High concentrations of glucose induce synthesis of argpyrimidine in retinal endothelial cells.
OBJECTIVE
To determine if high concentrations of glucose applied to cultured bovine retinal endothelial cells (BRE cells) would result in production of intracellular methylglyoxal (MG), and consequently, the synthesis of MG-derived advanced glycation end products (AGEs).


METHODS
BRE cells were incubated with 30 mM D-glucose or 30 mM L-glucose for 7 days. Cells incubated with medium that had 5-mM glucose served as controls. Cells were lysed and the lysate was centrifuged to get a supernatant and a pellet fraction. We measured argpyrimidine, a MG-derived fluorescent AGE in the two fractions by a competitive ELISA using a monoclonal antibody.


RESULTS
BRE cells incubated with 30 mM D-glucose produced significantly higher (P < 0.05) levels of intracellular MG than control cells or cells incubated with 30 mM L-glucose. Incubation with 30 mM D-glucose significantly (P < 0.05) enhanced the synthesis of argpyrimidine in both supernatant and pellet fractions when compared to control cells. Immunofluorescence studies confirmed results obtained by ELISA and showed higher levels of argpyrimidine in cells incubated with 30 mM D-glucose.


CONCLUSIONS
These results suggest that MG-mediated protein modification can occur in elevated glucose, and might contribute to endothelial cell changes associated with diabetic retinopathy.
Padayatti
P S
PS
Center for Vision Research, Department of Ophthalmology and Center for Diabetes Research, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH, USA.
Jiang
C
C
Glomb
M A
MA
Uchida
K
K
Nagaraj
R H
RH
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005947:Glucose / Q000494:pharmacology*; D017127:Glycation End Products, Advanced / Q000096:biosynthesis; D015416:Maillard Reaction; D018613:Microscopy, Confocal; D009952:Ornithine / Q000031:analogs & derivatives* / Q000096:biosynthesis*; D011743:Pyrimidines / Q000096:biosynthesis*; D011765:Pyruvaldehyde / Q000378:metabolism; D012171:Retinal Vessels / Q000187:drug effects* / Q000378:metabolism
D017127:Glycation End Products, Advanced; D011743:Pyrimidines; C107313:argpyrimidine; D011765:Pyruvaldehyde; D009952:Ornithine; D005947:Glucose