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. 2002 Feb;43(2):556-63.

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

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

Hany Naggar et al. Invest Ophthalmol Vis Sci. 2002 Feb.

Abstract

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 diabetes.

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Figures

FIGURE 1
FIGURE 1
Time course of attenuation of MTF uptake in the presence of high glucose. ARPE-19 cells were exposed to 45 mM glucose for various lengths of time, and the uptake of [3H]-MTF (3 nM) was determined. Parallel experiments were performed with cells cultured in the presence of 5 mM glucose plus 40 mM mannitol (osmolar control). Results are expressed as the percentage of MTF uptake measured in corresponding control cells not treated with high glucose. Data are the mean ± SEM of four determinations from two independent experiments. *Significantly different from control (P < 0.05).
FIGURE 2
FIGURE 2
Specificity of glucose-induced attenuation of MTF uptake. ARPE-19 cells were exposed to 45 mM glucose for 6 hours before measuring the uptake of [3H]-MTF (3 nM), [3H]-glutamine (25 nM), or [14C]-ascorbic acid (3 μM). Parallel experiments were performed with cells cultured for 6 hours in the presence of 5 mM glucose plus 40 mM mannitol (osmolar control). Results are expressed as the percentage of MTF uptake measured in corresponding control cells not treated with high glucose. Data are the mean ± SEM of four determinations from two independent experiments. *Significantly different from control (P < 0.05).
FIGURE 3
FIGURE 3
Dose–response relationship showing the effect of high glucose on the uptake of MTF. ARPE-19 cells were exposed for 6 hours to 15, 25, 35, or 45 mM glucose, after which the uptake of [3H]-MTF (3 nM) was measured. Parallel experiments were performed with cells cultured 6 hours in the presence of 5 mM glucose plus 10, 20, 30, or 40 mM mannitol (osmolar controls). Results are expressed as the percentage of MTF uptake measured in corresponding control cells not treated with high glucose. Data are the mean ± SEM of four determi-nations from two independent experiments. *Significantly different from control (P < 0.05).
FIGURE 4
FIGURE 4
Kinetic analysis of MTF uptake in ARPE-19 cells treated with high levels of glucose. ARPE-19 cells were treated with 45 mM glucose for 6 hours. Parallel experiments were performed with cells cultured 6 hours in the presence of 5 mM glucose plus 40 mM mannitol (osmolar control). Uptake of MTF was measured in these cells for 30 minutes over an MTF concentration range of 0.05 to 1 μM. Data are expressed as the mean ± SEM of three determinations from one independent experiment. Results are presented as plots describing the relationship between MTF concentration and MTF uptake rate and also as Eadie-Hofstee plots (inset; V/S versus V) V, MTF uptake in picomoles per milligram of protein per 30 minutes; S, MTF micromolar concentration.
FIGURE 5
FIGURE 5
Analysis of steady state levels of mRNA for RFT-1 and GAPDH in ARPE-19 cells exposed to high levels of glucose. ARPE-19 cells were treated with 45 or 5 mM glucose plus 40 mM mannitol (control) for 6, 12, and 24 hours at 37°C. Total RNA was then isolated from these cells and used for semiquantitative RT-PCR. Primer pairs specific for human RFT-1 and GAPDH mRNA were used. RT-PCR was performed with a wide range of PCR cycles (n = 9–32). The resultant products were run on a gel and then subjected to Southern hybridization with 32P-labeled cDNA probes specific for RFT-1 and GAPDH. The hybridization signals were quantified by phosphorescence imaging, and the intensities that were in the linear range with the PCR cycle number were used for analysis. (A) Representative Southern hybridization signal of bands from the 18th cycle for RFT-1 and the 15th cycle for GAPDH. (B) Relative band density (RFT-1/GAPDH) in cells treated with high glucose relative to that in control cells. The RFT-1-to-GAPDH ratio in control cells was taken as 1. Data are the mean ± SEM of three determinations from two independent experiments. *Significantly different from control (P < 0.05).
FIGURE 6
FIGURE 6
Western blot analysis of RFT-1 in ARPE-19 cells exposed to high glucose for 6 and 24 hours. ARPE-19 cells were exposed to 45 mM glucose for 6 or 24 hours and lysed and the lysate subjected to SDS-PAGE followed by immunoblot analysis with a polyclonal antibody recognizing RFT-1. Membranes were washed and reprobed with an antibody against β-actin. Parallel experiments were performed with cells cultured in the presence of 5 mM glucose plus 40 mM mannitol (osmolar control). The density of the bands was quantified with a phosphorescence imaging system. (A) Immunoblot from a representative experiment showing two loading concentrations: 10 and 20 μg. (B) Band intensity from densitometric scans (RFT-1/β-actin) in cells treated with high glucose relative to that in control cells for both loading concentrations. The RFT-1-to-β-actin ratio in control cells was taken as 1. Data are the mean ± SEM of two determinations from four independent experiments. *Significantly different from control (P < 0.05).
FIGURE 7
FIGURE 7
Uptake of MTF by RPE of normal and diabetic mice. C57BL/6 mice were made diabetic using three consecutive 75 mg/kg doses of STZ. At 12 weeks after onset of diabetes, the RPE was dissected from remaining ocular tissues. RPE from age-matched C57BL/6 mice was used as the control. Incubation of RPE was performed at 37°C for 30 minutes in folate-free RPMI 1640 medium supplemented with [3H]-MTF (3 nM). Results are the mean ± SEM (n = 12). Data are the mean ± SEM of six determinations from two independent experiments. *Significantly different from control (P < 0.05).
FIGURE 8
FIGURE 8
Analysis of steady state levels of mRNA for RFT-1 and GAPDH in RPE of control (C) and diabetic (D) mice. C57BL/6 mice were made diabetic using three consecutive 75-mg/kg doses of STZ. At 3, 6, and 12 weeks after onset of diabetes, mice were killed, and the RPE was dissected from the adjacent neural retina. Age-matched control mice were used in parallel. Total RNA was isolated from the RPE and used for semiquantitative RT-PCR. Primer pairs specific for mouse RFT-1 and mouse GAPDH mRNA were used, and RT-PCR performed as described in Figure 5. (A) Representative Southern hybridization signal showing bands from the 21st cycle of RT-PCR for RFT-1 and 15th cycle for GAPDH. (B) Band intensity (RFT-1/GAPDH) in diabetic RPE relative to that in control RPE. The RFT-1-to-GAPDH ratio in control cells was taken as 1. Data are the mean ± SEM of three determinations from two independent experiments. *Significantly different from control (P < 0.05).

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