Many cells in mammals exist in the state of quiescence, which is characterized by reversible exit from the cell cycle. Quiescent cells are widely reported to exhibit reduced size, nucleotide synthesis, and metabolic activity. Much lower glycolytic rates have been reported in quiescent compared with proliferating lymphocytes. In contrast, we show here that primary human fibroblasts continue to exhibit high metabolic rates when induced into quiescence via contact inhibition. By monitoring isotope labeling through metabolic pathways and quantitatively identifying fluxes from the data, we show that contact-inhibited fibroblasts utilize glucose in all branches of central carbon metabolism at rates similar to those of proliferating cells, with greater overflow flux from the pentose phosphate pathway back to glycolysis. Inhibition of the pentose phosphate pathway resulted in apoptosis preferentially in quiescent fibroblasts. By feeding the cells labeled glutamine, we also detected a “backwards” flux in the tricarboxylic acid cycle from α-ketoglutarate to citrate that was enhanced in contact-inhibited fibroblasts; this flux likely contributes to shuttling of NADPH from the mitochondrion to cytosol for redox defense or fatty acid synthesis. The high metabolic activity of the fibroblasts was directed in part toward breakdown and resynthesis of protein and lipid, and in part toward excretion of extracellular matrix proteins. Thus, reduced metabolic activity is not a hallmark of the quiescent state. Quiescent fibroblasts, relieved of the biosynthetic requirements associated with generating progeny, direct their metabolic activity to preservation of self integrity and alternative functions beneficial to the organism as a whole.
References
[1]
Werner-Washburne M, Braun E, Johnston G. C, Singer R. A (1993) Stationary phase in the yeast Saccharomycer cerevisiae. Microbiol. Rev. 57: 383–401.
[2]
Gray J. V, Petsko G. A, Johnston G. C, Ringe D, Singer R. A, et al. (2004) “Sleeping beauty”: quiescence in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 68: 187–206.
[3]
Noda T, Ohsumi Y (1998) Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast. J Biol Chem 273: 3963–3966.
[4]
Choder M (1991) A general topoisomerase I-dependent transcriptional repression in the stationary phase in yeast. Genes Dev 5: 2315–2326.
[5]
Fuge E. K, Braun E. L, Werner-Washburne M (1994) Protein synthesis in long-term stationary-phase cultures of Saccharomyces cerevisiae. J Bacteriol 176: 5802–5813.
[6]
Hedeskov C. J (1968) Early effects of phytohaemagglutinin on glucose metabolism of normal human lymphocytes. Biochem J 110: 373–380.
[7]
Sagone A. L Jr, LoBuglio A. F, Balcerzak S. P (1974) Alterations in hexose monophosphate shunt during lymphoblastic transformation. Cell Immunol 14: 443–452.
[8]
Bauer D. E, Harris M. H, Plas D. R, Lum J. J, Hammerman P. S, et al. (2004) Cytokine stimulation of aerobic glycolysis in hematopoietic cells exceeds proliferative demand. FASEB J 18: 1303–1305.
[9]
Frauwirth K. A, Riley J. L, Harris M. H, Parry R. V, Rathmell J. C, et al. (2002) The CD28 signaling pathway regulates glucose metabolism. Immunity 16: 769–777.
[10]
Brand K (1985) Glutamine and glucose metabolism during thymocyte proliferation. Pathways of glutamine and glutamate metabolism. Biochem J 228: 353–361.
[11]
Coller H. A, Sang L, Roberts J. M (2006) A new description of cellular quiescence. PLoS Biol 4: e83. doi:10.1371/journal.pbio.0040083.
[12]
Sang L, Coller H. A, Roberts J. M (2008) Control of the reversibility of cellular quiescence by the transcriptional repressor HES1. Science 321: 1095–1100.
[13]
Pollina E. A, Legesse-Miller A, Haley E. M, Goodpaster T, Randolph-Habecker J, et al. (2008) Regulating the angiogenic balance in tissues. Cell Cycle 7: 2056–2070.
[14]
Martin P (1997) Wound healing—aiming for perfect skin regeneration. Science 276: 75–81.
[15]
Iyer V. R, Eisen M. B, Ross D. T, Schuler G, Moore T, et al. (1999) The transcriptional program in the response of human fibroblasts to serum. Science 283: 83–87.
[16]
Kose O, Waseem A (2008) Keloids and hypertrophic scars: are they two different sides of the same coin? Dermatol Surg 34: 336–346.
[17]
Desmouliere A, Chaponnier C, Gabbiani G (2005) Tissue repair, contraction, and the myofibroblast. Wound Repair Regen 13: 7–12.
[18]
Holyoake T, Jiang X, Eaves C, Eaves A (1999) Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia. Blood 94: 2056–2064.
[19]
Ebben J. D, Treisman D. M, Zorniak M, Kutty R. G, Clark P. A, et al. (2010) The cancer stem cell paradigm: a new understanding of tumor development and treatment. Expert Opin Ther Targets 14: 621–632.
[20]
Yuan J, Fowler W. U, Kimball E, Lu W, Rabinowitz J. D (2006) Kinetic flux profiling of nitrogen assimilation in Escherichia coli. Nat Chem Biol 2: 529–530.
[21]
Munger J, Bajad S. U, Coller H. A, Shenk T, Rabinowitz J. D (2006) Dynamics of the cellular metabolome during human cytomegalovirus infection. PLoS Pathog 2: e132. doi:10.1371/journal.ppat.0020132.
[22]
Lu W, Kimball E, Rabinowitz J. D (2006) A high-performance liquid chromatography-tandem mass spectrometry method for quantitation of nitrogen-containing intracellular metabolites. J Am Soc Mass Spectrom 17: 37–50.
[23]
Sherr C. J, Roberts J. M (1999) CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13: 1501–1512.
[24]
Crissman H. A, Darzynkiewicz Z, Tobey R. A, Steinkamp J. A (1985) Normal and perturbed Chinese hamster ovary cells: correlation of DNA, RNA, and protein content by flow cytometry. J Cell Biol 101: 141–147.
[25]
Newsholme P, Lima M. M. R, Procopio J, Pithon-Curi T. C, Doi S. Q, et al. (2003) Glutamine and glutamate as vital metabolites. Braz J Med Biol Res 36: 153–163.
[26]
Association A. D (2001) Postprandial blood glucose. Diabetes Care 24: 775–778.
[27]
Vizan P, Alcarraz-Vizan G, Diaz-Moralli S, Solovjeva O. N, Frederiks W. M, et al. (2009) Modulation of pentose phosphate pathway during cell cycle progression in human colon adenocarcinoma cell line HT29. Int J Cancer 124: 2789–2796.
[28]
Naderi J, Hung M, Pandey S (2003) Oxidative stress-induced apoptosis in dividing fibroblasts involves activation of p38 MAP kinase and over-expression of Bax: resistance of quiescent cells to oxidative stress. Apoptosis 8: 91–100.
[29]
Boros L. G, Puigjaner J, Cascante M, Lee W. N, Brandes J. L, et al. (1997) Oxythiamine and dehydroepiandrosterone inhibit the nonoxidative synthesis of ribose and tumor cell proliferation. Cancer Res 57: 4242–4248.
[30]
Shantz L. M, Talalay P, Gordon G. B (1989) Mechanism of inhibition of growth of 3T3-L1 fibroblasts and their differentiation to adipocytes by dehydroepiandrosterone and related steroids: role of glucose-6-phosphate dehydrogenase. Proc Natl Acad Sci U S A 86: 3852–3856.
[31]
Bames D. J, Melo J. V (2006) Primitive, quiescent and difficult to kill: the role of non-proliferating stem cells in chronic myeloid leukemia. Cell Cycle 5: 2862–2866.
[32]
Winquist R. J, Boucher D. M, Wood M, Furey B. F (2009) Targeting cancer stem cells for more effective therapies: Taking out cancer's locomotive engine. Biochem Pharmacol 78: 326–334.
[33]
Reitzer L. J, Wice B. M, Kennell D (1979) Evidence that glutamine, not sugar, is the major energy source for cultured HeLa cells. J Biol Chem 254: 2669–2676.
[34]
Kovacevic Z, McGivan J. D (1983) Mitochondrial metabolism of glutamine and glutamate and its physiological significance. Physiol Rev 63: 547–605.
[35]
Zielke H. R, Zielke C. L, Ozand P. T (1984) Glutamine: a major energy source for cultured mammalian cells. Fed Proc 43: 121–125.
[36]
Wise D. R, DeBerardinis R. J, Mancuso A, Sayed N, Zhang X. Y, et al. (2008) Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction. Proc Natl Acad Sci U S A 105: 18782–18787.
[37]
DeBerardinis R. J, Lum J. J, Hatzivassiliou G, Thompson C. B (2008) The biology of cancer: metabolic reprogramming fuels cell growth and proliferation. Cell Metab 7: 11–20.
[38]
Portais J. C, Voisin P, Merle M, Canioni P (1996) Glucose and glutamine metabolism in C6 glioma cells studied by carbon 13 NMR. Biochimie 78: 155–164.
[39]
Yuneva M, Zamboni N, Oefner P, Sachidanandam R, Lazebnik Y (2007) Deficiency in glutamine but not glucose induces MYC-dependent apoptosis in human cells. J Cell Biol 178: 93–105.
[40]
Yoo H, Antoniewicz M. R, Stephanopoulos G, Kelleher J. K (2008) Quantifying reductive carboxylation flux of glutamine to lipid in a brown adipocyte cell line. J Biol Chem 283: 20621–20627.
[41]
Comte B, Vincent G, Bouchard B, Benderdour M, Des Rosiers C (2002) Reverse flux through cardiac NADP(+)-isocitrate dehydrogenase under normoxia and ischemia. Am J Physiol Heart Circ Physiol 283: H1505–H1514.
[42]
Ward P. S, Patel J, Wise D. R, Abdel-Wahab O, Bennett B. D, et al. (2010) The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer Cell 17: 225–234.
[43]
Fox C. J, Hammerman P. S, Thompson C. B (2005) Fuel feeds function: energy metabolism and the T-cell response. Nat Rev Immunol 5: 844–852.
[44]
Valentin M, Yang E (2008) Autophagy is activated, but is not required for the G0 function of BCL-2 or BCL-xL. Cell Cycle 7: 2762–2768.
[45]
Senger D. R, Destree A. T, Hynes R. O (1983) Complex regulation of fibronectin synthesis by cells in culture. Am J Physiol 245: C144–C150.
[46]
Ravandi F, Estrov Z (2006) Eradication of leukemia stem cells as a new goal of therapy in leukemia. Clin Cancer Res 12: 340–344.
[47]
Sehl M. E, Sinsheimer J. S, Zhou H, Lange K. L (2009) Differential destruction of stem cells: implications for targeted cancer stem cell therapy. Cancer Res 69: 9481–9489.
[48]
Legesse-Miller A, Elemento O, Pfau S. J, Forman J. J, Tavazoie S, et al. (2009) let-7 overexpression leads to an increased fraction of cells in G2/M, direct down-regulation of Cdc34, and stabilization of Wee1 kinase in primary fibroblasts. J Biol Chem 284: 6605–6609.
[49]
Crissman H. A, Darzynkiewicz Z, Tobey R. A, Steinkamp J. A (1985) Correlated measurements of DNA, RNA, and protein in individual cells by flow cytometry. Science 228: 1321–1324.
[50]
Bajad S. U, Lu W, Kimball E. H, Yuan J, Peterson C, et al. (2006) Separation and quantitation of water soluble cellular metabolites by hydrophilic interaction chromatography-tandem mass spectrometry. J Chromatogr A 1125: 76–88.
[51]
Luo B, Groenke K, Takors R, Wandrey C, Oldiges M (2007) Simultaneous determination of multiple intracellular metabolites in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle by liquid chromatography-mass spectrometry. J Chromatogr A 1147: 153–164.
[52]
Lu W, Bennett B. D, Rabinowitz J. D (2008) Analytical strategies for LC-MS-based targeted metabolomics. J Chromatogr B Analyt Technol Biomed Life Sci 871: 236–242.
[53]
Munger J, Bennett B. D, Parikh A, Feng X. J, McArdle J, et al. (2008) Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy. Nat Biotechnol 26: 1179–1186.
[54]
Feng X. J, Rabitz H (2004) Optimal identification of biochemical reaction networks. Biophys J 86: 1270–1281.
