Advertisement

Neurotoxicity Research

, Volume 3, Issue 2, pp 189–203 | Cite as

Reactive oxygen species, apoptosis and alte1red NGF-induced signaling in PC12 pheochromocytoma cells cultured in elevated glucose: AnIn Vitro cellular model for diabetic neuropathy

  • Efrat Lelkes
  • Brian R. Unsworth
  • Peter I. LelkesEmail author
Article

Abstract

Diabetic neuropathies, affecting the autonomic, sensory, and motor peripheral nervous system, are among the most frequent complications of diabetes. The symptoms of diabetic polyneuropathies are multi-faceted; the etiology and the underlying mechanisms are as yet unclear. Clinical studies established a significant correlation between the control of the patients’ blood glucose level and the severity of the damage to the peripheral nervous system. Recentin vitro studies suggest that elevated glucose levels induced dysfunction and apoptosis in cultured cells of neuronal origin, possibly through the formation of reactive oxygen species (ROS). Based on these results, we hypothesized that elevated glucose levels impair neuronal survival and function via ROS dependent intracellular signaling pathways. In order to test this hypothesis, we cultured neural crest-derived PC12 pheochromocytoma cells under euglycemic (5 mM) and hyperglycemic (25 mM) conditions. Continuous exposure of undifferentiated PC12 cells for up to 72 h to elevated glucose induced the enhanced generation of ROS, as assessed from the increase in the cell-associated fluorescence of the ROS-sensitive fluorogenic indicator, 2,7-dichlorodihydrofluorescein diacetate. In cells cultured in high glucose, both basal and secreta-gogue-stimulated catecholamine release were enhanced. Furthermore, high glucose, reduced (by ca. 30%) the rate of cell proliferation and enhanced the occurrence of apoptosis, as assessed by DNA fragmentation, TUNEL assay and the activation of an apoptosis-specific protease, caspase CCP32. Elevated glucose levels significantly attenuated nerve growth factor (NGF)-induced neurite extension, as quanti-tated by computer-aided image analysis. Culturing PC12 cells in high glucose resulted in alterations in basal and NGF-stimulated mitogen-activated protein kinase (MAPK) signaling pathways, specifically in a switch from the neuronal survival/differentiation-associated MAPKERK to that of apoptosis/stress-associated MAPKp38and JNK. Based on our results we present a model in which the prolonged, excess formation of ROS represents a common mechanisms for hyperglycemia-induced damage to neuronal cells. We propose that this simplein vitro system might serve as an appropriate model for evaluating some of the effects of elevated glucose on cultured cells of neuronal origin.

Keywords

PC12 Cell Hyperglycemia Nerve Growth Factor High Glucose Dorsal Root Ganglion Neuron 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Reference

  1. 1.
    Abu-Raya, S., Bloch-Schilderman, E., Lelkes, P.I., Trembovler, V., Shohami, E., Gutman, Y. and Lazarovici, P. Characterization of pardaxin-induced dopamine release from pheochromocytoma cells: Role of calcium and eicosanoids.J Pharmacol Exp Ther,288: 399–406, 1999.PubMedGoogle Scholar
  2. 2.
    Atkin, S.L., Masson, E.A. and Wilcox, D. Anin vitro model of diabetes. In Vitro Cell. Dev. Biol. Animal32: 379–381, 1996.CrossRefGoogle Scholar
  3. 3.
    Barber, A.J., Lieth, E., Khin, S.A., Antonetti, D.A., Buchanan, A.G. and Gardner, T.W. Neural apoptosis in the retina during experimental and human diabetes. Early onset and effect of insulin. J Clin Invest102:4 783–4791, 1998.PubMedCrossRefGoogle Scholar
  4. 4.
    Baumgartner-Parzer, S.M., Wagner, L., Pettermann, M., Grillari, J., Gressl, A. and Waldhausl, W. High-glucose-triggered apoptosis in cultured endothelial cells. Diabetes44: 11, 1323–1327, 1995.PubMedCrossRefGoogle Scholar
  5. 5.
    Burchiel, K.J, Russell L.C., Lee R.P., and Sima, A.A. Spontaneous activity of primary afferent neurons in diabetic BB/Wistar rats. A possible mechanism of chronic diabetic neuropathic pain. Diabetes34: 1210–3 1985.Google Scholar
  6. 6.
    Chan, J. and Greenberg, D.A. Effects of glucose on calcium channels in neural cells. Neurosci. Lett.121: 1–2 34.6, 1991.CrossRefGoogle Scholar
  7. 7.
    Chappey, O., Dosquet, C, Wautier, M.-P. and Wautier, J.-L. Advanced glycation end products, oxidant stress and vascular lesions. Eur. J. Clin. Invest.27: 97–108 1997.PubMedCrossRefGoogle Scholar
  8. 8.
    Cooper, G.M.The Cell: A Molecular Approach. Sunderland, MA, Sinauer Associates, 1997.Google Scholar
  9. 9.
    Cotter, M.A., Love, A., Watt, M.J., Cameron, N.E. and Dines, K.C. Effects of natural free radical scavengers on peripheral nerve and neurovascular function in diabetic rats. Diabetologia38: 1285–1294 1995.PubMedCrossRefGoogle Scholar
  10. 10.
    Davis, P.K., Dudek, S.M. and Johnson, G.V.W. Select alterations in protein kinases and phosphatases during apoptosis of differentiated PC12 cells. J. Neurochem.68: 2338–234, 1997.PubMedGoogle Scholar
  11. 11.
    Donnini, D., Zambito, A.M., Perella, G., Ambesi-Impiombato, F.S. and Curcio, F. Glucose may induce cell death through a free radical-mediated mechanism. Biochem Biophys Res Commun219: 2, 412–7, 1996.PubMedCrossRefGoogle Scholar
  12. 12.
    Doyle, J.W., Smith, R.M. and Roth, T.P. The effect of hyperglycemia and insulin on the replication of cultured human microvascular endothelial cells. Horm. Metab. Res.29: 43–45 1997.PubMedCrossRefGoogle Scholar
  13. 13.
    Dyck, P.J., and Thomas P.K. (eds.) Diabetic Neuropathy. Second edition. 560 pp., Philadelphia, W.B. Saunders, 1999.Google Scholar
  14. 14.
    Fine, E.L., Horal, M., Chang, T.I., Fortin, G. and Leoken, M.R. Evidence that elevated glucose causes altered gene expression, apoptosis, and neural tube defects in a mouse model of diabetic pregnancy. Diabetes48: 2454–2462 1999.PubMedCrossRefGoogle Scholar
  15. 15.
    Fink, K. and Goether, M. High glucose concentrations increase GABA release but inhibit release of norepinephrine and 5-hydroxytryptamine in rat cerebral cortex. Brain Research,618: 220–226, 1993.PubMedCrossRefGoogle Scholar
  16. 16.
    Fujii, J., Myint, T., Okado, A., Kaneto, H. and Taniguchi, N. Oxidative stress caused by glycation of Cu,Zn-superoxide dismutase and its effects on intracellular components. Nephrol. Dial. Transplant11: 34–40 1996.PubMedGoogle Scholar
  17. 17.
    Fujita, K., Lazarovici, P. and Guroff, G. Regulation of the differentiation of PC12 pheochromocytoma cells. Environ. Health Perspect.80: 17–142, 1989.CrossRefGoogle Scholar
  18. 18.
    Greene, D.A., Sima, A.A.F., Albers, J.W., and Pfeifer, M.A. Diabetic Neuropathy In: Diabetes Mellitus. Theory and practice. 4 edition (H. Rifkin and D. Porte, eds), pp 710–755, Elsevier, 1993.Google Scholar
  19. 19.
    Greene, DA, Stevens MJ, Obrosova I, Feldman EL Glucose-induced oxidative stress and programmed cell death in diabetic neuropathy. Eur J Pharmacol 1999375: 217–23, 1999.CrossRefGoogle Scholar
  20. 20.
    Hall, Karen E.; Slma, Anders A.F., and Wiley, John W. Opiate-mediated Inhibition of Calcium Signaling Is Decreased In Dorsal Root Ganglion Neurons from the Diabetic BB/W Rat. J Clin. Invest.97: 1165–1172 1996.PubMedCrossRefGoogle Scholar
  21. 21.
    Haas, M. and Kübler, W. Nicotine and sympathetic neurotransmission. Cardiovascular Drugs and Therapy.10: 657–665 1996.CrossRefGoogle Scholar
  22. 22.
    Haviv, R. and Stein, R. Nerve growth factor inhibits apoptosis induced by tumor necrosis factor in PC12 cells. J Neurosci Res,55 269–277, 1999.PubMedCrossRefGoogle Scholar
  23. 23.
    Holder, M., Holl, R.W., Bartz, J., Hecker, W., Heinze, E., Leichter, H.E. and Teller, W. Influence of long-term glycemic control on the development of cardiac autonomic neuropathy in pediatric patients with type I diabetes. Diabetes Care20: 1042–1043 1997.PubMedGoogle Scholar
  24. 24.
    Juntti-Berggren, L., Larsson, O., Rorsman, P., Ammala, C, Bokvist, K., Wahlander, K., Nicotera, P., Dypbukt, J., Orrenius, S., Hallberg, A. and Berggren, P.O. Increased activity of L-type Ca2+ channels exposed to serum from patients with type I diabetes. Science261: 86–90, 1997.CrossRefGoogle Scholar
  25. 25.
    Kahn, C.R., and Weir, G.C. (eds.) Joslin’s Diabetes Mellitus, 13 edition. Lea and Febiger, Philadelphia, 1994.Google Scholar
  26. 26.
    Kaplan, D.R. and Miller, F.D. Signal transduction by the neurotrophin receptors. Current Opinion in Cell Biology9: 213–221 1997.PubMedCrossRefGoogle Scholar
  27. 27.
    Kostyuk, E., Pronchuk, N. and Shmigol, A. Calcium signal prolongation in sensory neurons of mice with experimental diabetes. Neuroreport6: 1010–2 1995.PubMedCrossRefGoogle Scholar
  28. 28.
    Kyriakis, J.M. and Avruch, J. Protein kinase cascades activated by stress and inflammatory cytokines. Bioessays18: 567–577, 1996.PubMedCrossRefGoogle Scholar
  29. 29.
    Lelkes, P.I., Ramos, E.M., Nikolaychik, V., Unsworth, B.R. and Goodwin, T. GTSF: a new, versatile cell culture medium for diverse normal and transformed mammalian cells, In Vitro Cell Dev Biol33: 344–351, 1997.CrossRefGoogle Scholar
  30. 30.
    Levine, M. Ascorbic acid specifically enhances dopamine β-monooxygenase activity in resting and stimulated chromaffin cells. J. Biol. Chem.261: 7347–7356, 1986.PubMedGoogle Scholar
  31. 31.
    Malone, J.I., Lowitt, S., Korthals, J.K., Salem, A. and Miranda, C. The effect of hyperglycemia on nerve conduction and structure is age dependent. Diabetes45: 209–215 1996.PubMedCrossRefGoogle Scholar
  32. 32.
    McConkey, D.J. and Orrenius, S. Signal transduction pathways to apoptosis. Trends Cell Biol.4: 370–374, 1994.PubMedCrossRefGoogle Scholar
  33. 33.
    Mesner, P.K., Eating, C.L., Hearty, J.M. and Green, S.H. A timetable of events during programmed cell death induced by trophic factor withdrawal from neuronal PC12 cells. The Journal of Neuroscience15: 7357–7366, 1995.PubMedGoogle Scholar
  34. 34.
    Mohamed, A.K., Bierhaus, A., Schiekofer, S., Tritschler, H., Ziegler, H. and Nawroth, P.P. The role of oxidative stress and NF-KB activation in late diabetic complications. BioFactors10: 175–169 1999.CrossRefGoogle Scholar
  35. 35.
    Mills, E.M., Gunasekar, P.G., Pavlokovic, G., Isom, G.M. Cyanide-induced apoptosis and oxidative stress in differentiated PC12 cells. J. Neurochem.67: 1039–1046, 1996.PubMedCrossRefGoogle Scholar
  36. 36.
    Mizutani, M., Kern, T.S. and Lorenzi, M. Accelerated death of retinal microvascular cells in human and experimental diabetic retinopathy. J. Clin. Invest.97: 2883–2890 1996.PubMedCrossRefGoogle Scholar
  37. 37.
    Morigi, M., Angiolett, S., Imberti, B., Donadelli, R., Micheletti, G., Figliuzzi, M., Remuzzi, A., Zoja C, and Remuzzi, G. Leukocyte-endothelial interaction is augmented by high glucose concentrations and hyperglycemia in a NF-KB-dependent fashion. J Clin Invest 1998 May 1101:9 1905–15.PubMedCrossRefGoogle Scholar
  38. 38.
    Naftolin, F., Pinter, E., Reece, E.A. Hyperglycemia and the developing neuronal tube. Dev. Brain Dysfunt.9: 187–197 1996.Google Scholar
  39. 39.
    Nikolaychik, V.V., Samet, MM. and Lelkes, P.I. A new cryoprecipitate based coating for improved endothelial cell attachment and growth on medical grade artificial surfaces. ASAIO J.40: M846-M852 1994.PubMedCrossRefGoogle Scholar
  40. 40.
    O’Brien, B.A., Harmon, B.V., Cameron, D.P. and Allan, D.J. Apoptosis is the mode of beta-cell death responsible for the development of IDDM in the nonobese diabetic (NOD) mouse. Diabetes46: 750–757, 1997.PubMedCrossRefGoogle Scholar
  41. 41.
    Ortiz, A., Ziyadeh, F.N. and Neilson, E.G. Expression of apoptosis-regulatory genes in renal proximal tubular epithelial cells exposed to high ambient glucose and in diabetic kidneys. J. Investig. Med.45: 50–56 1997.PubMedGoogle Scholar
  42. 42.
    Papadimitriou, E. and Lelkes, P.I. Measurement of cell numbers in microtiter culture plates using the fluorescent dye Hoechst 33258. J. Immunol. Methods162: 41–45, 1993.PubMedCrossRefGoogle Scholar
  43. 43.
    Peng, Y., Finley, B.E., and Fechtel, K. Hyperglycemia delays rostral initiation sites during neural tube closure. Int. Devel. Neurosci.12: 189–196 1994.Google Scholar
  44. 44.
    Phelan, S.A., Ito, M. and Loeken, M.R. Neural tube defects in embryos of diabetic mice: role of the Pax-3 gene and apoptosis. Diabetes46: 1189–1197, 1997.PubMedCrossRefGoogle Scholar
  45. 45.
    Pieper, G.M., Siebeneich, W., Roza, A.M., Jordan, M. and Adams, M.B. Chronic treatment in vivo with dimethylthiourea, a hydroxyl radical scavenger, prevents diabetes-induced endothelial dysfunction. J. Car-diovasc. Pharmacol.28: 741–745 1996.CrossRefGoogle Scholar
  46. 46.
    Pitkänen, O.M., Martin, J.M., Hallman, M., Åkerblom, H.K., Sariola, H., and Andersson, S.M. Free radical activity during development of insulin-dependent diabetes mellitus in the rat. Life Sci.50: 335–339 1991.CrossRefGoogle Scholar
  47. 47.
    Rasouly, D., Shavit, D., Zuniga, R., Elejalde, R.B., Unsworth, B.R., Yayon, A., Lazarovici, P., and Lelkes, P.I. Staurosporine induces neurite outgrowth in neuronal hybrids (PC12EN) lacking NGF receptors. J. Cell Biochemistry62: 356–371 1996.CrossRefGoogle Scholar
  48. 48.
    Russell, JW, Feldman EL Insulin-like growth factor-I prevents apoptosis in sympathetic neurons exposed to high glucose. Horm Metab Res 199931: 90–9, 1999.CrossRefGoogle Scholar
  49. 49.
    Russell, J.W., Sullivan, K.A., Windebank, A.J., Herrmann, D.N., Feldman, EX. Neurons undergo apoptosis in animal and cell culture models of diabetes. Neurobiol Dis.6: 347–363 1999.PubMedCrossRefGoogle Scholar
  50. 50.
    Santini, F. and Beaven, M.A. Tyrosine phosphorylation of a mitogen-activated protein kinase-like protein occurs at a late step in exocytosis. J. Biol. Chem.268: 22716–22722 1993.PubMedGoogle Scholar
  51. 51.
    Schleicher, E. and Nerlich, A. The role of hyperglycemia in the Development of Diabetic Complications Horm. Metab. Res.28:367–373, 1996.CrossRefGoogle Scholar
  52. 52.
    Shimazaki, Y., Nishiki, T., Omori, A., Sekiguchi, M., Kamata, Y., Kozaki, S. and Takahashi, M. Phosphorylation of 25-kDa synaptosome-associated protein-Possible involvement in protein kinase C-mediated regulation of neurotransmitter release. J. Biol. Chem.271:14548–14553, 1996.PubMedCrossRefGoogle Scholar
  53. 53.
    Suzuki, N., Svensson, K. and Eriksson, U.J. High glucose concentration inhibits migration of rat cranial neural crest cells in vitro. Diabetologia39: 401–411 1996.PubMedCrossRefGoogle Scholar
  54. 54.
    Tesfamariam, B. Free radicals in diabetic endothelial cell dysfunction. Free Red. Biol. Med.16: 383–391 1994.CrossRefGoogle Scholar
  55. 55.
    Tesfamariam, B. and Cohen, R.A. Free radicals mediate endothelial cell dysfunction caused by elevated glucose. Am. J. Physiol.263: H321-H326 1992.PubMedGoogle Scholar
  56. 56.
    Tischler, A.S. and Green, L.A. Morphological and cytochemical properties of a clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Lab. Invest.34: 77–89 1978.Google Scholar
  57. 57.
    Vinik A.I. Diabetic neuropathy: pathogenesis and therapy. Am J Med107: 17S-26S, 1999.PubMedCrossRefGoogle Scholar
  58. 58.
    Wilke, R.A., Riley D.A., Lelkes P.I., and Hillard C.J. Decreased catecholamine secretion from the adrenal medullae of chronically diabetic BB-Wistar rats. Diabetes42: 862–8 1993.PubMedCrossRefGoogle Scholar
  59. 59.
    Zhang, W., Khanna, P., Chan, L.L., Campbell, G. and Ansari, N.H. Diabetes-induced apoptosis in rat kidney. Biochem. Mol. Med.61: 58–62 1997.PubMedCrossRefGoogle Scholar
  60. 60.
    Zulueta, J.J., Sawhney, R., Yu, F.S., Cote, C.C. and Hassoun, P.M. Intracellular generation of reactive oxygen species in endothelial cells exposed to anoxia-reoxygenation. Am. J. Physiol.272: L897-L902 1997.PubMedGoogle Scholar
  61. 61.
    Suzuki, N., Svensson, K. and Eriksson, U.J. High glucose concentration inhibits migration of rat cranial neural crest cells in vitro. Diabetologia39: 401–411 1996.PubMedCrossRefGoogle Scholar

Copyright information

© Springer 2001

Authors and Affiliations

  • Efrat Lelkes
    • 1
    • 2
  • Brian R. Unsworth
    • 3
  • Peter I. Lelkes
    • 1
    Email author
  1. 1.Department of MedicineUniversity of Wisconsin Medical SchoolMadison
  2. 2.Medical Scholars ProgramUniversity of Wisconsin Medical SchoolMadison
  3. 3.Department of BiologyMarquette UniversityMilwaukee

Personalised recommendations