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

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.

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Lelkes, E., Unsworth, B.R. & Lelkes, P.I. Reactive oxygen species, apoptosis and alte1red NGF-induced signaling in PC12 pheochromocytoma cells cultured in elevated glucose: AnIn Vitro cellular model for diabetic neuropathy. neurotox res 3, 189–203 (2001). https://doi.org/10.1007/BF03033191

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Keywords

  • PC12 Cell
  • Hyperglycemia
  • Nerve Growth Factor
  • High Glucose
  • Dorsal Root Ganglion Neuron