalytic cleft despite the latter's remoteness from the mutation site. We hypothesize that the Arg230His SDHB mutation rewires metabolism, reminiscent of metabolic reprogramming in cancer. Our tractable model provides a novel tool to investigate the metastatic propensity of this familial cancer and our approach could illuminate wider SDH pathology.This article has an associated First Person interview with the first author of the paper.

scholarly journals HISTOCHEMICAL INVESTIGATIONS ON THE IN VIVO EFFECTS OF FLUORIDE ON TRICARBOXYLIC ACID CYCLE DEHYDROGENASES FROM PELARGONIUM ZONALE: PART II

1967 ◽  
Vol 15 (4) ◽  
pp. 202-206
Author(s):  
C. JAMES LOVELACE ◽  
GENE W. MILLER
Keyword(s):  
Sodium Fluoride ◽  
Reductase Activity ◽  
Tricarboxylic Acid Cycle ◽  
Leaf Tissue ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Pelargonium Zonale ◽  
Acid Cycle ◽  
In Vivo Effects

In vivo effects of fluoride on tricarboxylic acid (TCA) cycle dehydrogenase enzymes of Pelargonium zonale were studied using p-nitro blue tetrazoleum chloride. Plants were exposed to 17 ppb HF, and enzyme activities in treated plants were compared to those in controls. Leaves of control plants were incubated in 5 x 10–3 M sodium fluoride. Injuries observed in fumigation and solution experiments were similar. Leaf tissue subjected to HF or sodium fluoride evidenced less succinic p-nitro blue tetrazoleum reductase activity than did control tissue. Other TCA cycle dehydrogenase enzymes were not observably affected by the fluoride concentrations used in these experiments. Excised leaves cultured in 5 x 10–3 M sodium fluoride exhibited less succinic p-nitro blue tetrazoleum reductase activity after 24 hr than did leaves cultured in 5 x 10–3 M sodium chloride.

scholarly journals Loss of Rb1 Enhances Glycolytic Metabolism in Kras-Driven Lung Tumors In Vivo

Cancers ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 237
Author(s):  
Lindsey R. Conroy ◽  
Susan Dougherty ◽  
Traci Kruer ◽  
Stephanie Metcalf ◽  
Pawel Lorkiewicz ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Tricarboxylic Acid Cycle ◽  
Genetic Alterations ◽  
Lung Tumors ◽  
Metabolic Reprogramming ◽  
Tracer Studies ◽  
Gene Encoding ◽  
Cycle Progression ◽  
Glucose Carbon

Dysregulated metabolism is a hallmark of cancer cells and is driven in part by specific genetic alterations in various oncogenes or tumor suppressors. The retinoblastoma protein (pRb) is a tumor suppressor that canonically regulates cell cycle progression; however, recent studies have highlighted a functional role for pRb in controlling cellular metabolism. Here, we report that loss of the gene encoding pRb (Rb1) in a transgenic mutant Kras-driven model of lung cancer results in metabolic reprogramming. Our tracer studies using bolus dosing of [U-13C]-glucose revealed an increase in glucose carbon incorporation into select glycolytic intermediates. Consistent with this result, Rb1-depleted tumors exhibited increased expression of key glycolytic enzymes. Interestingly, loss of Rb1 did not alter mitochondrial pyruvate oxidation compared to lung tumors with intact Rb1. Additional tracer studies using [U-13C,15N]-glutamine and [U-13C]-lactate demonstrated that loss of Rb1 did not alter glutaminolysis or utilization of circulating lactate within the tricarboxylic acid cycle (TCA) in vivo. Taken together, these data suggest that the loss of Rb1 promotes a glycolytic phenotype, while not altering pyruvate oxidative metabolism or glutamine anaplerosis in Kras-driven lung tumors.

scholarly journals Dynamic regulation of mitochondrial pyruvate metabolism Is necessary for orthotopic pancreatic tumor growth

2021 ◽  
Author(s):  
Nancy P Echeverri Ruiz ◽  
Vijay Mohan ◽  
Jinghai Wu ◽  
Sabina Scott ◽  
McKenzie Kreamer ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Tca Cycle ◽  
Metabolic Reprogramming ◽  
Flux Analysis ◽  
Dynamic Regulation ◽  
Environmental Signals ◽  
Substitution Mutations ◽  
Deficient Mice

Abstract BackgroundPyruvate dehydrogenase complex (PDC) plays a central role in carbohydrate metabolism, linking cytoplasmic glycolysis to the mitochondrial tricarboxylic acid (TCA) cycle. PDC is a conserved E1-E2-E3 dehydrogenase with a PDHA1 and PDHB heterotetramer functioning as the E1 subunit. PDHA1 contains three serine residues that can be reversibly phosphorylated by a dedicated family of four inhibitory pyruvate dehydrogenase kinases (PDHK1-4) and two reactivating phosphatases (PDP1,2). Hypoxia induces the expression of PDHK1 and PDHK3 and hyperphosphorylates PDHA1. The role of PDC in metabolic reprogramming and tumor progression appears to be for the integration of oncogenic and environmental signals which supports tumor growth. MethodsTo isolate the function of the serine-dependent regulation pf PDC, we engineered MiaPaca2 cells to express PDHA1 protein with either intact serines at positions 232, 293 and 300, or all the combinations of non-phosphorylatable alanine substitution mutations. These lines were compared in vitro for biochemical response to hypoxia by western blot, metabolic activity by biochemical assay and Seahorse XF flux analysis, and growth in media with reduced exogenous metabolites. The lines were also tested for growth in vivo after orthotopic injection into the pancreata of immune-deficient mice. ResultsIn this family of cells with non-phosphorylatable PDHA1 we found reduced hypoxic phosphorylation of PDHA1, decreased PDH enzymatic activity in normoxia and hypoxia, decreased mitochondrial function by Seahorse flux assay, reduced in vitro growth of cells in media depleted of lipids, and reduced growth of tumors after orthotopic transplantation of cells into the pancreata of immune-deficient mice. ConclusionsWe found that any substitution of alanine for serine at regulatory sites generated a hypomorphic PDC. However, the reduced PDC activity was insensitive to further reduction in hypoxia. These cells had very modest reduction of growth in vitro, but were significantly compromised in their growth as tumors, indicating that dynamic PDC adaptation to microenvironmental conditions is necessary for optimal pancreatic cancer growth in vivo.

scholarly journals Metabolic characterization of aggressive breast cancer cells exhibiting invasive phenotype: Impact of non-cytotoxic doses of 2-DG on diminishing invasiveness

2020 ◽  
Author(s):  
Mayumi Fujita ◽  
Kaori Imadome ◽  
Veena Somasundaram ◽  
Miki Kawanishi ◽  
Kumiko Karasawa ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Membrane Potential ◽  
Cell Lines ◽  
Cancer Metabolism ◽  
Tricarboxylic Acid Cycle ◽  
Prognostic Significance ◽  
Tca Cycle ◽  
Metabolic Reprogramming ◽  
Cancer Therapies ◽  
The Impact

Abstract BackgroundMetabolic reprogramming is being recognized as a fundamental hallmark of cancer, and efforts to identify drugs that can target cancer metabolism are underway. In this study, we used human breast cancer (BC) cell lines and established their invading phenotype (INV) collected from transwell inserts to compare metabolome differences and evaluate prognostic significance of the metabolome in aggressive BC invasiveness. MethodsThe invasiveness of seven human BC cell lines were compared using the transwell invasion assay. Among these, INV was collected from SUM149, which exhibited the highest invasiveness. Levels of metabolites in INV were compared with those of whole cultured SUM149 cells (WCC) using CE-TOFMS. The impact of glycolysis in INV was determined by glucose uptake assay using fluorescent derivative of glucose (2-NBDG), and significance of glycolysis, or tricarboxylic acid cycle (TCA) and electron transport chain (ETC) in the invasive process were further determined in aggressive BC cell lines, SUM149, MDA-MB-231, HCC1937, using invasion assays in the presence or absence of inhibitors of glycolysis, TCA cycle or ETC. ResultsSUM149 INV sub-population exhibited a persistent hyperinvasive phenotype. INV were hyper-glycolytic with increased glucose (2-NBDG) uptake; diminished glucose-6-phosphate (G6P) levels but elevated pyruvate and lactate, along with higher expression of phosphorylated-pyruvate dehydrogenase (pPDH) compared to WCC. Notably, inhibiting of glycolysis with lower doses of 2-DG (1 mM), non-cytotoxic to MDA-MB-231 and HCC1937, was effective in diminishing invasiveness of aggressive BC cell lines. In contrast, 3-Nitropropionic acid (3-NA), an inhibitor of succinate dehydrogenase, the enzyme that oxidizes succinate to fumarate in TCA cycle, and functions as complex II of ETC, had no significant effect on their invasiveness, although levels of TCA metabolites or detection of mitochondrial membrane potential with JC-1 staining, indicated that INV cells originally had functional TCA cycles and membrane potential. ConclusionsHyper-glycolytic phenotype of invading cells caters to rapid energy production required for invasion while TCA cycle/ETC cater to cellular energy needs for sustenance in aggressive BC. Lower, non-cytotoxic doses of 2-DG can hamper invasion and can potentially be used as an adjuvant with other anti-cancer therapies without the usual side-effects associated with cytotoxic doses.

scholarly journals Lipotoxicity in steatohepatitis occurs despite an increase in tricarboxylic acid cycle activity

2016 ◽  
Vol 310 (7) ◽  
pp. E484-E494 ◽  
Author(s):  
Rainey E. Patterson ◽  
Srilaxmi Kalavalapalli ◽  
Caroline M. Williams ◽  
Manisha Nautiyal ◽  
Justin T. Mathew ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Tricarboxylic Acid Cycle ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Hepatic Insulin Resistance ◽  
Cellular Respiration ◽  
Simple Steatosis ◽  
Triglyceride Accumulation ◽  
The Tca Cycle

The hepatic tricarboxylic acid (TCA) cycle is central to integrating macronutrient metabolism and is closely coupled to cellular respiration, free radical generation, and inflammation. Oxidative flux through the TCA cycle is induced during hepatic insulin resistance, in mice and humans with simple steatosis, reflecting early compensatory remodeling of mitochondrial energetics. We hypothesized that progressive severity of hepatic insulin resistance and the onset of nonalcoholic steatohepatitis (NASH) would impair oxidative flux through the hepatic TCA cycle. Mice (C57/BL6) were fed a high- trans-fat high-fructose diet (TFD) for 8 wk to induce simple steatosis and NASH by 24 wk. In vivo fasting hepatic mitochondrial fluxes were determined by 13C-nuclear magnetic resonance (NMR)-based isotopomer analysis. Hepatic metabolic intermediates were quantified using mass spectrometry-based targeted metabolomics. Hepatic triglyceride accumulation and insulin resistance preceded alterations in mitochondrial metabolism, since TCA cycle fluxes remained normal during simple steatosis. However, mice with NASH had a twofold induction ( P < 0.05) of mitochondrial fluxes (μmol/min) through the TCA cycle (2.6 ± 0.5 vs. 5.4 ± 0.6), anaplerosis (9.1 ± 1.2 vs. 16.9 ± 2.2), and pyruvate cycling (4.9 ± 1.0 vs. 11.1 ± 1.9) compared with their age-matched controls. Induction of the TCA cycle activity during NASH was concurrent with blunted ketogenesis and accumulation of hepatic diacylglycerols (DAGs), ceramides (Cer), and long-chain acylcarnitines, suggesting inefficient oxidation and disposal of excess free fatty acids (FFA). Sustained induction of mitochondrial TCA cycle failed to prevent accretion of “lipotoxic” metabolites in the liver and could hasten inflammation and the metabolic transition to NASH.

scholarly journals Systems-Level Metabolic Flux Profiling Elucidates a Complete, Bifurcated Tricarboxylic Acid Cycle in Clostridium acetobutylicum

2010 ◽  
Vol 192 (17) ◽  
pp. 4452-4461 ◽  
Author(s):  
Daniel Amador-Noguez ◽  
Xiao-Jiang Feng ◽  
Jing Fan ◽  
Nathaniel Roquet ◽  
Herschel Rabitz ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Genome Annotation ◽  
Clostridium Acetobutylicum ◽  
Metabolic Flux ◽  
Tricarboxylic Acid Cycle ◽  
Anaerobic Bacteria ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Fermentation Products

ABSTRACT Obligatory anaerobic bacteria are major contributors to the overall metabolism of soil and the human gut. The metabolic pathways of these bacteria remain, however, poorly understood. Using isotope tracers, mass spectrometry, and quantitative flux modeling, here we directly map the metabolic pathways of Clostridium acetobutylicum, a soil bacterium whose major fermentation products include the biofuels butanol and hydrogen. While genome annotation suggests the absence of most tricarboxylic acid (TCA) cycle enzymes, our results demonstrate that this bacterium has a complete, albeit bifurcated, TCA cycle; oxaloacetate flows to succinate both through citrate/α-ketoglutarate and via malate/fumarate. Our investigations also yielded insights into the pathways utilized for glucose catabolism and amino acid biosynthesis and revealed that the organism's one-carbon metabolism is distinct from that of model microbes, involving reversible pyruvate decarboxylation and the use of pyruvate as the one-carbon donor for biosynthetic reactions. This study represents the first in vivo characterization of the TCA cycle and central metabolism of C. acetobutylicum. Our results establish a role for the full TCA cycle in an obligatory anaerobic organism and demonstrate the importance of complementing genome annotation with isotope tracer studies for determining the metabolic pathways of diverse microbes.

scholarly journals Tricarboxylic Acid Cycle-Dependent Attenuation of Staphylococcus aureus In Vivo Virulence by Selective Inhibition of Amino Acid Transport

2009 ◽  
Vol 77 (10) ◽  
pp. 4256-4264 ◽  
Author(s):  
Yefei Zhu ◽  
Yan Q. Xiong ◽  
Marat R. Sadykov ◽  
Paul D. Fey ◽  
Mei G. Lei ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Tricarboxylic Acid Cycle ◽  
Inverse Correlation ◽  
Tca Cycle ◽  
Selective Inhibition ◽  
Tricarboxylic Acid ◽  
Glutamine Transporter ◽  
Cycle Dependent

ABSTRACT Staphylococci are the leading causes of endovascular infections worldwide. Commonly, these infections involve the formation of biofilms on the surface of biomaterials. Biofilms are a complex aggregation of bacteria commonly encapsulated by an adhesive exopolysaccharide matrix. In staphylococci, this exopolysaccharide matrix is composed of polysaccharide intercellular adhesin (PIA). PIA is synthesized when the tricarboxylic acid (TCA) cycle is repressed. The inverse correlation between PIA synthesis and TCA cycle activity led us to hypothesize that increasing TCA cycle activity would decrease PIA synthesis and biofilm formation and reduce virulence in a rabbit catheter-induced model of biofilm infection. TCA cycle activity can be induced by preventing staphylococci from exogenously acquiring a TCA cycle-derived amino acid necessary for growth. To determine if TCA cycle induction would decrease PIA synthesis in Staphylococcus aureus, the glutamine permease gene (glnP) was inactivated and TCA cycle activity, PIA accumulation, biofilm forming ability, and virulence in an experimental catheter-induced endovascular biofilm (endocarditis) model were determined. Inactivation of this major glutamine transporter increased TCA cycle activity, transiently decreased PIA synthesis, and significantly reduced in vivo virulence in the endocarditis model in terms of achievable bacterial densities in biofilm-associated cardiac vegetations, kidneys, and spleen. These data confirm the close linkage of TCA cycle activity and virulence factor production and establish that this metabolic linkage can be manipulated to alter infectious outcomes.

scholarly journals The Discovery of Highly Potent THP Derivatives as OCTN2 Inhibitors: From Structure-Based Virtual Screening to In Vivo Biological Activity

2020 ◽  
Vol 21 (19) ◽  
pp. 7431 ◽  
Author(s):  
Francesca Di Cristo ◽  
Anna Calarco ◽  
Filomena Anna Digilio ◽  
Maria Stefania Sinicropi ◽  
Camillo Rosano ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Biological Activities ◽  
Tricarboxylic Acid Cycle ◽  
Tca Cycle ◽  
Organic Cation Transporter ◽  
Acid Oxidation ◽  
High Plasticity

A mismatch between β-oxidation and the tricarboxylic acid cycle (TCA) cycle flux in mitochondria produces an accumulation of lipid metabolic intermediates, resulting in both blunted metabolic flexibility and decreased glucose utilization in the affected cells. The ability of the cell to switch to glucose as an energy substrate can be restored by reducing the reliance of the cell on fatty acid oxidation. The inhibition of the carnitine system, limiting the carnitine shuttle to the oxidation of lipids in the mitochondria, allows cells to develop a high plasticity to metabolic rewiring with a decrease in fatty acid oxidation and a parallel increase in glucose oxidation. We found that 3-(2,2,2-trimethylhydrazine)propionate (THP), which is able to reduce cellular carnitine levels by blocking both carnitine biosynthesis and the cell membrane carnitine/organic cation transporter (OCTN2), was reported to improve mitochondrial dysfunction in several diseases, such as Huntington’s disease (HD). Here, new THP-derived carnitine-lowering agents (TCL), characterized by a high affinity for the OCTN2 with a minimal effect on carnitine synthesis, were developed, and their biological activities were evaluated in both in vitro and in vivo HD models. Certain compounds showed promising biological activities: reducing protein aggregates in HD cells, ameliorating motility defects, and increasing the lifespan of HD Drosophila melanogaster.

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&lt; 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.

scholarly journals Absence of retbindin blocks glycolytic flux, disrupts metabolic homeostasis, and leads to photoreceptor degeneration

2021 ◽  
Vol 118 (6) ◽  
pp. e2018956118
Author(s):  
Tirthankar Sinha ◽  
Jianhai Du ◽  
Mustafa S. Makia ◽  
James B. Hurley ◽  
Muna I. Naash ◽  
...  
Keyword(s):  
Retinal Degeneration ◽  
Tca Cycle ◽  
Underlying Mechanism ◽  
Neural Retina ◽  
Pentose Phosphate ◽  
Metabolic Reprogramming ◽  
Glycolytic Flux ◽  
Acid Oxidation ◽  
Atp Production ◽  
Almost All

We previously reported a model of progressive retinal degeneration resulting from the knockout of the retina-specific riboflavin binding protein, retbindin (Rtbdn−/−). We also demonstrated a reduction in neural retinal flavins as a result of the elimination of RTBDN. Given the role of flavins in metabolism, herein we investigated the underlying mechanism of this retinal degeneration by performing metabolomic analyses on predegeneration at postnatal day (P) 45 and at the onset of functional degeneration in the P120 retinas. Metabolomics of hydrophilic metabolites revealed that individual glycolytic products accumulated in the P45 Rtbdn−/− neural retinas along with the elevation of pentose phosphate pathway, while TCA cycle intermediates remained unchanged. This was confirmed by using 13C-labeled flux measurements and immunoblotting, revealing that the key regulatory step of phosphoenolpyruvate to pyruvate was inhibited via down-regulation of the tetrameric pyruvate kinase M2 (PKM2). Separate metabolite assessments revealed that almost all intermediates of acylcarnitine fatty acid oxidation, ceramides, sphingomyelins, and multiple toxic metabolites were significantly elevated in the predegeneration Rtbdn−/− neural retina. Our data show that lack of RTBDN, and hence reduction in flavins, forced the neural retina into repurposing glucose for free-radical mitigation over ATP production. However, such sustained metabolic reprogramming resulted in an eventual metabolic collapse leading to neurodegeneration.

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