Downregulation of reduced-folate transporter by glucose in cultured RPE cells and in RPE of diabetic mice.
OBJECTIVE
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 diabetes.
Naggar
Hany
H
Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia 30912-2000, USA.
Ola
M Shamsul
MS
Moore
Pamela
P
Huang
Wei
W
Bridges
Christy C
CC
Ganapathy
Vadivel
V
Smith
Sylvia B
SB
D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism*; D015536:Down-Regulation; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013763:Tetrahydrofolates / Q000378:metabolism
D002352:Carrier Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; C120043:SLC19A2 protein, human; C492292:Slc19a2 protein, mouse; D013763:Tetrahydrofolates; D005947:Glucose; C005984:5-methyltetrahydrofolate