The role of local renin-angiotensin system on high glucose-induced cell toxicity, apoptosis and reactive oxygen species production in PC12 cells

Document Type: Original Article


1 Neurocognitive Research Center and Department of Physiology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran 2 Sleep Disorders Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran

2 Pharmacological Research Center of Medicinal Plants and Department of Pharmacology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

3 Neurocognitive Research Center and Department of Physiology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

4 Applied Physiology Research Center and Department of Physiology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

5 Immunogenetic and Cell Culture Department, Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran North Khorasan University of Medical Sciences, Bojnurd, Iran

6 Department of Physiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran


Objective(s): Hyperglycemia, oxidative stress and apoptosis have key roles in pathogenesis of diabetic neuropathy. There are local renin-angiotensin systems (RASs) in different tissues such as neural tissue. Local RASs are involved in physiological and pathophysiological processes such as inflammation, proliferation and apoptosis. This study aimed to investigate the role of local renin-angiotensin system on high glucose-induced cell toxicity, apoptosis and reactive oxygen species (ROS) production in PC12 cells, as a cell model of diabetic neuropathy.
Materials and Methods: PC12 cells were exposed to a high glucose concentration (27 mg/ml), captopril (ACE inhibitor), telmisartan and losartan (AT1 antagonists), and also PD123319 (AT2 antagonist) were administered before and after induction of high glucose toxicity. Then cell viability was assessed by MTT assay and apoptotic cells and intracellular ROS production were detected by annexin V-propidium iodide and DCFDA, respectively, using flow cytometry.
Results: High glucose concentration decreased cell viability, and increased apoptotic cells. Intracellular ROS production was also increased. In PC12 cells pretreatment and treatment by the drugs showed a significant improvement in cell viability and reduced apoptosis in captopril, telmisartan and PD123319 but only captopril and telmisartan were able to reduce ROS production. Losrtan significantly lowered ROS but didn’t show any improvements in cell viability and apoptotic cells.
Conclusion:  The results of the present study showed that RAS inhibitors reduced cell toxicity and apoptosis and ROS production was induced by high glucose. It may be suggested that local RAS has a role in high glucose toxicity.


1. Kamboj SS, Vasishta RK, Sandhir R. N-acetylcysteine inhibits hyperglycemia-induced oxidative stress and apoptosis markers in diabetic neuropathy. J Neurochem 2010; 112:77-91.

2. Zhang WF, Xu YY, Xu KP, Wu WH, Tan GS, Li YJ, et al. Inhibitory effect of selaginellin on high glucose-induced apoptosis in differentiated PC12 cells: role of NADPH oxidase and LOX-1. Eur J Pharmacol 2012; 694:60-68.

3. Mousavi SH, Tayarani NZ, Parsaee H. Protective effect of saffron extract and crocin on reactive oxygen species-mediated high glucose-induced toxicity in PC12 cells. Cell Mol Neurobiol 2010; 30:185–191.

4. Tamaddonfard E, Farshid AA, Asri-Rezaee S, Javadi S, Khosravi V, Rahman B, et al. Crocin improved learning and memory impairments in streptozotocin-induced diabetic rats. Iran J Basic Med Sci 2013; 16:91-100.

5. Peters J. Local renin-angiotensin systems in the adrenal gland. Peptides 2012; 34:427-432.

6. Sharifi AM, Eslami H, Larijani B, Davoodi J. Involvement of caspase-8, -9, and -3 in high glucose-induced apoptosis in PC12 cells. Neurosci Lett 2009; 459:47-51.

7. Moghadami M, Moghimi A, Jalal R, Behnam-Rasouli M, Mahdavi-Shahri N. Effects of infantile repeated hyperglycemia on neuronal density of hippocampus and pentylentetrazol induced convulsions in male wistar rats. Iran J Basic Med Sci 2012; 15:951-957.

8. Hsueh WA, Wyne K. Renin-Angiotensin-aldosterone system in diabetes and hypertension. J Clin Hypertens (Greenwich) 2011; 13:224-237.

9. Steckelings UM, Rompe F, Kaschina E, Unger T. The evolving story of the RAAS in hypertension, diabetes and CV disease: moving from macrovascular to microvascular targets. Fundam Clin Pharmacol 2009; 23:693-703.

10. Greene DA, Stevens MJ, Obrosova I, Feldman EL. Glucose-induced oxidative stress and programmed cell death in diabetic neuropathy. Eur J Pharmacol 1999; 375:217-223.

11. Coppey LJ, Davidson EP, Rinehart TW, Gellett JS, Oltman CL, Lund DD, et al. ACE inhibitor or angiotensin II receptor antagonist attenuates diabetic neuropathy in streptozotocin-induced diabetic rats. Diabetes 2006; 55:341-348.

12. Piga R, Naito Y, Kokura S, Handa O, Yoshikawa T. Protective effect of serotonin derivatives on glucose-induced damage in PC12 rat pheochromocytoma cells. Br J Nutr 2010; 103:25-31.

13. Sachse A, Wolf G. Angiotensin II-induced reactive oxygen species and the kidney. J Am Soc Nephrol 2007; 18:2439-2446.

14. Nishikawa T, Araki E. Impact of mitochondrial ROS production in the pathogenesis of diabetes mellitus and its complications. Antioxid Redox Signal 2007; 9:343-353.

15. Yu T, Robotham JL, Yoon Y. Increased production of reactive oxygen species in hyperglycemic conditions requires dynamic change of mitochondrial morphology. Proc Natl Acad Sci U S A 2006; 103:2653-2658.

16. Pop-Busui R, Sima A, Stevens M. Diabetic neuropathy and oxidative stress. Diabetes Metab Res Rev 2006; 22:257-273.

17. Edwards JL, Vincent AM, Cheng HT, Feldman EL. Diabetic neuropathy: mechanisms to management. Pharmacol Ther 2008; 120:1-34.

18. Sharifi AM, Mousavi SH, Farhadi M, Larijani B. Study of high glucose-induced apoptosis in PC12 cells: role of bax protein. J Pharmacol Sci 2007; 104:258-262.

19. Baltatu OC, Campos LA, Bader M. Local renin-angiotensin system and the brain--a continuous quest for knowledge. Peptides 2011; 32:1083-1086.

20. Paul M, Poyan Mehr A, Kreutz R. Physiology of local renin-angiotensin systems. Physiol Rev 2006; 86:747-803.

21. Wang JM, Slembrouck D, Tan J, Arckens L, Leenen FH, Courtoy PJ, et al. Presence of cellular renin-angiotensin system in chromaffin cells of bovine adrenal medulla. Am J Physiol Heart Circ Physiol 2002; 283:H1811-1818.

22. Wright JW, Harding JW. Brain renin-angiotensin--a new look at an old system. Prog Neurobiol 2011; 95:49-67.

23. Leung PS. The physiology of a local renin-angiotensin system in the pancreas. J Physiol 2007; 580:31-37.

24. Kumar R, Yong QC, Thomas CM, Baker KM. Intracardiac intracellular angiotensin system in diabetes. Am J Physiol Regul Integr Comp Physiol 2012; 302:R510-517.

25. Neutel JM. Effect of the renin-angiotensin system on the vessel wall: using ACE inhibition to improve endothelial function. J Hum Hypertens 2004; 18:599-606.

26. Chawla T, Sharma D, Singh A. Role of the renin angiotensin system in diabetic nephropathy. World J Diabetes 2010; 1:141-145.

27. Herr D, Bekes I, Wulff C. Local Renin-Angiotensin System in the Reproductive System. Front Endocrinol (Lausanne) 2013; 4:150.

28. Frigolet ME, Torres N, Tovar AR. The renin-angiotensin system in adipose tissue and its metabolic consequences during obesity. J Nutr Biochem 2013; 24:2003-2015.

29. Wolf G. "The road not taken": role of angiotensin II type 2 receptor in pathophysiology. Nephrol Dial Transplant 2002; 17:195-198.

30. Yorek MA. The potential role of angiotensin converting enzyme and vasopeptidase inhibitors in the treatment of diabetic neuropathy. Curr Drug Targets 2008; 9:77-84.

31. Forstermann U. Oxidative stress in vascular disease: causes, defense mechanisms and potential therapies. Nat Clin Pract Cardiovasc Med 2008; 5:338-349.

32. Yamagishi S, Amano S, Inagaki Y, Okamoto T, Inoue H, Takeuchi M, et al. Angiotensin II-type 1 receptor interaction upregulates vascular endothelial growth factor messenger RNA levels in retinal pericytes through intracellular reactive oxygen species generation. Drugs Exp Clin Res 2003; 29:75-80.

33. Hsieh TJ, Zhang SL, Filep JG, Tang SS, Ingelfinger JR, Chan JS. High glucose stimulates angiotensinogen gene expression via reactive oxygen species generation in rat kidney proximal tubular cells. Endocrinology 2002; 143:2975-2985.

34. Okamura T, Clemens DL, Inagami T. Generation of angiotensins in cultured pheochromocytoma cells. Neurosci Lett 1984; 46:151-156.

35. Meffert S, Stoll M, Steckelings UM, Bottari SP, Unger T. The angiotensin II AT2 receptor inhibits proliferation and promotes differentiation in PC12W cells. Mol Cell Endocrinol 1996; 122:59-67.

36. Wolf G, Harendza S, Schroeder R, Wenzel U, Zahner G, Butzmann U, et al. Angiotensin II's antiproliferative effects mediated through AT2-receptors depend on down-regulation of SM-20. Lab Invest 2002; 82:1305-1317.

37. Wolf G, Wenzel U, Burns KD, Harris RC, Stahl RA, Thaiss F. Angiotensin II activates nuclear transcription factor-kappaB through AT1 and AT2 receptors. Kidney Int 2002; 61:1986-1995.

38. Kamper M, Tsimpoukidi O, Chatzigeorgiou A, Lymberi M, Kamper EF. The antioxidant effect of angiotensin II receptor blocker, losartan, in streptozotocin-induced diabetic rats. Transl Res 2010; 156:26-36.

39. Bravenboer B, Kappelle AC, Hamers FP, van Buren T, Erkelens DW, Gispen WH. Potential use of glutathione for the prevention and treatment of diabetic neuropathy in the streptozotocin-induced diabetic rat. Diabetologia 1992; 35:813-817.

40. Allen DA, Yaqoob MM, Harwood SM. Mechanisms of high glucose-induced apoptosis and its relationship to diabetic complications. J Nutr Biochem 2005; 16:705-713.

41. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 2001; 414:813-820.

42. Brownlee M. The pathobiology of diabetic complications: a unifying mechanism. Diabetes 2005; 54:1615-1625.

43. Kasznicki J, Kosmalski M, Sliwinska A, Mrowicka M, Stanczyk M, Majsterek I, et al. Evaluation of oxidative stress markers in pathogenesis of diabetic neuropathy. Mol Biol Rep 2012; 39:8669-8678.

44. Malik RA, Williamson S, Abbott C, Carrington AL, Iqbal J, Schady W, et al. Effect of angiotensin-converting-enzyme (ACE) inhibitor trandolapril on human diabetic neuropathy: randomised double-blind controlled trial. Lancet 1998; 352:1978-1981.

45. Singh VP, Le B, Khode R, Baker KM, Kumar R. Intracellular angiotensin II production in diabetic rats is correlated with cardiomyocyte apoptosis, oxidative stress, and cardiac fibrosis. Diabetes 2008; 57:3297-3306.

46. Miller JA. Impact of hyperglycemia on the renin angiotensin system in early human type 1 diabetes mellitus. J Am Soc Nephrol 1999; 10:1778-1785.

47. Ali Q, Sabuhi R, Hussain T. High glucose up-regulates angiotensin II subtype 2 receptors via interferon regulatory factor-1 in proximal tubule epithelial cells. Mol Cell Biochem 2010; 344:65-71.

48. Shao J, Nangaku M, Inagi R, Kato H, Miyata T, Matsusaka T, et al. Receptor-independent intracellular radical scavenging activity of an angiotensin II receptor blocker. J Hypertens 2007; 25:1643-1649.

49. Saito M, Shinohara Y, Sasaki H, Netsu Y, Yoshida M, Nakahata N. Type 1 angiotensin receptor (AT1-R)-mediated decrease in type 2 angiotensin receptor mRNA level is dependent on Gq and extracellular signal-regulated kinase 1//2 in AT1-R-transfected PC12 cells. J Neuroendocrinol 2008; 20:299-308.

50. Goyal SN, Bharti S, Bhatia J, Nag TC, Ray R, Arya DS. Telmisartan, a dual ARB/partial PPAR-gamma agonist, protects myocardium from ischaemic reperfusion injury in experimental diabetes. Diabetes Obes Metab 2011; 13:533-541.

51. Yoshida T, Yamagishi S, Matsui T, Nakamura K, Ueno T, Takeuchi M, et al. Telmisartan, an angiotensin II type 1 receptor blocker, inhibits advanced glycation end-product (AGE)-elicited hepatic insulin resistance via peroxisome proliferator-activated receptor-gamma activation. J Int Med Res 2008; 36:237-243.

52. Garrido-Gil P, Joglar B, Rodriguez-Perez AI,
Guerra MJ, Labandeira-Garcia JL. Involvement of PPAR-gamma in the neuroprotective and anti-inflammatory effects of angiotensin type 1 receptor inhibition: effects of the receptor antagonist telmisartan and receptor deletion in a mouse MPTP model of Parkinson's disease. J Neuroinflammation 2012; 9:38.

53. Cianchetti S, Del Fiorentino A, Colognato R, Di Stefano R, Franzoni F, Pedrinelli R. Anti-inflammatory and anti-oxidant properties of telmisartan in cultured human umbilical vein endothelial cells. Atherosclerosis 2008; 198:22-28.

54. Yau H, Rivera K, Lomonaco R, Cusi K. The future of thiazolidinedione therapy in the management of type 2 diabetes mellitus. Curr Diab Rep 2013; 13:329-341.

55. Yahiro E, Miura S, Imaizumi S, Uehara Y, Saku K. Chymase inhibitors. Curr Pharm Des 2013;19:3065-3071.

56. White AT, Murphy AN. Administration of thiazolidinediones for neuroprotection in ischemic stroke: a pre-clinical systematic review. J Neurochem 2010; 115:845-853.

57. Kapadia R, Yi JH, Vemuganti R. Mechanisms of anti-inflammatory and neuroprotective actions of PPAR-gamma agonists. Front Biosci 2008; 13:1813-1826.

58. Fuenzalida K, Quintanilla R, Ramos P, Piderit D, Fuentealba RA, Martinez G, et al. Peroxisome proliferator-activated receptor gamma up-regulates the Bcl-2 anti-apoptotic protein in neurons and induces mitochondrial stabilization and protection against oxidative stress and apoptosis. J Biol Chem 2007; 282:37006-37015.