Uncoupling Proteins: Regulation by IGF-1 and Neuroprotection during Hyperglycemia in Vitro

Diabetic neuropathy is believed to arise due to oxidative stress following hyperglycemic situations. Uncoupling proteins (UCPs) constitute a subgroup of mitochondrial transporter proteins with putative antioxidant properties. By dissipating the proton gradient over the mitochondrial inner membrane, these proteins reduce the mitochondrial inner membrane potential (MMP), and thereby, the mitochondrial production of reactive oxygen species (ROS) is decreased. In this thesis I have examined the regulation of UCP2, UCP3, and UCP4 by the neuroprotective hormone insulin-like growth factor type 1 (IGF1). I have also investigated the possible involvement of UCP3 in IGF-1mediated neuroprotection following high glucose treatments. All studies were performed using human neuroblastoma SH-SY5Y cells as an in vitro cell model. The major findings were as follows: i. Native SH-SY5Y cells expressed UCP2, UCP3, and UCP4. ii. UCP3 was upregulated by IGF-1 via activation of the IGF-1 receptor. IGF1 increased UCP3 mRNA and protein levels primarily via activation of the “classical” anti-apoptotic phosphatidyl inositol 3 (PI3)-kinase signaling pathway, as shown by incubation with specific inhibitors of the PI3-kinase and mitogen activated protein (MAP) kinase signaling pathways. iii. UCP2 and UCP4 protein levels were only marginally or not at all regulated by IGF-1. These UCPs are probably not involved in IGF-1mediated neuroprotection. iv. High glucose concentrations reduced the UCP3 protein levels in highly differentiated SH-SY5Y cells. Concomitantly, the MMP and the levels of ROS and glutathione increased, whereas the number of neurites per cell was reduced. This supports an antioxidant and neuroprotective role of UCP3 v. IGF-1 prevented the glucose-induced reduction in UCP3 protein levels. In parallel, the effects on MMP, levels of ROS and glutathione, and number of neurites per cell were abolished or significantly reduced. These data suggest that UCP3 is involved in IGF-1-mediated neuroprotection. LIST OF PUBLICATIONS This thesis is based on the following papers, which are referred to by Roman numerals (I-IV) in the text: I Insulin-like growth factor type 1 up-regulates uncoupling protein 3. Gustafsson H., Adamson L., Hedander J., Walum E. and Forsby A. (2001) Biochem Biophys Res Commun. 287(5), 1105-1111. II Signalling pathways for insulin-like growth factor type 1-mediated expression of uncoupling protein 3. Gustafsson H., Tamm C. and Forsby A. (2004) J Neurochem. 88, 462-468. III Expression of uncoupling protein 2 and 4 in human neuroblastoma SH-SY5Y cells. Gustafsson H. and Forsby A. (2004) Manuscript. IV Insulin-like growth factor type 1 prevents hyperglycemia-induced uncoupling protein 3 down-regulation and oxidative stress. Gustafsson H., Söderdahl T., Jönsson G., Bratteng J.-O. and Forsby A. (2004) J Neurosci Res In press. Additional publications Development of an in vitro test battery for the estimation of acute human systemic toxicity: An outline of the EDIT project. Evaluationguided Development of New In Vitro Test Batteries. Clemedson C., Nordin-Andersson M., Bjerregaard H.-F., Clausen J., Forsby A., Gustafsson H., Hansson U., Isomaa B., Jorgensen C., Kolman A., Kotova N., Krause G., Kristen U., Kurppa K., Romert L., Scheers E. (2002) Altern Lab Anim 30(3), 313-21.