Molecular Biology

, Volume 52, Issue 3, pp 419–429 | Cite as

AICAR-Dependent Activation of AMPK Kinase Is Not Accompanied by G1/S Block in Mouse Embryonic Stem Cells

  • B. B. Grigorash
  • I. I. Suvorova
  • V. A. Pospelov
Molecular Cell Biology


Embryonic stem cells (ESCs) have the capacity for self-renewal and pluripotency. Due to high proliferative activity, ESCs use a specific pathway of the formation of ATP molecules, which can lead to the development of the adaptive metabolic response under the conditions of energy deficiency (which is different from the response of differentiated cells). It is known that metabolic signals are integrated with the cell cycle progression; however, the signaling pathways that connect the availability of nutrients with the regulation of cell cycle in ESCs are insufficiently studied. We have studied the effect of the AICAR agent, which imitates an increase in AMP level and induces the activation of the metabolic sensor AMPK, on proliferation, cell cycle distribution, and pluripotency of mouse ESCs (mESCs). It has been demonstrated that cells treated with AICAR do not stop at the control G1/S point of the cell cycle, since they do not accumulate P21/WAF1 (G1/S checkpoint regulator), despite P53 activation. On the contrary, AICAR increases the rate of mESC proliferation, which correlates with increased expression of pluripotency marker genes (OCT3/4, NANOG, SOX2, KLF4, ESRRB, PRDM14). In addition, an increase in the transcription of the HIF1α gene (a key regulator of the cell proliferation and viability, as well as glucose metabolism under stress) was detected. An increase in the expression of glycolytic enzyme genes (LDHA, ALDOA, PCK2, GLUT4) under the effect of AICAR indicates a change in mESC metabolism towards increased glycolysis. Thus, AICAR-dependent AMPK activation as one of possible mechanisms of the mESC adaptive response to the emergence of energetic imbalance is not accompanied by a cell cycle arrest at the G1/S checkpoint, but involves the processes of increasing glycolytic activity.


embryonic stem cells AMPK AICAR P53-P21/WAF1 G1/S checkpoint cell cycle differentiation pluripotency metabolism 



5-aminoimidazole-4-carboxamide ribonucleotide


AMP-activated protein kinase


lactate dehydrogenase A


aldolase A


phosphoenolpyruvate carboxykinase 2


glucose transporter types 4 and 1


retinoic acid


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  1. 1.
    Malashicheva A.B., Kisliakova T.V., Pospelov V.A. 2002. Embryonal stem cells do not undergo cell cycle arrest upon exposure to damaging factors. Tsitologiya. 44, 649–655.Google Scholar
  2. 2.
    Chuykin I.A., Lianguzova M.S., Pospelova T.V., Pospelov V.A. 2008. Activation of DNA damage response signaling in mouse embryonic stem cells. Cell Cycle. 7, 2922–2928.CrossRefPubMedGoogle Scholar
  3. 3.
    Becker K.A., Ghule P.N., Therrien J.A., et al. 2006. Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase. J. Cell. Physiol. 209, 883–893.CrossRefPubMedGoogle Scholar
  4. 4.
    Suvorova I.I., Katolikova N.V., Pospelov V.A. 2012. New insights into cell cycle regulation and DNA damage response in embryonic stem cells. Int. Rev. Cell. Mol. Biol. 299, 161–198.CrossRefPubMedGoogle Scholar
  5. 5.
    Prigione A., Adjaye J. 2010. Modulation of mitochondrial biogenesis and bioenergetic metabolism upon in vitro and in vivo differentiation of human ES and iPS cells. Int. J. Dev. Biol. 54, 1729–1741.CrossRefPubMedGoogle Scholar
  6. 6.
    Varum S., Rodrigues A.S., Moura M.B., et al. 2011. Energy metabolism in human pluripotent stem cells and their differentiated counterparts. PLoS One. 6, e20914.CrossRefGoogle Scholar
  7. 7.
    Folmes C.D., Nelson T.J., Martinez-Fernandez A., et al. 2011. Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming. Cell. Metab. 14, 264–271.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Zhang J., Khvorostov I., Hong J.S., et al. 2011. UCP2 regulates energy metabolism and differentiation potential of human pluripotent stem cells. EMBO J. 30, 4860–4873.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Warburg O. 1956. On the origin of cancer cells. Science. 123, 309–314.CrossRefPubMedGoogle Scholar
  10. 10.
    Gardner D.K. 2015. Lactate production by the mammalian blastocyst: Manipulating the microenvironment for uterine implantation and invasion? Bioessays. 37, 364–371.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Wu M., Neilson A., Swift A.L., et al. 2007. Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells. Am. J. Physiol.: Cell Physiol. 292, 125–136.CrossRefGoogle Scholar
  12. 12.
    Facucho-Oliveira J.M., Alderson J., Spikings E.C., et al. 2007. Mitochondrial DNA replication during differentiation of murine embryonic stem cells. J. Cell Sci. 120, 4025–4034.CrossRefPubMedGoogle Scholar
  13. 13.
    Todd L.R., Damin M.N., Gomathinayagam R., et al. 2010. Growth factor erv1-like modulates Drp1 to preserve mitochondrial dynamics and function in mouse embryonic stem cells. Mol. Biol. Cell. 21, 1225–1236.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Mandal S., Lindgren A.G., Srivastava A.S., et al. 2011. Mitochondrial function controls proliferation and early differentiation potential of embryonic stem cells. Stem Cells. 29, 486–495.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Rafalski V.A., Mancini E., Brunet A. 2012. Energy metabolism and energy-sensing pathways in mammalian embryonic and adult stem cell fate. J. Cell. Sci. 125, 5597–608.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Cherepkova M.Y., Sineva G.S., Pospelov V.A. 2016. Leukemia inhibitory factor (LIF) withdrawal activates mTOR signaling pathway in mouse embryonic stem cells through the MEK/ERK/TSC2 pathway. Cell Death Dis. 14, e2050.CrossRefGoogle Scholar
  17. 17.
    Chen H., Liu X., Chen H., et al. 2014. Role of SIRT1 and AMPK in mesenchymal stem cells differentiation. Ageing Res. Rev. 13, 55–64.CrossRefPubMedGoogle Scholar
  18. 18.
    Qu J., Lu D., Guo H., et al. 2016. MicroRNA-9 regulates osteoblast differentiation and angiogenesis via the AMPK signaling pathway. Mol. Cell. Biochem. 411, 23–33.CrossRefPubMedGoogle Scholar
  19. 19.
    Jones R.G., Plas D.R., Kubek S., et al. 2005. AMPactivated protein kinase induces a p53-dependent metabolic checkpoint. Mol. Cell. 18, 283–293.CrossRefPubMedGoogle Scholar
  20. 20.
    Dasgupta B., Milbrandt J. 2009. AMP-activated protein kinase phosphorylates retinoblastoma protein to control mammalian brain development. Dev. Cell. 16, 256–270.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Imamura K., Ogura T., Kishimoto A., et al. 2001. Cell cycle regulation via p53 phosphorylation by a 5'-AMP activated protein kinase activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, in a human hepatocellular carcinoma cell line. Biochem. Biophys. Res. Commun. 287, 562–567.CrossRefPubMedGoogle Scholar
  22. 22.
    Zang Y., Yu L.F., Nan F.J., et al. 2009. AMP-activated protein kinase is involved in neural stem cell growth suppression and cell cycle arrest by 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside and glucose deprivation by down-regulating phospho-retinoblastoma protein and cyclin D. Biol. Chem. 284, 6175–6184.CrossRefGoogle Scholar
  23. 23.
    Rattan R., Giri S., Singh A.K., Singh I. 2005. 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside inhibits cancer cell proliferation in vitro and in vivo via AMP-activated protein kinase. J. Biol. Chem. 280, 39582–39593.CrossRefPubMedGoogle Scholar
  24. 24.
    Liang X., Wang P., Gao Q., Tao X. 2014. Exogenous activation of LKB1/AMPK signaling induces G1 arrest in cells with endogenous LKB1 expression. Mol. Med. Rep. 9, 1019–1024.CrossRefPubMedGoogle Scholar
  25. 25.
    Teng H., Sui X., Zhou C., et al. 2016. Fatty acid degradation plays an essential role in proliferation of mouse female primordial germ cells via the p53-dependent cell cycle regulation. Cell Cycle. 15, 425–431.CrossRefPubMedGoogle Scholar
  26. 26.
    Cai X., Hu X., Tan X., et al. 2015. Metformin induced AMPK activation, G0/G1 phase cell cycle arrest and the inhibition of growth of esophageal squamous cell carcinomas in vitro and in vivo. PLoS One. 10, e0133349.Google Scholar
  27. 27.
    van Meerloo J., Kaspers G., Cloos J. 2011. Cell sensitivity assays: The MTT assay. Meth. Mol. Biol. 731, 237–245.CrossRefGoogle Scholar
  28. 28.
    Vallier L. 2015. Cell cycle rules pluripotency. Cell Stem Cell. 17, 131–132.CrossRefPubMedGoogle Scholar
  29. 29.
    Wu S.B., Wei Y.H. 2012. AMPK-mediated increase of glycolysis as an adaptive response to oxidative stress in human cells: implication of the cell survival in mitochondrial diseases. Biochim. Biophys. Acta. 182, 233–247.CrossRefGoogle Scholar
  30. 30.
    Shestov A.A., Mancuso A., Leeper D.B., Glickson J.D. 2013. Metabolic network analysis of DB1 melanoma cells: How much energy is derived from aerobic glycolysis? Adv. Exp. Med. Biol. 765, 265–271.CrossRefPubMedGoogle Scholar
  31. 31.
    Schuster S., Boley D., Möller P., et al. 2015. Mathematical models for explaining the Warburg effect: A review focussed on ATP and biomass production. Biochem. Soc. Trans. 43, 1187–1194.CrossRefPubMedGoogle Scholar
  32. 32.
    Watford M., Hod Y., Chiao Y.B., et al. 1981. The unique role of the kidney in gluconeogenesis in the chicken. The significance of a cytosolic form of phosphoenolpyruvate carboxykinase. J. Biol. Chem. 256, 10023–10027.PubMedGoogle Scholar
  33. 33.
    Leithner K., Hrzenjak A., Trötzmüller M., et al. 2015. PCK2 activation mediates an adaptive response to glucose depletion in lung cancer. Oncogene. 34, 1044–1050.CrossRefPubMedGoogle Scholar
  34. 34.
    Zhou W., Choi C., Margineantu D., et al. 2012. HIF1α induced switch from bivalent to exclusively glycolytic metabolism during ESC-EpiSC transition. EMBO J. 31, 2103–2116.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Lu H., Forbes R.A., Verma A. 2002. Hypoxia-inducible factor 1 activation by aerobic glycolysis implicates the Warburg effect in carcinogenesis. J. Biol. Chem. 277, 23111–23115.CrossRefPubMedGoogle Scholar
  36. 36.
    Vazquez-Martin A., Vellon L., Quirós P.M., et al. 2012. Activation of AMP-activated protein kinase (AMPK) provides a metabolic barrier to reprogramming somatic cells into stem cells. Cell Cycle. 11, 974–989.CrossRefPubMedGoogle Scholar
  37. 37.
    Suvorova I.I., Grigorash B.B., Chuykin I.A., Pospelova T.V., Pospelov V.A. 2016. G1 checkpoint is compromised in mouse ESCs due to functional uncoupling of p53-p21Waf1 signaling. Cell Cycle. 15, 52–63.CrossRefPubMedGoogle Scholar
  38. 38.
    Suvorova I.I., Pospelov V.A. 2014. Analysis of irradiation-induced repair foci in mouse embryonic stem cells. Tsitologiya. 56, 340–345.Google Scholar
  39. 39.
    Jang H., Kim Tae W., Yoon S., et al. 2012. O-GlcNAc regulates pluripotency and reprogramming by directly acting on core components of the pluripotency network. Cell Stem Cell. 11, 62–74.CrossRefPubMedGoogle Scholar
  40. 40.
    Panopoulos A.D., Yanes O., Ruiz S., et al. 2012. The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming. Cell Res. 22, 168–177.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Mathieu J., Zhou W., Xing Y., et al. 2014. Hypoxiainducible factors have distinct and stage-specific roles during reprogramming of human cells to pluripotency. Cell Stem Cell. 14, 592–605.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Kim H., Jang H., Kim T.W., et al. 2015. Core pluripotency factors directly regulate metabolism in embryonic stem cell to maintain pluripotency. Stem Cells. 33, 2699–2711.CrossRefPubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Inc. 2018

Authors and Affiliations

  • B. B. Grigorash
    • 1
  • I. I. Suvorova
    • 1
  • V. A. Pospelov
    • 1
  1. 1.Institute of CytologyRussian Academy of SciencesSt. PetersburgRussia

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