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

Hany Naggar, M. Shamsul Ola, Pamela Moore Martin, Wei Huang, Christy C. Bridges, Vadivel Ganapathy, Sylvia B Smith

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)556-563
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume43
Issue number2
StatePublished - Feb 11 2002

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Folic Acid Transporters
Reduced Folate Carrier Protein
Retinal Pigments
Down-Regulation
Epithelial Cells
Glucose
Messenger RNA
Polymerase Chain Reaction
Nitric Oxide
Western Blotting
Mannitol
Streptozocin
Folic Acid
Hyperglycemia
Proteins

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Downregulation of reduced-folate transporter by glucose in cultured RPE cells and in RPE of diabetic mice. / Naggar, Hany; Shamsul Ola, M.; Martin, Pamela Moore; Huang, Wei; Bridges, Christy C.; Ganapathy, Vadivel; Smith, Sylvia B.

In: Investigative Ophthalmology and Visual Science, Vol. 43, No. 2, 11.02.2002, p. 556-563.

Research output: Contribution to journalArticle

Naggar, Hany ; Shamsul Ola, M. ; Martin, Pamela Moore ; Huang, Wei ; Bridges, Christy C. ; Ganapathy, Vadivel ; Smith, Sylvia B. / Downregulation of reduced-folate transporter by glucose in cultured RPE cells and in RPE of diabetic mice. In: Investigative Ophthalmology and Visual Science. 2002 ; Vol. 43, No. 2. pp. 556-563.
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abstract = "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.",
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T1 - Downregulation of reduced-folate transporter by glucose in cultured RPE cells and in RPE of diabetic mice

AU - Naggar, Hany

AU - Shamsul Ola, M.

AU - Martin, Pamela Moore

AU - Huang, Wei

AU - Bridges, Christy C.

AU - Ganapathy, Vadivel

AU - Smith, Sylvia B

PY - 2002/2/11

Y1 - 2002/2/11

N2 - 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.

AB - 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.

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