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Douglas N. Henry, Robert N. Frank, Seth R. Hootman, Sandra E. Rood, Charles W. Heilig, Julia V. Busik; Glucose-Specific Regulation of Aldose Reductase in Human Retinal Pigment Epithelial Cells In Vitro. Invest. Ophthalmol. Vis. Sci. 2000;41(6):1554-1560.
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purpose. To test the hypothesis that pathophysiological levels of glucose
regulate aldose reductase (AR2) gene expression, protein
production, and activity in human retinal pigment epithelial (RPE)
cells in vitro.
methods. Primary cultures of human RPE cells were grown for up to 72 hours
in media supplemented with various concentrations of glucose (5, 20, or
75 mM), or in 5 mM glucose containing media supplemented with one of
the following: galactose, the transported but nonmetabolized glucose
analogue 3-O-methylglucose (3-OMG), or the impermeant
hexitol mannitol—so that the final hexose concentrations were
equimolar to those of the various glucose concentrations used. Changes
in the transcript levels for AR2 mRNA, AR2 protein content, and AR2
enzyme activity were determined. RPE glucose utilization and lactate
production were determined in media containing 5 and 20 mM glucose.
results. Glucose utilization and lactate production increased 4.8-fold and
4.4-fold, respectively, when RPE cells were grown in media containing
20 mM versus 5 mM glucose. Glucose was more effective than any other
hexose in the induction of AR2 mRNA or increased AR2 protein
expression. When RPE cells were grown in media containing 20 mM
mannitol, 3-OMG, or galactose they had lower levels of AR2 mRNA
expression than when cells were grown in medium containing 5 mM
glucose. RPE cells grown in medium supplemented with 20 or 75 mM
galactose did not show a greater increase in AR2 protein expression
than cells grown in medium containing 5 mM glucose. Hyperosmotic
induction of AR2 mRNA was the same in medium containing 75 mM glucose
or 75 mM mannitol, but was at least 50% lower when RPE cells were
grown in 75 mM galactose or 3-OMG.
conclusions. These data indicate that elevations in ambient glucose result in
greater metabolism of glucose through glycolysis and polyol metabolism.
Induction of AR2 was greatest when RPE cells were grown in
pathophysiological concentrations of glucose. Hyperosmolar stress is
not a necessary determinant of AR2 mRNA, AR2 protein, or AR2 protein
activity in cells that form the outer blood–retinal barrier. Increased
facilitative glucose transport or glucose metabolism appears to be
requisite for glucose-specific and nonosmotic regulation of AR2 in the
RPE cell in vitro.
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