High glucose concentration induces elevated expression of anti-oxidant and proteolytic enzymes in cultured human retinal pigment epithelial cells.
We investigated the differential protein expression patterns of retinal pigment epithelial (RPE) cells exposed to increased glucose concentrations. Cultured human RPE cells (ARPE-19) were exposed for 4 days with normal blood glucose concentration (5.5 mM D-glucose), followed by exposure to either normal (5.5 mM) or high (33 mM) concentrations of D-glucose for 48h. Protein extracts of glucose-treated RPE cells were then subjected to comparative proteome analysis based on 2-D gel electrophoresis. Protein spots were visualized by silver staining. The differentially expressed proteins were excised and digested in-gel with trypsin, then analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The expression levels of cathepsin B, glutathione peroxidase and heat shock protein 27 were increased, and that of protein disulfide isomerase decreased in high glucose treated RPE compared to normal glucose. The isoelectric point of copper/zinc-containing superoxide dismutase (Cu/Zn-SOD) shifted toward acidic region in response to high glucose. Cu/Zn-SOD activity in high glucose group was significantly lower than that in normal glucose group (P<0.05, Mann-Whitney U-test). Systematic survey of protein expression has revealed that RPE cells respond to acute, pathologically high glucose levels by the elevated expression of anti-oxidant and proteolytic enzymes.
Yokoyama
Tomoko
T
Department of Ophthalmology and Visual Science, Graduate School of Biomedical Sciences, Hiroshima University 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
Yamane
Ken
K
Minamoto
Atsushi
A
Tsukamoto
Hidetoshi
H
Yamashita
Hidetoshi
H
Izumi
Shunsuke
S
Hoppe
George
G
Sears
Jonathan E
JE
Mishima
Hiromu K
HK
D000975:Antioxidants / Q000032:analysis*; D015153:Blotting, Western; D002401:Cathepsin B / Q000032:analysis; D002460:Cell Line; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D005947:Glucose / Q000494:pharmacology*; D005978:Glutathione / Q000032:analysis; D006360:Heat-Shock Proteins / Q000032:analysis; D006801:Humans; D007091:Image Processing, Computer-Assisted; D010447:Peptide Hydrolases / Q000032:analysis*; D010449:Peptide Mapping; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000201:enzymology*; D019704:Protein Disulfide-Isomerases / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018709:Statistics, Nonparametric; D013482:Superoxide Dismutase / Q000032:analysis
D000975:Antioxidants; D003470:Culture Media; D006360:Heat-Shock Proteins; D013482:Superoxide Dismutase; D010447:Peptide Hydrolases; D002401:Cathepsin B; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D005947:Glucose