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K.G. Shadrach, P. Senanayake, K. Nishiyama, J.W. Lee, J.G. Hu, A. Calabro, D. Bok, J.G. Hollyfield; Glucose Utilization by Human RPE Cultures . Invest. Ophthalmol. Vis. Sci. 2003;44(13):394.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To evaluate the distribution of glucose transporters (GLUT) and glucose utilization by confluent retinal pigment epithelium (RPE) cultures that have established high resistance junctions Methods: . Human RPE was cultured in Millicell- [PCF] culture plates in medium containing 1 mg/ml glucose. Efficiency of separation of the apical and basal compartments was determined by transepithelial resistance measurements. Glucose utilization was evaluated with glucose either in the apical (1) or basal (2) medium or both (3). Cells with associated matrices (CM), and apical and basal media (Am, Bm) were collected, digested with proteinase K, ethanol precipitated and derivatized with the fluorescent tag 2-aminoacridone (AMAC). After separation by electrophoresis, the AMAC labeled bands were digitized and their fluorescent intensities quantitated. GLUT 1, 3 and 5 expression were evaluated with RT-PCR and GLUT 1, 2 and 3 were localized with immunocytochemistry using confocal imaging. Results: (1), glucose decreased by 50% from the Am in 1 h and was depleted in 24 h. Glucose reached the Bm in 1 h and remained detectable for 15 h. (2), glucose decreased by 50% from the Bm in 2 h and was depleted in 48 h. Glucose reached the Am in 2 h and remained detectable in the for 65 h. (3), glucose decreased by 50% from the Am and Bm in 16 and 7 h respectively and was depleted from both in 72 h. Expression of GLUT 1, 3 and 5 were detected by RTR-PCR . GLUT 1 was the dominant isoform expressed. GLUT 3 and 5 showed very low levels of expression. Confocal imaging also showed that GLUT 1, GLUT 2 and GLUT 3 were present in the RPE cultures. The transporters were localized preferentially on the apical side of the RPE cells. The density of GLUT 1 was significantly higher than GLUT 2 and 3. Conclusions: Glucose is utilized rapidly from both Am and Bm. Restricting glucose to either Am or Bm increased the rapidity of its utilization by the RPE cells. These data demonstrate bi-directional transport of glucose by the RPE cell cultures. Preferential apical localization of GLUT transporters may account for the increased, sustained levels of glucose in the Am when glucose is applied only to the basal surface of the RPE
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