Diabetes Metab J > Volume 30(5); 2006 > Article
Korean Diabetes Journal 2006;30(5):336-346.
DOI: https://doi.org/10.4093/jkda.2006.30.5.336    Published online September 1, 2006.
Inducible Nitric Oxide Synthase (iNOS) Expression in the Hypoxic Injury to Pancreatic Beta (MIN6) Cells.
Seung Hyun Ko, Seung Bum Kim, Kyung Ryul Ryu, Ji Won Kim, Yu Bai Ahn, Sung Dae Moon, Sung Rae Kim, Jung Min Lee, Hyuk Snag Kwon, Kun Ho Yoon, Ki Ho Song
Division of Endocrinology and Metabolism, Department of Internal Medicine, The Catholic University of Korea, Korea.
Abstract
BACKGROUND
Islet transplantation is an alternative potential strategy to cure type 1 diabetes mellitus. However, two or more donors are usually needed for one recipient because a substantial part of the graft becomes nonfunctional due to several factors including hypoxia. Though hypoxic exposure of pancreatic beta cells has been reported to induce apoptotic cell death, the molecular processes involved in hypoxia-induced cell death are poorly understood. In type I diabetes, Nitric Oxide (NO) is known as an important cytokine, involved in the pathogenesis of beta cell dysfunction. Pancreatic beta cells are sensitive to the induction of inducible nitric oxide synthase (iNOS) when stimulated by TNF-a or IL-1beta. But contribution of iNOS in response to hypoxia is not yet fully understood. METHODS: Mouse insulinoma cells (MIN6) were incubated in an anaerobic chamber (75% N2/15% CO2/5% H2) for up to 12 hours. Cell viability was measured after AO/PI staining. Caspase-3 activation was also determined using Western blot analysis. Nitric Oxide (NO) release into culture medium was measured using a Griess reagent. The expression of iNOS and PDX-1 mRNA and iNOS protein was examined using real time PCR and Western blot analysis. RESULTS: Marked cell death was observed within 6 hours after hypoxic exposure of MIN6 cells (control, < 5%; 2 hr, 11.0+/-7.6%; 6 hr, 46.2+/-12.8%, P < 0.05). Immunoreactivity to activated caspase-3 was observed at 2, 4 and 6 hrs. NO production was increased in a time dependent manner. Expression of iNOS mRNA and protein was significantly increased at 4 and 6 hour after hypoxia. iNOS expression was confirmed by immunostaining. Of note, Pdx-1 mRNA expression was markedly attenuated by hypoxic treatment. Pretreatment with a selective iNOS inhibitor, 1400 W, significantly prevented beta cell death induced by hypoxic injury. CONCLUSION: Our data suggest that iNOS-NO play an important role in hypoxic injury to MIN6 cells. Therefore, iNOS-NO might be a potential therapeutic target for improving engraftment of the transplanted islets and suppression of iNOS would be helpful for prevention of beta cells damage to hypoxic injury.
Key Words: Caspase-3, Cell death, Diabetes mellitus, Hypoxia, Inducible nitric oxide synthase (iNOS), Islet transplantation
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