Applied Microbiology and Biotechnology

, Volume 94, Issue 1, pp 57–67 | Cite as

Quercetin treatment changes fluxes in the primary metabolism and increases culture longevity and recombinant α1-antitrypsin production in human AGE1.HN cells

  • Jens Niklas
  • Yannic Nonnenmacher
  • Thomas Rose
  • Volker Sandig
  • Elmar HeinzleEmail author
Biotechnological Products and Process Engineering


Addition of the flavonoid quercetin to cultivations of the α1-antitrypsin (A1AT) producing human AGE1.HN.AAT cell line resulted in alterations of the cellular physiology and a remarkable improvement of the overall performance of these cells. In a first screening in 96-well plate format, toxicity and the effect of quercetin on the lactate/glucose ratio was analyzed. It was found that quercetin treatment reduced the lactate/glucose ratio dose dependently. An increase in culture longevity, viable cell density (160% of control), and A1AT concentration (from 0.39 g/L in the control to 0.76 g/L with quercetin, i.e., 195% of the control) was observed in batch cultivation with 10 μM quercetin compared to the control. A detailed analysis of quercetin effects on primary metabolism revealed dose-dependent alterations in metabolic fluxes. Quercetin addition resulted in an improved channeling of pyruvate into the mitochondria accompanied by reduced waste product formation and stimulation of TCA cycle activity. The observed changes in cellular physiology can be explained by different properties of quercetin and its metabolites, e.g., inhibition of specific enzymes, stimulation of oxidation of cytoplasmic, and mitochondrial NADH resulting in reduced NADH/NAD+ ratio, and cytoprotective activity. The present study shows that the addition of specific effectors to the culture medium represents a promising strategy to improve the cellular metabolic phenotype and the production of biopharmaceuticals. The provided results contribute, additionally, to an improved understanding of quercetin action on the metabolism of human cells in a general physiological context.


Flavonoid Biopharmaceutical Therapeutic protein Metabolic flux Neuronal cell Mammalian cell 



This work has been financially supported by the BMBF project SysLogics—Systems Biology of Cell Culture for Biologics (FKZ 0315275A-F). We thank Michel Fritz for the valuable assistance with the analytics.

Supplementary material

253_2011_3811_MOESM1_ESM.pdf (82 kb)
Table S1 (PDF 81 kb)


  1. A1AT Group (1998) Survival and FEV1 decline in individuals with severe deficiency of alpha1-antitrypsin. The Alpha-1-Antitrypsin Deficiency Registry Study Group. Am J Respir Crit Care Med 158(1):49–59Google Scholar
  2. Altamirano C, Paredes C, Cairo JJ, Godia F (2000) Improvement of CHO cell culture medium formulation: simultaneous substitution of glucose and glutamine. Biotechnol Prog 16(1):69–75CrossRefGoogle Scholar
  3. Blanchard V, Liu X, Eigel S, Kaup M, Rieck S, Janciauskiene S, Sandig V, Marx U, Walden P, Tauber R, Berger M (2011) N-glycosylation and biological activity of recombinant human alpha1-antitrypsin expressed in a novel human neuronal cell line. Biotechnol Bioeng 108(9):2118–2128CrossRefGoogle Scholar
  4. Boulton DW, Walle UK, Walle T (1999) Fate of the flavonoid quercetin in human cell lines: chemical instability and metabolism. J Pharm Pharmacol 51(3):353–359CrossRefGoogle Scholar
  5. Buss GD, Constantin J, de Lima LC, Teodoro GR, Comar JF, Ishii-Iwamoto EL, Bracht A (2005) The action of quercetin on the mitochondrial NADH to NAD(+) ratio in the isolated perfused rat liver. Planta Med 71(12):1118–1122CrossRefGoogle Scholar
  6. Chan T, Galati G, O'Brien PJ (1999) Oxygen activation during peroxidase catalysed metabolism of flavones or flavanones. Chem Biol Interact 122(1):15–25CrossRefGoogle Scholar
  7. Cherlet M, Marc A (2000) Stimulation of monoclonal antibody production of hybridoma cells by butyrate: evaluation of a feeding strategy and characterization of cell behaviour. Cytotechnology 32(1):17–29CrossRefGoogle Scholar
  8. Deshpande RR, Koch-Kirsch Y, Maas R, John GT, Krause C, Heinzle E (2005) Microplates with integrated oxygen sensors for kinetic cell respiration measurement and cytotoxicity testing in primary and secondary cell lines. Assay Drug Dev Technol 3(3):299–307CrossRefGoogle Scholar
  9. Fiorani M, Guidarelli A, Blasa M, Azzolini C, Candiracci M, Piatti E, Cantoni O (2010) Mitochondria accumulate large amounts of quercetin: prevention of mitochondrial damage and release upon oxidation of the extramitochondrial fraction of the flavonoid. J Nutr Biochem 21(5):397–404CrossRefGoogle Scholar
  10. Gasparin FR, Salgueiro-Pagadigorria CL, Bracht L, Ishii-Iwamoto EL, Bracht A, Constantin J (2003a) Action of quercetin on glycogen catabolism in the rat liver. Xenobiotica 33(6):587–602CrossRefGoogle Scholar
  11. Gasparin FR, Spitzner FL, Ishii-Iwamoto EL, Bracht A, Constantin J (2003b) Actions of quercetin on gluconeogenesis and glycolysis in rat liver. Xenobiotica 33(9):903–911CrossRefGoogle Scholar
  12. Gildea TR, Shermock KM, Singer ME, Stoller JK (2003) Cost-effectiveness analysis of augmentation therapy for severe alpha1-antitrypsin deficiency. Am J Respir Crit Care Med 167(10):1387–1392CrossRefGoogle Scholar
  13. Grisolia S, Rubio V, Feijoo B, Mendelson J (1975) Inhibition of lactic dehydrogenase and of pyruvate kinase by low concentrations of quercetin. Physiol Chem Phys 7(5):473–475Google Scholar
  14. Harris D, Li L, Chen M, Lagunero F, Go V, Boros L (2011) Diverse mechanisms of growth inhibition by luteolin, resveratrol, and quercetin in MIA PaCa-2 cells: a comparative glucose tracer study with the fatty acid synthase inhibitor C75. Metabolomics 1–10Google Scholar
  15. Ishige K, Schubert D, Sagara Y (2001) Flavonoids protect neuronal cells from oxidative stress by three distinct mechanisms. Free Radic Biol Med 30(4):433–446CrossRefGoogle Scholar
  16. John GT, Klimant I, Wittmann C, Heinzle E (2003) Integrated optical sensing of dissolved oxygen in microtiter plates: a novel tool for microbial cultivation. Biotechnol Bioeng 81(7):829–836CrossRefGoogle Scholar
  17. Karnaukhova E, Ophir Y, Golding B (2006) Recombinant human alpha-1 proteinase inhibitor: towards therapeutic use. Amino Acids 30(4):317–332CrossRefGoogle Scholar
  18. Kelly E, Greene CM, Carroll TP, McElvaney NG, O'Neill SJ (2010) Alpha-1 antitrypsin deficiency. Respir Med 104(6):763–772CrossRefGoogle Scholar
  19. Kromer JO, Fritz M, Heinzle E, Wittmann C (2005) In vivo quantification of intracellular amino acids and intermediates of the methionine pathway in Corynebacterium glutamicum. Anal Biochem 340(1):171–173CrossRefGoogle Scholar
  20. Liu CH, Chen LH (2007) Enhanced recombinant M-CSF production in CHO cells by glycerol addition: model and validation. Cytotechnology 54(2):89–96CrossRefGoogle Scholar
  21. Martin HJ, Kornmann F, Fuhrmann GF (2003) The inhibitory effects of flavonoids and antiestrogens on the Glut1 glucose transporter in human erythrocytes. Chem Biol Interact 146(3):225–235CrossRefGoogle Scholar
  22. Matsuo M, Sasaki N, Saga K, Kaneko T (2005) Cytotoxicity of flavonoids toward cultured normal human cells. Biol Pharm Bull 28(2):253–259CrossRefGoogle Scholar
  23. Metodiewa D, Jaiswal AK, Cenas N, Dickancaite E, Segura-Aguilar J (1999) Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal product. Free Radic Biol Med 26(1–2):107–116CrossRefGoogle Scholar
  24. Middleton E Jr, Kandaswami C, Theoharides TC (2000) The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev 52(4):673–751Google Scholar
  25. Niklas J, Heinzle E (2011) Metabolic flux analysis in systems biology of mammalian cells. Adv Biochem Eng Biotechnol. doi: 10.1007/1010_2011_1099
  26. Niklas J, Noor F, Heinzle E (2009) Effects of drugs in subtoxic concentrations on the metabolic fluxes in human hepatoma cell line Hep G2. Toxicol Appl Pharmacol 240(3):327–336CrossRefGoogle Scholar
  27. Niklas J, Priesnitz C, Rose T, Sandig V, Heinzle E (2011a) Primary metabolism in the new human cell line AGE1.HN at various substrate levels: increased metabolic efficiency and α1-antitrypsin production at reduced pyruvate load. Appl Microbiol Biotechnol. doi: 10.1007/s00253-011-3526-6
  28. Niklas J, Sandig V, Heinzle E (2011b) Metabolite channeling and compartmentation in the Human cell line AGE1.HN determined by 13C labeling experiments and 13C metabolic flux analysis. J Biosci Bioeng. doi: 10.1016/j.jbiosc.2011.07.021
  29. Niklas J, Schrader E, Sandig V, Noll T, Heinzle E (2011c) Quantitative characterization of metabolism and metabolic shifts during growth of the new human cell line AGE1.HN using time resolved metabolic flux analysis. Bioprocess Biosyst Eng 34(5):533–545CrossRefGoogle Scholar
  30. Noor F, Niklas J, Muller-Vieira U, Heinzle E (2009) An integrated approach to improved toxicity prediction for the safety assessment during preclinical drug development using Hep G2 cells. Toxicol Appl Pharmacol 237(2):221–231CrossRefGoogle Scholar
  31. Oh HK, So MK, Yang J, Yoon HC, Ahn JS, Lee JM, Kim JT, Yoo JU, Byun TH (2005) Effect of N-acetylcystein on butyrate-treated Chinese hamster ovary cells to improve the production of recombinant human interferon-beta-1a. Biotechnol Prog 21(4):1154–1164CrossRefGoogle Scholar
  32. Petrache I, Hajjar J, Campos M (2009) Safety and efficacy of alpha-1-antitrypsin augmentation therapy in the treatment of patients with alpha-1-antitrypsin deficiency. Biologics 3:193–204Google Scholar
  33. Ren W, Qiao Z, Wang H, Zhu L, Zhang L (2003) Flavonoids: promising anticancer agents. Med Res Rev 23(4):519–534CrossRefGoogle Scholar
  34. Rodriguez J, Spearman M, Huzel N, Butler M (2005) Enhanced production of monomeric interferon-beta by CHO cells through the control of culture conditions. Biotechnol Prog 21(1):22–30CrossRefGoogle Scholar
  35. Salter DW, Custead-Jones S, Cook JS (1978) Quercetin inhibits hexose transport in a human diploid fibroblast. J Membr Biol 40(1):67–76CrossRefGoogle Scholar
  36. Seddon AP, Douglas KT (1981) Photo-induced covalent labelling of malate dehydrogenase by quercetin. Biochem Biophys Res Commun 102(1):15–21CrossRefGoogle Scholar
  37. Shisheva A, Shechter Y (1992) Quercetin selectively inhibits insulin receptor function in vitro and the bioresponses of insulin and insulinomimetic agents in rat adipocytes. Biochemistry 31(34):8059–8063CrossRefGoogle Scholar
  38. Spencer JP, Kuhnle GG, Williams RJ, Rice-Evans C (2003) Intracellular metabolism and bioactivity of quercetin and its in vivo metabolites. Biochem J 372(Pt 1):173–181CrossRefGoogle Scholar
  39. Stephanopoulos GN, Aristidou AA, Nielsen J (1998) Metabolic engineering: principles and methodologies. Academic, San DiegoGoogle Scholar
  40. Strigun A, Noor F, Pironti A, Niklas J, Yang TH, Heinzle E (2011) Metabolic flux analysis gives an insight on verapamil induced changes in central metabolism of HL-1 cells. J Biotechnol 155(3):299–307CrossRefGoogle Scholar
  41. Wlaschin KF, Hu WS (2007) Engineering cell metabolism for high-density cell culture via manipulation of sugar transport. J Biotechnol 131(2):168–176CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Jens Niklas
    • 1
  • Yannic Nonnenmacher
    • 1
  • Thomas Rose
    • 2
  • Volker Sandig
    • 2
  • Elmar Heinzle
    • 1
    Email author
  1. 1.Biochemical Engineering InstituteSaarland UniversitySaarbrückenGermany
  2. 2.ProBioGen AGBerlinGermany

Personalised recommendations