Comparison of inducible nitric oxide synthase activity in pancreatic islets of young and aged rats

Document Type: Original Article


1 Department of Physiology and Biophysics, Faculty of Medicine, Baqiyatallah University of Medical Sciences, Tehran, Iran

2 Department of Marine Living Sciences, Iranian National Institute for Oceanography and Atmospheric Sciences (INIOAS), Tehran, Iran

3 Endocrine Physiology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran

4 Chemical Injuries Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran


Objective(s):Some pathologic situations such as diabetes and metabolic syndrome are associated with alternation in nitric oxide level. Incidence of these condition increases with aging. On the other hand, insulin secretion is modulated by nitric oxide, and nitric oxide synthase (NOS) activity is also altered in diabetes. In this study, modification in the enzyme activity associated with aging and also optimized procedure for islet NOS assay was investigated.
Materials and Methods: Male Wistar rats were randomly divided in two experimental groups: A: adult rats; were 4 month old and B: old rats; were 12 month old. In all groups, plasma glucose, insulin and NOX (nitrite + nitrate = NOX) were measured, and also insulin secretion in isolated pancreatic islet with or without L-NAME was investigated. Furthermore, the inducible NOS activity with L-citrulline measurement in islets was measured.
Results: L-citrulline was quantified using one step HPLC column. Aging induced hyperglycemia (P<0.05) and excess plasma NOX (17.74 ± 1.664 and 26.25 ± 2.166 μmol/l in A and B groups respectively, P<0.05) with unaltered plasma insulin. Islet insulin secretion was significantly reduced in aging rats. L-NAME induced islet insulin secretion especially in aging rats (P=0.003). Inducible NOS activity in islets of aging rats was significantly higher than adult rats (1.082 ± 0.084 and 6.277 ± 0.475 pmol/min per mg protein in adult and aging rats, respectively, P<0.001).
Conclusion: These findings show that decreased in islet insulin secretion may be related to increase in iNOS activity in islets, which follows impaired carbohydrate metabolism in aging.


1. Nathan C. Inducible nitric oxide synthase: what difference does it make? J Clin Invest 1997; 100:2417-2423.

2. Knowles RG, Moncada S. Nitric oxide synthases in mammals. Biochem J 1994; 298:249-258.

3. Lajoix AD, Reggio H, Chardes T, Peraldi-Roux S, Tribillac F, Roye M, et al. A neuronal isoform of nitric oxide synthase expressed in pancreatic beta-cells controls insulin secretion. Diabetes 2001; 50:1311-1323.

4. Soskic SS, Dobutovic BD, Sudar EM, Obradovic MM, Nikolic DM, Djordjevic JD, et al. Regulation of inducible nitric oxide synthase (inos) and its potential role in insulin resistance, diabetes and heart failure. Open Cardiovasc Med J 2011; 5:153-163.

5. Bedoya FJ, Salguero-Aranda C, Cahuana GM, Tapia-Limonchi R, Soria B, Tejedo JR. Regulation of pancreatic beta-cell survival by nitric oxide: clinical relevance. Islets 2012; 4:108-118.

6. Quintana-Lopez L, Blandino-Rosano M, Perez-Arana G, Cebada-Aleu A, Lechuga-Sancho A, Aguilar-Diosdado M, et al. Nitric oxide is a mediator of antiproliferative effects induced by proinflammatory cytokines on pancreatic beta cells. Mediators Inflamm 2013; 2013:905175.

7. Zhou JL, Fang HS, Peng H, Hu QJ, Liu SQ, Ming JH, et al. PKCa agonists enhance the protective effect of hyaluronic acid on nitric oxide-induced apoptosis of articular chondrocytes in vitro. Iran J Basic Med Sci 2013; 16:1276-1281.

8. Ghasemi A, Zahediasl S. Is nitric oxide a hormone? Iran Biomed J 2011; 15:59-65.

9. Kazeem A, Olubayo A, Ganiyu A. Plasma nitric oxide and acute phase proteins after moderate and prolonged xercises. Iran J Basic Med Sci 2012; 15:602-607.

10. Muller DC, Elahi D, Tobin JD, Andres R. Insulin response during the oral glucose tolerance test: the role of age, sex, body fat and the pattern of fat distribution. Aging (Milano) 1996; 8:13-21.

11. Wu D, Yue F, Zou C, Chan P, Alex Zhang Y. Analysis of glucose metabolism in cynomolgus monkeys during aging. Biogerontology 2012; 13:147-155.

12. Salehi A, Meidute Abaraviciene S, Jimenez-Feltstrom J, Ostenson CG, Efendic S, Lundquist I. Excessive islet NO generation in type 2 diabetic GK rats coincides with abnormal hormone secretion and is counteracted by GLP-1. PLoS One 2008; 3: e2165.

13. McCann SM, Mastronardi C, de Laurentiis A, Rettori V. The nitric oxide theory of aging revisited. Ann N Y Acad Sci 2005; 1057:64-84.

14. Zamora R, Vodovotz Y, Billiar TR. Inducible nitric oxide synthase and inflammatory diseases. Mol Med 2000; 6:347-373.

15. Nussler AK, Billiar TR. Inflammation, immunoregulation, and inducible nitric oxide synthase. J Leukoc Biol 1993; 54: 171-178.

16. Shafiee-Nick R, Ghorbani A, Vafaee Bagheri F, Rakhshandeh H. Chronic administration of a combination of six herbs inhibits the progression of hyperglycemia and decreases serum lipids and aspartate amino transferase activity in diabetic rats. Adv Pharmacol Sci 2012; 2012:789796.

17. Hindlycke M, Jansson L. Glucose tolerance and pancreatic islet blood flow in rats after intraperitoneal administration of different anesthetic drugs. Ups J Med Sci 1992; 97:27-35.

18. Carlberg M. Assay of neuronal nitric oxide synthase by HPLC determination of citrulline. J Neurosci Methods 1994; 52:165-167.

19. Zardooz H, Zahedi Asl S, Gharib Naseri MK, Hedayati M. Effect of chronic restraint stress on carbohydrate metabolism in rat. Physiol Behav 2006; 89:373-378.

20. Akesson B, Henningsson R, Salehi A, Lundquist I. Islet constitutive nitric oxide synthase and glucose regulation of insulin release in mice. J Endocrinol 1999; 163:39-48.

21. Miranda KM, Espey MG, Wink DA. A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide 2001; 5:62-71.

22. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248-254.

23. Chang AM, Halter JB. Aging and insulin secretion. Am J Physiol Endocrinol Metab 2003; 284:E7-12.

24. Alusik S, Jedlickova V, Paluch Z, Zecova S. Plasma levels of nitrite/nitrate and inflammation markers in elderly individuals. Bratisl Lek Listy 2008; 109:289-292.

25. Ghasemi A, Zahedi Asl S, Mehrabi Y, Saadat N, Azizi F. Serum nitric oxide metabolite levels in a general healthy population: relation to sex and age. Life Sci 2008; 83:326-331.

26. Reckelhoff JF, Kellum JA, Blanchard EJ, Bacon EE, Wesley AJ, Kruckeberg WC. Changes in nitric oxide precursor, L-arginine, and metabolites, nitrate and nitrite, with aging. Life Sci 1994; 55: 895-902.

27. Basu R, Breda E, Oberg AL, Powell CC, Dalla Man C, Basu A, et al. Mechanisms of the age-associated deterioration in glucose tolerance: contribution of alterations in insulin secretion, action, and clearance. Diabetes 2003; 52:1738-1748.

28. Iozzo P, Beck-Nielsen H, Laakso M, Smith U, Yki-Jarvinen H, Ferrannini E. Independent influence of age on basal insulin secretion in nondiabetic humans. European Group for the Study of Insulin Resistance. J Clin Endocrinol Metab 1999; 84:863-868.

29. Perfetti R, Rafizadeh CM, Liotta AS, Egan JM. Age-dependent reduction in insulin secretion and insulin mRNA in isolated islets from rats. Am J Physiol 1995; 269:E983-980.

30. Reaven E, Wright D, Mondon CE, Solomon R, Ho H, Reaven GM. Effect of age and diet on insulin secretion and insulin action in the rat. Diabetes 1983; 32:175-180.

31. Henningsson R, Salehi A, Lundquist I. Role of nitric oxide synthase isoforms in glucose-stimulated insulin release. Am J Physiol Cell Physiol 2002; 283:C296-304.

32. Yang B, Larson DF, Watson RR. Modulation of iNOS activity in age-related cardiac dysfunction. Life Sci 2004; 75:655-667.

33. Chou TC, Yen MH, Li CY, Ding YA. Alterations of nitric oxide synthase expression with aging and hypertension in rats. Hypertension 1998; 31:643-648.

34. Broniowska KA, Oleson BJ, Corbett JA. beta-cell responses to nitric oxide. Vitam Horm 2014; 95:299-322.

35. Alonso-Fernandez P, De la Fuente M. Role of the immune system in aging and longevity. Curr Aging Sci 2011; 4:78-100.

36. Salter M, Knowles RG. Assay of NOS activity by the measurement of conversion of oxyhemoglobin to methemoglobin by NO. Methods Mol Biol 1998; 100:61-65.

37. Ekelund M, Qader SS, Jimenez-Feltstrom J, Salehi A. Selective induction of inducible nitric oxide synthase in pancreatic islet of rat after an intravenous glucose or intralipid challenge. Nutrition 2006; 22:652-660.

38. Wu G, Meininger CJ. Analysis of citrulline, arginine, and methylarginines using high-performance liquid chromatography. Methods Enzymol 2008; 440: 177-189.

39. Mohammadpour AH, Ramezani M, Tavakoli Anaraki N, Malaekeh-Nikouei B, Amel Farzad S, Hosseinzadeh H. Development and Validation of HPLC Method for Determination of Crocetin, a constituent of Saffron, in Human Serum Samples. Iran J Basic Med Sci 2013; 16:47-55.

40. Schwarz EL, Roberts WL, Pasquali M. Analysis of plasma amino acids by HPLC with photodiode array and fluorescence detection. Clin Chim Acta 2005; 354:83-90.