Increased JNK phosphorylation and oxidative stress in response to increased glucose flux through increased GLUT1 expression in rat retinal endothelial cells.
OBJECTIVE
To investigate whether increased glucose flux through increased glucose transporter1 (GLUT1) expression results in increased oxidative stress and increased c-jun N-terminal kinase (JNK) phosphorylation.


METHODS
GLUT1-overexpressing cells were established using a rat retinal endothelial cell line. The intracellular reactive oxygen species was detected by the oxidation of 5- (and -6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2-DCFDA). Western blot was performed to determine JNK phosphorylation and lipid peroxidation. Differentially expressed genes were detected by cDNA microarray analysis and confirmed by Northern blot analysis.


RESULTS
Clones overexpressing GLUT1 showed an approximate four- to eightfold increase in GLUT1 expression and a 44% increase in intracellular glucose concentrations. GLUT1-overexpressing cells had a 80% increase in DCF fluorescence and increased lipid peroxidation, as well as increased JNK phosphorylation. Analysis of differentially expressed genes in GLUT1-overexpressing cells showed increased expression of JNK interacting protein (JIP)-1, a scaffold protein necessary for JNK activation. Northern blot analysis confirmed upregulation of JIP-1. Immunoprecipitation showed that phosphorylated JNK, but not total JNK, coimmunoprecipitated with JIP-1 protein. At the cellular level, JIP-1 was predominantly localized in cytoplasm, especially in the perinuclear area in retinal endothelial cells.


CONCLUSIONS
GLUT1 overexpression and increased glucose flux result in increased oxidative stress and JNK phosphorylation in immortalized rat retinal endothelial cells. Further studies are needed to understand molecular events after increased glucose flux in retinal endothelial cells and the relation between increased oxidative stress and JNK phosphorylation.
Zhou
Jie
J
Departments of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Deo
Baljit K
BK
Hosoya
Kenichi
K
Terasaki
Tetsuya
T
Obrosova
Irina G
IG
Brosius
Frank C
FC
Kumagai
Arno K
AK
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D020869:Gene Expression Profiling; D005947:Glucose / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D015227:Lipid Peroxidation; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D018384:Oxidative Stress; D010766:Phosphorylation; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D012171:Retinal Vessels / Q000166:cytology*
D048868:Adaptor Proteins, Signal Transducing; D051272:Glucose Transporter Type 1; C110528:Mapk8ip1 protein, rat; D009004:Monosaccharide Transport Proteins; D017382:Reactive Oxygen Species; C495679:Slc2a1 protein, rat; D048031:JNK Mitogen-Activated Protein Kinases; D005947:Glucose