Inducible nitric oxide synthase-nitric oxide plays an important role in acute and severe hypoxic injury to pancreatic beta cells.
BACKGROUND
Islet transplantation is a potential strategy to cure type 1 diabetes mellitus. However, a substantial part of the islet graft becomes nonfunctional due to several factors including hypoxia. However, the precise mechanism of cell damage is largely unknown in hypoxic exposure to pancreatic beta cells. The aim of the present study was to investigate whether acute and severe hypoxic injury could involve inducible nitric oxide synthase (iNOS)-nitric oxide (NO) signaling in beta cells.


METHODS
The rat beta cell line (INS-1) and primary rat islets were incubated in an anoxic chamber. Cell viability was determined by propium iodide staining or cell counting kit. The expression of iNOS mRNA and protein was examined using reverse-transcription polymerase chain reaction and Western blot analysis. NO production was measured as nitrite accumulation by Griess reagent method.


RESULTS
After hypoxic exposure, marked cell death occurred in INS-1 cells and rat islets, accompanied by increase in activated caspase-3 expression. NO production was increased in the culture medium in a time-dependent manner. Increase in expression of iNOS mRNA and protein was found. Pretreatment with a selective iNOS inhibitor, 1400W, significantly prevented cell death during hypoxia. In addition, hypoxia activated c-Jun N-terminal kinase (JNK) significantly, but the addition of 1400W inhibited hypoxia-induced JNK phosphorylation.


CONCLUSIONS
Our data suggest that iNOS-NO plays an important role in acute and severe hypoxic injury to pancreatic beta cells. Therefore, iNOS-NO might be a potential therapeutic target for preserving beta cell survival in islet transplantation through prevention of hypoxia-mediated cell death.
Ko
Seung-Hyun
SH
Division of Endocrinology & Metabolism, Department of Internal Medicine, The Catholic University of Korea, Seoul, Korea.
Ryu
Gyeong Ryul
GR
Kim
SeungBum
S
Ahn
Yu-Bae
YB
Yoon
Kun-Ho
KH
Kaneto
Hideaki
H
Ha
Hunjoo
H
Kim
Yu Seun
YS
Song
Ki-Ho
KH
D000818:Animals; D053148:Caspase 3 / Q000378:metabolism; D015687:Cell Hypoxia; D002469:Cell Separation; D002470:Cell Survival; D002478:Cells, Cultured; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000201:enzymology*; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
C497444:Hif1a protein, rat; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D020928:Mitogen-Activated Protein Kinases; D053148:Caspase 3