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Glucose-Specific Regulation of Aldose Reductase in Human Retinal Pigment Epithelial Cells In Vitro

From the Departments of ¹ Physiology and ² Pediatrics and Human Development, College of Human Medicine, Michigan State University, East Lansing; ³ Kresge Eye Institute, Wayne State University School of Medicine, Detroit, Michigan; and ⁴ Department of Internal Medicine, Division of Nephrology, Johns Hopkins School of Medicine, Baltimore, Maryland.

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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