Journal of Molecular Neuroscience

, Volume 34, Issue 1, pp 77–87 | Cite as

Neuroprotective Role of Antidiabetic Drug Metformin Against Apoptotic Cell Death in Primary Cortical Neurons

  • Mohamad-Yehia El-Mir
  • Dominique Detaille
  • Gloria R-Villanueva
  • Maria Delgado-Esteban
  • Bruno Guigas
  • Stephane Attia
  • Eric Fontaine
  • Angeles Almeida
  • Xavier Leverve


Oxidative damage has been reported to be involved in the pathogenesis of diabetic neuropathy and neurodegenerative diseases. Recent evidence suggests that the antidiabetic drug metformin prevents oxidative stress-related cellular death in non-neuronal cell lines. In this report, we point to the direct neuroprotective effect of metformin, using the etoposide-induced cell death model. The exposure of intact primary neurons to this cytotoxic insult induced permeability transition pore (PTP) opening, the dissipation of mitochondrial membrane potential (ΔΨm), cytochrome c release, and subsequent death. More importantly, metformin, together with the PTP classical inhibitor cyclosporin A (CsA), strongly mitigated the activation of this apoptotic cascade. Furthermore, the general antioxidant N-acetyl-l-cysteine also prevented etoposide-promoted neuronal death. In addition, metformin was shown to delay CsA-sensitive PTP opening in permeabilized neurons, as triggered by a calcium overload, probably through its mild inhibitory effect on the respiratory chain complex I. We conclude that (1) etoposide-induced neuronal death is partly attributable to PTP opening and the disruption of ΔΨm, in association with the emergence of oxidative stress, and (2) metformin inhibits this PTP opening-driven commitment to death. We thus propose that metformin, beyond its antihyperglycemic role, can also function as a new therapeutic tool for diabetes-associated neurodegenerative disorders.


Cytochrome c release Etoposide Metformin Neuronal apoptosis Mitochondrial permeability transition pore 



mitochondrial membrane potential


AMP-activated protein kinase


cyclosporin A




propidium iodide


permeability transition pore


tetramethyl-rhodamine methyl ester



The authors are very grateful to Drs. Juan P. Bolanos and Nicolas Wiernsperger for a stimulating discussion and to Mrs. MC Alguero Martín for her helpful technical assistance in flow cytometry. This work was partially supported by the JCyL (Grant SA062/03; Spain), INSERM, and Merck.


  1. Almeida, A., Moncada, S., & Bolanos, J. P. (2004). Nitric oxide switches on glycolysis through the AMP protein kinase and 6-phosphofructo-2-kinase pathway. Nature Cell Biology, 6, 45–51.PubMedCrossRefGoogle Scholar
  2. Andersen, J. K. (2004). Oxidative stress in neurodegeneration: Cause or consequence? Nature Medicine, 10, S18–S25.PubMedCrossRefGoogle Scholar
  3. Baines, C. P., Kaiser, R. A., & Purcell, N. H., et al. (2005). Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature, 434, 658–662.PubMedCrossRefGoogle Scholar
  4. Barrett, L. E., Van Bockstaele, E. J., Sul, J. Y., Takano, H., Haydon, P. G., & Eberwine, J. H. (2006). Elk-1 associates with the mitochondrial permeability transition pore complex in neurons. Proceedings of the National Academy of Sciences of the United States of America, 103, 5155–5160.PubMedCrossRefGoogle Scholar
  5. Batandier, C., Guigas, B., & Detaille, D., et al. (2006). The ROS production induced by a reverse-electron flux at respiratory chain complex I is hampered by metformin. J. Biomembr. Bioenerg., 38, 33–42.CrossRefGoogle Scholar
  6. Bolanos, J. P., Almeida, A., & Stewart, V., et al. (1997). Nitric oxide-mediated mitochondrial damage in the brain: Mechanisms and implications for neurodegenerative diseases. Journal of Neurochemistry, 68, 2227–2240.PubMedCrossRefGoogle Scholar
  7. Brunmair, B., Staniek, K., & Gras, F., et al. (2004). Thiazolidinediones, like metformin, inhibit respiratory complex I: A common mechanism contributing to their antidiabetic action. Diabetes, 53, 1052–1059.PubMedCrossRefGoogle Scholar
  8. Chauvin, C., De Oliveira, F., Ronot, X., Mousseau, M., Le, , verve, X., & Fontaine, E. (2001). Rotenone inhibits the mitochondrial permeability transition-induced cell death in U937 and KB cells. Journal of Biological Chemistry, 276, 41394–41398.PubMedCrossRefGoogle Scholar
  9. Chong, Z. Z., Li, F., & Maiese, K. (2005). Oxidative stress in the brain: Novel cellular targets that govern survival during neurodegenarative disease. Progress in Neurobiology, 75, 207–246.PubMedCrossRefGoogle Scholar
  10. Custodio, J. B., Cardoso, C. M., & Almeida, L. M. (2002). Thiol protecting agents and antioxidants inhibit the mitochondrial permeability transition promoted by etoposide: Implications in the prevention of etoposide-induced apoptosis. Chemico-Biological Interactions, 140, 169–184.PubMedCrossRefGoogle Scholar
  11. Delgado-Esteban, M., Martin-Zanca, D., Andres-Martin, L., Almeida, A., & Bolanos, J. P. (2007). Inhibition of PTEN by peroxynitrite activates the phosphoinositide-3-kinase/akt neuroprotective signaling pathway. Journal of Neurochemistry, 102, 194–205.PubMedCrossRefGoogle Scholar
  12. Detaille, D., Guigas, B., & Chauvin, C., et al. (2005). Metformin prevents high glucose-induced endothelial cell death through a mitochondrial permeability transition-dependent process. Diabetes, 54, 2179–2187.PubMedCrossRefGoogle Scholar
  13. Diaz-Hernandez, J. I., Moncada, S., Bolanos, J. P., & Almeida, A. (2007). Poly(ADP-ribose) polymerase-1 protects neurons against apoptosis induced by oxidative stress. Cell Death and Differentiation, 14, 1211–1221.PubMedCrossRefGoogle Scholar
  14. Duchen, M. R. (2004). Roles of mitochondria in health and disease. Diabetes, 53, S96–S102.PubMedCrossRefGoogle Scholar
  15. Dyck, P. J., Kratz, K. M., & Lehman, K. A., et al. (1991). The rochester diabetic neuropathy study: Design, criteria for types of neuropathy, selection bias, and reproducibility of neuropathic tests. Neurology, 41, 799–807.PubMedGoogle Scholar
  16. El-Mir, M. Y., Nogueira, V., Fontaine, E., Averet, N., Rigoulet, M., & Leverve, X. (2000). Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I. Journal of Biological Chemistry, 275, 223–228.PubMedCrossRefGoogle Scholar
  17. Fontaine, E., Eriksson, O., Ichas, F., & Bernardi, P. (1998). Regulation of the permeability transition pore in skeletal muscle mitochondria. Modulation by electron flow through the respiratory chain complex I. Journal of Biological Chemistry, 273, 12662–12668.PubMedCrossRefGoogle Scholar
  18. Forte, M., & Bernardi, P. (2005). Genetic dissection of the permeability transition pore. Journal of Bioenergetics and Biomembranes, 37, 121–128.PubMedCrossRefGoogle Scholar
  19. Gillessen, T., Grasshoff, C., & Szinicz, L. (2002). Mitochondrial permeability transition can be directly monitored in living neurons. Biomedicine & Pharmacotherapy, 56, 186–193.CrossRefGoogle Scholar
  20. Gilman, C. P., Chan, S. L., Guo, Z., Zhu, X., Greig, N., & Mattson, M. P. (2003). p53 is present in synapses where it mediates mitochondrial dysfunction and synaptic degeneration in response to DNA damage, and oxidative and excitotoxic insults. Neuromolecular Medecine, 3, 159–172.CrossRefGoogle Scholar
  21. Green, D. R., & Reed, J. C. (1998). Mitochondria and apoptosis. Science, 281, 1309–1312.PubMedCrossRefGoogle Scholar
  22. Guigas, B., Detaille, D., & Chauvin, C., et al. (2004). Metformin inhibits mitochondrial permeability transition and cell death: A pharmacological in vitro study. Biochemical Journal, 382, 877–884.PubMedCrossRefGoogle Scholar
  23. Hawley, S. A., Gadalla, A. E., Olsen, G. S., & Hardie, D. G. (2002). The antidiabetic drug metformin activates the AMP-activated protein kinase cascade via an adenine nucleotide-independent mechanism. Diabetes, 51, 2420–2425.PubMedCrossRefGoogle Scholar
  24. Karpinich, N. O., Tafani, M., Rothman, R. J., Russo, M. A., & Farber, J. L. (2002). The course of etoposide-induced apoptosis from damage to DNA and p53 activation to mitochondrial release of cytochrome c. Journal of Biological Chemistry, 277, 16547–16552.PubMedCrossRefGoogle Scholar
  25. Karpinich, N. O., Tafani, M., Schneider, T., Russo, M. A., & Farber, J. L. (2006). The course of etoposide-induced apoptosis in Jurkat cells lacking p53 and bax. Journal of Cellular Physiology, 208, 55–63.PubMedCrossRefGoogle Scholar
  26. Kroemer, G., & Reed, J. C. (2000). Mitochondrial control of cell death. Nature Medicine, 6, 513–519.PubMedCrossRefGoogle Scholar
  27. Kurosu, T., Fukuda, T., Miki, T., & Miura, O. (2003). Bcl6 overexpression prevents increase in reactive oxygen species and inhibits apoptosis induced by chemotherapeutic reagents in B-cell lymphoma cells. Oncogene, 22, 4459–4468.PubMedCrossRefGoogle Scholar
  28. Leverve, X. M., Guigas, B., & Detaille, D., et al. (2003). Mitochondrial metabolism and type-2 diabetes: A specific target of metformin. Diabetes & Metabolism, 29, 6S88–6S94.CrossRefGoogle Scholar
  29. Ma, T. C., Buescher, J. L., & Oatis, B., et al. (2007). Metformin therapy in a transgenic mouse model of Huntington’s disease. Neuroscience Letters, 411, 98–103.PubMedCrossRefGoogle Scholar
  30. Mattson, M. P., & Kroemer, G. (2003). Mitochondria in cell death: novel targets for neuroprotection and cardioprotection. Trends in Molecular Medicine, 9, 196–205.PubMedCrossRefGoogle Scholar
  31. Nakajima, M., Kashiwagi, K., & Ohta, J., et al. (1994). Etoposide induces programmed death in neurons cultured from the fetal rat central nervous system. Brain Research, 641, 350–352.PubMedCrossRefGoogle Scholar
  32. Owen, M. R., Doran, E., & Halestrap, A. P. (2000). Evidence that metformin exerts its anti-diabetic effects through inhibition of complex I of the mitochondrial respiratory chain. Biochemical Journal, 348, 607–614.PubMedCrossRefGoogle Scholar
  33. Panov, A. V., Gutekunst, C.-A., & Leavitt, B. R., et al. (2002). Early mitochondrial calcium defects in Huntington’s disease are a direct effect of polyglutamines. Nature Neuroscience, 5, 731–736.PubMedGoogle Scholar
  34. Pham, N.-U., & Hedley, D. W. (2001). Respiratory chain-generated oxidative stress following treatment of leukemic blasts with DNA-damaging agents. Experimental Cell Research, 264, 345–352.PubMedCrossRefGoogle Scholar
  35. Petronilli, V., Miotto, G., & Canton, M., et al. (1999). Transient and long-lasting openings of the mitochondrial permeability pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophysical Journal, 76, 725–734.PubMedCrossRefGoogle Scholar
  36. Pirart, J. (1977). Diabetes mellitus and its degenerative complications: A prospective study of 4,400 patients observed between 1947 and 1973. Diabetes & Metabolism, 3, 245–255.Google Scholar
  37. Precht, T. A., Phelps, R. A., & Linseman, D. A., et al. (2005). The permeability transition pore triggers Bax translocation to mitochondria during neuronal apoptosis. Cell Death and Differentiation, 12, 255–265.PubMedCrossRefGoogle Scholar
  38. Rapin, J. R., Lamproglou, I., Jacques, V., & Leponcin, M. (1988). Effects of metformin on metabolic indices of cerebral and peripheral ischemia. Diabetes & Metabolism, 14(Suppl 4bis), 587–590.Google Scholar
  39. Robertson, J. D., Gogvadze, V., Zhivotovsky, B., & Orrenius, S. (2000). Distinct pathways for stimulation of cytochrome c release by etoposide. Journal of Biological Chemistry, 275, 32438–32443.PubMedCrossRefGoogle Scholar
  40. Schinzel, A. C., Takeuchi, O., & Huang, Z., et al. (2005). Cyclophilin D is a component of mitochondrial permeability transition and mediates neuronal cell death after focal cerebral ischemia. Proceedings of the National Academy of Sciences of the United States of America, 102, 12005–12010.PubMedCrossRefGoogle Scholar
  41. Wei, M. C., Zong, W. X., & Cheng, E. H., et al. (2001). Proapoptotic Bax and Bak: a requisite gateway to mitochondrial dysfunction and death. Science, 292, 727–730.PubMedCrossRefGoogle Scholar
  42. Yuan, J., & Yankner, B. A. (2000). Apoptosis in the nervous system. Nature, 407, 802–809.PubMedCrossRefGoogle Scholar
  43. Zhou, G., Myers, R., & Li, Y., et al. (2001). Role of AMP-activated protein kinase in mechanism of metformin action. Journal of Clinical Investigation, 108, 1167–1174.PubMedCrossRefGoogle Scholar
  44. Zou, M. H., Kirkpatrick, S. S., & Davis, B. J., et al. (2004). Activation of the AMP-activated protein kinase by the antidiabetic drug metformin in vivo. Role of mitochondrial reactive nitrogen species. Journal of Biological Chemistry, 279, 43940–43951.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Mohamad-Yehia El-Mir
    • 1
  • Dominique Detaille
    • 2
    • 3
  • Gloria R-Villanueva
    • 1
  • Maria Delgado-Esteban
    • 4
  • Bruno Guigas
    • 2
    • 3
  • Stephane Attia
    • 2
    • 3
  • Eric Fontaine
    • 2
    • 3
  • Angeles Almeida
    • 5
  • Xavier Leverve
    • 2
    • 3
  1. 1.Departamento de Fisiología y FarmacologíaUniversidad de SalamancaSalamancaSpain
  2. 2.INSERM U884 Bioénergétique Fondamentale et AppliquéeGrenoble CedexFrance
  3. 3.Université Joseph FourierGrenobleFrance
  4. 4.Departamento de Bioquímica y Biología MolecularUniversidad de SalamancaSalamancaSpain
  5. 5.Hospital Clinico Universitario de SalamancaSalamancaSpain

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