Downregulation of reduced-folate transporter by glucose in cultured RPE cells and in RPE of diabetic mice

PURPOSE. The polarized distribution of reduced-folate transporter (RFT)-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]-N⁵-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 diabetes.