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Hany Naggar, M. Shamsul Ola, Pamela Moore, Wei Huang, Christy C. Bridges, Vadivel Ganapathy, Sylvia B. Smith; Downregulation of Reduced-Folate Transporter by Glucose in Cultured RPE Cells and in RPE of Diabetic Mice. Invest. Ophthalmol. Vis. Sci. 2002;43(2):556-563.
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purpose. The polarized distribution of reduced-folate transporter (RFT)-1 to the
apical retinal pigment epithelial (RPE) membrane was demonstrated
recently. Nitric oxide (NO) significantly decreases the activity of
RFT-1 in cultured RPE cells. NO is elevated in diabetes, and therefore
in the present study the alteration of RFT-1 activity in RPE under
conditions of high glucose was investigated.
methods. Human ARPE-19 cells were incubated in media containing 5 mM glucose
plus 40 mM mannitol (control) or 45 mM glucose for varying periods and
the activity of RFT-1 was assessed by determining the uptake of[ 3H]-N 5-methyltetrahydrofolate
(MTF). The levels of mRNA encoding RFT-1 were determined by RT-PCR and
protein levels by Western blot analysis. The activity of RFT-1 and
expression of mRNA encoding RFT-1 were analyzed also in RPE of
streptozotocin-induced diabetic mice.
results. Exposure of RPE cells to 45 mM glucose for as short an incubation time
as 6 hours resulted in a 35% decrease in MTF uptake. Kinetic analysis
showed that the hyperglycemia-induced attenuation was associated with a
decrease in the maximal velocity of the transporter with no significant
change in the substrate affinity. Semiquantitative RT-PCR demonstrated
that the mRNA encoding RFT-1 was significantly decreased in cells
exposed to high glucose, and Western blot analysis showed a significant
decrease in protein levels. The uptake of [3H]-MTF in RPE
of diabetic mice was reduced by approximately 20%, compared with that
in nondiabetic, age-matched control animals. Semiquantitative RT-PCR
demonstrated that the mRNA encoding RFT-1 was decreased significantly
in RPE of diabetic mice.
conclusions. These findings demonstrate for the first time that hyperglycemic
conditions reduce the expression and activity of RFT-1 and may have
profound implications for the transport of folate by RPE in
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