Quiescent Fibroblasts Exhibit High Metabolic Activity

Lemons, Johanna M. S.; Xiao, Feng; Bennett, Bryson D.; Legesse‐Miller, Aster; Johnson, Elizabeth L.; Raitman, Irene; Pollina, Elizabeth A.; Rabitz, Herschel; Rabinowitz, Joshua D.; Coller, Hilary A.

doi:10.1371/journal.pbio.1000514

Cited by 342 publications

(380 citation statements)

References 54 publications

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“…Our Intracellular metabolites were then extracted, and 13 C enrichment in cellular citrate was determined by GC-MS. Data shown are the mean ± SD of three independent cultures from a representative of three independent experiments. ***P < 0.001. data do not exclude a complementary role for cytosolic IDH1 in impacting reductive glutamine metabolism, potentially through its oxidative function in an IDH2/IDH1 shuttle that transfers high energy electrons in the form of NADPH from mitochondria to cytosol (16,24).…”

Section: Discussionmentioning

confidence: 78%

Hypoxia promotes isocitrate dehydrogenase-dependent carboxylation of α-ketoglutarate to citrate to support cell growth and viability

Wise

Ward

Shay

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

875

801

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Citrate is a critical metabolite required to support both mitochondrial bioenergetics and cytosolic macromolecular synthesis. When cells proliferate under normoxic conditions, glucose provides the acetyl-CoA that condenses with oxaloacetate to support citrate production. Tricarboxylic acid (TCA) cycle anaplerosis is maintained primarily by glutamine. Here we report that some hypoxic cells are able to maintain cell proliferation despite a profound reduction in glucose-dependent citrate production. In these hypoxic cells, glutamine becomes a major source of citrate. Glutamine-derived α-ketoglutarate is reductively carboxylated by the NADPH-linked mitochondrial isocitrate dehydrogenase (IDH2) to form isocitrate, which can then be isomerized to citrate. The increased IDH2-dependent carboxylation of glutamine-derived α-ketoglutarate in hypoxia is associated with a concomitant increased synthesis of 2-hydroxyglutarate (2HG) in cells with wild-type IDH1 and IDH2. When either starved of glutamine or rendered IDH2-deficient by RNAi, hypoxic cells are unable to proliferate. The reductive carboxylation of glutamine is part of the metabolic reprogramming associated with hypoxia-inducible factor 1 (HIF1), as constitutive activation of HIF1 recapitulates the preferential reductive metabolism of glutaminederived α-ketoglutarate even in normoxic conditions. These data support a role for glutamine carboxylation in maintaining citrate synthesis and cell growth under hypoxic conditions. C itrate plays a critical role at the center of cancer cell metabolism. It provides the cell with a source of carbon for fatty acid and cholesterol synthesis (1). The breakdown of citrate by ATP-citrate lyase is a primary source of acetyl-CoA for protein acetylation (2). Metabolism of cytosolic citrate by aconitase and IDH1 can also provide the cell with a source of NADPH for redox regulation and anabolic synthesis. Mammalian cells depend on the catabolism of glucose and glutamine to fuel proliferation (3). In cancer cells cultured at atmospheric oxygen tension (21% O 2 ), glucose and glutamine have both been shown to contribute to the cellular citrate pool, with glutamine providing the major source of the four-carbon molecule oxaloacetate and glucose providing the major source of the two-carbon molecule acetyl-CoA (4, 5). The condensation of oxaloacetate and acetyl-CoA via citrate synthase generates the 6 carbon citrate molecule. However, both the conversion of glucose-derived pyruvate to acetyl-CoA by pyruvate dehydrogenase (PDH) and the conversion of glutamine to oxaloacetate through the TCA cycle depend on NAD + , which can be compromised under hypoxic conditions. This raises the question of how cells that can proliferate in hypoxia continue to synthesize the citrate required for macromolecular synthesis.This question is particularly important given that many cancers and stem/progenitor cells can continue proliferating in the setting of limited oxygen availability (6, 7). Louis Pasteur first highlighted the impact of hypoxia on nutrient metabol...

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Section: Discussionmentioning

confidence: 78%

Hypoxia promotes isocitrate dehydrogenase-dependent carboxylation of α-ketoglutarate to citrate to support cell growth and viability

Wise

Ward

Shay

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

875

801

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“…However, there is a growing appreciation for the role of TCA metabolism in energy production, cofactor regeneration, and biosynthesis for cell growth in both tumors and nontransformed cells (Wise et al 2008;Lemons et al 2010;Weinberg et al 2010;Cheng et al 2011). As such, detailed knowledge describing how oncogenes, mitogens, and environmental factors coordinate flux through glycolysis and the TCA cycle will facilitate the exploitation of these metabolic changes to target neoplastic tissues.…”

Section: Discussionmentioning

confidence: 99%

Erk regulation of pyruvate dehydrogenase flux through PDK4 modulates cell proliferation

Grassian¹,

Metallo²,

Coloff³

et al. 2011

Genes Dev.

171

137

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Loss of extracellular matrix (ECM) attachment leads to metabolic impairments that limit cellular energy production. Characterization of the metabolic alterations induced by ECM detachment revealed a dramatic decrease in uptake of glucose, glutamine, and pyruvate, and a consequent decrease in flux through glycolysis, the pentose phosphate pathway, and the tricarboxylic acid (TCA) cycle. However, flux through pyruvate dehydrogenase (PDH) is disproportionally decreased, concomitant with increased expression of the PDH inhibitory kinase, PDH kinase 4 (PDK4), and increased carbon secretion. Overexpression of ErbB2 maintains PDH flux by suppressing PDK4 expression in an Erk-dependent manner, and Erk signaling also regulates PDH flux in ECMattached cells. Additionally, epidermal growth factor (EGF), a potent inducer of Erk, positively regulates PDH flux through decreased PDK4 expression. Furthermore, overexpression of PDK4 in ECM-detached cells suppresses the ErbB2-mediated rescue of ATP levels, and in attached cells, PDK4 overexpression decreases PDH flux, de novo lipogenesis, and cell proliferation. Mining of microarray data from human tumor data sets revealed that PDK4 mRNA is commonly down-regulated in tumors compared with their tissues of origin. These results identify a novel mechanism by which ECM attachment, growth factors, and oncogenes modulate the metabolic fate of glucose by controlling PDK4 expression and PDH flux to influence proliferation.

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“…The other portion was resuspended in 100 μL 80:20 methanol:water and derivatized by adding 10 μL triethylamine and 2 μL benzyl chloroformate, to enhance the measurement sensitivity of amino acids. Samples were analyzed by multiple LC-MS systems (each from Thermo Scientific and fed by electrospray ionization), as described previously (42)(43)(44). Briefly, a stand-alone orbitrap mass spectrometer (Exactive) operating in negative-ion mode was coupled to reversed-phase ion-pairing chromatography and used to scan from m/z 85-1,000 at 1 Hz and 100,000 resolution; A TSQ Quantum Discovery triple-quadrupole mass spectrometer operating in negative-ion mode was coupled to reversephase ion-pairing chromatography and used to analyze selected compounds by multiple reaction monitoring.…”

Section: Methodsmentioning

confidence: 99%

Pyruvate kinase M2 promotes de novo serine synthesis to sustain mTORC1 activity and cell proliferation

Mancuso

Tong

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

342

321

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Despite the fact that most cancer cells display high glycolytic activity, cancer cells selectively express the less active M2 isoform of pyruvate kinase (PKM2). Here we demonstrate that PKM2 expression makes a critical regulatory contribution to the serine synthetic pathway. In the absence of serine, an allosteric activator of PKM2, glycolytic efflux to lactate is significantly reduced in PKM2-expressing cells. This inhibition of PKM2 results in the accumulation of glycolytic intermediates that feed into serine synthesis. As a consequence, PKM2-expressing cells can maintain mammalian target of rapamycin complex 1 activity and proliferate in serinedepleted medium, but PKM1-expressing cells cannot. Cellular detection of serine depletion depends on general control nonderepressible 2 kinase-activating transcription factor 4 (GCN2-ATF4) pathway activation and results in increased expression of enzymes required for serine synthesis from the accumulating glycolytic precursors. These findings suggest that tumor cells use serinedependent regulation of PKM2 and GCN2 to modulate the flux of glycolytic intermediates in support of cell proliferation.amino acid synthesis | glucose | metabolism | nucleotide biosynthesis

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Quiescent Fibroblasts Exhibit High Metabolic Activity

Abstract: Metabolomics technology reveals that fibroblast that have exited the proliferative cell cycle nevertheless utilize glucose throughout central carbon metabolism and rely on the pentose phosphate pathway for viability.

Cited by 342 publications

References 54 publications

Hypoxia promotes isocitrate dehydrogenase-dependent carboxylation of α-ketoglutarate to citrate to support cell growth and viability

Hypoxia promotes isocitrate dehydrogenase-dependent carboxylation of α-ketoglutarate to citrate to support cell growth and viability

Erk regulation of pyruvate dehydrogenase flux through PDK4 modulates cell proliferation

Pyruvate kinase M2 promotes de novo serine synthesis to sustain mTORC1 activity and cell proliferation

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